Aquatic feed additive for relieving heat stress of aquatic animals, feed and preparation method thereof

Abstract

The invention discloses an aquatic feed additive and feed for relieving heat stress of aquatic animals and a preparation method thereof, and the aquatic feed additive comprises the following components in parts by weight: 3-10 parts of a yeast culture, 0.5-2 parts of a dandelion extract, 0.5-2 parts of a citrus peel extract, 1-3 parts of beer yeast hydrolysate and 1-3 parts of a grape seed extract. Through optimal combination and synergistic effect of the yeast culture and various plant extracts, the comprehensive capability of the aquatic animals for resisting heat stress is improved in a multi-way and multi-level manner, adverse effects such as survival rate reduction caused by heat stress are effectively relieved, and the production performance of the aquatic animals is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of aquatic feed, and in particular to an aquatic feed additive for alleviating heat stress of aquatic animals, feed and a preparation method thereof. Background Art

[0002] The Chinese mitten crab (Eriocheir sinensis), an important economic crustacean, possesses unique physiological characteristics. During its growth, the crab undergoes multiple molting cycles to adapt to its increasing size. Studies have shown that heat stress can affect the molting process of the crab. Some studies have found that under high-temperature conditions, the molting frequency of crabs may increase, possibly due to changes in hormone levels and accelerated metabolism caused by heat stress. However, frequent molting can also lead to excessive energy consumption, affecting the crab's growth and survival. Furthermore, crabs are territorial and aggressive, making them prone to fighting under high-density culture conditions. Heat stress can exacerbate crabs' aggressive behavior, leading to individual injuries or even death, thus affecting aquaculture efficiency.

[0003] The intestine is a special functional organ in aquatic animals. It not only digests food and absorbs nutrients, but also has an intestinal mucosal barrier function, which can prevent harmful substances such as bacteria and toxins in the cavity from invading the body. It is an important innate immune barrier in the body and is closely related to the health and growth performance of aquatic animals. Studies have shown that heat stress can cause intestinal damage and oxidative stress in aquatic animals, and can cause pathogens to invade the intestine, leading to inflammation and disease. In addition, heat stress may also cause changes in the structure of the intestinal flora, resulting in a decrease in beneficial bacteria and an increase in harmful bacteria, further affecting the health of the host. Therefore, maintaining intestinal integrity, improving antioxidant capacity, and regulating the balance of intestinal flora are important strategies to alleviate heat stress in Chinese mitten crabs. Summary of the Invention

[0004] In view of the problems existing in the above-mentioned prior art, the present invention provides an aquatic feed additive, feed and preparation method thereof for alleviating heat stress in aquatic animals. Through the optimized combination and synergistic effect of yeast culture and multiple plant extracts, the feed improves the comprehensive ability of aquatic animals to resist heat stress in multiple ways and at multiple levels, effectively alleviates the adverse effects such as decreased survival rate caused by heat stress, and significantly improves the production performance of aquatic animals.

[0005] In a first aspect, the present invention provides an aquatic feed additive, comprising, by weight: 3-10 parts of yeast culture (YC), 0.5-2 parts of dandelion extract, 0.5-2 parts of citrus peel extract, 1-3 parts of brewer's yeast hydrolyzate, and 1-3 parts of grape seed extract.

[0006] Preferably, the aquatic feed additive comprises, by weight: 5-8 parts of yeast culture (YC), 0.5-1.5 parts of dandelion extract, 0.5-1.5 parts of citrus peel extract, 1-2 parts of brewer's yeast hydrolyzate and 1.5-2 parts of grape seed extract.

[0007] Preferably, the aquatic feed additive comprises, by weight, 4.5 parts of yeast culture (YC), 1 part of dandelion extract, 1.5 parts of citrus peel extract, 1 part of brewer's yeast hydrolyzate and 2.0 parts of grape seed extract.

