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.     

Bioprotection of Golden Delicious apples and Iceberg lettuce against foodborne bacterial pathogens by lactic acid bacteria

Lactic acid bacteria were isolated from fresh vegetables and fruit and its ability to inhibit the growth of foodborne human pathogens (Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Salmonella typhimurium, and Staphylococcus aureus) was tested using the agar spot assay. Eighteen isolates showed a strong antagonistic capacity and were further characterised and identified using 16S rDNA sequencing and API 50CH. Most of them pertained to Leuconostoc spp. and Lactobacillus plantarum, and a few corresponded to Weissella spp. and Lactococcus lactis. Growth and efficacy of control of foodborne pathogen test bacteria by selected strains were tested in wounded Golden Delicious apples and Iceberg lettuce leaf cuts. The strains grew on the substrates and did not cause negative effects on the general aspect of tissues of apple or lettuce. Treatment of apple wounds and lettuce cuts with the antagonistic strains reduced the cell count of S. typhimurium and E .coli by 1 to 2 log cfu/wound or g, whereas the growth of L. monocytogenes was completely inhibited. Results support the potential use of lactic acid bacteria as bioprotective agents against foodborne human pathogens in ready-to-eat fresh fruit and vegetable products.     

驼乳制品中抑制α-淀粉酶和α-葡萄糖苷酶活性乳酸菌的筛选及益生特性研究

目的:本研究以新疆阿勒泰地区的驼乳制品为研究对象,筛选对α-淀粉酶和α-葡萄糖苷酶有抑制活性的优良乳酸菌。方法:采用稀释涂布法分离纯化菌株,DNS和pNPG法筛选对α-淀粉酶和α-葡萄糖苷酶有抑制活性的菌株,通过耐酸性、耐胆盐、模拟胃肠道环境耐受性、抑菌性、抗氧化活性实验评价菌株的益生特性。结果:从驼乳制品中共计获得34株菌株,其中6株对α-淀粉酶和α-葡萄糖苷酶有抑制活性,经过形态学和16S rRNA分子鉴定,确定有4株为植物乳杆菌Lactiplantibacillus plantarum,2株为副干酪乳杆菌Lactiplantibacillus paracasei。6株乳酸菌对α-淀粉酶的抑制活性都达到50%以上,其中X34对α-淀粉酶的抑制率最高达到88.59%。本实验中筛选的乳酸菌对α-葡萄糖苷酶的抑制率在11%~16%之间,X31抑制率最高为15.43%。6株乳酸菌在不同pH（1.0、2.0、3.0）和不同浓度胆盐（1、2、3 g/L）的培养基中均可存活。6株乳酸菌在模拟胃液和肠液中的存活率分别达到83%和90%以上。对羟自由基和超氧阴离子自由基的清除率超过90%以上,其中X29对羟自由基的清除率最高为92.43%,对超氧阴离子自由基清除率最强的是X33清除率达到94.04%。6株乳酸菌对大肠杆菌、沙门氏菌和金黄色葡萄球菌有一定的抑制作用,其中X31对大肠杆菌的最大抑菌圈直径为19.63 mm,X23对沙门氏菌抑菌圈最大达到19.85 mm,菌株X33对金黄色葡萄球菌的抑菌圈直径最大为19.17 mm。结论:从驼乳制品中筛选到6株具有潜在降糖活性的乳酸菌,对强酸、胆盐和胃肠液有一定的耐受性,抗氧化能力较强,具有抑制致病菌的效果,为后期研发功能性降糖饮品提供益生菌株。     

Lactobacillus fermentum isolated from human colonic mucosal biopsy inhibits the growth and adhesion of enteric and foodborne pathogens

A number of Lactobacillus species are used as probiotic strains in order to benefit health. We have isolated L. fermentum from human colonic mucosal biopsy samples that possess antimicrobial activities against entroinvasive and foodborne pathogens such as Escherichia coli, Salmonella paratyphi A, Shigella sonnei, Staphylococcus aureus, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, and Vibrio sp. In addition to lactic acid, L. fermentum secretes antimicrobial proteinacious compound(s) that was found to be active even at neutral pH (pH 7.0). The compound was sensitive to heat treatment and trypsin digestion. Lactobacillus fermentum inhibited the adhesion of enteropathogens to intestinal epithelial cells in vitro. Isolated cell surface associated proteins (SAPs) from L. fermentum were sufficient for the adhesion exclusions of enteropathogenic E. coli. Our results indicate that L. fermentum produces antimicrobial compounds and SAPs to inhibit the growth and adhesion of enteropathogens, respectively.     

