Lactobacillus rhamnosus ZKLGG0310, its Dendrobium officinale fermentation liquid and application in preparation of hypoglycemic drugs

Abstract

This invention relates to the field of microbial technology, specifically to a strain of *Lactobacillus rhamnosus* ZKLGG0310, its *Dendrobium officinale* fermentation broth, and its application in the preparation of hypoglycemic drugs. *Lactobacillus rhamnosus* (ZKLGG0310) Lactobacillus rhamnosus Lactobacillus rhamnosus ZKLGG0310 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 13, 2024, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 30547. This invention utilizes Lactobacillus rhamnosus ZKLGG0310 to ferment Dendrobium officinale extract, significantly improving the hypoglycemic effect of Dendrobium officinale while regulating lipid metabolism and alleviating complications of type 2 diabetes.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a strain of Lactobacillus rhamnosus ZKLGG0310, its Dendrobium officinale fermentation broth, and its application in the preparation of hypoglycemic drugs. Background Technology

[0002] In recent years, the incidence of diabetes has been on the rise, with type 2 diabetes (T2DM) being the most common, accounting for over 90% of all diabetes cases. Type 2 diabetes is a chronic disease caused by insufficient insulin use or reduced insulin efficiency, commonly affecting adults and also known as adult-onset diabetes. Type 2 diabetes is generally caused by a combination of genetic factors and environmental factors such as unhealthy lifestyle habits, overnutrition, and insufficient physical activity. The disease often presents with mild symptoms in its early stages, making it easy to overlook. As the disease progresses, it is usually only discovered and diagnosed after complications develop. The accumulation of complications increases the difficulty and cost of treatment. Complications of type 2 diabetes mainly include cardiovascular disease, kidney disease, neuropathy, eye disease, and infectious diseases. These complications severely impact the patient's quality of life, and if blood sugar is not controlled for a long period, they can even be life-threatening.

[0003] Currently, type 2 diabetes is mainly controlled through medication. Common oral hypoglycemic agents include sulfonylureas, meglitinides, thiazolidinediones, biguanides, alpha-glucosidase inhibitors, dipeptidyl peptidase-4 inhibitors, and sodium-glucose cotransporter-2 inhibitors. While oral hypoglycemic agents can lower blood sugar, they are prone to causing side effects such as functional damage to pancreatic islet cells and gastrointestinal disorders.

[0004] The *Huangdi Neijing* (Yellow Emperor's Inner Classic) records the main treatment method for diabetes as "nourishing yin and generating fluids, balancing yin and yang." According to the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), *Dendrobium officinale* "nourishes the five internal organs, relieves weakness and fatigue, and strengthens yin." The 2010 edition of the *Chinese Pharmacopoeia* records its effects of benefiting the stomach and generating fluids, nourishing yin and clearing heat, and it can be used for yin deficiency and fluid depletion, dry mouth and thirst, etc. Modern pharmacological research shows that *Dendrobium officinale* has a certain hypoglycemic effect, but it usually needs to be used in combination with other oral hypoglycemic drugs to control blood sugar within the normal range. Multiple drugs can interact during combined use, posing a potential risk of side effects. Summary of the Invention

[0005] To address the technical problem of potential side effects associated with the combined use of Dendrobium officinale with other oral hypoglycemic drugs, this invention provides a strain of Lactobacillus rhamnosus ZKLGG0310, its Dendrobium officinale fermentation broth, and its application in the preparation of hypoglycemic drugs. By using Lactobacillus rhamnosus ZKLGG0310 to ferment Dendrobium officinale extract, the hypoglycemic effect of Dendrobium officinale is significantly improved, while lipid metabolism is regulated, alleviating complications of type 2 diabetes.

[0006] In a first aspect, the present invention provides a strain of Lactobacillus rhamnosus ZKLGG0310, Lactobacillus rhamnosus ( Lactobacillus rhamnosus ZKLGG0310 was deposited on May 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.30547.

[0007] Secondly, the present invention provides a fermentation broth of Dendrobium officinale containing Lactobacillus rhamnosus ZKLGG0310, which is made from Dendrobium officinale extract with a content of 1.0 g / mL of fermented Lactobacillus rhamnosus ZKLGG0310 as described above.

[0008] Furthermore, the preparation method of Dendrobium officinale extract includes the following steps:

[0009] Step S1: Crush Dendrobium officinale to make Dendrobium officinale powder;

[0010] Step S2: Add 1 part by weight of Dendrobium officinale powder to 8 parts by weight of water to soak, and obtain Dendrobium officinale soaking solution;

[0011] Step S3: After boiling the Dendrobium officinale soaking solution, continue to simmer at a gentle boil, and then separate the liquid and residue;

[0012] Step S4: The liquid is boiled and concentrated to obtain Dendrobium officinale extract with a crude drug content of 1.0 g / mL.

[0013] Furthermore, in step S1, the particle size of the Dendrobium officinale powder is 70-300 mesh.

[0014] Furthermore, in step S2, the Dendrobium officinale powder is placed into a decoction bag and soaked.

[0015] Furthermore, in step S2, the soaking time is 0.5-1.5 hours, preferably 1 hour.

[0016] Furthermore, in step S3, the simmering time is 1.5-2.5 hours, preferably 2 hours.

