Method for producing phloretin by fermenting eurotium cristatum

Abstract

The invention belongs to the technical field of biology, and discloses a method for producing phloretin through fermentation of eurotium cristatum. On the basis of pretreatment of lithocarpus litseifolius, eurotium cristatum is used as a fermentation inoculant, and after aerobic solid fermentation is performed on the eurotium cristatum, trilobatin and phlorizin in the lithocarpus litseifolius are converted into phloretin. According to the method, eurotium cristatum is inoculated into lithocarpus litseifolius by utilizing a microbial solid-state fermentation technology, high-content phloretin is obtained after fermentation is finished, and the method is simple, convenient, short in production period and suitable for subsequent large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and relates to a method for producing phlorizin using *Aspergillus cristatus* fermentation. Specifically, it relates to a method for producing phlorizin using *Aspergillus cristatus* fermentation of *Litsea cubeba* leaves. Background Technology

[0002] Phloretin belongs to the dihydrochalcone class of compounds and possesses various physiological functions, including anti-inflammatory, hypoglycemic, and antioxidant effects, and has been widely used in cosmetic and skincare products. Studies have shown that phloretin is mainly distributed in apple peels, root bark, and other plants. However, research indicates that the content of phloretin in plants is extremely low, posing a significant challenge to its subsequent isolation and extraction.

[0003] Litsea cubeba is a versatile evergreen tree widely distributed in southern provinces of my country, with abundant resources. Studies have shown that Litsea cubeba plants (leaves and buds) contain a large amount of dihydrochalcone compounds, including phlorizin and trifolin. Phlorizin and trifolin can be converted into phlorin by lactase-phlorizin hydrolase and β-glucosidase. However, due to the difficulty in large-scale isolation and extraction of these enzymes, there is currently no research on the enzyme-catalyzed formation of phlorin from phlorizin and trifolin. Summary of the Invention

[0004] To overcome existing problems, this invention provides a method for significantly increasing the phlorizin content in *Litsea cubeba* through solid-state fermentation using *Aspergillus cristatus*. The method utilizes microbial solid-state fermentation technology, inoculating *Aspergillus cristatus* into *Litsea cubeba*, and yielding a high phlorizin content after fermentation. The method is simple, has a short production cycle, and is suitable for subsequent large-scale production.

[0005] The above-mentioned objective of this invention is achieved through the following technical solution: A method for producing phlorin using *Aspergillus cristatus* fermentation involves using *Aspergillus cristatus* as a fermentation agent after pretreatment of *Litsea cubeba*, and then converting trifolin and phlorizin in *Litsea cubeba* into phlorin through aerobic fermentation.

[0006] A method for producing phlorizin using *Aspergillus cristatus* fermentation specifically includes the following steps: (1) Pretreatment of Litsea cubeba: First, the fresh Litsea cubeba tea leaves are put into a fixation machine at a fixation temperature of 280-330℃. After fixation, the Litsea cubeba leaves are put into a rolling machine for rolling. The rolled Litsea cubeba leaves are then heated and dried to obtain the pretreated Litsea cubeba leaves. (2) Preparation of *Aspergillus cristatus*: On a clean bench, *Aspergillus cristatus* was inoculated into PDA solid medium using an inoculation loop and cultured at 30°C for 7 days; after culture, the spores were washed away with sterile physiological saline to prepare a spore suspension with a concentration of 1 x 10. 8 ~5x10 8 CFU / mL.

[0007] (3) Aerobic solid fermentation: Take the tea leaves that have been pretreated in step (1), mix them with pure water at a weight ratio of 1:1, sterilize them with steam at 121℃ for 10 minutes, cool them to room temperature after sterilization, and sterilize them with ultraviolet light on a clean bench for 30 minutes. Inoculate the *Aspergillus cristatus* spore suspension prepared in step (2) into 10 g of tea leaves after ultraviolet sterilization, and stir evenly after inoculation. The inoculation amount is 2-3% of the volume mass percentage, and 3-5 mL of sterile water is added. Under the condition of 30-35℃, aerobic fermentation is carried out for 8-10 days to obtain fermentation products containing phloretin.

