Cultural conditions for the production of bacteriocin by a native isolate of Lactobacillus delbruecki ssp. bulgaricus CFR 2028 in milk medium

The effect of growth parameters and the molecular basis for antibacterial activity by a natural isolate of Lactobacillus delbruecki ssp. bulgaricus CFR 2028 was studied. The inhibition was tested against a toxigenic strain of Bacillus cereus F 4810. When grown in milk medium, the activity was highest at an incubation temperature of 37 °C in 48 h. The antibacterial activity appeared to be produced between late logarithmic and early stationary phases. The active principle was proteinaceous in nature (bacteriocin) and stable to low pH (3·8–5·0) and heat (75 °C for 30 min). There was also the possible role of hydrogen peroxide in bringing about inhibition. The strain of Lact. delbruecki ssp. bulgaricus CFR 2028 revealed the presence of plasmid DNA bands of 9·4 and 6·5 kbp, respectively, in agarose gel electrophoresis. The above strain has the potential to be used as a biopreservative in popular Indian fermented foods.     

Cryoprotectants lead to phenotypic adaptation to freeze-thaw stress in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T

This study was conducted to investigate the ability of cryoprotective chemicals to induce phenotypic cryoadaptation in Lactobacillus delbrueckii ssp. bulgaricus CIP 101027T. Tolerance to negative temperature stress (freezing at -20 degrees C and thawing at 37 degrees C) was induced by pretreatment with Me(2)SO, glycerol, lactose, sucrose, and trehalose. Interestingly, Me(2)SO has a significantly greater cryoprotective effect than glycerol. Furthermore, lactose, sucrose, and trehalose, often referred to as osmotica, were shown to have greater cryoadaptive than cryoprotective properties. These results suggest that bacteria such as L. delbrueckii ssp. bulgaricus could be phenotypically adapted to freezing and thawing by an osmotic stress applied prior to freeze-thaw stress.     

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

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

郑州部分市售酸奶中乳酸菌的分离鉴定与益生特性的研究

目的调查郑州部分市售的酸奶中嗜热乳酸链球菌与保加利亚乳酸杆菌存在情况与益生特性。方法无菌采取样品进行乳酸菌分离,并对分离菌进行形态学、生化特性、产酸性、耐酸性、耐胆汁性、高温耐受性和抑菌性等生物学特性研究。结果从8种品牌中分离到4株保加利亚乳酸杆菌、7株嗜热链球菌,且分离菌株均能产酸、对高温具有一定的耐受性,在pH 3.0以上均能生长良好,而且分离菌的抑菌物质具有一定的热稳定性,保加利亚乳酸杆菌经70、100℃热处理抑菌活性基本不变。嗜热链球菌经70℃热处理后保留90%以上的抑菌活性,但经100℃高温处理后抑菌活性有所降低。分离菌产生的抑菌物质对胰蛋白酶敏感,保加利亚乳酸杆菌在偏中性条件下抑菌活性较高,而嗜热链球菌在偏中性条件下丧失抑菌活性。结论本研究为河南市售酸奶质量以及为消费者提供有利信息奠定基础。     

An X-prolyl dipeptidyl aminopeptidase from Lactococcus lactis: cloning, expression in Escherichia coli, and application for removal of N-terminal Pro-Pro from recombinant proteins

A novel pepX gene was cloned from isolated DNA of Lactococcus lactis by PCR. The deduced amino acid sequence of the 89-kDa protein showed 94, 93, 65, and 44% identity with the pepX protein from Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, Lactobacillus delbruecki subsp. bulgaricus, and Lactobacillus helveticus, respectively, and contained a serine protease G-K-S-Y-L-G consensus motif. The pepX gene has been cloned into pET17b and was expressed at a high level in Escherichia coli BL21 (DE3) LysS. PepX was purified to approximate homogeneity with ammonium sulfate precipitation and DEAE Sephadex A-50 chromatography. Optimal pepX activity was observed at pH 8.0 and 37 degrees C. According to SDS-PAGE analysis, pepX has a molecular mass of approximately 89 kDa. The peptidase can remove completely the unwanted X-Pro from the N-terminal of the target protein, releasing the naturally active protein and peptide, revealing a prospective application of pepX in large-scale production of pharmaceutical protein and peptide products.     

