酶法制备天然牛奶风味基料及牛奶香精的研究
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摘要
乳和乳制品在食品行业中占据重要地位,其不仅有较高的营养价值,口感和香气亦令人十分喜爱,因而乳品香精是香精香料领域中投资最大,开发研究最为活跃的香精之一。利用酶法生产奶类香精,不仅能够产生更加复杂的风味体系,模拟目标产品的天然风味,还能够通过对不同来源脂肪酶的水解特性进行选择,强化酶解产物的某种特征风味,从而使得到的成品香精香气柔和留香持久。此外利用复合风味蛋白酶对乳蛋白进行适当酶解不但可以提高原料利用率,还能显著增强酶解产物中的浓厚感滋味,从而使香精品质得到进一步提高。因此酶技术在乳品增香的研究应用领域中有着非常广阔的前景。
本论文以奶油为原料,分别利用脂肪酶和复合风味蛋白酶对其进行水解,并对脂肪酶酶解产物中特征挥发性风味物质进行鉴定;同时对复合蛋白酶酶解产物中呈浓厚感的呈味肽进行分离纯化和结构鉴定,并对其呈味特征进行进一步研究。主要研究内容如下:
分别采用橄榄油和奶油为底物测定脂肪酶酶活,对七种不同商品脂肪酶进行筛选,最终选择脂肪酶Palatase 20,000L作为酶解奶油增香反应用酶。由单因素试验结合响应面分析确定脂肪酶酶解奶油的最佳工艺条件为:底物浓度为92%(w/v),加酶量为2000U/g底物,温度48℃,pH8.3。同时根据不同酶解时间下酶解产物的风味剖面特征分析和偏好性感官评定试验结果,确定获得最佳感官品质奶油酶解产物的酶解时间为4小时。
利用SPME-GC-MS法对酶解不同时间的酶解产物中主要挥发性风味组分进行分析,共检出118种挥发性风味组分,其中酶解时间为4小时的酶解产物中短碳链的脂肪酸类、甲基酮类、酯类和内酯类化合物含量达到最大,因而其感官品质最佳。同时采用SPME-GC-MS法从酶解4小时的酶解产物中检出55种风味物质,主要是一些长链脂肪酸类化合物,此类化合物对酶解产物挥发性风味贡献较小,主要是呈味方面的贡献。
分别采用SPME-GC-O法和风味值分析法对具有最佳感官品质的酶解产物中的主要特征挥发性风味组分进行分析和鉴定,其中SPME-GC-O法鉴定得到丁酸乙酯、2-庚酮、柠檬烯、己酸乙酯、辛醛、辛酸乙酯、乙酸、丁酸、癸酸乙酯、δ-己内酯、己酸、δ-辛内酯、辛酸、癸酸等14种化合物为酶解产物中的特征风味组分;而风味值法分析得到的特征风味组分与SPME-GC-O法分析得到的化合物略有差别,按其风味贡献大小依次为:己酸、丁酸、丁酸乙酯、2-十五酮、十一酸、δ-辛内酯、癸酸、δ-十二内酯、己酸乙酯和辛酸等。
为了得到香气更加柔和的天然牛奶香精,采用SPME-GC-MS法对稀奶油、商品天然牛奶香精样品和合成牛奶香精样品中的挥发性风味物质进行分析,然后根据分析结果将酶解产物进行适当风味修饰得到自制天然牛奶香精。同时利用电子鼻对不同香精样品、不同品种类的奶酪样品和全脂奶粉样品中的挥发性风味组分进行分析,主成分分析结果表明三种商品天然牛奶香精和自制天然牛奶香精的风味最为接近,并且其均与奶酪样品中切达奶酪的风味最为相似。此外为了更好的对自制天然牛奶香精品质进行评定,将自制天然牛奶香精和其它市售牛奶香精一起分别加入复原乳和主食面包中,进行相应的感官评定试验。感官评分结果表明添加自制天然牛奶酶解香精与商品天然牛奶香精的样品同添加合成牛奶香精的样品感官品质有显著差异,而添加自制天然牛奶香精的面包的感官偏好性要高于添加有商品天然牛奶香精和黄油的面包样品。
选择复合风味蛋白酶酶解奶油中的乳蛋白生成呈浓厚味的滋味组分,采用单因素试验结合响应面优化试验,得到复合酶水解乳蛋白生成呈味肽的最佳工艺条件为:F酶的添加量为3.5% (w/w),N酶的添加量为2.25% (w/w),底物浓度为2.5%(w/v),温度45℃,初始pH8.0,反应时间为7.6h。反应生成肽百分含量为52.7%,并且水解产物感官评价浓厚感滋味浓郁,无苦味。
使用Sephadex G-15凝胶柱对乳蛋白酶解产物水提物进行分离纯化,得到6个分离峰组分(P1、P2、P3、P4、P5、P6),分别对其进行感官评定,得到三个浓厚感较强的分离峰组分(P3、P4、P5),并分别对其进行阈值测定和电子舌分析,确定浓厚感强度最高的组分为P4。采用RP-HPLC对P4组分进行进一步分离纯化,然后利用MALDI-TOF-MS对其中主要呈味肽组分的氨基酸序列进行鉴定,鉴定得到11种可能呈浓厚感的肽的一级结构,其中大部分肽链来自于β-酪蛋白的酶解产物,并且鉴定得到肽链序列中Glu和Cys的含量较高。然后根据鉴定得到肽的氨基酸序列的呈味特点和Q值分析,选择了其中可能呈浓厚感滋味的8种肽进行合成,并对这8种呈味肽通过感官评定进行阈值分析,得到序列为VPNSAEER的呈味肽阈值较低,浓厚感最强。此外根据评价员对这8种呈味肽的感官描述得到浓厚味与适当强度的苦味或涩味之间具有协同关系。最后对合成得到的呈味肽通过感官评定和电子舌分析,进一步对其呈味特点进行进一步确定。
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.
