Ecology, Diversity, Antibiotic Resistance Of Lactic Acid Bacteria (LAB) Isolated From Plant Sources And Their Role In Natural Fermentation Of Tempoyak

In recent years, bioprospecting of lactic acid bacteria (LAB) from plant sources contributes to the diversity of LAB and the discovery of novel application of LAB in food industries. As LAB play an important role in food fermentation, there are concern of dissemination of antibiotic resistance determinants harboured by the indigenous microbiota present in fresh fruits and fermented food through the food chain. The objectives of this study were to investigate the diversity and antibiotic resistance of LAB presence in fresh tropical fruits and flowers, as well as tempoyak produced by natural fermentation; to characterise the bacteriocin-like substances (BLIS) produced by these LAB isolates, and other technological properties

Cytotoxic and Antiproliferative Properties of Metabolites Produced by Six Strains of Lactobacillus Plantarum on Human Cancer Cells

Whole cells, cytoplasmic fractions and fermented products of LAB have been tested for anticancer effect. However, limited information is available for the metabolites produced by Lactobacillus plantarum. In this study, the anticancer effect of metabolites produced by six strains of L. plantarum (UL4, TL1, RS5, RG14, RG11 and RI11) isolated from Malaysian fermented foods was evaluated. All metabolites exhibited in vitro cytotoxic effect on the tested cancer cells (breast, colorectal, cervical, liver and leukemia cancer cell lines). An increased cytotoxic effect was observed with increased dose of metabolites used and time of incubation. In particular, metabolites UL4 exerted the most potent cytotoxicity against human breast carcinoma cells MCF-7 in a dose- and time-dependent manner in MTT assay, with inhibition concentration of 50 % growth (IC50) value of 15, 12 and 10% (v/v) for 24, 48 and 72 hours of incubation, respectively. In contrast, no cytotoxicity was detected in primary human peripheral blood mononuclear cells, mouse splenocytes, thymocytes and bone marrow cells for all the six metabolites tested. However, limited cytotoxicity was detected in nonmalignant human glandular epithelium cells MCF-10A when treated with UL4 and RG14 metabolites. Additionally, UL4 metabolites did not cause haemolysis, indicating cytotoxic effect of metabolites of six strains of L. plantarum is selective for malignant cells but spared on normal cells. Antiproliferative effect was focused on MCF-7 and colon cancer cell line (HT-29). In BrdU cell proliferation assay, all tested metabolites inhibited DNA synthesis of MCF-7 and HT-29 cells. An increased antiproliferative effect was observed with increased dose of metabolites used and time of incubation. In particular, UL4 metabolites exhibited 100% proliferation inhibition on MCF-7, whereas RG14 metabolites exhibited 89% proliferation inhibition on HT-29 for 72 hours of incubation. Growth arrest study showed significant cell growth inhibition (P < 0.05) in MCF-7 treated with UL4 metabolites and HT-29 cells treated with RG14 metabolites. Mode of cell death induced by UL4 metabolites on MCF-7 cells was elucidated. Results obtained in trypan blue dye exclusion assay suggested that UL4 metabolites did not cause necrosis. Induction of apoptosis rather than necrosis by UL4 metabolites was evident by the presence of most characteristics of apoptosis such as cell shrinkage, blebbing of cell membrane and fragmentation of DNA and nucleus. Annexin V/PI staining showed that substantial early apoptotic cells were detected in MCF-7 cells treated with UL4 metabolites compared to untreated control group. Cells treated with UL4 metabolites showed growth arrest at G0/G1 cell phase at 24 hours, followed by the increment of cells in sub-G0/G1 in DNA cell cycle analysis. In addition, the TUNEL assay showed that remarkable TUNEL-positive cells were detected in UL4 metabolites-treated MCF-7 cells. The results obtained in this study indicate the potential use of LAB metabolites as a promising antiproliferative and apoptosis induction agent as an alternative in nutraceutical industry and cancer therapy

Cytotoxic effects of commercial wheatgrass and fiber towards human acute promyelocytic leukemia cells (HL60).

