1株拮抗水稻纹枯病菌鼠乳杆菌的筛选与鉴定

采用平板对峙法从浙江省金华市水稻田土壤中筛选获得1株拮抗水稻纹枯病菌的细菌QT-0304菌株,最大拮抗带宽达到20 mm。根据形态特征、生理生化特征、16S rRNA基因比对及进化树构树分析,鉴定QT-0304菌株为鼠乳杆菌(Lactobacillus murinus)。抗菌谱实验结果表明:QT-0304菌株对苹果腐烂病菌(Valsa mali)和杨树溃疡病病菌(Botryosphaeria dothidea)均有较好的抑制作用,拮抗带宽分别达到34.5 mm和23.0 mm。

乳酸菌抗热保护剂的优化组合

以干酪乳杆菌(L.casei)、鼠李乳杆菌和3号菌为目标菌株,对喷雾干燥乳酸菌粉抗热保护剂进行了研究。结果:高温驯化后干酪乳杆菌、鼠李乳杆菌和3号菌最高适应温度为50℃,通过L9(34)正交试验,得出干酪乳杆菌高温驯化抗热保护剂最佳配比是在MRS液体培养基中添加明胶1.5 g/dL、蔗糖6 g/dL、甘油3 mL/dL;鼠李乳杆菌的最佳配比为:明胶1 g/dL、蔗糖4 g/dL、甘油4 mL/dL;3号菌的最佳配比为明胶1.5 g/dL、蔗糖6 g/dL、甘油5 mL/dL。结论:此条件下得到的复合抗热保护剂可以很好的保护干酪乳杆菌、鼠李乳杆菌和3号菌的最佳存活率,以满足将进一步进行的喷雾干燥法制备乳酸菌粉发酵剂的需要,所生产的乳酸菌粉的活性能够达到生产泡椒的要求,进而缩短了泡椒发酵的时间,保证了泡椒的良好风味。

Construction and Application of Plasmid pUC19-CM-D

[Objective] The aims were to construct a new suicide plasmid of Lactobacillus and gene deletion engineering bacteria of Lactobacillus with pUC19 vector. [Methods] pUC19-CM was constructed by inserting a chloramphenicol resistant gene into the multi-cloning site of pUC19,and then two homologous fragments were cloned into each side of the pUC19-CM to construct suicide plasmid pUC19-CM-D. [Results] A replacement mutant strain,whose target gene was replaced by resistant gene,could be obtained by transforming the suicide plasmid pUC19-CM-D into Lactobacillus for resistance screening. [Conclusion] The construction and application of pUC19-CM-D provided a fast and efficient means of construction of gene deletion engineering bacteria of Lactobacillus,and laid a foundation for study of gene function of Lactobacillus.

Effects of the viability of Lactobacillus rhamnosus GG on rotavirus infection in neonatal rats

AIM:To study the effects of live and dead Lactobacillus rhamnosus GG(GG) on rotavirus infection in a neonatal rat model.METHODS:At the age of 2 d,suckling Lewis rat pups were supplemented with either live or dead GG and the treatment was continued daily throughout the experi-ment.At the age of 5 and 6 d the pups received oral rotavirus(RV) SA-11 strain.The pups were sacrificed at the age of 7 or 8 d by decapitation.The gastrointestinal tract was removed and macroscopic observations were done.The consistency of feces in the colon was classified using a four-tier system.RV was detected from the plasma,small intestine,colon and feces by real-time quantitative polymerase chain reaction(PCR).RESULTS:In this neonatal rat model,RV induced a mild-to-moderate diarrhea in all except one pup of the RV-inoculated rats.RV moderately reduced body weight development from day 6 onwards.On day 7,after 2 d of RV infection,live and dead GG groups gained significantly more weight than the RV group without probiotics [36%(P = 0.001) and 28%(P = 0.031),respectively].In addition,when compared with the RV control group,both live and dead GG reduced the weight ratio of colon/animal body weight to the same level as in the healthy control group,with reductions of 22%(P = 0.002) and 28%(P < 0.001),respectively.Diarrhea increased moderately in both GG groups.However,the diarrhea incidence and severity in the GG groups were not statistically significantly different as compared with the RV control group.Moreover,observed diarrhea did not provoke weight loss or death.The RV control group had the largest amount of RV PCR-positive samples among the RV-infected groups,and the live GG group had the smallest amount.Rats receiving live GG had significantly less RV in the colon(P = 0.027) when compared with the RV control group.Live GG was also more effective over dead GG in reducing the quantity of RV from plasma(P = 0.047).CONCLUSION:Both live and dead GG have beneficial effects in RV infection.GG may increase RV clearance from the body and reduce colon swelling.

