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.     

Role of cysteine in the improvement of γ-aminobutyric acid production by nonproteolytic Levilactobacillus brevis in coculture with Streptococcus thermophilus

Previous research has showed that nonproteolytic Levilactobacillus brevis 145 (L) in coculture with Streptococcus thermophilus 1275 (S), not Lactobacillus delbrueckii ssp. bulgaricus (Lbu), was able to produce γ-aminobutyric acid (GABA) during milk fermentation in the presence of monosodium glutamate (MSG). It was assumed that differences of casein hydrolysis patterns between Strep. thermophilus 1275 and L. bulgaricus caused the phenomenon. Moreover, the GABA content was low and residual MSG was high in SL-fermented milk. In our research, comparison of peptide profiles determined by liquid chromatography/tandem mass spectrometry showed that α_S2-casein, β-casein, and κ-casein degradation by L. bulgaricus and Strep. thermophilus varied. Importantly, the peptide number in the L and Lbu coculture group increased compared with the Lbu monoculture group, whereas the peptide number in the SL coculture group decreased in comparison with S monoculture group, suggesting that L. bulgaricus was not able to provide peptides for the growth of Lb. brevis 145. Furthermore, we found that after supplementation with cysteine (50 mg/L) during milk fermentation by SL, 10 g/L MSG was converted into 4.8 g/L GABA with a minimum level of residual MSG, viable cell counts of Lb. brevis and lactic acid production were increased, and the casein hydrolysis pattern was not influenced. Moreover, sulfhydryl group-containing chemicals including cystine, reduced glutathione, and oxidized glutathione showed effects similar to that of cysteine in improving GABA production. Finally, when L. bulgaricus YIB2 was combined with SL, supplementation of cysteine was also able to significantly improve GABA production.     

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.     

Effects of Lactobacillus fermentum HY01 on the quality characteristics and storage stability of yak yogurt

Lactobacillus fermentum HY01 is a probiotic strain screened from traditional yak yogurt, which can effectively relieve enteritis and constipation. This study aimed to evaluate the effects of HY01 as an adjunct starter on the quality and storage of yak yogurt. A total of 36 main volatile flavor substances were detected in all samples. In particular, more aldehydes, esters, and alcohols were detected in yak yogurt prepared by mixed fermentation of L. fermentum HY01 and starter MY105 (including Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus). The rheological results showed that the yak yogurt prepared by mixed fermentation of L. fermentum HY01 and starter MY105 had higher apparent viscosity and lower tan δ value compared with compared with traditional yak yogurt, yak yogurt with only L. fermentum HY01, and cow yogurt with L. fermentum HY01 and starter MY105. Meanwhile, the conjugated linoleic acid in the yak yogurt prepared by mixed fermentation of L. fermentum HY01 and starter was significantly higher than those in the HY01 group or the yogurt starter group alone. After 28 d of storage at 4°C, the number of HY01 in the yak yogurt prepared by mixed fermentation of L. fermentum HY01 and starter was still higher than 10⁷ cfu/mL, its acidity was lower than 110°T, and its syneresis was the lowest. The results indicated that L. fermentum HY01 could improve the flavor, texture, and storage properties of yak yogurt.     

The critical role of urease in yogurt fermentation with various combinations of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus

The protocooperation between Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus relies on metabolite exchanges that accelerate acidification during yogurt fermentation. Conflicting results have been obtained in terms of the effect of the Strep. thermophilus urease and the NH₃ and CO₂ that it generates on the rate of acidification in yogurt fermentation. It is difficult to perform a systematic study of the effects of urease on protocooperation because it is necessary to distinguish among the direct, indirect, and strain-specific effects resulting from the combination of the strains of both species. To evaluate the direct effects of urease on protocooperation, we generated 3 urease-deficient mutants (ΔureC) of fast- and slow-acidifying Strep. thermophilus strains and observed the effects of NH₃ or CO₂ supplementation on acidification by the ΔureC strains. Further, we examined 5 combinations of 3 urease-deficient ΔureC strains with 2 CO₂-responsive or CO₂-unresponsive strains of L. bulgaricus. Urease deficiency induced a shortage of ammonia nitrogen and CO₂ for the fast- and slow-acidifying Strep. thermophilus and for the CO₂-responsive L. bulgaricus, respectively. Notably, the shortage of ammonia nitrogen had more severe effects than that of CO₂ on yogurt fermentation, even if coculture with L. bulgaricus masked the effect of urease deficiency. Our work established (1) that urease deficiency inhibits the fermentative acceleration of protocooperation regardless of the Strep. thermophilus and L. bulgaricus strain combinations, and (2) that urease is an essential factor for effective yogurt acidification.     

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.     

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.     

Short communication: effect of oxygen on symbiosis between Lactobacillus bulgaricus and Streptococcus thermophilus

Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) and Streptococcus thermophilus are traditionally used for the manufacture of yogurt. It is said that a symbiotic relationship exists between Strep. thermophilus and L. bulgaricus and this decreases fermentation time. It is well known that L. bulgaricus is stimulated by the formate produced by Strep. thermophilus, and Strep. thermophilus is stimulated by free amino acids and peptides liberated from milk proteins by L. bulgaricus in symbiotic fermentation. We found that acid production by starter culture LB81 composed of L. bulgaricus 2038 and Strep. thermophilus 1131 was greatly accelerated by decreasing dissolved oxygen (DO) to almost 0 mg/kg in the yogurt mix (reduced dissolved oxygen fermentation) and that DO interferes with the symbiotic relationship between L. bulgaricus 2038 and Strep. thermophilus 1131. We attributed the acceleration of acid production of LB81 by reduced dissolved oxygen fermentation mainly to the acceleration of formate production and the suppression of acid production of LB81 by DO mainly to the suppression of formate production.     

