WO2022063295A1 - 一种用于提高水产养殖动物成活率的添加剂及其应用 - Google Patents

一种用于提高水产养殖动物成活率的添加剂及其应用 Download PDF

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Publication number
WO2022063295A1
WO2022063295A1 PCT/CN2021/120944 CN2021120944W WO2022063295A1 WO 2022063295 A1 WO2022063295 A1 WO 2022063295A1 CN 2021120944 W CN2021120944 W CN 2021120944W WO 2022063295 A1 WO2022063295 A1 WO 2022063295A1
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feed
bacillus
additive
aquaculture
carp
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PCT/CN2021/120944
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English (en)
French (fr)
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李阳
王海燕
张广民
余璐璐
彭翔
蔡辉益
李爽
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天津博菲德科技有限公司
北京挑战农业科技有限公司
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Publication of WO2022063295A1 publication Critical patent/WO2022063295A1/zh

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof

Definitions

  • the invention relates to the technical field of aquaculture, in particular to a feed additive which is added in aquaculture and can effectively improve the survival rate of aquatic animals and its application.
  • 5-Aminolevulinic acid and its derivatives or salts are an active substance that widely exists in animal, plant and microbial cells, and is a biosynthesis of chlorophyll, heme, porphyrin and vitamin B12. It is an important precursor substance of tetrapyrrole compounds, which is non-toxic to humans and animals, easily degraded in the environment, and has no residues. As an endogenous substance, it plays an important role in the life process. Its application in livestock feed has been There are reports, but nothing about the application of additives in aquafeed.
  • Micro-ecological preparations play an important role in the decomposition, transformation and water quality regulation of aquaculture biological excrement and residual bait, and are a new type of green feed additive.
  • Bacillus is a ubiquitous type of aerobic bacteria. It has the advantages of good stability, strong stress resistance and high survival rate. It can metabolize and produce a variety of digestive enzymes, degrade some anti-nutritional factors in the feed, and improve the feed conversion rate. ; Bacillus can also improve the water quality of aquaculture water by reducing the content of nitrate and nitrite in the water body. HadiZokaeifar et al.
  • the purpose of the present invention is to aim at the situation that the survival rate of high-density fish and shrimp culture is reduced due to insufficient oxygen and poor water quality. On the one hand, it can improve the hypoxia condition, and on the other hand, by improving the water quality and promoting the health of aquatic animals, it can synergistically improve the survival rate in the process of high-density fish and shrimp farming.
  • the present invention provides an additive for improving the survival rate of aquaculture animals, comprising an active substance and Bacillus, wherein the active substance is any compound in 5-aminolevulinic acid, its derivatives or its salts.
  • Bacillus is selected from at least one of Bacillus subtilis, Bacillus licheniformis and Bacillus coagulans.
  • the 5-aminolevulinic acid and Bacillus in the additive are uniformly mixed with other aquatic feed components and then granulated; wherein, the mass of 5-aminolevulinic acid and Bacillus: effective viable bacteria ratio is 1mg: 10 4 -10 7 cfu.
  • the ratio of active substance and bacillus in the additive is 10 6-8 cfu bacillus per 20 mg of 5-aminolevulinic acid.
  • the present invention also provides a feed comprising the additive.
  • the additive amount in the feed is 1 mg-1000 mg/Kg.
  • the added amount of the active substance in the aquatic feed is 1 mg-100 mg/Kg; the added amount of the Bacillus is 10 4 -10 8 cfu/Kg feed.
  • the addition amount of the Bacillus is 10 5 -10 7 cfu/Kg feed.
  • the invention provides an aquaculture method, which uses the feed additive or the feed to feed aquatic animals.
  • the additive application method is to add 5-ALA and Bacillus into the feed together for uniform granulation and then feed; or add 5-ALA to the feed to uniformly granulate, and sprinkle the Bacillus evenly while feeding in aquaculture water.
  • the dosage of the Bacillus is 10 2 -10 5 cfu/mL of aquaculture water.
  • the Bacillus injection amount is 10 3 -10 4 cfu/mL of aquaculture water.
  • the aquatic animal is at least one of vannamei, white shrimp, river shrimp, crucian carp, grass carp, carp, silver carp, large yellow croaker, crayfish or tilapia.
