Black mulberry fermented liquid, preparation method and application thereof

Abstract

The application relates to a black mulberry fermentation liquor and a preparation method and application thereof, and belongs to the technical field of plant processing. The preparation method of the black mulberry fermentation liquor comprises the following steps: inoculating Zymomonas mobilis liquid into a culture medium containing black mulberry homogenate to form a mixed liquid, carrying out fermentation, and carrying out solid-liquid separation on fermented material obtained after the fermentation; the inoculation volume of the Zymomonas mobilis liquid is 5%-10% of the volume of the mixed liquid. Active components such as flavones and anthocyanins in the black mulberry can be efficiently extracted, the black mulberry fermentation liquor obtained through the fermentation has high flavone and anthocyanin contents, and has good antioxidant and anti-aging effects.

Description

Technical Field

[0001] This application relates to the field of plant processing technology, and in particular to a black mulberry fermentation broth, its preparation method, and its application. Background Technology

[0002] Anthocyanins are a class of compounds formed by the glycosidic bond between anthocyanins and sugars. They are widely found in the cell sap of plant flowers, fruits, stems, leaves, and root organs, giving them various colors from red and purplish-red to blue. Anthocyanins belong to the group of flavonoids—a class of substances based on flavonoid nuclei—that exhibit red coloration. Due to their unique functionalities, they are used to scavenge free radicals, promote lutein production, and have anti-tumor, anti-cancer, anti-inflammatory, lipid peroxidation and platelet aggregation inhibition, diabetes prevention, weight loss, and vision protection effects. As a natural pigment, anthocyanins are safe, non-toxic, and have many health benefits for the human body, and have been applied in the food, health product, cosmetic, and pharmaceutical industries.

[0003] Currently, anthocyanins are generally extracted from plants containing anthocyanins. However, the current extraction rate of anthocyanins from plants is low, which hinders their application. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present application aims to provide a black mulberry fermentation broth, its preparation method, and its applications. This method can produce a black mulberry fermentation broth with high flavonoid and anthocyanin content, which can be effectively used for antioxidant and anti-aging applications.

[0005] In a first aspect, embodiments of this application provide a method for preparing black mulberry fermentation broth, comprising: inoculating a culture medium containing black mulberry homogenate with a broth containing *C. motile* to form a mixture, fermenting, and separating the fermented material obtained after fermentation into solid and liquid components; the inoculation volume of the *C. motile* broth is 5%-10% of the volume of the mixture.

[0006] This application utilizes an appropriate amount of *Fermentomonas motilityis* as the fermentation strain to ferment black mulberry homogenate. The black mulberry homogenate and *Fermentomonas motilityis* work synergistically to promote the growth of *Fermentomonas motilityis*, thereby efficiently extracting active ingredients such as flavonoids and anthocyanins from black mulberries. The resulting fermented black mulberry broth has a high content of flavonoids and anthocyanins, exhibiting good antioxidant and anti-aging effects.

[0007] In some embodiments of this application, the culture medium contains 5%-15% by weight of black mulberry homogenate.

[0008] This application utilizes a suitable mass percentage of black mulberry homogenate in a culture medium, along with an appropriate amount of *Mammotrophic motility-promoting* bacteria for fermentation. This allows for the efficient extraction of active ingredients such as flavonoids and anthocyanins from black mulberries, resulting in a fermented black mulberry broth with high flavonoid and anthocyanin content and good antioxidant and anti-aging effects.

[0009] In some embodiments of this application, the preparation of black mulberry homogenate before fermentation is further included, and the black mulberry homogenate is sterilized. The preparation of black mulberry homogenate includes: mixing dried black mulberry fruit with water at a mass-to-volume ratio of 5%-15% and then beating the mixture to obtain black mulberry homogenate.

[0010] This application obtains a black mulberry homogenate of suitable mass percentage by mixing and pulping a suitable mass percentage of dried black mulberries with an appropriate volume of water. Using this homogenate, along with an appropriate amount of *Mammotrophic motility-promoting bacteria*, fermentation can be carried out to efficiently extract active ingredients such as flavonoids and anthocyanins from black mulberries. This results in a fermented black mulberry broth with high flavonoid and anthocyanin content, exhibiting good antioxidant and anti-aging effects.

[0011] In some embodiments of this application, the culture medium also includes 8%-15% glucose by mass.

[0012] This application utilizes the addition of an appropriate percentage of glucose to the culture medium to promote the growth of *Mammotrophic motility-promoting* bacteria, thereby effectively extracting active ingredients such as flavonoids and anthocyanins from black mulberries.

