Rosa roxburghii tratt and myotonin compound liquid and preparation method thereof

Abstract

The invention provides rosa roxburghii tratt and myotonin compound liquid and a preparation method thereof, and belongs to the technical field of beverage processing. The preparation method comprises the following steps: soaking myotonin, mixing the soaked myotonin with water, sequentially pulping, gelatinizing, and carrying out high-temperature alpha-amylase enzymolysis to obtain myotonin enzymatic hydrolysate; the coix seed enzymatic hydrolysate is inoculated with a probiotic BLH1 seed solution for fermentation; and after fermentation is completed, adding roxburgh rose juice into the fermentation liquid, and fermenting again to obtain the roxburgh rose and coix seed composite liquid. According to the invention, the sharp acerbity of the roxburgh rose and the grain flavor of the myotonin are successfully converted into a fermented beverage which is soft in sourness, extremely low in astringency, prominent in palatable taste, mellow in aftertaste, beneficial in efficacy and coordinated in flavor by virtue of microbial fermentation, and the effects of reducing acidity, reducing astringency, reducing blood sugar and improving sensory characteristics are realized.

Description

Technical Field

[0001] This invention relates to the field of beverage processing technology, and in particular to a prickly pear and coix seed compound liquid and its preparation method. Background Technology

[0002] Prickly pear has garnered attention for its extremely high vitamin C content and abundant polyphenols, polysaccharides, and other functional components; however, its strong astringent taste severely limits the product's acceptability and in-depth development. Job's tears, on the other hand, is a traditional grain resource, rich in polysaccharides, proteins, and various trace elements, possessing health benefits such as strengthening the spleen and removing dampness; however, its flavor is monotonous, and its added value is limited.

[0003] Combining prickly pear with Job's tears can achieve nutritional complementarity and flavor harmony, but simple physical mixing often fails to overcome the inherent astringency, excessive acidity, and thin taste of prickly pear. Summary of the Invention

[0004] The purpose of this invention is to provide a prickly pear and coix seed compound liquid and its preparation method, so as to solve the problems existing in the prior art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: One of the technical solutions of this invention is a method for preparing a prickly pear and coix seed compound liquid, comprising the following steps: (1) Soak the Job's tears and mix them with water, then grind and gelatinize them in sequence, and then hydrolyze them with α-amylase at high temperature to obtain Job's tears hydrolysate; (2) The coix seed hydrolysate was inoculated with probiotic BLH1 seed liquid for fermentation; (3) After fermentation, prickly pear juice is added to the fermentation liquid for further fermentation to obtain the prickly pear and coix seed compound liquid.

[0006] The second technical solution of the present invention is the prickly pear and coix seed compound liquid prepared by the above preparation method.

[0007] The present invention discloses the following technical effects: This invention utilizes microbial fermentation to successfully transform the sharp sourness of prickly pear and the grain flavor of Job's tears into a fermented beverage with a mild sour taste, very low astringency, prominent umami flavor, mellow aftertaste, and probiotic effects, achieving the effects of reducing acidity, astringency, and sugar, as well as enhancing sensory characteristics.

[0008] The total acid content of the prickly pear and coix seed compound liquid provided by this invention is reduced by 13.52%, the tannin content is significantly reduced compared with the unfermented prickly pear and coix seed compound liquid, the polysaccharide and GABA content is increased, the sugar content is reduced, the sweetness is mitigated, and the overall sensory evaluation reaches 80.45. It is a high-quality and flavorful new prickly pear and coix seed beverage. Attached Figure Description

[0009] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0010] Figure 1 These are the measurement results of the electronic tongue of the present invention. Detailed Implementation

[0011] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0012] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0013] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0014] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0015] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0016] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0017] This invention provides a method for preparing a prickly pear and coix seed compound liquid, comprising the following steps: (1) Soak the Job's tears and mix them with water, then grind and gelatinize them in sequence, and then hydrolyze them with α-amylase at high temperature to obtain Job's tears hydrolysate; (2) The coix seed hydrolysate was inoculated with probiotic BLH1 seed liquid for fermentation; (3) After fermentation, prickly pear juice is added to the fermentation liquid for further fermentation to obtain the prickly pear and coix seed compound liquid.

