Actinomucor elegans and application of actinomucor elegans in solid fermentation based on cape jasmine and fermented soybean soup

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses actinomucor elegans which is preserved in the China Center for Type Culture Collection on March 27, 2025, the preservation number is CCTCC NO: M2025595, the classification name is actinomucor elegans FPE, the preservation address is No. 299 of eight road, Wuchang District, Wuhan, Hubei Province, and the postcode is 430072. Based on the cape jasmine and fermented soybean soup, the actinomucor elegans is used as a fermentation strain, a bidirectional solid fermentation technology is applied, cape jasmine and aquatic feed raw materials are fused, the amino acid composition of plant protein raw materials can be optimized, the crude protein content of the plant protein raw materials can be increased, and the nitrogen content can be increased; in the aspect of conversion of active ingredients of gardenia, the conversion efficiency can be improved, and the content of gardenia blue is promoted to be increased.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to Actinobacillus elegans and application thereof in solid fermentation based on Gardenia and Fermented Black Bean Soup. Background Art

[0002] Gardenia, the fruit of Gardenia jasminoides Ellis, a plant of the Rubiaceae family, is cultivated in many parts of my country. As a traditional Chinese herbal medicine with multiple biological activities, gardenia is widely used in modern pharmacology and has attracted much research attention. In actual clinical application, gardenia is often used in combination with other Chinese herbal medicines. This combination can not only reduce the adverse reactions that gardenia itself may cause, but also significantly enhance its medicinal efficacy. The Gardenia and Fermented Soya Bean Soup recorded in the "Treatise on Febrile Diseases" is a classic combination method. It has the effects of eliminating chest heat, treating irritability and internal heat, and improving sleep quality. Gardenia and Fermented Soya Bean Soup is made by decocting gardenia with light fermented black beans. The active ingredients contained in gardenia, such as geniposide and genipin, as well as the microorganisms and amino acids provided by light fermented black beans, are the key material basis for the function of this prescription. Among them, the microorganisms and amino acids in light fermented black beans not only participate in the transformation of the active ingredients of gardenia but also create the conditions for their binding reactions. Modern pharmacological and pharmacokinetic studies have shown that geniposide, the main compound in gardenia, is converted to genipin by β-glucosidase, a enzyme secreted by microorganisms in fermented black beans. However, genipin is chemically unstable and further combines with amino acids in fermented black beans to form complexes such as gardenia blue. Studies have confirmed that these complexes are more stable and have a wider range of applications than geniposide and genipin.

[0003] However, the traditional decoction of Gardenia and Fermented Soya Beans has certain limitations during the actual decoction process. Because the high temperature environment during decoction is not the optimal condition for microorganisms to exert β-glucosidase activity, some microorganisms may even die due to the high temperature, which significantly affects the subsequent conversion efficiency of Gardenia jasminoides and reduces the efficacy of the prescription. Bidirectional solid-state fermentation technology provides a new approach to solving this problem. This technology combines a medicinal substrate with a microorganism for solid-state fermentation. During this process, the medicinal substrate provides the necessary nutritional support for microbial growth, while the microorganism secretes specific enzymes to modify and transform the chemical components of the substrate. Currently, bidirectional solid-state fermentation technology has been successfully applied to the processing of various Chinese herbal medicines such as red yeast rice and mulberry leaves, and has achieved remarkable results. Based on this principle, bidirectional solid-state fermentation technology can be introduced to optimize and reorganize these components during the fermentation process. For example, CN110339251A discloses a method for preparing Gardenia and Fermented Soya Beans fermentation bacteria, which solves the problem of large-scale destruction of β-glucosidase and significant reduction in enzyme activity during the traditional decoction process. It also changes the dosage form of the drug and reduces the dosage.

[0004] As a fermented bean product, light fermented black beans play a crucial role in the efficacy of Gardenia and Fermented Black Bean Soup, thanks to the microorganisms within them and the nutrients derived from fermented beans. Essentially, light fermented black beans, as a plant-based nutrient source, can be composed of a substance rich in similar plant-based components with the addition of microorganisms. In the aquaculture feed industry, plant-based raw materials are plentiful and in high demand. If common aquaculture feed plant-based raw materials were used in the fermentation process of Gardenia and Fermented Black Bean Soup to replace light fermented black beans, they would possess specialized properties related to the active ingredients of the Chinese herbal medicine Gardenia jasminoides, not found in traditional raw materials. This would not only enhance the nutritional value of these raw materials but also promote their application in aquaculture. However, relevant research is currently lacking. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of the present invention is to provide an Actinomucor elegantis and its application in the solid fermentation of Gardenia and Fermented Black Bean Soup, and to use it to construct a two-way solid fermentation system of Gardenia and Fermented Black Bean Soup in which the plant-based raw materials of aquatic feed replace light fermented black beans, so as to improve the conversion efficiency of the active ingredients of Gardenia and the value of the plant-based raw materials of aquatic feed.

