Scholarly article on topic 'Antifungal activity and identification of Lactobacilli, isolated from traditional dairy product “katak”'

Antifungal activity and identification of Lactobacilli, isolated from traditional dairy product “katak” Academic research paper on "Animal and dairy science"

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Abstract of research paper on Animal and dairy science, author of scientific article — Rositsa Tropcheva, Dilyana Nikolova, Yana Evstatieva, Svetla Danova

Abstract Filamentous moulds are the main spoilage microorganisms, responsible for significant economic losses and several healthy risks in human food chain. The lactic acid bacteria (LAB), especially lactobacilli could be a natural antagonist of these dangerous organisms. In Bulgaria, a very limited data exists on the antifungal activity of LAB microbiota of fermented dairy products. In the present study, four active strains were isolated from traditional fermented curd/yogurt-like product “katak”, produced in Bulgaria from centuries. The new isolates KR3, KR4, KR51 and KR53 were identified by API 50 CH biochemical test and different molecular methods (species-specific PCR, RAPD-PCR and 16S rDNA sequence analysis) as Lactobacillus brevis. According to our knowledge, this is the first data on the molecular characterization of the Lactobacillus microbiota of “katak”. A broad spectrum of antifungal activity of the four L. brevis KR strains against test-cultures representatives of carcinogenic, toxigenic, deteriorative and allergenic fungi from the genera Aspergillus, Fusarium, Penicillium and Trichoderma was estimated. Strains L. brevis KR3, KR4 and KR51 completely suppress the growth of Penicillium claviforme, Aspergillus awamori and Aspergillus niger. With regard to Aspergillus flavus and Trichoderma viride, a lower and strain-specific inhibitory activity was observed. The antifungal activity of our new L. brevis isolates seems to be a promising advantage of these four strains, suggesting their potential applications in different food technologies as bio-preservative agents against moulds.

Academic research paper on topic "Antifungal activity and identification of Lactobacilli, isolated from traditional dairy product “katak”"

Anaerobe xxx (2014) 1-7

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Anaerobe

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Pathogenesis and toxins

Antifungal activity and identification of Lactobacilli, isolated from traditional dairy product "katak"

Rositsa Tropcheva a, Dilyana Nikolova b, Yana Evstatieva b, Svetla Danova a' *

a Department of General Microbiology, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26, Acad. G. Bontchev Str., 1113 Sofia, Bulgaria

b Department of Biotechnology, Faculty of Biology, Sofia University St. Kliment Ohridski, 8, Dragan Tsankov Blvd., 1164 Sofia, Bulgaria

ARTICLE INFO

Article history: Received 12 February 2014 Received in revised form 19 May 2014 Accepted 21 May 2014 Available online xxx

Keywords: Antifungal activity Lactobacillus brevis Katak Aspergillus Fusarium Trichoderma Penicillium

ABSTRACT

Filamentous moulds are the main spoilage microorganisms, responsible for significant economic losses and several healthy risks in human food chain. The lactic acid bacteria (LAB), especially lactobacilli could be a natural antagonist of these dangerous organisms. In Bulgaria, a very limited data exists on the antifungal activity of LAB microbiota of fermented dairy products. In the present study, four active strains were isolated from traditional fermented curd/yogurt-like product "katak", produced in Bulgaria from centuries. The new isolates KR3, KR4, KR51 and KR53 were identified by API 50 CH biochemical test and different molecular methods (species-specific PCR, RAPD-PCR and 16S rDNA sequence analysis) as Lactobacillus brevis. According to our knowledge, this is the first data on the molecular characterization of the Lactobacillus microbiota of "katak". A broad spectrum of antifungal activity of the four L. brevis KR strains against test-cultures representatives of carcinogenic, toxigenic, deteriorative and allergenic fungi from the genera Aspergillus, Fusarium, Penicillium and Trichoderma was estimated. Strains L. brevis KR3, KR4 and KR51 completely suppress the growth of Penicillium claviforme, Aspergillus awamori and Aspergillus niger. With regard to Aspergillus flavus and Trichoderma viride, a lower and strain-specific inhibitory activity was observed. The antifungal activity of our new L. brevis isolates seems to be a promising advantage of these four strains, suggesting their potential applications in different food technologies as bio-preservative agents against moulds.

© 2014 Published by Elsevier Ltd.

