A method for preparing a bidirectional ferment of *Lycium barbarum* and its application

Abstract

This invention discloses a method for preparing a *Ligustrum lucidum*-*Lycium barbarum* bidirectional ferment and its application, belonging to the field of microbial fermentation and treatment of chemotherapy-induced intestinal mucosal damage. The *Ligustrum lucidum*-*Lycium barbarum* bidirectional ferment is prepared by bidirectional fermentation of *Ligustrum lucidum* mycelium and a basal culture medium supplemented with *Lycium barbarum*. Functional evaluation was conducted using a 5-FU-induced intestinal mucosal damage model in colon cancer-bearing mice. Experimental results showed that the *Ligustrum lucidum*-*Lycium barbarum* bidirectional ferment can significantly alleviate weight loss and diarrhea symptoms caused by chemotherapy in the model animals, significantly reduce serum diamine oxidase (DAO) levels, and effectively improve intestinal mucosal damage.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and treatment of chemotherapy-induced intestinal mucosal damage, specifically relating to a method for preparing a bidirectional ferment of *Lycium barbarum* and its application. Background Technology

[0002] Armillaria tabescens (Scop. ex Fr.) Sing, scientifically known as *Armillariella tabescens*, is a large medicinal fungus that produces various metabolites during its growth, including sclerotin A and B, amino acids, polypeptides, polysaccharides, and mannitol. Current research reports indicate that *Armillariella tabescens* has a beneficial effect on intestinal damage, including ulcerative colitis and chemotherapy-induced intestinal mucosal damage, demonstrating its potential in the treatment of gastrointestinal diseases. However, the limited natural resources of *Armillariella tabescens* and the low metabolite yield and insufficient biological activity in laboratory monomeric culture systems restrict its development and utilization.

[0003] In recent years, two-way fermentation technology, as an important strategy to improve the fermentation efficiency of fungi, has become a research hotspot, providing a new path for the industrial development of fungi. This technology involves adding an appropriate amount of traditional Chinese medicine (TCM) herbs to the fermentation medium to form a complete fermentation substrate. Fungi and TCM herbs are co-cultured in a liquid fermentation system, creating a two-way interactive fermentation environment. In this system, fungi not only utilize the carbon, nitrogen, and mineral nutrients provided by the TCM substrate to promote their own growth and metabolism, but also secrete various enzyme systems, such as glycoside hydrolases and oxidoreductases, which can structurally modify and transform the components of the TCM herbs, thereby activating or enhancing the activity of existing components and even producing new active substances. This two-way interaction provides an effective pathway to improve the overall bioavailability of fermentation products.

[0004] The applicant used *Ligustrum lucidum* to conduct bidirectional fermentation with various traditional Chinese medicines, including wolfberry, astragalus, polygonatum, ginseng, and dendrobium officinale. The fermented products were screened for activity, and it was found that the *Ligustrum lucidum*-wolfberry bidirectional fermentation broth showed outstanding performance in improving chemotherapy-induced intestinal mucosal damage, with effects comparable to melasmazine, a clinically positive drug. Currently, there are no reports on the use of *Ligustrum lucidum*-wolfberry bidirectional fermentation broth to alleviate chemotherapy-induced intestinal mucosal damage. Summary of the Invention

[0005] This invention aims to provide a method for preparing a bidirectional ferment of *Glechoma heliotropium* and *Lycium barbarum*, and its application. The bidirectional ferment of *Glechoma heliotropium* and *Lycium barbarum* is prepared by bidirectional fermentation of *Glechoma heliotropium* mycelium and a basal culture medium supplemented with *Lycium barbarum*. It can significantly improve weight loss and diarrhea in model animals; significantly reduce the activity level of the intestinal barrier marker DAO; improve the pathological morphology of small intestinal tissue; and has good activity in improving chemotherapy-induced intestinal mucosal damage. It can be used to prepare novel pharmaceutical formulations.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] The method for preparing the bifidobacteria-goji berry dual fermentation product of the present invention includes the following steps:

[0008] Step 1: Pretreatment of Goji Berries

[0009] Goji berries are dried and pulverized to obtain goji berry powder, which is then set aside.

[0010] Step 2: Activation of the *Bacillus subtilis* strain

[0011] Selected and preserved *Gymnocypris lucida* mycelia were inoculated onto PDA solid medium under aseptic conditions for activation at 28°C for 8-10 days to obtain activated *Gymnocypris lucida* mycelia.

[0012] Step 3: Prepare the basic culture medium containing wolfberry

[0013] The wolfberry powder obtained in step 1 was added to the basic culture medium at a ratio of 1 g: 10 mL to obtain a basic culture medium containing wolfberry. The basic culture medium consisted of 25 g / L glucose and 200 g / L potato. The specific preparation process was as follows: 200 g of peeled potato was chopped, 1 L of water was added, and the mixture was boiled for 30 min. The mixture was filtered through 8 layers of gauze, 25 g of glucose was added, and the mixture was sterilized at 121℃ for 30 min.

