CN114190472A - 一种免疫调节微生态制剂及其制备方法和应用 - Google Patents
一种免疫调节微生态制剂及其制备方法和应用 Download PDFInfo
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- CN114190472A CN114190472A CN202111254468.XA CN202111254468A CN114190472A CN 114190472 A CN114190472 A CN 114190472A CN 202111254468 A CN202111254468 A CN 202111254468A CN 114190472 A CN114190472 A CN 114190472A
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- fermentation liquor
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Classifications
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- A—HUMAN NECESSITIES
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- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- C—CHEMISTRY; METALLURGY
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A—HUMAN NECESSITIES
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
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Landscapes
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Zoology (AREA)
- Health & Medical Sciences (AREA)
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- General Health & Medical Sciences (AREA)
- Biodiversity & Conservation Biology (AREA)
- Insects & Arthropods (AREA)
- Birds (AREA)
- Sustainable Development (AREA)
- Fodder In General (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种免疫调节微生态制剂及其制备方法和应用。所述制备方法包括分别制备控制病原体感染剂和复合益生菌剂;所述控制病原体感染剂的制备为将酿酒酵母发酵液、抗菌肽、噬菌体与营养强化剂混合;所述营养强化剂包括稻壳、维生素C、维生素E、腺嘌呤和海藻酸钠;所述复合益生菌剂的制备为将乳酸菌发酵液、芽孢杆菌发酵液、酵母菌发酵液与水产饲料混合。本发明将治疗和免疫调节相结合,控制病原体感染剂中噬菌体配合抗菌肽联合作用,高效中和水体内的病原体,再配合营养强化剂,可以在养殖水体内水生动物发病之后,快速降低病害的传播,从而保证了养殖水体内水产品质量。
Description
技术领域
本发明涉及水产养殖技术领域,具体涉及一种免疫调节微生态制剂及其制备方法和应用。
背景技术
