CN111394275B - 一株解淀粉芽孢杆菌及其应用、水产饲料和水产养殖方法 - Google Patents
一株解淀粉芽孢杆菌及其应用、水产饲料和水产养殖方法 Download PDFInfo
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- CN111394275B CN111394275B CN202010109464.1A CN202010109464A CN111394275B CN 111394275 B CN111394275 B CN 111394275B CN 202010109464 A CN202010109464 A CN 202010109464A CN 111394275 B CN111394275 B CN 111394275B
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Abstract
本发明公开了一株解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其特征在于,所述解淀粉芽孢杆菌为解淀粉芽孢杆菌SS1,且保藏于中国典型培养物保藏中心,保藏编号为:CCTCCM2020024,保藏日期为2020年1月7日。本发明中的解淀粉芽孢杆菌安全性高,且具有提高鱼类碳水化合物利用率、促进鱼类生长、改善鱼类血糖稳态、缓解鱼类脂肪异常沉积、增加体蛋白沉积以及提高抗病能力等功能。
Description
技术领域
本发明涉及水产养殖领域,尤其涉及一株解淀粉芽孢杆菌及其应用、水产饲料和水产养殖方法。
背景技术
随着我国水产养殖规模的不断扩大和产量的逐年提高,鱼粉和鱼油资源日益短缺,因此,提高现有饲料原料的利用效率是缓解饲料原料短缺的重要方向。碳水化合物是鱼类重要的代谢供能底物之一,也是水产饲料中较为廉价的能量来源。有研究表明饲料中添加适量淀粉,不仅有利于饲料颗粒成型,而且还有助于节约饲料蛋白质,降低饲料成本以及减少鱼体氮排放。但是与高等动物相比,鱼类对淀粉等碳水化合物的消化利用效率偏低,长时间摄入高碳水化合物水平饲料会导致鱼体出现生长受阻、饲料利用效率较低、脂肪异常沉积、甚至高死亡率等营养性病害问题。因此,提高鱼类对碳水化合物的利用效率可以促进鱼类的生长,节约饲料蛋白,降低养殖成本,提高经济效益。
近年来,已有很多研究表明鱼肠道微生物在促进宿主健康方面发挥着重要作用。肠道微生物不仅影响饲料消化、营养物质吸收和能量供应,还调控宿主正常生理功能及疾病的发生与发展。因此,作为抗生素的替代品,益生菌被广泛应用于畜牧养殖、水产养殖、医药等领域,其能够抑制有害菌的生长,促进免疫***的发育,产生有益代谢产物,为机体提供营养成分和能量,调节代谢紊乱,保护肠道粘膜屏障等。
目前在水产养殖过程中,将益生菌添加至饲料或水体中,以此用来调节机体代谢稳态和提高抗病能力的研究越来越多,其中使用较多的有芽孢杆菌、乳酸菌。其中芽胞杆菌是微生物库中活性产物来源最丰富、最重要的菌种之一,生长周期短,繁殖速度非常快,在生长繁殖过程中产生种类繁多的代谢产物。
但是,目前水产用芽孢杆菌的功效主要集中于提高抗病性,而对其在鱼体代谢调节方面,尤其是在促进鱼体对于碳水化合物利用方面的作用鲜有人报道。
发明内容
本发明的目的是提供一株解淀粉芽孢杆菌及其应用、水产饲料和水产养殖方法,所述解淀粉芽孢杆菌安全性高,且具有提高鱼类碳水化合物利用率、促进鱼类生长、改善鱼类血糖稳态、缓解鱼类脂肪异常沉积、增加体蛋白沉积以及提高抗病能力等功能。
为了实现上述目的,本发明采用了如下技术方案:
