姬松茸综合利用关键技术研究与应用
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摘要
姬松茸是重要的食药用菌，含有丰富的蛋白质、多糖、多不饱和脂肪酸及膳食纤维，具有抑制肿瘤、降血糖、减少低密度胆固醇沉积、提高机体免疫力等功能。长期以来，对姬松茸的深加工主要是提取姬松茸多糖，而提取多糖后的姬松茸蛋白、姬松茸膳食纤维等副产物未得到充分利用，造成大量优质蛋白、膳食纤维的损失和浪费。另外，食用菌功能性油脂的研究和开发更未见报道。姬松茸资源利用率低，加工深度不够，缺乏高科技含量、高附加值及市场潜力大的新型产品。本研究在对国内外食用菌深加工现状及发展趋势进行研究的基础上，充分考虑原料的综合利用及加工过程对环境的影响，对姬松茸中特异性成分提取关键技术、功能因子制取关键技术及各种功能因子的功能特性与应用进行了深入的研究，实现原料全利用，符合国家倡导的资源节约型及环境友好型生产模式，可为姬松茸乃至其他食用菌的综合利用与精深加工提供理论依据及实践性的参考与借鉴。
主要研究内容及结果如下：
1．超临界CO_2流体选择性萃取分离技术提取姬松茸ω-6多不饱和脂肪酸。经正交试验分析确定最佳工艺条件为萃取压力25MPa，温度50℃，时间60min，CO_2流量25L·h~(-1)。萃取量为4．03g·(100g)~(-1)，萃取后姬松茸含脂率0．01％。为姬松茸多糖的提取及姬松茸蛋白肽等功能因子的提取或制取提供良好的前提条件。
2．姬松茸超临界CO_2流体萃取物的脂肪酸组成特征为ω-6多不饱和脂肪酸型，亚油酸含量为52．4％，γ-亚麻酸21．12％，油酸12．89％，软脂酸7．98％，硬脂酸未检出，ω-6多不饱和脂肪酸总量为73．52％，可作为保健食品姬松茸ω-6多不饱和脂肪酸(Agaricus blazeiMurrillω-6 polyunsaturated fatty acid，ω-6APFA)的加工原料。
3．大鼠60只，随机分为空白对照组、模型组、天然大豆磷脂胶囊(NSPC)0．70 g·kg~(-1)剂量组和ω-6APFA 0．28、0．14、0．07 g·kg~(-1)3个剂量组(n=10)。除空白对照组外均连续给予高脂饲料14d，于第14d起开始连续灌胃给药14d，对照组和模型组给予等容积蒸馏水10 mL·kg~(-1)。实验结束后麻醉大鼠，腹主动脉取血测血清总胆固醇(T-CHO)、甘油三酯(TG)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)。同时测肝脏超氧化物歧化酶(SOD)活性、肝脏丙二醛(MDA)含量及腹腔脂肪蓄积系数。雄性小鼠72只，随机分为空白对照组、模型组、NSPC 1．0g·kg~(-1)剂量组、ω-6APFA 0．4、0．2、0．1 g·kg~(-1)3个剂量组(n=12)，连续给药7d，末次给药前16h除空白对照组，均腹腔注射75％蛋黄生理盐水液0．5 mL，并开始饥饿，末次给药后1h，于眼球采血，测血清总胆固醇及甘油三酯含量。试验结果：与模型组相比，NSPC 0．70 g·kg~(-1)和ω-6APFA 0．28 g·kg~(-1)剂量组大鼠血清总胆固醇、甘油三酯均明显降低，血清HDL-C升高(P＜0．001)，NSPC 0．70 g·kg~(-1)和ω-6APFA 0．28、0．14 g·kg~(-1)剂量组大鼠肝组织SOD活性升高(P＜0．05、P＜0．001及P＜0．05)，MDA降低(P＜0．01、P＜0．001及P＜0．01)，大鼠腹腔脂肪系数均降低(P＜0．01)。与模型组相比，NSPC 1．0 g·kg~(-1)、ω-6APFA 0．4、0．2 g·kg~(-1)组急性高血脂小鼠T-CHO和TG明显降低(P＜0．05、P＜0．01及P＜0．05)。试验结果表明ω-6APFA对高血脂鼠有明显降血脂作用。
4．60只大鼠随机分为空白对照组(n=12)、天然大豆磷脂胶囊(NSPC)0．70 g·kg~(-1)·d~(-1)剂量组(n=12)和ω-6APFA 0．07、0．14、0．28g·kg~(-1)·d~(-1)3个剂量(n=12)组，各组均连续灌胃给药7d，末次给药后1h于动物腹腔注射300mg·kg~(-1)水合氯醛麻醉，背位固定，分离右侧颈总动脉，应用实验性体内血栓形成测定仪，检测血栓形成时间。另取60只小鼠，随机分为空白对照组(n=12)、NSPC 1．0 g·kg~(-1)·d~(-1)剂量组(n=12)和ω-6APFA0．1、0．2、0．4 g·kg~(-1)·d~(-1)3个剂量组(n=12)，各组均连续灌胃给药7d，于末次给药后1h小鼠眶静脉采血，采用玻片法测定凝血时间。结果NSPC 0．7 g·kg~(-1)d~(-1)剂量组及ω-6APFA0．14、0．28g·kg~(-1)·d.(-1)剂量组，均对大鼠体内血栓形成有明显抑制作用，与对照组相比P＜0．05，且ω-6APFA0．14、0．28g·kg~(-1)·d.(-1)剂量组优于NSPC 0．7 g·kg~(-1)·d.(-1)剂量组。NSPC 1．0 g·kg~(-1)d~(-1)剂量组及ω-6APFA0．2、0．4g·kg~(-1)·d.(-1)剂量组，均能延长小鼠凝血时间，与对照组相比，NSPC 1．0 g·kg~(-1)剂量组P＜0．05，ω-6APFA 0．2、0．4g·kg~(-1)·d.(-1)剂量组P＜0．01，说明ω-6APFA0．2、0．4g·kg~(-1)·d.(-1)剂量组优于NSPC 1．0 g·kg~(-1)·d.(-1)剂量组。试验结果表明ω-6APFA有很好的抑制血栓形成及活血化瘀作用。
5．采用超临界萃取脱脂处理的姬松茸为原料，采用微波萃取：技术提取姬松茸多糖，大幅度缩短萃取时间，降低液固比，减少后续浓缩工艺能耗。微波萃取姬松茸活性多糖时微波萃取功率影响最大，其次是微波萃取时间，然后是物料粒度，固液比影响最小，最佳工艺条件为微波萃取功率600W，微波萃取时间6min，固液比1：10，物料粒度0．147mm。按此工艺进行萃取，姬松茸多糖提取率高达14．2％。与有关报道相比多糖得率提高29．68％，用水量为其1／3～1／2．浸提时间为其1／30，实现节水省时降耗。
6．用D315树脂脱蛋白纯化姬松茸粗多糖效果理想，控制流出速度0．5mL·min~(-1)，蛋白脱除率达79．26％，多糖回收率达86．3％。
7．采用葡聚糖凝胶G75柱层析对脱蛋白的姬松茸多糖进一步纯化、分离，控制流速0．3mL·min~(-1)，得到分子量10万左右的β-葡聚糖，纯度为85％，此分子量范围的多糖生物活性较高。
8．考察AL蛋白酶、木瓜蛋白酶、胰蛋白酶、FW蛋白酶四种酶制剂对姬松茸蛋白的水解情况，根据对蛋白水解能力及酶解液分子量分布确定使用AL蛋白酶和FW蛋白酶对原料进行水解制取低聚肽。
9．利用AL蛋白酶和FW蛋白酶对姬松茸蛋白进行分步水解，AL蛋白酶最佳酶解条件为pH8．5，温度55℃，酶与底物比2％，底物浓度5％，水解时间2h，肽得率74．7％。以AL蛋白酶酶解产物为反应底物加入FW蛋白酶进行第2次酶解，最佳条件为pH值7．0，温度50℃，酶与底物比4％，水解时间1．5h，肽得率80．6％。
10．检测结果表明姬松茸低聚肽粉中肽含量为94．2％，总糖0．1％，脂肪未检出，水分5．2％，灰分0．5％。姬松茸低聚肽富含脯氨酸、赖氨酸、苯丙氨酸，必需氨基酸含量高达50．91％。
11．通过Sephadex G-25凝胶色谱分离，酶解物被分成3个组分，分子量集中在5600Da以下。
12．功能试验结果表明姬松茸低聚肽能明显降低高血脂模型动物血清T-CHO和TG含量，对高血脂模型大鼠血清中HDL-C含量降低有明显的抑制作用；对高血脂模型大鼠血清中LDL-C含量升高有明显的降低作用；并能明显的提高动物肝脏SOD水平，降低肝脏MDA含量。姬松茸低聚肽对高脂饮食所致小鼠血脂升高和DL-E所致肝脂肪蓄积有明显的改善作用；姬松茸低聚肽能明显增加小鼠脾、胸腺脏器系数，增加小鼠单核巨噬细胞系统吞噬功能，延长小鼠在水中的存活时间。说明姬松茸低聚肽具有降脂、保肝、提高免疫力及抗疲劳作用。
