Extraction of β-glucan of Hericium erinaceus, Avena sativa L., and Saccharomyces cerevisiae and in vivo evaluation of their immunomodulatory effects

The word ‘glucan’ refers to polymers of glucose that include cellulose (β-1,4-glucan). They are branched or unbranched, short or long, soluble or particulate, and in α or β isomeric forms. Beta-glucan (BG) is one of the most common types of polysaccharides (Goodridge et al., 2009; Chan et al., 2009). Fungal BG, in particular the BG of Saccharomyces cerevisiae, have variable numbers of 1, 6 branches, and the forms in which the refined glucan molecules exist range from glucose dimers to large insoluble particles. BG from yeast (S. cerevisiae), mushroom (Sclerotium glucanicum and others), bacteria (curdlan from Alcaligenes faecalis), and seaweed (laminarin from Laminaria digitata) have been reported for their proor anti-inflammatory effects (Goodridge et al., 2009).


Introduction
The word 'glucan' refers to polymers of glucose that include cellulose (β-1,4-glucan). They are branched or unbranched, short or long, soluble or particulate, and in α or β isomeric forms. Beta-glucan (BG) is one of the most common types of polysaccharides (Goodridge et al., 2009;Chan et al., 2009). Fungal BG, in particular the BG of Saccharomyces cerevisiae, have variable numbers of 1, 6 branches, and the forms in which the refined glucan molecules exist range from glucose dimers to large insoluble particles. BG from yeast (S. cerevisiae), mushroom (Sclerotium glucanicum and others), bacteria (curdlan from Alcaligenes faecalis), and seaweed (laminarin from Laminaria digitata) have been reported for their pro-or anti-inflammatory effects (Goodridge et al., 2009).
The host's immune receptors will recognize the carbohydrate moiety of BG and elucidate the defensive response (Brown & Gordon, 2005). The immunomodulatory and cancerostatic properties of BG are established both in vitro and in vivo against breast, lung, colorectal (Dongowski et al., 2002), and gastrointestinal cancers. BG also has antioxidant properties, and carry out wound healing activities (Petravic-Tominac et al., 2010). Further, it prevents coronary heart disease (Wang et al., 2002), decreases blood glucose levels in diabetic patients, ameliorates insulin resistance (Hallfrisch et al., 2003;Hlebowicz et al., 2008), controls serum cholesterol levels (Smith et al., 2008), and influences the gut microflora (Tungland, 2003).
Dther than the medical field, BG is involved in many other areas, like the food and cosmetic industries. BG can be used in the making of sauces, soups, and beverages. Moreover, it may act as a thickening agent, stabilizer, and emulsifier (Burkus & Temelli, 2000). BG serves as moisture retention agents in cosmetics, especially in creams and lotions for sensitive and irritated skin (Wheatcroft et al., 2002). BG accounts for a significant part of yeast biomass, and is necessary in the performance of cell functions (Bacic et al., 2009). Yeast-BG is also used as an adjuvant with anti-infective or antineoplastic agents, after radiotherapy (Petravic-Tominac et al., 2010). An in vitro study revealed that the BG of S. cerevisiae can promote TNF-α production and trigger the mononuclear cells, and neutrophils (Dlson et al., 1996). Ot is proven that the consumption of whole grain, low glycemic index, and high dietary fiber diets reduce the development of type 2 diabetes and heart diseases. Thus, oats and barley are the primary choice of cereals enriched with BG. The serum cholesterol-lowering property of the oat and barley is related to their molecular weight, and the solubility of the BG (Wood, 2007).
Mushrooms are well-known immune-modulators because of the richness of their polysaccharide fractions, primarily BG. Fungal BG can activate the leukocytes, and edible mushrooms have some effects on enterocytes (Volman et al., 2010). Hericium erinaceus is better identified for its antimicrobial, antioxidant, antihypertensive, antidiabetic nature and enrichment of protein (Woraharn et al., 2015). H. erinaceus is commonly used for the production of fermented mushroom juices, and Lactic Acid Bacteria mediated fermented H. erinaceus juices are enriched with L-glutamic acid and γ-aminobutyric acid (Woraharn et al., 2016). Even though the bioactivity of the mushroom depends on the configuration, and content of the BG, information about the BG of H. erinaceus is very limited. The structure, quality, and functionally of extracted BG is majorly influenced by the extraction method (Ahmad et al., 2012). The changes in the total antioxidant capacity and antioxidant enzymes level during BG exposure to the host system are not yet illustrated clearly, even though the antioxidant potential of BG has been reported (Kofuji et al., 2012). Thus, the current work deals with the extraction of BG from yeast (Saccharomyces cerevisiae HOO31), oat (Avena sativa L.), and mushroom (Hericium erinaceus) and in vivo analysis of their immunomodulatory and antioxidant properties. Moreover, changes in the antioxidant capacity of mice during BG supplementation were also studied.

