DETERMINATION OF ENNIATINS AND BEAUVERICIN BY NON-AQUEOUS CAPILLARY ELECTROPHORESIS–TIME OF FLIGHT MASS SPECTROMETRY IN ALL IONS MODE

DETERMINATION OF ENNIATINS AND BEAUVERICIN BY NON-AQUEOUS CAPILLARY ELECTROPHORESIS–TIME OF FLIGHT MASS SPECTROMETRY IN ALL IONS MODE

M.M. Delgado-Povedano, Francisco J. Lara, Emilio Borrego-Marín, Laura Gámiz-Gracia, Ana M. García-Campaña

Avda. de Fuente Nueva s/n, 18071, Granada (University of Granada, Faculty of Sciences, Department of Analytical Chemistry) Spain

mmdelgado@ugr.es

Enniatins (ENN) and beauvericin (BEA) are considered emerging mycotoxins that have been traditionally determined by liquid chromatography coupled to tandem mass spectrometry (MS/MS). However, to the best of our knowledge, no analytical methods based on capillary electrophoresis (CE)-MS/MS has been reported so far. Despite the absence of easily ionizable groups, the ionophoric character of these compounds makes them compatible with CE separation. Due to their apolar nature, in this work, a non-aqueous CE (NACE) method coupled to QTOF-MS is proposed for the first time to identify and quantify these mycotoxins. Separation was achieved in 4 min under optimum conditions: 40 mM ammonium acetate in 80:20 (v/v) MeCN-MeOH (buffer), 30 kV (voltage), 80 cm (capillary length), 20 ºC (capillary temperature) and 50 mbar × 20 s (injection). Higher selectivity can be achieved when compared with LC due to the formation of exclusive CE adducts such as [M+CH3CH2NH3]+. The “All ions” acquisition mode was selected as it allows the quantification of the usual enniatins, for which standards are available, and the identification of unusual enniatins, for which standards are not available. The method was validated for wheat samples, obtaining LOQs between 5 and 10 μg/kg, recovery values >87.3%, and intra- and inter-day precision values (RSDs) <15.2% in all cases. The method was applied to 29 wheat samples, finding 25 positives for ENNB (16.1-1480 μg/kg), 19 for ENNB1 (19.9-550 μg/kg), 7 for ENNA (10-55μg/kg), 4 for ENNA1 (12.677 μg/kg) and 4 for BEA (12.3-16.4 μg/kg) and two other ENNs (tentatively identity confirmed).

Acknowledgement: Financial support of the Spanish Ministry of Science, Innovation and Universities (RTI2018-097043-B-I00) and the Junta de Andalucía - Programa Operativo FEDER (BAGR-202-UGR20). M.M.D.P. is grateful for a postdoctoral contract to the Junta de Andalucía (DOC_00230).

Keywords: enniatins, NACE–QTOF MS/MS, beauvericin, all-ions, wheat.

25

ANTIFUNGAL VOLATILE COMPOUNDS PRODUCED BY Debaryomyces hansenii AGAINST Penicillium nordicum IN DRY-CURED FERMENTED SAUSAGES

M. Álvarez1, J. Delgado1, E. Roncero1, F. Gómez1, M. J. Andrade1

1University of Extremadura, Institute of Meat and Meat Products, Faculty of Veterinary Science, Food Hygiene and Safety. Cáceres, Spain.

maalvarezr@unex.es

Debaryomyces hansenii FHSCC 253H has been reported as an efficient biocontrol agent against toxigenic moulds, including Penicillium nordicum, the main ochratoxin A (OTA) producer in drycured meat products. Within the yeast’s antifungal mode of action, the production of volatile antifungal compounds (VOCs) has been reported. The aim of this study was to evaluate the involvement of the VOCs produced by D. hansenii in the OTA reduction during the ripening of drycured fermented sausages. For this, a batch containing 106 cells/g of D. hansenii in the meat dough before stuffing was manufactured. A non-inoculated batch was also made. Both batches were then superficially inoculated with P. nordicum. After their ripening using the usual conditions in industries, OTA was extracted by QuEChERS and measured through uHPLC-MS/MS-QqQ. The VOCs were extracted by SPME using a DVB/CAR/PDMS fibre and analysed by GS-MS. D. hansenii significantly decreased the OTA production at the same time it significantly increased the concentration of acetic acid, 3-methylbutanol and 2-phenyletanol respect to the non-inoculated batch. These compounds have previously shown antifungal activity against Penicillium and Aspergillus spp. (de Souza et al., 2018; Hua et al., 2014; Jin et al., 2021; Núñez et al., 2015; Quiao et al., 2020). Other compounds were only detected in the batch containing the yeast, such as 2-methylpropanol, 3methylbutanal and acetoin, which have also demonstrated antifungal effect (Delgado et al., 2021; Gao et al., 2018; Gergolet Diaz et al., 2021). Therefore, the production of specific VOCs seems to take part in the antifungal mode of action of D. hansenii, although more studies are necessary to fully elucidate their target site and the possibility of other involved mechanisms.

