CN116855399A - 复合菌剂及其应用 - Google Patents
复合菌剂及其应用 Download PDFInfo
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- CN116855399A CN116855399A CN202310365229.4A CN202310365229A CN116855399A CN 116855399 A CN116855399 A CN 116855399A CN 202310365229 A CN202310365229 A CN 202310365229A CN 116855399 A CN116855399 A CN 116855399A
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- lactobacillus plantarum
- pediococcus pentosaceus
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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Abstract
本发明涉及微生物技术领域,尤其涉及复合菌剂及其应用。本发明将植物乳杆菌、戊糖片球菌和产朊假丝酵母进行组合,通过对菌种的筛选、对配比的调整和对制备方法的改进,最终获得了具有良好效果的复合菌剂。该复合菌剂能够稳定水体中的氨氮、溶氧、pH三项指标,并能够抑制致病菌、调节水产动物肠道菌群。
Description
技术领域
本发明涉及微生物技术领域,尤其涉及复合菌剂及其应用。
背景技术
随着集约化、高密度养殖规模日益扩大,集中投饵造成养殖水体中积累大量的残饵、粪便,造成有害物质的不断积累,促使养殖水中致病菌大量繁殖,导致疾病爆发。
养殖水体中氮盐的主要来源就是是残饵、生物排泄物和残骸等蛋白质污染物,这些污染物的分解使水中氮元素发生非正常变化并产生有害物质,主要为氨氮、亚硝酸盐等。养殖水体中高浓度的氮盐不仅直接危害养殖生物,而且还会引起养殖动物发生病害。
微生物可迅速降解水体中的残存饲料、鱼类的粪便及其它有机物,特别是清除池塘长时间的养殖水体底部,如海边老虾池底部积累的残余饲料、排泄废物、动植物残体;同时,还能吸收利用水体中的氨、亚硝酸盐、硫化氢等有害物质,能有效避免固体有机物和有害物质的积累。
现有技术中的微生物净水菌剂通常由多种微生物复配而成,但不同的菌剂效果不同,适用的环境也不同,而大多数菌剂并无法同时起到降低水中有害物质并抑制有害菌的作用。
发明内容
有鉴于此,本发明要解决的技术问题在于提供降低水产养殖水体中有害物质并抑制有害菌的复合菌剂及其应用。
本发明提供的复合菌剂由植物乳杆菌、戊糖片球菌和产朊假丝酵母组成。
一些实施例中,所述复合菌剂中的植物乳杆菌包括:植物乳杆菌(1),菌种保藏号为CGMCC No.18401;植物乳杆菌(2),菌种保藏号为CGMCC 1.2158。
一些实施例中,所述复合菌剂中的戊糖片球菌为保藏编号为CGMCC 1.2441的戊糖片球菌。
一些实施例中,所述复合菌剂中的产朊假丝酵母为保藏编号为CGMCCNo.7691的产朊假丝酵母。
本发明中,所述复合菌剂中的植物乳杆菌、戊糖片球菌和产朊假丝酵母的菌液体积比为1:1:1。
本发明将植物乳杆菌、戊糖片球菌和产朊假丝酵母进行组合,形成的复合菌种彼此协调配合,具有良好的去除水体有害物质和致病菌的作用。同等剂量下,四个菌种的组合后的效果优于单一菌种的作用,也优于省略或替换其中一个或多个菌种的组合,说明本发明的复合菌剂在对组分进行选择和复配后,起到了良好的增效协同作用。
本发明所述复合菌剂的制备方法,包括将活化后的植物乳杆菌、戊糖片球菌和产朊假丝酵母,接种入共培养培养基进行共培养,获得所述复合菌剂。
