Rosa roxburghii enzyme beverage

Abstract

The invention provides a roxburgh rose enzyme beverage, the roxburgh rose enzyme beverage is prepared by fermenting roxburgh rose, the total acid content in the roxburgh rose enzyme beverage is 4-5g / L, and the condensed tannin content is 1000-1200 mg / 100g. The roxburgh rose enzyme beverage provided by the invention is obtained through a specific fermentation process, and physicochemical and sensory detection results show that sour, sweet and bitter taste are significantly different before and after fermentation. Specifically, after fermentation, the sour taste score is reduced from 24.95 to 19.43, the bitter taste score is reduced from 23.10 to 18.62, meanwhile, the sweet taste score is obviously increased from 8.52 to 12.19, the bad smell of part of roxburgh rose juice is obviously improved through fermentation, and the roxburgh rose juice becomes a popular enzyme beverage.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of beverage preparation, and specifically relates to a fermented beverage and a preparation method thereof. BACKGROUND

[0002] Rosa roxburghii Tratt. Rosa roxburghii Rosa roxburghii Tratt. (Roxb.), a deciduous shrub of Rosaceae, is mainly distributed in the southwest of China, especially in Guizhou, Yunnan, Sichuan and other places, and is most concentrated in Guizhou. The content of vitamin C in Rosa roxburghii Tratt. is as high as 800-2500 mg / 100g, far exceeding that of apples, oranges and kiwis, etc., and therefore it is known as the "king of natural vitamin C". In addition, Rosa roxburghii Tratt. is also rich in superoxide dismutase, polyphenols, flavonoids, dietary fiber and trace elements such as zinc, selenium and iron, and has extremely high nutritional value and functional food development potential.

[0003] Studies have shown that flavonoids and polyphenols in Rosa roxburghii Tratt. can effectively scavenge free radicals, enhance antioxidant enzyme activity, and play a role through the regulation of oxidative stress-related transcription factors and signaling pathways; vitamin C and polyphenols also have a synergistic antioxidant effect, which can delay cell aging (Wan Z, Cheng WJ, Peng L, et al. Research progress of inhibition of oxidative stress by Rosa roxburghii Tratt. [J]. Journal of Hubei University of Science and Technology (Medical Edition), 2024, 38(1): 74-78.). Animal and cell experiments have further shown that Rosa roxburghii Tratt. extract has potential effects on tumor inhibition and immune regulation, providing scientific basis for its application in the fields of functional food and medicine (YANG Y, LI W, XIAN W, et al. Free and bound phenolic profiles of Rosa roxburghii Tratt. leaves and their antioxidant and inhibitory effects on α-glucosidase [J]. Frontiers in Nutrition, 2022, 9: 922496.).

[0004] Rosa roxburghii Tratt. has a unique flavor, "sour and astringent", and the substances that mainly contribute to its flavor include organic acids, phenols and volatile aromatic compounds. Organic acids are the main source of sourness in R. roxburghii Tratt. Studies have shown that the total content of organic acids in R. roxburghii Tratt. can reach 15.3 g / kg, with malic acid (52.1%) and citric acid (31.4%) being the main components, and the rest including quinic acid (8.2%), tartaric acid (5.3%) and so on (LI T T, HUANG M Z, TANG W Y, et al. Analysis of organic acids and aroma components in Rosa roxburghii Tratt. fruit[J]. Food Chemistry, 2021, 362: 130196.). Phenolic substances, especially ellagic acid, catechin and proanthocyanidins, are the main source of astringency. The total phenol content of R. roxburghii Tratt. is generally between 16.4~21.7 mg / g fresh weight, and ellagic acid can account for more than 22.3% of the total phenols. In terms of aroma, R. roxburghii Tratt. fruit is rich in a variety of aldehydes, esters, alcohols and terpene volatile aromatic substances.

[0005] R. roxburghii Tratt. is rich in tannins and polyphenols, which gives it a relatively obvious bitter and astringent taste, affecting the acceptance of consumers. Current research attempts to remove astringency from juice through enzymatic and masking methods, but due to high cost and difficulty in standardization, it is difficult to promote in the industry (ZHANG Y, LUO Y, LIU F S, DING Z H. Analysis of flavor quality of R. roxburghii juice treated by different methods to remove bitterness and astringency[J]. Food Science, 2016(04): 21-26.). SUMMARY

[0006] In view of the deficiencies in the prior art, the present application provides a R. roxburghii Tratt. enzyme beverage, which explores a mixed fermentation process of Oenococcus oeni ( Oenococcus oeni ) and Lactobacillus plantarum ( Lactobacillus plantarum ) to improve the sensory quality of R. roxburghii Tratt. juice by degrading malic acid and condensing tannins.

