Exopolysaccharides produced by mixed culture of yeast Rhodotorula rubra GED10 and yogurt bacteria (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11)

AIMs: The studies of the production of exopolysaccharides by lactose-negative yeast and a yogurt starter co-cultivated in a natural substrate containing lactose may be considered of interest because they reveal the possibilities for high-efficiency synthesis of biopolymers by mixed cultivation. METHODS AND RESULTS: The mixed culture Rhodotorula rubra GED10 + (Streptococcus thermophilus 13a + Lactobacillus bulgaricus 2-11) was cultivated in cheese whey ultrafiltrate (WU) (44.0 g lactose l(-1)) at initial pH 6.0, 28 degrees C, under intensive aeration (air-flow rate 1.0 l l(-1) min(-1), agitation 220 rev min(-1)) in a MBR AG fermentor. The mixed culture manifested the highest activity for synthesis of exopolysaccharides (19.3 g l(-1)) and cell mass (21.0 g l(-1)) at the 84th hour. The yogurt starter synthesized neutral exopolysaccharides, while the mixed culture yeast + yogurt starter produced acidic exopolysaccharides containing uronic acid (6%). The neutral sugar composition was identified as mannose, glucose, galactose, xylose and arabinose. Mannose dominated in the polymer composition (83%) that was produced only by the yeast (97%). CONCLUSIONS: Lactose in the WU can be effectively utilized by a co-culture of lactose-negative yeast-yogurt starter for synthesis of exopolysaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The present findings propose an alternative use of WU as a cost-effective carbohydrate substrate, and suggest that the lactose-negative yeast Rhodotorula rubra can have industrial application as producers of exopolysaccharides.     

Improvement of carotenoid-synthesizing yeast Rhodotorula rubra by chemical mutagenesis

A mutant Rhodotorula rubra with enhanced carotenoid-synthesizing activity for synthesizing total carotenoids and beta-carotene was obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. When co-cultivated with yogurt starter bacteria (Lactobacillus bulgaricus + Streptococcus thermophilus) in whey ultrafiltrate it produced 15.7 mg total carotenoids l(-1) culture fluid or 946 microg total carotenoids g(-1) dry cells of which 71% was beta-carotene. Grown as a monoculture in glucose substrate, the mutant shown 1.4 times lower carotenoid-synthesizing activity, and the relative share of beta-carotene in the total carotenoids was lower (63%). The individual pigments torulene and torularhodin were identified, whose mass fractions were (29% and 7%) and (24% and 4%), respectively, for the mutant grown as a monoculture and as a mixed culture with the yogurt bacteria.     

Synthesis of carotenoids by<Emphasis Type="Italic"> Rhodotorula rubra</Emphasis> GED8 co-cultured with yogurt starter cultures in whey ultrafiltrate

Two cultures, a yeast (Rhodorula rubra GED8) and a yogurt starter (Lactobacillus bulgaricus 2–11+Streptococcus thermophilus 15HA), were selected for associated growth in whey ultrafiltrate (WU) and active synthesis of carotenoids. In associated cultivation with the yogurt culture L bulgaricus 2–11+S. thermophilus 15HA under intensive aeration (1.3 l^–1min^–1 air-flow rate) in WU (45 g lactose l^–1), initial pH 5.5, 30 °C, the lactose-negative strain R. rubra GED8 synthesized large amounts of carotenoids (13.09 mg l^–1 culture fluid). The carotenoid yield was approximately two-fold higher in association with a mixed yogurt culture than in association with pure yogurt bacteria. The major carotenoid pigments comprising the total carotenoids were -carotene (50%), torulene (12.3%) and torularhodin (35.2%). Carotenoids with a high -carotene content were produced by the microbial association 36 h earlier than by Rhodotorula yeast species. No significant differences were notd in the ratio between the pigments synthesized by R. rubra GED8+L. bulgaricus 2–11, R. rubra GED8+S. thermophilus 15HA, and R.rubra GED8+yogurt culture, despite the fact that the total carotenoid concentrations were lower in the mixed cultures with pure yogurt bacteria.     

