Method for improving nobiletin and hesperetin by co-fermenting dried orange peel with two microorganisms

Abstract

The invention relates to a technology for shortening the aging time of dried orange peel, and provides a method for improving nobiletin and hesperetin by co-fermenting dried orange peel with two microorganisms, which comprises the following steps: preparing a mixed bacteria solution by using Aspergillus sp. And Exophiala similia; mixing the sterilized pericarpium citri reticulatae powder and the sterilized rice meal to serve as a fermentation substrate, adding the mixed bacterial liquid and the sterilized water into the fermentation substrate to serve as a reaction system, fully stirring the reaction system, and placing the reaction system in a constant-temperature incubator for fermentation. By adopting the method disclosed by the invention, the contents of nobiletin and hesperetin in the dried tangerine or orange peel can be efficiently increased within a relatively short time, the aging time of the dried tangerine or orange peel is shortened, and the conversion efficiency of medicinal effect substances in the dried tangerine or orange peel is improved.

Description

Technical Field

[0001] The invention relates to a technology for shortening the aging of tangerine peel, which is a method for increasing the flavonoid content of tangerine peel by co-fermentation of two microorganisms. Background Art

[0002] Tangerine peel, the dried mature peel of the citrus plant and its cultivated varieties, has been widely used in traditional Chinese medicine since ancient times. Its unique pharmacological effects are mainly attributed to the flavonoids in it. This technology explores the use of two fungi co-fermentation methods to increase the content of flavonoids in tangerine peel to enhance its medicinal value and economic benefits.

[0003] The aging process of tangerine peel is not only a traditional Chinese medicinal material processing method, but also a process of improving the quality of medicinal materials through time accumulation. In this process, the content and diversity of flavonoids are significantly improved.

[0004] Both nobiletin and tangeretin are important components of flavonoids, and their functions are:

[0005] Nobiletin is a flavonoid compound with multiple pharmacological effects, especially in anti-inflammatory and antioxidant aspects. It can inhibit the expression of inflammatory factors, reduce cell damage, and inhibit the proliferation of tumor cells by regulating cell signaling pathways. In addition, nobiletin also shows a protective effect on neurodegenerative diseases, which may be related to its antioxidant and anti-inflammatory properties.

[0006] As a natural flavonoid compound, tangeretin is widely found in the peel of citrus fruits. It not only has strong antioxidant activity and can remove free radicals in the body, but also has the effects of regulating blood lipids, resisting thrombosis and protecting the liver. These properties of tangeretin make it a potential candidate for research and development of new drugs, especially in the prevention and treatment of cardiovascular and metabolic diseases.

[0007] The invention of CN113980821B, "A strain of Aspergillus niger for converting hesperidin and its application", discloses that Aspergillus niger CP-2 (accession number: GDMCC No: 61088) is isolated from tangerine peel and has the function of converting hesperidin into hesperidin, and the yield of hesperidin can reach more than 60%.

[0008] The invention of CN115413779B "A method for accelerating tangerine peel aging through microbial fermentation" informs: by selecting Mucor circinelloides as the inoculant, through pile fermentation, the tangerine peel aging time is greatly shortened, achieving an effect similar to natural aging, and the effect is better than Aspergillus niger (Aspergillus niger), red yeast (Rhodotorula), and microsporus Rhizopus (Rhizopusmicrosporus). Summary of the invention

[0009] The technical problem to be solved by the invention is to provide a method for improving the flavonoid content of tangerine peel by co-fermenting tangerine peel with Aspergillus sp. and Exophiala.

