AFAB-Vol.1-Issue 1

INAUGURAL ISSUE ISSN: 2159-8967 www.AFABjournal.com

Volume 1, Issue 1 May 2011

EDITORIAL BOARD Sooyoun Ahn

W.K. Kim

Arkansas State University, USA

University of Manitoba, Canada

Walid Q. Alali

M.B. Kirkham

University of Georgia, USA

Kansas State University, USA

Kenneth M. Bischoff

Todd Kostman

NCAUR, USDA-ARS, USA

University of Wisconsin, Oshkosh, USA

Claudia S. Dunkley

Y.M. Kwon

University of Georgia, USA

University of Arkansas, USA

Lawrence Goodridge

Maria Luz Sanz

Colorado State University, USA

MuriasInstituto de Quimica Organic General, Spain

Leluo Guan

Melanie R. Mormile

University of Alberta, Canada

Missouri University of Science and Tech., USA

Joshua Gurtler

Rama Nannapaneni

ERRC, USDA-ARS, USA

Mississippi State University, USA

Yong D. Hang

Jack A. Neal, Jr.

Cornell University, USA

University of Houston, USA

Divya Jaroni

Benedict Okeke

Southern University, USA

Auburn University at Montgomery, USA

Weihong Jiang Shanghai

John Patterson

Institute for Biol. Sciences, P.R. China

Purdue University, USA

Michael Johnson

Toni Poole

University of Arkansas, USA

FFSRU, USDA-ARS, USA

Timothy Kelly

Marcos Rostagno

East Carolina University, USA

LBRU, USDA-ARS, USA

William R. Kenealy

Roni Shapira

Mascoma Corporation, USA

Hebrew University of Jerusalem, Israel

Hae-Yeong Kim

Kalidas Shetty

Kyung Hee University, South Korea

University of Massachusetts, USA

2 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

EDITORIAL STAFF EDITOR-IN-CHIEF

MANAGING EDITOR

Steven C. Ricke University of Arkansas, USA

Ellen J. Van Loo Fayetteville Arkansas, USA

EDITORS

TECHNICAL EDITOR

Todd R. Callaway FFSRU, USADA-ARS, USA

Jessica C. Shabatura Eureka Springs Arkansas, USA

ONLINE EDITION EDITOR

Cesar Compadre University of Arkansas for Medical Sciences, USA

C.S. Shabatura Eureka Springs Arkansas, USA

Philip G. Crandall University of Arkansas, USA

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TABLE OF CONTENTS ARTICLES 6

Using Mortality Compost in Vegetable Production: A comparison Between Summer and Winter Composting and its Use in Cabbage Production C. S. Dunkley, D. L. Cunningham, C. W. Ritz, K. D. Dunkley, and A. Hinton

15

Determination of Antifungal Activity of Pseudomonas sp. A3 Against Fusarium oxysporum

by High Performance Liquid Chromatography (HPLC) P. Velusamy, H. S. Ko, and K. Y. Kim

24

Multi Food Functionalities of Kalmi Shak (Ipomoea aquatica) Grown in Bangladesh H.U. Shekhar, M. Goto, J. Watanabe, I. Konishide-Mikami, Md. L. Bari and Y. Takano-Ishikawa

33

Using Hydrogen- Limited Anaerobic Continuous Culture to Isolate Low Hydrogen Threshold Ruminal Acetogenic Bacteria P. Boccazzi, and J. A. Patterson

45

Effect of Plant-based Protein Meal Use in Poultry Feed on Colonization and Shedding of Salmonella Heidelberg in Broiler Birds W. Q. Alali, C. L. Hofacre, G. F. Mathis, and A. B. Batal

54

Optimization of Fermentative Production of Keratinase From Bacillus Subtilis NCIM 2724 S. M. Harde, I. B. Bajaj, and R. S. Singhal

REVIEWS 66

An Overview of Stress Response Proteomes in Listeria monocytogenes K. A. Soni, R. Nannapaneni, and T. Tasara

EXTRAS 86

Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

4 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

LETTER FROM THE EDITOR INTRODUCTION TO THE INAUGURAL ISSUE

W

elcome to the inaugu-

as the journal’s philosophy is to

ral issue of Agriculture,

encourage awareness of an inter-

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Bacteriology (AFAB). The editorial

that may be unique to many read-

staff is pleased to provide a high

ers. This is also encouraged via

and

quality forum for the publication

the open access digital publica-

INAUGURAL ISSUE

and dissemination of the latest scientific discoveries in agricul-

Volume 1, Issue 1 May 2011

tion model of the journal. At our interactive journal website (www.

tural and food research. The journal will serve

AFABjournal.com), interested readers around

as a peer reviewed scientific forum for the lat-

the world can freely access and download all

est advancements in bacteriology research on

articles we publish. Because of this open ac-

a wide range of topics including food safety,

cess approach, we believe that Agriculture,

food microbiology, gut microbiology, biofuels,

Food and Analytical Bacteriology will have a

bioremediation, environmental microbiology,

major and immediate impact on advancing

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scientific research that will only increase as the

crobiology. The journal will welcome all as-

journal grows. Please enjoy this inaugural is-

pects of research ranging from genomics and

sue complements of the editorial staff and we

proteomics to optimizing cultivation media,

cordially invite you to consider submitting your

isolation studies and growth kinetics. All types

scientific manuscripts to Agriculture, Food and

of manuscripts are encouraged for consider-

Analytical Bacteriology.

ation including full and brief original research papers, comprehensive and mini review papers, symposia reviews and rapid communications. Topics of regional interest are welcomed

Steven C. Ricke Editor-in-Chief

Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

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www.afabjournal.com Copyright © 2011 Agriculture, Food and Analytical Bacteriology

Using Mortality Compost in Vegetable Production: A Comparison Between sSummer and Winter Composting and its Use in Cabbage Production C. S. Dunkley1, D. L. Cunningham2, C. W. Ritz2, K. D. Dunkley3, and A. Hinton4 Department of Poultry Science, University of Georgia,Tifton, Georgia 31793-0478 Department of Poultry Science, University of Georgia, Athens, Georgia 30602-2772 3 School of Science and Math, Abraham Baldwin Agricultural College, Tifton, Georgia, 31793 4 USDA ARS, Russell Research Center, Athens Georgia, 30605 1

2

ABSTRACT A study was conducted to determine the effectiveness of composting to breakdown the carcasses of daily poultry mortality and in the process destroy pathogenic microorganisms that may be present. The study was conducted during the summer and repeated in the winter to determine whether the time of year would affect the temperature profile or the length of time required for the process to be completed. Daily mortalities were collected from a nearby producer and layered in a compost bin each day for four days.

Samples were collected from the litter before it was placed in the bin. Compost samples were collected every other day for a week after the bin was compiled and then once per week until the process was completed. The samples were evaluated for microbial content. Temperature was taken and recorded at random points in the bins on a daily basis. Upon completion of the composting process, the material was used as a soil amendment in two vegetable plots while a third plot without compost material served as the control.

Soil samples were collected from each of the plots prior to application of the compost material. Cabbage seedlings were then planted in each of the plots. Vegetative samples and soil samples were collected and evaluated for microbial presence prior to planting and at week, 1, 3, 7, and again at reaping.

The summer compost had the highest temperature of 156°F on d 9 during the primary phase while the winter compost had the highest temperature of 156°F on d 42 during the secondary phase of the compost.

The summer compost samples were Salmonella enterica (SE) negative from d 2 of the trial but mixed bacterial colonies remained for the duration of the study. The vegetative samples in plot 1 had coliform levels of 3.48 log10/gm at wk10 but the levels was not considered significantly different from the other two plots (p<0.05). The results show that while winter composting can effectively breakdown poultry carcasses and attain high temperatures, summer compost is more efficient and had consistently higher temperatures.

Keywords: poultry, winter compost, summer compost, daily mortality Agric. Food Anal. Bacteriol. 1: 6-14, 2011

Received: August 31, 2010, Accepted: October 19, 2010.

Released Online Advance Publication: May, 2011.

Correspondence: Claudia S. Dunkley, cdunkley@uga.edu Tel: +1 -229-386-3363 Fax: +1-229-86-3239

6 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

Introduction Composting is considered a positive alternative method of processing dead birds in an environmentally safe manner (Ritz and Worley, 2005). It is a natural biological decomposition process that takes place under aerobic and thermophilic conditions (Wilkinson, 2007), furthermore, it is an aerobic process that destroys rather than creates odor causing volatile compounds (Grewal et al., 2005). Composting poultry mortality in the USA began in the 1980’s and the process has been described as the above ground burial of dead animals in mounds of supplemental carbon such as sawdust, litter, straw or wood shavings (Kalbasi et al., 2005). The composting process generally follows two phases; the primary phase is considered the heating or developing phase and the secondary phase is considered the curing or maturation phase (Wilkinson, 2007). The process reduces the carcasses to nutrient rich humus which can be used as a soil amendment. Composting poultry mortalities can also be considered a value added product because instead of burying the birds (which could result in environmental pollution), the product can be sold as an organic fertilizer.

Even though composting is an effective way to dispose of daily mortalities, food safety concerns from the general public have limited the use of the mortality composted materials. There is considerable concern regarding the potential for contamination of agricultural products for human and animal consumption with pathogens that may be present in animal carcasses and manure from compost (Jones, 1999; Keener et al., 2000). Composting has, however, been established as a pathogen reduction technology (Wilkinson, 2007). Research has shown that the process will control nearly all pathogenic viruses, bacteria, fungi, protozoa (including cysts) and helminth ova to acceptable levels. Endospore forming bacteria such as Bacillus anthracis and prions such as bovine spongiform encephalopathy are exceptions (Kalbasi et al., 2005). The inactivation of pathogenic micro-organisms in the compost is dependent upon several mechanisms during composting. Exposure to heat, microbial antagonism, production of

organic acids and ammonia, competition for nutrients, physical composition of composting material and bedding type are all factors that will determine the fate of the microbes (Epstein, 1997; Hess et al., 2004; Turner, 2002). Temperature is considered the most important factor in pathogen inactivation. Not to be ignored, however, is the effect of time since inactivation is a function of both temperature and the length of time the pathogens are exposed to the high temperatures. Haug (1993) reported that pathogen exposure at temperatures of 131°F to 140°F for a couple of days were enough to kill a vast majority of enteric pathogens. Other reports have shown a 99.9% elimination of total coliform and Escherichia coli (E. coli) organisms from beef feedlot manures in the first seven days of composting with the average temperature being 92.3°F to 106.7°F (Larney et al., 2003).

The majority of cases of food borne illnesses observed in human are from the consumption of animal food products, although a variety of pathogens have been recovered from vegetables (Beuchat, 1996).

The most likely source of pathogens observed in vegetables would occur from the cross-contamination of the produce from animal manure or improper composted manure that has been used to amend the soil. Presently consumer demand for organically produced vegetables is on the increase; these are usually fertilized using animal manure. However, recent outbreaks of Salmonella and E. coli infections have demonstrated the need to properly compost animal wastes prior to use in vegetable production.

Islam et al. (2004) used composted poultry manure and composted dairy cattle manure to amend soil which was later planted with carrots and radish; they observed that Salmonella Typhimurium survived for up to 231 days after the soil was amended. In the study conducted by Islam et al. (2004), the compost process and the irrigation water that was used in the study was also inoculated with S. Typhimurium.

The use of mortality compost has been limited due to fear of contamination and/or recontamination of the compost by pathogenic microorganisms.

It has been used to amend pasture land but is not approved to be applied to land that is used to pro-

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7

duce food directly for human consumption. Senne et al. (1994) investigated the survival of Highly Pathogenic Avian Influenza Virus (HPAIV) and the adenovirus in composted poultry carcasses and observed that HPAIV was completely inactivated after 10 days of composting. The adenovirus was inactivated after a further 10 days of composting. Conner et al. (1991) failed to recover enteric microorganisms infused into poultry carcasses after 14 days of primary composting or after 14 days of secondary composting. Presently there are limited scientific reports on the safety of poultry mortality composting and even less on the use of this compost on soils used for growing food for human consumption. While work such as that conducted by Islam et al. (2004) provides us an indication of the potential length of time some pathogenic microorganisms can survive in the soil, it is still unclear as to the survival or the re-emergence of these microorganisms from the composted product after it has been incorporated or top-dressed to production soils.

With this in mind the objectives of this study were to evaluate the temperature profile of both a summer and winter compost, determine and characterize microbes present in the compost bin during the process of carcass composting and to evaluate the presence of “post compost” microorganisms in the soil and on cabbage plants fertilized using composted materials.

Materials and Methods The study was conducted in the summer (2008) and winter (2009) in two phases. Phase I involved the composting of the daily mortality and Phase II involved the application of the composted product to soil in which cabbage seedlings were planted.

Phase I For the first phase of the project, the daily mortality was collected from a nearby four-house commercial broiler farm. Daily mortality was collected for four days averaging thirty-eight birds each day. Cloacal swabs were taken from six birds from each day’s collection and the swabs were sent to the lab for microbial analysis. Litter obtained from a local broiler farm

was used as the carbon source for the compost. A remodeled swine barn was used as the compost facility which consisted of three bins; one primary bin, one secondary bin and a storage bin. The compost pile was layered as described in Ritz and Worley (2005).

Primary Phase During the primary phase of the trial the temperature was taken using a 48” compost thermometer.

The temperature was taken from the core of the pile and two other random areas within the bin. The ambient temperature was also taken on a daily basis to determine an effect on the bin temperature. The temperatures taken each day were averaged and used as the temperature for that particular day. Samples were randomly collected in triplicates every other day from the primary bin to evaluate the presence or absence of SE or E. coli in the bin. The samples were placed in plastic bags and taken immediately to the lab for evaluation. The duration of the primary phase would be dependent on the time/day the temperature of the pile dropped below 129°F. The compost pile was turned for a second heating phase when the temperature dropped below 129°F. Secondary Phase The pile was turned by flipping the contents of the primary bin into the secondary bin; this was done using a “skid loader”. Again, the temperature was taken daily from the core of the pile and two other random areas in the pile using a 48” compost thermometer.

Compost samples were collected in triplicates randomly from the pile every three days and immediately transported to the lab for evaluation. The end of the secondary phase would be indicated by the drop in the temperature below 100ºF. At the end of the secondary phase the composted material remained in the bin until it was transported to the vegetable plot to be used in the second phase of the study.

Phase II Phase II of the study involved the application of the composted material to plots of soil. Three 3’ X 20’ plots were designated as treatment plots were

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during this phase; in plot 1 the soil was top-dressed with the composted carcasses. In plot 2 the compost material was incorporated in the soil. Plot 3 was not treated with the composted material and was considered the control plot. Three soil samples were collected from each of the experimental plots prior to allocation of treatments and evaluated for microbial content. Compost samples were collected from three areas in the finished composted pile and were evaluated for microbial content prior to application to the experimental plots. Cabbage seedlings were planted at a distance of 12” X 8” in each plot. Nine seedlings were randomly selected and evaluated for microbial content prior to planting. Three vegetative and soil samples were randomly collected from each of the three experimental plots after the first, third and seventh week of planting. Three soil and three vegetative samples were also collected after the tenth week of planting. All the samples that were collected were evaluated for microbial content.

and Co.) and incubated for 24 hr. at 42°C. A 10 mm loopful from each broth sample was plated onto XLT4 agar (Becton-Dickinson and Co.) and Brilliant green sulfa agar (3). Plates were then incubated 24 – 48 hr. at 35°C. Suspect Salmonella colonies were biochemically tested using Triple Sugar Iron and Lysine Iron agar (Becton-Dickinson and Co.) and serology was performed using Difco Bacto Poly O antiserum (Becton-Dickinson and Co.).

Vegetative analysis Vegetative material was weighed and 2 X volume of buffered peptone water was added. The samples were then stomached for 2 min. on high and soaked at room temperature for 1.5 hr. before diluting for E.coli determination. Procedures for E. coli, total coliforms and Salmonella enrichment were performed as was done with the compost samples.

Statistical analysis Compost analysis Twenty-five g compost material was weighed and transferred to a sterile Tekmar bags (Seward Laboratory Systems, Inc., Bohemia, NY 11716) then blended in 100 ml buffered peptone water using a Seward Stomacher Laboratory blender on high for 2 minutes.

Serial dilutions of the suspension were made in 0.1% peptone (w/v). Escherichia coli and total coliforms were enumerated by transferring 1 ml from serial dilutions onto 3M Petrifilm (3M Microbiology, St. Paul, MN,55144) and incubating at 35°C for 24-48 hr. E. coli were identified as blue colonies with trapped gas, and total coliforms were determined by counting red colonies with trapped gas in addition to blue colonies.

Presence of salmonellae was determined by preenrichment of the remaining stomached compost material in buffered peptone water by incubation at 35°C for 24 hr. After incubation, 0.1 ml of the suspension was transferred to 9.9 ml Rappaport-Vassiliadis broth (Becton-Dickinson and Co. , Sparks, MD 21030) and 0.5 ml was transferred to 9.5 ml Tetrathionate broth with brilliant green-iodine (Becton-Dickinson

Statistical analyses were performed using GraphPad InStat® version 3.05 for Windows 95 (GraphPad Software, San Diego, CA, USA). One-way Analysis of Variance (ANOVA) with Tukey-Kramer Multiple Comparison tests was performed to determine significant differences in group means. The P value for all ANOVA tests was < 0.05.

Results and Discussion Temperature Profile Summer Trial Temperature changes were monitored on a daily basis during the primary and secondary phases of the experiment. Monitoring the compost temperature is essential in timing the turning of the pile (Malone, 2006) and temperature is considered the most important factor in pathogen inactivation (Wilkinson, 2007).

During the summer compost (Fig. 1), it was observed that the temperature in the compost bin increased to 121° F on d 1 of the study and continued an upward trend with a high temperature of 156° F on d 9. The

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9

Figure 1: Temperature profile for daily mortality compost in the summer and winter trials and the ambient temperature during both summer and winter trials.

180 160 140 120 100 80 60 40 20 0 0

10

20

30

40

50

60

Daily Summer Temp

Summer Ambient Temp

Daily Winter Temp

Winter Ambient Temp

temperature in the bin remained elevated above 130° F from d 2 until d 17. Pathogen inactivation is a function of both temperature and time. Haug (1993) stated that exposure to an average temperature of 131 to 140°F for two days is usually enough to kill a vast majority of enteric pathogens. After d 17 the temperature began a decline and on d 18 the temperature was 128° F. Ritz and Worley (2005) stated that when oxygen becomes limited in the pile the temperature would begin to fall. This would be about seven to twenty one days after the compost is capped when they recommended that the pile be turned. In the summer study the pile was turned for a second heating phase on d 19. The compost pile quickly reheated to a high of 147° F (Fig. 1) on day 24 (five days after the pile was turned). Similar elevations in temperature were observed by Murphy and Carr (1981) after they turned the compost pile when the heat in the compost dropped below 125° F. We observed the pile was not maintaining the heat during this secondary heating as evidenced by

70

80

the fluctuation of the heat in the pile and the low temperature at the base of the pile. For these reasons the pile was turned again on d 46 when the temperature had fallen to 129° F (Fig. 1). During this second turn we observed that the pile was extremely wet at the bottom of the pile and concluded that this resulted in the falling temperature within the pile. Ritz and Worley (2005) stated that the desirable moisture levels in the compost should be 40 to 60% and that too much water could make the compost pile soggy and anaerobic. Litter material was incorporated in the compost during this second turn and it was observed that the temperature within the pile rose steadily to a high of 155° F on the fifth day after the pile was turned. The temperature in the pile gradually declined but remained above 140° F until d 66 after which it continued a steady downward trend to 129° F on d 71. The temperature dropped below 100° F after d 80 and upon examination of the pile no carcass materials were observed. With the temperature below 100°F (84°F) and the absence of

10 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

mal-odors from the composted material, d 80 was marked as the end of the second stage. Mukhtar et al. (2004) stated that the end of the second phase is marked by a reduction of the internal temperature 77 to 86° F, a reduction in the bulk density and the lack of an unpleasant odor when the pile is turned.

Table 1. Temperature profile comparison between summer and the winter trials.

Primary Phase avg. temp.

141.14°F

130.9°F

Winter Trial Ambient temperature should have little or no effect on the compost temperature. However, we were interested in the effects of the external temperature on the length of time for each phase of the compost process, as well as the temperature profile of the pile. The winter compost trial began in November and the temperature was monitored for eighty days. The temperature in the pile rose from 76°F on d 0 to 131°F on d 3. The pile was turned on d 35 when the temperature was 129°F. The highest temperature during the primary phase of the winter compost was 136°F on d 4 compared to a high of 156°F on d 9 in the summer compost (Fig. 1).

The ambient temperature on d 4 during the winter trial was 73°F compared to 93°F during the summer trial. The 20° difference in the ambient temperature does not necessarily mean there will be a higher compost temperature during the summer trial especially because a high temperature of 156°F was recorded during the winter compost in the secondary phase when the ambient temperature was 43°F which was the coldest day during the trial. Because the summer compost had two turns we could not justifiably compare the duration of the primary phase in the summer compost to the primary phase in the winter compost. However, the summer compost had higher temperatures on average during the primary and secondary phases when compared to the primary phase during the winter compost (Table 1). When comparing the two trials the highest temperature observed during the secondary phase was 156°F in the winter compost compared to a high of 155°F during the summer trial (Table 1). Gonzalez and Sanchez (2005) observed temperatures above 140°F during their summer trials but did not see temperatures above 140°F during their winter trials.

Secondary Phase avg. temp.

145.19°F

137°F

Primary Phase high temp.

156°F

136°F

Secondary Phase high temp.

155°F

156°F

Summer Compost

Winter 1

Compost 2

Represents the temperatures in the summer compost. (This data references the temperature based on the second time the bin was turned during the summer trial).

1

2

Represents the temperature in the winter compost.

Microbial Profile Cloacal swabs were collected from six birds on each of the days bird carcasses were collected.

The results from the summer compost show that none of the birds were SE positive. However five of the six birds which were sampled on day 1 and 2 were E. coli positive and all the birds tested positive for coli-form. Only three birds were E. coli positive on days 3 and 4 but the numbers on day four were too numerous to count. Similar results were observed in the winter compost with none of the birds testing positive for SE, all coli-form positive and some of the birds testing positive for E. coli, these result however, were too numerous to count.

The compost piles from both the summer and winter trials were sampled and analyzed for microbial content (Table 2). Even though none of the sample birds tested positive for Salmonella, the summer compost tested positive for SE on day 1 of the trial and E. coli positive on day 1 and 2 but was negative for both of these microbes for the duration of the trial. The winter compost tested negative for both SE and E. coli for the duration of the trial. Both the

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Table 2. A comparison of microbial content between the summer compost and the winter compost.

Summer Compost 2

1

Winter Compost 3

Day 1

Salmonella

E. Coli

Coliform

Salmonella

E. Coli

Coliform

1

+

+

+

-

-

+

2

-

+

+

-

-

+

4

-

-

+

-

-

+

6

-

-

+

-

-

+

12

-

-

+

-

-

+

24

-

-

+

-

-

+

Day of the trial samples was collected from the summer and winter composts.

2

Days during the trial when summer compost tested positive or negative for Salmonella, E. coli or coliform.

3

Days during the trial when winter compost tested positive or negative for Salmonella, E. coli or coliform.

summer and winter samples were positive for coliform for the duration of the trial. Temperature is considered the most important factor in pathogen inactivation (Wilkinson, 2007) but the length of time exposure to high temperature also functions in the inactivation of pathogens (Haug, 1993). In the summer trial SE was not observed in the compost on d 2 when the temperature in the pile was 137°F neither was E. coli isolated on d 3 when the temperature was 137°F. Haug (1993) stated that two days is usually enough to kill the vast majority of enteric pathogens. Grewal et al. (2005) detected E. coli, SE and Listeria in manure on d 0 of a trial but after 3 days of composting at 55°F they did not detect any of these pathogens.

Even though SE and E. coli were not observed in the compost at the end of Phase 1 of the trial, we wanted to determine if there was any evidence of re-growth after the temperatures returned to more favorable levels. Under certain conditions, enteric pathogens have been known to re-grow in composted organic material when the temperature declines to sub lethal levels (Wilkinson, 2007). Also, there is the potential of Salmonella or E. coli colonies to survive the thermophilic conditions of the compost in clumped materials in the pile. Millner et al. (1987) showed that SE was not suppressed

in compost taken from 158°F compost pile zones.

When Johannessen et al. (2004) investigated the influence of manure on the hygienic quality of lettuce, E. coli O157:H7 was isolated from the manure that was used to fertilize and also from the soil one week after fertilizing. However, they did not isolate the E. coli from lettuce that was grown in the soil. In this study neither SE nor E. coli was isolated from the cabbage samples throughout the trial (Table 3). However, coliforms were isolated from treatment plots 1 and 2 during each of the weeks that samples were collected. There were numerical differences between the treatment plots but the differences were not considered significant (p<0.05). Coliforms were isolated from the control plot in weeks 1 and 3 of the trial but were not detected thereafter, the levels were not significantly different from the other two treatments (p<0.05). Islam et al. (2004) and Natvig et al. (2002) observed that pathogens could be transferred from manure to the surface of vegetables by way of contaminated soil. In this trial neither SE nor E. coli was detected in the soil samples for the duration of the trial but coliform was isolated from all three plots (data not shown). This result was an expected because coliform bacteria are commonly found in the environment.

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Week

1

Table 3. Microbial presence on cabbage samples.

1

Treatment 1 2 Coliform Salmonella E. Coli 5 Log10

Treatment 2 3 Coliform Salmonella E. Coli Log10

Control 4 Salmonella E. Coli

Coliform 5 Log10

1

-

-

1.70±.01

-

-

1.22±.01

-

-

0±0

3

-

-

1.73±.01

-

-

1.25±.01

-

-

1.52±.01

7

-

-

1.78±.02

-

-

1.26±.01

-

-

0±0

10

-

-

3.48±.03

-

-

1.30±.01

-

-

0±0

Week of the trial when cabbage samples were collected.

2 Presence or absence of Salmonella, E. coli or coliform on cabbage plants sampled from treatment 1compost top-dressed 3 Presence or absence of Salmonella, E. coli or coliform on cabbage plants sampled from treatment 2compost incorporated.

4 Presence or absence of Salmonella, E. coli or coliform on cabbage plants sampled from the control plot- no compost was added.

5

Log10 coliform numbers isolated on from the cabbage plants sampled from each of the experimental plots.

Conclusions Recently there has been an increased awareness of fruits and vegetables as potential sources of pathogenic microbes that causes human illness. Animal manure is an economical fertilizer for crop producers. However, animal manure frequently contains enteric pathogenic microbes (Pell, 1997) and spreading it on the land can result in pathogen entry into the food chain. Composting daily mortality can be done effectively and efficiently during the summer or winter. The temperature profiles that are attained in the winter compost compares favorably to that of the summer compost. Results from this study show that composting bird carcasses can eliminate the pathogenic microbes that may be present in the birds or the litter. Using the composted material to fertilize vegetable plots did not result in contamination of the vegetables by pathogenic microorgansisms.

Also there were no indications of re-emergence of SE or E. coli in the soil.

Composting as a means of dead bird disposal is effective no matter what time of the year it is practiced. It is an economical means of disposal as it uses everything in the production of chickens, nothing is wasted and the composted material can be used as a nutrient rich soil amendment.

Acknowledgement The authors would like to acknowledge Dr. S. Rajeev for her assistance in conducting the microbial analyses of the project samples. We also thank young scholar Tyler Reeves for working on this project.

References Beuchat, L. R. 1996. Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59:204216.

Conner, D. E., J. P. Blake, and J. O. Donald. 1991.

Microbiological safety of composted poultry farm

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mortalities. ASAE/CSAE Meeting Paper No. 914053. St. Joseph, MO: The American Society of Agricultural Engineers.

Epstein, E. 1997. The science of composting. Technomic Publishing AG, Lancaster, PA. 487 p.

González, J. L. and M. Sánchez. 2005. Treatment of poultry mortalities on poultry farms. Compost Sci. Util. 13:136-140.

Grewal, S. K., S. Rajeev, S. Sreevatsan and F. C. Michel, Jr. 2005. Persistence of Mycobacterium avium subsp. paratuberculosis and other zoonotic pathogens during simulated composting, manure packing, and liquid storage of dairy manure. Appl. Environ. Microbiol. 72:565-574.

Haug, R. T. 1993. The practical handbook of compost engineering. CRC Press, Boca Raton, FL. 717 p.

