【作者】 Moses Vernonxious Chamba；

【导师】 Yufei Hua；

【作者基本信息】 江南大学 ， Food Nutrition and Safety， 2014， 博士

【摘要】 随着人们对自身健康和生活环境的关注越来越多，人们对消费的（动物食用的）各种各样的产品（包括食品）产生了严重的担忧，因为越来越多的化学合成物质被使用。又由于新的健康威胁的不断出现和先前可治愈或缓解的疾病（如癌症、肺结核、心血管疾病等）的抗性的出现，对人们的生命威胁更加严重。使得这种怀疑进一步加深。现在，人们在质疑这些合成物质的安全性，特别是当他们与人体或动物相互作用后产生的未知的影响。因此，偏爱于选择消费天然的或近天然加工的产品的人们正以惊人的速度不断增加。这样一来，如果厂家不积极迅速投入到研究生产消费者可接受的加工技术，太多的需求得不到处理的时代将要来临。在许多工业食品系统，大豆分离蛋白（SPI）是一种基础的广泛使用的营养成分。然而，传统生产SPI的工艺中使用了许多非食品的合成化学品，比如正己烷、氢氧化钠,95%酒精和盐酸，如果不采取预防措施，许多都会产生一些已知的危害，消费者将会怀疑食品（SPI被作为基本成分）的安全性和对生态系统的安全性。本研究的目的是：（1）、以天然大豆粉（全脂）为原料，用纯天然的由食品植物萃取的化学物质生产天然的SPI，按照传统的等电点沉淀技术，尝试来解决消费者对健康的关心和担心。（2）、将由天然化学物质制得的SPI和传统用合成化学物质制得的SPI的品质和特性进行比较分析。一般地，溶液pH>12.5的碱溶液由去离子水和苋菜灰混合，同样的，pH <2.5的溶液由成熟的柠檬萃取得到，来替代传统使用的合成物质NaOH和HCl，这些天然物质用来从全脂豆粉中分离天然蛋白标号为NCFF，样品用来与由FF或DF为原料用天然物质（苋菜灰和柠檬萃取物）或合成物质制得的SPI进行对比，分别编号为：NCDF，SCFF，SCDF。开头的两个字母指的是使用的化学物质的类别，后两个字母指的是豆粉的类别。第一部分的对比是关于得率、组成、和蛋白质量（基于蛋白质氨基酸消化率相关性得分）。纯天然的SPI（FF为原料用天然化学物质）干基最少含粗蛋白质91.21%，因此可以被称为“分离蛋白”。结果显示：蛋白得率为43.62%（比传统方法低了7.54%），PDCAAS为0.77，比传统方法制得的SPI的PDCAAS高了22%。天然SPI中的甲硫氨酸和半胱氨酸含量明显变高。然而，在合成物质制得的SPI中谷氨酰胺和丝氨酸的含量比较高。其他的氨基酸含量没有显著变化。抗营养因子（胰蛋白酶抑制剂和植酸）含量显著低于传统的SPI。天然SPI中的许多矿物质含量都比较高，其中钾离子最高，钠在传统方法制得的SPI中比较高。第二部分，功能性质、总多酚含量和抗氧化活性进行了对比。天然的SPI的乳化性稳定性有明显提高。化学合成物质得到的SPI在吸油性（0.66±0.02%,0.50±0.01%）、乳化性（56.53±0.57%,55.50±0.39%）、起泡性（11.33±0.61%,10.40±0.40%）方面性能都比天然的SPI好，但只是很小的区别。在吸水性方面没有明显的差别。天然的SPI虽然总多酚含量比较低但DPPH值表明抗氧化能力有所提高。因此，与合成物质得到的SPI相比，天然SPI在一些功能特性方面还是比较有优势的。在感官评价系统方面，采用红外光谱分析仪分析研究能够显示天然蛋白、传统蛋白、商业蛋白具有显著区别。在特征向量负载量≥0.3，二维空间的组件基础上，主成分分析（PCA）的结果占样本变异的90.11％;与传统SPI对比的因素包括不透明度、酸、甜、苦（主要成分1，55.14%）；但不包括传统的和商业的SPI的特性如色泽、硬度、灰分、涩、咸和粘度（主要成分2,36.53%）。这些发现表明：传统的SPI没有比较特殊的影响其在食品系统中应用的特色。最后，随着天然的和合成物质在DF和FF中的反应，其对最终蛋白产品的氧化性和分子结构的改变进行了研究。LOX酶活测定表明：与脱脂豆粕（1.28x103±10.93for NCFF and1.15x103±18.92for SCDF）相比，由全脂豆粉制（5.28x103±132.45for NCFF and6.57x103±143.28for SCFF）得的蛋白的酶活明显较高，其中SCFF的酶活最高。由脱脂豆粕制得的样品之间没有区别。氧化程度、游离和总巯基含量和内荧光结果表明与DF制得的蛋白相比，由FF制得的蛋白的结构变化有所增加。圆二色谱表明存在α-螺旋和β-折叠的蛋白二级结构。然而，DF样品比FF样品无序结构的构象变化。表面疏水性的性质变化、分子排阻色谱和SDS-PAGE结果与使用的萃取化学试剂的关联比所用的豆粉的类型更大。可以看出SC制备的蛋白样品有所增加。这些分子结构的改变可以解释一些SPI的功能和感官特性的变化。因此，可以得出结论：仅使用苋菜灰和柠檬提取物作为天然植物来源的化学物质，可以用等电点沉淀方法生产具有优良营养质量、功能特性和感官特性的纯天然SPI，以解决由于使用较多非食品的合成物质对健康和环境的担忧。使用这些化学物质不会引起阻碍所得的SPI产品应用到其他食品系统。更多还原

【Abstract】 As many people are becoming more conscious about their health and theenvironment in which they live, there have been grave concerns about the ever-increasing use of synthetic chemicals in various products for human or animalconsumptions including food. This skepticism has been exacerbated by thecontinuous emergence new health conditions and development of therapeuticallyresistance of the previously curable or alleviable ones, such as cancer, tuberculosis,cardiovascular, and many others, which are critically putting human life at greaterrisk than before. People are now questioning the safety of these synthetic chemicals,especially their unknown effects when they interact with one another in the humanor animal bodies. As such, the number of people opting for consumption of naturalor near-naturally produced products is increasing at an alarming rate, such that ifmanufacturers do not swiftly