Preparation method of clostridium butyricum fermentation metabolite and application of clostridium butyricum fermentation metabolite in aquatic animal breeding

Abstract

The invention belongs to the technical field of aquaculture feed additives, and particularly relates to a preparation method of a clostridium butyricum fermentation metabolite and application of the clostridium butyricum fermentation metabolite in aquatic animal breeding. According to the clostridium butyricum fermentation medium disclosed by the invention, the fermentation medium is improved, and components such as methyl-beta-cyclodextrin, betaine and algal polysaccharides are added into the clostridium butyricum fermentation medium, so that accumulation of fermentation metabolites of the clostridium butyricum is facilitated, the fermentation effect of the clostridium butyricum is remarkably improved, and moreover, the fermentation effect of the clostridium butyricum is improved. The obtained clostridium butyricum fermentation metabolite is compounded with clostridium butyricum to prepare a feed additive, after the feed additive is used for culturing penaeus vannamei boone, the weight gain of the penaeus vannamei boone is obvious, the weight gain of the penaeus vannamei boone is increased from 7.3 g / head to 15.8 g / head, and meanwhile, the survival rate of the penaeus vannamei boone reaches up to 95%.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of aquatic feed additives, and particularly relates to a preparation method of Clostridium butyricum fermentation metabolites and application thereof in aquatic animal breeding. BACKGROUND

[0002] In recent years, with the continuous expansion of aquatic animal breeding scale and the improvement of the degree of intensification, many problems have gradually emerged, such as the aggravation of breeding water pollution, the frequent occurrence of aquatic animal diseases, and the low feed conversion rate, which seriously restricts the sustainable development of the industry.

[0003] In the face of the severe breeding situation, antibiotics have been widely used in the prevention and treatment of aquatic diseases. However, long-term and large-scale use of antibiotics not only leads to the continuous enhancement of pathogenic bacteria resistance, which greatly reduces the prevention and treatment effect of antibiotics, but also destroys the ecological balance of water, causes the residue of antibiotics in aquatic products, and through the food chain transmission poses a potential threat to human health.

[0004] In order to avoid the abuse of antibiotics, microbial agent feed additives have emerged, among which Clostridium butyricum, Brevibacillus laterosporus, Lactobacillus plantarum and other microbial strains have been widely applied in the field of aquatic breeding.

[0005] Among them, Clostridium butyricum belongs to gram-positive anaerobic Bacillus, which can colonize in the intestinal tract of aquatic animals, compete with harmful bacteria for living space, produce short-chain fatty acids such as butyric acid and acetic acid, reduce the pH value of the intestinal tract, effectively inhibit the growth and reproduction of harmful bacteria such as Vibrio and Aeromonas, and maintain the intestinal microecological balance. In addition, it can also secrete various digestive enzymes such as amylase and protease, promote the decomposition of macromolecular nutrients in feed, improve the feed conversion rate, improve the growth performance of aquatic animals, and reduce the feed coefficient. It is a good feed additive.

[0006] The fermentation process of Clostridium butyricum can be mainly divided into two parts. In the early growth stage, the cell obtains more ATP to meet its own growth needs, and the metabolic pathway is biased towards the acetic acid production pathway because more ATP can be produced by generating acetic acid than by generating butyric acid. In the late growth stage, in order to reduce the toxicity of high hydrogen ion concentration to the cell, the metabolism is biased towards the butyric acid metabolic pathway which produces less hydrogen gas. Therefore, the whole metabolic process of Clostridium butyricum shifts from acetic acid production to butyric acid production, which is specifically manifested as the gradual decrease of acetic acid concentration and the gradual increase of butyric acid concentration in the cell. Therefore, by optimizing the fermentation process of Clostridium butyricum and directing the metabolic pathway to promote the efficient production of butyric acid, the quality of the fermentation product can be improved, and the application efficiency of Clostridium butyricum in aquatic breeding can be significantly enhanced. SUMMARY

[0007] To address the aforementioned technical problems, this invention provides a method for preparing Clostridium butyricum fermentation metabolites and their application in aquaculture.