Effects of protective agents on membrane fluidity of freeze-dried Lactobacillus delbrueckii ssp. bulgaricus

AIMS: To evaluate the effect of protective agents upon survival of Lactobacillus delbrueckii ssp. bulgaricus during freeze-drying and storage, and selective amino acids on cell membrane fluidity. METHODS AND RESULTS: The protective effect of amino-acids and sugars at different concentrations was studied by determining the viability of lyophilized cells after storage under air at 30 degrees C. Survival following freeze-drying was improved by all compounds. During storage, neither proline nor maltose had protective effects on lyophilized Lact. delbrueckii ssp. bulgaricus. Glutamate 5% and aspartate 5% showed similar protection capability during freeze-drying (94-95%) and after storage (92-99%). Fluorescence probes (DPH and TMA-DPH) were used to study the effect of both amino acids on membrane fluidity. Polarization decreased with increasing concentrations of glutamate or aspartate. Lowest values were observed with TMA-DPH. CONCLUSIONS: Glutamate 5% and aspartate 5% allowed maintaining high viability rates during freeze-drying and storage of Lact. delbrueckii ssp. bulgaricus because of an increase of the membrane fluidity by inserting in the interfacial region of bacterial plasma membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These results show the first evidence of the mechanisms underlying glutamate and aspartate as lyoprotectors.     

Glutamate uptake in Lactobacillus delbrueckii subsp. bulgaricus CNRZ 208 and its enhancement by a combination of Mn2+ and Mg2+

AIMS: To demonstrate the mechanism of glutamate uptake in the dairy strain Lactobacillus delbrueckii subsp. bulgaricus CNRZ 208, and to characterize key aspects of the system. METHODS AND RESULTS: Glutamate uptake proceeded via an active transport system requiring an exogenous source of energy. The system also transported aspartate and glutamine. It was unique, with a Kt of 2.8 micro mol l-1 and a Vmax of 900 micro mol s-1 (g dry weight)-1. The activity was optimal at pH 7.3 and 50 degrees C, was independent of the glutamate charge, and was enhanced by Mn2+ + Mg2+ in combination. Inhibition of the activity by uncouplers and ionophores showed that transport was driven by an ATP-dependent mechanism involving the proton-motive force. This inhibition was partially abolished in the presence of both Mn2+ and Mg2+. CONCLUSIONS: We demonstrated for the first time that an active transport system governs the uptake of the essential amino acid glutamate in Lact. delbrueckii subsp. bulgaricus CNRZ 208, the activity of which is enhanced by a combination of Mn2+ and Mg2+. SIGNIFICANCE AND IMPACT OF THE STUDY: The potential of the findings is discussed with reference to the growth of Lact. delbrueckii subsp. bulgaricus in mixed-strain cultures for the dairy industry.     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.     

Structural diversity and endotoxic activity of the lipopolysaccharide of Yersinia pestis

The endotoxic activity of the lipopolysaccharides (LPS) with defined chemical structure from Yersinia pestis strains of various subspecies differing in their epidemic potential was studied. The LPS of two strains of Y. pestis ssp. caucasica and ssp. altaica, whose structures have not been studied earlier, were analyzed by high-resolution mass spectrometry. In addition to reported structural changes, an increase in the degree of LPS phosphorylation was observed when strain I-2377 (ssp. altaica) was cultivated at an elevated temperature. A high tumor necrosis factor alpha(TNF-alpha)-inducing activity observed for LPS samples from Y. pestis cultures grown at 25 degrees C correlated with an increased degree of lipid A acylation, particularly, with the presence of the hexaacyl form of lipid A, which was absent from the LPS when bacteria were cultivated at 37 degrees C. No correlation was found between the lethal toxicity of the LPS in vivo and its ability to induce TNF-alpha production in vitro.     

Combined influence of fermentation and drying conditions on survival and metabolic activity of starter and probiotic cultures after low-temperature vacuum drying

The influence of low temperature vacuum drying process parameters on the survival, metabolic activity and residual water content of three different bacterial strains (Lactobacillus paracasei ssp. paracasei, Lactobacillus delbrueckii ssp. bulgaricus and Bifidobacterium lactis) was investigated. Shelf temperature and chamber pressure were varied and optimized by response surface methodology with regard to survival and residual water content. It is shown that the survival rate after low temperature vacuum drying is comparable to that of freeze drying. Based on the optimization experiments the combined influence of fermentation pH and drying process parameters was studied for the most detrimental and the best process condition, respectively. The results show that interactions between process and fermentation conditions have to be taken in account and that these influences are highly strain specific.     

