Isolation and application of lactobacillus fermentum

Abstract

The present application relates to a lactobacillus fermentum strain and its application. Specifically, the present application provides a lactobacillus fermentum, its preparation and application. The lactobacillus fermentum of the present application has a preservation number of CGMCC No.33042. Adding fermented soybean meal produced by the strain of the present application to the feed of sea bass can replace raw materials such as soybean meal or corn protein without having a negative effect on the growth of fish, and can increase the content of total muscle collagen and total free amino acids.

Description

Technical Field

[0001] This invention relates to the field of feed applications, specifically to the isolation and application of a strain of Lactobacillus fermentum. Background Technology

[0002] With the development of the aquaculture industry, the demand for various protein sources in the aquatic feed industry is gradually increasing. Among them, fishmeal has a high protein content, a balanced amino acid composition, and meets the growth requirements of fish. However, fishmeal is expensive, and resources cannot meet the needs of the entire feed industry. At the same time, in order to reduce aquaculture costs, the aquatic feed industry is trending towards diversification and higher cost-effectiveness in its protein source requirements.

[0003] Soybean meal, a byproduct of oil extraction, is relatively nutritious, with a balanced and complete amino acid composition. Its wide availability, stable quality, and easy accessibility make it an important choice for aquatic feed. However, soybean meal contains various anti-nutritional factors, limiting its use in feed. To improve the application of soybean meal in aquatic feed and enhance animal digestibility and absorption of nutrients, research is being conducted on deep-processed soybean meal products to reduce anti-nutritional factor content, increase protein digestibility, and ensure fish growth performance. Yihai Kerry's fermented soybean meal breaks through the traditional boundaries of feed ingredients using biotechnology. It is a deep-processed soybean product produced through a fully automated process combining modern biotechnology and traditional solid-state fermentation technology.

[0004] Currently, largemouth bass is one of the important freshwater aquaculture species in my country. The amount of fishmeal in its commercial feed is generally 30-50%, which leads to high feed costs during bass farming. Therefore, based on the nutritional requirements of bass, research is being conducted to add other protein sources to bass feed to replace fishmeal in order to reduce farming costs. From a market demand perspective, one of the factors influencing consumers' preference for aquatic animals is the quality of the muscle, mainly reflected in its taste and flavor.

[0005] Therefore, this research aims to study the effects of using fermented soybean meal fermented with Lactobacillus fermentum as a substitute for soybean meal / corn protein / fishmeal on the growth and muscle quality of sea bass, and to develop new strains for application to reduce aquatic feed costs, improve fish muscle quality, and increase product value. Summary of the Invention

[0006] The first aspect of this invention provides a *Lactobacillus fermentatus*, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33042. In an exemplary embodiment, the 16S RNA sequence of the *Lactobacillus fermentatus* described herein is shown in SEQ ID NO:1.

[0007] The second aspect of the present invention provides a culture of Lactobacillus fermentum with accession number CGMCC No. 33042.

[0008] In one or more embodiments, the culture further comprises a culture medium.

[0009] In one or more embodiments, the culture medium is MRS culture medium.

[0010] In one or more embodiments, the culture medium contains peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, triammonium citrate, sodium acetate, glucose, Tween 80, magnesium sulfate, and manganese sulfate.

[0011] In one or more embodiments, the culture medium contains 5-15 g / L peptone, 2-10 g / L beef extract, 3-8 g / L yeast extract, 1-5 g / L dipotassium hydrogen phosphate, 1-5 g / L triammonium citrate, 3-8 g / L sodium acetate, 1-8 g / L glucose, 0.5-2 mL / L Tween 80, 0.1-0.5 g / L magnesium sulfate, and 0.01-0.2 g / L manganese sulfate.

[0012] A third aspect of the present invention provides a formulation containing Lactobacillus fermentum with accession number CGMCC No. 33042.

[0013] In one or more embodiments, the formulation is a microbial formulation.

[0014] The fourth aspect of the present invention provides a fermentation raw material containing Lactobacillus fermentum with accession number CGMCC No. 33042.

[0015] In some embodiments, the fermentation feedstock also contains meal and enzymes. In some embodiments, the fermentation feedstock also contains water.

[0016] In one or more embodiments, the meal is a by-product of oilseed or grain processing or processing, selected from one or more of soybean meal, rapeseed meal, cottonseed meal, peanut meal, sunflower meal, sesame meal, flaxseed meal, rice bran meal, tea meal, flaxseed meal, safflower meal, palm kernel meal, coconut meal, chili meal, olive meal, corn distillers grains with solubles, and beet meal, preferably one or more of soybean meal, rice bran meal, wheat bran meal, and palm meal.

[0017] In one or more embodiments, the enzyme is selected from one or more of proteases, pectinases, xylanases, α-galactosidases, and cellulases. Preferably, the enzyme is a protease, such as one or more of alkaline proteases, neutral proteases, and acidic proteases.

[0018] In one or more embodiments, the total amount of enzyme added is more than 100U per gram of meal, such as 100-1000U, 500-2000U or 100-500U.

[0019] In one or more embodiments, the amount of water used is 25-85% of the mass of the meal.

[0020] In one or more embodiments, the weight ratio of the meal to water is not higher than 10.

[0021] In one or more embodiments, the water content is 20-60% by total mass of the fermentation feedstock, for example 20-40%, 35-50%, or 35-40%.

[0022] In one or more embodiments, 0.1-10 mL of Lactobacillus fermentation solution is added per 100 g of meal.

[0023] In one or more embodiments, the OD value of the fermented Lactobacillus broth is 3-10, 5-10, or 8-10.

[0024] In one or more embodiments, the pH value of the fermented Lactobacillus broth is ≤4.5.

[0025] In one or more embodiments, the viable count of the fermented Lactobacillus broth is 3.0 * 10⁻⁶. 10 CFU / mL - 3.5*10 10 CFU / mL.

[0026] The fifth aspect of the present invention provides a fermentation composition containing Lactobacillus fermentans with accession number CGMCC No. 33042.

[0027] In one or more embodiments, the fermentation composition further contains an enzyme.

[0028] In one or more embodiments, the meal and / or enzymes in the fermentation composition are as described in any embodiment of the fourth aspect of the invention.

