Lactobacillus rhamnosus ZKLGG0310, dendrobium officinale fermentation liquor thereof and application of lactobacillus rhamnosus ZKLGG0310 in preparation of hypoglycemic drugs

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus rhamnosus ZKLGG0310, dendrobium officinale fermentation liquor of the lactobacillus rhamnosus ZKLGG0310 and application of the lactobacillus rhamnosus ZKLGG0310 to preparation of hypoglycemic drugs, the lactobacillus rhamnosus ZKLGG0310 is preserved in the China General Microbiological Culture Collection Center on May 13, 2024, the preservation address is No. 3, No.1 yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO. The preservation number is CGMCC (China General Microbiological Culture Collection Center) The lactobacillus rhamnosus ZKLGG0310 is used for fermenting the dendrobium officinale extracting solution, the blood glucose reducing effect of dendrobium officinale is remarkably improved, meanwhile, lipid metabolism is adjusted, and complications of type 2 diabetes mellitus are relieved.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus rhamnosus ZKLGG0310, its Dendrobium officinale fermentation liquid and application thereof in preparing hypoglycemic drugs. Background Art

[0002] In recent years, the incidence of diabetes has been on the rise, among which type 2 diabetes (T2DM) is the most common, accounting for more than 90% of all people with diabetes. Type 2 diabetes is a chronic disease caused by insufficient use of insulin or reduced efficiency. It is common in adults and is also called adult-onset diabetes. Type 2 diabetes is generally caused by genetic factors and environmental factors such as bad living habits, overnutrition, and lack of physical activity. The disease has mild symptoms in the early stages and is easy to be ignored. As the disease progresses, it is usually discovered and diagnosed only after complications occur. The accumulation of complications increases the difficulty of treatment and increases the cost of treatment. The complications of type 2 diabetes mainly include cardiovascular and cerebrovascular diseases, kidney diseases, neuropathy, eye diseases, and infectious diseases. These complications will seriously affect the quality of life of patients. If blood sugar is not controlled for a long time, it may even be life-threatening.

[0003] At present, type 2 diabetes is mainly controlled by drug therapy. Common oral hypoglycemic drugs include sulfonylureas, glinides, thiazolidinediones, biguanides, α-glucosidase inhibitors, dipeptidyl peptidase-4 inhibitors and sodium-glucose cotransporter-2 inhibitors, etc. Although oral hypoglycemic drugs can lower blood sugar, they are prone to side effects such as functional damage to pancreatic islet cells and gastrointestinal diseases.

[0004] The Yellow Emperor's Classic of Internal Medicine records the main treatment method for diabetes: "nourishing yin and promoting fluid production, balancing yin and yang". According to Shennong's Classic of Materia Medica, Dendrobium candidum "replenishes the weakness of the five internal organs and strengthens yin". The 2010 edition of the Chinese Pharmacopoeia records that it has the effects of nourishing the stomach and promoting fluid production, nourishing yin and clearing heat, and can be used for yin damage and fluid deficiency, dry mouth and thirst, etc. Modern pharmacological studies have shown that Dendrobium candidum has a certain blood sugar-lowering 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 will interact with each other during combined use, which has potential side effects. Summary of the invention

[0005] In view of the technical problem that there is a potential risk of side effects when Dendrobium candidum is used in combination with other oral hypoglycemic drugs, the present invention provides a Lactobacillus rhamnosus ZKLGG0310, a Dendrobium candidum fermentation liquid thereof and an application thereof in preparing a hypoglycemic drug. The Dendrobium candidum extract is fermented by using Lactobacillus rhamnosus ZKLGG0310, which not only significantly improves the hypoglycemic effect of Dendrobium candidum, but also regulates lipid metabolism and alleviates the 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 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.

[0007] In a second aspect, the present invention provides a Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid, which is prepared from the Dendrobium officinale extract having a crude drug content of 1.0 g / mL fermented by Lactobacillus rhamnosus ZKLGG0310 as described above.

[0008] Furthermore, 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 Dendrobium officinale soaking liquid and continuing to boil it in a slightly boiling state, 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.

[0009] Furthermore, in step S1, the particle size of the Dendrobium officinale powder is 70-300 mesh.

[0010] Furthermore, in step S2, the Dendrobium officinale powder is put into a decoction bag and soaked.

[0011] Furthermore, in step S2, the soaking time is 0.5-1.5 h, preferably 1 h.

[0012] Furthermore, in step S3, the decocting time is continued for 1.5-2.5 hours, preferably 2 hours.

