Licorice feed fermentation agent and application thereof

Abstract

The application discloses a liquorice feed fermentation agent and application thereof. It is found that Inonotus obliquus XJ-1 has the ability to produce cellulase, can fully decompose the fiber components in liquorice residues which are not easily digested by animals, and release part of the flavonoids which are not easily utilized and embedded in the residue cells, so that the bioactivity in the liquorice residues can be better exerted. After fermentation, the liquorice residue fermentation product of Inonotus obliquus XJ-1 is obtained, and when the product is added in the daily ration, the normal growth and metabolism of rumen microorganisms are not affected, and the rumen microorganisms can be promoted to fully digest the daily ration and metabolize the nutrient substances and the activity of the rumen microorganisms is promoted. The fermentation product can be used as a feed additive, can provide a theoretical basis for developing and utilizing rare fungi and local resources in Xinjiang Uygur Autonomous Region and helping the green and sustainable development of the animal husbandry in Xinjiang Uygur Autonomous Region.

Description

Technical Field

[0001] This invention relates to the technical field of microbial fermentation, specifically to the technical field of a licorice feed fermentation agent and its application. Background Technology

[0002] Inonotus obliquus, also known as birch polypore, is a rare medicinal fungus that grows on birch trees. It contains various bioactive components, such as polysaccharides, triterpenoids, and polyphenols, and possesses effects including lowering blood sugar, anti-tumor activity, anti-oxidation, and boosting immunity. Inonotus obliquus is scarce and has high medicinal value. It is mainly distributed in cold regions between 45° and 50° north latitude, including North America, Finland, Poland, Russia, Western Siberia, eastern Russia, the Kamchatka Peninsula, and the Lesser Khingan Mountains in Heilongjiang Province and the Changbai Mountains in Jilin Province of Northeast China, as well as Hokkaido, Japan. Wild Inonotus obliquus grows very slowly and is expensive. The sclerotia need to grow in the wild for 10-15 years to develop good medicinal value. Liquid fermentation, on the other hand, has advantages such as a short cultivation cycle, high yield, and is not limited by season or environment. The polysaccharides, polyphenols, and triterpenoids obtained from the fermentation products also have antioxidant, anti-tumor, and blood sugar-lowering functions, greatly enhancing the practical value of large-scale production of Inonotus obliquus.

[0003] Licorice is a perennial erect herb belonging to the genus Glycyrrhiza (Fabaceae). Its rhizome is an important traditional Chinese medicine with a history of use in my country spanning thousands of years. It possesses the effects of tonifying qi and strengthening the spleen, clearing heat and detoxifying, moistening the lungs and relieving cough, relieving pain, and harmonizing the properties of other herbs. It also has anti-ulcer, antiviral, anti-inflammatory, antispasmodic, analgesic, immune-regulating, and endocrine-regulating functions. Glycyrrhizic acid, the active ingredient, is usually extracted from licorice. The residue after glycyrrhizic acid extraction still contains a significant amount of flavonoids, polysaccharides, and other active substances that can exert biological activity. However, when existing strains are used in the fermentation technology of licorice residue, these active substances are easily decomposed, resulting in reduced activity. Furthermore, there are currently no reports on the application of Inonotus obliquus (Birch Inonotus) in the fermentation of licorice residue. Therefore, the rational utilization of strains in the fermentation of licorice residue is of great significance for improving the resource utilization rate of licorice medicinal materials and increasing economic benefits. Summary of the Invention

[0004] To address the issue that existing strains applied to licorice residue fermentation technology often result in the decomposition and reduced activity of active substances such as flavonoids and polysaccharides, and given the lack of reports on the application of *Inonotus obliquus* in licorice residue fermentation, this invention aims to provide a licorice feed fermentation agent and its application. This invention isolates and purifies a strain, *Inonotus obliquus* XJ-1, from *Inonotus obliquus* sclerotium tissue in Altay, Xinjiang Uygur Autonomous Region. It was found that this strain can effectively decompose the indigestible fiber components in licorice residue, while simultaneously releasing some flavonoids embedded within the residue cells that are difficult to utilize, thus better exerting their biological activity. Furthermore, the polysaccharides produced through fermentation with *Inonotus obliquus* XJ-1 increase the crude polysaccharide content in licorice residue, resulting in a better release and enhancement of flavonoid and polysaccharide active substances.