  • the aquacultured aquatic animals are at least one of vannamei, white shrimp, river shrimp, crucian carp, grass carp, common carp, silver carp, large yellow croaker, crayfish or tilapia.
  • the aquacultured aquatic animals are at least one of vannamei, white shrimp, river shrimp, crucian carp, grass carp, common carp, silver carp, large yellow croaker, crayfish or tilapia.
  • the following examples facilitate a better understanding of the present invention, but do not limit the present invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.
  • the quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.
  • the feed additive in the present invention is applied to high-density crucian carp culture, and the pond area is 0.08hm 2 .
  • the test crucian carp was temporarily cultured in the cement pond for 2 weeks before the start of the experiment. After the temporary culture, it was weighed and put into a grid. The size of the grid was 4m ⁇ 4m ⁇ 1.5m. The test was carried out in 18 grids. 2700 crucian carp (43.88 ⁇ 1.36g) with similar initial weight were set up into 4 treatment groups, 1 control group and 5 test groups, each treatment group had 3 replicates, and each replicate had 150 crucian carp, and the experiment time was 21 days .
  • the control group was fed with basal conventional feed, treatment 1 was fed with 5-ALA (10mg/Kg feed) and Bacillus licheniformis (2 ⁇ 10 6 cfu/Kg feed) on the basis of the control group feed, and treatment 2 was fed with control 5-ALA (20mg/Kg feed) and Bacillus licheniformis (2 ⁇ 10 6 cfu/Kg feed) were added to the feed of the group, and 5-ALA (30mg/Kg feed) and 5-ALA (30mg/Kg feed) and Bacillus licheniformis (2 ⁇ 10 6 cfu/Kg feed), 4 groups were fed the control group feed and added 5-ALA (40mg/Kg feed) and Bacillus licheniformis (2 ⁇ 10 6 cfu/Kg feed) at the same time, Treatment 5 was fed with the control group feed and added 5-ALA (50mg/Kg feed) and Bacillus licheniformis (2 ⁇ 10 6 cfu/Kg feed), of which the specification of Bacillus licheniformis
  • Feeding method 2 times a day (9:00 and 15:00). The feeding amount is 3% of the weight of the crucian carp.
  • the experimental conditions and feeding management were carried out routinely. At the end of the test, all surviving fish from each treatment were picked up, counted, and the survival rate was calculated; at the same time, 10 fish were randomly selected from each test pen, and the hemoglobin content in blood was determined by the Wenzi liquid method, and the Folin-phenol method was used to determine the content of hemoglobin in the blood.
  • Activity of protease in the intestine Amylase activity in the intestine was determined by the amylase-iodometric method.
  • treatment groups 1, 2, 3, 4 and 5 were The survival rates of high-density cultured crucian carp reached 98.26%, 99.20%, 98.42%, 97.56% and 97.17% respectively, which were 7.13, 8.07, 7.29, 6.43 and 6.04 percentage points higher than that of the basic diet group.
  • the improvement of the hypoxic state of aquatic animals will cause the oxygen-carrying capacity of the blood.
  • the oxygen-carrying capacity can be achieved by increasing the number of red blood cells in the blood and increasing the concentration of hemoglobin.
  • the anoxic state of crucian carp in the process of high-density aquaculture is generally believed that the activity of digestive enzymes in aquatic animals will have a great impact on their ability to absorb and utilize food, thereby promoting their growth.
  • administration of the additives in the present invention significantly increased hemoglobin content by 5.44%, 6.93%, 4.75%, 5.16% and 3.41%, and significantly increased intestinal protease activity by 12.22%, 14.24%, 13.43%, 9.81% and 5.84%, the amylase activity was increased by 7.41%, 13.68%, 8.14%, 2.35% and 3.86%, respectively (Table 1).
  • Bacillus can stimulate immunity by producing antibodies and promoting phagocytosis, stimulate humoral immunity and cellular immunity, and enhance the immune activity of animals.