Efficacy and safety profile of LCR35 complete freeze-dried culture in irritable bowel syndrome:A randomized,double-blind study

AIM:To assess the effects and safety of Lactobacillus casei rhamnosus LCR35 complete freeze-dried culture(LCR35) in patients suffering from irritable bowel syndrome(IBS).METHODS:A randomized,double-blind pilot study was performed in 50 patients complaining of IBS symptoms complying with RomeⅢcriteria.Patients were allocated to receive either LCR35(n = 25) at a minimum daily dose of 6 × 108 colony forming units or placebo(n = 25) for 4 wk.At inclusion,after treatment and 2 wk later,patients completed the IBS severity scale.Change from baseline in the IBS severity score at the end of treatment was the primary efficacy criterion.Changes were compared between groups in the whole population and in IBS subtypes(IBS with predominance of constipation,IBS with predominance of diarrhoea,mixed IBS,unsubtyped IBS).The presence of lactobacillus casei rhamnosus in stools was investigated at inclusion and at the end of treatment.The gastrointestinal quality of life questionnaire and the hospital anxiety and depression(HAD) scale were also completed.RESULTS:Both groups were balanced for baseline characteristics.In 85% of patients,stool analyses showed that lactobacillus casei rhamnosus able to survive in the digestive tract.In the whole population,improvements in the IBS severity score did not differ significantly between treatments with a 25% decrease after 4-wk treatment,and a 15% decrease from baseline 2 wk later in both groups.In IBS subgroups,statistical analysis could not be performed due to small sample size,but a clinical response in favour of LCR35 was observed in IBS patients with predominance of diarrhoea:no change in the symptom severity score was seen with the placebo after 4 wk treatment,whereas a clinically relevant decrease occurred with LCR35(-37% vs-3%).Furthermore,in spite of an increase in symptom intensity,the IBS severity score was maintained below the baseline value 2 wk later with LCR35(-19% from baseline),whilst a slight 5% increase from baseline was observed with placebo.In the IBS subgroup with predominance of diarrhoea only,a clinically relevant decrease in abdominal pain severity score(-36%)was observed with LCR35,whereas no change occurred with placebo.In mixed IBS patients,the 20% and 30% decreases in the IBS severity score observed after treatment with LCR35 and placebo,respectively,were maintained 2 wk later in both groups.A clinical response slightly in favour of placebo was observed at the end of the treatment period in IBS patients with predominance of constipation(-41% vs-20%) and unsubtyped IBS patients(-47% vs-17%),with the same value maintained 2 wk later.In both groups,no clinically relevant changes were observed either for the gastrointestinal quality of life index or HAD score.Thus,these results suggest that sub-grouping of IBS patients may be important for optimizing treatment responses by the physician.CONCLUSION:This pilot study suggests that LCR35 could have some efficacy in IBS patients complaining of diarrhoea.These preliminary results need to be conf irmed in larger studies.

Probiotic Lactobacillus rhamnosus downregulates FCER1 and HRH4 expression in human mast cells

AIM:To investigate the effects of four probiotic bacteria and their combination on human mast cell gene expression using microarray analysis.METHODS:Human peripheral-blood-derived mast cells were stimulated with Lactobacillus rhamnosus (L.rhamnosus) GG (LGG),L.rhamnosus Lc705 (Lc705),Propionibacterium freudenreichii ssp.shermanii JS (PJS) and Bifidobacterium animalis ssp.lactis Bb12 (Bb12) and their combination for 3 or 24 h,and were subjected to global microarray analysis using an Affymetrix GeneChip Human Genome U133 Plus 2.0 Array.The gene expression differences between unstimulated and bacteria-stimulated samples were further analyzed with GOrilla Gene Enrichment Analysis and Visualization Tool and MeV Multiexperiment Viewer-tool.RESULTS:LGG and Lc705 were observed to suppress genes that encoded allergy-related high-affinity IgE receptor subunits α and γ (FCER1A and FCER1G,respectively) and histamine H4 receptor.LGG,Lc705 and the combination of four probiotics had the strongest effect on the expression of genes involved in mast cell immune system regulation,and on several genes that encoded proteins with a pro-inflammatory impact,such as interleukin (IL)-8 and tumour necrosis factor alpha.Also genes that encoded proteins with anti-inflammatory functions,such as IL-10,were upregulated.CONCLUSION:Certain probiotic bacteria might diminish mast cell allergy-related activation by downregulation of the expression of high-affinity IgE and histamine receptor genes,and by inducing a pro-inflammatory response.

