Microbial antioxidant pigment feed additive for aquaculture and preparation method thereof

Abstract

The invention provides a microbial source anti-oxidation pigment feed additive for aquaculture and a preparation method of the microbial source anti-oxidation pigment feed additive, the microbial source anti-oxidation pigment feed additive for aquaculture is prepared by the following steps: carrying out low-temperature plasma physical mutagenesis on talaromyces albuminosus CY-G to generate a mutant strain, namely talaromyces albuminosus CY110, and then carrying out semi-solid state fermentation on the mutant strain, namely talaromyces albuminosus CY110 to produce wine red pigment. The pigment product obtained by the invention is high in yield of haematochrome, orange pigment and yellow pigment, low in citrinin content, stable in property and relatively strong in oxidation resistance, can be used as an aquaculture microorganism source anti-oxidation pigment feed additive, and has a wide application prospect. The preparation method disclosed by the invention is simple to operate, low in equipment requirement, high in production efficiency and suitable for large-scale production.

Description

Technical Field

[0001] The invention belongs to the technical field of pigment production, and in particular relates to a microbial source antioxidant pigment feed additive for aquaculture and a preparation method thereof. Background Art

[0002] my country is the world's leading aquaculture country, and also the country with the largest production and use of aquatic feed. Aquatic feed is rich in important nutrients such as carbohydrates, proteins and oils. Among them, oils are an important source of energy for fish, especially n-3 and n-6 ​​series of polyunsaturated fatty acids that cannot be synthesized by themselves, which play an important role in the growth, development and reproduction of aquatic animals. However, the oils in the feed are very easy to oxidize during use and storage, which will cause the feed to produce a "rancid smell" and generate harmful substances such as aldehydes, ketones, and alcohols, thereby inhibiting the growth of aquatic animals, reducing the digestion and absorption rate of nutrients, and affecting the efficiency of feed utilization. To prevent oil oxidation, companies usually add synthetic antioxidants (such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and ethoxyquinoline) during feed processing. However, the potential harm of synthetic antioxidants to animals has attracted widespread attention. The European Commission issued Regulation (EU) 2017 / 962, suspending the use of ethoxyquinoline as an animal feed additive. Therefore, companies are gradually phasing out synthetic antioxidants and switching to safer and more efficient natural antioxidants.

[0003] Natural pigments can not only be used as feed colorants, but also strong antioxidants. The production of natural edible pigments by microbial fermentation has the advantages of abundant raw materials, no dependence on seasonal changes, low production cost, high product concentration, easy purification, etc., and it is safer than synthetic pigments, and the market demand is huge. Polyketide pigments have a series of excellent physiological activities such as antioxidant, anti-mutagenic, anti-corrosion and preservation. At present, the main strains that can produce such pigments are Monascus and various Penicillium. However, these strains will produce a large amount of fungal toxins, citrinin, while producing pigments. Citrinin can cause kidney enlargement, tubular dilatation and epithelial cell necrosis, which is extremely harmful to humans and aquatic animals. my country has revised the limit standard of citrinin in food and issued a unified standard GB 5009.222-2016. The new standard specifically stipulates the content of citrinin in chili powder, rice and rice products, with the quantitative limit set at 25μg / kg and the detection limit at 8μg / kg; the quantitative limit of products related to Monascus is set at 80μg / kg and the detection limit is 25μg / kg.

[0004] Talaromyces albobiverticillius (T.albobiverticillus) belongs to the genus Talaromyces. It is one of the fungi that have been reported to produce large quantities of polyketide pigments without producing mycotoxins. It is considered to be one of the most promising sources of food pigments. In addition, the pigment also exhibits strong antioxidant activity (M, Umesh, et al., 2023). Chinese patent CN116144710A recently disclosed a method for high production of extracellular pigments using Talaromyces albobiverticillius. The method was carried out by liquid fermentation of soluble starch peptone medium for 10 days, and the yield of orange pigment and red pigment in the supernatant of the fermentation broth was measured, reaching 0.52g / L and 1.68g / L, respectively. However, the patent only describes the potential of Talaromyces albobiverticillius in pigment production. The yield under the existing culture technology conditions is low, which is not enough for large-scale application. In addition, due to the complex composition of the liquid fermentation system of filamentous fungi, it contains a large amount of impurities such as mycelium, organic acids, polysaccharides, proteins, etc., and has many problems such as high viscosity, difficulty in separation and purification, and low product purity, which limits the actual application effect of the pigment. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a microbial source antioxidant pigment feed additive for aquaculture and a preparation method thereof in view of the deficiencies of the above-mentioned prior art. A mutant strain Talaromyces albobiverticillius CY110 is produced by physical mutagenesis of white two-wheeled basket fungus Talaromyces albobiverticillius CY-G through low-temperature plasma, and then the mutant strain Talaromyces albobiverticillius CY110 is used to produce wine red pigment by semi-solid fermentation. The wine red pigment can be used as a microbial source antioxidant pigment feed additive for aquaculture.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0007] A mutant strain of Talaromyces albobiverticillius CY-G produced by low-temperature plasma physical mutagenesis, named Talaromyces albobiverticilliusCY110, was deposited in the China Center for Type Culture Collection (Wuhan, China) on November 12, 2024, with the deposit number CCTCCNO: M 20242539.

