CN116622526B - 一株米根霉菌株及其在生产菌丝体蛋白中的应用 - Google Patents
一株米根霉菌株及其在生产菌丝体蛋白中的应用 Download PDFInfo
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- CN116622526B CN116622526B CN202310893766.6A CN202310893766A CN116622526B CN 116622526 B CN116622526 B CN 116622526B CN 202310893766 A CN202310893766 A CN 202310893766A CN 116622526 B CN116622526 B CN 116622526B
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Abstract
本发明涉及一株米根霉菌株及其在生产菌丝体蛋白中的应用。所述菌株的保藏编号为CGMCC NO.40497,其用于生产菌丝体蛋白的方法包括从所述米根霉的发酵物中获得菌丝体蛋白的步骤。本发明发酵获得的菌丝体蛋白中粗蛋白含量达50%以上,是一种优质的蛋白原料,可用于制备蛋白棒、即食膨化食品等高蛋白产品,可为低成本、绿色的单细胞蛋白生产提供技术支撑,在发酵蛋白领域具有广阔的应用前景。
Description
技术领域
本发明属于微生物发酵及食品技术领域,具体涉及一株米根霉及其在生产菌丝体蛋白中的应用。
背景技术
传统的农业生产对全球环境造成了极大的负面影响,消耗了大量的资源。据估计,人类活动造成的水足迹70-85%与农业活动有关,农业部门的温室气体排放占人类排放总量的30%以上,其中一半以上与肉类生产相关。这些因素包括温室气体排放、水体富营养化以及土地紧缺等,给传统的蛋白质生产方式带来了巨大压力。随着全球人口不断增长,获得健康、营养、可持续的饮食将变得比以往任何时候都更加重要。
为了实现可持续饮食,减少肉类消费是其中的一项关键措施。采用替代蛋白来源,可有效应对全球蛋白紧缺的难题,同时最大限度地减少食物对环境的影响。替代蛋白来源包括基于植物(如大豆、豌豆和燕麦)、昆虫或微生物(如菌丝体蛋白)。菌丝体蛋白作为替代蛋白的其中一个选项,无需占用土地资源,在生物反应器内即可高效合成。同时,菌丝体蛋白作为一种低脂肪和高纤维的优质蛋白质来源,有助于实现饮食均衡。
米根霉(Rhizopus oryzae)是一种毛霉目真菌,常被用于生产有机酸、酒精饮料、传统食品、酶等,具有很高的商业价值。在专利文献CN104818221A中,披露了一株名为JHSW01的米根霉菌株,可用于将农林废弃物进行固态发酵,从而产生糖化酶、纤维素酶和有机酸等物质,有效促进纤维素和半纤维素的分解和转化。在专利文献CN114085778A中,披露了一株名为JYH-4-23的米根霉,其发酵产生的糖苷酶粗酶液可用于提取金银花中的绿原酸,提取率比常规方法高出41.5%。在专利文献CN102634460A中,披露了一株名为RH1-5的米根霉,其糖化能力高、耐酸、耐高温,可以提高和稳定大曲在酿酒过程中的糖化效果。
然而,目前还需要进一步研究使用米根霉进行液态发酵以高效生产菌丝体蛋白的方法。
发明内容
本发明的目的之一是提供一种高产菌丝体蛋白的米根霉(Rhizopus oryzae)FF215及其应用。
本发明提供了一种高产蛋白的丝状真菌,所述丝状真菌为米根霉(Rhizopus oryzae)FF215,保藏时间为2023年02月10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),编号为CGMCC No. 40497。
