A composition for improving the ammonia nitrogen stress resistance of largemouth bass, a preparation method and applications thereof

Abstract

The present application relates to the technical field of biotechnology, and particularly relates to a composition for improving the ammonia nitrogen stress resistance of largemouth bass, a preparation method and application thereof.The composition provided by the present application comprises, by weight, 28-32 parts of curcuma longa, 28-32 parts of ampelopsis grossedentata and 8-12 parts of mulberry leaves.When the largemouth bass is under ammonia nitrogen stress, the composition can significantly reduce the mortality of the largemouth bass, effectively maintain the osmotic pressure balance of the fish, and reduce the contents of cortisol and lactic acid in serum, thereby effectively relieving the stress response of the largemouth bass caused by ammonia nitrogen stress, and improving the energy metabolism state of the largemouth bass.The composition also has a significant effect of improving hematological indexes, is helpful to reducing the metabolic disorder phenomenon caused by ammonia nitrogen stress in the largemouth bass, protecting the liver tissue from damage caused by ammonia nitrogen stress, ensuring the normal structure and function of liver cells, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to a composition for improving the ammonia nitrogen stress resistance of Micropterus salmoides, a preparation method and application thereof. BACKGROUND

[0002] In aquaculture, ammonia nitrogen is considered as one of the key environmental factors affecting the health and growth of aquatic animals, especially under high-density intensive farming mode. Under this mode, the feces of aquatic animals, leftover feed and excessive fertilizers are rich in a large amount of protein, which is decomposed by microorganisms in the water to form ammonia nitrogen, resulting in a significant increase in the concentration of ammonia nitrogen in the water. Ammonia nitrogen mainly exists in the form of molecular ammonia (NH3, non-ionic ammonia) and enters the fish body through the gills, skin and intestinal mucosa, etc., posing a potential threat to fish.

[0003] The negative effects of ammonia nitrogen poisoning on farmed fish are widespread and far-reaching, including but not limited to reduced growth rate, low immune system function, increased oxidative stress and frequent inflammatory reactions. These physiological disorders not only inhibit the normal growth and development of fish, but also can cause metabolic disorders, tissue damage and even death. The concentration of ammonia nitrogen in the aquaculture water can fluctuate rapidly, causing acute stress to fish, resulting in gill filament hyperplasia, mucus cell layer thickening, and weakening of blood oxygen carrying capacity and liver function, showing symptoms such as tissue edema.

[0004] The high-density intensive farming mode of Micropterus salmoides also faces the challenge of ammonia nitrogen pollution. This farming mode exacerbates the rapid rise of ammonia nitrogen concentration in the water, posing a serious threat to the growth and development of Micropterus salmoides. Studies have shown that ammonia nitrogen stress not only interferes with the ammonia excretion mechanism of Micropterus salmoides, reduces its catabolic activity, but also damages important tissue structures such as gills and liver, increases serum transaminase activity, and inhibits its immune and antioxidant capacity.

[0005] Currently, the measures to deal with the high concentration of ammonia nitrogen in aquaculture water are mostly taken after fish death or detection of ammonia nitrogen exceeding the standard, using probiotics, organic acids or adsorbents as water quality regulators for intervention. However, such lag processing strategies often fail to reverse the damage caused by ammonia nitrogen stress to fish, and the resistance of Micropterus salmoides is thus reduced, making it more susceptible to pathogenic microorganisms. Therefore, it is of great significance to develop a composition that can effectively improve the ammonia nitrogen stress resistance of Micropterus salmoides for preventing ammonia nitrogen poisoning, ensuring the healthy growth of fish and reducing the loss of aquaculture. SUMMARY

[0006] In view of this, the present application provides a composition for improving the ammonia nitrogen stress resistance of Micropterus salmoides, a preparation method and application thereof.

[0007] The technical solution of the present application is implemented as follows:

[0008] In a first aspect, the present application provides a preparation method of a composition for improving the ammonia nitrogen stress resistance of large-mouth bass, comprising the following steps:

[0009] (S1) 8 times the volume of 85% v / v ethanol solution is added to 28-32 parts by weight of turmeric, and reflux extraction is performed 2-4 times, each time for 1.5-3 hours. The extract is collected and concentrated to obtain a thick paste. 1 times the volume of 70% v / v ethanol solution is added to the thick paste, and crystallization is performed at 4°C. The supernatant is collected after standing for 12-36 hours, and concentrated to a relative density of 1.10-1.15 to obtain turmeric oleoresin;

