Insight into the Postbiotic Potential of the Autochthonous Bacteriocin-Producing Enterococcus faecium BGZLM1-5 in the Reduction in the Abundance of Listeria monocytogenes ATCC19111 in a Milk Model.

This study aimed to explore the probiogenomic characteristics of artisanal bacteriocin-producing Enterococcus faecium BGZLM1-5 and its potential application in reducing Listeria monocytogenes in a milk model. The BGZLM1-5 strain was isolated from raw cow's milk from households in the Zlatar Mountain region. The whole genome sequencing approach and bioinformatics analyses reveal that the strain BGZLM1-5 is non-pathogenic to humans. Bacteriocin-containing supernatant was thermally stable and antimicrobial activity retained 75% of the initial activity compared with that of the control after treatment at 90 °C for 30 min. Antimicrobial activity maintained relative stability at pH 3-11 and retained 62.5% of the initial activity compared with that of the control after treatment at pH 1, 2, and 12. The highest activity of the partially purified bacteriocin was obtained after precipitation at 40% saturation with ammonium sulfate and further purification by mixing with chloroform. Applying 3% and 5% (v/v) of the bacteriocin-containing supernatant and 0.5% (v/v) of the partially purified bacteriocin decreased the viable number of L. monocytogenes ATCC19111 after three days of milk storage by 23.5%, 63.5%, and 58.9%, respectively.

Evaluation of Post-Larval Diets for Indoor Weaned Largemouth Bass (Micropterus salmoides).

This study aimed to evaluate different commercial diets (Otohime C1, Aller Futura (AF), Biomar Inicio Plus (BIP)) and one experimental feed (EF) in terms of their effectiveness as post-larval diets for indoor weaned largemouth bass, LMB (Micropterus salmoides). Key variations in the content of nutritive values were monounsaturated fatty acid (MUFA) and highly unsaturated FA (HUFA) ω3. Fish were fed with one of four tested diets from the 33rd to the 40th day post-hatch (DPH). Biometric indices, digestive enzyme-specific activities, thyroid hormone status, and mRNA expression of genes coding for skeleton, neuron, and muscle growth were analyzed. The lowest skeletal deformity rate and highest survival among the treatments were seen in BIP-fed fish. Dietary lipids, with an appropriate balance between MUFA and polyunsaturated FA (PUFA), alongside amino acid balance, were shown to be the main contributors to the growth of the skeleton and/or fish survival. On the other hand, fish growth is correlated with fish digestive capacity and feed moisture percent rather than feed quality. Unexpectedly, BIP-fed fish were attributed with the lowest expression of skeleton differentiation markers, which may reflect the sacrifice of scale and/or cranium growth at the expense of somatic growth. This study highlights the role of non-marine ingredients in the nutrition of post-larval LMB.

Assessment of stability and bioactive compounds in yogurt containing novel natural starter cultures with the ability to promote longevity in Caenorhabditis elegans.

Yogurt represent one of the oldest fermented foods containing viable lactic acid bacteria and many bioactive compounds that could exhibit beneficial effects on human health and train our immune system to better respond to invading pathogens. Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus are commonly used for yogurt preparation under controlled temperature and environmental conditions. In this study, we investigated probiotic features of S. thermophilus BGKMJ1-36 and L. bulgaricus BGVLJ1-21 strains isolated from artisanal sour milk and yogurt by using Caenorhabditis elegans as an in vivo model system. Further, we evaluated content of total fat, saturated fatty acids, proteins, and lactose, as well as vitamins and AA of yogurt prepared from above-mentioned starter cultures during 21 d of storage at 4°C to get insights of final product stability. We showed that S. thermophilus BGKMJ1-36 and L. bulgaricus BGVLJ1-21 strains applied in combination upregulated the expression of autophagy-related genes in C. elegans. Beside autophagy, we observed activation of TIR-1-dependent transcription of lysozyme-like antimicrobial genes involved in the immune defense of C. elegans. Upregulation of these genes strongly correlates with an increase in the longevity of the worms fed with yogurt culture bacteria. Further, we showed that yogurt prepared with S. thermophilus BGKMJ1-36 and L. bulgaricus BGVLJ1-21, as a final product, is rich with vitamin B2 and dominant AA known by their prolongevity properties. Taken together, our study pointed to the beneficial features of the tested starter cultures and yogurt and highlighted their potential to be used as a fermented food with added-value properties.

