Methylobacterium strain JC03 for producing single-cell protein and application of methylobacterium strain JC03 in field of aquaculture

Abstract

The invention provides a methylobacterium strain JC03 for producing single-cell protein and application of the methylobacterium strain JC03 in the field of aquaculture, and belongs to the technical field of microorganisms. The methylobacterium strain JC03 is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.32616. The strain is high in growth efficiency, high in thallus crude protein content, rich in amino acid spectrum, high in essential amino acid content and rich in functional components for improving the immunocompetence of aquatic animals; the single-cell protein produced by the strain can completely or partially replace fish meal protein in feeds of marine fishes such as turbot, pseudosciaena crocea, grouper and rainbow trout; vegetable protein in feed of freshwater fish such as grass carp can be completely replaced; animal and plant proteins in feeds for shrimps and crabs, such as penaeus vannamei, can be completely replaced, and animal and plant proteins in stichopus japonicus feeds can be completely replaced; the immunocompetence of fed aquatic animals can be obviously improved. The invention provides a microbial strain for efficiently converting methanol to produce single-cell protein, and the microbial strain has a wide application prospect in the field of aquaculture.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a methylbacterium strain JC03 producing single-cell protein and application thereof in the field of aquaculture. Background Art

[0002] Fishmeal is a feed material made from one or more fish species, which has been degreased, dehydrated and crushed. It is an excellent protein raw material for aquatic animal feeds due to its high protein content, good palatability, easy absorption and balanced composition of essential amino acids. The protein content of high-quality imported fishmeal is above 60%. Aquatic animals have a higher requirement for protein content, especially in special aquatic feeds, and are more dependent on fishmeal.

[0003] Traditional non-fishmeal protein sources, whether plant-based or animal-based, have some replacement problems, such as nutritional antagonism, amino acid imbalance, and lack of active ingredients, which lead to low feed production conversion rate, palatability deviation, and even intestinal inflammation in aquatic animals. Using microorganisms to produce single-cell protein as a substitute for fishmeal is an effective solution. Compared with animal and plant protein sources, single-cell protein requires fewer resources, has high production efficiency, and high yield per unit area. The production process is not affected by the environment and climate, the production and recycling process is simple and pollution-free, and the product quality is stable. In particular, single-cell protein with one-carbon non-grain raw materials such as methane, methanol, carbon monoxide and carbon dioxide as substrates has lower production costs and greater potential for sustainable development.

[0004] The output of traditional methanol produced from coal still occupies a dominant position in the methanol industry. However, the weak demand in the downstream of the methanol industry chain has led to high inventory, which has plunged the entire methanol industry into a structural overcapacity crisis. The industrialization of new technologies marks the rapid development of methanol manufacturing, which has injected new impetus into the promotion of clean energy and low-carbon economy. Therefore, the development of the single-cell protein industry using methanol as raw material can not only alleviate the crisis of methanol overcapacity, promote dual carbon emission reduction, but also meet the growing demand for feed protein in the aquaculture industry.

[0005] At present, methanol single-cell protein includes two types: bacterial source and yeast source. Among them, bacterial source single-cell protein represented by Methylobacterium has the advantages of using methanol as the only carbon source and rich in highly active immune factors. It is an important germplasm resource for producing methanol single-cell protein. KnipBio in the United States has developed and utilized Methylobacterium extorquens ( Methylobacterium extorquensPA1) as the chassis strain, which was adaptively evolved and rationally designed to enhance the synthesis of active substances carotenoids, and the relevant invention patent was applied for in 2014. However, compared with other commercially developed bacterial single-cell proteins in China, such as the single-cell protein of Methylococcus capsulatus using natural gas as the substrate and the single-cell protein of Clostridium ethanolum using steel plant exhaust gas as the substrate, the currently reported Methylobacterium extorquens still has low protein content (accounting for less than 60% of the cell dry weight) and slow growth rate (specific growth rate less than 0.15 h -1 ) and low biomass yield (less than 0.3 g bacteria / g methanol) limit the reduction of single-cell protein production costs and the improvement of product quality.

[0006] There are abundant Methylobacterium germplasm resources in nature, which are mainly located in the leaves of plants and use the methanol released during the synthesis of pectin by the cell wall to grow. There are great differences in the methanol metabolism ability, protein content and methanol conversion rate of different strains of Methylobacterium. Therefore, we should explore and screen Methylobacterium strains with fast growth rate, high protein content and high biomass yield, which are suitable for replacing fish meal feed in aquaculture. They can be used to efficiently produce methanol single-cell protein with independent intellectual property rights, which is of great significance to the sustainable development of my country's aquaculture industry. Summary of the invention

[0007] The purpose of the present invention is to provide a single-cell protein-producing Methylobacterium strain JC03 and its application in the field of aquaculture. The present invention screened a methylotrophic strain that can grow rapidly using methanol from radish leaves, identified as Methylobacterium, further improved its methanol tolerance and utilization efficiency through adaptive evolution, and screened a Methylobacterium evolved strain JC03 with significantly improved methanol growth rate and methanol conversion rate through deep-well plate culture. The single-cell protein produced by the strain with methanol as the only carbon source can effectively replace protein feeds such as fish meal in aquaculture feeds and has good immune activity, providing an important industrial strain for single-cell protein used in aquaculture.

[0008] In order to solve the above technical problems, the technical solution to be adopted in the present invention is: The present invention provides a methylbacterium strain JC03 that produces single-cell protein using methanol as the sole carbon source. The classification name of the methylbacterium evolutionary strain JC03 is methylbacterium Methylobacterium sp., its accession number is: CGMCC No.32616.

[0009] Furthermore, the sequence of 16S rRNA of the Methylobacterium JC03 is shown in SEQ ID NO.1.

[0010] Furthermore, the Methylobacterium strain JC03 is dark red, round, protruding upward, and has neat edges.

[0011] The present invention provides a bacterial agent containing the methylbacterium strain JC03.

[0012] The present invention provides the use of the methylobacterium strain JC03 or the bacterial agent in producing single-cell protein using methanol as a carbon source.

[0013] Furthermore, the Methylobacterium strain JC03 is grown on a culture medium with methanol as the sole carbon source, and single-cell protein is obtained by shake flask culture or semi-continuous culture in a fermenter.

[0014] Furthermore, the specific conditions for the shake flask culture are: 25-35°C, 150-250 rpm, and an initial methanol concentration of 0.5-1.5% (V / V) in the HM medium.