[0018] In a preferred embodiment of the present invention, the ratio of Job's tears to water is 1:(12-18) w / v; In a preferred embodiment of the present invention, the gelatinization temperature is 85-95 °C and the time is 15-30 min.

[0019] In a preferred embodiment of the present invention, the amount of the high-temperature α-amylase used is 170-230 U / g; the enzymatic hydrolysis temperature is 85-95 °C, and the time is 30-60 min.

[0020] In a preferred embodiment of the present invention, the inoculation amount of the probiotic BLH1 seed liquid is 3-5% v / v; the fermentation conditions are: closed fermentation at 37-39 °C in a carbon dioxide environment for 10-14 h.

[0021] In this invention, the probiotic BLH1 is a subspecies of Bifidobacterium animalis BLH1 (accession number: CCTCC M 20221979) isolated from Guizhou Hongsuantang.

[0022] In a preferred embodiment of the present invention, in step (3), the volume ratio of fermentation liquid to prickly pear juice is (3:7)-(5:5).

[0023] In a preferred embodiment of the present invention, the conditions for the second fermentation in step (3) are: 37-39 °C, 150-200 r / min shaker culture for 28-36 h.

[0024] The present invention also provides a prickly pear and coix seed compound liquid prepared by the above preparation method.

[0025] The materials and strains used in the embodiments of this invention are as follows: Prickly pear (Longli County, Guizhou Province); Job's tears (Guizhou Renxin Agricultural Development Co., Ltd.). Bifidobacterium animalis subspecies BLH1 (accession number: CCTCC M 20221979) was isolated from Hongsuantang, Guizhou. BLH1 was anaerobically cultured in PTYG liquid medium and MRS solid medium at 37 ℃ for 48 h.

[0026] High-temperature α-amylase (Jiangsu Ruiyang Biotechnology Co., Ltd.)

[0027] PTYG medium: 5 g soybean peptone, 5 g tryptone, 10 g glucose, 10 g yeast extract, 1 mL Tween 80, 0.05 g L-cysteine ​​hydrochloride, 4 mL salt solution, 1000 mL distilled water. Salt solution (0.2 g anhydrous calcium chloride, 0.48 g magnesium sulfate heptahydrate, 1 g dipotassium hydrogen phosphate, 1 g potassium dihydrogen phosphate, 10 g sodium carbonate, 2 g sodium chloride, 1000 mL distilled water, refrigerated for later use).

[0028] MRS medium: 10 g peptone, 20 g glucose, 5 g beef meal, 4 g yeast extract, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate heptahydrate, 5 g sodium acetate trihydrate, 2 g triammonium citrate, 0.2 g magnesium sulfate heptahydrate, 0.05 g manganese sulfate tetrahydrate, 20 g / L agar, 1000 mL distilled water.

[0029] In the statistical analysis of the data in this embodiment, all experimental samples were performed in triplicate, and the results are expressed as mean ± standard deviation. Statistical analysis of all data was performed using SPSS 27.0 and Excel. Tukey's test was used to analyze the significance of differences, with different letters representing significant differences. P< 0.05 indicates a significant difference. The graph was generated using Origin 2021.

[0030] Example 1 1. Materials and Methods 1.1 Preparation and fermentation of prickly pear and coix seed compound liquid Take out the fresh prickly pear fruit frozen at -20 ℃, thaw it, wash it three times, remove the leaves and stems, juice it with a juicer, strain it to obtain prickly pear juice, and store it in the dark for later use.

