Pericarpium citri reticulatae efficacy-oriented microbial aging preparation method and application thereof

Abstract

The invention discloses an efficacy-oriented microbial aging preparation method for pericarpium citri reticulatae and application of the efficacy-oriented microbial aging preparation method. The invention discloses a microorganism for improving the quality and the efficacy of citrus chachiensis hortorum, and the microorganism comprises bacillus aryabhattai, bacillus velezensis, bacillus velezensis and / or aspergillus fumigatus, and the microorganism comprises bacillus aryabhattai, bacillus velezensis, bacillus velezensis and / or aspergillus fumigatus, and the microorganism comprises bacillus aryabhattai, bacillus velezensis and / or aspergillus fumigatus. According to the invention, microbiological spectrums and metabolite spectrums of Xinhui dried orange peel in different aging years are compared through microbiomics and metabonomics to screen out the microorganisms for improving the content of 4-vinyl guaiacol (4-VG) in the dried orange peel, and the microorganisms act on the dried orange peel, so that oriented aging of the dried orange peel can be controlled; the dried orange peel for powerfully treating insulin resistance and diabetes mellitus is prepared.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a tangerine peel efficacy-oriented microbial aging preparation method and application thereof. Background Art

[0002] As a representative of "old medicine" that needs to be aged, the quality and efficacy of Guangdong dried tangerine peel depend to a certain extent on the control conditions of the aging process. In the aging process of dried tangerine peel, there are both natural fermentation biochemical reactions, natural volatilization physical processes and natural oxidation chemical reactions involved, which leads to the complexity of the aging process. Among them, due to the biological activity and fermentation performance of microorganisms, they play a leading role in the aging process of dried tangerine peel. Although it has been reported that microorganisms such as Bacillus and Mucor and their preparations can affect the aging process of dried tangerine peel, these technologies mainly focus on studying the effects of microorganisms on the changes in flavonoids and volatile oil components during the aging process of dried tangerine peel at the genus level, but do not involve the evaluation of specific strains on the aging of dried tangerine peel and its health effects. It is difficult to explain the scientific connotation of Guangdong dried tangerine peel's "older is better", and it is also impossible to fully reflect the purpose and significance of dried tangerine peel aging. Summary of the invention

[0003] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides a microorganism for increasing the content of 4-vinylguaiacol (4-VG) in dried tangerine peel.

[0004] The invention also provides an aging method for improving the quality and efficacy of tangerine peel.

[0005] The invention also provides tangerine peel prepared by the aging method.

[0006] The invention also provides the application of the dried tangerine peel.

[0007] The present invention also proposes the use of 4-VG in preparing products for preventing and / or treating insulin resistance and / or diabetes.

[0008] According to a first aspect of the present invention, a microorganism for increasing the 4-VG content in tangerine peel is provided, wherein the microorganism comprises at least one of P. aryabhattai, B. velezensis and A. fumigatus.

[0009] In some embodiments of the present invention, the microorganism comprises at least two or three of P. aryabhattai, B. velezensis and A. fumigatus.

[0010] In some embodiments of the present invention, the tangerine peel includes tangerine peel from tea branch tangerine (Guang tangerine peel), Wenzhou mandarin orange (Zhe tangerine peel), early tangerine (Zhe tangerine peel), lucky tangerine (Jian tangerine peel), Sichuan tangerine (Sichuan tangerine peel), Dahongpao (Sichuan tangerine peel), Nanfeng tangerine (Gan tangerine peel), etc.

[0011] According to the second aspect of the present invention, any of the following applications of the above-mentioned microorganisms is proposed:

[0012] (1) Increase the 4-VG content in dried tangerine peel;

[0013] (2) Control the aging process of tangerine peel;

[0014] (3) Improve the quality and efficacy of dried tangerine peel;

[0015] (4) Preparation of products for preventing and / or treating insulin resistance;

[0016] (5) Preparation of products for the prevention and / or treatment of diabetes;

[0017] (6) preparing products for promoting glucose uptake by cells;

[0018] (7) preparing products for increasing the expression of pAKT, P-GSK-3β and / or 4-VG;

[0019] (8) Preparation of products for lowering cholesterol levels in serum;

[0020] (9) Prepare a product for reducing the triglyceride content in serum.

[0021] In some embodiments of the present invention, the diabetes includes type I diabetes, type II diabetes, and gestational diabetes.

[0022] According to a second aspect of the present invention, an aging method for improving the quality and efficacy of dried tangerine peel is proposed, the aging method comprising the following steps: allowing the above-mentioned microorganisms to act on the dried tangerine peel to regulate the 4-VG content in the dried tangerine peel.

[0023] In some embodiments of the present invention, the regulation is to increase the 4-VG content in tangerine peel.

[0024] In some embodiments of the invention, the method of action comprises fermentation.

[0025] In some embodiments of the present invention, the concentration of the microorganisms inoculated in the fermentation is 1×10 6 -1×10 8 CFU / mL.

[0026] In some embodiments of the present invention, the fermentation temperature is 28-35°C.

[0027] According to a third aspect of the present invention, a dried tangerine peel for preventing and / or treating diabetes or insulin resistance is provided. The dried tangerine peel is prepared by the following method: allowing the above-mentioned microorganism to act on the dried tangerine peel.

[0028] In some embodiments of the invention, the method of action comprises fermentation.

[0029] In some embodiments of the present invention, the concentration of the microorganisms inoculated in the fermentation is 1×10 2 -1×10 11 CFU / mL.

[0030] In some embodiments of the present invention, the concentration of the microorganisms inoculated in the fermentation is 1×10 5 -1×10 9 CFU / mL.

[0031] In some embodiments of the present invention, the concentration of the microorganisms inoculated in the fermentation is 1×10 6 -1×10 8 CFU / mL.

