Multi-dimensional analysis and evaluation method for quality of distiller's yeast

Abstract

The invention discloses a multi-dimensional analysis and evaluation method for the quality of distiller's yeast, which comprises the following steps: S1, carrying out sensory evaluation on the distiller's yeast, and calculating the total score of sensory evaluation of the distiller's yeast; s2, performing physicochemical property determination on the distiller's yeast, and determining two core physicochemical indexes, namely moisture, volatile component content and color gradation, of the distiller's yeast; s3, determining the biochemical performance of the distiller's yeast, determining the liquefying power of the distiller's yeast, and quantifying the biochemical catalytic performance of the distiller's yeast; s4, screening through a GC-MS (Gas Chromatography-Mass Spectrometer) analysis and principal component analysis method, determining a characteristic marker of the quality of the distiller's yeast, optimizing an extraction process of the characteristic marker by adopting a Box-Behnken response surface method, and determining an optimal extraction process condition; s5, extracting the flavor compounds in the distiller's yeast under the optimal extraction condition, performing qualitative and quantitative analysis on the flavor compounds in the distiller's yeast, and calculating the relative proportion and content of the characteristic markers and the flavor compounds; and S6, calculating the comprehensive quality score of the distiller's yeast by adopting a weight scoring algorithm.

Description

Technical Field

[0001] This invention relates to the fields of food analytical chemistry and food processing technology, specifically a multidimensional analytical evaluation method for the quality of yeast starter. Background Technology

[0002] Yellow wine, one of the world's three ancient wines, embodies a profound Chinese brewing culture. Among them, Fangxian yellow wine, a geographical indication product, is brewed using a small-batch fermentation process. Its quality is closely related to the quality of the small-batch fermentation starter (xiaoqu), which directly determines the final flavor, taste, and quality grade of the yellow wine. As the core saccharifying and fermenting agent in the yellow wine brewing process, the starter is rich in various microorganisms (such as molds, yeasts, and bacteria) and enzymes (such as liquefying enzymes and saccharifying enzymes). It is the "soul" of yellow wine brewing; its quality not only directly affects the fermentation efficiency and yield but also significantly influences the chemical composition, flavor characteristics, and product stability of the yellow wine.

[0003] However, the rice wine industry currently faces numerous challenges in controlling the quality of yeast starter: the sources of yeast starter are complex, and the quality of yeast starter from different production areas and batches varies; the yeast starter production process lacks a unified standardized process, and the production process parameters fluctuate greatly, resulting in unstable yeast starter quality; more importantly, the evaluation of yeast starter quality lacks scientific and unified quantitative standards. Traditional yeast starter quality control mainly relies on the personal experience of brewing technicians, evaluating it by observing subjective indicators such as the appearance of the yeast starter, smelling its odor, or observing mycelial growth, or inferring the quality of the yeast starter based on the quality test results of the rice wine produced after fermentation. This evaluation method is highly subjective, uncertain, and lagging, making it difficult to guarantee the uniformity and stability of yeast starter quality from different batches and production areas, and also failing to meet the needs of the rice wine industry for standardized and large-scale development.

[0004] In recent years, although some local standards have been issued to regulate the production technology of rice wine starter, the starter production process involves multiple raw materials and technological steps. Changes in any step (such as raw material ratios, fermentation temperature, and fermentation time) can significantly affect the chemical composition, enzyme activity level, and flavor characteristics of the finished starter. Existing local standards have not yet solved the core problem of quantitative evaluation of starter quality. At the same time, there is limited research on the precise quantitative analysis of flavor substances in starter, the screening of indicative components, and the optimization of extraction processes. A multi-dimensional evaluation system covering sensory, physicochemical, biochemical, and chemical composition has not been established, and the quality level of starter cannot be comprehensively and objectively reflected.

[0005] Current technologies for evaluating the quality of yeast starters primarily focus on baijiu (Chinese white liquor) starters or the sensory evaluation of baijiu. For example, some studies use principal component analysis to screen sensory descriptors and create flavor profiles to assess baijiu quality, or they establish comprehensive evaluation models (F-values) incorporating multiple physicochemical and biochemical indicators to classify starter grades. However, these methods do not address the specific application of huangjiu (yellow wine) starters, nor do they identify indicative characteristic components from chemical fingerprints, and they do not systematically incorporate safety warning indicators into the evaluation system. Therefore, developing a multidimensional quantitative evaluation method specifically for huangjiu starters is crucial. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a multidimensional analysis and evaluation method for the quality of rice wine starter. This method comprehensively, objectively, and systematically quantifies the quality of rice wine starter from four core dimensions: sensory attributes, physicochemical properties, biochemical functions, and chemical composition. This fills the gap in existing technologies and provides technical support for the standardized production and quality control of rice wine starter.

[0007] To achieve the above objectives, the technical solution adopted by this invention is: a multidimensional analysis and evaluation method for the quality of yeast starter, comprising the following steps: Step S1. Performing sensory evaluation on the yeast starter and calculating the total sensory evaluation score; Step S2. Determining the physicochemical properties of the yeast starter, measuring the moisture and volatile component content and color gradation of the yeast starter as two core physicochemical indicators; Step S3. Determining the biochemical performance of the yeast starter, measuring the liquefaction power of the yeast starter, and quantifying the biochemical catalytic performance of the yeast starter; Step S4. Screening by GC-MS analysis and principal component analysis to determine the characteristic markers of yeast starter quality, and using the Box-Behnken response surface methodology to optimize the extraction process of the characteristic markers and determine the optimal extraction process conditions; Step S5. Extracting flavor compounds from the yeast starter under the optimal extraction conditions, and performing qualitative and quantitative analysis on the flavor compounds in the yeast starter, calculating the characteristic markers and the relative proportions and contents of each flavor compound; Step S6. Calculating the comprehensive quality score of the yeast starter using a weighted scoring algorithm.

[0008] Based on the above technical solution, the yeast is a small yeast starter for rice wine; step S1 specifically includes the following steps: Step S101. Collect the original descriptive words of the yeast, use the approximate estimation method to calculate the frequency F and intensity I of each descriptive word, calculate the contribution index M = √(F×I), filter the descriptive words with M value greater than the preset threshold, and after merging and sorting, establish a dedicated sensory descriptive word library for yeast containing three dimensions: appearance, aroma and texture. Step S102. Observe the color of mycelium on the surface of the yeast, the uniformity of mycelium coverage, and the surface cracks. Use a 0-10 score method to quantitatively evaluate the appearance. Step S103. After longitudinally cutting along the center of the yeast, observe the mycelial morphology, color, and porosity of the cut surface, and use a 0-10 score method to quantitatively evaluate the cross-sectional morphology. Step S104. Use vernier calipers to randomly select multiple test points and measure the thickness of the unfermented raw starch layer on the surface of the yeast starter. Take the average value as the evaluation basis for the yeast starter skin thickness, and use a 0-10 score method to quantitatively evaluate the yeast starter skin thickness. Step S105. Smell the aroma of the cross-section of the yeast starter. Based on the yeast starter-specific sensory description dictionary, identify the purity of the aroma, the intensity of the aroma, and whether there are any off-flavors. Use a 0-10 scoring method to quantitatively evaluate the aroma characteristics. Step S106. Add the scores of the four dimensions together and use the consistency ratio (CR) to test the consistency among the evaluators. When CR < 0.1, the evaluation results are considered reliable.

[0009] Based on the above technical solution, step S2, the determination of moisture and volatile component content specifically includes the following steps: Step S201. Take a clean, dry glass dish to constant weight, weigh it and record its mass as m0; Step S202. Accurately add the yeast sample, weigh the total mass of the glass dish and the sample, and record it as m1; Step S203. Place the glass dish containing the yeast sample in a drying oven at 100±5 ℃ and dry it to constant weight. Weigh the total mass of the glass dish and the dried sample at this point and record it as m2. Step S204. Calculate the moisture and volatile component content according to the formula W = (m1 - m2) / (m1 - m0) × 100%, where W is the moisture and volatile component content, m0 is the mass of the glass dish, m1 is the total mass of the sample and glass dish before drying, and m2 is the total mass of the sample and glass dish after drying. Step S205. The weighing accuracy of the yeast sample is 0.0001 g. Three parallel samples are set for each yeast sample, and the average value is taken as the final measurement result to ensure the reliability of the measurement data.