13．以脱脂、提多糖、制取低聚肽之后剩余的姬松茸残渣为原料，制取高品质食用菌膳食纤维，提高姬松茸原料的综合利用价值。采用高温高压挤出技术对原料进行处理，改善物料的理化特性，最佳处理条件为物料含水量80％、挤出温度145℃、进料速度20Kg·h~(-1)，螺杆转速220r·min~(-1)。未进行挤出改性处理的普通姬松茸纤维(ADF)的可溶性膳食纤维(SDF)含量为2．4％，膨胀力为4．0 mL·g~(-1)，持水力为3．1 g·g~(-1)，结合水力为2．7g·g~(-1)；挤出处理后SDF含量为15．1％，膨胀力为18．3mL·g~(-1)，持水力为9．4g·g~(-1)，结合水力为6．4g·g~(-1)，成为品质优良的高品质膳食纤维(HQADF)。
14．采用正常小鼠和燥结型便秘小鼠模型，灌服给药，并监测HQADF对正常小鼠胃小肠运动功能的推进作用，及对正常和燥结型便秘小鼠的排便量及排便时间的影响。结果：HQADF3．50、1．75g·kg~(-1)剂量组对正常小鼠的胃肠运动功能都有明显的促进作用，推进率分别提高17．80％、16．85％；3．50、1．75g·kg~(-1)bw剂量组都能明显增加正常小鼠的排便量，缩短小鼠的排黑便时间，排便量分别增加54．32％、32．28％，排黑便时间分别缩短26．87％、19．70％；能明显增加燥结型便秘小鼠的排便量，以3．50g·kg~(-1)bw剂量组效果最好，口服4h内排便量增加73．44％。试验结果表明HQADF具有明显的抗便秘作用。
15．小鼠50只随机分为空白对照组、四氧嘧啶模型组和HQADF 3．50、1．75、0．88g·kg~(-1)体重3个剂量组，每天给予HQADF 1次，连续15d，于第6d除空白对照组外，各组小鼠均尾静脉注射四氧嘧啶生理盐水溶液80mg·kg~(-1)体重，空白对照组尾静脉注射同体积生理盐水。末次给药前16h将所有各组小鼠饥饿，末次给药后1h，于小鼠眼球采血，分离血清，采用全自动生化分析仪测血糖含量；立即处死小鼠，取肝脏测小鼠肝糖元含量。另取60只小鼠，分组方法同上，每天给予HQADF 1次，连续10d。除空白对照组外，其余各组均在末次给予HQADF后1h腹腔注射盐酸肾上腺素250μg·kg~(-1)体重，30min后断头取血，测血糖含量，取肝组织测肝糖元含量。结果：HQADF 3．50g·kg~(-1)体重对四氧嘧啶糖尿病小鼠(模型鼠)血糖升高有明显的抑制作用(P＜0．05)；对四氧嘧啶糖尿病小鼠(模型组)肝糖元含量有明显的降低作用(P＜0．05)，且3．50 g·kg~(-1)体重优于1．75 g·kg~(-1)体重。3．50、1．75、0．88g·kg~(-1)体重剂量组对肾上腺素小鼠血糖(模型组)升高均有明显的抑制作用(P＜0．001、P＜0．01和P＜0．05)；对肾上腺素造成小鼠(模型组)肝糖元降低有明显的升高作用，且3．50g·kg~(-1)体重优于1．75g·kg~(-1)体重。试验结果表明HQADF对四氧嘧啶致糖尿病小鼠及肾上腺素血糖升高小鼠具有明显的降血糖作用。
16．取80只小鼠，雌雄各半，随机分为5组：空白对照组，肥胖模型组，HQADF 3．50 g·kg~(-1)、1．75 g·kg~(-1)、0．88 g·kg~(-1)3个剂量组，除空白对照组给正常饲料外，其余各组均服用肥胖饲料，每天给药1次，连续给药35天，每隔7天称1次体重。另取80只小鼠，雌雄各半，分组、饲喂及称量体重方法同上，末次给药后24小时，将小鼠处死，取腹部脂肪称湿重，计算脂肪指数。再另取80只小鼠，雌雄各半，分组、饲喂及称量体重方法同上，末次给药后24小时，摘除小鼠眼球取血，采用日立7080全自动生化分析仪检测有关生化指标。试验结果表明：HQADF能明显降低肥胖小鼠体重、脂肪指数、总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)、血糖(GLU)含量，对肥胖小鼠血清中高密度脂蛋白(HDL-C)有明显的升高作用，证明HQADF具有明显减肥降脂作用。
17．姬松茸膳食纤维(ADF)用于面包生产时必需经过适当方法处理才能改善面包品质。超临界流体萃取纯化处理、微波萃取多糖及制取姬松茸低聚肽后进行挤出是处理姬松茸纤维较为理想的方法。姬松茸膳食纤维采用挤出处理，粒度为0．175mm，用量为15％时面包综合品质最好，且面包添加剂用量最少(1．0％)。由于挤出处理改善了姬松茸膳食纤维的品质，不但可在面团中大量添加这种优良的食用菌膳食纤维，赋予面包产品特殊的保健功能，而且由于纤维的粒度并非越小越好，降低了纤维制粉的能耗。
Agaricus blazei Murill , a kind of important medical and edible mushroom, it is rich in proteins ,polysaccharides , polyunsaturated fatty acids and dietary fibers ,it also has the effect of inhibiting tumour,depressing the blood glucose , decreasing deposition of low density cholesterol and boosting organismimmunity and so on. For a long time, deep processing on Agaricus blazei Murrill was extraction ofpolysaccharides mainly, and proteins of Agaricus blazei, fibers of Agaricus blazei and the other by-productswere not utilized fully, the results caused large losses or wastes of quality protein and fiber. In addition, thestudy and development on functional oil in edible fungus was not reported even more.Availability of Agaricusblazei Murill is low, processing depth is insufficient, lacking high technology content, lacking high addedvalue and new product with great market potential. This research is on the base of the research to the domesticand foreign edible mushroom deep processing present situation and the development tendency, fullyconsidering the raw material comprehensive utilization and the influence of processing to environment, alsocritical technique of extracting Agaricus blazei Murill specificity ingredient and function factor, each kind offactor functional characteristic and the application has conducted the thorough research, implementing rawmaterial entire use, conforming to national initiative resource conservation and the environment friendlyproduction pattern, will provide the theory basis and the practical reference and the model for Agaricus blazeiMurill and even other edible mushroom comprehensive utilizations and the profound processing.