Identification and antagonistic activity of lactic acid bacteria occurring in porcine blood from industrial slaughterhouses--a preliminary study

Ninety-seven lactic acid bacteria (LAB) were isolated from slaughterhouse porcine blood in order to select autochthonous LAB strains for use as biopreservatives of this by-product. They were identified by 16S rDNA sequencing; and their inhibition capacity was determined against four bacterial species frequently found in contaminated blood, i.e. Staphylococcus aureus, Escherichia coli, Pseudomonas fluorescens and Bacillus spp. The taxonomic study showed an unexpected low diversity of LAB in blood, i.e. only 8 different species were found, from which just 4, i.e. Enterococcus raffinosus, Lactobacillus murinus, Lactobacillus reuteri and Lactococcus garvieae, amounted to more than 90% of all isolates. Inhibition tests in solid culture media proved that S. aureus and Bacillus spp. were inhibited by most LAB strains obtained from porcine blood. E. coli was the indicator less affected by the isolated LAB species. Several isolates efficiently inhibited the growth of all tested indicators.     

ISOLATION AND CHARACTERIZATION OF BACTERIOCIN-PRODUCING MICROORGANISMS FROM AGOS-OS

Agos-os, a fermented meat and sweetpotato mixture, was produced and analyzed for its microbial characteristics. pH decreased during fermentation. Mold and anaerobic bacterial counts increased while yeasts and aerobic bacterial counts decreased during the third and seventh day of fermentation. Six isolates with the widest zones of inhibition on the indicator lawn were selected for bacteriocin production. These isolates had exactly the same morphological, physiological and biochemical characteristics. The ribosomal RNA sequence was 99.5% identical with Enterococcus faecalis VRE 1492. The identification was confirmed through DNA homology test by the EMBL Genbank, Canada. This bacterium produced the L-isomer lactic acid. The amount of bacteriocin produced by the bacterium was optimized by growing the bacterium at different growth media, initial pH and fermentation time. Maximum production of bacteriocin was achieved in MRS (De Man Rugosa and Sharpe) medium (with glucose) at pH 7.50. The crude bacteriocin inhibited the growth of gram-positive bacteria such as Lactobacillus sake 15521 and Listeria innocua. The gram-negative bacteria such as Escherichia coli DH 5-alpha (with plasmid, PUC) , Salmonella typhii and Staphylococcus aureus were weakly inhibited. Other microorganisms such as Lactobacillus curvatus D31685 , Lactobacillus confusius M23036 , Lactococcus lactis MG1363 , Leuconostoc paramesenteroides S67831 , Pediococcus pentosaceus M58834 , Saccharomyces cerevisiae SS553 (wild type) and Escherichia coli JM109 (no plasmid) were not inhibited.     

Characterization of the lactic acid bacteria in ewe's milk and cheese from northwest Argentina

Indigenous lactic acid bacteria in ewe's milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.     

传统发酵牦牛酸乳中细菌素产生菌的筛选与鉴定

从西藏羊八井地区、云南香格里拉的传统牦牛酸乳中分离出26株乳酸菌,以枯草芽孢杆菌、大肠杆菌、金黄色葡萄球菌以及酿酒酵母为指示菌。采用牛津杯双层琼脂扩散法检测菌株的抑菌谱大小,并经过有机酸排除、过氧化氢排除、蛋白酶水解检测试验,最终筛选得到5株产细菌素的菌株,编号分别为3、23、24、21和25。通过微生物形态学与16SrDNA序列同源性分析,鉴定这些菌株分别为干酪乳杆菌、植物乳杆菌和乳酸乳球菌乳球亚种。抑菌谱试验表明,这些细菌素能够抑制部分食源性革兰氏阳性菌和阴性致病菌,对真菌无抑菌活性。     

