Toxicological Evaluation of a Probiotic-Based Delivery System for P8 Protein as an Anti-Colorectal Cancer Drug.

PURPOSE: This study aimed to toxicological evaluate a probiotics-based delivery system for p8 protein as an anti-colorectal cancer drug. INTRODUCTION: Lactic acid bacteria (LAB) have been widely ingested for many years and are regarded as very safe. Recently, a Pediococcus pentosaceus SL4 (PP) strain that secretes the probiotic-derived anti-cancer protein P8 (PP-P8) has been developed as an anti-colorectal cancer (CRC) biologic by Cell Biotech. We initially identified a Lactobacillus rhamnosus (LR)-derived anti-cancer protein, P8, that suppresses CRC growth. We also showed that P8 penetrates specifically into CRC cells (DLD-1 cells) through endocytosis. We then confirmed the efficacy of PP-P8, showing that oral administration of this agent significantly decreased tumor mass (~42%) relative to controls in a mouse CRC xenograft model. In terms of molecular mechanism, PP-P8 induces cell-cycle arrest in G2 phase through down-regulation of Cyclin B1 and Cdk1. In this study, we performed in vivo toxicology profiling to obtain evidence that PP-P8 is safe, with the goal of receiving approval for an investigational new drug application (IND). METHODS: Based on gene therapy guidelines of the Ministry of Food and Drug Safety (MFDS) of Korea, the potential undesirable effects of PP-P8 had to be investigated in intact small rodent or marmoset models prior to first-in-human (FIH) administration. The estimated doses of PP-P8 for FIH are 1.0×1010 - 1.0×1011 CFU/person (60 kg). Therefore, to perform toxicological investigations in non-clinical animal models, we orally administered PP-P8 at doses of 3.375 × 1011, 6.75 × 1011, and 13.5×1011 CFU/kg/day; thus the maximum dose was 800-8000-fold higher than the estimated dose for FIH. RESULTS: In our animal models, we observed no adverse effects of PP-P8 on clinicopathologic findings, relative organ weight, or tissue pathology. In addition, we observed no inflammation or ulceration during pathological necropsy. CONCLUSION: These non-clinical toxicology studies could be used to furnish valuable data for the safety certification of PP-P8.

Encapsulation of Purified Pediocin of Pediococcus pentosaceus into Liposome Based Nanovesicles and its Antilisterial Effect.

AIMS: To encapsulate a purified bacteriocin into a nanovesicles and check its antibacterial effect. BACKGROUND: Although the use of nano-encapsulated bacteriocins in food matrices is poorly reported, encapsulated nisin can reduce L. monocytogenes counts in whole and skimmed milk and in soft cheese. OBJECTIVE: The present study deals with the extraction and purification of a bacteriocin from an isolated strain Pediococcus pentosaceus KC692718. A comparative study of the effect of free pediocin and liposome encapsulated pediocin against Listeria sp. was performed. METHODS: The purification of the extracted cell free supernatant was subjected to ammonium sulphate precipitation, cation exchange chromatography followed by gel permeation chromatography. The bacteriocin activity and protein concentration were determined using Lowry's method. The characterization of the pure pediocin was done. Liposome like nanovesicle was constructed and the stability of the liposome encapsulated pediocin was checked. Finally, the antibacterial effect was comparatively studied of the free pediocin, liposome, and liposome encapsulated pediocin simultaneously. RESULTS: The pediocin of 3.6kDa was purified with a specific activity of 898.8. AU/mg. It remained stable from pH 2.0-8.0 was found to be moderately stable above 80°C and remain stable for one month when stored at -20°C. The encapsulated pediocin showed stability since it retained 50% of its initial activity. The encapsulated pediocin showed 89% of encapsulation efficiency. CONCLUSION: The encapsulated pediocin not only improved pediocin stability but also enhanced the controlled release of the antimicrobial substances, enough for inhibiting the foodborne pathogen L. monocytogenes.

Combined effects of enzymes and probiotics on hemato-biochemical parameters and immunological responses of juvenile Siberian sturgeon (Acipenser baerii).

