Prunus triloba ferment and use thereof

Abstract

The present application relates to the technical field of microorganisms, and provides a fermentation product of Prunus triloba with antioxidant and anti-inflammatory effects and application thereof, in particular, a Bifidobacterium bifidum 669, which was preserved in the China General Microbiological Culture Collection Center on August 8, 2025, and the preservation number is CGMCC No. 35548. The Bifidobacterium bifidum 669 according to the embodiment of the present application can ferment various substrates, especially Prunus triloba, the content of dihydrocaffeic acid in the obtained fermentation product is very high, the content of flavones is improved, the DPPH free radical clearance rate is high, the hydroxyl free radical clearance rate is high, the tyrosinase inhibition rate is obviously increased, and the SOD enzyme activity is increased. The fermentation product has good antioxidant activity and anti-inflammatory effect, the fermentation method is simple in operation, easy in industrial production, and low in production cost, and the strain has high application value.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a fermented product of Kanzan cherry blossoms with antioxidant and anti-inflammatory effects and its applications. Background Technology

[0002] Kanzan cherry blossoms, belonging to the genus *Prunus* of the Rosaceae family, are commonly known as "red tassels." Besides their ornamental value, they also contain antioxidants such as flavonoids and polyphenols, as well as polysaccharides, organic acids, and natural vitamins A, B, and E. They can be used as a vegetable or food ingredient. However, directly processing Kanzan cherry blossoms into food or using conventional extraction processes like water extraction often fails to fully extract their active ingredients. While existing fermentation broths from Kanzan cherry blossoms exhibit some activity, the specific characteristic active components within the broth remain unknown. Furthermore, although there are reports of using microorganisms for fermentation extraction, many of these microorganisms are inedible, raising concerns about the safety of the resulting fermentation products.

[0003] Therefore, it is still necessary to further explore fermentation strains that can ferment efficiently and with high safety, and to develop fermentation methods for Guanshan cherry blossoms in order to extract the effective components as completely as possible and to identify their active ingredients. Summary of the Invention

[0004] This invention aims to at least partially solve at least one of the technical problems existing in the prior art. The invention was discovered through the following understanding: an edible Bifidobacterium bifidum strain 669 was screened and discovered. Through fermentation of *Sakura kanzan* cherry blossoms using this strain, for example, the effective components of *Sakura kanzan* cherry blossoms are extracted microbially, and new active components are released by utilizing microbial metabolism, thereby improving the bioavailability and viability of *Sakura kanzan* cherry blossoms, while simultaneously increasing the amount of microbial metabolites with potential beneficial functions to the human body. The active components in the fermentation broth were analyzed using LC-MS / MS technology, revealing the presence of the active ingredient dihydrocaffeic acid. Based on the activity characteristics of the components, the efficacy of the fermentation product was analyzed, confirming that the fermented composition has good antioxidant activity and can be used in oral foods, health products, beauty products, and cosmetics, etc.

[0005] Therefore, in a first aspect of the present invention, the present invention provides a strain of Bifidobacterium bifidum (Bifidobacterium bifidum). Bifidobacterium bifidumAccording to an embodiment of the present invention, Bifidobacterium bifidum 669 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on August 8, 2025, with accession number CGMCC No. 35548. The strain according to this embodiment is an edible strain, whose fermentation product has a high content of dihydrocaffeic acid and an increased flavonoid content. It exhibits high DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, significantly increased tyrosinase inhibition rate, and increased SOD activity, demonstrating good antioxidant activity. It can be used in oral foods, health products, beauty products, and cosmetics.

[0006] Preservation information: Strain name: Bifidobacterium bifidum ( Bifidobacterium bifidum 669 Deposit date: August 8, 2025 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Accession number: CGMCC No. 35548 In a second aspect, the present invention provides a fermentation broth. According to an embodiment of the present invention, the fermentation broth is obtained by fermentation of *Bifidobacterium bifidum* 669 as described in the first aspect. As previously stated, the strain according to the embodiment of the present invention is the edible strain *Bifidobacterium bifidum* 669, whose fermentation product has a high content of dihydrocaffeic acid and an increased flavonoid content. It exhibits high DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, significantly increased tyrosinase inhibition rate, and increased SOD enzyme activity, demonstrating good antioxidant, anti-inflammatory, and whitening activities. It can be used in oral foods, health products, beauty products, and cosmetics.

[0007] It should be noted that the "fermentation broth" of the present invention refers to the solution obtained after culturing Bifidobacterium bifidum 669 for a period of time, which mainly contains Bifidobacterium bifidum 669 and its metabolites; or the supernatant after further treatment by centrifugation, filtration and other means, which mainly contains the metabolites of Bifidobacterium bifidum 669.

[0008] In a third aspect, the present invention provides a bacterial suspension. According to an embodiment of the present invention, the bacterial suspension comprises *Bifidobacterium bifidum* 669 as described in the first aspect. The strain according to an embodiment of the present invention is the edible strain *Bifidobacterium bifidum* 669. The bacterial suspension obtained from its fermentation has a high content of dihydrocaffeic acid and an increased flavonoid content. It exhibits high DPPH free radical scavenging rate, hydroxyl free radical scavenging rate, significantly increased tyrosinase inhibition rate, and increased SOD enzyme activity, demonstrating good antioxidant, anti-inflammatory, and whitening activities. It can be used in oral foods, health products, beauty products, and cosmetics.

[0009] It should be noted that the bacterial suspension can be obtained by processing the above-mentioned fermentation broth through centrifugation, resuspension and other means.

[0010] In a fourth aspect, the present invention provides a fermentation method. According to an embodiment of the invention, the fermentation method includes fermenting *Bifidobacterium bifidum* 669 as described in the first aspect with *Sakura chinensis* or its products.

