Microbial antioxidant pigment feed additive for aquaculture and preparation method thereof

Abstract

The application provides a microbial source antioxidant pigment feed additive for aquaculture and a preparation method thereof, and comprises the following steps: generating a mutant strain Talaromyces albobiverticillius CY110 by subjecting Talaromyces albobiverticillius CY-G to low-temperature plasma physical mutagenesis, and then producing wine red pigment by utilizing the mutant strain Talaromyces albobiverticillius CY110 to carry out semi-solid state fermentation, the pigment product obtained by the application has high yield of red pigment, orange pigment and yellow pigment, low content of citrinin, stable properties, and strong antioxidant capacity, and can be used for microbial source antioxidant pigment feed additives for aquaculture, and has a wide application prospect. The preparation method of the application is simple in operation, low in equipment requirement, and high in production efficiency, and is suitable for large-scale production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pigment production, and particularly relates to a microbial source antioxidant pigment feed additive for aquaculture and a preparation method thereof. BACKGROUND

[0002] China 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. Oils are an important energy source for fish, especially n-3 and n-6 series polyunsaturated fatty acids, which cannot be synthesized by themselves, and play an important role in the growth, development and reproduction of aquatic animals. However, the oils in feed are prone to oxidation during use and storage, which can cause the feed to produce "harla smell", and generate harmful substances such as aldehydes, ketones and alcohols, thereby inhibiting the growth of aquatic animals, reducing the nutrient digestion and absorption rate, and affecting the feed utilization efficiency. To prevent oil oxidation, enterprises usually add synthetic antioxidants (such as dibutyl hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and ethoxyquin) during feed processing. However, the potential hazards of synthetic antioxidants to animals have attracted widespread attention. The European Commission has issued Regulation (EU) 2017 / 962, which has suspended the use of ethoxyquin as an animal feed additive. Therefore, enterprises are gradually phasing out synthetic antioxidants and turning to safer and more efficient natural antioxidants.

[0003] Natural pigments are not only used as feed colorants, but also are strong antioxidants. The production of natural edible pigments by microbial fermentation has the advantages of abundant raw materials, independence from seasonal changes, low production cost, high product concentration, easy purification, and higher safety than synthetic pigments, and has a huge market demand. Polyketide pigments have a series of outstanding physiological activities such as antioxidant, anti-mutation, and preservation. The main strains that can produce such pigments are Monascus and various penicillium. However, these strains will produce a large amount of mycotoxin - citrinin while producing pigments. Citrinin can cause kidney enlargement, renal tubular dilation and epithelial cell necrosis, and poses a great danger to humans and aquatic animals. China has revised the limit standard of citrinin in food and issued GB 5009.222-2016, which specifically regulates the content of citrinin in chili powder, rice and rice products. The quantitative limit is set at 25 μg / kg, and the detection limit is 8 μg / kg. The quantitative limit of Monascus related products is set at 80 μg / kg, and the detection limit is 25 μg / kg.

[0004] Talaromyces albobiverticillius, belonging to the genus Talaromyces, is one of the fungi that can produce a large amount of polyketide pigments and does not produce mycotoxins, and is considered one of the most promising sources of food pigments. In addition, the pigment also shows strong antioxidant activity (M, Umesh, et al., 2023). Chinese patent CN116144710A recently disclosed a method for high-yield extracellular pigment production by Talaromyces albobiverticillius. The method was liquid fermentation for 10 days by soluble starch peptone medium, and the yield of orange pigment and red pigment in the supernatant of the fermentation broth was determined, reaching 0.52 g / L and 1.68 g / L, respectively. However, the patent only describes the potential of Talaromyces albobiverticillius in pigment production, and the yield under the existing culture conditions is low, which is not sufficient for large-scale application. In addition, due to the complexity of the liquid fermentation system of filamentous fungi, it contains many impurities such as mycelium, organic acids, polysaccharides, and proteins, which has high viscosity, difficulty in separation and purification, low product purity, and many other problems, which limits the actual application effect of the pigment. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a microbial source antioxidant pigment feed additive for aquaculture and a preparation method thereof, which addresses the shortcomings of the prior art. The mutant strain Talaromyces albobiverticillius CY110 is produced by low-temperature plasma physical mutagenesis of Talaromyces albobiverticillius CY-G, and the mutant strain Talaromyces albobiverticillius CY110 is used for semi-solid state fermentation to produce wine red pigment, which can be used for microbial source antioxidant pigment feed additive for aquaculture.

