植物乳杆菌Lactobacillu plantarumY1菌株胆盐水解酶基因(bsh)的克隆及重组表达

经同源蛋白比对分析设计引物,PCR扩增获得植物乳杆菌Y1菌株bsh基因(975 bp),首先克隆至表达载体pET-28a,转化E.coli BL21(DE3)菌株,IPTG诱导表达,SDS-PAGE分析结果显示表达的重组蛋白为包涵体.选择能为大肠杆菌稀有密码子提供额外tRNA的E.coli Rosetta(DE3)作为宿主菌,仍旧没有改善表达产物的可溶性.但是,选择含IF2融合蛋白标签的pLS-IF2质粒构建表达载体,SDS-PAGE分析及Western blot鉴定结果显示表达的融合重组蛋白IF2-BSH具有可溶性.该结果为进一步研究乳酸菌胆盐水解酶的生物活性,结构功能关系的研究奠定了基础.

羊源Lactobacillus plantarum对腹泻羔羊空肠菌群及肠道黏膜屏障的调控作用

旨在研究Lactobacillus plantarum对腹泻羔羊血清抗氧化能力和空肠菌群及空肠黏膜屏障损伤的调控作用。本试验选用24只刚断奶的羔羊,采用完全随机试验设计随机分为4组,分别为对照组(C组)、腹泻组(D组)、腹泻+抗生素处理组(DA组)、腹泻+植物乳杆菌处理组(DL组),每组6个重复,在正式试验第1天开始对D组、DA组和DL组羔羊分别灌服20 mL ETEC K99菌液,持续8 d。在第9天早上,DA组羔羊注射氟苯尼考,DL组羔羊灌服植物乳杆菌菌液,持续4 d。试验第1、9、13天空腹采集羔羊血液用于检测羔羊血清抗氧化指标以及二胺氧化酶(DAO)和D-乳酸(D-LA)浓度;屠宰后采集羔羊空肠内容物以测定菌群组成,采集空肠组织用于检测肠道黏膜屏障相关蛋白和黏膜蛋白,阿利新蓝(AB)染色研究酸性黏液的变化。结果表明:1)大肠杆菌处理8 d后,D、DA和DL三组羔羊粪便均不成形,且三组羔羊血清中DAO和D-LA浓度显著高于C组(P<0.05),说明腹泻模型构建成功;2)在第9天,与C组相比,D、DA和DL组羔羊血清中T-AOC、SOD显著降低(P<0.05),MDA含量显著升高(P<0.05);与第9天相比,DA和DL组羔羊在第13天时其抗氧化能力显著增加(P<0.05),DAO、D-LA浓度和MDA显著降低(P<0.05);3)与C组相比,D组羔羊空肠组织Occludin、Claudin1和MUC2蛋白表达量显著下降(P<0.05);与D组相比,DA组羔羊空肠组织Occludin蛋白表达量显著提高(P<0.05),但Claudin1蛋白和MUC2蛋白的表达量有所降低(P>0.05);DL组羔羊空肠组织Occludin蛋白、Claudin1蛋白以及MUC2蛋白表达量显著高于DA组;4)与C组相比,D和DA组羔羊空肠内容物中菌群Chao值和Shannon值均显著降低(P<0.05),DL羔羊空肠内容物中菌群Chao值和Shannon值有所增加(P>0.05)。C、D和DL组羔羊空肠中Firmicutes丰度均显著高于DA组(P<0.05)。C、D和DA组Actinobacteriota丰度都显著高于DL组(P<0.05)。D组羔羊空肠中Proteobacteria丰度略高于C和DL组(P>0.05),DA组羔羊空肠中Proteobacteria丰度显著高于C、D和DL组(P<0.05)。经相关性分析发现,与MUC2表达相关的微生物较多,Proteobacteria与空肠MUC2的表达显著负相关(P<0.05),Firmicutes和Verrucomicrobiota与空肠MUC2的表达显著正相关(P<0.05),NK4A214_group与MUC2表达量显著正相关(P<0.05),Escherichia-Shigella与MUC2的表达显著负相关(P<0.05)。综上表明,ETEC K99诱发羔羊腹泻,腹泻羔羊血清抗氧化能力和空肠内容物微生物多样性降低、空肠黏膜屏障蛋白表达量降低。相比于抗生素治疗,灌服Lactobacillus plantarum增加了空肠内容物菌群多样性,促进空肠酸性黏液及黏膜组织屏障蛋白的表达。总体而言,Lactobacillus plantarum对羔羊腹泻改善效果更佳。