  • the control group was fed with basal conventional feed, treatment 1 was fed with 5-ALA (20 mg/Kg feed) on the basis of the control group feed, and treatment 2 was fed with the control group feed supplemented with Bacillus subtilis (1 ⁇ 10 3 cfu/mL) water body), treatment 3 was fed with the control group feed supplemented with 5-ALA (20 mg/Kg feed) and Bacillus subtilis (1 ⁇ 10 2 cfu/mL water body), treatment 4 was fed with the control group feed supplemented with 5- ALA (20mg/Kg feed) and Bacillus subtilis (1 ⁇ 10 3 cfu/mL water body), treatment 5 was fed the control group feed supplemented with 5-ALA (20 mg/Kg feed) and Bacillus subtilis (1 ⁇ 10 4 ) cfu/mL water body), treatment 6 was fed the control group feed on the basis of adding 5-ALA (20mg/Kg feed) and Bacillus subtilis (1 ⁇ 10 5 cfu/mL water body).
  • the amount of 5-ALA added is 20mg/Kg feed, which is uniformly mixed with other feed components, and then pelleted and fed;
  • inoculum with a mass of 0, 5, 0.5, 5, 50, 500g) and a bacterial concentration of 2 ⁇ 10 8 cfu/g were poured into each 1000L bucket, once a week).
  • the water temperature was 25°C and the salinity was 30ppt.
  • Feeding method 4 times a day (6:00, 10:00, 15:00, 20:00).
  • the feeding amount was 5% of the shrimp weight.
  • the experimental conditions and feeding management were carried out routinely. At the end of the experiment, all the surviving shrimp in each bucket were fished, weighed, and the survival rate and FCR were calculated.
  • Feed conversion ratio (FCR) feed intake/net weight gain
  • the results show that (Table 2) the additive composition of treatment 1 and treatment group 2 is 5-ALA (addition amount is 20mg/Kg feed) and Bacillus subtilis Bacillus subtilis (the addition amount was Bacillus subtilis 1 ⁇ 10 3 cfu/mL water body), the results showed that the survival rate of Penaeus vannamei increased by 5.37 and 5.06 percentage points, respectively, and the feed conversion rate decreased from 1.13 to 1.08 and 1.08, respectively; Groups 4, 5 and 6 were respectively fed the additive composition of the present invention that 5-ALA and different amounts of Bacillus subtilis were added synergistically, and the survival rates of Penaeus vannamei reached 96.02%, 98.06%, 96.80% and 94.99% respectively.
  • the feed additive in the present invention is applied to tilapia culture, and the pond area is 0.08hm 2 .
  • the test fish were temporarily cultured in the cement pond for 2 weeks before the start of the experiment. After the temporary culture, they were weighed and put into the enclosure. The size of the enclosure was 4m ⁇ 4m ⁇ 1.5m.
  • 5400 juveniles (average body weight 2.59 ⁇ 0.51g, average body length 4.42 ⁇ 0.31cm) were selected and randomly assigned to 18 aquariums, and the 18 aquariums were randomly divided into 6 Treatment, 3 replicates for each treatment, 300 tails for each replicate, the treatment group was the control group as the basic diet; the treatment group 1 was 20mg/Kg 5-ALA+100g/ton Bacillus subtilis, that is, the amount of Bacillus subtilis added was 10 7 cfu/Kg feed; treatment group 2 was supplemented with 20mg/Kg 5-ALA+100g/ton Bacillus coagulans, that is, the addition amount of Bacillus was 10 7 cfu/Kg feed; treatment group 3 was 20mg/Kg 5-ALA+ 5g/ton of Bacillus subtilis and 5g/ton of Bacillus coagulans are compounded and added, that is, the amount of Bacillus added is 10 6 cfu/Kg feed; 4 groups are treated with 20mg/Kg 5-ALA+
  • the application method of 5-ALA and Bacillus is uniformly mixed with other feed components and prepared pellet feeding.
  • Feeding method 3 times a day (6:00, 13:00 and 20:00), the feeding amount is 3% of the tilapia weight.
  • the experimental conditions and feeding management were carried out as usual, and the animals were fed for 42 days.
  • all the surviving fish from each treatment were picked up, counted and the survival rate was calculated; the stomach and intestines of the Gift tilapia were separated, accurately weighed, and homogenized in an ice bath at a weight-to-volume ratio of 1:9.
  • the dopase assay was performed by the Folin phenol method.
  • the efficiency of fish and shrimp farming is positively related to the digestion and absorption capacity of nutrients and the activity of digestive enzymes.
  • the results of this example show (Table 3) that, relative to the control group fed the basal diet, the tilapia in treatment 1, treatment 2, treatment 3, treatment 4 and treatment 5 were treated with the feed additive of the present invention.
