Feed additive composition for relieving toxicity of algal toxin as well as preparation and application of feed additive composition

Abstract

The invention discloses a feed additive composition for relieving microcystin toxicity and preparation and application thereof.The composition is composed of eucommia ulmoides extract, astragalus membranaceus extract, radix bupleuri extract and macleaya cordata extract.The plant extract composition is added into feed, so that the growth performance of aquatic organisms can be promoted, the oxidation resistance and immunity are enhanced, and the microcystin toxicity can be relieved. The survival rate under the stress of the microcystic toxins is improved. The raw materials are non-medication parts of Chinese herbal medicines, and are low in price, ecological, environment-friendly, healthy and pollution-free.

Description

Technical Field

[0001] This invention relates to the field of aquaculture technology, and more specifically to a feed additive composition for mitigating the toxicity of algal toxins, its preparation, and its application. Background Technology

[0002] Cyanobacterial blooms are a common microbial hazard in aquaculture and have become a serious global public health and environmental problem. The algal toxins produced by cyanobacteria severely endanger the health of freshwater farmed animals and the safety of aquatic products. Algal toxins (MCs) have more than 100 different homologues and are classified as hepatotoxins. Microcystin-releasing agents (MC-LRs) are the most common and potent cyanobacterial toxins. MC-LRs can cause damage to the hepatopancreas, head, kidneys, and spleen of aquatic animals, and can also cause intrahepatic hemorrhage and centrilobular necrosis. Furthermore, MC-LRs also have immunotoxicity. MC stimulation can alter several immune-related genes and pathways in animals, thereby affecting the organism's immune system.

[0003] In recent years, with the rapid development of intensive aquaculture, environmental problems in shrimp farming have led to a decline in survival rates. Eutrophication causes excessive growth of cyanobacteria, producing macrophytes (MCs), which harm shrimp health. In aquatic ecosystems, MCs are a major cause of environmental stress and a potential health hazard. Farmers often use drugs to alleviate the environmental stress on farmed shrimp. However, with increasingly stringent environmental regulations both domestically and internationally, the illegal use of antibiotics and chemical drugs has been explicitly prohibited. Therefore, finding healthy, environmentally friendly, and highly effective antibiotic alternatives in feed additives is urgently needed. Healthy and pollution-free plant extracts will become the preferred choice for screening feed additives.

[0004] Plants contain numerous bioactive components that are safe and environmentally friendly, possessing immense potential application value and making them the preferred resource for developing antibiotic alternatives in feed additives. Furthermore, traditional Chinese herbal medicines generate many unused byproducts during their utilization, yet these byproducts also contain various active ingredients. These byproducts are inexpensive and readily available; utilizing them to develop feed additives is not only environmentally friendly but also conserves biological resources. Therefore, the research and screening of plant-derived antibiotic alternative feed additives will undoubtedly become a research hotspot. Summary of the Invention

[0005] In view of this, the present invention provides a plant extract composition for resisting microcystin stress, its preparation method and application, which can improve the survival rate of aquatic organisms and other organisms under microcystin stress, and enhance their antioxidant capacity and immunity.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] This invention provides a feed additive composition for mitigating the toxicity of algal toxins, comprising Eucommia ulmoides extract, Astragalus membranaceus extract, Bupleurum chinense extract, and Lysimachia christinae extract.

[0008] Preferably, the additive composition is added to the feed at the following amounts: 1 g / kg of Eucommia ulmoides extract, 0.5 g / kg of Astragalus membranaceus extract, 0.8 g / kg of Bupleurum chinense extract, and 0.04 g / kg of Angelica sinensis extract.

[0009] Preferably, the additive composition is added to the feed at the following amounts: 1 g / kg of Eucommia ulmoides extract, 1 g / kg of Astragalus membranaceus extract, 0.8 g / kg of Bupleurum chinense extract, and 0.04 g / kg of Angelica sinensis extract.