[0010] In order to solve the above technical problems, the present invention provides a method for increasing nobiletin and hesperidin by co-fermenting tangerine peel with two microorganisms, comprising the following steps:

[0011] 1) Prepare a mixed bacterial solution using Aspergillus sp. and Exophiala similis;

[0012] 2) After short-term storage (shelf life is 1 ± 0.5 years), the dried tangerine peel is sterilized by ultraviolet light and crushed (through a 40-mesh sieve) to obtain sterilized dried tangerine peel powder;

[0013] The rice is crushed (passed through a 40-mesh sieve) and sterilized with high-temperature steam to obtain sterilized rice flour;

[0014] 3) Mixing sterilized tangerine peel powder and sterilized rice flour as a fermentation substrate, wherein the weight ratio of the sterilized tangerine peel powder to sterilized rice flour is 3.5 to 4.5:1;

[0015] After adding the mixed bacterial solution and sterilized water to the fermentation substrate as a reaction system, the reaction system is fully stirred and placed in a constant temperature incubator for fermentation at a fermentation temperature of 25 to 35° C. (preferably 30° C.) for 6 to 12 days (preferably 10 days);

[0016] The weight ratio of the fermentation substrate to the reaction system = 30% to 60% (preferably 50%);

[0017] Mixed bacterial solution: reaction system = 10% to 20% (preferably 15%) by weight.

[0018] As an improvement of the method of using two microorganisms to co-ferment tangerine peel to increase nobiletin and hesperidin of the present invention, the step 1) is:

[0019] Pipette 1mL of a 1×10 5 ~1×10 7CFU / mL of Aspergillus sp. spore suspension was added to 50±2mL of PDB liquid culture medium, and cultured at 30±1℃ and 180±30rpm for 30±1h to obtain Aspergillus sp. bacterial solution;

[0020] Pipette 1mL of a 1×10 5 ~1×10 7 CFU / mL of Exophiala similis spore suspension was added to 50±2mL of YPD liquid culture medium, and cultured at 30±1℃ and 180±30rpm for 30±1h to prepare Exophiala similis bacterial solution;

[0021] The Aspergillus sp. bacterial solution and the Exophiala similis bacterial solution were mixed at a volume ratio of 1.9 to 2.1:1 to obtain a mixed bacterial solution.

[0022] As a further improvement of the method of utilizing two microorganisms to co-ferment tangerine peel to increase nobiletin and hesperidin of the present invention:

[0023] The concentration of Aspergillus sp. spore suspension is (1.8~2.2)×10 6 CFU / mL;

[0024] The concentration of Exophiala similis spore suspension is (0.8~1.2)×10 6 CFU / mL.

[0025] As a further improvement of the method of utilizing two microorganisms to co-ferment tangerine peel to increase nobiletin and hesperidin of the present invention:

[0026] Under sterile conditions, the Aspergillus sp. in the storage tube was activated for two generations in a PDA plate to prepare an Aspergillus sp. spore suspension; the activation condition was 30±1°C and the culture time was 7 days;

[0027] Under sterile conditions, the Exophiala similis in the storage tube was activated for two generations on a YPD plate to prepare an Exophiala spore suspension; the activation conditions were 30±1°C and the culture was carried out for 7 days.

[0028] As a further improvement of the method of utilizing two microorganisms to co-ferment tangerine peel to increase nobiletin and hesperidin of the present invention:

[0029] In the step 2):

[0030] The UV sterilization of dried tangerine peel is as follows: sterilize the dried tangerine peel with UV for (40±5) min, turn it over and sterilize it with UV again for (40±5) min, the UV radiation intensity is 70~90μW / cm 2 ;

[0031] High temperature steam sterilization: (121±1)℃ sterilization for 20±1min.

[0032] As a further improvement of the method of utilizing two microorganisms to co-ferment tangerine peel to increase nobiletin and hesperidin of the present invention:

[0033] The fermented product is dried and passed through a 60-mesh sieve to obtain fermented tangerine peel.

[0034] The Aspergillus sp. and Exophiala similis involved in the present invention are purchased from China Center for Type Culture Collection, and the strain collection numbers are CCTCC AF 2019015 and CCTCC AF 2021058. The present invention provides an application method for increasing the content of flavonoid substances nobiletin and tangeretin in tangerine peel and accelerating aging by co-fermentation.