[0015] Furthermore, the semi-continuous culture conditions of the fermentation tank are as follows: the inoculation amount of HM culture medium is 10-20%, the temperature is controlled at 25-35°C, the pH is regulated at 6.5-7.5 by ammonia water, the ventilation volume is controlled at 0.5-2.0 vvm, the dissolved oxygen is controlled at 10-30% by a cascade stirring speed of 300-1200 rpm, the methanol concentration is controlled at 0.1-1.5%, and the ammonia nitrogen concentration is controlled at 0.1-3.0 g / L ammonium sulfate. When the OD grows to 150-250, 20-80% of the fermentation liquid of the culture volume is released, and 70-10% of the sterile HM culture medium is supplemented accordingly, the semi-continuous fermentation cycle is continued, and the bacteria are collected.

[0016] The present invention provides the use of the single-cell protein produced by the Methylobacterium strain JC03 or the bacterial agent using methanol as a carbon source in preparing feed for aquaculture animals.

[0017] Furthermore, the single cell protein contains aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, proline and tryptophan.

[0018] Furthermore, the aquaculture animals include marine fish, freshwater fish, shrimps, crabs and sea cucumbers.

[0019] Furthermore, the marine fish include flounder, large yellow croaker, grouper, and rainbow trout; the freshwater fish include grass carp; the shrimps and crabs include white shrimp, and the sea cucumbers include sea cucumbers.

[0020] Furthermore, the application method is to use the single-cell protein to partially or completely replace the fish meal protein and / or plant protein in the feed.

[0021] Furthermore, the single cell protein replaces fish meal protein in turbot feed within 30%; Furthermore, the single cell protein replaces fish meal protein in large yellow croaker feed within 30%; Furthermore, the single cell protein replaces the fish meal protein in grouper feed within 50%; Further, the single cell protein replaces fish meal protein in rainbow trout feed within 50%; Furthermore, the single cell protein replaces soybean meal protein in feed for freshwater fish such as grass carp within 100%; Furthermore, the single cell protein replaces the fish meal protein in the feed of white shrimp within 100%; Furthermore, the single cell protein replaces the Sargassum protein in the sea cucumber feed within 100%.

[0022] The present invention provides application of the single-cell protein produced by the methylbacterium strain JC03 using methanol as a carbon source in preparing an aquatic feed additive for improving the antioxidant function and immune function of aquaculture animals.

[0023] Furthermore, the single cell protein contains high content of carotene, ergothioneine, PQQ, PHB and vitamin B12.

[0024] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention obtains the Methylobacterium JC03 by adaptively evolving the strains selected under natural conditions by increasing the methanol concentration during the culture process. The evolved Methylobacterium strain JC03 has the advantages of fast growth rate, high methanol bacterial protein conversion rate, high bacterial protein content, high essential amino acid content, and rich immune function components. With methanol as the only carbon source, the specific growth rate of the Methylobacterium evolved strain JC03 exceeds 0.30 h -1 The biomass yield is greater than 0.39 g / g, the protein content in the bacteria exceeds 72%, the total amino acid content is greater than 65%, and the essential amino acid content is greater than 32%.

[0025] Therefore, the single-cell protein produced by the Methylbacterium strain JC03 can replace fish meal protein in marine fish and shrimp, such as fish meal protein in giant flounder, large yellow croaker, grouper, rainbow trout and white shrimp, and soybean meal protein in freshwater fish such as grass carp. It can also replace the protein source (generally plant protein) in sea cucumbers, which is beneficial to reduce costs and promote the development of aquaculture.

[0026] 2. The Methylobacterium strain JC03 provided by the present invention contains a variety of antioxidant and immune active functional components such as carotenoids, ergothioneine, pyrroloquinoline quinone (PQQ) and poly-β-hydroxybutyric acid (PHB) in its cells, which can improve the immune activity of a variety of aquatic products such as large flounder, large yellow croaker, grouper, rainbow trout, grass carp, white shrimp and sea cucumber, increase the survival rate of aquatic pathogens, reduce the use of antibiotics, and play a positive role in reducing breeding costs and improving the quality of aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0028] Figure 1 is a colony morphology diagram of the Methylobacterium strain JC03; Figure 2 This is a growth comparison diagram of the Methylobacterium strain JC03 and its wild-type control strain on a deep-well plate; Figure 3 A comparison chart of the methanol biomass conversion rate of the Methylobacterium strain JC03 and its wild-type control strain; Figure 4 is the crude protein content of different batches of the Methylobacterium strain JC03 and its wild-type control strain during semi-continuous fermentation; Figure 5 The whole amino acid profile of the Methylobacterium strain JC03 and its wild-type control strain and commercial fish meal is compared; Figure 6 The essential amino acid composition of the Methylobacterium strain JC03, its wild-type control strain and commercial fish meal in giant flounder is compared; Figure 7 Analysis of antioxidant components of the Methylobacterium strain JC03 and its wild-type control strain; Figure 8 Analysis of immune function active components of the Methylobacterium strain JC03 and its wild-type control strain; Fig. 9 The weight gain rate, feed conversion rate, albinism rate and immune protection rate of each experimental group of turbot; Fig.10 Weight gain rate, feed conversion rate and immune protection rate of each experimental group of large yellow croaker; Fig.11 Weight gain rate, feed conversion rate and immune protection rate of each experimental group of grouper; Fig.12 Weight gain rate, feed conversion rate and immune protection rate of each experimental group of rainbow trout; Fig.13 Weight gain rate, feed conversion rate and immune protection rate of each experimental group of grass carp; Fig.14Weight gain rate, feed conversion rate and immune protection rate of each experimental group of Penaeus vannamei; Fig.15 Weight gain rate, feed conversion rate and immune protection rate of each experimental group of sea cucumber. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with embodiments. The following embodiments are for explanation of the present invention but the present invention is not limited to the following embodiments.

[0030] Example 1: Isolation and purification of wild-type strains of Methylobacterium The samples were from the leaves of Radix Astragali in the fields of Pingluo County, Shizuishan City, Ningxia Hui Autonomous Region. Weigh 5 g of Radix Astragali leaves and put them into a sterile shake flask. Add 50 mL of HM liquid culture medium, and then place the shake flask in a shaker for enrichment culture for 4-6 h. The shaker temperature was 30°C and the speed was 200 rpm. After the enrichment was completed, the enriched culture solution was aspirated and diluted by 10 -1 , 10 -2 and 10 -3 Three concentration gradients were prepared, and 100 μL of the dilution was spread on HM solid culture medium. The culture dish was inverted and cultured in a 30°C incubator for 2-6 days. Single colonies with faster growth, larger colonies and redder color were picked and purified three times to obtain the pure culture wild-type strain JC03.

[0031] The 16S rDNA sequence of strain JC03 was determined using primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGTTACCTTGTTACGACTT-3'). The 16S rDNA sequence is shown in SEQ ID NO.1. By BLAST sequence comparison, the wild-type strain obtained was screened to be Methylobacterium ( Methylobacterium sp.).