[0008] Preferably, the aquatic feed additive comprises, by weight: 6.0 parts of yeast culture (YC), 0.5 parts of dandelion extract, 0.5 parts of citrus peel extract, 1.5 parts of brewer's yeast hydrolyzate and 1.5 parts of grape seed extract.

[0009] Among them, the yeast culture is prepared by fermenting a mixture of brewer's yeast (Saccharomyces cerevisiae), soybean meal, glucose and gluten matrix, which contains a variety of active substances, such as β-glucan, mannooligosaccharide, nucleotides and functional proteins, which have the effects of improving animal immunity, improving intestinal health and enhancing anti-stress ability; dandelion extract can be prepared by water extraction or ethanol extraction of dandelion roots, leaves or whole herbs, and contains a variety of active ingredients such as inulin, chicoric acid, dandelion sterols, triterpenoid saponins, etc., and has pharmacological effects such as antioxidant, choleretic and digestive promotion; citrus peel extract can be prepared by water extraction or supercritical CO2 extraction, and mainly contains flavonoids such as limonin and hesperidin, and has biological activities such as antioxidant and anti-inflammatory; grape seed extract can be prepared by ethanol extraction, and its main components are proanthocyanidins and other multi-ingredients. Phenolic compounds have strong antioxidant capacity; the active ingredients of the above components are natural and safe, and can produce multiple synergistic effects after mutual compounding: on the one hand, the immune polysaccharides and bioactive peptides such as β-glucan and mannooligosaccharide provided by yeast culture can directly enhance the body's immunity and anti-stress ability; on the other hand, plant-derived antioxidants such as polyphenols and flavonoids can remove reactive oxygen free radicals produced by heat stress and maintain the body's redox balance; ingredients such as inulin and taraxasterol can regulate digestive function and promote nutrient absorption; nutrients such as polypeptides, amino acids, and nucleotides provide the necessary nutrients for cell repair and regeneration; multiple active ingredients work synergistically to comprehensively enhance the ability of aquatic animals to resist heat stress from multiple aspects such as anti-oxidation, immune regulation, intestinal protection, and nutrient supply.

[0010] In a second aspect, the present invention provides an aquatic compound feed, which comprises 8-10 parts by weight of the above-mentioned aquatic feed additive and 800-1000 parts by weight of a basic feed.

[0011] Preferably, the basic feed comprises, by weight percentage, 15% fish meal, 25% soybean meal, 25% cottonseed meal, 2% fish oil, 1.9% soybean oil, 0.5% soybean lecithin oil, 0.5% cholesterol, 0.5% choline chloride, 0.1% butylated p-cresol, 12% pregelatinized starch, 4% vitamin premix, 2% mineral premix, 2% sodium alginate, 1% betaine, 1% monocalcium phosphate and 7.5% cellulose.

[0012] In a third aspect, the present invention provides a method for preparing the above-mentioned aquatic feed, which comprises the following steps:

[0013] S1. Take all the raw materials of the above-mentioned aquatic feed additives by weight, grind all the raw materials, and sieve them through a 60-mesh sieve;

[0014] S2. Fully mixing all the sieved raw materials to obtain the aquatic feed additive;

[0015] S3. Take the basic feed and aquatic feed additives in the above-mentioned aquatic feed by weight, add deionized water at a ratio of 100-200 mL / kg of basic feed, and mix thoroughly to obtain a feed mixture;

[0016] S4, using a twin-screw extruder to make pellet feed with a diameter of 2-3 mm;

[0017] S5. Drying the pellet feed at room temperature;

[0018] S6. Sieve the dried pellet feed using 10, 14, and 16 mesh sieves in sequence;

[0019] S7. The feeds screened in step S6 are respectively put into ziplock bags and stored in a -20°C environment.

[0020] In a fourth aspect, the present invention provides a use of the above-mentioned aquatic compound feed in improving the heat stress resistance of aquatic animals.

[0021] Preferably, improving the heat stress resistance of aquatic animals includes: regulating the intestinal microbial community structure, improving the intestinal antioxidant capacity, alleviating heat stress damage, and maintaining the integrity of the intestinal barrier. One or more of the following.

