CN116376730B - 一种红法夫酵母菌及其用途 - Google Patents
一种红法夫酵母菌及其用途 Download PDFInfo
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- CN116376730B CN116376730B CN202310584236.3A CN202310584236A CN116376730B CN 116376730 B CN116376730 B CN 116376730B CN 202310584236 A CN202310584236 A CN 202310584236A CN 116376730 B CN116376730 B CN 116376730B
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Abstract
本发明涉及一种红法夫酵母菌及其用途。本发明从松树林的土壤中筛选分离获得该红法夫酵母菌（Phaffia rhodozyma）保藏号为：CGMCC NO.26861。发酵后菌体通过酶解破壁制备红法夫酵母水解物，多糖含量42%，蛋白含量30.2%，油脂含量18.5%，虾青素含量为1.67mg/g，核酸含量8.2%。因此，本发明的可用于车间制造模式生产富含虾青素和油脂的酵母水解物，突破依赖植物提取、时空生产效率受限的技术瓶颈，极具产业化前景。
Description
技术领域
本发明属于微生物领域和生物技术领域，具体涉及一种红法夫酵母菌及其用途。
背景技术
类胡萝卜素( carotenoids)是一类重要的天然色素的总称, 普遍存在于动物、高等植物、真菌、藻类的黄色、橙红色或红色的色素之中。类胡萝卜素是体内维生素A的主要来源, 同时还具有抗氧化、免疫调节、抗癌、延缓衰老等功效。
尤其是，虾青素是一种广泛存在的橘红色的类胡萝卜素，具有极强的抗氧化活性。虾青素是类胡萝卜素合成的最高级别产物，虾青素的抗氧化能力是维生素C的6000倍， 维生素E的1000倍，是白藜芦醇的3000倍，辅酶Q10的800倍；是一氧化氮的1800倍； 纳豆的3100倍；是花青素的700倍；β-胡萝卜素功效的100倍；是叶黄素功效的200倍；是番茄红素功效的10倍；茶多酚功效的320倍。动物实验表明，虾青素具有抑制肿瘤发生、增强免疫功能等方面的生理作用；也可以作为防治神经性疾病，如老年痴呆和帕金森综合征等，开发为抗衰老和预防动脉硬化等疾病的药物及保健品；也可以用于防治眼科疾病尤其是对糖尿病眼疾的发病率具有显著降低的效果。
虾青素在饲料领域中有一定应用，不仅仅是水产养殖动物的着色剂，也是水产动物必须的“维生素”，可以提高动物的免疫力和存活力，且能大量沉积在动物的皮肤及肌肉中沉积，增色的同时增加其营养价值。
法夫酵母属于担子菌门，红法夫酵母属，是生产虾青素的主要微生物之一。体细胞呈椭圆形、单个、成对。与其他酵母相似，红法夫酵母发酵所需的碳源进行发酵，需要的温度相对较低，在好氧发酵过程中产酸，释放二氧化碳同时合成虾青素。发酵液中氧含量有利于提升菌体及虾青素的合成。
红法夫酵母菌体细胞，其不仅富含蛋白质、氨基酸、维生素等多种营养物质，还富含胡萝卜素和虾青素等天然色素，能显著提高动物免疫力、净化环境和减少抗生素用量，是生态养殖的优良添加剂，具有很高的应用价值。
在海参、中华绒螯蟹等幼体育苗中可直接投喂红法夫酵母作为开口或幼体培育的饵料，主流产品分为发酵后的液体剂型和将发酵液喷雾干燥后的粉剂，虽然已在水产养殖中使用多年，但一直面临一些产品问题。但目前红法夫酵母培养生产虾青素的产量不高，不能适应工业化大生产的需求。发酵后喷干的粉剂产品遇水成团、融水慢、现场使用时操作效率低的问题。虽然液体产品更受养殖者的青睐，但遇到易变质的问题，一般需要低温储藏，需要在养殖区域配备冰箱或者恒温储藏，给养殖者带来很多不便。迫切需要开发具有自主知识产权的、高产虾青素含量的菌株，及其工艺方法。
发明内容
本发明涉及一种红法夫酵母菌及其用途，该红法夫酵母菌是红法夫酵母RH-A，分类命名为红法夫酵母Phaffia rhodozyma，其保藏编号为CGMCC NO. 26861，于2023年3月23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。该菌株是发明人从松树林的土壤中筛选分离并进行诱变获得的。