[0017] Furthermore, the preparation method of *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth includes the following steps:

[0018] Step 1: Add yeast extract to Dendrobium officinale extract and mix well. Then sterilize at 121℃ for 15 minutes to obtain sterilized Dendrobium officinale extract. The amount of yeast extract is 0% (g / mL)-8% (g / mL), preferably 4% (g / mL).

[0019] Step 2: Prepare a live bacteria count of 1×10⁻⁶.11 A CFU / g Lactobacillus rhamnosus preparation, ZKLGG0310, was inoculated into sterilized Dendrobium officinale extract at an inoculum concentration of 0.1 g / L. After shaking well, the mixture was incubated at 37°C with aerobic conditions for 24 h to obtain the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth. The viable count of the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth was 1.0 × 10⁻⁶. 9 -1.81×10 9 CFU / mL. The pH of the fermentation broth of *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* is ≤4.5.

[0020] Furthermore, the Lactobacillus rhamnosus ZKLGG0310 formulation is in the form of powder, tablets, granules or drops, preferably powder, and more preferably lyophilized powder.

[0021] Thirdly, the present invention also provides the application of the *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth described above in the preparation of hypoglycemic drugs, including its application in the preparation of drugs for treating type 2 diabetes. Drugs prepared using the *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth can inhibit the activity of α-amylase and α-glucosidase, achieving in vitro hypoglycemia; reduce the risk of complications in type 2 diabetes; regulate glucose and lipid metabolism; and improve insulin sensitivity.

[0022] The beneficial effects of this invention are as follows:

[0023] This invention provides a strain of *Lactobacillus rhamnosus* ZKLGG0310, its fermentation broth for *Dendrobium officinale*, and its application in the preparation of hypoglycemic drugs. The acidic fermentation broth of *Lactobacillus rhamnosus* ZKLGG0310 for *Dendrobium officinale* extract, prepared by fermenting *Lactobacillus rhamnosus* ZKLGG0310 with *Dendrobium officinale*, has the following effects: 1. Lowering the pH value of the intestine, which is beneficial to maintaining the microecological balance in the intestine; 2. Inhibiting the activity of α-amylase and α-glucosidase, achieving in vitro hypoglycemia; 3. Improving insulin sensitivity by significantly increasing insulin levels and reducing insulin resistance; 4. Controlling blood glucose levels within the normal range for a longer period, reducing the risk of complications of type 2 diabetes; 5. Regulating glucose and lipid metabolism, reducing hyperlipidemia complications caused by high serum total cholesterol and / or triglycerides. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1This is a diagram of the colony morphology cultivated on the enrichment medium plate in Example 1.

[0026] Figure 2 These are microscopic images of the dried colonies from Example 1.

[0027] Figure 3 This is a line graph showing the pH and viable cell count of *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth 1 and *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth 5 in Example 3. Figure 3 In the diagram, the square broken line represents pH, and the circle broken line represents the number of viable bacteria.

[0028] Figure 4 This is a statistical graph showing the fasting blood glucose concentrations of mice in the NC, MC, DO, FDO, and DMBG groups in Example 5. Figure 4 In the text, different letters represent significant differences, P < 0.05.

[0029] Figure 5 The concentration of glycated hemoglobin in the serum of mice in the NC, MC, DO, FDO, and DMBG groups in Example 5 is shown. Figure 5 In the text, different letters represent significant differences, P < 0.05.

[0030] Figure 6 The values ​​represent the average blood glucose concentrations of mice in the NC, MC, DO, FDO, and DMBG groups after gavage administration of glucose aqueous solution at 0 min, 30 min, 60 min, 90 min, and 120 min in Example 5.

[0031] Figure 7 This is a statistical graph showing the area under the glucose tolerance curve for mice in the NC, MC, DO, FDO, and DMBG groups in Example 5. Figure 7 In the text, different letters represent significant differences, P < 0.05.

[0032] Figure 8 This is a statistical chart showing the average serum insulin concentrations of mice in the NC, MC, DO, FDO, and DMBG groups in Example 5. Figure 8 In the text, different letters represent significant differences, P < 0.05.

[0033] Figure 9 This is a statistical chart showing the insulin resistance index of mice in the NC, MC, DO, FDO, and DMBG groups in Example 5. Figure 9 In the text, different letters represent significant differences, P < 0.05.

[0034] Figure 10This is a statistical chart showing the average serum TC levels of mice in the NC, MC, DO, FDO, and DMBG groups in Example 5. Figure 10 In the text, different letters represent significant differences, P < 0.05.

[0035] Figure 11 This is a statistical chart showing the average serum TG levels of mice in the NC, MC, DO, FDO, and DMBG groups in Example 5. Figure 11 In the text, different letters represent significant differences, P < 0.05. Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solutions of this invention, the technical solutions of the embodiments of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this invention.

[0037] Example 1 Isolation and Identification of Strains

[0038] 1. Strains screening and purification

[0039] (1) Source of strain: yak cheese, collected in December 2013 in Ganzi Tibetan Autonomous Prefecture, Sichuan Province, China.

[0040] (2) Sample preparation: The collected Sichuan Ganzi yak cheese was serially diluted with physiological saline to 10. -1 10 -2 10 -3 10 -4 10 -5 The samples were then numbered sequentially as Sample 1, Sample 2, Sample 3, Sample 4, and Sample 5, and were prepared for use.

[0041] (3) Preparation of MRS-CaCO3 solid culture medium: Sterilize the MRS solid culture medium with 1.5% CaCO3 at 121℃ for 15 min to obtain MRS-CaCO3 solid culture medium for later use.

