A systematic evaluation method for the physical and chemical qualities of a hair grape fine beer

Abstract

A kind of physical and chemical quality systematic evaluation method of hairy grape refined beer belongs to beer quality detection and evaluation technical field.The present application is to solve the technical problems that the existing traditional beer national standard detection method is poor in adaptability to hairy grape refined beer matrix,there is no exclusive systematic quality evaluation system,there is no precise synchronous detection method for characteristic functional components and flavor substances,constructs a whole-process evaluation system covering four dimensions of fermentation process dynamic monitoring,basic physical and chemical index detection,characteristic functional component quantification and core flavor substance analysis,optimizes the exclusive operation parameters and chromatographic conditions of each detection index.The detection results of the method are accurate and the detection efficiency is high,which can realize the whole-process standardized evaluation of hairy grape refined beer from fermentation control to finished product grading,provides scientific support for process optimization and quality standard formulation of this category.

Description

Technical Field

[0001] This invention relates to the field of beer physicochemical testing technology, and in particular to a systematic method for evaluating the physicochemical quality of wild grape craft beer. Background Technology

[0002] Craft beer is currently a core development direction in the beer industry. With consumers' increasing demand for personalized and healthy beverages, fruit-flavored craft beer, with its unique flavor and rich nutritional characteristics, has become a key area of ​​innovation in the craft beer category. Wild grapes, as a distinctive wild grape variety, are rich in various polyphenols, organic acids, and other functional components. Introducing wild grapes into beer brewing not only imparts a unique fruity aroma and rich acidity, broadening the flavor profile of beer, but also enhances its antioxidant and other health potential, possessing extremely high research and development value and promising prospects for industrial application.

[0003] Currently, the quality testing of traditional beer mainly relies on GB / T 4928-2008 "Methods for Beer Analysis," which provides a standardized basis for the testing of basic physicochemical indicators of traditional beer. Existing research has also explored optimizations for the testing of single indicators in some fruit-flavored beers. However, existing standards lack systematic component testing and comprehensive evaluation methods for specialty products like wild grape beer, particularly methods for precise qualitative and quantitative analysis of its characteristic organic acid profile, functional phenolic substances, and volatile flavor compounds. Furthermore, the national standard methods are designed for the traditional beer matrix. The large amount of exogenous organic acids, polyphenols, and sugars introduced from wild grapes form a complex matrix, interfering with the national standard testing methods for core indicators such as total acidity, bitterness, and color, leading to systematic biases in the test results and failing to accurately reflect the true quality of the product. Therefore, developing a systematic quality evaluation method suitable for wild grape craft beer has become an urgent technical problem to be solved in this field. Summary of the Invention

[0004] The purpose of this invention is to solve the above-mentioned problems in the prior art and provide a systematic evaluation method for the physicochemical quality of wild grape craft beer, providing a scientific and technological solution for the research and development and quality control of this category, improving the quality evaluation system of specialty fruit-flavored craft beer, and empowering the deep processing industry chain of wild grapes to achieve the dual development of agriculture and craft beer industry.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A systematic evaluation method for the physicochemical quality of wild grape craft beer includes the following steps:

[0007] 1) Dynamic monitoring of the fermentation process: The sugar-lowering ability of yeast during the fermentation of wild grape craft beer is monitored by carbon dioxide loss method, and the main fermentation endpoint is determined based on the carbon dioxide release rate;

[0008] 2) Basic physicochemical index testing: Quantitative testing of total acidity, bitterness, color, free amino nitrogen content, total sugar content, and alcohol content of wild grape craft beer;

[0009] 3) Quantitative analysis of characteristic functional components: The characteristic functional components in the craft beer made from wild grapes were qualitatively and quantitatively detected by high performance liquid chromatography. The characteristic functional components include six organic acids: citric acid, tartaric acid, malic acid, succinic acid, lactic acid, and acetic acid, as well as chlorogenic acid.

[0010] 4) Quantitative analysis of core flavor compounds: Headspace gas chromatography was used to simultaneously and quantitatively detect the core volatile flavor compounds in wild grape craft beer. The core volatile flavor compounds include five higher alcohols: n-propanol, isobutanol, isoamyl alcohol, 2-methyl-1-butanol, and 2-phenylethanol, as well as five esters: ethyl acetate, ethyl lactate, amyl acetate, ethyl hexanoate, and phenylethyl acetate.

