CN113598285A - 一种提高水产养殖动物肠道免疫的中药发酵饲料及其制备方法 - Google Patents
一种提高水产养殖动物肠道免疫的中药发酵饲料及其制备方法 Download PDFInfo
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- CN113598285A CN113598285A CN202110782971.6A CN202110782971A CN113598285A CN 113598285 A CN113598285 A CN 113598285A CN 202110782971 A CN202110782971 A CN 202110782971A CN 113598285 A CN113598285 A CN 113598285A
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Abstract
本发明公开了一种提高水产养殖动物肠道免疫的中药发酵饲料及其制备方法,属于水产养殖技术领域,本发明的中药发酵饲料是由枯草芽孢杆菌、植物乳杆菌和地衣芽孢杆菌组成的复合微生物菌剂发酵而成。本发明提供的水产养殖动物肠道免疫力的中药发酵饲料,通过发酵将中药的药性融入到饲料中,同时增加了饲料中的益生菌数量,提高水产养殖动物肠道菌群平衡。
Description
技术领域
本发明涉及水产养殖技术领域,具体的涉及一种提高水产养殖动物肠道免疫的中药发酵饲料及其制备方法。
背景技术
随着养殖水平的不断提升,水产养殖集约化程度的不断加深;但是这也带来了养殖环境的不断恶化以及药物滥用等问题,冲击了水产养殖业的生产,造成了不可估量的经济损失。随着市场对传统水产养殖饲料逐渐的摒弃,新型的环保健康功能性饲料越来越受欢迎。
肠道是重要的消化吸收器官,同时也是重要免疫器官。水产动物肠道壁结构薄,容易遭受损伤特别是饲料中的霉菌毒素、过氧化物质等都可以直接损伤肠道内壁的黏膜细胞,造成肠道内壁的机能下降,引起各种肠道疾病的发生,而且有毒有害物质更容易侵入。
由此发酵饲料在水产养殖中越来越受到大家的重视,目前的发酵饲料大都使用的是单一菌种发酵,虽然也有复合菌种发酵,但是使用哪些菌种进行复合对发酵后饲料的效果会有很大影响。
近年来,中药在水产养殖中发挥的作用逐渐收到大家的重视,中药的添加有些可以改善水产动物的肉质品质、有些可以提高水产动物的体液免疫水平,有些可以提高水产动物的溶菌酶活性和白细胞吞噬活性,但是对于水产养殖动物来说,肠道免疫水平是影响水产动物的存活率、生长速度、疾病发病率的重要因素。因此,在减少化合物药物使用的条件下,如何通过饲料来提高水产动物的肠道免疫能力是水产养殖业的发展方向。
发明内容
针对水产养殖动物养殖过程中容易发生肠道疾病的问题,本发明提供了一种有效提高水产养殖动物肠道免疫的中药发酵饲料。
为实现上述目的,本发明采用技术方案为:
一种提高水产养殖动物肠道免疫的中药发酵饲料,将复合微生物菌剂添加至含有中药的发酵饲料原料中经发酵获得;
其中,发酵饲料原料按质量份数计,
鱼粉25-35份、虾粉5-15份、花生粕5-15份、豆粕10-15份、乌贼粉1-5份、麦麸10-20份、大豆磷脂1-2份、鱼油2-4份、红糖20份、硫酸铵2份、磷酸二氢钾2份、雨生红球藻粉2份、中药粉6-8份;
优选比例为:
鱼粉25份、虾粉5份、花生粕15份、豆粕15份、乌贼粉3份、麦麸20份、大豆磷脂2份、鱼油4份、红糖20份、硫酸铵2份、磷酸二氢钾2份、雨生红球藻粉2份、中药粉8份;
所述中药粉按质量份数的成分:
茵陈3份、姜黄10份、打火草8份、蒲公英8份、穿心莲8份、银杏叶8份、水飞蓟3份、甘草15份。
所述复合微生物菌剂为枯草芽孢杆菌、植物乳杆菌和地衣芽孢杆菌按体积比为1:1:1的比例混合获得。
所述复合微生物菌剂中将菌数含量≥108CFU/mL的植物乳杆菌菌液接种到含有葡萄糖的LB液体培养基中,菌液加入量为培养基体积的10%;32℃条件下发酵30h;
