Candida and application thereof in fermentation of camellia yunnanensis

Abstract

The invention belongs to the technical field of microorganisms, and discloses candida and application thereof in fermentation of camellia yunnanensis. Candida ralensis BTN-HB-M5 is obtained by separating wild edible fungi (white boletus) in Yunnan, the candidida ralensis BTN-HB-M5 is preserved in Guangdong Microbial Culture Collection Center on September 20, 2024, and the preservation number is GDMCC No: 65164. The camellia yunnanensis fermentation extract can be obtained by utilizing the candida sp. BTN-HB-M5 through proper culture and collection processes, and the whole fermentation process is green and efficient; the prepared camellia japonica fermentation extract is a product fermented only by using Candida railensis, can be used in food and daily chemical products, keeps natural active substances of camellia japonica to the greatest extent, is free of other additional components and irritant by-products, and can be applied to the daily chemical products. The camellia yunnanensis fermentation extract has excellent anti-oxidation, anti-saccharification and anti-aging effects, can obviously relieve skin aging indexes through oxidation resistance, and has a great application prospect in the field of daily chemical products.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and particularly relates to a Candida yeast and its application in the fermentation of Yunnan camellia. Background Art

[0002] Yunnan camellia is a plant of the genus Camellia in the family Theaceae native to China, mainly growing in montane broad-leaved forests or mixed forests at an altitude of 1500 - 2800 meters and widely distributed in Yunnan region. Records in medical classics such as "Yunnan Chinese Herbal Medicine" and "Chinese Medicine Dictionary" show that Yunnan camellia can be used for medicinal purposes and has many beneficial effects. At the same time, there are also some current studies on the efficacy activities of extracts from different parts of Yunnan camellia, such as Yunnan camellia leaves and flowers, using different biochemical extraction processes.

[0003] The application of microbial technology has gradually occupied a place in the development of raw materials for efficacy skin care products. A large number of studies have proven that fermentation can enhance the active substances in plant raw materials and strengthen the efficacy of plant raw materials. At the same time, microbial fermentation extraction also has the characteristics of energy conservation, emission reduction and pollution reduction. In some cases, it can also reduce the adverse toxic substances in plant raw materials. Therefore, some studies have also tried to use microbial fermentation methods for the biological extraction of camellia. At present, there are mainly two technical routes in the field of camellia fermentation: The first is compound strain fermentation. This process often requires the addition of exogenous nutrients (such as glucose, peptone, etc.) and a multi-stage fermentation process, such as Chinese Patent CN115569096B and Chinese Patent CN119318609A. The former (Chinese Patent CN115569096B) discloses a camellia fermentation filtrate and a fermentation process. By using lactic acid bacteria to ferment camellia and adding carbon source, vitamins, inorganic salts, fructooligosaccharides externally, a camellia fermentation filtrate rich in tea polyphenols, total flavonoids and small molecule natural nutrients is obtained. The latter (Chinese Patent CN119318609A) discloses a camellia ferment and its preparation method and application. First, camellia is enzymolyzed by lytic enzyme, and then fermented with a compound yeast seed solution (Logos yeast and Candida krusei), and carbon source, nitrogen source, alkaloids, phosphates, ammonium salts and acidity regulators are added externally to obtain a camellia ferment with the effects of repair, soothe and oil control. However, similar technologies lead to increased production costs due to the multi-stage fermentation process, and there is a risk of uncontrollable metabolites in the compound strains. The second technical route in the field of camellia fermentation is to ferment with a single common yeast, such as Chinese Patent CN116785361A. It ferments camellia with Saccharomyces cerevisiae to obtain a camellia fermentation filtrate with the effects of wrinkle reduction and sebum resistance. Although the technical process is simplified, its strain Saccharomyces cerevisiae is a widely used strain with well-known characteristics and is derived from a conventional industrial strain library, lacking uniqueness and innovation. In addition, the existing research on fermented camellia mainly focuses on effects such as moisturizing, antioxidant, anti-wrinkle and skin brightening, but lacks the exploration of anti-glycation and anti-aging effects at the cellular level.

[0004] As can be seen from the above, the current camellia fermentation process patents do not involve functional strains from special ecological environments, lack strains that can specifically enhance the efficacy characteristics of the unique Yunnan camellia, and there is no process technology to enhance the anti-glycation and cellular aging synergistic mechanism of Yunnan camellia. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the present invention provides a special Candida strain isolated and purified from wild large edible fungi in Yunnan ( Candida railenensis), and designed a simplified Yunnan Camellia fermentation process according to the biological characteristics of the strain, and the fermentation filtrate obtained by fermenting Yunnan Camellia with the Candida strain. The present invention aims to provide a safe, environmentally friendly, economical, and beneficial (anti-glycation and cell aging) Yunnan Camellia microbial extraction method, so as to achieve the extraction of a filtrate rich in Yunnan Camellia active ingredients by microbial fermentation, and enrich the original functional effects of Yunnan Camellia.

[0006] The innovation of the present invention is that it provides for the first time a Candida strain isolated and purified from a large wild edible fungus in Yunnan ( Candida railenensis ), and designed a targeted bio-fermentation process based on the strain specificity; under this fermentation process, this Candida strain can enhance the original antioxidant, anti-glycation and anti-aging activities of Camellia yunnanensis, and the final Camellia yunnanensis fermentation extract can be used as a functional raw material in the cosmetics field.