[0031] After rinsing high-quality Job's tears with clean water, soak them overnight at room temperature, then grind them into a paste at a material-to-water ratio of 1:15 (w / v, g / mL). The paste is then gelatinized in a 90 ℃ water bath for 20 min, followed by enzymatic hydrolysis with high-temperature α-amylase (200 U / g) at 90 ℃ for 45 min. The resulting Job's tears hydrolysate is refrigerated for later use.

[0032] The prickly pear juice and coix seed enzymatic hydrolysate were sterilized at 90 ℃ (10 min) and 121 ℃ (20 min), respectively. After cooling, a portion of the coix seed enzymatic hydrolysate was inoculated with 4% (v / v) probiotic BLH1 seed culture (concentration 5×10⁻⁶). 8CFU was fermented in a sealed CO2 incubator at 37 ℃ for 12 h, and then prickly pear juice (the volume ratio of coix seed hydrolysate to prickly pear juice was 4:6) was added. The mixture was then cultured in a shaker at 37 ℃ and 180 r / min for 32 h and then refrigerated for later use.

[0033] 1.2 pH determination The pH value of the samples was recorded using a digital pH meter (Shanghai Lichen Bangxi Instrument Technology Co., Ltd.), and was calibrated with standard buffer solutions of pH 4 and 7 before use.

[0034] 1.3 Determination of total acidity Centrifuge 10 ml of sample (10000 rpm, 10 min), dilute the supernatant 10 times with deionized water and shake well, titrate with sodium hydroxide standard solution (0.1 mol / L) in the presence of phenolphthalein, and express the titration acidity result as a percentage of citric acid.

[0035] 1.4 Determination of Vitamin C The method for determining vitamin C in the sample was based on high performance liquid chromatography (HPLC) as specified in GB 5009.86-2016, "National Food Safety Standard - Determination of Ascorbic Acid in Food," with some modifications.

[0036] (1) Chromatographic conditions The chromatographic column was an Agilent C18 column (4.6 × 250 mm inner diameter, 5 μm particle size); the detector was a UV detector; the mobile phase was 0.1% phosphoric acid solution-100% methanol (98:2), filtered through a 0.45 μm filter membrane, and sonicated for 25 min; the flow rate was 0.7 mL / min; the detection wavelength was 245 nm; the column temperature was 25 ℃; the injection volume was 20 μL; and the reaction time was 10 min.

[0037] (2) Preparation of Vitamin C Standard Solution Weigh 0.01 g of L-ascorbic acid standard and dilute to 10 mL with metaphosphoric acid solution (20 g / L). This solution can be stored for one week at 2–8 °C protected from light. Pipette 0 mL, 0.005 mL, 0.05 mL, 0.1 mL, 0.25 mL, 0.5 mL, and 1.0 mL of vitamin C standard solution, respectively, and dilute to 10 mL with metaphosphoric acid solution (20 g / L) to obtain a series of working solutions of vitamin C standard at different concentrations. Prepare and use immediately. The vitamin C standard curve obtained by the above method is as follows: y = 138.19x + 61.121 R 2 = 0.9998 (3) Sample determination Weigh 1 g of sample into a beaker, add metaphosphoric acid solution (20 g / L) to a final volume of 100 mL, sonicate for 10 min, centrifuge (10000 rpm, 10 min), filter the supernatant through a 0.22 μm filter membrane, and then analyze it.

[0038] 1.5 Determination of Tannins (1) Determination of standard curve Accurately weigh 0.1 g (accurate to 0.001 g) of gallic acid standard into a 100 ml volumetric flask and dilute to volume. The concentration of this gallic acid solution is 1000 mg / L. Pipette 0, 10, 20, 30, 40, and 50 μL of the 1000 mg / L gallic acid standard solution, respectively, and add 1000, 990, 980, 970, 960, and 950 μL of water to make a volume of 1 ml, respectively, to obtain standard solutions with gallic acid concentrations of 0, 10, 20, 30, 40, and 50 mg / L. Add 5 ml of water, 1 ml of Folin-Ciocalteu reagent, and 3 ml of 20% sodium carbonate solution to each solution, heat in boiling water for 2 min, mix well, develop color, and let stand for 2 h. Measure the absorbance of the standard solution at 760 nm as the ordinate to obtain the standard curve: y = 0.0078x + 0.0381 R 2 = 0.9992 (2) Sample determination Take 1 ml of sample and add 20 ml of 75% ethanol at a ratio of 1:20. Extract by sonication at 60 °C for 40 min, then centrifuge (8000 rpm, 10 min). Store the supernatant at low temperature for later use. Take 1 ml of the supernatant to be tested and perform the determination according to the above standard curve determination procedure.

