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The presence of surface (S)-layer proteins as a rare property of lactic acid bacteria plays an important role in the expression of the probiotic properties of the producer strain and protects the cells from unfavourable environmental conditions. Therefore, the aim of this work was to evaluate the role of S-layer proteins of four Levilactobacillus brevis MB1, MB2, MB13, and MB20 strains, previously isolated from human milk. The results showed better survival under simulated gastrointestinal tract (GIT) conditions and better adhesion to Caco-2 cells, achieved when S-proteins were present on the surface of the examined probiotic Levilactobacillus brevis strains. Free cells and cells microencapsulated in alginate, with or without galactooligosaccharides (GOS) and fructooligosaccharides (FOS), survived in high numbers during freeze-drying and one-month storage at 4°C, as well as during exposure to simulated GIT conditions (>108 CFU/g), thus fulfilling the primary technological criterion for the selection of probiotic strains. Higher survival of freeze-drying of encapsulated cells than free cells, can be attributed to the protective effect of alginate, GOS and FOS as prebiotic agents. However, GOS was more protective than FOS in maintaining the viability of microencapsulated Levilactobacillus brevis MB1, MB2, MB13 and MB20 cells in alginate during storage and under the harsh conditions of GIT and freeze-drying. [ABSTRACT FROM AUTHOR]

Bioactive peptides (BPs) are molecules of paramount importance with great potential for the development of functional foods, nutraceuticals or therapeutics for the prevention or treatment of various diseases. A functional BP-rich dairy product was produced by lyophilisation of bovine milk fermented by the autochthonous strains Lactococcus lactis subsp. lactis ZGBP5-51, Enterococcus faecium ZGBP5-52 and Enterococcus faecalis ZGBP5-53 isolated from the same artisanal fresh cheese. The efficiency of the proteolytic system of the implemented strains in the production of BPs was confirmed by a combined high-throughput mass spectrometry (MS)-based peptidome profiling and an in silico approach. First, peptides released by microbial fermentation were identified via a non-targeted peptide analysis (NTA) comprising reversed-phase nano-liquid chromatography (RP nano-LC) coupled with matrix-assisted laser desorption/ionisation-time-of-flight/time-of-flight (MALDI-TOF/TOF) MS, and then quantified by targeted peptide analysis (TA) involving RP ultrahigh-performance LC (RP-UHPLC) coupled with triple-quadrupole MS (QQQ-MS). A combined database and literature search revealed that 10 of the 25 peptides identified in this work have bioactive properties described in the literature. Finally, by combining the output of MS-based peptidome profiling with in silico bioactivity prediction tools, three peptides (75QFLPYPYYAKPA86, 40VAPFPEVFGK49, 117ARHPHPHLSF126), whose bioactive properties have not been previously reported in the literature, were identified as potential BP candidates. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine how storage in the frozen state affects the quality of mare milk. Therefore, physicochemical properties such as pH, total dissolved solids (TDS), electrical conductivity (EC), particle size distribution and near-infrared (NIR) spectroscopy, as well as changes in antioxidant activity and the total phenol content were determined during 6 months of storage at -18°C. All of the determined parameters were compared to those in raw mare milk prior to freezing. According to the obtained results, frozen storage caused certain changes in mare milk quality, including increase in pH, electrical conductivity and TDS values, changes in protein structure recorded by NIR spectroscopy and SDS PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis), as well as changes in particle size distribution. There was also a continuous decrease in the antioxidant activity and the total phenol content of frozen mare milk throughout the 6 months of frozen storage. Thus, after 6 months of frozen storage, a decrease in the DPPH value of approximately 59%, in the FRAP value of approximately 26% and in the TPC of approximately 70% was observed, respectively. In conclusion, freezing and frozen storage affected the quality of mare milk, which might have negative consequences on its therapeutic value. [ABSTRACT FROM AUTHOR]

