一株耐受低pH、高浓度乳酸及琥珀酸菌株的筛选与鉴定

【目的】通过构建的人工耐酸系统,筛选耐受低pH值、乳酸及琥珀酸的菌株。【方法】构建人工耐酸系统长期驯化菌株,利用不同p H的酸性平板进行筛选,从环境中筛选出一株对低p H值、高浓度乳酸以及琥珀酸有很好耐受性的菌株。通过形态学特征、生理生化特征研究,并结合18S rDNA基因序列分析及分子系统发育树的构建结果,确定菌株的种类。【结果】经过酸性人工系统的长期驯化,筛选分离出一株耐受低pH值、高浓度乳酸以及琥珀酸的菌株WJ-2,经鉴定该菌株为酿酒酵母(Saccharomyces cerevisiae),其最适生长温度为30°C。酸性平板实验显示该菌株能够耐受pH2.5的酸性环境,同时对9%的乳酸及8%的琥珀酸也有很好的耐受性。另外,耐酸菌株WJ-2在pH 2.5、9%乳酸和8%琥珀酸的培养环境中仍能保持相对中性的细胞内pH值。【结论】通过构建人工酸性系统,成功筛选出一株对低pH值、高浓度乳酸以及琥珀酸具有耐受性的菌株——酿酒酵母菌WJ-2,该方法可为筛选具有特定耐受能力菌株提供一个新思路。     

基因组改组（genome－shuffling）提高

首先采用紫外线与亚硝基胍两种传统微生物诱变方法对干酪乳杆菌进行诱变,经低pH平板、碳酸钙平板和摇瓶试验获得了5株耐酸性提高的突变菌株。以获得的突变菌株为出发菌株,应用灭活双亲原生质体融合后致死损伤得到互补获得活性融合子的方法,对其进行基因组改组,经过低pH平板、碳酸钙平板和摇瓶筛选,获得4株可以在pH3.8平板上旺盛生长且产酸量较高的改组菌株。将改组菌株与原始菌株分别于pH 3.8和3.4的YE液体培养基中培养,改组菌株能够在原始菌株无法生存的pH条件（pH 3.4）下生长。在pH 3.8的条件下,对改组菌株与原始菌株的发酵特征进行比较,37℃发酵48小时后,改组菌株产酸量为原始菌株的2.4倍,表明基因组改组技术能有效提高多基因调控表型的进化。     

Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration.

The growth rate responses of Escherichia coli M23 (a nonpathogenic strain) to suboptimal pH and lactic acid concentration were determined. Growth rates were measured turbidimetrically at 20 degrees C in the range of pH 2.71 to 8.45. The total concentration of lactic acid was fixed at specific values, and the pH was varied by the addition of a strong acid (hydrochloric) or base (sodium hydroxide) to enable the determination of undissociated and dissociated lactic acid concentrations under each condition. In the absence of lactic acid, E. coli grew at pH 4.0 but not at pH 3.7 and was unable to grow in the presence of > or = 8.32 mM undissociated lactic acid. Growth rate was linearly related to hydrogen ion concentration in the absence of lactic acid. In the range 0 to 100 mM lactic acid, growth rate was also linearly related to undissociated lactic acid concentration. A mathematical model to describe these observations was developed based on a Bĕlehrádek-like model for the effects of water activity and temperature. This model was expanded to describe the effects of pH and lactic acid by the inclusion of novel terms for the inhibition due to the presence of hydrogen ions, undissociated lactic acid, and dissociated lactic acid species. Preliminary data obtained for 200 and 500 mM total lactic acid concentrations show that the response to very high lactic acid concentrations was less well described by the model. However, for 0 to 100 mM lactic acid, the model described well the qualitative and quantitative features of the response.     

Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH

Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid’s pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH₄OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.     

Salmonella enterica serovar Typhimurium and Listeria monocytogenes acid tolerance response induced by organic acids at 20 degrees C: optimization and modeling

An acid tolerance response (ATR) has been demonstrated in Listeria monocytogenes and Salmonella enterica serovar Typhimurium in response to low pH poised (i.e., adapted) with acetic or lactic acids at 20 degrees C and modeled by using dynamic differential equations. The ATR was not immediate or prolonged, and optimization occurred after exposure of L. monocytogenes for 3 h at pH 5.5 poised with acetic acid and for 2 h at pH 5.5 poised with lactic acid and after exposure of S. enterica serovar Typhimurium for 2 h at pH 5.5 poised with acetic acid and for 3 h at pH 5.5 poised with lactic acid. An objective mechanistic analysis of the acid inactivation data yielded estimates of the duration of the shoulder (t(s)), the log-linear decline (k(max)), and the magnitude of a critical component (C). The magnitude of k(max) gave the best agreement with estimates of conditions for optimum ATR induction made from the raw data.     