Hess, T. F., I. Grdzelishvili, H. Q. Sheng and C. J. Hovde. 2004. Heat inactivation of E. coli during manure composting. Compost Sci. Util. 12:314322.

Islam, M., J. Morgan, M. P. Doyle, S. C. Phatak, P. Millner and X. Jiang. 2004. Fate of Salmonella enterica serovar Typhimurium on carrots and radishes grown in fields treated with contaminated manure composts or irrigation water. Appl. Environ. Microbiol. 70:2497-2502.

Johannessen, G. S., G. B. Bengtsson, B. T. Heier, S. Bredholt, Y. Wasteson and L. M. Rørvik. 2004. Potential uptake of Escherichia coli O157:H7 from organic manure in Crisphead lettuce. Appl. Environ.

Microbiol. 71:2221-2225.

Jones, D. L. 1999. Potential health risks associated with the persistence of Escherichia coli O157 in agricultural environments. Soil Use Manag. 15:76-83.

Kalbasi, K., S. Mukhtar, S. E. Hawkins, and B. W. Auvermann. 2005. Carcass composting for management of farm mortalities: A review. Compost Sci. Util. 13:180-193.

Keener, H. M., W. A. Dick and H. A. J. Hoitink.

2000. Composting and beneficial utilization of composted by-product materials. In: J. F. Power and W. A. Dick. Eds. Land application of agricultural, industrial, and municipal by-products. Soil Science Society of America, Madison, Wisconsin. p 316-341.

Larney, F. J., L. J. Yanke, J. J. Miller and T. A. McAllister. 2003. Fate of coliform bacteria in composted beef cattle feedlot manure. J. Environ. Qual.

32:1508-1515.

Malone, G. S. 2006. Mass mortality composting programs. Pages 26-31 in Proc. Natl. Poult. Waste Manage. Symp., Springdale, AR. Millner, P. D., K. E. Powers, N. K. Enkiri and W. D. Burge. 1987. Microbially mediated growth suppression and death of Salmonella in composted sewage sludge. Microb. Ecol. 14:255-265.

Mukhtar, S., A. Kalasi and A. Ahmed. 2004.

Composting in carcass disposal: A comprehensive review. USDA APHIS Cooperative Agreement Project, Carcass Disposal Working Group.

Murphy, D. W. and L. E. Carr. 1981. Composting dead birds. FS-537. University of Maryland, Cooperative Extension Service, College Park, MD. Natvig, E. E., S. C. Ingham, B. H. Ingham, L. R. Cooperband, and T. R. Roper. 2002. Salmonella enterica serovar Typhimurium and Escherichia coli contamination of root and leaf vegetables grown in soils with incorporated bovine manure. Appl.

Environ. Microbiol. 68:2737-2744.

Pell, A. N. 1997. Manure and microbes: public and animal health problem? J. Dairy Sci. 80:2673-2610.

Ritz, C. W., and J. W. Worley. 2005. Poultry mortality composting management guide. Bulletin 1266.

The University of Georgia, Cooperative Extension Service, Athens, GA. Senne, D. A., B. Panigraphy and R. Morgan. 1994.

Effect of composting poultry carcasses on survival of exotic avian viruses: HPAI virus and adenovirus of egg drop syndrome- 76. Avian Dis. 38:733-737.

Turner, C. 2002. The thermal inactivation of E. coli in straw and pig manure. Bioresour. Technol. 84:5761.

Wilkinson, K. G. 2007. The biosecurity of on-farm mortality composting. J. Appl. Microbiol. 102:609618.

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www.afabjournal.com Copyright © 2011 Agriculture, Food and Analytical Bacteriology

Determination of antifungal activity of Pseudomonas sp. A3 against Fusarium oxysporum by high performance liquid chromatography (HPLC) P. Velusamy1, H. S. Ko2, K. Y. Kim2 Department of Biotechnology, School of Bioengineering, SRM University, Chennai-603 203, India Division of Applied Bioscience and Biotechnology, Chonnam National University, Gwangju 500-757, Korea 1

2

ABSTRACT It has frequently been reported that chitinolytic soil bacteria, in particular biocontrol strains, can lyse viable fungal hyphae and thereby release potential substrates for bacterial growth. The present work was carried out with an objective to get a better understanding of the relationship between chitinolytic and antifungal properties of bacteria that occur naturally in coastal soils, i.e. without artificial selection. Among the bacterial, strain A3 was identified as Pseudomonas sp. A3 based on morphologic observation and 16S rRNA analysis. Strain A3 exhibited a maximum chitinase production of 1.44 U/ml in CC broth after 3 days of cultivation. Besides having chitinolytic activity, the molecular weight of the crude enzyme was estimated to be 56 kDa by SDS-PAGE and zymogram. In vitro assays revealed that the crude chitinase inhibited activity of Fusarium oxysporum as identified by dual plate assay and microscopic methods. Hydrolysis products of the fungal cell wall by the crude enzymes of Pseudomonas sp. A3 were analyzed by high-pressure liquid chromatography (HPLC) and identified as oligosaccharides, which included monomers (GlcNAc), dimers (GlcNAc)2, and trimers (GlcNAc)3 using chitin oligomer standards. The crude chitinase isolated from strain A3 can be directly applied for suppressing growth of viable fungal hyphae.

Keywords: Antifungal activity, Pseudomonas, Fusarium oxysporum, chitinase, fungus, 16S rRNA, Zymogram, HPLC Agric. Food Anal. Bacteriol. 1: 15-23, 2011

Introduction Biological control of plant pathogens by soil bacteria is a well established phenomenon and chitinase production has been shown to play an important role in the suppression of various diseases (Chernin et al., 1995; Hong and Hwang, 2005; Hoster et al., 2005). Chitin (C8H13O5N)n is an unbranched long Received: September 3, 2010, Accepted: November 26, 2010.

Released Online Advance Publication: March 1, 2011.

Correspondence: P. Velusamy, vels@ktr.srmuniv.ac.in Tel: - +91-44-22127331 , Fax: +91-44-22121155

chain polymer of glucose derivatives, composed of ß-1,4 linked units of the amino sugar N-acetyl-Dglucosamine (NAGA), which is speculated to play a vital role in fungal defense against toxic stresses.

The interest in chitin degrading enzymes and their application in management of fungal pathogens are significant. Chitinases (EC 3.2.1.14), a group of antifungal proteins, catalyse the hydrolytic cleavage of the ß-1,4-glycoside bond present in the biopolymers of N-acetyl-D-glucosamine, mainly in chitin.

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Antagonistic bacteria are considered as ideal biological control agents that mediate one or several mechanism of disease suppression. Among these, hyper parasitism relies on chitinase for degradation of the cell walls of fungi (Chet et al., 1990). The soil-borne Enterobacter agglomerans IC1270 has a broad spectrum of antifungal activity and secretes a number of chitinolytic enzymes, including two Nacetyl-ß-D-glucosaminidases and chitinase. Its biocontrol activity has been demonstrated with Rhizoctonia solani in cotton using Tn5 mutants deficient in chitinolytic activity (Chernin et al., 1995). Hence, chitinolytic enzyme might be considered to have an important role in biological control of fungal pathogens.

It has frequently been reported that chitinase producing microorganisms, in particular biocontrol strains, can lyse viable fungal hyphae, thereby releasing substantial level of oligomers and other substances (Cohen-Kupiec and Chet, 1998; Dahiya et al., 2006; De Boer et al., 2001). In the present work, we report a new strain Pseudomonas sp. A3 possessing strong chitinolytic activity, which exhibited an antagonism toward F. oxysporum. Moreover, the antiungal activity of the crude chitinase from strain A3 was also partially characterized.

Materials and Methods Screening of bacteria Five soil samples were obtained from different sites of the coastal soils enriched with crab shells in Buan area, Korea. Soils were serially diluted with sterile water until a dilution of 106 colony forming units (CFU) g-1 of soils, inoculated on colloidal chitin (CC) agar medium containing 0.5% colloidal chitin, 0.2% Na2HPO4, 0.1% KH2PO4, 0.05% NaCl, 0.1% NH4Cl, 0.05% MgSO4 7H2O, 0.05% CaCl2 2H2O, 0.05% yeast extract and 2% agar, and incubated at 30°C for 3 days. Strains exhibiting a clear zone (degradation of chitin) around the colony were picked and further subjected to antifungal activity against F. oxysporum f. sp. cucumerinum (KACC 40032, Korean

Agricultural Culture Collection, Suwon, Korea) grown on potato dextrose agar (PDA) medium containing 0.5% colloidal chitin at 30°C for 7 days.

Bacterial identification To identify the bacterium, a polymerase chain reaction (PCR) was performed to amplify the 16S rRNA gene from the genomic DNA of strain A3 using universal primers fD1 (5’-GAGTTTGATCCTGGCTCA-3’) and rP2 (5’-ACGGCTACCTTGTTACGACTT-3’) as described earlier (Weisburg et al., 1991). The PCR product was cloned in a pGEM-T easy vector (Promega, Madison, WI, USA). The nucleotide sequence of the 16S rRNA gene was determined by an ABI Prism 377 DNA sequencer (PE Applied Biosystems, Foster City, CA, U.S.A) and compared with published 16S rRNA sequences using Blast search at Genbank data base of NCBI (Bethesda, MD, USA).

Chitinase assay For determination of chitinase activities, strain A3 was grown in CC broth at 30°C, and samples were taken at 1, 2, 3, 4, and 5 days. Each sample was centrifuged at 8000 ×g for 5 min and the supernatant was used for enzyme activities. Chitinase activity was determined by incubating 1 ml of culture supernatant with 1 ml of 1% colloidal chitin in a 0.05M phosphate buffer, pH 7.0 at 37°C for 1 h. After centrifugation of reaction mixture, the amount of N-acetyl-d-glucosamine released in the supernatant was determined by the standard method (Lingappa and Lockwood, 1962) using N-acetyl-d-glucosamine (GlcNAc) as a standard. GlcNAc present in 0.5 mL of aliquot of supernatant was determined by adding 0.1 ml of K2B4O7 and then boiled for 3 min in a water bath.

The tubes were cooled and 3 ml of p-dimethylaminobenzaldehyde was added. Absorbance was read within 10 min at 585 nm against the blank prepared with distilled water without the enzyme present. One unit of chitinase is defined as the amount of enzyme which releases 1 μM N-acetyl-d-glucosamine per hour under the conditions of the study.

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Preparation of crude enzyme Strain A3 was cultured in a 2000 ml Erlenmeyer flask containing 1000 ml of CC broth at 30°C for 3 days in a shaking incubator (180 rpm). After centrifugation of the broth culture at 8000 ×g for 30 min, ammonium sulfate was added to the supernatant at 50% saturation, and the mixture was left overnight at 4°C. The precipitate was centrifuged at 12 000 ×g for 30 min and the pellet was resuspended in 50 mM TrisHCl buffer [pH 8.0], and dialyzed against the same buffer for overnight. The dialyzate was concentrated by lyophilization, and the concentration of protein was determined using bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) as the standard (Bradford, 1976).

Electrophoresis The concentrated enzyme sample was subjected to electrophoresis in 12% SDS-PAGE, according to the method described previously (Laemmli, 1970).

Subsequently, zymogram was demonstrated by copolymerizing 0.01% of glycol chitin (Sigma) in SDSPAGE for the detection of chitinase activity (Trudel and Asselin, 1989).

ed as control. In order to determine the deformation of hyphae, the experiment was carried out at varying conditions such as pH, temperatures, incubation time, and different buffers.

Hydrolysis of fungal hyphae To determine the hydrolysis of F. oxysporum by crude chitinase (as described in the section on microscope analysis) after 24 h, the reaction was stopped by addition of 200 μl 1 M NaOH. The reaction product was centrifuged at 6000 ×g for 30 min, and the supernatant was passed through a 0.22 μm membrane filter (Nalgene, Rochester, NY, U.S.A).

The enzyme hydrolysate was analyzed by high-performance liquid chromatography (HPLC). The HPLC was performed with acetonitrile:water (70:30, v/v) as the mobile phase at the flow rate of 1 m/min and detected at 210 nm with NH2P50-4E column (Shodex, Tokyo, Japan) (Kuk et al., 2005). The retention times for the peaks obtained in the crude samples of hydrolytic products were compared with the chitin oligomer standard.

Results and Discussion Isolation and identification of antagonistic bacterium

Antifungal activity The crude chitinase was assayed for antifungal activity against F. oxysporum by well diffusion assay on a PDA plate. A fungal plug (6 mm diameter) was removed from the 5 day old culture. The plug was transferred onto the center of the PDA plate, which had been loaded with chitinase in the right well and the left well was loaded with the same volume of buffer. The plate was incubated for 5 days at 30ºC and was monitored for a zone of inhibition around the well. However, the antifungal effect was also observed by a light microscope (Nikon, Tokyo, Japan).

Two milliliters of the F. oxysporum suspension with crude chitinase (final concentrations of 2.5 U/ml) in 50 mM of sodium acetate buffer [pH 6.0] was added into a 12 well plates (12 mm, Corning, NY, USA). A mixture of hyphae suspension and buffer was treat-

In our pilot scale screening, various microbial colonies were able to degrade chitin on CC agar medium. Among these, a bacterial isolate that exhibited the maximum halo zone around the colonies, was designated to be the strain A3. Subsequent antimicrobial activity was examined through dual plate assays using various phytopathogens. Interestingly, strain A3 exhibited a strong antifungal activity against F. oxysporum (Fig. 1). From the morphologic observation, strain A3 was found to be a Gram-negative, rod-shaped and polar-flagella bacterium with permissive temperature ranging between 20 ºC to 37 ºC with an optimum at 30 ºC. The genomic DNA of the strain A3 was amplified with universal primers and 16S rRNA gene sequence was analyzed. Alignment of this sequence (1464 bp) through matching

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Figure 1. Inhibition of the growth of F. oxysporum by Pseudomonas sp. A3 on potato dextrose agar (PDA) medium containing 0.5% of colloidal chitin at 30ºC for 7 days.

strain A3 was identified as a member of Pseudomonas sp, and designated as Pseudomonas sp. A3 (Genbank accession number EU784845).

Members of the genus Pseudomonas are ubiquitous in soil microorganisms. They are believed to serve as a promising group of biocontrol agents and have been widely evaluated with the production of chitinases (Ajit et al., 2006; Choi et al., 2006; Fogliano et al., 2002; Folders et al., 2001; Neiendam and Sorensen, 1999).

Determination of chitinolytic activity Strain A3 was investigated for the production of extracellular chitinase in CC broth by spectrophotometry. At 24 h intervals, aliquots of cell cultures were taken, and the chitinase activity was determined by a standard method. The results from culture filtrate of strain exhibited maximum chitinase activity of 1.44 U/ml after 3 days of cultivation and gradually decreased thereafter (Fig. 3). with reported 16S rRNA gene sequences in the Genbank showed high similarity (99 to 100%) to Pseudomonas sp. The phylogenetic tree determined by the Neighbor-joining method showed that Pseudomonas sp. and P. aeruginosa were most closely related to strain A3 (Fig. 2). On the basis of these results,

SDS-PAGE and zymogram The molecular weight of crude chitinase was determined by gel electrophoresis using a standard marker (iNtRON Biotech, Inc., Gyeonggi-Do, Korea).

Figure 2. Phylogenetic location of strain A3 based on 16S rRNA sequences by Neighbor-joining method program. Phylogenetic tree based on 16S rRNA sequences displaying the relationship between strain A3 and that of the other species. Reference species with accession numbers were obtained from Genbank databases. Bar indicates 0.10 nucleotide substitutions per site.

Pseudomonas otitidis isolate WL15 (EF687744) Pseudomonas sp. M11 (EU375657) 97

97

Pseudomonas aeruginosa strain MML2212 (EU344794)

98

Pseudomonas aeruginosa strain CMG860 (EU037096) Pseudomonas sp. G3DM-81 (EU037286)

99

Strain A2 Pseudomonas sp. AHL 2 (AY379974)

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Chitinase Activity (units/ml)

Figure 3. Determination of chitinolytic activity by Pseudomonas sp. A3 in CC broth medium at 30ºC for 7 days. Mean values were 3 replicates. Bars represent standard error.

Period of Incubation (days)

Figure 4. SDS-PAGE and zymogram of crude chitinase from culture supernatant of Pseudomonas sp. A3 by ammonium sulfate precipitation.

Lanes 1 and 2 were obtained from 12% SDS-PAGE of Coomassie brilliant blue R-250 where lane 1 is molecular weight (MW) of standard markers in kilodaltons, and lane 2 is crude chitinase sample. Lane 3 indicates zymogram demonstrated by copolymerizing 0.1% of glycol chitin in 12% SDS-PAGE. The chitinase activity was detected by visualization under UV light.

MW (kDa)

1

Lane 2

3

144 84 63 56 kDa

45

34

26.5

The crude samples of strain A3 revealed several bands on 12% SDS-PAGE and the chitinase activity were identified as 56 kDa by zymogram (Fig. 4).

The molecular weights of bacterial chitinases ranged from 20,000 to 120,000, with little consistency. Different molecular masses have been reported for other bacterial chitinases as well (Jung et al., 2002; Wang and Chang, 1997).

Relationship between chitinase production and F. oxysporum suppression by Pseudomonas sp A3 The inhibitory effect on the growth of hyphae of F. oxysporum was investigated under different concentrations of crude chitinase by a well diffusion assay on a PDA plate and a concentration of 50 μl (2.5 U/ml) yielded the maximum inhibition against F. oxysporum. However, in the control well, the same volume of sodium acetate buffer did not inhibit the pathogen (data not shown). Microscopic observation revealed a morphology of hyphae that appeared as swelled, fragmented and distorted in the wells treated with the enzyme, whereas the hyphae from the control were normal and intact without any distortion (Fig. 5). The results presented here support antibiosis as the mechanism of antagonism against F. oxysporum by strain A3 mediated through chitinase production. Several studies have demonstrated that

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Figure 5. Morphological study of the hyphae of F. oxysporum in sodium acetate buffer supplemented with crude chitinase at a concentration 2.5 U/ml incubated at 40 °C for 24 h. (A) Control: hyphae of F. oxysporum + buffer, (B) Treatment: hyphae of F. oxysporum + crude chitinase

chitinases of potential biocontrol strains can cause deformation of viable hyphae and result in inhibition of hyphae of the test fungi (Fogliano et al., 2002; Giambattista et al., 2001; Mathivanan et al., 1998).

When the crude chitinase was further assayed for antimicrobial activity against various microorganisms.

As listed in the Table 1, the percentage of inhibitory effect of crude chitinase against R. solani and D. bryoniae were recorded to be approximately 50% and the remaining microorganisms were not significant.

Analysis of antifungal activity

Table 1. Inhibitory effects of crude chitinase against various microorganisms by in vitro dual plate assays

Name of Organisms Bacteria Pectobacterium carotovorum subsp. carotovorum KACC 10057 Xanthomonas oryzae pv. oryzae KACC 10378

ND

-

Fungi Phytophthora capsici KACC 40483

Fusarium wilt is a widespread plant disease caused by many forms of the soil-inhabiting fungus F. oxysporum. Several attempts have been made to exploit the biological control of F. oxysporum by chitinase producing bacteria (Chung and Kim, 2007; De la Vega et al., 2006; Giambattista et al., 2001). To investigate the antifungal effect of crude chitinase, samples of treated hyphae of F. oxysporum was analyzed by HPLC. As shown in Fig. 6, various products of chitin oligosaccharides such as monomers (GlcNAc), dimers (GlcNAc)2, and trimers (GlcNAc)3 were identified using chitin oligomer standards. Other peaks that were detected may have belonged to chitosan or glucan oligomers.

However, there was no detectable number of peaks found in control. Yet, in the present study, chitinolytic bacterium strain A3 may have an important role in the hydrolysis of fungal hyphae and release of substantial

Inhibition Ratio*

Didymella bryoniae KACC 40900 Botrytis cinerea KACC 40573 Rhizoctonia solani KACC 40117 Colletotrichum gloeosporioides KACC 40689

+ ++ ± ++

-

*Antimicrobial effect of crude enzyme 50 μl (2.5 U/ ml) was assayed by well diffusion assay on agar medium.

The percentage of inhibition of growth was calculated from the mean values as: % Inhibition = (A-B)/A x 100, where A = microorganism growth in control, and B = microorganism growth in chitinase.

The inhibition was reported as (ND) for any undetected inhibition of growth from below 5%, (-) between 5% and 15%, (±) between 15% and 25%, (+) between 25% and 35%, (++) between 35% and 50%. Triplicates were run simultaneously to obtain each value.

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Figure 6. HPLC chromatograms of hydrolysis products from the hyphae of F. oxysporum and crude chitinase mixture after 24 h of incubation at 40 °C. [A] Chitin oligomer standard (GlcNAc)n, [B] Hydrolytic products of crude sample obtained from hyphae of F. oxysporum + crude chitinase. Peak numbers are referred as 1- monomer (GlcNAc) ; 2- dimer (GlcNAc)2 ; 3- trimer (GlcNAc)3 ; 4- tetramer (GlcNAc)4 ; and 5- pentamer GlcNAc)5.

(1) [A]

Standard (GlcNAc)n (2) (3) (4)

[B]

(5)

Hyphae + Crude chitinase

(2) (1) (3)

Retention Time (min.)

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levels of chitin oligomers. A previous study reported that chitinase ChiA71 from Bacillus thuringiensis subsp. pakistani completely hydrolyzed colloidal chitin to GlcNAc monomers after incubation for 24 h (Thamthiankul et al., 2001). More recently, Van et al. (2008) suggested that chitinases from Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9 could release different oligosaccharides after hydrolysis from the hyphae of Fusarium solani.

CONCLUSION From the results presented in this experiment, a positive correlation can be inferred between the production of chitinase and suppression of the growth of F. oxysporum. However, it is necessary to study the secretion of other lytic enzymes as well, especially cellulase, ß-1,3-glucanase, and laminarinase, as chitinase may combine with other lytic enzymes to exhibit synergism, and result in high levels of antifungal activity. Therefore, we suggest that Pseudomonas sp. A3 may be an optimal candidate for use as a biocontrol agent of Fusarium wilt in tomato, but further studies are needed to evaluate more extensively this possibility.

Acknowledgement This study was supported by the Korean Research Foundation - second stage of BK21, and National Research Laboratory (NRL) Program from the Ministry of Science and Technology (MOST), Korea.

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Neiendam, N. M., and J. Sorensen. 1999. Chitinolytic activity of Pseudomonas fluorescens isolates from barley and sugarbeet rhizosphere. FEMS Microbiol. Ecol. 30:217-227.

Thamthiankul, S., S. Suan-Ngay, S. Tantimavanich, W. Panbangred. 2001. Chitinase from Bacillus thuringiensis subsp. pakistani. Appl. Microbiol. Biotechnol. 56:395-401.

Trudel, J. and A. Asselin. 1989. Detection of chitinase activity after polyacrylamide gel electrophoresis.

Anal. Biochem. 178:362-366.

Van, N. N., Y. J. Kim, K. T. Oh, W. J. Jung, R. D. Park.

2008. Antifungal activity of chitinases from Trichoderma aureoviride DY-59 and Rhizopus microsporus VS-9. Curr. Microbiol. 56:28-32.

Wang, S. L. and W. T. Chang. 1997. Purification and characterization of two bifunctional chitinase/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl. Environ. Microbiol. 63:380-386.

Weisburg, W. G., S. M. Barns, D. J. Lane. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173:697-703.

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www.afabjournal.com Copyright © 2011 Agriculture, Food and Analytical Bacteriology

Multi Food Functionalities of Kalmi Shak (Ipomoea aquatica) Grown in Bangladesh H. U. Shekhar1, M. Goto1, J. Watanabe1, I. Konishide-Mikami1, Md. L. Bari2 and Y. Takano-Ishikawa1 National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki 305-8642, Japan Center for Advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh 1

2

ABSTRACT Kalmi Shak or water spinach (Ipomoea aquatica) is a Bangladeshi indigenous green leafy vegetable and herbaceous aquatic or semi aquatic perennial plant. A primary study was conducted to elucidate the multi functionalities of this vegetable. Extract of Kalmi Shak exhibited high antioxidant properties with hydrophilic-oxygen radical absorbance capacity (H-ORAC) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity being 341.92 ± 1.32 and 37.67 ± 2.63 µmol Trolox equivalent / gram of dry weight (TE/g DW), respectively. The total polyphenols content was estimated to be 12.56 ± 0.08 mg gallic acid equivalent / gram of dry weight (mg GAE/g DW), and moisture content was found to be 85%. The extract also showed anti-mutagenic effect on Trp-P2 induced mutagenicity to Salmonella Typhimurium TA98, and antitumor activity to mouse myeloma cell line P388. The extract of this vegetable also exhibited anti-bacterial activities against several spoilage and pathogenic bacteria. The multi functionalities, economic price and availability during the entire year have made this indigenous Bangladeshi vegetable important from both medicinal and industrial aspects.

Keywords: Kalmi Shak, water spinach, antioxidant, anti-mutagenic activity, anti-tumor activity, antibacterial activity Agric. Food Anal. Bacteriol. 1: 24-32, 2011

Introduction Leafy vegetables have been extensively investigated as new sources of natural antioxidants as well as other bioactive compounds of human health benefits (Lakshmi and Vimala, 2000). Epidemiological studies have shown that consumption of vegetables is associated with reduced risk of chronic diseases. It has been reported that leafy vegetable extracts could Received: September 7, 2010, Accepted: October 21, 2010.

Released Online Advance Publication: March 25, 2011.

Correspondence: Hossain Uddin Shekhar, hossain@affrc.go.jp, Tel: - + 81-298-38-8055, Fax: +81-298-38-7996

be used to reduce blood sugar level (Villansennor et al., 1998) and as an antibiotic against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis and other microorganisms (Bhakta et al., 2009). Increased consumption of vegetables containing high levels of phytochemicals has been recommended to prevent chronic diseases related to oxidative stress in the human body (Chu et al., 2002). Natural antioxidants increase the antioxidant capacity of the plasma and reduce the risk of certain diseases such as cancer, heart diseases and stroke (Prior and Cao, 2000). The secondary metabolites including phenolics and

24 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

flavonoids from plants have been reported to be potent free radical scavengers (Chiang et al., 2004).

They are found in all parts of plants such as leaves, fruits, seeds, roots and bark (Mathew and Abraham, 2006). There are many synthetic antioxidants in use, however, it is reported that they have several side effects, such as risk of liver damage and carcinogenesis in laboratory animals (Gao et al., 1999; Ito et al., 1983; Osawa and Namiki, 1981). Therefore, a search for natural antioxidants from plant may help to find safer, more potent, less toxic and cost effective antioxidants.

Kalmi Shak, a semi-aquatic plant water spinach (Ipomoea aquatica) belongs to Convolvulaceae family, not only grows wild but is also cultivated throughout Southeast Asia, and is one of the widely consumed vegetable in the region (Huang et al., 2002). It is a tender, trailing or floating perennial aquatic plant, found in most soils along the margins of fresh water, ditches, marshes and wet rice field. It is usually found year round and treated as a leafy vegetable unlike other common vegetables in Bangladesh which are mostly seasonal. Kalmi Shak represents one of the richest sources of carotenoids and chlorophylls (Chen and Chen, 1992). The leaves contain adequate quantities of most of the essential amino acids in accordance with the WHO recommendation pattern for an ideal dietary protein (Prasad et al., 2008). Consequently, when compared with conventional food crops such as soybeans or whole egg, it has potential for utilization as a food supplement. Ayurveda, a system of traditional medicine native to the Indian subcontinent, has identified many medicinal properties of Kalmi Shak, and it is effectively used against nosebleeds and high blood pressure (Perry, 1980). However, very limited scientific studies have been conducted on its functional aspects.

Most of the studies have focused on the inhibition of prostaglandin synthesis (Tseng et al., 1992), effects on liver diseases (Badruzzaman and Husain, 1992), constipation (Samuelsson et al., 1992) and hypoglycemic effects (Malalavidhane et al., 2003). There have been no reports on the systematic study of the indigenous Kalmi Shak of Bangladesh to evaluate its potentiality as a functional food or food supplement. The objective of this study was to investigate the antioxidant activity,

total phenolic content, anti-tumor, anti-mutagenic, and antimicrobial properties of the extracts of indigenous fresh green Kalmi Shak.