The biodiversity of predominant lactic acid bacteria in dolo and pito wort for the production of sorghum beer

AIM: To quantify and identify the predominant lactic acid bacteria (LAB) in dolo and pito wort processing, and to examine their biodiversity at strain level. MATERIALS AND RESULTS: The processing of dolo and pito wort was studied at four production sites in Burkina Faso and Ghana. The succession of dominant micro-organisms, pH and titratable acidity were determined from sorghum malt through mashing and acidification to final wort. In the sorghum malt and during mashing, the LAB counts were 5.7-7.5 log CFU g(-1). Similar levels of yeasts and gram-negative, catalase-positive bacteria were observed. These levels decreased to 3.7-4.5 log CFU g(-1) and相似文献   

Media and process parameters affecting the growth, strain ratios and specific acidifying activities of a mixed lactic starter containing aroma-producing and probiotic strains

AIMS: The effects of medium-composition and fermentation parameters on the properties of mixed mesophilic starters were studied. The starter was composed of Lactococcus lactis ssp. lactis (L. lactis), Lactococcus lactis ssp. cremoris (L. cremoris), Lactobacillus rhamnosus (Lact. rhamnosus) and Leuconostoc mesenteroides ssp. cremoris (Leuc. cremoris). METHODS AND RESULTS: The media used were reconstituted skim milk (RSM), and whey-based media with either citrate or phosphate buffers. The fermentation parameters were incubation temperature (22 degrees C or 32 degrees C), no pH control, and pH control in pH zones of either pH 6.0-5.8 or pH 6.0-5.2. The starter properties were strain ratio, specific acidifying activity (SAA), total population, residual carbohydrates and organic acids produced. The growth of L. lactis was favoured under pH control in whey-based media. High concentrations of Lact. rhamnosus were favoured in whey-based media prepared at 32 degrees C. The highest contents of Leuc. cremoris were obtained in starters prepared in RSM at 22 degrees C without pH control. Starters prepared under pH control gave the highest populations and made it possible for significantly lower inoculation rates (IR) to be used to carry out subsequent milk fermentations. However, the SAA of starters prepared under pH control were lower than the SAA of starters grown without any pH control. CONCLUSIONS: None of the conditions enabled the strain ratio at inoculation to be maintained. The data show that it is possible to prepare a mesophilic starter that has a significant probiotic Lact. rhamnosus content; this starter could be used in the preparation of probiotic-containing cheeses or in Leuc. cremoris for aroma production in fermented milks. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data on what should be expected with respect to strain ratios and IR if cheesemakers decide to shift their aroma-producing starter production method from the traditional 'milk-based without pH control' method to whey-based media used with pH-zone control strategies.     

Partial purification and characterization of protease of Bacillus proteolyticus CFR3001 isolated from fish processing waste and its antibacterial activities

Bacillus proteolyticus CFR3001 isolated from fish processing wastes (both fresh water and marine) produced an alkaline protease. The optimum conditions for cell growth and protease production were 37 degrees C, 96 h, agitation speed of 100 rpm and medium pH 9. The partially purified protease obtained from had specific activity of 22.05 at 37 degrees C was active between 40 degrees C and 50 degrees C and lost >20% of its activity around 60 degrees C. Its molecular weight was approximately 29 kDa and it inhibited the growth of several pathogenic organisms such as Escherichia coli, Listeria monocytogenes, Bacillus cereus and Yersinia enterocolytica. The scanning electron microscopy (SEM) studies revealed that the protease produced by B. proteolyticus CFR3001 lysed the cells of these pathogenic bacteria.     

Exopolysaccharides produced by lactic acid bacteria of kefir grains

A Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with high exopolysaccharide activity was selected from among 40 strains of lactic acid bacteria, isolated from kefir grains. By associating the Lactobacillus delbrueckii subsp. bulgaricus HP1 strain with Streptococcus thermophilus T15, Lactococcus lactis subsp. lactis C15, Lactobacillus helveticus MP12, and Sacharomyces cerevisiae A13, a kefir starter was formed. The associated cultivation of the lactobacteria and yeast had a positive effect on the exopolysaccharide activity of Lactobacillus delbrueckii subsp. bulgaricus HP1. The maximum exopolysaccharide concentration of the starter culture exceeded the one by the Lactobacillus delbrueckii subsp. bulgaricus HP1 monoculture by approximately 1.7 times, and the time needed to reach the maximum concentration (824.3 mg exopolysacharides/l) was shortened by 6 h. The monomer composition of the exopolysaccharides from the kefir starter culture was represented by glucose and galactose in a 1.0:0.94 ratio, which proves that the polymer synthesized is kefiran.     

