Selective enumeration of Lactobacillus casei in yoghurt-type fermented milks based on a 15°C incubation temperature

A procedure was developed to enumerate selectively Lactobacillus casei populations in yoghurt-type fermented milks that can also contain strains of Streptococcus thermophilus, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus acidophilus and Bifidobacterium infantis. Commercial LBS agar was acidified to pH 5.4, and the plates were incubated at 15°C for 14 days under anaerobic conditions. Acidification prevented the development of streptococci, and incubation at 15°C limited the development of the lactobacilli and the bifidobacteria. L. casei formed colonies on HHD medium which were different from those obtained with L. bulgaricus. Counts of L. casei on HHD confirmed results obtained on LBS - pH 5.4 medium and incubated at 15°C. L. casei did not form colonies on M17, nor did L. acidophilus or L. bulgaricus.     

Lactic acid bacteria isolated from dairy products inhibit genotoxic effect of 4-nitroquinoline-1-oxide in SOS-chromotest

Antigenotoxic activity against 4-nitroquinoline-1-oxide (4-NQO) of lactic acid bacteria isolated from commercial dairy products was studied using SOS-Chromotest. The supernatants from bacteria-genotoxin co-incubations in general exhibited a strong suppression on SOS-induction produced by 4-NQO on the tester organism Escherichia coli PQ37 (sfiA::lacZ). High genotoxicity inhibition (>75%) was found for 31/67 of the examined bacteria and the maximum values of some strains within the species were as follows: Lactobacillus casei, 99.1%; L. plantarum, 93.3%; L. rhamnosus, 93.4%; L. acidophilus, 90.9%; L. delbrueckii subsp. bulgaricus, 85.7% and Bifidobacterium bifidum, 89.6%; Strains with low antigenotoxicity (5-60%) were evidenced in both L. acidophilus and L. delbrueckii subsp. bulgaricus, whereas some inactive strains were found only in L. casei and L. delbrueckii subsp. bulgaricus. Cell exposure to 100 degrees C for 15 min prevented antigenotoxicity and no effect was evidenced for cell-free spent media. The active strains survived at 0.1 mM 4-NQO exposure and generally presented some relevant functional properties, such as tolerance to bile (0.5%) or acid environment (pH 2.0) and adherence to Caco-2 enterocytes. Antigenotoxicity was always associated with modification of the 4-NQO absorbance profile.     

Characterization and antimicrobial spectrum of bacteriocins produced by lactic acid bacteria isolated from traditional Bulgarian dairy products

Aims:  To isolate bacteriocin-producing lactic acid bacteria (LAB) with high wide spectrum antibacterial activity and to characterize their inhibitory peptides.
Method and Results:  Seven LAB strains [ Lactobacillus casei ssp. rhamnosus (PC5), Lactobacillus delbrueckii ssp. bulgaricus (BB18), Lactococcus lactis ssp. lactis (BCM5, BK15), Enterococcus faecium (MH3), Lactobacillus plantarum (BR12), Lactobacillus casei ssp. casei (BCZ2)], isolated from authentic Bulgarian dairy products were capable of producing bacteriocins, inhibiting the widest range of pathogenic bacteria. The bacteriocins were resistant to heating at 121°C for 15 min, stable at pH 2–10, sensitive to protease, insensitive to α-amylase and lipase. Two of bacteriocins produced by Lact. bulgaricus BB18 (bulgaricin BB18) and E. faecium MH3 (enterocin MH3) were purified and the molecular masses were determined. The N -terminal amino acid sequence of bulgaricin BB18 did not show strong homology to other known bacteriocins.
Conclusions:  Lactobacillus bulgaricus BB18 and E. faecium MH3 produce two novel bacteriocins highly similar to the pediocin-like nonlantibiotics.
Significance and Impact of the Study:  The two bacteriocins are potential antimicrobial agents and, in conjunction with their producers, may have use in applications to contribute a positive effect on the balance of intestinal microflora. Furthermore, bulgaricin BB18 strongly inhibits Helicobacter pylori .     