[0006] To solve the above technical problems, the technical solution adopted by the present application is:

[0007] A Talaromyces albobiverticillius mutant strain produced by low-temperature plasma physical mutagenesis of Talaromyces albobiverticillius CY-G, named Talaromyces albobiverticillius CY110, was deposited at the China Center for Type Culture Collection (China. Wuhan) on November 12, 2024, with the accession number CCTCC NO: M 20242539.

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

[0009] S1, preparation of spore suspension

[0010] The strain Talaromyces albobiverticillius CY110 is inoculated on PDA medium and cultured at 30°C for 5-7 days, and then washed with sterile water, and then prepared for use by aliquoting;

[0011] S2, preparation of honeycomb array semi-solid medium

[0012] A mixture of potato powder 5 g / L, glucose 20 g / L, agar 20 g / L, and food-grade glycerol 5 g / L is prepared, high-temperature high-pressure sterilized, and then poured into multiple 30 cm x 30 cm sterile stainless steel plates after air flow stirring, and then the multiple sterile stainless steel plates are accumulated together to form a honeycomb array semi-solid medium, and then cooled;

[0013] S3, pigment fermentation

[0014] The spore suspension obtained in S1 is uniformly sprayed onto the surface of the honeycomb semi-solid medium obtained in S2, and after inoculation, a sterile breathable film or kraft paper is covered, and then placed in a 30°C environment for fermentation for 10 days;

[0015] S4, pigment extraction and analysis

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

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

[0018] Preferably, the extraction in S4 is placed in a shaker at room temperature under the condition of 200 rpm for 2 h or static extraction for 24 h.

[0019] The application also provides a wine red pigment prepared by the above method, wherein the yield of 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 citrinin content in the liquid sample of the wine red pigment is 0.21±0.02 ng / mL, and the citrinin content in the solid sample is 4.20±0.40 ng / g.

[0020] The application also provides the wine red pigment.

[0021] The application has the following technical effects:

[0022] 1. The application provides a mutant strain Talaromyces albobiverticillius CY110 of Talaromyces albobiverticillius CY-G generated through low-temperature plasma physical mutagenesis, wherein the mutant strain Talaromyces albobiverticillius CY110 can produce extracellular pigments and has good passage stability.

[0023] 2. The application provides a method for producing wine red pigments through semi-solid fermentation of the mutant strain Talaromyces albobiverticillius CY110, which is simple in operation, low in equipment requirement, high in production efficiency and suitable for large-scale production.

[0024] 3. The wine red pigments produced through semi-solid fermentation of the mutant strain Talaromyces albobiverticillius CY110 have strong antioxidant capacity and can be used for microbial source antioxidant pigment feed additives for aquaculture, thus having wide application prospects.

[0025] The application will be further described in detail in combination with the accompanying drawings and examples. DESCRIPTION OF DRAWINGS

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

[0027] Figure 2 is the influence of 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 mutant strains under different mutagenic times in Example 1;

[0029] Figure 4 is the extracellular pigment diffusion result of mutant strain T. albobiverticilllus CY110, starting strain T. albobiverticilllus CY-G and M. purpureus 40805 after 10 days of fermentation in Example 1; wherein, 1: mutant strain T. albobiverticilllus CY110, 2: starting strain T. albobiverticilllus CY-G, 3: M. purpureus 40805;

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

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

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

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

[0034] Example 1

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

[0036] The starting strain Talaromyces albobiverticillius CY-G in the application is isolated from a pond water sample in the Shiqi Aquaculture Technology Demonstration Park in Nansha District, Guangzhou. The fungal 18s universal primer (ITS1: SEQ ID NO. 1, ITS4: SEQ ID NO. 2) is used to amplify the genomic DNA, the product is sequenced, and the obtained ITS sequence is shown as SEQ ID NO. 3. Through comparison with the NCBI database, and in combination with the morphological and molecular biological identification according to the Fungus Identification Manual, it is identified as Talaromyces albobiverticillius, named T.albobiverticillus CY-G, and its sequence is uploaded to the NCBI database, and the accession number is: 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 is inoculated on a PDA plate by punch method, and cultured at a culture temperature of 30°C for 72h. 10mL of sterile water is aspirated to elute the mycelium surface, and the spore concentration is counted to be 10 6 -10 8 individuals / mL (the spore suspension with a concentration of 10 7 individuals / mL is better) by hemocytometer counting.