Materials and Methods Materials RPMI-1640, penicillin-streptomycin solution (Hybri-Max®), Dulbecco’s phosphate buffered saline (PBS), DPPH, and 0.4% Trypan Blue solution, 6-hydroxy-2,5,7,8,-tetramethylchroman-2-carboxylic acid (Trolox), and fluorescein sodium salt (FL) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Fetal calf serum (FCS) and Folin-Ciocalteau (F-C) reagent were purchased from JRH Biosciences (Lenexa, KS, USA) and MP Biomedical, LLC (Illkirch, France), respectively. S9-mix (rat liver homogenate containing rat liver microsome S9 fraction) was obtained from Kikkoman Co. Ltd. (Tokyo, Japan). Cell proliferation reagent WST-1 was purchased from Takara Bio Inc. (Siga, Japan). Methanol, acetone, gallic acid, 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P2), 2,2’-azobis(2-amidinopropane) dihydrochloride (AAPH) and dimethyl sulfoxide (DMSO) and all other chemicals were purchased from Wako Pure Chemical Co. (Osaka, Japan).

Plant material and sample preparation Fresh Kalmi Shak was collected within 24 hour of harvest from the Dhaka (Dhaka is the capital of Bangladesh and one of the major cities of south Asia) new market during the summer period (mid April to June onwards). One hundred grams of green leaves and veins were cleaned with water, and finally freezedried and kept at -20°C until use. One gram of the freeze-dried sample was sequentially extracted with hexane: dichloromethane (1:1) (v/v) and with methanol: water: acetic acid (MWA) solvent at the ratio of 90:9.5:0.5 (v/v/v) using an automatic accelerated solvent extraction system (ASE 350; Dionex, Sunnyvale, CA, USA). Lipohilic fraction was collected (3 times) by hexane: dichloromethane at 70°C, 5 min stand at 1500 psi. Hydrophilic fraction was collected thrice by MWA

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solvent at 80°C, 5 minute stand at 1500 psi. The resulting MWA extract of the Kalmi Shak which was used for subsequent experiments was filled up to 50 ml by MWA. For cell culture and microbiological analyses, MWA fraction was dried in vacuo and dissolved in DMSO.

Determination of hydrophilic-oxygen radical absorbance capacity (H-ORAC) H-ORAC assay was performed according to the method described by Cao et al. (1993), and Prior et al. (2003) with slight modifications. In brief, MWA extracts or Trolox standard solution diluted with 75 mmol/L potassium phosphate buffer (pH 7.4) were added to a 96-well microplate (#3072, Becton Dickinson, NJ, USA). Following the addition of 115 µl of 111 nmol/ LFL to the wells, the plates were incubated at 37°C for 10 min. After the addition of 50 µl of 31.7 mmol/l AAPH to the wells, fluorescence intensities were measured every two min. for 90 min. by a microplate reader (Powerscan HT; DS Pharma Biomedical, Osaka, Japan) with excitation wavelength of 485 nm and emission wave length of 530 nm. H-ORAC values were expressed as micromole Trolox equivalent per gram of dry sample weight (µmol TE/g DW).

All measurements were done in triplicate.

Measurement of total polyphenols content Total polyphenols content was measured by the Folin-Ciocalteu assay according to Sun et al. (2005) and Velioglu et al. (1998) with slight modifications.

Briefly, three volumes of F-C reagent was diluted by five volume of water before use. Reaction mixture containing 80 µl of samples or gallic acid standard (diluted with MWA) and 56 µl of diluted F-C reagent was placed in 96 well-microplate (Sumilon, Sumitomobakelite, Tokyo, Japan), and incubated for five min at room temperature. After the addition of 120 µl of 2% (w/v) sodium carbonate, the plate was allowed to stand for 15 min at room temperature. Absorbance at 750 nm was measured by a microplate reader (Powerscan HT; DS Pharma Biomedical). Total polyphenols content was expressed as milligram

gallic acid equivalent per gram of dry sample weight (mg GAE/g DW). All measurements were conducted in triplicate.

DPPH radical (DPPH-RSA)

scavenging

activity

DPPH-RSA of MWA extract was examined according to the method of Oki et al. (2001) with slight modifications. Briefly, the same volume of 10% methanol and MWA extract were mixed, and the mixture was further diluted with 50% methanol. A 50 µl of diluted MWA extract and 50 µl of 0.2 M morpholinoethanesulfonic acid (MES) buffer (pH 6.0) were subsequently placed in a 96-well microplate (Sumilon, Sumitomobakelite). The reaction was initiated by adding 50 µl of 800 µM DPPH in ethanol. After incubation for 20 min. at room temperature, the absorbance at 520 nm was measured using a microplate reader (Powerscan HT; DS Pharma Biomedical).

DPPH radical scavenging activity was expressed as micromole Trolox equivalent per gram of dry sample weight (µmol TE/g DW). All the determinations were conducted in triplicate.

Determination of moisture content The moisture content was determined by drying the samples in a drying oven at 105°C for 24 h (AOAC, 1984). The leaf and vein (edible portion) of fresh Kalmi Shak (5.0 g) were cut by dual razor blades into small pieces, subsequently placed in aluminum cups and weighed before and after drying. The percentages of moisture content were calculated by subtracting the two values. At least 10 samples per experiment were replicated, and mean values for each replicate were calculated.

Determination of the anti-mutagenic effect on Trp-P2 induced mutagenicity to Salmonella Typhimurium TA98 The assay was carried out according to the modified Ames test (Ames et al., 1975) with Salmonella Typhimurium TA98. In brief, TA98 strain was cultured

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aerobically in Nutrient broth no. 2 (Oxoid Ltd., Basingstoke, UK) at 37°C for 12-14h. Trp-P2 was dissolved in DMSO to give a working concentration of 100 ng/ml. The reaction mixture consisted of 0.7 ml of 0.1 mol/l phosphate buffer (pH 7.0), 50 µl of sample (40 µg/ml in DMSO), 100 µl of S-9 mix, 50 µl of Trp-P2 and 100 µl of S. Typhimurium TA98. The positive control contained the same concentration of perilla leaves extract in DMSO instead of the sample. Following incubation at 37°C for 20 min in water bath shaker, two milliliters of soft agar containing histidine and biotin was added, and the mixture was immediately plated on a minimal glucose agar.

After incubation at 37°C for two days, the number of developed revertants was scored. The experiment was performed in triplicate and the mean values are presented. Anti-mutagenic activities of the Kalmi Shak extract were calculated according to the equation described by Hosoda et al. (1992).

Anti-tumor effects to mouse myeloma P388 cells

lar Devices Co., Tokyo, Japan). Results were reported as percentage of the inhibition of cell viability, where the optical density measured from DMSO-treated control cells was considered to be 100% of viability.

Percentage of inhibition of cell viability was calculated as follows:

(-

)

1 Aexp group Acontrol

x 100

Test organisms Fifteen strains/species of frequently reported food borne pathogens or food spoilage bacteria were used in the study (Table 2). The stock cultures of the test organisms in 20% glycerol (Sigma) containing medium in cryogenic vials were maintained at -84°C. Working cultures were kept at 4°C on Trypto Soy Agar (TSB) slants (Nissui Chemical Co. Ltd, Tokyo, Japan) and were periodically transferred to fresh slants.

Anti-microbial sensitivity testing Anti-tumor activities were measured by the viabilities of myeloma P388 cells using WST-1 cell proliferation reagent (Shinmoto et al., 2001). In brief, P388 cells (Japan Health Sciences Foundations, Osaka, Japan) were seeded in 96-well culture plates (#353072, Falcon) at a density of 5,000 cells (100 µl) per well in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) and 100 units/ml penicillin and 100 μg/ml streptomycin and incubated at 37°C in a humidified atmosphere with 5% CO2. DMSO solutions of Kalmi Shak with various concentrations were added to each well (final concentrations of 0 (negative control), 50, 100 and 200 μg/ml). Final concentration of DMSO was 0.4%.

Rosemary (Rosmarinus officinalis) extract at varying concentrations (50 to 200 μg/ml) in DMSO were used for positive control. After 48 hours incubation, 10 µl of premixed WST-1 cell proliferation assay reagent was added to each well. Two hours after the addition of WST-1, the degree of cell viability was measured by the absorbance at 450-650 nm of the cell culture media using microplate reader (Thermomax, Molecu-

The anti-microbial activity of the Kalmi Shak extracts was done according to the method of Bauer et al. (1966). The 8 mm in diameter discs (Toyo Roshi Kaisha, Ltd. Tokyo, Japan) were impregnated with 50 µl of different concentration of Kalmi Shak extract before being placed on the inoculated agar plates.

The inocula of the test organisms were prepared by transferring a loopful of respective bacterial culture into 9 ml of sterile TSB medium and incubated at 37°C for 5 to 6 h. The bacterial culture was compared with McFarland (Jorgensen et al., 1999) turbidity standard (108 CFU/ml) and streaked evenly in three planes maintaining a 60° angle onto the surface of the Mueller Hinton agar plate (5 x 40 mm) with sterile cotton swab. Surplus suspensions were removed from the swabs by rotation against the side of the tube before the plate was inoculated. After the inocula dried, the impregnated discs were placed on the agar using an ethanol dipped and flamed forceps and were gently pressed down to ensure contact.

Plates were kept at refrigeration temperature (4°C)

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for 30 to 60 min for better absorption, during which microorganisms should not grow but absorption of extracts should take place. Negative controls were prepared using the same solvent without the plant extract. Reference antibiotics (streptomycin, gentamycin, and rifampicin) were used as positive control.

The inoculated plates containing the impregnated discs were incubated in an upright position at 37°C for overnight and/or 24 to 48 h (depending on the appearance of colonies). The results were expressed as positive/negative depending on the zone of inhibition.

Table 1. Anti-oxidative activity, total polyphenols content and moisture content of indigenous Kalmi Shak in Bangladesh

Statistical analysis Statistical analysis was performed using Microsoft Excel (2007). The data were expressed as means ± standard deviation (SD) for foods having sample numbers greater than 2.

Results and Discussion It was observed that Kalmi Shak possessed 341.92 ± 1.32 µmol TE/g DW of H-ORAC value (Table 1).

From the moisture content, the H-ORAC value in fresh weight basis can be calculated as 51.28 µmol TE/g fresh weight (FW). Wu et al. (2004) reported that H-ORAC values of common vegetables in USA were between 0.87 (cucumber) and 145.39 (small red beans) µmol TE/g FW. The most values were in a range from 5 to 20 µmol TE/g FW. It is suggested that H-ORAC value of water spinach is relatively high when compared with those of common vegetables and fruits.

Mikami et al. (2009) studied antioxidant activities of 11 crops from Ibaraki prefecture, Japan, and found that DPPH-RSA ranged from 0.38 (melon) to 91.0 (ginger) µmol TE/g FW. Pellegrini et al. (2003) studied 34 vegetables and found that spinach exhibited the highest antioxidant capacity (8.49 µmol TE/g FW). The DPPH-RSA of water spinach in our study (Table 1) was nearly equal to that of spinach, though the methodologies of determination were slightly different.

It has been reported that the total polyphenols contents of 10 vegetables examined by Cieslik

ac µmol Trolox equivalent (TE)/g DW ± SD bd µmol Trolox equivalent (TE)/g FW e mg galic acid equivalent (GAE)/g DW ± SD f mg galic acid equivalent(GAE)/g FW g Percentage

et al. (2006) were between 0.59 to 2.90 mg GAE/g FW of samples. Wu et al. (2004) observed that total polyphenols of 23 vegetables were between 0.24 ± 0.05 (cucumber) and 12.47 (red kidney beans). Water spinach is available in Bangladesh during the entire year and our collection period originated from early onset of summer. It has been reported that leaves harvested in the spring exhibited much higher levels of total polyphenols content and ORAC value than the leaves harvested in the fall (Howard et al., 2002).

Consequently, further study should be undertaken to see the seasonal variation of antioxidant content of this leafy vegetables.

MWA extract of Kalmi Shak exhibited anti-mutagenic effects on Trp-P2 induced mutagenicity to S. Typhimurium TA98 when tested with perilla as positive control (Fig. 1). Kanazawa et al. (1995) reported

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Figure 1. Anti-mutagenic activity on Trp-P2 induced mutagenicity to Salmonella Typhimurium TA98

Concentration μg/ml

Figure 2. Anti-tumor activity on mouse myeloma P388 cells

that flavonoids were very strong anti-mutagens against Trp-P2. In our study, anti-mutagenic activities against Trp-P2 were observed to be 52.62% and 50.79% for perilla and Kalmi Shak, respectively (Fig. 1). However, other established mutagens such as MNNG, AF-2, AB1 etc. were not tested. Therefore, it is necessary to check the anti-mutagenic activities of Kalmi Shak extract on the mutagenicity of these agents in future studies.

MWA extract of Kalmi Shak yielded detectable anti-tumor activity in the mouse myeloma P388 cell line. Rosemary extract was used as a positive control in this experiment, since rosemary leaves exhibit potent anti-tumor and anti-inflammation effects (Peng et al., 2007). Dose-dependent increase on the antitumor activity was observed in both Kalmi Shak and rosemary extract (Fig. 2). At a concentration of 50 µg/ml, the corresponding cell viability of rosemary and Kalmi Shak was 61.36% and 67.56 %, respectively (Fig. 2). At a concentration of 200 µg/ml, cell viability of rosemary was 1.66%. On the other hand, that of Kalmi Shak was 47.59%. Since Kalmi Shak inhibited the cell viability by more than 50% cell at this concentration, and almost 100% cells were not viable in the case of rosemary. We reported, here, that Kalmi Shak extract is capable of working against P388 cell viability.

MWA extract of Kalmi Shak exhibited anti-microbial activities against several spoilage and food borne pathogenic bacteria within tested fifteen selected bacteria. The result is presented in Table 2. The extract of Kalmi Shak exhibited in vitro anti-microbial activities against spoilage bacteria P. aeroginosa, P. putida, and pathogenic bacteria such as E. coli O157:H7 and C. freundii. The result of this study also suggests that Kalmi Shak extracts include compounds possessing anti-microbial properties that may be useful to control food borne pathogens and spoilage organisms.

Further studies need to be done with other food borne pathogens and spoilage organisms to see the anti-microbial activities of Kalmi Shak. It would also be of interest to apply this extract to actual food to assess the microbiological condition of the particular food or food products with an extended shelf life.

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CONCLUSION In conclusion, the results from in vitro experiments, including H-ORAC, DPPH-RSA, total polyphenols content, anti-mutagenic activity, anti-tumor activity, and anti-bacterial activity demonstrated that Bangladeshi water spinach variety possessed potent anti-oxidative

and anti-tumor activities. Hence water spinach can be used as an easy accessible source of natural antioxidants, as a food supplement or in the pharmaceutical or medical industries. Further work should be performed to isolate and identify the anti-oxidative, antimutagenic, anti-cancer, and anti-bacterial components of this indigenous vegetable of Bangladesh.

Table 2. Test organisms used, their source and antibacterial activity of DMSO suspended MWA extract of Kalmi Shak against selected food borne pathogens and spoilage bacteria Test Organisms

Origin

Anti-microbial activity

Spoilage bacteria Lactobacillus planterum (ATCC 8014)

Mexican style cheese

-

Perdicoccus pentosaceus(JCM 5890)

Dried American beer yeast

-

Lactoccus lactis (IFO 12007)

Unknown

-

Salmonella Enteritidis (SE1)

Chicken feces

-

Pseudomonas aeroginosa (PA 01)

Unknown

+

Enterobacter faecalis (NFRI 010618-8)

Unknown

-

Klebsilla pneumonia (JCM 1662)

Trevisan 1887

-

Bacillus subtilis (IFO 13719)

Wound

-

Pseudomonas putida (KT 2440)

Unknown

+

Escherichia coli (NFRI 080618-8)

Celery

+

Escherichia coli O157:H7 (CR 3)

Bovine feces

+

Escherichia coli O157:H7 (MY 29)

Bovine feces

+

Citrobacter freundi (JCM 1657)

Werkman and Gillen 1932

+

Bacillus cereus (IFO 3457)

Unknown

-

Alcaligenes faecalis (IFO 12669)

Unknown

-

Pathogenic bacteria

+ , - : indicates positive and no positive activity found in preliminary screening, respectively 30 Agric. Food Anal. Bacteriol. â&#x20AC;˘ www.AFABjournal.com â&#x20AC;˘ Vol. 1, Issue 1, 2011

Acknowledgement This research work was supported by Kirin Holdings Co., Ltd. (former Kirin Brewery Co., Ltd.) Tokyo, during UNU-Kirin fellowship at National Food Research Institute, Tsukuba, Japan in 2010-11, and its Follow-up Project in 2011-2013. Authors expressed their sincere gratitude to the authorities of the NFRI for providing laboratory facilities and logistic supports to carry out this investigation.

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32 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

www.afabjournal.com Copyright © 2011 Agriculture, Food and Analytical Bacteriology

Using Hydrogen- Limited Anaerobic Continuous Culture to Isolate Low Hydrogen Threshold Ruminal Acetogenic Bacteria P. Boccazzi¹,² and J. A. Patterson² ¹ Current address: Massachusetts Institute of Technology, Department of Biology and Health Sciences and Technology, 77 Massachusetts Ave. Room 68-370, Cambridge, MA 02139 ² Department of Animal Sciences, Purdue University, 1026 Poultry Building, Room 115, West Lafayette, IN 47907-1026

ABSTRACT Hydrogen—limited continuous culture was used to isolate autotrophic acetogenic bacteria from rumen contents of cattle on either a high roughage or a high concentrate diet. Twenty bacterial isolates were obtained and were presumptively identified as acetogenic bacteria. They were able to use H2:CO2 and they produced acetic acid as their sole end—product. Two isolates were selected for further studies based upon their low hydrogen threshold values. The acetogenic strain H3HH was a strictly anaerobic gram positive coccus with a hydrogen threshold of 1390 ppm. The acetogenic strain Al0 was a facultatively anaerobic gram positive coccus with a hydrogen threshold of 209 ppm. The use of H2 limited continuous culture to isolate low H2 threshold ruminal acetogens suggests that not only do acetogens with these properties exist in the rumen but this approach could be used in other ecosystems as well.

Keywords: Methane, greenhouse gasses, methanogens, acetogens, ruminants, rumen, hydrogen, continuous culture, carbon dioxide Agric. Food Anal. Bacteriol. 1: 33-44, 2011

Introduction Ruminants are characterized by having a four compartment stomach (Russell and Rychlik, 2001). The largest compartment, the rumen, has a volume of nearly 80 liters and is located before the gastric compartment (Weimer et al., 2009). The rumen ecosystem is essentially isothermal, there is a constant flux of feed and H2O and the fermentation of substrates Received: September 12, 2010, Accepted: November 17, 2010.

Released Online Advance Publication: April 2011.

Correspondence: John Patterson, jpatters@purdue.edu Tel: +1 -765-494-4826 Fax: +1-765-494-9347

results in the production of a large amount of acids (Weimer et al., 2009). Functionally important rumen microorganisms representing a varied and mixed population of bacteria, archaea, protozoa, and fungi hydrolyze complex and soluble feedstuffs primarily to sugars and other hydrolysis products such as ammonia (Ricke et al., 1996; Stevenson and Weimer, 2007; Uyeno et al., 2007). Glucose is subsequently fermented in the rumen by rumen microorganisms to short chain volatile fatty acids (VFA) with the end products of fermentation including acetate, H2, CO2, and reduced fermentation products (lactate, butyr-

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ate, propionate, ethanol) along with microbial cells (Stevenson and Weimer, 2007; Weimer et al., 2009).

Hydrogen and formate are produced by many microorganisms in the rumen; however, methanogens are also present in the rumen and convert H2, and CO2 to CH4 (Wright et al., 2006). Methanogenesis represents the primary H2 consumer in the rumen and energy captured as methane escapes the rumen via eructation (Boadi et al., 2004, Martin et al., 2010).

Energy lost as methane represents a 2 to 7% loss in gross energy intake energy of the animal (Branine and Johnson, 1990) and a loss of 10 to 15% of the apparently digestible feed energy to the host animal (Blaxter and Clapperton, 1965). However, direct inhibition of rumen methane production also results in energy loss in the form of eructated H2 and reduced microbial protein (Chalupa, 1980).

Chemo-lithoautotrophic acetogens are bacteria that utilize CO2 as their sole source of carbon and reduce it to acetate with H2 as the source of energy (Drake et al., 2008; Ragsdale, 2008). Acetogens are known to be present in the rumen but they are less numerous and considered to be less efficient than methanogens for utilization of hydrogen as a substrate (Martin et al., 2010). Replacement of methanogenesis with acetogenesis could decrease energy losses and increase the efficiency of ruminant production. Consequently, research on acetogenesis in ruminant animals has been focused toward two related areas of interest and application. First of all, since methane formed as a result of ruminal fermentation is subsequently eructated and is lost to the animal; thus, it would increase energetic efficiency of the host animal if this loss of feed energy and carbon could be minimized (Boadi et al., 2004; Martin et al., 2010). Secondly, there is increasing interest in global warming forced by the production of greenhouse gasses such as CO2, CH4, and NO2 (Boadi et al., 2004; Morrison, 2009). Reductive acetogenesis is a means for developing alternative H2 sinks away from methanogens that produce CH4 (Joblin, 1999).

Acetogenesis may provide an important model to find solutions for limiting CH4 emissions from livestock and livestock wastes (Morrison, 2009). Efforts to enhance in vivo acetogenesis in the rumen have

not been as successful as in vitro studies (Fonty et al., 2007). Methanogens are thought to outcompete acetogens because methanogens have a lower hydrogen threshold (Martin et al., 2010); however, most acetogens have been isolated in batch culture in the presence of high hydrogen concentrations and have not been selected for low hydrogen thresholds. A key may be a better understanding of hydrogen use by acetogens. The objective of this study was to use H2-limited continuous culture to demonstrate that it could be used to isolate ruminal acetogenic bacteria able to grow on low threshold concentrations of H2 utilizing CO2 as their sole carbon source.

Materials and Methods Source of Organisms Acetogenic bacterial strains were isolated either from rumen contents collected either from a ruminally fistulated Angus steer fed a diet of alfalfa and orchard grass hay at maintenance or of a ruminally fistulated lactating Holstein Friesian dairy cow consuming a 60:40 percent hay and corn silage: corn grain diet at 2.6% of her body weight. Rumen contents were used to inoculate H2-limiting continuous cultures. Individual strains were isolated after at least 8 turnovers of the continuous culture.

Media and Growth Conditions All media were prepared by the anaerobic techniques of (Hungate, 1966) as modified by (Balch and Wolfe, 1976; Bryant, 1972). The basal semidefined acetogen medium used for growth and nutritional studies and the methanogen medium are listed in Table 1. The medium was boiled under a stream of oxygen-free CO2, sealed, and autoclaved (120°C, 18 Ib/in², 15 min). The pH of the medium was adjusted to 6.8 with NaOH before boiling. The cooled medium was transferred into an anaerobic glove box (Coy Laboratories, Ann Arbor, MI) containing 95% CO2: 5% H2. For all media, the reducing agents, carbonate buffer and vitamins were added separately to the medium in the anaerobic glove box as sterile

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Table 1. Media Composition

Components

Acetogen (g/L or ml/L)

Methanogen (g/L or ml/L)

0.24 0.24 0.24 0.48 0.1 0.07 0.54 4

0.3 0.3 0.3 0.6 0.13 0.008 1 5

0.25

0.25

0.25 0.5 0.001 0.0001 -

0.25 2 0.001 0.001 2 0.01 0.2

Clarified Rumen Fluid (CRF)

50.0ml

100.0ml

Vitamin Sol.b Trace Min. Sol.

10.0ml 10.0ml

10.0ml 10.0ml

Wolf's Trace Min. Sol.c

10.0ml

-

KH2PO4 (200nM, pH=7)

-

50.0ml

VFA-Mc

-

10.0ml

K2HPO4 KH2PO4 (NH4)SO4 NaCl MgS04·7H2O CaCl2·2H20 NH4Cl Na2CO3a Cys·HCl a Na2S·9H2O a Yeast Extract Resazurin Hemin Trypticase CoM FeSO4·7H2O

a Na2CO3 (8% w/v), Cys·HCl (2.5% w/v) and Na2S·9H2O (2.5% w/v) were added separately as sterile anaerobic solutions, to autoclaved and cooled medium. b Greening and Leedle (1989) c Balch and Wolf (1976)

anaerobic solutions. The medium was subsequently dispensed into 120 ml serum bottles or into 20 ml serum tubes which were then closed with sterile black butyul rubber serum stoppers and aluminum crimp closures (BellCo Inc., Vineland, NJ). Solid medium for isolation of pure cultures consisted of acetogen medium with the addition of 2% (w/v) agar (Difco).

Continuous culture medium was the same as the acetogen medium, but the rumen fluid was previously incubated at 37°C for 6 days to remove carbo-

hydrates (Greening and Leedle, 1989). For chemolithoautotrophic growth of bacterial cultures, serum bottles (120 ml) and Erlenmeyer flasks (330 ml) were flushed for 30 seconds and then pressurized at 2.0 atm with either a H2:CO2 (80:20) or a N2:CO2 (75:25) gas phase, by insertion of sterile disposable needles through the black butyl stoppers. In all growth and nutritional studies cultures were incubated at 39°C.

Isolation Procedures Hydrogen-limited continuous cultures were utilized in an attempt to isolate acetogenic bacteria with low H2 thresholds from the bovine rumen. The isolation medium contained 5 mM 2-bromoethanesulfonic acid (BES) (LeVan et al., 1998) to inhibit methanogens. The growth vessel (200 ml) was initially half filled with isolation medium, and BES was added to give an initial concentration of 40 mM for the full volume of the growth vessel. The inoculum, 40 ml of rumen fluid, was collected from either the steer or the lactating dairy cow prior to morning feeding and strained through a bilayer of cheesecloth under a stream of CO2 and was added to the growth vessel.

The medium pump was started immediately after inoculation and the medium contained 5 mM BES. The reservoir and growth vessel of the continuous culture were flushed with a stream of humidified oxygen-free 100% CO2 gas through a glass diffusion stone. Although the flow rate was not measured, humidified oxygen-free 100% H2 gas was bubbled into the growth vessel at a rate to provide 5 to 10 bubbles/ minute. The dilution rate of the continuous culture was 0.06 h-1 during isolation of acetogenic bacteria from the steer and 0.28 h-1 during isolation of acetogenic bacteria from the lactating dairy cow.

The growth vessels were incubated at 39°C. After 8 fluid volume turnovers, 1 ml of fermentation fluid from the growth vessel was serially diluted in anaerobic dilution solution. Each dilution was plated in triplicate on solid acetogen medium containing 5 mM BES. Plates were subsequently incubated anaerobically under 1.5 atm of H2:CO2 (80:20) for 6 days at 39°C. Ten single colonies from each continuous culture were selected at random and transferred

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anaerobically to 10 ml of acetogen medium in duplicate serum bottles. The bottles were pressurized at 2 atm of either H2:CO2 (80:20) or N2:CO2 (75:25) and incubated on their sides in a rotatory shaker (New Brunswick Scientific Co., Inc. Model M52, Edison, NJ) operating at 200 rpm for 3 days at 39°C.

Volatile Fatty Acid (VFA) Assay After incubation, the supernatant of each culture was analyzed by gas chromatography using a Varian 3700 (Varian, Inc., Palo Alto, CA) gas chromatograph, to determine VFA composition. Bacterial isolates producing at least a 4 fold increase in acetate in bottles containing H2:CO2 over that produced in bottles containing N2:CO2 were retained for further characterization.

H2 Threshold Assay In order to isolate acetogenic strains with low H2 thresholds, a series of three experiments were performed. The general protocol was to grow cultures in a complex medium to increase cell number, then adapt the cells to H2:CO2 flush excess H2:CO2, and determine H2 thresholds using lower concentrations of H2. Culture vessels were incubated at 39°C on their sides in a rotatory shaker operating at 200 rpm.

Experiment 1 Triplicate cultures of each acetogenic isolate were grown in acetogen medium containing 27.8 mM glucose for 60 h. Serum bottles were pressurized to 1.5 atm with H2:CO2 (80:20) and the cultures were incubated an additional 60 h. Cultures were subsequently flushed and pressurized to 1.5 atm with N2:CO2 (75:25) and incubated for 36 h to lower residual H2 concentration. Cultures were subsequently flushed and pressurized to 1.5 atm with H2:CO2:N2 (1:24:75) and incubated for 60 h. Methanogens were grown on methanogen medium for 120 h on H2:CO2 (80:20) at 1.5 atm, then were flushed with N2:CO2 (75:25) and incubated with H2:CO2:N2 (1:24:75) at 1.5 atm for 60 h.

