Screening of lactic acid bacteria and bifidobacteria for potential probiotic use in Iberian dry fermented sausages

The purpose of this study was to select lactic acid bacteria and bifibobacteria strains as potential probiotic cultures during the processing of Iberian dry fermented sausages. A total of 1000 strains were isolated from Iberian dry fermented sausages (363), and human (337) and pig faeces (300) in different culture media. Around 30% of these strains, mainly isolated from Iberian dry fermented sausages in LAMVAB agar, were pre-selected for testing as potential probiotics by their ability to grow adequately at the pH values and NaCl concentrations of these meat products during the ripening process. Of the in vitro investigations used to predict the survival of a strain in conditions present in the gastro intestinal tract, exposure to pH 2.5 showed itself to be a highly discriminating factor with only 51 out of 312 pre-selected strains resisting adequately after 1.5h of exposure. All acid-resistant isolates identified as lactobacilli originated from human faeces (Lactobacillus casei and Lactobacillus fermentum) and pig faeces (Lactobacillus reuteri, Lactobacillus animalis, Lactobacillus murinus, and Lactobacillus vaginalis). Pediococcus acidilactici strains were isolated from Iberian dry fermented sausages and pig faeces, whereas the greatest number of Enterococcus strains were identified as Enterococcus faecium, with this species being isolated from Iberian dry fermented sausages, and human and pig faeces. Most of these strains are promising probiotic meat culture candidates suitable for Iberian dry fermented sausages.     

Antibiotic resistance in non-enterococcal lactic acid bacteria and bifidobacteria

Over the last 50 years, human life expectancy and quality of life have increased dramatically due to improvements in nutrition and the use of antibiotics in the fight against infectious diseases. However, the heyday of antibiotic treatment is on the wane due to the appearance and spread of resistance among harmful microorganisms. At present, there is great concern that commensal bacterial populations from food and the gastrointestinal tract (GIT) of humans and animals, such as lactic acid bacteria (LAB) and bifidobacteria, could act as a reservoir for antibiotic resistance genes. Resistances could ultimately be transferred to human pathogenic and opportunistic bacteria hampering the treatment of infections. LAB species have traditionally been used as starter cultures in the production of fermented feed and foodstuffs. Further, LAB and bifidobacteria are normal inhabitants of the GIT where they are known to exert health-promoting effects, and selected strains are currently been used as probiotics. Antibiotic resistance genes carried by LAB and bifidobacteria can be transferred to human pathogenic bacteria either during food manufacture or during passage through the GIT. The aim of this review is to address well-stated and recent knowledge on antibiotic resistance in typical LAB and bifidobacteria species. Therefore, the commonest antibiotic resistance profiles, the distinction between intrinsic and atypical resistances, and some of the genetic determinants already discovered will all be discussed.     

In vitro testing of commercial and potential probiotic lactic acid bacteria

Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host. The objective of this study was to investigate the diversity of selected commercial and potential probiotic lactic acid bacteria using common in vitro screening assays such as transit tolerance in the upper human gastrointestinal tract, adhesion capacity to human intestinal cell lines and effect on epithelial barrier function. The selected bacteria include strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus farciminis, Lactobacillus sakei, Lactobacillus gasseri, Lactobacillus rhamnosus, Lactobacillus reuteri and Pediococcus pentosaceus. Viable counts after simulated gastric transit tolerance showed that L. reuteri strains and P. pentosaceus tolerate gastric juice well, with no reduction of viability, whereas L. pentosus, L. farciminis and L. sakei strains lost viability over 180min. All strains tested tolerate the simulated small intestinal juice well. The bacterial adhesion capacity to human intestinal cells revealed major species and strain differences. Overall, L. plantarum MF1298 and three L. reuteri strains had a significant higher adhesion capacity compared to the other strains tested. All strains, both living and UV-inactivated, had little effect on the epithelial barrier function. However, living L. reuteri strains revealed a tendency to increase the transepithelial electrical resistance (TER) from 6 to 24h. This work demonstrates the diversity of 18 potential probiotic bacteria, with major species and strain specific effects in the in vitro screening assays applied. Overall, L. reuteri strains reveal some interesting characteristics compared to the other strains investigated.     

