Method for extracting saccharicterpenin through low-temperature fermentation

Abstract

The invention discloses a method for extracting saccharicterpenin through low-temperature fermentation, and relates to the technical field of feed additives. The method comprises the following steps: by taking oil tea meal as a fermentation raw material, adding a composition obtained by mixing a compound enzyme preparation consisting of bacillus licheniformis liquid, mannase and xylanase and magnesium sulfate into the fermentation raw material, and fermenting and purifying to obtain the saccharicterpenin. The efficient release of saccharicterpenin in the oil tea meal is realized through the cascade reaction of bacillus licheniformis microbial fermentation, compound enzyme preparation enzymolysis and metal salt activation, so that the content of total saponin and total sugar in the saccharicterpenin is increased. Specifically, in the saccharicterpenin prepared by the method disclosed by the invention, the content of total saponin of camellia oleifera can reach 76.8%, and the content of total sugar can reach 33.6%.

Description

Technical Field

[0001] This invention relates to the field of feed additive technology, and in particular to a method for extracting glycoterpenes by low-temperature fermentation. Background Technology

[0002] Glycoterpenes are a mixture of triterpenoid saponins and sugars extracted from the seed cake of Camellia plants. They are pure natural bioactive extracts composed of sugars, ligand sugars, and organic acids, containing no chemically synthesized components. Glycoterpenes can regulate the intestinal microecological balance and the body's signal transduction system, modulate immunity, improve feed conversion rate, animal production performance, and stress resistance. They are often used as feed additives to reduce the overuse of antibiotics in animal husbandry.

[0003] Currently, extraction methods for glycoterpenes include water extraction, methanol extraction, conversion synthesis, and fermentation. Among these, water extraction yields glycoterpenes with a high concentration of water-soluble impurities and a low yield. Methanol extraction increases the yield by 80% compared to water extraction, but methanol is volatile and toxic. Conversion synthesis requires separate extraction of tea saponins and tea polysaccharides, followed by a proportional conversion process. While this method yields high-purity glycoterpenes, it is complex, has a low yield, and is unsuitable for industrial production. Compared to these extraction methods, fermentation offers significant advantages in reducing impurities, increasing yield, and improving purity.

[0004] In existing technologies, the study "Research on the Extraction of Glycoterpenes from Camellia Oil Meal by Microbial Fermentation" (Diao Huan, China Oils and Fers, 2016 Vol.41 No.4) discloses the use of Aspergillus niger as a fermentation strain in a solid culture medium composed of camellia oil meal, magnesium sulfate, potassium dihydrogen phosphate, and blood meal. After fermentation at 30°C for 4 days, glycoterpenes are obtained through purification. However, using Aspergillus niger as a fermentation strain for glycoterpenes preparation involves excessively long fermentation times and complex subsequent purification processes. CN115521943A, a method for extracting glycoterpenes from camellia oil meal, discloses a combination of "ethanol purification-ultrasonic treatment-enzymatic hydrolysis" to maximize the extraction of glycoterpenes from the camellia oil meal. However, while ethanol can extract free glycoterpenes, ultrasonic treatment only partially breaks down cell walls, and enzymatic hydrolysis using acidic proteases only effectively utilizes the protein in the camellia oil meal, which contains only 10%-20% protein. It is evident that although the stepwise purification method described in the aforementioned patent can improve the yield and purity of glycoterpenes, the preparation process is complex, time-consuming, and energy-intensive. Furthermore, it cannot fully utilize the components in the camellia oil meal and still suffers from the drawback of low glycoterpenes yield.

[0005] Therefore, it is particularly important to develop a rapid method for preparing glycoterpenes and to ensure the content of total saponins and total sugars in the prepared glycoterpenes. Summary of the Invention