[0010] Preferably, the additive composition is added to the feed at the following amounts: 2 g / kg of Eucommia ulmoides extract, 1 g / kg of Astragalus membranaceus extract, 0.6 g / kg of Bupleurum chinense extract, and 0.04 g / kg of Angelica sinensis extract.

[0011] Furthermore, the feed comprises, by weight percentage, the following components:

[0012] Peanut bran 18%, blood meal 2%, soybean meal 15%, fish meal 26%, corn starch 10%, wheat flour 12%, cellulose 4.5%, fish oil 5%, shell powder 1%, calcium dihydrogen phosphate 1.5%, sodium carboxymethyl cellulose 1%, sodium alginate 2%, vitamin premix 1%, and mineral premix 1%.

[0013] Furthermore, per kilogram of basal feed, the vitamin premix includes: 45g biotin, 0.3g folic acid, 18g niacin, 7g calcium pantothenate, 8g vitamin C, 0.02g vitamin B12, 5g vitamin B6, 5g vitamin B2, 2g vitamin B12, 2g vitamin K3, 18IU vitamin E, 1000IU vitamin D3, and 9000IU vitamin A;

[0014] The mineral premix includes: Na2SeO3 0.02 g, CoSO4·7H2O 0.1 g, KI 0.04 g, FeSO4·H2O 25 g, MnSO4·H2O 15 g, ZnSO4·H2O 25 g and CuSO4·5H2O 2 g.

[0015] The present invention also provides a method for preparing the feed additive composition for mitigating algal toxin toxicity, comprising the following steps:

[0016] The stems and leaves of Eucommia ulmoides, Astragalus membranaceus, Bupleurum chinense, and Boletus edulis were extracted by hot reflux extraction, with 8, 6, and 6 times the volume of water extracted for 1 hour respectively. The filtrates were combined and concentrated under reduced pressure at 50°C to form an extract.

[0017] The concentrated extract was mixed with 4 times its volume of anhydrous ethanol and allowed to stand for one day. Then it was filtered and concentrated under reduced pressure at 40°C to form a paste to recover the ethanol.

[0018] The concentrated extract was dried at 40°C for 8 hours and then vacuum dried at 40°C for 6 hours. Finally, it was ground through an 80-mesh sieve and sterilized by irradiation to obtain the plant extract, which was stored at -20°C.

[0019] The present invention also provides the application of the feed additive composition for mitigating algal toxin toxicity, namely, its application in aquaculture to improve the growth performance, antioxidant capacity, immunity and resistance to algal toxin stress of aquatic organisms.

[0020] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a feed additive composition for mitigating algal toxin toxicity and its preparation method, which has the following beneficial effects:

[0021] Adding appropriate amounts of plant extracts, including Eucommia ulmoides extract, Astragalus membranaceus extract, Bupleurum chinense extract, and Clematis chinensis extract, to aquatic organism feed can improve the survival rate of aquatic organisms under microcystin stress and enhance their antioxidant capacity and immunity. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0023] Figure 1 The effect of dietary additives on the expression of antioxidant genes in red swamp crayfish;

[0024] Figure 2 The effect of adding a composition to the diet on the expression level of immune genes in redclaw crayfish. Detailed Implementation

[0025] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1

[0027] Taking the farming process of redclaw crayfish as an example,

[0028] A method for preparing a feed additive composition that mitigates the toxicity of algal toxins includes the following steps:

[0029] The stems and leaves of Eucommia ulmoides, Astragalus membranaceus, Bupleurum chinense, and Artemisia capillaris were extracted for 1 hour with 8, 6, and 6 times the amount of water, respectively, using a hot reflux extraction method. The filtrates were combined and concentrated under reduced pressure at 50°C to form a paste. The concentrated extract was mixed with 4 times the volume of anhydrous ethanol and allowed to stand for one day. Then it was filtered, concentrated under reduced pressure at 40°C to form a paste, and the ethanol was recovered. The concentrated extract was dried at 40°C for 8 hours and then vacuum dried at 40°C for 6 hours. Finally, it was ground through an 80-mesh sieve and sterilized by irradiation to obtain the plant extract, which was stored at -20°C for later use.

