WO2022083804A2 - 一种茵红李果酒及其酿造方法 - Google Patents

一种茵红李果酒及其酿造方法 Download PDF

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WO2022083804A2
WO2022083804A2 PCT/CN2022/072866 CN2022072866W WO2022083804A2 WO 2022083804 A2 WO2022083804 A2 WO 2022083804A2 CN 2022072866 W CN2022072866 W CN 2022072866W WO 2022083804 A2 WO2022083804 A2 WO 2022083804A2
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yinhong
acid
fermentation
plum
fruit wine
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WO2022083804A3 (zh
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李东
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四川轻化工大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/024—Preparation of other alcoholic beverages by fermentation of fruits other than botanical genus Vitis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C12G3/026—Preparation of other alcoholic beverages by fermentation with health-improving ingredients, e.g. flavonoids, flavones, polyphenols or polysaccharides, added before or during the fermentation stage; with flavouring ingredients added before or during the fermentation stage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
    • C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
    • C12H1/02—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages combined with removal of precipitate or added materials, e.g. adsorption material
    • C12H1/04—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages combined with removal of precipitate or added materials, e.g. adsorption material with the aid of ion-exchange material or inert clarification material, e.g. adsorption material
    • C12H1/0408—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages combined with removal of precipitate or added materials, e.g. adsorption material with the aid of ion-exchange material or inert clarification material, e.g. adsorption material with the aid of inorganic added material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
    • C12H1/00—Pasteurisation, sterilisation, preservation, purification, clarification, or ageing of alcoholic beverages
    • C12H1/22—Ageing or ripening by storing, e.g. lagering of beer

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  • the invention relates to the technical field of food engineering, in particular to a Yinhong plum fruit wine and a brewing method thereof.
  • Yinhong plum is an excellent plum species in the genus Prunus L..
  • Yinhong plum fruit is moderately sweet and sour, crisp and juicy, yellow-green flesh, crispy, thin skin, off the core, fragrant and refreshing.
  • Plum fruit is rich in polyphenols, flavonoids, flavonoids and other biologically active substances, which have positive effects in anti-oxidation, anti-inflammatory, cognitive improvement, prevention and treatment of cardiovascular diseases and bone health.
  • Organic acid is a very important component in fruit wine, which is less in the wine, but plays a vital role in the flavor of the wine.
  • Organic acids have obvious taste effects, are important components of fruit wine aftertaste, and are also precursors to form corresponding esters.
  • the type and content of their corresponding esters determine the flavor and style of wine.
  • Yinhong Li finished fruit wine there is no Yinhong Li finished fruit wine on the market, and the type and content of organic acids in the wine are still unclear. Therefore, using Yinhong plum as raw material to study its brewing process, as well as the type and content of organic acids in fruit and fruit wine, can provide a basis for further development of Yinhong plum products.
  • the object of the present invention is to provide a kind of optimal fermentation process of Yinhong plum fruit wine brewing based on fermentation time, fermentation temperature, initial sugar content, potassium metabisulfite addition and yeast addition,
  • the invention discloses a high-quality Yinhong plum fruit wine and a brewing method thereof.
  • the technical scheme is a brewing method of Yinhong plum fruit wine, comprising the following steps:
  • yeast is inoculated into fermentation broth to carry out constant temperature fermentation
  • step (3) no less than 0.03 g/L of food-grade pectinase is added for enzymatic hydrolysis for at least 2 hours.
  • step (3) pectinase enzymatic hydrolysis is performed at a temperature not higher than 45°C.
  • step (4) add a mixed acid-reducing agent of food-grade calcium carbonate and potassium bicarbonate to adjust acid; the concentration of the mixed acid-reducing agent is 2g/L; add white granulated sugar to adjust the sugar content to not higher than 23° Bx.
  • step (5) a food-grade potassium metabisulfite bacteriostatic agent of not more than 0.09 g/L is added.
  • the activated yeast is inoculated into the fermentation broth for constant temperature fermentation for 5-11 d, and the yeast cell concentration is not less than 0.4 g/L.
  • step (6) constant temperature fermentation is performed in a constant temperature box; the constant temperature temperature is 24°C-27°C.
  • step (8) the fermented wine body is sealed in a full tank for aging.
  • step (9) bentonite is added to the aged liquor for clarification.
  • the invention also discloses Yinhong plum fruit wine, which is prepared by the brewing method of the Yinhong plum fruit wine described in the above scheme, and contains oxalic acid, pyruvic acid, malic acid, lactic acid, acetic acid, citric acid, succinic acid and tartaric acid And fumaric acid 9 kinds of organic acids.
  • the invention provides an optimal fermentation process for Yinhong plum fruit wine brewing based on fermentation time, fermentation temperature, initial sugar content, potassium metabisulfite addition amount and yeast addition amount, so as to obtain Yinhong plum fruit wine of good quality and improve Yinhong plum fruit wine. added value.
