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%.     

Next issue (April 2007): Milk Processing

Edited by Prof. Knut Heller, Karlsruhe, Germany. Highlight articles: – Bacteriophages in dairy products: Pros and cons – Liberation of bioactive peptides from milk proteins – Lactobacillus delbrueckii subsp. bulgaricus for yoghurt with mild taste – Prebiotic galacto-oligosaccharides production using Lactobacillus reuteri – Lactobacillus kefiri biotransformation of pentoxifylline – Modification of milk proteins by microbial transglutaminase ... and much more: Read the next issue of BTJ!     

Exopolysaccharides production in Lactobacillus bulgaricus and Lactobacillus casei exploiting microfiltration

The physiology of Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus casei, extensively used in the dairy industry, was studied in order to evaluate key parameters in the synthesis of exopolysaccharides and to improve their production through novel fermentation processes. Selected strains were studied in shake flasks and in fermentor experiments using glucose and lactose as main carbon sources and bacto casitone as the only complex component, in a temperature range between 35 and 42°C. The production of exopolysaccharides was monitored and correlated to the growth conditions using both a colorimetric assay and chromatographic methods. Fermentor experiments in batch mode yielded 100 mg l⁻¹ of EPS from L. bulgaricus and 350 mg l⁻¹ from L. casei. Moreover, the use of a microfiltration (MF) bioreactor resulted in exopolysaccharides (EPS) concentrations threefold and sixfold those of batch experiments, respectively. The monosaccharidic composition of the two analyzed polymers differed from those previously reported. The optimization of the production of EPSs using the MF fermentation strategy could permit the use of these molecules produced by generally recognised as safe (GRAS) microorganisms in the place of other polysaccharides in the food industry.     

The effects of the novel bifidogenic trisaccharide,neokestose, on the human colonic microbiota

The potential prebiotic effect of the fructo-trisaccharide, neokestose, on intestinal bacteria was investigated. Bifidobacterium sp. utilized neokestose to a greater extend and produced more biomass from neokestose than facultative anaerobes under anaerobic conditions in batch culture. Lactobacillus salivarius utilized glucose but negligible amounts of neokestose. L. salivarius and the facultative anaerobes produced significantly more biomass from glucose than from neokestose, whereas the biomass yields obtained with bifidobacteria on neokestose and glucose, respectively, were not significantly different. Static batch cultures inoculated with faeces supported the prebiotic effect of neokestose, which had been observed in the pure culture investigations. Bifidobacteria and lactobacilli were increased while potentially detrimental coliforms, clostridia and bacteroides, decreased after 24 h fermentation with neokestose. In addition, this effect was more pronounced with neokestose than with a commercial prebiotic fructo-oligosaccharide. It was concluded that neokestose has potential as a novel bifidogenic substance and that it might have advantages over the commercially available sources currently used.     

Effect of medium composition on the ultrastructure of Lactobacillus strains

The growth of some locally isolated Lactobacillus strains forming D(-) or L(+) lactic acid, Lactobacillus helveticus ATCC 15009 and Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 was examined in different media. L. helveticus and Lactobacillus LBL strains formed atypical protoplast-like cells in LAPT medium, sensitive to SDS and proteinase. Specific morphological changes in the cell wall structure of these variants were revealed by transmission and scanning electron microscopy. The effect of glucose and various salts on their appearance was investigated. The prevalent role of metal cations, especially of Mg²⁺, was established.     

Response Surface Optimization of Lyoprotectant for Lactobacillus bulgaricus During Vacuum Freeze-Drying

The individual and interactive effects of skimmed milk powder, lactose, and sodium ascorbate on the number of viable cells and freeze-drying survival for vacuum freeze-dried powder formulation of Lactobacillus bulgaricus were studied by response surface methodology, and the optimal compound lyoprotectant formulations were gained. It is shown that skim milk powder, lactose, and sodium ascorbate had a significant impact on variables and survival of cultures after freeze-drying. Also, their protective abilities could be enhanced significantly when using them as a mixture of 28% w/v skim milk, 24% w/v lactose, and 4.8% w/v sodium ascorbate. The optimal freeze-drying survival rate and the number of viable cells of Lactobacillus bulgaricus were observed to be (64.41 ± 0.02)% and (3.22 ± 0.02) × 10¹¹ colony-forming units (CFU)/g using the optimal compound protectants, which were very close to the expected values 64.47% and 3.28 × 10¹¹ CFU/g.     