Acknowledgement: This research was funded by Junta de Extremadura-Consejería de Economía, Ciencia y Agenda Digital-, Fondo Europeo de Desarrollo Regional-“Una manera de hacer Europa” [IB16045 project and GR18056 grant]. M. Álvarez is recipient of a fellowship from the Spanish Ministerio de Economía, Industria y Competitividad [BES-2017-081340].

Keywords: Debaryomyces hansenii, Penicillium nordicum, volatile compounds, dry-cured fermented sausages

26

EMPLEO DE LOS MEDIOS FERMENTADOS DE HARINA Y SALVADO DE MOSTAZA AMARILLA POR BACTERIAS ÁCIDO-LÁCTICAS PARA LA BIOPRESERVACIÓN DE TOMATES

Nazareth TM 1 , Torrijos R 1 , Mañes J 1, Meca G 1

1Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Ave. Vicent Andrés Estellés s/n, 46100, Burjassot.

raquel.torrijos@uv.es

La contaminación fúngica postcosecha de hortofrutícolas supone importantes pérdidas económicas y un riesgo por la síntesis de micotoxinas. Actualmente, en la Industria Agroalimentaria existe una creciente demanda por reemplazar los fungicidas sintéticos por alternativas vegetales para garantizar la seguridad de los consumidores. El empleo de bacterias ácido-lácticas (BAL) representa una estrategia prometedora en la bio-preservación de alimentos. En este estudio se evaluó la actividad antifúngica cualitativa de los medios fermentados de harina de mostaza amarilla (YMF) y salvado de mostaza amarilla (YMB) con BAL frente a hongos de los géneros Aspergillus, Penicillium, Fusarium y Botrytis. Se seleccionaron Lactobacillus plantarum 5H1 y 5L1 por su mayor potencial antifúngico, determinándose su Concentración Mínima Inhibitoria (MIC) y la Concentración Fungicida Mínima (MFC) frente a los hongos. El medio YMF mostró una actividad antifúngica mayor, obteniéndose valores de MFC entre 25 y 100 g/L en las cepas más sensibles, Penicillium verrucosum VTT D-01847, Fusarium verticillioides ISPA 12052 y Botrytis cinerea CECT 20973. Además, se determinaron el ácido láctico y el ácido feniláctico (PLA), entre otros metabolitos antifúngicos, en los medios fermentados. La aplicación del medio liofilizado de 1% YMF fermentado por Lactobacillus plantarum 5H1 y 5L1 evidenció su efectividad frente al crecimiento de F. verticillioides ISPA 12052 y B. cinerea CECT 20973 en tomates, obteniéndose una reducción fúngica de 5,04 log UFC/g y 3,13 log UFC/g, así como una inhibición completa de la producción de Fumonisina B1 por F. verticillioides ISPA 12052 y de Ocratoxina A por P. verrucosum VTT D01847 en tomates.

Keywords: Bacterias ácido-lácticas, Lactobacillus plantarum, harina de mostaza amarilla, actividad antifúngica, tomate

27

AISLAMIENTO DE MICROORGANISMOS COMO POTENCIALES AGENTES DE BIOCONTROL DE HONGOS PRODUCTORES DE MICOTOXINAS EN VIÑEDO ECOLÓGICO