没食子酸对植物乳杆菌Lactobacillus plantarum的抑制和促进生长

该文以微生物生长曲线、多酚浓度变化和培养液pH值变化为指标,研究了没食子酸(GallicAcid,GA)和表没食子儿茶素没食子酸酯(Epigallocatechin Gallate,EGCG)对植物乳杆菌CICC 6253生长的影响。结果表明GA和EGCG对植物乳杆菌生长的影响具有浓度依赖性,可表现出抑制和促进的双向调节作用,且二者的作用存在构效差异性。在GA和EGCG分别在5 mg/mL和7 mg/mL以上时,两者都抑制了植物乳杆菌的生长,而当GA和EGCG分别低于4 mg/mL和6 mg/mL时二者都促进了细胞的增殖,且随质量浓度增加其促进作用增大。GA可作为碳源被植物乳杆菌迅速利用,而EGCG(4 mg/mL)因其良好的细胞亲和性表现出显著的促进作用,高效地促进了植物乳杆菌细胞的增殖,菌落数在28h内由7.42lgCFU/mL增加到12.54lgCFU/mL,与空白对照组相比增加了2个数量级以上。本研究表明,GA和EGCG均表现出对植物乳杆菌的双向调节作用,其中EGCG表现出更佳的益生特性。本文研究结果可为GA和EGCG及类似食源性多酚在益生菌生长调节和相关食品研发中提供参考。

基于转录组学探究Lactobacillus plantarum 6A促进二乙基亚硝胺形成的机制

乳酸菌是食品工业和人体肠道常见微生物,可促进强致癌物亚硝胺的合成,而其促进机制尚待揭示,严重制约了发酵食品安全的提升和益生菌产品的推广。该文考察植物乳杆菌(Lactobacillus plantarum)6A对二乙基亚硝胺(N-nitrosodiethylamnie,NDEA)促进作用在不同环境中的变化趋势,以转录组技术进一步探究促进作用的关键基因和形成途径,构建其机制网络。结果表明,NDEA胁迫L.plantarum 6A通过上调嘌呤核苷酸合成/代谢(关键基因purL、purK、purC、purB、purA)、核黄素合成/代谢(ribBA、ribH、ribE)、双组分系统途径(dltD、dltB、dltA)的基因表达,产生NDEA前体物,如富含氮元素的嘌呤、核黄素及丙氨酸,从而促进NDEA形成;还可能通过核黄素合成/代谢以及双组分系统途径间接催化亚硝化反应,加速NDEA的形成。

Effect of dietary supplement of inactivated Lactobacillus plantarum Ep-M 17 on growth performance,immune response,disease resistance,and intestinal microbiota in Penaeus vannamei