Experiment 2 The format was similar to experiment 1 in incuba-

tion times and sequence of gas phases. Differences were duplicate cultures were used and the initial medium contained 0.2% (w/v) Brain Heart Infusion Broth (BHI) instead of glucose. Cultures were incubated for 130 h under H2:CO2:N2 (1:24:75) after flushing with N2:CO2 (75:25).

Experiment 3 The format was similar to experiment 2 where bacterial cultures were initially grown in BHI and then flushed with N2:CO2 (75:25), except that 10 ml fresh acetogen medium (without glucose or BHI) was added prior to pressurizing with H2:CO2:N2 (1:24:75).

After incubation, the head space of each culture vessel was analyzed by gas chromatography using a varian 3700 gas chromatograph, to determine H2 concentration. Bacterial isolates with the lowest H2 thresholds were retained for further characterization. Selected strains were further purified on solid acetogen medium under H2:CO2 (80:20) and stored as broth cultures in glycerol at -4°C as described by (Teather, 1982).

Characterization studies Gram stain, flagella stain, optimum pH, and heat test for spore determination were performed according to (Holdeman et al., 1977). Optimum temperature of growth was determined by growing cultures in acetogen medium containing 5.6 mM glucose at the respective temperatures for 48 h with optimum temperature being defined as that temperature that yielded the highest OD measured at 660 nm at 48 hours. Oxygen sensitivity was tested by three methods: a) degree of growth throughout stab cultures in acetogen medium containing 0.5% (w/v) glucose and 0.4% (w/v) agar, in which the topmost layer was allowed to oxidize; b) zone of growth in PYG molten agar medium (Holdeman et al, 1977); and c) growth on non-reduced, aerobically prepared solid acetogen medium or in aerobic acetogen broth containing 0.5% (w/v) glucose. For colony formation, isolates were plated on solid acetogen medium containing 5.6 mM glucose and incubated aerobically or anaerobically at 39° C. Nitrate reduction, catalase, oxidase, esculin hydrolysis and utilization, and

36 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

starch-hydrolysis tests were performed according to (Holdeman et al., 1977). GC-fatty acid methyl ester (FAME) analysis was performed on strains H3HH and A10 grown in acetogen medium (Table 1) by Microbe Inotech Laboratories (St. Louis, MO). Similarity and distance coefficients were evaluated between strains A10 and H3HH and known bacterial species using the anaerobe database (Moore, ver. 3.7).

Nutritional Studies The ability of isolates to utilize organic substrates as energy sources was determined using acetogen medium containing 0.5% (w/v) of the substrate tested.

Each organic substrate tested was added to the medium as a sterile anaerobic stock solution. Substrate utilization was assessed by an increase in OD, 660 nm, after 36 h of growth at 39°C. Determination of cell dry mass was performed directly on cells washed in saline solution (NaCl, 0.1% w/v) and harvested from distilled water. For molar growth yields, cell net dry weight of 500 ml cultures were compared with the amount of substrate consumed. Glucose concentration was measured enzymatically using glucose oxidase reagents from Sigma Chemical Co. (St. Louis, MO).

The requirement of isolates for rumen fluid and yeast extract was determined using Erlenmeyer flasks (300 ml, BellCo Inc., Vineland, NJ) modified by addition of a side arm (130 mm x 16 mm) and a serum bottle (20 mm) closure at the top. The bottles were filled with 20 ml of acetogen medium and then were pressurized to 2 atm with either H2:CO2 (80:20) or N2:CO2 (75:25). The inoculum was 0.2 ml (1%, w/v) of a third transfer of a culture grown under H2:CO2 (80:20). The flasks were incubated upright at 39°C and agitated at 200 rpm on a rotatory shaker. The growth of each organism was followed by measuring the increase in optical density at 660 nm with time.

Growth Studies For the assessment of growth and stoichiometry of acetic acid production and H2 consumption, serum bottles (120 ml, BellCo Inc.) were filled with either 10 ml of basal acetogen medium or with acetogen

medium containing 5.6 mM glucose and pressurized to 2 atm with either H2:CO2 (80:20) or N2:CO2 (75:25).

Cultures from each isolate were transferred three times in medium containing the test substrate and then a 0.1 ml of the culture was used to inoculate serum bottles for growth determination. Serum bottles were incubated on their sides and agitated at 200 rpm on a rotatory shaker (New Brunswick Scientific Co.). Growth of each isolate was measured as an increase in OD. Hydrogen utilization was determined by measuring reduction in gas volume with a system similar to that described by (Balch and Wolfe, 1976).

For each sample time, a 4 ml sample of the culture liquid from each culture was frozen (-4°C) for subsequent VFA analysis.

Analytical Methods Optical density was measured at 660 nm using a Spectronic 70 spectrophotometer (Bausch & Lomb, Rochester, NY). Volatile fatty acid production by isolates was measured by gas-liquid chromatography (GLC) (Holdeman et al., 1977). The frozen samples were thawed and centrifuged at 15,000 rpm for 5 min, the supernatant was subsequently acidified by adding 20% (w/v) of methaphopsphoric acid (25%, w/v) and analyzed. A 3 ft long column, packed with SP 1220 (Supelco, Bellefonte, PA, USA), was used in a Varian 3700 GLC with a flame ionization detector.

Oven temperature was 130° C (isothermal), injector temperature was 170° C, and detector temperature was 180°C, with carrier gas (N2) flowing at rate of 30 ml per minute.

For the measurement of H2 uptake and CH4 production, gas samples were analyzed using a Varian 3700 gas chromatograph equipped with a thermal conductivity detector and a silica gel column (Supelco).

Temperatures of the injector, oven and detector were room temperature, 130°C, and 120°C respectively, with carrier gas (N2) flowing at 30 ml per minute.

Microscopy Determination of cell morphology and presence of spores and flagella were assessed by phase con-

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trast microscopy (Carl Zeiss D-7082, Oberkochen, Germany). Scanning electron micrographs were prepared from cells grown to midlog or early stationary phase in acetogen medium containing 27.8 mM glucose. For scanning electron microscopy, a poly-Dlysine coated cover slip was immersed in culture fluid for one hour. Each coverslip was then fixed for 3 h with 5% (w/v) glutaraldehyde and 1% (w/v) osmium tetroxide in 0.1 M phosphate buffer, pH = 6.8. The material was dehydrated by a series of graded ethanol solutions (Lamed et al., 1987). The cells on the cover slips were critical point dried with liquid CO2.

The samples were sputter coated with gold palladium in a Technics Hummer I and viewed with a JEOL JSMM840 scanning electron microscope (JEOL Ltd. Tokyo, Japan).

DNA Base Composition For determination of mole percent guanine plus cytosine, chromosomal DNA was extracted from bacterial cells using the procedure described by (Marmur, 1961). The mole percentage guanine plus cytosine was calculated from the inflection point of the temperature melting profile of isolated DNA with DNA from Escherichia coli strain K12 as the reference (Marmur and Doty, 1962). The temperature melting profiles were analyzed using a Perkin-Elmer Lambda 3A spectrophotometer (Norwalk, CT) equipped with a thermal cuvette.

Results and Discussion Isolation of Bacteria

On the basis of morphology, growth characteristics, and H2 threshold values, acetogenic strains H3HH, H3HP, A10, A2, A4, and A9 were selected for further characterization. Hydrogen threshold of strain A10 and H3HH were the closest to those of the methanogen strain NI4A (Table 2) and were more completely characterized. These threshold values are lower than those reported by (LeVan et al., 1998) for other ruminal acetogens and are comparable to the values for non-ruminal acetogens (Cord-Ruwisch et al., 1998).

Table 2. Hydrogen threshold values of methanogen strain NI4A and of acetogenic strains A10, A2, A9, A4, and H3HH. H2 Concentration (ppm)

Culture

EXP 1a

EXP 2b

EXP 3c

Initial NI4A A10 A2 A9 A4 H3HH

10702 92 1284 2516 5383 8007 1390

9993 90 994 1852 66157

9993

Acetogenic isolates initially grown in 10 ml acetogen medium containing 27.8 mM glucose then flushed and incubated with 1% H2 for an additional 60 h

a

Acetogenic isolates initially grown in 0.2% (w/v) BHI then flushed and incubated with 1% H2 for an additional 130 h b

Acetogenic isolates initially grown in 0.2% (w/v) BHI with 10 ml fresh medium added prior to incubation with 1% H2 for additional 130 h c

d

Twenty strains of acetogenic bacteria were isolated from rumen contents of either an Angus steer fed a high forage diet or a lactating dairy cow (Holstein Friesian) fed a 40% concentrate diet. All isolates produced at least five fold more acetate under H2:CO2 than under N2:CO2. Acetate production ranged from 40 to 75 mM on H2:CO2 and from 2 to 8 mM on N2:CO2. The production of other short chain VFA was minimal for all strains designated.

NDd ND

NDd 208 1383 1619 ND ND

ND=not done

Bacterial Characterization All isolates stained gram positive. Strain A4 and A9 were short rods while strains A2, A10, H3HH, and H3HP were oval cocci (Table 3). However, H3HH was pleomorphic, especially during exponential growth on a rich carbohydrate medium. No flagella were ob-

38 Agric. Food Anal. Bacteriol. â&#x20AC;˘ www.AFABjournal.com â&#x20AC;˘ Vol. 1, Issue 1, 2011

Table 3. Morphological characteristics of isolates.

Gram stain

Shape

H3HH

+

cocci-pleom

H3HP

+

A10

+

A2

+

Culture

Cat. = catalase

Cell size Oxygen Cat. Spore Motile µm Sens.

0.6-0.8 x anaerobe No 1.0-1.2 0.6-0.8 x oval-cocci anaerobe No 1.0-1.3 0.6-0.8 x facultative + No oval-cocci 1.0-1.4 0.6-0.8 x anaerobe No oval-cocci 1.0-1.5 Sens. = sensitivity pleom. = pleomorphic

Optimum G+C Optimum Temp.

Mol pH (°C) %

No

39

6.8-7.0

ND

No

39

7.5

ND

No

39

6.8-7.5

51.5

No

39

6.8-7.5

ND

served by either electron microscopy (Figures 1 and 2 for A10 and H2HH) or by standard staining procedures using light microscopy. No spores were observed by phase contrast microscopy and no spores were produced by heating the cultures at 80° C for 30 min (Holdeman et al., 1976).

All strains when grown in H2:CO2 or glucose were mesophilic. Strains H3HH, A10 and A2 grown in acetogen medium plus 0.1 (w/v) glucose, reached higher OD at 30° C, however all strains grew faster at 39°C (Table 4). Thus 39°C was considered the optimum temperature of growth. Cells grew within a temperature range of 17° to 45°C. strains H3HH and A2 reached higher OD at 30°C and grew poorly or did not grow at

17°C. Strain A10 grew at almost the same rate at 30° C and 39°C and was uniquely different from strain H3HH and A2 in that it was able to grow at 17° C. The optimum pH for growth in acetogen medium plus 0.5% (w/v) glucose was 7.0 for strains A10 and H3HH and 7.5 for strains A2 and H3HP (Table 3). The pH range for growth was 5.5 to 8.0 (data not shown). While strains H3HH, H3HP, and A2 were strict anaerobes, strain A10 was considered to be facultative, because it grew in all the media and conditions used to determine oxygen sensitivity (Table 3). Most acetogens isolated have been more strict anaerobes although several can tolerate low concentrations of O2 after exhibiting a lag phase in growth (Karnholz et al., 2002).

Figure 1. Picture A10: Morphology of strain A10 at late log-phase grown in acetogen medium containing 27.8 mM glucose (Scanning Electron Microscopy, x 11, 000).

Figure 2. Picture H3HH: Morphology of strain H3HH at late log-phase grown in acetogen medium containing 27.8 mM glucose (Scanning Electron Microscopy, x 11, 000).

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Table 4. Growtha of selected acetogenic strains on various substrates.

Substrateb

A10

H3HP

H3HH

A2

Arabinose Cellobiose Fructose Galactose Glucose Lactose Maltose Sucrose Casein Esculin Glycerol Mannitol Pectin Starch Glutamic acid

0.13a 3.1 1.54 1.38 1.5 2.16 1.97 2.58 0.33 0.59 0.22 0.38 0.23 1.5

0.09 1.71 0.89 0.76 1.1 1.28 1.25 1.23 0.34 0.72 1.04 0.36 0.25 1.35

0.12 2.6 1.29 1.93 2.1 2.48 2.13 2.14 0.37 0.45 0.69 1.19 0.28 0.12

0.08 2.39 1.56 1.35 1.7 2.12 1.56 1.99 0.43 0.23 1.2 0.21 0.2 0.15

0.11

0.05

0.18

0.09

0.17 0.48 0.17 0.1 Formic acid Fumaric 0.08 0.03 0.03 0.05 acid 0.13 0.17 0.03 0.13 Lactic acid Succinic 0.05 0.08 0.09 0.05 acid a Absorbance (660 nm) values represent the increase in OD after 36 hours of incubation at 39°C. Growth tests were carried out in acetogen medium plus 0.5% (w/v) of the desired substrate. Substrates were added separately as sterile anaerobic solutions to the autoclaved and cooled medium.

b

After 5 days of autotrophic growth on solid acetogen medium under H2:CO2, strain H3HH colonies were 1 mm in diameter, entire, slightly convex with a regular margin and the colonies were white in color.

Colonies of strain A10 were approximately 0.7 mm in diameter, the colonies were entire with a regular margin and were transparent. When incubated aerobically on solid acetogen medium containing 27.8 mM glucose, strain A10 colonies were 1.2 mm in diameter, were convex with a regular margin and were white in color (data not shown). The FAME composition of strain A10 and H3HH was not found to be similar to any of the species existing in the anaerobe

database (Moore, ver. 3.7). Species of interest closest to these isolates that were in the FAME database included Peptostreptococcus productus, Clostridium thermoaceticum, Clostridium thermoautotrophicum, and Eubacterium limosum. The mol% G+C for strain A10 was 51.5% but was not determined for the other isolates.

Nutrition Studies, Growth Studies, and Fermentation Yeast extract, rumen fluid, or both were required to support initial growth of strains A10 and H3HH (data not shown). Both strains reached higher optical density when growing on a basal acetogen medium plus yeast extract, but initially they grew faster when both yeast extract and rumen fluid were added to the basal acetogen medium. All strains utilized a wide range of carbohydrates as an energy source (Table 4). Cellobiose, lactose, and sucrose supported the highest growth. No strain was able to utilize arabinose. Esculin was utilized poorly even though all strains were able to hydrolyze it. Pectin and casein were poorly utilized. Strain A10 was the only strain that did not utilize glycerol. Simple acids listed in Table 4 were either poorly or not utilized.

Strains H3HH, H3HP, and A2 were all oxidase and catalase negative (Table 3). Strain A10 was oxidase negative, but showed a weakly positive response in the catalase test. Strains A10, A2, H3HH, and H3HP were able to hydrolyze esculin and both strains A10 and H3HH were able to reduce nitrate (Table 5).

Strain A10, but not strain H3HH, was able to hydrolyze starch.

Strain H3HH differed from the ruminal acetogen Acetitomaculum ruminis, in cell shape, absence of flagella, and substrate utilization (Greening and Leedle, 1989). Strain H3HH also differed from the ruminal acetogen Eubacterium limosum, in substrate utilization (Genthner et al., 1981; Genthner and Bryant, 1982, 1987) and the ability to reduce nitrate to nitrite. The species that most closely resembled strain H3HH was Peptostreptococcus productus which had been isolated from the calf rumen (Bryant et al., 1958). However, strain H3HH was not

40 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

Table 5. Growth and fermentation characteristics of isolates

O.D. H2:CO2

Doub.

Time H2:CO2

0.81 0.73 0.84

13h 15h 16h

35 62 68.8

1390 ND 209

0.51 ND 0.67

+ + +

+ ND +

ND ND

0.43

24h

30

1383

ND

+

ND

ND

Culture H3HH H3HP A10 A2

Doub. = doubling Prod. = production

Acetate y (H2) H2 Nitrate Esculin Prod. Threshold (g Hydrol. Reduction (mM) (ppm) DW/mole)

Starch Hydrol.

Hydrol. = hydrolysis ND = Not Determined

related to P. productus based on GC-FAME analysis.

Strain H3HH differed from Clostridium thermoaceticum and C. thermoautotrophicum in cell shape and because it did not form spores (Fontaine et al., 1941; Wiegel et al., 1981). Both strain H3HH and A. kivui lack flagella and do not produce spores but strain H3HH differed from A. kivui in cell shape, growth temperature range and substrate utilized (Klemps et al., 1987; Leigh et al., 1981; Leigh and Wolfe, 1983).

Strain A10 differed from strain H3HH in substrate utilization profile (Table 4), and because strain A10 is catalase positive, could also hydrolyze starch, and is a facultative anaerobe. Strain A10 differed from the ruminal acetogen Acetitomaculum ruminis, in cell shape, absence of flagella, substrates utilized, and DNA base composition (Greening and Leedle, 1989). Strain A10 also differed from the ruminal acetogen Eubacterium limosum, in being able to reduce nitrate and in substrate utilization profile (Genthner et al., 1981; Genthner and Bryant, 1982, 1987). Strain A10 was not related to P. productus based on GCFAME analysis. Strain A10 differed from Clostridium thermoaceticum, and C. thermoautotrophicum in cell shape, lack of flagella and a lower optimum temperature for growth (Fontaine et al., 1941; Wiegel et al., 1981). Strain A10 differed from Acetogenium kivui in cell shape, growth temperature range, and substrate utilization profile ( Klemps et al., 1987; Leigh et al., 1981; Leigh and Wolfe, 1983).

Acetate was the major VFA produced when strains A10 and H3HH were growing in H2:CO2, glucose, or

glucose plus H2:CO2 When strains A10 and H3HH were grown under H2:CO2 (80:20) strain A10 achieved a maximum OD of 0.84 and a doubling time of 16 h (Table 5), strain H3HH achieved a maximum OD of 0.81 and a doubling time of 13 h. These OD values are lower than those achieved by E. limosum but the doubling time is shorter (Genthner et al., 1981; Genthner and Bryant, 1982, 1987). The maximum acetate production of strains A10 and H3HH growing under H2:CO2 (80:20) was 69 and 35 mM respectively. Both C. thermoautotrophicum and E. limosum have been shown to produce slightly more acetate (Genthner et al., 1981; Genthner and Bryant, 1982, 1987; Wiegel et al., 1981). When strains A10 and H3HH were grown on glucose (5.6 mM) strain A10 achieved a maximum OD of 0.79 and a doubling time of 1.4 h, strain H3HH achieved a maximum OD of 0.65 and a doubling time of 1 h. The maximum acetate production of strains A10 and H3HH growing on glucose (5.6 mM) was 14 and 15 mM respectively. When grown on glucose plus H2:CO2 strain A10 and H3HH achieved a maximum OD of 1.27 and 1.22 respectively.

When grown on H2:CO2 as an energy source, H2 consumption by strain A10 and H3HH was close to the theoretical stoichiometry: 4H2 + 2CO2 g 1CH3COOH + 2H2O (Table 6) Molar growth yields (g dry weight cell/mol substrate consumed) for strain A10 and strain H3HH were 0.67 and 0.51 g/mole respectively which were

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Table 6. Growth yields and stoichiometry of fermentation of strains A10 and H3HH, grown on H2 + CO2(80:20)

Culture

Acetate Cell Dry Acetate H2 uptake Assimilatedb Produced mM a mM Weight mg/ml mM

%H2 Recovery

c

Y(H2) g/mole

A10

421.6

0.28

5.83

68.86

71

0.67

H3HH

128.3

0.07

1.36

35

114

0.51

a Cell dry weights were determined from 500 ml cultures grown in acetogen medium under a H2:CO2 (80:20) gas atmosphere.

Assimilation of acetate into cell material was calculated by the equation: 17C2H3O2 + 11H20 g 8<C4H7O3> + 2HCO3 + 150H; thus, 20.6 µmol acetate was required for 1.0 mg of cell dry matter (Eichler and Schink, 1984). b

c

H2 present in fermentation products as percentage of H2 consumed

lower than the values reported for C. thermoautotrophicum and E. limosum (Genthner et al., 1981; Genthner and Bryant, 1982, 1987; Wiegel et al., 1981). When grown on glucose as energy source, glucose consumption by strain A10 and H3HH was consistent with the theoretical stoichiometry: C6H12O6 (glucose)g 3CH3COOH (Table 7) Molar yields (g dry weight cell/mol substrate consumed) were 36.9 for strain A10 and 47.4 for strain H3HH.

CONCLUSION Ruminants are one of the many sources of biogenic methane, hence the interest in reducing emissions (Boadi et al, 2004; Fonty et al., 2007; Morrison, 2009). Ruminants may provide an important model to enhance the animal production efficiency while at the same time reduce global warming effects. In addition, assuming that the energy lost as methane by ruminants represents a loss of potential energy to the animal (Branine and Johnson, 1990), significant savings in feed cost to the producer could

Table 7. Growth yields and stoichiometry of fermentation of strains A10 and H3HH, grown in acetogen medium containing 5.6 mM glucose.

Initial Glucose Conc. mM Culture

Cell Dry a

Acetate

Weight mg/ml

Assimilated mM

b

Acetate Produced mM

%Carbon Recovery

Y(Glc) g/mole

A10

5.6

0.22

4.54

14.37

113

36.9

H3HH

5.6

0.26

5.42

11.18

99.6

47.4

Cell dry weights were determined from 500 ml cultures grown in acetogen medium under a H2:CO2 (80:20) gas atmosphere.

a

Assimilation of acetate into cell material was calculated by the equation: 17C2H3O2 + 11H20 g 8<C4H7O3> + 2HCO3 + 150H; thus, 20.6 µmol acetate was required for 1.0 mg of cell dry matter (Eichler and Schink, 1984). b

42 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

be realized if the potential energy presently lost as methane is captured as fermentation products. Consequently, methanogenesis in the rumen could be inhibited and acetogenesis in the rumen enhanced sufficiently to act as an electron sink and convert energy in H2 to acetate, which in turn can be utilized by the animal (Boadi et al., 2004; Morrison, 2009).

Several limitations remain in the study because unfortunately these isolates have now been lost. First, the taxonomy of the isolates was not resolved since standard nutritional and physiological methods were used instead of molecular methods. A next step would have been to use 16S rRNA gene sequence analysis of these isolates to provide phylogenetic identification of the isolates. Such information would have allowed design of FISH probes or PCR primers for quantifying these acetogens both in vivo and in vitro, thus expanding greatly the direction of future research in this area. Unfortunately, the current isolates were lost due to freezer malfunction and further phylogenetic characterization is not possible. Despite these limitations, the current study does demonstrate that H2 limited continuous culture is a possible approach for isolating low H2 threshold isolates from the rumen or other anaerobic ecosystems.

Acknowledgement We thank Kenneth Maciorowski, Purdue University, for performing the scanning electron micrographs.

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Mitigation strategies to reduce enteric methane emissions from dairy cows: update review. Can. J. Anim. Sci. 84:319-335.

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Studies on the composition of the ruminal flora and fauna of young calves. J. Dairy Sci. 14:17471767.

Chalupa, W. 1980. Chemical control of rumen microbial metabolism. In: Y. Ruckebusch and P. Thivend.

Eds. Digestive physiology and metabolism in ruminants. MTP Press, Lancaster, England. p 325-347.

Cord-Ruwisch, R., H.J. Setiz, R. Conrad. 1998. The capacity of hydrogenotrophic anaerobic bacteria to compete for traces of hydrogen depends on the redox potential of the terminal electron acceptor.

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Drake, H.L., A.S. Gößner, S.L. Daniel. 2008. Old acetogens, new light. Ann. N.Y. Acad. Sci. 1125:100128.

Eichler, B. and B. Schink. 1984. Oxidation of primary aliphatic alcohols by Acetobacterium carbinolicum sp. nov., a homoacetogenic anaerobe. Arch. Microbiol. 140:147-152.

Fontaine, F.E., W.H. Peterson, E. McCoy, M.A. Johnson. 1941. A new type of glucose fermentation.

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43:701-715.

Fonty, G.F., K. Joblin, M. Chavarot, G.N. Roux, F. Michallon. 2007. Establishment and development of ruminal hydrogenotrophs in methanogen-free lambs. Appl. Environ. Microbiol. 73:6391-6403.

Genthner, B.R.S., C.L. Davis, M.P. Bryant. 1981. Features of rumen and sewage strains of Eubacterium limosum, a methanol and H2-CO2-utilizing species. Appl. Environ. Microbiol. 42:12-19.

Genthner, B.R.S., M.P. Bryant. 1982. Growth of Eubacterium limosum with carbon monoxide as the energy source. Appl. Environ. Microbiol. 43:70-74.

Genthner, B.R.S., M.P. Bryant. 1987. Additional characteristics of one-carbon-compound utilization by Eubacterium limosum and Acetobacterium wodii.

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Appl. Environ. Microbiol. 53:471-476.

Greening, R.C., J.A.Z. Leedle. 1989. Enrichment and isolation of Acetitomaculum ruminis, gen. nov., sp. nov.: acetogenic bacteria from the bovine rumen.

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Holdeman, L.V., I.J. Good, W.E.C. Moore. 1976. Human fecal flora: variation in bacterial composition within individuals and a possible effect of emotional stress. Appl. Environ. Microbiol. 31:359-375.

Holdeman, L.V., E.P. Cato, W.E.C. Moore. 1977. Anaerobic cocci. In: Anaerobe laboratory manual. 4th ed. Virginia Polytechnic Institute and State University, Blacksburg, VA. p 12-21.

Hungate, R.E. 1966. The rumen and its microbes.

Academic Press, New York, NY. 533 p.

Joblin, K.N. 1999. Ruminal acetogens and their potential to lower ruminant methane emissions. Australian. J. Agric. Res. 50:1307-1313.

Karnholz, A., K. Kusel, A. Gobner, A. Schramm, H. Drake. 2002. Tolerance and metabolic response of acetogenic bacteria toward oxygen. Appl. Environ.

Microbiol. 68:1005-1009.

Klemps, R, S.M. Shoberth, H. Sahm. 1987. Production of acetic acid by Aetogenium Kivui. Appl. Microbiol. Biotechnol. 27:229-234.

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1987. Specialized cell surface structures in cellulolytic bacteria. J. Bacteriol. 169:3792-3800.

Leigh, J.A., F. Mayer, R.S. Wolfe. 1981. Acetogenium kivui, a new thermophilic hydrogen-oxidizing, acetogenic bacterium. Arch. Microbiol. 129:275-280.

Leigh, J.A., R.S. Wolfe. 1983. Acetogenium kivui gen. nov., sp.nov., a thermophilic acetogenic bacterium.

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Morrison, K. 2009. Towards greener grazing. Nat. Rep. Clim. Change 3:104-106.

Ragsdale, S. 2008. Enzymology of the Wood-Ljungdahl pathway of acetogenesis. Ann. N.Y. Acad. Sci. 1125:139-136.

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Stevenson, D.M., P.J. Weimer. 2007. Dominance of Prevotella and low abundance of classical ruminal bacteria species in the bovine rumen revealed by relative quantification real-time PCR. Appl. Microbiol. Biotechnol. 75:165-174.

Teather, R.M. 1982. Maintenance of laboratory strains of obligately anaerobic rumen bacteria. Appl. Environ. Microbiol. 44:499-501.

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Weimer, P.J., J.B. Russell, R.E. Muck. 2009. Lessons from the cow: what the ruminant animal can teach us about consolidated bioprocessing of cellulosic biomass. Bioresour. Technol. 100:5323-5331.

Wiegel, J., M. Braun, G. Gottschalk. 1981. Clostridium thermoautotrophicum species novum, a thermophile producing acetate from molecular hydrogen and carbon dioxide. Curr. Microbiol. 5:255-260.

