1.Laboratory of Molecular Sensory Science,Beijing Technology and Business University;2.Hunan Province Jiapinjiawei Biotechnology Co,Ltd
为研究制备热反应牛骨香精的较佳酶解工艺，以牛骨素为原料，选用复合酶，利用单因素和响应面试验考察酶解温度、酶解时间、复合酶加酶量及复合酶加酶比例4因素对酶解液水解度和由酶解液与糖、氨基酸经美拉德反应制备的热反应牛骨香精的感官评分的影响，并利用高效液相色谱法测定游离氨基酸质量比、利用全二维气相色谱-嗅闻-质谱技术分析牛骨素酶解前后制备的热反应牛骨香精关键气味化合物的质量比。结果表明，牛骨素的较佳酶解工艺为酶解温度50℃、酶解时间4 h、加酶量1250 U/g、复合风味蛋白酶与复合蛋白酶的加酶比例为1: 1，在此条件下感官评分为8.24。与牛骨素相比，酶解液中有15种游离氨基酸的质量比显著增加；使用电子鼻和电子舌测定牛骨素和酶解液的气味轮廓和滋味轮廓，发现酶解液的氮氧化物气味特征增强，酶解液的鲜味、浓厚味、咸味特征更明显。使用固相微萃取结合全二维气相色谱-嗅闻-质谱技术，对牛骨素、酶解液分别制备的热反应牛骨香精的关键气味化合物进行分析，共鉴定出挥发性气味化合物164种，包含27种醛类、30种酮类、12种醇类、16种酸类、7种含硫化合物、10种酯类、18种吡嗪类、10种呋喃类、6种噻唑类、4种噻吩类和24种其它类化合物，通过气相-嗅闻（gas chromatography-olfactometry, GC-O）分析，嗅闻到74种气味活性化合物；香气活性值（odor activity value, OAV）大于1的关键气味化合物有22种，牛骨素与酶解液分别制备的热反应牛骨香精中最重要的关键气味化合物均为双(2-甲基-3-呋喃基)二硫醚（OAV值分别为13078和8052）；与牛骨素制备的热反应牛骨香精相比，酶解液制备的热反应牛骨香精中出现了(E,E)-2,4-癸二烯醛、2-甲基-3-呋喃硫醇、甲基糠基二硫醚、己酸甲硫醇酯、β-石竹烯5种新的关键气味化合物。研究旨在为热反应牛骨香精工业化生产中的酶解工艺提供理论依据。
In order to study the optimal enzymatic hydrolysis process for preparing thermal reaction bovine bone flavoring, bovine bone extract (BBE) was used as raw material, and compound enzyme was selected. The effects of enzymatic hydrolysis temperature, enzymatic hydrolysis time, compound enzyme addition amount and compound enzyme addition ratio on the degree of hydrolysis of enzymatic hydrolysate and the sensory score of thermal reaction bovine bone flavoring prepared by Maillard reaction of enzymatic hydrolysate with sugar and amino acid were investigated by single factor and response surface test. The mass ratio of free amino acid was determined by high performance liquid chromatography (HPLC), and the mass ratio of key odor compounds of thermal reaction bovine bone flavoring prepared before and after enzymatic hydrolysis of BBE was analyzed by comprehensive two-dimensional gas chromatography-olfactometry-mass spectrometry (GC×GC-O-MS). The results showed that the better enzymatic hydrolysis process of BBE was enzymatic hydrolysis temperature of 50°C, enzymatic hydrolysis time of 4 h, enzyme addition amount of 1250 U/g, and the ratio of compound flavor protease to compound protease was 1: 1. Under this condition, the sensory score was 8.24. Compared with BBE, the mass ratio of 15 free amino acids in the enzymatic hydrolysate increased significantly. The odor profile and taste profile of BBE and enzymatic hydrolysate were measured by electronic nose and electronic tongue. It was found that the nitrogen oxide odor characteristics of enzymatic hydrolysate were enhanced, and the umami, richness and salty taste characteristics of enzymatic hydrolysate were more obvious. The key odor compounds of thermal reaction bovine bone flavoring prepared by BBE and enzymatic hydrolysate were analyzed by solid phase microextraction (SPME) combined with GC×GC-O-MS. A total of 164 volatile odor compounds were identified, including 27 aldehydes, 30 ketones, 12 alcohols, 16 acids, 7 sulfur-containing compounds, 10 esters, 18 pyrazines, 10 furans, 6 thiazoles, 4 thiophenes and 24 other compounds. Through gas chromatography-olfactometry (GC-O) analysis, 74 odor-active compounds were detected. There were 22 key odor compounds with odor activity value (OAV) greater than 1. The most important key odor compounds in the thermal reaction bovine bone flavoring prepared by BBE and enzymatic hydrolysate were bis(2-methyl-3-furanyl) disulfide (OAV was 13078 and 8052, respectively). Compared with the thermal reaction bovine bone flavoring prepared by BBE, 5 new key odor compounds (E,E)-2,4-decadienal, 2-methyl-3-furanthiol, methyl furfuryl disulfide, S-methyl thiohexanoate and β-caryophyllene appeared in the thermal reaction bovine bone flavoring prepared by enzymatic hydrolysate. The purpose of this study was to provide a theoretical basis for the enzymatic hydrolysis process in the industrial production of thermal reaction bovine bone flavoring.