CN114470084A - 环保高效防治罗非鱼白便的组合物及其制备方法和应用 - Google Patents
环保高效防治罗非鱼白便的组合物及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种环保高效防治罗非鱼白便的组合物及其制备方法和应用，组合物包括白术、茯苓、黄芪、山药、甘草，制备方法采用复合酶酶解方法和水提醇沉方法相结合，通过复合酶把细胞膜、细胞质内的淀粉多糖、糖蛋白酶解，使得药用多糖更好地释放，再采用水提醇沉的方法把药用多糖浸提、浓缩、沉淀分离出来，最终得到效价高、方便使用、便于保存的提取物。本发明通过从一种中药组合物中提取植物多糖，作为益生元，常温易保存，稳定性好，可用于饲料加工，直达肠道，作为肠道菌群的碳源促进肠道有益菌的增殖、抑制有害菌，从而改善肠道微生态菌群，维持平衡的菌群结构，有利于防治罗非鱼白便和肠炎问题。
Description
技术领域
本发明属于鱼类养殖技术领域，具体涉及一种环保高效防治罗非鱼白便的组合物及其制备方法和应用。
背景技术
罗非鱼，具有生长快、食性杂、料比低、出肉多、产量高的优势，是我国重要的经济养殖鱼类。然而，随着高密度集约化养殖模式的发展，各种疾病也频繁发生。罗非鱼由于摄食量大，在春夏交替、高温季节，经常会出现鱼体肛门“拖便”现象，鱼塘投料机周围、下风口漂浮大量白色絮状粪便，可判断消化道出现问题，肠道发炎，肠粘膜脱落。肠道不仅是鱼体重要的消化和吸收器官，也是鱼体最大的免疫和代谢器官，同时肠道也是机体内与外界环境接触表面积最大的器官，是机体伤害的始发器官，因此保护肠道健康对保护鱼体健康具有重要作用。然而，目前对普通淡水鱼来说，终端养殖户并不注重鱼体肠道健康的日常护理，往往都是在发现死鱼、爆发鱼病后，才寻找紧急特效药，如使用抗生素等化学药物，这导致了耐药菌株增加、水体污染、药物残留等环境问题和食品安全问题。研究表明，肠道菌群在动物营养吸收、免疫反应、生长发育发挥重要的生理作用，因此可通过提高肠道有益微生物数量、维持肠道微生物群落平衡，修复肠道损伤。相关技术公开了一种预防罗非鱼肠炎的饲料添加剂，通过添加柠檬酸、胆汁酸、沸石粉、乳酸链球菌、乳酸杆菌、地衣芽孢杆菌，改善肠道黏膜厚度、抑制肠道有害微生物的繁殖和促进有害物质的排放。该技术通过在商品饲料中拌入该添加剂发挥效果，主要是因为活菌本身存在活性保留技术难点，在实际生产应用受到一定限制。
针对罗非鱼白便问题，可通过调节肠道菌群来防治。但益生菌要求低温保存条件，不适用于直接添加到饲料生产、拌料也加大养殖户劳动力，且直接饲用后到达肠道的活性保留率降低。
发明内容
本发明旨在至少解决上述现有技术中存在的技术问题之一。为此，本发明提出一种环保高效防治罗非鱼白便的组合物及其制备方法和应用，主要通过从一种中药组合物中提取植物多糖，作为益生元，常温易保存，稳定性好，可用于饲料加工，直达肠道，作为肠道菌群的碳源促进肠道有益菌的增殖、抑制有害菌，从而改善肠道微生态菌群，维持平衡的菌群结构，有利于防治罗非鱼白便和肠炎问题。
根据本发明的一个方面，提出了一种环保高效防治罗非鱼白便的组合物，包括以下组分：白术、茯苓、黄芪、山药、甘草；所述组合物由以下方法制得：
S1：将白术、茯苓、黄芪、山药和甘草粉碎过筛，加水浸泡一段时间，得到组合物浆料；取复合酶加入温水进行活化，得到复合酶溶液；
