CN107223983A - 鸡腿菇固态转化山药的发酵物及其应用 - Google Patents
鸡腿菇固态转化山药的发酵物及其应用 Download PDFInfo
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- CN107223983A CN107223983A CN201710554596.3A CN201710554596A CN107223983A CN 107223983 A CN107223983 A CN 107223983A CN 201710554596 A CN201710554596 A CN 201710554596A CN 107223983 A CN107223983 A CN 107223983A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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Abstract
本发明涉及一种鸡腿菇固态转化山药的发酵物,本发明人以大米山药为主要原料,通过鸡腿菇菌体液态摇瓶培养、液体种子罐扩大培养获得含有鸡腿菇液体菌种。利用该液体菌种与灭过菌的大米山药和辅料混合,平摊于固态发酵床上,进行固态发酵。本发明的发酵物可以显著降低血糖,是一种纯天然制剂,与现有降糖药物相比不仅见效快、效果显著,并且服用后不会导致低血糖、有益无害。
Description
技术领域
本发明涉及一种鸡腿菇固态转化山药的发酵物,属于生物工程技术领域。
背景技术
随着我国经济的发展、人民生活水平的提高以及人们饮食结构的变化,尤其是高脂膳食摄入的增多,高血压、高和高血糖等“三高”疾病的发病率逐年上升。“三高”疾病已成为影响国民身体健康的头号大敌,每年因“三高”疾病死亡的人数居各种疾病死亡人数的首位。控制和预防“三高”,西药虽然效果较好,但因副作用大、价格相对较高,并非每个病人都能接受,因此开发疗效更好、副作用更小的药品及保健食品有着良好的发展前景。
山药为薯预科薯蓣属中能形成地下肉质块茎的栽培种,一年生或多年生缠绕性藤本植物,别名淮山、薯损、怀山药等。山药是一种药食兼用的高产、高效经济作物,干制入药为滋补强壮剂,对虚弱、慢性肠炎、糖尿病等有辅助疗效。山药富含蛋白质、氨基酸、淀粉等营养物质以及多糖、皂苷、维生素、尿囊素、多巴胺、山药碱、多酚氧化酶等生物活性成分。《本草纲目》山药治诸虚百损,疗五劳七伤,止腰痛、除烦热、补心气不足,开达心孔、多记事、强筋骨、益肾气、健脾胃、止泻痢、化痰涎、润皮毛等功效,《医学衷中参西录》记载山药具有止消渴(糖尿病)。现代医学研究证明山药具有调节或增强机体免疫、降低血压和功效以及抗肿瘤、抗氧化、抗衰老和抗突变等作用。山药最初食用方法是去皮、晒干、磨粉后长期保藏,通常将山药粉通过蒸煮做成各种食品或者将山药直接煲成汤来食用。近些年来,随着科学技术的进步,人们对山药的开发也逐渐的向深层次发展,有关山药产品研究开发的报道很多:将山药直接干燥制成全粉、精粉,将山药直接打浆制成系列饮料,将山药淀粉制成营养米粉,将山药和纯奶、大米等复合发酵成山药酸奶和山药酒等。但质量不稳定,而且山药还具有中药的共性,见效慢。将山药的有效成分直接提取出来开发成具有保健功能的食品正逐渐成为山药开发利用的新思路。
鸡腿菇(Coprinus comatus)是药用真菌的一种,又名鸡腿蘑,学名毛头鬼伞,隶属于真菌门(Eumycota),担子菌亚门(Basidiomycotina),层菌纲(Hymenomycetes),伞菌目(Agaricales),鬼伞科(Coprinaceae),鬼伞属(Coprinus)。鸡腿菇不仅具有多种营养功能,还具有抑制肿瘤、增强肌体免疫、降低血糖,预防治疗高血压、冠心病及动脉硬化等多方面的药学作用。
