Subspecies Bulgaricus Research Articles

Subspecies-level genome comparison of Lactobacillus delbrueckii

Lactobacillus delbrueckii comprises six subspecies, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. delbrueckii subsp. jakobsenii, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. sunkii, and L. delbrueckii subsp. indicus. We investigated the evolution of the six subspecies of L. delbrueckii using comparative genomics. While the defining feature of the species was the gene number increment driven by mobile elements and gene fragmentation, the repertoire of subspecies-specific gene gains and losses differed among the six subspecies. The horizontal gene transfer analyses indicated that frequent gene transfers between different subspecies had occurred when the six subspecies first diverged from the common ancestor, but recent gene exchange was confined to a subspecies implying independent evolution of the six subspecies. The subspecies bulgaricus is a homogeneous group that diverged from the other subspecies a long time ago and underwent convergent evolution. The subspecies lactis, jakobsenii, delbrueckii, and sunkii were more closely related to each other than to other subspecies. The four subspecies commonly show increasing genetic variability with increasing genome size. However, the four subspecies were distinguished by specific gene contents. The subspecies indicus forms a branch distant from the other subspecies and shows an independent evolutionary trend. These results could explain the differences in the habitat and nutritional requirements of the subspecies of L. delbrueckii.

Optimization of biofunctional jaggery yogurt: It’s physicochemical and antioxidantproperties

In addition to the standard healthy diet needs, balance food is taught to be a healthier diet that will promote human health. It’s also known as functional food, which is food that clearly claims to have health advantages as well as the ability to enhance the immune system. Yoghurt is a fermented dairy product that is in great demand for its nutrient content, bacterial activity and wide product range in terms of flavours and textures. In this study, physicochemical, microbiological and sensory differences of probiotic set yoghurt incorporated with different concentration of Jaggery were evaluated, as well as the changes taking place during storage at 4°C for 21 days. During the storage, the addition of jaggery improved survival of Lactobacillus delbrueckii subspecies bulgaricus (ABT-7) and Streptococcus thermophiles (YoFlex Express 1.0) from CHR HANSEN, Denmark were used as starter culture for yogurt preparation. All yogurts exhibited a decrease in pH accompanied by an increase in titratable acidity during storage. Therefore, this study revealed that jaggery could be used to produce probiotic set yoghurt with improved physicochemical, microbiological and sensorial attributes.

Edamame protein hydrolysis using Lactococcus lactis, Lactobacillus bulgaricus and Lactobacillus paracasei produce short peptides with higher antioxidant potential

Abstract. Anggraeni SL, Jayus J, Ratnadewi AAI, Nurhayati N. 2022. Edamame protein hydrolysis using Lactococcus lactis, Lactobacillus bulgaricus and Lactobacillus paracasei produce short peptides with higher antioxidant potential. Biodiversitas 23: 3604-3613. The high demand for protein sources for functional vegetable foods, especially proteins that have ACE (Angiotensin-Converting Enzyme) inhibitory activity has encouraged efforts to meet this need through exploration of potential protein sources and the development of food processing technology to modify the protein into derivative compounds in the form of short peptides to promote higher bioactivity than the native protein. This research focused on the hydrolysis of edamame proteins into shorter peptides through a fermentation process using a group of Lactic Acid Bacteria (LABs), that is Lactococcus lactis, Lactobacillus bulgaricus and L. paracasei, to gain a higher functional activity of its protein hydrolysate. Edamame milk was fermented with that LABs as starter culture until 24 hours at 37ºC. The activity of the protease released by the LABs was confirmed using the agar diffusion method based on the emergence of a clear zone surrounding the culture, while the antioxidant activity was confirmed with the ABTS and hydroxyl radical scavenging methods. The results exhibited that the population density of starter culture in edamame milk reached 108 CFU/mL, and the pH decreased from 6.75 to 4.15. All LABs used in fermentation were able to hydrolyze protein as indicated by the increasing degree of hydrolysis and changes in protein profile of SDS-PAGE analysis. In addition, when compared to the unfermented edamame milk, the protein hydrolysate from LABs fermentation showed an increase of ABTS and hydroxyl radical inhibitory activity, in which the highest IC50 values ??of 8.96 and 13.33 g/mL of each were found in edamame milk fermented by Lc. lactis InaCC B187. An increase in ACE inhibitory activity was also observed in edamame milk fermented using this microbe (9.80 g/µL). These findings indicate that Lc. lactis InaCC B187, L. delbrueckii subspecies bulgaricus FNCC41, and L. paracasei InaCC B145 have the potency as an effective starter culture to increase bioactive peptides in edamame soybeans potential for functional food sources.