S2：将所述组合物浆料进行加热，并加入柠檬酸-柠檬酸钠缓冲体系调节pH至4.5-5.0，然后加入所述复合酶溶液进行酶解；
S3：所述酶解结束后，升温灭酶活，再冷却，补充水后进行水浴提取，得到提取后液；
S4：将所述提取后液进行固液分离，所得液体进行浓缩得到浓缩液，向所述浓缩液中加入无水乙醇，静置，得到沉淀物，干燥后即得所述组合物；
其中，所述复合酶由纤维素酶、果胶酶、中温α-淀粉酶和中性蛋白酶组成。
在本发明的一些实施方式中，所述组分按以下重量份计：白术22.5-25份、茯苓22.5-25份、黄芪18-22份、山药15-20份、甘草10-15份。
在本发明的一些实施方式中，所述组分按以下重量份计：白术22.5份、茯苓22.5份、黄芪20份、山药20份、甘草15份。
在本发明的一些实施方式中，所述纤维素酶、果胶酶、中温α-淀粉酶和中性蛋白酶的质量比为(2-4)：(1-2)：(0.5-1)：(0.5-1)。
在本发明的一些实施方式中，步骤S1中，所述组合物浆料的料液比为1：(10-15)；所述浸泡的时间为45-60min。
在本发明的一些实施方式中，步骤S1中，所述过筛的目数为100-150目。
在本发明的一些实施方式中，步骤S1中，所述复合酶的用量为所述白术、茯苓、黄芪、山药和甘草总质量的0.3-0.5％。
在本发明的一些实施方式中，步骤S1中，所述温水的用量为所述复合酶质量的5-10倍；所述温水的温度为40-45℃，所述活化的时间为10-15min。
在本发明的一些实施方式中，步骤S2中，所述加热的温度为45-50℃；所述酶解的时间为120-180min。
在本发明的一些实施方式中，步骤S3中，所述水浴提取的温度为80-85℃，水浴提取的时间为180-210min。
在本发明的一些实施方式中，步骤S3中，所述升温的温度为85-95℃，所述灭酶活的时间为10-15min。
在本发明的一些实施方式中，步骤S3中，所述补充水后浆料的的固液比为1：(30-40)。
在本发明的一些实施方式中，步骤S4中，所述浓缩为真空浓缩，浓缩至原体积的1/15-1/10。
在本发明的一些实施方式中，步骤S4中，所述无水乙醇的用量为所述浓缩液的3-5倍。
在本发明的一些实施方式中，步骤S4中，所述静置的时间为20-30h。
在本发明的一些实施方式中，步骤S4中，所述沉淀物再干燥前还依次用无水乙醇、丙酮、乙醚进行洗涤；进一步优选的，所述洗涤的次数为两次。
在本发明的一些实施方式中，步骤S4中，所述干燥的温度为60-80℃，干燥后所得组合物的水分为5％以下。
本发明还提供一种罗非鱼饲料，由基础饲料和所述的组合物组成，所述组合物的添加量为所述基础饲料质量的0.3-0.7％。进一步地，所述基础饲料包括豆粕、加籽菜粕、小麦、向日葵仁粕、玉米DDGS、面粉、米糠粕、大麦、鱼粉、豆油、磷酸二氢钙、罗非鱼预混合饲料、食盐和氯化胆碱。
根据本发明的一种优选的实施方式，至少具有以下有益效果：