[0008] After fermentation, 10 g of fermentation product was soaked in 500 L of hot water for 20–40 minutes, then filtered through a 0.22 μm microporous membrane, and the supernatant was collected. High-performance liquid chromatography (HPLC) was used to qualitatively and quantitatively analyze phlorizin, phlorizin, and trifolin based on the peak times of the standards. The highest content of phlorizin was 1.68 mg / mL.

[0009] Furthermore, in step (1), the fixation time is 5 minutes. Fresh tea leaves and tender branches are selected.

[0010] Furthermore, in step (1), the specific conditions for kneading are: the room temperature is maintained between 20℃ and 24℃, the rotation speed is 45 rpm, and the kneading time is 10 minutes.

[0011] Furthermore, in step (1), the specific conditions for heating and drying are: the temperature is 40℃, the thickness of the spread leaves is 4~5cm, the leaves are turned over every 10 minutes or so, and the drying time is 30 minutes until the moisture content is 4%~6%.

[0012] Furthermore, in step (1), the tea leaves are Litsea cubeba tea leaves, preferably Litsea cubeba sweet tea.

[0013] Furthermore, in step (2), *Eurotium cristatum* is... E. cristatum Purchased from the China Industrial Microbial Culture Collection Center (CICC); culture collection number: CICC41041.

[0014] Furthermore, in step (2), the PDA solid culture medium preparation includes: 200 g of potato, 20 g of glucose, 15 g of agar and 500 mL of purified water.

[0015] Furthermore, in step (2), the specific preparation steps of sterile saline are as follows: 100 mL of saline is steam sterilized at 121℃ for 20 min and then sterilized by ultraviolet light in a clean bench for 30 min.

[0016] The advantages of this invention compared to the prior art are: The method provided by this invention, compared with unfermented Litsea cubeba, results in a large amount of enzymatic hydrolysis of the main components of dihydrochalcone, phlorizin and trifolin, in fermented Litsea cubeba. After fermentation, the content of phlorizin and trifolin is significantly reduced, and abundant phlorizin is produced. Attached Figure Description

[0017] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. Figure 1 This is a colony morphology diagram of *Aspergillus cristatus* seven days after inoculation into PDA medium according to the present invention.

[0018] Figure 2 This is a morphological diagram of the *Aspergillus cristatus* spore suspension after fermentation on *Litsea cubeba* for ten days.

[0019] Figure 3 The graph shows the contents of phlorizin, trifolin, and phloretin in Example 1 and Comparative Example 1 of this invention. Detailed Implementation

[0020] The present invention is described in detail below through specific embodiments, but this does not limit the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0021] This invention uses *Aspergillus cristatus* to ferment *Litsea cubeba*, directly converting the phlorizin and trifolin dihydrochalcone compounds contained therein into phlorizin through live-cell solid-state fermentation technology.

[0022] This invention provides a technical method for preparing phlorizin through bio-fermentation using Litsea cubeba as raw material and *Aspergillus cristatus* as the fermentation strain. In the specific embodiments of this invention, the *Aspergillus cristatus* used in the following examples is... E. cristatum Purchased from the China Industrial Microbial Culture Collection Center (CICC); the culture collection number is CICC41041, which is used in this specific embodiment.

[0023] This invention provides a method for preparing phlorizin by fermenting Lithocarpus rubrum with Corynebacterium cristatum, comprising the following steps: S1. Litsea cubeba was purchased from Dalian Youchuang Technology Development Co., Ltd., and after pretreatment, the material to be fermented was obtained.

[0024] S2. Preparation of *Eurotium cristatum* to obtain fermentation broth.

[0025] S3. *Eurotium cristatum* performs aerobic fermentation on pretreated and sterilized *Litsea cubeba* to obtain the fermentation product.

[0026] S4. Soak and test the fermented material to obtain the specific content of phloretin after fermentation.

[0027] In this invention, the *Eurotium cristatum* spore suspension is preferably inoculated into sterilized *Litsea cubeba* in liquid form, with an inoculation volume preferably of 2-3 mL.

