CN116162554B - 一株间型脉胞菌菌株及其在替代蛋白中的应用 - Google Patents
一株间型脉胞菌菌株及其在替代蛋白中的应用 Download PDFInfo
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- CN116162554B CN116162554B CN202310344908.3A CN202310344908A CN116162554B CN 116162554 B CN116162554 B CN 116162554B CN 202310344908 A CN202310344908 A CN 202310344908A CN 116162554 B CN116162554 B CN 116162554B
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Abstract
本发明涉及微生物和食品技术领域，公开了一株间型脉胞菌菌株及其在替代蛋白中的应用。该菌株可以利用简单廉价的底物快速生长并产生大量的菌丝体，其中菌丝体蛋白含量高达65%以上，且氨基酸种类丰富，同时富含膳食纤维，不饱和脂肪酸和多种矿物质。本发明的菌株可通过发酵生产优质的微生物蛋白，克服了传统蛋白生产成本高和对环境负面影响等问题，在替代蛋白领域中具有广泛的应用前景。
Description
技术领域
本发明涉及食品技术领域，具体涉及到一株产菌丝蛋白的间型脉胞菌及其在生产替代蛋白中的应用。
背景技术
动物肉富含优质蛋白质、必需维生素和矿物质以及其他营养物质，在人类饮食中扮演着重要角色。然而随着全球人口的快速增长，粮食需求急剧增加，传统畜牧业不足以满足未来人们对蛋白质的需求。此外，随着传统的肉制品和乳制品消费量不断增加，其生产过程对环境和动物福利的影响越来越明显。替代蛋白作为一种环保、对动物友好和健康的选择，正逐渐成为人们的关注焦点。然而，替代蛋白的生产成本高和品质难以保证是其发展的主要障碍。
目前，植物蛋白因其来自可再生资源、对环境无害、健康营养等优点作为替代蛋白备受青睐。但植物蛋白的产量也受限于植物原料的生长条件和收获周期等因素，同时植物生产过程对环境和土地资源的影响也是不可忽视的。因此，寻找一种更加环保、高效的生产替代蛋白的途径尤为重要。20世纪60年代研究人员提出了开发真菌蛋白，以应对可能出现的食物蛋白质短缺。70年代中期，人们对真菌蛋白进行了测试，以评估它是否适合人类食用，以及是否可以大规模培养用于生产和销售。1985年，英国政府批准真菌蛋白在食品中使用，随后该类产品在其他欧洲国家开始销售，并在2000年后进入了美国市场。目前全球范围内含真菌蛋白的产品中绝大多数来自于一株Fusarium venenatum通过工业发酵而得。
事实上，间型脉胞菌是一种天然存在的真菌，由于它能够分解复杂的碳水化合物和蛋白质，在食品工业中得到了广泛的应用。另外，间型脉胞菌是印度尼西亚发酵食物Oncom的主要成分，巴西土著部落使用脉胞菌属来加工木薯制作发酵饮料，婆罗洲的伊班人从烧毁的山坡稻田中采集橙色脉胞菌作为食物，脉胞菌也常出现在法国南部采用传统方法制备的羊乳干酪中。脉胞菌属的使用在人类食品中具有悠久的历史，同时它可以在玉米糖浆、木薯等廉价碳源中生长，并且能够产生大量的蛋白质。因此，未来通过脉胞菌属生产真菌蛋白在替代蛋白领域具有很大的应用前景。
发明内容
针对现实的需求，本发明人经过分离筛选获得一株高效生产菌丝蛋白的间型脉胞菌菌株，其能够利用简单的底物，转化为主要成分为蛋白的菌丝体产物。
因此，本发明首先提供一株丝状真菌FF171，该菌株从普洱茶中分离获得，经鉴定是间型脉胞菌Neurospora intermedia。所述菌株FF171的保藏编号为CGMCC NO. 40496，分类命名为间型脉胞菌Neurospora intermedia，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（保藏地址为：北京市朝阳区北辰西路1号院3号）。
