仿刺参(Apostichopus japonicus)肠道源乳酸乳球菌(Lactococcus lactis)的分离鉴定及其益生特性分析

利用MRS固体培养基从南移仿刺参肠道中分离得到1株优势乳酸菌,命名为AJC-XP-15,其最适生长温度为30℃,最适生长pH为6.5;其表面疏水性和自聚率分别为34.56%和35.61%,经16S RNA基因序列分析,并结合形态学和生理生化特性将其鉴定为乳酸乳球菌(Lactococcus lactis)。拮抗实验结果显示,菌株AJC-XP-15及其发酵上清液对塔氏弧菌(Vibriotubiashii)、哈维氏弧菌(V.harveyi)、副溶血弧菌(V.parahaemolyticus,VP)和嗜水气单胞菌(Aeromonas hydrophila)等病原菌具有很好的抑制作用;体外益生实验表明,菌株AJC-XP-15对人工胃肠液的耐受性很好,在pH3.0的模拟人工胃液处理3 h后的存活率为71.43%,在pH 6.8的模拟人工肠液处理3 h后的存活率为92.1%;抗氧化能力结果显示,菌株AJC-XP-15的无细胞提取物对DPPH自由基和羟自由基的清除能力最强,分别为(82.67±6.92)%和(15.36±2.95)%,其发酵上清液对超氧阴离子自由基的清除能力最强,为(26.36±2.58)%;药物敏感性分析结果显示,菌株AJC-XP-15对四环素、恩诺沙星、盐酸多西环素等抗生素敏感,对氟苯尼考、复方新诺明、庆大霉素等抗生素不敏感。研究结果可为仿刺参肠道源乳酸菌的分离鉴定和候选益生菌种筛选提供参考依据。

Lactococcus lactis谷氨酸脱羧酶的分离纯化及部分酶学性质

采用溶菌酶处理、超声破碎、硫酸铵分级、3次DEAE SepharoseCL 6B层析、SephacrylS 200凝胶过滤等手段,从乳酸菌细胞中分离纯化得到谷氨酸脱羧酶(GAD;EC4.1.1.15).纯化酶的比活力为14.4U/mg,纯化倍数31,回收率3.8%,SDS PAGE得到亚基的相对分子质量为65000.L 谷氨酸是测试的18种氨基酸中的惟一作用底物,表明乳酸菌GAD具有高度的底物专一性.酶在pH值3.6～5.4时具有活性,pH值4.7时活性最高,pH值6.0以上时没有活力.耐热性实验表明,pH值4.7条件下处理5h后,60℃时该酶仍能保持80%以上的活性,80℃以上迅速失活,由Lineweaver Burk作图得到的GAD的Km值为1.9mmol/L.

酸胁迫对乳酸链球菌素产生菌Lactococcus lactis LN26的影响

为研究乳酸链球菌素产生菌Lactococcus lactis LN26在酸胁迫条件下的应激反应,本试验测定了在不同pH值梯度条件下,Lactococcus lactis LN26菌体浓度、胞内pH、H+-ATPase活性、胞内ATP浓度、细菌素产量。结果表明:随着酸度的增加Lactococcus lactis LN26生长受到明显抑制,在pH值3.0条件下,菌体浓度最低;酸胁迫增强了H+-ATPase活性,在pH值3.0处理时,H+-ATPase活性最高;酸胁迫对乳酸链球菌素的合成具有抑制作用。pH值为6.0时最适合乳酸链球菌素产生菌Lactococcus lactis LN26的生长以及乳酸链球菌素的合成。

过量表达purC基因对Lactococcus lactis NZ9000酸胁迫抗性的影响

通过在乳酸乳球菌Lactococcus lactis NZ9000中过量表达嘌呤代谢途径中编码磷酸核糖基氨基咪唑-琥珀酰胺合酶的pur C基因,测定了重组菌株在酸胁迫条件下的存活率,经p H 4. 0胁迫培养4 h后,重组菌株的存活率为对照菌株的83. 2倍。采用ATP测定试剂盒和高效液相色谱分别考察菌株胞内ATP和氨基酸含量。结果表明,重组菌株在酸胁迫条件下维持了更高的胞内ATP和氨基酸(天冬氨酸、苏氨酸、谷氨酸和γ-氨基丁酸)含量,分别通过为细胞提供能量和消耗胞内质子的方式,帮助乳酸乳球菌抵御酸胁迫。研究发现,在乳酸乳球菌中过表达pur C基因能够明显提高菌株的酸胁迫抗性,同时为进一步通过改造嘌呤代谢途径提高乳酸菌酸胁迫耐受性提供了新的思路。

