CN114437950B - 一种改善水产动物体色的红法夫酵母goy1及其培养物的制备方法和应用 - Google Patents
一种改善水产动物体色的红法夫酵母goy1及其培养物的制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种改善水产动物体色的红法夫酵母GOY1及其培养物的制备方法和应用。所述红法夫酵母GOY1的分类命名为红法夫酵母Phaffia rhodozyma,保藏编号为CGMCC No.22504。红法夫酵母GOY1培养物的制备方法包括步骤:红法夫酵母GOY1种子液培养、固体培养基预酶解、固体培养、细胞破壁、烘干。红法夫酵母GOY1及其培养物都能用于制备水产养殖动物饲料添加剂,能够提高南美白对虾的增重率和存活率,并降低饵料系数,改善其体色,进而提高其非特异性免疫力,改善肠道菌群结构。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种改善水产动物体色的红法夫酵母GOY1及其培养物的制备方法和应用。
背景技术
红法夫酵母是真菌界、真菌门、半知菌亚门、隐球酵母科、红法夫酵母属的唯一一种,可产类胡萝卜素,其中45~90%为虾青素,且虾青素是红法夫酵母的主要色素,为胞内产物。
目前,红法夫酵母的应用技术多与虾青素的合成和提取相关。例如,CN112063539A公开了一种红法夫酵母发酵产类胡萝卜素的培养基及培养方法,并发酵产生出了3种天然类胡萝卜素:虾青素、角黄素和β-胡萝卜素;CN109593811A公开了一种转基因红法夫酵母高产3S,3’S虾青素的制备方法;CN113292469A公开了一种利用β-葡聚糖酶与几丁质酶提取红法夫酵母胞内虾青素的方法。
但红法夫酵母的培养物并不仅仅只包含虾青素,现有技术局限了红法夫酵母培养物更多的制备方法和使用途径,且目前也并无红法夫酵母培养物的相关技术被公开。而为了更好的利用红法夫酵母及其培养物,不仅需要开拓全新的菌株,还需要深入研究红法夫酵母培养物的制备方法和更广阔的应用途径。
发明内容
本发明提供了一种改善水产动物体色的红法夫酵母GOY1及其培养物的制备方法和应用。红法夫酵母培养物可以提高水产养殖动物的增重率和成活率,降低饵料系数,提高非特异性免疫力。
为实现上述发明目的,本发明通过下述技术方案予以实现:
本发明提供了一种改善水产动物体色的红法夫酵母GOY1,其分类命名为红法夫酵母Phaffia rhodozyma,保藏编号为CGMCC No.22504。
进一步的,所述红法夫酵母GOY1的菌落大而湿润,隆起,橙色至红色,表面光滑无褶皱;其菌体为不规则椭圆形,互相独立分散。
本发明还提供了所述的红法夫酵母GOY1的培养物的制备方法,包括以下步骤:
(1)种子液制备:以所述红法夫酵母GOY1为菌种,以5%~15%的接种量接种于液体培养基中培养,得到红法夫酵母GOY1种子液;
(2)固体培养基预酶解:配置固体培养基并调整其含水量至30%~40%,加入淀粉酶和糖化酶后,高温处理后得到预酶解固体培养基;
(3)固体培养:将步骤(1)的种子液以5%~15%的接种量接种于步骤(2)的预酶解固体培养基中培养,得到红法夫酵母GOY1固体培养物;
(4)细胞破壁和烘干:将步骤(3)的固体培养物继续发酵后,细胞自溶破壁释放得到的发酵物烘干至含水量≤10%,最终得到红法夫酵母GOY1培养物。