Elucidation of the mechanism of lactic acid growth inhibition and production in batch cultures of Lactobacillus rhamnosus

Batch cultures of Lactobacillus rhamnosus were carried out at different pH values in order to study the limitation of growth and lactic acid production by the hydrogen ion, non-dissociated lactic acid and internal lactate concentrations. The effect of pH between 5 and 6.8 was studied at non-limiting concentrations of glucose; this is more significant for the lactic acid fermentation rate than for the maximum specific growth rate, as shown by the incomplete substrate consumption at lower values of medium pH and by the constant maximum cell mass obtained within the range of pH values studied. To check whether these results were a direct consequence of the different concentrations of the non-dissociated form of lactic acid at different external pH values, specific growth rates and lactic acid productions rates were calculated for each external pH value. The same specific growth rates were observed at the same non-dissociated lactic acid concentrations only at pH values of 5 and 5.5. For higher values of pH (pH > 6) the specific growth rate falls to zero as the non-dissociated lactic acid concentration decreases. This shows that generalisations made from studies performed within very narrow ranges of pH are not valid and that the non-dissociated form of lactic acid is not the only inhibiting species. The internal pH was measured experimentally for each external pH value in order to calculate the internal lactate ion concentration. This form is described to be the inhibitory one. The results obtained confirmed that the specific growth rate reached zero at approximately the same lactate concentration for all the pH values studied. Received: 31 January 1997 / Received revision: 15 May 1997 / Accepted: 19 May 1997     

Improvement of lactic acid production in Saccharomyces cerevisiae by cell sorting for high intracellular pH

Yeast strains expressing heterologous L-lactate dehydrogenases can produce lactic acid. Although these microorganisms are tolerant of acidic environments, it is known that at low pH, lactic acid exerts a high level of stress on the cells. In the present study we analyzed intracellular pH (pHi) and viability by staining with cSNARF-4F and ethidium bromide, respectively, of two lactic-acid-producing strains of Saccharomyces cerevisiae, CEN.PK m850 and CEN.PK RWB876. The results showed that the strain producing more lactic acid, CEN.PK m850, has a higher pHi. During batch culture, we observed in both strains a reduction of the mean pHi and the appearance of a subpopulation of cells with low pHi. Simultaneous analysis of pHi and viability proved that the cells with low pHi were dead. Based on the observation that the better lactic-acid-producing strain had a higher pHi and that the cells with low pHi were dead, we hypothesized that we might find better lactic acid producers by screening for cells within the highest pHi range. The screening was performed on UV-mutagenized populations through three consecutive rounds of cell sorting in which only the viable cells within the highest pHi range were selected. The results showed that lactic acid production was significantly improved in the majority of the mutants obtained compared to the parental strains. The best lactic-acid-producing strain was identified within the screening of CEN.PK m850 mutants.     

Kinetic study on pH dependence of growth and death of Streptococcus faecalis

A chemostat culture was used for lactic acid fermentation with Streptococcus faecalis at various pH values (8.0, 7.0, 6.0, 5.5, 5.0) and glucose concentrations (10, 20, 30 g/l). At every pH value, the reciprocals of the specific consumption rate of glucose and the specific production rate of lactic acid were linearly correlated to the reciprocal of the specific growth rate. The product, lactic acid, caused non-competitive inhibition of the specific growth rate at every pH value. Moreover, it was found that the cell death rate was dependent on pH and lactic acid. The death rate was smallest at pH 7.0 and increased with increasing lactic acid concentration. The kinetic equations of growth and death are proposed in a broader pH range. Correspondence to: H. Ohara     

转运蛋白提高凝结芽孢杆菌酸耐受性

光学纯乳酸作为可降解生物材料——聚乳酸(polylactic acid,PLA)的前体物质,正在受到广泛关注。乳酸发酵过程中酸性产物的积累会影响菌株的生长,提高菌株酸耐受性具有重要意义。本研究以乳酸生产菌株凝结芽孢杆菌(Bacillus coagulans) DSM1为出发菌株,通过对凝结芽孢杆菌DSM1及其乳酸脱氢酶双敲除菌株(DldhL1DldhL2)进行比较转录分析,筛选酸耐受相关的转运蛋白基因。对关键基因RS16330、RS06895、RS16325、RS10595、RS00500、RS07275、RS10635及RS01930进行实时定量PCR分析,发现基因RS06895、RS10595、RS00500和RS10635在发酵12 h和24 h转录水平显著增强。过表达RS10595基因的菌株,在中性(pH 6.0)条件下生长状况和发酵性能均受到抑制,但在酸性条件下(pH 4.6),其乳酸生成相比对照组显著提高。上述结果表明,RS10595基因与菌株DSM1的酸耐受性密切相关。本研究有助于进一步探究凝结芽孢杆菌酸耐受的机制,也为构建耐酸菌株提供了基础。     