[0011] According to the fermentation method of this invention, the fermentation product has a high content of dihydrocaffeic acid and an increased flavonoid content. It exhibits a high DPPH free radical scavenging rate (91%), a hydroxyl free radical scavenging rate (85.2%), a significantly increased tyrosinase inhibition rate (93.20%), and an SOD activity increased to 1078 U / mL. This fermentation product possesses excellent antioxidant, anti-inflammatory, and skin-whitening activities; moreover, the fermentation method is simple to operate, easy to industrialize, has low production costs, and high application value.

[0012] According to embodiments of the present invention, the above-described fermentation method may further include at least one of the following additional technical features: According to an embodiment of the present invention, the fermentation product contains dihydrocaffeic acid. The present invention has identified dihydrocaffeic acid in the fermentation product, which possesses multiple effects including antioxidant, anti-inflammatory, chondroprotective, lipid-regulating, hypoglycemic, and anti-tumor properties.

[0013] According to an embodiment of the present invention, the fermentation treatment is carried out at a temperature of 30°C to 37°C under anaerobic or facultative anaerobic conditions for 2 to 6 days. Exemplarily, the fermentation treatment is carried out at a temperature of 30°C to 37°C under anaerobic or facultative anaerobic conditions for 2, 3, 4, 5, or 6 days, or for any number of days within the range of any two of the above values. According to a preferred embodiment of the present invention, the fermentation treatment is carried out at a temperature of 37°C under anaerobic conditions for 4 days.

[0014] According to an embodiment of the present invention, the culture medium for fermentation treatment contains yeast peptone, and the working concentration of the yeast peptone is 0.5% to 3%. Exemplarily, the working concentration of the yeast peptone is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of the yeast peptone is 0.5% to 2%. According to a specific embodiment of the present invention, the working concentration of the yeast peptone is 1%.

[0015] According to an embodiment of the present invention, the culture medium for fermentation contains glucose, and the working concentration of the glucose is 0.1% to 2%. Exemplarily, the working concentration of the glucose is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of the glucose is 0.3% to 1%. According to a specific embodiment of the present invention, the working concentration of the glucose is 0.5%.

[0016] According to an embodiment of the present invention, the culture medium for the fermentation treatment contains Tween-80, and the working concentration of Tween-80 is 0.05% to 1%. Exemplarily, the working concentration of Tween-80 is 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of Tween-80 is 0.05% to 0.5%. According to a specific embodiment of the present invention, the working concentration of Tween-80 is 0.1%.

[0017] According to an embodiment of the present invention, the culture medium for fermentation treatment contains dipotassium hydrogen phosphate, and the working concentration of dipotassium hydrogen phosphate is 0.05% to 1%. Exemplarily, the working concentration of dipotassium hydrogen phosphate is 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of dipotassium hydrogen phosphate is 0.1% to 0.5%. According to a specific embodiment of the present invention, the working concentration of dipotassium hydrogen phosphate is 0.2%.

[0018] According to an embodiment of the present invention, the culture medium for fermentation treatment contains sodium acetate, and the working concentration of the sodium acetate is 0.1% to 2%. Exemplarily, the working concentration of the sodium acetate is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of the sodium acetate is 0.3% to 1%. According to a specific embodiment of the present invention, the working concentration of the sodium acetate is 0.5%.

[0019] According to an embodiment of the present invention, the culture medium for the fermentation treatment contains ammonium citrate, and the working concentration of the ammonium citrate is 0.05% to 1%. Exemplarily, the working concentration of the ammonium citrate is 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of the ammonium citrate is preferably 0.1% to 0.5%. According to a specific embodiment of the present invention, the working concentration of the ammonium citrate is 0.2%.

[0020] According to an embodiment of the present invention, the culture medium for fermentation contains cysteine, and the working concentration of cysteine ​​is 0.01% to 1%. Exemplarily, the working concentration of cysteine ​​is 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of cysteine ​​is 0.03% to 0.5%. According to a specific embodiment of the present invention, the working concentration of cysteine ​​is 0.05%.

[0021] According to an embodiment of the present invention, the Kanzan cherry blossom product includes at least one of Kanzan cherry blossom powder, Kanzan cherry blossom extract, and Kanzan cherry blossom aqueous extract.

[0022] It should be noted that "aqueous extract" refers to a liquid extracted using the solubility of plant components in water. The main principle is that many highly polar compounds (such as polysaccharides, amino acids, and inorganic salts) have high solubility in water, and therefore can be extracted from plant tissues by soaking in water or boiling. The main procedures for aqueous extraction are: pulverizing the plant material and soaking it in an appropriate amount of water; improving extraction efficiency through methods such as boiling and reflux extraction; and obtaining the extract through filtration and concentration. Those skilled in the art will understand that the type of "Kansai cherry blossom aqueous extract" in this invention is not particularly limited; aqueous extracts obtained under different conditions, and Kansai cherry blossom aqueous extracts intended for further fermentation extraction, are all within the scope of protection of this invention.

[0023] According to an embodiment of the present invention, the working concentration of the Guanshan cherry blossom product in the fermentation medium is 3% to 8%. For example, the working concentration of the Guanshan cherry blossom product in the fermentation medium is 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, or 8.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of the Kanzan cherry blossom product in the fermentation medium is 3% to 5%. According to a specific embodiment of the present invention, the working concentration of the Kanzan cherry blossom product in the fermentation medium is 5%.

[0024] According to an embodiment of the present invention, during the fermentation treatment, the inoculum amount of Bifidobacterium bifidum 669 is 1% to 15%. Exemplarily, the inoculum amount of Bifidobacterium bifidum 669 is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the inoculum amount of Bifidobacterium bifidum 669 is 2% to 13%. According to a specific embodiment of the present invention, the inoculum amount of Bifidobacterium bifidum 669 is 2% to 10%.

[0025] According to an embodiment of the present invention, before carrying out the fermentation treatment, the Bifidobacterium bifidum 669 is pre-cultured as a seed culture for at least one stage.

