Liver health feed for cultured fishes and preparation method of cape jasmine and fermented soybean soup solid fermentation product

Abstract

The present invention belongs to the field of aquaculture feeds, and particularly discloses a cultured fish liver health feed and a preparation method of a cape jasmine and fermented soybean soup solid fermentation product, the feed comprises 90-95 wt% of a basal feed and 5-10 wt% of the cape jasmine and fermented soybean soup solid fermentation product, and the chemical composition comprises 45-50 wt% of crude protein, 8-10 wt% of crude fat, 5-10 wt% of soybean meal, and 5-10 wt% of soybean meal. The cape jasmine and fermented soybean soup solid fermentation product is obtained by taking cape jasmine decoction and a plant protein source as matrixes, inoculating actinomucor elegans and fermenting at room temperature. The cape jasmine and fermented soybean soup solid leavening is added into the basal feed, so that the utilization efficiency of the feed can be improved; in addition, by regulating and controlling related enzyme activity and gene expression, the anti-oxidation and anti-inflammatory capacity of the liver can be enhanced, oxidative damage and inflammatory response caused by high-dose GD are effectively relieved, and the liver health of the micropterus salmoides is protected.

Description

Technical Field

[0001] The invention belongs to the field of aquaculture feed, and particularly relates to a preparation method of a healthy liver feed for cultured fish and a solid fermentation product of gardenia and fermented black beans soup. Background Art

[0002] Largemouth bass (Micropterus salmoides) belongs to the order Perciformes, family Heliopsidae, and genus Micropterus. Since its introduction to my country in the 1980s, it has gradually developed into a major freshwater aquaculture species due to its rapid growth and strong adaptability. However, in recent years, a series of problems have become increasingly prominent, such as excessive stocking density, overfeeding, deteriorating water quality, and inadequate disease control, which have seriously hampered the healthy development of the largemouth bass aquaculture industry. The application of functional feed additives provides an effective strategy to address these common problems, therefore, in-depth research on functional feed additives is particularly necessary.

[0003] Gardenia jasminoides Ellis (GD), a plant of the Rubiaceae family, is widely cultivated in various regions of China. As one of the first medicinal and edible substances recorded in the Pharmacopoeia of the People's Republic of China, gardenia plays a crucial role in both culinary and health-care settings. GD is rich in a variety of functional components, including geniposide, genipin, crocin, volatile oils, terpenes, and organic acids. These functional components participate in various physiological processes in the body, endowing GD with biological activities such as antioxidant, anti-inflammatory, hypoglycemic, antidepressant, and sleep-improving properties. Gardenia can be used as a feed additive, positively impacting the growth and health of farmed animals. Studies have shown that adding 300 mL / kg of GD extract to feed improves the growth performance and yellowness of large yellow croaker (Pseudosciaena crocea) and increases the nutritional value of muscle. Adding 100 mg / kg of GD to the diet of Xianghuang chickens improves egg quality and enhances immune function. Furthermore, in a study of fattening Xiangcun pigs, the addition of 50g / kg and 100g / kg of GD to feed improved their antioxidant capacity without negatively impacting growth performance or meat quality. However, the practical application of GD as an aquaculture feed additive still has certain limitations. GD contains natural compounds such as gardenia yellow, which pose a toxicity risk at certain dosages. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the purpose of the present invention is to provide a feed for the healthy liver of farmed fish and a method for preparing a solid fermentation product of Gardenia and Fermented Soya Bean Soup, which can enhance the antioxidant and anti-inflammatory ability of the liver of farmed fish and protect the liver health.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The invention provides a feed for healthy livers of farmed fish, which comprises 90-95 wt% of a basic feed and 5-10 wt% of a solid fermentation product of a gardenia and fermented black beans soup. The chemical composition of the feed comprises 45-50 wt% of crude protein and 8-10 wt% of crude fat. The solid fermentation product of the gardenia and fermented black beans soup is obtained by inoculating Actinobacillus elegans with a decoction of gardenia and a plant protein source as a matrix and fermenting at room temperature.

[0007] Preferably, the mass ratio of the gardenia to the plant protein source is 3:10.

