Exploring the industrial potential of Lactobacillus delbrueckii ssp. bulgaricus by population genomics and genome-wide association study analysis

Lactobacillus delbrueckii ssp. bulgaricus is one of the most commonly used starter cultures for yogurt production. However, how its genetic background affects acid production capacity is unclear. This study investigated the industrial potential of L. delbrueckii ssp. bulgaricus using population genomics and GWAS analysis. To meet our goal, population genetics and functional genomics analyses were performed on 188 newly sequenced L. delbrueckii ssp. bulgaricus strains isolated from naturally fermented dairy products together with 19 genome sequences retrieved from the NCBI database. Four distinct clusters were identified, and they were correlated with the geographical sites where the samples were collected. The GWAS analysis about acidification fermentation results with sucrose-fortified reconstituted skim milk revealed a significant association between l-lactate dehydrogenase (lldD; Ldb2036) and the bacterial acid production rate. Our study has broadened the understanding of the population structure and genetic diversity of L. delbrueckii ssp. bulgaricus by identifying potential targets for further research, development, and use of lactic acid bacteria in the dairy industry.     

Proteolytic profiles of yogurt and probiotic bacteria

Nine strains of Streptococcus thermophilus, 6 strains of Lactobacillus delbrueckii ssp. bulgaricus, 14 strains of Lactobacillus acidophilus and 13 strains of Bifidobacterium spp. were screened for proteolytic, amino-, di-, tri- and endopeptidase activity by using the o-pthaldialdehyde-based spectrophotometric assay. Strains showing the highest and lowest proteolytic activity were further studied for their peptidase activities at the extracellular and intracellular levels. The amounts of free amino groups released by S. thermophilus, L. delbrueckii ssp. bulgaricus and L. acidophilus strains were higher than that by Bifidobacterium strains. Aminopeptidase activity was detected for all bacterial strains both at the extracellular and intracellular levels. The specific activity towards the six substrates studied was higher at the intracellular level for all strains. High dipeptidase activity was demonstrated by all bacterial strains for L. delbrueckii ssp. bulgaricus, L. acidophilus, and Bifidobacterium spp. whereas S. thermophilus had greater dipeptidase activity at the extracellular level. All bacterial cultures tested were able to hydrolyse large biologically active peptides, bradykinin, Ala–Ala–Ala–Ala–Ala and the tripeptide substrate Gly–Ala–Tyr at both the extracellular and intracellular levels. However, with the substrates ending with a C-terminal of phenylalanine, the hydrolysis only occurred at the intracellular level.     

Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia

Spontaneous milk fermentation has a long history in Mongolia, and beneficial microorganisms have been handed down from one generation to the next for use in fermented dairy products. The objective of this study was to investigate the diversity of lactic acid bacteria (LAB) communities in fermented yak, mare, goat, and cow milk products by analyzing 189 samples collected from 13 different regions in Mongolia. The LAB counts in these samples varied from 3.41 to 9.03 log cfu/mL. Fermented yak and mare milks had almost identical mean numbers of LAB, which were significantly higher than those in fermented goat milk but slightly lower than those in fermented cow milk. In total, 668 isolates were obtained from these samples using de Man, Rogosa, and Sharpe agar and M17 agar. Each isolate was considered to be presumptive LAB based on gram-positive and catalase-negative properties, and was identified at the species level by 16S rRNA gene sequencing, multiplex PCR assay, and restriction fragment length polymorphism analysis. All isolates from Mongolian dairy products were accurately identified as Enterococcus faecalis (1 strain), Enterococcus durans (3 strains), Lactobacillus brevis (3 strains), Lactobacillus buchneri (2 strains), Lactobacillus casei (16 strains), Lactobacillus delbrueckii ssp. bulgaricus (142 strains), Lactobacillus diolivorans (17 strains), Lactobacillus fermentum (42 strains), Lactobacillus helveticus (183 strains), Lactobacillus kefiri (6 strains), Lactobacillus plantarum ssp. plantarum (7 strains), Lactococcus lactis ssp. lactis (7 strains), Leuconostoc lactis (22 strains), Leuconostoc mesenteroides (21 strains), Streptococcus thermophilus (195 strains), and Weissella cibaria (1 strain). The predominant LAB were Strep. thermophilus and Lb. helveticus, which were isolated from all sampling sites. The results demonstrate that traditional fermented dairy products from different regions of Mongolia have complex compositions of LAB species. Such diversity of LAB provides useful information for further studies of probiotic strain selection and starter culture design, with regard to the industrial production of traditional fermented milk.     

