Food Control 31 (2013) 539e545

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Food Control
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Antimicrobial activity of lactic acid bacteria against pathogenic and spoilage microorganism isolated from food and their control in wheat bread
Dalia Cizeikiene a, *, Grazina Juodeikiene a, Algimantas Paskevicius b, Elena Bartkiene c
a b c

Department of Food Technology, Kaunas University of Technology, Radvilenu Rd. 19, LT-50254 Kaunas, Lithuania Laboratory of Biodeterioration Research, Institute of Botany of Nature Research Centre, Zaliuju Ezeru Str. 49, LT-08406 Vilnius, Lithuania Department of Food Safety and Animal Hygiene, Lithuanian University of Health Sciences, Tilzes Str. 18, LT-47181 Kaunas, Lithuania

a r t i c l e i n f o
Article history: Received 30 April 2012 Received in revised form 23 November 2012 Accepted 1 December 2012 Keywords: Bacteriocins like inhibitory substance Food borne pathogens Lactic acid bacteria Fungi Food safety

a b s t r a c t
The evaluation of antimicrobial activities of Lactobacillus sakei KTU05-6, Pediococcus acidilactici KTU05-7, Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10 strains producing organic acids and bacteriocins like inhibitory substances (BLIS) against undesirable microorganisms in the food industry, were performed using an agar well diffusion assay method. The metabolites of lactic acid bacteria (LAB) inhibited the growth of pathogenic bacteria, belonging to Bacillus, Pseudomonas, Listeria and Escherichia genera in various degrees. The organic acids and BLIS of LAB show fungicidal and fungistatic activities against fungi and yeast such as Fusarium culmorum, Penicillium chrysogenum, Aspergillus fumigatus, Aspergillus versicolor, Penicillium expansum, Aspergillus niger, Debaryomyces hansenii and Candida parapsilosis. 20% of P. pentosaceus KTU05-9 sourdough in a bread recipe suppressed the bread ropiness in articially contaminated bread by Bacillus subtilis spores, until 6 days storage at 23  C. Moreover P. acidilactici KTU05-7, P. pentosaceus KTU05-8 and KTU05-10 single cell suspension sprayed on the bread surface, inhibited growing of fungi until 8 days of storage in polythene bags. The presence of BLIS and organic acids by tested LAB is an indication that these bacteria can be used widely in the food industry as bio-preservatives due to their broad inhibition spectrum. 2012 Elsevier Ltd. All rights reserved.

1. Introduction During the recent years health-conscious consumers are looking for natural foods without chemical preservatives that will t in their healthy lifestyle. Bio-preservation refers to extended shelf life and enhanced safety of foods using microorganisms or their metabolites (Ross, Morgan, & Hill, 2002). Lactic acid bacteria (LAB) play a key role in food fermentations where they not only contribute to the development of the desired sensory properties in the nal product but also to their microbiological safety (Smaoui et al., 2010). The antimicrobial effect of LAB is mainly related to the production of lactic- and acetic-acids, as well as propionic-, sorbic-, benzoic-acids, hydrogen peroxide, diacetyl, ethanol, phenolic- and proteinaceous-compounds however some strains are able to synthesize antimicrobial substances e bacteriocins (Dali, Deschamps, & Richard-Forget, 2010). During recent years the interest increased in bacteriocin-like inhibitory substances (BLIS)

* Corresponding author. Tel.: 370 37 300188. E-mail address: dalia.eidukonyte@gmail.com (D. Cizeikiene). 0956-7135/$ e see front matter 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodcont.2012.12.004

