Probiotic potential of lactic acid bacteria from fermented Malaysian food or milk products

This study investigated the probiotic characteristics of lactic acid bacteria (LAB) isolated from 14 Malaysian fermented food or milk products. In total, 22.3% (121 of 542) of the LAB isolated from the local fermented products exhibited antibacterial activity against Micrococcus luteus. Twelve LAB that demonstrated better antibacterial activity against M. luteus as compared to a commercial strain Lactobacillus casei strain Shirota (LABPC) were selected for further characterisation. Based on 16S rRNA gene sequence, the LAB were identified as pediococci (seven) and lactobacilli (five). All 12 LAB showed bile tolerance, but only eight were acid tolerant at pH ≥ 3.0. The highest level of adhesion to HT‐29 cells was observed among the Lactobacillus sp. LAB 1 and 10. The LAB also showed antimicrobial activity against Escherichia coli and Staphylococcus aureus through the production of organic acids. LAB isolated from Malaysian fermented food and milk products, especially fermented tapioca, contains potential probiotic candidates.     

我国传统食品中乳酸菌资源的开发

我国存在着丰富的乳酸菌资源,一些少数民族地区包括内蒙古、新疆、西藏、川西高原、青海等地的牧民仍然沿用传统而古老的方法来制造各种乳制品,包括酸牛乳、酸马奶、酸驼乳、马奶酒、开菲尔等,从而使当地自然环境中的有益微生物得到了很好的保存。泡菜、腌渍菜和腊肠也是蕴藏乳酸菌的良好载体。不同来源的制品中蕴藏着不同种类的乳酸菌,这很大程度上取决于制品的制作方法和地域。传统的乳酸菌鉴定方法主要是依据其形态学及生理生化特性进行,近年来采用分子生物学方法对乳酸菌进行鉴定已有较大进展。目前对传统食品中乳酸菌菌株的鉴定存在的最大问题就是缺乏一种通用的系统或平台来鉴定不同来源的乳酸菌。     

新疆不同地域发酵乳品中Lactobacillus多样性的研究

利用MRS,M17等5种不同培养基从12份采自新疆北部伊犁、博乐、塔城、阿勒泰地区牧民家庭传统方法制作的乳品中分离乳酸菌,并进行了生理生化表型特征鉴定。对这些乳酸菌进行16Sr RNA基因序列的测序,构建系统发育树,初步建立其属水平的进化地位,再利用乳杆菌种间特异性引物对其进行种水平的鉴定和分类。共分离164株疑似乳酸菌,大部分菌株对温度适应性较强。以杆菌为主,系统发育表明:样品中乳酸菌主要有7个属,其中Lactobacillus(78株)、Carrobacterium(3株)、Weissella(1株)、Lactococcus(22株)、Enterococcus(47株)、Streptococcus(8株)、Vagococcus(5株)。种特性扩增显示乳杆菌存在种水平的差异。主要有4个种。利用牛津杯从样品中筛选出了10株对大肠杆菌(Escherichia coli)、枯草芽孢杆菌(Bacillus subtilis)、李斯特氏菌(Listeria monocytogenes)金黄色葡萄球菌(Staphylococcus aureus)均具有明显抑制作用的乳杆菌,为乳酸菌作为生物型防腐剂应用到食品工业中奠定基础。     

Lactic acid fermentation as a tool for increasing the folate content of foods

Folate is an essential micronutrient involved in numerous vital biological reactions. The dietary consumption of naturally occurring vitamin B9 is often inadequate in many countries, and supplementation or fortification programs (using synthetic folic acid) are implemented to alleviate folate deficiency. Other food-based alternatives are possible, such as the use of lactic acid bacteria (LAB) to synthesize folate during fermentation. Many studies have been conducted on this topic, and promising results were reported for some fermented dairy products. However, in other studies, folate consumption by LAB or rather low folate production were observed, resulting in fermented foods that may not significantly contribute to the recommended B9 intake. In addition, the optimum conditions for folate biosynthesis by LAB are still not clear. The aim of this review was thus to (i) clarify the ability of LAB to produce folate in food products, (ii) check if the production of folate by LAB in various fermented foods is sufficient to meet human vitamin B9 requirements and (iii) suggest ways to optimize folate production by LAB in fermented food products.     

