Enzymatic properties of lipase and characteristics production byLactobacillus delbrueckii subsp.bulgaricus

Some properties of an extracellular lipase produced byLactobacillus delbrueckii subsp.bulgaricus were studied. Maximum enzyme activity was found against olive and butter oil as enzyme substrates. Addition of 9% acacia gum, 0.1% Na-deoxycholate and 0.01 M CaCl₂ to the enzyme reaction mixture increased-lipase activity from 5.3 to 14.5 (FFA/mg protein/minute) at pH 6.0 and at 40° C. Maximum lipase production was reached in the presence of glucose as a sole source of carbon, wheat bran as nitrogen source, olive oil as a sole lipid source and butyric acid as fatty acid supporting the growth medium. An initial pH value of the culture medium of 6.0 and a temperature of 35° C gave the highest lipolytic activity.     

Exopolysaccharide and extracellular metabolite production by Lactobacillus delbrueckii subsp. bulgaricus, grown on lactose in continuous culture

Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483, when grown on lactose in continuous culture, showed increasing specific yields and volumetric productivities of exopolysaccharide (EPS) with increasing dilution rate. Specific and volumetric productivities of lactate and galactose, as extracellular metabolites, increased in response to the incremental changes in the dilution rate up to 0.4 h^–1. Elevated Y_p/s values determined for EPS (0.025 g EPSg lactose^–1) at the dilution rates of 0.3 h^–1–0.4 h^–1, relative to those determined at lower dilution rates, suggest a diversion of carbon flux towards EPS being associated with the higher rates of growth.     

Partial characterization and dynamics of synthesis of high molecular mass exopolysaccharides from Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus

Exopolysaccharide (EPS) preparations from Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) strains LBB.B26 and LBB.B332 and Streptococcus thermophilus strains LBB.T54 and LBB.T6V were characterized using ion-exchange chromatography and gel filtration. All four preparations contained a neutral EPS with molecular mass in the range of 1.3−1.6 × 10⁶ Da (HMM-EPS). The EPS preparations from the two L. bulgaricus strains also contained an acidic low molecular mass EPS fraction (LMM-EPS) comprising from 10% to 34% of the total EPS yield. HMM-EPS preparations were subjected to High Pressure Liquid Chromatography (HPLC) analysis of monomer sugars after complete hydrolysis. Glucose, galactose and/or rhamnose in different ratios proved to be the principal sugars building the HMM-EPS from all four strains. The chemical composition of HMM-EPS was strictly strain-specific. The LMM-EPS contained galactose. The viscosifying properties of the four different HMM-EPS varied greatly with intrinsic viscosity in the range from 0.26 (strain B26) to 2.38 (strain T6V). For 24 h the two L. bulgaricus strains accumulated more HMM-EPS in milk (>70 mg l⁻¹) than S. thermophilus strains T54 and T6V (<30 mg l⁻¹), but maximal yields were reached earlier with cocci (8 h) than with rods (16–24 h). The contribution of HMM-EPS production to increased viscosity of fermented milk was demonstrated for all of the tested strains grown as monocultures or as mixed yogurt starters compared to non-EPS producing S. thermophilus LBB.A and poor EPS-producer L. bulgaricus LBB.B5. The extent of increased viscosity was strongly dependent on the nature of the produced HMM-EPS, rather than simply on polymer yield.     

Growth and Proteolytic Activity of Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus in Reduced Sodium Kashkaval Cheese

Summary Reduced sodium Kashkaval cheese was produced from cow’s milk. Mixtures of NaCl, NaCl:KCl (1:1, 2:1) and NaCl:KH₂PO₄ (1:1, 2:1) were used for hot brining and salting of the cheddarized cheese curd. There were no significant differences (P < 0.05) in the count of Lb. delbrueckii ssp. bulgaricus after aging of Kashkaval samples. At the end of the ripening process the counts of Lb. delbrueckii ssp. bulgaricus reached 10⁶ c.f.u./g and the counts of Streptococcus thermophilus varied from 10⁴ to 10⁵ c.f.u./g. Proteolysis during ripening of reduced sodium Kashkaval cheese, initiated by the starter microorganisms Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, was studied through the changes in the levels of non-casein and non-protein nitrogen. It was observed that non-casein and non-protein nitrogen increased significantly (P < 0.05) during ripening. The amounts of non-casein and non-protein nitrogen accumulated in the studied Kashkaval samples were similar. That indicates that the partial replacement of NaCl with KCl or KH₂PO₄ does not cause significant changes in the course of proteolysis of Kashkaval cheese by Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus.     

