Study On The Preparation Of Dairy Flavor Base By Enzymatic Hydrolysis From The Cream And The Analysis Of Aroma Components In The Dairy Products

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.