[0006] The present invention provides an Actinomucorelegans elegant actinopterygium, which has been deposited in the China Center for Type Culture Collection on March 27, 2025, with a deposit number of CCTCC NO: M 2025595, a classification name of Actinomucorelegans elegant actinopterygium (Actinomucorelegans FPE), and a deposit address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

[0007] The present invention also provides the application of the Actinomucor elegantis in solid fermentation based on Gardenia and Fermented Soya Bean Soup.

[0008] Specifically, the application is: using Gardenia jasminoides decoction and a plant protein source as a matrix, inoculating the Actinomucor elegans, and fermenting in a light-proof and ventilated room temperature environment.

[0009] Specifically, the gardenia decoction is obtained by crushing the gardenia into powder, adding 10 times the mass of water, decocting twice, each time for 1 hour, filtering, and combining the filtrates.

[0010] Preferably, the mass ratio of the gardenia to the plant protein source is 3:10.

[0011] Preferably, the plant protein source is selected from one of soybean meal, rapeseed meal or cottonseed protein concentrate.

[0012] Preferably, the inoculation concentration of Actinomucor elegans is 1×10 7 cfu / g.

[0013] Preferably, the fermentation time is 12 to 60 hours.

[0014] The present invention also provides a solid fermentation mixture based on Gardenia and Fermented Soya Bean Soup, the preparation method of which 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] Preferably, the mass ratio of the gardenia to the plant protein source is 3:10.

[0019] Preferably, the inoculation concentration of Actinomucor elegans is 1×10 7 cfu / g.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The Actinomucor elegans provided by the present invention has a strong ability to produce β-glucosidase. Based on Gardenia and Fermented Black Bean Soup, the present invention uses Actinomucor elegans as the fermentation strain and utilizes bidirectional solid-state fermentation technology to combine Gardenia with aquatic feed ingredients. This method optimizes the amino acid composition of the plant protein raw material, increasing its crude protein content and nitrogen content. Furthermore, it improves the conversion efficiency of the active ingredients in Gardenia, promoting an increase in the content of Gardenia Blue. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The colony map of Actinomucor elegantis provided in Example 1;

[0023] Figure 2 This is a comparison chart of the DNA test of Actinomucor elegans provided in Example 1;

[0024] Figure 3 The apparent state changes of each group of fermentation mixtures in Example 2 during the fermentation process;

[0025] Figure 4 The results of the changes in the content of geniposide in the fermentation mixtures of each group in Example 2 are shown;

[0026] Figure 5 The results of the changes in the genipin content in each group of fermentation mixtures in Example 2 are as follows;

[0027] Figure 6 The results of the change of the blue valence of Gardenia jasminoides in each group of fermentation mixtures in Example 2 are shown;

[0028] Figure 7 The results of the changes in crude protein content in each group of fermentation mixtures in Example 3 are as follows;

[0029] Figure 8 The results of the changes in nitrogen content in each group of fermentation mixtures in Example 3 are as follows;

[0030] Figure 9 The results show the change of the blue valence of Gardenia jasminoides in each group of fermentation mixtures in Example 3. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1

[0033] The Actinomucor elegantis provided in this embodiment was deposited in the China Center for Type Culture Collection on March 27, 2025, with a deposit number of CCTCC NO: M 2025595, a classification name of Actinomucorelegans elegantis (Actinomucorelegans FPE), and a deposit address of 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

[0034] Source: Isolated from homemade naturally fermented tempeh.

[0035] Isolation: Crush 1 gram of fermented fermented black beans with a sterile pestle, add it to a conical flask containing 50 mL of sterile saline, and shake it to fully disperse it to make a bacterial suspension; take 1 mL of the bacterial suspension and add it to 9 mL of sterile saline, and perform a 10-fold gradient dilution; take 0.1 mL of the diluted bacterial suspension and spread it on a potato dextrose agar (PDA) medium plate, place the plate in a constant temperature incubator at 28°C in the dark and incubate for 48 hours, pick single colonies of different morphologies, and separate them by the plate streak method until the colonies are purified to obtain the isolated and purified strain.

[0036] Culture: The isolated and purified strain was inoculated into potato dextrose broth (PDB) medium after autoclaving (115°C, 20 min) for culture.

