WO2021068718A1 - 肉制品滋味化合物剖面分析方法 - Google Patents

肉制品滋味化合物剖面分析方法 Download PDF

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WO2021068718A1
WO2021068718A1 PCT/CN2020/115617 CN2020115617W WO2021068718A1 WO 2021068718 A1 WO2021068718 A1 WO 2021068718A1 CN 2020115617 W CN2020115617 W CN 2020115617W WO 2021068718 A1 WO2021068718 A1 WO 2021068718A1
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meat product
sample
response signal
meat
average value
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French (fr)
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张春晖
韩东
李侠
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中国农业科学院农产品加工研究所
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/86—Signal analysis
    • G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information

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  • the invention relates to the field of food flavors. More specifically, the present invention relates to a method for profile analysis of flavor compounds in meat products.
  • Meat products refer to cooked meat products or semi-finished products made from livestock and poultry meat with seasonings added, such as sausages, ham, marinated meat, barbecued meat, etc.
  • Inland China (excluding Hong Kong, Macao and Taiwan) is generally divided into seven regions: North China, Northeast China, East China, Central China, South China, Southwest China, and Northwest China.
  • the psychology of dietary consumption believes that the objective environment is the prerequisite for the generation of the psychological habit of dietary consumption and the guarantee for the continuation of the psychological habit of dietary consumption. That is, based on the different dietary habits of people in various regions, different meat products adapted to local tastes are produced. There are significant differences in the taste of meat products in some regions, and the differences in taste are mainly determined by the different taste compounds of the meat products. That is, the taste of meat products has an important impact on the quality of meat products and the amount of consumer purchases.
  • An object of the present invention is to solve at least the above-mentioned problems and provide at least the advantages described later.
  • Another object of the present invention is to provide a meat product flavor compound profile analysis method, which can distinguish and identify meat products according to the overall flavor characteristics of the top-selling meat products in different regions and combined with principal component analysis.
  • the identification method can be effective Preliminarily judge the suitable sales area of a certain meat product, and further conduct a comprehensive analysis of the flavored amino acids and nucleotides in the meat product to provide an objective and reliable method for the identification of meat products.
  • a meat product flavor compound profile analysis method which includes the following steps: Step 1. According to the sales volume of meat products in different regions, select the top-ranked meat products after mashing as Sample meat products, where the sample meat products selected in each region include at least m species, m ⁇ 2;
  • Step 2 Weigh the sample solution obtained by sampling the meat product. Each sample solution is collected by the sensor of the electronic tongue respectively.
  • the response signals include: sour taste response signal, salty taste response signal, umami response signal, and sweet taste response. Signal, bitterness response signal, GPS composite response signal, SPS composite response signal;
  • 360°rotation forms the elliptical area based on the long axis and the short axis.
  • n 3
  • the three-dimensional omnidirectional 360° rotation forms an ellipsoidal area
  • Step 4 Take the meat product f to be tested, and obtain the matrix (X f1 ... X fn ) corresponding to the scatter points of the meat product to be tested on the principal component analysis diagram according to steps 2 and 3 in turn, and judge whether the scatter points fall into it An elliptical area or ellipsoidal area, if it is, it is determined that the meat product to be tested is suitable for the taste requirements of consumers in the area corresponding to the elliptical area or ellipsoidal area.
  • the meat product flavor compound profile analysis method further includes: step 5, through high performance liquid chromatography, quantitatively analyze the free amino acids and nucleotides in the meat product to be tested to obtain the taste of the meat product to be tested
  • Compound information includes free amino acid type and content, nucleotide type and content, and flavor activity value of nucleotide.
  • the response signal collection in step 2 specifically includes: collecting each sample at least three times, and obtaining the average value of at least 7 data after the collected data is stable each time a , Obtain the average value b of each sample according to the average value a as the response signal;
  • Step 3 also includes: calculating the average value c ⁇ standard error of each response signal according to the average value b of m sample meat products in each area, and taking the average value c ⁇ standard error as the fluctuation of the corresponding response signal of the sample meat products in the area range;
  • Step 4 is specifically: take the meat product f to be tested, and obtain the average value a of the meat product to be tested and the matrix (X f1 ... X fn ) of the corresponding scatter points on the principal component analysis diagram according to steps 2 and 3 in turn, according to The average value a obtains the average value c ⁇ standard error of each response signal of the meat product f to be detected as the response signal of the meat product to be detected;
  • the processing of the sample meat product in step 2 specifically includes: weighing the sampled meat product in a packaging bag, adding ultrapure water to the packaging bag, vacuum sealing and packaging, placing it in a 40°C water bath, leaching for 30 minutes, and adjusting Centrifuge at 3500r/min, temperature 4°C, centrifuge for 20min, repeat centrifugation 2-3 times to obtain supernatant, supernatant is filtered with Whatman No. 1 filter paper and then vacuum filtered to obtain sample solution, among which, sample meat
  • the dosage ratio of the product to ultrapure water is 1g:3-5mL, and the pore size of the vacuum filtration membrane is 0.45 ⁇ m.
  • the sampled meat product Preferably, weigh the sampled meat product and add ultrapure water to it, homogenize twice in an ice bath at a rate of 18000r/min, 10s each time, add 20mL of a 5% volume fraction of trichloroacetic acid aqueous solution, mix well, After standing at 4°C for 12h, adjust the centrifuge speed to 3700r/min, the temperature is 4°C, centrifuge for 20min, take the supernatant and filter it with Whatman No.1 filter paper, adjust the pH to 6.0 with 4mol/L KOH, and vacuum after constant volume
  • the solution to be subjected to quantitative analysis of free amino acids is obtained by suction filtration, wherein the dosage ratio of the sample meat product to the added ultrapure water is 1g:2mL, and the pore size of the vacuum filtration membrane is 0.45 ⁇ m.
  • the chromatographic conditions for quantitative analysis of free amino acids are: the chromatographic column is a Nova-Pak TM C18 amino acid analysis column, the column temperature is 37°C, the ultraviolet detection wavelength is 248 nm, the sample volume is 10 ⁇ L, and the flow rate is 1.0 mL/min.