[0013] In some embodiments of this application, the pH value of the culture medium is 6.5-7.5.

[0014] This application, by configuring the culture medium to a suitable pH value, can facilitate the growth of *Mammotrophic motility-promoting* bacteria, promote the fermentation of black mulberry homogenate, and effectively extract active ingredients such as flavonoids and anthocyanins from black mulberries.

[0015] In some embodiments of this application, the fermentation conditions include fermentation at 35-38°C for 24-36 hours.

[0016] This application utilizes a suitable temperature and fermentation time to facilitate the fermentation of black mulberry homogenate by *Mammotrophic motility*, effectively extracting active ingredients such as flavonoids and anthocyanins from black mulberries.

[0017] In some embodiments of this application, activated motile fermentation monoclonal bacteria are also included before fermentation.

[0018] In some embodiments of this application, activating *M. motile fermentum* includes: transferring *M. motile fermentum* to a solid culture medium, activating it by static incubation at 37°C until single colonies grow, transferring the single colonies grown on the solid culture medium to a liquid culture medium, and continuing to culture at 37°C for 10-18 hours to obtain *M. motile fermentum* broth.

[0019] This application utilizes the activation and extended culture time of *Fermentomonas motilityis* to obtain a suitable concentration of *Fermentomonas motilityis* broth, which facilitates efficient fermentation of black mulberry homogenate and effectively extracts active ingredients such as flavonoids and anthocyanins from black mulberry.

[0020] Secondly, embodiments of this application provide a black mulberry fermentation broth prepared by the preparation method provided in the first aspect.

[0021] The black mulberry fermentation broth prepared by the above-described method has a high content of flavonoids and anthocyanins, and exhibits good antioxidant and anti-aging effects.

[0022] Thirdly, embodiments of this application provide an application of black mulberry fermentation broth as provided in the second aspect in the preparation of cosmetics.

[0023] This application utilizes black mulberry fermentation broth, which has a high content of flavonoids and anthocyanins and exhibits good antioxidant and anti-aging effects, in the preparation of cosmetics, thereby enabling the cosmetics to also have good antioxidant and anti-aging effects. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0025] This application provides a method for preparing black mulberry fermentation broth, comprising: inoculating a culture medium containing black mulberry homogenate with a broth containing *C. motile* to form a mixture, fermenting, and separating the fermented material after fermentation into solid and liquid components; the inoculation volume of the *C. motile* broth is 5%-10% of the volume of the mixture.

[0026] Using an appropriate amount of *Fermentomonas motilityis* as the fermentation strain, black mulberry homogenate was fermented. The black mulberry homogenate and *Fermentomonas motilityis* worked synergistically to promote the growth of *Fermentomonas motilityis*, thereby efficiently extracting active ingredients such as flavonoids and anthocyanins from black mulberries. The fermented black mulberry broth obtained from the fermentation had a high content of flavonoids and anthocyanins, and had good antioxidant and anti-aging effects.

[0027] As an example, the inoculation volume of the *Morphozoa motifosum* broth can be, but is not limited to, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of the mixed broth volume.

[0028] In some embodiments of this application, the culture medium contains 5%-15% by weight of black mulberry homogenate. Exemplarily, the culture medium may contain, but is not limited to, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by weight of black mulberry homogenate. Using a suitable percentage of black mulberry homogenate in the culture medium, combined with an appropriate amount of *Fermentomonas motilityis* for fermentation, allows for efficient extraction of active ingredients such as flavonoids and anthocyanins from black mulberries. This results in a fermented black mulberry broth with high flavonoid and anthocyanin content, exhibiting good antioxidant and anti-aging effects.

[0029] In some embodiments of this application, the process further includes preparing a black mulberry homogenate before fermentation and sterilizing the homogenate. Preparing the black mulberry homogenate involves mixing dried black mulberries with water at a mass-to-volume ratio of 5%-15% and then blending the mixture to obtain the homogenate. As an example, the mass-to-volume ratio of dried black mulberries to water can be, but is not limited to, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. By mixing and pulping a suitable amount of dried black mulberries with an appropriate volume of water, a suitable percentage of black mulberry homogenate can be obtained. Using this homogenate, along with an appropriate amount of *Mammotrophic motility-promoting* bacteria, fermentation can be carried out to efficiently extract flavonoids and anthocyanins from the black mulberries. This results in a fermented black mulberry broth with high flavonoid and anthocyanin content, exhibiting good antioxidant and anti-aging effects.