[0032] In some embodiments of the present invention, the fermentation temperature is 28-40°C.

[0033] According to a fourth aspect of the present invention, the application of the above-mentioned dried tangerine peel in any of the following items is proposed:

[0034] 1) Preparation of products for preventing and / or treating insulin resistance;

[0035] 2) Preparation of products for preventing and / or treating diabetes;

[0036] 3) preparing products for promoting glucose uptake by cells;

[0037] 4) preparing products for increasing the expression of pAKT, P-GSK-3β and / or 4-VG;

[0038] 5) Preparation of products for reducing cholesterol levels in serum;

[0039] 6) Prepare products for reducing the content of low-density lipoprotein cholesterol in serum.

[0040] In some embodiments of the present invention, the insulin resistance disease includes at least one of type II diabetes, coronary heart disease and metabolic syndrome.

[0041] In some embodiments of the present invention, the product comprises a medicine or a food.

[0042] According to the fifth aspect of the present invention, the application of 4-VG in any one of the following (A1) to (A3) is proposed:

[0043] (A1) preparing products for preventing and / or treating insulin resistance and / or diabetes;

[0044] (A2) controlling the aging process of tangerine peel;

[0045] (A3) Improve the quality and efficacy of dried tangerine peel.

[0046] In some embodiments of the present invention, the insulin resistance diseases include type II diabetes, coronary heart disease, metabolic syndrome and other diseases.

[0047] In some embodiments of the present invention, the product is a product for promoting glucose uptake by cells.

[0048] In some embodiments of the present invention, the product is a product for increasing the expression of pAKT, P-GSK-3β and / or 4-VG.

[0049] In some embodiments of the present invention, the product is a product for reducing the cholesterol content in serum.

[0050] In some embodiments of the present invention, the product is a product for reducing the triglyceride content in serum.

[0051] In some embodiments of the present invention, the product comprises a medicine or a food.

[0052] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0053] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener and a flavoring agent.

[0054] In some embodiments of the invention, the excipient comprises water.

[0055] In some embodiments of the present invention, the filler comprises at least one of starch and sucrose.

[0056] In some embodiments of the present invention, the binder includes at least one of a cellulose derivative, alginate, gelatin and polyvinyl pyrrolidone.

[0057] In some embodiments of the invention, the humectant comprises glycerin.

[0058] In some embodiments of the present invention, the disintegrant comprises at least one of agar, calcium carbonate and sodium bicarbonate.

[0059] In some embodiments of the invention, the absorption enhancer comprises a quaternary ammonium compound.

[0060] In some embodiments of the invention, the surfactant comprises cetyl alcohol.

[0061] In some embodiments of the present invention, the adsorption carrier includes at least one of kaolin and bentonite.

[0062] In some embodiments of the present invention, the lubricant includes at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0063] In some embodiments of the present invention, the dosage form of the drug is in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension.

[0064] In some embodiments of the present invention, the formulation is in the form of tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, pellets, dry extracts, injections or infusions, transdermal agents, or transdermal microneedles.

[0065] In some embodiments of the present invention, the drug may be administered by any conventional method in the art, including but not limited to injection or oral administration.

[0066] In some embodiments of the present invention, the injection administration can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

[0067] In some embodiments of the present invention, the mass fraction of 4-VG in the product is 0.01% to 100%.

[0068] According to some preferred embodiments of the present invention, the mass fraction of 4-VG in the product is 0.05% to 95%.

[0069] According to some preferred embodiments of the present invention, the mass fraction of 4-VG in the product is 0.05% to 50%.

[0070] According to some embodiments of the present invention, at least the following beneficial effects are achieved: the present invention screened out microorganisms for increasing the 4-VG content in tangerine peel by comparing the microbial spectra and metabolite spectra of Xinhui tangerine peel of different aging years (1, 3, 5 and 7 years) through microbiomics and metabolomics, and applied the microorganisms to tangerine peel to control the efficacy-oriented aging of tangerine peel, thereby preparing tangerine peel that is effective in treating insulin resistance and diabetes.

[0071] At the same time, the present invention discovered for the first time that 4-VG can be used to treat insulin resistance and diabetes, and can also promote cellular glucose uptake, reduce the accumulation of cholesterol and triglycerides, and increase the phosphorylation level of the IRS-1 / Akt / GSK-3β pathway. 4-VG can be widely used in the prevention and treatment of insulin resistance-related diseases such as obesity, diabetes or diabetic complications. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0073] Figure 1 The appearance diagram of dried tangerine peel of different years in Example 1 of the present invention;

[0074] Figure 2 It is a graph showing the changes in essential oil components of dried orange peels from different years in Example 1 of the present invention;

[0075] Figure 3 14 is α-Terpineol, 15 is Decanal, 16 is Carveol, 18 is Perillaldehyde, 19 is Thymol, 20 is p-Thymol, 21 is Carvacrol, 22 is 4-Vinylguaiacol (4-VG), 23 is Methyl 2-(methylamino)benzoate, 24 is β-Caryophyllene, 29 is α-Sinensal, and B is the glucose uptake test result of C2C12 myotube cells. Compared with the normal group, " * " indicates P < 0.05," ** " indicates P < 0.01," *** " indicates P < 0.001," *** " indicates P < 0.0001; compared with the model group, & P<0.05, &&& P < 0.001;

[0076] Figure 4 This is a graph showing the results of detecting the contents of ferulic acid, 4-VG, vanillin and guaiacol in 5-year-old dried orange peel in Example 1 of the present invention;

[0077] Figure 5 This is a non-targeted metabolomics detection result diagram of the relative content changes of 4-VG, ferulic acid, vanillin and guaiacol in dried orange peel of different years in Example 1 of the present invention;

[0078] Figure 6 This is a graph showing the microbiome detection results of the changes in the abundance of the main microorganisms in dried orange peel of different years in Example 1 of the present invention;

