Rheological and physical characteristics of non-fat set yogurt prepared with EPS-producing Streptococcus thermophilus and an H+-ATPase-defective mutant Lactobacillus delbrueckii subsp. bulgaricus

The rheological and physical characteristics of non-fat set yogurt produced using co-cultures of ropy EPS-producing Streptococcus thermophilus S3.3 and Lactobacillus delbrueckii subsp. bulgaricus LTM and of Streptococcus thermophilus S3.3 and Lactobacillus delbrueckii subsp. bulgaricus H+-ATPase-defective mutant L6 under different conditions were studied. Yogurts produced with mutant L6 had a higher pH and water-holding capacity and better textural properties compared to those produced with the LTM strain. The highest storage modulus (G′) and thixotropic loops were found for yogurt made with L6 at 42°C on day 21 of cold storage. This yogurt contained a network of thick, continuous protein aggregates and large void spaces.     

Morphology,FTIR fingerprint and survivability of encapsulated lactic bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) in simulated gastric juice and intestinal juice

Microencapsulation of bacteria in hydrocolloid beads is known as a potential way to enhance and protect their survivability in the digestive tract. We encapsulated a mix of two lactic bacteria (Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) in sodium alginate (AG) and chitosan (CH) matrices. The morphological parameters of beads containing bacteria were comparatively measured. The FTIR fingerprint can discriminate the presence of bacteria into the beads, identifying specific absorption peaks for bacteria located at 1750, 2852 and 2926 cm^?1. To check the survivability of bacteria, beads were incubated in simulated gastric juice (pH 1.5) and intestinal juice (pH 7.2) for different periods of time, up to 120 min. The 2% AG beads, better than CH beads, provided best protection of bacterial survivability. Encapsulated mix of S. thermophilus and L. delbrueckii subsp. bulgaricus can behave as a probiotic bacteria, viable, surviving in the simulated gastric and intestinal juice.     

Probiotic strains: survival under simulated gastrointestinal conditions,in vitro adhesion to Caco-2 cells and effect on cytokine secretion

This study evaluated three probiotic strains (Lactobacillus paracasei subsp. paracasei LC-01, L. acidophilus LA-5, Bifidobacterium lactis Bb-12) and two yoghurt strains (L. delbrueckii subsp. bulgaricus LBY-27 and Streptococcus thermophilus STY-31) with regard to their resistance to simulated gastrointestinal stress, and their ability to interact with human intestinal epithelial cells. The viability of strains was analyzed by measurements of fluorescence-stained cells and their growth by plate colony-counts. The results reveal that for all tested strains, gastric emptying (above pH 3.0) would release a large number of viable cells ranging from 91% for L. paracasei to 53% for S. thermophilus into the intestinal tract, and that between 12 and 23% of them subsequently survive intestinal stress. Among them L. paracasei showed the highest resistance to gastric stress. All the bacteria adhered to the Caco-2 cell line, with the highest adhesions being observed for L. delbrueckii subsp. bulgaricus (9%) and L. acidophilus (7%). Binding of all strains to Caco-2 cells did not result in a significant increase in the production of IL-6 and IL-8 cytokines, suggesting that these bacteria do not trigger an overt inflammatory response in human intestine epithelial cells. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Functional and Sensory Properties of Salad Dressing Containing Fermented Peanut Milk

The effects of substituting peanut milk fermented with mixed cultures of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus for buttermilk on chemical, physical and sensory properties of ranch-style salad dressing were investigated. Increased amounts of fermented peanut milk in dressing resulted in decreased lightness, creamy flavor, oil emulsion capacity and consistency. Changes were dependent upon the commercial brand of dressing mix. Fermented peanut milk can be substituted for buttermilk in salad dressing at levels up to 25% without substantial reduction in sensory qualities.     

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.     

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.     

Shelf life of alginate beads containing lactobacilli and bifidobacteria: characterisation of microspheres containing Lactobacillus delbrueckii subsp. bulgaricus

Capsules, containing different microorganisms (Lactobacillus plantarum, L. casei, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. delbrueckii, L. reuteri, L. rhamnosus, Bifidobacterium breve and B. animalis subsp. lactis), were produced through a simple extrusion protocol and dipped in a CaCl₂ solution (0.5% or 8%). Beads were characterised, to assess the encapsulation yield (EY) and the shelf life at 4 °C (defined as the time to retain inside a cell count of 6 log CFU g^?1); then, L. delbrueckii subsp. bulgaricus was used as target for system optimisation, through the study of the kinetic of cell release, as a function of the concentration of CaCl₂ solution, agitation (150 rpm) and recycling. The EY was 50% or higher, and the shelf life was at least 30 days. Concerning beads containing L. delbrueckii subsp. bulgaricus, the release of cells from capsules followed a biphasic (diauxic) trend and the microsphere could be used successfully for two times, as only after third reuse, the amount of release cells decreased.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

Development of a spray dried probiotic yoghurt containing Lactobacillus paracasei NFBC 338