  • the survival rate, pepsin and intestinal protease activities all played a significant role in improving the survival rate of tilapia, 5-ALA and Bacillus synergistically improved the survival rate of tilapia, reaching 97.75%, 97.64%, and 98.15%, respectively.
  • the addition amount of Bacillus has a greater effect on the survival rate, protease and intestinal protease activity, and the effect is the best when the addition amount is 10 6 -10 8 cfu/Kg feed; and the survival rate of Bacillus subtilis and Bacillus coagulans compound addition slightly higher than that administered alone.
  • the additive of the invention can improve the anoxic condition of fish and shrimp in the process of fish and shrimp culture (especially high-density fish and shrimp culture), and can improve the water quality of culture water, thereby reducing the disease rate of fish and shrimp, and the survival rate of culture reaches 98%. % or more, reducing the cost of fish and shrimp breeding and improving the breeding efficiency.
  • 5-ALA can act alone or through a synergistic effect with Bacillus, on the one hand, it can enhance the immune response of aquatic animals, mainly because 5-ALA upregulates immunity and defense.

Abstract

一种用于提高水产养殖动物成活率的添加剂及其在水产养殖中的应用，所述添加剂包含活性物质5-氨基乙酰丙酸(5-ALA)、其衍生物或其盐中的任一种化合物和芽孢杆菌。所述添加剂一方面可以改善缺氧状况，另一方面通过改善水质和促进水产动物的机体健康，协同改善高密度鱼虾养殖过程中的成活率。

Description

一种用于提高水产养殖动物成活率的添加剂及其应用 技术领域
本发明涉及水产养殖技术领域，具体涉及一种在水产动物养殖中添加，可有效提升水产动物成活率饲料添加剂及其应用。
背景技术
5-氨基乙酰丙酸(5-ALA)及其衍生物或者盐类，是一种广泛存在于动物、植物和微生物细胞中的活性物质，是生物合成叶绿素、血红素、卟啉和维生素B 12等四吡咯化合物的重要前体物质，其对人畜无毒性，在环境中易降解、无残留，作为生物体内源性物质，在生命过程中发挥着重要的作用，其在家畜饲料中添加应用已经有报道，但是还没有任何关于在水产饲料添加剂的应用。