  • the mobile phase A is AccQ ⁇ Tag Eluent A, which is diluted with ultrapure water in a volume ratio of 1:10; the mobile phase B is chromatographic grade acetonitrile; the mobile phase C is ultrapure water for gradient elution.
  • the sampled meat product in a centrifuge tube, add 5% by mass perchloric acid aqueous solution, homogenize twice at a rate of 18000r/min, 10s each time, leave it at 4°C for 1h and centrifuge Take the supernatant and transfer it to a beaker, shake the residue with a 5% by mass perchloric acid solution for 5 minutes, centrifuge and combine the supernatant, use 1mol/L sodium hydroxide solution to adjust the pH to 6.5, by Whatman No.1 Filter with filter paper and vacuum filter to obtain the solution for nucleotide quantitative analysis after constant volume.
  • the ratio of the sample meat product to the twice-added perchloric acid is 1g:3mL:2mL.
  • the centrifugation is specifically a regulated centrifuge. Rotation speed is 3700r/min, temperature is 4°C, centrifugation is 15min, and the pore size of vacuum filtration membrane is 0.45 ⁇ m.
  • the meat products can be distinguished and identified.
  • the identification method the suitable sales promotion area of a certain meat product can be effectively judged initially;
  • Fig. 1 is a two-dimensional scatter diagram of the principal component analysis of the taste profile of a sample meat product in one of the techniques of the present invention
  • Figure 2 is a radar chart of a sample meat product of one of the technical solutions of the present invention.
  • AQC derivatization kit boric acid buffer, derivatizing agent powder, derivatizing agent diluent), acetate-phosphate buffer (AccQ ⁇ Tag Eluent A): Waters, USA;
  • Acetonitrile (chromatographic grade): American Fisher Company;
  • nucleotide standards 5’-AMP, 5’-GMP, 5’-IMP Sigma, USA;
  • ASTREE type electronic tongue (7 taste sensors, French Alpha Moss), of which 7 taste sensors are SRS-sourness sensors, mainly used to obtain sour taste response signals; STS-saltiness sensors, mainly used to obtain salty response Signal; UMS-umami sensor, mainly used to obtain umami response signals, sensitive to umami substances (such as guanylic acid, aspartic acid and glutamic acid); SWS sensor, mainly used to obtain sweet response signals, Sweet substances (such as serine, glycine, threonine, and alanine) are sensitive; BRS sensors are mainly used to obtain bitter taste response signals, and are sensitive to bitter compounds (such as histidine, tyrosine, leucine, and isoleucine). Acid and phenylalanine) sensitive; GPS sensor, which is a composite taste sensor, mainly used to obtain GPS composite response signals; SPS sensor, which is a composite taste sensor, mainly used to obtain SPS composite response signals.
  • SRS-sourness sensors mainly used to obtain
  • Aglient 1260 Infinity High Performance Liquid Chromatograph (Aglient, USA);
  • HH-6 digital display constant temperature water bath (Changzhou Zhiborui Instrument Manufacturing Co., Ltd.);
  • Type 101-2 electric heating blast drying oven (Shanghai Experimental Instrument Factory);
  • the method for profile analysis of taste compounds in meat products includes the following steps:
  • a sample of the above-mentioned original-flavored stewed beef was taken, and the frozen stew and tendons on the surface were removed, then mashed with a tissue masher, frozen at -20°C, and used as a sample meat product for later use.
  • Step two electronic tongue detection-obtain detection data
  • YP10002 electronic balance to sample the meat products, put them in a packaging bag after thawing, add ultrapure water to the packaging bag, and vacuum seal the package, place it in a HH-6 digital thermostat water bath, adjust the temperature to 40°C, Extract in a water bath for 30 minutes, adjust the centrifuge (X-12 series high-speed low-temperature centrifuge) at a speed of 3500r/min, a temperature of 4°C, centrifuge for 20 minutes, and repeat the centrifugation 2-3 times (each time the supernatant is obtained) to obtain the upper
  • the clear liquid, the supernatant liquid is filtered with gauze, and then filtered with Whatman No.1 filter paper, and then vacuum filtered to obtain a clear and transparent sample solution.
  • the weighed amount of the sample meat product is 50g, and the amount of ultrapure water is added 200mL, the pore size of the vacuum filtration membrane is 0.45 ⁇ m;
  • the sensor of the electronic tongue (ASTREE electronic tongue) is used to collect the response signal.
  • the acquisition time is set to 120s, and the sampling interval is 1s.
  • the response signals of all sensors tend to be stable after 100s.
  • the response signal includes: sour response signal, salty response signal, umami response signal, sweet response signal, bitter response signal, GPS composite Response signal, SPS composite response signal;
  • Principal component analysis method is used to reduce the dimensionality of the collected response signals and sort them according to the contribution rate. Among them, the contribution rate of the first principal component is 80.19%, and the contribution rate of the second principal component is 18.79%. The contribution rate is 98.98%, so the first and second principal components with the top 2 contribution rates are selected to construct a two-dimensional scatter plot, see Figure 1 for details;
  • Step 4 Take the meat product f to be tested and name it: OMJ;
  • Sample configuration Weigh the meat product f to be tested, add ultrapure water to it, homogenize 2 times in an ice bath at a rate of 18000r/min, 10s each time, add 20mL of a 5% volume fraction of trichloroacetic acid aqueous solution, and mix well After standing at 4°C for 12h, adjust the centrifuge speed to 3700r/min, the temperature is 4°C, centrifuge for 20min, take the supernatant and filter it with Whatman No.1 filter paper and adjust the pH to 6.0 with 4mol/L KOH, after constant volume
  • the solution to be subjected to quantitative analysis of free amino acids is obtained by vacuum filtration, wherein the weighed amount of the meat product f to be tested is 10.00g, the amount of ultrapure water added is 20mL, and the pore size of the vacuum filtration membrane is 0.45 ⁇ m;
  • AQC derivatizer pipet 1 mL of derivatizer diluent into a derivatizer bottle with derivatizer powder, cap and seal, place in a vortex shaker, vortex for 10 seconds, and heat at 55°C for 10 minutes until the derivatizer The powder is completely dissolved;
  • the chromatographic conditions are: the chromatographic column is a Nova-Pak TM C18 amino acid analysis column, the column temperature is 37°C, the ultraviolet detection wavelength is 248nm, the injection volume is 10 ⁇ L, the flow rate is 1.0mL/min, the mobile phase A is AccQ ⁇ Tag Eluent A, Diluted with ultrapure water at a volume ratio of 1:10; mobile phase B is chromatographic grade acetonitrile; mobile phase C is ultrapure water, and gradient elution is performed to obtain the free amino acid type and content of the meat product f to be tested, as follows Table 1 shows:
  • the pH of the solution was adjusted to 6.5, filtered through Whatman No.1 filter paper, and after constant volume, vacuum filtration was used to obtain the solution to be subjected to quantitative nucleotide analysis.