Some low homogenization pressures improve certain probiotic characteristics of yogurt culture bacteria and Lactobacillus acidophilus LA-K

Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus salivarius ssp. thermophilus, and Lactobacillus acidophilus are dairy cultures widely used in the manufacture of cultured dairy products. Commonly used homogenization pressures in the dairy industry are 13.80 MPa or less. It is not known whether low homogenization pressures can stimulate bacteria to improve their probiotic characteristics. Objectives were to determine the effect of homogenization at 0, 3.45, 6.90, 10.34, and 13.80 MPa on acid tolerance, bile tolerance, protease activity, and growth of L. delbrueckii ssp. bulgaricus LB-12, S. salivarius ssp. thermophilus ST-M5, and L. acidophilus LA-K. The cultures were individually inoculated in cool autoclaved skim milk (4°C) and homogenized for 5 continuous passes. Growth and bile tolerance of samples were determined hourly for 10 h of incubation. Acid tolerance was determined every 20 min for 120 min of incubation. Protease activity was determined at 0, 12, and 24 h of incubation. All homogenization pressures studied improved acid tolerance of L. delbrueckii ssp. bulgaricus LB-12 but had no beneficial effect on protease activity and had negative effects on growth and bile tolerance. A pressure of 6.90 MPa improved acid tolerance, bile tolerance, and protease activity of S. salivarius ssp. thermophilus ST-M5, but none of the homogenization pressures studied had an effect on its growth. Homogenization pressures of 13.80 and 6.90 MPa improved acid tolerance and bile tolerance, respectively, of L. acidophilus LA-K but had no effect on protease activity and its growth. Some low homogenization pressures positively influenced some characteristics of yogurt culture bacteria and L. acidophilus LA-K. Culture pretreatment with some low homogenization pressures can be recommended for improvement of certain probiotic characteristics.     

A selective medium for the enumeration and differentiation of Lactobacillus delbrueckii ssp. bulgaricus

Modified reinforced clostridial medium (mRCM) was developed and evaluated for the differential enumeration of Lactobacillus delbrueckii ssp. bulgaricus. Lactobacillus bulgaricus, an important species of lactic acid bacteria with health benefits, is used in the production of yogurt and other fermented foods. Our results showed that supplementing reinforced clostridial medium with 0.025% CaCl₂, 0.01% uracil, and 0.2% Tween 80 (mRCM) significantly enhanced the growth rate of L. bulgaricus RR and ATCC 11842 strains as measured by the optical densities of these strains after 12 h of incubation at 42°C. The bacterial populations (plate count) of the RR and ATCC 11842 strains were 0.76 and 0.77 log cfu/g higher in mRCM than in de Man, Rogosa, and Sharpe and reinforced clostridial medium media, respectively. Conversely, the population counts for other bacterial species (Bifidobacterium, Lactobacillus rhamnosus, and Lactobacillus reuteri) were significantly inhibited in the mRCM medium. The addition of aniline blue dye to mRCM (mRCM-blue) improved the selectivity of L. bulgaricus in mixed lactic bacterial cultures compared with de Man, Rogosa, and Sharpe medium and lactic agar with regard to colony appearance and morphology. The mRCM-blue performed better than the conventional medium in culturing, enumerating, and differentiating L. bulgaricus. Therefore, mRCM-blue could be used as a selective medium to enhance the growth and differentiation of L. bulgaricus in order to meet the increasing demand for this beneficial species of bacteria.     