[0026] According to an embodiment of the present invention, the seed culture is carried out at a temperature of 30℃~37℃ in an anaerobic or facultative anaerobic environment for 15~25 h. According to a preferred embodiment of the present invention, the seed culture is carried out at 37℃ in an anaerobic environment for 16~24 h.

[0027] Those skilled in the art will understand that the seed culture medium is based on the Kanzan cherry blossom product and its fermentation strain. Exemplarily, according to embodiments of the present invention, the seed culture medium includes, but is not limited to, MRS liquid culture medium or a modified version thereof.

[0028] In a fifth aspect, the present invention provides a fermentation broth. According to an embodiment of the invention, the fermentation broth is obtained using the fermentation method described above.

[0029] According to the fermentation method of this invention, the fermentation broth has a high dihydrocaffeic acid content and an increased flavonoid content, exhibiting a high DPPH free radical scavenging rate (91%), a hydroxyl free radical scavenging rate (85.2%), a significantly increased tyrosinase inhibition rate (93.20%), and an SOD enzyme activity increased to 1078 U / mL. This fermentation broth possesses excellent antioxidant, anti-inflammatory, and skin-whitening activities; moreover, the fermentation method is simple to operate, easy to industrialize, has low production costs, and high application value.

[0030] It should be noted that the "fermentation broth" of the present invention refers to the solution obtained after culturing Bifidobacterium bifidum 669 for a period of time, which mainly contains Bifidobacterium bifidum 669 and its metabolites; or the supernatant after further treatment by centrifugation, filtration and other means, which mainly contains the metabolites of Bifidobacterium bifidum 669.

[0031] In a sixth aspect, the present invention provides a composition. According to an embodiment of the invention, the composition comprises the aforementioned Bifidobacterium bifidum 669, fermentation broth, and bacterial suspension. The composition according to an embodiment of the invention contributes to antioxidant, anti-inflammatory, skin-whitening, and / or immune-boosting effects.

[0032] According to embodiments of the present invention, the composition may further include at least one of the following additional technical features: According to embodiments of the present invention, the composition further includes an ingestible excipient and / or a carrier.

[0033] According to an embodiment of the present invention, the excipient includes at least one selected from adhesives, disintegrants, lubricants, flow aids, stabilizers, fillers, diluents, and sustained-release agents.

[0034] According to an embodiment of the present invention, the carrier comprises at least one selected from sugars, cellulose and its derivatives, calcium phosphates, alkaline earth metal stearates, vegetable oils, nonionic surfactants, cationic surfactants, anionic surfactants, fatty alcohols, and hydrolyzed cereal solids.

[0035] According to embodiments of the present invention, the dosage form of the composition includes at least one selected from oral liquids, powders, granules, capsules, tablets, and pills.

[0036] In a seventh aspect of the invention, the invention provides for the use of the aforementioned Bifidobacterium bifidum 669, fermentation broth, bacterial suspension, or composition in the preparation of foods, beverages, health products, feeds, food additives, and / or feed additives that contribute to antioxidant activity and / or enhance immunity.

[0037] In an eighth aspect, the present invention provides a food, beverage, health product, feed, food additive, and / or feed additive. According to an embodiment of the invention, the food, beverage, health product, feed, food additive, and / or feed additive comprises the fermentation broth or composition described above. The food, beverage, health product, feed, food additive, and / or feed additive contributes to antioxidant activity and / or enhances immunity.

[0038] According to embodiments of the present invention, the aforementioned food, beverage, health product, feed, food additive and / or feed additive may further include at least one of the following additional technical features: According to embodiments of the present invention, it further includes excipients or carriers acceptable in food or health products, or excipients or carriers acceptable in animal feed.

[0039] In this article, "acceptable in food" refers to substances or compositions that are edible for human consumption, which may be adjusted according to the food requirements of different countries.

[0040] In this article, "acceptable in health supplements" refers to substances or compositions that can be consumed by humans, and these can be adjusted according to the health supplement requirements of different countries.

[0041] In this article, “acceptable adjuvants or carriers in animal feed” refers to substances or compositions that can be consumed by animals, and these can be adjusted according to the animal feed requirements of different countries.

[0042] According to embodiments of the present invention, at least one of the aforementioned Bifidobacterium bifidum 669, fermentation broth, bacterial suspension, and composition is added to or inoculated into food, beverage, health product, feed, food additive, and / or feed additive, thereby further obtaining food, beverage, health product, feed, food additive, and / or feed additive that contributes to antioxidant activity and / or enhances immunity.

[0043] For example, the aforementioned food products include, but are not limited to: Bifidobacterium bifidum 669 tablets, fermented dairy products (such as Bifidobacterium bifidum 669 yogurt), Bifidobacterium bifidum 669 solid beverages, Bifidobacterium bifidum 669 milk powder, Bifidobacterium bifidum 669 cheese, Bifidobacterium bifidum 669 soy products, Bifidobacterium bifidum 669 candy, Bifidobacterium bifidum 669 fermented vegetables, etc.

[0044] For example, the aforementioned health products include, but are not limited to: health products for human use and health products for veterinary use.

[0045] In a ninth aspect of the invention, the invention proposes the use of the aforementioned Bifidobacterium bifidum 669, fermentation broth, or composition in the preparation of antioxidant and whitening skin care products and cosmetics.

[0046] In a tenth aspect, the present invention provides a skin care product and / or cosmetic product. According to an embodiment of the invention, the skin care product and / or cosmetic product comprises the aforementioned Bifidobacterium bifidum 669, fermentation broth, bacterial suspension, or composition. The cosmetic product according to an embodiment of the invention contributes to antioxidant and whitening effects.

[0047] In this document, "cosmetics" is intended to describe substances intended for topical application to human skin, including leave-on and wash-off products. "Skin" includes the skin on the face, neck, chest, back, arms, hands, legs, and scalp.

[0048] According to an embodiment of the present invention, the cosmetic also includes excipients acceptable in the cosmetic field. These cosmetic-acceptable excipients refer to substances that can be applied to the skin, and can be tailored to different dosage forms and requirements.