[0022] Preferably, the aquatic animal includes Chinese mitten crab (Eriocheir sinensis).

[0023] In summary, the compound aquatic feed additive and its compound feed provided by the present invention, through the optimized combination and synergistic effect of yeast culture and various plant extracts, improve the comprehensive ability of aquatic animals to resist heat stress in multiple ways and at multiple levels, can effectively alleviate the adverse effects such as decreased survival rate caused by heat stress, and significantly improve the production performance of aquatic animals. The compound additive is easy to use, quick to take effect, broad-spectrum and efficient, and easy to promote and apply. At the same time, the raw materials of the present invention are natural, green and safe, the process route is simple and easy, and the production cost is low. It provides an economical, safe and effective solution for the aquaculture industry to cope with high temperature heat waves, and has important practical significance for maintaining the health of aquatic products, improving industrial benefits, and ensuring market supply. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the effect of the compound feed of the present invention on the weight gain rate, survival rate and molting frequency of heat-stressed Chinese mitten crabs;

[0025] Figure 2 This is a graph showing the effect of the compound feed of the present invention on molting-related hormones in heat-stressed Chinese mitten crabs;

[0026] Figure 3 This figure shows the effect of the compound feed of the present invention on the fighting behavior of heat-stressed Chinese mitten crabs;

[0027] Figure 4 This is a graph showing the effect of the compound feed of the present invention on the relative expression of heat shock protein genes in heat-stressed Chinese mitten crabs;

[0028] Figure 5 This is a graph showing the effect of the compound feed of the present invention on the antioxidant enzyme activity of heat-stressed Chinese mitten crab;

[0029] Figure 6 This figure shows the effect of the compound feed of the present invention on the intestinal tissue structure of heat-stressed Chinese mitten crabs;

[0030] Figure 7 This figure shows the effect of the compound feed of the present invention on the biomarkers of intestinal damage in heat-stressed Chinese mitten crabs.

[0031] Figure 8 This figure shows the effect of the compound feed of the present invention on the species richness of intestinal microorganisms of heat-stressed Chinese mitten crabs.

[0032] Figure 9 This figure shows the effect of the compound feed of the present invention on the intestinal microbial community structure of heat-stressed Chinese mitten crabs. DETAILED DESCRIPTION

[0033] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0034] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0035] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it constitute a separate or selective embodiment that is mutually exclusive of other embodiments.

[0036] Example:

[0037] 1. Preparation of experimental feed

[0038] A control group feed, experimental group feed 1, and experimental group feed 2 were prepared, respectively. The control group feed was the base feed, and experimental group feed 1 and experimental group feed 2 were both aquatic feeds according to the present invention, differing only in the ratio of aquatic feed additives. The formulas of the three feeds are shown in Table 1. Yeast culture (YC) was provided by Diamond V Biological & Fermentation Engineering Technologies (Shenzhen) Co., Ltd. and can be prepared by fermenting a mixture of Saccharomyces cerevisiae, soybean meal, glucose, and gluten.

[0039] Table 1 Experimental feed formula and its nutritional components

[0040] Raw material (g / kg) Control group feed Experimental group feed 1 Experimental group feed 2 fishmeal 150 150 150 soybean meal 250 250 250 cottonseed meal 250 250 250 Yeast culture (YC) 0 4.5 6.0 fish oil 20 20 20 soybean oil 19 19 19 Soy lecithin 5 5 5 cholesterol 5 5 5 Choline chloride 5 5 5 tert-Butyl-4-cresol 1 1 1 Pregelatinized starch 120 115 115 Vitamin premix 40 40 40 Mineral premix 20 20 20 Sodium alginate 20 20 20 Betaine 10 10 10 Calcium dihydrogen phosphate 10 10 10 cellulose 75 70 70 Dandelion extract 0 1 0.5 citrus peel extract 0 1.5 0.5 Brewer's yeast hydrolysate 0 1 1.5 grape seed extract 0 2 1.5

[0041] In Table 1, the vitamin premix and mineral premix are both existing technologies, and the raw material ratio composition thereof only needs to meet the growth requirements of Chinese mitten crab.

