Cetobacterium sp. YDC001 and its application

Abstract

The present invention discloses a Cetobacterium sp. YDC001 and its application. The Cetobacterium sp. YDC001 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration Committee on May 13, 2024, with a deposit number of CGMCC No. 30560. The present invention screened out a strain of Cetobacterium sp. YDC001 with good performance from the intestines of wild yellow catfish in Gaoyou area, Yangzhou City, Jiangsu Province. The strain belongs to fermentation bacteria and is a potential probiotic with excellent performance. It can be used to prepare aquatic fermented soft granular feed. As a Cetobacterium sp. fermented soft granular feed, it is applied to fish farming, and has the functions of increasing the proportion of Cetobacterium sp. in the intestines of fish, promoting growth, protecting the liver and intestines of fish, and enhancing the body's immunity and antioxidant capacity.

Description

Technical Field

[0001] The invention relates to a strain of Cetobacterium sp. YDC001 and application thereof in aquatic soft particle fermented feed, belonging to the field of aquatic fermented feed. Background Art

[0002] At present, green and healthy aquaculture technology with the goal of high-quality development of fisheries is being promoted in an orderly manner across the country, and higher requirements have been put forward for the urgent problems to be solved in fishery production, such as the reduction of aquaculture drugs, the treatment of aquaculture tailwater, and the replacement of compound feed. Against this background, aquatic soft-granular fermented feed characterized by green, healthy and safe came into being. Aquatic soft-granular fermented feed is a soft-granular feed formed by adding beneficial fermentation agents to complete granular feed and undergoing solid-state fermentation for a certain period of time. After fermentation, this type of feed often has the characteristics of good palatability, easy digestion, and contains special beneficial biomass. At present, the fermentation agents used to make aquatic soft-granular fermented feed are mainly lactic acid bacteria, fungi and spore-forming fungi, and are usually used in the form of composite agents. These strains are mainly isolated from species such as livestock and poultry or from water bodies, soil and other environments. For aquatic animals, they often cannot be well colonized and only play the role of a "passing" bacteria. In this case, the probiotic effect of fermented feed will be greatly reduced. In recent years, studies have found that Cetobacterium is the dominant bacteria in the intestinal flora of fish and is also an indigenous bacteria in the intestinal tract of fish. It plays an important regulatory role in the health of fish and has a certain inhibitory effect on harmful bacteria. At the taxonomic level, this bacterium belongs to the genus Cetobacterium of the phylum Fusobacterium, a Gram-negative bacterium, an anaerobic type, can grow on GAM medium, and produces acetic acid and other substances during the metabolic process. High abundance of Cetobacterium helps fish stabilize the intestinal flora and maintain intestinal health. In recent years, people have carried out useful attempts and made certain progress in the biological characteristics, antibacterial properties, metabolites and their effects on fish growth of Cetobacterium, but there has been no report on the direct application of a single Cetobacterium species in the production of fermented soft pellet feeds in complete pellet feeds. Summary of the invention

[0003] Purpose of the invention: The first purpose of the present invention is to provide a Cetobacterium sp. YDC001; the second purpose of the present invention is to provide a strain fermentation broth containing the Cetobacterium sp. YDC001; the third purpose of the present invention is to provide a microbial preparation containing the Cetobacterium sp. YDC001 or its strain fermentation broth; the fourth purpose of the present invention is to provide the use of the Cetobacterium sp. YDC001 or its strain fermentation broth or its biological preparation in the preparation of aquatic fermented soft pellet feed. The fifth purpose of the present invention is to provide the use of the Cetobacterium sp. YDC001 or its strain fermentation broth or its biological preparation in the feeding of aquatic animals with aquatic fermented soft pellet feed.

[0004] Technical solution: A Cetobacterium sp. YDC001 of the present invention, the Cetobacterium sp. YDC001 was deposited in the General Microbiology Center (CGMCC) of the China Microorganism Culture Collection Administration on May 13, 2024, with a deposit number of CGMCC No. 30560, and a deposit address of the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and is classified and named: Cetobacterium sp. YDC001.

[0005] Furthermore, the Cetobacterium sp. YDC001 was screened from the intestines of wild yellow catfish in Gaoyou area of ​​Yangzhou City, Jiangsu Province.

