Combination Treatment of Sugarcane (Sacccharum Officinarum) Stem for Microbial Safety nd Inhibit

International Journal of Bio-Technology and Research (IJBTR) ISSN (P): 2249-6858; ISSN (E): 2249-796X Vol. 10 Issue 1, Jun 2020, 63–78 © TJPRC Pvt. Ltd.

COMBINATION TREATMENT OF SUGARCANE (SACCCHARUM OFFICINARUM) STEM FOR MICROBIAL SAFETY AND INHIBITION OF POST EXTRACTION ENZYMATIC BROWNING IN FRESH JUICE BIBHUTI BHUSAN MISHRA1* & SATYENDRA GAUTAM2 1

Group Leader, Agroproduce & Beverage Technology Group

Food Flavour & Aroma Chemistry Section, Food Technology Division, B.A.R.C., Mumbai, India 2

Food Technology Division, Bhabha Atomic Research Centre, Mumbai, India

ABSTRACT Freshly extracted sugarcane juice is a highly nutritious, energy rich natural beverage, liked in most parts of the world.

However, presence of high microbial load on the stem may lead to contaminated juice, with risk of ill health upon consumption. Freshly extracted sugarcane juice is also having problem of rapid browning discoloration due high phenolic content and oxidative enzymatic activity. In the current study, chlorination (0.02-1%), hot water blanching (40-

kHz) processing and their combinations were evaluated for decontamination efficacy of stem. Here, we show that a multistep sequential dip treatment including chlorination (sodium hypochlorite 0.02%; 60°C, 10 min), hot water blanching (60°C,10 min), and ozone treatment (400 mg/h, 20 min) resulted microbial safety of juice and retained sensory quality. It improved extractability of soluble sugars, polyphenol content, flavonoids leading to enhanced antioxidant activity and overall nutritional value of juice. The treatment also inhibited post-extraction enzymatic browning of juice (by 59%) due to inactivation of enzymes such as polyphenol oxidase (PPO), peroxidase (POD), and phenylalanine

Original Article

80°C), ozonation (10-40 min), UV-C (254 nm, intensity, 125 µW sec/cm2) treatment, ultrasonication (frequency, 33 ± 3

ammonia lyase (PAL).

KEYWORDS: Antioxidant Activity; Enzyme Activity; Microbial Analysis; Juice Quality & Sugarcane

Received: Dec 17 2019; Accepted: Jan 07, 2020; Published: Feb 03, 2020; Paper Id.: IJBTRJUN20206

1 INTRODUCTION Sugarcane (Saccharum officinarum L.) is one of the important commercial crops and the major sources of sugar crystals, jaggery and molasses. Its major center of production is Brazil followed by India (Mondal, 2014).

Sugarcane juice is freshly extracted and consumed in many parts of the world as a highly nutritious and popular sweet beverage (Eggleston, 2002). Surface microbial contaminants on the stem, growth of bacteria, yeast and mold on cracked part of the stem, and high sugar content are the major cause of microbial load in the extracted juice.

Extraction and sale of the juice in open environment by street vendors further aggravate the safety problem(Yusof et al., 2000; Singh et al., 2002; Good burn and Wallace, 2013). Hence, hygienization of the sugarcane stem prior to extracting the juice is very much important. The issue can be addressed in two ways. First, to hygienize the sugarcane stem and store in proper condition and extract the juice when needed for fresh consumption. Another approach is, to extract the juice from hygienized stem and store in proper condition for consumption. Most of the studies addressed the concern by second approachi.e. extracting the juice from hygeinized stem and storing it (Eggleston, 2002). However, juice being liquid and rich in nutrients, spoilage is faster due to microbial fermentation www.tjprc.org

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and biochemical changes (Yusof et al., 2000). In the current study, first approach was followed, where different physico-chemical treatments and their combinations were evaluated for their potential to hygienize sugarcane stem. In current study, many processes were explored, which included chlorination, blanching, ozonation, sonication, UV treatment and their combinations. These methods individually have earlier shown promise in achieving microbialde contamination of different food samples (US FDA, 2004; Erkan et al., 2008; Rastogi, 2011; Horvitz and Cantalejo, 2012). Further, the effect of optimized combination process was evaluated on the organoleptic characteristics, enzymatic browning, and nutritional qualities of juice which included estimation of total sugars, phenolics, flavonoids, and in vitro antioxidant capacity.

2. MATERIALS AND METHODS 2.1 Physical and Chemical Treatments The sugarcane stems were procured from local market and were cleaned using potable water. The cut stem was subjected to different treatments (physical, chemical, or their combinations). The treatments included blanching at various temperatures and time, chlorination using sodium hypochlorite at different temperatures. The stem water ratio for blanching and chlorination was kept at 20:80. The UV treatment (30 watt lamp, G30T8, Sankyo Denki, Japan; 254 nm, intensity, 125 µW sec/cm2) was performed in a laminar air flow cabinet for different durations. The ozonation(Ozonator, Pure Natural Healthcare Pvt. Ltd., New Delhi, India) carried out in potable water (400 mg/h)for different time period. The sonication (Microclean-103, Oscar ultrasonic cleaner Pvt. Ltd., Mumbai, India; power 120 W) was undertaken in potable water(frequency, 33 ± 3 kHz) for different time period. The treatment specific conditions such as temperature, concentration, and duration have been detailed in results and discussion and corresponding Tables.

