Method for increasing content of effective components of rose by using edible fungus fermentation

Abstract

The application provides a method for increasing the content of effective components of rose by using edible fungus fermentation, and belongs to the technical field of biological fermentation.The method comprises the following steps: mixing rose powder, corn starch, glucose, potassium dihydrogen phosphate and water, adjusting the pH value, sterilizing, and obtaining a rose culture medium; inoculating the rose culture medium with a chrysophic acid fungus liquid, adding L-cysteine hydrochloride after 20-30h of fermentation culture, continuing the fermentation culture for 5-7d, and obtaining a rose fermentation product.The method can significantly increase the content of quercetin, quercetin-3-O-rutinose and polyphenol in the fermentation product by inoculating the rose with the chrysophic acid fungus for solid-state deep fermentation, has obvious tyrosinase inhibition capacity, is simple in process, low in production cost, and high in safety.The application further provides the rose fermentation product prepared by the method, the rose fermentation product, and the application of the rose fermentation product in the preparation of health care products.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a method for increasing the content of effective components in roses by fermenting edible fungi. Background Technology

[0002] Roses are perennial deciduous shrubs belonging to the genus Rosa in the family Rosaceae. They are traditionally used for both food and medicine, possessing high added value in ornamental, edible, medicinal, and daily chemical applications, with a wide range of industrial applications. Rose petals are rich in volatile oils, flavonoids, polyphenols, anthocyanins, polysaccharides, and other active ingredients, possessing multiple physiological functions such as anti-oxidation, anti-inflammatory and antibacterial properties, liver-soothing and mood-lifting effects, skin whitening and blemish-fading, and metabolism regulation. They are widely used in health foods, functional beverages, natural fragrances, cosmetics, and pharmaceuticals, with market demand continuing to rise.

[0003] Currently, the processing and utilization of rose raw materials mainly relies on traditional processes such as drying, brewing, extraction, brewing, and simple fermentation. Mainstream extraction methods include physicochemical techniques such as hot water extraction, ethanol reflux, ultrasound-assisted extraction, and microwave-assisted extraction. These conventional processing and extraction technologies share several common drawbacks: Firstly, the dense cell wall structure of roses, with large molecules such as cellulose and pectin encapsulating a large number of active ingredients, results in low dissolution rates of effective components and insufficient raw material utilization. Secondly, physicochemical extraction processes easily cause the inactivation and degradation of heat-sensitive active substances, and it is difficult to achieve targeted transformation and enrichment of components. Some processes also suffer from solvent residues, high energy consumption, and insufficient product purity. Existing rose fermentation technologies mostly use conventional microorganisms such as yeast and lactic acid bacteria as fermentation strains. While these can improve the dissolution rate of components to some extent, they suffer from limitations such as single function, limited component enhancement, and low added value of fermentation products, failing to efficiently achieve significant enrichment and structural optimization of effective rose components. Furthermore, traditional fermentation processes are prone to aroma loss and a sour taste, hindering the quality improvement and industrialization of deep-processed rose products. Therefore, developing an effective method to increase the content of active ingredients in roses, overcome the shortcomings of existing rose processing technology, improve raw material utilization and product added value, and meet the market demand for high-activity rose deep-processed products is of great practical significance and industrial application value. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the first objective of the present invention is to provide a method for increasing the content of effective components of rose by fermenting edible fungi, which can significantly increase the content of quercetin, quercetin-3-O-rutin glycoside and polyphenols in the fermentation product.

[0005] A second objective of this invention is to provide a rose fermentation product prepared by the above method.

[0006] A third objective of this invention is to provide a rose fermentation product.

[0007] The fourth objective of this invention is to provide the application of the rose fermentation product or rose fermentation product in the preparation of health-promoting products, thereby expanding the health benefits of roses.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for increasing the content of effective components in roses by fermenting with edible fungi, comprising the following steps: mixing rose powder, corn starch, glucose, potassium dihydrogen phosphate and water, adjusting the pH value and sterilizing to obtain a rose culture medium; inoculating the rose culture medium with Auricularia auricula-judae liquid, fermenting for 20-30 hours, adding L-cysteine ​​hydrochloride, and continuing fermentation for 5-7 days to obtain the rose fermentation product.

[0009] Preferably, the rose powder is obtained by drying, crushing, and sieving rosebuds.

[0010] Preferably, the mass ratio of rose powder to water is 1:(9~20); based on the total mass of rose powder and water, the mass of corn starch added is 2%~4%, the mass of glucose added is 0.8%~1.2%, and the mass of potassium dihydrogen phosphate added is 0.6%~1%.

[0011] Preferably, the pH value is adjusted to 5.5-6.0; the sterilization conditions are 115-121℃ for 5-15 minutes.

[0012] Preferably, the inoculum amount of *Auricularia auricula-judae* solution is 5% to 15% of the rose culture medium by volume; the viable count of *Auricularia auricula-judae* solution is 1 × 10⁻⁶. 6 ~1×10 7 CFU / ml; the auricularia auricula-judae culture was obtained by domestication in PDA medium containing rose pollen and propagation in PDB liquid.