本发明进而提供所述的米根霉菌株在生产菌丝体蛋白中的应用。
进一步提供一种利用所述的米根霉生产菌丝体蛋白的方法,其是从所述米根霉的发酵物中获得菌丝体蛋白。
具体地,所述米根霉进行发酵所采用的培养基中氮源为无机氮源或有机氮源,或其组合。
优选地,所述培养基中除碳源、氮源外,培养基其它组成为:KH2PO4 2-5 g/L、MgSO4×7H2O 0.5-1 g/L、EDTA 0.5-1 g/L、微量金属0.5-1.5 mL/L、维生素0.5-1.5 mL/L;其中,微量金属为:CaCl2×2H2O、ZnSO4×7H2O、FeSO4×7H2O、H3BO3、MnCl2×2H2O、Na2MoO4×2H2O、CoCl2×2H2O、CuSO4×5H2O、KI中的一种或多种组合;维生素为:氨基苯甲酸、烟酸、泛酸钙、维生素B6、维生素B1、生物素、肌醇中的一种或多种组合。
更优选地,所述培养基的pH在3.5-5.5范围内。
在具体实施方式中,所述米根霉进行液体发酵的条件:米根霉孢子按照1×104~106个/mL接种到液体发酵培养基中,于220rpm,35℃培养58-96h。
在另外的实施方式中,所述米根霉在发酵完成后,对发酵物采用真空抽滤泵过滤,无菌水清洗,将收集到的生物质滤饼进一步冷冻干燥,得到滤饼生物质或进一步通过研磨,得到粉末状生物质。
优选地,所述米根霉菌丝体蛋白中蛋白质含量为不低于50%,总膳食纤维含量不低于 20 %,总脂肪含量不低于4 %,碳水化合物含量不低于2 %。
本发明还提供所述的方法得到的米根霉菌丝体蛋白。
本发明进一步提供一种由所述的米根霉菌丝体蛋白制备的单细胞蛋白产品,所述单细胞蛋白产品为蛋白棒或即食膨化食品。
具体地,以米根霉菌丝体蛋白主要成分,加入糖类、脂类物质,进行混合、高温加热后,挤压成型,冷却,脱模,包装得到。
本发明的有益效果在于:本发明筛选出的菌株FF215,在液体发酵中产生较高的生物量和蛋白含量。同时菌丝体蛋白作为丰富蛋白含量的载体,氨基酸种类齐全,脂质、膳食纤维等营养全面,具有良好的食品应用基础。
附图说明
图1为米根霉FF215的菌落形态图。
生物材料保藏信息:本发明的米根霉FF215,分类命名为米根霉Rhizopus oryzae,于2023年02月10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),保藏单位地址为在北京市朝阳区北辰西路1号院3号,其保藏编号为CGMCC NO.40497。
具体实施方式
下面通过具体实施例对本发明的技术方案进行清楚、完整的描述。所描述的实施例仅是本发明一部分实施例,而不是全部实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1、根霉属菌株的获得
2022年5月在云南产茶基地收集普洱茶样品。分别将普洱茶样品在均质机粉碎,样品与样品间严格消毒。随后使用倾注法分离样品中的微生物菌株。
具体分离过程是指:使用均质机将茶叶样品打碎至粉末状,打碎时间控制在30 -60 s;称取0.1 g粉碎后的茶叶样品(每个样品3份),加入40 mL 0.1%蛋白胨水溶液中,28℃150r/min 振荡1h,倒入冷却至45℃的氯硝胺孟加拉红氯霉素(DRBC)琼脂基础培养基,每80mL培养基倒2个方形平板。将每个样品的平板置于35℃,黑暗环境下培养5-10 d。根据菌落表型特征(包括大小、形状、颜色、质地、有无渗透液、有无可溶性色素等)挑取根霉形态特征菌落的菌丝于DRBC平板上进行多次分离纯化,直至获得纯菌落,将获得菌落4℃保存,以备进行形态特征和分子鉴定。
所述氯硝胺孟加拉红氯霉素(DRBC)琼脂基础培养基为:蛋白胨 5 g,葡萄糖10 g,磷酸二氢钾1 g,氯硝胺0.002 g,硫酸镁0.5 g,孟加拉红0.025 g,氯霉素0.1 g,琼脂15 g,蒸馏水定容至1000 mL,pH5.6,121℃高压灭菌15分钟。