目前已经公开的鸡腿菇专利主要是如何获得鸡腿菇菌种、鸡腿菇的栽培或是鸡腿菇多糖的提取,没有利用其生产含有皂苷、甾醇、多酚和多糖的保健品或药品的报道,而且都是实验室规模,其工艺技术能否用于工业化还不能确定。
发明内容
本发明的目的在于:提供一种鸡腿菇固态转化山药的发酵物,将液体菌种扩大培养技术与固态生物转化相结合,以鸡腿菇为菌种,大米山药为主要原料,采用发酵与转化相结合,生产具有降血糖功能的保健食品和中药。
技术方案
本发明人经过深入的研究,摸索出一种以大米山药为主要原料,通过鸡腿菇菌体液态摇瓶培养、液体种子罐扩大培养获得含有鸡腿菇液体菌种。利用该液体菌种与灭过菌的大米山药和辅料混合,平摊于固态发酵床上,进行固态发酵。具体方案如下:
一种鸡腿菇固态转化山药的发酵物,其由如下方法制备而成:
a.试管扩大培养:将鸡腿菇菌种接种于马铃薯葡萄糖斜面培养基中,20-35℃下培养3-10d后,进行1-10℃保藏,制得鸡腿菇斜面菌种;
b.液体摇瓶培养:将步骤a制得的鸡腿菇斜面菌种切成3mm×3mm大小,挑取3~5块接种到装有液体摇瓶培养基的摇瓶中,20-35℃下培养3-10d后,制得液体摇瓶菌种;其中,每1L液体摇瓶培养基含有:葡萄糖10~60g,玉米粉10~50g,小麦麸皮5~20g,玉米皮5~25g,酵母浸膏5~30g,磷酸二氢钾1-8g,硫酸镁1~8g;
c.种子罐种子培养:将步骤b制得的液体摇瓶菌种接种到种子罐培养基,在种子罐内进行培养,制得种子罐菌种液体;其中,每1L种子罐培养基含有:葡萄糖10~60g,小麦麸皮5~20g,玉米皮5~80g,酵母浸膏5~30g,磷酸二氢钾1-8g,硫酸镁1~8g;
d.固体发酵培养:将种子罐菌种液体和固体发酵培养基混合均匀,摊在固态发酵床上,厚度6-20cm,宽度60-120cm,在固态发酵培养室中培养3-15天后,干燥至恒重,得到鸡腿菇固态转化山药的发酵物;
步骤d中,所述固体发酵培养基是通过将培养基原料进行灭菌制得,培养基原料按重量份计包括:100份大米,20~60份山药,10~50份玉米皮,60~150份水。
进一步,步骤c中,将液体摇瓶菌种接种到种子罐培养基的接种量为2-20v%。
进一步,种子罐中的培养条件为:温度为20-35℃,搅拌转速为50-160r/m,通气量为0.2-1.8:1v/v/m,培养18-200h。
进一步,步骤d中,接种量为:每100g固体发酵培养基中接种1-20ml种子罐菌种液体。
进一步,步骤d中,培养条件为:温度20-35℃,通气量为0.05-0.5:1v/v/m。
上述鸡腿菇固态转化山药的发酵物在降血糖和中的应用。
本发明的鸡腿菇固态转化山药的发酵物中,100克干的发酵物含有20~80毫克皂苷,10~100毫克多酚,10~50毫克甾醇,100~1000mg多糖。
可将本发明的鸡腿菇固态转化山药的发酵物制备醇提物和水提物,用于腹腔注射途径或口服途径。
鸡腿菇固态转化山药的发酵物的醇提物的制备方法:以1g鸡腿菇固态转化山药的发酵物计算,将鸡腿菇固态转化山药的发酵物加入10~60ml20~80wt%的乙醇溶液,于85℃提取2~6h,然后离心,滤渣重复此提取过程两次,三次提取滤液合并;然后真空浓缩至2-10ml,再加入10~20ml水,2℃沉淀;滤出浓缩沉淀物,用2℃水洗涤后烘干,得到鸡腿菇固态转化山药发酵物的醇提物。
鸡腿菇固态转化山药的发酵物的水提物的制备方法:以1g鸡腿菇固态转化山药的发酵物计算,将鸡腿菇固态转化山药的发酵物加入10~60ml水,煮沸提取2~6h,然后离心,滤渣重复此提取过程两次,三次提取滤液合并;三次提取合并滤液真空浓缩至2-10ml,再加入4~10ml乙醇,2℃沉淀;滤出浓缩沉淀物,用乙醇洗涤后烘干,得到鸡腿菇固态转化山药发酵物的水提物。