[0010] Based on the above technical solution, step S2, the determination of color gradation specifically includes the following steps: Step S211. Accurately weigh the yeast powder sample m, add 70% ethanol as the extraction solvent, and dilute to volume V after extraction. Step S212. Place the extract in a 60 ℃ water bath for 2 h and extract at a constant temperature. After cooling to room temperature, centrifuge to collect the supernatant and dilute it by F times as needed. Step S213. Using 70% ethanol as a blank control, measure the absorbance A of the diluted supernatant at the maximum absorption wavelength of the UV-Vis spectrophotometer. Step S214. Calculate the color level according to the formula L=(F×A×V) / m, where L is the absorbance value per unit mass of sample, F is the dilution factor of supernatant, V is the volume of extract, and m is the mass of yeast powder sample. Step S215. Set up 3 parallel samples for each sample, and take the average value as the final color scale result to reduce detection error.

[0011] Based on the above technical solution, step S3 specifically includes the following steps: Step S301. Based on the measured moisture and volatile component content W of the yeast, calculate and weigh a yeast sample equivalent to a certain weight of oven-dried yeast; add the sample to distilled water and an acetate-sodium acetate buffer solution with pH 4.6, and soak it at a constant temperature of 40 ℃ for 3 h. After soaking, filter and collect the filtrate as enzyme solution for later use. Step S302. Take a 2% soluble starch solution and preheat it to a constant temperature in a 35 ℃ water bath. Add the above enzyme solution to the preheated starch solution and start the reaction immediately. Take samples at regular intervals during the reaction and add dilute iodine solution to observe the color change of the solution. When the color of the solution is close to the color of the dilute iodine solution itself, record the reaction time T at this time. Step S303. Calculate the liquefaction force according to the formula Lq = 24 / T, where the unit of liquefaction force is g / (g·h), and T is the reaction time and 24 is the conversion factor. Step S304. By measuring the saccharification rate and alcohol production rate of different liquefaction kojis during the fermentation process of rice wine, establish the correlation equation between liquefaction force Lq and rice wine fermentation efficiency η: η = α×ln(Lq) + β, where α and β are regression coefficients, and the correlation coefficient R²≥0.90, which is used to predict the brewing performance of kojis.

[0012] Based on the above technical solution, in step S4, the different quality grades of Shaoxing rice wine starter, which were initially classified by sensory and core physicochemical indicators, were taken as the research object. The volatile component spectra of Shaoxing rice wine starter of different quality grades were analyzed by GC-MS. The principal component analysis method was used to extract characteristic variables, and the correlation coefficient r between each compound and the quality grade of the starter was calculated. Compounds with a correlation coefficient |r|≥0.85 and a coefficient of variation CV≥30% between different quality grades were screened as candidate indicators. Palmitic acid was determined to be the characteristic marker of starter quality by comparison.

[0013] Based on the above technical solution, in step S4, the Box-Behnken response surface methodology is used to optimize the extraction process of palmitic acid. Extraction time, extraction temperature, and material-to-liquid ratio are selected as three key factors, and palmitic acid extraction rate is used as the response value. Through a three-factor, three-level experimental design, a quadratic regression equation is established for the high starch matrix characteristics of Shaoxing rice wine koji. ; Where Y is the palmitic acid extraction rate, A is the extraction time, B is the extraction temperature, and C is the solid-liquid ratio. For constant terms, ~ The regression coefficients were used to verify the significance of the regression equation through analysis of variance (P < 0.01); the goodness of fit was R² ≥ 0.95; after eliminating insignificant factors, P > 0.05, the optimal extraction process conditions were determined.

[0014] Based on the above technical solution, step S5 specifically includes the following steps: Step S501. Chromatographic conditions: An HP-5 MS flexible quartz capillary column with dimensions of 30 m × 0.25 mm and a diameter of 0.25 μm was used; the injection port temperature was set to 250 ℃; the temperature program was as follows: initial column temperature of 60 ℃, held for 1 min; then increased to 220 ℃ at a rate of 20 ℃ / min, held for 1 min; then increased to 300 ℃ at a rate of 5 ℃ / min, held for 7 min; high-purity helium with a purity ≥99.999% was used as the carrier gas, and the carrier gas flow rate was set to 1 mL / min; a splitless injection method was used, with an injection volume of 1 μL. Step S502. Mass spectrometry conditions: The chromatographic-mass spectrometry interface temperature is set to 280 ℃; an electron impact source is used, with an ionization energy set to 70 eV; the ion source temperature is set to 230 ℃; qualitative analysis uses full scan mode, covering the characteristic ions of the target flavor compounds; quantitative analysis uses selected ion monitoring mode to specifically monitor the characteristic ions of palmitic acid and internal standards; the solvent delay time is set to 7 min to avoid interference from solvent peaks on the target peaks. Step S503. Quantitative Method: Quantitative analysis was performed using the internal standard method. The peak area of ​​the internal standard methyl palmitate was used as a reference. The correction factor f was calculated by the ratio of the peak areas of palmitic acid and the internal standard. The formula for calculating the correction factor is: f = (C_s × A_i) / (C_i × A_s), where C_s is the concentration of palmitic acid reference standard, A_i is the peak area of ​​the internal standard, and A_s is the peak area of ​​palmitic acid. The specific content of palmitic acid was calculated by combining the correction factor: C_sample = (A_sample × C_i × f) / A_i.

[0015] Based on the above technical solution, step S6 specifically includes the following steps: Step S601. Weighting: Sensory indicators account for 30% of the total score, liquefaction power accounts for 25%, ester ratio accounts for 20%, 2,4-di-tert-butylphenol safety index accounts for 15%, moisture and color scale account for 5%, palmitic acid and ketones account for 5%; Step S602. Scoring Rules: Within each evaluation indicator, all samples of yeast to be evaluated are ranked. The sample with the best ranking receives the full score corresponding to the weight of that indicator, the sample with the worst ranking receives 1 / 4 of the weight score corresponding to that indicator, and the samples in the middle of the ranking are scored using linear interpolation. Among them, 2,4-di-tert-butylphenol is used as a safety indicator. If its content exceeds the preset safety threshold, all weight scores for that indicator are deducted. Palmitic acid is used as an indicator component. If its content exceeds the preset reasonable range, some or all weight scores for that indicator are deducted according to the excess ratio. Step S603. Terminology definition: The alcohol-ester ratio specifically refers to the percentage of the sum of the chromatographic peak areas of alcohols, esters, and organic acids in the yeast sample relative to the total peak area of ​​all detectable flavor compounds in the yeast, used to quantify the harmony of the yeast flavor.

[0016] Based on the above technical solution, in step S6, the rice wine starter is divided into three grades according to the comprehensive score. Grade 1 brewing yeast: Overall score ≥ 85 points; Level 2 yeast: 70 points ≤ overall score < 85 points; Grade 3 brewing yeast: Overall score < 70 points; If the content of 2,4-di-tert-butylphenol exceeds the safety threshold, it will be directly judged as unqualified yeast and will not be included in the grading.

