益生菌Lactobacillus casei Zhang增殖培养基的优化

Lactobacillus casei Zhang是一株分离自传统酸马奶中的益生菌。本文研究了不同碳源、氮源、碳氮比例、微量元素及缓冲盐对Lactobacillus casei Zhang增殖培养的效果, 并采用响应面法对优选的碳源、氮源和缓冲盐类的组成含量进行优化, 得到Lactobacillus casei Zhang的增殖培养基为：葡萄糖 20.9 g/L、大豆蛋白胨10.45 g/L、酵母粉10.45 g/L、K2HPO4 3.5 g/L、醋酸钠14.6 g/L、柠檬酸钠2.3 g/L、MgSO4?7H2O 1 g/L、MnSO4?5H2O 54 mg/L、CuSO4?5H2O 10 mg/L、吐温80为1 g/L。Lactobacillus casei Zhang在此增殖培养基中经37℃18 h培养活菌数可达到4.78×109 CFU/mL, 比在MRS中(4.8×108 CFU/mL)提高近10倍。     

Effects of <Emphasis Type="Italic">Lactobacillus buchneri</Emphasis> and <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> on fermentation,aerobic stability,bacteria diversity and ruminal degradability of alfalfa silage

Silages are important feedstuffs. Homofermentative lactic acid bacterial inoculants are often used to control silage fermentation. However, some research pointed out those homofermentative lactic acid bacteria (LAB) impaired the aerobic stability of wheat, sorghum, and corn silages. Adding heterofermentative LAB can produce more acetic acid, thereby stabilizing silages during aerobic exposure. Alfalfa is difficult to ensile. The present work was to study the effects of L. buchneri (heterofermentative LAB), alone or in combination with L. plantarum (homofermentative LAB) on the fermentation, aerobic stability, bacteria diversity and ruminal degradability of alfalfa silage. After 90 days ensiling, the pH, NH₃-N/TN, butyric acid content and molds counts of control were the highest. The inoculated silages had more lactic acid, acetic acid content and more lactic acid bacteria than the control. Inoculating LAB inhibited harmful microorganisms, such as Enterobacterium and Klebsiella pneumoniae. The L. buchneri + L. plantarum-inoculated silage had more acetic acid and less yeasts than other three treatments (P < 0.05), and lower NH₃-N/TN than control (P < 0.05). The CO₂ production of L. buchneri + L. plantarum-inoculated silage was less than that of L. plantarum-inoculated silage (P < 0.05). Inoculating LAB in alfalfa silages can decrease pH, increase the production of lactic and acetic acids, reduce the number of yeasts and molds, and inhibit Enterobacterium and K. pneumoniae. Inoculating with L. buchneri or L. buchneri + L. plantarum can improve aerobic stability of alfalfa silages. A combination of L. buchneri and L. plantarum is preferable because it enhanced alfalfa silage quality and aerobic stability.     

基于耐酸性与抑菌性效果的益生菌候选菌株体外筛选研究

目的 基于筛选耐酸性强和抑菌效果好的菌株为靶点,为益生菌研发提供优良的候选菌株。考察菌株耐酸性与抑菌性的相关性,为益生菌快速筛选探索新方法。方法 选取62株不同乳杆菌、魏斯菌、粪肠球菌,在酸性程度不同的培养基中采用接种培养的方式研究受试菌株耐酸能力。用大肠埃希菌和金黄色葡萄球菌作为指示菌,采用牛津杯法研究受试菌株的抑菌能力。分析同种细菌的耐酸能力和抑菌能力的相关关系。结果 在酸性环境下,受试菌株存活率超过50%的有22株,耐酸性较强的菌株有DMLA16017、DMLA16116、DMLA16117、DMLA16118和DMLA16131。受试菌株对病原菌的抑菌力较强的有DMLA16017、DMLA16009、DMLA16118、DMLA16002和DMLA15015。结论 鼠李糖乳杆菌DMLA16017和发酵乳杆菌DMLA16118在耐酸性和抑菌性上表现出优良性状。可以通过受试菌株对酸性耐受能力指示受试菌株对病原菌抑制能力,实现对受试菌株的快速筛选。     