[0006] To address the aforementioned limitations of existing technologies, the present invention aims to provide a method for extracting glycoterpenes through low-temperature fermentation. This invention uses camellia oil meal as the fermentation raw material. A composite enzyme preparation consisting of Bacillus licheniformis bacterial culture, mannanase, and xylanase, along with magnesium sulfate, is added to the fermentation raw material. After fermentation and purification, glycoterpenes are obtained. The efficient release of glycoterpenes from camellia oil meal is achieved through a cascade reaction involving Bacillus licheniformis microbial fermentation, enzymatic hydrolysis by the composite enzyme preparation, and activation by metal salts, thereby increasing the content of total camellia oil saponins and total sugars in the glycoterpenes. Specifically, the glycoterpenes obtained using the method of this invention can contain up to 76.8% total camellia oil saponins and 33.6% total sugars.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for extracting glycoterpenes by low-temperature fermentation, comprising the following steps: After mixing with water, the camellia seed cake is sterilized to obtain fermentation raw material. A bacterial-enzyme-metal salt composition is added to the fermentation raw material for fermentation treatment. The fermented liquid is collected to obtain culture medium. The culture medium is purified to obtain glycoterpenoids. The bacterial-enzyme-metal salt composition is prepared by mixing Bacillus licheniformis bacterial solution, mannanase, xylanase and magnesium sulfate in a mass ratio of (1-2):(0.6-0.8):(0.5-1):(0.03-0.06).

[0008] As a preferred option, Bacillus licheniformis has the accession number ACCC 05186.

[0009] As a preferred method, the preparation method of Bacillus licheniformis bacterial culture is as follows: Bacillus licheniformis is inoculated into LB liquid medium and fermented at 30-37℃ for 15-20h to obtain Bacillus licheniformis bacterial culture.

[0010] Furthermore, the inoculum size of Bacillus licheniformis is 2%-5% of the volume of LB liquid medium; the viable count of Bacillus licheniformis in the bacterial culture solution is ≥1×10⁻⁶. 8 cfu / mL.

[0011] Preferably, the volume ratio of camellia seed cake to water is 1:(2.5-3.5).

[0012] Preferably, the sterilization temperature is 121℃ and the sterilization time is 10-20 minutes.

[0013] Preferably, the amount of the bacteria-enzyme-metal salt composition added is 0.2%-0.4% of the mass of the fermentation raw materials.

[0014] As a preferred method, the fermentation temperature is 37-40℃ and the fermentation time is 10-15h.

[0015] As a preferred method, the purification process is as follows: add anhydrous ethanol to the culture medium, stir, centrifuge, take the supernatant after centrifugation, remove the anhydrous ethanol from the supernatant, and dry it to obtain glycoterpenoids.

[0016] Furthermore, the method for removing anhydrous ethanol from the supernatant is thermal evaporation or concentration.

[0017] Furthermore, the volume ratio of culture medium to anhydrous ethanol is 1:(5-10).

[0018] Furthermore, the stirring speed is 50-100 r / min, and the stirring time is 45-60 min.

[0019] In a second aspect, the present invention provides a glycoterpene prepared by the above method.

[0020] The beneficial effects of this invention are: This invention uses camellia seed cake as a fermentation raw material. A composite enzyme preparation consisting of Bacillus licheniformis bacterial solution, mannanase, and xylanase, and magnesium sulfate is added to the fermentation raw material. After fermentation and purification, glycoterpenes are obtained. The efficient release of glycoterpenes from camellia seed cake is achieved through a cascade reaction of Bacillus licheniformis microbial fermentation, enzymatic hydrolysis by the composite enzyme preparation, and activation by metal salts, thereby increasing the content of total camellia saponins and total sugars in the glycoterpenes. Specifically, the glycoterpenes obtained by the method of this invention can reach a total camellia saponin content of up to 76.8% and a total sugar content of up to 33.6%.

[0021] In addition, the present invention utilizes the synergistic effect of Bacillus licheniformis microbial fermentation and compound enzyme preparation to increase the content of total saponins and total sugars in Camellia oleifera in glycoterpenoids. Detailed Implementation

[0022] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0023] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0024] While existing technologies disclose the use of Aspergillus niger as a strain to ferment Camellia oleifera meal for the preparation of glycoterpenes, the fermentation time is too long and the purification process is complex, making it unsuitable for industrial production. Therefore, a rapid fermentation method for preparing glycoterpenes that ensures the content of active ingredients in the glycoterpenes is currently a research hotspot.