[0017] 1. This invention establishes a multi-dimensional evaluation system for the quality of rice wine starter, covering five core dimensions: sensory characteristics, physicochemical properties, biochemical performance, chemical composition, and safety. It overcomes the limitations of traditional experience-based evaluation, which is highly subjective, has a single evaluation dimension, and lacks unified standards. It achieves a comprehensive, objective, and quantitative assessment of the quality of rice wine starter, and the evaluation results have good comparability and repeatability. 2. This invention uses GC-MS technology to perform precise qualitative and quantitative analysis of flavor compounds in rice wine starter, and screens palmitic acid as an indicator component for the quality of rice wine starter, filling the gap in the existing technology for the absence of indicator components in rice wine starter, and providing a new technical dimension and quantitative basis for the evaluation of starter quality. 3. This invention uses the Box-Behnken response surface methodology to optimize the extraction process of palmitic acid, determines the optimal extraction conditions, significantly improves the accuracy, stability and efficiency of the detection of indicative components, ensures the reliability of the detection data, and meets the requirements of "the technical solution is practical and operable" in patent examination. 4. This invention uses a scientific weighted scoring method for comprehensive evaluation. The weight allocation is reasonable and the scoring rules are standardized. It can intuitively and accurately reflect the comprehensive quality of the yeast, and provides operable technical support for the quality grading, standardized production and standardized quality control of rice wine yeast. 5. The method of the present invention is simple and easy to implement, with moderate detection cost and good repeatability. It does not require complex large-scale equipment (except for GC-MS, the other equipment are conventional equipment in the field of food testing), which makes it easy for rice wine brewing enterprises and food testing institutions to promote and apply it, and has significant industrial application value. 6. This invention effectively solves the technical problems of traditional yeast quality evaluation being highly subjective, having vague evaluation standards, lacking unified quantitative basis, and having incomparable evaluation results. It fills the gap in the multi-dimensional quantitative evaluation system for rice wine yeast, promotes the transformation of the rice wine industry from "experience-based brewing" to "standardized and scientific brewing", and has important theoretical significance and industrial application value. Attached Figure Description

[0018] Figure 1 This is a flowchart illustrating the construction of the comprehensive quality evaluation system for yeast used in this invention. Figure 2 Here are the GC-MS chromatograms of palmitic acid and methyl nonadecanoate reference standards; Figure 3 The GC-MS mass chromatograms of the yeast samples are shown. Sample numbers: (A) #1, (B) #2, (C) #3, (D) #4; Figure 4 This is a response surface plot showing the interaction of various factors affecting palmitic acid extraction rate. A: Temperature and liquid-to-solid ratio; B: Temperature and extraction time; C: Liquid-to-solid ratio and extraction time. Detailed Implementation

[0019] The following description, in conjunction with the accompanying drawings, further illustrates specific embodiments of the present invention, making the technical solution and its beneficial effects clearer and more explicit. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the invention.

[0020] See Figures 1-4 As shown, this invention provides a multidimensional analysis and evaluation method for the quality of Shaoxing rice wine starter, characterized by the following steps: Step (1) Sensory evaluation: After crushing and mixing the yeast sample, a systematic sensory evaluation is carried out from four indicators: appearance, aroma characteristics, yeast skin thickness and cross-sectional morphology. The 0~10 scoring method is used to quantify and score each dimension separately. The scores of all sensory indicators are summarized as the total sensory evaluation score to realize the quantification of sensory evaluation. Step (2) Physicochemical property determination: The two core physicochemical indicators of moisture and volatile components and color scale in the yeast were determined. Among them, the moisture and volatile component content were determined by the drying loss method, and the color scale was determined by 70% ethanol extraction-ultraviolet spectrophotometry. The specific detection conditions and calculation methods are as follows: the moisture content was determined by drying to constant weight at 100±5 ℃, and the color scale was determined by extracting in a 60 ℃ water bath for 2 h, and then measuring and calculating the absorbance at the maximum absorption wavelength. Step (3) Biochemical performance determination: The liquefaction power is determined by the iodine-starch reaction method. The specific calculation method is: the number of grams of soluble starch that 1 g of dry yeast can liquefy within 1 h under optimized reaction conditions of pH 4.6 and 35 ℃, and the quantitative standard of liquefaction power is clarified. Step (4) Optimization of the extraction process of index components: Palmitic acid was used as the index component of the yeast extraction process. The screening criteria for palmitic acid were as follows: taking different quality grades of rice wine yeast based on sensory and core physicochemical indicators as the research object, the volatile component spectra of different quality grades of rice wine yeast were analyzed by GC-MS, and the characteristic variables were extracted by principal component analysis (PCA). The correlation coefficient r between each compound and the quality grade of yeast was calculated. Compounds with a correlation coefficient |r|≥0.85 and a coefficient of variation CV≥30% between different quality grades were screened as candidate indicators. After comparison, palmitic acid was determined to be the characteristic marker of the quality of rice wine yeast. The Box-Behnken response surface design method was used to optimize the extraction process of palmitic acid. Extraction time, extraction temperature, and material-liquid ratio were selected as three key factors. The extraction rate of palmitic acid was used as the response value. Through a three-factor, three-level experimental design, a quadratic regression equation was established for the high starch matrix characteristics of rice wine yeast. .

[0021] Where Y is the palmitic acid extraction rate (%), A is the extraction time (h), B is the extraction temperature (°C), and C is the solid-liquid ratio (g / mL). For constant terms, ~ The regression coefficients were used to verify the significance (P<0.01) and goodness of fit (R²≥0.95) of the regression equation through analysis of variance. Insignificant factors (P>0.05) were eliminated, and the optimal extraction process conditions were optimized and determined.

[0022] Step (5) Qualitative and quantitative analysis of flavor compounds: Gas chromatography-mass spectrometry (GC-MS) combined with the NIST17 database was used to perform comprehensive qualitative and quantitative analysis of flavor organic compounds in the yeast. Flavor compounds were extracted from the yeast sample under the optimal extraction conditions determined in step (4). The specific content of palmitic acid was calculated using the internal standard method (with methyl nonadecanoate as the internal standard). At the same time, the peak area ratios of alcohols, organic acids, and esters relative to palmitic acid, as well as the peak area of ​​2,4-di-tert-butylphenol, were determined. Based on the ratio of the peak area to the palmitic acid content, the relative contents of each flavor compound and 2,4-di-tert-butylphenol were calculated. Step (6) Comprehensive evaluation: Select the total score of sensory indicators, liquefaction power, color scale, moisture and volatile component content, palmitic acid content, alcohol-ester ratio, and 2,4-di-tert-butylphenol content as comprehensive evaluation indicators, calculate the comprehensive score according to the preset weight allocation scheme, and realize the comprehensive quantitative evaluation of the quality of the yeast.

[0023] Specifically, the descriptive sensory analysis in step (1) adopts a standardized operating procedure, and a sensory descriptive vocabulary database specifically for Shaoxing rice wine koji is established. The specific method is as follows: (1) Establish a sensory descriptive word library for rice wine koji: recruit trained sensory evaluators (n≥15) to conduct sensory descriptions on no less than 30 different batches of rice wine koji samples, collect original descriptive words, use approximate estimation method to calculate the frequency F and intensity I of each descriptive word, calculate the contribution index M = √(F×I), screen descriptive words with M value greater than the preset threshold, and establish a sensory descriptive word library for rice wine koji containing three dimensions: appearance, aroma and texture after merging and sorting. (2) Appearance evaluation: Under the conditions of non-direct natural light or standard light source D65 with an illumination intensity of 500~1000 lux, observe the color of mycelium on the surface of the yeast, the uniformity of mycelium coverage and the surface cracks, and use the 0~10 score method for quantitative evaluation; (3) Evaluation of cross-sectional morphology: After longitudinally cutting along the center of the yeast, observe the morphology of the mycelium, the color of the cross-section and the condition of the porosity of the cross-section, and use a 0-10 score method for quantitative evaluation; (4) Evaluation of the thickness of the koji skin: Five test points were randomly selected using vernier calipers to measure the thickness of the unfermented raw starch layer on the surface of the koji. The average value was taken as the basis for evaluating the thickness of the koji skin and converted into a score of 0 to 10 according to the thickness range. (5) Aroma characteristics evaluation: Smell the aroma of the cross-section of the yeast, and identify the purity, intensity and presence of off-flavors of the aroma based on the sensory description dictionary for small yeast of rice wine. Use a 0-10 score method for quantitative evaluation. (6) Calculate the total sensory evaluation score: add the scores of the four dimensions together and use the consistency ratio (CR) to test the consistency among the evaluators. When CR < 0.1, the evaluation result is considered reliable.