[0042] (4) Cultivation: Take 100 µL of each of the samples 1#, 2#, 3#, 4# and 5# from step (2) and spread them evenly on MRS-CaCO3 solid medium. Invert the medium at 37℃ and anaerobic culture for 24-48h.

[0043] (5) Colony selection: After colony formation, select single colonies with calcium zones and perform three three-zone streaking tests. Repeat the streaking process multiple times until the colonies are purified. The indicators of colony purification are uniform colony morphology and uniform Gram staining morphology. Inoculate the purified strains into MRS liquid medium for culture. Take the culture medium of different strains and mix it with 50% sterile glycerol at a ratio of 1:1 (v / v), and store it in a -80℃ freezer.

[0044] The preparation method of MRS liquid culture medium is as follows: accurately weigh 10.0g of peptone; 5.0g of beef powder; 20.0g of glucose; 4.0g of yeast powder; 5.0g of sodium acetate; 2.0g of dipotassium hydrogen phosphate; 0.2g of magnesium sulfate; 2.0g of triammonium citrate; 0.05g of manganese sulfate; and 1mL of Tween 80. Add the contents to 1000mL of distilled water, shake well to dissolve completely, and sterilize at 121℃ for 15min before use.

[0045] 2. Identification

[0046] (1) Identification of colony morphology

[0047] ① The isolated and purified bacterial strain was cultured on MRS solid medium plates for 48 hours, and the colony morphology and color were observed and recorded. Colony morphology is as follows: Figure 1 As shown, the colonies are round, smooth, raised, milky white, occasionally light yellow, with neat edges and opaque.

[0048] ② Gram staining: Colonies cultured on MRS solid medium plates were smeared, fixed, primary stained with crystal violet, mordanted, destained, washed, counterstained with safranin, and dried. The dried colonies were then examined under an oil immersion microscope at 1000x magnification. The microscopic results are as follows: Figure 2 As shown. By Figure 2 As can be seen, under a microscope, the colonies are stained blue-purple and are short rod-shaped.

[0049] (2) 16S rDNA sequencing

[0050] ① Extraction of genomic DNA from the strain: Take the culture medium of the isolated and purified strain, centrifuge at 5000 r / min for 10 min, discard the supernatant after centrifugation, collect the pure culture cells to be tested, and extract the genomic DNA of the strain using a DNA extraction kit (purchased from Beijing Tiangen Biotech Co., Ltd.), following the instructions.

[0051] ②PCR amplification

[0052] Based on the conserved regions of the Lactobacillus 16S rDNA sequence, the universal bacterial primer 27F / 1492R was used.

[0053] The primer sequences are as follows:

[0054] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (as shown in Sequence 1);

[0055] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (as shown in Sequence 2).

[0056] The PCR amplification conditions were as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 30 s; annealing at 55℃ for 35 s; extension at 72℃ for 90 s; after 32 cycles, a final extension at 72℃ for 5 min.

[0057]

[0058] (3) Result of the appraisal

[0059] The sequencing results were compared with the NCBI database using BLAST to construct a phylogenetic tree, and the tested strain was identified as Lactobacillus rhamnosus. Lactobacillus rhamnosus Therefore, the strain to be tested was named *Lactobacillus rhamnosus* ZKLGG0310, and was deposited on May 13, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30547, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0060] Example 2

[0061] A Dendrobium officinale extract, the preparation method of which includes the following steps:

[0062] Step S1: Use a Chinese medicine pulverizer to pulverize Dendrobium officinale to make Dendrobium officinale powder with a particle size of 70-300 mesh.

[0063] Step S2: After putting 1 part by weight of Dendrobium officinale powder into a decoction bag, soak it in 8 parts by weight of water for 1 hour to obtain Dendrobium officinale soaking solution.

[0064] Step S3: First, bring the Dendrobium officinale soaking liquid to a boil over high heat, then simmer over low heat for 2 hours. During the simmering process, keep the Dendrobium officinale soaking liquid at a gentle boil. After the simmering is finished, filter to separate the liquid and residue.

[0065] Step S4: The liquid is boiled and concentrated to obtain Dendrobium officinale extract with a crude drug content of 1.0 g / mL.

[0066] Example 3

[0067] A fermentation broth of *Lactobacillus rhamnosus* ZKLGG0310 for *Dendrobium officinale* is prepared by the following steps:

[0068] Step 1: Take five equal portions of the Dendrobium officinale extract prepared in Example 2 and label them as Extract 1, Extract 2, Extract 3, Extract 4, and Extract 5, respectively. Add yeast extract powder at concentrations of 0% (g / mL), 2% (g / mL), 4% (g / mL), 6% (g / mL), and 8% (g / mL) to Extract 1 through Extract 5, respectively, and mix well. Then sterilize at 121℃ for 15 minutes to obtain sterilized Dendrobium officinale extract 1, sterilized Dendrobium officinale extract 2, sterilized Dendrobium officinale extract 3, sterilized Dendrobium officinale extract 4, and sterilized Dendrobium officinale extract 5.