提供了一株解淀粉芽孢杆菌(Bacillus amyloliquefaciens),所述解淀粉芽孢杆菌为解淀粉芽孢杆菌SS1,且保藏于中国典型培养物保藏中心,保藏编号为:CCTCC M2020024,保藏日期为2020年1月7日。
提供了一种上述解淀粉芽孢杆菌SS1在制备抑菌制剂中的应用。
提供了一种上述解淀粉芽孢杆菌SS1在促进鱼类生长中的应用。
提供了一种上述解淀粉芽孢杆菌SS1在改善鱼类血糖稳态中的应用。
提供了一种上述解淀粉芽孢杆菌SS1在降低鱼类脂肪沉积中的应用。
提供了一种上述解淀粉芽孢杆菌SS1在促进鱼体蛋白沉积中的应用。
提供了一种上述解淀粉芽孢杆菌SS1在改善鱼类抗病能力中的应用。
提供了一种水产饲料,其包括上述解淀粉芽孢杆菌SS1。
优选的,所述水产饲料中,所述解淀粉芽孢杆菌SS1的浓度为105-107CFU/g。
提供了一种水产养殖方法,其采用上述水产饲料对鱼类进行投喂,每天投喂1-2次,且每次按体重的4%投喂,养殖时间为5-10周。
本发明至少具备以下有益效果:
本发明中的解淀粉芽孢杆菌SS1可添加至鱼类养殖饲料中,以通过解淀粉芽孢杆菌SS1提高鱼类对碳水化合物的利用效率、提高消化酶活来促进生长;进一步的,所述解淀粉芽孢杆菌SS1还可提高鱼类肝脏糖酵解水平,以此改善血糖稳态,通过增加能量消耗缓解鱼类脂肪异常沉积,以及通过激活蛋白合成通路增加体蛋白沉积;此外,所述解淀粉芽孢杆菌SS1还具有抑菌特性,并且可通过保护机体免疫***提高鱼体抗病能力。
附图说明
为了更清楚地说明本发明实施例技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明的解淀粉芽孢杆菌SS1的菌落形态;
图2为本发明的解淀粉芽孢杆菌SS1在淀粉培养基上的生长状况、溶血状况、抑制嗜水气单胞菌状况以及在含0.3%牛胆盐的LB固体培养基上的生长状况;
图3为本发明的解淀粉芽孢杆菌SS1所产淀粉酶活力数据;
图4a为乙酸、丙酸、丁酸的气相色谱分离结果;
图4b为本发明的解淀粉芽孢杆菌SS1体外所产乙酸的气相色谱分离结果;
图5为本发明的解淀粉芽孢杆菌SS1对鱼体均重(a)、增重率(b)以及饵料系数(c)的影响;
图6为本发明的解淀粉芽孢杆菌SS1对鱼体空腹血糖水平(a)、糖耐受能力(b)以及AUC(c)的影响;
图7为本发明的解淀粉芽孢杆菌SS1对鱼体腹腔脂肪系数(Mesenteric fatindex,MFI)(a)、血清甘油三酯(TG)(b)、腹腔脂肪细胞大小(c)以及腹腔脂肪细胞大小评估(d)的影响;
图8为本发明的解淀粉芽孢杆菌SS1对鱼体肝脏脂肪含量(a)、肝脏TG(b)、肝脏脂肪细胞大小(c)以及肝脏脂肪面积评估(d)的影响;
图9为本发明的解淀粉芽孢杆菌SS1对鱼体躯壳比(a)、躯壳蛋白(b)以及mTOR基因表达量(c)的影响;
图10为本发明的解淀粉芽孢杆菌SS1对鱼体存活率(a)以及头肾巨噬细胞氧呼吸爆发活性(b)的影响。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合附图及实施例,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1:
本实施例提供了一株解淀粉芽孢杆菌(Bacillus amyloliquefaciens)SS1(以下为“SS1”),其保藏于中国典型培养物保藏中心,保藏编号为:CCTCC M 2020024,保藏日期为:2020年1月7日。
上述菌株信息如下:
1、菌株来源
该菌株分离自健康罗非鱼(Oreochromis mossambicus)肠道。
2、形态学特征
革兰氏染色阳性,如图1所示,在LB培养基平板上、28℃条件下倒置培养12h的菌落为白色不规则圆形,表面干燥,菌落中央有火山样白色***,类似煎蛋样。