[0013] Preferably, the fermentation culture temperature is 22~30℃.

[0014] Preferably, the amount of L-cysteine ​​hydrochloride added is 0.01% to 0.03% based on the dry weight of the rose culture medium.

[0015] The present invention also provides a rose fermentation product prepared by the above method.

[0016] The present invention also provides a rose fermentation product, wherein the above-mentioned rose fermentation product is dried at 40~60℃ to constant weight to obtain the rose fermentation product.

[0017] This invention also provides the application of the above-mentioned rose fermentation product or rose fermentation product in the preparation of health-promoting products.

[0018] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: The method described in this invention is simple and easy to operate, with low production costs. It can significantly increase the content of quercetin, quercetin-3-O-rutin glycoside and polyphenols in fermentation products, has obvious tyrosinase inhibition ability, and does not cause problems such as aroma loss, sour taste, or poor stability of active ingredients.

[0019] The fermentation strains used in this invention are edible fungi, and the strains are single and safe. Detailed Implementation

[0020] This invention provides a method for increasing the content of effective components in roses by fermenting with edible fungi, comprising the following steps: mixing rose powder, corn starch, glucose, potassium dihydrogen phosphate and water, adjusting the pH value and sterilizing to obtain a rose culture medium; inoculating the rose culture medium with Auricularia auricula-judae liquid, fermenting for 20-30 hours, adding L-cysteine ​​hydrochloride, and continuing fermentation for 5-7 days to obtain the rose fermentation product.

[0021] The rose powder of this invention is obtained by drying, crushing and sieving rosebuds; the drying is preferably done at 50~60℃ to constant weight, and the drying temperature is more preferably 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃ or 59℃; the sieving is preferably done through a 40-mesh sieve (more than 90% of the particles have a diameter ≥40 mesh); the dried rosebuds are preferably double-petaled red rosebuds.

[0022] The weight ratio of rose powder to water in this invention is 1:(9~20), preferably 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, or 1:19. Based on the total mass of rose powder and water, the added mass of corn starch is 2%~4%, preferably 3%; the added mass of glucose is 0.8%~1.2%, preferably 1%; and the added mass of potassium dihydrogen phosphate is 0.6%~1%, preferably 0.8%. This invention can be used to directly mix rose powder, corn starch, glucose, potassium dihydrogen phosphate, and water, or it can be used to mix rose powder and water evenly before adding corn starch, glucose, and potassium dihydrogen phosphate. After the rose culture medium is evenly mixed, the pH value is adjusted to 5.5~6.0, preferably 5.6, 5.7, 5.8, or 5.9. The sterilization conditions in this invention are 115~121℃ for 5~15 minutes.

[0023] The inoculation amount of *Auricularia auricula-judae* suspension described in this invention is 5% to 15% (v / v) of the rose culture medium volume, preferably 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, or 14%. The viable count of the *Auricularia auricula-judae* suspension is 1 × 10⁻⁶. 6 ~1×10 7CFU / ml, preferably 5×10 6 CFU / ml. The *Auricularia auricula-judae* spores described in this invention are obtained through domestication on PDA medium containing rose pollen and propagation in PDB liquid medium. Adaptation culture is first performed to ensure that the *Auricularia auricula-judae* strains gradually adapt to the rose substrate environment. The adaptation culture of this invention uses PDA medium containing rose pollen. Specifically, *Auricularia auricula-judae* strains (mycelia) with good growth and no contamination are selected and sequentially transferred to PDA medium containing 1%~5% (v / v) rose pollen, and cultured at 25℃~28℃ for 3~5 days. During the adaptation culture, the v / v concentration of rose pollen is increased in a gradient of 1%~5% (gradually adapting from low to high concentration), optionally increasing at 1%, 3%, and 5% respectively. Then, *Auricularia auricula-judae* strains (mycelia) with good growth after adaptation culture (dense mycelia, no discoloration, and uniform growth) are selected and transferred to PDB liquid medium for amplification culture. The amplification culture conditions are 25℃~28℃, shaking speed 200~250 r / min, and culture for 48~72 h to obtain a concentration of 1×10⁻⁶. 6 ~1×10 7 Prepare a CFU / ml solution of Auricularia auricula-judae for later use.

[0024] The PDA culture medium containing rose powder described in this invention comprises 200 g / L potato, 20 g / L glucose, 20 g / L agar, rose powder, and 1000 mL distilled water. The pH is natural, and the medium is sterilized at 121°C for 15 minutes and then cooled for later use. In the PDA culture medium containing 1% rose powder, the rose powder content is 1% (volume ratio); in the PDA culture medium containing 3% rose powder, the rose powder content is 3% (volume ratio); and in the PDA culture medium containing 5% rose powder, the rose powder content is 5% (volume ratio).