Proteases of Lactobacillus and Lactococcus strains catalyze casein degradation in fermented dairy products, which can result in the production of bioactive peptides. Proteolytic properties of a selection of lactic acid bacteria (LAB) strains previously isolated in Croatia, including Lactococcus lactis and Lactobacillus strains, are described. All strains of Lactobacillus and Lactococcus showed an Fmc+ phenotype that can be associated with efficient growth in milk. The significant caseinolytic effect, after incubation of culture supernatant or concentrated cell biomass, was observed for Levilactobacillus brevis D6 and Lactiplantilactobacillus plantarum D13 after growth in the optimal growth medium, while for Lactoccocus lactis ZGBP5-32 and Levilactobacillus brevis SF9B strains after growth in skimmed milk. To assess the LAB growth in skimmed milk, the acidification rate was monitored. Statistically, significant acidification capacity was determined for L. plantarum D13 in the optimal medium and by the proteolytic strain Lactobacillus helveticus M92 in skimmed milk. After extraction of proteinases from the strains with caseinolytic activity, protein samples were analysed by the SDS-PAGE. The protein extract of the Lc. lactis ZGBP5-32 and ZGZA7-10, retained proteolytic activity even at very low concentrations. The ultrafiltration improved protein extraction. The crude extract possibly contained putative protease, as a decrease in contaminating proteins was confirmed by SDSPAGE in samples of L. brevis D6 and SF9B, L. fermentum D12 and L. plantarum D13. [ABSTRACT FROM AUTHOR]

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The antibiotic susceptibility and antimicrobial activity, as food safety parameters important for application of autochthonous lactic acid bacteria (LAB), that previously satisfied technological criteria for functional starter cultures in fresh cheese production were examined. Soluble whole cell protein patterns of autochthonous LAB strains from fresh cheese, obtained by SDS-PAGE, revealed the presence of two predominant strains, which were identified as Lactobacillus fermentum A8 and Enterococcus faecium A7. These strains were not resistant and shown susceptibility to antibiotics: ampicillin, bacitracin, penicillin G, azithromycin, chloramphenicol, clarithromycin, clindamycin, spiramycin, tetracycline, streptomycin, neomycin, gentamicin, erythromycin, rifampicin and novo-biocin. Lb. fermentum A8 strain displayed phenotypic resistance to vancomycin, but this resistance is intrinsic, not transferable and it is acceptable from the safety aspect. The capacity of Lb. fermentum A8 and Ec. faecium A7 to inhibit growth of test-microorganisms Listeria monocytogenes ATCC 11911, Escherichia coli 3014, Salmonella enterica serovar Typhimurium FP1 and Staphylococcus au-reus 3048, was also analysed. According to obtained results, Lb. fermentum A8 and Ec. faecium A7 are safe from the aspect of spreading antibiotic resistance and could be useful as bioprotective cultures that inhibit common bacterial food contaminants, including L. monocytogenes. [ABSTRACT FROM AUTHOR]

The use of commercial starter cultures in fresh cheese production from pasteurized milk results in the loss of typical characteristics of artisan fresh cheese due to the replacement of complex native microbiota with a defined starter culture. Hence, the aim of this research is to isolate and characterize dominant lactic acid bacteria (LAB) in artisan fresh cheese and to evaluate their capacity as autochthonous starter cultures for fresh cheese production. Fifteen most prevalent Gram-positive, catalase-negative and asporogenous bacterial strains were selected for a more detailed characterization. Eleven lactic acid bacterial strains were determined to be homofermentative cocci and four heterofermentative lactobacilli. Further phenotypic and genotypic analyses revealed that those were two different LAB strains with high acidifying and proteolytic activity, identified as Lactobacillus fermentum A8 and Enterococcus faecium A7. These two autochthonous strains, alone or in combination with commercial starter, were used to produce different types of fresh cheese, which were evaluated by a panel. Conventional culturing, isolation, identification and PCR-denaturing gradient gel electrophoresis (PCR-DGGE) procedures, applied to the total fresh cheese DNA extracts, were employed to define and monitor the viability of the introduced LAB strains and their effect on the final product characteristics. Production of fresh cheese using a combination of commercial starter culture and selected autochthonous strains resulted in improved sensorial properties, which were more similar to the ones of spontaneously fermented fresh cheese than to those of cheese produced with only starter culture or selected strains. After 10 days of storage, that cheese retained the best sensorial properties in comparison with all other types of cheese. The presence of inoculated autochthonous and starter cultures and their identification was demonstrated by DGGE analysis. The obtained results indicate that autochthonous strains have a strong potential to enrich the flavour of industrially produced fresh type cheese under controlled conditions. [ABSTRACT FROM AUTHOR]