本发明提供一种红法夫酵母菌，其保藏编号为CGMCC NO. 26861。
进一步提供所述的红法夫酵母菌在生产虾青素中的应用。
相应本发明提供一种生产虾青素的方法，其包括如下步骤：培养所述的红法夫酵母菌，提取所产生的虾青素。
具体地，培养时采用的培养基以葡萄糖、蔗糖、纤维二糖为碳源。
优选地，培养时的培养基组成，单位g/L：葡萄糖10，酵母粉10，蛋白胨20，pH调节至6.0；基础摇瓶的培养基组成，单位g/L：葡萄糖10，蔗糖6， KH2PO4 1，NaCl 0.1，MgSO4·7H2O5，CaCl2·2H2O，酵母粉0.2，pH调节至6.0；培养基灭菌条件：蒸汽湿热灭菌115℃，20min。
进一步优选地，培养时分成种子培养和发酵培养，在种子培养期间，培养条件为：培养温度22 °C，摇床转速200 rpm，培养时间24 h；在发酵培养期间，培养条件为：控制发酵温度为22℃，通气量为2vvm，设置溶解氧与转速关联并控制溶解氧为30％以上，发酵培养时长60-300小时。
在一个实施方式中，提取所产生的虾青素的方法如下：将发酵培养的发酵液离心后，去除上清液，收集菌体，无菌水洗涤，将洗涤后的菌体与的二甲亚砜中，再剧烈震荡摇匀，在水浴锅中保温；加入甲醇与二氯甲烷按照体积比3:1混合得到的提取液，静置后离心，将离心得到的上清液转移到新的离心管中。
进一步地，对转移到新的离心管中上清液进一步与其同样条件下进行离心分离，其菌体沉淀物重复用二甲亚砜进行破壁提取，直到菌体为白色为止，汇集提取液。
更具体地，提取所产生的虾青素的方法如下：将发酵培养的发酵液，12000rpm离心后，去除上清液，收集菌体，无菌水洗涤两次后，将洗涤后的菌体与预热到60℃的二甲亚砜中，再剧烈震荡摇匀，在温度为60℃水浴锅中保温20分钟。加入1ml甲醇与二氯甲烷按照体积比3:1混合得到的提取液，静置3分钟后，2000rpm离心1分钟，将离心得到的上清液转移到新的离心管中，与其同样条件下进行离心分离，其菌体沉淀物重复用预热60℃的二甲亚砜进行破壁提取，直到菌体为白色为止，汇集提取液。
本发明通过筛选分离并进行诱变从松树林的土壤中得到的红法夫酵母菌能够高产虾青素，葡萄糖、木糖，乳糖，纤维二糖，蔗糖、阿拉伯糖为唯一碳源时，红法夫酵母均可以正常生长，说明其可以利用的碳源存在广谱性。并且该红法夫酵母生长速度快，培养温度22-28℃培养3d，菌落红色明显。本发明的菌株可用于车间制造模式生产富含虾青素和油脂的酵母水解物，突破依赖植物提取、时空生产效率受限的技术瓶颈，多糖含量42%，蛋白含量30.2%，油脂含量18.5%，虾青素含量为1.67mg/g，核酸含量8.2%，极具产业化前景。
附图说明
图1：红法夫酵母平板培养情况照片。
图2：不同碳源培养红法夫酵母Phaffia rhodozyma RH-A生长情况比较。
图3：红法夫酵母Phaffia rhodozyma RH-A发酵液高效液相检测结果。
图4：红法夫酵母Phaffia rhodozyma RH-A菌体中油脂种类及含量检测。
生物材料保藏信息
红法夫酵母RH-A，其保藏编号为CGMCC NO. 26861，分类命名为红法夫酵母Phaffia rhodozyma，于2023年3月23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。
具体实施方式
实施例一、菌株的筛选
筛选：在河北、天津等地区的松树林下采集31份土壤样品，利用YPD培养基(含50μg/mL的链霉素抑制大肠杆菌)进行筛选酵母菌株。具体步骤为，将每份土壤样品进行编号，取1g土壤样品溶于10mL0.9％NaCl溶液中，吸取100μL溶液至YPD培养基上，使用无菌涂布棒均匀涂布，置于22℃培养箱中培养96h。挑取多个颜色较红、菌落较大的菌株，利用YPD培养基进行纯化，接种到液体培养基中振荡培养，测定各组的虾青素含量，选择类胡萝卜素和虾青素含量最高的菌株进行进一步纯化。
纯化：使用无菌接种环，将筛选纯化的菌株在葡萄糖蛋白胨酵母粉琼脂培养基(YPD培养基)上划线纯化。培养温度22℃，培养时间48h，得如图1所示的菌落形态，菌落颜色为橘红色，单菌落直径2mm。
实施例二、菌种的鉴定