1. Introduction

Filamentous moulds and yeasts are the main spoilage organisms of various products such as fermented dairy foods (cheese, yogurt), bread, stored crops and feed hay and silage [1,2]. Moreover, the food/feed contamination with various types of toxigenic moulds is a serious problem nowadays [3]. The moulds' growth causes alterations in the texture and harms the external aspect of the products [2,4] which leads to significant economic losses. Five to ten percent of the world's food production is lost due to fungal contamination [5,6], 27% of the foods produced in U.S. are annually destroyed by fungi [7]. In addition, the toxigenic and spoilage fungi are responsible for numerous diseases and health risks [8], due to the potential production of mycotoxins or allergenic conidia, spores and mycelia [2].

* Corresponding author. Tel.: +359 2 9793119. E-mail addresses: std@microbio.bas.bg, stdanova@yahoo.com (S. Danova).

http://dx.doi.org/10.1016/j.anaerobe.2014.05.010 1075-9964/© 2014 Published by Elsevier Ltd.

The society wants to reduce the chemical additives in products and at the same time, there is a requirement of high quality, preservative-free safety food, with an extended shelf life [9]. Likewise, the problem of increasing antibiotic resistance is worsens and also there are new legislations which has restricted the use of some currently accepted preservatives in different food products [9]. All these important issues raise the need to seek alternative methods of foods/feeds producing and preserving of the human food chain. There is a centuries-old tradition of using lactic acid bacteria (LAB) in food processing. LAB represents the microbial group, which is the most commonly used as protective cultures [10]. Recently, several reports on LAB with antifungal activities and their possible applications as biopreservatives have been published [4,8,11—17]. The LAB belonging to the species Lactobacillus rhamnosus and Lactobacillus fermentum [18], Lactobacillus acidophilus, Lactobacillus plan-tarum strain VTT E78076, L. rhamnosus, Lactobacillus coryniformis subsp. coryniformis, Lactobacillus sanfrancisensis strain CB1, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus lactis subsp. cremoris [19], possess a strain-specific spectrum and mechanism of inhibitory activity against the different moulds

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1 and yeasts respectively. In vitro assays showed the presence of a with glycerol (20% v/v). Before the assays, the strains were twice 66

2 broad antifungal strain-specific spectrum of activity of lactobacilli, pre-cultured under anaerobic condition (BBL® Gas Pak Anaerobic 67

3 based on acids, hydrogen peroxides, diacetyl and/or proteinaceous System Envelopes, Becton Dickinson) in MRS broth, for 24 h at 68

4 active compounds [18—20]. Therefore, a large screening with 37 °C. Exponential Lactobacillus cultures in MRS broth were used as 69

5 different test-cultures is initially needed in order to find active LAB. inoculum for the antifungal tests as follows: aliquots of 100 mL 70

6 Moreover, some habitats are not well-studied in this aspect and sterile skimmed milk 10% w/v (Scharlau, Spain) were inoculated 71

7 they are a promising source of active bio-protective LAB strains. In (1% v/v) with cells suspension, containing harvested and washed 72

8 Bulgaria, a very limited data exists on antifungal activity of lactic (with PBS) Lactobacillus cells (~108 CFU mL-1 from exponential 73

9 acid microbiota of traditional fermented products [21,22]. Our culture of every single strain) and were incubated at 37 ° C for 12 h. 74

10 recent in vitro studies have showed the ability of lactobacilli from 75

11 traditional Bulgarian dairy products to inhibit the growth of 2.3. Characterization and identification of IAB isolates from katak 76

12 different pathogens, food-born and spoilage microorganisms [23] 77

13 and a rich laboratory collection of 219 newly isolated LAB have A combined polyphasic-taxonomic approach for species iden- 78

14 been created [24]. Concerning the species occurrence, L. plantarum tification of the "katak" isolates KR3, KR4, KR51, KR53 was applied 79

15 and L. delbrueckii were more frequently isolated species from arti- as followed: 80

16 sanal samples of Bulgarian white cheese and yogurt, respectively 81

17 [23,24], while in "katak" a variety in LAB microbiota was detected, 82

18 but the species identification was not completed yet. The artisanal 231 Phenotypic characterization of LAB strains 83

19 samples of cheeses, yogurts and "katak" have been proved as a The classical phenotypic characteristics were done according to 84

20 promising source for isolation of new Lactobacillus strains with established phenotypic criteria [27]. The ce» m°rphol°gy of the 85