集约化和规模化是水产养殖的高收益生产方式,因此广大水产养殖户均采用高密度养殖方式,但是高密度养殖会导致病害频繁发生,造成水体环境中污染因子增多,氮磷等营养富集后有机污染严重,水体内透明度下降;蓝绿藻等有害生物大量繁殖造成水体内溶解氧浓度下降、水体酸碱度发生变化,从而使得环境和水生动物机体内的有益菌和致病菌的微生态平衡失调,导致水生动物受到长期应激后感染疾病。为了在短时间内挽回损失,养殖户传统做法是将市面上各类化学药物及抗生素添加到养殖水体内,但是该种方法会导致动物体内菌群失调、产生病原菌耐药性以及抗生素残留问题,也不满足生态绿色养殖的发展需求。经过科技工作者多年实践研究和探索,水产养殖业正逐渐向可持续发展模式转型,微生态制剂作为新型渔药处理病害问题受到了广泛的关注,微生态制剂具有的无毒副作用、无残留和无耐药性的三无优点,使其已成为水产养殖中的主力生物防治渔药。
现有技术中关于微生态制剂的技术方案有很多,但在菌株的配合使用上存在很多不合理的地方,增加了制备成本且难以具有良好的防治效果。如中国发明专利CN108967742A公开了一种水产养殖用复合微生态制剂,其采用粪肠球菌、屎肠球菌、枯草芽孢杆菌、酿酒酵母菌制备成复合微生物,仍需额外添加包括蛋白酶、纤维素酶、脂肪酶的复合酶系统以及复合免疫增强因子,无疑为微生态制品增加了制作和使用的成本;如中国发明专利CN112624350A公开了一种用于淡水养殖的微生态制剂,其主要用膏剂代替市面上主流的粉剂和水剂,提高活菌浓度的同时还能更好地解决货架期的问题,但是该技术方案只是从单方面提高生产和使用上的便利性,难以提升治疗或防治的效果,实际水产养殖过程中应用该种微生物制品后,缓解水生动物病症的效果有限。
发明内容
有鉴于此,有必要针对上述的问题,提供一种免疫调节微生态制剂及其制备方法和应用。
为实现上述目的,本发明采取以下的技术方案:
第一方面,本发明提供一种免疫调节微生态制剂的制备方法,包括分别制备控制病原体感染剂和复合益生菌剂;所述控制病原体感染剂的制备为将酿酒酵母发酵液、抗菌肽、噬菌体与营养强化剂混合;所述营养强化剂包括稻壳、维生素C、维生素E、腺嘌呤和海藻酸钠;所述复合益生菌剂的制备为将乳酸菌发酵液、芽孢杆菌发酵液、酵母菌发酵液与水产饲料混合。
本发明的免疫调节微生态制剂中控制病原体感染剂含有酿酒酵母,其细胞壁富含葡聚糖类物质,其具有酵母天然合成的抗菌肽,可以在水体内与抗菌肽和噬菌体协同作用感染病害水体,起到中和水体内病原体的目的,被患病水产动物食用后,可以迅速激活T淋巴细胞,NK细胞和巨噬细胞,促进淋巴细胞产生抗体,分泌TNFα、IL和IFN等细胞因子,调节免疫细胞之间的相互作用,免疫反应加速和放大,终导致靶细胞坏死凋亡;复合益生菌剂可以调节水产动物的免疫系统,重点是对肠黏膜细胞进行修复,在乳酸菌等益生菌附着定值后,肠黏膜免疫系统受刺激处于合适稳定的激活状态,能够刺激巨噬细胞活力,增强细胞特异性免疫,活化淋巴组织,产生细胞因子,提高免疫力防止病毒、细菌入侵,维护健康的免疫调节功能。
进一步的,所述控制病原体感染剂按质量百分比计包括30%~80%酿酒酵母发酵液、1%~5%抗菌肽、1%~5%噬菌体和10%~60%营养强化剂。
优选的,所述控制病原体感染剂按质量百分比计包括57%酿酒酵母发酵液、1.6%抗菌肽、1.4%噬菌体和40%营养强化剂。
进一步的,所述酿酒酵母菌发酵液的制备方法为将酿酒酵母接种于100mL摇瓶YPD培养基中,30℃培养24h得到种子培养液,然后接种于1.8L发酵罐YPD培养基中,600rpm培养16h得到放大培养液;然后移种到18L发酵培养液中,350rpm培养14小时,罐压0.05Mpa,通风比1:1,酿酒酵母菌菌液浓度≥4.1logCFU/ml。
进一步的,酿酒酵母菌发酵液的发酵培养液包括葡萄糖5~20g/L,蛋白胨5~20g/L,酵母粉5~20g/L,磷酸二氢钾1~5g/L,硫酸镁0.1~1.0g/L,消泡剂0.1~1.0g/L,pH为6.5~7.8。
