Induction of the acid tolerance response in Leuconostoc mesenteroides ATCC 8293 by pre-adaptation in acidic condition

Leuconostoc mesenteroides is a commercially important lactic acid bacterium which is used as a starter culture in various fermentation processes. However, because of its sensitivity to acid stress, high cell-density culture has not been accomplished yet. Therefore, we investigated the effect of preadaptation of L. mesenteroides under mildly acidic conditions for resistance to normally lethal levels of acid stress. For this, the cells grown to the early-exponential phase at pH 7.0 were incubated at pH 6.5, 6.0, and 5.0 for 1 h, and then the survival rates of acid-adapted cells in pH 4.0 solution was determined. Acid-adapted cells at pH 5.0 exhibited maximum increase in tolerance, showing an increased survival of approximately 2,500 folds compared to the control (pH 7.0).     

Increased acid tolerance of Escherichia coli O157:H7 as affected by acid adaptation time and conditions of acid challenge

Three strains of Escherichia coli O157:H7, ATCC 43889, 43895 and 933 were subjected to acid adaptation in Tryptic Soy Broth (pH 5.0) for 1, 2, 3, 4 and 6 h. Acid tolerance of the adapted cells was determined in subsequent acid challenge at pH 3.0, 4.0 and 5.0 (acidified with HCl) and in the presence of lactic, acetic or propionic acid. It was found that acid adaptation increased acid tolerance of the E. coli O157:H7 strains tested and was dependent on strain, acid adaptation time and pH of the challenge. Among the acid adaptation times tested, 4 h of adaptation enabled the test organism, regardless of strains, to exhibit the most pronounced acid adaptation response which was most marked at pH 3.0, followed by pH 4.0 and 5.0. The extent of increased acid tolerance varied with the strains of E. coli O157:H7 and challenge of organic acid. The 4-h acid-adapted cells of ATCC 43889 and 933 showed an increase in acid tolerance in the presence of lactic, acetic and propionic acids. An increase in tolerance was also noted with ATCC 43895 in the presence of acetic and lactic acid, but not in the presence of propionic acid.     

Wound healing effect of cuttlebone extract in burn injury of rat

This study was designed to evaluate the morphological and physiological characteristics of high pressure (HP)-treated Listeria monocytogenes treated at different pHs. L. monocytogenes in phosphate buffered saline (PBS, pH 7.2) adjusted with HCl or lactic acid to pH 4.0, 5.0, and 6.0 was subjected to 300 MPa for 5 min. The numbers of HP-treated L. monocytogenes at pH 4.0 adjusted with HCl or lactic acid were reduced by more than 6 log CFU/mL, while the numbers of non-HP-treated L. monocytogenes were not significantly reduced at pH 4.0. The propidium iodide (PI) fluorescence intensity of HP-treated cells adjusted with lactic acid was increased from 65 (pH 6.0) to 78% (pH 4.0). The HP-induced inactivation of L. monocytogenes in low pH was not directly associated with the membrane disruption. The results provide valuable information for understanding the HP-induced inactivation mechanisms and enhancing microbial lethality in acidic food systems.     

Short communication: impact of pH and temperature on the acidifying activity of Carnobacterium maltaromaticum

The acidifying activity of Carnobacterium maltaromaticum LMA28, a strain isolated from French soft cheese, was studied in trypticase soy broth with yeast extract (TSB-YE) medium and in milk. In TSB-YE supplemented with lactose, glucose, or galactose, lactose and glucose were metabolized with a maximum growth rate of 0.32 h⁻¹ and galactose was not metabolized. During hydrolysis of lactose, the galactose moiety was not excreted. The major product was l(+) lactic acid, with no significant difference in the lactic acid yield. Glucose was not completely metabolized because cell growth stopped when pH values reached an average of 5.0. In sterilized UHT milk, the addition of 1 g/L of YE enhanced its coagulation. Compared with commercial starter lactic acid bacteria such as Lactococcus lactis DSMZ 20481 or Streptococcus thermophilus INRA 302, Carnobacterium maltaromaticum LMA 28 was shown to be a slow acidifying strain. However, in spite of this weak acidifying ability, C. maltaromaticum LMA 28 can sustain low pH values in coculture with Lc. lactis DSMZ 20481 or S. thermophilus INRA 302. The individual and interactive effects of initial pH values (5.2 to 8.0) and incubation temperatures (23 to 37°C) on acidifying activity were studied by response surface methodology. The 3 strains displayed different behaviors depending on pH and temperature. The psychrotrophic lactic acid strain C. maltaromaticum LMA 28 was able to grow at alkaline pH values and during storage conditions. It could be used as a potential ripening flora in soft cheese.     

