Evaluation of Probiotic Properties of Novel Brazilian Lactiplantibacillus plantarum Strains

Probiotics and Antimicrobial Proteins - Beneficial effects of Lactiplantibacillus plantarum strains have been widely reported. Knowing that the effects of probiotic bacteria are strain-dependent,...     

Genetic Variation of pln Loci Among Probiotic Lactobacillus plantarum Group Strains with Antioxidant and Cholesterol-Lowering Ability

Probiotics and Antimicrobial Proteins - In the present study, 14 different plantaricin-encoding genes of pln loci were studied and compared to available sequences from public domain database of...     

Bacillus amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for the production of Bikalga,an alkaline fermented food

Aims

To identify and screen dominant Bacillus spp. strains isolated from Bikalga, fermented seeds of Hibiscus sabdariffa for their antimicrobial activities in brain heart infusion (BHI) medium and in a H. sabdariffa seed‐based medium. Further, to characterize the antimicrobial substances produced.

Methods and Results

The strains were identified by gyrB gene sequencing and phenotypic tests as B. amyloliquefaciens ssp. plantarum. Their antimicrobial activity was determined by the agar spot and well assay, being inhibitory to a wide range of Gram‐positive and Gram‐negative pathogenic bacteria and fungi. Antimicrobial activity against Bacillus cereus was produced in H. sabdariffa seed‐based medium. PCR results revealed that the isolates have potential for the lipopeptides iturin, fengycin, surfactin, the polyketides difficidin, macrolactin, bacillaene and the dipeptide bacilysin production. Ultra‐high‐performance liquid chromatography‐time of flight mass spectrometry analysis of antimicrobial substance produced in BHI broth allowed identification of iturin, fengycin and surfactin.

Conclusions

The Bacillus amyloliquefaciens ssp. plantarum exhibited broad‐spectrum antifungal and antibacterial properties. They produced several lipopeptide antibiotics and showed good potential for biological control of Bikalga.

Significance and Impact of the Study

Pathogenic bacteria often occur in spontaneous food fermentations. This is the first report to identify indigenous B. amyloliquefaciens ssp. plantarum strains as potential protective starter cultures for safeguarding Bikalga.     

鸡源益生芽胞杆菌的筛选

根据菌体对低pH和高胆盐的耐受性、产酶特性和对病原菌的抑菌效果,对家鸡肠道中的芽胞杆菌进行了筛选。结果表明,有4株芽胞杆菌(A2、C5、C19和D8)能抑制金黄色葡萄球菌(Staphylococcus aureus)的生长。这4株菌在pH 3.0和高胆盐条件下的存活率都达到60%以上,产淀粉酶和蛋白酶的能力也较好,表明这些菌株具有作为益生菌的开发潜力。经形态学和生理生化反应鉴定,初步确定A2和D8为地衣芽胞杆菌(Bacillus licheniform is),C5和C19为蜡样芽胞杆菌(Bacillus cereus)。     

Probiotic Bacillus amyloliquefaciens SC06 Prevents Bacterial Translocation in Weaned Mice

Probiotic is a preparation containing microorganisms that confers beneficial effect to the host. This work assessed whether oral administration of Bacillus amyloliquefaciens SC06 (Ba) could decrease bacterial translocation in weaned mice. Weaned C57BL/6 were randomly allocated into three groups: group I as the control group, group II were treated with 0.85 % NaCl. Group III was administered with probiotic Ba 1 × 10⁹ CFU/day dissolved in 100 μl of 0.85 % NaCl for 30 days. Mice were then sacrificed, and tissue were cultured to determine bacterial translocation. Meanwhile, splenic CD4⁺T cells, CD8⁺T cells, B cells, and macrophages were analysised by FACS. Our results showed that probiotic Ba significantly reduced bacteria translocation compared with the control group and 0.85 % NaCl group (P < 0.05), lower levels of bacteria were detected in the MLN, liver, spleen, and kidney of mice. Moreover, significant increase in percentage and number of macrophages were observed in the spleen of Ba-treated mice compared with the control and 0.85 % NaCl groups. Together, these data indicated that Ba could decrease bacterial translocation in weaned mice. This effect seems to be correlated with the changes of macrophage numbers.     