Multistrain probiotic production by co‐culture fermentation in a lab‐scale bioreactor

Most commercial probiotic products intended for pharmaceutical applications consist of combinations of probiotic strains and are available in various forms. The development of co‐culture fermentation conditions to produce probiotics with the correct proportion of viable microorganisms would reduce multiple operations and the associated costs. The aim of this study was to develop a fermentation medium and process to achieve biomass comprising the desired proportion of two probiotic strains in co‐culture. Initially, a quantification medium was developed, and the method was optimized to allow the quantification of each strain's biomass in a mixture. The specific growth rates of Lactobacillus delbrueckii spp. bulgaricus and Lactobacillus plantarum were determined in media with different carbon sources. The inoculum volume was optimized to achieve equal proportion of biomass in co‐culture fermentation in test tubes. Next, fermentation was carried out in a 3‐L bioreactor. A biomass concentration of 2.06 g/L, with L. delbrueckii spp. bulgaricus and L. plantarum in the ratio of 47%:53% (by weight), was achieved with concomitant production of 12.69 g/L of lactic acid in 14 h. The results show that with careful manipulation of process conditions, it is possible to achieve the desired proportion of individual strains in the final biomass produced by co‐culture fermentation. This process may serve as a model to produce multistrain probiotic drugs at industrial scale.     

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.     

The Structure of Destruxin B,a Toxic Metabolite of Oospora destructor

The viability of freeze-dried Lactobacillus bulgaricus B-1 was affected by rehydration temperature, and maximum recovery of the viable cells was obtained when they were rehydrated at 20 to 25°C. Cellular ribonucleotides leaked out from the freeze-dried cells during rehydration, but there was no correlation between the viability of cells and the amount of leaked substances. Rehydration of the freeze-dried cells in the presence of RNase caused marked loss of viability. These results suggest that the cell surface was damaged by freeze-drying and its selective permeability was lost to some extent.     

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.     

Frontiers in mycoplasma pathogenicity

Four different strains ofLactobacillus delbrueckii subsp.bulgaricus (Ss₁ and Yop₁₂) andStreptococcus salivarius subsp.thermophilus (Ss₂ and Yop₉) were isolated from two different yogurt sources in Argentina. In medium containing different carbon sources: lactose, fructose, sucrose or glucose plus fructose, the growth of a mixed culture (Yop₁₂+Ss₂) shows stimulation ofS. thermophilus and inhibition ofL. bulgaricus with respect to pure cultures. Both microorganisms in mixed culture grew less well on glucose plus galactose. However, in medium with glucose or galactose, both microorganisms were stimulated.     

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.     

保加利亚乳杆菌ATCC11842 L-乳酸脱氢酶同工酶基因的克隆与表达分析

通过对保加利亚乳杆菌(Lactobacillus delbrueckii subsp.bulgaricus)L-乳酸脱氢酶(L-lactate dehydrogenase, L-LDH)同工酶基因的异源表达、酶活测定和摇瓶发酵研究L-LDH在乳酸合成中的作用。将保加利亚乳杆菌ATCC11842中L-乳酸脱氢酶基因ldb0120和ldb0094分别克隆至载体pET28a(+)中,构建重组表达载体pET28aldb0120和pET28aldb0094,并转化到大肠埃希菌(Escherichia coli) BL21(DE3)中进行表达。进一步对重组蛋白进行Ni-NTA柱亲和层析和酶学活性测定,结果显示,LDB0120和LDB0094的比活力分别为0和25 U/mg,表明LDB0094是具有低活性的L-乳酸脱氢酶,而LDB0120不具有活性。对两株重组菌分别进行好氧和微好氧发酵,重组菌E.coli BL21/pET28aldb0094在好氧和微好氧条件可以合成L-乳酸,浓度分别为41.9和227.9 mg/L,而菌株E.coli BL21/pET28aldb0120在两种培养条件下均基本不合...     

Changes in the cell membrane of Lactobacillus bulgaricus during storage following freeze-drying

Summary The mechanism of inactivation of freeze-dried Lactobacillus bulgaricus during storage in maltodextrin under controlled humidity was investigated. Evidence is presented supporting the hypothesis that membrane damage occurs during storage. A study on the lipid composition of the cells by gas chromatography showed a decrease in the unsaturated and saturated fatty acid content of the cell. Further evidence indicating membrane damage includes a decrease in membrane bound proton-translocating ATPase activity.     

Effect of fermented oatmeal soup on the cholesterol level and theLactobacillus colonization of rat intestinal mucosa

Rats were fed with freeze-dried oatmeal soup fermented by six differentLactobacillus strains from rat and man; the formula is intended for enteral feeding. The serum cholesterol levels after 10 d were lower for rats eating oatmeal as compared to a commercial product, Biosorb Sond. Colonizing ability of the administered strains were evaluatedin vivo. OnlyLactobacillus reuteri R21c were able to, effectively, colonizing the mucosa; it represented about 30% of theLactobacillus population 24 d after termination of the administration.L. reuteri R21c was easily recognized by the ability to produce a yellow pigment on agar plates. The identity was confirmed by carbohydrate fermentations (API 50CH), plasmid pattern and endonuclease restriction analysis of the chromosomal DNA.     

