Preparation method of Huizhou black rice wine

Abstract

The invention discloses a preparation method of Huizhou black rice wine. The preparation method comprises the steps of raw material treatment, ultrahigh pressure treatment, filtration separation, cooking gelatinization, saccharification, fermentation, filter pressing, color protection and sterilization. An ultrahigh pressure treatment process and a color protection process are added on the basis of a traditional rice wine making process, the content of total phenols, total flavonoids and anthocyanin in fermentation of the black rice wine can be remarkably increased by 1.95 times, 1.18 times and 2.48 times respectively, the heat resistance and storage stability of anthocyanin in the Huizhou black rice wine to high-temperature sterilization can be remarkably improved, and the quality of the Huizhou black rice wine is improved. If the black rice wine is stored for 90 days, the retention rate of anthocyanin in the black rice wine reaches up to 68.28% and is increased by 3.17 times. Meanwhile, lactobacillus sake subspecies is added in the fermentation process, specific alcohol, ester and other compounds can be formed in the black rice wine fermentation process, the relative content of ester substances is increased by 1.26 times, the unique fruit fragrance, flower fragrance, cream flavor and other flavors and tastes of Huizhou black rice wine are presented, the quality of the Huizhou black rice wine is greatly improved, and the black rice wine is suitable for being eaten by people. The method can be widely applied to the field of black rice wine production and processing.

Description

Technical Field

[0001] This invention relates to the field of rice wine manufacturing technology, and in particular to a method for preparing Huizhou black rice wine. Background Technology

[0002] Rice wine, also known as sweet wine or fermented rice, is a traditional Chinese fermented food. It is made primarily from glutinous rice through fermentation by microorganisms such as molds, yeasts, and bacteria. Huizhou rice wine, a specialty of Xiuning County, Huangshan City, Anhui Province, boasts a long history and unique regional flavor. Its unique brewing process has earned it a place on the list of intangible cultural heritage and it is a national geographical indication product. Black rice wine is an important category of Huizhou rice wine. It is made from black rice through processes such as steaming, mixing with yeast, saccharification, fermentation, pressing, filtering, and sterilization. Its color is purplish-red and crystal clear, with a sweet, mellow, and rich aroma. In addition to its own easily absorbed nutrients, fermented black rice wine is also rich in anthocyanins, polyphenols, amino acids, vitamins, and other bioactive components.

[0003] Anthocyanins are a class of natural, water-soluble flavonoids, readily soluble in water and polar solvents such as ethanol. Their basic structural unit is a 3,5,7-trihydroxy-2-phenylbenzopyran cation. Due to different substituent positions in the benzene ring, various anthocyanin monomers can be formed, mainly cyanidin, delphinidin, malvidin, pelargonidin, paeoniflorin, and petuniadin. However, the free anthocyanin monomers in black rice wine are relatively unstable. Environmental factors such as pH, light, temperature, and metal ions can all affect the stability and biological activity of anthocyanins, leading to deterioration in the color and reduction in anthocyanin activity during fermentation and storage of Huizhou black rice wine. This has become a key problem restricting the development of the black rice wine industry and urgently needs to be solved, greatly hindering the development of high-quality black rice wine. To improve the nutritional value of black rice wine, it is necessary to improve the existing rice wine-making process to enhance the stability of nutrients in black rice, especially anthocyanins, and improve the quality of the rice wine. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing Huizhou black rice wine, which solves the problems of poor nutritional components and color stability in black rice wine produced by existing rice wine processing techniques.

[0005] The technical solution adopted by this invention to solve its technical problem is: a method for preparing Huizhou black rice wine, comprising the following steps:

[0006] 1) Raw material processing: Select plump black rice and white glutinous rice without spoilage, wash them clean, put them into different containers for soaking, and add water at a mass ratio of 1:1 to 5 for 1 to 4 hours, with a water temperature of 20 to 25℃.

[0007] 2) Ultra-high pressure treatment: The black rice and water-soaked mixture obtained in step 1) are placed together in a high-pressure resistant bag, the air is removed, and the bag is sealed tightly. The bag is then placed in an ultra-high pressure chamber, with the pressure set to 50–200 MPa and the holding time to 5–15 minutes. The pressure increase rate is 8–35 MPa / s, the temperature used in the ultra-high pressure equipment is room temperature (20–25°C), and the pressure transmission medium is water. After reaching the set holding time, the pressure is rapidly released within 0.5–3 seconds (i.e., the depressurization rate is 100–250 MPa / s). This rapid pressure increase and decrease creates a pressure difference between the inside and outside of the black rice cell walls, and the high pressure accelerates cell wall rupture, promoting the release of internal substances.