Metabolism associated with raised metabolic flux to sugar nucleotide precursors of exopolysaccharides in Lactobacillus delbrueckii subsp. bulgaricus

Exopolysaccharide (EPS) metabolism was studied in a galactose-negative strain of Lactobacillus delbrueckii subsp. bulgaricus, using two different approaches. Firstly, using both the parent strain and a chemically induced mutant with higher yield and specific productivity of EPS than the parent, comparative information was obtained relating to enzyme activities and metabolite levels associated with EPS formation when grown on lactose. Under continuous culture conditions (D=0.10 h⁻¹), the higher metabolic flux towards EPS formation in the mutant strain relative to the parent appeared to be mediated by raised levels of UDP-glucose pyrophosphorylase (UGP). Marginally raised UDP-galactose 4-epimerase (UGE) activity in the mutant strain suggested that this enzyme could also play a role in EPS overproduction. The second approach involved investigating the effect of growth rate on sugar nucleotide metabolism in the parent, as it is known that EPS production is growth-associated in this strain. UGE activity in the parent strain appeared to increase when the growth rate was elevated from 0.05 to 0.10 h⁻¹, and further to 0.35 h⁻¹, conditions that can be associated with higher levels of metabolic flux to EPS formation. Concurrent with these increments, intracellular ATP levels in the cell were raised. In both investigations glucose-6-phosphate accumulated pointing to a constriction at this branch-point, and a limitation in the flow of carbon towards fructose-6-phosphate or glucose-1-phosphate. The changes in metabolism associated with enhanced flux to EPS provide guidance as to how the yield of Lactobacillus delbrueckii subsp. bulgaricus EPS can be improved.     

Fermentation performance of an exopolysaccharide-producing strain of<Emphasis Type="Italic"> Lactobacillus delbrueckii</Emphasis> subsp.<Emphasis Type="Italic"> bulgaricus</Emphasis>

The formation of exopolysaccharide (EPS) and extracellular metabolites was studied in a strain of Lactobacillus delbrueckii subsp. bulgaricus (NCFB 2483), grown under batch culture conditions in a semi-defined medium incorporating lactose and casein hydrolysate. Performance parameters were derived from the fermentation data, and kinetic models were applied in order to describe the production of EPS, extracellular metabolites, and biomass produced. Lactose was split intracellularly, with the resultant galactose being exported from the cell, and the glucose being metabolised further to EPS and lactic acid. Production of EPS, lactate, and galactose was closely growth-associated and followed a pattern of primary kinetics. A marginally lower galactose level relative to the modelled levels throughout most of the time course of the fermentation suggests that not all galactose is exported from the cell, and that a low level of flux to other metabolites, such as EPS, might exist.     

Structural determination of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B332 in skimmed milk was found to be composed of d-glucose, d-galactose, and l-rhamnose in a molar ratio of 1:2:2. Linkage analysis and 1D/2D NMR (1H and 13C) studies carried out on the native polysaccharide as well as on an oligosaccharide generated by a periodate oxidation protocol, showed the polysaccharide to consist of linear pentasaccharide repeating units with the following structure: -->3-alpha-D-Glcp-(1-->3)-alpha-D-Galp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->.     

Enhancement of lactic acid production with ram horn peptone by Lactobacillus casei

Ram horns are a waste material from the meat industry. The use of ram horn peptone (RHP) as a supplement for lactic acid production was investigated using Lactobacillus casei. For this purpose, first, RHP was produced. Ram horns were hydrolysed by treating with acids (3 M H₂SO₄ and 6 M HCl) and neutralizing the solutions to yield ram horn hydrolysate (RHH). The RHH was evaporated to yield RHP. The amounts of protein, nitrogen, ash, some minerals, total sugars, total lipids and amino acids of the RHP were determined and compared with a bacto-tryptone from casein. When the concentrations (1–6% w/v) of the RHP were used in bacterial growth medium as a supplement, 2% RHP (ram horn peptone medium) had a maximum influence on the production of lactic acid by L. casei. The content of lactic acid in the culture broth containing 2% RHP (43 g l^–1) grown for 24 h was 30% higher than that of the control culture broth (33 g l^–1) and 10% higher than that of 2% bacto-tryptone (39 g l^–1). RHP was demonstrated to be a suitable supplement for production of lactic acid. This RHP may prove to be a valuable supplement in fermentation technology.     