[0032] The formula of HM liquid medium is as follows: 3.32 g potassium hydrogen phosphate trihydrate, 1.68 g sodium dihydrogen phosphate dihydrate, 0.21 g magnesium sulfate heptahydrate, 0.5 g ammonium sulfate, 10 mg disodium ethylenediaminetetraacetic acid, 1 mg ferrous sulfate heptahydrate, 1.4 mg calcium chloride dihydrate, 1 mg manganese chloride tetrahydrate, 0.2 mg sodium molybdate dihydrate, 0.3 mg copper sulfate pentahydrate, 1.6 mg cobalt chloride hexahydrate, 4.4 mg zinc sulfate heptahydrate, add water to 1L, sterilize at high temperature, and add 5 mL of methanol filtered through a 0.22 μm filter membrane. For solid culture medium, add 25 g agar powder per liter on the basis of liquid culture medium, sterilize and dispense for use.

[0033] Example 2: Adaptive evolution of wild-type strains and isolation of JC03 strains In order to improve the tolerance, metabolic rate and biomass yield of the wild-type strain to methanol, and at the same time enhance the strain's ability to utilize and transform ammonia nitrogen, the wild-type strain was used as the ancestral strain and subcultured in HM liquid medium with a methanol concentration of 150 mM and an ammonium sulfate concentration of 3.8 mM, and the cells were transferred every 24 h (the initial OD 600 0.02), after 10 transfers, the highest OD of the strain under this culture condition 600 The value increased from 2.0 to 2.5, and the highest OD 600 The value remained stable. According to this evolutionary method, the methanol concentration was then increased gradually (the methanol concentration increased from 150 mM to 180 mM, 210 mM, 240 mM, 270 mM, 300 mM, 330 mM to 360 mM) and the ammonium sulfate concentration (the ammonium sulfate concentration increased from 3.8 mM to 4.56 mM, 5.32 mM, 6.08 mM, 6.84 mM, 7.6 mM, 8.36 mM to 9.12 mM) to increase the selection pressure of adaptive evolution. Finally, after about 250 generations of subculture, single colonies were screened on HM plates containing 360 mM methanol and 9.12 mM ammonium sulfate, and single colonies with faster growth, larger colonies and redder colors were selected and purified three times to obtain the pure cultured evolved strain JC03. The colony morphology of the JC03 evolved strain on HM solid culture medium is shown in the figure. Figure 1 As shown, the colony is dark red, round, protruding upward, and has neat edges.

[0034] The selected evolutionary JC03 strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration (CGMCC), address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; the deposit date is November 14, 2024, Methylobacterium Methylobacterium sp.’s deposit number is: CGMCC No.32616.

[0035] Example 3: Determination of growth curve of strain JC03 and analysis of methanol conversion rate 1. The growth curves of strain JC03 and wild-type ancestor control strain were evaluated using an automatic microbial growth curve analyzer. Monoclonal colonies cultured on HM solid medium for 5 days were inoculated into a 250 mL shake flask containing 50 mL HM liquid medium and cultured at 30°C and 200 rpm for 30 h to prepare seed solution. Dilute with fresh HM liquid medium to the initial OD 600 The concentration of HM medium was 0.05, and then the culture medium was plated in a 48-well plate. Three parallel wells were set up, 1 mL was added to each well, and uninoculated fresh HM medium was used as a blank control. The growth curve of the strain was measured at 30°C and 800 rpm.

[0036] From the results, it can be seen that ( Figure 2 ), JC03 strain had a faster growth rate when methanol was the only carbon source, and its specific growth rate was 0.32±0.01 h -1 The doubling time was about 2.2 hours, and the growth rate was faster than that of the wild-type control strain under the same culture conditions, with the growth rate increased by more than 12%. 600 It can reach above 4.3, and the maximum biomass is increased by more than 25% compared with the wild-type control strain. This shows that the JC03 strain can more efficiently utilize methanol as a carbon source through adaptive evolution, and can grow rapidly while efficiently assimilating methanol.

[0037] 2. In order to further measure the methanol conversion rate of JC03, the seed solution was first prepared according to the same method as above, and then diluted with fresh HM liquid medium to the initial OD 600 The concentration of 0.05 was inoculated in a 250 mL shake flask containing 50 mL HM liquid culture medium and cultured at 30°C and 200 rpm until the stable growth period of the strain. At this time, the methanol in the shake flask was completely consumed. The dry weight data of the bacteria were obtained after washing, centrifugation and freeze-drying, and the methanol conversion rate was calculated. Three parallels were set for each group.

[0038] According to the experimental results ( Figure 3 ), the methanol biomass conversion rate of strain JC03 reached 0.37±0.02 g / g, which was 27% higher than that of the wild-type control strain. The shake flask measurement results were consistent with the deep-well plate results, indicating that strain JC03 had a high methanol conversion rate and could reduce methanol usage and cost while obtaining the same bacterial protein yield, thus having the potential to be used as a methanol single-cell protein producer.

[0039] Example 4: Analysis of protein content and amino acid composition of strain JC03 The JC03 cells used for analysis of protein content and amino acid composition were collected by semi-continuous fermentation. The seed solution of semi-continuous fermentation was cultured by the method of Example 3. 600When the cell density grows to about 1.5-2.2, inoculation is performed with an inoculum size of 10-20%. The temperature is controlled at 25-35°C. The pH is adjusted to 6.5-7.5 using ammonia water or 1M sodium hydroxide solution. The ventilation volume is controlled at 0.5-2.0 vvm. The dissolved oxygen is maintained at 10-30% by cascade stirring speed (the speed is controlled at 300-1200 rpm) or by adding pure oxygen (the pure oxygen flow rate accounts for 0-60% of the air flow rate). The methanol concentration is controlled at 0.1-1.5%, and the ammonia nitrogen concentration is controlled at 0.1-3.0 g / L ammonium sulfate. When the OD grows to 150-300, 20-80% of the fermentation liquid in the fermenter is discharged, and 70-10% of the sterile HM culture medium is supplemented accordingly. The semi-continuous fermentation cycle is continued and the bacteria are collected.

[0040] The analysis of crude protein content was carried out in accordance with the National Standard of the People's Republic of China GB / T 6432-2018 "Determination of crude protein in feed by Kjeldahl method", and the amino acid content and composition were determined using Hitachi 8900 high-speed amino acid analyzer in accordance with the National Standard of the People's Republic of China GB / T 18246-2019 "Determination of amino acids in feed". Figure 4 The crude protein content of JC03 accounted for more than 70% of the dry weight of the bacteria, which was 9% higher than that of the control Methylobacterium strain. The amino acid composition analysis results showed that ( Figure 5 and 6 ), the single-cell protein produced by JC03 using methanol as the carbon source contains aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, proline and tryptophan.