[0010] (S2) 8 times the volume of 85% v / v ethanol solution is added to 28-32 parts by weight of kudzu vine, and reflux extraction is performed 2-4 times, each time for 1.5-3 hours. The extract is collected and concentrated to obtain kudzu vine extract;

[0011] (S3) 10 times the volume of deionized water is added to 8-12 parts by weight of mulberry leaves, and immersed in a boiling water bath for 2-4 times, each time for 1.5-3 hours. The extract is collected and concentrated to obtain mulberry leaf extract;

[0012] (S4) The kudzu vine extract and mulberry leaf extract are mixed, concentrated to a clear paste with a relative density of 1.10-1.15, and then the turmeric oleoresin is added. The mixture is concentrated and dried to obtain the composition.

[0013] In some specific embodiments, the raw materials of the composition include, in parts by weight: 30 parts of turmeric, 30 parts of kudzu vine, and 10 parts of mulberry leaves.

[0014] In a second aspect, the present application provides the use of the preparation method in the preparation of a feed or a medicine for improving the ammonia nitrogen stress resistance of large-mouth bass. The feed or medicine protects the fish body from damage caused by ammonia nitrogen stress through at least one of the following pathways (A1)-(A4):

[0015] (A1) repairing pathological damage to liver tissue;

[0016] (A2) maintaining the normal function of gill filaments;

[0017] (A3) regulating oxidative stress levels;

[0018] (A4) maintaining osmotic pressure balance.

[0019] In a third aspect, the present application provides a composition for improving the ammonia nitrogen stress resistance of large-mouth bass, which is prepared according to the preparation method.

[0020] In a fourth aspect, the present application provides use of the composition in the preparation of a feed for improving the ammonia nitrogen stress resistance of a largemouth bass.

[0021] In some specific embodiments, the composition for improving the ammonia nitrogen stress resistance of a largemouth bass is added to a basal feed. Further, the composition is added in an amount of 1% by weight of the basal feed.

[0022] In a fifth aspect, the present application provides a feed for improving the ammonia nitrogen stress resistance of a largemouth bass, comprising a basal feed and the composition for improving the ammonia nitrogen stress resistance of a largemouth bass. Further, the composition is added in an amount of 1% by weight of the basal feed.

[0023] The present application has at least the following advantages:

[0024] The composition provided by the present application exhibits an effective ability to reduce the mortality of largemouth bass under high-concentration ammonia nitrogen acute stress (water ammonia nitrogen concentration of 20.15 mg / L), and has a significant advantage in improving the survival ability of fish. After application of the composition, the Na + / K + ATPase activity in the gill filaments of largemouth bass is significantly improved, thereby effectively maintaining the osmotic pressure balance of fish. At the same time, the contents of cortisol and lactic acid in serum are significantly reduced, which indicates that the composition helps to alleviate the stress response of largemouth bass caused by ammonia nitrogen stress and improves the energy metabolism state of fish.

[0025] The experimental results also show that under low-concentration ammonia nitrogen (water ammonia nitrogen concentration of 4 mg / L) chronic long-term stress, hematological indicators such as red blood cell count, hemoglobin content and white blood cell count are also improved, indicating a possible enhancement of the immune function of fish, which is of great significance for fish to resist external environmental pressure and pathogen invasion. By detecting serum biochemical indicators, the present application further found that the application of the composition helps to maintain the metabolic balance of largemouth bass, and significantly reduces the metabolic disorder caused by chronic ammonia nitrogen stress, which is crucial for ensuring the normal growth and development of fish. The results of histopathological section observation show that the composition can significantly reduce the damage of chronic ammonia nitrogen stress to the liver tissue of largemouth bass, and effectively protect the normal structure and function of liver cells. Therefore, the present application not only has great potential in protecting important organs of fish, but also provides a new idea for solving the health problems of fish caused by ammonia nitrogen stress.

