一株具有良好粘附性的植物乳杆菌的生物学特性及应用

本文研究了植物乳杆菌AR326的最适生长温度、最适接种量、生长曲线、最适初始pH、胆汁耐受性、NaCl耐受性,并进一步探究了单菌株发酵酸奶的性能。结果显示植物乳杆菌AR326生长较快,4 h进入对数期,14 h进入稳定期,最适生长温度为30℃,在初始pH 3.0~7.0范围可生长,适宜的接种量为1.5%~2.0%,耐受胆盐浓度达0.2%,耐高渗透压能力强,可在含NaCl 8%的MRS培养基中生长。发酵乳中菌落数和对照组一致,脱水收缩性优于对照组,因而可用于商业上生产功能性酸奶。     

Utilization of amino acids and dipeptides by Lactobacillus plantarum from orange in nutritionally stressed conditions

Aims:  To investigate amino acid and dipeptide utilization by Lactobacillus plantarum N4 isolated from orange peel, in a nutritionally depleted medium based on MRS (Mann, Rogosa, Sharpe).
Methods and Results:  In MRS with 0·1 g l⁻¹ of meat extract and without peptone and yeast extract, growth increased when essential and stimulatory amino acids and nonessential amino acid were added to the medium. Replacement of the essential amino acid, leucine, and the nonessential amino acid, glycine, by leucyl-leucine (Leu-Leu) and/or glycyl-glycine (Gly-Gly) significantly enhanced growth. Essential amino acids were mainly consumed and the dipeptides were almost completely used at the end of growth. Leucine and glycine accumulated internally from the peptides were higher than from the free amino acids. Glucose utilization increased in the media containing dipeptides compared with the medium containing free amino acids.
Conclusions:  In a N-depleted medium, Leu-Leu and/or Gly-Gly were more effective than the respective amino acids in supporting growth of the micro-organism. The more efficient internal accumulation of glycine and especially leucine from dipeptides confirmed the ability of the strain to assimilate mainly complex nitrogen molecules rather than simple ones.
Significance and Impact of the Study:  The ability of Lact. plantarum N4 to efficiently use dipeptides could contribute to spoilage development in the natural medium of the organism, orange juice.     

Development of a minimal growth medium for Lactobacillus plantarum

Aim:  A medium with minimal requirements for the growth of Lactobacillus plantarum WCFS was developed. The composition of the minimal medium was compared to a genome-scale metabolic model of L. plantarum .
Methods and Results:  By repetitive single omission experiments, two minimal media were developed: PMM5 (true minimal medium) and PMM7 [a pseudominimal medium, supporting proper biomass formation of 350 mg l⁻¹ dry weight (DW)]. The specific growth rate of L. plantarum on PMM7 was found to be 50% and 63% lower when compared to growth on established growth media (chemically defined medium and MRS, respectively). Using a genome-scale metabolic model of L. plantarum , it was predicted that PMM5 and PMM7 would not support the growth of L. plantarum . This is because the biosynthesis of para- aminobenzoic acid ( p ABA) was predicted to be essential for growth. The discrepancy in simulated growth and experimental growth on PMM7 was further investigated for p ABA; a molecule which plays an important role in folate production. The growth performance and folate production were determined on PMM7 in the presence and absence of p ABA. It was found that a 12 000-fold reduction in folate pools exerted no influence on formation of biomass or growth rate of L. plantarum cultures when grown in the absence of p ABA.
Conclusion:  Largely reduced folate production pools do not have an effect on the growth of L. plantarum , showing that L. plantarum makes folate in a large excess.
Significance and Impact of the study:  These experiments illustrate the importance of combining genome-scale metabolic models with growth experiments on minimal media.     

