Safety and probiotic functionality of isolated goat milk lactic acid bacteria

Purpose

Lactic acid bacteria (LAB) are traditionally employed in the food industry. LAB strains from goat milk may also present probiotic potential, and it is fundamental to study the safety and functionality aspects which are desirable for their use in food. The objective of this study was to verify the probiotic potential of lactic bacteria isolated from goat milk.

Methods

The presence of safety-related virulence factors (hemolytic activity, gelatinase production, coagulase, and sensitivity to antibiotics) as well as functionality (exopolysaccharide (EPS) production, proteolytic activity, autoaggregation, gas production, survival in the gastrointestinal tract, and antimicrobial activity against bacteria that impair oral health) were determined.

Result

The selected LAB strains are safe against the evaluated parameters and have characteristics of possible probiotic candidates. Especially L. plantarum (DF60Mi) and Lactococcus lactis (DF04Mi) have potential to be added to foods because they have better resistance to simulated gastrointestinal conditions. In addition, they are isolated with already proven antimicrobial activity against Listeria monocytogenes, an important food-borne pathogen. DF60Mi was able to produce EPS (exopolysaccharides). LS2 and DF4Mi strains, both Lactococcus lactis subsp. lactis, demonstrated antimicrobial activity against S. mutans ATCC 25175, a recurrent microorganism in oral pathologies, mainly caries.

Conclusion

This study provides subsidies for future exploration of the potentialities of these LAB strains for both the development of new functional foods and for application in oral health.

     

Safety of industrial lactic acid bacteria.

Lactic acid bacteria (LAB) are ubiquitous in fermented and non-fermented foods and are common components of the human commensal microflora. This long history of human exposure and consumption has led to the reasonable conclusion that they are generally safe. Recent attention has also focused on their possible role as probiotic bacteria, promoting beneficial health effects. There have, however, been a number of reports of human infections caused by LAB and these are reviewed. In most cases, the source of the infection was the commensal LAB flora rather than ingested bacteria and the patient had some underlying disease or predisposing condition. Even as opportunistic pathogens, the LAB, with the notable exception of the enterococci, are much less successful than a number of other members of the commensal microflora. The use of new strains for probiotic use is likely to require more detailed evidence for their safety, particularly if the strains have been genetically modified or have been derived from animals. Procedures that have been proposed for assessing the safety of new strains are described.     

Genetic engineering as a powerful tool to improve probiotic strains

Over the last decade, there has been increasing interest in the use of probiotic microorganisms. However, certain doubts have arisen around probiotics, because of the beneficial effects of these microorganisms are not clear yet, and in many occasions those beneficial effects have not been proven. Therefore, it would be of interest if these probiotic strains were able to acquire new attributes to allow them improve and increase their beneficial characteristics. Genetic engineering can be used for human applications; for instance, the resistance to antibiotics is removed and the probiotic bacteria are modified in its own DNA. This process can be achieved by: (1) the use of food-grade vectors derived from lactic acid bacteria and/or bifidobacteria cryptic plasmids, (2) the genes integration or deletion in the chromosome of the probiotic strain, by site-specific recombination using the attP/integrase system, or by homologous recombination, using either suicide vectors, (3) using the clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (Cas) nuclease. Through genetic engineering, the knowledge of probiotic strains as well as its use could be improved, and the doubts about probiotics could be crumped.     

Rapid identification of Lactobacillus and Bifidobacterium in probiotic products using multiplex PCR

Lactic acid bacteria (LAB) are beneficial for the gastrointestinal tract and reinforce immunity in human health. Recently, many functional products using the lactic acid bacteria have been developed. Among these LAB, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum are frequently used for probiotic products. In order to monitor these LAB in commercial probiotic products, a multiplex PCR method was developed. We designed four species-specific primer pairs for multiplex PCR from the 16S rRNA, 16S-23S rRNA intergenic spacer region, and 23S rRNA genes in Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium longum, and Bifidobacterium bifidum. Using these primer pairs, 4 different LAB were detected with high specificity in functional foods. We suggest that the multiplex PCR method developed in this study would be an efficient tool for simple, rapid, and reliable identification of LAB used as probiotic strains.     

