乳酸菌遗传育种研究现状

乳酸菌是一大类发酵糖产生大量乳酸的细菌的通称 ,主要包括乳杆菌、双歧杆菌、乳球菌、链球菌、肠球菌、明串珠菌、片球菌、芽胞杆菌等属。乳酸菌在医药、工业、农业等与人类生活密切相关的重要领域有很高的应用价值 ,如乳杆菌、双歧杆菌、嗜热链球菌发酵的酸奶 ,应用于保健食品 ;植物乳杆菌应用于青贮饲料 ;乳杆菌、双歧杆菌等应用于临床医疗。但目前很多菌种都是野生型的 ,其某些性状、功能不太令人满意。因此 ,许多研究者通过微生物遗传育种来改良野生型菌株的某些遗传性状 ,以更适合开发利用其价值。1　传统的乳酸菌菌种改良方法1 1　…     

具高抗氧化能力乳酸菌菌株的筛选与鉴定

目的筛选抗氧化能力强的乳酸菌菌株并进行菌种鉴定。方法通过清除DPPH自由基、清除超氧阴离子自由基、还原能力等实验,筛选出高抗氧化能力的乳酸菌菌株。运用16SrDNA序列分析方法,对抗氧化能力强的菌株进行鉴定。结果本研究筛选得到的抗氧化能力较强的菌株有长双歧杆菌NQ1501、La8、La5和保加利亚乳杆菌NQ2508,经16SrDNA鉴定和系统进化分析,La5鉴定为发酵乳杆菌(Lactobacillus fermentum),La8鉴定为鼠李糖乳杆菌(Lactobacillus rhamnosus)。结论本研究筛选出四株具有高抗氧化活性的乳酸菌,分别为长双歧杆菌NQ1501、鼠李糖乳杆菌La8、发酵乳杆菌La5和保加利亚乳杆菌NQ2508。     

林麝肠道乳酸菌及肠杆菌多态性分析及耐药性研究

为研究林麝肠道中乳酸菌和肠杆菌基因型及表型多样性并对其耐药性进行分析,本研究从10头健康成年林麝粪便样品中分离到22株乳酸菌和16株肠杆菌,通过16S rDNA进化分析和生理生化测定对分离菌株进行了鉴定,采用脉冲场凝胶电泳(PFGE)技术分析了分离菌株的基因型并利用药敏纸片法进行了分离菌株的耐药性研究。本研究共分离到了11种细菌,分别为大肠杆菌Escherichia coli、阴沟肠杆菌Enterobacter cloaca、河生肠杆菌E.amnigenus、肺炎克雷伯氏菌Klebsiella pneumoniae、蒙氏肠球菌Enterococcus mundtii、耐久肠球菌E.durans、明串球菌Leuconostoc fallax、植物乳杆菌Lactobacillus plantarum、嗜酸乳杆菌L.acidipiscis、屎肠球菌E.faecium和食窦魏斯氏菌Weissella cibaria。其中大肠杆菌(n=11,28.9%)是林麝肠道中的优势菌群,肠球菌(n=10,26.3%)是林麝肠道中的优势乳酸菌群。PFGE分型结果表明肠杆菌分为7个基因型,乳酸菌分为9个基因型。本研究首次分析了林麝肠道中可培养细菌的基因型及表型并且对其耐药性进行了测定,结果表明林麝肠道中的原生菌群多态性明显,并且对目前常用的抗生素敏感,未检测到耐药菌株。     

具有降胆固醇功能益生菌的筛选研究

目的通过体外实验从发酵制品及人体肠道内分离筛选出安全高效的降胆固醇乳酸菌,并研究其生理生化特性,为今后功能性乳制品的开发提供优良菌种。方法应用MRS．胆固醇培养基液体发酵法进行目的菌株筛选,并测定其耐酸及耐胆盐等能力。结果筛选出4株具有降胆固醇能力的乳酸菌,其降解率分别为DM8403163．1％、DM8403470．3％、DM850381．1％、DM8605663．1％;同时4株乳酸菌均表现出不同程度的耐酸及耐胆盐能力,其中DM86056能力最强,其他3株乳酸菌次之;经鉴定,DM84031为保加利亚乳杆菌、DM84034为嗜热链球菌、DM8503为詹氏乳杆菌、DN86056为屎肠球菌。结论经上述实验综合比较,DM86056降胆固醇能力比较强且耐酸及耐胆盐等能力也较强,因此有待于将其应用于动物体内进行深入研究。     