Extraction
The source materials (yeast, oat, and mushroom) were suspended in 5 fold of 1.0 M NaDH and incubated at 80 °C. After shaking for 2 h, centrifugation was carried out at 6,000 × g, for 25 min at 4 °C. The pellets were washed with distilled water 3 times by centrifugation at 6,000 × g, for 25 min at 4 °C for cleaning purpose. The obtained pellets were suspended in 5 fold of 1.0 M acetic acid and incubated at 80 °C for 2 h with constant stirring by a stirrer force. After incubation, the suspensions were centrifuged at 6,000 × g, for 25 min at 4 °C. The process was repeated three times. The collected pellets were dried in absolute ethanol and hot air oven at 60 °C. The dried glucan pellets were stored at 4 °C until analysis (Pengkumsri et al., 2017a).

Glucan determination and FT-IR
The glucan content of the sample was determined by glucan assay kit as per the manufacturer's instructions (yeast & mushroom, Megazyme, Oreland). The component and ratio of glucan in the extracts were analyzed by Fourier Transform Onfrared Spectroscopy (FT-OR). The Spectra (from 400 to 4,000 cm -1 at 4 cm -1 resolution) were recorded for glucan extracts with FT-OR spectrometer (Nicolet Nexus 470 FT-OR instrument, USA); an average of 32 scans was computed for each sample. All measurements were acquired in duplicate normalization. The spectra were pre-processed (baseline correction, normalization, and second derivative) with DMNOC 32 software. The Drigin Pro 8.0 software was used to calculate the area with curve-fitting method (Pengkumsri et al., 2017a, b).

Evaluation of cytokines in the serum samples
The levels of the selected cytokines (OL-6, OL-10, OL-17, OFN-γ, and TGF-β) in the serum samples collected from each experimental group were determined by ELOSA as per the manufacturer's instructions (R&D system, USA).

Preparation of liver extract and determination of protein
The extracted mice lives were prepared as detailed by Guo et al. (2013). Briefly, 1.0 g of a freshly-collected mouse liver was ground in cool condition with 10 mL of Phosphate-buffered saline (PBS; pH 7.0). The liver suspension was centrifuged at 3,500 rpm for 30 min at 4 °C. Then, the supernatant was collected and used for antioxidant capacity, antioxidant enzymes, and oxidation of lipid determinations. Afterward, the protein contents of the samples were determined by Lowry's method with bovine serum albumin (BSA) (20 -200 µg/mL) as a substrate for standard curve preparation (Lowry et al., 1951).

Evaluation of lipid oxidation, antioxidant capacity, and antioxidant enzymes
The lipid oxidation was measured by determining the concentration of malondialdehyde (MDA) as detailed in a previous report (Pengkumsri et al., 2015). The MDA product was measured using a Multimode Detector (Beckman coulter, DTX 880, USA) at 540 nm. The ng/mg protein of the liver extract was reported.
The catalase (CAT), superoxide dismutase (SDD), and glutathione peroxidase (GPx) activities were measured in the liver extract as reported by Pengkumsri et al. (2015). All experiments were carried out in triplicates, and the values were represented as U/mg protein.

Statistical analysis
Analysis of variance (ANDVA) was performed, with the confident interval at 95% (p < 0.05). The statistical analysis was performed in SPSS 17.0 to check the significant differences in the glucan content of the extracts and the cytokine levels in the serum samples. The Least Significant Difference (LSD) post hoc test was performed to analyze the significant differences in antioxidant activities and p < 0.05 was considered as significant.