[0008] The present invention also provides a method for producing wine red pigment by semi-solid fermentation using the Talaromyces albobiverticillius CY110, comprising the following steps:

[0009] S1. Preparation of spore suspension

[0010] The strain Talaromyces albobiverticillius CY110 was inoculated on PDA medium, cultured at 30°C for 5-7 days, fully eluted with sterile water, and divided for use;

[0011] S2. Preparation of semi-solid culture medium for honeycomb arrays

[0012] A mixture of 5 g / L potato extract powder, 20 g / L glucose, 20 g / L agar, and 5 g / L food-grade glycerol is prepared, sterilized at high temperature and high pressure, stirred by air flow, poured into multiple 30 cm×30 cm sterile stainless steel plates while hot, and multiple sterile stainless steel plates are accumulated together to form a honeycomb array semi-solid culture medium, and cooled;

[0013] S3, pigment fermentation

[0014] The spore suspension obtained in S1 is evenly sprayed onto the surface of the honeycomb semi-solid culture medium obtained in S2. After inoculation, it is covered with a sterile breathable film or kraft paper and placed at 30°C for fermentation for 10 days.

[0015] S4. Pigment extraction and analysis

[0016] The fermented honeycomb semi-solid culture medium is extracted with a 50% by mass ethanol aqueous solution, and then filtered under pressure. The filtrate is diluted and then subjected to ultraviolet full spectrum scanning to quantitatively analyze the pigment content and citrinin content.

[0017] Preferably, the ratio of the fermented honeycomb semi-solid culture medium to 50% by mass ethanol aqueous solution in S4 is 1 g:20 mL.

[0018] Preferably, the leaching in S4 is performed by placing the mixture in an oscillator at room temperature and shaking it at 200 rpm for 2 hours or standing it for leaching for 24 hours.

[0019] The present invention also provides a wine red pigment prepared by the above method, wherein the yield of red pigment in the wine red pigment is 167.14±1.21 mg / g, the yield of orange pigment is 130.07±1.11 mg / g, the yield of yellow pigment is 165.21±1.54 mg / g, the content of citrinin in the liquid sample of the wine red pigment is 0.21±0.02 ng / mL, and the content of citrinin in the solid sample is 4.20±0.40 ng / g.

[0020] The present invention also provides an application of the wine red pigment, which can be used as an antioxidant pigment feed additive of aquaculture microorganisms.

[0021] The present invention has significant technical effects due to the adoption of the above technical solution:

[0022] 1. The present invention provides a mutant strain Talaromyces albobiverticillius CY110 produced by physical mutagenesis of Talaromyces albobiverticillius CY-G through low-temperature plasma. The mutant strain Talaromyces albobiverticillius CY110 can produce extracellular pigments and has good stability in passage.

[0023] 2. The present invention provides a method for producing wine red pigment by semi-solid fermentation using mutant strain Talaromyces albobiverticillius CY110. The method is simple to operate, has low equipment requirements, high production efficiency, and is suitable for large-scale production. The yield of red pigment in the wine red pigment produced by semi-solid fermentation of strain Talaromyces albobiverticillius CY110 is 167.14±1.21mg / g, the yield of orange pigment is 130.07±1.11mg / g, the yield of yellow pigment is 165.21±1.54mg / g, the content of citrinin in the liquid sample of wine red pigment is 0.21±0.02ng / mL, and the content of citrinin in the solid sample is 4.20±0.40ng / g. The pigment yield is high, the content of citrinin is low, and the property is stable, which not only enriches the resources of talaromyces pigment, but also improves the actual application effect of the pigment.

