Potential bioactivities via anticancer, antioxidant, and immunomodulatory properties of cultured mycelial enriched ß-D-glucan polysaccharides from a novel fungus Ophiocordyceps sinensis OS8.

Background: Ophiocordyceps sinensis is well-known worldwide as a traditional medicine. An alternative natural source of O. sinensis is provided by mycelial cultivation. However, the bioactivities of cultured mycelial-enriched ß-D-glucan polysaccharides from a novel fungus O. sinensis OS8 are still unknown. Methods: We investigated the potential bioactivities via anticancer, antioxidant, and immunomodulatory polysaccharides (OS8P) produced from cultured mycelia of O. sinensis OS8. This strain is a novel fungus isolated from natural O. sinensis, which is further cultured by submerged mycelial cultivation for polysaccharide production. Results: The yield of mycelial biomass was 23.61 g/l, and it contained 306.1 mg/100 g of adenosine and 3.22 g/100 g of polysaccharides. This OS8P was enriched with ß-D-glucan at 56.92% and another form of α-D-glucan at 35.32%. The main components of OS8P were dodecamethyl pentasiloxane, 2,6-bis (methylthiomethyl) pyridine, 2-(4-pyrimidinyl)-1H-Benzimidazole, and 2-Chloro-4-(4-nitroanilino)-6-(O-toluidino)-1,3,5-triazine at the rates of 32.5, 20.0, 17.5, and 16.25%, respectively. The growth of colon cancer cells (HT-29) was significantly inhibited by OS8P, with IC50 value of 202.98 µg/ml, and encouraged apoptosis in HT-29 cells as confirmed by morphological change analysis via AO/PI and DAPI staining, DNA fragmentation, and scanning electron microscopic observations. In addition, significant antioxidant activity was demonstrated by OS8P through DPPH and ABTS assays, with IC50 values of 0.52 and 2.07 mg/ml, respectively. The OS8P also exhibited suitable immunomodulatory activities that significantly enhanced (P< 0.05) the induction of splenocyte proliferation. Conclusion: The OS8P enriched with ß-D-glucan polysaccharides and produced by submerged mycelial culture of a new fungal strain of O. sinensis OS8 strongly inhibited the proliferation of colon cancer cells without any cytotoxicity against normal cells. The potential effect of the OS8P on the cancer cells was due to the stimulation of apoptosis. Also, the OS8P exhibited good antioxidant and immunomodulatory activities. The results indicate that OS8P has promising applications in the functional food industry and/or therapeutic agents for colon cancer.

Bioactive Earthworm Peptides Produced by Novel Protease-Producing Bacillus velezensis PM 35 and Its Bioactivities on Liver Cancer Cell Death via Apoptosis, Antioxidant Activity, Protection Against Oxidative Stress, and Immune Cell Activation.

Earthworms have long been used as traditional medicine. The purposes of this research were to create bioactive peptides from the unique Amynthas arenulus earthworm (PAAEs) and test their potentials on liver cancer bioprophylactic activity, antioxidant, oxidative stress protection, and immune cell activation. This earthworm had a high protein content ratio, at 55.39%. Besides, PM 35 is one out of 58 bacteria isolated from the earthworm carcasses that exhibited the highest protease and yield protein production which was chosen as the protease-producing bacteria to hydrolyze the protein. The genera were identified by 16S rRNA and 16S-23S rRNA comparison and confirmed as Bacillus velezensis PM 35. The response surface methodology was applied to optimize these hydrolysis parameters, i.e., the enzyme/substrate (E/S) concentration ratio [1%-3% (v/v)] and time (1-3 h) of the hydrolyzing earthworm's proteins. The optimal hydrolyzing conditions were 3% (v/v) of E/S concentration ratio and 3 h of hydrolysis time, which found protein-hydrolysate yield (24.62%) and degree of hydrolysis (85.45%) as the highest. After being challenged in the gastrointestinal tract-resistant model, these PAAEs (MW <3 and 3-5 kDa) induced liver cancer cell (HepG2) death via apoptotic action modes (cell morphological change and DNA fragmentation). The PAAEs (MW <3 kDa) exhibited significant antioxidant activity via DPPH, ABTS, and FRAP with IC50 values of 0.94, 0.44, and 6.34 mg/ml, respectively. The PAAEs (MW < 3 kDa) were non-cytotoxic and protected the mouse fibroblast cells (L929) against oxidative stress. These PAAEs (MW < 3 kDa, 0.2 mg/ml) stimulated the B lymphocytes (122.3%), and T lymphocytes (126.7%) proliferation. This research suggests that PAAEs can be used in a variety of applications, especially in the food and pharmaceutical industries.