[0008] The specific technical solution of the present invention is as follows: This invention provides a method for preparing Clostridium butyricum fermentation metabolites, comprising the following steps: Activation of Clostridium butyricum S1; Cultivation of S2 Clostridium butyricum seed culture: The Clostridium butyricum suspension obtained from S1 was inoculated into seed culture medium and cultured in primary and secondary anaerobic conditions at 32-38℃ and 60-80 r / min for 12-48 h to obtain secondary seed culture. The seed culture medium consists of the following components: glucose 8-15 g / L, soybean meal hydrolysate 5-20 g / L, yeast extract 3-12 g / L, dipotassium hydrogen phosphate 0.2-0.8 g / L, magnesium sulfate heptahydrate 0.1-0.5 g / L, manganese sulfate 0.05-0.15 g / L, calcium chloride 0.5-2 g / L, ferrous sulfate 1-4 g / L, L-cysteine ​​hydrochloride 0.3-0.8 g / L, sodium butyrate 0.05-0.1 g / L, with a pH of 6.5±0.2. S3 Fermentation Culture: Add the fermentation medium to the fermenter, sterilize and cool to 35-38℃, inoculate the secondary seed liquid of Clostridium butyricum obtained in S2 into the fermenter at a volume ratio of 1%-5% for fermentation culture, and recover the supernatant of the culture medium and the biomass precipitate separately. The fermentation medium consists of the following components: glucose 6-8 g / L, methyl-β-cyclodextrin 1-5 g / L, betaine 0.01-1 g / L, corn cob extract 0.2-0.6 g / L, soybean meal hydrolysate 5-15 g / L, yeast powder 10-20 g / L, sodium bicarbonate 0.1-0.5 g / L, dipotassium hydrogen phosphate 0.2-0.8 g / L, magnesium sulfate heptahydrate 0.1-0.3 g / L, calcium citrate 0.5-2 g / L, seaweed polysaccharide 1-5 g / L, vitamin B1 0.01-0.02 g / L, and pH 6.2±0.1. S4 is resuspended in sterile water containing 0.85% NaCl in the biomass precipitate obtained in S3, and centrifuged 1-2 times to obtain purified bacterial cell precipitate. The purified bacterial cell precipitate is diluted with sterile water to a solid content of 5%-8%, and then homogenized under high pressure at 30-60 MPa 2-3 times to obtain lysate. S5 involves mixing the lysate from S4 with the supernatant obtained in S3 to obtain the fermentation metabolites of Clostridium butyricum.

[0009] In the above preparation method, preferably, in S2, the seed culture medium consists of the following components: glucose 12 g / L, soybean meal hydrolysate 8 g / L, yeast powder 10 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.3 g / L, manganese sulfate 0.1 g / L, calcium chloride 1 g / L, ferrous sulfate 3 g / L, L-cysteine ​​hydrochloride 0.5 g / L, sodium butyrate 0.08 g / L, and pH 6.5 ± 0.2.

[0010] Preferably, the inoculation amount of seed liquid in S3 is 2%-4%.

[0011] Preferably, in S3, the fermentation medium consists of the following components: glucose 7 g / L, methyl-β-cyclodextrin 5 g / L, betaine 0.5 g / L, corn cob extract 0.5 g / L, soybean meal hydrolysate 10 g / L, yeast powder 12 g / L, sodium bicarbonate 0.2 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate heptahydrate 0.2 g / L, calcium citrate 0.8 g / L, seaweed polysaccharide 3 g / L, vitamin B1 0.01 g / L, and pH 6.2 ± 0.1.

[0012] As a preferred option, in S3, the fermentation temperature is 35-38℃.

[0013] As a preferred option, in S3, the fermentation temperature is 36°C.

[0014] Preferably, in S4, the centrifugation speed is 2500-3500 r / min and the centrifugation time is 8-20 min.

[0015] In addition, products containing Clostridium butyricum fermentation metabolites prepared by the above method are also the key technical content protected by this invention.

[0016] Preferably, the product form can be any one of nanoemulsion, lyophilized powder, tablets, capsules, or gel candy.

[0017] Preferably, the application of the product in the preparation of feed additives is also a key area of ​​protection for this invention.

[0018] Furthermore, the present invention also provides a feed additive comprising Clostridium butyricum and its fermentation metabolites, wherein the total number of Clostridium butyricum in the feed additive is ≥1.0 × 10⁻⁶. 7 CFU / mL.

[0019] Preferably, the total number of Clostridium butyricum in the feed additive is ≥1.0×10⁻⁶. 9 CFU / mL.