微生态制剂对养殖生物排泄物、残饵的分解、转化、水质的调节有重要作用，是一种新型绿色饲料添加剂。芽孢杆菌是普遍存在的一类好氧性细菌，其具有稳定性好、抗逆性强、成活率高等优点，可代谢产生多种消化酶，降解饲料中的部分抗营养因子，提高饲料转化率；芽孢杆菌还可以通过降低水体中硝酸盐、亚硝酸盐的含量，改善养殖水体水质。HadiZokaeifar等(Zokaeifar，2014)向白虾养殖水体中投加枯草芽孢杆菌制剂8周后，NH 4 +-N和NO 2 --N浓度显著降低(P<0.05)，保持养殖过程中良好的水质。
在水产(包括鱼类、虾蟹类)养殖中，为了在单位面积和单位时间内获得更高的经济效益，水产类的养殖密度逐步提高。爱尔兰Hatch Aquaculture Accelerator的调查显示，6个养虾大国中，中国的养殖密度最高，平均达到220-300尾虾/平米成活率在低至60％左右。养殖密度过大时，鱼、虾等水生生物的呼吸增加，耗氧量也增加，容易造成水体中溶解氧的缺乏，水产动物成活率下降。此外，大量的排泄物以及残余饵料会在养殖池底聚集，从而会导致底泥缺氧以及病原菌滋生，对水产生物成活及生长造成威胁。如何有效的改善高密度鱼虾养殖过程中缺氧状况，保持养殖过程中良好的水质，提高养殖成活率，是目前水产养殖面临的主要问题之一，现有研究中效果较好的添加剂有维生素C、核苷酸、腐殖酸、海带、甜菜碱、小肽、虾青素、柠檬苦素类似物、肉毒碱盐酸盐、糖萜素、几丁聚糖和中药添加剂等，均可一定程度上缓解养殖应激，提高生产性能及成 活率，但是，对于在高密度养殖氧气缺乏的条件下未见有较好效果；另一方面解决鱼虾高密度养殖成活率的方法集中在改善养殖设备或者降低养殖密度，有效的饲料添加剂尚未见研究报道。
发明公开
本发明的目的就是针对高密度鱼虾养殖由于氧气不足和水质变差导致的成活率降低的情况，通过在饲料中添加含有5-ALA或(芽孢杆菌与5-ALA合用)作为饲料添加剂，一方面可以改善缺氧状况，另一方面通过改善水质和促进水产动物的机体健康，可协同改善高密度鱼虾养殖过程中的成活率。
本发明提供一种用于提高水产养殖动物成活率的添加剂，包含活性物质和芽孢杆菌，所述活性物质为5-氨基乙酰丙酸、其衍生物或其盐中的任一种化合物。
其中，所述芽孢杆菌选自枯草芽孢杆菌、地衣芽孢杆菌和凝结芽孢杆菌中的至少一种。
其中，所述添加剂中的5-氨基乙酰丙酸和芽孢杆菌是与其他水产饲料组分均匀混合后制粒；其中，5-氨基乙酰丙酸与芽孢杆菌的质量：有效活菌比为1mg：10 4-10 7cfu。
其中，所述添加剂中活性物质和芽孢杆菌的配比为每20mg5-氨基乙酰丙酸：10 6-8cfu芽孢杆菌。
本发明还提供一种饲料，所述饲料包含所述的添加剂。
其中，所述饲料中添加剂的添加量为1mg-1000mg/Kg。
其中，所述活性物质在水产饲料中的添加量为1mg-100mg/Kg；所述芽孢杆菌的添加量为10 4-10 8cfu/Kg饲料。
其中，所述芽孢杆菌的添加量为为10 5-10 7cfu/Kg饲料。
本发明提供一种水产养殖方法，使用所述的饲料添加剂或者所述的饲料投喂水产动物。
其中，所述添加剂施用方式为将5-ALA与芽孢杆菌一起添加到饲料中均匀制粒后投喂；或者将5-ALA添加到饲料中均匀制粒，在投喂的同时将 芽孢杆菌均匀泼洒于养殖水体中。
其中，所述芽孢杆菌投放量为10 2-10 5cfu/mL养殖水体。
其中，所述芽孢杆菌投放量为10 3-10 4cfu/mL养殖水体。
其中，所述水产动物为南美白对虾、海白虾、河虾、鲫鱼、草鱼、鲤鱼、鲢鱼、大黄鱼、小龙虾或罗非鱼中的至少一种。
所述添加剂在水产养殖中的应用也应在本发明的保护范围之内。
所述水产养殖的水产动物为南美白对虾、海白虾、河虾、鲫鱼、草鱼、鲤鱼、鲢鱼、大黄鱼、小龙虾或罗非鱼中的至少一种。
所述饲料在水产养殖中的应用也应在本发明的保护范围之内。
所述水产养殖的水产动物为南美白对虾、海白虾、河虾、鲫鱼、草鱼、鲤鱼、鲢鱼、大黄鱼、小龙虾或罗非鱼中的至少一种。
实施发明的最佳方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
下述实施例中的5-ALA、地衣芽孢杆菌、枯草芽孢杆菌和凝结芽孢杆菌均购自北京挑战生物技术有限公司。
实施例1
