Preliminary evaluation of probiotic potential of Lactobacillus plantarum strains isolated from Italian food products

The aim of this study was to investigate some probiotic properties of 42 wild Lactobacillus plantarum strains isolated from different Italian foods of animal origin. The strains were first screened for their antibiotic resistance profile (chloramphenicol, erythromycin, gentamicin, and tetracycline), subsequently they were tested for their in vitro resistance to lysozyme (100 mg L^?1), low pH (3.0, 2.5 and 2.0) and bile salts (0.3, 0.5 and 1.0 %). Moreover, agglutination property was studied (adhesion to Saccharomyces cerevisiae cells), as well as the presence of bsh and msa genes. The strains with the best characteristics were subjected to a further trial in order to evaluate their ability to survive to multiple stresses over time (lysozyme, low pH and bile salts) and the effect of these treatments on adhesion to yeast cells. All the strains were susceptible to chloramphenicol, erythromycin and gentamicin, while 6 strains were excluded from further evaluation because of their resistant phenotype against tetracycline. All the strains were able to grow in presence of lysozyme, as well as in MRS broth at pH 3.0. Only 4 strains showed a growth rate lower than 80 % when grown in MRS broth at pH 2.5, while a relevant growth rate decrease was observed after exposure to pH 2.0. Bile salts didn’t affect the viability of the L. plantarum cells. Twenty-one strains out of 33 tested strains were able to adhere to S. cerevisiae cells. Presence of both bsh and msa genes was detected in 6 strains. The strains resistant to all the stresses, positive to agglutination with S. cerevisiae and showing bsh and msa genes were selected for further evaluation and subjected to different stress treatments over time. The assessment of growth rates showed that exposure to lysozyme significantly increased low pH resistance in L. plantarum. This increase ranged from 2.35 to 15.57 %. The consequential lysozyme and low pH exposures didn’t affect the growth rate values after bile salts treatment, as well as the ability of the strains to adhere to yeast cells wasn’t modified by previous treatments (lysozyme, low pH and bile salts). The present work allows to increase knowledge about non starter lactic acid bacteria from Italian food products. The studied L. plantarum strains showed a good potential for their use as probiotic cultures. However, more in vivo tests are necessary to confirm this potentiality.     

The probiotic properties and potential of vaginal Lactobacillus spp. isolated from healthy women against some vaginal pathogens

During the last decade, probiotic research has progressed considerably and significant advances have been made in the selection and characterization of specific probiotic strains. The most studied probiotics belong to the genus Lactobacillus. In this study, 80 Lactobacillus spp. isolated from healthy women tolerated low pH and were able to grow in the presence of bile salts. RAPD PCR technique resulted in the identification of 38 different types. These isolates were then evaluated based on adhesion capacity, antibiotic susceptibility and tolerance in simulated gastrointestinal tract. Species-specific PCR and detection of bacteriocin-related genes were also surveyed. Among the isolates, five strains—Lacticaseibacillus rhamnosus NO21, Lacticaseibacillus casei NO1, Lactiplantibacillus plantarum NO4, Lactobacillus acidophilus NO7 and Lactobacillus gasseri NO38—presented acceptable antibiotic susceptibility pattern. Further analysis showed antimicrobial activity of Lacticaseibacillus culture against various bacterial pathogens and real-time PCR showed all five strains were able to prevent the colonization of bacterial pathogens. All five selected strains produced organic acids, hydrogen peroxide and were resistant to the spermicide. In addition, they lacked haemolytic activity with the ability of hydrophobicity, auto-aggregation and co-aggregation with pathogens. These results suggest that the vaginal microbiome could be a good source for the isolation of probiotics and the strains of this study may be considered as good probiotic candidates.     

