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A strain of Escherichia coli (FMJ144) deficient for pyruvate formate lyase and lactate dehydrogenase (LDH) was complemented with a genomic DNA library from Lactobacillus delbrueckii subsp. bulgaricus. One positive clone showed LDH activity and production of D(−)lactate was demonstrated. The nucleotide sequence of the D-LDH gene (ldhA) revealed the spontaneous insertion of an E. coli insertion sequence IS2 upstream of the gene coding region. The open reading frame encoded a 333-amino acid protein, showing no similarity with known L-LDH sequences but closely related to L. casei D-hydroxyisocaproate dehydrogenase (D-HicDH).

Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 is able to hydrolyze X-proline- para -nitroanilides and X-proline-β-naphthylamides (X for alanyl- or glycyl-). A single metal-independent cytoplasmic enzyme with a molecular weight estimated to be 82,000 is responsible for these activities and was named X-prolyl-dipeptidyl aminopeptidase (X-Pro-DPAP). Isolation and analysis of mutants totally deficient for X-Pro-DPAP activity showed that a total lack of this enzyme induces (i) a decrease in the growth rate; (ii) an increase in cell wall proteinase activity; (iii) the loss of three cell wall proteins with respective molecular masses of 16, 40, and 52 kilodaltons; and (iv) enhancement of a cell wall protein with a molecular mass of 150 kilodaltons. The involvement of X-Pro-DPAP in casein catabolism is discussed.

Cells of Streptococcus salivarius subsp. thermophilus and Lactococcus lactis subsp. lactis entrapped in k -carrageenan-locust bean gum gel performed similarly to free cells in the conversion of lactose to lactic acid. Bead diameter influenced the fermentation rate. Cells entrapped in smaller beads (0.5 to 1.0 mm) showed higher release rates, higher lactose, glucose, and formic acid utilization, higher galactose accumulation, and higher lactic acid production than did cells entrapped in larger beads (1.0 to 2.0 mm). Values for smaller beads were comparable with those for free cells. Immobilization affected the fermentation rate of lactic acid bacteria, especially Lactobacillus delbrueckii subsp. bulgaricus. Entrapped cells of L. delbrueckii subsp. bulgaricus demonstrated a lower lactic acid production than did free cells in batch fermentation. The kinetics of the production of formic and pyruvic acids by L. lactis subsp. lactis and S. salivarius subsp. thermophilus are presented.

The type strains of Lactobacillus delbrueckii, Lactobacillus lactis, Lactobacillus leichmannii und Lactobacillus bulgaricus were found to exhibit DNA-DNA homologies of 90-100% among each other. The strains are thus considered as belonging to one and the same species which, according to the rules of priority retains the name Lactobacillus delbrueckii. On the basis of different habitats and different ranges of fermented sugars, the two subspecies, Lactobacillus delbrueckii subsp. lactis comb, nov., and Lactobacillus delbrueckii subsp. bulgaricus comb. nov. are described.

SUMMARY: An improved growth medium for lactobacilli is described. It supports good growth of lactobacilli generally and also is particularly useful for a number of fastidious strains which grow only poorly in other general media. In addition, tomato juice, a highly variable material, is not required. In a slightly modified form, it can also be used as a basal medium for fermentation tests.

In this report, it is shown that the rolling circle replicon pG+host and the theta replicon pIP501 are thermosensitive in Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus bulgaricus). Using a pIP501 derivative as a delivery vector for six insertion sequences originating from lactic acid bacteria, it is shown that IS1223 and IS1201 transpose in L. bulgaricus.

Texture profile analysis (TPA) of stirred and set cultured milk were evaluated, using an exopolysaccharide producing strain of Lactobacillus delbrueckii subsp. bulgaricus (NCFB 2772) and a protein enriched substrate (retentate). In both cases, samples were compared respectively with a nonropy strain (NCFB 1489) and reconstituted skim milk substrate. The retentate fermented products were firmer in comparison with skim milk products, both at 10% of total solids. A loss of structure occurred when the retentate products were stirred causing a nine-fold decrease in hardness, whereas in cultured milk products the loss was of around three-fold. The main differences were found in adhesiveness and fracture characteristics. Set retentate ropy product was five times more adhesive than the nonropy one, whereas in stirred retentate and set skim milk products the difference was two-fold. No differences in adhesiveness were detected when skim milk products were stirred using either strain. The fracture force on ropy retentate samples increased after fracture while the nonropy ones showed a decrease, implying more structural breakdown in the latter. On set products, cohesiveness increased slightly due to the ropy strain. Changes in texture observed between ropy and nonropy strains can be attributed to exopolysaccharide attachment to the casein matrix being increased when the protein content is higher. Le sujet de cette etude est d'evaluer la difference entre des laits fermentes brasses ou non, en determinant la contribution de souches de lactobacillus delbrueckii subsp. Bulgaricus NCFB 2772, productrices d'exopolysaccharides, sur la consistance particuliere de lait ecreme fermente et de retentat d'ultrafiltration de lait et en comparant les souches productrices aux souches non productrices. Elle etudie en particulier l'interaction entre ces polysaccharides et les proteines laitieres conduisant a un effet synergique.