producing LAB because of their potential as to produce natural antimicrobial agents to enhance the safety of food products. BLIS from LAB are antimicrobial compounds that possess bacteriocin capacities requisites but that have not been characterized for their amino acid sequence (Jack, Tagg, & Ray, 1995) yet. Bacteriocins from the generally recognized as safe LAB, have received signicant attention as a novel approach to the control of pathogens in foods (Klaenhammer, 1993; Settani, Massitti, Van Sinderen, & Corsetti, 2005). During the last few years, a large number of new LAB bacteriocins have been identied and characterized. However, only a few studies have been described bacteriocins activities against gram-negative bacteria, fungi and yeast (Altuntas, Cosansu, & Ayhan, 2010; Gerez, Torino, Rolln, & Valdez, 2009; Hassan & Bullerman, 2008; Smaoui et al., 2010; Todorov, Nyati, Meincken, & Dicks, 2007). The prevention of foods by natural and microbiological compounds may be a satisfactory approach solving economic losses due to microbial spoilage of raw materials and food products, to reduce the incidence of food borne illnesses (Galvez, Lucas-Lopez, & Abriouel, 2008). Ropiness is prevalent in the bacterial spoilage of bread mainly caused by Bacillus subtilis (Collins, Kirschner, & Von Holy, 1991)

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D. Cizeikiene et al. / Food Control 31 (2013) 539e545 Table 1 Indicator microorganism used in this study and their isolation source. Microorganism Bacillus thuringiensis 1.1 Bacillus subtilis subsp. subtilis Bacillus subtilis subsp. spizizenii Bacillus cereus ATCC 10876 Pseudomonas gladioli pv. aliicola 3.1 Pseudomonas cepacia 1.1 Pseudomonas uorescens biovar. V 3.3 Pseudomonas marginalis 3.5 Pseudomonas uorescens biovar. V 1.2 Pseudomonas facilis 4.1 Pseudomonas aureofaciens biovar. III 2.4.1 Pseudomonas marginalis 4.2 Pseudomonas cichorii 7.3.1 Pseudomonas uorescens biovar. III 2.1.1.1.1 Pseudomonas pseudoalcaligenes 11 Pseudomonas cepacia 7.2 Pseudomonas uorescens biovar. I 9.2 L. monocytogenes 1.1 L. monocytogenes 1.5 Escherichia coli 1.10 Escherichia coli ATCC 25922 Salmonella typhimurium ATCC 14028 Staphylococcus aureus ATCC 25923 Rhizopus stolonifer 2-KA Penicillium expansum 23L Aspergillus niger MD-029 Aspergillus versicolor MI-130 Aspergillus fumigatus KO-5 Penicillium chrysogenum 48-L Alternaria alternata 1-4U Penicillium cyclopium 21AL Aspergillus tereus S-1 Fusarium culmorum L-2 Fusarium solani 4-SA Pichia brutonii P.3 Saccharomycopsis capsularis S.1 Zygosaccharomyces bailii Z.1(T) Kluyveromyces marxianus K.7.1(T) Saccharomyces cerevisiae Sa.1.5(T) Candida parapsilosis C.7.2 Yarrowia lipolytica Y.C.6.1 Debaryomyces hansenii Deb.4(T) Isolation source Water Daily products Daily products Daily products Onion Onion Onion Potato Potato Potato Beetroots Beetroots Carrot Meat Fat Carrot Carrot Fish Fish Water Daily products Daily products Daily products Cake Apple Bread Cake Grains Grains Carrot Grains Grains Grains Grains Apple Carrot Jam Cheese Juice Meat Meat Cabbage