[0025] This invention involves inoculating *Auricularia auricula-judae* into a rose culture medium for fermentation. The fermentation temperature is 22-30℃, preferably 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, or 29℃. After 20-30 hours of fermentation, L-cysteine ​​hydrochloride is added, preferably after 21, 22, 23, 24, 25, 26, 27, 28, or 29 hours. Fermentation continues for 5-7 days after the addition of L-cysteine ​​hydrochloride to obtain the rose fermentation product. The amount of L-cysteine ​​hydrochloride added, based on the dry weight of the rose culture medium, is 0.01%-0.03%, preferably 0.02%. The preferred fermentation time is 6 days.

[0026] This invention, by adding L-cysteine ​​hydrochloride, can effectively scavenge free radicals generated during fermentation, prevent oxidative stress damage to *Auricularia auricula-judae* cells, and thus ensure the stable operation of the quercetin synthesis pathway. The addition of L-cysteine ​​hydrochloride participates in the metabolic process of *Auricularia auricula-judae*, promoting the synthesis of quercetin precursors such as phenylalanine and tyrosine, providing sufficient raw materials for quercetin biosynthesis. Furthermore, the addition of L-cysteine ​​hydrochloride, as a cofactor for various key enzymes in quercetin synthesis, can enhance the catalytic efficiency of quercetin synthases, thereby accelerating the key steps in the quercetin synthesis pathway and increasing the quercetin content in the fermentation products.

[0027] The present invention also provides a rose fermentation product prepared by the above method. The present invention involves solid-state deep fermentation by inoculating auricularia aurea strain on a rose culture medium. The bioactive component quercetin in the rose, which has multiple effects such as anti-oxidation and anti-inflammation, is significantly increased compared with that before fermentation. At the same time, the bioactivity of rose polyphenols is enhanced, thereby improving the quality of roses and deepening the health benefits of roses.

[0028] This invention also provides a rose fermentation product, obtained by drying the above-mentioned rose fermentation product at 40-60°C to constant weight. The drying temperature is preferably 45-55°C, more preferably 50°C. After drying, the product is preferably pulverized and sieved to obtain the rose fermentation product. The rose fermentation product of this invention has high contents of quercetin, quercetin-3-O-rutin glycoside, and polyphenols, and exhibits a high tyrosinase inhibition rate.

[0029] This invention also provides the application of the above-mentioned rose fermentation product or rose fermentation product in the preparation of health-promoting products.

[0030] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0031] In a specific embodiment of the present invention, *Auricularia auricula-judae* was purchased from Beijing Kezhan Biotechnology Co., Ltd.; *Auricularia auricula-judae* was purchased from Yunnan Yunjun Technology (Group) Co., Ltd.; and *Cordyceps militaris* was purchased from Yunnan Yunjun Technology (Group) Co., Ltd.

[0032] Unless otherwise specified, the following embodiments are all conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1 A method for increasing the content of active ingredients in roses using fermentation with edible fungi: Rose powder: Take dried rosebuds (double-petaled red roses), wash them, dry them at 60℃ to constant weight, pulverize them and pass them through a 40-mesh sieve (more than 90% of the particles have a diameter ≥ 40 mesh).

[0035] (1) Adaptation culture of edible fungi strain (Auricularia auricula-judae): Preparation of PDA medium containing rose powder: The components include 200 g / L potato, 20 g / L glucose, 20 g / L agar, rose powder, and 1000 mL distilled water. The pH is natural. The medium is sterilized at 121℃ for 15 min and then cooled for later use. The rose powder addition amounts in the medium are 1%, 3%, and 5%, corresponding to PDA medium containing 1% rose powder, 3% rose powder, and 5% rose powder, respectively.

[0036] Healthy, uncontaminated *Auricularia auricula-judae* mycelia were selected and transferred to PDA medium containing 1% rose pollen and cultured at 25°C for 3 days. Subsequently, they were transferred to PDA medium containing 3% rose pollen and cultured at 25°C for 3 days. Finally, they were transferred to PDA medium containing 5% rose pollen and cultured at 25°C for 3 days to obtain healthy *Auricularia auricula-judae* mycelia. This mycelium was then transferred to PDB liquid medium and cultured at 25°C with a shaking speed of 200 rpm for 48 hours to obtain a concentration of 1×10⁻⁶. 6 Prepare a CFU / ml solution of Auricularia auricula-judae for later use.

[0037] (2) Fermentation process of edible fungi: Mix rose powder and water thoroughly at a weight ratio of 1:9 (approximately 11% by weight). Then add 3 wt% corn starch, 1 wt% glucose, and 0.8 wt% potassium dihydrogen phosphate to the system. After mixing thoroughly, adjust the pH to 5.5. Place the culture medium in an autoclave and sterilize at 121°C and 0.1 MPa for 15 minutes. Cool to 25°C to obtain the rose culture medium for later use.