  • the centrifugation specifically refers to adjusting the centrifuge speed to 3700r/min and the temperature to 4°C. ,Centrifuge for 15min, the pore size of the vacuum filter membrane is 0.45 ⁇ m;
  • chromatographic column Intersil ODS-3 chromatographic column Intersil ODS-3, temperature 30°C, UV detection wavelength 254nm, injection volume 100 ⁇ L, flow rate 1.0mL/min, mobile phase A is chromatographic grade methanol, mobile phase B is 0.05% phosphoric acid, isocratic Elution, the specific data is shown in Table 2:
  • the specific response signal collection in step two is: each sample is collected three times, and each time the collected data is stable, the 10 data are obtained and the average value a is obtained, and the average value a of each sensor is obtained, based on the total average value of each sensor. For the value a, take the average value b of each sensor corresponding to each sample as the response signal;
  • Step 3 also includes: calculating the average value c ⁇ standard error of each response signal (calculated from multiple corresponding average values b) based on the average value b of m sample meat products in each region, and taking the average value c ⁇ standard
  • the error is taken as the fluctuation range of the response signal corresponding to the sample meat products in the region, that is, the fluctuation range is (average value-standard error, average value + standard error), as shown in Table 3 below:
  • Step 4 is specifically: take the meat product f to be detected, and obtain the average value a of the meat product to be detected according to step 2 and step 3 (each sensor corresponds to three average values a) and the corresponding scatter points on the principal component analysis chart The matrix (485.535,254.474) of, according to the average value a to obtain the average value c ⁇ standard error of each response signal of the meat product f to be detected (the corresponding three average values a are obtained), as the response signal of the meat product to be detected;
  • the average value of the response signals of the same sensor of multiple samples in the same area is taken as the response signal of the sample meat products in the area to the sensor. They are evenly arranged on the circumference, and each branch represents a sensor to form a radar chart to obtain the flavor profile of the original sauce stewed beef in each region, as shown in Figure 2;
  • the salty response signal obtained by the STS-saltiness sensor there are significant differences in the salty response signal obtained by the STS-saltiness sensor, the umami response signal obtained by the UMS-umami sensor, and the GPS composite response signal obtained by the GPS sensor.