Synthesis of cyclopropane fatty acid and its effect on freeze-drying survival of Lactobacillus bulgaricus L2 at different growth conditions

In order to correlate cyclopropane fatty acid of the membrane of Lactobacillus bulgaricus L2 with freeze-drying survival at different growth conditions, fatty acid methyl esters (FAME) from extracts grown at difference fermentation pH (5.0, 5.5, 6.0, 6.5) and temperature (30, 35, 37, 39°C) were obtained and analyzed. Results showed that cultures grown at 30°C and pH 5.0, 35°C and pH 5.0, 39°C and pH 6.0 exhibited more resistance to the freeze-drying process than cultures grown in other conditions, cells cultured at 30°C and pH 5.0 had a highest survival rate. On the other hand, cells grown at 37°C displayed poor resistance to adverse conditions possible because of the lower cycC19:0 content. It was concluded that the improved cryotolerance observed during freeze-drying would be associated with an increase in cycC19:0 content and cycC19:0/SFA ratio and vice versa.     

Galacto-oligosaccharides as protective molecules in the preservation of Lactobacillus delbrueckii subsp. bulgaricus

In this work, the protective capacity of galacto-oligosaccharides in the preservation of Lactobacillus delbrueckii subsp. bulgaricus CIDCA 333 was evaluated.Lactobacillus bulgaricus was freeze-dried or dried over silica gel in the presence of three commercial products containing galacto-oligosaccharides. The freeze-dried samples were stored at 5 and 25 °C for different periods of time. After desiccation, freeze-drying or storage, samples were rehydrated and bacterial plate counts were determined.According to the results obtained, all galacto-oligosaccharides assays demonstrated to be highly efficient in the preservation of L. bulgaricus. The higher content of galacto-oligosaccharides in the commercial products was correlated with their higher protective capacity.Galacto-oligosaccharides are widely known by their prebiotic properties. However, their role as protective molecules have not been reported nor properly explored up to now. In this work the protective capacity of galacto-oligosaccharides in the preservation of L. bulgaricus, a strain particularly sensitive to any preservation process, was demonstrated.The novel role of galacto-oligosaccharides as protective molecules opens up several perspectives in regard to their applications. The supplementation of probiotics with galacto-oligosaccharides allows the production of self-protected synbiotic products, galacto-oligosaccharides exerting both a prebiotic and protecting effect.     

Optimization of a protective medium for enhancing the viability of freeze-dried Lactobacillus delbrueckii subsp. bulgaricus based on response surface methodology

Response surface methodology (RSM) was used to optimize a protective medium for enhancing the cell viability of Lactobacillus delbrueckii subsp. bulgaricus LB14 during freeze-drying. Using a previous Plackett–Burman design, it was found that sucrose, glycerol, sorbitol and skim milk were the most effective freeze-drying protective agents for L. bulgaricus LB14. A full factorial central composite design was applied to determine the optimum levels of these four protective agents. The experimental data allowed the development of an empirical model (P<0.0001) describing the inter-relationships between the independent and dependent variables. By solving the regression equation, and analyzing the response surface contour and surface plots, the optimal concentrations of the agents were determined as: sucrose 66.40 g/L, glycerol 101.20 g/L, sorbitol 113.00 g/L, and skim milk 130.00 g/L. L. bulgaricus LB14 freeze-dried in this medium obtained a cell viability of up to 86.53%.     

A new methodology for rapid detection of Lactobacillus delbrueckii subsp. bulgaricus based on multiplex PCR

In this study we present a novel multiplex PCR assay for rapid and efficient detection of Lactobacillus delbrueckii subsp. bulgaricus. The accuracy of our method was confirmed by the successful identification of L. delbrueckii subsp. bulgaricus in commercial yoghurts and food supplements and it may be readily applied to the food industry.     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

Caseicin 80: purification and characterization of a new bacteriocin from Lactobacillus casei

When grown in complex or synthetic media, Lactobacillus casei B 80 synthesizes a mitomycin C-inducible polypeptide with very specific bactericidal activity against the sensitive strain Lactobacillus casei B 109. The amount of secreted bacteriocin in the culture solution was low, about 1 mg/l. The bacteriocin which we called caseicin 80, was also detectable in cell extracts, although only 2% of the total activity was retained intracellularly. Caseicin 80 was concentrated by ultrafiltration and purified by cation exchange chromatography with Cellulose SE-23 and Superose. The molecular weight was in the range of M _r=40,000–42,000 and the isoelectric point was pH 4.5.     