Screening from pickled vegetables the potential probiotic strains of lactic acid bacteria able to inhibit the Salmonella invasion in mice

Aims: To screen from pickled vegetables the potential probiotic lactic acid bacteria (LAB) strains with antagonistic activity against Salmonella invasion in host. Methods and Results: Probiotic properties including acid and bile tolerance as well as inhibition on pathogenic bacteria were used for screening of LAB strains from pickled vegetables. Two strains, i.e Pediococcus pentosaceus MP12 and Lactobacillus plantarum LAP6, were selected and further assayed for their activities against Salmonella invasion in mouse liver and spleen. For these two LAB strains, strain LAP6 was able to adhere to the mouse intestinal epithelium cells. Conclusions: In screening of the probiotic strains able to inhibit the Salmonella invasion in host, factors other than the adherence to host intestinal epithelium may contribute some roles. Significance and Impact of the Study: Probiotic LAB strains with activity against Salmonella invasion in host could be isolated from vegetable origins. These strains may be used for vegetable processing.     

Probiotic characteristics of lactic acid bacteria isolated from kimchi

Aims: The present work was aimed at identifying strains of lactic acid bacteria (LAB) from kimchi, with properties suitable for use as starter cultures in yogurt fermentation. Methods and Results: A total of 2344 LAB strains were obtained from two different sources, one group consisted of commercial LAB strains from kimchi, and the second group consisted of those strains isolated from various types of kimchi. The LAB strains from both groups were screened for resistance to biological barriers (acid and bile salts), and the four most promising strains were selected. Further analysis revealed that KFRI342 of the four selected strains displayed the greatest ability to reduce the growth of the cancer cells, SNU‐C4. The in vivo efficacy of strains in quinone reductase induction assay was evaluated, and the extent of DNA strand breakage in individual cells was investigated using the comet assay. Strain KFRI342 was identified as Lactobacillus acidophilus by 16S rRNA sequence analysis, showed protection against tumour initiation and imparted immunostimulation as well as protection against DNA damage. Conclusions: Strain KFRI342, which showed probiotic characteristics reducing cancer cell growth, could be a suitable starter culture for yogurt fermentation because of its strong acid production and high acid tolerance. Significance and Impact of the Study: This is the first report to describe a bacterium, isolated from kimchi, Lact. acidophilus KFRI342 which has the probiotic characteristics and the acid tolerance needed for its use as a starter culture in yogurt fermentation.     

Quantitative Appraisal of the Probiotic Attributes and In Vitro Adhesion Potential of Anti-listerial Bacteriocin-producing Lactic Acid Bacteria

Estimation of bile tolerance, endurance to gastric and intestinal environment and adhesion potential to intestinal cells are significant selection criteria for probiotic lactic acid bacteria (LAB). In this paper, the probiotic potential of native bacteriocin-producing LAB isolated previously from indigenous source has been determined through quantitative approaches. Among fifteen anti-listerial bacteriocin-producing native LAB, ten strains were found to be bile tolerant. The presence of bile salt hydrolase (bsh) gene in native Lactobacillus plantarum strains was detected by PCR and confirmed by nucleic acid sequencing of a representative amplicon. Interestingly, three native LAB strains exhibited significant viability in simulated gastric fluid, analogous to the standard LAB Lactobacillus rhamnosus GG, while an overwhelming majority of the native LAB strains demonstrated the ability to survive and remain viable in simulated intestinal fluid. Quantitative adhesion assays based on conventional plating method and a fluorescence-based method revealed that the LAB isolates obtained from dried fish displayed significant in vitro adhesion potential to human adenocarcinoma HT-29 cells, and the adhesion level was comparable to some of the standard probiotic LAB strains. The present study unravels putative probiotic attributes in certain bacteriocin-producing LAB strains of non-human origin, which on further in vivo characterization could find specific applications in probiotic food formulations targeted for health benefits.     

Probiotic properties of Lactobacillus strains with high cinnamoyl esterase activity isolated from jeot-gal,a high-salt fermented seafood

Cinnamoyl esterases (CEs) improve the bioavailability of caffeic acid, a potent antioxidant with beneficial health effects. This study aimed to characterize the probiotic properties of 14 strains of CE-producing lactic acid bacteria (LAB) isolated from jeot-gal, a high-salt fermented seafood. We evaluated properties of the probiotic LAB with high CE activity, including tolerance to low pH and bile salts, antimicrobial activity, surface hydrophobicity, adhesion, and immunomodulatory effects, in vitro. All LAB tested tolerated pH 2.0 and 3% Oxgall, i.e., conditions comparable with those in the gastrointestinal environment. Three isolates, Lactobacillus paracasei JBCC10650, Lactobacillus pentosus JBCC10659, and Lactobacillus plantarum JBCC10543, showed stronger adherence to epithelial cells (12.3, 9.6, and 9.4%) than a commercial probiotic Lactobacillus rhamnosus GG (9.1%; p < 0.05), and exhibited broad antibacterial activity against putative pathogens. Most of the 14 LAB strains were able to regulate mRNA expression of pro- and anti-inflammatory cytokines in macrophages, indicating their potential immunomodulatory effects. Our findings suggest that the newly isolated CE-producing probiotics may show beneficial health effects by supporting the host immune system.     

