【作者】 李宁；

【导师】 孙宝国；

【作者基本信息】 陕西科技大学 ， 应用化学， 2011， 博士

【摘要】 随着我国经济的发展,人民生活水平的提高,人们对食品风味的需求日益上升。乳及各种乳制品已经逐步成为我国日常膳食的重要组成部分,对于这类具有明显乳品特征的食品,人们为了弥补其在加工过程中香气的损失,要借助于奶味香精的使用;另外,一些需要和奶味联系在一起的食品,如咖啡、巧克力、糖果、烘焙食品等,需要在其风味中具有奶油感、丰满感、厚重感等乳制品气味,也可以借助于奶味香精的使用。因此,在食品添加剂的开发和研究领域中,奶味香精是非常具有市场价值的一类产品。由于生物科技的迅速发展,同时也为了满足人们对食品越来越多的天然性方面的诉求,利用酶解乳脂等生物技术手段制备的奶味香基,因其具备加工成本低、易于贮藏、货架期较长等优点,在国外的奶味香精加工工业中已经被广泛应用。自2000年以来,在国内利用酶解乳脂技术制备奶味香基的研究已被广泛开展。在酶解乳脂制备奶味香基的过程中,脂肪酸是奶味香基的重要香味成份,在一定范围内,乳脂水解程度越大,则脂肪酸含量越高,酸值越高,香气越强烈。目前,国内文献报道所使用的脂肪酶大多为进口脂肪酶,酶的价格较高,并且,在较优条件下,乳脂经12h水解后,酸值仅在8.0mg/g左右。本文选用了一种我国自主研制的由假丝酵母（Candida sp.）产酶菌株进行发酵制备的高效脂肪水解酶,将其用于奶味香基的制备,以酶解乳脂产物的酸值为评价指标,分别对酶解时间（1～10h）、酶解温度（30～50℃）、酶的添加量（500～1500U/g）进行单因素实验后,再经过正交试验,确定了该酶在5h,40℃,1250U/g条件下可获得酸值为51.90±5.74mg/g的乳脂酶解产物,即奶味香基。通过感官实验,我们确定了这种酶解乳脂制备的奶味香基能提高加香样品的奶香味和香气强度。目前对酶解乳脂产物酸值的测定方法一般使用的是酸碱滴定法,但此法需要大量有机溶剂,且操作较繁琐。本文建立了一个基于法国Alpha Mos的FOX3000型电子鼻的快速可行的酶解乳脂产物酸值的测定方法,用主成分分析法确定了各检测参数对乳脂酸值测定结果影响大小依次是:产生时间>进样针进样量>样品上样量>产生温度,较优的测定参数是进样针进样量为2500μL,产生时间为900s,产生温度为60℃,样品上样量为5g。该方法对不同酶解程度的乳脂做酸值线性模型分析,建立最小回归定量分析模式（PLS）酸值标准曲线,对未知酸值的乳脂快速检测,准确性为93.85%,此方法方便、快捷、准确,便于酶解乳脂制备奶味香基的质量监测控制效率。另一方面我们发现,仅局限于酶解乳脂制备奶味香基的研究是不能做出被市场认可的奶味香精的,这里面最主要的原因是任何香精的生产过程中,香精的调配都占有极其重要的地位,而在香精的调配工作中,关键性奶味香料化合物的确定又是调香工作的重要基础和关键性步骤。为了解制备奶味香精所需要使用的奶香味香原料,本文剖析了1种国外奶味香精中的挥发性成份,从中获得一些相关数据;本文还剖析了2种具有特殊风味的奶制品——新疆发酵骆驼奶和西藏酥油。选择这两种奶制品,是因为新疆发酵骆驼奶是中国新疆维吾尔自治区的哈萨克、蒙古、柯尔克孜等民族群众喜爱的一种饮食,它不仅味道甘醇,并且具有淡淡的葡萄干、水果、甜味和奶酪的气味;而酥油是中国藏区特有的具有悠久历史的食品之一,它因具有独特的风味,奶味浓郁,口感极佳而深受藏区群众喜爱。新疆发酵骆驼奶和西藏酥油不仅是风味极佳具有奶香味研究价值的乳制品,并且是具有中国特色的,在新疆和西藏地区的食品消费中也占有很重要地位的食品。因此,研究确定新疆发酵骆驼奶和西藏酥油的挥发性香味成份,其研究成果不仅可以为奶味香精的调香工作提供重要参考,提高奶香型香精的像真度,为关键性奶香味化合物的研究提供重要的科学依据,还对传承和弘扬中国的特色民族饮食文化具有重要意义。本文通过同时蒸馏萃取法、固相微萃取法、溶剂辅助香味物质蒸发法等3种样品前处理方法从新疆发酵骆驼奶和西藏酥油中萃取出可挥发性物质,并通过GC/MS进行了可挥发性化合物的鉴定,进而使用GC/O对其香味化合物进行嗅闻分析,并确定其结构,从而确定了它们各自的特征风味化合物。无论是国外的奶味香精,还是新疆发酵骆驼奶或是西藏酥油,都属于复杂基质样品,其挥发性物质含于大量的水、脂肪、蛋白质等物质中,并且它们的挥发性香味成份往往组成复杂,并且有些挥发性香味成份是热敏性、受热易氧化变性的;有些挥发性香味成份的含量很低,难以提取,因此,选择合适的样品前处理方法,并优化前处理方法的实验条件参数都是真实地、准确地揭示样品中复杂香味成份的首要实验基础。我们选择了同时蒸馏萃取法、固相微萃取法、溶剂辅助香味物质蒸发法等3种样品前处理方法,以已知配比的已知奶味香料化合物组成的模拟样品为试材,以样品萃取率为主要评价指标,分别对每种样品前处理方法进行主要影响因素的优化。这些方法包括:1)同时蒸馏萃取法:对萃取溶剂（二氯甲烷、乙醚、戊烷/二氯甲烷、戊烷/乙醚）和萃取时间（1～6h）2个主要影响因素进行考察,结合GC/MS技术,用内标法进行定量,以各种奶香味化合物萃取率为主要评价指标,得到同时蒸馏萃取法对奶香味化合物的较优萃取条件:以二氯甲烷为萃取溶剂,萃取时间为3h;2)固相微萃取法:以同样的实验思路和方法对样品的预热温度（30～80℃）,萃取时间（10～180min）,样品量（1～11mL）和纤维种类（65μm CWAX/DVB,65μm PDMS/DVB,75μm CAR/PDMS和50/30μm CAR/PDMS/DVB）等4个主要影响因素进行考察,结果表明,PDMS/DVB纤维对奶味香精中挥发性化合物的选择性最好,灵敏度最高,其次是CAR/PDMS/DVB;优化的固相微萃取条件是:预热温度50℃,萃取时间40min,样品量5mL;3)溶剂辅助香味物质蒸发法:以奶味香料化合物组成的模拟体系为试材,采用溶剂辅助香味物质蒸发法提取,结合GC/MS技术,对比其定量结果与样品实际组成之间的差异,对不同种类奶香味化合物的萃取率进行分析。此项关于样品前处理的研究获得了大量、系统的基础数据,这些数据对奶制品中挥发物成分鉴定和呈香成份分析有重要的参考价值,对复杂基质样品前处理处理方法的选择有一定的指导意义。用上述优化的3种样品前处理方法结合GC/MS、GC/O技术用于确定国外的奶味香精、新疆发酵骆驼奶和西藏酥油中的挥发性香味化合物。结果鉴定出奶味香精中含有的香味化合物共计有70个;新疆发酵骆驼奶中的可挥发性物质总计有133个化合物,通过GC/O分析,共嗅闻出71个香味化合物;西藏酥油中的可挥发性香味物质总计有83个化合物,通过GC/O分析,共嗅闻出25个香味化合物。在鉴定了新疆发酵骆驼奶和西藏酥油中关键性奶香味化合物的结构之后,本文欲探索这些化合物的分子结构与香味之间是否有联系,具有什么分子结构的化合物对奶香味有贡献。化学结构与香味的关系是现代和当代香料化学家都十分关切的问题,但是基于嗅觉感知的复杂性和嗅质（香味物质）化学结构的多样性和复杂性,目前尚未有一种完善的理论被公认,多是实验和经验的总结。从嗅觉认知机理出发,对嗅质如何引起嗅觉的具有一定认知度的理论可概括为2种,即分子识别理论和振动理论。我们对新疆发酵骆驼奶和西藏酥油中分析检测出来的香味化合物的分子结构和气味描述进行分类,选择了16个合适的分子（分子中碳数少于6个）基于振动理论、简化的非弹性电子散射光谱（IETS）理论和预估的嗅觉传感器的能量分辨率,通过半经验量子力学计算,进行了分子立体结构的优化。在优化的立体结构中,计算奶香味分子的振动频率和耦合强度,通过正态分布函数卷积计算以后得到分子的振动谱图。并对关键性奶味香料化合物振动频率与其香味之间的关系做对比,研究发现了2个关键性奶味香料化合物的振动频率与香味之间的关系:1)在2800-3000 cm^-1振动区域,如果分子的振动频率增加,化合物分子振动频率发生蓝移,则该化合物的刺激性气味增强;2)化合物的振动谱带和耦合强度决定化合物的气味类型。更多还原

