ß-Glucan bioactivities from Cystobasidium benthicum in Totoaba macdonaldi thymus cells.

The overuse of antibiotics in aquaculture has led to serious concerns on microbial resistance and chemical residues. Novel sources of immunostimulants could help to solve this problem by stimulating the immune system to fight against pathogens. Therefore, this study aims to explore the immunostimulant potential of Cystobacidium benthicum-ß-glucans (Cb-ßG) using thymus cells from Totoaba (Totoaba macdonaldi), a recently farmed fish species in Mexico. The Cb-ßG was characterized and tested for its own antioxidant capacity. Then, a Cb-ßG safety experiment was carried out in thymus cells by evaluating the effects on immune parameters and immune-related genes. Cb-ßG had a molecular weight of 2.32 kDa, comprised of ß-1,3-1,6-glucan (53.4%), and showed strong antioxidant capacity compared to that of the positive antioxidant control. Cb-ßG had no toxic effects of thymus cells and enhanced phagocytic, respiratory burst, myeloperoxidase and superoxide activities. Additionally, immune-related genes implicated in recognition and effector functions of yeast glucans were up-regulated (Toll like receptor 2, C-type lectin family 17 member A, colony-stimulating factor 1 receptor 2, macrophage mannose receptor 1, and Interleukin-1ß). In conclusion, the glucan -characterized physically-chemically from the yeast C. benthicum (Cb-ßG)- was safe, had strong antioxidant capacity to scavenge free radicals, and stimulated immune parameters and immune-related gene expressions on thymus cells from Totoaba macdonaldi.

ß-D-glucan from marine yeast Debaryomyces hansenii BCS004 enhanced intestinal health and glucan-expressed receptor genes in Pacific red snapper Lutjanus peru.

Previous studies have shown that marine yeast Debaryomyces hansenii BCS004 (also known as Dh004) has a potential biotechnological application. The aim of this study was to investigate the structural characterization, antioxidant properties and possible health inductor of dietary ß-D-glucan BCS004. In this study, a glucan BCS004 was obtained containing (1-6)-branched (1-3)-ß-D-glucan with low molecular weight and a high purity of 90 and 91.7% for one and 4 h, respectively. ß-D-glucan BCS004 showed higher antioxidant activity, including DPPH radical and superoxide anion scavenging, ß-carotene bleaching inhibition, and iron chelation activity. An in vitro study showed that ß-D-glucan BCS004 was safe for peripheral blood leukocytes inducing proliferative effects. Moreover, in an in vivo study using ß-D-glucan BCS004 no histopathological damages or intestinal inflammation were observed in fish. The gene expression analysis highlighted that dietary ß-D-glucan BCS004 could also up-regulate glucan and macrophage receptor genes in intestine, such as C-type lectin (CTL) and macrophage mannose receptors (MMR). Overall, the results demonstrated that ß-D-glucan from D. hansenii BCS004 could be an immunostimulant with antioxidant properties and beneficial effects on intestinal health in fish.

Dietary supplementation of marine yeast Yarrowia lipolytica modulates immune response in Litopenaeus vannamei.

The immunostimulatory potential of the marine yeast Yarrowia lipolytica (D1 and N6 strains) administered orally was evaluated in the white shrimp Litopenaeus vannamei. Yeasts and commercial glucans were mixed with a commercial feed to formulate diets with a 1.1% concentration of immunostimulants. The shrimp were fed daily for a period of 21 days. Weekly determinations were performed for immunological parameters in hemolymph, such as total hemocyte count (THC), lysozyme activity (LYZ), prophenoloxidase activity, antioxidant enzymatic activities (superoxide dismutase [SOD], catalase [CAT], and peroxidases), and bactericidal activity against Vibrio parahaemolyticus. Expression profiles of penaeidin (PEN), lysozyme (LYZ), and prophenoloxidase (proPO) immune genes were evaluated in hemocytes. In general, an increase in the immune parameters was observed in shrimp fed yeast diet compared to glucan and the control diets. Yarrowia lipolytica, especially strain N6, provided maximum immunostimulatory effects evidenced by the increase of immune parameters (THC, LYZ, SOD, CAT) and gene expression profile. In conclusion, this study demonstrated that Y. lipolytica had immunostimulatory effects and increased bactericidal activity in L. vannamei hemocytes against V. parahaemolyticus. These findings open the path for the potential application of Y. lipolytica-based immunostimulant for shrimp aquaculture.

[Synthesis of cello-oligosaccharides which promotes the growth of intestinal probiotics by multi-enzyme cascade reaction].