The main contents and results as follow:
1.ω-6 polyunsaturated fatty acids were extracted by supercritical fluid extraction technology from Agaricusblazei Murill , through the analysis of orthogonal test, the optimum condition was determined: extractingpressure was 25 MPa, extracting temperature was 50℃, extracting time was 60 min, CO_2 volume was 25L·h~(-1).The yields of extraction were 4.03g·(100g)~(-1), lipid contents in Agaricus blazei Murill was 0.01% afterextraction. This research results provided good precondition for extracting and producing Agaricus blazeiMurill peptide and other functional factors.
2. Compositional characteristic of Agaricus blazei Murill fatty acid after supercritical fluid extraction was typeofω-6 polyunsaturated fatty acid, and the content of linoleic acid was 52.4%, the content ofγ-linolenic acidwas 21.12%, the content of oleic acid was 12.89%, the content of palmitic acid was 7.98%, stearic acid wasnot detected out, the total cintents ofω-6 polyunsaturated fatty acids were 73.52%,it may be as the materialfor producing health food which was Agaricus blazei Murrillω-6 polyunsaturated fatty acid(ω-6APFA).
3. Sixty rats wererandomly divided into control group, model group, natural soybean phospholipids capsule(NSPC)0.70g·kg~(-1) group,ω-6APFA 0.28, 0.14, 0.07 g·kg~(-1) groups(n=10). The other groups excepting control group were given high-fat diets for 14 d, on the fourteenth the rats began to be administered orally, the controlgroup and model group were administered distilled water 10ml·kg~(-1) at the same volume, after 14 days ofcontinuously administration ,rats were anesthetized, the blood extracted in abdominal artery , T-CHO, TG,HDL-C, LDL-C in sera were determined. At the same time, the activity of SOD in liver and the content ofMDA was determined, the fat accumulated coefficient was calculated. 72 male mice were randomly dividedinto control group, model group, NSPC 1.0g·kg~(-1) group,ω-6APFA 0.4, 0.2, 0.1 g·kg~(-1) groups (n=12). Micewere administered continuously,16h before the last administration ,except oontrol group, the other groups wasinjected 75% yolk physiological salt solution 0.5mL through the abdominal cavity, and began to starve ,1 hafter the last administration, blood was extracted in eyeball , sera total cholesterol and triglyceride weredetermine. Compare with model group, T-CHO and TG in rat of NSPC 0.70g·kg~(-1) andω-6APFA 0.28 g·kg~(-1) allreduced and HDL-C raised obviously(P＜0.001), activity liver SOD of NSPC 0.70g·kg~(-1) andω-6APFA0.28、0.14 g·kg~(-1) obvious raised (P＜0.05, P＜0.001 and P＜0.05), MDA reduced obviously(P＜0.01, P＜0.001, P＜0.01), fat accumulation coefficient of NSPC 0.70g·kg~(-1) andω-6APFA reduced obviously. Comparewith model mice, T-CHO and TG in acute hyperlipemia mice of NSPC 1.0g·kg~(-1) andω-6APFA0.4、0.2 g·kg~(-1)reduced obviously(P＜0.05, P＜0.01 and P＜0.05).The results indicate thatω-6APFA has the function oflowering blood fat on hyperlipemia rats and mice.
4. Sixty rats were randomly divided into control group, natural soybean phospholipids capsule(NSPC)0.70g·kg~(-1)·d~(-1)group ,ω-6APFA 0.07, 0.14,0.28g·kg~(-1)·d~(-1)groups (n=12).Rats in each group were exhibitedonce a day for 7 d,1 h after the last exhibition ,rats were injected chloral hydrate 300g·kg~(-1) in abdominal cavityto anesthetize, the back were fixed, the carotid of the right neck were separated, and the time of thrombosisformation were determined by experimental intracorporeal thrombosis surveyor. Another 60 mice wererandomly divided into control group, NSPC 1.0g·kg~(-1)·d~(-1)group,ω-6APFA0.1, 0.2,0.4 g·kg~(-1)·d~(-1) groups(n=12).Mice were exhibited once a day for 7 d continuously, 1h after the last exhibition, blood was extracted in theorbit vein, the clotting time were determined through glass slides method. NSPC 0.7 g·kg~(-1)·d~(-1) andω-6APFA0.14,0.28g·kg~(-1)·d~(-1)all had obvious inhibitory effect on thrombus formation in blood stasis mice,compare with model group, P＜0.05, andω-6APFA0.14,0.28g·kg~(-1)·d~(-1) were superior to NSPC 0.7g·kg~(-1)·d~(-1).NSPC 1.0 g·kg~(-1)·d~(-1) andω-6APFA0.2,0.4g·kg~(-1)·d~(-1) all could prolong the clotting time ,compare with modelgroup, NSPC 1.0 g·kg~(-1)·d~(-1) was P＜0.05,ω-6APFA0.2, 0.4g·kg~(-1)·d~(-1) were P＜0.01,the result showed thatω-6APFA0.2, 0.4g·kg~(-1)·d~(-1) were superior to NSPC 1.0g·kg~(-1)·d~(-1) group. The results indicate thatω-6APFAhad the function of inhibiting thrombus formation, promoting blood flow ability and lowering blood viscosity.
5. Used defatted Agaricus biazei Murill through Supercritical fluid extraction as material, and used themicrowave extract technology to extract the Agaricus blazei Murill polysaccharides, the extract time wasshorten substantially, proportion of fluid to solid was reduced, and the following concentration energyconsumption was decreased. When Agaricus blazei Murill active polysaccharides were extracted through microwave technique, the influence of microwave power was biggest, next was the microwave time, and thenwas the material size, the influence of proportion of solid to fluid was the smallest, the optimum condition wasdetermined: microwave power was 600W, microwave time was 6 min, the proportion of solid to fluid was1 : 10, material size was 0.147 mm. According this technology to extract Agaricus blazei Murill activepolysaccharides, the extraction rate was 14.2%.The yield was raised 29.68% compared with reported before,and the volume of water was 1/3 or 1/2 of reported before, the extractive times were 1/30 of reported before,so the water ,times and energy were saved all.