[0014] Step 4: Inoculate the activated mycelium obtained in Step 2 into 500 mL of basal culture medium containing wolfberry at an inoculation rate of 0.4% (v / v), and incubate statically in a constant temperature incubator at 28℃ for 21 days.

[0015] Step 5: Filter the bidirectional fermentation broth obtained in Step 4, centrifuge to collect the supernatant, concentrate under reduced pressure, and freeze-dry under vacuum to obtain the *Lycium barbarum*-goji berry bidirectional fermentation product powder. The filtration conditions are: filtration through a 60-mesh sieve; the centrifugation conditions are: centrifugation at 5000 rpm for 10-15 min.

[0016] The present invention relates to the application of the bifidobacteria-goji berry dual fermentation product in the preparation of pharmaceutical formulations.

[0017] The drug formulation has excellent efficacy in improving chemotherapy-induced intestinal mucosal damage, can significantly alleviate weight loss and diarrhea symptoms caused by chemotherapy, significantly reduce serum diamine oxidase (DAO) levels, and effectively improve intestinal mucosal damage.

[0018] The beneficial effects of this invention are reflected in:

[0019] 1. A method for preparing bidirectional fermentation products of *Lycium barbarum* and *Lycium barbarum* is provided, which is characterized by simple operation, economic efficiency, and excellent results.

[0020] 2. The present invention is used to prepare a pharmaceutical formulation for improving chemotherapy-induced intestinal mucosal damage, wherein the formulation has outstanding efficacy in improving chemotherapy-induced intestinal mucosal damage. Attached Figure Description

[0021] Figure 1 This represents the changes in body weight of mice in each group.

[0022] Figure 2 The degree of diarrhea in each group of mice.

[0023] Figure 3 This represents the DAO activity level in the serum of mice in each group.

[0024] Figure 4 HE staining of the ileum of mice in each group. A: blank group; B: tumor-bearing group; C: model group; D: melasmacin group; E: *Glechoma heliotropium* fermentation group; F: *Glechoma heliotropium*-*Glechoma heliotropium* culture medium group; G: *Glechoma heliotropium*-*Glechoma heliotropium* bidirectional fermentation group; H: *Glechoma heliotropium*-*Glehnia littoralis* bidirectional fermentation group; I: *Glechoma heliotropium*-*Glehnia littoralis* bidirectional fermentation group; J: *Glechoma heliotropium*-*Glehnia littoralis* bidirectional fermentation group; K: *Glechoma heliotropium*-*Dendrobium officinale* bidirectional fermentation group. For simplicity, the term "bidirectional" is omitted from the names of groups G and K in the figure.

[0025] Figure 5 The values ​​are the intestinal villus height (b) and crypt depth (a) of each group of mice. Detailed Implementation

[0026] The following describes a method for preparing and applying a bidirectional fermentation product of *Gynostemma pentaphyllum* and *Lycium barbarum*. The examples given are for illustrative purposes only and are not intended to limit the scope of protection of this invention. Unless otherwise specified, the following test and detection methods are conventional methods; the reagents and raw materials mentioned are commercially available unless otherwise specified.

[0027] Source of strain:

[0028] The strain of Armillaria tabescens (Scop. ex Fr.) Sing used in this invention was purchased from the China Agricultural Microbial Culture Collection Center, with the strain number ACCC51415.

[0029] Example 1: Preparation of bidirectional fermentation product of *Bacillus subtilis* and wolfberry

[0030] 1. Pretreatment of wolfberries: Dry and pulverize wolfberries to obtain wolfberry powder for later use;

[0031] 2. Activation of *Bacillus lucida* strain: Select the preserved *Bacillus lucida* mycelium and inoculate it onto PDA solid medium under aseptic conditions for activation. The activation conditions are 28℃ and 10 days to obtain activated mycelium.

[0032] 3. Preparation of basal culture medium containing wolfberry: The wolfberry powder obtained in step 1 was added to the basal culture medium at a material-to-liquid ratio of 1 g:10 mL to obtain the basal culture medium containing wolfberry. The composition of the basal culture medium was: glucose 25 g / L and potato 200 g / L. The specific preparation process was as follows: 200 g of peeled potato was chopped, 1 L of water was added, and the mixture was boiled for 30 min. The mixture was filtered through 8 layers of gauze, 25 g of glucose was added, and the mixture was sterilized at 121℃ for 30 min.

[0033] 4. Cut a 2 × 2 cm piece from the activated mycelium obtained in step 2 using an inoculation spatula. 2 The mycelium blocks were inoculated into 500 mL of basic culture medium containing wolfberry and incubated statically in a constant temperature incubator at 28℃ for 21 days.