Soluble cello-oligosaccharide with 2-6 oligosaccharide units is a kind of oligosaccharide with various biological functions, which can promote the proliferation of intestinal probiotics such as Bifidobacteria and Lactobacillus paracei. Therefore, it has a regulatory effect on human intestinal microbiota. In this study, a Cc 01 strain was constructed by expressing cellodextrin phosphorylase (CDP) in Escherichia coli. By combining with a previously constructed COS 01 strain, a three-enzyme cascade reaction system based on strains COS 01 and Cc 01 was developed, which can convert glucose and sucrose into cello-oligosaccharide. After optimization, the final titer of soluble cello-oligosaccharides with 2-6 oligosaccharide units reached 97 g/L, with a purity of about 97%. It contained cellobiose (16.8 wt%), cellotriose (49.8 wt%), cellotetrose (16.4 wt%), cellopentaose (11.5 wt%) and cellohexose (5.5 wt%). When using inulin, xylo-oligosaccharide and fructooligosaccharide as the control substrate, the biomass (OD600) of Lactobacillus casei (WSH 004), Lactobacillus paracei (WSH 005) and Lactobacillus acidophilus (WSH 006) on cello-oligosaccharides was about 2 folds higher than that of the control. This study demonstrated the efficient synthesis of cello-oligosaccharides by a three-enzyme cascade reaction and demonstrated that the synthesized cello-oligosaccharides was capable of promoting intestinal microbial proliferation.

Yeast ß-Glucans as Fish Immunomodulators: A Review.

Administration of immunostimulants in fish is a preventive method to combat infections. A wide variety of these biological molecules exist, among which one of the yeast wall compounds stands out for its different biological activities. The ß-glucan that forms the structural part of yeast is capable of generating immune activity in fish by cell receptor recognition. The most frequently used ß-glucans for the study of mechanisms of action are those of commercial origin, with doses recommended by the manufacturer. Nevertheless, their immune activity is inefficient in some fish species, and increasing the dose may show adverse effects, including immunosuppression. Conversely, experimental ß-glucans from other yeast species show different activities, such as antibacterial, antioxidant, healing, and stress tolerance properties. Therefore, this review analyses the most recent scientific reports on the use of yeast ß-glucans in freshwater and marine fish.

Conjugation of ß-glucans on heat-stable enterotoxin (ST) to enhance the immunogenic response in mouse leucocytes.

Enterotoxigenic Escherichia coli (ETEC) is an important diarrhea-causing pathogen for humans. Heat-stable enterotoxin (ST) plays a crucial role in triggering diarrhea and ETEC pathogenesis. However, ST is a small peptide that lacks immunogenic activity itself but becomes immunogenic when it is coupled to a carrier molecule. In this study, the ß-glucans (BG) from yeasts have been used to test their immunomodulatory activity and adjuvant effect on the properties of ST. This study aimed to synthesize and characterize a conjugate of yeast-derived ß-glucan with the ST enterotoxin (BG-ST) and evaluate the antigenic and antioxidant activities in mouse splenocytes. Fourier transform infrared spectroscopy and scanning electron microscopy analysis showed new bands and changes in morphology, respectively, confirming ST was successfully coupled to beta glucan. Additionally, according to the enzyme-linked immunosorbent assay (ELISA), conjugation efficiency was almost 90%. Cellular viability, phagocytic cell proportion, and respiratory burst enhanced splenocytes stimulated by BG-ST. In addition, nitric oxide production and antioxidant enzymes increased in cells stimulated with BG-ST, BG and ST. In conclusion, the results revealed the successful conjugation of ß-glucan with ST peptide enhancing immune and antioxidant parameters to a greater extent than their individual components.

Short-Chain Cello-oligosaccharides: Intensification and Scale-up of Their Enzymatic Production and Selective Growth Promotion among Probiotic Bacteria.

Short-chain cello-oligosaccharides (COS; degree of polymerization, DP ≤ 6) are promising water-soluble dietary fibers. An efficient approach to their bottom-up synthesis is from sucrose and glucose using glycoside phosphorylases. Here, we show the intensification and scale up (20 mL; gram scale) of COS production to 93 g/L product and in 82 mol % yield from sucrose (0.5 M). The COS were comprised of DP 3 (33 wt %), DP 4 (34 wt %), DP 5 (24 wt %), and DP 6 (9 wt %) and involved minimal loss (≤10 mol %) to insoluble fractions. After isolation (≥95% purity; ≥90% yield), the COS were examined for growth promotion of probiotic strains. Benchmarked against inulin, trans-galacto-oligosaccharides, and cellobiose, COS showed up to 4.1-fold stimulation of cell density for Clostridium butyricum, Lactococcus lactis subsp. lactis, Lactobacillus paracasei subsp. paracasei, and Lactobacillus rhamnosus but were less efficient with Bifidobacterium sp. This study shows the COS as selectively functional carbohydrates with prebiotic potential and demonstrates their efficient enzymatic production.