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www.afabjournal.com Copyright © 2011 Agriculture, Food and Analytical Bacteriology

Effect of Plant-based Protein Meal Use in Poultry Feed on Colonization and Shedding of Salmonella Heidelberg in Broiler Birds W. Q. Alali1, C. L. Hofacre2, G. F. Mathis3, A. B. Batal4 Center for Food Safety, University of Georgia, Griffin, Georgia Department of Population Health, Poultry Diagnostic and Research Center, University of Georgia, Athens, Georgia 3 Southern Poultry Research, Athens, Georgia 4 Department of Poultry Science, University of Georgia, Athens, Georgia 1

2

ABSTRACT The objective of this experiment was to determine the effect of plant-based protein meals (soybean and canola) in poultry feed on colonization and shedding of Salmonella Heidelberg in broiler birds over a 42day period. One-day old chicks were randomly assigned to 4 different dietary treatments (n=360 birds per treatment) with 6 replicates per treatment, 60 birds per replication. Three all plant protein meal diets and one commercial diet containing animal protein meal (meat and bone) were used in the study. Half of the birds (n=30) per pen were challenged with nalidixic acid-resistant (NA) S. Heidelberg on day one (called seeders), and the remaining unchallenged birds were called contacts. Drag swabs were collected from all pens on days 0 (prior to placement), 14, and 42. Ceca samples were collected from 20 birds per pen (10 seeders and 10 contacts) on day 42. Drag swabs and ceca samples were examined for NA- S. Heidelberg using enrichment and enumeration/enrichment, respectively. All drag swabs were negative on day 0, but positive for S. Heidelberg on both days 14 and 42. Within seeder and contact birds, there was no significant differences in:1) NA-S. Heidelberg concentration (cfu/g of ceca), and 2) proportions of positive ceca among the treatment groups. It can be concluded that all plant-based protein meal diets did not significantly reduce the environmental contamination with S. Heidelberg nor did it reduce the concentration and proportion of positive S. Heidelberg in contact and seeder birds compared to commercial diet containing animal protein meal.

Keywords: Salmonella Heidelberg, broiler, canola, soybean, performance, plant-based protein meal, animal protein meal, shedding, colonization

Agric. Food Anal. Bacteriol. 1: 45-53, 2011

Introduction Foodborne salmonellosis is a major public health concern in the United States. Poultry continues to be Received: Sepetember 22, 2010, Accepted: October 19, 2010.

Released Online Advance Publication: May 6, 2011.

Correspondence: W. Q. Walid , walali@uga.edu Tel: +1 (770) 467-6066 Fax: +1-(770) 229-3216

an important frequent vehicle of Salmonella transmission to humans, mainly via contaminated chicken meat. Therefore, reducing Salmonella colonization and shedding in live chickens and consequently chicken meat contamination can lead to a decline in the burden of salmonellosis in humans. Poultry feed is at the beginning of the food safety chain in

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45

the farm-to-table continuum. Broiler bird infection, colonization and subsequent fecal shedding of Salmonella is prevalent ( Bailey et al., 2001; Liljebjelke et al., 2005; Renwick et al., 1992; Rodriguez et al., 2006). Several studies have examined the effect of various on-farm management practices (e.g., vaccine, use of antibiotics and probiotics, cereal grains) on colonization and shedding of Salmonella in poultry (Barrow et al., 1984; Bjerrum et al., 2005; Engberg et al., 2004; Hofacre et al., 2007; Van Immerseel et al., 2005). However, very little is known about how different feed rations/ingredients; specifically, how the type of protein meals in broiler feed affect the colonization and shedding of Salmonella in broiler birds.

Globally, the feed industry is considering alternatives (e.g., soybean, cottonseed meal, fishmeal, and legumes) to the rising cost of animal protein meals (e.g., meat and bone meal and animal by-products) and the worldwide growth in intensive poultry production (FAO, 2004). Furthermore, the high prevalence of contaminants (e.g., Salmonella spp.) detected in the animal protein meal ( Hacking et al., 1978; Hofacre et al., 2001; Isa et al., 1963; Veldman et al., 1995) is another reason to look for suitable alternatives. Soybean and canola meals (i.e., rapeseed) are the largest protein meals produced worldwide (USDA, 2010a). These meals are high in protein and can be used in broiler feed to provide up to 60% of the crude protein in a typical diet (Newkirk and Classen, 2002). Canola meal has lower protein content than soybean meal (34 to 38 % compared to 44 to 49 %) and lower concentration of essential amino acids. Canola meal is considered a protein source for animals that do not have high energy demand.

It has been largely fed to cattle and pigs as part of their diet rations (Brzoska, 2008; Caine et al., 2008).

Additionally, canola meal is used in broiler diet as a protein source (up to 10%) (Dale, 1996; MontazerSadegh, et al. 2008). The U.S. produced 41 million tons of soybean meal in 2009 (valued at $12.5 billion) and 787,000 tons canola meal in the same year (valued at $196 million) (USDA, 2010b). On the other hand, the U.S. imported 1.9 million tons of canola meal, mainly from Canada, in 2009 valued at $469 million (USDA, 2010b).

This study was conducted to compare the effect of plant-based protein meals (soybean and canola) in poultry feed on colonization and shedding of Salmonella Heidelberg in broiler birds over a 42-day period compared to broiler fed animal protein meals (meat and bone). We also compared the effect of the diets used in the study on the broiler birds performance (feed conversion ratio [FCR] and weight gain) and mortality percentages.

Materials and Methods Study Design Chicks were assigned randomly to 4 different dietary treatments (n=360 birds per treatment) with 6 replicates per treatment, 60 birds per replication.

Treatment 1 was an all plant-based feed containing diet, 35% soybean protein meal (SBM). Treatment 2 was an all plant-based diet containing 30% SBM and 10% canola meal (CM). Treatment 3 was an all plant-based diet containing 10% SBM and 40% CM. Treatment 4 (control diet) was a commercial diet containing animal protein meal (10% meat and bone) and plant-based protein meal (17% SBM and 10% CM). The study consisted of 24 pens starting with 60 male broiler chickens. The birds were CobbĂ&#x2014;Cobb one-day-old male broilers purchased from CobbVantress hatchery (Cleveland, GA). All birds were spray vaccinated with Coccivac-B on day one. Sixty birds were housed in each floor pen (0.77 ft2/bird, stocking density) that contained litter of approximately 4 inches of fresh pine shavings. Feed and water were available ad libitum.

Feed Formulation All diets were formulated to meet or exceed the animal nutrient requirements (NRC, 1994). Three diet phases were used: a starter (0 to 14 days of age), grower (14 to 28 days), and finisher (28 to 42 days).

Tables 1 through 3 show the composition of the starter, grower, and finisher diets used in this study.

Broiler diets were fed as crumbles (starter feed) or as pellets (grower and finisher). All feed contained

46 Agric. Food Anal. Bacteriol. â&#x20AC;˘ www.AFABjournal.com â&#x20AC;˘ Vol. 1, Issue 1, 2011

Table 1. Composition of dietary treatments (as-fed basis) for broiler starter (0-14 days of age)

35% SBM

30% SBM +10% CM

10% SBM +40% CM

10% Animal protein meal

57.68

54.23

45.2

58.78

36.45

28.95

9.95

20.19

Canola

_

10

35.55

10

Poultry meat & bone meal

_

_

_

7

Fat

2.57

3.61

6.32

1.86

Dicalcium phosphate

1.17

1.12

0.99

Limestone

0.93

0.87

Salt

0.6

Methionine

Table 2. Composition of dietary treatments (as-fed basis) for broiler grower (14-28 days of age)

35% SBM

30% SBM +10% CM

10% SBM +40% CM

10% Animal protein meal

65.11

61.67

50.83

64.34

28.66

21.12

9.8

15.82

Canola

_

10

50.83

9

Poultry meat & bone meal

_

_

_

6

Fat

2.82

3.87

6.46

2.46

0.17

Dicalcium phosphate

1.07

1.02

0.89

0.2

0.7

0.62

Limestone

0.94

0.87

0.73

0.66

0.34

0.32

0.38

Salt

0.37

0.35

0.32

0.39

0.29

0.25

0.29

0.24

Methionine

0.3

0.26

0.15

0.24

Vitamin 1 premix

0.25

0.25

0.25

0.25

Vitamin premix 1

0.25

0.25

0.25

0.25

Lysine

0.08

0.15

0.16

0.29

Lysine

0.23

0.29

0.31

0.36

Trace mineral premix 2

0.08

0.08

0.08

0.08

Trace mineral premix 2

0.08

0.08

0.08

0.08

Coban 3

0.05

0.05

0.05

0.05

Coban 3

0.05

0.05

0.05

0.05

0.05

0.05

0.05

0.05

BMD

4

0.05

0.05

0.05

0.05

_

_

0.04

0.02

L-Threonine

0.04

0.06

0.05

0.07

0.02

0.02

0.02

0.02

TBCC 5

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

Ronozyme

0.02

0.02

0.02

0.02

Ingredient Corn, yellow, ground Soybean Meal (49)

BMD

4

L-Threonine TBCC

5

Ronozyme

Ingredient Corn, yellow, ground Soybean Meal (49)

Vitamin mix provided the following (per kilogram of diet): vitamin A, 2481 IU; vitamin D3, 496 IU; vitamin E, 4.96 IU; vitamin B12, 5.46 μg; riboflavin, 2.0 mg; niacin, 19.8 mg, d-Pantothenic acid, 4.96 mg; choline, 86.1 mg; menadione, 0.33 mg; folic acid, 0.25 mg; thiamine, 0.99 mg, pyridoxine, 0.99 mg; biotin, 0.49 mg; and ethoxyquin, 57.1 mg.

1

Trace mineral mix provided the following (per kilogram of diet): calcium, 0.022 mg; iron, 0.02 mg; magnesium, 0.02 mg; maganese, 0.1 mg; zinc, 0.08 mg; copper, 4000 ppm; iodine, 1000 ppm; and selemium, 400 ppm.

2

Coban: coccidiostat

3

BMD: bacitracin methylene disalycilate

4

TBCC: tribasic copper chloride

5

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Table 3. Composition of dietary treatments (asfed basis) for broiler grower (28-42 days of age)

30% SBM +10% CM

10% SBM +40% CM

10% Animal protein meal

69.6

66.85

58.73

69.06

25.16

19.14

6.36

16.26

_

8

26.95

7

_

_

_

2.89

2.86

3.7

5.88

2.87

0.43

0.39

28

_

0.7

0.65

0.52

0.56

Salt

0.44

0.43

0.4

0.45

Methionine Vitamin premix1 Lysine Trace mineral premix 2 Coban 3

0.19

0.16

0.08

0.16

0.25

0.25

0.25

0.25

0.15

0.2

0.28

0.25

0.08

0.08

0.08

0.08

0.05

0.05

0.05

0.05

BMD

4

0.05

0.05

0.05

0.05

L-Threonine

_

0.02

0.04

0.03

0.02

0.02

0.02

0.02

0.02

0.02

0.02

0.02

Ingredient Corn, yellow, ground Soybean Meal (49) Canola Poultry meat & bone meal Fat Dicalcium phosphate Limestone

TBCC

5

Ronozyme

35% SBM

Vitamin mix provided the following (per kilogram of diet): vitamin A, 2481 IU; vitamin D3, 496 IU; vitamin E, 4.96 IU; vitamin B12, 5.46 μg; riboflavin, 2.0 mg; niacin, 19.8 mg, d-Pantothenic acid, 4.96 mg; choline, 86.1 mg; menadione, 0.33 mg; folic acid, 0.25 mg; thiamine, 0.99 mg, pyridoxine, 0.99 mg; biotin, 0.49 mg; and ethoxyquin, 57.1 mg.

1

Trace mineral mix provided the following (per kilogram of diet): calcium, 0.022 mg; iron, 0.02 mg; magnesium, 0.02 mg; maganese, 0.1 mg; zinc, 0.08 mg; copper, 4000 ppm; iodine, 1000 ppm; and selemium, 400 ppm.

2

Coban: coccidiostat

3

BMD: bacitracin methylene disalycilate

4

TBCC: tribasic copper chloride

5

bacitracin methylene disalycilate (BMD) antibiotic.

Bird weights (kg) by pen were recorded on days 1 and 42. Feed intake was measured at 21, 35, and 42 days of age. Feed conversion ratios (feed intake/ weight gain) were also calculated.

Salmonella Challenge and Sample Collection Nalidixic-acid (NA) Salmonella Heidelberg isolate was grown for 6 h in Tryptic Soy Broth (TSB; Difco), whereafter the number of colony-forming units (cfu) per ml was determined by plating 10-fold dilutions of the bacterial suspension on Xylose Lys Tergitol-4 (XLT-4; Difco) medium. Then, a bacterial colony was diluted in Phosphate Buffered Saline (PBS) solution to reach the inoculation titer (5×107 cfu/ml).

Chick-box liners (i.e., paper pads) were collected, and stored in sterile sampling bags for Salmonella analysis. At day 1, prior to placement, 30 of the 60 chicks per pen (all treatments) were orally administered by gavage 0.1 ml of a NA (25 µg/ml) S. Heidelberg (5×107 cfu/ml). Each of these 30 chicks challenged with S. Heidelberg were also wing-banded and were called seeders, whereas the remaining 30 chicks were called contacts. Contact birds in our study model the horizontal transmission of Salmonella in broiler houses (i.e., infected broilers with Salmonella horizontally spread this organism to other birds (contact) in the same poultry house).

Drag swab samples were tested for Salmonella environmental contamination from all pens on days 0 (prior to placement), 14, and 42. Dragging sterile gauze swabs soaked in double strength skim milk across the birds bedding material is considered to be the most sensitive method of environmental sampling by the National Poultry Improvement Plan (NPIP) (USDA, 1996). To reduce the possibility of cross contaminating a sample, gloves were changed between completing each drag swab. Appropriate dress in clean coveralls and plastic shoe covers was used on entry to any pen by the farm workers. Self contained Solar-Cult sterile drag swabs (Solar Biologicals Inc., Ogdensburg, NY) were used. Samples were transported on ice within 24 h to Center for

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Food Safety Laboratory (Griffin, GA).

On day 42, 20 birds per pen (10 seeders and 10 contacts) were randomly selected, euthanized by cervical dislocation, weighed, and the ceca were aseptically removed and then placed into sterile plastic sampling bags for Salmonella isolation. Samples were transported on ice within 24 h to Center for Food Safety Laboratory.

Salmonella Analysis Chick paper pads were enriched in 400 ml tetrathionate broth (TT- Difco™) containing 0.001% aqueous brilliant green and 2 ml of iodine solution (25% iodine and 30% potassium iodide), then incubated at 42ºC for 24 h. A loopful (~10 µL) of the broth was streaked on XLT4 agar without and with NA supplement (final concentration was 25 parts per million [ppm]). The XLT4 plates were then incubated at 37ºC for 24 h.

Drag swab samples were enriched with 100 ml of TT as above and incubated (42ºC, 24 h). One loopful was streaked onto XLT4 agar supplemented with NA. The XLT4 plates were then incubated at 37ºC for 24 h, and one black colony per plate was streaked on blood agar (Remel, Lenexa, KS). One colony per blood agar plate was tested against Salmonella O group B antiserum (Difco) and confirmed to be S. Heidelberg if agglutination was noted. If no black colonies were present from a sample, the enriched drag swab was held at room temperature for 5 days and a delayed secondary enrichment was performed as follow: 1:10 dilution into new TT broth was made and then incubated at 37ºC for 24 h. A loopful of the mixture was streaked onto XLT4 agar supplemented with NA. The remaining isolation and confirmation of S. Heidelberg was carried out similarly as the primary enrichment.

Fifty milliliters of phosphate buffered saline (PBSFisher) solution was added to the ceca, and then mechanically homogenized using a stomacher (Technar Company, Cincinnati, OH) for 20 seconds.

A serial dilution (10 fold) was performed using 0.5 ml portion of the stomacher bag content added to 4.5 ml of saline solution. From each dilution, a 500

μl aliquot was spread plated onto XLT4 plate containing NA (25 ppm). After incubation (37ºC, 24 h), black colonies were counted. One colony per plate was randomly selected and struck onto blood agar (Remel, Lenexa, KS). One colony per blood agar plate was tested against Salmonella O group B antiserum (Difco) and confirmed to be S. Heidelberg if agglutination was noted. Five milliliters of TT broth containing brilliant green and iodine solution was then added to the ceca and PBS mixture, and incubated at 42ºC for 24 h. One loopful per sample was streaked on XLT4 containing NA, incubated (37ºC, 24 h), and black colonies were identified as described earlier. If no black colonies were present from a sample, the enriched ceca were held at room temperature for an additional 7 days and a delayed secondary enrichment was tested for Salmonella as for the drag swabs.

Statistical Analysis A sample was considered positive if Salmonella was recovered from direct plating, primary or secondary enrichments. A randomized complete block design was used in the study with 6 blocks and 4 treatments. The experimental unit was the pen. The study outcomes were: 1) the proportion of Salmonella isolates from drag swabs or ceca, 2) Salmonella counts (cfu/g) from ceca samples, and 3) performance data (FCR, weight gain, and mortality).

First, proportion of Salmonella isolates were compared between treatment groups by contact and seeders using a Generalized Linear Model (GLM) with binomial distribution and a logit link (GENMOD procedure, SAS Inst. version 9.1.3, Inc., Cary, NC). Second, Salmonella counts (concentrations) were compared between treatment groups by contacts and seeders using MIXED procedure in SAS software. Third, performance data were compared between treatment groups using MIXED procedure in SAS software. Treatment was a fixed effect and the block was a random effect in the model. Counts (cfu/g) were logarithmically transformed by use of log base 10 to approximate normality. Treatment effects were considered significant if P < 0.05.

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Results and Discussion Data on FCR, average weight gain, and percentage of mortality for the 4 treatment groups are shown in Table 4.

Table 4. Performance results and mortality percentages in broilers following exposure to Salmonella Heidelberg1

Treatment

FCR (0 to 42 d)

Average weight gain

Mortality (%)

35% SBM

1.754a

2.306a

4.67a

30% SBM +10% CM 10% SBM +40% CM

1.776a

2.287a

1.67a

1.789b

2.119b

2.33a

10% Animal protein

1.710c

2.345c

2.33a

meal

2

Means in a column with no common superscript letters are significantly different (P <0.05)

a-c

Means represent 6 pens per treatment, with 60 birds per pen.

1

The 10% Animal protein meal treatment included plant-based protein meal (17% SBM and 10% CM).

2

The FCR and average weight gain for birds in treatments 3 and 4 were significantly different (P <0.05). The highest FCR was observed in treatment group 3 (10% SBM and 40% CM), and lowest FCR was in group 4 (animal protein diet); whereas average weight gain was highest in group 4 and lowest in group 3. This may indicate that higher percentage of CM in the diet significantly increased FCR in broiler birds in our study. However, the higher weight gain in group 4 was due to an increase in feed consumption compared to other treatment groups (data not shown). This is in contrast to Newkirk and Classen (2002) findings that replacement of up to 60% of SBM with CM in broiler diets has no adverse effect on growth performance. There was no significant dif-

ference in mortality percentages in our study. This indicates that all plant-based protein meal diets did not adversely affect the bird mortality compared to the animal protein diet. The BMD used in the feed diets in our study would have no or little effect on S. Heidelberg since it was applied across all the treatments (with the same amount), and it would have no effect on performance. This antibiotic was used to prevent necrotic enteritis infections caused by Clostridium perfringens and as a growth promoter; a common practice by the poultry industry.

Paper pads were Salmonella negative which indicated that chicks were Salmonella-free prior the inoculation. Feed was not tested for Salmonella prior consumption by chicks. However, we used Salmonella inoculum that was nalidixic acid resistant and the media we used for culture had nalidixic acid in it. Therefore, even if the feed had a small amount of Salmonella, the counts reported in this study were only NA-S. Heidelberg strain that we confirmed with nalidixic acid supplement XLT4 and Salmonella O group B antiserum.

All drag swabs collected on day 0 from the 24 pens (i.e., 4 treatment groups) were negative for NA-S. Heidelberg; whereas swabs collected on both days 14 and 42 were all positives. This suggests that none of the treatment groups in our study lowered the shedding of S. Heidelberg throughout the 42day study period to a level below the detection limit of the enrichment method used in this study. Newly hatched chicks can be colonized with Salmonella early in life (i.e., during hatching process and first few days post hatching) and that could lead to presence of this organism in their intestinal tract and subsequent shedding during the grow out period (Cason et al., 1994).

There was no significant difference in percentage of cecal samples positive for S. Heidelberg, by challenge type (seeder or contact birds) among the treatment groups (Table 5). Furthermore, there was no significant difference in S. Heidelberg populations (cfu/g of ceca), by challenge type, among the treatment groups (Table 6). We challenge the birds (i.e., seeders) with 5 log cfu of NA-S. Heidelberg, however, both proportions (percentages) of positive cecal

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Table 5. Mean percentages of positive Salmonella Heidelberg ceca culture results in broilers following exposure to Salmonella Heidelberg1

All Treatment (seeders+ Seeders2 contacts)

Contacts3

Table 6. Mean Salmonella Heidelberg enumeration results per gram of cecal content in broilers following exposure to Salmonella Heidelberg1

Treatment

Seeder2

Contacts3

35% SBM

70

36.67

33.33

35% SBM

872.2

63.9

30% SBM +10% CM

63.33

31.67

31.67

30% SBM +10% CM

409.7

44

10% SBM +40% CM

70

35

35

10% SBM +40% CM

136.1

111.1

76.67

40

36.67

291.1

1212.2

10% Animal protein meal4

10% Animal protein meal4

Means represent 6 pens per treatment, with 60 birds per pen. At day 42, 20 birds per pen (10 seeders and 10 contacts) were selected and the ceca were removed for S. Heidelberg isolation. There were no significant differences detected among treatments within seeder and contact birds. Statistical analysis was conducted using MIXED procedure in SAS software (GENMOD procedure, SAS Inst., Inc., Cary, NC). Treatment was a fixed effect and the block was a random effect in the model.

1

Seeders were chicks challenged with S. Heidelberg and wing-banded (n=30 per pen).

2

Contacts were chicks unchallenged with S. Heidelberg (i.e., the remaining 30 chicks per pen).

3

The 10% Animal protein meal treatment included plant-based protein meal (17% SBM and 10% CM).

4

samples and the concentrations of NA-S. Heidelberg were low. Birds with fully functioning T-cell immune systems will naturally begin to clear Salmonella from their ceca over time after exposure (i.e., initial inoculation). Furthermore, even though we challenged them with 5 logs NA-S. Heidelberg, that does not mean all 5 logs colonized. It has been reported in several studies that different types of feed may influence Salmonella populations in multiple parts of the gastrointestinal tract (GIT) of broiler birds. Bjerrum et al. (2005) found that broiler birds fed pelleted commercial feed had higher Salmonella Typhimurium population in the gizzard and ileum compared to birds fed whole wheat based diet. Similarly, whole wheat based diet lowered Lactobacillus, enterococci, and Clostridium perfringens populations in broiler bird GIT compared to pelleted commercial feed (Engberg et al., 2004).

In another study, triticale-soybean based diet fed to broilers reduced Salmonella cecal colonization compared to corn-based diet (Santos et al., 2008). The authors used a cocktail of 4 Salmonella serotypes (Typhimurium, Newport, Heidelberg, and Kentucky) for their inoculum. It is difficult to assess whether Salmonella populations in the ceca samples were Kentucky, Heidelberg, Typhimurium, Newport or a combination of serotypes. Furthermore, since Salmonella is present in the environment, there is no way to distinguish between the sources of Salmonella (inoculated versus environmental). In our study, we used NA- S. Heidelberg (one serotype) as a surrogate for the following reasons: 1) the ability to differentiate the source of Salmonella (inoculated versus environmental) which could confound the results and 2) inoculation with one serotype could reduce the possibility of multiple

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serotypes interaction which could confound the results (treatment effect versus interaction effect).

To the best of our knowledge, this is the first study to investigate the effect of canola and soybean in combination in all plant-based protein diet on the colonization and shedding of Salmonella in broiler birds. The results obtained herein suggests that plant-based protein meal diets did not significantly reduced the environmental contamination with S. Heidelberg nor did it reduce the concentration and proportion of positive S. Heidelberg in contact and seeder birds compared to commercial diet containing animal protein meal.

Acknowledgement This study was supported in part by the University of Georgia, Center for Food Safety seed grant. We thank Rebekah Turk, Aparna Petkar, Kim Hortz, and Christine Lobsinger for the technical assistance in processing samples and performing microbiological assays at the laboratory.

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Bjerrum, L., K. Pedersen, R. M. Engberg. 2005. The influence of whole wheat feeding on Salmonella infection and gut flora composition in broilers.

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Cason, J. A., N. A. Cox, J. S. Bailey. 1994. Transmission of Salmonella typhimurium during hatching of broiler chicks. Avian Dis. 38:583-588.

Dale, N. 1996. Variation in feed ingredient quality: Oilseed meals. Anim. Feed Sci. Technol. 59:129135.

Engberg, R. M., M. S. Hedemann, S. Steenfeldt, B. B. Jensen. 2004. Influence of whole wheat and xylanase on broiler performance and microbial composition and activity in the digestive tract. Poult.

Sci. 83:925-938.

Food and Agriculture Organization of the United Nations (FAO). 2004. Protein sources for the animal feed industry. ftp://ftp.fao.org/docrep/fao/007/ y5019e/y5019e00.pdf. Accessed on September 8, 2010.

Hacking, W. C., W. R. Mitchell, H. C. Carlson. 1978.

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Hofacre, C. L., G. F. Mathis, S. H. Miller, M. W. LaVorgna. 2007. Use of bacitracin and roxarsone to reduce Salmonella Heidelberg shedding following a necrotic enteritis challenge model. J. Appl. Poult.

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Liljebjelke, K. A., C. L. Hofacre, T. R. Liu, D. G. White, S. Ayers, S. Young, J. J. Maurer. 2005. Vertical and horizontal transmission of Salmonella within integrated broiler production system. Foodborne Pathog. Dis. 2:90-102.

Montazer-Sadegh, R., Y. Ebrahim-Nezhad, N. Maheri-Sis. 2008. Replacement of Different Levels of Rapeseed Meal with Soybean Meal on Broilers

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Performance. Asian J. Anim. Vet. Adv. 3:278-285.

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Newkirk, R. W. and H. L. Classen. 2002. The effects of toasting canola meal on body weight, feed conversion efficiency, and mortality in broiler chickens.

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69:2576-2580.

Santos, F. B. O., B. W. Sheldon, A. A. Santos, P. R. Ferket. 2008. Influence of housing system, grain type, and particle size on Salmonella colonization and shedding of broilers fed triticale or corn-soybean meal diets. Poult. Sci. 87:405-420.

United States Department of Agriculture Animal and Plant Health Inspection Service. 1996. The National Poultry Improvement Plan. Auxiliary provisions on National Poultry Improvement Plan. Subpart B. Bacteriological examination procedure. 147-12. procedures for collection, isolations, and identification of Salmonella from environmental samples, cloacal swabs, chick box papers, and meconium samples p. 914-915. United States Department of Agriculture, Washington, D.C. United States Department of Agriculture (USDA).

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www.afabjournal.com Copyright © 2011 Agriculture, Food and Analytical Bacteriology

Optimization of Fermentative Production of Keratinase From Bacillus Subtilis NCIM 2724 S. M. Harde1, I. B. Bajaj1, R. S. Singhal1 Food Engineering and Technology Department, Institute of Chemical Technology,

1

Matunga, Mumbai, Maharashtra, India, 400 019

ABSTRACT Microbial keratinases have become biotechnologically important since they target the hydrolysis of highly rigid, strongly cross-linked structural polypeptide “keratin” recalcitrant to the commonly known proteolytic enzymes trypsin, pepsin and papain. Keratinases are produced in a medium containing keratinous substrates such as feathers and hair. This paper reports on the optimization of keratinase production

by Bacillus subtilis NCIM 2724. One factor-at-a-time method was used to investigate the effect of carbon sources, nitrogen sources and pH on keratinase production. An L8 orthogonal array design was adopted to select the most important fermentation parameters influencing the yield of keratinase. Response surface methodology (RSM) was used to develop a mathematical model to identify the optimum concentrations of the key parameters for higher keratinase production, and confirm its validity experimentally. The effect of various amino acids on the production of keratinase was also studied. The final optimized medium gave a maximum yield of 12.32 KU ml-1 of keratinase. Keratinases are commercially important among the proteases that have been studied since they attack the keratin residues and hence find application in developing cost-effective feather by-products for feeds and fertilizers.

Keywords: Keratinase, Fermentation, Bacillus subtilis, Optimization, Orthogonal Array Design, Response surface methodology Agric. Food Anal. Bacteriol. 1: 54-65, 2011

Introduction Keratin is a fibrous and insoluble structural protein extensively cross linked with hydrogen, disulphide and hydrophobic bonds. It forms a major component of the epidermis and its appendages viz. hair, feathers, nails, horns, hoofs, scales and wool (Anbu et al., Received: September 27, 2010, Accepted: October 29, 2010.

Released Online Advance Publication: May 6, 2011.