进一步的,所述营养强化剂按质量百分比计包括60%~90%稻壳、0.1%~5%维生素C、0.1%~5%维生素E、0.1%~5%腺嘌呤和2%~20%海藻酸钠。
优选的,所述营养强化剂按质量百分比计包括87%稻壳、1%维生素C、1%维生素E、1%腺嘌呤和10%海藻酸钠。
进一步的,所述抗菌肽包括菌丝霉素和金环蛇抗菌肽。菌丝霉素是一种真菌抗菌肽,也称宿主防御肽,抗菌谱包括革兰氏阳性菌、临床耐药细菌、寄生虫、包膜病毒和肿瘤细胞。金环蛇抗菌肽(Bungarus fasciatus-30)是一种脊椎动物抗菌肽,具有广谱抗微生物活性,抗菌谱包括革兰氏阳性菌、革兰氏阴性菌、真菌及病毒及临床耐药细菌,其时间迅速,没有溶血活性和细胞毒性。
优选的,按质量比计,所述菌丝霉素和金环蛇抗菌肽的比例为1:1。
进一步的,所述营养强化剂中的稻壳需先在45~80℃水中加热0.5~5小时后,再与其他组分混合。
优选的,所述营养强化剂中的稻壳需先在50℃水中加热2小时后,再与其他组分混合。
进一步的,所述复合益生菌剂按质量百分比包括1%~5%乳酸菌发酵液、1%~5%芽孢杆菌发酵液、1%~5%酵母菌发酵液和80%~99%水产饲料。
优选的,所述复合益生菌剂按质量百分比包括4%乳酸菌发酵液、3%芽孢杆菌发酵液、3%酵母菌发酵液和90%水产饲料。
进一步的,所述乳酸菌发酵液包括植物乳杆菌、粪肠球菌、干酪乳杆菌和乳酸乳杆菌;所述芽孢杆菌发酵液包括凝结芽孢杆菌、地衣芽孢杆菌和枯草芽孢杆菌;所述酵母菌发酵液包括酿酒酵母和产朊假丝酵母。
进一步的,所述乳酸菌发酵液的制备方法为分别将植物乳杆菌、粪肠球菌、干酪乳杆菌和乳酸乳杆菌接种于100mL摇瓶MRS培养基中,37℃培养18h到24h得到种子培养物,然后接种于1.8L发酵罐MRS培养基中,200rpm培养12小时得到放大培养物,然后移种到18L发酵培养液中,120rpm培养12小时,罐压0.05Mpa,通风比1:0.6,菌液浓度≥6.3logCFU/ml;将分别得到的植物乳杆菌发酵液、粪肠球菌发酵液、干酪乳杆菌发酵液和乳酸乳杆菌发酵液混合在一起,得到乳酸菌发酵液。
进一步的,乳酸菌发酵液的发酵培养液包括红糖14~21g/L,蛋白胨1~10g/L,牛肉膏1~10g/L,酵母浸粉0.5~5.0g/L,硫酸镁0.1~2.0g/L,硫酸锰0.001~0.010g/L,pH为6.5-7.8。
进一步的,所述芽孢杆菌发酵液的制备方法为分别将凝结芽孢杆菌、地衣芽孢杆菌和枯草芽孢杆菌接种于100mL摇瓶BPY培养基中,32℃培养24h得到种子培养物,然后接种于1.8L发酵罐BPY液体中,400rpm培养16h得到放大培养物;然后移种到18L发酵培养液中,250rpm培养14小时,罐压0.05Mpa,通风比1:1,菌液浓度≥6.9logCFU/ml;将分别得到凝结芽孢杆菌发酵液、地衣芽孢杆菌发酵液和枯草芽孢杆菌发酵液混合在一起,得到芽孢杆菌发酵液。
进一步的,芽孢杆菌发酵液的发酵培养液包括蔗糖5~20g/L,蛋白胨5~20g/L,酵母粉5~20g/L,硫酸铵1~5g/L,硫酸镁0.1~1.0g/L,硫酸锰0.001~0.010g/L,消泡剂0.1~1.0g/L,pH为6.5~7.8。
进一步的,所述酵母菌发酵液的制备方法为分别将酿酒酵母和产朊假丝酵母接种于100mL摇瓶YPD培养基中,30℃培养24h得到种子培养液,然后接种于1.8L发酵罐YPD培养基中,600rpm培养16h得到放大培养液;然后移种到18L发酵培养液中,350rpm培养14小时,罐压0.05Mpa,通风比1:1,菌液浓度≥4.1logCFU/ml;将分别得到的酿酒酵母菌发酵液和产朊假丝酵母菌发酵液混合在一起,得到酵母菌发酵液。
进一步的,酵母菌发酵液的发酵培养液包括葡萄糖5~20g/L,蛋白胨5~20g/L,酵母粉5~20g/L,磷酸二氢钾1~5g/L,硫酸镁0.1~1.0g/L,消泡剂0.1~1.0g/L,pH为6.5~7.8。