[0006] Furthermore, the nucleotide sequence of the Cetobacterium sp. YDC001 is as shown in SEQ ID NO.1.

[0007] The strain fermentation broth of the present invention comprises the Cetobacterium sp. YDC001 of the present invention.

[0008] The microbial preparation of the present invention comprises the strain Cetobacterium sp. YDC001 of the present invention or the fermentation liquid of the strain of the present invention.

[0009] The present invention also includes the use of the Cetobacterium sp. YDC001 or the strain fermentation liquid or the microbial preparation in the preparation of aquatic fermented soft granular feed.

[0010] The present invention also includes a method for preparing aquatic fermented soft pellet feed, which is prepared by using the Cetobacterium sp. YDC001 or the strain fermentation liquid or the microbial preparation of the present invention, and comprises the following steps:

[0011] The pH of the complete pellet feed for aquaculture is adjusted by using acid or alkali, and the Cetobacterium sp. YDC001 or the fermentation liquid of the strain or the microbial preparation is added, stirred and mixed, and sealed for fermentation and culture.

[0012] Furthermore, the addition amount of the Cetobacterium sp. YDC001 of the present invention or the fermentation liquid of the strain of the present invention or the microbial preparation of the present invention is 0.1-1% of the weight of the complete pellet feed for aquaculture.

[0013] Furthermore, the complete pellet feed for aquaculture is yellow catfish feed, California seabass feed or channel catfish feed, etc.

[0014] Furthermore, the pH is adjusted to 7.0-9.0.

[0015] Furthermore, during sealed fermentation, a liquid culture medium containing cysteine ​​and having a pH value of 7.0 to 9.0 is added, and the sealed fermentation is carried out at 25 to 40° C. for 48 to 96 hours.

[0016] Furthermore, when using the Cetobacterium sp. YDC001 described in the present invention, the Cetobacterium sp. YDC001 described in the present invention is activated and inoculated into a Cetobacterium sp. fermentation liquid culture medium, and sealed and statically cultured at 25-40° C. for 8-12 hours. The formula of 1L of the Cetobacterium sp. fermentation liquid culture medium is as follows: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L sodium chloride, 0.5 g / L cysteine, pH 7.0-9.0.

[0017] Furthermore, complete pellet feed for aquaculture: 1kg of cysteine: 0.4-1.6g.

[0018] The invention discloses an application of aquatic fermented soft granular feed obtained by the preparation method in feeding aquatic animals.

[0019] Furthermore, during feeding, the aquatic fermented soft granular feed is mixed with the commercial feed, and the added amount of the aquatic fermented soft granular feed is 50-100% of the weight of the commercial feed.

[0020] The soft granules fermented with cetacean are used for aquaculture animals, and have the functions of increasing the proportion of cetacean in the intestinal tract of fish, promoting growth, protecting the liver and intestinal tract of fish, and enhancing the immunity and antioxidant capacity of the body.

[0021] The Cetobacterium sp. YDC001 of the present invention belongs to a fermentative bacterium, which produces gas and acidic substances by fermentation, and can utilize compounds such as glucose, maltose, cellobiose, fusin (trehalose), sucrose, etc., and is non-pathogenic to yellow catfish. At the same time, the metabolomics analysis of the soft pellet feed fermented by the Cetobacterium sp. of the present invention found that the content of beneficial substances such as bile acid, nicotinic acid and D-glucuronic acid was significantly increased, and these substances are helpful to regulate the nutritional physiological process of aquatic animals.

[0022] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0023] (1) The Cetobacterium sp. YDC001 screened by the present invention can grow in fermented soft pellet feed, and can be applied to aquatic fermented soft pellet feed to increase the proportion of Cetobacterium sp. in the intestine of fish.

[0024] (2) The soft pellet feed fermented with Bacillus cereus used in the present invention has the effects of promoting fish growth, protecting fish liver and intestines, and enhancing the body's immunity and antioxidant capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The colony morphology of Cetobacterium sp. YDC001 and the results of Gram staining microscopic observation;

[0026] Figure 2 A phylogenetic diagram comparing the 16S rDNA sequences of Cetobacterium sp. YDC001 and other Fusobacteria;

[0027] Figure 3 This is the result of the hemolysis test of Cetobacterium sp. YDC001;

[0028] Figure 4 This is a schematic diagram of the state of soft pellet feed fermented by Bacillus cereus before and after fermentation;

[0029] Figure 5 This is the VIP value analysis diagram of the KEGG compound metabolic set in soft pellet feed fermented by Bacillus cereus;

[0030] Figure 6 This is a bar chart showing the effect of soft pellet feed fermented with Bacillus cereus on the foregut flora of yellow catfish at the genus level.