本发明由此提供所述的间型脉胞菌在生产替代蛋白的应用。
进一步地，本发明提供利用所述的间型脉胞菌生产菌丝蛋白的方法，其通过所述间型脉胞菌的发酵以产生菌丝蛋白，其中所述间型脉胞菌优选的在已知成分的培养基中生长或培养。
在一些具体的实施方式中，所述培养基中无机氮源为磷酸铵、硫酸铵或氯化铵。更优选的，所述无机氮源为硫酸铵。进一步优选的，硫酸铵在发酵中提供1.5-3.5 g/L的氮含量，更优选的添加量为11.80 g/L。
在一些具体的实施方式中，所述碳源为葡萄糖，进一步优选的，所述间型脉胞菌能够耐受 4-12 g/L的碳源。更优选的，碳源添加量在4g/L。
在另一些具体的实施方式中，所述发酵培养基pH范围广，进一步优选的发酵培养基pH 范围在3.5-7.0，更优选的pH为7.0。
在一些具体实施方式中，所述间型脉胞菌的蛋白质含量高于65%，氨基酸组成丰富，富含人体所需的9种必需氨基酸，且必需氨基酸占比43.32%。
在另一些实施方式中，所述间型脉胞菌产物在替代蛋白产品中作为优质脂肪的来源，其中总脂肪在干重中占比4.22%，进一步地，不饱和脂肪和饱和脂肪比例为4.22:1。
在另一些实施方式中，所述间型脉胞菌产物在替代蛋白产品中作为矿物质元素钙、镁、磷、铁、钾来源。
在另一些实施方式中，所述间型脉胞菌产物含有丰富的膳食纤维。
本发明的间型脉胞菌来源于我国传统发酵食品普洱茶。其能够耐受35℃以上高温、适应广泛的pH环境，利用简单的碳源、氮源快速生长，生物质转化率高，获得的菌丝体中菌丝蛋白的含量高于65%，氨基酸种类丰富，且未检测出毒素，具有良好的食品应用基础。该间型脉胞菌产物种类丰富，可发酵获得包括蛋白质、脂质、膳食纤维、矿物质等多种营养成分，具有广泛的应用价值。本发明的间型脉胞菌真菌生物质可用于肉类替代品、肉类增量剂、富含蛋白质的食品、富含纤维的食品或新型含真菌蛋白的食品。
附图说明
图1为基于QMA、TMI、TML、DMG序列构建的脉胞菌多基因系统发育树。
图2为间型脉胞菌FF171菌株的菌落形态图。
图3为间型脉胞菌FF171菌株的菌丝体形态图。
生物材料保藏信息：本发明的丝状真菌FF171，分类命名为间型脉胞菌Neurospora intermedia，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：北京市朝阳区北辰西路1号院3号，其保藏编号为CGMCCNO.40496。
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：普洱茶中丝状真菌的筛选
2022年04月在云南西双版纳地区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。
倾注法具体是指称取0.1 g粉碎后的茶叶样品，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40 mL双料DRBC培养基混匀，每80 mL培养基倒2个方形平板。将处理后的平板分别置于28℃、35℃、45℃培养5-10d。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）挑取菌落于PDA平板上进行分离纯化。分离纯化次数不少于3次，直至获得纯菌落。
实施例2：真菌基因组的提取及鉴定
收集PDA平板上生长的菌丝体，通过真菌基因组提取试剂盒提取总DNA，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后测序。采用BLAST方式将其与GenBank数据库中的序列进行比对。鉴定后的菌株保存在10%甘油中，于-80℃保藏。
在3个不同的温度（28℃、35℃、45℃）下共分离到真菌195株，初步鉴定分别来自Schizophyllum sp.、Neurospora sp.、Irpex sp.、Trametes sp. 、Aspergillus sp.、lichtheimia sp.、Penicillium sp.、Rhizopus sp.8个属。进一步的，其中在35℃分离纯化及初步鉴定为Neurospora sp.的菌株17株，命名为NEU-1～NEU-17。