[0029] In one or more embodiments, the fermentation temperature is 37-50°C, for example 37-45°C or 40-50°C.

[0030] In one or more embodiments, the fermentation time is 24-96 hours, for example 24-72 hours or 48-72 hours.

[0031] In one or more embodiments, after fermentation, a drying and pulverizing step is also included.

[0032] In one or more embodiments, the drying temperature is 60-70°C or 65-80°C.

[0033] In one or more embodiments, the drying time is 5-10 hours or 6-12 hours.

[0034] In one or more embodiments, the fermentation composition is pulverized to a fineness of 40 mesh or higher, for example, 60 mesh or 80 mesh.

[0035] In one or more embodiments, the fermentation composition, by weight of the total fermentation composition, has a protein content of 50-55%, a pH value of 4.0-5.0, an acid-soluble protein content of 8.5-9.0%, and a live lactic acid bacteria count of 1*10^6. 3 -3*10 3 CFU / g or 2.0*10 3 -2.5*10 3 CFU / g, with a moisture content of 8-12% or 10-15%.

[0036] The sixth aspect of the present invention provides feed containing a fermentation composition as described in any embodiment of the fifth aspect of the present invention.

[0037] In one or more embodiments, the fermentation composition is present in the feed at a content of 5-10%, 5-15%, or 8-10%.

[0038] In one or more embodiments, the feed is aquatic animal feed.

[0039] The seventh aspect of the present invention provides a method for improving the growth performance of aquatic animals and / or improving the quality of aquatic animal products, the method comprising feeding aquatic animals with feed as described in any embodiment of the sixth aspect herein.

[0040] The eighth aspect of the present invention provides the use of fermenting Lactobacillus with accession number CGMCC No. 33042, fermentation raw materials as described in any embodiment of the fourth aspect of the present invention, fermentation compositions as described in any embodiment of the fifth aspect of the present invention, or feeds as described in any embodiment of the sixth aspect of the present invention in the preparation of animal feed.

[0041] In one or more embodiments, the animal feed is aquatic animal feed.

[0042] In one or more embodiments, the application includes improving the growth performance of aquatic animals and / or improving the appearance of aquatic animal products, and / or improving the appearance and taste of aquatic animal products, and / or improving the flavor of aquatic animal products, and / or improving the nutritional content of aquatic animal products, and / or improving the appearance of aquatic animal products after refrigeration, and / or improving the taste of aquatic animal products after refrigeration.

[0043] In one or more embodiments, the aquatic animal products include cooked aquatic animal products, raw aquatic animal products, pickled aquatic animal products, salted aquatic animal products, dried aquatic animal products, surimi products, aquatic seasonings, and aquatic animal fats and oils and their products. Detailed Implementation

[0044] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0045] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0046] In this document, the terms “contains,” “includes,” “containing,” and similar terms encompass the meanings of “basically composed of” and “composed of.” For example, when this document discloses “A contains B and C,” “A is basically composed of B and C” and “A is composed of B and C” should be considered as having been disclosed in this document.

[0047] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0048] Unless otherwise specified, percentages refer to mass percentages and proportions refer to mass ratios in this article.

[0049] In this article, the sum of the percentages of all components in the composition is 100%.

[0050] In this document, when describing embodiments or examples, it should be understood that it is not intended to limit the invention to those embodiments or examples. Rather, all alternatives, modifications, and equivalents of the methods and materials described herein are covered within the scope defined by the claims.

[0051] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0052] The inventors collected naturally fermented shrimp paste from Hainan and screened out a strain of *Lactobacillus fermentans* F. After testing the fermentation performance of *Lactobacillus fermentans* F, it was found that it can be used for fermenting oilseed meals, increasing the content of small peptides and organic acids in the meal. More unexpectedly, when the fermented samples were applied to the feed of aquatic animals, it had a palatability-enhancing effect in sea bass feed, replacing soybean meal, improving fish growth performance, increasing muscle collagen content, improving muscle hardness and shear strength, and increasing the content of flavor amino acids. This completes the invention.

[0053] Lactobacillus fermentation for meal fermentation

[0054] This article first provides a *Lactobacillus fermentatus*, which is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 33042. Exemplarily, the 16S rDNA sequence of the *Lactobacillus fermentatus* described in this article is shown in SEQ ID NO:1.

[0055] Microbial strains are generally preserved in the form of inoculum (such as powder or liquid) and usually require activation before use. Depending on the strain type and preservation method, a suitable activation method can be selected. The fermenting Lactobacillus can be cultured using conventional methods, such as MRS medium. A typical MRS medium includes peptone, yeast, sodium acetate, Tween 80, magnesium sulfate heptahydrate, beef extract, glucose, diammonium citrate, potassium dihydrogen phosphate, manganese sulfate heptahydrate, and water. For solid MRS medium, an appropriate amount of agar powder can be added to this formula. The proportions of these ingredients can be adjusted according to actual needs. Culture conditions can be conventional, such as incubating at 25-40℃ or 35-40℃ for 10-20 hours or 15-20 hours. Single colonies of the bacterial strain can be directly transferred to a suitable fresh solid culture medium (such as MRS medium). After growth and reproduction for a period of time (e.g., 2-5 days), they can be inoculated into liquid culture medium. The inoculation amount can be selected according to actual needs, for example, two to three loops can be inoculated into 10-50 mL or 15-30 mL of liquid culture medium. Activation is then performed in the liquid culture medium at 30-40℃ or 35-40℃ for about 20-24 hours to obtain an activated bacterial solution (seed culture). In the exemplary embodiment, the activated bacterial solution of *Lactobacillus fermentum* in this article has an OD value of 3-10, 5-10, or 8-10, a pH value ≤ 4.5, and a viable count of 3.0*10⁻⁶. 10 CFU / mL - 3.5*10 10 CFU / mL.

[0056] In some embodiments, this document also provides a culture of *Lactobacillus fermentum* with accession number CGMCC No. 33042. This culture contains the *Lactobacillus fermentum* described herein and a culture medium. The culture medium can be any commonly used culture medium for culturing *Lactobacillus fermentum* known in the art, including media used for preparing *Lactobacillus fermentum* seed culture or activated culture, and media used for fermentation using *Lactobacillus fermentum*.

