CN105166602B - 一种利用黑芝固体发酵降解花生粕中黄曲霉毒素的方法 - Google Patents
一种利用黑芝固体发酵降解花生粕中黄曲霉毒素的方法 Download PDFInfo
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Abstract
本发明公开了一种利用黑芝固体发酵降解花生粕中黄曲霉毒素的方法,包括原料处理、菌种活化、固体发酵、黄曲霉毒素降解以及黄曲霉毒素的测定等步骤,本发明提供了一种高效安全降解花生粕中黄曲霉毒素的方法,降解后的花生粕可用于食品和饲料生产加工,同时还可增加花生粕的营养价值;该方法操作简单,易于工业化。
Description
技术领域
本发明涉及一种利用黑芝固体发酵降解花生粕中黄曲霉毒素的方法,属于花生食品安全领域。
背景技术
我国是花生生产大国,每年花生榨油后可得到约300万吨的花生粕。花生粕中含有丰富的营养成分,如花生蛋白、黄酮类、糖类、灰分等。其中花生蛋白与应用较广的大豆蛋白相比,具有含肠胃胀气因子和抗营养因子较少的优点;与菜籽、棉籽蛋白相比,具有所含毒性物质较少的优点。但花生粕利用率低,主要作为饲料,价格低廉,现在约2600元/吨,远低于大豆粕的价格(3200元/吨)。限制价格和广泛应用的主要因素是其易感染黄曲菌产生黄曲霉毒素。
黄曲霉毒素(Aflatoxins,AFT)主要是由黄曲霉(Aspergillus flavus)和寄生曲霉(Aspergillus parasiticus)等多种真菌产生的毒性代谢产物,无色、无嗅、无味,含有一个双呋喃环和一个氧杂萘邻酮(香豆素),前者为基本毒素结构,后者与致癌有关。因其极强的致癌、致突变和致畸性,被国际癌症研究机构(IARC)确认为I类致癌物。此外,黄曲霉毒素还是一种剧毒物,其急性毒性为砒霜的68倍,***的10倍,可在短期内使肝脏严重受损而造成死亡。因此黄曲霉毒素是世界各国食品检验和进出口检验的重要指标,并规定食品中黄曲霉毒素允许含量安全标准,超标的食品一律销毁,造成巨人的损失,据统计,全世界每年平均有2%的谷物由于黄曲霉毒素污染而不能食用。因此花生粕中黄曲霉毒素的降解迫在眉睫。
对于食品和饲料中黄曲霉毒素的去除方法主要有物理法、化学法和生物降解法。物理方法去毒主要有水洗法、挑除法、吸附法、晾晒去毒法、溶剂提取法、加热去毒法、辐射法等。化学方法去毒主要是使用化学试剂除去毒素。使用的化学试剂有次氯酸钠、臭氧、过氧化氢、氢氧化钠、氨水以及氯气等。
传统的理化方法去毒都存在一些问题,如导致食品和饲料中的营养损失,影响感官品质,有些方法所需要的设备价格昂贵等等;其次有些方法的反应机制是可逆的,黄曲霉毒素的毒性会重新出现,这些都限制了传统理化方法在实际生产中的应用。
生物降解脱毒是黄曲霉毒素分子的毒性基团被微生物产生的次级代谢产物或者所分泌的胞外胞内酶分解破坏,同时产生无毒降解产物的过程,该法不但处理条件温和,不会破坏产品品质,而且还能增加产品营养价值。
国内外生物降解脱除黄曲霉毒素的研究主要集中在微生物菌株直接作用于黄曲霉毒素B1,以及把菌株产生的酶做成制剂作用于黄曲霉毒素。而对于复杂基质如花生粕中黄曲霉毒素的降解研究较少。
生物降解所涉及的菌种目前主要涉及国内的假蜜环菌、粘细菌、嗜麦芽窄食单胞菌以及国外研究的橙色黄杆菌和红串红球菌等,但存在着菌株在花生粕中不易生长繁殖、降解率低、有些菌株不能在食品生产中应用,有一定的安全隐患等问题。我们前期通过大量的研究探索,发现黑芝能够高效降解复杂基质花生粕中的黄曲霉毒素,且黑芝在花生粕中生长茂盛,能够快速生长繁殖,同时可抑制黄曲霉菌的生长。