[0013] Furthermore, 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), preferably 4% (g / mL); Step 2: Set the number of viable bacteria to 1×10 11CFU / 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. The pH of the fermentation liquid of Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale was ≤4.5.

[0014] Furthermore, the Lactobacillus rhamnosus ZKLGG0310 preparation is in the form of powder, tablet, granule or drop, preferably powder, more preferably freeze-dried powder.

[0015] In a third aspect, the present invention also provides an application of the above-mentioned Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid in the preparation of hypoglycemic drugs, including the application in the preparation of drugs for treating type 2 diabetes. The drug prepared using the fermentation liquid of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale can inhibit the activity of α-amylase and α-glucosidase, achieve in vitro hypoglycemia; reduce the risk of complications of type 2 diabetes; regulate sugar metabolism and fat metabolism; and improve insulin sensitivity.

[0016] The beneficial effects of the present invention are: The invention provides a Lactobacillus rhamnosus ZKLGG0310, a Dendrobium officinale fermentation liquid thereof and an application thereof in preparing a hypoglycemic drug. The Dendrobium officinale extract is fermented by using the Lactobacillus rhamnosus ZKLGG0310 to prepare an acidic Dendrobium officinale fermentation liquid, which 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 activities of α-amylase and α-glucosidase to achieve in vitro hypoglycemia; 3. improving insulin sensitivity by significantly increasing the insulin level and reducing insulin resistance; 4. controlling the blood sugar level within a normal range for a long time, reducing the risk of complications of type 2 diabetes; 5. regulating sugar metabolism and lipid metabolism, and reducing hyperlipidemia complications caused by high serum total cholesterol and / or triglycerides. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 This is a picture of the colony morphology cultured on the enriched medium plate in Example 1.

[0019] Figure 2 This is a microscopic picture of the bacterial colony after drying in Example 1.

[0020] Figure 3 is a line graph of pH and viable count of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 1-Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 5 in Example 3, Figure 3 In the figure, the square line represents pH, and the circle line represents the number of viable bacteria.

[0021] Figure 4 The fasting blood glucose concentration statistics of mice in the NC group, MC group, DO group, FDO group and DMBG group in Example 5 are shown in FIG. Figure 4 Different letters represent significant differences, P < 0.05.

[0022] Figure 5 is the serum glycated hemoglobin concentration of mice in the NC group, MC group, DO group, FDO group and DMBG group in Example 5, Figure 5 Different letters represent significant differences, P < 0.05.

[0023] Figure 6 It is the average blood glucose concentration of mice in the NC group, MC group, DO group, FDO group and DMBG group in Example 5 at 0 min, 30 min, 60 min, 90 min and 120 min after gavage with glucose aqueous solution.

[0024] Figure 7 The area under the glucose tolerance curve of the mice in the NC group, MC group, DO group, FDO group and DMBG group in Example 5 is a statistical graph. Figure 7 Different letters represent significant differences, P < 0.05.

[0025] Figure 8 This is a statistical graph of the mean values ​​of serum insulin concentrations of mice in the NC group, MC group, DO group, FDO group, and DMBG group in Example 5. Figure 8 Different letters represent significant differences, P < 0.05.

[0026] Fig. 9 This is a statistical graph of the insulin resistance index of mice in the NC group, MC group, DO group, FDO group and DMBG group in Example 5, Fig. 9 Different letters represent significant differences, P < 0.05.

[0027] Fig.10 This is a statistical diagram of the average TC content in the serum of mice in the NC group, MC group, DO group, FDO group and DMBG group in Example 5, Fig.10Different letters represent significant differences, P < 0.05.

[0028] Fig.11 This is a statistical graph of the average TG content in the serum of mice in the NC group, MC group, DO group, FDO group and DMBG group in Example 5, Fig.11 Different letters represent significant differences, P < 0.05. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0030] Example 1 Isolation and identification of strains 1. Strain screening and purification (1) Strain source: Yak cheese, collected in Ganzi Tibetan Autonomous Prefecture, Sichuan Province, China in December 2013.

[0031] (2) Sample preparation: Sichuan Ganzi yak cheese was collected and diluted with physiological saline to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 times, and label them in sequence as 1# sample, 2# sample, 3# sample, 4# sample, and 5# sample for later use.

[0032] (3) Preparation of MRS-CaCO3 solid culture medium: Sterilize MRS solid culture medium with 1.5% CaCO3 added at 121°C for 15 min to obtain MRS-CaCO3 solid culture medium for later use.