A 10-week feeding trial was run to investigate the separate and simultaneous effects of exogenous enzymes (Enz), probiotics (Pro), and Pro-Enz mixtures on the hematology indices, serum biochemical parameters, and innate-immunity status of juvenile Siberian sturgeon. The fish (138.06 ± 3.64 g) were randomly dispersed into 12 tanks (20 individuals per tank) and fed with Enz (Phytase, protease, and xylanase), Pro (Pediococcus pentosaceus and Lactococcus lactis), and Pro-Enz cocktail. At the end of the feeding bioassay, the highest values of red blood cell count, hemoglobin concentration, hematocrit level, and lymphocyte percentage followed by the lowest neutrophil percentage were obtained in Pro-Enz treatment (P < 0.05). Despite a significantly lower level of alkaline phosphatase in the fish fed with Pro supplemented diet (P < 0.05), no significant difference was found in the serum level of alanine aminotransferase and aspartate aminotransferase among the experimental groups (P > 0.05). Total protein content was significantly upregulated in serum and skin mucus samples from those fed with supplemented diets compared to the control group (P < 0.05). In both serum and skin mucus samples, higher immune responses in terms of lysozyme activity, immunoglobulin M, total protein was seen in Pro-Enz treatment compared to the control group followed by the serum complement components (P < 0.05). The results indicate that the combinational supplementation of Siberian sturgeon diet with the exogenous enzymes and probiotics modulates the physiometabolic responses and innate immune system to a higher grade than their individual supplementation.

Pediococcus pentosaceus, a future additive or probiotic candidate.

BACKGROUND: Pediococcus pentosaceus, a promising strain of lactic acid bacteria (LAB), is gradually attracting attention, leading to a rapid increase in experimental research. Due to increased demand for practical applications of microbes, the functional and harmless P. pentosaceus might be a worthwhile LAB strain for both the food industry and biological applications. RESULTS: As an additive, P. pentosaceus improves the taste and nutrition of food, as well as the storage of animal products. Moreover, the antimicrobial abilities of Pediococcus strains are being highlighted. Evidence suggests that bacteriocins or bacteriocin-like substances (BLISs) produced by P. pentosaceus play effective antibacterial roles in the microbial ecosystem. In addition, various strains of P. pentosaceus have been highlighted for probiotic use due to their anti-inflammation, anticancer, antioxidant, detoxification, and lipid-lowering abilities. CONCLUSIONS: Therefore, it is necessary to continue studying P. pentosaceus for further use. Thorough study of several P. pentosaceus strains should clarify the benefits and drawbacks in the future.

Antibacterial and antifungal activity of crude and freeze-dried bacteriocin-like inhibitory substance produced by Pediococcus pentosaceus.

Pediococcus pentosaceus LBM 18 has shown potential as producer of an antibacterial and antifungal bacteriocin-like inhibitory substance (BLIS). BLIS inhibited the growth of spoilage bacteria belonging to Lactobacillus, Enterococcus and Listeria genera with higher activity than Nisaplin used as control. It gave rise to inhibition halos with diameters from 9.70 to 20.00 mm, with Lactobacillus sakei being the most sensitive strain (13.50-20.00 mm). It also effectively suppressed the growth of fungi isolated from corn grain silage for up to 25 days and impaired morphology of colonies by likely affecting fungal membranes. These results point out that P. pentosaceus BLIS may be used as a new promising alternative to conventional antibacterial and antifungal substances, with potential applications in agriculture and food industry as a natural bio-controlling agent. Moreover, cytotoxicity and cell death induction tests demonstrated cytotoxicity and toxicity of BLIS to human colon adenocarcinoma Caco-2cells but not to peripheral blood mononuclear cells, with suggests possible applications of BLIS also in medical-pharmaceutical applications.

Physicochemical, bioactive and rheological properties of an exopolysaccharide produced by a probiotic Pediococcus pentosaceus M41.