which has been reported to originate from the raw materials, the bakery atmosphere and equipment surfaces (Bailey & Von Holy, 1993) and is still of major economic concern in the baking industry (Nielsen, 2003). During bread baking only vegetative forms of microorganisms are destroyed, while both bacterial spores and fungi spores remain in bread (Nowicki, Gaba, Dexter, Matsuo, & Clear, 1988). The most common spoiling fungi isolated from grain products belong to Penicillium, Aspergillus, Fusarium and Rhizopus genera (Legan, 1993), whereas yeast of bakery products and ingredients belong to Zygosaccharomyces, Saccharomycopsis, Pichia, Candida and Debaryomyces genera (Legan & Voysey, 1991). Baked goods can be protected from fungal and yeast spoilage by destroying spores which have contaminated the products, using LAB with high and wide antimicrobial activities as starters for sourdough preparation (Lavermicocca et al., 2000). This paper describes therefore the antimicrobial activities of LAB producing metabolites as well as BLIS against (i) various pathogenic bacteria, (ii) fungi and yeast, isolated from food and water, as well as (iii) LAB adaptation in wheat bread production to decrease the spores of rope producing B. subtilis and moulding caused fungi, thus to obtain more safe bread products with an extended shelf life. 2. Materials and methods 2.1. LAB preparation for antimicrobial activity testing Lactobacillus sakei KTU05-6, Pediococcus acidilactici KTU05-7, Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10 previously isolated from spontaneous Lithuanian rye sourdoughs and selected due to their preliminary inhibiting properties (Digaitiene, Hansen, Juodeikiene, & Josephsen, 2005) were obtained from the culture collection of Kaunas University of Technology, Department of Food Science and Technology. The LAB were grown in de Man Rogosa Sharpe (MRS) medium (Oxoid, Milan, Italy) at their optimal temperatures 25  C (KTU05-8 and KTU05-9), 30  C (KTU05-6) or 35  C (KTU05-10 and KTU05-7). 2% of LAB cells were inoculated into a fresh medium and propagated for 18 h. The cells have been harvested by centrifugation (6000 g, 10 min, 4  C). The culture supernatants were ltered through a 0.2 mm sterile Millipore lter to remove all cells and were divided into two parts. One part of supernatants were used for the determination of antimicrobial activities of LAB supernatant (total metabolites), whereas the other part of supernatant samples were adjusted to pH 6.5 with 5 mol l1 NaOH to eliminate the organic acids effect and thus to evaluate potential production of BLIS. 2.2. Culture conditions Bacterial strains isolated from various vegetables, meat, fats and water were used in this study and their isolation sources are listed in Table 1. Bacteria strains belong to the species Bacillus thuringiensis, Pseudomonas gladioli, Pseudomonas cepacia, Pseudomonas uorescens, Pseudomonas marginalis, Pseudomonas facilis, Pseudomonas aureofaciens, Pseudomonas cichorii, Pseudomonas pseudoalcaligenes, Listeria monocytogenes, Escherichia coli 1.10 and E. coli were obtained from the Institute of Botany of Nature Research Centre, while B. subtilis, Bacillus cereus, Streptococcus aureus, Salmonella typhimurium and E. coli ATCC 25922 were obtained from the collection of the Food Institute of Kaunas University of Technology and were used as indicator microorganisms for antimicrobial activity testing. Antifungal activities were determined against Pichia brutonii, Saccharomycopsis capsularis, Saccharomyces cerevisiae, Zygosaccharomyces bailii, Kluyveromyces marxianus, Candida parapsilosis, Yarrowia lipolytica, Debaryomyces hansenii, Rhizopus stolonifer, Penicillium

expansum, Penicillium chrysogenum, Penicillium cyclopium, Aspergillus niger, Aspergillus versicolor, Aspergillus fumigatus, Aspergillus tereus, Alternaria alternata, Fusarium solani isolated from grain-based food, fruit, vegetables and meat (Table 1) which were obtained from the collection of the Institute of Botany of Nature Research Centre, whereas Fusarium culmorum were previously isolated from contaminated wheat grain. Strains were propagated as follows: Pseudomonas spp., were cultured at 28  C in a nutritional medium (per 1 L used: 5 g peptone, by 1.5 g meat and yeast extract and 5 g NaCl); L. monocytogenes, S. aureus, S. typhimurium and E. coli were cultured on the same medium at 37  C, whereas Bacillus spp. were grown in a brain heart infusion (BHI) broth (OXOID) at 30  C. All fungi were cultured on YEPD medium (per 1 L: 10 g yeast extract, 20 g peptone, 20 g glucose and 18 g agar) at 25  C. 2.3. Determination of antimicrobial activity The ability of LAB strains to produce antimicrobial metabolites was tested by an agar well diffusion assay by Schillinger and Lcke (1989). The appropriate solid medium was used for each indicator microorganism. The fungi and yeasts were growing in an incubator at 25  C on YEPD media for 3e7 days, whereas bacteria were propagated 3 days at optimal temperatures (is given above). Spores

