奶制品中蛋白质的检测仪器和方法研究
摘要
本文针对国内奶制品中蛋白质含量测定与质量控制过程中长期存在的检测仪器成本较高、检测方法耗时较长以及对掺假（伪）物识别能力较差等难题，研制了3种小型化检测仪器和4种快速检测方法，可用于快速测定原奶总蛋白和奶制品中真蛋白、皮革水解蛋白、植物蛋白的含量，基本上满足了奶制品中蛋白质主要掺假（伪）物检测的需求，具有显著的实用价值。
基于近红外光谱法，研制开发出一种原奶中总蛋白含量测定专用的新型低成本短波近红外漫透射光谱测量仪器，该仪器基于卤钨灯光源—固定光栅—阵列检测光电传感器设计，可用于原奶样品的现场或在线检测，无需样品前处理操作，不消耗化学试剂，单样品检测时间在15s以内；检测结果与凯氏定氮法相比无显著性差异。
采用有机染料作为探针，建立了一种奶制品中真蛋白含量的褪色—比色测定方法，并研制出与该方法配套的专用小型仪器检测系统。对显色温度、时间和非蛋白氮干扰物质等测量影响因素进行了系统考察研究，结果表明，该检测系统测定结果准确、检测速度快，单样品的处理和检测时间在10min以内，并可避免常见非蛋白氮物质的干扰。
根据皮革水解蛋白中羟脯氨酸含量较高的特点，通过对皮革水解蛋白质的高温催化消解和羟脯氨酸显色系统研究，建立了奶制品中皮革水解蛋白专用检测方法，并研制出多通道高温催化消解设备和皮革水解蛋白检测仪；通过对催化消解条件的优化，使单批样品（25个）的前处理时间在35min以内完成，检测结果与国标方法吻合。
基于胶束电动毛细管色谱法对不同尺寸蛋白质分子的分离能力，提出了一种可对奶制品中特征乳蛋白和掺杂大豆蛋白分布情况进行快速筛查的新方法。通过对分离条件的优化，实现了对奶制品中特征乳蛋白含量的定量测定和大豆蛋白的定性分析。
Dairy products are natural nutritional foods of humans, especially for infant. Thequality of dairy products is related to types and concentrations of proteins in dairyproducts and can affect on health of consumers. In recent years, the dairy productswere adulterated with illegal materials by some manufactures in China for economicbenefit. In this thesis,4kinds of methods and3kinds of instruments for rapiddetermination of proteins in dairy products were developed. The determination andidentification of total protein, true protein, hydrolyzed animal protein and soybeanprotein in dairy products was investigated systematically. The instruments developedin this thesis, including near infrared total protein analyzer for milk, GDYN-200S trueprotein analyzer and GDYN-300S hydrolyzed animal protein analyzer for qualitycontrol of dairy products, the last two instruments have been produced by ChangchunJilin University Little Swan Instruments Co., Ltd and widely used in dairy plants andadministration departments of Chinese government.
In the introduction, the composition and characteristics of typical proteins indairy products were summarized. The routine methods and instruments for thedetermination of different type proteins in dairy products were reviewed. The qualityof dairy products in Chinese dairy plants was analyzed. The instruments and methodsfor rapid detection of typical proteins in dairy products were proposed.
In chapter2, a new type of portable short-wave near-infrared analyzer for rapiddetermination of total protein in raw milk was developed. The carefully numericalsimulation computation and design of optical and electrical systems were carried out, followed by the research on chemometric methods for outlier sample eliminating, datapretreatment, and construction of multivariable calibration model. When the Kjeldahlmethod was used as reference method, the correlation coefficient androot-mean-square errors of prediction (RMSEP) of the present method were0.9606and0.114, respectively. The halogen lamp, fixed grating and charge-coupled device(CCD), were used in the developed analyzer. The built-in32-bits ARMmicroprocessor and firmware can guarantee the computing capability of the analyzer.The analyzer and chemometric methods developed in this work are rapid, reagent-free,non-destructive, relatively inexpensive, and suitable for the filed or on-linedetermination of total protein in raw milk.
