Identification and characterization of a new antifungal peptide in fermented milk product containing bioprotective Lactobacillus cultures.

Mold and yeast contamination constitutes a major problem in food commodities, including dairy products, hence new natural preventive measures are in high demand. The aim of the current study is to identify and characterize novel antifungal peptides produced by lactic acid bacteria (LAB) in sour cream. By the use of a newly developed image-based 96-well plate fungal growth inhibition assay targeting Debaryomyces hansenii, combined with a range of analytical tools comprising HPLC-high-resolution mass spectrometry, ultrahigh-performance liquid chromatography-Triple Quadrupole MS and nuclear magnetic resonance spectroscopy, we successfully identified a new antifungal peptide (DMPIQAFLLY; 1211 Da) in sour cream enriched with two bioprotective LAB strains. This peptide represents a fragment of casein, the most abundant protein in milk. Presumably, the proteolytic activity of these bioprotective strains results in the observed 4-fold higher concentration of the peptide during storage. Both bioprotective strains are able to generate this peptide in concentrations up to 0.4 µM, independently of the sour cream starter culture employed. The peptide attenuates the growth rate of D. hansenii at concentrations ≥35 µM, and results in smaller cells and more compact colonies. Hence, the peptide is likely contributing to the overall preserving effect of the investigated bioprotective LAB strains.

Novel Variants of Streptococcus thermophilus Bacteriophages Are Indicative of Genetic Recombination among Phages from Different Bacterial Species.

Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCEStreptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.

The changes of proteins fractions shares in milk and fermented milk drinks.

BACKGROUND: The aim of this research was to observe the changes which take place in the electrophoretic picture of milk proteins after pasteurisation and inoculation with different starter cultures (both traditional and probiotic). After incubation, the yoghurt, kefir, acidified milk, fermented Bifidobacterium bifidum drink and Lactobacillus acidophillus drink were chilled for 14 days to observe the changes which occurred. METHODS: The research materials were raw and pasteurised milk, as well as fermented milk- based drinks. The raw milk used for research came from Polish Holstein-Fresian black and white cows. The milk was sampled 3 times and divided into 5 parts, each of which was pasteurised at 95°C for 10 min and then cooled for inoculation: yoghurt to 45°C, kefir and acidified milk to 22°C and drinks with Bifidobacterium bifidum and Lactobacillus acidophillus to 38°C. Milk was inoculated with lyophilised, direct vat starter cultures, in an amount equal to 2% of the working starter. For the production of fermented drinks, the subsequent starters were applied: "YC-180" Christian Hansen for yoghurt, "D" Biolacta-Texel-Rhodia for kefir, CH-N--11 Christian Hansen for acidified milk, starter by Christian Hansen for the probiotic Bifidobacterium bifidum milk, starter by Biolacta-Texel-Rhodia for the probiotic Lactobacillus acidophillus milk. The analyses were conducted in raw, pasteurised and freshly fermented milk as well as in milk drinks stored for 14 days. The total solid content was estimated by the drying method; the fat content by the Gerber method; the lactose content by the Bertrand method; the protein content by the Kjeldahl method with Buchi apparatus; the density of milk was measured with lactodensimeter; acidity with a pH-meter; and potential acidity by Soxhlet-Henkl method (AOAC, 1990). The electrophoretic separation of proteins in raw and pasteurised milk, as well as in freshly produced milk drinks and those stored for 14 days, was performed with SDS-PAGE (on polyacrylamid gel) basing on procedure described by Laemmli (1970). RESULTS: It was shown that, in comparison with raw milk, the pasteurised milk had smaller amounts of αs-, ß- and κ-casein, whereas the shares of γ-casein and peptides were greater, and there were no changes in immunoglobulin, α-lactalbumin or ß-lactoglobulin levels, which indicated that hydrolysis of caseins had occurred. In all freshly fermented milk drinks, a drop in αs- and ß-casein was observed relative to raw milk. An increase in peptides and γ-casein was also noticed (with the exception of acidified milk). There were differences in α-lactalbumin and ß-lactoglobulin levels between the different drinks: raw, pasteurised or freshly fermented milk. It was shown that kefir, compared to the other drinks, had the lowest levels of αs- and ß-casein, α-lactalbumin and of peptides, as well as the highest level of γ-casein, which is evidence of an increased rate of hydrolysis in that drink. It was stated that, during the storage of fermented milk drinks, the levels of lactoferrin, serum albumin and peptides significantly increased whereas the content of κ-casein diminished. The proportions of serum albumin and lactoferrin in fermented milk drinks increased relative to raw milk and/or after storage, which is evidence of aggregation of proteins of low molecular mass into bigger conglomerates. CONCLUSIONS: The observed differences between fermented milks, including during chilled storage, in the amounts of individual proteins proves the different proteolytic abilities of starter cultures used in fermented milk production. α-lactoalbumin and ß-lactoglobulin are, besides caseins, the most allergenic milk proteins. So, kefir, because of its low α-lactoalbumin content, and Bifidobacterium bifidum milk, with the lowest content of ß-lactoglobulin, were the most advantageous and least allergenic drinks examined.

