生长抑制剂所诱导的乳酸乳球菌MG1363的自溶表型及其生长阶段特异的性质

【目的】自溶是细菌在压力环境下通过自身裂解而获得的一种生理适应现象,研究的目的是全面探讨乳酸菌株在生长抑制剂条件下的自溶表型及机理。【方法】对多种来源的乳酸菌株的自溶能力进行检测,通过在不同生长条件和抑制剂压力条件下乳酸乳球菌MG1363的生长检测对其自溶表型进行分析。【结果】在葡萄糖严格受限的培养基中,氨苄青霉素的加入能够显著诱导MG1363的自溶,而且该自溶现象只发生在葡萄糖耗尽的时间点,展现出一种狭窄的生长时期依赖的特征。与此同时,因为氨苄青霉素的加入,4种主要的自溶酶的表达都发生了不同程度的显著改变。此外,所有受试的抑制剂都削弱了MG1363在非营养条件下的自溶,表明该菌株可能具有一种涉及细胞壁合成和降解酶的共同调控的模式。【结论】乳酸菌株在不同生长抑制剂条件下的自溶表型存在很大差异,且该自溶体现出营养条件和生长时期严格依赖的特征。     

Partial characterization of bacteriocins produced by human Lactococcus lactis and Pediococccus acidilactici isolates

AIMS: The aim of this study was to isolate bacteriocin-producing lactic acid bacteria (LAB) from human intestine. METHODS AND RESULTS: A total of 111 LAB were isolated from human adult stool and screened for their bacteriocin production. Neutralized cell-free supernatants from Lactococcus lactis subsp. lactis MM19 and Pediococcus acidilactici MM33 showed antimicrobial activity. The antimicrobials in the supernatant from a culture of L. lactis inhibited Enterococcus faecium, various species of Lactobacillus and Staphylococcus aureus; while those in the supernatant from a culture of P. acidilactici inhibited Enterococcus spp., some lactobacilli and various serotypes of Listeria monocytogenes. The antimicrobial metabolites were heat-stable and were active over a pH range of 2-10. The antimicrobial activities of the supernatants of both bacteria were inhibited by many proteases but not by catalase. The plate overlay assay allowed an approximation of size between 3.5 and 6 kDa for both antimicrobial substances. CONCLUSIONS: As the antagonistic factor(s) produced by L. lactis MM19 and P. acidilactici MM33 were sensitive to proteolytic enzymes, it could be hypothesized that bacteriocins were involved in the inhibitory activities. Inhibition spectrum and biochemical analysis showed that these bacteria produced two distinct bacteriocins. SIGNIFICANCE AND IMPACT OF THE STUDY: We are the first to isolate bacteriocin-producing strains of Pediococcus and Lactococcus from human intestine. These strains might be useful for control of enteric pathogens.     

Mutation of the oxaloacetate decarboxylase gene of Lactococcus lactis subsp. lactis impairs the growth during citrate metabolism

Aims:  Citrate metabolism generates metabolic energy through the generation of a membrane potential and a pH gradient. The purpose of this work was to study the influence of oxaloacetate decarboxylase in citrate metabolism and intracellular pH maintenance in relation to acidic conditions.
Methods and Results:  A Lactococcus lactis oxaloacetate decarboxylase mutant [ILCitM (pFL3)] was constructed by double homologous recombination. During culture with citrate, and whatever the initial pH, the growth rate of the mutant was lower. In addition, the production of diacetyl and acetoin was altered in the mutant strain. However, our results indicated no relationship with a change in the maintenance of intracellular pH. Experiments performed on resting cells clearly showed that oxaloacetate accumulated temporarily in the supernatant of the mutant. This accumulation could be involved in the perturbations observed during citrate metabolism, as the addition of oxaloacetate in M17 medium inhibited the growth of L. lactis .
Conclusions:  The mutation of oxaloacetate decarboxylase perturbed citrate metabolism and reduced the benefits of its utilization during growth under acidic conditions.
Significance and impact of the study:  This study allows a better understanding of citrate metabolism and the role of oxaloacetate decarboxylase in the tolerance of lactic acid bacteria to acidic conditions.     