[0007] The technical scheme for achieving the above-mentioned purposes of the present application is as follows: A R. roxburghii Tratt. enzyme beverage, which is prepared by fermenting R. roxburghii Tratt., has a total acid content of 4~5 g / L and a condensed tannin content of 1000~1200.

[0008] Preferably, the R. roxburghii Tratt. enzyme beverage has a total acid content of 4.3~4.7 g / L and a condensed tannin content of 1080~1120 mg / 100 g.

[0009] Further, the R. roxburghii Tratt. enzyme beverage has a vitamin C content of 600~700 mg / 100 g and a sugar acid ratio (SAR) of 13~14.

[0010] The application further provides a preparation method of the roxburgh rose enzyme beverage. 1) sterilizing and cooling roxburgh rose original juice; 2) activating Oenococcus oeni and Lactobacillus plantarum on culture medium respectively to obtain bacterial liquid with a cell concentration of 5-10×10 9 CFU / mL independently; 3) adjusting the initial pH of the cooled roxburgh rose juice to 3.4-4.2, inoculating the Oenococcus oeni and Lactobacillus plantarum bacterial liquid into the roxburgh rose juice, and fermenting for 6-30 hours.

[0011] The roxburgh rose original juice is squeezed from roxburgh rose (without adding water), has a total acid of 7-8 g / L, and a condensed tannin content of 1850-2000 mg / 100 g.

[0012] In step 2), the Oenococcus oeni is cultured on ATB culture medium, and the Lactobacillus plantarum is cultured on lactic acid bacterial liquid culture medium, and the obtained bacterial liquid is centrifuged, resuspended in normal saline, and the cell concentration is 5-10×10 9 CFU / mL.

[0013] When the culture medium is cultured, a shaker at 180 rpm can be used. The centrifugation condition can be 5000-10000 rpm for 3-8 min; after centrifugation, the supernatant is discarded, and the cell concentration can be resuspended in 0.9% normal saline to 5-10×10 9 CFU / mL.

[0014] In step 3), the volume ratio of the Oenococcus oeni and Lactobacillus brevis bacterial liquid is 1-2:1-2.

[0015] In step 3), the fermentation temperature is 25-37℃, and the inoculation amount is 1-9%.

[0016] In step 3), the fermentation time is 24-25 hours.

[0017] Preferably, in step 3), the initial pH is 4, and the fermentation temperature is 28℃.

[0018] Compared with the prior art, the application has the following beneficial effects: Rosa roxburghii Tratt. is highly regarded for its extremely high vitamin C and polyphenol content, but its high levels of organic acids and condensed tannins result in a sour and astringent taste, severely impacting product acceptance. Traditional physicochemical methods for reducing acidity often lead to nutrient loss and flavor imbalance, while bio-fermentation technology can directionally transform undesirable components through microbial metabolism, offering the dual advantages of being both green and mild, and improving flavor. *C. tartrazine* and *Lactobacillus plantarum*, as lactic acid bacteria capable of degrading organic acids, have potential regulatory value in the synergistic effect on the metabolism of citric acid and malic acid, as well as the biotransformation of bitter substances in rosa roxburghii juice.

[0019] Regarding the selection of microbial strains, this invention employs a mixed fermentation of *C. chrysogenum* and *Lactobacillus plantarum* in a specific ratio. *C. chrysogenum* possesses malic-lactic acid fermentation capabilities, degrading malic acid through the malic-lactic acid pathway, transforming the sharp-tasting malic acid into a milder, more flavorful lactic acid, thus improving the overall taste and flavor complexity of the juice. *Lactobacillus plantarum* has the ability to degrade condensed tannins, generating monomeric tannins and small-molecule phenolic substances through hydrolysis and condensation, reducing the astringency and bitterness caused by high-molecular-weight tannins, and improving the acceptability of the prickly pear juice. The synergistic effect of these microorganisms enhances both the flavor and the product's functional characteristics and stability.

[0020] The prickly pear enzyme beverage proposed in this invention is obtained through a specific fermentation process. Physicochemical and sensory testing results show significant differences in sourness, sweetness, and bitterness before and after fermentation. Specifically, after fermentation, the sourness score decreased from 24.95 to 19.43, the bitterness score decreased from 23.10 to 18.62, while the sweetness score increased significantly from 8.52 to 12.19. Fermentation significantly improved some of the unpleasant odors of the prickly pear juice, making it a popular enzyme beverage. Attached Figure Description

[0021] Figure 1 Single-factor experiment - malic acid change curve.