[0031] Figure 7 This is a process flow chart for preparing soft pellet feed fermented by Bacillus subtilis. Implementation

[0032] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0033] Example 1

[0034] 1. Isolation and screening of Cetobacterium sp. YDC001

[0035] Cetobacterium sp. YDC001 was purified and isolated from the intestine of wild yellow catfish provided by Jiangsu Gaoyou Lake Ecological Fishery Development Co., Ltd.

[0036] Under sterile conditions, the hind intestine of the yellow catfish was taken, cut longitudinally, and the intestinal contents were gently rinsed with sterile saline. The cleaned hind intestine of the yellow catfish was ground at 70Hz for 120s using a high-throughput tissue grinder. The modified GAM medium (Qingdao Haibo Biological, catalog number HB8518) was used for streaking, and cultured at 37°C for 24h under anaerobic conditions. Single colonies were selected and identified by 16s rDNA. After confirmation, the colonies were expanded and preserved.

[0037] 2. Description of biological characteristics of Cetobacterium sp. YDC001

[0038] 2.1 Observation of morphological characteristics

[0039] The plate streak method was used to separate single colonies and observe the morphological characteristics of single bacterial colonies. A small amount of bacterial liquid was taken for smear and Gram staining, and the bacterial morphology and staining characteristics were observed under an optical microscope. Figure 1 . Figure 1 The following are the colony morphology characteristics and Gram staining microscopic observation results of Cetobacterium sp. YDC001, where a is the colony morphology characteristics and b is the Gram staining microscopic image. Figure 1 It can be seen that the colony of Cetobacterium sp. YDC001 is white and round, with smooth edges, raised and opaque. Gram staining is negative, short rod-shaped, blunt at both ends, 1.5 to 2.5 μm in length, and 0.7 to 1.0 μm in diameter.

[0040] 2.2 Physiological and biochemical reaction experiments

[0041] The physiological and biochemical experiments on Bacillus cetaceus were carried out using biochemical reaction tubes (Qingdao Haibo Biotechnology Co., Ltd.), and the measured indicators were: decomposition of glucose, maltose, mannitol experiment, lysine decarboxylation reaction, oxidation fermentation OF, catalase test and gelatin, etc. The results are shown in Table 1.

[0042] Table 1 Physiological and biochemical reaction results of Cetobacterium sp. YDC001

[0043]

[0044]

[0045] 3. Molecular biological identification information and construction of phylogenetic tree

[0046] The genomic DNA of Ceti was extracted using a bacterial genomic DNA extraction kit (DP302-02, Tiangen Biochemical Technology Co., Ltd.). PCR amplification was performed using bacterial 16S rDNA universal primers, 27F: 5'AGAGTTTGATCCATGGCTCAG-3' (SEQ ID NO.2), 1429R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO.3). The amplified product was sent to Qingke Biotechnology Co., Ltd. for sequencing. By 16S rDNA sequencing, the following sequence SEQ ID NO.1 was obtained:

[0047]

[0048] The 16S rDNA sequences of 16 strains in the same family (Fusobacteriaceae) as the strain Cetobacterium sp. YDC001 in the GenBank database were selected, and the phylogenetic tree was constructed using the Neighbor-joining method on MEGA 7.0. The Kimura two-parameter model was used for analysis, and 500 bootstrap samplings were performed. The sequence was found to be similar to the strain Cetobacterium somerae CBA7533 (GenBank accession number MN646996.1) with clear species information, with a QueryCover value of 92%, an E value of 0.0, and a Per.Ident value of 98.06%. Phylogenetic tree (see Figure 2) The results showed that the isolated strain was closest to Cetobacterium sp. ZF1NB02, and on the same branch, it was the closest to Cetobacterium sp. ZF1NH01. It was preliminarily identified as Cetobacterium sp. and named Cetobacterium sp. YDC001. The strain was sent to the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) in Beijing, China for preservation on May 13, 2024, with the preservation number CGMCC No. 30560.

