B-group vitamin production by lactic acid bacteria--current knowledge and potential applications

Although most vitamins are present in a variety of foods, human vitamin deficiencies still occur in many countries, mainly because of malnutrition not only as a result of insufficient food intake but also because of unbalanced diets. Even though most lactic acid bacteria (LAB) are auxotrophic for several vitamins, it is now known that certain strains have the capability to synthesize water-soluble vitamins such as those included in the B-group (folates, riboflavin and vitamin B(12) amongst others). This review article will show the current knowledge of vitamin biosynthesis by LAB and show how the proper selection of starter cultures and probiotic strains could be useful in preventing clinical and subclinical vitamin deficiencies. Here, several examples will be presented where vitamin-producing LAB led to the elaboration of novel fermented foods with increased and bioavailable vitamins. In addition, the use of genetic engineering strategies to increase vitamin production or to create novel vitamin-producing strains will also be discussed. This review will show that the use of vitamin-producing LAB could be a cost-effective alternative to current vitamin fortification programmes and be useful in the elaboration of novel vitamin-enriched products.     

Lactic acid bacteria producing B-group vitamins: a great potential for functional cereals products

Wheat contains various essential nutrients including the B group of vitamins. However, B group vitamins, normally present in cereals-derived products, are easily removed or destroyed during milling, food processing or cooking. Lactic acid bacteria (LAB) are widely used as starter cultures for the fermentation of a large variety of foods and can improve the safety, shelf life, nutritional value, flavor and overall quality of the fermented products. In this regard, the identification and application of strains delivering health-promoting compounds is a fascinating field. Besides their key role in food fermentations, several LAB found in the gastrointestinal tract of humans and animals are commercially used as probiotics and possess generally recognized as safe status. LAB are usually auxotrophic for several vitamins although certain strains of LAB have the capability to synthesize water-soluble vitamins such as those included in the B group. In recent years, a number of biotechnological processes have been explored to perform a more economical and sustainable vitamin production than that obtained via chemical synthesis. This review article will briefly report the current knowledge on lactic acid bacteria synthesis of vitamins B2, B11 and B12 and the potential strategies to increase B-group vitamin content in cereals-based products, where vitamins-producing LAB have been leading to the elaboration of novel fermented functional foods. In addition, the use of genetic strategies to increase vitamin production or to create novel vitamin-producing strains will be also discussed.     

Recent research on 3-phenyllactic acid, a broad-spectrum antimicrobial compound

3-Phenyllactic acid (PLA), which is an organic acid widely existing in honey and lactic acid bacteria fermented food, can be produced by many microorganisms, especially lactic acid bacteria. It was proved as an ideal antimicrobial compound with broad and effective antimicrobial activity against both bacteria and fungi. In addition, it could be used as feed additives to replace antibiotics in livestock feeds. This article presented a review of recent studies on the existing resource, antimicrobial activity, and measurement of PLA. In addition, microorganism strains and dehydrogenases producing PLA were reviewed in detail, the metabolic pathway and regulation of PLA synthesis in LAB strains were discussed, and high-level bioproduction of PLA by microorganism fermentation was also summarized.     

Immunobiosis and probiosis: antimicrobial activity of lactic acid bacteria with a focus on their antiviral and antifungal properties

Lactic acid bacteria (LAB), a heterogeneous group of bacteria that produce lactic acid as the main product of carbohydrate degradation, play an important role in the production and protection of fermented foods. Moreover, beside the technological use of these microorganisms added to control and steer food fermentations, their beneficial healthy properties are largely overt. Thus, numerous LAB strains have obtained the probiotic status, which entails the ability to maintain and promote a good health of consumers. In particular, increasing consideration is being focused on probiotic microorganisms that can improve the human immune response against dangerous viral and fungal enemies. For such beneficial microbes, the term “immunobiotics” has been coined. Together with an indirect host-mediated adverse effect against undesirable microorganisms, also a direct antagonistic activity of several LAB strains has been largely demonstrated. The purpose of this review is to provide a fullest possible overview of the antiviral and antifungal activities ascribed to probiotic LAB. The interest in this research field is substantiated by a large number of studies exploring the potential application of these beneficial microorganisms both as biopreservatives and immune-enhancers, aiming to reduce and/or eliminate the use of chemical agents to prevent the development of pathogenic, infectious, and/or degrading causes.

