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Enumeration and isolation of Streptococcus salivarius subsp. thermophilus from cheese is challenging, due to the relatively high number of species it may host. We describe medium SPY9.3 for the cultivation of S. salivarius subsp. thermophilus from cheese. The medium and related incubation conditions (SPY) was compared with 2 other protocols, M17 and ST: sensitivity was assessed by parallel cultivation of 55 strains of S. salivarius subsp. thermophilus, and selectivity by (i) parallel cultivation of 60 strains belonging to 20 different non-target species and sub-species and (ii) isolating bacteria from 3 raw-milk cheeses. Colony counts were similar on SPY9.3 and M17 (mean difference 0.07 log(cfu/mL), p > 0.001) and significantly higher on ST than on M17 and SPY9.3 (mean differences 0.42 and 0.48 log(cfu/mL), respectively, p < 0.001). SPY was more specific than ST and M17, with respectively 20%, 40%, and 50% of the investigated non-target species able to grow. S. salivarius subsp. thermophilus, Enterococcus spp., and Staphylococcus aureus were indistinguishable using all 3 protocols. Only SPY avoided growth of Lactobacillus delbrueckii subsp. lactis. Finally, ST and SPY displayed higher recoveries of S. salivarius subsp. thermophilus colonies from cheese than M17 (5.6, 5.5, and 3.0 adjusted log(cfu/mL), respectively) and the lowest proportion of non-specific isolates. The protocol described here and based on SPY9.3 presents a promising alternative to existing protocols for the enumeration and isolation of S salivarius subsp. thermophilus from cheese or other complex fermented products., (Copyright © 2020 Agroscope. Published by Elsevier Ltd.. All rights reserved.)

Electrospun nanofiber membranes are widely investigated in the past few decades as candidates for tissue engineering, which can mimic natural extracellular matrix (ECM) and improve cell adhesion, proliferation, and expression on nanofiber membranes. However, the formation of bacterial biofilms on nanofiber membranes and application of the biofilm-integrated nanofiber membranes remain largely unknown. Here, electrospun cellulose acetate nanofiber membranes are first utilized as scaffold materials for Lactobacillus plantarum ( L. plantarum) biofilm formation. Nanofiber membranes proved to be an excellent scaffold for bacteria biofilm with high stability, where biofilms were interlocked with nanofibers forming a cohesive structure. In comparison with planktonic bacteria, L. plantarum biofilms on nanofiber membranes show excellent gastrointestinal resistance. Instead of decreasing, the number of viable cells increased after 3 h digestion in vitro. The L. plantarum biofilm-integrated nanofiber membranes were used as reusable starter cultures for fermented milk production showing excellent fermentative ability and higher survival of L. plantarum during shelf life. The viable cells in fermented milk remained at 11 log CFU/g throughout the reusable batches, which is far above the required value of 7 log CFU/g in commercial products. In addition, the produced fermented milk possesses shorter fermentation time and higher survival of probiotics during shelf life. The results suggest electrospun nanofiber membranes are ideal scaffold materials for bacteria biofilms immobilization in biotechnology and fermentation engineering, which broaden the potential use of electrospun nanofiber membranes in microbiology and strengthen the application of biofilms in fermentation engineering.

Lactic acid bacteria alone or with special adjunct probiotic strains are inevitable for the preparation of various specific functional foods. Moreover, because of their growth and metabolism, the final products are preserved for a certain time. Thus, growth dynamics of the lactic acid bacteria of the Fresco DVS 1010 culture ( Lactococcus lactis spp. lactis, Lactococcus lactis spp. cremoris, Streptococcus salivarius spp. thermophilus) during liquid-state fermentation of soya mashes and pH values within the process were analyzed in this study. Although milk is the most typical growth medium for the lactic acid bacteria, presumable viable counts of Fresco culture reached levels 10 9  CFU ml -1 after 8 h, representing 2-3 log increase in comparison to initial state (specific growth rates ranged from 1.06 to 1.64 h -1 ). After 21 days of storage period, the pH levels in the products were reduced to 4.50-4.70, representing a decrease of about 1.5-1.7 units. All prepared soybean products contained detectable amounts of raffinose-series oligosaccharides (0.25-0.68 g per 100 g) that were reduced in average by about 30.5% during period of 21 days.