Effect of grape polyphenols on lactic acid bacteria and bifidobacteria growth: resistance and metabolism

Food polyphenols are able to selectively modify the growth of susceptible micro-organisms. This study describes the effect of a flavan-3-ol enriched grape seed extract (GSE) on the growth of several lactic acid bacteria (LAB) and bifidobacteria and the ability of the resistant strains to metabolize these compounds. Streptococcus thermophilus, Lactobacillus fermentum, Lactobacillus acidophilus and Lactobacillus vaginalis strains showed a remarkable sensitivity to the phenolic extracts assayed, including a GSE fraction consisting mainly in (+)-catechin and (−)-epicatechin (GSE-M). On the other hand, Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus bulgaricus strains reached maximal growth with the GSE fractions, including a rich-oligomeric (GSE-O) fraction. Within bifidobacteria, Bifidobacterium lactis BB12 showed the highest sensitivity to the phenolic extracts assayed, whereas Bifidobacterium breve 26M2 and Bifidobacterium bifidum HDD541 reached maximum growth in presence of GSE-O and GSE-M fractions. Metabolism of flavan-3-ols by LAB and bifidobacteria resistant strains was investigated in vitro. The results revealed that only L. plantarum IFPL935 was able to metabolize the polyphenols studied by means of galloyl-esterase, decarboxylase and benzyl alcohol dehydrogenase activities that led to the formation of gallic acid, pyrogallol and catechol, respectively. An unknown metabolite that does not exhibit a phenolic-acid-type structure was also detected, which suggests a new enzyme activity in L. plantarum IFPL935 able to degrade flavan-3-ol monomers.     

潜在益生乳酸菌分离和鉴定研究进展

利用益生菌提高人类健康水平是当前营养健康领域最具前景的方向之一。乳酸菌是食品中常用的益生菌,迄今为止,联合国粮食及农业组织（FAO）和世界卫生组织（WHO）推荐仅从人的胃肠道分离的乳酸菌可在食品中使用。而越来越多的研究从食品来源中分离出具有益生功能的菌株以扩大益生菌的选择范围。随着分子生物学和检测分析等技术的发展,基质辅助激光解吸电离-飞行时间质谱（MALDI-TOF MS）、全基因组测序、实时荧光定量聚合酶链式反应（RT-FQ-PCR）等技术可以鉴定到亚种及菌株水平上的益生菌。鉴于分离鉴定技术是益生菌安全规范应用的先决条件,该文综述了近年来针对潜在益生乳酸菌有效分离和鉴定技术的研究进展,以期为我国益生乳酸菌自主开发利用提供借鉴。     

Screening of bile resistance and bile precipitation in lactic acid bacteria and bifidobacteria.

A modification of the ecometric method was developed for a rapid screening of bile resistance in lactic acid bacteria and bifidobacteria. Validation of the MEM bile assay (modified ecometric method) was performed comparing the bile resistance index (RIbile) and the bile resistance ratio (R%). Most Bifidobacterium strains assayed were bile sensitive (83.3%), while only 62.1% of lactobacilli showed that behavior. Some bifidobacterial strains (55.6%) showed a crystalline precipitate when grown on solid medium supplemented with 0.5% ox bile. The crystalline structures produced by B. pseudolongum CIDCA 531 were isolated and analyzed by optical and scanning electron microscopy, thin-layer chromatography, melting point, and specific cholesterol reactions. Those studies confirmed the presence of cholesterol in these crystalline structures. On the other hand, none of the lactobacilli and streptococci studied had the ability to produce crystalline precipitates.     

Screening of lactic acid bacteria isolated from fermented table olives with probiotic potential

The aim of this work was to study the potential probiotic properties of lactobacilli associated with table olives. From a total of 111 isolates from spontaneously fermented green olive brines, 109 were identified at species level by multiplex PCR amplifications of the recA gene. One hundred and seven of these were identified as Lactobacillus pentosus, one as Lactobacillus plantarum, and another as Lactobacillus paraplantarum. Repetitive bacterial DNA element fingerprinting (rep-PCR) with GTG₅ primer revealed a higher variability within the L. pentosus isolates and nine different clusters were obtained. Most of them showed high autoaggregation ability, low hydrophobicity properties, and lower survival to gastric than to pancreatic digestion; however, no isolate showed bacteriocin, haemolytic or bile salt hydrolase activities. A multivariate analysis based on results from phenotypic tests led to the segregation of some L. pentosus isolates with promising potential probiotic characteristics, which are even better than probiotic reference strains. Due to the autochthonous origin of the strains, their use as starter cultures may contribute to improving natural fermentation and the nutritional characteristics of table olives.     