黑芝也称“中国灵芝”,味甘,性平。主要含麦角甾醇、有机酸、氨基葡萄糖、多糖类、树脂、甘露醇、多糖醇、脂肪酸,又含生物碱、内酯、香豆精、水溶性蛋白质和多种酶类。
黑芝具有如下功效:
增强人体免疫:表现在通过服用黑芝能增加胸腺、脾脏重量,增强白细胞活性和吞噬功能,促进淋巴细胞增殖和转化增强细胞的***与再生。
增强学习记忆能力:临床观察,服用黑芝能提高人体多巴胺含量,从而加强大脑的单胺类介质,促进人脑的注意力、反应力和记忆力。
促进核酸、蛋白质的合成:黑芝所含有的多糖类化合物能促进人体蛋白质的合成,改善造血功能,有利于增强人体的防御机制。
保肝、解毒:黑芝的醇类化合物以及黑芝酸具有很强的保肝活性,降低肝脏三酰甘油的蓄积,提高肝细胞的代谢能力和再生修复能力。
5)延缓细胞衰老:黑芝所特含的多糖肽能有效的清楚人体自由基的能力,亦减少心、脑中脂褐质的生产,提高细胞的活性与代谢能力,预防老年痴呆症的产生,并使皮肤细胞保持紧肌肤紧致,亮泽。
6)治疗高血压和高血脂症:黑芝醇类化合物、多糖类能增强心肌收缩力,软化血管、减慢心率,达到降压的作用;同时还能降低血糖,提升胰岛素,帮助糖尿病人恢复健康。
因此黑芝在花生粕中固体发酵,不仅能显著降解花生粕中的黄曲霉毒素,还能提高花生粕的营养价值,增加花生粕保健和药用价值。
发明内容
为了高效降解花生粕中的黄曲霉毒素,提高花生粕的营养价值和利用率,本发明提供了一种利用黑芝固体发酵降解花生粕中黄曲霉毒素的方法,利用黑芝固体发酵花生粕产生降解酶,利用产生的降解酶对花生粕中黄曲霉毒素进行酶解,降解花生粕中的黄曲霉毒素。
为达到上述目的,本发明所采用的技术方案有如下步骤:
(1)原料处理:将花生粕粉碎,过40目筛;向花生粕粉中加入一定量的蒸馏水,调节花生粕pH值3-5,灭菌;
(2)菌种活化:将花生壳粉碎成5mm左右的粒装,加入2-3倍重量的水,灭菌,接种黑芝,30-35℃发酵10-12d,得到活化菌种。
(3)固体发酵:向灭菌的花生粕中接种一定量活化后的黑芝菌种,25-40℃固体发酵12-18d;
(4)黄曲霉毒素降解:发酵后的培养基加入一定比例的蒸馏水,30-40℃振荡酶解3-6h;
(5)黄曲霉毒素的测定:酶解液100℃灭酶处理10min,用高效液相色谱测定酶解液中黄曲霉毒素含量。
优选地,一种利用黑芝固体发酵降解花生粕中黄曲霉毒素的方法步骤(1)所述的花生粕粉和蒸馏水的添加比例为1:1-1:2。
优选地,一种利用黑芝固体发酵降解花生粕中黄曲霉毒素的方法步骤(2)所述的黑芝的接种量为:10g花生壳接种2-5菌种塞(7mm直径)的菌株。
优选地,一种利用黑芝固体发酵降解花生粕中黄曲霉毒素的方法步骤(3)所述的活化黑芝菌种的接种量为:花生粕和活化菌种的比例为10:1-10:3。
优选地,一种利用黑芝固体发酵降解花生粕中黄曲霉毒素的方法步骤(4)所述的发酵后的培养基和蒸馏水的重量比例为1:1-1:3。
本发明的有益效果如下:
(1)本发明通过黑芝固体发酵花生粕可以高效降解花生粕中的黄曲霉毒素;该发明能将黄曲霉毒素含量在100ppb以下的花生粕降解为黄曲霉毒素含量低于20ppb的花生粕,低于食品中黄曲霉毒素含量的国家限量标准(20ppb),可用于食品和饲料加工生产;该发明能将黄曲霉毒素含量在100ppb-200ppb的花生粕降解为黄曲霉毒素含量低于50ppb的花生粕,低于饲料中黄曲霉毒素含量的国家限量标准(50ppb),可用于饲料加工生产。
(2)黑芝在花生壳培养基上活化后,活力提高。此外将用花生壳活化的黑芝接种到花生粕中,因花生壳含有丰富的膳食纤维和黄酮,增加了花生粕的营养成分。
(3)黑芝在花生粕中生长茂盛,可以抑制黄曲霉菌的生长和产毒;