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and cells were harvested from slants after growing, to prepare inoculums containing w105 spores/cells ml1 of fungi and w108 cells ml1 of bacteria. Solid media were prepared by adding 1.5% (w/v) agar to the broth medium. Into Petri dishes 100 ml of the indicator strain suspension was poured and overlaid with a soft agar medium cooled to 45  C and mixed. A culture supernatant (50 ml) was added to each well (6 mm in diameter) punched in the cooled agar plates and incubated for 48 h at the optimal growth temperature (is given above). The antimicrobial activities against bacteria were determined by measuring the inhibition zones (mm). Data of antimicrobial activities against fungi were recorded according to the following scale: () no inhibition, () delay of spore formation, () delay of spore formation with a small clear zone of inhibition around the punched well, () a very good inhibition of mycelium growth and sporulation with large clear zones around the punched well. This method was used to evaluate the antimicrobial activities of LAB supernatants (total metabolites) and BLIS. 2.4. Effect of proteolytic enzymes on BLIS Filter sterilized (0.22 mm) culture supernatant uids (adjusted to pH 6.5) were tested to proteolytic enzymes. Sensitivity to trypsin, pepsin, chymotrypsin and proteinase K was detected by incubation at 37  C for 30 min (with 0.5 mg ml1 nal concentration). 2.5. Wheat bread contaminated with B. subtilis manufacture and storage The LAB strain P. pentosaceus KTU05-9 which showed a high inhibitory activity against rope producing B. subtilis was further tested to determine its anti-rope activity during bread storage. Wheat bread samples were prepared using LAB as a starter for sourdough preparation according to Juodeikiene et al. (2011). B. subtilis was cultivated in a Brain heart infusion broth (BHI) at 30  C over night. Then the suspension of B. subtilis was heat-treated for 15 min. 80  C to destroy vegetative cells so that only spores were left. The dough was articially contaminated by adding 5 104 spores of B. subtilis g1 into the mixing bowl. Wheat bread samples containing sourdough prepared with P. pentosaceus KTU05-9 (moisture content of 65%) 10, 15 and 20% of the quantity of our were prepared using a laboratory baking device (automatic Germatic BM-2 oven, AFK, Hamburg, Germany) under the following program: mixing for 10 min, rst proong for 25 min at 25  C, mixing for 15 min at 30  C, second proong for 35 min at 32  C, third proong for 70 min at 38  C, baking for 55 min at 121  C. Bread samples prepared without the addition of LAB sourdough and using only commercial 5 107 colony forming units of S. cerevisiae g1, were analysed as a control sample. After baking the bread samples were stored at 23  C and 30  C in polythene bags. For 6 days the samples were evaluated by sensory examination for the presence of typical unpleasant sweet fruity odour which characterizes the rst stage of ropiness. 2.6. Screening for anti-mould activity of LAB on the bread surface The LAB strains were tested for anti-mould activities on the bread surface. The LAB were propagated in MRS broth at optimal temperatures for 18 h. 2% culture was inoculated into fresh MRS media and cultivated for 24 h. The single LAB cell suspension (w5 104 CFU/cm2) was sprayed on the surface of the baked wheat bread. For the control sample the LAB suspension was not used. The wheat bread samples were stored for 8 days at 15e16  C in polythene bags. The loaves were examined externally for presence of mould according to the following scale: () no mould; () scarce and small colonies; () large colonies.

