Effect of strains and extraction methods on β -glucan production, antioxidant properties, and FTIR Spectra from Mushroom fruiting bodies of Schizophyllum commune Fr . in Thailand

: Schizophyllum commune Fr, a native mushroom of Thailand, has a high nutrition value and it classified as a mushroom with medicinal properties, which can neutralize the growth of many cancer cells. Thus, the aim of this research was studied effect of S. commune strain and the extraction method on the quantity and properties of β -glucan. The five S. commune Fr strains consisted of Chanthaburi, 85-022, 85-023, 85-031, and 85- 043, which used in this research. The β -glucan extraction method compared two different extraction: hot water (M1) and hot alkali extraction (M2), with control (native-MR). It found that Chanthaburi strain has the highest in β -glucan content 49.20 + 0.35 % (w/w), and high potential antioxidant activity (79.14 +0.77 DPPH % and 50.92 + 0.48 ABTS %) (p < 0.05). The extraction methods had no effect on the yie ld of β -glucan, except antioxidant properties and chemical structure of extract substance. The extract substance from M2 has significant the highest potential antioxidant activity (80.22 + 0.51 drink mushroom juice in can by using 1-day-old MR and adjust pH more than 7 can increase antioxidant properties of product. Mushroom fruiting bodies, Schizophyllum commune Fr polysaccharide, β -glucan, β -glucan extraction method, FTIR Spectra

industries, such as the food, pharmaceutical or cosmetics industries, especially local food production from natural mushroom. Because in this research, it is an adaptation of the extraction method of β-glucan by using filtering instead of centrifugation, which is a low-cost technology that can be produced by the community.

Materials
Five Pure mycelial culture strain of S. commune Fr were obtained from Chanthaburi mushroom farm (1 strain), and Department of Agriculture (4 strains), Thailand. The culture collection number from Chanthaburi mushroom farm was Chanthaburi, and Department of Agriculture was 85-022, 85-023, 85-031, and 85-043. The mycelium grown on sterile culture food bag at 30 0 C for 7 days or until the mushroom fibers start to gather to grow into a mushroom. The sample used in the experiment was 1-day-old mushroom fruiting body, which came from the preliminary experiment; it found that the mushroom fruiting body had glucan content than in the mushroom mycelium and at the 1 day mushroom fruiting body the most. The mushroom samples were dried in hot air oven at 70 + 5 0 C until 2.50 + 0.02 % moisture content. The dry samples were ground, sifted through an 80 mesh, and stored at room temperature in aluminum bag for further analysis.

Glucan content determination
The Glucan contents was determined by using a β-Glucan Assay Kit (Megazyme International, Wicklow, Ireland). The principle of Mushroom and Yeast β-Glucan Assay Kit based on the determination of total glucan, which consists of α-Glucan and β-Glucan linkages. The bond of (1 3,1 6) -β -D-Glucan, (1 3) -β-Glucan and α-Glucan are dissolved and cut by concentrate hydrochloric acid at 100 0 C for 2 h, and then the solution was incubated with exo-1, 3-β-glucanase and β-glucosidase in order to get complete D-glucose for analysis total glucan content. For α-Glucan, it was digested to be glucose with amyloglucosidase plus invertase, using GOPOD reagent to measure glucose content.

(1) Total glucan content
For total glucan content,10 mg of native-MR powder placed in Eppendorf tube then added 0.15 ml of 37% hydrochloric acid. The solution was mixed and incubated at 30 0 C for 45 min (vortexed every 15 min). Then, 1 ml of distilled water was added, mixed and incubated at 100 0 C for 2 h before added with 0.5 ml of 4 M KOH. The 200 µl solution was taken, adjusted volume to 1 ml with sodium acetate buffer pH 5 (800 µl) and mixed.
After that, the mixtures were centrifuged at 13,000 x g for 5 min. Samples (20 µl) were taken to each well (in duplicates) before added with 10 µl of a mixture of exo-1, When ΔE is the absorbance F is the factor to convert the absorbance to µg of D-glucose W is the weight of sample (g)

Total phenolic compounds determination
The total phenolic content was determined, which modified method from dark at room temperature for 30 min to complete the reaction. The absorbance measured at 517 nm. The scavenging effect of DPPH free radicals calculated as follows:

Effect of extraction method
To study the effect of extraction method performed by modified from Suwanno et al., 2005 [19] and Mizuno and Nishitani (2013)

Statistical analysis
The data collected from triplicates. Analysis was perform by statistical package SPSS 17 for windows, p < 0.05 (two-tailed) was consider as statistically significant. All of data analyzed with Analysis of Variance (ANOVA) and multiple comparison F-test. Chanthaburi strains was the most suitable for use as a raw material for the β-glucan production. Therefore, Chanthaburi was select to study the effect of extraction method on the amount and quality of β-glucan. The values are the mean of three replications ± standard deviation.

Production of β-glucan
Different letters in each column represent significant differences between treatments (p < 0.05).

Effect of β-glucan extraction method.
In this research, the researcher would like to study the effect of the extraction method on the obtained glucan content and antioxidant properties, that will lead to the development of community canned mushroom juice products from S. commune Fr, Thailand. The researcher chose to use the filter paper method instead of sedimentation by centrifugation to extract the glucan. β-glucan Most of glucan content in both dried extraction substrates and native-MR dried powder were β-glucan (48.90 -49.23 g/100 g of native-MR dried powder), which consistent with the research of cm -1 , 1405-1409 cm -1 , and 878-876 cm -1 . The result of FT-IR spectroscopy showed that heat and alkalinity had an effect on the structure of S. commune Fr β-glucan, which was consistent with the research of Gieroba et al. [3] that found that the 1,3-β-D-glucan polymer gelled at 80 0 C has a distinctly different structure than the matrix gelled at 90 0 C. The important FT-IR absorption spectra region of polysaccharides consisted of the sugar region (1200-950 cm -1 ) and anomeric region (950-750 cm -1 ) [16], which heat used for extraction or the pH has an effect on the structure of the extracted β-glucan, thus effect on its antioxidant properties. The values are the mean of three replications ± standard deviation.

Conclusions
In this research, it found that the strains of S. commune Fr in Thailand had effect on the amount of extracted β-glucan. The 1-day-old MR S. commune Fr from Chanthaburi was the good source for β-glucan production. For the effect of extraction method, it found that temperature and pH in extraction have effect on the structure and antioxidant properties of β-glucan. The extraction 1-day-old MR S. commune Fr from Chanthaburi with 1 M of NaOH at 100 0C for 24 h yielded of β-glucan with more antioxidant properties than β-glucan from hot water extraction at 121 0 C for 15 min. The results of this research led to the development of ready-to-drink mushroom juice in can by using 1-day-old MR S. commune Fr and adjust to pH more than 7 can increase antioxidant properties of product.