21 antibacterial [23—25] and antifungal activity [26]. "Katak" (or strains was determined and the strains were tested for Gram and 86

22 krutmatch, kutmatch) is a fermented curd/yogurt-like product with catalase reactions, gas production from glucose and growth ability 87

23 a specific salted milk-acid taste. The recipe is inherited from the in MRS broth at 15 °C for 7 days at 30 °C, 37 °C for 2 days. In 88

24 proto-Bulgarians. Nowadays, the long-lasting "katak" is made in addition, carbohydrate fermentation patterns were established 89

25 some rural regions from leavened ewe's milk. There is no valuable using the API 50CH system (Biomerieux, France). Results from the 90

26 information on its starter/non-starter microbiota. tests were analyzed by the APILAB program version 5.0 and 91

27 In the present study 4 active strains, isolated from traditional compared with the database. 92

28 product "katak" were pre-selected for further characterization. The 93

29 aim was to identify them and to characterize their antifungal 2.3.2. Molecular identification and genotyping of LAB strains 94

30 properties. The taxonomic characterization and identification of the Molecular characterization and genotyping, based on species- 95

31 isolates was an important part of our work, due to the lack of data specific PCR, 16S rDNA sequence analysis and randomly amplified 96

32 on the molecular identification of the microbiota of this traditional polymorphic DNA (RAPD) fingerprinting were applied. Chromo- 97

33 and still insufficiently studied milk product. We examined in vitro somal DNA was isolated according to the modified method of 98

34 their ability to inhibit the mould's growth, including different Delley et al., 1991 [28]. Concentration and DNA purity were deter- 99

35 deteriorative food moulds and toxin-producing aspergilli. mined by absorbance readings at 260/280 nm (NanoDrop 1000, 100

36 Thermo Scientific). One mL of DNA from each strain was used as a 101

37 2. Materials and methods template in 25 mL reaction mixtures, using Ready To Go™ PCR 102

38 beads (Amersham, Biosciences) and PCR cycler (Techne, UK) for all 103

39 2.1. Test-cultures, media and cultivation conditions PCR analyses. The protocol of Guarneri et al. (2001), was applied for 104

40 the Lactobacillus brevis species-specific PCR [29]. RAPD-PCR anal- 105

41 Six fungal species: Aspergillus flavus NBIMCC 916 (National Bank ysis with a primer M13V (MWG-Biotech, Germany) was performed 106

42 for Industrial Microorganisms and Cell Cultures, Sofia, Bulgaria), by the method of Ehrmann et al. [30]. 107

43 Fusarium graminearum ATCC 24373, Aspergillus awamori K1 from DNA from the strains KR3, KR4, KR51, KR53 was amplified using 108

44 the Collection of Department of Biotechnology, Faculty of Biology, the primer set fD1 and rD1, according to the method of Weisburg 109

45 Sofia University, (DB-SU), Aspergillus niger A3 (DB-SU), Trichoderma et al. [31]. Obtained 16S rDNA- PCR products were purified by the 110

46 viride (DB-SU) and Penicillium claviforme (from the laboratory GFX Genomic Blood DNA Purification Kit (Amersham Biosciences) 111

47 collection of The Stephan Angeloff Institute of Microbiology, BAS) and the DNA was used as a template for the standard sequencing 112

48 were used as test-cultures in antifungal assays. They were main- procedure (Macrogen Inc, Seoul, Republic of Korea). The sequences 113

49 tained on Potato Dextrose Agar (PDA, Oxoid, Hampshire, UK) plates were edited to exclude the PCR primer-binding site and manually 114

50 at 29 °C and subcultured on a monthly basis until sporulation. The corrected with Sequence Scanner 1.0 (Applied Biosystems) and 115

51 spores were harvested after establishing a good growth rate of each were compared with the available nucleotide database from the 116

52 of the fungal cultures and were filtered with sterile cotton filter, to NCBI GenBank using the BLAST program. A similarity of >98% to the 117

53 avoid the presence of conidia and mycelia. The spore's suspensions 16S rDNA sequence of the reference L. brevis strain was used as a 118

54 in PBS (pH — 7.0) were adjusted to the final concentrations in the criterion for the identification. The 16S rDNA sequences obtained in 119

55 range of 105—106 spores/mL (A. awamori, A. niger A3, T. viride and this study have been deposited in the NCBI GenBank database. A 120