[0030] In some embodiments of this application, the culture medium further includes 8%-15% glucose by mass. Exemplarily, the culture medium may, but is not limited to, include 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% glucose by mass. Adding an appropriate percentage of glucose to the culture medium can help promote the growth of *Mammotrophic motility-promoting bacteria*, effectively extracting active components such as flavonoids and anthocyanins from black mulberries.

[0031] In some embodiments of this application, the pH value of the culture medium is 6.5-7.5. As an example, the pH value of the culture medium may be, but is not limited to, 6.5, 6.8, 7.0, 7.2, or 7.5. Configuring the culture medium to a suitable pH value can facilitate the growth of *Mammotrophic motility-promoting* bacteria, promote the fermentation of black mulberry homogenate, and effectively extract active components such as flavonoids and anthocyanins from black mulberries.

[0032] In some embodiments of this application, the fermentation conditions include fermentation at 35-38℃ for 24-36 hours. Using a suitable temperature and fermentation time facilitates the fermentation of black mulberry homogenate by *Mammotrophic motility-promoting* bacteria, effectively extracting active ingredients such as flavonoids and anthocyanins from black mulberries.

[0033] In some embodiments of this application, activated motile fermentation monoclonal bacteria are also included before fermentation.

[0034] In some embodiments of this application, activating *Fermentomonas motilityis* includes: transferring *Fermentomonas motilityis* to a solid culture medium, activating it by static incubation at 37°C until single colonies grow, transferring the single colonies grown on the solid culture medium to a liquid culture medium, and continuing to culture at 37°C for 10-18 hours to obtain *Fermentomonas motilityis* broth. Appropriate activation and culture time for *Fermentomonas motilityis* can yield a suitable concentration of *Fermentomonas motilityis* broth, which is beneficial for efficient fermentation of black mulberry homogenate and effective extraction of active components such as flavonoids and anthocyanins from black mulberries.

[0035] This application provides a black mulberry fermentation broth prepared by the preparation method described above.

[0036] The black mulberry fermentation broth prepared by the above method has a high content of flavonoids and anthocyanins, and has good antioxidant and anti-aging effects.

[0037] This application provides an example of the use of the black mulberry fermentation liquid as described above in the preparation of cosmetics.

[0038] Black mulberry fermentation broth, which has a high content of flavonoids and anthocyanins and has good antioxidant and anti-aging effects, is used in the preparation of cosmetics, so that the cosmetics also have good antioxidant and anti-aging effects.

[0039] The features and performance of this application will be further described in detail below with reference to the embodiments.

[0040] The *Mammotrophin* strain used in this application was obtained from the Guangdong Provincial Center for Microbial Culture Collection, with the number GDMCC1.133.

[0041] The liquid culture medium used in this embodiment is prepared as follows: 100g glucose; 5g yeast extract; 1g potassium dihydrogen phosphate; 0.5g magnesium sulfate; 1g ammonium sulfate; 1000mL water; pH adjusted to 7.0.

[0042] Solid culture medium is prepared by adding 1.5%-5% agar to the liquid culture medium.

[0043] Liquid and solid culture media can be commercially available media of the corresponding types.

[0044] Example 1

[0045] This embodiment provides a method for preparing black mulberry fermentation broth, including the following steps:

[0046] (1) Add dried black mulberries to water, blend to obtain a black mulberry homogenate, and sterilize it;

[0047] (2) Transfer the motile fermentation monoclonal bacteria to a solid culture medium and activate it at 37°C until single colonies grow. Transfer the single colony bacteria that grow on the solid culture medium to a liquid culture medium and continue to expand the culture at 37°C for 14 hours to obtain the motile fermentation monoclonal bacteria liquid.

[0048] (3) The black mulberry homogenate was used as the substrate of the culture medium, which included the following substances by mass percentage: 10% black mulberry homogenate, 10% glucose, and pH 7.0.

[0049] (4) Inoculate the culture medium containing black mulberry homogenate with motile fermentation monoclonal bacteria to form a mixture. The inoculation volume of motile fermentation monoclonal bacteria is 10% of the volume of the mixture. Let it stand at 37°C for 24 hours to ferment. Centrifuge the fermented material after fermentation to obtain black mulberry fermentation broth.

[0050] The black mulberry fermentation broth prepared by the method provided in this embodiment is denoted as A.