[0037] The morphology of the strains Figure 1 As shown. After DNA extraction, sequencing, NCBI alignment results are shown Figure 2 Based on the results of morphological and molecular biological identification, it was determined to be Actinomucor elegans.

[0038] Example 2

[0039] This example compares the fermentation activities of several strains.

[0040] The strains were all isolated from naturally fermented fermented black beans, and a total of 8 strains were selected: strain 1 was Mucor macrocor, strain 2 was Mucor racemosus, strain 3 was Aspergillus QHL15, strain 4 was Actinomucor elegante of Example 1, strain 5 was Aspergillus oryzae, strain 6 was Aspergillus oryzae and Aspergillus QHL15, strain 7 was Aspergillus oryzae, and strain 8 was Aspergillus oryzae.

[0041] The fermentation matrix is ​​soybean meal plus Gardenia jasminoides decoction, with the mass ratio of Gardenia jasminoides to soybean meal being 3:10. The preparation method of the fermentation matrix is ​​as follows:

[0042] The gardenia is crushed into powder, decocted with 10 times the weight of water for 1 hour, filtered, and the residue is again decocted with 10 times the weight of water for 1 hour, filtered, and the filtrates from the two decoctions are combined to obtain a gardenia decoction; the gardenia decoction is mixed with soybean meal and stirred evenly to obtain a fermentation substrate.

[0043] The fermentation matrix was inoculated with a concentration of 1×10 7 cfu / g of 8 strains were placed in a light-proof and ventilated room temperature environment for fermentation.

[0044] Sampling was carried out every 12 hours, and high performance liquid chromatography (HPLC) was used to detect the changes in geniposide and genipin, the main components of Gardenia jasminoides.

[0045] Sample pretreatment: The fermentation mixture was dried and crushed into powder. 3.0 g of each sample was weighed and placed in a stoppered conical flask. 50 mL of 50% methanol by volume was added and the weight was recorded. The solid was dissolved by ultrasonication for 30 min and the sample was weighed again after cooling. The lost weight was made up with 50% methanol by volume. The sample was centrifuged at 4000 r / min for 15 min, 10 mL of the supernatant was taken, and the volume was made up to 50 mL with 50% methanol by volume. The sample was shaken and filtered through a 0.45 μm filter membrane for later use.

[0046] Preparation of reference solutions: Gardeniaside and genipin reference substances were purchased from Zhumo Technology Co., Ltd. (Chengdu, China). They were each prepared into a 400 μg / mL reference solution in 50% by volume methanol. Subsequent gradient dilutions were also made using a 50% by volume methanol solution and filtered through a 0.45 μm filter.

[0047] HPLC operating conditions: the chromatographic column is a Symmetry C18 column (4.6mm*150mm, 5μm); the mobile phase A is methanol, and the mobile phase B is 0.5% glacial acetic acid; the flow rate is 1mL / min, the detection wavelength is 254nm, the injection volume is 10μL, and the column temperature is 40℃; the gradient elution program setting is based on mobile phase A: 0-2min, 10%; 3-8min, 22%; 9-13min, 30%; 14-40min, 60%; 41-42min, 30%; 43-50min, 10%.

[0048] Figure 3 Figure 2 shows the changes in the apparent state of each fermentation mixture during the fermentation process. It can be seen that the color and moisture content of the fermentations of the eight strains changed significantly during the fermentation process, but the degree of change was different.

[0049] Figure 4 、 Figure 5 、 Figure 6 The results of the changes in the content of geniposide, genipin and gardenia blue value in the fermentation mixture of each group are shown. It can be seen that the conversion rate of geniposide and genipin in strain 4 is the fastest. After 48 hours of fermentation, the content of geniposide and genipin both dropped to 0, and the gardenia blue value was the highest.

[0050] Example 3

[0051] This example studies the results of mixed fermentation of three plant protein sources (soybean meal, rapeseed meal, cottonseed protein concentrate) and Gardenia jasminoides by Actinobacillus elegans.

[0052] The mass ratio of gardenia to plant protein source is 3:10. The preparation method of the fermentation matrix is ​​as follows:

[0053] The gardenia is crushed into powder, decocted with 10 times the weight of water for 1 hour, filtered, and the residue is again decocted with 10 times the weight of water for 1 hour, filtered, and the filtrates from the two decoctions are combined to obtain a gardenia decoction; the gardenia decoction is mixed with a plant protein source and stirred evenly to obtain a fermentation matrix.

[0054] The fermentation matrix was inoculated with a concentration of 1×10 7 cfu / g of Actinomucor elegans was placed in a dark, ventilated room temperature environment for fermentation. Samples were collected and tested every 12 hours.

