Rose flower fermented beverage and preparation method thereof

Abstract

The application provides a rose flower fermentation beverage and a preparation method thereof, and belongs to the technical field of food. The preparation method of the rose flower fermentation beverage comprises the following steps: after dry rose petals are soaked and beaten, enzyme hydrolysis is performed under the action of pectinase and cellulase, and then the obtained enzyme hydrolysis product is fermented under the action of yeast and lactobacillus plantarum in sequence; and the liquid phase in the rose flower fermentation product is separated to obtain the rose flower fermentation beverage. The rose flower fermentation beverage prepared by the method has bright red color, and retains the special aroma and fermentation flavor of roses.

Description

Technical Field

[0001] This invention belongs to the field of food technology, specifically relating to a rose-fermented beverage and its preparation method. Background Technology

[0002] Roses are rich in anthocyanins, flavonoids and other functional components, which have antioxidant, anti-tumor, anti-radiation, antibacterial and anti-aging functions. They also contain polysaccharides, alcohols, esters, terpenes, aldehydes, vitamins, alkaloids, rare linoleic acid, amino acids, dietary fiber, trace elements and proteins. These components determine that roses have great nutritional and medicinal value.

[0003] Fermented beverages refer to drinks made from fermented raw materials (fruits, vegetables, plants, etc.) through fermentation with lactic acid bacteria, yeast, or other strains permitted by the state, with an alcohol content of 0.5% or less. Microbial fermentation of plants can maintain and improve the inherent nutritional and sensory qualities of the raw materials, and also plays an important role in regulating intestinal flora, improving digestion, and lowering cholesterol. Existing patents mostly use fruits and vegetables to prepare fermented beverages; reports on fermented beverages made from flowers are relatively few. Furthermore, fermentation usually affects the color and aroma of flowers, making it difficult to obtain flower-based fermented beverages that combine both good taste and appealing color. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide a rose-fermented beverage with high sensory scores and good stability.

[0005] To achieve the above objectives, the present invention provides a method for preparing a rose-fermented beverage, comprising the following steps: Dried rose petals are soaked in water and then pulped. The resulting rose petal pulp is then enzymatically hydrolyzed under the action of a compound enzyme to obtain the enzymatic hydrolysis product. The compound enzyme includes cellulase and pectinase. The enzymatic hydrolysate is fermented for the first time under the action of yeast, the yeast is then inactivated, and then fermented for the second time under the action of Lactobacillus plantarum to obtain rose fermentation product. The liquid phase of the rose fermentation product is separated and clarified to obtain a rose fermented beverage.

[0006] Preferably, the mass ratio of the dried rose petals to the water is 1:(25~50).

[0007] Preferably, the pectinase is added at a mass of 0.08% to 0.2% of the rose petal slurry, the cellulase is added at a mass of 0.08% to 0.2% of the rose petal slurry, the enzymatic hydrolysis temperature is 48 to 60 °C, and the enzymatic hydrolysis time is 1.5 h to 3 h.

[0008] Preferably, the first fermentation time is 22-26 hours; the temperature of the first fermentation is 20-30°C. The second fermentation time is 45-75 hours; the temperature of the second fermentation is 30-40℃.

[0009] Preferably, the inoculation concentrations of the yeast and the *Lactobacillus plantarum* are independently 6 log CFU / mL to 8 log CFU / mL; The yeast includes *Corydalis churnb.*

[0010] Preferably, the process before the first fermentation further includes adjusting the soluble solids concentration and pH value of the enzymatic hydrolysis product and sterilizing the enzymatic hydrolysis product. The concentration of soluble solids in the enzymatic hydrolysis product is adjusted to 1.2-1.7 g / L, and the pH value of the enzymatic hydrolysis product is adjusted to 4.8-6.2. The sterilization process includes a boiling water bath; the sterilization time is 4-6 minutes.

[0011] Preferably, the method for inactivating yeast includes a boiling water bath; the temperature for inactivating yeast is 95~105℃; and the time for inactivating yeast is 4~6 min.

[0012] Preferably, the clarification treatment agent includes polyvinylpyrrolidone; The added treatment agent accounts for 0.01% to 0.03% of the mass of the liquid phase.

[0013] Preferably, the clarification treatment temperature is 20 ℃ to 40 ℃, and the clarification treatment time is 1 h to 3 h.

[0014] This invention provides a rose-fermented beverage prepared by the method described above, wherein the rose-fermented beverage has a pH value > 4.1, a total sugar concentration > 78 mg / mL, a total phenol concentration > 2.9 mg / mL, a total flavonoid concentration > 1.4 mg / mL, a tannin concentration < 3.9 mg / mL, an anthocyanin concentration of 0.13~0.14 mg / mL, and a clarity > 21%.