益生乳酸菌对氟喹诺酮类药物耐药机制的研究进展

益生乳酸菌对抗菌药物的耐药性机制是生物医药研究的重要领域。概述了益生乳酸菌对氟喹诺酮类药物的3种耐药性机制(靶位突变、外排泵作用、质粒介导)及其可能出现的耐药机制。显示出国内关于益生乳酸菌对氟喹诺酮类药物的耐药机制研究甚少的现状,提出对益生乳酸菌的耐药机制进行全面深入的研究的重要性。     

Antioxidative activities of soymilk fermented with lactic acid bacteria and bifidobacteria

To further the goal of developing a probiotic dietary adjunct using soymilk, soymilk is fermented with lactic acid bacteria (Lactobacillus acidophilus CCRC 14079 or Streptococcus thermophilus CCRC 14085) and bifidobacteria (Bifidobacterium infantis CCRC 14633 or Bifidobacterium longum B6) individually, and in conjunction. We investigate several antioxidative activities including the inhibition of ascorbate autoxidation, the scavenging effect of superoxide anion radicals and hydrogen peroxide, and the reducing activity exerted by different varieties of fermented soymilks. In addition, the effect of spray-drying and freeze-drying on changes in antioxidative activity is examined. We find that in fermented soymilk both the inhibition of ascorbate autoxidation, and the reducing activity and scavenging effect of superoxide anion radicals varied with the starters used, but nevertheless are significantly higher than those found in unfermented soymilk. In general, antioxidative activity in soymilk fermented with lactic acid bacteria and bifidobacteria simultaneously is significantly higher (P < 0.05) than that fermented with either individually. Moreover, antioxidative activity increases as the fermentation period is extended. However, unfermented soymilk shows an H2O2-scavenging effect, while there is no scavenging effect except for the accumulation of H2O2 in fermented soymilk. Finally, we find that freeze-drying causes a significantly lesser (P < 0.05) reduction in the antioxidative activity of soymilk than does spray-drying. Irrespective of the drying method and the starters used for fermentation. The antioxidative activity of fermented soymilk reduces after drying yet remains higher than that of dried unfermented soymilk.     

Competition mechanisms of lactic acid bacteria and bifidobacteria: Fermentative metabolism and colonization

Enzyme activities (α- and β-glucosidases, α- and β-galactosidases and β-fructofuranosidase) and organic acid production of four strains of lactic acid bacteria (LAB; Streptococcus thermophilus STY-31, Lactobacillus delbrueckii subsp. bulgaricus LBY-27, Lactobacillus casei LC-01 and Lactobacillus acidophilus LA-5) and Bifidobacterium lactis BB-12 were tested on milk and MRS fermentation broth with glucose, lactose or fructooligosaccharides (FOS) as carbon source. The highest β-galactosidase activity was found in L. acidophilus growing on milk. As compared to milk, α-glucosidase activity was increased with FOS by B. lactis, L. acidophilus and L. casei. The analysis of organic acids and short-chain fatty acids in the medium growth showed that lactate and acetate were the major fermentation metabolites produced by LAB and bifidobacteria, respectively. However, a metabolic shift towards more acetate and formate production, at the expense of lactate production, was observed during growth of L. casei on FOS. When grown on FOS as sole carbon source, L. acidophilus showed the highest production of lactate among the species tested. In addition, L. acidophilus demonstrated resistance to colonization against the intestinal pathogens Escherichia coli and Salmonella enterica in competition assays.     

