樟芝深层液体发酵工艺及其多糖特性的研究
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摘要
樟芝(Antrodia camphorata)是新近发现的一种食药用真菌,因为其子实体具有的生物活性作用,引起广泛的关注；出于对保护环境、自然资源匮乏和子实体栽培技术尚未成功等因素的考虑,近年来,樟芝菌丝体深层发酵培养的研究进展迅速。多糖是樟芝中主要的生物活性因子,对樟芝深层发酵营养保健产品研究和开发有着重要的意义。本文研究内容包括樟芝深层发酵生物学特性、樟芝多糖和菌丝体产量为指标的最佳培养基配方的筛选、樟芝发酵工艺、樟芝菌丝体多糖提取工艺、樟芝深层发酵菌丝体营养成分分析,以及樟芝菌丝体多糖的理化特性、功能学特性等品质学性质的研究,并在此基础上开发出樟芝菌丝体多糖口服液。主要研究结果如下：
(1)樟芝菌株鉴定rDNA序列分析
樟芝菌株rDNA的ITS序列在NCBI上BLAST,进行同源性比对达99%以上,可判断所用菌株确实为樟芝菌株Antrodia camphorata。
(2)樟芝菌丝体深层发酵生物学特性的研究
十五种碳源对比实验结果表明：采用天然多糖类物质(米粉、面粉、玉米粉、甘薯粉、木薯粉)为培养基碳源,其樟芝菌丝体产量,明显高于双糖和单糖(麦芽糖、蔗糖、葡萄糖、果糖、木糖、半乳糖、阿拉伯糖、甘露糖),也高于化学制剂多糖(可溶性淀粉、微晶纤维素)。
三类氮源,对樟芝菌丝体产量的影响结果显示：有机氮高于铵态氮,铵态氮高于硝态氮。即有机氮(黄豆粉>玉米粉>蛋白胨)>铵态氮(酒石酸铵>氯化铵>磷酸氢二铵>硫酸铵)>硝态氮,樟芝不能利用硝态氮。
樟芝菌丝生长最适合的碳氮比(C/N)为50：1～100：1；常量元素钠离子和镁离子对樟芝菌丝生长并不是必需的；钙、硫、磷、钾离子是樟芝菌丝生长所必需的,缺乏钾离子将显著地影响菌丝生长。微量元素Co对菌丝生长有抑制作用；分别添加6种维生素(生物素、核黄素、硫胺素、尼克酸、叶酸、抗坏血酸)对樟芝菌丝生长没有显著影响；樟芝菌丝适于酸性和中性条件下生长,pH4-8之间菌丝生长速度差异不显著,都生长较好；pH9以上菌丝不能生长；光照与黑暗下樟芝菌丝的生长没有显著差异。
(3)樟芝菌丝体深层发酵培养基配方和发酵动力学的研究
以樟芝菌丝体含量和樟芝多糖含量作为指标,采用L27(313)五因素三水平的正交设计,确定樟芝菌的深层发酵最佳的发酵培养基配方为：马铃薯20%+(黄豆粉0.5%+玉米粉0.5%)+(酵母膏0.15%+蛋白胨0.15%)+麸皮0.2%+(KH2PO40.05%+MgSO40.035%)。
以樟芝菌丝体含量和樟芝多糖含量作为指标,采用U12(1212)三因素四水平的均匀设计,确定最佳的发酵参数：接种量为15%(V/V),种子菌龄为8d,培养温度为26℃。同时获得樟芝菌丝体干重为11克/升发酵液。通过发酵动力学的研究,掌握了樟芝发酵过程各项指标的变化规律：发酵4天以后,菌丝开始快速生长,氨态氮水平降低。在发酵后的第16天,菌丝生长到达了稳定期,之后有些老菌丝开始自溶,使氨态氮又有所回升。这一转折点作为菌丝对数生长转入稳定期的标志。
(4)樟芝多糖提取工艺研究
樟芝菌丝体中粗脂肪含量为11.61%(m/m),其中饱和脂肪酸占12.6%(v/v),不饱和脂肪酸占87.4%(v/v)；樟芝菌丝体中粗蛋白含量为34.20%(m/m),总氨基酸含量为270.68mg/kg (m/m),达FAO标准的26.6倍,含有人体所需的8种必需氨基酸;樟芝菌丝体含常量元素K、Na、P、Ca、Mg和多种人体所需的微量元素Fe、Zn/ Mn、Cu、Mo、Cr、Se、Sr等；樟芝菌丝体中麦角甾醇的含量为19.55mg/g (m/m),总三萜的含量为2.81mg/g (m/m)。
樟芝菌丝浸提的次数为2次时,可提取其中多糖的94%；利用超低温冷冻方法处理(冻融破壁作用)对樟芝多糖的提取具有很好的效果,其它方法处理的效果(超声波处理、液氮处理、冷冻干燥处理等)都比对照菌丝干燥法有不同程度的提高。
利用响应面法优化樟芝多糖提取工艺和条件。建立了樟芝多糖提取方法的二次多项数学模型,验证了模型的有效性,并探讨了温度、时间、水料比三个因子的交互作用及其最佳水平范围,优化出樟芝多糖提取的外界条件为：温度120℃,时间110.4min,水料比.1：1.89。
(5)樟芝菌丝体、发酵液多糖性质的研究