  • the prepared Yinhong plum fruit wine contains 9 kinds of organic acids, oxalic acid, pyruvic acid, malic acid, lactic acid, acetic acid, citric acid, succinic acid, tartaric acid and fumaric acid, and has obvious taste effect.
  • Fig. 2 in the specific embodiment of the present invention, the influence of different initial sugar content on alcohol content and reducing sugar;
  • Fig. 6 HPLC chromatogram of organic acid standard mixed solution in the specific embodiment of the present invention.
  • the labels in the figure 1 oxalic acid, 2 tartaric acid, 3 pyruvic acid, 4 malic acid, 5 lactic acid, 6 shikimic acid, 7 acetic acid, 8 citric acid, 9 succinic acid, 10 fumaric acid.
  • Yinhong Plum Yibin City; FX10 Yeast: French LAFFORT Company; EX-V Pectinase: French LALLEMAND Company; Potassium Metabisulfite (Food Grade): French OENOFRANCE Company; Calcium Carbonate (Food Grade): Dexing City Mingyuan Chemical Materials Co., Ltd.; white sugar: commercially available.
  • LB80T hand-held sugar content meter Guangdong Suwei Electronic Technology Co., Ltd.; LHS-250SC constant temperature and humidity box: Shanghai Qixin Scientific Instrument Co., Ltd.; FA2204B Analytical Balance: Shanghai Youke Instrument Co., Ltd.; Electrical Appliance Manufacturing Co., Ltd.; 0 ⁇ 40%Vol alcohol meter: Huaou Instrument Factory, Wuqiang County, Hebei City; 1260infinity II High Performance Liquid Chromatograph: Agilent, USA; 0.22 ⁇ m Water Filter: Tianjin Jinteng Test Equipment Co., Ltd. .
  • the fermentation time (A), the initial sugar content (B), the fermentation temperature (C), the amount of potassium metabisulfite (D) and the amount of yeast added (E) were selected respectively. 5 factors, 3 levels were selected for each factor, and L 18 (3 7 ) was used as an index to optimize the optimal fermentation conditions of Yinhong plum wine.
  • Alcohol content and reducing sugar Refer to GB/T15038-2006 "General Analysis Method for Wine and Fruit Wine” to determine the alcohol content by alcohol meter, and direct titration method for reducing sugar;
  • Chromatographic conditions Take 5mL of the original liquor before use, filter it through a 0.22 ⁇ m nylon filter, and then inject it for analysis.
  • the pH value was adjusted with PO 4 , the mobile phase B was methanol 5%, the injection volume was 15 ⁇ L, the flow rate was 0.4 mL/min, and the column temperature was 25 °C.
  • Solution preparation prepare a 0.05mol/L KH 2 PO 4 solution, adjust the pH value to 2.4 with H 3 PO 4 as the mobile phase, and then sonicate for use after suction filtration.
  • Single standard stock solution Accurately weigh an appropriate amount of pyruvic acid, malic acid, shikimic acid, oxalic acid, lactic acid, acetic acid, citric acid, fumaric acid, succinic acid, and tartaric acid and place them in a 5 mL volumetric flask, dissolve them in ultrapure water Dilute to the mark to obtain standard solutions with mass concentrations of 1.1, 0.66, 0.46, 0.78, 0.98, 0.92, 0.44, 0.46, 0.46, and 0.40 mg/mL, respectively, and store them at 4°C in the dark for future use.
  • Pretreatment of Yinhong plum fruit Take a certain amount of Yinhong plum pulp to make pulp, take 10g of homogenate, transfer it to a 50mL volumetric flask, add ultrapure water, heat and extract in a 75°C water bath for 45min, cool to room temperature and set the volume. Filter after 30 min of ultrasonic extraction. Take a certain amount of filtrate and centrifuge at 4000r/min for 30min to separate the interfering substances such as precipitated protein and pectin. Take the supernatant and filter it with a 0.45 ⁇ m pore size filter membrane. Nylon filter.
  • the alcohol content firstly increased and then tended to balance with the fermentation time.
  • the alcohol content increased with time at 5 days before fermentation, and the alcohol content tended to increase after 5 days. stable (P>0.05).
  • the fermentation time is 7d, the alcohol content reaches the maximum, which is 12.8%.
  • the content of reducing sugar in the fermented fruit wine gradually decreased, and the reducing sugar content of 9-11d tended to be stable (P>0.05), while the reducing sugar content of 3-7d changed greatly.
  • the optimum fermentation time was initially determined to be 7d.
  • Fermentation time affects the alcohol content of fruit wine mainly because the yeast can fully grow and multiply with the extension of time, and the alcoholic fermentation starts after anaerobic conditions, and when the alcohol reaches a certain amount, it will inhibit the alcoholic fermentation of the yeast. effect, so the alcohol content will stabilize after a period of time.