[0039] 1.6 Determination of Polysaccharides The polysaccharide content was determined using the phenol-sulfuric acid reagent method.

[0040] (1) Determination of standard curve Pipette 0, 0.2, 0.4, 0.6, 0.8, and 1 mL of glucose standard solution (0.1 mg / mL) into test tubes, bring the volume to 1 mL with distilled water, add 1 mL of 6% phenol, vortex for 5 s, then carefully and slowly add 5 mL of concentrated sulfuric acid. Transfer the test tubes to boiling water, vortex for 10 s, then boil in a water bath for 15 min. Cool to room temperature and measure the absorbance at 490 nm. Plot a standard curve with the glucose standard solution concentration on the x-axis and the corresponding absorbance on the y-axis. The equation of the standard curve is as follows: y = 5.5376x + 0.078 R 2 = 0.9934 (2) Sample determination Centrifuge the sample (12000 rpm, 10 min). Dilute the supernatant 100 times with water and centrifuge again (10000 rpm, 10 min). Reserve the supernatant. Pipette 5 mL of the 100-fold diluted supernatant into a centrifuge tube, add 20 mL of anhydrous ethanol, refrigerate and allow to stand for 16 h for alcohol precipitation, then centrifuge again (10000 rpm, 10 min). Discard the supernatant, and reconstitute the residue with water to 5 mL. Pipette 1 mL of this solution into a test tube and proceed with the standard curve determination procedure described above. Substitute the measured absorbance value into the standard curve equation to obtain the polysaccharide content in the sample.

[0041] 1.7 Determination of γ-aminobutyric acid (GABA) The γ-aminobutyric acid (GABA) in the sample was determined by high performance liquid chromatography.

[0042] (1) Chromatographic conditions Column: Acclaim TM 120 C18 column (4.6 × 250 mm, 5 μm); UV detector; mobile phase: 0.12% phosphoric acid solution: 100% acetonitrile (50:50), filtered through a 0.45 μm filter membrane, sonicated for 25 min; flow rate: 1 mL / min; detection wavelength: 370 nm; column temperature: 35 ℃; injection volume: 10 μL; detection time: 10 min.

[0043] (2) Preparation of GABA standard solution GABA standards were prepared at concentrations of 0 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, 100 μg / mL, and 200 μg / mL. 0.4 mL of each standard solution was placed in a 10 mL brown centrifuge tube, followed by the addition of 0.4 mL of sodium bicarbonate (0.5 mol / L) and 0.4 mL of 1% 2,4-dinitrofluorobenzene solution (FDNB). The FDNB solution was dissolved in acetonitrile and stored in a brown bottle. The mixture was then placed in a 60°C water bath in the dark for 1 h to induce derivatization. After the reaction, 2.8 mL of 0.12% phosphoric acid solution was added, the mixture was vortexed for 10 s, and filtered through a 0.22 μm filter membrane. The filtrate was then analyzed. The resulting GABA standard curve equation is as follows: y = 10.667x + 1.6858 R 2 = 0.9999 (3) Sample determination Pipette 4 ml of sample into a centrifuge tube and centrifuge (10000 rpm, 10 min). Filter the supernatant through a 0.45 μm filter membrane. Then, take 0.4 mL of this supernatant and perform the derivatization reaction as described above, and filter it through a membrane for instrumentation.