P. de la Huerta Bengoechea1, J. Gil-Serna1, B. Patiño Álvarez1, C. Vázquez1 . 1 Departamento de Genética, Fisiología y Microbiología, Universidad Complutense de Madrid, Madrid, España

pauladeh@ucm.es

La presencia en viñedos de hongos del género Aspergillus productores de micotoxinas, como la

ocratoxina A, supone un problema para la seguridad alimentaria y la economía. El uso de

microorganismos como agentes de control biológico (ACB) es una de las estrategias más

prometedoras para evitar el crecimiento fúngico y la producción de toxinas en estos cultivos. En este

trabajo, se aislaron 542 microorganismos procedentes de suelos de viñedos ecológicos de diferentes

regiones de España. Se identificaron 11 hongos entre los que había importantes productores de

micotoxinas y un posible ACB (Trichoderma sp.). Con las 480 bacterias y 33 levaduras aisladas se

realizó un cribado secuencial para seleccionar aquellas con las características más adecuadas para ser

utilizadas como ACB. Los 16 aislamientos que cumplieron todos los requisitos se identificaron

mediante secuenciación. Se descartaron todas las levaduras por pertenecer al género Cryptococcus y

se seleccionaron 9 aislamientos de 4 especies bacterianas para ensayar su potencial para controlar a

los tres principales hongos toxígenos en uvas: A. carbonarius, A. welwitschiae y A. flavus.

Arthrobacter sp., Rhodococcus sp. y Mycetocola sp. destacaron por su capacidad de reducir el

crecimiento de los hongos indicados.

Keywords: micotoxinas, agentes de biocontrol, Aspergillus, actinobacterias, viñedos.

28

REDUCCIÓN DEL CRECIMIENTO FUNGICO EN QUESO MEDIANTE EL USO DE UN RECUBRIMIENTO NATURAL BASADO EN SUERO DE LECHE FERMENTADO

M. Vitali, P. Vila. F. Illueca, J. Calpe, J.M. Quiles, G. Meca

Laboratorio de Toxicología, Departamento de Medicina Preventiva y Salud Pública, Ciencias de la Alimentación, Toxicología y Medicina Legal, Facultat de Farmàcia, Universitat de València, España.

mattvi@alumni.uv.es

Este trabajo muestra como la reutilización de suero lácteo como medio fermentable por bacterias

acido lácticas (BAL) puede ser una alternativa para biopreservar el queso. Para ello se aislaron BAL

de derivados lácteos y se comprobó de forma cualitativa la actividad antifúngica de cada BAL y de

forma semicuantitativa la actividad antifúngica de los liofilizados de caldo MRS y suero lácteo

fermentado por estas BAL frente a cepas fúngicas del género Penicillium. Se determinó

cuantitativamente la Concentración Mínima Inhibitoria (MIC) y la Concentración Mínima Fungicida

(MFC) de los liofilizados de suero lácteo fermentado. Con el objetivo de estudiar la viabilidad de P.

commune CECT 20767 en un recubrimiento formado por hidroxipropilmetilcelulosa (HPMC) y

liofilizado de suero lácteo fermentado, en el ensayo de MIC y MFC se seleccionó el liofilizado de

suero lácteo fermentado con mayor actividad antifúngica frente a este hongo. En el ensayo de MIC y

MFC los liofilizados de suero lácteo fermentado por KK13, KB2, KB3 y KB4 fueron los que mayor

actividad antifúngica presentaron. El liofilizado de suero lácteo fermentado por KB3 fue

seleccionado por presentar la menor MFC, concretamente, 15.23 mg/mL frente a P. commune CECT

20767. Los recubrimientos de HPMC y 100 mg/mL de liofilizado de suero lácteo fermentado por

KB3 inhibieron el crecimiento de P. commune CECT 20767.

Keywords: bacterias acido lácticas, actividad antifúngica, Penicillium, suero lácteo.

29

CHARACTERIZATION OF Penicillium expansum MUTANTS DEFECTIVE IN PATULIN PRODUCTION AS POTENTIAL BIOCONTROL AGENTS

B. Llobregat, L. González-Candelas, A.R. Ballester*

Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC). Calle Catedrático Agustín Escardino 7, Paterna 46980. Spain.

ballesterar@iata.csic.es

Due to its high pathogenicity on harvested fruits, Penicillium expansum is one of the most studied

molds in the genus Penicillium. It causes the common postharvest disease known as blue mold rot.

Patulin is the most researched and well-documented mycotoxin among the many secondary

metabolites (SM) produced by P. expansum. This polyketide-derived mycotoxin has a wide range of

toxic effects, including cytotoxicity or genotoxicity. This fact has generated intense concern in the

society, particularly in recent years, due to the health and economic risks it represents. The gene

cluster responsible for patulin biosynthesis in P. expansum is composed of 15 genes (patA - patO);

among them is the patK gene, which encodes 6-methylsalicylic acid synthase, the first enzyme of the

patulin pathway. In fungi, both pathway-specific gene clusters and global regulatory factors are

known to regulate secondary metabolism. In particular, the VELVET family of regulatory proteins

(VeA, VelB, LaeA, among others) work to coordinate fungal growth and SM production. In the

present study, we have characterized the competitive ability of P. expansum veA- and patK-gene

deleted mutants against the wild-type strain and their effect on patulin production. The knockout

mutants displaced the wild-type strain during in vitro growth and, consequently, there was a

reduction in patulin levels. This preliminary study raises the possibility of using non-mycotoxigenic

strains of P. expansum as biocontrol agents.