从不同普洱茶样品中共分离到7株根霉属(Rhizopus spp.)菌株,菌株编号分别为RHI-1至RHI-7。
实施例2、菌株筛选
7株根霉属(Rhizopus spp.)菌株在PDA平板上生长5-10天,使用无菌水冲洗收集孢子。分别以106个/mL孢子接种到2种不同液体发酵培养基中,35℃,220rpm发酵72h。发酵结束,过滤收集菌丝体,干燥至恒重,测定真菌生物质含量和蛋白浓度。不同发酵条件下根霉(Rhizopus spp.)产蛋白能力如表1所示。
所述液体发酵培养基1:葡萄糖20 g/L、酵母提取物5 g/L、 (NH4)2SO4 7.5 g/L、KH2PO4 3.5 g/L、MgSO4×7H2O 0.75 g/L、微量金属溶液10 mL/L、维生素1 mL/L,pH5.5。
所述液体发酵培养基2:葡萄糖10 g/L、酵母提取物5 g/L、 (NH4)2SO4 7.5 g/L、KH2PO4 3.5 g/L、MgSO4×7H2O 0.75 g/L、微量金属溶液10 mL/L、维生素1 mL/L,pH4.5。
表1不同发酵条件下根霉(Rhizopus spp.)产蛋白能力
本发明筛选到的根霉属菌株,发酵后蛋白浓度在36.59%-56.33%,总蛋白含量在1.52-4.10 g/L。特别是RHI-4在液体发酵培养基1中达到56.33%的蛋白浓度和4.10 g/L的总蛋白含量,在液体发酵培养基2中达到53.47%的蛋白浓度和2.48 g/L的总蛋白含量,因此将菌株RHI-4正式命名为FF215,以作进一步的研究。
实施例3、FF215的形态特征
本发明的根霉属菌株FF215分离自普洱茶生茶样品,主要生物学特征为:在PDA平板上菌落呈棉白色(图1),后来变成带有孢子囊的斑点,最后呈现黑灰色,通过假根和匍匐茎扩散到整个培养基平板。在光学显微镜下观察,孢子囊大部分直立,不分枝,壁光滑,亚透明至棕色,单个或成群,无隔膜,通常3-5个一组由匍匐茎产生。孢子囊呈球形,呈灰褐色,有许多直径为70-200 μm的孢子。小柱呈灰褐色、亚透明、亚球形至卵圆形。匍匐茎和假根呈黑褐色。孢子囊孢子数量多,不规则且不等长,呈亚球形或椭圆形。
实施例4、FF215基因组的提取及鉴定
收集PDA平板上生长的FF215菌丝体,通过真菌基因组提取试剂盒提取总DNA,以其为模板,然后采用真菌ITS通用引物扩增真菌ITS区域。扩增产物经1%琼脂糖凝胶电泳分离后,采用胶回收试剂盒回收,并测序。将菌株测序结果与GenBank数据库中的序列进行Blast比对。BLAST分析表明,FF215与Rhizopus oryzaeATCC 44467 (GenBank: GU256757.1)在ITS的同源性高于99%,因此,结合形态学特征,FF215鉴定为Rhizopus oryzae。
该菌株于2022年02月10日藏保于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为CGMCC NO. 40497。
实施例5、不同发酵条件对FF215的影响
将FF215孢子接种于不同pH、氮源种类、氮源含量的液体发酵培养基中,评估其产蛋白能力。除无机氮源、pH外,培养基其它组成为酵母提取物5-10 g/L、KH2PO4 1-5 g/L、MgSO4·7H2O 0.1-1 g/L、微量金属元素 1 mL/L、维生素1 mL/L。
培养基1:葡萄糖10 g/L、酒石酸铵7.89 g/L,pH3.5,接种1×106个/mL孢子,35℃发酵72 h,生物质含量4.90 g/L,蛋白浓度53.27%,总蛋白含量2.60 g/L。