[0038] Under aseptic conditions, 5% by volume of Auricularia auricula-judae mycelium was added to the rose culture medium and placed in a constant temperature incubator at 22℃ and 60% humidity for static culture. After 24 hours of culture, 0.02% of L-cysteine ​​hydrochloride by dry weight of the rose culture medium was added and cultured for another 5 days until the Auricularia auricula-judae mycelium covered the entire culture medium, and the fermentation product was obtained.

[0039] Example 2 A rose fermentation product: The fermentation product obtained in Example 1 was taken out, dried at 40°C with forced air until constant weight, and then pulverized through a 40-mesh sieve to obtain the rose fermentation product.

[0040] Example 3 A method for increasing the content of active ingredients in roses using fermentation with edible fungi: Rose powder: Take dried rosebuds (double-petaled red roses), wash them, dry them at 55℃ to constant weight, pulverize them and pass them through a 40-mesh sieve (more than 90% of the particles have a diameter ≥ 40 mesh).

[0041] (1) Adaptation culture of edible fungi strain (Auricularia auricula-judae): Preparation of PDA culture medium containing rose powder: Same as in Example 1.

[0042] Healthy, uncontaminated *Auricularia auricula-judae* mycelia were selected and transferred to PDA medium containing 1% rose pollen, and cultured at 27°C for 4 days. Subsequently, they were transferred to PDA medium containing 3% rose pollen and cultured at 27°C for 4 days. Finally, they were transferred to PDA medium containing 5% rose pollen and cultured at 27°C for 4 days to obtain healthy *Auricularia auricula-judae* mycelia. This mycelium was then transferred to PDB liquid medium and cultured at 27°C with a shaking speed of 220 rpm for 60 hours to obtain a concentration of 5 × 10⁻⁶. 6 Prepare a CFU / ml solution of Auricularia auricula-judae for later use.

[0043] (2) Fermentation process of edible fungi: Rose powder and water were thoroughly mixed at a weight ratio of 1:15 (approximately 6.7% by mass). Then, 3 wt% corn starch, 1 wt% glucose, and 0.8 wt% potassium dihydrogen phosphate were added to the system. After mixing thoroughly, the pH was adjusted to 5.8. The culture medium was then placed in an autoclave and sterilized at 121°C and 0.1 MPa for 15 minutes. After cooling to 28°C, the rose culture medium was obtained and ready for use.

[0044] Under aseptic conditions, 10% by volume of Auricularia auricula-judae mycelium was added to the rose culture medium and placed in a constant temperature incubator at 26°C and 65% humidity for static culture. After 24 hours of culture, 0.02% of L-cysteine ​​hydrochloride by dry weight of the rose culture medium was added and cultured for another 6 days until the Auricularia auricula-judae mycelium covered the entire culture medium, and the fermentation product was obtained.

[0045] Example 4 A rose fermentation product: The fermentation product obtained in Example 3 was taken out, dried at 50°C with forced air until constant weight, and then pulverized through a 40-mesh sieve to obtain the rose fermentation product.

[0046] Example 5 A method for increasing the content of active ingredients in roses using fermentation with edible fungi: Rose powder: Take dried rosebuds (double-petaled red roses), wash them, dry them at 50℃ to constant weight, pulverize them and pass them through a 40-mesh sieve (more than 90% of the particles have a diameter ≥ 40 mesh).

[0047] (1) Adaptation culture of edible fungi strain (Auricularia auricula-judae): Preparation of PDA culture medium containing rose powder: Same as in Example 1.

[0048] Healthy, uncontaminated *Auricularia auricula-judae* mycelia were selected and transferred to PDA medium containing 1% rose pollen, and cultured at 28°C for 5 days. Subsequently, they were transferred to PDA medium containing 3% rose pollen and cultured at 28°C for 5 days. Finally, they were transferred to PDA medium containing 5% rose pollen and cultured at 28°C for 5 days to obtain healthy *Auricularia auricula-judae* mycelia. This mycelium was then transferred to PDB liquid medium and cultured at 28°C with a shaking speed of 250 rpm for 72 hours to obtain a concentration of 1×10⁻⁶. 7 Prepare a CFU / ml solution of Auricularia auricula-judae for later use.

[0049] (2) Fermentation process of edible fungi: Mix rose powder and water thoroughly at a weight ratio of 1:20 (approximately 5% by mass). Then add 3 wt% corn starch, 1 wt% glucose, and 0.8 wt% potassium dihydrogen phosphate to the system. After mixing thoroughly, adjust the pH to 6.0. Place the culture medium in an autoclave and sterilize at 121°C and 0.1 MPa for 15 minutes. Cool to 30°C to obtain the rose culture medium for later use.

[0050] Under aseptic conditions, 15% by volume of Auricularia auricula-judae mycelium was added to the rose culture medium and placed in a constant temperature incubator at 30°C and 70% humidity for static culture. After 24 hours of culture, 0.02% of L-cysteine ​​hydrochloride by dry weight of the rose culture medium was added and cultured for another 7 days until the Auricularia auricula-judae mycelium covered the entire culture medium, and the fermentation product was obtained.