有益效果:本发明的鸡腿菇固态转化山药的发酵物中,100克干的发酵物含有20~80毫克皂苷,10~100毫克多酚,10~50毫克甾醇;100~1000mg多糖。本发明人经反复试验证实,鸡腿菇固态转化山药发酵物口服途径及其醇提物和水提物腹腔注射途径或口服途径给予高血糖大鼠可以显著降低其血糖。而且本发明的发酵物是一种纯天然制剂,与现有降糖药物相比不仅见效快、效果显著,并且服用后不会导致低血糖、有益无害。
具体实施方式
下面结合具体实施例对本发明作进一步说明。
实施例1
a.试管扩大培养:将鸡腿菇菌种接种于马铃薯葡萄糖斜面培养基中,20℃下培养10d后,进行10℃保藏,制得鸡腿菇斜面菌种;
b.液体摇瓶培养:将步骤a制得的鸡腿菇斜面菌种切成3mm×3mm大小,挑取5块接种到装有液体摇瓶培养基的摇瓶中,35℃下培养3d后,制得液体摇瓶菌种;其中,每1L液体摇瓶培养基含有:葡萄糖60g,玉米粉50g,小麦麸皮20g,玉米皮25g,酵母浸膏30g,磷酸二氢钾8g,硫酸镁8g;
c.种子罐种子培养:将步骤b制得的液体摇瓶菌种按照体积比20%接种到种子罐培养基,在温度为35℃,搅拌转速为50r/m,通气量为0.2:1v/v/m的条件下,培养18h,制成种子罐菌种;其中,每1L种子罐培养基含有:葡萄糖10g,小麦麸皮5g,玉米皮5g,酵母浸膏5g,磷酸二氢钾1g,硫酸镁1g;
d.固体发酵培养:将种子罐菌种和固体发酵培养基混合均匀(每100g固体发酵培养基中接种20ml种子罐菌种液体),摊在固态发酵床上,厚度20cm,宽度60cm,长度不限,在温度20℃,通气量为0.05:1固态发酵培养室中培养15d后,干燥至恒重,得到鸡腿菇固态转化山药发酵物;固体发酵培养基通过将培养基原料进行灭菌制得,固体发酵培养基以每100Kg大米添加的其他各成分的重量为:山药20Kg,玉米皮10Kg,水60kg。
实施例2
a.试管扩大培养:将鸡腿菇菌种接种于马铃薯葡萄糖斜面培养基中,25℃下培养6d后,进行5℃保藏,制得鸡腿菇斜面菌种;
b.液体摇瓶培养:将步骤a制得的鸡腿菇斜面菌种切成3mm×3mm大小,挑取3块接种到装有液体摇瓶培养基的摇瓶中,20℃下培养10d后,制得液体摇瓶菌种;其中,每1L液体摇瓶培养基含有:葡萄糖10g,玉米粉10g,小麦麸皮5g,玉米皮5g,酵母浸膏5g,磷酸二氢钾1g,硫酸镁1g;
c.种子罐种子培养:将步骤b制得的液体摇瓶菌种按照体积比2%接种到种子罐培养基,在温度为20℃,搅拌转速为160r/m,通气量为1.8:1v/v/m的条件下,培养200h,制成种子罐菌种;其中,每1L种子罐培养基含有:葡萄糖60g,小麦麸皮20g,玉米皮80g,酵母浸膏30g,磷酸二氢钾8g,硫酸镁8g;
d.固体发酵培养:将种子罐菌种和固体发酵培养基混合均匀(每100g固体发酵培养基中接种1ml种子罐菌种液体),摊在固态发酵床上,厚度6cm,宽度60cm,长度不限,在温度35℃,通气量为0.05:1固态发酵培养室中培养10d后,干燥至恒重,得到鸡腿菇固态转化山药发酵物;固体发酵培养基通过将培养基原料进行灭菌制得,固体发酵培养基以每100Kg大米添加的其他各成分的重量为:山药60Kg,玉米皮50Kg,水150kg。
实施例3
a.试管扩大培养:将鸡腿菇菌种接种于马铃薯葡萄糖斜面培养基中,35℃下培养3d后,进行1℃保藏,制得鸡腿菇斜面菌种;
b.液体摇瓶培养:将步骤a制得的鸡腿菇斜面菌种切成3mm×3mm大小,挑取3块接种到装有液体摇瓶培养基的摇瓶中,28℃下培养7d后,制得液体摇瓶菌种;其中,每1L液体摇瓶培养基含有:葡萄糖30g,玉米粉30g,小麦麸皮12.5g,玉米皮12.5g,酵母浸膏18g,磷酸二氢钾5g,硫酸镁4g;
c.种子罐种子培养:将步骤b制得的液体摇瓶菌种按照体积比10%接种到种子罐培养基,在温度为28℃,搅拌转速为110r/m,通气量为1:1v/v/m的条件下,培养100h,制成种子罐菌种;其中,每1L种子罐培养基含有:葡萄糖30g,小麦麸皮12.5g,玉米皮42g,酵母浸膏16g,磷酸二氢钾5g,硫酸镁5g;