第二方面,本发明提供一种免疫调节微生态制剂,采用上述制备方法制备而成。
第三方面,本发明提供一种免疫调节微生态制剂在水产养殖中的应用,将控制病原体感染剂投入水体3~5天后,再添加复合益生菌剂。
进一步的,复合益生菌剂在混合均匀后放置2小时后再添加到水体中。
本发明的有益效果为:
1、本发明的免疫调节微生态制剂能替代化学药物和抗生素使用,可以快速调节水生动物免疫力,并且降低应激带来的摄食减少等异常情况;本发明可以无需大量换水的情况下高效增强水生动物抵抗病原体感染,节约用水保护生态环境,减少了养殖户的劳动强度;本发明的免疫调节微生态制剂操作方便而且对环境无污染,不会产生耐药性,也不会有残留物质危害食品安全。
2、本发明的免疫调节微生态制剂包括控制病原体感染剂和复合益生菌剂,将治疗和免疫调节相结合,控制病原体感染剂中噬菌体配合抗菌肽联合作用,会高效中和水体内的病原体,再配合营养强化剂,可以在养殖水体内水生动物发病之后,快速降低病害的传播,初步改善得病水生动物的肠道功能;复合益生菌剂可以调节水产动物的免疫系统,重点是对肠黏膜细胞进行修复,在乳酸菌等益生菌附着定值后,肠黏膜免疫系统受刺激处于合适稳定的激活状态,能够刺激巨噬细胞活力,增强细胞特异性免疫,活化淋巴组织,产生细胞因子,提高免疫力防止病毒、细菌入侵,维护健康的免疫调节功能,增强消化系统能力,使更多的饲料能量转化为肌肉组织,从而保证了养殖水体内水产品质量。本发明通过调整控制病原体感染剂和复合益生菌剂的时间,避免二者之间产生影响,抑制菌体活性。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将结合本发明实施例,对本发明的技术方案作进一步清楚、完整地描述。需要说明的是,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。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。
本发明实施例中所述水产饲料为本领域水产养殖过程中常见市售产品即可,本发明不作特别限定。
实施例1
一种免疫调节微生态制剂的制备方法,包括分别制备控制病原体感染剂和复合益生菌剂;所述控制病原体感染剂的制备为将质量百分比计的57%酿酒酵母菌发酵液、1.6%抗菌肽(0.8%菌丝霉素和0.8%金环蛇抗菌肽)、1.4%噬菌体与40%营养强化剂混合;所述营养强化剂按质量百分比计包括88%稻壳、1.2%维生素C、1.2%维生素E、1.2%腺嘌呤和8.4%海藻酸钠;所述复合益生菌剂的制备为将质量百分比计的4%乳酸菌发酵液、3%芽孢杆菌发酵液、3%酵母菌发酵液与90%水产饲料混合。
其中,所述乳酸菌发酵液的制备方法为分别将植物乳杆菌、粪肠球菌、干酪乳杆菌和乳酸乳杆菌接种于100mL摇瓶MRS液体中,37℃培养18h到24h得到种子培养物,然后接种于1.8L发酵罐MRS培养基中,200rpm培养12小时得到放大培养物,然后移种到18L发酵培养液中,120rpm培养12小时,罐压0.05Mpa,通风比1:0.6,菌液浓度≥6.3logCFU/ml;将分别得到的植物乳杆菌发酵液、粪肠球菌发酵液、干酪乳杆菌发酵液和乳酸乳杆菌发酵液混合在一起,得到乳酸菌发酵液。乳酸菌发酵液的发酵培养液为红糖15g/L,蛋白胨4g/L,牛肉膏4g/L,酵母浸粉2g/L,硫酸镁0.8g/L,硫酸锰0.004g/L,pH为6.8。
其中芽孢杆菌发酵液的制备方法为分别将凝结芽孢杆菌、地衣芽孢杆菌和枯草芽孢杆菌接种于100mL摇瓶BPY培养基中,32℃培养24h得到种子培养物,然后接种于1.8L发酵罐BPY液体中,400rpm培养16h得到放大培养物;然后移种到18L发酵培养液中,250rpm培养14小时,罐压0.05Mpa,通风比1:1,菌液浓度≥6.9logCFU/ml;将分别得到凝结芽孢杆菌发酵液、地衣芽孢杆菌发酵液和枯草芽孢杆菌发酵液混合在一起,得到芽孢杆菌发酵液。芽孢杆菌发酵液的发酵培养液为蔗糖11g/L,蛋白胨9g/L,酵母粉7g/L,硫酸铵3g/L,硫酸镁0.9g/L,硫酸锰0.008g/L,消泡剂0.7g/L,pH为6.7。
