CN107811279A - 一种药食用真菌发酵核桃青皮的方法及其发酵制品与应用 - Google Patents
一种药食用真菌发酵核桃青皮的方法及其发酵制品与应用 Download PDFInfo
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- CN107811279A CN107811279A CN201710871252.5A CN201710871252A CN107811279A CN 107811279 A CN107811279 A CN 107811279A CN 201710871252 A CN201710871252 A CN 201710871252A CN 107811279 A CN107811279 A CN 107811279A
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- walnut
- green peel
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- fungi
- edible fungal
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Abstract
本发明公开一种药食用真菌发酵核桃青皮的方法及其发酵制品与应用。所述药食用真菌发酵核桃青皮的方法包括核桃青皮制备、菌种活化、液体种子培养、真菌发酵、发酵终点判定步骤。所述发酵制品经药食用真菌发酵核桃青皮的方法制备得到。所述应用是将药食用真菌发酵核桃青皮发酵制品制备食品、保健食品、化妆品和/或药品。本发明采用药食同源的大型真菌对核桃青皮发酵,利用真菌具有降解纤维素、果胶、淀粉等物质的酶系,将核桃青皮的纤维素、木质素等分解为小分子碳源,又保留了核桃青皮中的酚类、黄烷酮类、萘醌类等有益活性成分,还增加了真菌代谢产物,使得发酵制品含有丰富的真菌多糖、皂苷类、多酚类、黄酮类等生物活性物质,应用范围广泛。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一种药食用真菌发酵核桃青皮的方法及其发酵制品与应用。
背景技术
药食同源的真菌由于富含萜类化合物(倍半萜、二萜类化合物和三萜类化合物、四萜类化合物)、多糖类化合物、蛋白多肽、生物碱、甾醇类化合物、核苷类化合物等活性物质,大多具有抗菌、抗病毒、抗癌、抗氧化、提高免疫力、降血压、降血糖、降胆固醇等功效。由于受生理状态特殊性和复杂性以及外部环境的制约,造成药食同源的真菌在自然界中形成子实体稀少,特别是形成可用子实体需多年。而人工栽培极其困难,培养条件苛刻,且生长周期长达,难以满足日益膨胀的市场需要。现已有多种大型真菌能通过液体发酵,在短时间(例如5~45d)内获得大量的菌丝体,并在发酵液中包含其多种药用成分。
核桃青皮(walnut green husk)又名胡桃青龙,是核桃的外果皮,始载于《开宝本草》,《本草纲目》记载的胡桃青皮苦涩,无毒。现代药理研究表明核桃青皮具有抗肿瘤、镇痛、抗菌消炎及抗氧化等活性。具民间记载,青皮核桃泡酒有治疗胃病的功能,也有人用核桃青皮治疗癌症。核桃青皮作为抗癌民间药方,历史渊源。白桃汤剂为民间祖传秘方,曾为不少病人解除病痛,而核桃青皮就是白桃汤剂中的主要成分之一。在中医验方中,核桃青皮可用于治疗皮肤瘙痒、疮疡肿痛、白癜风等病症。
植物源天然抗氧化剂的研究与开发已成为当今功能食品与添加剂领域及化妆品原料的重要发展方向,其抗氧化性能的稳定直接影响着抗氧化的功效。核桃青皮富含多种活性物质,具有显著的抗氧化功效。但在核桃果实采收后大量青皮成为垃圾堆放在田间、地头或沟边,会造成严重的环境污染,如果对其加以利用,不仅可以减少环境污染,还可以增加果农收入。