[0024] Specifically, the method for determining the moisture and volatile component content in step (2) adopts a precise operation, specifically as follows: take a clean, dried glass dish to constant weight, weigh it and record its mass as m0; precisely add the yeast sample, weigh the total mass of the glass dish and the sample and record it as m1; place the glass dish containing the sample in a drying oven at 100±5 ℃ and dry it to constant weight (the mass difference between two consecutive weighings is ≤0.3 mg), weigh the total mass of the glass dish and the dried sample at this time and record it as m2; calculate the moisture and volatile component content according to the formula W = (m1 - m2) / (m1 - m0) × 100%, where W is the moisture and volatile component content (%), m0 is the mass of the glass dish (g), m1 is the total mass of the sample and the glass dish before drying (g), and m2 is the total mass of the sample and the glass dish after drying (g); the sample weighing accuracy is 0.0001 g, and three parallel samples are set for each sample. The average value is taken as the final measurement result to ensure the reliability of the measurement data.

[0025] Specifically, the method for determining the color level in step (2) adopts a standardized extraction and detection process, which is as follows: accurately weigh the yeast powder sample m (g, weighing accuracy 0.0001 g), add 70% ethanol as the extraction solvent, and dilute to volume V (mL) after extraction; place the extract in a 60 ℃ water bath for constant temperature extraction for 2 h, cool to room temperature, centrifuge and take the supernatant, and dilute it by F times as needed; use 70% ethanol as a blank control, and measure the absorbance A of the diluted supernatant at the maximum absorption wavelength of the UV-Vis spectrophotometer; calculate the color level according to the formula L=(F×A×V) / m, where L is the absorbance value per unit mass of sample (u / g), F is the dilution factor of the supernatant, V is the volume of the extract (mL), and m is the mass of the yeast powder sample (g); set up 3 parallel samples for each sample, and take the average value as the final color level result to reduce detection error.

[0026] Specifically, the method for determining liquefaction force in step (3) employs optimized conditions tailored to the characteristics of Shaoxing wine starter, and establishes a correlation model between liquefaction force and Shaoxing wine fermentation efficiency, as follows: (1) Enzyme solution preparation: Based on the moisture content W of the yeast determined in step (2), calculate and weigh the sample mass equivalent to 5 g of oven-dried yeast (the calculation formula is m = 5 / (1-W)); add the sample to 90 mL of distilled water and 10 mL of pH 4.6 acetate-sodium acetate buffer solution, and soak it at a constant temperature of 40 ℃ for 3 h. After soaking, filter and collect the filtrate as enzyme solution for later use. (2) Liquefaction reaction: Take 20 mL of 2% soluble starch solution and preheat it to a constant temperature in a 35 ℃ constant temperature water bath; add 10 mL of the above enzyme solution to the preheated starch solution and start the reaction immediately; take samples at regular intervals during the reaction, add dilute iodine solution to observe the color change of the solution, and record the reaction time T (min) when the solution color is close to the color of the dilute iodine solution itself. (3) Calculation of liquefaction force: The liquefaction force is calculated according to the formula Lq = 24 / T. The unit of liquefaction force is g / (g·h), where T is the reaction time (min) and 24 is the conversion factor. (4) Establish a correlation model between liquefaction force and rice wine fermentation efficiency: By measuring the saccharification rate and alcohol production rate of koji with different liquefaction forces during rice wine fermentation, establish a correlation equation between liquefaction force Lq and rice wine fermentation efficiency η: η = α×ln(Lq) + β, where α and β are regression coefficients, and the correlation coefficient R²≥0.90, which is used to predict the brewing performance of koji.

[0027] Specifically, the palmitic acid extraction process in step (4) is systematically optimized using the Box-Behnken response surface methodology, which is tailored to the high starch and high protein matrix characteristics of Shaoxing rice wine koji. The specific optimization process is as follows: (1) Analysis of the matrix characteristics of rice wine koji: The starch content in rice wine koji is 40%~60% and the protein content is 15%~25%. The high starch and high protein matrix will encapsulate lipids and affect the extraction efficiency of palmitic acid. (2) Single-factor experiments: The effects of extraction solvent type (methanol, ethanol, ethyl acetate, n-hexane), extraction time (0.5~3.0 h), extraction temperature (40~80 ℃), and material-liquid ratio (1:5~1:15 g / mL) on palmitic acid extraction rate were investigated to determine the appropriate range of each factor; (3) Box-Behnken response surface experimental design: Extraction time (A), extraction temperature (B), and material-liquid ratio (C) were used as three factors (independent variables), and palmitic acid extraction rate (Y) was used as the response value (dependent variable). A three-factor, three-level experimental design was carried out. The factor level codes are shown in Table 1.

[0028] (4) Establishing a regression model: The experimental data were fitted and analyzed using Design-Expert software to establish a quadratic regression equation for the matrix characteristics of Shaoxing rice wine koji. Y = -156.28 + 18.56A + 3.42B + 12.35C - 0.15AB - 0.08AC - 0.05BC -5.62A² - 0.02B² - 0.68C². The negative coefficients of A² and C² in the equation indicate that there are extreme points in the influence of extraction time and the solid-liquid ratio on the extraction rate, which is consistent with the encapsulation and release kinetics of lipids by the high-starch matrix.

[0029] (5) Analysis of variance and model validation: Analysis of variance was performed on the regression equation. The model F value was >50 and P <0.0001, indicating that the model was highly significant; the lack of fit term P >0.05, indicating that the lack of fit was not significant; the coefficient of determination R² ≥0.95 and the adjusted coefficient of determination R²_adj ≥0.90, indicating that the model fit was good. (6) The optimal extraction process conditions were obtained by optimization: anhydrous methanol was used as the extraction solvent, the extraction time was 1.5 h, the material-liquid ratio was 1:9 (g / mL), and the extraction temperature was 65 ℃. Under these optimal process conditions, the palmitic acid extraction rate was ≥90%, and the extraction results were stable and reproducible (relative standard deviation RSD≤5%).

[0030] Specifically, the GC-MS analysis method for palmitic acid in step (5) employs... Precision instrument conditions, specifically including chromatographic and mass spectrometric conditions, have been optimized to ensure detection accuracy: optimized instrument conditions specifically for the flavor characteristics of Shaoxing rice wine starter. Specifically, it includes: (1) Chromatographic conditions: HP-5 MS flexible quartz capillary column (30 m × 0.25 mm, 0.25 μm) was used; the injection port temperature was set to 250 ℃; the temperature program was as follows: the initial column temperature was 60 ℃, held for 1 min; then the temperature was increased to 220 ℃ at a rate of 20 ℃ / min, held for 1 min; then the temperature was increased to 300 ℃ at a rate of 5 ℃ / min, held for 7 min; high-purity helium (purity ≥99.999%) was used as the carrier gas, and the carrier gas flow rate was set to 1 mL / min; splitless injection was used, and the injection volume was 1 μL. (2) Mass spectrometry conditions: The temperature of the chromatography-mass spectrometry interface was set to 280 °C; an electron impact source (EI) was used, with an ionization energy of 70 eV; the ion source temperature was set to 230 °C; the qualitative analysis used full scan mode, covering the characteristic ions of the target flavor compounds; the quantitative analysis used selected ion monitoring (SIM) mode to specifically monitor the characteristic ions of palmitic acid and internal standard; the solvent delay time was set to 7 min to avoid interference of the solvent peak on the target peak; (3) Quantitative method: The internal standard method was used for quantitative analysis. The peak area of ​​the internal standard methyl nonadecanoate was used as a reference. The correction factor f was calculated by the ratio of the peak area of ​​the analyte (palmitic acid) to that of the internal standard. The formula for calculating the correction factor is: f = (C_s × A_i) / (C_i × A_s). Where C_s is the concentration of palmitic acid reference standard, A_i is the peak area of ​​internal standard, C_i is the concentration of internal standard, and A_s is the peak area of ​​palmitic acid; the specific content of palmitic acid is calculated by combining the correction factor: C_sample = (A_sample × C_i × f) / A_i.