2.2 Microbiological Analysis It was performed as per the standard methods detailed earlier for sugarcane (Mishra et al., 2011). Total bacterial load of juice was determined by serial dilution using sterile saline (0.85%) and pour plating on plate count agar (Himedia, India)plates, which were incubated at 30ºC for 36 h. The yeast and mold count (YMC) was determined similarly using potato dextrose agar (PDA, Himedia, India) (pH 3.5) plates and incubating at 28ºC for 36h. Presumptive coliform (PC) was determined on violet red bile agar (VRBA, Himedia, India) plates by incubating at 37ºC for 24 pathogenic Staphylococcus(coagulase positive) was enumerated on Baird- Parker agar (Himedia, India) plates supplemented with egg yolk tellurite emulsion by incubating at 37 ºC for 24 h.

2.3 Biochemical Analysis The sugarcane juice was extracted using crusher (Model J-10, Akshar Engineering, Rajkot, India) and clarified at 6,000 x g for 10 minat 15°C. The supernatant termed as “Clear Juice Supernatant (CSJ)” and was used for all biochemical assays.

2.3.1 Total Soluble Solids and pH TSS (Brix) was measured using a hand-held refractometer (Model Master- α, Atago, Japan) which gave values in degrees.

The pH of the juice was measured using a pH meter(Elico pH analyzer, ElicoPvt Ltd., Mumbai, India).

2.3.2 Estimation of Total and Reducing Sugar Content The total and reducing sugar content was estimated by DNSA (3, 5-Dinitrosalicylic acid) method (Mishra et al., 2011). For total and reducing sugar estimation, the CSJ(25 µl) was acid hydrolyzed using 2NHCl (17 µl) at 68ºC in a water bath for 10 min. The reaction mixture was cooled to ambient temperature and neutralized with 5NNaOH. Its aliquot(25 µl) was Impact Factor (JCC): 5.9084

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diluted to 1 ml with milli Q water, mixed with DNSA reagent (1 ml), and incubated at 100ºC for 10 min in a water bath.

The absorbance was measured at 540 nm (Spectrophotometer, ModelUV4, Unicam, and Cambridge, UK). The sugar content was calculated from slope of the curve of standard glucose (Himedia, India) and expressed as mg/ml of juice.

2.3.3 Estimation of Sucrose using HPLC The sucrose was quantified in CSJ using a HPLC (Waters Corporation, MA, USA) system equipped with dual gradient pump (model 515), auto sampler (model 717 plus), refractive index (RI) detector (model 2410) and analytical amino (NH2) column (50376, Lichrosorb,250 x 4.6 mm, particle size 5 µm, pore size 0.01µm,). The CSJ was diluted (1:100), membrane filtered (0.22µm) and loaded using auto sampler. Isocratic elution was performed with 80% acetonitrile (in milliQ water) at flow rate of 0.9 ml/min for 9 min. The sucrose content was calculated from slope of the curve of standard sucrose (Himedia, India) and expressed as mg/ml of juice.

2.3.4 Estimation of Total Soluble phenolics (TSP) and Flavonoids Contents The TSP content was determined by Folin Ciocalteu reagent as detailed earlier (Mishra et al., 2011) using20µl of CSJ. Gallic acid (Himedia, India) was used as a standard and the TSP content was expressed as mg GAE/ml. The flavonoid content was determined by aluminum chloride colorimetric method using 250µl of CSJ (Mishra et al., 2011). The catech in(Sigma-Aldrich) was used as standard and the flavonoids content was expressed as catech in equivalents (mg CE/ml).

2.4 Antioxidant Capacity: DPPH and ABTS Radical Scavenging Activities The DPPH (2, 2-diphenyl-1-picrylhydrazyl) radical scavenging activity was determined as described earlier (Mishra et al., 2011). CSJ (0.1 ml) was diluted to 0.5 ml with ethanol (70%), mixed with 0.5 ml of DPPH solution (0.2 mM), and incubated at ambient temperature for 15 min. The decrease in the absorbance (at 517 nm)due to addition of juice was calculated and expressed as percentage DPPH radical scavenging activity.

The ABTS [2, 2′-azino-bis-(3-ethylbenzothiazoline-6- sulfonic acid)] radical scavenging assay was performed as described by Re et al. (1999). An aliquot (950 µl) of fresh ABTS solution prepared by mixing ammonium per sulfate (4.9 mM, 5 ml) and ABTS (14 mM, 5 ml), was mixed with 50 µl of CSJ, incubated for 1 min and absorbance was measured at 734 nm. The decrease in the absorbance due to juice was expressed as percentage ABTS radical scavenging activity.

2.5 Enzyme Activities 2.5.1 Polyphenol Oxidase (PPO) Activity The PPO activity in CSJ was determined using 4-methylcatechol as substrate, where PPO catalyzed oxidation of 4-methylcatechol was measured at 410 nm as described earlier (Mishra et al., 2012). The assay mixture consisted of 0.1M phosphate buffer (pH 6.8), 0.1 ml 4-methylcatechol (0.05 mM) and 0.04 ml of CSJ. 2.5.2 Peroxidase (POD) Activity It is based on the oxidation of guaiacol to tetraguaiacol polymer, catalyzed by POD which was measured at 470 nm as described earlier (Mishra et al., 2012). The assay mixture consisted of 0.8 ml of phosphate buffer (0.05 M, pH 6.8), 0.05 ml of H2O2 (30%), 0.05ml of guaiacol(30%), and the CSJ (0.025 ml).