6. Removed proteins and purified Agaricus blazei Murill polysaccharides by D315 resin, and geted the idealresult, rate of removing proteins is 79.26%, rate of recovering in polysaccharides is 86.3%.
7. PurifIed and separated Agaricus blazei Muril polysaccharides by Sephadex G75 gelatum, and removedproteins further, controled speed of flow, the flow was 0.3mL·min~(-1), obtainedβ-the glucosan which molecularweight was about 100000, the purity was 85%, the polysaccharides belong to this molecular weight scope hadhigher biological activity.
8. Inspected the hydrolysis of the AL proteinase, the papain, the trypsin, the FW proteinase on Agaricus blazeiMuril protein, according to proteolysis ability and the molecular weight of enzymolysis product, the ALproteinase and the FW proteinase were determined to manufacture oligopeptide.
9. Hydrolyzed the Agaricus blazei Muril protein with AL proteinase and the FW proteinase though two step.The best enzymolysis condition in AL proteinase was determined: pH was 8.5, the temperature was 55℃, theenzyme to the substrate was 2%, the substrate concentration was 5%, and hydrolisis time was 2h, the yield ofpeptide was 74.7%. Then used above hydrolysis product as material to hydrolyze through the FW proteinase,and pH was 7.0, the temperature was 50℃, the enzyme to substrate was 4%, the hydrolysis time was 1.0h,peptides yield were 80.6%.
10. The examinating result indicated that the content of peptides was 94.2%, the total glucose was 0.1%, thefat has not pick out, moisture content was 5.2%, ash was 0.5% in Agaricus blazei Muril oligopeptides powder.Agaricus blazei Muril oligopeptides was rich in proline, the lysine, the phenylalanine, and the essential aminoacids content reached 50.91%.
11. The finally enzymolysis products were purified by Sephadex G-25 gel filtration chromatography, theywere divided into three components, the molecular weight was lower than 5600Da.
12. The functional test results showed T-CHO content and the TG content of blood serum were reducedobviously in high blood fat model animal due to Agaricus blazei Muril oligopeptides (ABO), and the decreaseof HDL-C content in the high blood fat model rats were inhibited obviously by ABO, the increase of bloodserum LDL-C content in the high blood fat model rats were reduced obviously. SOD level in the animal ABOwere raised obviously, and the liver MDA content were reduced. The situation of hyperlipaemia mousebecause of high fat diet and fat cumulation in liver due to DL-E were obvious improved by ABO. The coefficient of viscera in mouse spleen and the thymus were increased obviously by ABO, inchondriosis ofmononuclear phagocyte system in mice was enhanced, and mouse's survival times in water were lengthened.The results showed ABO had functions which of lowing fat, protecting the liver, enhancing the immunity, andlessening fatigue.
13. The Agaricus blazei Muril by-product which after defatted, polysaccharides were extracted andoligopeptides were produced were used as material to manufacture high quality edible mushroom dietaryfibers, comprehensive utilization value of Agaricus blazei Muril was enhanced. Material physics andchemistry characteristic were improved by extrusive technology with high temperature and high pressure, theoptimum process condition was that moisture was 80%, extrusive temperature was 145℃, rate of feed was20Kg·h~(-1), speed of screw rotational was 220r·min~(-1). Content of soluble dietary fiber(SDF)in Agaricus blazeiMuril fibers without extrusion was 2.4%, the expansive power was 4.0 mL·g~(-1), holding water power was 3.1g·g~(-1) the bound water power was 2.7g·g~(-1); The SDF content after extrusion was 15.1%, the expansive powerwas 18.3mL·g~(-1), holding water power was 9.4 g·g~(-1), bounding water power was 6.4 g·g~(-1), and became highquality Agaricus blazei dietary fibers (HQADF).
14. The normal mice and constipation model mice were selected and fed with HQADE The propellingfunction of HQADF to the gastrointestinal vermicular motion was monitored. The influence of the HQADF tothe defecation frequency and the time of the two group mice were detected. Results : the normal mice'sgastrointestinal vermicular motion was promoted obviously in dose of 3.50, 1.75g·kg~(-1), their defecationfrequency was improved and defecation time was shorted. The constipation mice's defecation frequency wasevidently improved and the most efficient dose was 3.5g·kg~(-1). The results showed that HQADF hadconspicuously anti-constipation function.
15. The selected 50 mice were randomly divided into control group, alloxan model group, HQADF 3.50,1.75,0.88g·kg~(-1) dose group(n=10). Mice in each group were exhibited once a day for 15 days , on the sixthday ,mice in every group were injected alloxan physiological salt solution 80mg·kg~(-1) in caudal vena exceptcontrol group, mice in control group were injected physiological salt solution that is equal volume. 16 hbefore the last exhibition, mice in every group were starved.1h before the last exhibition, extracted blood inwhere eyeballs, separated sera and the contents of blood sugar were determined by auto biochemistry analyzer,mice were killed at once. The liver samples were obtained and the contents of liver glycogen were determined.Another 60 mice were divided into 5 groups with the method mentioned above. Each group was exhibitedonce day for 10 days continuously, 1h after the last exhibition, except control group, the others were injectedadrenalin hydrochloride 250μg·kg~(-1) through abdominal cavity, after 30 min, blood was extracted bydecapfitation, and the contents of blood sugar and the contents of liver glycogen were determined all by autobiochemistry analyzer. Results: the HQADF 3.50g·kg~(-1) had obvious inhibitory effect on the raise of bloodsugar in diabetes mice ( model group ) (P＜0.05 ) , it had obvious action on reducing the contents of liver glycogen in diabetes mice ( model group ) (P＜0.05 ) ,and 3.50g·kg~(-1) was superior to 1.75 g·kg~(-1) . TheHQADF with different dose had obvious action on inhibiting the raise of blood sugar in adrenine mice( model group ) (P＜0.001, P＜0.01, P＜0.05 ) ,and had obvious action on increasing the reduction of liverglycogen on adrenine mice ( model group ) ,and 3.50g·kg~(-1) was superior to 1.75g·kg~(-1). The results showedthat HQADF had the function of falling blood sugar in alloxan diabetes mice and hyperglycaemia miceinduced by adrenine.
16. The selected 80 mice (half male, half female) were randomly divided into 5 groups: control group,adiposity model group, HQADF 3.50,1.75,0.88g·kg~(-1) dose group. Each group except the control group wasexhibited once a day for 35 days and was weighed once every 7 days. Another 80 mice (half male, half female)were grouped, fed and weighed in the same way as the above, 24 hours after the last exhibition, the mice werekilled, abdominal fat was weighed and the fat index number was calculated. Another 80 mice (half male, halffemale) were grouped, fed and weighed in the same way as the above, 24 hours after the last exhibition, theireyeballs were removed and the blood was extracted. Its biochemical indexes were determined byHITACHI7080 auto analyzer. The results show that: the HQADF can obviously reduce the adiposity mice'sweight, fat Index, the content of TC, TG, LDL-C and GLU, and improve their HDL-C in blood serum, soHQADF had great functions of losing weight and fat.