Three types of lactic starters of Streptococcus salivarius ssp. thermophilus 404 and Lactobacillus delbrueckii ssp. bulgaricus 398 have been produced by controlled pH batch cultures: pure starters, mixings of pure starters and mixed starters. They were compared according to their viability and their acidification activity after the different steps of their production and during storage. At the end of the batch cultures, the acidification activity, characterized by the maximum acidification rate in milk, was 40% higher for the starters issued from the mixed cultures or the mixings of pure cultures with regard to the pure starters. During the concentration, freezing and freeze-drying processes, tea significant alteration of the activity of the starters was observed. The viability of the starters, during 24 wk of preservation after freezing (-75°C) or freeze-drying (4°C), decreased continuously. This decrease was slightly faster in the case of the starter obtained from a mixed culture. The starters were more resistant during storage in a frozen state than in a freeze-dried state. Finally, thanks to the use of a well normalized procedure, the acidification activity was not altered during the storage of each type of starter.

Five residues involved in catalysis and coenzyme binding have been identified in D-2-hydroxy-4-methylvalerate dehydrogenase from Lactobacillus delbrueckii subsp. bulgaricus by using biochemical and genetical methods. Enzyme inactivation with diethylpyrocarbonate indicated that a single histidine residue was involved in catalysis. Since H296 is the only conserved histidine in the whole family of NAD-dependent D-2-hydroxyacid dehydrogenases, we constructed the H296Q and H296S mutants and showed that their catalytic efficiencies were reduced 10(5)-fold compared with the wild-type enzyme. This low residual activity was shown to be insensitive to diethylpyrocarbonate. Taken together these data demonstrate that H296 is responsible for proton exchange in the redox reaction. Two acidic residues (D259 and E264) were candidates for maintaining H296 in the protonated state and their roles were examined by mutagenesis. The D259N and E264Q mutant enzymes both showed similar and large reductions in their Kcat/K(m) ratios (200-800-fold, depending on pH), indicating that either D259 or E264 (or both) could partner H296. The conserved R235 residue was a candidate for binding the alpha-carboxyl group of the substrate and it was changed to lysine. The R235K mutant showed a 104-fold reduced Kcat/K(m) due to both an increased K(m) and a reduced Kcat for 2-oxo-4-methylvalerate. Thus R235 plays a role in binding the substrate carboxylate similar to R171 in the L-lactate dehydrogenases. Finally, we constructed the H205Q mutant to test the role of this partially conserved histidine residue (in 10/13 enzymes of the family). This mutant enzyme displayed a 7.7-fold increased Kcat and a doubled catalytic efficiency at pH 5, was as sensitive to diethylpyrocarbonate as the wild-type but showed a sevenfold increased K(m) for NADH and a 100-fold increase in Kd for NADH together with 10-30-fold lower substrate inhibition. The transient kinetic behaviour of the H205Q mutant is as predicted from our previous study on the enzymatic mechanism of D-2-hydroxy-4-methylvalerate dehydrogenase which showed that coenzyme binding is highly pH dependent and indicated that release of the oxidised coenzyme is a significant component of the rate-limiting processes in catalysis at pH 6.5.

Yoghurt is fermented milk which results from the exclusive action of Lactobacillus delbrueckii subsp bulgaricus and Streptococcus thermophilus. However, a disruption of the bacterial growth can lead to significant industrial losses. Currents monitoring methods, based on the checking of the pH during the fermentation step are not always satisfactory. The NeOse Pro system, a portable electronic nose, is a mean to obtain immediately aromatic profiles. In this work, we applied this technology to the yoghurt ecosystem, a highly hydrated product. The profiles obtained allowed us to discriminate yoghurts before and after fermentation. In detail, the discrepancy between the two bacteria cultured alone was slight. It was also the case when we compared different initial bacterial ratios. However, two different mixes of bacteria led to clearly distinguishable profiles. A GC/MS analysis performed on products fermented 7 h allowed us to explain the detection of acetaldehyde by the stimulation of some captors of the apparatus. NeOse Pro is so convenient to study the fermentation of yogurt.