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2.5.3 Phenylalanine-Ammonia lyase (PAL) Activity The PAL activity of juice was determined based on the principle, where the enzyme catalyzed deamination of L-phenylalanine yields trans-cinnamic acid i.e. measured at 290 nmas described earlier (Mishra et al., 2012). The assay mixture consisted of 0.9 ml sodium borate buffer (0.1M,pH 8.8), 0.1 ml of L-phenylalanine (0.1M) and 0.05ml of CSJ. For all these assays, one unit of activity was measured as0.01 change in absorbance/min, measured up to 4 min and averaged. Protein content of the CSJ was determined by the method using bovine serum albumin (BSA; Sigma Chemical, St. Louis, USA) as a standard.

2.6 Organoleptic Evaluation It was performed by 20 panelists in a Taste Panel Laboratory, in individually partitioned compartments under a controlledenvironment condition. The juice extracted from fresh (unprocessed)as well as processed sugarcane stem was evaluated by the panelists for quality attributes such as appearance, color, aroma, taste, and overall acceptance on a 9-point hedonic scale (Mishra et al., 2011).

2.8 Statistical Analysis All experiments including microbial, biochemical, enzymatic and organoleptic analysis were repeated three times (each time with three replicates). The means and standard deviations (SD) were calculated based upon all these readings and statistically analyzed using Bio Stat 2009 (5.8.0.0, Analyst Soft Inc., Canada). Two way ANOVA, using Tukey test were performed to analyze the data (P≤0.05).

3 RESULTS AND DISCUSSIONS 3.1 Potable Water Wash was not Enough for Hygiene of Sugarcane Stem Although potable water wash is the only treatment routinely used for hygienisation of the sugarcane stem before extracting the juice for fresh consumption, it was not found to significantly improve the hygiene of the extracted juice. The presumptive coliform (PC) and Staphylococcus count (SC)were still in the range of 2 log cfu/ml (Figure 1) which is quite similar to control uncleaned sample (2.4 log cfu/ml). In general, complete elimination in pathogen counts below detectable level is recommended upon processing to ensure the safety of juices (US FDA, 2004).The TBC and YMCin control samples were 4.1 and 3.9 log cfu/ml, which after 20 min of potable water washing reduced to 3.5 and 2.2 log cfu/ml, respectively (Table S1). Other earlier reports too have shown the inability of potable water wash in drastically reducing the microbial and pathogen load in agricultural produces (Kumar et al., 2015). Further, increasing the duration of washing from 20 to 80 min did not eliminate the PC and SC (Table S1), although some reduction in total microbial load was observed.

The findings thus indicate that in case of sugarcane stem potable water wash does not provide a solution to achieve hygienisation in the extracted juice although it is the only standard practice particularly being followed in the developing countries.

3.1.1 Chlorination at Permissible Level could not Hygienize Sugarcane Chlorination with sodium hypochlorite has been approved at the concentration of 200 ppm or below for hygienisation of agricultural produce. At this concentration chlorination of sugarcane stem at ambient temperature (28 ±2°C) was not found to be very effective. Even increasing the treatment duration for 80 min could not eliminate the pathogens (PC and SC). The Impact Factor (JCC): 5.9084

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presumptive pathogens were detectable (<0.1 log cfu/ml) in juice (Table S1), and hence considered unacceptable because they can cause illness even if present in small numbers. However, the treatment could reduce the TBC and YMC significantly to 1.6 and 0.5 log cfu/ml, respectively (Table S1). To increase the efficiency of chlorination, the treatment was performed at high temperature due to possible synergistic effect. In certain earlier studies too, hypochlorite treatment has been found to be highly effective at higher temperature (Gil et al., 2009; Kumar et al., 2015).At 50 (±2) °C, the effect of chlorination (200 ppm) on TBC and YMC was found to be insignificant. However, at 60 (±2) °C the TBC and YMC were reduced to 1.5 and 1.6 log cfu/ml, respectively. The chlorination at further high temperature ≥70 ±2°C) reduced the microbial count including coliforms to below detection limit.

3.0 a

Counts (log cfu/ml)

2.5

a Presum ptive coliforms Staphylococcus spp

2.0 1.5 1.0 b

0.5

b

0.0 Control

T1

* *

* *

T1+T2

T1+T2+T3

Treatm ents (a)

(b) Figure 1: The Effect of Combination Treatment of Sugarcane Stem on (A) Pathogenic Load in Freshly Extracted Juice. Control – Potable Water Wash; T1- (0.02% Sodium Hypochlorite 60°C 10 Min); T2- Blanching (60°C, 10 Min); T3- Ozonation (20 Min). * The Pathogen Counts were Below Detectable Level (10 Cfu/Ml). The Columns with Different Letter Superscript Indicated Statistically Significant Difference in the Mean (P ≤0.05). (B) Scheme of Processing Steps for Effective Hygienisation Treatment of Sugarcane Stem for Juice Extraction.