[0033] (4) Cultivation: 100 µL of each of sample #1, sample #2, sample #3, sample #4, and sample #5 in step (2) was evenly spread on MRS-CaCO3 solid culture medium, inverted and anaerobically cultured at 37°C for 24-48 h.

[0034] (5) Colony selection: After the colonies are formed, single colonies with calcium rings are selected for three-zone streaking, and the streaking separation is repeated many times until the colonies are purified. The indicators of colony purification are single colony morphology and single Gram staining morphology. The purified strains are inoculated into MRS liquid culture medium for culture, and the culture medium of different strains and 50% sterile glycerol are mixed in a ratio of 1:1 (v / v) and stored in a -80℃ refrigerator.

[0035] The preparation method of MRS liquid culture medium is as follows: accurately weigh 10.0 g of peptone, 5.0 g of beef powder, 20.0 g of glucose, 4.0 g of yeast powder, 5.0 g of sodium acetate, 2.0 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 2.0 g of triammonium citrate, 0.05 g of manganese sulfate, and 1 mL of Tween 80, add to 1000 mL of distilled water, shake well to fully dissolve, and sterilize at 121°C for 15 min for use.

[0036] 2. Identification (1) Colony morphology identification ① Culture the isolated and purified strain on MRS solid medium plates for 48 hours, and observe and record the colony morphology and color. Figure 1 As shown, the colonies are round, smooth, convex, milky white, occasionally light yellow, with neat edges and opaque.

[0037] ② Gram staining: Select the colonies cultured on the MRS solid medium plate and carry out smearing, fixation, crystal violet primary staining, mordanting, decolorization, water washing, safranin counterstaining and drying in sequence. Then examine the dried colonies under an oil microscope with a magnification of 1000 times. The results of the microscopic examination are as follows: Figure 2 As shown. Figure 2 It can be seen that under the microscope, the colonies are stained blue-purple and are short rod-shaped.

[0038] (2) 16S rDNA sequence determination ① Extraction of genomic DNA of strains: Take the culture medium of the strain obtained after separation and purification, centrifuge at a centrifugal speed of 5000r / min for 10min, discard the supernatant after centrifugation, collect the pure culture bacteria to be tested, and use a DNA extraction kit (purchased from Beijing Tiangen Biochemical Technology Co., Ltd.) to extract the genomic DNA of the strain, and operate according to the instructions.

[0039] ②PCR amplification Based on the conserved region of Lactobacillus 16S rDNA sequence, bacterial universal primers 27F / 1492R were used.

[0040] The primer sequences are as follows: 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′ (as shown in sequence 1); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (as shown in sequence 2).

[0041] The PCR amplification conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 30 s; annealing at 55°C for 35 s; extension at 72°C for 90 s; after 32 cycles, the final extension was at 72°C for 5 min.

[0042]

[0043] (3) Identification results The sequencing results were compared with the NCBI database by BLAST, and a phylogenetic tree was constructed to determine that the strain to be tested was Lactobacillus rhamnosus ( Lactobacillus rhamnosus ). Therefore, the strain to be tested was named Lactobacillus rhamnosus ZKLGG0310, and was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on May 13, 2024, with the deposit number CGMCC No.30547, and the deposit address was No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0044] Example 2 A Dendrobium officinale extract, the preparation method comprising the following steps: Step S1: using a traditional Chinese medicine grinder to grind the Dendrobium officinale to prepare a Dendrobium officinale powder with a particle size of 70-300 meshes.

[0045] Step S2: After putting 1 weight part of Dendrobium officinale powder into a decoction bag, the bag is put into 8 weight parts of water and soaked for 1 hour to obtain Dendrobium officinale soaking liquid.

[0046] Step S3: first boil the Dendrobium officinale soaking liquid over high heat, then simmer over low heat for 2 hours, keeping the Dendrobium officinale soaking liquid slightly boiling during the simmering process, and after the simmering is completed, filtering and separating the liquid and the residue.

[0047] Step S4: boiling and concentrating the feed liquid to obtain a Dendrobium officinale extract having a crude drug content of 1.0 g / mL.

[0048] Example 3 A Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid, the preparation method comprising the following steps: Step 1: Take five equal portions of the Dendrobium officinale extract prepared in Example 2, and mark them as extract 1, extract 2, extract 3, extract 4 and extract 5, respectively, add 0% (g / mL), 2% (g / mL), 4% (g / mL), 6% (g / mL) and 8% (g / mL) of yeast extract powder to extracts 1 to 5, respectively, mix them evenly, and then sterilize them at 121° C. for 15 min 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.

