Leuconostoc mesenteroides subsp. Mesenteroides FTCM002 and application thereof in preparation of dendrobium officinale leavening with effects of reducing blood sugar, nourishing stomach and resisting inflammation

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to leuconostoc mesenteroides subsp. Mesenteroides FTCM002 and application thereof in preparation of dendrobium officinale leavening with the effects of reducing blood sugar, nourishing the stomach and resisting inflammation. Dendrobium officinale is subjected to enzymolysis and inoculated with the strain for fermentation, and the obtained fermented dendrobium officinale has the effect of inhibiting alpha-glucosidase; the gastric mucosal injury can be relieved, the expression of VEGF, IL-6, IL-8 and TNF-alpha in the gastric mucosal injury is reduced, and the effect of nourishing the stomach is achieved; the cell phagocytic rate can be increased, the NO concentration and the content of immune factors IL-6 and TNF-alpha can be increased, and the effect of enhancing the immunity is achieved; the expression quantity of IL-6, IL-1beta and TNF-alpha in inflammatory cells can be reduced, and the anti-inflammatory effect is achieved.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a subspecies of Leuconostoc mesenteroides FTCM002 and its application in the preparation of Dendrobium officinale fermentation products with hypoglycemic, stomach-nourishing and anti-inflammatory effects. Background Technology

[0002] Dendrobium is a perennial epiphytic herb belonging to the Orchidaceae family, with a rich variety of species distributed in China. Common species include Dendrobium huoshanense, Dendrobium officinale, and Dendrobium nobile. As a precious traditional Chinese medicine used for both medicinal and edible purposes, Dendrobium is hailed as the first of the "Nine Immortal Herbs of China." Among them, Dendrobium officinale is widely used due to its high medicinal value and well-established large-scale cultivation. Dendrobium officinale is slightly cold in nature and sweet in taste, entering the stomach and kidney meridians. It can nourish stomach yin, promote body fluid production and quench thirst, and also has the effects of clearing stomach heat. It is a traditional and precious Chinese medicine, known as "the gold among medicines." Dendrobium officinale is rich in various bioactive components, including polysaccharides, alkaloids, flavonoids, and polyphenolic compounds. Among them, Dendrobium officinale polysaccharides are the most abundant active ingredient, hence there is a large body of literature on its research.

[0003] For example, Chinese patent CN118725154A discloses a method for preparing low molecular weight Dendrobium officinale polysaccharide with both antioxidant and anti-inflammatory effects. The method includes: (1) washing, drying, and cutting fresh Dendrobium officinale stems into sections; blanching, pulping, filtering, and sterilizing to obtain Dendrobium officinale juice; (2) adding lactic acid bacteria powder to physiological saline solution for activation to obtain an activated solution; and (3) inoculating the Dendrobium officinale juice into the activated solution to obtain fermented Dendrobium officinale juice. The selected lactic acid bacteria are a mixed powder of Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus acidophilus. The Dendrobium officinale polysaccharide obtained by this method has anti-inflammatory and antioxidant effects. This patent focuses on Dendrobium officinale polysaccharide, with effects limited to anti-inflammatory and antioxidant properties, and the fermentation agent is a complex bacterial strain. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a Leuconostoc mesenteroides subsp. mesenteroides FTCM002 strain, which can significantly improve the hypoglycemic, stomach-nourishing, anti-inflammatory and immune-boosting effects of Dendrobium officinale fermentation.

[0005] The second objective of this invention is to provide a Dendrobium officinale fermentation product that can significantly improve the hypoglycemic effect.

[0006] The third objective of this invention is to provide a Dendrobium officinale fermented product that has a stomach-nourishing effect.

[0007] The fourth objective of this invention is to provide a Dendrobium officinale fermented product that enhances immunity.

[0008] The fifth objective of this invention is to provide a Dendrobium officinale fermentation product that has anti-inflammatory effects.

[0009] In this invention, "bacterial activity", "bacterial viability", "live count", "live content", and "live quantity" have the same meaning under certain conditions.

[0010] In this invention, "enzyme activity", "enzyme vitality", "enzyme activity" and "specific activity of enzyme" have the same meaning under certain conditions.

[0011] In order to solve the above-mentioned technical problems / achieve the above-mentioned objectives, or at least partially solve the above-mentioned technical problems / achieve the above-mentioned objectives, as a first aspect of the present invention, a Leuconostoc mesenteroides subsp. mesenteroides FTCM002 is provided, which is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 35016 and deposit date of June 26, 2025.

[0012] As a second aspect of the invention, the use of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 in the fermentation of *Dendrobium officinale* or the preparation of *Dendrobium officinale* fermented products is provided. In some embodiments of the invention, the use of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 as the sole fermentation strain in the preparation of *Dendrobium officinale* fermented products is provided.

[0013] As a third aspect of the present invention, a Dendrobium officinale ferment is provided, which is prepared by enzymatic hydrolysis of Dendrobium officinale and fermentation by inoculation with Leuconostoc mesenteroides subsp. FTCM002.

[0014] Optionally, the accession number of the Leuconostoc mesenteroides subsp. mesenteroides FTCM002 is CGMCC No.35016.

[0015] Optionally, the inoculation amount of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 is 1-5%. In some embodiments of the present invention, the inoculation amount of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 is 1%, 2%, 3%, 4%, 5%, or any value between two of these.

[0016] Optionally, the viability count of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 is 1.0 × 10⁻⁶. 9 CFU / mL or higher.

[0017] Optionally, the enzyme preparations used in the Dendrobium officinale enzymatic hydrolysate include cellulase, pectinase, and papain.

[0018] In some embodiments of the present invention, the amount of cellulase added is 0.3-0.7%. In other embodiments of the present invention, the amount of cellulase added is 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, or any value between the two.

[0019] In some embodiments of the present invention, the amount of pectinase added is 0.2-0.6%. In other embodiments of the present invention, the amount of pectinase added is 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, or any value between the two.

[0020] In some embodiments of the present invention, the amount of papain added is 0.1-0.4%. In other embodiments of the present invention, the amount of papain added is 0.1%, 0.2%, 0.3%, 0.4%, or any value between two of these.

[0021] Optionally, the cellulase has an enzyme activity of 10,000-30,000 U / g, the pectinase has an enzyme activity of 50,000-100,000 U / g, and the papain has an enzyme activity of 100,000-150,000 U / g. In some embodiments of the present invention, the cellulase has an enzyme activity of 10,000 U / g, the pectinase has an enzyme activity of 60,000 U / g, and the papain has an enzyme activity of 100,000 U / g.