The aim of this study was to evaluate the competitive effects of three bacteriocin producing strains of Lactococcus lactis subsp. lactis against two aminobiogenic lactic acid bacteria, i.e. the tyramine producing strain Enterococcus faecalis EF37 and the histamine producing strain Streptococcus thermophilus PRI60, inoculated at different initial concentrations (from 2 to 6 log cfu/ml). The results showed that the three L. lactis subsp. lactis strains were able to produce bacteriocins: in particular, L. lactis subsp. lactis VR84 and EG46 produced, respectively, nisin Z and lacticin 481, while for the strains CG27 the bacteriocin has not been yet identified, even if its peptidic nature has been demonstrated. The co-culture of E. faecalis EF37 in combination with lactococci significantly reduced the growth potential of this aminobiogenic strain, both in terms of growth rate and maximum cell concentration, depending on the initial inoculum level of E. faecalis. Tyramine accumulation was strongly reduced when E. faecalis EF37 was inoculated at 2 log cfu/ml and, to a lesser extent, at 3 log cfu/ml, as a result of a lower cell load of the aminobiogenic strain. All the lactococci were more efficient in inhibiting streptococci in comparison with E. faecalis EF37; in particular, L. lactis subsp. lactis VR84 induced the death of S. thermophilus PRI60 and allowed the detection of histamine traces only at higher streptococci inoculum levels (5-6 log cfu/ml). The other two lactococcal strains did not show a lethal action against S. thermophilus PRI60, but were able to reduce its growth extent and histamine accumulation, even if L. lactis subsp. lactis EG46 was less effective when the initial streptococci concentration was 5 and 6 log cfu/ml. This preliminary study has clarified some aspects regarding the ratio between bacteriocinogenic strains and aminobiogenic strains with respect to the possibility to accumulate BA and has also showed that different bacteriocins can have different effects on BA production on the same strain. This knowledge is essentially aimed to use bacteriocinogenic lactococci as a predictable strategy against aminobiogenic bacteria present in cheese or other fermented foods., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricus was investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and (1)H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product., (Copyright © 2014 Elsevier B.V. All rights reserved.)

For the isolation of probiotic cultures of Streptococcus thermophilus from dahi, we collected 120 samples from the southern regions of Punjab, Pakistan. Eleven isolates were obtained, and six were scrutinized for antibacterial activities against food-borne pathogens. The carbohydrate fermentation profile of these six strains was determined by the API50 CHL system. Additionally, these strains were amplified for their 16S rRNA regions to confirm their genotypic relationship. Furthermore, phenotypic characteristics among these strains were established by S-layer protein analysis of their cell walls by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and by plasmid profiling. The outer cell wall layers of these strains have 6-14 different sizes of protein bands of 27, 34, 37, 40, 45 and 60 kDa molecular weight. Similarly, except S02FT, all strains have a single prominent plasmid of 23 kbp, whereas S02FT has an additional plasmid of 9 kbp. On the basis of this unique feature and a wide spectrum of killing patterns against pathogenic bacteria, S. thermophilus S02FT was further characterized. This culture showed an optimum antibacterial activity of 800 AU/ml at pH 5.0-5.5 and a temperature of 30-37°C. It grows well in in vitro acidic conditions and tolerates bile salt up to 2% concentration. It was resistant to nalidixic acid, ciprofloxacin, gentamicin and sulphamethoxazol, but showed intermediate behaviour to vancomycin and erythromycin.

