Growth and energy generation by Enterococcus faecium FAIR-E 198 during citrate metabolism

Citrate metabolism by Enterococcus faecium FAIR-E 198, isolated from Greek Feta cheese, was studied in various growth media containing citrate either in the presence of glucose, or as the sole carbon source, both under aerobic and anaerobic conditions. In de Man-Rogosa-Sharpe (MRS) broth with increasing citrate concentrations, cometabolism of citrate and glucose took place. Glucose was stoichiometrically converted into lactate, while citrate into acetate. Glucose consumption and biomass yield were enhanced with increasing initial citrate concentrations, even though maximum specific growth rate was not. When citrate was used as the sole carbon source in increasing initial concentrations, the main end product was acetate. Small amounts of lactate, formate, ethanol, and acetoin were also produced. In all cases, no significant differences were observed between aerobic and anaerobic conditions. However, when citrate was used as sole carbon source, formate production was favored in the absence of oxygen. The present work shows that E. faecium is able to utilize citrate in synthetic media, either in the presence of glucose or as the sole carbon source, resulting in energy production and the formation of aroma compounds.     

环境因素对屎肠球菌产酪胺的影响

酪胺是发酵食品中广泛存在的、毒性较强的一种生物胺。屎肠球菌作为发酵剂或污染菌广泛存在于发酵食品中,大多数具有产生酪胺的能力,成为发酵食品的潜在安全隐患。该研究以一株黄酒酒曲中分离的产酪胺屎肠球菌为对象,选取葡萄糖、酪氨酸、温度、pH值和酒精含量5个因素,研究这些因素对该菌生长和酪胺合成的影响。结果表明,葡萄糖或酪氨酸添加量对菌体的生长影响不大,温度、环境pH值和酒精含量对菌体生长的影响较显著。酪氨酸对菌体产酪胺促进作用明显,温度＜20 ℃,环境pH值＞5,酒精含量＞10%vol时都会减少酪胺的产生。     

Factors influencing the synthesis of an extracellular proteinase by Enterococcus faecalis subsp. liquefaciens.

Studies were conducted on optimum temperature, pH and the requirement for an energy source, amino acids, casein, Zn2+ and Ca2+ during the synthesis of an extracellular acid proteinase by Enterococcus faecalis var. liquefaciens. Synthesis was monitored using cells grown to mid-logarithmic phase and resuspended at high density in fresh growth medium. Proteinase production was optimal at 30 degrees C and pH 7.0. Proteinase synthesis, being energy-dependent, occurred only in glycolysing cells. The synthesis was high when lactose but not glucose was utilized as a source of energy, indicating that phospho-beta-galactosidase gene might probably be located directly upstream the proteinase gene on a plasmid. Good induction of proteinase synthesis could be achieved by 0.2-0.5% of either yeast extract or tryptic digested casein, perhaps due to its content of a wide variety of free amino acids. Casein was essential for preventing proteinase autolysis and sustaining the enzyme production. Zn2+ and Ca2+ were required for the formation of an active extracellular proteinase. The synthesis immediately ceased after addition of chloramphenicol or EDTA. EDTA inactivated the preformed proteinase as well. Sodium chloride at a concentration of 6.5% inhibited both proteinase synthesis and glycolysis.     

The metabolism of several carboxylic acids by lactic acid bacteria

The anaerobic metabolism of citrate, fumarate, gluconate, malate, 2-oxoglutarate and pyruvate by 137 strains of 23 species of lactic acid bacteria was investigated. The bacteria were from various sources (plant material, meat and dairy products, dough and wine) and belonged to the genera Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus. The ability of metabolize the acids was determined by thin layer chromatography or by enzymatic analysis after growth of the strains in a glucose-containing medium. All strains metabolized pyruvate and only 12 mainly heterofermentative strains were malate negative. These strains were also unable to decompose citrate. This acid was fermented by 23 strains, all of which metabolized malate. Many lactic acid bacteria reduced 2-oxoglutarate to hydroxyglutarate. The strains of Lactobacillus plantarum did not metabolize 2-oxoglutarate whereas all strains of Leuconostoc oenos decarboxylated this acid and formed 4-hydroxybutyrate and succinate. Gluconate was fermented by 52 mainly heterofermentative strains. No correlation was observed between the ability to ferment citrate, malate or gluconate.     