The effect of microencapsulated probiotic Lactobacillus helveticus M92 cells and transglutaminase addition on the probiotic set yoghurt properties was investigated. Addition of probiotic bacteria, either free or microencapsulated in sodium caseinate, decreased the fermentation time and significantly enhanced the appearance and consistency of probiotic set yoghurt. Better survival of microencapsulated than free probiotic bacteria in produced yoghurts during storage, as well as during exposure to simulated gastrointestinal conditions, emphasises the efficiency of microencapsulation in the cell protection. Pretreatment of the milk with transglutaminase increased the gel strength and decreased the syneresis, which resulted in a better appearance and consistency of probiotic set yoghurts. [ABSTRACT FROM AUTHOR]

Probiotic strain Lactobacillus plantarum L4 and strain Leuconostoc mesenteroides LMG 7954 were applied for the controlled fermentation of cabbage heads. The parameters of the controlled and spontaneous fermentations, including antimicrobial effect of cabbage brines obtained at the end of both fermentations, were monitored. To check out the influence of starter culture strains, 10 randomly chosen lactic acid bacteria, isolated at the end of controlled cabbage heads fermentation were identified by API 50CHL test, and the presence of the probiotic culture was confirmed by Pulsed Field Gel Electrophoresis. The starter cultures applied for cabbage heads fermentation allowed lowering of NaCl concentrations from 4.0% to 2.5% (w/v), considerably accelerated fermentation process by 14 d, and improved the product quality. The produced sauerkraut heads are considered probiotic product as viable probiotic cells count in final product was higher than 10 colony-forming units (CFU) per gram of product. The results of this research could be applied in the production of fermented cabbage heads with added functional (probiotic) value and with lower NaCl concentration with expected shortened fermentation time. This could not only be of economic but also of ecological importance. [ABSTRACT FROM AUTHOR]

The antimicrobial activity of industrially important lactic acid bacteria as starter cultures and probiotic bacteria is the main subject of this review. This activity has been attributed to the production of metabolites such as organic acids (lactic and acetic acid), hydrogen peroxide, ethanol, diacetyl, acetaldehyde, acetoine, carbon dioxide, reuterin, reutericyclin and bacteriocins. The potential of using bacteriocins of lactic acid bacteria, primarily used as biopreservatives, represents a perspective, alternative antimicrobial strategy for continuously increasing problem with antibiotic resistance. Another strategy in resolving this problem is an application of probiotics for different gastrointestinal and urogenital infection therapies. [ABSTRACT FROM AUTHOR]

In this research the application of bacterial strains Lactobacillus acidophilus M92, Lactobacillus plantarum L4 and Enterococcus faecium L3 in the production of fermented probiotic beverages from milk permeate enriched with 10 % (v/v) whey retentate, was investigated. In the previous researches of probiotic concept, probiotic properties of these three strains of lactic acid bacteria have been defined. At the end of controlled fermentation, probiotic strains have produced 7.4 g/L lactic acid, pH was decreased to 4.7, and number of live cells was around 108 CFU/mL. Number of viable count of probiotic bacteria, which were identified with RAPD (RandomAmplifiedPolymorphicDNA)(Random Amplified Polymorphic DNA) method, was maintained at around 107 CFU/mL during 28 days of the preservation at 4 °C. Furthermore, a spontaneous fermentation of milk permeate enriched with 10 % (v/v) of whey retentate was carried out and lactic acid bacteria present in these substrates were isolated. All of these bacterial strains have rapidly acidified the growth media and have shown antibacterial activity against chosen test-microorganisms, what are important properties of potential starter cultures for the fermentation of dairy products. The results of biochemical API analysis have identified isolated strains as Lactococcus lactis subsp. lactis and Lactobacillus helveticus. [ABSTRACT FROM AUTHOR]