[0008] 3) Filtration and separation: Pour the soaked black rice and water after step 2) into a 200-mesh screen for filtration and separation to obtain black rice and black rice water extract respectively. Store the black rice water extract at 4°C for later use.

[0009] 4) Steaming and gelatinization: Mix the soaked white glutinous rice from step 1) with the black rice processed in step 3) at a ratio of 1:1 to 3, and steam at a high temperature of 100℃ to 105℃ for 30 to 50 minutes. Since black rice has a high anthocyanin content, which is not conducive to fermentation, adding white glutinous rice helps with fermentation, and the resulting Huizhou black rice wine has a smoother taste.

[0010] 5) Saccharification: Stir and cool the cooked and gelatinized raw materials to 45℃~60℃, add 0.1%~0.2% α-amylase by mass and treat for 1~6h; continue stirring and cooling, maintain the temperature to 25℃~30℃, add 0.2%~0.5% yeast (composed of rice flour and Rhizopus, purchased from Jiangxi Shouchang Microbial Technology Development Co., Ltd.) by mass and saccharify for 3~4d.

[0011] 6) Fermentation: After saccharification, add 1-3.5 times the weight of black rice water extract and 0.1%-0.25% *Lactobacillus sakei* subsp. *sakei* suspension. After thorough stirring, set the temperature to 15℃-25℃ and ferment for 8-10 days. The *Lactobacillus sakei* subsp. *sakei* CMCI-8 has been deposited at the China Center for Type Culture Collection (CCTCC) on May 13, 2022, with accession number CCTCC NO.M 2022621 and a suspension concentration of 10. 6 ~10 8CFU / mL. The black rice water extract after ultra-high pressure treatment contains a large amount of active ingredients such as anthocyanins. The high temperature in the cooking gelatinization and saccharification steps can greatly affect the structure and activity of anthocyanins. Therefore, separating the black rice water through filtration and then adding it during fermentation is beneficial to the stability of the anthocyanin structure and the maintenance of anthocyanin activity.

[0012] 7) Press filtration: After fermentation, the fermentation product is pressed and filtered through a 200-mesh screen to obtain the filtrate. The filtrate is then added to a centrifuge and centrifuged at 8000-10000 r / min for 10-20 min to obtain the supernatant of black rice wine.

[0013] 8) Color protection: Add one or more of quercetin, gallic acid, syringic acid, and L-tryptophan to the supernatant, stir well, fill the bottle to the brim, and seal the bottle. The amount of quercetin added is 0.1–0.5 g / L, the amount of gallic acid added is 0.1–0.3 g / L, the amount of syringic acid added is 0.1–0.3 g / L, and the amount of L-tryptophan added is 0.5–3 g / L.

[0014] 9) Sterilization: Place the black rice wine treated in step 8) into a sterilization chamber and sterilize at 65℃~75℃ for 15~30 minutes. Store after natural cooling.

[0015] The beneficial effects of this invention patent are as follows: By improving the rice wine production process, this invention adds ultra-high pressure treatment and color protection processes to the traditional rice wine making process. This not only significantly increases the content of total phenols, total flavonoids, and anthocyanins in black rice wine fermentation by 1.95 times, 1.18 times, and 2.48 times, respectively, but also significantly improves the heat resistance and storage stability of anthocyanins in Huizhou black rice wine to high-temperature sterilization. For example, after 90 days of storage, the anthocyanin retention rate in black rice wine reaches 68.28%, an increase of 3.17 times. Simultaneously, this invention also adds *Lactobacillus sakei* subsp. to the fermentation process, which can form unique alcohols and esters during black rice wine fermentation. The relative content of esters increases by 1.26 times, and it presents the unique fruity, floral, and creamy flavors and taste of Huizhou black rice wine, significantly improving its quality.

[0016] The present invention will be described in more detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1a This is a GC-MS total ion chromatogram of the volatile components of black rice wine in Comparative Example 1 of the present invention.

[0018] Figure 1b This is a GC-MS total ion chromatogram of the volatile components of black rice wine in Example 1 of the present invention.

[0019] Figure 2 The sensory evaluation results of Huizhou black rice wine after fermentation according to the present invention are as follows.

[0020] Figure 3 This is a comparison of the content of the main active substances in Huizhou black rice wine after fermentation according to the present invention.

[0021] Figure 4 The variation of anthocyanin content in Huizhou black rice wine under different storage times is shown in this invention. Detailed Implementation

[0022] Example 1: A method for preparing Huizhou black rice wine, comprising the following steps:

[0023] 1) Raw material processing: Select plump black rice and white glutinous rice without spoilage, wash them clean, put them into different containers for soaking, and add water at a mass ratio of 1:3 for 4 hours, with a water temperature of 20-25℃.