[0035] In the present invention:

[0036] Aspergillus sp. and Exophiala have different contributions to increasing the content of flavonoids. Aspergillus sp. can produce a series of enzymes, such as cellulase, protease and polyphenol oxidase, which can not only promote the synthesis of flavonoids in tangerine peel, but also catalyze the conversion of precursors into more active flavonoids. At the same time, Exophiala, as a salt-tolerant and acid-tolerant microorganism, can survive under extreme conditions, and its metabolites may have a positive effect on the stability and biological activity of flavonoids in tangerine peel. During the co-fermentation process, the complementary effects of metabolites between the two fungi can optimize the fermentation environment and create more favorable conditions for the synthesis of flavonoids. For example, the enzymes produced by Aspergillus sp. may catalyze the conversion of certain intermediates in tangerine peel into flavonoids, while Exophiala may provide an optimal environment for the activity of these enzymes by adjusting the pH value of the fermentation liquid.

[0037] The benefits of promoting aging of tangerine peel by co-fermentation with Aspergillus sp. and Exophiala are not only reflected in the increase of the content of flavonoids, but also in the improvement of the bioavailability and bioactivity of these compounds.

[0038] During the co-fermentation process, enzymes and other metabolites produced by microorganisms may promote the release of flavonoids from tangerine peel, while enhancing their stability, making them easier to be absorbed and utilized by the human body. In addition, co-fermentation may also trigger new biochemical reactions in tangerine peel, producing new flavonoids or their derivatives, which may have stronger biological activity or unique pharmacological effects than the original compounds.

[0039] During the co-fermentation process, enzymes and other metabolites produced by microorganisms may promote the release of flavonoids from tangerine peel, while enhancing their stability, making them easier to be absorbed and utilized by the human body. In addition, co-fermentation may also trigger new biochemical reactions in tangerine peel, producing new flavonoids or their derivatives, which may have stronger biological activity or unique pharmacological effects than the original compounds.

[0040] In the current context of growing demand for natural medicines and healthy foods, increasing the content and activity of flavonoids in tangerine peel by microbial co-fermentation technology can not only enhance the market value of tangerine peel, but also bring new inspiration to the food industry and drug development. Therefore, in-depth research and optimization of the technology of co-fermentation of Aspergillus sp. and Exophiala to promote aging of tangerine peel has important scientific significance and application value for the development of new drugs and health products.

[0041] In summary, the present invention provides a method for solid co-fermentation of tangerine peel by two microorganisms Aspergillus sp. and Exophialacancerae, which can efficiently increase the content of nobiletin and hesperidin in tangerine peel in a short time, shorten the aging time of tangerine peel, and improve the conversion efficiency of medicinal substances in tangerine peel. DETAILED DESCRIPTION

[0042] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0043] Aspergillus sp. and Exophiala similis are CCTCC AF 2019015 and CCTCC AF2021058, respectively.

[0044] Example 1: A method for increasing nobiletin and tangeretin by co-fermenting tangerine peel with two microorganisms (i.e., a method for solid-state fermentation of tangerine peel using Aspergillus sp. and Exophiala similis), wherein the following steps are performed in sequence:

[0045] 1) Spread the dried tangerine peel (stored for 1 year) on the bottom of the culture dish, sterilize it with ultraviolet light for 40 minutes, turn it over, and sterilize it with ultraviolet light for 40 minutes. The ultraviolet radiation intensity is 70-90μW / CM 2 ; Then grind into powder (pass through a 40-mesh sieve) to obtain sterilized tangerine peel powder;

[0046] The rice is ground into powder (passed through a 40-mesh sieve) and then sterilized with high-temperature steam in an autoclave (121° C., 20 min) to obtain sterilized rice powder;

[0047] 2) Preparation of fermentation solution:

[0048] Under sterile conditions, the Aspergillus sp. in the storage tube was activated for two generations on a PDA plate to prepare a concentration of 2×10 6 CFU / mL of spore suspension; activation conditions are 30°C and culture time is 7d; then, 1mL of the spore suspension is pipetted into a 100mL Erlenmeyer flask containing 50mL of PDB liquid culture medium and cultured at 30°C and 180rpm for 30h to obtain Aspergillus sp. bacterial solution.