Funding: RTI2018-093392-A-I00 (AEI/FEDER, UE), JAEIntro2019-CSIC (JAEINT_19_01896), PRE2019-089326 y RYC-2017-22009.

Keywords: Penicillium expansum, patulin, competitive ability, patK, veA

30

USO DE HIDROLIZADOS DE CASEINAS EN LA BIO-PRESERVACIÓN DEL PAN CONTRA HONGOS TOXIGÉNICOS

D’ Opazo, V., Luz, C., Calpe J., Quiles, J.M., Illueca, F., Vitali, M., Meca G.

Laboratory of Food Chemistry and Toxicology. Faculty of Pharmacy. University of Valencia. Spain

victor.dopazo@uv.es

En la actualidad los hongos son una de las mayores preocupaciones para la industria alimentaria

debido a su capacidad de contaminar alimentos y de afectar la salud humana. Una de las industrias

más afectadas por este tipo de contaminación es la industria panadera, la cual sufre cuantiosas

pérdidas anualmente causadas por estos microorganismos. La respuesta común a este tipo de

contaminaciones es el uso de antifúngicos sintéticos. Pero ante el miedo del consumidor a este tipo

de preservantes la industria busca nuevas alternativas, como el uso de péptidos bioactivos. Siguiendo

esta tendencia en este trabajo se evaluó la capacidad antifúngica de la caseína hidrolizada con

pancreatina y tripsina. Para ello también se estudió el grado de hidrolisis de estas proteínas en

contacto con estas enzimas y su potencial como antioxidantes. Finalmente, se realizaron panes con

las caseínas como ingrediente donde se determinaron las propiedades tecnológicas de las masas y

panes, además de, su capacidad de ser usadas para la bio-preservación de panes frente a hongos del

género Aspergillus, Penicillium y Fusarium. Los ensayos de actividad antifúngica demostraron

inhibición del crecimiento de hongos pertenecientes al género Aspergillus y un retraso en la

esporulación de todos los hongos testados. A mayor tiempo en contacto con la enzima aumentó el

grado de proteólisis. La caseína hidrolizada por tripsina evidenció el mayor aumento de la actividad

antioxidante de todos los tratamientos estudiados. Finalmente, los panes con caseína como

ingrediente no presentaron diferencias significativas en las propiedades tecnológicas respecto a los

controles, no obstante, se observó una reducción en el crecimiento de los hongos en los panes con un

2 % de caseína y con un 0.5 % de caseína hidrolizada con pancreatina.

Keywords: caseínas, hidrólisis enzimática, hongos, pan, enzimas, péptidos antifúngicos

31

EFFECT OFINTERACTIONS AMONG STRAINS OFBLACK ASPERGILLI ON OCHRATOXINAPRODUCTION

G. Castellá, M.R. Bragulat, F.J. Cabañes

Veterinary Mycology Group, Department of Animal Health and Anatomy, Veterinary Faculty, Universitat Autònoma de Barcelona, Barcelona, Spain.

Gemma.Castella@uab.cat

Ochratoxin A (OTA) is a nephrotoxic mycotoxin that can be found in a variety of common foods and beverages. After cereals, grapes and their derivatives are the second most contaminated products with OTA, being Aspergillus carbonarius the main source of this mycotoxin. In previous studies, we isolated and characterized three atypical and unique non-OTA producing strains of A. carbonarius [1, 2, 3, 4]. Biological control using microbial antagonists has emerged as a promising approach for control of mycotoxins in both pre- and post-harvested crops. As an example, one of the more promising strategies for control of aflatoxins in crops involves the use of atoxic strains of A. flavus as biological control agents [5]. In this study, in vitro interactions between ochratoxigenic strains of A. carbonarius and A. niger and non-ochratoxigenic strains of A. carbonarius and A. tubingensis were assessed in order to evaluate their potential for controlling OTA production. Two non-OTAproducing strains, A. carbonarius (Ac-) and A. tubingensis (At), used as potential biocontrol agents, were screened for their ability to inhibit or reduce OTA production in co-inoculation with an OTAproducing strain of A. carbonarius (Ac+) and an OTA-producing strain of A. niger (An+). The effect of the potential biological agent on OTA production was evaluated using different mixed spore suspensions of OTA-producing strain:non-OTA-producing strain ratios. In all cases of coinoculation, the presence of Ac- and At decreased the production of OTA at different percentages, depending on their inoculum load. Of the two non-ochratoxigenic species assayed, the non-OTAproducing strain of A. carbonarius gave the best control resulting in practically complete inhibition of OTA production in co-inoculation with an ochratoxigenic strain of A. niger and high percentages of OTA reduction (74%) with an ochratoxigenic strain of A. carbonarius. This study opens new possibilities for using non-ochratoxigenic strains of A. carbonarius as biocontrol agents in grapes.