[0041] The total amino acid content of the single-cell protein produced by strain JC03 exceeded 65%, which was 12% higher than that of the control Methylobacterium strain. The essential amino acids required for the growth of giant flounder included lysine, methionine, valine, threonine, histidine, phenylalanine, leucine, isoleucine and arginine, with a total content of more than 32%, which was 20% higher than that of the control Methylobacterium strain and 9% higher than that of a commercial fish meal. Except for histidine, the composition of other essential amino acids for giant flounder in JC03 bacterial protein was close to or exceeded that of commercial fish meal.

[0042] Example 5: Analysis of antioxidant and immune function active components of strain JC03 The JC03 bacterial culture used to analyze the antioxidant and immune function active ingredients was based on the shake flask fermentation method in Example 3. The extraction and quantification method of carotenoids is as follows: the cultured bacteria are collected by centrifugation, 1 mL of 65°C methanol is added and vortexed for 1 minute, 0.4 mL of water and 0.3 mL of chloroform are added and vortexed for 2 minutes, the lower organic phase is extracted and recovered, 1 mL of methanol and 0.4 mL of water are added, the lower organic phase is extracted and recovered again, 2 mL of acetone is added and allowed to stand at -20°C overnight, the supernatant is recovered by centrifugation and spin-dried, 0.1 mL of DMSO is added, and 30 μL is taken for HPLC quantitative analysis. The HPLC column is Spherisorb 5.0 μmODS2 (4.6 mm×250 mm), the mobile phase A is acetonitrile aqueous solution (acetonitrile: water = 9:1, V / V), the mobile phase B is methanol isopropanol solution (methanol: isopropanol = 3:2, V / V), and the mobile phase elution program is as follows: 0-10 min 100%A-5%A, 10-20 min 5%A, 20-25 min 5%A-100%A, flow rate of 1mL / min, detection wavelength of 460nm, use carotenoid standards to make a standard curve under the same chromatographic conditions, and put the sample measurement results into the standard curve to calculate the carotenoid content per unit cell.

[0043] The extraction and quantitative method of ergothioneine is as follows: 100 mg of cultured cells are collected by centrifugation, 1 mL of sterile water is added, and the cells are vortexed and resuspended, and the cells are water-bathed at 90 ° C for 30 min, and the supernatant is centrifuged for HPLC detection. The HPLC column uses an ODS-BP column, and the mobile phase A is a diammonium phosphate solution (1.15 g of diammonium phosphate is weighed and added to 400 mL of water, and the pH is adjusted to 5.0 with ammonia water, and then water is added to make the volume 500 mL), and the mobile phase B is acetonitrile, and the mobile phase A: mobile phase B = 99: 1, the flow rate is 1 mL / min, and the detection wavelength is 258 nm. The standard curve is prepared by using ergothioneine standard products with reference to the same chromatographic conditions, and the sample measurement results are brought into the standard curve to calculate the content of ergothioneine in the unit cell.

[0044] The quantitative analysis method of PHB is as follows: weigh 30 mg of bacteria, add 2 mL of methanol containing 3% sulfuric acid and 0.1% benzoic acid, vortex and mix, then add 2 mL of chloroform, and water bath at 100 ° C for 4 h, mixing every half an hour during the reaction. After the water bath, cool to room temperature, add 1 mL of 1M sodium chloride solution, vortex and mix, and let stand. Take the lower layer of solution for gas chromatography-mass spectrometry quantitative analysis. The chromatographic column is Shimadzu's SH-Rxi-5Sil capillary column. The column temperature is initially maintained at 50 ° C for 5 min, and then increased to 300 ° C at a heating rate of 30 ° C / min. Weigh different masses of PHB standards for methyl esterification reaction under the same conditions. After gas chromatography-mass spectrometry analysis, the ratio of the PHB monomer peak area to the internal standard peak area is used as the standard peak area, and a standard curve is made with the PHB mass. The ratio of the PHB monomer peak area measured in the sample to the internal standard peak area is brought into the standard curve to calculate the PHB content.

[0045] The quantitative method of PQQ is detected by NBT method. Take NBT powder and dissolve it in pure water to prepare 3 g / L mother solution. Take glycine to prepare 2 M solution and adjust the pH to 10 with sodium hydroxide to prepare glycine mother solution. Prepare PHB solution with pH 7, take 20 μL of extraction sample or PQQ standard, add 180 μL of glycine mother solution, 80 μL of PBS buffer and 20 μL of NBT mother solution, mix well and react in the dark for 1 h, measure the absorbance of the reaction solution at 530 nm, and calculate the PQQ content by the standard curve prepared by PQQ standard. The analysis of vitamin B12 content is carried out in accordance with the national standard of the People's Republic of China GB / T 17819-2017 "High Performance Liquid Chromatography Method for the Determination of Vitamin B12 in Additive Premix Feed".

[0046] Methylobacterium contains some antioxidant active ingredients, mainly including carotenoids, ergothioneine and PQQ. Carotene is a type of fat-soluble pigment with antioxidant function, which can improve the body color of aquatic economic animals, improve the antioxidant status and immune system function of aquatic animals, and improve their growth performance, disease resistance and survival rate. It is one of the ideal functional ingredients in aquaculture; ergothioneine and PQQ, as a natural antioxidant, have multiple physiological functions such as scavenging free radicals, detoxification, anti-inflammation, maintaining DNA biosynthesis and immunity.

[0047] According to the measurement results ( Figure 7), the carotenoid content of the evolved strain JC03 was 0.70±0.04 mg / g, which was 40% higher than that of the control Methylobacterium strain; the ergothioneine content of the evolved strain JC03 was 3.12±0.02 mg / g, which was 24% higher than that of the control Methylobacterium strain; the PQQ content of the evolved strain JC03 was 2.3±0.1 mg / g, which was more than 50% higher than that of the control Methylobacterium strain. Therefore, JC03 has better antioxidant function and is more helpful in improving the body color of aquatic economic animals such as fish and shrimp.

[0048] In addition to antioxidant components, Methylobacterium can naturally synthesize some immune function-related active ingredients, such as PHB and vitamin B12. PHB and monomeric β-hydroxybutyric acid can inhibit Vibrio virulence factors and stimulate the body's immune function, such as Figure 8 As shown, the PHB content of the evolved strain JC03 was 2.1%, and its content was affected by the carbon-nitrogen ratio in the culture conditions, and had a certain degree of negative correlation with the crude protein content. The PHB content of the evolved strain JC03 was not significantly lower than that of the control strain; Vitamin B12 is closely related to the formation of red blood cells and the integrity of the nervous system. It is necessary for the normal growth, red blood cell maturation, DNA synthesis and nerve tissue health of aquatic animals. For aquatic animals that cannot synthesize vitamin B12 from feed, their needs must be met through food or feed supplements. The vitamin B12 content of the evolved strain JC03 was 24±2 μg / g, which was 10% higher than that of the control Methylobacterium strain.