[0031] Specifically, the comprehensive evaluation in step (6) adopts a scientific weighting scheme and standardized scoring rules, as follows: (1) Weighting: Sensory indicators account for 30% of the total score, liquefaction power accounts for 25%, alcohol and ester ratio accounts for 20%, 2,4-di-tert-butylphenol safety index accounts for 15%, moisture and color scale account for 5%, palmitic acid and ketones account for 5%; The above weighting is based on the analysis of key driving factors of rice wine brewing quality and is determined by using the analytic hierarchy process (AHP) combined with the experience of rice wine brewing experts, ensuring the scientificity and practicality of the evaluation system. (2) Scoring rules: Within each evaluation index, all samples of yeast to be evaluated are ranked. The sample with the best ranking gets the full score of the corresponding weight of the index, the sample with the worst ranking gets 1 / 4 of the corresponding weight of the index, and the samples in the middle of the ranking are scored by linear interpolation. Among them, 2,4-di-tert-butylphenol is used as a safety index. If its content exceeds the preset safety threshold, all weight of the index will be deducted. Palmitic acid is used as an index component. If its content exceeds the preset reasonable range (too high or too low), part or all weight of the index will be deducted according to the excess ratio. (3) Definition of terms: The alcohol-ester ratio specifically refers to the percentage of the sum of the chromatographic peak areas of alcohols, esters and organic acids in the yeast sample relative to the total peak area of ​​all detectable flavor compounds in the yeast, used to quantify the harmony of the yeast flavor.

[0032] Specifically, establish a quality grading standard for Shaoxing rice wine starter: divide Shaoxing rice wine starter into three grades based on a comprehensive score. Grade 1 brewing yeast: Overall score ≥ 85 points; Level 2 yeast: 70 points ≤ overall score < 85 points; Grade 3 brewing yeast: Overall score < 70 points; If the content of 2,4-di-tert-butylphenol exceeds the safety threshold, it will be directly judged as unqualified yeast and will not be included in the grading.

[0033] The present invention will be further illustrated by an embodiment below. Example

[0034] Embodiment 1 of this invention provides a multi-dimensional comprehensive analysis and evaluation method for the quality of koji (fermentation starter) used in Shaoxing wine brewing. Four samples of koji from different batches of Shaoxing wine (numbered #1, #2, #3, and #4) were selected as evaluation objects. The specific implementation steps are as follows: 1. Instruments and reagents 1.1 Instruments: TU-1901 UV-Vis spectrophotometer (Beijing Purkinje General Instrument Co., Ltd.), used for colorimetric determination; TQ8040 triple quadrupole gas chromatograph-mass spectrometer (GC-MS, Shimadzu Corporation), used for qualitative and quantitative analysis of flavor compounds; DF-101S heated magnetic stirrer (Wuhan Keer Instrument Equipment Co., Ltd.), used for constant temperature stirring of the extract; Lynx 4000 Label high-speed refrigerated centrifuge (Thermo Fisher Scientific Co., Ltd.), used for centrifugation of the extract; 101-3AB electric heating drying oven (Tianjin Tester Instrument Co., Ltd.), used for determination of moisture and volatile component content; SQP analytical balance (Sartorius Scientific Instruments Beijing Co., Ltd.), accuracy 0.0001 g, used for precise weighing of samples and reagents; vernier calipers (accuracy 0.01 mm), used for measuring skin thickness.

[0035] 1.2 Reagents: Chromatographically pure ethanol, ethyl acetate, and methanol (purity ≥99.9%) were used as extraction solvents and for preparing reference solutions; palmitic acid reference standard (purity ≥98%) was used for standard curve plotting and quantitative reference; methyl nonadecanoate internal standard (purity ≥98%) was used for internal standard quantification; HP-5 MS capillary column (30 m × 0.25 mm, 0.25 μm) was used for GC-MS analysis; dilute iodine solution (iodine-potassium iodide solution) was used for liquefaction power determination; pH 4.6 acetate-sodium acetate buffer was used to maintain the activity of liquefying enzymes; 2% soluble starch solution was used for liquefaction power determination; all reagents met the standards for food analysis reagents, and the experimental water was ultrapure water.

[0036] 2. Descriptive sensory analysis Fifteen volunteers were recruited who had received systematic sensory evaluation training (the training included the definition of sensory evaluation indicators, scoring criteria, and operating procedures, ensuring consistency among evaluators). Four types of yeast samples (5.0 g each) were crushed, mixed, and randomly numbered (#1, #2, #3, #4), and presented to each evaluator. Sensory evaluation was conducted under standard light source conditions of 500–1000 lux, following the procedure below: (1) Establish a sensory descriptive word library for rice wine koji: recruit trained sensory evaluators (n≥15) to conduct sensory descriptions on no less than 30 different batches of rice wine koji samples, collect original descriptive words, use the approximate estimation method to calculate the frequency F and intensity I of each descriptive word, calculate the contribution index M = √(F×I), screen descriptive words with M value greater than 0.05, and establish a sensory descriptive word library for rice wine koji containing three dimensions: appearance, aroma and texture after merging and sorting. (2) Appearance evaluation: Observe the color of mycelium on the surface of the yeast, the uniformity of mycelium coverage, and the condition of surface cracks; (3) Evaluation of cross-sectional morphology: After longitudinally cutting along the center of the yeast, observe the morphology of the hyphae, the color of the cross-section and the condition of the pores in the cross-section; (4) Evaluation of the thickness of the koji skin: Five test points were randomly selected using vernier calipers to measure the thickness of the unfermented raw starch layer on the surface of the koji, and the average value was taken as the basis for evaluating the thickness of the koji skin; (5) Aroma characteristics evaluation: smell the aroma of the cross-section of the yeast to identify the purity of the aroma, the intensity of the aroma, and whether there are any off-flavors.

[0037] Based on the pre-set standardized scoring criteria (four dimensions: appearance, aroma characteristics, peel thickness, and cross-sectional morphology, with each dimension scored from 0 to 10 points), the evaluators quantified and scored each dimension of each sample. The total sensory evaluation score for each sample = appearance score + aroma characteristics score + peel thickness score + cross-sectional morphology score (total score 40 points). The average of the scores from 15 evaluators was taken as the final sensory evaluation score for the sample, reducing subjective errors of the evaluators.

[0038] 3. Determination of physicochemical properties 3.1 Determination of Moisture and Volatile Components: Take a clean, dried glass dish to constant weight, weigh it and record its mass as m0; accurately add 5.0000 g of the yeast sample, weigh the total mass of the glass dish and the sample and record it as m1; place the glass dish containing the sample in an electric heating drying oven at 100±5 ℃ and dry it to constant weight (the difference in mass between two consecutive weighings ≤ 0.3 mg), weigh the total mass of the glass dish and the dried sample at this point and record it as m2; calculate the moisture and volatile component content according to the formula X = (m1 - m2) / (m1 - m0) ×100%, where X is the moisture and volatile component content (%), m0 is the mass of the glass dish (g), m1 is the total mass of the sample and glass dish before drying (g), and m2 is the total mass of the sample and glass dish after drying (g); set up 3 parallel samples for each sample, and take the average value as the final determination result, with a relative standard deviation (RSD) ≤ 5%.

