Method for increasing content of total flavonoids and hesperidin in dried orange peel by fermenting dried orange peel with alternaria alternata

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to application of utilizing alternaria alternata to ferment pericarpium citri reticulatae so as to improve the content of total flavonoids and hesperidin in pericarpium citri reticulatae, which comprises the following steps: culturing alternaria alternata spore suspension in a PDB liquid culture solution to obtain alternaria alternata inoculation liquid; uniformly mixing the sterilized pericarpium citri reticulatae powder and the sterilized rice powder, adding the alternaria alternata inoculation liquid, fully and uniformly mixing, and then adding sterilized water; after uniform stirring, the mixture is placed in a constant-temperature incubator to be fermented and co-cultured, the fermentation temperature is 25-35 DEG C, and the fermentation time is 5-12 days. According to the method disclosed by the invention, the efficient capability of the alternaria alternata in the aspect of converting flavonoid components in the pericarpium citri reticulatae can be fully exerted, so that the content of total flavonoids and hesperidin in the pericarpium citri reticulatae is remarkably increased.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an application of fermenting tangerine peel with Alternaria alternata to increase the content of total flavonoids and hesperidin in tangerine peel. Background Art

[0002] Tangerine peel originates from the ripe peel of Rutaceae plants and their cultivated varieties. After being dried, it becomes this ancient Chinese medicinal material. Tangerine peel is listed as a top-grade medicinal material because of its significant effects on regulating qi and strengthening the spleen, drying dampness and resolving phlegm. In traditional Chinese medicine theory, there is a saying that "the older the better", which means that the medicinal effect of tangerine peel will gradually increase with the increase of storage time. Tangerine peel is widely used in a variety of compound preparations. Its unique aroma and slightly bitter taste, coupled with its positive effects on the digestive system, respiratory system and cardiovascular system, such as relieving asthma, antitussive and anti-allergic inflammation.

[0003] The flavonoids in tangerine peel, especially hesperidin, nobiletin and tangeretin, are at the core of its efficacy. These bioactive molecules have a variety of positive effects on human health. In particular, hesperidin, nobiletin and tangeretin in tangerine peel have been shown to have powerful antioxidant properties, which can neutralize free radicals, slow down cell aging and reduce the risk of chronic diseases. These ingredients have also shown significant effects in anti-inflammatory, inhibiting the production of inflammatory mediators, thereby combating a variety of inflammatory diseases such as arthritis and certain cardiovascular diseases. In addition to antioxidant and anti-inflammatory effects, flavonoids also show anti-tumor potential. Studies have shown that they can interfere with the growth and spread of tumor cells and promote apoptosis of cancer cells. In terms of cardiovascular protection, flavonoids help to enhance the elasticity of blood vessels, lower blood pressure, and reduce the formation of atherosclerosis, so they play an important role in preventing heart disease and stroke. In addition, the flavonoids in tangerine peel have also shown benefits for the respiratory system, relieving asthma and coughing and reducing sputum production, making tangerine peel a traditional medicinal material for the treatment of respiratory diseases. Flavonoids also have a certain antibacterial effect, which can help resist certain bacterial and viral infections and enhance human immunity. Therefore, the flavonoids in tangerine peel are not only the key to its medicinal value, but also the basis for it as a health product.

[0004] The flavonoid active substances described herein include hesperidin, nobiletin and tangeretin.

[0005] Hesperidin, English name Hesperidin, molecular formula C28H34O15, chemical structure formula is as follows:

[0006]

[0007] Nobiletin, also known as tangerine, has the following chemical structure:

[0008]

[0009] Tangeretin, also known as 5,6,7,8,4'-pentamethoxyflavone, English name Tangeretin, molecular formula C20H20O7, chemical structure formula is as follows:

[0010]

[0011] In the prior art, the aging process of dried orange peel usually depends on long-term natural storage, and this process is not only time-consuming, but also greatly affected by environmental conditions, resulting in low efficiency of the promotion of flavonoids content in dried orange peel, and unstable quality. In addition, traditional aging methods are difficult to accurately control, so that the conversion efficiency and content of the active substance in dried orange peel are limited. Therefore, how to improve the content of flavonoids in dried orange peel in a short time, accelerate the aging process, and ensure the stability and safety of the active substance at the same time, has become a technical problem to be solved. Summary of the invention

[0012] The technical problem to be solved by the present invention is to provide a method for increasing the content of total flavonoids and hesperidin in tangerine peel by utilizing Alternaria alternata.