A double-blind placebo controlled investigation was carried out to study the effect of peroral colonization. Human volunteers were given mixtures of bifidobacteria and lactic acid bacteria. Measurements were made over a 1 week treatment period and for another week after the end of the treatment. Two different bacteriological preparations were used, one consisted of Enterococcus faecium and Bifidobacterium longum (a total of 6.4 x 10(8) cfu d-1); the other consisted of Lactobacillus acidophilus, Bif. bifidum, Lact. delbrueckii ssp. bulgaricus, and Streptococcus thermophilus (a total of 9 x 10(9) cfu d-1). Together with a placebo preparation, they were given to 24 healthy controls (eight in each group). Microbiological examinations of jejunal aspirates showed that viable counts of most species were below the detection limit. However, the test preparation containing Ent. faecium and Bif. longum significantly reduced the anaerobe: aerobe ratio in faeces by a factor of three during treatment (P = 0.03), and increased it by a factor of 30 during the following week (P < 0.02). This study shows that peroral administration of certain bacterial cultures may affect the distal intestinal microflora.

A novel single-step microbial transformation process for the production of testosterone from cholestrol by Lactobacillus bulgaricus in an aerated fermenter was investigated. The metabolism of glucose possibly supplying reducing power as NADH was necessary for the reduction of androst-4-en-3,17-dione (AD) to testosterone (TS). The growth period for the accumulation of testosterone in maximal amount and the residual glucose increased in parallel with the amount of glucose supplement in growth cultures. After the glucose in the fermentation culture was completely exhausted, most of the testosterone was oxidized to AD. Addition of a larger amount of glucose could prevent oxidation of testosterone to AD. The biotransformation of cholestrol was significantly increased in the presence of cyclodextrin (CD) in the fermenting medium. The addition of 0.1% CD to the growth medium facilitated the transport of the steroid substrate through the microbial cell wall.

Aims: To investigate the diversity in specificity of cell-bound extracellular proteinases in Lactobacillus helveticus and Lactobacillus delbrueckii subsp. bulgaricus. Methods and Results: HPLC analysis of whole-cell preparations of 14 Lact. delbrueckii subsp. bulgaricus and eight Lact. helveticus strains incubated with αs1-casein (f 1–23) detected at least six distinct proteolytic patterns. Differences between groups were found in both the primary and secondary specificity toward αs1-casein (f 1–23) and its breakdown products. No correlation was found between the o-phthaldialdehyde (OPA) general proteolysis analysis and αs1-casein (f 1–23) cleavage profiles. Conclusions, Significance and Impact of Study: Using the αs1-CN (f 1–23) method, six patterns of proteolysis were found in the dairy lactobacilli tested. Understanding the influence of Lactobacillus proteinase specificity on casein degradation should facilitate efforts to develop starter cultures that predictably improve the functional properties of Mozzarella cheese.

A synergistic set of the strains Lactobacillus delbrueckii subsp. bulgaricus 151 and Streptococcus thermophilus MK-10 was preserved with the method of spray drying. Studies were performed on the effect of outlet air temperature in range of 60-80 degrees C upon the survival of yoghurt cultures, as well as the moisture content and sensoric properties of yoghurt powder. Survival of yoghurt cultures was the highest at 60 and 65 degrees C, but excessive moisture (10.2%) of yoghurt powder had a negative effect on its texture. Moisture content of the powder was low at 80 degrees C (4.4%), unfortunately sensoric faults appeared at this temperature, and survival of bacteria cultures decreased considerably. Temperatures within the range 70-75 degrees C ensured satisfactory survival of yoghurt cultures (L. delbrueckii subsp. bulgaricus--13.7-15.8%; S. thermophilus--51.6-54.7%), maintained proportion of the strains (L:S--1:3), satisfactory moisutre content (5.1-6.3%), and good sensoric properties of yoghurt powder.

