1株产细菌素植物乳杆菌的筛选及其细菌素抑菌性质研究

从广西巴马地区取样的米粉中共分离出31株乳酸菌,以单核细胞增生李斯特菌为指示菌,采用牛津杯平板扩散法检测抑菌物质,筛选得到1株产细菌素的乳酸菌M23。通过微生物形态学与16S rRNA基因序列分析,鉴定该菌株为植物乳杆菌。植物乳杆菌M23代谢产生的乳酸菌细菌素在p H 2.0~10.0范围内具有良好的抑菌活性,121℃处理15 min的条件下依然具有抑菌活性,表明其具有良好的酸碱稳定性和热稳定性。发酵试验表明,该乳酸菌素的最佳收获时间为乳酸菌培养14 h。抑菌谱试验表明,该乳酸菌素能够有效抑制部分食源性革兰氏阳性、阴性致病菌。     

Coculture-inducible bacteriocin activity of Lactobacillus plantarum strain J23 isolated from grape must

Detection and characterization of bacteriocin production by Lactobacillus plantarum strain J23, recovered from a grape must sample in Spain, have been carried out. Bacteriocin activity was degraded by proteolytic enzymes (trypsin, alfa-chymotrypsin, papaine, protease, proteinase K and acid proteases), and it was stable at high temperatures (121 degrees C, 20min), in a wide range of pH (1-12), and after treatment with organic solvents. L. plantarum J23 showed antimicrobial activity against Oenococcus oeni, and a range of Lactobacillus and Pediococcus species. Bacteriocin production was detected in liquid media only when J23 was cocultivated with some inducing bacteria, and induction took place when intact cells or 55 degrees C heated cells of the inducer were cocultivated with J23, but not with their autoclaved cells. Bacteriocin activity of J23 was not induced by high initial J23 inocula, and it was detected in cocultures during the exponential phase. The presence of ethanol or acidic pH in the media reduced bacteriocin production in the cocultures of J23 with the inducing bacteria. The presence of plantaricin-related plnEF and plnJ genes was detected by PCR and sequencing. Nevertheless, negative results were obtained for plnA, plnK, plNC8, plS and plW genes.     

泡菜中产细菌素菌株的分离及细菌素性质的初步研究

从四川泡菜中分离得到一株产细菌素的菌株,经形态学、生理生化鉴定等试验,初步鉴定此菌株为乳酸菌。对其所产细菌素做温度、pH、蛋白酶的敏感性试验,实验结果表明,发酵上清液中的抑菌物质对胃蛋白酶、木瓜蛋白酶较敏感,菌株所产细菌素在高温(121℃20min)和酸性条件(pH 3.0～5.0)下均有较强的抑菌活性,可以较好地抑制革兰氏阳性、阴性菌,筛选得到的菌株所产细菌素是一类具有广谱抑菌活性的细菌素。     

植物乳杆菌的研究进展

本文综述了植物乳杆菌的生物学特性、生理学功能以及其在食品工业中的应用.     

植物乳杆菌的生理功能与应用

本综述了植物乳杆菌的生物学特性、生理学功能以及其在食品工业中的应用。     

植物乳杆菌L5降解亚硝酸盐机理的初步研究

为了研究植物乳杆菌(Lactobacillus plantarum)L5降解亚硝酸盐的机理,采用盐酸萘乙二胺法、十二烷基硫酸钠聚丙烯酰胺凝胶电泳和扫描电镜分别对亚硝酸盐存在条件下L5的降解亚硝酸盐能力、蛋白的表达和菌体形态等进行了初步研究。结果表明,在正常培养条件下,发酵36 h,L5对亚硝酸盐的降解率为56.25%;在控制p H 6.8,消除酸对亚硝酸盐降解作用的条件下,发酵36 h,L5对亚硝酸盐的降解率为70.59%;在有亚硝酸盐存在的条件下,L5能分泌一种分子质量约为200 k Da的a蛋白,且分子质量为50 k Da左右的b蛋白、分子质量为40 k Da左右的c蛋白、分子质量为34 k Da左右的d蛋白的表达均上调;L5在以亚硝酸盐为唯一氮源的培养基中仍然能生长,但亚硝酸盐的存在使L5的菌体形态发生了改变。     

Short communication: Complete genome sequence of Lactobacillus plantarum J26, a probiotic strain with immunomodulatory activity