【Abstract】 With the development of China’s economy and the improvement of the people’s living standards, the demands for the flavor of the food are increasing rapidly. Milk and dairy products have gradually become a most important part of people’s diet in our country. In order to make up for the flavor losses of the food that has the obvious dairy flavor characteristics in the processing, dairy flavoring is needed; in addition, dairy flavoring is required as well to give and enrich the dairy flavor in the production of the food that has some correlation with dairy flavor and needs the aroma of dairy products, such as coffee, chocolate, candy, baked food, etc.. Therefore, dairy flavoring is a kind of product possessing market value in the research and development in the field of the food additives.Due to the rapid development of biotechnology and the increasing demands for natural quality of the food, the biotechnology of preparing dairy flavor base by enzymatic hydrolysis from the cream has been widely used in dairy flavoring processing industry abroad because of its low cost, easy storage and long shelf-life. Since 2000, the research about the biotechnology of preparing dairy flavor base by enzymatic hydrolysis from the cream has been widely carried out in China. In the process of preparing dairy flavor base by enzymatic hydrolysis from the cream, fatty acids are an important aroma component in the dairy flavor base and within a certain range, the higher degree of hydrolysis of the cream is, the higher the content of the fatty acids and the acid value will be, and as a result, the stronger the aroma will be. At present, most of the lipase used in the domestic research is imported and the price is usually very high with only about 8.0mg/g acid value after 12h hydrolysis of the cream under optimum conditions. In this study, a kind of lipase, invented and produced domestically, derived from the fermentation from Candida sp. enzyme - producing strain, was chosen to make dairy flavor base; the acid value of the enzymatic hydrolyzed cream was taken as evaluation; after the single-factor experiment of the enzymatic time （1¹0h）, enzymatic temperature （30⁵0℃）, and the enzyme dosage （500¹500U/g） respectively and then the orthogonal tests, it was decided that the enzymatic hydrolyzed cream, ie., the dairy flavor base, with the acid value of 51.90±5.74mg/g, could be obtained under the condition of 5h,40℃,1250U/g. The sensory tests proved that this dairy flavor base obtained from enzymatic hydrolysis from the cream could enhance the dairy flavor, the odor intensity the flavored sample. At present, the measurement method usually adopted to determine the acid value of the enzymatic hydrolyzed cream is acid-base titration method, which needs a large amount of organic solvents and is complicated to