降胆固醇乳酸菌的应用及其机理研究进展

乳酸菌除了能够改善肠道微生态环境促进人体健康外,有些乳酸菌还具有能降低人体血清胆固醇的特性。近年来许多研究都已证明某些乳杆菌、双歧杆菌及肠球菌等具有调节血脂代谢、降低人体血清胆固醇的能力。本文综述了降胆固醇乳酸菌的体外筛选模型及其降胆固醇的作用机理,以期为进一步开发相关产品提供技术依据。     

饲料乳酸菌的分离鉴定及优良菌株筛选

对从饲料玉米、高粱、麦秆及棉花中筛选出的乳酸菌进行分类鉴定和综合性分析。用MRS+CaCO3固体培养基从棉花中分离出乳酸菌18株、高粱中30株、饲料玉米中18株、麦秆中18株。经形态学、生理生化试验进行初步鉴定并按产酸试验,耐盐及耐酸试验挑选出32株产酸率强的乳酸菌对其进行16S rDNA分子鉴定。结果显示,32株菌都具有良好的耐盐、耐酸能力;经生理生化和16S rDNA基因序列鉴定可知32株乳酸菌分属于两个属,即乳杆菌属、肠球菌属,4个种,即干酪乳杆菌(Lactobacilluscasei)、肠道球菌(Entercoccus faecium)、植物乳杆菌(Lactobacillus plantarum)、海氏肠球菌(Entercoccus hirae)。4种饲料原料中肠道球菌普遍存在。除了这种乳酸菌以外,棉花有干酪乳杆菌、植物乳杆菌、海氏肠球菌,玉米和麦秆内有植物乳杆菌。从饲料中筛选出4株具有较强产酸能力的乳酸菌,可进一步研发成青贮饲料添加剂。     

肠道菌群与肠易激综合征分型及患者血清细胞间黏附分子 1表达的关系

目的分析肠道菌群与IBS分型及患者血清细胞间黏附分子 1(ICAM 1)表达的关系,为后续研究提供参考。方法选择2018年12月至2020年1月我院收治的152例符合标准的肠易激综合征(IBS)患者作为观察组,并依照罗马Ⅲ标准将观察组患者分为腹泻型组(62例)、便秘型组(56例)、混合型组(34例);选择同期我院健康体检者30例为对照组。检测受试者粪便中双歧杆菌、乳杆菌、肠杆菌和肠球菌数量,同时检测受试者血清ICAM 1水平。采用Pearson相关性分析患者肠道菌群与血清ICAM 1关系,并采用Logistics回归模型分析肠道菌群与IBS各亚型的关系。结果观察组患者血清ICAM 1、肠道双歧杆菌、乳杆菌水平显著低于对照组,肠道肠杆菌和肠球菌水平显著高于对照组,差异均有统计学意义(均P<0.05)。不同亚型IBS患者血清ICAM 1、肠道双歧杆菌、乳杆菌、肠杆菌和肠球菌水平存在明显差异,其中混合组患者血清ICAM 1、肠杆菌、肠球菌数量最高,双歧杆菌、乳杆菌数量最低;腹泻型组肠道肠杆菌、肠球菌数量最低,双歧杆菌、乳杆菌数量最高(均P<0.05)。肠道中双歧杆菌、乳杆菌、肠杆菌、肠球菌数量是IBS分型的独立影响因素(均P<0.05)。患者肠道双歧杆菌、乳杆菌数量与ICAM 1水平呈显著负相关,而肠道肠杆菌和肠球菌数量与ICAM 1水平呈显著正相关(均P<0.05)。结论肠道菌群可影响IBS患者分型,且肠道中部分菌群与ICAM 1水平存在显著相关关系。     