Lactobacillus plantarum J26, a significant probiotic isolated from Chinese traditional fermented dairy products, exerts a positive immunomodulatory effect by regulating the expression of immune-related genes. We investigated expression of the cytokines IL-1α, IL-1β, IL-6, and tumor necrosis factor-α in the intestinal tract of mice stimulated by L. plantarum J26. In vivo, these cytokines were upregulated, peaked on d 5, and then decreased to the control level, indicating that L. plantarum J26 could induce expression of the genes encoding these proinflammatory cytokines. Teichoic acids produced by L. plantarum are recognized as key immunomodulatory molecules involved in the regulation of the host immune response. To better understand the genetic basis of this immunomodulatory mechanism, we sequenced and analyzed the whole genome of L. plantarum J26. The genome of L. plantarum J26 contains a circular chromosome and 4 circular plasmids. Lactobacillus plantarum J26 was predicted to synthesize ribitol-type backbones of wall teichoic acid. Furthermore, orthologous average nucleotide identity (OrthoANI) values showed that the genome was highly similar (>98.00%) to other L. plantarum strains, especially to L. plantarum ST-III and JDM1. The genomic data of L. plantarum J26 provide a genetic basis to further elucidate its mechanism of immunoregulation and will facilitate its application in the functional dairy food industry.     

Use of the gfp gene in monitoring bacteriocin-producing Lactobacillus plantarum N014, a potential starter culture in nham fermentation

Lactobacillus plantarum N014 is a bacteriocin-producing lactic acid bacteria originally isolated from nham, a traditional Thai fermented sausage, and in the process of development to be used as a starter culture for nham fermentation. During the fermentation process, there is a need to identify the starter culture among several naturally occurring bacteria. In this study, a new plasmid carrying the gfp (green fluorescent protein) gene was constructed based on pGKV210, an Escherichia coli/ Lactococcus shuttle vector containing an erythromycin resistance marker. The gfp gene derived from pGFPuv was placed under the control of an L-lactate dehydrogenase promoter and then inserted at the EcoRI site of pGKV210, leading to pN014-GFP. The novel plasmid was used to transform L. plantarum N014, which is a bacteriocin-producing lactic acid bacteria isolated from nham. The resulting transformant, L. plantarum N014-GFP+, was brightly fluorescent and harbored the expected plasmid. A plasmid stability test revealed that pN014-GFP was stable after 100 generations of growth under nonselective pressure. L. plantarum N014-GFP+ and its parent strain were shown to be very similar in growth rate, bacteriocin production, and lactate production. L. plantarum N014-GFP+ was able to survive in a nham model. The survival clones were still fluorescent and harbored pN014-GFP.     

植物乳杆菌6003对肌原纤维蛋白的分解能力

本文研究了植物乳杆菌6003活细胞(Cell)、细胞裂解液(Cell extracs,CE)以及两者混合液(Cell+CE)对肌原纤维提取液中的蛋白质的分解作用.通过蛋白质、肽、游离氨基酸分析,表明各实验组对肌原纤维提取液中蛋白、肽分解、游离氨基酸的增加作用较弱,其中Cell+CE组中作用稍为显著,CE组对肌原纤维提取液中蛋白质的分解几乎不起作用.     

植物乳杆菌、戊糖片球菌对产肠毒素金黄色葡萄球菌生长的影响

研究食盐含量、pH值、温度对产肠毒素金黄色葡萄球菌的生长的影响,并主要研究植物乳杆菌和戊糖片球菌对产肠毒素和不产肠毒素金黄色葡萄球菌生长的影响以及之间的差别.结果表明:植物乳杆菌和戊糖片球菌均能抑制金黄色葡萄球菌的生长,植物乳杆菌的抑菌活性显著高于戊糖片球菌(P＜0.05),植物乳干菌和戊糖片球菌对不产肠毒素金黄色葡萄球菌的抑菌效果显著高于产肠毒素金黄色葡萄球菌(P＜0.05).     

植物乳杆菌和发酵乳杆菌发酵对蓝莓花色苷的影响

选用植物乳杆菌（Lactobacillus plantarum）FM-LP-9和发酵乳杆菌（Lactobacillus fermentum）FM-LP-SR6分别对蓝莓花色苷进行发酵,考察发酵过程中菌落总数、pH值、色差、总黄酮含量、总糖含量、总花色苷含量、单体花色苷含量、有机酸含量等指标的变化规律。结果表明,经48 h发酵后,两种乳杆菌的菌落总数均能达到9.0 lg（CFU/mL）以上,发酵液的pH值降至3.6,总糖含量相较于发酵前分别降低了48.65%和58.97%,总黄酮含量分别增加至11.4、11.8 mg/L,总花色苷含量分别降低了48.63%和46.06%,色差值升高,颜色变暗。两株乳杆菌均能代谢花色苷和转化酚类物质,发酵过程中花色苷含量呈下降趋势,主成分分析得出两株乳杆菌发酵花色苷后的单体花色苷组分差异较大,主要来自飞燕草素-3-O-葡萄糖苷和矢车菊素-3-O-葡萄糖苷。此外,两株菌发酵后,发酵液中的乳酸、乙酸含量升高,草酸、苹果酸、柠檬酸含量降低。     