[0024] 2) Ultra-high pressure treatment: The black rice and water soaking solution obtained in step 1) are placed together in a high-pressure resistant packaging bag, the air in the bag is removed, and the bag is sealed tightly. The bag is then placed in an ultra-high pressure chamber, with the pressure set at 200 MPa and the holding time at 12 minutes. Rapid pressure increase and decrease are used to create a pressure difference between the inside and outside of the black rice in a short time, which helps to break down the cell walls of the black rice cells and release nutrients. The optimal pressure increase rate is 20 MPa / s, the temperature used in the ultra-high pressure equipment is room temperature (i.e., 20–25°C), and the pressure transmission medium is water. After reaching the set holding time, the pressure is rapidly released within 2 seconds (i.e., the depressurization rate is 100 MPa / s).

[0025] 3) Filtration and separation: Pour the soaked black rice and water after step 2) into a 200-mesh screen for filtration and separation to obtain black rice and black rice water extract respectively. Store the black rice water extract at 4°C for later use.

[0026] 4) Steaming and gelatinization: Mix the soaked white glutinous rice from step 1) with the black rice from step 3) in a 1:2 ratio, and steam at 100℃ for 50 minutes.

[0027] 5) Saccharification: Stir and cool the cooked and gelatinized raw materials to 55°C, add 0.1% α-amylase by mass and treat for 4 hours; continue stirring and cooling, maintain the temperature to 25°C, add 0.2% yeast (composed of rice flour and Rhizopus, purchased from Jiangxi Shouchang Microbial Technology Development Co., Ltd.) by mass and carry out saccharification for 4 days.

[0028] 6) Fermentation: After saccharification, add 2.5 times the amount of black rice water extract and 0.1% *Lactobacillus sakei* subspecies suspension by weight. After thorough stirring, set the temperature to 20℃ and ferment for 10 days. The *Lactobacillus sakei* subspecies has been deposited at the China Center for Type Culture Collection (CCTCC) on May 13, 2022, with the strain accession number CCTCC NO.M2022621 and a suspension concentration of 10. 8 CFU / mL.

[0029] 7) Filtration: After fermentation, the fermentation product is pressed and filtered through a 200-mesh screen to obtain the filtrate. The filtrate is then added to a centrifuge and centrifuged at 8000 r / min for 20 min to obtain the supernatant of black rice wine.

[0030] 8) Color protection: Add quercetin and L-tryptophan to the supernatant, stir well, fill the bottle to the brim and seal. The amount of quercetin added is 0.1 g / L and the amount of L-tryptophan added is 1 g / L.

[0031] 9) Sterilization: Place the black rice wine treated in step 8) into a sterilization chamber and sterilize at 72℃ for 25 minutes. After natural cooling, store it.

[0032] Comparative Example 1: A method for preparing Huizhou black rice wine, comprising the following steps:

[0033] 1) Raw material processing: Select plump black rice and white glutinous rice without spoilage, wash them clean, put them into different containers for soaking, and add water at a mass ratio of 1:3 for 4 hours, with a water temperature of 20-25℃.

[0034] 2) Ultra-high pressure treatment: The black rice and water soaking solution obtained in step 1) are placed together in a high-pressure resistant packaging bag, the air in the bag is removed, and the bag is sealed tightly. The bag is then placed in an ultra-high pressure chamber, with the pressure set at 200 MPa and the holding time at 12 minutes. Rapid pressure increase and decrease are used to create a pressure difference between the inside and outside of the black rice in a short time, which helps to break down the cell walls of the black rice cells and release nutrients. The optimal pressure increase rate is 20 MPa / s, the temperature used in the ultra-high pressure equipment is room temperature (i.e., 20–25°C), and the pressure transmission medium is water. After reaching the set holding time, the pressure is rapidly released within 2 seconds (i.e., the depressurization rate is 100 MPa / s).

[0035] 3) Filtration and separation: Pour the soaked black rice and water after step 2) into a 200-mesh screen for filtration and separation to obtain black rice and black rice water extract respectively. Store the black rice water extract at 4°C for later use.

[0036] 4) Steaming and gelatinization: Mix the soaked white glutinous rice from step 1) with the black rice from step 3) in a 1:2 ratio, and steam at 100℃ for 50 minutes.