[0008] Preferably, the Actinomucor elegans was deposited in the China Center for Type Culture Collection on March 27, 2025, with a deposit number of CCTCC NO: M 2025595 and a Latin name of Actinomucor elegans FPE.

[0009] Preferably, the plant protein source is selected from one of soybean meal, rapeseed meal or cottonseed protein concentrate.

[0010] Preferably, the inoculation concentration of Actinomucor elegans is 1×10 7 cfu / g.

[0011] Preferably, the basic feed is composed of the following raw materials in parts by mass: 30-35 parts of fish meal, 10-12 parts of chicken meal, 5-6 parts of rapeseed meal, 10-12 parts of cottonseed protein concentrate, 4-6 parts of gluten, 10-12 parts of cassava starch, 4-6 parts of fish oil, 4-5 parts of cellulose, 1.5-2.5 parts of multivitamin and multimineral premix, 1-2 parts of calcium dihydrogen phosphate, 0.3-0.5 parts of choline chloride, 0.3-0.5 parts of lysine, and 0.1-0.3 parts of methionine.

[0012] Preferably, the multi-dimensional multi-mineral premix is composed of the following raw materials in parts by mass: 200-210 parts of magnesium sulfate heptahydrate, 195-200 parts of iron sulfate heptahydrate, 50-55 parts of manganese sulfate heptahydrate, 0.05-0.1 parts of potassium iodide, 95-100 parts of copper sulfate pentahydrate, 440-450 parts of zinc sulfate heptahydrate, 0.5-0.8 parts of sodium selenite, 1-2 parts of cobalt sulfate heptahydrate, 1700-2000 parts of vitamin A, 3300-3500 parts of vitamin D, 25-30 parts of vitamin E, 2-3 parts of vitamin K3, 5-8 parts of vitamin B1, 5-8 parts of vitamin B2, 5-8 parts of vitamin B6, 0.02-0.08 parts of vitamin B12, and 0.1-0.2 parts of vitamin C. 65-72 parts of calcium pantothenate 12-18 parts of niacinamide 42-48 parts of folic acid 1-2 parts of d-biotin 0.03-0.08 parts of inositol 35-42 parts of.

[0013] Preferably, the farmed fish is largemouth bass.

[0014] The preparation method of the solid fermentation product of Gardenia and Fermented Soya Bean Soup provided by the present invention is as follows:

[0015] Grind the Gardenia into powder, add 10 times the weight of water and boil for 1 hour, filter, add 10 times the weight of water to the residue and boil for 1 hour again, filter, combine the filtrates of the two decoctions to obtain the Gardenia decoction;

[0016] The decoction of Gardenia jasminoides is mixed with the plant protein source and stirred evenly to obtain a fermentation matrix;

[0017] Inoculate Actinomucor elegans into the fermentation substrate and ferment for 12 to 60 hours in a light-proof and ventilated room temperature environment.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. The present invention adds the solid fermentation product of Gardenia and Fermented Soya Bean Soup to the basic feed, which can improve feed utilization efficiency; and can enhance the liver's antioxidant and anti-inflammatory capabilities by regulating related enzyme activities and gene expression, effectively alleviating the oxidative damage and inflammatory response caused by high-dose GD, and playing a protective role on the liver health of largemouth seabass.

[0020] 2. The addition of GSPD to the feed of the present invention can improve feed utilization efficiency. Regarding liver health, the addition of 10g / kg of GD to feed can increase oxidative stress and inflammation levels in the liver of largemouth bass, causing substantial damage. However, the addition of GSPD to feed can enhance the liver's antioxidant and anti-inflammatory capacity by regulating related enzyme activities and gene expression, effectively alleviating the oxidative damage and inflammatory response caused by high-dose GD, and thus protecting the liver health of largemouth bass.