Application of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and L. helveticus for sourdough bread making

The application of Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) as starter cultures for sourdough bread making was examined. Production of lactic and acetic acids, bread rising, volatile composition, shelf-life and organoleptic quality of the sourdough breads were evaluated. The amount of starter culture added to the flour, the dough fermentation temperature and the amount of sourdough used were examined in order to optimise the bread making process. The use of mixed cultures led to higher total titratable acidities and lactic acid concentrations compared to traditionally made breads. Highest acidity (3.41 g lactic acid/kg of bread) and highest resistance to mould spoilage were observed when bread was made using 50% sourdough containing 1% K. marxianus and 4% L. delbrueckii ssp. bulgaricus. The use of these cultures also improved the aroma of sourdough breads, as shown by sensory evaluations and as revealed by GC–MS analysis.     

Antibacterial activity of selenium-enriched lactic acid bacteria against common food-borne pathogens in vitro

Selenium (Se) is an essential trace element for human health and animal nutrition. The aim of this study was to evaluate the inhibitory activities of Se-enriched lactic acid bacteria (LAB), Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus, against pathogenic Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes in vitro. The results indicated that the accumulation amount of Se by Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus reached 12.05 ± 0.43 µg/mL and 11.56 ± 0.25 µg/mL, respectively, accompanied by the relative maximum living cells when sodium selenite was 80 µg/mL. Oxford cup double plate assay showed that bacterial culture solution and cell-free culture supernatant (CFCS) from Se-enriched LAB exerted stronger antibacterial activity than those from the non-Se strains. The growth of pathogenic bacterial culture with CFCS at any growth stages was worse than that without CFCS; moreover, the inhibiting effect of CFCS of Se-enriched LAB was more significant than that of non-Se strains. Results from a scanning electron microscope equipped with energy dispersion X-ray spectrometry showed that elemental Se nanoparticles, which characteristically energy peak around 1.42 keV, were deposited on the cell surfaces of Lactobacillus delbrueckii ssp. bulgaricus. In addition, CFCS of Se-enriched LAB induced more serious cell structure damage of pathogenic bacteria than did non-Se LAB.     

Characterization of volatile compounds in fermented milk using solid-phase microextraction methods coupled with gas chromatography-mass spectrometry

Lactic acid bacteria (LAB) are industrially important bacteria that are widely used in the fermented food industry, especially in the manufacture of yogurt. Characteristic flavors are produced by LAB during fermentation and storage that affect the quality and acceptability of fermented milk products. In this study, the volatile compounds in milk fermented by Streptococcus thermophilus IMAU80842 alone, Lactobacillus delbrueckii ssp. bulgaricus IMAU20401 alone, or both species together were identified using solid-phase microextraction methods coupled with gas chromatography-mass spectrometry. A total of 53, 43, and 32 volatile compounds were identified in milk fermented by S. thermophilus alone, L. delbrueckii ssp. bulgaricus alone, or both species together, respectively. The presence of some important flavor compounds was confirmed: acetic acid, acetaldehyde, acetoin, 2,3-butanedione, ethanol, and 1-heptanol. Our results demonstrate that the composition of the volatile compounds in fermented milk depends on the species of LAB used and whether they are used alone or in combination. This is important for the selection of appropriate starter cultures for the production of different types of fermented milk product with particular flavors.     