[0039] 3.2 Color Scale Determination: Accurately weigh 0.2000 g (m) of the yeast powder sample and place it in a centrifuge tube. Add 70% ethanol as the extraction solvent, sonicate to dissolve, and then bring the volume to 50 mL (V). Place the centrifuge tube in a 60 ℃ water bath and extract at a constant temperature for 2 h, gently shaking every 15 min to ensure thorough extraction. After extraction, cool to room temperature and place the extract in a high-speed refrigerated centrifuge. Centrifuge at 8000 r / min for 10 min and collect the supernatant. Dilute the supernatant to an appropriate factor (dilution factor F) based on its absorbance. Use 70% ethanol as a blank control and measure the absorbance at the maximum absorption wavelength of the UV-Vis spectrophotometer (preliminarily determined to be 280 nm). The absorbance A of the diluted supernatant was measured (nm). The color gradation was calculated using the formula L=(F×A×V) / m, where L is the absorbance value per unit mass of sample (u / g), F is the dilution factor of the supernatant, V is the volume of the extract (mL), and m is the mass of the yeast powder sample (g). Three parallel samples were set up for each sample, and the average value was taken as the final color gradation result. The relative standard deviation RSD ≤ 5%.

[0040] 4. Liquefaction power measurement Based on the moisture content W of the yeast determined in step 3.1, calculate and weigh the sample mass equivalent to 5 g of oven-dried yeast using the formula m = 5 / (1-W) (g). Place the sample in a beaker, add 90 mL of ultrapure water and 10 mL of pH 4.6 acetate-sodium acetate buffer solution, stir well, and then soak in a 40 ℃ constant temperature water bath for 3 h, stirring once every 30 min to ensure that the enzyme solution is fully extracted. After soaking, filter with qualitative filter paper, collect the extract as enzyme solution for later use, and discard the filter residue.

[0041] Take 20 mL of 2% soluble starch solution and place it in an Erlenmeyer flask. Preheat the flask in a 35 ℃ constant temperature water bath for 10 min to ensure the starch solution reaches the reaction temperature. Add 10 mL of the above enzyme solution to the preheated starch solution and start the reaction immediately. During the reaction, take one drop of the solution every 5 min and drop it onto a white porcelain plate pre-filled with dilute iodine solution, and observe the color change of the solution. When the solution color is close to the color of the dilute iodine solution itself (pale yellow), stop the timing and record the reaction time T (min). Calculate the liquefaction force according to the formula Lq=24 / T, where the unit of liquefaction force is g / (g·h), where T is the reaction time (min) and 24 is the conversion factor (the conversion is based on the fact that 20 mL of 2% starch solution contains 0.4 g of soluble starch, which is equivalent to the number of grams of soluble starch that 1 g of oven-dried yeast can liquefy in 1 h). Set up 3 parallel samples for each sample and take the average value as the final liquefaction force result. The relative standard deviation RSD ≤ 5%.

[0042] 5. Optimize palmitic acid extraction process (Box-Behnken response surface methodology) Based on the single-factor experiments (investigating the effects of extraction time, extraction temperature, and solid-liquid ratio on palmitic acid extraction rate), three factors with significant effects on extraction rate, namely extraction time (A), extraction temperature (B), and solid-liquid ratio (C), were selected. A three-factor, three-level Box-Behnken experiment was designed using Design-Expert 10.0 software. The experimental factors and levels are shown in Table 2 below (-1, 0, 1 levels are: extraction time 1.0 / 1.5 / 2.0 h, extraction temperature 65 / 70 / 75 ℃, solid-liquid ratio 1:7 / 1:9 / 1:11 g / mL, respectively).

[0043] Using palmitic acid extraction rate as the response value (Y), three parallel samples were set for each experimental condition, and the average value was taken as the response value data. The experimental data were fitted and analyzed using Design-Expert software to establish a quadratic regression equation between the extraction rate and the three independent variables. The significance of the regression equation (P<0.01 was considered highly significant, and P<0.05 was considered significant) and the goodness of fit (R²≥0.95) were verified by analysis of variance (ANOVA). Insignificant factors were eliminated, and the interaction of the three factors was analyzed by response surface plots and contour plots to optimize the optimal extraction process conditions for palmitic acid.

[0044] The optimization results show that the optimal extraction conditions are: anhydrous methanol as the extraction solvent, extraction time of 1.5 h, a solid-liquid ratio of 1:9 (g / mL), and an extraction temperature of 65 ℃. Under these optimal conditions, the palmitic acid extraction rate is 92.3%, with a relative standard deviation (RSD) of 2.1%, indicating that the extraction process is stable and reliable, and can meet the requirements for high-efficiency palmitic acid extraction.

[0045] 6. GC-MS Analysis 6.1 Preparation of reference solutions: Accurately weigh 10.0 mg of palmitic acid reference standard and 10.0 mg of methyl nonadecanoate internal standard, and place them in 10 mL volumetric flasks respectively. Add ethyl acetate to dissolve and dilute to the mark, and shake well to obtain a palmitic acid standard stock solution and a methyl nonadecanoate internal standard stock solution with a concentration of 1 mg / mL. Accurately measure 0.1 mL of each of the above standard stock solution and internal standard stock solution, place them in 10 mL volumetric flasks, add ethyl acetate to dilute and dilute to the mark, and shake well to obtain a reference solution with a concentration of 10 μg / mL (containing palmitic acid reference standard and methyl nonadecanoate internal standard). Store in a refrigerator at 4 ℃ for later use. The shelf life is 7 days.

[0046] 6.2 Preparation of the test solution: After pulverizing the yeast sample, pass it through a 40-mesh sieve and place it in a vacuum drying oven at 60 ℃ for 72 h to remove moisture and volatile impurities; accurately weigh 3.0000 g of the dried yeast sample and place it in a 250 mL round-bottom flask. Extract it using the optimal extraction process optimized in step 5 (extraction solvent: anhydrous methanol, material-to-liquid ratio: 1:9 g / mL, extraction temperature: 65 ℃, extraction time: 1.5 h) by hot reflux extraction; after extraction, cool to room temperature and transfer the extract to a 50 mL volumetric flask. Wash the round-bottom flask three times with anhydrous methanol, combine the washings in the volumetric flask, add anhydrous methanol to the mark, and shake well; place the extract in a high-speed refrigerated centrifuge and centrifuge at 8000 r / min for 10 min. Take the supernatant and filter it through a 0.22 μm organic phase filter membrane to obtain the test solution. Store it in a refrigerator at 4 ℃ for later use, with a shelf life of 3 days.

[0047] 6.3 GC-MS Conditions: The chromatographic column used was an HP-5 MS flexible quartz capillary column (30 m × 0.25 mm, 0.25 μm); the injection port temperature was set to 250 ℃; the temperature program was as follows: initial column temperature 60 ℃, held for 1 min; then increased to 220 ℃ at a rate of 20 ℃ / min, held for 1 min; then increased to 300 ℃ at a rate of 5 ℃ / min, held for 7 min; high-purity helium (purity ≥99.999%) was used as the carrier gas, with a flow rate of 1 mL / min; splitless injection was used, with an injection volume of 1 μL; the GC-MS interface temperature was set to 280 ℃; an electron impact source (EI) was used, with an ionization energy set to 70 eV; the ion source temperature was set to 230 ℃; qualitative analysis was performed using full scan mode, with a scan range of 40–500 μV. m / z; Quantitative analysis was performed using selected ion monitoring (SIM) mode, with palmitic acid characteristic ion at m / z 256 and methyl nonadecanoate characteristic ion at m / z 298; Solvent delay time was set to 7 min to avoid interference from solvent peaks on target peaks.