使用天根生化科技(北京)有限公司的真菌基因组DNA提取试剂盒(离心柱型)对纯化完成的菌株HFF501进行基因组DNA的提取作为模板，以ITS基因引物为引物，进行PCR，程序为：94℃，5min；94℃，30s，54℃，30s，72℃，50s，35个循环；72℃，5min。PCR产物送至擎科有限公司进行测序。将测序结果在NCBI数据库中进行比对，结果与红法夫酵母（phaffia rhodozyma strain NRRLY17430）的相似性达到100%，因此菌株鉴定为红法夫酵母Phaffia rhodozyma。
实施例三、菌株诱变
对上述筛选获得红法夫酵母进行诱变及分选：
a. 诱变时间确定：采用100µl新鲜的红法夫酵母细胞，孢子浓度为105, 诱变不同时间。当诱变设置0s，60 s，90 s，120 s，150 s的诱变时间，分别凃板统计每个诱变时间的致死率，以70%致死率为理想诱变时间(0s情况作为对照)。
b.诱变后菌落孔板法评价：诱变后菌落挑入24孔板内，22℃、130 rpm培养1d，测定其OD600判断诱变后菌落生长速度。挑选生长速度最快的菌株（编号RH-A），将获得菌落4℃保存。
实施例四：菌种的培养
培养基：培养基组成（g/L）：葡萄糖10，酵母粉10，蛋白胨20，pH调节至6.0。基础摇瓶培养基：培养基组成（g/L）：葡萄糖10，蔗糖6， KH2PO4 1， NaCl 0.1， MgSO4·7H2O 5，CaCl2·2H2O，酵母粉0.2， pH调节至6.0。培养基灭菌条件：蒸汽湿热灭菌115℃，20min。
种子培养: 在无菌条件下，从完成活化的固体培养基中挑取为红法夫酵母RH-A单菌落，接入种子培养基中,种子摇瓶装液量50 mL/250 mL,培养温度22 °C,摇床转速200rpm,培养时间24 h。
发酵培养：在无菌条件下，以10%的接种量从种子摇瓶中吸取菌液转接到5L发酵罐培养基，接种量为10％，控制发酵温度为22℃，通气量为2vvm，设置溶解氧与转速关联并控制溶解氧为30％以上。
实施例五、虾青素的提取
1ml采用实施例三的法夫酵母RH-A菌株的发酵液，12000rpm离心后，去除上清液，收集菌体，无菌水洗涤两次后，将洗涤后的菌体与预热到60℃的二甲亚砜中，再剧烈震荡摇匀，在温度为60℃水浴锅中保温20分钟。加入1ml甲醇与二氯甲烷按照体积比3:1混合得到的提取液，静置3分钟后，2000rpm离心1分钟，将离心得到的上清液转移到新的离心管中，与其同样条件下进行离心分离，其菌体沉淀物重复用预热60℃的二甲亚砜进行破壁提取，直到菌体为白色为止，汇集提取液。
高压液相色谱法提取虾青素的含量：上清液利用HPLC检测，UV3000紫外检测器，使用ZY104型液相色谱柱C18色谱柱，流动相是甲醇乙腈二氯甲烷（21：21：8），流速为1.0ml/min，检测波长为476nm，检测物的浓度根据标准品的标准曲线确定。
总油脂及脂肪酸检测：取菌体干重1g，加入10mL正己烷溶液，加塞充分摇匀10h，静置分层，取上层有机相，用正己烷溶液重复洗3~5次，直至上层有机相无色，将有机相合并，充氮挥发除去溶剂即得脂肪酸，称重计算。加入0.5mol/L的KOH甲醇溶液1mL，60℃水浴中振荡皂化15min，冷却后加入14%BF3-甲醇溶液2ml，于60℃水浴中振荡2min，放至室温，加入正己烷1ml，振荡，加入饱和NaCL溶液1ml，加入无水硫酸钠，取上清液980μL，通过气相色谱分析脂肪酸的种类及含量。
实施例六、菌株对不同糖源利用情况
葡萄糖、木糖，乳糖，纤维二糖，蔗糖、阿拉伯糖为唯一碳源时，红法夫酵母均可以正常生长，说明其可以利用的碳源存在广谱性。尤其是，葡萄糖、蔗糖、纤维二糖为唯一碳源时，红法夫酵母生长速度快，培养温度22-28℃培养3d，菌落红色明显(图2)。
红法夫酵母phaffia rhodozyma RH-A完成5L发酵罐放大培养，由于溶氧和补料的有效控制，摇瓶中红法夫酵母phaffia rhodozyma RH-A生长速度比摇瓶水平更快。随着发酵时间延长，红法夫酵母phaffia rhodozyma RH-A菌体量不断积累。当发酵时间达到70h时，OD600可以达到43，菌体积累量达到最大。但类胡萝卜素含量在90h达到最大，在菌体干重中类胡萝卜素产量可以达到1.67mg/g。类胡萝卜产物进行高效液相分析后发现红法夫酵母phaffia rhodozyma RH-A可以高效合成虾青素单一产物（图3，11.3分钟出峰），β-胡萝卜素和番茄红素均无积累。当发酵继续进行，菌体达到稳定期，OD略有升高，类胡萝卜素在菌体干重中含量继续积累。