[0022] Figure 2 Single-factor experiment - condensed tannin change curve.

[0023] Figure 3 Response surface optimization surface - malic acid. Figure 3 The vertical axis of the response surface in the figure is malic acid. In the figure, A: temperature, B: inoculum size, C: pH, and D: time.

[0024] Figure 4 Response surface optimization surface - condensed tannin. Figure 4 The vertical axis of the response surface in the figure is tannin. In the figure, A: temperature, B: inoculum size, C: pH, and D: time.

[0025] Figure 5Total ion chromatogram of volatile compounds in prickly pear juice.

[0026] Figure 6 Total ion chromatogram of volatile aroma compounds in fermented prickly pear juice Figure 7 Sensory evaluation results, with the left side showing a comparison of the five basic flavors and the right side showing the quality of fermented prickly pear juice.

[0027] Figure 8 Comparison of vitamin C, total phenols and total flavonoids content before and after fermentation. Detailed Implementation

[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention.

[0029] In the examples, some of the raw materials were sourced from: Prickly pear juice: obtained by directly juicing prickly pear (without adding water), provided by Guizhou Liupanshui Ciliwang Biotechnology Co., Ltd., with a vitamin C content of 1135.62±3.68 mg / 100 g and a condensed tannin content of 1935.69289 mg / 100 g.

[0030] Chlorella vulgaris mL34 ( Oenococcus oeni Lactobacillus plantarum Lp01 (accession number CICC 6057) Lactobacillus plantarum All samples (accession number CICC 6545) were purchased from the China Industrial Microbial Culture Collection Center. ATB medium: Shandong Top Biotechnology; Lactic acid bacteria liquid medium: Beijing Aoboxing.

[0031] Some detection methods in the examples: (a) Determination of malic acid degradation rate: First, the malic acid content in the prickly pear juice before and after fermentation was determined by WST colorimetric method.

[0032] (ii) The degradation rate of condensed tannins was determined by the protein precipitation method (PP method).

[0033] Add 500 μL of prickly pear juice to 1.00 mL of BSA (bovine serum albumin) solution, incubate at room temperature for 15 min, centrifuge at 16,200 rpm for 5 min, discard the supernatant, add 1.00 mL of triethanolamine (TEA) buffer, and vortex for 20 min. Measure the absorbance at 510 nm as a control. Then add 875 μL of TEA buffer (pH 9.4), vortex for 20 min, add 125 μL of 10 mM FeCl3, vortex at room temperature for 10 min, and record the final absorbance at 510 nm.

[0034] When calculating absorbance, the final absorbance value is subtracted from the control value. At this point, the tannin content is (+)-catechin equivalent.

[0035] Example 1: (1) Sterilization: The Rosa roxburghii juice was placed in a conical flask and pasteurized in a water bath at 85°C for 40 min.

[0036] (2) Strain activation and culture: 5.2 g of ATB medium and 3.5 g of lactic acid bacteria liquid medium were weighed into 100 mL of deionized water, respectively, and heated to dissolution on an electromagnetic stove. The mixture was sterilized at 121°C for 15 min, and then cooled to room temperature. After sterilization, 10 mL of the mixture was injected into a 50 mL centrifuge tube using a pipette. 1 μL of the freeze-dried bacterial powder was taken with a inoculation loop and dispersed in the corresponding medium (Oenococcus oeni-ATB medium; Lactobacillus plantarum-lactic acid bacteria liquid medium) on a shaker at 180 rpm and 37°C for 1 day. When the medium became turbid, the initial activated bacterial solution was inoculated into new medium at a 10% inoculation rate, and cultured under the same conditions for 1 day. The resulting bacterial solution was centrifuged (8000 rpm, 5 min), and the supernatant was discarded. The cells were resuspended in 0.9% saline to a concentration of 1×10 9 CFU / mL.

[0037] (3) Inoculation and fermentation: Oenococcus oeni and Lactobacillus plantarum were inoculated into cooled Rosa roxburghii juice at a certain ratio, and then placed in a set constant-temperature incubator for fermentation. After a certain period of time, the mixture was taken out, frozen at −80°C, and stored for later use.

[0038] Specifically, Oenococcus oeni and Lactobacillus brevis were mixed at different volume ratios (1:0, 2:1, 1:1, 1:2, 0:1, and 1:4) at an inoculation rate of 5%, and incubated at 31°C. Samples were taken every 6 h to determine the malate degradation rate and condensed tannin degradation rate. The data were analyzed by SPSS software.