[0044] 1.8 Determination of Carbohydrates (1) Chromatographic conditions Chromatographic column: Amino acid column (4.6 mm × 250 mm, 5 μm); Mobile phase: Acetonitrile + Water = 70 + 30 (v / v); Flow rate: 1.0 mL / min; Column temperature: 40 °C; Injection volume: 10 μL; Differential spectrometer refractive index detector conditions: 40 °C.

[0045] (2) Preparation of standard solutions Weigh out 1 g each of fructose dried at 90 ℃±2 ℃ for 2 h and glucose, sucrose, maltose, and lactose dried at 96 ℃±2 ℃ for 2 h (accurate to 0.001 g), dissolve them in water, transfer them to a 50 mL volumetric flask, add 2.5 mL of acetonitrile, and dilute to the mark with water. Store at 0 ℃ -4 ℃ in a sealed container. Shelf life is 3 months.

[0046] Pipette 0.100, 1.00, 2.00, 3.00 and 5.00 mL of the mixed standard stock solution (20.0 mg / mL) into a 10-liter volumetric flask, and dilute to the mark with water to prepare mixed standard working solutions with mass concentrations of fructose, glucose, sucrose, maltose and lactose of 0.200, 2.00, 4.00, 6.00 and 10.00 mg / mL, respectively. Prepare and use immediately.

[0047] (3) Sample determination Pipette 1 mL of sample into a 10 mL centrifuge tube, add 5 mL of water, then slowly add 0.5 mL of zinc acetate solution (1 mol / L) and 0.5 mL of potassium ferrocyanide solution (0.25 mol / L), vortex to mix, sonicate for 30 min, add water to make up to 10 mL, mix well and let stand.

[0048] After centrifuging the above sample (1000 rpm, 10 min) to obtain the supernatant, it was filtered through a membrane for analysis. The results were calculated using equation (2): (2) In the formula, X is the content of fructose, glucose, sucrose, maltose and lactose in the sample (mg / mL); ρ is the mass concentration of fructose, glucose, sucrose, maltose and lactose in the sample obtained according to the standard curve (mg / mL); V is the volume of the sample (mL); f is the dilution factor of the sample; and V0 is the amount of sample taken.

[0049] 1.9 Sensory Evaluation Referring to the evaluation criteria of GB7101-2022 "National Food Safety Standard for Beverages", and combining the sensory characteristics of prickly pear and Job's tears, 20 food professionals scored the prickly pear and Job's tears compound liquid from four aspects: color, aroma, taste, and state, with a total score of 100. The sensory scoring criteria for the compound liquid are shown in Table 1 below.

[0050] Table 1 Sensory Evaluation Criteria for Prickly Pear and Coix Seed Compound Liquid

[0051] 1.10 Measurement of electronic tongue The electronic tongue of the sample was measured using the SA402B electronic tongue from Insent Corporation of Japan, simulating the taste perception mechanism of a living organism. The sensor was first cleaned in a cleaning solution for 90 s, then in a reference solution for 120 s, followed by another 120 s of cleaning with a different reference solution. The sensor was then zeroed at its equilibrium position for 30 s. The testing time was 30 s. After a 3-sip cleaning with the reference solution, the sensor was immersed in a new reference solution for a 30-sip aftertaste test. The average of three tests was taken as the result.

[0052] 2. Results Table 2. Effects of probiotic fermentation on the quality of prickly pear and coix seed compound liquid

[0053] Note: Different letters in the same row of the table indicate significant differences. P <0.05); Y0: Job's tears liquid; CO: prickly pear juice; Y12: Job's tears liquid fermented for 12 hours; YC0: unfermented prickly pear and Job's tears compound liquid; Y12C32: prickly pear and Job's tears compound liquid of the present invention.