[0048] 6.4 Qualitative and Quantitative Analysis: Qualitative Analysis: The GC-MS total ion chromatogram of the test solution was compared with that of the reference solution. Combined with NIST mass spectrometry library search, the types of palmitic acid and other flavor compounds (alcohols, organic acids, esters, ketones, etc.) in the yeast sample were determined. Quantitative Analysis: Quantitative analysis was performed using the internal standard method. Methyl nonadecanoate was used as the internal standard. A precise amount of palmitic acid standard stock solution of a certain concentration was measured, and an appropriate amount of methyl nonadecanoate internal standard stock solution was added to prepare a standard working solution with a certain concentration gradient. The solution was injected and analyzed under the GC-MS conditions described above, and the concentrations of palmitic acid and other flavor compounds were recorded. The peak area ratio of methyl nonadecanoate was used as a correction factor to calculate the palmitic acid content in the test solution, which was then converted to obtain the palmitic acid content (%) in the yeast sample. Simultaneously, the peak areas of alcohols, organic acids, and esters were recorded, and their ratios to the palmitic acid peak area were calculated, along with the peak area of ​​2,4-di-tert-butylphenol. Based on the ratios of these peak areas to palmitic acid content, the relative contents of each flavor compound and 2,4-di-tert-butylphenol were calculated. Three parallel samples were prepared for each sample, and the average value was taken as the final determination result, with a relative standard deviation (RSD) ≤ 5%.

[0049] 7. Overall Evaluation The four yeast samples were comprehensively evaluated according to the preset weighting scheme and scoring rules, as follows: (1) Weighting: Sensory indicators total score 30%, liquefaction power 25%, alcohol ester ratio 20%, 2,4-di-tert-butylphenol safety index 15%, moisture and color gradation 5%, palmitic acid and ketones 5%; (2) Scoring rules: Within each evaluation index, the four yeast samples are ranked. The best-ranked sample receives the full score corresponding to the weight of the index, and the worst-ranked sample receives 1 / 4 of the weight score corresponding to the index. The scores of the samples in the middle of the ranking are calculated by linear interpolation. The calculation formula is: Single score = Lowest score + (Rank value of the sample - 1) × (Full score - Lowest score) / (Total number of samples - 1); Among them, 2,4-di-tert-butylphenol is used as a safety index, and its preset safety threshold is 0.005%. If its content in the sample exceeds the threshold, all weight scores of the index will be deducted; the preset reasonable range of palmitic acid is 0.02%~0.25%. If its content in the sample exceeds the range, part or all weight scores of the index will be deducted according to the excess ratio; The alcohol-ester ratio specifically refers to the percentage of the sum of the chromatographic peak areas of alcohols, esters and organic acids in the yeast sample relative to the total peak area of ​​all detectable flavor compounds in the yeast. (3) Calculation of comprehensive score: Comprehensive score = Sensory index score × 30% + Liquefaction power score × 25% + Alkyd ester ratio score × 20% + 2,4-Di-tert-butylphenol score × 15% + Moisture and color scale score × 5% + Palmitic acid and ketone score × 5%; (4) Evaluation results: The comprehensive evaluation scores and rankings of the four yeast samples are shown in Table 7 below. Among them, sample #4 had the highest comprehensive score (90.25 points) and ranked first, indicating that its comprehensive quality was the best. Sample #2 had the lowest comprehensive score (43.5 points) and ranked fourth. The main reason was that its 2,4-di-tert-butylphenol content exceeded the safety threshold, and all weighted points of this indicator were deducted.

[0050] Four representative yeast samples were randomly selected and evaluated. The results of their tests and evaluations are shown in Table 2-8 below:

[0051]

[0052]

[0053]

[0054]

[0055]

[0056]

[0057] Through the above implementation methods, a scientific, objective, and comprehensive evaluation of the quality of yeast used in rice wine brewing can be achieved, providing reliable technical support for yeast quality control.

[0058] This invention discloses a multidimensional analytical evaluation method for the quality of Shaoxing rice wine starter, relating to the fields of food analytical chemistry and food processing technology. This method, targeting the fermentation characteristics and unique matrix (high starch, high protein) of Shaoxing rice wine starter, for the first time constructs a five-dimensional evaluation system covering "sensory perception, physicochemical properties, biochemical properties, chemical fingerprinting, and safety." It innovatively screens palmitic acid as an indicator component for the quality of Shaoxing rice wine starter and establishes a Box-Behnken response surface optimization model based on the matrix characteristics of Shaoxing rice wine starter. This effectively solves the technical problems of traditional starter quality evaluation, such as strong subjectivity, vague evaluation standards, lack of unified quantitative basis, and incomparable evaluation results. It provides scientific, objective, and operable technical support for the standardized production, standardized quality control, and quality grading of Shaoxing rice wine starter, filling the gap in the multidimensional quantitative evaluation system for Shaoxing rice wine starter.

[0059] In the description of this specification, references to terms such as "an embodiment," "preferred," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. Illustrative expressions of the above terms in this specification do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0060] This invention is not limited to the embodiments described above. Those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention. Contents not described in detail in this specification are prior art known to those skilled in the art.

Claims

1. A multidimensional analysis and evaluation method for the quality of yeast starter, characterized in that, Includes the following steps: Step S1. Conduct a sensory evaluation of the yeast and calculate the total sensory evaluation score of the yeast; Step S2. Perform physicochemical property testing on the yeast, measuring the moisture and volatile component content and color gradation of the yeast, which are two core physicochemical indicators. Step S3. Conduct biochemical performance tests on the yeast, measure the liquefaction power of the yeast, and quantify the biochemical catalytic performance of the yeast; Step S4. Screening was performed using GC-MS analysis and principal component analysis to determine the characteristic markers of yeast quality, and the extraction process of the characteristic markers was optimized using the Box-Behnken response surface methodology to determine the optimal extraction process conditions; Step S5. Extract flavor compounds from the yeast under the optimal extraction conditions, and perform qualitative and quantitative analysis on the flavor compounds in the yeast, and calculate the characteristic markers and the relative proportions and contents of each flavor compound; Step S6. Calculate the overall quality score of the yeast using a weighted scoring algorithm.

2. The multidimensional analysis and evaluation method for the quality of yeast as described in claim 1, characterized in that: The yeast starter is a small yeast starter for rice wine; step S1 specifically includes the following steps: Step S101. Collect the original descriptive words of the yeast, use the approximate estimation method to calculate the frequency F and intensity I of each descriptive word, calculate the contribution index M = √(F×I), filter the descriptive words with M value greater than the preset threshold, and after merging and sorting, establish a dedicated sensory descriptive word library for yeast containing three dimensions: appearance, aroma and texture. Step S102. Observe the color of mycelium on the surface of the yeast, the uniformity of mycelium coverage, and the surface cracks. Use a 0-10 score method to quantitatively evaluate the appearance. Step S103. After longitudinally cutting along the center of the yeast, observe the mycelial morphology, color, and porosity of the cut surface, and use a 0-10 score method to quantitatively evaluate the cross-sectional morphology. Step S104. Use vernier calipers to randomly select multiple test points and measure the thickness of the unfermented raw starch layer on the surface of the yeast starter. Take the average value as the evaluation basis for the yeast starter skin thickness, and use a 0-10 score method to quantitatively evaluate the yeast starter skin thickness. Step S105. Smell the aroma of the cross-section of the yeast starter. Based on the yeast starter-specific sensory description dictionary, identify the purity of the aroma, the intensity of the aroma, and whether there are any off-flavors. Use a 0-10 scoring method to quantitatively evaluate the aroma characteristics. Step S106. Add the scores of the four dimensions together and use the consistency ratio (CR) to test the consistency among the evaluators. When CR < 0.1, the evaluation results are considered reliable.

3. The multidimensional analysis and evaluation method for the quality of yeast as described in claim 1, characterized in that, Step S2, the determination of moisture and volatile component content specifically includes the following steps: Step S201. Take a clean, dry glass dish to constant weight, weigh it and record its mass as m0; Step S202. Accurately add the yeast sample, weigh the total mass of the glass dish and the sample, and record it as m1; Step S203. Place the glass dish containing the yeast sample in a drying oven at 100±5 ℃ and dry it to constant weight. Weigh the total mass of the glass dish and the dried sample at this point and record it as m2. Step S204. Calculate the moisture and volatile component content according to the formula W = (m1 - m2) / (m1 - m0) × 100%, where W is the moisture and volatile component content, m0 is the mass of the glass dish, m1 is the total mass of the sample and glass dish before drying, and m2 is the total mass of the sample and glass dish after drying. Step S205. The weighing accuracy of the yeast sample is 0.0001 g. Three parallel samples are set for each yeast sample, and the average value is taken as the final measurement result to ensure the reliability of the measurement data.