[0049] Step 2: The Lactobacillus rhamnosus ZKLGG0310 isolated and purified in Example 1 was freeze-dried at -50°C for 40 h to obtain a viable cell count of 1×10 11CFU / g of Lactobacillus rhamnosus ZKLGG0310 freeze-dried powder, Lactobacillus rhamnosus ZKLGG0310 freeze-dried powder was diluted to 0.1 g / L was inoculated into sterilized Dendrobium officinale extract 1-sterilized Dendrobium officinale extract 5, respectively, and the mixture was shaken and aerobically cultured at 37°C for 24 h. After the culture, Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 1, Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 2, Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 3, Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 4 and Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 5 were obtained. The pH and viable count of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 1-Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 5 were tested, respectively. The test results are as follows: Figure 3 shown.

[0050] Depend on Figure 3 It can be seen that when the dosage of yeast extract powder is 0% (g / mL)-4% (g / mL), the number of live bacteria in the fermentation liquid of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale increases with the increase of the dosage of yeast extract powder, and the pH of the fermentation liquid of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale decreases with the increase of the dosage of yeast extract powder. When the dosage of yeast extract powder exceeds 4% (g / mL), the number of live bacteria and pH of the fermentation liquid of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale gradually tend to be stable, among which the pH of the fermentation liquid of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale 3 is 3.57, and the number of live bacteria is 1.81×10 9 CFU / mL, the number of viable bacteria is large and the pH is stable, indicating that the fermentation effect is better when the dosage of yeast extract is 4% (g / mL). Therefore, Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 3 is used in the following examples.

[0051] Example 4 Application of Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale fermentation broth in in vitro hypoglycemic The enzyme content of α-amylase in this example is 14 U / mg, and the enzyme content of α-glucosidase is 50 U / mg.

[0052] α-Amylase, α-glucosidase, soluble starch, 3,5-dinitrosalicylic acid (DNS) reagent, acarbose, and nitrophenolpyranoglucoside (PNPG) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0053] Solution preparation method Preparation of DNS color developer: 6.3g of 3,5-dinitrosalicylic acid, 5g of phenol and 5g of anhydrous sodium sulfite, 182g of potassium sodium tartrate, dissolved in 262mL of 2mol / L NaOH, mixed, and distilled water to 500mL. Place at room temperature away from light for 7 days before use.

[0054] Preparation of PBS buffer: NaCl 8g, KCl 0.2g, Na2HPO4•12H2O 2.88g, KH2PO40.2g, dissolve in distilled water, adjust pH to 7.3-7.4 and make up to 1L, sterilize at high temperature and high pressure and store at 4℃ Preparation of 30U / mL α-amylase solution: weigh 0.2143g α-amylase, dilute to 100mL with PBS buffer, sonicate and filter.

[0055] Preparation of 0.5% soluble starch solution: Accurately weigh 0.5 g of soluble starch, dissolve it in distilled water to make up to 100 mL, and store at 4°C.

[0056] 1.5 U / mL α-glucosidase solution: weigh 3 mg of α-amylase, dilute to 100 mL with PBS buffer, sonicate and filter.

[0057] Acarbose solution: Take acarbose tablets, grind them into powder, accurately weigh 0.1 g, dilute to 100 mL with distilled water, and prepare a 1 mg / mL solution.

[0058] 20 mmol / L PNPG solution: Accurately weigh 0.6025 g PNPG, dissolve it in PBS buffer to make up to 100 mL, and store at 4°C.

[0059] (1) Test of α-amylase activity inhibition rate Preparation and testing of fermentation group sample 1: First, 100 μL of the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth 3 in Example 3 was mixed with 100 μL of 30 U / mL α-amylase solution, incubated at 37°C with shaking for 10 min, then 200 μL of 0.5% (w / v) soluble starch solution was added, and the incubation was continued at 37°C for 10 min. Finally, 1.6 mL of DNS reagent was added, heated in a boiling water bath for 5 min, and fermentation group sample 1 was obtained after cooling. 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 liquid 3 in the preparation method of fermentation group sample 1 was replaced with an equal volume of PBS buffer to obtain a blank control sample of fermentation group sample 1; the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 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 a calibration sample of fermentation group sample 1. Using acarbose solution as a positive control, the ultraviolet absorbance values ​​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.