对樟芝菌丝体提取液、樟芝发酵液在不同乙醇浓度下沉淀得到的多糖的理化性质、红外光谱和凝胶色谱特征等进行研究,结果显示：樟芝菌丝体多糖为棕褐色蓬松粉末,质轻,无特殊气味,易溶于水,不溶于乙醇、乙醚、丙酮等有机溶剂,其比旋光度为48.31；樟芝菌丝体多糖与苯酚-硫酸试剂反应呈红色,与蒽酮试剂反应呈蓝绿色,与间苯二酚试剂反应呈紫红色,与KI-12不显色。红外光谱测定表明,不同乙醇浓度梯度沉淀的樟芝菌丝体、发酵液多糖具有多糖的特征吸收,并且其糖环为吡喃环。分步沉淀的樟芝菌丝体、发酵液多糖经高效液相色谱GPC柱分离各得到3-4个峰,计算得各组分的数均分子量和重均分子量。不同浓度乙醇沉淀处理对樟芝菌丝体、发酵液多糖分子量的影响表现在低浓度(65%)乙醇沉淀出较大分子量的多糖组分,而高浓度(75%、85%)乙醇沉淀出较小分子量的多糖组分。较高浓度乙醇沉淀的多糖,不管是樟芝发酵液多糖还是樟芝菌丝体多糖,体外试验表明都具有较高的清除超氧自由基的活性。
樟芝菌丝体多糖ACP1和樟芝发酵液多糖ACP2的多糖含量分别为31.76%和38.09%,樟芝菌丝体多糖、发酵液多糖的提取率分别为1.59%和4.89%。樟芝菌丝体多糖ACP1和樟芝发酵液多糖ACP2的特性粘度[η]为1.982和.1.657。
樟芝菌丝体多糖ACP1和樟芝发酵液多糖ACP2经高效液相色谱GPC柱分离后,各得到三个峰,高效液相色谱测定证明樟芝菌丝体、发酵液多糖中已完全除去蛋白质和小分子还原糖,因此,凝胶色谱图之间的色谱峰为大分子多糖。采用GPC软件计算样品各组分的数均分子量和重均分子量。樟芝菌丝体多糖ACP1和樟芝发酵液多糖ACP2为灰白色疏松纤维状粉末,红外光谱图及谱峰归属分析得出两种多糖样品均具有多个多糖特征吸收峰。
樟芝菌丝体多糖ACP1和樟芝发酵液多糖ACP2的浓度达3g/L时,超氧自由基的清除率分别达到74.8%和76.9%。ACP1和ACP2经凝胶葡聚糖sephadexG-200柱层析分别洗脱出的精多糖单一主峰ACP1-1和ACP2-1,其超氧自由基的清除率分别达到54.7%和61.6%,说明樟芝菌丝体多糖和发酵液多糖具有较强的体外抗氧化能力。
(6)樟芝菌丝体、发酵液多糖生物活性的研究
樟芝菌丝体多糖和发酵液多糖对小鼠宫颈癌U14有显著抑制作用,其肿瘤抑制率分别为44.7%和77.2%,樟芝发酵液多糖的抑制率显著高于樟芝菌丝体多糖的抑制率。
选择樟芝菌丝体多糖(胞内ACP1)和樟芝发酵液多糖(胞外ACP2)的低、中、高剂量组,分别为100、400、1600mg/kgBW三个剂量,连续灌胃30天,进行小鼠免疫功能试验。试验结果说明樟芝菌丝体多糖和樟芝发酵液多糖(ACP1、ACP2)对小鼠的细胞免疫、体液免疫、单核-巨噬细胞功能、NK细胞活性四个方面均有增强作用,且有剂量效应关系；樟芝发酵液多糖这四个方面的免疫功能都高于樟芝菌丝体多糖。
(7)樟芝多糖口服液的研制
以感官评价分数作为评定指标,采用正交实验设计,优化樟芝发酵多糖口服液的制备工艺。口服液中添加混合糖9.5%(白糖5%+果葡糖浆4.5%)、柠檬酸0.06%、黄原胶0.04%、乙基麦芽酚0.006%时,樟芝多糖口服液感官评价分最高,达92分。樟芝发酵多糖口服液中多糖含量达3.3g/100mL。
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 rDNA
The 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 camphorata
Among 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 dynamics
The 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 camphorata
To 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 Camphorata
The 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 polysaccharide
The 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.