2.7. Statistical analyses All the experiments were performed at least in two independent experiments. The means and standard deviations of the data were calculated. 3. Results 3.1. Effect of enzymes on BLIS activity All ve LAB produced BLIS were found to be sensitive to proteolytic enzymes as the activities were totally destroyed by pepsin, trypsin, chymotrypsin and proteinase K. 3.2. The LAB antimicrobial activities Antimicrobial activities of LAB were evaluated in this study both: the LAB supernatants (Table 2) and their produced BLIS (Table 3). The lactic acid bacteria strains were shown the different antimicrobial activities. All ve LAB strains supernatants effectively inhibited the growth of gram positive B. subtilis strains, B. thuringiensis, and gram negative S. typhimurium, P. gladioli pv. aliicola 3.1, P. cepacia 1.1, P. uorescens biovar. V 3.3, P. marginalis 3.5, P. uorescens biovar. V 1.2, P. facilis 4.1, P. aureofaciens biovar. III 2.4.1, P. marginalis 4.2, P. cichorii 7.3.1, P. uorescens biovar. III 2.1.1.1.1 strains in varies degree (the diameters of the inhibition zones varied between 6.5 0.6 mm and 22.5 0.6 mm). Highest antimicrobial activity show P. pentosaceus KTU05-10 against P. pseudoalcaligenes 11 and P. cepacia 7.2 (inhibition zone diameter was 24.0 0.7 mm and 27.5 0.7, respectively). P. acidilactici KTU05-7 inhibited the L. monocytogenes 1.1 and B. cereus growth (inhibition zone diameter was 17.0 0 mm and 9.5 0.6 mm, respectively). P. acidilactici KTU05-7 and L. sakei KTU05-6 show antimicrobial activities against E. coli 1.10 (inhibition zone diameter was 14.3 0.6 mm and 13 0 mm, respectively), whereas E. coli ATCC 25922 was inhibited by P. acidilactici KTU05-7, L. sakei KTU05-6 and P. pentosaceus KTU05-10 (inhibition zone diameter observed 8.3 0.5 mm, 7.0 0 mm and 8.8 0.5 mm, respectively). Antimicrobial activities of L. sakei KTU05-6 supernatants against some Pseudomonas spp. and B. thuringiensis after 48 h cultivation is shown in Fig. 1. 3.3. Antimicrobial activities of LAB produced BLIS L. sakei KTU05-6 produced BLIS show wide-ranging antimicrobial activities against gram positive and gram negative strains. L. sakei KTU05-6 produced BLIS inhibited both B. subtilis substrains: B. subtilis subsp. subtilis and B. subtilis subsp. spizizenii (inhibition zone diameter was 15.8 0.4 mm and 13.5 0.5 mm, respectively). Among gram negative bacteria 7 from 13 Pseudomonas spp. strains were inhibited by L. sakei KTU05-6 produced BLIS, whereas other tested pathogenic bacteria strains were not affected by this BLIS. P. acidilactici and P. pentosaceus strains produce BLIS were active against gram positive B. subtilis substrains only, whereas P. pentosaceus KTU05-10 produced BLIS additionally inhibited the P. uorescens biovar. V 3.3 growth (inhibition zone diameter was 10.0 0 mm). Antimicrobial activities of LAB produced BLIS are given in Table 3. 3.4. The LAB and BLIS antimicrobial activities against fungi and yeast BLIS producing LAB show fungistatic activities against P. expansum, A. versicolor, A. fumigatus, P. chrysogenum and

542 Table 2 Inhibition of the growth of several bacteria by LAB. Microorganisms

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Zone of inhibition/mm KTU05-6 KTU05-7 17.3 0.5 18.8 0.5 16.5 0.6 15.3 0.5 14.2 0.4 14.8 1.0 12.8 0.4 12.2 0.4 13.2 0.4 14.8 0.8 11.5 0.5 11.5 0.5 14.5 0.5 e 15 0 e 17 0 e 14.3 0.6 8.3 0.5 7.8 0.5 e 9.5 0.6 KTU05-8 14.7 0.5 21.5 0.6 18.5 0.6 14.3 0.5 14.7 0.8 15.2 0.4 15.8 0.8 13.7 0.5 12.5 0.5 13.8 0.4 10.2 0.4 9.2 0.4 12.7 0.5 e e e e e e e 9.5 0.6 e e KTU05-9 17.2 0.4 21.5 0.6 18.5 0.6 14.5 0.5 15.5 0.5 14.7 0.5 13.0 0.9 9.8 0.4 11.7 0.5 16.0 0.6 80 12.3 0.5 16.7 0.5 11 0 27 0 e e e e e 8 0.8 e e KTU05-10 15.7 0.5 22.5 0.6 18.5 0.6 16.5 0.5 15.0 0.6 14.0 0 12.2 0.4 10.3 0.5 13.3 0.5 17.0 0.6 10.5 0.5 12.2 0.8 13.0 0.6 24.0 0.7 27.5 0.7 12 0 e e e 8.8 0.5 6.5 0.6 e e