In vitro activity of potential probiotic Lactobacillus murinus isolated from the dog

Aims:  The aim of this study was to isolate and identify Lactobacillus spp. isolates from faeces of a healthy dog, and to characterize their potential as probiotics in order to evaluate their possible use as probiotics for dogs.
Methods and Results:  An in vitro approach was used to characterize the isolates as potential probiotics including the evaluation of pH and bile salts tolerance, production of antimicrobial substances, biofilm formation on glass and polystyrene surfaces, aggregation ability and adhesion to canine intestinal mucus. The isolates survived to different pH and bile salts conditions, inhibited the in vitro growth of Escherichia coli and Clostridium perfringens , and adhered to glass and intestinal mucus.
Conclusions:  The properties shown by these isolates may indicate that they could colonize and persist in the gastrointestinal tract and induce beneficial effects to the host.
Significance and Impact of Study:  The evaluation of native canine isolates and future exprimental feeding assays may be useful tools to develop probiotics to improve animal health and reduce the risk of gastrointestinal disorders.     

Assessment and comparison of probiotic potential of four Lactobacillus species isolated from feces samples of Iranian infants

The probiotic potential of Lactobacillus species isolated from infant feces was investigated. For this study, the antibiotic susceptibility, tolerance in gut‐related conditions, antimicrobial activity, and ability to adhere to a human colorectal adenocarcinoma cell line (Caco‐2 cells) of four common Lactobacillus species (Lactobacillus paracasei [n = 15], Lactobacillus rhamnosus [n = 45], Lactobacillus gasseri [n = 20] and Lactobacillus fermentum [n = 18]) were assessed. Most isolates that which were sensitive to imipenem, ampicillin, gentamycin, erythromycin and tetracycline were selected for other tests. L. gasseri isolates had the greatest sensitivity to gastric and intestinal fluids (<10% viability). L. fermentum (FH5, FH13 and FH18) had the highest adhesion to Caco‐2 cells. The lowest antibacterial activity against pathogenic bacteria was shown by L. gasseri strains in spot tests. Furthermore, non‐adjusted cell‐free culture supernatants with low pH had greater antimicrobial activity, which was related to organic acid. The results showed that some isolates of L. rhamnosus and L. fermentum are suitable for use as a probiotic.     

植物乳杆菌发酵、冻干工艺及其益生特性的研究

目的 以Lactobacillus plantarum SQ-2506为目标,研究该菌株的发酵、冻干工艺及其益生特性。方法 通过对培养基中C源、N源和刺激因子的浓度改变考察对活菌数的影响,从而确定培养基的最佳配方;在确定最佳培养基后做出该菌的生长曲线以确定最佳发酵时间点;同时考察冻干保护剂的配方和预冷时间对菌粉活菌数的影响;此外,对植物乳杆菌进行产酸、产H2O2、生物膜形成能力、抑菌特性以及抗氧化能力的检测。结果 最佳MRS培养基中葡萄糖浓度为0.8%、酪蛋白胨为0.4%、牛肉粉为0.6%、吐温为0.06%;植物乳杆菌的生长曲线在5 h时达到稳定期,此时发酵液活菌数为3.16×109 CFU/mL,发酵液的pH为4.45。最佳冻干保护剂的配方：脱脂乳100 g/L,蔗糖120 g/L,抗坏血酸20 g/L,谷氨酸钠30 g/L;冻干前对上机液预冻时间为2 h,此时菌粉冻干存活率为70.21%。该菌株具有产酸、产H2O 2能力,并对大肠埃希菌、金黄色葡萄球菌和白色假丝酵母均有一定的抑制作用,形成膜能力较强,且具有一定的抗氧化能力。结论 通过培养基成分、发酵条件和冻干工艺的优化以及对其益生特性的研究,为下一步新药开发和规模化生产奠定基础。     

Genomic characterization and probiotic potential of Lactobacillus casei IDCC 3451 isolated from infant faeces

Probiotics play an important role in health benefits on the host. However, they also possess potentials for infectivity or in situ toxin production; thus, requiring a comprehensive assessment of their safety. In this study, we report genomic characteristics of a newly isolated Lactobacillus casei IDCC 3451 from infant faeces. Phenotypic assays based on enzyme activities and carbohydrate fermentation profiles represented metabolic features of the strain. Safety evaluation for antimicrobial resistance, biogenic amines production and cytotoxicity to a murine mouse model suggested its safe use as a probiotic strain. Our findings on the genetic background of L. casei IDCC 3451 and its potential features provide a promising functional and safe probiotic strain for the human consumption.     