[0025] Based on this, the present invention utilizes the synergistic fermentation of camellia seed cake with microorganisms and enzymes to prepare glycoterpenes. Specifically, *Bacillus licheniformis* is used as the fermentation strain, and a combination of mannanase and xylanase is used as a complex enzyme preparation. The combined use of microbial culture and fermentation enzyme preparation maximizes the conversion of components in camellia seed cake into glycoterpenes, thus exhibiting a synergistic effect in increasing the total saponin and total sugar content of the glycoterpenes. Furthermore, to further enhance the activity of the microbial enzymes, magnesium sulfate is added, utilizing the metal salt to improve enzyme activity and thereby enhance the efficiency of glycoterpene preparation.

[0026] In addition, CN107259101A discloses a method for fermenting camellia seeds to produce feed. Specifically, after hot water extraction of camellia seed meal, protease, pectinase, xylanase, acid cellulase, and glucose oxidase are added for enzymatic hydrolysis. After adjusting the pH, Bacillus licheniformis, Lactobacillus plantarum, and Saccharomyces cerevisiae are added for anaerobic fermentation. The fermentation products are collected and a compound enzyme preparation is added to obtain the feed. Although the above method involves compound enzyme preparations and Bacillus licheniformis for bacterial and enzyme treatment of camellia seed meal, the above method is not suitable for the preparation of glycoterpenes because the hot water extraction of camellia seed meal in the above patent is used to remove camellia saponins from the camellia seed meal. Camellia oleifera saponin is a raw material for preparing pentacyclic triterpenoids and oligosaccharides in glycoterpenoids. The mixture of the two is glycoterpenoids (paper "Research on the Process of Preparing Glycoterpenoids from Camellia oleifera Seed Meal", Chen Xinxin, Wuhan University of Light Industry, June 2013). It can be seen that the extraction process in the above patent removes the raw materials in Camellia oleifera seed meal that can be used to prepare glycoterpenoids. Even if the subsequent processing uses a bacterial enzyme composition including Bacillus licheniformis and xylanase to ferment Camellia oleifera seed meal, the role played by the bacterial enzyme composition is completely different from the role played by the bacterial enzyme composition in this invention.

[0027] The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and can be purchased through commercial channels.

[0028] In this invention, the LB medium consisted of 10 g / L peptone, 5 g / L NaCl, and 1.5 g / L glucose, with a pH of 6.8-7.2. *Bacillus licheniformis* strains were purchased from the China Agricultural Microbiological Culture Collection Center, strain number ACCC 05186. Xylanase was purchased from Taian Xindeli Biotechnology Co., Ltd., and mannanase was purchased from Shandong Nuojie Biotechnology Co., Ltd. The xylanase activity was 3 × 10⁻⁶. 5 The enzyme activity of mannanase (U / g) is 3×10⁻⁶. 4 U / g.

[0029] Example 1: Method for extracting glycoterpenes by low-temperature fermentation (1) Inoculate Bacillus licheniformis into LB liquid medium at an inoculation rate of 3% (v / v), ferment at 35°C for 18 h, centrifuge, collect the supernatant to obtain Bacillus licheniformis bacterial culture, and adjust the viable count in the Bacillus licheniformis bacterial culture to 1×10⁻⁶. 8 cfu / mL; A bacterial-enzyme-metal salt composition was obtained by mixing Bacillus licheniformis bacterial solution, mannanase, xylanase and magnesium sulfate in a mass ratio of 1.5:0.7:0.8:0.045. (2) Take 1000g of camellia seed cake, mix the camellia seed cake and water in a volume ratio of 1:3, heat at 121℃ for 15min for sterilization, and cool down to 38℃ to obtain fermentation raw material; add bacteria-enzyme-metal salt composition to fermentation raw material, wherein the amount of bacteria-enzyme-metal salt composition added is 0.3045% of the mass of fermentation raw material, ferment at 38℃ for 12h, collect the fermented liquid to obtain culture medium; (2) Add 7 times the volume of anhydrous ethanol to the culture medium, stir at 75 r / min for 55 min, centrifuge, collect the supernatant after centrifugation, use vacuum concentration to remove the anhydrous ethanol in the supernatant, dry, and obtain glycoterpenoid.