Results and discussion
The yeast, mushroom, and oat samples were subjected to total glucan extraction. About 68 ± 3, 25 ± 2, and 26 ± 2% of yield were recorded in the yeast, mushroom, and oat samples, respectively. The total glucan content of the extracts were found as 97 ± 4, 92 ± 4, and 89 ± 4% in the yeast, mushroom, and oat samples, respectively. About 91 ± 5, 87 ± 3, and 84 ± 4% of β-glucan (BG) were recorded in the yeast, mushroom, and oat samples, respectively, with 4-5% of α-glucan (AG) in all the samples (Table 1). The results suggest that yeast cells have more BG than other tested samples with respect to total yield and percentage of BG content (Table 1).
The immunomodulatory properties of the extracted BG samples were assessed. The levels of inflammatory and anti-inflammatory cytokines were assessed. The levels of OL-6 were 685 ± 62, 659 ± 50, and 682 ± 39 pg/mL for the 200 mg/kg yeast-BG, oat-BG, and mushroom-BG interventions, respectively. The levels of OL-17 were 155 ± 15, 145 ± 15, and 151 ± 16 pg/mL for the yeast-BG, oat-BG, and mushroom-BG interventions, respectively. The OFN-γ levels were 238 ± 26, 203 ± 16, and 214 ± 15 pg/mL in mice serum for the yeast-BG, oat-BG, and mushroom-BG intervention-, respectively ( Table 4). The data indicate that the yeast-BG stimulated the expression of OL-6, OL-17, and OFN-γ more effectively than oat and mushroom-BG. Onterestingly, the oat-BG mediated induction was not enhanced effectively with increased concentration, even though statistically different from the other group.
The lipopolysaccharide (LPS) mediated enteritis induced in rat, supplemented with high molecular oat-BG (2,179,700 g/mol) showed an improvement in stress oxidative parameters in the spleen, and reduced the lipid superoxides, 7-ketocholesterol level and glutathione disulfide activity (Blaszczyk et al., 2015). The LPS induced enteritis in rat treated with partially purified (75%) low MW oat-BG showed a reduction in the T and B lymphocytes, granulocytes and Tc lymphocytes.
The high MW Dat-BG (2,180,000 g/mol) intervention effectively reduced the lipid peroxidation in naïve control rats, whereas the low MW BG (70,000 g/mol) did the same in the enteritis groups, which suggest that low MW BG supplementation enhanced the antioxidant capacity of the host and facilitates disease recovery . The studies proves that yeast-BG can stimulate the level of TNF-α, OL-6, OL-10, and TGF-β (Dlson et al., 1996;Tzianabos, 2000;Engstad et al., 2002). The involvement of BG in immune regulation of colitis condition has been reported (Roda et al., 2011). The water extract of the fruiting body of Dyster mushroom (Pleurotus ostreatus) can induce the production of reactive oxygen species and modulate the defense system (Shamtsyan et al., 2004). NF-kB is a transcription factor which is involved in immune response and regulation. Agaricus bisporus polysaccharide, possibly BG, affects and lowers the NF-kB transactivation in Caco-2 cells (Volman et al., 2010). The structure, composition and the biological properties of oat-BG have been reviewed (Daou & Zhang, 2012). The changes in the immune activation property were due to the compositional differences among the samples. The glucan compounds are either homo or hetero glucans with β (1, 3), β (1,4) and β (1,6) glycosidic bond that plays a vital role in some bioactivity of mushrooms. On cereals (oat and barley), BG is made-up of mixed-linkage (1,3), (1,4)-β-d-glucose units, whereas it is composed of mixed-linkage of (1,3), (1,6)-β-d glucose units in yeasts and mushrooms. The FT-OR and band area analysis also reveal that the oat-BG is composed more of β-1,4 glucans, and β-1,6 glucans, whereas yeast and mushroom BG are mainly composed of β-1, 3 glucan, and β-1, 6 glucans (Table 1, 2, 3).
The antioxidant capacity of BG supplemented mice was determined. All the BG-treated mice showed an increase in the trolox equivalent of antioxidant capacity (TEAC), in a dose-dependent manner. The maximum TEAC in yeast BG-200 mg group (4.0 ± 0.3 ng/mg of protein) and a slight increase in TEAC in oat-BG-100 mg group (2.7 ± 0.1 ng/mg of protein) were observed while the TEAC of naïve control was 2.5 ± 0.1 ng/mg of protein. The mushroom glucan displayed a moderate level of enhancement of TEAC in experimental mice ( Figure 1A). The ability of the studied BG extracts to inhibit lipid peroxidation was assessed. Like TEAC, yeast-BG groups, oat-BG groups, and mushroom-BG groups showed maximum, minimum, and a moderate level of suppression of lipid peroxidation regarding MDA formation ( Figure 1B).