(1)本发明的组合物组分白术、茯苓、黄芪、山药、甘草，是在传统中药方剂“四君子汤”(出自宋代《太平惠民和剂局方》)基础上进行加减，其中白术甘苦性温，具益气健脾利水之效；茯苓甘淡性平，具渗湿健脾宁心的功效；两药具有健脾祛湿的共同功效，由于脾喜燥而恶湿，白术苦能燥湿，茯苓淡能利湿，两药陪用，一燥一渗，使水湿除而脾气健。黄芪性甘微温，补中益气，偏补脾阳，可治脾虚泄泻；山药性微涩，补脾健胃，滋阴固肾，可治泄泻便溏；山药与黄芪相合，有固涩升提之效，二药配伍一滋阴一升阳，对治疗泄泻具有相互增益作用。甘草味甘性平，归心、肺、脾、胃经，具有补脾益气、祛痰止咳、缓急止痛、清热解毒、调和诸药的作用。脾是动物体主要免疫器官之一，对抵抗病菌炎症具有重要作用，白术、茯苓具有健脾益气渗湿的功效，为君药；黄芪健脾补中，山药能渗湿健脾、兼并抑制腹泻之效，两药同用是为臣药。甘草主要用作使药，调和以上四药。君药同臣药相称运用，使药使其合力祛邪，本组合物方剂在调节胃肠道运动、改善营养物质消化吸收等方面有很好的调节作用，可在不同程度上改善脾胃气虚、胃肠功能紊乱。
(2)传统中药大多采用水煎煮口服的给药方式，近些年来，也有一些终端养殖户采用此方法煎煮中药给鱼类保健治病，也取得一定疗效，实际上可溶性多糖是中药水煎液的重要组分，也是中药发挥疗效的重要物质基础。但是简单的煎煮，并不能把有效成分更好地提取出来，本发明的制备方法采用复合酶酶解方法和水提醇沉方法相结合，通过纤维素酶、果胶酶先把细胞壁破解，通过中温α-淀粉酶、中性蛋白酶把细胞膜、细胞质内的淀粉多糖、糖蛋白酶解，使得药用多糖更好地释放，再采用水提醇沉的方法把药用多糖浸提、浓缩、沉淀分离出来，最终得到效价高、方便使用、便于保存的提取物。
(3)本发明的组分经过其制备方法提取后，主要成分是植物多糖类物质，动物体缺乏多糖水解酶，除淀粉外的多糖不能被直接消化和吸收，而肠道菌群起着重要的桥梁作用，多糖则作为肠道菌群的益生元，促进肠道有益菌的增殖，抑制有害菌的繁殖。实验证明，本发明的组合物可以改善罗非鱼肠道菌群微生态环境，增强罗非鱼的抗病力，减少罗非鱼白便发生率，从而减少养殖用药物，保护水质环境。
具体实施方式
以下将结合实施例对本发明的构思及产生的技术效果进行清楚、完整地描述，以充分地理解本发明的目的、特征和效果。显然，所描述的实施例只是本发明的一部分实施例，而不是全部实施例，基于本发明的实施例，本领域的技术人员在不付出创造性劳动的前提下所获得的其他实施例，均属于本发明保护的范围。
实施例1
本实施例制备了一种环保高效防治罗非鱼白便的组合物，每100份组合物中包括如下重量份的组分：白术22.5份、茯苓22.5份、黄芪20份、山药20份、甘草15份，具体制备过程为：
将上述原料分别粉碎过100目筛，按组分比例称取组合物，搅拌混匀，按料液比1：12加入12倍量蒸馏水，浸泡30min，得组合物浆料；同时，按组合物干重0.4％称取复合酶，加5倍量40℃温水活化10-15min，得复合酶溶液；使用水浴方式、柠檬酸-柠檬酸钠缓冲体系调节组合物浆料温度45-50℃，pH为4.5-5.0，保持此反应条件，缓缓加入复合酶溶液并轻轻搅拌，恒温酶解120min左右(据组合物重量而定)；酶解结束后，加热至85℃水浴灭酶活15min，冷却至室温，补充蒸馏水使浆料固液比1：40，搅拌均匀，80℃水浴提取180min，抽滤得滤液，真空浓缩至1/10左右，浓缩液加4倍量无水乙醇，静置24h，收集沉淀物，依次用无水乙醇、丙酮、乙醚洗涤两次，60℃真空干燥至水分5％以下便于保存，得到组合物。
上述复合酶是由纤维素酶、果胶酶、中温α-淀粉酶、中性蛋白酶按2：1：0.5：0.5比例组成。