Antagonistic action of Lactobacillus lactis toward Salmonella spp. and Escherichia coli O157:H7 during growth and refrigerated storage

Cells of Lactobacillus lactis were added to trypticase soy broth that contained cells of Escherichia coli O157:H7 or cells of Salmonella spp. in order to determine if L. lactis inhibited the pathogens. The inhibition of all pathogens was examined during growth at 37 degrees C for 24 h. Inhibition of Salmonella spp. was also examined at refrigeration temperatures (6 degrees C) for 5 days. One strain each of E. coli O157:H7, Salmonella Typhimurium, and Salmonella Enteritidis was examined. E. coli was enumerated on violet red bile agar, and Salmonella spp. were enumerated on brilliant green agar. In all experiments at 37 degrees C, the L. lactis completely inhibited all pathogens, producing numbers that were not detectable after 24 h of incubation. There were significant (P > 0.05) increases in numbers of the pathogens in the control samples containing no L. lactis. There were significant (P < 0.05) declines in the pH of both control and L. lactis inoculated samples. There was a significantly (P < 0.05) larger decline in the pH of samples inoculated with L.lactis. Interaction studies with pH-neutralized broth indicated that acid production by L lactis was primarily responsible for the inhibition. Numbers of Salmonella spp. incubated at 6 degrees C did not decline significantly (P > 0.05) for control or inoculated samples, which suggests that this strain of L. lactis does not inhibit Salmonella spp. at refrigeration temperatures. Additionally, there were no significant (P > 0.05) changes in pH or in numbers of L. lactis during refrigerated storage.     

抗食源性致病菌益生菌的筛选及其抑菌活性评价

采用交叉划线法、径向划线法、琼脂扩散法及液体共培养法评价各益生菌菌株及其代谢产物对4 株具代表性食源性致病菌的抑菌作用,并从20?株益生菌中筛选出同时对单核细胞性李斯特菌和金黄色葡萄球菌（革兰氏阳性致病菌）及大肠杆菌和鼠伤寒沙门菌（革兰氏阴性致病菌）具有良好抑菌活性的益生菌菌株。将筛选出的益生菌添加到羟丙基纤维素、丙三醇及魔芋粉的高分子复合材料中制备生物活性抑菌膜,并对抑菌膜中益生菌的存活情况及抑菌膜的抗菌效果进行评价。交叉划线法及径向划线法实验结果一致：乳杆菌属和肠球菌属的菌株,特别是Lactobacillus paracasei CICC 20241菌株对4?株目标致病菌均具有较高的抗菌活性;琼脂扩散法实验表明：L. paracasei CICC 20241菌株的培养液、上清液和菌悬液均具有较好的抑菌效果;液体共培养法实验表明：乳酸菌L. paracasei CICC 20241先于致病菌接种时表现出更高的生长活性和拮抗活性。此外,本实验制备的抑菌膜是L. paracasei CICC 20241菌株的良好载体,适宜该菌株的生长存活并对目标致病菌表现出持续的抗菌活性,对于开发鲜食食品的包装材料具有较大的应用潜力。     

嗜酸乳杆菌和乳酸链球菌混合培养及产物分析

嗜酸乳杆乳菌(LA)和酸链球菌(SL)在33℃培养48 h的发酵液中抑菌(金黄色葡萄球菌ATCC25923)效果最好;2菌混培20h后生物量和体系总产酸量远大于各自纯培养,乳酸含量一直上升,到24 h达23.175 mg/L,乙酸含量一直稳定在12 mg/L左右,变化不大,乳酸与抑菌关系密切。LA和SL两菌混培接种比例的抑菌优先顺序为(1:1)、(2:1)、(1:2)。     