     

Bacteriocinogenic LAB Strains for Fermented Meat Preservation: Perspectives,Challenges, and Limitations

Over the last decades, much research has focused on lactic acid bacteria (LAB) bacteriocins because of their potential as biopreservatives and their action against the growth of spoilage microbes. Meat and fermented meat products are prone to microbial contamination, causing health risks, as well as economic losses in the meat industry. The use of bacteriocin-producing LAB starter or protective cultures is suitable for fermented meats. However, although bacteriocins can be produced during meat processing, their levels are usually much lower than those achieved during in vitro fermentations under optimal environmental conditions. Thus, the direct addition of a bacteriocin food additive would be desirable. Moreover, safety and technological characteristics of the bacteriocinogenic LAB must be considered before their widespread applications. This review describes the perspectives and challenges toward the complete disclosure of new bacteriocins as effective preservatives in the production of safe and “healthy” fermented meat products.     

Genetic screening of functional properties of lactic acid bacteria in a fermented pearl millet slurry and in the metagenome of fermented starchy foods

Lactic acid bacteria (LAB) (n = 152) in African pearl millet slurries and in the metagenomes of amylaceous fermented foods were investigated by screening 33 genes involved in probiotic and nutritional functions. All isolates belonged to six species of the genera Pediococcus and Lactobacillus, and Lactobacillus fermentum was the dominant species. We screened the isolates for the abilities to survive passage through the gastrointestinal tract and to synthesize folate and riboflavin. The isolates were also tested in vitro for their abilities to survive exposure to bile salts and to survive at pH 2. Because the ability to hydrolyze starch confers an ecological advantage on LAB that grow in starchy matrixes as well as improving the nutritional properties of the gruels, we screened for genes involved in starch metabolism. The results showed that genes with the potential ability to survive passage through the gastrointestinal tract were widely distributed among isolates and metagenomes, whereas in vitro tests showed that only a limited set of isolates, mainly those belonging to L. fermentum, could tolerate a low pH. In contrast, the wide distribution of genes associated with bile salt tolerance, in particular bsh, is consistent with the high frequency of tolerance to bile salts observed. Genetic screening revealed a potential for folate and riboflavin synthesis in both isolates and metagenomes, as well as high variability among genes related to starch metabolism. Genetic screening of isolates and metagenomes from fermented foods is thus a promising approach for assessing the functional potential of food microbiotas.     

Genotypic and phenotypic diversity of Pediococcus pentosaceus strains isolated from food matrices and characterisation of the penocin operon

Lactic acid bacteria (LAB) are widely used in the food industry. Pediococcus spp. belong to the LAB group and include several species that are essential for the quality of fermented food. Pediococcus pentosaceus is the species that is most frequently isolated from fermented food and beverages but its uncontrolled growth during food fermentation processes can contribute to undesired flavours. Hence, the characterisation of these bacteria at the strain level is of great importance for the quality of fermented products. Despite their importance, misidentification at the species level is common for members of the genus Pediococcus. To clarify the taxonomic relationships among strains, a multilocus sequencing approach was developed for the characterisation of a collection of 29 field strains, 1 type strain and 1 reference strain of P. pentosaceus isolated from food. These strains were also tested for several phenotypic properties of technological interest and for the production of bacteriocins. The chromosomal operon involved in the synthesis of the bacteriocin penocin was also investigated. The present study enabled a good genomic characterisation, identifying 17 sequence types, with an overview of phenotypic characteristics related to different technological abilities, and also provides a thorough characterisation of the operon involved in penocin production.     