[0028] In this invention, the preparation of the fermentation broth of *Aspergillus cristatus* includes: inoculating *Aspergillus cristatus* into PDA solid medium and culturing it at 30°C for 7 days.

[0029] In this invention, the preferred conditions for aerobic fermentation of Litsea cubeba tea by *Aspergillus cristatus* are: a temperature of 30°C and a fermentation time of 10 days.

[0030] The present invention involves drying the obtained fermented material to obtain the fermented product. In this invention, the preferred drying conditions are: a temperature of 120°C and a drying time of 6 hours.

[0031] The technical solutions in the examples of this invention will be clearly and completely described below.

[0032] Pretreatment of Litsea cubeba leaves; First, fresh Litsea cubeba leaves are blanched in a blanching machine at a temperature of 280-330℃. After blanching, the Litsea cubeba leaves are placed in a rolling machine and rolled. The rolled Litsea cubeba leaves are then heated and dried to obtain pre-treated Litsea cubeba leaves. Preparation of *Aspergillus cristatus*; Place the PDA solid culture medium on a clean bench and sterilize it under UV light for 30 minutes. After UV sterilization, place *Aspergillus cristatus* into the clean bench. Use a sterile inoculation loop to pick up *Aspergillus cristatus* bacterial suspension and streak it onto the PDA medium. After streaking, place it in an incubator and incubate at 30°C for 7 days. Figure 1 After cultivation, the bacterial cells were washed with sterile physiological saline in a clean bench under ultraviolet light and counted to prepare a *Aspergillus cristatus* spore suspension with a concentration of 1 x 10⁻⁶. 8 ~5x10 8 CFU / mL.

[0033] Solid-state aerobic fermentation of Litsea cubeba by *Eurotium cristatum* after pretreatment and sterilization; Weigh 10 g of pretreated tea leaves, place them in a glass bottle, add 10 mL of deionized water, and steam sterilize at 121℃ for 20 min. Place the sterilized tea leaves in a clean bench for UV sterilization for 30 min. After sterilization, inoculate the tea leaves with a suspension of *Aspergillus cristatus* spores. Using different examples (fermentation conditions), solid-state fermentation of *Litsea cubeba* tea leaves was carried out under different conditions using *Aspergillus cristatus*. The fully fermented tea leaves were then dried in a forced-air dryer at 120℃ for 6 h. The resulting fermented product was obtained after drying.

[0034] The different fermentation conditions are described below. Example

[0035] The *Eurotium cristatum* spore suspension was inoculated into a solid substrate at a rate of 3% (v / w), and 5 mL of sterile water was added. The substrate was then placed in an incubator at 30°C for aerobic fermentation for 8 days. Example

[0036] The *Eurotium cristatum* spore suspension was inoculated into a solid substrate at an inoculation rate of 3% (v / w), 3 mL of sterile water was added, and the substrate was placed in an incubator for aerobic fermentation at 30°C for 10 days. Example

[0037] The *Eurotium cristatum* spore suspension was inoculated into a solid substrate at an inoculation rate of 2% (v / w), 3 mL of sterile water was added, and the substrate was placed in an incubator for aerobic fermentation at 30°C for 10 days. Example

[0038] The *Eurotium cristatum* suspension was inoculated into a solid substrate at an inoculation rate of 3% (v / w), and 3 mL of sterile water was added. The substrate was then placed in an incubator for aerobic fermentation at 35°C for 10 days.

[0039] Comparative Example 1 Untreated Litsea cubeba tea leaves. The phlorizin content was determined. The results are shown in Table 1.

[0040] The dried fermented products from Examples 1-4 were added to boiling water at a ratio of 1:50 and extracted for 50 minutes. The content of phloretin produced in the final fermentation of Examples 1-4 was then tested. The test results are shown in Table 1.

[0041] Table 1 shows the changes in the content of dihydrochalcone compounds in Examples 1-4 and Comparative Example 1.