培养基2:葡萄糖10g/L、磷酸二氢铵13.11 g/L,pH 3.5,接种1×106个/mL孢子,35℃发酵48 h,生物质含量4.5 g/L,蛋白浓度58.59%,总蛋白含量2.70 g/L。
培养基3:葡萄糖10 g/L、硫酸铵 5.66 g/L,pH 4.5,接种1×107个/mL孢子,35℃发酵48h,生物质含量5.7 g/L,蛋白浓度47.08%,总蛋白含量2.70 g/L。
培养基4:蔗糖10 g/L、硫酸铵 5.66 g/L,pH 4.5,接种1×107个/mL孢子,35℃发酵72h,生物质含量5.90 g/L,蛋白浓度49.69%,总蛋白含量2.90 g/L。
培养基5:葡萄糖10 g/L、磷酸二氢铵9.83 g/L,pH 5.5,接种5×106个/mL孢子,35℃发酵48h,生物质含量5.40 g/L,蛋白浓度48.21%,总蛋白含量2.60 g/L。
FF215能够适应葡萄糖、蔗糖等无机碳源,酒石酸铵、硫酸铵、磷酸二氢铵等无机氮源,pH3.5-5.5,在不同的发酵条件下迅速生长,达到5g/L以上的生物质含量,碳源转化率达到0.5g生物质/g底物。
实施例6、FF215菌丝体蛋白的生产及营养成分分析
将FF215以106 个孢子/mL接种到2L液体发酵培养基中。220 rpm、35℃培养2d。在发酵结束时,真菌生物质产量达到 5 g/L。
所述发酵培养基组成为:葡萄糖10 g/L、酵母提取物5 g/L、 磷酸二氢铵13.14 g/L、KH2PO4 3.5 g/L、MgSO4·7H2O 0.75 g/L、微量金属溶液10 mL/L、维生素1 mL/L;pH 3.5。
1、FF215的生物质基本营养成分
FF215生物质的基本营养成分蛋白质含量为 54.67%,总膳食纤维含量 22.67 %,总脂肪含量5.43 %,碳水化合物含量2.05 %,能量13.46 KJ/g。
菌丝体是纤维的重要来源,含有天然的膳食纤维。FF215菌丝体中膳食纤维含量高于全麦面包(9.89%)、玉米(8.33%)、杏仁(11.28%)和黑豆(4.20%)。
同时FF215生物质中饱和脂肪占比1.59%,单不饱和脂肪占比1.57%,多不饱和脂肪占比2.24%,不饱和脂肪与饱和脂肪比例为2.4:1。
2、FF215生物质中氨基酸含量
FF215干重中蛋白含量为 54.67%,高于动物源蛋白生猪肉(43.44%)、生牛肉(36.95%)和植物源蛋白黄豆(40%)(数据来源:美国农业部食品数据库)。
蛋白质来源的质量还取决于其所含氨基酸的丰度和种类,米根霉的氨基酸组成如表2所示。人类饮食中有9种必需氨基酸,因为这些氨基酸不能轻易地从哺乳动物的其他前体合成。FF215含有所有必需氨基酸,占总蛋白质含量的 45.67%。这意味着与大多数其他植物性和动物性蛋白质来源相比,菌丝体蛋白具有更高百分比的必需氨基酸,包括大豆(36%)、牛奶(41%)、家禽(45%)甚至乳清蛋白(43%)(数据来源:美国农业部食品数据库)。
同时FF215的菌丝体蛋白中支链氨基酸(亮氨酸、缬氨酸、异亮氨酸)占比6.05%,高于鸡胸肉(5.9%)、鸡蛋(2.5%)、鹰嘴豆(1.1%)(数据来源:美国农业部食品数据库)。这些数据均表明FF215菌丝体蛋白是一种具备高附加值属性的食用蛋白质来源。
表2 FF215生物质氨基酸组成
3、FF215生物质的微量元素
FF215的微量元素如表3所示。FF215生物质中相比动物源蛋白质和植物源蛋白质含有更多的镁、磷、钾、锌、锰(数据来源:美国农业部食品数据库)。
表3 矿物质成分组成
4、FF215生物质的真菌毒素含量
基于SN/T 3136-2012,使用液相色谱-质谱/质谱的方法对FF215生物质中11种真菌毒素进行检测。如表4所示,对于所检测的11种真菌毒素,均低于检测限(ND:未检出或低于方法检测限)。表明FF215生物质中不含真菌毒素或远低于国家标准。