本论文以奶油为原料,分别利用脂肪酶和复合风味蛋白酶对其进行水解,并对脂肪酶酶解产物中特征挥发性风味物质进行鉴定;同时对复合蛋白酶酶解产物中呈浓厚感的呈味肽进行分离纯化和结构鉴定,并对其呈味特征进行进一步研究。主要研究内容如下:
分别采用橄榄油和奶油为底物测定脂肪酶酶活,对七种不同商品脂肪酶进行筛选,最终选择脂肪酶Palatase 20,000L作为酶解奶油增香反应用酶。由单因素试验结合响应面分析确定脂肪酶酶解奶油的最佳工艺条件为:底物浓度为92%(w/v),加酶量为2000U/g底物,温度48℃,pH8.3。同时根据不同酶解时间下酶解产物的风味剖面特征分析和偏好性感官评定试验结果,确定获得最佳感官品质奶油酶解产物的酶解时间为4小时。
利用SPME-GC-MS法对酶解不同时间的酶解产物中主要挥发性风味组分进行分析,共检出118种挥发性风味组分,其中酶解时间为4小时的酶解产物中短碳链的脂肪酸类、甲基酮类、酯类和内酯类化合物含量达到最大,因而其感官品质最佳。同时采用SPME-GC-MS法从酶解4小时的酶解产物中检出55种风味物质,主要是一些长链脂肪酸类化合物,此类化合物对酶解产物挥发性风味贡献较小,主要是呈味方面的贡献。
分别采用SPME-GC-O法和风味值分析法对具有最佳感官品质的酶解产物中的主要特征挥发性风味组分进行分析和鉴定,其中SPME-GC-O法鉴定得到丁酸乙酯、2-庚酮、柠檬烯、己酸乙酯、辛醛、辛酸乙酯、乙酸、丁酸、癸酸乙酯、δ-己内酯、己酸、δ-辛内酯、辛酸、癸酸等14种化合物为酶解产物中的特征风味组分;而风味值法分析得到的特征风味组分与SPME-GC-O法分析得到的化合物略有差别,按其风味贡献大小依次为:己酸、丁酸、丁酸乙酯、2-十五酮、十一酸、δ-辛内酯、癸酸、δ-十二内酯、己酸乙酯和辛酸等。
为了得到香气更加柔和的天然牛奶香精,采用SPME-GC-MS法对稀奶油、商品天然牛奶香精样品和合成牛奶香精样品中的挥发性风味物质进行分析,然后根据分析结果将酶解产物进行适当风味修饰得到自制天然牛奶香精。同时利用电子鼻对不同香精样品、不同品种类的奶酪样品和全脂奶粉样品中的挥发性风味组分进行分析,主成分分析结果表明三种商品天然牛奶香精和自制天然牛奶香精的风味最为接近,并且其均与奶酪样品中切达奶酪的风味最为相似。此外为了更好的对自制天然牛奶香精品质进行评定,将自制天然牛奶香精和其它市售牛奶香精一起分别加入复原乳和主食面包中,进行相应的感官评定试验。感官评分结果表明添加自制天然牛奶酶解香精与商品天然牛奶香精的样品同添加合成牛奶香精的样品感官品质有显著差异,而添加自制天然牛奶香精的面包的感官偏好性要高于添加有商品天然牛奶香精和黄油的面包样品。
选择复合风味蛋白酶酶解奶油中的乳蛋白生成呈浓厚味的滋味组分,采用单因素试验结合响应面优化试验,得到复合酶水解乳蛋白生成呈味肽的最佳工艺条件为:F酶的添加量为3.5% (w/w),N酶的添加量为2.25% (w/w),底物浓度为2.5%(w/v),温度45℃,初始pH8.0,反应时间为7.6h。反应生成肽百分含量为52.7%,并且水解产物感官评价浓厚感滋味浓郁,无苦味。
使用Sephadex G-15凝胶柱对乳蛋白酶解产物水提物进行分离纯化,得到6个分离峰组分(P1、P2、P3、P4、P5、P6),分别对其进行感官评定,得到三个浓厚感较强的分离峰组分(P3、P4、P5),并分别对其进行阈值测定和电子舌分析,确定浓厚感强度最高的组分为P4。采用RP-HPLC对P4组分进行进一步分离纯化,然后利用MALDI-TOF-MS对其中主要呈味肽组分的氨基酸序列进行鉴定,鉴定得到11种可能呈浓厚感的肽的一级结构,其中大部分肽链来自于β-酪蛋白的酶解产物,并且鉴定得到肽链序列中Glu和Cys的含量较高。然后根据鉴定得到肽的氨基酸序列的呈味特点和Q值分析,选择了其中可能呈浓厚感滋味的8种肽进行合成,并对这8种呈味肽通过感官评定进行阈值分析,得到序列为VPNSAEER的呈味肽阈值较低,浓厚感最强。此外根据评价员对这8种呈味肽的感官描述得到浓厚味与适当强度的苦味或涩味之间具有协同关系。最后对合成得到的呈味肽通过感官评定和电子舌分析,进一步对其呈味特点进行进一步确定。
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.
引文
1.齐军茹,苏景如,彭志英.生物技术在食品风味物质生产中的应用[J].食品科技, 2002, 8: 9-11.
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