[0026] The application also determines the best ratio of turmeric, rattan tea and mulberry leaves as raw materials of the composition, fully realizes the significant synergistic effect among the components, ensures the good comprehensive benefits of the composition in reducing mortality, improving enzyme activity, relieving stress reaction, reducing tissue damage, enhancing anti-stress capacity and protecting liver and the like, and therefore has wide application prospect and market value. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0028] Figure 1 For the embodiment of the present application, the Na + / K + -ATPase activity change of gill of Micropterus salmoides under acute ammonia nitrogen stress; the abscissa represents the time length of ammonia nitrogen stress (24h, 48h, 72h and 96h); if the columnar charts of the same time point are marked with different lowercase letters (such as a, b, c), it indicates that there is significant difference (P<0.05, the same below) between different treatment groups;

[0029] Figure 2 For the embodiment of the present application, the cortisol content change in serum of Micropterus salmoides under acute ammonia nitrogen stress;

[0030] Figure 3 For the embodiment of the present application, the lactic acid content change in serum of Micropterus salmoides under acute ammonia nitrogen stress;

[0031] Figure 4 For the embodiment of the present application, the liver tissue pathological section (HE staining, 200 times) of Micropterus salmoides; wherein: the arrow “→” represents the liver sinusoid; indicates that the liver tissue structure is disordered, and the cell boundary is not clear;

[0032] Figure 4 a is a normal control group;

[0033] Figure 4 b is a medication group;

[0034] Figure 4 c is a negative control group. DETAILED DESCRIPTION

[0035] In order to make the objectives, technical solutions and advantages of the present application clearer, the technical solutions in the present application will be clearly and completely described below. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application. If the specific conditions are not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturers are adopted. If the manufacturers of the reagents or instruments are not specified, the conventional products that can be purchased in the market are adopted.

[0036] Example 1 Anti-ammonia nitrogen acute stress test

[0037] 1. Preparation of compound preparation

[0038] The preparation process of the compound preparations 1-5 (hereinafter, the compound preparation is referred to as compound) is as follows:

[0039] (1) Preparation of turmeric oleoresin:

[0040] 30.0 g of turmeric was weighed, 8 times the volume (240 mL) of 85% (v / v) ethanol solution was added, and 3 times of reflux extraction was carried out, each for 2 h. After the extraction was completed, the obtained extract was combined and concentrated under reduced pressure to prepare a thick paste 3 g. Then, 30 mL of 70% (v / v) ethanol was added to the thick paste, and crystallization was carried out at 4℃ for 24 h. The supernatant was continuously concentrated to a relative density of 1.13 (measured at a temperature of 50℃) to obtain the oleoresin, which was the turmeric oleoresin.

[0041] (2) Preparation of Ampelopsis grossedentata extract:

[0042] 30.0 g of Ampelopsis grossedentata was weighed, 8 times the volume (240 mL) of 85% (v / v) ethanol solution was added, and 3 times of reflux extraction was carried out, each for 2 h. After the extraction was completed, the obtained extract was combined to obtain the Ampelopsis grossedentata extract.

[0043] (3) Preparation of mulberry leaf extract:

[0044] 10.0 g of mulberry leaf was weighed, and 10 times the volume (100.0 mL) of deionized water was added. It was placed in a boiling water bath environment and extracted 3 times, each for 2 h. After the extraction was completed, the extract was combined to obtain the mulberry leaf extract.

[0045] (4) Preparation of compound preparation:

[0046] After the Ampelopsis grossedentata extract and the mulberry leaf extract were mixed, they were concentrated under reduced pressure to prepare a clear paste (the relative density of the clear paste was 1.10, measured at a temperature of 50℃). The turmeric oleoresin was added to the clear paste, and it was fully stirred and homogenized. Finally, it was concentrated and dried to obtain the final compound preparation powder.

[0047] (5) The raw material ratio of turmeric oleoresin, Ampelopsis grossedentata extract and mulberry leaf extract in compound preparations 1-5 is as follows (by weight):

[0048] Compound 1: turmeric 30 parts, Ampelopsis grossedentata 30 parts, mulberry leaf 10 parts;

[0049] Compound 2: turmeric 30 parts, Ampelopsis grossedentata 30 parts, mulberry leaf 30 parts;

[0050] Compound 3: turmeric 30 parts, Ampelopsis grossedentata 30 parts, mulberry leaf 20 parts;

[0051] Compound 4: turmeric 30 parts, Ampelopsis grossedentata 20 parts, mulberry leaf 10 parts;

[0052] Compound 5: turmeric 30 parts, Ampelopsis grossedentata 20 parts, mulberry leaf 20 parts.

