Method for increasing content of cubene in fen-flavor liquor by adding fructus amomi in brewing process

Abstract

The invention relates to the technical field of baijiu brewing, and particularly discloses a method for increasing the content of cobaene in fen-flavor baijiu by adding fructus amomi in the brewing process, which comprises the following steps: adding fructus amomi powder into green-skin Guanyin koji according to 20% of the total mass of the koji for mixed fermentation; the green coating white clay koji is prepared from 1-3 parts of rice bran, 1-3 parts of mold koji and 5-10 parts of white clay, the mold koji is prepared by inoculating and culturing mother koji according to 1.5-2.5% of the mass of rice flour, and the mother koji comprises rhizopus oryzae, saccharomyces cerevisiae and Wickerhasenula polymorpha. The efficient synthesis of the cubene in a non-transgenic way is realized in the fen-flavor liquor for the first time.

Description

Technical Field

[0001] The invention relates to the technical field of liquor brewing, and specifically provides a method for increasing the content of copaene in light-fragrance liquor by adding Amomum villosum during a brewing process. Background Art

[0002] Light-fragrance baijiu (Chinese liquor) is popular among consumers for its pure fragrance, mellow sweetness, natural harmony, and refreshing aftertaste. In 2016, Academician Sun Baoguo proposed that the development direction of baijiu should be "dual-oriented in flavor and health," and healthy baijiu has become a new trend in Chinese baijiu.

[0003] Cubanene (C 15 H 24 ) belongs to the terpene class of substances, has a unique fragrance, as well as antioxidant, anti-aging, anti-inflammatory, analgesic, blood pressure lowering, anti-tumor activity and other effects. It exists naturally in many medicinal and aromatic plants and is widely used in food, medicine, agriculture and other fields. It is an ingredient with a unique flavor and certain health care functions. Adding Chinese medicinal materials through the process of koji making, fermentation and aging to introduce healthy ingredients into liquor is one of the means of brewing healthy liquor. There has been no comprehensive and systematic study on Cubanene in various types of liquor. At present, the presence of this ingredient has only been detected in Bamboo Leaf Green Liquor, and the specific content is unknown. It mainly comes from Amomum villosum and Patchouli added to the wine. However, there are two major technical bottlenecks in its introduction into liquor: Limitations of the exogenous addition method: For example, Bamboo Leaf Green Liquor introduces cubanene by soaking herbs such as Amomum villosum and Patchouli. However, this method results in a pronounced medicinal taste, destroying the pure flavor of the light-fragrant liquor, and the ingredient is easily lost due to volatility. Microbial synthesis is hindered: The genetically engineered yeast developed by Hubei University of Technology can synthesize α-cubane, but the "Terms of the Liquor Industry" (GB / T 15109) clearly stipulates that the use of genetically modified strains in liquor production is prohibited, and consumers have a low acceptance of genetically modified ingredients.

[0004] Therefore, adding the Chinese medicinal herb Amomum villosum containing cubanene during the brewing process to increase the cubanene content in the liquor and achieve the endogenous generation of cubanene in the light-fragrance liquor is a method that is currently feasible and complies with industry regulations. Summary of the Invention

[0005] In view of this, the present invention proposes a method for adding Amomum villosum during the brewing process to increase the content of copaene in light-fragrance liquor. The Chinese medicinal material Amomum villosum is applied to the brewing process of light-fragrance liquor, and after the fermentation is completed, the content of copaene in the liquor is significantly increased, and the quality and flavor of the liquor are improved.

[0006] The technical solution of the present invention is achieved as follows: The present invention provides a method for adding Amomum villosum during a brewing process to increase the content of copaene in light-fragrance liquor, comprising: adding Amomum villosum powder to green-coated Guanyin koji and mixing, and the mixed koji is used for fermenting light-fragrance liquor, wherein the green-coated Guanyin koji is prepared from 1-3 parts of rice bran, 1-3 parts of seed koji, and 5-10 parts of Guanyin soil, wherein the seed koji is a mother koji inoculated into rice flour at 1.5-2.5% of the mass of rice flour, and is prepared by culturing at 28-32°C for 48-72 hours, and the mother koji contains Rhizopus oryzae, Saccharomyces cerevisiae, and Wilcoxonella oxysporum.

[0007] In some embodiments, the amount of Amomum villosum powder added is 20% of the mass of the koji.

[0008] In some embodiments, the method for preparing the green-clothed Guanyin earthenware comprises the following steps: (1) Mix rice bran, koji and Guanyin soil in proportion, add water and knead into a sphere with a diameter of 8-10 cm; (2) Cultivate the spheres at 25-30°C and 55-75% humidity for 7 days; (3) Dry until the moisture content is ≤5.0%.