3、产酶特性
1)用牙签蘸取SS1单菌落,点在淀粉培养基上,28℃倒置培养12h后,在培养板上滴加卢戈氏碘液,静置3-5min,观察到SS1能够在淀粉培养基上产生透明圈,结果如图2①所示。由此证明SS1可在体外产生淀粉酶。
2)DNS还原糖法测淀粉酶活性
标准葡萄糖溶液(2mg/mL)制备:准确称取0.2g葡萄糖,溶于蒸馏水中并用容量瓶准确定容至100mL,混匀后放于4℃冰箱中保存备用。
3,5-二硝基水杨酸(DNS)试剂制备:将6.3g DNS和262mL 2mol/L NaOH溶液加入到500mL含有185g酒石酸钾钠的热水溶液中,再加入5g结晶酚和5g Na2SO3,搅拌溶解,冷却后加蒸馏水定容至1L,放入棕色瓶中避光保存一周后备用。
具体操作方法为:
取6支洁净的刻度试管分别编号,用标准葡萄糖溶液(2mg/mL)分别配制浓度为0mg/mL、0.4mg/mL、0.8mg/mL、1.2mg/mL、1.6mg/mL、1mg/mL的葡萄糖溶液,分别取1mL不同浓度的葡萄糖溶液与1.5mL DNS试剂混合,沸水浴加热5min后立即置于冰上,最后用蒸馏水定容至10mL,上下颠倒混匀后于OD 540nm处测定反应混合物的吸光度值,最后以吸光度值(OD540)为横坐标,葡萄糖浓度(mg/mL)为纵坐标作图,绘制葡萄糖标准曲线。
取1mL粗酶液(即SS1菌液)以及1mL培养基(作为对照)分别与底物溶液(1%可溶性淀粉溶液)充分混匀后于45℃水浴中反应30min,设立三个平行,反应结束后立即吸取1mL反应液与1.5mL DNS试剂混合,沸水浴5min后立即置于冰上,最后用蒸馏水定容至10mL,上下颠倒混匀后于OD 540nm处测定反应混合物的吸光度值。
葡萄糖标准曲线为:y=0.1227x-0.0073(R2=0.9938)
淀粉酶活(U/mL)=k×OD×1000×n/(180×30)
其中,k为葡萄糖标准曲线的斜率;OD为在540nm处吸光值;1000为单位换算倍数;n为稀释倍数;180为葡萄糖的分子量;30为反应时间(min)。
结果如图3所示,SS1体外产生的淀粉酶具有较高的淀粉酶活性(约为1.0U/L)。
4、产乙酸特性
标准品制备:将乙酸、丙酸、丁酸用蒸馏水稀释至1000倍的混合标准酸,加入100μL50%硫酸酸化,涡旋振荡30s,加入400μL***,涡旋振荡15s,静置2min萃取,4℃12000rpm/min离心5min;吸取上层有机相至1mL棕色小瓶中。
样品制备:取200μL SS1菌液或相应培养基作为对照,加入100μL 50%硫酸酸化,涡旋振荡30s,加入400μL***,涡旋振荡15s,静置2min萃取,4℃12000rpm/min离心5min;吸取上层有机相至1mL棕色小瓶中。
气相色谱条件:色谱柱初温100℃,保持2min,5℃每分钟升至180℃,保持2min,控制色谱柱流量为1mL/min;进样口温度为220℃,不分流,进样1μL;检测器温度为200℃,空气200mL/min,氢气32mL/min,氮气24mL/min。
如图4a所示,3种短链脂肪酸都可以被气相色谱柱有效的分离,其中乙酸保留时间为7.019min,丙酸保留时间为8.622min,丁酸保留时间为10.422min;此外,如图4b所示,SS1在7.020min出现较为明显的峰,由此即说明SS1可以在体外发酵淀粉产生乙酸,而乙酸除了能够为机体提供能量,并且对降低血糖水平和脂肪沉积有重要作用。
5、致病性