[0057] In some embodiments, the culture medium is MRS medium. More preferably, the medium contains peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, triammonium citrate, sodium acetate, glucose, Tween 80, magnesium sulfate, and manganese sulfate. In some embodiments, the medium contains 5-15 g / L peptone, 2-10 g / L beef extract, 3-8 g / L yeast extract, 1-5 g / L dipotassium hydrogen phosphate, 1-5 g / L triammonium citrate, 3-8 g / L sodium acetate, 1-8 g / L glucose, 0.5-2 mL / L Tween 80, 0.1-0.5 g / L magnesium sulfate, and 0.01-0.2 g / L manganese sulfate. When a solid culture medium is required, an appropriate amount (e.g., 10-20 g / L) of agar powder can be added to the above medium.

[0058] The *Lactobacillus fermentum* described herein can be used as a probiotic additive; therefore, this article also provides a formulation containing *Lactobacillus fermentum* with accession number CGMCC No. 33042. In some embodiments, the formulation is a microbial preparation.

[0059] Fermentation raw materials, fermentation methods for oilseed meals, and fermentation compositions

[0060] The *Lactobacillus fermentans* described in this article belongs to the lactic acid bacteria family and can be used to ferment roughage, grains, and other animal and plant by-products, such as rice flour, corn flour, rice bran, wheat bran, livestock and poultry manure (including chicken manure), straw, swill, forage, cottonseed hulls, peanut shells, various oilseed cakes, miscellaneous meals, and dregs, thereby preparing fermentation raw materials. Therefore, this article also provides a fermentation raw material, *Lactobacillus fermentans* with accession number CGMCC No. 33042. Preferably, this fermentation raw material also contains oilseed cakes, enzymes, and optionally water.

[0061] In this article, the fermentation raw material uses *Lactobacillus fermentatus* in the form of an inoculum, such as a liquid or powder. As mentioned earlier, the *Lactobacillus fermentatus* inoculum can be activated and then added to the fermentation raw material. The amount of activated inoculum can be conventional, such as 0.1-10 mL of activated *Lactobacillus fermentatus* inoculum per 100 g of meal, for example, 0.5-10 mL, 1.0-8.0 mL, or 3.0-5.0 mL per 100 g of meal. The activated *Lactobacillus fermentatus* in this article can be diluted before being added to the meal raw material, for example, diluted 3-80 times, 5-40 times, or 8-20 times.

[0062] In this article, the fermentation raw materials can be various crop straw powder, leaf and weed powder, melon vine powder, fruit pulp, dried sugarcane bagasse, rice husk powder, rice bran, distiller's grains, brewer's grains, sugar residue, vinegar residue, starch residue, cassava residue, citric acid residue, soy sauce residue, monosodium glutamate residue, edible fungus residue, powder residue, tofu residue, medicinal residue, oil residue, oil cake meal, bran, cottonseed meal, moldy feed, slaughterhouse by-products, swill, leftover food, chicken manure, and other waste. In an exemplary implementation scheme, the fermentation raw materials in this article are meal-like substances. The term "oil meal" in this article refers to high-protein oil meals commonly used in animal feed, typically byproducts of oilseed or grain processing, such as defatted or partially defatted oilseed cakes or meals, including but not limited to soybean meal, rapeseed meal, cottonseed meal, peanut meal, sunflower meal, sesame meal, flaxseed meal, rice bran meal, tea meal, flaxseed meal, safflower meal, palm kernel meal, coconut meal, chili meal, olive meal, corn distillers' grains with solubles (DDGS), corn gluten meal, and beet meal, with soybean meal being preferred. Preferably, the oil meal content, based on the total weight of the fermented raw materials, can be 70-80% or 72-75%.

[0063] The enzymes suitable for use in this invention can be various enzymes conventionally used in the art for fermenting and enzymatically hydrolyzing soybean meal, including but not limited to one or more of proteases, pectinases, xylanases, α-galactosidases, and cellulases. In some embodiments, the invention uses proteases, such as one or more of alkaline proteases, neutral proteases, and acidic proteases. Typically, the amount of enzyme used can be the conventional amount used in soybean meal fermentation in the art, for example, more than 100U per gram of meal (such as soybean meal), such as 100-2000U, 500-2000U, or 100-500U of enzyme.

[0064] The amount of water used is typically 25-85% of the weight of the meal. In some embodiments, the weight ratio of meal to water is not higher than 10, for example not higher than 5, such as 1-3 or 2.5-3.0. In some embodiments, the water content is 20-60% based on the total weight of the fermentation feedstock, for example 20-40%, 35-50%, or 35-40%.

[0065] This document also provides a method for fermenting meal, which includes the step of fermenting meal using *Lactobacillus fermentatus* with accession number CGMCC No. 33042. Therefore, the method of this document includes the step of fermenting a fermentation feedstock containing *Lactobacillus fermentatus*, meal, enzymes, and water. In some embodiments, the method of this document includes the step of mixing the *Lactobacillus fermentatus*, meal, enzymes, and water described herein. Typically, during fermentation, the meal and enzymes are first mixed, and then the *Lactobacillus fermentatus* and water described herein are added to form the fermented meal.

[0066] Typically, the fermentation temperature is 37-50℃, for example, 37-45℃. The fermentation time can be 24-96 hours, for example, 24-72 hours or 48-72 hours. The fermentation environment can be a closed environment. After fermentation, a drying and pulverizing step may be included, which can be done at 60-70℃ or 65-80℃ for 5-10 hours or 6-12 hours. Usually, the fermented meal is pulverized to a fineness of 40 mesh or higher, for example, 60 mesh or 80 mesh.

[0067] In some embodiments, this document also provides a fermentation composition containing meal, *Lactobacillus fermentum* with accession number CGMCC No. 33042, or obtained by fermentation from the fermentation feedstock described in any embodiment of this document. The type and amount of meal, the type and amount of protease, and the amount of water, etc., can be as described above. The fermentation composition of the present invention can be a mixture prepared for fermentation (i.e., not yet fermented) (i.e., the fermentation feedstock described herein), a mixture during fermentation, or a mixture after fermentation. Preferably, the fermentation composition described herein is prepared by the method described in any embodiment of this document.