产细菌素屎肠球菌TRS5特性分析及其编码基因的扩增

屎肠球菌TRS5在37℃、p H 6.5的MRS培养基中经过24 h的培养后,其细菌素生成量达到最大。培养基中添加胰蛋白胨或葡萄糖有利于促进TRS5细菌素的生成,而添加麦芽糖、乳糖或甘露糖(20 g/L)后细菌素活性减少50%。外源添加5 g/L的甘油和吐温-80会抑制TRS5细菌素的产生,而添加K_2HPO_4或VB_1、VB_2、VB_6、VC则对细菌素的生成没有影响。药敏实验证实屎肠球菌TRS5对红霉素、氯霉素、万古霉素、替考拉宁、四环素、青霉素敏感。聚合酶链式反应及测序结果证实屎肠球菌TRS5含有肠球菌素enterocin P和类L50的结构基因。     

凝结芽孢杆菌混合碳源乳酸发酵研究

为了更好的研究工业乳酸发酵中的分解代谢物阻遏效应,在实验室规模反应器对凝结芽孢杆菌混合碳源乳酸发酵进行不同通气量、p H、中和剂等条件的发酵特性研究,并研究了不同金属离子、渗透压等条件对饥饿状态下凝结芽孢杆菌自溶的影响。结果表明：凝结芽孢杆菌葡萄糖+海藻糖混合碳源乳酸发酵呈现明显的分解代谢物阻遏效应,且发酵可分为葡萄糖消耗阶段、有机酸消耗阶段和海藻糖消耗阶段。不同发酵条件在葡萄糖消耗阶段对菌体生长和乳酸生成影响较小,但对发酵后期海藻糖利用阶段菌体生长和乳酸合成有明显影响。在海藻糖消耗阶段,通气量为7.2 L/h时有较高的底物消耗速率和产酸速率;用NaOH作为中和剂在p H6.5时海藻糖消耗速率和乳酸生成速率均高于pH6.0,而p H5.5时不利用海藻糖且不生产乳酸;与NaOH作为中和剂相比,在p H6.5条件下Ca(OH)₂作为中和剂使海藻糖消耗速率和乳酸生成速率分别提高21.0%和28.3%。发现Ca₂₊、Mg₂₊等二价离子确实对菌体自溶有一定的缓解作用,且Na₊能在饥饿状态下维持菌体的活性,为研...     

Identification and characterization of Enterococcus species isolated from forage crops and their influence on silage fermentation

Forty-eight strains of lactic acid bacteria were isolated from forage crops, and their identification, characterization and influence on silage fermentation were studied. All isolates were Gram-positive, short-chain-forming, catalase-negative, and facultatively anaerobic cocci that did not produce gas from glucose, only formed L-lactic acid, and were able to grow at pH 9.6%, in 6.5% NaCl, or with 40% bile, but not below pH 4.5. These isolates were commonly isolated from forage crops, and they were divided into four groups (A, B, C, and D) according to sugar fermentation characteristics. Selected strains of FA 5, FA 27, FA 45, and FA 57 were identified as Enterococcus hirae, E. faecalis, E. casseliflavus, and E. mundtii, respectively, on the basis of DNA-DNA homology, Strains FA 5, FA 27, FA 45, and FA 57 and two strains from commercial inoculants, LC 10 (Lactobacillus casei) and LP 15 (L. plantarum), were used as additives to alfalfa and guinea grass silages. Alphalfa and guinea grass silages inoculated with LC 10, LP 15, FA 5 + LC 10, and FA 5 + LP 15-treatments in alfalfa and guinea grass silages had significantly lower pH values, contents of butyric acid and ammonia nitrogen, gas production, and dry matter losses compared with the control silage after 60 d of fermentation. However, the E. hirae FA 5, E. faecalis FA 27, E. casseliflavus FA 45, and E. mundtii FA 57-inoculated silages gave similar values to the control in both types of silage. The FA 27 + LC 10 and FA 27 + LP 15-inoculated silages did not differ in fermentation quality from LC 10 and LP 15-inoculated silages alone. The results confirmed that Enterococcus species were not able to improve silage quality.     

