Sorbitol synthesis by an engineered Lactobacillus casei strain expressing a sorbitol-6-phosphate dehydrogenase gene within the lactose operon

Sorbitol is claimed to have important health-promoting effects and Lactobacillus casei is a lactic acid bacterium relevant as probiotic and used as a cheese starter culture. A sorbitol-producing L. casei strain might therefore be of considerable interest in the food industry. A recombinant strain of L. casei was constructed by the integration of a d-sorbitol-6-phosphate dehydrogenase-encoding gene (gutF) in the chromosomal lactose operon (strain BL232). gutF expression in this strain followed the same regulation as that of the lac genes, that is, it was repressed by glucose and induced by lactose. (13)C-nuclear magnetic resonance analysis of supernatants of BL232 resting cells demonstrated that, when pre-grown on lactose, cells were able to synthesize sorbitol from glucose. Inactivation of the l-lactate dehydrogenase gene in BL232 led to an increase in sorbitol production, suggesting that the engineered route provides an alternative pathway for NAD(+) regeneration.     

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.     

Thymine transport in Lactobacillus casei

Abstract A transport system for thymine was investigated in a Lactobacillus casei mutant lacking thymidine phosphorylase activity (the first enzyme required for thymine utilization). Transport was dependent on an energy supply; transport was inhibited by the uncouplers carbonylcyanide-m-chlorophenylhydrazone (CCCP) and 2,4 dinitrophenol (DNP), and also by sodium azide and N,N'-dicyclohexylcarbodiimide (DCCD). Thymine transport was inhibited by some uracil and adenosine derivatives and by thymidine, but was not affected by guanosine, deoxycytidine or azacytidine. Inhibition by p-chloromercurybenzoate (PCMB) was reversed by dithiothreitol (DTT).     

Lactobacillus casei NY1301 Increases the Adhesion of Lactobacillus gasseri NY0509 to Human Intestinal Caco-2 Cells

In the presence of Lactobacillus casei NY1301, the adhesion of Lactobacillus gasseri NY0509 to cultured human intestinal Caco-2 cells was significantly increased (P<0.01). In contrast, L. gasseri NY0509 did not affect the adhesion of L. casei NY1301. A heat-stable cell component of L. casei NY1301 was involved in this increase of adhesion. These results suggest that a combination of these strains may have synergistic effects of adhesion to human intestinal mucosa.     

环丙沙星预先处理牙鲆消化道对干酪乳杆菌定植的影响

利用1株干酪乳杆菌,通过实验研究用环丙沙星预先处理牙鲆消化道后乳杆菌的定植和演替规律。在投喂含有1.2×10^9CFU/g乳杆菌的饲料5 d后,消化道定植的乳杆菌超过106CFU/g,其后维持在10^6～10^8CFU/g动态平衡中。同时随着乳杆菌的投喂,不经环丙沙星预先处理牙鲆消化道的正常组,鱼消化道的弧菌数从10^7～8CFU/g降低到10^6CFU/g左右;而经环丙沙星预先处理牙鲆消化道的药饵组,鱼胃、小肠和盲囊的弧菌数则是先增加,然后显著下降。停喂乳杆菌7 d后,2个实验组鱼消化道的乳杆菌均从10^5～6CFU/g下降到10^4CFU/g,干酪乳杆菌正常组鱼盲囊中弧菌从10^5CFU/g回升到10^6CFU/g,胃和小肠中弧菌数量基本不变。干酪乳杆菌药饵组则有所不同：除胃中弧菌数量则基本不变外,盲囊和小肠中弧菌数量继续下降,其原因有待进一步研究。实验结果表明,干酪乳杆菌能在牙鲆消化道内定植,而用预先处理牙鲆消化道后,更有利于乳杆菌的定植;乳杆菌的投喂和定植,使牙鲆消化道中的弧菌数量明显下降。     

Inducible resistance against nisin in Lactobacillus casei

Most strains of Lactobacillus casei tested were found to be nisin-resistant. The addition of nisin to a growing culture of a resistant strain stopped growth for several hours; however, growth then resumed at the previous rate. Nisin induced a resistance mechanism that was lost by one passage in nisin-free medium. During induction with nisin, the cells produced an anionic, phosphate-containing polysaccharide with the subunits rhamnose and galactose. This polysaccharide protected sensitive cells of L. casei against the bactericidal action of nisin. Received: 27 July 1995 / Accepted: 30 October 1995     