[0068] In the fermentation composition described in this article, the protein content can be 50-55%, the pH can be 4.0-5.0, the acid-soluble protein content (as a percentage of dry matter) can be 8.5-9.0%, 8.8-9.0%, or 8.8-8.9%, and the viable lactic acid bacteria count is 1*10^6. 3 -3*10 3 CFU / g or 2.0*10 3 -2.5*10 3 The concentration of CFU / g and the moisture content can be 8-12% or 10-15%. Typically, the fermentation composition is dried and pulverized before its protein content, pH, acidity, acid-soluble proteins, and viable lactic acid bacteria count are determined.

[0069] Feed containing fermented composition

[0070] Feed typically includes energy sources, protein sources, mineral sources, vitamin sources, and optional additives, wherein the protein sources include fermentation compositions. Therefore, in some embodiments, this document also provides a feed containing the fermentation composition described in any embodiment herein.

[0071] Protein raw materials can be those commonly used in the field that contain a high protein content (e.g., 40-50% or 40-60%), such as one or more of animal protein, plant protein, and / or microbial protein. Animal protein can be by-products of fish processing or processing, or by-products of livestock or poultry processing or processing. By-products of fish processing or processing include fishmeal. By-products of livestock or poultry processing or processing include products obtained by high-temperature cooking, defatting, drying, or pulverizing inedible carcasses, bones, blood, plasma, etc., from livestock or poultry, such as fishmeal, meat and bone meal, and plasma protein powder. Plant proteins can be plant protein extracts, byproducts of oilseed crop oil extraction, fermentation products of byproducts of oilseed crop oil extraction, etc. Preferred byproducts of oilseed crop oil extraction include one or more of the following: soybean meal, cottonseed meal, peanut meal, rapeseed meal, sesame meal (sesame residue), sunflower seed cake, flaxseed cake, coconut meal, walnut meal, and palm kernel meal. The byproducts of oilseed crop oil extraction in the protein raw material can be the same as or different from the meal, and the fermentation products of byproducts of oilseed crop oil extraction can be the same as or different from the byproducts of oilseed crop oil extraction. Plant protein extracts include legume protein extracts, cereal protein extracts, hemp protein extracts, and algae protein extracts, preferably selected from soy protein concentrate, soy protein isolate, pea protein concentrate, pea protein isolate, broad bean protein concentrate, broad bean protein isolate, wheat gluten protein (gluten), wheat protein isolate, zein, flaxseed protein, hemp seed protein, spirulina protein, etc.

[0072] The specific protein sources and amounts in the protein feed can be adjusted according to the actual feeding species. For example, if the feed is fish feed, the protein feed may include byproducts of fish processing or processing, plant protein extracts, byproducts of oilseed crop extraction, and fermented products of oilseed crop extraction byproducts. In some embodiments, based on the total weight of the feed, the content of fish processing or processing byproducts in the protein feed is 30-40% or 35-45%, the content of plant protein extracts is 10-15% or 15-20%, the content of oilseed crop extraction byproducts is 10-12% or 10-15%, and the content of fermented products of oilseed crop extraction byproducts is 5-10% or 5-8%. In some embodiments, the protein feed in this document includes fishmeal, soybean meal, fermented soybean meal, soybean protein concentrate, and wheat gluten. Preferably, the fishmeal content is 30-40% or 35-45% by total feed weight; preferably, the fermented soybean meal content is 10-20% or 5-15%; preferably, the soybean meal content is 10-12% or 12-15%; preferably, the corn protein content is 5-7% or 7-10%; preferably, the soybean protein concentrate content is 8-10% or 8-12%; preferably, the wheat gluten content is 5-10% or 5-12%. For example, if the feed is poultry feed, the protein ingredients may include by-products from oilseed crop extraction and fermented products of these by-products. In some embodiments, the feed described herein contains 1-2% or 1-5% by-products from oilseed crop extraction and 5-8% or 3-8% fermented products of these by-products by weight. In some embodiments, the feed described herein contains soybean meal and fermented soybean meal as protein ingredients. The protein content of raw materials can be 40-70%, 50-80%, or 75-85% based on the total weight of the feed.

[0073] Energy feed ingredients can be commonly used energy-providing raw materials, such as grains, grain processing by-products, and oils. Grains can be those with high carbohydrate content or containing a large amount of easily digestible and absorbable starch, including but not limited to corn, wheat, barley, sorghum, rice, and oats. Grain processing by-products include but are not limited to corn gluten meal, corn germ meal, corn bran, wheat bran, barley bran, barley germ meal, sorghum bran, sorghum germ meal, rice bran, rice husk powder, oat bran, and oat germ meal. The amount of grains added can be 10%, 12%, or 15% or more, for example, 10-15%. The amount of grain processing by-products added can be 1-5%, for example, 2-5% or 3-4%. Adding an appropriate amount of oil (such as 1-3% or 1.0-1.5%) to the feed can increase the energy content of the feed and also improve its palatability. The oils and fats can be vegetable oils and / or animal oils. Vegetable oils include, but are not limited to, palm oil, soybean oil, sunflower seed oil, peanut oil, rapeseed oil, corn oil, safflower seed oil, sesame oil, rice bran oil, rice bran oil, flaxseed oil, olive oil, hazelnut oil, pumpkin seed oil, grapeseed oil, evening primrose oil, Sichuan pepper seed oil, almond oil, wheat germ oil, perilla seed oil, pecan oil, almond oil, cashew oil, macadamia nut oil, pistachio oil, palm kernel oil, and coconut oil, preferably one or more of soybean oil, corn oil, rapeseed oil, palm oil, and coconut oil. Animal oils include, but are not limited to, lard, tallow, mutton tallow, duck tallow, chicken tallow, fish oil, and shrimp oil, preferably one or more of fish oil, lard, mutton tallow, and tallow. The amount of energy source added can be selected according to actual needs. Based on the total weight of the feed, the oil content can be 1.5-4.0% or 3.5-5.0%, wherein the content of vegetable oils and animal oils can be equal or unequal.

[0074] Mineral feed ingredients provide laying hens with calcium, phosphorus, sodium, and trace elements. Calcium sources are typically limestone powder (mainly calcium carbonate) and shell powder; phosphorus sources are typically dicalcium phosphate and calcium dihydrogen phosphate; sodium sources can be table salt; and trace elements mainly include iron, copper, zinc, manganese, iodine, and selenium. By total feed weight, calcium content can be 3-4%, phosphorus content can be 2-6% or 2-3%, sodium content can be 3-4%, and trace element additions are minimal. By total feed weight, calcium dihydrogen phosphate is the primary mineral feed ingredient, with a content of 2-6% or 2-3%.