[0054] 2.1 Effects of fermentation on pH and total acidity of prickly pear and coix seed extract Changes in pH can reflect changes in the growth rate, types and yields of metabolites, and microbial community structure during fermentation, which affect the flavor and texture of the product. As shown in Table 2, the pH of the fermented prickly pear and coix seed compound liquid (Y12C32) increased from 3.52 ± 0.01 to 3.56 ± 0.01 compared to the unfermented prickly pear and coix seed liquid (YC0). The total acid content of sample Y12C32 decreased from 6.88 ± 0.15 g / L in YC0 to 5.95 ± 0.24 g / L, a reduction of 13.52%. This is because BLH1 fermentation can degrade some organic acids in the prickly pear and coix seed compound liquid, indicating that the probiotic BLH1 fermentation of prickly pear and coix seed liquid has an acid-reducing effect.

[0055] 2.2 Effect of fermentation on vitamin C in prickly pear and Job's tears liquid Table 2 shows that the vitamin C in the compound solution mainly comes from prickly pear, which is an important bioactive substance of prickly pear. Water-soluble vitamins such as vitamin C are easily degraded when exposed to oxygen, moisture, light, heat, and pH changes during food manufacturing and storage. The vitamin C content of YC0, the unfermented prickly pear and Job's tears compound solution (669.13 ± 1.85 mg / 100g), is significantly higher than that of the fermented Y12C32 sample (584.53 ± 1.37 mg / 100g). This difference is mainly due to the instability of vitamin C in the fermentation environment. During fermentation, even in a closed environment, the metabolic activities of microorganisms consume nutrients and antioxidants in the system. Vitamin C is likely to be consumed directly by the microorganisms as a carbon source or to maintain intracellular redox balance. Simultaneously, trace amounts of reactive oxygen species or metabolites generated during fermentation may also accelerate the chemical oxidation of vitamin C. Therefore, the decrease in vitamin C content in the Y12C32 sample is the result of the combined effects of microbial metabolic consumption and chemical degradation in the fermentation environment.

[0056] 2.3 Effect of fermentation on tannins in prickly pear and coix seed extract Tannins, as polyphenolic compounds, are extremely high in prickly pear juice (C0), which perfectly matches the characteristic of prickly pear fruit being rich in polyphenols and is also the main source of its typical astringent taste. In contrast, the tannin content of coix seed extract (Y0) and its fermentation product (Y12) is extremely low, confirming that coix seed itself is not the main source of tannins. The tannin content of prickly pear and coix seed fermentation broth (Y12C32) (6484.62 ± 44.41 mg / L) is significantly lower than that of unfermented YC0 (6988.89 ± 64.53 mg / L), revealing the transformation and degradation of tannins by microbial fermentation. This is because tannins or other enzymes secreted by microorganisms can hydrolyze the ester bonds in tannins, breaking them down into gallic acid or other phenolic acids with smaller molecular weights and weaker astringency; in addition, tannins may also be consumed by microorganisms as part of a carbon source to resist oxidative stress. This process not only directly reduces the absolute content of tannins but may also alter their molecular structure and sensory properties, thereby helping to improve the astringent taste of the final product. This change has significant practical implications, demonstrating that through specific fermentation processes, it is possible to effectively reduce the tannin content that contributes to the strong astringency of prickly pear while retaining its beneficial polyphenols. This allows for the improvement of the taste and acceptability of complex beverages without the use of physical or chemical de-astringency methods.