Identification and functional traits of lactic acid bacteria isolated from Ciauscolo salami produced in Central Italy

Lactic acid bacteria (LAB) from Ciauscolo salami produced in Marche Region of Central Italy, and LAB strains belonging to our laboratory collection were examined for their capability to survive at low pH and bile, to adhere to Caco-2 cells, and for antibiotic resistance. LAB from Ciauscolo were identified by ARDRA and RAPD-PCR. Our study showed that all LAB strains had good adaptation to gastric juice and moderate tolerance to bile. The adhesiveness was variable among strains but significantly lower in LAB from food. Antibiotic resistance was broadly spread among food strains, with level of resistance exceeding 15% for all the antibiotics tested. The resistance determinants erm(B) and tet(M) were found in nine strains of food origin (21.4%) while tet(L) in one strain of our collection (5%). Our work suggests that fermented foods are valuable sources of bacterial strains with functional traits of intestinal lactobacilli. These bacteria may be further studied for their use in probiotic applications.     

Tetracycline resistance associated with commensal bacteria from representative ready-to-consume deli and restaurant foods

Proper knowledge of antibiotic resistance (AR) dissemination is essential for effective mitigation. This study examined the profiles of tetracycline-resistant (Tetr) commensal bacteria from representative ready-to-consume food samples from salad bars at local grocery stores and restaurants. Out of 900 Tetr isolates examined, 158 (17.6%) carried one or more of tetM, tetL, tetS, and tetK genes by conventional PCR, 28 harbored more than one Tetr determinants. The most prevalent genotype was tetM, which was detected in 70.9% of the AR gene carriers, followed by tetL (31.6%), tetS (13.9%), and tetK (2.5%). Identified AR gene carriers included Enterococcus, Lactococcus, Staphylococcus, Brochothrix, Carnobacterium, Stenotrophomonas, Pseudomonas, and Sphingobacterium, by 16S rRNA gene sequence analysis. AR determinants were successfully transmitted, and led to resistance in Streptococcus mutans via natural gene transformation and Enterococcus faecalis via electroporation, suggesting the functionality and mobility of the AR genes from the food commensal bacteria. In addition, the AR traits in many isolates are quite stable, even in the absence of the selective pressure. The identification of new commensal carriers for representative AR genes revealed the involvement of a broad spectrum of bacteria in the horizontal transmission of AR genes. Meanwhile, the spectrum of the antibiotic-resistant bacteria differed from the spectrum of the total bacteria (by denaturing gradient gel electrophoresis) associated with the food items. Our data revealed a common avenue in AR exposure and will assist in proper risk assessment and the development of comprehensive mitigation strategies to effectively combat AR.     

Phenotypic and genotypic identification of lactic acid bacteria isolated from ethnic fermented bamboo tender shoots of North East India

Mesu, soidon, soibum and soijim are ethnic fermented bamboo tender shoot products prepared by the people in North East India. Microbiological analysis of mesu, soidon, soibum and soijim showed the population dominated by lactic acid bacteria (LAB) ranging up to 10(8) cfu g(-1). The phenotypic characterisation of predominant LAB isolated from the fermented bamboo shoot products was based on general morphology, physiological tests, API and Biolog systems. The genotypic characterisation of LAB was based on RAPD-PCR, rep PCR, species-specific PCR techniques, 16S rRNA gene sequencing and DNA-DNA hybridisation. Predominant functional LAB strains associated with the fermented bamboo shoot products were identified as Lactobacillus brevis, Lb. plantarum, Lb. curvatus, Pediococcus pentosaceus, Leuconostoc mesenteroides subsp. mesenteroides, Leuc. fallax, Leuc. lactis, Leuc. citreum and Enterococcus durans.     

Diversity of lactic acid bacteria associated with traditional fermented dairy products in Mongolia

Spontaneous milk fermentation has a long history in Mongolia, and beneficial microorganisms have been handed down from one generation to the next for use in fermented dairy products. The objective of this study was to investigate the diversity of lactic acid bacteria (LAB) communities in fermented yak, mare, goat, and cow milk products by analyzing 189 samples collected from 13 different regions in Mongolia. The LAB counts in these samples varied from 3.41 to 9.03 log cfu/mL. Fermented yak and mare milks had almost identical mean numbers of LAB, which were significantly higher than those in fermented goat milk but slightly lower than those in fermented cow milk. In total, 668 isolates were obtained from these samples using de Man, Rogosa, and Sharpe agar and M17 agar. Each isolate was considered to be presumptive LAB based on gram-positive and catalase-negative properties, and was identified at the species level by 16S rRNA gene sequencing, multiplex PCR assay, and restriction fragment length polymorphism analysis. All isolates from Mongolian dairy products were accurately identified as Enterococcus faecalis (1 strain), Enterococcus durans (3 strains), Lactobacillus brevis (3 strains), Lactobacillus buchneri (2 strains), Lactobacillus casei (16 strains), Lactobacillus delbrueckii ssp. bulgaricus (142 strains), Lactobacillus diolivorans (17 strains), Lactobacillus fermentum (42 strains), Lactobacillus helveticus (183 strains), Lactobacillus kefiri (6 strains), Lactobacillus plantarum ssp. plantarum (7 strains), Lactococcus lactis ssp. lactis (7 strains), Leuconostoc lactis (22 strains), Leuconostoc mesenteroides (21 strains), Streptococcus thermophilus (195 strains), and Weissella cibaria (1 strain). The predominant LAB were Strep. thermophilus and Lb. helveticus, which were isolated from all sampling sites. The results demonstrate that traditional fermented dairy products from different regions of Mongolia have complex compositions of LAB species. Such diversity of LAB provides useful information for further studies of probiotic strain selection and starter culture design, with regard to the industrial production of traditional fermented milk.     