[0005] To achieve the above technical effects, the present invention is implemented through the following technical solution.

[0006] In a first aspect, the present invention provides a licorice feed fermentation agent, wherein the fermentation agent is obtained by fermentation with Inonotus obliquus XJ-1, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41419.

[0007] The fermentation preparation conditions described in this invention are as follows: Inoculation of Inonotus obliquus XJ-1 seed liquid into the fermentation medium at a 10% inoculation rate, cultured at 150 r / min and 30℃ for 10 days.

[0008] The fermentation agent described in this invention is prepared using a culture medium containing 20.0 g / L glucose, 4.0 g / L peptone, 1.0 g / L MgSO4, and 1.0 g / L KH2PO4.

[0009] The application of the aforementioned feed fermentation agent in the preparation of fermented feed.

[0010] Preferably, the application of the present invention involves inoculating the fermentation broth of Inonotus obliquus XJ-1 into pulverized licorice residue, fermenting for 10 days, and then drying the fermented licorice residue at 50°C to obtain the fermented product of Inonotus obliquus XJ-1 licorice residue.

[0011] The method for preparing the licorice residue fermentation product involves inoculating pulverized licorice residue with 10% of Inonotus obliquus XJ-1 fermentation broth, which has a water content of 60% and a temperature of 28℃.

[0012] Preferably, the fermented product prepared by the method described above is used in feed preparation.

[0013] Through the above technical solutions, the present invention achieves the following technical effects:

[0014] (1) The pure culture strain Inonotus obliquus XJ-1 provided by this invention was isolated from the sclerotium of Inonotus obliquus in Altay, Xinjiang Uygur Autonomous Region. It was found that this strain has the ability to produce cellulase, which can fully decompose the fiber components in licorice residue that are difficult for animals to digest. At the same time, it releases some of the flavonoids that are embedded in the residue cells and are not easily utilized, thus better exerting their biological activity. On the other hand, the polysaccharide substances produced by fermentation of Inonotus obliquus XJ-1 increase the crude polysaccharide content in licorice residue, and the flavonoids and polysaccharide active substances are better released and enhanced.

[0015] (2) The present invention provides a licorice feed fermentation agent and its application. Adding licorice residue Inonotus obliquus XJ-1 fermented product to the diet will not affect the normal growth and metabolism of rumen microorganisms. It can also promote the full digestion of the diet, the metabolism of nutrients, and the activity of rumen microorganisms. Adding 1.5% of the fermented product can achieve significant effects. Using the fermented product as a feed additive can provide a theoretical basis for developing and utilizing rare fungi and local resources in Xinjiang Uygur Autonomous Region to help promote the green and sustainable development of animal husbandry in Xinjiang Uygur Autonomous Region. Attached Figure Description

[0016] Figure 1 This is a colony morphology diagram of Inonotus obliquus XJ-1.

[0017] Figure 2 A phylogenetic tree was constructed based on the ITS rDNA sequence of the XJ-1 gene of Inonotus obliquus. Detailed Implementation

[0018] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0019] In this embodiment, the plant flavonoid content detection kit manufactured by Solarbio Chemical Reagent Kit Co., Ltd. was used to determine the flavonoid content according to the instructions.

[0020] The total mixed ration used in this embodiment was purchased from Xinjiang Tiankang Feed Co., Ltd.

[0021] Example 1: Taxonomic identification of Inonotus obliquus XJ-1 based on ITS sequence analysis

[0022] This invention isolated and purified a strain of Inonotus obliquus, numbered XJ-1, from sclerotium tissue of Inonotus obliquus in Altay, Xinjiang Uygur Autonomous Region. The colony morphology is shown in the appendix. Figure 1 As shown, the colony color gradually changed from light yellow to dark brown initially. Hyphae grew slowly on PDA plates, reaching a diameter of 8 cm after 15 days of cultivation at the optimal temperature of 28℃. The *Inonotus obliquus* strain XJ-1 was identified using ITS primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The ITS rDNA sequence analysis results were submitted to the GenBank database, and BLAST online alignment was performed. A phylogenetic tree was constructed using MEGA 11.0 software with the Neighbor-joining method (1000 bootstrap) for homology analysis.