A multiplex real time PCR (mRealT-PCR) useful to rapidly screen microbial composition of thermophilic starter cultures for hard cooked cheeses and to compare samples with potentially different technological properties was developed. Novel primers directed toward pheS gene were designed and optimized for multiple detection of Lactobacillus helveticus, Lactobacillus delbrueckii, Streptococcus thermophilus and Lactobacillus fermentum. The assay was based on SYBR Green chemistry followed by melting curves analysis. The method was then evaluated for applications in the specific detection of the 4 lactic acid bacteria (LAB) in 29 different natural whey starters for Parmigiano Reggiano cheese production. The results obtained by mRealT-PCR were also compared with those obtained on the same samples by Fluorescence in Situ Hybridization (FISH) and Length-Heterogeneity PCR (LH-PCR). The mRealT-PCR developed in this study, was found to be effective for analyzing species present in the samples with an average sensitivity down to less than 600 copies of DNA and therefore sensitive enough to detect even minor LAB community members of thermophilic starter cultures. The assay was able to describe the microbial population of all the different natural whey starter samples analyzed, despite their natural variability. A higher number of whey starter samples with S. thermophilus and L. fermentum present in their microbial community were revealed, suggesting that these species could be more frequent in Parmigiano Reggiano natural whey starter samples than previously shown. The method was more effective than LH-PCR and FISH and, considering that these two techniques have to be used in combination to detect the less abundant species, the mRealT-PCR was also faster. Providing a single step sensitive detection of L. helveticus, L. delbrueckii, S. thermophilus and L. fermentum, the developed mRealT-PCR could be used for screening thermophilic starter cultures and to follow the presence of those species during ripening of derived dairy products. A major increase in understanding the starter culture contribution to cheese ecosystem could be harnessed to control cheese ripening and flavor formation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Bacterial communities of fermented foods are usually investigated by culture-dependent methods. Real-time quantitative PCR (qPCR) and reverse transcription (RT)-qPCR offer new possibilities to quantify the populations present and their metabolic activity. The aim of this work was to develop qPCR and RT-qPCR methods to assess the metabolic activity and the stress level of the two species used as ripening cultures in Emmental cheese manufacture, Propionibacterium freudenreichii and Lactobacillus paracasei. Three small scale (1/100) microbiologically controlled Emmental cheeses batches were manufactured and inoculated with Lactobacillus helveticus, Streptococcus thermophilus, P. freudenreichii and L. paracasei. At 12 steps of cheese manufacture and ripening, the populations of P. freudenreichii and L. paracasei were quantified by numerations on agar media and by qPCR. 16S, tuf and groL transcript levels were quantified by RT-qPCR. Sampling was carried out in triplicate. qPCR and RT-qPCR assessments were specific, efficient and linear. The quantification limit was 10(3) copies of cells or cDNA/g of cheese. Cell quantifications obtained by qPCR gave similar results than plate count for P. freudenreichii growth and 0.5 to 1 log lower in the stationary phase. Bacterial counts and qPCR quantifications showed that L. paracasei began to grow during the pressing step while P. freudenreichii began to grow from the beginning of ripening (in the cold room). Tuf cDNA quantification results suggested that metabolic activity of L. paracasei reached a maximum during the first part of the ripening (in cold room) and decreased progressively during ripening (in the warm room). Metabolic activity of P. freudenreichii was maximum at the end of cold ripening room and was stable during the first two weeks in warm room. After lactate exhaustion (after two weeks of warm room), the number of tuf cDNA decreased reflecting reduced metabolic activity. For L. paracasei, groL cDNA were stable during ripening. For P. freudenreichii, groL1 gene was highly-expressed during acidification, while groL2 gene highly expression was only observed at the end of the ripening stage after lactate (carbon substrate of P. freudenreichii) exhaustion. The potential use of 16S and tuf genes for the normalization of cDNA quantification throughout an Emmental cheese manufacture is discussed. For the first time, specific gene expression was performed by RT-qPCR yielding metabolic activity and stress response evaluation for L. paracasei and P. freudenreichii in cheese., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Enhancement of nutritional or vitamin content of foods is commonly touted as a major benefit of probiotics. In this paper, we examined the ability of three probiotic bacteria either alone or in combination to enhance nutritional content. Pure and mixed cultures of Streptococcus thermophilus ST5 and Lactobacillus helveticus R0052 or Bifidobacterium longum R0175 were used to prepare fermented soy beverages. The effects of strain, acidification and mixed cultures on the deconjugation of soy isoflavones were examined. Acidification to pH 4.7 alone resulted in a 7% drop in isoflavone levels. Deconjugation levels varied between the different glucosides. Fermentation by L. helveticus R0052 resulted in the 50% reduction in total glucosides with O-malonyl glucosides being reduced 64%. Fermentation by S. thermophilus ST5 or B. longum R0175 had no significant effect on isoflavone levels. Combining a S. thermophilus strain with a L. helveticus culture reduced the effectiveness of the latter. Fermentation did not significantly modify vitamin B1 or B6 levels., (Crown Copyright 2010. Published by Elsevier Ltd. All rights reserved.)