试验例1
(1)实验鱼及饲养管理
实验用罗非鱼来自广东顺德顺爷水产有限公司，在正式开始实验前，放于暂养池中使用基础饲料训食2周。挑选体质健壮、规格均匀的罗非鱼(平均体重170.53±2.31g)，随机分配到20个圆形养殖桶中(直径＝180cm，高＝100cm，盛水体积为1800L)，每桶30尾，每组饲料4个重复。实验期间，每天8：30和16：30分别饱食投喂一次，每次投喂量以实验鱼20min内吃完为准。采用室内循环水养殖系统，水温变化范围为28.3-32.6℃，溶解氧大于6.8mg.L-1,氨氮低于0.5mg.L-1，光照周期为自然光照。养殖周期8周。
(2)实验饲料
实验用豆粕、加籽菜粕、小麦、向日葵仁粕、玉米DDGS、面粉、米糠粕、大麦、鱼粉、豆油、磷酸二氢钙、罗非鱼预混合饲料、食盐、氯化胆碱均来自佛山市顺德区旺海饲料实业有限公司；白术、茯苓、黄芪、山药、甘草购自广州市药材公司。纤维素酶、果胶酶、中温α-淀粉酶、中性蛋白酶购自酶制剂公司。实验饲料配方及化学成分如表1所示。
表1饲料配方和营养成分(％)
其中，组合物的添加含量分别为0(对照组)、0.1％、0.3％、0.5％、0.7％，作为1个对照组、4个实验组进行养殖实验。
(3)样品采集与处理
养殖实验结束后，每桶随机取鱼5尾，用MS-222麻醉后，分别称重，采用头皮针从心脏静脉窦取血置于2ml无菌离心管中，4℃静置待其凝血后，3000r/min离心10min，取上清液保存于-20℃，用于血清酶活的测定(使用南京建成试剂盒)。取血后的鱼，分离脾脏称重并记录，以计算各实验组的脾脏指数。
每桶另取鱼3尾，在超净工作台上，酒精灯旁，迅速解剖取出鱼肠，置于消毒后的干净托盘上，快速剪开中肠，刮取0.5g肠道内容物，放到灭菌处理的10mL离心管中，加入4.5mL无菌水，震荡混匀，制成浓度为10-1样品，再依次用无菌水倍比稀释至10-8样品，用移液枪吸取合适稀释度的样品100uL涂布到大肠杆菌、双歧杆菌的选择性培养基上，每个稀释度做三个平行，37℃培养24h，培养结束后进行活菌计数(每克鱼肠标本的活菌数Ig CFU/g)。
(4)数据处理
用SPSS 19.0 for windows对所得的数据进行独立样本T检验，比较均值的显著性差异，显著性水平为P<0.05。
表2脾脏指数(％)
表3血清酶活(U/mL)
表4罗非鱼肠道微生物(Ig CFU/g)
注：表中数值后不同上标英文字母表示差异显著(P<0.05)。
由表2-4的结果表明，添加本发明组合物的实验组饲料养殖的罗非鱼免疫器官(脾脏指数)、血清非特异性免疫酶活性均比无添加组合物的对照组高，且以添加量为0.3％-0.7％最为显著。罗非鱼肠道微生物菌群也受影响，添加组合物的实验组鱼体内肠道有益菌(双歧杆菌)含量上升、有害菌(大肠杆菌)含量下降，有益菌/有害菌的比值有所提升。可见，本发明组合物经制备后，能增强罗非鱼免疫功能，且改善鱼体肠道菌群环境，促进肠道健康。
试验例2
经试验例1的初步评估后，按0.5％的比例添加组合物到商品饲料配方中，生产出大塘试验料，以同配方无添加组合物的正常商品饲料作为对照组。在惠州、茂名各邀请10位养殖户同时试用对比，即两地分别挑选5个塘饲喂试验料、5个塘饲喂正常商品饲料(对照组)，从6月中下旬养至10月份出鱼，规格从20尾左右养至1斤上，高温期7、8月发生链球菌的塘数记录如表5，整个养殖阶段白便发生的严重程度记录如表6，最终出鱼包产如表5。