Administration of dietary prebiotics improves growth performance and reduces pathogen colonization in broiler chickens.

Dietary prebiotics are thought to be potentially important alternatives to antibiotic growth promoters in poultry production because of their beneficial performance and health effects. The administration of dietary prebiotics has been demonstrated to improve animal health, growth performance, and microbial food safety in poultry production. In this study, we evaluated the effects of Saccharomyces-derived prebiotic refined functional carbohydrates (RFC) with yeast culture on growth performance and gastrointestinal and environmental microbiota when administered in-feed and through drinking water to broiler chickens. Broilers were administered 2 doses of prebiotic in-feed through 42 d of production and prebiotic-treated water in the final 72 h. Administration of prebiotic RFC improved ADG and decreased cecal Campylobacter counts, while the high dose also increased final BW. Additionally, significant main effects of prebiotic RFC dose were observed with the high dose improving ADG and ADFI over the finisher phase and final BW. Although the effects were not significant, the prevalence of Campylobacter in the cecum after feed withdrawal was 17% lower when broilers were administered the high prebiotic dose, and recovery of Campylobacter from litter was up to 50% lower when broilers were administered prebiotic RFC. Our results suggest that co-administration of RFC with yeast culture as a prebiotic can be used to improve growth performance and reduce human foodborne pathogens in poultry.

Protective effect of ß-D-glucan and glutamine on the genomic instability induced by Cytarabine/Ara-C in BALB/c mice.

Prophylactic antibiotics and growth promoters have been substituted, mainly for livestock, by immunomodulators and intestinal health promoters - such as ß-D-glucans and glutamine. The aim of this study was to verify the beneficial effects of ß-D-glucans and glutamine against Cytarabine/Ara-C, evaluating the DNA damage in leukocytes, the leukogram, and the mitotic index of intestinal crypts cells. Balb/C mice received treatment with ß-D-glucan (80 mg/Kg), glutamine (150 mg/Kg), or both, for 21 days. On the last two days of this period, Ara-C was administered (1.8 mg/animal) by intraperitoneal injection every 12 h. The animals submitted to the treatment with Ara-C presented the highest genotoxic index, a significant leukopenia, and a decrease in the mitotic index of the intestinal crypts cells. Treatment with ß-D-glucan protected the leukocytes against DNA fragmentation induced by Ara-C. Glutamine alone promoted maintenance of the mitotic index and, in association with ß-Dglucan, reduced leukopenia. Thus, the use of ß-D-glucan and glutamine proved to be beneficial to intestinal tropism. This can happen once the damage to the genetic material, prevented by the treatments with ß-D-glucan and glutamine, can result in genotoxicity. Not only this, but it might be capable of turning into a mutagenesis, with consequential physiopathological alterations.

Effect of refined functional carbohydrates from enzymatically hydrolyzed yeast on the presence of Salmonella spp. in the ceca of broiler breeder females.

Broiler breeders hatched from Salmo-nella negative grandparents received either zero or 50 g/MT of refined functional carbohydrates (RFC) in their diets from d of placement to end of lay. There were no other treatments used. Pullets and cockerels were reared separately in an enclosed litter-floor house to 21 wk of age when 28 randomly selected pullets from each diet were transferred to individual cages for an additional 14 d before they were killed, and their ceca were excised aseptically and tested for Salmonella spp. The remaining birds were transferred to a two-thirds slat and one-third litter curtain-sided laying house. There were 8 pens of 60 to 65 females and 8 to 18 males, depending upon flock age and housing type, fed each diet, and there was no effort made to isolate pens from typical daily foot traffic between pens. At 51 wk of age, male progeny broiler chicks were hatched and received either zero or 50 g/MT of RFC to complete a 2 × 2 design with 4 replicate pens of 12 males per interaction. All broilers were tested for cecal Salmonella spp. at 34 d of age. Ceca were collected from 30 breeder hens from each treatment at 64 wk of age and tested for Salmonella spp. Of the ceca sampled at 23 wk from the control pullets, 71.4% were found to contain Salmonella spp., while none of the ceca from the RFC pullets tested positive. Of the ceca sampled from the control hens at 64 wk, 40% were found to contain Salmonella spp., while none of the ceca from the RFC hens tested positive. Salmonella spp. was isolated from broilers in one pen of the control broilers that were also progeny of control breeders out of 4 replicates but not from any pens in which the breeders had been fed RFC. These data demonstrated that RFC reduced natural Salmonella spp. colonization of broiler breeder hen and broiler progeny ceca during a complete production cycle.