B. thuringiensis 1.1 B. subtilis subsp. subtilis B. subtilis subsp. spizizenii P. gladioli pv. aliicola 3.1 P. cepacia 1.1 P. uorescens biovar. V 3.3 P. marginalis 3.5 P. uorescens biovar. V 1.2 P. facilis 4.1 P. aureofaciens biovar. III 2.4.1 P. marginalis 4.2 P. cichorii 7.3.1 P. uorescens biovar. III 2.1.1.1.1 P. pseudoalcaligenes 11 Pseudomonas cepacia 7.2 P. uorescens biovar. I L. monocytogenes 1.1 L. monocytogenes 1.5 E. coli 1.10 E. coli ATCC 25922 S. typhimurium ATCC 14028 St. aureus ATCC 25923 B. cereus ATCC 10876

14.2 0.4 17.5 0.6 15.8 0.4 16 0 17 0 14.5 0.5 17.5 0.5 10.2 0.4 13 0 14.2 1.0 8.7 0.5 12.2 0.4 12.3 0.5 e 15.3 0.5 e e e 13 0 70 8.5 0.6 e e

F. culmorum, whereas fungicidal activities were observed against A. niger (Table 4). A very good fungicide inhibition of mycelial growth and sporulation with large clear zones around the punched well against F. culmorum were observed of all tested LAB supernatants and their produced BLIS. Fungistatic activities of all tested LAB supernatants and produced BLIS were observed against A. niger (Fig. 1). Four of ve LAB supernatants and BLIS fungistatic activities were determined against P. expansum, P. chrysogenum. A. fumigatus was suppressed by P. pentosaceus KTU05-8 and KTU05-10 and their produced BLIS. A. alternata, P. cyclopium, A. tereus and R. stolonifer growth were not suppressed by LAB.

L. sakei KTU05-6, P. pentosaceus KTU05-8 and KTU05-10 and their produced BLIS were active against C. parapsilosis, besides L. sakei KTU05-6 and P. pentosaceus KTU05-10 show fungistatic activities against D. hansenii. 3.5. Anti-rope activity of LAB against B. subtilis in baked bread The addition of sourdough prepared with P. pentosaceus KTU059 started had a signicant effect on reducing the bacterial spoilage of bread in compare with bread samples prepared without LAB sourdough. The B. subtilis inoculums level of 5 104 cfu g1 caused rope spoilage in the control bread sample after 1 and 1.5 days of

Table 3 Inhibition of the growth of several bacteria by LAB produced BLIS (sakacin 05-6, pediocin Ac05-7, pediocin 05-8, pediocin 05-9, pediocin 05-10). Microorganisms Zone of inhibition/mm Sakacin 05-6 B. thuringiensis 1.1 B. subtilis subsp. subtilis B. subtilis subsp. spizizenii P. gladioli pv. aliicola 3.1 P. cepacia 1.1 P. uorescens biovar. V 3.3 P. marginalis 3.5 P. uorescens biovar. V 1.2 P. facilis 4.1 P. aureofaciens biovar. III 2.4.1 P. marginalis 4.2 P. cichorii 7.3.1 P. uorescens biovar. III 2.1.1.1.1 P. pseudoalcaligenes 11 Pseudomonas cepacia 7.2 P. uorescens biovar. I L. monocytogenes 1.1 L. monocytogenes 1.5 E. coli 1.10 E. coli ATCC 25922 S. typhimurium ATCC 14028 St. aureus ATCC 25923 B. cereus ATCC 10876 e 15.8 0.4 13.5 0.5 12.5 0.5 7.3 0.5 13 0 13.2 0.8 e 7.3 0.5 13.5 0.5 e 10.2 0.4 e e e e e e e e e e e Pediocin Ac05-7 e 17.5 0.6 13.8 0.5 e e e e e e e e e e e e e e e e e e e e Pediocin 05-8 e 19.3 0.5 14 0 e e e e e e e e e e e e e e e e e e e e Pediocin 05-9 e 19.5 0.6 16.2 0.5 e e e e e e e e e e e e e e e e e e Pediocin 05-10 e 20 0 16.3 0.5 e e 10 0 e e e e e e e e e e e e e e e e e

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Fig. 1. Antimicrobial activity of L. sakei KTU05-6 against Pseudomonas gladioli pv. aliicola 3.1 (A), P. uorescens biovar. V 3.3 (B), B. thuringiensis (C) and A. niger (D) supernatants (top) and BLIS (bottom) after 48 h cultivation.

storage at 30  C and 23  C, respectively (Table 5), whereas bread prepared with 20% P. pentosaceus KTU05-9 as starter did not show visual roping until 4 and 6 days of storage, respectively, at 30  C and 23  C.