[0060] Preparation and testing of mixed group sample 1: 10 mL of the extract of Dendrobium officinale prepared in Example 2 was mixed with 1×10 11 The lyophilized powder of Lactobacillus rhamnosus ZKLGG0310 with a concentration of CFU / mL was mixed evenly to obtain a mixed solution, wherein the amount of lyophilized powder of Lactobacillus rhamnosus ZKLGG0310 was 0.181 g. First, 100 μL of the mixed solution was mixed with 100 μL of α-amylase with a concentration of 30 U / mL, incubated at 37°C for 10 min with shaking, then 200 μL of 0.5% (w / v) soluble starch solution was added, and incubated at 37°C for 10 min, and finally 1.6 mL of DNS reagent was added, heated in a boiling water bath for 5 min, and mixed group sample 1 was obtained after cooling. 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 mixed solution in the preparation method of mixed group sample 1 was replaced with an equal volume of PBS buffer to obtain a blank control sample of mixed group sample 1; the mixed solution 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 a calibration sample of mixed group sample 1. Using acarbose solution as a positive control, the ultraviolet absorbance values ​​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.

[0061] The α-amylase activity inhibition rates of fermentation group sample 1, mixed group sample 1 and acarbose were calculated according to formula 1: α-amylase activity inhibition rate (%) = [1-(A3-A4) / (A1-A2)] × 100% (Formula 1), In formula 1, A3 is the absorbance of the samples to be tested, namely, fermentation group sample 1 and mixed group sample 1; A4 is the absorbance of the control sample corresponding to the sample to be tested; A1 is the absorbance of the blank control sample corresponding to the sample to be tested; A2 is the absorbance of the calibration sample corresponding to the sample to be tested. The inhibition rates of α-amylase activity of fermentation group sample 1, mixed group sample 1 and acarbose solution are shown in Table 1.

[0062] Table 1 α-amylase activity inhibition rate of fermentation group sample 1, mixed group sample 1 and acarbose

[0063] Note: Different letters in the table represent significant differences, P < 0.05.

[0064] (2) Test of α-glucosidase activity inhibition rate Preparation and testing of fermentation group sample 2: First, 200 μL of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation broth 3 in Example 3 was mixed evenly with 50 μL of 1.5 U / mL α-glucosidase solution, incubated at 37°C for 5 min, then 75 μL of 20 mmol / L PNPG solution was added and mixed evenly, and the mixture was incubated at 37°C for 15 min. Finally, 400 μL of 0.2 mol / L Na2CO3 solution was added to terminate the reaction to obtain fermentation group sample 2. 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 liquid 3 in the preparation method of fermentation group sample 2 was replaced with an equal volume of PBS buffer to obtain a blank control sample of fermentation group sample 2; the Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 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 a calibration sample of fermentation group sample 2. Acarbose was used as a positive control, and the ultraviolet absorbance values ​​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.

[0065] Preparation and testing of mixed group sample 2: First, 200 μL of the mixed solution was mixed evenly with 50 μL of 1.5 U / mL α-glucosidase solution, incubated at 37°C for 5 min, then 75 μL of 20 mmol / L PNPG solution was added and mixed evenly, and incubated at 37°C for 15 min. Finally, 400 μL of 0.2 mol / L Na2CO3 solution was added to terminate the reaction, and mixed group sample 2 was obtained. The α-glucosidase solution in the preparation method of mixed group sample 2 was replaced with an equal volume of distilled water as the control sample of mixed group sample 2; the mixed solution in the preparation method of mixed group sample 2 was replaced with an equal volume of PBS buffer to obtain the blank control sample of mixed group sample 2; the mixed solution in the preparation method of mixed 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 mixed group sample 2. Using acarbose as a positive control, the ultraviolet absorbance values ​​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 were measured at 405 nm.

[0066] The α-glucosidase activity inhibition rates of fermentation group sample 2, mixed group sample 2 and acarbose were calculated according to formula 2: α-glucosidase activity inhibition rate (%) = [1-(A3'-A4') / (A1'-A2')] × 100% (Formula 2) In formula 2, A3' is the absorbance of the samples to be tested - fermentation group sample 2 and mixed group sample 2; A4' is the absorbance of the control sample corresponding to the sample to be tested; A1' is the absorbance of the blank control sample corresponding to the sample to be tested; A2' is the absorbance of the calibration sample corresponding to the sample to be tested. The inhibition rates of α-glucosidase activity of fermentation group sample 2, mixed group sample 2 and acarbose are shown in Table 2.

[0067] Table 2 α-glucosidase activity inhibition rate of fermentation group sample 2, mixed group sample 2 and acarbose

[0068] Note: Different letters in the table represent significant differences, P < 0.05.