[0020] Preferably, the feed additive is an aquatic animal aquaculture feed additive, and the aquatic animal is any one or more of the following: whiteleg shrimp, Chinese shrimp, sea bass, California bass, silver carp, and swimming crab.

[0021] Preferably, the feed additive can be directly mixed with aquatic animal feed and fed to aquatic animals, or dissolved in water and mixed before being directly sprinkled into the pond.

[0022] The beneficial effects of this invention are as follows: (1) This invention improves the fermentation medium by adding methyl-β-cyclodextrin, betaine, and seaweed polysaccharide to the fermentation medium of Clostridium butyricum. The addition of these components not only helps to regulate the permeability of the Clostridium butyricum cell membrane, thereby mediating the earlier migration of the fermentation process of Clostridium butyricum from the acetic acid metabolic pathway to the butyric acid metabolic pathway, which is beneficial to the increase of Clostridium butyricum fermentation metabolites, but also provides a continuous energy supply for the metabolism of Clostridium butyricum. Experimental results show that after fermentation, the viable number of Clostridium butyricum reaches 1.3 × 10⁻⁶. 9 The concentration of CFU / mL is above 63.5 g / L, and the butyric acid content is as high as 63.5 g / L. (2) This invention provides a feed additive containing Clostridium butyricum fermentation metabolites. When the feed additive is used to supplement ordinary feed for feeding aquatic animals such as Litopenaeus vannamei, the weight gain of the shrimp is more significant. The weight of the shrimp increases from 7.3g / head to 15.8g / head, nearly doubling the weight, while the survival rate of the shrimp is as high as 95%. Attached Figure Description

[0023] Figure 1 The figure shows the effect of feed additives on the intestines of Litopenaeus vannamei in this invention. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0025] Example 1 A fermentation metabolite of Clostridium butyricum was prepared using the following method: Activation of Clostridium butyricum S1; S2 Cultivation of Clostridium butyricum seed culture: The Clostridium butyricum suspension obtained in step S1 was inoculated into seed culture medium and cultured in a primary anaerobic environment at 32℃ and 60 r / min for 24 h to obtain the primary seed culture. Next, the primary seed culture was inoculated into seed culture medium for secondary seed culture. The culture conditions were 36℃ and 80 r / min for 36 h to obtain the secondary seed culture. The seed culture medium consisted of the following components: glucose 12 g / L, soybean meal hydrolysate 8 g / L, yeast extract 10 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.3 g / L, manganese sulfate 0.1 g / L, calcium chloride 1 g / L, ferrous sulfate 3 g / L, L-cysteine ​​hydrochloride 0.5 g / L, sodium butyrate 0.08 g / L, with a pH of 6.5 ± 0.2. S3 Fermentation Culture: The fermentation medium was added to the fermenter, sterilized and cooled to 36℃. The secondary seed culture of Clostridium butyricum obtained in S2 was inoculated into the fermenter at a volume ratio of 3% for 48 h. Then, it was centrifuged at 4℃ and 4000 r / min for 10 min, and the supernatant of the culture medium and the biomass precipitate were recovered respectively. The fermentation medium consists of the following components: glucose 7 g / L, methyl-β-cyclodextrin 5 g / L, betaine 0.5 g / L, corn cob extract 0.5 g / L, soybean meal hydrolysate 10 g / L, yeast powder 12 g / L, sodium bicarbonate 0.3 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate heptahydrate 0.2 g / L, calcium citrate 0.8 g / L, seaweed polysaccharide 3 g / L, vitamin B1 0.01 g / L, and pH 6.2±0.1. The preparation method of corn cob extract is as follows: corn cobs are dried and pulverized into powder. First, 0.2 mol / L NaOH solution is added at a mass-volume ratio of 1g:10mL for pretreatment for 1 h. After pretreatment, the corn cobs are repeatedly washed with water until the pH is 6.0-7.0. Then, a mixed enzyme preparation of cellulase and hemicellulase is used for enzymatic hydrolysis at 50℃ and 150 r / min for 48 h. After centrifugation, the hydrolysate is dried. The mixed enzyme preparation accounts for 10% of the substrate (by dry weight). The mass ratio of cellulase to hemicellulase is 2:1. The corn cob extract is obtained. S4. The biomass precipitate obtained in step S3 is resuspended in sterile water containing 0.85% NaCl, and centrifuged twice (3000 r / min, 10 min) to obtain purified bacterial cell precipitate. The purified bacterial cell precipitate is taken, diluted with sterile water to a solid content of 5%, and then homogenized twice under high pressure at 50 MPa to obtain lysate. S5. The lysate from step S4 is mixed evenly with the supernatant obtained in step S3 to obtain the fermentation metabolites of Clostridium butyricum.