Dominant lactic acid bacteria and their technological properties isolated from the Himalayan ethnic fermented milk products

Ethnic people of the Himalayan regions of India, Nepal, Bhutan and China consume a variety of indigenous fermented milk products made from cows milk as well as yaks milk. These lesser-known ethnic fermented foods are dahi, mohi, chhurpi, somar, philu and shyow. The population of lactic acid bacteria (LAB) ranged from 10(7) to 10(8) cfu/g in these Himalayan milk products. A total of 128 isolates of LAB were isolated from 58 samples of ethnic fermented milk products collected from different places of India, Nepal and Bhutan. Based on phenotypic characterization including API sugar test, the dominant lactic acid bacteria were identified as Lactobacillus bifermentans, Lactobacillus paracasei subsp. pseudoplantarum, Lactobacillus kefir, Lactobacillus hilgardii, Lactobacillus alimentarius, Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris and Enterococcus faecium. LAB produced a wide spectrum of enzymes and showed high galactosidase, leucine-arylamidase and phosphatase activities. They showed antagonistic properties against selected Gram-negative bacteria. None of the strains produced bacteriocin and biogenic amines under the test conditions used. Most strains of LAB coagulated skim milk with a moderate drop in pH. Some strains of LAB showed a high degree of hydrophobicity, suggesting these strains may have useful adhesive potential. This paper is the first report on functional lactic acid bacterial composition in some lesser-known ethnic fermented milk products of the Himalayas.     

Bacteriocins from lactic acid bacteria: production, purification, and food applications

In fermented foods, lactic acid bacteria (LAB) display numerous antimicrobial activities. This is mainly due to the production of organic acids, but also of other compounds, such as bacteriocins and antifungal peptides. Several bacteriocins with industrial potential have been purified and characterized. The kinetics of bacteriocin production by LAB in relation to process factors have been studied in detail through mathematical modeling and positive predictive microbiology. Application of bacteriocin-producing starter cultures in sourdough (to increase competitiveness), in fermented sausage (anti-listerial effect), and in cheese (anti-listerial and anti-clostridial effects), have been studied during in vitro laboratory fermentations as well as on pilot-scale level. The highly promising results of these studies underline the important role that functional, bacteriocinogenic LAB strains may play in the food industry as starter cultures, co-cultures, or bioprotective cultures, to improve food quality and safety. In addition, antimicrobial production by probiotic LAB might play a role during in vivo interactions occurring in the human gastrointestinal tract, hence contributing to gut health.     

Biodiversity of lactic acid bacteria in Romanian dairy products

Traditionally fermented dairy products are still a very important part of the daily food in Romania, especially for people living in the countryside. To study the biodiversity of lactic acid bacterium strains of these products, 110 samples (raw and fermented milk, sour cream, and cheese) were collected from farm houses, monasteries, and local markets throughout Romania. Lactic acid bacteria (LAB) were isolated using six different cultivation conditions. All 599 isolates were tested for their Gram reaction, catalase activity, and morphology. A rep-PCR fingerprinting technique with the (GTG)5 primer and, in some cases SDS-PAGE of total cell proteins and 16S rRNA gene sequencing were used to cluster and/or identify the LAB. The biodiversity of the isolated strains was correlated with the type of product and/or technology applied. The most frequent LAB found in Romanian raw milk and fermented dairy products were Lactococcus lactis, Leuconostoc spp., and Enterococcus spp. Among the latter, a new species E. saccharominimus was found.     

Isolation of lactic acid bacteria from Malaysian foods and assessment of the isolates for industrial potential

Two traditional fermented food 'tapai' (fermented tapioca) and 'tempoyak' (fermented durian flesh), chilli puree and fresh goat's milk were used as sources for the isolation of lactic acid bacteria (LAB). A total of 126 isolates were obtained and by sequential screening for catalase activity and Gram-staining, 55 were determined to be LAB out of which 16 were established to be homofermentative by the gel plug test. Seven isolates were identified by use of the API 50CHL kit and two lactobacilli strains and one lactococci strain were selected to study their growth and lactic acid production profiles in a time course experiment. The lactobacilli strains, both isolated from 'tapai', produced higher amounts of cells and lactic acid from glucose as compared to the lactococci strain isolated from fresh goat's milk.     