(1)樟芝菌株鉴定rDNA序列分析
樟芝菌株rDNA的ITS序列在NCBI上BLAST,进行同源性比对达99%以上,可判断所用菌株确实为樟芝菌株Antrodia camphorata。
(2)樟芝菌丝体深层发酵生物学特性的研究
十五种碳源对比实验结果表明：采用天然多糖类物质(米粉、面粉、玉米粉、甘薯粉、木薯粉)为培养基碳源,其樟芝菌丝体产量,明显高于双糖和单糖(麦芽糖、蔗糖、葡萄糖、果糖、木糖、半乳糖、阿拉伯糖、甘露糖),也高于化学制剂多糖(可溶性淀粉、微晶纤维素)。
三类氮源,对樟芝菌丝体产量的影响结果显示：有机氮高于铵态氮,铵态氮高于硝态氮。即有机氮(黄豆粉>玉米粉>蛋白胨)>铵态氮(酒石酸铵>氯化铵>磷酸氢二铵>硫酸铵)>硝态氮,樟芝不能利用硝态氮。
樟芝菌丝生长最适合的碳氮比(C/N)为50：1～100：1；常量元素钠离子和镁离子对樟芝菌丝生长并不是必需的；钙、硫、磷、钾离子是樟芝菌丝生长所必需的,缺乏钾离子将显著地影响菌丝生长。微量元素Co对菌丝生长有抑制作用；分别添加6种维生素(生物素、核黄素、硫胺素、尼克酸、叶酸、抗坏血酸)对樟芝菌丝生长没有显著影响；樟芝菌丝适于酸性和中性条件下生长,pH4-8之间菌丝生长速度差异不显著,都生长较好；pH9以上菌丝不能生长；光照与黑暗下樟芝菌丝的生长没有显著差异。
(3)樟芝菌丝体深层发酵培养基配方和发酵动力学的研究
以樟芝菌丝体含量和樟芝多糖含量作为指标,采用L27(313)五因素三水平的正交设计,确定樟芝菌的深层发酵最佳的发酵培养基配方为：马铃薯20%+(黄豆粉0.5%+玉米粉0.5%)+(酵母膏0.15%+蛋白胨0.15%)+麸皮0.2%+(KH2PO40.05%+MgSO40.035%)。
以樟芝菌丝体含量和樟芝多糖含量作为指标,采用U12(1212)三因素四水平的均匀设计,确定最佳的发酵参数：接种量为15%(V/V),种子菌龄为8d,培养温度为26℃。同时获得樟芝菌丝体干重为11克/升发酵液。通过发酵动力学的研究,掌握了樟芝发酵过程各项指标的变化规律：发酵4天以后,菌丝开始快速生长,氨态氮水平降低。在发酵后的第16天,菌丝生长到达了稳定期,之后有些老菌丝开始自溶,使氨态氮又有所回升。这一转折点作为菌丝对数生长转入稳定期的标志。
(4)樟芝多糖提取工艺研究
樟芝菌丝体中粗脂肪含量为11.61%(m/m),其中饱和脂肪酸占12.6%(v/v),不饱和脂肪酸占87.4%(v/v)；樟芝菌丝体中粗蛋白含量为34.20%(m/m),总氨基酸含量为270.68mg/kg (m/m),达FAO标准的26.6倍,含有人体所需的8种必需氨基酸;樟芝菌丝体含常量元素K、Na、P、Ca、Mg和多种人体所需的微量元素Fe、Zn/ Mn、Cu、Mo、Cr、Se、Sr等；樟芝菌丝体中麦角甾醇的含量为19.55mg/g (m/m),总三萜的含量为2.81mg/g (m/m)。
樟芝菌丝浸提的次数为2次时,可提取其中多糖的94%；利用超低温冷冻方法处理(冻融破壁作用)对樟芝多糖的提取具有很好的效果,其它方法处理的效果(超声波处理、液氮处理、冷冻干燥处理等)都比对照菌丝干燥法有不同程度的提高。
利用响应面法优化樟芝多糖提取工艺和条件。建立了樟芝多糖提取方法的二次多项数学模型,验证了模型的有效性,并探讨了温度、时间、水料比三个因子的交互作用及其最佳水平范围,优化出樟芝多糖提取的外界条件为：温度120℃,时间110.4min,水料比.1：1.89。
(5)樟芝菌丝体、发酵液多糖性质的研究