Non-Fusion and Fusion Expression of β-Galactosidase from Lactobacillus bulgaricus in Lactococcus lactis

Objective To construct four recombinant Lactococcus lactis strains exhibiting high β-galactosidase activity in fusion or non-fusion ways, and to study the influence factors for their protein expression and secretion. Methods The gene fragments encoding β-galactosidase from two strains of Loctobacillus bulgaricus, wch9901 isolated from yogurt and 1.1480 purchased from the Chinese Academy of Sciences, were amplified and inserted into lactococcal expression vector pMG36e. For fusion expression, the open reading frame of the β-galactosidase gene was amplified, while for non-fusion expression, the open reading frame of the β-galactosidase gene was amplified with its native Shine-Dalgarno sequence upstream. The start codon of the β-galactosidase gene partially overlapped with the stop codon of vector origin open reading frame. Then, the recombinant plasmids were transformed into Escherichia coli DH5α and Lactococcus lactis subsp, lactis MG1363 and confirmed by determining β-galactosidase activities. Results The non-fusion expression plasmids showed a significantly higher β-galactosidase activity in transformed strains than the fusion expression plasmids. The highest enzyme activity was observed in Lactococcus lactis transformed with the non-fusion expression plasmids which were inserted into the β-galactosidase gene from Lactobacillus bulgaricus wch9901. The β-galactosidase activity was 2.75 times as high as that of the native counterpart. In addition, β-galactosidase expressed by recombinant plasmids in Lactococcus lactis could be secreted into the culture medium. The highest secretion rate （27.1%） was observed when the culture medium contained 20 g/L of lactose. Conclusion Different properties of the native bacteria may have some effects on the protein expression of recombinant plasmids. Non-fusion expression shows a higher enzyme activity in host bacteria. There may be a host-related weak secretion signal peptide gene within the structure gene of Lb. bulgaricus β-galactosidase, and its translation product may introduce the enzyme secretion out of cells in special hosts.

Influence of Maternal and Larval Immunisation against <i>Lactococcus garviae</i>Infection in Rainbow Trout <i>Oncorhynchus mykiss</i>(Walaum) Lysozyme Activity and IgM Level

This study evaluated efficacy of maternal and larval immunisation against Lactococcus garviae infection and on the lysozyme and immunoglobulin (IgM) levels in rainbow trout Oncorhynchus mykiss (Walaum). Forty-eight-day-old larvae (mean weight 96 mg) originating from injected weekly with letrozole and immunised, only immunised and non-immunised parents were experimentally infected with the L. garvieae, and the mortality rate was recorded daily. Larvae were vaccinated by immersion at 58 days post hatch with live L. garvieae (109 cells/mL) for 15 min. Every third day post larvae vaccination, two larvae from each group were collected for analysis lysozyme (by a method based on the ability of lysozyme to lyse the bacterium Micrococcus lysodeikticus) and IgM (by enzyme-linked immunosorbent assay (ELISA)) parameters. Vaccinated and control larvae were tested for protection against L. garvieae 30 days post larvae immunization when the larvae were 88 days old. Larvae were challenged by bath exposure with live L. garvieae (109 cells/mL) for 2 min and monitored for mortality for at least 10 days following challenge. The challenge experiment with L. garvieae showed a significant reduction in larvae from immunised (54.44% ± 0.64%) and injected weekly with letrozole and immunised fish (52.96% ± 0.97%) compared to larvae from control fish (62.96% ± 2.22%). Vaccinated larvae originated from injected weekly with letrozole and immunised parents showed significantly higher lysozyme activity compared to other fish groups. Vaccinated larvae showed significantly less mortality compared to controls. The relative percent survival (RPS) values of larvae from only immunised, injected weekly with letrozole and immunised and non-immunised parents vaccinated with L. garvieae were 67.36% ± 0.9%, 68.05% ± 0.66% and 48.27% ± 2.79% respectively. The results indicate that the effect of maternal immunization rainbow trout against L. garvieae infection by eliciting the immune responses as indicated by an increase in the IgM level and lysozyme activity.