In vitro growth inhibition of Helicobacter pylori by lactobacilli belonging to the Lactobacillus plantarum group

AIMS: The aim of this study was to test and locate the in vitro anti-Helicobacter activity of seven Lactobacillus strains belonging to Lactobacillus plantarum group. METHODS AND RESULTS: Growth inhibition of H. pylori was tested using a well-plate assay. Of the strains displaying the strongest growth inhibition, a L. plantarum isolated from sauerkraut (MLBPL1) was chosen for further studies. The detected anti-Helicobacter activity of MLBPL1 was mainly associated with cell wall, and to a minor extent with the culture supernatant. The active component, which was determined to be between 3 and 10 kDa in size, retained its activity after 10 min treatment at 100 degrees C. The activity was present when MLBPL1 was cultivated in rich laboratory cultivation medium MRS and in different food matrices. CONCLUSIONS: The strains belonging to L. plantarum group showed anti-Helicobacter activity in vitro. The main activity seemed to be associated with cell wall rather than culture supernatant or intracellular fraction. SIGNIFICANCE AND IMPACT OF THE STUDY: In view of the rapid spread of resistant H. pylori strains caused by antibiotic therapy, addition of a fermented food containing L. plantarum to the conventional antibiotic treatment of Helicobacter infection could establish a potential complementary means to suppress the infection.     

Isolation and partial characterization of a bacteriocin produced by Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product

Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product was found to produce a novel bacteriocin that is active against a wide range of gram‐positive and gram‐negative bacteria. Production of the bacteriocin BM‐1 started early in the exponential phase and its maximum activity (5120 AU/mL) was recorded early during the stationary phase (16 hr). Bacteriocin BM‐1 is sensitive to proteolytic enzymes but stable in the pH range of 2.0–10.0 and heat‐resistant (15 min at 121°C). This bacteriocin was purified through pH‐mediated cell adsorption–desorption and cation‐exchange chromatography on an SP Sepharose Fast Flow column. The molecular weight of the purified bacteriocin BM‐1 was determined to be 4638.142 Da by electrospray ionization Fourier transform mass spectrometry. Furthermore, the N‐terminal amino acid sequence was obtained through automated Edman degradation and found to comprise the following 15 amino acid residues: H₂N‐Lys‐Tyr‐Tyr‐Gly‐Asn‐Gly‐Val‐Tyr‐Val‐Gly‐Lys‐His‐Ser‐Cys‐Ser. Comparison of this sequence with that of other bacteriocins revealed that bacteriocin BM‐1 contains the consensus YGNGV amino acid motif near the N‐terminus. Based on its physicochemical characteristics, molecular weight, and N‐terminal amino acid sequence, plantaricin BM‐1 is a novel class IIa bacteriocin.     

Examination of Lactobacillus plantarum lactate metabolism side effects in relation to the modulation of aeration parameters

AIMS: The characterization of global aerobic metabolism of Lactobacillus plantarum LP652 under different aeration levels, in order to optimize acetate production kinetics and to suppress H2O2 toxicity. METHODS AND RESULTS: Cultures of L. plantarum were grown on different aeration conditions. After sugar exhaustion and in the presence of oxygen, lactate was converted to acetate, H2O2 and carbon dioxide with concomitant ATP production. Physiological assays were performed at selected intervals in order to assess enzyme activity and vitality of the strain during lactic acid conversion. The maximal aerated condition led to fast lactate-to-acetate conversion kinetics between 8 and 12 h, but H2O2 immediately accumulated, thus affecting cell metabolism. Pyruvate oxidase activity was highly enhanced by oxygen tension and was responsible for H2O2 production after 12 h of culture, whereas lactate oxidase and NADH-dependent lactate dehydrogenase activities were not correlated to metabolite production. Limited NADH oxidase (NOX) and NADH peroxidase (NPR) activities were probably responsible for toxic H2O2 levels in over-aerated cultures. CONCLUSION: Modulating initial airflow led to the maximal specific activity of NOX and NPR observed after 24 h of culture, thus promoting H2O2 destruction and strain vitality at the end of the process. SIGNIFICANCE AND IMPACT OF THE STUDY: Optimal aeration conditions were determined to minimize H2O2 concentration level during growth on lactate.     

1株植物乳杆菌生物学特性的研究

研究1株植物乳杆菌(N3)的生物学特性,实验结果表明该菌能耐受80~85℃的高温和0.20kg/cm2蒸汽压力;直接分解玉米淀粉的乳酸产率为7.05%(36 h)和8.19%(48 h);能耐受pH为4.5的酸性环境;在人工胃液中的活菌数为4.1×106CFU/g;对金霉素、土霉素、痢特灵和氟哌酸等抗生素的敏感性强,而对防霉剂和脱霉素不敏感。植物乳杆菌(N3)是1株优良的益生素生产菌。     