[0051] Example 6 A rose fermentation product: The fermentation product obtained in Example 5 was taken out, dried at 60°C with forced air until constant weight, and then pulverized through a 40-mesh sieve to obtain the rose fermentation product.

[0052] Comparative Example 1 The only difference between this comparative example and Example 4 is that L-cysteine ​​hydrochloride is not added.

[0053] Comparative Example 2 The only difference between this comparative example and Example 4 is that after 0 hours of cultivation, 0.02% of L-cysteine ​​hydrochloride by dry weight of rose culture medium was added.

[0054] Comparative Example 3 The only difference between this comparative example and Example 4 is that after culturing for 72 hours, 0.02% of L-cysteine ​​hydrochloride by dry weight of rose culture medium was added.

[0055] Comparative Example 4 The only difference between this comparative example and Example 4 is that Auricularia auricula-judae (mycelium) was used instead of Auricularia auricula-judae (mycelium).

[0056] Comparative Example 5 The only difference between this comparative example and Example 4 is that Cordyceps militaris (mycelium) was used instead of Auricularia auricula-judae (mycelium).

[0057] Comparative Example 6 This comparative example uses a traditional fermentation method, differing from Example 4 only in that it uses *Saccharomyces cerevisiae* (5 × 10⁻⁶ viable cells). 8 Replace the *Auricularia auricula-judae* with CFU / ml, without adding L-cysteine ​​hydrochloride, and ferment for 7 days (without adding any adjuvants).

[0058] Experimental Example 1 The rose fermentation products obtained in Examples 2, 4, and 6, and the rose fermentation products obtained in Comparative Examples 1 to 6, were tested for quercetin content, quercetin-3-O-rutin glycoside content, and polyphenol content in the rose powder before fermentation. The tyrosinase inhibition rate of the 80% ethanol extract of the fermentation products was also measured. The quercetin and quercetin-3-O-rutin glycoside contents were determined using high-performance liquid chromatography (HPLC), and the polyphenol content was determined using spectrophotometry.

[0059] Experimental method for tyrosinase inhibition: Preparation of 80% ethanol extract stock solution: Accurately weigh 1.0 g of dried rose fermentation product (or rose powder before fermentation), pulverize and pass through a 40-mesh sieve to ensure uniform sample particle size; place the sample in a 50 mL sterile centrifuge tube, accurately add 20 mL of 80% ethanol solution, tighten the centrifuge tube cap and shake thoroughly to ensure complete contact between the sample and the ethanol solution. Place the centrifuge tube in an ultrasonic extractor and ultrasonically extract for 30 min at room temperature. Set the ultrasonic power to 200 W and the frequency to 40 kHz. Shake the centrifuge tube every 10 min during extraction to ensure thorough extraction. After extraction, place the centrifuge tube in a high-speed refrigerated centrifuge, set the speed to 8000 r / min and the temperature to 4℃, and centrifuge for 15 min. After centrifugation, carefully aspirate the supernatant with a pipette and transfer it to a clean 50 mL volumetric flask. Retain the residue after centrifugation in the original centrifuge tube, add 20 mL of 80% ethanol solution again, and repeat the above ultrasonic extraction and centrifugation operation, repeating the extraction twice in total. Combine all the supernatants obtained from the three extractions into the volumetric flask. The combined supernatants from the volumetric flasks were placed in a rotary evaporator and concentrated under reduced pressure until no alcohol odor remained (the concentration temperature was controlled not to exceed 50°C to avoid degradation of the active ingredients). After concentration, the volume was brought to 10 mL with 80% ethanol solution and thoroughly mixed. The diluted extract solution was then filtered through a 0.22 μm organic filter membrane to remove minute impurities and suspended particles, yielding an 80% ethanol extract stock solution. The stock solution was stored in a sealed container at 4°C for later use.

[0060] Preparation of experimental reagents and instruments: Prepare 0.1 mol / L phosphate buffer (pH 6.8, used to maintain the acidity and alkalinity of the experimental system), 200 U / mL tyrosinase solution (prepared with phosphate buffer before use, use immediately after preparation), and 0.03 mol / L L-DOPA solution (substrate, prepared before use, stored in the dark); prepare 96-well microplates, microplate reader, constant temperature water bath, pipettes (10 μL, 20 μL, 50 μL), sterile centrifuge tubes, etc. All instruments should be sterilized in advance to avoid interference from bacteria in the experimental results.

[0061] Sample gradient dilution: Take the 80% ethanol extract stock solution prepared above and dilute it with 0.1 mol / L phosphate buffer to prepare sample solutions with five concentration gradients of 10, 20, 40, 80, and 160 μg / mL. Set up 3 parallel samples for each concentration gradient. At the same time, set up a blank control group, a sample blank group, and a blank blank group. Set up 3 parallel wells for each group to ensure experimental repeatability.