[0037] 5) Saccharification: Stir and cool the cooked and gelatinized raw materials to 55°C, add 0.1% α-amylase by mass and treat for 4 hours; continue stirring and cooling, maintain the temperature to 25°C, add 0.2% yeast (composed of rice flour and Rhizopus, purchased from Jiangxi Shouchang Microbial Technology Development Co., Ltd.) by mass and carry out saccharification for 4 days.

[0038] 6) Fermentation: After saccharification, add 2.5 times the amount of black rice water extract according to the mass ratio, stir thoroughly, set the temperature to 20℃, and ferment for 10 days.

[0039] 7) Filtration: After fermentation, the fermentation product is pressed and filtered through a 200-mesh screen to obtain the filtrate. The filtrate is then added to a centrifuge and centrifuged at 8000 r / min for 20 min to obtain the supernatant of black rice wine.

[0040] 8) Color protection: Add quercetin and L-tryptophan to the supernatant, stir well, fill the bottle to the brim and seal. The amount of quercetin added is 0.1 g / L and the amount of L-tryptophan added is 1 g / L.

[0041] 9) Sterilization: Place the black rice wine treated in step 8) into a sterilization chamber and sterilize at 72℃ for 25 minutes. After natural cooling, store it.

[0042] Comparative Example 2: A method for preparing Huizhou black rice wine, comprising the following steps:

[0043] 1) Raw material processing: Select plump black rice and white glutinous rice without spoilage, wash them clean, put them into different containers for soaking, and add water at a mass ratio of 1:3 for 4 hours, with a water temperature of 20-25℃.

[0044] 2) Filtration and separation: Pour the soaked black rice and water after step 1) into a 200-mesh screen for filtration and separation to obtain black rice and black rice water extract respectively. Store the black rice water extract at 4°C for later use.

[0045] 3) Steaming and gelatinization: Mix the soaked white glutinous rice from step 1) with the black rice from step 2) in a 1:2 ratio, and steam at 100℃ for 50 minutes.

[0046] 4) Saccharification: After cooking and gelatinizing, the raw materials are stirred and cooled to 55°C. 0.1% α-amylase is added and treated for 4 hours. Stirring and cooling are continued, and the temperature is maintained at 25°C. 0.2% yeast (composed of rice flour and Rhizopus, purchased from Jiangxi Shouchang Microbial Technology Development Co., Ltd.) is added and saccharification is carried out for 4 days.

[0047] 5) Fermentation: After saccharification, add 2.5 times the amount of black rice water extract and 0.1% *Lactobacillus sakei* subspecies suspension by weight. After thorough stirring, set the temperature to 20℃ and ferment for 10 days. The *Lactobacillus sakei* subspecies has been deposited at the China Center for Type Culture Collection (CCTCC) on May 13, 2022, with the strain accession number CCTCC NO. M2022621 and a suspension concentration of 10. 8 CFU / mL.

[0048] 6) Filtration: After fermentation, the fermentation product is pressed and filtered through a 200-mesh screen to obtain the filtrate. The filtrate is then added to a centrifuge and centrifuged at 8000 r / min for 20 min to obtain the supernatant of black rice wine.

[0049] 7) Sterilization: Place the supernatant of the black rice wine treated in step 6) into a sterilization chamber and sterilize at 72℃ for 25 minutes. After natural cooling, store it.

[0050] Comparative Example 3: A method for preparing Huizhou black rice wine, comprising the following steps:

[0051] 1) Raw material processing: Select plump black rice and white glutinous rice without spoilage, wash them clean, put them into different containers for soaking, and add water at a mass ratio of 1:3 for 4 hours, with a water temperature of 20-25℃.

[0052] 2) Filtration and separation: Pour the soaked black rice and water after step 1) into a 200-mesh screen for filtration and separation to obtain black rice and black rice water extract respectively. Store the black rice water extract at 4°C for later use.

[0053] 3) Steaming and gelatinization: Mix the soaked white glutinous rice from step 1) with the black rice from step 2) in a 1:2 ratio, and steam at 100℃ for 50 minutes.

[0054] 4) Saccharification: After cooking and gelatinizing, the raw materials are stirred and cooled to 55°C. 0.1% α-amylase is added and treated for 4 hours. Stirring and cooling are continued, and the temperature is maintained at 25°C. 0.2% yeast (composed of rice flour and Rhizopus, purchased from Jiangxi Shouchang Microbial Technology Development Co., Ltd.) is added and saccharification is carried out for 4 days.

[0055] 5) Fermentation: After saccharification, add 2.5 times the amount of black rice water extract and 0.1% *Lactobacillus sakei* subspecies suspension by weight. After thorough stirring, set the temperature to 20℃ and ferment for 10 days. The *Lactobacillus sakei* subspecies has been deposited at the China Center for Type Culture Collection (CCTCC) on May 13, 2022, with the strain accession number CCTCC NO. M2022621 and a suspension concentration of 10. 8 CFU / mL.