  • the growth of yeast and anaerobic respiration in the early stage of fermentation will consume sugars, and the amount of reducing sugar will be more, and the content of reducing sugar will decrease sharply at the beginning and then slowly decrease.
  • the alcohol content when the initial sugar content increases, the alcohol content first increases and then decreases.
  • the highest alcohol content is 13.7% when the initial Brix is 23°Bx.
  • Sugar can be an essential nutrient for the growth of yeast. In a certain concentration range, the yeast is in the reproductive stage and can quickly convert sugar into alcohol, which can promote the fermentation of alcohol content to a certain extent. Excessive sugar will affect the metabolism of yeast, and even cause yeast to die, thus reducing the alcohol content.
  • the reducing sugar content gradually increased. At 23°Bx, the reducing sugar content was relatively low at 6.875g/L.
  • the alcohol content did not change much (P>0.05) under different additions of potassium sulfite, and when the addition of potassium metabisulfite was 0.09 g/L, the alcohol content was up to 12.8%;
  • the reducing sugar first decreased and then increased with the increase of potassium metabisulfite content.
  • the addition amount of potassium metabisulfite was 0.09g/L
  • the reducing sugar content was the least, and when the addition amount of potassium metabisulfite was greater than 0.09g/L
  • the content of reducing sugar increased, it was preliminarily determined that the optimum addition amount of potassium metabisulfite was 0.09g/L. This is because potassium metabisulfite will decompose into SO 2 under certain conditions.
  • the alcohol content showed a trend of rising first and then falling.
  • the yeast addition was 0.4 g/L, the alcohol content was the highest at 14.05%. If the amount of yeast added is too small, the utilization of nutrients will be reduced, the self-reproduction and metabolism will be slow, the growth of miscellaneous bacteria will be fast, and the alcohol production will be small. Metabolized to alcohol content but decreased. At the same time, when the amount of yeast added gradually increased, the content of reducing sugar gradually decreased. When the amount of yeast added was 0.7g/L, the content of reducing sugar reached the lowest level.
  • the relationship between the factors affecting the alcohol content is fermentation time (A) > fermentation temperature (C) > initial sugar content (B) > potassium metabisulfite addition (D) > yeast addition (E).
  • the optimal process parameters are A 2 B 2 C 3 D 3 E 2 , that is, the fermentation time is 7d, the initial sugar content is 23°Bx, the fermentation temperature is 27°C, the addition amount of potassium metabisulfite is 0.12g/L, and the addition amount of yeast is 0.4g/L. Since the best experimental group did not appear in the experimental design, repeated verification experiments were carried out under this condition.
  • the most organic acid is malic acid, reaching 6.976g/kg, followed by oxalic acid content of 0.409g/kg, the least acid is fumaric acid content of 0.004g/kg, and the other four kinds of acids are all below 0.1g/kg .
  • 10 kinds of organic acids were detected, a total of 9 kinds were detected, and no shikimic acid was detected.
  • the detected organic acids were oxalic acid, pyruvic acid, malic acid, lactic acid, acetic acid, and citric acid. , succinic acid, tartaric acid, fumaric acid.
  • the content of malic acid, lactic acid and succinic acid is relatively high, with the highest content reaching 4.503g/L, 3.37g/L and 0.832g/L respectively, while the content of oxalic acid, acetic acid, citric acid, pyruvic acid, tartaric acid and fumaric acid All were kept below 0.5g/L, indicating that malic acid, lactic acid and succinic acid were the main organic acids in Yinhong plum wine.
  • the main organic acid in the fruit of Yinhong plum is malic acid, which has the highest content.
  • the fermentation process of residual sugar will trigger malolactic fermentation, malic acid will be converted into lactic acid, and finally the lactic acid content will increase.
  • the lactic acid also comes from the alcoholic fermentation of yeast and becomes the acid with the highest content except malic acid; succinic acid is the tricarboxylic acid of yeast.
  • Intermediate product of the acid cycle (TCA); pyruvate is produced by the glycolytic pathway.
  • the acetic acid content is as low as 0.023g/L, which is produced by the oxidation-reduction of acetaldehyde in the process of alcohol fermentation, and a small amount of acetic acid is produced by the acetic acid fermentation of a very small amount of acetic acid bacteria.
  • citric acid in Yinhong plum wine is more than that in the fruit. This is because citric acid is produced in both aerobic and anaerobic conditions of yeast. Under aerobic conditions, it is generated by TCA cycle; under anaerobic conditions, it is obtained by reduction of oxaloacetic acid. External conditions have a great influence on the metabolism of citric acid, so the content of citric acid in wine is more or less than that in fruit.
  • oxalic acid Compared with Yinhong plum fruit, the content of oxalic acid in Yinhong plum fruit wine is lower, and oxalic acid generates CO 2 through oxidation, decarboxylation and acetylation; shikimic acid is not detected in Yinhong plum fruit wine, and shikimic acid passes through the shikimic acid pathway. It is broken down into phenylalanine, tyrosine, and tryptophan for use by yeast.
