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.     

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.     

Effect of Various Inhibitors on Lipase Action of Thermophilic Fungus Humicola lanuginosa S-38

The hydrolysis of olive oil by the Humicola lipase was inhibited by the addition of n-alcohols, fatty acids and surface active agents. The inhibition of n-alcohols was overcomed by the addition of more substrate but not by the addition of more enzyme. The inhibition of fatty acids and bile salts was eliminated by adding calcium ion. It was concluded that the inhibition of the Humicola lipase by n-alcohols, fatty acids and bile salts was not due to inactivation of the enzyme directly but due to the displacing of the substrate from the oil/water interface, thus blocking the enzyme from the substrate.     

Physical and Chemical Properties of the Lipase of Thermophilic Fungus Humicola lanuginosa S-38

Some physical and chemical properties of the extracellular lipase from the thermophilic fungus, Humico la lanuginosa S–38, were investigated. The results were as follows: Sedimentation coefficient was 2.4 × 10^?13 (cm-g/sec-dyne); diffusion coefficient was 8.8 × 10^?7 (cm²/sec); and frictional coefficient was 1.22. Molecular weight was 27,500±500 and α-helix content was 18.9%. The number of amino acid residues contained in 1 mole of protein of Humicola lipase was 224. Sugar and lipid were not detected. The effect of calcium ion and denaturing reagents, such as urea, sodium dodecyl sulfate and dithiothreitol, on the thermostability of Humicola lipase was examined. It was concluded that the thermostability of Humicola lipase was not influenced by protective cofactors but was attributable to the enzyme itself. Some properties of enzyme structure which were concerned with the thermostability of Humicola lipase are also discussed.     

Substrate Specificity and Mode of Action of the Lipase of Thermophilic Fungus Humicola lanuginosa S-38

Substrate specificity of the lipase of thermophilic fungus, Humicola lanuginosa S–38, was investigated. It was found that the lipolytic activity was greatly influenced by the structure of both fatty acid and alcohol moieties of the substrate. It was concluded that the hydrolysis of both water soluble and water insoluble ester was catalyzed by the Humicola lipase itself. The Humicola lipase showed no positional specificity and split ester bonds on all positions of triolein at about the same rate. Both palmitic acid (α) and linoleic acid (β) ester bonds of phosphatidyl-ethanolamine were split indicating no positional specificity of fatty acid ester bonds. From above results, it was made clear that mode of action of Humicola lipase on triolein and on phosphatidyl-ethanolamine is identical. The Humicola lipase had no activity of lipoprotein lipase.     

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).     

Purification and Properties of a Chromobacterium Lipase with a High Molecular Weight

A lipase with a high molecular weight was purified from Chromobacterium viscosum by chromatography using the Amberlite CG–50 and Sephadex G–75. The purified lipase (Lipase A) was found to be homogeneous by disc electrophoresis.

Lipase A had an optimum pH around 7 for lipolysis of olive oil and the enzyme was stable at the range of pH 4 to 9 and below 50°C. Zn²⁺, Cu²⁺, Fe³⁺ and high concentrations of l-cysteine, iodoacetic acid and NBS had remarkable inhibitory effects. Bile salts were activator. Lipase A was more active on water insoluble esters than water soluble esters. The isoelectric point of the enzyme was pH 4.7.     

Antilisterial activity of lactic acid bacteria isolated from rigouta, a traditional Tunisian cheese

AIMS: Screening for lactic acid bacteria (LAB) producing bacteriocins and other antimicrobial compounds is of a great significance for the dairy industry to improve food safety. METHODS AND RESULTS: Six-hundred strains of LAB isolated from 'rigouta', a Tunisian fermented cheese, were tested for antilisterial activity. Eight bacteriocinogenic strains were selected and analysed. Seven of these strains were identified as Lactococcus lactis and produced nisin Z as demonstrated by mass spectrometry analysis of the purified antibacterial compound. Polymerase chain reaction experiments using nisin gene-specific primers confirmed the presence of nisin operon. Plasmid profiles analysis suggests the presence of, at least, three different strains in this group. MMT05, the eighth strain of this antilisterial collection was identified, at molecular level, as Enterococcus faecalis. The purified bacteriocin produced by this strain showed a molecular mass of 10 201.33 +/- 0.85 Da. This new member of class III bacteriocins was termed enterocin MMT05. CONCLUSIONS: Seven lactococcal strains producing nisin Z were selected and could be useful as bio-preservative starter cultures. Additional experiments are needed to evaluate the promising strain MMT05 as bio-preservative as Enterococci could exert detrimental or beneficial role in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: Only a few antibacterial strains isolated from traditional African dairy products were described. The new eight strains described herein contribute to the knowledge of this poorly studied environment and constitute promising strains for fermented food safety.     

PURIFICATION AND CHARACTERIZATION OF PULLULANASE FROM Lactococcus lactis

This paper describes a simple and efficient method of isolation of a plullulanase type I from amylolytic lactic acid bacteria (ALAB). Extracellular pullulanase type I was purified from a cell-free culture supernatant of Lactococcus lactis IBB 500 by using ammonium sulfate fractionation and dialysis (instead of ultrafiltration), and ion-exchange chromatography with CM Sepharose FF followed by gel filtration chromatography with Sephadex G-150 as the final step. A final purification factor of 14.36 was achieved. The molecular mass of the enzyme was estimated as 73.9 kD. The optimum temperature for the enzyme activity was 45°C and the optimum pH was 4.5. Pullulanase activity was increased by addition Co²⁺ and completely inhibited by Hg²⁺. The enzyme activity was specifically directed toward α-1,6 glycosidic linkages of pullulan giving maltotriose units. Enzymatic hydrolysis of starch and amylose produced a mixture of maltose and maltotriose.     

