CN114522156B - 一种牛磺酸的应用 - Google Patents
一种牛磺酸的应用 Download PDFInfo
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- CN114522156B CN114522156B CN202210094520.8A CN202210094520A CN114522156B CN 114522156 B CN114522156 B CN 114522156B CN 202210094520 A CN202210094520 A CN 202210094520A CN 114522156 B CN114522156 B CN 114522156B
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Abstract
本发明涉及生物技术领域,具体涉及一种牛磺酸的应用,牛磺酸作为制备增加甲壳动物抗副溶血弧菌能力药物的应用,以天然代谢产物增加甲壳动物的抗副溶血弧菌能力,避免甲壳动物因AHPND引起大面积死亡,且无毒无副作用,为甲壳动物种苗饲料提供对抗病原刺激的绿色高效环保的饲料添加剂。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种牛磺酸的应用。
背景技术
甲壳类动物是海洋生态系统的重要组成部分。其中,虾蟹构成了供人类食用的水产养殖业的很大比例。在过去的十年里,虾蟹养殖业迅速发展,带来了巨大的经济效益。然而,随之而来的虾蟹病害问题严重制约着该产业的健康、可持续发展。特别是急性肝胰腺坏死病(Acute Hepatopancreatic necrosis Disease,AHPND)是近年来严重危害对虾养殖的一种细菌性疾病。目前研究证实,AHPND是由携带pVA1质粒(表达pirA和pirB毒素蛋白)的副溶血弧菌(Vibrio parahaemolyticus)感染引起的,它已导致亚洲许多国家的养殖对虾大面积死亡,每年因其导致的损失高达10亿美元。甲壳类作为无脊椎动物,缺乏适应性免疫系统,主要利用先天免疫系统进行自我防御和保护。免疫系统从营养物质中获取能量和代谢物,为此,通过代谢组学方法筛选和鉴定具有抗AHPND活性的代谢物,可为绿色高效环保水产种苗饲料的开发提供候选靶位和科学依据。
牛磺酸,作为具有抗氧化能力的氨基酸,以高浓度的形式存在于甲壳动物组织中,特别是肝胰腺、心脏、肌肉、大脑、肠、血浆和血细胞中。因此,其功能包括抗氧化、解毒、调节免疫反应、钙转运、心肌收缩、胆汁酸代谢、渗透调节等。
在现有技术中,甲壳动物饲料未见牛磺酸添加的报道,鱼饲料的牛磺酸主要用作蛋白质来源,可以提高蛋白质的利用率,也可以缓解热应激,但在对抗病原刺激方面尚无研究报道。
发明内容
本发明的目的在于,解决甲壳动物抗副溶血弧菌能力弱,容易因为AHPND引起大面积死亡的问题。
为了解决上述技术问题,本发明提供了一种可在养殖现场通过典型的AHPND临床体征和组织病理特征确认AHPND疾病的方法。
本发明还提供了一种通过GC-MS测定对虾肝胰腺代谢谱的方法,用于对虾疾病鉴定的应用。
本发明还提供了一种代谢物,牛磺酸,外源牛磺酸能调节凡纳对虾的代谢,提高对虾在AHPND致病原——带有pVA1质粒副溶血弧菌(Vibrio parahaemolyticus)刺激下的存活率。添加剂量为200~750μg/6g。
采用如下技术方案:
一种牛磺酸的应用,牛磺酸在制备增加甲壳动物抗副溶血弧菌能力药物中的应用。
优选的,所述甲壳动物为虾。
优选的,所述牛磺酸的添加量为90~750μg/6g。
优选的,所述牛磺酸的添加量为200~750μg/6g。
一种牛磺酸的应用,牛磺酸作为甲壳动物的饲料添加剂。
一种含牛磺酸的添加剂功能包,包括如下质量分数的组分:10~50%牛磺酸,20~40%维生素E,20~55%维生素C,5%~50%填充剂。
填充剂包括糊精、玉米粉、小麦粉、稻壳粉中一种或多种。
