Study On The Materials Of Natural Milk Flavor And Milk Flavoring By Enzymes

For their high nutrition value and excellent flavor, dairy products have been more and more popular and taken a great proportion of food manufacture. Therefore, the dairy flavoring has been one of most important flavorings in flavoring and fragrance manufacture. Recently in China the research of enzymatic milk flavoring, which could produce more complicated and ripe flavor and greatly imitate the natural flavor of the aim dairy samples, have gradually earn more attentions. Moreover, the enzymatic production of milk protein of cream hydrolyzed by flavor protease could not only dramatically increase the application ratio of material, but also noticeably increase its kokumi flavor.Cream produced from New Zealand which respectively hydrolyzed by lipase and protease was used as raw material. The major volatile compounds and taste composition in lipoyzed production were identified. In addition the structure of kokumi taste-active peptides of hydrolyzed milk protein flavor was identified, and the taste features of these peptides were also studied by sensory analysis and electronic tongue. The main contents of this paper were as following.Olive oil and cream were individually conducted as substrates to determine the lipase activity. Based the result of these lipase activities, the optimal lipase was selected from seven kinds of commercial lipases. Then the single factor test and response surface analysis were used to confirm the optimum condition of lipolyzed reactions. Results showed that the optimum condition was reaction temperature 48 oC, starting pH 8.3, the dose of lipase was 2000U/g substrate and the substrate concentration was 92% (w/v). And on the basis of descriptive quantitative analysis and hedonic scale tests, the optimum reaction time of lipolyzed production having the best sensory quality was 4 hours.The volatile compounds of lipolyzed production having different reaction time were analyzed by solid-phase micro-extraction (SPME) method, and 118 volatile compounds were identified. The hydrolyzed production with 4 hours reaction time have the most of short and medium chain fatty acids, methyl ketones, esters and lactones, which validate the result of sensory tests. Besides that the solvent extracting method was also conducted to analyze the volatiles of lipolyzed production during 4 hours, and 55 volatiles were determined. Most of them were long chain fatty acids, which may have little contribution to the aroma flavor of lipolyzed production but for its taste flavor.The SPME gas chromatograph olfactometry (SPME-GC-O) method and the odor unit value (OUV) analysis method were applied to identify major characteristic volatile compounds of hydrolyzed production having best sensory characteristic. The result of SPME-GC-O indicate the most important volatiles are as following: ethyl butyrate, 2-heptanone, limonene, ethyl caproate, octanal, ethyl caprylate, acetic acid, butanoic acid, hexanoic acid, octanoic acid, decanoic acid, ethyl decanoate,δ-hexalactone andδ-octalactone. While the result of OUV have a little different with the result of SPME-GC-O, the order of these main volatiles were arranged with their flavor contribution as following, hexanoic acid, butanoic acid, ethyl butyrate, 2-pentadecanone, undecanoic acid,δ-octalactone, decanoic acid,δ-dodecalactone, ethyl caproate and octanoic acid.For the purpose of getting the self-made natural milk flavoring which could be directly applied in foods, the SPME-GC-MS analysis was selected to analyze different samples, such as cream, three kinds of commercial natural milk flavorings and commercial synthetic milk flavoring. Based on the result of SPME-GC-MS the flavor treating process was conducted. The electronic nose and its relating statistical analysis were used to evaluate the similarities of different samples, such as self-made natural milk flavoring, different commercial natural milk flavorings, cheeses and whole fat milk powders. The results of electronic nose indicate that the aroma flavor of self-made natural milk flavoring is similar with the three commercial natural milk flavorings which were clustered on the PCA figure. And all of these nature flavorings were having the similar flavor with the Cheddar cheese. Moreover, aiming at better assessing the flavor characteristic of self-made natural milk flavoring, the self-made natural milk flavoring and other commercial natural milk flavorings were added in skim milk and bread. The sensory statistic results show that the sample added with self-made natural milk flavoring didn't have the significant difference with the sample added with natural milk flavoring, but have significant difference with the sample added with synthetic milk flavoring.The single factor test and response surface analysis was used to hydrolyze the milk protein separated form cream to get the kokumi composition. The statistic results showed that the optimum conditions were the dose of Flavorzyme 3.5% (w/w) and Neutrase 2.25% (w/w), reaction temperature 45 oC, starting pH 8.0, the substrate content was 2.5% (w/v), and the reaction time was 7.6 hours. Moreover, the rough peptide content of hydrolyzed product was 52.7%, and the hydrolysate had the highest scores in the taste of sensory evaluation without any bitterness.Based on the sensory characteristic of 6 gel permeation chromatography (GPC) fractions (named P1, P2, P3, P4, P5 and P6) separated from the milk protein hydrolyzed compounds, there are three GPC fractions (P3, P4, P5) have clear"kokumi"flavor compared with the skimmed fresh milk. Moreover, according to the sensory thresholds and electronic tongue analysis, P4 fraction was identified as the composition which has the strongest kokumi flavor. Then the P4 fraction was further separated by semi-preparation reverse phase high performance liquid chromatography (RP-HPLC). The identified results of the matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) on P4 fraction separated by RP-HPLC show that there are 11 peptides existing in the P4 fraction, most of which have 8 ~ 9 amino acids and originated fromβ-casein. And most of their amino acid sequences have Glu and Cys which were identical with the kokumi peptide researches of other people. Moreover, on the basis of their sequence characteristic and Q value analysis, 8 peptides were selected and synthesized. Then the taste characteristics of these synthesized peptides were evaluated by the sensory analysis and electronic tongue. Finally one of these peptides with the sequence of Val-Pro-Asn-Ser-Ala-Glu- Glu-Arg has the greatest kokumi flavor, and the sensory result also shows that kokumi flavor would have the synergism with the bitterness and astringent flavor.