[0013] In order to solve the above technical problems, the present invention provides a method for increasing the total flavonoids and hesperidin content of tangerine peel by fermenting tangerine peel with Alternaria alternata (i.e., a method for shortening the aging process of tangerine peel), comprising the following steps:

[0014] 1) sterilize the short-term stored tangerine peel (storage period is 1±0.5 years) by ultraviolet light, and then grind it into powder (powder through a 60-mesh sieve); obtain sterilized tangerine peel powder;

[0015] Grinding rice into powder (powder that passes through a 40-mesh sieve), and then sterilizing with high-temperature steam to obtain sterilized rice powder;

[0016] 2) preparing Alternaria seed solution;

[0017] The spore suspension of Alternaria alternata is cultured in PDB liquid culture medium to obtain an inoculum of Alternaria alternata;

[0018] 3), after 40g sterilized tangerine peel powder and 10±1g sterilized rice flour are mixed, 5~10ml (preferably 6ml) of Alternaria alternata inoculum is added and fully mixed, and then 40~100ml of sterilized water is added; Stir and place a constant temperature incubator to ferment and cultivate together, the fermentation temperature is 25~35 ℃ (preferably 30 ℃), and the fermentation time is 5~12 days (preferably 8~12 days, more preferably 10 days).

[0019] As an improvement of the method of the present invention for increasing the total flavonoids and hesperidin content of tangerine peel by fermenting tangerine peel with Alternaria alternata, the method further includes the following subsequent steps:

[0020] The fermentation product of step 3) is filtered, dried at a constant temperature (55° C. to constant weight), and passed through a 60-mesh sieve to obtain fermented tangerine peel.

[0021] As a further improvement of the method of the present invention for increasing the total flavonoids and hesperidin content of tangerine peel by fermenting tangerine peel with Alternaria alternata, the step 2) is:

[0022] First prepare a concentration of 1×10 5 ~1×10 7 CFU / mL (preferably (2±0.2)×10 6 CFU / mL) of Alternaria spore suspension;

[0023] Then, under sterile conditions, take 1 ml of the spore suspension and inoculate it into (50±5) mL of PDB liquid culture medium. Culture it at (30±1)°C and (180±30rpm) for (30±1) h to obtain the inoculum solution.

[0024] As a further improvement of the method of increasing the total flavonoids and hesperidin content of tangerine peel by fermenting tangerine peel with Alternaria alternata, in the step 2), the preparation method of the spore suspension of Alternaria alternata is as follows: culturing Alternaria alternata in a PDA solid culture medium (aerobic culture) at an incubator temperature of 30±1° C. for 7±1 days;

[0025] After the culture was completed, the spores were taken into sterile water and counted on a hemocytometer to prepare a concentration of 1×10 5 ~1×10 7 CFU / mL (preferably (2±0.2)×10 6 CFU / mL) of spore suspension.

[0026] As a further improvement of the method of increasing the total flavonoids and hesperidin content of tangerine peel by fermenting tangerine peel with Alternaria alternata, in step 1):

[0027] UV sterilization: sterilize the dried tangerine peel (short-term stored tangerine peel) with UV irradiation for (40±5) min, turn it over and sterilize it with UV irradiation for (40±5) min again, with the UV radiation intensity of 70~90μW / cm 2 ;

[0028] High temperature steam sterilization: (121±1)℃ sterilization for 20±1min.

[0029] The present invention utilizes Alternaria alternata to carry out solid-state fermentation of tangerine peel, showing significant advantages. The unique metabolic ability of Alternaria alternata enables it to effectively transform the bioactive substances in tangerine peel and promote the biosynthesis and accumulation of flavonoid compounds. This method not only shortens the aging period of tangerine peel, but also significantly increases the content of effective ingredients in tangerine peel and enhances the pharmacological activity of tangerine peel.

[0030] The present invention utilizes solid-state fermentation of Alternaria alternata and tangerine peel to effectively increase the content of flavonoid compounds in tangerine peel in a shorter time under an optimal fermentation environment compared with a traditional natural aging process, thereby ensuring full utilization and improvement of the active ingredients of tangerine peel and providing new possibilities for its application in the food and pharmaceutical industries.