[0062] Blank control group (no extract added, all other reagents are the same): 50 μL phosphate buffer + 20 μL tyrosinase solution + 10 μL L-DOPA solution; Sample blank group (without tyrosinase, all other reagents are the same): 50 μL phosphate buffer + 20 μL sample solution + 10 μL L-DOPA solution; Blank control group (without tyrosinase and extract, only buffer and substrate added): 70 μL phosphate buffer + 10 μL L-DOPA solution; Sample group: 50 μL phosphate buffer + 20 μL sample solution + 20 μL tyrosinase solution + 10 μL L-DOPA solution.

[0063] Experimental system sample loading: L-DOPA was used as the substrate. The samples were loaded sequentially into the 96-well microplate, and the volume of each sample was strictly controlled. The specific loading order was as follows: First, add 50 μL of 0.1 mol / L phosphate buffer to each well. Then, add 20 μL of the corresponding concentration of sample solution to the sample group wells and the sample blank group wells. Add 20 μL of phosphate buffer (to replace the sample solution) to the blank control group wells and the blank blank group wells.

[0064] Incubation and reaction: Add 20 μL of 200 U / mL tyrosinase solution to all wells except the sample blank group and the blank-control group (sample wells and blank control group wells). Gently shake the microplate for 1 min to ensure thorough mixing. Place the microplate in a 37°C water bath and incubate for 15 min to allow the tyrosinase to fully react with the sample. After incubation, accurately add 10 μL of 0.03 mol / L L-DOPA solution to all wells of the 96-well microplate. Gently shake the microplate again to mix, and continue incubating in a 37°C water bath in the dark for 20 min to allow the substrate and tyrosinase to fully react and generate a colored product.

[0065] Absorbance measurement: After incubation, immediately remove the microplate and place it in the microplate reader. Set the detection wavelength to 475 nm and measure the absorbance value (OD value) of each well. Keep the microplate in the dark during the measurement process. Measure each well three times consecutively and take the average value as the final absorbance value of that well. Record the experimental data.

[0066] Inhibition rate calculation: Based on the absorbance values ​​measured for each well, the tyrosinase inhibition rate is calculated using the following formula. The calculation result is rounded to two decimal places. After calculating the inhibition rate for all parallel samples, the average value is taken as the final inhibition rate for that concentration of sample. The formula for calculating the tyrosinase inhibition rate is: Inhibition rate (%) = [1 - (Absorbance of sample group - Absorbance of blank group) / (Absorbance of blank control group - Absorbance of blank control group)] × 100%.

[0067] Experimental repeatability and error control: Each experiment was repeated three times in parallel. Experimental conditions such as sample volume, incubation temperature and time, and ultrasonic extraction parameters were strictly controlled to avoid human error. All reagents were stored in the dark during the experiment, and tyrosinase solution was prepared and used immediately to prevent reagent failure from affecting the experimental results. When measuring absorbance, the microplate reader was preheated and calibrated to ensure accurate and reliable detection data.

[0068] The results of the detection of quercetin content, quercetin-3-O-rutin content, polyphenol content and tyrosinase inhibition rate are shown in Table 1 below.

[0069] Table 1. Component content and tyrosinase inhibition rate of rose before and after fermentation

[0070] Note: Different lowercase letters in the same column indicate significant differences between groups (P<0.05); P<0.05, P<0.01, P<0.001, all compared to rose powder before fermentation.

[0071] The results showed that the rose fermentation products of Examples 2, 4, and 6 of this invention exhibited excellent performance in terms of core active ingredients and tyrosinase inhibition rate, with significant differences compared to the comparative examples (P<0.05): the quercetin content reached 7.2~9.5 mg / g, an increase of 1.06~1.71 times compared to before fermentation (3.5 mg / g), with Example 6 showing the largest increase, up 1.71 times compared to before fermentation; the quercetin-3-O-rutin content reached 5.9~7.1 mg / g, compared to before fermentation... The content of quercetin (2.8 mg / g) increased by 1.11 to 1.54 times, achieving a doubling of growth; the polyphenol content reached 25.8 to 30.1 mg / g, an increase of 1.05 to 1.39 times compared to before fermentation (12.6 mg / g), also achieving a significant increase; the tyrosinase inhibition rate reached 62.5% to 73.6%, an increase of 1.21 to 1.60 times compared to before fermentation (28.3%), and increased synchronously with the increase of active ingredients such as quercetin and polyphenols, showing a clear positive correlation. The above data fully demonstrate that the method of the present invention can directionally promote the simultaneous enrichment of quercetin, quercetin-3-O-rutin glycoside and polyphenols in roses, significantly enhance their tyrosinase inhibition ability, and strengthen the health benefits of roses.