[0021] 3. Adding 10 g / kg of GD to the feed of the present invention will disrupt the inflammatory balance of the liver of largemouth bass; while adding GSPD to the feed can alleviate liver inflammation, which has a positive significance for maintaining the liver's anti-inflammatory ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 These are the plasma biochemical indicators of the largemouth bass in each group according to the embodiments of the present invention;

[0023] Figure 2 The liver morphology and tissue structure of each group of largemouth bass in the examples of the present invention;

[0024] Figure 3 The antioxidant enzyme activities in the liver of the largemouth bass in each group of the examples of the present invention;

[0025] Figure 4 is the relative expression level of antioxidant-related genes in the liver of each group of largemouth bass in the examples of the present invention;

[0026] Figure 5The relative expression levels of liver inflammation-related genes in each group of largemouth bass in the examples of the present invention. DETAILED DESCRIPTION

[0027] In order to enable those skilled in the art to more clearly understand the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are further described in detail below in conjunction with the accompanying drawings and preferred embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the invention. In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products familiar to those skilled in the art; unless specifically specified, the technical means used are conventional means familiar to those skilled in the art.

[0028] The solid fermentation product of Gardenia and Fermented Soya Bean Soup in the embodiment of the present invention is prepared by the following method:

[0029] Strains: The experimental strains were isolated from naturally fermented fermented black beans and cultured on potato dextrose agar (PDA) and potato dextrose broth (PDB). A strain with strong β-glucosidase production was screened and identified as Actinomucor elegans. It was deposited with the China Center for Type Culture Collection on March 27, 2025, with the deposit number CCTCCNO: M 2025595 and the taxonomic name Actinomucor elegans FPE. The deposit address is: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

[0030] Gardenia jasminoides (GD): provided by Fuding Gardenia Association.

[0031] Soybean meal: Fengyuan brand soybean meal produced by Yihai (Lianyungang) Grain and Oil Industry Co., Ltd.

[0032] Preparation process:

[0033] Prepare GD and soybean meal at a mass ratio of 3:10 in advance. Grind gardenia into powder, add 10 times the mass of water, and boil for 1 hour. Filter. Add 10 times the mass of water to the residue and boil for 1 hour again. Filter. Combine the filtrates from the two decoctions to obtain a gardenia decoction. Mix the gardenia decoction with soybean meal and stir evenly to obtain a fermentation medium. Inoculate the fermentation medium with a concentration of 1×10 7 cfu / g of the strain, and fermented it in a dark, ventilated room temperature environment for 48 h to obtain Gardenia jasminoides fermented soybean meal (GSPD).

[0034] The present invention designs three types of isonitrogenous and isolipid feeds: a group using soybean meal as raw material and without the addition of GD is set as the control group (CON); a group using soybean meal as raw material and adding 10g / kg GD is set as the gardenia-added group (GD); and a group using the same dose of GD added in the form of GSPD is set as the gardenia-fermented and fermented black bean soup simulation group (GSPD). In terms of the selection of feed raw materials, the main protein sources are fish meal, chicken meal, soybean meal and cottonseed protein concentrate, and the main fat source is fish oil. Methionine and lysine are additionally added to make their content meet the nutritional requirements of largemouth bass. The specific formula is shown in Table 1. In the feed preparation stage, the various raw materials are first crushed and the crushed raw materials are sieved through an 80-mesh sieve; then, the sieved raw materials are mixed with an appropriate amount of water and processed using a twin-screw extruder to produce an extruded feed with a particle size of about 3mm; the prepared feed is dried at 65°C to a moisture content of about 5%, and finally stored in a refrigerator at 4°C for future use.

[0035] Table 1 Experimental feed formula and chemical composition

[0036]

[0037]

[0038] Among them, the multivitamin premix is a commercially available product and is composed of the following raw materials in parts by mass: 205.39 parts of magnesium sulfate heptahydrate, 198.57 parts of iron sulfate heptahydrate, 50.36 parts of manganese sulfate heptahydrate, 0.07 parts of potassium iodide, 98.30 parts of copper sulfate pentahydrate, 442.46 parts of zinc sulfate heptahydrate, 0.66 parts of sodium selenite, 1.67 parts of cobalt sulfate heptahydrate, 1800 parts of vitamin A, 390 parts of vitamin D3, 30 parts of vitamin E, 2.8 parts of vitamin K3, 6 parts of vitamin B1, 6 parts of vitamin B2, 6 parts of vitamin B6, 0.04 part of vitamin B12, 70 parts of vitamin C, 15 parts of calcium pantothenate, 45 parts of niacinamide, 2 parts of folic acid, 0.05 part of d-biotin, and 40 parts of inositol.

