CN111117894B - 一株米曲霉菌株及其应用 - Google Patents
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Abstract
本发明公开了一株米曲霉CGMCC No.18109，其具有较高的产纤维素酶的活性和淀粉酶活性，相比传统的沪酿3.042，其纤维素酶活性是其1.5倍左右，能够有效的应用于富含纤维素原料的发酵和饲料的制备，将其应用于橘皮果渣中可溶性膳食纤维、陈皮甙、陈皮多糖含量都较发酵前含量显著增加。
Description
技术领域
本发明属于生物领域，涉及微生物发酵，具体涉及一株米曲霉菌株及其应用。
背景技术
膳食纤维已被国内外医学和营养学专家视为“第七营养素”，具有重要的作用：促进肠胃蠕动，改善肠道发育；增加饱腹感；调节肠道微生物平衡，改善肠道健康；提高胰岛素的敏感性，稳定血糖深度，降低血液胆固醇水平；减少病原菌从后肠向前肠转移，吸附病原菌及霉菌毒素等“清道夫”作用；提供能量；满足咀嚼等生理行为需要。可溶性膳食纤维具有多种生理功能，包括预防心血管疾病，糖尿病及抗肿瘤作用。不可溶性膳食纤维主要包括纤维素和半纤维素，它们可增加食物通过消化道的速率。不可溶性膳食纤维能够增加胃肠里的食物体积，抵抗胃肠消化液的侵袭，完好无损地抵达大肠，最后排出体外。它们能够吸收食物中的有害物质，预防便秘并弱化消化道中细菌排出的毒素，对改善母猪尤其是妊娠后期母猪临床常见的便秘情况有显著效果。
陈皮是一种传统的中药材，其在动物营养学上也具有广泛的作用，具理气降逆、调中开胃、燥湿化痰之功。主治脾胃气滞湿阻、胸膈满闷、脘腹胀痛、不思饮食、呕吐秽逆、二便不利、肺气阻滞、咳嗽痰多，亦治乳痈初起。但是传统的药材陈皮成本昂贵，尤其是陈皮苷含量较高的多年陈皮，其属于名贵中药材，不适合在饲料领域大规模应用。橘皮果渣是橘子或柠檬皮提取果胶后的废弃物，水分含量40-60％。柑橘皮和柠檬皮在中医上被认为有健胃消食的功效，应用在动物饲料中也有很好促进采食的作用，同时柑橘皮和柠檬皮还是优质的膳食纤维，尤其能够有效解决母猪的便秘问题。但是现有技术中缺乏有效的降解橘皮果渣中不溶性膳食纤维，并将其有效的转化为可溶性膳食纤维的饲料生产菌株，因此，分离和纯化合适的菌株，以提高橘皮果渣的利用率，降低陈皮类饲料成分的经济成本，具有重要的社会和经济意义。
发明内容
为解决以上技术问题，本发明只在提供一株米曲霉菌株，其具有相对较高的发酵活力，能够有效的降解橘皮果渣中的不溶性膳食纤维，将其转化为可溶性膳食纤维，具有极高的饲料利用价值。
为解决以上技术问题，本发明采用如下技术方案：
本发明提供一株米曲霉(Aspergillus oryzae)菌株，保藏号为：CGMCC No.18109。
本发明的米曲霉(Aspergillus oryzae)菌株分离自自然发酵的黄豆酱中，具体分离过程如下：
1)增菌培养：将采集到的自然发酵黄豆酱样品稀释后接种于增殖液体培养基中，在30℃恒温培养增殖3-5天；
2)分离纯化：对步骤1)增菌培养物在增殖琼脂培养基上进行稀释、涂布、培养，从中挑取具有特征形态的单菌落，进行编号，再划线培养，直至为纯菌落为止，通过革兰氏染色镜检，观察菌体形态是否单一，否则重新划线分离、纯化；将疑似菌落进行16S rDNA检测，将经鉴定为米曲霉的菌落接种于试管斜面中增殖，然后将斜面菌种保藏于4℃冰箱中，待用；
3)将鉴定出的米曲霉(Aspergillus oryzae)于2019年7月19日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址：北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所，保藏编号为：CGMCC No.18109。
其中所述增殖培养基为PDA培养基、察氏培养基中的一种。
本发明还请求保护所述米曲霉菌株在饲料加工中的应用，所述饲料原料含丰富的纤维素成分，优选的，所述饲料原料为橘皮果渣。
本发明还请求保护一种橘皮果渣的发酵方法，其特征在于：发酵剂中包含米曲霉CGMCC No.18109。
所述橘皮果渣的发酵方法包括如下步骤：
(1)活化：在无菌条件下，接种1环米曲霉于PDA斜面培养基上，置30℃恒温箱内培养3d，待斜面上长满绿色孢子以后取出备用；