[0034] 5. Filter the bidirectional fermentation broth obtained in step 4, centrifuge to collect the supernatant, concentrate under reduced pressure and freeze dry under vacuum to obtain the bidirectional fermentation product powder of *Lycium barbarum* and *Lycium barbarum*. The filtration conditions are: filtration through a 60-mesh sieve; the centrifugation conditions are: centrifugation at 5000 rpm for 15 min.

[0035] Comparative Example 1: Preparation of *Bacillus lucida* ferment broth

[0036] The difference from Example 1 is that the fermentation medium does not contain the traditional Chinese medicine ingredient wolfberry, while the rest of the steps are the same as in Example 1.

[0037] Comparative Example 2: Preparation of Culture Medium Containing Lycium barbarum

[0038] The difference from Example 1 is that the fermentation medium was not inoculated with *Bacillus lucida* mycelium, but the rest of the steps were the same as in Example 1.

[0039] Comparative Example 3: Preparation of bidirectional fermentation products of *Bacillus lucida* and *Astragalus membranaceus*

[0040] The difference from Example 1 is that the traditional Chinese medicine component in the fermentation culture medium was replaced with Astragalus membranaceus, while the rest of the steps were the same as in Example 1.

[0041] Comparative Example 4: Preparation of bidirectional fermentation products of *Ligustrum lucidum* and *Polygonatum sibiricum*

[0042] The difference from Example 1 is that the traditional Chinese medicine component in the fermentation culture medium was replaced with Polygonatum sibiricum, while the rest of the steps were the same as in Example 1.

[0043] Comparative Example 5: Preparation of bidirectional fermentation product of *Gynostemma pentaphyllum* and ginseng

[0044] The difference from Example 1 is that the traditional Chinese medicine component in the fermentation culture medium was replaced with ginseng, while the rest of the steps were the same as in Example 1.

[0045] Comparative Example 6: Preparation of bidirectional fermentation products of *Bacillus thuringiensis* and *Dendrobium officinale*

[0046] The difference from Example 1 is that the traditional Chinese medicine component in the fermentation culture medium was replaced with Dendrobium officinale, while the rest of the steps were the same as in Example 1.

[0047] Example 2: Determination of the efficacy of fermentation broth in improving chemotherapy-induced intestinal mucosal damage in Examples 1, 1, 2, 3, 4, 5, and 6.

[0048] Using the ferments obtained in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 as raw materials, and employing a 5-FU-induced intestinal mucosal injury model in colon cancer-bearing mice, the efficacy of the ferments in Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5 and Comparative Example 6 in improving chemotherapy-induced intestinal mucosal injury was evaluated.

[0049] Step 1: Animal Model Establishment and Grouping: Male BALB / c mice (4-5 weeks old, weight range 17±1g) were acclimatized for one week, and then CT26 cells (4 × 10⁻⁶) were injected subcutaneously. 6 (Number of cells) were inoculated into the right buttock region of mice. When the tumor diameter reached 0.5 cm, the animals were randomly divided into 11 groups (n=6): blank group, tumor-bearing group, model group, melasrazine group, *Ligustrum lucidum* fermentation group, *Lycium barbarum* culture medium group, *Ligustrum lucidum*-Lycium barbarum bidirectional fermentation group, *Ligustrum lucidum*-Astragalus membranaceus bidirectional fermentation group, *Ligustrum lucidum*-Polygonatum sibiricum bidirectional fermentation group, *Ligustrum lucidum*-Ginseng bidirectional fermentation group, and *Ligustrum lucidum*-Dendrobium officinale bidirectional fermentation group. The grouping and treatment were as follows: The blank group was not inoculated with tumor cells, but received intraperitoneal injection of normal saline for 7 days, followed by oral administration of normal saline for 10 days. The tumor-bearing group was inoculated with tumor cells, received intraperitoneal injection of normal saline for 7 days, followed by oral administration of normal saline for 10 days. The model group, melasrazine group, *Ligusticum striatum* fermentation group, *Ligusticum striatum*-*Ligusticum striatum*-*Astragalus membranaceus* fermentation group, *Ligusticum striatum*-*Polygonatum sibiricum* fermentation group, *Ligusticum striatum*-*Ginseng* fermentation group, and *Ligusticum striatum*-*Dendrobium officinale* fermentation group all received intraperitoneal injection of 5-FU (50 mg / kg / d) for 7 days, and were respectively administered normal saline, melasrazine (10 mg / kg / d), *Ligusticum striatum* fermentation (400 mg / kg / d), *Ligusticum striatum*-*Ligusticum striatum* fermentation (400 mg / kg / d), *Ligusticum striatum*-*Astragalus membranaceus* fermentation (400 mg / kg / d), and respectively. The bacteria were administered by gavage for 10 days, including *Ligustrum lucidum*-Polygonatum odoratum fermentation product (400 mg / kg / d), *Ligustrum lucidum*-Ginseng fermentation product (400 mg / kg / d), and *Ligustrum lucidum*-Dendrobium officinale fermentation product (400 mg / kg / d).