Our previous study found that feeding with Lactobacillus plantarum Ep-M17 could effectively affect the growth performance,immune response,and gut microbiota of Penaeus vannamei.However,high temperature and pressure during feed pelletizing is the main problem that can lead to a decrease in the activity of probiotics or cause their inactivation.Further investigation needs to investigate whether inactivated Ep-M17 can exert similar effects as live Ep-M17.Therefore,we evaluated the effects of inactivated L.plantarum Ep-M17 on growth performance,immune response,disease resistance,and gut microbiota in P.vannamei.Results show that adding inactivated Ep-M17 to the feed also promoted body weight gain and increased relative immune protection in shrimp.Also,histological examination revealed that the administration of inactivated Ep-M17 led to improvements in the density and distribution of microvilli in the intestines and enhancements in the abundance of B and R cells in the hepatopancreas.Additionally,the inactivated Ep-M17 supplementation resulted in increased activity levels of nutrient immune-related enzymes in both the shrimp hepatopancreas and intestines.Moreover,it stimulated the expression of Lvlec,PEN-3a,Crustin,LGBP,Lysozyme,and proPo genes in both the hepatopancreas and intestines.Furthermore,the inactivated Ep-M17 also increased bacterial diversity in the gut of shrimp and promoted the abundance of specific flora,facilitating the host organism’s metabolism and immunity to improve the disease resistance of shrimp.Therefore,supplementation of inactivated L.plantarum Ep-M17 in shrimp diets can exert similar effects as live L.plantarum Ep-M17 effectively improving growth performance,gut microbiota,immune response,and disease resistance in P.vannamei.

Klebsiella as an α-tocopherol source facilitating Lactobacillus plantarum fermentation in rice straw silage

Silage serves as the indispensable diet of ruminants,the increasing mechanism of α-tocopherol during silage making is unclear.Rice straw lacks chlorophyll after harvesting the grain,this can eliminate the impact of tocopherols formed by the breakdown of chlorophyll.Here,we explored the α-tocopherol source,its influencing factor,and its relationship with dominant lactic acid bacteria in rice straw silage treated without or with different additives(sodium benzoate,Lactobacillus plantarum,cell wall degrading enzymes,the combination of L.plantarum and cell wall degrading enzymes)and vacuum times(5,8,11,and 14 s)after ensiling for 42 d.We found that the pathogenic Klebsiella was traced as the source of increasedα-tocopherol in rice straw silage.The residue air in the silo,pH value,and additive variety had impacts on Klebsiella activity,which was strongly active at levels of residue air in the silo and pH that were high.As an acidic niche creator,L.plantarum was more effective than sodium benzoate in restraining Klebsiella.Despite having a low acidity tolerance,Klebsiella was still present in rice straw silage treated with L.plantarum.The relationship between Klebsiella and L.plantarum was that Klebsiella could affordα-tocopherol to the multiplication of L.plantarum and residue capsular polysaccharide protected Klebsiella from escaping the extinction in rice straw silage.

Lactobacillus plantarum AR495 improves stress-induced irritable bowel syndrome in rats by targeting gut microbiota and Mast cell-PAR2-TRPV1 signaling pathway

Probiotics have great potential in regulating intestinal pain.In this study,the effects of Lactobacillus plantarum AR495 on the visceral sensitivity and gut microbiota of irritable bowel syndrome(IBS)rats were studied.The results showed that tryptase released after mast cell activation and degranulation plays a key role in visceral pain,and L.plantarum AR495 reduced the stimulation of colonic mast cells and the expression of protease-activated receptor 2(PAR2)and TRPV1 in dorsal root ganglia.Research further showed that supplementation with L.plantarum AR495 increased the level of short-chain fatty acids(SCFAs)and enhanced the barrier function of the colon.In addition,the microbiota analysis of the colon indicated that L.plantarum AR495 promoted the proliferation of Bifidobacterium and inhibited the proliferation of Lachnospiraceae,which alleviated the imbalance of the intestinal microbiota caused by IBS to a certain extent.In total,L.plantarum AR495 might reduce visceral sensitivity through the Mast cell-PAR2-TRPV1 signaling pathway by maintaining the homeostasis of the intestinal barrier.

Improved viability of trehalose on Lactobacillus plantarum embedded with whey protein concentrate/pullulan in simulated gastrointestinal conditions and its application in acid juice

Trehalose(TRE)was used to improve the gastrointestinal tolerance of Lactobacillus plantarum embedded with whey protein concentrate/pullulan(WPC/PUL)hydrogel and the embedded L.plantarum was applied to juice.The study indicated that 5%TRE significantly increased the viable counts of L.plantarum embedded in WPC/PUL hydrogel from(8.83±0.03)to(9.14±0.04)(lg(CFU/g))in simulated gastric juice(SGJ)and from(9.13±0.04)to(9.38±0.04)(lg(CFU/g))in simulated intestinal juice,respectively.The addition of TRE improved the glass transition temperature of WPC/PUL hydrogel and decreased the hardness and its solubility in SGJ,which may be responsible for the improved protection of WPC/PUL hydrogels on L.plantarum.In addition,TRE increased the viable counts of L.plantarum in WPC/PUL probiotic microcapsule juice at low pH and high temperature during storage.