[0022] As a fourth aspect of the present invention, a method for preparing Dendrobium officinale fermentation product of the present invention is provided, comprising the following steps: (1) after mixing Dendrobium officinale powder with water, adding an enzyme preparation for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, performing enzyme inactivation and sterilization to obtain an enzymatic hydrolysate; (2) inoculating Leuconostoc mesenteroides subsp. entericae FTCM002 into the enzymatic hydrolysate and performing fermentation to obtain a fermentation product.

[0023] Optionally, the accession number of the Leuconostoc mesenteroides subsp. mesenteroides FTCM002 is CGMCC No.35016.

[0024] Optionally, the preparation method further includes activating the *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 before fermentation. In some embodiments of the present invention, the activation method of the *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 is as follows: the strain is inoculated into MRS medium and cultured at 37°C for 24 hours, and passaged three times consecutively to fully activate the strain to a viable count of 1.0 × 10⁻⁶. 9 CFU / mL or higher.

[0025] In some other embodiments of the present invention, the MRS culture medium comprises: 10g peptone, 5g beef extract powder, 4g yeast extract powder, 20g glucose, 1g Tween-80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate (MgSO4·7H2O), and 0.5g manganese sulfate (MnSO4·4H2O).

[0026] Optionally, the ratio of Dendrobium officinale powder to water is 1:(10-30); in some embodiments of the present invention, the ratio of Dendrobium officinale powder to water can be 1:10, 1:15, 1:20, 1:25, 1:30 or any value between the two.

[0027] Optionally, the enzymatic hydrolysis time is 1-3 hours, and the enzymatic hydrolysis temperature is 45-55°C; in some embodiments of the present invention, the enzymatic hydrolysis temperature is 50°C, and the enzymatic hydrolysis time is 2 hours.

[0028] Optionally, the enzyme inactivation time is 8-12 minutes; in some embodiments of the present invention, the enzyme inactivation is performed in a boiling water bath for 10 minutes.

[0029] Optionally, the sterilization temperature is 110-120℃ and the time is 15-25 min; in some embodiments of the present invention, the sterilization temperature is 115℃ and the time is 20 min.

[0030] Optionally, the fermentation temperature is 35-42℃ and the fermentation time is 48-72h; in some embodiments of the present invention, the fermentation temperature is 37℃ and the time is 48h.

[0031] As a fifth aspect of this application, the application of the above-mentioned Dendrobium officinale fermentation product provided by the present invention or the Dendrobium officinale fermentation product obtained by the above preparation method in the preparation of Dendrobium officinale functional products is provided.

[0032] Optionally, the Dendrobium officinale functional products include products for lowering blood sugar, enhancing immunity, anti-inflammatory effects, and protecting the gastric mucosa.

[0033] As a sixth aspect of this application, a microbial preparation is provided, the active ingredient of which includes Leuconostoc mesenteroides subsp. enterica FTCM002.

[0034] Optionally, the microbial preparation may also include adjuvants for maintaining the preservation of the microorganisms.

[0035] Compared with the prior art, the present invention has the following advantages:

[0036] 1. The single strain of Leuconostoc mesenteroides FTCM002 provided by this invention can significantly improve the α-glucosidase inhibition rate of Dendrobium officinale fermentation products, thereby enhancing its stomach-nourishing, anti-inflammatory, and immune-enhancing effects.

[0037] 2. This invention provides a Dendrobium officinale fermentation product with a high α-glucosidase inhibition rate, which can be applied to hypoglycemic products.

[0038] 3. The Dendrobium officinale fermentation product provided by this invention can significantly improve the cell survival rate of GES-1 cells damaged by gastric mucosa and alleviate cell inflammation. It has been verified by rat animal experiments that it can reduce gastric mucosal damage and reduce the levels of TNF-α, IL-1β and IL-6 inflammatory factors in gastric tissue. Moreover, the effect is better than that of Dendrobium officinale polysaccharide after fermentation. It can be applied to products for nourishing the stomach and treating gastric ulcers and gastric mucosal damage.

[0039] 4. The Dendrobium officinale fermentation product provided by this invention can increase cell phagocytosis rate and increase the release of immune factors TNF-α, IL-6 and NO, thereby enhancing immunity and can be applied to products that enhance immunity.

[0040] 5. The Dendrobium officinale fermentation product provided by this invention can reduce the levels of inflammatory factors TNF-α, IL-1β, and IL-6, thereby achieving anti-inflammatory effects and can be applied to anti-inflammatory products.

[0041] Preservation Information

[0042] Leuconostoc mesenteroides subsp. mesenteroides FTCM002 was deposited on June 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35016. The depository is located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Attached Figure Description

[0043] Figure 1 The figure shows the effect of Dendrobium officinale samples provided by this invention on the survival rate of human gastric mucosal epithelial cell line GES-1; different letters represent significant differences between them, P<0.05;

[0044] Figure 2 The effects of fermentation supernatants of Dendrobium officinale obtained from fermentation with different lactic acid bacteria on the expression of inflammatory factors IL-6, IL-8, TNF-α, and VEGF in GES-1 cells were investigated. Different letters represent significant differences between them (P < 0.05).

[0045] Figure 3The effects of Dendrobium officinale polysaccharides obtained from different lactic acid bacteria fermentation on the expression of inflammatory factors IL-6, IL-8, TNF-α, and VEGF in GES-1 cells were investigated; different letters represent significant differences between them, P<0.05;

[0046] Figure 4 The figure shows the number and scoring results of gastric ulcers in rats obtained by fermenting Dendrobium officinale subsp. enterica FTCM002.

[0047] Figure 5 Macroscopic observation of gastric mucosal damage caused by Dendrobium officinale samples fermented with Leuconostoc mesenteroides subsp. FTCM002;

[0048] Figure 6 HE staining results of gastric tissue from rats with a gastric mucosal injury model, obtained by fermentation of Dendrobium officinale subsp. enterica FTCM002;

[0049] Figure 7 The results show the effects of Dendrobium officinale samples fermented from Leuconostoc mesenteroides subsp. FTCM002 on inflammatory factors IL-6, IL-1β, and TNF-α in the gastric tissue of rats with a gastric mucosal injury model; different letters represent significant differences between them, P<0.05;

[0050] Figure 8 The results show the effects of Dendrobium officinale samples obtained by fermentation of Leuconostoc mesenteroides subsp. enterica FTCM002 on serum inflammatory factors IL-6, IL-1β, and TNF-α in rats with a gastric mucosal injury model; different letters represent significant differences between them, P<0.05;

[0051] Figure 9 The molecular weight peak diagram of Dendrobium officinale polysaccharides obtained before fermentation and fermentation with Leuconostoc mesenteroides subsp. FTCM002 is shown.