A Study of the Antimicrobial Activity of Combined Black Pepper and Cinnamon Essential Oils against Escherichia fergusonii in Traditional African Yoghurt.

The antimicrobial activity of the essential oils of black pepper (BPE) and cinnamon bark (CE) extracts against E. fergusonii was assessed in pasteurized full cream milk during and post-fermentation. The milk was fermented with 1% (v/v) of Lactobacillus delbrueckii subspecies bulgaricus (NCIMB 11778) and Streptococcus thermophilus (NCIMB 10387) (approx. 106 cfu/mL each) and incubated and stored at 25 °C for 5 days (144 h) or at 43 °C for 24 h and then stored at 25 °C for 120 h. The milk was spiked with E. fergusonii at the start of fermentation by the lactic acid bacteria (pre-fermentation contamination) for after fermentation (post fermentation contamination). BPE and CE were applied at concentrations based on their minimum inhibitory concentration of 0.5% and 0.25% respectively as follows: 0.5% BPE alone; 0.125% BPE with 0.1875% CE; 0.25% BPE with 0.125% CE; 0.375% BPE with 0.0625% CE; 0.25% CE alone. Results showed that during fermentation at 25 °C, E. fergusonii grew to a similar level (approx. 109 CFU/mL) in control samples and 108 CFU/mL when BPE or CE were added alone. Whereas, in the samples with the combined essential oils, the bacterium grew to 106–107 CFU/mL only. During the milk fermentation at 43 °C, E. fergusonii grew to approx. 109 CFU/mL in samples without treatment. However, it was not detected in samples containing mixed BPE with CE after 8, 10 and 12 h of fermentation. Subsequent storage at 25 °C resulted in undetectable levels of the bacterium in all the samples treated with BPE or CE after 24 h of storage. These results indicated that BPE in combination with CE reduced growth during fermentation and was bactericidal during storage.

Selection of raw cow’s milk thermophilic lactic acid bacteria obtained from southwest Parana, Brazil, with potential use as autochthonous starter

Abstract This work aimed to isolate and identify Streptococcus and Lactobacillus species from raw cow’s milk obtained from Southwest Paraná - Brazil. We used randomly amplified polymorphic DNA (RAPD)-PCR to identify and type 58Streptococcus and 48 Lactobacillus isolates, of which 04 Streptococcus thermophilus and 02 Streptpcoccus macedonicus were confirmed by species-specific PCR and by sequencing of the 16S ribosomal RNA of 02Streptococcus lutetiensis/infantarius, 10 Lactobacillus fermentum, 03 Lactobacillus delbrueickii subspecies bulgaricus, 01 Lactobacillus rhamnosus/casei and 02 Lactobacillus helveticus. The results indicated predominance of Streptococcus thermophillus and Lactobacillus fermentum. Streptococcus thermophilus and Lactobacillus delbrueickii subspecies bulgaricus strains were tested on the basis of their acidification kinetics. Considerable variation between the Streptococcus thermophilus was observed for the maximum rate of acidification (Vm), with a maximum of -4.5 and minimum of -4.2 pH milliunits min-1. The Lactobacillus delbrueickii subspecies bulgaricus showed values between -8.4 and -7.1 pH milliunits min-1. These results suggest that strains characterized as having a high acidifying capacity, could be used as starters in cheesemaking. The ferments presented an excellent performance in the acidification process, generating adequate curves, characteristics of a starter culture.

A Double-Blind Controlled Study to Evaluate the Effects of Yogurt Enriched with Lactococcus lactis 11/19-B1 and Bifidobacterium lactis on Serum Low-Density Lipoprotein Level and Antigen-Specific Interferon-γ Releasing Ability.