3.6. Inhibition of fungi by the LAB on bread surface The P. acidilactici KTU05-7, P. pentosaceus KTU05-8 and KTU0510 single cell suspension, sprayed on bread surface, inhibited the

Table 4 Inhibition of sporulation and growth of several fungi by LAB and antifungal preparations. Microorganisms Inhibitory spectrum of LAB supernatants (S) and neutralized to pH 6.5 supernatants (BLIS)a KTU05-6 S Fungus R. stolonifer 2-KA P. expansum 23L A. niger MD-029 A. versicolor MI-130 A. fumigatus KO-5 P. chrysogenum 48-L A. alternata 1-4U P. cyclopium 21AL A. tereus S-1 F. culmorum L-2 F. solani 4-SA P. brutonii P.3 S. capsularis S.1 Z. bailii Z.1(T) K. marxianus K.7.1(T) S. cerevisiae Sa.1.5(T) C. parapsilosis C.7.2 Y. lipolytica Y.C.6.1 D. hansenii Deb.4(T) BLIS KTU05-7 S BLIS KTU05-8 S BLIS KTU05-9 S BLIS KTU05-10 S BLIS

Yeast

a The following scale was used: () no inhibition, () delay of spore formation, () delay of spore formation with small clear zone of inhibition around the cut well, () a very good inhibition of mycelial growth and sporulation with large clear zones around the cut well.

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Table 5 The effect of P. pentosaceus KTU05-9 as starter for bread roping during 8 days of storage. Amount of sourdough used for bread making, % 10 15 20 0 (Control) Time until rst visual roping, days 23  C 2 3 6 1.5 30  C 2 2 4 1

growing of fungi during 8 days of storage (Table 6), whereas on the control bread sample fungi colonies were detected large after 8 days storage. 4. Discussion Bacterial and fungal spoilage is the main cause of essential economic damage in bakeries and in the food industry (Saranraj & Geetha, 2012). Fungal spoilage is regarded as a source of mycotoxins, involving human health problems (Legan, 1993; Legan & Voysey, 1991). As alternative for reduction of microbial contamination, LAB with high antimicrobial activities may be considered as a bio-conservator. In this study, P. pentosaceus KTU05-10, P. pentosaceus KTU05-9, P. pentosaceus KTU05-8, P. acidilactici KTU05-7 and L. sakei KTU05-6 were screened for antimicrobial activities against pathogenic bacteria and fungi. Results evidenced that the antifungal and antibacterial abilities of LAB were depended on the LAB strain and on the indicator microorganism species and strain. The destruction of the total BLIS activities after treatment with proteinase K, trypsin, pepsin and chymotrypsin indicates that antimicrobial substances produced by the tested LAB have a proteinaceous nature. They might be bacteriocins because protease sensitivity is a key criterion in the characterization of antimicrobial substances as BLIS (Klaenhammer, 1993). In these studies, BLIS were designated as sakacin 05-6, pediocin Ac05-7, pediocin 05-8, pediocin 05-9 and pediocin 05-10. Pediococcus produced BLIS shows a narrow killing spectrum against bacteria and inhibit only B. subtilis strains, whereas L. sakei KTU05-7 produced sakacin 05-6 shows a broad inhibition spectrum against pathogenic gram-positive (B. subtilis) and gram-negative (Pseudomonas spp.) bacteria. L. sakei KTU05-7 and P. acidilactici KTU05-7 inhibit E. coli 1.10 and two tested P. cepacia strains which are able to cause a dangerous illness such as pneumonia (Mahenthiralingam, Urban, & Goldberg, 2005). P. acidilactici KTU05-7 suppressed the growth of L. monocytogenes which is able to causes the disease listeriosis (Ramaswamy et al., 2007). Lavermicocca et al. (2000) studies show that Lactobacillus alimentarius, Lactococcus lactis subsp. lactis, Leuconostoc citreum and Lactobacillus plantarum producing acids were able to inhibit some species of fungi. Strom, Sjogren, Broberg, and Schnurer (2002) isolated L. plantarum producing antimicrobial compounds active against some fungi. Hassan and Bullerman (2008) investigated
Table 6 The effect of LAB suspension for bread surface moulding after 8 days storage. LAB P. acidilactici KTU05-7 L. sakei KTU05-6 P. pentosaceus KTU05-8 P. pentosaceus KTU05-9 P. pentosaceus KTU05-10 Control Mould intensity