表5链球菌发生塘数、出鱼包产
表6白便发生的严重程度
注：由养殖户自行评估整个养殖过程的白便严重程度，以*的颗数代表严重程度，以5颗*为最严重，认为正常或可忽略不计的为无。
由表5和表6的大塘试用结果表明，添加本发明的组合物0.5％的饲料组，高温期在两个地区的链球菌发生率都比对照组的低，白便发生程度也较对照组低，可有效防止罗非鱼白便发生，最终对饲料效果表达有利好影响。今年气温高，普遍塘头链球菌、白便发病率较大，而试验料组的养殖户表示，此造鱼吃料比周围养户好，发病率比往年大大降低，额外用药保健量大大减少，基本不用治病类药物。
上面对本发明实施例作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。
Claims (10)
1.一种环保高效防治罗非鱼白便的组合物，其特征在于，包括以下组分：白术、茯苓、黄芪、山药、甘草；所述组合物由以下方法制得：
S1：将白术、茯苓、黄芪、山药和甘草粉碎过筛，加水浸泡一段时间，得到组合物浆料；取复合酶加入温水进行活化，得到复合酶溶液；
S2：将所述组合物浆料进行加热，并加入柠檬酸-柠檬酸钠缓冲体系调节pH至4.5-5.0，然后加入所述复合酶溶液进行酶解；
S3：所述酶解结束后，升温灭酶活，再冷却，补充水后进行水浴提取，得到提取后液；
S4：将所述提取后液进行固液分离，所得液体进行浓缩得到浓缩液，向所述浓缩液中加入无水乙醇，静置，得到沉淀物，干燥后即得所述组合物；
其中，所述复合酶由纤维素酶、果胶酶、中温α-淀粉酶和中性蛋白酶组成。
2.根据权利要求1所述的组合物，其特征在于，所述组分按以下重量份计：白术22.5-25份、茯苓22.5-25份、黄芪18-22份、山药15-20份、甘草10-15份。
3.根据权利要求1所述的组合物，其特征在于，所述组分按以下重量份计：白术22.5份、茯苓22.5份、黄芪20份、山药20份、甘草15份。
4.根据权利要求1所述的组合物，其特征在于，所述纤维素酶、果胶酶、中温α-淀粉酶和中性蛋白酶的质量比为(2-4)：(1-2)：(0.5-1)：(0.5-1)。
5.根据权利要求1所述的组合物，其特征在于，步骤S1中，所述组合物浆料的料液比为1：(10-15)；所述浸泡的时间为45-60min。
6.根据权利要求1所述的组合物，其特征在于，步骤S1中，所述复合酶的用量为所述白术、茯苓、黄芪、山药和甘草总质量的0.3-0.5％。
7.根据权利要求1所述的组合物，其特征在于，步骤S2中，所述加热的温度为45-50℃；所述酶解的时间为120-180min。
8.根据权利要求1所述的组合物，其特征在于，步骤S3中，所述水浴提取的温度为80-85℃，水浴提取的时间为180-210min。
9.根据权利要求1所述的组合物，其特征在于，步骤S4中，所述无水乙醇的用量为所述浓缩液的3-5倍。
10.一种罗非鱼饲料，其特征在于，由基础饲料和权利要求1-9任一项所述的组合物组成，所述组合物的添加量为所述基础饲料质量的0.3-0.7％。
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