In order to clarify the effects of the Lactococcus lactis (L. lactis) 11/19-B1 strain, a double-blind controlled study of yogurt fermented with the strain was carried out. For the study, two kinds of yogurt, the control and test yogurt, were prepared; the control yogurt was fermented with Streptococcus thermophiles, Lactobacillus delbrueckii subspecies bulgaricus, and Lactobacillus acidophilus, and the test yogurt was enriched with L. lactis 11/19-B1 and Bifidobacterium lactis (B. lactis) BB-12 strains. Seventy-six volunteers who had not received treatment with pharmaceuticals were randomly divided into two groups with each group ingesting 80 g of either the test or control yogurt every day for 8 weeks. Before and after yogurt intake, fasting blood was taken and blood sugar, blood lipids, and anti-cytomegalovirus cellular immunity were estimated. In the test yogurt group, low-density lipoprotein (LDL) was significantly decreased (159.1 ± 25.7 to 149.3 ± 24.4; p = 0.02), but this effect was not observed in the control yogurt group. When the test yogurt group was divided into two groups based on LDL levels of over or under 120 mg/dL, this effect was only observed in the high LDL group. No LDL-lowering effect of B. lactis BB-12 strain was previously reported; therefore, the hypocholesterolemic effects observed in this study are thought to be caused by the L. lactis 11/19-B1 strain alone or its combination with the B. lactis BB-12 strain.

Metabolic engineering of Bacillus subtilis for production of D-lactic acid.

Poly lactic acid (PLA) based plastics is renewable, bio-based, and biodegradable. Although present day PLA is composed of mainly L-LA, an L- and D- LA copolymer is expected to improve the quality of PLA and expand its use. To increase the number of thermotolerant microbial biocatalysts that produce D-LA, a derivative of Bacillus subtilis strain 168 that grows at 50°C was metabolically engineered. Since B. subtilis lacks a gene encoding D-lactate dehydrogenase (ldhA), five heterologous ldhA genes (B. coagulans ldhA and gldA101, and ldhA from three Lactobacillus delbrueckii) were evaluated. Corresponding D-LDHs were purified and biochemically characterized. Among these, D-LDH from L. delbrueckii subspecies bulgaricus supported the highest D-LA titer (about 1M) and productivity (2 g h-1 g cells-1 ) at 37°C (B. subtilis strain DA12). The D-LA titer at 48°C was about 0.6 M at a yield of 0.99 (g D-LA g-1 glucose consumed). Strain DA12 also fermented glucose at 48°C in mineral salts medium to lactate at a yield of 0.89 g g-1 glucose and the D-lactate titer was 180 ± 4.5 mM. These results demonstrate the potential of B. subtilis as a platform organism for metabolic engineering for production of chemicals at 48°C that could minimize process cost.

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

Survival and bioactivities of selected probiotic lactobacilli in yogurt fermentation and cold storage: New insights for developing a bi-functional dairy food

In previous work, we demonstrated that two probiotic strains, namely Lactobacillus casei PRA205 and Lactobacillus rhamnosus PRA331, produce fermented milks with potent angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities. Here, we tested these strains for the survivability and the release of antihypertensive and antioxidant peptides in yogurt fermentation and cold storage. For these purposes three yogurt batches were compared: one prepared using yogurt starters alone (Lactobacillus delbrueckii subspecies bulgaricus 1932 and Streptococcus thermophilus 99), and the remaining two containing either PRA205 or PRA331 in addition to yogurt starters. Despite the lower viable counts at the fermentation end compared to PRA331, PRA205 overcame PRA331 in survivability during refrigerated storage for 28 days, leading to viable counts (>108 CFU/g) higher than the minimum therapeutic threshold (106 CFU/g). Analyses of in vitro ACE-inhibitory and antioxidant activities of peptide fractions revealed that yogurt supplemented with PRA205 displays higher amounts of antihypertensive and antioxidant peptides than that produced with PRA331 at the end of fermentation and over storage. Two ACE-inhibitory peptides, Valine-Proline-Proline (VPP) and Isoleucine-Proline-Proline (IPP), were identified and quantified. This study demonstrated that L. casei PRA205 could be used as adjunct culture for producing bi-functional yogurt enriched in bioactive peptides and in viable cells, which bring health benefits to the host as probiotics.