1. Cabañes FJ, Bragulat MR Castellá G. Characterization of nonochratoxigenic strains of Aspergillus carbonarius from grapes. Food Microbiol 36: 135-141. 2013. 2. Cabañes, F. J., Sanseverino, W., Castellá, G., Bragulat, M. R., Cigliano, R. A., Sanchez, A., 2015. Rapid genome resequencing of an atoxigenic strain of Aspergillus carbonarius. Sci. Rep. 5, 9086. 3. Castellá, G., Bragulat, M.R., Puig, L., Sanseverino, W., Cabañes, F.J., 2018. Genomic diversity in ochratoxigenic and non ochratoxigenic strains of Aspergillus carbonarius. Sci. Rep. 8, 5439. 4. Castellá, G., Bragulat, M.R., Cigliano, R.A., Cabañes, F.J., 2020. Transcriptome analysis of non-ochratoxigenic Aspergillus carbonarius strains and interactions between some black aspergilli species. Int. J. Food Microbiol. 317, 108498. 5. Ehrlich, K.C. 2014. Non-aflatoxigenic Aspergillus flavus to prevent aflatoxin contamination in crops: advantages and limitations. Frontiers in Microbiology 5, 50.

Acknowledgments: This research was supported by the Ministerio de Economía y Competitividad of the Spanish Government (AGL2014-52516-R). Keywords: Aspergillus carbonarius, ochratoxin A, fungal interactions, biological control agents.

32

LA BIOPRESERVACIÓN DE MASAS DE PANADERÍA Y PANES MEDIANTE EL EMPLEO DE BACTERIAS ÁCIDO LÁCTICAS

F. Illueca, V. D'Opazo, JM Quiles, C.Luz, M.Vitali, J.Calpe, G.Meca

Universitat de València. Facultat de Farmàcia. Departamento de Medicina Preventiva y Salud Pública, Ciencias de la Alimentación, Toxicología y Medicina Legal. Av. Vicente Andrés Estellés s/n 46100 Burjassot, Valencia, España.

franillueca_31@hotmail.com

Currently, one of the most studied fields in food bioconservation is the application of lactic acid

bacteria (LAB). In this work, the antifungal capacity of eight strains of Lactobacillus plantarum

against fungi contaminating bread of the genera Botrytis, Alternaria, Aspergillus and Penicillium was

studied. In this way, the antifungal activity of different types of fermented milk serum (SLF) was

characterized by BAL using qualitative methods, such as agar diffusion and subsequently

quantitative, such as the minimum inhibitory concentration and minimum fungicidal concentration

(MIC-MFC) method. After that, the antioxidant capacity was also studied. Finally, the WF with the

BAL that had shown the highest efficacy against fungal growth was used as a treatment in bread.

Thus, the impact of the treatment on the properties of the food and the bioconservation capacity

against Aspergillus flavus and Penicillium verrucosum was studied. The results obtained in the agar

diffusion showed that all the fermented media showed activity against fungi, especially against

Aspergillus and Penicillium. The MIC-MFC highlighted that SL100 fermented by L. plantarum 5L1

presented the highest antifungal activity and the fungi most sensitive to the treatment were A. flavus

and P. verrucosum. The FW presented a low antioxidant activity. Finally, the treatment used on

the bread did not generate great changes in its properties, but it did extend the useful life and reduce

the fungal load in A. flavus and more clearly in P. verrucosum.

Keywords: Lactic acid bacteria, fermented whey, bread, Aspergillus and Penicillium.