[0053] 2. Test animals and breeding management

[0054] The initial body weight of the experimental fish, Micropterus salmoides, used in the present application was (18.20 ± 0.81) g, which was purchased from Hubei Weilicheng Agricultural Technology Co., Ltd. Before the start of the experiment, the fish were temporarily fed with basic feed (protein content 48% m / m, purchased from Tongwei Co., Ltd., Cat. No.: 20240305) for one week. The test fish were randomly divided into 7 groups, one normal control group (without drug feeding, without stress), one negative control group (without drug feeding, with stress), and 5 drug groups (with drug feeding, with stress). Each group had 3 replicates, and each replicate had 30 fish. The fish were placed in a glass breeding system (57.8 cm x 38.2 cm x 22.7 cm). The water temperature was maintained at 26 ± 2°C, the dissolved oxygen content was about 6 mg / L, the pH value was 6.8 ± 0.3, and the ammonia nitrogen content was <0.05 mg / L during the feeding period. The fish were fed with basic feed at 8:30-9:00 am and 17:00-18:00 pm every day. The breeding tank was cleaned every day in the afternoon, and the filter tank, filter screen and filter stones were cleaned every three days, and one-third of the water in the breeding system was replaced.

[0055] 3. Experimental design

[0056] The drug was administered by mixing it with the feed. The addition amount of the compound preparation in the feed was 1% (m / m). The normal control group and the negative control group were fed with basic feed, and the drug groups were fed with drug-containing feed containing 1% (m / m) of compound preparations 1-5. The feeding was continued for 30 days.

[0057] After 30 days of feeding, all test fish were deprived of food for 24 hours to adapt to the environment. The negative control group and the drug groups were subjected to 96-hour ammonia nitrogen stress, while the normal control group was observed synchronously without stress. All groups were not fed during the stress period. The 96-hour LC50 of ammonia nitrogen to Micropterus salmoides was determined by pre-experiment to be 0.5 mg / L. 5020.15 mg / L, so 20.15 mg / L was used as the stress concentration of ammonia nitrogen. NH4Cl solution was added in the negative control pool and the drug pool to make the ammonia nitrogen concentration of the water body 20.15 mg / L, and the ammonia nitrogen stress test was carried out for 96 h, and the death of the test fish was recorded.

[0058] 4、Na + / K + -ATPase activity, cortisol and lactic acid index determination

[0059] At 24 h, 48 h, 72 h and 96 h of anti-ammonia nitrogen stress test, 5 fish were taken from each parallel, blood was taken from the tail vein, and the blood was placed at 4°C overnight, centrifuged at 3000 r / min for 10 min, and the supernatant was stored at -80°C for standby, and the gills were separated. Serum cortisol and lactic acid, gill Na + / K + -ATPase, according to the kit method; cortisol (Cat.No.: 240402), lactic acid (Cat.No.: 20240316) and Na + / K + -ATPase (Cat.No.: 20240418) test kits were purchased from Nanjing Jiancheng Biological Engineering Institute. The data are expressed as "mean ± standard deviation", and the Excel 2017 and SPSS19.0 statistical software were used for drawing and data analysis.

[0060] 5、Results

[0061] (1) The death results are as follows:

[0062] Table 1 Death of largemouth bass caused by acute ammonia nitrogen stress (96 h ammonia nitrogen stress)

[0063]

[0064]

[0065] Under the condition of acute ammonia nitrogen stress, specific behavioral responses and physiological state changes of largemouth bass were observed, which were as follows: the test fish swam around the experimental barrel wall, as the exposure time to ammonia nitrogen toxicity increased, the fish gradually showed convulsive symptoms, the abdomen was turned upside down, the overall vitality decreased significantly, and the abnormal swimming mode was sometimes hidden in the water bottom and sometimes jumped out of the water surface. This state lasted for about 4-6 min, and then the fish finally slowly sank to the bottom of the water, the mouth and gills were open, and finally died. The mortality rate of the negative control group was as high as 53.33% within 96 h.

[0066] In contrast, the mortality rate of fish treated with the compound preparation showed varying degrees of reduction across all groups. Particularly noteworthy was the lowest mortality rate, at only 18.89%, observed in compound preparation group 1. This result indicates that the combination of compound preparation 1 (referring to the rational combination of two or more drugs based on pathological needs and drug characteristics, aiming to enhance efficacy, reduce toxicity, or broaden the therapeutic spectrum) exhibits optimal effects and can effectively reduce fish mortality induced by acute ammonia nitrogen stress.

[0067] (2) The effect of compound preparations on Na in the gill filaments of largemouth bass under acute ammonia nitrogen stress + / K + Effects of ATPase activity

[0068] Acute ammonia nitrogen stress affects the Na content of the gill filaments of the largemouth bass. + / K + -ATPase activity showed a significant inhibitory effect (P < 0.05). At each sampling time point, the enzyme activity in all groups exposed to ammonia nitrogen stress (including the negative control group and the compound preparation treatment group) was significantly lower than that in the normal control group. This result is consistent with previous studies and further confirms that ammonia nitrogen stress interferes with the osmotic regulation mechanism of fish, affects the function of chlorine-secreting cells in the gills and proteases on organelle membranes, thereby disrupting the osmotic pressure balance of fish.