56 P. claviforme -105; A. flavus and F. graminearum -106 spores/mL). phylogenetic tree was generated from the alignment of the 121

57 deposited sequences by the neighbor-joining method using Sea- 122

58 2.2. Lactic acid bacteria isolates View version 4 [32]. 123

59 Gel electrophoresis (1 and 2.5% w/v agarose gels, Sigma type II, 124

60 Four Lactobacillus strains, called KR3, KR4, KR51 and KR53, from USA) and ethidium bromide staining were done to visualize the 125

61 our laboratory collection of dairy LAB, were pre-selected for the DNA preparations and PCR (species-specific, 16S rDNA gene and 126

62 present study. They were isolated from samples of home-made RAPD) products. A GenLadder 100 bp + 1.5 kbp (Gennaxon, Ger- 127

63 dairy product "katak" from the Rodopa Mountain, region of Tri- many) was used. RAPD profiles were compared using Bionumerics 128

64 grad [26]. The pure cultures were stored at -20 °C in de Man, software (version 6.6, Applied Maths, Canada) and similarities were 129

65 Rogosa and Sharpe (MRS, Merck, Germany) broth, supplemented expressed based on Dice coefficient. 130

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2.4. In vitro antifungal test

A modified protocol of agar-layer diffusion method was applied as followed: Overnight Lactobacillus cultures on skimmed milk were mixed with temperate PDA agar at 45 ° C in equal volumes and poured into plates. The agar plates, with sterile skimmed milk instead of LAB cultures, were prepared as a control. After drying of the plates, 3 ml of mould spores suspension was inoculated as a discrete spot onto the centre of the surface of the agar layer in each plate. The plates were incubated aerobically in an upright position at 29 °C. Diameters of the growing mould colonies were measured daily until the mould in the control sample completely filled the volume of the petri dishes. Experiments were performed in triplicate.

2.5. Statistical analysis

Statistical evaluation of the moulds growth changes, between the control and the samples (with L. brevis KR cultures), was carried out using two tailed Student's t-test analysis and GraphPad Prism

4.0. A p-value below 0.05 was considered to be statistically significant.

3. Results

3.1. Identification and molecular typing of LAB isolates

The four LAB (KR3, KR4, KR51 and KR53), isolated from "katak", were identified by a simultaneous application of classical and different molecular methods, according to the polyphasic taxonomy. The strains were characterized as Gram-positive, small rod shape, catalase-negative, non-motile and non-spore forming bacteria. They produce gas from glucose and grow well in MRS broth (pH 5.4—6.5) at temperature of 37 °C in microaerophilic and anaerobic conditions, which correspond well to the group III — heterofermentative lactobacilli [27]. The four KR strains were classified to the species L. brevis, based on their carbohydrate fermentation API 50CHL patterns (Table 1). Despite of the differences of the probability (from low to excellent) of species identification, the results allowed the selection of primers and type of appropriate molecular methods for further species identification.

The species-specific PCR analysis (according to Guarneri et al., 2001) [29] showed amplified products with a size of about 1340 bp, which are identical to the amplified product of the L. brevis ATCC 27305 (Fig. 1). The phenotypic similarity and the outcome of this analysis allow us to confirm the initial affiliation of the isolates KR3, KR4, KR51 and KR53 to the species L. brevis. However, each of our strains generated a weak additional PCR product (~800 bp) which was not detected for the type culture (Fig. 1). In addition, a strain characteristic distinction of the reference culture L. brevis ATCC 27305 and simultaneously a high degree of genetic similarity between the isolates KR3 and KR53, KR4 and KR51 respectively were observed from the RAPD-PCR analysis (Fig. 2).

The 16S rDNA sequence analysis was the final step in our identification protocol of the isolates from "katak". BLAST analyses

Fig. 1. Agarose gel electrophoresis (1% w/v) of the PCR products obtained with species-specific primers for L. brevis (Guarneri at al., 2001): 1 — L. brevis ATCC 27305T; 2 — KR3; 3 — KR4; 4 — GenLadder 100 bp + 1.5 kbp (Gennaxon, Germany); 5 — KR51; 6 — KR53; 7 — a PCR negative control (no DNA added).

of the resulting sequences with the available nucleotide database in the GenBank, showed a 100% similarity of the KR strains with the species L. brevis (Table 1 and Fig. 3). The comparative analysis of the L. brevis KR sequences with published sequences of different Lactobacillus species, using the SeaView v. 4 program confirmed this similarity with the L. brevis ATCC14869 type strain, and demonstrated the phylogenetic distances in a generated neighbor-joining tree (Fig. 3).