(4)黑芝在花生粕中固体发酵,可以增加花生粕的营养价值,使发酵后的花生粕具有增强机体免疫力、增强学习记忆能力、促进核酸、蛋白质的合成、保肝、解毒以及治疗高血压和高血脂症等功效
(5)黑芝固体发酵花生粕可以广泛应用在食品加工和饲料生产中,提高花生粕的利用率和经济价值。
(6)黑芝固体发酵花生粕降解黄曲霉毒素,操作方法简单,易于工业化操作;
附图说明
图1是食用菌与不同培养基的颜色反应图
图2是温度对漆酶酶活的影响图
图3是最适温度下漆酶的酶解曲线图
图4是pH对漆酶酶活的影响图
图5是最适pH下漆酶的酶解曲线图
图6是金属离子对漆酶酶活的影响图
图7是黑芝在花生粕上的长势图
具体实施方式
实施例1----黑芝固体发酵花生粕能高效降解花生粕中的黄曲霉毒素
一.降解黄曲霉毒素的食用菌菌株的筛选
1.产降解黄曲霉毒素菌株的初筛
所选择的菌株在GLDS和NLDS这两种选择培养基上进行培养时,产生的漆酶是不同种类的。漆酶在不同的选择培养基上的反应是不同的。跟愈创木酚反应时,会产生红褐色变色圈,和α-萘酚反应时,会产生蓝黑色的变色圈(如图1),变色圈的直径和颜色随着培养时间的增加而变大和变深。菌株在愈创木酚培养基上的变色圈的颜色比α-萘酚培养基的颜色更深,但是它产生的变色圈的直径的大小没有在α-萘酚培养基的大,推测这是因为选取的菌种分泌的为多种同工漆酶,而且这些漆酶对GLDS和NLDS的催化的专一性有所不同。同时王剑峰等通过探究后发现,GLDS对于漆酶生物合成反应的诱导性作用会更加明显,这就让培养的菌株在GLDS中变色圈的的反应不同,变得颜色加深,直径却减小。
表1食用菌在选择平板培养基上的培养结果
2.高产降解黄曲霉毒素菌株的复筛
表2食用菌发酵花生粕降解黄曲霉毒素的能力
上表中各菌种对于黄曲霉毒素的降解率可以看出,黑芝、香菇808、中华黑平的降解率是复筛中最高的三种,而黑芝又是其中最高的,明显高于另外两种,所以黑芝能够高效降解花生粕中黄曲霉毒素。
二.黑芝产漆酶酶学性质的研究
加热灭活后的酶液对照吸光度结果为:0.034、0.037、0.036,酶活为3.148、3.426、3.333。
1.温度对漆酶酶活的影响
由图2可以看出,在35℃之前,随着温度的上升,酶的活性也随之上升,在35℃之后,酶活呈现大幅度的下降,并在40℃后趋于平缓,所以35℃为此漆酶的最适温度。
2.最适温度下漆酶的酶解曲线
由图3可以看出,在35℃时,当反应时间不断延长,开始是快速上升,慢慢涨势减弱,趋于平缓。说明此漆酶在35℃的条件下,随反应时间的增加,酶活性不断增加,最后保持在一个较高的水平上。
3.pH对漆酶酶活的影响
在图4中在从pH 2.0到pH 3.0时,酶的活性快速增长,在pH 3.0之后开始下降,到pH为7.0时,酶活已经很低,近似于热灭活后的酶液吸光度的结果,说明此漆酶在此时酶活受到很大抑制,接近失活状态。由此可以推断此漆酶为嗜酸性酶,最适pH为3.0,趋于碱性的环境会抑制酶的活性,但对于太低的pH环境还是不耐受。
4.最适pH下漆酶的酶解曲线
从图5可以得出,在60min内,漆酶的酶活呈现直线增长,且涨势良好。一小时后,曲线趋于平直,保持在一个较高酶活的水平。
5.金属离子对漆酶酶活的影响
由图6可明显看出,除了Cu2+对它有一定程度的激活作用之外,其他的那些金属离子全都对此漆酶的酶活都有抑制作用,而且还很强。
实施例2----黑芝在花生壳培养基上活化后,活力提高。
10g花生粕加入10ml的水,接种1g花生壳活化后的黑芝菌种,30℃培养14天;同时作为对照,10g花生粕加入10ml的水,接种1g玉米杆活化后的黑芝菌种,30℃培养14天;再加入20ml的蒸馏水,30℃酶解4h,测定酶解液中黄曲霉毒素的含量,接种花生壳活化的黑芝菌种黄曲霉毒素的降解率为76.2%,而接种玉米杆活化的黑芝菌种黄曲霉毒素的降解率为62.2%,黄曲霉毒素的降解率提高22.5%。