对樟芝菌丝体提取液、樟芝发酵液在不同乙醇浓度下沉淀得到的多糖的理化性质、红外光谱和凝胶色谱特征等进行研究,结果显示：樟芝菌丝体多糖为棕褐色蓬松粉末,质轻,无特殊气味,易溶于水,不溶于乙醇、乙醚、丙酮等有机溶剂,其比旋光度为48.31；樟芝菌丝体多糖与苯酚-硫酸试剂反应呈红色,与蒽酮试剂反应呈蓝绿色,与间苯二酚试剂反应呈紫红色,与KI-12不显色。红外光谱测定表明,不同乙醇浓度梯度沉淀的樟芝菌丝体、发酵液多糖具有多糖的特征吸收,并且其糖环为吡喃环。分步沉淀的樟芝菌丝体、发酵液多糖经高效液相色谱GPC柱分离各得到3-4个峰,计算得各组分的数均分子量和重均分子量。不同浓度乙醇沉淀处理对樟芝菌丝体、发酵液多糖分子量的影响表现在低浓度(65%)乙醇沉淀出较大分子量的多糖组分,而高浓度(75%、85%)乙醇沉淀出较小分子量的多糖组分。较高浓度乙醇沉淀的多糖,不管是樟芝发酵液多糖还是樟芝菌丝体多糖,体外试验表明都具有较高的清除超氧自由基的活性。
樟芝菌丝体多糖ACP1和樟芝发酵液多糖ACP2的多糖含量分别为31.76%和38.09%,樟芝菌丝体多糖、发酵液多糖的提取率分别为1.59%和4.89%。樟芝菌丝体多糖ACP1和樟芝发酵液多糖ACP2的特性粘度[η]为1.982和.1.657。
樟芝菌丝体多糖ACP1和樟芝发酵液多糖ACP2经高效液相色谱GPC柱分离后,各得到三个峰,高效液相色谱测定证明樟芝菌丝体、发酵液多糖中已完全除去蛋白质和小分子还原糖,因此,凝胶色谱图之间的色谱峰为大分子多糖。采用GPC软件计算样品各组分的数均分子量和重均分子量。樟芝菌丝体多糖ACP1和樟芝发酵液多糖ACP2为灰白色疏松纤维状粉末,红外光谱图及谱峰归属分析得出两种多糖样品均具有多个多糖特征吸收峰。
樟芝菌丝体多糖ACP1和樟芝发酵液多糖ACP2的浓度达3g/L时,超氧自由基的清除率分别达到74.8%和76.9%。ACP1和ACP2经凝胶葡聚糖sephadexG-200柱层析分别洗脱出的精多糖单一主峰ACP1-1和ACP2-1,其超氧自由基的清除率分别达到54.7%和61.6%,说明樟芝菌丝体多糖和发酵液多糖具有较强的体外抗氧化能力。
(6)樟芝菌丝体、发酵液多糖生物活性的研究
樟芝菌丝体多糖和发酵液多糖对小鼠宫颈癌U14有显著抑制作用,其肿瘤抑制率分别为44.7%和77.2%,樟芝发酵液多糖的抑制率显著高于樟芝菌丝体多糖的抑制率。
选择樟芝菌丝体多糖(胞内ACP1)和樟芝发酵液多糖(胞外ACP2)的低、中、高剂量组,分别为100、400、1600mg/kgBW三个剂量,连续灌胃30天,进行小鼠免疫功能试验。试验结果说明樟芝菌丝体多糖和樟芝发酵液多糖(ACP1、ACP2)对小鼠的细胞免疫、体液免疫、单核-巨噬细胞功能、NK细胞活性四个方面均有增强作用,且有剂量效应关系；樟芝发酵液多糖这四个方面的免疫功能都高于樟芝菌丝体多糖。
(7)樟芝多糖口服液的研制
以感官评价分数作为评定指标,采用正交实验设计,优化樟芝发酵多糖口服液的制备工艺。口服液中添加混合糖9.5%(白糖5%+果葡糖浆4.5%)、柠檬酸0.06%、黄原胶0.04%、乙基麦芽酚0.006%时,樟芝多糖口服液感官评价分最高,达92分。樟芝发酵多糖口服液中多糖含量达3.3g/100mL。
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 rDNA
The 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 camphorata
Among 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 dynamics
The 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 camphorata
To 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 Camphorata
The 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 polysaccharide
The 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.
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