[0049] Similarly, under sterile conditions, the Exophiala similis in the storage tube was activated for two generations on YPD plates to make a concentration of 1×10 6 CFU / mL of spore suspension; activation conditions are 30°C and culture for 7 days; then, 1 mL of the spore suspension is pipetted into a 100 mL Erlenmeyer flask containing 50 mL of YPD liquid culture medium and cultured at 30°C and 180 rpm for 30 hours to obtain Exophialasimilis bacterial solution.

[0050] The Aspergillus sp. bacterial solution and the Exophiala similis bacterial solution were mixed in a volume ratio of 2:1 to obtain a mixed bacterial solution.

[0051] 3) Set up the fermentation group and the control group separately:

[0052] Fermentation group: Add fermentation substrate (consisting of 40g sterilized tangerine peel powder and 10g sterilized rice powder) and 15g mixed bacterial solution into a 250ml conical flask, and then add 35g sterilized water; that is, adjust the water content to 50%; mix thoroughly and mark with numbers.

[0053] Control group: 15 g of mixed bacterial solution was replaced with 15 g of sterile water, and the rest was the same as the fermentation group.

[0054] The fermentation group and the control group were cultured at 30°C for 10 days. Three parallels were set up for each.

[0055] When the fermentation group is prepared, it is named as the product before fermentation of the fermentation group, and after the fermentation of the fermentation group, it is named as the product after fermentation of the fermentation group;

[0056] Similarly,

[0057] When the preparation of the control group is completed, it is named as the control group pre-fermentation result, and after the control group is fermented, it is named as the control group post-fermentation result.

[0058] Experiment 1, HPLC determination of nobiletin and tangeretin in tangerine peel samples:

[0059] 1.1 Preparation of standard solution

[0060] Accurately weigh 20.00 mg of Nobiletin and 20.00 mg of Hesperidin standard, dilute to 50 mL with methanol, ultrasonically treat (power 300 W, frequency 35 kHz) for 45 min, and shake well. Accurately pipette 1, 2, 3, 4, 5 mL of the above standard solution and dilute to 20 mL with methanol and shake well to obtain Nobiletin and Hesperidin standard solutions with concentrations of 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL.

[0061] 1.2 Chromatographic conditions are as follows:

[0062] The chromatographic column was Agilent C18 (4.6×200mm, 5μm); the mobile phase consisted of 0.05% by volume phosphoric acid aqueous solution and acetonitrile, and the elution was performed according to the following gradient program:

[0063] Time: 0-5min, acetonitrile: 20%-25%;

[0064] Time: 5-10 minutes, acetonitrile: 25%-30%;

[0065] Time: 10-20 minutes, acetonitrile: 30%-50%;

[0066] Time: 20-30 minutes, acetonitrile: 50%-60%;

[0067] Time: 30-35 minutes, acetonitrile: 60%-90%;

[0068] Time: 35-40 minutes, acetonitrile: 90%-100%;

[0069] Column temperature: 30℃; flow rate: 0.7mL·min-1; detection wavelength: 335nm; injection volume: 5.0μL.

[0070] Note: Taking "0-5min, acetonitrile: 20%-25%" as an example, it means: at 0min, the mobile phase is composed of 20% acetonitrile and 80% 0.05% phosphoric acid aqueous solution; at 5min, the mobile phase is composed of 25% acetonitrile and 75% 0.05% phosphoric acid aqueous solution. The above % is volume %.