[0026] Example 2 A fermentation metabolite of Clostridium butyricum was prepared using the following method: Activation of Clostridium butyricum S1; S2 Cultivation of Clostridium butyricum seed culture: The Clostridium butyricum suspension obtained in step S1 was inoculated into seed culture medium and cultured in a primary anaerobic environment at 34℃ and 80 r / min for 24 h to obtain the primary seed culture. Next, the primary seed culture was inoculated into seed culture medium for secondary seed culture. The culture conditions were 34℃, 80 r / min and cultured for 36 h to obtain the secondary seed culture. The composition of the seed culture medium is the same as in Example 1; S3 Fermentation Culture: The fermentation medium was added to the fermenter, sterilized and cooled to 34℃. The secondary seed culture of Clostridium butyricum obtained in S2 was inoculated into the fermenter at an inoculation rate of 3% and fermented for 48 h. Then, it was centrifuged at 4℃ and 4000 r / min for 10 min, and the supernatant of the culture medium and the biomass precipitate were recovered respectively. The fermentation medium consists of the following components: glucose 8 g / L, methyl-β-cyclodextrin 3 g / L, betaine 0.2 g / L, corn cob extract 0.3 g / L, soybean meal hydrolysate 15 g / L, yeast powder 12 g / L, sodium bicarbonate 0.2 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate heptahydrate 0.2 g / L, calcium citrate 0.8 g / L, seaweed polysaccharide 5 g / L, vitamin B1 0.01 g / L, and pH 6.2±0.1. S4-S5 are the same as in Example 1, obtaining fermentation metabolites of Clostridium butyricum.

[0027] Example 3 A fermentation metabolite of Clostridium butyricum, prepared by a method different from that in Example 1, the fermentation conditions in step S3 are as follows: The fermentation medium was added to the fermenter, sterilized, and then cooled to 36°C. The secondary seed culture of Clostridium butyricum obtained in S2 was inoculated into the fermenter at an inoculation rate of 2% and fermented for 48 h. Then, it was centrifuged at 4°C and 4000 r / min for 10 min, and the supernatant of the culture medium and the biomass precipitate were recovered respectively. The fermentation medium consists of the following components: glucose 8 g / L, methyl-β-cyclodextrin 2 g / L, betaine 0.5 g / L, corn cob extract 0.5 g / L, soybean meal hydrolysate 10 g / L, yeast powder 10 g / L, sodium bicarbonate 0.2 g / L, dipotassium hydrogen phosphate 0.4 g / L, magnesium sulfate heptahydrate 0.2 g / L, calcium citrate 0.8 g / L, seaweed polysaccharide 1 g / L, vitamin B1 0.01 g / L, and pH 6.2±0.1.

[0028] Comparative Example 1 In step S3, no methyl-β-cyclodextrin or betaine was added to the fermentation medium, and the glucose content was adjusted to 12.5 g / L. All other aspects were the same as in Example 1.

[0029] Comparative Example 2 In step S3, no seaweed polysaccharide was added to the fermentation medium, and the glucose content was adjusted to 10 g / L.

[0030] Comparative Example 3 Unlike Example 1, corn cob extract was not added to the fermentation medium in step S3, but everything else was the same as in Example 1.

[0031] Comparative Example 4 Unlike Example 1, betaine was not added to the fermentation medium in step S3, and the glucose content was adjusted to 15 g / L. All other aspects were the same as in Example 1.

[0032] The butyric acid content in *Clostridium butyricum* and its metabolites obtained from fermentation in Examples 1-3 and Comparative Examples 1-4 was detected, and the results are shown in Table 1 below. The method for determining the *Clostridium butyricum* content refers to standard T / CSWSL006-2019.