具有抑制大肠杆菌作用的乳酸菌的初步筛选及其益生潜能的研究

为了筛选出具有抑制致病性大肠杆菌作用的乳酸菌,本实验以大肠杆菌ATCC25922作为指示菌,抑菌实验采用牛津杯法。通过发酵上清液对大肠杆菌ATCC25922的抑菌实验,初步筛选出三株乳酸菌发酵上清液对大肠杆菌ATCC25922具有抑制作用,经过对16S rRNA基因进行系统发育分析,发现这三株乳杆菌均为嗜酸乳杆菌。随后又对这三株嗜酸乳杆菌进行了耐酸、耐胆盐及抗生素敏感性实验。结果表明:KLDS1.0901酸耐受性最强,而KLDS1.0902和KLDS1.1003与Lactobacillus acidophilus NCFM酸耐受性接近;对照菌株Lactobacillus acidophilus NCFM胆盐耐受能力最强,KLDS1.0901和KLDS1.1003与Lactobacillus acidophilus NCFM胆盐耐受能力差异不显著,KLDS1.0902胆盐耐受能力最弱;对于抗生素敏感性,三株嗜酸乳杆菌展现出类似的结果,对氨基糖苷类的抗生素展现出耐受;相比于KLDS1.0902,KLDS1.0901和KLDS1.1003有着更好的益生作用潜能,因此随后将对这两株菌在细胞和动物模型中对抗病原菌的作用进行进一步的研究。      

Bioinformatics and its role in the study of the evolution and probiotic potential of lactic acid bacteria

Food Science and Biotechnology - Due to their numerous well-established applications in the food industry, there have been many studies regarding the adaptation and evolution of lactic acid...     

Genetic screening of lactic acid bacteria of oenological origin for bacteriocin-encoding genes

A total of 330 lactic acid bacteria isolated from South African red wines during alcoholic and malolactic fermentations and 9 commercial malolactic bacteria starter cultures were screened for antimicrobial activity. Of the entire screened isolates, 26 strains, belonging to the species Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus hilgardii and Oenococcus oeni, showed activity towards various wine-related and non-wine-related indicator strains. A PCR-based screening revealed the presence of the plantaricin encoding genes plnA, plnEF, plnJ and plnK in five selected Lb. plantarum strains. Furthermore, a co-culture experiment with Lb. plantarum and Enterococcus faecalis was performed. A complete inhibition of cell growth of Ent. faecalis was observed within 72 h. Four putative bacteriocin-encoding genes in the genome of O. oeni were identified and sequenced.     

鲊广椒细菌多样性评价及其对风味的影响

本研究使用Illumina MiSeq测序技术解析兴山和保靖鲊广椒细菌群落结构。α多样性分析表明,兴山鲊广椒群落多样性及丰度均低于保靖鲊广椒（P<0.05）。两地鲊广椒中细菌主要是隶属于厚壁菌门（Firmicutes）的Lactobacillus（乳酸杆菌属）,其在兴山鲊广椒中含量为50.24%,在保靖鲊广椒中为88.26%。β多样性分析发现,兴山和保靖鲊广椒细菌群落结构整体上具有显著差异（P<0.01）,同时发现11个差异显著的细菌类群,其中得分较高的为Lactobacillus和Chishuiella（赤水河菌属）。表型预测发现,兴山鲊广椒细菌类群在可移动原件、生物膜形成、革兰氏阴性、致病潜力和氧化胁迫耐受等表型上表达显著,而保靖鲊广椒细菌类群则在革兰氏阳性表型上表达显著。     

分离自藏灵菇的乳酸菌的益生特性

从藏灵菇中分离纯化5株乳酸菌,初步鉴定2株为嗜酸乳杆菌,3株为乳酸乳球菌。选取其中2株菌研究其益生特性。结果表明,从藏灵菇中分离出的乳酸菌具有良好的益生特性,2株乳酸菌在pH值为4～6可生长良好;耐热范围为30～60℃;胆盐耐受性为0.1%～0.5%;发酵液对金黄色葡萄球菌、沙门氏菌、大肠杆菌等肠道病原菌有抑制作用;对抗生素有不同程度的耐药性。     

Mutagenicity and antimutagenic effect of soymilk fermented with lactic acid bacteria and bifidobacteria