[0069] Depend on Figure 1 The results showed that compound preparations 1, 2, and 3 significantly increased enzyme activity in largemouth bass under ammonia stress (P < 0.05). Compared with the negative control group, these compound preparations effectively reduced the effect of ammonia stress on Na+. + / K + The adverse effects of ammonia nitrogen stress on ATPase activity were mitigated, and the relative stability of gill filament regulatory function was maintained. Compound preparation 1 showed the best performance, suggesting that its component ratio or synergistic effect may be more beneficial in alleviating the damage caused by ammonia nitrogen stress to the fish. In contrast, compound preparations 4 and 5 did not significantly increase enzyme activity in the treatment groups (P > 0.05), indicating that these two compound preparations had limited effect in alleviating the inhibitory effect of ammonia nitrogen stress on enzyme activity. This may be related to the fact that the components or ratios of the preparations failed to effectively counteract the effects of ammonia nitrogen stress.

[0070] The changes in enzyme activity within all groups over time showed a slight increase in enzyme activity at 48 h, followed by a gradual decrease, with no significant difference in enzyme activity at 72 h and 96 h (P > 0.05). This pattern of change may reflect the adaptive response of fish in the early stages of ammonia nitrogen stress, namely, attempting to increase enzyme activity through active osmotic regulation by activating the body's osmotic mechanisms. However, as ammonia nitrogen continues to damage the gill tissue, Na... + / K +-ATPase activity gradually decreased and reached a relatively stable low level under long-term stress, indicating that the Na + / K + -ATPase activity gradually decreased and reached a relatively stable low level under long-term stress, indicating that the Na

[0071] (3) Effects of compound preparations on serum cortisol content of largemouth bass under acute ammonia-nitrogen stress

[0072] From Figure 2 It can be seen that ammonia-nitrogen stress significantly increased the cortisol content in the serum of largemouth bass (P<0.05), and at each sampling time point, the cortisol content of all groups exposed to ammonia-nitrogen stress (including the negative control group and the compound preparation treatment group) was significantly higher than that of the normal control group. Studies have shown that cortisol levels in fish increase under stress conditions, and the severity of stress can be represented by the degree and duration of this increase. This result shows that acute ammonia-nitrogen stress causes largemouth bass to be in an acute stress state, and the stress state lasts for 96 h. From the change of cortisol content in all groups over time, it can be seen that the cortisol content has a small upward trend after 48 h, and the increase in cortisol content is not significant after 72 h, and there is no significant difference compared with the 96 h value (P>0.05). Acute ammonia-nitrogen stress causes largemouth bass to exhibit a stress response, and the cortisol in the blood rapidly increases, reaching a peak at 72 h, and then stabilizing.

[0073] At the same time point, the cortisol content in the serum of compound 1 and compound 2 was significantly lower than that of the negative control group (P<0.05), indicating that compound 1 and compound 2 can well alleviate the stress response under acute ammonia-nitrogen stress, and compound 1 has the best effect, and it is speculated that the component ratio or the synergistic effect between components may be more conducive to relieving the stress caused by ammonia-nitrogen stress. In contrast, the alleviating effect of compound preparations 3, 4, and 5 in the treatment group was not significant (P>0.05), indicating that these three compound preparations have limited effect in alleviating the stress caused by ammonia-nitrogen stress. This may be related to the fact that the components or ratios of the preparations cannot effectively counteract the effects of ammonia-nitrogen stress.

[0074] (4) Effects of compound preparations on serum cortisol content of largemouth bass under acute ammonia-nitrogen stress

[0075] From Figure 3It can be seen that acute ammonia nitrogen stress significantly increased the lactic acid content in the serum of the largemouth bass (P<0.05). At each sampling time point, the lactic acid content of all groups exposed to ammonia nitrogen stress (including the negative control group and the compound preparation treatment groups) was significantly higher than that of the normal control group. Lactic acid is the product of anaerobic respiration energy metabolism and can reflect the mode of respiratory metabolism. When fish are subjected to ammonia nitrogen stress, the gill tissue is damaged to a certain extent, affecting gas exchange, and thus the fish body is hypoxic, the aerobic metabolism of the body is blocked, and anaerobic metabolism is started, so the lactic acid content in the blood increases. This result shows that acute ammonia nitrogen stress can cause damage to the gill filament tissue of the largemouth bass. From the change of the intracortical cortisol content of all groups over time, it can be seen that the lactic acid in the serum of the largemouth bass has an increasing trend since 48h, and the lactic acid content increases insignificantly after 72h, and there is no significant difference compared with the 96h value (P>0.05). This indicates that acute ammonia nitrogen stress causes damage to the gill filament of the largemouth bass, and the influence of the damage on the gas exchange of the largemouth bass will last for 96h.