d.固体发酵培养:将种子罐菌种和固体发酵培养基混合均匀(每100g固体发酵培养基中接种10ml种子罐菌种液体),摊在固态发酵床上,厚度8cm,宽度110cm,长度不限,在温度20℃,通气量为0.1:1固态发酵培养室中培养15天后,干燥至恒重,得到鸡腿菇固态转化山药发酵物;固体发酵培养基通过将培养基原料进行灭菌制得,固体发酵培养基以每100Kg大米添加的其他各成分的重量为:山药40Kg,玉米皮30Kg,水100kg。
试验测试:
1、将实施例1-3制得的鸡腿菇固态转化山药的发酵物进行多酚、皂苷、多糖的含量测试,测试方法如下:
1)多酚的含量测试
(1)标准曲线制作:准确量取1.00、2.00、3.00、4.00、5.00、6.00、7.00、8.00mL0.01mg/mL没食子酸标准溶液,分别置于10mL容量瓶中,在每个容量瓶内分别加入5.0mL 10%福林酚试剂摇匀,反应8min内,加入4.0mL7.5%Na2CO3溶液,加水定容至刻度、摇匀。室温下放置60min。用10mm比色皿、用蒸馏水作为空白,在765nm波长条件下测定吸光度,制作没食子酸浓度-吸光度标准曲线。
(2)多酚的提取:取经80℃干燥至恒重的鸡腿菇转化山药样品1.0g250mL三角瓶中,加入25mL 70%乙醇溶液,于70℃的水浴中进行振荡提取,3h后过滤,滤渣用25mL二次70%乙醇二次重复提取取物。两次滤液合并经0.45微米滤膜过滤,获得茶多酚浸提液。
(3)样品中多酚浓度的测定:准确吸取多酚提取液1.0mL共3份,分别置10mL容量瓶,在每个容量瓶内分别加入5.0mL 10%福林酚试剂摇匀,反应8min内,加入4.0mL 7.5%Na2CO3溶液,加水定容至刻度、摇匀。室温下放置60min。用10mm比色皿、在765nm波长条件下,以水作空白对照,测定吸光度(A),根据标准曲线计算样品多酚的含量。
2)皂苷的含量测试
(1)标准曲线制作:吸取标准液0.1、0.15、0.20、0.25、0.30、0.35和0.40mL1mg/mL用无水乙醇配置的薯蓣皂甙标准溶液,分别置于10mL具塞试管中,70℃水浴挥干溶剂,加入0.2mL的5%香草醛-冰醋酸溶液,再加入0.8mL的高氯酸,摇匀,于60℃水浴中加热20min,立即取出用流水冷却至室温,加入冰醋酸5mL,摇匀,于505nm处测定其吸光值OD。绘制薯蓣皂甙吸光度-浓度标准曲线。
(2)样品皂苷的提取:取经80℃干燥至恒重的鸡腿菇转化山药样品2.5g,用乙醚经索氏提取器脱脂风干。准确称取脱脂风干的鸡腿菇山药粉0.2g于10mL具塞试管中,准确加入4mL含1%氨水乙醇,加塞后放入超声波仪中超声1h,然后取出试管,置于离心机中以3000转/分离心15min,上清液作为待测液。
(3)样品中皂苷浓度的测定:吸取上述样品皂苷提取液各0.4mL,3个重复,分别置于10mL具塞试管中,70℃水浴挥干溶剂,加入0.2mL的5%香草醛-冰醋酸溶液,再加入0.8mL的高氯酸,摇匀,于60℃水浴中加热20min,立即取出用流水冷却至室温,加入冰醋酸5mL,摇匀,于505nm处测定其吸光值OD。
3)多糖的含量测试
a.多糖准曲线测定:准确量取0.1、0.2、0.3、0.4、0.5、0.6和0.7mL1mg/mL的葡萄糖标准品溶液于定量刻度试管中,蒸馏水定容到2mL,分别加入1.0mL 5%的苯酚溶液,快速加入5.0mL浓硫酸并摇匀,沸水浴15min,流动水冷却至室温,以2mL蒸馏水代替标准液,冷却后,用水代替标准葡萄糖溶液作为空白对照。于波长490nm处测量吸光值,然后绘制标准葡萄糖浓度-吸光度曲线图。
b.多糖的提取:称取1g样品,放入试管,加入10mL蒸馏水,塞入试管塞,在80℃下水浴3h,转入离心管离心,残渣再加入30mL蒸馏水,沸水浴1h,取上清液,蒸馏水定容至10mL保存在4℃冰箱中,用于分析多糖含量。