Influence of pH on antimicrobial activity of organic acids against rabbit enteropathogenic strain of<Emphasis Type="Italic">Escherichia coli</Emphasis>

Susceptibility of the rabbit enteropathogenic strain Escherichia coli C6 (O128 serogroup) to C6-C14 fatty acids, oleic, citric, lactic and fumaric acid at 5 mg/mL was determined by the plating technique in the near-neutral pH region (pH approximately 6.5), and in a weakly acid and acid environment (pH 5.4 +/- 0.1 and 2.2-2.5, respectively). In the near-neutral pH region caproic and caprylic acid reduced the concentration of viable cells by 3 and 6 orders, respectively. At lower pH the bactericidal effect of caproic acid remained similar, but caprylic acid decreased the concentration of viable cells to < 100/mL. The bactericidal activity of capric acid was low at pH 6.5 but increased at pH 5.3. High environmental acidity was intrinsically bactericidal and at very low pH the effects of fatty acids were thus less pronounced. Citric acid reduced the counts of viable cells to 1/10. Antimicrobial activity of other acids examined was marginal or absent. Medium-chain fatty acids, caprylic and, to a lesser extent, also caproic and capric acid were better antimicrobials than other organic acids examined; the antimicrobial activity of fatty acids toward the C6 strain was pH-dependent. Beneficial effects of citric, lactic and fumaric acid reported by animal nutritionists are thus probably related to factors other than their direct antimicrobial action.     

Arginine- and Polyamine-Induced Lactic Acid Resistance in Neisseria gonorrhoeae

Microbe-derived lactic acid protects women from pathogens in their genital tract. The purpose of this study was to determine lactic acid susceptibility of Neisseria gonorrhoeae, and identify potential acid resistance mechanisms present in this pathogen. Tested in vitro, lactic acid killed all 10 gonococcal strains analyzed in a low pH-dependent manner. Full inactivation occurred at pH 4.5. At low pH, lactic acid treatment resulted in the entry of the DNA-binding fluorochrome propidium iodide into the microbial cells, suggesting that hydrogen ions from lactic acid compromise the integrity of the bacterial cell wall/membrane. Most likely, hydrogen ions also inactivate intracellular proteins since arginine rendered significant protection against lactic acid presumably through action of the gonococcal arginine decarboxylase, an enzyme located in the bacterial cytoplasm. Surprisingly, arginine also lessened lactic acid-mediated cell wall/membrane disruption. This effect is probably mediated by agmatine, a triamine product of arginine decarboxylase, since agmatine demonstrated a stronger protective effect on GC than arginine at equal molar concentration. In addition to agmatine, diamines cadaverine and putrescine, which are generated by bacterial vaginosis-associated microbes, also induced significant resistance to lactic acid-mediated GC killing and cell wall/membrane disruption. These findings suggest that the arginine-rich semen protects gonococci through both neutralization-dependent and independent mechanisms, whereas polyamine-induced acid resistance contributes to the increased risk of gonorrhea in women with bacterial vaginosis.     

Influence of pH and lactic acid concentration on Clostridium tyrobutyricum during continuous growth in a pH-auxostat

The aim of this project was to establish the minimal inhibitory concentration (MIC) of lactic acid for growth of Clostridium tyrobutyricum. A pH-auxostat was used to maintain a constant pH and to allow continuous growth at the highest possible rates at fixed, but adjustable concentrations of lactate. By raising the concentration of lactic acid and keeping the pH constant, the growth rate was shown to decrease linearly with increasing lactic acid concentration. The p K _a of lactic acid, measured in the actual growth medium at 37°C, was 3.40 (±0.03). Based on this value, the MIC_undiss values for each pH were estimated. The MIC of total lactic acid (MIC_tot) ranged from 150 mmol l⁻¹ to 1510 mmol l⁻¹ at pH 4.6–6.25, respectively. The corresponding MIC values of undissociated lactic acid (MIC_undiss) ranged from 8.9 to 2.1 mmol l⁻¹ at the same pH values. These results emphasize the importance of a rapid pH decrease and an equally rapid initial lactic acid fermentation of the ensilage, in order to sufficiently suppress clostridial growth.     