Impact of Dietary Lactobacillus plantarum Postbiotics on the Performance of Layer Hens under Heat Stress Conditions

This experiment was conducted to determine the performance of heat-stressed layers fed a diet containing the probiotic Lactobacillus plantarum RS5 or its products of fermentation (postbiotics). Twenty-week-old Isa White layers, were subdivided into six treatments of 32 individually caged birds. Half of the birds were reared under regular temperature conditions, while the other half was subjected to cyclic daily heat stress. Layers were offered one of three diets: 1) Control;2) Control + Lactobacillus plantarum RS5 probiotic;3) Control + Lactobacillus plantarum RS5 postbiotics. Birds were tested for performance and visceral organ development for 5 months. Heat stress negatively affected the birds’ feed intake, egg weight, shell weight percentage, Haugh unit, shell thickness, yolk color, body weight and spleen weight percentage. Postbiotics significantly increased egg production (p < 0.05) in comparison to the control and the probiotic fed group (94.8% vs 92.6% vs 93.1%, respectively). Birds under probiotic or postbiotic diet showed a significantly higher (p < 0.05) feed intake and egg weight, although the probiotic had a more pronounced and gradual effect. Specific gravity, yolk weight percentage and shell thickness didn’t show differences among dietary groups. The Haugh Unit was significantly higher (p < 0.05) in probiotic group which also showed a significantly lower yolk color index (p < 0.05). The different feed treatments did not impact the bird’s viscera weight percentage, except for the ileum that was significantly lower (p < 0.05) under postbiotic supplementation. Both probiotics and postbiotics could be used as a potential growth promoters and might alleviate heat stress impact in poultry industry.

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(P<0.05);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(P<0.05),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(P<0.05)。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。

植物乳杆菌(Lactobacillus plantarum)NDC75017产γ-氨基丁酸的影响因素及其代谢机制

为探索植物乳杆菌(Lactobacillus plantarum)NDC75017产γ-氨基丁酸(gamma-aminobutyric acid,GABA)的影响因素及相关代谢机制,首先采用响应面法,对菌体积累GABA的发酵条件进行优化。其次,分别采用高效液相色谱和实时荧光定量反转录聚合酶链式反应(real-time reverse transcript polymerase chain reaction)测定了不同时间点GABA产量和谷氨酸脱羧酶(glutamic acid decarboxylase,GAD)编码基因gad B的相对表达量。L.plantarum NDC75017发酵过程中产GABA的最佳底物(谷氨酸钠)、辅酶(5’-磷酸吡哆醛)的添加量和发酵初始p H值分别为68.03 mmol/L、20.92μmol/L和4.65。底物与辅酶之间、辅酶与发酵初始p H值之间的交互作用能够显著地影响GABA的积累(P<0.05)。发酵48 h内,基因gad B表达量呈现先升高后下降的趋势,与GABA产量的变化趋势并不完全一致。上述结果表明:底物、辅酶和发酵初始p H值以及它们之间的交互作用是影响L.plantarum NDC75017产生GABA的关键因素。同时GABA的积累不仅受到GAD和gadB的影响,其他与GABA支路(GABA shunt)相关的酶和基因也参与了GABA的代谢。