17. The Agaricus blazei dietary fibers (ADF) through suitable processing can improve the bread quality. Theideal way was that extracted polysaccharide by microwave after supercritical fluid extraction or purify, thenoligopeptides were manufactured, the by-products were extruded. When the HQADF size was 0.175mm, theHQADF amount was 15%, the bread quality was the best, and the amount of bread additive was the least(1.0%). Because exrusion processing improved ADF quality, not only can add a great quantity dietarymushroom fibers in the dough, but also provided special health function for bread, in addition, the fiber sizewas not the smaller the better, so the energy consumption because mill fiber powder was reduced.
主要研究内容及结果如下：
1．超临界CO_2流体选择性萃取分离技术提取姬松茸ω-6多不饱和脂肪酸。经正交试验分析确定最佳工艺条件为萃取压力25MPa，温度50℃，时间60min，CO_2流量25L·h~(-1)。萃取量为4．03g·(100g)~(-1)，萃取后姬松茸含脂率0．01％。为姬松茸多糖的提取及姬松茸蛋白肽等功能因子的提取或制取提供良好的前提条件。
2．姬松茸超临界CO_2流体萃取物的脂肪酸组成特征为ω-6多不饱和脂肪酸型，亚油酸含量为52．4％，γ-亚麻酸21．12％，油酸12．89％，软脂酸7．98％，硬脂酸未检出，ω-6多不饱和脂肪酸总量为73．52％，可作为保健食品姬松茸ω-6多不饱和脂肪酸(Agaricus blazeiMurrillω-6 polyunsaturated fatty acid，ω-6APFA)的加工原料。
3．大鼠60只，随机分为空白对照组、模型组、天然大豆磷脂胶囊(NSPC)0．70 g·kg~(-1)剂量组和ω-6APFA 0．28、0．14、0．07 g·kg~(-1)3个剂量组(n=10)。除空白对照组外均连续给予高脂饲料14d，于第14d起开始连续灌胃给药14d，对照组和模型组给予等容积蒸馏水10 mL·kg~(-1)。实验结束后麻醉大鼠，腹主动脉取血测血清总胆固醇(T-CHO)、甘油三酯(TG)、高密度脂蛋白(HDL-C)、低密度脂蛋白(LDL-C)。同时测肝脏超氧化物歧化酶(SOD)活性、肝脏丙二醛(MDA)含量及腹腔脂肪蓄积系数。雄性小鼠72只，随机分为空白对照组、模型组、NSPC 1．0g·kg~(-1)剂量组、ω-6APFA 0．4、0．2、0．1 g·kg~(-1)3个剂量组(n=12)，连续给药7d，末次给药前16h除空白对照组，均腹腔注射75％蛋黄生理盐水液0．5 mL，并开始饥饿，末次给药后1h，于眼球采血，测血清总胆固醇及甘油三酯含量。试验结果：与模型组相比，NSPC 0．70 g·kg~(-1)和ω-6APFA 0．28 g·kg~(-1)剂量组大鼠血清总胆固醇、甘油三酯均明显降低，血清HDL-C升高(P＜0．001)，NSPC 0．70 g·kg~(-1)和ω-6APFA 0．28、0．14 g·kg~(-1)剂量组大鼠肝组织SOD活性升高(P＜0．05、P＜0．001及P＜0．05)，MDA降低(P＜0．01、P＜0．001及P＜0．01)，大鼠腹腔脂肪系数均降低(P＜0．01)。与模型组相比，NSPC 1．0 g·kg~(-1)、ω-6APFA 0．4、0．2 g·kg~(-1)组急性高血脂小鼠T-CHO和TG明显降低(P＜0．05、P＜0．01及P＜0．05)。试验结果表明ω-6APFA对高血脂鼠有明显降血脂作用。
4．60只大鼠随机分为空白对照组(n=12)、天然大豆磷脂胶囊(NSPC)0．70 g·kg~(-1)·d~(-1)剂量组(n=12)和ω-6APFA 0．07、0．14、0．28g·kg~(-1)·d~(-1)3个剂量(n=12)组，各组均连续灌胃给药7d，末次给药后1h于动物腹腔注射300mg·kg~(-1)水合氯醛麻醉，背位固定，分离右侧颈总动脉，应用实验性体内血栓形成测定仪，检测血栓形成时间。另取60只小鼠，随机分为空白对照组(n=12)、NSPC 1．0 g·kg~(-1)·d~(-1)剂量组(n=12)和ω-6APFA0．1、0．2、0．4 g·kg~(-1)·d~(-1)3个剂量组(n=12)，各组均连续灌胃给药7d，于末次给药后1h小鼠眶静脉采血，采用玻片法测定凝血时间。结果NSPC 0．7 g·kg~(-1)d~(-1)剂量组及ω-6APFA0．14、0．28g·kg~(-1)·d.(-1)剂量组，均对大鼠体内血栓形成有明显抑制作用，与对照组相比P＜0．05，且ω-6APFA0．14、0．28g·kg~(-1)·d.(-1)剂量组优于NSPC 0．7 g·kg~(-1)·d.(-1)剂量组。NSPC 1．0 g·kg~(-1)d~(-1)剂量组及ω-6APFA0．2、0．4g·kg~(-1)·d.(-1)剂量组，均能延长小鼠凝血时间，与对照组相比，NSPC 1．0 g·kg~(-1)剂量组P＜0．05，ω-6APFA 0．2、0．4g·kg~(-1)·d.(-1)剂量组P＜0．01，说明ω-6APFA0．2、0．4g·kg~(-1)·d.(-1)剂量组优于NSPC 1．0 g·kg~(-1)·d.(-1)剂量组。试验结果表明ω-6APFA有很好的抑制血栓形成及活血化瘀作用。
5．采用超临界萃取脱脂处理的姬松茸为原料，采用微波萃取：技术提取姬松茸多糖，大幅度缩短萃取时间，降低液固比，减少后续浓缩工艺能耗。微波萃取姬松茸活性多糖时微波萃取功率影响最大，其次是微波萃取时间，然后是物料粒度，固液比影响最小，最佳工艺条件为微波萃取功率600W，微波萃取时间6min，固液比1：10，物料粒度0．147mm。按此工艺进行萃取，姬松茸多糖提取率高达14．2％。与有关报道相比多糖得率提高29．68％，用水量为其1／3～1／2．浸提时间为其1／30，实现节水省时降耗。
6．用D315树脂脱蛋白纯化姬松茸粗多糖效果理想，控制流出速度0．5mL·min~(-1)，蛋白脱除率达79．26％，多糖回收率达86．3％。
7．采用葡聚糖凝胶G75柱层析对脱蛋白的姬松茸多糖进一步纯化、分离，控制流速0．3mL·min~(-1)，得到分子量10万左右的β-葡聚糖，纯度为85％，此分子量范围的多糖生物活性较高。
8．考察AL蛋白酶、木瓜蛋白酶、胰蛋白酶、FW蛋白酶四种酶制剂对姬松茸蛋白的水解情况，根据对蛋白水解能力及酶解液分子量分布确定使用AL蛋白酶和FW蛋白酶对原料进行水解制取低聚肽。
9．利用AL蛋白酶和FW蛋白酶对姬松茸蛋白进行分步水解，AL蛋白酶最佳酶解条件为pH8．5，温度55℃，酶与底物比2％，底物浓度5％，水解时间2h，肽得率74．7％。以AL蛋白酶酶解产物为反应底物加入FW蛋白酶进行第2次酶解，最佳条件为pH值7．0，温度50℃，酶与底物比4％，水解时间1．5h，肽得率80．6％。
10．检测结果表明姬松茸低聚肽粉中肽含量为94．2％，总糖0．1％，脂肪未检出，水分5．2％，灰分0．5％。姬松茸低聚肽富含脯氨酸、赖氨酸、苯丙氨酸，必需氨基酸含量高达50．91％。
11．通过Sephadex G-25凝胶色谱分离，酶解物被分成3个组分，分子量集中在5600Da以下。
12．功能试验结果表明姬松茸低聚肽能明显降低高血脂模型动物血清T-CHO和TG含量，对高血脂模型大鼠血清中HDL-C含量降低有明显的抑制作用；对高血脂模型大鼠血清中LDL-C含量升高有明显的降低作用；并能明显的提高动物肝脏SOD水平，降低肝脏MDA含量。姬松茸低聚肽对高脂饮食所致小鼠血脂升高和DL-E所致肝脂肪蓄积有明显的改善作用；姬松茸低聚肽能明显增加小鼠脾、胸腺脏器系数，增加小鼠单核巨噬细胞系统吞噬功能，延长小鼠在水中的存活时间。说明姬松茸低聚肽具有降脂、保肝、提高免疫力及抗疲劳作用。