[0039] Table 1. Malate degradation rate (%) at different strain ratios

[0040] Table 2. Condensed tannin degradation rate (%) at different strain ratios

[0041] Results and analysis: As shown in Tables 1 and 2, Oenococcus oeni dominated malate degradation in mixed fermentation, while Lactobacillus brevis mainly promoted condensed tannin degradation. As the proportion of Lactobacillus brevis increased, the malate degradation rate gradually decreased, while the condensed tannin degradation rate slightly increased. When the ratio reached 1:4, the malate degradation rate decreased to about 20% or less, similar to the pure Lactobacillus brevis group, indicating that the insufficient number of Oenococcus oeni led to insufficient malate-lactic acid conversion.

[0042] The experimental data showed that the optimal conditions were inoculation of Oenococcus oeni and Lactobacillus plantarum at a ratio of 2:1 and fermentation for 24 h. In terms of degradation rate of malic acid, the 2:1 ratio group reached 24.35±0.27% at 24 h, which was higher than the two pure bacterial groups (1:0 group 23.67±0.87% and 0:1 group 21.22±0.51%), and had obvious advantages compared with the 1:1 group peak 22.38±0.16%. At 30 h, the malic acid degradation rate of the 2:1 ratio group decreased slightly to 23.03±0.19%, but still maintained a high level, indicating that its fermentation performance was relatively stable.

[0043] In terms of condensed tannin degradation rate, the 2:1 ratio group reached 28.58±0.12% at 24 h, which was the highest among all experimental groups, indicating that the strain activity was best under this condition. Although the strain ratios of 1:2 and 0:1 groups showed similar degradation rates (28.36±0.24% and 27.19±0.30%) after 30 h of fermentation, the 2:1 group had reached a degradation effect close to the peak at 24 h, with the highest time efficiency. From the previous literature search results, Lactobacillus plantarum played a major role in degrading condensed tannin, but when the ratio of Oenococcus oeni to Lactobacillus plantarum was 1:2 and 0:1, the fermentation efficiency was not as good as that of the 2:1 group.

[0044] In summary, fermentation at a ratio of 2:1 for 24 h can balance the degradation efficiency and fermentation time, not only ensuring high malic acid and tannin degradation rates, but also avoiding the efficiency decline that may be caused by excessive fermentation, fully exerting the synergistic effect of Oenococcus oeni and Lactobacillus plantarum, optimizing the economic efficiency of the fermentation process while ensuring the degradation effect.

[0045] Example 2 Single factor experiment This example optimizes the fermentation process through single factor experiment. The following experiments are included: Effect of initial pH on total acid degradation rate of rose hip juice: Under the conditions of fermentation temperature 31℃, inoculum size 5%, and fermentation time 18 h, adjust the initial pH of rose hip juice to 3.4, 3.6, 3.8, 4.0, and 4.2, and study the effect of different pH on malic acid and condensed tannin degradation rate; (2) Effect of fermentation temperature on total acid degradation rate of rose hip juice: Fix the initial pH at 3.8, inoculum size at 5%, and fermentation time at 18 h, and record the changes in malic acid and condensed tannin degradation rate at fermentation temperatures of 25, 28, 31, 34, and 37℃; (3) Effect of inoculum size on total acid degradation rate of rose hip juice: Under the conditions of initial pH 3.8, fermentation temperature 31℃, and fermentation time 18 h, set the inoculum size gradient at 1%, 3%, 5%, 7%, and 9%, and explore its effect on malic acid and condensed tannin degradation rate; (4) Effect of fermentation time on the total acid degradation rate of prickly pear juice: With the initial pH 3.8, fermentation temperature 31℃ and inoculum amount 0.05% fixed, the changes in malic acid degradation rate and condensed tannin degradation rate were investigated when the fermentation time ranged from 6 h to 30 h.

[0046] As shown in Figure 1, when the inoculum size was 5%, the malic acid degradation rate increased sharply from 19.7% to 25.92%, and then the growth rate slowed down significantly. When the inoculum size increased to 9%, the malic acid degradation rate reached 27.69%, showing a positive correlation between the increase in malic acid degradation rate and the increase in inoculum size. The reason for this pattern may be that at an inoculum size of 5%, the number of microorganisms and their enzyme activity were sufficient to significantly promote the degradation of malic acid. However, when the inoculum size increased to 9%, the microbial density was too high, leading to insufficient resources such as growth factors and nutrients. This intensified competition among microorganisms, thus limiting the further acceleration of the degradation process. At an initial pH of 4, the malic acid degradation rate reached its highest value of 23.41% across all levels. When the fermentation time was extended to 24 h, the degradation rate peaked at 24.81%. The malic acid degradation rate first increased and then decreased with increasing fermentation temperature, stabilizing at 26.33% at 28℃. This indicates that the degradation rate peaked at pH 4 and fermentation temperature of 28℃, suggesting that the effect of microbial metabolism on the target substance was strongest at this temperature. The peak at 24 h of fermentation time may be due to side reactions or depletion of nutrients caused by prolonged fermentation.

