【作者】 王大为；

【导师】 图力古尔；

【作者基本信息】 吉林农业大学 ， 植物病理学， 2008， 博士

【摘要】 蒙古口蘑自古以来既被视为席上珍品而深受广大消费者喜爱。其味道鲜美，富含蛋白质、维生素B₁、B₂、B₆、PP及钙、磷、铁等矿物质，具有很高的营养价值。除此之外，蒙古口蘑还含有大量对人体具有医疗保健作用的成分，如多糖、多肽、多不饱和脂肪酸、核糖核酸、腺嘌呤、甾体等，能促进和调控人体的新陈代谢，提高机体的免疫能力，拮抗肿瘤，起到预防疾病，强身健体的作用。长期以来，人们对蒙古口蘑的研究多集中在其驯化栽培、生态学及促生长激素样作用等方面，而在其功能性成分提取技术及产品开发领域的研究极少。本研究对蒙古口蘑的基础成分进行了测定，依据蒙古口蘑的成分组成特点，本研究以蒙古口蘑为原料，采用超临界CO₂流体萃取、复合抗氧化、微波震荡提取及有限改性挤出等高新技术提取与制取蒙古口蘑ω-6多不饱和脂肪酸、多糖、高F值寡肽等功能因子，研究筛选最佳提取纯化工艺，应用功能因子制取功能食品，并进行功能试验，为蒙古口蘑的精深加工及高附加值综合利用奠定理论与实践基础。本研究主要内容有：采用超临界CO₂流体选择性萃取分离技术提取蒙古口蘑ω-6多不饱和脂肪酸，即可得到功能性油脂，又能为蒙古口蘑多糖及蒙古口蘑高F值寡肽的提取及制取提供良好的前提条件，经正交试验确定最佳萃取工艺条件。以蒙古口蘑ω-6多不饱和脂肪酸为原料，采用软胶囊内容物及囊材同时进行抗氧化处理的复合抗氧化技术制作终端产品ω-6多不饱和脂肪酸软胶囊，大幅度提高产品的抗氧化性与稳定性。经正交试验确定最佳复合抗氧化条件。以提取ω-6多不饱和脂肪酸后的蒙古口蘑粉为原料，采用微波震荡萃取技术提取蒙古口蘑多糖，使细胞间质松散化、细胞壁被迅速破碎，加快目的物向溶剂中的扩散速度，大大提高多糖提取速率和多糖得率，经正交试验确定最佳微波震荡萃取工艺条件。采用乙醇醇沉、离子交换层析及葡聚糖凝胶柱层析法对多糖进行纯化处理。将提取ω-6多不饱和脂肪酸及多糖后的蒙古口蘑粉渣采用可控有限改性挤出技术对原料进行预处理，打开蒙古口蘑子实体蛋白质的紧密结构，使组织中蛋白质大分子裂解为小分子，降低其疏水性，增加其溶解性，使酶解更易于进行，提高水解度及产品得率。研究确定最佳挤出工艺条件。采用两段式酶解法制取蒙古口蘑高F值寡肽。首先使用碱性蛋白酶对挤出处理后的蒙古口蘑粉渣进行酶解制成蒙古口蘑肽，再应用木瓜蛋白酶切下肽链末端的芳香族氨基酸，使芳香族氨基酸游离。经正交试验确定最佳酶解条件。选用载留相对分子量1000Da的超滤膜对酶解液进行超滤处理，以达到纯化目的。采用活性炭吸附芳香族氨基酸，筛选最佳吸附条件，达到在吸附芳香族氨基酸的同时最大限度的保留支链氨基酸组分。本研究各项结果如下：1、经检测可知，蒙古口蘑干品中含总糖47.61％，含还原糖12.77％，含多糖34.84％，含蛋白质37.96％，含水分8.04％，含灰分3.85％，含脂肪2.50％。2、超临界CO₂流体可选择性萃取蒙古口蘑ω-6多不饱和脂肪酸，萃取压力对萃取率影响最大，其次为萃取时间，然后是萃取温度，最后为CO₂流量；最佳萃取工艺条件为萃取压力为25MPa，萃取温度为45℃，萃取时间为120min，CO₂流量为20L·h^-1；此条件下可将蒙古口蘑所含脂肪萃取出96.9％，为蒙古口蘑多糖及高F值寡肽的提取及制取创造良好的前提条件。3、蒙古口蘑超临界CO₂流体萃取物的脂肪酸组成特征是以ω-6多不饱和脂肪酸为主，其中亚油酸含量为52.589％，γ-亚麻酸含量为7.646％，油酸含量为28.797％。4、采用内容物及囊材同时进行抗氧化处理的复合抗氧化技术制作ω-6多不饱和脂肪酸软胶囊，在内容物中加入V_E、BHA和TBHQ作为抗氧化剂，囊材中加入V_C作为抗氧化增效剂时，V_E加入量对抗氧化性能影响影响最大，其次为BHA加入量，然后是TBHQ加入量，最后为V_C加入量；当V_E、BHA、TBHO及V_C加入量分别为160mg·kg^-1、60 mg·kg^-1、40 mg·kg^-1、180 mg·kg^-1时软胶囊内容物过氧化值达到200mmol·kg^-1需用45d，产品抗氧化效果最好。5、以超临界CO₂流体萃取脱脂处理的蒙古口蘑为原料，采用微波震荡萃取技术提取蒙古口蘑多糖，微波萃取功率对提取效果影响最大，其次是微波萃取时间，然后是物料粒度，料液比影响最小；最佳工艺条件为物料粒度为0.147mm，固液比为1∶15，微波萃取功率为900W，微波萃取时间为4min。按此工艺进行萃取，蒙古口蘑多糖提取率达30.14％。6、采用95％乙醇醇沉法进行蒙古口蘑多糖的初步纯化处理，最佳醇沉时间为6h，最佳乙醇加入量为粗提液体积的3倍，最佳醇沉条件下多糖得率为92.07％。7、采用离子交换层析法脱除多糖中的蛋白效果较好，最佳条件为选用D315树脂装柱，洗脱液流速为1.0mL·min^-1，此时蛋白脱除率达77.08％，多糖损失率为5.50％。8、采用葡聚糖凝胶G75柱层析法对脱蛋白的蒙古口蘑多糖进一步纯化处理，当洗脱液流出速度为0.5mL·min^-1时，蛋白脱出率达78.86％，多糖损失率为2.81％。9、采用紫外可见分光光度法对本研究所得多糖进行检测，结果表明，经微波震荡提取、醇沉、树脂纯化、凝胶分离纯化后所得蒙古口蘑多糖中主要成分为β-葡聚糖，其分子量约为10万Da，纯度为95.22％，多糖得率为71.36％。10、功能试验结果表明，蒙古口蘑多糖具有抑制肿瘤作用及明显的降血糖作用。11、将提取多不饱和脂肪酸及多糖后的蒙古口蘑渣料进行可控有限改性挤出处理可疏松蒙古口蘑致密的组织结构，增加其可溶性蛋白含量，对改性影响程度大小依次为挤出温度＞物料粒度＞物料含水量＞进料速度，可控有限改性最佳条件为挤出温度为130℃、物料粒度为0.147mm、物料含水量为55％、进料速度为25kg·h^-1，经此条件处理的蒙古口蘑其可溶性蛋白含量达5.25％，有利于下一步酶解的进行。12、采用两段式酶解法制取蒙古口蘑高F值寡肽效果理想。首先使用碱性蛋白酶进行酶解制成蒙古口蘑肽，再应用木瓜蛋白酶切下肽链末端的芳香族氨基酸，使芳香族氨基酸游离。对碱性蛋白酶酶解效果影响程度大小依次为酶解液pH值＞酶解温度＞酶加入量＞底物浓度，碱性蛋白酶酶解最佳条件为pH值为8.5、温度为60℃、碱性蛋白酶加入量为1％、底物浓度为4％，酶解时间3h，此时耗碱量最大，酶解效果最好。