Bioprophylactic potential of novel human colostrum probiotics via apoptotic induction of colon cancer cells and cell immune activation.

The potential and modes of action of novel human colostrum probiotics were investigated with the aim of studying their application as an alternative in the bioprophylactic and biotherapy of colon cancer. A total of 218 isolates of Gram-positive rod and cocci bacteria obtained from the colostrum of 50 healthy lactating females were collected. Beneficial probiotic criteria from these isolates, which included growth inhibition against seven foodborne pathogens (Helicobacter pylori, Escherichia coli, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes), no blood haemolysis and tolerance to acid and bile salt conditions, resulted in only eight probiotic bacteria successfully inhibiting the proliferation of colon cancer cells at rates of 32.47-61.21%. Two probiotic bacteria with higher anticancer efficacy (Streptococcus salivarius CP163 and S. salivarius CP208) were identified using 16S rRNA sequences. High rates of cell surface hydrophobicity, autoaggregation and coaggregation were obtained from both probiotics. The probiotic mode of action involved synergic probiotic adhesion to colon cancer cells that triggered SCFA bioproduction. Apoptotic induction of colon cancer cell death through caspase-2 activity, DNA fragmentation and morphological change as assessed by AO/PI staining were also observed. Immune stimulation by S. salivarius CP163 and S. salivarius CP208 resulted in B and T-cell lymphocyte activation. This study suggests that these novel human colostrum probiotics could be applied as a functional food to facilitate a bioprophylactic strategy in colon cancer.

Influences of Bacillus subtilis and fructooligosaccharide on growth performances, immune responses, and disease resistance of Nile tilapia, Oreochromis niloticus.

The present study investigated the effects of Bacillus subtilis and fructooligosaccharide (FOS) on growth performances, immunity improvement, and disease resistance of Nile tilapia (Oreochromis niloticus). The fish (24.5 ± 1.6 g) were fed a basal diet (G1), diets supplemented with 1 g/kg (G2), 3 g/kg (G3) and 5 g/kg (G4) of FOS as well as diets supplemented with 1 × 109 CFU/g (G5), 3 × 109 CFU/g (G6) and 5 × 109 CFU/g (G7) of B. subtilis for 56 days. After the feeding trial, the complement C3, IL-1ß, TNF-α, IFN-γ, hsp70 gene expression in the liver was then analyzed by a quantitative Real-time PCR. Then, fish were infected with Streptococcus agalactiae, and the survival rate was recorded. The results showed that FOS and B. subtilis had no significant effect (P > 0.05) on growth performances and survival rate. Lysozyme activity was significantly greater in the G4, G5, G6, and G7 groups. Also, all fish fed FOS and B. subtilis showed significantly (P < 0.05) higher respiratory burst activity than other groups. The expressions of complement C3, IL-1ß, TNF-α, IFN-γ, and hsp-70 in the liver were significantly higher for fish fed 5 g/kg of FOS as well as for fish that received any concentration level of B. subtilis (P < 0.05) used in the study. After the S. agalactiae challenge test, the survival rate of fish-fed diets supplemented with FOS and B. subtilis was slightly higher than for the control group. The results indicated that FOS and B. subtilis could stimulate immune responses and immune-related genes in tilapia. However, further investigation of other prebiotics or herbs in combination with B. subtilis is encouraged at molecular levels and screening for beneficial metabolites that may increasingly improve digestive enzymes, growth performances, and health benefits in tilapia. In addition, on-farm experiments are needed.

Characterization of Streptococcus salivarius as New Probiotics Derived From Human Breast Milk and Their Potential on Proliferative Inhibition of Liver and Breast Cancer Cells and Antioxidant Activity.

Breast milk is well known as the abundant source of beneficial bacteria. A new alternative source of human probiotic origin from breast milk is in demand and currently of interest for both the functional food industry and biopharmaceuticals. The aim in this study was to investigate the anticancer and antioxidant efficacies of the new potential probiotics isolated from human breast milk. Three strains of lactic acid bacteria (LAB) have shown their potential probiotic criteria including antimicrobial activity, non-hemolytic property, and survival in acid and bile salt conditions. These strains showed high abilities on cell surface hydrophobicity, auto-aggregation, and co-aggregation. The genera identification by 16S rRNA sequencing and comparison revealed that they were Streptococcus salivarius BP8, S. salivarius BP156, and S. salivarius BP160. The inhibition of liver cancer cells (HepG2) and breast cancer cells (MCF-7) proliferation by these probiotic strains using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was 44.83-59.65 and 29.85-37.16%, respectively. The probiotic action mode was inducted via apoptotic mechanisms since they stimulate the liver and breast cancer cell death through DNA fragmentation and positive morphological changes by acridine orange (AO) and propidium iodide (PI) staining. The antioxidant activity of these probiotics in the form of intact cells, cell free supernatant (CFS), and heat-killed cells was evaluated by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, resulting in the scavenging activity rates of 16.93-25.43, 15.47-28.03, and 13.67-23.0%, respectively. These S. salivarius probiotic strains protected the L929 mouse fibroblasts against oxidative stress with very high survival rates at 94.04-97.77%, which was significantly higher (P < 0.05) than L-ascorbic acid at 75.89-78.67% in the control groups. The results indicated that S. salivarius BP8 and S. salivarius BP160 probiotic strains could be applied as functional foods or new alternative bioprophylactics for treating liver and breast cancers.