In chapter3, a novel determination method for true protein in dairy productsusing organic dyes as molecular probes was investigated to eliminate the interferencefrom non-protein nitrogen in samples. The miniature colorimeter was developed basedon light-emitting diode (LED) and narrow-band interference filters. With the carefulchoosing of devices such as LED light source, glass cell and photoelectric sensor, andthe design optimization of controlling system, the miniature colorimeter developed inthis work was simple and has some advantages such as low production cost, highstability and short operation time(10min). The method and instrument were applied tothe determination of true protein in500samples, including fresh milk, drinkscontaining milk, milk powder, soybean powder, soybean milk and eggs. The relativeerror of protein concentration were less than±3.0%compared with proteinconcentration in powdered milk standard reference substance. The relative deviationof results of true protein in fresh milk and milk powder were less than±1.5%compared with these obtained with similar foreign instruments. The relative deviationof results of protein concentration in samples not containing non-protein nitrogenwere less than±5.0%compared with these obtained by Kjeldahl method.
In chapter4, a method for detecting hydrolyzed animal proteins in dairy productsbased on the determination of the amino acid4-hydroxyproline (4-Hyp) wasestablished. An analyzer for detecting hydrolyzed animal proteins was developed. The analyzer was coupled with a special equipment for high-temperature hydrolysis ofdairy samples with25channels. The hydrolytic conditions of hydrolysis includingvolume and concentration of sulfuric acid, amount of catalyst, temperature and timeof hydrolysis were optimized. The results showed that samples can be hydrolyzedcompletely in5mL sulfuric acid (6mol/L) and300mg catalyst for35min (150℃).Compared with the GB standard hydrolysis method, the hydrolytic time wasshortened from16h to35min.Compared with the commonly used spectrophotometerthe analyzer is simpler, lighter and more suitable for the detection in the field.
In chapter5, the preliminary study on the method for rapid separation anddetermination of milk proteins in dairy products using the micellar electrokineticcapillary chromatography was carried out. The separation of the milk proteins andsoybean proteins in dairy products was investigated. The effects of several separationconditions, such as concentrations of surfactant and viscosity-controlling agent,voltage, column temperature and time of separation were examined. The optimalconditions are as follows:120mg/L tween-20was used as surfactant,4.8mol/L ureawas used as viscosity-controlling agent, voltage column temperature and time ofseparation were25kV,30℃and30min, respectively. Limits of detection of5milkproteins in dairy product were in the range of2.50-5.61μg/L. The adulteratingsamples with concentration of soybean protein more than3.13mg/g or0.15mg/mLcould be distinguish properly. The recoveries of5milk proteins in3spiked samplesincluding skim milk powder, full-cream milk powder and pure milk are from89.16%to109.99%and the relative standard deviations are less than4%.
基于近红外光谱法，研制开发出一种原奶中总蛋白含量测定专用的新型低成本短波近红外漫透射光谱测量仪器，该仪器基于卤钨灯光源—固定光栅—阵列检测光电传感器设计，可用于原奶样品的现场或在线检测，无需样品前处理操作，不消耗化学试剂，单样品检测时间在15s以内；检测结果与凯氏定氮法相比无显著性差异。
采用有机染料作为探针，建立了一种奶制品中真蛋白含量的褪色—比色测定方法，并研制出与该方法配套的专用小型仪器检测系统。对显色温度、时间和非蛋白氮干扰物质等测量影响因素进行了系统考察研究，结果表明，该检测系统测定结果准确、检测速度快，单样品的处理和检测时间在10min以内，并可避免常见非蛋白氮物质的干扰。
根据皮革水解蛋白中羟脯氨酸含量较高的特点，通过对皮革水解蛋白质的高温催化消解和羟脯氨酸显色系统研究，建立了奶制品中皮革水解蛋白专用检测方法，并研制出多通道高温催化消解设备和皮革水解蛋白检测仪；通过对催化消解条件的优化，使单批样品（25个）的前处理时间在35min以内完成，检测结果与国标方法吻合。
基于胶束电动毛细管色谱法对不同尺寸蛋白质分子的分离能力，提出了一种可对奶制品中特征乳蛋白和掺杂大豆蛋白分布情况进行快速筛查的新方法。通过对分离条件的优化，实现了对奶制品中特征乳蛋白含量的定量测定和大豆蛋白的定性分析。
Dairy products are natural nutritional foods of humans, especially for infant. Thequality of dairy products is related to types and concentrations of proteins in dairyproducts and can affect on health of consumers. In recent years, the dairy productswere adulterated with illegal materials by some manufactures in China for economicbenefit. In this thesis,4kinds of methods and3kinds of instruments for rapiddetermination of proteins in dairy products were developed. The determination andidentification of total protein, true protein, hydrolyzed animal protein and soybeanprotein in dairy products was investigated systematically. The instruments developedin this thesis, including near infrared total protein analyzer for milk, GDYN-200S trueprotein analyzer and GDYN-300S hydrolyzed animal protein analyzer for qualitycontrol of dairy products, the last two instruments have been produced by ChangchunJilin University Little Swan Instruments Co., Ltd and widely used in dairy plants andadministration departments of Chinese government.