灰树花(Grifolarondosa(Dicks.:Fr.)S.F.Gray)是药食同源覃菌,属担子菌亚门,层菌纲,非褶菌目,多孔菌科,树花菌属。富含多糖、维生素、微量元素等功能性成分,能预防白癜风、贫血、坏血病,同时具有抗肿瘤、降血糖等功效,近代医学进一步证明,还能防止动脉硬化、提高免疫力、抗衰老等多种作用。
云芝或称彩绒革盖菌(CorilusversicolorQuel.),隶属担子菌亚门,层菌纲,非褶菌目,多孔菌科,云芝属或革盖菌属。云芝在我国被广泛用于癌症和免疫缺陷等疾病的治疗,被民间视为治病神药。研究表明,云芝多糖具有显著的抗氧化活性,对超氧阴离子自由基和DPPH自由基具有显著的清除作用。
猴头(Hericium erinaceus(RullexF.)Pers.)是药食同源覃菌,具有抗溃疡、抗炎症、抗肿瘤、抗衰老、抗疲劳、提高机体耐缺氧能力、增加心肌血液输出量、加速机体血液循环、降血糖、保肝护肝和降血脂、降血压等作用。
凸圆灵芝又称白芝(Fuscoporiapunctata(Fr.)Cum),又名桦剥管孔菌,多孔菌科,滴孔菌属,属桦木属树木专性木腐菌,引起木质部形成褐色腐朽,分布于我国黑龙江、吉林、辽宁、内蒙古等地区,具有重要的实用价值和药用价值。
茯苓(Poria cocos)隶属于真菌门,是药食同源覃菌。茯苓为多孔菌科真菌茯苓的干燥菌核,具有利尿、镇静、抗肿瘤、保护防脏组织、增强心律等作用。
红灵芝(Ganoderma lucidium)是一种药食同源的真菌,中医认为能够扶正固本、增强免疫功能和提高机体抵抗力,具有降血糖,降血压,抗神经衰弱,抗肿瘤,保肝解毒,抗衰老等功效。
申请号为2010105338675专利公开了一种用核桃青皮、松针发酵酒的生产方法。将灭菌后的核桃青皮打浆,松针磨浆,按比例调配、加热、过滤取其滤液,采用葡萄酒干酵母发酵得原酒液,经倒灌、后发酵、陈酿制得。实现了核桃青皮和松针的综合利用,通过利用核桃青皮和松针的有益成分,达到强身健体的功效。
申请号为2010101386646专利公开了一种利用核桃青皮制备生发乌发剂的方法。采用核桃青皮所含有的“胡桃醌”物质与其它物质配伍综合的一种利用核桃青皮制备生发乌发剂的方法,以达到生发乌发的医疗功效。
申请号为201310195534X专利公开了一种核桃青皮提取物及其制备方法,以及其体外抗氧化功效的测定。将核桃青皮烘干粉碎后用污水甲醇浸提,然后用丙酮-水溶解以石油醚、氯仿、乙酸乙酯、正丁醇萃取,得到乙酸乙酯提取物作为抗氧化剂使用。
申请号为2011100208010专利公开了一种新的茯苓菌株及其液体发酵方式,经发酵的茯苓,提高了菌丝产量和胞外多糖的产量,增加了茯苓功效。
申请号为2013104016324专利公开了灰树花发酵液分离菌丝体或固体发酵后的菌丝体经一系列工艺后,提取的灰树花发酵菌丝体粗多糖对禽流感病毒具有明显的抑制作用。
发明内容
本发明的第一目的在于提供一种方法简单、过程安全且不受季节限制、有利于环境保护的药食用真菌发酵核桃青皮的方法;第二目的在于提供一种第一目制备得到的发酵制品;第三目的在于提供一种第二目的的发酵制品的应用。
本发明的第一目的是这样实现的:包括核桃青皮制备、菌种活化、液体种子培养、真菌发酵、发酵终点判定步骤,具体步骤如下:
A、核桃青皮制备:将新鲜带青皮的核桃用流水洗净,自然晾干后取下青皮,切小块备用;
B、菌种活化:将保藏的药食同源真菌菌种转接到斜面固体培养基上于28~30℃下培养5~10d;
C、液体种子培养:将上述经活化的药食同源真菌菌种切取一块接种于装有100mL液体培养基的300mL三角瓶中,在28~30℃条件下以140~150r/min震荡培养7~10d,直到形成肉眼可见的子实体;