将菌数含量≥108CFU/mL的枯草芽孢杆菌菌液接种到含有葡萄糖的LB液体培养基中,菌液加入量为培养基体积的5%;37℃条件下发酵40h;
将菌数含量≥109CFU/mL的地衣芽孢杆菌菌液接种到含有葡萄糖的LB液体培养基中,菌液加入量为培养基体积的5%;37℃条件下发酵40h;
将上述各菌株的发酵液按体积比为1:1:1混合。
所述含有葡萄糖的LB液体培养基为向灭菌后的LB液体培养基中加入葡萄糖水溶液,高温灭菌后备用;其中,LB液体培养基与葡萄糖水溶液按100:5的体积比混合;LB液体培养基为:蛋白胨10.0g、酵母膏3.0g、NaCl5.0g、蒸馏水1.0L;pH7.0,葡萄糖水溶液为质量分数为20%葡萄糖水溶液。
一种提高水产养殖动物肠道免疫的中药发酵饲料的制备方法,
将中药粉碎至80目,粉碎后按上述比例将所有的原料混匀;
加入原料总重量85%的水,搅拌均匀后蒸汽灭菌,冷却;
向冷却后的原料中加入复合微生物菌剂,复合微生物菌剂加入量为加入后总重量的10%,在35℃条件下发酵15天。
所述饲料作为对虾的养殖中的肠道免疫的中药发酵饲料的应用。
本发明技术方案的优点
本发明提供的水产养殖动物肠道免疫力的中药发酵饲料,利用中草药茵陈、姜黄、打火草、蒲公英、穿心莲、银杏叶、水飞蓟、甘草等作为菌株的发酵培养底物,通过发酵将中药的药性融入到饲料中,而后将其在动物饲养的过程中通过食源性的饲料大幅提高水产动物的肠道免疫力,减少水产养殖动物疾病暴发的几率,提高水产养殖动物的成活率和生长速度,提高水产养殖经济效益,对于推动水产养殖业的发展具有积极的作用。本发明的中药发酵饲料安全、无毒副作用,不会使水产养殖动物产生药物依赖。本发明的发酵饲料中保留了微生物,增加了饲料中的益生菌数量,提高水产养殖动物肠道菌群平衡。
具体实施方式
以下的实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何方式限制本发明。
下述实施例中饲料原料均为市购产品。
实施例1
复合微生物菌剂的制备
本发明实施例菌种均购置于中国普通微生物保藏管理中心;其中植物乳杆菌(Lactobacillus plantarum)CGMCC1.573;枯草芽孢杆菌(Bacillus subtilis)CGMCC1.8715;地衣芽孢杆菌(Bacillus licheniformis)CGMCC1.518。
制备方法:
(1)培养基制备
取LB液体培养基(蛋白胨10.0g、酵母膏3.0g、NaCl 5.0g、蒸馏水1.0L;pH7.0),加入20%葡萄糖水溶液,高温灭菌后备用;
(2)菌剂制备
将植物乳杆菌按常规方式活化而后将含量≥108CFU/g的菌液接种到上述灭菌后的培养基中,菌液加入量为培养基体积的10%;32℃条件下发酵30h;
将枯草芽孢杆菌按常规方式活化而后将含量≥108CFU/mL的菌液接种到上述灭菌后的培养基中,菌液加入量为培养基体积的5%;37℃条件下发酵40h;
将地衣芽孢杆菌按常规方式活化而后将含量≥109CFU/mL的菌液接种到上述灭菌后的培养基中,菌液加入量为培养基体积的5%;37℃条件下发酵40h;
(3)将步骤(2)制备的菌剂按照1:1:1混合。
实施例2
一种提高水产养殖动物肠道免疫的中药发酵饲料,发酵饲料原料由以下质量的原料制备而成:
鱼粉25kg、虾粉5kg、花生粕15kg、豆粕15kg、乌贼粉3kg、麦麸20kg、大豆磷脂2kg、鱼油4kg、红糖20kg、硫酸铵2kg、磷酸二氢钾2kg、雨生红球藻粉2kg、中药粉8kg;
所述中药粉包含以下质量份数的成分:
茵陈3份、姜黄10份、打火草8份、蒲公英8份、穿心莲8份、银杏叶8份、水飞蓟3份、甘草15份。
制备方法:
按量取原料,将中药粉碎至80目,粉碎后将所有的原料混匀;
加入原料总重量85%的水,搅拌均匀后蒸汽灭菌,冷却;
向冷却后的原料中加入复合微生物菌剂,菌液加入量为加入后总重量的10%,在35℃条件下发酵15天。
发酵结束后低温条件下干燥,使用常规的饲料制备工艺造粒。