优选的,所述添加剂功能包在基础饲料中的添加量为1~25kg/吨。
牛磺酸可作为甲壳动物的饲料添加剂,提高对虾在带有pVA1质粒副溶血弧菌刺激下的存活率。但如果饲料中添加量过大,则会影响对虾对饲料的摄食,导致在病原菌刺激下,牛磺酸对凡纳对虾存活率的影响不显著。实验表明,以添加剂功能包中的牛磺酸添加量计算,牛磺酸的最佳添加浓度为0.01~2%,可以抑制AHPND,显著提高对虾的存活率,从4%提高至27.5%~37.8%。
一种牛磺酸抑制细菌生长的方法,包括如下步骤:采用细菌菌液与牛磺酸溶液按体积比1:1混合,之后恒温培育。
优选的,包括如下步骤:采用所述细菌菌液与所述牛磺酸溶液按体积比1:1混合,37℃培养箱孵育2小时;取30μL上述孵育后的混合液涂布TSB或LB平板,每组3个生物学重复,每个生物学重复有3个平行;将涂布后的平板在37℃培养箱培养12小时后,进行拍照并记录平板菌落数。
以溶液混合的形式先孵育2小时,此时没有培养基,给牛磺酸与细菌充足的时间和空间接触。之后再涂布培养12小时,细菌可以在培养基上生长,验证牛磺酸的抑制细菌效果。
优选的,所述细菌包括副溶血性弧菌、哈维氏弧菌、美人鱼发光杆菌、溶藻酸弧菌、大肠杆菌、海豚链球菌、金黄色葡萄球菌中的一种或多种。
优选的,所述牛磺酸溶液的浓度为2500μg/100μl。
与现有技术相比较,实施本发明,具有如下有益效果:
以天然代谢产物增加甲壳动物的抗副溶血弧菌能力,避免甲壳动物因AHPND引起大面积死亡,且无毒无副作用,为甲壳动物种苗饲料提供对抗病原刺激的绿色高效环保的饲料添加剂。
附图说明
图1A为在养殖场里采集凡纳对虾的图片(Ⅰ)作为阴性对照组的健康对虾,可见健康肝胰腺和健康的肠道(Ⅳ);(Ⅱ)AHPND存活组对虾,可见肝胰腺苍白(箭头标记)和空肠(菱形)等症状(Ⅴ);(Ⅲ)AHPND死亡组对虾,可见肝胰腺苍白(箭头标记)和空肠(菱形)等症状(Ⅵ)。图1B为苏木精伊红染色后对虾肝胰腺切片图,比例尺:100μm。(Ⅰ,Ⅱ)健康组对虾,结构正常;(Ⅲ,Ⅳ)AHPND存活组对虾,肝小管上皮细胞(三角形标记)脱落;(Ⅴ,Ⅵ)AHPND死亡组对虾,未见正常肝小管。
图2为健康组、AHPND存活组和AHPND死亡组肝胰腺样本聚类分析热图。
图3为基于GC-MS检测的总代谢物类别图。
图4为对健康组、AHPND存活组和AHPND死亡组PCA分析图。
图5为健康组与AHPND存活组相关S-plot分析图。
图6为健康组与AHPND死亡组相关S-plot分析图。
图7为AHPND存活组与AHPND死亡组相关S-plot分析图。
图8为健康组、AHPND存活组、AHPND死亡组差异代谢物途径分析和统计富集分析图。
图9A为基于GC-MS分析的牛磺酸表达量比较分析;B.为基于ELISA分析的牛磺酸表达量比较分析。*表示存在显著性差异(p<0.05),**表示存在极显著差异(p<0.01)。
图10为在带有pVA1质粒的副溶血弧菌刺激下,添加不同剂量的牛磺酸对对虾存活率的影响。
图11为不同浓度的牛磺酸对带有pVA1质粒的副溶血弧菌的抑菌率分析。
图12为不同浓度的牛磺酸对哈维氏弧菌的抑菌率分析。
图13为不同浓度的牛磺酸对美人鱼发光杆菌的抑菌率分析。
图14为不同浓度的牛磺酸对溶藻酸弧菌的抑菌率分析。
图15为不同浓度的牛磺酸对大肠杆菌的抑菌率分析。
图16为不同浓度的牛磺酸对海豚链球菌的抑菌率分析。
图17为不同浓度的牛磺酸对金黄色葡萄球菌的抑菌率分析。
图18为牛磺酸对带有pVA1质粒的副溶血弧菌的最佳抑菌浓度分析。
图19为牛磺酸对哈维氏弧菌的最佳抑菌浓度分析。
图20为牛磺酸对美人鱼发光杆菌的最佳抑菌浓度分析。
图21为牛磺酸对溶藻酸弧菌的最佳抑菌浓度分析。
图22为牛磺酸对大肠杆菌的最佳抑菌浓度分析。
图23为牛磺酸对海豚链球菌的最佳抑菌浓度分析。
图24为牛磺酸对金黄色葡萄球菌的最佳抑菌浓度分析。
图25为实施例7牛磺酸功能包对虾的存活率影响分析图。
图26为实施例8牛磺酸功能包对虾的存活率影响分析图。