Construction and Immunogenicity of Recombinant Lactococcus lactis Expressing S1 Protein of Porcine Epidemic Diarrhea Virus(PEDV)

To evaluate the specific immune responses induced by recombinant Lactococcus lactis（L.lactis） which expresses porcine epidemic diarrhea virus（PEDV） S1 protein through oral administration,the spike gene fragment of PEDV was amplified from PEDV SDLY strain to construct p MG36 e-S1 recombinant plasmid.The recombinant plasmid was then electro-transferred into competent cells of L.lactis MG1363,to prepare the recombinant L.lactis expressing S1 protein of PEDV.The expression of target protein was identified by SDS-PAGE and Western-blot.New Zealand white rabbits were orally administered with the recombinant strain;the antibody titer in intestinal mucosa and serum was detected by neutralizing test;and the specific Ig G in serum was evaluated by indirect ELISA.The results showed that the recombinant L.lactis could effectively induce high level of Ig G in serum and high level of mucosal immune antibody.The recombinant L.lactis is qualified to be a potential oral vaccine because it could successfully stimulate both humoral and mucosal immune responses against PEDV.

Inhibition mechanisms of secretome proteins from Paenibacillus polymyxa Kp10 and Lactococcus lactis Gh1 against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus

Objective:To determine the inhibition mechanisms of secretome protein extracted from Paenibacillus polymyxa Kp10(Kp10)and Lactococcus lactis Gh1(Gh1)against methicillin-resistant Staphylococcus aureus(MRSA)and vancomycin-resistant Enterococcus(VRE).Methods:The sensitivity and viability of MRSA and VRE treated with secretome proteins of Kp10 and Gh1 were determined using minimal inhibitory concentration,minimum bactericidal concentration,and time-to-kill assays.The morphological changes were observed using scanning electron microscopy and transmission electron microscopy.To elucidate the antimicrobial mechanism of secretome protein of Kp10 and Gh1 against MRSA and VRE,2D gel proteomic analysis using liquid chromatography-mass spectrometry was run by comparing upregulated and downregulated proteins,and the proton motive force study including the efflux of ATP,pH gradient,and the membrane potential study were conducted.Results:MRSA and VRE were sensitive to Kp10 and Gh1 secretome protein extracts and displayed apparent morphological and internal composition changes.Several proteins associated with cellular component functions were either downregulated or upregulated in treated MRSA and VRE by changing the membrane potential gradient.Conclusions:Kp10 and Gh1 secretome proteins reduce the growth of VRE and MRSA by damaging the cell membrane.Cell division,cell wall biosynthesis,and protein synthesis are involved in the inhibition mechanism.

NisP Is Related to Nisin Precursor Processing and Possibly to Immunity in Lactococcus Lactis

In this study, a plasmid was integrated into nisP, creating the first defined mutation in a nisin biosynthetic gene. The mutant strain secreted fully modified nisin with the N-terminal leader still attached.The presence of the leader was confirmed by N-terminal sequencing of the purified precursor. The dehydration and lanthionine formation of the precursor were already completed as active nisin could be formed by cleaving the leader from the inactive precursor by a trypsin treatment or by incubation with wild type cells. Nisin immunity of the NisP mutant strain was lowered to about 10% of the wild type immunity. The results show that NisP is needed for precursor processing and for development of high immunity of nisin.