(2)米曲霉种子菌液的制备：将活化好的米曲霉菌株接种于PDA液体培养基，于28～30℃、150rpm摇床培养3天，即为米曲霉种子菌液，测定活菌数>1.0×109cfu/mL；
(3)橘皮果渣发酵：将1L米曲霉菌液与100kg果渣充分混合均匀，装入呼吸式发酵袋中，置于35℃，60％的恒温恒湿发酵车间中静置发酵72-96h。
基于以上技术方案，本发明具有如下优点和有益效果：
本发明公开了一株米曲霉CGMCC No.18109，其具有较高的产纤维素酶的活性和淀粉酶活性，相比传统的沪酿3.042，其纤维素酶活性是其1.5倍左右，能够有效的应用于富含纤维素原料的发酵和饲料的制备，将其应用于橘皮果渣中可溶性膳食纤维、陈皮甙、陈皮多糖含量都较发酵前含量显著增加。
附图说明
图1：米曲霉中淀粉酶的活力变化。
图2：米曲霉中纤维素酶的活力变化。
具体实施方式
实施例1：米曲霉CGMCC No.18109的分离和保藏
1)增菌培养：将采集到的自然发酵黄豆酱样品稀释后接种于增殖液体培养基中，在30℃恒温培养增殖3-5天；
2)分离纯化：对步骤1)增菌培养物在增殖琼脂培养基上进行稀释、涂布、培养，从中挑取具有特征形态的单菌落，进行编号，再划线培养，直至为纯菌落为止，通过革兰氏染色镜检，观察菌体形态是否单一，否则重新划线分离、纯化；将疑似菌落进行16S rDNA检测，将经鉴定为米曲霉的菌落接种于试管斜面中增殖，然后将斜面菌种保藏于4℃冰箱中，待用；
3)将鉴定出的米曲霉(Aspergillus oryzae)于2019年7月19日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址：北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所，保藏编号为：CGMCC No.18109。
实施例2:米曲霉发酵活力的测定
试验菌株：米曲霉沪酿3.042，由中国农业大学赠送；米曲霉CGMCC No.3.7190，购自中国微生物菌种保藏管理委员会普通微生物中心；米曲霉CGMCC No.18109。
试验方法：纤维素酶和淀粉酶活性测定
(1)菌株的活化，分别取保存的米曲霉沪酿3.042、米曲霉CGMCC3.7190和米曲霉CGMCC No.18109，使用马铃薯葡萄糖琼(PDA)培养基作为活化培养基和种子培养基。配方为：葡萄糖2％，马铃薯2％，琼脂1.5％～2.0％，在115℃条件下灭菌30min。
在无菌条件下，接种1环米曲霉于PDA斜面培养基上，置30℃恒温箱内培养3d，待斜面上长满绿色孢子以后取出备用。
(2)种子液的制备：用10mL生理盐水将活化培养的米曲霉菌株制成1×107个孢子/mL的孢子悬浮液，各取1mL分别接种于100mL液体种子培养基中接种于PDA液体培养基，于28～30℃、150rpm摇床培养3天，即为米曲霉种子菌液。
(3)发酵产酶：将米曲霉种子菌液5mL接种至50g大曲培养基(豆粕：麸皮：水重量份为60:40:100，拌匀分装，每个500mL三角瓶分装50g培养基，121℃高温灭菌)中，每个菌株分别接种3瓶，于28℃培养24h、48h和72h时分别取出，按照20:1(mL/g)加入蒸馏水，40℃恒温水浴1h，6层纱布过滤，8000rpm离心10min，取上清液即粗酶液于4℃冰箱中保存备用。
(4)酶活测定：以可溶性淀粉为底物，采用二硝基水杨酸(DNS)法测定淀粉酶活力；以CMC为底物，采用二硝基水杨酸(DNS)法测定纤维素酶活力，具体结果参见图1和图2。
(5)结果分析：基于图1的结果可知，随着发酵时间的延长，米曲霉中的淀粉酶活力均表现为上升趋势，本发明的米曲霉CGMCC No.18109活性高于传统的米曲霉菌株沪酿3.042和米曲霉CGMCC No.3.7190，发酵72h后的酶活高达1150U/g(发酵基质)。
基于图2的结果可知，随着发酵时间的延长，发酵基质中的纤维素酶活力也逐渐上升，本发明米曲霉CGMCC No.18109的活力最高，在72h时，其酶活是传统的米曲霉菌株沪酿3.042的1.5倍，是米曲霉CGMCC No.3.7190纤维素酶活的2倍左右，说明，本发明筛选得到的米曲霉能够产生丰富的纤维素酶，适合高纤维素物料的发酵。
实施例3：橘皮果渣发酵
