Immunomodulatory effects of polysaccharides produced by Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1

The extracellular polysaccharides (EPS) produced by lactic acid bacteria (LAB) are associated with the rheology, texture, and mouthfeel of fermented milk products, including yogurt. This study investigated the immunomodulatory effects of EPS purified from the culture supernatant of Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) OLL1073R-1. The crude EPS were prepared from the culture supernatant of L. bulgaricus OLL1073R-1 by standard chromatographic methods, and were fractionated into neutral EPS and acidic EPS (APS). Acidic EPS were further fractionated into high molecular weight APS (H-APS) and low molecular weight APS (L-APS). High molecular weight APS were shown to be phosphopolysaccharides containing D-glucose, D-galactose, and phosphorus. Stimulation of mouse splenocytes by H-APS significantly increased interferon-γ production, and, moreover, orally administered H-APS augmented natural killer cell activity. Oral administration of yogurt fermented with L. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059 to mice showed a similar level of immunomodulation as H-APS. However, these effects were not detected following administration of yogurt fermented with the starter combination of L. bulgaricus OLL1256 and S. thermophilus OLS3295. We conclude from these findings that yogurt fermented with L. bulgaricus OLL1073R-1, containing immunostimulative EPS, would have an immunomodulatory effect on the human body.     

Fat-free yogurt made using a galactose-positive exopolysaccharide-producing recombinant strain of Streptococcus thermophilus

To prevent textural defects in low-fat and fat-free yogurts, fat substitutes are routinely added to milk. In situ production of exopolysaccharides (EPS) by starter cultures is an acknowledged alternative to the addition of biothickeners. With the aim of increasing in situ EPS production, a recombinant galactose-positive EPS⁺Streptococcus thermophilus strain, RD-534-S1, was generated and compared with the parent galactose-negative EPS⁺ strain RD-534. The RD-534-S1 strain produced up to 84 mg/L of EPS during a single-strain milk fermentation process, which represented 1.3 times more than the EPS produced by strain RD-534. Under conditions that mimic industrial yogurt production, the starter culture consisting of RD-534-S1 and (EPS⁻) Lactobacillus bulgaricus L210R strain (RD-534-S1/L210R) led to an EPS production increase of 1.65-fold as compared with RD-534-S1 alone. However, the amount of EPS produced did not differ from that found in yogurts produced using an isogenic starter culture that included the parent S. thermophilus strain RD-534 and Lb. bulgaricus L210R (RD-534/L210R). Moreover, the gel characteristics of set-style yogurt and the rheological properties of stirred-style yogurt produced using RD-534-S1/L210R were similar to the values obtained for yogurts made with RD-534/L210R. In conclusion, it is possible to increase the production of EPS by ropy S. thermophilus strains through genetic engineering of galactose metabolism. However, when used in combination with Lb. bulgaricus for yogurt manufacture, the EPS overproduction of recombinant strain is not significant.     

Isolation and identification of exopolysaccharide producer lactic acid bacteria from Turkish yogurt

Lactic acid bacteria (LAB) were isolated from traditional yogurt samples and genotypic characterization of these isolates revealed the presence of 21 distinct LAB strains belonging to Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Leuconostoc mesenteroides, and Lactobacillus plantarum as new LAB strains. Determination of the exopolysaccharide (EPS) production characteristics of the selected strains of each species revealed that all strains possessed at least one gene required for both homopolymeric‐ and heteropolymeric‐type EPS production. Structural analysis of the EPSs showed that L. delbrueckii subsp. bulgaricus Y39 and S. thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuc. mesenteroides Y35 and L. plantarum Y36 produced homopolymeric glucan‐type EPS. The level of EPS production in these strains was found to be in a similar range. These strains with EPS production characteristics are good candidates for future studies as new LAB for yogurt production.

Practical applications

Recent trends in yogurt production technology have led to an increased use of ropy starter cultures in yogurt production due to the technological roles of exopolysacharides (EPS) produced by these cultures. The main role of EPS in yogurt production is to improve the textural properties of yogurt as an in situ produced natural polymer. In addition to the yogurt starter cultures, use of adjunct cultures during production of yogurt is also of special interest to enhance the technological and nutritional characteristics of yogurt. Therefore, in this study, potential yogurt starter and adjunct cultures from traditional yogurt samples with EPS production characteristics were isolated. From these isolates, Lactobacillus delbrueckii subsp. bulgaricus Y39 and Streptococcus thermophilus Y102 produced heteropolymeric EPS containing glucose and galactose, whereas Leuconostoc mesenteroides Y35 and Lactobacillus plantarum Y36 produced homopolymeric glucan.     