This study aimed to investigate the physicochemical properties, health-promoting benefits and rheological properties of an EPS produced by a novel probiotic Pediococcus pentosaceus M41 isolated from a marine source. P. pentosaceus M41 was able to produce an EPS with average molecular weight of 682.07 kDa. EPS-M41 consisted of arabinose, mannose, glucose and galactose with a molar ratio of 1.2:1.8:15.1:1.0. EPS-M41 structure could be proposed as →3)α-D-Glc(1→2)ß-D-Man(1→2)α-D-Glc(1→6)α-D-Glc(1→4)α-D-Glc(1→4)α-D-Gal(1→ with arabinose linked at the terminals. At concentration of 10 mg.ml-1, the antioxidant capacity was 76.5% and 48.9% for DPPH and ABTS, respectively. EPS-M41 inhibited 86.8% and 90.8% of the α-amylase and α-glucosidase activities, respectively, at 100 µg.ml-1. A 77.5% and 46.4% of antitumor inhibition occurred by EPS-M41 against Caco-2 and MCF-7 cells, respectively. The apparent viscosity (ƞ) of all EPS-M41 solutions decreased with shear rate increases. Salt type and pH value had an impact on the rheological properties of EPS-M41.

Expression of coagulin A with low cytotoxic activity by Pediococcus pentosaceus ST65ACC isolated from raw milk cheese.

AIMS: We aimed to evaluate some specific conditions for growth of Pediococcus pentosaceus ST65ACC and its bacteriocin expression through ABC transporters; to purify the bacteriocin and determine its sequence; and to evaluate the cytotoxicity potential of the purified bacteriocin(s). METHODS AND RESULTS: The results presented for growth behaviour of P. pentosaceus ST65ACC showed that the bacterial growth was slightly influenced when cultured in MRS broth with different amounts of inoculum: 1, 2, 5 and 10%. The bacteriocin activity increased when 5 and 10% inocula were used. The carbon source (glucose) used in different amounts (1, 2, 3 or 4%) had no significant effect on growth and bacteriocin production. The studied strain P. pentosaceus ST65ACC was able to metabolize xylooligosaccharide (XOS) as the sole carbon source, resulting in the production of an antimicrobial peptide. The genes involved in the ABC transport system and sugar metabolism of P. pentosaceus ST65ACC were expressed at different levels. The bacteriocin produced by P. pentosaceus ST65ACC was partially purified by precipitation with ammonium sulphate (40% saturation), followed by reversed-phase liquid chromatography, resulting in the identification of an active bacteriocin. Tandem mass spectrometry was used to identify the partial sequence KYYGNGVTCGKHSCSVDWGK sharing high similarity to coagulin A. The semi-purified bacteriocin had low cytotoxicity based on estimated values for maximal nontoxic concentration (MNC) and cytotoxicity concentration (CC50 ). CONCLUSIONS: The bacteriocin produced by P. pentosaceus ST65ACC is similar to coagulin, with low cytotoxicity, strong antimicrobial activity and possible additional metabolite routes in the producer cell. In addition to MRS broth, bacteriocin was produced also in medium containing XOS (as the single carbon source). SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of evaluation of the role of ABC transporters in the expression of bacteriocin by P. pentosaceus, cultured in MRS and XOS.

Lactic acid bacteria, Enterococcus faecalis Y17 and Pediococcus pentosaceus G11, improved growth performance, and immunity of mud crab (Scylla paramamosain).

Mud crabs (Scylla paramamosain), a commercially important cultured species in the southeastern region of China, is usually infected by Vibriosis or parasites, causing great economic losses in cultured farms. Previous studies have demonstrated that probiotics benefited in enhancing the immune response against invading pathogens in aquatic animals. In this study, the effects of dietary administration of lactic acid bacteria (LAB) (Enterococcus faecalis Y17 and Pediococcus pentosaceus G11) on growth performance and immune responses of mud crab were assessed. Both strains (Y17 and G11) showed an inhibitory activity against bacterial pathogens (Aeromonas hydrophila, Vibrio parahaemolyticus, Vibrio alginolyticus, Staphylococcus aureus, and ß Streptococcus), and a wide pH tolerance range of 2-10. In vivo, mud crabs were fed a control diet and experimental diets supplemented with 109 cfu g-1 diet either Y17 or G11 for 6 weeks before subjecting to a challenge test with V. parahaemolyticus for 12 h. The probiotic-supplemented diets had significant effects on weight gain and specific growth rate during the feeding trial. Increased serum enzyme activities of phenoloxidase, lysozyme, and SOD were observed in the hemolymph of mud crab in Y17 and G11-supplemented groups compared to that in the controls (P < 0.01). The significantly up-regulated expression of gene CAT, LYS, proPO, and SOD could be seen in hepatopancreas in G11-supplemented groups. After the pathogenicity test, the survival rate of Y17 + and G11 + V. parahaemolyticus groups was 66.67% and 80.00%, respectively, compared with 53.33% for the control groups. Taken together, dietary supplementation of Y17 and G11 strains were beneficial in mud crab, which could increase growth performance, modulate immune system and protect the host against V. parahaemolyticus infection.