Enterococci from Raw-Milk Cheeses: Current Knowledge on Safety, Technological, and Probiotic Concerns.

The present study is focused on the safety, technological characteristics, and probiotic evaluation of Enterococcus species from different artisanal raw milk dairy products, mainly cheeses with ripening. Apart from proteolytic and lipolytic activities, most enterococci show the ability to metabolize citrate and convert it to various aromatic compounds. Long-ripened cheeses therefore have a specific flavor that makes them different from cheeses produced from thermally treated milk with commercial starter cultures. In addition, enterococci are producers of bacteriocins effective against spoilage and pathogenic bacteria, so they can be used as food preservatives. However, the use of enterococci in the dairy industry should be approached with caution. Although originating from food, enterococci strains may carry various virulence factors and antibiotic-resistance genes and can have many adverse effects on human health. Still, despite their controversial status, the use of enterococci in the food industry is not strictly regulated since the existence of these so-called desirable and undesirable traits in enterococci is a strain-dependent characteristic. To be specific, the results of many studies showed that there are some enterococci strains that are safe for use as starter cultures or as probiotics since they do not carry virulence factors and antibiotic-resistance genes. These strains even exhibit strong health-promoting effects such as stimulation of the immune response, anti-inflammatory activity, hypocholesterolemic action, and usefulness in prevention/treatment of some diseases.

Antioxidant and Antiproliferative Activity of Allium ursinum and Their Associated Microbiota During Simulated in vitro Digestion in the Presence of Food Matrix.

In this study, for the first time, the comprehensive analysis of antiproliferative and antioxidant activities of ramson, followed by the analysis of its associated microbiota and health-promoting effects of lactic acid bacteria (LAB), was performed. Ramson (Allium ursinum) is recognized as a medicinal plant with a long history of use in traditional medicine due to its antimicrobial and antioxidant activity. In this study the influence of in vitro gastrointestinal digestion on the cytotoxic activity of A. ursinum extracts against human malignant cell lines was demonstrated. Seven sulfur compounds, the degradation products of thiosulfinates, including diallyl disulfide were shown to inhibit proliferation of malignant cells by inducing accumulation within G2/M phase as well as to induce apoptosis through activation of caspase-3 and mitochondrial signaling pathway. Further, the A. ursinum microbiota, particularly LAB with potential probiotic effects, was analyzed by culture-dependent method and culture-independent method [denaturing gradient gel electrophoresis (DGGE)]. The obtained results revealed that the most abundant genera were Streptococcus, Lactobacillus, and Bacillus. The Lactobacillus genus was mainly represented by L. fermentum. The pulsed-field gel electrophoresis (PFGE) analysis revealed the presence of two PFGE pulsotypes. The probiotic potential of the strain L. fermentum BGSR163 belonging to PFGE pulsotype 1 and the strain L. fermentum BGSR227 belonging to the PFGE pulsotype 2 was characterized. The results revealed that both strains are safe for human use, successfully survive the simulated gastrointestinal conditions, have potential to transiently colonize the gastrointestinal tract (GIT) and have a protective immunomodulatory effect, inducing the production of proinflammatory cytokine IL17 and regulatory cytokine IL10, while decreasing the production of proinflammatory cytokine IFN-γ. In conclusion, the results of this study suggest that consumption of A. ursinum might have health-promoting properties, including anticancer effects, while L. fermentum strains isolated from A. ursinum leaves could be used as probiotics for human consumption.

Yogurt Produced by Novel Natural Starter Cultures Improves Gut Epithelial Barrier In Vitro.