Proteolytic action of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 reduces antigenic response to bovine β-lactoglobulin

The whey protein β-lactoglobulin (BLG) is highly allergenic. Lactic acid bacteria can degrade milk proteins. The capacity of Lactobacillus delbrueckii subsp. bulgaricus CRL 656 to hydrolyse the major BLG epitopes (V41–K60; Y102–R124; L149–I162) and decrease their recognition by IgE of allergic patients was evaluated. The intensity of BLG degradation was analysed by Tricine SDS–PAGE and RP-HPLC. Peptides released were identified by LC–MS/MS and the hydrolysates were tested for their capacity to inhibit IgE binding by ELISA test. L. delbrueckii subsp. bulgaricus CRL 656 degraded BLG (35%, 8 h). The sequence analysis of the released peptides indicated that this strain degraded three main BLG epitopes. BLG-positive sera (3–5 year old children) were used for testing IgE binding inhibition of BLG hydrolysates from the Lactobacillus strain. The hydrolysates were less immuno-reactive (32%) than the heated BLG. L. delbrueckii subsp. bulgaricus CRL 656 could be used for developing hypoallergenic dairy products.     

Productions and monomer compositions of exopolysaccharides by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains isolated from traditional home-made yoghurts and raw milk

In this study, a total of forty‐five strains of lactobacilli and streptococci were determined exopolysaccharide (EPS) production in skim milk and Man Rogosa and Sharpe (MRS)/M17 medium, viscosity and proteolytic activity. The exopolysaccharide production by lactobacilli strains during growth in MRS medium was twenty‐one to 211 mg L^?1, while in skim milk was to thirty‐six to 315 mg L^?1. The EPS production by streptococci strains during growth in M17 medium was sixteen to 114 mg L^?1, while in skim milk was to twenty‐four to 140 mg L^?1. The EPS production of strains was lower in MRS/M17 medium than skim milk. Results showed that it was not clear correlation between the viscosity and EPS production of some strains. All strains were shown proteolytic activity. Positive correlations between exopolysaccharide production and proteolytic activity in skim milk were found some strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. These results indicated that the high exocellular protease‐producing strains can produce high EPS in skim milk. The monomer compositions of the EPSs formed by selected five strains were analysed. Mannose dominated (99–100%) on the EPS produced by L. delbrueckii subsp. bulgaricus and S. thermophilusstrains (except L. delbrueckii subsp. bulgaricus 22) in skim milk and MRS/M17 medium. Besides, the EPSs of strains in skim milk contained small amount of lactose.     

Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants

Antimicrobial behavior of lactic acid bacteria (LAB) has been explored since many years to assess their ability to produce bacteriocin, a natural preservative, to increase the shelf life of food. This study aims to characterize bacteriocin producing strains of lactic acid bacteria isolated from acidic to slightly acidic raw vegetables including tomato, bell pepper and green chili and to investigate their potential to inhibit food related bacteria. Among twenty nine LAB screened for antimicrobial activity, three exhibited antagonism against closely related bacterial isolates which was influenced by varying temperature and pH. They were identified up to strain level as Lactococcus lactis subsp. lactis TI-4, L. lactis subsp. lactis CE-2 and L. lactis subsp. lactis PI-2 based on 16S rRNA gene sequence. Their spectrum of inhibition was observed against food associated strains of Bacillus subtilis and Staphylococcus aureus. Moreover, L. lactis subsp. lactis PI-2 selected on the basis of higher antimicrobial activity was further evaluated for bacteriocin production which was detected as nisin A and nisin Z. These findings suggest the possible use of L. lactis strains of vegetable origin as protective cultures in slightly acidic as well as slightly alkaline food by the bio-preservative action of bacteriocins.     

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.     

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.     

Gelation and resistance to shearing of fermented milk: Role of exopolysaccharides

Milk was fermented with the exopolysaccharide-producing (EPS⁺) strains Lactococcus lactis subsp. cremoris, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and with the non-EPS-producing strain (EPS⁻) L. lactis subsp. cremoris. The kinetics of gelation and the behaviour of set fermented milk during and after shearing were studied using rheometry and confocal scanning laser microscopy. The time of gelation of milk depended on the kinetics of acidification of strains whereas the pH of gelation depended mostly on the presence of exopolysaccharides (EPS). In set fermented milk with EPS⁺ strains, bacteria were observed in protein-free areas likely filled with EPS. Phase-separated EPS and caseins contributed to induce the gelation of fermented milk at pH 5.6. The high resistance to shearing of milk fermented with the EPS⁺ strain L. lactis subsp. cremoris might be due to the negative charge of the exopolysaccharide allowing an attractive interaction with caseins.     