3.2. In vitro tests for moulds' growth inhibition

The newly identified L. brevis strains were selected after a preliminary screening for antibacterial and antifungal activity of several LAB isolates from traditional Bulgarian milk products [25,26]. In the current work, we estimate the spectrum of antifungal activity of the four L. brevis strains (KR3, KR4, KR51 and KR53) against members of mould genera Aspergillus, Penicillium, Fusarium and Trichoderma. In order to approximate the test's conditions to real dairy product — the world famous Bulgarian yogurt, an optimized laboratory protocol was applied. As it is indicated in Fig. 4, a strong antifungal activity of L. brevis KR3, KR4, KR51 and KR53 against A. awamori (Fig. 4A) and P. claviforme (Fig. 4B) was detected in triplicate repeated tests. A full inhibition in most of the samples with A. niger (with the exception of L. brevis KR53 (Fig 5A) and F. graminearum (with the exception of L. brevis KR4) (Fig. 5D) was also obtained. A retarded T. viride spores' germination and

Table 1

Identification of the newly isolated lactobacilli from "katak" by classical biochemical test and 16S rDNA sequence analysis.

60 Strain Identified as: Probability [%] according Probability [%] based on the NCBI-GenBank acc. number

61 to APILAB identification BLAST analysis of the sequences

62 KR3 Lactobacillus brevis 99.4 (very good) 100 HM568883

63 KR4 Lactobacillus brevis 99.9 (excellent) 100 HM568884

64 KR51 Lactobacillus brevis 72.2 (low) 100 HM568885

65 KR53 Lactobacillus brevis 99.9 (excellent) 100 HM568886

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Fig. 2. (A) DNA fingerprints obtained with M13V primer in RAPD-PCR analysis and visualized in 2% agarose gel, after ethidium bromide staining: 1 — GenLadder 100 bp + 1.5 kbp (Gennaxon, Germany); 2 — L. brevis ATCC 27305T; 3 — KR53; 4 — KR4; 5 — KR3; 6 — KR51. (B) A dendrogram derived from computer cluster analyses of the digitalized images.

colony growth was observed (Fig. 5C). The four lactobacilli retain the fungal colonies less than 1 cm by the end of the experiment in which the control fills the entire surface of the petri dish (Fig. 5C). Moreover, L. brevis KR strains prevent the abundant conidial germination (sporogenesis) which is well-demonstrated in the control samples of T. viride, A. niger, A. awamori, P. claviforme and A. flavus (Figs. 4 and 5).

Fig. 3. Phylogenetic tree based on the Lactobacillus 16S rDNA sequences of the four KR isolates from traditional dairy product "katak". The scale bar represents relative sequence similarities.

4. Discussion

4.1. Identification of LAB from "katak"

The current study presents results of the characterization of lactobacilli with antifungal activity from traditional Bulgarian dairy product "katak". There is no systematic study on the lactic micro-biota of this appetizing product. Thus, the characterization of LAB's diversity and their antagonistic properties is a challenge for keeping the centuries-old tradition of safety foods. The polyphasic approach was applied as the basis of a contemporary identification of Lactobacillus species. The biochemical API 50 CH tests matched the isolates to L. brevis with a variable and insufficient degree of probability (in regard to strain KR51) (Table 1). The reliability of these tests in the case of LAB isolates from not-well studied habitats has been questioned and some controversial results were reported [33]. Thus, more discriminative molecular approaches are also needed. The species-specific PCR is one of the methods with a proven effectiveness in bacterial identification and differentiation of phenotypically similar species that are difficult to distinguish by the conventional microbiological approaches [34]. We applied the method of Guarneri et al., 2001 and RAPD-PCR analysis, which is applied with success in the identification of lactobacilli from the species L. brevis [29,35,36]. The discrepancy in PCR profiles of L. brevis, as those that we obtained (Figs. 1 and 2), have been reported in the literature and their additional analysis led to identification of two new species [35]. Therefore, we combine species-specific PCR (Fig. 1) with a high-discriminative RAPD-PCR (Fig. 2) and subsequently 16S rDNA sequencing analysis (Table 1). RAPD-PCR analysis was applied to generate species/strain-specific DNA profiles. Depending on the type of primers and conditions for the RAPD-PCR, it is possible to generate adequate amplification profiles through which can be identified the new isolates to the level of genus, species and even strain [37—39]. Identification of L. brevis by RAPD-PCR has been reported for isolates from cheese, beer [40], dry fermented sausages [36] and eggplants [41]. Our results from RAPD-PCR analysis suggest very satisfactory differentiation on strain and species level (Fig. 2A and B). Cluster analysis of RAPD fingerprints grouped our isolates with a low degree of similarity (D < 50) with the type culture L. brevis ATCC 27305T (Fig. 2B). RAPD-PCR is a commonly used method for typing of lactobacilli in different ecological niches [36,42]. Based on RAPD segregation as a primary genotyping method, Valcheva et al. [35] identified isolates from sourdough as L. brevis — like. Later the same cultures have been reclassified as members of the new species Lactobacillus nantensis who is phylogenetically closely to L. brevis [35]. There are other cases of reclassification of strains which were considered to be L. brevis as other type heterofermentative species like