[0076] At the same time point, compound 1-3 can significantly reduce the increase of lactic acid content in the serum (P<0.05), compared with the negative control group, these compound preparations can effectively alleviate the adverse effects of ammonia nitrogen stress on the gill filament, and maintain the relative stability of the gill filament gas exchange function. Compound 1 has the best effect, and it is speculated that the component ratio or the synergistic effect between the components may be more conducive to alleviating the damage to the gill filament caused by ammonia nitrogen stress. In contrast, the alleviating effect of compound preparations 3, 4 and 5 in the treatment group is not significant (P>0.05), indicating that these three compound preparations have limited effect on alleviating the damage to the gill filament caused by ammonia nitrogen stress.

[0077] Example 2 Anti-ammonia nitrogen chronic stress test

[0078] 1. Preparation of compound preparation

[0079] The preparation process of the compound preparation (hereinafter referred to as compound) is as follows:

[0080] (1) Preparation of turmeric oleoresin:

[0081] Take 30.0g of turmeric and add 8 times the volume (240mL) of 85%(v / v) ethanol solution, and perform 3 times of reflux extraction, each for 2h. After the extraction is completed, the obtained extract is combined and concentrated under reduced pressure to prepare a thick paste 3g. Then, 30mL of 70%(v / v) ethanol is added to the thick paste, and it is placed at 4℃ for 24h to crystallize. The supernatant is continuously concentrated to a relative density of 1.13 (measured at a temperature of 50℃), and oleoresin is obtained, which is turmeric oleoresin.

[0082] (2) Preparation of Ampelopsis grossedentata extract:

[0083] Take 30.0 g of R. loxophylla, add 8 times the volume (240 mL) of 85% (v / v) ethanol solution, and perform 3 times of reflux extraction, each for 2 h. After the extraction is completed, combine the obtained extract to obtain the R. loxophylla extract.

[0084] (3) Preparation of mulberry leaf extract:

[0085] Take 10.0 g of mulberry leaves, add 10 times the volume (100.0 mL) of deionized water. Place in a boiling water bath environment, and perform 3 times of immersion extraction, each for 2 h. After the extraction is completed, combine the extract to obtain the mulberry leaf extract.

[0086] (4) Preparation of compound preparation:

[0087] After mixing the R. loxophylla extract and the mulberry leaf extract, concentrate under reduced pressure to prepare a clear paste (the relative density of the clear paste is 1.10, measured at a temperature of 50°C). Add turmeric oleoresin to the clear paste, and fully stir to homogenize. Finally, perform concentration and drying treatment to obtain the final compound preparation powder.

[0088] (5) The raw material ratio of turmeric oleoresin, R. loxophylla extract, and mulberry leaf extract in the compound preparation is as follows (in weight parts): turmeric 30 parts, R. loxophylla 30 parts, and mulberry leaves 10 parts.

[0089] 2. Test animals and breeding management

[0090] The initial body weight of the experimental fish, large-mouth bass, used in the present application was (18.20 ± 0.81) g, and was purchased from Hubei Weilicheng Agricultural Technology Co., Ltd. Before the start of the experiment, the experimental fish was temporarily raised for one week using a basic feed (protein content 48% m / m, purchased from Tongwei Co., Ltd., Cat. No.: 20240507), and was randomly divided into 7 groups, one normal control group (not fed with medicine, not stressed), one negative control group (not fed with medicine, stressed), and 5 medicine groups (fed with medicine, stressed), each with 3 parallels, 30 fish in each parallel, and was placed in a glass breeding system (57.8 cm x 38.2 cm x 22.7 cm). During the experimental period, the water temperature was maintained at 26 ± 2°C, the dissolved oxygen content was about 6 mg / L, the pH value was 6.8 ± 0.3, and the ammonia nitrogen content was <0.05 mg / L. The fish was fed with the test feed at 8:30-9:00 am and 17:00-18:00 pm every day. The feces in the breeding tank was cleaned every day in the afternoon, and the filter tank, filter screen, and filter stone were cleaned every three days, and 1 / 3 of the water in the breeding system was replaced.