Selection of starter cultures forthe fermentation of soya milk

The growth of four cultures selected on the basis of their ability to produce lactic acid in soya milk and/or utilize oligosaccharides, namelyStreptococcus thermophilus, Lactobacillus delbrueckiisubspbulgaricus, Lactobacillus fermentiandLactobacillus fermentum, was examined in reconstituted, low fat, spray-dried soya milk powder (12% total solids). The single culture ofS. thermophilusproduced a drop in pH from 6.5 to 4.7 over a 10-h period, and reduced the level of stachyose from 8.5 mg ml^-;1in the original milk to 3.2 mg ml^-;1; after 24 h fermentation, pH fell to 4.5, and the stachyose concentration to 3.0 mg ml^-;1. The paired culture ofS. thermophilusandL. fermentumbehaved in a similar fashion, but with only a slight improvement in stachyose utilization. When yeast extract (0.3%) and glucose (1.0%) were added to the soya milk, acid production by all the cultures increased dramatically, andL. delbrueckiisubspbulgaricusalone or in combination withS. thermophiluslowered the pH of the milk to 4.3 over 10 h. This combination ofS. thermophilusandL. delbrueckiisubspbulgaricuswas considered to be a likely combination for the production of a fermented product from soya milk or modified soya milk, as neitherL. fermentinorL. fermentumwere appreciably more effective in lowering the concentrations of oligosaccharides.     

Probiotic viability and storage stability of yogurts and fermented milks prepared with several mixtures of lactic acid bacteria

Currently, the food industry wants to expand the range of probiotic yogurts but each probiotic bacteria offers different and specific health benefits. Little information exists on the influence of probiotic strains on physicochemical properties and sensory characteristics of yogurts and fermented milks. Six probiotic yogurts or fermented milks and 1 control yogurt were prepared, and we evaluated several physicochemical properties (pH, titratable acidity, texture, color, and syneresis), microbial viability of starter cultures (Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus) and probiotics (Lactobacillus acidophilus, Lactobacillus casei, and Lactobacillus reuteri) during fermentation and storage (35 d at 5°C), as well as sensory preference among them. Decreases in pH (0.17 to 0.50 units) and increases in titratable acidity (0.09 to 0.29%) were observed during storage. Only the yogurt with S. thermophilus, L. delbrueckii ssp. bulgaricus, and L. reuteri differed in firmness. No differences in adhesiveness were determined among the tested yogurts, fermented milks, and the control. Syneresis was in the range of 45 to 58%. No changes in color during storage were observed and no color differences were detected among the evaluated fermented milk products. Counts of S. thermophilus decreased from 1.8 to 3.5 log during storage. Counts of L. delbrueckii ssp. bulgaricus also decreased in probiotic yogurts and varied from 30 to 50% of initial population. Probiotic bacteria also lost viability throughout storage, although the 3 probiotic fermented milks maintained counts ≥10⁷ cfu/mL for 3 wk. Probiotic bacteria had variable viability in yogurts, maintaining counts of L. acidophilus ≥10⁷ cfu/mL for 35 d, of L. casei for 7 d, and of L. reuteri for 14 d. We found no significant sensory preference among the 6 probiotic yogurts and fermented milks or the control. However, the yogurt and fermented milk made with L. casei were better accepted. This study presents relevant information on physicochemical, sensory, and microbial properties of probiotic yogurts and fermented milks, which could guide the dairy industry in developing new probiotic products.     

Fatty acid profile, trans-octadecenoic, α-linolenic and conjugated linoleic acid contents differing in certified organic and conventional probiotic fermented milks

Development of dairy organic probiotic fermented products is of great interest as they associate ecological practices and benefits of probiotic bacteria. As organic management practices of cow milk production allow modification of the fatty acid composition of milk (as compared to conventional milk), we studied the influence of the type of milk on some characteristics of fermented milks, such as acidification kinetics, bacterial counts and fatty acid content. Conventional and organic probiotic fermented milks were produced using Bifidobacterium animalis subsp. lactis HN019 in co-culture with Streptococcus thermophilus TA040 and Lactobacillus delbrueckii subsp. bulgaricus LB340. The use of organic milk led to a higher acidification rate and cultivability of Lactobacillus bulgaricus. Fatty acids profile of organic fermented milks showed higher amounts of trans-octadecenoic acid (C18:1, 1.6 times) and polyunsaturated fatty acids, including cis-9 trans-11, C18:2 conjugated linoleic (CLA-1.4 times), and α-linolenic acids (ALA-1.6 times), as compared to conventional fermented milks. These higher levels were the result of both initial percentage in the milk and increase during acidification, with no further modification during storage. Finally, use of bifidobacteria slightly increased CLA relative content in the conventional fermented milks, after 7 days of storage at 4 °C, whereas no difference was seen in organic fermented milks.     