High genetic and phenotypic variability of Streptococcus thermophilus strains isolated from artisanal Yuruk yoghurts

Streptococcus thermophilus is a commonly used starter bacterium in dairy industry. It reduces the pH of milk rapidly and equilibrates the medium for the growth of Lactobacillus delbrueckii subsp. bulgaricus during yoghurt fermentation. Efforts to increase the diversity of artisanal yoghurt starters are not only important to bring new strains with novel and desirable characteristics, but also for the preservation of natural diversity which diminishes with the overuse and spread of industrial starters to natural resources. In the present study, 14 artisanal yoghurt samples were processed for the isolation of promising strains for yoghurt starter culture production and 66 strains were subsequently characterized. They were all identified as S. thermophilus using species-specific PCR and 16S rRNA gene sequencing. Genotypic diversity at the strain level was investigated by pulsed field gel electrophoresis (PFGE), and 22 homology groups were obtained. Further phenotypic characterization unearthed a significant phenotypic heterogeneity within homology groups, mostly with atypical novel character. Only 7 out of 66 strains showed S. thermophilus type-strain like phenotypic traits. Majority of the isolates were determined to be protease positive and fast milk acidifier to be used as yoghurt starter culture.     

Comparison of Associative Growth and Proteolytic Activity of Yogurt Starters in Whole Milk from Camels and Cows

Growth and proteolytic activities were studied using yogurt starter cultures incubated in pasteurized whole milk from camels and cows at 42°C as single and mixed cultures. In general, the growth of four strains of Streptococcus thermophilus and three strains of Lactobacillus delbrueckii ssp. bulgaricus was higher in cow milk than in camel milk. However, proteolysis was higher in camel milk than in cow milk. Lactobacillus delbrueckii ssp. bulgaricus LB12 in combination with streptococci had lowered pH more than did the other lactobacilli. Mixed cultures released the same amount of free amino groups as the corresponding single cultures, except for L. delbrueckii ssp. bulgaricus LB12.     

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.     

Characterization of probiotic bacteria involved in fermented milk processing enriched with folic acid

Yogurt products fermented with probiotic bacteria are a consumer trend and a challenge for functional food development. So far, limited research has focused on the behavior of the various probiotic strains used in milk fermentation. In the present study, we characterized folic acid production and the sensory and textural characteristics of yogurt products fermented with probiotic bacteria. Yogurt fermented with Lactobacillus plantarum had improved nutrient content and sensory and textural characteristics, but the presence of L. plantarum significantly impaired the growth and survival of Lactobacillus delbrueckii ssp. bulgaricus during refrigerated storage. Overall, L. plantarum was a good candidate for probiotic yogurt fermentation; further studies are needed to understand the major metabolite path of lactic acid bacteria in complex fermentation.     

Production of exopolysaccharides by Lactobacillus and Bifidobacterium strains of human origin, and metabolic activity of the producing bacteria in milk

This work reports on the physicochemical characterization of 21 exopolysaccharides (EPS) produced by Lactobacillus and Bifidobacterium strains isolated from human intestinal microbiota, as well as the growth and metabolic activity of the EPS-producing strains in milk. The strains belong to the species Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus vaginalis, Bifidobacterium animalis, Bifidobacterium longum, and Bifidobacterium pseudocatenulatum. The molar mass distribution of EPS fractions showed 2 peaks of different sizes, which is a feature shared with some EPS from bacteria of food origin. In general, we detected an association between the EPS size distribution and the EPS-producing species, although because of the low numbers of human bacterial EPS tested, we could not conclusively establish a correlation. The main monosaccharide components of the EPS under study were glucose, galactose, and rhamnose, which are the same as those found in food polymers; however, the rhamnose and glucose ratios was generally higher than the galactose ratio in our human bacterial EPS. All EPS-producing strains were able to grow and acidify milk; most lactobacilli produced lactic acid as the main metabolite. The lactic acid-to-acetic acid ratio in bifidobacteria was 0.7, close to the theoretical ratio, indicating that the EPS-producing strains did not produce an excessive amount of acetic acid, which could adversely affect the sensory properties of fermented milks. With respect to their viscosity-intensifying ability, L. plantarum H2 and L. rhamnosus E41 and E43R were able to increase the viscosity of stirred, fermented milks to a similar extent as the EPS-producing Streptococcus thermophilus strain used as a positive control. Therefore, these human EPS-producing bacteria could be used as adjuncts in mixed cultures for the formulation of functional foods if probiotic characteristics could be demonstrated. This is the first article reporting the physicochemical characteristics of EPS isolated from human intestinal microbiota.     