[0079] Figure 7 4-VG, ferulic acid, vanillin and guaiacol in the dried orange peel of Example 1 of the present invention are analyzed for the correlation between the relative contents of guaiacol and microorganisms, wherein A is an analysis diagram of the correlation between ferulic acid, vanillin and guaiacol and different strains; B is a correlation analysis diagram of the relative content of ferulic acid and the abundance of the genus Bacillus; C is a correlation analysis diagram of the relative content of guaiacol and the abundance of the genus Bacillus; D is a correlation analysis diagram of the relative content of vanillin and the abundance of the genus Bacillus; E is a correlation analysis diagram of the relative content of ferulic acid and the abundance of the genus Aspergills; F is a correlation analysis diagram of the relative content of guaiacol and the abundance of the genus Aspergills; G is a correlation analysis diagram of the relative content of vanillin and the abundance of the genus Aspergills;

[0080] Figure 8 The graphs are the test results of 4-VG enhancing the glucose uptake of insulin-stimulated C2C12 myotube cells in Example 2 of the present invention, wherein A is the test result of 4-VG toxicity to C2C12 myotube cells detected by MTT method; B is the test result of 4-VG enhancing the glucose uptake of insulin-stimulated C2C12 myotube cells in a dose-dependent manner; C is the test result of 4-VG activating the insulin signaling pathway of C2C12 myotube cells; the data are expressed as mean ± SD (n = 3), compared with the control group, " & " indicates P < 0.05, compared with the model group, " * " indicates P < 0.05," *** ” indicates P < 0.0001;

[0081] Fig. 9The figure is a test result diagram of 4-VG in Example 2 of the present invention to improve the phenotype of diabetic mice induced by high-fat diet / streptozotocin (HFD / STZ); wherein A is an experimental grouping and flow chart; B is a test result diagram of weight changes of experimental mice; C is a test result diagram of final weight of mice at the end of the experiment; D is a test result diagram of average weekly energy intake of mice; E is a test result diagram of weekly fasting blood glucose of mice; F is a test result diagram of fasting blood glucose of mice after 4-VG intervention for 4 weeks; G is a glucose tolerance test and AUC value after 4-VG treatment for 4 weeks; H is a result diagram of Western blot detection of key protein expression of gastrocnemius insulin signaling pathway; I is a test result diagram of serum total cholesterol (TC); J is a test result diagram of serum total triglycerides (TG); K is a test result diagram of serum low-density lipoprotein cholesterol (LDL-C); data are expressed as mean ± SD (n = 6 to 7), compared with the normal group, * P<0.05, **** P<0.0001; compared with the model group, & P<0.05, && P<0.01, &&& P < 0.001, &&&& P<0.0001. DETAILED DESCRIPTION

[0082] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0083] Example 1 A microorganism for increasing the content of 4-VG in dried tangerine peel

[0084] This embodiment provides a microorganism for increasing the 4-VG content in dried tangerine peel, including P. aryabhattai, B. velezensis and / or A. fumigatus. The screening and verification methods of the specific microorganisms are as follows:

[0085] 1. GC-MS analysis of the main chemical components in tangerine peel volatile oil

[0086] Grind 20g of dried tangerine peel from different years into powder (1, 3, 5, 7 years old dried tangerine peel powder respectively, the morphology of dried tangerine peel from different years is as follows Figure 1As shown) and after passing through a 60-mesh sieve, it was placed in a three-necked flask equipped with a volatile oil extractor. 10 times distilled water was added, and the mixture was refluxed and extracted twice at 95-100°C, each extraction for 2 hours. Subsequently, the extracted volatile oils were combined, and the extraction yield was about 0.3%. In order to perform GC-MS analysis on the volatile oil, a DB-5 capillary column (Agilent) was used for chromatographic analysis. Chromatographic conditions: initial temperature 70°C, 2 minutes, the temperature was increased to 160°C at a rate of 5°C / min, and then further increased to 240°C at a rate of 20°C / min, and maintained for 4 minutes. The total running time of the analysis was 26 minutes. The injection temperature was set to 230°C, and the flow rate was maintained at 1.3mL / min using high-purity helium as the carrier gas. The split ratio used was 10:1.

[0087] Table 1

[0088]

[0089]

[0090]

[0091] Changes in the main chemical components of essential oils in different years Figure 2 As shown in Table 1, a total of 29 components were identified, including 15 monoterpenes, 6 sesquiterpenes, 5 aromatic hydrocarbons, 2 fatty aldehydes and 1 alkaloid. Monoterpenes D-limonene and γ-terpinene are the main components of the volatile oil of D. tangerine peel. The contents of alkaloids 2-(methylamino)benzoic acid methyl ester, monoterpenes β-cis-butylene, α-pinene, β-pinene and β-myrcene, and a sesquiterpene α-octenal were higher than those of other components. Among them, the relative content of the aromatic compound 4-VG increased with age.

[0092] 2. Effects of the main components of tangerine peel essential oil on glucose uptake activity of C2C12 myoblasts

[0093] (1) Cultivation of C2C12 cells: Mouse C2C12 myoblasts (purchased from ATCC, RRID: CVCL_0188) were routinely cultured in DMEM complete medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S).

[0094] (2) C2C12 differentiation induction: When C2C12 cells reached about 100% confluence, they were incubated with DMEM containing 2% heat-inactivated horse serum and 1% P / S for 4 days.