In hard cooked cheeses, any interactions between the thermophilic starters as they grow during the cheese-making are critical, since they modify bacterial growth kinetics and acidification kinetics, so affecting the ripening process and the final characteristics of the cheese. Twenty-four experimental hard cooked cheeses were made under controlled conditions, the milk being inoculated with various combinations of thermophilic strains of Streptococcus thermophilus, Lactobacillus helveticus and Lactobacillus delbrueckii. Over the first day of manufacturing we recorded a wide range of different growth kinetics for each starter species used, and a wide range of pH kinetics, depending on the starter combination. Most of the bacterial variability could be statistically explained by the nature, quantity, and/or presence or absence of the different strains inoculated. Four main interactions between the three species were evidenced during cheese-making. There was antagonism between L. helveticus and L. delbrueckii. The lactobacilli had a positive effect on S. thermophilus, which was reciprocal for L. helveticus. L. helveticus had a negative effect on S. thermophilus cultivability. And the combination of S. thermophilus inoculated in large quantities and L. helveticus strain H2 had a negative effect on the growth of the L. delbrueckii strain D2. While the positive effect of L. delbrueckii on S. thermophilus probably corresponds to interactions in milk that have already been described and published, the other interactions were hitherto unknown. These interactions are of major importance for the growth kinetics of streptococci and thermophilic lactobacilli during cheese-making.

The objective of this study was to examine the effect of Versagel on the growth and proteolytic activity of Streptococcus thermophilus 1275 and Lactobacillus delbrueckii ssp. bulgaricus 1368 and angiotensin-I converting enzyme inhibitory activity of the peptides generated thereby as well as on the physical properties of low-fat yogurt during a storage period of 28 d at 4 degrees C. Three different types of low-fat yogurts, YV0, YV1, and YV2, were prepared using Versagel as a fat replacer. The fermentation time of the low-fat yogurts containing Versagel was less than that of the control yogurt (YV0). The starter cultures maintained their viability (8.68 to 8.81 log CFU/g of S. thermophilus and 8.51 to 8.81 log CFU/g of L. delbrueckii ssp. bulgaricus) in all the yogurts throughout the storage period. There was some decrease in the pH of the yogurts during storage and an increase in the concentration of lactic acid. However, the proteolytic and ACE-inhibitory potential of the starter cultures was suppressed in the presence of Versagel. On the other hand, the addition of Versagel had a positive impact on the physical properties of the low-fat yogurt, namely, spontaneous whey separation, firmness, and pseudoplastic properties.