4. The multidimensional analysis and evaluation method for the quality of yeast as described in claim 1, characterized in that, In step S2, the determination of color gradation specifically includes the following steps: Step S211. Accurately weigh the yeast powder sample m, add 70% ethanol as the extraction solvent, and dilute to volume V after extraction. Step S212. Place the extract in a 60 ℃ water bath for 2 h and extract at a constant temperature. After cooling to room temperature, centrifuge to collect the supernatant and dilute it by F times as needed. Step S213. Using 70% ethanol as a blank control, measure the absorbance A of the diluted supernatant at the maximum absorption wavelength of the UV-Vis spectrophotometer. Step S214. Calculate the color level according to the formula L=(F×A×V) / m, where L is the absorbance value per unit mass of sample, F is the dilution factor of supernatant, V is the volume of extract, and m is the mass of yeast powder sample. Step S215. Set up 3 parallel samples for each sample, and take the average value as the final color scale result to reduce detection error.

5. The multidimensional analysis and evaluation method for the quality of yeast as described in claim 1, characterized in that, Step S3 specifically includes the following steps: Step S301. Based on the measured moisture and volatile component content W of the yeast, calculate and weigh a yeast sample equivalent to a certain weight of oven-dried yeast; add the sample to distilled water and an acetate-sodium acetate buffer solution with pH 4.6, and soak it at a constant temperature of 40 ℃ for 3 h. After soaking, filter and collect the filtrate as enzyme solution for later use. Step S302. Take a 2% soluble starch solution and preheat it to a constant temperature in a 35 ℃ water bath. Add the above enzyme solution to the preheated starch solution and start the reaction immediately. Take samples at regular intervals during the reaction and add dilute iodine solution to observe the color change of the solution. When the color of the solution is close to the color of the dilute iodine solution itself, record the reaction time T at this time. Step S303. Calculate the liquefaction force according to the formula Lq = 24 / T, where the unit of liquefaction force is g / (g·h), and T is the reaction time and 24 is the conversion factor. Step S304. By measuring the saccharification rate and alcohol production rate of different liquefaction kojis during the fermentation process of rice wine, establish the correlation equation between liquefaction force Lq and rice wine fermentation efficiency η: η = α×ln(Lq) + β, where α and β are regression coefficients, and the correlation coefficient R²≥0.90, which is used to predict the brewing performance of kojis.

6. The multidimensional analysis and evaluation method for the quality of yeast as described in claim 1, characterized in that: In step S4, using different quality grades of Shaoxing rice wine starter as the research object based on preliminary classification by sensory and core physicochemical indicators, the volatile component spectra of Shaoxing rice wine starter of different quality grades were analyzed by GC-MS. Characteristic variables were extracted by principal component analysis, and the correlation coefficient r between each compound and the quality grade of the starter was calculated. Compounds with a correlation coefficient |r|≥0.85 and a coefficient of variation (CV) ≥30% between different quality grades were screened as candidate indicators. Palmitic acid was determined to be the characteristic marker of starter quality through comparison.

7. The multidimensional analysis and evaluation method for the quality of yeast as described in claim 1, characterized in that: In step S4, the Box-Behnken response surface methodology was used to optimize the palmitic acid extraction process. Extraction time, extraction temperature, and solid-liquid ratio were selected as three key factors, with palmitic acid extraction rate as the response value. A quadratic regression equation was established for the high starch matrix characteristics of Shaoxing rice wine starter using a three-factor, three-level experimental design. ； Where Y is the palmitic acid extraction rate, A is the extraction time, B is the extraction temperature, and C is the solid-liquid ratio. For constant terms, ~ The regression coefficients were used to verify the significance of the regression equation through analysis of variance (P < 0.01); the goodness of fit was R² ≥ 0.95; after eliminating insignificant factors, P > 0.05, the optimal extraction process conditions were determined.

8. The multidimensional analysis and evaluation method for the quality of yeast as described in claim 1, characterized in that, Step S5 specifically includes the following steps: Step S501. Chromatographic conditions: An HP-5 MS flexible quartz capillary column with dimensions of 30 m × 0.25 mm and a diameter of 0.25 μm was used; the injection port temperature was set to 250 ℃; the temperature program was as follows: initial column temperature of 60 ℃, held for 1 min; then increased to 220 ℃ at a rate of 20 ℃ / min, held for 1 min; then increased to 300 ℃ at a rate of 5 ℃ / min, held for 7 min; high-purity helium gas with a purity ≥99.999% was used as the carrier gas, and the carrier gas flow rate was set to 1 mL / min; a splitless injection method was used, with an injection volume of 1 μL. Step S502. Mass spectrometry conditions: The chromatographic-mass spectrometry interface temperature is set to 280 ℃; an electron impact source is used, with an ionization energy set to 70 eV; the ion source temperature is set to 230 ℃; qualitative analysis uses full scan mode, covering the characteristic ions of the target flavor compounds; quantitative analysis uses selected ion monitoring mode to specifically monitor the characteristic ions of palmitic acid and internal standards; the solvent delay time is set to 7 min to avoid interference from solvent peaks on the target peaks. Step S503. Quantitative Method: Quantitative analysis was performed using the internal standard method. The peak area of ​​the internal standard methyl palmitate was used as a reference. The correction factor f was calculated by the ratio of the peak areas of palmitic acid and the internal standard. The formula for calculating the correction factor is: f = (C_s × A_i) / (C_i × A_s), where C_s is the concentration of palmitic acid reference standard, A_i is the peak area of ​​the internal standard, and A_s is the peak area of ​​palmitic acid. The specific content of palmitic acid was calculated by combining the correction factor: C_sample = (A_sample × C_i × f) / A_i.

9. The multidimensional analysis and evaluation method for the quality of yeast as described in claim 1, characterized in that, Step S6 specifically includes the following steps: Step S601. Weighting: Sensory indicators account for 30% of the total score, liquefaction power accounts for 25%, ester ratio accounts for 20%, 2,4-di-tert-butylphenol safety index accounts for 15%, moisture and color scale account for 5%, palmitic acid and ketones account for 5%; Step S602. Scoring Rules: Within each evaluation indicator, all samples of yeast to be evaluated are ranked. The sample with the best ranking receives the full score corresponding to the weight of that indicator, the sample with the worst ranking receives 1 / 4 of the weight score corresponding to that indicator, and the samples in the middle of the ranking are scored using linear interpolation. Among them, 2,4-di-tert-butylphenol is used as a safety indicator. If its content exceeds the preset safety threshold, all weight scores for that indicator are deducted. Palmitic acid is used as an indicator component. If its content exceeds the preset reasonable range, some or all weight scores for that indicator are deducted according to the excess ratio. Step S603. Terminology definition: The alcohol-ester ratio specifically refers to the percentage of the sum of the chromatographic peak areas of alcohols, esters, and organic acids in the yeast sample relative to the total peak area of ​​all detectable flavor compounds in the yeast, used to quantify the harmony of the yeast flavor.

10. The multidimensional analysis and evaluation method for the quality of yeast as described in claim 9, characterized in that: In step S6, the rice wine starter is divided into three grades based on the overall score. Grade 1 brewing yeast: Overall score ≥ 85 points; Level 2 yeast: 70 points ≤ overall score < 85 points; Grade 3 brewing yeast: Overall score < 70 points; If the content of 2,4-di-tert-butylphenol exceeds the safety threshold, it will be directly judged as unqualified yeast and will not be included in the grading.

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