Immobilization of Lactobacillus rhamnosus TISTR108 on Crude Pectin of Krung Kha Mao Leaves （Cissampe/os pareira L.） to Produce Lactic Acid in Longan Juice

L-（＋）-lactic acid production was studied by immobilized Lactobacillus rhamnosus T1STR108 on crude pectin from Krung Kha Mao Leaves. Central composite design was employed to determine the maximum lactic acid production of 42.88 g L-1 in predicted model with the factors at 4.11 g L1 of pectin, 6.05 mLLl inoculum and 1.09 mm of bead diameter. Statistical analyses demonstrated very high significance for the regression model, since the F-value computed 116.09 was much higher than the tabulated F-value 2.08 for the lactic acid production at 5% level for linear and quadratic polynomial regression models. The highest experimental lactic acid production was 43.57 g L^-1 at 96 h of fermentation, 1.58% higher than the predicted value.

Lactobacillus crispatus M206119 exacerbates murine DSS-colitis by interfering with inflammatory responses

AIM： TO investigate the role of Lactobacillus crispatus （L. crispatus） strain China Center for Type Culture Col- lection （CCTCC） M206119 in intestinal inflammation.METHODS： Forty 8-wk-old Balb/c mice （20± 2 g） were divided into four groups of 10 mice each. Three groups that had received dextran sulfate sodium （DSS） were administered normal saline, sulfasalazine or CCTCC M206119 strain, and the fourth group received none of these. We assessed the severity of colitis using a disease activity index, measured the colon length and weight, collected stools and mesenteric lymph nodes for bacterial microflora analysis. One centimeter of the proximal colon, middle colon and distal colon were collected and fixed in 10% buffered formalin, dehydrated in ethanol, and embedded in paraffin. Interleukin （IL）- 1β, IL-6 and tumor necrosis factor （TNF）-α expression was detected using reverse transcription polymerase chain reaction. Protective factors zonula occludens （ZO）-1 and β-defensin 2 were detected by immunoblot-ting. The features of CCTCC M206119 strain were identified based on morphology, biochemical profile, and 16S RNA sequencing.RESULTS： DSS-colitis animals treated with CCTCC M206119 had markedly more severe disease, with greater weight loss, diarrhea, fecal bleeding, and shortened colon length. In addition, the CCTCC-M206119- treated group had comparatively higher histologi- cal scores and more neutrophil infiltration than the controls. Expression of protective factors ZO-1 and β-defensin 2 was downregulated due to destruction of the mucosal barrier after CCTCC M206119 strain treatment. An in vitro assay demonstrated that CCTCC M206119 strain increased the nuclear translocation of nuclear factor-κB in epithelial cells. Intestinal proinflam- matory or anti-inflammatory cytokine responses were evaluated. Proinflammatory colonic cytokine （IL-Iβ, IL-6 and TNF-α） levels were clearly increased in CCTCC- M206119-treated animals, whereas anti-inflammatory colonic cytokine （IL-10） level was lowered compared with saline or 5-aminosalicylic-acid-treated DSS-colitis mice. Next, CCTCC M206119 strain was characterized as 1. crispatus by microscopic morphology, biochemical tests and 16S rRNA gene level.CONCLUSION： Not all lactobacilli are beneficial for in- testinal inflammation, and L. crispatus CCTCC M206119 strain is involved in exacerbation of intestinal inflamma- tion in DSS-colitis mice.

Effects of Probiotic Lactobacillus gasseri Strains of Breast-Fed Infant in a Murine Model

The ingestion of probiotic lactic acid bacteria has been evaluated and noted that it has an effect on the balance of desirable microbiota in the gastrointestinal tract. Lactobacillus gasseri demonstrates good survival in the gastrointestinal tract, and it has been associated with a variety of probiotic activities and roles, including the reduction of fecal mutagenic enzymes, the production of bacteriocins and the stimulation of macrophages immunomodulation. The aim of the study was to evaluate the effects of a pool of L. gasseri strains isolated from the feces of breastfed infants added in the human milk of healthy women. The milk was both pasteurized and unpasteurized, to verify the cell cytotoxicity of macrophages and to quantify the production of immunologic mediators such as IL-4, IL-6, IFN-γ, TNF-α, NO and oxygen intermediary compounds （H2O2）. The administration of raw human milk and pasteurized human milk to infants is a regular, encouraged practice in units of intensive therapy （UITs） and our present investigation verified the beneficial effect of addition of a pool of L. gasseri to pasteurized human milk （PHML）. Our results show that probiotic supplementation helped to maintain cell viability, reduced IL-6 and IFN-γ production and stimulated TNF-α, NO, H2O2, IL-4 production. Nevertheless, the results indicate that the addition of lactobacillus to human milk was not a determinant in the production of TNF-α. L. gasseri added to breast milk did not present a cytotoxic risk, and the addition ofL. gasseri to pasteurized milk of human milk bank would benefit newborns that depend on milk banks for the colonization of more desirable microbiota.