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

[0040] (3) Screening and isolation of excellent pigment-producing mutant strains: the suspension after mutagenesis in step (2) is diluted to a certain gradient and plated on PDA plates. Under this condition, the lethality and mutation rate of the starting strain T.albobiverticillus CY-G are as follows Figure 2The results are shown (lethality 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] Through plate culture screening: under the condition of gradient mutagenesis time, a variety of mutant strains can be obtained by comparing the color depth, radius size and mycelium radius size of the colonies and the color of the starting strain, such as Figure 3 At 110s, it can be observed that a mutant strain produces extracellular pigment faster than the starting strain under the same culture conditions. After isolation and purification, the Talaromyces strain is obtained and named Talaromyces albobiverticillus CY110 (T. albobiverticillus CY110), which is preserved in the China Center for Type Culture Collection, with the preservation number CCTCC NO: M20242539.

[0042] (4) Stability evaluation of the mutant strain: the starting strain T. albobiverticillus CY-G, the mutant strain T. albobiverticillus CY110 continuously transmitted for 15 generations, and Monascus purpureus (M. purpureus) (collection number: CICC 40805) are cultured on PDA medium at 30°C for 10 days, and the colony map is shown in Figure 4 It can be obtained that under the same culture conditions, the mutant strain T. albobiverticillus CY110 can produce more extracellular pigment than the starting strain T. albobiverticillus CY-G and M. purpureus 40805, which verifies that the mutant strain T. albobiverticillus CY110 has good stability in transmission. In addition, the T. albobiverticillus CY110 strain has great differences in morphological characteristics from the starting strain T. albobiverticillus CY-G, mainly including: the colony morphology is large after being cultured on PDA plate for 10 days; the colony grows outward in annual ring shape, with complete and round edges; in the late growth stage, a large amount of wine red exudate is accompanied, and a large amount of wine red pigment is exuded outward to the culture medium, so that the whole culture medium is dyed wine red.

[0043] Example 2

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

[0045] (1) Spore suspension preparation: the mutant strain T. albobiverticillus CY110 was inoculated on PDA medium and cultured at 30°C for 5-7 days, and then 100 mL sterile water was used to fully elute and sub-packaged in 10 mL quantitative seed spray bottles for standby.

[0046] (2) Preparation of honeycomb array semi-solid medium: according to the accurate weighing of potato powder 5 g / L, glucose 20 g / L, agar 20 g / L, and food-grade glycerol 5 g / L, after high-temperature high-pressure sterilization, the whole liquid was filled with small air bubbles through air flow stirring, and then poured into a 30 cm*30 cm sterile stainless steel plate while hot, to form a multi-array semi-solid medium.

[0047] (3) Pigment fermentation: after the medium was cooled, the seed spray bottle containing spore suspension was evenly sprayed on the surface of the honeycomb semi-solid medium, and after inoculation, a sterile breathable film or cowhide paper was covered and placed at 30°C for 10 days.

[0048] (4) Pigment extraction and analysis: the fermented honeycomb semi-solid medium was fully scraped into a conical flask, and 50% ethanol aqueous solution was added at a solid-liquid ratio of 1 g:20 mL, and then placed in a shaker at room temperature for 2 h at 200 rpm or static extraction for 24 h. The oscillation liquid was poured into a stainless steel positive pressure filter with 0.22 μm organic filter membrane for pressure filtration, and the filtrate was collected to obtain a liquid pigment sample, which was diluted and subjected to ultraviolet full spectrum scanning, pigment content and citrinin absolute quantitative analysis; the filtrate was dried to obtain a wine red powder, which was a solid pigment sample, and was analyzed as above.

[0049] Comparative Example 1

[0050] The starting strain T. albobiverticillus CY-G was used for semi-solid fermentation to produce pigments, and the method was the same as that of Example 2.

[0051] Comparative Example 2

[0052] M. purpureus 40805 was used for semi-solid fermentation to produce pigments, and the method was the same as that of Example 2.

[0053] Figure 5 The ultraviolet full spectrum scanning diagrams of the mutant strain T. albobiverticillus CY110, the starting strain T. albobiverticillus CY-G and M. purpureus 40805 fermented pigments.