[0052] Figure 10 The results show the effects of Dendrobium officinale fermentation broth provided by this invention on IL-6, IL-1β, and TNF-α in RAW264.7 cells; different letters represent significant differences between them, P<0.05;

[0053] Figure 11 The results show the effects of the Dendrobium officinale fermentation broth provided by this invention on the phagocytic rate, NO concentration, IL-6, IL-1β, and TNF-α of RAW264.7 cells; different letters represent significant differences between them, P<0.05. Detailed Implementation

[0054] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0055] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0056] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all chemical reagents used in the embodiments of the present invention are obtained through conventional commercial channels. Products from different manufacturers do not have a significant impact on the effectiveness.

[0057] Dendrobium officinale (Taining Dendrobium officinale, produced in Taining County, Sanming City, Fujian Province, a Chinese National Geographical Indication Product); cellulase was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., with an enzyme activity of 10,000 U / g; pectinase was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., with an enzyme activity of 60,000 U / g; papain was purchased from Guangzhou Solarbio Biotechnology Co., Ltd., with an enzyme activity of 100,000 U / g.

[0058] Leuconostoc mesenteroides subsp. XZ9302, obtained from the China Center for Type Culture Collection (CCTCC) with accession number CCTCC LB 2008044, is a commercially available strain.

[0059] Lactobacillus plantarum GXL94 has been disclosed in Chinese patent CN202311711349.1.

[0060] Leuconostoc mesenteroides XD054 is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M20241728. It has been published in Chinese patent CN119752679A.

[0061] Preparation Example 1: Activation of strain

[0062] (1) The composition and preparation method of MRS medium are as follows: 10g peptone, 5g beef extract powder, 4g yeast extract powder, 20g glucose, 1g Tween-80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate (MgSO4·7H2O), 0.5g manganese sulfate (MnSO4·4H2O), 1L distilled water, pH 6.2±0.2, sterilized at 121℃ for 20min.

[0063] (2) Activation of lactic acid bacteria: The fermentation strains are Leuconostoc mesenteroides FTCM002, Leuconostoc mesenteroides XD054, Leuconostoc mesenteroides XZ9302; and Lactobacillus plantarum GXL94.

[0064] The above-mentioned bacteria were inoculated separately into MRS medium and incubated at 37°C for 24 hours. The culture was then subcultured three times to fully activate the strains to a viable count of 1.0 × 10⁻⁶. 9 CFU / mL or higher.

[0065] Example

[0066] This embodiment provides a Dendrobium officinale fermentation product, which specifically includes the following steps:

[0067] (1) After mixing Dendrobium officinale powder and water at a ratio of 1:15, 0.5% cellulase, 0.5% pectinase and 0.1% papain were added, homogenized for 2 min, and enzymatically hydrolyzed in a 50℃ water bath for 2 h. After enzymatic hydrolysis, the enzyme was inactivated by boiling in a water bath for 10 min. Then, it was sterilized at 115℃ for 20 min to obtain Dendrobium officinale enzymatic hydrolysate;

[0068] (2) Dendrobium officinale enzymatic hydrolysate was inoculated with strains according to Table 1 (keeping the number of viable cells consistent during inoculation) and fermented at 37°C for 48 hours to obtain fermentation product.

[0069] Table 1 Inoculated strains

[0070]

[0071] Experimental Example 1: Determination of α-glucosidase inhibition rate

[0072] 1 Experimental Sample

[0073] The Dendrobium officinale fermentation product obtained in the example was centrifuged at 8000 rpm for 10 minutes and the supernatant was collected to obtain the Dendrobium officinale fermentation supernatant liquid, which was used as the sample to be tested.

[0074] Centrifuge the Dendrobium officinale enzymatic hydrolysate obtained in step (1) of the example at 8000 rpm for 10 minutes and collect the supernatant to obtain the Dendrobium officinale enzymatic hydrolysate supernatant, which is used as the sample to be tested;

[0075] 2 Experimental Methods

[0076] Take 0.2 mL of sample and add 1 mL of α-glucosidase solution (0.1 U / mL). Incubate at 37℃ for 10 min. Add 0.5 mL of 5 mmol / mL PNPG (4-nitrophenyl-β-D-glucopyranoside) solution and incubate at 37℃ for 10 min. Finally, add 1 mL of 0.1 mol / L Na₂CO₃ solution to terminate the reaction and measure the absorbance at 405 nm. The positive control is 0.5 µg / mL acarbose solution. The formula for calculating the α-glucosidase inhibition rate is as follows, and the detection results are shown in Table 2.

[0077] R4=[1-((AB) / (CD))]×100%

[0078] In the formula: R4: α-glucosidase inhibition rate; A: absorbance of the experimental group; B: absorbance of the experimental background group (pure water replaces enzyme solution); C: absorbance of the sterile, uninoculated group (pure water replaces sample); D: absorbance of the blank background group (pure water replaces sample and enzyme solution).

[0079] 3 Experimental Results

[0080] The results of fermentation with different lactic acid bacteria are shown in Table 2. As can be seen from Table 2, the α-glucosidase inhibition rate of the fermentation supernatant of Dendrobium officinale was improved in all cases. Among them, the effect of Leuconostoc mesenteroides subsp. enterica FTCM002 was the most significant, with its α-glucosidase inhibition rate increasing by 70.67% compared with the supernatant of the enzyme hydrolysate, and there was no significant difference compared with acarbose. This indicates that fermentation of Dendrobium officinale with Leuconostoc mesenteroides subsp. enterica FTCM002 can significantly improve the hypoglycemic effect of Dendrobium officinale fermentation broth.

[0081] The fermentation supernatant of Dendrobium officinale obtained from fermentation with Lactobacillus plantarum GXL94, Leuconostoc mesenteroides XD054, and Leuconostoc mesenteroides subsp. XZ9302 showed a significantly lower α-glucosidase inhibition rate than the fermentation broth of Dendrobium officinale obtained from fermentation with Leuconostoc mesenteroides subsp. FTCM002, indicating that not all lactic acid bacteria can achieve a superior α-glucosidase inhibition rate.

[0082] The α-glucosidase inhibition rate of the supernatant of *Dendrobium officinale* obtained by fermentation with a mixed strain of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 and *Lactobacillus plantarum* GXL94 was significantly lower than that of the supernatant of *Dendrobium officinale* obtained by fermentation with *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 alone was the optimal α-glucosidase inhibition rate.

[0083] Table 2. α-glucosidase inhibition rate of different Dendrobium officinale fermentation broths

[0084]

[0085] Note: Different letters in the same column represent significant differences, P<0.05.