Genetic diversity and population structure of Lactobacillus delbrueckii subspecies bulgaricus isolated from naturally fermented dairy foods.

Lactobacillus delbrueckii subsp. bulgaricus is one of the most widely used starter culture strains in industrial fermented dairy manufacture. It is also common in naturally fermented dairy foods made using traditional methods. The subsp. bulgaricus strains found in naturally fermented foods may be useful for improving current industrial starter cultures; however, little is known regarding its genetic diversity and population structure. Here, a collection of 298 L. delbrueckii strains from naturally fermented products in Mongolia, Russia, and West China was analyzed by multi-locus sequence typing based on eight conserved genes. The 251 confirmed subsp. bulgaricus strains produced 106 unique sequence types, the majority of which were assigned to five clonal complexes (CCs). The geographical distribution of CCs was uneven, with CC1 dominated by Mongolian and Russian isolates, and CC2–CC5 isolates exclusively from Xinjiang, China. Population structure analysis suggested six lineages, L1–L6, with various homologous recombination rates. Although L2–L5 were mainly restricted within specific regions, strains belonging to L1 and L6 were observed in diverse regions, suggesting historical transmission events. These results greatly enhance our knowledge of the population diversity of subsp. bulgaricus strains, and suggest that strains from CC1 and L4 may be useful as starter strains in industrial fermentation.

Thesis Abstract Fermented milk elaborated with Camellia sinensis.

This study aimed to develop and to characterize fermented dairy beverage formulated with Camellia sinensis. The infusion was elaborated with the addiction of dehydrated leaves of C. sinensis in whey (1g/100g) which added in sweetened milk (10% sucrose w/w) coagulated by Streptococcus salivarius subspecies thermophilus and Lactobacillus delbrueckii subspecies bulgaricus in proportions of 10, 20, 30 and 40% (v/w). The control treatment consisted of yogurt added with sucrose (10% w/w). Analysis were performed to quantify dry mass, moisture, ash, protein, fat, sodium, acidity, total quantification of lactic acid bacteria, total antioxidant activity and viscosity at the initial time of production and at 15 and 30 days of storage. Chromatographic determination of volatile compounds and sensory tests of acceptance and consumption intention were conducted at the initial time of production. Dry matter content, moisture, ash and total count of lactic acid bacteria from fermented milk drink formulations were not significantly affected by the amount of infusion of C. sinensis. However, the content of protein, fat and sodium were significantly lower with the increase of the proportion of infusion incorporated into the product. Significant reduction in apparent viscosity occurs with the increase in the amount of infusion added. The total antioxidant activity of the formulations was significantly higher as higher were the amount of added infusion. The addition of infusion contributed to the diversification of volatile aroma and taste makers in the product. The formulation of fermented dairy drink with addition of 30% infusion C. sinensis was better evaluated in sensory tests, with greater acceptance and greater consumer intent of consumption.

Influence of culture conditions and preconditioning on survival of Lactobacillus delbrueckii subspecies bulgaricus ND02 during lyophilization

The cryotolerance of Lactobacillus delbrueckii ssp. bulgaricus is weak during vacuum freeze-drying. Many factors affect cryoresistance of these bacteria, such as cryoprotectant composition, the lyophilization technology used, and the intrinsic characteristics of the bacteria. In this research, we explored the fermentation technology and other preconditioning treatments of cells in improving the cryoresistance of Lactobacillus delbrueckii ssp. bulgaricus strains during lyophilization. The addition of yeast extract in the propagation medium exerted a negative effect on the cryotolerance of these bacteria and decreased survival during lyophilization. The count of the freeze-dried cells from medium containing a high level (4%) of yeast extract was only 4.1×109 cfu/g, indicating a death rate as high as 88%, compared with the culture medium without yeast extract, with a lower death rate of 44.7%. When Lactobacillus delbrueckii ssp. bulgaricus ND02 was propagated in yeast extract-free de Man, Rogosa, and Sharpe broth at a set pH value of 5.1, the cells showed unexpectedly higher survival after freeze-drying. Viable counts of the lyophilized cell of strain ND02 cultivated at pH 5.1 could reach 1.05×1011cfu/g and survival of the freeze-drying process was 68.3%, whereas at pH 5.7, survival was only 51.2%. We also examined the effects of pretreatment of cells on survival of the bacteria after vacuum freeze-drying. By analyzing the effect of pretreatment conditions on the expression of cold- and heat-shock genes, we established 2 pretreatments that improved survival of cells after lyophilization. Optimal fermentation conditions and pretreatment of the cell-cryoprotectant mixture at 10°C for 2h or 37°C for 30min improved the cryoresistance of 4 strains of Lactobacillus delbrueckii ssp. bulgaricus to varying degrees. Cells of IMAU20269 and IMAU20291 that were pretreated showed enhanced survival of 16.06 and 16.82%, respectively, after lyophilization. Expression of cold- and heat-shock genes for pretreated strains ND02, IMAU80423, IMAU20269, and IMAU20291 was analyzed by using quantitative PCR. From the expression of 2 cold shock-induced genes (cspA and cspB) and 6 heat shock-induced genes (groES, hsp, hsp20, hsp40, hsp60, and hsp70), strain ND02 showed a higher relative quantity of gene expression and displayed superior resistance to cold-induced stress during the freeze-drying process.