[0049] The above experimental results show that the evolved strain JC03 has undergone adaptive evolution, and its intracellular antioxidant and immune function activity groups, such as carotene, ergothioneine, PQQ and vitamin B12, have increased to varying degrees compared with the control strain. Therefore, using the evolved strain JC03 as a substitute protein in aquatic feed can be more conducive to improving the immune function of aquatic animals and reducing the risk of disease.

[0050] Example 6: Turbot test This example designs an experiment for turbot ( Scophthalmus maximus) were grown under five different experimental diet conditions and their different protection rates after pathogen challenge were used to determine whether JC03 is a suitable feed ingredient for marine model fish and whether it can provide effective protection and replace antibiotics. The five diets consisted of the following: (1) a standard commercial turbot diet; (2) an experimental diet; (3) an experimental diet containing 10% of the fish meal replaced by JC03 single cell protein; (4) a diet with 30% of the fish meal replaced by JC03; (5) a diet with 50% of the fish meal replaced by JC03. Each condition was tested in triplicate (15 aquariums), with 10 fish in each tank, for a total of 150 fish. The Vibrio anguillarum and Streptococcus paramammary required for the challenge were rejuvenated, isolated, and 16S rRNA identified before the experiment, and were cultured after confirmation of the species. Ten fish were taken from each of the five experimental diet groups for each challenge strain experiment. Vibrio anguillarum and Streptococcus parauberis were divided into 1*10 6 / L of the challenge concentration for 20 minutes. The blank control was recorded as 0h, and the mortality of turbot challenged with the virus was recorded within 72 hours and 15 days thereafter. The fish weight gain rate, feed conversion rate, albinism rate and immune protection efficiency were finally evaluated.

[0051] Fig. 9 Growth, feed conversion, albinism and immune protection of turbot using 5 experimental diets are shown. During this experiment, the turbot were fed 1.5% of body weight in the morning and evening using the 5 experimental diets for a total of 60 days. The weight gain of the turbot in the standard commercial turbot diet group was 118%, the weight gain of the turbot in the experimental diet group was 97%, the weight gain of the turbot in the experimental diet group containing 10% of the fish meal component replaced by JC03 single cell protein was 101%, the weight gain of the turbot in the diet group with 30% of the fish meal replaced by JC03 was 85%, and the weight gain of the turbot in the diet group with 50% of the fish meal replaced by JC03 was 88%.

[0052] The feed conversion rate of the standard commercial turbot diet group was 1.242, the feed conversion rate of the experimental diet group was 1.279, the feed conversion rate of the experimental diet group containing 10% of the fish meal component replaced by JC03 single cell protein was 1.259, the feed conversion rate of the turbot in the diet group with 30% of the fish meal replaced by JC03 was 1.088, and the feed conversion rate of the turbot in the diet group with 50% of the fish meal replaced by JC03 was 1.043. There was no significant difference in the final weight gain and feed conversion rate of turbot fed with 10% JC03 bacterial powder for 60 days.

[0053] After 60 days of feeding turbot feed containing less than 30% of single cell protein JC03 powder instead of fish meal, the growth and survival rate of turbot was comparable to that of turbot fed a standard diet containing 60% fish meal.

[0054] The proportion of white body color in the standard commercial turbot diet group was 58%, the proportion of white body color in the experimental diet group was 53%, the proportion of white body color in the experimental diet group in which 10% of the fish meal was replaced by JC03 single-cell protein was 37%, the proportion of white body color in the diet group in which 30% of the fish meal was replaced by JC03 was 27%, and the proportion of white body color in the diet group in which 50% of the fish meal was replaced by JC03 was 13%. The data proves that the addition of JC03 single-cell protein can reduce the albinism rate of turbot.

[0055] After being challenged with the Gram-negative pathogen Vibrio anguillarum and the Gram-positive pathogen Streptococcus paramammary, the immune protection rates of turbot fed the five experimental diets were as follows: 72 hours after challenge with Vibrio anguillarum, the immune protection rate of the turbot in the standard commercially available turbot diet group was 0%, the immune protection rate of the turbot in the experimental diet group was 0%, the immune protection rate of the turbot in the experimental diet group containing 10% of the fish meal replaced by JC03 single-cell protein was 20%, the immune protection rate of the turbot in the diet group with 30% of the fish meal replaced by JC03 was 20%, and the immune protection rate of the turbot in the diet group with 50% of the fish meal replaced by JC03 was 60%. Fifteen days after challenge with S. parauberis, the immune protection rate of the turbot in the standard commercial diet group was 10%, the immune protection rate of the turbot in the experimental diet group was 0%, the immune protection rate of the turbot in the experimental diet group containing 10% of the fish meal component replaced by JC03 single-cell protein was 10%, the immune protection rate of the turbot in the diet group with 30% of the fish meal replaced by JC03 was 10%, and the immune protection rate of the turbot in the diet group with 50% of the fish meal replaced by JC03 was 40%.

[0056] Example 7: Large yellow croaker test Buy a large yellow croaker weighing 17g ( Larimichthys crocea ) fry were first kept temporarily for two weeks, and then healthy fry with similar weight were randomly divided into 4 groups, with 3 replicates in each group and 10 fish in each replicate, and they were raised in 12 breeding tanks (90L / tank).

[0057] Four groups of yellow croaker were fed the following feeds: (1) experimental diet; (2) experimental diet in which 10% of the fish meal was replaced by JC03 single cell protein; (3) diet in which 30% of the fish meal was replaced by JC03; (4) diet in which 50% of the fish meal was replaced by JC03. They were recorded as control group, 10% group, 30% group, and 50% group respectively. The experimental conditions controlled the temperature to be maintained at 20 degrees Celsius and the salinity to be 31‰. The fish were fed twice a day until full, and the feeding time was 5:00 and 17:00. The feeding experiment lasted for 90 days. After that, a Vibrio challenge test was carried out. The Vibrio harveyi required for the challenge was rejuvenated, isolated, and 16SrRNA identified before the experiment. After confirming that it was this type of bacteria, it was cultured and used. For each challenge strain experiment, 10 yellow croakers were taken from each of the four experimental diet groups. Vibrio harveyi was added at a rate of 1*10 6 / L of the challenge concentration for 20 minutes. The blank control was recorded as 0h, and the mortality of the challenged large yellow croaker was recorded within 72 hours and 15 days thereafter. The weight gain rate, feed conversion rate and immune protection efficiency of large yellow croaker were finally evaluated.