In the introduction, the composition and characteristics of typical proteins indairy products were summarized. The routine methods and instruments for thedetermination of different type proteins in dairy products were reviewed. The qualityof dairy products in Chinese dairy plants was analyzed. The instruments and methodsfor rapid detection of typical proteins in dairy products were proposed.
In chapter2, a new type of portable short-wave near-infrared analyzer for rapiddetermination of total protein in raw milk was developed. The carefully numericalsimulation computation and design of optical and electrical systems were carried out, followed by the research on chemometric methods for outlier sample eliminating, datapretreatment, and construction of multivariable calibration model. When the Kjeldahlmethod was used as reference method, the correlation coefficient androot-mean-square errors of prediction (RMSEP) of the present method were0.9606and0.114, respectively. The halogen lamp, fixed grating and charge-coupled device(CCD), were used in the developed analyzer. The built-in32-bits ARMmicroprocessor and firmware can guarantee the computing capability of the analyzer.The analyzer and chemometric methods developed in this work are rapid, reagent-free,non-destructive, relatively inexpensive, and suitable for the filed or on-linedetermination of total protein in raw milk.
In chapter3, a novel determination method for true protein in dairy productsusing organic dyes as molecular probes was investigated to eliminate the interferencefrom non-protein nitrogen in samples. The miniature colorimeter was developed basedon light-emitting diode (LED) and narrow-band interference filters. With the carefulchoosing of devices such as LED light source, glass cell and photoelectric sensor, andthe design optimization of controlling system, the miniature colorimeter developed inthis work was simple and has some advantages such as low production cost, highstability and short operation time(10min). The method and instrument were applied tothe determination of true protein in500samples, including fresh milk, drinkscontaining milk, milk powder, soybean powder, soybean milk and eggs. The relativeerror of protein concentration were less than±3.0%compared with proteinconcentration in powdered milk standard reference substance. The relative deviationof results of true protein in fresh milk and milk powder were less than±1.5%compared with these obtained with similar foreign instruments. The relative deviationof results of protein concentration in samples not containing non-protein nitrogenwere less than±5.0%compared with these obtained by Kjeldahl method.
In chapter4, a method for detecting hydrolyzed animal proteins in dairy productsbased on the determination of the amino acid4-hydroxyproline (4-Hyp) wasestablished. An analyzer for detecting hydrolyzed animal proteins was developed. The analyzer was coupled with a special equipment for high-temperature hydrolysis ofdairy samples with25channels. The hydrolytic conditions of hydrolysis includingvolume and concentration of sulfuric acid, amount of catalyst, temperature and timeof hydrolysis were optimized. The results showed that samples can be hydrolyzedcompletely in5mL sulfuric acid (6mol/L) and300mg catalyst for35min (150℃).Compared with the GB standard hydrolysis method, the hydrolytic time wasshortened from16h to35min.Compared with the commonly used spectrophotometerthe analyzer is simpler, lighter and more suitable for the detection in the field.
In chapter5, the preliminary study on the method for rapid separation anddetermination of milk proteins in dairy products using the micellar electrokineticcapillary chromatography was carried out. The separation of the milk proteins andsoybean proteins in dairy products was investigated. The effects of several separationconditions, such as concentrations of surfactant and viscosity-controlling agent,voltage, column temperature and time of separation were examined. The optimalconditions are as follows:120mg/L tween-20was used as surfactant,4.8mol/L ureawas used as viscosity-controlling agent, voltage column temperature and time ofseparation were25kV,30℃and30min, respectively. Limits of detection of5milkproteins in dairy product were in the range of2.50-5.61μg/L. The adulteratingsamples with concentration of soybean protein more than3.13mg/g or0.15mg/mLcould be distinguish properly. The recoveries of5milk proteins in3spiked samplesincluding skim milk powder, full-cream milk powder and pure milk are from89.16%to109.99%and the relative standard deviations are less than4%.
引文
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