[0049] The cosmetic formulation can be a face mask, serum, toner, lotion, cream, etc.

[0050] In an eleventh aspect, the present invention provides a medicament. According to an embodiment of the invention, the medicament comprises the aforementioned Bifidobacterium bifidum 669, fermentation broth, bacterial suspension, or a combination thereof. The medicament according to an embodiment of the invention is capable of effectively preventing and / or treating diseases associated with oxidative stress, such as inflammatory diseases, metabolic syndrome, diabetes, and / or hypertension.

[0051] In a twelfth aspect, the present invention provides a method for producing dihydrocaffeic acid. According to an embodiment of the invention, the method includes fermenting *Bifidobacterium bifidum* 669 onto *Chlorophytum comosum* or its products to obtain dihydrocaffeic acid. As previously described, after fermentation of *Chlorophytum comosum* or its products with *Bifidobacterium bifidum* 669 according to an embodiment of the present invention, the obtained fermentation product is identified to contain dihydrocaffeic acid. This substance has multiple effects such as antioxidant, anti-inflammatory, chondroprotective, lipid-regulating, hypoglycemic, and antitumor properties, and has high application value.

[0052] According to embodiments of the present invention, the above-described method for producing dihydrocaffeic acid may further include at least one of the following additional technical features: According to an embodiment of the present invention, the fermentation treatment is carried out at a temperature of 30°C to 37°C under anaerobic or facultative anaerobic conditions for 2 to 6 days. Exemplarily, the fermentation treatment is carried out at a temperature of 30°C to 37°C under anaerobic or facultative anaerobic conditions for 2, 3, 4, 5, or 6 days, or for any number of days within the range of any two of the above values. According to a preferred embodiment of the present invention, the fermentation treatment is carried out at a temperature of 37°C under anaerobic conditions for 4 days.

[0053] According to an embodiment of the present invention, the culture medium for fermentation treatment contains yeast peptone, and the working concentration of the yeast peptone is 0.5% to 3%. Exemplarily, the working concentration of the yeast peptone is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, or 3.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of the yeast peptone is 0.5% to 2%. According to a specific embodiment of the present invention, the working concentration of the yeast peptone is 1%.

[0054] According to an embodiment of the present invention, the culture medium for fermentation contains glucose, and the working concentration of the glucose is 0.1% to 2%. Exemplarily, the working concentration of the glucose is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of the glucose is 0.3% to 1%. According to a specific embodiment of the present invention, the working concentration of the glucose is 0.5%.

[0055] According to an embodiment of the present invention, the culture medium for the fermentation treatment contains Tween-80, and the working concentration of Tween-80 is 0.05% to 1%. Exemplarily, the working concentration of Tween-80 is 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of Tween-80 is 0.05% to 0.5%. According to a specific embodiment of the present invention, the working concentration of Tween-80 is 0.1%.

[0056] According to an embodiment of the present invention, the culture medium for fermentation treatment contains dipotassium hydrogen phosphate, and the working concentration of dipotassium hydrogen phosphate is 0.05% to 1%. Exemplarily, the working concentration of dipotassium hydrogen phosphate is 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of dipotassium hydrogen phosphate is 0.1% to 0.5%. According to a specific embodiment of the present invention, the working concentration of dipotassium hydrogen phosphate is 0.2%.

[0057] According to an embodiment of the present invention, the culture medium for fermentation treatment contains sodium acetate, and the working concentration of the sodium acetate is 0.1% to 2%. Exemplarily, the working concentration of the sodium acetate is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of the sodium acetate is 0.3% to 1%. According to a specific embodiment of the present invention, the working concentration of the sodium acetate is 0.5%.

[0058] According to an embodiment of the present invention, the culture medium for the fermentation treatment contains ammonium citrate, and the working concentration of the ammonium citrate is 0.05% to 1%. Exemplarily, the working concentration of the ammonium citrate is 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of the ammonium citrate is preferably 0.1% to 0.5%. According to a specific embodiment of the present invention, the working concentration of the ammonium citrate is 0.2%.

[0059] According to an embodiment of the present invention, the culture medium for fermentation contains cysteine, and the working concentration of cysteine ​​is 0.01% to 1%. Exemplarily, the working concentration of cysteine ​​is 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of cysteine ​​is 0.03% to 0.5%. According to a specific embodiment of the present invention, the working concentration of cysteine ​​is 0.05%.

[0060] According to an embodiment of the present invention, the Kanzan cherry blossom product includes at least one of Kanzan cherry blossom powder, Kanzan cherry blossom extract, and Kanzan cherry blossom aqueous extract.

[0061] It should be noted that "aqueous extract" refers to a liquid extracted using the solubility of plant components in water. The main principle is that many highly polar compounds (such as polysaccharides, amino acids, and inorganic salts) have high solubility in water, and therefore can be extracted from plant tissues by soaking in water or boiling. The main procedures for aqueous extraction are: pulverizing the plant material and soaking it in an appropriate amount of water; improving extraction efficiency through methods such as boiling and reflux extraction; and obtaining the extract through filtration and concentration. Those skilled in the art will understand that the type of "Kansai cherry blossom aqueous extract" in this invention is not particularly limited; aqueous extracts obtained under different conditions, and Kansai cherry blossom aqueous extracts intended for further fermentation extraction, are all within the scope of protection of this invention.

[0062] According to an embodiment of the present invention, the working concentration of the Guanshan cherry blossom product in the fermentation medium is 3% to 8%. For example, the working concentration of the Guanshan cherry blossom product in the fermentation medium is 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6.0%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7.0%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, or 8.0%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the working concentration of the Kanzan cherry blossom product in the fermentation medium is 3% to 5%. According to a specific embodiment of the present invention, the working concentration of the Kanzan cherry blossom product in the fermentation medium is 5%.