Structure of a neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26

The neutral exopolysaccharide produced by Lactobacillus delbrueckii ssp. bulgaricus LBB.B26 in skimmed milk was found to be composed of d-glucose and d-galactose in a molar ratio of 2:3. Linkage analysis and 1D/2D NMR ((1)H and (13)C) studies performed on the native polysaccharide, and on an oligosaccharide obtained from a partial acid hydrolysate of the native polysaccharide, showed the polysaccharide to consist of branched pentasaccharide repeating units with the following structure. [structure: see text]     

Lactic acid production by mixed cultures of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus helveticus

Lactic acid production using Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) individually or as mixed culture on cheese whey in stirred or static fermentation conditions was evaluated. Lactic acid production, residual sugar and cell biomass were the main features examined. Increased lactic acid production was observed, when mixed cultures were used in comparison to individual ones. The highest lactic acid concentrations were achieved when K. marxianus yeast was combined with L. delbrueckii ssp. bulgaricus, and when all the strains were used revealing possible synergistic effects between the yeast and the two lactic acid bacteria. The same synergistic effects were further observed and verified when the mixed cultures were applied in sourdough fermentations, proving that the above microbiological system could be applied in the food fermentations where high lactic acid production is sought.     

Characterisation of lactic dehydrogenase fromLactobacillus casei

Lactic dehydrogenase fromLactobacillus casei ATCC 7469 has been purified to homogeneity by a two-step affinity chromatography procedure which gave an yield of 35%. The enzyme specifically catalysed the conversion of pyruvate to lactate. The enzyme was maximally active at pH 4.6, which was shifted to 5.4 in the presence of fructose 1,6-biphosphate. The enzyme had a molecular weight of 70,800 with two identical subunits, unlike the lactic dehydrogenase from other sources. Histidine and primary amino groups appeared to be involved in catalysis.     

Comparative Genomic Analysis of Two-Component Signal Transduction Systems in Probiotic Lactobacillus casei

Lactobacillus casei has traditionally been recognized as a probiotic, thus needing to survive the industrial production processes and transit through the gastrointestinal tract before providing benefit to human health. The two-component signal transduction system (TCS) plays important roles in sensing and reacting to environmental changes, which consists of a histidine kinase (HK) and a response regulator (RR). In this study we identified HKs and RRs of six sequenced L. casei strains. Ortholog analysis revealed 15 TCS clusters (HK–RR pairs), one orphan HKs and three orphan RRs, of which 12 TCS clusters were common to all six strains, three were absent in one strain. Further classification of the predicted HKs and RRs revealed interesting aspects of their putative functions. Some TCS clusters are involved with the response under the stress of the bile salts, acid, or oxidative, which contribute to survive the difficult journey through the human gastrointestinal tract. Computational predictions of 15 TCSs were verified by PCR experiments. This genomic level study of TCSs should provide valuable insights into the conservation and divergence of TCS proteins in the L. casei strains.     

Inhibition of proton-translocating ATPases of Streptococcus mutans and Lactobacillus casei by fluoride and aluminum

One of the major effects of fluoride on oral bacteria is a reduction in acid tolerance, and presumably also in cariogenicity. The reduction appears to involve transport of protons across the cell membrane by the weak acid HF to dissipate the pH gradient, and also direct inhibition of the F₁F₀, proton-translocating ATPases of the organisms, especially for Streptococcus mutans. This direct inhibition by fluoride was found to be dependent on aluminum. The dependence on aluminum was indicated by the protection against fluoride inhibition afforded by the Al-chelator deferoxamine and by loss of protection after addition of umolar levels of Al³⁺, which were not inhibitory for the enzyme in the absence of fluoride. The F₁ form of the enzyme dissociated from the cell membrane previously had been found to be resistant to fluoride in comparison with the F₁F₀ membrane-associated form. However, this difference appeared to depend on less aluminum in the F₁ preparation in that the sensitivity of the F₁ enzyme to fluoride could be increased by addition of umolar levels of Al³⁺. The effects of Al on fluoride inhibition were apparent when enzyme activity was assayed in terms of phosphate release from ATP or with an ATP-regenerating system containing phosphoenolpyruvate, pyruvate kinase, NADH and lactic dehydrogenase. Also, Be²⁺ but not other metal cations, e.g. Co²⁺, Fe²⁺, Fe³⁺, Mn², Sn²⁺, and Zn²⁺, served to sensitize the enzyme to fluoride inhibition. The differences in sensitivities of enzymes isolated from various oral bacteria found previously appeared also to be related to differences in levels of Al. Even the fluoride-resistant enzyme of isolated membranes of Lactobacillus casei ATCC 4646 could be rendered fluoride-sensitive through addition of Al³⁺. Thus, the F₁F₀ ATPases of oral bacteria were similar to E₁E₂ ATPases of eukaryotes in being inhibited by Al-F complexes, and the inhibition presumably involved formation of ADP-Al-F _inf3 ^sup- complexes during catalysis at the active sites of the enzymes.