Sequence-based analysis of the microbial composition of water kefir from multiple sources.

Water kefir is a water-sucrose-based beverage, fermented by a symbiosis of bacteria and yeast to produce a final product that is lightly carbonated, acidic and that has a low alcohol percentage. The microorganisms present in water kefir are introduced via water kefir grains, which consist of a polysaccharide matrix in which the microorganisms are embedded. We aimed to provide a comprehensive sequencing-based analysis of the bacterial population of water kefir beverages and grains, while providing an initial insight into the corresponding fungal population. To facilitate this objective, four water kefirs were sourced from the UK, Canada and the United States. Culture-independent, high-throughput, sequencing-based analyses revealed that the bacterial fraction of each water kefir and grain was dominated by Zymomonas, an ethanol-producing bacterium, which has not previously been detected at such a scale. The other genera detected were representatives of the lactic acid bacteria and acetic acid bacteria. Our analysis of the fungal component established that it was comprised of the genera Dekkera, Hanseniaspora, Saccharomyces, Zygosaccharomyces, Torulaspora and Lachancea. This information will assist in the ultimate identification of the microorganisms responsible for the potentially health-promoting attributes of these beverages.

The microbial diversity of water kefir.

The microbial diversity of water kefir, made from a mixture of water, dried figs, a slice of lemon and sucrose was studied. The microbial consortia residing in the granules of three water kefirs of different origins were analyzed. A collection of 453 bacterial isolates was obtained on different selective/differential media. Bacterial isolates were grouped with randomly amplified polymorphic DNA (RAPD)-PCR analyses. One representative of each RAPD genotype was identified by comparative 16S rDNA gene sequencing. The predominant genus in water kefirs I and II was Lactobacillus, which accounted for 82.1% in water kefir I and 72.1% in water kefir II of the bacterial isolates. The most abundant species in water kefirs I and II were Lactobacillus hordei and Lb. nagelii followed by considerably lower numbers of Lb. casei. Other lactic acid bacteria (LAB) were identified as Leuconostoc mesenteroides and Lc. citreum in all three water kefirs. The most abundant species in water kefir III was Lc. mesenteroides (28%) and Lc. citreum (24.3%). A total of 57 LAB belonging to the species of Lb. casei, Lb. hordei, Lb. nagelii, Lb. hilgardii and Lc. mesenteroides were able to produce exopolysacchrides from sucrose. Non LABs were identified as Acetobacter fabarum and Ac. orientalis. The Acetobacter species were more prevalent in consortium III. Cluster analyses of RAPD-PCR patterns revealed an interspecies diversity among the Lactobacillus and Acetobacter strains. Aditionally, Saccharomyces cerevisiae, Lachancea fermentati, Hanseniaospora valbyensis and Zygotorulaspora florentina were isolated and identified by comparison of partial 26S rDNA sequences and FTIR spectroscopy.