[0011] In step 1), the carbon dioxide loss method includes: periodically weighing the mass of the fermentation system during the fermentation process, calculating the amount of carbon dioxide lost per unit time, and using this loss as an indicator of the yeast fermentation rate; when the daily carbon dioxide loss is lower than a preset threshold for a continuous period of time, the main fermentation stage is determined to be over.

[0012] In step 2), the total acid content is determined by alkaline titration and expressed as tartaric acid or lactic acid; the bitterness is determined by organic solvent extraction-ultraviolet spectrophotometry.

[0013] In step 2), the colorimetry is determined by spectrophotometry, with wavelengths including 430 nm and 700 nm, and the colorimetry value is calculated based on the absorbance.

[0014] In step 2), the free amino nitrogen content is determined by the ninhydrin colorimetric method.

[0015] In step 2), the total sugar content is determined by the phenol-sulfuric acid colorimetric method or other sugar colorimetric methods.

[0016] In step 2), the alcohol content is determined by high performance liquid chromatography, gas chromatography, or distillation-density method.

[0017] In step 3), the high performance liquid chromatography conditions for the six organic acids are as follows: cation exchange column, dilute sulfuric acid solution as mobile phase, and ultraviolet detector.

[0018] In step 3), the high performance liquid chromatography (HPLC) conditions for chlorogenic acid are as follows: reversed-phase C18 column, gradient elution with acidic aqueous solution and organic solvent as mobile phase, detection with ultraviolet detector, and sample pretreatment before HPLC detection, which includes degassing, centrifugation and filtration.

[0019] In step 4), the headspace injection conditions for the headspace gas chromatography method include: an equilibrium temperature of 60~90℃ and an equilibrium time of 20~40 min.

[0020] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0021] This invention establishes for the first time a unique physicochemical quality evaluation system for wild grape craft beer, addressing the core pain points of strong matrix interference from directly applying existing national standard methods and the long-standing lack of a dedicated evaluation standard for this category, thus filling a gap in the industry. This invention provides core support for process optimization and quality control in wild grape craft beer, and also offers a referable template for the quality evaluation of other specialty fruit beers. Furthermore, it broadens the channels for deep processing of wild grapes, increasing the added value of agricultural products. Attached Figure Description

[0023] Figure 1 The daily CO2 loss of Nottingham yeast varies with fermentation time;

[0024] Figure 2 For FAN content standard curve;

[0025] Figure 3 This is the standard curve for total sugar content;

[0026] Figure 4 This is the standard curve for alcohol content.

[0027] Figure 5 Standard curve for the content of major organic acids;

[0028] Figure 6 This is a standard curve for chlorogenic acid content;

[0029] Figure 7 This is a standard curve for the content of the main flavor compounds. Detailed Implementation

[0031] To make the technical problems, technical solutions and beneficial effects of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0032] This invention addresses the core deficiencies of existing technologies. The current GB / T 4928-2008 "Methods for Analysis of Beer" is not sufficiently adapted to the complex matrix of wild grape craft beer, and the industry lacks a systematic physicochemical quality evaluation system for the entire chain of this product. There is no accurate and simultaneous detection method for its characteristic organic acids, functional phenols and volatile flavor substances. This invention provides a set of exclusive systematic physicochemical quality evaluation methods adapted to wild grape craft beer.

[0033] A systematic evaluation method for the physicochemical quality of wild grape craft beer includes the following steps:

[0034] 1) Dynamic monitoring of the fermentation process: The CO2 weight loss method was used to dynamically monitor the yeast's sugar-reducing ability in the fermentation liquid of wild grape craft beer to determine the endpoint of the primary fermentation.

[0035] 2) Basic physicochemical index testing: Quantitative testing of total acidity, bitterness, color, free amino nitrogen (FAN) content, total sugar content, and alcohol content of wild grape craft beer;

[0036] 3) Quantitative analysis of characteristic functional components: The characteristic functional components in the craft beer made from wild grapes were qualitatively and quantitatively detected by high performance liquid chromatography. The characteristic functional components include six organic acids: citric acid, tartaric acid, malic acid, succinic acid, lactic acid, and acetic acid, as well as chlorogenic acid.