Yogurt is a traditional fermented dairy product, prepared with starter cultures containing Streptococcus thermophilus and Lactobacillus bulgaricus that has gained widespread consumer acceptance as a healthy food. It is widely accepted that yogurt cultures have been recognized as probiotics, due to their beneficial effects on human health. In this study, we have characterized technological and health-promoting properties of autochthonous strains S. thermophilus BGKMJ1-36 and L. bulgaricus BGVLJ1-21 isolated from artisanal sour milk and yogurt, respectively, in order to be used as functional yogurt starter cultures. Both BGKMJ1-36 and BGVLJ1-21 strains have the ability to form curd after five hours at 42 °C, hydrolyze αs1-, ß-, and κ- casein, and to show antimicrobial activity toward Listeria monocytogenes. The strain BGKMJ1-36 produces exopolysaccharides important for rheological properties of the yogurt. The colonies of BGKMJ1-36 and BGVLJ1-21 strains that successfully survived transit of the yogurt through simulated gastrointestinal tract conditions have been tested for adhesion to intestinal epithelial Caco-2 cells. The results reveal that both strains adhere to Caco-2 cells and significantly upregulate the expression of autophagy-, tight junction proteins-, and anti-microbial peptides-related genes. Hence, both strains may be interesting for use as a novel functional starter culture for production of added-value yogurt with health-promoting properties.

Diversity of non-starter lactic acid bacteria in autochthonous dairy products from Western Balkan Countries - Technological and probiotic properties.

The aim of this review was to summarize the data regarding diversity of non-starter lactic acid bacteria (NSLAB) isolated from various artisanal dairy products manufactured in Western Balkan Countries. The dairy products examined were manufactured from raw cow's, sheep's or goat's milk or mixed milk, in the traditional way without the addition of commercial starter cultures. Dairy products such as white brined cheese, fresh cheese, hard cheese, yogurt, sour cream and kajmak were sampled in the households of Serbia, Croatia, Slovenia, Bosnia and Herzegovina, Montenegro, and North Macedonia. It has been established that the diversity of lactic acid bacteria (LAB) from raw milk artisanal dairy products is extensive. In the reviewed literature, 28 LAB species and a large number of strains belonging to the Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Pediococcus, Leuconostoc and Weissella genera were isolated from various dairy products. Over 3000 LAB strains were obtained and characterized for their technological and probiotic properties including: acidification and coagulation of milk, production of aromatic compounds, proteolytic activity, bacteriocins production and competitive exclusion of pathogens, production of exopolysaccharides, aggregation ability and immunomodulatory effect. Results show that many of the isolated NSLAB strains had one, two or more of the properties mentioned. The data presented emphasize the importance of artisanal products as a valuable source of NSLAB with unique technological and probiotic features important both as a base for scientific research as well as for designing novel starter cultures for functional dairy food.

Solid state treatment with Lactobacillus paracasei subsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 improves nutrient bioavailability in granular fish feed.

The aim of this research was to improve nutritive value of fishmeal-based feed by lactobacilli in order to achieve satisfactory nutrient availability needed to support fish development. Feed was solid-state treated at a laboratory scale with the combination of Lactobacillus paracasei subsp. paracasei BGHN14 and Lactobacillus rhamnosus BGT10 in different experimental settings, which included the variation of strain ratio, total lactobacilli concentration, percentage of moisture and duration of incubation. Short peptides, soluble proteins, phospho-, neutral and unsaturated lipids were quantified. Differences among treated and control feeds were evaluated by Student t-test, while Gaussian process regression (GPR) modeling was employed to simulate the incubation process and define the optimal treatment combination in the context of overall feed nutritional profile. Treatment duration was shown to be the critical determinant of final outcome, either as single factor or via interaction with strain ratio. Optimal nutrient balance was achieved with 12 h incubation period, 260% moisture, 75:25 and 50:50 BGHN14:BGT10 ratios and 200 mg of lactobacilli per g of dry feed. This study should serve as the basis for large-scale tests which would simulate on-farm production of both fishmeal-based and unconventional, lower cost aquafeed with added value.

The Influence of Heat-Killed Enterococcus faecium BGPAS1-3 on the Tight Junction Protein Expression and Immune Function in Differentiated Caco-2 Cells Infected With Listeria monocytogenes ATCC 19111.