[0091] 3. Experimental design

[0092] At the start of the experiment, NH4Cl solution was added to the water in the negative control group and the medicine groups to make the ammonia nitrogen concentration 4 mg / L (96 h LC 5020.15 mg / L, 20% of the safety concentration is 4 mg / L, the water ammonia nitrogen concentration was measured every other day, and timely adjustment was made to maintain the ammonia nitrogen concentration at 4 mg / L during the entire test period (30d), and no NH4Cl solution was added to the water body in the normal control group.

[0093] During the test period, the drug was administered by mixing with feed, and the addition amount of the compound preparation in the feed was 1% (m / m) respectively. The normal control group and the negative control group were fed with basic feed, and the drug group was fed with mixed drug feed containing 1% (m / m) compound preparation, and the continuous feeding lasted for 30d.

[0094] 4. Hematology index and serum biochemical index determination

[0095] 24h after the end of the feeding test, 5 fish were randomly taken from each tank, and blood was taken from the tail vein. Part of the blood was injected into an anticoagulant tube to prepare anticoagulated blood, which was stored at 4°C for determination of hematology indexes; the other part was injected into an EP tube and stored at 4°C overnight, then centrifuged at 3000r / min for 10min, and the upper layer serum was taken and stored at-80°C for determination of serum biochemical indexes.

[0096] The hematology indexes were red blood cell count (RBC), white blood cell count (WBC), and hemoglobin (HGB), which were determined by Mindray animal automatic blood cell analyzer (model: BC-2800vet).

[0097] The serum determination indexes were malondialdehyde (MDA) and superoxide dismutase (SOD), which were determined according to the kit method. SOD kit (Cat. No.: 20240520), MDA kit (Cat. No.: 20240603) were purchased from Nanjing Jiancheng Reagent Co., Ltd.

[0098] The data are expressed as "mean ± standard deviation", and the Excel 2017 and SPSS19.0 statistical software were used for drawing and data analysis.

[0099] 5. Liver tissue pathological section observation

[0100] After blood sampling, the livers of 3 fish randomly taken from each tank were fixed with 4% neutral formaldehyde solution, paraffin sections were prepared, HE staining was performed, and microscopic examination and photography were carried out.

[0101] 6. Experimental results

[0102] (1) Effect of compound preparation on hematology index of chronic ammonia nitrogen stressed largemouth bass

[0103] As shown in Table 2, the red blood cell count and hemoglobin content of the negative control group and the drug group were significantly less than that of the normal control group (P<0.05), and the red blood cell count and hemoglobin content of the drug group were significantly greater than that of the negative control group (P<0.05); the white blood cell count of the negative control group was significantly less than that of the normal control group and the drug group (P<0.05), and there was no significant difference between the normal control group and the drug group (P>0.05). Due to long-term stress of ammonia nitrogen, the red blood cell count, white blood cell count and hemoglobin content of the test fish of the negative control group and the drug group were less than that of the normal control group, and the indexes of the drug group were higher than those of the negative control group, among which the white blood cell count had no significant difference with the normal group, indicating that ammonia nitrogen stress affects the hematopoietic function and immune system of fish, leading to changes in the number of red blood cells, white blood cells and hemoglobin, and the compound preparation can alleviate the damage of ammonia nitrogen stress to the fish body by reducing the toxicity of ammonia nitrogen and improving the immunity and metabolic function of fish.

[0104] Table 2 Changes in hematological indexes

[0105] Group Red blood cell count (xlO 12 / L) White blood cell count (x 10 10 / L) Hemoglobin (x g / L) Normal control group 3.68 ± 0.27 a ]] 4.54 ± 0.24 a ]] 74.58 ± 6.84 a ]] Negative control group 2.16 ± 0.21 c ]] 3.51 ± 0.31 b ]] 43.46 ± 6.49 c ]] Drug group 3.28 ± 0.26 b ]] 4.03 ± 0.30 a ]] 62.44 ± 5.24 b ]]

[0106] Note: Different letters on the right shoulder of each column indicate significant difference (P<0.05).