  • the sensor can clearly distinguish the region of the original sauce braised beef sample.
  • 5'-AMP adenylic acid
  • 5'-IMP inosine acid
  • 5'-GMP guanylic acid
  • taste thresholds of 5'-AMP, 5'-IMP, and 5'-GMP are 50mg/ 100mL, 25mg/100mL, 12.5mg/100mL.
  • TAV is a frequently used method to determine the intensity of taste in foods and determine the contribution of a compound to the overall taste.
  • the compound with a TAV value greater than 1 in the original sauce braised beef is 5'-IMP, indicating that the flavoring nucleotide has a greater contribution to the overall flavor of the original sauce braised beef; however, the 5'-IMP in the original sauce braised beef
  • the TAV value of AMP is much lower than 1, indicating that the compound has little effect on the taste of the original sauce braised beef.

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Abstract

一种肉制品滋味化合物剖面分析方法，包括以下步骤：步骤一、选取样本肉制品；步骤二、样本肉制品用电子舌的传感器进行响应信号的采集；步骤三、采用主成分分析法对采集的响应信号进行降维处理，构建n维散点图，分别计算长轴、短轴和第二长轴，当n＝2时，以长轴和短轴为基准360°旋转构成椭圆区域，当n≥3时，以长轴、短轴和第二长轴为基准，立体全方位360°旋转构成椭球区域；步骤四，取待检测肉制品f，判断该散点是否落入其中一个椭圆区域或椭球区域，若是，确定f适于对应地区消费者滋味要求。肉制品滋味化合物剖面分析方法可以初步判断肉制品的适宜销售地区，为肉制品滋味化合物信息鉴别、鉴定提供一个客观、可靠的方法。

Description

肉制品滋味化合物剖面分析方法 技术领域
本发明涉及食品滋味领域。更具体地说，本发明涉及一种肉制品滋味化合物剖面分析方法。
背景技术
随着国民经济的快速发展，肉类食品逐渐成为人们饮食结构的重要组成部分，肉制品加工业也成为了市场消费的主导产业。我国是世界上肉类生产的第一大国，也是肉类产品消费总量最多的国家。肉制品指的是以畜禽肉为原料，经添加调味料制作的熟肉制成品或半成品，如香肠、火腿、酱卤肉、烧烤肉等。
中国内陆(不包括港澳台)一般按地区划分为华北、东北、华东、华中、华南、西南、西北7个地区。饮食消费心理学认为，客观环境是饮食消费心理习惯产生的前提，又是饮食消费心理习惯得以延续的保证，即基于各地区人们不同的饮食习惯，生产出不同的适应当地口味的肉制品，导致在某些地区间肉制品在口感上存在显著差异，而口感上的差异主要取决于肉制品滋味化合物的不同。即肉制品的滋味对肉制品的质量和消费者购买量具有重要影响。
目前，各地区肉制品鉴别和区分大多数都是通过人的感官审评来评定的。但是，人的感官评价的灵敏度和准确性常常受到外界因素的干扰，同时评审人员的主观意识不可避免地会产生一些人为误差。如何初步有效鉴别不同地区肉制品，依据该鉴别方法初步判断某种肉制品的适宜销售地区，并进一步鉴定该肉制品，为肉制品滋味化合物信息鉴别、鉴定提供一个客观、可靠的方法，是目前急需解决的问题。
发明内容
本发明的一个目的是解决至少上述问题，并提供至少后面将说明的优点。
本发明还有一个目的是提供一种肉制品滋味化合物剖面分析方法，其根据不同地区销量排名在前肉制品整体滋味特性，结合主成分分析将肉制品进行区分和鉴别，依据该鉴别方法能够有效初步判断某种肉制品的适宜销售地区，进一步肉制品中呈味氨基酸和核苷酸 进行全面的分析，为肉制品鉴别提供一个客观、可靠的方法。
为了实现根据本发明的这些目的和其它优点，提供了一种肉制品滋味化合物剖面分析方法，包括以下步骤：步骤一、依据不同地区的肉制品销量，选取排名在前的肉制品捣碎后作为样本肉制品，其中，每个地区选取的样本肉制品至少包括m种，m≥2；
步骤二、称取样本肉制品处理得样品溶液，每种样品溶液分别用电子舌的传感器进行响应信号的采集，响应信号包括：酸味响应信号、咸味响应信号、鲜味响应信号、甜味响应信号、苦味响应信号、GPS复合响应信号、SPS复合响应信号；
步骤三、采用主成分分析法对采集的响应信号进行降维处理，并按照贡献率由大至小排序，按贡献率排名由前至后选取n个主成分至贡献率总和超过85％，并依据n个主成分构建n维散点图，获得每个地区m个样本肉制品对应散点在主成分分析图上的矩阵
Figure PCTCN2020115617-appb-000001
其中，
Figure PCTCN2020115617-appb-000002
代表该地区m个样品在F1上的矩阵，
Figure PCTCN2020115617-appb-000003
代表该地区m个样品在Fn上的矩阵，求矩阵
Figure PCTCN2020115617-appb-000004
挑选散点到矩阵Y最长距离的矩阵A＝(X a1 … X an)、最短距离的矩阵B＝(X b1 … X bn)和第二长距离的矩阵C＝(X c1 … X cn)，分别计算长轴D＝A-Y、短轴E＝B-Y和第二长轴F＝C-Y，当n＝2时，以长轴和短轴为基准360°旋转构成椭圆区域，当n≥3时，以长轴、短轴和第二长轴为基准，立体全方位360°旋转构成椭球区域；