[0049] Example 2 Safety Evaluation of Cetobacterium sp. YDC001

[0050] 1. Hemolytic ability test

[0051] Cetobacterium sp. YDC001 was streaked onto the surface of Columbia blood agar medium (Changde Beekman Biotechnology Co., Ltd., China), placed in an anaerobic bag, and cultured at 37.0°C for 48 h. Figure 3 As shown. Figure 3 It can be seen that there is neither a grass-green ring (α-hemolysis) nor a transparent ring (β-hemolysis) around the colonies of Cetobacterium sp. YDC001, which belongs to non-toxic γ-hemolysis, that is, non-hemolysis.

[0052] 2. Acute gavage test

[0053] There are 4 kinds of gavage solutions: sterile saline, modified GAM broth, Cetobacterium suspension and Cetobacterium fermentation supernatant. Saline and modified GAM broth were made by high pressure sterilization and used as the control group. Take 100ml of GAM broth cultured with Cetobacterium sp. YDC001 for 8 hours and centrifuge at 12000rpm for 2min, discard the supernatant, add saline to wash, 12000rpm for 2min again, repeat the washing twice, add 10ml of saline to prepare Cetobacterium suspension, at this time, each milliliter contains about 1 to 5×10 9The number of bacteria in CFU / ml, stored at 4℃, used up within 2 hours. Take another 100ml of GAM broth cultured with Cetobacterium sp. YDC001 for 18-24 hours, centrifuge at 12000rpm for 2min, take the supernatant as the fermentation supernatant of Cetobacterium sp., store at 4℃, and use up within 2 hours. For gavage, use a stainless steel gavage needle with a handheld continuous syringe, and sterilize at high temperature before use. When gavage, wear model gloves, hold the dorsal fin and pectoral fin of the yellow catfish, so that the head of the yellow catfish is tilted upward, and when the yellow catfish opens its mouth slightly, insert the gavage needle into the stomach of the yellow catfish, and inject 0.5ml of gavage solution. If vomited, gavage again. The yellow catfish used for gavage were healthy all-male yellow catfish, about 50g, with 10 fish in each group. The gavage time was 6:00-7:00 a.m. every day. The water was changed once after gavage, and a small amount of feed was fed at 6:00 p.m. During the period, the activity of the yellow catfish was observed. After 7 consecutive gavage tests for 7 days, no death occurred in the yellow catfish in each group, and the activities were normal.

[0054] Example 3 Preparation of soft pellet feed fermented by Bacillus cereus

[0055] The process flow of soft pellet feed fermented by Bacillus cereus Figure 7 As shown, the details are as follows:

[0056] 1. Preparation of Cetiella fermentation agent

[0057] Take a small amount of Cetobacterium sp. YDC001 strain and inoculate it into 10ml of modified GAM liquid medium, under anaerobic conditions, activate it at 37℃ for 24h. Then inoculate the activated bacterial liquid into Cetobacterium fermentation liquid medium at a ratio of 1:100, seal it and culture it for 8h to make Cetobacterium fermentation agent, refrigerate it at 4℃ for use, and it is effective within 7 days. The formula of 1L Cetobacterium fermentation liquid medium is as follows: 10g / L tryptone, 5g / L yeast extract, 5g / L sodium chloride, 0.5g / L cysteine, pH 8.0.

[0058] 2. Fermentation parameters of soft pellet feed fermented by Bacillus subtilis.

[0059] Fermentation time: 48-96h

[0060] Initial fermentation pH: 7.0~9.0

[0061] Starter inoculation amount: 0.1%~1.0%

[0062] Fermentation moisture content: 35%~45%

[0063] Fermentation protective agent: 0.4-1.6 g / kg cysteine

[0064] Fermentation container: vacuum sealed special plastic fermentation bag

[0065] Fermented feed type: extruded pellet feed

[0066] 3. The fermentation process of soft pellet feed fermented by Bacillus subtilis.