Preventive Effect of Lactococcus lactis subsp. lactis JCM 5805 Yogurt Intake on Influenza Infection among Schoolchildren

Objective: A community-based intervention study was conducted to examine the effect of consumption of JCM 5805 yogurt on influenza incidence rates and the cumulative incidence rates among schoolchildren in Iwate Prefecture, Japan. Methods: Schoolchildren and their parents in Shizukuishi town were told of the purpose, frequency and duration of JCM 5805 yogurt administration. The number of elementary schoolchildren in Shizukuishi town was 780 while that of junior high school students in Shizukuishi town numbered 475. The number of elementary schoolchildren in neighboring town A was 208 and that of junior high school students in town A was 121. JCM 5805 yogurt was delivered three times a week to all elementary schools and junior high schools in Shizukuishi town from January 16 through March 18, 2015. The incidence rate was calculated every week as the maximum case number divided by the number of schoolchildren in each school. The cumulative incidence rate was calculated as the total case number during the period when JCM 5805 yogurt was delivered divided by the number of schoolchildren in each school. Results: JCM 5805 yogurt intake was associated with a two-thirds reduction in influenza incidence rates in Shizukuishi town schoolchildren compared with those of town A. Furthermore, the cumulative incidence rates of the elementary school and combined data from the elementary school and junior high school were significantly lower than those of neighbor town A. Conclusion: JCM 5805 yogurt intake reduced both the incidence rates and cumulative incidence rates of influenza.

Use of <i>Lactococcus lactis</i>Subsp. <i>Lactis</i>Strains to Inhibit the Development of Pathogens

Bovine mastitis affects the udder health and thus causing significant economic losses. Probiotic products based on the use of lactic acid bacteria (LAB) to limit pathogens multiplication and pre-infection risks can be an interesting alternative to post infection allopathic treatment with antibiotics. Lactococcus lactis is one of the most important bacteria used in dairy technology. In this work, a total of 21 Lactococcus lactis subsp. Lactis strains, 20 from goat milk whey and one strain from cow milk were used to evaluate their antibacterial activity against four pathogenic germs responsible for mastitis: Escherichia coli, Staphylococcus aureus, Streptococcus uberis and Streptococcus agalactiae. The nisin-producing cow milk strain was active against St. uberis and Str. Agalactiae using the well diffusion method. For the strains isolated from goat milk whey, no antimicrobial effect was observed against these pathogens. However, a different approach based on the growth of pathogenic bacteria interacting with the Lactococcus lactis strains in a minimum medium was used to study the barrier effect of LAB. The Lactococcus lactis strains S1 and S2 from goat milk whey depleted the growth of Sa. aureus, St. uberis and E. coli during 8 h and stopped the development of St. agalactiae.

Safety Evaluation of Excessive Intake of <i>Lactococcus lactis</i>Subsp. <i>lactis</i>JCM 5805: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Group Trial

Background/Aims: Administration of a lactic acid bacterial strain, Lactococcus lactis subsp. lactis JCM 5805 (LC-Plasma), is reported to prevent viral infection via activation of plasmacytoid dendritic cells in mouse and human studies. As it is assumed that LC-Plasma is taken in excess when it is commercially provided as a supplement, we conducted a trial using capsules to give 250 mg LC-Plasma (5 times the effective anti-viral dose) every day for four weeks to healthy volunteers to investigate the safety of excessive intake of LC-Plasma. Trial Design: A randomized, double-blind, placebo-controlled, parallel-group trial was conducted. Methods: Forty healthy subjects were randomly assigned to the LC-Plasma group (daily intake of five capsules containing 50 mg heat-killed LC-Plasma cells per capsule) or the placebo group (daily intake of five placebo capsules with no LC-Plasma). Physical, hematological, biochemical and urinary examinations and medical interviews were used to evaluate safety. Results: No abnormal differences were observed after excessive intake of LC-Plasma capsules when compared to the intake of placebo capsules. Conclusions: There are no safety concerns associated with the excessive intake of heat-killed LC-Plasma capsules.