[0047] From the degradation rate of condensed tannins ( Figure 2 The horizontal axis of the upper right figure is the same as... Figure 1 As shown in the upper right figure, when the inoculum size was 5%, the degradation rate of condensed tannins reached 31.84%, then gradually decreased and stabilized at 32.22%. When the fermentation time was 24 hours, the degradation rate was 28.96%, and then the degradation rate tended to stabilize, indicating that degradation was close to saturation within 24 hours of fermentation. The experimental results presented by these two factors can also be explained by the competitive relationship in terms of microbial numbers. At an initial pH of 4, the degradation rate of condensed tannins was 32.72%, significantly higher than under other pH conditions. At a temperature of 28℃, the degradation rate was 31.65%, also the highest among the five levels, indicating that this condition was most suitable for the metabolic reaction system of microbial degradation of condensed tannins.

[0048] From the analysis of the degradation rates of malic acid and condensed tannin, inoculation amount, initial pH, fermentation time and fermentation temperature can be used as key factors affecting the degradation process. The degradation rate of malic acid can be significantly improved and the degradation rate of condensed tannin can reach the peak value when the inoculation amount is 5%. The degradation rates of malic acid and condensed tannin both reach the peak value when the initial pH is 4 and the fermentation temperature is 28℃. The stability and efficiency of the two degradation reactions are better at the temperature of 28℃, so the conditions of the temperature of 28℃, the inoculation amount of 5%, the initial pH of 4 and the fermentation time of 24 h are selected for the response surface optimization experiment to further explore the best roxburgh rose juice fermentation process.

[0049] Example 3 Response surface optimization of roxburgh rose juice fermentation process With temperature (A), inoculation amount (B), initial pH (C) and fermentation time (D) as factors and the degradation rates of malic acid and condensed tannin (%) as response values, Box-Behnken response surface optimization design is carried out, and the test scheme and results are shown in Table 3.

[0050] Table 3 Response surface test design and results of roxburgh rose juice acid reduction and bitterness reduction process

[0051] Through multiple regression fitting, the quadratic regression equation (ignoring insignificant terms) of each factor and the degradation rates of malic acid (Y1) and condensed tannin (Y2) is obtained: Y1=-3259.40+3.15B+1337.58C+0.37D-1.47AC+0.08AD+1.00BC+1.14CD-0.69A²-0.58B²-165.78C²- 0.15D2 Y2=-3253.07+38.70A+4.20B+1354.2C+0.05AD+2.44BC+0.08BD-0.73A²-1.40B²-171.94C²-0.11D² Table 4 Analysis of variance of regression model- malic acid Note: * means P the observed results are statistically significant; ** means P the observed results are extremely statistically significant.

[0052] From Table 4, the model P <0.0001, extremely significant, the regression model of the misfit term P =0.0726>0.05, the model correction determination coefficient R 1Adj= 0.9907, the determination coefficient R1 value was 0.9954, indicating that the model and the experimental results fitted well. From the F value, the order of the influence of each factor on the degradation rate of malic acid in rose apple juice was B > C > D > A, i.e. inoculum > initial pH > fermentation time > fermentation temperature. From the perspective of quadratic terms, AC, AD, CD and the square terms of each factor had a significant effect on the degradation rate of malic acid, and the square of the initial pH had the greatest impact, followed by the square of the temperature and the square of the time.

[0053] Table 5 Regression model analysis of variance results - condensed tannin Note: * means P value ≤ 0.05, the observed results were statistically significant; ** means P value ≤ 0.01, the observed results were extremely statistically significant.

[0054] From Table 5, the model P <0.0001, extremely significant, the regression model of misfit P = 0.261 > 0.05, the model correction determination coefficient R 2Adj = 0.9838, the determination coefficient R2 value was 0.9919, indicating that the model and the experimental results fitted well. From the F value, the order of the influence of each factor on the degradation rate of condensed tannin in rose apple juice was B > A > C > D, i.e. initial temperature > inoculum > initial pH > time. From the perspective of quadratic terms, AD, BC, BD and the square terms of each factor had a significant effect on the degradation rate of malic acid, and the square of the initial pH had the greatest impact, followed by the square of the temperature and the square of the inoculum.