[0086] Experiment Example 2: Cell Experiment - Ethanol-induced GES-1 Cell Damage in Mouse Gastric Mucosal Cells

[0087] 1. Sample to be tested:

[0088] The Dendrobium officinale fermentation product obtained in the example was centrifuged at 8000 rpm for 10 minutes and the supernatant was collected to obtain Dendrobium officinale fermentation supernatant.

[0089] Centrifuge the Dendrobium officinale enzymatic hydrolysate obtained in step (1) of the example at 8000 rpm for 10 minutes and collect the supernatant to obtain Dendrobium officinale enzymatic hydrolysate supernatant;

[0090] The *Dendrobium officinale* fermentation product obtained in the example was centrifuged at 8000 rpm for 10 minutes to collect the supernatant. The filtrate was concentrated by rotary evaporation. Four times the volume of 95% ethanol was added to the concentrated liquid, and the mixture was centrifuged at 4000 rpm for 15 minutes to obtain a precipitate, which was then washed three times with anhydrous ethanol. The precipitate was dissolved in an appropriate amount of ultrapure water to prepare a crude polysaccharide solution. An equal volume of Sevag reagent (chloroform: n-butanol = 4:1) was added to the crude polysaccharide solution, and the mixture was vortexed vigorously for 5 minutes. The mixture was then centrifuged at 4000 rpm for 15 minutes, and the supernatant was collected. Sevag reagent was added again, and the process was repeated 2-3 times until no protein layer was visible. The resulting supernatant was concentrated and freeze-dried to obtain *Dendrobium officinale* polysaccharide after fermentation.

[0091] The *Dendrobium officinale* enzymatic hydrolysate obtained in step (1) of the example was centrifuged at 6000 rpm for 10 min to collect the supernatant. After concentration by rotation, anhydrous ethanol was added to the supernatant until the ethanol concentration was 80% (v / v). The solution was allowed to stand at 4℃ for 12 h, and then centrifuged at 8000 rpm for 20 min to collect the precipitate. The precipitate was reconstituted with pure water (600 mL) and then deproteinized using the Sevag method. 1 / 4 volume of Sevag reagent (chloroform: n-butanol, V / V=4:1) was added to the crude polysaccharide solution, and the mixture was thoroughly mixed and vigorously shaken for 30 min. The liquid was then transferred to a centrifuge and centrifuged at 8000 rpm for 10 min to remove the intermediate denatured monolayer. The upper and lower layers were combined, and the deproteinization step was repeated until no protein precipitation occurred. The filtrate was collected and concentrated in a rotary evaporator under reduced pressure at 50℃. The filtrate was then freeze-dried to obtain the pre-fermentation *Dendrobium officinale* polysaccharide.

[0092] 2. Cell Culture Methods

[0093] The human gastric mucosal epithelial cell line GES-1 is an immortalized cell line. It was cultured in a complete culture medium containing 1640 medium and 10% fetal bovine serum at 37°C and 5% CO2 saturated humidity. When the cells reached a healthy state, with a monolayer covering 80%-90% of the culture flask wall, they were washed with PBS, digested with 0.25% trypsin solution, and passaged 2-3 times per week.

[0094] 3. Experimental grouping and modeling

[0095] Take GES-1 cells in good growth condition at a ratio of 100 μL per well and 1 × 10⁻⁶ cells per well. 4 Cells were seeded into each well of a 96-well plate and incubated in a CO2 incubator for 24 h. The cells were divided into three groups: normal group, model group, ranitidine group, Dendrobium officinale fermentation supernatant group, Dendrobium officinale polysaccharide group after fermentation, Dendrobium officinale enzymatic hydrolysis supernatant group, and Dendrobium officinale polysaccharide group before fermentation, with three replicates for each group.

[0096] After cell adhesion, complete culture medium was added to the normal group of GES-1 cells, while 8% ethanol was added to the other experimental groups. The cells were incubated in a CO2 incubator for 6 hours to establish the cell model. The supernatant was aspirated, and the cells were washed twice with PBS. After modeling, 100 μL of drug-free culture medium was added to the normal and model groups, while 100 μL of ranitidine-containing culture medium (160 μg / mL) was added to the ranitidine group. The drug-treated groups were incubated with 100 μL of drug-containing culture medium containing either pre-fermentation Dendrobium officinale polysaccharide (25 μg / mL), post-fermentation Dendrobium officinale polysaccharide (25 μg / mL), or Dendrobium officinale fermentation supernatant (2.5%), or Dendrobium officinale enzymatic hydrolysis supernatant (2.5%), respectively. All cells were then incubated in a CO2 incubator for another 24 hours.

[0097] 4. Determination of cell viability

[0098] After culture, the viability of GES-1 cells was measured by the MTT assay, and the cell survival rate was calculated. The above experiment was repeated 3 times.

[0099] Cell viability (%) = (OD of experimental group / OD of control group) × 100%

[0100] 5. Inflammatory factor measurement

[0101] After culturing, the normal group, model group, ranitidine group, *Dendrobium officinale* fermentation supernatant group, *Dendrobium officinale* enzymatic hydrolysis supernatant group, *Dendrobium officinale* polysaccharide group before fermentation, and *Dendrobium officinale* polysaccharide group after fermentation were centrifuged at 4000 r / min for 10 min, and the cell pellet was collected. Total mRNA was extracted using a kit (Nanjing Novizan, RC113-01), and cDNA was obtained by reverse transcription (Nanjing Novizan, RT01-01) and stored at -20℃. Using cDNA as a template, qPCR amplification was performed with SYBR Green I (20 μL system) to determine the expression levels of interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor (VEGF). GAPDH was used as an internal reference gene, and 2... -ΔΔCT The relative quantification of gene expression was calculated using a method, and the primer sequences are shown in Table 3.

[0102] Table 3 Gene Primer Sequences

[0103]

[0104] 6 Experimental Results

[0105] Adding ethanol to GES-1 cells (human gastric mucosal epithelial cells) causes gastric mucosal damage, and the core significance of cell viability is to quantify the degree of ethanol damage to the gastric mucosa. In this invention, ethanol is first added to the cells to establish a model, followed by the addition of different test samples, and the cell viability is measured. Figure 1 It can be seen that, compared with the model group, both the enzymatic hydrolysis supernatant and the fermentation supernatant significantly improved the survival rate of gastric mucosal cells damaged by ethanol. Among them, the cell survival rate of the supernatant of *Leuconostoc mesenteroides* subsp. *enteroides* fermented with *Dendrobium officinale* was the highest, indicating that the supernatant obtained by fermenting *Dendrobium officinale* subsp. *enteroides* with *Leuconostoc mesenteroides* subsp. *enteroides* was the most effective in alleviating ethanol-induced gastric mucosal damage.