Remote Regions Involved in Phosphoenolpyruvate Binding to Lactobacillus Delbreuckii Phosphofructokinase

Phosphofructokinase from Lactobacillus delbrueckii subspecies bulgaricus (LbPFK), unlike that from Thermus thermophilus (TtPFK) and Bacillus stearothermophilus (BsPFK), exhibits weak binding affinity for the allosteric inhibitor phospho(enol)pyruvate (PEP). LbPFK has 57% sequence identity, with 75% similarity, to BsPFK and 48% identity, with 65% similarity, to TtPFK. A comparison of crystal structures between apo-LbPFK and apo-BsPFK indicates an overall conservation in structure except for the allosteric binding site. The two regions within the allosteric binding site which differ between LbPFK and other PFKs include the end of an a-helix containing residues 55-59 and a loop containing residues 211-215. Individual mutations were made in both of these regions to the corresponding residues from either TtPFK or BsPFK, with no enhancement in PEP binding. Therefore, chimeric substitutions were introduced into LbPFK in which all the residues in these regions were replaced with those from TtPFK, since TtPFK binds PEP 18,000-fold tighter when compared to LbPFK. Tt(52-61)/LbPFK and Tt(206-218)/LbPFK resulted in no enhancement in PEP binding when compared to LbPFK. They were also combined to form Tt(52-61,206-218)/LbPFK, which again exhibits similar PEP binding to LbPFK. These results indicate that the weak PEP binding in LbPFK cannot be explained as due solely to the residues which directly interact with the ligand upon binding. Another region of interest is the a-helix containing residues 227-236, which exhibits little sequence identity between the various PFKs. Currently Tt(222-242)/LbPFK is being characterized to determine its potential role in PEP binding. The only mutation to show enhancement in PEP binding in LbPFK is D12A (5-fold), which is located on the active site interface approximately 16 Å from the allosteric binding site. The role of D12A is LbPFK is currently under investigation. Funding provided by NIH grant GM33216 and Welch Foundation grant A1548.

Weak Binding of Phospho(enol)pyruvate to Phosphofructokinase from Lactobacillus delbrueckii

Most prokaryotic phosphofructokinases (PFKs), like those from Bacillus stearothermolphilus (BsPFK) and E.coli (EcPFK), exhibit both tight binding affinity for the allosteric inhibitor phosphoenolpyruvate (PEP) and strong inhibition. PFK from Lactobacillus delbrueckii subspecies bulgaricus (LbPFK), which is similar in structure and sequence to BsPFK and EcPFK, exhibits weak PEP binding, with the strength of the inhibition being indeterminate. D12, found along the active site interface, is conserved in over 150 prokaryotic PFKs, and it forms hydrogen bonds with highly conserved residues across this interface. Mutating D12 to alanine in BsPFK enhances PEP binding by 100-fold with no change in the extent of PEP inhibition. When D12A was introduced into LbPFK only a 5-fold enhancement in PEP binding was observed. Crystal structures of D12A BsPFK and D12A LbPFK were solved to 2.4 A resolution. Comparison of D12A and wild-type BsPFK with fructose 6-phosphate bound shows a quaternary shift along the active site interface, breaking the hydrogen bonds involving D12. By contrast, D12A LbPFK exhibits no major change in structure relative to wild type BsPFK. In hopes of further enhancing PEP binding, the following mutations of non-conserved residues in the allosteric site were made to the corresponding residues in either EcPFK or BsPFK, respectively. H59D, E55Y, D187E and S319Y combined showed no enhancement in PEP binding. S211R, D214K and G216S alone and in combination also had no effect on PEP binding. All these mutations suggest that the diminished PEP binding affinity to LbPFK is the consequence of more that just the residues in the allosteric site, likely involving the resistance of this enzyme to undergoing the quaternary shift. Funding came from NIH grant GM33216, NIH CBI training grant, and the Welch Foundation grant A1543.