不同香型大曲酯化酶的特性分析

本文主要研究了不同酸浓度和pH值下清香型、浓香型和酱香型大曲的酯化酶催化合成乙酸乙酯、乳酸乙酯、己酸乙酯和丁酸乙酯的不同特性。结果显示,随着酸浓度的升高,三种香型大曲催化合成乙酸乙酯、己酸乙酯的酶活力均是先升高后降低的趋势,催化合成乳酸乙酯的酶活力随乳酸浓度的升高降低;而催化合成丁酸乙酯的酶活力变化趋势三种香型大曲有所不同,清香型和浓香型大曲的酶活力随丁酸浓度的升高先升高后降低,酱香型大曲的酶活力则是逐渐升高。清香型、浓香型和酱香型大曲催化合成乙酸乙酯的最适pH均为4.0,催化合成乳酸乙酯的最适pH分别为7.0、7.0、8.0,催化合成己酸乙酯的最适pH分别为5.0、4.0、4.5,催化合成丁酸乙酯的最适pH分别为4.5、4.0和3.0。综上,不同香型大曲酯化酶的催化特性各有不同,且每种酯的合成都有其合适的酸浓度和pH范围。     

Acid or erythromycin stress significantly improves transformation efficiency through regulating expression of DNA binding proteins in Lactococcus lactis F44

Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation. However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. lactis F44 showed a 56.1-fold increase in acid condition (pH 5.0); meanwhile, erythromycin stress (0.04 μg/mL) promoted the transformation efficiency more significantly (76.9-fold). Notably, the transformation efficiency of F44e (L. lactis F44 harboring empty pLEB124) increased up to 149.1-fold under the synergistic stresses of acid and erythromycin. In addition, the gene expression of some DNA binding proteins (DprA, RadA, RadC, RecA, RecQ, and SsbA) changed correspondingly. Especially for radA, 25.1-fold improvement was detected when F44e was exposed to pH 5.0. Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L. lactis through regulating gene expression of DNA binding proteins. We have proposed a simple but promising strategy for improving the transformation efficiency of L. lactis and other hard-transformed microorganisms.     

Effects of yeast extract and different amino acids on the dynamics of some components in cabbage juice during fermentation with Bifidobacterium lactis BB-12

Various amino acids and the yeast extract, in amounts of 0.1%(w/v), were separately tested for their influence on the analytical parameters of lactic acid fermentation of cabbage juice with Bifidobacterium animalis subsp. lactis BB-12. Compared with the control, cysteine supplementation led to a decrease of the time to reach pH 5.0 of 6 times and an increase of lactic acid productivity of 1.22 times. After 48 h the ascorbic acid content was by 360.73% higher, the fermented cabbage juices being assigned into a distinct group applying both factor analysis (FA) and cluster analysis (CA). Tryptophan contributed to better values for lactic and acetic acid yield, while lysine and yeast extract especially for acetic acid yield. Valine and leucine were not able to improve the fermentation progress, estimated through the analyzed variables. This work would provide some helpful information for the development of various lacto-fermented vegetable juices using probiotic bacteria.     

Natural populations of lactic acid bacteria isolated from vegetable residues and silage fermentation

Natural populations of lactic acid bacteria (LAB) and silage fermentation of vegetable residues were studied. Fifty-two strains of LAB isolated from cabbage, Chinese cabbage, and lettuce residues were identified and characterized. The LAB strains were gram-positive and catalase-negative bacteria, which were divided into 6 groups (A to F) according to morphological and biochemical characteristics. The strains in group A were rods that did not produce gas from glucose and formed the d and l isomers of lactate. Groups B and C were homofermentative cocci that formed l-lactic acid. Groups D, E, and F were heterofermentative cocci that formed d-lactic acid. Based on 16S rDNA gene sequence analysis, group A to F strains were identified as Lactobacillus plantarum, Lactococcus piscium, Lactococcus lactis, Leuconostoc citreum, Weissella soli and Leuconostoc gelidum, respectively. The prevalent LAB, predominantly homofermentative lactobacilli, consisted of Lactobacillus plantarum (34.6%), Weissella soli (19.2%), Leuconostoc gelidum (15.4%), Leuconostoc citreum (13.5%), Lactococcus lactis (9.6%), and Lactococcus piscium (7.7%). Lactobacillus plantarum was the dominant member of the LAB population in 3 types of vegetable residues. These vegetable residues contained a high level of crude protein (20.2 to 28.4% of dry matter). These silages prepared by using a small-scale fermentation system were well preserved, with low pH and a relatively high content of lactate. This study suggests that the vegetable residues contain abundant LAB species and nutrients, and that they could be well preserved by making silage, which is a potentially good vegetable protein source for livestock diets.     

Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants

Antimicrobial behavior of lactic acid bacteria (LAB) has been explored since many years to assess their ability to produce bacteriocin, a natural preservative, to increase the shelf life of food. This study aims to characterize bacteriocin producing strains of lactic acid bacteria isolated from acidic to slightly acidic raw vegetables including tomato, bell pepper and green chili and to investigate their potential to inhibit food related bacteria. Among twenty nine LAB screened for antimicrobial activity, three exhibited antagonism against closely related bacterial isolates which was influenced by varying temperature and pH. They were identified up to strain level as Lactococcus lactis subsp. lactis TI-4, L. lactis subsp. lactis CE-2 and L. lactis subsp. lactis PI-2 based on 16S rRNA gene sequence. Their spectrum of inhibition was observed against food associated strains of Bacillus subtilis and Staphylococcus aureus. Moreover, L. lactis subsp. lactis PI-2 selected on the basis of higher antimicrobial activity was further evaluated for bacteriocin production which was detected as nisin A and nisin Z. These findings suggest the possible use of L. lactis strains of vegetable origin as protective cultures in slightly acidic as well as slightly alkaline food by the bio-preservative action of bacteriocins.     

清香型大曲酯化酶活力的研究

研究了乳酸质量浓度和pH对清香型大曲酯化酶活力的影响,结果表明,乳酸质量浓度对大曲酯化酶活力有很大影响,当乳酸质量浓度较低时,大曲中的酯化酶表现为合成作用,分别在低于8 g/L和16 g/L时催化乳酸乙酯和乙酸乙酯的合成;当乳酸质量浓度较高时,酯化酶表现为水解作用,将酯分解为相应酸和醇;pH对大曲酯化酶酯分解活力影响显著,在pH值为2.0时,酯的分解主要为化学分解,在pH值为3.0～6.0时,化学分解和酯化酶分解都起作用。实验发现在pH值为4.0时大曲酯化酶催化乙酸乙酯合成的活力最强,100 h时乙酸乙酯质量浓度达188.8 mg/L;pH值为5.0时催化乳酸乙酯合成的活力最强,100 h时乳酸乙酯质量浓度达182.6 mg/L。     

Production of flavour-active methionol from methionine metabolism by yeasts in coconut cream

Yeasts Candida kefyr NCYC143, Candida utilis CUM, Kluyveromyces lactis KL71, Saccharomyces bayanus SB1, Saccharomyces cerevisiae EC1118, Saccharomyces chevalieri CCICC1028, Candida famata (previously Torulopsis candida) CCICC1041 and Williopsis saturnus var. saturnus CBS254 were screened for their ability to produce flavour-active methionol (3-methylthio-1-propanol) in coconut cream supplemented with l-methionine. The yeasts varied with their ability to produce methionol from methionine with Saccharomyces cerevisiae EC1118 producing the most, followed by Kluyveromyces lactis KL71. Little methionol was produced by the other yeasts. Methionol production by Kluyveromyces lactis KL71 was subjected to further studies under different conditions of initial pH (4.0-6.3), temperature (20-33 °C), l-methionine concentration (0.05-0.25%) and yeast extract concentration (0-0.50%); optimal conditions were established at pH 5.0, 33.0 °C, 0.15% l-methionine and 0.05% yeast extract. CharmAnalysis™ using SPME-GC-MS was conducted on the coconut cream ferment; methional (3-methylthio-1-propanal), methionol and 2-phenylethyl acetate were found to be the most potent aroma-active compounds. The product of coconut cream fermentation by Kluyveromyces lactis KL71 may be considered as a novel, plant-based, natural and complex flavoring bioingredient in food applications.     