[0106] Both before and after fermentation, Dendrobium officinale polysaccharides can improve the survival rate of gastric mucosal cells to a certain extent. Among them, the cell survival rate of Dendrobium officinale polysaccharides fermented by Leuconostoc mesenteroides FTCM002 is the highest. In summary, the fermentation supernatant and fermented Dendrobium officinale polysaccharides obtained by fermenting Dendrobium officinale with Leuconostoc mesenteroides FTCM002 have the best effect on alleviating gastric mucosal cell damage, and Leuconostoc mesenteroides FTCM002 is the best strain to improve gastric mucosal damage.

[0107] The gastric mucosa acts as a barrier against external stimuli. Damage to this barrier directly triggers an inflammatory response. Therefore, the addition of ethanol to GES-1 cells (human gastric mucosal epithelial cells) also causes the release of inflammatory factors (VEGF, IL-6, TNF-α, IL-8). Figure 2 It was found that both the fermentation supernatant and the enzymatic hydrolysis supernatant (enzymatic hydrolysate) inhibited the expression of inflammatory factors VEGF, IL-6, IL-8, and TNF-α genes. Among them, the expression levels of inflammatory factors VEGF, IL-6, IL-8, and TNF-α in the *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 fermentation supernatant group were significantly lower than those in other fermentation supernatant groups and the enzymatic hydrolysis supernatant group, and showed no significant difference compared to the positive control group (ranitidine group). This indicates that the *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 fermentation supernatant of *Dendrobium officinale* has the best efficacy in inhibiting the expression of inflammatory factors in gastric mucosal injury. *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 is the optimal strain for fermenting *Dendrobium officinale*, used to inhibit the expression of inflammatory factors, alleviate gastric mucosal inflammation, and achieve the effect of nourishing and protecting the stomach.

[0108] Depend on Figure 3 It was found that the polysaccharide from Dendrobium officinale fermented with Leuconostoc mesenteroides FTCM002 had the most significant inhibitory effect on the gene expression of inflammatory factors VEGF, IL-6, IL-8, and TNF-α, indicating that Leuconostoc mesenteroides FTCM002 is the optimal strain for alleviating intracellular inflammation of gastric mucosal cells, and that fermented Dendrobium officinale polysaccharide has the best effect on nourishing and protecting the stomach.

[0109] In summary, in in vitro cell experiments, the supernatant of Dendrobium officinale obtained by single-strain fermentation of Leuconostoc mesenteroides FTCM002 and the fermented Dendrobium officinale polysaccharide selected in this invention can alleviate gastric mucosal damage and reduce inflammatory factors, and the effect is better than that of the enzymatic hydrolysis group and other lactic acid bacteria fermentation groups.

[0110] Experiment Example 3: Animal Experiments on Stomach Nourishment

[0111] 1. Samples to be tested: fermentation supernatant of Dendrobium officinale obtained by fermentation of Leuconostoc mesenteroides FTCM002; enzymatic hydrolysis supernatant of Dendrobium officinale; Dendrobium officinale polysaccharide before fermentation; Dendrobium officinale polysaccharide after fermentation of Leuconostoc mesenteroides FTCM002. The preparation method is the same as in Experiment Example 2.

[0112] 2. Experimental animals, grouping, and administration

[0113] Eight-week-old male SD rats were used to conduct a gastrointestinal health-promoting experiment with Dendrobium officinale. The animals were divided into a control group, a model group, a Dendrobium officinale enzymatic hydrolysis supernatant group (20 mL / kg), a Dendrobium officinale supernatant fermented with Leuconostoc mesenteroides FTCM002 group (20 mL / kg), a Dendrobium officinale polysaccharide group before fermentation (200 mg / kg), and a Dendrobium officinale polysaccharide group after fermentation with Leuconostoc mesenteroides FTCM002 (200 mg / kg). Ten rats were administered the drugs to each group. The drugs were administered once daily. Rats in all groups were strictly fasted for 24 hours after gavage on day 13. Except for the control group, the other groups underwent model induction with 5 mL / kg anhydrous ethanol 2 hours after drug administration on day 14.

[0114] Gavage dosage: The gavage dosage of the fermentation supernatant was obtained by doubling the daily dosage of 100 mL of fermented beverage for humans. During the experiment, the inventors tested the polysaccharide content of the fermentation supernatant of Dendrobium officinale obtained by fermenting Leuconostoc mesenteroides FTCM002. They found that the polysaccharide content in 20 mL of fermentation supernatant was less than 200 mg. Therefore, in the rat experiment, 200 mg was used as the gavage dosage of fermented Dendrobium officinale polysaccharide to evaluate the stomach-nourishing effect of fermentation supernatant and fermented Dendrobium officinale polysaccharide.

[0115] 3. Detection of gastric mucosal ulcer sites and gastric mucosal damage

[0116] The gastric mucosal injury score, ulcer sites, and related pathological indicators were assessed. Specific scoring criteria: 0 points: No bleeding or erosion observed in the gastric mucosa. 1 point: 1-2 sites of bleeding or erosion observed in one gastric region. 2 points: 3-5 sites of bleeding or erosion observed in one gastric region. 3 points: Bleeding or erosion observed in two gastric regions, or 6 or more sites of bleeding or erosion observed in one gastric region but no more than 10 sites in the entire stomach. 4 points: Bleeding or erosion observed in 3 or more gastric regions, or 11 or more sites of bleeding or erosion observed in the entire stomach.

[0117] 4. Measurement of inflammatory factors

[0118] One hour after modeling, rats were anesthetized with isoflurane, and blood was collected from the abdominal aorta. After the blood was allowed to stand for 2 hours, it was centrifuged at 4°C, 1500×g for 15 minutes, and the serum was aspirated. The levels of TNF-α, interleukin-1β (IL-1β), and IL-6 in the blood of rats in each group were measured by enzyme-linked immunosorbent assay (ELISA).

[0119] Take an appropriate amount of gastric tissue, weigh it, grind it into powder on ice, add physiological saline at a mass:volume ratio of 1:9 to obtain a 10% tissue homogenate, centrifuge at 5000×g for 10 min at 4℃, collect the supernatant, and use enzyme-linked immunosorbent assay (ELISA) to determine the contents of TNF-α, IL-1β and IL-6 in the gastric tissue of rats in each group. The detection steps are all performed according to the kit instructions (Beijing Solarbio Science & Technology Co., Ltd., SEKM-0034(HS), SEKM-0002(HS), SEKM-0007).