13．以脱脂、提多糖、制取低聚肽之后剩余的姬松茸残渣为原料，制取高品质食用菌膳食纤维，提高姬松茸原料的综合利用价值。采用高温高压挤出技术对原料进行处理，改善物料的理化特性，最佳处理条件为物料含水量80％、挤出温度145℃、进料速度20Kg·h~(-1)，螺杆转速220r·min~(-1)。未进行挤出改性处理的普通姬松茸纤维(ADF)的可溶性膳食纤维(SDF)含量为2．4％，膨胀力为4．0 mL·g~(-1)，持水力为3．1 g·g~(-1)，结合水力为2．7g·g~(-1)；挤出处理后SDF含量为15．1％，膨胀力为18．3mL·g~(-1)，持水力为9．4g·g~(-1)，结合水力为6．4g·g~(-1)，成为品质优良的高品质膳食纤维(HQADF)。
14．采用正常小鼠和燥结型便秘小鼠模型，灌服给药，并监测HQADF对正常小鼠胃小肠运动功能的推进作用，及对正常和燥结型便秘小鼠的排便量及排便时间的影响。结果：HQADF3．50、1．75g·kg~(-1)剂量组对正常小鼠的胃肠运动功能都有明显的促进作用，推进率分别提高17．80％、16．85％；3．50、1．75g·kg~(-1)bw剂量组都能明显增加正常小鼠的排便量，缩短小鼠的排黑便时间，排便量分别增加54．32％、32．28％，排黑便时间分别缩短26．87％、19．70％；能明显增加燥结型便秘小鼠的排便量，以3．50g·kg~(-1)bw剂量组效果最好，口服4h内排便量增加73．44％。试验结果表明HQADF具有明显的抗便秘作用。
15．小鼠50只随机分为空白对照组、四氧嘧啶模型组和HQADF 3．50、1．75、0．88g·kg~(-1)体重3个剂量组，每天给予HQADF 1次，连续15d，于第6d除空白对照组外，各组小鼠均尾静脉注射四氧嘧啶生理盐水溶液80mg·kg~(-1)体重，空白对照组尾静脉注射同体积生理盐水。末次给药前16h将所有各组小鼠饥饿，末次给药后1h，于小鼠眼球采血，分离血清，采用全自动生化分析仪测血糖含量；立即处死小鼠，取肝脏测小鼠肝糖元含量。另取60只小鼠，分组方法同上，每天给予HQADF 1次，连续10d。除空白对照组外，其余各组均在末次给予HQADF后1h腹腔注射盐酸肾上腺素250μg·kg~(-1)体重，30min后断头取血，测血糖含量，取肝组织测肝糖元含量。结果：HQADF 3．50g·kg~(-1)体重对四氧嘧啶糖尿病小鼠(模型鼠)血糖升高有明显的抑制作用(P＜0．05)；对四氧嘧啶糖尿病小鼠(模型组)肝糖元含量有明显的降低作用(P＜0．05)，且3．50 g·kg~(-1)体重优于1．75 g·kg~(-1)体重。3．50、1．75、0．88g·kg~(-1)体重剂量组对肾上腺素小鼠血糖(模型组)升高均有明显的抑制作用(P＜0．001、P＜0．01和P＜0．05)；对肾上腺素造成小鼠(模型组)肝糖元降低有明显的升高作用，且3．50g·kg~(-1)体重优于1．75g·kg~(-1)体重。试验结果表明HQADF对四氧嘧啶致糖尿病小鼠及肾上腺素血糖升高小鼠具有明显的降血糖作用。
16．取80只小鼠，雌雄各半，随机分为5组：空白对照组，肥胖模型组，HQADF 3．50 g·kg~(-1)、1．75 g·kg~(-1)、0．88 g·kg~(-1)3个剂量组，除空白对照组给正常饲料外，其余各组均服用肥胖饲料，每天给药1次，连续给药35天，每隔7天称1次体重。另取80只小鼠，雌雄各半，分组、饲喂及称量体重方法同上，末次给药后24小时，将小鼠处死，取腹部脂肪称湿重，计算脂肪指数。再另取80只小鼠，雌雄各半，分组、饲喂及称量体重方法同上，末次给药后24小时，摘除小鼠眼球取血，采用日立7080全自动生化分析仪检测有关生化指标。试验结果表明：HQADF能明显降低肥胖小鼠体重、脂肪指数、总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL-C)、血糖(GLU)含量，对肥胖小鼠血清中高密度脂蛋白(HDL-C)有明显的升高作用，证明HQADF具有明显减肥降脂作用。
17．姬松茸膳食纤维(ADF)用于面包生产时必需经过适当方法处理才能改善面包品质。超临界流体萃取纯化处理、微波萃取多糖及制取姬松茸低聚肽后进行挤出是处理姬松茸纤维较为理想的方法。姬松茸膳食纤维采用挤出处理，粒度为0．175mm，用量为15％时面包综合品质最好，且面包添加剂用量最少(1．0％)。由于挤出处理改善了姬松茸膳食纤维的品质，不但可在面团中大量添加这种优良的食用菌膳食纤维，赋予面包产品特殊的保健功能，而且由于纤维的粒度并非越小越好，降低了纤维制粉的能耗。
Agaricus blazei Murill , a kind of important medical and edible mushroom, it is rich in proteins ,polysaccharides , polyunsaturated fatty acids and dietary fibers ,it also has the effect of inhibiting tumour,depressing the blood glucose , decreasing deposition of low density cholesterol and boosting organismimmunity and so on. For a long time, deep processing on Agaricus blazei Murrill was extraction ofpolysaccharides mainly, and proteins of Agaricus blazei, fibers of Agaricus blazei and the other by-productswere not utilized fully, the results caused large losses or wastes of quality protein and fiber. In addition, thestudy and development on functional oil in edible fungus was not reported even more.Availability of Agaricusblazei Murill is low, processing depth is insufficient, lacking high technology content, lacking high addedvalue and new product with great market potential. This research is on the base of the research to the domesticand foreign edible mushroom deep processing present situation and the development tendency, fullyconsidering the raw material comprehensive utilization and the influence of processing to environment, alsocritical technique of extracting Agaricus blazei Murill specificity ingredient and function factor, each kind offactor functional characteristic and the application has conducted the thorough research, implementing rawmaterial entire use, conforming to national initiative resource conservation and the environment friendlyproduction pattern, will provide the theory basis and the practical reference and the model for Agaricus blazeiMurill and even other edible mushroom comprehensive utilizations and the profound processing.