[0055] From the response surface optimization three-dimensional graph ( Figure 3 and Figure 4 ), the surface of the interaction term of each factor had a maximum value, indicating that there was a point that made the degradation rate of malic acid and condensed tannin reach the maximum value, which was consistent with the results in Table 4 and Table 5, i.e. the square terms of each factor had a significant effect on the response value.

[0056] The preferred fermentation conditions were predicted as follows: fermentation temperature 27.935 ℃, inoculation amount 5.315%, initial pH 4.009, and fermentation time 24.135 h, with the goal of maximizing the degradation rate of malic acid and condensed tannin in the roxburgh rose juice. Under these conditions, the predicted value of the degradation rate of malic acid in the roxburgh rose juice was 37.19%, and the predicted value of the degradation rate of condensed tannin in the roxburgh rose juice was 43.19%. Considering the convenience of actual operation, the fermentation parameters were adjusted as follows: fermentation temperature 28 ℃, inoculation amount 5%, initial pH 4, and fermentation time 24 h. Two parallel experiments were conducted under the above conditions, and the measured average values were 36.57% and 42.65%, respectively, with an error of no more than 2%, indicating that the model fitting degree was good.

[0057] Example 4 1) Sterilize and cool the roxburgh rose juice; 2) Activate Oenococcus oeni and Lactobacillus plantarum on the culture medium to obtain bacterial liquid with a cell concentration of 1×10 9 Steps 1) and 2) were the same as in Example 1. 3) Adjust the initial pH of the cooled roxburgh rose juice to 4.0, inoculate Oenococcus oeni and Lactobacillus plantarum in the roxburgh rose juice at a ratio of 2:1, fermentation temperature 28 ℃, inoculation amount 5%, and fermentation time 24 h.

[0058] The tannin content of the roxburgh rose ferment beverage obtained after fermentation was 1109.7 mg / 100 g, and the degradation rate of condensed tannin in the roxburgh rose juice was 42.65%.

[0059] First, the roxburgh rose juice and the fermented roxburgh rose juice of Example 4 were subjected to physicochemical analysis and sensory evaluation: The SPME extraction head was inserted into the headspace portion of the sample through the bottle cap to extract the sample, and then the extraction head was inserted into the GC-MS sample inlet, and the chromatographic column was DB-WAX (30.0 m×250 μm, 0.25 μm). Qualitative and quantitative analysis of the sample was performed: the detected components were qualitatively analyzed using the MS database NIST20 and retention time; the column loss peaks were excluded from the database screening results. Quantitative analysis was performed using the area normalization method and internal standard semi-quantitative method.

[0060] The composition and content of volatile compounds in the before and after fermentation of the roxburgh rose juice are shown in Table 6. The total ion chromatograms of the volatile compounds in the before and after fermentation of the roxburgh rose juice are shown in Figures 5 and 6. A total of 42 aroma compounds were detected in the roxburgh rose original juice, with a total concentration of 32.5 mg / L, of which octanoic acid 27.2 mg / L, cyclohexanone 271.4 mg / L and Z-3-hexenol 8.6 mg / L were the main components. After fermentation, 38 substances were detected in the sample, with a total concentration of 22.8 mg / L, forming a new balance system mainly composed of Z-3-hexenol 17.2 mg / L, octanoic acid 15.9 mg / L and phenethyl alcohol 0.5 mg / L.

[0061] Referring to Table 6, in the original juice, acid substances accounted for an absolute advantage of 93.5%, with a total concentration of 30.4 mg / L, and octanoic acid as a characteristic component, with a content of 27.2 mg / L. Alcohols accounted for 3.2%, with a total concentration of 1.03 mg / L, with Z-3-hexenol as the representative, with a content of 8.6 mg / L. Ester content was the lowest, accounting for only 1.4%, with a total concentration of 0.45 mg / L, mainly derived from dioctyl phthalate 0.40 mg / L. After fermentation, the proportion of acids decreased to 76.8%, and the total concentration decreased to 17.5 mg / L, but still maintained a dominant position, and the content of the highest octanoic acid decreased to 15.9 mg / L. At the same time, the proportion of alcohols increased significantly to 18.4%, with a total concentration of 4.2 mg / L, and the concentration of Z-3-hexenol doubled to 17.2 mg / L. Not only did the total concentration of ester substances increase by 44% to 0.65 mg / L, but the proportion also increased to 2.8%, the number of species increased from 8 to 12, and new substances such as butanediol diacetate 0.15 mg / L significantly changed the original ester composition characteristics.