Exopolysaccharides produced by lactic acid bacteria of kefir grains

A Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains. By associating the Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp. lactis C15, Lactobacillus helveticus MP12, and Sacharomyces cerevisiae A13, a kefir starter was formed. The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp. bulgaricus HP1. The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp. bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h. The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.     

Carotenoid production by lactoso-negative yeasts co-cultivated with lactic acid bacteria in whey ultrafiltrate

Two strains were selected--the lactoso-negative yeast Rhodotorula rubra GED2 and the homofermentative Lactobacillus casei subsp. casei Ha1 for co-cultivation in cheese whey ultrafiltrate (WU) and active synthesis of carotenoids. Under conditions of intensive aeration (1.0 l/l min, 220 rpm), a temperature of 30 degrees C, WU with 55.0 g lactose/l, initial pH = 5.5, the carotenoid content in the cells reached a maximum, when the growth of the cultures had come to an end, i.e. in the stationary phase of the yeast. The maxima for dry cell accumulation (27.0 g/l) and carotenoid formation (12.1 mg/l culture medium) did not coincide on the 5th and 6th day, respectively. A peculiarity of the carotenoid-synthesizing Rh. rubra GED2 strain, co-cultivated with L. casei Ha1, was the production of carotenoids with high beta-carotene content (46.6% of total carotenoids) and 10.7% and 36.9% for torulene and torularhodin, respectively.     

Growth and activity of Bulgarian yogurt starter culture in iron-fortified milk

Bulgarian yogurts were manufactured and fortified with 8, 15 and 27 mg of iron kg(-1) of yogurt. The growth and acidifying activity of the starter culture bacteria Streptococcus thermophilus 13a and Lactobacillus delbrueckii subsp. bulgaricus 2-11 were monitored during milk fermentation and over 15 days of yogurt storage at 4 degrees C. Fortifying milk with iron did not affect significantly the growth of the starter culture during manufacture and storage of yogurt. Counts of yogurt bacteria at the end of fermentation of iron-fortified milks were between 2.1 x 10(10) and 4.6 x 10(10) CFU ml(-1), which were not significantly different from numbers in unfortified yogurts. In all batches of yogurt, the viable cell counts of S. thermophilus 13a were approximately three times higher than those of L. delbrueckii subsp. bulgaricus 2-11. Greater decrease in viable cell count over 15 days of storage was observed for S. thermophilus 13a compared to L. delbrueckii subsp. bulgaricus 2-11. Intensive accumulation of lactic acid was observed during incubation of milk and all batches reached pH 4.5 +/- 0.1 after 3.0 h. At the end of fermentation process, lactic acid concentrations in iron-fortified yogurts were between 6.9 +/- 0.4 and 7.3 +/- 0.5 g l(-1). The acidifying activity of starter culture bacteria in the control and iron-fortified milks was similar. There was no increase in oxidized, metallic and bitter off-flavors in iron-fortified yogurts compared to the control. Iron-fortified yogurts did not differ significantly in their sensorial, chemical and microbiological characteristics with unfortified yogurt, suggesting that yogurt is a suitable vehicle for iron fortification and that the ferrous lactate is an appropriate iron source for yogurt fortification.     

Effect of aeration on the production of carotenoid pigments by Rhodotorula rubra-lactobacillus casei subsp. casei co-cultures in whey ultrafiltrate

Under intensive aeration (1.3 l/l min) the associated growth of Rhodotorula rubra GED2 and Lactobacillus casei subsp. casei in cheese whey ultrafiltrate (55 g lactose/l) proceeded effectively for both cultures with production of maximum carotenoids (12.4 mg/l culture fluid). For maximum amount of carotenoids synthesized in the cell, the yeast required more intensive aeration than the aeration needed for synthesis of maximum concentration of dry cells. Maximum concentration of carotenoids in the cell (0.49 mg/g dry cells) was registered with air flow rate at 1.3 l/l min, and of dry cells (27.0 g/l) at 1.0 l/l min. An important characteristic of carotenogenesis by Rhodotorula rubra GED2 + Lactobacillus casei subsp. casei was established--the intensive aeration (above 1.0 l/l min) stimulated beta-carotene synthesis (60% of total carotenoids).     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.     