  • N.D means not detected.
  • the organic acids in Yinhong plum fruit and its fermented wine were determined by HPLC. Seven kinds of acids were quantitatively detected in the fruit, among which the content of malic acid accounted for 92.5% of the total acid detected in the fruit, and the content of the other six kinds of organic acids did not exceed 0.5g/kg; 9 kinds of acids were quantitatively detected in Yinhong plum wine. Among the organic acids, malic acid had the highest content, accounting for 47.2% of the total detected acids, followed by lactic acid with 35.3% and succinic acid with 8.7%. After Yinhong plum fruit was fermented into Yinhong plum wine, the content of malic acid decreased and the content of lactic acid increased, but the content of malic acid was still higher than that of other types of fruit wine.
  • the wine brewing process for Yinhong plum fruit can also be applied to other plum varieties.

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Abstract

本发明公开了一种茵红李果酒及其酿造方法，包括以下步骤：挑选无虫害、无霉烂的茵红李，清洗并沥干水分；对茵红李进行肉核分离，将果肉打浆；加入食品级果胶酶酶解；加入降酸剂调酸，加入砂糖调节糖度；加入抑菌剂；将酵母接种到发酵液中进行恒温发酵；残糖发酵；将发酵好的酒体密封陈酿；在陈酿好的酒液中加入澄清剂进行澄清，过滤，制得茵红李果酒。本发明提供一种基于发酵时间、发酵温度、初始糖度、焦亚硫酸钾添加量和酵母添加量的茵红李果酒酿造最佳发酵工艺，制得品质好的茵红李果酒。

Description

一种茵红李果酒及其酿造方法
本申请要求于2021年04月08日提交中国专利局、申请号为“202110377931.3”、发明名称为“一种茵红李果酒及其酿造方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及食品工程技术领域，尤其涉及一种茵红李果酒及其酿造方法。
背景技术
茵红李是李属(PrunusL.)中一个优良的李子品种。茵红李果质酸甜适中，脆嫩多汁，果肉黄绿，肉脆，皮薄，离核，清香爽口。李果实中含有丰富的多酚、黄青素、黄酮等生物活性物质，在抗氧化、抗炎、改善认知、防治心血管疾病和骨骼健康等方面有积极作用，把茵红李加工成果酒不仅能提高茵红李的附加值，还能增加茵红李鲜果的销售途径。有机酸是果酒中一类非常重要的成分，在酒中含量较少，但对酒的风味起至关重要的作用。有机酸具有明显的呈味作用，是果酒后味的重要组分，也是形成相应酯类的前驱物质，与其对应酯的种类及含量决定了酒的香型与风格。目前，市面上没有茵红李成品果酒，对该酒中有机酸种类与含量均尚不清楚。因此，以茵红李为原料，研究其酿造工艺，以及果实和果酒中有机酸的种类和含量，可为进一步开发茵红李产品提供依据。
发明内容
为了克服上述所存在的技术缺陷，本发明的目的在于提供一种本发明基于发酵时间、发酵温度、初始糖度、焦亚硫酸钾添加量和酵母添加量的茵红李果酒酿造最佳发酵工艺，制得品质好的茵红李果酒及其酿造方法。
为了达到上述目的，本发明通过以下技术方案实现：