[0063] According to an embodiment of the present invention, during the fermentation treatment, the inoculum amount of Bifidobacterium bifidum 669 is 1% to 15%. Exemplarily, the inoculum amount of Bifidobacterium bifidum 669 is 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or a range between any two of the above values. According to a preferred embodiment of the present invention, the inoculum amount of Bifidobacterium bifidum 669 is 2% to 13%. According to a specific embodiment of the present invention, the inoculum amount of Bifidobacterium bifidum 669 is 2% to 10%.

[0064] According to an embodiment of the present invention, before carrying out the fermentation treatment, the Bifidobacterium bifidum 669 is pre-cultured as a seed culture for at least one stage.

[0065] According to an embodiment of the present invention, the seed culture is carried out at a temperature of 30℃~37℃ in an anaerobic or facultative anaerobic environment for 15~25 h. According to a preferred embodiment of the present invention, the seed culture is carried out at 37℃ in an anaerobic environment for 16~24 h.

[0066] Those skilled in the art will understand that the seed culture medium is based on the Kanzan cherry blossom product and its fermentation strain. Exemplarily, according to embodiments of the present invention, the seed culture medium includes, but is not limited to, MRS liquid culture medium or a modified version thereof.

[0067] Compared with the prior art, the present invention has at least the following beneficial effects: (1) The Bifidobacterium bifidum 669 obtained by screening in this invention is an edible strain, which has a high fermentation efficiency for Guanshan cherry blossoms.

[0068] (2) The fermentation product obtained by the fermentation method of the present invention is prepared by fermentation of natural and safe edible substrate (Kanshan cherry blossom or its products) with Bifidobacterium bifidum 669 obtained by screening. It is suitable for a wider range of people, has no obvious toxic side effects, is non-irritating, and has high safety.

[0069] (2) The fermentation product obtained by the fermentation method of the present invention contains dihydrocaffeic acid, which has good antioxidant activity, whitening and anti-inflammatory effects. It can also be used to prevent and / or treat related diseases caused by oxidative stress, such as inflammatory diseases, metabolic syndrome, diabetes and / or hypertension, and has high application value.

[0070] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0071] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0072] Figure 1 The image shows the liquid phase results of the blank culture medium according to Example 2 of the present invention, with a peak time of 8.118 min.

[0073] Figure 2 The figure shows the liquid phase results of the fermentation broth of the Guanshan cherry blossom 669 strain according to Example 2 of the present invention, with a peak time of 7.329 min.

[0074] Figure 3 The liquid chromatography result of the dihydrocaffeic acid standard according to Example 2 of the present invention is shown in the figure. The peak time is 7.330 min.

[0075] Figure 4 This is a graph showing the linear relationship between the concentration of different rutin standard solutions and the absorbance value according to Example 2 of the present invention.

[0076] Figure 5 This is a linear relationship graph between different gallic acid concentration solutions and absorbance values ​​according to Example 2 of the present invention.

[0077] Figure 6 This is a primary mass spectrum of dihydrocaffeic acid according to Example 2 of the present invention.

[0078] Figure 7 This is a secondary mass spectrum of dihydrocaffeic acid according to Example 2 of the present invention. Detailed Implementation

[0079] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0080] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "multiple" or "a plurality of" means at least two, two types, such as two, two, three, three, etc., unless otherwise explicitly specified.

[0081] In this document, the terms “comprising,” “having,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0082] In this document, the term “optionally” generally means that an event or condition described below may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0083] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0084] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined herein. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0085] In this article, "dihydrocaffeic acid" is also known as caffeic acid, and its chemical formula is C8H. 10 O4, with the structural formula shown in formula (Ⅰ) and CAS No.: 1078-61-1, can scavenge intracellular ROS, increase the activity of nitric oxide synthase, reduce the phosphorylation level of MAPKp38, prevent UV-induced skin damage, and has antioxidant, anti-inflammatory, and anti-cartilage degradation activities.

[0086] Equation (Ⅰ).

[0087] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0088] Example 1: Optimization of the fermentation method for Kanzan cherry blossoms 1.1 Screening of Bifidobacterium bifidum 669 In this embodiment, specific species of Bifidobacterium bifidum 669 were screened. Based on the screening results, it was found that Bifidobacterium bifidum 669 strain had a better fermentation effect on Kanzan cherry blossoms. Therefore, Bifidobacterium bifidum 669 strain was used in subsequent experiments below.

[0089] 1.2 Screening of Fermentation Medium Formulation This invention screens the following two formulations, wherein the components in each formulation are expressed and described as mass-volume percentages (g / 100ml).

[0090] Fermentation formula 1: 1% yeast peptone (FP103), 0.5% glucose, 0.1% Tween-80, 0.2% dipotassium hydrogen phosphate, 0.5% sodium acetate, 0.2% ammonium citrate, 0.05% cysteine, 5% Kanzan cherry blossom powder, pH 7.0±0.2.

[0091] Fermentation formula 2: 1% glucose, 5% cherry blossom powder.

[0092] (1) Fermentation inoculum size of the strain: 2%~10%.

[0093] (2) Cultivation conditions: Cultivation temperature 30℃~37℃, fermentation time 2~6 days, anaerobic and facultative anaerobic during cultivation, static and shaking cultivation.

[0094] (3) Sterilization conditions for fermentation medium: 121℃ or 115℃, sterilization for 20-30 min.

[0095] (4) Fermentation broth treatment method: filter or centrifuge to remove bacteria.

[0096] (5) Dihydrocaffeic acid content: The dihydrocaffeic acid content of the fermentation broth shall not be less than 0.7 mg / ml.