33

EVALUATION OF THE ANTIFUNGAL ACTIVITY OF CLOVE ESSENTIAL OIL AGAINST Penicillium nordicum IN DRY-CURED FERMENTED SAUSAGES

E. Roncero1, M.J. Andrade1, M.M. Álvarez1, E. Cebrián1, J.C. Pulido1, J. Delgado1

1Food Hygiene and Safety, Meat and Meat Products Research Institute, Faculty of Veterinary Science, University of Extremadura, Avda. de las Ciencias s/n. 10003 Cáceres, Spain

eroncerob@unex.es

Microbial contamination occurs during the processing of dry-cured meat products, being one of the main concerns for the meat industry. Focusing on products such as dry-cured fermented sausages, moulds predominate in the final stages of their processing, during which the most worrying hazard is the contamination with ochratoxin A (OTA)-producing species, such as Penicillium nordicum and Penicillium verrucosum. As an alternative to the strategies used in the industry to control their mycotoxin production, the application of plant extracts, such as essential oils, is being analysed. The objective of this study was to evaluate the antifungal activity of clove essential oil (CEO) against three strains of P. nordicum (Pn92, Pn15 and Pn856) in two meat products. Sterile portions of raw chorizo and salchichón were inoculated with each of the three moulds together with the CEO (250 μL/mL). A negative control without antifungal agent, and two positive controls based on a commercial antifungal at subinhibitory level (AF1: 150 μL and AF2: 50 μL) were also prepared. After 15 days under similar conditions to those of the curing process, OTA was quantified using a Qexactive mass spectrometer. In the negative control, it was observed that salchichón was more favorable for OTA production than chorizo for the three strains. AF2 did not affect OTA production, whereas AF1 significantly increased OTA production in Pn92. A significant reduction of OTA levels was obtained for the three strains in salchichón in the presence of CEO, which was less effective in reducing OTA in chorizo. Pn15 and Pn856 similarly behaved in both substrates in response to the applied treatments. Consequently, CEO could be proposed as a potential effective antifungal agent in salchichón.

Acknowledgments: Research funded by Junta de Extremadura and FEDER “Una manera de hacer Europa” [IB16045 project; GR18056 grant]. Q-Exactive mass spectrometer was funded by MINECO (Ref. UNEX-AE-3394).

Keywords: Ochratoxin A, mould, clove essential oil, dry-cured fermented sausages

34

SCREENING OF ISOLATED BACTERIA FOR ANTIFUNGAL ACTIVITY AGAINST MYCOTOXIGENIC FUNGI OF CITRUS FRUITS

C. Stracquadanio1, J. Calpe1, C. Luz1, G. Meca1

1 Departemento de Medicina Preventiva, Facultat de Framacia, Universitat de Valencia, Av. Vincent Andrés Estellés s/n, 46100, Burjassot, Valencia, Spain.

claudia.stracquadanio@uv.es

Fungal rots are the leading cause of postharvest losses (about 30%) of citrus and can greatly reduce

its shelf life. Some postharvest fungal pathogens of citrus fruits produce mycotoxins that can pass

through and be found in juices. The aims of this study were to screen bacteria from different

matrices, explored their antagonistic properties against the main mycotoxigenic pathogens of citrus

fruits, belonging to Aspergillus sp., Penicillium sp., Fusarium oxysporum and Alternaria

alternata. Isolated bacteria were tested against the pathogens with overlay assay that showed

inhibition by direct contact. Active bacteria were identified based on their protein and peptide

fingerprints by MALDI-TOF/MS and fermented in MRS broth to produce secondary metabolites

with antifungal activity. Preliminarily, the 13 cell-free supernatants (CFS) were evaluated by agar

diffusion assay in which the activity was established by the presence of the inhibition halo.

Subsequently, the minimum inhibition concentration and the minimum fungicidal concentration

(MIC & MFC) were determined. In addition, volatile organic compounds (VOCs) present in CFS

were identified by GC-MS. A total of 30 strains were isolated of which 13 isolates, identified

belonging to the genera Pediococcus, Lactobacillus and Leuconostoc, showed clear zones of

inhibition on the tested pathogens. The cell-free supernatants (CFS) of 13 isolates inhibited the

growth of most tested pathogens, in particular three CFS of isolates 5H1, 5L1 and N2B2 showed

good activity with MIC values between 15.6-125 mg/mL and MFC values between 15.6-250 mg/mL.

Keywords: antifungal activity, isolated bacteria, mycotoxigenic pathogen, fermentation

35