operate. While this study, based on the use of electronic nose FOX3000, set up a fast and practicable method to determine the acid value of the enzymatic hydrolyzed cream. Different influencing factors during the measuring process were studied through principal component analysis （PCA） and the sequence of the effecting degrees of different influencing factors was: incubation time > inject volume > sample volume > incubation temperature, and the results showed that the optimal condition was: inject volume 2500μl, incubation time 900s, incubation temperature 60℃, and sample volume 5g. In this method, the acid value linear model of the cream of different enzymatic hydrolysis degrees was analyzed, the acid value standard curve of partial least square （PLS） was built and the unknown acid value of the cream could be determined rapidly and the accuracy was 93.85%. This method was convenient, fast, accurate and it was convenient for on-line monitoring in the industrial production, and could increase the quality-monitored control efficiency in the preparing of dairy flavor base by enzymatic hydrolysis from the cream.We also found that the market-recognized dairy flavoring could not be made if we only limited the research only to the preparation of the dairy flavor base by enzymatic hydrolysis from the cream, the most important reason of which is that in the process of producing any flavoring, the adjustment of the flavoring holds the most important position, while in this process, the determination of the key dairy flavor components is the basic and crucial step.In order to know well the raw materials with dairy flavor needed to produce dairy flavoring, the volatile components of a dairy flavoring from abroad were analyzed in this study to gain the related data; more over, the volatile components and aroma-active components of two dairy products with special flavors---- Chinese Sinkiang fermented camel milk and Tibetan Yak butter ---- were analyzed. These two dairy products were chosen. Because the fermented camel milk is very popular with the nomadic tribes in the Sinkiang Uygur Autonomous Region, such as Kazakh, Mongolian and Kirgiz nationalities, and it has luscious taste, yoghourt note, weak raisin-like, fruity, sweet, and cheesy odor; while the yak butter, as one of the typical foods in Tibetan area, has a long history, strong and distinctive flavor, a luscious taste and is people’s favorite food there. Chinese Sinkiang fermented camel milk and Tibetan Yak butter are not only the dairy products with special flavors worthy of dairy flavor research, but also a kind of food of Chinese characteristics which occupy very important positions in the food consumption in Sinkiang and Tibetan areas. Therefore, the volatile aromas in Chinese Sinkiang fermented camel milk and Tibetan Yak butter were analyzed and detected, the achievement of which could not only provide important reference for the adjusting of the dairy flavoring, increase the similarity between the aroma of the dairy flavoring and the aroma of the dairy products, provide important scientific basis for the