D、真菌发酵:取制备得到的核桃青皮小块90g置于250mL三角瓶或180g置于500mL三角瓶中,按3:1~10:1的料液质量体积比加入生长促进剂,灭菌后将C步骤培养好的真菌液体种子按核桃青皮重量10%的比例接种到核桃青皮中,恒温发酵直到形成肉眼可见的子实体和/或菌球、菌丝体;
E、发酵终点判定:在真菌发酵过程中每天对核桃青皮进行取样,进行DPPH清除率、羟基自由基清除率及还原力大小测定,以还原力达到最高点时作为发酵终点。
本发明的第二目的是这样实现的:药食用真菌发酵核桃青皮的方法制备得到的药食用真菌发酵核桃青皮发酵制品。
本发明的第三目的是这样实现的:药食用真菌发酵核桃青皮发酵制品在制备食品、保健食品、化妆品和/或药品中的应用。
本发明与现有技术相比具有以下有益效果:
1)通过药食同源的真菌来发酵核桃青皮,即提高了资源利用率,也减少了环境污染,整过生产过程安全、可控且不受季节限制,可规模化生产;制成的核桃青皮发酵制品有益成分丰富、还原力强、抗氧化性能好、吸收利用率高,可作为食品、化妆品和/或药品添加剂,营养丰富。
2)本发明采用药食同源的真菌灰树花、茯苓、猴头、凸圆灵芝、云芝或红灵芝对新鲜核桃青皮进行低温发酵,利用真菌具有分解纤维素、淀粉、蛋白质、脂类等物质的强大酶系,以真菌对核桃青皮基质进行深层发酵分解为小分子碳源,又能保留核桃青皮中的酚类、黄烷酮类、萘醌类等有益成分,同时核桃青皮中的某些成分(如醌类、黄酮类等)可促进或抑制真菌的生长及代谢产物的生产,不仅增加了原料有效成分利用率,而且增加了其抗氧化能力,可作为抗氧化剂的有效成分广泛用于食品、保健、化妆品及医药领域。
3)本发明以药食同源的真菌发酵核桃青皮,真菌在生长中自身产生的代谢产物如真菌多糖、生物碱、核苷类化合物等积累,这些化合物具有抗菌、抗病毒、提高免疫力等作用,由于核桃青皮与真菌的代谢产物双重作用,使得制备的核桃青皮发酵制品含有丰富的真菌多糖、皂苷类、多酚类、黄酮类等生物活性物质。
4)本发明采用的核桃青皮价格低廉、来源广泛;采用培养基培养和液体发酵的方法,采收菌丝和肉眼可见的子实体获得真菌的药用活性成分,因而具有周期短、节省劳力、成本低以及受外部环境影响小。
附图说明
图1为实施例1与对照例不同菌种核桃青皮发酵制品的抗氧化能力测定结果;
图中:DPPH—1,1-二苯基-2-三硝基苯肼,别名1,1-二苯基-2-苦肼基(自由基);OH—羟基自由基;还原力—还原氢,包括NADH(还原型辅酶Ⅱ)、NADPH(还原型辅酶Ⅰ)、FADH(黄素腺嘌呤二核苷酸)。
具体实施方式
下面结合附图和实施例对本发明作进一步的说明,但不以任何方式对本发明加以限制,基于本发明教导所作的任何变更或改进,均属于本发明的保护范围。
本发明之药食用真菌发酵核桃青皮的方法,包括核桃青皮制备、菌种活化、液体种子培养、真菌发酵、发酵终点判定步骤,具体步骤如下:
A、核桃青皮制备:将新鲜带青皮的核桃用流水洗净,自然晾干后取下青皮,切小块备用;
B、菌种活化:将保藏的药食同源真菌菌种转接到斜面固体培养基上于28~30℃下培养5~10d;
C、液体种子培养:将上述经活化的药食同源真菌菌种切取一块接种于装有100mL液体培养基的300mL三角瓶中,在28~30℃条件下以140~150r/min震荡培养7~10d,直到形成肉眼可见的子实体;
D、真菌发酵:取制备得到的核桃青皮小块90g置于250mL三角瓶或180g置于500mL三角瓶中,按3:1的料液质量体积比加入生长促进剂,灭菌后将C步骤培养好的真菌液体种子按核桃青皮重量10%的比例接种到核桃青皮中,恒温发酵直到形成肉眼可见的子实体和/或菌球、菌丝体;
E、发酵终点判定:在真菌发酵过程中每天对核桃青皮进行取样,进行DPPH清除率、羟基自由基清除率及还原力大小测定,以还原力达到最高点时作为发酵终点。