[0037] 4) Quantitative analysis of core flavor compounds: Headspace gas chromatography was used to simultaneously and quantitatively detect the core volatile flavor compounds in wild grape craft beer. The core volatile flavor compounds include five higher alcohols: n-propanol, isobutanol, isoamyl alcohol, 2-methyl-1-butanol, and 2-phenylethanol, as well as five esters: ethyl acetate, ethyl lactate, amyl acetate, ethyl hexanoate, and phenylethyl acetate.

[0038] In step 1), the specific operation of the CO2 loss method is as follows: add wort to the sterilized fermentation bottle, inoculate with yeast, install the fermentation plug, and place it in a constant temperature environment of 20 ℃ for fermentation; weigh the total mass of the fermentation bottle every 24 hours, calculate the amount of CO2 loss within 24 hours, and use the daily CO2 loss as the evaluation index of yeast fermentation rate; when the cumulative CO2 release within 24 consecutive hours is less than 0.20 g, the main fermentation stage is determined to be over.

[0039] In step 2), the total acid content was determined by indicator titration. Specifically, 100.0 mL of distilled water that had been boiled to remove carbon dioxide was added to 10.0 mL of beer sample that had been degassed by ultrasound. The mixture was heated to a boil again and then cooled to room temperature. Phenolphthalein indicator was added, and the solution was titrated with 0.1 mol / L sodium hydroxide standard titrant until a light pink color was obtained and the color did not fade for 30 seconds. The total acid content of the sample was then calculated.

[0040] In step 2), bitterness was determined using isooctane extraction-ultraviolet spectrophotometry. Specifically, 10.0 mL of undegassed beer sample at 10 ℃ was taken, octanol defoamer, 1.0 mL of 3 mol / L hydrochloric acid solution and 20.0 mL of isooctane were added, shaken for 15 min and centrifuged at 4000 r / min for 10 min. The absorbance of the upper isooctane extract was measured at a wavelength of 275 nm, and the bitterness value of the sample was calculated.

[0041] In step 2), the colorimetry is determined using a spectrophotometer. Specifically, after ultrasonic degassing and centrifugation at 4000 r / min for 10 min, the supernatant is taken and its absorbance is measured at wavelengths of 430 nm and 700 nm. If A 430 ×0.039>A 700 According to the formula S=A 430 Calculate the sample colorimetry using ×25×n, in EBC; where n is the sample dilution factor.

[0042] In step 2), the content of free amino nitrogen (FAN) was determined by the ninhydrin colorimetric method. Specifically, 200 μL of diluted sample was taken, 100 μL of ninhydrin colorimetric reagent was added, the mixture was mixed, heated in a boiling water bath for 16 min, cooled in a 20 ℃ water bath for 20 min, 500 μL of potassium iodate diluent was added and mixed, and the absorbance was measured at a wavelength of 570 nm. The FAN content was calculated by using the glycine standard curve.

[0043] In step 2), the total sugar content was determined using the phenol-sulfuric acid colorimetric method. Specifically, 200 μL of the diluted sample was taken, and 100 μL of 6% phenol solution and 500 μL of concentrated sulfuric acid were added sequentially. After vortexing and mixing, the sample was allowed to stand at room temperature for 5 min, heated in a water bath at 40 ℃ for 30 min, cooled to room temperature, and the absorbance was measured at a wavelength of 490 nm. The total sugar content was calculated using the glucose standard curve.

[0044] In step 2), the high-performance liquid chromatography (HPLC) detection conditions for alcohol content are as follows: an Aminex HPX-87H column is used, the injection volume is 10 μL, the column temperature is 35 ℃, the mobile phase is 5 mmol / L sulfuric acid solution, the flow rate is 0.6 mL / min, and the detection is performed using a differential refractive index detector. Qualitative analysis is performed by retention time, and quantitative analysis is performed by external standard method.