[0050] Step 2: During the experiment, mouse weight and diarrhea status were recorded every 2 days. The severity of diarrhea was assessed using the Bowen scoring system, which was divided into four levels based on stool consistency: 0: normal stool; 1: slightly soft stool, mild diarrhea; 2: wet and unformed stool, moderate diarrhea; 3: watery stool, severe diarrhea.

[0051] The results are as follows Figure 1 and Figure 2 As shown, the mice in the model group experienced a significant decrease in body weight and a continuous increase in diarrhea severity, while the 5-FU-induced weight loss and diarrhea in the *Ligusticum striatum*-*Lycium barbarum* bidirectional fermentation broth group significantly improved the 5-FU-induced weight loss and diarrhea in the mice.

[0052] Step 3: The day before the end of the experiment, mice in each group were fasted overnight but allowed free water for 12 hours. Mice were anesthetized with isoflurane, and blood was collected from behind the eye sockets. The blood was allowed to stand at room temperature for 30 minutes, then centrifuged at 3000 rpm for 20 minutes to separate the serum. The serum samples were aliquoted and stored at -80°C. The serum samples were then removed from the freezer and thawed on ice. The DAO content in the serum was measured strictly according to the kit instructions.

[0053] The results are as follows Figure 3 As shown, compared with the blank group, the DAO intestinal mucosal barrier index in the serum of mice in the model group was significantly increased, while the DAO activity level in the *Lycium barbarum*-*Lycium barbarum* bidirectional fermentation group was significantly decreased.

[0054] Step 4: After the experiment, mice were euthanized by cervical dislocation, and fresh ileal tissue was collected. The tissue was washed with physiological saline, fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin, and prepared into 8 μm sections. These sections were then stained with hematoxylin and eosin (H&E) for 5 min, dehydrated, and sealed with neutral glue. Pathological changes in the mouse ileal tissue were observed using an optical microscope, and villus length and crypt depth were recorded.

[0055] The results are as follows Figure 4 and Figure 5 As shown, the small intestinal tissue of the model group mice was damaged, the intestinal mucosal epithelium was not intact, the villi were broken, the villi height was reduced, and the crypt depth was increased. In the group of mice with the bifidobacteria-goji berry bidirectional fermentation product, the small intestinal villi were more intact, the villi height was significantly increased, and the crypt depth was significantly reduced.

[0056] Note: Data values ​​are expressed as mean ± SD. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001 is compared with the model group.

Claims

1. A method for preparing a bidirectional fermentation product of *Lycium barbarum* and *Lycium chinense*, characterized in that: The activated mycelium of *Bacillus lucida* was inoculated into a basic culture medium containing wolfberry, and after fermentation, a two-way fermentation broth was obtained. The two-way fermentation broth was filtered and centrifuged to collect the supernatant, which was then concentrated under reduced pressure and freeze-dried under vacuum to obtain *Bacillus lucida*-wolfberry two-way fermentation product powder. The basal culture medium containing wolfberry was prepared by the following method: Goji berry powder was added to the basal culture medium at a ratio of 1 g: 10 mL to obtain a basal culture medium containing goji berries. The preparation process of the basal culture medium is as follows: 200 g of peeled potatoes were chopped, 1 L of water was added, boiled for 30 min, filtered through 8 layers of gauze, 25 g of glucose was added, and sterilized at 121℃ for 30 min. Activation method of Glowing fungus: Select the preserved Glowing fungus mycelium and inoculate it onto PDA solid medium under aseptic conditions for activation. The activation conditions are 28℃ and 8-10 days to obtain activated Glowing fungus mycelium. The fermentation process was carried out at a constant temperature of 28℃ for 21 days.

2. The preparation method according to claim 1, characterized in that: The activated mycelium of *Gynostemma pentaphyllum* was inoculated into a basal culture medium containing wolfberry at an inoculation rate of 0.4%.

3. The preparation method according to claim 1, characterized in that: The filtration conditions for the two-way fermentation broth are: filtration through a 60-mesh sieve.

4. The preparation method according to claim 1, characterized in that: The centrifugation conditions are: 5000 rpm for 10-15 min.

5. The application of the *Lycium barbarum*-Lycium barbarum bidirectional ferment obtained by any one of the preparation methods of claims 1-4 in the preparation of pharmaceutical formulations, characterized in that: The drug formulation can improve chemotherapy-induced intestinal mucosal damage.

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