[0007] The present invention provides the following technical solutions: In the first aspect, the present invention provides a Candida isolated from a wild edible mushroom (Boletus edulis) in Yunnan, the strain of Candida is numbered BTN-HB-M5 (hereinafter referred to as M5), and is classified as Candida railenensis , the deposit date is September 20, 2024. The deposit unit is Guangdong Microbiological Culture Collection Center located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit number is GDMCC No: 65164.

[0008] Preferably, the ITS sequence of Candida is shown in SEQ NO: 1 in the sequence listing.

[0009] Preferably, in the present invention, the specific method for obtaining the Candida M5 strain is as follows: (1) Select wild white boletus samples grown in Yunnan, wash them, soak them in sterile water at 4°C for 18 h~24 h, apply appropriate soaking liquid on YPD solid culture medium, culture them at 25°C~37°C for 24 h~30 h, pick out colonies of different morphologies on the YPD solid culture medium, and purify them by streaking on the surface of the YPD solid culture medium; (2) Observe the single colonies on different plates after purification under a microscope, select different strains with yeast morphology for identification, and preserve the selected strains.

[0010] In a second aspect, the present invention provides a fermented Yunnan Camellia syrup obtained by fermenting Yunnan Camellia using Candida with a preservation number of GDMCC No: 65164 as a fermentation bacterium.

[0011] Thirdly, the present invention provides a fermented extract of Camellia reticulata Lindl., using Candida sp. with the preservation number of GDMCC No: 65164 as the fermentation bacterium to ferment Camellia reticulata Lindl. to obtain a fermented Camellia reticulata Lindl. pulp, and filtering the fermented Camellia reticulata Lindl. pulp to obtain the fermented extract of Camellia reticulata Lindl. This fermented extract of Camellia reticulata Lindl. is also called the fermented filtrate of Camellia reticulata Lindl. This fermented extract of Camellia reticulata Lindl. is a single Candida sp. fermentation product, which can be used in food and daily chemical products, further enhancing and enriching the skin care effects of Camellia reticulata Lindl., without adding other extra components and having no irritating by-products, and can be applied in daily chemical products. This fermented extract of Camellia reticulata Lindl. has superior antioxidant, anti-glycation and anti-aging effects, can significantly relieve skin aging indicators through antioxidant action, and has great application prospects in the field of daily chemical products.

[0012] Fourthly, the present invention provides a preparation method of a fermented extract of Camellia reticulata Lindl., comprising the following steps: Step S1, inoculating Candida sp. with the preservation number of GDMCC No: 65164 into a YPD liquid medium for activation to obtain a strain seed solution of Candida sp. M5; Step S2, inoculating the strain seed solution into a Camellia reticulata Lindl. fermentation medium for fermentation extraction at an inoculation amount of 1% - 3% to obtain a fermented Camellia reticulata Lindl. pulp; Step S3, centrifuging the fermented Camellia reticulata Lindl. pulp first to remove insoluble substances such as solid residues and bacterial cells, taking the supernatant after centrifugation, and filtering the supernatant to obtain a fermented extract of Camellia reticulata Lindl. prepared by microbial means.

[0013] Preferably, in step S1, the preserved Candida sp. M5 is streaked on a YPD plate and cultured at 25°C - 37°C for 24 h to complete strain resuscitation; single colonies are picked from the resuscitated Candida sp. M5 plate and inoculated into a YPD liquid medium for activation, and cultured at 25°C - 30°C for 18 h - 24 h during activation, and activated for 1 - 2 generations to obtain a strain seed solution of Candida sp. M5.

[0014] Preferably, in step S2, the fermentation temperature is 25°C - 37°C, the fermentation time is 24 h - 48 h, and the rotation speed of the shaker culture is set at 150 rpm - 200 rpm.

[0015] Preferably, in step S3, the centrifugation conditions are 5000 rpm - 8000 rpm, and the centrifugation time is 15 min - 30 min; then the supernatant obtained by centrifugation is filtered with a 0.22 μm filter membrane to obtain a fermented extract of Camellia reticulata Lindl.

[0016] Preferably, the preparation method of the Camellia reticulata Lindl. fermentation medium is as follows: Select Camellia reticulata Lindl. as the raw material and perform comminution treatment; then add Camellia reticulata Lindl. powder to ultrapure water at an addition amount of 5% by mass, and sterilize it with steam at 121 °C for 20 min to obtain it.

[0017] Fifthly, the present invention provides the above-mentioned Camellia reticulata Lindl. fermentation extract as an antioxidant, anti-glycation, and anti-aging component in daily chemical products.

[0018] Sixthly, the present invention provides a daily chemical product containing the above-mentioned Camellia reticulata Lindl. fermentation extract.

[0019] Preferably, the addition amount of the Camellia reticulata Lindl. fermentation extract in the daily chemical product is 3% by volume ratio. Through experimental verification, it is safe, non-toxic and has significant effects at this addition amount.

[0020] Preferably, the daily chemical product can be of various types, such as shampoo, hair care products, cosmetics, etc.

[0021] Preferably, the cosmetics can be lotion, skin care cream, skin care lotion, skin care essence, eye cream, facial mask, makeup remover oil, facial cleanser, body wash, shampoo, etc.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: 1. The present invention has screened a yeast strain suitable for fermenting Camellia reticulata Lindl., Candida sp. BTN-HB-M5 (abbreviated as M5). Using this strain to carry out biological fermentation on Camellia reticulata Lindl., this strain can specifically enhance the efficacy of Camellia reticulata Lindl., filling the gap in the current Camellia reticulata Lindl. fermentation extraction technology that does not involve strains from special ecological environments.