Correspondence: Ishwar B. Bajaj, ishbajaj1@gmail.com Tel: - +91 22 24145616, Fax: +91 22 24145614

2007). Feather keratin exhibits an elevated content of several amino acids such as glycine, alanine, serine, cysteine and valine. The intensive cross-linkage in keratins hinders their degradation by commonly known proteolytic enzymes (Gupta and Ramnani, 2006). Degradation of feathers will not only decrease the environmental problem caused due to their accumulation but could also act as source of some nutritionally important amino acids.

Currently, some industries have produced feather

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meal by steam pressure cooking. This technique requires high energy input and may degrade amino acids. The enzymatic hydrolysis of feather may be a viable alternative to steam pressure cooking (Grazziotin et al., 2006). The use of crude enzymes from Bacillus species particularly Bacillus licheniformis and Bacillus subtilis have been extensively studied due to their effectiveness in terms of feather degradation (Manczinger et al., 2003).

Keratinases [EC.3.4.99.11] belong to the group of serine proteases capable of degrading keratin. It is an extracellular enzyme produced in a medium containing keratinous substrates such as feathers and hair. Keratinases have applications in traditional industrial sectors including feed, detergent, medicine, cosmetics and leather manufacturers (Farag and Hassan, 2004), they also find application in more recent fields such as prion degradation for treatment of the dreaded mad cow disease (Langeveld et al., 2003), biodegradable plastic manufacture and feather meal production and thus can be appropriately called “modern proteases”. The use of keratinases to enhance drug delivery in some tissues and hydrolysis of prion proteins arise as novel potentially high impact applications for these enzymes (Brandelli, 2007). Although many applications of keratinases are still in the stage of infancy, a few have found their way to commercialization, particularly the use of Bioresource International’s (BRI) Versazyme for feather meal production. The crude enzyme can also serve as a nutraceutical product, leading to significant improvement in broiler performance (Odetallah et al., 2003). The most promising application of keratinase is in the production of nutritious, cost effective and environmentally benign feather meal (Gupta and Ramnani, 2006). Nutritional enhancement can be achieved by hydrolysis of feather meal/raw feather using keratinase which significantly increases the levels of essential amino acids methionine, lysine and arginine (Williams et al., 1991).

The present work focuses on trying to produce keratinase from nonpathogenic microorganisms and utilization of chicken feathers as a sole carbon source. Several bacteria produce keratinase as an extracellular material. Most of these belong to the

genus Bacillus. These bacteria use keratinous substrates such as chicken feathers as carbon sources for the production of keratinase. Aspergillus fumigatus was previously reported to be able to use chicken feather flour as carbon and nitrogen source (Santos et al., 1996). Addition of glucose, sucrose and lactose resulted in strong inhibition of keratinase production (Brandelli, 2007). The production of keratinase is usually most noticeable when chicken feathers are used as a sole carbon source (Williams et al., 1990). Farag and Hassan (2004) used chicken feathers as a sole carbon, nitrogen and sulphur sources for keratinase production and observed 26.69 U/mg of keratinase activity. Lin et al. (1992) used chicken feathers as a sole carbon, nitrogen and energy sources for keratinase production. Suntornsuk and Suntornsuk (2003) reported that keratinase activity increased upto 0.9 U/ml by using chicken feathers as a substrate and sole carbon source from Bacillus sp. FK 46. They also varied the feather concentration for production of keratinase and observed that higher feather concentrations cause substrate inhibition or repression of keratinase production, resulting in a low percentage of feather degradation. ElRefai et al. (2005) used different substrates for keratinase production from Bacillus pumilus FH9 like feather, muscle protein and wool. They observed that wool gave the maximum keratinase activity of 647 U/ml. According to Kim et al. (2001), B. cereus gave the maximum keratinase activity of 117 U/ml by using feathers as a carbon source. Anbu et al.

(2007) produced keratinase from Scopulariopsis brevicaulis by using glucose and feather as carbon sources and observed 1% glucose and 1.5% feather to achieve a maximum keratinase activity of 6.2 KU/ml.

Besides carbon sources, factors such as nitrogen sources (Thyes et al., 2006) and medium pH (Suntornsuk and Suntornsuk, 2003) can influence the productivity of keratinase. B. licheniformis produced keratinase at neutral pH (Wang and Shih, 1999). Anbu et al. (2007) studied the effect of several organic and inorganic nitrogen sources on keratinase production and found maximum

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production in the presence of 1.5 to 2% sodium nitrate (6.2 KU/ml) followed by peptone (6 KU/ml) and potassium nitrate (5.5 KU/ml). Sodium nitrate below 1.0 to 1.5% permitted enzyme synthesis, but was inhibitory above 2%. Thyes et al. (2006) studied the effect of feather meal, soybean, gelatin, casein, yeast extract, cheese whey and peptone at 10 g/L for production of protease from Microbacterium arborescenes. Among the various nitrogen sources studied, maximum keratinase was produced in feather meal (96.5 U/ml), followed by soybean protein (73.8 U/ml) and gelatin (45.8 U/ml).

The initial pH of the medium greatly affects the bacterial growth, percentage of feather degradation and keratinase production (Suntornsuk and Suntornsuk, 2003). It was observed that Bacillus species is most active under neutral or basic conditions. The optimum pH for B. cereus was 7.0 (Kim et al., 2001), while that for B. pumilus was 8.0 (El-Refai et al., 2005). For B. subtilis, highest enzyme production was obtained over a broad range of pH 5 to 9.

According to Wang and Shih (1999) maximum growth rate and keratinase productivity of B. subtilis occurred at 42°C instead of 37°C, and the fermentation time could also be shortened. However, the maximum keratinase activity was observed at 37°C. Elevated temperature increased cell growth, but not enzyme production. The temperature differential effect on growth versus keratinase production was more obvious in B. licheniformis, where cells grew best at 50°C, but keratinase production was best at 37°C. High temperature may increase the protein turnover rate. According to El-Refai et al. (2005) the optimal reaction temperature recorded for B. pumilus FH9 keratinase is higher than those reported for other B. pumilus strains.

This paper reports on optimization of keratinase production using a statistical approach. Effects of pH, carbon source and nitrogen source were investigated by using one factor at-a-time method. Initial screening of the medium components was done by using an L8 orthogonal array design to understand the significance of their effect on the product formation, and then a few of the more significant parameters were selected for further optimization using response surface methodology (RSM).

Materials and Methods Materials Chicken feathers were collected from the Devgiri poultry farm, Wadegavhaon, Pune, India. Chicken feathers were washed three times with distilled water followed by defattening with chloroform: methanol (1:1), dried and ground. All chemicals used were of the AR grade and were purchased from Hi Media Limited, Mumbai, India.

Bacterial strain and medium A bacterial strain of Bacillus subtilis NCIM 2724 was used in the present study. The medium used for the growth and maintenance contained (g L-1), ammonium chloride, 0.5; magnesium sulphate, 0.1; yeast extract, 0.1; sodium chloride, 0.5; dipotassium hydrogen phosphate, 0.3; potassium hydrogen phosphate, 0.3; feathers, 10 (pH 7.5 ± 0.2). Bacterial cells in agar slants were incubated at 37°C for 24 h and stored at 4°C. The medium was sterilized in an autoclave for 15 min at 121°C. For the production of keratinase, a medium reported by El-Refai et al. (2005) was used, which contained (g L-1) Feather, 10; Yeast extract, 0.1; MgSO4, 0.1; NH4Cl, 0.5; K2HPO4, 0.3; KH2PO4, 0.3; NaCl, 0.5.

Initial pH of the medium was adjusted to 7.5 ± 0.2 with Tris–HCl buffer. The medium was sterilized in an autoclave for 15 min at 121°C.

Inoculum and fermentation One ml cell suspension from a slant was transferred to 20 ml of the seed medium containing (g L-1) peptone, 5; yeast extract, 1.5; beef extract, 1.5 and sodium chloride, 5; (pH 7 ± 0.2) and incubated at 37°C and 200 rpm for 24 h. This was used as the inoculum. Fermentation was carried out in 250 ml Erlenmeyer flasks, each containing 50 ml of the sterile production medium. The medium was inoculated with 5% (v/v) of 12 h old B. subtilis culture containing approximately 2×106 cells/ml. The flasks were inoculated on a rotary shaker at 37 ± 2 °C and 200 rpm for 48 h.

56 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

All the experiments were carried out at least in triplicate.

on the production of keratinase, fermentation medium containing chicken feathers was supplemented with additional carbon sources, viz., glycerol, sucrose, soluble starch, maltose, lactose, fructose, glucose. All carbon sources were used at 10 g L-1.

Optimization of fermentation medium using one factor-at-a-time method In order to investigate the effect of initial pH of medium on keratinase production, fermentation runs were carried out by adjusting initial pH of the medium in the pH range of 5 to 8, and analyzing the samples for keratinase production after 48 h. To study the effects of different nitrogen sources on keratinase production, yeast extract in the medium was replaced with different organic nitrogen sources, such as peptone, malt extract, and beef extract at 0.1 g L-1, and ammonium chloride was replaced with different inorganic nitrogen sources, such as sodium nitrate, potassium nitrate, ammonium sulphate or ammonium nitrate at 0.5 g L-1 and fermen-

Optimization of fermentative production by using Orthogonal Array Design An L8 orthogonal array method was used for screening of the most significant fermentation parameters influencing keratinase production. The design for the L8 orthogonal array was developed and analyzed using MINITAB 13.30 software (Pennsylvania State university, University Park, Pennsylvania). The L8 orthogonal array design is shown in Table 1. Seven factors at two levels were studied viz. chicken feather, ammonium chloride, beef extract, potassium dihydrogen phosphate, potassium hydrogen phosphate,

tation was carried out as described in the previous section. To check the effect of additional carbon sources

Table 1. Orthogonal project design for 2 levels of 7 variables used for media optimization for keratinase production.

Run

Feathers Beef extract

MgSO4

KH2PO4

K2HPO4

NaCl

NH4Cl

Keratinasea -1 (KU ml-1)

1

1 (5)

1 (0.05)

1 (0.05)

1 (0.1)

1 (0.1)

1(0.15)

1 (0.15)

1.33 ± 0.04

2

1 (5)

1 (0.05)

1 (0.05)

2 (0.5)

2 (0.5)

2 (0.75)

2 (0.75)

2.37 ± 0.1

3

1 (5)

2 (0.25)

2 (0.25)

1 (0.1)

1 (0.1)

2 (0.75)

2 (0.75)

1.1 ± 0.23

4

1 (5)

2 (0.25)

2 (0.25)

2 (0.5)

2 (0.5)

1 (0.15)

1 (0.15)

1.05 ± 0.04

5

2 (25)

1 (0.05)

2 (0.25)

1 (0.1)

2 (0.5)

1 (0.15)

2 (0.75)

3.66 ± 0.07

6

2 (25)

1 (0.05)

2 (0.25)

2 (0.5)

1 (0.1)

2 (0.75)

1 (0.15)

1.90 ± 0.25

7

2 (25)

2 (0.25)

1 (0.05)

1 (0.1)

2 (0.5)

2 (0.75)

1 (0.15)

2.88 ± 0.2

8

2 (25)

2 (0.25)

1 (0.05)

2 (0.5)

1 (0.1)

1 (0.15)

2 (0.75)

4.20 ± 0.08

a Results are mean ± SD of three determinations

Values in the parenthesis indicate the real values of variables

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magnesium sulphate and sodium chloride for their significance in production of keratinase by B. subtilis NCIM 2724.

Optimization of concentrations of the selected medium components using Response Surface Methodology (RSM) Response surface methodology is an empirical statistical modeling technique employed for multiple regression analysis using quantitative data obtained from properly designed experiments to solve multivariable equations simultaneously (Puri et al., 2002). RSM was used to determine the optimum nutrient concentrations for the production of keratinase. A central composite design (CCD) for four independent variables was used to obtain the combination of values that optimizes the response within the region of three dimensional observation spaces, which allows one to design a minimal number of experiments. The experiments were designed using the software, Design Expert Version 6.0.10 trial version (State Ease, Minneapolis, MN).

The medium components (independent variables) selected for the optimization were chicken feather, ammonium chloride, magnesium sulphate, and dipotassium hydrogen phosphate. Regression analysis was performed on the data obtained from the design experiments. Coding of the variables was done according to the following equation:

xi =

(X -X ) i

∆Xi

cp

i= 1,2,3,... k

where: xi, dimensionless value of an independent variable; Xi, real value of an independent variable; Xcp, real value of an independent variable at the center point; and ∆Xi, step change of real value of the variable i corresponding to a variation of a unit for the dimensionless value of the variable i.

The experiments were carried out at least in triplicate, which was necessary to estimate the variability of measurements, i.e. the repeatability of the phenomenon. Replicates at the center of the

domain in three blocks permit the checking of absence of bias between several sets of experiments.

The relationship of the independent variables and the response was calculated by the second order polynomial equation: Y is the predicted response; ß0 a constant; ßi the k

k

k

k

i=1

i=1

i<j

i<j

linear coefficient; ßii the squared coefficient; and ßij the cross-product coefficient, k is number of factors.

The second order polynomial coefficients were calculated using the software package Design Expert Version 6.0.10 to estimate the responses of the dependent variable. Response surface plots were also obtained using Design Expert Version 6.0.10.

Effect of amino acids on keratinase production by B. subtilis NCIM 2724 To study the effect of amino acids on keratinase production, various amino acids including L-cysteine, L-serine, L-valine, L-alanine, L-methionine, L-glutamic acid, L-threonine, L-histidine, L-arginine and Llysine were added individually at 0.05 g L-1, 0.10 g L-1 and 0.50 g L-1 in the RSM optimized medium.

Keratinase assay Keratinase activity was determined by the method reported by Yu et al. (1968). Chicken feathers (20 mg) were suspended in 3.8 ml of 100 mM Tris–HCl buffer (pH 7.8), to which 0.2 ml of the culture filtrate (enzyme source) was added.

The reaction mixture was incubated at 37°C for 1 h. After incubation, the assay mixture was dipped into the ice cold water for 10 min and the remaining feathers were filtered out by Whatman filter paper (Whatman® Schleicher and Schuell, Mumbai, India). The absorbance of the clear mixture was measured at 280 nm. The keratinase activity was expressed as one unit of the enzyme corresponding to an increase in the absorbance value 0.1 (1KU= 0.100 corrected absorbance).

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Results and Discussion Optimization using one-factor-at-a-time An initial pH of 8.0 supported maximum production of keratinase of 3.3 KU ml-1. pH is a significant factor that influences the physiology of a microorganism by affecting nutrient solubility and uptake, enzyme activity, cell membrane morphology, byproduct formation and oxidative-reductive reactions. During production of keratinase, keratin utilization occurs more rapidly and to a great extent at pH 7.5 (Suntornsuk and Suntornsuk, 2003). Friedrich and Antranikian (1996) described maximum keratinase production at alkaline pH. Alkaline pH favors keratin degradation at higher pH, probably by modifying the cystine residues to lanthionine and making it accessible for keratinase action. The optimum pH reported for keratinase production by B. cereus is 7.0 (Kim et al., 2001), chryseobacterium sp. is 9.0 (Casarin et al., 2008), while that by B. pumilus FH9 is 8.0 (El-Refai et al., 2005). For B. subtilis, highest enzyme production has been reported over a range of pH of 7 to 9. It was observed that maximum keratinase production occurs at alkaline pH. It was found that ammonium chloride and beef extract supported maximum keratinase activity of 4.05 KU ml-1 and 4.15 KU ml-1 respectively. These results are in accordance with the results obtained by El-Refai et al. (2005), where ammonium chloride and yeast extract supported maximum keratinase production in B. pumilus FH9. Some researchers have considered feather meal as a nitrogen source for keratinase production (Thyes et al., 2006).

B. subtilis NCIM 2724 produced keratinase in presence of chicken feathers as the sole carbon source which supported a maximum production of 4.14 KU ml-1. Addition of simple carbon sources reduced the production of keratinase. A decrease in the keratinase production due to the addition of conventional carbon sources is reported in literature. Addition of fructose and maltose in medium decreased the keratinase production in Trichophyton rubrum (Meevootisom and Niederpruem, 1979) and B. licheniformis (Sen and Satyanarayana, 1993), respectively. These

results may be due to the catabolic repression of keratinase (Anbu et al., 2007; Ignatova et al., 1999; Yamamura et al., 2002; Santos et al., 1996). It has been reported that chicken feathers act as the best carbon source for keratinase production.

Statistical media optimization Optimization of fermentative production by using Orthogonal Array Design Once the best carbon and nitrogen sources were selected, the medium was subjected to screening of the most significant parameters for keratinase production using the L8 orthogonal array. The responses for means (larger is better) and for signal to noise ratios obtained using the L8 orthogonal array are shown in Table 2. The last two rows in the tables show delta values and ranks for the system.

Rank and delta values help in assessing which factors have the greatest effect on the response characteristic of interest. Delta measures the size of the effect by taking the difference between the highest and lowest characteristic average for a factor. A higher delta value indicates a greater effect of that component. Rank orders the factors from the greatest effect (on the basis of the delta values) to the least effect on the response characteristic. The order in which the individual components affected the fermentation process were feather > ammonium chloride > magnesium sulphate > dipotassium hydrogen phosphate > sodium chloride > beef extract > potassium dihydrogen phosphate suggesting that feathers had a major effect, while K2HPO4 had the least effect on keratinase production by B. subtilis NCIM 2724.

Optimization by RSM Based on the L8 orthogonal array design, feather (A), ammonium chloride (B), magnesium sulphate (C) and dipotassium hydrogen phosphate (D) were selected for further optimization by RSM. To examine the combined effect of these medium components (independent variables) on keratinase production, a central composite factorial design of 24 =16 plus 6 center points and (2 × 4 = 8) star points lead-

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Table 2. Response table for means and S/N ratio.

Level

A Feathers S/N

Mean

B Beef Extract S/N

C MgSO4

Mean

S/N

Mean

D KH2PO4 S/N

Mean

E K2HPO4 S/N

Mean

F NaCl S/N

Mean

G NH4CL S/N

Mean

1

2.82

2.82

6.70

2.31

7.71

2.60

5.94

2.24

5.14

2.04

6.48

2.47

4.43

1.79

2

9.42

9.42

5.54

2.21

4.54

1.92

6.31

2.29

7.11

2.49

5.77

2.06

7.81

2.74

Delta 6.60

6.60

1.15

0.09

3.17

0.67

0.37

0.04

1.96

0.44

0.70

0.40

3.38

0.95

Rank

1

6

3

ing to a total of 30 experiments were performed.

A CCRD matrix of independent variables along with responses of each experimental trial is given in Table 3.

The ANOVA of the quadratic regression model indicated the model to be significant (P < 0.05) (Table 4). The P values were used as a tool to check the significance of each of the coefficients, which, in turn, are necessary to understand the pattern of the mutual interactions among the test variables.

The smaller the magnitude of the P, the more significant is the corresponding coefficient (Thys et al., 2006). Among the test variables used in the study, A (feather), B (ammonium chloride), C (magnesium sulphate), D (dipotassium hydrogen phosphate), A2 (feather2), B2 (ammonium chloride2) and D2 (dipotassium hydrogen phosphate2) are significant model terms. Interactions between B (ammonium chloride) and C (magnesium sulphate); B (ammonium chloride) and D (dipotassium hydrogen phosphate); and C (magnesium sulphate) and D (dipotassium hydrogen phosphate) are also significant. Other interactions were found to be insignificant.

The corresponding second-order response model found after analysis for the regression was keratinase (KU ml-1) = 3.66 + 1.09 * feather + 0.51 * ammonium chloride + 0.72* magnesium sulphate + 1.26 * dipotassium hydrogen phosphate + 1.20

7

4

5

2

* feather2 + 0.36 * ammonium chloride2 + 0.12 * magnesium sulphate2 + 0.36 * dipotassium hydrogen phosphate2 - 0.084 * feather * ammonium chloride + 0.15 * feather *magnesium sulphate - 0.23 * feather * dipotassium hydrogen phosphate - 0.24 * ammonium chloride * magnesium sulphate + 0.68 * ammonium chloride * dipotassium hydrogen phosphate + 0.43 * magnesium sulphate * dipotassium hydrogen phosphate.

The fit of the model was also expressed by the coefficient of regression R2, which was found to be 0.98, indicating that 98.0% of the variability in keratinase yield could be explained by the model.

Other parameters of ANOVA for response surface quadratic model were also studied. The ‘Pred RSquared’ of 0.92 is in reasonable agreement with the ‘Adj R-Squared’ of 0.96. ‘Adeq Precision’ measures the signal to noise ratio.

The special features of the RSM tool, “contour plot generation” and “point prediction” were also studied to find optimum value of the combination of the four media constituents. It was observed that medium containing (g L-1), feather, 60.0; ammonium chloride, 1.0; magnesium sulphate, 0.08; and dipotassium hydrogen phosphate, 0.2 yielded maximum (10.6 KU ml-1) keratinase.

Accordingly, three-dimensional graphs were generated for the pair-wise combination of the four factors,

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Table 3. The CCRD matrix of independent variables in coded form and actual values with their corresponding response in terms of production of keratinase by B. subtilis NCIM 2724.

Sr. no.

Feather (g L-1)

NH4CL (g L-1)

MgSO4 (g L-1)

K2HPO4 (g L-1)

Keratinase a (KU ml-1)

1

-1 (30)

-1 (0.5)

-1 (0.04)

-1 (0.1)

2.77 ± 0.14

2

1 (60)

-1 (0.5)

-1 (0.04)

-1 (0.1)

5.5 ± 0.11

3

-1 (30)

1 (1.0)

-1 (0.04)

-1 (0.1)

3.1 ± 0.20

4

1 (60)

1 (1.0)

-1 (0.04)

-1 (0.1)

5.13 ± 0.13

5

-1 (30)

-1 (0.5)

1 (0.08)

-1 (0.1)

3.65 ± 0.10

6

1 (60)

-1 (0.5)

1 (0.08)

-1 (0.1)

6.81 ± 0.18

7

-1 (30)

1 (1.0)

1 (0.08)

-1 (0.1)

3.41 ± 0.13

8

1 (60)

1 (1.0)

1 (0.08)

-1 (0.1)

5.75 ± 0.17

9

-1 (30)

-1 (0.5)

-1 (0.04)

1 (0.2)

3.45 ± 0.16

10

1 (60)

-1 (0.5)

-1 (0.04)

1 (0.2)

4.59 ± 0.14

11

-1 (30)

1 (1.0)

-1 (0.04)

1 (0.2)

6.36 ± 0.17

12

1 (60)

1 (1.0)

-1 (0.04)

1 (0.2)

7.75 ± 0.23

13

-1 (30)

-1 (0.5)

1 (0.08)

1 (0.2)

6.14 ± 0.20

14

1 (60)

-1 (0.5)

1 (0.08)

1 (0.2)

8.24 ± 0.18

15

-1 (30)

1 (1.0)

1 (0.08)

1 (0.2)

7.91 ± 0.24

16

1 (60)

1 (1.0)

1 (0.08)

1 (0.2)

9.93 ± 0.13

17

-2 (15)

0 (0.75)

0 (0.06)

0 (0.15)

6.18 ± 0.21

18

2 (75)

0 (0.75)

0 (0.06)

0 (0.15)

10.84 ± 0.8

19

0 (45)

-2 (0.25)

0 (0.06)

0 (0.15)

4.15 ± 0.04

20

0 (45)

2 (1.25)

0 (0.06)

0 (0.15)

6.12 ± 0.03

21

0 (45)

0 (0.75)

-2 (0.02)

0 (0.15)

3.14 ± 0.20

22

0 (45)

0 (0.75)

2 (0.1)

0 (0.15)

5.21 ± 0.20

23

0 (45)

0 (0.75)

0 (0.06)

-2 (0.05)

2.13 ± 0.05

24

0 (45)

0 (0.75)

0 (0.06)

2 (0.25)

8.14 ± 0.17

25

0 (45)

0 (0.75)

0 (0.06)

0 (0.15)

3.55 ± 0.11

26

0 (45)

0 (0.75)

0 (0.06)

0 (0.15)

2.75 ± 0.25

27

0 (45)

0 (0.75)

0 (0.06)

0 (0.15)

4.12 ± 0.04

28

0 (45)

0 (0.75)

0 (0.06)

0 (0.15)

3.86 ± 0.02

29

0 (45)

0 (0.75)

0 (0.06)

0 (0.15)

3.86 ± 0.17

30

0 (45)

0 (0.75)

0 (0.06)

0 (0.15)

3.8 ± 0.20

a Results are mean ± SD of three determinations

Values in the parenthesis indicate the real values of variables

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Table 4. Analysis of variance (ANOVA) for the experimental results of the central-composite design (Quadratic Model).

Factor a

Coefficient Estimate

Sum of squares

Standard Error

DF b

F value

p

Intercept

3.66

139.52

0.18

1

53.27

< 0.0001

A

1.09

28.67

0.088

1

153.24

< 0.0001

B

0.51

6.13

0.088

1

32.77

< 0.0001

C

0.72

12.51

0.088

1

66.89

< 0.0001

D

1.16

38.18

0.088

1

204.07

< 0.0001

A2

1.20

39.46

0.083

1

210.94

< 0.0001

B2

0.36

3.47

0.083

1

18.55

0.0006

C2

0.12

0.37

0.083

1

1.96

0.1815

D2

0.36

3.47

0.083

1

18.55

0.0006

AB

-0.084

0.11

0.11

1

0.61

0.4474

AC

0.15

0.34

0.11

1

1.81

0.1981

AD

-0.23

0.81

0.11

1

4.35

0.0544

BC

-0.24

0.94

0.11

1

5.0

0.0409

BD

0.68

7.38

0.11

1

39.47

< 0.0001

CD

0.43

3.02

0.11

1

16.14

0.0011

a A = Feathers, B = NH Cl, C = MgSO , D =K HPO 4 4 2 4 b Degree of freedom

Figure 1. Contour plot for keratinase production (-Effect of MgSO4 and NH4Cl).

Figure 2. Contour plot for keratinase production (Effect of K2HPO4 and NH4Cl).

62 Agric. Food Anal. Bacteriol. • www.AFABjournal.com • Vol. 1, Issue 1, 2011

while keeping the other two at their center point levels.

Graphs for interactions are given here to highlight the roles played by these factors (Figure 1 and Figure 2).

From the central point of the contour plot the optimal process parameters were identified. The keratinase yield (10.6 KU/ml) in the present study is quite high as compared to the literature reports. The maximum keratinase production reported till date by using most widely used strain Bacillus subtilis S1 MTCC 2616 is 4.89 KU/ml and Scopulariopsis brevicaulis MTCC 2170 is 6.2 KU/ml.

The effect of amino acids on keratinase production by B. subtilis NCIM 2724 Effect of amino acids on production of keratinase is shown in Figure 3. All of the amino acids examined supported keratinase production, but the maximum keratinase activity of 12.32 KU ml-1 was observed with 0.5 g L-1 of L-valine. Further increases in L-valine concentration did not increase keratinase activity (Data not shown).

Addition of amino acids is of considerable importance in the protease synthesis in terms of metabolic

driving force. Feather keratin is composed of various amino acids including glycine, alanine, serine, cysteine and valine that are extensively cross linked with hydrogen, disulphide and hydrophobic bonds.

Degraded feathers may act as source of some nutritionally important amino acids and also serves as an inducer for keratinase production.

CONCLUSION Statistical nutrient optimization was done to optimize keratinase production from B. subtilis NCIM 2724. Taguchi design (L8 orthogonal array) demonstrated the effect of feather, ammonium chloride, K2HPO4 and MnSO4 to be significant. Further optimization of the most significant factors by RSM revealed complex nutrient interactions among them, and also increased the production of keratinase by B. subtilis NCIM 2724 from 3.0 KU ml-1 to 10.6 KU ml-1. All amino acids supported keratinase production and the maximum keratinase activity of 12.32 KU ml-1 was observed with 0.5 g L-1 of L-valine.

Figure 3. Effect of amino acids on keratinase production by B. subtilis NCIM 2724.