[0075] Vitamin raw materials can be commonly used, such as fat-soluble vitamins A, D, E, and K, and B vitamins (such as vitamins B1, B2, B6, B12, B23, B24, B25, B26, B28, B29, B20 ... 12 (etc.) and water-soluble vitamins such as vitamin C.

[0076] The most commonly used additives are amino acid additives, such as methionine and / or lysine. They can also be enzyme preparations such as amylase, protease, and cellulase, probiotics (such as lactic acid bacteria and bifidobacteria) and prebiotics, antioxidants (such as ethoxyquinoline and butylated hydroxyanisole) or antifungal agents (such as calcium propionate and sodium diacetate), and nutritional additives (such as phospholipids, especially soybean phospholipids and squid paste).

[0077] In some implementations, the moisture content can be 9-10% by total feed weight, the protein content can be 50-60% or 50-55%, the fat content can be 10-15% or 10-12%, and the ash content can be 9-10% or 5-12%.

[0078] The feed described in this article can be in conventional form, but can take various forms depending on the species being fed. For example, fish feed is usually in powder or pellet form with good buoyancy or sinking properties, and is stable in water. Poultry feed is usually in larger pellets or powder form, which is easy for poultry such as chickens, ducks, and geese to peck at. Poultry feed is usually in block, pellet, or powder form with a mesh size of 4-10 or larger.

[0079] The feed described in this article can be prepared using conventional feeding methods. You can refer to the industry standards for animal husbandry, such as feeding once or twice a day, or once or twice a week. The amount of feed can be 1-3% of the animal's body weight.

[0080] In some implementations, the feed described herein is aquatic animal feed, preferably fish feed.

[0081] In this article, aquatic animals include, but are not limited to, fish, shrimp, crabs, crustaceans, shellfish, cephalopods, amphibians, reptiles, mollusks, echinoderms, coelenterates, and zooplankton, such as freshwater fish and / or marine fish, shrimp, crabs, lobsters, oysters, mud snails, scallops, clams, mussels, squid, octopuses, frogs, salamanders, sea turtles, sea slugs, starfish, sea urchins, sea cucumbers, sea anemones, corals, kelp, laver, jellyfish, seahorses, and krill.

[0082] Applications and methods

[0083] This document also provides the application of *Lactobacillus fermentatus* with accession number CGMCC No. 33042, fermentation feedstocks as described in any embodiment of this document, fermentation compositions as described in any embodiment of this document, and / or fermentation methods for meal as described in any embodiment of this document in the preparation of aquatic animal feed. In some embodiments, the application includes improving the quality of aquatic products corresponding to aquatic animals.

[0084] In some implementations, the application includes improving the appearance of aquatic animal products, and / or improving the appearance and texture of aquatic animal products, and / or improving the flavor of aquatic animal products, and / or improving the nutritional content of aquatic animal products, and / or improving the appearance of aquatic animal products after refrigeration, and / or improving the texture of aquatic animal products after refrigeration.

[0085] In some implementations, the application includes one or more of the following aspects: increasing the content of total collagen and / or heat-soluble collagen in the muscle of aquatic animal products; increasing the firmness and / or shear strength of the muscle of aquatic animal products; increasing the content of free amino acids and / or amino acids in aquatic animal products; increasing the firmness of the aquatic animal body; improving the flavor of aquatic animal products; improving the brightness of aquatic animal products; improving the juiciness of aquatic animal products; improving the appeal of aquatic animal products; slowing down the rate of quality deterioration of aquatic animal products; extending the cold storage period of aquatic animal products; reducing the fishy smell of aquatic animal products after refrigeration; improving the color of aquatic animal products after refrigeration; improving the gloss of the cut surface of aquatic animal products after refrigeration; improving the texture of the muscle tissue of aquatic animal products; improving the juiciness of aquatic animal products after refrigeration; and improving the tenderness of aquatic animal products after refrigeration.

[0086] In some embodiments, this document provides the use of fermentation methods for Lactobacillus fermentation, fermentation feedstocks, fermentation compositions and / or meal as described in any embodiment of this document in improving the growth performance of aquatic animals and / or improving the quality of aquatic animal products.

[0087] In some embodiments, this document also provides a method for improving the growth performance of aquatic animals and / or improving the quality of aquatic animal products, the method comprising feeding aquatic animals with a feed containing a fermented composition as described in any embodiment of this document.

[0088] In some implementation schemes, aquatic animal products include cooked aquatic animal products, raw aquatic animal products, pickled aquatic animal products, salted aquatic animal products, dried aquatic animal products, surimi products, aquatic condiments, aquatic animal fats and their products, such as drunken crab, pickled shrimp, crab paste, mud snails, sashimi, raw fish meat, sashimi, wasabi octopus, caviar, etc.

[0089] In this article, "flavor amino acids (DAA)" refers to amino acids that can present a special taste sensation. They mainly include umami amino acids, sweet amino acids, bitter amino acids, and sour amino acids. Umami amino acids mainly include glutamic acid and aspartic acid, sweet amino acids mainly include glycine and alanine, bitter amino acids mainly include arginine and histidine, and sour amino acids mainly include asparagine and glutamine.

[0090] In this article, "fish" refers to common fish species that can be fed with feed, including freshwater fish and / or marine fish. Common freshwater fish include crucian carp, common carp, grass carp, black carp, silver carp, catfish, snakehead, mandarin fish, perch, catfish, silver carp, tilapia, loach, and eel. Common marine fish include yellow croaker, ribbonfish, pomfret, lizardfish, conger eel, filefish, saury, flounder, salmon, and grouper. Preferably, the fish in this article belong to the order Perciformes, including but not limited to the genera *Perciformes* (such as spotted perch and seven-star perch), the genera *Perciformes* (such as perch, also known as red perch or five-striped perch), the genera *Perciformes* (such as white perch), and the genus *Perciformes* (such as *Perciformes spp.*) of the family Perciformes.