[0023] 2. The present invention can obtain the Camellia reticulata Lindl. fermentation extract (fermentation filtrate) by using Candida sp. BTN-HB-M5 through a suitable culture and collection process. The overall fermentation process is green and efficient; the prepared Camellia reticulata Lindl. fermentation extract has the ability to scavenge DPPH free radicals and ABTS free radicals in vitro, inhibit the activity of hyaluronidase, and inhibit the generation of AGEs, and has good in vitro anti-glycation, antioxidant, and anti-aging efficacy activities. At the same time, it has safety and can be widely used in the cosmetics field.

[0024] 3. After using Candida sp. M5 to ferment Camellia reticulata Lindl., the present invention has improved the efficacy of Camellia reticulata Lindl. in terms of antioxidant, anti-glycation, and anti-aging, so the efficacy of its use in daily chemical products can be enhanced, making the product have higher performance: 1) Compared with the unfermented extract of Camellia reticulata Lindl., the DPPH scavenging rate of the Camellia reticulata Lindl. fermentation extract prepared with Candida sp. M5 has increased by 11.96%; 2) Compared with the unfermented extract of Camellia reticulata Lindl., the ABTS scavenging rate of the fermented extract of Camellia reticulata Lindl. prepared with Candida M5 increased by 6.46%; 3) Compared with the unfermented extract of Camellia reticulata Lindl., the hyaluronidase inhibition rate of the fermented extract of Camellia reticulata Lindl. prepared with Candida M5 increased by 32.06%; 4) Compared with the unfermented extract of Camellia reticulata Lindl., the AGEs inhibition rate of the fermented extract of Camellia reticulata Lindl. prepared with Candida M5 increased by 13.20%; 5) Compared with the unfermented extract of Camellia reticulata Lindl., the fermented extract of Camellia reticulata Lindl. prepared with Candida M5 had a more significant growth-promoting effect on skin fibroblasts. After intervention with 3% (v / v) of the fermented extract of Camellia reticulata Lindl. prepared with Candida M5, the viability of skin fibroblasts reached 105.99%; 6) At an addition amount of 3%, compared with the unfermented extract of Camellia reticulata Lindl., the expression level of MMP3 mRNA in skin fibroblasts in the H2O2 aging model was significantly reduced by 46.53% for the fermented extract of Camellia reticulata Lindl. prepared with Candida M5.

[0025] The preparation method of the present invention is simple to operate, and the operation process is economical, green, and pollution-free. All the devices used are easy to clean. At the same time, the fermentation method enhances the efficacy and activity of the extract of Camellia reticulata Lindl., and it is also non-toxic and has good performance in in vitro skin cell experiments. These characteristics indicate that this technology is not only applicable to large-scale industrial production but also has great potential in the development of raw materials for skin care products. Brief Description of the Drawings

[0026] Figure 1 It is the result diagram of the fluorescence AGEs inhibition experiment of Test Example 4 of the present invention; Figure 2 It is the result diagram of the evaluation experiment of the in vitro HFF cell proliferation of Test Example 5 of the present invention; Figure 3 It is the result diagram of the expression of skin fibroblast senescence-related genes of Test Example 6 of the present invention. Detailed Embodiments

[0027] The technical solution of the present invention will be further described below in conjunction with the drawings and through specific embodiments. However, the following examples are only simple examples of the present invention and do not represent or limit the scope of the protection of the rights of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0028] Unless otherwise specified, the raw materials and materials used in the embodiments of the present invention are purchased through general commercial channels.

[0029] The source information of the relevant raw materials, materials, and instruments involved in the following examples or comparative examples is as follows: Yeast M9: Self-developed, which has been described in the examples; Yeast M6: Self-developed, as described in the examples; Bacillus L25: Self-developed, as described in the examples; Lactic acid bacterium M4: Self-developed, as described in the examples; YPD liquid medium: Qingdao Haibo Biotech; DPPH (1,1-diphenyl-2-picrylhydrazyl), Aladdin; ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt), Aladdin; Hyaluronidase, Yuanye Bio-Technology; Sodium hyaluronate, Aladdin; Human foreskin fibroblasts (HFF cells), purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences; DMEM culture medium, Eva cell; Fetal bovine serum, Gibco; Penicillin-Streptomycin (10,000 U / mL) (hereinafter referred to as: double antibody); Complete medium: DMEM medium supplemented with 10% (v / v) FBS fetal bovine serum and 1% (v / v) double antibody; MTT test solution, Aladdin; Sample solvent, sterile water.