木瓜蛋白酶酶解条为pH值7.0、底物浓度为7.0％，酶加入量为2.0％，温度为50℃，酶解时间4h。13、本研究选用载留相对分子量1000Da的超滤膜对酶解液进行超滤处理，以达到纯化目的。当超滤压力控制在0.10～0.15MPa时超滤效果理想，这时污染物在过滤面附近湍流的作用下对膜的吸附作用变缓，可有效延长超滤膜的使用时间。14、采用活性炭吸附芳香族氨基酸，其最佳吸附条件为固液比1：10、温度为25℃、pH值为3.0。在此条件下活性炭去除芳香族氨基酸的能力较强，同时可最大限度的保留支链氨基酸组分。由于活性炭还具有较好的脱色和脱苦效果，使最终得到的寡肽溶液澄清透明、无异味、无苦味。15、检测及计算结果表明，本研究制得的蒙古口蘑肽的F值为86.33，其分子量在1000Da以下，为典型的高F值寡肽，色泽淡黄，纯度为95.6％。采用本研究方法制取蒙古口蘑高F值寡肽，产品得率为41.2％。16、功能试验结果表明，蒙古口蘑高F值寡肽具有一定的降脂、保肝、提高免疫力及抗疲劳作用。更多还原

【Abstract】 intercellular substances were loosed, the cell wall were broken rapidly, aim products were diffused intosolvent sharply, rate of extraction and yield were increased significantly, the optimum condition ofmicrowave treatment method was determined through orthogonal test.The extracted polysaccharides were purified by precipitain in ethanol, ion exchange chromatographyand glucan gel filtration chromatography. The materials were pretreated through limited modified extrusion,broke the tight protein structure in Tricholoma mongolicum fruiting body, the giant molecules were brokeninto small molecules, the hydrolysis was decreased, the solubility was increased, so the enzyme process waseven more easy to carry on, the hydrolysis and product yield were enhanced all. The optimum extrusiontechnique was determined though study and research.High Fischer oligopeptides of Tricholoma mongolicum were manufactured by enzymic hydrolysis intwo step method. First, Tricholoma mongolicum peptides were produced through hydrolysis in alkalinityproteinase, then the aromatic amino acids were cut away with papain, the optimum condition wasdetermined by orthogonal test. The enzymolysis products were treated in ultrafiltration method which theultrafitration membrance could catch the materials of relative molecular was 1000Da, and the products werepurified. The aromatic amino acids were adsorbed in acticarbon, and the optimum condition was determinedout, the branched chain amino acids were reserved extremely when the aromatic amino acids were adsorbed.The results as follow:1. In dried Tricholoma mongolicum fruiting body, the total carbohydrate were 47.61%, reducing sugarwere 12.77%, polysaccharides were 34.84%, protein was 37.96%, water was 8.04%, ash was 3.85%, fat was2.50%.2. During Tricholoma mongolicumω-6 polyunsaturated fatty acids extraction, the rate of extractionwas affected seriously due to pressure, next was time, then was temperature, the co₂ volume was the last;the optimum technique was determined, the pressure was 25MPa, temperature was 45℃, time was 120 min.,co₂ volume was 25L·h^-1; the extraction rate of fat was 96.9%, provided good precondition formanufacturing polysaccharides and producing high Fischer value peptides.3. Compositional characteristic