步骤四，取待检测肉制品f，依次依据步骤二和步骤三获得该待检测肉制品对应散点在主成分分析图上的矩阵(X f1 … X fn)，判断该散点是否落入其中一个椭圆区域或椭球区域，若是，确定该待检测肉制品适于该椭圆区域或椭球区域对应地区消费者滋味要求。
优选的是，所述的肉制品滋味化合物剖面分析方法，还包括：步骤五，通过高效液相色谱，对待检测肉制品中的游离氨基酸、核苷酸进行定量分析，获得待检测肉制品的滋味化合物信息，滋味化合物信息包括游离氨基酸种类及含量、核苷酸种类及含量、核苷酸的滋味活性值。
优选的是，所述的肉制品滋味化合物剖面分析方法，步骤二中进行响应信号的采集具体为：每个样品采集至少三次，每次获取采集数据稳定后的至少7个数据求取平均值a，依据平均值a获取每个样品的平均值b作为响应信号；
步骤三还包括：依据每个地区m个样本肉制品的平均值b计算获得每种响应信号的平均值c±标准误差，以平均值c±标准误差作为该地区样本肉制品对应响应信号的波动范围；
步骤四具体为：取待检测肉制品f，依次依据步骤二和步骤三获得该待检测肉制品的平均值a及对应散点在主成分分析图上的矩阵(X f1 … X fn)，依据平均值a获得待检测肉制品f每种响应信号的平均值c±标准误差，作为该待检测肉制品响应信号；
判断该散点是否落入其中一个椭圆区域或椭球区域，若是，判断该待检测肉制品的响应信号是否与该椭圆区域或椭球区域对应地区样本肉制品对应响应信号的波动范围内有交集，若是，确定该待检测肉制品适于该椭圆区域或椭球区域对应地区消费者滋味要求。
优选的是，步骤二中样本肉制品处理具体为：称取样本肉制品置于包装袋中，向包装袋中加入超纯水后真空密封包装，置于40℃水浴中，浸提30min，调节离心机转速3500r/min、温度为4℃，离心20min，重复离心2-3次，获取上清液，上清液用Whatman No.1滤纸过滤后经真空抽滤获得样品溶液，其中，样本肉制品与超纯水的用量比为1g:3-5mL，真空抽滤滤膜的孔径为0.45μm。
优选的是，称取样本肉制品向其中加入超纯水，冰浴中于18000r/min速率匀浆2次，每次10s，加入20mL体积分数为5％的三氯乙酸水溶液，混合均匀，于4℃下静置12h后调节离心机转速3700r/min、温度为4℃，离心20min，取上清液用Whatman No.1滤纸过滤后用4mol/L KOH调pH至6.0，定容后经真空抽滤获得待进行游离氨基酸定量分析的溶液，其中，样本肉制品与加入的超纯水的用量比为1g:2mL，真空抽滤滤膜的孔径为0.45μm。
优选的是，进行游离氨基酸定量分析的色谱条件为：色谱柱为Nova-Pak TM C18氨基酸分析柱，柱温为37℃，紫外检测波长为248nm，进样量为10μL，流速1.0mL/min，流动相A为AccQ·Tag Eluent A，用超纯水按体积比为1:10稀释而得；流动相B为色谱级乙腈；流动相C为超纯水，进行梯度洗脱。
优选的是，称取样本肉制品置于离心管中，加入质量百分比为5％的高氯酸水溶液，于18000r/min速率匀浆2次，每次10s，于4℃下静置1h后离心取上清液转入烧杯中，残渣用质量百分比为5％的高氯酸溶液振荡5min，离心合后并上清液，使用1mol/L氢氧化钠溶液调pH至6.5，经Whatman No.1滤纸过滤，定容后经真空抽滤获得待进行核苷酸 定量分析的溶液，其中，样本肉制品与两次加入的高氯酸的用量比为1g:3mL:2mL，离心具体为调节离心机转速为3700r/min、温度为4℃，离心15min，真空抽滤滤膜的孔径为0.45μm。
本发明至少包括以下有益效果：
第一、根据不同地区销量排名在前肉制品的整体滋味特性，结合主成分分析将肉制品进行区分和鉴别，依据该鉴别方法能够有效初步判断某种肉制品的适宜销售推广地区；
进一步全面解析了肉制品滋味活性化合物，将其映射到不同肉制品滋味雷达图上，结合滋味雷达图，设定比较肉制品对应响应信号的波动范围配合判断某种肉制品的适宜销售推广地区，提高判断准确性；
再进一步对肉制品中呈味氨基酸和核苷酸进行全面的分析，为肉制品鉴别提供一个客观、可靠的方法；
第二、同时基于滋味活性物质剖面分析及不同地区肉制品滋味雷达图对比分析，能够有效为肉制品滋味发育提供理论参考，即为调控肉制品滋味提供理论依据。
本发明的其它优点、目标和特征将部分通过下面的说明体现，部分还将通过对本发明的研究和实践而为本领域的技术人员所理解。
附图说明
图1为本发明的其中一种技术中样本肉制品滋味轮廓主成分分析二维散点图；
图2为本发明的其中一种技术方案样本肉制品雷达图。
具体实施方式
下面结合实施例对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。
1试验材料与仪器
1.1、试验材料
17种氨基酸混合标准溶液、AQC衍生试剂盒(硼酸缓冲液、衍生剂粉末、衍生剂稀释液)、醋酸盐-磷酸盐缓冲液(AccQ·Tag Eluent A)：美国Waters公司；
乙腈(色谱级)：美国Fisher公司；
α-氨基丁酸(内标物)、核苷酸标准品5’-AMP、5’-GMP、5’-IMP：美国Sigma公司；
甲醇(色谱级)：德国Merck公司；
三氯乙酸、高氯酸、氢氧化钾、氢氧化钠、磷酸等：分析纯，国药集团化学试剂有限公司。
1.2、试验仪器
ASTREE型电子舌(7个味觉传感器，法国阿尔法莫斯公司)，其中，7个味觉传感器分别为SRS-sourness传感器，主要用于获取酸味响应信号；STS-saltiness传感器，主要用于获取咸味响应信号；UMS-umami传感器，主要用于获取鲜味响应信号，对鲜味物质(如鸟苷酸、天冬氨酸和谷氨酸)敏感；SWS传感器，主要用于获取甜味响应信号，对甜味物质(如丝氨酸、甘氨酸、苏氨酸和丙氨酸)敏感；BRS传感器，主要用于获取苦味响应信号，对苦味化合物(如组氨酸、酪氨酸、亮氨酸、异亮氨酸和苯丙氨酸)敏感；GPS传感器，其为复合味觉传感器，主要用于获取GPS复合响应信号；SPS传感器，其为复合味觉传感器，主要用于获取SPS复合响应信号。