Listeria monocytogenes, the common foodborne pathogenic bacteria species, compromises the intestinal epithelial barrier, leading to development of the listeriosis, a severe disease especially among immunocompromised individuals. L. monocytogenes infection usually requires antibiotic treatment, however, excessive use of antibiotics promotes emergence of antibiotic resistance and the destruction of gut microbiota. Probiotics, including lactic acid bacteria (LAB), have been repeatedly proven as an alternative approach for the treatment of various infections. We have analyzed the potential of Enterococcus faecium BGPAS1-3, a dairy isolate exhibiting strong direct antilisterial effect, to modulate the response of differentiated Caco-2 intestinal epithelial cells to L. monocytogenes ATCC 19111 infection. We showed that the molecule with antilisterial effect is a bacterial cell-wall protein that is highly resistant to the high-temperature treatment. When we tested the antilisterial potential of heat-killed BGPAS1-3, we found that it could prevent tight junction disruption in differentiated Caco-2 monolayer infected with L. monocytogenes ATCC 19111, induce antilisterial host response mechanisms, and stimulate the production of protective TGF-ß in intestinal epithelial cells. We also showed that the modulation of MyD88 dependent TLR2 and TLR4 pathways by BGPAS1-3 are involved in host response against L. monocytogenes ATCC 19111. Since heat-killed BGPAS1-3 possess strong antilisterial effects, such postbiotic could be used as a controllable and safe therapeutic.

AggLr, a novel aggregation factor in Lactococcus raffinolactis BGTRK10-1: its role in surface adhesion.

The ability of lactic acid bacteria to form multi-cellular aggregates via self-aggregation is regarded as an important mechanism for stress tolerance, adhesion, colonization and genetic material exchange. The novel aggLr gene encoding for the auto-aggregation promoting protein (AggLr) of Lactococcus raffinolactis BGTRK10-1 was cloned. Heterologous expression of AggLr enabled auto-aggregation, higher hydrophobicity and collagen and fibronectin binding of the carrier strains. Domain analysis and the type of aggregates formed by cells expressing AggLr confirmed that this aggregation factor belongs to the family of high molecular weight proteins that the authors propose to be called Snow-flake Forming Collagen Binding Aggregation Factors (SFCBAF). An additional feature of SFCBAF is that they are rich in threonine and lysine and are free of cysteine in all of the aggregation factors described so far. In contrast to previously discovered SFCBAF, the gene encoding for AggLr is located on the chromosome in the strain BGTRK10-1.

New Insight into Biofilm Formation Ability, the Presence of Virulence Genes and Probiotic Potential of Enterococcus sp. Dairy Isolates.

Enterococci have controversial status due to their emerging role in nosocomial infections and transmission of antibiotic resistance genes, while some enterococci strains are used as probiotics for humans and animals and starter cultures in dairy industry. In order to improve our understanding of factors involved in the safe use of enterococci as potential probiotics, the antibiotic susceptibility, virulence and probiotic traits of 75 dairy enterococci isolates belonging to Enterococcus durans (50), En. faecium (15), En. faecalis (6), En. italicus (3), and En. hirae (1) were evaluated. The results revealed that ciprofloxacin resistance and biofilm formation are correlated with isolates originated from Golija mountain (Serbia), while gelatinase activity was more common in isolates from Prigorje region (Croatia), pointing to uncontrolled use of antibiotics and anthropogenic impact on dairy products' microbiota in these regions. The virulence genes were sporadically present in 13 selected dairy enterococci isolates. Interestingly, biofilm formation was correlated with higher ability of strains to reduce the adhesion of E. coli and Salmonella Enteritidis to HT29-MTX cells. To our knowledge this is the first study reporting the presence of the esp gene (previously correlated with pathogenesis) in dairy enterococci isolates, mostly associated with the genes involved in adhesion property. Hence, the results of this study revealed that the virulence genes are sporadically present in dairy isolates and more correlated to adhesion properties and biofilm formation, implicating their role in gut colonization rather than to the virulence traits.

Promotion of Early Gut Colonization by Probiotic Intervention on Microbiota Diversity in Pregnant Sows.