[0051] Example 2

[0052] This embodiment provides a method for preparing black mulberry fermentation broth, including the following steps:

[0053] (1) Add dried black mulberries to water, blend to obtain a black mulberry homogenate, and sterilize it;

[0054] (2) Transfer the motile fermentation monoclonal bacteria to a solid culture medium and activate it at 37°C until single colonies grow. Transfer the single colony bacteria that grow on the solid culture medium to a liquid culture medium and continue to expand the culture at 37°C for 14 hours to obtain the motile fermentation monoclonal bacteria liquid.

[0055] (3) The black mulberry homogenate was used as the substrate of the culture medium, which included the following substances by mass percentage: 10% black mulberry homogenate, 10% glucose, and pH 7.0.

[0056] (4) Inoculate the culture medium containing black mulberry homogenate with motile fermentation monoclonal bacteria to form a mixture. The inoculation volume of motile fermentation monoclonal bacteria is 5% of the volume of the mixture. Let it stand at 37°C for 36 hours to ferment. Centrifuge the fermented material after fermentation to obtain black mulberry fermentation broth.

[0057] The black mulberry fermentation broth prepared by the method provided in this embodiment is denoted as B.

[0058] Example 3

[0059] This embodiment provides a method for preparing black mulberry fermentation broth, including the following steps:

[0060] (1) Add dried black mulberries to water, blend to obtain a black mulberry homogenate, and sterilize it;

[0061] (2) Transfer the motile fermentation monoclonal bacteria to a solid culture medium and activate it at 37°C until single colonies grow. Transfer the single colony bacteria that grow on the solid culture medium to a liquid culture medium and continue to expand the culture at 37°C for 14 hours to obtain the motile fermentation monoclonal bacteria liquid.

[0062] (3) The black mulberry homogenate was used as the substrate of the culture medium, which included the following substances by mass percentage: 10% black mulberry homogenate, 10% glucose, and pH 7.0.

[0063] (4) Inoculate the culture medium containing black mulberry homogenate with motile fermentation monoclonal bacteria to form a mixture. The inoculation volume of motile fermentation monoclonal bacteria is 7.5% of the volume of the mixture. Let it stand at 37°C for 30 hours to ferment. Centrifuge the fermented material after fermentation to obtain black mulberry fermentation broth.

[0064] The black mulberry fermentation broth prepared by the method provided in this embodiment is denoted as C.

[0065] Example 4

[0066] This embodiment provides a method for preparing black mulberry fermentation broth, including the following steps:

[0067] (1) Add dried black mulberries to water, blend to obtain a black mulberry homogenate, and sterilize it;

[0068] (2) Transfer the motile fermentation monoclonal bacteria to a solid culture medium and activate it at 37°C until single colonies grow. Transfer the single colony bacteria that grow on the solid culture medium to a liquid culture medium and continue to expand the culture at 37°C for 14 hours to obtain the motile fermentation monoclonal bacteria liquid.

[0069] (3) The black mulberry homogenate was used as the substrate of the culture medium, which included the following substances by mass percentage: 15% black mulberry homogenate, 10% glucose, and pH 7.0.

[0070] (4) Inoculate the culture medium containing black mulberry homogenate with motile fermentation monoclonal bacteria to form a mixture. The inoculation volume of motile fermentation monoclonal bacteria is 10% of the volume of the mixture. Let it stand at 37°C for 24 hours to ferment. Centrifuge the fermented material after fermentation to obtain black mulberry fermentation broth.

[0071] The black mulberry fermentation broth prepared by the method provided in this embodiment is denoted as D.

[0072] Example 5

[0073] This embodiment provides a method for preparing black mulberry fermentation broth, including the following steps:

[0074] (1) Add dried black mulberries to water, blend to obtain a black mulberry homogenate, and sterilize it;

[0075] (2) Transfer the motile fermentation monoclonal bacteria to a solid culture medium and activate it at 37°C until single colonies grow. Transfer the single colony bacteria that grow on the solid culture medium to a liquid culture medium and continue to expand the culture at 37°C for 14 hours to obtain the motile fermentation monoclonal bacteria liquid.

[0076] (3) The black mulberry homogenate was used as the substrate of the culture medium, which included the following substances by mass percentage: 15% black mulberry homogenate, 10% glucose, and pH 7.0.

[0077] (4) Inoculate the culture medium containing black mulberry homogenate with motile fermentation monoclonal bacteria to form a mixture. The inoculation volume of motile fermentation monoclonal bacteria is 5% of the volume of the mixture. Let it stand at 37°C for 36 hours to ferment. Centrifuge the fermented material after fermentation to obtain black mulberry fermentation broth.

[0078] The black mulberry fermentation broth prepared by the method provided in this embodiment is denoted as E.