[0009] In some embodiments, the Amomum villosum is a local medicinal material in the production area of ​​Jinpai Co., Ltd., specifically the Amomum villosum grown in the production area of ​​Huangshi City, Hubei Province, Jinpai Co., Ltd., preferably Amomum villosum, which is crushed into 80-100 mesh powder before use.

[0010] In some embodiments, the fermentation process comprises: After the cooked grains are air-dried to 30℃, add green Guanyin koji containing Amomum villosum and saccharify for 24 hours; The saccharified mash and sterilized distiller's grains are mixed in equal proportions and fermented under oxygen for 7 days; Distill the wine.

[0011] In a second aspect, the present invention further provides an application of green-coated Guanyin earthenware containing Amomum villosum in the production of light-fragrance liquor, and the green-coated Guanyin earthenware mixture containing Amomum villosum is fermented and brewed according to the light-fragrance liquor fermentation process.

[0012] In a third aspect, the present invention also provides a light-fragrance liquor obtained by fermenting green-clothed Guanyin koji containing Amomum villosum, and introducing the healthy ingredient cubane into the light-fragrance liquor, thereby achieving the endogenous growth of the healthy ingredient cubane in the light-fragrance liquor.

[0013] The present invention has the following beneficial effects compared to the prior art: While minimizing the impact on the quality of light-flavor liquor, the present invention achieved a 112.49 micrograms per liter (µg / L) copaene content in the resulting liquor sample when Amomum villosum was added at a level of 20% of the total koji (no copaene was detected in the control liquor sample). The results showed no significant impact on n-propanol and fusel oil, while increasing ethyl acetate by 34.83% and liquor yield by 4.23%. This approach achieves the endogenous production of the healthy ingredient copaene in light-flavor liquor and significantly improves its quality. DETAILED DESCRIPTION

[0014] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the present invention belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with definitions set forth in the patents, patent applications, published patent applications, and other publications incorporated herein by reference, the definitions listed in this section take precedence over the definitions incorporated herein by reference.

[0016] Unless otherwise specified, the methods used in the following examples are conventional methods. The materials, reagents, and instruments used are conventional materials, reagents, and instruments in the art, unless otherwise specified, and can be obtained commercially by those skilled in the art.

[0017] When an amount, concentration or other value or parameter is expressed as a range, a preferred range or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any upper range limit or preferred value with any lower range limit or preferred value, regardless of whether the range is disclosed alone. For example, when a range "1 to 5" is disclosed, the described range should be interpreted as including the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range. In the present specification and claims, range definitions may be combined and / or interchanged, and if not otherwise stated, such ranges include all subranges contained therein.

[0018] Examples 1-5 Production of koji Preparation of seed koji: Take the seed koji of light-fragrance liquor (containing Rhizopus oryzae, Saccharomyces cerevisiae and Wilcoxon yeast) as the mother koji, inoculate it into rice flour at 2% of the mass of rice flour, add water to adjust the moisture content to 45%, and culture it at a temperature of 28-32℃ for 60h to obtain a new seed koji. After testing, it was found that the new seed koji contained Rhizopus oryzae, Saccharomyces cerevisiae and Wilcoxon yeast.

[0019] Prepare rice bran, seed koji, and Guanyin soil in the ratio of 2 parts rice bran, 2 parts seed koji, and 8 parts Guanyin soil. Mix thoroughly, add appropriate amount of water and stir, and then shape into balls with a diameter of 8-10 cm after stirring evenly. Place the shaped koji balls in the box bed. Select the koji balls in the central area of ​​the box bed as the monitoring point and insert them into the temperature and humidity recorder to automatically record the temperature values ​​every hour. Open the box about 24 hours after entering the box. Wait for the temperature of the koji in the box to naturally cool to room temperature. Transfer the koji to the koji bending machine. Control the temperature of the incubator between 25-30℃ and the humidity of the incubator between 65±10%. After 7 days of incubation, dry the koji balls until the moisture content of the koji is less than or equal to 5.0%. Then, take them out of the drying room and put them into storage for future use.

[0020] Brewing of Medicinal Qu Liquor 280 g of glutinous sorghum was weighed and soaked in 60°C hot water overnight. Steaming began 24 hours later, with the initial steaming conditions being 115°C for 20 min. After the initial steaming, the grain was simmered in 60°C hot water for 10 min, and then re-steamed at 110°C for 10 min. After steaming, 1 kg of cooked grain was weighed and aired to approximately 30°C. 5.0 g of green-clothed guanyin (a Chinese medicinal herb) was added as a control, while Examples 1-5 were added with varying amounts of Huangshi-produced Amomum villosum (10% (Example 1), 20% (Example 2), 30% (Example 3), 40% (Example 4), and 50% (Example 5). The mixture was thoroughly mixed and saccharified at 30°C for one day. After saccharification, the mash was mixed with sterilized distiller's grains in equal proportions. After 7 days of oxygen-free fermentation, the liquor was distilled and sampled over low heat. The contents of ethyl acetate, fusel oil, and n-propanol were determined by gas chromatography, and the changes were calculated based on the control (change = (a certain indicator in the experimental group - a certain indicator in the control group) / a certain indicator in the control group × 100%). The content of cubane in the liquor was determined by headspace solid-phase microextraction-gas chromatography-mass spectrometry.