本技术方案为一种茵红李果酒的酿造方法，包括以下步骤：
(1)挑选无虫害、无霉烂的茵红李，清洗并沥干水分；
(2)对茵红李进行肉核分离，将果肉打浆；
(3)加入食品级果胶酶酶解；
(4)加入降酸剂调酸，加入砂糖调节糖度；
(5)加入抑菌剂；
(6)将酵母接种到发酵液中进行恒温发酵；
(7)残糖发酵；
(8)将发酵好的酒体密封陈酿；
(9)在陈酿好的酒液中加入澄清剂进行澄清，过滤，制得茵红李果酒。
优选的，步骤(3)中，加入不少于0.03g/L的食品级果胶酶酶解至少2h。
优选的，步骤(3)中，在不高于45℃的温度下进行果胶酶酶解。
优选的，步骤(4)中，加入食品级碳酸钙和碳酸氢钾的混合降酸剂进行调酸；混合降酸剂的浓度为2g/L；加入白砂糖将糖度调节至不高于23°Bx。
优选的，步骤(5)中，加入不大于0.09g/L的食品级焦亚硫酸钾抑菌剂。
优选的，步骤(6)中，将活化后的酵母接种到发酵液中恒温发酵5-11d，酵母的菌体浓度不小于0.4g/L。
优选的，步骤(6)中，在恒温箱中进行恒温发酵；恒温温度为24℃-27℃。
优选的，步骤(8)中，将发酵好的酒体密封满罐陈酿。
优选的，步骤(9)中，在陈酿好的酒液中加入皂土进行澄清。
本发明还公开了一种茵红李果酒，由上述方案所述的一种茵红李果酒的酿造方法制得，含有草酸、丙酮酸、苹果酸、乳酸、乙酸、柠檬酸、琥珀酸、酒石酸和富马酸9种有机酸。
本发明的有益效果是：
本发明提供一种基于发酵时间、发酵温度、初始糖度、焦亚硫酸钾添加量和酵母添加量的茵红李果酒酿造最佳发酵工艺，制得品质好的茵红李果酒，提升茵红李的附加值。制得的茵红李果酒中含有草酸、丙酮酸、苹果酸、乳酸、乙酸、柠檬酸、琥珀酸、酒石酸和富马酸9种有机酸，具有明显的呈味作用。
附图说明
图1本发明的具体实施例中不同发酵时间对酒精度和还原糖的影响；
图2本发明的具体实施例中不同初始糖度对酒精度和还原糖的影响；
图3本发明的具体实施例中不同发酵温度对酒精度和还原糖的影响；
图4本发明的具体实施例中不同焦亚硫酸钾添加量对酒精度和还原 糖的影响；
图5本发明的具体实施例中不同酵母添加量对酒精度和还原糖的影响；
图6本发明的具体实施例中有机酸标准混合溶液HPLC色谱图。其中，图示中标号：1草酸，2酒石酸，3丙酮酸，4苹果酸，5乳酸，6莽草酸，7乙酸，8柠檬酸，9琥珀酸，10富马酸。
具体实施方式
下面对本发明作详细的说明。
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例
材料与方法
材料与试剂：
茵红李：宜宾市市购；FX10酵母：法国LAFFORT公司；EX-V果胶酶：法国LALLEMAND公司；焦亚硫酸钾(食品级)：法国OENOFRANCE公司；碳酸钙(食品级)：德兴市明缘化工材料有限责任公司；白砂糖：市售。
仪器
LB80T手持糖度仪：广东市速为电子科技有限公司；LHS-250SC恒温恒湿箱：上海齐欣科学仪器有限公司；FA2204B分析天平：上海佑科仪器仪表有限公司；MJ-WBL2531H搅拌机：广东美的生活电器制造有限公司；0～40％Vol酒精计：河北省武强县华欧仪器仪表厂；1260infinity II高效液相色谱仪：美国安捷伦公司；0.22μm水系滤膜：天津市津腾试验设备有限公司。
方法
工艺流程及操作要点
茵红李→挑选、清洗→打浆→果胶酶酶解→调酸、调糖→添加焦亚硫酸钾→酵母活化→发酵→残糖发酵→陈酿→澄清过滤→灌装→检验→成品。
挑选无虫害、无霉烂的茵红李，清洗、打浆。加入0.03g/L果胶酶45℃下酶解2小时，2.0g/L的碳酸钙和碳酸氢钾的混合降酸剂调酸，用市售白砂糖调节糖度，加入一定量的焦亚硫酸钾抑菌剂。将活化后的酵母接种到发酵液中于恒温箱中发酵7d。主发酵结束后进行残糖发酵20d左右。将发酵好的酒体密封满罐陈酿2～3个月后，在陈酿好的酒液中加入皂土进行澄清，并再次过滤，得到清澈、透亮的酒体。
茵红李果酒发酵单因素试验
确定发酵时间，初始糖度，发酵温度，焦亚硫酸钾添加量，酵母添加量作为因素进行单因素实验。以上各因素的水平梯度见表1。
表1 茵红李果酒单因素试验因素和水平表
Figure PCTCN2022072866-appb-000001
茵红李果酒发酵工艺优化正交试验
在茵红李果酒发酵单因素试验的基础上，分别选用发酵时间(A)，初始糖度(B)，发酵温度为(C)，焦亚硫酸钾添加量(D)，酵母添加量(E)5个因素，每个因素选择3个水平，以酒精度作为指标，选用L 18(3 7)进行正交设计来优化最佳茵红李果酒发酵工艺条件。