Fermentation of a milk-soymilk and Lycium chinense Miller mixture using a new isolate of Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum

A milk–soymilk mixture was fermented using Lactobacillus paracasei subsp. paracasei NTU101 and Bifidobacterium longum BCRC11847 at different inoculum ratios (1:1, 1:2, 1:5, 2:1, and 5:1). When the inoculum ratio was 1:2, the cell numbers of both strains were balanced after 12 h of cultivation. The pH and titratable acidity were very similar at the various inoculum ratios of cultivation. The milk–soymilk mixture was supplemented with 5, 10, 15, and 20% Lycium chinense Miller juice and fermented with Lactobacillus paracasei subsp. paracasei NTU101 and B. longum BCRC11847. Sensory evaluation results showed that supplementation with 5% Lycium chinense Miller juice improved the acceptability of the fermented milk–soymilk. The fermented beverage was stored at 4°C for 14 days; variations in pH and titratable acidity were slight. The cell numbers of L. paracasei subsp. paracasei NTU101 and B. longum BCRC11847 in the fermented beverage were maintained at 1.2×10⁹ CFU/ml and 6.3×10⁸ CFU/ml, respectively, after 14 days of storage.     

Optimization of the green fluorescent protein (GFP) expression from a lactose-inducible promoter in Lactobacillus casei

An expression vector for Lactobacillus casei has been constructed containing the inducible lac promoter and the gene encoding ultraviolet visible green fluorescent protein (GFP(UV)) as reporter. Different conditions to grow L. casei were assayed and fluorescence as well as total protein synthesized were quantified. The maintenance of neutral pH had the greatest incidence on GFP(UV) expression, followed by aeration and a temperature of 30 degrees C. Environmental factors favoring GFP(UV) accumulation did not exactly correlate with those enhancing fluorescence. Therefore, oxygenation, by stirring the culture, had the greatest influence on the proportion of fluorescent protein, which is in accordance with the structural requirements of this protein. The highest yield obtained was 1.3 microg of GFP per mg of total protein, from which 55% was fluorescent.     

Isolation and characterization of bacteriocin‐producing bacteria from the intestinal microbiota of elderly Irish subjects

Aims

To isolate and characterize bacteriocins produced by predominant species of lactic acid bacteria from faeces of elderly subjects.

Methods and Results

Screening over 70 000 colonies, from faecal samples collected from 266 subjects, using the indicator organisms Lactobacillus bulgaricus LMG 6901 and Listeria innocua DPC 3572, identified 55 antimicrobial‐producing bacteria. Genomic fingerprinting following ApaI digestion revealed 15 distinct strains. The antimicrobial activities associated with 13 of the 15 strains were sensitive to protease treatment. The predominant antimicrobial‐producing species were identified as Lactobacillus salivarius, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus crispatus and Enterococcus spp. A number of previously characterized bacteriocins, including ABP‐118 and salivaricin B (from Lact. salivarius), enterocin B (Enterococcus faecium), lactacin B (Lact. acidophilus), gassericin T and a variant of gassericin A (Lact. gasseri), were identified. Interestingly, two antimicrobial‐producing species, not generally associated with intestinally derived microorganisms were also isolated: Lactococcus lactis producing nisin Z and Streptococcus mutans producing mutacin II.

Conclusion

These data suggest that bacteriocin production by intestinal isolates against our chosen targets under the screening conditions used was not frequent (0·08%).

Significance and Impact of the Study

The results presented are important due to growing evidence indicating bacteriocin production as a potential probiotic trait by virtue of strain dominance and/or pathogen inhibition in the mammalian intestine.     