植物乳杆菌Lactobacillus plantarum GUO安全性研究

对实验室筛选得到一株具有降胆固醇作用的植物乳杆菌GUO(Lactobacillus plantarum GUO)进行致突变性试验和毒力试验,以测定该菌株的食用安全性。致突变性试验结果表明:在加大鼠肝微粒体酶S9和不添加的条件下,与自发回变菌落数的差异皆不显著,且与剂量效应无关,而阳性对照的回变菌落数均显著增加(P<0.05),说明Lactobacillus plantarum GUO无致突变作用;毒力试验结果表明:Lactobacillus plantarum GUO低、高浓度培养液与各自对照组相比,对小鼠体重增加的影响无统计学意义(P>0.05),且受试小鼠无毒力性反应或死亡。

利用PCR-DGGE技术监测Lactobacillus plantarum WCFS1对小鼠肠道菌群的动态变化

利用传统培养法和变性梯度凝胶电泳技术(PCR-DGGE)评价Lactobacillus plantarum WCFS1对小鼠肠道微生物数量和种类的影响。结果表明,乳酸杆菌数量在灌胃期和恢复期较空白期显著升高(P<0.05),而肠杆菌数量在灌胃期和恢复期较空白期显著下降(P<0.01);DGGE图谱及多样性分析显示小鼠灌胃期间菌群多样性显著提高(P<0.01),但在停止灌胃7d后与空白期相比无显著性差异(P>0.05)。此外,DGGE条带测序表明Lacto-bacillus plantarum WCFS1在灌胃期间可在大部分小鼠肠道内占据优势地位。

Lactobacillus plantarum 163产细菌素食品级培养基筛选及发酵条件优化

为获得Lactobacillus plantarum 163最佳食品级培养基,首先筛选出Lb.plantarum 163食品级培养基配方,即白菜汁200?m L/L、番茄汁50?m L/L、葡萄糖10?g/L、K2HPO4 2?g/L,蒸馏水补足至1?L。Plackett-Burman试验设计筛选出Lb.plantarum 163食品级培养基配方的关键因子,即接种量、K2HPO4添加量、p H值和大白菜汁添加量。通过Box-Behnken试验构建了Lb.plantarum 163食品级培养基二次多项式模型,得到理想发酵条件,即K2HPO4 1.89?g/L、大白菜汁341.5?m L/L、接种量3.56%、p H?6.95,其抑菌活性比优化前增加30%以上。

植物乳杆菌Lactobacillus plantarum(FJAT-7926)生物学特性研究

分离到1株植物乳杆菌FJAT-7926,并对其进行生物学特征研究,结果表明:该乳酸菌的最适生长温度为37℃,最适pH为6。接种量为3%,培养36h后,pH达到3.98,产乳酸量为25.4g·L-1。应用该菌株发酵大豆豆浆,研究发酵后的植物性乳酸菌饮品特性,结果显示:饮品乳酸菌含量高达1×109cfu·mL-1,感官评价良好。

Streptococcus thermophilus S10复合Lactobacillus plantarum P-8在发酵豆乳中的应用

以商业豆乳发酵剂A和B为对照组,Streptococcus thermophilus S10和Lactobacillus plantarum P-8(S10+P-8)复合发酵豆乳为实验组,对42℃发酵过程及4℃贮藏期间活菌数、酸度、黏度、持水性、活性大豆异黄酮含量、基本味觉特征和质构特性进行测定,对感官进行品鉴。结果表明:3种样品到达发酵终点的时间、酸度、黏度和持水性差异不显著;发酵结束后,实验组和发酵剂A组样品中活性大豆异黄酮含量变化显著(P <0. 05);发酵完成到贮藏结束实验组样品益生菌Lactobacillus plantarum P-8活菌数高于1. 0×109CFU/g;贮藏结束时,样品pH在4. 30左右,滴定酸度均低于74. 00°T; 6种基本滋味和3种回味的相对强度及差异性分析表明:3种样品的苦味、涩味、咸味和甜味差异显著(P <0. 05);贮藏过程中,实验组感官得分显著高于对照组(P <0. 05),28 d时为93. 7分。豆乳发酵剂S10+P-8在发酵豆乳制品开发中具有良好的应用潜力。