[0033] Table 1. Fermentation effect of Clostridium butyricum in each experimental group Experimental group Clostridium butyricum number (CFU / mL) Butyric acid content (g / L) Example 1 1.3 x 10 9 ]]> 63.5 Example 2 1.16 x 10 9 ]]> 58.4 Example 3 1.10 x 10 9 ]] 53.2 Comparative Example 1 8.31 x 10 8 ]]> 32.5 Comparative Example 2 9.92 x 10 8 ]] 50.2 Comparative Example 3 9.65 x 10 8 ]] 40.9 Comparative Example 4 8.03 x 10 8 ]]> 28.6

[0034] The experimental results in Table 1 above show that, in Example 1 of this invention, the use of gradient temperature rise combined with a specific fermentation medium for the fermentation culture of Clostridium butyricum resulted in a maximum viable count of 1.3 × 10⁻⁶ cells after fermentation. 9 The CFU / mL concentration of the fermentation metabolites was as high as 63.5 g / L, with butyric acid content reaching 63.5 g / L.

[0035] Example 2 uses fixed fermentation conditions to ferment Clostridium butyricum, while the content of components such as methyl-β-cyclodextrin and corn cob extract is slightly reduced, and the content of butyric acid in the fermentation metabolites is reduced.

[0036] In Comparative Examples 1-4, the fermentation medium for Clostridium butyricum was adjusted by omitting components such as corn cob extract, methyl-β-cyclodextrin, seaweed polysaccharide, and betaine. The results showed that both the bacterial count and the yield of butyric acid were significantly reduced, with the adjustment of seaweed polysaccharide having the least impact on the fermentation effect.

[0037] The addition of components such as methyl-β-cyclodextrin and betaine not only helps to improve the utilization rate of carbon sources by Clostridium butyricum, but also regulates the exchange capacity between cells and the environment, thereby mediating the metabolism of Clostridium butyricum towards the production of butyric acid, thus obtaining metabolites with higher butyric acid content.

[0038] Application Example 1 The fermentation metabolites prepared in Examples 1-3 and Comparative Examples 1-4 were mixed with Clostridium butyricum to prepare a feed additive, which was then fed to aquatic animals simultaneously with ordinary feed. The prepared feed additive contained the fermentation metabolites prepared in each example and each comparative example, as well as Clostridium butyricum, with a Clostridium butyricum content of 1.3 × 10⁻⁶. 9 CFU / mL, the volume-to-mass ratio of feed additive to feed is 1 mL: 100 kg.

[0039] The specific experimental procedure is as follows: 1100 healthy juvenile whiteleg shrimp aged 15-20 days were randomly selected and divided into 11 groups of 100 shrimp each, with an initial average weight of 5.2 g / s. The control group was fed regular feed without any feed additives. Experimental groups 1-7 are feed additives prepared from fermented metabolites of Examples 1-3 and Comparative Examples 1-4, respectively. Control group 1 was fed regular feed, with the same amount of Clostridium butyricum solution added as the feed additive; Control group 2 was fed ordinary feed, while a certain amount of butyric acid was added to the feed, the amount of butyric acid added was the same as in Example 1; Control group 3 was fed with ordinary feed and the fermented metabolites prepared in Example 1.

[0040] Eleven groups of Litopenaeus vannamei were reared for 40 days, with no other medications used during the entire feeding process, and all other rearing conditions were the same. Mortality rates and average individual weights of the Litopenaeus vannamei in each group were recorded, and the results are shown in Table 2 below.

[0041] Table 2. Growth status of Litopenaeus vannamei. Final weight (g) Average weight (g / head) Survival rate (%) Blank group 297 7.3 42 Experimental group 1 1501 15.8 95 Experimental group 2 1255.6 14.6 86 Experimental group 3 1074.2 13.1 82 Experimental group 1 798 11.4 70 Experimental group 2 912 12.0 76 Experimental group 3 828 11.5 72 Experimental group 4 636 10.6 60 Control group 1 526.4 9.4 56 Control group 2 550.8 10.8 51 Control group 3 875 12.5 70

[0042] As shown in Table 2, the experimental results indicate that when the butyric acid bacteria fermentation metabolites obtained by the culture method of this invention are combined with butyric acid bacteria and used in aquaculture, the shrimp gain weight significantly and the survival rate is higher.

[0043] in addition, Figure 1 The images show the intestinal condition of shrimp after 2 days of feeding, specifically the control group (left) and experimental group 1 (right). As can be seen from the images, the shrimp's intestines were empty when no feed additive was used, while the shrimp's intestines were full and intact after the feed additive was used.

