Flow Cytometric Assessment of Viability of Lactic Acid Bacteria

The viability of lactic acid bacteria is crucial for their applications as dairy starters and as probiotics. We investigated the usefulness of flow cytometry (FCM) for viability assessment of lactic acid bacteria. The esterase substrate carboxyfluorescein diacetate (cFDA) and the dye exclusion DNA binding probes propidium iodide (PI) and TOTO-1 were tested for live/dead discrimination using a Lactococcus, a Streptococcus, three Lactobacillus, two Leuconostoc, an Enterococcus, and a Pediococcus species. Plate count experiments were performed to validate the results of the FCM assays. The results showed that cFDA was an accurate stain for live cells; in exponential-phase cultures almost all cells were labeled, while 70°C heat-killed cultures were left unstained. PI did not give clear live/dead discrimination for some of the species. TOTO-1, on the other hand, gave clear discrimination between live and dead cells. The combination of cFDA and TOTO-1 gave the best results. Well-separated subpopulations of live and dead cells could be detected with FCM. Cell sorting of the subpopulations and subsequent plating on agar medium provided direct evidence that cFDA labels the culturable subpopulation and that TOTO-1 labels the nonculturable subpopulation. Applied to cultures exposed to deconjugated bile salts or to acid, cFDA and TOTO-1 proved to be accurate indicators of culturability. Our experiments with lactic acid bacteria demonstrated that the combination of cFDA and TOTO-1 makes an excellent live/dead assay with versatile applications.     

Effect of Compatible Solutes on Survival of Lactic Acid Bacteria Subjected to Drying

Four strains of lactic acid bacteria were investigated to determine if a relationship exists between accumulation of compatible solutes and the ability of cells to survive drying. Betaine was the major solute found in these lactic acid bacteria subjected to salt stress. Survival of cultures subjected to drying was considerably enhanced when this solute was accumulated by cells.     

Moisture Requirements for Growth and Metabolite Production by Lactic Acid Bacteria

The effect of water activity (a_w) reduction on growth and acid and diacetyl production by three lactic streptococci was studied. In addition, the influence of low moisture conditions on several bacteria of significance in the fermentation of sauerkraut was examined. The minimal a_w supporting growth of dairy lactics was 0.93 in a medium adjusted with glycerol. Media adjusted with sucrose generally were more inhibitory than those in which glycerol was the humectant. Titratable acidity, although not related to the type of humectant, did depend on the a_w of the medium and was directly related to the extent of growth. Diacetyl concentration increased in cultures of reduced a_w when the media were adjusted with both humectants; however, the effect was greatest with glycerol. A lactic strain associated with sauerkraut fermentation appeared to grow at a lower minimal a_w in a glycerol-adjusted medium than in a system adjusted with NaCl; however, none of the sauerkraut organisms grew at a_w levels of <0.95 when NaCl was the solute. Acid production appeared to be related to the presence and extent of growth at all of the a_w levels studied.     

猪源乳酸菌产乳酸及其抑菌特性研究

研究了5株(L1、12、L3、L5和L7)分离自仔猪肠道的乳酸菌的产乳酸能力及抑菌特性。结果表明：L5菌株产乳酸的速度最快,培养液中乳酸含量最高,L5菌株培养液pH值的下降速度最快,终末pH值最低,而L1菌株产乳酸的速度最慢,培养液乳酸含量最低。5株乳酸菌对大肠杆菌K88、K99、987P、O141和大肠杆菌E1及金黄色葡萄球菌均有不同程度的抑制作用;排除酸的影响后仍有22％～53％抑菌效果;经热处理后保持有92％以上的抑菌效果;蛋白酶处理后保持85％以上的抑菌效果。     

Development of Freeze-Dried Bacteriocin-Containing Preparations from Lactic Acid Bacteria to Inhibit Listeria monocytogenes and Staphylococcus aureus