Lactobacillus paracasei ssp. tolerans isolated from sourdough bread did not inuence A. niger formation, whereas in our studies LAB showed fungistatic activities, when conidia formation of A. niger was delayed. According to literature the LAB produce organic acids and other small compounds with a low molecular weight during fermentation processes. Schillinger and Villarreal (2010) determinate antimicrobial activity of LAB against Penicillium nordicum, but no conrmed exactly that antifungal activity shows BLIS or bacteriocins. Our tested LAB has been produced organic acids as well as BLIS with antifungal activities in various degree (fungicidal or fungistatic) against most common contaminants from grains such as from e P. expansum, A. niger, A. versicolor, A. fumigatus, P. chrysogenum, F. culmorum, C. parapsilosis, D. hansenii. The presence of BLIS and organic acids by L. sakei KTU05-6, P. acidilactici KTU05-7, P. pentosaceus KTU05-8, KTU05-9 and KTU05-10 is an indication that these bacteria can be used widely in the food industry as a probiotic and as bio-preservatives due to their broad inhibition spectrum. The anti-rope activity of the LAB as to prolong the shelf life of bread is well known and has an impressive record. As shown by a well diffusion assay, the inhibition of B. subtilis strains, causing bread roping, was observed by all ve tested LABs. Antirope activity screening tests in bread, during storage, previously articially contaminated by B. subtilis spores, conrm that the use of an antirope starter made of selected LAB may be considered as a natural and effective method to prevent rope spoilage in wheat bread. These results are in agreement with other authors (Katina, Sauri, Alakomi, & Mattila-Sandholm, 2002; Pepe, Blaiotta, Moschetti, Greco, & Villani, 2003), that by using of microbial cultures in starter preparation the shelf life and safety of bakery products is increased. The results obtained using sprayed LAB suspension on bread surface indicate that effective inhibition of undesirable microorganism could be controlled by LAB for preventing bread moulding. This indicates that P. acidilactici KTU05-7, P. pentosaceus KTU05-8 and KTU05-10 strains might have a potential to use not only as anti-ropiness but moreover as anti-fungal agents in the bread industry. Another obvious application is that it can serve as natural replacement of synthetic food preservatives. Bread moulding suppression using LAB as a starter appeared in literature (Lavermicocca et al., 2000). Instead of using LAB in a starter we sprayed a LAB cells suspension on the bread surface after baking which showed a positive result by prolonging the shelf life of bread to 8 days. The search of a new LAB with a wider spectrum of antimicrobial activities which can be used in human health, agriculture, and food industry is being studied by several groups (Gerez et al., 2009; Schillinger & Villarreal, 2010) and it is outstanding importance. 5. Conclusion According to a large inhibitory spectrum and strong inhibitory activity, our results show that L. sakei KTU05-6, can be applied as food preservative and/or in the protection of human health. The LAB application is wide-ranging and most promising if one considers that the indicator microorganisms isolated from varies vegetables, grain, fruit, meat, oil and water were suppressed. These studies highlight the possibility that food safety and food quality can dramatically be improved by using the LAB with antimicrobial activities as starter for food fermentation. Acknowledgements The research was funded by a grant (project SVE-09/2011 BIOFITAS) from Research Council of Lithuania.

Mould colony description: () no mould; () scarce and small; () large.

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