[0045] In step 3), the high-performance liquid chromatography (HPLC) detection conditions for the six organic acids were as follows: an Aminex HPX-87H column was used, the injection volume was 10 μL, the column temperature was 30 ℃, the mobile phase was 3 mmol / L sulfuric acid solution, the flow rate was 0.3 mL / min, the UV detection wavelength was 215 nm, and qualitative analysis was performed by retention time, while quantitative analysis was performed by external standard method.

[0046] In step 3), the high-performance liquid chromatography (HPLC) detection conditions for chlorogenic acid were as follows: a reversed-phase C18 column was used, with an injection volume of 10 μL, a column temperature of 45 ℃, mobile phase A being chromatographic methanol, and mobile phase B being 0.1% formic acid aqueous solution, with a flow rate of 0.6 mL / min. Segmented low-pressure gradient elution was used: 0–10 min, A:B = 5:95; 10–30 min, A:B = 1:1; 30–40 min, A:B = 5:95. Ultraviolet detection was performed at a wavelength of 306 nm. Qualitative analysis was conducted using retention time, and quantification was performed using the external standard method.

[0047] In step 3), the sample pretreatment is uniformly as follows: take a sample of raw grape craft beer, degas it by sonication for 15 min, centrifuge it at 4 ℃, take the supernatant, filter it through a 0.45 μm organic phase filter membrane and set it aside.

[0048] In step 4), the headspace injection conditions are: equilibrium temperature 80 ℃, sampling needle temperature 95 ℃, valve box temperature 110 ℃, transfer tube temperature 120 ℃; equilibrium time 30 min, purge flow rate 15 mL / min.

[0049] In step 4), the gas chromatography detection conditions are as follows: a Kroamt KB-1 capillary column and an FID detector are used; the carrier gas is high-purity nitrogen, constant flow mode is used, the column flow rate is 1.5 mL / min, and the split ratio is 15:1; the temperature program is as follows: the initial temperature is 90 ℃ and held for 5 min, the temperature is increased to 180 ℃ at 15 ℃ / min and held for 5 min, and then the temperature is increased to 240 ℃ at 20 ℃ / min and held for 15 min.

[0050] In step 4), the 10 target flavor compounds are qualitatively analyzed by retention time, and each compound is quantitatively analyzed by external standard method.

[0051] The following are specific examples.

[0052] Example 1

[0053] The complete implementation of a systematic evaluation method for the physicochemical quality of wild grape craft beer involves the following steps:

[0054] S1. Dynamic Monitoring of Fermentation Process and Determination of Primary Fermentation Endpoint: The CO2 loss method was used to dynamically monitor the yeast's sugar-lowering ability in the fermentation broth of wild grape craft beer. 300 mL of wort was added to a sterilized fermentation bottle, followed by the addition of activated Nottingham yeast activating solution at an inoculation rate of 0.5 g / L wort. After installing the fermentation plug and water seal, the bottle was placed in a 20 ℃ constant temperature incubator for fermentation. The total mass of the fermentation bottle was weighed every 24 hours, and the CO2 loss during that time period was calculated. A yeast fermentation rate curve was plotted with fermentation time (h) on the x-axis and daily CO2 loss (g) on ​​the y-axis. When the cumulative CO2 release was less than 0.20 g over 24 consecutive hours, the primary fermentation stage was considered essentially complete.

[0055] S2. Sample pretreatment: Take the raw grape craft beer sample after the main fermentation is completed and divide it into 3 parallel samples: the first sample is used for direct detection of basic physicochemical indicators; the second sample is degassed by ultrasonication for 15 min, centrifuged at 4 ℃ for 10 min, and the supernatant is filtered through a 0.45 μm organic phase filter membrane for high performance liquid chromatography detection; the third sample is degassed by ultrasonication for 60 min after being warmed to room temperature and then used for headspace gas chromatography detection.

[0056] S3. Quantitative Detection of Basic Physicochemical Indicators: For the basic quality evaluation of wild grape craft beer, quantitative detection of 6 core basic physicochemical indicators is set up, including total acidity, bitterness, color, free amino nitrogen (FAN), total sugar, and alcohol content.