Influence of compounds associated with fermented dairy products on the growth of lactic acid starter and probiotic bacteria

The growth of 24 strains of lactic acid starter bacteria (Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus and Lactococcus lactis) and 24 strains of probiotic bacteria (Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus and bifidobacteria) in liquid media containing different substances was assessed. The substances used were salts (NaCl and KCl); sugars (sucrose and lactose); sweeteners (acesulfame and aspartame); aroma compounds (diacetyl, acetaldehyde and acetoin); natural colorings for fermented milk (red, yellow and orange colorings); flavoring agents (strawberry, vanilla, peach and banana essences); flavoring–coloring agents (strawberry, vanilla and peach); nisin, natamycin and lysozyme. Bacterial growth in the presence of natural fruit juices (green apple, kiwi, pineapple, peach and strawberry) with or without neutralization and cell viability in lactic acid acidified (pH 4 and 5) milk for 4 weeks at 5°C were also studied.Some compounds (KCl, sweeteners, aroma compounds, natamycin, flavoring agents and the peach flavoring–coloring agent) did not influence the growth of the strains in the concentrations commonly used in the dairy industry. The effect of other substances (especially flavoring–coloring agents) on the growth of lactic acid starters and probiotic bacteria was strain-dependent. Natural fruit juices weakly inhibited mainly S. thermophilus strains. Cell viability during cold storage in acidified milk was satisfactory for L. delbrueckii subsp. bulgaricus and L. casei group strains. For L. acidophilus and Bifidobacterium, the decreases in cell counts at pH 5 were negligible. Nevertheless, decreases from 1.6 to 6.2 and from 0.1 to 7.6 log orders, respectively were observed at pH 4.     

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.     

Evolution of aroma volatiles during storage of sourdough breads made by mixed cultures of Kluyveromyces marxianus and Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus helveticus

Two mixed starter cultures were used for sourdough bread making to evaluate their ability to improve quality and increase bread shelf-life: Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus helveticus mixed with the lactose fermenting yeast Kluyveromyces marxianus as alternative baker’s yeast. Control sourdough breads (K. marxianus) without the addition of bacteria, were also prepared. The changes on the headspace aroma volatiles during storage were assessed using solid-phase microextraction (SPME) GC–MS analysis. The effect of these changes on bread flavour was evaluated by consumer preference evaluations and the results were co-evaluated with those from the GC–MS analysis. The obtained results showed differences in the volatile composition of the different types of breads examined, as well as dramatic decreases of the number and the amount of volatiles after five days of storage. The sourdough breads made with K. marxianus and L. bulgaricus, had a more complex aroma profile, longer shelf-life and achieved the highest scores in the sensory tests.     

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.     

Effect of lactose hydrolysis on the milk-fermenting properties of Lactobacillus delbrueckii ssp. bulgaricus 2038 and Streptococcus thermophilus 1131

Yogurt is a well-known nutritious and probiotic food and is traditionally fermented from milk using the symbiotic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. However, yogurt consumption may cause health problems in lactose-intolerant individuals, and the demand for lactose-free yogurt has been increasing. The standard method to prepare lactose-free yogurt is to hydrolyze milk by lactase; however, this process has been reported to influence the fermentation properties of starter strains. This study aimed to investigate the fermentation properties of an industrial starter culture of L. bulgaricus 2038 and S. thermophilus 1131 in lactose-hydrolyzed milk and to examine the metabolic changes induced by glucose utilization. We found that the cell number of L. bulgaricus 2038, exopolysaccharide concentration, and viscosity in the coculture of L. bulgaricus 2038 and S. thermophilus 1131 was significantly increased in lactose-hydrolyzed milk compared with that in unhydrolyzed milk. Although the cell number of S. thermophilus 1131 showed no difference, production of formic acid and reduction of dissolved oxygen were enhanced in lactose-hydrolyzed milk. Further, in lactose-hydrolyzed milk, S. thermophilus 1131 was found to have increased the expression of NADH oxidase, which is responsible for oxygen reduction. These results indicated that glucose utilization promoted S. thermophilus 1131 to rapidly reduce the dissolved oxygen amount and produce a high concentration of formic acid, presumably resulting in the increased cell number of L. bulgaricus 2038 in the coculture. Our study provides basic information on the metabolic changes in starter strains in lactose-hydrolyzed milk, and demonstrates that lactose-free yogurt with increased cell number of L. bulgaricus can be prepared without delay in fermentation and decrease in the cell number of S. thermophilus.