取上述SS1冻存菌和分离得到的嗜水气单胞菌Aeromonas hydrophila CS4(A.hydCS4),分别按1:1000接种至LB液体培养基中复苏,28℃、220rpm/min条件下摇菌12h,用接种环蘸取上述嗜水气单胞菌菌液,在LB培养基平板上划线,28℃倒置培养12h,用牙签蘸取SS1单菌落,点在血平板上,28℃倒置培养12h。
结果如图2②所示,SS1(即图2②中的“1”)在血平板上无溶血现象,而致病菌A.hydCS4(即图2②中的“2”和“3”)在血平板上有溶血圈的产生,由此说明SS1无明显致病性,安全性较高。
6、体外抑菌特性
将上述SS1菌液用PBS缓冲液调整至浓度为1×106CFU/mL,将上述嗜水气单胞菌液以12000rpm/min离心,收集沉淀用PBS缓冲液重悬,调整浓度至1×109CFU/mL,取100μL涂布到LB培养基上,待培养基表面干燥,用打孔器在培养基上打孔,用无菌针头挑出多余的琼脂,在孔内加入SS1菌液20μL,于28℃培养箱中正置培养12h。
从图2③可以观察到SS1可以抑制嗜水气单胞菌的生长,产生抑菌透明圈,说明该菌具有一定的抑菌作用。
7、抗逆特性
配制含0.3%牛胆盐的LB固体培养基,用无菌牙签蘸取SS1在LB固体培养基上的单菌落,点在含0.3%牛胆盐的LB固体培养基上,28℃倒置培养12h。
从图2④可以观察到SS1能够在含0.3%牛胆盐的LB固体培养基生长,产生耐胆盐透明圈,说明该菌具有较好的耐胆盐能力。
实施例2:
本实施例提供了实施例1中所述解淀粉芽孢杆菌SS1(以下为“SS1”)的分离方法,其包括如下步骤:
S1、按照胰蛋白胨5g/L,酵母提取物2g/L,KH2PO42 g/L,MgSO4·7H2O 2g/L,NaCl5g/L,可溶性淀粉10g/L,琼脂糖20g/L的配比配制淀粉培养基,高压灭菌后于超净台内倒板,以获得淀粉培养基固体平板;
S2、取鱼类(如罗非鱼)肠道内容物与PBS缓冲液按1:9(w/v,g/ml)的比例混匀,1500rpm/min条件下离心,取上清液100μL涂布到所述淀粉培养基固体平板上;
S3、于28℃条件下倒置培养24h后根据菌落的大小、形状、颜色筛选菌落,且对筛选出的菌落划斜面进行保存并编号,同时对筛选出的菌株进行16S rRNA分子鉴定,以获得所述解淀粉芽孢杆菌SS1。
实施例3:
本实施例提供了一种采用实施例1中所述的解淀粉芽孢杆菌SS1(以下为“SS1”)进行水产养殖的方法,其具体包括如下步骤:
S1、鱼苗暂养:购买鱼苗(如罗非鱼鱼苗)600尾,用商业饲料暂养2周后投入28℃曝气水中暂养,每两天换一次水,待鱼苗适应生长环境,确定无病害后结束暂养;鱼苗分为五组,正常组(CON)、高淀粉组(HCD)、益生菌组1(HCB1)、益生菌组2(HCB3)、益生菌组3(HCB3),每组三个平行,每个平行30尾,均重1.6±0.1g;
S2、SS1菌液制备:取上述解淀粉芽孢杆菌SS1按1:1000(w/v,g/ml)接种至LB液体培养基中进行复苏,28℃、220rpm/min条件下摇菌12h,得复苏菌液;取复苏菌液按1:100(v/v,ml/ml)接种至LB液体培养基中进行扩大培养,28℃、220rpm/min条件下摇菌24h,4℃、12000rpm/min条件下离心20min,收集菌体;用PBS缓冲液重悬,以获得重悬菌液;
S3、饲料制备:将所述重悬菌液与高淀粉饲料(按重量比计,含45%淀粉)按1:2(v/w,ml/g)混合均匀,使饲料中解淀粉芽孢杆菌SS1的浓度分别为105CFU/g、106CFU/g、107CFU/g,以获得三种不同菌种浓度的益生菌饲料(也即水产饲料),再将所述益生菌饲料切粒;