Cytotoxicity, the possible selective activity upon HL60 as well as the anti-proliferation effect of local health supplement wheatgrass and mixture of fibers were investigated in vitro using various cancerous cell line and normal blood cell culture. The IC(50) of wheatgrass-treated HL60 (17.5 ± 1.1, 12.5 ± 0.3, and 16 ± 0.5 microgram/ml for 24, 48 and 72 h, respectively) and fibers-treated HL60 (86.0 ± 5.5, 35.0 ± 2.5, and 52.5 ± 4.5 microgram/ml for 24, 48 and 72 h, respectively) showed that both extracts possessed optimum effect after 48 hours of treatment. No significant cytotoxic effect was observed on other type of cells. For trypan blue dye exclusion method, wheatgrass reduced the number of viable cells by 13.5% (±1.5), 47.1% (±3.6), and 64.9% (±2.7) after 24, 48 and 72 h exposure, respectively. Mixture of fibers reduced the number of viable cells by 36.4% (±2.3), 57.1% (±3.1), and 89.0% (±3.4) after 24, 48 and 72 h exposure, respectively, indicated that necrosis is also an alternative to the apoptotic mechanism of cell death. Annexin-V/propidium iodide staining revealed that both extracts induced apoptosis where early apoptosis had been detected concurrently with the reduction of percentage of cell viability. Cell cycle analysis revealed that in HL60, the percentage of apoptosis increased with time (wheatgrass: 16.0% ± 2.4, 45.3% ± 3.4 and 39.6% ± 4.1; mixture of fibers: 14.6% ± 1.8, 45.4% ± 2.3 and 45.9% ± 1.2) after exposure for 24, 48 and 72 h, respectively at the concentration of 100 microgram/ml and showed optimum effect at 48 hours. Thus, these health products can be a potential alternative supplement for leukaemia patients

Updates on Antiobesity Effect of Garcinia Origin (−)-HCA

Garcinia is a plant under the family of Clusiaceae that is commonly used as a flavouring agent. Various phytochemicals including flavonoids and organic acid have been identified in this plant. Among all types of organic acids, hydroxycitric acid or more specifically (−)-hydroxycitric acid has been identified as a potential supplement for weight management and as antiobesity agent. Various in vivo studies have contributed to the understanding of the anti-obesity effects of Garcinia/hydroxycitric acid via regulation of serotonin level and glucose uptake. Besides, it also helps to enhance fat oxidation while reducing de novo lipogenesis. However, results from clinical studies showed both negative and positive antiobesity effects of Garcinia/hydroxycitric acid. This review was prepared to summarise the update of chemical constituents, significance of in vivo/clinical anti-obesity effects, and the importance of the current market potential of Garcinia/hydroxycitric acid

Prevalence of Salmonella in poultry processing environments in wet markets in Penang and Perlis, Malaysia

Aim: The aim of this study was to determine the prevalence of various Salmonella serotypes in chickens, carcass contact surfaces as well as environmental samples collected from wet markets and small scale processing plant. Materials and Methods: A total of 182 poultry and environmental samples were collected at random on separate occasions from wet markets and small scale processing plant, during the period of October 2014 to July 2015 in Penang and Perlis, Malaysia. The samples were analyzed for the presence of Salmonella using ISO 6579:2002 conventional culture-based method. Presumptive Salmonella colonies were subjected to various biochemical tests (such as triple sugar iron and lysine iron test), serologically confirmed using polyvalent O and H antisera and further serotyped at Public Health Laboratory, Ministry of Health, Perak, Malaysia. Results: Salmonella serotypes were isolated from 161 out of 182 samples (88.46%) with 100% prevalence in the whole chicken carcass and chicken cuts - as well as transport crate, cage, drum, knife, chopping board, display table, floor, bench wash water, wash water, and drain water. Salmonella was isolated from 91.67%, 83.33%, and 66.67% of defeathering machines, drain swabs, and apron, respectively. 17 serotypes were isolated in this study with Salmonella Albany (57/161), Salmonella Corvallis (42/161), and Salmonella Brancaster (37/161) being the predominant serovars. Conclusion: The most carcass contact and environmental samples collected along the wet market chicken processing line were consistently contaminated with Salmonella. This indicates that Salmonella has established itself in poultry processing environments by colonizing the surfaces of the equipment and survives in these environments by establishing biofilms. Our results highlight the need of implementing strict hygiene and sanitation standards to reduce the incidence of Salmonella. The prevalence of Salmonella in poultry can be reduced effectively by identifying and eliminating the sources and contamination sites during slaughter and processing of poultry