[0030] The feed formulation is shown in Table 1.

[0031] Table 1. Composition of basic feed for redclaw crayfish (by weight %)

[0032]

[0033]

[0034] Note: 1 g / kg of vitamin premix contains the following per kilogram of feed: Biotin (45 g), Folic acid (0.3 g), Niacin (18 g), Calcium pantothenate (7 g), Vitamin C (8 g), Vitamin B12 (0.02 g), Vitamin B6 (5 g), Vitamin B2 (5 g), Vitamin B1 (2 g), Vitamin K3 (2 g), Vitamin E (18 IU), Vitamin D3 (1000 IU), and Vitamin A (9000 IU);

[0035] The mineral premix 2 (g / kg) includes: Na2SeO3 (0.02g), CoSO4·7H2O (0.1g), KI (0.04g), FeSO4·H2O (25g), MnSO4·H2O (15g), ZnSO4·H2O (25g) and CuSO4·5H2O (2g).

[0036] Experimental Example

[0037] Based on the composition and basic feed determined by the preparation method in Example 1, the following experiments were conducted:

[0038] The control group was fed a basal diet. The experimental groups were named Mix1, Mix2, Mix3, Mix4, Mix5, Mix6, Mix7, Mix8, and Mix9.

[0039] Mix 1 group was supplemented with 1g / kg Eucommia ulmoides extract, 0.5g / kg Astragalus membranaceus extract, 0.6g / kg Bupleurum chinense extract, and 0.04g / kg Clematis chinensis extract in the basic feed.

[0040] Mix2 group was supplemented with 1g / kg Eucommia ulmoides extract, 0.5g / kg Astragalus membranaceus extract, 0.8g / kg Bupleurum chinense extract, and 0.04g / kg Clematis chinensis extract in the basal feed.

[0041] Mix3 group was supplemented with 1g / kg Eucommia ulmoides extract, 1g / kg Astragalus membranaceus extract, 0.6g / kg Bupleurum chinense extract, and 0.04g / kg Clematis chinensis extract in the basic feed.

[0042] Mix4 group was supplemented with 1g / kg Eucommia ulmoides extract, 1g / kg Astragalus membranaceus extract, 0.8g / kg Bupleurum chinense extract, and 0.04g / kg Clematis chinensis extract in the basal feed.

[0043] Mix5 group was supplemented with 2g / kg Eucommia ulmoides extract, 0.5g / kg Astragalus membranaceus extract, 0.6g / kg Bupleurum chinense extract, and 0.04g / kg Angelica sinensis extract in the basic feed.

[0044] Mix6 group was supplemented with 2g / kg Eucommia ulmoides extract, 0.5g / kg Astragalus membranaceus extract, 0.8g / kg Bupleurum chinense extract, and 0.04g / kg Angelica sinensis extract in the basic feed.

[0045] Mix7 group was supplemented with 2g / kg Eucommia ulmoides extract, 1g / kg Astragalus membranaceus extract, 0.6g / kg Bupleurum chinense extract, and 0.04g / kg Clematis chinensis extract in the basic feed.

[0046] Mix8 group was supplemented with 2g / kg Eucommia ulmoides extract, 1g / kg Astragalus membranaceus extract, 0.8g / kg Bupleurum chinense extract, and 0.04g / kg Clematis chinensis extract in the basic feed.

[0047] All the basic feed and extract raw materials were crushed and filtered through an 80-mesh sieve before being mixed. Then, oil and water were added and the mixture was ground into 1.5mm diameter pellets using a feed mill. Finally, the pellets were dried in an oven at 40℃ and stored at 4℃ for later use.