S4、投喂:采用市售商业饲料、高淀粉饲料(按重量比计,含45%淀粉)以及三种不同菌种浓度的益生菌饲料对应投喂上述正常组(CON)、高淀粉组(HCD)、益生菌组1(HCB1)(投喂浓度105CFU/g益生菌饲料)、益生菌组2(HCB3)(投喂浓度106CFU/g益生菌饲料)、益生菌组3(HCB3)(投喂浓度107CFU/g益生菌饲料)鱼苗,每天投喂1-2次,且每次按体重的4%投喂;养殖5-10周(优选为8周)后饥饿24h,每组取10尾鱼进行称重、采血和组织样品采集。
经检测,市售商业饲料、高淀粉饲料(按重量比计,含45%淀粉)以及所述益生菌饲料原料配方如表1所示。
表1饲料原料配方表(g)
其中:1、复维(mg or IU/kg):维生素A:500,000I.U.(国际单位);维生素D3:50,000I.U.;维生素E:2500mg;维生素K3:1000mg;维生素B1:5000mg;维生素B2:5000mg;维生素B6:5000mg;维生B125000μg;肌醇:25,000mg;叶酸:1000mg;泛酸:10,000mg;生物素:250mg;胆碱:100,000mg;烟酸:25,000mg;维生素C:10,000mg。
2、复矿(g/kg):碳酸钙:314.0g;磷酸二氢钾:469.3g;七水硫酸镁:147.4g;氯化钠:49.8g;葡萄糖酸亚铁:10.9g;一水硫酸锰3.12g;钼酸铵:0.06g;七水硫酸锌:4.67g;无水硫酸铜:0.62g;六水氯化钴:0.08g;***钠:30.02g。
所述解淀粉芽孢杆菌SS1功能:
(1)SS1促进鱼体生长(包括增重)的作用
养殖结束后,分别称各组鱼体总重,计算每组均重、增重率以及饵料系数。
其中,均重(g/尾)=总重(g)/尾数,增重率(%)=(末重-初重)×100/初重。
如图5(a)所示,与CON组相比,HCD组均重显著增加,同时HCB组均重相对HCD组也显著增加,同时如图5(b)所示,CON组与HCD组增重率无显著差异,但HCB组增重率相对HCD组显著增加,由此说明本发明中的解淀粉芽孢杆菌SS1能显著、快速促进鱼体体重增长。进一步的,如图5(c)所示,HCB组饵料系数相对CON组、HCD组均有所降低,尤其相对HCD组降低明显,
如上所述,解淀粉芽孢杆菌SS1具有较高的淀粉酶活性,可以提高碳水化合物的利用率,由此,可通过在水产饲料中运用本发明中的解淀粉芽孢杆菌SS1来提高消化酶活性,如淀粉酶的活性,促进鱼体生长,同时可减少成本,增加经济收益。
注:图5中“*”代表组之间的差异,且*:P<0.05,**:P<0.01,***:P<0.001,且(a)-(c)中HCB组数据均为HCB1、HCB2以及HCB3三组数据的平均值。
(2)解淀粉芽孢杆菌SS1对改善鱼体血糖稳态的作用
1)采集血液,收集血清,用葡萄糖试剂盒测定空腹血糖浓度。
2)糖耐受试验
每组取5尾鱼,配制500mg/mL的葡萄糖溶液,每尾腹腔注射葡萄糖浓度为200mg/kg,于0h、0.5h、1.5h和3h时分别采血,收集血清,用葡萄糖试剂盒测定血糖浓度并计算曲线下面积(AUC),结果如图6所示。
从图6(a)中可以看出,与CON组相比,HCD组空腹血糖水平显著增加,但与HCD组相比,HCB组空腹血糖显著降低,说明通过添加解淀粉芽孢杆菌SS1可显著降低鱼体血糖水平,同时图6(b)中的糖耐受实验结果以及图6(c)中的AUC证明,HCB组鱼体具有较强的糖耐受能力。
因此,通过运用解淀粉芽孢杆菌SS1可提高鱼类肝脏糖酵解水平,且通过产生乙酸降低鱼体内的血糖水平,从而当鱼体长时间摄入高碳水化合物水平饲料时,有助于稳定鱼体内的血糖水平,提高糖耐受能力,改善鱼体血糖稳态。
注:图6中“*”代表组之间的差异,且*:P<0.05,**:P<0.01,***:P<0.001;图6(b)中“#”代表HCB组与HCD组之间的差异,且#:P<0.05,##:P<0.01,###:P<0.001,且(a)-(c)中HCB组数据均为HCB1、HCB2以及HCB3三组数据的平均值。