[0097] 1.3 Optimized Post-Fermentation Method Based on the above screening of strains, fermentation formulas, and fermentation conditions, the present invention obtains a preferred fermentation method, the specific method of which is as follows: (1) Preparation of MRS solid plate culture medium: The MRS solid plate culture medium consists of the following components: 10g peptone, 5g beef meal, 4g yeast powder, 2g K2HPO4·7H2O, 2g triammonium citrate, 20g glucose, 5g CH3COONa·3H2O, 0.2g MgSO4·7H2O, 0.05g MnSO4·4H2O, 1mL Tween-80, 15.0g agar, 1000mL distilled water, and natural pH. The MRS culture medium is sterilized by moist heat at 121℃ for 15 minutes. After cooling to an appropriate temperature, plates are poured, with each plate containing 20-30ml.

[0098] (2) Activation of the strain: Take the glycerol tube of Bifidobacterium bifidum strain 669 stored at -80℃, take 0.1 ml~0.2 ml and spread it on the MRS solid plate. Incubate anaerobically at 37±1℃ for 24h. After the bacterial growth on the solid plate, pick an appropriate amount of bacterial growth and spread it on a new MRS plate. Incubate anaerobically at 37±1℃ for 24h to obtain the activated strain of Bifidobacterium bifidum strain 669.

[0099] (3) The activated Bifidobacterium bifidum strain 669 was anaerobic cultured on an MRS plate and a single colony was grown.

[0100] (4) Prepare seed culture medium (MRS liquid medium), pick the single colonies obtained in step (3) into the seed culture medium, and culture anaerobicly at 37℃ for 16-24h; scrape 0.5cm*0.5cm activated bacterial growth of Bifidobacterium bifidum strain 669 from the mature slant and place it in an Erlenmeyer flask, culture anaerobicly at 37℃ for 18-24h, and the viable count is greater than 1*10 9 The concentration of CFU / ml was normal, and microscopic examination showed no contaminants, meeting the requirements for seed culture. The seed culture medium consisted of the following components: 10g peptone, 5g beef meal, 4g yeast extract, 2g K₂HPO₄·7H₂O, 2g triammonium citrate, 20g glucose, 5g CH₃COONa·3H₂O, 0.2g MgSO₄·7H₂O, 0.05g MnSO₄·4H₂O, 1mL Tween-80, and 1000mL distilled water at natural pH. The above materials were weighed and dissolved in water according to the specified proportions, dispensed into Erlenmeyer flasks, and sterilized at 121℃ for 30 minutes.

[0101] (5) Prepare fermentation medium. Inoculate the above-mentioned Bifidobacterium bifidum strain 669, which has been expanded by seed culture, into the fermentation medium at an inoculation rate of 2%. Anaerobically culture at 37℃ and 100 rpm for 2 days to obtain the fermentation product of Kanzan cherry blossoms. The fermentation medium includes the following components: yeast peptone (FP103) 1%, glucose 0.5%, Tween-80 0.1%, dipotassium hydrogen phosphate 0.2%, sodium acetate 0.5%, ammonium citrate 0.2%, cysteine ​​0.05%, Kanzan cherry blossom powder 5%, pH 7.0±0.2. The preparation method of Kanzan cherry blossom powder is as follows: 5% cherry blossom powder aqueous solution, ultrasonically break the cell wall for 30 minutes, centrifuge the supernatant, and dry or freeze-dry at low temperature to obtain cherry blossom extract.

[0102] (6) Centrifuge the obtained Guanshan cherry blossom fermentation product at 12000 rpm for 10 min and take the fermentation supernatant.

[0103] Example 2: Detection of active ingredients and efficacy of Guanshan cherry blossom fermentation liquid Based on the fermentation supernatant of Kanzan cherry blossoms obtained using the optimized method of Example 1, the components and their effects in the fermentation supernatant were detected, and the specific details are as follows: 2.1 High-performance liquid chromatography-mass spectrometry (HPLC-MS) detection of fermentation broth components This experiment used ultra-high performance liquid chromatography-triple quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF-MS) to rapidly analyze the chemical composition of the fermentation supernatant of the above-mentioned *Cherry Blossom from Kanzan*. The fermentation medium was used as a blank control. The specific experimental procedures are as follows: UPLC-Triple-TOF / MS System: UPLC high-performance liquid chromatograph (Waters Corporation, USA), TripleTOF 5600 + The time-of-flight mass spectrometer is equipped with an electrospray ionization source (AB SCIEX, USA).

[0104] Chromatographic conditions: The liquid chromatograph was a UPLC (High Performance Liquid Chromatography) system; the column was an Agilent ZORBAX Eclipse XDB-C. 18 Column (5.0 μm, 4.6 × 250 mm); using 0.1% formic acid aqueous solution as mobile phase A and methanol as mobile phase B, linear gradient elution: 0 min 20% B; 10 min 50% B; 20 min 80% B; 25 min 20% B; flow rate 1 ml / min; column temperature 40 °C. C; Detection wavelength: 285nm, injection volume: 5µL.

[0105] Mass spectrometry conditions: UPLC-Triple-TOF 5600 + Time-of-Flight LC-MS: Positive and negative ion scanning modes; Scan range: m / z 100-1500; Nebulizer gas (GS1): 55 psi; Nebulizer gas (GS2): 55 psi; Curtain gas (CUR): 35 psi; Ion source temperature (TEM): 550℃ (negative); Ion source voltage (IS): -4500 V (negative); First-stage scan: Declustering voltage (DP): 100 V; Focusing voltage (CE): 10 V; Second-stage scan: Mass spectrometry data acquired using TOF MS-Product Ion-IDA mode, CID energy -40 Before injection, use a CDS pump to perform mass axis calibration at 20 eV to ensure that the mass axis error is less than 2 ppm.