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www.afabjournal.com Copyright © 2011 Agriculture, Food and Analytical Bacteriology

REVIEW

An Overview of Stress Response Proteomes in Listeria monocytogenes K. A. Soni1, R. Nannapaneni1*, and T. Tasara2 Department of Food Science, Nutrition and Health Promotion, Mississippi State University, Mississippi State, MS 39762, USA 2 Institute for Food Safety and Hygiene, Vetsuisse Faculty University of Zurich, Zurich, Switzerland 1

ABSTRACT Listeria monocytogenes adapts to diverse stress conditions including cold, osmotic, heat, acid, and alkali stresses encountered during food processing and preservation which is a serious food safety threat. In this review, we have presented the major findings on this bacterium’s stress response proteomes to date along with the different approaches used for its proteomic analysis. The key proteome findings on cold, heat shock, salt, acid, alkaline and HHP stresses illustrate that the cellular stress responses in this organism are a culmination of multiple protein expression changes in response to a particular stress stimuli. Moreover, a number of key proteins may be involved in conferring the cross protective effects against various stress environments. As an example, ferritin-like protein (designated as Fri or Flp) is induced during cold, heat, and HHP stresses. Similarly, general stress protein Ctc is induced in cold and osmotic stresses while molecular chaperones such as GroEL and DnaK are induced in cold and heat stresses. Furthermore, a number of stress proteins also contribute towards L. monocytogenes virulence and pathogenicity. Future research may lead to understanding the stress proteomes of this pathogen induced on various food matrices and processing environments in which it can persist for long periods of time.

Keywords: Listeria monocytogenes, proteome, cold stress, osmotic stress, heat stress, acid stress, alkali stress.

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Introduction Listeria monocytogenes is an important foodborne pathogen with significant public health threats and economic impacts on the food industry. It causes Received: November 22, 2010, Accepted: April 9, 2011.

Released Online Advance Publication: April 1, 2011.

Correspondence: Ramakrishna Nannapaneni, nannapaneni@fsnhp.msstate.edu.

Tel: +1 -662-325-7697 Fax: +1-662-325-8728

“listeriosis” in humans, which is associated with a variety of symptoms ranging from flu-like illness to severe life threatening meningitis as well as high mortality (Lennon et al., 1984). Epidemiological studies estimate that listeriosis to be responsible for approximately 19 % of food-related deaths in the United States annually (Scallan et al., 2011). Suspected L. monocytogenes contamination is also among the leading causes of food recalls resulting in significant

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financial losses to the food industry due to the “zero tolerance” standard adopted for the ready-to-eat food products in the USA (Kramer et al., 2005; Marsden, 2001; Teratanavat and Hooker, 2004).

The prevalence of L. monocytogenes is mainly due to its wide-spread distribution and its ability to withstand adverse environmental conditions. This includes the ability of this pathogen to survive and grow at low temperatures, and resistance to high osmolarity, acidic and alkaline environments. Cold adaptation of this organism is of growing concern due to the changing life styles over the years that have increased the consumption of refrigerated and minimally processed food products. Besides cold storage, elevated salt concentrations are an alternative means of food preservation, but L. monocytogenes is also highly salt tolerant and has been documented to grow in the presence of as high as 10% NaCl (McClure et al., 1991). Jensen et al.

(2007) recently showed that L. monocytogenes cells can display increased aggregation and biofilm formation when exposed to NaCl stress. Additionally this bacterium exhibits acid tolerance responses (ATR), which significantly increases its resistance to a subsequent lethal acid (pH 3.0-3.5) stress exposure after an initial encounter with the non-lethal acidic (pH 5.0-5.5) conditions. As an example, 4-log higher survival was observed in L. monocytogenes cells exposed to acid stress at pH 3.5 for 6 h after an initial 90 minute exposure to a mild acidic condition at pH 5.5 (Koutsoumanis et al., 2004). Similarly, L. monocytogenes may also acquire an increased alkaline stress tolerance subsequent to sublethal alkaline stress exposure (Mendonca et al., 1994). During food processing and preservation, L. monocytogenes cells may become exposed to multiple forms of sublethal stresses, leading to “stress hardening”.

Consequently, L. monocytogenes exposure to mild forms of particular stresses may inadvertently induce cross protection against subsequent exposures to lethal levels of other unrelated stresses. For example, it has been shown that acid (pH 4.5 for 1 h) or cold (10°C for 4 h) stressed L. monocytogenes LO28 (serotype 1/2c) cells tend to be more resistant to high hydrostatic pressure (HHP) in comparison

to the non-stress adapted cells (Wemekamp-Kamphuis et al., 2002). Lou and Yousef (1997) reported that the heat stress of L. monocytogenes results in cell-hardening and subsequent osmoprotection and higher resistance of these cells to ethanol treatment. Likewise, L. monocytogenes cells were also found to be more thermotolerant after a combined acid and heat shock or after osmotic and heat shock treatments (Skandamis et al., 2008).

Stress adaptation events in L. monocytogenes, as in other microorganisms, includes coordinated induction of different stress protection systems within the affected cells. Proteomics and transcriptomics are both invaluable tools in delineation of the different mechanisms of stress response in microbes.

Transcriptome analysis technologies while important in deciphering the global mRNA expression changes during stress responses, fail to capture all aspects of these molecular responses since mRNA transcripts changes may not directly correlate with protein expression due to the fact that transcripts produced in abundance may be rapidly degraded, translated poorly, or influenced through post-translational modifications. Therefore complementation of the transcriptome based analysis of stress responses with the proteome studies is important to get a clearer picture as proteins are the key functional units involved in physiological stress responses. As a result of new developments in microbial cell global protein profiling based on the protein identification approaches and bioinformatics, researchers are now also able to monitor and determine the importance of stress induced proteins in L. monocytogenes during its adaptation to diverse conditions. A number of proteome profiling studies performed on this organism so far have already provided extensive preliminary insights into gene and protein expression changes that are associated with the environmental stress adaptation in this bacterium. The purpose of this review is to discuss the significant developments in proteomic analysis of the stress-adaptation in L. monocytogenes with focus on cold, heat, osmotic, acid, alkaline, and HHP adaptation along with cross linking between stress proteins and virulence.

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Proteomic Technologies Applied in L. monocytogenes Analysis The summary of different gel-based and non-gel based techniques and their assay principles are discussed in-depth in recent review articles by Haynes et al. (2007) and Nesatyy and Suter (2007). For L. monocytogenes, the most commonly applied protocols to date have used two-dimensional gel electrophoresis (2DE) for protein separation (Folio et al., 2004; Mujahid et al., 2007; Ramnath et al., 2003; Schaumburg et al., 2004). The majority of the L. monocytogenes stress proteome studies utilized soluble cellular proteins (excluding the extracellular fraction) that were fractionated using mechanical disruption alone or protocols combining mechanical disruption and enzymatic lysis. In the earlier studies most of the proteins were not identified (; Bayles et al., 1996; Phan-Thanh and Gormon, 1995), although in later studies a significant number of the 2DE separated proteins were identified by mass spectrophotometry (MS) (Abram et al., 2008b; Dumas et al., 2008; Folio et al., 2004; Mujahid et al., 2007; 2008; Phan-Thanh and Jansch, 2006; Schaumburg et al., 2004 ). More recently however, non-gel based approaches that combine liquid chromatography (LC) separation and MS (LC-MS/MS) are increasingly used. The fractionated complex bacterial protein mixtures are digested into peptides, separated by liquid chromatography and analyzed in MS, taking advantage of the advances in bioinformatics to identify even larger numbers of the fractionated proteins (Abram et al., 2008b; Calvo et al., 2005; Trost et al., 2005).

The majority of studies that have compared protein expression between normal versus stress exposed L. monocytogenes cells using 2DE gel-based protein separation with or without subsequent application of MS to identify separated proteins (Bayles et al., 1996; Duche et al., 2002a; Esvan et al., 2000; PhanThanh and Gormon, 1995; 1997; Phan-Thanh and Mahouin, 1999; Wemekamp-Kamphuis et al., 2004a).

A 2DE reference map covering an estimated 28.8% of potential gene products was generated from the soluble subproteome of L. monocytogenes EGDe serotype 1/2a strain (Folio et al., 2004). Ramnath et

al. (2003) also used this approach and detected two proteins found in L. monocytogenes EGDe but were absent in some food isolates. The identification of these proteins revealed they were involved in glycolytic pathway and metabolism of coenzymes, but the relevance of their differential expression specifically in such food isolates remains unknown. The drawbacks of gel based 2DE proteomics include poor reproducibility in separation of highly basic or hydrophobic proteins, gel-to-gel variations and poor resolution of high molecular weight protein complexes.

Attempts to overcome these drawbacks include the recent use of 2D-DIGE (Two dimensional-difference gel electrophoresis) based proteomics analysis. By using different fluorescent dyes such as Cy2, Cy3 or Cy5 for protein labeling, such approaches allow protein mixtures of different origins to be analyzed within the same gel run. Thus these approaches are more amenable to stress proteome response studies where protein expression patterns of stress-adapted cells and control samples can be directly compared within the same gel run to minimize the influence of gel-to-gel variations. Folsom and Frank (2007) used a 2DE-DIGE based proteomics approach to analyze protein expression changes associated with chlorine resistance and biofilm formation in a hypochlorous acid tolerant variant of the L. monocytogenes Scott A (4b) strain. They found 19 proteins that were differentially expressed between planktonic and biofilm cells of a hypochlorous acid tolerant cultural variant of this strain (Folsom and Frank, 2007). Six of these differentially expressed proteins were subsequently identified by peptide-mass mapping. They included three ribosomal proteins (L7, L10 and L12), peroxide resistance protein (Dpr/Flp/Fri), sugar-binding protein (Lmo0181), and a putative protein Lmo1888 of yet unknown function. This study also revealed that peroxide stress resistance proteins Fri that is involved in multitude of other stresses was expressed 2.2-fold times higher in biofilm than in planktonic cells. At phenotypic level it was observed that L. monocytogenes cells present in biofilm mass were more resistant to sanitization treatments compared to planktonic cells (Pan et al., 2006). Although not yet widely adapted for L. monocytogenes analysis, LC

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based techniques seem capable of detecting even higher numbers of proteins compared to traditional 2DE gel-based techniques. As an example when the same protein fraction of cell free supernatant (extracellular) of L. monocytogenes EGDe was analyzed, 105 proteins were identified using LC-MS/MS compared to 58 detected by 2DE (Trost et al., 2005).

Forty-five of the detected proteins were found to be common between the two methods. The analysis of differential protein expression between L. monocytogenes 10403S and its σB null mutant strain using the LC-MS/MS with iTRAQ (isotope tag for relative and absolute quantification) identified 35 σB regulated proteins, whereas the 2DE approach only managed to detect 13 proteins. Four proteins were common between the two methods (Abram et al., 2008b). A combination of SDS-PAGE and LC-MS/MS detected 301 membrane associated proteins of L. monocytogenes EGDe (Wehmhoner et al., 2005). This was greater than 79 proteins detected using the 2DE approach by Mujahid et al. (2007). One possible reason for increased protein detection with SDS-PAGE/LCMS/MS might be increased protein solubilization in the SDS-PAGE sample buffer in comparison to the urea based sample buffer applied in the 2DE-MS approach (Haynes and Roberts, 2007).

In another example LC-LC-MS/MS combination also called the multidimensional protein identification technique (MudPIT), has also been used for proteome analysis of L. monocytogenes cells. Fifteen proteins that covalently bound the LPXTG motif were identified in the subproteome fraction of cell wall associated proteins of L. monocytogenes strain EGDe (Calvo et al., 2005). The SrtA and SrtB enzymes anchor surface proteins to the cell wall. Surface proteins recognized by these two sortases were also analyzed using LC-LC-MS/MS in the EGDe strain. A total of 13 and 2 LPXTG-containing proteins were identified in srtA and srtB null mutant strains (Pucciarelli et al., 2005). Recently, MudPIT was used to study the differences that exist between serotype 1/2a (strain EGD) and 4b (strain F2365) (Donaldson et al., 2009).

In total, 1754 EGD proteins and 1427 F2365 proteins were detected representing 50-60% of total Listeria proteome coverage. In total 1077 proteins were

common to both serotypes and of these 413 proteins displayed significantly differential expression level between the two serotypes.

Proteome Analysis in StressAdapted L. monocytogenes Cells The ability of L. monocytogenes to sense and respond to a particular stress factor has implications for both survival and virulence properties of this bacterium. Stress exposure elicits various fundamental changes in this organism’s cellular physiology. These changes are mediated via multiple and specific changes in gene and protein expression profiles in cells. Proteins associated with cold, heat, osmotic, acid, and high hydrostatic pressure stress adaptation will be discussed in the following sections.

Cold stress adaptation The growth of L. monocytogenes on cold preserved food products is one of its important food safety challenges. In addition to decreased metabolic capacity, cold stress exposed microorganisms are faced with a wide range of structural and functional impediments in membrane structures, nucleic acids (DNA and RNA), and macromolecular assemblies such as ribosomes (Schumann, 2009).

The putative integral membrane protein PgpH, whose deletion leads to impaired cold growth, has been proposed as a possible cold sensing factor in L. monocytogenes (Liu et al., 2006). Based on the proposed model, environmental cold stress sensed through membrane bound PgpH proteins is conveyed intracellularly through homeodomain dependent signaling pathways.

Using 2DE gel-based proteome analysis, initial studies revealed modulation in expression of between 10 to 38 proteins in association with cold stress adaptation of this organism (Bayles et al., 1996; Hebraud and Guzzo, 2000; Phan-Thanh and Gormon, 1995). Of these differentially expressed proteins visualized, the predominating cold shock protein was subsequently identified through microsequencing as ferritin (Fri) (designated as Flp or Fri) (Hebraud and

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Guzzo, 2000). The role of this protein in cold adaptation was also later phenotypically confirmed when Dussurget et al. (2005) created a fri null mutant strain in L. monocytogenes EGDe, which exhibited a cold sensitive phenotype. Although physiological and cold adaptation roles of Fri are not yet well understood, it is hypothesized that it might facilitate alleviation of oxidative stress environments developing in cold stress exposed L. monocytogenes cells (Liu et al., 2002; Tasara and Stephan, 2006). WemekampKamphuis et al. (2002) described four approximately 7 kDa protein that were cold inducible in L. monocytogenes LO28 as determined by using a combination of 2DE gel electrophoresis and immunoblotting. These proteins designated Csp1-Csp4, were described as the L. monocytogenes cold shock family proteins based on their cross reactivity with anti-B. subtilis CspB polyclonal antibodies. Although their identity as such was not confirmed by peptide mass fingerprinting (PMF) in this work, genomic information show that L. monocytogenes harbors three proteins of the cold shock domain protein family (Glaser et al., 2001). Two of these L. monocytogenes Csp proteins, CspL (CspA) and CspD have now been confirmed to be functionally vital for efficient cold growth in this bacterium (Schmid et al., 2009). CspA and CspD proteins, based on knowledge from other microorganisms, are also presumed to facilitate cold growth possibly through nucleic acid (DNA and RNA) chaperone-like functions (Horn et al., 2007).

This facilitates DNA replication and gene expression events that may otherwise be hampered through secondary structures that tend to form in bacterial cells at low temperatures.

Meanwhile, a more comprehensive cold adaptation proteome analysis in this bacterium has been recently described. Cacace et al. (2010) performed detailed proteome analysis on L. monocytogenes cells grown for 13 days at 4°C with subsequent MALDI (Matrix-assisted laser desorption/ionization) analysis. Proteome analysis revealed that 57 proteins in total were over-expressed and eight were repressed in cold grown cells compared to cells cultivated at 37°C. Proteome changes detected in this study indicated the increased synthesis of proteins linked to

energy production, oxidative stress resistance, nutrient uptake, lipid synthesis, and protein synthesis and folding. Cold stress adaptation proteins identified by this study that are of particular interest include: OppA, Ctc, GroEL and DnaK. The OppA protein, which facilitates accumulation of short peptide substrates, is important for efficient cold growth in this bacterium and at phenotypic level oppA null mutant of this bacterium was unable to grow at low temperature (5°C) (Borezee et al., 2000). Ctc is a general stress protein which has been found to promote the adaptation of L. monocytogenes cells to high osmolarity conditions (Gardan et al., 2003b). The GroEL and DnaK proteins are molecular chaperones that promote proteins refolding and degradation of stress damaged proteins that accumulate under different suboptimal conditions including heat stress (Sokolovic et al., 1990). The cold growth associated induction of the Ctc, GroEL and DnaK proteins, which have been previously associated with adaptation to other stresses (i.e. Ctc for cold and osmotic stress and GroEL-DnaK for cold and heat stress) conditions may thus indicate commonality of some stress adaptive responses in this bacterium (Cacace et al., 2010; Duche et al., 2002a,b; Gardan et al., 2003b; Sokolovic et al., 1990).

The accumulation of compatible solutes especially glycine, betaine, and carnitine also promotes cold growth in various bacteria including L. monocytogenes (Mendum and Smith, 2002; Smith, 1996; Wemekamp-Kamphuis et al., 2004b). There are no enzymatic systems for the de novo synthesis of main cryoprotective compatible solutes glycine, betaine, and carnitine in L. monocytogenes, but transport systems (Gbu, BetL and OpuC) that accumulate them from environmental sources are present, and deletion of genes coding for these transporters has confirmed that they facilitate efficient cold growth of this bacterium (Angelidis et al., 2002; Ko and Smith, 1999; Sleator et al., 1999). Analysis of cold-sensitive mutants in which Lmo1078 (Chassaing and Auvray, 2007), and LtrC (Chan et al., 2007) proteins are inactivated also indicates that these proteins functionally contribute to cold adaptation processes in L. monocytogenes. The Lmo1078 protein is a UDP-

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glucose pyrophosphorylase proposed to promote cold adaptation through enhanced UDP-glucose production at low temperatures. UDP glucose is an essential substrate in lipoteichoic acid production and might facilitate maintenance of architectural integrity in cell wall and membrane structures leading to protection of bacterial cells from cold stress damage (Chassaing and Auvray, 2007).

Heat stress adaptation The understanding of heat stress adaptation in food-borne pathogens is an important issue since heating constitutes one of the major food processing and preservation methods. The heat shock response is one of the most universal and extensively studied physical stress responses in living organisms.

This process involves increased production of various cell protective protein systems, which ultimately promotes general environmental stress resistance and enhanced thermal tolerance (Gandhi and Chikindas, 2007; Klinkert and Narberhaus, 2009; Muga and Moro, 2008; van der Veen et al., 2007). Similar to other bacteria, L. monocytogenes synthesizes a highly conserved set of proteins, also defined as heat shock proteins (Hsps), upon exposure to high temperatures (>45°C). Hsps include highly conserved molecular chaperones and proteases that functionally prevent nonproductive protein aggregations under different stress environments. GroEL and DnaK are major Hsps that promote refolding and degradation of damaged proteins through ATPdependent mechanisms (Kandror et al., 1994; Sherman and Goldberg, 1996; van der Veen et al., 2007).

These two proteins are highly conserved among living organisms and also constitute as the main Hsp chaperones observed in L. monocytogenes (Gahan et al., 2001; Hanawa et al., 2000) Using proteome analysis, the induction of up to 15 Hsps in response to heat shock (48°C/30 min) was observed using SDS-PAGE (Sokolovic et al., 1990). Of these, two Hsps were identified as GroEL and DnaK in L. monocytogenes CLIP 54149 (serotype 1/2a) based on immunological detection. In another study the induction of as many as 32 Hsps was observed

using preparative 2DE gels of L. monocytogenes EGD in response to a temperature shock of 49°C/15 min (Phan-Thanh and Gormon, 1995). One identified predominant protein, Fri, with molecular weight 18 kDa and pI of 5.1 showed 50.6-fold inductions due to heat shock. This very same protein spot was 10.5fold induced in response to cold shock (Phan-Thanh and Gormon, 1995). Similarly, other researchers have also observed the transcriptional induction of fri transcripts in response to heat (Hebraud and Guzzo, 2000; van der Veen et al., 2007) and cold stress (Dussurget et al., 2005). Phenotypically fri gene null L. monocytogenes EGDe cells also failed to reach the maximal optical density compared to the wild type strain during growth under heat at 45°C (Dussurget et al., 2005). These findings together suggest that ferritin-like protein is important for high and low temperature adaptation in L. monocytogenes.

Recently, Agoston et al. (2009) compared the effect of mild and prolonged heat treatments on L. monocytogenes cells using 2DE analysis. In line with the reduced metabolic activity at suboptimal temperature, large numbers of metabolic proteins were suppressed during heat exposure in this study which is also consistent with the observation from other studies (Phan-Thanh and Gormon, 1995; Phan-Thanh and Jansch, 2006). Importantly, L. monocytogenes stress protein DnaN, a beta subunit of polymerase III, was highly induced in response to different heat shock treatments. Observed induced expression of DnaN, involved in DNA synthesis process, may indicate its role in increased synthesis of some HSPs.

Osmotic stress adaptation The osmotolerance of L. monocytogenes is another property crucial to survival and growth of this pathogen at high salt levels and low water activity environments encountered in conserved food products. Osmotic stress adaptation in microorganisms depends on the modulation of both ionic and organic solute pools so as to sustain cytoplasmic water and turgor pressure at levels, which are compatible with cell viability and growth at low water activity (Booth and Louis, 1999; Wood, 2007).

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L. monocytogenes cells cope with elevated levels of osmolarity through appropriate changes in protein expression levels. Significant modulation (approximately 32 proteins) in protein expression under hyper osmotic conditions (3.5% to 6.5% NaCl concentration) was first visualized in preparative 2DE gels (Esvan et al., 2000) and some of the salt stress adaptation proteins were later identified (Duche et al., 2002a,b). Identified osmotic stress proteins include those related to general stress (Ctc and DnaK), transporters (GbuA and AppA), ribosomal proteins (RpsF, 30S ribosomal protein S6), as well as proteins involved in general metabolism processes (Ald, CcpA, CysK, TufA (EF-Tu), Gap, GuaB, PdhA, PdhD, and Pgm) (Duche et al., 2002a,b). Among the salt stress induced proteins, the role of Ctc in osmotolerance was further characterized by Gardan et al.

(2003b), who demonstrated that ctc gene is involved in L. monocytogenes osmotolerance. They found that growth of the ctc mutant strain was significantly impaired compared to its isogenic wild type L. monocytogenes LO28 strain in minimal medium with 3.5% NaCl. Other than the differential expression of salt stress proteins, increased uptake of glycine betaine and carnitine osmolytes via betL, gbu, and opuC encoded transporter porters is crucial under hyper-osmotic conditions. Accumulation of these osmolytes prevents the intracellular water loss by counteracting external osmolarity and keeping the macromolecular structure of the cells intact. Indeed, the induced expression (>2-fold) of GbuA transporter protein under high osmolarity (3.5% NaCl) has been observed in 2DE analysis of L. monocytogenes LO28 (Duche et al., 2002a). Meanwhile the induction of compatible solute transporter encoding genes, betL, gbu, and opuC in response to higher osmolarity has been reported at the transcriptional level in L. monocytogenes cells (Fraser et al., 2003). Interestingly these transporter systems expressed under hyper-osmotic stress conditions are the same as the ones expressed under cold stress (Mendum and Smith, 2002; Smith, 1996; Wemekamp-Kamphuis et al., 2004b), suggesting that some of the mechanisms counteracting osmotic and cold stress may be common in L. mono-

cytogenes. Moreover, the cold shock protein CspD also facilitates both osmotic and cold stress adaptation in L. monocytogenes and a mutant strain lacking cspD gene also display a stress sensitive phenotype under NaCl salt stress conditions (Schmid et al., 2009). Other important proteins in L. monocytogenes salt stress adaptation are HtrA (Wonderling et al., 2004) and Lmo 1078 (Chassaing and Auvray, 2007). The HtrA protein is a general stress response serine protease that contributes to osmotic stress adaptation functions through its role in degradation of salt stress damaged proteins. At the phenotypic level the L. monocytogenes htrA null mutant displays diminished growth in presence of NaCl stress. The Lmo1078 promotes both cold and osmotic tolerance based on its proposed functional contribution to maintenance of cell wall and membrane architectural integrity in this bacterium. The CstR transcriptional repressor protein is also involved in modulation of L. monocytogenes osmotic stress tolerance functions since a CstR null mutant of this bacterium displays improved growth under NaCl salt stress conditions (Nair et al., 2000b).

Acid stress adaptation The adaptation of microorganisms to acid stress environments includes significant gene and protein expression changes associated with, among other response, the mobilization of cellular mechanisms that consume acids and generate basic amines (Foster, 2004; Merrell and Camilli, 2002). L. monocytogenes cells face acid stress conditions in low pH foods and at various stages during human infection.

L. monocytogenes counteracts acidic stress conditions by production of various acid stress response proteins (ASPs). ASPs were initially designated based on their location on the preparative 2DE gels (Davis et al., 1996; Oâ&#x20AC;&#x2122;Driscoll et al., 1997) and some were later identified by PMF (Phan-Thanh and Mahouin, 1999; Wemekamp-Kamphuis et al., 2004a). Some of the identified ASPs include: proteins involved in respiration (enzyme dehydrogenases and reductases), osmolyte transport (GbuA), protein folding and repair (Chapronin, GroEL, ClpP), general stress re-

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sponse (sigma H homologous of B. subtilis), flagella synthesis (FlaA), and metabolism (Pfk, GalE) (PhanThanh and Mahouin, 1999; Wemekamp-Kamphuis et al., 2004a).

The acid tolerance response (ATR) is characterized by increased microbial cell resistance to lethal acid after an exposure to mild acidic condition (Koutsoumanis et al., 2004). This phenomenon has been examined by a number of studies in L. monocytogenes cells. When the synthesis of ASPs in L. monocytogenes LO28 produced under both mild (pH 5.5 for 2 h) and lethal (pH 3.5 for 15 min) acidic conditions were compared to a normal pH (~7.2), a total of 37 proteins were induced under mild acidic treatment and 47 under lethal acidic treatment, with 23 of the induced proteins being common under both conditions (Phan-Thanh and Mahouin, 1999).

The different aspects of acid stress adaptive mechanisms in L. monocytogenes are well elucidated from acid stress adaptation mechanisms studies in this bacterium (Abram et al., 2008a; Ferreira et al., 2003; Phan-Thanh and Jansch, 2006; Ryan et al., 2008b ). In brief, when exposed to a lower external pH, bacterial cells attempt to maintain their cytoplasmic pH by decreasing the membrane permeability to protons, buffering their cytoplasm, and by equilibrating the external pH through catabolism (Phan-Thanh and Jansch, 2006). One of the ways that limit the bacterial permeability to proton is through changes in the lipid bilayer of cell membrane. Giotis et al. (2007) suggested that there was an increased concentration of straight chain fatty acids and decreased concentration of branched chain fatty acids in L. monocytogenes 10403S cells grown under acidic conditions (pH 5.0 to 6.0) compared to neutral pH. Another important approach that the bacterial cells use for dispelling the protons outside the cells is to accelerate electron transferring reactions through enhanced oxidation reduction potential. The ASPs identified as dehydrogenases (GuaB, PduQ and lmo0560) and reductases (YcgT) together with respiratory enzymes are implicated to play an important role in maintaining pH homeostasis by active proton transport (Phan-Thanh and Jansch, 2006).

Organic acid salts such as sodium lactate and

sodium diacetate are extensively used in ready-toeat (RTE) meat products as antiListerial food preservatives. Recently Mbandi et al. (2007) used 2DE to evaluate the protein induction in L. monocytogenes Scott A by these organic salts. Experiments were conducted in defined medium with either sodium lactate (2.5%) or sodium diacetate (0.2%) or in combination. Some of the proteins that showed substantial up or down regulation (>10 fold) were identified using PMF. Oxidoreductase and lipoproteins were upregulated whereas DNA-binding proteins, alpha amylase and SecA were repressed during exposure to these organic acid salts. Identified enzyme protein oxidoreductase in L. monocytogenes has been previously suggested to be involved in dispelling proton molecules to maintain cell homeostasis (Phan-Thanh and Jansch, 2006).

The glutamate decarboxylase (GAD) and arginine deiminase (ADI) are well described major acid adaptive mechanisms in L. monocytogenes. L. monocytogenes LO28 strain with a mutation in genes of GAD proteins GadA, GadB and GadC displayed higher acid stress sensitivity in an acidified reconstituted skim milk background (Cotter et al., 2001b) and gastric fluid (Cotter et al., 2001a). The L. monocytogenes ADI system includes proteins ArcA, ArcB and ArcC and ArcD for the conversion and transfer of arginine into ornithine and deletion in functional genes of ADI leads impaired growth in mildly acidic conditions (pH 4.8) and survival in lethal pH conditions (pH 3.5) (Ryan et al., 2009).