表4 FF215真菌毒素含量检测结果
。
应用例1:制作替代蛋白棒
蛋白棒的原料包括酪蛋白水解物(10-100份)、糖类(150-500份)、脂类(20-100份)和纯净水(5-15份),以及FF215的菌丝体蛋白(150-600份)。除菌丝体蛋白外,所有使用的原料均为市售购得。
FF215生物质经过发酵后,在65℃下处理30分钟,并在90℃下保持30分钟,以去除细胞内RNA并杀灭真菌细胞。随后,通过过滤洗涤除去异味,收集生物质。然后进行冻干脱水,并使用多功能粉碎机进行粉碎,再通过筛子筛选,筛下物即为FF215菌丝体蛋白粉成品,备用。
按照上述配比,将酪蛋白水解物和菌丝体蛋白粉称量后混合均匀,制成蛋白料。同时,将脂类物质如大豆磷脂等,在60℃水浴锅中预融混合均匀,制成油脂料。然后,将麦芽糖浆、果葡糖浆、白砂糖等加入适量纯净水中,与预融好的油脂料一起在锅中混合,熬煮至105℃,冷却至70℃,制成浆料。将蛋白料加入浆料中,混合搅拌后挤压成型并切割成棒状体。对棒体涂层,涂层材料可为花生酱、水果酱、巧克力等或其复合物。将制备好蛋白棒进行包装,即可获得替代蛋白棒成品。本产品特点为富含优质蛋白、膳食纤维、矿物质等,且具有良好的口感,可满足不同人群对高蛋白健康食品的需求。
应用例2:制作即食替代蛋白膨化产品
按照50g大豆蛋白粉、50g FF215菌丝体蛋白粉和30g水的重量份数称取原料,然后将它们投入搅拌机中,在400rpm的速度下搅拌3分钟,制成粉团;将粉团投入挤出熟化机中成型,得到大小一致、厚度均匀的薄片坯料;随后采用160℃的大豆油高温油炸膨化,炸制时间为15秒;脱油过程中,使用低速离心机,在1500rpm的转速下离心10分钟,将油炸过的坯料脱油;脱油后的坯料加入适量白糖、食盐、香辛料和谷氨酸钠等调味料进行调味;最后采用真空包装机进行包装,即可得到替代蛋白膨化成品。产品色泽金黄、味香少油、口感酥脆、可长时间储存,且提供人体所需的优质蛋白质来源,是一种营养的新型膨化食品。
Claims (8)
1.一株产蛋白的米根霉(Rhizopus oryzae),其特征在于,其保藏编号为CGMCC NO.40497。
2.如权利要求1所述的米根霉菌株在生产菌丝体蛋白中的应用。
3.一种利用权利要求1所述的米根霉生产菌丝体蛋白的方法,其特征在于,从所述米根霉的发酵物中获得菌丝体蛋白。
4.如权利要求3所述的方法,其特征在于,所述米根霉进行发酵所采用的培养基中氮源为无机氮源或有机氮源,或其组合。
5. 如权利要求4所述的方法,其特征在于,所述培养基中除碳源、氮源外,培养基其它组成为:KH2PO4 2-5 g/L、MgSO4×7H2O 0.5-1 g/L、微量金属0.5-1.5 mL/L、维生素0.5-1.5mL/L;其中,微量金属为:CaCl2×2H2O、ZnSO4×7H2O、FeSO4×7H2O、H3BO3、MnCl2×2H2O、Na2MoO4×2H2O、CoCl2×2H2O、CuSO4×5H2O、KI中的一种或多种组合;维生素为:烟酸、泛酸钙、维生素B6、维生素B1、生物素、肌醇中的一种或多种组合。
6.如权利要求4或5所述的方法,其特征在于,所述培养基的pH在3.5-5.5范围内。
7.如权利要求3至5任一项所述的方法,其特征在于,所述米根霉进行液体发酵的条件:米根霉孢子按照1×104~106个/mL接种到液体发酵培养基中,于220rpm,35℃培养58-96h。
8.如权利要求3至5任一项所述的方法,其特征在于,所述米根霉在发酵完成后,对发酵物采用真空抽滤泵过滤,无菌水清洗,将收集到的生物质滤饼进一步冷冻干燥,得到滤饼生物质或进一步通过研磨,得到粉末状生物质。
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