实施例3：脉胞菌的多基因鉴定
由于ITS不能很好地区分粗糙脉胞菌和间型脉胞菌，使用ACPTOOLS软件构建了QMA、TMI、TML、DMG四个序列的脉胞菌多基因系统发育树。首先在NCBI上下载脉胞菌属标准菌株的QMA、TMI、TML、DMG序列，通过MAFFT进行序列比对，随后使用Gblocks对序列进行修剪，修剪后的序列进行多基因拼接。拼接后的序列用于构建脉胞菌MrBayes进化树，经过比对筛选到的17株脉胞菌均为间型脉胞菌（图1）。
实施例4：高产蛋白的间型脉胞菌菌株筛选
17株脉胞菌在PDA平板上生长3-5d，然后挑取菌块接种到100 mL液体培养基中进行发酵，以评估其蛋白生产能力（表1）。所述液体发酵培养基组成：葡萄糖 10 g/L、酵母提取物5g/L、(NH4)2SO411.80 g/L、KH2PO43.5 g/L、MgSO4·7H2O0.75 g/L、微量金属元素 1mL/L、维生素1 mL/L，pH7.0。发酵在35℃下以220 rpm振荡培养48h。
发酵结束，过滤收集间型脉胞菌菌丝体，70℃干燥24h至恒重，测定真菌生物质含量和蛋白浓度。
表1、间型脉胞菌菌株发酵后生物质含量及蛋白浓度
本发明筛选到的间型脉胞菌具有很高的底物转化能力，其中15株间型脉胞菌发酵48h后的生物质产量在4g/L以上。这些菌株中有10株间型脉胞菌菌株有高于60%的蛋白浓度。表现更优的是间型脉胞菌NEU-6（Neurospora intermediaFF171），发酵后生物质产量为4.65 g/L，蛋白浓度为64.08%，总蛋白含量为2.98 g/L。
实施例5：N. intermediaFF171的形态分析
N. intermediaFF171在PDA平板上的菌落形态如图2所示。PDA上的菌落在35℃下快速生长，1d内直径达到85mm，2d时菌丝变厚，避光环境下菌丝始终为白色。35℃，自然光环境下，气生菌丝顶端呈橙红色，并伴有分生孢子的产生。菌落颜色呈灰黄色，菌落背面呈淡黄色。菌落表面平薄，菌丝沉没，气生菌丝稀疏。
N. intermediaFF171在显微镜下的的菌丝体形态结构如图3所示。显微镜下菌丝体由透明至浅棕色菌丝组成，有隔膜，分枝和网状，光滑。分生孢子梗呈橙红色，球形或椭圆形，直径10-12μm，分生孢子表面光镜下呈不规则纹饰。
实施例6：不同发酵条件对N. intermediaFF171真菌蛋白的影响
比较不同的无机氮源种类、pH、葡萄糖含量、接种方式等因素对N. intermediaFF171发酵生物质蛋白含量的影响。除碳源、无机氮源、pH外，培养基其它组成为酵母提取物5-10 g/L、(NH4)2SO4 10-15 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、磷酸二氢铵20.54g/L，pH3.5，接种6×105个/mL孢子，25℃发酵48 h，生物质含量4.23g/L，蛋白浓度52.58%，总蛋白含量2.65g/L。
培养基2：葡萄糖10g/L、硝酸钾18.05 g/L，pH5.5，接种6×105个/mL孢子，30℃发酵48 h，生物质含量1.86g/L，蛋白浓度41.20%，总蛋白含量0.77g/L。
培养基3：葡萄糖10 g/L、硫酸铵 11.80 g/L，pH7.0，接种6×105个/mL孢子，35℃发酵48h，生物质含量1.59g/L，蛋白浓度60.20%，总蛋白含量0.95g/L。
上述实验结果表明，接种孢子时，pH和氮源种类对蛋白浓度和总蛋白含量的影响最大。pH 3.5时，蛋白浓度和总蛋白含量最高；磷酸氢二铵对总蛋白含量产生积极的影响，硫酸铵对蛋白浓度产生积极的影响，硝酸钾是差的氮源。
培养基4：葡萄糖10g/L、硫酸铵 11.80 g/L，pH7.0，接种菌块，35℃发酵48 h，生物质含量4.33g/L，蛋白浓度62.16%，总蛋白含量2.69g/L。
培养基5：葡萄糖10g/L、磷酸二氢铵20.54 g/L，pH3.5，接种菌块，35℃发酵48 h，菌株不生长。