[0024] 3. The wine red pigment produced by semi-solid fermentation of the mutant strain Talaromyces albobiverticillius CY110 of the present invention has strong antioxidant capacity and can be used as an antioxidant pigment feed additive of aquaculture microorganisms, and has broad application prospects.

[0025] The present invention is further described in detail below with reference to the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is a schematic diagram of the atmospheric pressure glow discharge low temperature plasma mutagenesis process of the T. albobiverticillus CY-G strain in Example 1;

[0027] Figure 2 The effect of the mutagenesis time on the lethality and mutation rate of the T. albobiverticillus CY strain in Example 1;

[0028] Figure 3 is a morphological difference diagram of the mutant strains at different mutagenesis times in Example 1;

[0029] Figure 4 The extracellular pigment diffusion results of the mutant strain T.albobiverticillus CY110, the starting strain T.albobiverticillus CY-G and Monascus purpureus 40805 in Example 1 after 10 days of fermentation; wherein, 1: mutant strain T.albobiverticillus CY110, 2: starting strain T.albobiverticillusCY-G, 3: Monascus purpureus 40805;

[0030] Figure 5 It is a UV full spectrum scanning diagram of the fermentation pigments of the mutant strain T.albobiverticillus CY110, the starting strain T.albobiverticillus CY-G and Monascus purpureus 40805 in Example 2 and Comparative Examples 1-2;

[0031] Figure 6 The absolute quantitative analysis results of pigments of the fermentation pigment samples of the mutant strain T.albobiverticillus CY110, the starting strain T.albobiverticillus CY-G and the Monascus purpureus 40805 in Example 2 and Comparative Examples 1-2, and the liquid pigment sample diagram;

[0032] Figure 7 is the DPPH free radical scavenging rate result of T.albobiverticillus CY110 wine red pigment in Example 3;

[0033] Figure 8 This is the ABTS free radical scavenging rate result of T.albobiverticillus CY110 wine red pigment in Example 3. DETAILED DESCRIPTION

[0034] Example 1

[0035] This example is the isolation and identification of Talaromyces albobiverticillius CY110 (T.albobiverticillus CY110).

[0036] The starting strain Talaromyces albobiverticillius CY-G in the present invention was isolated from a pond water sample in the Seventeenth East Wei High-tech Aquaculture Technology Demonstration Park of the Fishery Industrial Park in Nansha District, Guangzhou. The genomic DNA was amplified using fungal 18s universal primers (ITS1: SEQ ID NO.1, ITS4: SEQ ID NO.2), and the product was sequenced to obtain an ITS sequence as shown in SEQ ID NO.3. By comparing with the NCBI database, and referring to the "Handbook of Fungal Identification", combined with morphology and molecular biology, it was identified as Talaromyces albobiverticillius and named T.albobiverticillusCY-G, and its sequence was uploaded to the NCBI database to obtain the accession number: PP663690.

[0037] Low-temperature plasma physical mutagenesis of T.albobiverticillus CY-G:

[0038] (1) Preparation of spore suspension: The starting strain T. albobiverticillus CY-G was inoculated on a PDA plate by punching, cultured at 30°C for 72 h, 10 mL of sterile water was drawn to fully wash the surface of the bacterial lawn, and the spores were counted by microscopic examination using a hemocytometer until the spore concentration reached 10. 6 -10 8 / mL (diluted to a concentration of 10 7 Spore suspensions with a concentration of spores / mL are more effective).

[0039] (2) Atmospheric pressure glow discharge low temperature plasma mutagenesis: 10 μL of the spore suspension obtained in step (1) was pipetted onto a stainless steel disc with a diameter of 10 mm and placed directly below the low temperature plasma source. The low temperature plasma source was excited by a high voltage pulse power supply at a power of 6 to 8 W, a frequency of 20 to 40 Khz, and a distance of 3 to 4 cm for 10 to 150 seconds. The schematic diagram of the mutagenesis process is shown in FIG. Figure 1 During the atmospheric pressure glow discharge process, a large number of active particles (including free electrons, oxygen free radicals and nitrogen free radicals) generated by the low-temperature plasma source will cause diverse damage to the fungal DNA structure, thereby forming a large number of mutation sites.