Lactobacillaceae and cell adhesion: genomic and functional screening

The analysis of collections of lactic acid bacteria (LAB) from traditional fermented plant foods in tropical countries may enable the detection of LAB with interesting properties. Binding capacity is often the main criterion used to investigate the probiotic characteristics of bacteria. In this study, we focused on a collection of 163 Lactobacillaceace comprising 156 bacteria isolated from traditional amylaceous fermented foods and seven strains taken from a collection and used as controls. The collection had a series of analyses to assess binding potential for the selection of new probiotic candidates. The presence/absence of 14 genes involved in binding to the gastrointestinal tract was assessed. This enabled the detection of all the housekeeping genes (ef-Tu, eno, gap, groEl and srtA) in the entire collection, of some of the other genes (apf, cnb, fpbA, mapA, mub) in 86% to 100% of LAB, and of the other genes (cbsA, gtf, msa, slpA) in 0% to 8% of LAB. Most of the bacteria isolated from traditional fermented foods exhibited a genetic profile favorable for their binding to the gastrointestinal tract. We selected 30 strains with different genetic profiles to test their binding ability to non-mucus (HT29) and mucus secreting (HT29-MTX) cell lines as well as their ability to degrade mucus. Assays on both lines revealed high variability in binding properties among the LAB, depending on the cell model used. Finally, we investigated if their binding ability was linked to tighter cross-talk between bacteria and eukaryotic cells by measuring the expression of bacterial genes and of the eukaryotic MUC2 gene. Results showed that wild LAB from tropical amylaceous fermented food had a much higher binding capacity than the two LAB currently known to be probiotics. However their adhesion was not linked to any particular genetic equipment.     

Activity of crude and fractionated extracts by lactic acid bacteria (LAB) isolated from local dairy,meat, and fermented products against Staphylococcus aureus

This study aimed to evaluate anti-staphylococcal properties of crude and fractionated extracts of lactic acid bacteria (LAB) isolated from local meat, dairy, and fermented products. A total of 36 LAB isolates were obtained and identified via 16S rDNA sequencing. Cell-free supernatant (CFS) of all isolates exhibiting a statistically significant inhibition against Staphylococcus aureus (ρ < 0.05), with six LAB isolates exhibiting a more prevalent inhibition. The inhibition effects of cell wall and intracellular extracts from the six prevalent isolates were evaluated. Lactobacillus plantarum USM8613 was the most prominent isolate with both CFS and cell wall extract exhibiting the most prevalent inhibition against S. aureus. Scanning electron micrographs showed alteration of S. aureus membrane morphology upon CFS treatment, suggesting an anti-staphylococcal effect via membrane destruction. Confocal laser scanning micrographs showed inhibition against biofilm formations by S. aureus in porcine skins upon CFS treatment. The CFS from L. plantarum USM8613 was separated into protein, lipid, and polysaccharide fractions for evaluation of anti-staphylococcal activity and chemical characterization. All fractions inhibited growth of S. aureus (ρ < 0.05), with protein fractions exhibiting stronger inhibition effect. Data from our present study showed that extracts from LAB could be applied as biopreservatives in the food industries and/or as an antimicrobial agent against bacterial infections for cosmeceutical and pharmaceutical uses.     

Engineering of Synechococcus sp. strain PCC 7002 for the photoautotrophic production of light-sensitive riboflavin (vitamin B2)

Due to their capability of photosynthesis and autotrophic growth, cyanobacteria are currently investigated with regard to the sustainable production of a wide variety of chemicals. So far, however, no attempt has been undertaken to engineer cyanobacteria for the biotechnological production of vitamins, which is probably due to the light-sensitivity of many of these compounds. We now describe a photoautotrophic bioprocess to synthesize riboflavin, a vitamin used as a supplement in the feed and food industry. By overexpressing the riboflavin biosynthesis genes ribDGEABHT from Bacillus subtilis in the marine cyanobacterium Synechococcus sp. PCC 7002 riboflavin levels in the supernatant of the corresponding recombinant strain increased 56-fold compared to the wild-type. Introduction of a second promoter region upstream of the heterologous ribAB gene – coding for rate-limiting enzymatic functions in the riboflavin biosynthesis pathway – led to a further increase of riboflavin levels (211-fold compared to the wild-type). Degradation of the light-sensitive product riboflavin was prevented by culturing the genetically engineered Synechococcus sp. PCC 7002 strains in the presence of dichromatic light generated by red light-emitting diodes (λ = 630 and 700 nm). Synechococcus sp. PCC 7002 naturally is resistant to the toxic riboflavin analog roseoflavin. Expression of the flavin transporter pnuX from Corynebacterium glutamicum in Synechococcus sp. PCC 7002 resulted in roseoflavin-sensitive recombinant strains which in turn could be employed to select roseoflavin-resistant, riboflavin-overproducing strains as a chassis for further improvement.     