The importance of lactic acid bacteria for the prevention of bacterial growth and their biogenic amines formation: A review

Foodborne pathogens (FBP) represent an important threat to the consumers' health as they are able to cause different foodborne diseases. In order to eliminate the potential risk of those pathogens, lactic acid bacteria (LAB) have received a great attention in the food biotechnology sector since they play an essential function to prevent bacterial growth and reduce the biogenic amines (BAs) formation. The foodborne illnesses (diarrhea, vomiting, and abdominal pain, etc.) caused by those microbial pathogens is due to various reasons, one of them is related to the decarboxylation of available amino acids that lead to BAs production. The formation of BAs by pathogens in foods can cause the deterioration of their nutritional and sensory qualities. BAs formation can also have toxicological impacts and lead to different types of intoxications. The growth of FBP and their BAs production should be monitored and prevented to avoid such problems. LAB is capable of improving food safety by preventing foods spoilage and extending their shelf-life. LAB are utilized by the food industries to produce fermented products with their antibacterial effects as bio-preservative agents to extent their storage period and preserve their nutritive and gustative characteristics. Besides their contribution to the flavor for fermented foods, LAB secretes various antimicrobial substances including organic acids, hydrogen peroxide, and bacteriocins. Consequently, in this paper, the impact of LAB on the growth of FBP and their BAs formation in food has been reviewed extensively.     

Application of molecular methods for the classification and identification of lactic acid bacteria

Phylogenetic analysis has revealed that the typical lactic acid bacteria (LAB) belong to the Gram-positive bacteria with a low guanine plus cytosine DNA content. The genera Lactobacillus, Leuconostoc and Pediococcus can be traditionally differentiated on the basis of morphological and physiological properties but phylogentically they are intermixed. The former genus Streptococcus has been split up into the four genera Enterococcus, Lactococcus, Streptococcus and Vagococcus.

The traditional phenotypic identification of LAB is rather tedious and not always reliable. Nucleic acid probe technology may be an alternative for a faster and more reliable differentiation. 16S or 23S rRNA-targeted oligonucleotides have been used for the specific identification of LAB. It is even now possible to identify various LAB in fermented food, without any preceding enrichment or cultivation step, at the species level within one working day. Application of fluorescently labelled oligonucleotides also allowed the in situ detection and identification of whole cells of lactococci and enterococci. DNA restriction fragment analysis and ribotyping have been used to distinguish LAB at the strain level or groups of closely related strains. Specific hybridization probes are also useful tools for the identification of genetically modified nucleic acids and the corresponding organisms.     

Isolation of an exopolysaccharide-producing <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> from Algerian raw cow milk

Lactic acid bacteria (LAB) play a key role in the development of the organoleptic and textural qualities of fermented dairy products. The exopolysaccharides (EPS) produced by LAB are of great technological interest since they improve the rheological characteristics of these products and help reduce syneresis. In the present work, an EPS-producing Streptococcus thermophilus strain was isolated from Algerian raw cow milk. Its identity was confirmed by biochemical and molecular methods. The presence in its genome of a priming glycosyltransferase gene was confirmed by PCR. The strain’s growth and EPS production in milk was analysed and the molar mass of the EPS produced determined by HPLC.     

Characterization of volatile compounds in fermented milk using solid-phase microextraction methods coupled with gas chromatography-mass spectrometry

Lactic acid bacteria (LAB) are industrially important bacteria that are widely used in the fermented food industry, especially in the manufacture of yogurt. Characteristic flavors are produced by LAB during fermentation and storage that affect the quality and acceptability of fermented milk products. In this study, the volatile compounds in milk fermented by Streptococcus thermophilus IMAU80842 alone, Lactobacillus delbrueckii ssp. bulgaricus IMAU20401 alone, or both species together were identified using solid-phase microextraction methods coupled with gas chromatography-mass spectrometry. A total of 53, 43, and 32 volatile compounds were identified in milk fermented by S. thermophilus alone, L. delbrueckii ssp. bulgaricus alone, or both species together, respectively. The presence of some important flavor compounds was confirmed: acetic acid, acetaldehyde, acetoin, 2,3-butanedione, ethanol, and 1-heptanol. Our results demonstrate that the composition of the volatile compounds in fermented milk depends on the species of LAB used and whether they are used alone or in combination. This is important for the selection of appropriate starter cultures for the production of different types of fermented milk product with particular flavors.     