本发明中,所述共培养培养基经过对组分和浓度的优化,能够对复合菌种中的四个菌种同时起到良好的培养效果。相对其他培养基而言,本发明所述的共培养培养基对复合菌种进行培养,能够在短时间内获得更大的菌种密度。本发明所述的共培养培养基包括水和糖蜜5~10g/L,红糖60~90g/L,蛋白胨2~10g/L,磷酸二氢钾0.01~0.40g/L,柠檬酸氢二铵0.1~0.3g/L,氢氧化钠1.0~1.85g/L。
一些实施例中,所述共培养培养基由水和糖蜜5g/L,红糖60g/L,蛋白胨2g/L,磷酸二氢钾0.01g/L,柠檬酸氢二铵0.1g/L,氢氧化钠1.0g/L,pH4.5-6.0。
另一些实施例中,所述共培养培养基由水和糖蜜5g/L,红糖60g/L,蛋白胨2g/L,磷酸二氢钾0.01g/L,柠檬酸氢二铵0.1g/L,氢氧化钠1.0g/L,pH4.5-6.0。
另一些实施例中,所述共培养培养基由水和糖蜜10g/L,红糖90g/L,蛋白胨10g/L,磷酸二氢钾0.40g/L,柠檬酸氢二铵0.3g/L,氢氧化钠1.85g/L,pH4.5-6.0。
本发明所述制备方法中,所述植物乳杆菌和戊糖片球菌的活化培养基为MRS培养基,活化条件包括35±2℃,静置培养24~26h;
所述产朊假丝酵母的活化培养基为YPD培养基,活化条件包括35±2℃,170rpm振荡培养24~26h。
本发明所述制备方法的所述接种中,各菌种的接种量皆为2%~8%。一些实施例中,接种量为5%,
本发明所述制备方法中,所述共培养的条件包括:温度35℃,转速100~200rpm,pH值为4.5~6.0,溶解氧25~30mg/L。
本发明所述制备方法中,所述共培养的时长为24~26h,培养至培养液中菌体密度为28亿cfu/mL以上。
本发明还提供了一种水产养殖水体的净化方法,其包括施用本发明所述的复合菌剂或施用本发明所述制备方法制得的复合菌剂。
本发明中,所述施用包括:每5~10天按照3~5L/亩水面的用量使用1次,连续使用。
本发明中,所述水产养殖水体为南美对虾养殖的水体。所述水体包括养殖箱或养殖池塘。
本发明将植物乳杆菌、戊糖片球菌和产朊假丝酵母进行组合,通过对菌种的筛选、对配比的调整和对制备方法的改进,最终获得了具有良好效果的复合菌剂。该复合菌剂能够稳定水体中的氨氮、溶氧、pH三项指标,并能够抑制致病菌、调节水产动物肠道菌群。
具体实施方式
本发明提供了复合菌剂及其应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。
研究表明,乳酸菌能通过氧化、固氮、氨化、硝化和反硝化等作用,将养殖水体中的残饵、粪便、死藻等有害有机物转化为磷酸盐、硝酸盐和二氧化碳等营养物质,从而达到净化水质的目的;同时提高水体中的溶氧量,保持养殖水体的洁净,促进水产动物的健康生长。并且,乳酸菌能产生淀粉酶、脂肪酶、蛋白酶等多种酶类,将机体不易吸收的大分子量蛋白质降解为易于吸收的游离氨基酸和多肽,合成机体所需的烟酸、泛酸、叶酸、维生素B1、维生素B6及维生素B12等多种物质,为水产动物提供营养,并能产生乙酸、乳酸和促生长因子。乳酸菌还能促进维生素D的吸收和脂类的消化,提高矿物质磷、钙和铁的利用率。本发明中,所述植物乳杆菌和戊糖片球菌都是乳酸菌。其中,植物乳杆菌包括:保藏编号为CGMCCNo.18401的植物乳杆菌和/或保藏编号为CGMCC 1.2158的植物乳杆菌。戊糖片球菌为保藏编号为CGMCC 1.2441的戊糖片球菌。实验表明,在组合物中添加保藏编号为CGMCCNo.18401的植物乳杆菌、保藏编号为CGMCC 1.2158的植物乳杆菌以及保藏编号为CGMCC1.2441的戊糖片球菌的效果优于其他菌种的组合。适宜菌种的选择使组合物能够更好的去除有害物质,并抑制致病菌。
另有研究表明,酵母菌能为乳酸菌的生长繁殖提供氨基酸、维生素、丙酮酸盐等营养因子,促进乳酸菌的生长。酵母菌属于兼性厌氧菌,可消耗水产动物胃肠道内的氧气,促进乳酸菌生长繁殖,并抑制需氧菌的生长,从而促进水产动物肠道微生态平衡。此外,β-葡聚糖和甘露寡糖是酵母菌细胞壁的有效成分。β-葡聚糖能激活巨噬细胞,增强水产动物的非特异性免疫系统功能。本发明组合物中使用的酵母菌为产朊假丝酵母,具体为保藏编号为CGMCCNo.7691的产朊假丝酵母。相对于其他菌种,保藏编号为CGMCCNo.7691的产朊假丝酵母在组合物中能够更好的与乳酸菌配合,产生良好的去除有害物质和致病菌的作用。