In vitro study of potentially probiotic lactic acid bacteria strains isolated from kimchi

The objective of the present study was to investigate lactic acid bacteria (LAB) isolated from kimchi for their potential probiotic use. Ten preselected LAB strains were evaluated for their functionality and safety. Examined characteristics included acid and bile tolerance, cell adhesion, antimicrobial activity against pathogens, hemolytic activity, undesirable biochemical characteristics, and antibiotic resistance. Results indicated that consumption of these 10 strains does not pose any health risk, as they were not hemolytic and exhibited no undesirable biochemical activity or antibiotic resistance. In particular, three strains, Lactobacillus plantarum NO1, Pediococcus pentosaceus MP1, and Lactobacillus plantarum AF1, showed high degrees of acid and bile tolerance, adherence to Caco-2 and HT-29 cells, and antimicrobial activity against four pathogens (Staphylococcus aureus, Escherichia coli O157:H7, Salmonella typhi, and Listeria monocytogenes). These results suggest that LAB strains from kimchi may have potential use as novel probiotics.     

Characterization of the Properties of Human- and Dairy-Derived Probiotics for Prevention of Infectious Diseases in Fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

In Vitro Evaluation of the Protective Role of Lactobacillus StrainsAgainst Inorganic Arsenic Toxicity

Inorganic arsenic [iAs, As(III) + As(V)] is considered a human carcinogen. Recent studies show that it has also toxic effects on the intestinal epithelium which might partly explain its systemic toxicity. The aim of this study is to evaluate the protective role of lactic acid bacteria (LAB) against the toxic effects of iAs on the intestinal epithelium. For this purpose, the human colonic cells Caco-2 were exposed to As(III) in the presence of various LAB strains or their conditioned medium. Results showed that some strains and their conditioned media partially revert the oxidative stress, the production of pro-inflammatory cytokines, the alterations of the distribution of tight junction proteins, and the cell permeability increases caused by As(III). These results show that both soluble factors secreted or resulting from LAB metabolism and cell-cell interactions are possibly involved in the beneficial effects. Therefore, some LAB strains have potential as protective agents against iAs intestinal barrier disruption.

     

Engineering lactic acid bacteria for increased industrial functionality

Based on their spoilage-preventing and flavor-contributing characteristics, lactic acid bacteria (LAB) are employed as starter cultures for the fermentation of foods and feeds. In addition, several specific LAB strains are marketed on basis of their beneficial effects on the consumer's health, representing an explosively growing market for the products containing these so-called probiotics. Due to this extensive industrial use there is a strong interest in unraveling the molecular mechanisms involved in industrial robustness, cognate stress resistance, and health-promoting phenotypes of these LAB that may vary drastically between different starter and probiotic strains currently marketed. This review describes some of the post-genomic tools developed, as well as their employment for the identification of bacterial effector molecules involved in the aforementioned industrially relevant phenotypes. Furthermore, it addresses possible strategies to exploit such knowledge into the rational design of LAB strains with increased industrial functionality.     

Use of mixed cultures for the fermentation of cereal-based substrates with potential probiotic properties

The production of a new cereal-based probiotic foods with suitable aroma, flavor and pH using mixed culture fermentation has been investigated. This required the selection of suitable types of cereal grains and probiotic microorganisms. In a medium of 5% (w/v) malt suspension the effects of yeast presence on the fermentation of a lactic acid bacterium (LAB), Lactobacillus reuteri, was studied. With different inoculum ratios between the yeast and the LAB, the characteristics of the fermentation broth including pH and the contents of free amino nitrogen (FAN), reducing sugar, lactic acid and ethanol were investigated. It was found that LAB growth was enhanced by the introduction of the yeast. In mixed culture broth pH was lowered and the production of lactic acid and ethanol were increased in comparison against pure LAB culture.     