[0120] 5. Observation of gastric mucosal tissue morphology (hematoxylin-eosin staining)

[0121] The most severely damaged 1cm×1cm gastric mucosa was cut from each group of rats, fixed with tissue fixative, embedded in paraffin, and prepared into 4μm sections using standard methods. The sections were stained with hematoxylin and eosin (H&E) and observed under a microscope.

[0122] 6 Experimental Results

[0123] Figure 4-8 In the above, the water extract treatment group was the supernatant of Dendrobium officinale enzymatic hydrolysis, the unfermented polysaccharide treatment group was the Dendrobium officinale polysaccharide group before fermentation, the fermentation broth treatment group was the supernatant of Dendrobium officinale fermented with Leuconostoc mesenteroides FTCM002, and the fermented polysaccharide treatment group was the Dendrobium officinale polysaccharide group after fermentation with Leuconostoc mesenteroides FTCM002.

[0124] Gastric mucosal injury is a basic pathological condition. Bleeding and ulcers are different manifestations of gastric mucosal injury. Bleeding is an accompanying symptom after the injury breaks through blood vessels, while ulcers are a type of lesion after the injury reaches a certain depth. The gastric ulcer injury score is a standardized quantitative scoring system that integrates the two and other injury characteristics to objectively assess the severity of gastric mucosal lesions.

[0125] Macroscopic observation images of gastric mucosal injury, including gastric mucosal injury scores, ulcer sites, and gastric mucosal tissue morphology. Figure 4 and 5 .

[0126] Depend on Figure 4 and Figure 5 It can be seen that bleeding was clearly observed in the gastric tissue of the model group, with a darker color, and the number and score of gastric ulcers were relatively high, indicating that the model group had symptoms such as gastric mucosal damage and bleeding.

[0127] The gastric tissue of the *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 fermentation supernatant group showed no bleeding points and was lighter in color. Furthermore, the gastric ulcer index and gastric ulcer point score were significantly lower than those of the model group. This means that the *Dendrobium officinale* supernatant obtained from the fermentation of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 provided by this invention can effectively alleviate gastric ulcers and gastric damage, and may even completely avoid alcohol damage to the gastrointestinal mucosa.

[0128] Furthermore, the gastric tissue structure, number of gastric ulcers, and gastric ulcer score of the fermentation supernatant group of Leuconostoc mesenteroides FTCM002 were all superior to those of the fermented Dendrobium officinale polysaccharide group and the Dendrobium officinale enzymatic hydrolysis supernatant group. This indicates that the protective effect of the fermentation supernatant group of Leuconostoc mesenteroides FTCM002 on the gastric mucosa of rats is superior to that of Dendrobium officinale polysaccharide.

[0129] Results of HE staining of gastric tissue are shown in Figure 6 , Figure 6 The control group showed no obvious gastric mucosal damage. The aqueous extract treatment group showed inflammatory cell infiltration (+), the unfermented polysaccharide treatment group showed inflammatory cell infiltration (++), the model group showed gastric wall congestion and inflammatory cell infiltration (+++), the fermentation broth treatment group showed inflammatory cell infiltration (+), and the fermented polysaccharide treatment group showed inflammatory cell infiltration (+). The results indicate that the fermentation supernatant of Leuconostoc mesenteroides FTCM002 has a good alleviating effect on gastric mucosal damage and inflammation in rats.

[0130] Results of ELISA assays in rat gastric tissue and blood are shown below. Figure 7-8 ,

[0131] Figure 7-8 In this study, the blank group was the model group, the pre-fermentation polysaccharide was the pre-fermentation Dendrobium officinale polysaccharide group, the post-fermentation polysaccharide was the post-fermentation Dendrobium officinale polysaccharide group of Leuconostoc mesenteroides FTCM002, the pre-fermentation supernatant was the Dendrobium officinale enzymatic hydrolysis supernatant group, and the post-fermentation supernatant was the Dendrobium officinale fermentation supernatant group of Leuconostoc mesenteroides FTCM002.

[0132] Depend on Figure 7 The results of inflammatory factor detection in gastric tissue showed that the contents of TNF-α, IL-1β, and IL-6 in the supernatant after fermentation were significantly lower than those in the model group, the supernatant before fermentation, and the polysaccharide group after fermentation. This indicates that the fermentation broth of Dendrobium officinale obtained by fermenting Leuconostoc mesenteroides FTCM002 can reduce the level of inflammatory factors in damaged gastric tissue, and its effect is better than that of the Dendrobium officinale polysaccharide group and the Dendrobium officinale enzymatic hydrolysis supernatant group after fermentation of Leuconostoc mesenteroides FTCM002.

[0133] Depend on Figure 8The results of serum inflammatory factor detection showed that the TNF-α content in the pre-fermentation supernatant and post-fermentation polysaccharide was not significantly lower than that in the model group, indicating that the fermentation of Dendrobium officinale polysaccharide from Leuconostoc mesenteroides FTCM002 had no effect on the TNF-α content in the serum of rats with gastric injury. However, the TNF-α content in the post-fermentation supernatant was significantly lower than that in the model group, indicating that the fermentation broth of Dendrobium officinale obtained from the fermentation of Leuconostoc mesenteroides FTCM002 can reduce the TNF-α content in the serum of rats with gastric injury.

[0134] The results of in vivo rat experiments showed that the fermentation supernatant of Dendrobium officinale obtained by fermenting Leuconostoc mesenteroides subsp. enterica FTCM002 could alleviate gastric mucosal damage, significantly inhibit the secretion of TNF-α, IL-1β, and IL-6 in rat gastric tissue, and the secretion of TNF-α in serum, and the effects were better than those of Dendrobium officinale polysaccharide after fermentation.

[0135] In conclusion, the fermentation supernatant obtained from the fermentation of Leuconostoc mesenteroides subsp. mesenteroides FTCM002 can reduce the expression levels of inflammatory factors TNF-α, IL-1β, and IL-6 in a rat model of gastric mucosal injury, reduce the body's inflammatory response, improve ethanol-induced gastric mucosal injury and inflammatory cell infiltration, and achieve a protective effect on the gastric mucosa.

[0136] Experiment Example 4: Effect of Fermentation on the Molecular Weight of Dendrobium officinale

[0137] 1 Experimental Sample

[0138] Polysaccharides from Dendrobium officinale after fermentation and before fermentation of *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002.