[0057] S4. Precise Quantification of Characteristic Functional Components: High-performance liquid chromatography (HPLC) is used to perform qualitative and quantitative detection of characteristic functional components in wild grape craft beer. The target substances include six characteristic organic acids: citric acid, tartaric acid, malic acid, succinic acid, lactic acid, and acetic acid, as well as chlorogenic acid, a characteristic marker of wild grapes.

[0058] S5. Quantitative Detection of Core Flavor Compounds: Headspace gas chromatography was used to simultaneously and quantitatively detect the core volatile flavor compounds in wild grape craft beer. The target compounds included five higher alcohols: n-propanol, isobutanol, isoamyl alcohol, 2-methyl-1-butanol, and 2-phenylethanol; and five esters: ethyl acetate, ethyl lactate, pentyl acetate, ethyl hexanoate, and phenylethyl acetate, totaling 10 core flavor compounds.

[0059] Example 2

[0060] The methodological verification of the evaluation method of this invention was carried out, and the specific steps and results are as follows:

[0061] S1. Preparation of standard solutions: Prepare a series of gradient standard working solutions for each detection index, with the concentration range completely covering the detection linear range set by this invention. The specific range is shown in Table 1.

[0062] S2. Detection and Standard Curve Plotting: Perform three parallel analyses on each series of standard working solutions according to the detection method. Plot a standard curve with the target concentration on the x-axis and the corresponding absorbance or average peak area on the y-axis. Fit a linear regression equation and calculate the coefficient of determination R. 2 .

[0063] S3. Verification Results: The standard curves for all detection indicators in this invention exhibit excellent linearity, fully meeting the industry standard requirements for quantitative food testing. Specific results are as follows: Figures 2-7 As shown in Table 1.

[0064] Table 1 Retention times and standard curve parameters of different substances

[0065] Example 3.

[0066] The verification and implementation of the dynamic monitoring method for the fermentation process of this invention, including the specific steps and results, are as follows:

[0067] S1. Sample preparation: Add 300 mL of 12 °P wort to a sterilized fermentation flask, add activated Nottingham yeast at an inoculation rate of 0.5 g / L wort, and ferment at a constant temperature of 20 °C. Weigh the total mass of the fermentation flask every 24 h according to the method in Example 1, calculate the daily CO2 loss, and plot the yeast fermentation rate curve.

[0068] S2. Verification Results: Using the CO2 weight loss method of this invention, the complete metabolic process of yeast fermentation can be accurately captured, such as... Figure 1 As shown in the figure, the yeast is most active during the fermentation period from 0 to 72 hours, with the daily CO2 loss continuously increasing and reaching a peak at 72 hours. During this stage, the yeast consumes fermentable sugars in the wort at the fastest rate, and the intensity of sugar metabolism continues to increase. From 72 to 192 hours, the daily CO2 loss gradually decreases from the peak, and readily available monosaccharides are basically exhausted. Meanwhile, the yeast's metabolic rate of sugars such as maltose slows down. At the same time, factors such as the decrease in substrate concentration and the accumulation of ethanol lead to an overall weakening of yeast metabolic activity. When fermentation reaches 192 hours, the cumulative CO2 release over 24 hours is less than 0.20 g, which meets the main fermentation endpoint determination criteria set in this invention and is completely consistent with the typical pattern of yeast sugar metabolism.

[0069] Specific measurement method

[0070] Determination of total acid content: The determination was performed using the indicator titration method optimized according to the national standard GB / T4928-2008. 100.0 mL of distilled water was accurately transferred to a 250 mL Erlenmeyer flask, heated to boiling on a hot plate, and maintained for 2 minutes to remove dissolved carbon dioxide. Then, 10.0 mL of ultrasonically degassed beer sample was accurately added, and heating continued for 1 minute, ensuring the solution remained boiling again within the last 30 seconds of heating to thoroughly remove residual carbon dioxide. The Erlenmeyer flask was immediately removed and allowed to cool naturally for 5 minutes. The outer wall of the flask was then quickly rinsed with running tap water to rapidly cool the solution to room temperature. 0.5 mL of 0.1% phenolphthalein indicator solution was added to the cooled solution, and after mixing, titration was performed with 0.1 mol / L sodium hydroxide standard titrant until the solution turned a stable pale pink color that did not fade within 30 seconds. This was the titration endpoint. The volume of sodium hydroxide standard titrant consumed was recorded. The total acid content of the sample, calculated as tartaric acid content, is as follows: X = 75.045 × 0.1 × c² × V²

[0071] In the formula:

[0072] X—Total acid content of the sample (calculated as tartaric acid), in grams per liter (g / L);

[0073] c2—The actual concentration of the sodium hydroxide standard titrant, in moles per liter (mol / L).