Alkaline stress adaptation L. monocytogenes cells are more resistant to alkaline stress in comparison to other foodborne pathogens such as Salmonella Enteritidis and E. coli O157:H7 (Mendonca et al., 1994). At pH 12, L. monocytogenes F5069 (serotype 4b) cell concentrations decreased by only 1-log in 10 min compared to 8-log reductions observed for E. coli and S. Enteritidis within 15 s. Earlier, 2DE analysis of alkaline stressed (pH 10.0 for 35 min) L. monocytogenes EGDe cells by Phan-Thanh and Gormon (1997) showed induction of 16 proteins, synthesis of 11 novel proteins, and

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repression of nearly half of the total proteins in comparison to non-stressed cells. Recently, Giotis et al.

(2008) also reported the repression of a large number of proteins along with synthesis of 8 novel proteins in response to alkaline stress of L. monocytogenes 10403S strain. In addition to proteomic analysis, they also evaluated the alkaline stress adaptive mechanism using microarray transcriptional profiling and found 390 gene transcripts differentially expressed (Giotis et al., 2008). Protein identification of four differentially expressed proteins by peptide-mass mapping revealed induction of heat shock proteins DnaK and GroEL and repression of DdlA (alanine ligase) and AtpD (ATP synthase). These identified proteins spots were also found to be induced or repressed in microarray analysis. In addition, screening library of Tn917- lac insertional mutants in L. monocytogenes LO28 identified 12 mutants sensitive to alkaline conditions, though identification of transposition target suggest they all carried mutations in only putative transporter genes (Gardan et al., 2003a).

High hydrostatic pressure (HHP) stress adaptation L. monocytogenes cells undergo mechanical stress following HHP treatment. The usual pressure range employed in HHP is in the range of 200-600 MPa for 5-10 min depending on the food matrices.

Such high pressure damages the cell membrane and results in leakage of cell content along with dissociation of protein complexes (Gross and Jaenicke, 1994). However, HHP treated L. monocytogenes cells were found to be sublethally injured with their metabolic-activity largely maintained and had the potential for a gradual recovery (Ritz et al., 2006). In addition although L. monocytogenes cells in HHP treated cooked ham displayed a lag phase lasting up to 1.5 months, they subsequently recovered to grow more than 5-logs over 3 months (Aymerich et al., 2005).

To characterize the HHP induced proteins enabling resistance to mechanical stress, Jofre et al.

(2007) conducted 2DE analysis of L. monocytogenes CTC1011 (serotype 1/2c) after treatment with 400

MPa for 2 h and observed expression of 23 proteins being modulated. These high pressure induced proteins were related to ribosomal function (RplJ, RplL, RpsF, RpsB, IleS, GatA), transcription (GreA), protein degradation (PepF, PepT), protein folding (GroES), metabolism (PflB, Pta, Zwf, Ald,), general stress (Fri) and unknown functions. Of these high pressure induced proteins, chaperone GroES may be necessary in refolding of dissociated protein complexes following HHP treatment, and peptidases (PepF, PepT) may contribute to degradation of proteins that cannot be folded by molecular chaperones. Flp has been previously elucidated to have roles in cold, heat, and oxidative stress adaptation (Dussurget et al., 2005).

Moreover, L. monocytogenes shows increased resistant to HHP treatment following prior exposure to cold stress along with induced expression of cold shock proteins following HHP treatment.

Implications of L. monocytogenes stress adaptation to virulence responses The stress responses of L. monocytogenes are not only important in survival of hostile external and food-associated environments but also during host colonization processes. The pathogenicity of food-borne L. monocytogenes also depends on their physiological status at infection, which is determined by, among other factors, the environmental stress challenges encountered and stress responses activated prior to interaction with susceptible hosts.

Besides the fact that acid stress adaptation of this bacterium promotes survival in acidic food environments, this process has been also shown to modulate various aspects of virulence in this pathogen. As an example, the pathogenic potential of this bacterium can be increased through improved viability in the gastrointestinal tract, which includes increased survival of the gastric acid stress challenges. The increased expression of virulence genes as well as enhanced cell adhesion and invasion has been reported in association with acid stress adaptation of L. monocytogenes cells (Conte et al., 2000; Garner et al., 2006; Olsen et al., 2005; Werbrouck et al., 2009).

Conte et al. (2000) detected enhanced Caco-2 cell

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invasion ability, in addition to improved survival and proliferation in activated murine macrophages of L. monocytogenes cells preadapted by mild organic acid stress exposure. Werbrouck et al. (2009) also described increased cellular invasiveness and inlA mRNA levels in their analysis of acid stress adapted L. monocytogenes cells. In similar fashion there was an increased transcription of virulence genes such as prfA, inlA and inlB, as well as enhanced adhesion and invasion of Caco-2 cells in two L. monocytogenes strains adapted to prolonged acid stress (Olesen et al., 2009). Another stress commonly encountered by L. monocytogenes cells in food associated environments considered to potentially influence virulence of this bacterium is NaCl osmotic stress. NaCl stress exposure is associated with increased expression of various general stress resistance and virulence genes in this bacterium suggesting that osmotic stress adaptation events along the food supply chain may enhance subsequent pathogenicity (Kazmierczak et al., 2003; Olesen et al., 2009; Sue et al., 2004). Phenotypically increased cell adhesion and invasion in vitro has been observed in NaCl stress adapted L. monocytogenes cells (Garner et al., 2006; Olesen et al., 2009). The significance of these phenotypic observations however remains to be further examined. One study, which examined the growth of some food environment persistent strains and clinical isolates under NaCl osmotic stress, was not able to detect significant influence of this stress exposure on pathogenicity of these strains using several virulence models (Jensen et al., 2008). Similarly, Wałecka et al., (2011) did not find increased expression of internalins with salt stress and suggested that bacterial growth phase instead of salt stress was direct determinant of L. monocytogenes invasiveness. Hence the above mentioned reports determining the involvement of salt stress show conflicting findings and more work in this direction would be required to understand the factors that result in such differing view. The expression of prfA controlled virulence genes and cell invasion capacity of L. monocytogenes cells is temperature dependent and pathogenicity in some meat-processing plant derived strains of this bacterium was reported to decrease during

long term cold storage at 4°C (Duodu et al., 2010; Johansson et al., 2002; McGann et al., 2007). Similarly, cold stress exposed wild type and mutants lacking csp genes in the L. monocytogenes EGDe strain were significantly impaired in cell invasion relative to corresponding controls grown at 37°C (Loepfe et al., 2010). Temperature dependent virulence gene expression repression as well as membrane damage and cell surface modifications in these organisms exposed at low temperatures might lead to phenotypic virulence defects observed in cold adapted L. monocytogenes organisms.

Van de Velde et al. (2009) compared proteomes between L. monocytogenes cells grown in human THP-1 monocytes versus those growing extracellularly in TSB broth using 2D-DIGE. Down regulations of general stress protein Ctc and oxidative stress protein Sod was detected suggesting that compared to extra cellular environment the intracellular uptake by host cells may be more favorable environment for L. monocytogenes survival and adaptations. Shin et al. (2010) observed the increased σB activity, as measured by ß-galactosidase lacZ promoter assay, to vancomycin antibiotic stress. While subsequent proteome analysis of L. monocytogenes σB wild type and null mutant strains using LC-ESIMS/MS also revealed among other proteins the increased production of the virulence protein InlD. Fri protein is another general stress response protein with virulence promoting functions in L. monocytogenes. It has been shown by using both mice challenge and macrophage cell virulence models that fri null strains of L. monocytogenes are significantly impaired (Dussurget et al., 2005; Mohamed et al., 2006; Olsen et al., 2005). Proteome analysis of the fri mutant and wild type strain was compared to reveal repression in Hly (Listeriolysin O) and stress response proteins CcpA (Catabolite control protein A) and OsmC (Dussurget et al., 2005).

The stress induced chaperone proteins ClpB, ClpC, ClpE, ClpP have all been shown to provide virulence promoting activities in L. monocytogenes and thus it is possible that their induction in this bacterium in response to stress in food associated environments also increases the capacity of stress

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adapted organism to survive hostile host environments as well as enhance their pathogenicity (Chastanet et al., 2004; Gaillot et al., 2000; Nair et al., 1999; 2000a;). Meanwhile Olesen et al. (2009) in their recent study showed that acid exposed L. monocytogenes cells displaying increased Caco2 cell virulence also displayed increased expression of genes encoding the ClpC and ClpP. The RNA binding regulatory protein Hfq, is another general stress response modulating protein which has been shown to protects cells from osmotic and ethanol stress as well as facilitate enhanced pathogenicity in L. monocytogenes infected mice (Christiansen et al., 2004). Stack et al. (2005) found that the HrtA serine protease, which protects L. monocytogenes from various stresses including exposure to acidic conditions also contributes towards virulence capablities of this bacterium. The general stress response protein σB, which facilitates L. monocytogenes adaptation to multiple stresses has also been shown to promote virulence and cell invasiveness in this bacterium (Garner et al., 2006; Ivy et al., 2010). Recently it was shown that the importance of σB responses in these aspects might be lineage specific with its activity being important in lineage I, II, IIIB strains but not in IIIA (Oliver et al., 2010).

Role of alternative sigma factor (σB ) in L. monocytogenes stress adaptation In L. monocytogenes, σB is a major stress response regulator and mutant strain lacking σB activity shows increased sensitivity to a wide range of stresses including cold (Becker et al., 2000; Chan et al., 2007; 2008; Moorhead and Dykes, 2004; Raimann et al., 2009; Wemekamp-Kamphuis et al., 2004a; ), heat (Hu et al., 2007a,b; van der Veen et al., 2007), osmotic (Becker et al., 1998, Fraser et al., 2003; Okada et al 2008; Raimann et al., 2009), acid (Cotter et al., 2001a,b; Ryan et al., 2008a; Wemekamp-Kamphuis et al., 2004a), and HHP (Wemekamp-Kamphuis et al., 2004a). The main role of σB in L. monocytogenes is to regulate the expression of various stress response associated genes. As an

example, Flp is a general stress protein involved in cold, oxidative and heat stress adaptation. The expression of fri gene encoding Flp protein is partially regulated through σB-dependent pathways in L. monocytogenes 10403S (Chan et al., 2007).

To identify the proteins that show σB dependent expression in the acidic conditions, 2DE analysis of acid adapted (pH 4.5) and non-adapted cells (both wild type and σB mutant) was performed (Wemekamp-Kamphuis et al., 2004a). The expression of 9 proteins was dependent on σB during acid stress and some of these proteins were identified using PMF. The identified proteins with σB dependent expression in response to HHP stress included Pfk, GalE, ClpP, and Lmo1580. The Pfk (6-phosphofructokinase) and GalE are enzymes involved in glycolysis and sugar metabolism, respectively, and ClpP is the ATP-dependent chaperone protease that plays a role in preventing the accumulation of misfolded proteins. The induction of ClpP protein expression may be necessary in acidic conditions to help in resolution of protein aggregations that are likely to occur due to acid stress induced protein damage.

Recently, the role of σB regulon on L. monocytogenes 10403S cells grown to stationary phase in the presence or absence of 0.5 M NaCl was evaluated using both 2DE and iTRAQ (Abram et al., 2008b).

Using a combination of these two approaches a total of 38 proteins (17 induced and 21 repressed) were identified whose expression was σB dependent. Among these σB controlled proteins, 10 proteins (7 positively regulated and 3 negatively regulated by σB) were further classified based on their potential role in stress related functions. Of these 7 σB positively regulated proteins, two proteins OpuC and HtrA were previously conferred to have role in L. monocytogenes stress adaptation (Fraser et al., 2003; Wonderling et al., 2004). OpuC is involved in osmolyte transfer needed for osmotic and cold adaptation (Fraser et al., 2003) and HtrA serves as a protease whose deletion leads to growth defects under NaCL stress (Wonderling et al., 2004). Intracellular accumulation of glycine betaine and carnitine osmolytes is necessary in cold

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as well as osmotic stress. Expressions of the osmolyte transporter proteins, Gbu and Opu, have been shown to be at least partially dependent on the σB activity (Cetin et al., 2004; Fraser et al., 2003;). Also L. monocytogenes 10403S strain with a null mutation in the σB gene showed substantial defects in its ability to accumulate glycine betaine and carnitine osmolytes (Becker et al., 1998; 2000).

Moreover, σB deletion impairs the ability of L. monocytogenes 10403S cells to withstand against heat stress (55°C for 30-60 min) and class II heat shock genes, which also includes the osmolyte transporter gene opuC, are positively upregulated following heat shock (48°C for 3 min) in L. monocytogenes EGDe strain (Hu et al., 2007a; van der Veen et al., 2007). Using transcriptional analysis, Ryan et al. (2008a) reported the induction of the σB in response to sublethal levels of detergent stress.

In addition, following HHP treatment of 300 MPa of 20 min, the parent strain (EGDe) showed 100fold higher survival compared to σB mutant strain (Wemekamp-Kamphuis et al., 2004a).

Apart from σB, other sigma factors σc, σH, and L σ (RpoN) are also known to play important roles in stress adaptation of L. monocytogenes. L. monocytogenes strain lacking σB, σc, σH encoding proteins have been shown to have significantly impaired growth compared to wild type strain at 4°C for 12 days (Chan et al., 2008). Raimann et al. (2009) reported that L. monocytogenes strain lacking σL has impaired cold growth due to in part by the repressed transcript production of oligopeptide-binding OppA protein that facilitates accumulation of short peptide substrates which are also important for efficient cold growth in this bacterium (Borezee et al., 2000). Absence of σc increases the L. monocytogenes sensitivity to thermal treatment, thus highlighting the importance of this regulatory factor in conferring L. monocytogenes adaptation to heat stress (Zhang et al., 2005). σL aids in L. monocytogenes ability to grow at high salt concentrations (Okada et al., 2006) as well as control carbohydrate metabolism through its influence on expression of phosphotransferase system genes (Arous et al., 2004).

Conclusion and Future Perspectives The ability of L. monocytogenes cells to survive adverse physiological conditions is a serious food safety and public health concern. The physiological changes in response of environmental stress stimuli’s reflect key changes instituted by microbial cells at gene or protein expression levels. In the future an improved understanding of fundamental changes occurring at genes or proteins level in L. monocytogenes cells in response to adverse environmental conditions will provides new insights that can be harnessed in developing more effective practical food preservation approaches (Gandhi and Chikindas, 2007; Tasara and Stephan, 2006).

The physiological changes mounted in response to particular environmental stress stimuli in L. monocytogenes are a consequence of changes at gene transcription and/or protein expression levels. The cold adaptive nature of this organism is probably one of the most important concerns to food production due to the ability of this pathogen to grow and achieve high concentrations in long shelf life readyto-eat products preserved by refrigeration. Various cold adaptive mechanisms such as synthesis of conserved cold shock proteins (Schmid et al., 2009), increased uptake of cryoprotective osmolytes (Angelidis and Smith, 2003), increased membrane permiablity (Borezee et al., 2000), increased production of general stress proteins Fri (Dussurget et al., 2005), etc have been identified that may directly or indirectly confer this bacterium with an ability to multiply and/or survive at lower temperatures. However, at this stage it is unclear if these different mechanisms work in any coordinated manner or if they work on separate niches leading overall cold stress resistance of L. monocytogenes cells. Future experiments are warranted to understand the complex hierarchy between these different stress response mechanisms.

One way to do this would be to conduct gene knock out studies where the related genes/proteins of a particular stress adaptive mechanism (i.e. deletion of cold shock proteins) is deleted and use these strains to understand the modulations in genes/proteins of other stress mechanisms. The adaptation of this bac-

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terium to osmotic stress also involves complex sets of cellular responses. Notably some osmotic stress response mechanisms, such as compatible solute uptake systems as well as alternative sigma factors are also involved in cold stress adaptation (Fraser et al., 2003; Wemekamp-Kamphuis et al., 2004b), which suggests that some cellular response mechanism towards food related environmental (cold and osmotic) stresses in this bacterium are common.

The main limitation of current studies is that large numbers of genes/proteins are tabulated as being differentially expressed but there is little or no insight on what the modulations in these gene/ proteins mean. In any event, perturbation in physiology of living cells is likely to change the expression levels of various genes/proteins. Such information is of limited value without further functional characterizations of such putative stress adaptation genes or proteins. While it may not be practical to use such approach for hundreds of genes/proteins that are differentially expressed along with each stress, it is necessary to do follow-up studies on genes/proteins that exhibit substantially large changes in expression level. So far only in a few cases of stress proteins has the follow-up work been done in elucidating their molecular roles during stress adaptation of L. monocytogenes. Some key examples are: (a) Flp protein, first identified to be highly induced in cold and heat stress, and subsequently confirmed through fri mutant strain of L. monocytogenes EGDe, which is impaired under both stress conditions (Dussurget et al., 2005; Hebraud and Guzzo, 2000; Phan-Thanh and Gormon, 1995); (b) Ctc protein is induced under salt stress and L. monocytogenes LO28 ctc mutant strain is found defective in growth under NaCl stress conditions (Duche et al., 2002a; Gardan et al., 2003b); and (c) GbuA osmolyte transporter protein, induced under high osmolarity, (at 3.5% NaCl) was confirmed by gbu mutant strain of L. monocytogenes LTG59 as defective in growth in the absence of osmolyte uptake activity (Duche et al., 2002a; Mendum and Smith, 2002). Moreover most of the current stress adaptation findings are based on laboratory media and it is crucial that to design new experimental strategies that detect stress adaption response in L.

monocytogenes cells exposed to different food matrices. The experiments with food substrate may be designed to see how different food components and food preservatives modulate the expression of stress proteins identified using broth media.

Acknowledgement This research was supported in part by Food Safety Initiative award to RN by the Mississippi Agricultural and Forestry Experiment Station (MAFES), Mississippi State University.

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ing author. Author proofs should be read carefully and checked against the typed manuscript, because the responsibility for proofreading is with the author(s).

Corrections must be returned by e-mail. Changes sent by e-mail to the technical editor must indicate page, column, and line numbers for each correction to be made on the proof. Corrections can also be marked using “track changes” in Microsoft Word or using e-annotation tools for electronic proof correction in Adobe Acrobat to indicate necessary changes. Author alterations to proofs exceeding 5% of the original proof content will be charged to the author. All correspondence of proofs must be agreed to by the editorial office and the author within 48 hours or proof will be published as is and AFAB will assume no responsibility for errors that result in the final publication.

PUBLICATION CHARGES AFAB has two publication charge options: conventional page charges and rapid communication. The current charge for conventional publication is $25 per printed page in the journal. There is no additional charge for the publication of pages containing color images, micrographs or pictures. For authors who wish to have their papers processed as a rapid communication, authors will pay the rapid communication fee when proofs are returned to the editorial office in addition to twice the conventional page charges.

Charges for rapid communications are $1000 per manuscript for guaranteed peer review within one week and $100 per journal page.

HARD COPY OFFPRINTS If you are wishing to obtain a physical hard copy of the AFAB journal, offprints are available in any quantity at an additional charge: $100/page for black-white and $150/page for color prints. You may order your offprints at any time after publication on our website.

Scientific conference organizers may be expected to agree to a set number of offprints as a part of their agreement with AFAB.

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MANUSCRIPT CONTENT REQUIREMENTS Preparing the Manuscript File Manuscripts must be written in grammatically correct English. AFAB offers a fee based language service upon request (language@afabjournal.com).

Manuscripts should be typed double-spaced, with lines and pages numbered consecutively. All documents must be submitted in Microsoft Word (.doc or .docx, PC or Mac). All special characters (e.g., Greek, math, symbols) should be inserted using the symbols palette available in this font. Tables and figures should be placed in separate sections at the end of the manuscript (not placed in the text). Failure to follow these instructions will cause delays of the processing and review of the manuscript.

Title Page At the very top of the title page, include a title of not more than 100 characters. Format the title with the first letter of each word capitalized. No abbreviations should be used. Under the title, the authors names are listed. Use the author’s initials for both first and middle names with a period (full-stop) between initials (e.g., W. A. Afab). Underneath the authors, a list affiliations must be listed. Please use numerical superscripts after the author’s names to designate affiliation. If an authors address has changed since the research was completed, this new information must be designated as “Current address:”. The corresponding author should be indicated with an asterisk e.g., * Corresponding author. The title page shall include the name and full address of the corresponding author. Telephone and e-mail address must also be provided for the corresponding author, and emailaddresses must be provided for all authors.

at the beginning of the manuscript. In vivo, in vitro and bacterial names must be italicized (obligatory).

Authors must avoid single sentence paragraphs and merge such paragraphs appropriately. Authors must not begin sentences with “Figure or Table shows…” as these are inanimate objects and cannot “show” anything. When number are reported in text or in tables, always put a zero in front of decimal numbers: “0.10” instead of “.10”.

Manuscript Sections Abstract The abstract provides an abridged version of the manuscript. Please submit your abstract on a separate page after the title page. The abstract should provide a justification of your work, objectives, methods, results, discussion and implications of study or review findings . Your abstract must consist of complete sentences without references to other work or footnotes and must not exceed 250 words. On the same page as your abstract, please provide at least ten (10) keywords to be used for linking and indexing. Ideally, these keywords should include significant words from the title.

Introduction The introduction should clearly present the foundation of the manuscript topic and what makes the research or the review unique. The introduction should validate why this topic is important based on previously published literature, and the relevance of the current research. Overall goals and project objectives must be clearly stated in the final sentence of the last paragraphs of the introduction.

Materials and Methods Editing Author-derived abbreviations should be defined at first use in the abstract and again in the body of the manuscript. If abbreviations are extensive authors may need to provide a list of abbreviations

Information on equipment and chemicals used must include the full company name, city, and state (country if outside the United States or Province if in Canada) [i.e., (Model 123, ACME Inc., Afab, AR)].

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Variability, Replication, and Statistical Analysis To properly assess biological systems independent replication of experiments and quantification of variation among replicates is required by AFAB. Reviewers and/or editors may request additional statistical analysis depending on the nature of the data and it will be the responsibility of the authors to respond appropriately. Statistical methods commonly used in the bacteriology do not need to be described in detail, but an adequate description and/or appropriate references should be provided.

The statistical model and experimental unit must be designated when appropriate. The experimental unit is the smallest unit to which an individual treatment is imposed. For bacterial growth studies, the average of replicate tubes per single study per treatment is the experimental unit; therefore, individual studies must be replicated. Repeated time analyses of the same sample usually do not constitute independent experimental units. Measurements on the same experimental unit over time are also not independent and must not be considered as independent experimental units. For analysis of time effects, assess as a rate of change over time. Standard deviation refers to the variability in the biological response being measured and is presented as standard deviation or standard error according to the definitions described in statistical references or textbooks.

Results Results represent the presentation of data in words and all data should be described in same fashion. No discussion of literature is included in the results section.

Discussion The discussion section involves comparing the current data outcomes with previously published work in this area without repeating the text in the results section. Critical and in-depth dialogue is encouraged.

Results and Discussion Results and discussion can be under combined or separate headings.

Conclusions State conclusions (not a summary) briefly in one paragraph

Acknowledgments Acknowledgments of individuals should include institution, city, and state; city and country if not U.S.; and City or Province if in Canada. Copies being reviewed shall have authors’ institutions omitted to retain anonymity.

References a) Citing References In Text Authors of cited papers in the text are to be presented as follows: Adams and Harry (1992) or Smith and Jones (1990, 1992). If more than two authors of one article, the first author’s name is followed by the abbreviation et al. in italics. If the sentence structure requires that the authors’ names be included in parentheses, the proper format is (Adams and Harry, 1982; Harry, 1988a,b; Harry et al., 1993). Citations to a group of references should be listed first alphabetically then chronologically. Work that has not been submitted or accepted for publication shall be listed in the text as: “G.C. Jay (institution, city, and state, personal communication).” The author’s own unpublished work should be listed in the text as “(J. Adams, unpublished data).” Personal communications and unsubmitted unpublished data must not be included in the References section. Two or more publications by the same authors in the same year must be made distinct with lowercase letters after the year (2010a,b). Likewise when multiple author citations designated by et al. in the text have the same first author, then even if the other authors are different these references in the text and the references section must be identified by a letter. For example

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“(James et al., 2010a,b)” in text, refers to “James, Smith, and Elliot. 2010a” and “James, West, and Adams. 2010b” in the reference section.

Book Chapter: Author(s) of the chapter. Year. Title of the chapter. In: author(s) or editor(s). Title of the book. Edition or volume, if relevant. Publisher name, Place of publication.

b) Citing References In Reference Section In the References section, references are listed in alphabetical order by authors’ last names, and then chronologically. List only those references cited in the text. Manuscripts submitted for publication, accepted for publication or in press can be given in the reference section followed by the designation: “(submitted)”, “(accepted)’, or “(In Press), respectively. If the DOI number of unpublished references is available, you must give the number. The year of publication follows the authors’ names. All authors’ names must be included in the citation in the Reference section. Journals must be abbreviated. First and last page numbers must be provided. Sample references are given below. Consult recent issues of AFAB for examples not included in the following section.

Journal manuscript: Author(s). Year. Article title. Journal title [abbreviated].

Volume number:inclusive pages.

Examples: Chase, G. and L. Erlandsen. 1976. Evidence for a complex life cycle and endospore formation in the attached, filamentous, segmented bacterium from murine ileum. J. Bacteriol. 127:572-583.

Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van Doesburg, and A. J. M. Stams. 2009. A typical one-carbon metabolism of an acetogenic and hydrogenogenic Moorella thermioacetica strain.

Arch. Microbiol. 191:123-131.

Book: Examples: Author(s) [or editor(s)]. Year. Title. Edition or volume (if relevant). Publisher name, Place of publication. Number

Inclusive pages of chapter.

Examples: O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.

Assessing consumer concerns and perceptions of food safety risks and practices: Methodologies and outcomes. In: S. C. Ricke and F. T. Jones. Eds. Perspectives on Food Safety Issues of Food Animal Derived Foods. Univ. Arkansas Press, Fayetteville, AR. p 273-288.

Dissertation and thesis: Author. Date of degree. Title. Type of publication, such as Ph.D. Diss or M.S. thesis. Institution, Place of institution. Total number of pages.

Maciorowski, K. G. 2000. Rapid detection of Salmonella spp. and indicators of fecal contamination in animal feed. Ph.D. Diss. Texas A&M University, College Station, TX. Donalson, L. M. 2005. The in vivo and in vitro effect of a fructooligosacharide prebiotic combined with alfalfa molt diets on egg production and Salmonella in laying hens. M.S. thesis. Texas A&M University, College Station, TX. Van Loo, E. 2009. Consumer perception of ready-toeat deli foods and organic meat. M.S. thesis. University of Arkansas, Fayetteville, AR. 202 p.

Web sites, patents: Examples: Davis, C. 2010.

Salmonella. Medicinenet.com. http://www.medicinenet.com/salmonella /article. htm. Accessed July, 2010.

of pages.

Hungate, R. E. 1966. The rumen and its microbes.

Academic Press, Inc., New York, NY. 533 p.

Afab, F. 2010, Development of a novel process. U.S. Patent #_____

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Abstracts and Symposia Proceedings: Fischer, J. R. 2007. Building a prosperous future in which agriculture uses and produces energy efficiently and effectively. NABC report 19, Agricultural Biofuels: Tech., Sustainability, and Profitability. p.27 Musgrove, M. T., and M. E. Berrang. 2008. Presence of aerobic microorganisms, Enterobacteriaceae and Salmonella in the shell egg processing environment.

IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10) Vianna, M. E., H. P. Horz, G. Conrads. 2006. Options and risks by using diagnostic gene chips. Program and abstracts book , The 8th Biennieal Congress of the Anaerobe Society of the Americas. p. 86 (Abstr.)

Data Presentation in Tables and Figures Figures and tables to be published in AFAB must be constructed in such a fashion that they are able to “stand alone” in the published manuscript. This

means that the reader should be able to look at the figure or table independently of the rest of the manuscript and be able to comprehend the experimental approach sufficiently to interpret the data.

Consequently, all statistical analyses should be very carefully presented along with variation estimates and what constitutes an independent replication and the number of replicates used to calculate the averages presented in the table or figure.

Each table and figure must be on a separate page in the submitted paper. If your manuscript is accepted for publication, you will need to submit all data for charts, tables and figures in Excel spreadsheet format.

All figures should be clearly presented with well defined axis and units of measurement. Symbols, lines, and bars must be made distinct as “stand alone” black and white presentations. Stippling, dashed lines etc. are encouraged for multiple comparison but shades of gray are discouraged. Color images, micrographs, pictures are recommended and there is no additional fee for their submission.

AFAB Online Edition is Now Available!

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International Scientific Conference on Nutraceuticals and Functional Foods - FF 2 01 1 The next International Scientific Conference on Nutraceuticals and Functional Foods, Food and Function 2011, will be held during 25th-27th October 2011 in the university city of Kosice, Slovakia.

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