[0044] This is because, on the one hand, the fermentation process of Clostridium butyricum produces a large amount of short-chain fatty acid components such as butyric acid and acetic acid, as well as a variety of active ingredients, which can not only promote the repair of intestinal mucosa in aquatic animals, but also prevent enteritis diseases such as jejunum, white feces, and tarry feces, thus benefiting the maintenance of intestinal health.

[0045] On the other hand, the butyric acid bacteria added to the feed additives can colonize the anaerobic layer at the bottom of the aquaculture water, decompose organic matter such as uneaten feed and feces, further inhibit the proliferation of harmful bacteria at the bottom of the pond, and improve the bottom quality. Therefore, the survival rate of shrimp is greatly improved after the use of feed additives.

Claims

1. A method for preparing Clostridium butyricum fermentation metabolites, characterized in that, The steps include the following: Activation of Clostridium butyricum S1; Cultivation of S2 Clostridium butyricum seed culture: The Clostridium butyricum suspension obtained from S1 was inoculated into seed culture medium and cultured in primary and secondary anaerobic conditions at 32-38℃ and 60-80 r / min for 12-48 h to obtain secondary seed culture. The seed culture medium consists of the following components: glucose 8-15 g / L, soybean meal hydrolysate 5-20 g / L, yeast extract 3-12 g / L, dipotassium hydrogen phosphate 0.2-0.8 g / L, magnesium sulfate heptahydrate 0.1-0.5 g / L, manganese sulfate 0.05-0.15 g / L, calcium chloride 0.5-2 g / L, ferrous sulfate 1-4 g / L, L-cysteine ​​hydrochloride 0.3-0.8 g / L, sodium butyrate 0.05-0.1 g / L, with a pH of 6.5±0.

2. S3 Fermentation Culture: Add the fermentation medium to the fermenter, sterilize and cool to 35-38℃, inoculate the secondary seed liquid of Clostridium butyricum obtained in S2 into the fermenter at a volume ratio of 1%-5% for fermentation culture, and recover the supernatant of the culture medium and the biomass precipitate separately. The fermentation medium consists of the following components: glucose 6-8 g / L, methyl-β-cyclodextrin 1-5 g / L, betaine 0.01-1 g / L, corn cob extract 0.2-0.6 g / L, soybean meal hydrolysate 5-15 g / L, yeast powder 10-20 g / L, sodium bicarbonate 0.1-0.5 g / L, dipotassium hydrogen phosphate 0.2-0.8 g / L, magnesium sulfate heptahydrate 0.1-0.3 g / L, calcium citrate 0.5-2 g / L, seaweed polysaccharide 1-5 g / L, vitamin B1 0.01-0.02 g / L, with a pH of 6.2±0.

1. S4 is resuspended in sterile water containing 0.85% NaCl in the biomass precipitate obtained in S3, and centrifuged 1-2 times to obtain purified bacterial cell precipitate. The purified bacterial cell precipitate is diluted with sterile water to a solid content of 5%-8%, and then homogenized under high pressure at 30-60 MPa 2-3 times to obtain lysate. S5 involves mixing the lysate from S4 with the supernatant obtained in S3 to obtain the fermentation metabolites of Clostridium butyricum.

2. The preparation method according to claim 1, characterized in that, In S4, the centrifugation speed is 2500-3500 r / min, and the centrifugation time is 8-20 min.

3. A product containing Clostridium butyricum fermentation metabolites prepared by any one of the preparation methods in claims 1-2.

4. The product as described in claim 3, characterized in that, The product described is any one of nanoemulsion, lyophilized powder, tablets, capsules, or gel candy.

5. The use of the product as described in any one of claims 3-4 in the preparation of feed additives.

6. A feed additive, characterized in that, The feed additive includes Clostridium butyricum and its fermentation metabolites, wherein the fermentation metabolites are prepared according to any one of the methods described in claims 1-2, and the total number of Clostridium butyricum in the feed additive is ≥1.0 × 10⁻⁶. 7 CFU / mL.

7. The feed additive as described in claim 6, characterized in that, The feed additive is an aquatic animal aquaculture feed additive, and the aquatic animals are any one or more of the following: whiteleg shrimp, Chinese shrimp, sea bass, California bass, silver carp, and swimming crab.

8. The feed additive as described in claim 7, characterized in that, The feed additive can be directly mixed with the aquatic feed and fed to the aquatic animals, or the feed additive can be dissolved in water, mixed, and then directly sprinkled into the pond.

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