Kinetic and Structural Characterization of Phosphofructokinase from Lactobacillus bulgaricus

Phosphofructokinase from Lactobacillus delbrueckii subspecies bulgaricus (LbPFK) has been reported to be a nonallosteric analogue of phosphofructokinase from Escherichia coli at pH 8.2 [Le Bras et al. (1991) Eur. J. Biochem. 198, 683-687]. A reexamination of the kinetics of this enzyme shows LbPFK to have limited binding affinity toward the allosteric ligands, MgADP and PEP, with dissociation constants of approximately 20 mM for both. Their allosteric effects are observed only at high concentrations of these ligands, with both exhibiting inhibitory effects on substrate binding. No pH dependence was observed for the binding and the influence of MgADP and PEP on the enzyme. To attempt to explain these results, the crystal structure of LbPFK was solved using molecular replacement to 1.86 A resolution. A comparative study of the LbPFK structure with that of phosphofructokinases from E. coli (EcPFK) and Bacillus stearothermophilus (BsPFK) reveals a structure with conserved fold and substrate binding site. The effector binding site, however, shows many differences that could explain the observed decreases in binding affinity for MgADP and PEP in LbPFK as compared to the other two enzymes.

Exopolysaccharide-producing strains of thermophilic lactic acid bacteria cluster into groups according to their EPS structure.

To compare galactose-negative strains of Streptococcus thermophilus and Lactobacillus delbrueckii subspecies bulgaricus isolated from fermented milk products and known to produce exopolysaccharides (EPSs). The structures of the EPSs were determined using nuclear magnetic resonance (NMR) and their genetic relationships determined using restriction endonuclease analysis (REA) and random amplification of polymorphic DNA (RAPD). Similar groupings were apparent by REA and RAPD, and each group produced an EPS with a particular subunit structure. Although none of the strains assimilated galactose, all inserted a high proportion of galactose into their EPS when grown in skimmed milk, and fell into three distinct groups. This information should help in an understanding of genetic exchanges in lactic acid bacteria.

Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis by SDS-PAGE of cell-wall proteins.

In the present study, a method based on SDS-PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis dairy isolates. The two subspecies, identified by species-specific PCR, were characterized by different SDS-PAGE cell-wall protein profiles; subspecies bulgaricus showed one band of about 31 kDa which, in some cases, was observed at a doublet, and subspecies lactis showed one band of about 21 kDa or 18 kDa. The sensitivity of this procedure for discriminating between the two subspecies was very high. The different types of SDS-PAGE profile for cell-wall proteins of the strains studied in this work did not seem to be correlated to the different dairies of origin. The method appears to be an efficient taxonomic tool. It has the advantage of easy gel interpretation over fingerprinting of whole-cell protein extracts, and may be used as an alternative to established PCR-based techniques which, though rapid and safe, require expensive instruments and reagents.

DNA probe for Lactobacillus delbrueckii subsp. lactis.