[0021] Detection method Gas chromatography analysis method: Accurately transfer 1 mL of the base liquor into a 2 mL sample vial. Add 10 μL of a three-internal standard solution (tert-amyl alcohol (IS1): 17028.1 mg / L, n-amyl acetate (IS2): 16864 mg / L, and 2-ethylhexanol (IS3): 12104.2 mg / L). Cap the vial and shake thoroughly. Determine the contents of ethyl acetate, n-propanol, and fusel oil in the base liquor by gas chromatography. Chromatographic conditions: Agilent HP6890 High-Performance Gas Chromatograph, FID detector, CP-WAX capillary column (50 m x 0.2 μm x 0.25 mm). Temperature program: Maintain the column temperature at 40°C for 8 min, then increase the temperature to 150°C at a rate of 5°C / min. The injection port temperature was 250°C, the detector temperature was 260°C, the air and hydrogen flow rate ratio was 300:30, the carrier gas was nitrogen, the split ratio was 30:1, the column flow rate was 1.0 mL / min, and the injection mode was automatic injection.

[0022] Headspace solid-phase microextraction-gas chromatography (GC-MS): 1.0 mL of alcohol sample was added with 3 g of sodium chloride and 7.0 mL of ultrapure water, along with 10 μL of eugenol-d3 (50 mg / L) as an internal standard. The headspace vial was capped and shaken thoroughly. The solid-phase microextraction tip was inserted into the GC inlet and conditioned for 15 minutes at 250°C and a carrier gas flow rate of 3 mL / min. The headspace vial was then placed in an automated extraction apparatus, with the tip inserted to a depth of 23 mm. Sample equilibration time was 1 minute, extraction temperature was 50°C, extraction time was 45 minutes, and inlet desorption was performed for 5 minutes. Gas chromatography conditions included a DB-FFAP (60 m × 0.25 mm × 0.25 μm) capillary column at a flow rate of 1.0 mL / min; an inlet temperature of 250°C; and a splitless carrier gas of high-purity helium (>99.999%) in constant flow mode. The column temperature program was as follows: a 40°C initial temperature hold for 5 min, then a 10°C / min increase to 180°C, followed by a 20°C / min increase to 240°C, where the temperature was held for 2 min. Mass spectrometry conditions included an electron impact ionization (EI) source with an energy of 70 eV and a temperature of 230°C. The quadrupole temperature was 150°C, the auxiliary channel heater was at 280°C, and a solvent delay of 5 min. The characteristic ion scan (SIM) analysis yielded cubane quantifier ion 161, qualifier ions 119 and 105, and guaiacol-d3 quantifier ion 127.

[0023] Quantitative analysis: Establishment of an internal standard curve: Dissolve the standard compound of cubane in anhydrous ethanol to prepare a standard stock solution of known mass concentration. Gradual dilution is performed to form a series of mixed standard solutions (1, 5, 10, 50, 100 μg / L). Pre-treatment and detection are performed according to the above sample method. Finally, an internal standard standard curve is established with the peak area ratio of the analyte to the corresponding internal standard as the ordinate and the mass concentration ratio as the abscissa. The mass concentration of cubane in the sample is calculated based on this curve.

[0024] The new strains prepared in Examples 1-5 above were subjected to high-throughput sequencing combined with plate count method to calculate the percentage of viable bacteria, and the bacterial species percentage data shown in the following table were obtained:

[0025] Comparative Example 1 The method for preparing distiller's yeast is the same as that in Example 2, except that 20% cloves are added instead of Amomum villosum.

[0026] The method for brewing medicinal liquor is the same as that in Example 2.

[0027] The index detection method is the same as that in Example 2.

[0028] Comparative Example 2 The method for preparing distiller's yeast is the same as that in Example 2, except that 20% of Patchouli sinensis is added instead of Amomum villosum.

[0029] The method for brewing medicinal liquor is the same as that in Example 2.

[0030] The index detection method is the same as that in Example 2.

[0031] Comparative Example 3 The method for preparing distiller's yeast is the same as that in Example 2, except that 20% jasmine is added instead of Amomum villosum.

[0032] The method for brewing medicinal liquor is the same as that in Example 2.

[0033] The index detection method is the same as that in Example 2.