[0062] From the data, fermentation improved some bad odors: 2-methylhexanoic acid, valeric acid and other substances with a foul odor were completely degraded, and the concentration of phthalate, which may be derived from the pollution of roxburgh rose original juice packaging materials, was reduced.

[0063] Table 6 Comparison of aroma compounds in roxburgh rose juice before and after fermentation

[0064] Note: * indicates that cyclohexanone is an internal standard.

[0065] Two, sensory evaluation Four graduate students and seventeen undergraduate students were recruited to conduct sensory evaluation test on the product of Example 4. Basic taste evaluation was used to compare the differences in the five basic tastes of the roxburgh rose juice before and after fermentation, with a 30-point scoring system, 0-10 indicating very weak or almost no flavor; 11-20 indicating obvious flavor, but slightly unbalanced; 21-30 indicating outstanding flavor, balanced and coordinated. Table 7 was used to convert the subjective experience of color, aroma, taste, and organizational state into quantifiable scores, Table 7 Sensory evaluation table of fermented roxburgh rose juice After removing outliers, the absolute values of kurtosis and skewness of the five basic taste indicators of roxburgh rose juice before and after fermentation were less than 1, indicating that the data approximately followed a normal distribution, so the mean value could effectively represent the overall characteristics. Paired sample t-test was conducted by SPSS 26.0, and the results showed that there were significant differences in sour taste, sweet taste and bitter taste before and after fermentation. Specifically, the sour taste score decreased from 24.95 to 19.43 after fermentation, and the bitter taste score decreased from 23.10 to 18.62, which was consistent with the downward trend of malic acid and condensed tannin content detected by response surface optimization. At the same time, the sweet taste score increased significantly from 8.52 to 12.19, and this synergistic change in taste characteristics may improve product acceptance through sensory compensation effect. The fresh taste index did not show significant changes before and after fermentation.

[0066] Table 8 Comparison of basic taste differences of roxburgh rose juice before and after fermentation-t test

[0067] *** P <0.001 extremely significant, ** P <0.01 very significant, * P <0.05 significant, “-” not significant The sensory quality analysis of fermented roxburgh rose juice is shown in Table 9, which performs best in the organizational state with a score of 21.60±3.02, the juice body is uniform and consistent, without stratification, sedimentation phenomenon, and no visible impurities; the aroma is coordinated, with rich and pure fermentation flavor, with a score of 20.85±2.64, close to the ideal level; the taste is in the transition interval, with a score of 19.85±2.78, the sour, bitter and sweet are basically coordinated, the taste is acceptable, the roxburgh rose flavor is obvious but the aftertaste is short; the color score is relatively low, 18.75±2.92, the color is darker than the original roxburgh rose juice, but still within the acceptable range. The scores of each dimension show that the overall quality of the fermented product is good, and the taste and color have further optimization space, From the data distribution characteristics, the skewness of color is 0.36, and the kurtosis is -1.40, indicating that the subjects as a whole give a moderate evaluation, but there are a few evaluators with high scores, resulting in a slightly higher average than the median; the skewness of aroma is -0.85, and the kurtosis is -0.22, showing a left-skewed distribution, which may reflect that some subjects have a lower acceptance of the aroma of fermented Rosa roxburghii fruit juice, while most people evaluate it at a moderate or preferred level; the skewness of taste is -0.91, and the kurtosis is -0.45, indicating a left-skewed distribution, which means that some subjects are still not satisfied with the taste of fermented Rosa roxburghii fruit juice, reflecting the viscosity, turbidity, and uniformity of the juice, with a skewness of -0.42 and a kurtosis of -0.51, indicating that some subjects are not satisfied with the organization state, but the data distribution is wide, and the high segment still makes this dimension score the highest (see Figure 7 ).

[0068] Overall, the subjects gave a higher and stable evaluation of the color and organization state of the fermented Rosa roxburghii fruit juice.

[0069] Table 9 Analysis of sensory evaluation results of fermented Rosa roxburghii fruit juice

[0070] Three, total soluble solids, total acid, and sugar-acid ratio before and after fermentation As shown in the table below, the sugar-acid ratio of Rosa roxburghii is between 12.3 and 14.8, which is close to that of citrus fruits, but the content of malic acid and citric acid in the juice is relatively high, so the taste is still relatively sour. The experimental results show that the sugar content of fermented Rosa roxburghii fruit juice decreased from 10.16 ± 0.02 °Brix to 6.19 ± 0.01 °Brix, and the titratable acid content decreased from 7.39 ± 0.15 g / L to 4.54 ± 0.08 g / L, which is consistent with the results of response surface optimization and sensory evaluation. The calculated sugar-acid ratio was 13.75 and 13.63, respectively, with no significant difference, indicating that the sugar and acid content remained in dynamic balance during fermentation.