The aim of this work was to design a novel mixed probiotic culture for piglets and to evaluate its beneficial effect on the piglets' gut health. The possible mechanisms of probiotic activity, such as adhesion, competitive pathogen exclusion and influence on gut microbiota diversity were determined. Mixed probiotic starter culture is composed of three thermophilic lactic acid bacteria (LAB) strains: Lactobacillus helveticus BGRA43, Lactobacillus fermentum BGHI14 and Streptococcus thermophilus BGVLJ1-44. The strains BGVLJ1-44 and BGRA43 showed good technological properties (fast milk curdling, strong proteolytic activity). In addition, the strain BGVLJ1-44 produces exopolysaccharide (EPS), BGHI14 is heterofermentative LAB strain with significant immunomodulatory effect, while the strain BGRA43 showed strong antimicrobial activity against different pathogens and exhibited significantly higher level of adhesion to Caco-2 cells comparing to other two strains. Both lactobacilli strains BGRA43 and BGHI14 (p < 0.05), as well as probiotic combination (p < 0.01) significantly reduced the adhesion of Escherichia coli ATCC25922 to Caco-2 cells, while the strains BGVLJ1-44 (p < 0.01) and BGRA43 (p < 0.05) significantly reduced adhesion of Salmonella 654/7E (veterinary isolate). The results of farm trial revealed that treatment of sows with new fermented dairy probiotic influenced the piglets' gut colonization with beneficial bacteria and reduced the number of enterobacteriaceae in litters from some treated sows (no significant due to high variability among animals). Finally, this is the first study reporting that the treatment of sows with probiotic combination resulted in the improved microbiota diversity in neonatal piglets.

Novel Aggregation Promoting Factor AggE Contributes to the Probiotic Properties of Enterococcus faecium BGGO9-28.

The understanding of mechanisms of interactions between various bacterial cell surface proteins and host receptors has become imperative for the study of the health promoting features of probiotic enterococci. This study, for the first time, describes a novel enterococcal aggregation protein, AggE, from Enterococcus faecium BGGO9-28, selected from a laboratory collection of enterococcal isolates with auto-aggregation phenotypes. Among them, En. faecium BGGO9-28 showed the strongest auto-aggregation, adhesion to components of ECM and biofilm formation. Novel aggregation promoting factor AggE, a protein of 178.1 kDa, belongs to the collagen-binding superfamily of proteins and shares similar architecture with previously discovered aggregation factors from lactic acid bacteria (LAB). Its expression in heterologous enterococcal and lactococcal hosts demonstrates that the aggE gene is sufficient for cell aggregation. The derivatives carrying aggE exhibited the ten times higher adhesion ability to collagen and fibronectin, possess about two times higher adhesion to mucin and contribute to the increase of biofilm formation, comparing to the control strains. Analysis for the presence of virulence factors (cytolysin and gelatinase production), antibiotic resistance (antibiotic susceptibility) and genes (cylA, agg, gelE, esp, hylN, ace, efaAfs , and efaAfm ) showed that BGGO9-28 was sensitive to all tested antibiotics, without hemolytic or gelatinase activity. This strain does not carry any of the tested genes encoding for known virulence factors. Results showed that BGGO9-28 was resistant to low pH and high concentrations of bile salts. Also, it adhered strongly to the Caco-2 human epithelial cell line. In conclusion, the results of this study indicate that the presence of AggE protein on the cell surface in enterococci is a desirable probiotic feature.

In vitro and in vivo antagonistic activity of new probiotic culture against Clostridium difficile and Clostridium perfringens.

BACKGROUND: Genus Clostridium accompanies more than 200 known species and at least 30 among them are associated with human and animal diseases. At the moment, the treatment of clostridial infections is based on use of antibiotics. However, due to the European ban on the use of antibiotics in livestock production, novel therapeutic strategies for treatment of these hardly curable infections have been evaluated. Hence, in this study the antimicrobial effect of newly designed probiotic culture consisted of natural isolates Lactobacillus helveticus BGRA43, Lactobacillus fermentum BGHI14 and Streptococcus thermophilus BGVLJ1-44 against Clostridium difficile and Clostridium perfringens was analyzed. RESULTS: The probiotic culture showed strong in vitro antimicrobial effect on C. difficile (human clinical isolate). In addition, individual strains and the probiotic combination exhibited immunomodulatory activity. The probiotic combination significantly increased the proliferation of GALT lymphocytes. At the other hand, none of the bacterial treatments (individual strains and the combination) induced the production of proinflammatory cytokines IL-6 and IL-1ß by intestinal epithelial cells, Caco-2. Interestingly, Caco-2 cells exposed to the probiotic combination produced significantly elevated amount of TGFß pointing to potential protecting effect of the probiotic. In addition, the results of field trial on spontaneously infected goats revealed reduction of C. perfringens in goats (below the detection threshold) after the probiotic treatment. CONCLUSIONS: The results of this study indicated that the novel probiotic deserves to be further investigated as a promising antimicrobial agent against C. difficile and C. perfringens.