Cell wall modifications during osmotic stress in Lactobacillus casei

AIMS: To study the modification of the cell wall of Lactobacillus casei ATCC 393 grown in high salt conditions. METHODS AND RESULTS: Differences in the overall structure of cell wall between growth in high salt (MRS + 1 mol l(-1) NaCl; N condition) and control (MRS; C condition) conditions were determined by transmission electronic microscopy and analytical procedures. Lactobacillus casei cells grown in N condition were significantly larger than cells grown under unstressed C condition. Increased sensitivity to mutanolysin and antibiotics with target in the cell wall was observed in N condition. Purified cell wall also showed the increased sensitivity to lysis by mutanolysin. Analysis of peptidoglycan (PG) from stressed cells showed that modification was at the structural level in accordance with a decreased PG cross-link involving penicillin-binding proteins (PBP). Nine PBP were first described in this species and these proteins were expressed in low percentages or presented a modified pattern of saturation with penicillin G (Pen G) during growth in high salt. Three of the essential PBP were fully saturated in N condition at lower Pen G concentrations than in C condition, suggesting differences in functionality in vivo. CONCLUSIONS: The results show that growth in high salt modified the structural properties of the cell wall. SIGNIFICANCE AND IMPACT OF STUDY: Advances in understanding the adaptation to high osmolarity, in particular those involving sensitivity to lysis of lactic acid bacteria.     

益生菌Lactobacillus casei Zhang的研究简况

<正>用于发酵乳制品生产的益生乳酸菌菌种和发酵剂的研制对于我国相关产业的生存与发展至关重要。为了建立具有我国自主知识产权的益生乳酸菌菌种和发酵剂的相关技术,张和平等人分离并系统研究了一株     

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。     

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培养活茵数可达到4.78×109 CFU/mL,比在MRS中(4.8×108 CFU/mL)提高近10倍.     

Regulation of the synthesis and activity of thymidine phosphorylase in Lactobacillus casei

Abstract: Lactobacillus casei cells grown on excess thymine or on folic acid contained low levels of thymidine phosphorylase. On the other hand, thymine starved cells and also cells of a thymidine-monophosphate-kinase-defective mutant grown on excess thymine, possessed derepressed levels. These results suggest that the synthesis of thymidine phosphorylase is regulated by the end product of the thymidine-triphosphate-biosynthetic pathway. L. casei cells lacked 2-deoxyribose-1-phosphate-mutase activity and did not grow on 2-deoxyribose or thymidine as the sole-carbon source. Growth in the presence of thymidine did not result in induction of thymidine-phosphorylase synthesis, probably due to the inability of the cell to convert it to 2-deoxyribose-5-phosphate, which is known to act as an inducer in E. coli cells. Thymidine triphosphate inhibited non-competitively the activity of thymidine phosphorylase. It was also inhibited by dihydrofolic acid.     

Enhanced immune response to pneumococcal infection in malnourished mice nasally treated with heat-killed Lactobacillus casei

The present study analyzed whether nasal administration of viable and non-viable Lactobacillus casei CRL 431 to immunocompromised mice was capable of increasing resistance against Streptococcus pneumoniae. Weaned mice were malnourished after consuming a PFD for 21 days. Malnourished mice were fed a BCD for 7 days or BCD for 7 days with viable or non-viable L. casei nasal treatments on day 6 and day 7 (BCD+LcV and BCD+LcN, respectively). The MNC group received PFD whereas the WNC mice consumed BCD. MNC mice showed greater lung colonization, more severe lung injuries, impaired leukocyte recruitment and reduced antibodies and cytokine production when compared with WNC mice. Administration of L. casei increased the resistance of malnourished mice to the infection. Both BCD+LcV and BCD+LcN treatments prevented the dissemination of the pathogen to the blood and induced its lung clearance. BCD+LcV or BCD+LcN groups showed improved production of TNF-α and activity of phagocytes in the respiratory tract, an effect that was not observed in the BCD control group. In addition, IL-4 and IL-10 were significantly increased in BCD+LcV and BCD+LcN groups, which correlated with the increase in the levels of specific respiratory IgA. The nasal treatments with L. casei were also effective at stimulating the production of specific IgG at both the systemic and the respiratory levels. The comparative study between the viable and the non-viable bacteria demonstrated that viability would be an important factor to achieve maximum protective effects. However, the results from this study suggest that heat-killed lactic acid bacteria are also effective in the immunomodulation of the systemic and respiratory immune system.     