[0074] V2—The average volume of sodium hydroxide standard titrant consumed, in milliliters (mL);

[0075] 0.1 — Combined conversion factor, which converts the total acid content of 10 mL sample to the concentration of 1 L sample.

[0076] Bitterness determination: The isooctane extraction-UV spectrophotometry method as specified in GB / T 4928-2008 was followed. 10.0 mL of undegassed beer sample, kept at 10 °C, was accurately transferred to a 50 mL centrifuge tube, and octanol was added as an antifoaming agent. Then, 1.0 mL of 3 mol / L hydrochloric acid solution was added, followed by 20.0 mL of isooctane. The centrifuge tube cap was tightened to ensure a tight seal, and the tube was shaken on an electric shaker for 15 min until a homogeneous emulsion formed. After shaking, the tube was centrifuged at 4000 r / min for 10 min to allow complete separation of the emulsion. The clear upper layer of isooctane extract was carefully aspirated and injected into a 10 mm quartz cuvette. Using isooctane as a blank control, the absorbance at 275 nm was measured using a UV spectrophotometer. The bitterness of the sample was calculated using the formula: X = A 275 ×50. Where X is the bitterness (BU) of the sample; A 275The absorbance is the average absorbance of the isooctane extract of the sample measured at a wavelength of 275 nm; 50 is the conversion factor.

[0077] Colorimetric determination: The colorimetric method specified in GB / T 4928-2008 was followed by ultrasonic degassing and centrifugation at 4000 r / min for 10 min to remove suspended solids. An appropriate amount of supernatant was injected into a 10 mm glass cuvette. The instrument was zeroed using ultrapure water as a reference. The absorbance values ​​of the sample at wavelengths of 430 nm and 700 nm were then measured sequentially and recorded as A. 430 With A 700 If A 430 ×0.039>A 700 To determine if the sample solution is clear and transparent, use the formula S=A 430 Calculate the sample colorimetry using ×25×n; if A 430 ×0.039<A 700 This indicates that the sample is turbid and requires further centrifugation or filtration before re-testing; if A 430 If the value is greater than 0.8, the sample needs to be appropriately diluted with ultrapure water and re-measured. In the formula, S is the sample colorimetric index (EBC), and n is the sample dilution factor.

[0078] FAN content determination: The ninhydrin method was used for determination, and the specific steps are as follows:

[0079] Preparation of colorimetric reagent: Accurately weigh 10.0 g Na2HPO4・12H2O, 6.0 g KH2PO4, 0.5 g ninhydrin and 0.3 g fructose, dissolve in water and bring to a final volume of 100 mL, transfer to a brown reagent bottle and store in a refrigerator at 4 ℃ protected from light.

[0080] Preparation of KIO3 dilution solution: Accurately weigh 2.0 g KIO3, dissolve it in 600 mL of water, dilute to 1000 mL with 95% ethanol, shake well, and store in a refrigerator at 4 ℃.

[0081] Glycine standard stock solution (1 g / L): Accurately weigh 0.1 g of glycine, dissolve it in water and bring the volume to 100 mL, then store it in a refrigerator at 4℃.

[0082] Determination Procedure: Take 200 μL of appropriately diluted wort sample into a centrifuge tube, add 100 μL of ninhydrin colorimetric reagent, mix well, and heat accurately in a boiling water bath for 16 min. After the reaction is complete, immediately transfer the centrifuge tube to a 20 °C water bath to cool for 20 min. Then add 500 μL of potassium iodate diluent and mix thoroughly. Within 30 min, using deionized water as a blank reference, measure the absorbance at a wavelength of 570 nm, and calculate the FAN content of the sample using the glycine standard curve.