Interactions between Staphylococcus aureus and lactic acid bacteria: an old story with new perspectives

Staphylococcus aureus is a gram positive opportunistic pathogen and a major concern for both animal and human health worldwide. In some contexts where Lactic Acid Bacteria (LAB) are the normal dominant microbiota, such as in fermented food or in the vaginal ecosystem, S. aureus sometimes colonises, persists, expresses virulence factors and produces food poisoning or urogenital infections, respectively. Studies on the interactions between LAB and S. aureus began a few decades ago and were pursued to shed light on the inhibitory capabilities that LAB might have on S. aureus growth and/or enterotoxin production in fermented foodstuffs. These early studies had the aim of developing methods to prevent staphylococcal food poisoning, thus improving food safety. More recently, the concept of vaginal probiotic LAB has emerged as a promising way to prevent urogenital infections, S. aureus being one of the potential pathogens targeted. This review provides an up-to-date look at the current hypotheses of the mechanisms involved in the inhibition of S. aureus by LAB in both the vaginal ecosystem and in fermented food ecosystems. We also emphasise that post-genomic approaches can now be envisioned in order to study these diverse and complex interactions at the molecular level. Further works in this field will open up new avenues for methods of biocontrol by LAB and/or for biotechnological uses of LAB-compounds to fight against the long-standing, yet incumbent menace of staphylococcal infection.     

Diversity of lactic acid bacteria in fermented brines used to make stinky tofu

Stinky tofu is a kind of fermented tofu with a strong odor. Although stinky tofu is a very popular snack in the Asian region, the community of microbes, and especially lactic acid bacteria (LAB), indigenous to the fermented brine from which it is made remains poorly described. We examined 168 isolates obtained from the original fermented brine (brine A) and two brines in which the hard tofu (brine B) and soft tofu (brine C) had been soaked. Through random amplified polymorphic DNA (RAPD) analysis for typing and 16S rDNA sequencing, 136 representative strains were identified as belonging to 7 genera and 32 species: Enterococcus (2 species), Lactobacillus (14 species), Lactococcus (3 species), Leuconostoc (6 species), Pediococcus (1 species), Streptococcus (2 species), and Weissella (4 species). The LAB composition of brine A was the most diverse: 19 different species were isolated, and 9 of them were classified as Lactobacillus species. The 16S rDNA sequences of 9 strains (6 from brine A and 3 from brine C) showed low values of similarity (below 98%) with currently known species by analysis using the FASTA software. Thus, a wide variety of LAB strains were associated with the fermentation of stinky tofu brines.     

Diversity of lactic acid bacteria in suan-tsai and fu-tsai, traditional fermented mustard products of Taiwan

Fu-tsai and suan-tsai are spontaneously fermented mustard products traditionally prepared by the Hakka tribe of Taiwan. We chose 5 different processing stages of these products for analysis of the microbial community of lactic acid bacteria (LAB) by 16S rRNA gene sequencing. From 500 LAB isolates we identified 119 representative strains belonging to 5 genera and 18 species, including Enterococcus (1 species), Lactobacillus (11 species), Leuconostoc (3 species), Pediococcus (1 species), and Weissella (2 species). The LAB composition of mustard fermented for 3 days, known as the Mu sample, was the most diverse, with 11 different LAB species being isolated. We used sequence analysis of the 16S rRNA gene to identify the LAB strains and analysis of the dnaA, pheS, and rpoA genes to identify 13 LAB strains for which identification by 16S rRNA gene sequences was not possible. These 13 strains were found to belong to 5 validated known species: Lactobacillus farciminis, Leuconostoc mesenteroides, Leuconostoc pseudomesenteroides, Weissella cibaria, and Weissella paramesenteroides, and 5 possibly novel Lactobacillus species. These results revealed that there is a high level of diversity in LAB at the different stages of fermentation in the production of suan-tsai and fu-tsai.     