In vitro antifungal,probiotic and antioxidant properties of novel Lactobacillus plantarum K46 isolated from fermented sesame leaf

This study aimed to describe the diversity of antifungal lactic acid bacteria (LAB) in popular traditional Korean fermented food. A total of 22 LAB strains was selected and subjected to a monophasic identical approach using 16S rRNA gene sequence analysis. Antifungal LAB associated with fermented food was identified as Lactobacillus plantarum (9), Lactobacillus graminis (5), Lactobacillus pentosus (4), Lactobacillus sakei (2), Lactobacillus paraplantarum (1), and Leuconostoc mesenteroides subsp. mesenteroides (1). Novel Lactobacillus plantarum strain K46 exhibited comparatively better antifungal activity against several spoilage fungi, and was deposited in the Korean Collection for Type Cultures (KACC91758P). Antifungal substances from the spent medium in which K46 was cultivated were extracted with ethyl acetate. Antifungal activity was assessed using the broth micro dilution technique. Compounds were characterized based on infrared, ¹³C nuclear magnetic resonance (NMR), and ¹H NMR spectral data. The minimum inhibitory concentration (MIC) of the compounds against Aspergillus clavatus, Aspergillus oryzae, Penicillium chrysogenum and Penicillium roqueforti was 2.5 mg/mL and that against Aspergillus fumigatus, Aspergillus niger, Curvularia lunata and Gibberella moniliformis was 5.0 mg/mL. K46 was able to survive gastrointestinal conditions simulating the stomach and the duodenum passage with the highest percentage of hydrophobicity. In addition, its resistance to hydrogen peroxide and highest hydroxyl radical and 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activities, with inhibition rates of 43.53 % and 56.88 %, respectively, were to its advantage. An antimicrobial susceptibility pattern was an intrinsic feature of this strain, thus consumption does not represent a health risk to humans. The results showed the potential of K46 strain as an antifungal, probiotic and antioxidant culture, and hence it was determined to be suitable for application in functional foods.     

Antimutagenic activity of Lactobacillus plantarum KLAB21 isolated from kimchi Korean fermented vegetables

Antimutagenic activity of Lactobacillus plantarum KLAB21, isolated from Korean kimchi, was investigated against MNNG (N-methyl-N-nitro-N-nitrosoguanidine), NQO (4-nitroquinoline-1-oxide), NPD (4-nitro-O-phenylenediamine) and aflatoxin B1 using Salmonella typhimurium strains TA100 and TA98. Although all the cell fractions including the culture supernatant, dry cells and cell-free extract exhibited antimutagenic activity against MNNG and NQO, the culture supernatant possessed the highest activity. The antimutagenic ratio of the culture supernatant was 98.4% against MNNG on strain TA100 and 57.3% against NQO on strain TA98. Its antimutagenic activity was reconfirmed by a Bacillus subtilis spore-rec assay. Levels of the antimutagenic ratios of other lactic acid bacteria originating from fermented milk ranged between 26.8 to 53% against MNNG and 28.5 to 43.4% against NQO. The antimutagenic activities of the strain KLAB21 against NPD were 72.6% on TA100 and 62.8% on TA98, and those against aflatoxin B1 were 82.5% on TA100 and 78.2% on TA98.     

Evaluation of the probiotic and functional potential of Lactobacillus agilis 32 isolated from pig manure

Escherichia coli is a symbiotic bacterium in humans and animals and an important pathogen of humans and animals. Prevention and suppression of E. coli infection is of great concern. In this study, we isolated a strain of Lactobacillus agilis 32 from pig manure and evaluated its biological characteristics, and found that its bacterial survival rate was 25% after 4 h of treatment at pH 2, and under the condition of 0·5% bile concentration, its survival rate exceeds 30%. In addition, L. agilis 32 has a cell surface hydrophobicity of 77·8%, and exhibits 67·1% auto-aggregation and 63·2% aggregation with Enterotoxigenic E. coli 10 (ETEC 10). FITC fluorescence labelling showed that the fluorescence intensity of cecum was significantly higher than that of duodenum, jejunum or colon (P < 0·05), but no significant difference from ileum. Lactobacillus agilis 32 bacterial culture and CFS showed average inhibition zone diameters of 14·2 and 15·4 mm respectively. Lactobacillus agilis 32 CFS treatment can significantly reduce the pathogenicity of ETEC 10. These results show that L. agilis 32 is an active and potential probiotic, and it has a good antibacterial effect on ETEC10, which provides basic research for probiotics to prevent and treat intestinal diarrhoea pathogen infection.     