The changes in the major antioxidant enzymes such as catalase, SDD, and GPx during BG supplementations were studied. The levels of all the studied enzymes were increased upon yeast-and mushroom-BG interventions for all the concentrations (100 -200 mg), whereas oat BG required slightly high concentration (150 -200 mg) to enhance the enzymes level in mice compared to the naïve control. The maximum level of catalase, SDD, and GPx during yeast BG-200 mg intervention in mice were recorded as 28 ± 2, 113 ± 9, and 7 ± 1 U/mg of protein, respectively ( Figure 2).
Studies have shown the enhanced bioactivity of BG isolated from other sources like barley, and mushroom. Barley-BG showed a stronger antioxidant activity than the BG of black yeast and oats. The extraction method, and complexity (oligomers, macromolecules) of BG influence the free radical scavenging activity (Kofuji et al., 2012). The glucan of Geastrum saccatum (mushroom) protects cells from lipid peroxidation, and oxidative (hydroxyl and superoxide) stresses (Guerra Dore et al., 2007).  Dat-BG supplementation nullified the impact of bacterial LPS mediated enteritis, which was characterized by improved values of antioxidative markers, like total antioxidant status, SDD, glutathione reductase and GPx activity in rat colon .
The treatment of BALB/c mice with 50mg/kg/day of sulfated glucans from S. cerevisiae for 14 days improved the serum CAT, and GPx activities, and decreased MDA level. The glucan from S. cerevisiae showed the in vitro scavenging activity in 1,1-Diphenyl-2-picryl-hydrazyl assay, and against superoxide, and hydroxyl radicals (Lei et al., 2015). The chloroform: methanol extract of mushroom, Polyporus dermoporus, was reported for the suppression of 42.9, and 83.3% of lipid peroxidation, and superoxide radicals, respectively in BALB/c mice. Moreover, the anti-inflammatory property of P. dermoporus extract was also observed in terms of the decline in polymorphonuclear cells and nitric oxide, which are further evidenced by histopathological examination of ear edema (Dore et al., 2014).
The total antioxidant capacity of 1 mg of soluble branched β-d-glucan from Entoloma lividoalbum was determined to be equal to 70 ± 15 µg of ascorbic acid, and it has also been reported for its hydroxyl and superoxide radical scavenging ability . β-d-glucan of the mushroom Russula albonigra (Krombh.) Fr. has been reported for its antioxidant capacity and nitric oxide mediated macrophage activation, as well as its in vitro splenocytes, and thymocytes proliferation .
The results of the present study suggest that yeast-BG supplementation at a concentration of 200 mg enhanced the health status of experimental mice by improving the TAC, inhibiting lipid peroxidation, and increasing antioxidant enzymes more than other tested interventions (Figure 1, 2). The anti-colitis property of BG from S. cerevisiae HOO31 was recently reported by our group. We have also proved that yeast-BG supplementation and rice bran extract (rice phenolic acids, anthocyanins) significantly reduced the consequences of Dextran Sodium Sulphate (DSS) mediated colitis induction in rat model by regulating the serum cytokines, antioxidant capacity and antioxidant enzymes (Pengkumsri et al., 2017b).

Conclusion
The data collectively revealed that yeast-BG is a potent activator of inflammatory and anti-inflammatory cytokines, as well as antioxidant capacity of mice than oat-, and mushroom-BG. The bioactivity of BG was directly proportional to the concentration. Moreover, the extraction method plays a major role in the yield of the BG. Thus, a more detailed study is mandatory to explain the influence of extracted BG on the immune system and other biological parameters like antioxidant level variation, which help to develop functional food supplements. and (C) glutathione peroxidase in mice liver during experimental supplements. a-g represent the significant difference (p < 0.05) among the different groups. YG, DG, and MG indicate that yeast glucan, oat glucan, and mushroom glucan, respectively.