[0015] Beneficial effects This invention provides a method for preparing a rose-fermented beverage. Dried rose petals are soaked, pulped, and then enzymatically hydrolyzed using pectinase and cellulase. The resulting hydrolysate is then fermented sequentially using yeast and *Lactobacillus plantarum* to obtain a rose-fermented product. The liquid phase of the fermented product is separated to obtain the rose-fermented beverage. This invention, through enzymatic hydrolysis of roses, fully dissolves their nutrients. The hydrolysate is then fermented with yeast to inactivate the yeast, followed by fermentation with *Lactobacillus plantarum*, promoting the production of flavor compounds, reducing total sugar and tannin content, and significantly improving the nutritional value of the rose-fermented beverage. The rose-fermented beverage prepared using this method has a bright, uniform red color, is clear and transparent, and has a more appealing sweet-sour ratio and richer aroma, resulting in high sensory scores. This method promotes the development of rose deep-processing technology, enriches my country's flower fermented beverage market, and provides a reference for industrial production. Attached Figure Description

[0016] Figure 1 Images of rose-fermented beverage samples prepared in Examples 1-3; Figure 2 Images of rose-fermented beverage samples prepared for comparative examples 1-3. Detailed Implementation

[0017] This invention provides a method for preparing a rose-fermented beverage, comprising the following steps: Dried rose petals are soaked in water and then pulped. The resulting rose petal pulp is then enzymatically hydrolyzed under the action of a compound enzyme to obtain the enzymatic hydrolysis product. The compound enzyme includes cellulase and pectinase. The enzymatic hydrolysate is fermented for the first time under the action of yeast, the yeast is then inactivated, and then fermented for the second time under the action of Lactobacillus plantarum to obtain rose fermentation product. The liquid phase of the rose fermentation product is separated and clarified to obtain a rose fermented beverage.

[0018] This invention involves soaking dried rose petals in water and then pulping them. The resulting rose pulp is then enzymatically hydrolyzed under the action of a compound enzyme to obtain the enzymatic hydrolysis product.

[0019] In this invention, dried rose petals are soaked in water and then pulped when preparing a rose-fermented beverage. The dried rose petals are preferably free of mold or rot; they are easy to store in large quantities for long periods and are stable in properties during beverage preparation, ensuring the consistency of raw materials and the stability of fermentation. The preferred mass ratio of dried rose petals to water is 1:(25~50), more preferably 1:(28~40), further preferably 1:(29~35), and most preferably 1:30. Pulping at this specific mass ratio results in a richer rose flavor. The water preferably includes distilled water or purified water, more preferably purified water. The soaking time is preferably 10~60 min, more preferably 20~40 min, and most preferably 30 min. The pulping method preferably includes grinding the rose petals using a colloid mill to obtain the rose petal pulp. Soaking the dried rose petals in water and then pulping them results in a high degree of grinding, good dispersibility of the rose petal pulp, a large amount of water-soluble substances dissolved, and a good taste in the prepared rose-fermented beverage.

[0020] In this invention, the pectinase added is preferably 0.08%~0.2% of the rose petal slurry by mass, more preferably 0.09%~0.15%, and most preferably 0.1%. The cellulase added is preferably 0.08%~0.2% of the rose petal slurry by mass, more preferably 0.09%~0.15%, and most preferably 0.1%. The enzymatic hydrolysis temperature is preferably 48~60℃, more preferably 49~60℃, and most preferably 50℃. The enzymatic hydrolysis time is preferably 1.5~3 h, more preferably 1.8~2.6 h, further preferably 1.9~2.2 h, and most preferably 2 h. The enzymatic hydrolysis can degrade some phenolic substances in the rose petal slurry, and at the same time hydrolyze pectin and sugars in the rose petal slurry, causing tannins to lose the protective effect of pectin and other substances and precipitate together with other colloids, thereby reducing the tannin content and improving the flavor of the rose petal fermented beverage. In this embodiment of the invention, pectinase and cellulase were purchased from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd. One embodiment of the invention compared the effects of enzymatic hydrolysis treatment and omitting enzymatic hydrolysis treatment on rose-fermented beverages. The results showed that the rose-fermented beverage prepared by omitting enzymatic hydrolysis had higher total sugar and tannin content, resulting in an unbalanced sweet and sour taste, a prominent bitterness, low clarity, and a low sensory score. In contrast, the rose-fermented beverage prepared by enzymatic hydrolysis had a more vibrant and uniform red color, was clear and bright, and had a more palatable sweet and sour taste and richer aroma, resulting in the highest sensory score.