Mare milk is known for therapeutic properties due to the presence of bioactive components that are sensitive to thermal treatments and tend to lose their activity. Accordingly, mare milk is consumed raw or frozen, but for food safety reasons recent studies suggest mandatory pasteurization of mare milk before consumption. The aim of this study was to examine the effects of different heat treatments on mare milk quality and compare those to heat induced changes in cow milk. In all milk samples, the total dissolved solids (TDS), electrical conductivity (EC), antioxidant capacity (AC) by the DPPH and FRAP methods, changes in colour (ΔE*), concentration of 5-hydroxymethylfurfural (HMF) and the total phenols (TPC) were determined. Heat induced protein denaturation was monitored by SDS-PAGE, while NIR was applied for qualitative analysis of structural changes. In-bottle sterilization at 116 °C/16ʹ and 120 °C/5ʹ showed to be inappropriate for mare milk processing, since they resulted in the most intense colour changes (ΔE*>5), the highest free and total HMF concentrations (40.70 µmol/L free, 70.26 µmol/L total HMF at 116 °C/16ʹ; 35.49 µmol/L free, 58.12 µmol/L total HMF at 120 °C/5ʹ) and the most intense protein denaturation. On contrary, the applied HTST regimes (72 °C/20”, 85 °C/30”, 90 °C/5ʹ) and thermisation at 63 °C/15” were appropriate for mare milk processing considering the studied qualitative changes. Thereby, the HMF concentration, FRAP, TPC and ΔE* value, combined with NIR analysis showed a good potential to be used as rapid and useful tools for monitoring the intensity of heat induced changes in mare milk. [ABSTRACT FROM AUTHOR]

Bacteriocins produced by probiotic strains effectively contribute to colonization ability of probiotic strains and facilitate their establishment in the competitive gut environment and also protect the gut from gastrointestinal pathogens. Moreover, bacteriocins have received considerable attention due to their potential application as biopreservatives, especially in dairy industry. Hence, the objective of this research was to investigate antimicrobial activity of probiotic strains Lactobacillus helveticus M92, Lactobacillus plantarum L4 and Enterococcus faecium L3, with special focus on their bacteriocinogenic activity directed towards representatives of the same or related bacterial species, and towards distant microorganisms including potential food contaminants or causative agents of gut infections. In order to induce bacteriocin production, probiotic cells were cocultivated with Lactococcus lactis subsp. lactis LMG 9450, one of the most important starter cultures in cheese production. The presence of bacteriocin coding genes was investigated by PCR amplification with sequence-specific primers for helveticin and was confirmed for probiotic strain L. helveticus M92. All examined probiotic strains have shown bacteriocinogenic activity against Staphylococcus aureus 3048, Staphylococcus aureus K-144, Escherichia coli 3014, Salmonella enterica serovar T yphimurium FP1, Bacillus subtilis ATCC 6633, Bacillus cereus TM2, which is an important functional treat of probiotic strains significant in competitive exclusion mechanism which provides selective advantage of probiotic strains against undesirable microorganisms in gastrointestinal tract of the host. According to obtained results, living cells of starter culture Lc. lactis subsp. lactis LMG 9450 induced bacteriocin production by examined probiotic strains but starter culture itself was not sensitive to bacteriocin activity. [ABSTRACT FROM AUTHOR]