本发明将植物乳杆菌、戊糖片球菌和产朊假丝酵母进行组合通过对菌种的筛选、对配比的调整和对制备方法的改进,最终获得了具有良好效果的复合菌剂。该复合菌剂能够稳定水体中的氨氮、溶氧、pH三项指标,并能够抑制致病菌、调节水产动物肠道菌群。
本发明采用的试材皆为普通市售品,皆可于市场购得。
其中涉及的菌株,包括:保藏编号为CGMCC No.18401的植物乳杆菌、保藏编号为CGMCC No.1.2158的植物乳杆菌、保藏编号为CGMCC No.1.2441的戊糖片球菌、保藏编号为CGMCCNo.7691的产朊假丝酵母,皆购自中国普通微生物菌种保藏管理中心(China GeneralMicrobiological Culture Collection Center,CGMCC)。下面结合实施例,进一步阐述本发明:
实施例1
将植物乳杆菌ZR-1(保藏号为CGMCC No.18401),植物乳杆菌ZR-2(保藏号为CGMCC1.2158),戊糖片球菌OP-10(保藏号为CGMCC 1.2441)分别按照1%-2%的接种量接入MRS肉汤培养基中,35±2℃,静置培养24-26h,混合均匀,得乳酸菌菌液;
将假丝酵母CZ(保藏号为CGMCCNo.7691)按照5-10%的接种量接入YPD培养基中,35±2℃,170rpm,培养24-26h,得到假丝酵母菌液。
假丝酵母与乳酸菌菌种混合得共培养菌种;
按照糖蜜5g/L,红糖60g/L,蛋白胨2g/L,磷酸二氢钾0.01g/L,柠檬酸氢二铵0.1g/L,氢氧化钠1.0g/L,制备共培养培养基,接种乳酸菌菌种,接种量5%,温度35℃,转速100-200rpm,pH4.5-6.0,溶解氧25-30mg/L,24-26h检测菌量在28亿cfu/mL,54℃稳定性加速实验,菌量0.3亿cfu/mL。
实施例2
将植物乳杆菌ZR-1(保藏号为CGMCC No.18401)、菌种保藏号为CGMCC 1.2158植物乳杆菌(保藏号为CGMCC 1.2158)、和戊糖片球菌保藏号为CGMCC 1.2441)OP-10。分别按照1%-2%的接种量接入MRS肉汤培养基中,35±2℃,静置培养24-26h,混合均匀,得乳酸菌菌种;
将保藏号为CGMCCNo.7691的假丝酵母菌种按照5%-10%的接种量接入YPD培养基中,35±2℃,170rpm,培养24-26h,得假丝酵母菌种;
将假丝酵母与乳酸菌菌种混合得共培养菌种;
按照糖蜜5g/L,红糖60g/L,蛋白胨2g/L,磷酸二氢钾0.01g/L,柠檬酸氢二铵0.1g/L,氢氧化钠1.0g/L,制备共培养培养基,接种共培养菌种,接种量5%,温度35℃,转速100-200rpm,pH4.5-6.0,溶解氧25-30mg/L,24-26h检测菌量在34亿cfu/mL,54℃稳定性加速实验,菌量1亿cfu/mL。
实施例3
将保藏号为CGMCC No.18401的植物乳杆菌、保藏号为CGMCC 1.2158的植物乳杆菌;菌种保藏号为CGMCC 1.2441的戊糖片球菌,分别按照1%-2%的接种量接入MRS肉汤培养基中,35±2℃,静置培养24-26h,混合均匀,得乳酸菌菌种;
将假丝酵母:菌种保藏号为CGMCCNo.7691按照5-10%的接种量接入YPD培养基中,35±2℃,170rpm,培养24-26h,得假丝酵母菌种;
将,假丝酵母菌种与乳酸菌菌种混合得共培养菌种;
按照糖蜜糖蜜10g/L,红糖90g/L,蛋白胨10g/L,磷酸二氢钾0.40g/L,柠檬酸氢二铵0.3g/L,氢氧化钠1.85g/L,制备共培养培养基,接种共培养菌种,接种量5%,温度35℃,转速100-200rpm,pH4.5-6.0,溶解氧25-30mg/L,,24-26h检测菌量在45亿cfu/mL,54℃稳定性加速实验,菌量3亿cfu/mL。
效果验证
一、以实施例3制备的净水菌剂,进行净水效果实验。
地点:南美白对虾养殖池塘;