[0069] Step 2: The *Lactobacillus rhamnosus* ZKLGG0310 isolated and purified in Example 1 was freeze-dried at -50°C for 40 hours to obtain a viable count of 1×10⁻⁶. 11 CFU / g of Lactobacillus rhamnosus ZKLGG0310 lyophilized powder was prepared by adding 0.1 CFU / g of Lactobacillus rhamnosus ZKLGG0310 lyophilized powder. Inoculations of *Dendrobium officinale* extract 1 to 5 were performed at a concentration of g / L, respectively. After mixing, the mixtures were incubated at 37℃ for 24 hours under aerobic conditions. Following the inoculation, *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth 1, 2, 3, 4, and 5 were obtained. The pH and viable cell count of each fermentation broth were then measured. The results are shown below. Figure 3 As shown.

[0070] Depend on Figure 3 It was found that when the amount of yeast extract was 0% (g / mL)-4% (g / mL), the viable cell count of *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth increased with increasing yeast extract dosage, while the pH of the fermentation broth decreased with increasing yeast extract dosage. When the amount of yeast extract exceeded 4% (g / mL), the viable cell count and pH of the *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth gradually stabilized. Specifically, the pH of fermentation broth 3 of *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* was 3.57, and the viable cell count was 1.81 × 10⁻⁶. 9 The high CFU / mL count and stable pH indicate that the fermentation effect is better when the yeast extract concentration is 4% (g / mL). Therefore, the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth 3 was used in the following examples.

[0071] Example 4: Application of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth in in vitro hypoglycemic agents.

[0072] In this embodiment, the enzyme content of α-amylase is 14 U / mg, and the enzyme content of α-glucosidase is 50 U / mg.

[0073] α-Amylase, α-glucosidase, soluble starch, 3,5-dinitrosalicylic acid (DNS) reagent, acarbose, and nitrophenol glucopyranoside (PNPG) were all purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0074] Solution preparation method

[0075] DNS colorimetric reagent preparation: Dissolve 6.3g of 3,5-dinitrosalicylic acid, 5g of phenol, 5g of anhydrous sodium sulfite, and 182g of potassium sodium tartrate in 262mL of 2mol / L NaOH and mix well. Dilute to 500mL with distilled water. Let stand at room temperature in the dark for 7 days before use.

[0076] PBS buffer preparation: Dissolve 8g NaCl, 0.2g KCl, 2.88g Na₂HPO₄•12H₂O, and 0.2g KH₂PO₄ in distilled water, adjust the pH to 7.3-7.4, and bring the volume to 1L. Autoclave and store at 4°C.

[0077] Preparation of 30 U / mL α-amylase solution: Weigh 0.2143 g α-amylase, bring the volume to 100 mL with PBS buffer, sonicate to aid dissolution, and filter to obtain the solution.

[0078] Preparation of 0.5% soluble starch solution: Accurately weigh 0.5g of soluble starch, dissolve in distilled water and bring the volume to 100mL, store at 4℃.

[0079] 1.5 U / mL α-glucosidase solution: Weigh 3 mg α-amylase, bring the volume to 100 mL with PBS buffer, sonicate to aid dissolution, and filter to obtain the solution.

[0080] Acarbose solution: Take acarbose tablets, grind the tablets into powder, accurately weigh 0.1g, and dilute with distilled water to 100mL to prepare a 1mg / mL solution.

[0081] 20 mmol / L PNPG solution: Accurately weigh 0.6025 g PNPG, dissolve in PBS buffer and bring the volume to 100 mL, store at 4 °C.

[0082] (1) Test of α-amylase activity inhibition rate

[0083] Preparation and testing of fermentation sample 1: First, 100 μL of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth 3 from Example 3 was mixed with 100 μL of α-amylase solution with a concentration of 30 U / mL, and incubated with shaking at 37℃ for 10 min. Then, 200 μL of 0.5% (w / v) soluble starch solution was added, and incubation was continued at 37℃ for 10 min. Finally, 1.6 mL of DNS reagent was added, and the mixture was heated in a boiling water bath for 5 min. After cooling, fermentation sample 1 was obtained. The α-amylase solution in the preparation method of fermentation group sample 1 was replaced with an equal volume of distilled water to serve as the control sample of fermentation group sample 1. The *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth 3 in the preparation method of fermentation group sample 1 was replaced with an equal volume of PBS buffer to obtain the blank control sample of fermentation group sample 1. The *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth 3 in the preparation method of fermentation group sample 1 was replaced with an equal volume of PBS buffer, and the α-amylase solution was replaced with an equal volume of distilled water to obtain the calibration sample of fermentation group sample 1. Acarbose solution was used as a positive control, and the UV absorbance of fermentation group sample 1, the control sample of fermentation group sample 1, the blank control sample of fermentation group sample 1, and the calibration sample of fermentation group sample 1 were measured at 540 nm.