research of the key dairy flavor compounds, but also can play an important role in inheriting and developing China’s special ethnic dietetic culture. In this study, the volatile components of Chinese Sinkiang fermented camel milk and Tibetan Yak butter were isolated by simultaneous distillation extraction, solid-phase microextraction, solvent assisted flavor evaporation, the three pretreatment methods and were analyzed by GC/MS. Then, the aroma compounds were analyzed and identified by GC-O and were identified. Therefore, the characteristic flavor compounds of Chinese Sinkiang fermented camel milk and Tibetan Yak butter were identified.However, the dairy flavoring from abroad, Chinese Sinkiang fermented camel milk and Tibetan Yak butter are all complex matrix samples, the volatiles of which, existing in plenty of water, fat and protein, etc. are usually complex and some of them are heat-sensitive, easily oxidized when exposed to heat; some of the volatile aroma components are of low content and are difficult to extract, therefore, the primary experimental basis to truly and accurately reveal the complex aroma components in the samples is to choose the proper pretreatment method and optimize the pretreatment experimental condition parameters. In this study, three pretreatment methods, simultaneous distillation extraction, solid-phase microextraction and solvent assisted flavor evaporation were adopted, the mimic sample composed of the dairy flavor compounds of known formulation was taken as the testing materials, the sample extraction yield was taken as evaluation and the main influence factors of each pretreatment method were optimized. These methods include: 1) simultaneous distillation extraction: Two main influence factors, extraction solvents （dichloromethane, ether, pentane/dichloromethane, pentane/ether） and extraction time （1⁶h）, were investigated; together with GC/MS, quantified by internal standard, the extraction yield of the dairy flavor compounds was taken as evaluation, and the optimum condition for extracting the dairy flavor compounds in this method was obtained: dichloromethane as the extraction solvent, 3h as the extraction time. 2) solid-phase microextraction: as the similar design and operation process above, the four main influence factors, the incubation temperature （30～80℃）, the incubation time （10～180min）, the sample volume （1～11mL） and the fiber type （65μm CWAX/DVB, 65μm PDMS/DVB, 75μm CAR/PDMS and 50/30μm CAR/PDMS/DVB） were investigated. The result showed that PDMS/DVB fiber had the greater sensitivity to a more diverse range of volatile compounds followed by CAR/PDMS/DVB. The optimum condition for extracting the dairy flavor compounds in this method was: incubation temperature 50℃, the incubation time 40min, the sample volume 5mL; 3) solvent assisted flavor evaporation: The mimic sample composed of the dairy flavor compounds of known formulation was taken as the testing materials, solvent assisted flavor evaporation was adopted, coupled with GC/MS, the differences between the quantitative results and the real composition of the