[0030] <Example 1> Screening and identification of strains This example provides a screening method for Candida ( Candida railenensis ) M5 derived from wild edible mushrooms (Boletus edulis) in Yunnan. The specific steps are as follows: (1) Select wild Boletus edulis samples growing in Yunnan. After cleaning, soak them in sterile water at 4°C for 18 h to 24 h to obtain the soaking solution; then pipette an appropriate amount of the soaking solution and spread it on the YPD solid medium. Incubate at 25°C to 37°C for 24 h to 30 h, and then pick out colonies with different morphologies on the YPD solid medium. Streak and purify on the surface of the YPD solid medium, and perform continuous subculture for at least 2 times to obtain purified colonies; (2) Observe the single colonies on different purified plates under a microscope, and select different strains with yeast morphology for screening and identification. After identification, a strain of Candida ( Candida railenensis), named BTN-HB-M5 (hereinafter referred to as M5 for short). At the same time, the selected strain was preserved. The preservation unit is the Guangdong Microbial Culture Collection Center located on the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou. The preservation number is GDMCC No: 65164. The screening and verification method for this step is to sequence the obtained yeast, and the results show that the ITS sequence of this strain is as shown in the following sequence list SEQ NO:1: CCTGCGGAAGGATCATTACAGTATTCTTTTGCCAGCGCTTAATTGCGCGGCGAAAAACCTTACACACTATGTTTTTTTAATTTGAAACTATTGCTTTGGTCTGGCTTAgAAATAGGTTGGGCCAAAGGTTTTATCAAAACTTCAATATTTATTATTGAATTGTTATTTTTAATTTTATGTCAATTTGTTGATTAATATCAAAAATCTTCAAAACTTTCAACAACGGATCTCTTGGTTCTCGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATATGAATTGCAGATTTTCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTTTGGTATTCCAAAGGGCATGCCTGTTTGAGCGTCATTTCTCTCTCAAATCTTCGGATTTGGTTTTGAGTGATACTCTTAGTCAGACTAAGCGTTTGCTTGAAATGTATTGGCATGAGTGGTACTAGATAGTGCTGAACTGTTTTCAATGTATTAGGTTTATCCAACTCATTGACCAGTAAAGTATTTGTTTATTACACAGGCTCGGCCTTACAACAACAAACAAAGTTTGACCTCAAATCAGGTAGGACTACCCGCTGAACTTAAGCATATC <Example 2> This example provides an unfermented extract of Camellia reticulata Lindl. f. simplex Sealy, and the specific preparation method is as follows: Select dried flowers of Camellia reticulata Lindl. f. simplex Sealy as raw materials and crush them; add Camellia reticulata Lindl. f. simplex Sealy flower powder into ultrapure water at an addition amount of 5% by mass, and sterilize it at 121 °C with high-pressure steam for 20 min for use as a fermentation medium for Camellia reticulata Lindl. f. simplex Sealy.

[0031] Centrifuge the fermented medium of Camellia reticulata Lindl. at a rotational speed of 5000 rpm to 8000 rpm for 15 min to 30 min; then filter the supernatant through a 0.22 μm filter membrane to obtain an aqueous extract of unfermented Camellia reticulata Lindl. under high temperature and high pressure, simply referred to as the unfermented extract of Camellia reticulata Lindl.

[0032] <Example 3> This example provides a fermented extract of Camellia reticulata Lindl. (fermented filtrate of Camellia reticulata Lindl.), and its preparation method is as follows: Step S0, prepare the fermented medium of Camellia reticulata Lindl. according to Example 2.

[0033] Step S1, streak Candida M5 preserved in Example 1 on a YPD plate and culture it at 25°C to 30°C for 24 h to complete strain recovery; then pick a single colony from the recovered Candida M5 plate and inoculate it into a YPD liquid medium for activation. During activation, culture it at 25°C to 30°C for 24 h and activate it for 2 generations to obtain a strain seed solution of Candida M5.

[0034] Step S2, inoculate the strain seed solution of Candida M5 into the fermented medium of Camellia reticulata Lindl. at 1% (v / v) for fermentation to obtain the fermented Camellia reticulata Lindl. pulp. The fermentation temperature is 25°C and the fermentation time is 24 h. At the same time, set the rotational speed of the shaker culture to 150 rpm.

[0035] Step S3, first centrifuge the fermented Camellia reticulata Lindl. pulp to remove insoluble substances such as solid residues and bacterial cells. The centrifugation conditions are 5000 - 8000 rpm and the centrifugation time is 15 - 30 min; then filter the supernatant obtained by centrifugation through a 0.22 μm filter membrane to obtain a fermented extract of Camellia reticulata Lindl. prepared by microbial means.

[0036] <Example 4> This example provides a fermented extract of Camellia reticulata Lindl. (fermented filtrate of Camellia reticulata Lindl.). The difference in its preparation method from Example 3 lies in modifying the inoculation amount, fermentation temperature, fermentation time, and shaker rotational speed of the seed solution during the fermentation of Camellia reticulata Lindl. in Step S2. Other components, dosages, and preparation methods are the same as those in Example 3. After modification, Step S2 is as follows: Inoculate the strain seed solution of Candida M5 into the fermented medium of Camellia reticulata Lindl. at 2% (v / v) for fermentation. The fermentation temperature is 30°C and the fermentation time is 48 h. At the same time, set the rotational speed of the shaker culture to 200 rpm.

[0037] <Example 5> This example provides a fermented extract of Camellia reticulata Lindl. (fermented filtrate of Camellia reticulata Lindl.). The difference in its preparation method from that of Example 4 lies in modifying the inoculation amount of the seed liquid and the fermentation temperature during the fermentation of Camellia reticulata Lindl. The other components, dosages, and preparation methods are the same as those in Example 3. After modification, step S2 is as follows: The strain seed liquid of Candida sp. M5 was inoculated into the Camellia reticulata Lindl. fermentation medium at 3% (v / v) for fermentation. The fermentation temperature was 37 °C, the fermentation time was 48 h, and the rotation speed of the shaker culture was set at 200 rpm.