In this study, soymilk was first fermented with lactic acid bacteria (Streptococcus thermophilus, Lactobacillus acidophilus) and bifidobacteria (Bifidobacterium infantis, Bifidobacterium longum) both individually and simultaneously. Mutagenicity and the suppression of fermented soymilk against the mutagenesis induced by 4-nitroquinoline-N-oxide (4-NQO), a direct-acting mutagen, and 3,2'-dimethyl-4-amino-biphenyl (DMAB), an indirect-acting mutagen, on Salmonella typhimurium TA 100, was then investigated. It was found that the fermented soymilk shows no mutagenic activity on Sal. typhimurium TA 100. Fermentation, in general, significantly (p<0.05) enhanced the antimutagenicity of soymilk. The levels of increased antimutagenicity of fermented soymilk varied with the starter organism and the type of mutagen tested. Although unfermented soymilk exerted lower antimutagenic activity against DMAB than 4-NQO, the fermented soymilk, generally, showed a higher antimutagenic activity against DMAB than 4-NQO. Among the various fermented soymilk tested, soymilk fermented with both Str. themophilus and B. infantis simultaneously exhibited the highest antimutagenicity of 85.07% and 85.78%, respectively, against 4-NQO and DMAB Further investigation on this fermented soymilk revealed that both the antimutagenic factors formed during fermentation and the cells of the starter organisms contributed to the increased antimutagenic activity against DMAB, while the former led to the increased activity against 4-NQO.     

泡菜中益生性乳酸菌的筛选和鉴定

以筛选具益生特性的泡菜发酵剂为目的,从15个泡菜样品中通过比较溶钙圈的大小分离出59株产酸能力较强的菌株。通过测定发酵液的酸度以及耐酸性和耐胆汁盐试验,再从中筛选出2株产酸能力和在胃肠道生存能力都强的菌株A18和B17。经形态特征及16S rDNA序列分析,确定A18和B17均属于植物乳杆菌。     

乳酸菌的益生特性及应用研究进展

活性乳酸菌产品因其良好的益生功效越来越受到人们的青睐,市场需求量不断增大。然而,受乳酸菌厌氧和热敏感性的限制,以及加工中加热和氧气胁迫的影响,产品中活性乳酸菌数量大幅度下降。进入体内的活性乳酸菌在胃液高酸性和肠道高胆汁酸等复杂环境的胁迫下进一步降低,严重影响产品的益生功效。为此,人们在提高乳酸菌对热、氧气、胃肠环境耐受性方面进行了大量研究,并开发了系列新技术,但缺乏彼此间的综合对比与分析。本文围绕如何提高乳酸菌的加工存活率,以及胃肠耐受性和肠道递送问题,对静电纺丝、静电喷雾、乳滴技术、多酚纳米盔甲、热诱导预处理等新技术的作用效果进行了总结与对比,以期为相关研究和技术应用提供参考。     

In vitro evaluation of physiological probiotic properties of different lactic acid bacteria strains of dairy and human origin

Eleven lactic acid bacteria strains of importance to the dairy industry were subjected to in vitro analyses to determine their probiotic potential. Seven strains were isolated from ewe’s and cow’s milk (Enterococcus faecalis – five –, Lactococcus lactis and Lactobacillus paracasei). Four were obtained from American Type Culture Collection (ATCC), isolated from cheese (Lactobacillus casei 393), human feces (L. paracasei 27092 and Lactobacillus rhamnosus 53103) and used in cheese making (L. lactis 54104). Although none of the strains was able to degrade mucin, all E. faecalis showed, at least, one transferable antibiotic resistance, which excluded them as candidates for addition to foods. Of the remaining six safe strains, L. lactis strains were more tolerant to low pH than Lactobacillus spp.; all were tolerant to pancreatin and bile salts and showed antibacterial activity. The highest level of adhesion to Caco-2 cells was observed with L. lactis 660, even higher than L. rhamnosus ATCC 53103 (recognized probiotic and used as control). The physiological probiotic properties of these strains, mainly isolated from dairy sources, are interesting in view of their use in cheese productions as starter and non starter cultures. The five LAB safe strains studied may have potential as novel probiotics in the dairy foods.