所述药食同源真菌为食用后不会造成人体中毒且能形成肉质或胶质的肉眼可见子实体的真菌,包括灰树花、凸圆灵芝、茯苓、云芝、红灵芝或猴头。
所述灰树花为G2M510菌株(保藏号GIM5.10)、G2M563菌株(GIM5.63)、G2M5164菌株(GIM5.164),所述凸圆灵芝为G2M56菌株(GIM5.6),所述茯苓为G2M5219菌株(GIM5.219),所述云芝为G2M5178菌株(GIM5.178),所述红灵芝为G2M5250菌株(GIM5.250),所述猴头为G2M5208菌株(GIM5.208),上述菌株均来自广东省微生物菌种保藏中心(GDMCC)。
所述A步骤中核桃青皮切成长度低于1cm的小块。
所述A步骤中核桃青皮切小块后经灭菌置于-20℃冷藏备用。
所述B步骤中的斜面固体培养基为PDA斜面培养基,所述PDA斜面培养基成分按质量百分比包括:马铃薯20%、葡萄糖2%、琼脂1.5~2%及余量的水。
所述B步骤中的菌种接种于斜面固体培养基的斜面底端上方1/3处,所述菌种在斜面固体培养基上于28℃下培养至菌丝长满斜面即可。
所述C步骤中的液体培养基为PDA液体培养基,所述PDA液体培养基成分按质量百分比包括:马铃薯20%、葡萄糖2%及余量的水。
所述D步骤中的生长促进剂成分按质量百分比包括:0.1%的MgSO4,0.2%的KH2PO4、0.13%的蛋白胨及余量的水。
所述D步骤中核桃青皮恒温发酵的条件为:恒温温度28~30℃,发酵时间为7~14d。
所述D步骤中核桃青皮和生长促进剂置于三角瓶后灭菌是用八层纱布覆盖瓶口并包扎,外用旧报纸包裹瓶口,于121℃灭菌20min,然后自然冷却。
本发明之药食用真菌发酵核桃青皮的方法还包括冷冻干燥步骤,所述冷冻干燥步骤是将达到发酵终点的发酵制品经真空冷冻干燥后粉碎得到药食用真菌发酵核桃青皮发酵制品粉剂。
所述发酵制品包括已发酵的核桃青皮、真菌菌丝及子实体和/或发酵液。
本发明之药食用真菌发酵核桃青皮的方法制备得到的药食用真菌发酵核桃青皮发酵制品。
本发明之药食用真菌发酵核桃青皮发酵制品在制备食品、保健食品、化妆品和/或药品中的应用。
实施例1
S100:新鲜带青皮的核桃用流水洗净,自然晾干,取下青皮,切成1×1cm大小占80%的小块,放-20℃冷藏备用。
S200:将保藏的药食同源真菌菌种灰树花G2M510菌株(保藏号GIM5.10)、灰树花G2M563菌株(GIM5.63)、灰树花G2M5164菌株(GIM5.164)、凸圆灵芝G2M56菌株(GIM5.6)、茯苓G2M5219菌株(GIM5.219)、云芝G2M5178菌株(GIM5.178)、红灵芝G2M5250菌株(GIM5.250)、猴头G2M5208菌株(GIM5.208)(各菌种均来自广东省微生物菌种保藏中心GDMCC)分别从母种斜面试管中用接种铲切取1cm2大小的菌丝块,分别接种于各PDA斜面培养基的斜面底端上方1/3处,于28℃下培养5~10d至菌丝长满斜面即可;PDA斜面培养基成分按质量百分比包括:马铃薯20%、葡萄糖2%、琼脂2%及余量的水。
S300:将S200活化的药食同源真菌菌种,用接种铲切取1cm2的菌块接种于装有100mL液体培养基的300mL三角瓶中,在28~30℃条件下以150r/min震荡培养7~10d,直到形成肉眼可见的子实体和菌球。
S400:取S100制备的核桃青皮小块90g置于250mL三角瓶中,按3:1的料液比加入30mL生长促进剂,然后用八层纱布覆盖瓶口并用棉线包扎,外用旧报纸包裹瓶口,于121℃灭菌20min后自然冷却,整个过程在无菌操作台中进行;灭菌后将S300培养好的真菌液体种子取9g接种到核桃青皮中,于28℃恒温发酵7~14d,直到形成肉眼可见的子实体;营养液成分按质量百分比包括:0.1%的MgSO4,0.2%的KH2PO4、0.13%的蛋白胨及余量的水。
