CN106038614B - 蛹虫草多糖食用菌酵素复方饮液及其制备方法 - Google Patents
蛹虫草多糖食用菌酵素复方饮液及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种蛹虫草多糖食用菌酵素复方饮液，由以下组分组成：蛹虫草粗多糖18～22%，蔗糖3～8%，食用菌酵素15～25%，柠檬酸0.1～0.3%，羧甲基纤维素钠0.2～0.5%，山梨酸钾0.005～0.015%，余量为水，以重量份数计。本发明不仅能够改善肠胃消化系统功能，加速多糖的吸收，而且能够提高多糖的生物利用度，使得产品在水解吸收过程中产生更多的寡糖、低聚糖，利于与体内免疫系统发生作用，例如与粘膜上的细胞受体结合，从而激活一些信号通路、引起免疫反应，不但可以增多功效，而且还可以提高药理作用和临床疗效。
Description
技术领域
本发明涉及一种用药食同源的蛹虫草等原料配制成的营养蛹虫草多糖食用菌酵素复方饮液及其制备方法。
背景技术
冬虫夏草为麦角科真菌冬虫夏草[cordyceps inensis(Berk.)sacc.]寄生在蝙蝠科昆虫幼虫的子座复合体。我国是世界上冬虫夏草资源分布最多最广的国家。冬虫夏草主要分布在中国青海、西藏、四川、云南等地海拔3000～5100m的高寒草甸区。
随着人们生活水平的提高，人们的健康意识也随着逐步提高，越来越重视对健康的投资，所以市场上出现了大量的保健产品，但是虫草类保健产品的大量生产，使得人们对野生天然虫草的盗挖、采挖现象愈演愈烈，与此同时，虫草天然的繁殖生长环境的遭到极具破坏，自然产量随之骤减，日趋枯竭野生资源使得相关市场需求产生很大缺口。通过接种从天然冬虫夏草中分离出的菌株感染蜂蛹产生子实体，能够于某些程度上弥补因野生虫草不足而造成的资源缺口。大量相关药理、药化及临床应用表明：蛹虫草和天然冬虫夏草具有相似的化学成分、药理作用和临床疗效。两者都含有的蛋白质、脂肪、多种无机元素和维生素、氨基酸、虫草酸含量相近，并且蛹虫草中虫草素含量，特别是多糖大大高于冬虫夏草。目前，市面上主要出售虫酒、虫草药膳、虫草茶、虫草饮料和各类胶囊、片剂等产品。
蛹虫草多糖的功效佳，但是直接服用有很大的腥味，很多人接受不了，且人体能吸收的仅仅只有小部分。把蛹虫草打成粉末，提取其中的多糖，配制成口服液。
发明内容
为克服蛹虫草的腥味及其多糖的吸收效果，本发明研制蛹虫草多糖食用菌酵素复方饮液。
本发明的目的是通过以下措施实现的：
一种蛹虫草多糖食用菌酵素复方饮液，由以下组分组成：蛹虫草粗多糖18～22％，蔗糖3～8％，食用菌酵素15～25％，柠檬酸0.1～0.3％，羧甲基纤维素钠0.2～0.5％，山梨酸钾0.005～0.015％，余量为水，以重量份数计。
上述食用菌酵素，其制备方法包括以下步骤：选取重量份数为平菇1～2份、茶树菇1～2份、香菇1～2份、金针菇1～2份、杏鲍菇1～2份为原料，去杂，切成片状，加入1～3份蜂蜜，于121℃高温高压条件下灭菌15min，在无菌操作条件下，接种10wt％复合发酵菌液，进行发酵，发酵时间200天，发酵结束后于121℃高温高压条件下灭菌15min杀灭酵母菌并灭菌，得到含有菌肉和全部酵素的发酵液，过滤后液体为食用菌酵素。
上述复合发酵菌液是将乳酸菌、酵母菌、干酪乳杆菌的菌种活化培养制备而成。具体包括以下步骤：(1)将乳酸菌、酵母菌、干酪乳杆菌分别在LB平板上划线接种，放入30℃培养箱中培养至长满菌丝；(2)挑取平板上的菌落分别接入装有50ml培养液的三角培养瓶中，置于摇床，转速200rpm，温度30℃，恒温培养48～72小时，得到三种菌液；(3)将三种菌液按乳酸菌∶酵母菌∶干酪乳杆菌＝1∶1∶1比例配成浓度为每毫升含2.5×107个孢子的菌液，作为复合发酵液。
上述蛹虫草多糖食用菌酵素复方饮液的制备方法，包括以下步骤：
(1)蛹虫草粉以物料重量比为1∶15加蒸馏水，超声30min，在100℃下油浴提取2h，离心得提取液，将提取液浓缩，加入3-5倍无水乙醇进行醇沉过夜；
(2)离心并用无水乙醇洗沉淀，得到粗多糖，将蛹虫草粗多糖进行冷冻干燥，得到蛹虫草多糖粉；