[0083] Total sugar content determination: The phenol-sulfuric acid colorimetric method was used. A 6% (w / w) phenol aqueous solution was prepared and stored in the dark under refrigeration. 200 μL of the diluted wort sample was accurately transferred to a centrifuge tube, and 100 μL of phenol solution (restored to room temperature) and 500 μL of concentrated sulfuric acid were added sequentially. After vortexing to mix, the mixture was allowed to stand at room temperature for 5 min. The reaction system was then heated in a 40 ℃ water bath for 30 min. After cooling to room temperature, the absorbance was measured at 490 nm. The total sugar content of the sample was calculated using a standard curve.

[0084] Alcohol content determination: High performance liquid chromatography (HPLC) was used, with sample pretreatment identical to that in Example 1. Chromatographic conditions were as follows: Aminex HPX-87H column (300 mm × 7.8 mm, 4 μm); injection volume set to 10 μL; column and detector temperatures maintained at 35 ℃; mobile phase was 5 mmol / L sulfuric acid solution, prepared, filtered through a 0.45 μm filter, and degassed by sonication for 15 min; mobile phase flow rate set to 0.6 mL / min, using low-pressure gradient elution mode; detection was performed using a refractive index detector (RID), with qualitative analysis based on retention time; quantitative analysis was performed using the external standard method.

[0085] Determination of six characteristic organic acids: High performance liquid chromatography (HPLC) was used for determination, and the sample pretreatment was the same as in Example 1. Chromatographic conditions were as follows: An Aminex HPX-87H column (300 mm × 7.8 mm, 4 μm) was used; the injection volume was set to 10 μL; the column temperature and detector temperature were maintained at 30 ℃; the mobile phase was 3 mmol / L sulfuric acid solution, prepared, filtered through a 0.45 μm filter membrane, and degassed by sonication for 15 min; the mobile phase flow rate was set to 0.3 mL / min, using a low-pressure gradient elution mode; a UV detector was used with a detection wavelength of 215 nm; qualitative analysis was performed by retention time, and quantitative analysis was performed using the external standard method.

[0086] Chlorogenic acid content determination: High performance liquid chromatography (HPLC) was used, with sample pretreatment identical to that in Example 1. Chromatographic conditions were as follows: a reversed-phase C18 column (250 mm x 4.6 mm, 5 μm) was used; the injection volume was set to 10 μL; the column and detector temperatures were maintained at 45 ℃; the mobile phase consisted of chromatographic methanol (phase A) and 0.1% formic acid aqueous solution (phase B), filtered through a 0.45 μm filter and degassed by sonication for 15 min; the mobile phase flow rate was set to 0.6 mL / min, using a segmented low-pressure gradient elution mode: 0–10 min, phase A to phase B ratio of 5:95; 10–30 min, phase A to phase B ratio of 1:1; 30–40 min, phase A to phase B ratio of 5:95. An ultraviolet detector was used at a detection wavelength of 306 nm; qualitative analysis was performed by retention time, and quantitative analysis was performed using the external standard method.

[0087] Determination of core flavor compounds: Headspace gas chromatography was used for determination, with sample pretreatment the same as in Example 1. Headspace injection conditions: Headspace system equilibrium temperature 80 ℃, sampling needle temperature 95 ℃, valve box temperature 110 ℃, transfer tube temperature 120 ℃, equilibrium time 30 min, purge flow rate 15 mL / min. Gas chromatography conditions: A Kromat KB-1 capillary column (60 m × 0.53 mm × 10 μm) was used, with an FID detector; high-purity nitrogen was used as the carrier gas, constant flow mode was used, column flow rate was 1.5 mL / min, and split ratio was 15:1. The column oven was programmed to increase the temperature: the initial temperature was increased from room temperature to 90 ℃ and held for 5 min; then increased to 180 ℃ at a rate of 15 ℃ / min and held for 5 min; finally, increased to 240 ℃ at a rate of 20 ℃ / min and held for 15 min. Qualitative analysis was performed by retention time, and quantitative analysis was performed by external standard method.