Phenotypic variation in Lactococcus lactis subsp. lactis isolates derived from intestinal tracts of marine and freshwater fish

Aims:  We compared phenotypic characteristics of Lactococcus lactis subsp. lactis derived from different sources including the intestinal tract of marine fish and freshwater fish, and cheese starter culture.
Methods and Results:  In the phylogenetic analysis based on partial 16S rRNA gene nucleotide sequences (1371 bp), freshwater fish-, marine fish- and cheese starter culture-derived strains were identical to that of L. lactis subsp. lactis previously reported. Fermentation profiles determined using the API 50 CH system were similar except for fermentation of several sugars including l -arabinose, mannitol, amygdalin, saccharose, trehalose, inulin and gluconate. The strains did have distinct levels of halotolerance: marine fish-derived strains > cheese starter-derived strain > freshwater fish-derived isolate.
Conclusions:  Lactococcus lactis subsp. lactis showed extensive diversity in phenotypic adaptation to various environments. The phenotypic properties of these strains suggested that L. lactis subsp. lactis strains from fish intestine have additional functions compared with the cheese starter-derived strain that has previously described.
Significance and Impact of the Study:  The unique phenotypic traits of the fish intestinal tract-derived L. lactis subsp. lactis might make them useful as a probiotics in aquaculture, and contribute to the development of functional foods and novel food additives, since the strains derived from fish intestines might have additional functions such as antibacterial activity.     

Production of biological active murine IFN-γ by recombinant Lactococcus lactis

IFN-γ is a cytokine produced primarily by both T lymphocytes and natural killer cells and it is considered to be an attractive therapeutic molecule. In the present study, a DNA sequence encoding the mature murine IFN-γ (muIFN-γ) protein was cloned and expressed in the food-grade lactic acid bacterium Lactococcus lactis . The activity of recombinant muIFN-γ produced by genetically engineered L. lactis was confirmed in an antiviral assay using MoV cells infected with Vesicular Stomatitis Virus. The data provide the first demonstration that a Gram-positive bacterium, L. lactis , is able to produce functional muIFN-γ. This recombinant strain could lead to the development of a new, well-tolerated vector to deliver active muIFN-γ at the mucosal level.     

Alternatives for biosurfactants and bacteriocins extraction from Lactococcus lactis cultures produced under different pH conditions

Aims: Study of the potential of Lactococcus lactis CECT‐4434 as a biosurfactants and nisin (the only bacteriocin allowed to be used in the food industry) producer for industrial applications, exploiting the possibility of recovering separately both metabolites, taking into account that L. lactis is an interesting micro‐organism with several applications in the food industry because it is recognized as GRAS. Methods and Results: The results showed the ability of this strain to produce cell‐bound biosurfactants, under controlled pH, and cell‐bound biosurfactants and bacteriocins, when pH was not controlled. Three extraction procedures were designed to separately recover these substances. Conclusions: The strain L. lactis CECT‐4434 showed to be a cell‐bound biosurfactants and bacterocins producer when fermentations were carried out under uncontrolled pH. Both products can be recovered separately. Significance and Impact of the Study: Development of a convenient tool for the extraction of cell‐bound biosurfactants and bacteriocins from the fermentation broth.     

Biosynthetic characterization and biochemical features of the third natural nisin variant,nisin Q,produced by Lactococcus lactis 61‐14

Aims: To characterize the genetic and biochemical features of nisin Q. Methods and Results: The nisin Q gene cluster was sequenced, and 11 putative orfs having 82% homology with the nisin A biosynthesis gene cluster were identified. Nisin Q production was confirmed from the nisQ‐introduced nisin Z producer. In the reporter assay, nisin Q exhibited an induction level that was threefold lower than that of nisin A. Nisin Q demonstrated an antimicrobial spectrum similar to those of the other nisins. Under oxidizing conditions, nisin Q retained a higher level of activity than nisin A. This higher oxidative tolerance could be attributed to the presence of only one methionine residue in nisin Q, in contrast to other nisins that contain two. Conclusions: The 11 orfs of the nisin producers were identical with regard to their functions. The antimicrobial spectra of the three natural nisins were similar. Nisin Q demonstrated higher oxidative tolerance than nisin A. Significance and Impact of the Study:  Genetic and biochemical features of nisin Q are similar to those of other variants. Moreover, owing to its higher oxidative tolerance, nisin Q is a potential alternative for nisin A.     

Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis

Aims: To improve the production of sweet‐tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions. Methods and Results: The nisin‐controlled expression system was used for brazzein expression. The concentration of nisin for induction and the optical density (OD) at induction were therefore optimized, together with growth conditions (medium composition, pH, aerobic growth in the presence of hemin). Brazzein was assayed with ELISA on Ni‐NTA plates and Western blot. Use of the M‐17 medium, containing 2·5% glucose, anaerobic growth at pH 5·9 and induction with 40 ng ml^?1 nisin at OD 3·0 led to an approx. 17‐fold increase in brazzein per cell production compared to non‐optimized starting conditions. Aerobic growth in the presence of hemin did not increase the production. Conclusions: Considerable increase in brazzein per cell production was obtained at optimized fermentation conditions. Significance and Impact of the Study: Optimized growth conditions could be used in application of brazzein expression in L. lactis. The importance of pH and OD at induction contributes to the body of knowledge of optimal recombinant protein expression in L. lactis. The new assay for brazzein quantification was introduced.     

Increased stress tolerance of Bifidobacterium longum and Lactococcus lactis produced during continuous mixed-strain immobilized-cell fermentation

AIMS: The effect of immobilization and long-term continuous culture was studied on probiotic and technological characteristics of lactic acid and probiotic bacteria. METHODS AND RESULTS: A continuous culture in a two-stage system was carried out for 17 days at different temperatures ranging from 32 to 37 degrees C, with a first reactor containing Bifidobacterium longum ATCC 15707 and Lactococcus lactis subsp. lactis biovar. diacetylactis MD immobilized separately in gel beads, and a second reactor operated with free cells released from the first reactor. The tolerance of free cells from both strains produced in the effluent medium of both reactors to hydrogen peroxide, simulated gastric and intestinal juices, antibiotics and nisin, and freeze-drying markedly increased with culture time and was generally higher after 6 days than that of stationary-phase cells produced during free-cell batch fermentations. The reversibility of the acquired tolerance of B. longum, but not L. diacetylactis, to antibiotics was shown during successive free-cell batch cultures. CONCLUSIONS: Free cells produced from continuous immobilized-cell culture exhibited altered physiology and increased tolerance to various chemical and physico-chemical stresses. SIGNIFICANCE AND IMPACT OF THE STUDY: Continuous culture with immobilized cells could be used to produce probiotic and lactic acid bacteria with enhanced technological and probiotic characteristics.     

Improvement of phage defence in Lactococcus lactis by introduction of the plasmid encoded restriction and modification system LlaAI

AIMS: To study the ability of the plasmid-encoded restriction and modification (R/M) system LlaAI to function as a bacteriophage resistance mechanism in Lactococcus lactis during milk fermentations. METHODS AND RESULTS: Plasmid pAIcat4, carrying the R/M system LlaAI and a chloramphenicol resistance cassette, was introduced into the plasmid-free strain L. lactis MG1614 and the industrial strain L. lactis 964. By measuring changes in conductivity the influence of different phage on the growth was determined. CONCLUSIONS: The plasmid-encoded R/M system LlaAI significantly improves the bacteriophage resistance of L. lactis during milk fermentations. SIGNIFICANCE AND IMPACT OF THE STUDY: It is essential to determine the potential of a phage defence mechanism in L. lactis starter culture strains during growth in milk before steps are taken to improve starter cultures. This study shows that LlaAI is useful for improvement of starter cultures.     

Neutralization/recovery of lactic acid from Lactococcus lactis: effects on biomass,lactic acid,and nisin production

Besides lactic acid, many lactic acid bacteria also produce proteinaceous metabolites (bacteriocins) such as nisin. As catabolite repression and end-product inhibition limit production of both products, we have investigated the use of alternative methods of supplying substrate and neutralizing or extracting lactic acid to increase yields. Fed-batch fermentation trials using a stillage-based medium with pH control by NH4OH resulted in improved lactic acid (83.4 g/l, 3.18 g/l/h, 95% yield) and nisin (1,260 IU/ml, 84,000 IU/l/h, 14,900 IU/g) production. Removing particulate matter from the stillage-based medium increased nisin production (1,590 IU/ml, 33,700 IU/g), but decreased lactic acid production (58.5 g/l, 1.40 g/l/h, 96% yield). Removing lactic acid by ion exchange resins stimulated higher lactic acid concentrations (60 to 65 g/l) and productivities (2.0 to 2.6 g/l/h) in the filtered stillage medium at the expense of nisin production (1,500 IU/ml, 25,800 IU/g).     