[0042] The control, experimental, and 2 feeds were dried at 105°C until their mass remained constant. The moisture content of the feeds was analyzed, crude protein content was determined using the Kjeldahl method, and crude fat was extracted using petroleum ether. The feed powder samples were then thoroughly carbonized in a carbonization furnace and then calcined in a crucible furnace at 550°C for at least 6 hours to determine their ash content. The moisture, crude protein, crude fat, and ash content of the three feeds are shown in Table 2.

[0043] Table 2 Feed moisture and composition results based on dry basis (%)

[0044] name Moisture ash content crude protein crude fat Control group feed 9.02 8.07 36.52 7.08 Experimental group feed 1 9.08 8.12 36.68 7.11 Experimental group feed 2 9.09 8.15 36.80 7.12

[0045] It can be seen that there is no significant change in the crude protein content and crude fat content of the three feeds, and all meet the nutritional needs of the normal growth and development of Chinese mitten crab.

[0046] 2. Experimental Design and Sample Collection

[0047] Chinese mitten crabs (Eriocheir sinensis) were purchased and acclimated in the laboratory for two weeks. During this period, the water temperature was maintained at 24.0±0.2°C and the dissolved oxygen level was maintained at 6.8±0.2 mg / L. After the acclimation period, 720 healthy crabs (weighing 2.82±0.01 g) were divided into four groups and placed in corresponding aquaculture tanks for culture: control group 1, control group 2, experimental group 1, and experimental group 2, with 180 crabs in each group. Each group had six replicates (n=6). The aquaculture tanks were all 87×65.3×61.5 cm in size, and arched tiles and corrugated plastic pipes were placed at the bottom of the tanks to serve as shelter for the Chinese mitten crabs.

[0048] The water temperature in control group 1 was maintained at 24.0±0.2℃, and the fish were fed the control group feed. The water temperature in control group 2 was maintained at 30.0±0.2℃, and the fish were fed the control group feed. The water temperature in experimental group 1 and experimental group 2 was maintained at 30.0±0.2℃, and the fish were fed the experimental group feed 1 and experimental group feed 2 respectively. Other conditions, such as pH range of 7.3-8.4, ammonia nitrogen concentration below 0.05mg / L, dissolved oxygen concentration above 7.0mg / L, etc., were the same for the four groups.

[0049] The crabs in each group were cultured for 42 days. During this period, each group was fed three times daily (at 8:00 AM, 2:00 PM, and 8:00 PM), with each feeding amount representing 4-6% of the crab seedlings' body weight. Normal daily care was also performed during the culture period, including siphoning off uneaten feed and feces, removing dead crabs, and recording molting frequency.

[0050] 3. Behavioral Test

[0051] After the breeding experiment, the survival rate, body weight and weight gain rate of each group of crab seedlings were evaluated. Half of the crabs in each tank were used for behavioral testing, and the rest were anesthetized for subsequent anatomical sampling. A total of 20 crabs of similar size and sex were randomly selected from 6 parallel breeding tanks in each treatment group (control group 1, control group 2, experimental group 1, experimental group 2) (3-4 crabs were selected from each tank). The selected crabs were randomly paired to form 10 pairs for testing. During the test, the two paired crabs were placed in a test tank of the same specifications at the same time, and their behavioral responses were observed and recorded, including three types: fighting each other, fleeing the scene, or no response. The higher the fighting ratio, the stronger the aggressiveness. All test crabs were used only once.

[0052] 4. Biochemical index analysis

[0053] The crabs were fasted for 24 hours before sampling. Ten crabs were randomly selected from each tank and anesthetized on crushed ice. Hemolymph was carefully extracted from the base of the appendages of each anesthetized crab using a 1 mL syringe and stored in 1.5 mL centrifuge tubes in a 4°C refrigerator overnight.