应用例
一、生长和成活率试验:
使用上述实施例制备的饲料养殖南美白对虾(学名凡纳滨对虾,Penaeusvannamei)90天,统计南美对虾的生长和成活率。以传统日常饲料为对照组,结果如表1所示
表1
组别 | 始重(g/尾) | 终重(g/尾) | 成活率(%) |
实施例组 | 0.5±0.04 | 22.42±0.24 | 93.54 |
对照组 | 0.5±0.03 | 16.84±0.22 | 74.24 |
整个养殖期间,实施例组对虾的活动未见任何异常,虾体洁净透亮、触须完整,触碰反应明显,立即弹射躲避。
从表1可以看出,本发明的发酵饲料可以有效提高对虾的成活率,而且对虾生长更快速。
二、免疫力试验
试验用南美白对虾体重约1-2g,活泼、无伤残。试验虾暂养一周后被随机分为3组,每组设3个平行,每个平行30尾虾;分别为空白对照组(传统虾饲料喂养)、氨氮胁迫组(传统虾饲料喂养)、发酵饲料组(实施例2制备的虾饲料喂养);
3个组分别按照设计的喂养方式在不添加氯化铵的水体中喂养7天;7天后氨氮胁迫组和发酵饲料组的水体中添加氯化铵(90mg/L;1次/日),连续喂养3天,之后转入正常水体中继续喂养7天,试验结束。
实验结果:
临床表现及存活率
试验期间,每天观察对虾的外观体征、游泳情况、摄食情况、死亡时间和死亡数(触碰时无明显反应),计算各组的存活率。
整个试验期间,空白对照组对虾的活动未见任何异常,虾体洁净透亮、触须完整,触碰时反应明显,立即弹射躲避。
水体中添加氯化铵后氨氮胁迫组和发酵饲料组的南美白对虾活力均明显减弱,触碰时的弹射躲避行为较少,摄食活动也明显减少,部分对虾胃肠道空无食物,经氯化铵胁迫处理的第2天,氨氮胁迫组和发酵饲料组对虾均出现死亡情况,氨氮胁迫组死亡更多。试验结束后,成活率情况如表2。
表2
空白对照组 | 氨氮胁迫组 | 发酵饲料组 | |
成活率% | 97.78 | 75.56 | 91.11 |
免疫酶活性
分别于试验开始前和试验结束后从各组随机取10尾虾,用经抗凝剂处理过的注射器从心脏、胸足基部和腹节等处采集血淋巴样品。每组的样品在4℃条件下4000r/min离心10min,收集上清液后,测定酚氧化酶PO、总超氧化物歧化酶SOD、溶菌酶LZM和碱性磷酸酶ALP活性,每个指标平行测定三次,计算平均值,结果如表3所示。
表3
肠道菌群组成
试验试验结束后,分别从各组随机选取10尾虾,清洗消毒后,于超净工作台中剥离对虾的整条肠道;剥离后的肠道置于无菌容器中;每组剥离完成后,采用分析天平进行称重,记录重量;向肠道样品中加入一定量的无菌生理盐水进行匀浆并制备成一定体积的匀浆溶液;并采用10倍递增稀释法制备成不同梯度的悬液。采用平板菌落计数法;分别取上述制备的各组悬液0.1mL分别涂布于2216E培养基和TCBS培养基,涂布完成后,放入培养箱中培养5d;统计菌落数,计算虾肠道细菌总数和弧菌的数量。每组选取菌落数适量,且分布均匀的平板,从中随机挑取30个菌落进行分离纯化,并根据Shewan与Olier的检索图表将其鉴定到属。统计并计算各实验组对虾肠道菌群的组成情况,结果如表4和表5所示:
表4
由表4可知,投喂本发明发酵饲料组的对虾肠道总菌含量整体相对于空白对照组有所提高;氨氮胁迫组相对于空白对照组有所下降;弧菌被认为是对虾养殖中最常见的病原菌;各组对虾肠道菌群中弧菌含量相差不大;而弧菌在肠道总菌中的占比不同;其中,弧菌在发酵饲料组中的占比约为8.68%;显著低于空白对照组21.57%;而氨氮胁迫组中弧菌占比约为36.31%;相对于空白对照组显著提高。由此可见,本发明的发酵饲料可以显著降低弧菌在对虾肠道总菌群中的占比,调整对虾肠道菌群平衡,维持肠道健康。
表5