Acidic proteome of growing and resting Lactococcus lactis metabolizing maltose

The acidic proteome of Lactococcus lactis grown anaerobically was compared for three different growth conditions: cells growing on maltose, resting cells metabolizing maltose, and cells growing on glucose. In maltose metabolizing cells several proteins were up-regulated compared with glucose metabolizing cells, however only some of the up-regulated proteins had apparent relation to maltose metabolism. Cells growing on maltose produced formate, acetate and ethanol in addition to lactate, whereas resting cells metabolizing maltose and cells growing on glucose produced only lactate. Increased levels of alcohol-acetaldehyde dehydrogenase (ADH) and phosphate acetyltransferase (PTA) in maltose-growing cells compared with glucose-growing cells coincided with formation of mixed acids in maltose-growing cells. The resting cells did not grow due to lack of an amino acid source and fermented maltose with lactate as the sole product, although ADH and PTA were present at high levels. The maltose consumption rate was approximately three times lower in resting cells than in exponentially growing cells. However, the enzyme levels in resting and growing cells metabolizing maltose were similar, which indicates that the difference in product formation in this case is due to regulation at the enzyme level. The levels of 30S ribosomal proteins S1 and S2 increased with increasing growth rate for resting cells metabolizing maltose, maltose-growing cells and glucose-growing cells. A modified form of HPr was synthesized under amino acid starvation. This is suggested to be due to alanine misincorporation for valine, which L. lactis is auxotrophic for. L. lactis conserves the protein profile to a high extent, even after prolonged amino acid starvation, so that the protein expression profile of the bacterium remains almost invariant.     

硬脂酰-辅酶A脱氢酶基因在食品级乳酸菌中的表达

为了实现硬脂酰-辅酶A脱氢酶1编码基因在乳酸乳球菌中的表达,采用PCR技术扩增获得人类scd1的编码序列。Nco I和Xba I双酶切后定向插入到食品级表达载体pNZ8149中,构建表达载体pNZ8149-scd1。电转化乳酸乳球菌NZ3900,经菌落PCR和测序鉴定scd1基因成功插入到乳酸乳球菌中。在乳链菌肽诱导下进行scd1的表达,转化株提取脂肪酸,进行脂肪酸含量的气相色谱分析。结果显示,SCD1转化菌株中的C16∶1n-7和C18∶1n-7脂肪酸组分比转化pNZ8149的对照组乳酸菌分别提高了92%~169%和53%~127%。文中以scd1基因为例,尝试并证明了脂肪酸脱氢酶类基因能够在食品级乳酸菌中有效表达,为后续研究奠定了基础。     

Large increase in brazzein expression achieved by changing the plasmid /strain combination of the NICE system in Lactococcus lactis

Aims:  To evaluate brazzein production in Lactococcus lactis using the nisin-controlled expression (NICE) system. The approach is through analysis of different plasmid/strain combinations.
Methods and Results:  Two plasmid/strain combinations of the NICE system were used in brazzein expression: L. lactis NZ9000 harbouring plasmid pNZ8148, and L. lactis IL1403 harbouring plasmid pMSP3545. The former combination proved superior, with a >800-fold increase in His-tagged brazzein expression (to 1·65 mg l⁻¹ of fermentation broth), comparable to expression levels in Escherichia coli . Improved expression resulted in a minor increase in secretion to the medium with the use of the Usp45 signal peptide. The yield of wild-type brazzein corresponded to that of His-tagged brazzein. Wild-type brazzein was partially soluble and low-intensity sweetness was detected.
Conclusions:  The plasmid/strain combination of the NICE system has a significant impact on the expression of brazzein where a >800-fold increase was achieved. The greatly increased expression of brazzein resulted in minor improvement in secretion and low-intensity sweetness.
Significance and Impact of the Study:  The choice of the plasmid/strain combination of the NICE system was shown to be of extreme importance in brazzein expression.     

Conversion of methionine to methional by Lactococcus lactis

Lactic acid bacteria were screened for methional production from 4-methylthio-2-ketobutanoate. Only Lactococcus lactis IFPL730 produced high amounts of methional. It was demonstrated that production of this compound was an exclusively enzymatic reaction. The present work describes for the first time that L. lactis can convert enzymatically methionine to methional in a process mediated by aminotransferase and alpha-ketoacid decarboxylase activities. The activity seems to be strain dependent.     