[0139] 2. Polysaccharide extraction and purification methods

[0140] A 20 mg / mL polysaccharide solution was prepared using deionized water, and the precipitate was removed by centrifugation. The top of the chromatography column was opened, and the centrifuged, precipitate-free polysaccharide solution was slowly and evenly added to the column using a disposable dropper. Distilled water was used as the eluent, and the sample added to the column was eluted. The pump speed was adjusted to control the flow rate at approximately 1 mL / min. The eluent was collected using an automatic collector at a rate of one tube every 5 minutes, for a total of 35 tubes. After collection, the eluent in each tube was analyzed sequentially using the phenol-sulfuric acid method. The absorbance of the measured solution was measured at 490 nm, and an elution curve of the polysaccharide was plotted based on the experimental results. The elution results of each component were analyzed in conjunction with the elution curve, and fractions with the same characteristics were combined. After completion, the purified polysaccharide was obtained by freeze-drying.

[0141] 3. Polysaccharide molecular weight determination

[0142] A 1 mg / mL polysaccharide solution was prepared, filtered through a 0.22 μm filter to remove impurities, and then injected. High-performance size exclusion chromatography (HPLC) equipped with a multi-angle laser dispersive spectroscopy and differential refractive index detector was used to analyze the molecular weight distribution of the polysaccharide. Specific conditions were as follows: OHpakSB-806 HQ column (8.0 mm × 300 mm) in dual-column tandem; mobile phase: 0.1 M NaNO3 (dissolved in 0.02% NaN3 aqueous solution); flow rate: 0.6 mL / min; injection volume: 100 μL; column temperature: 35 °C.

[0143] 4 Experimental Results

[0144] Dextran gel column chromatography was used to separate and purify Dendrobium polysaccharides before and after fermentation, and their molecular weight was determined. The results are shown in the figure. Figure 9 Based on the glucose elution time, the molecular weight of Dendrobium officinale polysaccharide decreased from 4551.94 Da to 2111.62 Da before and after fermentation, a reduction of 53.6%. This indicates that fermentation can reduce the molecular weight of Dendrobium officinale polysaccharide, possibly due to the degradation of large polysaccharide molecules caused by a large amount of enzymes produced by Leuconostoc mesenteroides subsp. mesenteroides. The smaller the molecular weight of the polysaccharide, the smaller its volume. Smaller polysaccharides can easily penetrate cell membranes, which is beneficial for the polysaccharide to exert its own activity and can improve its absorption and utilization rate in the intestine.

[0145] Experiment Example 5: Cell Experiment - Anti-inflammatory Experiment

[0146] 1 Sample to be tested

[0147] The preparation methods for the fermentation supernatant and enzymatic hydrolysis supernatant of Dendrobium officinale are the same as those in Experiment Example 2.

[0148] 2 Experimental Methods

[0149] RAW264.7 cells were subcultured in DMEM complete medium at 37°C in a 5% CO2 incubator. The density was 5 × 10⁶ cells / year. 3RAW264.7 cells at a density of 500 μL / well were seeded into 24-well plates and cultured stably for 24 h. Groups were established: a control group, a model group, and experimental groups (Dendrobium officinale fermentation supernatant group and Dendrobium officinale enzymatic hydrolysis supernatant group). Except for the control group, all other groups were treated with lipopolysaccharide (LPS) solution (1 μg / mL) for 24 h. Subsequently, the experimental groups were pretreated with 0.78% (the highest mass concentration for 100% cell viability) of the test sample for 24 h. Cell supernatants were collected, and the concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit for mouse cytokines, according to the kit instructions (Beijing Solarbio Science & Technology Co., Ltd., SEKM-0034(HS), SEKM-0002(HS), SEKM-0007).

[0150] 3 Experimental Results

[0151] Results analysis:

[0152] LPS is a pro-inflammatory inducer. When added, it produces a strong inflammatory response in cells. It is necessary to inhibit the production of inflammatory factors to achieve anti-inflammatory effects. Figure 10 The results showed that in LPS-induced inflammatory macrophages, the levels of inflammatory factors TNF-α, IL-6, and IL-1β in the supernatant of fermented *Dendrobium officinale* were significantly lower than those in the model group, indicating that lactic acid bacteria fermentation can enhance the anti-inflammatory efficacy of *Dendrobium officinale* fermentation supernatant. Among these, the supernatant obtained from fermentation with *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 showed the strongest inhibitory effect on the levels of lipopolysaccharide-induced pro-inflammatory factors IL-1β, IL-6, and TNF-α, significantly lower than other lactic acid bacteria and the model group. This indicates that *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 is the optimal strain for enhancing the anti-inflammatory efficacy of *Dendrobium officinale* fermentation products.

[0153] Experiment Example 6: Cell Experiment - Immunostimulation Activity Assay

[0154] 1 Sample to be tested

[0155] The preparation methods for the fermentation supernatant and enzymatic hydrolysis supernatant of Dendrobium officinale are the same as those in Experiment Example 2.

[0156] 2. Experimental methods for detecting immune factors

[0157] RAW264.7 cells at a density of 5 × 10³ cells / mL were seeded at 500 μL per well in 24-well plates and cultured stably for 24 h. Groups were established: blank group, LPS group, and experimental groups (Dendrobium officinale fermentation supernatant group and Dendrobium officinale enzymatic hydrolysis supernatant group). Untreated cells served as the blank group (NC), cells treated with only 1 μg / mL LPS solution served as the LPS group, and cells treated with 0.78% of the test sample and 1 μg / mL LPS solution served as the experimental groups for 24 h. Cell supernatants were collected, and the concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and nitric oxide (NO) were measured using a commercial enzyme-linked immunosorbent assay (ELISA) kit for mouse cytokines, according to the kit instructions (Beijing Solarbio Science & Technology Co., Ltd., SEKM-0034(HS), SEKM-0002(HS), SEKM-0007, BC1475).

[0158] 3. Experimental methods for macrophage activity assay

[0159] RAW 264.7 cells (5 × 10⁻⁶) 3 RAW 264.7 cells were seeded in 96-well plates at 100 μL per well (cells / mL). After the cells resumed adherent growth, the experimental group was treated with the test sample (100 μL of 1640 complete medium containing 1 / 32 of the original test sample solution per well) and lipopolysaccharide (LPS 1.174 μg / mL); the control group received no treatment; and the LPS group received only LPS (1.174 μg / mL). After culturing RAW 264.7 cells for 24 h, neutral red dye was added and incubated for 1 h to remove the neutral red dye. The cells were washed three times with PBS at 37°C, and then lysed with acetic acid-ethanol. After incubation at 37°C for 2 h, the absorbance was measured at 540 nm.

[0160] 4 Experimental Results

[0161] LPS activates RAW264.7 cells, increasing their phagocytic rate (directly enhancing their ability to clear pathogens) and inducing the release of immune factors (such as TNF-α, IL-6, and IL-1β) and NO. These substances recruit more immune cells (such as T cells) to participate in the response, forming an "immunoenhancing effect."