Regulation of lactate production in Streptococcus bovis: A spiraling effect that contributes to rumen acidosis

When Streptococcus bovis was grown in batch culture with 6 g/L glucose at pH 6.7, maximum specific growth rate was 1.47 h(-1), and lactate was the primary fermentation product. In continuous culture at pH 6.7 and growth rate equal to .10 h(-1), little lactate was formed, and formate, acetate, and ethanol accounted for most of the product. When extra-cellular pH decreased to 4.7, intra-cellular pH declined to 5.4, and organisms switched back to lactate production. Intracellular concentration of fructose 1,6-diphosphate of batch culture cells was greater than 12 mM, a concentration that allowed maximal lactate dehydrogenase activity. When Streptococcus bovis was grown in continuous culture at pH 6.7, intracellular fructose-l,6-diphosphate declined to .4 mM, a concentration which gave little lactate dehydrogenase activity at pH 6.5 or greater. Decreasing pH of continuous culture to 4.7 increased intracellular fructose-1,6-diphosphate concentration to .8 mM. This concentration was still limiting if lactate dehydrogenase was assayed at pH 6.5, but nearly maximal activity was obtained when enzyme was assayed at pH 5.5. The small increase in fructose-l,6-diphosphate and decreased requirement of lactate dehydrogenase for fructose-l,6-diphosphate under acidic assay conditions, accounted for increased lactate production during low pH (4.7) continuous culture. These and other aspects of lactate regulation by Streptococcus bovis are discussed as factors leading to rumen acidosis. This pattern of regulation also helps to explain why rumen acidosis is difficult to reverse.     

Enterococci from Tolminc cheese: population structure, antibiotic susceptibility and incidence of virulence determinants

Microbiological analysis of ripened artisanal Tolminc cheese revealed the presence of an enterococcal population in numbers of up to 10(6) per g. All colonies, isolated from the citrate azide tween carbonate (CATC) enterococcal selective medium were Gram positive and coccal-shaped and were analysed with PhenePlate FS system. This system discriminated 10 PhP clusters among the 90 enterococcal isolates. From each cluster the most representative isolate for that particular type was selected for further study. The 10 representative enterococci were catalase negative and grew in the presence of NaCl (2%, 4% and 6.5%) and bile salts (0.06%). Genus specific primers confirmed all 10 enterococcal representatives as Enterococcus members, while species specific primers determined them further as strains of Enterococcus faecalis species. PCR for vanA and vanB genes detection, respectively, amplified no PCR products. The absence of van genes was confirmed with both disc and E-test, as isolates were susceptible to vancomycin according to the National Committee for Clinical Laboratory Standards (NCCLS). The results of disc tests with other antimicrobial agents (ampicillin, vancomycin, kanamycin, penicillin, erythromycin, neomycin, chloramphenicol, clindamycin, rifampin) did not differ much among the tested enterococci: they were all very resistant to clindamycin only. The incidence of enterococcus virulence determinants was as expected: all of the 10 E. faecalis strains tested possessed multiple determinants (between 7 and 11).     

EMPLOYMENT OF ORGANIC ACIDS TO ENHANCE ASTAXANTHIN FORMATION IN HETEROTROPHIC CHLORELLA ZOFINGIENSIS

The green alga Chlorella zofingiensis produces significant amounts of the valuable ketocarotenoid astaxanthin under heterotrophic growth conditions. In this study, pyruvate, citrate and malic acid were investigated, for the first time, to promote the formation of secondary carotenoids including astaxanthin in C. zofingiensis in the dark. The addition of pyruvate, citrate and malic acid into the culture medium at a concentration above 10 mM stimulated biosynthesis of astaxanthin and other secondary carotenoids. The addition of 100 mM pyruvate enhanced the yield of astaxanthin from 8.36 to 10.72 mg/L, representing an increase of 28.2%. Citrate and malic acid also had stimulatory effects on the formation of astaxanthin. The results showed that organic acid supplementation was potentially useful for commercial production of astaxanthin by C. zofingiensis in dark heterotrophic culture.

PRACTICAL APPLICATIONS

This article revealed, for the first time, that the organic acids including pyruvate, citrate and malic acid could enhance the production of secondary carotenoids including the highly valuable product astaxanthin in the green alga Chlorella zofingiensis using fermentation technology. This research contributed significantly to our understanding of the synthesis of astaxanthin in green algae in dark heterotrophic cultures which would facilitate scale-up of the process to an industrial scale. In practice, organic acids could be used to effectively induce the formation of astaxanthin by C. zofingiensis during fermentation using organic carbon substrates as carbon sources, and at the same time to help adjust pH values favorably for astaxanthin production by C. zofingiensis in the fermenter.     