[0106] Figures 1-3The liquid chromatography (LC) results for blank culture medium, fermentation broth of *Sakura kansanense* strain 669, and dihydrocaffeic acid standard are shown. The results, compared with the blank culture medium chromatogram, show that the fermentation broth of *Sakura kansanense* strain 669 newly produces a peak with a retention time of 7.329 min, consistent with the elution time of the dihydrocaffeic acid standard. Simultaneously, the quadrupole time-of-flight mass spectrometry (QTF) negative ion mode primary mass spectrum is shown below. Figure 6 As shown, the results indicate that m / z 181.0511 is [MH]. - The peak, m / z 363.1086, is [2M-H]. - The fitted molecular formula is C9H 10 O4, with an unsaturation RBD of 5 and a molecular weight deviation of 1.5 ppm. Based on quadrupole time-of-flight mass spectrometry in negative ion mode, 181.1 [MH]. - The secondary mass spectrum of the peaks shows that the main fragments are: 137.0610, 135.0454, 121.0295, 109.0295, 93.0346, 59.0139, etc. Figure 7 As shown, the above primary mass spectrum and dihydrogen... Caffeic acid molecular formula C9H 10 Corresponding to O4, each fragment ion in the secondary mass spectrum corresponds to a structural unit of dihydrocaffeic acid. The mass spectrometry results are consistent with the structural characteristics of dihydrocaffeic acid, confirming that the fermentation medium containing *Salvia kansanensis* produced dihydrocaffeic acid through specific transformation by strain 669. It should be noted that... Figure 6 and Figure 7 In the graph, the horizontal axis Mass / Charge,Da represents the mass-to-charge ratio of the ion (the ratio of mass to charge, in Daltons), and the vertical axis Intensity represents the (relative) strength of the ion.

[0107] 2.2 Detection of Free Radical Scavenging Capacity of Guanshan Cherry Blossom Fermentation Broth The fermentation broth of *Chlorophytum comosum* obtained by the optimized method in Example 1 was tested using a DPPH free radical scavenging ability test kit (Solepro) and a hydroxyl free radical scavenging ability test kit (Solepro). The fermentation medium group was used as a blank control group. The specific operation was carried out according to the instructions of the kits, and will not be described in detail here.

[0108] Table 1 shows the DPPH free radical scavenging rates of the blank culture medium group and the fermentation broth of strain 669. Compared to the blank culture medium group, the DPPH free radical scavenging rate was significantly higher. Bifidobacterium bifidum The fermentation extraction of Guanshan cherry blossom powder by strain 669 showed a significant increase in the DPPH free radical scavenging rate of the fermentation broth. This indicates that the Guanshan cherry blossom fermentation broth helps reduce free radical damage to cells and tissues in the body, and may, to some extent, delay aging and reduce the risk of oxidative stress-related diseases. Furthermore, Bifidobacterium bifidumThe hydroxyl radical scavenging rate of the fermentation broth of strain 669 was 85.2%.

[0109] Table 1

[0110] 2.3 Detection of Tyrosinase Inhibition Capacity in Guanshan Cherry Blossom Fermentation Broth 1. Solution preparation (1) Preparation of PBS buffer: 1.701g potassium dihydrogen phosphate, 2.177g dipotassium hydrogen phosphate, add purified water to make up to 500mL to obtain PBS buffer, store in the refrigerator at 4℃ protected from light for later use.

[0111] (2) Preparation of L-DOPA solution: Accurately weigh 0.0395 g of L-DOPA, dissolve it in the above-mentioned PBS buffer, transfer it to a 100 mL volumetric flask, make up to volume, shake well, and sonicate to obtain 2 mmol·L⁻¹ solution. -1 The L-DOPA solution should be prepared and used immediately.

[0112] (3) Preparation of TYR (tyrosine kinase) solution: Dissolve 25 KU of TYR in a small amount of PBS buffer, wash several times, transfer the washing solution to a 100 mL volumetric flask, make up to volume with PBS buffer, and shake well to obtain 250 U·mL -1 The TYR solution was then diluted with the stock solution to 125 U·mL. -1 Store in a refrigerator at -20℃ for later use.

[0113] (4) Preparation of α-arbutin: Weigh 0.2g of arbutin, add PBS to dissolve and make up to 100ml as a control.

[0114] 2. Sample preparation: Centrifuge the fermentation broth at 12000 rpm for 10 min, collect the supernatant and set aside.

[0115] 3. Determination of the inhibitory activity of the sample against TYR (tyrosine) The tyrosinase inhibitory activity of the fermentation broth obtained by the optimized method in Example 1 was tested. The reaction system shown in Table 2 was prepared, with 2 mmol·L⁻¹... -1 Using L-DOPA as the substrate, PBS buffer, sample solution, and substrate were added sequentially to 96-well plates numbered 1, 2, 3, and 4, with each well repeated three times. The temperature of the multi-functional microplate reader was set to 37℃. After linear shaking for 10 minutes, the plate was removed, TYR solution was added, and the plate was quickly replaced. The detection wavelength was 475 nm, and the total volume of the reaction system was 240 μL. The inhibition rate was calculated based on the absorbance. The inhibition rate was calculated using formula (1). When the calculated result is negative, the absolute value of the negative number is the activation rate.

[0116] Table 2: Composition of the reaction system

[0117] Formula (1); Wherein, A1: the absorbance of the reaction solution with TYR added but without sample, measured at 475 nm; A2: Absorbance measured at 475 nm for the reaction solution without sample and TYR; A3: The absorbance of the reaction solution with sample and TYR added was measured at 475 nm; A4: Absorbance measured at 475 nm for the reaction solution with sample but without TYR.

[0118] Table 3 shows the cherry blossoms of Kanzan. Bifidobacterium bifidum The results of tyrosinase inhibition rate of the fermentation broth of strain 669 showed that the inhibition rate of tyrosinase in the fermentation broth was as high as 93.2%, which was significantly higher than that in the blank culture medium and α-arbutin at a content of 0.2%.

[0119] Table 3

[0120] 2.4 Kanzan Cherry Blossom Fermentation Liquid The fermentation broth obtained by the optimized method in Example 1 was tested using a superoxide dismutase (SOD) activity assay kit (WST-1 method) (Solepro). The above-mentioned fermentation medium group was used as a blank control group. The specific operation was carried out in accordance with the kit instructions, and will not be described in detail here.