The aim of this study was to monitor viability of probiotic Lactobacillus paracasei NFBC 338 during: (a) two-stage yoghurt fermentation with starter cultures Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, (b) following spray drying, and (c) during storage for 42 days. During the initial fermentation phase (10 h), probiotic Lactobacillus numbers increased 7-fold to 3.9 × 10⁹ cfu g^?1 and these numbers were maintained during fermentation for a further 3 h in the presence of the yoghurt starters. Following spray-drying, the probiotic culture survived best, followed by S. thermophilus and L. delbrueckii subsp. bulgaricus (yielding 3.4 × 10⁸, 1.2 × 10⁸ and 4.0 × 10⁵ cfu g^?1 powder, respectively). L. paracasei NFBC 338 and S. thermophilus were stable during storage at 4 °C and 15 °C (for 42 days) with viable counts exceeding 10⁷ cfu g^?1, while viability of L. delbrueckii subsp. bulgaricus decreased considerably throughout storage.     

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.     

Optimization of the rheological properties of probiotic yoghurts supplemented with milk proteins

This study aimed to optimize the rheological properties of probiotic yoghurts supplemented with skimmed milk powder (SMP), whey protein concentrate (WPC) and sodium caseinate (Na-Cn) by using an experimental design type simplex-centroid for mixture modeling. It included seven batches/trials: three were supplemented with each type of the dairy protein used, three corresponding to the binary mixtures and one to the ternary one in order to increase protein concentration in 1 g 100 g⁻¹ of final product. A control experiment was prepared without supplementing the milk base. Processed milk bases were fermented at 42 °C until pH 4.5 by using a starter culture blend that consisted of Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Bifidobacterium animalis subsp. lactis. The kinetics of acidification was followed during the fermentation period as well the physico-chemical analyses, enumeration of viable bacteria and rheological characteristics of the yoghurts. Models were adjusted to the results (kinetic responses, counts of viable bacteria and rheological parameters) through three regression models (linear, quadratic and cubic special) applied to mixtures. The results showed that the addition of milk proteins affected slightly acidification profile and counts of S. thermophilus and B. animalis subsp. lactis, but it was significant for L. delbrueckii subsp. bulgaricus. Partially-replacing SMP (45 g/100 g) with WPC or Na-Cn simultaneously enhanced the rheological properties of probiotic yoghurts taking into account the kinetics of acidification and enumeration of viable bacteria.     

Characterization and Classification of Streptococcus Thermophilus and Lactobacillus Delbrueckii Subsp. Bulgaricus Strains Isolated from Traditional Greek Yogurts

ABSTRACT Streptococcus thermophilus (74 strains) and Lactobacillus delbrueckii subsp. bulgaricus (75 strains) isolates from traditional Greek yogurts were tested on the basis of their acidification kinetics, acidity, viscosity, and aroma compounds formation in milk pure cultures. Strains of Lb. bulgaricus presented greater viscosity values, higher acidification activity and produced more acetaldehyde at culture pH 4.6 than S. thermophilus strains. Acetoin was formed only by the latter strains, while diacetyl was not detected in either species. Application of cluster and discriminant analysis permitted definition of distinct groups of strains and of the most significant variables to discriminate among them. Finally, classification and regression tree analysis allowed clusters characterization by critical values of the most discriminant variables. Evaluation of results as above could be a useful tool to select, through a large number of screened strains, the suitable starters for specific purposes.     

Identification and characterization of lactic acid bacteria isolated from traditional cone yoghurt

Cone yoghurt is a yoghurt variety produced by adding only pine cones to milk without culture in a limited area of Turkey. The present study was conducted to identify and characterize lactic flora in traditional cone yoghurt. Morphological, cultural, physiological, biochemical, and genotypic characteristics were employed to identify lactic acid bacteria isolates from cones and cone yoghurts. Streptococcus salivarius subsp. thermophilus (S. thermophilus) and Lactobacillus delbrueckii subsp. bulgaricus (L. bulgaricus) were obtained from both cones and yoghurts. Among the isolates, L. plantarum was frequently isolated except for these two bacteria (S. thermophilus and L. bulgaricus). The results indicate that the cone yoghurt has a mixed microflora contrary to the yoghurt produced by the addition of a starter culture and S. thermophilus, and L. bulgaricus in cone yoghurt originates from the pine cones.     

Effects of glutathione on acid stress resistance and symbiosis between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

Streptococcus thermophilus SDMCC18 contains a novel bi-functional glutathione synthetase gene gshF, and it can produce glutathione (GSH). In the present study, we demonstrated that the produced GSH by S. thermophilus SDMCC18 could protect cells against acid challenge and be secreted into the medium. Moreover, using a gshF-defective mutant strain as the control, we found that the GSH conferred a protective role against acid stress to Lactobacillus delbrueckii subsp. bulgaricus ATCC11842. In addition, L. bulgaricus ATCC11842 could import the exogenous GSH into the cytoplasm, leading to an improved growth of strain ATCC11842. Taken together, our study reported a novel proto-cooperation relationship between S. thermophilus and L. bulgaricus in yoghurt fermentation.     