[0095] (3) The differentiated 5×10 6C2C12 cells / mL were inoculated into 96-well plates, 200 μL per well, and after overnight culture, the drugs were administered according to the following groups: drug administration group, blank control group, negative control group and positive control group. The drug administration group included drugs α-Pinene, β-Pinene, β-Myrcene, D-Limonene, γ-Terpinene, Terpinolene, Terpinen-4-ol, Linalool, α-Terpineol, Decanal, Carveol, Perillaldehyde, Thymol, p-Thymol, Carvacrol, 4-V inylguaiacol, Methyl2-(methylamino)benzoate, β-Caryophyllene, α-Sinensal or 0.1μM insulin; and groups with the addition of drugs ferulic acid, 4-VG, vanillin or guaiacol at a final concentration of 10μM, and 0.1μM insulin respectively; the blank control group was different from the drug-treated group only in that insulin and drugs were not added; the negative control group was different from the drug-treated group only in that drugs were not added; the positive control group was different from the drug-treated group only in that the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, final concentration of 20μM) was used to replace the drug.

[0096] Six replicate wells were set for each group. After 12 hours of drug incubation, the cells were washed with Krebs–Ringer phosphate (KRP) buffer (20mM HEPES, 137mM NaCl, 4.7mM KCl, 1.2mM MgSO4, 1.2mM KH2PO4, 2.5mM CaCl2, and 2mM pyruvate; pH 7.4) and incubated in KRP buffer containing 0.2% BSA for 2 hours. The cells were placed in suspension, 0.2mM 3H-labeled 2-deoxyglucose was added, and incubated at 37°C for 30 minutes. The intracellular 2-deoxyglucose content was detected using an enzyme-labeled instrument with an excitation wavelength of 475nm and an emission wavelength of 550nm.

[0097] The results are as follows Figure 3 As shown, from Figure 3As shown in Figure A, among the 19 components of tangerine peel essential oil, 4-VG has the highest activity in enhancing the glucose uptake of insulin-stimulated C2C12 cells, indicating that 4-VG is the key active ingredient in tangerine peel to improve insulin resistance; ferulic acid, 4-VG and vanillin are important aroma substances in tangerine peel. Among them, ferulic acid can generate 4-VG and vanillin through microbial action, and the latter can also be converted into guaiacol under the action of microorganisms. The cell model was further used to evaluate the effects of 4-VG, ferulic acid, vanillin and guaiacol on glucose uptake activity. The results are shown in the figure. Figure 3 As shown in Figure B, it can be seen that among the related compounds of 4-VG biotransformation, 4-VG can significantly enhance the glucose uptake of insulin-stimulated C2C12 cells, while the other three compounds have no significant activity.

[0098] 3. Metabolomics detection and analysis

[0099] Accurately weigh 50.0 mg of tangerine peel powder from different years (1, 3, 5, and 7 years, respectively), and add 1000 μL of methanol: acetonitrile: water (2:2:1) extraction solution. The mixture was vortexed for 30 seconds, then ground with a steel ball at 45 Hz for 10 minutes and treated with ultrasound in an ice-water bath for 10 minutes. Subsequently, the mixture was allowed to stand at -20°C for 1 hour and centrifuged at 12000 rpm for 15 minutes at 4°C. 500 μL was taken from the supernatant, concentrated and dried. The extract was resuspended by adding a 1:1 mixture of acetonitrile and water, and it was repeatedly vortexed and centrifuged. Finally, 120 μL of the supernatant was aliquoted into 2 mL injection vials, and quality control (QC) samples were prepared by mixing 10 μL of each sample.

[0100] The UPLC-Q-TOF / MS system for metabolomics analysis consisted of a Waters Acquity I-Class PLUS ultra-high performance liquid chromatography coupled to a Waters Xevo G2-XS QTof high-resolution mass spectrometer equipped with a Waters AcqutyUPLC-HSS T3 column (1.8 μm, 2.1 × 100 mm). The detection conditions were as follows: mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile, and the injection volume was 1 μL. Mass spectrometry conditions: Waters Xevo G2-XS QTOF high-resolution mass spectrometer, which can acquire primary and secondary mass spectrometric data in MSe mode under the control of acquisition software (MassLynx V4.2, Waters). In each data acquisition cycle, dual-channel data acquisition was performed simultaneously for low and high collision energies. The low collision energy was 2 V, the high collision energy range was 10–40 V, and the mass spectrometry scanning frequency was 0.2 s. The parameters of the ESI ion source are as follows: capillary voltage: 2000 V (positive ion mode) or -1500 V (negative ion mode); cone voltage: 30 V; ion source temperature: 150°C; solution gas temperature: 500°C; backwash gas flow rate: 50 L / h; desolvation gas flow rate: 800 L / h.

[0101] The metabolic data of 4-VG-related compounds, including ferulic acid, vanillin, and guaiacol, were extracted and analyzed from the metabolomics data.

[0102] The results are as follows Figure 4 and Figure 5 As shown in the figure, it can be seen that in tangerine peel of different years, the precursor of 4-VG, ferulic acid, decreases with the increase of aging years, while its conversion products, vanillin and guaiacol, increase with the increase of aging years. Quantitative detection by high-performance liquid chromatography shows that the content trend of these compounds is consistent with the results of metabolomics detection. At the same time, the content of 4-VG in tangerine peel decreases with the increase of aging years, further suggesting that ferulic acid is converted into 4-VG, vanillin and guaiacol by microorganisms during the aging process of tangerine peel, thus showing the characteristic of tangerine peel that "the older it is, the better it is".

[0103] 4. Microbiome sequencing and analysis

[0104] By grinding 5g of tangerine peel powder of different years (respectively 1, 3, 5, 7 years) with liquid nitrogen, and adding 100mL sterile saline (0.9% w / v NaCl) in a 500mL flask at 4°C, shaking at 220rpm / minute for 4 hours, the microorganisms in the tangerine peel samples were collected. After shaking, the tangerine peel particles were removed with four layers of gauze, and centrifuged at 8000rpm / minute for 10 minutes to collect microorganisms. The total DNA of the microbial samples was extracted using the TGuide S96 kit (DP812, Tiangen Biotechnology Co., Ltd., Beijing, China).