Cholesterol removal by some lactic acid bacteria that can be used as probiotic

In the present study, the relationship between exopolysaccharide production and cholesterol removal rates of five strains of Lactobacillus delbrueckii subsp. bulgaricus isolated from home‐made yoghurt was studied. Test strains were selected according to their exopolysaccharide production capacity. Influence of different bile concentrations on cholesterol removal was investigated. It was confirmed that B3, ATCC 11842 and G11 strains which produce high amounts of exopolysaccharide (211, 200 and 159 mg/l, respectively) were able to remove more cholesterol from the medium compared to those that produce low amounts of exopolysaccharide (B2, A13). The highest cholesterol removal (31%) was observed by strain L. delbrueckii subsp. bulgaricus B3, producing a high amount of exopolysaccharide, in 3 mg/ml bile concentration. Cholesterol removal by resting and dead cells was investigated and it was found to be 4%–14% and 3%–10%, respectively. Cholesterol removal by immobilized and free cells of the B3 strain was studied and it was determined that immobilized cells are more effective. Influence of cholesterol on exopolysaccharide production has also been tested and it was found that cholesterol increased the production of EPS. The results indicated that: (i) there is a correlation between cholesterol removal and EPS production; and (ii) L. delbrueckii subsp. bulgaricus B3 is regarded as a suitable candidate probiotic and adjunct culture.     

Preventive Effect of Lactobacillus delbrueckii subsp. bulgaricus on the Oxidation of LDL

Lactobacillus delbrueckii subsp. bulgaricus 2038 was examined for its activity to prevent the oxidation of the erythrocyte membrane in vitro, and the oxidation of LDL in vivo.

Strain 2038 produced radical scavengers that reacted with 1,1-diphenyl-2-picrylhydrazl (DPPH) during cultivation. Moreover, the ethereal extract from the supernatant of the culture prevented the oxidation of the erythrocyte membrane in vitro.

As an in vivo study, male F344 rats were fed on diets containing 20% fresh soybean oil (or 13% oxidized oil and 7% fresh oil) with 10% freeze-dried powder of the 2038 culture (or with skim milk powder) for 4 weeks. The level of thiobarbituric acid-reactive substances was lower in the low-density lipoproteins (per milligram of cholesterol) from rats fed on the oxidized oil with freeze-dried powder of the 2038 culture than without it. The level of vitamin E in the plasma was higher in the rats fed on the oxidized oil with the freeze-dried powder than without it.     

Effect of incubation temperature on acid production and proteolytic activity in milk by selected lactobacilli mutants

Lactobacillus bulgaricus 59 andL. casei RTS and their respective nitrosoguanidine-induced mutants were estimated for titratable and volatile acidities, production of biacetyl and acetoin and for proteolytic activity. Both the mutants showed a marked increase in proteolytic activity as compared to the parents.     

Analysis of β-galactosidase production and their genes of two strains of <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis>

A bacterial β-galactosidase delivery system is a potential therapy for lactose intolerance. Currently, two Lactobacillus bulgaricus strains with different biological characteristics are under consideration as potential sources. However, differences in these β-galactosidase genes and their resulting production levels are poorly characterized. The β-galactosidase ORF of L. bulgaricus yogurt isolate had high variability and was terminated at site 1924 due to a stop codon. However, the full 114 kDa β-galactosidase band was still resolved by SDS-PAGE, which may indicate that the interrupted ORF was translated into more than one peptide, and they together were folded into the complete enzyme protein that showed much higher β-galactosidase activity (6.2 U/mg protein) than the enzyme generated from L. bulgaricus reference strain (2.5 U/mg protein).     

Studies on the Proteolytic Action of Dairy Lactic Acid Bacteria

In sterilized skim milk or sterilized 10% solution of dry skim milk at 120°C for 15 min, Lactobacillus bulgaricus, Lactobacillus helveticus and Streptococcus lactis were cultivated for 7 days at given temperature.

Both NCN (non casein type nitrogen) content and pH in each culture of lactic acid bacteria were rapidly decreased until 2 days after cultivation, But NCN content increased and the pH change got small after 3 days cultivation.

Caseins prepared from the cultures of these three kinds of lactic acid bacteria were examined electrophoretically. From the results of electrophoresis of these caseins, we have concluded that α-casein could be hydrolyzed by these lactic acid bacteria. And, it seemed that β-casein could not be hydrolyzed by these lactic acid bacteria.

Rennet easily hydrolyzed casein treated with L. bulgaricus and L. helveticus but hardly hydrolyzed that treated with S. lactis compared with control-casein. Caseins treated with L. bulgaricus and L. helveticus were hydrolyzed easier than control-casein.

Particle weights of caseins prepared from fermented milk by lactic acid bacteria, Streptococcus cremoris, Streptococcus lactis, Lactobacillus bulgaricus and Lactobacillus helveticus, and of hydrolyzed casein by rennet, trypsin or pepsin were measured according to the light scattering experiment.

Particle weights of various treated caseins were larger than that of raw native casein at both pH 7.0 and 12.0. And the heating caused the polymerization of casein to large particle.