[0079] Example 6

[0080] This embodiment provides a method for preparing black mulberry fermentation broth, including the following steps:

[0081] (1) Add dried black mulberries to water, blend to obtain a black mulberry homogenate, and sterilize it;

[0082] (2) Transfer the motile fermentation monoclonal bacteria to a solid culture medium and activate it at 37°C until single colonies grow. Transfer the single colony bacteria that grow on the solid culture medium to a liquid culture medium and continue to expand the culture at 37°C for 14 hours to obtain the motile fermentation monoclonal bacteria liquid.

[0083] (3) The black mulberry homogenate was used as the substrate of the culture medium, which included the following substances by mass percentage: 15% black mulberry homogenate, 10% glucose, and pH 7.0.

[0084] (4) Inoculate the culture medium containing black mulberry homogenate with motile fermentation monoclonal bacteria to form a mixture. The inoculation volume of motile fermentation monoclonal bacteria is 7.5% of the volume of the mixture. Let it stand at 37°C for 30 hours to ferment. Centrifuge the fermented material after fermentation to obtain black mulberry fermentation broth.

[0085] The black mulberry fermentation broth prepared by the method provided in this embodiment is denoted as F.

[0086] Example 7

[0087] This embodiment provides a method for preparing black mulberry fermentation broth, including the following steps:

[0088] (1) Add dried black mulberries to water, blend to obtain a black mulberry homogenate, and sterilize it;

[0089] (2) Transfer the motile fermentation monoclonal bacteria to a solid culture medium and activate it at 37°C until single colonies grow. Transfer the single colony bacteria that grow on the solid culture medium to a liquid culture medium and continue to expand the culture at 37°C for 14 hours to obtain the motile fermentation monoclonal bacteria liquid.

[0090] (3) The black mulberry homogenate was used as the substrate of the culture medium, which included the following substances by mass percentage: 5% black mulberry homogenate, 10% glucose, and pH 7.0.

[0091] (4) Inoculate the culture medium containing black mulberry homogenate with motile fermentation monoclonal bacteria to form a mixture. The inoculation volume of motile fermentation monoclonal bacteria is 10% of the volume of the mixture. Let it stand at 37°C for 24 hours to ferment. Centrifuge the fermented material after fermentation to obtain black mulberry fermentation broth.

[0092] The black mulberry fermentation broth prepared by the method provided in this embodiment is denoted as G.

[0093] Example 8

[0094] This embodiment provides a method for preparing black mulberry fermentation broth, including the following steps:

[0095] (1) Add dried black mulberries to water, blend to obtain a black mulberry homogenate, and sterilize it;

[0096] (2) Transfer the motile fermentation monoclonal bacteria to a solid culture medium and activate it at 37°C until single colonies grow. Transfer the single colony bacteria that grow on the solid culture medium to a liquid culture medium and continue to expand the culture at 37°C for 14 hours to obtain the motile fermentation monoclonal bacteria liquid.

[0097] (3) The black mulberry homogenate was used as the substrate of the culture medium, which included the following substances by mass percentage: 5% black mulberry homogenate, 10% glucose, and pH 7.0.

[0098] (4) Inoculate the culture medium containing black mulberry homogenate with motile fermentation monoclonal bacteria to form a mixture. The inoculation volume of motile fermentation monoclonal bacteria is 5% of the volume of the mixture. Let it stand at 37°C for 36 hours to ferment. Centrifuge the fermented material after fermentation to obtain black mulberry fermentation broth.

[0099] The black mulberry fermentation broth prepared by the method provided in this embodiment is denoted as H.

[0100] Example 9

[0101] This embodiment provides a method for preparing black mulberry fermentation broth, including the following steps:

[0102] (1) Add dried black mulberries to water, blend to obtain a black mulberry homogenate, and sterilize it;

[0103] (2) Transfer the motile fermentation monoclonal bacteria to a solid culture medium and activate it at 37°C until single colonies grow. Transfer the single colony bacteria that grow on the solid culture medium to a liquid culture medium and continue to expand the culture at 37°C for 14 hours to obtain the motile fermentation monoclonal bacteria liquid.

[0104] (3) The black mulberry homogenate was used as the substrate of the culture medium, which included the following substances by mass percentage: 5% black mulberry homogenate, 10% glucose, and pH 7.0.

[0105] (4) Inoculate the culture medium containing black mulberry homogenate with motile fermentation monoclonal bacteria to form a mixture. The inoculation volume of motile fermentation monoclonal bacteria is 7.5% of the volume of the mixture. Let it stand at 37°C for 30 hours to ferment. Centrifuge the fermented material after fermentation to obtain black mulberry fermentation broth.