将本发明中的饲料添加剂应用于高密度鲫鱼养殖，池塘面积为0.08hm 2。试验鲫鱼在试验开始前先在水泥池中暂养2周，暂养结束后称重放入围格中，围格的规格为：4m×4m×1.5m，试验在18个网格中进行。将初重相近的鲫鱼(43.88±1.36g)2700条，设置4个处理组，1个对照组和5个试验组，每个处理组3个重复，每个重复150尾鲫鱼，试验时间为21天。对照组饲喂基础常规饲料，处理1饲喂的是在对照组饲料基础上加入5-ALA(10mg/Kg饲料)和地衣芽孢杆菌(2×10 6cfu/Kg饲料)，处理2饲喂对照组饲料基础上加入5-ALA(20mg/Kg饲料)和地衣芽孢杆菌 (2×10 6cfu/Kg饲料)，处理3饲喂对照组饲料基础上同时添加5-ALA(30mg/Kg饲料)和地衣芽孢杆菌(2×10 6cfu/Kg饲料)，处理4组饲喂对照组饲料基础上同时添加5-ALA(40mg/Kg饲料)和地衣芽孢杆菌(2×10 6cfu/Kg饲料)，处理5饲喂对照组饲料基础上同时添加5-ALA(50mg/Kg饲料)和地衣芽孢杆菌(2×10 6cfu/Kg饲料)，其中地衣芽孢杆菌规格为2×10 7cfu/g，使用时添加100g/吨，5-ALA和芽孢杆菌施用方式是与其他饲料组分均匀混合后，制粒投喂。饲喂方式：每天2次(9：00和15：00)。投喂量为鲫鱼重量的3％。试验条件和饲养管理按常规进行。试验结束时，捞取每个处理所有存活的鱼，计数并计算成活率；同时，每个试验围栏中随机抽取10条鱼，采用文齐氏液法测定血液中血红蛋白含量，采用Folin-酚法测定肠道中蛋白酶的活性，采用淀粉-碘量法测定肠道中的淀粉酶活性。
表1 5-ALA与芽孢杆菌对鲫鱼成活率、血红蛋白含量和肠道消化酶活性的影响
Figure PCTCN2021120944-appb-000001
Figure PCTCN2021120944-appb-000002
如表1所示，通过施加本发明中的提高鱼虾养殖成活率的饲料添加剂，实施例中设置了6个处理组中，与对照组相比，处理1、2、3、4和5组高密度养殖鲫鱼的成活率分别达到了98.26％、99.20％、98.42％、97.56％和97.17％，比基础日粮组提高了7.13、8.07、7.29、6.43和6.04个百分点。水产动物缺氧状态的改善，会引起血液载氧量，载氧量可以通过血液红细胞数增多、血红蛋白浓度增大等来实现，本实施例结果表明，鲫鱼血液中血红蛋白含量增加，可见该添加剂改善了鲫鱼在高密度养殖过程中的缺氧状态；一般认为，水产动物体内消化酶活性，对其吸收利用饵料的能力会有很大影响，从而促进其生长。与对照组相比，施用本发明中的添加剂，血红蛋白含量显著增加5.44％、6.93％、4.75％、5.16％和3.41％，肠道蛋白酶活性显著提高12.22％、14.24％、13.43％、9.81％和5.84％，淀粉酶活性分别提高7.41％、13.68％、8.14％、2.35％和3.86％(表1)。此外，芽孢杆菌还能通过产生抗体和促进噬菌作用等刺激免疫，激发体液免疫和细胞免疫，使动物免疫活性增强。
实施例2
选取南美白对虾，虾苗16800尾。虾苗暂养5天，称初始体重后开始试验。初始体重为1.18±0.37g。饲养设施为1000L的玻璃纤维桶，将16800尾虾苗平均分配。设置7个处理组，1个对照组和6个试验组，每个处理 组3个重复，每个重复800尾虾，试验时间为14天。对照组饲喂基础常规饲料，处理1饲喂在对照组饲料基础上加入5-ALA(20mg/Kg饲料)，处理2饲喂对照组饲料基础上添加枯草芽孢杆菌(1×10 3cfu/mL水体)，处理3饲喂对照组饲料基础上同时添加5-ALA(20mg/Kg饲料)和枯草芽孢杆菌(1×10 2cfu/mL水体)，处理4饲喂对照组饲料基础上添加5-ALA(20mg/Kg饲料)和枯草芽孢杆菌(1×10 3cfu/mL水体)，处理5饲喂对照组饲料基础上添加5-ALA(20mg/Kg饲料)和枯草芽孢杆菌(1×10 4cfu/mL水体)，处理6饲喂对照组饲料基础上添加5-ALA(20mg/Kg饲料)和枯草芽孢杆菌(1×10 5cfu/mL水体)。