一株植物乳酸杆菌的高密度发酵

目的研究一株植物乳酸杆菌的高密度发酵。方法通过单因素实验来确定此植物乳酸杆菌的最适生长温度、pH、碳源、氮源、缓冲盐等。结果最适生长温度是37℃、起始pH6.4、最适碳源为葡萄糖、最适氮源为牛肉膏,通过对缓冲盐的选择最终确定NaAc：CaCO3比为0.5：1.5;控制发酵条件,结合优化培养基,可使发酵后的培养液达到2.75×10^10CFU/ml。结论该缓冲盐能够有效的缓解发酵液pH的下降,延长对数生长期,使活菌数提高1倍,适合高密度发酵培养。     

降胆固醇植物乳杆菌的紫外诱变选育

通过对植物乳杆菌进行紫外线诱变处理,采用胆固醇梯度平板的初筛方法和体外降胆固醇能力的测试,结果得到二株降胆固醇能力强且性能比较稳定的植物乳杆菌突变菌株Lp-UVs 29和Lp-UVs 44,胆固醇的降解率分别为48.7%和44.2%,分别比诱变前提高了97.97%和79.67%.     

Identification and characterization of antibiotic resistance genes in Lactobacillus reuteri and Lactobacillus plantarum

Aims:  The study aimed to identify the resistance genes mediating atypical minimum inhibitory concentrations (MICs) for tetracycline, erythromycin, clindamycin and chloramphenicol within two sets of representative strains of the species Lactobacillus reuteri and Lactobacillus plantarum and to characterize identified genes by means of gene location and sequencing of flanking regions.
Methods and Results:  A tet (W) gene was found in 24 of the 28 Lact. reuteri strains with atypical MIC for tetracycline, whereas four of the six strains with atypical MIC for erythromycin were positive for erm (B) and one strain each was positive for erm (C) and erm (T). The two Lact. plantarum strains with atypical MIC for tetracycline harboured a plasmid-encoded tet (M) gene. The majority of the tet (W)-positive Lact. reuteri strains and all erm -positive Lact. reuteri strains carried the genes on plasmids, as determined by Southern blot and a real-time PCR method developed in this study.
Conclusions:  Most of the antibiotic-resistant strains of Lact. reuteri and Lact. plantarum harboured known plasmid-encoded resistance genes. Examples of putative transfer machineries adjacent to both plasmid- and chromosome-located resistance genes were also demonstrated.
Significance and Impact of the Study:  These data provide some of the knowledge required for assessing the possible risk of using Lact. reuteri and Lact. plantarum strains carrying antibiotic resistance genes as starter cultures and probiotics.     

Comparison of growth-phase-dependent cytosolic proteomes of two Lactobacillus plantarum strains used in food and feed fermentations

Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium highly adapted to a wide variety of environments and widely used in food and feed fermentations. Proteomes of two strains of L. plantarum, one isolated from spontaneously fermented cereal-based feed (strain REB1), and the other from white cabbage (strain MLBPL1), were studied to elucidate the strain-specific variation and the physiological changes occurring between the growth (lag, early-exponential, late-exponential and early-stationary) phases of this bacterium when cultivated in a standard rich medium. A total of 231 protein spots were identified by LC-MS/MS. These proteins showed that strain MLBPL1 had more proteins with growth phase-dependent expression than REB1, which possesses a more constant expression profile. The proteins with growth phase-dependent expression in REB1 and MLBPL1 were mainly associated with energy metabolism (glycolysis, phosphoketolase pathway and ribose metabolism), all having preferential expression in the early-exponential phase, confirming the use of different carbohydrates simultaneously. Indication of energy production was also seen in lag and early-stationary phases.     

一种快速定量检测益生菌制品中植物乳杆菌的方法

目的建立快速定量检测植物乳杆菌的方法,排除近缘菌的干扰。方法利用SMM系统筛选植物乳杆菌种特异序列,根据特异序列设计引物进行实时荧光定量PCR反应。结果设计的引物具有良好的种特异性,检测限为2．26×10^2CFU／mL。结论该方法快速灵敏,可以在实际生产中应用。     

植物乳杆菌环二腺苷酸合成酶的克隆表达与活性分析

[背景]环二腺苷酸(Cyclic Diadenosine Monophosphate,c-di-AMP)是一种主要存在于革兰氏阳性菌中的重要的第二信使分子,其参与细菌的生长、生存、抗逆性等多种生理活动,但目前关于乳酸菌中c-di-AMP的研究甚少.[目的]从植物乳杆菌(Lactobacillus plantarum)中...     