Potential probiotic lactic acid bacteria of human origin induce antiproliferation of colon cancer cells via synergic actions in adhesion to cancer cells and short-chain fatty acid bioproduction.

The activities and modes of probiotic action of lactic acid bacteria isolated from infant feces were investigated for alternative application in the prevention and biotherapy of colon cancer. From a total of 81 isolates of Gram-positive rod and cocci bacteria obtained from healthy infants, only 15 isolates had the probiotic criteria which included growth inhibition against eight food-borne pathogens, no blood hemolysis, and tolerance to gastrointestinal tract properties such as pH 2.5 and 0.3 % bile salt. Four probiotic bacteria showed antiproliferation of colon cancer cells with the use of MTT and Trypan blue exclusion assay at the rates of 17-35 %. Through comparison of probiotic 16S rRNA sequences, they were identified as Pediococcus pentosaceus FP3, Lactobacillus salivarius FP25, L. salivarius FP35, and Enterococcus faecium FP51. Finding the mechanism of proliferative inhibition of colon cancer cells in this study indicated synergic induction by probiotic bacteria directly adhered to these cancer cells and triggered the bioproduction of short-chain fatty acids, mainly butyric and propionic acids. This study suggested that the use of these probiotics may be suitable as an alternative bioprophylactic and biotherapeutic strategy for colon cancer.

Protection activity of a novel probiotic strain of Bacillus subtilis against Salmonella Enteritidis infection.

The activity of 240 bacterial isolates screened from the gastrointestinal tracts of native chickens were evaluated for use as a potential probiotic in food animal production in order to protect against animal diseases and reduce pathogenic contamination of human food products. In observing the antagonistic activity of 117 bacilli isolates, 10 of these isolates exhibited higher growth inhibition of seven foodborne pathogens, including Salmonella Enteritidis, Salmonella Typhimurium, Escherichia coli, Bacillus cereus, Staphylococcus aureus, Listeria monocytogenes, and Vibrio cholerae. Beneficial probiotic criteria from these isolates - which included non-pathogenicity, acid and bile salt tolerance, hydrophobicity, and adhesion to intestinal epithelial cells - exhibited that one isolate of NC11 had the most potential as a probiotic. 16S rRNA gene sequencing showed that this NC11 isolate was Bacillus subtilis. This B. subtilis NC11 was sensitive to all antibiotics and was not cytotoxic to intestinal epithelial cells. Reduction of S. Enteritidis attachment to the surfaces of intestinal epithelial cells via action of a cultured medium from B. subtilis NC11 was observed by scanning electron microscopy. B. subtilis NC11 cells, as well as the bacterial cultured medium or the cultured medium adjusted to pH 7, significantly inhibited S. Enteritidis invasion (P<0.01) of intestinal epithelial cells. This study indicates that B. subtilis NC11 has characteristics of a potential probiotic, and exhibits strong inhibition activity against S. Enteritidis infection to intestinal epithelial cells.

Probiotic potential of lactic acid bacteria isolated from fermented dairy milks on antiproliferation of colon cancer cells.

Fifty-four strains of lactic acid bacteria obtained from fermented dairy milks were investigated for possible use as probiotics and for colon cancer biological products. Five of these strains inhibited growth of eight food-borne pathogens including Helicobacter pylori, Escherichia coli, and Salmonella typhimurium. Three of these strains survived at pH 2.5 and in 0.3% bile salts. Additionally they produced no haemolysis, were resistant to kanamycin and adhered to Caco-2 cells. 16S rRNA gene sequences of probiotic strains indicated that RM11 and RM28 were Enterococcus faecium and Lactobacillus fermentum, respectively. Both the cultured medium and live whole cells from probiotic strains were tested for antiproliferation of colon cancer cells through MTT and Trypan Blue exclusion assays. The probiotic strains of E. faecium RM11 and L. fermentum RM28 also triggered antiproliferation of colon cancer cells at the rates of 21-29%, and 22-29%, respectively. This suggested that both strains could be used as potential probiotics in functional food or for colon cancer biological products.