[0031] The beneficial effects of the present invention are as follows: the method of solid fermentation of tangerine peel by Alternaria alternata can more quickly and efficiently transform total flavonoids and hesperidin substances in tangerine peel by Alternaria alternata and significantly increase their contents, which can not only improve the aging efficiency of tangerine peel but also promote the increase of the content of medicinal substances in tangerine peel.

[0032] In summary, the present invention provides a technology for treating tangerine peel by solid-state fermentation using Alternaria alternata, which can give full play to the high efficiency of Alternaria alternata in transforming flavonoid components in tangerine peel, thereby significantly increasing the content of total flavonoids and hesperidin in tangerine peel. Compared with the traditional aging method, the present method can significantly increase the active ingredients of tangerine peel in a shorter time, greatly improving the conversion efficiency and biological activity of the effective substances. DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0034] The Alternaria Sp. involved in the embodiments of the present invention is from China Center for Type Culture Collection, and the strain collection number is CCTCC AF 2019019.

[0035] Embodiment 1, utilize Alternaria alternata solid fermentation tangerine peel:

[0036] 1) Spread the dried tangerine peel on the bottom of the culture dish and 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 70-90 μW / cm 2 ; Then grind into powder (pass through a 60-mesh sieve) to obtain sterilized tangerine peel powder;

[0037] 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;

[0038] 2) Cultivate Alternaria in PDA solid medium (aerobic culture) at 30°C for 7 days. After the culture, take the spores into sterile water and count them with a hemocytometer. The concentration is 2×10 6 CFU / mL of spore suspension.

[0039] Preparation of inoculum: Under sterile conditions, take 2×10 6 1 ml of spore suspension with a CFU / mL was inoculated into a 100-mL Erlenmeyer flask containing 50 mL of PDB liquid culture medium and cultured at 30°C and 180 rpm for 30 h to obtain an inoculum (Alternaria alternata inoculum).

[0040] 3) Set up the fermentation group and the control group separately:

[0041] 40g of sterilized tangerine peel powder and 10g of sterilized rice powder were uniformly mixed to form a fermentation medium;

[0042] Fermentation group: Add 50 g of fermentation substrate, 6 ml of Alternaria inoculum into a 250 ml conical flask, and then add 44 ml of sterilized water; that is, adjust the water content to 50%; mix thoroughly and mark with numbers.

[0043] Control group: 6 ml of Alternaria inoculum was replaced with 6 mL of sterile water, and the rest was the same as the fermentation group.

[0044] The fermentation group and the control group were set up in triplicate; all were placed in a constant temperature incubator for fermentation at 30°C for 10 days.

[0045] 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;

[0046] Similarly, when the preparation of the control group is completed, it is named as the control group pre-fermentation product, and after the control group is fermented, it is named as the control group post-fermentation product.

[0047] Experiment 1: Detection of total flavonoids and flavonoid substances in dried tangerine peel

[0048] The dried tangerine peel used is stored for one year.

[0049] The total flavonoids and hesperidin content of the control group and the fermentation group were determined before and after fermentation, respectively. The determination method is as follows:

[0050] 1. Determination of total flavonoids content in fermented tangerine peel samples

[0051] 1.1 Preparation of standard solution

[0052] Accurately weigh 25.00 mg of hesperidin standard, dilute to 25 mL with methanol, then ultrasonically treat (power 300 W, frequency 35 kHz) for 45 min and shake well. Accurately pipette 1, 2, 3, 4, 5, 6 mL of the above standard solution and dilute to 5 mL with methanol and shake well to obtain standard solutions with concentrations of 0.2, 0.4, 0.6, 0.8, 1, and 1.2 mg / mL, and measure the absorbance at 283 nm. With the concentration of the hesperidin standard as the horizontal axis (X) and the absorbance value as the vertical axis (Y), a linear regression is performed, and the regression equation is Y=0.8988X+0.0904, R 2 =0.9944.

[0053] 1.2 Preparation of test solution

[0054] 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:

[0055] 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.

[0056] illustrate:

[0057] The pre-fermentation product is dried and passed through a 60-mesh sieve to obtain the pre-fermentation dried tangerine peel;

[0058] The fermented product is dried and passed through a 60-mesh sieve to obtain fermented tangerine peel.

[0059] 1.3 Determination of total flavonoids in fermented tangerine peel samples

[0060] Perform the following operations on the four test solutions:

[0061] Accurately measure 0.6 mL of the test solution and place it in a 25 mL volumetric flask, add methanol to dilute to the mark (i.e., add methanol to make up to 25 mL), and measure the absorbance at 283 nm. Then substitute it into the equation obtained in step 1.1 to obtain the total flavonoid content of the diluted test sample.