Biosorption of cadmium by potential probiotic Pediococcus pentosaceus using in vitro digestion model.

An exponential increase of heavy metal toxicant in the human body is a growing health-related problem. In the present study, Pediococcus pentosaceus FB145 and FB181 strains were isolated from fermented seafood and served as highly Cd-resistant strains. The unchanged structural cells of P. pentosaceus strains after treatment with Cd cations were investigated using a scanning electron microscope energy dispersive X-ray analysis. Furthermore, both P. pentosaceus strains showed strong human gastrointestinal tract resistance properties. The Cd biosorption results fit the pseudo-second-order kinetics with capacities for P. pentosaceus FB145 and FB181 for Cd were 52.8 and 50.3 mg/g, respectively, whereas the maximum biosorption capacities were 52.65 and 50.35 mg/g, respectively. The equilibrium data were well fitted to the Freundlich isotherm model. The binding of Cd to bacterial cells may be caused by the presence of different functional groups such as carboxyl, amide, and phosphate on cell surface, which was confirmed by the Fourier transform infrared-attenuated total reflectance spectra. Moreover, these strains decreased the Cd bioaccessibility by 44.7-46.8% in the in vitro digestion model. These findings indicate that P. pentosaceus FB145 and FB181 are novel potent biosorbent for preventing cadmium toxicity and reducing its absorption into the human body.

Purification and characterization of pediocin from probiotic Pediococcus pentosaceus GS4, MTCC 12683.

Pediococcus pentosaceus GS4 (MTCC 12683), a probiotic lactic acid bacterium (LAB), was found to produce bacteriocin in spent culture. Antibacterial and antagonistic potential of this bacteriocin against reference strains of Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 25619), and Listeria monocytogenes (ATCC 15313) was proven by double-layer and well diffusion methods wherein nisin and ampicillin were used as positive controls. Bacteriocin in supernatant was purified and analyzed by SDS-PAGE, RP-HPLC, and circular dichroism (CD). The physico-chemical properties of purified bacteriocin were characterized being treated at different temperatures (30 to 110 °C), pH (3.0 to 12.0), with different enzymes (α-amylase, pepsin, and lysozyme), and organic solvents (hexane, ethanol, methanol, and acetone) respectively. The molar mass of bacteriocin (named pediocin GS4) was determined as 9.57 kDa. The single peak appears at the retention time of 2.403 with area amounting to 25.02% with nisin as positive control in RP-HPLC. CD analysis reveals that the compound appears to have the helix ratio of 40.2% with no beta sheet. The antibacterial activity of pediocin GS4 was optimum at 50 °C and at pH 5.0 and 7.0. The pediocin GS4 was not denatured by the treatment of amylase and lysozyme but was not active in the presence of organic solvents. This novel bacteriocin thus m ay be useful in food and health care industry.

Membrane-acting bacteriocin purified from a soil isolate Pediococcus pentosaceus LB44 shows broad host-range.