[0091] "Refrigeration" is generally considered to be storage at 0-4℃. In this article, the refrigeration time can be 0-6 days or 3-6 days.

[0092] The present invention has the following beneficial effects:

[0093] This invention provides a fermented Lactobacillus and prepares a fermented soybean meal sample for feeding sea bass, which improves feed conversion rate, enhances the flavor of sea bass, and has an appetite-stimulating effect when used in sea bass feed as a substitute for soybean meal / corn protein. It also improves fish growth performance, increases muscle collagen content, enhances muscle hardness and shear strength, and increases the content of flavor amino acids.

[0094] The present invention will be further described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and reagents used in the embodiments are conventional methods and reagents in the art.

[0095] Reagent formulation and detection methods

[0096] Lactobacillus plantarum M was CGMCC 1.557, purchased from the Institute of Microbiology, Chinese Academy of Sciences.

[0097] MRS liquid culture medium formula: 10g peptone, 5g Angel yeast extract, 5g sodium acetate, 1g Tween 80, 0.2g magnesium sulfate heptahydrate, 10g beef extract, 20g glucose, 2g diammonium citrate, 2g potassium dihydrogen phosphate, 0.05g manganese sulfate heptahydrate, 1L water; MRS solid culture medium formula: based on MRS liquid culture medium, add 2% agar.

[0098] Moisture content was determined according to GB / T6435-2006; acid-soluble protein content was determined according to GB9005.5-2010.

[0099] Example 1: Screening and identification of Lactobacillus fermentum F

[0100] The strain of this invention was isolated from shrimp paste in Hainan. The specific steps are as follows:

[0101] Take 1g of shrimp paste sample and resuspend it in 9mL of sterile physiological saline, then shake well. Take more than 1mL of the liquid and add it to 9mL of sterile physiological saline, then shake well to obtain a dilution of 10. -1 The diluted solution. Repeat the above steps to perform serial dilutions, obtaining dilutions of 10-10. -2 10 -3 10 -4 10 -5 10 -6 10 -7 The dilution solution was selected. A dilution of 10 was chosen. -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 200 μL of the diluted solution was spread onto MRS solid medium, with two replicates for each dilution. All operations were performed under aseptic conditions. After spreading, the plates were incubated at 37°C. After 48 hours, the plates were removed, and single colonies were picked from the plates under aseptic conditions and incubated at 37°C for another 48 hours. Colony morphology was then observed. One colony was 1-2 mm in diameter, milky white, with raised, smooth, moist, and opaque surfaces and neat edges. Microscopic observation revealed a short rod-shaped bacterial strain.

[0102] PCR amplification of bacterial 16S rDNA was performed using bacterial culture. The amplification primers were those known in the art:

[0103] Upstream primer 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO:2) Downstream primer 1492R: 5'-TACGGYTACCTTGTTACGACTT-3' (SEQ ID NO:3)

[0104] The PCR system (50 μL) is as follows:

[0105] The PCR program was as follows: ① 94℃ for 5 min; ② 94℃ for 30 s, 50℃ for 30 s, 72℃ for 30 s, for 30 cycles; ③ 72℃ for 5 min.

[0106] After the PCR reaction was completed, the PCR products were confirmed by nucleic acid electrophoresis and then sent to Sangon Biotech (Shanghai Co., Ltd.) for sequencing. The 16S rDNA sequence of the sampled bacteria was obtained and compared with the 16S rDNA sequences indexed in the NCBI website. The isolated bacteria were identified as non-engineered wild-type Lactobacillus fermentum F.

[0107] The sequencing results of the 16S rRNA gene are as follows:

[0108]

[0109] The Lactobacillus fermentum strain was deposited on December 12, 2024, at the China General Microbiological Culture Collection Center (CGMCC, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China), with accession number CGMCC No. 33042.

[0110] Example 2:

[0111] 2.1 Preparation of Fermented Soybean Meal Samples

[0112] Lactic acid bacteria culture: A single colony of *Lactobacillus fermentum* was picked and inoculated into a sterile glass tube containing 15 ml of MRS liquid medium. The culture was incubated at 37°C for 16 hours. The cultured solution was then stored at 4°C for later use. At this point, the OD value of the bacteria was approximately 9, and the viable count was 3.5 x 10⁻⁶. 10 CFU / mL. The MRS medium formula used was: 10g peptone, 5g Angel yeast extract, 5g sodium acetate, 1g Tween 80, 0.2g magnesium sulfate heptahydrate, 10g beef extract, 20g glucose, 2g diammonium citrate, 2g potassium dihydrogen phosphate, 0.05g manganese sulfate heptahydrate, and 1L water.

[0113] Solid sample preparation: 100g of commercial soybean meal (from Dahai Grain and Oil Industry (Fangchenggang) Co., Ltd.) was placed in a self-sealing bag. Protease (200,000 U / g alkaline protease) was added at a ratio of 500 U / g soybean meal and mixed evenly to form sample a. 1mL of lactic acid bacteria solution and 37.6g of purified water were mixed evenly and then poured into sample a. The mixture was mixed again, the air in the bag was removed, and the bag was sealed. The sample was then placed in a 40℃ constant temperature incubator for 72h. After 72h of incubation, the sample was dried in a 65℃ oven for 6h, then pulverized and passed through a 60-mesh sieve. Protein, acid-soluble protein, pH, and acidity were measured. Acidity was determined by titration.

[0114] Moisture content was determined using the GB / T6435-2006 method; acid-soluble proteins were determined using the GB9005.5-2010 method; viable bacteria count was determined using the plate count method; and acidity was determined by titration.

[0115] The results are shown in Table 1 below. In Table 1, sample 1 was without strain; sample 2 was a fermented sample with *Lactobacillus fermentum* F; and sample 3 was a fermented sample with *Lactobacillus plantarum* M. The indicators for samples 1, 2, and 3 were measured.

[0116] Table 1: Detection indicators of various fermentation samples

[0117]

[0118] As shown in the table above, the samples prepared from *Lactobacillus fermentum* F have better performance in all indicators than other strains; after drying, the viable count of lactic acid bacteria in the samples is consistently above 10. 3 CFU / g, compared to commercially available lactic acid bacteria inoculants (live count 10). 9 The cfu / g ratio varies considerably.