Lactobacillus reuteri CRL 1098 prevents side effects produced by a nutritional vitamin B12 deficiency

Aims:  To evaluate the efficiency of the vitamin B_12-producing Lactobacillus reuteri CRL1098 strain in preventing the symptoms caused by a nutritional cobalamin-deficient diet in pregnant female mice and their weaned offspring.
Methods and Results:  Pregnant female mice were divided into three groups: animals fed with a B₁₂-deficient diet (DD), animals fed with DD plus L. reuteri CRL1098 and animals fed with a B₁₂-sufficient diet. The animals received the different feedings from the end of gestation up to weaning. At the end of the trials, they and their corresponding offspring were bled to determine haematological, immunological and histological parameters. The administration of the pseudovitamin B₁₂-producing strain prevented the symptoms observed in female and weaned young animals fed with a nutritional B₁₂-deficient diet.
Conclusions:  Our data suggest that the pseudovitamin B₁₂ produced by L. reuteri CRL1098 is biologically active and effective in preventing the pathologies caused by the nutritional deficiency of B₁₂ both in pregnant mice and their offspring.
Significance and Impact of the Study:  The ability of L. reuteri CRL1098 to prevent a nutritional vitamin deficiency was demonstrated for the first time. The addition of a GRAS micro-organism to complement the B₁₂ content in deficient foods is an interesting biotechnological alternative.     

Mode of antimicrobial action of vanillin against Escherichia coli, Lactobacillus plantarum and Listeria innocua

AIMS: To investigate the mode of action of vanillin, the principle flavour component of vanilla, with regard to its antimicrobial activity against Escherichia coli, Lactobacillus plantarum and Listeria innocua. METHODS AND RESULTS: In laboratory media, MICs of 15, 75 and 35 mmol l(-1) vanillin were established for E. coli, Lact. plantarum and L. innocua, respectively. The observed inhibition was found to be bacteriostatic. Exposure to 10-40 mmol l(-1) vanillin inhibited respiration of E. coli and L. innocua. Addition of 50-70 mmol l(-1) vanillin to bacterial cell suspensions of the three organisms led to an increase in the uptake of the nucleic acid stain propidium iodide; however a significant proportion of cells still remained unstained indicating their cytoplasmic membranes were largely intact. Exposure to 50 mmol l(-1) vanillin completely dissipated potassium ion gradients in cultures of Lact. plantarum within 40 min, while partial potassium gradients remained in cultures of E. coli and L. innocua. Furthermore, the addition of 100 mmol l(-1) vanillin to cultures of Lact. plantarum resulted in the loss of pH homeostasis. However, intracellular ATP pools were largely unaffected in E. coli and L. innocua cultures upon exposure to 50 mmol l(-1) vanillin, while ATP production was stimulated in Lact. plantarum cultures. In contrast to the more potent activity of carvacrol, a well studied phenolic flavour compound, the extent of membrane damage caused by vanillin is less severe. CONCLUSIONS: Vanillin is primarily a membrane-active compound, resulting in the dissipation of ion gradients and the inhibition of respiration, the extent to which is species-specific. These effects initially do not halt the production of ATP. SIGNIFICANCE AND IMPACT OF THE STUDY: Understanding the mode of action of natural antimicrobials may facilitate their application as natural food preservatives, particularly for their potential use in preservation systems employing multiple hurdles.     

乳杆菌DM9811发酵液提取物中RNA组分对人结肠癌细胞系HT-29增殖影响及分子机制研究

目的观察乳杆菌DM9811发酵液提取物中RNA组分对人结肠癌细胞系HT-29增殖的影响,探讨其对肠道肿瘤细胞的作用及其分子机制,为阐明乳杆菌与宿主相互作用规律的分子机制奠定基础。方法应用MTT方法研究不同时间不同浓度RNA组分对HT-29增殖的影响,应用流式细胞术、RT-PCR研究RNA组分对HT-29细胞周期的影响。结果乳杆菌DM9811发酵液中RNA组分能抑制HT-29细胞增殖,并呈现出时间-剂量依赖性;RNA组分作用于HT-29细胞24 h、48 h时,细胞周期G0/G1期所占比例明显上升,S期所占比例明显降低(P0.01),细胞周期调控因子CDK6、p27Kip1、p53的表达升高,CDK2、CDK4、PCNA的表达降低。结论乳杆菌DM9811代谢产物RNA在体外具有抑制癌细胞周期活性。