[0057] 2.4 Effect of fermentation on polysaccharides from prickly pear and coix seed extract Table 2 shows that the polysaccharide content of sample Y12C32 (1.69 ± 0.06 mg / ml) was significantly higher than that of sample YC0 (1.32 ± 0.07 mg / ml). This trend is mainly attributed to the deep transformation and synergistic effect of microbial fermentation on the raw material matrix. YC0, as an unfermented composite liquid, primarily derived its polysaccharide content from the inherent, easily extractable soluble polysaccharides in the prickly pear juice and Job's tears liquid. Due to the lack of prior biotransformation, many large polysaccharides encapsulated in cell wall structures or existing in bound forms were not effectively released into the solution. The higher polysaccharide content of Y12C32 may be attributed to the secretion of a series of extracellular enzymes (such as cellulase, hemicellulase, and pectinase) by microorganisms during fermentation to obtain carbon and energy. These enzymes effectively degrade the cell walls of prickly pear and Job's tears, breaking down their dense structure and releasing previously insoluble polysaccharides, colloids, and other large molecules, significantly increasing the measurable polysaccharide concentration in the solution. Secondly, during their growth and metabolism, microorganisms may also synthesize and secrete some extracellular polysaccharides. Therefore, the higher polysaccharide content in the Y12C32 sample is not simply the sum of raw materials, but rather the result of microbial-led biotransformation and biosynthesis.

[0058] 2.5 Effect of fermentation on γ-aminobutyric acid (GABA) in prickly pear and coix seed extract Table 2 shows that the GABA content in YC0 mainly originates from the inherent content of its raw materials. After microbial fermentation, the GABA content in sample Y12C32 (65.14 ± 1.27 ug / ml) was significantly higher than that in YC0 (56.41 ± 1.98 ug / ml). This result may be due to the activation and significant enhancement of the GABA biosynthesis pathway within the system during microbial fermentation. During fermentation, microorganisms activate their glutamate decarboxylase (GAD) system to maintain intracellular pH homeostasis in an acidic environment. The core function of this system is to catalyze the decarboxylation reaction of glutamate, thereby generating GABA. Therefore, fermentation is an effective biological strategy for enhancing the content of GABA, a specific functional component, in compound beverages.

[0059] 2.6 Effect of fermentation on the sugar content of prickly pear and Job's tears liquid As shown in Table 3, compared with the unfermented YC0 sample, the Y12C32 sample obtained by fermentation with Bifidobacterium animalis BLH1 showed a significant decrease in the content of various sugars. The most direct and fundamental reason for this phenomenon is that the microorganisms consumed and utilized these sugars on a large scale as the primary carbon and energy source during fermentation. During fermentation, in order to maintain their growth, reproduction, and vigorous metabolic activities, the microorganisms continuously ingest easily usable simple sugars, such as fructose and glucose, from the culture environment. These sugars are broken down through metabolic pathways such as glycolysis in the microorganisms and converted into various metabolites, thus leading to a decrease in the sugar concentration in the fermentation broth.

[0060] Therefore, the overall decrease in sugar content in sample Y12C32 is a direct result of microbial fermentation metabolism. This change not only proves the activity of the cells, but is also directly related to the reshaping of the final product flavor. That is, by reducing sugar content, the sweetness is mitigated, and in synergy with the reduced acidity, it creates a more balanced, mellow flavor profile that is different from that of unfermented samples.

[0061] Table 3 Sugar content of prickly pear and Job's tears fermentation liquid

[0062] 2.7 Effects of fermentation on the electronic tongue of prickly pear and coix seed liquid like Figure 1 As shown, sample Y12C32 showed significantly lower response values ​​in the sourness and astringency dimensions compared to YC0. This is a crucial change closely related to biotransformation during fermentation. During fermentation, *Bifidobacterium animalis* BLH1 likely metabolized some organic acids in the complex liquid system (such as citric acid and malic acid naturally present in prickly pear) as preferential carbon sources, leading to a decrease in total acid content, ultimately manifested as a reduction in sourness intensity on the electronic tongue. This perfectly aligns with the previous assessment of total acid content, jointly confirming that the fermentation process plays a role in "softening the sourness." The reduction in astringency may be attributed to the biodegradation of tannins and other polyphenols abundant in the prickly pear raw material by microorganisms. These substances are the main source of bitterness and astringency, and their reduction directly improves the product's taste. The increased abundance of Y12C32 may be related to polysaccharides and peptides secreted by microorganisms, which impart a richer, fuller texture to the beverage. In summary, the electronic tongue data accurately reveals the profound impact of the fermentation process: through microbial fermentation, the Y12C32 sample effectively reduced the inherent sour and astringent irritating taste of the prickly pear and coix seed compound liquid, while enhancing its richness and pleasant aftertaste, ultimately creating a fermented beverage with a more balanced flavor, a smoother mouthfeel, and superior quality.