[0030] Example 2: Method for extracting glycoterpenes by low-temperature fermentation (1) Inoculate Bacillus licheniformis into LB liquid medium at an inoculation rate of 2% (v / v), ferment at 30°C for 15 h, centrifuge, collect the supernatant to obtain Bacillus licheniformis bacterial culture, and adjust the viable count in the Bacillus licheniformis bacterial culture to 1×10⁻⁶. 8 cfu / mL; A bacterial-enzyme-metal salt composition was obtained by mixing Bacillus licheniformis bacterial solution, mannanase, xylanase and magnesium sulfate in a mass ratio of 1:0.6:0.5:0.03. (2) Take 1000g of camellia seed cake, mix the camellia seed cake and water at a volume ratio of 1:2.5, heat at 121℃ for 10min for sterilization, and cool down to 37℃ to obtain fermentation raw material; add bacteria-enzyme-metal salt composition to fermentation raw material, wherein the amount of bacteria-enzyme-metal salt composition added is 0.213% of the mass of fermentation raw material, ferment at 37℃ for 10h, collect the fermented liquid to obtain culture medium; (2) Add 5 times the volume of anhydrous ethanol to the culture medium, stir at 50 r / min for 40 min, centrifuge, collect the supernatant after centrifugation, use vacuum concentration to remove the anhydrous ethanol in the supernatant, dry, and obtain glycoterpenoid.

[0031] Example 3: Method for extracting glycoterpenes by low-temperature fermentation (1) Inoculate Bacillus licheniformis into LB liquid medium at an inoculation rate of 5% (v / v), ferment at 37°C for 20 h, centrifuge, collect the supernatant to obtain Bacillus licheniformis bacterial culture, and adjust the viable count in the Bacillus licheniformis bacterial culture to 1×10⁻⁶. 8 cfu / mL; A bacterial-enzyme-metal salt composition was obtained by mixing Bacillus licheniformis bacterial solution, mannanase, xylanase and magnesium sulfate in a mass ratio of 2:0.8:1:0.06. (2) Take 1000g of camellia seed cake, mix the camellia seed cake and water at a volume ratio of 1:3.5, heat at 121℃ for 20min for sterilization, and cool down to 40℃ to obtain fermentation raw material; add bacteria-enzyme-metal salt composition to fermentation raw material, wherein the amount of bacteria-enzyme-metal salt composition added is 0.386% of the mass of fermentation raw material, ferment at 40℃ for 15h, collect the fermented liquid to obtain culture medium; (2) Add 10 times the volume of anhydrous ethanol to the culture medium, stir at 100 r / min for 60 min, centrifuge, collect the supernatant after centrifugation, use vacuum concentration to remove the anhydrous ethanol in the supernatant, dry, and obtain glycoterpenoid.

[0032] Comparative Example 1: The difference between this comparative example and Example 1 is that Bacillus licheniformis bacterial solution, mannanase, and xylanase were not used. Specifically: Add 0.3045% (by mass) magnesium sulfate to the fermentation feedstock, react at 38°C for 12 hours, collect the reaction liquid, and purify it to obtain glycoterpenoids.

[0033] The specific operations for preparing and purifying the fermentation raw materials are the same as in Example 1.

[0034] Comparative Example 2: The difference between this comparative example and Example 1 is that mannanase and xylanase were not used. Specifically: Bacillus licheniformis bacterial culture and magnesium sulfate were mixed at a mass ratio of 1.5:0.045 to obtain a bacterial-metal salt composition. 0.3045% (mass ratio) of the bacterial-metal salt composition was added to the fermentation raw material, and fermentation was carried out at 38°C for 12 hours. The fermented liquid was collected to obtain a culture medium. The culture medium was purified to obtain glycoterpenoids.

[0035] The preparation of Bacillus licheniformis bacterial culture, the preparation of fermentation raw materials, and the purification of the culture medium are the same as in Example 1.

[0036] Comparative Example 3: The difference between this comparative example and Example 1 is that Bacillus licheniformis bacterial suspension was not used. Specifically: Mannanase, xylanase, and magnesium sulfate were mixed in a mass ratio of 0.7:0.8:0.045 to obtain an enzyme-metal salt composition. 0.3045% (mass ratio) of the enzyme-metal salt composition was added to the fermentation feedstock, and fermentation was carried out at 38°C for 12 hours. The fermented liquid was collected to obtain a culture medium. The culture medium was purified to obtain glycoterpenoids.