多肽抗生素apidaecin基因在乳酸乳球菌中的融合表达

利用乳链菌肽(nisin)诱导表达系统,以泛素(ubiquitin)融合蛋白的形式在乳酸乳球菌(Lactococcus lactis)中表达了多肽抗生素apidaecin。利用TricineSDSPAGE和Western blotting均可在诱导后的宿主菌中检测到特异蛋白带。表达产物的最高产量可达宿主菌可溶性蛋白的7.2%左右。在体外用泛素特异性蛋白酶UBPI从融合蛋白中切除泛素后,产物具有明显的抗菌活性。     

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。     

Nuclear Magnetic Resonance Study on 5-O-Methyl-pentoses and Related Isopropylidene Hexoses

The nuclear magnetic resonance spectra of erythrose, threose and the 5-O-methyl derivatives of arabinose, xylose and lyxose were examined. In certain cases α,β-anomeric ratios in deuterium oxide might be measurable. And with erythrose evidence was obtained which indicated that 55 % of it existed in a form other than furanose form. There was a correlation between the C-l proton signals of the 5-O-methyl-pentoses and those of the stereochemically similar isopropylidene hexoses, obtained from the corresponding isopropylidene diethyl thioacetal. The evidence indicated 5,6-substitution in the diethyl thioacetals of mannose, glucose and altrose.     

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.     

Potential application of the nisin Z preparation of Lactococcus lactis W8 in preservation of milk

Aims: The aim of the study is to evaluate the effectiveness of the preparation of nisin Z from Lactococcus lactis W8‐fermented milk in controlling the growth of spoilage bacteria in pasteurized milk. Methods and Results: Spoilage bacteria isolated from pasteurized milk at 8 and 15°C were identified as Enterococcus italicus, Enterococcus mundtii, Enterococcus faecalis, Bacillus thuringiensis, Bacillus cereus, Lactobacillus paracasei, Acinetobacter sp., Pseudomonas fluorescens and Enterobacter aerogenes. These bacteria were found to have the ability to survive pasteurization temperature. Except Enterobacter aerogenes, the spoilage bacteria were sensitive to the nisin Z preparation of the L. lactis W8. Addition of the nisin Z preparation to either the skim milk or fat milk inoculated with each of the spoilage bacteria reduced the initial counts (about 5 log CFU ml^?1) to an undetectable level within 8–20 h. The nisin Z preparation extended the shelf life of milk to 2 months under refrigeration. Conclusions: The nisin Z preparation from L. lactis W8‐fermented milk was found to be effective as a backup preservative to counteract postpasteurization contamination in milk. Significance and Impact of the Study: A rapid inhibition of spoilage bacteria in pasteurized skim and fat milk with the nisin Z preparation of L. lactis W8 is more significant in comparison with the commercially available nisin (nisin A). The nisin Z preparation can be used instead of commercial nisin, which is not effective in fat milk.     

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.     

Spontaneous resistance in Lactococcus lactis IL1403 to the lantibiotic lacticin 3147

The ability and frequency at which target organisms can develop resistance to bacteriocins is a crucial consideration in designing and implementing bacteriocin-based biocontrol strategies. Lactococcus lactis ssp. lactis IL1403 was used as a target strain in an attempt to determine the frequency at which spontaneously resistant mutants are likely to emerge to the lantibiotic lacticin 3147. Following a single exposure to lacticin 3147, resistant mutants only emerged at a low frequency (10(-8)-10(-9)) and were only able to withstand low levels of the bacteriocin (100 AU mL(-1)). However, exposure to increasing concentrations, in a stepwise manner, resulted in the isolation of eight mutants that were resistant to moderately higher levels of lacticin 3147 (up to 600 AU mL(-1)). Interestingly, in a number of cases cross-resistance to other lantibiotics such as nisin and lacticin 481 was observed, as was cross-resistance to environmental stresses such as salt. Finally, reduced adsorption of the bacteriocin in to the cell was documented for all resistant mutants.     

利用PCR技术克隆乳链菌肽前体基因

乳链菌肽(Nisin)是由核糖体合成,经翻译后修饰、加工而含有稀有氨基酸的一种小肽分子。它对许多革兰氏阳性菌,包括利斯特氏菌属、梭菌属和芽孢杆菌属这些腐败苗和病原菌有强烈抑制作用。 已被50多个国家和地区广泛应用于乳制品、罐头食品、高蛋白食品及乙醇饮料的防腐保鲜,是越来越 受到人们重视的一种无毒的天然食品防腐剂⑴。对乳链菌肽分子结构与功能及生物合成遗传控制的研 究不仅有应用价值,而且有其理论意义。本实验室以产乳链菌肽的乳酸乳酸球菌ATCC 11454总DNA 为模板,利用PCR技术成功地扩增了乳链菌肽前体基因,并对其核苷酸序列进行了测定。     

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.