We studied the dynamics of the microbial population during ripening of Cueva de la Magahá cheese using a combination of classical and molecular techniques. Samples taken during ripening of this Spanish goat's milk cheese in which Lactococcus lactis and Streptococcus thermophilus were used as starter cultures were analyzed. All bacterial isolates were clustered by using randomly amplified polymorphic DNA (RAPD) and identified by 16S rRNA gene sequencing, species-specific PCR, and multiplex PCR. Our results indicate that the majority of the 225 strains isolated and enumerated on solid media during the ripening period were nonstarter lactic acid bacteria, and Lactobacillus paracasei was the most abundant species. Other Lactobacillus species, such as Lactobacillus plantarum and Lactobacillus parabuchneri, were also detected at the beginning and end of ripening, respectively. Non-lactic-acid bacteria, mainly Kocuria and Staphylococcus strains, were also detected at the end of the ripening period. Microbial community dynamics determined by temporal temperature gradient gel electrophoresis provided a more precise estimate of the distribution of bacteria and enabled us to detect Lactobacillus curvatus and the starter bacteria S. thermophilus and L. lactis, which were not isolated. Surprisingly, the bacterium most frequently found using culture-dependent analysis, L. paracasei, was scarcely detected by this molecular approach. Finally, we studied the composition of the lactobacilli and their evolution by using length heterogeneity PCR.

This study investigated the influence of incorporating Raftiline HP on the pH, growth, proteolytic, and angiotensin-I converting enzyme inhibitory activities and on spontaneous whey separation, firmness, and rheological properties of low-fat yogurts during storage for 28 d at 4 degrees C. Three types of yogurts were prepared from skim milk containing 0% (YI0, control), 2% (YI2), and 3% (YI3) Raftiline HP, respectively. The incorporation of Raftiline HP improved the growth of starter organisms, particularly that of Lactobacillus delbrueckii ssp. bulgaricus, resulting in shorter fermentation time. There was a significant improvement in total proteolysis, which was highest in yogurt containing 3% Raftiline HP. The ACE inhibitory activity was maximal in YI3 compared to YI2 and YI0. Incorporation of Raftiline HP did not affect whey separation and firmness of the low-fat yogurts. All these products were more fluid like with distinct pseudoplastic properties and lesser ability to resist deformation upon applied shear.

Whey, a by-product of the cheese industry usually disposed as waste, is a source of biological and functional valuable proteins. The aim of this work was to evaluate the potentiality of three lactic acid bacteria strains to design a starter culture for developing functional whey-based drinks. Fermentations were performed at 37 and 42 degrees C for 24h in reconstituted whey powder (RW). Carbohydrates, organic acids and amino acids concentrations during fermentation were evaluated by RP-HPLC. Proteolytic activity was measured by the o-phthaldialdehyde test and hydrolysis of whey proteins was analyzed by Tricine SDS-PAGE. The studied strains grew well (2-3log cfu/ml) independently of the temperature used. Streptococcus thermophilus CRL 804 consumed 12% of the initial lactose concentration and produced the highest amount of lactic acid (45 mmol/l) at 24h. Lactobacillus delbrueckii subsp. bulgaricus CRL 454 was the most proteolytic (91 microg Leu/ml) strain and released the branched chain amino acids Leu and Val. In contrast, Lactobacillus acidophilus CRL 636 and S. thermophilus CRL 804 consumed most of the amino acids present in whey. The studied strains were able to degrade the major whey proteins, alpha-lactalbumin being degraded in a greater extent (2.2-3.4-fold) than beta-lactoglobulin. Two starter cultures were evaluated for their metabolic and proteolytic activities in RW. Both cultures acidified and reduced the lactose content in whey in a greater extent than the strains alone. The amino acid release was higher (86 microg/ml) for the starter SLb (strains CRL 804+CRL 454) than for SLa (strains CRL 804+CRL 636, 37 microg/ml). Regarding alpha-lactalbumin and beta-lactoglobulin degradation, no differences were observed as compared to the values obtained with the single cultures. The starter culture SLb showed high potential to be used for developing fermented whey-based beverages.