葡萄藤叶青贮中乳酸菌的分离与鉴定

为了筛选出性状优良的乳酸菌菌株,本试验以实验室纯培养方式从葡萄藤叶自然发酵的青贮中分离鉴定乳酸菌。经过菌落形态、细胞形态、生理生化鉴定和16S r DNA基因序列以及系统发育树的分析,从葡萄藤叶青贮中分离出5株乳酸菌,其中菌株P-1. 2和P-2. 2为戊糖乳杆菌(Lactobacillus pentosus),菌株P-1. 7为戊糖片球菌(Pediococcus pentosaceus),菌株P-2. 1为粪肠球菌(Enterococcus faecium),菌株P-2. 3为短乳杆菌(Lactobacillus brevis)。测定产酸速率和生长速率,发现菌株P-2. 1的产酸能力和生长性能最好。     

重组粪肠球菌gshF基因对副干酪乳杆菌(Lactobacillus paracasei)L14抗逆性能的影响

乳酸菌作为重要的食品工业微生物,其在工业生产应用过程中会受到多种非生物胁迫。已有研究表明部分乳酸菌可以吸收培养基中或是自身合成谷胱甘肽(Glutathione,GSH),提高对各种胁迫的抵抗作用。克隆了粪肠球菌(Enterococcus faecalis)中的谷胱甘肽合成酶基因gshF,通过构建乳杆菌重组表达质粒,实现了gshF在副干酪乳杆菌(Lactobacillus paracasei)L14菌株中的异源表达。通过对重组gshF-副干酪乳杆菌阳性菌株的抗逆性性测定,结果表明在过氧化氢、酸、冻干脱水和渗透等胁迫条件下,gshF重组菌株的存活率与对照菌株相比均有显著提高。     

活性乳酸菌饮料对人体肠道菌群影响的研究

目的 探讨一种活性乳酸菌饮料对人体肠道菌群的影响作用.方法 采用卫生部《保健食品检验与评价技术规范》调节肠道菌群功能检验方法进行检验.结果 试食组试验前后自身比较：肠道内双歧杆菌、乳杆菌数量明显增加（P＜0.01）,肠球菌数量增加（P＜0.05）,产气荚膜梭菌数量明显减少（P＜0.01）,肠杆菌和拟杆菌无明显变化;试验后对照组与试食组组间比较：双歧杆菌数量明显增加（P＜0.01）,乳杆菌的数量增加（P＜0.05）,肠杆菌、肠球菌、产气荚膜梭菌和拟杆菌无明显变化.结论 活性乳酸菌饮料具有调节人体肠道菌群、改善肠道内环境的作用.     

Foot-and-mouth disease virus Lb proteinase can stimulate rhinovirus and enterovirus IRES-driven translation and cleave several proteins of cellular and viral origin.