[0058] Fig.10 The growth, feed conversion rate, albino rate and immune protection rate of large yellow croaker using 4 kinds of experimental diets are shown. In the process of this experiment totaling 90 days, large yellow croaker was fed by 1.5% of body weight in the morning and evening using the 4 kinds of experimental diets. The weight gain rate of large yellow croaker in the experimental configuration diet group was 104%, the weight gain rate of large yellow croaker in the experimental configuration diet group containing 10% fish meal component replaced by JC03 single cell protein was 102%, the weight gain rate of large yellow croaker in the diet group with 30% fish meal replaced by JC03 was 105%, and the weight gain rate of large yellow croaker in the diet group with 50% fish meal replaced by JC03 was 84%.

[0059] The feed conversion rate of large yellow croaker in the experimental diet group was 1.579, the feed conversion rate of large yellow croaker in the experimental diet group containing 10% of fish meal replaced by JC03 single-cell protein was 1.559, the feed conversion rate of large yellow croaker in the diet group with 30% of fish meal replaced by JC03 was 1.588, and the feed conversion rate of large yellow croaker in the diet group with 50% of fish meal replaced by JC03 was 1.643.

[0060] After being challenged with the Gram-negative pathogen Vibrio harveyi, the immune protection rates of yellow croakers fed the four experimental diets were as follows: 72 hours after being challenged with Vibrio harveyi, the immune protection rate of yellow croakers in the experimental diet group was 0%, the immune protection rate of yellow croakers in the experimental diet group in which 10% of the fish meal was replaced by JC03 single-cell protein was 20%, the immune protection rate of yellow croakers in the diet group in which 30% of the fish meal was replaced by JC03 was 30%, and the immune protection rate of yellow croakers in the diet group in which 50% of the fish meal was replaced by JC03 was 60%.

[0061] Example 8: Grouper Test Buy brown spotted grouper weighing 17g ( Epinephelus fuscoguttatus ) fry were first fed with commercial feed for two weeks, and then healthy fry with similar weight were randomly divided into 4 groups, with 3 replicates in each group and 10 fish in each replicate, which were raised in 12 breeding tanks (90L / tank).

[0062] Four groups of groupers were fed the following feeds: (1) experimental diet; (2) experimental diet in which 10% of the fish meal was replaced by JC03 single cell protein; (3) diet in which 30% of the fish meal was replaced by JC03; (4) diet in which 50% of the fish meal was replaced by JC03. They were recorded as control group, 10% group, 30% group, and 50% group respectively. The experimental conditions controlled the temperature to be maintained at 28 degrees Celsius and the salinity to be 30‰. The fish were fed twice a day until full, and the feeding time was 8:00 and 17:00. The feeding experiment lasted for 8 weeks. After that, a Vibrio challenge test was carried out. The Vibrio harveyi required for the challenge was rejuvenated, isolated, and 16SrRNA identified before the experiment. After confirming that it was this type of bacteria, it was cultured and used. For each challenge strain experiment, 10 groupers were taken from each of the four experimental diet groups. Vibrio harveyi was added at a rate of 1*10 6 The fish were immersed in the challenge concentration of 100 mg / L for 20 minutes. The blank control was recorded as 0 h, and the mortality of the challenged grouper was recorded within 72 hours and 15 days thereafter. The weight gain rate, feed conversion rate and immune protection efficiency of the grouper were finally evaluated.

[0063] Fig.11 The growth, feed conversion rate, albinism rate and immune protection rate of grouper using 4 experimental diets are shown. In the process of this experiment totaling 60 days, grouper was fed at 1.5% of body weight in the morning and evening using the 4 experimental diets. The weight gain rate of grouper in the experimental configuration diet group was 547%, the weight gain rate of grouper in the experimental configuration diet group containing 10% fish meal component replaced by JC03 single cell protein was 542%, the weight gain rate of grouper in the diet group with 30% fish meal replaced by JC03 was 537%, and the weight gain rate of grouper in the diet group with 50% fish meal replaced by JC03 was 518%.

[0064] The feed conversion rate of grouper in the experimental diet group was 1.259, the feed conversion rate of grouper in the experimental diet group containing 10% of fish meal replaced by JC03 single cell protein was 1.129, the feed conversion rate of grouper in the diet group with 30% of fish meal replaced by JC03 was 1.118, and the feed conversion rate of grouper in the diet group with 50% of fish meal replaced by JC03 was 1.025.

[0065] After being challenged with the Gram-negative pathogen Vibrio harveyi, the immune protection rates of grouper in the four experimental diets were as follows: 72 hours after being challenged with Vibrio harveyi, the immune protection rate of grouper in the experimental diet group was 0%, the immune protection rate of grouper in the experimental diet group containing 10% of the fish meal replaced by JC03 single-cell protein was 20%, the immune protection rate of grouper in the diet group with 30% of the fish meal replaced by JC03 was 20%, and the immune protection rate of grouper in the diet group with 50% of the fish meal replaced by JC03 was 60%.

[0066] Example 9: Rainbow trout test This example designs an experiment for rainbow trout ( Oncorhynchus mykiss ) were grown under five different experimental diet conditions to determine whether JC03 is a suitable feed ingredient for replacing fish meal. The diets consisted of the following items: (1) a standard commercial rainbow trout diet (2) an experimental diet (3) an experimental diet in which 10% of the fish meal component was replaced with JC03 single cell protein (4) a diet in which 30% of the fish meal was replaced with JC03 (5) a diet in which 50% of the fish meal was replaced with JC03. The fish were randomly divided into 5 groups, each with three replicates, with 10 fish in each box, for a total of 150 fish. The salmonicidal Aeromonas ( Aeromonas salmonicida) and Lactococcus garvei ( Lactococcus garvieae) Before the experiment, the fish were rejuvenated, isolated, and identified by 16S rRNA. After confirmation, the fish were cultured and used. Ten fish were taken from each of the five experimental diet groups for each challenge strain experiment. Aeromonas salmonicida and Lactococcus garvei were added at a rate of 1*10 6 / L of challenge concentration for 20 minutes. The blank control was recorded as 0h, and the mortality of challenged rainbow trout was recorded in the following 7 days and 15 days. The fish weight gain rate, feed conversion rate and immune protection efficiency were finally evaluated.

[0067] Fig.12 Growth, feed conversion, and immune protection efficiency of rainbow trout using five experimental diets are shown. In this 60-day trial, rainbow trout were fed 3% of body weight in the morning and evening using the five experimental diets. The weight gain rate of rainbow trout in group 1 was 320%, the weight gain rate of rainbow trout in group 2 was 318%, the weight gain rate of rainbow trout in group 3 was 312%, the weight gain rate of rainbow trout in group 4 was 317%, and the weight gain rate of rainbow trout in group 5 was 326%.