Complete genome sequence of a plant associated bacterium Bacillus amyloliquefaciens subsp. plantarum UCMB5033

Bacillus amyloliquefaciens subsp. plantarum UCMB5033 is of special interest for its ability to promote host plant growth through production of stimulating compounds and suppression of soil borne pathogens by synthesizing antibacterial and antifungal metabolites or priming plant defense as induced systemic resistance. The genome of B. amyloliquefaciens UCMB5033 comprises a 4,071,167 bp long circular chromosome that consists of 3,912 protein-coding genes, 86 tRNA genes and 10 rRNA operons.     

Probiotic Bacillus amyloliquefaciens mediate M1 macrophage polarization in mouse bone marrow-derived macrophages

Depending on the microenvironment, macrophages can acquire distinct functional phenotypes, referred to as classically activated M1 and M2. M1 macrophages are considered potent effector cells that kill intracellular pathogens, and M2 macrophages promote the resolution of wound healing. In this study, we are interested to know whether probiotic Bacillus amyloliquefaciens (Ba) can induce macrophages polarization. Real-time fluorescence PCR analysis demonstrated that the expression of IL-1β, iNOS, TNF-α and IL-6 genes for M1 macrophages was significantly increased at 1.5 h after probiotic Ba treatment compared to the probiotic Ba-free treatment (P < 0.01), whereas the expression of M2 macrophage marker genes (Arg1, Fizz1, MR, Ym1) was decreased (P < 0.05). Furthermore, the phagocytic activity was dramatically increased in the Ba-treated BMDMs using a FITC-dextran endocytosis assay. Together, these findings indicated that probiotic Ba facilitated polarization of M1 macrophages and enhanced its phagocytic capacity. The results expanded our knowledge about probiotic function-involved macrophage polarization.     

Genetic manipulation of Bacillus amyloliquefaciens.

Application of modern gene technology to strain improvement of the industrially important bacterium Bacillus amyloliquefaciens is reported. Several different plasmid constructions carrying the alpha-amylase gene (amyE) from B. amyloliquefaciens were amplified in this species either extrachromosomally or intrachromosomally. The amyE gene cloned on a pUB110-derived high copy plasmid pKTH10 directed the highest yields both in rich laboratory medium and in crude industrial medium. The alpha-amylase activity, when compared with the parental strain, was enhanced up to 20-fold in the pKTH 10 transformant. This strain showed decreased activities for other exoenzymes, such as proteases and beta-glucanase suggesting common limiting resources in the processing of these enzymes. Deletions were made in vitro in genes encoding neutral (nprE), alkaline (aprE) protease and beta-glucanase (bglA). The engineered genes were cloned into the thermosensitive plasmid pE194, and the resulting plasmids were used to replace the corresponding wild type chromosomal genes in B. amyloliquefaciens by integration-excision at non-permissive temperature. The double mutant deficient in the major proteases (delta nprE delta aprE) showed about a 2-fold further enhancement in alpha-amylase production in the industrial medium compared with the relevant wild type backgroud, both when plasmid-free and when transformed with pKTH10; this strain also produced elevated levels of the chromosomally-encoded beta-glucanase; pKTH10 was stably maintained both in the wild type strain and in the delta nprE delta aprE mutant. We suggest that the higher yields in alpha-amylase and beta-glucanase in the delta nprE delta aprE strain are primarily due to improved access to limiting resources, and that decreased proteolytic degradation may have had a secondary role in retaining the high activity obtained.     