Phylogenetic study of Lactobacillus acidophilus group, L. casei group and L. plantarum group based on partial hsp60, pheS and tuf gene sequences

The partial nucleotide sequences encoding elongation factor Tu (tuf gene), 60-kDa heat shock protein (hsp60 gene) and phenylalanyl-tRNA synthase (pheS gene) were determined to assess the suitability as phylogenetic markers for discriminating the closely related species in Lactobacillus acidophilus group, L. casei group and L. plantarum group. A total of 234 lactobacilli were chosen from traditional fermented dairy products that were not exactly assigned to species based on biochemical tests and 16S rRNA gene sequences. The sequencing of partial tuf, hsp60 and pheS gene of all strains was performed, and then, the phylogenetic trees were constructed by neighbor-joining method. Phylogenetic tree revealed three genes provided better resolution of each Lactobacillus species than 16S rDNA, and all of strains were clearly identified as L. casei (63 strains), L. plantarum (58 strains) and L. helveticus (113 strains) by comparison of sequences with the type strains. From our results, the partial sequences of three genes had a higher discriminatory power than 16S rRNA gene sequences and were an alternative molecular tool for the taxonomical analysis of L. casei group, L. plantarum group and L. acidophilus group.     

Simulation of the effect of sausage ingredients and technology on the functionality of the bacteriocin-producing Lactobacillus sakei CTC 494 strain

Bacteriocin-producing lactic acid bacteria may be applied as novel functional starter cultures for sausage fermentation. In this way, safer and more standardised end products may be obtained. However, it is not clear how such strains behave under sausage fermentation conditions. In this study, the combined effects of typical sausage ingredients and process technology on the functionality of Lactobacillus sakei CTC 494 were simulated by a modelling approach. Under simulated sausage fermentation conditions, the strain was able to produce a considerable amount of bacteriocin. Model simulations indicate that sausage fermentation conditions of temperature and pH favour bacteriocin production, whereas salting and curing with sodium nitrite decrease growth and bacteriocin production. Sodium nitrite inhibits cell growth under its undissociated nitrous acid form, and its inhibitory effect seems to parallel lactic acid production. Whereas oxygen and magnesium levels did not influence bacterial functionality, manganese limitations severely decreased cell growth. Moreover, the presence of large amounts of fat, which is typical for a sausage environment, leads to an apparent bacteriocin inactivation, probably due to adsorption of the bacteriocin molecules from the water phase to the fat particles.     

植物乳杆菌melA基因的克隆及其作为食品级筛选标记的初步研究

以L. plantarum1.557的基因组DNA为模板,通过PCR技术成功克隆α-半乳糖苷酶基因(melA基因),与报道的melA基因序列同源性达到99%以上。再以大肠杆菌-乳酸菌穿梭表达载体pMG36e为基本骨架,将melA基因插入该载体,构建melA基因标记的穿梭表达载体pMG36e-melA。重组表达质粒pMG36e-melA经Sac I和Sph I双酶切和PCR鉴定与预期片段大小相符。结果表明已初步构建了以melA基因为食品级筛选标记的重组载体。     

Molecular Characterization of Lactobacilli Isolated from Piper betle L. var. Pachaikodi and Comparative Analysis of the Antimicrobial Effects of Isolate Lactobacillus plantarum KJB23 and Betel Leaves Extract

Traditionally Piper betle L. leaves have been used in India for fermenting certain foods. In our previous study, while fermenting Uttapam batter along with Piper betle L. var. Pachaikodi leaves led to suppression of gas formation and altered Lactic acid bacteria profile, especially in bacilli isolates compared to those observed in plain Uttapam batter fermentation. Hence, the aim of the present study was to understand the rationale behind these changes through characterization of Lactobacillus isolates from Piper betle L. var. Pachaikodi and also the leaves extracts for antimicrobial activity. Out of 72 isolates obtained from the betel leaves, only 10 isolates were bacilli which were chosen for their molecular characterization and to elucidate their inhibitory effects against major food borne pathogens and gas-forming bacteria and to compare with the effect of betel leaves extract. Random amplified polymorphic DNA (RAPD)-PCR and phenotype analysis was used to differentiate the isolates at strain level, 16S rRNA gene sequence for phylogenetic analysis and species-specific multiplex PCR analysis for sub-species identification. All isolates were identified as Lactobacillus plantarum subsp. plantarum. All Lactobacillus isolates were indigenous to leaf as they were resistant to betel leaves extract and showed maximum activity against some LAB and non-LAB indicator strains except Lactobacillus plantarum MTCC 6160 which was resistant. Among them, KJB23, 36 and 47 were the most effective. Particularly, the isolate KJB23 and ethanolic betal leaf extract showed rivaling inhibitory activity against major food borne pathogens, while dissimilar activity against gas forming bacteria. This study revealed that either Piper betle L. leaf or Lactobacillus strains from the leaves can be valuable for food applications when added to fermented products.     