[0039] The largemouth sea bass used in the experiments in the embodiments of the present invention were purchased from Hubei Zhenghao Aquaculture Company (Hubei, China). Before the formal experiment was carried out, the largemouth sea bass were fed and acclimated for a week using a control feed. 24 hours before the start of the formal experiment, the feeding of the largemouth sea bass was stopped. Subsequently, experimental fish with relatively consistent individual size and a body weight of (38.19±0.18) g were selected for a 7-week breeding experiment. At the beginning of the experiment, experimental fish with similar initial body weight were randomly assigned to 9 breeding tanks, and 15 experimental fish were placed in each breeding tank. The experiment set up 3 treatment groups, and 3 parallels were set up in each treatment group. During the experiment, the water temperature of the breeding water was maintained at 24.5-27°C, the dissolved oxygen level was maintained above 5.0 mg / L, and the pH value was controlled at 6.0-7.5.

[0040] After the experiment, all experimental fish were removed from each tank, anesthetized with MS-222 (Sigma-Aldrich), and weighed in batches. Three fish were randomly selected from each tank and stored in a −20°C freezer for subsequent body composition analysis. Next, two fish were randomly selected from each tank, and their body lengths were accurately measured. After these measurements, the fish were dissected, and the visceral masses and liver were carefully weighed. The ratio of each organ weight to body weight was then calculated. Additionally, three fish were randomly selected from each tank. After anesthesia, blood was collected from the caudal vein using a 2 mL syringe pre-soaked with sodium heparin anticoagulant. The blood was then transferred to a 1.5 mL centrifuge tube pre-soaked with anticoagulant. The tubes containing the blood were then centrifuged at 3500 rpm for 10 minutes to separate the plasma. At the same time, liver tissue samples were placed in 1.5 mL centrifuge tubes containing RNA preservation solution (R0118, Beyotime, China) and stored at -80°C for subsequent analysis. Additionally, a portion of liver tissue was fixed in 4% paraformaldehyde for subsequent histological analysis.

[0041] The data is processed as follows:

[0042] Weight gain rate (%) = (final weight - initial weight) / initial weight × 100;

[0043] Feed coefficient = dry matter intake / weight gain;

[0044] Protein efficiency = body weight gain / protein intake;

[0045] Survival rate (%) = final number of fish / initial number of fish × 100;

[0046] Liver to body ratio (%) = liver weight / body weight × 100;

[0047] Data were analyzed for normality and homogeneity of variance using IBM SPSS 23.0 statistical software, followed by one-way analysis of variance and Duncan's multiple comparisons. P < 0.05 indicated a significant difference between treatment groups. All data are presented as means ± standard error (SEM).

[0048] The growth performance and feed utilization of largemouth bass are shown in Table 2. Compared with the CON group, the addition of GD or GSPD to the diet had no significant effect on FBW, WGR, PER, and SR of largemouth bass (P>0.05). However, the FCR of the GSPD group was significantly reduced compared with the CON group (P<0.05).

[0049] Table 2 Growth performance and feed utilization of largemouth bass

[0050]

[0051]

[0052] The body composition of largemouth black bass in each treatment group was measured, and the results are shown in Table 3. The crude protein and crude fat content in the GSPD group were slightly higher than in the other two groups, but the differences were not significant (P>0.05). There were no significant differences in body moisture and ash content among the groups (P>0.05).

[0053] Table 3 Body composition of largemouth black bass

[0054]

[0055] To evaluate the effects of dietary GD and GSPD on liver health of largemouth bass, relevant plasma biochemical indices were measured. Figure 1 As shown in the figure, AST, ALT, and ALP levels were significantly increased in the GD group compared with the CON group (P < 0.05). AST and ALP levels were significantly decreased in the GSPD group compared with the GD group (P < 0.05). There were no significant differences in AST, ALT, and ALP levels between the GSPD group and the CON group (P > 0.05). There were no significant differences in TC, TG, Glu, ALB, and TP between the groups (P > 0.05).