由表5可知,各组对虾肠道菌群细菌群落分布情况中,弧菌属(Vibrio)的分布情况同表4中的数据大体一致;芽孢杆菌属(Bacillus spp.)为对虾肠道的益生菌,能够产生拮抗物质和多种消化酶,不仅能够抑制对虾肠道致病菌和有害菌的生长;还能够提高宿主肠道的消化吸收能力,维持肠道菌群平衡,提高抗病能力。发酵饲料组芽孢杆菌的数量显著高于空白对照组;且在对虾肠道中形成优势菌群;在氨氮胁迫组中,弧菌为肠道中的优势菌群。肠杆菌属(Enterobacteriaceae)、发光杆菌属(Photobacterium)、黄单孢菌属(Xanthomonas)、假单胞菌属(Pseudomonas)以及气单胞菌属(Aeromonas)在各处理组中均有分布,其中,肠杆菌属与气单胞菌属的菌大多数为病原菌,该两个属的菌在氨氮胁迫组好空白对照组中的占比高于本发明发酵饲料组;这进一步说明,本发明的发酵饲料能够调节对虾肠道菌群平衡,增加有益菌的生长繁殖,抑制有害菌的生长,进而达到防治对虾病害的目的。
仅为本发明的具体实施方式,并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。
Claims (6)
1.一种提高水产养殖动物肠道免疫的中药发酵饲料,其特征在于:将复合微生物菌剂添加至含有中药的发酵饲料原料中经发酵获得;
其中,发酵饲料原料按质量份数计,
鱼粉25-35份、虾粉5-15份、花生粕5-15份、豆粕10-15份、乌贼粉1-5份、麦麸10-20份、大豆磷脂1-2份、鱼油2-4份、红糖20份、硫酸铵2份、磷酸二氢钾2份、雨生红球藻粉2份、中药粉6-8份;
所述中药粉按质量份数的成分:
茵陈3份、姜黄10份、打火草8份、蒲公英8份、穿心莲8份、银杏叶8份、水飞蓟3份、甘草15份。
2.根据权利要求1所述的提高水产养殖动物肠道免疫的中药发酵饲料,其特征在于:所述复合微生物菌剂为枯草芽孢杆菌、植物乳杆菌和地衣芽孢杆菌按体积比为1:1:1的比例混合获得。
3.根据权利要求2所述的提高水产养殖动物肠道免疫的中药发酵饲料,其特征在于:所述复合微生物菌剂中将菌数含量≥108CFU/mL的植物乳杆菌菌液接种到含有葡萄糖的LB液体培养基中,菌液加入量为培养基体积的10%;32℃条件下发酵30h;
将菌数含量≥108CFU/mL的枯草芽孢杆菌菌液接种到含有葡萄糖的LB液体培养基中,菌液加入量为培养基体积的5%;37℃条件下发酵40h;
将菌数含量≥109CFU/mL的地衣芽孢杆菌菌液接种到含有葡萄糖的LB液体培养基中,菌液加入量为培养基体积的5%;37℃条件下发酵40h;
将上述各菌株的发酵液按体积比为1:1:1混合。
4.根据权利要求3所述的提高水产养殖动物肠道免疫的中药发酵饲料,其特征在于:所述含有葡萄糖的LB液体培养基为向灭菌后的LB液体培养基中加入葡萄糖水溶液,高温灭菌后备用;其中,LB液体培养基与葡萄糖水溶液按100:5的体积比混合;LB液体培养基为:蛋白胨10.0g、酵母膏3.0g、NaCl5.0g、蒸馏水1.0L;pH7.0,葡萄糖水溶液为质量分数为20%葡萄糖水溶液。
5.一种根据权利要求1所述的提高水产养殖动物肠道免疫的中药发酵饲料的制备方法,其特征在于,
将中药粉碎至80目,粉碎后按上述比例将所有的原料混匀;
加入原料总重量85%的水,搅拌均匀后蒸汽灭菌,冷却;
向冷却后的原料中加入复合微生物菌剂,复合微生物菌剂加入量为加入后总重量的10%,在35℃条件下发酵15天。
6.根据权利要求5所述的提高水产养殖动物肠道免疫的中药发酵饲料的制备方法,其特征在于,所述饲料作为对虾的养殖中的肠道免疫的中药发酵饲料的应用。
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CN117598398A (zh) * | 2023-11-27 | 2024-02-27 | 山东省海洋科学研究院(青岛国家海洋科学研究中心) | 一种水产动物无抗性配合饲料添加剂 |
CN117598398B (zh) * | 2023-11-27 | 2024-05-24 | 山东省海洋科学研究院(青岛国家海洋科学研究中心) | 一种水产动物无抗性配合饲料添加剂 |
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