Influence of addition of Raftiline HP on the growth, proteolytic, ACE- and α-glucosidase inhibitory activities of selected lactic acid bacteria and Bifidobacterium

This study examined the influence of Raftiline HP^®, added at the rate of 1, 2 and 3 g 100 mL⁻¹ to reconstituted skim milk, on the growth and biochemical activities of selected strains of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus casei, Lactobacillus acidophilus and Bifidobacterium longum. The growth of B. longum and S. thermophilus was improved in reconstituted skim milk (RSM) containing 1 g 100 mL⁻¹ Raftiline HP^®. All the organisms except for S. thermophilus produced more lactic acid and acetic acid in the presence of Raftiline HP^® than in the control. L. acidophilus and B. longum showed improvement in the proteolytic capabilities at all the three levels of Raftiline HP^® addition. L. delbrueckii ssp. bulgaricus showed maximum percent ACE inhibition in RSM containing 2 g 100 mL⁻¹ Raftiline HP^® while B. longum exhibited this potential in RSM containing 3 g 100 mL⁻¹ Raftiline HP^®. All organisms, except L. delbrueckii ssp. bulgaricus, however, showed improvement in the α-glucosidase inhibitory activity in RSM containing Raftiline HP^®.     

Effect of exopolysaccharides (EPSs) produced by Lactobacillus delbrueckii subsp. bulgaricus strains to bacteriophage and nisin sensitivity of the bacteria

Lactobacillus strains used in this study were isolated from village-type yogurt and raw milk. The isolates were identified as Lactobacillus delbrueckii subsp. bulgaricus by 16 s rDNA sequence analysis and API 50 CHL identification systems. The exopolysaccharide (EPS) production of the strains growth in skim milk were investigated. In addition sensitivity and insensitivity of these strains against domestic bacteriophages and nisin were examined. It was deduced that those strains which had relatively high EPS-producing capacity were insensitive against phages and nisin. Linear relationships were determined between EPS production of the bacteria and bacteriophage and nisin insensitivity of the bacteria.There was a negative correlation between EPS production quantity and phage and nisin sensitivity of the bacteria. Of all the strains, L. delbrueckii subsp bulgaricus B3 produced the highest EPS quantity, and it was insensitive against phages and nisin. Based on these results, it is suggested that L. delbrueckii subsp bulgaricus B3 can be used with the starter culture in dairy industry for stable and high-quality yogurt production.     

Effect of exopolysaccharide-producing starter cultures and post-fermentation mechanical treatment on textural properties and microstructure of low fat yoghurt

The microstructure and texture of yoghurts produced by four different exopolysaccharide (EPS)-producing starter cultures and mechanically treated post-fermentation at four levels of intensity (applied back-pressure) were studied. Two Lactobacillus delbrueckii ssp. bulgaricus (LB) strains were used in combination with two Streptococcus thermophilus (ST) strains and yoghurts were formulated by pairwise combining one LB and one ST strain. The choice of ST strain was the major determinant for the rheological properties of the yoghurts, since one of the ST strains conferred a ropy texture and resulted in yoghurts with decreased water holding capacity and an open microstructure. In addition, one of the LB strains used produced both aggregated and threadlike EPS and improved water holding. When combined with an ST strain that produced neglible amounts of EPS this LB strain resulted in yoghurt where a moderate mechanical treatment post-fermentation was able to further improve the water holding capacity.     