[0056] To further explore the changes in the liver of largemouth bass in different treatment groups, liver morphology and histology were evaluated. Figure 2 shown. Figure 2 Figure (A) shows the liver-to-body ratio of each treatment group, (B) shows the liver morphology of each treatment group, and (C) shows H&E-stained sections of each treatment group. Red arrows indicate cell swelling, black arrows indicate loss of cell boundaries, and blue arrows indicate nuclear atrophy. The liver-to-body ratio of the GD group, supplemented with 10 g / kg GD, was significantly increased compared to the CON and GSPD groups (P < 0.05). This result suggests that supplementing the diet with 10 g / kg GD causes liver enlargement and increased mass in largemouth bass, while GSPD mitigates this abnormal increase in liver-to-body ratio. Similar changes can be observed in the morphology and histology. Compared to the CON group, the liver volume of the GD group was significantly increased, and the liver cells showed abnormalities such as swelling, blurred cell boundaries, and nuclear atrophy. In contrast, the liver-to-body ratio of the GSPD group was not significantly different from that of the CON group (P > 0.05). The livers appeared plump and succulent, and the morphology and structure of the liver cells were intact. These results further indicate that the addition of GD to the diet can damage the liver tissue morphology of largemouth bass, while the addition of GSPD has no adverse effect on the liver tissue morphology of largemouth bass.

[0057] In order to further explore the intrinsic mechanism of the effects of GD and GSPD added to feed on the liver of largemouth bass, the activities of liver antioxidant-related enzymes were tested. The results are as follows: Figure 3 As shown. Regarding liver antioxidant enzyme activity, CAT activity and T-AOC in the GD group were significantly lower than those in the CON and GSPD groups (P < 0.05); SOD activity was also significantly lower in the GD group than in the GSPD group (P < 0.05). In contrast, while no significant differences were observed in the various antioxidant enzyme activity indicators in the GSPD group compared with the CON group (P > 0.05), all indicators were slightly higher. Furthermore, there was no significant difference in GSH-Px activity between the groups (P > 0.05). Regarding liver oxidative damage-related products, MDA and ROS levels were significantly higher in the GD group than in the CON and GSPD groups (P < 0.05); however, there were no significant differences in these two indicators between the GSPD group and the CON group (P > 0.05). Comprehensive enzyme activity test results indicate that the addition of 10 g / kg GD to the diet causes oxidative damage to the liver of largemouth bass; however, GD added in the form of GSPD can effectively reduce the harmful effects of GD alone on liver antioxidant enzyme systems.

[0058] The relative expression of liver antioxidant-related gene mRNA Figure 4 As shown. Compared with the CON and GSPD groups, the relative expression of the keap1 gene was significantly increased in the GD group, while the relative expression of the nqo-1 gene was significantly decreased (P < 0.05). Compared with the GSPD group, the relative expression of the nrf2, ho-1, sod, and gpx genes was significantly decreased in the GD group (P < 0.05). Furthermore, the relative expression of the keap1 gene was significantly decreased in the GSPD group compared with the CON group (P < 0.05). Consistent with the enzyme activity test results, the relative gene expression data indicate that the addition of 10 g / kg GD to the diet can cause oxidative damage to the liver by regulating the expression of liver antioxidant-related genes. Conversely, the addition of GSPD to the diet can alleviate the damage to antioxidant genes caused by the addition of 10 g / kg GD, thereby enhancing the antioxidant capacity of largemouth bass.

[0059] In order to explore the effects of adding GD and GSPD to the diet on liver inflammation of largemouth bass, the related inflammatory genes were detected. Figure 5As shown. Compared with the CON and GSPD groups, the relative gene expression levels of the pro-inflammatory factors nf-kb and il-β in the GD group were significantly increased (P<0.05), and the relative gene expression level of the anti-inflammatory factor il-10 was significantly decreased (P<0.05). In addition, compared with the GD group, the relative gene expression level of the pro-inflammatory factor il-6 in the GSPD group was significantly decreased (P<0.05). Compared with the CON group, the relative expression levels of all genes in the GSPD group were significantly decreased (P<0.05). Overall, the addition of 10g / kg GD to the feed disrupted the inflammatory balance of the liver of largemouth bass; while the addition of GSPD to the feed could reduce liver inflammation, which has a positive significance for maintaining the liver's anti-inflammatory ability.