Current status and emerging role of glutathione in food grade lactic acid bacteria

ABSTRACT: Lactic acid bacteria (LAB) have taken centre stage in perspectives of modern fermented food industry and probiotic based therapeutics. These bacteria encounter various stress conditions during industrial processing or in the gastrointestinal environment. Such conditions are overcome by complex molecular assemblies capable of synthesizing and/or metabolizing molecules that play a specific role in stress adaptation. Thiols are important class of molecules which contribute towards stress management in cell. Glutathione, a low molecular weight thiol antioxidant distributed widely in eukaryotes and Gram negative organisms, is present sporadically in Gram positive bacteria. However, new insights on its occurrence and role in the latter group are coming to light. Some LAB and closely related Gram positive organisms are proposed to possess glutathione synthesis and/or utilization machinery. Also, supplementation of glutathione in food grade LAB is gaining attention for its role in stress protection and as a nutrient and sulfur source. Owing to the immense benefits of glutathione, its release by probiotic bacteria could also find important applications in health improvement. This review presents our current understanding about the status of glutathione and its role as an exogenously added molecule in food grade LAB and closely related organisms.     

Short-chain fatty acid and vitamin production potentials of Lactobacillus isolated from fermented foods of Khasi Tribes,Meghalaya, India

Vitamins and SCFA (short-chain fatty acids) production from Lactobacillus isolates are studied due to its health benefits to the human hosts. Lactobacillus strains are widely used in fermented foods, and few of them are reported with vitamin and SCFA production potential. Therefore, in the present study, vitamins and SCFA production capability of isolates were studied to find the potent Lactobacillus cultures for value-added functional food product development. Five Lactobacillus strains, i.e., KGL2, KGL3A, KGL4, RNS4, and WTS4, were isolated from rice-based traditional fermented foods of Garo Hills, Meghalaya, India. All the well grown isolates were morphologically, physiologically, and genetically characterized. Then, vitamins and SCFA were estimated using HPLC based methods. Vitamins produced in vitamins free assay medium and SCFA in milk medium are produced by Lactobacillus. Lactic acid bacteria produce essential vitamins like riboflavin, folate, cobalamin, and SCFA which have health impacts (anti-obesity, anti-diabetics, anti-microbial, and other chronic diseases prevention) to the host. These vitamins are essential for cellular and metabolic growth of living system. In the study, five potent Lactobacillus isolates viz., KGL2 (Lactobacillus fermentum), KGL3A (Lactobacillus plantarum), KGL4 (Lactobacillus fermentum), RNS4 (Lactobacillus rhamnosus), and WTS4 (Lactobacillus fermentum) were considered for vitamins (B2, B12, and B9) and SCFA productions (lactate, butyrate, and acetate). However, KGL3A had shown highest B2 production (0.7 μg/ml) while KGL2 exhibited maximum B12 production (0.05 μg/ml) after 36 h. Moreover, WTS4 attributed highest folate production (0.09 μg/ml) after 24 h. In addition, RNS4 reported the maximum short-chain fatty acid production (0.77 g/l acetic acid, 0.26 g/l lactic acid, and 0.008 g/l butyric acid respectively). Potent Lactobacillus isolates from traditional fermented foods of Garo Hills, Meghalaya, India (North East Part of India) showed maximum production of B2, B9, and B12 as well as short-chain fatty acids and could be used for their application as health beneficial functional fermented dairy products.     