(3)取虫草粗多糖20％，蔗糖5％，食用菌酵素20％，柠檬酸0.2％，羧甲基纤维素钠0.3％，山梨酸钾0.01％，用去离子水加至100％混合，均质、瞬时灭菌，灭菌温度在100-120℃，时间在30-60分钟，灌装封口。
有益效果
1.本发明针对野生冬虫夏草越来越少的状况，以较容易获得的材料配制出与野生冬虫夏草的营养成分十分相仿且人体较易吸收的饮液。本发明的饮液长期服用具有增强免疫力的功效。
2.在蛹虫草多糖饮液的加工制备中，有效成分的生物活性易削弱或丧失，且多糖是大分子物质，其生物利用度极低。而本发明不仅能够改善肠胃消化系统功能，加速多糖的吸收，而且能够提高多糖的生物利用度，使得产品在水解吸收过程中产生更多的寡糖、低聚糖，利于与体内免疫系统发生作用，例如与粘膜上的细胞受体结合，从而激活一些信号通路、引起免疫反应，不但可以增多功效，而且还可以提高药理作用和临床疗效。
3.本发明的饮液含有多种多糖、蛋白质、有机酸、维生素和高浓度的锌、硒、镁等微量元素混合物。把食用菌制成酵素，配制成蛹虫草多糖食用菌酵素复方饮液，不但可以增多功效，而且还可以提高药理作用和临床疗效。
具体实施方式
下面对本发明做进一步详细说明。以下实施例仅限于说明本发明而不用于限制本发明的范围。
实施例1
一种蛹虫草多糖食用菌酵素复方饮液，其原料重量份数是：蛹虫草粗多糖20％，蔗糖5％，食用菌酵素20％，柠檬酸0.2％，羧甲基纤维素钠0.3％，山梨酸钾0.01％，去离子水54.49％，以重量百分比计。
制备方法的制备方法为：
(1)筛选优质原料蛹虫草，清洗，烘干，磨粉。热水回流侵提取，离心，浓缩，醇沉，离心，冷冻干燥，得蛹虫草粗多糖。
(2)选取平菇1份、茶树菇1份、香菇1份、金针菇1份、杏鲍菇1份为原料，去杂，打浆，过滤，调整糖度，杀灭空气中的微生物和杂菌，加入10wt％的复合发酵菌液，发酵，121℃高温高压条件下灭菌15min杀灭发酵菌终止发酵，得到含有菌肉和全部酵素的发酵液，过滤后液体为酵素。
上述复合发酵菌液是将乳酸菌、酵母菌、干酪乳杆菌的菌种活化培养制备成发酵菌液。具体包括以下步骤：①将乳酸菌、酵母菌、干酪乳杆菌分别在LB平板上划线接种，放入30℃培养箱中培养至长满菌丝；②挑取平板上的菌落分别接入装有50ml培养液的三角培养瓶中，置于摇床，转速200rpm，温度30℃，恒温培养48～72小时，得到三种菌液；③将三种菌液按乳酸菌∶酵母菌∶干酪乳杆菌＝1∶1∶1比例配成浓度为每毫升含2.5×107个孢子的菌液，作为复合发酵液。
(3)取蛹虫草粗多糖20％，蔗糖5％，食用菌酵素20％，柠檬酸0.2％，羧甲基纤维素钠0.3％，山梨酸钾0.01％，用去离子水加至100％混合，均质、瞬时灭菌，灭菌温度在100-120℃，时间在30-60分钟，最后灌装封口为本发明蛹虫草多糖食用菌酵素复方饮液。制得的产品效果验证如下：
1.保健功能试验：
(1)抗肿瘤作用
选取20只健康且体重相近(18±2g)的清洁型ICR小鼠，在小鼠背部皮下接种5×106个S180细胞。随机分为A(0.9％生理盐水作为对照组)、B(实施例1作为实验组)两组，每组10只。A组灌入0.4ml生理盐水，B组灌入0.4ml蛹虫草多糖液，每天同一时间灌胃1次，连续灌胃10天后放血处死，取瘤称重。实验组瘤重0.410±0.123g，对照组瘤重0.876±0.126g。
结论：本产品具有良好的抗肿瘤作用。
(2)抗氧化作用
首先配制好9mmol/LFeSO4、9mmol/L水杨酸乙醇溶液、8.8mmo L/L过氧化氢溶液和不同浓度梯度的本产品(分别为5mg/mL、10mg/mL、15mg/mL、20mg/mL、25mg/mL)。取15支试管分成5组依次做好编号，每组3个平行试验，每支试管中加入1mL 9mmol/L FeSO4、2mL9mmol/L水杨酸乙醇溶液，按照编号依次加入不同浓度的多糖溶液2mL，再分别加入2mL8.8mmol/L过氧化氢溶液，室温下反应1h后。蒸馏水空白调零，在510nm处测定各试管的吸光度，计算不同浓度本产品对羟自由基的清除率。
计算公式：羟自由基清除率(％)＝(A0-AS)/A0×100％