[0071] 1.3. Pipette 1 ml of the standard solution in step 1.1 through a 0.22 μm microporous filter membrane, and then perform HPLC analysis according to the chromatographic conditions described in step 1.2 above; obtain the regression equation:

[0072] Nobiletin Y=1×10 8 X+823828,R 2 =0.9992

[0073] Hesperidin Y=7×10 7 X+774709,R 2 =0.9995

[0074] The ordinate Y represents the peak area of ​​the standard, and the abscissa X represents the mass concentration (mg / mL);

[0075] Note: The peak area of ​​nobiletin was obtained when the retention time was about 26.32 min (about 26.1-26.6 min), and the peak area of ​​tangeretin was obtained when the retention time was about 30.35 min (about 30.1-30.7 min).

[0076] 1.4 Preparation of test solution

[0077] The pre-fermentation product of the fermentation group, the post-fermentation product of the fermentation group, the pre-fermentation product of the control group, and the post-fermentation product of the control group were subjected to the following operations respectively:

[0078] Dry at 55°C to constant weight, pass through a 60-mesh sieve, accurately weigh 0.2 g, add 25 mL of methanol, and the weighed weight is named the original weight. Ultrasonic treatment (power 300 W, frequency 35 kHz) for 45 min, cool to room temperature, weigh again, add solvent (methanol) to the original weight, shake well, and filter (through a 0.22 μm microporous filter membrane) to obtain 4 test solutions.

[0079] illustrate:

[0080] The pre-fermentation product is dried and passed through a 60-mesh sieve to obtain the pre-fermentation dried tangerine peel;

[0081] The fermented product is dried and passed through a 60-mesh sieve to obtain fermented tangerine peel.

[0082] 1.5. Detect the four test solutions obtained in step 1.4 respectively according to the chromatographic conditions of step 1.2; each batch of samples is measured in parallel 3 times, and the measured values ​​are averaged.

[0083] The details are as follows:

[0084] The corresponding test solution of the fermentation group before fermentation had obvious highest peaks at retention times of 26.32min and 30.35min at 335nm (26.32min for nobiletin and 30.35min for tangeretin), with peak areas of 3293828 and 2258709 μV*sec, which were subsequently entered into the regression equation of step 1.3 to obtain a nobiletin content of 0.0247mg / ml and a tangeretin content of 0.0212mg / ml. After conversion, the nobiletin content of the fermentation group before fermentation was 3.09mg / g and the tangeretin content was 2.65mg / g.

[0085] The corresponding test solution of the fermentation group after fermentation had obvious highest peaks at retention times of 26.19min and 30.67min at 335nm, with peak areas of 4253828 and 2636709 μV*sec, which were subsequently inserted into the regression equation of step 1.3 to obtain a nobiletin content of 0.0343 mg / ml and a tangeretin content of 0.0266 mg / ml. After conversion, the nobiletin content of the fermentation group after fermentation was 4.29 mg / g and the tangeretin content was 3.33 mg / g.

[0086] The corresponding test solution of the control group before fermentation had obvious peaks at retention times of 26.54min and 30.26min at 335nm, with peak areas of 3311828 and 2244709 microvolts*seconds, and the latter was put into the regression equation of step 1.3, and the content of nobiletin was 0.0249mg / ml and the content of tangeretin was 0.021mg / ml. After conversion, the content of nobiletin and tangeretin in the control group before fermentation was 3.11mg / g and 2.63mg / g.

[0087] The corresponding test solution of the control group after fermentation had obvious peaks at retention times of 26.21min and 30.16min at 335nm, with peak areas of 3323828 and 2244709 microvolts*seconds, and the latter was inserted into the regression equation of step 1.3 to obtain the content of nobiletin of 0.025mg / ml and the content of tangeretin of 0.021mg / ml. After conversion, the content of nobiletin and tangeretin of the fermentation matrix of the control group after fermentation was 3.1mg / g and 2.63mg / g.