发酵完成后，收集phaffia rhodozyma RH-A菌体，进行油脂分析检测，发现菌体中油脂含量占到18.5%。在脂肪组成中，以C18:1的油酸和C18:2的亚油酸为主，占到总脂肪酸的73.8%。
油酸可以降低低密度脂蛋白胆固醇（好胆固醇），同时不降低高密度脂蛋白胆固醇（好胆固醇），因此在预防高脂血症及心血管疾病方面很有帮助；而亚油酸是动物体的必需脂肪酸，也就是说维持健康必不可少，比如说亚油酸是合成前列腺素的前体，进而和血管扩张和收缩、神经刺激的传导等重要的生理功能有关。可见两种油脂成分都对动物体健康很重要。
发酵后菌体通过酶解破壁制备红法夫酵母phaffia rhodozyma RH-A水解物，多糖含量42%，蛋白含量30.2%，油脂含量18.5%，虾青素含量为1.67mg/g，核酸含量8.2%。
Claims (9)
1.一种红法夫酵母菌(Phaffia rhodozyma)RH-A，其特征在于，其保藏编号为CGMCCNO.26861。
2.如权利要求1所述的红法夫酵母菌在生产虾青素中的应用。
3.一种生产虾青素的方法，其特征在于，包括如下步骤：培养如权利要求1所述的红法夫酵母菌，提取所产生的虾青素。
4.如权利要求3所述的方法，其特征在于，培养时采用的培养基以葡萄糖、蔗糖或纤维二糖为唯一碳源。
5.如权利要求3所述的方法，其特征在于，培养时分成种子培养和发酵培养，在种子培养期间，培养条件为：培养温度22 °C，摇床转速200 rpm，培养时间24 h。
6.如权利要求3所述的方法，其特征在于，培养时分成种子培养和发酵培养，在发酵培养期间，培养条件为：控制发酵温度为22℃，通气量为2vvm，设置溶解氧与转速关联并控制溶解氧为30％以上，发酵培养时长60-300小时。
7.如权利要求6所述的方法，其特征在于，提取所产生的虾青素的方法如下：将发酵培养的发酵液离心后，去除上清液，收集菌体，用无菌水洗涤，将洗涤后的菌体加入到二甲亚砜中，再剧烈震荡摇匀，在水浴锅中保温；加入甲醇与二氯甲烷按照体积比3:1混合得到的提取液，静置后离心，将离心得到的上清液转移到新的离心管中。
8.如权利要求7所述的方法，其特征在于，将转移到新的离心管中的上清液，相同条件下，进一步进行离心分离，其菌体沉淀物重复用二甲亚砜进行破壁提取，直到菌体为白色为止，汇集提取液。
9.如权利要求8所述的方法，其特征在于，提取所产生的虾青素的方法如下：将发酵培养的发酵液，12000rpm离心后，去除上清液，收集菌体，无菌水洗涤两次后，将洗涤后的菌体加入到预热到60℃的二甲亚砜中，再剧烈震荡摇匀，在温度为60℃水浴锅中保温20分钟；
加入1ml甲醇与二氯甲烷按照体积比3:1混合得到的提取液，静置3分钟后，2000rpm离心1分钟，将离心得到的上清液转移到新的离心管中，相同条件下再次进行离心分离，其菌体沉淀物重复用预热到60℃的二甲亚砜进行破壁提取，直到菌体为白色为止，汇集提取液。
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| CN105861342A (zh) * | 2016-05-20 | 2016-08-17 | 吉林省希玛生物科技有限公司 | 一株富含虾青素的红法夫酵母菌株及其筛选方法和应用 |
| CN112358975A (zh) * | 2020-10-12 | 2021-02-12 | 厦门昶科生物工程有限公司 | 红法夫酵母菌及其用途 |
| CN113789322A (zh) * | 2021-10-09 | 2021-12-14 | 湖北绿科乐华生物科技有限公司 | 一种高产虾青素的红法夫酵母菌株及其选育方法与应用 |
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| CN105861342A (zh) * | 2016-05-20 | 2016-08-17 | 吉林省希玛生物科技有限公司 | 一株富含虾青素的红法夫酵母菌株及其筛选方法和应用 |
| CN112358975A (zh) * | 2020-10-12 | 2021-02-12 | 厦门昶科生物工程有限公司 | 红法夫酵母菌及其用途 |
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