[0054] After hemolymph collection, the 10 crabs were immediately dissected. The hepatopancreas of each crab was removed with forceps. The hepatopancreas tissue from four crabs was immediately placed in liquid nitrogen and stored at -80°C for subsequent gene expression analysis. The hepatopancreas tissue from the remaining six crabs was mixed into one sample, and a homogenate (i.e., a 10% tissue homogenate) was prepared at a ratio of 1:9 tissue mass (g) to 0.85% saline volume (mL). The homogenate was placed in a 0°C water bath and centrifuged at 1500g for 15 minutes. The supernatant was collected and stored at -80°C for determination of malondialdehyde content and glutathione peroxidase and superoxide dismutase activities.

[0055] At the same time, intestinal tissue was removed and pooled from 10 crabs in the same tank to form a single sample. Intestinal tissue homogenates were prepared using the same method as for the hepatopancreatic tissue homogenate preparation. The homogenates were centrifuged at 1500 g for 15 minutes, and the supernatant was collected and stored at −80°C for histamine determination. Simultaneously, hindgut tissue was removed from four additional crabs randomly selected from each tank and stored in Bouin's solution for subsequent histological analysis. Intestinal contents were carefully collected and pooled from all four crabs in the same tank to form a single sample. The samples were placed in a sterile 1.5 mL centrifuge tube, immediately snap-frozen in liquid nitrogen, and subsequently stored at −80°C for subsequent intestinal microbiome analysis.

[0056] Overnight hemolymph samples were fully crushed with a syringe needle until no obvious clots were found, and centrifuged at 7000 rpm and 4°C for 10 minutes. The supernatant was carefully aspirated, divided into 0.2 mL centrifuge tubes, and stored in a -80°C refrigerator.

[0057] 5. Gene Expression Analysis

[0058] Total RNA was extracted from preserved hepatopancreatic tissue samples using TRIzol reagent. RNA was reverse transcribed into cDNA using a reverse transcription kit. The reaction volume was 20 μL, including 10 μL of 2× SYBR Green PCR Master Mix, 0.8 μL of upstream and downstream primers (10 μM), 1 μL of cDNA template, and 8.2 μL of nuclease-free water. qRT-PCR was performed using a real-time fluorescence quantitative PCR system, using β-actin as an internal reference gene, to compare relative mRNA expression levels among the groups.

[0059] All data are expressed as mean ± standard error (SEM), and data statistical analysis was performed using SPSS20.0 software. Under the same water temperature conditions, one-way analysis of variance (One-wayANOVA) was used to determine the significant differences between the groups at 30±0.2℃. If the one-way ANOVA indicated a significant difference, the Duncan multiple comparison method was used to evaluate the significance between the groups. In addition, the t-test was used to compare the differences between control group 1 and control group 2, experimental group 1, and experimental group 2, respectively. When p<0.05, the difference was considered statistically significant. Values ​​with different superscripts on the same line indicate significant differences, and the asterisk indicates the significance level (* indicates p<0.05; ** indicates 0.001 <p<0.01)。

[0060] VI. Gut Microbiota Sequencing and Analysis

[0061] use Genomic DNA was extracted from intestinal content samples using the Soil DNA Kit (Omega Biotek, USA). The V3-V4 region of the bacterial 16S rRNA gene was amplified and sequenced using the Illumina platform. OTUs were defined using UPARSE software with a 97% similarity threshold. Species classification was performed using the RDP Classifier against the Silva 138 database, and alpha and beta diversity indices were calculated using Qiagen Imaging (QIIME).