[0084] Preparation and testing of mixed sample 1: 10 mL of Dendrobium officinale extract prepared in Example 2 was mixed with a sample containing 1×10⁻⁶ viable bacteria. 11 The lyophilized powder of *Lactobacillus rhamnosus* ZKLGG0310 (CFU / mL) was mixed evenly to obtain a mixture, wherein the amount of *Lactobacillus rhamnosus* ZKLGG0310 lyophilized powder used was 0.181 g. First, 100 μL of the mixture was mixed with 100 μL of α-amylase at a concentration of 30 U / mL and incubated at 37℃ with shaking for 10 min. Then, 200 μL of 0.5% (w / v) soluble starch solution was added, and the mixture was incubated at 37℃ for another 10 min. Finally, 1.6 mL of DNS reagent was added, and the mixture was heated in a boiling water bath for 5 min. After cooling, the mixed sample 1 was obtained. The α-amylase solution in the preparation method of mixed group sample 1 was replaced with an equal volume of distilled water to serve as the control sample of mixed group sample 1; the mixture in the preparation method of mixed group sample 1 was replaced with an equal volume of PBS buffer to obtain the blank control sample of mixed group sample 1; the mixture in the preparation method of mixed group sample 1 was replaced with an equal volume of PBS buffer, and the α-amylase solution was replaced with an equal volume of distilled water to obtain the calibration sample of mixed group sample 1. Acarbose solution was used as a positive control, and the UV absorbance of mixed group sample 1, the control sample of mixed group sample 1, the blank control sample of mixed group sample 1, and the calibration sample of mixed group sample 1 were measured at 540 nm.

[0085] The α-amylase activity inhibition rates of fermentation group sample 1, mixed group sample 1, and acarbose were calculated according to Equation 1:

[0086] α-Amylase activity inhibition rate (%) = [1-(A3-A4) / (A1-A2)]×100% (Equation 1).

[0087] In Equation 1, A3 represents the absorbance of the test samples—fermentation group sample 1 and mixed group sample 1; A4 represents the absorbance of the control sample corresponding to the test sample; A1 represents the absorbance of the blank control sample corresponding to the test sample; and A2 represents the absorbance of the calibration sample corresponding to the test sample. The α-amylase activity inhibition rates of fermentation group sample 1, mixed group sample 1, and acarbose solution are shown in Table 1.

[0088] Table 1. Inhibition rate of α-amylase activity of fermentation group sample 1, mixed group sample 1, and acarbose.

[0089]

[0090] Note: Different letters in the table represent significant differences, P < 0.05.

[0091] (2) Test of α-glucosidase activity inhibition rate

[0092] Preparation and testing of fermentation sample 2: First, 200 μL of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth 3 from Example 3 was mixed with 50 μL of α-glucosidase solution with a concentration of 1.5 U / mL and incubated at 37℃ for 5 min. Then, 75 μL of PNPG solution with a concentration of 20 mmol / L was added and mixed well, and incubated at 37℃ for another 15 min. Finally, 400 μL of Na2CO3 solution with a concentration of 0.2 mol / L was added to terminate the reaction, and fermentation sample 2 was obtained. The α-glucosidase solution in the preparation method of fermentation group sample 2 was replaced with an equal volume of distilled water to serve as the control sample of fermentation group sample 2. The *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth 3 in the preparation method of fermentation group sample 2 was replaced with an equal volume of PBS buffer to obtain the blank control sample of fermentation group sample 2. The *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth 3 in the preparation method of fermentation group sample 2 was replaced with an equal volume of PBS buffer, and the α-glucosidase solution was replaced with an equal volume of distilled water to obtain the calibration sample of fermentation group sample 2. Acarbose was used as a positive control, and the UV absorbance of fermentation group sample 2, the control sample of fermentation group sample 2, the blank control sample of fermentation group sample 2, and the calibration sample of fermentation group sample 2 were measured at 405 nm.

[0093] Preparation and testing of mixed sample 2: First, 200 μL of the mixture was mixed thoroughly with 50 μL of 1.5 U / mL α-glucosidase solution and incubated at 37℃ for 5 min. Then, 75 μL of 20 mmol / L PNPG solution was added and mixed thoroughly, and the mixture was incubated at 37℃ for another 15 min. Finally, 400 μL of 0.2 mol / L Na2CO3 solution was added to terminate the reaction, resulting in mixed sample 2. The α-glucosidase solution in the preparation method of mixed sample 2 was replaced with an equal volume of distilled water as the control sample of mixed sample 2. The mixture in the preparation method of mixed sample 2 was replaced with an equal volume of PBS buffer to obtain the blank control sample of mixed sample 2. The mixture in the preparation method of mixed sample 2 was replaced with an equal volume of PBS buffer, and the α-glucosidase solution was replaced with an equal volume of distilled water to obtain the calibration sample of mixed sample 2. Using acarbose as a positive control, the UV absorbance of mixed group sample 2, the control sample of mixed group sample 2, the blank control sample of mixed group sample 2, and the calibration sample of mixed group sample 2 was measured at 405 nm.

[0094] The inhibition rates of α-glucosidase activity of fermentation group sample 2, mixed group sample 2, and acarbose were calculated according to Equation 2:

[0095] α-glucosidase activity inhibition rate (%) = [1-(A3'-A4') / (A1'-A2')]×100% (Equation 2)

[0096] In Equation 2, A3' represents the absorbance of the test samples—fermentation group sample 2 and mixed group sample 2; A4' represents the absorbance of the control sample corresponding to the test sample; A1' represents the absorbance of the blank control sample corresponding to the test sample; and A2' represents the absorbance of the calibration sample corresponding to the test sample. The α-glucosidase activity inhibition rates of fermentation group sample 2, mixed group sample 2, and acarbose are shown in Table 2.

[0097] Table 2. Inhibition rate of α-glucosidase activity of fermentation group sample 2, mixed group sample 2, and acarbose.

[0098]

[0099] Note: Different letters in the table represent significant differences, P < 0.05.