Characterization of a 3944 Da bacteriocin, produced by Enterococcus mundtii ST15, with activity against Gram-positive and Gram-negative bacteria

Strain ST15, isolated from soy beans, and identified as Enterococcus mundtii, produces a 3944 Da bacteriocin that inhibits the growth of Lactobacillus sakei, Enterococcus faecalis, Bacillus cereus, Propionibacterium sp., Clostridium tyrobutyricum, Acinetobacter baumanii, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pneumoniae and Streptococcus caprinus. Bacteriocin ST15 is inactivated by proteinase K, pronase, pepsin, protease and Triton X-114, but not when treated with catalase, alpha-amylase, Triton X-100, SDS, Tween 20, Tween 80, urea and EDTA. No change in activity was recorded after 2 h at pH values between 2.0 and 12.0, and after treatment at 100 degrees C for 90 min. Activity was, however, lost after treatment at 121 degrees C for 20 min. The mode of activity is bactericidal. The highest level of activity (51200 AU ml(-1)) was recorded when cells were grown in MRS broth, pH 6.5. Bacteriocin ST15 differs from other broad-spectrum bacteriocins described for Enterococcus spp. by being active against Gram-negative bacteria and by being smaller.     

Organic acids produced by lactobacilli, enterococci and yeasts isolated from Picante cheese

 Four species of bacteria (Enterococcus faecium and Enterococcus faecalis, Lactobacillus plantarum and Lactobacillus paracasei) and three species of yeasts (Debaryomyces hansenii, Yarrowia lipolytica and Cryptococcus laurentii), previously isolated from Picante cheese, were cultured in ovine and in caprine milk and assayed for sugar and organic acids metabolism for 6 days. The results indicated that both milk types can be coagulated by the four strains of lactic acid bacteria. Lb. paracasei led to a faster and greater reduction in pH. Production of lactic acid correlated to lactose degradation, and was highest for Lb. paracasei followed by E. faecium; citrate metabolism was apparent for E. faecalis and, to a lesser extent, for E. faecium, Lb. plantarum and Lb. paracasei. Relatively high contents of formic acid were found when inoculation was with Enterococcus and with Lb. plantarum. Received: 4 February 1999     

Effect of Lactococcus garvieae, Lactococcus lactis and Enterococcus faecalis on the behaviour of Staphylococcus aureus in microfiltered milk

The effect of four strains of Lactococcus garvieae, three strains of Lactococcus lactis and one strain of Enterococcus faecalis on Staphylococcus aureus SA15 growth in microfiltered milk was evaluated. Lactococcus and Enterococcus strains were co-cultured with S. aureus in microfiltered milk and in medium buffered at pH 6.8. All Lactococcus and Enterococcus strains were able to inhibit S. aureus growth after 6h of incubation. Inhibition by L. lactis and E. faecalis strains could be partially attributed to the decrease in pH below 6.0 as it has been observed in medium buffered at pH 6.8. L. garvieae strains were the most effective to inhibit S. aureus growth without acidification. Inhibition of S. aureus could not be attributed neither to production of lactate, acetate or nor to antistaphylococcal substance. Amino acids competition was not involved in the inhibition by L. garvieae as addition of valine, isoleucine, threonine, methionine and phenylalanine did not suppress the inhibition of S. aureus.     

植酸酶YiAPPA在粪肠球菌中的高效组成型表达研究

旨在获得强组成型启动子来实现植酸酶YiAPPA在粪肠球菌EXW27中的高效组成型表达。本研究通过对比分析粪肠球菌中16S rRNA的启动子序列,将启动子中非保守区域替换为随机化序列,获得随机的人工启动子序列。然后利用大肠杆菌-乳酸菌穿梭质粒pSIP409中酶切位点构建人工启动子文库,并结合植酸酶活性的高通量筛选技术,筛选获得启动YiAPPA在粪肠球菌EXW27中高效组成型表达的强组成型启动子,实现YiAPPA在粪肠球菌EXW27中成功表达,并研究重组YiAPPA的酶学性质。结果表明,在组成型启动子p10的控制下,重组YiAPPA在粪肠球菌EXW27中成功表达,其表达量约占细胞内蛋白总量的15%。重组YiAPPA的最适反应pH为4.5,在pH1.0~10.0的范围内具有优良的pH稳定性,于55 ℃的绝对酶活高达3900 U/mg。本研究构建了既具有益生特性又具有植酸酶活性的转基因粪肠球菌,为研制新型转基因微生态制剂奠定了基础。     