OBJECTIVE: The goal of this study was to ferment yoghurt from milk using lactic acid bacteria found in pinecones and to examine the cell viability and lipid peroxidation levels of the fermented yoghurt on Vascular Endothelial Cell Line ECV304. METHODS: ECV304 was cultured in vitro. To determine cell viability, various concentration of yogurt extract fermented with pinecone were given to cultured cells using the MTT Assay. At the same doses as the MTT Assay, the Lipid Peroxidation (LPO) Assay was employed to evaluate the malondialdehyde (MDA) levels of cells. Acridine orange/Ethidium Bromide staining technique was applied to detect apoptosis. Gas Chromatography-Mass Spectrometry was used to identify the volatiles in yoghurt fermented with pinecone. RESULTS: All dosages of pinecone fermented yogurt enhanced cell viability of the human healthy vascular endothelial cell line ECV304 and decreased MDA levels, as validated by fluorescence microscopy pictures. The primary essential oils identified in yogurt fermented with pinecone were hexadecanoic acid, eicosanoic acid, stearic acid, and 2-palmitoylglycerol. CONCLUSIONS: In this work, the effects of yogurt fermented with pinecones on human healthy cell lines were examined for the first time. The study discovered that this yoghurt promotes the proliferation of healthy human cells while decreasing oxidative stress in these cells.

Recent work by our group has shown that an exopolysaccharide (EPS)-producing starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, can significantly increase moisture retention in low-fat mozzarella (D. B. Perry, D. J. McMahon, and C. J. Oberg, J. Dairy Sci. 80:799–805, 1997). The objectives of this study were to determine whether MR-1C, MR-1R, or both of these strains are required for enhanced moisture retention and to establish the role of EPS in this phenomenon. Analysis of low-fat mozzarella made with different combinations of MR-1C, MR-1R, and the non-EPS-producing starter culture strains S. thermophilus TA061 and Lactobacillus helveticus LH100 showed that S. thermophilus MR-1C was responsible for the increased cheese moisture level. To investigate the role of the S. thermophilus MR-1C EPS in cheese moisture retention, the epsE gene in this bacterium was inactivated by gene replacement. Low-fat mozzarella made with L. helveticus LH100 plus the non-EPS-producing mutant S. thermophilus DM10 had a significantly lower moisture content than did cheese made with strains LH100 and MR-1C, which confirmed that the MR-1C capsular EPS was responsible for the water-binding properties of this bacterium in cheese. Chemical analysis of the S. thermophilus MR-1C EPS indicated that the polymer has a novel basic repeating unit composed of d -galactose, l -rhamnose, and l -fucose in a ratio of 5:2:1.

We have developed a method to quantify the resistance to freezing and frozen storage of lactic acid starters, based on measuring the time necessary to reach the maximum acidification rate in milk (tm) using the Cinac system. Depending on the operating conditions, tm increased during the freezing step and storage. The loss of acidification activity during freezing was quantified by the difference (Δtm) between the tm values of the concentrated cell suspension before and after freezing. During storage at −20 °C, linear relationships between tm and the storage time were established. Their slope, k, allowed the quantitation of the decrease in acidification activity during 9–14 weeks of frozen storage. The method was applied to determine the resistance to freezing and frozen storage of four strains of lactic acid bacteria and to quantify the cryoprotective effect of glycerol.

The growth dynamics of the natural microbial community responsible for the fermentation of Scamorza Altamurana, a typical Southern Italian cheese made using backslopping, was investigated applying a polyphasic approach combining 1) microbial enumeration with culture media, 2) randomly amplified polymorphic DNA (RAPD) fingerprinting of microbial communities, 3) sequencing of partial 16S ribosomal DNA (rDNA) genes, and 4) physiological tests. Viable cell counts on different culture media showed that the cocci community prevailed during the 18 h of curd fermentation and the 6 d of cheese ripening. RAPD fingerprinting made it possible to isolate 25 different strains identified by 16S rDNA sequencing as belonging to five species of Lactobacillus, three species of Streptococcus, one species of Weissella, and one species of Enterococcus. The physiological analyses of all lactic acid bacteria strains revealed that the isolates belonging to Streptococcus genus were the most acidifying, whereas lactobacilli were most proteolytic. Streptococcus thermophilus C48W and Lactobacillus delbrueckii subsp. bulgaricus B15Z dominated all through the fermentation process. Furthermore, they seemed to be stable in a subsequent whey sample analyzed after 7 mo. The recovery of strains endowed with interesting technological features, such as acidifying and proteolytic activities, and surviving in natural whey could allow the upscaling of cheese processing safeguarding the organoleptic characteristics of Scamorza Altamurana and could possibly improve other fermented dairy products.