Relationship between lactic acid concentration and bacterial spoilage in ground beef.

Lactic acid concentration correlated with organoleptic spoilage of refrigerated, coarsely ground beef stored in casings with low oxygen permeability. The samples were assayed over time for lactic acid concentration, total aerobic plate count, percentage of gram-positive organisms, and pH. Lactic acid increased in all samples, as did the bacterial counts and percentage of gram-positive organisms in the total microflora, the latter representing an increase in the lactic acid-producing bacteria. pH was found to decrease in all samples, with the smallest decrease in pH being observed in the meat sample which maintained the lowest proportion of gram-positive organisms. With samples evaluated by a sensory panel, lactic acid levels were found to correlate inversely with odor acceptability.     

代谢工程改造野生耐酸酵母生产L-乳酸

以选育低pH条件下高产L-乳酸的酵母菌为目的,从自然样品中筛选分离得到一株能在pH 2.5 (乳酸调节) 的培养基中生长且不利用乳酸的酵母 (初步鉴定为木兰假丝酵母Candida magnolia);进一步将来源于米根霉As3.819的乳酸脱氢酶编码基因 (ldhA) 插入含有G418抗性基因的酵母穿梭载体,构建了重组质粒pYX212-kanMX-ldhA,电转化入野生型C. magnolia中,筛选获得了一株具有产L-乳酸能力的重组菌株C. magnolia-2;通过发酵实验表明,该重组菌产L-乳酸的最     

Demineralization of crab shells by chemical and biological treatments

To achieve demineralization of crab shell waste by chemical and biological treatments, lactic acid and lactic acid bacterium were applied. In 5.0 and 10% lactic acid, pH rapidly decreased from 6.8 to 4.2 and from 4.5 to 2.4 at day 3, respectively, and thereafter the pH remained at an almost constant level. In a 10% lactic acid bacterium inoculum, pH lowered to 4.6 at day 5. Relative residual ash content rapidly decreased to 49.1 and 16.4% in 5 and 10% lactic acid treatments, respectively, for the initial 12 h. In 2.5, 5 and 10% lactic acid bacterium inoculums, relative residual ash content rapidly decreased to 55.2, 40.9 and 44.7%, respectively, on the first day. Residual dry masses were 76.4, 67.8 and 46.6% in 2.5, 5 and 10% lactic acid treatments, respectively, for the initial 12 h. After a one-time exchange of the lactic acid solution, in the 5.0% lactic acid treatment, residual dry mass rapidly decreased from 66.0 to 41.4%. In 2.5, 5 and 10% lactic acid bacterium inoculums, residual dry masses decreased to 67.6, 57.4 and 59.6% respectively, on the first day. Protein contents after demineralization ranged from 51.3–54.7% in the chemical treatments and decreased to 32.3% in the lactic acid fermentation process. A negative relationship was shown between pH and demineralization rate in lactic acid and lactic acid bacterium treatments. These results suggest that lactic acid fermentation can be an alternative for demineralization of crab shells, even though the rate and efficiency of the demineralization is lower than the chemical treatment.     

Inhibition of enterobacteria and Listeria growth by lactic, acetic and formic acids

Minimum inhibitory concentrations (MIC) of undissociated lactic, acetic and formic acids were evaluated for 23 strains of enterobacteria and two of Listeria monocytogenes. The evaluation was performed aerobically and anaerobically in a liquid test system at pH intervals of between 4.2 and 5.4. Growth of the enterobacteria was inhibited at 2–11 mmol 1⁻¹, 0.5–14 mmol 1⁻¹ and 0.1–1.5 mmol 1⁻¹ of undissociated lactic, acetic and formic acids, respectively. The MIC value was slightly lower with anaerobic conditions compared with aerobic conditions. The influence of protons on the inhibition was observed for acetic acid at the low pH values. Undissociated lactic acid was 2 to 5 times more efficient in inhibiting L. monocytogenes than enterobacteria. Acetic acid had a similar inhibitory action on L. monocytogenes compared with enterobacteria. Inorganic acid (HCl) inhibited most enterobacteria at pH 4.0; some strains, however, were able to initiate growth to pH 3.8. The results indicate that the values of undissociated acid which occur in a silage of pH 4.1–4.5 are about 10–100 times higher than required in order to protect the forage from the growth of enterobacteria and L. monocytogenes.