[0121] Table 4 shows the SOD activity detection results of the fermentation broth of Sakura 669 strain from Guanshan. The SOD activity of this fermentation broth was as high as 1078 U / mL, which was significantly better than that of the blank culture medium group.

[0122] Table 4

[0123] 2.5 Kanzan Cherry Blossom Fermentation Liquid The fermentation broth obtained by the optimized method in Example 1 was tested using a plant flavonoid content assay kit (Solepro). The above-mentioned fermentation medium group was used as a blank control group. The specific operation was carried out in accordance with the kit instructions, and will not be described in detail here.

[0124] Figure 4 The linear relationship between the concentration of rutin standard solution and absorbance value was shown. Table 5 shows the total flavonoid concentration in the fermentation broth of Sakura guanshanensis strain 669, which was 2.79 mg / mL, higher than that of the blank culture medium group.

[0125] Table 5

[0126] 2.6 Cherry Blossoms of Kanzan The fermentation broth obtained by the optimized method in Example 1 was tested using a total plant phenol (TP) content detection kit (Solepro). The above-mentioned fermentation medium group was used as a blank control group. The specific operation was carried out in accordance with the instructions of the kit, and will not be described in detail here.

[0127] Figure 5 The linear relationship between gallic acid concentration and absorbance value is shown in Table 6. The total phenol concentration in the fermentation broth of Sakura guanshanensis strain 669 is 7.89 mg / mL, which is not significantly different from the blank culture medium group.

[0128] Table 6

[0129] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "embodiment," or "specific embodiment," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment. Furthermore, the specific features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments and features described in this specification.

[0130] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A strain of Bifidobacterium bifidum ( Bifidobacterium bifidum 669, characterized in that, The Bifidobacterium bifidum 669 was deposited at the China General Microbiological Culture Collection Center on August 8, 2025, with accession number CGMCC No. 35548.

2. A fermentation broth, characterized in that, The fermentation broth was obtained by fermenting Bifidobacterium bifidum 669 as described in claim 1.

3. A bacterial suspension, characterized in that, The bacterial suspension includes Bifidobacterium bifidum 669 as described in claim 1.

4. A fermentation method, characterized in that, This includes using Bifidobacterium bifidum 669 as described in claim 1 to ferment Guanshan cherry blossoms or their products.

5. The fermentation method according to claim 4, characterized in that, The fermentation product contains dihydrocaffeic acid; and / or The fermentation process is carried out at 30℃~37℃ under anaerobic or facultative anaerobic conditions for 2~6 days; and / or The culture medium for fermentation treatment contains at least one of the following: Yeast peptone, wherein the working concentration of the yeast peptone is 0.5% to 3%. Glucose, wherein the working concentration of the glucose is 0.1% to 2%. Tween-80, wherein the working concentration of Tween-80 is 0.05% to 1%. Dipotassium hydrogen phosphate, wherein the working concentration of dipotassium hydrogen phosphate is 0.05%~1%. Sodium acetate, wherein the working concentration of sodium acetate is 0.1%~2%. Ammonium citrate, wherein the working concentration of ammonium citrate is 0.05%~1%. Cysteine, wherein the working concentration of said cysteine ​​is 0.01% to 1%; and / or The Kanzan cherry blossom products include at least one of Kanzan cherry blossom powder, Kanzan cherry blossom extract, and Kanzan cherry blossom aqueous extract; and / or The working concentration of the Guanshan cherry blossom product in the fermentation medium is 3%~8%.

6. The method according to claim 4, characterized in that, During the fermentation process, the inoculum size of Bifidobacterium bifidum 669 is 1% to 15%; and / or Prior to the fermentation process, the *Bifidobacterium bifidum* 669 was pre-cultured using seed culture medium for at least one primary stage; and / or The seed culture was carried out at 30℃~37℃ under anaerobic or facultative anaerobic conditions for 15~25 h; and / or The seed culture medium includes MRS liquid medium.

7. A fermentation broth, characterized in that, The fermentation broth is obtained by the fermentation method described in any one of claims 4 to 6.

8. A composition, characterized in that, It comprises Bifidobacterium bifidum 669 as described in claim 1, the fermentation broth as described in claim 2, the bacterial suspension as described in claim 3, or the fermentation broth as described in claim 7.

9. Use of the Bifidobacterium bifidum 669 of claim 1, the fermentation broth of claim 2, the bacterial suspension of claim 3, the fermentation broth of claim 7, or the composition of claim 8 in the preparation of foods, beverages, health products, feeds, food additives, and / or feed additives that contribute to antioxidant activity and / or enhance immunity.

10. Use of the Bifidobacterium bifidum 669 of claim 1, the fermentation broth of claim 2, the bacterial suspension of claim 3, the fermentation broth of claim 7, or the composition of claim 8 in the preparation of skin care products and cosmetics that help with antioxidation.

11. A food, beverage, health product, feed, food additive, and / or feed additive, characterized in that, The mixture comprises Bifidobacterium bifidum 669 as described in claim 1, the fermentation broth as described in claim 2, the bacterial suspension as described in claim 3, the fermentation broth as described in claim 7, or the composition as described in claim 8.

12. A skincare and / or cosmetic product, characterized in that, The mixture comprises Bifidobacterium bifidum 669 as described in claim 1, the fermentation broth as described in claim 2, the bacterial suspension as described in claim 3, the fermentation broth as described in claim 7, or the composition as described in claim 8.

13. A drug, characterized in that, The mixture comprises Bifidobacterium bifidum 669 as described in claim 1, the fermentation broth as described in claim 2, the bacterial suspension as described in claim 3, the fermentation broth as described in claim 7, or the composition as described in claim 8.

14. A method for producing dihydrocaffeic acid, characterized in that, This includes using Bifidobacterium bifidum 669 as described in claim 1 to ferment Guanshan cherry blossoms or their products.

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