Rhinovirus and enterovirus 2A proteinases stimulate translation initiation driven from the cognate internal ribosome entry segment (IRES) (S. J. Hambidge and P. Sarnow, Proc. Natl. Acad. Sci. USA 89:10272-10276, 1992; H.-D. Liebig, E. Ziegler, R. Yan, K. Hartmuth, H. Klump, H. Kowalski, D. Blaas, W. Sommergruber, L. Frasel, B. Lamphear, R. Rhoads, E. Kuechler, and T. Skern, Biochemistry 32:7581-7588, 1993). Given the functional similarities between the foot-and-mouth disease virus (FMDV) L proteinase and these 2A proteinases (autocatalytic excision from the nascent viral polyprotein and cleavage of eIF-4 gamma), we investigated whether the FMDV L proteinase would also be able to stimulate translation initiation. We found that purified recombinant FMDV Lb proteinase could stimulate in vitro translation driven from a rhinovirus or enterovirus IRES by 5- to 10-fold. In contrast, stimulation of translation initiation on a cardiovirus IRES by this proteinase was minimal, and stimulation of translation driven from the cognate FMDV IRES could not be evidenced. Studies using an inhibitor or a mutant Lb proteinase indicated that stimulation of IRES-driven translation is mediated via proteolysis of some cellular component(s). Our studies also demonstrated that the Lb proteinase is capable of stimulating initiation of translation on an uncapped cellular message. Unexpectedly, and in contrast to the 2A proteinases, the Lb proteinase specifically cleaved the products of the two reporter genes used in this study: Xenopus laevis cyclin B2 and influenza virus NS. Therefore, we also set out to investigate the requirements for substrate recognition by the Lb proteinase. Purified recombinant Lb proteinase recognized at least one mengovirus polypeptide and specifically cleaved human cyclin A and poliovirus replicase-related polypeptides. In the latter case, the site(s) of cleavage was located within the N-terminal part of polypeptide 3D. Sequence comparisons revealed no significant primary sequence similarities between the target proteins and the two sites already known to be recognized by the FMDV L proteinase.     

Antibacterial activity of Lactobacillus sake isolated from meat

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Wide-Inhibitory Spectra Bacteriocins Produced by Lactobacillus gasseri K7

The aim of our study was to determine the genetic characterization and classification of Lb. gasseri K7 bacteriocins, comparison with bacteriocins of the Lb. gasseri LF221 strain and other related strains. Bacteriocin-encoding genes were amplified by PCR, subjected to DNA sequencing, and BLAST sequence analysis was performed to search the database for homologous peptides. Lb. gasseri K7 produces two two-peptide bacteriocins, named gassericin K7 A and gassericin K7 B. Their nucleotide sequences were deposited at GenBank, under accession numbers EF392861 for the gassericin K7 A and AY307382 for the gassericin K7 B. Analysis of gene clusters of bacteriocins in Lb. gasseri K7 strain revealed a 100 percent sequence identity with bacteriocins in LF221 strain. An active peptide of gassericin K7 B is homologous to the complementary peptide of gassericin T, and a complementary peptide of gassericin K7 B is homologous to the active peptide of gassericin T. Another surprising finding was that the sakacin T-beta peptide is partly homologous to the active peptide of gassericin K7 A, while the other sakacin T peptide (alfa) is partly homologous to the complementary peptide of gassericin K7 B. Gassericins of Lb. gasseri K7 strain were both classified as two-peptide bacteriocins. Human probiotic strains Lb. gasseri K7 and LF221 are different isolates but with identical bacteriocin genes. They produce wide-inhibitory spectra bacteriocins that are new members of two-peptide bacteriocins with some homologies to other bacteriocins in this group. Described bacteriocins offer a great potential in applications in food industry, pharmacy and biomedicine.     

Ferric Leghemoglobin in Plant-Attached Leguminous Nodules

Leghemoglobin (Lb) is essential for nitrogen fixation by intact leguminous nodules. To determine whether ferric Lb (Lb3+) was detectable in nodules under normal or stressed conditions, we monitored the status of Lb in intact nodules attached to sweet clover (Melilotus officinalis) and soybean (Glycine max [L.] Merr.) roots exposed to various conditions. The effects of N2 and O2 streams and elevated nicotinate levels on root-attached nodules were tested to determine whether the spectrophotometric technique was showing the predicted responses of Lb. The soybean and sweet clover nodules' Lb spectra indicated predominantly ferrous Lb and LbO2 in young (34 d) plants. As the nodule aged beyond 45 d, it was possible to induce Lb3+ with a 100% O2 stream (15 min). At 65 d without inducement, the nodule Lb status indicated the presence of some Lb3+ along with ferrous Lb and oxyferrous Lb. Nicotinate and fluoride were used as ligands to identify Lb3+. Computer-calculated difference spectra were used to demonstrate the changes in Lb spectra under different conditions. Some conditions that increased absorbance in the 626 nm region (indicating Lb3+ accumulation) were root-fed ascorbate and dehydroascorbate, plant exposure to darkness, and nodule water immersion.     