[0021] After obtaining the enzymatic hydrolysis product, the present invention performs a first fermentation of the enzymatic hydrolysis product under the action of yeast, then inactivates the yeast, and then performs a second fermentation under the action of Lactobacillus plantarum to obtain the rose fermentation product.

[0022] In this invention, the enzymatic hydrolysate, before the first fermentation, further includes adjusting the soluble solids concentration and pH value of the enzymatic hydrolysate and sterilizing the enzymatic hydrolysate. The soluble solids concentration of the enzymatic hydrolysate is preferably adjusted to 1.2-1.7 g / L, more preferably 1.3-1.6 g / L, and most preferably 1.5 g / L. The pH value of the enzymatic hydrolysate is preferably adjusted to 4.8-6.2, more preferably 5.2-6.1, and most preferably 6. The regulator for adjusting the soluble solids concentration of the enzymatic hydrolysate preferably includes soluble sugars, more preferably food-grade sucrose. The regulator for adjusting the pH value of the enzymatic hydrolysate preferably includes an acidity regulator, more preferably food-grade sodium citrate. In this invention, adjusting the soluble solids concentration and pH value of the enzymatic hydrolysate can provide a suitable environment for fermentation, thereby improving the taste and color of the rose fermented beverage. The sterilization method preferably includes boiling water bath sterilization. The sterilization time is preferably 4-6 min, more preferably 4.5-5.5 min, further preferably 4.8-5.2 min, and most preferably 5 min; the sterilization temperature is preferably 80-120 ℃, more preferably 90-110 ℃, and most preferably 100 ℃. In this invention, using a specific sterilization method to sterilize the enzymatic hydrolysis product can avoid the influence of miscellaneous bacteria and complex enzymes on subsequent fermentation, while preserving the taste and color of the rose fermented beverage.

[0023] In this invention, the inoculation concentration of the yeast is preferably 6 log CFU / mL to 8 log CFU / mL, more preferably 6 log CFU / mL to 7 log CFU / mL, and most preferably 6 log CFU / mL. The yeast preferably includes *Pichia kluferra*. The *Pichia kluferra* strain number is XT110, as described in patent publication CN 105695348A, entitled "A Method for Preparing Alcohol-Free Fermented Bayberry Juice from *Pichia kluferra*", published on June 22, 2016, and is a known strain in the prior art. The first fermentation time is 22 to 26 hours; more preferably 23 to 25 hours, and most preferably 24 hours; the first fermentation temperature is 20 to 30 °C, more preferably 25 to 29 °C, and most preferably 28 °C. Fermentation under the conditions specified in this invention can avoid the production of alcohols that affect the taste, aroma, and color of the rose fermented beverage, while reducing the tannin content of the rose fermented beverage, making it moderately sweet and sour, and improving sensory evaluation.

[0024] In this invention, the method for inactivating yeast preferably includes a boiling water bath. The temperature for inactivating yeast is preferably 95-105°C, more preferably 98-102°C, and most preferably 100°C. The inactivation time is preferably 4-6 min, more preferably 4.5-5.5 min, further preferably 4.8-5.2 min, and most preferably 5 min. After fermentation with *Pichia pastoris*, the inactivated yeast is then inoculated with *Lactobacillus plantarum*, which can control the ethanol content and improve the taste and color of the beverage. In the embodiments of this invention, the effects of different inactivated yeast conditions on rose fermented beverages were further compared. The results showed that compared with 100°C for 5 min, 90°C for 10 min and 80°C for 15 min resulted in a decrease in anthocyanin and total phenol content; and the color was not bright enough, with lower brightness and red-green component values. Therefore, the rose fermented beverage prepared by inactivating yeast at 100°C for 5 min has the best physicochemical properties.

[0025] In this invention, the inoculation concentration of *Lactobacillus plantarum* is 6 log CFU / mL to 8 log CFU / mL, more preferably 6.5 log CFU / mL to 7.5 log CFU / mL, and most preferably 7 log CFU / mL. In this embodiment, the *Lactobacillus plantarum* is *Lactobacillus plantarum* CICC 21796 purchased from the China Industrial Microbial Culture Collection Center. The second fermentation time is 45 to 75 h, more preferably 48 to 72 h, and most preferably 70 h; the second fermentation temperature is 30 to 40 °C, more preferably 35 to 38 °C, and most preferably 37 °C. The container for the first and second fermentations is preferably a vacuum filtration flask device, the filter nozzle of which is sealed with a sealing film, and the mouth of which is sealed with sterile physiological saline. The vacuum filtration flask device is preferably placed in an incubator, and the device is manually shaken once every other day. Fermentation under the conditions specified in this invention can reduce tannin content, improve the sensory score of rose fermented beverage, and avoid excessive acid production, resulting in an unbalanced sweet and sour ratio. The embodiments of the present invention compared the effects of sequential fermentation of yeast and Lactobacillus plantarum, fermentation of yeast alone, and fermentation of Lactobacillus plantarum alone on rose-flavored fermented beverages. The results showed that sequential fermentation of yeast and Lactobacillus plantarum could make the rose-flavored fermented beverages more bright red and uniform in color, clear and transparent, and the sweet and sour ratio and aroma richness of the taste more acceptable, and the sensory score was also the highest.