Design and synthesis of novel oridonin analogues as potent anticancer agents

To identify anticancer agents with higher potency and lower toxicity, a series of oridonin derivatives with substituted benzene moieties at the C17 position were designed, synthesised, and evaluated for their antiproliferative properties. Most of the derivatives exhibited antiproliferative effects against AGS, MGC803, Bel7402, HCT116, A549, and HeLa cells. Compound 2p (IC_50?=?1.05?µM) exhibited the most potent antiproliferative activity against HCT116 cells; it was more potent than oridonin (IC₅₀?=?6.84?µM) and 5-fluorouracil (5-FU) (IC_50?=?24.80?µM). The IC₅₀ value of 2p in L02 cells was 6.5-fold higher than that in HCT116 cells. Overall, it exhibited better selective antiproliferative activity and specificity than oridonin and 5-FU. Furthermore, compound 2p arrested HCT116 cells at the G2 phase of the cell cycle and increased the percentage of apoptotic cells to a greater extent than oridonin.     

Biological characteristics and whole-genome analysis of the potential probiotic,Lactobacillus reuteri S5

Lactic acid bacteria are micro-organisms used for probiotic purposes and form major parts of human and mammalian intestinal microbiota, exerting important health-promoting effects on the host. Here, we evaluated Lactobacillus reuteri strain S5 isolated from the intestines of healthy white feather broilers. Lactobacillus reuteri S5 grew best after 20 h of incubation in MRS medium. Lactic acid production was 1·42 mmol l⁻¹ at 24 h, which was well tolerated. Activities of T-AOC, GSH-Px and T-SOD in the cell-free fermentation supernatant of L. reuteri S5 were higher than those in the bacteria, and the strain showed good hydrophobicity in vitro. The dominant carbon and nitrogen sources of L. reuteri S5 were glucose and soybean meal. A high-quality complete genome map of L. reuteri S5 was obtained using a Pacbio nanopore third-generation sequencing platform. The results showed that L. reuteri S5 possesses a complete primary metabolic pathway, encoding the main functional enzymes of the glycolysis pathway and pentose phosphate pathway. The genome contains genes encoding antioxidants and conferring tolerance to inorganic salt ions, acids and bile salts. This study shows that L. reuteri S5 is a probiotic strain with excellent probiotic characteristics and has great potential for the development of feed additives to promote animal health.     

Genome sequence of Lactobacillus salivarius SMXD51, a potential probiotic strain isolated from chicken cecum, showing anti-campylobacter activity

We report the draft genome sequence of Lactobacillus salivarius SMXD51, isolated from the cecum of healthy chickens showing an activity against Campylobacter--the food-borne pathogen that is the most common cause of gastroenteritis in the European Union (EU)--and potentially interesting features for a probiotic strain, explaining our interest in it.     

The biotechnological potential of subtilisin‐like fibrinolytic enzyme from a newly isolated Lactobacillus plantarum KSK‐II in blood destaining and antimicrobials

An antimicrobial oxidative‐ and SDS‐stable fibrinolytic alkaline protease designated as KSK‐II was produced by Lactobacillus plantarum KSK‐II isolated from kishk, a traditional Egyptian food. Maximum enzyme productivity was obtained in medium containing 1% lactose and 0.5% soybean flour as carbon and nitrogen sources, respectively. Purification of enzyme increased its specific activity to 1,140‐fold with a recovery of 33% and molecular weight of 43.6 kDa. Enzyme activity was totally lost in the presence of ethylenediaminetetraacetic acid and was restored after addition of Fe²⁺ suggesting that KSK‐II is a metalloprotease and Fe²⁺ acts as cofactor. Enzyme hydrolyzed not only the natural proteins but also synthetic substrates, particularly Suc‐Ala‐Ala‐Pro‐Phe‐pNA. KSK‐II can hydrolyze the Lys‐X easier than Arg‐X; thus, it was considered as a subtilisin‐family protease. Its apparent K_m, V_max, and K_cat were 0.41 mM, 6.4 µmol mg^?1 min^?1, and 28.0 s^?1, respectively. KSK‐II is industrially important from the perspectives of its maximal activity at 50°C (stable up to 70°C), ability to function at alkaline pH (10.0), stability at broad pH ranges (7.5–12.0) in addition to its stability toward SDS, H₂O₂, organic solvents, and detergents. We emphasize for the first time the potential of fibrinolytic activity for alkaline proteases used in detergents especially in blood destaining. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:316–324, 2015     