[0037] The preparation of Bacillus licheniformis bacterial culture, the preparation of fermentation raw materials, and the purification of the culture medium are the same as in Example 1.

[0038] Experimental Example 1: The total saponin content and total sugar content of the glycoterpenes prepared in Example 1 and Comparative Examples 1-4 were detected, and the results are shown in Table 1.

[0039] Specifically, the total saponin content and total sugar content in camellia oleifera were determined by referring to the methods for determining the total saponin content and total sugar content in GB / T 25247-2010 "Feed Additives - Glycoterpenes". Table 1. Content of active ingredients in the glycoterpenes prepared in Example 1 and Comparative Examples 1-4 As shown in Table 1, the composition prepared by this invention using a complex enzyme consisting of Bacillus licheniformis, xylanase, and mannanase, along with magnesium sulfate, was used to ferment camellia seed meal to prepare glycoterpenes. The obtained glycoterpenes contained 75.8%-76.8% total saponins and 32.9%-33.6% total sugars. Compared to Comparative Example 1 (control group), Comparative Example 2, which used only Bacillus licheniformis and magnesium sulfate for fermentation to prepare glycoterpenes, only increased the total saponin content by 23.3% and the total sugar content by only 6.9%. Comparative Example 3, which used only a complex enzyme consisting of xylanase and mannanase with magnesium sulfate for fermentation to prepare glycoterpenes, increased the total saponin content by 19.9% ​​and the total sugar content by 5.6%. Therefore, it can be seen that the combined use of Bacillus licheniformis bacterial solution, xylanase and mannanase has a synergistic effect in increasing the total saponin content and total sugar content of Camellia oleifera.

[0040] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for extracting glycoterpenes by low-temperature fermentation, characterized in that, Includes the following steps: After mixing with water, the camellia seed cake is sterilized to obtain fermentation raw material. A bacterial-enzyme-metal salt composition is added to the fermentation raw material for fermentation treatment. The fermented liquid is collected to obtain culture medium. The culture medium is purified to obtain glycoterpenoids. The bacterial-enzyme-metal salt composition is prepared by mixing Bacillus licheniformis bacterial solution, mannanase, xylanase and magnesium sulfate in a mass ratio of (1-2):(0.6-0.8):(0.5-1):(0.03-0.06).

2. The method as described in claim 1, characterized in that, The preparation method of Bacillus licheniformis bacterial culture is as follows: Bacillus licheniformis is inoculated into LB liquid medium and fermented at 30-37℃ for 15-20h to obtain Bacillus licheniformis bacterial culture; the preservation number of Bacillus licheniformis is ACCC 05186.

3. The method as described in claim 2, characterized in that, The inoculum size of Bacillus licheniformis is 2%-5% of the volume of LB liquid medium; the viable count of Bacillus licheniformis in the bacterial culture solution is ≥1×10⁻⁶. 8 cfu / mL.

4. The method as described in claim 1, characterized in that, The volume ratio of camellia seed cake to water is 1:(2.5-3.5).

5. The method as described in claim 1, characterized in that, The amount of the bacteria-enzyme-metal salt composition added is 0.2%-0.4% of the mass of the fermentation raw materials.

6. The method as described in claim 1, characterized in that, The fermentation temperature is 37-40℃, and the fermentation time is 10-15 hours.

7. The method as described in claim 1, characterized in that, The specific purification process is as follows: after adding anhydrous ethanol to the culture medium, stir and centrifuge, take the supernatant after centrifugation, remove the anhydrous ethanol from the supernatant, and dry it to obtain glycoterpenoids.

8. The method as described in claim 7, characterized in that, The method for removing anhydrous ethanol from the supernatant is thermal evaporation or concentration.

9. The method as described in claim 7, characterized in that, The volume ratio of culture medium to anhydrous ethanol is 1:(5-10); the stirring speed is 50-100 r / min, and the stirring time is 45-60 min.

10. The glycoterpenoid prepared by the method according to any one of claims 1-9.

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