[0038] <Comparative Example 1> This comparative example provides a fermented extract of Camellia reticulata Lindl. (fermented filtrate of Camellia reticulata Lindl.). The difference in its preparation method from that of Example 4 lies in changing the strain used. Candida sp. M5 from Yunnan wild edible fungi (Boletus edulis) was replaced with a yeast M9 independently isolated from Yunnan wild edible fungi (Termitomyces albuminosus) by this laboratory. The other components, dosages, and preparation methods are the same as those in Example 4. The isolation method of yeast M9 is the same as that in Example 1.

[0039] <Comparative Example 2> This comparative example provides a fermented extract of Camellia reticulata Lindl. (fermented filtrate of Camellia reticulata Lindl.). The difference in its preparation method from that of Example 4 lies in changing the strain used; Candida sp. M5 from Yunnan wild edible fungi (Boletus edulis) was replaced with a yeast M6 independently isolated from Yunnan wild edible fungi (Boletus edulis) by this laboratory. The other components, dosages, and preparation methods are the same as those in Example 4. The isolation method of yeast M6 is the same as that in Example 1.

[0040] <Comparative Example 3> This comparative example provides a fermented extract of Camellia reticulata Lindl. (fermented filtrate of Camellia reticulata Lindl.). The difference in its preparation method from that of Example 5 lies in changing the strain used; Candida sp. M5 from Yunnan wild edible fungi (Boletus edulis) was replaced with a Bacillus sp. L25 independently isolated from Cypripedium wardianum in Haba Snow Mountain, Yunnan. The other components, dosages, and preparation methods are the same as those in Example 5. The isolation method of Bacillus sp. L25 is the same as that in Example 1, except that the medium was changed to LB medium and the culture temperature was changed to 37 °C.

[0041] <Comparative Example 4> This comparative example provides a fermented extract of Camellia reticulata Lindl. (fermented filtrate of Camellia reticulata Lindl.), and the difference in its preparation method from that of Example 5 lies in changing the strain used; Candida sp. M5 derived from Yunnan wild edible fungi (Boletus queletii Schulz.) is replaced with a Lactobacillus sp. M4 independently isolated from Yunnan wild edible fungi (Boletus queletii Schulz.) by this laboratory. Other components, dosages and preparation methods are the same as those in Example 5. The isolation method of Lactobacillus sp. M4 is the same as that in Example 1 except that the culture medium is changed to MRS medium and the culture temperature is changed to 37°C.

[0042] <Test Example 1> DPPH free radical scavenging experiment Referring to "Cosmetics - Free Radical (DPPH) Scavenging Experiment Method (T / SHRH006-2018)", the scavenging rates (%) of DPPH free radicals of the fermented extracts of Camellia reticulata Lindl., unfermented extracts of Camellia reticulata Lindl. and the sample blank control group prepared in Examples 2-5 and Comparative Examples 1-4 were detected respectively.

[0043] (1) Drug preparation: Weigh 10 mg of DPPH, dissolve it by ultrasonic wave with absolute ethanol and make the volume up to 100 mL for standby (0.1 mg / mL), store it in the dark, and obtain the DPPH solution.

[0044] (2) Experimental steps: Set up the sample group, sample blank control group, control group and control blank control group, with 3 replicates in each group. In the sample group, take 150 μL of the fermentation product filtrates of Examples 2-5 and Comparative Examples 1-4 in a 96-well plate as the test samples respectively, then add the test samples and the DPPH solution in equal volume at a ratio of 1:1, react at room temperature in the dark for 30 min, shake well, and measure the absorbance at a wavelength of 517 nm; in the sample blank control group, use absolute ethanol in equal volume to replace the DPPH solution, and other conditions are the same as those in the sample group; in the control group, use ultrapure water in equal volume to replace the fermentation product filtrate, and other conditions are the same as those in the sample group; in the control blank control group, use ultrapure water in equal volume to replace the fermentation product filtrate and use absolute ethanol to replace the DPPH solution, and other conditions are the same as those in the sample group.

[0045] (3) The calculation method of the DPPH (1,1-diphenyl-2-trinitrophenylhydrazine) scavenging rate is as follows: Scavenging rate (%) = [1 - (As - As0) / (Ac - Ac0)] × 100%; In the formula: As is the measured absorbance of the sample group; As0 is the measured absorbance of the sample blank group; Ac is the measured absorbance of the control group; Ac0 is the measured absorbance of the control blank group.

[0046] The test results of DPPH free radical scavenging are shown in Table 1.

[0047] <Test Example 2> ABTS (2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) radical scavenging experiment (1) Preparation of reagents: Weigh accurately 0.0284 g of ABTS and dissolve it in 10 mL of distilled water. Separately, weigh accurately 0.0067 g of potassium persulfate and dissolve it in 10 mL of distilled water. Mix the two solutions in a volume ratio of 1:1, and then place the mixture in the dark at room temperature for 16 h to 24 h to obtain the ABTS stock solution. When conducting the experiment, dilute the ABTS stock solution with 15 times its volume of distilled water to make its absorbance (λ = 734 nm) within the range of 0.7 ± 0.1, thus obtaining the ABTS working solution.