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,下面将结合附图对本发明作进一步地详细描述。
实施例1
急性肝胰腺坏死病(AHPND)的病虾采集与临床体征、组织病理分析
首先,从水产养殖场分别采集三组对虾(均重约6g),如图1A所示,(Ⅰ)为无AHPND症状的健康对虾(健康组)、(Ⅱ)为AHPND发病后的存活对虾(AHPND存活组)和(Ⅲ)为AHPND发病后的死亡对虾(AHPND死亡组);
继而,分别采用临床体征观察和组织病理分析等方法对上述三组对虾进行AHPND相关病理特征分析。临床体征观察发现,AHPND发病虾(包括AHPND存活组和死亡组)的肝胰腺可见明显的空胃、空肠以及肝胰脏苍白、萎缩等症状,而健康组对虾没有类似症状(图1A);随后,肝胰腺病理切片分析发现,健康组对虾的肝小管和上皮细胞结构正常(图1B-Ⅰ、Ⅱ),而AHPND存活组对虾肝小管上皮细胞脱落,同时在坏死的肝小管周围可见血细胞被包裹的现象(图1BⅢ、Ⅳ);此外,在AHPND死亡组中,发现所有对虾肝小管都已损伤,出现大面积的血细胞浸润现象(图1BⅤ、Ⅵ)。
实施例2
对虾肝胰腺代谢组谱分析
收集上述三组对虾的肝胰腺,采用GC-MS技术进行代谢组学分析;将上述三组对虾按每组13个生物重复和2个技术重复进行代谢组学分析。结果显示,对虾肝胰腺样品中存在308个代谢物图谱,其中健康组、AHPND存活组和AHPND死亡组分别存在108个、107和106个NIST库检索代谢物(图2)。随后,根据KEGG分析代谢产物的生物学作用(图3),其中占比最多的是氨基酸及其衍生物(29.6%),占比最少的是脂类化合物(3.7%)。上述研究结果提示,对虾感染带有pVA1质粒的副溶血弧菌后,AHPND存活组与死亡组对虾具有不同的代谢特征。
实施例3
与抗AHPND潜在代谢物的筛选
首先,通过对所有样品进行主成分分析(PCA)和偏最小二乘判别分析(PLS-DA),评估了GC-MS方法的稳定性和重现性。PCA结果(图4)表明三组之间可以很好地聚类,表明组内差异较小,说明样本稳定性较好。同时也说明三组的样本之间差异显著,提示AHPND可影响对虾的代谢物。
继而,通过创建OPLS-DA和S-plot模型来比较导致每个样本组分离的代谢物特征。基于三个S-plot,抗AHPND的关键代谢物出现在三个图中(图5-7)。这些结果表明,当AHPND发病时,代谢产物的变化与对虾是否能存活密切相关。
随后,分别将两组之间的差异代谢物通过通路分析和通路富集统计(图8),共富集到17条代谢通路。其中氨基酸代谢通路占比最大,如酪氨酸代谢,色氨酸代谢,苯丙氨酸、酪氨酸和色氨酸代谢,苯丙氨酸代谢,甘氨酸、丝氨酸和苏氨酸代谢,半胱氨酸和蛋氨酸代谢,精氨酸和脯氨酸代谢,丙氨酸、天冬氨酸和谷氨酸代谢。结果提示,氨基酸代谢物(图8)可能参与对虾对AHPND的免疫防御。
实施例4
与抗AHPND潜在代谢物的筛选验证
为了分析上述代谢组学方法所筛选的氨基酸代谢物在AHPND存活组和AHPND死亡组中是否存在显著性差异,我们进一步采用ELISA技术对其进行验证。结果如图9B所示,牛磺酸在肝胰腺中的结果与代谢组学结果(图9A)一致,即在AHPND存活组的丰度比AHPND死亡组的丰度显著增多。
实施例5
对虾体内牛磺酸抗AHPND的作用研究
为了探究牛磺酸是否具有抗AHPND的功能。将健康的凡纳对虾(均重约6g)随机分为六组,第一组和第二组是对照组(肌肉注射生理盐水),试验组三、试验组四和试验组五分别每条虾肌肉注射90~160μg、200~310μg、350~750μg牛磺酸,每组30条。24小时后对第二、三、四、五组肌肉注射带有pVA1质粒的副溶血性弧菌5×105CFU/虾,而对第一组注射生理盐水,并感染后10天内监测对虾的累计死亡情况。结果如图10所示,没有注射牛磺酸的对照组经过带有pVA1质粒的副溶血弧菌攻击后,对虾在9.5天存活率为0%;而注射了牛磺酸的试验组三、试验组四和试验五的存活率分别为28.6%,52.4%和41.7%。提示外源牛磺酸能调节凡纳对虾的代谢,提高对虾在带有pVA1质粒副溶血弧菌刺激下的存活率。