[0055] Figure 7 The results of the change of crude protein content in the fermentation mixture show that the crude protein content in dry matter of the soybean meal group increased the most, followed by the cottonseed protein concentrate group, and the rapeseed meal group showed a downward trend.

[0056] Tables 1, 2, and 3 show the changes in hydrolyzed amino acid content in each group. It can be seen that the total amino acid content in the soybean meal group increased within 60 hours of fermentation, reaching its highest level at 24 hours. Asp, Ser, Glu, Ala, Ile, and Phe increased by 6%, 4.6%, 6%, 10.4%, 7.7%, and 22.11%, respectively. The total amino acid content in the rapeseed meal group reached its highest level at 24 hours. At 24 hours of fermentation, Met, Pro, and Asp increased by 185.92%, 87%, and 20%, respectively, while (Cys)2 decreased by 34%. The total amino acid content in the cottonseed protein concentrate group reached its highest level at 12 hours of fermentation. Asp, Glu, Phe, Arg, and Pro showed an increasing trend within the first 24 hours of fermentation, and then gradually decreased with the extension of fermentation time.

[0057] Table 1 Changes in hydrolyzed amino acid content in soybean meal group

[0058]

[0059] Table 2 Changes in hydrolyzed amino acid content in rapeseed meal group

[0060]

[0061] Table 3 Changes in hydrolyzed amino acid content of cottonseed protein concentrate

[0062]

[0063]

[0064] Figure 8 Figure 2 is the change of nitrogen content in the fermentation mixture. It can be seen that after 60 hours of fermentation, the nitrogen content of the soybean meal group, rapeseed meal group, and cottonseed protein concentrate group increased by 9.49%, 4.97%, and 8.68%, respectively.

[0065] Figure 9 Figure 3 is the change result of the Gardenia Blue value, a fermentation marker of Gardenia in the fermentation mixture. It can be seen that the Gardenia Blue value showed an overall upward trend in the soybean meal group, reaching the highest at 36 hours; the rapeseed meal group showed an overall trend of first increasing and then decreasing, reaching the highest at 12 hours; the cottonseed protein concentrate group showed an overall upward trend, reaching the highest at 48 hours.

[0066] 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. An Actinomucor elegantis, characterized in that The Actinomucor elegans was deposited in the China Center for Type Culture Collection on March 27, 2025, with the collection number CCTCC NO: M2025595, the Latin name Actinomucor elegansFPE, and the collection address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code: 430072.

2. An application of Actinomucor elegantis according to claim 1 in solid fermentation based on Gardenia and Fermented Soya Bean Soup, characterized in that: The application comprises the following steps: using Gardenia jasminoides decoction and a plant protein source as a matrix, inoculating the Actinomucor elegans, and fermenting in a light-proof and ventilated room temperature environment.

3. The use of Actinomucor elegantis in solid fermentation based on Gardenia and Fermented Soya Bean Soup according to claim 2, characterized in that: The gardenia decoction is prepared by crushing the gardenia into powder, adding 10 times the weight of water, decocting twice, each time for 1 hour, filtering, and combining the filtrates.

4. The use of Actinomucor elegantis in solid fermentation based on Gardenia and Fermented Soya Bean Soup according to claim 3, characterized in that: The mass ratio of the gardenia to the plant protein source is 3:

10.

5. The use of Actinomucor elegantis in solid fermentation based on Gardenia and Fermented Soya Bean Soup according to claim 4, characterized in that: The plant protein source is selected from one of soybean meal, rapeseed meal or cottonseed protein concentrate.

6. The use of Actinomucor elegantis in solid fermentation based on Gardenia and Fermented Soya Bean Soup according to any one of claims 2 to 4, characterized in that: The inoculation concentration of Actinomucor elegans was 1×10 7 cfu / g.

7. The use of Actinomucor elegantis in solid fermentation based on Gardenia and Fermented Soya Bean Soup according to claim 6, characterized in that: The fermentation time is 12 to 60 hours.

8. A solid fermentation mixture based on Gardenia and Fermented Soya Bean Soup, characterized in that: Its preparation method is: 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; The Actinomucor elegantis described in claim 1 is inoculated into a fermentation substrate, and fermented for 12 to 60 hours in a light-proof and ventilated room temperature environment to obtain the product.

9. The solid fermentation mixture based on Gardenia and Fermented Soya Bean Soup according to claim 8, characterized in that: The mass ratio of the gardenia to the plant protein source is 3:

10.

10. The solid fermentation mixture based on Gardenia and Fermented Soya Bean Soup according to claim 9, characterized in that: The inoculation concentration of Actinomucor elegans was 1×10 7 cfu / g.

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