培养基6：葡萄糖10 g/L、磷酸二氢铵20.54 g/L，pH7.0，接种菌块，35℃发酵48 h，菌株不生长。
上述实验结果表明，接种菌块时，氮源种类对菌株生长产生最大的影响。接种菌块时，硫酸铵是最合适的氮源，磷酸二氢铵不适合菌块的生长。
培养基7：葡萄糖10g/L、氯化铵9.55g/L，pH4.0，接种1%活化48h的种子液，35℃发酵48 h，生物质含量4.31 g/L，蛋白浓度58.67%，总蛋白含量2.53 g/L。
培养基8：葡萄糖30g/L、硫酸铵 11.80 g/L，pH5.5，接种1%活化48h的种子液，35℃发酵48 h，生物质含量4.28 g/L，蛋白浓度56.08%，总蛋白含量2.4 g/L。
培养基9：葡萄糖20 g/L、磷酸二氢铵20.54 g/L，pH 7.0，接种1%活化48h的种子液，35℃发酵48 h，生物质含量0.24g/L，蛋白浓度29.58%，总蛋白含量0.07g/L。
上述实验结果表明，接种种子液时，pH和氮源种类对菌株蛋白浓度产生最大的影响。生物量和蛋白含量随着pH的升高降低，氯化铵对蛋白浓度和总蛋白含量产生积极地影响，磷酸二氢铵导致最低的蛋白浓度和总蛋白含量。
实施例7：不同廉价有机氮源对N. intermediaFF171真菌蛋白的影响
探究N. intermediaFF171使用廉价有机氮源脱脂豆粉、胰蛋白胨、花生饼粉、玉米浆干粉发酵真菌蛋白的能力，按照实施例4所述方法配置液体发酵培养基。接种1%种子液，在35℃下以220 rpm振荡培养24h。发酵结束，过滤收集间型脉胞菌菌丝体，70℃干燥24h至恒重，测定真菌生物质含量和蛋白浓度，如表2所示。
表2、N. intermediaFF171利用不同有机氮源发酵比较
上述实验结果表明，N. intermedia FF171能够利用多种廉价可再生有机氮源快速发酵，转化为高蛋白浓度的生物质，表明N. intermedia FF171发酵生产替代蛋白具有经济、环保等特点。
实施例8：N. intermediaFF171单细胞蛋白的营养价值
使用实施例4所述培养基生产N. intermediaFF171菌丝蛋白。将N. intermediaFF171接种至2 L培养基中，以220rpm的转速在35℃的摇床内培养48h。然后使用真空抽滤泵过滤菌丝体，同时用3倍体积的无菌水洗涤。将收集到的菌丝体滤饼进一步冷冻干燥并研磨成粉末状生物质。
1、N. intermediaFF171生物质基本营养成分
N. intermediaFF171生物质营养组成的检测方法和结果如表3所示。
表3、N. intermediaFF171生物质的基本营养成分
如表3所示，本发明的间型脉胞菌生物质具有高蛋白和富含膳食纤维的营养价值，在其干物质中含有65.53%的蛋白以及22.27%的总膳食纤维。进一步地，N. intermediaFF171生物质的脂肪含量为4.22%，不饱和脂肪和饱和脂肪的占比达到4.22:1。
如表4所示，动物源性食品的蛋白含量以干重计为36.95%至43.44%，植物源性食品的蛋白含量以干重计为40%至55.72%。然而，在本发明的发酵工艺下，N. intermedia FF171生物质的蛋白质含量显著高于这些食物来源，为65.53%。
表4人类主要食物中的蛋白质含量
2、N. intermediaFF171的氨基酸谱
利用高效液相色谱法检测N. intermediaFF171生物质的氨基酸组成，如表5所示。N. intermedia FF171真菌蛋白含有9种必需氨基酸，且必需氨基酸占比达到43.32%，是优质的蛋白质来源。
表5、N. intermediaFF171生物质中氨基酸组成
为必需氨基酸
3、N. intermediaFF171的矿物质含量
如表6所示，按照国家标准《GB 5009.268-2016 食品中多元素的测定》检测N. intermediaFF171生物质含有多种矿物质。
表6、N. intermediaFF171生物质中矿物质含量（mg/kg）
其中表中所有数据均基于干重。