其中5-ALA加入量为20mg/Kg饲料，与其他饲料组分均匀混合后，制粒投喂；枯草芽孢杆菌粉剂兑水均匀泼洒相应质量(处理1,2,3,4,5,6在1000L水中分别泼洒质量为0,5,0.5,5,50,500g)的菌量规格2×10 8cfu/g的菌剂于每个1000L的桶中，每周泼洒一次)。试验中水体温度25℃，盐度30ppt。投喂方式：每天4次(6：00，10：00，15：00，20：00)。投喂量为虾重量的5％。试验条件和饲养管理按常规进行。试验结束时，捞取每个桶中所有存活的虾，称重，计算成活率和FCR。
成活率(％)＝末期对虾数量/初始对虾数量×100
饲料转化率(FCR)＝摄入饲料量/净增重量
表2 5-ALA与芽孢杆菌对南美白对虾成活率和FCR的影响
Figure PCTCN2021120944-appb-000003
通过施加本发明中的提高鱼虾养殖成活率的饲料添加剂，结果表明(表2)处理1和处理2组分别饲喂的添加剂组成为5-ALA(添加量为 20mg/Kg饲料)和枯草芽孢杆菌(添加量为枯草芽孢杆菌1×10 3cfu/mL水体)，结果显示南美白对虾的成活率分别提高了5.37和5.06个百分点，饲料转化率从1.13分别降低至1.08和1.08；处理3、4、5和6组分别饲喂本发明中的添加剂组成是5-ALA与不同量的枯草芽孢杆菌协同添加，南美白对虾的成活率分别达到了96.02％、98.06％、96.80％和94.99％，比基础日粮组分别提高了7.36、9.40、8.14、和6.33个百分点；同时，饲料转化率由1.13降低至1.04、1.01、1.03和1.04，在成活率和饲料转化率两个指标上都表现出协同效应。此外，通过成活率和饲料转化率的改善，可明显降低生产成本，进而提高了鱼虾养殖收益。
实施例3
将本发明中的饲料添加剂应用于罗非鱼养殖，池塘面积为0.08hm 2。试验鱼在试验开始前先在水泥池中暂养2周，暂养结束后称重放入围格中，围格的规格为：4m×4m×1.5m。选择5400尾(平均体重为2.59±0.51g，平均体长为4.42±0.31cm)驯养后的吉富罗非鱼幼鱼，随机分配到18个水族箱中，将18个水族箱随机分为6个处理，每个处理3个重复，每个重复300尾，处理组分别为对照组为基础日粮；处理1组为20mg/Kg 5-ALA+100g/吨枯草芽孢杆菌，即芽孢杆菌添加量为10 7cfu/Kg饲料；处理2组为添加20mg/Kg 5-ALA+100g/吨凝结芽孢杆菌，即芽孢杆菌添加量为10 7cfu/Kg饲料；处理3组为20mg/Kg 5-ALA+5g/吨枯草芽孢杆菌与5g/吨凝结芽孢杆菌复合添加，即芽孢杆菌添加量为10 6cfu/Kg饲料；处理4组为20mg/Kg5-ALA+50g/吨枯草芽孢杆菌与50g/吨凝结芽孢杆菌复合添加，即添加量为10 7cfu/Kg饲料；处理5组为20mg/Kg 5-ALA+500g/吨枯草芽孢杆菌与500g/吨凝结芽孢杆菌复合添加，即添加量为10 8cfu/Kg饲料；其中枯草芽孢杆菌规格为1×10 8cfu/g，凝结芽孢杆菌规格为1×10 8cfu/g，5-ALA和芽孢杆菌施用方式是与其他饲料组分均匀混合后，制粒投喂。投喂方式：每天3次(6：00，13：00和20:00)，投喂量为罗非鱼重量的3％。试验条件和饲养管理按常规进行，饲养42d。试验结束时，捞取每个处理所有存活的 鱼，计数并计算成活率；将吉富罗非鱼胃和肠道分离，准确称重，按重量体积比1∶9进行冰浴匀浆，胃和肠道蛋白酶测定采用福林酚法。
表3 5-ALA协芽孢杆菌对罗非鱼成活率、消化道消化酶活性的影响
Figure PCTCN2021120944-appb-000004
鱼虾养殖效益与营养物质的消化吸收能力与消化酶活性的强弱成正相关。本实施例结果表明(表3)，相对于饲喂基础日粮的对照组，施用本发明所述饲料添加剂后，处理1、处理2、处理3、处理4和处理5中的罗非鱼的成活率、胃蛋白酶和肠道蛋白酶的活性都起到了显著改善的作用，5-ALA和芽孢杆菌协同改善罗非鱼的成活率，分别达到了97.75％、97.64％、98.15％、同时表明，芽孢杆菌的添加量对成活率、蛋白酶和肠道蛋白酶的活性作用较大，在添加量为10 6-10 8cfu/Kg饲料时效果最好；且枯草芽孢杆菌与凝结芽孢杆菌复合添加的成活率稍高于单独施用。