[0062] Then, the total flavonoid content in Table 1 was obtained through the following conversion relationship W=c×v×n / M.

[0063] W is the total flavonoids content (%), c is the total flavonoids mass concentration (mg / ml), v is the volume of the extract, n is the dilution multiple, and M is the mass of the tangerine peel powder sample.

[0064] Each batch of samples was measured three times in parallel, and the measured values ​​were averaged to calculate the total flavonoids content in each batch of fermented tangerine peel.

[0065] Statistical methods: spss software, t-test analysis.

[0066] 1.4. The results are as follows:

[0067] Table 1. Total flavonoids content in dried orange peel before and after fermentation

[0068]

[0069] As shown in Table 1, the total flavonoids content in the dried tangerine peel samples of the control group did not change significantly; while the total flavonoids content in the dried tangerine peel samples of the fermentation group increased significantly.

[0070] 2. HPLC determination of hesperidin content before and after fermentation of tangerine peel

[0071] 2.1. Chromatographic conditions are as follows:

[0072] 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:

[0073] Time: 0-5min, acetonitrile: 20%-25%;

[0074] Time: 5-10 minutes, acetonitrile: 25%-30%;

[0075] Time: 10-20 minutes, acetonitrile: 30%-50%;

[0076] Time: 20-30 minutes, acetonitrile: 50%-60%;

[0077] Time: 30-35 minutes, acetonitrile: 60%-90%;

[0078] Time: 35-40 minutes, acetonitrile: 90%-100%;

[0079] Column temperature: 30℃; flow rate: 0.7mL·min-1; detection wavelength: 283nm; injection volume: 5.0μL.

[0080] 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 %.

[0081] 2.2 Preparation of standard solution

[0082] Accurately weigh 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, and 5 mL of the above standard solution, dilute to 20 mL with methanol, and shake well to obtain standard solutions with concentrations of 0.02, 0.04, 0.06, 0.08, and 0.10 mg / mL.

[0083] 1 ml of the standard solution was filtered through a 0.22 μm microporous membrane and analyzed by HPLC according to the chromatographic conditions described in step 2.1 above to obtain the regression equation Y = 2 × 10 7 X+353075,R 2 =0.9993, the ordinate Y represents the peak area of ​​the standard, and the abscissa X represents the mass concentration (mg / mL).

[0084] 2.3 Preparation of test solution

[0085] Same as 1.2 above; obtain 4 test sample solutions.

[0086] 2.4. Detect the four test solutions according to the chromatographic conditions of step 2.1 respectively; each batch of samples is measured three times in parallel, and the measured values ​​are averaged.

[0087] The corresponding test solution of the fermentation group before fermentation had an obvious peak at a retention time of 10.33 min at 283 nm, with a peak area of ​​9153075 μV*s, which was later inserted into the regression equation of step 2.2 to obtain a hesperidin content of 0.44 mg / ml. After conversion, the hesperidin content of the fermentation group before fermentation was 55 mg / g.

[0088] The corresponding test solution of the fermentation group after fermentation has an obvious peak at a retention time of 10.35min at 283nm, with a peak area of ​​11393075 microvolts*seconds, and the hesperidin content is 0.552mg / ml after entering the regression equation of step 2.2. After conversion, the hesperidin content of the fermentation group after fermentation is 69mg / g.

[0089] The corresponding test solution of the control group before fermentation had an obvious peak at a retention time of 10.14 min at 283 nm, with a peak area of ​​8953075 μV*s, which was later inserted into the regression equation of step 2.2 to obtain a hesperidin content of 0.43 mg / ml. After conversion, the hesperidin content of the control group before fermentation was 54 mg / g.

[0090] The corresponding test solution of the control group after fermentation has an obvious peak at a retention time of 10.21min at 283nm, with a peak area of ​​8673075 microvolts*seconds, and the hesperidin content is 0.416mg / ml after entering the regression equation of step 2.2. The hesperidin content of the control group after fermentation is 52mg / g.

[0091] In summary, the HPLC test results are shown in Table 2:

[0092] Table 2 Hesperidin content in tangerine peel before and after fermentation

[0093]

[0094]

[0095] As shown in Table 2, the hesperidin content in the dried tangerine peel samples of the control group did not change significantly; while the hesperidin content in the dried tangerine peel samples of the fermentation group increased significantly.