指标测定
酒精度、还原糖：参照GB/T15038-2006《葡萄酒、果酒通用分析方法》酒精计法测定酒精度、还原糖运用直接滴定法；
糖度：采用手持折光仪测定。
有机酸测定
色谱条件：取5mL原酒液临用前经0.22μm尼龙滤膜过滤后进样分析。依照有机酸测定方法，HPLC条件为：Agilent ZORBAX SB-Aq(4.6mm×250mm，5μm)色谱柱，检测波长：210nm，流动相A为磷酸二氢钾(pH＝2.4)95％，并用H 3PO 4调节pH值，流动相B为甲醇5％，进样量15μL，流速0.4mL/min，柱温25℃。
溶液配制：配置0.05mol/L的KH 2PO 4溶液，用H 3PO 4调节pH值至2.4作为流动相，抽滤后超声备用。单一标准储备液：精确称取适量丙酮酸、苹果酸、莽草酸、草酸、乳酸、乙酸、柠檬酸、富马酸、琥珀酸、酒石酸标准品分别置于5mL容量瓶中，用超纯水溶解稀释至刻度，得到质量浓度分别为1.1、0.66、0.46、0.78、0.98、0.92、0.44、0.46、0.46、0.40mg/mL的标准品溶液，置于4℃条件下避光保存备用。混合标准工作液：由于丙酮酸、莽草酸、富马酸、草酸的峰值较大，将其混合成一组母液，另外6种混合成一组母液，分别移取适量的各单一标准储备液混合，分别取母液200、400、600、800、1000uL，加超纯水到1mL，得到梯度混合标准液。
茵红李果实前处理：取一定量茵红李果肉打浆，取10g匀浆，转移到50mL容量瓶中，加入超纯水，于75℃水浴加热浸提45min，冷却至室温后定容。超声提取30min后过滤。取一定量的滤液，以4000r/min离心30min，分离沉淀蛋白质、果胶等干扰物质，取上清液用0.45μm孔径的滤膜过滤，滤液即为样液，进样前再过一次0.22μm尼龙滤膜。
数据统计分析：应用Microsoft Office Excel 2016软件进行数据处理，SPSS 24进行显著性分析，采用Origin 2017进行图表的绘制；所有试验均重复3次。
结果与分析：发酵时间对茵红李果酒酒精度和还原糖的影响
如图1中所示，在不同的发酵时间下，酒精度随发酵时间的推移呈现先上升然后趋于平衡的趋势，在发酵前5d酒精度随时间增加而上升，而5d后酒精度趋于稳定(P>0.05)。当发酵时间为7d的时候，酒精度达到最大，为12.8％。随着时间的延长，发酵后的果酒中的还原糖的含量逐渐减少，其中9～11d的还原糖趋于稳定(P>0.05)，而3～7d的还原糖含量变化较大。由此初步而确定最适宜的发酵时间为7d。发酵时间影响果酒酒精 度主要是因为随着时间的延长酵母菌能够得到充分的生长与繁殖，待无氧条件后开始进行酒精发酵，而当酒精到达一定的量时会对酵母的酒精发酵产生抑制作用，因此酒精度在一段时间后会趋于稳定。同时发酵初期酵母菌的生长和无氧呼吸都要消耗糖类物质，利用还原糖的量也就越多，还原糖的含量会出现初期急剧下降，而后缓慢下降的趋势。
初始糖度对茵红李果酒酒精度和还原糖的影响
如图2中所示，当初始糖度增加时酒精度先升高在下降。当初始糖度为23°Bx的时候酒精度最高为13.7％。糖能够为酵母菌的生长必需的营养物质，在一定浓度范围内酵母菌处于繁殖阶段，能很快的将糖转化为酒精，对酒精度的发酵起到一定的促进作用。过高的糖分会影响酵母菌的代谢，甚至造成酵母死亡，由此酒精度有所降低。同时，初始糖度的增加，还原糖含量逐渐增大，在23°Bx时，还原糖含量相对较低为6.875g/L。这是由于适合的糖含量会对酵母菌的生长有一定的促进作用，而高糖会造成的渗透压的升高会在一定程度上抑制酵母的生长繁殖，不利于发酵，所以还原糖的含量会增加显著(p<0.05)。由此确定最适宜的初始糖度为23°Bx。
发酵温度对茵红李果酒酒精度和还原糖的影响
如图3中所示，在不同的温度下，酒精度随温度的上升呈现先后稳定的趋势，当温度在24℃时酒精度的值达到11.1％。随着温度的增加，酵母菌的代谢的能力增强，导致酵母菌的发酵速度加快，过高的温度会造成酵母的早衰现象，对果酒的风味影响较大，导致在27℃之后酒精度变化不大(P>0.05)。同时，温度对还原糖的影响很大，温度在18～24℃，还原糖有个骤减的趋势(P<0.05)，这是由于温度较低时，酵母生产缓慢，对糖的需求量减少，当温度在24℃时，达到该酵母所合适的温度，因此把大部分的糖转化成了酒精，还原糖的含量急剧下降，当温度过高时，大部分酵母菌停止生长，所以残留的还原糖趋于稳定(P>0.05)，由此初步确定最适宜的发酵温度为24℃。