of Tricholoma mongolicum fatty acid after supercritical fluid extractionwas type ofω-6 polyunsaturated fatty acid, and the content of linoleic acid was 52.589%, the content ofγ-linolenic acid was 7.646%, the content of oleic acid was 28.797%.4. During soft capsules withω-6 polyunsaturated fatty acid were produced, the technique of compoundanti-oxidation was utilized that the contents in capsule and wall materials were treated in oxidation-resistant at once. V_E, BHA and TBHQ were added in contents as antioxidant, V_C was added in wall materials asbuilder, another, amount of V_E was major factor affecting capability of oxidant-resistant, nest was amount ofBHA, then was amount of TBHQ, amount of V_C was the last; when amounts of V_E, BHA, TBHQ and V_Cwere 160mg·kg^-1, 60 mg·kg^-1, 40 mg·kg^-1, 180 mg·kg^-1, when the amount of perhydride of the contentsin the capsules was 200mmol·kg^-1, demanded 45 days, so the anti-oxidation ability of the products wasstrong and the best.5. Tricholoma mongolicum after Supercritical fluid extracted were used as materials, microwave shaketechnique was utilized to extract Tricholoma mongolicum polysaccharides, the power of microwave wasmajor factor influencing the rate of extraction, next was microwave time, then was size of materials, theration of solid to liquid was 1:15, power of microwave was 900W, microwave time was 30.14% accordingto this technique.6. Tricholoma mongolicum polysaccharides were purified roughly using 95% ethanol solution forprecipitating, the optimum time was 6h, the amounts of ethanol were 3 times to rough extracted liquid, theyield of polysaecharides was 92.07%.7. Ion exchange chromatography method was utilized to take away proteins in the polysaccharides, theefficency was best, the optimum condition was that D315 resins were utilized, the flow rate of eluant was1.0mL·min^-1, the elimination rate of protein was 77.08%, loss ratio of polysaccharides was 5.50%.8. Glucan gel G75 filtration chromatography method was utilized to purify Tricholoma mongolicumpolysaccharides after took away protein further more, when the flow rate of eluant was 0.5mL·min^-1, theelimination rate of protein was 78.86%, loss ratio of polysaccharides was 2.81%.9. Ultraviolet-visible spectrophotometry was ultilized to detect polysaccharides, the results showed theTricholoma mongolicum polysaccharides got through microwave extraction precipitated with precipitain,purified with resin, and gel separation major composition wasβ-glucan, its molecular weight was 100000Da,purity was 95.22%, yield of polysaccharides was 71.36%.10. Functional test showed, Tricholoma mongolicum polysaccharides had obvious inhibitory functionon tumor and had obvious