c.样品中多糖测定:准确量取多糖提取液2mL,快速加入5.0mL浓硫酸并摇匀,沸水浴15min,流动水冷却至室温,以2mL蒸馏水代替标准液,冷却后,用水代替标准葡萄糖溶液作为空白对照。于波长490nm处测量吸光值。
d.样品多糖含量分析结果:实施例1、实施例2和实施例3的多糖含量分别为102mg/100g干基质,449mg/100g干基质和998mg/干基质。
测试结果见下表:
实施例1 | 实施例2 | 实施例3 | |
多酚,mg/100g发酵物 | 10.02 | 99.6 | 58.7 |
皂苷,mg/100g发酵物 | 20.1 | 44.6 | 79.9 |
多糖,mg/100g发酵物 | 102 | 449 | 998 |
2、降血糖功效评价
试验方法:100只小鼠以基础饲料适应喂养5d后,腹腔注射180mg/kg体重的四氧嘧啶,饲喂普通小鼠饲料(12%酪蛋白,60.98%玉米淀粉,15%蔗糖,7%玉米油,1%维生素糖粉,4%矿物盐,0.02%鱼肝油)五天后,取尾血用血糖试剂盒测定血糖含量,以血糖含量高于13mmol/mL的模型鼠随机分为6组:正常对照组、高血糖对照组、实施例1样品组、实施例2样品组和实施例3样品组,所有实验鼠都饲喂基础饲料。各组小鼠均自由摄食和饮水,动物室温度18~22℃。连续给药四周后,每隔七天尾部静脉取血,分析血糖含量,试验结果见图1。
由图1可以看出,在整个试验期间,正常组和阳性对照组血糖值基本不变,而实施例1、实施例2和实施例3组血糖值显著降低。
Claims (6)
1.一种鸡腿菇固态转化山药的发酵物,其特征在于,其由如下方法制备而成:
a.试管扩大培养:将鸡腿菇菌种接种于马铃薯葡萄糖斜面培养基中,20-35℃下培养3-10d后,进行1-10℃保藏,制得鸡腿菇斜面菌种;
b.液体摇瓶培养:将步骤a制得的鸡腿菇斜面菌种切成3mm×3mm大小,挑取3~5块接种到装有液体摇瓶培养基的摇瓶中,20-35℃下培养3-10d后,制得液体摇瓶菌种;其中,每1L液体摇瓶培养基含有:葡萄糖10~60g,玉米粉10~50g,小麦麸皮5~20g,玉米皮5~25g,酵母浸膏5~30g,磷酸二氢钾1-8g,硫酸镁1~8g;
c.种子罐种子培养:将步骤b制得的液体摇瓶菌种接种到种子罐培养基,在种子罐内进行培养,制得种子罐菌种液体;其中,每1L种子罐培养基含有:葡萄糖10~60g,小麦麸皮5~20g,玉米皮5~80g,酵母浸膏5~30g,磷酸二氢钾1-8g,硫酸镁1~8g;
d.固体发酵培养:将种子罐菌种液体和固体发酵培养基混合均匀,摊在固态发酵床上,厚度6-20cm,宽度60-120cm,在固态发酵培养室中培养3-15d后,干燥至恒重,得到鸡腿菇固态转化山药的发酵物;
步骤d中,所述固体发酵培养基是通过将培养基原料进行灭菌制得,培养基原料按重量份计包括:100份大米,20~60份山药,10~50份玉米皮,60~150份水。
2.如权利要求1所述的鸡腿菇固态转化山药的发酵物,其特征在于,步骤c中,将液体摇瓶菌种接种到种子罐培养基的接种量为2-20v%。
3.如权利要求1所述的鸡腿菇固态转化山药的发酵物,其特征在于,步骤c中,种子罐中的培养条件为:温度为20-35℃,搅拌转速为50-160r/m,通气量为0.2-1.8:1v/v/m,培养18-200h。
4.如权利要求1所述的鸡腿菇固态转化山药的发酵物,其特征在于,步骤d中,接种量为:每100g固体发酵培养基中接种1-20ml种子罐菌种液体。
5.如权利要求1或2或3或4所述的鸡腿菇固态转化山药的发酵物,其特征在于,步骤d中,培养条件为:温度20-35℃,通气量为0.05-0.5:1v/v/m。
6.权利要求1-5任一项所述的鸡腿菇固态转化山药的发酵物在降血糖中的应用。
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