[0106] The black mulberry fermentation broth prepared by the method provided in this embodiment is denoted as I.

[0107] Comparative Example 1

[0108] This comparative example provides a method for preparing grape fermentation broth, including the following steps: The difference from Example 1 is that black mulberry homogenate is replaced with grape homogenate.

[0109] The grape fermentation broth prepared by the method provided in this comparative example is denoted as M1.

[0110] Comparative Example 2

[0111] This comparative example provides a method for preparing black rice fermentation liquid, including the following steps: The difference from Example 1 is that the black mulberry homogenate is replaced with black rice homogenate.

[0112] The black rice fermentation liquid prepared by the method provided in this comparative example is denoted as M2.

[0113] Comparative Example 3

[0114] This comparative example provides a method for preparing purple sweet potato fermentation liquid, including the following steps: The difference from Example 1 is that black mulberry homogenate is replaced with purple sweet potato homogenate.

[0115] The purple sweet potato fermentation broth prepared by the method provided in this comparative example is denoted as M3.

[0116] Comparative Example 4

[0117] This comparative example provides a method for preparing blueberry fermentation broth, including the following steps: The difference from Example 1 is that the black mulberry homogenate is replaced with blueberry homogenate.

[0118] The blueberry fermentation broth prepared by the method provided in this comparative example is denoted as M4.

[0119] Comparative Example 5

[0120] This comparative example provides a method for preparing black goji berry fermentation liquid, including the following steps: The difference from Example 1 is that black mulberry homogenate is replaced with black goji berry homogenate.

[0121] The black goji berry fermentation liquid prepared by the method provided in this comparative example is denoted as M5.

[0122] The preparation methods of the fermentation broth provided in Examples 1-9 and Comparative Examples 1-5 are shown in Table 1.

[0123] Table 1. Preparation methods of fermentation broth

[0124]

[0125] Experimental Example 1

[0126] In this experiment, the total flavonoid content of the black mulberry fermentation broth AI prepared in Examples 1-9 and the fermentation broth M1-M5 prepared in Comparative Examples 1-5 was determined by spectrophotometry. The specific method is as follows:

[0127] Transfer 2.4 mL each of the stock solutions of rutin standards at various concentrations, water (as a blank control), and each fermentation broth into stoppered test tubes. Add 0.4 mL of 5% sodium nitrite solution to each tube, shake well, and react for 6 min; add 0.4 mL of 10% aluminum nitrate solution, shake well, and react for 6 min; add 1.0 mL of 4% sodium hydroxide solution and 0.7 mL of water, shake well, and react for 15 min. Measure the absorbance using a spectrophotometer at 510 nm using a 10 mm cuvette. Plot a standard curve from the absorbance values ​​of the rutin standards at various concentrations: y = 0.0016x + 0.0422, R0. 2 =0.9987. The flavonoid content was calculated from the absorbance values ​​of each fermentation broth using the standard curve. The results are shown in Table 2.

[0128] Table 2 Flavonoid content of fermentation broth

[0129] Fermentation broth sample Flavonoid content (mg / mL) A 1.152 B 1.014 C 0.969 D 1.079 E 0.973 F 0.957 G 0.881 H 0.978 I 0.775 M1 0.043 M2 0.063 M3 0.218 M4 0.103 M5 0.561

[0130] As shown in Table 2, comparing the fermented black mulberry broths prepared in Examples 1-9 with those prepared in Comparative Examples 1-5, the flavonoid content in the fermented black mulberry broths prepared in Examples 1-9 was greater than 0.7 mg / mL, while the flavonoid content in the fermented broths prepared in Comparative Examples 1-5 was less than 0.6 mg / mL, and the flavonoid content in the fermented broths prepared in Comparative Examples 1-2 was even less than 0.1 mg / mL. This indicates that this application uses an appropriate amount of *Fermentomonas motilityis* as the fermentation strain to ferment an appropriate amount of black mulberry homogenate. The black mulberry homogenate and *Fermentomonas motilityis* work synergistically to promote the growth of *Fermentomonas motilityis*, thereby efficiently extracting flavonoids from black mulberries and resulting in a high flavonoid content in the fermented black mulberry broth.