哈族传统乳制品中乳酸菌多样性及抑菌性研究

从新疆阿勒泰地区哈萨克族传统发酵乳制品中分离得到11株乳酸菌,采用生理生化特性和16S rDNA序列同源性分析,对其进行鉴定。通过纸片法筛选,大多数乳酸菌对大肠杆菌(Escherichia coli)、金黄色葡萄球菌(Staphylococcus aureus)、铜绿假单胞菌(Pseudomonas aerugonosa)、枯草芽孢杆菌(Bacillus subtilis)和李斯特氏菌(Listeria monocytogenes)这5株指示菌有抑菌效果,其中1株干酪乳杆菌(TNG2)和4株瑞士乳杆菌(NS2-a,TNS1-1,NS1-1和NS2-1)对5株指示菌均有抑菌效果。     

酵素中具有抑菌能力乳酸菌的筛选及鉴定

该研究探讨了从酵素中分离纯化具有抗菌性能的12株乳酸菌，对其进行生理生化实验和API鉴定。利用牛津杯法对阪崎大肠杆菌、鼠伤寒沙门氏菌和宋内志贺氏菌、大肠杆菌、金黄色葡萄球菌进行抑菌特性研究，并探讨了乳酸菌是否产生物胺能力和偶氮还原酶。结果表明，酵素源的12株乳酸菌对肠道致病菌均具有不同程度的抑菌活性，其抑菌圈直径为14~22 mm，对大肠杆菌的抑菌效果较好，其抑菌圈直径为15~23 mm，对金黄色葡萄球菌的抑菌圈直径为14~24 mm，12株乳酸菌都不具有产生物胺和偶氮还原酶的能力。通过生理和API生化鉴定确定XZJ003、XZJ006、XZJ015均为副干酪乳杆菌，通过16S rDNA和同源性确证XZJ015为副干酪乳杆菌，该研究结果对今后益生菌的深入研究和功能性食品的开发具有一定的作用。     

Effect of mannoproteins on the growth, gastrointestinal viability, and adherence to Caco-2 cells of lactic acid bacteria

Yeast cell wall (YCW) preparations and yeast mannoprotein extracts have been effective against some enteropathogenic bacteria as Campylobacter jejuni, Escherichia coli, and Salmonella, and they can affect the population of beneficial lactic acid bacteria (LAB). In this work, we studied the effect of a mannoprotein extract on five strains of LAB. This extract was metabolised by the bacteria, enhancing their survival in simulated gastrointestinal juice, and increasing the adherence of Lactobacillus plantarum, L. salivarius, and Enterococcus faecium to Caco-2 cells. Yeast mannoproteins are promising naturally occurring compounds that could be used to enhance LAB intestinal populations and control pathogens.     

Isolation, identification, and selection of lactic acid bacteria from alfalfa sprouts for competitive inhibition of foodborne pathogens

Several studies have investigated the control of pathogens on alfalfa sprouts, and some treatments have been shown to be effective in reducing pathogen populations. However, control methods investigated thus far only provide pathogen control at a given point in the sprouting process and can affect germination. Competitive inhibition of pathogens with lactic acid bacteria might provide pathogen control throughout the sprouting process and up to consumption. The purpose of this study was to isolate and identify lactic acid bacteria from alfalfa sprouts to inhibit the growth of foodborne pathogens. Fifty-eight lactic acid bacteria isolates were obtained from alfalfa seeds and sprouts. These isolates were evaluated for inhibitory action against Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes by agar spot tests. All pathogens were inhibited by 32 (55%) of the isolates, S. enterica by 56 (97%), E. coli O157:H7 by 49 (84%), and L. monocytogenes by 41 (71%). The isolates were identified by the Analytical Profile Index evaluation of carbohydrate utilization. Isolates obtained from a sample of alfalfa seeds and identified as Lactococcus lactis subsp. lactis showed zones of inhibition of 4.0 mm or greater for all pathogens. One of these isolates, Lactococcus lactis subsp. lactis (L7), and an isolate previously obtained, Pediococcus acidilactici (D3), were evaluated for competitive inhibition of S. enterica, E. coli O157:H7, and L. monocytogenes in deMan Rogosa Sharpe agar and broth. Pathogen populations were significantly reduced by day 5. The selected isolates will be further evaluated in future studies for inhibitory action toward S. enterica, E. coli O157:H7, and L. monocytogenes during sprouting.     

Studies on antimutagenic effect of milk cultured with lactic acid bacteria on the Trp-P2-induced mutagenicity to TA98 strain of Salmonella typhimurium.