[0088] The HPLC test results are shown in Table 1:

[0089] Table 1 Contents of nobiletin and tangeretin in tangerine peel before and after fermentation

[0090]

[0091] As shown in Table 1, the contents of nobiletin and tangeretin in the dried tangerine peel samples of the control group did not change significantly; while the contents of nobiletin and tangeretin in the dried tangerine peel samples of the fermentation group increased significantly.

[0092] The experimental results of the present invention show that the tangerine peel treated by Aspergillus sp. and Exophialasimilis co-fermentation under optimal fermentation conditions has a significantly increased content of flavonoids. Specifically, the content of nobiletin in the tangerine peel of the experimental group after co-fermentation increased from 3.1 mg / g to 4.29 mg / g, with an increase rate of 38.4%, and the content of tangerine peel increased from 2.61 mg / g to 3.33 mg / g, with an increase rate of 27.6%, while the content of the control group was almost unchanged. This significant difference proves that the co-fermentation method effectively promotes the biosynthesis and accumulation of flavonoid compounds nobiletin and tangerine peel in tangerine peel.

[0093] Comparative Example 1: The "15 g mixed bacterial solution" in Example 1 was changed to "15 g Aspergillus sp. bacterial solution", and the rest was the same as Example 1. The reaction product was tested according to the above experiment.

[0094] The results obtained were as follows: the content of nobiletin in the fermentation group increased by about 7.9%, and the content of tangeretin increased by about 6.3%, which were significantly lower than those in Example 1.

[0095] Comparative Example 2: "15 g of mixed bacterial solution" in Example 1 was changed to "15 g of Exophiala similis bacterial solution", and the rest was the same as Example 1. The reaction product was tested according to the above experiment.

[0096] The results obtained were as follows: the content of nobiletin in the fermentation group increased by about 6.5%, and the content of tangeretin increased by about 5.7%, which were significantly lower than those in Example 1.

[0097] Comparative Example 3: In the mixed bacterial solution, the volume ratio of Aspergillus sp. bacterial solution to Exophiala similis bacterial solution was changed from 2:1 to 1:1; the amount of the mixed bacterial solution remained unchanged (still 15 g); the rest was the same as in Example 1. The reaction product was tested according to the above experiment.

[0098] The results obtained were as follows: the content of nobiletin in the fermentation group increased by about 13.5%, and the content of tangeretin increased by about 13.8%, which were significantly lower than those in Example 1.

[0099] Comparative Example 4: In the mixed bacterial solution, the volume ratio of Aspergillus sp. bacterial solution to Exophiala similis bacterial solution was changed from 2:1 to 3:1; the amount of the mixed bacterial solution remained unchanged (still 15 g); the rest was the same as in Example 1. The reaction product was tested according to the above experiment.

[0100] The results obtained were as follows: the content of nobiletin in the fermentation group increased by about 16.7%, and the content of tangeretin increased by about 15.5%, which were significantly lower than those in Example 1.

[0101] Comparative Example 5: The amount of sterile water in Example 1 was changed from 35 g to 100 g, that is, the water content was adjusted to about 70%; the rest was the same as Example 1. The reaction product was tested according to the above experiment.

[0102] The results obtained were as follows: the content of nobiletin in the fermentation group increased by about 22.8%, and the content of tangeretin increased by about 21.4%. Therefore, the increase was obviously lower than that in Example 1.

[0103] Comparative Example 6: The amount of sterile water in Example 1 was changed from 35 ml to 10 ml, that is, the water content was adjusted to about 33%; the rest was the same as Example 1. The reaction product was tested according to the above experiment.

[0104] The results obtained were as follows: the content of nobiletin in the fermentation group increased by about 12.8%, and the content of tangeretin increased by about 12.5%. Therefore, the increase was obviously lower than that in Example 1.