[0048] (2) Experimental procedure: First, add 180 μL of the ABTS working solution to a 96-well plate, and then add 20 μL of the sample to be tested to each well. After reacting for 180 min, shake well and measure the absorbance at 724 nm.

[0049] The samples to be tested are the fermented extracts of Camellia reticulata Lindl., the unfermented extracts of Camellia reticulata Lindl., and the blank control group (ultrapure water) prepared in Examples 2 - 5 and Comparative Examples 1 - 4.

[0050] (3) The calculation method for the ABTS scavenging rate is as follows: Scavenging rate (%) = 1 - As / Ac × 100%; Where: As is the measured absorbance of the sample group; Ac is the measured absorbance of the blank control group.

[0051] The test results of ABTS radical scavenging are shown in Table 1.

[0052] <Test Example 3> Hyaluronidase inhibition experiment (1) Preparation of reagents: Dissolve hyaluronidase with 0.1 mM acetate buffer to prepare a concentration of 0.25 mg / mL; Dissolve sodium hyaluronate with 0.1 mM acetate buffer to prepare a concentration of 1 mg / mL; Take 0.6 g of calcium chloride and add ultrapure water to make up to 50 g to prepare a 12.5 mM calcium chloride solution; Take 0.8 g of sodium hydroxide and add ultrapure water to 50 g to prepare a 0.4 M sodium hydroxide solution; One day in advance, take 6.1 g of potassium borate and add water to 50 g to prepare a 4 M potassium borate solution; Take 0.8 g of p-dimethylaminobenzaldehyde, add it to 20 ml of acetic acid, and then add 5 ml of concentrated hydrochloric acid (store in the dark) to obtain the color reagent.

[0053] (2) Set up groups: Sample group (Group A): The samples to be tested are the fermented extracts and unfermented extracts of Camellia reticulata Lindl. prepared in Examples 2 - 5 and Comparative Examples 1 - 4. In this group, the samples to be tested, hyaluronidase, and sodium hyaluronate will be added simultaneously. Sample blank group (Group B): The samples to be tested are the fermented extracts and unfermented extracts of Camellia reticulata Lindl. prepared in Examples 2 - 5 and Comparative Examples 1 - 4. In this group, only the samples to be tested are added, without adding hyaluronidase and sodium hyaluronate. Control group (Group C): The sample to be tested is the sample solvent. In this group, the sample solvent, hyaluronidase, and sodium hyaluronate are added. Control blank group (Group D): The sample to be tested is the acetate buffer solution. In this group, only the sample solvent and acetate buffer solution are added.

[0054] (3) Experimental steps: Step (a): According to the grouping requirements, add 0.5 mL of the samples to be tested to Groups A, B, C, and D respectively; then add 0.5 mL of acetate buffer solution to Groups A, B, and C, and add 0.5 mL of sample solvent to Group D, and incubate at 37 °C for 20 min.

[0055] Step (b): After step (a) is completed, add 100 μL of calcium chloride to each group and incubate at 37 °C for 20 min.

[0056] Step (c): After step (b) is completed, add 0.5 mL of sodium hyaluronate to Groups A and B respectively, and add 0.5 mL of acetate buffer solution to Groups C and D, and incubate at 37 °C for 40 min.

[0057] Step (d): After step (c) is completed, add 100 μL of sodium hydroxide and 100 μL of potassium borate to each group, then place in a water bath at 85 °C for 5 min, then in an ice bath for 2 min, and finally leave at room temperature for 5 min to obtain the reaction solution.

[0058] Step (e): After the reaction solution obtained in step (d) is shaken well, take 100 μL from each and add them to a 96 - well plate, and then add 100 μL of the chromogenic agent to each well.

[0059] Step (f): Quickly place the 96 - well plate in an enzyme - linked immunosorbent assay (ELISA) reader and oscillate at 37 °C for 1 minute. 10 minutes after adding the chromogenic agent, measure the absorbance at 585 nm.

[0060] (4) The calculation method of the hyaluronidase (HAS) inhibition rate is: HAS inhibition rate (%) = [(C - D)-(A - B)] / (C - D) × 100% In the formula: A, B, C, and D are the absorbances measured at 585 nm for Groups A, B, C, and D respectively.

[0061] The results of the HAS inhibition rate test are shown in Table 1.

[0062] Table 1. Test results of test cases 1 to 3

[0063] As can be seen from Table 1, the fermented Camellia japonica extract obtained by fermentation with Candida BTN-HB-M5 (Camellia japonica fermentation filtrate, Examples 3-5) has better DPPH scavenging ability, ABTS scavenging ability and HAS inhibition ability than the unfermented Camellia japonica extract (Example 2), indicating that the fermentation method using Candida BTN-HB-M5 can enhance the in vitro antioxidant and anti-aging effects of Camellia japonica extract.

[0064] Further comparing Examples 3-5 with the Yunnan Camellia fermentation extracts of other self-developed strains (Yunnan Camellia fermentation filtrate, Comparative Examples 1-4), it was found that although the Yunnan Camellia fermented with other strains had a slightly improved in vitro antioxidant and anti-aging effect compared with the unfermented Yunnan Camellia, the improvement effect of the fermentation with other strains was not as good as the Yunnan Camellia fermentation filtrate of Candida BTN-HB-M5; This indicates that Candida BTN-HB-M5 is a strain that is more suitable for fermentation to enhance the efficacy of Yunnan Camellia.