The effect of six wild strains on the volatile profile of the PS cheese was investigated and compared to that generated from industrial starters generally used to produce PS cheese. All cheeses were subjected to microbiological, physicochemical, and volatile compounds analyses. The DGGE of the 16S rDNA analysis was also applied. The volatile compounds generated during ripening were studied through the SPME and the GC-MS methods. No difference was detected between the experimental and control cheeses throughout chemical and microbiological analyses, while the DGGE results showed the presence of Streptococcus thermophilus in all cheeses, and the dominance of Enterococcus durans, Lactobacillus rhamnosus, and Lactobacillus casei in most of the experimental cheeses. Moreover, the presence of Lactococcus lactis species as in the control and in the experimental P2 and P4 cheeses was also revealed. The SPME results showed more pronounced volatile compounds in the experimental cheese samples than in the control ones.

Aims: Considering the significant rise in the probiotic market in Columbia, and given the lack of reports concerning the microbial population and strain performance in products from different producers, this study aims at determining the number of viable starter bacteria and probiotics in bio-yoghurts available at the Columbian market, identifying the species and analysing the performance of the isolated strains in bile acid resistance, antagonistic activity against pathogens, and adherence capacity to human intestinal epithelial cells., Methods and Results: Seven bio-yoghurts were analysed for the bacterial species present. Species identification was carried out using 16S rRNA gene targeted PCR. The cultured bacteria were tested for bile acid resistance, adherence to a human intestinal epithelial cell line, and antagonism against the pathogen Salmonella enterica serovar Typhimurium. A total of 17 different strains were identified. Based on plate counting, all bio-yoghurts have at least total viable cells of approximately 10(7) CFU ml(-1). Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus were the most frequently isolated bacteria. Viable Bifidobacterium was only recovered from one product. However, after PCR analysis, DNA of this genus was confirmed in five out of seven products. Major differences were found for S. typhimurium antagonism. The adherence capacity to Caco-2 cells was observed in 10 of the isolated strains. In general, low survival to simulated gastric juice was observed., Conclusions: Some of the isolated strains have probiotic potential, although not all of them were present in the advised amount to exert beneficial health effects. However, the full correct scientific name of the isolated bacteria and their viable counts were not included on the product label., Significance and Impact of the Study: This is the first report describing the identification and functionality of starter bacteria and probiotics present in dairy products on the Columbian market.

The viability of Lactobacillus paracasei and its effect on growth of the microbiota in potentially probiotic and synbiotic fresh cheeses during storage at 4 +/- 1 degree C was investigated. Three cheese-making trials (T1, T2, and T3) were prepared in quadruplicate, all supplemented with a Streptococcus thermophilus culture. L. paracasei subsp. paracasei was added to cheeses in T1 and T2, and inulin was added to cheeses in T2. Counts of L. paracasei, S. thermophilus, coliforms, Escherichia coli, Staphylococcus spp., DNase-positive Staphylococcus, and yeasts and molds were monitored during storage for up to 21 days. Viable counts of L. paracasei in probiotic (T1) and synbiotic (T2) cheeses remained above 7 log CFU/g during the entire storage period, whereas counts of S. thermophilus remained above 9.5 log CFU/g for cheeses from TI, T2, and T3. Populations of coliforms, Staphylococcus spp., and DNase-positive Staphylococcus were higher in T3 cheese and differed significantly from those in cheeses from T1 and T2 (P < 0.05). Inhibition of contaminants prevailed when both L. paracasei and S. thermophilus were present in fresh cream cheese and probably was due to acid production by both strains; bacteriocin production was not found. Addition of inulin in T2 did not impact microbial viability (P > 0.05). L. paracasei subsp. paracasei in coculture with S. thermophilus was inhibitory against microbial contaminants in fresh cream cheese with or without the addition of inulin, indicating the potential use of this combination in a probiotic and synbiotic product.