[0026] After obtaining the rose fermentation product, the present invention separates the liquid phase from the rose fermentation product, clarifies the liquid phase, and obtains a rose fermented beverage.

[0027] In this invention, the method for separating the liquid phase from the rose fermentation product preferably includes filtration. The filtration material preferably includes filter cloth. The pore size of the filtration material is preferably 750-850 mesh, more preferably 770-830 mesh, further preferably 790-810 mesh, and most preferably 800 mesh. The clarification agent preferably includes polyvinylpyrrolidone; the added mass of the clarification agent accounts for 0.01%-0.03% of the mass of the liquid phase, more preferably 0.02%-0.03%, and most preferably 0.03%. The clarification temperature is preferably 20-40℃, more preferably 24-36℃, further preferably 28-32℃, and most preferably 30℃. The clarification time is 1-3 h, more preferably 1.5-2.5 h, further preferably 1.8-2.2 h, and most preferably 2 h. The clarifying agent polyvinylpyrrolidone (PVP) has complexing ability, which can react with polyphenols to precipitate them, changing the color and taste of the system. Specifically, it increases the clarity of the system, brightens the color, reduces bitterness, and makes the taste more acceptable. In the embodiments of this invention, the effects of different amounts of PPVP added to the liquid phase on rose fermented beverages were compared. The results showed that when the amount added was low (less than 0.01% of the liquid phase mass), the clarity was low, the tannin content was high, the bitterness was prominent, and the sensory score was low.

[0028] After the clarification treatment, the supernatant is preferably obtained by centrifugation. The centrifugation speed is preferably 4500~5500 r / min, more preferably 4800~5200 r / min, and most preferably 5000 r / min. The centrifugation time is preferably 7~13 min, more preferably 9~11 min, and most preferably 10 min. After obtaining the supernatant, it is preferably bottled, sealed, and then pasteurized. The pasteurization temperature is preferably 62~68 ℃, more preferably 64~66 ℃, and most preferably 65 ℃. The pasteurization time is preferably 28~35 min, more preferably 29~32 min, and most preferably 30 min.

[0029] This invention provides a rose-fermented beverage prepared by the method described above, wherein the rose-fermented beverage has a pH value > 4.1, a total sugar concentration > 78 mg / mL, a total phenol concentration > 2.9 mg / mL, a total flavonoid concentration > 1.4 mg / mL, a tannin concentration < 3.9 mg / mL, an anthocyanin concentration of 0.13~0.14 mg / mL, and a clarity > 21%.

[0030] The rose-fermented beverage of this invention has a delicate and smooth taste, a balanced sweet and sour ratio, a slightly astringent flavor, a bright red color with a uniform hue, and a rich rose aroma. Fermentation further enhances its richness and harmony of flavor, resulting in a high sensory score. Furthermore, the rose-fermented beverage of this invention is rich in anthocyanins, phenols, and flavonoids, possessing antioxidant and immune-boosting functions.

[0031] To further illustrate the present invention, a rose-fermented beverage and its preparation method provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] The determination method involved in this invention is as follows: 1. pH measurement: pH meter is used for measurement.

[0033] 2. Clarity determination: The determination was carried out in accordance with existing technology (Zhong Xuan, Li Youmei, Huang Wensheng, et al. Study on the clarification effect of pectinase on passion fruit juice [J]. Food Safety Guide, 2024, (07): 134-136+176. DOI: 10.16043 / j.cnki.cfs.2024.07.055.).

[0034] 3. Determination of total sugar content: The phenol-sulfuric acid method was used, referring to existing technology (Zhu Yan, Li Xianzhe, Yan Ling, et al. Analysis and research on determination of polysaccharide content in health wine by phenol-sulfuric acid method [J]. Brewing Technology, 2023, (02): 118-121+127. DOI: 10.13746 / j.njkj.2022140.). 1 mL of the supernatant after centrifugation was placed in a colorimetric tube, 1 mL of 5% phenol solution was added, followed by 5 mL of concentrated sulfuric acid. The mixture was shaken well, allowed to stand for 5 min, and then incubated in a 30 ℃ water bath for 15 min. The absorbance was measured at 490 nm. The total sugar content in the sample solution was calculated based on the glucose standard curve.