[0068] The feed conversion rate of rainbow trout in group 1 was 1.041, that of rainbow trout in group 2 was 1.035, that of rainbow trout in group 3 was 1.117, that of rainbow trout in group 4 was 1.229, and that of rainbow trout in group 5 was 1.246. There was no significant difference in final weight gain and feed conversion rate among rainbow trout fed with different levels of JC03 powder for 60 days.

[0069] After 60 days of feeding rainbow trout with a diet containing less than 30% of fish meal replaced by JC03 single cell protein powder, the growth and survival rates of rainbow trout were comparable to those of rainbow trout fed a standard diet containing 60% fish meal. Seven days after challenge with A. salmonicida, the immune protection rates of rainbow trout in the standard commercial diet group were 0%, those in the experimental diet group were 0%, those in the experimental diet group containing 10% of the fish meal component replaced by JC03 single cell protein were 10%, those in the diet group with 30% of the fish meal replaced by JC03 were 20%, and those in the diet group with 50% of the fish meal replaced by JC03 were 60%. Fifteen days after the challenge with Lactococcus garvei, the immune protection rate of the rainbow trout in the standard commercially available diet group was 0%, the immune protection rate of the rainbow trout in the experimental diet group was 0%, the immune protection rate of the rainbow trout in the experimental diet group containing 10% of the fish meal replaced by JC03 single-cell protein was 20%, the immune protection rate of the rainbow trout in the diet group with 30% of the fish meal replaced by JC03 was 20%, and the immune protection rate of the rainbow trout in the diet group with 50% of the fish meal replaced by JC03 was 50%.

[0070] Example 10: Freshwater fish experiment (grass carp farming) This embodiment designs an experiment for grass carp ( Ctenopharyngodon idella ) were grown under five different experimental diet conditions to determine whether JC03 is a suitable feed ingredient for replacing fish meal. The diets consisted of the following items: (1) a standard commercial grass carp diet (2) an experimental diet (3) an experimental diet in which 30% of the soybean meal components were replaced with JC03 single cell protein (4) a diet in which 70% of the soybean meal was replaced with JC03 (5) a diet in which 100% of the soybean meal was replaced with JC03. The fish were randomly divided into 5 groups, each with three replicates, with 10 fish in each box, for a total of 150 fish. The intestinal type of Aeromonas punctata required for the attack ( Aeromonas punotata f.intestinalis) Before the experiment, the fish were rejuvenated, isolated, and identified by 16S rRNA. After confirmation, the fish were cultured and used. Ten fish were taken from each of the five experimental diet groups for each challenge strain experiment. 6 / L of the challenge concentration for 20 minutes. The blank control was recorded as 0h, and the mortality of the challenged grass carp was recorded in the following 7 days and 15 days. The fish weight gain rate, feed conversion rate, and immune protection efficiency were finally evaluated.

[0071] Fig.13The growth, feed conversion rate, and immune protection efficiency of grass carp using 5 experimental diets are shown. In this 60-day trial, the grass carp were fed 3% of their body weight in the morning and evening using the 5 experimental diets. The weight gain rate of grass carp in group 1 was 60.42%, the weight gain rate of grass carp in group 2 was 55.25%, the weight gain rate of grass carp in group 3 was 58.56%, the weight gain rate of grass carp in group 4 was 59.06%, and the weight gain rate of grass carp in group 5 was 51.09%.

[0072] The feed conversion rate of grass carp in group 1 was 1.17, the feed conversion rate of grass carp in group 2 was 1.27, the feed conversion rate of grass carp in group 3 was 1.20, the feed conversion rate of grass carp in group 4 was 1.19, and the feed conversion rate of grass carp in group 5 was 1.23. There was no significant difference in final weight gain and feed conversion rate between grass carp fed with different levels of JC03 powder for 90 days. After 90 days of feeding grass feed with single cell protein JC03 powder instead of soybean meal, the growth and survival rate of grass carp were comparable to those fed with standard feed.

[0073] Seven days after the infection with intestinal Aeromonas punctata, the immune protection rate of grass carp in the standard commercially available diet group was 0%, the immune protection rate of grass carp in the experimental diet group was 0%, the immune protection rate of grass carp in the experimental diet group in which 30% of the soybean meal components were replaced by JC03 single-cell protein was 20%, the immune protection rate of grass carp in the diet group in which 70% of the soybean meal was replaced by JC03 was 50%, and the immune protection rate of grass carp in the diet group in which 100% of the soybean meal was replaced by JC03 was 60%.

[0074] Example 11: Whiteleg shrimp test 0.5 cm shrimp fry were purchased and temporarily raised in indoor breeding tanks. One week later, 225 healthy and uniform shrimp fry (0.5±0.05 g / fish) were selected and randomly divided into 4 groups, with 3 replicates in each group and 15 shrimps in each replicate. They were raised in 12 breeding tanks (50 L / tank).

[0075] Four groups of whiteleg shrimp were fed the following feeds: (1) basic feed + 15% fish meal, (2) basic feed + 10% fish meal + 5% JC03 bacterial powder (3) basic feed + 5% fish meal + 10% JC03 bacterial powder, (4) basic feed + 15% JC03 bacterial powder, which were recorded as control group, 5% group, 10% group, and 15% group, respectively. The experimental conditions controlled the temperature to be maintained at 22 degrees Celsius and the salinity to be 26‰. The shrimp were fed 5 times a day until full. Two hours after feeding, any uneaten feed was drawn out of the water tank, collected using a plankton net, dried at 65 degrees Celsius for 24 hours, and then weighed to calculate the feed intake. The feeding time was 7:00, 11:00, 16:00, and 22:00, and the water was changed to maintain a transparency of 40cm. The feeding experiment lasted for 60 days. After that, the Vibrio challenge test was conducted. The Vibrio parahaemolyticus required for the challenge was rejuvenated, isolated, and 16SrRNA identified before the experiment. After confirming that it was this type of bacteria, it was cultured and used. For each challenge strain experiment, 10 shrimps were taken from each of the four experimental diet groups. The Vibrio parahaemolyticus was divided into 1*10 6 / L of the challenge concentration for 20 minutes. The blank control was recorded as 0h, and the mortality of the challenged white shrimp was recorded within 72 hours and 15 days thereafter. The weight gain rate, feed conversion rate and immune protection efficiency of white shrimp were finally evaluated.