Aglient 1260 Infinity高效液相色谱仪(美国Aglient公司)；
X-12系列高速低温离心机(美国贝克曼库尔特公司)；
IKA T10匀浆机、旋涡震荡器(德国IKA公司)；
HH-6型数显恒温水浴锅(常州智博瑞仪器制造有限公司)；
FE-20实验室pH计(梅特勒-托利多仪器(上海)有限公司)；
YP10002电子天平(上海越平科学仪器有限公司)；
101-2型电热鼓风干燥箱(上海市实验仪器总厂)；
探针式食品温度计(深圳市乐格电子有限公司)。
<实施例1>
肉制品滋味化合物剖面分析方法，包括以下步骤：
步骤一、样本肉制品准备
获取西南、西北、华中、华北、华东5个地区的原味酱卤牛肉的近一年的销量信息，选取排名在前的肉制品(按照排名由前至后选取)，获得如下13种原味酱卤牛肉(真空包装)，产品购于北京物美超市、天猫官方旗舰店，其中，西南地区的2种，分别命名为LSC 和NTP；西北地区的2种，分别命名为JYX和YBLX；华中地区的3种，分别命名为LSF、MZ和ZJK；华北地区的3种，分别命名为PY、DLS和TFH，华东地区的3种，分别命名为WFZ、WL和YONGSZ；
取上述原味酱卤牛肉样品，去除表面冻卤、筋腱后，用组织捣碎机捣碎后，于-20℃条件下冷冻，作为样本肉制品备用。
步骤二、电子舌检测-获取检测数据
利用YP10002电子天平称取样本肉制品，解冻后置于包装袋中，向包装袋中加入超纯水后真空密封包装，置于HH-6型数显恒温水浴锅中，调节温度为40℃，水浴浸提30min，调节离心机(X-12系列高速低温离心机)转速3500r/min、温度为4℃，离心20min，重复离心2-3次(每次离心均获得上清液)，获取上清液，上清液用纱布过滤后，再用Whatman No.1滤纸过滤，然后经真空抽滤获得澄清透明的样品溶液，其中，样本肉制品的称取量为50g，加入超纯水的量为200mL，真空抽滤滤膜的孔径为0.45μm；
每种样品溶液分别用电子舌(ASTREE型电子舌)的传感器进行响应信号的采集，设定采集时间为120s，采样时间间隔为1s，采集过程中发现在100s以后所有传感器的响应信号趋于稳定，取最后采集的10个数据求取平均值作为对应传感器的响应信号，其中，响应信号包括：酸味响应信号、咸味响应信号、鲜味响应信号、甜味响应信号、苦味响应信号、GPS复合响应信号、SPS复合响应信号；
步骤三、主成分分析
采用主成分分析法对采集的响应信号进行降维处理，并按照贡献率大小排序，其中，第一主成分的贡献率为80.19％、第二主成分的贡献率为18.79％，两者的总贡献率为98.98％，故选取贡献率排名前2的第一主成分、第二主成分构建二维散点图，具体参见图1；
获得每个地区m个样本肉制品对应散点在主成分分析图上的矩阵
Figure PCTCN2020115617-appb-000005
其中，
Figure PCTCN2020115617-appb-000006
代表该地区m个样品在F1上的矩阵，
Figure PCTCN2020115617-appb-000007
代表该地区m个样品在Fn上的矩阵，求矩阵
Figure PCTCN2020115617-appb-000008
挑选散点到矩阵Y最长距离的矩阵A＝(X a1 … X an)、最短距离的矩阵B＝(X b1 … X bn)和第二长距离的矩阵 C＝(X c1 … X cn)，分别计算长轴D＝A-Y、短轴E＝B-Y和第二长轴F＝C-Y，当n＝2时，以长轴和短轴为基准360°旋转构成椭圆区域，当n≥3时，以长轴、短轴和第二长轴为基准，立体全方位360°旋转构成椭球区域；
其中，华东地区3个肉制品样品对应的散点矩阵分别为r1＝(597.990,285.779)，r2＝(514.527,242.582)和r3＝(476.206,309.582)，求得矩阵
Figure PCTCN2020115617-appb-000009
挑选散点到矩阵Y的最长距离的矩阵A＝r3，对应的长轴D为：D＝r3-Y＝(-53.321,30.34)，散点到矩阵Y的最长距离的矩阵B＝r2，对应的短轴E为：E＝r2-Y＝(-15.047,-36.66)，以长轴D和短轴E为基准360°旋转构成椭圆区域。
进一步，由图1可知，每个地区的不同样品间的距离较近，说明同一地区的不同原味酱卤牛肉制品的品质特性相似，5个地区的原味酱卤牛肉制品分成了3大区域，分别位于第一、二和四象限，其中，西北地区和华东地区的原味酱卤牛肉有部分重叠，说明这两个地区原味酱卤牛肉滋味成分较为接近，华中地区和西南地区同理有部分重叠；华北地区原味酱卤牛肉与其他4个地区相距较远，说明华北地区原味酱卤牛肉滋味成分与它们存在显著不同。
步骤四，取待检测肉制品f，命名为：OMJ；
按照<步骤二、电子舌检测-获取检测数据>的相同方式获取OMJ对应散点在主成分分析图上的矩阵(485.535,254.474)；
判断该散点落入华东地区和西北地区对应的椭圆区域，确定该待检测肉制品适于华东地区和西北地区消费者的滋味要求。
步骤五：
五(1)、游离氨基酸测定
样品的配置：称取待检测肉制品f向其中加入超纯水，冰浴中于18000r/min速率匀浆2次，每次10s，加入20mL体积分数为5％的三氯乙酸水溶液，混合均匀，于4℃下静置12h后调节离心机转速3700r/min、温度为4℃，离心20min，取上清液用Whatman No.1滤纸过滤后用4mol/L KOH调pH至6.0，定容后经真空抽滤获得待进行游离氨基酸定量分析的溶液，其中，待检测肉制品f的称取量为10.00g，加入的超纯水的用量为20mL，真空抽滤滤膜的孔径为0.45μm；
AQC衍生剂的配制：吸取1mL衍生剂稀释液放入具有衍生剂粉末的衍生剂瓶中，加盖密封，置于旋涡震荡器中旋涡震荡10s，于55℃条件下，加热10min，直至衍生剂粉末全部溶解；
色谱条件为：色谱柱为Nova-Pak TM C18氨基酸分析柱，柱温为37℃，紫外检测波长为248nm，进样量为10μL，流速1.0mL/min，流动相A为AccQ·Tag Eluent A，用超纯水按体积比为1:10稀释而得；流动相B为色谱级乙腈；流动相C为超纯水，进行梯度洗脱，获得待检测肉制品f的游离氨基酸种类及含量，如下表1所示：
表1
Figure PCTCN2020115617-appb-000010