New Potentially Probiotic Strains Isolated from Humans – Comparison of Properties with Strains from Probiotic Products and ATCC Collection

Lactic acid bacteria are used in various types of probiotic products. Due to the constantly growing probiotics market, new strains with pro-health properties are sought. The present study compared 39 strains of Lactobacillus, Lacticaseibacillus, and Lactiplantibacillus, isolated from probiotic products and healthy people. The current research aimed to search for new, potentially probiotic strains. For this purpose the relationship between Lactobacillaceae strains was carried out; moreover, the basic properties of probiotic microorganisms, such as survival at low pH and bile salt environment, antibiotic susceptibility, aggregation and antagonism were estimated. The properties of these isolates were also compared with the properties of probiotic strains from the ATCC collection. In comparing the genetic relationship (PFGE method) between the tested isolates, it was observed that some of them show a high degree of similarity. All tested strains tolerated an environment with a pH value of 3.0, and the addition of 0.3% bile salt; showed auto-aggregation properties and displayed antagonism against pathogenic microorganisms. In the present study, the bacteria were susceptible to tetracycline, chloramphenicol and ampicillin; the resistance to vancomycindepended on the bacteria type. All the properties were strain-depended. Most of the tested strains had properties comparable to the reference strains. Three L. acidophilus strains isolated from cervical swabs seem to be promising candidates for probiotic strains. Key words: lactobacilli, PFGE, antimicrobial susceptibility, antagonism     

Probiotic Properties of Lactobacillus Strains Isolated from Tibetan Kefir Grains

The objective of this study was to evaluate the functional properties of lactic acid bacteria (LAB) isolated from Tibetan kefir grains. Three Lactobacillus isolates identified as Lactobacillus acidophilus LA15, Lactobacillus plantarum B23 and Lactobacillus kefiri D17 that showed resistance to acid and bile salts were selected for further evaluation of their probiotic properties. The 3 selected strains expressed high in vitro adherence to Caco-2 cells. They were sensitive to gentamicin, erythromycin and chloramphenicol and resistant to vancomycin with MIC values of 26 µg/ml. All 3 strains showed potential bile salt hydrolase (BSH) activity, cholesterol assimilation and cholesterol co-precipitation ability. Additionally, the potential effect of these strains on plasma cholesterol levels was evaluated in Sprague-Dawley (SD) rats. Rats in 4 treatment groups were fed the following experimental diets for 4 weeks: a high-cholesterol diet, a high-cholesterol diet plus LA15, a high-cholesterol diet plus B23 or a high-cholesterol diet plus D17. The total cholesterol, triglyceride and low-density lipoprotein cholesterol levels in the serum were significantly (P<0.05) decreased in the LAB-treated rats compared with rats fed a high-cholesterol diet without LAB supplementation. The high-density lipoprotein cholesterol levels in groups B23 and D17 were significantly (P<0.05) higher than those in the control and LA15 groups. Additionally, both fecal cholesterol and bile acid levels were significantly (P<0.05) increased after LAB administration. Fecal lactobacilli counts were significantly (P<0.05) higher in the LAB treatment groups than in the control groups. Furthermore, the 3 strains were detected in the rat small intestine, colon and feces during the feeding trial. The bacteria levels remained high even after the LAB administration had been stopped for 2 weeks. These results suggest that these strains may be used in the future as probiotic starter cultures for manufacturing novel fermented foods.     

Probiotic Potential of Bacillus Strains Isolated from an Acidic Fermented Food Idli

Present study is intended to assess the probiotic properties of Bacillus spp. isolated from idli batter, a traditional fermented food of Southern India and Sri Lanka. A total of 32 isolates were screened for potential pathogenic behaviour through haemolysis assay, DNase activity and antibiotics sensitivity. Two of the isolates were found to be potentially safe and identified as Bacillus spp. These strains were characterized for in vitro probiotic attributes and antioxidant activity. Both the strains showed strong acid and bile tolerance, transit tolerance, lysozyme tolerance, cell surface hydrophobicity, auto-aggregation, co-aggregation, biofilm formation potential and adhesion to human colon adenocarcinoma (HT 29) cell line demonstrating potential probiotic ability. These strains also exhibited considerable cholesterol binding, thermostability, β-galactosidase production, proteolytic, amylolytic and lipolytic activity. Cell-free supernatant inhibited the biofilm formation by Pseudomonas aeruginosa (KT266804) to 90%. Intact cells showed significant DPPH (41%), hydroxyl (31%), radical scavenging activity and lipid peroxidation inhibition (20.38%), while cell-free extracts exhibited significant superoxide anion radical scavenging activity (16.25%). Results revealed that isolates could be potential probiotic candidate after further assessment of in vivo probiotic properties and safety evaluation and could be utilised as starter cultures in functional foods.