[0131] Experimental Example 2

[0132] In this experiment, the anthocyanin content of black mulberry fermentation broth AI prepared in Examples 1-9 and fermentation broth M1-M5 prepared in Comparative Examples 1-5 was determined using the pH differential method. When pH = 1.0, anthocyanins exhibit a characteristic absorption peak at 530 nm. When pH = 4.5, anthocyanins are converted to a colorless chalcone form, resulting in no absorption peak at 530 nm. Utilizing this characteristic, the absorbance values ​​at 530 nm and 700 nm were measured under different pH conditions to quantitatively detect the anthocyanin content in plants. Specific measurement methods and data processing were performed according to the instructions of the BOXBIO anthocyanin detection kit (Beijing Box Biotechnology Co., Ltd.). The results are shown in Table 3.

[0133] Table 3 Anthocyanin content of fermentation broth

[0134] Fermentation broth sample Anthocyanin content (μmol / mL) A 0.5680 B 0.4755 C 0.5415 D 0.4562 E 0.5038 F 0.5172 G 0.4934 H 0.3684 I 0.4031 M1 0.0100 M2 0.0159 M3 0.0589 M4 0.0004 M5 0.0000

[0135] As shown in Table 3, comparing the fermented black mulberry broths prepared in Examples 1-9 with those prepared in Comparative Examples 1-5, the anthocyanin content in the fermented black mulberry broths prepared in Examples 1-9 was greater than 0.3 μmol / mL, while the anthocyanin content in the fermented broths prepared in Comparative Examples 1-5 was less than 0.05 μmol / mL, and the anthocyanin content in the fermented broths prepared in Comparative Examples 4-5 was almost zero. This indicates that this application uses an appropriate amount of *Fermentomonas motilityis* as the fermentation strain to ferment an appropriate amount of black mulberry homogenate. The black mulberry homogenate and *Fermentomonas motilityis* work synergistically to promote the growth of *Fermentomonas motilityis*, thereby efficiently extracting both flavonoids and anthocyanins from black mulberries, resulting in fermented black mulberry broths with high flavonoid and anthocyanin content.

[0136] Experimental Example 3

[0137] This experiment determined the DPPH free radical scavenging rate of the black mulberry fermentation broth prepared in Examples 1-9 using AI. The specific method is as follows:

[0138] Pipette 2.0 mL of black mulberry fermentation broth sample (or anhydrous ethanol) and 2.0 mL of DPPH solution into stoppered test tubes, mix well, and react in the dark for 30 min. Measure the absorbance at 517 nm. The DPPH free radical scavenging rate is calculated using the following formula:

[0139]

[0140] In the formula:

[0141] A0: 2 mL anhydrous ethanol + 2 mL DPPH;

[0142] A1: 2 mL of sample + 2 mL of DPPH;

[0143] A2: 2 mL of anhydrous ethanol + 2 mL of sample.

[0144] The results are shown in Table 4.

[0145] Table 4. Scavenging rate of DPPH free radicals by black mulberry fermentation broth

[0146]

[0147]

[0148] As shown in Table 4, the black mulberry fermentation broths prepared in Examples 1-9 all achieved a DPPH free radical scavenging rate of over 55%. This indicates that by using an appropriate amount of *Fermentomonas motilityis* as the fermentation strain and fermenting an appropriate amount of black mulberry homogenate, flavonoids and anthocyanins in black mulberries can be efficiently extracted, resulting in a fermentation broth with high flavonoid and anthocyanin content, thus exhibiting good DPPH free radical scavenging ability. Therefore, it can be used as an antioxidant in cosmetics.

[0149] Test Example 4

[0150] This experiment determined the scavenging rate of ABTS+ free radicals in the black mulberry fermentation broth prepared in Examples 1-9 using AI. The specific method is as follows:

[0151] (1) Take 7 mmol / L ABTS solution and dilute it 60 times with 10 mmol / L PBS (pH=7.4) to obtain ABTS working solution;

[0152] (2) Take 190 μL of ABTS working solution (or PBS), add 10 μL of black mulberry fermentation broth sample (or PBS), shake for 10 s, let stand at 30℃ for 6 min, and measure the absorbance at 734 nm. The scavenging rate of ABTS+ free radicals is calculated according to the following formula:

[0153]

[0154] In the formula:

[0155] A0: 10 μL PBS + 190 μL ABTS;

[0156] A1: 10 μL sample + 190 μL ABTS;

[0157] A2: 190 μL PBS + 10 μL sample.

[0158] The results are shown in Table 5.