(3)解淀粉芽孢杆菌SS1对降低鱼体肝脏和腹腔脂肪沉积的作用
1)取不同组鱼相同部位和重量的脂肪组织置于波恩氏固定液中,用于制作HE切片,收集腹腔脂肪组织并称重;取相同部位和重量的肝脏组织置于波恩氏固定液中,用于制作HE切片,收集肝脏组织并称重。
2)HE切片
常规石蜡切片制作方法及HE染色。
3)肝脏脂肪含量检测和TG检测
氯仿甲醇法测定肝脏脂肪含量:3个处理组,每组6个平行,需两套共36个10ml的干净的带刻度玻璃试管,标记后称重去皮,每个管中加0.5g左右肝脏样品,在通风厨中加入6ml的氯仿甲醇(氯仿:甲醇=2:1),拧紧管盖,4℃静置过夜。取出样品在旋涡振荡器上震荡30s,放入4℃冰箱里静置1h,然后在通风厨中加入0.37mol/L的氯化钾,再次震荡后在低速离心机里以1500rpm/min离心10min,用巴斯德吸管将下层透明液体吸出,经漏斗过滤后转移至新的已称重并标记过的玻璃管中,并用三氯甲烷冲洗滤纸,将试管放入真空干燥箱中,待三氯甲烷挥发完后,取出玻璃管放入干燥器冷却后称管重,减去之前记录的管重,即为脂肪重,计算肝脏脂肪含量。
血清和肝脏TG检测按照南京建成试剂盒说明书进行操作。
从图7中可以看出,CON组与HCD组腹腔脂肪组织指数(Mesenteric fat index,MFI)无显著差异,但与HCD组相比,HCB组MFI显著降低;且与CON组相比,HCD组血清TG显著升高,但与HCD组相比,HCB组血清TG显著降低;鱼体腹腔脂肪组织HE切片以及腹腔脂肪细胞大小评估(Average area value of adipocyte)结果说明,与CON组相比,HCD组腹腔脂肪细胞大小显著增加,但相对于HCD组,HCB组脂肪细胞大小显著减小。
进一步的,从图8中可以看出,与CON组相比,HCD组肝脏脂肪含量、肝脏TG含量均显著增加,但与HCD组相比,HCB组肝脏脂肪、肝脏TG含量均显著降低;此外鱼体肝脏HE切片以及肝脏脂肪面积评估(Lipid droplet area value of liver)结果说明,与HCD组相比,HCB组脂肪细胞大小与肝脏脂肪面积均显著减小。
注:图7-8中“*”代表组之间的差异,且*:P<0.05,**:P<0.01,***:P<0.001,(a)、(b)及(d)中HCB组数据为HCB1、HCB2以及HCB3三组数据的平均值;图7(c)中HCB组HE切片为HCB3组(SS1浓度为107CFU/g)鱼的腹腔脂肪组织切片,图8(c)中HCB组HE切片为HCB3组(SS1浓度为107CFU/g)罗非鱼的肝脏切片。
由此,通过添加解淀粉芽孢杆菌SS1可通过促进鱼体对于碳水化合物(如淀粉)的利用能力,增加能量消耗来显著降低鱼类肝脏和腹腔脂肪的TG含量,以避免鱼类肝脏和腹腔脂肪异常沉积。
(4)解淀粉芽孢杆菌SS1对鱼体蛋白沉积的作用
将各组鱼体去除头、尾和内脏的部分称重并记录,计算躯壳比及躯壳蛋白含量,采用凯氏定氮法测定蛋白含量以及采用实时荧光定量法检测蛋白合成标记基因mTOR的表达量,结果如图9所示。
结果表明,CON组与HCD组躯壳比无显著差异,但HCB组躯壳比增加明显,说明蛋白含量显著增加;同时躯壳蛋白含量测定以及mTOR表达量结果发现,与HCD组相比,HCB组躯壳蛋白含量、mTOR表达量均有显著增加。
注:图9中“*”代表组之间的差异,且*:P<0.05,**:P<0.01,***:P<0.001,且(a)-(c)中HCB组数据均为HCB1、HCB2以及HCB3三组数据的平均值。