[0040] (3) Screening and separation of excellent pigment-producing mutant strains: The suspension after mutagenesis in step (2) was diluted to a certain gradient and then spread on a PDA plate. Under this condition, the lethality and mutation rate of the starting strain T. albobiverticillus CY-G were as follows: Figure 2As shown in (lethal rate = (number of spores before glow discharge treatment - number of spores after glow discharge treatment / number of spores before glow discharge treatment) * 100%, mutation rate = (number of mutant spores / number of spores after glow discharge treatment) * 100%);

[0041] Screening by plate culture: Under the condition of gradient mutagenesis time, various mutant strains can be obtained by comparing the depth of pigment color, radius size and hyphae radius size produced by the colonies with those of the starting strains, such as Figure 3 At 110 seconds, it was observed that a mutant strain produced extracellular pigment faster than the starting strain under the same culture conditions. After separation and purification, a Talaromyces strain was obtained and named Talaromyces albobiverticillius CY110 (T.albobiverticillus CY110), which was deposited in the China Center for Type Culture Collection with the deposit number of CCTCC NO: M20242539.

[0042] (4) Evaluation of the stability of mutant strains: The starting strain T.albobiverticillus CY-G, the mutant strain T.albobiverticillus CY110 after 15 generations, and Monascus purpureus (M.purpureus) (collection number: CICC 40805) were cultured on PDA medium at 30°C for 10 days. The colony patterns were as shown in Figure 2. Figure 4 As shown. It can be obtained that under the same culture conditions, the mutant strain T.albobiverticillus CY110 can produce more extracellular pigments than the starting strain T.albobiverticillusCY-G and Monascus purpureus 40805, verifying that the mutant strain T.albobiverticillus CY110 has good passage stability. In addition, the morphological characteristics of the T.albobiverticillus CY110 strain and the starting strain T.albobiverticillus CY-G are quite different, mainly including: after 10 days of culture on the PDA plate, the colony morphology is larger; the colony grows outward in the shape of annual rings, with complete and rounded edges; in the late growth stage, it is accompanied by a large amount of wine red exudate, and a large amount of wine red pigment is exuded into the culture medium, making the entire culture medium dyed wine red.

[0043] Example 2

[0044] This example is a method for producing wine red pigment by semi-solid fermentation using the mutant strain T.albobiverticillus CY110 obtained in Example 1, and the steps are as follows:

[0045] (1) Preparation of spore suspension: The mutant strain T. albobiverticillus CY110 was inoculated on PDA medium and cultured at 30°C for 5-7 days. The spore suspension was fully eluted with 100 mL of sterile water and dispensed into 10 mL quantitative seed spray bottles for later use.

[0046] (2) Preparation of honeycomb array semi-solid culture medium: 5 g / L potato extract powder, 20 g / L glucose, 20 g / L agar, and 5 g / L food-grade glycerol were accurately weighed and prepared, sterilized at high temperature and high pressure, stirred by air flow to make the entire liquid filled with tiny bubbles, and then poured into a 30 cm*30 cm sterile stainless steel plate while hot to form a multi-array semi-solid culture medium.

[0047] (3) Pigment fermentation: After the culture medium has cooled, use a seed spray bottle containing spore suspension to evenly spray the surface of the honeycomb semi-solid culture medium. After inoculation, cover with a sterile breathable film or kraft paper and place at 30°C for fermentation for 10 days.

[0048] (4) Pigment extraction and analysis: Scrape the fermented honeycomb semi-solid culture medium thoroughly into a conical flask, add a 50% ethanol aqueous solution at a solid-liquid ratio of 1g:20mL, place in an oscillator at 200rpm for 2h at room temperature or leave to extract for 24h. Pour the oscillated liquid into a stainless steel positive pressure filter equipped with a 0.22μm organic filter membrane for pressure filtration, collect the filtrate to obtain a liquid pigment sample, dilute it, perform UV full spectrum scanning, pigment content and citrinin absolute quantitative analysis; then dry the filtrate to obtain a wine red powder, which is a solid pigment sample, and perform the same analysis as above.

[0049] Comparative Example 1

[0050] The starting strain T.albobiverticillus CY-G was used to carry out semi-solid fermentation to produce pigments in the same manner as in Example 2.

[0051] Comparative Example 2

[0052] The pigment was produced by semi-solid fermentation using Monascus purpureus 40805, in the same manner as in Example 2.