The inhibitory effects of cultured milk using 76 strains of lactic acid bacteria isolated from milk products were investigated on the mutagenicity of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P2), a tryptophan pyrolysate for Salmonella typhimurium TA98. Each cultured milk sample displayed its characteristic antimutagenic effect against the mutagenicity of Trp-P2. The milk cultured with Lactobacillus acidophilus LA106 (LA2) showed the highest inhibition of 82.1% among the strains used. Milk samples cultured with Lactococcus lactis subsp. lactis, Lll103 (10-3) and Lll102 (KM) also exhibited higher inhibition percentages.     

Isolation of lactic acid bacteria from Lighvan cheese, a semihard cheese made from raw sheep milk in Iran

The lactic acid bacteria contributing to Lighvan cheese ripening during the different stages of production were investigated. Isolated strains from different culture media were identified phenotypically to species and subspecies level. In total, 413 strains were isolated from raw milk, 1-day-old cheese and fully ripened cheese. The most abundant species belonged to Enterococcus faecium (87 isolates), Lactococcus lactis ssp. lactis (68 isolates), Enterococcus faecalis (55 isolates) and Lactobacillus plantarum (48 isolates). E. faecium, Lc. lactis and Lb. plantarum were the predominantly isolated strains from ripened cheese. Therefore, they may contribute considerably to the aroma and flavour development of Lighvan cheese.     

Isolation of bacteriocinogenic Lactobacillus plantarum strains from ben saalga, a traditional fermented gruel from Burkina Faso

A collection of lactic acid bacteria isolated from ben saalga, a traditional fermented gruel from Burkina Faso, was screened for bacteriocin production. Seven isolates were selected for their broad antimicrobial spectra, which overall included strains of Bacillus cereus, Bacillus licheniformis, Enterococcus faecalis, Listeria innocua, Listeria monocytogenes, Staphylococcus aureus, Escherichia coli and Salmonella enterica. Cluster analysis of RAPD-PCR patterns revealed that six of the isolates represent different strains. The six selected strains were identified as Lactobacillus plantarum by 16S rDNA sequencing, species-specific PCR and multiplex PCR of the recA gene. PCR amplification revealed the presence of genes of the plantaricin cluster described in L. plantarum C11. Among them, strain 5.2.2 carried the largest number of genes from this cluster.     

INHIBITION OF LISTERIA MONOCYTOGENES BY LACTOCOCCUS LACTIS SUBSP. LACTIS ISOLATED FROM ITALIAN RAW HAM

A total of 168 strains of lactic acid bacteria were isolated from Italian raw ham and screened for antagonistic activity against Listeria monocytogenes by using an agar spot assay. Only one strain of Lactococcus lactis subsp . lactis produced antagonistic effects other than inhibition by low pH and hydrogen peroxide. The proteinaceous nature of the compound produced by L. lactis B10 was demonstrated by its inactivation by proteolytic enzymes. The cell-free culture super-natants (filtered and heat-treated) showed a bactericidal mode of action. Bacteriocin produced by L. lactis B10 was inhibitory to other lactic acid bacteria and one strain of Staphylococcus aureus, but not to the gram-negative bacteria tested .     

东北酸菜优良乳酸菌筛选及其抑菌特性研究

该实验从东北酸菜中分离纯化并鉴定出40种乳酸菌,主要为植物乳杆菌（Lactobacillus plantarum）、融合魏斯氏菌（Weissella confuse）、副干酪乳杆菌（Lactobacillus paracasei）、干酪乳杆菌（Lactobacillus casei）、食窦魏斯氏菌（Weissella cibaria）。通过测定乳酸菌乳酸产量及其抑制大肠杆菌（Escherichia coli）的能力,探究乳酸菌的产酸量与其抑菌作用之间相互关系。结果表明,菌株C1、E8、A5产酸能力较强,发酵12 h后发酵上清液中乳酸质量浓度＞12 g/L,抑菌圈直径＞12 mm,对大肠杆菌抑制能力强。表明东北酸菜中乳酸菌的产酸量与抑菌作用存在紧密的联系。