五(2)、呈味核苷酸测定
称取5.00g待检测肉制品f置于50mL离心管中，加入15mL质量百分比为5％的高氯酸水溶液，置于IKA T10匀浆机中，于18000r/min速率匀浆2次，每次10s，于4℃下静置1h后离心取上清液转入烧杯中，残渣用质量百分比为5％的高氯酸溶液振荡5min，离心合后并上清液，使用1mol/L氢氧化钠溶液调pH至6.5，经Whatman No.1滤纸过滤，定容后经真空抽滤获得待进行核苷酸定量分析的溶液，其中，离心具体为调节离心机转速 为3700r/min、温度为4℃，离心15min，真空抽滤滤膜的孔径为0.45μm；
色谱条件为：色谱柱Intersil ODS-3，温度30℃，紫外检测波长254nm，进样量为100μL，流速1.0mL/min，流动相A为色谱级甲醇，流动相B为0.05％磷酸，等度洗脱，具体数据如表2所示：
表2
Figure PCTCN2020115617-appb-000011
<实施例2>
在实施例1的基础上：
步骤二中进行响应信号的采集具体为：每个样品采集三次，每次获取采集数据稳定后的10个数据求取平均值a，获得每个传感器的平均值a，依据每个传感器的全部平均值a求取每个样品对应的每个传感器的平均值b作为响应信号；
步骤三还包括：依据每个地区m个样本肉制品的平均值b计算获得每种响应信号的平均值c±标准误差(由多个对应的平均值b计算获得)，以平均值c±标准误差作为该地区样本肉制品对应响应信号的波动范围，即波动范围为(平均值-标准误差，平均值+标准误差)，具体如下表3所示：
Figure PCTCN2020115617-appb-000012
Figure PCTCN2020115617-appb-000013
步骤四具体为：取待检测肉制品f，依次依据步骤二和步骤三获得该待检测肉制品的平均值a(每种传感器对应三个平均值a)及对应散点在主成分分析图上的矩阵(485.535,254.474)，依据平均值a获得待检测肉制品f每种响应信号的平均值c±标准误差(对应的三个平均值a获得)，作为该待检测肉制品响应信号；
其中，待检测肉制品f的响应信号，如下表4所示：
表4
Figure PCTCN2020115617-appb-000014
判断OMJ散点落入华东地区和西北地区对应的椭圆区域，进一步判断该OMJ的响应信号(范围)与该椭圆区域对应地区样本肉制品对应响应信号的波动范围内有交集，确定该待检测肉制品适于华东地区和西北地区消费者的滋味要求。
<实施例3>
样本肉制品雷达图分析
为了更直观的分析电子舌对不同原味酱卤牛肉的响应，求取同一地区的多个样品的同种传感器的响应信号的平均值，作为该地区的样本肉制品针对该传感器的响应信号，将其均匀的排列在圆周上，每个分支代表一个传感器，形成雷达图，获得每个地区的原味酱卤牛肉的滋味轮廓，具体如图2所示；
由图2可知，华中地区和西南地区的原味酱卤牛肉滋味轮廓性状相似，西北地区和华东地区的原味酱卤牛肉滋味轮廓性状相似，进一步印证说明它们的滋味成分较为接近。华北地区原味酱卤牛肉的滋味轮廓不同其他原味酱卤牛肉，表明其在滋味呈现方面存在差异， 即将西南、西北、华中、华北、华东5个地区的原味酱卤牛肉的分成3个大类，其与主成分分析的结果相照应。其中，对于三大类滋味轮廓而言，STS-saltiness传感器获取的咸味响应信号、UMS-umami传感器获取的鲜味响应信号和GPS传感器获取的GPS复合响应信号存在显著差异，这说明这三个传感器可以比较清晰区分原味酱卤牛肉样品的地区。
<实施例4>
样本肉制品游离氨基酸测定
依据<五(1)、游离氨基酸测定>中记载的游离氨基酸测定的方式，获得不同区域样品肉制品的游离氨基酸种类及含量，如下表5所示：
表5不同区域样品肉制品游离氨基酸含量(mg/100g)
Figure PCTCN2020115617-appb-000015
由表5可知，华中地区、西南地区和西北地区7种原味酱卤牛肉中共鉴定出18种滋味活性物质，依次为鲜味(鸟苷酸、谷氨酸)，甜味(丝氨酸、甘氨酸、苏氨酸、丙氨酸、精氨酸和脯氨酸)，苦味(组氨酸、酪氨酸、亮氨酸、异亮氨酸、苯丙氨酸、半胱氨酸、缬氨酸、甲硫氨酸和赖氨酸)，因其对原味酱卤牛肉总体滋味具有显著贡献，被认定为华中地区、西南地区和西北地区原味酱卤牛肉的关键滋味物质。
华东地区和华北地区6种原味酱卤牛肉样品中，共同检出19种滋味活性物质，依次为鲜味(鸟苷酸、天冬氨酸、谷氨酸)，甜味(丝氨酸、甘氨酸、苏氨酸、丙氨酸精氨酸和脯氨酸)，苦味(组氨酸、酪氨酸、亮氨酸、异亮氨酸、苯丙氨酸、半胱氨酸、缬氨酸、甲硫氨酸和赖氨酸)，这些物质对原味酱卤牛肉的整体风味具有较大贡献，因此被认定为华东地区和华北地区原味酱卤牛肉的关键滋味物质。
进一步，由表1可知，华东地区原味酱卤牛肉中总游离氨基含量最高为1582.76mg/100g，其次为华北和西北地区，最后为西南和华中地区最低，且三个大区域中总游离氨基酸差异较大。
<实施例5>
样本肉制品呈味核苷酸测定
依据<五(2)、呈味核苷酸测定>中记载的呈味核苷酸的方式，获得不同区域样品肉制品的呈味核苷酸含量(mg/100g)和滋味活性值(TAV)，具体数据如表6所示：
表6 5个地区原味酱卤牛肉呈味核苷酸含量(mg/100g)和滋味活性值(TAV)
Figure PCTCN2020115617-appb-000016
注：5’-AMP:腺苷酸、5’-IMP:肌苷酸、5’-GMP:鸟苷酸；5’-AMP、5’-IMP、5’-GMP的味道阈值分别是50mg/100mL、25mg/100mL、12.5mg/100mL。
由表6可知，三种呈味核苷酸(5’-AMP、5’-IMP、5’-GMP)在5个地区原味酱卤牛肉中均有检出，5’-IMP在三种呈味核苷酸中含量最大。华东地区中呈味核苷酸含量最高，其次为西北地区和华中地区，华北地区和西南地区含量最低，且它们之间存在差异性显著。
对于食品中滋味强度的判定及确定某个化合物在整体滋味中的贡献时，TAV是经常用到的方法。在不同地区原味酱卤牛肉中TAV值大于1的化合物为5’-IMP，说明该呈味核苷酸对整体原味酱卤牛肉的滋味贡献较大；然而，在原味酱卤牛肉中5’-AMP的TAV值远远低于1，表明该化合物对原味酱卤牛肉的滋味影响作用较小。
尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节和这里示出与描述的图例。

Claims (8)

  1. 肉制品滋味化合物剖面分析方法，其特征在于，包括以下步骤：
    步骤一、依据不同地区的肉制品销量，选取排名在前的肉制品捣碎后作为样本肉制品，其中，每个地区选取的样本肉制品至少包括m种，m≥2；
    步骤二、称取样本肉制品处理得样品溶液，每种样品溶液分别用电子舌的传感器进行响应信号的采集，响应信号包括：酸味响应信号、咸味响应信号、鲜味响应信号、甜味响应信号、苦味响应信号、GPS复合响应信号、SPS复合响应信号；
    步骤三、采用主成分分析法对采集的响应信号进行降维处理，并按照贡献率由大至小排序，按贡献率排名由前至后选取n个主成分至贡献率总和超过85％，并依据n个主成分构建n维散点图，获得每个地区m个样本肉制品对应散点在主成分分析图上的矩阵
    Figure PCTCN2020115617-appb-100001
    其中，
    Figure PCTCN2020115617-appb-100002
    代表该地区m个样品在F1上的矩阵，
    Figure PCTCN2020115617-appb-100003
    代表该地区m个样品在Fn上的矩阵，求矩阵
    Figure PCTCN2020115617-appb-100004
    挑选散点到矩阵Y最长距离的矩阵A＝(X a1…X an)、最短距离的矩阵B＝(X b1…X bn)和第二长距离的矩阵C＝(X c1…X cn)，分别计算长轴D＝A-Y、短轴E＝B-Y和第二长轴F＝C-Y，当n＝2时，以长轴和短轴为基准360°旋转构成椭圆区域，当n≥3时，以长轴、短轴和第二长轴为基准，立体全方位360°旋转构成椭球区域；
    步骤四，取待检测肉制品f，依次依据步骤二和步骤三获得该待检测肉制品对应散点在主成分分析图上的矩阵(X f1…X fn)，判断该散点是否落入其中一个椭圆区域或椭球区域，若是，确定该待检测肉制品适于该椭圆区域或椭球区域对应地区消费者滋味要求。
  2. 如权利要求1所述的肉制品滋味化合物剖面分析方法，其特征在于，还包括：步骤五，通过高效液相色谱，对待检测肉制品中的游离氨基酸、核苷酸进行定量分析，获得待检测肉制品的滋味化合物信息，滋味化合物信息包括游离氨基酸种类及含量、核苷酸种类及含量、核苷酸的滋味活性值。
  3. 如权利要求1所述的肉制品滋味化合物剖面分析方法，其特征在于，步骤二中进行响应信号的采集具体为：每个样品采集至少三次，每次获取采集数据稳定后的至少7个 数据求取平均值a，依据平均值a获取每个样品的平均值b作为响应信号；
    步骤三还包括：依据每个地区m个样本肉制品的平均值b计算获得每种响应信号的平均值c±标准误差，以平均值c±标准误差作为该地区样本肉制品对应响应信号的波动范围；
    步骤四具体为：取待检测肉制品f，依次依据步骤二和步骤三获得该待检测肉制品的平均值a及对应散点在主成分分析图上的矩阵(X f1…X fn)，依据平均值a获得待检测肉制品f每种响应信号的平均值c±标准误差，作为该待检测肉制品响应信号；
    判断该散点是否落入其中一个椭圆区域或椭球区域，若是，判断该待检测肉制品的响应信号是否与该椭圆区域或椭球区域对应地区样本肉制品对应响应信号的波动范围内有交集，若是，确定该待检测肉制品适于该椭圆区域或椭球区域对应地区消费者滋味要求。
  4. 如权利要求1所述的肉制品滋味化合物剖面分析方法，其特征在于，步骤二中样本肉制品处理具体为：称取样本肉制品置于包装袋中，向包装袋中加入超纯水后真空密封包装，置于40℃水浴中，浸提30min，调节离心机转速3500r/min、温度为4℃，离心20min，重复离心2-3次，获取上清液，上清液用Whatman No.1滤纸过滤后经真空抽滤获得样品溶液，其中，样本肉制品与超纯水的用量比为1g:3-5mL，真空抽滤滤膜的孔径为0.45μm。
  5. 如权利要求2所述的肉制品滋味化合物剖面分析方法，其特征在于，步骤五中，称取样本肉制品向其中加入超纯水，冰浴中于18000r/min速率匀浆2次，每次10s，加入20mL体积分数为5％的三氯乙酸水溶液，混合均匀，于4℃下静置12h后调节离心机转速3700r/min、温度为4℃，离心20min，取上清液用Whatman No.1滤纸过滤后用4mol/L KOH调pH至6.0，定容后经真空抽滤获得待进行游离氨基酸定量分析的溶液，其中，样本肉制品与加入的超纯水的用量比为1g:2mL，真空抽滤滤膜的孔径为0.45μm。
  6. 如权利要求5所述的肉制品滋味化合物剖面分析方法，其特征在于，进行游离氨基酸定量分析的色谱条件为：色谱柱为Nova-Pak TMC18氨基酸分析柱，柱温为37℃，紫外检测波长为248nm，进样量为10μL，流速1.0mL/min，流动相A为AccQ·Tag Eluent A，用超纯水按体积比为1:10稀释而得；流动相B为色谱级乙腈；流动相C为超纯水，进行梯度洗脱。
  7. 如权利要求2所述的肉制品滋味化合物剖面分析方法，其特征在于，步骤五中，称取样本肉制品置于离心管中，加入质量百分比为5％的高氯酸水溶液，于18000r/min速 率匀浆2次，每次10s，于4℃下静置1h后离心取上清液转入烧杯中，残渣用质量百分比为5％的高氯酸溶液振荡5min，离心合后并上清液，使用1mol/L氢氧化钠溶液调pH至6.5，经Whatman No.1滤纸过滤，定容后经真空抽滤获得待进行核苷酸定量分析的溶液，其中，样本肉制品与两次加入的高氯酸的用量比为1g:3mL:2mL，离心具体为调节离心机转速为3700r/min、温度为4℃，离心15min，真空抽滤滤膜的孔径为0.45μm。
  8. 如权利要求6所述的肉制品滋味化合物剖面分析方法，其特征在于，进行核苷酸定量分析的色谱条件为：色谱柱Intersil ODS-3，温度30℃，紫外检测波长254nm，进样量为100μL，流速1.0mL/min，流动相A为色谱级甲醇，流动相B为分析纯磷酸，等度洗脱。
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