The main contents and results as follow:
1.ω-6 polyunsaturated fatty acids were extracted by supercritical fluid extraction technology from Agaricusblazei Murill , through the analysis of orthogonal test, the optimum condition was determined: extractingpressure was 25 MPa, extracting temperature was 50℃, extracting time was 60 min, CO_2 volume was 25L·h~(-1).The yields of extraction were 4.03g·(100g)~(-1), lipid contents in Agaricus blazei Murill was 0.01% afterextraction. This research results provided good precondition for extracting and producing Agaricus blazeiMurill peptide and other functional factors.
2. Compositional characteristic of Agaricus blazei Murill fatty acid after supercritical fluid extraction was typeofω-6 polyunsaturated fatty acid, and the content of linoleic acid was 52.4%, the content ofγ-linolenic acidwas 21.12%, the content of oleic acid was 12.89%, the content of palmitic acid was 7.98%, stearic acid wasnot detected out, the total cintents ofω-6 polyunsaturated fatty acids were 73.52%,it may be as the materialfor producing health food which was Agaricus blazei Murrillω-6 polyunsaturated fatty acid(ω-6APFA).
3. Sixty rats wererandomly divided into control group, model group, natural soybean phospholipids capsule(NSPC)0.70g·kg~(-1) group,ω-6APFA 0.28, 0.14, 0.07 g·kg~(-1) groups(n=10). The other groups excepting control group were given high-fat diets for 14 d, on the fourteenth the rats began to be administered orally, the controlgroup and model group were administered distilled water 10ml·kg~(-1) at the same volume, after 14 days ofcontinuously administration ,rats were anesthetized, the blood extracted in abdominal artery , T-CHO, TG,HDL-C, LDL-C in sera were determined. At the same time, the activity of SOD in liver and the content ofMDA was determined, the fat accumulated coefficient was calculated. 72 male mice were randomly dividedinto control group, model group, NSPC 1.0g·kg~(-1) group,ω-6APFA 0.4, 0.2, 0.1 g·kg~(-1) groups (n=12). Micewere administered continuously,16h before the last administration ,except oontrol group, the other groups wasinjected 75% yolk physiological salt solution 0.5mL through the abdominal cavity, and began to starve ,1 hafter the last administration, blood was extracted in eyeball , sera total cholesterol and triglyceride weredetermine. Compare with model group, T-CHO and TG in rat of NSPC 0.70g·kg~(-1) andω-6APFA 0.28 g·kg~(-1) allreduced and HDL-C raised obviously(P＜0.001), activity liver SOD of NSPC 0.70g·kg~(-1) andω-6APFA0.28、0.14 g·kg~(-1) obvious raised (P＜0.05, P＜0.001 and P＜0.05), MDA reduced obviously(P＜0.01, P＜0.001, P＜0.01), fat accumulation coefficient of NSPC 0.70g·kg~(-1) andω-6APFA reduced obviously. Comparewith model mice, T-CHO and TG in acute hyperlipemia mice of NSPC 1.0g·kg~(-1) andω-6APFA0.4、0.2 g·kg~(-1)reduced obviously(P＜0.05, P＜0.01 and P＜0.05).The results indicate thatω-6APFA has the function oflowering blood fat on hyperlipemia rats and mice.
4. Sixty rats were randomly divided into control group, natural soybean phospholipids capsule(NSPC)0.70g·kg~(-1)·d~(-1)group ,ω-6APFA 0.07, 0.14,0.28g·kg~(-1)·d~(-1)groups (n=12).Rats in each group were exhibitedonce a day for 7 d,1 h after the last exhibition ,rats were injected chloral hydrate 300g·kg~(-1) in abdominal cavityto anesthetize, the back were fixed, the carotid of the right neck were separated, and the time of thrombosisformation were determined by experimental intracorporeal thrombosis surveyor. Another 60 mice wererandomly divided into control group, NSPC 1.0g·kg~(-1)·d~(-1)group,ω-6APFA0.1, 0.2,0.4 g·kg~(-1)·d~(-1) groups(n=12).Mice were exhibited once a day for 7 d continuously, 1h after the last exhibition, blood was extracted in theorbit vein, the clotting time were determined through glass slides method. NSPC 0.7 g·kg~(-1)·d~(-1) andω-6APFA0.14,0.28g·kg~(-1)·d~(-1)all had obvious inhibitory effect on thrombus formation in blood stasis mice,compare with model group, P＜0.05, andω-6APFA0.14,0.28g·kg~(-1)·d~(-1) were superior to NSPC 0.7g·kg~(-1)·d~(-1).NSPC 1.0 g·kg~(-1)·d~(-1) andω-6APFA0.2,0.4g·kg~(-1)·d~(-1) all could prolong the clotting time ,compare with modelgroup, NSPC 1.0 g·kg~(-1)·d~(-1) was P＜0.05,ω-6APFA0.2, 0.4g·kg~(-1)·d~(-1) were P＜0.01,the result showed thatω-6APFA0.2, 0.4g·kg~(-1)·d~(-1) were superior to NSPC 1.0g·kg~(-1)·d~(-1) group. The results indicate thatω-6APFAhad the function of inhibiting thrombus formation, promoting blood flow ability and lowering blood viscosity.
5. Used defatted Agaricus biazei Murill through Supercritical fluid extraction as material, and used themicrowave extract technology to extract the Agaricus blazei Murill polysaccharides, the extract time wasshorten substantially, proportion of fluid to solid was reduced, and the following concentration energyconsumption was decreased. When Agaricus blazei Murill active polysaccharides were extracted through microwave technique, the influence of microwave power was biggest, next was the microwave time, and thenwas the material size, the influence of proportion of solid to fluid was the smallest, the optimum condition wasdetermined: microwave power was 600W, microwave time was 6 min, the proportion of solid to fluid was1 : 10, material size was 0.147 mm. According this technology to extract Agaricus blazei Murill activepolysaccharides, the extraction rate was 14.2%.The yield was raised 29.68% compared with reported before,and the volume of water was 1/3 or 1/2 of reported before, the extractive times were 1/30 of reported before,so the water ,times and energy were saved all.
6. Removed proteins and purified Agaricus blazei Murill polysaccharides by D315 resin, and geted the idealresult, rate of removing proteins is 79.26%, rate of recovering in polysaccharides is 86.3%.
7. PurifIed and separated Agaricus blazei Muril polysaccharides by Sephadex G75 gelatum, and removedproteins further, controled speed of flow, the flow was 0.3mL·min~(-1), obtainedβ-the glucosan which molecularweight was about 100000, the purity was 85%, the polysaccharides belong to this molecular weight scope hadhigher biological activity.