There has been a recent movement to produce and consume ??minimally processed?? and more ??natural?? foods through the use of fewer chemical preservatives. The shift to more ??natural?? foods has resulted in a great interest in the use of bacteriocins from lactic acid bacteria as natural biopreservatives. The objective of this comparative study was to identify bacteriocins that can be produced in low-cost or no-cost dairy-based media (DBM), concentrated using freeze-drying, and applied to Cheddar cheese samples to concurrently inhibit Listeria monocytogenes and Staphylococcus aureus. Select bacteriocin producers were grown in DBM, their cell-free supernatants (CFS) were frozen, and the frozen CFS samples were freeze-dried to produce bacteriocin-containing powders. Cheddar cheese samples were challenged with L. monocytogenes or Staph. aureus cells. The challenged samples were exposed to buffered solutions of freeze-dried powders containing bacteriocins, incubated at 4?°C for 24?C72?h, and plated onto appropriate selective media. All freeze-dried bacteriocin-containing powders tested were active against L. monocytogenes and Staph. aureus. Our research findings indicated that low-cost or no-cost DBM could successfully be used for production of bacteriocin-containing preparations. In addition, freeze-drying was determined to be a feasible approach to prepare concentrated and stable bacteriocin-containing powders for prospective food applications. The prevention of even a very small percentage of foodborne illnesses via the use of bacteriocins as natural biopreservatives would help reduce the number of foodborne illness-related hospitalizations, deaths, and financial loss due to medical expenses, lost income/productivity, cost of litigation/penalties, and loss of trade.     

Medium for the Selective Enumeration of Lactic Acid Bacteria from Foods

A nitrite actidione polymyxin agar was developed for the enumeration of lactic acid bacteria. It was effective in recovering organisms from pure cultures and from foods.     

Malt Extract as Protective Medium for Lactic Acid Bacteria in Cryopreservation

S ummary : Malt extracts prepared from barley and sorghum malts were used as media for preserving bacteria at -196°. The results are compared with those obtained with other protective substances such as dimethylsulphoxide, glycerol, malic acid, sucrose and sodium glutamate. The malt extract medium is recommended for use in the food industry.     

Effect of Lactic Acid Bacteria on Growth of Staphylococcus aureus

Cultures of lactic acid bacteria, mostly from foods, were tested for their effect on the growth of Staphylococcus aureus in Trypticase Soy Broth (BBL). Some of the effectors, e.g., Streptococcus faecalis, S. faecium, Lactobacillus lactis, L. brevis, and Leuconostoc mesenteroides, stimulated growth of S. aureus during early hours of growth, especially at higher temperatures of incubation, but most cultures were inhibitory, and some (S. faecium and L. mesenteroides) were even killing by the time of attainment of the maximal phase of growth of the Staphylococcus. Low-temperature meat lactobacilli and Leuconostoc dextranicum inhibited S. aureus at 10, 15, 20, and 25 C throughout its growth. Streptococcus faecalis var. liquefaciens inhibited at these temperatures and at 30 and 37 C, as well. When the ratio of effectors to staphylococci in the inoculum was 100:1, the three enterococci, the meat Lactobacillus, and L. dextranicum prevented the attainment of 5 x 10(6) staphylococci per milliliter at 15 C, and all but the meat Lactobacillus did so at 22 C. A ratio of 1:1 accomplished similar results at 15 C, except that S. aureus was only delayed for 12 hr by S. faecalis. A ratio of 1:100 usually was ineffective. In general, the more effector bacteria there were in the inoculum, the greater was the overall inhibition (or stimulation) of S. aureus. Inhibition was most effective at 10 or 15 C, less so at 20 or 25 C, and least at 30 or 37 C, whereas stimulation during early growth was greater at the higher temperatures. Results with different strains of the effectors and with two strains of S. aureus were similar, for the most part.     

乳酸菌酸胁迫反应机制研究进展

乳酸菌可发酵糖类产生乳酸,并广泛应用于食品、药物和饲料等工业。由于有机酸的积累,乳酸菌大部分的生长代谢都在低pH的酸性环境中进行,具有酸胁迫反应。pH的自我平衡、ATR反应机制、对大分子的保护和修复作用及细胞膜的变化等是乳酸菌酸胁迫反应的主要机制,其中,pH自我平衡包括F0F1-ATPase质子泵、精氨酸脱氨酶途径(ADI)和谷氨酸脱羧酶途径(GAD)等。由此可见,乳酸菌酸胁迫反应机制涉及到基因和蛋白的表达调控等,是非常复杂的网络调控体系。     

Protective Effect of Adonitol on Lactic Acid Bacteria Subjected to Freeze-Drying

The protective effects of glycerol, adonitol, and four other related polyhydric alcohols on lactic acid bacteria subjected to freeze-drying were examined. The presence of adonitol in the suspending medium markedly protected the viabilities of the 12 stains tested. Dulcitol, mannitol, m-inositol, and sorbitol were found to provide little or no protection.     