不同分离源植物乳杆菌的群体基因组分析

【背景】植物乳杆菌(Lactobacillus plantarum)广泛存在于植物、乳制品、肉制品、哺乳动物和昆虫的肠道等多种生态环境中。【目的】探究不同分离源L. plantarum基因组与其所在环境是否存在潜在的联系。【方法】利用比较基因组学对126株分离自植物、乳制品、肉制品、果蝇及哺乳动物肠道和口腔等部位的L. plantarum菌株基因组进行系统发育分析和功能基因组分析,解析不同分离源菌株间的亲缘关系和进化历程。【结果】果蝇分离株的基因组大小显著高于植物、哺乳动物肠道、肉制品和乳制品分离株(P0.05),植物和哺乳动物肠道、口腔等部位与肉制品分离株的基因组大小和编码基因数量无显著差异(P0.05)。基于单拷贝基因串联和核心基因系统发育树分析均发现,果蝇分离株和乳制品分离株分别集中聚集分布在某一分支中,其余分离源均匀分布在各个分支中。附属基因分析结果与系统发育树分析结果一致。功能基因注释结果发现,果蝇分离株的环境特异性基因参与低聚果糖和几丁质代谢,乳制品分离株的环境特异性基因参与mazEF毒素-抗毒素系统和CRISPR系统。【结论】植物乳杆菌分离株为适应较为独特的果蝇和乳制品生境而发生了适应性进化。本研究为植物乳杆菌适应性进化提供了新见解,同时为解析菌株的进化历程提供了理论基础。     

Transformation of Lactobacillus plantarum with the plasmid pTV1 by electroporation

Abstract Lactobacillus plantarum ATCC 8014 was transformed with pTV1 by electroporation using a modification of a procedure described for Escherichia coli . The plasmid pTV1 which contains the pE194 replicon from Staphylococcus aureus and transposon Tn917 from Streptococcus faecalis was shown to replicate as a high copy number plasmid in L. plantarum , and the two encoded antibiotic resistance traits were expressed. Tn917 transposed with a high frequency into plasmid DNA of L. plantarum as shown by restriction enzyme analysis and Southern hybridization studies. There are no previous reports on transposition in the lactobacilli. This system may prove to be an important tool in further work on the genetics of these organisms.     

植物乳杆菌LpT1和LpT2体外降胆固醇机制

【目的】初步探讨植物乳杆菌LpT1和LpT2的体外降胆固醇机制。【方法】以MRS、MRS+CH、MRS+CH+S和MRS+CH+N四种培养基为基础,接种植物乳杆菌LpT1和LpT2进行培养,通过分析比较培养基上清液、菌体沉淀和菌体细胞内部胆固醇含量以及接种和未接种两种情况上清液、沉淀和细胞内胆固醇总量变化,推测植物乳杆菌体外降胆固醇机制。【结果】乳酸菌体外降胆固醇存在非代谢和代谢降解两条途径,非代谢途径与共沉淀作用和菌体吸收有关。代谢降解是由于植物乳杆菌在生长过程中产生了特殊的酶系,从而将胆固醇降解成其他物质,导致其含量降低。【结论】研究结果为进一步研究植物乳杆菌体外降胆固醇的机制奠定了良好基础。     

植物乳杆菌粘附性与其表面特征关系的探究

本文通过16s rDNA鉴定获得4株植物乳杆菌,并以HT29细胞为体外黏附筛选模型,进一步探讨了这些菌株粘附能力与表面疏水性、自聚共聚能力等表型特征的相关性。结果表明,植物乳杆菌AR326菌株对HT29细胞的粘附性最强,并显示高度的自聚性(25%)和共聚性(25%),但其表面疏水性偏低(15%);通过相关性分析发现,植物乳杆菌的自聚性和共聚性与HT29细胞粘附性呈显著相关性(r=1.0和0.8,p0.05),但表面疏水性、自凝聚性和共聚性两两之间并无显著相关性(p0.05)。本研究结果为建立快速筛选高粘附性植物乳杆菌的方法及其菌株在体内定植和分布研究提供一定参考依据。