Relationship between Lactobacillus bulgaricus and Streptococcus thermophilus under whey conditions: Focus on amino acid formation

In this study, we performed microarray experiments using Lactobacillus delbrueckii subsp. bulgaricus 2038 cultured with or without Streptococcus thermophilus in whey medium to investigate nitrogen utilisation of these two strains, since Lb. bulgaricus 2038 use whey as their nitrogen before casein. For the utilisation of environmental free amino acids and peptides in co-culture, Lb. bulgaricus 2038 imported the types of peptides necessary for growth more effectively compared with monoculture because S. thermophilus primarily preferred different types of peptides than Lb. bulgaricus, but has lost the ability to prioritise the use of free branched-chain amino acids. Considering the ratio of nitrogen versus carbon being lower during co-culture, Lb. bulgaricus 2038 was induced to stop converting aspartate into carbon-skeleton intermediates which is catalysed by phosphoenolpyruvate carboxykinase. Despite mutualism, Lb. bulgaricus underwent competitive co-evolution with S. thermophilus to optimise nitrogen utilisation during co-cultivation.     

Identification of peptides in traditional and probiotic sheep milk yoghurt with angiotensin I-converting enzyme (ACE)-inhibitory activity

Two sets of traditional Greek sheep milk yoghurt were produced: the first one (YC) using normal yoghurt culture (Lactobacillus delbrueckii subsp. bulgaricus ?10.13 and Streptococcus thermophilus ?10.7) and the second (PR) with the same normal culture mixed with Lactobacillus paracasei subsp. paracasei DC412. YC and PR had similar physicochemical properties and proteolysis patterns throughout storage. Both products showed similar peptide profiles by RP-HPLC but quantitative differences were observed in respect to storage time. Single-strain cultures of the microorganisms used showed similar peptide profiles for both lactobacilli, yet L. delbrueckii subsp. bulgaricus was the most proteolytic of all three microorganisms. The peptide content and the ACE-inhibitory activity of the water-soluble extracts of yoghurts, YC and PR, increased throughout storage. Major peptides were identified from yoghurt PR and from the separate cultures of L. delbrueckii subsp. bulgaricus and L. paracasei subsp. paracasei. Most of these peptides were derived from β-casein. A peptide, β-CN f114-121, with well-established ACE-inhibitory and opiate-like activity was identified in yoghurt PR. Further identified peptides were regarded as potential ACE-inhibitors according to their sequence.     

Isolation and characterisation of a Lactobacillus delbrueckii subsp. bulgaricus mutant with low H+‐ATPase activity

Lactobacillus delbrueckii subsp. bulgaricus is an acid‐tolerant species used in yoghurt production and considered important in postacidification reductions in palatability of yoghurt during storage. In this study, a variant (ND06‐2) of L. delbrueckii subsp. bulgaricus ND06 was selected using neomycin sulphate. ND06‐2 exhibited sensitivity to acids and lower H⁺‐ATPase activity compared with ND06. High‐pressure liquid chromatography (HPLC) was used to demonstrate that the capacity of ND06‐2 to produce lactic acid was significantly lower than for ND06 during fermentation or storage. These results suggest that the L. delbrueckii subsp. bulgaricus variant could be used in yoghurt starters and have a lower tendency to cause postacidification.     

The effect of wild lactic acid bacteria on the production of goat’s milk soft cheese

A blend of four wild strains of lactic acid bacteria, Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus paracasei subsp. paracasei and Enterococcus faecalis, was used as starter to produce a goat’s milk soft cheese. The cheese was analysed microbiologically, physicochemically and organoleptically through a 30‐day period of ripening. Counts of the starter cultures increased in the first 24 h and then remained stable during ripening. Yeasts and coliforms were found at low numbers initially and gradually decreased to undetectable levels. Micrococcal counts were high throughout ripening. The end product was a soft cheese characterised by a mild, aromatic taste as well as a smooth structure.     

Microbial and physiochemical changes in yoghurts containing different Lactobacillus delbrueckii subsp. bulgaricus strains in association with Lactobacillus plantarum as an adjunct culture

Two strains of slow and normal acid‐producing Lactobacillus delbrueckii subsp. bulgaricus, mixed separately with Streptococcus thermophilus, were used in the preparation of yoghurt with or without Lactobacillus plantarum A7. Viable cell counts of lactic bacteria, pH, lactic acid and acetic acid concentrations, soluble nitrogen content, spontaneous syneresis, viscosity and firmness were studied at selected points during the refrigerated storage period. Results showed that survival of L. plantarum A7 in yoghurt was independent of the acidification capability of L. delbrueckii subsp. bulgaricus strains which had already been characterised in their pure culture. Thus, in the preparation of probiotic yoghurt, using slow acid‐producing L. delbrueckii subsp. bulgaricus strain in combination with nondairy lactobacilli postfermentation acidification is not restricted but instability in other physicochemical properties of the yoghurt may be expected.