[0048] Healthy, disease-free redclaw crayfish in their molting stage were selected for the experiment. Before the experiment, the crayfish were temporarily housed in the culture system for two weeks. Redclaw crayfish with an average weight of (0.28±0.02) g were randomly assigned to culture tanks (500L), with three replicates per group and 30 crayfish per replicate. To prevent cannibalism during culture, 40 PVC pipes were placed parallel to each tank. The crayfish were fed to satiation at 8:00 AM and 6:00 PM daily. The culture period was 8 weeks.

[0049] After the aquaculture experiment, samples were collected from the starved swamp crayfish 24 hours later. All crayfish in the experimental groups were harvested, counted, and weighed. Blood samples from the redclaw crayfish were drawn from the basal sinus of the third pair of appendages using a 2.5 mL syringe containing an anticoagulant (26.3 g / L NaCl, 18.0 g / L glucose, 6.3 g / L citric acid, 6.7 g / L sodium citrate, 2.9 g / L EDTA). The drawn blood was centrifuged (3000 rpm, 10 min, 4 °C), and the supernatant was separated and stored at -80 °C for later use. Redclaw crayfish hepatopancreas and intestinal samples were separated, flash-frozen in liquid nitrogen, and stored at -80 °C for later use. Redclaw crayfish dorsal muscle samples were stored at -20 °C for muscle nutrient composition determination.

[0050] The experimental statistics and measurement calculation results are as follows:

[0051] 1) Effects on growth performance

[0052] As shown in Table 2, compared with the control group, the weight gain rate and specific growth rate of the Mix2, Mix4, Mix7 and Mix8 groups were significantly increased (P<0.05).

[0053] Table 2. Effects of different extract complexes on the growth performance of redclaw crayfish

[0054]

[0055] 2) Antioxidant capacity

[0056] As shown in Table 3, compared with the control group, the total antioxidant capacity, superoxide dismutase, glutathione peroxidase, and catalase activities of Mix2, Mix4, and Mix7 groups were significantly increased (P<0.05), while the malondialdehyde content was significantly decreased.

[0057] Table 3. Effects of different extract complexes on the antioxidant capacity of the hepatopancreas of red swamp crayfish.

[0058]

[0059] 3) Immunity

[0060] Table 4 shows that, compared with the control group, the addition of extract complexes (Mix1-8) significantly increased the acid phosphatase activity of crayfish (P<0.05). The alkaline phosphatase activities of Mix2, Mix3, Mix4, Mix5, Mix6, Mix7, and Mix8 groups were significantly higher than those of the control group (P<0.05). The phenol oxidase activities of Mix2, Mix4, and Mix7 groups were significantly increased compared with the control group (P<0.05).

[0061] Table 4. Effects of different extract complexes on serum immune indicators of redclaw crayfish

[0062]

[0063]

[0064] 4) Expression of antioxidant and immune-related genes

[0065] Antioxidant gene expression

[0066] like Figure 1 As shown, compared with the control group, the expression levels of superoxide dismutase (SOD), thioredoxin 1 (Trx1), and glutathione S-transferase 1 (GST1) genes in the Mix2, Mix4, and Mix7 groups were significantly increased (P < 0.05).

[0067] Immune gene expression

[0068] like Figure 2 As shown, compared with the control group, the expression level of lysozyme (LZM) in Mix4 and Mix5 groups was significantly increased (P < 0.05); the expression levels of anti-lipopolysaccharide factor (ALF) and nuclear factor κB inhibitory protein α (NFKBIA) genes in Mix4 and Mix7 were significantly increased (P < 0.05).

[0069] 5) Resistance to algal toxin stress

[0070] Compared with the control group, the survival rate of crayfish in the Mix2, Mix4 and Mix7 groups was significantly increased under microcystin stress (P < 0.05).

[0071] Table 5. Effects of different extract complexes on the survival rate of redclaw crayfish under algal toxin stress.