Bacteriocin LB44 was purified from cell-free supernatant (CFS) of Pediococcus pentosaceus LB44 using activity-guided chromatography techniques. It was stable up to 121 °C, pH 2.0-6.0, sensitive to proteinase K, papain and trypsin, and retained complete activity in the presence of organic solvents tested. The molecular weight of bacteriocin was ∼6 kDa and initial ten amino acid residues (GECGMCXECG) suggested a new compound. The loss in viable cell count and K+ ion efflux of target cells of Micrococcus luteus suggested bactericidal activity. The cell membrane of bacteriocin-treated cells was found to be ruptured which was further confirmed by Fourier Transform Infrared (FTIR) analysis suggesting interaction of bacteriocin with phospholipids in cell membrane. It showed broad host-range and inhibited the growth of Lactobacillus delbrueckii NRRL B-4525, L. plantarum NRRL B-4496, L. acidophilus NRRL B-4495, Enterococcus hirae LD3, Weissella confusa LM85, Staphylococcus aureus, Salmonella typhi ATCC 13311, Serratia marcescens ATCC 27137, Pseudomonas aeruginosa ATCC 27853, Proteus vulgaris ATCC 29905, Haloferax larsenii HA1, HA3, HA8, HA9 and HA10. These properties suggested a new bacteriocin from soil isolate P. pentosaceus LB44 which may offers possible applications in food-safety and therapeutics.

Isolation of Endotoxin Eliminating Lactic Acid Bacteria and a Property of Endotoxin Eliminating Protein.

Recently, many scholars have reported lactic acid bacteria (LAB) functions, such as anticancer activity and anti-inflammatory activity for intestines. To decrease inflammatory substances such as endotoxins, LAB consumed safely with meals were isolated from food and food ingredients. First, LAB were isolated as 168 strains of bacillus LAB (49 strain) and coccus LAB (119 strains) from food ingredients and fermented foods such as rice, rice bran, malt, grains, miso soy paste, and some pickles. Their LAB (168 strains) were cultivated in medium containing endotoxin from Escherichia coli O18 LPS at 15 and 30 °C for 64 h to identify endotoxin-eliminating LAB. Consequently, the AK-23 strain was screened as an endotoxin-eliminating LAB strain. The strain decreased endotoxin in YP medium without sugar at 30 °C for 64 h until 9% of endotoxin. The strain was identified as Pediococcus pentosaceus according to morphological characteristics such as its cell shape, physiological characteristics related to its fermentation type, assimilation of sugars, pH tolerance, optimum growth temperature, and molecular biological characteristics as its homology to 16S rRNA. To investigate the location of the endotoxin-eliminating substance, 4 fractions were separated from AK-23 cells as extracellular, cell wall digestion, cytoplasm, and cell membrane fractions. The endotoxin-decreasing substance, located on a cell wall, was identified as a 217 kDa protein.

Exopolysaccharide fraction from Pediococcus pentosaceus KFT18 induces immunostimulatory activity in macrophages and immunosuppressed mice.

AIMS: Exopolysaccharide fraction from Pediococcus pentosaceus KFT18 (PE-EPS), a lactic acid bacteria isolated from Kimchi (a Korean fermented vegetable product), was preliminary characterized and its immunostimulating effects were analysed. METHODS AND RESULTS: In this study, we used interferon-γ (IFN-γ)-primed RAW 264·7 macrophages and CD3/CD28-stimulated splenocytes to determine the immunotimulatory activities of PE-EPS. Upon exposure to PE-EPS, IFN-γ-primed RAW 264·7 macrophages showed significant increases in the expressions of inducible nitric oxide synthase (iNOS), tumour necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß. Molecular data using reporter gene assay and electrophoretic mobility shift assay (EMSA) revealed that PE-EPS upregulated transcriptional activity, DNA binding and the nuclear translocation of nuclear factor-κB (NF-κB). Furthermore, PE-EPS enhanced anti-CD3/CD28-specific proliferation and the productions of IL-2 and IFN-γ in primary splenocytes. In cyclophosphamide-induced immunosuppressed mice, pretreatment with PE-EPS (5, 15 or 45 mg kg(-1) day(-1), p.o.) increased thymus and spleen indices, and improved lymphocyte and neutrophil counts. CONCLUSION: PE-EPS stimulated the IFN-γ-primed macrophages and primary splenocytes to induce immune responses and improved the cyclophosphamide-induced immunosuppression in mice. SIGNIFICANCE AND IMPACT OF THE STUDY: The results in this study improved our understanding of immunostimulating activity of PE-EPS and supported its potential treatment option as a natural immunostimulant.