[0056] 6) Filtration: After fermentation, the fermentation product is pressed and filtered through a 200-mesh screen to obtain the filtrate. The filtrate is then added to a centrifuge and centrifuged at 8000 r / min for 20 min to obtain the supernatant of black rice wine.

[0057] 7) Color protection: Add quercetin and L-tryptophan to the supernatant, stir well, fill the bottle to the brim and seal. The amount of quercetin added is 0.1 g / L and the amount of L-tryptophan added is 1 g / L.

[0058] 8) Sterilization: Place the supernatant of the black rice wine treated in step 6) into a sterilization chamber and sterilize at 72℃ for 25 minutes. After natural cooling, store it.

[0059] To illustrate the advantages of this invention, a comparative analysis is conducted from the following aspects:

[0060] 1. Determination of Physicochemical Indicators of Huizhou Black Rice Wine

[0061] (1) Experimental methods

[0062] Ethanol content determination: Refer to the alcohol meter method in GB 5009.225—2023 "Determination of ethanol concentration in wine and edible alcohol".

[0063] pH value determination: Take 10 mL of Huizhou black rice wine sample and measure the pH value using a pH meter at 25℃.

[0064] Total acid content determination: Refer to the acid-base indicator titration method in GB 12456—2021 "Determination of total acid in food".

[0065] Total sugar content determination: The anthrone-sulfuric acid colorimetric method was used. Glucose (0, 0.1, 0.2, 0.3, 0.4, 0.5 g / L) were used as standards. A standard curve was plotted with absorbance at 620 nm as the ordinate and glucose concentration as the abscissa. The regression equation was Y = 3.865X + 0.1153(R²). 2 =0.9989). The total sugar concentration (in g / L) in the Huizhou black rice wine sample was calculated using a glucose standard curve.

[0066] Reducing sugar content determination: The 3,5-dinitrosalicylic acid method was used. Glucose (0, 0.2, 0.4, 0.6, 0.8, 1.0 g / L) was used as a standard. A standard curve was plotted with absorbance at 540 nm as the ordinate and glucose concentration as the abscissa, yielding the regression equation Y = 2.2117X + 0.0284(R²). 2 =0.9951). The mass concentration of reducing sugar in the Huizhou black rice wine sample was calculated (in g / L) using a glucose standard curve.

[0067] (2) Experimental Results and Analysis

[0068] As shown in Table 1, compared to Comparative Example 1, the ethanol content of Huizhou black rice wine in Example 1 increased to (15.33±0.45)% after fermentation, while the pH value decreased to (4.33±0.10). Total acid content is an important indicator of alcoholic beverages, contributing significantly to their quality and flavor. The total acid concentration of Huizhou black rice wine in Example 1 was significantly higher than that in Comparative Example 1, and its trend was consistent with the pH value trend. Furthermore, compared to Comparative Example 1, the total sugar and reducing sugar concentrations of Huizhou black rice wine in Example 1 were significantly reduced, decreasing to (7.39±1.72) g / L and (6.43±0.22) g / L, respectively. The above results indicate that the *Lactobacillus sakei* subsp. added in this invention can grow and multiply in large quantities during the fermentation of black rice wine, consuming fermentable sugars and converting them into organic acids, amino acids, and ethanol. This results in a decrease in pH, a reduction in total sugar and reducing sugar content, an increase in total acid content, and an increase in the volume fraction of ethanol, thus promoting the complete fermentation of black rice wine.

[0069] Table 1 Comparison of physicochemical properties of Huizhou black rice wine after fermentation

[0070]

[0071] 2. Determination of volatile components in Huizhou black rice wine

[0072] (1) Experimental methods

[0073] Insert the CAR / PDMS extraction head into the GC-MS inlet and activate it at 250℃ for 5 min. Take 8 mL of Huizhou black rice wine sample and place it in a headspace vial, insert the activated extraction head, and adsorb at 80℃ for 30 min. After completion, remove the extraction head, insert it into the GC-MS inlet, desorb at 250℃ for 5 min, and then perform GC-MS detection and analysis.

[0074] GC conditions: Capillary column: HP-5MS (30m × 0.25mm, 0.25μm); Inlet temperature: 250℃. Temperature program: Initial temperature 50℃, hold for 2 min; increase to 115℃ at 2℃ / min, hold for 3 min; increase to 200℃ at 4℃ / min; increase to 230℃ at 6℃ / min, hold for 10 min. Carrier gas: High-purity He; Flow rate: 1 mL / min; Splitless injection. MS conditions: Ionization method: Electron ionization source; Electron energy: 70 eV; Ion source temperature: 230℃; Mass scan range m / z 30–400. Finally, the sample was matched with the experimental mass spectrometry library in the NIST98 database, retaining substances with a matching degree >80, and identifying the volatile components of the Huizhou black rice wine sample.