[0119] 2.2 Effect of different amounts of added Lactobacillus F on sample quality

[0120] Lactic acid bacteria culture: see Example 2.

[0121] Solid sample preparation: see Example 2.

[0122] The experimental formulation is shown in Table 2 below.

[0123] Table 2: Experimental Formula

[0124]

[0125]

[0126] The results are shown in Table 3.

[0127] Table 3: Detection of various indicators in fermentation samples

[0128]

[0129] As shown in the table above, the effect of adding 3% Lactobacillus fermentum F is better than other addition amounts, and this condition is preferred for subsequent sample preparation.

[0130] Example 3: Design of Feeding Formula

[0131] 1. Experimental formulation and grouping: Four feed formulations were designed based on the nutritional requirements of sea bass. The control group contained 40% fishmeal, 12% soybean meal, and 8% corn protein. The treatment groups consisted of three groups, which were different from the control group. The treatment groups were: the group with 10% fermented soybean meal (without added bacteria) replacing 12% soybean meal, the group with 10% fermented soybean meal (fermented lactobacillus F group) replacing 12% soybean meal, and the group with 10% fermented soybean meal (plant lactobacillus group) replacing 12% soybean meal. The detailed formulation composition is shown in Table 2.

[0132] 2. Experimental species: Bass (largemouth bass).

[0133] 3. Experimental Management: During the rearing period, feed twice daily (08:00 and 16:00). For the first two weeks of the experiment, all net cages were fed the same weight of feed (1-3% of the total weight). Afterward, the feed amount was adjusted according to weather, water temperature, and the fish's feeding behavior. During the rearing period, approximately one-third of the freshwater was changed every three days, and the bottom of the pond was cleaned with a siphon every six days to remove feces. The water temperature during the rearing period was maintained at 27.5±2.5℃, dissolved oxygen at 6.0-7.0 mg / L, pH at 7.0-7.5, ammonia nitrogen content ≤0.2 mg / L, and nitrite content ≤0.1 mg / L.

[0134] 4. Testing indicators:

[0135] Feed intake (g / tail / day) = FI(g) / [(FFN+IFN) / 2] / days;

[0136] Weight gain rate (%) = [FBW(g) - IBW(g)] / IBW(g) × 100;

[0137] Feed conversion ratio = FI(g) / [FBW(g)-IBW(g)];

[0138] Specific growth rate (%) / day = (lnFBW(g) - lnIBW(g)) / days × 100;

[0139] FFN, final number of tails; IFN, initial number of tails; FBW, final body weight; IBW, initial body weight; IBP, initial body protein.

[0140] The feed formulation composition and nutritional levels are shown in Table 4 below.

[0141] Table 4: Feed Formulation Composition and Nutritional Levels (g / kg)

[0142]

[0143]

[0144] Note: Premix ingredients: VA, 3000 IU, VD3, 1500 IU, VE, 40 mg, VK3, 4.5 mg, VB, 22.515 mg, VC, 110 mg; Biotin, 0.15 mg, Inositol, 40 mg, Folic Acid, 1.3 mg; Iodine, 1.2 mg, Manganese, 8.5 mg, Molybdenum, 1 mg, Copper, 6.5 mg, Zinc, 53 mg; Selenium, 0.35 mg, Iron, 45 mg.

[0145] Example 4: Experimental Results – Growth Performance

[0146] As shown in Table 5 below, compared with the control group, the Lactobacillus fermentation group F significantly increased the feed intake and weight gain rate of sea bass, and showed a trend of decreasing the feed conversion ratio. The other groups had no significant effect on weight gain rate and feed conversion ratio.

[0147] Table 5: Growth Performance

[0148]

[0149] Note: In the same column, different lowercase letters indicate significant differences (P<0.05), different uppercase letters indicate extremely significant differences (P<0.01), and the same letter or no letter indicates no significant differences (P>0.05).

[0150] Example 5: Experimental Results – Analysis of Muscle Composition and Collagen Indicators

[0151] The quality of sea bass muscle was tested, and the results are shown in Table 6 below.

[0152] Moisture content was determined using the 105℃ drying method; crude protein was determined using the Kjeldahl nitrogen determination method; crude fat was determined using the chloroform-methanol extraction method; and crude ash was determined using the high-temperature ignition method. The content of total muscle collagen and heat-soluble collagen was determined using the hydroxyproline method. Heat-soluble collagen was determined according to the method of Kong et al. (see Kong F, Tang J, Lin M et al. Thermal effects on chicken and salmon muscles: Tenderness, cook loss, aeroashrinkage, collagen solubility and microstructure). The content of heat-insoluble collagen was the difference between total muscle collagen and heat-soluble collagen. Three fish were randomly selected, the skin of the back muscles was removed, and the white muscle (red muscle removed) above the lateral line behind the head was gently pressed with a colorimeter, and the L*, a*, and b* values ​​were recorded. Two muscle pieces were taken from each side of the back muscles for texture and shear force analysis. Steaming loss: Meat pieces of the same size and location were steamed for 3 minutes, dried, and weighed for calculation. Freezing loss: Meat pieces of the same size and location were frozen at -20℃ for 24 hours and then weighed for calculation. The textural properties (hardness, elasticity, cohesiveness, chewiness) and shear force of the muscle were tested using a Universal TA texture analyzer (Tengba Company).

[0153] Table 6: Typical muscle composition, collagen, color difference, water retention and texture

[0154]

[0155]

[0156] Table 6 shows that the quality testing of sea bass muscle revealed no significant differences in brightness (L*), redness (a*), yellowness (b*), evaporation loss, freezing loss, elasticity, and cohesiveness among the groups, indicating no significant differences in the conventional components of sea bass muscle among the groups. Furthermore, the *Lactobacillus fermentum* F group exhibited significantly higher hardness and shear strength than the control group, and its total collagen and heat-soluble collagen content were also significantly higher than other groups. Therefore, the addition of fermented soybean meal (*Lactobacillus fermentum* F) can increase the content of total collagen and heat-soluble collagen in sea bass muscle, thereby improving its hardness and shear strength.

[0157] Example 6: Experimental Results – Analysis of Free Amino Acids

[0158] The content of free amino acids and flavor amino acids in sea bass meat was detected, and the results are shown in Table 7.