[0035] 4. Determination of total phenol content: The Folin-Ciocalteu method was used, referring to existing techniques (Qun Yu, Liuping Fan, et al. Ultrasound and heating treatments improve the antityrosinase ability of polyphenols [J]. Food Chemistry, 2020, 317: 126415.). 1 mL of the supernatant after centrifugation was placed in a colorimetric tube, 0.5 mL of Folin-Ciocalteu reagent was added, and the mixture was thoroughly mixed. Then, 1.5 mL of 7.5% Na₂CO₃ solution was added, and the mixture was incubated in a 70 °C water bath for 30 min. After cooling, the solution was diluted to 10 mL with deionized water, and the absorbance was measured at 760 nm. The total phenol content in the sample solution was calculated based on the gallic acid standard curve.

[0036] 5. Determination of anthocyanin content: The pH differential method was used, referring to existing technology (Yuan Y, Tian Y, Gao S, et al. Effects of environmental factors and fermentation on red raspberry anthocyanins stability[J]. LWT, 2023, 173:114252-.DOI:10.1016 / j.lwt.2022.114252.). 1 mL of the supernatant after centrifugation was placed in a colorimetric tube, and the volume was adjusted to 10 mL with pH 1.0 and pH 4.5 buffer solutions, respectively. The anthocyanin content was calculated based on the absorbance measured at 520 nm and 700 nm.

[0037] 6. Determination of tannin content: Spectrophotometry was used, referring to existing technology (Liu Yuxia, Zhang Ling, Zhang Xiaojun, et al. Classification and evaluation of the taste quality of walnut inner seed coat based on electronic tongue technology [J]. Food and Fermentation Industries, 2020, 46(19):258-263.DOI:10.13995 / j.cnki.11-1802 / ts.024127.). 1 mL of the supernatant after centrifugation was placed in a colorimetric tube, 0.5 mL of Folin-Ciocalteu reagent was added, followed by 7.5 mL of deionized water. After reacting for 3 min, 1 mL of saturated Na2CO3 solution was added, and the reaction was allowed to proceed at room temperature for 1 h, inverting the tube three times during the reaction. The absorbance was measured at 725 nm. The tannin content in the sample solution was calculated based on the tannic acid standard curve.

[0038] 7. Determination of total flavonoid content: The aluminum chloride colorimetric method was used, referring to existing techniques (Qun Yu, Jinwei Li, Liuping Fan. Effect of drying methods on the microstructure, bioactivity, substances, and antityrosinase activity of asparagus stems [J], Journal of Agricultural and Food Chemistry, 2019, 67: 1537-1545.). 1 mL of the supernatant after centrifugation was placed in a colorimetric tube, and 0.5 mL of 5% sodium nitrite was added. The reaction was allowed to proceed for 5 min, followed by the addition of 0.3 mL of 10% aluminum nitrate. The reaction was allowed to proceed for 6 min, and then 4 mL of 4% NaOH was added. The solution was then diluted to 10 mL with deionized water and reacted at room temperature for 12 min. The absorbance was measured at 510 nm. The total flavonoid content in the sample solution was calculated based on the rutin standard curve.

[0039] Example 1 A method for preparing a rose-fermented beverage includes the following steps: (1) Soaking: Select dried rose petals without mold or rot for soaking; (2) Pulping: Add the soaked rose petals and purified water to a colloid mill at a mass ratio of 1:30 and grind them to obtain rose petal pulp; (3) Enzymatic hydrolysis: Add 0.1% pectinase and 0.1% cellulase by weight of rose petal sap to the rose petal sap and hydrolyze at 50 °C for 2 h. (4) Preparation: Add food-grade sucrose to the enzymatically hydrolyzed rose petal liquid to adjust its soluble solids to 15%, and add food-grade sodium citrate to adjust its pH to 6.00; (5) Sterilization: Sterilize the prepared rose petal liquid in a boiling water bath for 5 minutes. (6) Fermentation: 6.00 logCFU / mL of Pichia pastoris was added to the sterilized rose petal liquid and fermented at 28 ℃ for 1 day. The yeast was then inactivated at 100 ℃ for 5 min. 7.00 logCFU / mL of Lactobacillus plantarum was added and fermented at 37 ℃ for 3 days. The fermentation product was obtained after a total of 4 days of fermentation. (7) Filtration: The fermentation product is filtered through an 800-mesh filter cloth to separate the liquid phase; (8) Clarification and centrifugation: Add 0.03% polyvinylpyrrolidone by mass to the liquid phase, clarify at 30°C for 2 hours, centrifuge at 5000 r / min for 10 minutes, and take the supernatant after centrifugation; (9) Filling and pasteurization: The supernatant is filled and sealed, and then pasteurized at 65 °C for 30 min to obtain rose fermented beverage.