Dual PINK Mutant and A<i>β</i>42-Dependent Lifespan Shorten and Flight Impairment in Transgenic Drosophila Partially Alleviates by a <i>Lactococcus lactis</i>Supplemented Diet

Oxidative stress has been strongly related with Parkinson disease (PD) and Alzheimer disease pathogenesis. We determined the effects of Lactococcus lactis (LAL) supplementation on the generated loss-of-function mutants of PINK1 B9, an AR-JP-linked gene and Aβ42 induced phenotypes in a Drosophila melanogaster model of PD/AD. Enhanced mutant PINK1 B9 and Aβ42 expression in D. melanogaster dopaminergic (DA) neurons can curtail lifespan, flight muscle accompanied by locomotive defects and we have observed longevity methods to assay the effects of LAL on D. melanogaster survival. Furthermore, flies expressing mutant PINK1 B9 and Aβ42 in their brain fed LAL had up to the two weeks, or 25%, greater median lifespan than those fed a standard sucrose diet. In addition, LAL improved mutant PINK1 B9 and Aβ42-induced flight impairments in the Drosophila wing. Our microscopy analyses revealed that individuals fed LAL had improved atypical ommatidia as well as an increased thirteen percentage of flight ability than those fed a control diet. We propose that LAL, rich in naturally occurring probiotics and antioxidants, promotes the survival of neurons in brain and wing muscle tissues with increased levels of mutant PINK1 B9 and Aβ42 via a protective cell survival mechanism.

Improvement of Poplar Bark Extract on Physiological Characters of Lactococcus lactis in Intestine of White Feather Broilers

[Objective]The paper was to study the improvement of poplar bark extract on intestinal Lactococcus lactis of white feather broilers.[Method]Totally 450 Ross 308 white-feather broilers were randomly divided into five groups:control group,low dose group,medium dose group,high dose group,and antibiotic group(oxytetracycline hydrochloride).The feeding duration was 45 d.The probiotics were screened and isolated through homology,and the physiological and biochemical characteristics of chicken intestinal bacteria in different groups were compared to determine the properties of bacterial strain.The drug resistance,antibacterial ability,proliferation ability,acid resistance and bile salt resistance of isolated strain were tested,and a strain of L.lactis was obtained.[Result]The isolated L.lactis was sensitive to other drugs except natural tetracyclines,and there was no significant difference among the four groups except oxytetracycline group;as the concentration of extract increased,the inhibition of L.lactis against Salmonella sp.increased;the medium dose extract had the largest increase in the ability to tolerate the proliferation of L.lactis.[Conclusion]Feeding poplar bark extract will produce positive effects on the physiological characters of intestinal L.lactis in broiler chicken,which will provide potential probiotic strain for chicken production.

Molecular Verification of Two Potent Bacteria Isolated from Darfiyeh Cheese: <i>Lactococcus lactis</i>Subsp. <i>Lactis</i>and <i>Lactobacillus plantarum</i>

Raw goat milk cheeses are known for their natural microflora linked to many biodiversity factors such as the use of raw milk. That microflora serves probiotic attribution conferring a beneficial health impact on the consumer. Darfiyeh is an artisanal raw goat milk cheese manufactured traditionally in Northern Lebanese Mountains. To emphasize its clinical significance in both digestive and immune system, and to provide health remunerations to the consumer, the cheese microbiota will be investigated. To serve that purpose, the presence of the two potent probiotic Lactic Acid Bacteria (LAB): Lactococcus lactis subsp. lactis and Lactobacillus plantarum will be investigated. For bacterial identification, selection and isolation: culture-dependent techniques that imply the use of laboratory media will be implemented, and culture independent techniques: Polymerase Chain Reaction (PCR) will be applied for further validation. Both bacteria were further verified as Lactococcus lactis subsp. lactis and Lactobacillus plantarum by implementing specie-specific primers for the qualitative PCR.

Synergistic Effect of Watermelon Powder and Lactococcus lactis subsp lactis Supplemented Diet Partially Ameliorates Aβ42-Dependent Lifespan Shorten and Flight Impairment in Transgenic Drosophila

Oxidative stress has been strongly related with Alzheimer disease pathogenesis. We determined the effects of watermelon powder (WMP) and Lactococcus lactis subsp lactis (LAL) supplementation on the generated Aβ42-induced phenotypes in a Drosophila melanogaster model of AD. Enhanced Aβ42 expression in D. melanogaster neurons can diminish lifespan and flight ability. We have observed longevity methods to assay the effects of WMP and LAL on D. melanogaster survival. Furthermore, flies expressing Aβ42 in their body fed WMP and LAL had up to 90 days, or 35% longer median lifespan than those fed a control diet. In addition, synergistic effect of WMP and LAL improved Aβ42-induced flight impairments in the Drosophila wing tissues. Our microscope experiments revealed that individuals fed synergistic effect of WMP and LAL had ameliorated Aβ42 expression as well as increment of flight ability than Aβ42-induced flies. We propose that WMP is typically rich in L-citrulline and LAL, rich in naturally occurring probiotics and antioxidants, and that it promotes the survival of neurons in brain and wing muscle tissues with increased levels of Aβ42 via a protective cell survival mechanism.