Production and monomer composition of exopolysaccharides by yogurt starter cultures

As components of starter cultures for Bulgarian yogurt, Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus revealed extensive exopolysaccharide (EPS) production activity when cultivated in whole cow's milk. The polymer-forming activity of thermophilic streptococci was lower (230-270 mg EPS/L) than that of the lactobacilli (400-540 mg EPS/L). Mixed cultures stimulated EPS production in yogurt manufacture, and a maximum concentration of 720-860 mg EPS/L was recorded after full coagulation of milk. The monomer structure of the exopolysaccharides formed by the yogurt starter cultures principally consists of galactose and glucose (1:1), with small amounts of xylose, arabinose, and/or mannose.     

Isolation and characterization of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus from plants in Bulgaria

One of the traditional ways of preparation of yogurt starter in Bulgaria is placing a branch of a particular plant species into boiled sheep's milk maintained at about 45°C, which is further incubated until a dense coagulum is obtained. To investigate the possible origin of the yogurt starter bacteria, Lactobacillus delbrueckii ssp. bulgaricus ( L. bulgaricus ) and Streptococcus thermophilus ( S. thermophilus ), the traditional way of yogurt-starter preparation was followed. Hundreds of plant samples were collected from four regions in Bulgaria and incubated in sterile skim milk. The two target bacteria at low frequencies from the plant samples collected were successfully isolated. Phenotypic and genotypic characteristics of these bacterial isolates revealed that they were identified as L. bulgaricus and S. thermophilus . Twenty isolates of L. bulgaricus and S. thermophilus , respectively, were selected from the isolated strains and further characterized with regard to their performance in yogurt production. Organoleptic and physical properties of yogurt prepared using the isolated strains from plants were not significantly different from those prepared using commercial yogurt-starter strains.
It was therefore suggested that L. bulgaricus and S. thermophilus strains widely used for commercial yogurt production could have originated from plants in Bulgaria. To our knowledge, this is the first report on the isolation and characterization of L. bulgaricus and S. thermophilus strains from plants.     

Production of natural folates by lactic acid bacteria starter cultures isolated from artisanal Argentinean yogurts

Folate is a B-group vitamin that cannot be synthesized by humans and must be obtained exogenously. Although some species of lactic acid bacteria (LAB) can produce folates, little is known about the production of this vitamin by yogurt starter cultures. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains were isolated from artisanal Argentinean yogurts and were grown in folate-free culture medium (FACM) and nonfat milk after which intracellular and extracellular folate production were evaluated. From the initial 92 isolated LAB strains, 4 L. delbrueckii subsp. bulgaricus and 32 S. thermophilus were able to grow in the absence of folate. Lactobacillus delbrueckii subsp. bulgaricus CRL 863 and S.?thermophilus CRL 415 and CRL 803 produced the highest extracellular folate levels (from 22.3 to 135?μg/L) in FACM. In nonfat milk, these strains were able to increase the initial folate concentrations by almost 190%. This is the first report where native strains of L. delbrueckii subsp. bulgaricus were shown to produce natural folate. The LAB strains identified in this study could be used in developing novel fermented products bio-enriched in natural folates that could in turn be used as an alternative to fortification with the controversial synthetic chemical folic acid.     

Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus survive gastrointestinal transit of healthy volunteers consuming yogurt

To date, there is significant controversy as to the survival of yogurt bacteria (namely, Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus) after passage through the human gastrointestinal tract. Survival of both bacterial species in human feces was investigated by culture on selective media. Out of 39 samples recovered from 13 healthy subjects over a 12-day period of fresh yogurt intake, 32 and 37 samples contained viable S. thermophilus (median value of 6.3 x 10(4) CFU g(-1) of feces) and L. delbrueckii (median value of 7.2 x 10(4)CFU g(-1) of feces), respectively. The results of the present study indicate that substantial numbers of yogurt bacteria can survive human gastrointestinal transit.     