S500:在真菌发酵过程中每天对核桃青皮进行取样,进行DPPH清除率、羟基自由基清除率及还原力大小测定,以还原力达到最高点时作为发酵终点。真菌在核桃青皮中的菌丝生长情况如表1。
S600:将达到发酵终点的核桃青皮、真菌菌丝及子实体和发酵液等发酵制品经真空冷冻干燥后粉碎,得到药食用真菌发酵核桃青皮发酵制品粉剂。
S500中DPPH清除率按《基于DPPH、FRAP法的薄荷药材抗氧化谱效关系研究》,徐晶晶等,北京中医药大学学报,2015,38(6):405-410所述方法进行测定;羟基自由基(OH)清除率按《紫色胡萝卜汁体外抗氧化活性的研究》,虎春艳等,中国酿造,2015,34(1):85-88所述方法进行测定;还原力按《红茶菌发酵过程中抗氧化能力的变化规律》,朱晓庆等,生产与科研经验,2010,36(11):111-114所述方法进行测定。
表1真菌在核桃青皮中的菌丝生长情况
实施例2
S100:新鲜带青皮的核桃用流水洗净,自然晾干,取下青皮,切成1×0.5cm大小占80%的小块,放-20℃冷藏备用。
S200:将保藏的药食同源真菌菌种灰树花G2M510菌株(保藏号GIM5.10)、灰树花G2M563菌株(GIM5.63)、灰树花G2M5164菌株(GIM5.164)、凸圆灵芝G2M56菌株(GIM5.6)、茯苓G2M5219菌株(GIM5.219)、云芝G2M5178菌株(GIM5.178)、红灵芝G2M5250菌株(GIM5.250)(各菌种均来自广东省微生物菌种保藏中心)分别从母种斜面试管中用接种铲切取1cm2大小的菌丝块,分别接种于各PDA斜面培养基的斜面底端上方1/3处,于30℃下培养5~10d至菌丝长满斜面即可;PDA斜面培养基成分按质量百分比包括:马铃薯20%、葡萄糖2%、琼脂1.5%及余量的水。
S300:将S200活化的药食同源真菌菌种,用接种铲切取1cm2的菌块接种于装有100mL液体培养基的300mL三角瓶中,在30℃条件下以140r/min震荡培养7~10d,直到形成肉眼可见的子实体。
S400:取S100制备的核桃青皮小块90g置于250mL三角瓶中,按5:1的料液比加入18mL生长促进剂,然后用八层纱布覆盖瓶口并用棉线包扎,外用旧报纸包裹瓶口,于121℃灭菌20min后自然冷却,整个过程在无菌操作台中进行;灭菌后将S300培养好的真菌液体种子取9g接种到核桃青皮中,于30℃恒温发酵7~14d,直到形成肉眼可见的子实体;营养液成分按质量百分比包括:0.1%的MgSO4,0.2%的KH2PO4、0.13%的蛋白胨及余量的水。
S500:在真菌发酵过程中每天对核桃青皮进行取样,按实施例1的方法进行DPPH清除率、羟基自由基清除率及还原力大小测定,以还原力达到最高点时作为发酵终点。真菌在核桃青皮中的菌丝生长情况如表2。
S600:将达到发酵终点的核桃青皮、真菌菌丝及子实体和发酵液等发酵制品经真空冷冻干燥后粉碎,得到药食用真菌发酵核桃青皮发酵制品粉剂。
表2真菌在核桃青皮中的菌丝生长情况
1d | 3d | 5d | 7d | 9d | 11d | 13d | 15d | |
灰树花G2M510 | — | — | — | + | ++ | ++ | +++ | +++ |
灰树花G2M563 | — | — | — | + | ++ | ++ | ++ | +++ |
灰树花G2M5164 | — | — | — | + | ++ | ++ | ++ | +++ |
凸圆灵芝G2M56 | — | — | + | ++ | ++ | ++ | +++ | +++ |