[0100] Alpha-amylase and alpha-glucosidase hydrolyze the α-1,4-glycosidic bonds of starch and glycogen, as well as the α-1,6-glycosidic bonds of oligosaccharides, helping the body convert ingested carbohydrates into glucose for absorption in the intestines. The inhibitory effect of these two digestive enzymes, alpha-amylase and alpha-glucosidase, has always been an important indicator for evaluating in vitro hypoglycemic activity. Inhibiting the activity of alpha-amylase and alpha-glucosidase can reduce the rate of carbohydrate breakdown into glucose, thereby slowing down the postprandial rise in blood glucose. Tables 1 and 2 show that the mixture of *Lactobacillus rhamnosus* ZKLGG0310 fermentation broth of *Dendrobium officinale*, *Dendrobium officinale* extract, and *Lactobacillus rhamnosus* ZKLGG0310 lyophilized powder exhibits strong inhibitory effects on both alpha-amylase and alpha-glucosidase in vitro. The inhibitory effect of *Lactobacillus rhamnosus* ZKLGG0310 fermentation broth on *Dendrobium officinale* was significantly stronger than that of *Lactobacillus rhamnosus* + *Dendrobium officinale* extract (P < 0.05), and also higher than that of acarbose. This indicates that fermentation of *Dendrobium officinale* extract with *Lactobacillus rhamnosus* ZKLGG0310 can significantly improve its in vitro hypoglycemic ability.

[0101] Example 5: The Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth prepared in Example 3 was used to treat type 2 diabetic mice.

[0102] (1) Modeling: Sixty male C57BL / 6J mice were acclimatized to a normal diet for one week. Twelve mice were randomly selected as the control group (NC group) and forty-eight mice were selected as the high-fat, high-sugar group (HFD group). Mice in the control group were fed a normal diet every day; mice in the high-fat, high-sugar group were fed a high-fat, high-sugar diet every day. The high-fat, high-sugar diet was purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0103] Mice in the high-fat, high-glucose group were fed continuously for 4 weeks, then fasted for 12 hours, and then injected intraperitoneally with STZ prepared with citrate buffer. STZ was purchased from Sigma-Aldrich, USA, and was freshly prepared before use. It was prepared with 0.1 mol / L citrate buffer solution at pH 4.2 to a 1% concentration. The injection dose of STZ was 50 mg / kg mouse body weight per day. STZ injection was repeated for 3 days. One week after the last injection, the tail was cut off and blood was collected to measure the fasting blood glucose level of the mice. A fasting blood glucose level ≥11.1 mmol / L was used as the standard for successful establishment of the type 2 diabetes pathological model. Mice that failed to establish the model were removed from the high-fat, high-glucose group, resulting in forty successfully modeled type 2 diabetes mice.

[0104] (2) Grouping: Forty successfully modeled type 2 diabetic mice were randomly divided into four groups, which were labeled as the hyperglycemic model group (MC group), Lactobacillus rhamnosus ZKLGG0310 mixed group (DO group), Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation group (FDO group) and metformin positive control group (DMBG group). The number of mice in each group was ten.

[0105] (3) Administration:

[0106] NC and MC groups: 0.2 mL of sterile saline was administered by gavage daily.

[0107] DO group: 0.2 mL of a mixture of Dendrobium officinale extract and Lactobacillus rhamnosus ZKLGG0310 was administered by gavage daily. The viable count of Lactobacillus rhamnosus ZKLGG0310 in the mixture was 1.81 × 10⁻⁶. 9 CFU / mL.

[0108] FDO group: 0.2 mL of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth prepared in Example 3 was administered by gavage daily.

[0109] DMBG group: Mice were given 200 mg / kg of metformin by gavage daily.

[0110] Mice in each group were administered the drug via gavage for four consecutive weeks according to the above administration method. During the administration period, mice had free access to food and water. Except for the control group mice which were fed normal feed, the mice in other groups were fed a high-fat, high-sugar diet.

[0111] (4) Sample collection and index detection:

[0112] ① Body weight and fasting blood glucose: After one week of acclimatization, the body weight of mice in the NC, MC, DO, FDO, and DMBG groups was monitored and recorded weekly. The trend of body weight change in each group was statistically analyzed, and the trend of body weight change in each group is shown in Table 3. During the experimental period after the acclimatization period, the fasting blood glucose concentration of each group of mice was measured every 2 weeks using a blood glucose meter. The average value of the fasting blood glucose concentration of each group of mice was taken each time. Before measuring the fasting blood glucose concentration, each group of mice was fasted for 16 hours but allowed free access to water. Blood was collected from the tail of the mice when measuring the fasting blood glucose concentration. The statistical graph of the last fasting blood glucose concentration of each group of mice before slaughter is shown in the figure. Figure 4 As shown.

[0113] Table 3. Changes in body weight of mice in each group (unit: g)

[0114]

[0115] Table 3 shows that the NC group mice maintained a stable weight gain throughout the experimental period and had normal biochemical status. The MC group mice experienced a continuous decrease in weight after one week of adaptive feeding, and weight loss is one of the symptoms of type 2 diabetes. The DO, FDO, and DMBG groups showed a slower rate of weight loss after drug administration, and the FDO and DMBG groups showed a slight increase in weight in the fourth week after drug administration. This indicates that the *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth had a stronger inhibitory effect on weight loss in type 2 diabetic mice than the mixture of *Dendrobium officinale* extract and *Lactobacillus rhamnosus* ZKLGG0310, and its effect was comparable to that of metformin.