sample were compared, and the extraction yield of different dairy flavor compounds were analyzed. This study on the pretreatment methods gained a lot of and systematic primary data, which had great reference value in the determination of the volatile components in the dairy products and the analysis of the aroma components, and an instructive importance in choosing the pretreatment method of the complex matrix samples. Then, the three optimized pretreatment methods, together with GC/MS and GC/O were adopted to determine the volatile aroma compounds in the dairy flavoring from abroad, Chinese Sinkiang fermented camel milk and Tibetan Yak butter. A total of 70 dairy flavor compounds were identified in the dairy flavoring from abroad; all together 133 volatile compounds were identified in the Sinkiang fermented camel milk and 71 aroma compounds were detected by GC/O; in the Tibetan Yak butter, 83 volatile compounds were identified and 25 aroma compounds were detected by GC/O.After the identification of the structures of the key dairy flavor compounds in Sinkiang fermented camel milk and Tibetan Yak butter, this study tried to make it clear whether there was any relationship between the molecular structure and odor, and with what kind of molecular structure could the compound contribute to the dairy flavor. The relationship between the molecular structure and odor is what the modern and contemporary flavor chemists much cared about. However, based on the complex of the olfactory perception and the diversity and complex of the molecular structure of the odorants, nowadays, there is not a perfect theory that has gained the universal recognition, most of which are experiment and experience summaries. Two theories with certain recognition are summarized, molecular recognition theory and the molecular vibration theory. The molecular structures of the aroma compounds detected in the Sinkiang fermented camel milk and the Tibetan Yak butter and the descriptions of the aroma were classified and 16 proper molecules （Carbon number less than 6 in a molecule） were selected. Based on the vibration theory, simplified inelastic electron tunnelling spectroscopy （IETS） theory and pre-assessed energy resolution of the olfactory sensor, through the semi-empirical quantum mechanical calculations, spatial molecular structures were optimized. In the optimized spatial molecular structures, the vibration frequency and the coupling strength were calculated and the molecular vibration spectrum was obtained through the convolution calculation of the normal distribution function. At last, the relationships between the molecular vibration spectrum of the key dairy flavor compounds and their aroma were compared and the relationships between the structural characteristics of 2 key dairy flavor compounds and their aroma were discovered: 1) in the range of 2800-3000cm^-1, if the molecular vibration frequency has a blue–shift, then the pungent odor of the molecules increased; 2) The charactor of molecule’s flavor is determined by it’s vibrational bands and the intensity of the vibration.更多还原