In Vitro and In Vivo Toxicity of Garcinia or Hydroxycitric Acid: A Review

Obesity is one of the pandemic chronic diseases commonly associated with health disorders such as heart attack, high blood pressure, diabetes or even cancer. Among the current natural products for obesity and weight control, Garcinia or more specifically hydroxycitric acid (HCA) extracted from Garcinia has been widely used. The evaluation of the potential toxicity of weight control supplement is of the utmost importance as it requires long term continuous consumption in order to maintain its effects. Majority of reports demonstrated the efficacy of Garcinia/HCA without any toxicity found. However, a few clinical toxicity reports on weight-loss diet supplements of which some were combinations that included Garcinia/HCA as an active ingredient showed potential toxicity towards spermatogenesis. Nonetheless, it cannot be concluded that Garcinia/HCA is unsafe. Those products which have been reported to possess adverse effects are either polyherbal or multi-component in nature. To date, there is no case study or report showing the direct adverse effect of HCA. The structure, mechanism of action, long history of the use of Garcinia/HCA and comprehensive scientific evidence had shown &quot;no observed adverse effect level (NOAEL)&quot; at levels up to 2800 mg/day, suggesting its safety for use

Postbiotic metabolites produced by Lactobacillus plantarum strains exert selective cytotoxicity effects on cancer cells

Background: Lactobacillus plantarum, a major species of Lactic Acid Bacteria (LAB), are capable of producing postbiotic metabolites (PM) with prominent probiotic effects that have been documented extensively for rats, poultry and pigs. Despite the emerging evidence of anticancer properties of LAB, very limited information is available on cytotoxic and antiproliferative activity of PM produced by L. plantarum. Therefore, the cytotoxicity of PM produced by six strains of L. plantarum on various cancer and normal cells are yet to be evaluated. Methods: Postbiotic metabolites (PM) produced by six strains of L. plantarum were determined for their antiproliferative and cytotoxic effects on normal human primary cells, breast, colorectal, cervical, liver and leukemia cancer cell lines via MTT assay, trypan blue exclusion method and BrdU assay. The toxicity of PM was determined for human and various animal red blood cells via haemolytic assay. The cytotoxicity mode was subsequently determined for selected UL4 PM on MCF-7 cells due to its pronounced cytotoxic effect by fluorescent microscopic observation using AO/PI dye reagents and flow cytometric analyses. Results: UL4 PM exhibited the lowest IC50 value on MCF-7, RG14 PM on HT29 and RG11 and RI11 PM on HL60 cell lines, respectively from MTT assay. Moreover, all tested PM did not cause haemolysis of human, dog, rabbit and chicken red blood cells and demonstrated no cytotoxicity on normal breast MCF-10A cells and primary cultured cells including human peripheral blood mononuclear cells, mice splenocytes and thymocytes. Antiproliferation of MCF-7 and HT-29 cells was potently induced by UL4 and RG 14 PM respectively after 72 h of incubation at the concentration of 30% (v/v). Fluorescent microscopic observation and flow cytometric analyses showed that the pronounced cytotoxic effect of UL4 PM on MCF-7 cells was mediated through apoptosis. Conclusion: In conclusion, PM produced by the six strains of L. plantarum exhibited selective cytotoxic via antiproliferative effect and induction of apoptosis against malignant cancer cells in a strain-specific and cancer cell type-specific manner whilst sparing the normal cells. This reveals the vast potentials of PM from L. plantarum as functional supplement and as an adjunctive treatment for cancer