[0067] Preparation process Figure 7 As shown, taking the puffed pellet feed of yellow catfish as an example, it takes about 0.5 kg of sodium hydroxide tablets to adjust the pH of 100 kg puffed pellet feed (Yangzhou Hongda Feed Co., Ltd., puffed feed for adult yellow catfish 8249) to 8.0. 0.5 kg of sodium hydroxide tablets (or sodium hydroxide solution of equivalent concentration) are dissolved in 30 L of pure water to form a pH regulating solution. A multifunctional dry and wet dual-purpose stainless steel mixer is used to mix the acid / alkali solution and 100 kg puffed pellet feed evenly. At the same time, 1 L of the cetacean fermentation agent obtained in step 1 is mixed with 29 L of an aqueous solution containing 80 g of cysteine ​​to prepare a cetacean suspension. After the cetacean suspension and the above feed are stirred and mixed again, they are put into a special plastic fermentation bag, vacuumed, and the fermentation agent inoculation amount is 1%. The bag is sealed and fermented at 35 ° C for 96 hours to prepare a cetacean fermented soft pellet feed. According to the different raw material compositions of the puffed feed, the added moisture content can be appropriately adjusted within a certain range, with the principle of softness, particle formation, and ease of feeding as the principle. Other fermented feed production equipment can also be used for large-scale production after scientific debugging. Figure 4 shown.

[0068] Example 4 Metabolomics analysis of soft pellet feed fermented by Bacillus cereus

[0069] Take the feed samples before and after fermentation in Example 3 (see Figure 4 ). Figure 4 This is a schematic diagram of the state of soft pellet feed fermented by Cetiella fermented before and after fermentation, where a is a schematic diagram of the state before fermentation, and b is a schematic diagram of the state after fermentation. Take a small amount at the moment of opening the bag and place it in a 2 ml cryotube, quick-freeze it with liquid nitrogen, and store it at -80°C as a metabolome analysis sample. The metabolomics samples were transported to Shanghai Meiji Biotechnology Co., Ltd., China using dry ice, and the metabolome composition was analyzed by LC-MS using the UHPLC-Q Exactive HF-X system with ultra-high performance liquid chromatography tandem Fourier transform mass spectrometry. A total of 2099 substances were found in this metabolomics test. Compared with the metabolites of the feed before fermentation, 244 compounds were significantly upregulated in the fermented feed, 246 compounds were significantly downregulated, and 1607 compounds had no significant changes. After comparison and annotation with the KEGG Compound database, a total of 42 differentially functional metabolites were annotated. The differentially functional metabolites mainly belong to nucleic acids, carbohydrates, lipids, peptides, vitamins, steroids, etc. From Figure 5It can be seen that the metabolomics analysis of fermented feed found that the contents of beneficial substances such as bile acid, niacin and D-glucuronic acid were significantly increased, which help regulate the nutritional and physiological processes of yellow catfish.

[0070] Example 5 Study on the probiotic properties of soft pellet feed fermented by Bacillus cereus

[0071] The soft pellet feed fermented by cetacean prepared in Example 3 was used to carry out a breeding experiment on yellow catfish. The test fish species was "Quanxiong No. 1" yellow catfish (Tachysurus fulvidraco), purchased from Dong's Special Aquatic Products Co., Ltd., Gaoyou, Yangzhou City, Jiangsu Province. Before the formal experiment began, commercial feed was used for temporary domestication for 2 weeks. After fasting for 24 hours, 192 healthy yellow catfish with an initial weight of 60.58±12.12g were randomly divided into 16 breeding barrels (300L), 12 fish per barrel, and 4 treatment groups were set up, each with four replicates. The fermented soft pellet feed was mixed with commercial feed at 0%, 10%, 50% and 100% dry weight, and recorded as the control group, C10 group, C50 group and C100 group. The experimental breeding cycle was 70 days. After the experiment, the growth performance, liver and intestinal antioxidant indicators, and intestinal flora of the test fish were tested.

[0072] The growth performance data are shown in Table 2.

[0073] Table 2 Effects of different addition ratios of soft pellet feed fermented with Cetacea on the growth performance of Pelteobagrus fulvidraco (n=4)

[0074]

[0075] Note: Data are expressed as mean±SD. Values ​​with different superscript letters in the same row indicate significant differences (P<0.05), while values ​​with the same or no superscript letters in the same row indicate no significant differences (P>0.05), the same below.