[0065] From the analysis of the efficacy results of the fermentation products obtained by different processes of preparing the Yunnan Camellia Fermentation Extract (Yunnan Camellia Fermentation Filtrate) by Candida BTN-HB-M5 in Examples 3-5 in Table 1, it can be seen that the Yunnan Camellia Fermentation Extract (Yunnan Camellia Fermentation Filtrate) obtained by the fermentation process of Example 4 has the best comprehensive antioxidant and anti-aging effects, and is also better than the activity of the Yunnan Camellia Fermentation Extract (Yunnan Camellia Fermentation Filtrate, Comparative Examples 1-4) obtained by fermentation of other strains. Therefore, it is the best choice to select Candida BTN-HB-M5 to prepare the Yunnan Camellia Fermentation Extract under the fermentation process conditions of Example 4.

[0066] <Test Example 4> Fluorescence AGEs inhibition experiment (1) Drug preparation: prepare 0.2 mol / L PBS phosphate buffer (pH=7.4); weigh bovine serum albumin and dissolve it in PBS buffer to make a 0.8 mg / mL BSA solution; weigh D-glucose and dissolve it in PBS buffer to make a 200 mM Glu solution.

[0067] (2) Test samples: the unfermented extract of Camellia japonica in Example 2 and the fermented extract of Camellia japonica prepared in Example 4; the blank control group was replaced with PBS buffer.

[0068] (3)Experimental procedure: Add 1.0 mL of each of the above-mentioned test samples, BSA solution, and Glu solution into a sterile centrifuge tube, and then add 1.0 mL of PBS buffer and mix well. Measure the initial fluorescence value of each group of test samples at an excitation wavelength (Ex) of 370 nm and an emission wavelength (Em) of 420 nm. The fluorescence value of the sample group is denoted as Fs0, and that of the blank control group is denoted as Fc0.

[0069] After incubation in a metal bath at 60 °C for 24 h, measure the fluorescence value Fs of the advanced glycation end products at an excitation wavelength (Ex) of 370 nm and an emission wavelength (Em) of 420 nm, which represents the content of total fluorescent AGEs. The fluorescence value of the sample group is denoted as Fs, and that of the blank control group is denoted as Fc.

[0070] (4)Fluorescent AGEs inhibition rate (%) = [1 - (Fs - Fs0) / (Fc - Fc0)] × 100% The test results of the fluorescent AGEs inhibition rate are as Figure 1 shown. As can be seen from Figure 1 this, the fluorescent AGEs inhibition rate of the unfermented extract of Camellia reticulata Lindl. is 45.15%, and that of the fermented extract of Camellia reticulata Lindl. is 51.11%, indicating that the fermentation of Camellia reticulata Lindl. by Candida BTN-HB-M5 improves the anti-glycation performance of Camellia reticulata Lindl.

[0071] <Test Example 5> Evaluation experiment on the proliferation of HFF cells in vitro (1)Cells and culture medium: Select HFF cells from the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, and culture them under the condition of DMEM culture medium (10% fetal bovine serum).

[0072] (2)Test samples: The unfermented extract of Camellia reticulata Lindl. in Example 2 and the fermented extract of Camellia reticulata Lindl. prepared in Example 4, with the blank control group replaced by PBS buffer.

[0073] (3)Effect of the test substance on the proliferation of HFF cells: Select HFF cells with good morphology and in the logarithmic growth phase for inoculation in a 96-well plate and incubate in an incubator for 24 h. The test substances are all at a concentration of 3% (v / v), diluted with complete culture medium, and 6 replicate wells are set for each concentration gradient and co-incubated in an incubator for 24 h. Remove the original supernatant, add MTT test solution to each well, incubate in an incubator for 2 h, measure the absorbance at an absorption wavelength of 570 nm with an enzyme-labeled instrument, and calculate the cell viability. The results are as Figure 2 shown. Figure 2 In this figure, * indicates that the other sample groups are compared with the blank control group, * indicates p < 0.05, and ** indicates p < 0.01. As can be seen from Figure 2It can be seen that the 3% fermented extract of Camellia reticulata Lindl. has more significant proliferation-promoting activity than the 3% unfermented extract of Camellia reticulata Lindl.

[0074] <Test Example 6> Anti-aging efficacy evaluation experiment of in vitro HFF cell H2O2-induced aging model (1) Cells and culture medium: HFF cells from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences were selected and cultured under the condition of DMEM culture medium (10% FBS fetal bovine serum).

[0075] (2) Samples to be tested: The unfermented extract of Camellia reticulata Lindl. in Example 2 (experimental concentration of 3% (v / v)), the fermented extract of Camellia reticulata Lindl. prepared in Example 4 (experimental concentration of 3% (v / v)), and the blank control group was replaced with PBS buffer.

[0076] (3) Select HFF cells in the logarithmic growth phase with good morphology and inoculate them into a 6-well plate, set the inoculation concentration at 2.5×10 5 cells / well and incubate in an incubator for 24 h.