Thermostability of allicin determined by chemical and biological assays

The garlic-derived antibacterial principle, alk(en)yl sulfinate compounds, has long been considered as very short-lived substance. However, there are some data showing a rather more stable nature of allicin. We determined here the thermostability of allicin by a systematic analyses employing chemical quantification and an antibacterial activity assay. Allicin in an aqueous extract of garlic was degraded stoichiometrically in proportion to the temperature; we estimated the half-life of allicin to be about a year at 4 degrees C (from 1.8 mg/ml to 0.9 mg/ml) and 32 d at 15 degrees C, but only 1 d at 37 degrees C (from 2.0 mg/ml to 1.0 mg/ml). The half-life values for antibacterial activity showed a similar trend in results: 63 d or more at 4 degrees C for both antibacterial activities, 14 d for anti-staphylococcal activity, and 26 d for anti-escherichia activity at 15 degrees C, but only 1.2 d and 1.9 d for the respective activities at 37 degrees C. Such antibacterial activities were attributable to the major allicin, allyl 2-propenylthiosulfinate. Surprisingly, the decline in the quantity of allicin was not accompanied by its degradation; instead, allicin became a larger molecule, ajoene, which was 3-times larger than allicin.     

Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus

A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested.     

Thuricin: the bacteriocin produced by Bacillus thuringiensis

Bacillus thuringiensis serovar, thuringiensis (HD-2) demonstrated antibacterial activity against 48 of 56 strains of B. thuringiensis and against some other Gram-positive species but not against Gram-negative species. The antibacterial activity was not inducible by mitomycin C or by ultraviolet irradiation, and additional activity was not liberated from cells by sonication. Upon dilution of the antibacterial substance, zones of inhibition diminished without the appearance of plaques. Gel filtration chromatography indicated an Mr greater than 950,000 for the bacteriocin (thuricin) in its native form. The native thuricin was sedimented by ultracentrifugation, but electron microscopy of the pellet failed to reveal phage particles or phage components. Nondenaturing polyacrylamide gel electrophoresis (PAGE) of thuricin demonstrated the association of bacteriocin activity with a protein band which migrated only slightly into a 5% gel. Sodium dodecyl sulfate (SDS)-PAGE of partially purified thuricin revealed five major bands. Thuricin activity was substantially reduced by treatment with chymotrypsin, pronase, subtilisin, trypsin, and heat at 96 degrees C but not by treatment with lysozyme, phospholipase C, papain, peptidase, or organic solvents. It exhibited a bactericidal and bacteriolytic effect on a sensitive strain, B. thuringiensis serovar, canadensis (MF4). Partially purified preparations of thuricin had phospholipase A activity which was adsorbed by sensitive cells but not by cells which were insensitive to thuricin. Antibacterial activity was blocked by preincubation of thuricin with phospholipid. Loss of a 150-mDa plasmid was correlated with loss of thuricin production.     

Action of trehalose on the preservation of Lactobacillus delbrueckii ssp. bulgaricus by heat and osmotic dehydration

AIM: This work determines the efficiency of trehalose on the preservation by heat or osmotic drying of a strain of Lactobacillus delbrueckii ssp. bulgaricus. Cell recovery at different trehalose concentrations during drying correlated with the surface properties and osmotic response of cells after rehydration. METHODS AND RESULTS: Bacteria were dried in the presence of glycerol, trehalose, sucrose at 70 degrees C and at 20 degrees C. Trehalose attenuates the loss of viability at 0.25 m. At this concentration, the osmotic response and zeta potential of the bacteria were comparable with the nondried ones. CONCLUSIONS: Trehalose diminishes significantly the damage produced by dehydration both when the bacteria are dried by heating or subjected to osmotic dehydration. This effect appears related to the preservation of the permeability to water and the surface potential of the bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Dehydration occurring during heating or during osmosis appears to have similar effects. As dehydration-induced damage is in correlation with osmotic response recovery and is hindered or buffered by the presence of trehalose, it may be related to water eliminated from biological structures involved in water permeation.     

Antibacterial properties of serum from the American alligator (Alligator mississippiensis)

Treatment of alligator (Alligator mississippiensis) and human serum samples with Escherichia coli resulted in a time- and concentration-dependent inhibition of bacterial proliferation. When inoculated with E. coli, alligator serum exhibited 10-fold lower bacterial survival rates after 1 h than human serum. In addition, the antibacterial spectrum of alligator serum was shown to be much broader than that of human serum, with growth inhibition occurring in 100% of bacterial strains tested (compared to only 35% for human serum). Additional results showed that the antimicrobial activities of alligator serum could be completely inhibited by preincubation with proteases, indicating the proteinaceous nature of the antimicrobial activities. Furthermore, incubation of alligator serum at 56 degrees C for 30 min (classical human serum complement inactivation conditions) obliterated all antimicrobial properties of the alligator serum. The antibacterial activities occurred relatively quickly in vitro, with significant activity occurring within 5 min of inoculation with E. coli and maximal activity at 20 min. Also, the antimicrobial activity exhibited temperature dependence, with a substantial decrease in activity below 15 degrees C. These data suggest that the antimicrobial properties of alligator serum may be due to an active serum complement system.