焦亚硫酸钾对茵红李果酒酒精度和还原糖的影响
如图4中所示，在不同的偏重亚硫酸钾的添加量下酒精度变化不大(P>0.05)，当焦亚硫酸钾添加量为0.09g/L时，酒精度最高为12.8％；还原糖随焦亚硫酸钾量的增加先减少后增加，当焦亚硫酸钾的添加量为 0.09g/L时，还原糖的含量最少，而当偏重亚硫酸钾的添加量大于0.09g/L时，还原糖的含量有所增加，由此初步确定最适宜的焦亚硫酸钾添加量为0.09g/L。这是由于焦亚硫酸钾在一定的条件下会分解成SO 2，由于SO 2具有杀菌作用，造成此现象的原因是当游离的SO 2达到一定的量时，不仅会杀死其他微生物，对酵母菌也有一定的杀伤能力，甚至会杀灭一些较弱的酵母菌，因此果酒中的还原糖被利用的就少。
酵母添加量对茵红李果酒酒精度和还原糖的影响
如图5中所示，随着酵母添加量的增加，酒精度呈现先上升在下降的趋势，当酵母添加量为0.4g/L时酒精度最高为14.05％。酵母的添加量过少会导致营养物质的利用降低，自身繁殖代谢慢，杂菌生长快，产酒精量少；接种量过大，酵母菌繁殖过旺，需要消耗大量的糖分用于自身生长，代谢为的酒精含量反而降低。同时，当酵母的添加量逐渐增加时，还原糖的含量逐渐减小。当酵母的添加量为0.7g/L的时候，还原糖的含量达到最低。酵母的添加量越多果酒发酵中对营养物质的需求越大，剩余的还原糖的含量就越小，所以还原糖的含量随着酵母菌的添加量的增加而减小。虽然酵母的添加量越多会影响其生长，但对还原糖的消耗仍然会继续。由此选0.4g/L做为酵母最适宜的添加量。
正交试验结果
在茵红李果酒发酵单因素试验的基础上，选出以上因素的较优范围，以酒精度作为指标，选用L 18(3 7)进行正交设计来优化茵红李果酒发酵工艺条件。因素和水平表见表2，试验方案和分析结果见表3。
表2 正交试验水平因素
Figure PCTCN2022072866-appb-000002
Figure PCTCN2022072866-appb-000003
由表3知，影响酒精度得因素大小关系依次为发酵时间(A)＞发酵温度(C)＞初始糖度(B)＞焦亚硫酸钾添加量(D)>酵母添加量(E)。最佳工艺参数为A 2B 2C 3D 3E 2，即发酵时间为7d，初始糖度23°Bx，发酵温度为27℃，焦亚硫酸钾添加量为0.12g/L，酵母添加量为0.4g/L。由于最佳试验组未出现在试验设计内，因此按此条件进行了重复验证试验，在此优化条件下发酵7d后，茵红李果酒的酒精度(以体积分数计)为14.5％，高于其它各发酵组，可见该工艺结果可靠，由此确定茵红李果酒最佳发酵工艺组合为A 2B 2C 3D 3E 2。
表3 正交试验结果
Figure PCTCN2022072866-appb-000004
Figure PCTCN2022072866-appb-000005
有机酸测定结果
有机酸标准品混合溶液色谱图及有机酸含量结果如图6和表4中所示。由图6可知，10种混合有机酸能较好地分离，峰形较好，没有重叠和拖尾现象，表明本试验色谱条件适用于茵红李果实和果酒中有机酸的测定。 由表4可知，茵红李果实中一共检出7种酸，分别是苹果酸、草酸、柠檬酸、酒石酸、莽草酸、丙酮酸、富马酸。最多的有机酸是苹果酸，达到6.976g/kg，其次是草酸含量为0.409g/kg，最少的酸是富马酸含量为0.004g/kg，其余4种酸含量均在0.1g/kg以下。茵红李果酒残糖发酵结束后对10种有机酸进行检测，共检出9种，未检出莽草酸，检出的有机酸分别为草酸、丙酮酸、苹果酸、乳酸、乙酸、柠檬酸、琥珀酸、酒石酸、富马酸。其中苹果酸、乳酸、琥珀酸的含量较高，最高含量分别达到4.503g/L、3.37g/L、0.832g/L，而草酸、乙酸、柠檬酸、丙酮酸、酒石酸、富马酸的含量均保持在0.5g/L以下，表明苹果酸、乳酸、琥珀酸是茵红李果酒中的主要有机酸。
茵红李果实中的主要有机酸为苹果酸，含量最高。残糖发酵过程会触发苹果酸乳酸发酵，苹果酸会转化成乳酸，最终乳酸含量增加，乳酸还来源于酵母的酒精发酵，成为除苹果酸以外含量最高的酸；琥珀酸是酵母菌的三羧酸循环(TCA)的中间产物；丙酮酸由糖酵解途径产生。乙酸含量较低为0.023g/L，它是酒精发酵过程中乙醛经氧化还原作用产生，也是极少量的醋酸菌进行了醋酸发酵产生了少量的乙酸。