ComX improves acid tolerance by regulating the expression of late competence proteins in Lactococcus lactis F44

ComX can improve bacterial competence by modulating global gene expression. Although competence induction may also be a protective mechanism under stress, this has not been investigated in detail. Here, we demonstrated that ComX improved the acid tolerance and nisin yield of Lactococcus lactis, which is an important gram-positive bacterium increasingly used in modern biotechnological applications. We found that overexpression of comX could improve the survival rate up to 36.5% at pH 4.0, compared with only 5.4% and 1.1% with the wild-type and comX knockout strains, respectively. Moreover, quantitative real-time PCR results indicated that comX overexpression stimulated the expression of late competence genes synergistically with exposure to acid stress. Finally, electrophoretic mobility shift assay demonstrated the binding of purified ComX to the cin-box in the promoters of these genes. Taken together, our results reveal a regulation mechanism by which ComX and acid stress can synergistically modulate the expression of late competence genes to enhance cells' acid tolerance and nisin yield.     

IN VITRO SURVIVAL AT LOW pH AND ACID ADAPTATION RESPONSE OF CAMPYLOBACTER JEJUNI AND CAMPYLOBACTER COLI

The survival of Campylobacter jejuni and Campylobacter coli at pH 7.0, 6.0, 5.0 and 4.0 for up to 24 h, and the induction of an acid adaptation response in 12 C. jejuni and 10 C. coli strains in early exponential or late stationary phases in tryptic soy broth (TSB) and Brucella broth were investigated. C. coli strains were more sensitive than C. jejuni in media adjusted to pH 5.0 or 4.0 with hydrochloric acid. Five log₁₀ cfu/mL of C. coli were inhibited at 12 h in pH 5.0, but only 2.5 log₁₀ cfu/mL of C. jejuni cells were inhibited under the same conditions. No viable cells were detected at 4 h in pH 4.0 from an inoculum of 6 log₁₀ cfu/mL of C. coli. Late stationary phase cells of C. jejuni exposed to pH 5.0 for 4 h in TSB exhibited a significant (P ≤ 0.05) acid tolerance response when compared to nonexposed cells. Late stationary phase cells of C. coli exposed to pH 5.0 for 3 h in TSB and late stationary and early exponential phase cells exposed to pH 5.0 for 3 h in Brucella broth showed a significant (P ≤ 0.05) acid tolerance response when compared to nonexposed cells. The acid tolerance responses observed for C. jejuni and C. coli conferred adapted cells no more than 1 log₁₀ cfu/mL survival advantage over nonadapted cells. Brucella broth appeared to be more protective than TSB for the survival of C. jejuni and C. coli at pH 4.0.     

Metabolism of lactic acid in fermented cucumbers by Lactobacillus buchneri and related species,potential spoilage organisms in reduced salt fermentations

Recent evidence suggests that Lactobacillus buchneri may play an important role in spoilage-associated secondary fermentation of cucumbers. Lactic acid degradation during fermented cucumber spoilage is influenced by sodium chloride (NaCl) concentration, pH, and presence of oxygen. Objectives were to evaluate these factors on lactic acid utilization by L. buchneri, and to compare the biochemical changes to those which occur during fermented cucumber spoilage. Effects of NaCl (0, 2, 4, and 6% w/w), pH (3.8 vs 5.0), and aerobic environment were investigated using fermented cucumber media (FC) inoculated with spoilage microorganisms. At pH 3.8, L. buchneri degraded lactic acid in all NaCl concentrations. The highest rate of lactic acid utilization occurred in FC with 2% NaCl (P < 0.05). Lactic acid utilization was nearly identical under aerobic and anaerobic conditions, indicating that oxygen does not influence lactate metabolism by L. buchneri. Lactic acid utilization was accompanied by increases in acetic acid and 1,2-propanediol, and Lactobacillus rapi was able to convert 1,2-propanediol to propionic acid and propanol. L. buchneri initiated spoilage in a wide range of environmental conditions that may be present in commercial cucumber fermentations, and L. rapi may act syntrophically with L. buchneri to produce the commonly observed spoilage metabolites.     