[0076] Depend on Figure 2 It can be seen that the terminal mean weight (FBW), weight gain rate (WGR), and specific growth rate (SGR) of each supplement group were significantly higher than those of the control group (P<0.05), and the WGR of the C50 group was the highest, which was 26% higher than that of the control group. The feed conversion rate (FCR) of each supplement group was significantly lower than that of the control group (P<0.05), among which the feed conversion rate of the C50 group was 25% lower than that of the control group. The survival rate (SR) of each group was 100%. There were no significant differences in body shape indicators such as fatness (CF), liver-to-body ratio (HSI) and mesenteric fat index (MATI) among the groups (P>0.05).

[0077] The data of liver and intestinal antioxidant enzymes are shown in Table 3.

[0078] Table 3 Effects of different addition ratios of soft pellet feed fermented with Cetacea on antioxidant enzymes in yellow catfish (n=4)

[0079]

[0080] As shown in Table 3, in the liver, the superoxide dismutase (SOD) activity of the C10 group and the C50 group was significantly higher than that of the C100 group (P<0.05), and the SOD activity of the C100 group was significantly higher than that of the control group (P<0.05); there were no significant differences in the total antioxidant capacity (T-AOC), catalase (CAT), malondialdehyde (MDA) and glutathione peroxidase (GSH-px) among the groups (P>0.05). In the intestine, the T-AOC activity of the C10 group was significantly higher than that of the control group; the CAT activity of the C100 group was significantly higher than that of the control group (P<0.05); the MDA content of the C50 group was significantly lower than that of the control group (P<0.05); there were no significant differences in the SOD and GSH-px indicators among the groups (P>0.05).

[0081] The results of intestinal flora analysis were as follows Figure 6 Displayed by Figure 6 It can be seen that at the genus level, the relative abundance of Cetiella in group C100 was significantly higher than that in group C50 (P<0.05), and the relative abundance of Cetiella in group C50 was significantly higher than that in group C10 (P<0.05).

Claims

1. A cetacean ( Cetobacterium sp. )YDC001, characterized by, The Ceti Cetobacterium sp. )YDC001 was deposited in the China General Microbiology Center (CGMCC) on May 13, 2024, with the deposit number CGMCC No. 30560.

2. A strain fermentation broth, characterized in that: comprising the Cetiaceae described in claim 1 ( Cetobacterium sp. )YDC001.

3. A microbial preparation, characterized in that: comprising the bacillus cetaceus of claim 1 ( Cetobacterium sp. ) YDC001 or the fermentation broth of the strain described in claim 2.

4. A method for preparing aquatic fermented soft granular feed, characterized in that: Utilizing the bacillus cetaceus described in claim 1 ( Cetobacterium sp. ) The preparation of the fermentation broth of the strain YDC001 or claim 2 or the microbial preparation according to claim 3 comprises the following steps: The pH of the complete pellet feed for aquaculture is adjusted by using acid or alkali, and the cetacean described in claim 1 is added ( Cetobacterium sp. ) YDC001 or the fermentation broth of the strain described in claim 2 or the microbial preparation described in claim 3, stir and mix, and then seal and ferment to obtain the product.

5. The preparation method according to claim 4, characterized in that: The cetacean according to claim 1 ( Cetobacterium sp. ) The amount of YDC001 or the fermentation broth of the strain described in claim 2 or the microbial preparation described in claim 3 added is 0.1 to 1% by weight of the complete pellet feed for aquaculture.

6. The preparation method according to claim 4, characterized in that: The complete granular feed for aquaculture is yellow catfish feed, California seabass feed or channel catfish feed, and the pH is adjusted to 7.0-9.

0. During sealed fermentation, a liquid culture medium containing cysteine ​​and a pH of 7.0-9.0 is added, and the sealed fermentation is carried out at 25-40°C for 48-96 hours.

7. Use of the aquatic fermented soft granular feed obtained by the preparation method according to any one of claims 4 to 6 in fish breeding.

8. The use according to claim 7, characterized in that: During feeding, aquatic fermented soft granular feed is mixed with commercial feed, and the added amount of aquatic fermented soft granular feed is 50-100% of the weight of commercial feed.

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