[0105] The V3-V4 region of the bacterial 16S rRNA gene was amplified using forward primer 338F (5′-ACCTACGGGAGGCAGCA-3′ (SEQ ID NO: 5)) and reverse primer 806R (5′-GACTACCGGGTATCTAAT-3′ (SEQ ID NO: 6)). The fungal ITS1 region was amplified using forward primer ITS1 (5′-CTTGTCATTTAGAGAAGTAA-3′ (SEQ ID NO: 7)) and reverse primer ITS2 (5′-GTGCGTTCTTCATCGATGC-3′ (SEQ ID NO: 8)). The PCR reaction included: genomic DNA (5 to 50 ng), forward primer (0.3 μL), reverse primer (0.3 μL), KOD FX Neo buffer (5 μL), dNTP mix (2 μL) and KOD FX Neo (0.2 μL) and ddH2O added to a final volume of 10 μL. The PCR reaction conditions were: 95°C for 5 minutes (initial denaturation), 95°C for 30 seconds (denaturation), 50°C for 30 seconds, and 72°C for 40 seconds, repeated 25 cycles. TM The total PCR product was quantified by dsDNAHS reagent, and high-throughput double-end sequencing was performed using the Illumina Hiseq 2500 platform (Beijing Biomarker Biotechnology Co., Ltd.). The original fastq files were quality filtered by Trimmomatic, and operational taxonomic units (OTUs) were clustered using UPARSE at 97% similarity. The different microbial species and relative contents in tangerine peel samples were analyzed.

[0106] Changes in microbial abundance in dried tangerine peel in different years Figure 6 As shown in the figure, it can be seen that with the increase of aging time, the relative abundance of Bacillus and Enterobacter in tangerine peel increases, while the relative abundance of Paecilomyces decreases with the increase of aging time, indicating that the structure of bacteria and fungi changes significantly during the aging process of tangerine peel.

[0107] 5. Isolation and identification of microorganisms

[0108] Under sterile conditions, 10g of 5-year-old tangerine peel sample was minced with sterile scissors, and 1.0g was placed in a 50mL centrifuge tube. 5mL of sterile saline solution was added, and the mixture was shaken at 200rpm / min for 1 hour at 28°C to obtain a microbial suspension. 400μL of the suspension was added to solid culture plates of Luria Bertani (LB), yeast protein glycoside (YPD) and PDA, respectively. LB plates were incubated at 37°C, while YPD and PDA plates were incubated at 30°C. After 24 hours, colonies showing different morphologies were selected from these plates and separated by repeated streaking. The single colonies were then transferred to the corresponding liquid culture medium, cultured at 37°C or 30°C, and colony samples Z-1, X-1, and X-2 were isolated.

[0109] The steps for strain identification are as follows: the colony suspension was transferred to a 2 mL centrifuge tube and centrifuged at 12000 rpm / min at 4 ° C for 1 minute. The total DNA of the colony was extracted using a genomic extraction kit suitable for bacterial and fungal DNA, and PCR amplification was performed after the purity and concentration were determined. The primers 27F (5'-AGAGTTTTGATCCTGTCTCCAG-3' (SEQ ID NO: 1)) and 1492R (5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 2)) were used for bacterial amplification; the primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3' (SEQ ID NO: 3)) and ITS4 (5'-tcCTCCGTTATTGATATGC-3' (SEQ ID NO: 4)) were used for fungal amplification. After amplification, the products were electrophoresed on a 1% agarose gel in 1×TBE buffer at 110 V for 35 min, using DS2000 as a molecular weight marker, a bacterial band at about 1500 bp and a fungal band between 500-750 bp, and gel imaging. The PCR amplification products were sent for sequencing.

[0110] The PCR products were purified and sequenced, and the obtained sequences were submitted to GenBank for BLAST analysis, and three microorganisms with high homology were identified, including Priestia aryabhattai (P. aryabhattai), Bacillus velezensis (B. velezensis), and Aspergillus fumigatus (A. fumigatus).

[0111] 6. Microbial transformation of 4-VG-related compounds

[0112] The sources of the strains used for microbial transformation and fermentation are as follows: Priestia aryabhattai strain was purchased from Beina Biotechnology with a BNCC number of BNCC374507; Bacillus velezensis strain was purchased from Beina Biotechnology with a BNCC number of BNCC352076; Aspergillus fumigatus strain was purchased from Beina Biotechnology with a BNCC number of BNCC340016.

[0113] For bacterial transformation, P.aryabhattai and B.velezensis strains were inoculated into 50mL centrifuge tubes containing 20mL LB broth, and incubated for 18 hours on a shaking table (180rpm, 37°C, pH=7). 1.0mL of the culture medium was transferred to 10mL containing a final concentration of 0.3g / L ferulic acid or 0.4g / L 4-VG, and incubated for 72 hours on a shaking table (180rpm, 37°C, pH=7). The culture medium was filtered and subjected to three freeze-thaw cycles, centrifuged at 10000rpm / for 10 minutes. Supernatant was collected and filtered with a 0.22μm microporous membrane, and the filtrate was used for subsequent use.

[0114] For fungal transformation, A. fumigatus strains were inoculated onto YPD solid agar plates and cultured in a constant temperature incubator (30°C, pH = 7) for 36 hours. 10 mL of culture medium containing a final concentration of 0.3 g / L ferulic acid or 0.4 g / L 4-VG was transferred to a 50 mL centrifuge tube, a single culture was scraped off the solid agar plate and added to the tube, and incubated on a shaker (180 rpm, 37°C, pH = 7) for 108 hours. The culture medium was filtered out, and the solution for transformation was prepared as described above.

[0115] The contents of ferulic acid, 4-vinylguaiacol, vanillin and guaiacol in the conversion solution were determined by high performance liquid chromatography.