Characterization of the properties of human- and dairy-derived probiotics for prevention of infectious diseases in fish

The present study aimed to investigate the potential probiotic properties of six lactic acid bacteria (LAB) intended for human use, Lactobacillus rhamnosus ATCC 53103, Lactobacillus casei Shirota, Lactobacillus bulgaricus, L. rhamnosus LC 705, Bifidobacterium lactis Bb12, and Lactobacillus johnsonii La1, and one for animal use, Enterococcus faecium Tehobak, for use as a fish probiotic. The strains for human use were specifically chosen since they are known to be safe for human use, which is of major importance because the fish are meant for human consumption. The selection was carried out by five different methods: mucosal adhesion, mucosal penetration, inhibition of pathogen growth and adhesion, and resistance to fish bile. The adhesion abilities of the seven LAB and three fish pathogens, Vibrio anguillarum, Aeromonas salmonicida, and Flavobacterium psychrophilum, were determined to mucus from five different sites on the surface or in the gut of rainbow trout. Five of the tested LAB strains showed considerable adhesion to different fish mucus types (14 to 26% of the added bacteria). Despite their adhesive character, the LAB strains were not able to inhibit the mucus binding of A. salmonicida. Coculture experiments showed significant inhibition of growth of A. salmonicida, which was mediated by competition for nutrients rather than secretion of inhibitory substances by the probiotic bacteria as measured in spent culture liquid. All LAB except L. casei Shirota showed tolerance against fish bile. L. rhamnosus ATCC 53103 and L. bulgaricus were found to penetrate fish mucus better than other probiotic bacteria. Based on bile resistance, mucus adhesion, mucus penetration, and suppression of fish pathogen growth, L. rhamnosus ATCC 53103 and L. bulgaricus can be considered for future in vivo challenge studies in fish as a novel and safe treatment in aquaculture.     

Lactic acid bacteria as probiotics

A number of Lactobacillus species, Bifidobacterium sp, Saccharomyces boulardii, and some other microbes have been proposed as and are used as probiotic strains, i.e. live microorganisms as food supplement in order to benefit health. The health claims range from rather vague as regulation of bowel activity and increasing of well-being to more specific, such as exerting antagonistic effect on the gastroenteric pathogens Clostridium difficile, Campylobacter jejuni, Helicobacter pylori and rotavirus, neutralising food mutagens produced in colon, shifting the immune response towards a Th2 response, and thereby alleviating allergic reactions, and lowering serum cholesterol (Tannock, 2002). Unfortunately, most publications are case reports, uncontrolled studies in humans, or reports of animal or in vitro studies. Whether or not the probiotic strains employed shall be of human origin is a matter of debate but this is not a matter of concern, as long as the strains can be shown to survive the transport in the human gastrointestinal (GI) tract and to colonise the human large intestine. This includes survival in the stressful environment of the stomach - acidic pH and bile - with induction of new genes encoding a number of stress proteins. Since the availability of antioxidants decreases rostrally in the GI tract production of antioxidants by colonic bacteria provides a beneficial effect in scavenging free radicals. LAB strains commonly produce antimicrobial substance(s) with activity against the homologous strain, but LAB strains also often produce microbicidal substances with effect against gastric and intestinal pathogens and other microbes, or compete for cell surface and mucin binding sites. This could be the mechanism behind reports that some probiotic strains inhibit or decrease translocation of bacteria from the gut to the liver. A protective effect against cancer development can be ascribed to binding of mutagens by intestinal bacteria, reduction of the enzymes beta-glucuronidase and beta-glucosidase, and deconjugation of bile acids, or merely by enhancing the immune system of the host. The latter has attracted considerable interest, and LAB have been tested in several clinical trials in allergic diseases. Characteristics ascribed to a probiotic strain are in general strain specific, and individual strains have to be tested for each property. Survival of strains during production, packing and storage of a viable cell mass has to be tested and declared.     

Identification and probiotic properties of lactobacilli isolated from two different fermented beverages

Purpose

Scientific information regarding the microbial content and functional aspects of fermented beverages traditionally produced in certain parts of Europe are scarce. However, such products are believed to have some health benefits and might contain functional bacterial strains, such as probiotics. The aim of the study was to identify such lactic acid bacteria strains isolated from water kefir and, for the first time, from braga, a Romanian fermented beverage made of cereals.

Methods

Lactic acid bacteria (LAB) were identified to species level based on (GTG)₅-PCR fingerprinting and 16S rRNA gene sequencing. Selected strains were screened for their antibacterial activity and probiotic potential.

Results

Eight isolates belonging to seven Lactobacillus species were recovered from the two drinks. The identification of LAB involved in the fermentation of braga (Lactobacillus plantarum, Lactobacillus fermentum, and Lactobacillus delbrueckii) is firstly reported here. Five of the Lactobacillus isolates showed antibacterial activity against pathogenic bacteria, including Listeria monocytogenes, Escherichia coli, Staphylococcus aureus, and Salmonella enterica. Moreover, most of them showed a good resistance to pH 2.5 and some survived at high concentrations of bile salts (up to 2%). Two L. plantarum isolates were able to inhibit all the indicator strains, and showed the best viability (about 70%) after a sequential treatment simulating the passage through the gastrointestinal tract.