[0069] α-amylase and α-glucosidase can hydrolyze the α-1,4-glycosidic bonds of starch and glycogen, as well as the α-1,6-glycosidic bonds of oligosaccharides, to help the human body convert edible carbohydrates into glucose for intestinal absorption. The inhibitory effect on the activity of these two digestive enzymes, α-amylase and α-glucosidase, has always been an important evaluation index of hypoglycemic activity in vitro. By inhibiting the activity of α-amylase and α-glucosidase, the conversion rate of carbohydrates into glucose can be reduced, thereby slowing down the rise in blood sugar after a meal. As can be seen from Tables 1 and 2, the mixture of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid, Dendrobium officinale extract and Lactobacillus rhamnosus ZKLGG0310 freeze-dried powder has a strong inhibitory effect on α-amylase and α-glucosidase in vitro. The inhibitory effect of the fermented liquid of Dendrobium officinale with Lactobacillus rhamnosus ZKLGG0310 was significantly stronger than that of the extract of Dendrobium officinale with Lactobacillus rhamnosus ZKLGG0310 (P < 0.05), and was higher than that of acarbose. This indicates that the fermentation of Dendrobium officinale extract with Lactobacillus rhamnosus ZKLGG0310 can significantly improve its hypoglycemic ability in vitro.

[0070] Example 5 The fermentation liquid of Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale prepared in Example 3 was used to treat type 2 diabetic mice (1) Modeling: Sixty male C57BL / 6J mice were fed with a normal diet for one week, and then twelve mice were randomly selected as the control group (NC group) and forty-eight mice were randomly selected as the high-fat and high-sugar group (HFD group). The mice in the control group were fed with a normal diet every day; the mice in the high-fat and high-sugar group were fed with a high-fat and high-sugar diet every day. The high-fat and high-sugar diet was purchased from Beijing Huafukang Biotechnology Co., Ltd.

[0071] After being fed continuously for 4 weeks, the mice in the high-fat and high-sugar group were fasted for 12 h and then intraperitoneally injected with STZ prepared in citrate buffer. STZ was purchased from Sigma Company in the United States and was freshly prepared before use. It was made into a 1% concentration with 0.1 mol / L citrate buffer solution with a pH of 4.2. The injection dose of STZ was 50 mg / kg of mouse body weight per day. STZ injection was repeated for 3 days, and one week after the last injection, the tails were cut and blood was collected to measure the fasting blood glucose levels of the mice. The fasting blood glucose level ≥11.1 mmol / L was used as the standard for the successful establishment of the type 2 diabetes pathological model. After the mice with unsuccessful modeling were removed from the high-fat and high-sugar group, forty type 2 diabetic mice with successful modeling were obtained.

[0072] (2) Grouping: Forty successfully modeled type 2 diabetic mice were randomly divided into four groups, namely, the hyperglycemia model group (MC group), the Lactobacillus rhamnosus ZKLGG0310 mixed group (DO group), the Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale fermentation group (FDO group), and the metformin positive control group (DMBG group), with ten mice in each group.

[0073] (3) Administration: NC group and MC group were gavaged with 0.2 mL of sterile saline every day.

[0074] DO group: 0.2 mL of the mixture of Dendrobium officinale extract and Lactobacillus rhamnosus ZKLGG0310 was intragastrically administered every day. The number of viable Lactobacillus rhamnosus ZKLGG0310 in the mixture was 1.81×10 9 CFU / mL.

[0075] FDO group: 0.2 mL of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermentation liquid 3 prepared in Example 3 was administered intragastrically every day.

[0076] DMBG group: mice were gavaged with 200 mg / kg of metformin daily.

[0077] Each group of mice was gavaged continuously for four weeks according to the above-mentioned administration method. During the administration period, they were free to eat and drink water. Except for the mice in the control group who were fed with ordinary feed, the mice in other groups were fed with high-fat and high-sugar feed.

[0078] (4) Sample collection and index testing: ① Body weight and fasting blood glucose: After one week of adaptive feeding, the body weights of mice in the NC group, MC group, DO group, FDO group and DMBG group were monitored and recorded weekly, and the body weight change trends of mice in each group were statistically analyzed. The body weight change trends of mice in each group are shown in Table 3; during the experimental period after the end of the adaptation period, the fasting blood glucose concentration of mice in each group was measured once every 2 weeks using a blood glucose meter, and the average value of the fasting blood glucose concentration of mice in each group was taken for each measurement. Before measuring the fasting blood glucose concentration, the mice in each group fasted but not watered for 16 hours. When measuring the fasting blood glucose concentration, blood was collected from the tail of the mice. The statistical graph of the last fasting blood glucose concentration of mice in each group before slaughter is shown in the figure below. Figure 4 shown.