[0072] Comparative Example 1 (without L-cysteine ​​hydrochloride) showed significantly lower performance across all key indicators compared to Example 4 (P<0.05). Specifically, the quercetin content was only 4.8 mg / g, a 45.5% decrease compared to Example 4 (8.8 mg / g); the quercetin-3-O-rutinoside content was 3.2 mg / g, a 52.2% decrease compared to Example 4 (6.7 mg / g); the polyphenol content was 16.9 mg / g, a 41.5% decrease compared to Example 4 (28.9 mg / g); and the tyrosinase inhibition rate was 35.7%, a 47.7% decrease compared to Example 4 (68.3%). This demonstrates that L-cysteine ​​hydrochloride, as a fermentation aid, plays a crucial role in the enrichment of quercetin, quercetin-3-O-rutinoside, and polyphenols, while also significantly enhancing the tyrosinase inhibition rate. Its absence leads to a substantial decrease in overall fermentation efficiency, further confirming the necessity of this aid in this invention.

[0073] The core indicators of Comparative Example 2 (with L-cysteine ​​hydrochloride added in advance) and Comparative Example 3 (with L-cysteine ​​hydrochloride added late) were significantly lower than those of Example 4 (P<0.05), and there was no significant difference between the two. Specifically, the quercetin content of Comparative Example 2 was 5.3 mg / g, quercetin-3-O-rutin glycoside was 3.6 mg / g, polyphenols were 18.9 mg / g, and tyrosinase inhibition rate was 39.2%, which were reduced by 39.8%, 46.3%, 34.6%, and 42.6% respectively compared with Example 4; the above four indicators of Comparative Example 3 were 5.1 mg / g, 3.4 mg / g, 17.5 mg / g, and 37.5% respectively, which were reduced by 42.0%, 49.3%, 39.4%, and 45.1% respectively compared with Example 4. This indicates that the addition time of L-cysteine ​​hydrochloride must be strictly controlled within 20-30 hours of fermentation. Adding it earlier or later will significantly inhibit the synthesis and enrichment of quercetin, quercetin-3-O-rutin glycoside and polyphenols, reduce the inhibitory ability of tyrosinase, and further highlight the rationality and rigor of the process parameters of this invention.

[0074] The core indicators of Comparative Example 4 (using Auricularia auricula-judae instead of Auricularia auricula-judae) and Comparative Example 5 (using Cordyceps militaris instead of Auricularia auricula-judae) were all at low levels (P<0.05), showing only a slight improvement compared to before fermentation, and far lower than those of Example 4. Comparative Example 4 contained 4.2 mg / g of quercetin, 2.9 mg / g of quercetin-3-O-rutin, 14.8 mg / g of polyphenols, and a tyrosinase inhibition rate of 32.1%. Comparative Example 5 contained 4.0 mg / g, 2.7 mg / g, 14.2 mg / g, and 30.5% of the above four indicators, respectively. Notably, the quercetin content in Comparative Example 5 was only 45.5% of that in Example 4, and the tyrosinase inhibition rate was only 44.7% of that in Example 4. This indicates that Auricularia auricula-judae is significantly superior to Auricularia auricula-judae and Cordyceps militaris, and its metabolic characteristics can directionally promote the synchronous synthesis and release of quercetin, quercetin-3-O-rutin, and polyphenols in roses, significantly enhancing tyrosinase inhibition ability.

[0075] The core indicators of Comparative Example 6 (fermentation with Saccharomyces cerevisiae, without the addition of L-cysteine ​​hydrochloride) were significantly lower than those of Example 4 (P<0.05) and far lower than those of other examples. Specifically, the quercetin content was 4.5 mg / g, quercetin-3-O-rutin glycoside was 3.0 mg / g, polyphenols were 15.6 mg / g, and the tyrosinase inhibition rate was 34.2%, representing reductions of 48.9%, 55.2%, 46.0%, and 49.9% respectively compared to Example 4, only slightly higher than the pre-fermentation levels. This further demonstrates that the process of using Auricularia aurea fermentation combined with L-cysteine ​​hydrochloride regulation, compared to traditional yeast fermentation processes, has significant advantages in the enrichment of quercetin, quercetin-3-O-rutin glycoside, and polyphenols, while also significantly improving tyrosinase inhibition capacity, effectively overcoming the shortcomings of traditional fermentation processes.

[0076] Experimental Example 2 The rose fermentation products of Example 4 and Comparative Example 6 were used as test samples, and the rose powder before fermentation was used as a blank control. The aroma and taste sensory evaluation tests were carried out respectively.

[0077] 1. Aroma sensory evaluation Five individuals familiar with the characteristics of rose fragrance were selected and evaluated blindly. The samples were scored on a scale of 10 points (higher scores indicate better quality) based on three dimensions: fragrance intensity, fragrance purity, and fragrance persistence. The average score of the five individuals was used as the final evaluation result. The evaluation criteria are as follows: (1) Aroma intensity: 8~10 points (rich aroma, you can clearly smell the unique sweet aroma of rose); 5~7 points (medium aroma, you can smell the rose aroma but it is not strong); 1~4 points (weak aroma, or almost no rose aroma).