茯苓G2M5219 | — | — | + | ++ | ++ | +++ | +++ | +++ |
云芝G2M5178 | — | — | + | +++ | +++ | +++ | +++ | +++ |
红灵芝G2M5250 | — | — | + | ++ | ++ | +++ | +++ | +++ |
猴头G2M5208 | — | — | + | ++ | ++ | ++ | +++ | +++ |
实施例3
S100:新鲜带青皮的核桃用流水洗净,自然晾干,取下青皮,切成0.8×0.8cm大小占80%的小块,放-20℃冷藏备用。
S200:将保藏的药食同源真菌菌种灰树花G2M510菌株(保藏号GIM5.10)、灰树花G2M563菌株(GIM5.63)、灰树花G2M5164菌株(GIM5.164)、凸圆灵芝G2M56菌株(GIM5.6)、茯苓G2M5219菌株(GIM5.219)、云芝G2M5178菌株(GIM5.178)、红灵芝G2M5250菌株(GIM5.250)(各菌种均来自广东省微生物菌种保藏中心)分别从母种斜面试管中用接种铲切取1cm2大小的菌丝块,分别接种于各PDA斜面培养基的斜面底端上方1/3处,于28℃下培养5~10d至菌丝长满斜面即可;PDA斜面培养基成分按质量百分比包括:马铃薯20%、葡萄糖2%、琼脂2%及余量的水。
S300:将S200活化的药食同源真菌菌种,用接种铲切取1cm2的菌块接种于装有100mL液体培养基的300mL三角瓶中,在28℃条件下以150r/min震荡培养7~10d,直到形成肉眼可见的子实体。
S400:取S100制备的核桃青皮小块180g置于500mL三角瓶中,按10:1的料液比加入18mL生长促进剂,然后用八层纱布覆盖瓶口并用棉线包扎,外用旧报纸包裹瓶口,于121℃灭菌20min后自然冷却,整个过程在无菌操作台中进行;灭菌后将S300培养好的真菌液体种子取9g接种到核桃青皮中,于28℃恒温发酵7~14d,直到形成肉眼可见的子实体;营养液成分按质量百分比包括:0.1%的MgSO4,0.2%的KH2PO4、0.13%的蛋白胨及余量的水。
S500:在真菌发酵过程中每天对核桃青皮进行取样,按实施例1的方法进行进行DPPH清除率、羟基自由基清除率及还原力大小测定,以还原力达到最高点时作为发酵终点。真菌在核桃青皮中的菌丝生长情况如表3。
S600:将达到发酵终点的核桃青皮、真菌菌丝及子实体和发酵液等发酵制品经真空冷冻干燥后粉碎,得到药食用真菌发酵核桃青皮发酵制品粉剂。
表3真菌在核桃青皮中的菌丝生长情况
1d | 3d | 5d | 7d | 9d | 11d | 13d | 15d | |
灰树花G2M510 | — | — | + | + | ++ | ++ | +++ | +++ |
灰树花G2M563 | — | — | + | + | ++ | ++ | ++ | +++ |
灰树花G2M5164 | — | — | + | + | ++ | ++ | ++ | +++ |
凸圆灵芝G2M56 | — | — | + | ++ | ++ | ++ | +++ | +++ |
茯苓G2M5219 | — | — | + | ++ | ++ | +++ | +++ | +++ |
云芝G2M5178 | — | — | + | ++ | +++ | +++ | +++ | +++ |
红灵芝G2M5250 | — | — | + | ++ | ++ | +++ | +++ | +++ |
猴头G2M5208 | — | — | + | ++ | ++ | ++ | +++ | +++ |
对照例
S100:新鲜带青皮的核桃用流水洗净,自然晾干,取下青皮,切成1×1cm大小占80%的小块,放-20℃冷藏备用。