[0023] Based on ITS DNA sequence phylogenetic analysis and microbiological characterization, strain XJ-1 belongs to the genus *Inonotus obliquus*. Gene sequencing of this species was performed, and the resulting sequences were compared and analyzed on the NCBI website. The sequences were then submitted to the GenBank database, obtaining accession number PQ285365. See the appendix for the phylogenetic tree. Figure 2 As shown, comparative analysis revealed that the ITSDNA gene sequence of the Inonotus obliquus strain XJ-1 had the highest homology (99.86%) with Inonotus obliquus HS819. It was named Inonotus obliquus XJ-1 and has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41419 and deposit date of August 5, 2024.

[0024] Example 2: Preparation of Inonotus obliquus XJ-1 inoculum

[0025] The bacterial strain preserved on a PDA slant at 4℃ was inoculated onto a PDA plate and statically cultured at 30℃ for 7-14 days. After two activation cycles, four 1cm diameter mycelial cakes were punched out of the PDA plate and inoculated into seed culture medium. The seed culture medium consisted of: glucose 30.0 g / L, peptone 5.0 g / L, MgSO4 0.5 g / L, KH2PO4 2.0 g / L, and natural pH. After culturing at 30℃ and 150 rpm for 3.5 days, the seed culture was inoculated into fermentation medium at a 10% inoculum and cultured at 150 rpm and 30℃ for 10 days. The fermentation medium consisted of: glucose 20.0 g / L, peptone 4.0 g / L, MgSO4 1.0 g / L, KH2PO4 1.0 g / L, and natural pH.

[0026] Example 3: Solid fermentation product of licorice residue

[0027] Inoculate pulverized licorice residue with 10% Inonotus obliquus XJ-1 inoculum and ferment for 10 days at 28°C with a moisture content of 60%. Dry the fermented licorice residue at 50°C for later use.

[0028] Example 4: Determination of acidic and neutral detergent fiber content in licorice residue before and after fermentation

[0029] The content of neutral detergent fibers was determined by washing method in accordance with national standard GB / T 20806-2022, and the content of acid detergent fibers was determined by washing method in accordance with agricultural industry standard NY / T 1459-2022.

[0030] Example 5: Determination of polysaccharide content in licorice residue before and after fermentation

[0031] (1) Sample preparation: Weigh 0.5g of licorice residue before and after fermentation, add 20mL of dH2O, place it in a shaker and shake it thoroughly, then remove it, filter it through filter paper, centrifuge it at 4500r / min for 10min, and take the supernatant for later use.

[0032] (2) Crude polysaccharide = total polysaccharide - reducing sugar

[0033] A. Total polysaccharides in solution were determined using the phenol-sulfuric acid method: 300 μL of concentrated sulfuric acid was added to 100 μL of supernatant, and the mixture was quickly mixed. Then, 60 μL of 5% phenol solution was added. The mixture was reacted in a 90°C water bath for 5 min, followed by a 5 min water bath at room temperature. The absorbance was then measured at 490 nm.

[0034] B. Reducing sugars were measured using the DNS method: 300 μL of DNS was added to 100 μL of supernatant solution, mixed thoroughly, heated in boiling water for 5 min, cooled with running water, and then 400 μL of distilled water was added to the test tube. The mixture was mixed thoroughly and the absorbance was measured at 540 nm.

[0035] Example 6: Comparative analysis of the contents of acid detergent fiber, neutral detergent fiber, total flavonoids, and polysaccharides in licorice residue before and after fermentation.

[0036] As shown in Table 1, fermentation of licorice residue with *Inonotus obliquus* XJ-1 significantly reduced the contents of acid and neutral detergent fibers while increasing the total flavonoid content. This indicates that the pure culture strain of *Inonotus obliquus* XJ-1 possesses the ability to produce cellulase, which can effectively decompose the fibrous components in licorice residue that are difficult for animals to digest, while simultaneously releasing some flavonoids that are embedded in cells and difficult to utilize, thus better exerting their biological activity. Furthermore, the polysaccharides produced by fermentation with *Inonotus obliquus* XJ-1 increased the crude polysaccharide content in the licorice residue, resulting in a better release and enhancement of flavonoids and polysaccharide active substances.