本发明N. intermediaFF171生物质中矿物质含量与其它真菌蛋白产物和生肉（猪肉（美国农业部食品数据库NDB Number:10219）、牛肉（美国农业部食品数据库NDB Number:13330））相比，有更高的Mg、P、K和Fe和较低的Na含量。这些矿物质对人体的正常运作至关重要，因为磷和镁对人体组织细胞和骨骼的组成很重要，铁用于运输和储存氧气以及维持正常的造血功能和免疫力，钾在维持人体酸碱性、维持细胞正常含水量和维持人体的新陈代谢中发挥重要作用。
4、N. intermediaFF171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N. intermedia FF171生物质中11种真菌毒素进行检测，如表7所示。实验表明，FF171生物质中不含检测的11种常见的真菌毒素。
表7、N. intermedia FF171生物质中真菌毒素检测
应用例1：发酵后生物质的处理
按照实施例8中所述方法发酵获得生物质。在真菌生物质发酵完成后，对其进行热处理，首先将其置于65℃下处理30 min，以去除细胞内RNA。然后，将发酵后的生物质进一步加热至90℃保持30min，以灭活真菌细胞。之后，通过100目的纱布进行过滤，并使用5倍体积的无菌水洗涤以完全除去培养基。获得水分含量为85%的生物质，可用于替代蛋白产品开发。
应用例2：含有N. intermediaFF171生物质的替代肉类产品的生产
根据应用例1得到含水量约85%的糊状生物质，将约180g的糊状物质加入高速厨房搅拌机，加水混合样品3-5 min，直至混合物均匀。以湿基为基准，加入2%的蛋清蛋白和肉味香精（鸡肉风味、猪肉风味、牛肉风味）混合均匀。混合均匀后，将混合物放入模具中，在蒸锅中以100℃蒸煮20 min，使蛋清蛋白凝固。将蒸好的生物质转移到速冻机冷却2h，然后放入-20℃冷库冷冻至少24h。冷冻后的混合物可在0-10℃解冻，切片、切块，调味制成各种肉类替代品。
应用例3：含有N. intermediaFF171生物质的甜点
根据应用例1得到含水量约85%的糊状生物质，加入调味后可均质冷冻制作冰激凌甜品。含有真菌生物质的冰激凌甜品具有减少脂肪摄入，降低胆固醇和影响饱腹感的优点。具体而言，该方法包括使用高速剪切机将糊状生物物质与糖、葡萄糖浆、牛奶和淡奶油按照一定比例混合，然后进一步加工后冷冻制成含有真菌生物质的冰激凌。
Claims (8)
1.一株间型脉胞菌（Neurospora intermedia），其特征在于，其保藏编号为CGMCC NO.40496。
2.如权利要求1所述的间型脉胞菌在生产替代蛋白中的应用。
3.一种利用权利要求1所述的间型脉胞菌生产替代蛋白的方法，其特征在于，从所述间型脉胞菌的发酵物中获得替代蛋白。
4.如权利要求3所述的间型脉胞菌生产替代蛋白的方法，其特征在于，所述间型脉胞菌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所述的间型脉胞菌生产替代蛋白的方法，其特征在于，所述无机氮源选自硫酸铵或氯化铵；所述有机氮源选自酵母提取物、脱脂豆粉、玉米浆干粉。
6. 如权利要求5所述的间型脉胞菌生产替代蛋白的方法，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 0-1 g/L、EDTA 0-1 g/L、微量金属1 mL/L、维生素1 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。
7.如权利要求4至6任一项所述的间型脉胞菌生产替代蛋白的方法，其特征在于，所述培养基的pH在3.5-7.0范围内。
8.如权利要求4至6任一项所述的间型脉胞菌生产替代蛋白的方法，其特征在于，在所述发酵完成后，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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