Construction of a food-grade host/vector system for Lactococcus lactis based on the lactose operon

Abstract A plasmid-based food-grade vector system was developed for Lactococcus lactis by exploiting the genes for lactose metabolism. L. lactis MGS267 is a plasmid-free strain containing the entire lactose operon as a chromosomal insertion. The lacF gene was deleted from this strain by a double cross-over homologous recombination event. The lacF -deficient strain produced a Lac⁻ phenotype on indicator agar. A cloned copy of the lacF gene expressed on a plasmid was capable of complementing the lacF -deficient strain resulting in a Lac⁺ phenotype. This stably maintained system fits the requirements of a self-selecting vector system and has the potential to be exploited in the food industry.     

Response of Lactobacillus casei BL23 to phenolic compounds

Aims: To determine the inhibitory effect of phenolic compounds on Lactobacillus casei BL23, the role of two component signal transduction systems (TCS) and the response of Lact. casei BL23 to p‐coumaric acid. Methods and Results: Growth of Lact. casei BL23 and 17 derivative strains defective in each TCS harboured by this strain in the presence of p‐coumaric acid, ferulic acid, caffeic acid or methyl gallate was monitored. Furthermore, changes in the protein content of Lact. casei BL23 when exposed to p‐coumaric acid were evaluated by 2D‐SDS‐PAGE. Eleven proteins differentially expressed in the presence of p‐coumaric acid were detected. Six of them could be identified: ClpP and HtrA, involved in protein turnover and folding, acetyl‐CoA carboxylase, involved in lipid metabolism, and an arginyl‐tRNA synthetase were more abundant, whereas PurL and PurN, involved in purine biosynthesis, were less abundant. Conclusions: No significant differences were observed between the parental strain and the TCS‐defective mutants. p‐Coumaric acid elicited a response against membrane and cytoplasmic damages. Significance and Impact of the Study: The inhibitory effect of phenolic compounds on Lact. casei BL23 has been determined. For the first time, cytoplasmic proteins presumably involved in the response of Lact. casei BL23 against p‐coumaric acid have been identified.     

益生乳酸菌Lactobacillus casei Zhang和Lactobacillus plantarum P8对全价饲料pH及微生物类群变化的研究

研究了益生乳酸菌干酪乳杆菌Zhang(Lactobacillus casei Zhang)和植物乳杆菌P8(Lactobacillus planta-rum P8)对全价饲料pH及微生物类群变化的影响。分别将L.casei Zhang、L.plantarum P8单一菌种及复合菌种(11)以6.30 lg cfu/g的接种总量发酵全价饲料,测定25℃10 d发酵期间全价饲料pH和微生物类群的变化,应用选择培养基测定发酵饲料中的乳酸菌及杂菌(酵母菌、霉菌、大肠菌群、芽胞杆菌和梭状芽胞杆菌)的动态变化,应用RT-PCR技术测定试验组中的L.casei Zhang和L.plantarum P8的动态变化。结果显示,试验组pH下降显著,发酵10 d时,L.casei Zhang、L.plantarum P8单一菌种和复合菌种发酵饲料的pH分别为4.23、4.24和4.22,显著低于对照组(P0.05);L.casei Zhang、L.plantarum P8单一菌种和复合菌种发酵饲料中的L.casei Zhang、L.plantarum P8活菌数分别为8.91、8.89、6.58和8.69 lg cfu/g。发酵期间,试验组中酵母菌、霉菌、大肠菌群、芽胞杆菌及梭状芽胞杆菌活菌数显著低于对照组(P0.05),其中L.plantarum P8单一菌种发酵和复合菌种发酵对杂菌抑制效果显著优于L.casei Zhang单一菌种发酵(P0.05)。结果表明,全价饲料经L.casei Zhang、L.plantarum P8发酵可以显著降低其pH,抑制其中杂菌的生长,同时L.casei Zhang、L.plantarum P8在饲料中具有良好的稳定性。