植物乳杆菌(Lactobacillus plantarum)8-6产细菌素发酵条件的优化

对植物乳杆菌(Lactobacillus plantarum)8-6产细菌素的发酵条件进行了优化,分别研究了培养时间、温度、接种量、培养基起始pH值、培养基碳源、氮源等因素对细菌素产生的影响,通过单因素水平试验和正交试验,确定产细菌素的最佳培养基组合和最佳发酵条件为葡萄糖3%,胰蛋白胨2%,蛋白胨1%,酵母膏1%,硫酸镁0.058%,吐温-80 0.2%,30℃培养24h,培养基起始pH值为6.5,接种量2%。乳杆菌8-6优化后效价为1825.56IU/mL,比优化前提高了373.15%。

益生菌Lactobacillus plantarum4在豆乳中发酵特性的评价

以大豆为原料,利用益生菌Lactobacillus plantarum4发酵豆乳,通过测定豆乳在发酵及贮藏期间酸度、蛋白水解能力、活菌数、脱水收缩性等指标,对L.plantarum 4的发酵特性及贮藏稳定性进行研究,同时比较不同处理方法对大豆抗营养因子(脂肪氧化酶和胰蛋白酶抑制剂)的抑制作用。结果显示,L.plantarum4在豆乳中生长良好,发酵结束时,滴定酸度为68.91°T,活菌数可达到2.33×108 CFU/mL,蛋白水解能力和持水力都显著增加,感官品质较佳;低温贮藏时各指标均无明显变化,贮藏稳定性明显优于室温贮藏;同时得出热磨法是抑制大豆抗营养因子的最佳方法。研究表明L.plantarum4具备益生菌所应有的发酵特性,为其进一步应用于实际生产提供了理论依据。

植物乳杆菌Lactobacillus plantarum 15对不同品种柑橘全果汁的发酵及其挥发性物质分析

采用植物乳杆菌Lactobacillus plantarum 15对蜜桔、胡柚、脐橙和春见4种不同品种柑橘全果汁进行深度发酵,比较其发酵过程中主要成分的变化规律及其发酵前后挥发性物质。结果显示,植物乳杆菌L. plantarum15发酵过程中4种全果汁p H呈现基本稳定-快速下降-逐渐平缓的趋势,伴随着总酸含量的增加,总糖、还原糖和总黄酮等含量总体上下降。采用顶空固相微萃取气质联用法(HS-SPME-GC-MS),检出蜜桔、胡柚、脐橙和春见4种全果汁及发酵全果汁的挥发性物质共46种,均以柠檬烯为主;植物乳杆菌L. plantarum 15发酵明显降低了己醛、辛醛、癸醛、乙醇和芳樟醇等相对含量,但并未能丰富其酯类和酮类。研究表明,原料品种明显影响着柑橘全果汁的发酵进程和挥发性物质。相比于胡柚、脐橙和春见3种全果汁,蜜桔全果汁更适合作为植物乳杆菌L. plantarum 15的发酵基质。

Lactobacillus plantarum M616生长及代谢特性研究

植物乳杆菌(Lactobacillus plantarum)是酸面团中优势菌群之一,为提供植物乳杆菌M616在酸面团发酵中应用的理论依据,采用比浊方法测定该菌株的生长曲线、产酸能力以及耐酸、耐盐、耐胆盐特性,并对其抑菌性能进行分析。结果表明:植物乳杆菌M616为革兰氏阳性菌,细胞呈直或弯短杆状;在37℃时MRS摇瓶中发酵30 h,p H 3.89,具有良好的产酸能力;在pH值为2.0、3.0的条件下培养6 h,OD600 nm分别为0.03、0.05;胆盐质量浓度为0.3 g/100 m L时培养4 h,OD600 nm为0.03;当NaCl质量分数达到8%以上时,菌株几乎不能生长。在抑菌方面,该菌株对大肠杆菌、金黄色葡萄球菌以及单增李斯特菌均有抑制效果,抑菌圈直径分别达14、15、17 mm。因此,植物乳杆菌M616具备较强的产酸能力,代谢性能良好,可作为酸面团发酵用菌株。