[0116] Table 2

[0117]

[0118]

[0119] The results are shown in Table 2 and Figure 7As shown in Table 2, it can be seen that the conversion rates of ferulic acid to 4-VG by P.aryabhattai and B.velezensis are 69.58% and 31.65%, respectively, and the conversion rate of ferulic acid to 4-VG by A.fumigatus is 45.68%. The conversion rates of 4-VG to vanillin by P.aryabhattai and B.velezensis are 2.315% and 2.491%, respectively, and the conversion rate of 4-VG to guaiacol by A.fumigatus is 6.665%. It can be seen that P.aryabhattai, B.velezensis and A.fumigatus can effectively convert ferulic acid to 4-VG, mediating the metabolic pathway from ferulic acid to 4-VG, and then to vanillin and guaiacol.

[0120] from Figure 7 The Spearman correlation between the relative abundance of microorganisms and the compound content showed that the relative abundance of Bacillus and Enterobacter was negatively correlated with the relative content of ferulic acid, and positively correlated with the relative content of guaiacol and vanillin, the microbial transformation products of ferulic acid. Although the relative abundance of Aspergillus fumigatus did not change significantly with the increase in the storage time of tangerine peel, it was positively correlated with the relative content of ferulic acid and negatively correlated with the relative content of guaiacol. These findings suggest that the decrease of ferulic acid and the increase of guaiacol and vanillin in tangerine peel are aging time-dependent, which may be due to microbial transformation.

[0121] 7. Effects of different microbial combinations on the aging process and 4-VG content of tangerine peel

[0122] The conventionally cultured P. aryabhattai, B. velezensis and A. fumigatus strains were prepared at a concentration of 10 7 CFU / mL spore suspension, and P. aryabhattai, B. velezensis, and A. fumigatus strains were prepared in a ratio of 5:1:4 to a concentration of 1×10 7 CFU / mL mixed spore suspension; about 50g 3-year-old tangerine peel samples were placed in glass culture dishes, sprayed with 50mL / dish of the above different spore suspensions or distilled water. The treated samples were then placed in a constant temperature and humidity incubator (temperature 30±2°C, humidity 75±5%) for fermentation culture. When cultured for 30 and 60 days, the content (mass percentage) of 4-VG in the sample tangerine peel was determined by high performance liquid chromatography. See Table 3.

[0123] Table 3

[0124]

[0125] The test results of 4-VG content in the dried tangerine peel fermented by microorganisms are shown in Table 3. After the three strains of single bacteria or combined fermentation of dried tangerine peel, the 4-VG content in the dried tangerine peel increases over time, suggesting that these microbial strains can cause the change of 4-VG content during the aging process of dried tangerine peel, and different strains have different effects on the conversion efficiency of 4-VG, wherein the conversion efficiency of 4-VG in the dried tangerine peel by the three mixed bacteria is higher than that of the single bacteria. Therefore, the combination of three different strains can be optimized to prepare the dried tangerine peel with high 4-VG content by aging.

[0126] Example 2 Application of 4-VG in the preparation of drugs for treating insulin resistance and diabetes

[0127] This example provides the use of 4-VG in the preparation of a drug for treating insulin resistance and diabetes. The specific verification process is as follows:

[0128] 1. Effect of 4-VG on C2C12 cell viability

[0129] C2C12 myoblasts were seeded into 96-well culture plates at 0.5 × 10 cells per well. 4 Cells (100 μL culture medium) were cultured with DMEM complete culture medium containing 10% fetal bovine serum for 12 hours, and the original culture medium was discarded. The cells were treated with complete culture medium containing 0, 2.5, 5, 10, 20 and 40 μM 4-VG for 24 hours, and 3 replicates were set for each concentration, with 200 μL in each well. After the incubation was completed, 10 μL of freshly prepared MTT solution with a final concentration of 5 mg / mL was added to each well and cultured for 4 hours. The original culture medium was discarded, and 100 μL of dimethyl sulfoxide (DMSO) was added to each well. The cells were shaken on a plate shaker for 10 minutes to fully dissolve the crystals. The absorbance was immediately measured on an ELISA reader with a detection wavelength of 490 nm. The cell survival rate was calculated according to the following formula.

[0130] Cell viability (%) = (OD of drug-treated cells - OD of blank cells) / (OD of control cells - OD of blank cells) × 100%.

[0131] The experimental results are as follows Figure 8 As shown in Figure A, it can be seen that within 0-40 μM, 4-VG has little effect on the viability of C2C12 cells.

[0132] 2. 4-VG enhances glucose uptake in insulin-stimulated C2C12 myoblasts

[0133] C2C12 cells were cultured in DMEM complete medium until the cell proliferation was about 100%, and then DMEM containing 2% heat-inactivated HS and 1% P / S was added for 4 days to induce differentiation. 6The cells were inoculated into a 96-well plate at a concentration of 100 μM / mL, with 200 μL in each well, and the drugs were administered according to the following groups: drug-administered group, blank control group, negative control group and positive control group, wherein the drug-administered group was added with 4-VG and 0.1 μM insulin at final concentrations of 5, 10 and 20 μM, respectively; the blank control group was different from the drug-administered group only in that insulin and 4-VG were not added; the negative control group was different from the drug-administered group only in that 4-VG was not added; the positive control group was different from the drug-administered group only in that AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, 20 μM) was used to replace 4-VG.

[0134] Six replicate wells were set for each group. After 12 hours of drug incubation, the cells were washed with Krebs–Ringer phosphate (KRP) buffer (20mM HEPES, 137mM NaCl, 4.7mM KCl, 1.2mM MgSO4, 1.2mM KH2PO4, 2.5mM CaCl2, and 2mM pyruvate; pH 7.4) and incubated in KRP buffer containing 0.2% BSA for 2 hours. The cells were placed in suspension, 0.2mM 3H-labeled 2-deoxyglucose was added, and incubated at 37°C for 30 minutes. The intracellular 2-deoxyglucose content was detected using an enzyme-labeled instrument with an excitation wavelength of 475nm and an emission wavelength of 550nm.