进一步的,所述步骤(1)中所述红法夫酵母GOY1在液体培养基中的培养条件为:温度18~22℃、转速200rpm,pH 5.0~6.0、培养时间48小时;所述液体培养基的组分为蔗糖35g/L~38g/L、硫酸铵3.0g/L~3.3g/L、酵母提取物1.6g/L~1.9g/L、CaCl2 0.05g/L~0.15g/L、KH2PO4 1.5g/L~2.5g/L、MgSO4 0.4g/L~0.6g/L、NaCl 0.05g/L~0.15g/L。
进一步的,所述步骤(2)中固体培养基的组分为,以重量份数计,玉米粉50%、麸皮35%、豆粕15%,糖蜜45mL/kg;所述淀粉酶和糖化酶的添加量相同,均为15U/g;所述高温处理的温度为70℃~90℃,处理时间为40min。
进一步的,所述步骤(3)中固体培养的条件为:培养基厚度3cm~6cm、pH 5.0~6.0、温度18~22℃,超过22℃时翻料,培养96h。
进一步的,所述步骤(4)中固体培养物继续发酵的条件为:温度50℃~55℃,发酵时间12h~24h。
经过条件优化后,所述的红法夫酵母GOY1的培养物的制备方法,包括以下步骤:
(1)种子液制备:以所述红法夫酵母GOY1为菌种,以10%的接种量接种于液体培养基中,pH 5.5、温度20℃、转速200 rpm培养48小时,得到红法夫酵母GOY1种子液;
(2)固体培养基预酶解:配置固体培养基并调整其含水量至35%,加入等量的15U/g的淀粉酶和糖化酶后,90℃处理40min得到预酶解固体培养基;
(3)固体培养:将步骤(1)的种子液以10%的接种量接种于步骤(2)的预酶解固体培养基中,培养基厚度3cm、pH 5.5、温度20℃培养96h,得到红法夫酵母GOY1固体培养物;
(4)细胞破壁和烘干:将步骤(3)的固体培养物55℃继续发酵24h后,细胞自溶破壁释放得到的发酵物烘干至含水量≤10%,最终得到红法夫酵母GOY1培养物。
本发明还提供了所述的红法夫酵母GOY1或所述的红法夫酵母GOY1培养物在用于制备提高水产养殖动物免疫力及改善体色的饲料添加剂中的应用。
进一步的,将所述红法夫酵母GOY1培养物以0.5%~5%的重量比添加到水产养殖动物饲料后,投喂水产养殖动物15d~90d,能够提高水产养殖动物的增重率和存活率、降低饵料系数和致病菌数量、改善体色,进而提高水产养殖动物的非特异性免疫力并改善其肠道菌群结构。
进一步的,所述水产养殖动物包括南美白对虾。
与现有技术相比,本发明的优点和有益技术效果是:
1、本发明得到了一株新的红法夫酵母GOY1,并利用该菌结合本发明的制备方法制得了红法夫酵母培养物,其含有更全面的营养成分,可用作饲料添加剂,不仅提高养殖动物增重率和成活率、降低饵料系数、提高非特异性免疫力、改善肠道菌群结构,还便于保存和运输。
2、本发明采用低温发酵制备红法夫酵母培养物的方法,有利于虾青素等类胡萝卜素的积累,并促进其他成分的析出,提高产品的使用效果,在水产养殖动物饲料中添加,可改善体色。
3、本发明采用的固体发酵培养基预酶解的处理方法,使培养基更容易被微生物利用,加快生长繁殖速度,缩短发酵时间,提升红法夫酵母的菌量,从而制备获得更多的培养物。
附图说明
图1是红法夫酵母GOY1在YPD培养基上的菌落、镜检菌体图。
图2为固体培养基经不同温度处理后的还原糖含量。
图3为不同厚度固体培养基发酵后红法夫酵母GOY1菌量的影响。
图4为投喂红法夫酵母培养物的南美白对虾与对照组的血清免疫酶活对比。
图5为投喂红法夫酵母培养物的南美白对虾与对照组的肠道菌群结构对比。
图6为投喂红法夫酵母培养物的南美白对虾与对照组的体色对比结果。
具体实施方式
结合以下具体实例对本发明的技术方案作进一步详细的说明。
下述实施例中,如无特殊说明,所使用的实验方法均为常规方法,所用材料、试剂等均可从生物或化学试剂公司购买。