Diversity and antibiotic susceptibility of autochthonous dairy enterococci isolates: are they safe candidates for autochthonous starter cultures?

Enterococci represent the most controversial group of dairy bacteria. They are found to be the main constituent of many traditional Mediterranean dairy products and contribute to their characteristic taste and flavor. On the other hand, during the last 50 years antibiotic-resistant enterococci have emerged as leading causes of nosocomial infections worldwide. The aim of this study was to determine the diversity, technological properties, antibiotic susceptibility and virulence traits of 636 enterococci previously isolated from 55 artisan dairy products from 12 locations in the Western Balkan countries (WBC) of Serbia, Croatia and Bosnia and Herzegovina. All strains were identified both by microbiological and molecular methods. The predominant species was Enterococcus durans, followed by Enterococcus faecalis and Enterococcus faecium. Over 44% of the isolates were resistant to ciprofloxacin and erythromycin, while 26.2% of the isolates were multi-resistant to three or more antibiotics belonging to different families. 185 isolates (29.1%) were susceptible to all 13 of the antibiotics tested. The antibiotic-susceptible isolates were further tested for possible virulence genes and the production of biogenic amines. Finally, five enterococci isolates were found to be antibiotic susceptible with good technological characteristics and without virulence traits or the ability to produce biogenic amines, making them possible candidates for biotechnological application as starter cultures in the dairy industry.

Characterization of lactic acid bacteria isolated from artisanal Travnik young cheeses, sweet creams and sweet kajmaks over four seasons.

The aim of this study was to investigate the composition of lactic acid bacteria (LAB) in autochthonous young cheeses, sweet creams and sweet kajmaks produced in the Vlasic mountain region of central Bosnia and Herzegovina near the town of Travnik over a four season period. These three products were made from cow's milk by a traditional method without the addition of a starter culture. Preliminary characterization with phenotype-based assays and identification using rep-PCR with a (GTG)5 primer and 16S rDNA sequence analysis were undertaken for 460 LAB isolates obtained from all the examined samples. Fifteen species were identified as follows: Lactococcus lactis, Lactococcus raffinolactis, Lactococcus garviae, Lactobacillus casei, Lactobacillus plantarum, Lactobacillus helveticus, Enterococcus faecium, Enterococcus durans, Enterococcus faecalis, Enterococcus italicus, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Leuconostoc lactis, Streptococcus thermophilus and Streptococcus mitis. A wide genotypic and phenotypic heterogeneity of the species was observed, particularly within the Lc. lactis strains. In all of the tested dairy products across four seasons, a significantly positive correlation (r = 0.690) between the presence of lactococci and enterococci and a negative correlation (r = 0.722) between the presence of lactococci and leuconostocs were recorded. Forty-five percent of the lactobacilli and 54.4% of the lactococci exhibited proteolytic activity, whereas 18.7% of the total LAB isolates exhibited antimicrobial activity.

Evaluation of lactic acid bacteria and yeast diversity in traditional white pickled and fresh soft cheeses from the mountain regions of Serbia and lowland regions of Croatia.

The goal of this study was the characterisation of indigenous lactic acid bacteria (LAB) and yeasts isolated from nine white pickled (BG) and nine fresh soft (ZG) artisanal cheeses collected in Serbia and Croatia. While LAB were present in all of the cheeses collected, yeasts were found in all BG cheeses but only in three ZG cheese samples. High LAB and yeast species diversity was determined (average H'(L)=0.4 and H'(Y)=0.8, respectively). The predominant LAB species in white pickled (BG) cheeses were Lactococcus lactis, Lactobacillus plantarum, and Leuconostoc mesenteroides, while in fresh soft (ZG) cheeses the most dominant LAB species were L. lactis, Enterococcus faecalis, and Leuconostoc pseudomesenteroides. Among the 20 yeast species found, Debaryomyces hansenii, Candida zeylanoides, and Torulaspora delbrueckii were found to be predominant in BG cheeses, while Yarrowia lipolytica was predominant in ZG cheeses. The characterisation of metabolic and technological potentials revealed that 53.4% of LAB isolates produced antimicrobial compounds, 44.3% of LAB strains showed proteolytic activity, while most of the yeast species possessed either lipolytic or proteolytic activity. In conclusion, the results obtained in this study showed that the composition of LAB and yeast populations in white pickled and fresh soft cheeses is region specific. The knowledge gained in this study could eventually be used to select region specific LAB and yeast strains for the production of white pickled and fresh soft artisanal cheeses with geographically specific origins under controlled conditions.