[0042]

[0043] The method provided by this invention, utilizing the optimal conditions for fermentation of Litsea cubeba tea by *Aspergillus cristatus*, resulted in a decrease in phlorizin and trifolin from 8.18 mg / mL to 1.93 mg / mL and 14.37 mg / mL to 2.11 mg / mL, respectively, while phlorizin increased from 0 mg / mL to 1.68 mg / mL. Figure 3 ).

[0044] The embodiments described above are merely preferred embodiments of the present invention, and not all feasible embodiments of the present invention. Any obvious modifications made by those skilled in the art without departing from the principles and spirit of the present invention should be considered to be included within the scope of protection of the claims of the present invention.

Claims

1. A method for producing phloretin by fermentation using Eurotium cristatum, characterized by, On the basis of the processing of Lithocarpus glaber, Eurotium cristatum is used as a fermentation inoculum, and after aerobic fermentation, phlorin and phloridzin in Lithocarpus glaber are converted into phloretin.

2. The method for producing phloretin by fermentation using Eurotium cristatum according to claim 1, characterized by, Specifically, the following steps are included: (1) Lithocarpus glaber pretreatment: fresh tea leaves are first subjected to fixation in a fixation machine at a fixation temperature of 280-330 DEG C, and the fixed tea leaves are then rolled in a rolling machine, and the rolled tea leaves are heated and dried; thus, pretreated tea leaves are obtained; (2) Eurotium cristatum preparation: in a clean bench, Eurotium cristatum is inoculated into PDA solid medium using an inoculation loop, and cultured in a 30 DEG C incubator for 7 days; after the culture, spores are eluted using sterile saline to prepare a spore suspension; (3) aerobic solid fermentation: the tea leaves pretreated in step (1) are mixed with pure water at a weight ratio of 1:1, and after the mixing, the mixture is sterilized at 121 DEG C for 10 minutes, and after the sterilization, the mixture is cooled to room temperature, and then subjected to ultraviolet sterilization in a clean bench for 30 minutes; 10 g of the tea leaves after the ultraviolet sterilization are inoculated with the spore suspension prepared in step (2) at a volume-to-mass percentage of 2-3%, and 3-5 mL of sterile water is added, and after the inoculation, the mixture is stirred uniformly, and subjected to aerobic fermentation at 30-35 DEG C for 8-10 days; thus, a fermentation product containing phloretin is obtained.

3. The method for producing phloretin by fermentation using Eurotium cristatum according to claim 2, characterized by, In step (1), the fixation time is 5 minutes.

4. The method for producing phloretin by fermentation using Eurotium cristatum according to claim 2, characterized by, In step (1), the rolling conditions are as follows: the indoor temperature is maintained at 20-24 DEG C, the rotating speed is 45 rpm, and the rolling time is 10 minutes.

5. The method for producing phloretin by fermentation using Eurotium cristatum according to claim 2, wherein In step (1), the heating and drying conditions are as follows: the temperature is 40 DEG C, the leaf thickness is 4-5 cm, the leaves are turned over every 10 minutes or so, and the heating and drying time is 30 minutes until the water content is 4-6%.

6. The method for producing phloretin by fermentation using Eurotium cristatum according to claim 2, wherein In step (2), Eurotium cristatum is E. cristatum ; purchased from China General Microbiological Culture Collection Center (CICC); and the strain preservation number is CICC 41041.

7. The method for producing phloretin by fermentation using Eurotium cristatum according to claim 2, wherein In step (2), the PDA solid medium is prepared as follows: 200 g of potatoes, 20 g of glucose, 15 g of agar, and 500 mL of pure water.

8. The method for producing phloretin by fermentation using Eurotium cristatum according to claim 2, wherein In step (2), the sterile saline is prepared as follows: 100 mL of saline is sterilized at 121 DEG C for 20 minutes, and then subjected to ultraviolet sterilization in a clean bench for 30 minutes.

9. The method for producing phloretin by fermentation using Eurotium cristatum according to claim 2, wherein Further, in step (2), the spore suspension concentration is 1 x 10 8 5 x 10 8 CFU / mL.

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