[0116] Depend on Figure 4 It was found that the fasting blood glucose concentration of mice in the MC group was significantly higher than that in the NC group (P < 0.05). The fasting blood glucose concentration of mice in the DO, FDO, and DMBG groups was significantly lower than that in the MC group after administration (P < 0.05). Moreover, the fasting blood glucose concentration of mice in the FDO group was significantly lower than that in the DO group (P < 0.05) and close to that in the DMBG group. This indicates that the fermentation broth of *Lactobacillus rhamnosus* ZKLGG0310 and *Dendrobium officinale* had a stronger effect on reducing fasting blood glucose in type 2 diabetic mice than the mixture of *Dendrobium officinale* extract and *Lactobacillus rhamnosus* ZKLGG0310, and its effect was comparable to that of metformin.

[0117] ② Glycated Hemoglobin: The serum glycated hemoglobin concentration of each group of mice was determined using a glycated hemoglobin kit (purchased from Nanjing Jiancheng). Before slaughter, blood was collected from the mice via ocular sampling. The blood was centrifuged at 4500 rpm for 3 minutes to obtain mouse serum. Following the supplier's instructions on the kit, the glycated hemoglobin content in the serum of each group of mice was measured. The serum glycated hemoglobin concentrations of each group of mice are shown below. Figure 5 As shown.

[0118] Glycated hemoglobin (HbA1c) concentration is less affected by environmental factors, thus it can stably reflect the body's average blood glucose level over the past 2-3 months. Figure 5 The results showed that the glycated hemoglobin (HbA1c) concentration in the MC group was significantly higher than that in the NC group (P < 0.05), while the HbA1c concentrations in the DO, FDO, and DMBG groups were significantly lower than those in the MC group (P < 0.05). The HbA1c concentration in the FDO group was significantly lower than that in the DO group (P < 0.05) and close to that in the DMBG group. This indicates that the *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth had a stronger effect on reducing HbA1c concentration in type 2 diabetic mice than the mixture of *Dendrobium officinale* extract and *Lactobacillus rhamnosus* ZKLGG0310, and its effect was comparable to that of metformin. The *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* fermentation broth can control blood glucose levels within the normal range for a longer period, reducing the risk of complications in type 2 diabetes.

[0119] ③ Oral Glucose Tolerance Test (OGTT): Four weeks after administration, mice in each group were fasted for 16 hours but allowed free access to water. They were then administered a 40% (w / v) glucose solution via gavage at a dose of 2 g / kg of mouse body weight. Blood was collected from the tail vein of the mice at 0, 30, 60, 90, and 120 minutes after gavage. The average blood glucose concentration was measured using a glucometer and matching test strips, and the mean concentration for each group was calculated. After blood collection, the puncture sites were wiped with iodine to prevent infection. The area under the cerve (AUC) was calculated by plotting blood glucose concentrations at each time point. The AUC value directly reflects the glucose tolerance of each group of mice. The average blood glucose concentrations of each group of mice at 0, 30, 60, 90, and 120 minutes after gavage are shown below. Figure 6 As shown in the figure, the area under the glucose tolerance curve for each group of mice is statistically analyzed. Figure 7 As shown.

[0120] like Figure 6 As shown, after gavage administration of glucose solution, the blood glucose concentration of mice in all groups first increased and then decreased, reaching its maximum at 30 min. Although the blood glucose concentration of mice in the MC group decreased slightly between 30 and 120 min, it remained at a relatively high level. Compared with the MC group, the blood glucose concentrations of mice in the DO, FDO, and DMBG groups decreased, with the blood glucose concentration of the FDO group being lower than that of the DO group and close to that of the DMBG group. This indicates that the fermentation broth of *Lactobacillus rhamnosus* ZKLGG0310 *Dendrobium officinale* has a stronger effect on controlling blood glucose in type 2 diabetic mice than *Dendrobium officinale* extract, and its effect is comparable to that of metformin.

[0121] like Figure 7 As shown, the AUC of MC group mice was significantly higher than that of NC group mice (P < 0.05). Four weeks after administration, compared with the MC group, the AUC of DO group, FDO group, and DMBG group mice was significantly decreased (P < 0.05), and the AUC of FDO group mice was significantly lower than that of DO group (P < 0.05), and close to that of positive DMBG group. This indicates that Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth can significantly improve glucose tolerance in type 2 diabetic mice and repair impaired glucose tolerance.

[0122] ④ Calculation of Insulin Levels and Insulin Resistance Index: After the last administration, mice in each group were fasted for 16 hours. Body weight was then measured, and blood was collected by enucleation. The supernatant was collected by centrifugation to obtain serum from each group of mice. The serum insulin concentration of each group was measured using an insulin kit (purchased from Nanjing Jiancheng), and the average value was calculated. The average serum insulin concentration of each group of mice is shown below. Figure 8 As shown.

[0123] Based on the fasting blood glucose concentration measured in ① and the average serum insulin concentration of each group of mice, the insulin resistance index (HOMA-IR) of each group of mice was calculated according to Equation 3. The insulin resistance index of each group of mice is as follows: Figure 9 As shown.

[0124] HOMA-IR = [serum insulin concentration (mIU / L) × fasting blood glucose concentration (mmol / L)] / 22.5 (Equation 3).