工业应用
本发明所述的添加剂可通过改善鱼虾养殖(特别是高密度鱼虾养殖)过程中鱼虾机体的缺氧状况，并可改善养殖水体水质，从而降低鱼虾生病率，养殖成活率达到98％以上，降低鱼虾养殖成本，提高养殖效益。
所述提高水产动物成活率的饲料添加剂的作用效果反应机理：5-ALA可单独作用或与芽孢杆菌通过协同作用，一方面增强水产动物的免疫反应，主要由于5-ALA上调了免疫和防御相关基因表达，并增强了有氧能量代谢能力，进而可以增加细胞内ATP水平，同时，5-ALA的添加也会影响其他一些途径(Yamada et.al，2017)，线粒体合成的血红素通过线粒体膜运输，调节一系列有氧代谢过程，如转录、翻译、运输、加工和细胞分化等；另一方面，芽孢杆菌可将养殖水体中的污染物降解转化，同时其自身微生物代谢过程中产生的有益因子，可促进水产动物的生长和发育；通过5-ALA和芽孢杆菌对水产动物自身免疫和生长水质的改善，起到协同增效的作用。

Claims (17)

  1. 一种用于提高水产养殖动物成活率的添加剂，其特征在于，包含活性物质和芽孢杆菌，所述活性物质为5-氨基乙酰丙酸、其衍生物或其盐中的任一种化合物。
  2. 如权利要求1所述的添加剂，其特征在于，所述芽孢杆菌选自枯草芽孢杆菌、地衣芽孢杆菌和凝结芽孢杆菌中的至少一种。
  3. 如权利要求1或2的添加剂，其特征在于，所述添加剂中的5-氨基乙酰丙酸和芽孢杆菌是与其他水产饲料组分均匀混合后制粒；其中，5-氨基乙酰丙酸与芽孢杆菌的质量：有效活菌比为1mg：10 4-10 7cfu。
  4. 如权利要求3所述的添加剂，其特征在于，所述添加剂中活性物质和芽孢杆菌的配比为每20mg5-氨基乙酰丙酸：10 6-8cfu芽孢杆菌。
  5. 一种饲料，其特征在于，所述饲料包含权利要求1-4任一所述的添加剂。
  6. 如权利要求5所述的饲料，其特征在于，所述饲料中添加剂的添加量为1mg-1000mg/Kg。
  7. 如权利要求6所述的饲料，其特征在于，所述活性物质在水产饲料中的添加量为1mg-100mg/Kg；所述芽孢杆菌的添加量为10 4-10 8cfu/Kg饲料。
  8. 如权利要求7所述的饲料，其特征在于，所述芽孢杆菌的添加量为为10 5-10 7cfu/Kg饲料。
  9. 一种水产养殖方法，其特征在于，使用权利要求1-4任一所述的饲料添加剂或者权利要求5-8任一所述的饲料投喂水产动物。
  10. 如权利要求9所述的方法，其特征在于，所述添加剂施用方式为将5-ALA与芽孢杆菌一起添加到饲料中均匀制粒后投喂；或者将5-ALA添加到饲料中均匀制粒，在投喂的同时将芽孢杆菌均匀泼洒于养殖水体中。
  11. 如权利要求10所述的方法，其特征在于，所述芽孢杆菌投放量 为10 2-10 5cfu/mL养殖水体。
  12. 如权利要求10所述的方法，其特征在于，所述芽孢杆菌投放量为10 3-10 4cfu/mL养殖水体。
  13. 如权利要求9-12任一所述的方法，其特征在于，所述水产动物为南美白对虾、海白虾、河虾、鲫鱼、草鱼、鲤鱼、鲢鱼、大黄鱼、小龙虾或罗非鱼中的至少一种。
  14. 如权利要求1-4中任一项所述添加剂在水产养殖中的应用。
  15. 如权利要求14所述的应用，其特征在于，所述水产养殖的水产动物为南美白对虾、海白虾、河虾、鲫鱼、草鱼、鲤鱼、鲢鱼、大黄鱼、小龙虾或罗非鱼中的至少一种。
  16. 如权利要求5-8任一所述饲料在水产养殖中的应用。
  17. 如权利要求16所述的应用，其特征在于，所述水产养殖的水产动物为南美白对虾、海白虾、河虾、鲫鱼、草鱼、鲤鱼、鲢鱼、大黄鱼、小龙虾或罗非鱼中的至少一种。
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