其中酵母菌发酵液的制备方法为分别将酿酒酵母和产朊假丝酵母接种于100mL摇瓶YPD培养基中,30℃培养24h得到种子培养液,然后接种于1.8L发酵罐YPD培养基中,600rpm培养16h得到放大培养液;然后移种到18L发酵培养液中,350rpm培养14小时,罐压0.05Mpa,通风比1:1,菌液浓度≥4.1logCFU/ml;将分别得到的酿酒酵母菌发酵液和产朊假丝酵母菌发酵液混合在一起,得到酵母菌发酵液。酵母菌发酵液的发酵培养液为葡萄糖10g/L,蛋白胨12g/L,酵母粉6g/L,磷酸二氢钾2g/L,硫酸镁0.8g/L,消泡剂0.8g/L,pH为6.8。
其中酿酒酵母菌发酵液的制备方法与酵母菌发酵液的制备方法相同,只是接种过程中只接种酿酒酵母菌即可。
其中营养强化剂中的稻壳先经50℃水加热2小时后,再与其他组分混合制备营养强化剂。
实施例2
一种免疫调节微生态制剂的制备方法,包括分别制备控制病原体感染剂和复合益生菌剂;所述控制病原体感染剂的制备为将质量百分比计的65%酿酒酵母菌发酵液、1.5%抗菌肽(0.75%菌丝霉素和0.75%金环蛇抗菌肽)、1.5%噬菌体与32%营养强化剂混合;所述营养强化剂按质量百分比计包括87%稻壳、1%维生素C、1%维生素E、1%腺嘌呤和10%海藻酸钠;所述复合益生菌剂的制备为将质量百分比计的5%乳酸菌发酵液、5%芽孢杆菌发酵液、5%酵母菌发酵液与85%水产饲料混合。
其中,所述乳酸菌发酵液的制备方法为分别将植物乳杆菌、粪肠球菌、干酪乳杆菌和乳酸乳杆菌接种于100mL摇瓶MRS液体中,37℃培养18h到24h得到种子培养物,然后接种于1.8L发酵罐MRS培养基中,200rpm培养12小时得到放大培养物,然后移种到18L发酵培养液中,120rpm培养12小时,罐压0.05Mpa,通风比1:0.6,菌液浓度≥6.3logCFU/ml;将分别得到的植物乳杆菌发酵液、粪肠球菌发酵液、干酪乳杆菌发酵液和乳酸乳杆菌发酵液混合在一起,得到乳酸菌发酵液。乳酸菌发酵液的发酵培养液为红糖16g/L,蛋白胨5g/L,牛肉膏5g/L,酵母浸粉3g/L,硫酸镁0.7g/L,硫酸锰0.005g/L,pH为6.9。
其中芽孢杆菌发酵液的制备方法为分别将凝结芽孢杆菌、地衣芽孢杆菌和枯草芽孢杆菌接种于100mL摇瓶BPY培养基中,32℃培养24h得到种子培养物,然后接种于1.8L发酵罐BPY液体中,400rpm培养16h得到放大培养物;然后移种到18L发酵培养液中,250rpm培养14小时,罐压0.05Mpa,通风比1:1,菌液浓度≥6.9logCFU/ml;将分别得到凝结芽孢杆菌发酵液、地衣芽孢杆菌发酵液和枯草芽孢杆菌发酵液混合在一起,得到芽孢杆菌发酵液。芽孢杆菌发酵液的发酵培养液为蔗糖10g/L,蛋白胨10g/L,酵母粉5g/L,硫酸铵2g/L,硫酸镁0.6g/L,硫酸锰0.004g/L,消泡剂0.6g/L,pH为6.6。
其中酵母菌发酵液的制备方法为分别将酿酒酵母和产朊假丝酵母接种于100mL摇瓶YPD培养基中,30℃培养24h得到种子培养液,然后接种于1.8L发酵罐YPD培养基中,600rpm培养16h得到放大培养液;然后移种到18L发酵培养液中,350rpm培养14小时,罐压0.05Mpa,通风比1:1,菌液浓度≥4.1logCFU/ml;将分别得到的酿酒酵母菌发酵液和产朊假丝酵母菌发酵液混合在一起,得到酵母菌发酵液。酵母菌发酵液的发酵培养液为葡萄糖8g/L,蛋白胨6g/L,酵母粉12g/L,磷酸二氢钾3g/L,硫酸镁0.8g/L,消泡剂0.6g/L,pH为6.7。