Note: evaluation of selective media for the enumeration of Bifidobacterium sp. in milk

Pure cultures of three species of bifidobacteria (Bifidobacterium longum, Bif. adolescentis and Bif. bifidum), Lactobacillus acidophilus and a mixed culture of Lact. delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus were each enumerated on two differential media and six selective media for the enumeration of bifidobacteria. The appearance of the colonies on the differential media was as expected but when mixed cultures were present, it proved extremely difficult to distinguish one species from another. Of the selective media, AMC, RMS, NPNL and BL-OG performed well in that they gave good recoveries of bifidobacteria and were inhibitory to the growth of Lact. delbrueckii subsp. bulgaricus, Strep. salivarius subsp. thermophilus and Lact. acidophilus. However, of these four media, AMC was most convenient as it is based on a commercially available medium, whereas the others must be made up from individual constituents. The AMC agar is thus a good choice for the routine enumeration of bifidobacteria from mixed cultures.     

Classification of lactic acid bacteria with UV-resonance Raman spectroscopy

UV-resonance Raman spectroscopy is applied as a method for the identification of lactic acid bacteria from yogurt. Eight different strains of bacteria from Lactobacillus acidophilus, L. delbrueckii ssp. bulgaricus, and Streptococcus thermophilus were investigated. At an excitation wavelength of 244 nm signals from nucleic acids and proteins are selectively enhanced. Classification was accomplished using different chemometric methods. In a first attempt, the unsupervised methods hierarchical cluster analysis and principal component analysis were applied to investigate natural grouping in the data. In a second step the spectra were analyzed using several supervised methods: K-nearest neighbor classifier, nearest mean classifier, linear discriminant analysis, and support vector machines.     

Metabolism of pyruvate and citrate in lactobacilli

Lactobacillus acidophilus, L. bulgaricus, L. casei, L. delbrueckii , L. lactis and L. plantarum contained a pyruvate oxidase for the oxidation of pyruvate to acetyl phosphate and acetate. The presence of an acetate kinase converted the acetyl phosphate to acetate. L. casei and L. plantarum produced lactate and acetoin, in addition to acetate, under the conditions used while L. casei also produced diacetyl. L. casei and L. plantarum were the only species to utilize citrate. L. helveticus and L. helveticus subsp. jugurti did not utilize pyruvate under the conditions used.     

Identification and characterization of a strain-dependent cystathionine β/γ-lyase in Lactobacillus casei potentially involved in cysteine biosynthesis

The trans -sulfuration pathways allow the interconversion of cysteine and methionine with the intermediary formation of cystathionine and homocysteine. The genome database of Lactobacillus casei ATCC 334 provides evidence that this species cannot synthesize cysteine from methionine via the trans -sulfuration pathway. However, several L. casei strains use methionine as the sole sulfur source, which implies that these strains can convert methionine to cysteine. Cystathionine synthases and lyases play a crucial role in the trans -sulfuration pathway. By applying proteomic techniques, we have identified a protein in cell-free extracts of L. casei , which showed high homology to a gene product encoded in the genome of Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus and Lactobacillus helveticus but not in the genome of L. casei ATCC 334. The presence of the gene was only found in strains able to grow on methionine as the sole sulfur source. Moreover, two gene variants were identified. Both gene variants were cloned and expressed heterologously in Escherichia coli . The recombinant enzymes exhibited cystathionine lyase activity in vitro and also cleaved cysteine, homocysteine and methionine releasing volatile sulfur compounds.     