[0037] Table 1: Comparison of the content of four components in licorice residue before and after fermentation

[0038]

[0039] Note: The data in the table are mean ± standard deviation. The t-test was used. Different uppercase letters in the subscript indicate that the difference is extremely significant (p < 0.01) and different lowercase letters indicate that the difference is significant (p < 0.05).

[0040] Example 7: External fermentation of sheep rumen liquid from licorice residue fermented with Inonotus obliquus XJ-1

[0041] (1) Preparation of artificial rumen fluid

[0042] Composition of the artificial rumen nutrient solution: Trace element solution A: MnCl2·4H2O 10.0g, CaCl2·2H2O 13.2g, FeCl3·6H2O 8.0g, CoCl2·6H2O 1.0g, 100mL distilled water; Buffer solution B: NH4HCO3 4.0g, NaHCO3 35.0g, 1000mL distilled water; Macro element solution C: KH2PO4 6.2g, Na2HPO4 5.7g, MgSO4·7H2O 0.6g, 1000mL distilled water; Resazurin solution: Resazurin 0.1g, 100mL distilled water; Reducing agent solution: Na2S·9H2O 625.0mg, 1mol / L NaOH 4.0mL, 95mL distilled water. The nutrient solution composition ratio is as follows: 0.1 mL of trace element solution A, 200 mL of buffer solution B, 200 mL of macro element solution C, 1 mL of resazurin solution, 40 mL of reducing agent solution, and 400 mL of distilled water (H2O).

[0043] Add the prepared solution to the bottle in the order of the nutrient solution ratio, mix well, introduce CO2, preheat in a 39°C water bath until the mixture becomes colorless and transparent.

[0044] (2) Preparation of fermentation broth

[0045] Sheep rumen fluid was collected using a vacuum gastric tube rumen fluid sampler and filtered through four layers of gauze to remove impurities. The sample was then placed in a sealed thermos filled with CO2 and preheated to 39°C. The sample was immediately taken back to the laboratory and transferred to a 39°C constant temperature water bath, where CO2 was continuously introduced to maintain anaerobic conditions.

[0046] Artificial rumen nutrient solution and fresh sheep rumen fluid were mixed in a 2:1 ratio to prepare an in vitro fermentation liquid, which was then placed in a volumetric flask filled with CO2 at 39°C and stirred until homogeneous before use.

[0047] (3) Extra-rumen liquid fermentation

[0048] Accurately weigh 0.4 g of total mixed diet. The control group received no Inonotus obliquus XJ-1 fermentation broth; the low-dose treatment group received 1% Inonotus obliquus XJ-1 fermentation broth; and the high-dose treatment group received 1.5% Inonotus obliquus XJ-1 fermentation broth. All three treatment groups were placed in preheated culture flasks, and 60 mL of fermentation broth was quickly added before sealing and fermentation. The flasks were incubated at 39℃ for 48 h, with three replicates per group. Gas production was measured at 8, 24, and 48 h of fermentation. After fermentation, pH, rumen ammonia nitrogen, microbial protein (MCP), and volatile fatty acid content were measured. The pH of the fermentation broth was determined using a portable pH meter; the ammonia nitrogen (NH3-N) content of the fermentation broth was determined by colorimetry; the MCP content was determined by the Coomassie brilliant blue method; and the volatile fatty acid content in the rumen fluid was determined by gas chromatography.

[0049] Example 8: Effect of licorice residue Inonotus obliquus XJ-1 fermentation product on in vitro fermentation of sheep rumen fluid

[0050] pH is the comprehensive response of rumen microorganisms to nutrient metabolism and utilization, with a normal range of 6.0–7.5. In this experiment, the pH of the fermentation broth remained within the suitable rumen pH range after 48 hours of fermentation, indicating that adding Inonotus obliquus XJ-1 fermented product from licorice residue to the diet does not affect the normal growth and metabolism of rumen microorganisms. As shown in Table 2, the pH of the 1.5% treatment group was significantly lower than that of the control group, indicating that adding 1.5% fermented product promoted rumen digestion, allowing the diet to be fully degraded to form more volatile fatty acids, thus providing more energy for the animal body.