实施例1:红法夫酵母GOY1的获取及其培养物的制备
一、红法夫酵母GOY1的获取
取青岛胶州海参养殖水,梯度稀释后涂布于YPD培养基上,多次分离纯化后得到单菌落,命名为GOY1,保存。
菌株GOY1的菌落形态如图1a所示,菌落大而湿润,隆起,橙色至红色,表面光滑无褶皱;其菌体如图1b均为不规则椭圆形,互相独立分散。
提取菌株GOY1的DNA作为模板,使用18S rRNA通用引物进行扩增,扩增片段进行序列测定,将得到的菌株GOY1的18S rDNA测序结果与GenBank中的序列进行比对分析,结果显示菌株GOY1与Phaffia rhodozyma同源性最高,因此确定该菌株GOY1为红法夫酵母。
将筛选到的菌株GOY1进行菌种保藏,所述红法夫酵母GOY1的保藏单位:中国微生物菌种保藏管理委员会普通微生物中心(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;保藏日期:2021年05月11日;红法夫酵母Phaffia rhodozyma的保藏编号为CGMCC No.22504。
红法夫酵母GOY1在25℃~35℃温度范围内能够生长繁殖,但其最适合生长温度在30℃,在pH 5~9的改良YPD培养基内均能正常生长,最适生长pH范围为6.5~8,该菌最高可耐受5% NaCl。该菌可利用葡萄糖、麦芽糊精、木糖、海藻糖、七叶苷等多种碳水化合物产酸,还可经过发酵分泌较多的酵母胞外多糖或产生丰富的色素类代谢物,如虾青素、番茄红素等。
二、红法夫酵母培养物的制备
1、培养基:液体培养基包含蔗糖36g/L、硫酸铵3.2g/L、酵母提取物1.7g/L、CaCl20.1g/L、KH2PO4 2g/L、MgSO4 0.5g/L、NaCl 0.1g/L,蒸馏水配制;固体培养基由以下原料组成:玉米粉50、麸皮35、豆粕15,以上为重量份数,糖蜜45mL/kg。
2、红法夫酵母种子液制备:以红法夫酵母GOY1为菌种,接种于液体培养基中,接种量10%、pH 5.5、温度20℃、转速200 rpm,培养48小时。
3、固体培养基预酶解:调整固体培养基含水量为35%,等量添加15U/g的淀粉酶和糖化酶,90℃处理40min。
4、固体培养:将红法夫酵母种子液按10%的接种量接种到预酶解后的固体培养基中,培养基厚度3cm、pH 5.5、温度20℃(超过22℃时翻料),培养96h。
5、细胞破壁:取上述固体培养的红法夫酵母培养物在55℃条件下发酵24h,使红法夫酵母细胞自溶破壁,释放细胞内容物,得发酵物。
7、烘干:将上述的发酵物烘干至含水量≤10%,得红法夫酵母培养物。
实施例2:红法夫酵母培养物制备过程中的影响因素
1、固体培养基预处理的影响
同实施例1中的步骤,将添加了15U/g淀粉酶和15U/g糖化酶且含水量为30~40%的固体培养基分别置于50℃,60℃,70℃,80℃,90℃的培养箱中酶解40min,按照国标的分光光度法检测培养基中还原糖总量。
结果(图2)表明,酶解温度为90℃时,产生的还原糖总量最高,可达到101.9 mg/mL,相对对照组,还原糖总量提高了大约150%,这为红法夫酵母GOY1的增殖提供了充足的快速碳源,提高生长繁殖速度。
2、铺料厚度对红法夫酵母GOY1菌量的影响
同实施例1中的步骤,将培养红法夫酵母培养物的固体培养基设置三个厚度梯度:3cm、8cm、16cm,接种10%的红法夫酵母种子液,在相同培养条件下培养,对比发酵后的红法夫酵母GOY1菌量。
结果(图3)表明,培养基厚度为3cm时,培养物中红法夫酵母GOY1的菌量最高,随着铺料厚度的增加,红法夫酵母GOY1菌量显著降低。
实施例3:南美白对虾室内养殖试验