[0075] (2) Experimental Results and Analysis

[0076] Figure 1 shows the GC-MS total ion chromatograms of volatile components in the black rice wine of Comparative Example 1 and Example 1. As shown in Table 2, the relative contents of volatile compounds detected in the Huizhou black rice wine samples of Comparative Example 1 and Example 1 using solid-phase microextraction combined with GC-MS were 87.44% and 96.71%, respectively. A total of 21 volatile compounds were detected after fermentation, including esters, alcohols, acids, and ketones, among which alcohols and esters were the main volatile substances. Alcohols are important aroma compounds in wine; an appropriate amount of volatile alcohols can increase the aroma of the wine and contribute significantly to its fragrance. Compared to Comparative Example 1, the relative content of alcohols in the Huizhou black rice wine of Example 1 was higher, at 89.69%. Besides ethanol, four other alcohols were detected in the Huizhou black rice wine of Example 1 during fermentation: isobutanol, isoamyl alcohol, 2,3-butanediol, and phenylethanol, with relative contents of 0.81%, 1.99%, 0.26%, and 0.77%, respectively. Esters are substances generated from the esterification reactions of fatty acids and alcohols during microbial metabolism and aging during fermentation, and are the most important flavor compounds in alcoholic beverages. Ten esters were detected during the fermentation of the Huizhou black rice wine of Example 1, mainly including ethyl acetate, isoamyl acetate, phenethyl acetate, ethyl myristate, ethyl palmitate, ethyl linoleate, and ethyl octanoate, among other volatile components. Compared to Comparative Example 1, the relative ester content of the black rice wine in Example 1 was 2.62%, an increase of 1.26 times. Although the relative content of esters is relatively low, their low threshold values ​​make them major contributors to the floral and fruity aromas of alcoholic beverages. Studies have reported that ethyl acetate has a pineapple aroma and a sweet, refreshing taste; ethyl caprylate mainly exhibits pear and pineapple aromas, giving Miao rice wine a unique flavor; ethyl palmitate has a creamy flavor; and isoamyl acetate and phenylethyl acetate have banana and floral aromas, respectively. These results indicate that the *Lactobacillus sakei* subsp. *sake* added in this invention can form abundant alcohols and esters during the fermentation of black rice wine, presenting the unique fruity, floral, and creamy flavors and taste of Huizhou black rice wine.

[0077] Table 2 Comparison of volatile components in Huizhou black rice wine after fermentation

[0078]

[0079] Note: Retention index reference website: https: / / webbook.nist.gov / chemistry / ; - This substance was not found or detected.

[0080] 3. Sensory evaluation of Huizhou black rice wine

[0081] (1) Experimental methods

[0082] ①Sensory evaluation: A sensory evaluation team of 10 professionally trained personnel was formed. Huizhou black rice wine from Comparative Example 1 and Example 1 were selected and evaluated based on five indicators: color, taste, aroma, clarity, and acceptability. The evaluation criteria are shown in Table 3.

[0083] Table 3 Sensory Scoring Criteria for Huizhou Black Rice Wine

[0084]

[0085] (2) Experimental Results and Analysis

[0086] Figure 2 The sensory evaluation results are as follows: The sensory evaluation was based on a comprehensive assessment by the evaluation panel members using five indicators: color, taste, aroma, clarity, and acceptability. Compared to Comparative Example 1, the Huizhou black rice wine prepared using this invention in Example 1 exhibited higher sensory acceptability, a more uniform and bright color (deep, full-bodied, and pure blackish-purple, purplish-red, or ruby ​​red), a mellow taste with a high balance of sweetness, acidity, and alcohol content, a rich and layered aroma that clearly presented the unique aroma of black rice, along with subtle fruity and floral notes, and a clear and transparent body. The results indicate that the *Lactobacillus sakei* subsp. added in this invention significantly enhances the taste and aroma of the processed Huizhou black rice wine.

[0087] 4. Determination of the content of main active substances in Huizhou black rice wine

[0088] (1) Experimental methods

[0089] Total phenol content determination: The Folin-Ciocalteu method was used. Tannic acid (0, 50, 100, 150, 200, 250 mg / L) was used as a standard. A standard curve was plotted with absorbance at 750 nm as the ordinate and tannic acid concentration as the abscissa, yielding the regression equation Y = 0.002X + 0.0332(R²). 2 =0.9938). The total phenol concentration (in mg / L) in different Huizhou black rice wine samples (Comparative Example 1, Comparative Example 3 and Example 1) was calculated using the tannin standard curve.