[0079] Table 3 Changes in body weight of mice in each group (unit: g)

[0080] As shown in Table 3, the weight of mice in the NC group increased steadily throughout the experimental period, and their biochemical status was normal; the weight of mice in the MC group continued to decrease after one week of adaptive feeding, and one of the symptoms of type 2 diabetes is weight loss. The weight loss rate of mice in the DO, FDO and DMBG groups slowed down after administration, and the weight of the FDO and DMBG groups increased slightly in the fourth week after administration, indicating that the inhibitory effect of the fermented liquid of Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale on the weight loss symptoms of type 2 diabetic mice was stronger than that of the mixed liquid of Dendrobium officinale extract and Lactobacillus rhamnosus ZKLGG0310, and was equivalent to that of metformin.

[0081] Depend on Figure 4 It can be seen that the fasting blood glucose concentration of mice in MC group was significantly higher than that in NC group (P<0.05), and the fasting blood glucose concentrations of mice in DO group, FDO group and DMBG group were significantly lower than that in MC group after administration (P<0.05), and the fasting blood glucose concentration of mice in FDO group was significantly lower than that in DO group (P<0.05), and close to that in DMBG group, indicating that the effect of Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale fermentation liquid on reducing fasting blood glucose in type 2 diabetic mice is stronger than that of the mixed liquid of Dendrobium officinale extract and Lactobacillus rhamnosus ZKLGG0310, and is equivalent to that of metformin.

[0082] 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 killing the mice, blood was obtained by taking blood from the eyeballs of the mice, and the blood was centrifuged at 4500r / min for 3min to obtain mouse serum. The glycated hemoglobin content in the serum of each group of mice was detected according to the steps indicated by the supplier on the kit. The serum glycated hemoglobin concentration of each group of mice was as follows: Figure 5 shown.

[0083] The concentration of glycosylated hemoglobin is less affected by the environment, so it can stably reflect the average blood sugar level of the body in the past 2-3 months. Figure 5 It can be seen that the glycated hemoglobin concentration of mice in the MC group was significantly higher than that in the NC group (P < 0.05), and the glycated hemoglobin concentration of mice in the DO group, FDO group and DMBG group was significantly lower than that in the MC group (P < 0.05). The glycated hemoglobin concentration of mice in the FDO group was significantly lower than that in the DO group (P < 0.05), and was close to that in the DMBG group, indicating that the effect of Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale fermentation liquid on reducing the glycated hemoglobin concentration of type 2 diabetic mice was stronger than the mixed liquid of Dendrobium officinale extract and Lactobacillus rhamnosus ZKLGG0310, and was equivalent to that of metformin. Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale fermentation liquid can control blood sugar levels within the normal range for a long time and reduce the risk of complications of type 2 diabetes.

[0084] ③ Oral glucose tolerance test (OGTT): After four weeks of administration, mice in each group were fasted but not watered for 16 h, and gavaged with a glucose aqueous solution at a volume of 2 g / kg of mouse body weight. The concentration of the glucose aqueous solution was 40% (w / v). Blood was collected from the tail vein of the mice at 0 min, 30 min, 60 min, 90 min, and 120 min after gavage with the glucose aqueous solution. The blood glucose concentration of each group of mice was measured and calculated using a blood glucose meter and matching blood glucose test strips. After blood sampling, the acupuncture site of the mouse was wiped with iodine to prevent infection. The blood glucose concentration at each time point was plotted and the area under the glucose tolerance curve (AUC) was calculated. The numerical value of AUC directly reflects the glucose tolerance of each group of mice. The average blood glucose concentrations of each group of mice at 0 min, 30 min, 60 min, 90 min, and 120 min after gavage with the glucose aqueous solution are shown in the figure. Figure 6 The area under the glucose tolerance curve of each group of mice is shown in the figure below. Figure 7 shown.

[0085] like Figure 6 As shown in the figure, after intragastric administration of glucose solution, the blood glucose concentration of mice in each group increased first and then decreased, and all reached the maximum value at the 30th minute. Although the blood glucose concentration of mice in the MC group decreased from 30 to 120 minutes, it was still at a high level. Compared with the mice in the MC group, the blood glucose concentration of mice in the DO group, FDO group and DMBG group decreased, and the blood glucose concentration of mice in the FDO group was lower than that in the DO group and close to that in the DMBG group, indicating that the control effect of Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermented liquid on blood glucose in type 2 diabetic mice was stronger than that of Dendrobium officinale extract, and was equivalent to that of metformin.