谷胱甘肽在乳酸乳球菌抵抗氧胁迫中的保护作用

为研究谷胱甘肽(GSH)在乳酸乳球菌NZ9000抗氧胁迫中的生理作用,以能够生物合成GSH的重组菌NZ9000(pNZ3203)为实验菌株进行了研究。结果表明,在较高H2O2胁迫剂量(150mmol/L H2O2,15min)下,前培养3h、5h和7h(即乳酸链球菌素诱导1h、3h和5h)时的重组菌细胞的存活率分别是处于相应生长时期对照菌NZ9000(pNZ8148)的1.8±0.1倍、2.6±0.1倍和2.9±0.3倍。表明GSH可以提高宿主菌NZ9000对H2O2所引发氧胁迫的抗性。GSH还可以提高宿主菌NZ9000对其它化学物质(如超氧阴离子自由基生成剂———甲萘醌)所引发氧胁迫的抗性。这表现在经20mmol/L甲萘醌处理60min后,前培养5h(即乳酸链球菌素诱导3h)时重组菌细胞的存活率是对照菌的6.2±0.1倍。由此表明,通过代谢工程手段在菌株NZ9000中引入GSH合成能力,可以提高宿主菌对氧胁迫的抗性。     

Probiotic properties of Lactococcus lactis ssp. lactis HV219, isolated from human vaginal secretions

AIMS: To determine the resistance of Lactococcus lactis ssp. lactis HV219 to acids, bile, antibiotics, inflammatory drugs and spermicides, compare adsorption of the strain to bacteria and Caco-2 cells under stress, and evaluate the antimicrobial activity of bacteriocin HV219. METHODS AND RESULTS: Bacteriocin HV219 activity against Gram-positive and Gram-negative bacteria was confirmed by leakage of DNA and beta-galactosidase, and atomic force microscopy. Adsorption of bacteriocin HV219 to bacteria is influenced by pH, temperature, surfactants and salts. Initially, only 3% of HV219 cells adhered to Caco-2 cells. However, after 2 h, adherence increased to 7%. Strain HV219 and Listeria monocytogenes ScottA did not compete for colonization. Strain HV219 is sensitive to most antibiotics tested, but resistant to amikacin, ceftazidime, nalidixic acid, metronidazole, neomycin, oxacillin, streptomycin, sulphafurazole, sulphamethoxazole, sulphonamides, tetracycline and tobramycin. Ibuprofen, ciprofloxacin, diklofenak and nonoxylol-9 inhibited the growth of strain HV219. CONCLUSION: Strain HV219 is resistant to hostile conditions in the intestinal tract, including therapeutic levels of specific antibiotics and binds to Caco-2 cells, but not in competition with L. monocytogenes. SIGNIFICANCE AND IMPACT OF THE STUDY: Strain HV219 will only be effective as probiotic if taken with specific antibiotics and not with anti-inflammatory drugs and spermicides.     

Rapid plasmid DNA isolation from Lactococcus lactis using overnight cultures

A simple and quick method has been developed to isolate plasmid DNA from Lactococcus lactis using overnight or stationary-phase cultures which therefore eliminates the need for subculturing for generating log-phase cultures that are necessary with existing methods. The new method was effective in isolating plasmids, from 1.4 to 64 kb, from the three subspecies of Lactococcus lactis. The resultant DNA was of high yield and purity and therefore no additional purification steps were required for down-stream molecular procedures.     

Proteomic analysis of Lactococcus lactis,a lactic acid bacterium

Lactococcus lactis is a Gram-positive bacteria, which belongs to the group of lactic acid bacteria among which several genera play an essential role in the manufacture of food products. Cytosolic proteins of L. lactis IL1403 cultivated in M17 broth have been resolved by two-dimensional gel electrophoresis using two pH gradients (pH 4-7, 4.5-5.5). More than 230 spots were identified by peptide mass fingerprints, corresponding to 25% of the predicted acid proteome. The present study made it possible to describe at the proteome level a significant number of cellular pathways (glycolysis, fermentation, nucleotide metabolism, proteolysis, fatty acid and peptidoglycan synthesis) related to important physiological processes and technological properties. It also indicated that the fermentative metabolism, which characterizes L. lactis is associated with a high expression of glycolytic enzymes. Thirty-four proteins were matched to open reading frames for which there is no assigned function. The comparison at the proteome level of two strains of L. lactis showed an important protein polymorphism. The comparison of the proteomes of glucose- and lactose-grown cells revealed an unexpected link between the nature of the carbon source and the metabolism of pyrimidine nucleotides.