(Table S2). However, such high temperature affected the sensory qualities, induced browning in the extracted juice, and also could induce unwanted jaggery flavor in sugarcane juice (Mishra et al., 2011). Besides, extreme high temperature treatment couldinduce HMF formation as reported in case of many other foods (Singh et al., 2002).

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3.2 UV-C Treatment too did not Eliminate Presumptive Pathogens The exposure of sugarcane stem to 3.0 kJ m-2UV treatments reduced the TBCand YMC to2.7 and 0.8 log cfu/ml, respectively (Table 1). Further exposure of 7.5 kJ m-2UV treatment could reduce the TBC and YMC significantly to1.1 and 0.5 log cfu/ml, respectively. However, even after 7.5 kJ m-2UV treatment the presumptive coliform and Staphylococcus count were not entirely eliminated and the counts were found to be in the range of 0.4 - 0.7 log cfu/ml (Table 1), which restricts its acceptability (>0.1 log cfu/ml). Park and Kim (2015) have showed that 2kJm−2UV-C resulted partial reduction in microbial load in garlic. Fonseca and Rushing (2006) have shown similar results with 4.1kJm−2UV-C in fresh cut water melon. Short wavelength (254 nm) ultraviolet (UV) is well known for its efficiency to kill micro organisms, and hence quite frequently used in a variety of applications for hygienisation of food, air, and water (Erkan et al., 2008). However, in this study it was not found to be sufficient for microbial decontamination of sugarcane stem.

3.3 Ultrasonication Treatment was also not Effective for Sugarcane The ultrasonication treatment (frequency, 33 ± 3 kHz) for 10 min could reduce the TBC and YMC in sugarcane stem only to2.5 and 1.6 log cfu/ml, respectively (P ≤0.05). Further increase in treatment period up to 50 min resulted in reduction of TBC and YMC to 1.0 and 0.5 log cfu/ml, respectively (Table 1). However, even after 50 min of treatment the presumptive coliform and staphylococcus count could not be eliminated completely (Table 1). Ultrasonication works on the principle, where powerful generator produced ultrasonic oscillations were converted into mechanical vibrations. This produced intense scrubbing action and pulls out microbial contaminants from food items dipped for cleaning (Rastogi, 2011). Similar to the earlier findings in strawberry (Cao et al., 2010), ultrasonication lowered microbial count in sugarcane stem, but not eliminated the pathogenic contaminations.

3.4 Ozonation could not Eliminate Microbial Load In earlier studies, ozone has been reported to be a strong antimicrobial agent with high reactivity and penetrability, which spontaneously decompose toa non-toxic product (Sharma and Hudson, 2008). Hence, it was checked for its efficiency to decontaminate sugarcane stem. The ozonationtreatment (400 mg/h) for 20 min reduced the TBC and YMCto 3.4 and 1.7 log cfu/ml, respectively(P ≤0.05) (Table 1). Further increase in treatment period up to 40 min resulted in further reduction in TBC and YMC, respectively. The coliform and staphylococcus count also similarly decreased to 0.4 and 0.6 log cfu/ml, respectively. In earlier studies, researchers have shown that treatment with ozone appears to have a beneficial effect in extending the storage life of fresh cut lettuce and red bell peppers due to its antimicrobial action (Rico et al., 2006; Horvitzand Cantalejo, 2012). However, similar to current findings, Fonseca and Rushing(2006) also have shown unsatisfactorily results with ozone for decontamination of fresh cut water melon.

3.5 A Combination of Chlorination, Blanching, and Ozonation as an Effective Hygienisation Treatment Since some of the commonly used individual treatment was not found to be effective in achieving desired hygienisation where load of pathogenic microorganism should get reduced to undetectable level, combination treatments were explored.

While selecting a combination, minimum requirement of combination was set as a criterion to make the process cost effective during

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Table 1: Comparative Evaluation of UV Radiation, Sonication and Ozonation Treatments of the Sugarcane Stem on Microbial Quality of Extracted Juice.

SC (log Treatment Types Time (min) TBC (log cfu/ml) YMC (log cfu/ml) PC (log cfu/ml) cfu/ml) Unprocessed 0 4.2a ±0.4 2.8a ±0.3 1.8a ±0.4 1.9a ±0.3 UV 0.75 10 3.7a ±0.3 1.4a ±0.3 2.3a ±0.4 1.5a ±0.2 (radiant energy 3.0 40 2.7b ±0.4 0.8b ±0.1 1.5b ±0.2 0.7b ±0.1 b b b density, 4.5 60 2.5 ±0.3 0.7 ±0.2 1.4 ±0.3 0.6b ±0.2 -2 c b b kJ m ) 6.0 90 1.6 ±0.4 0.6 ±0.1 1.4 ±0.2 0.4b ±0.3 c b c 7.5 120 1.1 ±0.3 0.5 ±0.3 0.7 ±0.3 0.4b ±0.2 b b a 10 2.5 ±0.4 1.6 ±0.4 1.5 ±0.4 1.6a ±0.2 Sonication b b a 20 2.2 ±0.5 1.4 ±0.3 1.4 ±0.3 1.5a ±0.3 (Frequency, 33 ± 3 c c b 40 1.2 ±0.2 0.7 ±0.3 0.6 ±0.1 0.7b ±0.2 kHz) c c b 50 1.0 ±0.1 0.5 ±0.3 0.4 ±0.2 0.4b ±0.3 a a a 10 3.8 ±0.2 2.4 ±0.3 1.6 ±0.3 1.7a ±0.4 a b a 20 3.4 ±0.4 1.7 ±0.2 1.2 ±0.4 1.6a±0.4 Ozonation (400 b b b 30 2.5 ±0.3 1.5 ±0.3 0.7 ±0.1 0.8b ±0.2 mg/h) 40 2.2b ±0.4 1.3b ±0.3 0.4b ±0.3 0.6b ±0.2 d c Multistep Comb 1* 0.5 ±0.2 0.4 ±0.2 ND ND Multistep Comb 2# ND ND ND ND NaClO- Sodium hypochlorite *Combination 1: Chlorination (0.02% NaClO, 60°C, 10 min) + Blanching (60°C, 10 min) #