在循环水养殖系统,以240尾体长为4~5cm的南美白对虾为研究对象,分成2组,每组6个重复,每个重复20尾虾,对照组与试验组投喂量相同,对照组只投喂配合饲料,试验组投喂的饲料中含有1%实施例1制备的红法夫酵母培养物的等量替代,试验周期为30d。每天记录对虾摄食量、存活率和健康状况;养殖周期结束后,测定每缸对虾总体重,计算增重率、存活率、饵料系数,观察对虾体色,并通过高通量测序分析肠道菌群,抽取血清检测主要免疫酶活性。
结果表明:试验组30天增重率比对照组提高5.30%,成活率提高10.98%,饵料系数降低0.05(表1);试验组南美白对虾体色较对照组鲜艳(图6),血清中碱性磷酸酶AKP、溶菌酶LYS含量显著提升(图4);肠道菌群分析(图5)显示,试验组对虾肠道中弧菌属在总菌群中的占比明显降低,菌群结构得到改善。
表1 红法夫酵母培养物改善南美白对虾的生长性能
组号 | 增重率/% | 饵料系数 | 成活率/% |
对照 | 325.17±13.56ab | 1.31±0.16a | 84.36±6.80a |
试验组 | 330.47±10.34ab | 1.26±0.26a | 95.34±9.56b |
综合以上结果,表明在南美白对虾饲料中添加本发明制备的红法夫酵母培养物,可提高对虾增重率和成活率,降低饵料系数,同时可以抑制弧菌、改善肠道菌群结构、提升免疫酶活。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (4)
1.一种红法夫酵母(Phaffia rhodozyma)GOY1,其特征在于,其保藏编号为CGMCCNo.22504。
2.权利要求1所述的红法夫酵母GOY1的培养物的制备方法,其特征在于,包括以下步骤:
(1)种子液制备:以所述红法夫酵母GOY1为菌种,以10%的接种量接种于液体培养基中培养,得到红法夫酵母GOY1种子液;所述红法夫酵母GOY1在液体培养基中的培养条件为:温度20℃、转速200 rpm,pH 5.5、培养时间48小时;所述液体培养基的组分为蔗糖36g/L、硫酸铵3.2g/L、酵母提取物1.7g/L、CaCl2 0.1g/L、KH2PO4 2g/L、MgSO4 0.5g/L、NaCl 0.1g/L;
(2)固体培养基预酶解:配置固体培养基并调整其含水量至30%~40%,加入淀粉酶和糖化酶后,高温处理后得到预酶解固体培养基;
所述固体培养基的组分为,以重量份计,玉米粉50%、麸皮35%、豆粕15%,糖蜜45mL/kg;所述淀粉酶和糖化酶的添加量相同,均为15U/g;所述高温处理的温度为90℃,处理时间为40min;
(3)固体培养:将步骤(1)的种子液以10%的接种量接种于步骤(2)的预酶解固体培养基中培养,得到红法夫酵母GOY1固体培养物;所述固体培养的条件为:培养基厚度为3cm、pH5.5、温度20℃,超过22℃时翻料,培养96h;
(4)细胞破壁和烘干:将步骤(3)的固体培养物在55℃条件下继续发酵24h后,细胞自溶破壁释放得到的发酵物烘干至含水量≤10%,最终得到红法夫酵母GOY1培养物。
3.权利要求1所述的红法夫酵母GOY1或权利要求2中所述的红法夫酵母GOY1培养物在用于制备改善水产养殖动物体色的饲料添加剂中的应用,其特征在于,所述水产养殖动物为南美白对虾。
4.根据权利要求3所述的应用,其特征在于,将所述红法夫酵母GOY1培养物以0.5%~5%的重量比添加到水产养殖动物饲料后,投喂水产养殖动物15d~90d。
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