[0053] Figure 5 This is a UV full spectrum scan of the fermentation pigments of the mutant strain T.albobiverticillus CY110, the starting strain T.albobiverticillusCY-G and Monascus purpureus 40805.

[0054] The pigments in the fermentation broths of the mutant strain T.albobiverticillus CY110, the starting strain T.albobiverticillus CY-G and Monascus purpureus 40805 in Example 2 and Comparative Examples 1-2 were analyzed by absolute quantitative analysis, wherein:

[0055] Red pigment yield (mg / g 湿重 )=((Y 510nm吸光度 -0.0433))*V 浸提液体积 * dilution factor) / (2.3853*wet weight);

[0056] Orange pigment yield (mg / g 湿重 )=((Y 470nm吸光度 -0.0973))*V 浸提液体积 * dilution factor) / (2.4852*wet weight);

[0057] Yellow pigment yield (mg / g 湿重 )=((Y 420nm吸光度 -2.9254))*V 浸提液体积 * dilution factor) / (0.1124*wet weight).

[0058] Figure 6 The results of absolute quantitative analysis of pigments in the fermentation samples of mutant strain T.albobiverticillus CY110, starting strain T.albobiverticillusCY-G and Monascus purpureus 40805 are shown in the figure. The small picture shows the liquid pigment sample. The yields of red pigment, orange pigment and yellow pigment in the pigment samples obtained by semi-solid fermentation of mutant strain T.albobiverticillus CY110 were 167.14±1.21mg / g, 130.07±1.11mg / g and 165.21±1.54mg / g, respectively, which were much higher than those of the fermentation pigment samples of starting strain T.albobiverticillus CY-G and Monascus purpureus 40805.

[0059] The absolute quantification of citrinin was analyzed using the citrinin Elisa kit (limit of quantification was 0.025 ng / g). The citrinin content in the liquid sample (ng / mL) = (10 (Ln(Y百分度值 / 0.0726)) / (-1.263) )*dilution factor, citrinin content in solid sample (ng / g) = ((10 (Ln(Y百分度值 / 0.0726)) / (-1.263) )*dilution factor*V 溶液体积) / pigment dry weight). The results of the determination of citrinin content in liquid and solid pigment samples of mutant strain T.albobiverticillus CY110, starting strain T.albobiverticillus CY-G and Monascus purpureus 40805 are shown in Table 1.

[0060] Table 1 Citrinin content in pigment samples of T.albobiverticillus CY110, T.albobiverticillus CY-G and M.purpureus40805

[0061] strains Liquid sample (ng / mL) Solid sample (ng / g) T.albobiverticillus CY110 0.21±0.02 4.20±0.40 T.albobiverticillus CY-G 0.25±0.01 5.00±0.20 M.purpureus40805 72.19±0.16 1443.80±3.2

[0062] Example 3

[0063] This example is an application of the wine red pigment produced by semi-solid fermentation of the mutant strain T.albobiverticillus CY110 in Example 2. The pigment can be used as an antioxidant pigment feed additive derived from aquaculture microorganisms.

[0064] First, the free radical scavenging ability of the wine red pigment produced by semi-solid fermentation of the mutant strain T.albobiverticillus CY110 in Example 2 was determined by the DPPH method, and the total antioxidant capacity was determined by the ABTS method, while Vc was used as a control. The results are shown in Table 2. Figure 7 and Figure 8 , indicating that the wine red pigment produced by the present invention has good antioxidant ability.

[0065] Then, the wine red pigment produced by the present invention is applied to feed additives, and the method is as follows: the wine red pigment is added in an amount of 0.02% to commercially available fish meal feed that does not contain antioxidants, and is fully stirred, and a proper amount of water is added to form a state of "kneading into a ball by hand and loosening when lightly pressed", and a feed extruder is used to extrude it into particles of appropriate size, and finally the particle is placed in a freeze dryer for freeze drying to obtain a granular feed.