[0077] Set up a sample group, a control group and a blank group, with 3 replicate wells in each group; the sample group contains two groups. The first group is added with complete medium containing 3% unfermented extract of Camellia reticulata Lindl. in Example 2, and the second group is added with DMEM complete medium containing 3% fermented extract of Camellia reticulata Lindl. prepared in Example 4. After treating the cells in both groups for 4 h, 400 μM H2O2 solution was added and induced for 2 h. Then, DMEM complete medium containing 3% filtrate of the fermentation product in Example 3 was added again, and the incubation was continued in a 37°C, 5% CO2 incubator for 24 h; in the control group, the filtrate of the fermentation product was not added, and the other conditions were the same as those in the sample group; in the blank group, the filtrate of the fermentation product was not added, and H2O2 solution was not used for induction, and the other conditions were the same as those in the sample group.

[0078] (3) After the incubation, RNA was extracted and its content was measured, cDNA was reverse transcribed, and finally a fluorescence quantitative PCR experiment was carried out. The internal reference gene was β-actin, and the method was used for result determination. The experimental results are as Figure 3 shown, where indicates p < 0.001 compared with the blank, and ** indicates p < 0.01 compared with H2O2.

[0079] (4) Expression results of skin fibroblast senescence-related genes: MMP3, as the core mediator of matrix degradation, its overexpression is related to multiple aging mechanisms such as photoaging and glycosylation aging. From Figure 3It can be seen that the mRNA level of MMP3 in HFF cells increases under the induction of H2O2. However, after the intervention of the unfermented extract and the fermented extract of Camellia reticulata Lindl. at a concentration of 3%, the expression level of matrix metalloproteinase decreases and aging is alleviated. Moreover, the anti-aging ability of the fermented extract of Camellia reticulata Lindl. by Candida sp. M5 is improved, and the mRNA level of MMP3 is significantly lower compared with the group only induced by H2O2. Considering the close relationship between glycation and oxidative damage, this result fully demonstrates that the fermentation by Candida sp. M5 not only enhances the anti-aging ability of Camellia reticulata Lindl. and is effective in reducing the expression of MMP3, but also further exhibits a powerful anti-glycation and anti-aging effect by alleviating glycation damage under oxidative stress, which echoes the improvement of the anti-glycation function of Camellia reticulata Lindl. by Candida sp. M5 fermentation in vitro.

[0080] The applicant declares that the above is only the specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by any person skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A Candida yeast strain ( Candida railenensis ), characterized in that It was deposited in the Guangdong Microbial Culture Collection Center on September 20, 2024, with the deposit number GDMCC No: 65164.

2. The Candida as claimed in claim 1, wherein, The ITS sequence of the Candida is shown in Sequence Listing SEQ NO:

1.

3. A fermented Camellia reticulata Lindl. flower pulp, characterized in that, It is obtained by fermenting Camellia reticulata Lindl. with the Candida with the deposit number GDMCC No: 65164 as the fermenting bacterium.

4. A fermented extract of Camellia reticulata Lindl., characterized in that, Using the Candida with the deposit number GDMCC No: 65164 as the fermenting bacterium to ferment Camellia reticulata Lindl. to obtain the fermented Camellia reticulata Lindl. pulp, and filtering the fermented Camellia reticulata Lindl. pulp to obtain the Camellia reticulata Lindl. fermentation extract.

5. The preparation method of the fermented extract of Camellia reticulata Lindl. as described in claim 4, wherein, It includes the following steps: Step S1, inoculating the Candida with the deposit number GDMCC No: 65164 into the YPD liquid medium for activation to obtain the strain seed liquid of Candida M5; Step S2, inoculating the strain seed liquid with an inoculation amount of 1% - 3% into the Camellia reticulata Lindl. fermentation medium for fermentation extraction to obtain the fermented Camellia reticulata Lindl. pulp; Step S3, centrifuging the fermented Camellia reticulata Lindl. pulp and taking the supernatant, and filtering the supernatant to obtain the Camellia reticulata Lindl. fermentation extract.

6. The preparation method of the fermented extract of Camellia reticulata Lindl. as described in claim 5, wherein, In step S1, during activation, it is cultured at 25°C - 30°C for 18 h - 24 h, and activated for 1 - 2 generations to obtain the strain seed liquid of Candida M5; In step S2, the fermentation temperature is 25°C - 37°C, the fermentation time is 24 h - 48 h, and at the same time, the rotation speed of the shaker culture is set to 150 rpm - 200 rpm; In step S3, the centrifugation conditions are 5000 rpm - 8000 rpm, and the centrifugation time is 15 min - 30 min; then the supernatant obtained by centrifugation is filtered with a 0.22 μm filter membrane to obtain the Camellia reticulata Lindl. fermentation extract.

7. The preparation method of the fermented extract of Camellia reticulata Lindl. as claimed in claim 5, characterized in that The preparation method of the Camellia reticulata Lindl. fermentation medium is: selecting Camellia reticulata Lindl. as the raw material and performing comminution treatment; then adding Camellia reticulata Lindl. powder into ultrapure water with an addition amount of 5% by mass percentage, and sterilizing with steam at 121°C for 20 min to obtain it.

8. Use of the fermented extract of Camellia reticulata Lindl. as described in claim 4 in a daily chemical product, characterized in that, The Camellia reticulata Lindl. fermentation extract is used as an antioxidant, anti - glycation and anti - aging ingredient in daily chemical products.

9. A daily chemical product, characterized in that, It contains the Camellia reticulata Lindl. fermentation extract as described in claim 4.

10. A daily chemical product according to claim 9, characterized in that, The addition amount of the Camellia reticulata Lindl. fermentation extract in daily chemical products is 3% by volume ratio.

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