[0090] Total flavonoid content determination: The aluminum trichloride colorimetric method was used. Rutin (0, 40, 80, 120, 160, 200 mg / L) were used as standards. A standard curve was plotted with absorbance at 420 nm as the ordinate and rutin concentration as the abscissa, yielding the regression equation Y = 0.0017X + 0.0304(R²). 2 =0.9919). The total flavonoid concentration (in mg / L) in different Huizhou black rice wine samples (Comparative Example 1, Comparative Example 3 and Example 1) was calculated using the rutin standard curve.

[0091] Anthocyanin content determination: Take 1 mL of different Huizhou black rice wine samples (Comparative Example 1, Comparative Example 3, and Example 1), and add 9 mL of potassium chloride buffer (pH 1.0) and sodium acetate buffer (pH 4.5), respectively. After equilibration for 40 min, measure the absorbance at wavelengths of 511 nm and 700 nm using an ELISA reader. Distilled water was used as a blank control. The total anthocyanin concentration was calculated as cyanidin-3-O-glucoside, in mg / L.

[0092] (2) Experimental Results and Analysis

[0093] Polyphenols and flavonoids are the main bioactive substances in rice wine. For example... Figure 3 As shown, compared to Comparative Examples 2 and 3, the total phenol concentration in Example 1 increased to (537.02±20.23) mg / L, representing increases of 1.95 times and 0.47 times, respectively. The total flavonoid concentration in Example 1 reached (279.25±7.65) mg / L, representing increases of 1.18 times and 0.85 times, respectively, compared to Comparative Examples 2 and 3. Anthocyanins are the main coloring substances in black rice wine, giving it a vibrant color, and also possess antioxidant, antitumor, anti-inflammatory, and cardiovascular protective physiological effects. Figure 3 As shown, compared to Comparative Example 2, the anthocyanin concentration in Comparative Example 3 increased by 1.73 times, indicating that the color-protecting treatment technology of the present invention can significantly improve the heat resistance of the anthocyanin to high-temperature sterilization. This may be due to the fact that the added quercetin and L-tryptophan can form hydrogen bonds, hydrophobicity, van der Waals forces, and ionic interactions with the anthocyanins in black rice wine, thereby improving the stability of the anthocyanins. Compared to Comparative Examples 2 and 3, the anthocyanin concentration in Example 1 increased to (507.76±3.12) mg / L, an increase of 2.48 times and 0.27 times, respectively. Furthermore, compared to the anthocyanin content of 140.44 mg / L obtained in the fermented black rice wine obtained in "A method for brewing black rice wine with high anthocyanin content and enhanced flavor" disclosed in CN 113583783 B, the anthocyanin concentration in Example 1 was (507.76±3.12) mg / L, an increase of 2.62 times. The above results indicate that the ultra-high pressure processing combined with color protection technology used in this invention can significantly increase the content of total phenols, total flavonoids and anthocyanins in black rice wine fermentation. This may be related to the fact that ultra-high pressure processing can destroy the cell wall of black rice seed coat, allowing more total phenols, total flavonoids and anthocyanins to be released, and that the added quercetin and L-tryptophan can significantly improve the heat resistance and stability of anthocyanins in black rice wine to high-temperature sterilization.

[0094] 5. Determination of anthocyanin content changes in Huizhou black rice wine after different storage times

[0095] (1) Experimental methods

[0096] Anthocyanin content determination: Take 1 mL of different Huizhou black rice wine samples stored for different times (0, 30, 60, 90 days), and add 9 mL of potassium chloride buffer (pH 1.0) and sodium acetate buffer (pH 4.5), respectively. After equilibration for 40 min, measure the absorbance at wavelengths of 511 nm and 700 nm using an ELISA reader. Distilled water was used as a blank control. The total anthocyanin concentration was calculated as cyanidin-3-O-glucoside, in mg / L.