Combination 2: Chlorination (0.02% NaClO, 60°C, 10 min) + Blanching (60°C, 10 min) + Ozonation (20 min).

TBC- Total bacterial count, YMC - Yeast and mold count, PC - presumptive coliform, SC – Staphylococcus count, ND- not detectable.The values in columns with different superscript indicated statistically significant difference in the mean (P ≤0.05).

Actual application. Different trials (data not shown) resulted in a combination comprising of potable water wash, chlorination, blanching (60°C, 10 min) and ozonation (20 min) which resulted in effective hygienisation. Amongst the treatments studied, washing of sugarcane stem with hypochlorite (0.02%) at temperature of 60°C for 10 min (T1) could reduce the bacterial and yeast mold count to 1.5 and 1.6 log cycles, respectively (Table S2).

Sodium hypochlorite treatment is most effective within 10 min and hence its use was restricted to this time point only(Kumar et al., 2015). When the sodium hypochlorite washed samples were further subjected to hot water blanching for additional 10 min (T2) the coliform and Staphylococcus count reduced to below detectable level (Figure 1). When ozonation was included in the combination for 20 min (T3), the total microbial load reduced to below detection level.

Although complete removal of total microbial load is not needed in regular application, it is essentially required if food has to be served to the patients with weak immunity. Besides being helpful in reducing microbial load, ozonation also helped in controlling post extraction browning of sugarcane juice (Figure 2a and b). The combination of these three treatments {chlorination (T1), hot water blanching (T2) and ozonation (T3)} did not affect the extractability and yield of juice. The average content of juice was found to be in the range of 52±4 to 58±5 ml/100g of stem in the unprocessed samples, which remained unchanged in processed samples.Similarly, the total soluble solids (TSS) and pH also remained unaffected and was found to be in the range of 17±2 brix and 5.6±0.3, respectively.

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3.6 Inhibition of PPO, POD and PAL Enzyme Activities in Processed Stem and Reduced Browning in Extracted Juice The browning, which deteriorates the quality of extracted juice was found to be inhibited by the optimized treatment of the stem (Figure 2). The chlorination (T1), blanching (T2), and ozonation (T3) have reduced browning by 18, 16, and 31%, respectively. However, the combination of chlorination, blanching, and ozonation was found to be more effective and reduced browning by 59%. Compared to the individual treatments the significant inhibition by the combination could have been due to the synergistic effect. The browning inhibition was found to be due to inactivation of browning enzymes such as PPO, POD and PAL (Figure 3). In litchi fruit, similar reduction in enzyme activity due to chlorination was reported earlier (Kumar et al., 2012). In earlier studies too, ozonation have reduced PPO and PAL activities in case of lettuce (Rico et al., 2006). The chlorination treatment significantly lowered the PPO and PAL activity (Figure 3).

The kinetics of browning in extracted juice from combination treated stem was compared with that of untreated control. The browning was not found to increase in combination treated samples, whereas, the others showed significant increase in discoloration (Figure S1).

3.7 Shelf Life of Hygienized Stem Affected by Storage Temperature The aseptically processed and LDPE (low density polyethylene), packed sugarcane stem can be stored for 9 days at low temperature of 4±2 ºC, whereas, at 10±2ºC and ambient temperature (26±2ºC) the storage life is 5 and 1day, respectively (Figure S2). During this storage period the juice extracted from the processed stem was found to be free from bacterial, yeast mold, coliform, and staphylococcus counts. The shelf life of combination treated sugarcane stem could not be extended beyond this period as significant reduction in juice yield (<45 ±5 ml/100g) was observed due to dehydration. The browning index of the extracted juice was found to be higher (>22 ±4) due to extensive discoloration in the extracted juice, which made it unacceptable. Also, gradual visible mold growth was observed during further extended storage period beyond 10 days.

In developing countries, where use of freshly extracted sugarcane juice from street side venders is a common practice, particularly in tropics, could be the cause of associated disease outbreaks, which is usually not reported due to poor documentation of theincidence. Hence, health conscious individuals generally avoid consumption of sugarcane juice extracted in street side stalls (US FDA, 2004).