8. Inspected the hydrolysis of the AL proteinase, the papain, the trypsin, the FW proteinase on Agaricus blazeiMuril protein, according to proteolysis ability and the molecular weight of enzymolysis product, the ALproteinase and the FW proteinase were determined to manufacture oligopeptide.
9. Hydrolyzed the Agaricus blazei Muril protein with AL proteinase and the FW proteinase though two step.The best enzymolysis condition in AL proteinase was determined: pH was 8.5, the temperature was 55℃, theenzyme to the substrate was 2%, the substrate concentration was 5%, and hydrolisis time was 2h, the yield ofpeptide was 74.7%. Then used above hydrolysis product as material to hydrolyze through the FW proteinase,and pH was 7.0, the temperature was 50℃, the enzyme to substrate was 4%, the hydrolysis time was 1.0h,peptides yield were 80.6%.
10. The examinating result indicated that the content of peptides was 94.2%, the total glucose was 0.1%, thefat has not pick out, moisture content was 5.2%, ash was 0.5% in Agaricus blazei Muril oligopeptides powder.Agaricus blazei Muril oligopeptides was rich in proline, the lysine, the phenylalanine, and the essential aminoacids content reached 50.91%.
11. The finally enzymolysis products were purified by Sephadex G-25 gel filtration chromatography, theywere divided into three components, the molecular weight was lower than 5600Da.
12. The functional test results showed T-CHO content and the TG content of blood serum were reducedobviously in high blood fat model animal due to Agaricus blazei Muril oligopeptides (ABO), and the decreaseof HDL-C content in the high blood fat model rats were inhibited obviously by ABO, the increase of bloodserum LDL-C content in the high blood fat model rats were reduced obviously. SOD level in the animal ABOwere raised obviously, and the liver MDA content were reduced. The situation of hyperlipaemia mousebecause of high fat diet and fat cumulation in liver due to DL-E were obvious improved by ABO. The coefficient of viscera in mouse spleen and the thymus were increased obviously by ABO, inchondriosis ofmononuclear phagocyte system in mice was enhanced, and mouse's survival times in water were lengthened.The results showed ABO had functions which of lowing fat, protecting the liver, enhancing the immunity, andlessening fatigue.
13. The Agaricus blazei Muril by-product which after defatted, polysaccharides were extracted andoligopeptides were produced were used as material to manufacture high quality edible mushroom dietaryfibers, comprehensive utilization value of Agaricus blazei Muril was enhanced. Material physics andchemistry characteristic were improved by extrusive technology with high temperature and high pressure, theoptimum process condition was that moisture was 80%, extrusive temperature was 145℃, rate of feed was20Kg·h~(-1), speed of screw rotational was 220r·min~(-1). Content of soluble dietary fiber(SDF)in Agaricus blazeiMuril fibers without extrusion was 2.4%, the expansive power was 4.0 mL·g~(-1), holding water power was 3.1g·g~(-1) the bound water power was 2.7g·g~(-1); The SDF content after extrusion was 15.1%, the expansive powerwas 18.3mL·g~(-1), holding water power was 9.4 g·g~(-1), bounding water power was 6.4 g·g~(-1), and became highquality Agaricus blazei dietary fibers (HQADF).
14. The normal mice and constipation model mice were selected and fed with HQADE The propellingfunction of HQADF to the gastrointestinal vermicular motion was monitored. The influence of the HQADF tothe defecation frequency and the time of the two group mice were detected. Results : the normal mice'sgastrointestinal vermicular motion was promoted obviously in dose of 3.50, 1.75g·kg~(-1), their defecationfrequency was improved and defecation time was shorted. The constipation mice's defecation frequency wasevidently improved and the most efficient dose was 3.5g·kg~(-1). The results showed that HQADF hadconspicuously anti-constipation function.
15. The selected 50 mice were randomly divided into control group, alloxan model group, HQADF 3.50,1.75,0.88g·kg~(-1) dose group(n=10). Mice in each group were exhibited once a day for 15 days , on the sixthday ,mice in every group were injected alloxan physiological salt solution 80mg·kg~(-1) in caudal vena exceptcontrol group, mice in control group were injected physiological salt solution that is equal volume. 16 hbefore the last exhibition, mice in every group were starved.1h before the last exhibition, extracted blood inwhere eyeballs, separated sera and the contents of blood sugar were determined by auto biochemistry analyzer,mice were killed at once. The liver samples were obtained and the contents of liver glycogen were determined.Another 60 mice were divided into 5 groups with the method mentioned above. Each group was exhibitedonce day for 10 days continuously, 1h after the last exhibition, except control group, the others were injectedadrenalin hydrochloride 250μg·kg~(-1) through abdominal cavity, after 30 min, blood was extracted bydecapfitation, and the contents of blood sugar and the contents of liver glycogen were determined all by autobiochemistry analyzer. Results: the HQADF 3.50g·kg~(-1) had obvious inhibitory effect on the raise of bloodsugar in diabetes mice ( model group ) (P＜0.05 ) , it had obvious action on reducing the contents of liver glycogen in diabetes mice ( model group ) (P＜0.05 ) ,and 3.50g·kg~(-1) was superior to 1.75 g·kg~(-1) . TheHQADF with different dose had obvious action on inhibiting the raise of blood sugar in adrenine mice( model group ) (P＜0.001, P＜0.01, P＜0.05 ) ,and had obvious action on increasing the reduction of liverglycogen on adrenine mice ( model group ) ,and 3.50g·kg~(-1) was superior to 1.75g·kg~(-1). The results showedthat HQADF had the function of falling blood sugar in alloxan diabetes mice and hyperglycaemia miceinduced by adrenine.
16. The selected 80 mice (half male, half female) were randomly divided into 5 groups: control group,adiposity model group, HQADF 3.50,1.75,0.88g·kg~(-1) dose group. Each group except the control group wasexhibited once a day for 35 days and was weighed once every 7 days. Another 80 mice (half male, half female)were grouped, fed and weighed in the same way as the above, 24 hours after the last exhibition, the mice werekilled, abdominal fat was weighed and the fat index number was calculated. Another 80 mice (half male, halffemale) were grouped, fed and weighed in the same way as the above, 24 hours after the last exhibition, theireyeballs were removed and the blood was extracted. Its biochemical indexes were determined byHITACHI7080 auto analyzer. The results show that: the HQADF can obviously reduce the adiposity mice'sweight, fat Index, the content of TC, TG, LDL-C and GLU, and improve their HDL-C in blood serum, soHQADF had great functions of losing weight and fat.
17. The Agaricus blazei dietary fibers (ADF) through suitable processing can improve the bread quality. Theideal way was that extracted polysaccharide by microwave after supercritical fluid extraction or purify, thenoligopeptides were manufactured, the by-products were extruded. When the HQADF size was 0.175mm, theHQADF amount was 15%, the bread quality was the best, and the amount of bread additive was the least(1.0%). Because exrusion processing improved ADF quality, not only can add a great quantity dietarymushroom fibers in the dough, but also provided special health function for bread, in addition, the fiber sizewas not the smaller the better, so the energy consumption because mill fiber powder was reduced.
引文
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