Antagonism of Lactic Acid Bacteria against Phytopathogenic Bacteria

A variety of lactic acid bacteria, isolated from plant surfaces and plant-associated products, were found to be antagonistic to test strains of the phytopathogens Xanthomonas campestris, Erwinia carotovora, and Pseudomonas syringae. Effective “in vitro” inhibition was found both on agar plates and in broth cultures. In pot trials, treatment of bean plants with a Lactobacillus plantarum strain before inoculation with P. syringae caused a significant reduction of the disease incidence.     

Effect of Sodium Nitrite and Sodium Chloride on Growth of Lactic Acid Bacteria

The effect of NaNO2 and NaCl on the growth of 24 lactic acid bacteria strains isolated from vacuum-packed cooked ring sausages were examined by analyzing different growth parameters with Bioscreen. NaNO2 had a very limited effect on the growth of lactic acid bacteria at 50 and 100 mg/l but at 400 mg/l a more pronounced inhibitory effect was found. Bacterial growth was enhanced by 1–2% (w/v) of added NaCl, while NaCl concentrations above 3% (w/v) had a clear inhibitory effect. Leuconostoc isolates seemed to be more sensitive to sodium nitrite and sodium chloride than homofermentative lactobacilli strains. Among homofermentative lactobacilli, the strains resembling Lactobacillus curvatus were more sensitive to NaCl than those resembling Lactobacillus sake.     

一组鸡源乳酸菌产乳酸及其耐受特性研究*

研究了12株（K9、D17、C1、C12、D11、D14、C2、D9、K6、C21、D1和D7）分离自肉鸡肠道的乳酸菌的产乳酸能力及其中3株产酸能力强的菌株的耐受特性。12株乳酸菌产乳酸结果表明：12h内,K6产乳酸速度最快,其次为K9和C1,24h时,D17乳酸浓度最高,48h时C1终乳酸浓度最高。K9、D17和C1的耐受试验结果表明：C1菌株耐酸能力最强,pH2时,C1菌株培养3h后还能检测到活菌,D17和K9菌株培养1h后就已经检测不到活菌。在胆盐浓度0．08％-0．40％范围内,C1、D17和K9均有一定的耐受能力,随着胆盐浓度的升高,C1、D17和K9的存活数呈现缓慢的下降趋势。3株菌中D17耐热能力最强,经80％处理后仍有10^4.9／mL存活数,而K9和C1已检测不到活菌;C1对热最敏感,65℃处理后存活数由10^8／mL降为10^3／mL。     

Antioxidant Properties of Lactic Acid Bacteria

Microbiology - Lactic acid bacteria (LAB) are widely used in fermentation processes for the preparation of various foodstuffs, including dairy, meat and vegetable products. In the course of...     

Studies on Lipolysis of Dairy Lactic Acid Bacteria

Cell-free extracts were prepared by Hughes’ type disintegrator from cells of Lactobacillus casei, L. plantarum, L. helveticus and Streptococcus diacetilactis. Lipolytic activities of these extracts were measured by titration of total acids liberated from tributyrin and butterfal emulsions at given temperature. More acids were liberated from tributyrin than from butterfat, olive oil and composite butter. Lipolytic activities of all extracts for tributyrin were approximately the same at reaction temperature of 30°C, 37°C and 45°C, and were higher at pH 6 to 8. Free fatty acids of C₁₀, C₁₂, C₁₄, C₁₆ and C₁₈ liberated by extracts of L. casei and L. plantarum, were tentatively identified on gas-liquid chromatograms.     

乳酸菌在基因工程领域的应用

乳酸菌是工业微生物中一类非常重要的细菌,也是众所周知的维持人及动物健康的重要益生菌群.在最近十几年来,乳酸菌的遗传学和分子生物学特性很受关注,通过对它们进行遗传修饰改造取得了重要成果,扩大了它们的应用.