[0086] like Figure 7 As shown in the figure, the AUC of mice in the MC group was significantly higher than that of mice in the NC group (P < 0.05). After four weeks of administration, compared with the MC group, the AUC of mice in the DO group, FDO group and DMBG group was significantly reduced (P < 0.05), and the AUC of mice in the FDO group was significantly lower than that in the DO group (P < 0.05), and was close to the positive DMBG group. This indicates that the fermented liquid of Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale can significantly improve the glucose tolerance of mice with type 2 diabetes and repair the impaired glucose tolerance.

[0087] ④ Calculation of insulin level and insulin resistance index: After the last administration, mice in each group were fasted for 16 h, then their body weight was measured and their eyeballs were removed for blood collection. The supernatant was obtained by centrifugation to obtain the serum of mice in each group. The serum insulin concentration of mice in each group was measured using an insulin kit (purchased from Nanjing Jiancheng) and the average value was taken. The average serum insulin concentration of mice in each group is shown in the figure. Figure 8 shown.

[0088] According to 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 formula 3. The insulin resistance index of each group of mice is as follows: Fig. 9 shown.

[0089] HOMA-IR=[serum insulin concentration (mIU / L) × fasting blood glucose concentration (mmol / L)] / 22.5 (Formula 3).

[0090] Insulin is secreted by pancreatic β cells and is the only hormone in the body that lowers blood sugar. The level of insulin is a key factor affecting blood sugar levels. Figure 8 As shown in the figure, the average insulin concentration of mice in the MC group was significantly lower than that of mice in the NC group (P < 0.05). After four weeks of administration, compared with the MC group, the average serum insulin concentration of mice in the DO group, FDO group and DMBG group increased significantly (P < 0.05), and the average serum insulin concentration of mice in the FDO group was significantly higher than that in the DO group (P < 0.05), and was close to that in the DMBG group. This indicates that the fermented liquid of Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale can significantly increase the serum insulin level of mice with type 2 diabetes.

[0091] like Fig. 9 As shown in the figure, the HOMA-IR of mice in the MC group was significantly higher than that of mice in the NC group (P < 0.05). After four weeks of administration, compared with the MC group, the HOMA-IR of mice in the DO group, FDO group and DMBG group was significantly reduced (P < 0.05), and the FDO group was significantly lower than the DO group (P < 0.05), and close to the DMBG group, indicating that the fermented liquid of Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale can significantly reduce the insulin resistance of type 2 diabetic mice and improve the insulin sensitivity.

[0092] ⑤ Blood lipid index detection: The serum total cholesterol (TC) kit (purchased from Nanjing Jiancheng) was used to measure the TC content in the serum of each group of mice and the average value was taken. The average statistical graph of the TC content in the serum of each group of mice is shown in the figure below. Fig.10 The triglyceride (TG) kit (purchased from Nanjing Jiancheng) was used to measure the TG content in the serum of each group of mice. The average statistical graph of the TG content in the serum of each group of mice is shown in Fig.11 shown.

[0093] Insulin resistance in patients with type 2 diabetes can cause fat metabolism disorders in the body, accelerate the oxidative decomposition of fat by body cells, and lead to increased blood lipid levels. Fig.10 and Fig.11As shown in the figure, the TC and TG of mice in the MC group were significantly higher than those in the NC group (P < 0.05). After four weeks of administration, compared with the MC group, the TC and TG concentrations of mice in the DO group, FDO group and DMBG group were significantly reduced (P < 0.05), and the FDO group was significantly lower than the DO group (P < 0.05), and close to the DMBG group. This indicates that the fermented liquid of Lactobacillus rhamnosus ZKLGG0310 and Dendrobium officinale can significantly reduce the blood lipid indexes of type 2 diabetic mice and regulate the lipid metabolism of diabetic mice.

[0094] In summary, Lactobacillus rhamnosus ZKLGG0310 Dendrobium officinale fermented liquid has a significant hypoglycemic effect on type 2 diabetic mice compared with unfermented Dendrobium officinale extract (P < 0.05), can significantly increase insulin levels (P < 0.05), significantly reduce insulin resistance (P < 0.05), and regulate lipid metabolism.

[0095] Although the present invention has been described in detail with reference to the accompanying drawings and in combination with the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection 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: Including the use in preparing medicines for treating type 2 diabetes.

Images