响应面法优化粪肠球菌包被发酵培养基

本研究旨在优化粪肠球菌包被发酵培养基,以获得高密度包被粪肠球菌。以包被培养活菌含量为检测指标,采用Plackett-Burman(PB)试验分析培养基中对粪肠球菌发酵影响最重要的主要因素,结合响应面法(RSM)对主要因素进行了优化,并研究了最优条件下包被培养的粪肠球菌冻干粉的稳定性。结果表明,最优包被发酵培养基配方为:葡萄糖4.93%、蛋白胨 1%、牛肉膏 0.8%、酵母粉 0.6%、柠檬酸铵 0.3%、K₂HPO₄·7H₂O 0.2%、MgSO₄·7H₂O 0.06%、MnSO₄·H₂O 0.045%、NaAc·3H₂O 0.6%、吐温80 0.1%、海藻酸钠 1.87%、纳米碳酸钙 3.65%。在此条件下,粪肠球菌包被活菌数达6.83×109 CFU/mL,与预测值误差仅4.12%。贮存稳定性试验表明,制备成的冻干粉在37 ℃条件下贮存90 d后仍保留80%以上的存活率,说明包被培养粪肠球菌冻干粉具有较高的稳定性。     

Heme-dependent catalase activity of lactobacilli

The heme-dependent catalase in Lactobacillus pentosus, L. sake, L. delbrueckii and Enterococcus faecalis was studied. The catalase was formed by cells grown aerobically in the presence of hematin or for lactobacilli when grown without added hematin, after incubation of buffered cells in the presence of hematin. The kinetics of the production of catalase revealed maximum activity for L. pentosus and E. faecalis at late stationary and late logarithmic growth phase, respectively. The physiological role of catalase was studied with L. sake. The presence of hematin allows higher growth yields, since it protects the cells against hydrogen peroxide formed endogenously up to concentrations of 4.6 mmol/l.     

Amino acid decarboxylation by Lactobacillus curvatus CTC273 affected by the pH and glucose availability

The aim of the present study was to investigate the aminogenic behaviour of Lactobacillus curvatus strain CTC273, isolated from a fermented pork sausage, and showing its ability to produce tyramine, putrescine, phenylethylamine and lower amounts of cadaverine. Besides the amine production by growing cells under different pH, glucose availability and aero-/anaerobiosis, the effect of these environmental conditions on the activity of decarboxylase enzymes was also studied in resting cells. Tyrosine decarboxylation occurred during the active growth phase and followed the acidification curve. Glucose and oxygen availability had little influence on tyramine production, although anaerobiosis seemed to favour the enzyme activity. The decarboxylation of other amino acids occurred later and was more pronounced during the stationary phase. Compared to aromatic amines, the more acidic pH inhibited the production of putrescine. Slightly higher amounts of putrescine were reached during aerobic growth at lower glucose concentration. Thus, the function of ornithine-decarboxylase in this bacterium would not seem to be a mechanism to neutralise the acid environment, but it may play a role in supplying metabolic energy.     

发酵香肠中分离纯化的三株乳酸菌产酸特性研究

为了研究从发酵香肠中分离纯化的3株乳酸菌粪肠球菌(Enterococcus faecalis)、戊糖片球菌(Pediococcus pentosaceus)和肠膜明串珠菌(Leuconostoc mesenteroides)的产酸性能,对这3株菌在不同pH、温度、NaCl、NaNO2条件下的生长情况及产酸情况进行了测定。研究结果显示:这3株菌中,肠膜明串珠菌和戊糖片球菌的生长特性较好;粪肠球菌的生长特性虽不如肠膜明串珠菌和戊糖片球菌,但产酸能力最好,戊糖片球菌耐盐性最好、肠膜明串珠菌耐亚硝酸盐的特性最好;在不同温度和pH条件的测试中,肠膜明串珠菌的生长能力最好,粪肠球菌次之。这3株乳酸菌在发酵肉制品中均产生乳酸。总之,这3株菌均具有用于发酵制备乳酸的能力。     

Effects of environmental factors on leucine catabolism by Carnobacterium piscicola

The metabolites from leucine degradation are involved in dry fermented sausage aroma. The catabolism of leucine by a strain of Carnobacterium piscicola was studied directly in the growth medium with 3H-labelled leucine to investigate the effect of five parameters: phase of growth, pH, oxygen, glucose and alpha-ketoisocaproic acid. Resting cells (RC) were also incubated with 3H-labelled leucine. The radioactive metabolites from leucine catabolism were analysed by high performance liquid chromatography (HPLC). At pH 5.4 and 7.2, the main metabolites detected were 3-methyl butanal, 3-methyl butanol and alpha-ketoisocaproic acid. At pH 6.5, the leucine catabolism was maximum and was characterised by a high production of 3-methyl butanoic acid. Leucine catabolism was most important during the exponential phase of growth. The addition of alpha-ketoisocaproic acid at 1%, glucose at levels of 0.5% to 2% and shaking of the growth medium increased leucine catabolism.