[0072]

[0073]

[0074] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on its differences from other embodiments. Similar or identical parts between embodiments can be referred to interchangeably. For the apparatus disclosed in the embodiments, since they correspond to the methods disclosed in the embodiments, the description is relatively simple; relevant parts can be referred to the method section.

[0075] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A feed additive composition for mitigating the toxicity of algal toxins, characterized in that, It includes extracts of Eucommia ulmoides, Astragalus membranaceus, Bupleurum chinense, and Clematis chinensis.

2. The feed additive composition for mitigating algal toxin toxicity according to claim 1, characterized in that, The additive composition is added to the feed at the following amounts: 1 g / kg of Eucommia ulmoides extract, 0.5 g / kg of Astragalus membranaceus extract, 0.8 g / kg of Bupleurum chinense extract, and 0.04 g / kg of Angelica sinensis extract.

3. The feed additive composition for mitigating algal toxin toxicity according to claim 1, characterized in that, The additive composition is added to the feed at the following amounts: 1 g / kg of Eucommia ulmoides extract, 1 g / kg of Astragalus membranaceus extract, 0.8 g / kg of Bupleurum chinense extract, and 0.04 g / kg of Angelica sinensis extract.

4. The feed additive composition for mitigating algal toxin toxicity according to claim 1, characterized in that, The additive composition is added to the feed at the following amounts: 2 g / kg of Eucommia ulmoides extract, 1 g / kg of Astragalus membranaceus extract, 0.6 g / kg of Bupleurum chinense extract, and 0.04 g / kg of Angelica sinensis extract.

5. The feed additive composition for mitigating algal toxin toxicity according to any one of claims 1-4, characterized in that, The feed comprises the following components by weight percentage: Peanut bran 18%, blood meal 2%, soybean meal 15%, fish meal 26%, corn starch 10%, wheat flour 12%, cellulose 4.5%, fish oil 5%, shell powder 1%, calcium dihydrogen phosphate 1.5%, sodium carboxymethyl cellulose 1%, sodium alginate 2%, vitamin premix 1%, and mineral premix 1%.

6. The feed additive composition for mitigating algal toxin toxicity according to claim 5, characterized in that, Its features are, Based on per kilogram of basal feed, the vitamin premix includes: 45g biotin, 0.3g folic acid, 18g niacin, 7g calcium pantothenate, 8g vitamin C, 0.02g vitamin B12, 5g vitamin B6, 5g vitamin B2, 2g vitamin B12, 2g vitamin K3, 18IU vitamin E, 1000IU vitamin D3, and 9000IU vitamin A; The mineral premix includes: Na2SeO3 0.02 g, CoSO4·7H2O 0.1 g, KI 0.04 g, FeSO4·H2O 25 g, MnSO4·H2O 15 g, ZnSO4·H2O 25 g and CuSO4·5H2O 2 g.

7. A method for preparing a feed additive composition for mitigating algal toxin toxicity according to any one of claims 1-4, characterized in that, Includes the following steps: The stems and leaves of Eucommia ulmoides, Astragalus membranaceus, Bupleurum chinense, and Boletus edulis were extracted by hot reflux extraction, with 8, 6, and 6 times the volume of water extracted for 1 hour respectively. The filtrates were combined and concentrated under reduced pressure at 50°C to form an extract. The concentrated extract was mixed with 4 times its volume of anhydrous ethanol and allowed to stand for one day. Then it was filtered and concentrated under reduced pressure at 40°C to form a paste to recover the ethanol. The concentrated extract was dried at 40°C for 8 hours and then vacuum dried at 40°C for 6 hours. Finally, it was ground through an 80-mesh sieve and sterilized by irradiation to obtain the plant extract, which was stored at -20°C.

8. The application of a feed additive composition for mitigating algal toxin toxicity according to any one of claims 1-4 in aquaculture, characterized in that, It is used to improve the growth performance, antioxidant capacity, immunity and resistance to algal toxin stress of aquatic organisms.

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