实施例3----黑芝在花生粕中生长茂盛,可以抑制黄曲霉菌的生长和产毒
10g花生粕加入10ml的水,接种3菌种塞的黑芝菌株,30℃培养14天,黑芝在花生粕中生长旺盛(见图7);同时作对照,10g花生粕加入10ml的水,不接种黑芝,30℃培养14天,接种黑芝的花生粕中黄曲霉毒素的含量为46.5ppb,而对照花生粕中曲霉毒素的含量为325.9ppb。
实施例4---黑芝在花生粕中固体发酵,可以增加花生粕的营养价值
表3黑芝发酵后的花生粕营养成分
黑芝发酵后的花生粕 | 未发酵的花生粕 | |
蛋白(%) | 49.98 | 48.76 |
脂肪(%) | 0.81 | 0.80 |
可溶性总糖(%) | 7.2 | 5.6 |
麦角甾醇(%) | 0.5 | 0 |
氨基葡萄糖(%) | 0.25 | 0 |
黑芝多糖(%) | 0.2 | 0 |
膳食纤维(%) | 0.35 | 0.15 |
黄酮(%) | 0.25 | 0.1 |
实施例5
10g花生粕(黄曲霉毒素含量为52ppb)加入15ml的水,接种2g花生壳活化后的黑芝菌种,35℃培养12天;加入15ml的蒸馏水,35℃酶解5h,测定酶解液中黄曲霉毒素的含量为0.5ppb,黄曲霉毒素的降解率为86.3%,发酵后花生粕黄曲霉毒素含量为7.28ppb的花生粕,发酵后的花生粕可用于食品和饲料生产加工。
实施例6
10g花生粕(黄曲霉毒素含量为102ppb)加入15ml的水,接种2g花生壳活化后的黑芝菌种,37℃培养16天;加入15ml的蒸馏水,40℃酶解6h,黄曲霉毒素的降解率为81.5%,发酵后花生粕黄曲霉毒素含量为18.8ppb,发酵后的花生粕可用于食品和饲料生产加工。
实施例7
10g花生粕(黄曲霉毒素含量为152ppb)加入15ml的水,接种2g花生壳活化后的黑芝菌种,30℃培养18天;加入15ml的蒸馏水,30℃酶解6h,黄曲霉毒素的降解率为74.8%,发酵后花生粕黄曲霉毒素含量为38.3ppb的花生粕,发酵后的花生粕可用于饲料生产加工。
实施例8
10g花生粕(黄曲霉毒素含量为201ppb)加入15ml的水,接种2g花生壳活化后的黑芝菌种,25℃培养18天;加入15ml的蒸馏水,30℃酶解6h,黄曲霉毒素的降解率为76.4%,发酵后花生粕黄曲霉毒素含量为47.3ppb的花生粕,发酵后的花生粕可用于饲料生产加工。
以上所述,仅为本发明的具体实施方式,并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。
Claims (1)
1.一种利用黑芝固体发酵降解花生粕中黄曲霉毒素的方法,其特征在于,步骤如下:
(1)原料处理:将花生粕粉碎,过40目筛;向花生粕粉中按照添加比例1:1-1:2加入蒸馏水,调节花生粕pH值3-5,灭菌;
(2)菌种活化:将花生壳粉碎成5mm左右的粒状 ,加入2-3倍重量的水,灭菌,接种黑芝,30-35℃发酵10-12d得到活化菌种;黑芝的接种量为:10g花生壳接种2-5菌种塞的菌株,菌种塞直径7mm;
(3)固体发酵:向灭菌的花生粕中接种一定量活化后的黑芝菌种,黑芝的接种量为:花生粕和活化菌种的比例为10:1-10:3,25-40℃固体发酵12-18d;
(4)黄曲霉毒素降解:发酵后的培养基按照重量比例为1:1-1:3加入蒸馏水,30-40℃振荡酶解3-6h;
(5)黄曲霉毒素的测定:酶解液100℃灭酶处理10min,用高效液相色谱测定酶解液中黄曲霉毒素含量。
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