     

Cloning and expression of alpha-amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactobacillus plantarum

Lactobacillus plantarum is used in a wide range of agricultural and food fermentations. In this paper we report the introduction of alpha-amylase into the organism from Bacillus amyloliquefaciens on a stable recombinant plasmid. The genetically manipulated organism grew on MRSB medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations.     

Unrelatedness of Bacillus amyloliquefaciens and Bacillus subtilis

Eight strains of highly amylolytic, sporeforming bacilli (hereafter referred to as Bacillus amyloliquefaciens) were compared with respect to their taxonomic relationship to B. subtilis. The physiological-biochemical properties of these two groups of organisms showed that B. amyloliquefaciens differed from B. subtilis by their ability to grow in 10% NaCl, characteristic growth on potato plugs, increased production of alpha-amylase, and their ability to ferment lactose with the production of acid. The base compositions of the deoxyribonucleic acid (DNA) of the B. subtilis strains consistently fell in the range of 41.5 to 43.5% guanine + cytosine (G + C), whereas that of the B. amyloliquefaciens strains was in the 43.5 to 44.9% G + C range. Hybrid formation between B. subtilis W23 and B. amyloliquefaciens F DNA revealed only a 14.7 to 15.4% DNA homology between the two species. Transducing phage, SP-10, was able to propagate on B. subtilis W23 and B. amyloliquefaciens N, and would transduce B. subtilis 168 (indole(-)) and B. amyloliquefaciens N-10 (arginine(-)) to prototrophy with a frequency of 3.9 x 10(-4) and 2.4 x 10(-5) transductants per plaque-forming unit, respectively. Attempts to transduce between the two species were unsuccessful. These data show that Bacillus amyloliquefaciens is a valid species and should not be classified as a strain or variety of B. subtilis.     

In Vitro Assessment of Bio-Functional Properties from Lactiplantibacillus plantarum Strains

In recent years, alongside the conventional screening procedures for the evaluation of probiotics for human usage, the pharmaceutical and food industries have encouraged scientific research towards the selection of new probiotic bacterial strains with particular functional features. Therefore, this study intended to explore novel functional properties of five Lactiplantibacillus plantarum strains isolated from bee bread. Specifically, antioxidant, antimicrobial and β-glucosidase activities, exopolysaccharides (EPS) production and the ability to synthesize γ-aminobutyric acid (GABA) were evaluated. The results demonstrated that the investigated L. plantarum strains were effective in inhibiting the growth of some human opportunistic pathogens in vitro (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Enterococcus faecalis and Staphylococcus aureus). Moreover, the evaluation of antioxidant and β-glucosidase activity and of EPS and GABA production, revealed a different behavior among the strains, testifying how these properties are strongly strain-dependent. This suggests that a careful selection within a given species is important in order to identify appropriate strains for specific biotechnological applications. The results highlighted that the five strains of L. plantarum are promising candidates for application as dietary supplements in the human diet and as microbial cultures in specific food productions.     

Properties of Thymineless Strains of Bacillus megaterium

Both Bacillus megaterium KM:T(-)R(1), a strain partially resistant to thymineless death, and strain KM:T(-), the parent strain, can satisfy their thymine requirement with either thymidine, 5-methyldeoxycytidine, or 5-methyluridine. Neither strain can use 5-methylcytosine, 5-hydroxymethylcytosine, 5-hydroxymethyluracil, or 5-aminouracil for this purpose. Strain KM:T(-)R(1) requires as little as 0.01 mM thymine for maximum growth, whereas strain KM:T(-) requires 0.10 to 0.20 mM thymine. Lysogenic KM:T(-)R(1) dies more rapidly in the presence of mitomycin C than the corresponding phage-sensitive strain. Unexpectedly, the lysogenic strain was found to be less sensitive to thymineless death than the phage-sensitive strain. Lysogenic KM:T(-)R(1) is induced by exposure to mitomycin C and by thymineless incubation. It is concluded that thymineless death occurs by a mechanism which is unrelated to phage induction and that a major lethal effect of mitomycin C is probably a consequence of phage induction.     