EFFECT OF COLD STORAGE ON CULTURE VIABILITY AND SOME RHEOLOGICAL PROPERTIES OF FERMENTED MILK PREPARED WITH YOGURT AND PROBIOTIC BACTERIA

ABSTRACT

We examined the effect of storage time on culture viability and some rheological properties (yield stress, storage modulus, loss modulus, linear viscoelastic region, structural recuperation and firmness) of fermented milk made with Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus (LA) and Bifidobacterium animalis ssp. lactis in coculture with Streptococcus thermophilus (ST). Acidification profiles and factors that affect viability (postfermentation acidification, acidity and dissolved oxygen) were also studied during 35 days at 4C. Fermented milk prepared with a coculture of ST and Bifidobacterium lactis gave the most constant rheological behavior and the best cell viability during cold storage; it was superior to ST plus LA for probiotic fermented milk production.

PRACTICAL APPLICATIONS

Probiotic cultures should grow quickly in milk, provide adequate sensory and rheological properties to the product, and remain viable during storage. Commercially, it is very common to use yogurt starter culture (i.e. Streptococcus thermophilus[ST] and Lactobacillus delbrueckii ssp. bulgaricus) in combination with the probiotic bacteria in order to reduce fermentation time. However, LB tends to post acidify fermented milk, which reduces the viability of the probiotic bacteria; thus, it is recommended to use starter cultures devoid of this species. We found that the technological properties and the viability of the probiotic bacterium Bifidobacterium animalis ssp. lactis BL O4 in coculture with ST make it suitable for probiotic fermented milk production; it produces rheological characteristics similar to those of yogurt.     

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.     

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.     

Quality characteristics of yogurts fermented with short-chain fatty acid-producing probiotics and their effects on mucin production and probiotic adhesion onto human colon epithelial cells

Probiotics can ferment nondigestible carbohydrates and produce short-chain fatty acids (SCFA; acetate, propionate, and butyrate) in the human colon. In this study, the levels of SCFA were determined in the following yogurts fermented with different combinations of probiotics: (1) cocultures of Streptococcus thermophilus and Lactobacillus bulgaricus (control, C); (2) S. thermophilus, L. bulgaricus, and Bifidobacterium bifidum (C-Bb); (3) S. thermophilus, L. bulgaricus, and Lactobacillus acidophilus (C-La); and (4) S. thermophilus, L. bulgaricus, and Lactobacillus gasseri (C-Lg). Results showed that the acetate levels were significantly higher in C-Bb, C-La, and C-Lg yogurts than in C yogurt. Fermentation and physicochemical characteristics of all yogurts were identical. Treatment of mucus-secreting colon epithelial cells (HT29-MTX) with C-Bb, C-La, and C-Lg yogurt supernatants resulted in an increase in the expression of MUC2 and CDX2 and the production of mucin proteins. The adhesion of probiotics onto HT29-MTX cells increased following treatment with C-Bb, C-La, and C-Lg yogurt supernatants. Our data suggest that a yogurt diet rich in acetate improves the protective function of the intestinal epithelium.     

Effect of epigallocatechin gallate on the fermentative and physicochemical properties of fermented milk

Yogurt, a traditional fermented dairy product, is made with a starter that contains Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. The aim of this study was to investigate the effects of different concentrations of epigallocatechin gallate (EGCG; 0, 0.5, 1.0, 3.0, and 5.0 mg/mL) on the growth, metabolism, and acid production of lactic acid bacteria, as well as the texture, stability, and antioxidant activity of fermented milk (yogurt). The results showed that a low concentration of EGCG had no significant effect on the acid production capacity of the starter or on the water-holding capacity of the yogurt but did increase its viscosity. A high concentration (5.0 mg/mL) of EGCG delayed the acid production rate of the starter and decreased the water-holding capacity, but significantly increased the antioxidant activity of yogurt. The addition of EGCG significantly increased the hardness of yogurt. Therefore, EGCG can improve the texture of fermented milk and enhance its antioxidant activity and stability, thus improving the overall quality of yogurt.     

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.     

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.     

Probiotic Strains as Starter Cultures Improve Angiotensin-converting Enzyme Inhibitory Activity in Soy Yogurt

Suitability of soy yogurt as a system for delivering probiotics and other bioactive compounds was assessed by fermenting soy milk using starter culture containing Lactobacillus delbrueckii ssp. bulgaricus Lb1466, Streptococcus thermophilus St1342, and probiotic organisms (Lactobacillus acidophilus LAFTI® L10, Bifidobacterium lactis LAFTI® B94, and Lactobacillus paracasei LAFTI® L26). Fermentations were terminated at different pH of 4.50, 4.55, and 4.60 and metabolic patterns of cultures (viability, proteolytic activity, organic acids production, angiotensin‐converting enzyme (ACE) inhibitory activity) were investigated during 28 d of storage at 4 °C. The presence of probiotics enhanced the growth of L. delbrueckii ssp. bulgaricus Lb1466 and S. thermophilus St134 in soy yogurt in comparison to the control produced by sole yogurt culture. In general, different termination pH had no effect (P > 0.05) on the viability of probiotic organisms that maintained good viability in soy yogurt during cold storage. Higher levels of essential growth factors in the form of peptides and amino acids in soy yogurts may have promoted the growth of L. acidophilus LAFTI® L10, B. lactis LAFTI® B94, and L. paracasei LAFTI® L26. The use of probiotic strains as a part of starter culture in soy yogurt resulted in a substantial increase in in vitro ACE inhibitory activity compared with the control produced by yogurt culture only. This improvement of ACE inhibition in soy yogurt is partly due to higher proteolytic activity of probiotics.     

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.