[0063] 2.8 Effect of fermentation on sensory evaluation of prickly pear and coix seed liquid As shown in Table 2, the Y12C32 sample scored significantly higher than the YC0 in sensory evaluation, a direct reflection of its comprehensive optimization after microbial fermentation. Fermentation significantly improved the flavor, texture, and functional components of the compound liquid, thus creating more appealing product characteristics.

[0064] Fermentation plays a crucial role in influencing flavor and mouthfeel, which affect sensory acceptance. The total acid content of Y12C32 is lower than that of YC0, while its pH value slightly increases, effectively reducing the sharp acidity of the original prickly pear juice. More importantly, the tannin content of Y12C32 is significantly reduced, meaning that the key substances causing astringency have been effectively degraded by microorganisms, greatly reducing the astringency of the product. This change is corroborated by the reduced bitterness and astringency signals in electronic tongue analysis. Although fermentation consumes some simple sugars, which may reduce sweetness, it is precisely this readjustment of the sweet-acid ratio, combined with the reduced astringency, that creates a more refreshing and harmonious overall flavor. The polysaccharide content of Y12C32 is significantly higher than that of YC0, thanks to the degradation of macromolecules in the raw materials by microorganisms and the synthesis of their own extracellular polysaccharides, thus giving the beverage a fuller, smoother texture. At the same time, the GABA content of Y12C32 is also significantly increased. The enrichment of this functional component with health benefits not only increases the nutritional value of the product, but its umami flavor also contributes to the overall flavor richness.

[0065] In summary, Y12C32 demonstrates a comprehensive improvement in sensory quality compared to YC0. This improvement is attributed to the effective reduction of astringent tannins and moderate adjustment of acidity through microbial fermentation, while enriching the flavor with a long-lasting and pleasant aftertaste supported by abundant polysaccharides and a rich umami flavor. Ultimately, a prickly pear and Job's tears compound fermented beverage with superior taste, flavor, and some functional properties was developed.

[0066] 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 preparing a prickly pear and coix seed compound liquid, characterized in that, Includes the following steps: (1) Soak the Job's tears and mix them with water, then grind and gelatinize them in sequence, and then hydrolyze them with α-amylase at high temperature to obtain Job's tears hydrolysate; (2) The coix seed hydrolysate was inoculated with probiotic BLH1 seed liquid for fermentation; (3) After fermentation, prickly pear juice is added to the fermentation liquid for further fermentation to obtain the prickly pear and coix seed compound liquid.

2. The preparation method according to claim 1, characterized in that, The ratio of Job's tears to water is 1:(12-18) w / v.

3. The preparation method according to claim 1, characterized in that, The gelatinization temperature is 85-95 °C and the time is 15-30 min.

4. The preparation method according to claim 1, characterized in that, The amount of the high-temperature α-amylase used is 170-230 U / g; the enzymatic hydrolysis temperature is 85-95 °C, and the time is 30-60 min.

5. The preparation method according to claim 1, characterized in that, The inoculation amount of the probiotic BLH1 seed liquid is 3% v / v-5% v / v; the fermentation conditions are: closed fermentation at 37-39 °C in a carbon dioxide environment for 10-14 h.

6. The preparation method according to claim 1, characterized in that, In step (3), the volume ratio of fermentation liquid to prickly pear juice is (3:7)-(5:5).

7. The preparation method according to claim 1, characterized in that, In step (3), the conditions for the second fermentation are: 37-39 °C, 150-200 r / min shaker culture for 28-36 h.

8. The prickly pear and coix seed compound liquid prepared by the preparation method according to any one of claims 1-7.

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