[0051] Gas production is an important indicator for evaluating rumen microbial fermentation in rumen fermentation systems of ruminants, and to a certain extent, it indicates the overall trend of rumen microbial fermentation activity. In this experiment, the gas production of the 1.5% treatment group was significantly higher than that of the control group in the first 8 hours of fermentation; from 24 to 48 hours of fermentation, the gas production of the 1.0% and 1.5% treatment groups was significantly higher than that of the control group, with the highest gas production in the first 24 hours. This indicates that the fermented product promoted the full fermentation of the diet and the metabolic function of its nutrients and the activity of rumen microorganisms, with the 1.5% treatment group showing the best effect.

[0052] However, the fermentation product of Inonotus obliquus XJ-1 in licorice residue had no significant effect on the content of ammonia nitrogen, MCP and most volatile fatty acids, while the 1.0% treatment group significantly increased the content of butyric acid.

[0053] Table 2. Effects of licorice residue fermentation by Inonotus obliquus XJ-1 on external fermentation of sheep rumen fluid.

[0054]

[0055]

[0056] Note: The data in the table are mean ± standard deviation. Analysis of variance and Duncan's test were used. Different lowercase letters indicate that p < 0.05 is statistically significant.

[0057] In summary, the pure culture strain of Inonotus obliquus XJ-1 has the ability to produce cellulase, which can fully decompose the fibrous components in licorice residue that are difficult for animals to digest. At the same time, it releases some flavonoids that are embedded in the cells and are not easily utilized. There is no significant degradation of crude polysaccharides in fermented licorice, and the flavonoid and polysaccharide active substances are released and enhanced in a better way.

[0058] The results of the in vitro fermentation experiment of licorice residue and Inonotus obliquus XJ-1 fermentation product in sheep rumen showed that the fermentation product not only did not affect the normal growth and metabolism of rumen microorganisms, but also promoted digestion and full fermentation of the diet, as well as the metabolic function of its nutrients and the activity of rumen microorganisms. The effect of adding 1.5% fermentation product was significant.

[0059] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but rather to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. The application of a licorice feed fermentation agent in feed fermentation, characterized in that, The application described is to use Inonotus obliquus ( Inonotus obliquus The XJ-1 fermentation broth was inoculated into pulverized licorice residue and fermented for 10 days. The fermented licorice residue was then dried at 50℃ to obtain Inonotus obliquus (Bauhinia purpurea). Inonotus obliquus XJ-1 Licorice Residue Fermentation Product; The fermentation broth uses Inonotus obliquus (Inonotus obliquus) Inonotus obliquus Obtained by fermentation of XJ-1, this Chaga mushroom ( Inonotus obliquus Strain XJ-1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41419; The fermentation broth preparation conditions are as follows: Inonotus obliquus (… Inonotus obliquus XJ-1 seed culture was inoculated into fermentation medium at a 10% inoculum and cultured at 150 r / min and 30℃ for 10 days. The fermentation broth was prepared using a culture medium containing 20.0 g / L glucose, 4.0 g / L peptone, 1.0 g / L MgSO4, and 1.0 g / L KH2PO4. The licorice residue is the residue after extracting glycyrrhizic acid.

2. A method for preparing a fermented product of licorice residue, characterized in that, The method for preparing the licorice residue fermentation product uses Inonotus obliquus (Inonotus obliquus) Inonotus obliquus The XJ-1 fermentation broth was inoculated with 10% of crushed licorice residue, which had a water content of 60% and a temperature of 28℃. The fermentation broth uses Inonotus obliquus (Inonotus obliquus) Inonotus obliquus Obtained by fermentation of XJ-1, this Chaga mushroom ( Inonotus obliquus Strain XJ-1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41419; The fermentation broth preparation conditions are as follows: Inonotus obliquus (… Inonotus obliquus XJ-1 seed culture was inoculated into fermentation medium at a 10% inoculum and cultured at 150 r / min and 30℃ for 10 days. The fermentation broth was prepared using a culture medium containing 20.0 g / L glucose, 4.0 g / L peptone, 1.0 g / L MgSO4, and 1.0 g / L KH2PO4. The licorice residue is the residue after extracting glycyrrhizic acid.

3. The application of the fermented product obtained by the method for preparing licorice residue fermented product as described in claim 2 in feed preparation.

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