虾苗:采购海大幼苗,养殖户自己完成标粗,实验时虾苗800头/500g左右。
养殖密度:每亩水面3-5万尾。
净水菌剂使用方法:
根据水质情况,每5-10天按照3-5L/亩水面的用量使用1次,连续使用。一般在早上或傍晚使用,按照使用量要求量取净水菌剂,均匀泼洒在水面上。
以不施用任何净水药物的池塘为对照组。
表1实验水质指标
注:总弧菌数为TCBS固体培养基上绿弧菌和黄弧菌之和。
表2虾肠道微生物指标检测结果
检测项目 | 对照组 | 实验组 |
肠道总菌量(104cfu/ml) | 650 | 748 |
乳酸菌量(104cfu/ml) | 301 | 489 |
大肠杆菌量(104cfu/ml) | 75 | 69 |
体长(cm) | 11.5 | 13 |
水质检测结果显示,养殖过程中氨氮、溶氧、pH三项指标基本稳定在安全范围内,但是实验组数据整体优于对照组;亚硝酸盐和绿弧菌差异较明显;绿弧菌为较强致病菌,实验结果显示,实验组比对照组降低64.5%;
肠道乳酸菌菌量实验组明显高于对照组,同时大肠杆菌菌量实验组明显低于对照组,说明本申请所述的一种共培养净水菌剂,可促进肠道益生菌定植,抑制有害菌,起到调节肠道功能的作用。
二、以实施例1制备的净水菌剂,进行净水效果实验。
地点:南美白对虾养殖池塘;
虾苗:采购海大幼苗,养殖户自己完成标粗,实验时虾苗800头/500g左右。
养殖密度:每亩水面3-5万尾。
净水菌剂使用方法:
根据水质情况,每5-10天按照3-5L/亩水面的用量使用1次,连续使用。一般在早上或傍晚使用,按照使用量要求量取净水菌剂,均匀泼洒在水面上。以不施用任何净水药物的池塘为对照组。
结果表明,与以实施例3为受试品的虾塘类似,养殖过程中氨氮、溶氧、pH三项指标基本稳定在安全范围内,但实施例1的复合菌剂对致病菌的抑制效果略差,其中,以绿弧菌为例,实验组仅比对照组降低17.3%。
以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (10)
1.复合菌剂,其特征在于,由植物乳杆菌、戊糖片球菌和产朊假丝酵母制得。
2.根据权利要求1所述复合菌剂,其特征在于,
所述植物乳杆菌为保藏编号为CGMCC No.18401和保藏编号为CGMCCNo.CGMCC 1.2441的植物乳杆菌;
所述戊糖片球菌为保藏编号为CGMCC 1.2441的戊糖片球菌;
所述产朊假丝酵母为保藏编号为CGMCCNo.7691的产朊假丝酵母。
3.权利要求1或2所述的复合菌剂的制备方法,其特征在于,包括将所述植物乳杆菌、戊糖片球菌和产朊假丝酵母共培养,获得所述复合菌剂。
4.根据权利要求3所述的制备方法,其特征在于,所述共培养的培养液包括:糖蜜5~10g/L,红糖60~90g/L,蛋白胨2~10g/L,磷酸二氢钾0.01~0.40g/L,柠檬酸氢二铵0.1~0.3g/L,氢氧化钠1.0~1.85g/L和水。
5.根据权利要求4所述的制备方法,其特征在于,所述共培养前还包括活化的步骤,
所述植物乳杆菌和戊糖片球菌的活化培养基为MRS培养基,活化条件包括35±2℃,静置培养24~26h;
所述产朊假丝酵母的活化培养基为YPD培养基,活化条件包括35±2℃,170rpm振荡培养24~26h。
6.根据权利要求4所述的制备方法,其特征在于,所述共培养步骤中,各菌种的接种量皆为2%~8%。
7.根据权利要求4所述的制备方法,其特征在于,所述共培养的条件包括:温度35℃,转速100~200rpm,pH值为4.5~6.0,溶解氧25~30mg/L。
8.根据权利要求4所述的制备方法,其特征在于,所述共培养的时长为24~26h,培养至培养液中菌体密度为28亿cfu/mL以上。
9.一种水产养殖水体的净化方法,其特征在于,包括施用权利要求1或2所述的复合菌剂或施用权利要求3~8任一项所述制备方法制得的复合菌剂。
10.根据权利要求9所述的净化方法,其特征在于,所述施用包括:每5~10天按照3~5L/亩水面的用量使用1次,连续使用。
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