[0097] (2) Experimental Results and Analysis

[0098] The results of changes in anthocyanin content in Huizhou black rice wine at different storage times are as follows: Figure 4 As shown. In Comparative Example 2, the anthocyanin content of Huizhou black rice wine at different storage times (0, 30, 60, 90 days) was (146.11±22.15), (81.37±3.44), (52.39±7.41), and (23.93±4.93) mg / L, respectively, with anthocyanin loss rates of 44.31%, 64.14%, and 83.62%, respectively. Compared to Example 2, the anthocyanin content of Huizhou black rice wine in Example 1 at different storage times (0, 30, 60, 90 days) was (507.76±3.12), (438.41±7.87), (383.21±4.46), and (346.72±11.61) mg / L, respectively, with anthocyanin loss rates of only 13.66%, 24.53%, and 31.72%, respectively. In contrast, the anthocyanin loss rates of Huizhou black rice wine in Example 3 at different storage times (30, 60, 90 days) were 19.41%, 25.71%, and 32.38%, respectively. Therefore, the anthocyanin retention rate of the Huizhou black rice wine prepared by this invention is better, reaching as high as 68.28% within 90 days, which is 3.17 times higher than the group without color-protecting treatment (Comparative Example 2). The above results indicate that the color-protecting technology used in this invention can significantly improve the stability of anthocyanins in Huizhou black rice wine at different storage times, and is beneficial to delaying the deterioration of the color of black rice wine, thus better preserving its flavor and quality.

[0099] The present invention has been described above by way of example with reference to the accompanying drawings. Obviously, the specific implementation of the present invention is not limited to the above-described manner. Any non-substantial improvements made using the inventive concept and technical solution of the present invention; or the direct application of the inventive concept and technical solution of the present invention to other situations without modification, are all within the protection scope of the present invention.

Claims

1. A method for preparing Huizhou black rice wine, characterized in that, Includes the following steps: 1) Raw material processing: Select plump black rice and white glutinous rice without spoilage, wash them clean, and soak them in water with a mass ratio of 1:1 to 5 for 1 to 4 hours at a water temperature of 20 to 25℃. 2) Ultra-high pressure treatment: The black rice and water soaking solution obtained in step 1) are put into a high-pressure resistant packaging bag and placed in a pressure chamber for pressure treatment. The pressure is set to 50-200 MPa and the pressure holding time is 5-15 min. 3) Filtration and separation: The black rice and water soaking material after ultra-high pressure treatment are poured into a 200-mesh screen for filtration and separation to obtain black rice and black rice water extract respectively. The black rice water extract is stored at 4℃ for later use. 4) Steaming and gelatinization: Mix the soaked white glutinous rice from step 1) with the black rice processed in step 3) in a ratio of 1:1 to 3, and steam at high temperature for 30 to 50 minutes at a temperature of 100℃ to 105℃. 5) Saccharification: Stir and cool the cooked and gelatinized raw materials to 45℃~60℃, add 0.1%~0.2% α-amylase by mass for 1~6h; continue stirring and cooling, maintain the temperature to 25℃~30℃, add 0.2%~0.5% yeast by mass for saccharification for 3~4d; 6) Fermentation: After saccharification, add 1-3.5 times the amount of black rice water extract and 0.1%-0.25% of *Lactobacillus sakei* subsp. *sake* suspension according to weight, with a suspension concentration of 10. 6 ~10 8 CFU / mL, after stirring completely, set the temperature to 15℃~25℃ and ferment for 8~10 days; 7) Press filtration: After fermentation, the fermentation product is pressed and filtered through a 200-mesh screen to obtain the filtrate. The filtrate is then added to a centrifuge and centrifuged at 8000-10000 r / min for 10-20 min to obtain the supernatant of black rice wine.

2. The method for preparing Huizhou black rice wine according to claim 1, characterized in that: The Lactobacillus sakei subspecies mentioned in step 6) is a Lactobacillus sakei subspecies deposited at the China Center for Type Culture Collection (CCTCC) on May 13, 2022, with the accession number CCTCC NO.M 2022621.

3. The method for preparing Huizhou black rice wine according to claim 1, characterized in that: Following step 7), a further step 8) is performed for color protection: one or more of quercetin, gallic acid, syringic acid, and L-tryptophan are added to the supernatant, stirred thoroughly, and then filled to the brim and sealed. The amount of quercetin added is 0.1–0.5 g / L, the amount of gallic acid added is 0.1–0.3 g / L, the amount of syringic acid added is 0.1–0.3 g / L, and the amount of L-tryptophan added is 0.5–3 g / L.

4. The method for preparing Huizhou black rice wine according to claim 1, characterized in that: After step 8), there is a step 9) sterilization: put the black rice wine treated in step 8) into a sterilization box and sterilize it at 65℃~75℃ for 15~30 minutes, and then store it after natural cooling.

5. The method for preparing Huizhou black rice wine according to claim 1, characterized in that: In step 2), the pressurization pressure is 200 MPa, the pressure holding time is 12 min, the pressurization rate is 20 MPa / s, and the depressurization rate is 100 MPa / s.

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