function in falling blood sugar.11. Tricholoma mongolicum was treated by limited modified extrusion, broke the tight proteinstructure, the content of soluble protein was increased, the influence of extrusion temperature on modifywas serious, next was size of materials and moisture, the influence of feed speed was the weakest, theoptimum conditions were that temperature was 130℃, size of materials was 0.47m, moisture was 55%, feed speed was 25kg·h^-1, contents of soluble protein were 5.25%, had good for next hydrolysis.12. High Fischer oligopeptides of Tricholoma mongolicum were manufactured by enzymic hydrolysisin two step method. First, Tricholoma mongolicum peptides were produced through hydrolysis in alkalinityproteinase, then the aromatic amino acids were cut away with papain, the influence of pH on hydrolysis inalkalinity proteinase was serious, next was temperature and the content of enhzyme, the influence ofsubstrate density was the weakest, the optimum conditions were that pH was 8.5, temperature was 60℃, thecontent of alkalinity proteinase was 1%, substrate density was 4%, time was 3h, at this time theconsumption alkali was most, and enzymic effect was best. When hydrolysis by papain, pH was 7.0, thecontent of papain was 2%, substrate density was 7.0%, temperature was 50℃, time was 3h.13. The enzymolysis products were treated in ultrafiltration method which the ultrafitration membrancecould catch the materials of relative molecular was 1000Da, and the products were purified. The pressure ofultrafiltration was controlled 0.10 to 0.15MPa, the effect was ideal, the adsorption of pollutants uponmembrance was slow, because of turbulent flow near filtration area. The affective time of membrance waslengthened.14. The aromatic amino acids were adsorbed in acticarbon, the ration of solid to liquid was 1:10,temperature was 25℃, pH was 3.0, the aromatic amino acids were adsorbed in acticarben, the aromaticamino acids were adsorbed obviously, at once, the branched chain amino acids were reserved extremely.Because acticarbon had better effect of discolor and diabitter, so oligopeptides solution was clear andtransparent, and hadn’t strange smell and bitter.15. Results of detection and calculation showed that, the Fischer value of Tricholoma mongolicumpeptides was 86.33, it was high Fischer value oligopeptides, the molecular weight was lower than 1000 Da,the color was light yellow, purity was 95.6%. The yield of high Fischer value oligopeptide was 41.2%according to this technique.16. Foundtional fest showed that Tricholoma mongolicum high Fischer value oligopeptides hadfunction in lowing fat, protecting the liver, enhancing the immunity, and lessening fatigue.更多还原