酸浆果籽油的提取研究

研究浸泡法、超声波辅助萃取法和索氏萃取法对黑龙江产酸浆果籽萃取的影响,分别对乙酸乙酯,乙醚和正己烷的提取效果进行比较.结果表明,浸泡法的籽油产率为乙酸乙酯的18.68%、乙醚的17.68%以及正己烷的14.30%;超声波辅助萃取法的油脂产率为乙酸乙酯的18.51%、乙醚的19.55%以及正己烷的17.26%;索氏提取法的油脂产率为乙酸乙酯的19.53%、乙醚的19.70%以及正己烷的18.83%.GC-MS分析表明3种方法获得的果籽油成分均以亚油酸和油酸为主.     

Simulation of the effect of sausage ingredients and technology on the functionality of the bacteriocin-producing Lactobacillus sakei CTC 494 strain

Bacteriocin-producing lactic acid bacteria may be applied as novel functional starter cultures for sausage fermentation. In this way, safer and more standardised end products may be obtained. However, it is not clear how such strains behave under sausage fermentation conditions. In this study, the combined effects of typical sausage ingredients and process technology on the functionality of Lactobacillus sakei CTC 494 were simulated by a modelling approach. Under simulated sausage fermentation conditions, the strain was able to produce a considerable amount of bacteriocin. Model simulations indicate that sausage fermentation conditions of temperature and pH favour bacteriocin production, whereas salting and curing with sodium nitrite decrease growth and bacteriocin production. Sodium nitrite inhibits cell growth under its undissociated nitrous acid form, and its inhibitory effect seems to parallel lactic acid production. Whereas oxygen and magnesium levels did not influence bacterial functionality, manganese limitations severely decreased cell growth. Moreover, the presence of large amounts of fat, which is typical for a sausage environment, leads to an apparent bacteriocin inactivation, probably due to adsorption of the bacteriocin molecules from the water phase to the fat particles.     

转基因大米TT51-1品系精准定量检测方法的比较研究

基于大米蔗糖磷酸合成酶(SPS)基因和TT51-1品系特异性基因序列筛选适用于数字PCR的内、外源基因特异性引物探针并建立转基因大米TT51-1品系的双重数字PCR定量方法。其定量的绝对灵敏度和相对灵敏度分别达2 copies/μL和0.1%。当样品中TT51-1转基因大米成分含量低至0.1%时,6次定量值的相对标准偏差在7.30%~18.63%之间,偏差在-8.77%~9.62%之间,精密度和稳定性均较为理想,而同样的引物探针所建立的实时荧光PCR方法定量的相对灵敏度仅达到1%。为促进该方法的标准化应用,将微滴式数字PCR平台上建立的定量方法在芯片式数字PCR平台上进行室内验证,结果表明该方法的定量精密度和准确性符合要求。该方法可应用于大米、稻谷及其初加工产品中TT51-1转基因大米成分的精准定量检测。     

应用RT-PCR技术定量检测发酵乳中Lactobacillus plantarum活菌数

根据发酵乳中常见乳酸菌种的16S rRNA基因序列设计了L.plantarum的种属特异性引物,应用该引物对植物乳杆菌的模式菌株L.plantarum ATCC14917、L.plantarum非同源性对照菌株及采自西藏地区的自然发酵乳样品进行了种属特异性PCR检测,并以样品RNA转录的cDNA为模板,对检出含有L.plantarum的发酵乳样品进行了Real-Time PCR定量检测。通过Real-Time PCR图谱分析,准确地检测出了该样品中L.plantarum活菌数的含量为6.6lgcfu/mL。结果表明该方法能够简便、快速地检测出发酵乳中是否含有L.plantarum,并能够对其活菌数进行准确地定量,对发酵乳的生产和监管提供了重要的理论和实践依据。