Characterization and association of bacterial communities and nonvolatile components in spontaneously fermented cow milk at different geographical distances

In the ecosystem of spontaneously fermented cow milk, the characteristics and relationship of bacterial communities and nonvolatile components at different scales of geographical distances (provincial, county, and village levels) are unclear. Here, 25 sampling sites from Xin Jiang and Tibet, 2 provinces of China, were selected based on the distribution of spontaneously fermented cow milk and used for metagenomic and metabolomic analysis. At the provincial geographical distance, the same predominant species, Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus, were detected in Xin Jiang and Tibet. Further, the richness of the bacterial composition of samples from Tibet was higher than those from Xin Jiang; specifically, at the species level, 28 species were identified in Tibet samples but only 7 species in Xin Jiang samples. At the provincial geographical level, we detected significant differences in bacterial structure, shown in principal coordinate analysis plots, and significant differences (Simpson index) in bacterial diversity were also detected. However, at the county and village levels, no significant differences were detected in bacterial communities and diversity, but a difference in bacterial compositions was detectable. This indicates that bacterial communities and diversity of spontaneously fermented milk dissimilarity significantly increased with geographic distance. For the nonvolatile component profiles, the partial least squares discriminant analysis plot (R²Y > 0.5 and Q² > 0.5 for the goodness-of-fit and predictive ability parameter, respectively) showed that samples from different geographical distances (provincial, county, and village) were all separated, which indicated that all the discriminations in nonvolatile components profiles were from different geographical distances. Investigating relationships between lactic acid bacteria and discriminatory nonvolatile components at the county level showed that 9 species were positively correlated with 16 discriminatory nonvolatile components, all species with low abundance rather than the predominant species L. delbrueckii ssp. bulgaricus and Strep. thermophilus, which indicates the importance of the selection of autochthonous nonpredominant bacteria.     

Influence of culture conditions and preconditioning on survival of Lactobacillus delbrueckii subspecies bulgaricus ND02 during lyophilization

The cryotolerance of Lactobacillus delbrueckii ssp. bulgaricus is weak during vacuum freeze-drying. Many factors affect cryoresistance of these bacteria, such as cryoprotectant composition, the lyophilization technology used, and the intrinsic characteristics of the bacteria. In this research, we explored the fermentation technology and other preconditioning treatments of cells in improving the cryoresistance of Lactobacillus delbrueckii ssp. bulgaricus strains during lyophilization. The addition of yeast extract in the propagation medium exerted a negative effect on the cryotolerance of these bacteria and decreased survival during lyophilization. The count of the freeze-dried cells from medium containing a high level (4%) of yeast extract was only 4.1 × 10⁹ cfu/g, indicating a death rate as high as 88%, compared with the culture medium without yeast extract, with a lower death rate of 44.7%. When Lactobacillus delbrueckii ssp. bulgaricus ND02 was propagated in yeast extract-free de Man, Rogosa, and Sharpe broth at a set pH value of 5.1, the cells showed unexpectedly higher survival after freeze-drying. Viable counts of the lyophilized cell of strain ND02 cultivated at pH 5.1 could reach 1.05 × 10¹¹ cfu/g and survival of the freeze-drying process was 68.3%, whereas at pH 5.7, survival was only 51.2%. We also examined the effects of pretreatment of cells on survival of the bacteria after vacuum freeze-drying. By analyzing the effect of pretreatment conditions on the expression of cold- and heat-shock genes, we established 2 pretreatments that improved survival of cells after lyophilization. Optimal fermentation conditions and pretreatment of the cell-cryoprotectant mixture at 10°C for 2 h or 37°C for 30 min improved the cryoresistance of 4 strains of Lactobacillus delbrueckii ssp. bulgaricus to varying degrees. Cells of IMAU20269 and IMAU20291 that were pretreated showed enhanced survival of 16.06 and 16.82%, respectively, after lyophilization. Expression of cold- and heat-shock genes for pretreated strains ND02, IMAU80423, IMAU20269, and IMAU20291 was analyzed by using quantitative PCR. From the expression of 2 cold shock-induced genes (cspA and cspB) and 6 heat shock-induced genes (groES, hsp, hsp20, hsp40, hsp60, and hsp70), strain ND02 showed a higher relative quantity of gene expression and displayed superior resistance to cold-induced stress during the freeze-drying process.     