其中：A0——空白对照管的吸光度；
AS——加入样品后的吸光度
结果表明本产品多糖浓度为20mg/mL时，羟自由基的抑制率达到最高80％以上，试验表明本产品具有良好的还原能力。
结论：本产品具有良好的体外抗氧化作用。
(3)降血糖
选取12只健康且体重相近(18±2g)的清洁型ICR小鼠，以多次低剂量链脲霉素(multiple low-dose streptozotocin，MLD-STZ)方法腹腔注射BALB/c小鼠，成功诱导出糖尿病小鼠。随后随机分成A、B两组，每组6只。每天同一时间，分别给A组小鼠注射本产品300mL/kg、B组小鼠注射饮用蒸馏水，每天注射一次，连续注射6周，每周断尾取血，用血糖仪测定血糖浓度。实验组血糖浓度由17.96±2.74(mmol/L，n＝6)变成12.32±3.68(mmol/L，n＝6)，对照组由17.98±2.76(mmol/L，n＝6)变成19.20±3.56(mmol/L，n＝6)。
结论：本产品对糖尿病小鼠有较好的降糖作用。
(4)护肝作用。
选取30只健康且体重相近(18±2g)的清洁型ICR小鼠，随机分成正常对照组(饲喂正常饲料)、急性CCl4肝损伤模型组(简称模型对照组，正常饲料+CCl4)、产品组(正常饲料+CCl4+本产品为500mg/kg多糖)，每组10只。每天晚上7：00，产品组灌胃0.2mL，正常对照组与模型对照组灌胃等体积0.2mL生理盐水，连续灌胃7天，末次灌胃2小时后，正常对照组注射0.2mL花生油，其他各组每只鼠腹腔注射0.2％CCl4花生油溶液0.2mL。禁食16h，断头取血约3mL，室温放置2h，3000r/min离心10min后取血清，-20℃密封保存备用；取肝左叶用生理盐水制成10％的组织匀浆。取肝右叶，用10％甲醛溶液固定，冰冻保存。用考马斯亮蓝法测定组织中蛋白含量，采用黄嘌呤氧化酶法测定SOD活性。取甲醛固定的肝右叶组织，常规石蜡切片，HE染色、20×10倍光镜镜检。正常对照组SOD活性31.27±4.05(U/mg pro)，MDA含量2.48±2.44(nmol/mg pro)，TP含量64.21±6.82(mg/mL pro)；模型对照组SOD活性15.04±5.02(U/mg pro)，MDA含量8.98±2.28(nmol/mg pro)，TP含量56.87±7.53(mg/mL pro)；产品组SOD活性26.02±6.88(U/mg pro)，MDA含量3.52±1.02(nmol/mg pro)，TP含量61.01±7.65(mg/mL pro)。
结论：模型对照组SOD水平显著低于正常对照组(P＜0.01)，说明肝细胞已受到破坏。产品组SOD水平显著高于模型对照组(P＜0.01)，表明产品组起到了抑制SOD降低的作用。模型对照组MDA含量显著高于正常对照组(P＜0.01)，说明CCl4诱导的急性肝损伤模型成功。产品组MDA水平显著低于模型对照组(P＜0.01)，表明本产品起到了抑制MDA升高的作用。因此，本产品具有护肝作用。
(5)保护肾功能
健康witsra大鼠12只，雌、雄各半，50天龄，雌性体重160～180g，雄性体重170～200g，随机分成三组，正常对照组、模型组对照组、产品组，每组4只。模型对照组、产品组分别在水合氯醛麻醉下行左侧肾脏2/3切除术。术后大鼠单独饲养3天后分组置饲养笼群养。第三天开始进行千预治疗：(A)产品组，4只，每天给予本产品500mg/kg灌胃；(B)模型对照组，4只，(C)正常对照组，4只。模型组和正常对照组每大给予等量的饮用水灌胃，大鼠自山饮水、进食。次术后4周各组分别处死大鼠2只。术后9周处死剩余的2只。开放肾脏静脉，用4℃预冷的生理盐水冲洗肾脏直至变白，摘下肾脏，将其剖开，取部分肾组织(兼顾皮、髓质)置10％福尔马林固定液固定24小时，流水冲洗2小时，常规剃度酒精脱水，二甲苯透明，石蜡包埋，3pm厚连续切片，用HE染色、PSA染色，检查肾小球硬化指数。采用半定量方法评估肾小球硬化程度，每张切片均观察20个完整肾小球。4周时肾小球硬化指数，正常对照组2.48±1.13、模型对照组28.03±3.98、蛹虫草多糖组25.12±3.67，9周时肾小球硬化指数，正常对照组2.67±1.24、模型对照组44.53±9.95、产品组29.89±7.86。