identify alternative consumer-acceptable productiontechniques, time will come when demand will be too great to manage.Soy protein isolate (SPI) is one of the fundamental ingredients applicable in manymanufactured food systems. However, the conventional technique of producing SPIinvolves use of strong non-food synthetic chemicals (SC), such as n-hexane, sodiumhydroxide (NaOH),95%alcohol and hydrochloric acid (HCl), many of which havewell known hazards if handled without taking precautions, a thing which makesconsumers doubt the safety of the food products in which SPI is used as aningredient and to the ecosystem upon disposal.The aim of this study was to (1) produce a natural soy protein isolate using purelynatural, food-plant-based extraction chemicals from native (full-fat) soy flour,following the usual isoelectric precipitation technique, in an attempt to address theconcerns and fears of the healthy conscious food consumers,(2) compare the qualityand characteristics of these natural-chemicals extracted SPIs to those producedconventionally using the synthetic chemicals. Briefly, an alkaline solution of pH>12.5was prepared by filtering distilled waterthrough amaranth (Amarathus tricolor L.) ash, while the acid of pH <2.5wasextracted from ripe lemon (Citrus limon) fruits, to substituted the conventionallyused synthetic NaOH and HCl, respectively. These natural chemicals (NC) wereused to isolate native protein from the full-fat soybean flour (FF) coded NCFF.Samples for comparison were prepared from either the FF or n-hexane-defattedflours (DF) using either the natural chemicals (amaranth ash and lemon fruitextracts) or the synthetic ones and were coded NCDF, SCFF and SCDF, with thefirst two letters referring to the type of chemical used and the last two, to the type offlour.The first set of comparisons was on yield, composition and protein quality, basingon protein digestibility correlation amino acid score (PDCAAS). On dry basis thepurely-natural SPI (prepared from FF using the natural chemicals) contained aminimum of91.21%crude protein, thereby qualifying to be called a ‘proteinisolate’. It showed a protein yield of43.62%(about7.54%lower than theconventional SPI) and the PDCAAS of0.77, which was about22%higher than thatof the laboratory prepared conventional SPI, but still lower than the maximum of0.99reported by some literature. Methionine and cystein-s were significantly higherin the natural SPI while glutamine and serine were higher in synthetic SPI. The restof amino acids did not differ significantly. Anti-nutritional factors (Trypsininhibitors and phytic acid) were considerably lower in the natural than the syntheticSPIs. Many of the determined minerals were higher in the natural SPI withpotassium being the highest. Sodium was very high in the synthetic SPI.Secondly, functional properties, total polyphenol content, and antioxidant activity ofthe natural and conventional synthetic SPIs were compared. The natural SPI showedsignificant increase in emulsion stability (p <0.05). While higher values withnarrow margins were shown by the synthetic than the