[0105] Conclusion: The present invention provides a novel method for increasing the content of flavonoid compounds in tangerine peel by microbial co-fermentation. This method can not only significantly improve the medicinal value of tangerine peel in a relatively short period of time, but also shorten the aging period of tangerine peel and improve the conversion efficiency of effective substances. The co-fermentation of Aspergillus sp. and Exophiala has opened up a new way for the processing and utilization of tangerine peel and has important industrial application potential.

[0106] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for increasing nobiletin and hesperidin by co-fermenting tangerine peel with two microorganisms, characterized in that The following steps are involved: 1) Prepare a mixed bacterial solution using Aspergillus sp. and Exophiala similis; 2) sterilizing the short-term stored tangerine peel by ultraviolet light and crushing it to obtain sterilized tangerine peel powder; The rice is crushed and then sterilized with high temperature steam to obtain sterilized rice flour; 3) Mixing sterilized tangerine peel powder and sterilized rice flour as a fermentation substrate, wherein the weight ratio of the sterilized tangerine peel powder to sterilized rice flour is 3.5 to 4.5:1; After adding the mixed bacterial solution and sterilized water to the fermentation substrate as a reaction system, the reaction system is fully stirred and placed in a constant temperature incubator for fermentation at a fermentation temperature of 25 to 35° C. for 6 to 12 days; The weight ratio of the fermentation substrate to the reaction system is 30% to 60%; Mixed bacterial solution: reaction system = 10% to 20% by weight.

2. The method for increasing nobiletin and hesperidin by co-fermenting tangerine peel with two microorganisms according to claim 1, characterized in that The step 1) is: Pipette 1mL of a 1×10 5 ~1×10 7 CFU / mL of Aspergillus sp. spore suspension was added to 50±2mL of PDB liquid culture medium, and cultured at 30±1℃ and 180±30rpm for 30±1h to obtain Aspergillus sp. bacterial solution; Pipette 1mL of a 1×10 5 ~1×10 7 CFU / mL of Exophiala similis spore suspension was added to 50±2mL of YPD liquid culture medium, and cultured at 30±1℃ and 180±30rpm for 30±1h to prepare Exophiala similis bacterial solution; The Aspergillus sp. bacterial solution and the Exophiala similis bacterial solution were mixed at a volume ratio of 1.9 to 2.1:1 to obtain a mixed bacterial solution.

3. The method for increasing nobiletin and hesperidin by co-fermenting tangerine peel with two microorganisms according to claim 2, characterized in that: The concentration of Aspergillus sp. spore suspension is (1.8~2.2)×10 6 CFU / mL; The concentration of Exophiala similis spore suspension is (0.8~1.2)×10 6 CFU / mL.

4. The method for increasing nobiletin and hesperidin by co-fermenting tangerine peel with two microorganisms according to claim 2 or 3, characterized in that: Under sterile conditions, the Aspergillus sp. in the storage tube was activated for two generations in a PDA plate to prepare an Aspergillus sp. spore suspension; the activation condition was 30±1°C and the culture time was 7 days; Under sterile conditions, the Exophiala similis in the storage tube was activated for two generations on a YPD plate to prepare an Exophiala spore suspension; the activation conditions were 30±1°C and the culture was carried out for 7 days.

5. The method for increasing the production of nobiletin and hesperidin by co-fermenting tangerine peel with two microorganisms according to any one of claims 1 to 4, characterized in that In the step 2): The UV sterilization of dried tangerine peel is as follows: sterilize the dried tangerine peel with UV for (40±5) min, turn it over and sterilize it with UV again for (40±5) min, the UV radiation intensity is 70~90μW / cm 2 ; High temperature steam sterilization: (121±1)℃ sterilization for 20±1min.

6. The method for increasing the content of nobiletin and hesperidin by co-fermenting tangerine peel with two microorganisms according to any one of claims 1 to 5, characterized in that: The fermented product is dried and passed through a 60-mesh sieve to obtain fermented tangerine peel.

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