实施例6
体外牛磺酸的抗菌作用研究
为了探究牛磺酸在体外是否具有抗菌的功能,取60μL(A)带有pVA1质粒的副溶血性弧菌(2×104CFU/mL)、(B)哈维氏弧菌(2×104CFU/mL)、(C)美人鱼发光杆菌(5×103CFU/mL)、(D)溶藻酸弧菌(5×103CFU/mL)、(E)大肠杆菌(3×104CFU/mL)、(F)海豚链球菌(5×103CFU/mL)、(G)金黄色葡萄球菌(5×103CFU/mL)的悬液,与等体积的不同浓度(100μg/100μL,500μg/100μl,2500μg/100μl)的牛磺酸混合,37℃培养箱孵育2h;取30μL上述孵育后的混合液涂布TSB或LB平板,每组3个生物学重复,每个生物学重复有3个平行。37℃培养箱培养12h后,拍照并记录平板菌落数。细菌抑制率=(空白对照菌落数-实验组菌落数)/空白对照菌落数×100%。结果如图11-图24所示,一定浓度的牛磺酸能够抑细菌的生长,当浓度为2500μg/100μl时,对(图11)带有pVA1质粒副溶血性弧菌(2×104CFU/mL)、(图12)哈维氏弧菌(2×104CFU/mL)、(图13)美人鱼发光杆菌(5×103CFU/mL)、(图14)溶藻酸弧菌(5×103CFU/mL)、(图15)大肠杆菌(3×104CFU/mL)、(图16)海豚链球菌(5×103CFU/mL)、(图17)金黄色葡萄球菌(5×103CFU/mL)等的抑制率分别为100%、99.87%、-96.03%、-6.84%、17.80%、89.96、30.81%。但是当牛磺酸的浓度为100μg/100μl时,不但不能抑制(图12)哈维氏弧菌(2×104CFU/mL)、(图13)美人鱼发光杆菌(5×103CFU/mL)、(图14)溶藻酸弧菌(5×103CFU/mL)、(图15)大肠杆菌(3×104CFU/mL)、(图16)海豚链球菌(5×103CFU/mL)、(图17)金黄色葡萄球菌(5×103CFU/mL)的生长,反而促进致病菌的生长。
实施例7
为了解决AHPND对对虾养殖的危害,增加虾的抗菌功能,现设计出一种含牛磺酸的功能包(牛磺酸:10~50%,维生素E:20~40%,维生素C:20~55%,糊精或玉米粉或小麦粉或稻壳粉:5%~50%)。
以牛磺酸最低剂量的功能包(牛磺酸:10%,维生素E:20%,维生素C:20%,糊精:50%)为例,检测其对凡纳对虾存活率的影响。首先将健康的凡纳对虾(均重约6g)随机分为五组,第一组为对照组(饲喂不含功能包的饲料),第二组、第三组、第四组和第五组为实验组,进行5倍梯度实验,即每吨全价料分别含有1公斤、5公斤、25公斤和125公斤的功能包,每组50条虾。然后分别饲喂72小时饲料后,使用带有pVA1质粒的副溶血弧菌(5×105/虾)刺激48小时,记录对虾的存活数。结果如图25所示,在饲喂72小时饲料后,注射病原菌之前,与对照组(存活率为74.0%)相比,试验组1、实验组2和实验组3的存活率分别提升至80.0%、90.0%和80.0%。同时,每吨全价料中添加1~25公斤的功能包后,对虾在带有pVA1质粒的副溶血弧菌刺激48小时后,与对照组(存活率为5.4%)相比,试验组1~3的存活率显著增加,分别为35.0%(p=0.004)、37.8%(p<0.001)和27.5%(p=0.037)。由此可见,从存活率的结果上看,试验组2,即添加量为5公斤是最优的选择,但试验组1与试验组2的结果差异并不显著,因此从性价比角度分析,可以选择添加量更少且同样有效的试验组1的配方。但如果在饲料中添加125公斤的功能包后,对虾在采食48小时后,对虾的采食量骤减。在饲喂72小时饲料后,注射病原菌之前,与对照组(存活率为74.0%)相比,试验组4的存活率反而降至60.0%,且在带有pVA1质粒的副溶血弧菌刺激48小时后,试验组4(存活率为20.0%)与对照组的存活率差异不显著(p=0.931)。说明此饲料中功能包的添加量过大,会影响对虾对饲料的摄食,导致在病原菌刺激下,虽然最终仍对凡纳对虾存活率有帮助,但影响不显著。以上结果表明,该功能包添加量在1~25公斤可以显著提高虾在带有pVA1质粒的副溶血弧菌刺激下的存活率。