(a)

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Combination Treatment of Sugarcane (Sacccharum Officinarum) Stem for Microbial Safety and Inhibition of Post Extraction Enzymatic Browning in Fresh Juice

Browning index (100-L)

50

71

a b

b

40

c

c

30

d

20 10 0 Control

T1

T2

T3

T+T2 T1+T2+T3

Treatments

(b) Figure 2: The Effect of Combination Treatment of Sugarcane Stem on Inhibition of Browning Discoloration in Freshly Extracted Juice (a) and its Colorimetric Estimation (b).

Control – Non Processed Stem; T1- (0.02% Sodium Hypochlorite 60°C 10 min); T2Blanching (60°C 10 min); T3- Ozonation (20 min). The Columns with Different Letter Superscript Indicated Statistically Significant Difference in the Mean (P ≤0.05).

850

a

a

a

PPO POD PAL

750 (Units/mg protein)

Specific enzyme activities

800

b

700

b

650 600

c

550 500 150 100 50 0

d

e

Control

e

e

T1

e

T2

e

e

T3

Treatments

e

f

T1+T2

f

g

h

T1+T2 +T3

Figure 3: The Effect of Combination Treatments of the Sugarcane Stem on Polyphenol Oxidase (PPO), Peroxidase (POD), and Phenylalanine Ammonia lyase (PAL) Specific Enzyme Activities of Extracted Juice. T1- (0.02% Sodium Hypochlorite 60°C 10 min); T2- Blanching (60°C 10 min); T3- Ozonation (20 min).

The Columns with Different Superscript Indicated Statistically Significant Difference in the Mean (P ≤0.05).

3.8. The Combination Treatments Resulted in Increased Extractability of Nutrients The total and reducing sugar contents in the juice was found to be approximately 158 and 3.2 mg/ml, respectively (Table 2). The HPLC determination showed that total sugar mainly included sucrose, which was found to be 152 ±9 mg/ml. The optimized combination treatment (chlorination, blanching and ozonation) resulted in significant increase in total (0.24 fold) and reducing (0.46 fold) sugars (Table 2). The chlorination at high temperature followed by blanching also resulted in 0.22 fold increase in total sugar content(P ≤0.05). This could be due to higher temperature mediated hydrolysis or conversion of residual starch to simple sugars through residual enzyme hydrolytic activitiesin the extracted juice.

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Similar findings have been reported earlier in case of kiwiand melon fruit (Aguayo et al., 2008; Shahkoomahally and Ramezanian, 2013). The sucrose content (estimated using HPLC, Figure S3) in samples treated by chlorination and blanching was found to be 171±10 and 178±12 mg/ml, respectively. The combination treatment resulted in increase in sucrose content to 186 ±9 mg/ml, respectively (data not shown). However, the ozonation treatment alone was not found to have any significant effect on the extractability of total soluble sugar (Table 2).

The combination treatment resulted in significant increase (P ≤0.05) in the total soluble phenolics content to 1.26 ±0.02 mg/ml, which initially was 0.89±0.06 mg/ml, probably due to increased extractability of phenolics (Table 2). Such observation has been observed earlier in case of sweet corn (Kumar et al., 2015). It was reported earlier that hot water treatment changes the microstructure of tissue resulting in easier extractability of nutrients (Pedreschi et al., 2011).

Amongst the combination, chlorination and blanching were found to be the primary contributor to the observed increase in total soluble phenolics. The ozonation treatment alone for 20 min did not significantly affect the soluble phenolics content (P ≤0.05) (Table 2). In case of iceberg lettuce upon ozonation and chlorination, similar observation was earlier noticed (Baur et al, 2004).

Table 2: The Effect of Optimized Combination Treatment of the Sugarcane Stem on Biochemical and Antioxidant Properties of Extracted Juice Total Sugar (mg/ml)

Reducing Sugar (mg/ml)

Phenolics (mg/ml)

Non-treated control

158b ±10

3.2b ±0.12

0.89b ±0.06

T1- Chlorination (NaClO, 0.02%) at high temperature (60°C, 10 min) T2- Blanching in potable water (60°C, 10 min) T3- Ozonation (20 min) in potable water

185a ±11

4.4a ±0.16

1.23a ±0.03

189a ±13

4.5a ±0.11

1.25a ±0.06

147b ±10

3.6b ±0.12

0.83b ±0.05

(T1 +T2) Multistep Combination 1*

193a ±12

4.6a ±0.13

1.22a ±0.03

(T1 + T2 +T3) Multistep Combination 2 # The values represent mean ±SD. NaClO- Sodium hypochlorite

196a±10

4.7a ±0.17

1.26a ±0.02

Treatment Condition

Flavonoids (mg/ml)

0.17b ±0.02 0.26a ±0.01 0.29a ±0.02 0.14b ±0.02 0.30a ±0.02 0.27a ±0.03

DPPH Scavenging Activity (%)

ABTS Scavenging Activity (%)

74b ±5

53b ±4

86a ±5

63a ±3

89a ±5

65a ±3

73b ±6

57b ±2

88a ±3

65a ±3

90a ±4

68a ±4

*Multistep Combination 1: Chlorination (0.02% NaClO, 60°C, 10 min) + Blanching (60°C, 10 min) #

Multistep Combination 2: Chlorination (0.02% NaClO, 60°C, 10 min) + Blanching (60°C, 10 min) +

Ozonation (20 min).