[0066] The antioxidant activity of pellet feed with 0.02% wine red pigment was evaluated, and the pellet feed without pigment was used as negative control, and the pellet feed with 0.02% BHA was used as positive control. The evaluation method was to conduct Schaal oxidation experiment: the Schaal oxidation simulation system was used to accelerate the oxidation of fats and oils, and 100g of feed pellet samples were placed in a heat-resistant glass bottle, opened and placed in an oven at 60±2℃ for continuous heating for 20 days, fully stirred every 5 days, and sampled in 100mL conical flasks at 20 days of reaction, and placed in a -80℃ refrigerator for freezing and storage, waiting for testing. The determination of peroxide value refers to GB 5009.227-2016, and the determination of anise safety value and total oxidation value refers to GB / T 24304-2009. The results are shown in Table 2.

[0067] Table 2 Evaluation of antioxidant activity of feed supplemented with T.albobiverticillus CY110 wine red pigment and feed supplemented with BHA

[0068]

[0069] The results show that the wine red pigment produced by semi-solid fermentation of the mutant strain T.albobiverticillus CY110 of the present invention has strong antioxidant capacity and has broad application prospects for being used as an antioxidant pigment feed additive of aquaculture microorganisms.

[0070] The physical mutagenesis of the present invention is only one of the mutation methods, and similar effects can also be produced by other mutagenesis methods well known to those skilled in the art, which can cause changes in the gene coding of living cells, enzyme activity characteristics and morphological changes. These methods include physical methods such as radiation, particles, lasers, ultraviolet light, and chemical mutagenesis methods such as alkylating agents, base analogs, hydroxylamine, and acridine pigments. Mutagenesis can be multi-generation mutagenesis of one or more of the above methods, and is not limited to these methods. Based on the strain provided by the present invention, it is also possible to further perform physical and chemical breeding, and its genome can also be changed, including but not limited to knock-in and knock-out, and the enzyme production performance of the obtained mutants and transformants can be further improved.

[0071] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A white two-wheeled basket fungus, characterized in that The mutant strain generated by low-temperature plasma physical mutagenesis of Talaromyces albobiverticillius CY-G was named Talaromyces albobiverticillius CY110 and deposited in China Center for Type Culture Collection with the deposit number of CCTCC NO: M 20242539.

2. A method for producing wine red pigment by semi-solid fermentation of Talaromyces albobiverticillius CY110 according to claim 1, characterized in that: The following steps are involved: S1. Preparation of spore suspension The strain Talaromyces albobiverticillius CY110 was inoculated on PDA medium, cultured at 30°C for 5-7 days, fully eluted with sterile water, and aliquoted for use; S2. Preparation of semi-solid culture medium for honeycomb arrays A mixture of 5 g / L potato extract powder, 20 g / L glucose, 20 g / L agar, and 5 g / L food-grade glycerol is prepared, sterilized at high temperature and high pressure, stirred by air flow, poured into multiple 30 cm×30 cm sterile stainless steel plates while hot, and multiple sterile stainless steel plates are accumulated together to form a honeycomb array semi-solid culture medium, and cooled; S3, pigment fermentation The spore suspension obtained in S1 is evenly sprayed onto the surface of the honeycomb semi-solid culture medium obtained in S2. After inoculation, it is covered with a sterile breathable film or kraft paper and placed at 30°C for fermentation for 10 days. S4. Pigment extraction and analysis The fermented honeycomb semi-solid culture medium is extracted with a 50% by mass ethanol aqueous solution, and then filtered under pressure. The filtrate is diluted and then subjected to ultraviolet full spectrum scanning to quantitatively analyze the pigment content and citrinin content.

3. The method according to claim 2, characterized in that The ratio of the fermented honeycomb semi-solid culture medium to the ethanol aqueous solution with a mass fraction of 50% in S4 is 1 g:20 mL.

4. The method according to claim 2, characterized in that: The extraction in S4 is to place the mixture in an oscillator at room temperature and shake it at 200 rpm for 2 hours or to allow it to stand and extract for 24 hours.

5. The method according to claim 2, characterized in that: The yield of red pigment in the wine red pigment described in S4 is 167.14±1.21 mg / g, the yield of orange pigment is 130.07±1.11 mg / g, and the yield of yellow pigment is 165.21±1.54 mg / g.

6. The method according to claim 2, characterized in that The content of citrinin in the liquid sample of S4 wine red pigment is 0.21±0.02 ng / mL, and the content of citrinin in the solid sample is 4.20±0.40 ng / g.

7. A wine red pigment prepared by the method according to any one of claims 2 to 6.

8. An application of the wine red pigment according to claim 7, characterized in that: The wine red pigment is used as an antioxidant pigment feed additive of aquaculture microorganisms.

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