[0062] 7. Results Analysis

[0063] 1. Effects of aquatic feed on weight gain, survival rate and molting frequency of heat-stressed Chinese mitten crab

[0064] Depend on Figure 1It can be concluded that compared with the control group 1, after feeding the basic feed in a high temperature environment of 30°C, the weight gain rate of the Chinese mitten crab in the control group 2 increased significantly, but the survival rate decreased. After using the aquatic compound feed of the present invention, the survival rate of the Chinese mitten crab in the experimental group 1 and the experimental group 2 was significantly improved, while the weight gain rate remained unchanged. At the same time, the molting rate of the crabs in the experimental group 1 and the experimental group 2 was significantly reduced. This shows that the yeast culture in the compound feed of the present invention is compounded with other natural extracts, which can effectively alleviate the adverse effects of heat stress on the survival rate of the Chinese mitten crab through multiple mechanisms, while maintaining its growth performance, for example, it can keep the weight gain rate unchanged in a high temperature environment, and reduce the molting rate of the crab in a high temperature environment.

[0065] 2. Effects of aquatic feed on molting-related hormones in heat-stressed Chinese mitten crabs

[0066] Depend on Figure 2 It can be concluded that compared with the control group 1, the mRNA expression levels of crustacean hyperglycemic hormone and molting hormone in the hepatopancreas of the Chinese mitten crab in the control group 2 were significantly upregulated, while the mRNA expression level of molting-inhibiting hormone was significantly downregulated. When the aquatic feed additive of the present invention was added to the basic feed under a high temperature environment of 30°C, the mRNA expression levels of crustacean hyperglycemic hormone and molting hormone in the hepatopancreas of the Chinese mitten crab were significantly reduced, and their expression levels were similar to those of the control group 1, while the mRNA expression level of molting-inhibiting hormone was significantly upregulated. This shows that the synergistic effect of the various functional ingredients in the aquatic feed of the present invention can effectively alleviate the effect of heat stress on the abnormal increase in molting of the Chinese mitten crab at the molecular level.

[0067] 3. Effects of aquatic feed on the fighting behavior of heat-stressed Chinese mitten crabs

[0068] Depend on Figure 3 It can be concluded that compared with the control group 1, the number of Chinese mitten crabs showing fighting behavior in the control group 2 increased, while the number of escape scenes did not change significantly. When the aquatic feed additive of the present invention was added to the basic feed in a high temperature environment of 30°C, the number of Chinese mitten crabs showing fighting behavior gradually decreased. It can be seen that after the yeast culture is compounded with brewer's yeast hydrolysate, grape seed extract, dandelion extract, and citrus peel extract, the active ingredients produced, such as polypeptides and amino acids contained in brewer's yeast hydrolysate, can reduce the fighting behavior of Chinese mitten crabs by improving the function of the central nervous system, and have a significant heat stress relief effect at the behavioral level.

[0069] 4. Effect of aquatic feed on the relative expression of heat shock protein genes in heat-stressed Chinese mitten crabs

[0070] Depend on Figure 4It can be concluded that compared with the control group 1, the mRNA expression levels of the heat shock protein genes hsp70 (i.e., "heat shock 70") and hsp90 (i.e., "heat shock 90") in the hepatopancreas of the Chinese mitten crab in the control group 2 were significantly upregulated. After the aquatic feed additive of the present invention was added to the basal feed, the mRNA expression levels of hsp70 and hsp90 in the hepatopancreas of the Chinese mitten crab were significantly reduced, and the expression levels were similar to those in the control group 1. This result shows that the aquatic feed additive of the present invention can significantly reduce the expression of heat shock protein genes at the molecular level. For example, it can indirectly regulate the expression of heat shock proteins by inhibiting related signal pathways through flavonoids and polyphenols in citrus peel extract and grape seed extract, so as to alleviate the effects of heat stress on the Chinese mitten crab.