[0040] Example 2 Example 2: The preparation method of rose-fermented beverage is the same as that of Example 1, except that in step (6), fermentation is carried out for 2 days after inoculation with Lactobacillus plantarum.

[0041] Example 3 Example 3: The preparation method of the rose fermented beverage is the same as that of Example 1, except that the amount of polyvinylpyrrolidone added is 0.01% of the mass of the liquid phase.

[0042] Example 4 The effect of different clarifying agents on rose-fermented beverages.

[0043] After preparing the liquid phase according to the steps (1) to (7) of Example 1, different clarifying agents were added, and the liquid phase was clarified at 30 °C for 2 h. After clarification, the liquid phase was centrifuged at 5000 r / min for 10 min. The supernatant was then collected. The supernatant was filled and sealed, and then pasteurized at 65 °C for 30 min to obtain the rose fermented beverage.

[0044] The clarity, anthocyanin content, and tannin content of rose-fermented beverages obtained by adding different clarifying agents were determined, and the results are shown in Table 1.

[0045] Of these, sample 1 contained no clarifying agent; sample 2 contained 0.01% chitosan by weight of the liquid phase; sample 3 contained 0.02% gelatin by weight of the liquid phase; sample 4 contained 0.02% polyvinylpyrrolidone by weight of the liquid phase; sample 5 contained 0.01% chitosan and 0.02% polyvinylpyrrolidone by weight of the liquid phase; and sample 6 contained 0.02% gelatin and 0.02% polyvinylpyrrolidone by weight of the liquid phase.

[0046] Table 1. Effects of different clarifying agents on rose-flavored fermented beverages

[0047] As shown in Table 1, the beverage with the highest clarity and the greatest reduction in tannin content was achieved after the addition of polyvinylpyrrolidone, which means it has the effect of reducing the astringency of the beverage.

[0048] The effects of adding different mass percentages of polyvinylpyrrolidone to the liquid phase on the clarity, anthocyanin content, and tannin content of rose-fermented beverage were further compared. The test results are shown in Table 2.

[0049] Table 2. Effects of different amounts of polyvinylpyrrolidone added on rose-fermented beverages

[0050] Table 2 shows that when the amount of polyvinylpyrrolidone added is 0.03%, the tannin content is the lowest and the clarity is relatively high. When the amount of polyvinylpyrrolidone added is 0.01%, the anthocyanin content is relatively high.

[0051] Comparative Example 1 The preparation method of the rose fermented beverage in Comparative Example 1 is the same as that in Example 1, except that the enzymatic hydrolysis treatment is omitted.

[0052] Comparative Example 2 The preparation method of the rose-flavored fermented beverage in Comparative Example 2 was the same as that in Example 1, except that fermentation began after inoculation with *Bacillus krusei* and continued for 4 days, without inoculation with *Lactobacillus plantarum*.

[0053] Comparative Example 3 The preparation method of the rose-flavored fermented beverage in Comparative Example 3 was the same as that in Example 1, except that after sterilization, Lactobacillus plantarum was directly inoculated to start fermentation until the end, and fermentation lasted for 4 days. Other conditions were the same as in Example 1.

[0054] Comparative Example 4 The preparation method of the rose-fermented beverage in Comparative Example 4 was the same as that in Example 1, except that the yeast sterilization conditions were 90 °C for 10 min.

[0055] Comparative Example 5 The preparation method of the rose-fermented beverage in Comparative Example 5 was the same as that in Example 1, except that the yeast sterilization conditions were 80 °C for 15 min.

[0056] Example 5 The physicochemical properties and volatile compound content of Examples 1-3 and Comparative Examples 1-3 were tested, and their sensory evaluation was performed. The physicochemical properties of Examples 1-3 and Comparative Examples 1-3 are shown in Table 3. The volatile compound content (containing 58 volatile compounds, including 16 esters, 18 alcohols, 8 acids, 5 aldehydes, 4 phenols, 2 ketones, 3 alkenes, and 2 others) is shown in Table 4. The sensory evaluation criteria are shown in Table 5, and the sensory scores for each group are shown in Table 6. The comparison results of the physicochemical properties of Examples 1 and Comparative Examples 4-5 are shown in Table 7.