Genome Analysis of Bacillus amyloliquefaciens Subsp. plantarum UCMB5113: A Rhizobacterium That Improves Plant Growth and Stress Management

The Bacillus amyloliquefaciens subsp. plantarum strain UCMB5113 is a Gram-positive rhizobacterium that can colonize plant roots and stimulate plant growth and defense based on unknown mechanisms. This reinforcement of plants may provide protection to various forms of biotic and abiotic stress. To determine the genetic traits involved in the mechanism of plant-bacteria association, the genome sequence of UCMB5113 was obtained by assembling paired-end Illumina reads. The assembled chromosome of 3,889,532 bp was predicted to encode 3,656 proteins. Genes that potentially contribute to plant growth promotion such as indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis and siderophore production were identified. Moreover, annotation identified putative genes responsible for non-ribosomal synthesis of secondary metabolites and genes supporting environment fitness of UCMB5113 including drug and metal resistance. A large number of genes encoding a diverse set of secretory proteins, enzymes of primary and secondary metabolism and carbohydrate active enzymes were found which reflect a high capacity to degrade various rhizosphere macromolecules. Additionally, many predicted membrane transporters provides the bacterium with efficient uptake capabilities of several nutrients. Although, UCMB5113 has the possibility to produce antibiotics and biosurfactants, the protective effect of plants to pathogens seems to be indirect and due to priming of plant induced systemic resistance. The availability of the genome enables identification of genes and their function underpinning beneficial interactions of UCMB5113 with plants.     

In vivo Testing of Functional Properties of Three Selected Probiotic Strains

Summary Lactobacillus acidophilus M92, Lactobacillus plantarum L4 and Enterococcus faecium L3 were previously selected as probiotic strains on the base of in vitro selection criteria. To investigate functional properties of these three probiotic strains in vivo, Swiss albino mice were used as animal model. Survival, competition, adhesion and colonization were monitored in the gastrointestinal tract, as well as the immunomodulating capability of L. acidophilus M92, L. plantarum L4 and E. faecium L3. During the feeding of mice with probiotic strains with daily dose of 2 × 10¹⁰ rifampicin-resistant cells, the number of lactic acid bacteria in the faeces increased and reduction of enterobacteria and sulphite-reducing clostridia was observed. Rifampicin-resistant colonies of probiotic strains could be reisolated from the faeces of mice fed with the rifampicin-resistant cells. The similar results were obtained in homogenates of small and large intestine of mice on the first and fourteenth days after feeding with L. acidophilus M92, L. plantarum L4 and E. faecium L3. The adherence of the probiotic strains obtained in vitro correlated with their capability to adhere to mouse ileal epithelial cells in vivo. After oral immunization of mice with viable cells of L. acidophilus M92, L. plantarum L4 and E. faecium L3 with a daily dose of 2 × 10¹⁰ cells, the concentrations of serum IgA, IgG and IgM antibodies from all groups of mice were significantly higher in comparison to the control.     

几株乳酸菌益生潜力及降胆固醇的研究

采用体外试验方法研究7株乳酸菌和双歧杆菌对人体消化道不良环境的抵抗能力、在肠道上皮的黏附定植能力及其降胆固醇潜力。结果表明:受试菌对CCL-187细胞粘附能力存在显著差异(P<0.05),粪肠球菌M1粘附能力最强;各菌对胃液和胰液都有较强耐受能力,在模拟胃液和胰液中分别培养3h和4h后仍能保证95%以上存活;在含胆汁培养基中受到不同程度抑制,但都能存活;受试菌在生长过程中能够使环境中胆固醇降低从25%～54%不等,粪肠球菌A30和A31和嗜酸乳杆菌A878和PB1能使胆固醇降低40%以上,是良好的降胆固醇备选菌株。