Lactobacilli are of great commercial value because they are widely used as starters in food fermentation. The species Lactobacillus delbrueckii, which comprises the three subspecies bulgaricus, lactis and delbrueckii, is important in dairy products and vegetables. The subspecies bulgaricus is present in yogurt, and subspecies lactis is recovered from whey, starter cultures and cheeses (Stiles & Holzapfel, 1997). It is unusual to recover Lb. delbrueckii subsp. delbrueckii (Lb. delbrueckii) from dairy products, from which Lb. delbrueckii subsp. lactis (Lb. lactis) and subsp. bulgaricus (Lb. bulgaricus) are typically isolated. However, given the similarity of these two latter subspecies, a clear identification of isolates on the basis of phenotype criteria alone is often problematic (Dellaglio, 1989; Millière et al. 1996).The use of DNA probes and methods based on polymerase chain reaction (PCR) have greatly facilitated diagnostic identification of bacteria. For the lactobacilli, DNA probes have been described for Lb. helveticus, Lb. acidophilus, Lb. fermentum, Lb. plantarum and most of the other Lactobacillus species (Pot et al. 1994; Tailliez et al. 1994; Quere et al. 1997). For Lb. delbrueckii, an EcoRI DNA fragment of the plasmid pY85 was used as a probe for this species, although it was not able to discriminate its three subspecies (Delley et al. 1990).In our laboratory, a specific amplification of a DNA fragment of ∼1·7 kbp using universal primers for the amplification of ribosomal RNA (rRNA) genes was found only for dairy isolates of Lb. lactis and not for Lb. bulgaricus, Lb. delbrueckii, Lb. helveticus and Lb. acidophilus (G. Giraffa, P. de Vecchi and L. Rossetti, unpublished results). This prompted us to test the possible use of this fragment as a specific DNA probe for Lb. lactis. To this end, Southern and dot blot hybridization experiments were carried out with total DNA of several strains belonging to different lactic acid bacteria (LAB) species.

Structural characterisation of the exopolysaccharide produced by Lactobacillus delbrückii subspecies bulgaricus rr grown in skimmed milk

The exopolysaccharide of Lactobacillus delbrückii subsp. bulgaricus rr. isolated from skimmed milk, is a heteropolymer of d-galactopyranosyl, d-glucopyranosyl, and l-rhamnopyranosyl residues in the molar ratio 5:1:1. The structure was established by linkage analysis and 1D and 2D NMR spectroscopy of the native polysaccharide, in combination with characterisation of oligosaccharide fragments, obtained by Smith degradation and partial acid hydrolysis, using methylation analysis, EIMS, and 1D and 2D 1H NMR spectroscopy. The polysaccharide has a branched heptasaccharide repeating unit with the following structure: → 2)-[β- d-Gal p-(1 → 3)]-α- d-Gal p-(1 → 3)-β- d-Glc p-(1 → 3)-β- d-Glc p-(1 → 4)]-β- d-Gal p-(1 → 4)-[α- l-Rha p-(1 → 3)]-α- d-Gal p-(1 →.

Purification and properties of the phosphofructokinase from Lactobacillus bulgaricus

Phosphofructokinase, the enzyme which catalyzes the conversion of fructose 6-phosphate into fructose 1,6-bisphosphate in Lactobacillus bulgaricus (Lactobacillus delbrueckii, subspecies bulgaricus) has been purified to homogeneity and some of its structural and functional properties have been studied. The enzyme is a tetramer composed of four 35-kDa subunits. Its N-terminal sequence determined on 38 residues is homologous to those of the major allosteric enzymes from Escherichia coli and Bacillus stearothermophilus, suggesting that the three proteins have closely related structures. The maximum velocity of the enzyme from L. bulgaricus increases with pH according to the ionization of a group with a pK of 6.2. At all pH values, the saturation by fructose 6-phosphate is hyperbolic. At the optimum pH of 8.2, the maximum velocity and the affinities for the ATP and fructose 6-phosphate substrates are not modified by the presence of ADP or GDP nor by phosphoenolpyruvate. Partial inhibition by phosphoenolpyruvate exists at acidic pH, but is not related to an allosteric mechanism similar to that in E. coli. This inhibition results from a shift from 6.2 to 7.1 of the pK of an ionizable group which controls Vmax. Protection against thermal denaturation shows that the enzyme binds phosphoenolpyruvate and not GDP. The phosphofructokinase from L. bulgaricus appears as a structural analog of the E. coli enzyme which does not undergo an allosteric transition between two states R and T, but instead remains in a unique conformational state, intermediate between the R and T states; the active sites have an R-like conformation since they bind fructose 6-phosphate, whereas the regulatory sites have a T-like conformation since they bind phosphoenolpyruvate and not GDP.