[0162] The experimental results on the immune-boosting effect on macrophages are shown in Figure 11Macrophages not only initiate innate immune responses but also participate in cellular immune responses. Activating macrophages is the first step in the immune response, and enhanced phagocytosis is a key indicator of improved macrophage-mediated immunity, reflecting their ability to engulf and clear exogenous pathogens and endogenous dead cells. Results showed that the phagocytic rate of the supernatant from fermented *Dendrobium officinale* was significantly higher than that of the enzymatically hydrolyzed supernatant. Among these, the supernatant from fermented *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 showed the strongest promotion of macrophage phagocytosis, with no significant difference compared to the LPS group. This indicates that the supernatant obtained from fermenting *Dendrobium officinale* with *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 best phagocytosed and eliminated exogenous antigens and enhanced cellular immunity.

[0163] The release of cytotoxic molecules such as NO is the second step in cellular defense against external pathogens, and its release level is typically used to assess the immunomodulatory activity of macrophages. Macrophages can produce NO, which reacts with superoxide anion free radicals to form peroxynitrosoanions, assisting the body in resisting foreign antigens and thus achieving immunomodulatory effects. Figure 11 It can be seen that the supernatant obtained from the fermentation of Dendrobium officinale by Leuconostoc mesenteroides FTCM002 significantly increased the NO concentration in RAW264.7 cells compared to the enzymatic digestion group and other groups, but showed no significant difference compared to the LPS group. This indicates that the supernatant obtained from the fermentation of Dendrobium officinale by Leuconostoc mesenteroides FTCM002 can promote the release of NO, thereby achieving the effects of resisting foreign antigens and immunomodulation.

[0164] Interleukins (IL-6, IL-8) and tumor necrosis factor are key substances regulating immune activity. Interleukins primarily participate in regulating the proliferation of immune cells, while tumor necrosis factor is mainly responsible for regulating cell proliferation, differentiation, and effector functions. Figure 11 It can be seen that the contents of IL-6 and TNF-α in the supernatant of Dendrobium officinale after fermentation are significantly higher than those in the Dendrobium officinale enzymatic hydrolysate group. Among them, the contents of IL-6 and TNF-α in the supernatant of Dendrobium officinale after fermentation of Leuconostoc mesenteroides FTCM002 are the highest. The contents of IL-6 are not significantly different from those in the LPS group, indicating that the supernatant of Dendrobium officinale fermented by Leuconostoc mesenteroides FTCM002 has strong immune-enhancing activity.

[0165] In summary, the supernatant of Dendrobium officinale obtained by fermentation of Leuconostoc mesenteroides subsp. enterica FTCM002 enhances the immune activity by increasing cell phagocytosis rate, NO concentration, IL-6 and TNF-α content.

[0166] In summary, this invention provides *Leuconostoc mesenteroides* subsp. *enteroides* FTCM002 as a preferred strain. The fermented *Dendrobium officinale* broth obtained from its fermentation exhibits several advantages: First, it inhibits the inhibition rate of α-glucosidase, thus lowering blood sugar. Second, in vivo rat experiments revealed that it can inhibit the production of inflammatory factors TNF-α, IL-6, and IL-1β, alleviating gastric mucosal damage, with effects superior to *Dendrobium officinale* polysaccharides. Third, in vitro cell experiments showed that it can inhibit IL-6, IL-8, TNF-α, and VEGF, reducing inflammation and demonstrating anti-inflammatory effects. Fourth, cell experiments revealed that it can enhance phagocytosis, increase NO concentration, IL-6 and TNF-α levels, and enhance immune activity.

[0167] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. Leuconostoc mesenteroides subsp. mesenteroides FTCM002, characterized in that, The preservation number of the Leuconostoc mesenteroides subsp. mesenteroides FTCM002 is CGMCC No. 35016, which is preserved in the China General Microbiological Culture Collection Center on June 26, 2025.

2. The application of the Leuconostoc mesenteroides subsp. mesenteroides FTCM002 in the preparation of a Dendrobium officinale fermentation product or fermented Dendrobium officinale according to claim 1.

3. A Dendrobium candidum fermentate, characterized by, The Dendrobium officinale fermentation product is prepared by enzymatic hydrolysis of Dendrobium officinale and fermentation of the Leuconostoc mesenteroides subsp. mesenteroides FTCM002, and the preservation number of the Leuconostoc mesenteroides subsp. mesenteroides FTCM002 is CGMCC No. 35016.

4. The Dendrobium candidum ferment according to claim 3, characterized in that, The enzyme preparation used for the enzymatic hydrolysis includes cellulase, pectinase, and papain; The addition amount of the cellulase is 0.3-0.7%, the addition amount of the pectinase is 0.2-0.6%, and the addition amount of the papain is 0.1-0.4%.

5. The Dendrobium candidum ferment according to claim 3, characterized in that, The inoculation amount of the Leuconostoc mesenteroides subsp. mesenteroides FTCM002 is 1-5%. The viable cell count of Leuconostoc mesenteroides subsp. mesenteroides FTCM002 is above 1.0 x 10 9 CFU / mL.

6. A method for preparing the Dendrobium candidum fermentate according to any one of claims 3 to 5, characterized by, It includes: (1) After the Dendrobium officinale powder is mixed with water, enzyme preparation is added for enzymatic hydrolysis, and after the enzymatic hydrolysis is completed, enzyme inactivation and sterilization are performed to obtain an enzymatic hydrolysate; (2) The Leuconostoc mesenteroides subsp. mesenteroides FTCM002 is inoculated in the enzymatic hydrolysate for fermentation to obtain a fermentation product; and the preservation number of the Leuconostoc mesenteroides subsp. mesenteroides FTCM002 is CGMCC No. 35016.

7. The preparation method according to claim 6, characterized in that, The enzymatic hydrolysis time is 1-3 h, and the enzymatic hydrolysis temperature is 45-55℃; the enzyme inactivation time is 8-12 min; The sterilization temperature is 110-120℃, and the sterilization time is 15-25 min; The fermentation temperature is 35-42℃, and the fermentation time is 48-72 h.

8. The application of the Dendrobium officinale fermentation product according to any one of claims 3-5 or prepared by the preparation method according to any one of claims 6-7 in the preparation of a Dendrobium officinale functional product.

9. Use according to claim 8, characterized in that, The Dendrobium officinale functional product includes a blood glucose-lowering product, an immunity-enhancing product, an anti-inflammatory product, and a gastric mucosa-protecting product.

10. A microbial preparation, characterized in that, The active ingredient includes the Leuconostoc mesenteroides subsp. mesenteroides FTCM002 according to claim 1.

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