The values in columns with different superscript indicated statistically significant difference (P ≤0.05) The flavonoid content in the freshly extracted sugarcane juice was found to be 0.17 mg/ml, which was not affected by the combination treatment significantly (P ≤0.05) (Table 2). Insignificant change in flavonoid content could be due to its overall content which was quite lesser than the total soluble phenolics.

3.9. Antioxidant Capacity Increased in Juice Extracted from Combination Treated Stem The juice from unprocessed sugarcane stem showed 74% DPPH radical scavenging activity and 53% ABTS radical scavenging activity (Table 2). The combination treatment resulted significant (P ≤0.05) increase in DPPH (to 90%) and Impact Factor (JCC): 5.9084

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ABTS (to 68%) radical scavenging activity which was primarily due to chlorination and blanching treatment (Table 2).

This seems to be due to increasedlevel of soluble phenolics in such samples, as phenolics have been reported to contribute to the antioxidant capacity in various agro produce such askiwifruitand litchi (Mishra et al., 2012; Shahkoomahally and Ramezanian, 2013).

3.11. Organoleptic Evaluation of Extracted Juice from the Combination Processed Stem Showed Good Acceptability The organoleptic qualities of the extracted juice from the sugarcane stem processed with combination of chlorination (T1), blanching (T2) and ozonation (T3) were found to be well acceptable (P ≤0.05) by the panelists, and the score was ranged between ‘like very well (8)’ to ‘like fairlywell (6)’. The overall acceptance in processed sample was quite similar to the unprocessed fresh sample (Table 3). In a similar study in iceberg lettuce, washing with chlorinated and ozonated water shown to improve the appeal and hence overall acceptability (Bauret al., 2004). As discussed above the ozonation treatment in the combination helped in retaining colorof the extracted juice (Figure 2b) due to reduced browning (by ~59%), which eventually improved its sensory scores. The chlorination treatment and blanching did not have any adverse effect the taste.

Table 3: Sensory Evaluation@ of Sugarcane Juice Extracted from Sugarcane Stem after Different Treatments Treatment Condition

Non-treated control T1 - Chlorination (NaClO, 0.02%) at high temperature (60°C, 10 min) T2 -Blanching in potable water (60°C, 10 min) T3 -Ozonation in potable water (20 min) (T1 +T2) Multistep Combination 1* (T1 +T2+T3) Multistep Combination 2# The values represent mean ±SD.

Appearance

Color

Aroma

Taste

Overall Acceptance

7.2a ±1.1

7.1a ±1.2

6.5a ±1.1

7.5a ±0.9

6.9a ±1.1

7.2a ±0.7

7.2a ±0.9

7.1a ±1.1

7.5a ±0.8

6.8a ±0.8

6.6a ±0.5

6.6a ±0.5

7.5a ±1.1

7.2a ±0.8

7.0a ±0.6

7.3a ±0.7 6.5a ±1.1 7.7a ±0.9

7.6a ±0.8 6.7a ±1.0 7.7a ±0.9

7.3a ±1.1 6.7a ±0.9 7.6a ±1.4

7.1a ±1.4 7.2a ±0.5 8.0a ±0.8

7.5a ±1.0 6.8a ±0.9 7.9a ±0.8

*Combination 1: Chlorination (0.02% NaClO, 60°C, 10 min) + Blanching (60°C, 10 min) #

Combination 2: Chlorination (0.02% NaClO, 60°C, 10 min) + Blanching (60°C, 10 min) + Ozonation (20 min).

NaClO- Sodium hypochlorite @

A 9-point hedonic scale (9-like extremely, 8- like very well, 7- like moderately, 6-like fairly well, 5-like slightly,

4-dislike slightly, 3-dislike moderately, 2-dislike very much, and 1 -dislike extremely) for scoring the parameters.

The values in columns with different superscript indicated statistically significant difference (P ≤0.05).

4. CONCLUSIONS A multistep dip treatment, including potable water wash followed by washing with sodium hypochlorite at high temperature(60°C, 10 min), extended hot water blanching(60°C, 10 min) and ozonation (20 min) reduced the pathogenic load of sugarcane stem to below detectable level, and also overall bacterial and yeast mold load to remarkably low level.

The treatment resulted in the extracted juice having better hygiene with acceptable sensory, nutritional attributes and reduced browning. The treatment, thus helped in ensuring the overall safety of the extracted sugarcane juice for fresh consumption.

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Bibhuti Bhusan Mishra & Satyendra Gautam

ACKNOWLEDGEMENT Authors acknowledge the technical help provided by Mr. Rohit Waghole, a student from Mumbai University, India.