[0125] Insulin, secreted by pancreatic beta cells, is the only hormone in the body that lowers blood sugar; its level is a key factor affecting blood sugar levels. Figure 8 As shown, the average insulin concentration in the MC group mice was significantly lower than that in the NC group mice (P < 0.05). Four weeks after administration, compared with the MC group, the average serum insulin concentrations in the DO, FDO, and DMBG groups were significantly increased (P < 0.05), and the average serum insulin concentration in the FDO group was significantly higher than that in the DO group (P < 0.05), and close to that in the DMBG group. This indicates that the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth can significantly increase serum insulin levels in type 2 diabetic mice.

[0126] like Figure 9 As shown, the HOMA-IR of MC group mice was significantly higher than that of NC group mice (P < 0.05). Four weeks after administration, compared with MC group, HOMA-IR of DO group, FDO group and DMBG group mice was significantly decreased (P < 0.05), and FDO group was significantly lower than DO group (P < 0.05) and close to DMBG group, indicating that Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth can significantly reduce insulin resistance and improve insulin sensitivity in type 2 diabetic mice.

[0127] ⑤ Blood lipid index detection: The serum TC content of mice in each group was measured using a serum total cholesterol (TC) kit (purchased from Nanjing Jiancheng), and the average value was taken. The statistical graph of the average serum TC content of mice in each group is shown in the figure below. Figure 10 As shown in the figure. The serum TG levels of mice in each group were measured using a triglyceride (TG) kit (purchased from Nanjing Jiancheng). The statistical graph of the average serum TG levels of mice in each group is shown in the figure. Figure 11 As shown.

[0128] In patients with type 2 diabetes, insulin resistance can cause disordered fat metabolism, accelerating the oxidative breakdown of fat by cells and leading to elevated blood lipid levels. For example... Figure 10 and Figure 11As shown, the TC and TG levels in the MC group mice were significantly higher than those in the NC group mice (P < 0.05). Four weeks after administration, compared with the MC group, the TC and TG concentrations in the DO, FDO, and DMBG groups were significantly lower (P < 0.05), with the FDO group showing significantly lower levels than the DO group (P < 0.05), and close to those in the DMBG group. This indicates that the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth can significantly reduce blood lipid levels in type 2 diabetic mice and regulate lipid metabolism in diabetic mice.

[0129] In conclusion, the fermented broth of *Dendrobium officinale* ZKLGG0310 showed a significant hypoglycemic effect (P<0.05) in type 2 diabetic mice compared with the unfermented *Dendrobium officinale* extract, significantly increasing insulin levels (P<0.05), significantly reducing insulin resistance (P<0.05), and regulating lipid metabolism.

[0130] Although the present invention has been described in detail with reference to the accompanying drawings and preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made to the embodiments of the present invention by those skilled in the art without departing from the spirit and essence of the invention, and such modifications or substitutions should all be within the scope of the present invention. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should also be covered within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus ZKLGG0310, characterized in that: Lactobacillus rhamnosus ( Lactobacillus rhamnosus ) ZKLGG0310 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on May 13, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.30547.

2. A Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale fermentation liquid, characterized in that: The invention is prepared from the Dendrobium officinale extract having a crude drug content of 1.0 g / mL fermented by Lactobacillus rhamnosus ZKLGG0310 as claimed in claim 1.

3. The Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid according to claim 2, characterized in that: The preparation method of the Dendrobium officinale extract comprises the following steps: Step S1: crushing the Dendrobium officinale to prepare Dendrobium officinale powder; Step S2: adding 1 part by weight of the Dendrobium officinale powder into 8 parts by weight of water for soaking to obtain a Dendrobium officinale soaking liquid; Step S3: boiling the soaking liquid of Dendrobium officinale and then continuing to decoct it, and then separating the liquid and the residue; Step S4: boiling and concentrating the feed liquid to obtain a Dendrobium officinale extract having a crude drug content of 1.0 g / mL.

4. The Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid according to claim 3, characterized in that: In step S1, the particle size of the Dendrobium officinale powder is 70-300 mesh.

5. The Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid according to claim 3, characterized in that: In step S2, the Dendrobium officinale powder is put into a decoction bag for soaking.

6. The Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid according to claim 3, characterized in that: In step S2, the soaking time is 0.5-1.5h.

7. The Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid according to claim 3, characterized in that: In step S3, the decocting time is continued for 1.5-2.5 hours.

8. The Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid according to claim 3, characterized in that: The preparation method of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid comprises the following steps: Step 1: adding yeast extract powder to the Dendrobium officinale extract and mixing evenly, and then sterilizing at 121° C. for 15 minutes to obtain a sterilized Dendrobium officinale extract, wherein the amount of yeast extract powder is 0% (g / mL)-8% (g / mL); Step 2: Set the number of viable bacteria to 1×10 11 CFU / g of Lactobacillus rhamnosus ZKLGG0310 preparation was inoculated into the sterilized Dendrobium officinale extract at an inoculum amount of 0.1 g / L, and then shaken and cultured aerobically at 37°C for 24 h to obtain the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid. The number of viable bacteria in the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid was 1.0×10 9 -1.81×10 9 CFU / mL.

9. The Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid according to claim 8, characterized in that: The preparation of Lactobacillus rhamnosus ZKLGG0310 is in the form of powder, tablet, granule or drop.

10. Use of the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid as claimed in any one of claims 2 to 9 in the preparation of hypoglycemic drugs, characterized in that: Application in the preparation of medicines for treating type 2 diabetes.

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