[0135] The results are as follows Figure 8 As shown in Figure B, it can be seen that 4-VG can enhance the glucose uptake of insulin-stimulated C2C12 myoblasts, and the uptake capacity increases with the increase of concentration. It is worth noting that at a concentration of 20 μM, 4-VG significantly increased the insulin-mediated glucose uptake level (increased by about 275%), which is significantly higher than the 160% of 20 μM positive drug AICAR.

[0136] 3.4-VG improves insulin resistance in C2C12 myoblasts

[0137] The differentiated C2C12 cells were cultured at 5×10 6 / mL concentration was inoculated into a 96-well plate, 200 μL in each well, and divided into the following groups: a drug-treated group, a blank control group, a negative control group, and a positive control group. The drug-treated group was added with 4-VG and 0.1 μM insulin at final concentrations of 5, 10, and 20 μM, respectively; the blank control group was different from the drug-treated group only in that insulin and 4-VG were not added; the negative control group was different from the drug-treated group only in that 4-VG was not added; the positive control group was different from the drug-treated group only in that AMPK activator 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, 20 μM) was used to replace 4-VG.

[0138] After incubation of cells with drugs for 24 h, cell extract proteins were collected and the protein concentration of each sample was quantified by BCA protein assay kit. Equal amounts of denatured proteins (30-50 μg) were separated on SDS-polyacrylamide gel (SDS-PAGE) and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA) and blocked with TBST buffer (100 mM NaCl, 10 mM Tris–HCl, pH 7.5, and 0.1% Tween 20) containing 5% skim milk powder for 2 h at room temperature. After washing three times with TBST, specific primary antibodies including p-AKT, AKT, GSK-3β, p-GSK-3β, IRS-1, p-IRS-1, and GAPDH (1:2000, CST, USA) were added and incubated overnight at 4°C. After washing four times with TBST buffer, HRP-conjugated secondary antibodies (1:5000, CST, USA) were added and incubated for 2 h at room temperature. The cells were washed four times with TBST buffer and a 1:1 mixture of ECL chemiluminescent reagent A and B was added in a light-proof environment. Protein bands were visualized on a ChemiDoc MP imaging system and quantified using Image Lab 5.1 (Bio-Rad).

[0139] Test results such as Figure 8 As shown in Figure C, it can be seen from the figure that insulin stimulation leads to the phosphorylation of IRS-1, AKT and GSK-3β in C2C12 cells, and insulin combined with 4-VG treatment significantly further increases the phosphorylation levels of these proteins. These changes are basically consistent with the changes in their glucose uptake ability, indicating that 4-VG enhances insulin-stimulated glucose uptake in C2C12 myotubes by activating the IRS-1 / Akt / GSK-3β signaling pathway. In other words, 4-VG can improve the insulin resistance of C2C12 myotubes and increase the cell's ability to uptake glucose, thereby achieving the effect of lowering blood sugar and reducing the damage caused by high sugar.

[0140] 4. 4-VG effectively improves the diabetic phenotype of HFD / STZ-induced mouse model

[0141] All animal experiments were approved by the Animal Ethics and Welfare Committee of Jiangmen Big Health International Innovation Institute (Jiangmen, China), and all animal experimental procedures were performed in strict accordance with the guidelines and regulations of this institution.

[0142] Male C57BL / 6J mice (7 weeks old, 18-20 g) were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. and routinely housed in a standard specific pathogen-free (SPF) environment (22±4°C, 12-h light-dark cycle) with free access to food. All animals were acclimatized to the environment for 1 week before the experiment.

[0143] The method for constructing the HFD / STZ-induced diabetic mouse model can be found in Fig. 9 Figure A in the figure, the specific steps are as follows: the NC group was fed with a normal diet (calories, 3.5kcal / g), and the HFD group was fed with 45% HFD (calories, 16.8kcal / g). After 5 weeks of HFD feeding, the mice were fasted for 12 hours and then intraperitoneally injected with STZ (40mg / kg, 10mL / kg) prepared with citric acid-sodium citrate buffer solution for 7 consecutive days. The NC group was intraperitoneally injected with citric acid-sodium citrate buffer (0.1mM, pH4.5, 10mL / kg) and set as the blank control group. On the 10th day after STZ injection, a blood glucose meter was used to screen out mice that met the diabetes criteria with fasting blood glucose higher than 11.1mM, higher than 13mM on the 12th day, and higher than 13mM on the 14th day. The diabetic mice were randomly divided into 3 groups, namely, the negative control group HFD / STZ, the experimental group HFD / STZ+4-VG, and the positive control group HFD / STZ+ACA. The doses of 4-VG and acarbose (ACA) were 50 mg / kg and 50 mg / kg, respectively, and they were gavaged once a day for 4 consecutive weeks. Glucose tolerance test (GTT) was performed after 25 days of 4-VG and ACA treatment. That is, after fasting for 12 hours, mice were gavaged with glucose at a dose of 2.0 g / kg, and blood glucose levels at 0, 15, 30, 60, 90 and 120 minutes were measured by Roche blood glucose meter. 24 hours after the last gavage, orbital blood was collected, euthanasia was performed, and gastrocnemius muscles were dissected. The levels of total TC, TG and HDL-C in serum were analyzed using commercial kits (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China).