[0159] Method for determining free amino acids in muscle: The trichloroacetic acid hydrolysis method was used, and the processed samples were measured using an ultra-high-speed automatic amino acid analyzer (Hitachi LA8080, Japan).

[0160] Table 7

[0161]

[0162]

[0163] As shown in Table 7, the total free amino acid content of the Lactobacillus F fermentation group was higher than that of other groups, and its content of flavor amino acids was also the highest. Therefore, adding fermented soybean meal (Lactobacillus F fermentation) can increase the content of free amino acids and flavor amino acids in fish meat.

[0164] Example 7: Experimental Results – Flavor Analysis

[0165] The results of the tasting test of sea bass muscle are shown in Table 8 below.

[0166] Table 8

[0167]

[0168] Note: Two fish were randomly selected from each group for steaming. After steaming, eight evaluators tasted and evaluated the fish. The scores were 1, 2, 3, 4, 5, and 6, with 1 being the weakest and 6 being the strongest.

[0169] As shown in the table above, the tasting test on sea bass muscle revealed that the Lactobacillus F fermented group had higher body firmness, and its flavor, juiciness, and overall satisfaction were higher than the other three groups. This indicates that adding fermented soybean meal (Lactobacillus F fermented) can improve muscle firmness, fillet shine, increase juiciness and flavor, and improve overall fish satisfaction.

[0170] Example 8: Experimental Results – Storage and Tasting

[0171] Sensory evaluation was conducted on sea bass that were refrigerated at 4℃ for 3 and 6 days. Ten people with sensory evaluation experience were selected to conduct the sensory evaluation. Each indicator was scored out of 10. The evaluation criteria are shown in Table 9 below, and the evaluation results are shown in Table 10.

[0172] Table 9

[0173]

[0174] The sensory evaluation results of the storage quality are shown in the table below.

[0175] Table 10

[0176]

[0177] Table 10 shows that the sensory test on cold-frozen sea bass muscle revealed that the aroma, color, texture, juiciness, and tenderness of the Lactobacillus fermentum F group were significantly higher than those of the other groups. While the overall muscle quality declined with increasing refrigeration time, the decline was slower in the Lactobacillus fermentum F group. This indicates that adding fermented soybean meal (Lactobacillus fermentum F group) can slow down the rate of muscle quality deterioration.

Claims

1. Lactobacillus fermentum with accession number CGMCC No.33042.

2. A culture of Lactobacillus fermentum with accession number CGMCC No. 33042.

3. The culture as described in claim 2, characterized in that, The culture also contains a culture medium; preferably, the culture medium is MRS medium; more preferably, the culture medium contains peptone, beef extract, yeast extract, dipotassium hydrogen phosphate, triammonium citrate, sodium acetate, glucose, Tween 80, magnesium sulfate, and manganese sulfate; preferably, the culture medium contains 5-15 g / L peptone, 2-10 g / L beef extract, 3-8 g / L yeast extract, 1-5 g / L dipotassium hydrogen phosphate, 1-5 g / L triammonium citrate, 3-8 g / L sodium acetate, 1-8 g / L glucose, 0.5-2 mL / L Tween 80, 0.1-0.5 g / L magnesium sulfate, and 0.01-0.2 g / L manganese sulfate.

4. A formulation containing Lactobacillus fermentum with accession number CGMCC No. 33042; preferably, the formulation is a microbial formulation.

5. A fermentation feedstock containing *Lactobacillus fermentatus* with accession number CGMCC No. 33042; preferably, the fermentation feedstock further contains meal and enzymes; Preferably, the meal is a byproduct of oilseed or grain processing, and is preferably selected from one or more of soybean meal, rapeseed meal, cottonseed meal, peanut meal, sunflower meal, sesame meal, flaxseed meal, rice bran meal, tea meal, flaxseed meal, safflower meal, palm kernel meal, coconut meal, chili meal, olive meal, corn distillers' grains meal, and beet meal. More preferably, it is selected from one or more of soybean meal, rice bran meal, wheat bran meal, and palm meal. Preferably, the enzyme is selected from one or more of proteases, pectinases, xylanases, α-galactosidases, and cellulases; more preferably, the enzyme is a protease, such as one or more of alkaline proteases, neutral proteases, and acidic proteases. Preferably, the total amount of enzyme added is more than 100U per gram of meal, such as 100-1000U, 500-2000U or 100-500U.

6. A fermentation composition, characterized in that, The fermentation composition is obtained by fermentation of the fermentation raw material according to claim 5; preferably, the fermentation composition includes fermented meal; Preferably, the fermentation temperature is 37-50℃, for example 37-45℃ or 40-50℃. Preferably, the fermentation time is 24-96 hours, for example, 24-72 hours or 48-72 hours. Preferably, after fermentation, the process further includes a drying and pulverizing step. Preferably, the drying temperature is 60-70℃ or 65-80℃. Preferably, the drying time is 5-10 hours or 6-12 hours. Preferably, the fermentation composition is pulverized to a mesh size of 40 or larger, for example, 60 mesh or 80 mesh.

7. A feed containing the fermentation composition of claim 6; preferably, the fermentation composition is fermented meal; preferably, the content of the fermentation composition in the feed is 5-10%, 5-15%, or 8-10%; preferably, the feed is aquatic animal feed.

8. A method for improving the growth performance of aquatic animals and / or improving the quality of aquatic animal products, the method comprising feeding the aquatic animals with the feed as described in claim 7.

9. The use of Lactobacillus fermentum with accession number CGMCC No. 33042, the fermentation raw material as described in claim 5, the fermentation composition as described in claim 6, or the feed as described in claim 7 in the preparation of animal feed.

10. The application as described in claim 9, characterized in that, The animal feed is aquatic animal feed. Preferably, the application includes improving the growth performance of aquatic animals and / or improving the appearance of aquatic animal products, and / or improving the appearance and taste of aquatic animal products, and / or improving the flavor of aquatic animal products, and / or improving the nutritional content of aquatic animal products, and / or improving the appearance of aquatic animal products after refrigeration, and / or improving the taste of aquatic animal products after refrigeration. Preferably, the aquatic animal products include cooked aquatic animal products, raw aquatic animal products, pickled aquatic animal products, salted aquatic animal products, dried aquatic animal products, surimi products, aquatic seasonings, and aquatic animal fats and their products.

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