Redox potential to discriminate among species of lactic acid bacteria

AIMS: To verify to what degree reducing capacity is a characterizing parameter of a species, and of the strains themselves within a given species, of lactic acid bacteria. METHODS AND RESULTS: Eighty-eight strains belonging to 10 species of lactic acid bacteria (LAB) isolated from traditional Italian cheeses were studied for their reduction activity: Enterococcus faecalis, Enterococcus faecium, Enterococcus durans, Streptococcus thermophilus, Lactococcus lactis ssp. lactis, Lactobacillus paracasei ssp. paracasei, Lactobacillus plantarum, Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus and Pediococcus pentosaceus. It was observed that the lactococci reached minimum redox potential before the lactobacilli. The reduction rate of Enterococcus spp. and L. lactis ssp. lactis was higher than that of the streptococci and Lactobacillus spp. All the P. pentosaceus strains had poor reduction activity compared with the other species. CONCLUSIONS: The evolution of the redox potential in milk over a time span of 24 h has been found to be a parameter that characterizes a species: the different courses corresponding to the species in question are clearly evident, and interesting differences can also be noted within the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: The reduction aptitude of strains might be used to select and adapt appropriate strains for use as starters for dairy products.     

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.     

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.     

Antiproliferative effects of homogenates derived from five strains of candidate probiotic bacteria.

Unheated and heat-treated homogenates were separately prepared from candidate probiotic bacteria, including Lactobacillus rhamnosus GG, Bifidobacterium lactis, Lactobacillus acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Streptococcus thermophilus. We compared the phytohemagglutinin-induced proliferation of mononuclear cells in the presence of homogenates and in the presence of a control containing no homogenate by assessing thymidine incorporation in cell cultures. All homogenates suppressed proliferation, whether the enzymatic activity was inactivated or not inactivated by heating. When the proliferation assays were repeated with cytoplasmic and cell wall extracts derived from the homogenate of L. rhamnosus GG, the cytoplasmic extract but not the cell wall extract was suppressive. These findings indicate that candidate probiotic bacteria possess a heat-stable antiproliferative component(s). These bacteria may be used to generate microbiologically nonviable yet immunologically active probiotic food products that are easier to store and have a longer shelf life.     

Prediction of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus populations in yoghurt by Curie point pyrolysis-mass spectrometry.

We evaluated the potential of pyrolysis-mass spectrometry (PyMS) for quantifying the binary mixed population of Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. bulgaricus in yoghurt. For this purpose, a new analytical approach was developed. The yoghurt was transparised and its total bacterial population was recovered by centrifugation and estimated by turbidimetric measurement. The quantity of each population (L. bulgaricus, S. thermophilus) was then estimated in the pellet by PyMS, and the data were analysed by artificial neural networks (ANNs). In parallel, streptococci and lactobacilli were numerated on SYL agar and these data were used as reference values to predict the bacterial counts of each population by PyMS. A close correlation was established between the streptococci and the lactobacilli counts on SYL agar and PyMS measurements (r(2)=0.98 for S. thermophilus and r(2)=0.96 for L. bulgaricus). Combined turbidimetric measurement and PyMS/ANNs seemed to be a powerful method for obtaining rapid counts of binary mixtures of bacteria in yoghurt.     

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.     

EZAL-MY96乳酸菌种菌株分离鉴定

目的：对引进法国罗地亚EZAL-MY96直投式酸奶发酵剂(DVS)进行菌株分离鉴定。方法：利用半选择性培养基进行发酵剂菌株分离,并采用糖发酵实验和RAPD指纹图谱进行鉴定。结果：EZAL-MY96中球菌的平板菌落与光镜显微观察、糖发酵实验和RAPD指纹图谱均只有1种情况,而杆菌均有2种情况。结论：从EZAL-MY96菌种中共分离到3株菌,其中1株鉴定为嗜热链球菌,1株为德氏乳杆菌保加利亚亚种,另1株为未知的乳杆菌。     

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.     

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.     

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.     