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Fig. 4. Antifungal activity of skimmed milks, fermented with L. brevis KR3, KR4, KR51, KR53, against Aspergillus awamori (A), Penicillium claviforme (B), as determined by hyphal radial growth inhibition after 5—11 days of incubation at 29 °C compared with a control. Results are presented as mean (SD), n = 3.

Lactobacillus hilgardii, Lactobacillus kefir, Lactobacillus confuses and Lactobacillus colinoides [43]. Obtained RAPD-PCR results (Fig. 2) confirmed our initial identification of Lactobacillus KR3, KR4, KR51 and KR53 and the observed differences (Fig. 2A) in the strains profiles are probably due to the reported diversity within the species L. brevis.

16S rDNA sequence analysis uniquely identified the strains KR3, KR4, KR51 and KR53 as representatives of the species L. brevis with probability of 100% (Table 1 and Fig. 3). For a successful inclusion in the species, more than 98% similarity to the consensus sequence of the 16S rRNA gene is required [44]. The phylogenetic tree showed the high similarity with the type strain L. brevis ATCC 11842 and the evolutionary distance with several Lactobacillus species (Fig. 3).

To our knowledge, this is the first data on the molecular characterization of Lactobacillus microbiota of "katak" and additional complex evaluation of the LAB of this product is still in progress. This traditional Bulgarian product is poorly studied and the presence of enterococci only has been reported [22].

4.2. Inhibition of mould growth

A broad spectrum of antifungal activity of the four newly identified as L. brevis strains - KR3, KR4, KR51 and KR53 was estimated. Our in vitro studies were performed with fermented milks, in order to simulate a real dairy product (yogurt) and to assess the bio-protective potential of the tested lactobacilli. The used test-cultures are representatives of carcinogenic, toxigenic, deteriorative and allergenic fungi from the genera Aspergillus, Fusarium, Penicillium and Trichoderma. They are associated with the food chain contaminations and responsible for several human's health problems [2,16]. Fermented dairy products, like many others foods, are exposed to spoilage organisms and pathogens. The low pH values caused by the growth of LAB in fermented milk make these foods a very suitable habitat for the fungus proliferation [45]. The conducted experiments with a modified mono-layer agar plate method showed a strain-specific capacity of the tested lactobacilli to prevent the growth of some of these spoilage microorganisms.

Fig. 5. Antifungal activity of skimmed milks, fermented with L. brevis KR3, KR4, KR51, KR53, against Aspergillus niger (A), Aspergillus flavus (B), Trichoderma viride (C) Fusarium graminearum (D), as determined by hyphal radial growth inhibition after 5—6 days of incubation at 29 °C compared with a control. Results are presented as mean (SD), n = 3, t-test *p < 0.05 and **p < 0.001.

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Strains L. brevis KR3, KR4 and KR51 completely suppress the growth of P. claviforme, A. awamori and A. niger (Figs. 4 and 5A). While, in the sample with L. brevis KR3, a retarded and weak growth of A. niger (Fig. 5A) was observed. However, the spore germination and the colony growth started only on the fifth day of the mould-lactobacilli co-cultivation, which also should be considered as a good result.