[0060] These results indicate that the addition of GSPD to feed can improve feed utilization efficiency. Regarding liver health, the addition of 10g / kg of GD to feed increased oxidative stress and inflammation in the liver of largemouth bass, causing substantial damage. However, the addition of GSPD to feed can enhance the liver's antioxidant and anti-inflammatory capacity by regulating relevant enzyme activities and gene expression, effectively alleviating the oxidative damage and inflammatory response caused by high-dose GD, and thus protecting the liver health of largemouth bass.

[0061] It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A feed for healthy liver of farmed fish, characterized in that: The feed comprises 90-95 wt% of basic feed and 5-10 wt% of solid fermentation product of gardenia and fermented black beans soup. Its chemical composition contains 45-50 wt% of crude protein and 8-10 wt% of crude fat. The solid fermentation product of gardenia and fermented black beans soup is obtained by inoculating Actinobacillus elegans with gardenia decoction and a plant protein source as a matrix and fermenting at room temperature.

2. The farmed fish liver health feed according to claim 1, characterized in that: The mass ratio of the gardenia to the plant protein source is 3:

10.

3. The farmed fish liver health feed according to claim 2, characterized in that: The Actinomucor elegans was deposited in the China Center for Type Culture Collection on March 27, 2025, with the deposit number CCTCC NO: M 2025595 and the Latin name Actinomucor elegans FPE.

4. The farmed fish liver health feed according to claim 3, characterized in that: The plant protein source is selected from one of soybean meal, rapeseed meal or cottonseed protein concentrate.

5. The farmed fish liver health feed according to claim 4, characterized in that: The inoculation concentration of Actinomucor elegans was 1×10 7 cfu / g.

6. The farmed fish liver health feed according to any one of claims 1 to 5, characterized in that: The basic feed is composed of the following raw materials in parts by mass: 30-35 parts of fish meal, 10-12 parts of chicken meal, 5-6 parts of rapeseed meal, 10-12 parts of cottonseed protein concentrate, 4-6 parts of gluten, 10-12 parts of cassava starch, 4-6 parts of fish oil, 4-5 parts of cellulose, 1.5-2.5 parts of multivitamin and multimineral premix, 1-2 parts of monocalcium phosphate, 0.3-0.5 parts of choline chloride, 0.3-0.5 parts of lysine, and 0.1-0.3 parts of methionine.

7. The farmed fish liver health feed according to claim 6, characterized in that: The multi-dimensional multi-mineral premix is composed of the following raw materials in parts by mass: 200-210 parts of magnesium sulfate heptahydrate, 195-200 parts of iron sulfate heptahydrate, 50-55 parts of manganese sulfate heptahydrate, 0.05-0.1 parts of potassium iodide, 95-100 parts of copper sulfate pentahydrate, 440-450 parts of zinc sulfate heptahydrate, 0.5-0.8 parts of sodium selenite, 1-2 parts of cobalt sulfate heptahydrate, 1700-2000 parts of vitamin A, 3300-3500 parts of vitamin D, 25-30 parts of vitamin E, 2-3 parts of vitamin K3, 5-8 parts of vitamin B1, 5-8 parts of vitamin B2, 5-8 parts of vitamin B6, 0.02-0.08 parts of vitamin B12, and 0.1-0.2 parts of vitamin C. 65-72 parts of calcium pantothenate 12-18 parts of niacinamide 42-48 parts of folic acid 1-2 parts of d-biotin 0.03-0.08 parts of inositol 35-42 parts of.

8. The farmed fish liver health feed according to any one of claims 1 to 5, characterized in that: The cultured fish is largemouth bass.

9. A method for preparing a solid fermentation product of Gardenia and Fermented Soya Bean Soup, characterized in that: include: Grind the Gardenia into powder, add 10 times the weight of water and boil for 1 hour, filter, add 10 times the weight of water to the residue and boil for 1 hour again, filter, combine the filtrates of the two decoctions to obtain the Gardenia decoction; The decoction of Gardenia jasminoides is mixed with the plant protein source and stirred evenly to obtain a fermentation matrix; Inoculate Actinomucor elegans into the fermentation substrate and ferment for 12 to 60 hours in a light-proof and ventilated room temperature environment.

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