Studies On Submerged Culture Fermentation Technology And Polysaccharide Prperties Of Antrodia Camphorata

Antrodia camphorata is recently discovered fungi specie for food and medicinal purposes, because of bioactive function of its fruit body, it has been widely attended. Submerged culture fermentation technology of Antrodia camphorata has developed very fast in recent years under the consideration of environmental protection, natural resource deficiency and unfruitful development of its fruit body cultural technology. Polysaccharide is the main bioactive functional factor in Antrodia camphorata, it is very important in submerged culture fermentation and in the study of its nutritional value. In this paper, biological characteristic of submerged cultured Antrodia camphorata mycelia has been studied, the most suitable culture medium formula has been screened with the highest polysaccharide content and mycelium output; nutritional value of Antrodia camphorata mycelia has been analyzed, as well as the physics and chemistry characteristic, function characteristic of polysaccharide. In this study, the technological method of polysaccharide oral fluid product has also been developed. The main findings were as follows:(1) The sequence analysis of rDNAThe result of rDNA sequence analysis on NCBI showed that its isogenous rate was 99%, the strain was tentatively identified as Antrodia camphorata.(2)Studies on biological characteristic of Antrodia camphorataAmong 15 different kinds of carbon sources, natural polysaccharides (rice powder, wheat powder, corn powder, sweet potato powder and cassava powder) resulted in obviously higher mycelia concentration than disaccharides (maltose, sucrose), monosaccharides (D-glucose, fructose, D-xylose, D-galactose, L-arabinose, D-mannitol) and chemical polysaccharides (soluble starch and.crystallite fiber).Among 3 different kinds of nitrogen sources, it was found that organic nitrogen sources (soybean powder>corn powder> peptone) have resulted in higher mycelia concentration than ammonium nitrogen (ammonium tartrate>ammonium chloride>ammonium secondary phosphate>ammonium sulfate), which was higher than nitric nitrogen. In this study, Antrodia camphorata could not utilize nitric nitrogen.The most suitable carbon and nitrogen ratio was 50:1 to 100:1. Sodium and magnesium were not essential for the fermentation of Antrodia camphorata while calcium, sulfur, phosphorus, potassium were essential. Potassium deficient effected obviously in Antrodia camphorata fermentation. Trace mineral cobalt inhibited the growth of Antrodia camphorata. There was no significant effect in fermentation of Antrodia camphorata when added six kinds of vitamins (biotin, riboflavin, thiamine, niacin, folic acid, and ascorbic acid). Results of the study showed that culturing mycelia could grow well at acidity and neutrality condition and couldn't grow at pH above 9.0 respectively. Its growth rate was the same regardless whether it was exposed to light or not.(3) Studies on culture medium formula and fermentation dynamicsThe fermentation culture medium for Antrodia camphorata was studied by orthogonal experiment design to get the highest polysaccharide content and mycelium output. Researches on five ingredients such as potato, soy bean powder, corn powder, yeast extract, peptone, wheat bran and KH2PO4+MgSO4·7H2O were conducted in three levels using the orthogonal form of L27(313). Variance of the experimental results was analyzed and the best fermentation culture medium was established. The results showed that the optimal fermentation culture medium for Antrodia camphorata was:potato 20%, soy bean 0.5%, corn powder 0.5%, yeast extract 0.15%, peptone 0.15%, wheat bran 0.2%, KH2PO4 0.05% and MgSO4 0.035%.The fermentation parameter for Antrodia camphorata was studied by even design with three factors at four levels to get the highest polysaccharide content and mycelium output. The optimal fermentation parameter were determined:the most suitable growing temperature of mycelium was 26℃, the best inoculation volume was 15%, the optimum cultural length was 8days. The highest dry weight of mycelia of Antrodia camphotrata was obtained up to 11g/L. The fermentation path of Antrodia camphotrata was grasped through the study of fermentation dynamics, the growth of mycelia was faster while the level of ammonia nitrogen was decreased on the 4th day When the growth of mycelia reached its stable stage, some mycelia dissolved and the concentration of ammonia nitrogen was increased on the 16th day. It was the signal that mycelia growth from rapidly growing period into a stable growing period.(4)Studies on polysaccharide extraction technologies of submerged cultured Antrodia camphorataTo determine the content of functional components within Antrodia Camphorata:fatty acids, amino acids, minerals, ergosterol, total triterpene, the amount of crude fat in Antrodia Camphorata was 11.61%(m/m). Among the fatty acid, saturated fatty acid accounts for 12.6%(v/v), while unsaturated fatty acid accounts for 87.4%(v/v). The content of crude protein was 34.20%(m/m), total amount of amino acids were 270.68 mg/kg (m/m) Antrodia. Camphorata also had many kinds of minerals, especially trace minerals, there were Potassium, sodium, phosphorus, calcium, magnesium, iron, zinc, manganese, copper, molybdenum, chromium, selenium and strontium. The content of ergosterol was 19.55mg/g (m/m), and total triterpene was 2.8125mg/g (m/m) respectivily. The determination of functional components of Antrodia Camphorata would be