Production of β-carotene by a mutant of Rhodotorula glutinis

Wild strains of Rhodotorula glutinis and R. rubra were investigated concerning their carotenoid production, proportion of beta-carotene and cell mass yield. R. glutinis NCIM 3353 produced 2.2 mg carotenoid/l in 72 h; and the amount of beta-carotene was 14% (w/w) of the total carotenoid content (17 microg/g cell dry weight). It was subjected to mutagenesis using UV radiation for strain improvement. Out of 2,051 isolates screened, the yellow coloured mutant 32 produced 120-fold more beta-carotene (2,048 microg/g cell dry weight) than the parent culture in 36 h, which was 82% (w/w) of the total carotenoid content. Mutant 32 was grown on different carbon and nitrogen sources. The best yield of beta-carotene (33+/-3 mg/l) was obtained when glucose and yeast extract were supplied as carbon and nitrogen sources, respectively. Divalent cation salts further increased the total carotenoid content (66+/-2 mg/l) with beta-carotene as the major component (55+/-2%, w/w).     

Survival of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the terminal ileum of fistulated Göttingen minipigs

The ability of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus administered in yogurt to survive the passage through the upper gastrointestinal tract was investigated with G?ttingen minipigs that were fitted with ileum T-cannulas. After ingestion of yogurt containing viable microorganisms, ileostomy samples were collected nearly every hour beginning 3 h after food uptake. Living L. delbrueckii subsp. bulgaricus and S. thermophilus were detected in the magnitude of 10(6) to 10(7) per gram of intestinal contents (wet weight) in all animals under investigation. A calculation of the minimum amount of surviving bacteria that had been administered is presented. Total DNA extracted from ileostomy samples was subjected to PCR, which was species specific for L. delbrueckii and S. thermophilus and subspecies specific for L. delbrueckii subsp. bulgaricus. All three bacterial groups could be detected by PCR after yogurt uptake but not after uptake of a semisynthetic diet. One pig apparently had developed an endogenous L. delbrueckii flora. When heat-treated yogurt was administered, L. delbrueckii was detected in all animals. S. thermophilus or L. delbrueckii subsp. bulgaricus was not detected, indicating that heat-inactivated cells and their DNAs had already been digested and their own L. delbrueckii flora had been stimulated for growth.     

Application of the extracellular alpha-amylase gene from Streptococcus bovis 148 to construction of a secretion vector for yogurt starter strains.

Streptococcus thermophilus ATCC 19258, Lactobacillus delbrueckii subsp. bulgaricus T-11, and Lactococcus lactis subsp. lactis IL1403 were transformed with the alpha-amylase gene (amyA) from Streptococcus bovis 148 by using a wide host-range vector, and all the transformants secreted the alpha-amylase successfully. Since the promoter and the secretion signal of the amyA gene were functional in these strains, we constructed a secretion vector using the expression elements of amyA. Trials to secrete foreign enzymes in yogurt starter strains were performed using this novel secretion vector.     

Molecular analysis of the glutamate decarboxylase locus in Streptococcus thermophilus ST110

γ-aminobutyric acid (GABA) is generated from glutamate by the action of glutamic acid decarboxylase (GAD) and characterized by hypotensive, diuretic, and tranquilizing effects in humans and animals. The production of GABA by lactic acid starter bacteria would enhance the functionality of fermented dairy foods including cheeses and yogurt. The survey of 42 strains of the yogurt starter culture Streptococcus thermophilus by PCR techniques indicated the presence of a glutamate decarboxylase gene (gadB) in 16 strains. DNA sequencing data indicated that the GAD/GABA antiporter locus (gadB/gadC) in GAD(+) S. thermophilus strains is flanked by transposase elements (5' and 3') and positioned between the luxS (5') and the HD-superfamily hydrolase genes (3'). The PCR amplification product of a ca. 2-kb genomic fragment that included the gadB and its putative promoter region was inserted into a shuttle vector, which was used to transform Escherichia coli DH5α. Subsequently, the recombinant plasmid pMEU5a-1/gadB (7.24 kb) was electrotransformed into the GAD-negative strain S. thermophilus ST128. The ST128 transformants carrying the plasmid-encoded gadB produced functional GAD enzyme as evidenced by the conversion of glutamate to GABA at a rate similar to strains with the gadB/gadC operon located on the chromosome. The results demonstrated the potential to impart to non-GABA-producing strains of S. thermophilus and other lactic acid bacteria the GAD(+) phenotype that improves their appeal in possible applications in the development of health-promoting functional foods.     