Conclusion

Based on the results, the most promising candidates for designing new probiotic products are: L. plantarum BR9 from braga and L. plantarum CR1 from water kefir.

     

Complete glutathione system in probiotic <Emphasis Type="Italic">Lactobacillus fermentum</Emphasis> ME-3

There is much information about glutathione (GSH) in eukaryotic cells, but relatively little is known about GSH in prokaryotes. Without GSH and glutathione redox cycle lactic acid bacteria (LAB) cannot protect themselves against reactive oxygen species. Previously we have shown the presence of GSH in Lactobacillus fermentum ME-3 (DSM14241). Results of this study show that probiotic L. fermentum ME-3 contains both glutathione peroxidase and glutathione reductase. We also present that L. fermentum ME-3 can transport GSH from environment and synthesize GSH. This means that it is characterized by a complete glutathione system: synthesis, uptake and redox turnover ability that makes L. fermentum ME-3 a perfect protector against oxidative stress. To our best knowledge studies on existence of the complete glutathione system in probiotic LAB strains are still absent and glutathione synthesis in them has not been demonstrated.     

Lactic acid bacteria isolated from canine faeces

AIMS: Lactic acid bacteria (LAB) were isolated and sequenced from the faeces of healthy dogs. Five of these strains were selected and further characterized to clarify the potential of these strains as probiotics for canine. METHODS AND RESULTS: LAB were found in 67% (14/21) of the canine faeces samples when plated on Lactobacilli Selective Media without acetic acid. Out of 13 species identified with partial 16S rRNA gene sequencing, Lactobacillus fermentum LAB8, L. mucosae LAB12, L. rhamnosus LAB11, L. salivarius LAB9 and Weissella confusa LAB10 were selected as candidate probiotic strains based on their frequency, quantity in faeces, growth density, acid tolerance and antimicrobial activity. The minimal inhibitory concentration values of these isolates were determined for 14 antibiotics. L. salivarius LAB9, W. confusa LAB10 and L. mucosae LAB12 were viable in pH 2 for 4 h (mLBS), indicating tolerance to acidity and thus the potential to survive in gastrointestinal tract of the canine. The LAB8-LAB12 strains showed antimicrobial activity against Micrococcus luteus A1 NCIMB86166. CONCLUSIONS: Thirteen different LAB species were found from the faecal microbiota of the healthy canines. Five acid tolerant and antimicrobially active LAB strains with the capacity to grow to high densities both aerobically and anaerobically were chosen to serve as candidate probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: The selected LAB strains are among the first host-specific LAB with antimicrobial activity isolated from canines that could serve as potential probiotics for canine use.     

Abundance of Lactobacillus plantarum Strains with Beneficial Attributes in Blackberries (Rubus sp.), Fresh Figs (Ficus carica), and Prickly Pears (Opuntia ficus-indica) Grown and Harvested in Algeria

This first study performed on traditional fruits consumed in North Africa reveals their richness in microorganisms with beneficial attributes like cholesterol lowering capabilities. Blackberries (Rubus sp.), fresh figs (Ficus carica), and prickly pears (Opuntia ficus-indica) are fruits largely and traditionally consumed in Kabylia, a beautiful northern Algerian region. Here, 85 lactic acid bacteria (LAB)-isolates were isolated and identified by MALDI-TOF mass spectrometry. The identified species belong to Lactobacillus and Leuconostoc genera. These 85 LAB-isolates were then assessed for their capabilities to grow under conditions mimicking the gastrointestinal tract, and the resulting data were statistically treated with principal component analysis (PCA). After which, only 26 LAB-isolates were selected and characterized for their genetic relatedness using random amplified polymorphic DNA (RAPD) method. Following the genetic relatedness assessment, only 10 LAB-strains, among which nine Lactobacillus plantarum and one Lactobacillus paracasei were studied for their pathoproperties and some probiotic features. Interestingly, all of these 10 LAB-strains were devoid of adverse effects, but capable to adhere to human epithelial colorectal adenocarcinoma Caco-2 cells. Of note, these 10 LAB-strains exhibited an important in vitro hypocholesteromia effect, in strain-dependent manner. Moreover, the Lactobacillus strains exhibited a high bile salt hydrolase (BSH) activity which was correlated with expression of bsh2, bsh3 and bsh4 genes.