Lactic Acid Production from Longan Juice （Euphora Iongana Lam.） by Crude Pectin of Immobilized Lactobacillus rhamnosus TISTRI08

L-（＋）-lactic acid production was studied by immobilized Lactobacillus rhamnosus TISTR108 on crude pectin from Krung Kha Mao （Cissampelospareira L.） leaves. Central composite design was employed to determine the maximum lactic acid production of 45.40 g/L in predicted model （Y = 43.98 - 2.43X1 ＋ 1.02X2 ＋ 2.96X3 - 8.72X1^2 - 3.99X2^2 - 1.74X3^2） with the factors at 5.9 of cultural medium pH, 37.6 ℃ of process temperature and 202 rpm of liquid agitation. Statistical analyses demonstrated very high significance for the regression model fitted the data adequately and explained the lactic acid production, since the F-value computed 54.89 was much higher than the tabulated F-value 2.08 for the lactic acid production at 5% level for linear and quadratic polynomial regression models. The highest experimental lactic acid production was 46.91 g/L at 72 h of fermentation.

Antimicrobial Activity of Probiotics on Swiss Mice

Salmonella enterica serovar typhimurium is the first cause identified of food poisoning in humans; new data indicate that the resistant strains of Salmonella sp. can be inhibited by a bacterial population present in foods have been called probiotics. Our study was conducted on Swiss albino mice in order to know the effect of probiotics or biotherapeutic agents, and suggest the different therapeutic possibilities of intestinal complications related to the presence of Salmonella enterica serovar typhimurium; Reference strains, Lactobacillus delbrueckii subsp, bulgaricus, Lactobacillus acidophilus and Lactobacillus paracasei in combination with Bifidobacterium sp. were tested for their health benefits. The number of Salmonella enterica serovar typhimurium decreases in intestine, in the stomach and in the liver for group 2, 3 in case of preventive treatment and group 4, 5 in case of a therapeutic treatment due to the use of a cuRure of probiotics, in comparison with group 1 where we recorded a high number. The bio-therapeutic treatment was finally confirmed by macroscopic and microscopic observations of different target organs before and after treatment for the experiment period.

清肠化湿汤对肠道细菌增殖影响的体外研究

目的:探索清肠化湿汤对5种肠道细菌(双歧杆菌、鼠李唐乳杆菌、大肠杆菌、金黄色葡萄球菌、沙门氏菌)体外增殖与抑制效果,为清肠化湿汤治疗溃疡性结肠炎提供实验依据。方法:针对溃疡性结肠炎活动期紊乱的肠道菌群,采用光电比色法测定清肠化湿汤对双歧杆菌、鼠李唐乳杆菌益生菌的增殖的影响;同时通过试管二倍稀释法测定清肠化湿汤对大肠杆菌、金黄色葡萄球菌、沙门氏菌致病菌最低抑菌浓度(MIC)与最低杀菌浓度(MBC)。结果:清肠化湿汤可明显促进双歧杆菌、鼠李唐乳杆菌增殖,抑制大肠杆菌、金黄色葡萄球菌及沙门氏菌,并有效杀灭大肠杆菌、金黄色葡萄球菌。结论:清肠化湿汤可通过促进益生菌生,抑制并杀灭致病菌,恢复肠道菌群平衡,治疗溃疡性结肠炎。

Oral administration of Lactobacillus rhamnosus GG to newborn piglets augments gut barrier function in pre-weaning piglets

To understand the effects of Lactobacillus rhamnosus GG(ATCC 53103)on intestinal barrier function in pre-weaning piglets under normal conditions,twenty-four newborn littermate piglets were randomly divided into two groups.Piglets in the control group were orally administered with 2 mL 0.1 g/mL sterilized skim milk while the treatment group was administered the same volume of sterilized skim milk with the addition of viable L.rhamnosus at the 1st,3rd,and 5th days after birth.The feeding trial was conducted for 25 d.Results showed that piglets in the L.rhamnosus group exhibited increased weaning weight and average daily weight gain,whereas diarrhea incidence was decreased.The bacterial abundance and composition of cecal contents,especially Firmicutes,Bacteroidetes,and Fusobacteria,were altered by probiotic treatment.In addition,L.rhamnosus increased the jejunal permeability and promoted the immunologic barrier through regulating antimicrobial peptides,cytokines,and chemokines via Toll-like receptors.Our findings indicate that oral administration of L.rhamnosus GG to newborn piglets is beneficial for intestinal health of pre-weaning piglets by improving the biological,physical,and immunologic barriers of intestinal mucosa.