[0076] Fig.14 (Bar graph) shows the growth, feed conversion rate, and immune protection efficiency of white shrimp using the four experimental diets. The weight gain of the four groups of white shrimp were as follows: (1) 3.78g, (2) 3.77g, (3) 3.81g, (4) 4.01g, and the feed conversion rate (intake / weight gain) of the four groups of white shrimp were as follows: (1) 1.26 (2) 1.21 (3) 1.21 (4) 1.15. There were no significant differences in weight gain, feed consumption, and feed conversion rate among white shrimp fed with different levels of JC03 bacterial powder for 60 days. The growth and survival rate of Pacific white shrimp fed with single-cell protein JC03 bacterial powder instead of fish meal for 60 days was comparable to that of shrimp fed with a standard diet containing 15% fish meal. Specifically, the feed conversion rate, specific growth rate, and overall weight gain of shrimp fed with bacterial powder were greater than those of shrimp fed with a standard diet. Importantly, the methanotrophic bacterial powder reduced the Vibrio spp. load in the hepatopancreas and improved survival when shrimp were exposed to a V. parahaemolyticus challenge.

[0077] Fig.14(Broken line) shows the immune protection rate of Penaeus vannamei in four experimental diets after challenge with Gram-negative pathogen Vibrio parahaemolyticus. 72 hours after challenge with Vibrio parahaemolyticus, the immune protection rate of Penaeus vannamei in the experimental diet group was 0%, the immune protection rate of Penaeus vannamei in the experimental diet group containing 10% of the fish meal component replaced by JC03 single cell protein was 20%, the immune protection rate of Penaeus vannamei in the diet group with 30% of the fish meal replaced by JC03 was 30%, and the immune protection rate of Penaeus vannamei in the diet group with 50% of the fish meal replaced by JC03 was 60%.

[0078] Example 12: Sea cucumber experiment This example designs an experiment for sea cucumber ( Apostichopus japonicus ) were grown under five different experimental diet conditions to determine whether JC03 is a suitable feed ingredient for replacing seaweed ingredients. The diet consisted of the following items: (1) 70% sea mud plus 30% Sargassum (2) 70% sea mud plus 22.5% Sargassum plus 7.5% JC03 bacterial powder (3) 70% sea mud plus 15% Sargassum plus 15% JC03 bacterial powder (4) 70% sea mud plus 7.5% Sargassum plus 22.5% JC03 bacterial powder and (5) 70% sea mud plus 30% JC03 bacterial powder. They were randomly divided into 5 groups, each with three replicates, with 10 sea cucumbers in each box, for a total of 150 sea cucumbers. Vibrio splendens required for the challenge ( Vibrio splendidus Before the experiment, the bacteria were rejuvenated, isolated, and identified by 16S rRNA. After confirmation, they were cultured and used. Ten bacteria were taken from each of the five experimental diet groups for each challenge strain experiment. 6 / L of challenge concentration for 20 minutes. The blank control was recorded as 0h, and the mortality of sea cucumbers challenged with poison was recorded in the following 7 days and 15 days. Finally, the weight gain rate, feed conversion rate and immune protection efficiency of sea cucumbers were evaluated.

[0079] Fig.15 The growth, feed conversion rate, and immune protection efficiency of sea cucumbers using the five experimental diets are shown. In this 60-day experiment, sea cucumbers were fed 4% of their body weight in the morning and evening using the five experimental diets. The weight gain rate of sea cucumbers in group 1 was 34.39%, the weight gain rate of sea cucumbers in group 2 was 37.37%, the weight gain rate of sea cucumbers in group 3 was 39.3%, the weight gain rate of sea cucumbers in group 4 was 33.03%, and the weight gain rate of sea cucumbers in group 5 was 31.96%.

[0080] The feed conversion rate of sea cucumbers in group 1 was 8.01, the feed conversion rate of sea cucumbers in group 2 was 7.75, the feed conversion rate of sea cucumbers in group 3 was 7.31, the feed conversion rate of sea cucumbers in group 4 was 8.46, and the feed conversion rate of sea cucumbers in group 5 was 8.12. There was no significant difference in the final weight gain and feed conversion rate of sea cucumbers fed with different levels of JC03 powder for 90 days. After feeding with single cell protein JC03 powder instead of Sargassum for 90 days, the growth and survival rate of sea cucumbers fed with standard diet were comparable.

[0081] Seven days after the attack with Vibrio splendens, the immune protection rate of group 1 was 0%, the immune protection rate of group 2 was 0%, the immune protection rate of group 3 was 10%, the immune protection rate of group 4 was 20%, and the immune protection rate of group 5 was 40%.

[0082] The above examples show that the single-cell protein produced by the Methylobacterium evolved strain JC03 can be used as a substitute protein for a variety of aquatic animal feeds, thereby promoting cost reduction and efficiency improvement in the aquaculture industry.

[0083] In addition, it should be noted that the shapes and names of the parts and components of the specific embodiments described in this specification may be different. Any equivalent or simple changes made based on the structure, features and principles described in the patent concept of the present invention are included in the protection scope of the patent of the present invention. The technicians in the technical field of the present invention can make various modifications or supplements to the specific embodiments described or replace them in a similar manner, as long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. A Methylobacterium strain JC03 that produces single-cell protein using methanol as the sole carbon source, characterized in that: The classification name of the Methylobacterium strain JC03 is Methylobacterium Methylobacterium sp., its accession number is: CGMCC No.32616.

2. The Methylobacterium strain JC03 according to claim 1, characterized in that: The Methylobacterium strain JC03 is dark red, round, protruding upward, and has neat edges.

3. A bacterial agent containing the Methylobacterium strain JC03 according to claim 1.

4. Use of the Methylobacterium strain JC03 according to claim 1 or the bacterial agent according to claim 3 in producing single-cell protein using methanol as a carbon source.

5. The use according to claim 4, characterized in that: The application method is as follows: the methylbacterium strain JC03 is grown on a culture medium with methanol as the sole carbon source, and single-cell protein is obtained through shake flask culture or semi-continuous culture in a fermenter.

6. Use of the single-cell protein produced by the Methylobacterium strain JC03 according to claim 1 or the bacterial agent according to claim 3 using methanol as a carbon source in the preparation of aquaculture animal feed.

7. The use according to claim 6, characterized in that: The aquaculture animals include flounder, large yellow croaker, grouper, rainbow trout, grass carp, white shrimp and sea cucumber.

8. The use according to claim 6, characterized in that: The application method is to use the single cell protein to partially or completely replace the fish meal protein and / or plant protein in the feed.

9. Use of the single-cell protein produced by the Methylobacterium strain JC03 according to claim 1 using methanol as a carbon source in the preparation of an aquatic feed additive for improving the antioxidant function and immune function of aquaculture animals.

10. The use according to claim 9, characterized in that: The single cell protein contains high content of carotene, ergothioneine, PQQ, PHB and vitamin B12.

Images