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SUPPLEMENTARY MATERIALS Table S1: The Effect of Potable Water Washing and Chlorination (NaClO) Treatment of Sugarcane Stem on the Microbial Quality of Extracted Juice Concentration (%) Time (min) TBC (log cfu/ml) YMC (log cfu/ml) PC (log cfu/ml) SC (log cfu/ml) Unprocessed 0 4.1a ±0.5 2.9a ±0.5 1.9a ±0.4 1.8a ±0.1 a a b 10 3.7 ±0.2 2.4 ±0.2 1.2 ±0.2 1.0b ±0.3 a b c Potable water 40 3.3 ±0.2 1.7 ±0.2 0.9 ±0.3 0.9c ±0.2 b b c 80 2.1 ±0.1 1.4 ±0.1 0.8 ±0.4 0.7c ±0.3 Sodium hypochlorite (NaClO) 10 3.5a±0.2 2.2a±0.2 0.7c ±0.1 0.8c ±0.1 b b d 0.02% 40 2.0 ±0.4 1.4 ±0.1 0.4 ±0.1 0.5d ±0.1 b c d 80* 1.6 ±0.1 0.5 ±0.1 0.3 ±0.2 0.4d ±0.2 b b 10 2.8 ±0.2 1.3 ±0.2 ND ND 0.5% 40* 1.8b ±0.2 0.7c ±0.2 ND ND 80* 0.8c ±0.1 0.5c ±0.1 ND ND b 10 2.0 ±0.2 1.2b±0.1 ND ND 1.0% 40* 1.8b ±0.1 0.7c ±0.2 ND ND 80* 0.6c ±0.1 0.4c ±0.1 ND ND TBC- Total bacterial count, YMC - Yeast and mold count, PC - presumptive coliform, SC – Staphylococcus count, ND- not detectable.

The values in columns with different superscript indicated statistically significant difference in the mean (P ≤0.05). *Organoleptically not acceptable Table S2: The Effect of Chlorination (NaClO) of Sugarcane Stem (10 min) at Different Temperature on the Microbial Quality of Extracted Juice.

PC (log SC (log TBC (log YMC (log Temperature Concentration cfu/ml) cfu/ml) cfu/ml) cfu/ml) (°C) (%) a a a 0 4.0 ±0.3 2.7 ±0.2 1.7 ±0.4 1.9a ±0.3 a a a 0.02 3.8 ±0.1 2.6 ±0.3 1.6 ±0.3 1.4a ±0.2 40 ±2°C a b 0.5* 3.2 ±0.2 1.6 ±0.1 ND ND 1* 1.6b ±0.1 0.8c ±0.1 ND ND 0 3.9a ±0.3 2.7a ±0.2 0.6b ±0.2 0.8b ±0.3 a a 0.02 3.5 ±0.3 2.5 ±0.2 ND 0.4b ±0.2 50 ±2°C 0.5* ND ND ND ND 1* ND ND ND ND 0 3.1a ±0.3 1.8b ±0.2 ND ND b 0.02 1.5 ±0.2 1.6b ±0.2 ND ND 60 ±2°C 0.5* ND ND ND ND 1* ND ND ND ND 0 1.5b ±0.3 1.3b ±0.2 ND ND c 70 ±2°C* 0.02 0.6 ±0.2 ND ND ND 0.2 ND ND ND ND 0 0.5c ±0.1 0.4c ±0.1 ND ND 80 ±2°C* 0.02 ND ND ND ND 0.2 ND ND ND ND TBC- Total bacterial count, YMC - Yeast and mold count, PC - presumptive coliform, SC – Staphylococcus count, ND- not detectable. *Organoleptically not well acceptable. The values in columns with different superscript indicated statistically significant difference in the mean (P ≤0.05).

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Browning index (100-L)

50 45

a

C o n tr o l

c

T1

b

40 d

35 f

30

e

T1+T 2

f

g

25

h

20

h

h

T1+T 2+T 3

h

15

0

1

2 T im e (h )

3

4

Figure S1: The Effect of Combination Treatment of Sugarcane Stem on Browning Discoloration of Juice as a Function of Time after Extraction. Control – Non Processed Stem; T1- (0.02% Sodium Hypochlorite 60°C 10 min); T2- Blanching (60°C 10 min); T3- Ozonation (20 min).as a Function of Time. The Values with Different Superscript Indicated Statistically Significant Difference in the Mean (P ≤0.05).

12

Days of storage

10

a

8 6

b

4 2

c

0 4±2

1 0 ±2 2 6 ±2 o S torage tem peratu re ( C )

Figure S2: The Shelf Life of Sugarcane Stem at Different Storage Temperature upon Treatment with Optimized Combination Process. The Values with Different Superscript Indicated Statistically Significant Difference in the Mean (P ≤0.05).

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160.00

MV

128.00 96.00 64.00 32.00 0.00 0.00

2.00

4.00 6.00 Minutes (a)

8.00

2.00

4.00 6.00 Minutes

8.00

160.00

MV

128.00 96.00 64.00 32.00 0.00 0.00

(b) Figure S3. The HPLC Chromatogram of Sucrose Standard (a) and Extracted Sugarcane Juice Sample (b) Showing the Sucrose as the Single Major Sugar Compound.

AUTHORâ&#x20AC;&#x2122;S PROFILE

Dr.Bibhuti Bhusan Mishra, Group Leader, Agroproduce & Beverage Technology Group, Food Flavour & Aroma Chemistry Section, Food Technology Division, Bhabha Atomic Research Centre, Mumbai.

Dr. Satyendra Gautam, Head, Food Science and Safety Section, Food Technology Division, Bhabha Atomic Research Centre, Mumbai.

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