[0057] Table 3 Physicochemical properties of rose-fermented beverages from Examples 1-3 and Comparative Examples 1-3

[0058] Table 4. Volatile compound content (μg / mL) of rose fermented beverages in Examples 1-3 and Comparative Examples 1-3

[0059] Table 5 Sensory Scoring Criteria

[0060] Table 6 Sensory Rating Table

[0061] Table 7 Physicochemical properties of rose-fermented beverages from Example 1 and Comparative Examples 4-5

[0062] According to the results in Tables 3-7, the rose-fermented beverages prepared by the methods in Examples 1-3 had a pH > 4.1, total sugar content > 78 mg / mL, total phenol content > 2.9 mg / mL, total flavonoid content > 1.4 mg / mL, tannin content < 3.9 mg / mL, anthocyanin content of 0.13-0.14 mg / mL, and clarity > 21%. The results of the volatile compound content determination showed that the rose-fermented beverages prepared by the methods in Examples 1-3 had high alcohol and lipid content. Among them, the rose-fermented beverage in Example 1 had an alcohol content of 57.36% and an ester content of 29.7%, indicating that the co-fermentation of *Pichia pastoris* and *Lactobacillus plantarum* is conducive to the production of aroma substances such as alcohols and esters, such as ethyl phenylacetate (rich and sweet honey aroma), phenylethanol (pleasant and lasting rose aroma), α-terpineol (pine and clove-like aroma), and rose ether (rose and grassy aroma). Sensory evaluation results showed that the rose-flavored fermented beverages prepared by the methods in Examples 1-3 had a delicate and smooth taste, a balanced sweet and sour ratio, a slightly astringent flavor, a bright red color with uniform hue, and a rich and harmonious rose aroma, resulting in high sensory scores. Therefore, the preparation methods of this invention can effectively improve the nutritional quality and sensory properties of rose-flavored fermented beverages.

[0063] In contrast, the rose-flavored fermented beverages prepared by methods 1 to 3 had an unbalanced sweet-sour ratio, high tannin content, prominent bitterness, weak rose aroma, and lacked flavor, resulting in low sensory scores.

[0064] Specifically, compared to Example 1, Comparative Example 1 showed a 9.15% increase in total sugar content in its rose-fermented beverage, resulting in a prominent sweetness and an unbalanced sweet-sour ratio. The total phenol content increased by 4.95%, and the tannin content increased by 7.01%. The increased tannin content led to a prominent bitterness in the beverage, masking the rose aroma. Clarity decreased by 43.57%, resulting in slight sedimentation and a low sensory score. The low content of esters and alcohols in the volatile compounds, combined with the sensory evaluation, indicated a weak overall flavor, suggesting that enzymatic hydrolysis promotes the interaction between aroma components and enhances the richness of the beverage's flavor. Therefore, adding pectinase and cellulase for enzymatic hydrolysis can make the rose-fermented beverage more vibrant and uniform in color, clearer and brighter, with a more palatable sweet-sour ratio and richer aroma, resulting in the highest sensory score.

[0065] Compared to Example 1, Comparative Example 2 produced less acid, consumed more sugar, and increased the total phenol content by 11.76% and tannin content by 5.66%. This resulted in a more pronounced bitter taste in the rose fermented beverage. The volatile compound content analysis showed a high alcohol content, leading to a strong alcoholic flavor that masked the aroma, resulting in a poor taste; a pungent odor with a very faint rose fragrance; a dark reddish-brown color; a 70.61% decrease in clarity; the formation of a small amount of sediment; and a low sensory score. Therefore, sequential fermentation with *Pichia pastoris* and *Lactobacillus plantarum* produces a more vibrant and uniform red color, a clearer and brighter appearance, a more palatable sweet-sour ratio, and a richer aroma, resulting in the highest sensory score.

[0066] Compared to Example 1, Comparative Example 3 produced more acid, resulting in an overly acidic rose-flavored fermented beverage with an unbalanced sweet-sour ratio. The total phenol content increased by 8.36%, and the tannin content also increased, leading to a more pronounced bitter taste. Furthermore, the single-strain fermentation lacked the unique aroma and characteristic fragrance produced by co-fermentation, resulting in a lackluster flavor and a low sensory score. The volatile compound content determination results showed a high acid content, causing an imbalance in the sweet-sour ratio and an unpleasant taste. Therefore, sequential fermentation with *Pichia pastoris* and *Lactobacillus plantarum* produces a more vibrant and uniform red color, a clear and bright appearance, a more palatable sweet-sour ratio and richer aroma, and the highest sensory score.

[0067] Compared to Comparative Example 2, Example 1 showed a decrease in total phenolic and tannin content in the rose fermented beverage, which reduced its bitterness. Furthermore, the total sugar content decreased more significantly during yeast fermentation alone, weakening the masking effect of sugar on bitterness and making it more prominent. Compared to Comparative Example 3, the decrease in total phenolic and tannin content further reduced bitterness; the increase in total flavonoid content will enhance its antioxidant properties. The rose fermented beverage obtained in Example 1 had a uniform, bright red color, was clear and transparent, and had a balanced sweet and sour taste. Fermentation enriched its flavor, and the slight astringency was within an acceptable range. It possessed a rich rose aroma without any off-flavors, thus achieving the highest sensory score.