Selective Lactococcus Enumeration in Raw Milk

The Lactococcus diversity in cow and goat raw milk was investigated. To do so, a protocol had to be established for the specific enumeration of lactococci. Eight agar media and one control medium were analysed to compare their proficiency in evaluating the Lactococcus population in raw milk: M17 Nal, Elliker, modified Elliker, PCA + milk, modified KCA, modified Chalmers, Turner, FSDA. The M17 medium was used as reference. Eighteen pure strains were tested on these media for their selectivity towards lactococci: six Lactococcus species or subspecies, three Leuconostoc, three Enterococcus, two Lactobacillus, one Streptococcus thermophilus, one Pseudomonas fluorescens, one Escherichia coli and one Staphylococcus aureus. All these bacteria were chosen for their regular presence in raw milk. The KCA medium proved to be the most selective towards lactococci, on condition that 1) we discriminated the colonies using the catalase test and 2) we subtracted the Enterococcus population counted on BEA. However, it was not possible to separate the Streptococcus from the Lactococcus colonies on KCA. The “Lactococcus-like” population including these two genera was estimated at a mean level of 3.18 log(cfu)/mL and 4.14 log(cfu)/mL in cow and goat raw milk respectively. This is consistent with the data already published.

Influence of the Backslopping Practice on the Microbial Diversity of the Lactococcus Population in a Model Cheesemaking

The objective of this work was to study the microbial diversity of natural whey starters (NWS), with a special focus on Lactococcus strains, during 10 successive days of backslopping. Each day, whey samples were enumerated for their Lactococcus, Enterococcus, Leuconostoc and Lactobacillus levels. The diversity and dynamics of the Lactococcus population in whey were investigated using phenotypic methods such as acidifying aptitude and enzymatic activities. Molecular approach using REP-PCR, ERIC-PCR, plamid profiles, phages and prophages research and PFGE was also applied. Lactococci were the main population in whey. Strains from raw milk were sub-dominant. Based on PFGE and phenotypic results lactococci in whey displayed a more heterogeneous phenotype and pulsotype which may reflect greater variations than previously observed within starter. No phages and prophages were spotted on. Plasmids did not seem to be exchanged from strain to strain. The backslopping practice seemed to allow the strains of the starter to rapidly acquire a specificity of their own. The changes observed presumed a slow adaptation of the strains to the “back- slopping environment”. The study of NWS diversity constitutes an important step for the comprehension of acidification defects that recurrently occur in cheesemaking technologies using the backslopping practice.

精氨酸代谢途径抗酸关键基因对乳酸乳球菌Lactococcus lactis NZ9000胁迫抗性的影响

【目的】寻找精氨酸代谢途径中与酸胁迫相关的关键作用因素。【方法】通过在Lactococcus lactis NZ9000中分别过量表达来源于Lactobacillus casei Zhang的精氨酰琥珀酸合成酶(ASS)和精氨酰琥珀酸裂解酶(ASL)改变精氨酸代谢提高酸胁迫抗性。【结果】与对照菌株对比,重组菌株在环境胁迫下表现了较高的生长性能、存活率和发酵性能。生理学分析发现,酸胁迫环境下,重组菌株细胞有较高的胞内NH4+、ATP含量和H+-ATPase活性,并显著提高了精氨酸脱亚胺酶(ADI)途径中的氨基酸浓度。进一步的转录分析发现,天冬氨酸合成、精氨酸代谢相关的基因转录水平上调。【结论】在L.lactis NZ9000中过量表达ASS或ASL可以引发精氨酸代谢流量的上调,进而提高了细胞的多种胁迫抗性。精氨酸合成途径广泛存在于多种微生物中,为微生物,尤其是工业微生物提高胁迫抗性提供了新思路。