Heat shock-induced thermotolerance inListeria monocytogenes13-249 is dependent on growth phase,pH and lactic acid

The effect of heat shock on the thermotolerance ofListeria monocytogenes13-249 was compared as a function of growth phase (log and late stationary phase), pH (5.4, 5.8, 6.2) and heating medium (broth vs minced beef). Lactic acid was added to tryptic phosphate broth (TPB⁺) to simulate the level found in beef. Furthermore, the specific effect of lactic acid was investigated, comparing results obtained in broth with and without lactic acid with pH adjusted to 5.4 and 7.0. In late stationary cultures, heat shock produced only marginal differences in thermotolerance in TPB⁺and beef regardless of pH. For log phase cultures, the increase in heat shock induced thermotolerance (HSIT) depended on pH in both TPB⁺and beef with gradually larger increases at rising pH. At the highest pH tested (pH 7.050 in TPB⁺and pH 6.2 in beef) the increase in HSIT was 5.7-fold and 2.4-fold, respectively. These increases were highly significant (P< 0.001). If no lactic acid was present, the HSIT increased at both pH 5.4 for 7.0 for log phase cultures. This indicates that the presence of lactic acid in naturally occurring levels will have a specific effect on the response ofL. monocytogenesto heat shock. The D₆₀-values ofL. monocytogenes13-249 were generally higher (2—6-fold) in minced beef than in TPB⁺.     

Properties of Crude Ethylester-forming Enzyme Preparations from Some Lactic Acid and Psychrotrophic Bacteria

Ester-forming enzyme preparations were obtained from 48 h cultures of Streptococcus diacetilactis ATCC 15346, S. lactis ML3, Lactobacillus No. 81 and two strains of psychrotrophic bacteria belonging to the genus Pseudomonas (Strains No. 50 and 53).Optimum pH's of the enzyme preparations of Lactobacillus No. 81 and two strains of the lactic streptococci for the production of ethylbutyrate and ethylhexanoate were 6.5 and 7.0 to 7.5, respectively. Enzymes of Strains No. 50 and 53 had their maximum activities at pH 8.0. The optimum temperature was 32 C for all strains. Enzymes were generally stable below 35 C at pH 7.0, but their activities were rapidly lost at higher temperature. When the esterases were incubated at 4 C for 24 h, Lactobacillus No. 81, the lactic streptococci (Strains ML3 and ATCC 15346), and the psychrotrophic bacteria (Strains No. 50 and 53) were most stable at pH 6.0, 7.0, and 8.0, respectively. Copper, manganese, and cobalt salts inhibited all the enzymes. The inhibitory effect of cobalt on enzyme activities of Strains ML 3, No. 50 and 53 and that of copper on Strain No. 81 were especially significant.     

红曲霉和乳酸菌发酵低温猪肉火腿肠工艺优化及品质分析

为探究红曲霉和乳酸菌发酵工艺对低温猪肉火腿肠品质的影响,通过单因素研究红曲霉添加量、乳酸菌添加量、发酵温度对低温猪肉火腿肠理化指标、感官品质和质构的影响,以感官评分为响应值,通过Box-Behnken响应面设计优化发酵工艺。结果表明,低温猪肉火腿肠最佳发酵工艺为红曲霉添加量4.0%,乳酸菌添加量5.0%,发酵时间44 h。在此最佳发酵工艺条件下,低温猪肉火腿肠呈浅红色,口感柔和,感官评分为82.5分,pH值6.24,亚硝酸盐含量为4.9 mg/kg,且4.0%红曲霉和5.0%乳酸菌可替代84.31%亚硝酸盐对低温猪肉火腿肠进行发色。     

Effect of high hydrostatic pressure treatment on the viability and acidification ability of lactic acid bacteria

The effect of high pressures of 100–450 MPa combined with temperatures of 20–40 °C on Lactobacillus delbrueckii subsp. bulgaricus ACA-DC0105, Streptococcus thermophilus ACA-DC0022 and Lactococcus lactis ACA-DC0049 cell viability and acid production ability was studied. The rates of decrease in pH and cell viability were estimated for all the process combinations studied. The viability and acidification ability of the cells depended on the process conditions. More intense process conditions resulted in a lower number of viable cells and simultaneous reduction of lactic acid production correlated to lower rates of decrease of pH. Lb. bulgaricus appeared to be the microorganism most resistant to pressure, while Lc. lactis the most sensitive. Similar behaviour was observed for the acidification ability of these microorganisms, with Lc. lactis being least able to decrease the pH value of cheese. The HP-treated strains could be used as adjunct starters for cheese production.