[0034] Comparative Example 4 The method for preparing distiller's yeast is the same as that in Example 2, except that 20% Shennong Xiangju is added instead of Amomum villosum.

[0035] The method for brewing medicinal liquor is the same as that in Example 2.

[0036] The index detection method is the same as that in Example 2.

[0037] The results are shown in the following table:

[0038] The data in the table above show that the content of copainene in the liquor samples increases with the addition of Amomum villosum, but drops sharply at an addition of 50%. This may be due to the excessive addition, which affects the proliferation of microorganisms in the koji, thereby affecting the release of copainene in the liquor. Tasting results show that when the Amomum villosum content is too high, exceeding 20%, the medicinal taste becomes more pronounced, affecting the flavor of the light-fragrant liquor. Gas chromatography data shows that when the Amomum villosum content exceeds 20%, the n-propanol and fusel oil levels in the liquor increase significantly, which has a certain negative impact on the liquor quality. Therefore, without affecting the quality and flavor of the liquor, the optimal addition of Amomum villosum should be 20%. This increases the copainene content in the liquor by 112.49 μg / L, has no significant impact on n-propanol and fusel oil, and increases the ethyl acetate content by 34.83%, increasing the liquor yield by 4.23%.

[0039] The comparative examples are four medicinal materials that are reported in the literature to produce copaene. Compared with Example 2, their ability to increase the content of copaene in the light-fragrance liquor is not as good as adding 20% ​​Amomum villosum. Some medicinal materials have a greater negative effect on the n-propanol, fusel oil and alcohol rate in the liquor quality.

[0040] Comprehensive comparison shows that in order to increase the content of cubanene under the premise of having little impact on the quality of the liquor, when Amomum villosum is added at a rate of 20% of the total amount of koji, the cubanene content is increased to the highest level, reaching 112.49 μg / L, which improves the quality of the liquor and realizes the endogenous generation of cubanene in the light-fragrance liquor.

[0041] Example 6 On the basis of Example 2, the addition amount of Amomum villosum was changed to 15%, and the other steps and conditions were the same as Example 2.

[0042] Example 7 On the basis of Example 2, the addition amount of Amomum villosum was changed to 25%, and the other steps and conditions were the same as Example 2.

[0043] After testing the indicators of the obtained wine, the results are shown in the following table:

[0044] Examples 8-9 This example uses self-blended koji on the basis of Example 2, and the proportion of viable bacteria in the koji is as follows:

[0045] Other parameters are the same as those in Example 2. The indicators of the obtained wine are tested as shown in the following table:

[0046] Comparative Examples 5-6 This comparative example uses self-blended koji on the basis of Example 2, and the proportion of viable bacteria in the koji is as follows:

[0047] Other parameters are the same as those in Example 2. The indicators of the obtained wine are tested as shown in the following table:

[0048] Comparative Example 7 This comparative example is based on Example 2, but the step of adding 20% ​​Amomum villosum to the koji is deleted. An equal amount of Amomum villosum is added to the fermented mash before distillation, and then the wine is distilled. As a result, the content of cubane in the obtained wine is 38.51 μg / L, and the wine has a prominent medicinal flavor.

[0049] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for increasing the content of copaene in light-fragrance liquor by adding Amomum villosum during the brewing process, characterized in that The Amomum villosum powder is added to the green Guanyin koji and mixed. The mixed koji is used for the fermentation of light-fragrance liquor. The green Guanyin koji is prepared by 1-3 parts of rice bran, 1-3 parts of seed koji, and 5-10 parts of Guanyin soil. The seed koji is the mother koji, which is inoculated into the rice flour at 1.5-2.5% of the mass of the rice flour and cultured at 28-32°C for 48-72 hours. The mother koji contains Rhizopus oryzae, Saccharomyces cerevisiae, and Wilcoxon yeast.

2. The method according to claim 1, wherein The amount of Amomum villosum powder added is 20% of the mass of the koji.

3. The method according to claim 1, wherein The preparation method of the green-clothed Guanyin earthenware comprises the following steps: (1) Mix rice bran, koji and Guanyin soil in proportion, add water and knead into a sphere with a diameter of 8-10 cm; (2) Cultivate the spheres at 25-30°C and 55-75% humidity for 7 days; (3) Dry until the moisture content is ≤5.0%.

4. The method according to claim 1, wherein The Amomum villosum is a local medicinal material produced in the production area of ​​Jinpai Co., Ltd. and is crushed into 80-100 mesh powder before use.

5. The method according to claim 1, wherein The fermentation process includes: After the cooked grains are air-dried to 30℃, add green Guanyin koji containing Amomum villosum and saccharify for 24 hours; The saccharified mash and sterilized distiller's grains are mixed in equal proportions and fermented under oxygen for 7 days; Distill the wine.

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