乳酸菌合成叶酸的研究进展

叶酸普遍存在于各类食物中,但由于叶酸的不稳定性以及饮食习惯的差异性,各国叶酸缺乏现象普遍存在。叶酸是参与核酸合成及细胞分裂分化的重要物质,叶酸缺乏引起机体功能的混乱,由此引发癌症等一系列的疾病。大部分乳酸菌是叶酸缺陷型菌株,但越来越多的研究表明其很多种类都具有叶酸合成能力。本文主要概述产叶酸乳酸菌的分类,乳酸菌生物合成叶酸的机制,以及利用乳酸菌进行的叶酸基因工程研究进展。     

Functional relevance and health benefits of soymilk fermented by lactic acid bacteria

The growing interest of consumers towards nutritionally enriched, and health promoting foods, provoke interest in the eventual development of fermented functional foods. Soymilk is a growing trend that can serve as a low-cost non-dairy alternative with improved functional and nutritional properties. Soymilk acts as a good nutrition media for the growth and proliferation of the micro-organism as well as for their bioactivities. The bioactive compounds produced by fermentation of soymilk with lactic acid bacteria (LAB) exhibit enhanced nutritional values, and several improved health benefits including antihypertensive, antioxidant, antidiabetic, anticancer and hypocholesterolaemic effects. The fermented soymilk is acquiring a significant position in the functional food industry due to its increased techno-functional qualities as well as ensuring the survivability of probiotic bacteria producing diverse metabolites. This review covers the important benefits conferred by the consumption of soymilk fermented by LAB producing bioactive compounds. It provides a holistic approach to obtain existing knowledge on the biofunctional attributes of fermented soymilk, with a focus on the functionality of soymilk fermented by LAB.     

Lactococci and lactobacilli as mucosal delivery vectors for therapeutic proteins and DNA vaccines

Food-grade Lactic Acid Bacteria (LAB) have been safely consumed for centuries by humans in fermented foods. Thus, they are good candidates to develop novel oral vectors, constituting attractive alternatives to attenuated pathogens, for mucosal delivery strategies. Herein, this review summarizes our research, up until now, on the use of LAB as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Most of our work has been based on the model LAB Lactococcus lactis, for which we have developed efficient genetic tools, including expression signals and host strains, for the heterologous expression of therapeutic proteins such as antigens, cytokines and enzymes. Resulting recombinant lactococci strains have been tested successfully for their prophylactic and therapeutic effects in different animal models: i) against human papillomavirus type 16 (HPV-16)-induced tumors in mice, ii) to partially prevent a bovine β-lactoglobulin (BLG)-allergic reaction in mice and iii) to regulate body weight and food consumption in obese mice. Strikingly, all of these tools have been successfully transposed to the Lactobacillus genus, in recent years, within our laboratory. Notably, anti-oxidative Lactobacillus casei strains were constructed and tested in two chemically-induced colitis models. In parallel, we also developed a strategy based on the use of L. lactis to deliver DNA at the mucosal level, and were able to show that L. lactis is able to modulate the host response through DNA delivery. Today, we consider that all of our consistent data, together with those obtained by other groups, demonstrate and reinforce the interest of using LAB, particularly lactococci and lactobacilli strains, to develop novel therapeutic protein mucosal delivery vectors which should be tested now in human clinical trials.     

Biotechnological and in situ food production of polyols by lactic acid bacteria

Polyols such as mannitol, erythritol, sorbitol, and xylitol are naturally found in fruits and vegetables and are produced by certain bacteria, fungi, yeasts, and algae. These sugar alcohols are widely used in food and pharmaceutical industries and in medicine because of their interesting physicochemical properties. In the food industry, polyols are employed as natural sweeteners applicable in light and diabetic food products. In the last decade, biotechnological production of polyols by lactic acid bacteria (LAB) has been investigated as an alternative to their current industrial production. While heterofermentative LAB may naturally produce mannitol and erythritol under certain culture conditions, sorbitol and xylitol have been only synthesized through metabolic engineering processes. This review deals with the spontaneous formation of mannitol and erythritol in fermented foods and their biotechnological production by heterofermentative LAB and briefly presented the metabolic engineering processes applied for polyol formation.