natural SPIs in oil absorption(0.66±0.02%,0.50±0.01%, respectively), emulsion capacity (56.53±0.57%, 55.50±0.39%) and foam stability (11.33±0.61%,10.40±0.40%). No significantdifference was observed in water absorption capacity. The DPPH assay showedincreased antioxidant activity in the natural SPI although its total polyphenol contentwas lower. Thus, the natural SPI was superior in some functional propertiescompared to its synthetic counterpart.On sensory evaluation, the SpectrumTMdescriptive analysis revealed somedistinctive characteristics among the natural, conventional and some commercialSPIs. Principal component analysis (PCA) results accounted for90.11%of thesamples variability on a two dimensional component space based on the attributeeigenvector loadings of≥0.3, and associated the traditional SPI with opacity, pasta,sweet, sour, and bitter (principal component1,55.14%), but not color, cardboard,cereal, brothy, ashy, astringent, salty, and viscosity, which were also characteristicto the conventional and commercial SPIs (principal component2,36.53%). Thesefindings signified that the traditional SPI had comparatively no unique characteristicthat would affect its application in food systems.Finally, the effects of the natural and synthetic chemicals on oxidation and themolecular structure modification of the resultant protein products as they interactwith the proteins in the DF and FF systems were examined. Determination oflipoxygenase (LOX) activity showed that those prepared from full-fat flour hadsignificantly (p <0.05) higher values (5.28x103±132.45for NCFF and6.57x103±143.28for SCFF) compared to their defatted flour counterparts (1.28x103±10.93for NCFF and1.15x103±18.92for SCDF), with SCFF being the highest.There was no significant difference in samples prepared from defatted flours. Thedegree of oxidation, free and total sufhydryl content, and intrinsic fluorescenceindicated increased structural changes in the FF-based samples as compared to thoseprepared from DF. Circular dichroism spectroscopy showed existence of α-helicesand β-sheets protein secondary structures. However, DF samples showed increase inunordered structural conformation changes than the FF sample. Characteristic variability in surface hydrophobicity, size exclusion chromatography and SDS-PAGE results were more related to the extraction chemicals than the type of flourused, with samples prepared with SC showing an increase. These molecularstructure modifications could explain some functional and sensory characteristics incertain specific SPIs.It was therefore generally concluded that a purely natural SPI with preferablenutritional quality, functional properties and sensory characteristics can be producedisoelectrically, solely using amaranth ash and lemon extracts as natural food-plants-based chemicals, to address the ever-increasing health and environmental concernsregarding the use of strong non-food synthetic chemicals. The use of thesechemicals cannot cause serious distinctive changes that would prevent applicabilityof the resultant SPI products into other food systems.更多还原