[0068] Compared to Comparative Example 2, Example 2 showed a 17.45% reduction in total phenolic content and a 14.80% reduction in tannin content in the rose fermentation beverage, which reduced its bitterness. Furthermore, the reduction in total sugar during yeast fermentation alone further weakened the masking effect of sugar on bitterness, making the bitterness more prominent. Compared to Comparative Example 3, the total phenolic content decreased by 14.86% and the tannin content decreased by 10.70%, which also reduced its bitterness. The rose fermentation beverage obtained in Example 2 had a bright, uniform red color and was relatively clear and transparent, but its sweet and sour taste was unbalanced, lacked the unique flavor imparted by fermentation, and had a weak rose aroma, making it less effective than Example 1, resulting in a lower sensory score. Therefore, the sequential fermentation time of *Pichia pastoris* and *Lactobacillus plantarum* has a significant impact on the rose fermentation beverage.

[0069] Compared to Comparative Example 2, Example 3's preparation method consumes less sugar, better masks bitterness, has lower tannin content, weaker astringency, and higher clarity. Polyvinylpyrrolidone (PVP) has the ability to adsorb phenolic substances. Compared to Example 1, Example 3 has a darker red color, lower clarity, higher tannin content, and a more pronounced bitterness, masking the sweet and sour taste and rose aroma of the rose fermented beverage. The effect is not as good as Example 1, resulting in a lower sensory score. Therefore, the amount of PVP added after sequential fermentation of *Bacillus krusei* and *Lactobacillus plantarum* has a significant impact on the rose fermented beverage.

[0070] Compared to Example 1, Comparative Examples 4 and 5 showed lower temperatures and longer sterilization times for yeast, resulting in a decrease in anthocyanin content and total phenol content under continuous heating. The brightness and red-green component values ​​were also lower, leading to a less vibrant and bright color. Therefore, the temperature and time for yeast sterilization have a significant impact on rose-flavored fermented beverages.

[0071] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a rose-fermented beverage, characterized in that, Includes the following steps: Dried rose petals are soaked in water and then pulped. The resulting rose petal pulp is then enzymatically hydrolyzed under the action of a compound enzyme to obtain the enzymatic hydrolysis product. The compound enzyme includes cellulase and pectinase. The enzymatic hydrolysate is fermented for the first time under the action of yeast, the yeast is then inactivated, and then fermented for the second time under the action of Lactobacillus plantarum to obtain rose fermentation product. Separate the liquid phase from the rose fermentation product, clarify the liquid phase, and obtain a rose fermented beverage. The pectinase is added at a mass of 0.08% to 0.2% of the rose petal slurry, the cellulase is added at a mass of 0.08% to 0.2% of the rose petal slurry, the enzymatic hydrolysis temperature is 48 to 60°C, and the enzymatic hydrolysis time is 1.5 to 3 hours. The first fermentation time is 22-26 hours; the temperature of the first fermentation is 28°C. The second fermentation time is 48-72 hours; the temperature of the second fermentation is 37℃. The process before the first fermentation also includes adjusting the soluble solids concentration and pH value of the enzymatic hydrolysis product and sterilizing the enzymatic hydrolysis product. The sterilization process includes a boiling water bath; the sterilization time is 5 minutes. The clarification treatment agent includes polyvinylpyrrolidone; The mass of the treatment agent added accounts for 0.03% of the mass of the liquid phase; The mass ratio of dried rose petals to water is 1:(25~50). The inoculation concentrations of the yeast and the Lactobacillus plantarum were independently 6 log CFU / mL to 8 log CFU / mL; The yeast includes *Corydalis kulfibrio*. The concentration of soluble solids in the enzymatic hydrolysis product is adjusted to 1.2-1.7 g / L, and the pH value of the enzymatic hydrolysis product is adjusted to 4.8-6.

2.

2. The method according to claim 1, characterized in that, The method for inactivating yeast includes a boiling water bath; the temperature for inactivating yeast is 95~105℃; and the time for inactivating yeast is 4~6 minutes.

3. The method according to claim 1, characterized in that, The clarification treatment is performed at a temperature of 20℃ to 40℃ for 1 hour to 3 hours.

4. The rose-fermented beverage prepared by the method according to any one of claims 1 to 3, wherein the rose-fermented beverage has a pH value > 4.1, a total sugar concentration > 78 mg / mL, a total phenol concentration > 2.9 mg / mL, a total flavonoid concentration > 1.4 mg / mL, a tannin concentration < 3.9 mg / mL, an anthocyanin concentration of 0.13~0.14 mg / mL, and a clarity > 21%.

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