[0159] Table 5. Scavenging rate of ABTS+ free radicals by black mulberry fermentation broth

[0160] Black mulberry fermentation broth sample ABTS+ free radical scavenging rate (%) A 99.74 B 82.50 C 50.92 D 82.28 E 30.26 F 73.62 G 36.33 H 81.58 I 48.99

[0161] As shown in Table 5, the black mulberry fermentation broths prepared in Examples 1-9 all achieved a scavenging rate of over 30% for ABTS+ free radicals. This indicates that by using an appropriate amount of *Fermentomonas motilityis* as the fermentation strain and fermenting an appropriate amount of black mulberry homogenate, flavonoids and anthocyanins in black mulberries can be efficiently extracted, resulting in a fermentation broth with high flavonoid and anthocyanin content, thus exhibiting good scavenging ability against ABTS+ free radicals. Therefore, it can be used as an antioxidant in cosmetics.

[0162] Experimental Example 5

[0163] This experiment determined the hydroxyl radical scavenging rate of the black mulberry fermentation broth prepared in Examples 1-9 using AI. The specific method is as follows:

[0164] Add 20 μL of black mulberry fermentation broth sample (or pure water) and 180 μL of hydroxyl radical reaction colorimetric solution (or hydroxyl radical blank solution) to a 96-well plate, respectively, and mix well. Incubate at 37℃ for 15 min, and measure the absorbance at 510 nm. The hydroxyl radical scavenging rate is calculated using the following formula:

[0165] Hydroxyl radical scavenging rate (%) = 100% (A0 - (AX - AXO)) / A0

[0166] In the formula:

[0167] A0: 20 μL of pure water + 180 μL of reaction colorimetric solution;

[0168] A X : 20 μL sample + 180 μL reaction solution;

[0169] A xo : 20 μL sample + 180 μL blank solution.

[0170] The results are shown in Table 6.

[0171] Table 6. Scavenging rate of hydroxyl radicals by black mulberry fermentation broth

[0172] Black mulberry fermentation broth sample Hydroxyl radical scavenging rate (%) A 84.73 B 70.56 C 71.64 D 84.09 E 79.92 F 69.66 G 58.96 H 58.65 I 59.77

[0173] As shown in Table 6, the black mulberry fermentation broths prepared in Examples 1-9 all achieved a hydroxyl radical scavenging rate of over 55%. This indicates that by using an appropriate amount of *Fermentomonas motilityis* as the fermentation strain and fermenting an appropriate amount of black mulberry homogenate, flavonoids and anthocyanins in black mulberries can be efficiently extracted. This results in a fermentation broth with high flavonoid and anthocyanin content, thus exhibiting good hydroxyl radical scavenging ability. Therefore, it can be used as an antioxidant in cosmetics.

[0174] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A method for preparing black mulberry fermentation broth, characterized in that, The preparation method includes: inoculating a culture medium containing black mulberry homogenate with a broth containing *Saccharomyces cerevisiae* to form a mixture, fermenting the mixture, and separating the fermented material after fermentation into solid and liquid components; the inoculation volume of the broth containing *Saccharomyces cerevisiae* is 7.5%-10% of the volume of the mixture, and the culture medium contains 10%-15% by mass of black mulberry homogenate; the *Saccharomyces cerevisiae* is derived from the Guangdong Provincial Center for Microbial Culture Collection, with the number GDMCC 1.

133.

2. The preparation method according to claim 1, characterized in that, The process before fermentation also includes preparing the black mulberry homogenate and sterilizing the black mulberry homogenate. The preparation of the black mulberry homogenate includes mixing dried black mulberries with water at a mass-to-volume ratio of 10%-15% and then pulping the mixture to obtain the black mulberry homogenate.

3. The preparation method according to claim 1, characterized in that, The culture medium also includes 8%-15% glucose by mass.

4. The preparation method according to claim 1, characterized in that, The pH value of the culture medium is 6.5-7.

5.

5. The preparation method according to claim 1, characterized in that, The fermentation conditions include fermentation at 35-38℃ for 24-36 hours.

6. The preparation method according to claim 1, characterized in that, The fermentation process also includes activating motile fermentation monoclonal bacteria.

7. The preparation method according to claim 6, characterized in that, Activating the motile fermentation monoclonal bacteria includes: transferring the motile fermentation monoclonal bacteria to a solid culture medium, activating it by static incubation at 37°C until single colonies grow, transferring the single colonies grown on the solid culture medium to a liquid culture medium, and continuing to culture at 37°C for 10-18 hours to obtain the motile fermentation monoclonal bacteria broth.

8. A black mulberry fermentation broth prepared by any one of claims 1-7.

9. The application of the black mulberry fermentation liquid as described in claim 8 in the preparation of cosmetics.

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