useful to explore the mechanism of its functional effect.When the mycelia were extracted by hot water twice,94% of polysaccharide was extracted from Antrodia camphotrata mycelia. The highest amount of polysaccharide was extracted by ultra low temperature freezing method compared to other methods. Freezing and thawing of mycelia exert positive effect on polysaccharide extraction.The purpose of the research was to determine the optimum conditions of Antrodia Camphorata polysaccharide extraction by using the response surface methodology (RSM). Response surface methodology was employed and a second order quadratic equation for Antrodia Camphorata polysaccharide extraction was established. We found that extraction temperature, time, and the ratio of water for material were important factors by one factor tests. The applicability of the model equation for predicting the optimum response values was verified effectively by the validation data. By analyzing the response surface plots and their corresponding contour plots as well as solving the quadratic equation, the optimum process parameters for Antrodia Camphorata polysaccharide extraction were obtained when:temperature was 120℃, time 110.4 min and the ratio of water for material 1.89:1.(5) Studies on polysaccharide properties of Antrodia CamphorataThe physical and chemical properties of polysaccharides precipitated by different concentration of ethanol had been studied in this paper. Infrared spectrum analysis and HPLC-GPC analysis were used to detect the structure of those polysaccharides. Results showed that polysaccharides were dark brown, fluffy and odorless powder, it is easy to dissolve in water, but did not dissolve in organic solvent ethanol, ether and acetone. Reducing optical rotation was 48.31. The result of color reaction showed:when Antrodia Camphorata polysaccharides responded with phenol-sulfuric acid reagent, the color turned red, when it responded with anthrone reagent, the color turned cyan, while it responded with resorcinol reagent, the color turned purple red. It was colorless with KI-I2. The polysaccharides contained pyranride and had the characteristic of glycoconjugate by infrared spectrum analysis. The polysaccharide obtained three to four peaks after the high performance liquid chromatography GPC column separated, the computation obtains the number-average molecular weight and the heavy-average molecular weight. High molecular weight polysaccharide was gained by low concentration of ethanol (65% ethanol), while low molecular weight polysaccharide was gained by high concentration of ethanol (75% and 85% ethanol) respectively. Either mycelia or filtrate polysaccharides of Antrodia Camphorata had strong scavenging effect on superoxide radical.The content of polysaccharide from Antrodia camphorata mycelium (ACP1) and fermentation filtrate (ACP2) were 31.76% and 38.09%, and extraction rate of ACP1 and ACP2 were 1.59% and 4.89%. The reduced viscosities of polysaccharide ACP1 and ACP2 were [η]=1.982 and [η]=1.657.Mycelium polysaccharide (ACP1) and fermentation filtrate polysaccharide (ACP2) obtained three peaks after the highly efficient liquid chromatography GPC column separation. The protein and reducing sugar have been removed. There was polysaccharide left only by HPGPC. The computation obtained the number-average molecular weight and the heavy-average molecular weight by GPC soft ware.Mycelium polysaccharide (ACP1) and fermentation filtrate polysaccharide (ACP2) were gray and loose fibrous powder. This two polysaccharide had the characteristic of glycoconjugate by infrared spectrum analysis.When the concentration of mycelium polysaccharide (ACP1) and fermentation filtrate polysaccharide (ACP2) was 3g/L, the eliminate rate for oxygen free radical reached to 74.8% and 76.9%. After purification by molecular sieve chromatography on sephadex G-200, the refined polysaccharide from ACP1 and ACP2, named ACP1-1 and ACP2-1 were obtained. Its eliminate rate for oxygen free radical reached to 54.7% and 61.6%. The polysaccharides from Antrodia camphorata showed significant antioxidant activities in experiments in vitra. (6)Studies on bioactive function in Antrodia camphorata mycelium and fermentation filtrate polysaccharideThe purpose of the research was to observe the anti-tumor activity of Antrodia camphorata polysaccharide. Mycelium polysaccharide (ACP1) and fermentation filtrate polysaccharide (ACP2) had anti-tumor activity on cervical cancer U14 in mice. Tumor suppression ability of fermentation filtrate polysaccharide was obviously stronger than that of mycelium polysaccharide. The tumor suppression rates were 44.7% and 77.2%, respectively.The mice were fed with ACP1 and ACP2 at different concentration 100,400 and 1600 mg/kg·d for 30 days to observe the immune regulating effect in normal mice. There were remarkably enhance effects in mice cellular immunity function, body fluid immunity function, single nucleus -macrophage function, NK cell activeness and interferon-2 (IL-2) value. Fermentation filtrate polysaccharide (ACP2) showed a stronger immune function than mycelium polysaccharide(ACP1) in mice.(7) Study on technological method in polysaccharide orally fluid product.Sense scores were used as assessment targets to optimize technology of polysaccharide orally fluid product. Results showed that sense score was 92 when added 5% sugar,4.5% fructose and glucose syrup,0.06% citric acid,0.04% xanthan gum and 0.006% ethyl maltol respectively. The content of polysaccharide in orally fluid product was 3.3g/100mL.