[0078] (2) Aroma purity: 8~10 points (pure aroma, no off-flavors, sourness, or strange smells); 5~7 points (relatively pure aroma, with occasional slight off-flavors); 1~4 points (impure aroma, with obvious off-flavors).

[0079] (3) Fragrance persistence: 8~10 points (fragrance is persistent, and the rose fragrance can still be smelled after 30 minutes); 5~7 points (fragrance is moderately persistent, and the fragrance is significantly weakened after 15~30 minutes); 1~4 points (fragrance is short-lived, and the fragrance is basically lost within 15 minutes).

[0080] 2. Sensory evaluation of taste The same blind tasting method was used, with 5 people evaluating the samples. The samples were brewed with 80℃ warm water (sample to water ratio 1:50), and after standing for 5 minutes, they were scored from three dimensions: richness of taste, balance of sweet and sour, and presence of any sour or astringent taste (out of 10 points). The average score of the 5 people was taken as the final evaluation result. The evaluation criteria are as follows: (1) Richness of taste: 8~10 points (rich taste, no grainy texture, smooth mouth); 5~7 points (medium taste, slightly grainy texture, relatively smooth mouth); 1~4 points (rough taste, obvious grainy texture, dry mouth).

[0081] (2) Sweet and sour balance: 8~10 points (the sweet and sour taste is moderate and matches the flavor of the rose itself without any abruptness); 5~7 points (the sweet and sour taste is relatively balanced, with a slight tendency to be sour or sweet); 1~4 points (the sweet and sour taste is unbalanced, with a noticeable tendency to be sour or sweet).

[0082] (3) Whether there is a sour or astringent taste: 8~10 points (no sour or astringent taste, refreshing taste); 5~7 points (slightly sour or astringent taste, does not affect the overall taste); 1~4 points (obvious sour or astringent taste, affects the taste).

[0083] 3. Test Results The sensory evaluation results for aroma and taste are shown in Table 2 below: Table 2. Sensory evaluation results of aroma and taste

[0084] As shown in Table 2, the rose fermentation product of Example 4 scored 9.05 points, significantly higher than Comparative Example 6 (5.07 points) and the rose powder before fermentation (6.47 points). Example 4 demonstrated excellent performance in aroma intensity, purity, and persistence, with no off-flavors and a long-lasting aroma; its taste was mellow and smooth, without any sourness or astringency, effectively solving the problems of weak aroma, easy dissipation, obvious off-flavors, and sourness and poor smoothness inherent in traditional fermentation processes (Comparative Example 6). Comparative Example 6 (traditional fermentation) scored only 4.2 points in aroma persistence, with the aroma almost completely dissipating within 15 minutes, and exhibiting a noticeable sourness and astringency, resulting in a poor taste; the defects of traditional fermentation were quite evident.

[0085] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for increasing the content of effective components in roses using fermentation with edible fungi, characterized in that, Includes the following steps: Rose powder, corn starch, glucose, potassium dihydrogen phosphate, and water were mixed, the pH was adjusted, and the mixture was sterilized to obtain a rose culture medium. Auricularia auricula-judae broth was inoculated into the rose culture medium, and after fermentation for 20-30 hours, L-cysteine ​​hydrochloride was added, and fermentation was continued for 5-7 days to obtain the rose fermentation product.

2. The method according to claim 1, characterized in that, The rose powder is obtained by drying, crushing, and sieving rosebuds.

3. The method according to claim 1, characterized in that, The mass ratio of rose powder to water is 1:(9~20); based on the total mass of rose powder and water, the mass of corn starch added is 2%~4%, the mass of glucose added is 0.8%~1.2%, and the mass of potassium dihydrogen phosphate added is 0.6%~1%.

4. The method according to claim 1, characterized in that, The pH value is adjusted to 5.5-6.0; the sterilization conditions are 115-121℃ for 5-15 minutes.

5. The method according to claim 1, characterized in that, The inoculation amount of the *Auricularia auricula-judae* broth, by volume, is 5%–15% of the rose culture medium; the viable count of the *Auricularia auricula-judae* broth is 1 × 10⁻⁶. 6 ~1×10 7 CFU / ml; the auricularia auricula-judae culture was obtained by domestication in PDA medium containing rose pollen and propagation in PDB liquid.

6. The method according to claim 1, characterized in that, The fermentation culture temperature is 22~30℃.

7. The method according to claim 1, characterized in that, The amount of L-cysteine ​​hydrochloride added is 0.01% to 0.03% based on the dry weight of the rose culture medium.

8. The rose fermentation product prepared by the method according to any one of claims 1 to 7.

9. A rose fermentation product, characterized in that, The rose fermentation product described in claim 8 is dried at 40-60°C to constant weight to obtain the rose fermentation product.

10. The use of the rose fermentation product of claim 8 or the rose fermentation product of claim 9 in the preparation of health-promoting products.

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