[0071] 5. Effects of aquatic feed on antioxidant enzyme activity in heat-stressed Chinese mitten crabs

[0072] Depend on Figure 5 It can be concluded that compared with control group 1, the antioxidant enzyme activity of Chinese mitten crab in control group 2 is significantly reduced, which is specifically manifested as a reduction in superoxide dismutase (SOD) and glutathione peroxidase (GSH-px) activity, while a rise in malondialdehyde (MDA) content. And after adding the aquatic feed additive of the present invention to the basal feed, the SOD and GSH-px activities of Chinese mitten crab in experimental group 1 and experimental group 2 are significantly improved, while the MDA content is significantly reduced. It is illustrated that the aquatic feed additive in the present invention can be compounded with raw materials such as yeast culture and dandelion extract and grape seed extract, and can effectively enhance the antioxidant capacity of Chinese mitten crab under heat stress conditions through the synergistic effect of multiple antioxidant ingredients, and reduce the damage of oxidative stress to the body.

[0073] 6. Effects of aquatic feed on the intestinal tissue structure of heat-stressed Chinese mitten crabs

[0074] Depend on Figure 6 It can be concluded that compared with control group 1, the intestinal tissue of the Chinese mitten crab in control group 2 showed obvious damage, specifically manifested as shedding of the intestinal outer membrane. However, when fed the aquatic feed of the present invention in a high temperature environment of 30°C, the intestinal outer membrane of the Chinese mitten crab remained intact, indicating that the aquatic feed additive of the present invention can protect the intestinal tissue structure through active substances such as β-glucan provided by yeast culture and polypeptides provided by brewer's yeast hydrolysate, allowing the intestinal tissue of the Chinese mitten crab to maintain its tissue structure intact under heat stress conditions.

[0075] 7. Effects of aquatic feed on biomarkers of intestinal damage in heat-stressed Chinese mitten crabs

[0076] Depend on Figure 7It can be concluded that compared with control group 1, the diamine oxidase activity and lipopolysaccharide levels in the serum of Chinese mitten crabs in control group 2 were significantly increased, and the histamine content in the intestine also increased. The above three substances are all important biomarkers of intestinal damage. When the aquatic feed of the present invention was fed in a high temperature environment of 30°C, the diamine oxidase activity, lipopolysaccharide level, and histamine content all decreased. This shows that the aquatic feed additive of the present invention also achieves multiple protections for the intestine through multiple active substances, effectively reducing intestinal damage caused by heat stress and improving the intestinal physiological state of Chinese mitten crabs in a high temperature environment.

[0077] 8. Effects of aquatic feed on the species richness of intestinal microorganisms in heat-stressed Chinese mitten crabs

[0078] Depend on Figure 8 It can be seen that the species richness of the intestinal microorganisms of Chinese mitten crabs in control group 2 (ACE and Chao1 index) was significantly lower than that in control group 1 (P < 0.05). When fed with the aquatic feed of the present invention under a high temperature environment of 30°C, the species richness of the intestinal microorganisms of Chinese mitten crabs increased, indicating that the aquatic feed additives of the present invention have a significant effect on the richness of microbial species. Among them, the prebiotics contained in brewer's yeast hydrolysate can play a key role in regulating the diversity of intestinal flora. In addition, flavonoids in citrus peel extract and inulin in dandelion extract further optimize the intestinal microecology by providing a selective carbon source for beneficial bacteria, thereby indirectly enhancing the body's ability to resist heat stress.

[0079] 9. Effects of aquatic feed on the intestinal microbial community structure of heat-stressed Chinese mitten crabs

[0080] Figure 9 The results of principal component analysis (PCA) showed that there were significant differences between control group 2 and control group 1, experimental group 1, and experimental group 2, indicating that after feeding the aquatic feed of the present invention under a high temperature environment of 30°C, the aquatic feed additive of the present invention can have a significant effect on the structure of the microbial community. Among them, the flavonoids contained in the citrus peel extract can selectively regulate specific flora, such as promoting the growth of beneficial bacteria such as lactobacillus and bifidobacterium, and inhibiting potential pathogenic bacteria, thereby improving the intestinal flora structure and improving the body's ability to adapt to heat stress. At the same time, the prebiotics provided by yeast culture and brewer's yeast hydrolysate also provide the necessary nutritional basis for the colonization and metabolism of these beneficial bacteria.