其中酿酒酵母菌发酵液的制备方法与酵母菌发酵液的制备方法相同,只是接种过程中只接种酿酒酵母菌即可。
其中营养强化剂中的稻壳先经49℃水加热2.5小时后,再与其他组分混合制备营养强化剂。
实施例3
一种免疫调节微生态制剂的制备方法,包括分别制备控制病原体感染剂和复合益生菌剂;所述控制病原体感染剂的制备为将质量百分比计的57%酿酒酵母菌发酵液、1.6%抗菌肽(0.8%菌丝霉素和0.8%金环蛇抗菌肽)、1.4%噬菌体与40%营养强化剂混合;所述营养强化剂按质量百分比计包括87%稻壳、1%维生素C、1%维生素E、1%腺嘌呤和10%海藻酸钠;所述复合益生菌剂的制备为将质量百分比计的5%乳酸菌发酵液、4%芽孢杆菌发酵液、6%酵母菌发酵液与85%水产饲料混合。
其中,所述乳酸菌发酵液的制备方法为分别将植物乳杆菌、粪肠球菌、干酪乳杆菌和乳酸乳杆菌接种于100mL摇瓶MRS液体中,37℃培养18h到24h得到种子培养物,然后接种于1.8L发酵罐MRS培养基中,200rpm培养12小时得到放大培养物,然后移种到18L发酵培养液中,120rpm培养12小时,罐压0.05Mpa,通风比1:0.6,菌液浓度≥6.3logCFU/ml;将分别得到的植物乳杆菌发酵液、粪肠球菌发酵液、干酪乳杆菌发酵液和乳酸乳杆菌发酵液混合在一起,得到乳酸菌发酵液。乳酸菌发酵液的发酵培养液为红糖16g/L,蛋白胨4g/L,牛肉膏6g/L,酵母浸粉4g/L,硫酸镁0.8g/L,硫酸锰0.004g/L,pH为6.8。
其中芽孢杆菌发酵液的制备方法为分别将凝结芽孢杆菌、地衣芽孢杆菌和枯草芽孢杆菌接种于100mL摇瓶BPY培养基中,32℃培养24h得到种子培养物,然后接种于1.8L发酵罐BPY液体中,400rpm培养16h得到放大培养物;然后移种到18L发酵培养液中,250rpm培养14小时,罐压0.05Mpa,通风比1:1,菌液浓度≥6.9logCFU/ml;将分别得到凝结芽孢杆菌发酵液、地衣芽孢杆菌发酵液和枯草芽孢杆菌发酵液混合在一起,得到芽孢杆菌发酵液。芽孢杆菌发酵液的发酵培养液为蔗糖6g/L,蛋白胨5g/L,酵母粉5g/L,硫酸铵1.5g/L,硫酸镁0.6g/L,硫酸锰0.004g/L,消泡剂0.6g/L,pH为7.0。
其中酵母菌发酵液的制备方法为分别将酿酒酵母和产朊假丝酵母接种于100mL摇瓶YPD培养基中,30℃培养24h得到种子培养液,然后接种于1.8L发酵罐YPD培养基中,600rpm培养16h得到放大培养液;然后移种到18L发酵培养液中,350rpm培养14小时,罐压0.05Mpa,通风比1:1,菌液浓度≥4.1logCFU/ml;将分别得到的酿酒酵母菌发酵液和产朊假丝酵母菌发酵液混合在一起,得到酵母菌发酵液。酵母菌发酵液的发酵培养液为葡萄糖8g/L,蛋白胨6g/L,酵母粉8g/L,磷酸二氢钾2.5g/L,硫酸镁0.75g/L,消泡剂0.7g/L,pH为6.9。
其中酿酒酵母菌发酵液的制备方法与酵母菌发酵液的制备方法相同,只是接种过程中只接种酿酒酵母菌即可。
其中营养强化剂中的稻壳先经55℃水加热3小时后,再与其他组分混合制备营养强化剂。
对比例1
一种免疫调节微生态制剂的制备方法,其与实施例1的区别在于,对比例1中控制病原体感染剂中不含有抗菌肽和噬菌体,其余均与实施例1相同。
对比例2
一种免疫调节微生态制剂的制备方法,其与实施例1的区别在于,对比例2中复合益生菌剂不含有乳酸菌发酵液,其余均与实施例1相同。
对比例3
市售的EM菌剂,购自郑州某生物技术公司,其主要成分为光合菌群、乳酸菌群、酵母菌群、革兰氏阳性放线菌群和发酵系的丝状菌群。
试验对比