Effects of yogurt starter cultures on the survival of Lactobacillus acidophilus

Recognized to confer health benefits to consumers, probiotics such as Lactobacillus acidophilus are commonly incorporated into fermented dairy products worldwide; among which yogurt is a popular delivery vehicle. To materialize most of the putative health benefits associated with probiotics, an adequate amount of viable cells must be delivered at the time of consumption. However, the loss in their viabilities during refrigerated storage has been demonstrated previously. This study focused on the effects of yogurt starter cultures on the survival of five strains of L. acidophilus, with emphases on low pH and acid production. Differential survival behavior between L. acidophilus strains was further analyzed. To this end, viable cell counts of L. acidophilus were determined weekly during 4 °C storage in various types of yogurts made with Streptococcus thermophilus alone, L. delbrueckii ssp. bulgaricus alone, both species of the starter cultures, or glucono-delta-lactone (GDL). All yogurt types, except for pasteurized yogurts, were co-fermented with L. acidophilus. Yogurt filtrate was analyzed for the presence of any inhibitory substance and for the amount of hydrogen peroxide. Multiplication of L. acidophilus was not affected by the starter cultures as all strains reached high level on day 0 of the storage period. Throughout the 28-day storage period, cell counts of L. acidophilus PIM703 and SBT2062 remained steady (~ 6 × 10⁷ CFU/g) in yogurts made with both starter cultures, whereas those of ATCC 700396 and NCFM were reduced by a maximum of 3 and 4.6 logs, respectively. When starter cultures were replaced by GDL, all strains survived well, suggesting that a low pH was not a critical factor dictating their survival. In addition, the filtrate collected from yogurts made with starter cultures appeared to have higher inhibitory activities against L. acidophilus than that made with GDL. The presence of viable starter cultures was necessary to adversely affect the survival of some strains, as pasteurized yogurts had no effect on their survival. In particular, the inhibitory effect exerted by L. delbrueckii ssp. bulgaricus on L. acidophilus NCFM was highly pronounced than by S. thermophilus, nevertheless, the same effect was not observed on SBT2062. The inhibition against stationary-phase NCFM cells might be caused by the elevated level of hydrogen peroxide produced by L. delbrueckii ssp. bulgaricus. Delineating factors driving the differences in survival trait among probiotic strains will lead to a more efficacious delivery of health benefits in fermented dairy products through targeted technological interventions.     

Development of a pentaplex PCR assay for the simultaneous detection of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. helveticus, L. fermentum in whey starter for Grana Padano cheese

A pentaplex PCR assay for the rapid, selective and simultaneous detection of Lactobacillus helveticus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and L. fermentum, was developed. The target sequences were a group of genes coding for beta-galactosidase production (S. thermophilus and L. delbrueckii subsp. bulgaricus), for cell-enveloped associated proteinase synthesis (L. helveticus), for dipeptide transport system production (L. delbrueckii subsp. lactis) and for arginine-ornithine antiporter protein production (L. fermentum). The analytical specificity of the assay was evaluated with 5 reference strains and 140 lactic acid bacterial strains derived from raw milk cheeses and belonging to the Lactobacillus, Streptococcus, Lactococcus and Enterococcus genera. The identification limit for each target strain was 10³ CFU/ml. This new molecular assay was used to investigate the LAB population by direct extraction of DNA from the 12 whey cultures for Grana Padano. The pentaplex PCR assay revealed a good correspondence with microbiological analyses and allowed to identify even minor LAB community members which, can be out-competed in vitro by numerically more abundant microbial species.