[0144] The results are as follows Fig. 9 As shown:

[0145] from Fig. 9 As can be seen from the BD graph in the figure, compared with normal mice, the body weight of diabetic mice was significantly reduced, while 4-VG intervention for 4 weeks had no significant effect on the body weight and energy intake of diabetic mice, indicating that 4-VG has no obvious toxic side effects;

[0146] from Fig. 9 As can be seen from the EG figure, 4-VG can significantly reduce fasting blood glucose in diabetic mice and improve glucose tolerance, suggesting that 4-VG can effectively alleviate the diabetic phenotype of mice;

[0147] The results of IRS-1 / Akt / GSK-3β signaling pathway detection are as follows Fig. 9 As shown in Figure H, it can be seen from the figure that 4-VG intervention significantly upregulated the expression levels of p-IRS-1 / IRS-1, pAKT / AKT and p-GSK-3β / GSK-3β in the gastrocnemius of diabetic mice, indicating that 4-VG can effectively improve the insulin resistance of diabetic mice;

[0148] Serum total cholesterol TC content Fig. 9 As shown in I in the figure, it can be seen from the figure that 4-VG can significantly reduce the total cholesterol content in the serum of diabetic mice;

[0149] Serum biochemical index test results such as Fig. 9 As shown in the IK figure in , it can be seen from the figure that although 4-VG intervention has no significant effect on the total serum TG content of diabetic mice, it can effectively reduce the levels of serum total TC and LDL-C, indicating that 4-VG can significantly improve the lipid metabolism disorders in diabetic mice.

[0150] In summary, 4-VG can effectively enhance the glucose uptake of insulin-stimulated C2C12 myoblasts and improve the symptoms of glycolipid disorders in HFD / STZ-induced diabetic mice, which may be related to its regulation of the IRS-1 / Akt / GSK-3β signaling pathway.

[0151] Example 3 Preparation of Tangerine Peel with Strong Anti-diabetes and Insulin Resistance Effects by Microbial Aging

[0152] This embodiment provides a method for aging tangerine peel in a guided manner by microbial transformation to increase the 4-VG content in tangerine peel and prepare tangerine peel with strong anti-diabetic and insulin resistance improving effects, which specifically comprises the following steps: P. aryabhattai, B. velezensis and A. fumigatus strains are respectively prepared into a concentration of 1×10 7 CFU / mL spore suspension, and P. aryabhattai, B. velezensis, and A. fumigatus strains were prepared in a ratio of 5:1:4 to a concentration of 1×10 7 CFU / mL of mixed spore suspension; the above spore suspension was inoculated into 3-year-old tangerine peel at 50 mL / dish for fermentation culture, the culture temperature was 30±2°C, and the humidity was 75±5%.

[0153] The results are shown in Table 3. It can be seen from the table that P. aryabhattai, B. velezensis and / or A. fumigatus strains can effectively increase the 4-VG content in tangerine peel, thereby achieving control of the aging process of tangerine peel and enhancing the anti-diabetic effect of tangerine peel.

[0154] The aging method can be used to prepare tangerine peels with different aging degrees that have high anti-diabetic effects.

[0155] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A microorganism for increasing the 4-VG content in dried tangerine peel, characterized in that: The microorganism includes at least one of P. aryabhattai, B. velezensis and A. fumigatus.

2. Use of the microorganism according to claim 1 in any of the following: (1) Increase the 4-VG content in dried tangerine peel; (2) Control the aging process of tangerine peel; (3) Improve the quality and efficacy of dried tangerine peel; (4) Preparation of products for preventing and / or treating insulin resistance; (5) Preparation of products for the prevention and / or treatment of diabetes; (6) preparing products for promoting glucose uptake by cells; (7) preparing products for increasing the expression of pAKT, P-GSK-3β and / or 4-VG; (8) Preparation of products for lowering cholesterol levels in serum; (9) Prepare a product for reducing the triglyceride content in serum.

3. The use according to claim 2, characterized in that: The insulin resistance disease includes at least one of type II diabetes, coronary heart disease and metabolic syndrome.

4. An aging method for improving the quality and efficacy of dried orange peel, characterized in that: The aging method comprises the following steps: allowing the microorganism as claimed in claim 1 to act on tangerine peel to regulate the 4-VG content in the tangerine peel.

5. A tangerine peel for preventing and / or treating diabetes or insulin resistance, characterized in that: The tangerine peel is prepared by the following method: the microorganism as claimed in claim 1 is allowed to act on the tangerine peel.

6. Application of the dried tangerine peel described in claim 5 in any one of the following: 1) Preparation of products for preventing and / or treating insulin resistance; 2) Preparation of products for preventing and / or treating diabetes; 3) preparing products for promoting glucose uptake by cells; 4) preparing products for increasing the expression of pAKT, P-GSK-3β and / or 4-VG; 5) Preparation of products for reducing cholesterol levels in serum; 6) Preparation of products for reducing triglyceride levels in serum. 7.4-VG is used in any of the following (A1) to (A3): (A1) preparing products for preventing and / or treating insulin resistance and / or diabetes; (A2) controlling the aging process of tangerine peel; (A3) Improve the quality and efficacy of dried tangerine peel.

8. The use according to claim 7, characterized in that: The product is a product for promoting glucose uptake by cells; and / or, the product is a product for increasing the expression of pAKT, P-GSK-3β and / or 4-VG; and / or, the product is a product for reducing the cholesterol content in serum; And / or, the product is a product for reducing the triglyceride content in serum.

9. The use according to claim 7, characterized in that: The product includes a medicine or a food; Preferably, the drug further comprises a pharmaceutically acceptable excipient; More preferably, the pharmaceutically acceptable excipients include at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener and a flavoring agent; Preferably, the dosage form of the drug is tablets, capsules, soft capsules, granules, pills, oral liquids, dry suspensions, drop pills, dry extracts, injections or infusions, transdermal agents, transdermal microneedles; Preferably, the administration of the drug includes injection or oral administration.

10. The use according to claim 7, characterized in that: The mass fraction of the 4-VG in the product is 0.01% to 100%.

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