[0071] The Florida Citrus Commission in the United States stipulates that when the SAR (sugar-acid ratio) is 10-12, the sour taste is obvious, and when the SAR is greater than 15, the sweet taste dominates. The SAR of Rosa roxburghii fruit juice before and after fermentation is between 12 and 15, which is moderately sour and sweet, but after fermentation, the content of malic acid, which has the highest content and a sharp sour taste, is partially converted into lactic acid, which has a soft and long sour taste, and the content of condensed tannin with strong bitterness also decreases significantly, so the advantage of balanced sour and sweet is more prominent.

[0072] Table 10 Total soluble solids, total acid, and sugar-acid ratio before and after fermentation

[0073] Four, vitamin C, total phenol, and total flavonoid content before and after fermentation Referring to the table below, after the combined fermentation of the rosehip juice by Oenococcus oeni and Lactobacillus plantarum, the content of vitamin C decreased from 1135.62 ± 3.68 mg / 100g to 650.11 ± 0.99 mg / 100g, with a loss rate of 42.8%. Nevertheless, the content of vitamin C in the fermented rosehip juice was still higher than that in common fruits, such as kiwi (62 mg / 100g), orange (33 mg / 100g) and strawberry (47 mg / 100g), showing its potential as a natural high-vitamin C drink.

[0074] Table 11 Content of vitamin C in rosehip juice before and after fermentation

[0075] Referring to Figure 8 Before fermentation, the contents of total phenols and total flavonoids in the rosehip juice were 1227.81 mg / 100g and 136.38 mg / 100g, respectively, which decreased to 1088.14 mg / 100g and 108.25 mg / 100g after fermentation.

[0076] Although the present application has been described by way of examples, it should be understood that, without departing from the spirit and scope of the present application, improvements and modifications made to the present application by those skilled in the art shall all fall within the scope of protection of the present application.

Claims

1. A prickly pear enzyme beverage, characterized in that, The prickly pear enzyme beverage is made from prickly pear through fermentation. The total acid content of the prickly pear enzyme beverage is 4~5 g / L, and the condensed tannin content is 1000~1200mg / 100g.

2. The prickly pear enzyme beverage according to claim 1, characterized in that, The prickly pear enzyme beverage contains 4.3-4.7 g / L of total acid and 1080-1120 mg / 100g of condensed tannins.

3. The prickly pear enzyme beverage according to claim 1, characterized in that, The prickly pear enzyme beverage contains 600-700 mg / 100g of vitamin C and has a sugar-acid ratio of 13-14.

4. The prickly pear enzyme beverage according to claim 1, characterized in that, It is prepared by the following steps: 1) Sterilize and cool the original prickly pear juice; 2) *Lactobacillus chrysogenum* and *Lactobacillus plantarum* were activated separately on culture media to obtain cell concentrations that were independently determined to be 5–10 × 10⁻⁶ cells / day. 9 CFU / mL bacterial suspension; 3) Adjust the initial pH of the cooled prickly pear juice to 3.4~4.2, inoculate the prickly pear juice with *Lactobacillus oryzae* and *Lactobacillus plantarum* cultures, and ferment for 6~30 hours.

5. The prickly pear enzyme beverage according to claim 4, characterized in that, The prickly pear juice is obtained by pressing prickly pears, with a total acid content of 7-8 g / L and a condensed tannin content of 1850-2000 mg / 100g.

6. The prickly pear enzyme beverage according to claim 4, characterized in that, In step 2), the *Lactobacillus chrysogenum* was cultured on ATB medium and *Lactobacillus plantarum* on lactic acid bacteria liquid medium at 36-38°C for 1 day. The resulting bacterial culture was centrifuged and resuspended in physiological saline to achieve a cell concentration of 5-10 × 10⁻⁶ cells / mL. 9 CFU / mL.

7. The prickly pear enzyme beverage according to claim 4, characterized in that, In step 3), the volume ratio of the *Lactobacillus vinifera* and *Lactobacillus brevis* bacterial solutions is 1~2:1~2.

8. The prickly pear enzyme beverage according to claim 4, characterized in that, In step 3), the fermentation temperature is 25~37℃ and the inoculum size is 1~9%.

9. The prickly pear enzyme beverage according to any one of claims 4 to 8, characterized in that, In step 3), the fermentation time is 24-25 hours.

10. The prickly pear enzyme beverage according to any one of claims 4 to 8, characterized in that, In step 3), the initial pH is 4 and the fermentation temperature is 28℃.

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