[0096] The experimental results show that the present invention alleviates the technical problem of long aging time in the prior art by using Alternaria tenuissima to solid-ferment tangerine peel. The method of the present invention can significantly increase the total flavonoid content in tangerine peel in a relatively short period of time, and significantly increase the content of hesperidin, the main component of flavonoids. The experimental results show that the tangerine peel treated with Alternaria tenuissima solid fermentation has a total flavonoid content increase rate of 36.7%, of which the hesperidin content is increased by 32.69%.

[0097] Comparative Example 1: The "Alternaria Sp" of the present invention was replaced with "Alternaria alternata, aalt", and the rest was referred to the above Example 1. The reaction product was tested according to the above experiment.

[0098] The results obtained were: although the total flavonoid content in the fermentation group increased by 12%, the hesperidin content only increased by about 7.5%.

[0099] Comparative Example 2: The amount of sterile water in Example 1 was changed from 44 ml to 120 ml, that is, the water content was adjusted to about 72%; the rest was the same as Example 1. The reaction product was tested according to the above experiment.

[0100] The results obtained were: the total flavonoid content of the fermentation group increased by about 16.7%, and the hesperidin content increased by about 18.4%. Therefore, the increase was significantly lower than that of Example 1.

[0101] Comparative Example 3: The amount of sterile water in Example 1 was changed from 44 ml to 20 ml, that is, the water content was adjusted to about 34%; 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 total flavonoid content of the fermentation group increased by about 11.3%, and the hesperidin content increased by about 13.5%. Therefore, the increase was significantly lower than that of Example 1.

[0103] 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 the content of total flavonoids and hesperidin in dried tangerine peel by fermenting it with Alternaria alternata, characterized in that The following steps are involved: 1) sterilize the short-term stored tangerine peel by ultraviolet light, and then grind it into powder to obtain sterilized tangerine peel powder; The rice is ground into powder, and then sterilized by high temperature steam to obtain sterilized rice powder; 2) preparing Alternaria seed solution; The spore suspension of Alternaria alternata is cultured in PDB liquid culture medium to obtain an inoculum of Alternaria alternata; 3) Mix 40g of sterilized tangerine peel powder and 10±1g of sterilized rice powder, add 5-10ml of Alternaria inoculum and mix well, then add 40-100ml of sterilized water; stir well and place in a constant temperature incubator for fermentation and co-cultivation, the fermentation temperature is 25-35°C, and the fermentation time is 5-12 days.

2. The method for increasing the content of total flavonoids and hesperidin in tangerine peel by fermenting tangerine peel with Alternaria alternata according to claim 1, characterized in that The following follow-up steps are also included: The fermentation product of step 3) is filtered, dried at a constant temperature, and passed through a 60-mesh sieve to obtain fermented tangerine peel.

3. The method for increasing the total flavonoids and hesperidin content of tangerine peel by fermenting tangerine peel with Alternaria alternata according to claim 1 or 2, characterized in that The step 2) is: First prepare a concentration of 1×10 5 ~1×10 7 CFU / mL of Alternaria spore suspension; Then, under sterile conditions, 1 ml of the spore suspension was taken and inoculated into (50±5) mL of PDB liquid culture medium, and cultured at (30±1)° C. and (180±30 rpm) for (30±1) h to obtain an Alternaria inoculum solution.

4. The method for increasing the total flavonoids and hesperidin content of tangerine peel by fermenting tangerine peel with Alternaria alternata according to claim 3, characterized in that In the step 2), the preparation method of the Alternaria alternata spore suspension is as follows: culturing the Alternaria alternata in a PDA solid culture medium at an incubator temperature of 30±1° C. for 7±1 days; After the culture was completed, the spores were taken into sterile water and the concentration was prepared to 1×10 5 ~1×10 7 CFU / mL of spore suspension.

5. The method for increasing the content of total flavonoids and hesperidin in tangerine peel by fermenting tangerine peel with Alternaria alternata according to claim 4, characterized in that In the step 1): UV sterilization: sterilize the tangerine peel with UV for (40±5) min, turn it over and sterilize it with UV for (40±5) min again, with the UV radiation intensity of 70~90μW / cm 2 ; High temperature steam sterilization: (121±1)℃ sterilization for 20±1min.

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