乳酸菌抗氧化活性及其在发酵肉制品中的应用研究进展

乳酸菌作为发酵肉制品中的发酵剂,已被证实具有抗氧化活性,但在发酵过程中,容易受到各种环境胁迫因子的影响,这些胁迫因子能够诱发菌体胞内活性氧自由基的产生和积累,进一步影响菌株的抗氧化活性,很大程度上限制了其在发酵肉制品中的抗氧化作用。本文综述乳酸菌面临的环境胁迫因子,探究其对氧化胁迫的应答与防御机制和抗氧化机理,并阐述提升乳酸菌氧化胁迫抗性的策略及乳酸菌在发酵肉制品中的抗氧化作用。     

耐胆酸盐、降胆固醇乳酸菌的筛选

从乳酸发酵制品和发酵菌剂中分离纯化获得了25株菌，通过显微镜形态观察、革兰氏染色反应和石蕊牛奶试验证实其中10株为乳酸菌。进一步比较了这些菌株和Streptococcus thermophilis，Lactobaccillus bulgaricus的胆酸盐耐受能力以及对牛奶中胆固醇的作用效果。结果表明，多数乳酸菌可耐受低质量分数(0．1％)的胆酸盐，可以不同程度的降低牛奶中的胆固醇含量；但在含有质量分数为0．3％胆酸盐时，只有少数乳酸菌对牛奶中降胆固醇的能力有所提高。     

Safety assessment and antimicrobial properties of the lactic acid bacteria strains isolated from polish raw fermented meat products

Twenty-one strains of the genus Lactobacillus and the genus Pediococcus, isolated from Polish raw fermented meat products, were examined for the potential probiotic properties: resistance to simulated gastric and intestine conditions, safety assessment, and antimicrobial properties. Strains were resistant to gastric enzymes and low pH (3–6 log CFU/mL decrease) and intestinal enzymes and bile salts (1–3 log CFU/mL decrease). Most strains were resistant to gentamycin, streptomycin, vancomycin, tetracycline, ciprofloxacin, and kanamycin. Three of them (Lb. brevis BAL1, BAL10, and KL5) produced β-glucuronidase, which excludes them from qualifying as safe. Seven strains had the ability to produce bacteriocins or bacteriocin-like substances. Overall, strains Lb. brevis SCH6, Pd. pentosaceus BAL6, and KL14 revealed selected superior characteristics (resistance to the gastrointestinal conditions, safety assessment, and antimicrobial properties) as compared to the other LAB strains investigated, which made them a viable bioprotective culture that can be inoculated in raw fermented meat products as starter cultures.     

The rpoB gene as a target for PCR-DGGE analysis to follow lactic acid bacterial population dynamics during food fermentations

Culture-independent molecular techniques offer a valuable tool in the study and understanding of population dynamics in food fermentations. PCR-DGGE protocols have been developed based on the 16S rDNA gene; however, its heterogeneity complicates interpretation of the results. The rpoB gene, encoding the RNA polymerase beta subunit, usually exists in a single copy and offers a valuable alternative for PCR-DGGE analysis. In this study, PCR products generated from the rpoB gene of several strains of lactic acid bacteria (LAB), responsible for various fermentations, were analysed by DGGE, and a database of rpoB partial sequences was created. Unique DGGE profiles were obtained for rpoB sequences from the Lactobacillus, Lactococcus, Enterococcus, Streptococcus, Leuconostoc and Weisella strains tested, allowing their identification and differentiation in complex ecosystems, such as fermented food products. The application of rpoB targeted PCR-DGGE to tracking ecological changes in food systems during maturation was validated in fermented sausage and cheese.     

Antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products

Commercial starter culture bacteria are widely used in the production of dairy products and could represent a potential source for spread of genes encoding resistance to antimicrobial agents. To learn more about the antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products, a total of 189 isolates of lactic acid bacteria were examined for susceptibility to ampicillin, penicillin G, cephalothin, vancomycin, bacitracin, gentamicin, streptomycin, erythromycin, tetracycline, chloramphenicol, quinupristin/dalfopristin, ciprofloxacin, trimethoprim and sulphadiazine using Etest for MIC determination. Most of the isolates (140) originated from 39 dairy products (yoghurt, sour cream, fermented milk and cheese), while 49 were isolated directly from nine commercial cultures. The bacteria belonged to the genera Lactobacillus, Lactococcus, Leuconostoc and Streptococcus. Only one of the 189 isolates was classified as resistant to an antimicrobial agent included in the study. This isolate, a lactobacillus, was classified as high level resistant to streptomycin. The remaining isolates were not classified as resistant to the antimicrobial agents included other than to those they are known to have a natural reduced susceptibility to. Thus, starter culture bacteria in Norwegian dairy products do not seem to represent a source for spread of genes encoding resistance to antimicrobial agents.