由此,通过添加解淀粉芽孢杆菌SS1可通过增加蛋白合成相关基因,如mTOR的表达量来激活蛋白合成通路,进一步提高蛋白合成总量,增加体蛋白沉积。
(5)解淀粉芽孢杆菌SS1对改善鱼体抗病能力的作用
1)攻毒试验
取嗜水气单胞菌Aeromonas hydrophila CS4(A.hyd CS4)接种至LB液体培养基中复苏,28℃、220rpm/min条件下摇菌12h,取复苏菌液按1:100接种至LB液体培养基中进行扩大培养,28℃、220rpm/min条件下摇菌24h,4℃、12000rpm/min条件下离心20min,收集菌体,用PBS缓冲液重悬,调整浓度为109CFU/mL。每组取21尾鱼(如罗非鱼)进行攻毒实验,每尾鱼腹腔注射106CFU/g,观察并记录7天的死亡情况,计算存活率。
2)呼吸爆发试验
每组取5尾鱼,使用MS-222麻醉后,分离鱼体头肾巨噬细胞,使用Percoll密度梯度法分离头肾巨噬细胞,并使用台盼蓝进行细胞活性检测计数,以确保活细胞的数量大于90%,调整细胞浓度为1×107cell/mL。头肾巨噬细胞氧呼吸爆发活性的测定采用NBT法,在540nm下测定吸光值。结果如图10所示。
结果表明,HCD组存活率最低,通过添加SS1,HCB组存活率相对HCD组显著增加,7d后可基本恢复至与CON组相同;头肾巨噬细胞氧呼吸爆发结果显示,CON组与HCD组呼吸爆发活性无显著差异,与HCD组相比,HCB组呼吸爆发活性显著增加。
注:图10中“*”代表组之间的差异,且*:P<0.05,**:P<0.01,***:P<0.001,且(a)-(b)中HCB组数据均为HCB1、HCB2以及HCB3三组数据的平均值。
由此说明运用解淀粉芽孢杆菌SS1可有效提高鱼机体免疫细胞的活性,促进鱼类抗病毒、病菌(如嗜水气单胞菌)感染能力,增强鱼体综合免疫力及抗病能力,进一步提高存活率。
综上所述,本发明分离出的解淀粉芽孢杆菌SS1可作为安全性功能饲料添加剂添加至鱼类高淀粉饲料中,以通过解淀粉芽孢杆菌SS1提高鱼类对碳水化合物的利用效率、提高消化酶类酶活来促进生长;进一步的,所述解淀粉芽孢杆菌SS1还可提高鱼类肝脏糖酵解水平,以此改善血糖稳态,通过增加能量消耗缓解鱼类脂肪异常沉积,以及通过激活蛋白合成通路增加体蛋白沉积;此外,所述解淀粉芽孢杆菌SS1还具有抑菌特性,并且可通过保护机体免疫***提高鱼体抗病能力。
上述实施例1-3中的技术特征可进行任意组合,且组合而成的技术方案均属于本申请的保护范围。以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是本发明的原理,在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明的范围内。本发明要求的保护范围由所附的权利要求书及其等同物界定。
Claims (6)
1.一株解淀粉芽孢杆菌(Bacillus amyloliquefaciens),其特征在于,所述解淀粉芽孢杆菌为解淀粉芽孢杆菌SS1,且保藏于中国典型培养物保藏中心,保藏编号为:CCTCC M2020024,保藏日期为2020年1月7日。
2.权利要求1中的解淀粉芽孢杆菌SS1在制备抑菌制剂中的应用。
3.权利要求1中的解淀粉芽孢杆菌SS1在促进鱼类生长中的应用。
4.权利要求1中的解淀粉芽孢杆菌SS1在促进鱼体蛋白沉积中的应用。
5.一种水产饲料,其特征在于,包括权利要求1中的解淀粉芽孢杆菌SS1。
6.如权利要求5所述的水产饲料,其特征在于,所述水产饲料中,所述解淀粉芽孢杆菌SS1的浓度为105-107CFU/g。
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