Design,synthesis, anticancer evaluation,and molecular modelling studies of novel tolmetin derivatives as potential VEGFR-2 inhibitors and apoptosis inducers

Novel tolmetin derivatives 5a–f to 8a–c were designed, synthesised, and evaluated for antiproliferative activity by NCI (USA) against a panel of 60 tumour cell lines. The cytotoxic activity of the most active tolmetin derivatives 5b and 5c was examined against HL-60, HCT-15, and UO-31 tumour cell lines. Compound 5b was found to be the most potent derivative against HL-60, HCT-15, and UO-31 cell lines with IC₅₀ values of 10.32 ± 0.55, 6.62 ± 0.35, and 7.69 ± 0.41 µM, respectively. Molecular modelling studies of derivative 5b towards the VEGFR-2 active site were performed. Compound 5b displayed high inhibitory activity against VEGFR-2 (IC₅₀ = 0.20 µM). It extremely reduced the HUVECs migration potential exhibiting deeply reduced wound healing patterns after 72 h. It induced apoptosis in HCT-15 cells (52.72-fold). This evidence was supported by an increase in the level of apoptotic caspases-3, -8, and -9 by 7.808-, 1.867-, and 7.622-fold, respectively. Compound 5b arrested the cell cycle in the G0/G1 phase. Furthermore, the ADME studies showed that compound 5b possessed promising pharmacokinetic properties.     

Antiapoptotic role of heme oxygenase (HO) and the potential of HO as a target in anticancer treatment

Heme oxygenase-1 (HO-1) is an inducible enzyme that catalyzes oxidative degradation of heme to form biliverdin, carbon monoxide (CO), and free iron. Biliverdin is subsequently reduced to bilirubin by the enzyme biliverdin reductase. Increasing evidence has indicated the critical role of HO-1 in cytoprotection and more diverse biological functions. Induction of HO-1 by various chemical inducers that are primarily cell stress inducers or by HO-1 gene transfection confers a protective capacity to cultured cells as well as to cells in several in vivo animal models. In addition, HO-1-deficient mice exhibit a significant increase in susceptibility to tissue injury. The cytoprotective action of HO-1 seems to be mainly a function of the antiapoptotic effects of the enzyme. HO-1 is believed to exert this antiapoptotic action by multiple mechanisms: (a) decreased intracellular pro-oxidant levels, (b) increased bilirubin levels, and (c) elevated CO production. CO may produce an antiapoptotic effect by inhibiting both expression of p53 and release of mitochondrial cytochrome c. HO-1 may also be a target in antitumor therapy because the growth of most tumors depends on HO-1. Our preliminary studies with an HO inhibitor showed a promising antitumor effect. This preliminary work warrants continued investigation for possible novel anticancer chemotherapy.     

Exo‐ and surface proteomes of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM is a well‐known probiotic bacterium extensively studied for its beneficial health effects. Exoproteome (proteins exported into culture medium) and surface proteome (proteins attached to S‐layer) of this probiotic were identified by using 2DE followed by MALDI TOF MS to find proteins potentially involved in bacteria–host interactions. The exo‐ and surface proteomes included 43 and 39 different proteins from 72 and 49 successfully identified spots, respectively. Twenty‐two proteins were shared between the two proteomes; both contained the major surface layer protein that participates in host interaction as well as several well‐known and putative moonlighting proteins. The exoproteome contained nine classically‐secreted (containing a signal sequence) and ten nonclassically‐secreted proteins, while the surface proteome contained four classically‐secreted and eight nonclassically secreted proteins. Identification of exo‐ and surface proteomes contributes describing potential protein‐mediated probiotic–host interactions.