[0054] Absolute quantitative analysis of pigments in the fermentation broth of mutant strain T. albobiverticillus CY110, starting strain T. albobiverticillus CY-G and M. purpureus 40805 in Example 2 and Comparative Examples 1-2 was carried out, wherein,

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

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

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

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

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

[0060] Table 1 Citrinin content of T. albobiverticillus CY110, T. albobiverticillus CY-G and M. purpureus 40805 pigment samples

[0061] Strains Liquid samples (ng / mL) Solid samples (ng / g) T. albo biverticillus CY110 0.21±0.02 4.20±0.40 T. albo biverticillus CY-G 0.25±0.01 5.00±0.20 M. purpureus 40805 72.19±0.16 1443.80±3.2

[0062] Example 3

[0063] This example is the application of the wine-red pigment produced by semi-solid fermentation of mutant strain T. albobiverticillus CY110 in Example 2, which can be used in microbial source antioxidant pigment feed additives for aquaculture.

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

[0065] Then, the wine-red pigment produced by the present application was applied to feed additives, the method being: adding the wine-red pigment to commercially available fish meal feed without antioxidant at an addition amount of 0.02%, stirring thoroughly, adding an appropriate amount of water to form a state of "hand kneading into a ball, and light pressing to disperse", using a feed extruder to extrude into particles of appropriate size, and finally placing in a freeze-drying machine for freeze-drying to obtain granular feed.

[0066] The granular feed added with 0.02% wine red pigment is subjected to antioxidation evaluation, and the granular feed without the addition of pigment is used as a negative control, and the granular feed added with 0.02% BHA is used as a positive control. The evaluation method is to perform Schaal oxidation experiment: the Schaal oxidation simulation system is used to accelerate oil oxidation, 100g of the granular feed sample is taken in a heat-resistant glass bottle, and is placed in an oven at 60±2 DEG C for continuous heating for 20 days, is stirred once every 5 days, and is taken in a 100mL conical bottle at 20d, and is stored in a-80 DEG C refrigerator for freezing preservation, and is detected. Peroxide value determination is performed according to GB 5009.227-2016, aniseed value and total oxidation value determination is performed according to GB / T 24304-2009, and the results are shown in Table 2.

[0067] Table 2 Antioxidation evaluation of the feed added with T.albobiverticillus CY110 wine red pigment and the feed added with BHA

[0068]

[0069] The results show that the wine red pigment produced by the mutant strain T.albobiverticillus CY110 through semi-solid state fermentation has strong antioxidation capacity, and has wide application prospects for being used as a microbial source antioxidant pigment feed additive for aquaculture.

[0070] The physical mutagenesis of the present application is only one of the mutation methods, and similar effects can also be achieved through other mutagenesis methods well known to those skilled in the art, which can cause changes in gene coding, enzyme activity characteristics and morphology of living cells, and these methods include physical methods such as rays, particles, lasers and ultraviolet light, chemical mutagenesis methods such as alkylating agents, base analogs, hydroxylamine and acridine pigments, and mutagenesis can be one or multiple generations of mutagenesis, and is not limited to these methods. Based on the strain provided by the present application, further breeding through physical and chemical methods can be performed, and the 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 application, and does not limit the present application. Any simple modification, change and equivalent change made according to the technical essence of the present application to the above embodiment are still within the protection scope of the technical solution of the present application.

Claims

1. A type of white bivalve-shaped bacterium ( Talaromyces albobiverticillius ), characterized in that, The mutant strain produced by low-temperature plasma physical mutagenesis of Talaromyces albobiverticillius CY-G is named Talaromyces albobiverticillius CY110 and is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC No. M20242539.

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

3. The method of claim 2, wherein, The ratio of the fermented honeycomb semi-solid culture medium to the 50% ethanol aqueous solution in S4 is 1 g:20 mL.

4. The method of claim 2, wherein, The extraction in S4 is carried out at room temperature in a shaker at 200 rpm for 2 h or static extraction for 24 h.

5. The method of claim 2, wherein, The yield of red pigment in the wine red pigment 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 of claim 2, wherein, The content of citrinin in the wine red pigment liquid sample in S4 is 0.21±0.02 ng / mL, and the content of citrinin in the solid sample is 4.20±0.40 ng / g.

7. The method of claim 2, wherein, The wine red pigment is used for preparing an aquatic breeding microbial source antioxidant pigment feed additive.

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