Complete sequencing and pan-genomic analysis of Lactobacillus delbrueckii subsp. bulgaricus reveal its genetic basis for industrial yogurt production

Lactobacillus delbrueckii subsp. bulgaricus (Lb. bulgaricus) is an important species of Lactic Acid Bacteria (LAB) used for cheese and yogurt fermentation. The genome of Lb. bulgaricus 2038, an industrial strain mainly used for yogurt production, was completely sequenced and compared against the other two ATCC collection strains of the same subspecies. Specific physiological properties of strain 2038, such as lysine biosynthesis, formate production, aspartate-related carbon-skeleton intermediate metabolism, unique EPS synthesis and efficient DNA restriction/modification systems, are all different from those of the collection strains that might benefit the industrial production of yogurt. Other common features shared by Lb. bulgaricus strains, such as efficient protocooperation with Streptococcus thermophilus and lactate production as well as well-equipped stress tolerance mechanisms may account for it being selected originally for yogurt fermentation industry. Multiple lines of evidence suggested that Lb. bulgaricus 2038 was genetically closer to the common ancestor of the subspecies than the other two sequenced collection strains, probably due to a strict industrial maintenance process for strain 2038 that might have halted its genome decay and sustained a gene network suitable for large scale yogurt production.     

Characterization of dominant microbiota of a Ghanaian fermented milk product, nyarmie, by culture- and nonculture-based methods

AIMS: To characterize the predominant micro-organisms in a Ghanaian traditional fermented dairy product, nyarmie, made from cows' milk, using both culture- and nonculture-based methods. METHODS AND RESULTS: Samples of nyarmie were analysed from three production sites in Accra, by determining the counts on selective culture media. The microbial diversity occurring in nyarmie was also evaluated by 16S/18S ribosomal DNA PCR amplification and denaturing gradient gel electrophoresis. Results showed that nyarmie contained lactococci and lactobacilli in the range of 10(8) and 10(10) CFU ml(-1), respectively, and yeasts at around 10(7) CFU ml(-1). The pH ranged between 3.49 and 4.25. The predominant lactic acid bacteria (LAB) in nyarmie were Leuconostocmesenteroides ssp. mesenteroides, Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lact.helveticus, Lact. delbrueckii ssp. lactis and Lactococcus lactis, while Saccharomyces cerevisiae was the predominant yeast species. Lactobacillus delbrueckii ssp. delbrueckii was not detected by cultivation but its predominance was revealed by PCR-DGGE analysis. CONCLUSIONS: The flora in products from different producers varied in the LAB composition present and may result in variations in product quality. SIGNIFICANCE AND IMPACT OF THE STUDY: Development and use of starter cultures for nyarmie may be beneficial in improving the consistency of product quality.     

Scarce evidence of yogurt lactic acid bacteria in human feces after daily yogurt consumption by healthy volunteers

In a double-blind prospective study including 114 healthy young volunteers, the presence in human feces of the yogurt organisms Lactobacillus delbrueckii and Streptococcus thermophilus after repeated yogurt consumption (15 days) was analyzed by culture, specific PCR, and DNA hybridization of total fecal DNA. Detection of yogurt lactic acid bacteria in total fecal DNA by bacterial culture and PCR assay was consistently negative. DNA compatible with yogurt bacteria was found by hybridization experiments in only 10 (10.52%) of 96 individuals after consumption of fresh yogurt and in 2 (2.10%) of 96 individuals after consumption of pasteurized yogurt (P = 0.01).     

Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.