[0107] (2) Effect of the compound preparation on serum biochemical indexes of Micropterus salmoides under chronic ammonia nitrogen stress

[0108] As shown in Table 3, the Na + / K + -ATPase activity of the three groups had significant difference (P<0.05), among which the normal control group > the drug group > the negative control group, and the serum cortisol and lactic acid of the three groups had significant difference (P<0.05), among which the negative control group > the drug group > the normal control group. Na + / K + -ATPase is a kind of protease on the membrane of chloride cells and organelles in gill, which mainly participates in the process of osmoregulation in fish body. When the gill filament tissue is damaged, the enzyme activity will decrease; when fish are stressed by ammonia nitrogen, the gill tissue is damaged to a certain extent, affecting gas exchange, and thus the fish body is hypoxic, the aerobic metabolism of the body is blocked, and anaerobic metabolism is started, so the lactic acid content in the blood increases; the cortisol level in fish increases under stress conditions, and the severity of stress can be indicated by the degree and duration of this increase, so the cortisol content can be an important physiological index of stress level. As shown in Table 3, feeding the compound preparation can reduce the damage of long-term ammonia nitrogen stress to the gill filament of Micropterus salmoides and improve the ability of Micropterus salmoides to resist ammonia nitrogen stress.

[0109] Table 3 Changes in serum biochemical indexes

[0110]

[0111] Note: Different letters on the right side of each column indicate significant differences (P < 0.05).

[0112] (3) Histopathological sections

[0113] Depend on Figure 4 It can be seen that the normal control group ( Figure 4 a) A large number of hepatic sinusoids and full sinusoids indicate that the normal control group had high blood flow to the liver tissue and good liver tissue development. The drug-treated group ( Figure 4 b) The number of hepatic sinusoids was greater than that in the negative control group. Figure 4 c) Less than the normal control group; in the negative control group (c), the liver tissue of some areas showed disordered hepatocyte structure and unclear boundaries. Under long-term stress of low concentration of ammonia nitrogen, the liver tissue of the negative control group and the drug treatment group was damaged to a certain extent and the development was worse than that of the normal group. The compound preparation played a certain protective role on the liver of the largemouth bass, and the liver tissue condition was better than that of the negative control group.

[0114] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method of preparing a composition for improving the ability of Micropterus salmoides to withstand ammonia nitrogen stress, characterized by, The method comprises the following steps: (S1) adding 8 times volume of 85% v / v ethanol solution to 28-32 parts by weight of turmeric, refluxing and extracting 2-4 times, each time for 1.5-3 hours, collecting the extract and concentrating to obtain a thick paste; adding 1 times volume of 70% v / v ethanol solution to the thick paste, crystallizing at 4°C, standing for 12-36 hours, collecting the supernatant, concentrating to a relative density of 1.10-1.15 to obtain turmeric oleoresin; (S2) adding 8 times volume of 85% v / v ethanol solution to 28-32 parts by weight of Ampelopsis grossedentata, refluxing and extracting 2-4 times, each time for 1.5-3 hours, and combining the extract to obtain Ampelopsis grossedentata extract; (S3) adding 10 times volume of deionized water to 8-12 parts by weight of mulberry leaves, immersing and extracting 2-4 times in a boiling water bath, each time for 1.5-3 hours, and combining the extract to obtain mulberry leaf extract; (S4) mixing the Ampelopsis grossedentata extract and the mulberry leaf extract, concentrating to a clear paste with a relative density of 1.10-1.15, and then adding the turmeric oleoresin and concentrating to dryness to obtain the composition.

2. The preparation method according to claim 1, characterized in that, The raw materials of the composition comprise, by weight parts, 30 parts of turmeric, 30 parts of Ampelopsis grossedentata, and 10 parts of mulberry leaves.

3. A composition for improving the ability of Micropterus salmoides to withstand ammonia nitrogen stress, characterized in that it comprises a mixture of probiotics and prebiotics. The composition is prepared according to the preparation method of claim 1 or 2.

4. Use of the composition of claim 3 in the preparation of feed for improving the ammonia nitrogen stress resistance of large-mouth bass.

5. Use according to claim 4, characterized in that, The composition for improving the ammonia nitrogen stress resistance of large-mouth bass is added to the basic feed.

6. Use according to claim 5, characterized in that, The composition for improving the ammonia nitrogen stress resistance of large-mouth bass is added in an amount of 1% by weight of the basic feed.

7. A feed for improving the resistance of largemouth bass to ammonia nitrogen stress, characterized in that, The composition for improving the ammonia nitrogen stress resistance of large-mouth bass is prepared from the basic feed and the composition of claim 3.

8. The feed of claim 7, characterized in that, The composition for improving the ammonia nitrogen stress resistance of large-mouth bass is added in an amount of 1% by weight of the basic feed.

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