Characterization of lactic acid bacteria isolated from Malaysian foods

Lactic Acid Bacteria (LAB)are a group of Gram positive bacteria,which are industrially important microorganisms due to their vast applications in various industries. In this study, 10 LAB isolates namely CB1, I-1, OMR1, TB-1, UP2, TP5, GP8, RW18, B12-M9 and Big-2-Y isolated from Malaysian foods were characterized phenol typically and genotypically. The genotypic characterization technique employed in this study was 16SrDNA-Polymerase Chain Reaction amplification, where a pair of universal primers: 8FPL16S and 149216S was used to amplify the 16S rDNA. The amplicon with approximate size of 1.5kb was then purified and analysed for DNA sequence. Based on the results of 16Sr DNA sequence, the isolates of CB1,TB-1 and UP2 were identified as Pediococcus acidilactici, whereas isolates of I-1, OMR1 and B12-M-9 were identified as Pediococcus pentosaceus and the other 4 isolates namely TP5, GP8, RW18 and Big-2-Y were identified as Lactobacillus plantarum with 100% homology. Carbohydrate fermentation profiles were determined for phenotypic characteristics, where the results of API50CHL kits showed more than 90% homology for all tested isolates, which were in agreement with the results of 16Sr DNA analyses. Antibiotic resistance profile was also determined according to the Kirby-Baueragar diffusion test using antibiotic impregnated disks. All tested LAB isolates showed resistance to nalidixic acid, streptomycin, vancomycin, gentamycin, kanamycin and bacitracin. However, they were sensitive to tetracycline, chloramphenicol, erythromycin and penicillin G. It is important to determine the antibiotic resistance profile of LAB isolates prior to their application in respective industry since the results in his study showed that some LAB isolates are resistant to several antibiotics being used commonly in livestock industry as growth promoter and disease treatment

In Vitro

Obesity is one of the pandemic chronic diseases commonly associated with health disorders such as heart attack, high blood pressure, diabetes or even cancer. Among the current natural products for obesity and weight control, Garcinia or more specifically hydroxycitric acid (HCA) extracted from Garcinia has been widely used. The evaluation of the potential toxicity of weight control supplement is of the utmost importance as it requires long term continuous consumption in order to maintain its effects. Majority of reports demonstrated the efficacy of Garcinia/HCA without any toxicity found. However, a few clinical toxicity reports on weight-loss diet supplements of which some were combinations that included Garcinia/HCA as an active ingredient showed potential toxicity towards spermatogenesis. Nonetheless, it cannot be concluded that Garcinia/HCA is unsafe. Those products which have been reported to possess adverse effects are either polyherbal or multi-component in nature. To date, there is no case study or report showing the direct adverse effect of HCA. The structure, mechanism of action, long history of the use of Garcinia/HCA and comprehensive scientific evidence had shown “no observed adverse effect level (NOAEL)” at levels up to 2800 mg/day, suggesting its safety for use

Data on antibiogram and resistance genes harboured by Salmonella strains and their Pulsed-field gel electrophoresis clusters

This article describes the Pulsed-field gel electrophoresis clustering of the predominant Salmonella strains (Salmonella ser. Albany, Salmonella ser. Brancaster, and Salmonella ser. Corvallis) isolated from poultry and processing environment in wet market and small-scale processing plant in Penang and Perlis, the northern states of Malaysia. Agar disk diffusion assay was performed to determine the phenotypic antibiotic resistance of these Salmonella strains. The most common antibiograms among the three predominant Salmonella serovars were reported. The presence of integrase genes and antibiotic resistance genes conferring to resistance against β-lactams, aminoglycosides, tetracyclines, quinolones, sulphonamides and chloramphenicol, was detected via PCR amplification. Keywords: Multidrug resistance, Salmonella, Pulsed-field gel electrophoresis, Antibiotic resistance gene, Poultr