Technological and probiotic potential of BGRA43 a natural isolate of Lactobacillus helveticus.

Lactobacillus helveticus BGRA43 is a human intestinal isolate showing antimicrobial activity, amongst others, against Yersinia enterocolitica, Shigella sonnei, Shigella flexneri, and Streptococcus pneumoniae. BGRA43 produces PrtH proteinase with proteolytic activity on both casein and ß-lactoglobulin (BLG). BGRA43 is able to reduce the allergenicity of BLG. Bioactive peptides released in BGRA43 fermented milk are potent modulators of innate immunity by modulating the production of proinflammatory cytokines IL-6 and TNF-α. BGRA43 is able to survive in simulated gastric and intestinal conditions. The growth of BGRA43 in milk results in a fast acidification lowering the milk pH to 4.53 generating mild, homogeneous, and viscous yogurt-like product. The strain BGRA43 grows suitably in pure cow or goat's milk as well as in milk containing inulin or nutrim even when they are used as the sole carbon source. It is suggested that strain BGRA43 could be used as a single-strain culture for the preparation of yogurt-like products from bovine or caprine milk. Overall, L. helveticus BGRA43 could be considered as a potential probiotic candidate with appropriate technological properties attractive for the dairy industry.

Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat's milk cheese.

The Bukuljac cheese is traditionally homemade cheese, produced from heat-treated goat's milk without the addition of any bacterial starter culture. The presence of lactic acid bacteria (LAB) in Bukuljac cheese has been analyzed by using a polyphasic approach including microbiological and molecular methods such as rep-PCR with (GTG)5 primer. Lactobacillus paracasei subsp. paracasei represents a dominant strain in the microflora of analyzed cheese. Out of 55 Gram-positive and catalase-negative isolates, 48 belonged to L. paracasei subsp. paracasei species. Besides lactobacilli, five Lactococcus lactis subsp. lactis and two Enterococcus faecalis were found. Results of PCR-denaturing gradient gel electrophoresis (DGGE) of DNA extracted directly from the fresh cheese revealed the presence of Leuconostoc mesenteroides. Only lactobacilli showed a high proteolytic activity and hydrolyzed alpha(s1)- and beta-caseins. They are also producers of diacetyl. In addition, 34 out of 55 isolates, all determined as lactobacilli, showed the ability of auto-aggregation. Among 55 isolates, 50 also exhibited antimicrobial activity.

Characterization of microflora in homemade semi-hard white Zlatar cheese.

The Zlatar cheese belongs to the group of traditionally homemade cheeses, which are produced from nonpasteurized cow's milk, without adding of any bacterial starter culture. Changes were followed in lactic acid bacteria population and chemical composition during the ripening period of cheese up to 60 days. Results showed that the percentage of lactic acid cocci was higher in raw milk and one day old cheese and their percentage was gradually decreasing, whereas the number of lactobacilli was increasing. After 30 days of cheese ripening the number of cocci increased again, reaching the number of lactobacilli. The results of API 50 CH system and rep-PCR analysis showed that Lactobacillus paracasei subsp. paracasei, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcuus faecium and Enterococcus faecalis were the main groups present during the ripening of Zlatar cheese. Results revealed that in older cheeses (45 and 60 days old) enterococci were the main group present. It was also demonstrated that 57 isolates showed antimicrobial activity. The number of bacteria showing antimicrobial activity slowly decreased during the ripening period and in samples of 60 days old cheese producers of antimicrobial activities were not detected.