1、取停饲料半天的40日对虾,用400L水产养殖桶,每桶90尾,共18桶,并用弧菌悬液注射攻毒。将其分为不同的试验组和空白对照组,每组试验组和空白对照组均设置三个平行样本。统一养殖方法为:每天在9:30和18:00投放饲料,以每桶对虾的总体重为基准,每天投喂饲料总量在3-4%。每组试验组分别投喂实施例1~3、对比例1~2制备方法制备的免疫调节微生态制剂以及对比例3中的EM菌剂,具体为养殖过程中首先投入实施例1、2、3和对比例1、2的控制病原体感染剂和对比例3的EM菌,投入时间以及投入量均相同,连续投放三天后,停止使用。继续正常投喂饲料5天,随后相应的试验组别分别投入实施例1、2、3和对比例1、2中的复合益生菌剂,而对比例3仍是投入EM菌,连续五天后结束试验,检测水体内弧菌总数,并计算成活率。试验结果如表1所示,表1中的数据为三个平行样本的平均值。
表1
2、将一口20亩对虾塘出现病害后,将一整塘对虾平均转移至7个1亩塘内,分别使用本发明实施例1~3、对比例1~2的免疫调节微生态制剂和对比例3的EM菌进行处理。养殖过程中实施例1、2、3和对比例1、2的控制病原体感染剂的投入时间以及投入量均相同,而对比例3以EM菌替代投放,连续三天后,停止使用。正常投喂5天饲料后,相对应的塘内分别投放实施例1、2、3和对比例1、2的复合益生菌剂,对比例3继续投放EM菌,连续使用五天。之后正常投喂饲料直到收虾,计算成活率,成活率=养殖结束后的对虾尾数/转塘时的对虾初始尾数,数据如表2所示。
表2
由上述数据可知,本发明实施例1~3制备的免疫调节微生态制剂,能在发病水体内中和大部分病原体,科学合理地抑制病原体扩散,保护健康个体,并且能快速培养水产动物免疫力,提高免疫系统应答水平,修复肠道细胞并促进个体正常消化吸收,帮助农户将损失降到最低水平。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种免疫调节微生态制剂的制备方法,其特征在于,包括分别制备控制病原体感染剂和复合益生菌剂;所述控制病原体感染剂的制备为将酿酒酵母发酵液、抗菌肽、噬菌体与营养强化剂混合;所述营养强化剂包括稻壳、维生素C、维生素E、腺嘌呤和海藻酸钠;所述复合益生菌剂的制备为将乳酸菌发酵液、芽孢杆菌发酵液、酵母菌发酵液与水产饲料混合。
2.根据权利要求1所述的免疫调节微生态制剂的制备方法,其特征在于,所述控制病原体感染剂按质量百分比计包括30%~80%酿酒酵母发酵液、1%~5%抗菌肽、1%~5%噬菌体和10%~60%营养强化剂。
3.根据权利要求2所述的免疫调节微生态制剂的制备方法,其特征在于,所述控制病原体感染剂按质量百分比计包括57%酿酒酵母发酵液、1.6%抗菌肽、1.4%噬菌体和40%营养强化剂。
4.根据权利要求1所述的免疫调节微生态制剂的制备方法,其特征在于,所述营养强化剂按质量百分比计包括60%~90%稻壳、0.1%~5%维生素C、0.1%~5%维生素E、0.1%~5%腺嘌呤和2%~20%海藻酸钠。
5.根据权利要求1所述的免疫调节微生态制剂的制备方法,其特征在于,所述抗菌肽包括菌丝霉素和金环蛇抗菌肽。
6.根据权利要求1所述的免疫调节微生态制剂的制备方法,其特征在于,所述营养强化剂中的稻壳需先在45~80℃水中加热0.5~5小时后,再与其他组分混合。
7.根据权利要求1所述的免疫调节微生态制剂的制备方法,其特征在于,所述复合益生菌剂按质量百分比包括1%~5%乳酸菌发酵液、1%~5%芽孢杆菌发酵液、1%~5%酵母菌发酵液和80%~99%水产饲料。
8.根据权利要求7所述的免疫调节微生态制剂的制备方法,其特征在于,所述乳酸菌发酵液包括植物乳杆菌、粪肠球菌、干酪乳杆菌和乳酸乳杆菌;所述芽孢杆菌发酵液包括凝结芽孢杆菌、地衣芽孢杆菌和枯草芽孢杆菌;所述酵母菌发酵液包括酿酒酵母和产朊假丝酵母。
9.一种免疫调节微生态制剂,其特征在于,采用权利要求1~8任意一项所述的制备方法制备获得。
10.权利要求9所述的免疫调节微生态制剂在水产养殖中的应用,其特征在于,将控制病原体感染剂投入水体3~5天后,再添加复合益生菌剂。
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