Antibacterial activity of Lactobacillus sake isolated from meat.

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Effects of Leghaemoglobin Components on Nitrogen Fixation and Oxygen Consumption

The effects of purified oxyleghaemoglobin components added toa suspension of bacteroids from soybean and pea root noduleprepared anaerobically were studied in terms of nitrogen fixationand oxygen consumption. Soybean leghaemoglobin components (Lba and Lb c) and pea leghaemoglobin components (Lb I and Lb IV)have different O₂-binding affinities. Lb a and Lb IV showedhigher O₂-binding affinities than Lb c and Lb I. When anaerobicallyprepared bacteroids were incubated with these leghaemoglobincomponents separately under low oxygen tension and in the presenceof a reduction system, Lb a and Lb IV were more effective forboth nitrogen fixation and oxygen consumption than Lb c andLb I. These results suggest that leghaemoglobin components participatein more effective nitrogen fixation by controlling oxygen transportto bacteroids. (Received July 7, 1981; Accepted November 2, 1981)     

Physiological Role of Leghaemoglobin Heterogeneity in Pea Root Nodule Development

Disk-gel-electrophoresis of the leghaemoglobin isolated frompea root nodules revealed two major (Lb I and Lb IV) and threeminor components. The ratio of the two major components (LbI/Lb IV) decreased with increasing age. This ratio was higherin the distal than in proximal region when nodules were cutinto distal and proximal sections. In young nodules, the incorporationof radioactive leucine into Lb I was much higher than into LbIV. In older nodules, the radioactivities were much higher inLb IV than in Lb I. These data suggest that the change in thisratio is due mainly to differences in the rates of biosynthesis. Two major components (Lb I and Lb IV) which were isolated separatelyhad different O₂-binding affinities. The O₂-binding affinityof the component synthesized mainly in older nodules (Lb IV)was higher than that of the component synthesized mainly inyoung nodules (Lb I). These results indicate that changes inthe relative contents of leghaemoglobin during nodule developmentcontribute to more effective nitrogen fixation which is mediatedby changes in the capacities of oxygen transport. (Received July 7, 1981; Accepted November 2, 1981)     

Ultrastructural changes of the intracellular surfactant pool in a rat model of lung transplantation-related events

Background

Ischemia/reperfusion (I/R) injury, involved in primary graft dysfunction following lung transplantation, leads to inactivation of intra-alveolar surfactant which facilitates injury of the blood-air barrier. The alveolar epithelial type II cells (AE2 cells) synthesize, store and secrete surfactant; thus, an intracellular surfactant pool stored in lamellar bodies (Lb) can be distinguished from the intra-alveolar surfactant pool. The aim of this study was to investigate ultrastructural alterations of the intracellular surfactant pool in a model, mimicking transplantation-related procedures including flush perfusion, cold ischemia and reperfusion combined with mechanical ventilation.

Methods

Using design-based stereology at the light and electron microscopic level, number, surface area and mean volume of AE2 cells as well as number, size and total volume of Lb were determined in a group subjected to transplantation-related procedures including both I/R injury and mechanical ventilation (I/R group) and a control group.

Results

After I/R injury, the mean number of Lb per AE2 cell was significantly reduced compared to the control group, accompanied by a significant increase in the luminal surface area per AE2 cell in the I/R group. This increase in the luminal surface area correlated with the decrease in surface area of Lb per AE2. The number-weighted mean volume of Lb in the I/R group showed a tendency to increase.

Conclusion

We suggest that in this animal model the reduction of the number of Lb per AE2 cell is most likely due to stimulated exocytosis of Lb into the alveolar space. The loss of Lb is partly compensated by an increased size of Lb thus maintaining total volume of Lb per AE2 cell and lung. This mechanism counteracts at least in part the inactivation of the intra-alveolar surfactant.