橘皮果渣为柑橘皮和柠檬皮提取果胶后的废弃物，水分含量40-60％。柑橘皮和柠檬皮在中医上被认为有健胃消食的功效，应用在动物饲料中也有很好促进采食的作用，同时柑橘皮和柠檬皮还是优质的膳食纤维，尤其能够有效解决母猪的便秘问题。
试验菌株：选取经实施例2检测得到的发酵性能优越的米曲霉沪酿3.042和米曲霉CGMCC No.18109作为发酵菌株。
培养基：马铃薯葡萄糖琼(PDA)培养基，配方为：葡萄糖2％，马铃薯2％，琼脂1.5％～2.0％，在115℃条件下灭菌30min。
试验方法：
(1)活化：在无菌条件下，接种1环米曲霉于PDA斜面培养基上，置30℃恒温箱内培养3d，待斜面上长满绿色孢子以后取出备用。
(2)米曲霉种子菌液的制备：将活化好的米曲霉菌株接种于PDA液体培养基，于28～30℃、150rpm摇床培养3天，即为米曲霉种子菌液，测定活菌数>1.0×109cfu/mL。
(3)橘皮果渣发酵：将1L米曲霉菌液与100kg果渣充分混合均匀，装入呼吸式发酵袋中，置于35℃，60％的恒温恒湿发酵车间中静置发酵72h；根据GB/T 5009.88-2008《食品中膳食纤维的测定》方法检测可溶性膳食纤维，使用HPLC法检测陈皮甙含量，使用苯酚-硫酸比色法检测陈皮多糖含量。
表1米曲霉发酵果渣后各项营养指标的变化情况
注：同行相应数据肩标不同大写字母表示差异极显著(P<0.01)，不同小写字母表示差异显著(P<0.05)，字母相同表示差异不显著(P>0.05)。
试验结果发现，经过米曲霉的发酵，橘皮果渣中可溶性膳食纤维、陈皮甙、陈皮多糖含量都较发酵前含量显著增加。相比传统的米曲霉沪酿3.042，本发明的米曲霉(CGMCCNo.18109)发酵后，其可溶性膳食纤维、陈皮甙、陈皮多糖含量增加更为显著。
Claims (7)
1.一株米曲霉菌株，其已于2019年7月19日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址：北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所，保藏编号为：CGMCC No.18109。
2.根据权利要求1所述的米曲霉菌株，其特征在于：所述米曲霉(Aspergillus oryzae)菌株分离自自然发酵的黄豆酱中，具体分离过程如下：
1)增菌培养：将采集到的自然发酵黄豆酱样品稀释后接种于增殖液体培养基中，在30℃恒温培养增殖3-5天；
2)分离纯化：对步骤1)增菌培养物在增殖琼脂培养基上进行稀释、涂布、培养，从中挑取具有特征形态的单菌落，进行编号，再划线培养，直至为纯菌落为止，通过革兰氏染色镜检，观察菌体形态是否单一，否则重新划线分离、纯化；将疑似菌落进行16S rDNA检测，将经鉴定为米曲霉的菌落接种于试管斜面中增殖，然后将斜面菌种保藏于4℃冰箱中，待用；
3)将鉴定出的米曲霉(Aspergillus oryzae)于2019年7月19日保藏于中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC，地址：北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所，保藏编号为：CGMCC No.18109。
3.根据权利要求2所述的米曲霉菌株，其特征在于，所述增殖培养基为PD A培养基、察氏培养基中的一种。
4.根据权利要求1-3任一项所述的米曲霉菌株在饲料加工中的应用。
5.根据权利要求4所述的应用，所述饲料加工原料包括橘皮果渣。
6.一种橘皮果渣的发酵方法，其特征在于：发酵剂中包含米曲霉CGMCC No.18109。
7.根据权利要求6所述的方法，其特征在于，所述橘皮果渣的发酵方法包括如下步骤：
(1)活化：在无菌条件下，接种1环米曲霉于PDA斜面培养基上，置30℃恒温箱内培养3d，待斜面上长满绿色孢子以后取出备用；
(2)米曲霉种子菌液的制备：将活化好的米曲霉菌株接种于PDA液体培养基，于28～30℃、150rpm摇床培养3天，即为米曲霉种子菌液，测定活菌数>1.0×109cfu/mL；
(3)橘皮果渣发酵：将1L米曲霉种子菌液与100kg果渣充分混合均匀，装入呼吸式发酵袋中，置于35℃，60％的恒温恒湿发酵车间中静置发酵72-96h。
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