茵红李果酒中柠檬酸比果实中多，这是由于柠檬酸在酵母有氧和无氧状态都会产生，在有氧条件下，经TCA循环生成；无氧条件，由草酰乙酸还原得到。外界条件对柠檬酸代谢有很大的影响，因此柠檬酸在酒中含量比果实中含量多或少都是有的。与茵红李果实相比茵红李果酒中草酸的含量降低，草酸经过氧化、脱羧和乙酰化作用生成了CO 2；在茵红李果酒中莽草酸未检出，莽草酸是经过莽草酸途径被分解为苯丙氨酸，酪氨酸，色氨酸被酵母所利用。
表4 有机酸测定结果
Figure PCTCN2022072866-appb-000006
表4中，N.D表示未检测出。
由以上可知：通过单因素试验，在茵红李果酒发酵过程中，酵母添加量、初始糖度、发酵温度、发酵时间、焦亚硫酸钾添加量对酒精度、还原糖均有影响，通过正交设计优化得茵红李果酒发酵最优工艺为发酵时间7d，初始糖度23°Bx，发酵温度24℃，焦亚硫酸钾添加量0.09g/L，酵母添加量0.4g/L。
采用HPLC，对茵红李果实及其发酵果酒中有机酸进行测定。在果实中定量检测出来7种酸，其中苹果酸含量最高占果实检出总酸的92.5％，其他6种有机酸含量都不超过0.5g/kg；在茵红李果酒中定量检测出了9 种有机酸，苹果酸含量最高，占检出酸总和的47.2％，其次是乳酸占35.3％，琥珀酸占8.7％，其他6种酸检出量均低。茵红李果实经发酵成茵红李果酒后，苹果酸含量降低，乳酸含量增加，但苹果酸相较于其他种类水果果酒中含量还是较多的。
以上，针对茵红李果实的果酒酿造工艺还可以应用于其他李子品种中。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。

Claims (13)

  1. 一种茵红李果酒的酿造方法，其特征在于，包括以下步骤：
    (1)挑选无虫害、无霉烂的茵红李，清洗并沥干水分；
    (2)对茵红李进行肉核分离，将果肉打浆；
    (3)加入食品级果胶酶酶解；
    (4)加入降酸剂调酸，加入砂糖调节糖度；
    (5)加入抑菌剂；
    (6)将酵母接种到发酵液中进行恒温发酵；
    (7)残糖发酵；
    (8)将发酵好的酒体密封陈酿；
    (9)在陈酿好的酒液中加入澄清剂进行澄清，过滤，制得茵红李果酒。
  2. 根据权利要求1所述的一种茵红李果酒的酿造方法，其特征在于，步骤(3)为加入不少于0.03g/L的食品级果胶酶酶解至少2h。
  3. 根据权利要求2所述的一种茵红李果酒的酿造方法，其特征在于，步骤(3)中，在不高于45℃的温度下进行酶解。
  4. 根据权利要求1所述的一种茵红李果酒的酿造方法，其特征在于，步骤(4)中，所述降酸剂为食品级碳酸钙和碳酸氢钾的混合降酸剂；混合降酸剂的浓度为2g/L；所述砂糖为白砂糖，加入白砂糖将糖度调节至不高于23°Bx。
  5. 根据权利要求1所述的一种茵红李果酒的酿造方法，其特征在于，步骤(5)为加入不大于0.09g/L的食品级焦亚硫酸钾抑菌剂或者加入0.12g/L的食品级焦亚硫酸钾抑菌剂。
  6. 根据权利要求1所述的一种茵红李果酒的酿造方法，其特征在于，步骤(6)为将活化后的酵母接种到发酵液中恒温发酵5～11d，酵母的菌体浓度不小于0.4g/L。
  7. 根据权利要求6所述的一种茵红李果酒的酿造方法，其特征在于，步骤(6)中，所述恒温发酵的时间为7～9d。
  8. 根据权利要求6或7所述的一种茵红李果酒的酿造方法，其特征在于，步骤(6)中，在恒温箱中进行恒温发酵；恒温温度为24～27℃。
  9. 根据权利要求1所述的一种茵红李果酒的酿造方法，其特征在于， 步骤(8)为将发酵好的酒体密封满罐陈酿。
  10. 根据权利要求1所述的一种茵红李果酒的酿造方法，其特征在于，步骤(9)中，在陈酿好的酒液中加入皂土进行澄清。
  11. 根据权利要求1所述的一种茵红李果酒的酿造方法，其特征在于，所述茵红李替换为其他李子品种。
  12. 一种茵红李果酒，其特征在于，由权利要求1～11任一项所述的一种茵红李果酒的酿造方法制到，含有草酸、丙酮酸、苹果酸、乳酸、乙酸、柠檬酸、琥珀酸、酒石酸和富马酸9种有机酸。
  13. 根据权利要求12所述的茵红李果酒，其特征在于，所述苹果酸占检出酸总和的47.2％、乳酸占检出酸总和的35.3％、琥珀酸占检出酸总和的8.7％。
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