[0088] In summary, based on the current national standard testing methods for beer, this invention constructs a comprehensive evaluation system covering dynamic monitoring of the fermentation process, adaptation detection of basic physicochemical indicators, precise quantification of characteristic functional components, and simultaneous analysis of core flavor substances, targeting the complex matrix characteristics of wild grape craft beer. It optimizes the detection process and parameters to eliminate matrix interference and establishes a dedicated simultaneous detection method for characteristic substances of wild grape.

Claims

1. A systematic evaluation method for the physicochemical quality of wild grape craft beer, characterized in that, Includes the following steps: 1) Dynamic monitoring of the fermentation process: The sugar-lowering ability of yeast during the fermentation of wild grape craft beer is monitored by carbon dioxide loss method, and the main fermentation endpoint is determined based on the carbon dioxide release rate; 2) Basic physicochemical index testing: Quantitative testing of total acidity, bitterness, color, free amino nitrogen content, total sugar content, and alcohol content of wild grape craft beer; 3) Quantitative analysis of characteristic functional components: The characteristic functional components in the craft beer made from wild grapes were qualitatively and quantitatively detected by high performance liquid chromatography. The characteristic functional components include six organic acids and chlorogenic acid. The six organic acids include citric acid, tartaric acid, malic acid, succinic acid, lactic acid, and acetic acid. 4) Quantitative analysis of core flavor compounds: Headspace gas chromatography was used to simultaneously and quantitatively detect the core volatile flavor compounds in wild grape craft beer. The core volatile flavor compounds include n-propanol, isobutanol, isoamyl alcohol, 2-methyl-1-butanol, 2-phenylethanol, ethyl acetate, ethyl lactate, amyl acetate, ethyl hexanoate, and phenylethyl acetate.

2. The systematic evaluation method for the physicochemical quality of wild grape craft beer as described in claim 1, characterized in that, In step 1), the carbon dioxide loss method includes: periodically weighing the mass of the fermentation system during the fermentation process, calculating the amount of carbon dioxide lost per unit time, and using this loss as an indicator of the yeast fermentation rate; when the daily carbon dioxide loss is lower than a preset threshold for a continuous period of time, the main fermentation stage is determined to be over.

3. The systematic evaluation method for the physicochemical quality of wild grape craft beer as described in claim 1, characterized in that: In step 2), the total acid content is determined by acid-base titration and expressed as tartaric acid or lactic acid; the bitterness is determined by organic solvent extraction-ultraviolet spectrophotometry.

4. The systematic evaluation method for the physicochemical quality of wild grape craft beer as described in claim 1, characterized in that: In step 2), the colorimetry is determined by spectrophotometry, with wavelengths including 430 nm and 700 nm, and the colorimetry value is calculated based on the absorbance.

5. The systematic evaluation method for the physicochemical quality of wild grape craft beer as described in claim 1, characterized in that: In step 2), the free amino nitrogen content is determined by the ninhydrin colorimetric method.

6. The systematic evaluation method for the physicochemical quality of wild grape craft beer as described in claim 1, characterized in that: In step 2), the total sugar content is determined by the phenol-sulfuric acid colorimetric method or other sugar colorimetric methods.

7. The systematic evaluation method for the physicochemical quality of wild grape craft beer as described in claim 1, characterized in that: In step 2), the alcohol content is determined by high performance liquid chromatography, gas chromatography, or distillation-density method.

8. The systematic evaluation method for the physicochemical quality of wild grape craft beer as described in claim 1, characterized in that: In step 3), the high performance liquid chromatography conditions for the six organic acids are as follows: cation exchange column, dilute sulfuric acid solution as mobile phase, and ultraviolet detector.

9. The systematic evaluation method for the physicochemical quality of wild grape craft beer as described in claim 1, characterized in that: In step 3), the high performance liquid chromatography conditions for chlorogenic acid are as follows: reversed-phase C18 column, gradient elution with acidic aqueous solution and organic solvent as mobile phase, and detection with ultraviolet detector.

10. The systematic evaluation method for the physicochemical quality of wild grape craft beer as described in claim 1, characterized in that: In step 4), the headspace injection conditions for the headspace gas chromatography method include: an equilibrium temperature of 60~90℃ and an equilibrium time of 20~40 min.

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