S200:取S100制备的核桃青皮小块90g置于250mL三角瓶中,按3:1(g/mL)的料液比加入30mL生长促进剂,然后用八层纱布覆盖瓶口并用棉线包扎,外用旧报纸包裹瓶口,于121℃灭菌20min后自然冷却,整个过程在无菌操作台中进行;灭菌后将取9g无菌水接种到核桃青皮中,于28℃恒温发酵7~14d;营养液成分按质量百分比包括:0.1%的MgSO4,0.2%的KH2PO4、0.13%的蛋白胨及余量的水。
S300:按实施例1的方法对已发酵14d的核桃青皮进行取样测定DPPH清除率、羟基自由基清除率及还原力大小。
S400:将发酵14d的核桃青皮和发酵液等发酵制品经真空冷冻干燥后粉碎,得到核桃青皮发酵制品粉剂。
Claims (10)
1.一种药食用真菌发酵核桃青皮的方法,其特征在于包括核桃青皮制备、菌种活化、液体种子培养、真菌发酵、发酵终点判定步骤,具体步骤如下:
A、核桃青皮制备:将新鲜带青皮的核桃用流水洗净,自然晾干后取下青皮,切小块备用;
B、菌种活化:将保藏的药食同源真菌菌种转接到斜面固体培养基上于28~30℃下培养5~10d;
C、液体种子培养:将上述经活化的药食同源真菌菌种切取一块接种于装有100mL液体培养基的300mL三角瓶中,在28~30℃条件下以140~150r/min震荡培养7~10d,直到形成肉眼可见的子实体;
D、真菌发酵:取制备得到的核桃青皮小块90g置于250mL三角瓶或180g置于500mL三角瓶中,按3:1~10:1的料液质量体积比加入生长促进剂,灭菌后将C步骤培养好的真菌液体种子按核桃青皮重量10%的比例接种到核桃青皮中,恒温发酵直到形成肉眼可见的子实体和/或菌球、菌丝体;
E、发酵终点判定:在真菌发酵过程中每天对核桃青皮进行取样,进行DPPH清除率、羟基自由基清除率及还原力大小测定,以还原力达到最高点时作为发酵终点。
2.根据权利要求1所述药食用真菌发酵核桃青皮的方法,其特征在于所述药食同源真菌为灰树花、凸圆灵芝、茯苓、云芝、红灵芝或猴头。
3.根据权利要求2所述药食用真菌发酵核桃青皮的方法,其特征在于所述灰树花为G2M510菌株、G2M563菌株、G2M5164菌株,所述凸圆灵芝为G2M56菌株,所述茯苓为G2M5219菌株,所述云芝为G2M5178菌株,所述红灵芝为G2M5250菌株,所述猴头为G2M5208菌株,上述菌株均来自广东省微生物菌种保藏中心。
4.根据权利要求1所述药食用真菌发酵核桃青皮的方法,其特征在于所述A步骤中核桃青皮切小块后经灭菌置于-20℃冷藏备用。
5.根据权利要求1所述药食用真菌发酵核桃青皮的方法,其特征在于所述B步骤中的斜面固体培养基为PDA斜面培养基,所述PDA斜面培养基成分按质量百分比包括:马铃薯20%、葡萄糖2%、琼脂1.5~2%及余量的水。
6.根据权利要求1所述药食用真菌发酵核桃青皮的方法,其特征在于所述C步骤中的液体培养基为PDA液体培养基,所述PDA液体培养基成分按质量百分比包括:马铃薯20%、葡萄糖2%及余量的水。
7.根据权利要求1所述药食用真菌发酵核桃青皮的方法,其特征在于所述D步骤中的生长促进剂成分按质量百分比包括:0.1%的MgSO4,0.2%的KH2PO4、0.13%的蛋白胨及余量的水。
8.根据权利要求1或7所述药食用真菌发酵核桃青皮的方法,其特征在于所述D步骤中核桃青皮恒温发酵的条件为:恒温温度28~30℃,发酵时间为7~14d。
9.一种权利要求1至8任意一项所述药食用真菌发酵核桃青皮的方法制备得到的药食用真菌发酵核桃青皮发酵制品。
10.一种权利要求9所述药食用真菌发酵核桃青皮发酵制品在制备食品、保健食品、化妆品和/或药品中的应用。
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