结论：模型对照组肾小球硬化指数显著低于正常对照组(P＜0.01)，说明肾小球已受到破坏。产品组肾小球硬化指数显著低于模型对照组(P＜0.01)，表明产品组对肾小球起到了保护作用。模型对照组肾小球硬化指数显著高于正常对照组(P＜0.01)，说明肾小球损伤模型成功。因此，本产品具有能保护肾功能。
(6)改善肠胃消化系统
选取20只健康且体重相近(18±2g)的清洁型ICR小鼠，禁食20-24小时，随机分为2组，即产品组(20.0ml/kg灌胃)，对照组(等量生理盐水灌胃)，每组10只动物，用苦味酸标记。90分钟后各组给予0.3ml墨汁液灌胃。20分钟后，颈椎脱臼处死，打开腹腔分离肠膜，上端至幽门、下端至回盲部剪取肠管，置于托盘上。轻轻将小肠拉成直线，测量肠管长度作为小肠总长度。从幽门至墨汁前沿的距离作为“墨汁在肠内推进距离”。用公式计算墨汁推进百分率。墨汁推进率(％)＝墨汁在肠内推进距离(cm)/小肠全长(cm)×100％。各组小鼠小肠推动功能。正常对照组推进率64.431±7.047％，产品组推进率84.276±8.691％。
结论：本产品能改善肠胃消化系统。
2.吸收率试验验证：
选取20只健康且体重相近(18±2g)的清洁型ICR小鼠，随机分2组，即本专利产品组(简称实验组，本产品20.0ml/kg灌胃)，对照组(等量生理盐水灌胃)，每组10只动物，10分钟后每只小鼠灌入0.4mL2％葡聚糖蓝2000溶液，30分钟后颈椎脱臼处死动物，开腹取出全部胃肠，自幽门括约肌处取胃，将其内残留的葡聚糖蓝2000充分溶2mL去离子水中，3500rpm离心15分钟，取上清液，滤液用723型分光光度计，在620nm处测定吸光度，为胃内葡聚糖蓝2000残留量，并求出实验组均值。以对照组均值为100％，得实验组相对胃内色素残留率50.7％。
结论：本产品有助于机体对对营养的吸收。
3.感官评定试验选择20名专业评判人员，对本产品进行感官评价。表1为本产品感官评价指标，表2为评价结果。
表1实施例1的蛹虫草多糖食用菌酵素复方饮液感官评分标准
表2实施例1的蛹虫草多糖食用菌酵素复方饮液感官评价结果
由表2可以看出，本发明蛹虫草多糖食用菌酵素复方饮液酸甜味适中，基本无苦味，不含不溶性颗粒，澄清度较好，黏稠度适中，整体口感好，满足消费者需求。
Claims (2)
1.一种蛹虫草多糖食用菌酵素复方饮液，由以下组分组成：蛹虫草粗多糖18～22%，蔗糖3～8%，食用菌酵素15～25%，柠檬酸0 .1～0 .3%，羧甲基纤维素钠0 .2～0 .5%，山梨酸钾0 .005～0 .015%，余量为水，以重量份数计；
所述的食用菌酵素 原料为平菇1～2份、茶树菇1～2份、香菇1～2份、金针菇1～2份、杏鲍菇1～2份为原料，1～3份蜂蜜，以重量份数计；所述的食用菌酵素 制备方法包括以下步骤：以平菇、茶树菇、香菇、金针菇、杏鲍菇为原料，加入蜂蜜，于121℃高温高压条件下灭菌15min，在无菌操作条件下，接种10wt%复合发酵菌液，进行发酵，发酵时间200天，然后121℃高温高压条件下灭菌15min杀灭发酵菌，过滤后液体为酵素。
2.如权利要求1所述蛹虫草多糖食用菌酵素复方饮液的制备方法，包括以下步骤：
（1）蛹虫草粉以物料重量比为1：15加蒸馏水，超声30min，在100℃下油浴提取2h，离心得提取液，将提取液浓缩，加入3-5倍无水乙醇进行醇沉过夜；
（2）离心并用无水乙醇洗沉淀，得到蛹虫草粗多糖，将蛹虫草粗多糖进行冷冻干燥，得到蛹虫草多糖粉；
（3）取虫草粗多糖20%，蔗糖5%，食用菌酵素20%，柠檬酸0 .2%，羧甲基纤维素钠0 .3%，山梨酸钾0 .01%，用去离子水加至100%混合，均质、瞬时灭菌，灭菌温度在100-120℃，时间在30-60 分钟，灌装封口。
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