实施例8
为了解决AHPND对对虾养殖的危害,增加虾的抗菌功能,现设计出一种含牛磺酸的功能包(牛磺酸:10~50%,维生素E:20~40%,维生素C:20~55%,糊精或玉米粉或小麦粉或稻壳粉:5%~50%)。
以牛磺酸最高剂量的功能包(牛磺酸:50%,维生素E:25%,维生素C:25%)为例,检测其对凡纳对虾存活率的影响。首先将健康的凡纳对虾(均重约6g)随机分为五组,第一组为对照组(饲喂不含功能包的饲料),第二至五组为实验组,进行5倍梯度实验,即分别每吨全价料含有1公斤、5公斤、25公斤和125公斤的功能包,每组50条虾。然后分别饲喂72小时饲料后,使用带有pVA1质粒的副溶血弧菌(5×105/虾)刺激48小时内,记录对虾的存活数,结果如图26所示,在饲喂72小时饲料后,注射病原菌之前,与对照组(存活率为80.0%)相比,试验组1、实验组2和实验组3的存活率分别提升至84.0%、88.0%和82.0%。同时,每吨全价料中添加1~25公斤的功能包后,对虾在带有pVA1质粒的副溶血弧菌刺激48小时后,与对照组(存活率为2.0%)相比,试验组1~3的存活率显著增加,分别为30.0%(p<0.001)、34.0%(p<0.001)和24.0%(p=0.004)。可见,从存活率的结果上看,试验组1和试验组2,即添加量为1公斤和5公斤都是最优的选择,但从性价比的角度,可以选择添加量更少且同样有效的试验组1的配方。但如果饲料中添加125公斤的功能包后,对虾在采食48小时后,对虾的采食量骤减。同时,在饲喂72小时饲料后,注射病原菌之前,与对照组(存活率为80.0%)相比,试验组4的存活率反而降至64.0%,且在带有pVA1质粒的副溶血弧菌刺激48小时后,试验组4(存活率为16%)与对照组的存活率差异不显著(p=0.680)。说明此饲料中功能包的添加量过大,会影响对虾对饲料的摄食,导致在病原菌刺激下,虽然最终仍对凡纳对虾存活率有帮助,但影响不显著。以上结果表明,该功能包添加量在1~25公斤可以显著提高虾在带有pVA1质粒的副溶血弧菌刺激下的存活率。
从实施例7和实施例8的结果可以看出,本发明设计的含牛磺酸的功能包(牛磺酸:10~50%,维生素E:20~40%,维生素C:20~55%,糊精或玉米粉或小麦粉或稻壳粉:5%~50%),在牛磺酸为10%~50%范围内,在同样的饲料添加比例下,具有相似的提高对凡纳对虾存活率、特别是提高对凡纳对虾在带有pVA1质粒的副溶血弧菌刺激下存活率的功能,证明本发明设计的功能包安全有效。
以上所揭露的仅为本发明的较佳实施例而已,当然不能以此来限定本发明之权利范围,因此依本发明权利要求所作的等同变化,仍属本发明所涵盖的范围。
Claims (5)
1.一种牛磺酸的应用,其特征在于,牛磺酸在制备增加甲壳动物抗副溶血弧菌能力药物中的应用;所述甲壳动物为虾。
2. 根据权利要求1所述牛磺酸的应用,其特征在于,所述牛磺酸的添加量与所述甲壳动物的体重比为90~750 μg/6g。
3. 根据权利要求2所述牛磺酸的应用,其特征在于,所述牛磺酸的添加量与所述甲壳动物的体重比为200~750 μg/6g。
4.一种牛磺酸体外抑制细菌生长的方法,其特征在于,包括如下步骤:采用细菌菌液与牛磺酸溶液按体积比1:1混合,之后恒温培育;
所述细菌包括副溶血性弧菌、哈维氏弧菌、海豚链球菌中的一种或多种;
所述牛磺酸溶液的浓度为2500μg/100μl。
5. 根据权利要求4所述牛磺酸抑制细菌生长的方法,其特征在于,包括如下步骤:采用所述细菌菌液与所述牛磺酸溶液按体积比1:1混合,37℃ 培养箱孵育2小时;取30μL上述孵育后的混合液涂布TSB或LB平板,每组3个生物学重复,每个生物学重复3个平行;将涂布后的平板于37℃ 恒温培养箱培养12小时后,记录平板菌落数。
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