In the literature, most of the active antifungal strains in fermented milk products were related to the L. casei group [16]. The displayed strong ability to inhibit species of genus Aspergillus (Figs. 4 and 5A) is a good testimonial to our L. brevis isolates from "katak" since these moulds often show resistance [46] and the inhibition of their growth is usually a difficult task. In the scientific literature there is a little data on the inhibition of aspergilli by representatives of the species L. brevis, and especially a successful one [47—49].

The partial inhibition of mycelial growth and conidia germination of A. flavus by KR53 and especially by KR51 (Fig. 5B) is a very promising result, since A. flavus is now considered as the second leading cause of aspergillosis in human and among the most toxic and carcinogenic representatives of the micromycetes [50]. A successful control of A. flavus growth by L. brevis is reported by Cassandra De Muynck et al., 2004 [51].

In our experiments, it was found that the presence of four Lactobacillus strains, cultivated in skimmed milk, inhibit the P. claviforme growth (Fig. 4B). There are limited published data on the inhibition of Penicillium spp. by L. brevis [49,51].

Fusarium species are often found in foods and feeds, especially on cereal grains [52]. The demonstrated within this study ability of the new isolated LAB strains KR3, KR51 and KR53 to suppress the growth of F. graminearum (Fig. 5D) confirms the already published data on the successful inhibition of Fusarium spp. by L. brevis [48,53].

T. viride is one of three Trichoderma spp. (T. viride, T. harzianum, and T. koningii) that are usually found in indoor environments on buildings [54], which is a potential factor for occurrence of undesirable contamination of different foods. Trichoderma spp. has been reported to be allergenic and capable to produce toxins [54] and antibiotics which are toxic to humans, so from these perspectives the inhibition of the contaminants from this genera is also an important task. According to our knowledge, the results (Fig. 5C) with L. brevis KR51 (full inhibition) and KR3, KR4 and KR53 (partial inhibition and delay of sporogenesis) are the first published data on the inhibition of Trichoderma species by LAB cultures, in particular by species L. brevis. The prevention of conidia germination and sporogenesis is an important outcome since most spores are allergens.

The exponential L. brevis KR cultures in MRS broth demonstrated a complete suppression of the growth and sporogenesis of P. claviforme, T. viride and A. niger within our previous work, visualized by a modified dual-agar plate assays with MRS/PDA nutrient media [26]. In the present study the only difference is the replacement of the MRS broth with milk, fermented by the appropriate KR strain. Therefore, the incomplete sample inhibition (Fig. 5) already commented on, could be also a result of the specific metabolic activity of tested in milk LAB. Thus, the presence of some milk ingredients, or metabolites produced by lactic acid fermentation are suspected to function as a potential fungal growth factor. For example, contrary to most claims that lactic acid is an inhibiting factor, some authors demonstrate the hypothesis that it is a promoter for fungal growth in extreme environmental conditions [55,56].

L. brevis is a representative of the heterofermentative LAB [27] which are capable to produce a wide range of different active metabolites, mostly organic acids and ethanol. Therefore, the reported

strong antifungal activity is probably due to their synergistic effect. Eight different strains of L. brevis, from brewing barley and sourdough are producers of proteinaceous compound and organic acids and express a strain-specific broad spectrum of activity against A. flavus, Fusarium culmorum, Penicillium sp., Rhizopus oryzae, Eurotium repens, Trichophyton tonsurans [48,51,53,57].

Only a few papers report a broad spectrum of inhibitory effects of LAB against micromycetes [12,19,20]. Most of the studies revealed a strain-specific antifungal activity against species of one, maximum two major mould genera [48,58—62].

5. Conclusion

In this study four isolates KR3, KR4, KR51 and KR53 from the traditional Bulgarian dairy product "katak" have been identified as L. brevis and characterized as cultures with promising antifungal activity. Obtained results from the combined polyphasic taxonomic approach contribute to give new data on the microbial biodiversity of this not well-studied niche. The antifungal activity of our new isolates, identified as L. brevis, seems to be a promising advantage of these four strains, suggesting their potential applications in different food technologies. However, more experiments have to be conducted to clarify the nature and the mechanisms of the reported antifungal activity and they are still in progress. The combination of dairy origin and strong inhibitory activity of the KR strains is a prerequisite for their possible application as starters and/or bio-protective antifungal adjuncts.

Acknowledgements

The publication of this study was supported by grant BG051PO001-3.3-05/0001, Science Business Project, financed by "Human Resources Development" Operational Programme.

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