[0081] In summary, in the prior art, there are few studies on the application of multi-component compound additives in aquatic animals, especially aquatic animals under heat stress conditions. The present invention adopts a unique feed additive ratio, and through the action of multiple active substances and the synergistic enhancement between different ingredients, a multi-pathway, multi-target synergistic effect is formed, which effectively alleviates the adverse effects of heat stress on Chinese mitten crab from multiple aspects such as growth performance, endocrine regulation, antioxidant function, and intestinal health. In addition, the feed additive components used in the present invention are all natural, safe and non-toxic raw materials, which are more green and environmentally friendly than conventional heat stress relief methods such as drug addition, and are of great significance for guiding production practice and promoting industrial development.

Claims

1. An aquatic feed additive, characterized in that: The invention comprises, by weight: 3-10 parts of yeast culture, 0.5-2 parts of dandelion extract, 0.5-2 parts of citrus peel extract, 1-3 parts of brewer's yeast hydrolyzate and 1-3 parts of grape seed extract.

2. The aquatic feed additive according to claim 1, characterized in that: The aquatic feed additive comprises, by weight: 5-8 parts of yeast culture, 0.5-1.5 parts of dandelion extract, 0.5-1.5 parts of citrus peel extract, 1-2 parts of brewer's yeast hydrolyzate and 1.5-2 parts of grape seed extract.

3. The aquatic feed additive according to claim 1, characterized in that: The aquatic feed additive comprises, by weight, 4.5 parts of yeast culture, 1 part of dandelion extract, 1.5 parts of citrus peel extract, 1 part of brewer's yeast hydrolyzate and 2 parts of grape seed extract.

4. The aquatic feed additive according to claim 1, characterized in that: The aquatic feed additive comprises, by weight: 6.0 parts of yeast culture, 0.5 parts of dandelion extract, 0.5 parts of citrus peel extract, 1.5 parts of brewer's yeast hydrolyzate and 1.5 parts of grape seed extract.

5. A kind of aquatic feed, characterized in that: Calculated by weight, it comprises 8-10 parts of the aquatic feed additive according to any one of claims 1 to 4 and 800-1000 parts of basic feed.

6. The aquatic compound feed according to claim 5, characterized in that: The basic feed comprises, by weight percentage, 15% fish meal, 25% soybean meal, 25% cottonseed meal, 2% fish oil, 1.9% soybean oil, 0.5% soybean lecithin oil, 0.5% cholesterol, 0.5% choline chloride, 0.1% butylated p-cresol, 12% pregelatinized starch, 4% vitamin premix, 2% mineral premix, 2% sodium alginate, 1% betaine, 1% monocalcium phosphate and 7.5% cellulose.

7. The method for preparing aquatic compound feed according to claim 5, characterized in that: The steps include: S1. Take all the raw materials in the aquatic feed additive according to any one of claims 1 to 4 by weight, and The raw materials were ground and sieved through a 60-mesh screen; S2, fully mixing all the sieved raw materials to obtain the aquatic feed additive; S3. Take the basic feed and the aquatic feed additive in the aquatic feed according to claim 5 by weight, add deionized water at a ratio of 100-200 mL / kg of basic feed, and mix thoroughly to obtain a feed mixture; S4, using a double-rod screw extruder to make the feed mixture into pellet feed with a diameter of 2-3 mm; S5, drying the pellet feed; S6, sieving the dried pellet feed with 10, 14 and 16 mesh sieves in turn; S7. Put the feeds screened in step S6 into ziplock bags for storage.

8. Use of the aquatic compound feed according to claim 5 or 6 in improving the heat stress resistance of aquatic animals.

9. The use according to claim 8, characterized in that Improving the ability of aquatic animals to resist heat stress includes: regulating the structure of intestinal microbial communities, improving intestinal antioxidant capacity, alleviating heat stress damage, and maintaining one or more of the following:

10. The use according to claim 9, characterized in that The aquatic animals include Chinese mitten crab.

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