Hydrolysis of whey proteins by Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus grown in a chemically defined medium

AIMS: To evaluate the ability of themophilic lactic acid bacteria (LAB) to hydrolyse the whey proteins beta-lactoglobulin (BLG) and alpha-lactalbumin (ALA) in a chemically defined medium (CDM). METHODS AND RESULTS: The ability of three LAB strains to hydrolyse BLG and ALA was studied in a CDM supplemented with these proteins or whey protein concentrate (WPC). Protein hydrolysis was determined by Tricine/SDS-PAGE and RP-HPLC. Maximum BLG (21%) and ALA (26%) degradation by LAB was observed using WPC. Under starving conditions, BLG degradation was greater for Lactobacillus delbrueckii ssp. bulgaricus CRL 454 than for Lactobacillus acidophilus CRL 636 and Streptococcus thermophilus CRL 804. All three strains showed different peptide profiles and were not able to hydrolyse ALA under starvation. CONCLUSIONS: The assayed LAB strains were able to degrade BLG during growth in a CDM and under starving conditions. The different peptide profiles obtained indicate distinct protease specificities. SIGNIFICANCE AND IMPACT OF THE STUDY: These strains could be used as adjunct cultures to increase BLG digestibility in whey-based or whey-containing foods. To our knowledge, this is the first report on the ability of a Lact. acidophilus strain to degrade BLG.     

Utilization of UF-permeate for production of beta-galactosidase by lactic acid bacteria

Four lactobacilli strains (Lactobacillus bulgaricus, Lactobacillus acidophilus, Lactobacilus casei and Lactobacillus reuteri) were grown in MRS broth and three lactococci strains (Streptococcus thermophilus, Lactococcus lactis subsp. Lactis and Lactococcus lactis subsp. lactis biovar. diacetilactis) were grown in M17 broth. L. reuteri and S. thermophilus were chosen on the basis of the best mean beta-galactosidase activity of 10.44 and 10.01 U/ml respectively, for further studies on permeate-based medium. The maximum production of beta-galactosidase by L. reuteri was achieved at lactose concentration of 6%, initial pH 5.0-7.5, ammonium phosphate as nitrogen source at a concentration of 0.66 g N/L and incubation temperature at 30 degrees C/24 hrs to give 6.31 U/ml. While in case of S. thermophilus, maximum beta-galactosidase production was achieved at 10% lactose concentration of permeate medium, supplemented with phosphate buffer ratio of 0.5:0.5 (KH2PO4:K2HPO4, g/L), at initial pH 6.0-6.5, ammonium phosphate (0.66g N/L) as nitrogen source and incubation temperature 35 degrees C for 24 hrs to give 7.85 U/ml.     

Comparative sequence analysis of plasmids from Lactobacillus delbrueckii and construction of a shuttle cloning vector

While plasmids are very commonly associated with the majority of the lactic acid bacteria, they are only very rarely associated with Lactobacillus delbrueckii, with only four characterized to date. In this study, the complete sequence of a native plasmid, pDOJ1, from a strain of Lactobacillus delbrueckii subsp. bulgaricus was determined. It consisted of a circular DNA molecule of 6,220 bp with a G+C content of 44.6% and a characteristic ori and encoded six open reading frames (ORFs), of which functions could be predicted for three-a mobilization (Mob) protein, a transposase, and a fused primase-helicase replication protein. Comparative analysis of pDOJ1 and the other available L. delbrueckii plasmids (pLBB1, pJBL2, pN42, and pLL1212) revealed a very similar organization and amino acid identities between 85 and 98% for the putative proteins of all six predicted ORFs from pDOJ1, reflecting a common origin for L. delbrueckii plasmids. Analysis of the fused primase-helicase replication gene found a similar fused organization only in the theta replicating group B plasmids from Streptococcus thermophilus. This observation and the ability of the replicon to function in S. thermophilus support the idea that the origin of plasmids in L. delbrueckii was likely from S. thermophilus. This may reflect the close association of these two species in dairy fermentations, particularly yogurt production. As no vector based on plasmid replicons from L. delbrueckii has previously been constructed, an Escherichia coli-L. delbrueckii shuttle cloning vector, pDOJ4, was constructed from pDOJ1, the p15A ori, the chloramphenicol resistance gene of pCI372, and the lacZ polylinker from pUC18. This cloning vector was successfully introduced into E. coli, L. delbrueckii subsp. bulgaricus, S. thermophilus, and Lactococcus lactis. This shuttle cloning vector provides a new tool for molecular analysis of Lactobacillus delbrueckii and other lactic acid bacteria.