响应面法优化鸡肉蛋白酶解工艺

于亚辉; 陈沁雯; 李晓婷; 方婷

doi:10.19303/j.issn.1008-0384.2020.01.012

响应面法优化鸡肉蛋白酶解工艺

doi: 10.19303/j.issn.1008-0384.2020.01.012

福建农林大学食品科学学院，福建　福州　350002

基金项目: 国家自然科学基金项目(31401597、31601393)

详细信息

作者简介:
于亚辉（1993−），男，硕士，研究方向：食品风味化学（E-mail：2951588827@qq.com）

通讯作者:
方婷（1981−），女，博士，教授，研究方向：食品加工理论与应用（fangting930@163.com）

中图分类号: TS 254.1
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出版历程
- 收稿日期: 2019-09-22
- 修回日期: 2019-10-25
- 刊出日期: 2020-01-01

Optimized Proteolysis of Chicken Protein for Food Flavoring

College of Food Science, Fujian Agriculture and Forestry University, Fuzhou, Fujian　350002, China

摘要

摘要: 目的采用双酶分步酶解方法优化鸡肉蛋白酶解工艺，为制备品质较佳的鸡肉香精提供技术参考。方法以动物蛋白酶与复合蛋白酶为目标酶，游离氨基酸、多肽含量为评价指标，在单因素试验的基础上，采用Plackett-Burman设计筛选出重要影响因素。接着采用最陡爬坡试验逼近最大响应区域，确定响应面优化试验中心点，最后选取游离氨基酸、多肽含量以及热反应产物风味评分3个指标为评价依据，采用Box-Behnken响应面优化法，探讨鸡肉蛋白双酶分步酶解工艺及其对制备鸡肉香精风味的影响，确定重要参数的最佳水平。结果总加酶量、总时长、固液比为主要影响因素，通过响应面及岭脊分析确定参数值分别为0.82%、7.26 h、1 ∶ 4.40，预测综合得分为89.41，实际得分88.97，模型可靠。结论通过多指标响应面法优化了鸡肉蛋白酶解条件，提高酶解效果，并制备出风味较佳的鸡肉香精。
- 双酶 /
- 分步酶解工艺 /
- Plackett-Burman设计 /
- 响应面优化
Abstract: Objective The stepwise enzymatic hydrolysis was applied to hydrolyze chicken protein for the preparation of a food flavoring ingredient with improved quality. Method An animal and a compound proteolytic enzyme were used to digest the chicken protein. Free amino acid and peptide contents in the hydrolyzed material were employed as the criteria to initially identify the critical process evaluation parameters based on a single factor Plackett-Burman design. A steepest ascent test was conducted to approximate the maximum response area and arrive at the center point in a response surface experiment. Then, according to the selected indicators for the Box-Behnken experiment, optimized process conditions were determined. Result The selected indicators for the Box-Behnken optimization included the free amino acid and polypeptide contents as well as the flavor scores on the thermal reaction products of the hydrolyzed compounds. The optimal processing conditions were found to include the addition of 0.82% enzymes on the chicken protein substrate, a solid-liquid ratio of 1 ∶ 4.40, and a reaction duration of 7.26 h to achieve an actual composite evaluation score of 88.97 in comparison to the predicted 89.41. The method was deemed reliable. Conclusion The multi-indicator response surface method effectively optimized the enzymatic proteolysis process of chicken protein materials in producing a high quality flavoring ingredient for food applications.
- enzymatic proteolysis /
- stepwise hydrolysis /
- Plackett-Burman design /
- response surface optimization

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图 1 单因素试验结果

Figure 1. Single factor test results

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图 2 总加酶量、总时长和固液比交互作用对游离氨基酸浓度及多肽浓度影响的响应面和等高线

Figure 2. Response surface and contour lines on interactions among enzyme applied/process duration/solid-liquid ratio for process and free amino acid/peptide concentrations of hydrolysate

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表 1 鸡肉风味基料评价标准

Table 1. Evaluation standards on chicken flavor

评价项目 Evaluation project	满分标准 Full score standard	评价分数范围 Evaluation score range
香味 Fragrance	香气浓郁、饱满、圆润与炖煮鸡肉香气较为一致 The aroma is rich, full, round and the chicken aroma is more consistent	0～30
色泽 Color	红褐色、色泽均匀无明显异色 Reddish brown, uniform color, no obvious color	0～30
滋味 Taste	口感柔和具有鸡肉滋味、无苦味等不适感 The palate is soft and has a taste of chicken and no bitterness.	0～20
异味 Odor	无异味、焦糊味 No odor, burnt smell	0～20

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表 2 单因素试验设计

Table 2. Single factor test design

因素 Factor	处理组 Group
因素 Factor	1	2	3	4	5
总加酶量 Total enzyme amount /%	0.2	0.4	0.6	0.8	1.0
加酶比 Enzyme ratio	1∶1	1∶2	2∶1	1∶3	3∶1
固液比 Solid-liquid ratio	1∶1	1∶2	1∶3	1∶4	1∶5
总时长 Total length/h	4	5	6	7	8
时长比 Duration ratio	1∶1	1∶2	2∶1	2∶3	3∶2
NaCl添加量 NaCl addition amount/%	0.4	0.6	0.8	1.0	1.2

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表 3 Plackett-Burman设计试验参数和水平

Table 3. Factors and levels of PIackett-Burman design

编号 Code	变量 Variable	水平（−1） Level （−1）	水平（1） Level (1)
A	总加酶量 Total enzyme amount /%	0.4	0.8
B	加酶比 Enzyme ratio	1∶1	3∶1
C	总时长 Total length/h	5	7
D	时长比 Duration ratio	3∶2	2∶1
E	固液比 Solid-liquid ratio	1∶2	1∶4
F	NaCl添加量 NaCl addition amount/%	0.6	1.0

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表 4 Plackett-Burman试验设计及结果

Table 4. Plackett-Burman design and experimental results

试验号 Test number	A	B	C	D	E	F	氨基酸含量 Amino acid content /(mg·g⁻¹)	多肽含量 Peptide content/ (mg·hg⁻¹)	综合评分 Comprehensive score (Y)
1	1	1	−1	1	1	1	337.73	278.67	103.46
2	1	−1	−1	−1	1	−1	320.65	266.78	98.65
3	−1	−1	1	−1	1	1	257.71	225.08	81.33
4	−1	1	1	−1	1	1	265.43	235.96	84.56
5	−1	−1	−1	1	−1	1	222.03	168.44	65.19
6	1	−1	1	1	1	−1	342.45	280.19	104.45
7	−1	1	1	1	−1	−1	234.04	185.24	70.19
8	−1	1	−1	1	1	−1	247.69	215.66	78.04
9	1	1	1	−1	−1	−1	287.58	248.37	90.22
10	1	1	−1	−1	−1	1	263.58	221.64	81.54
11	1	−1	1	1	−1	1	300.36	245.71	91.6
12	−1	−1	−1	−1	−1	−1	207.75	156.49	60.78

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表 5 Plackett-Burman试验显著性因子选择模型方差分析

Table 5. ANOVA of Plackett-Burman test on selected factorial model

变异来源 Source of variation	自由度 Degree of freedom	效应 Effect	F值 F value	P值 P value	显著性 Significance
总模型 Total model	6	84.17	95.15	＜0.000 1	**
A	1	10.82	361.09	＜0.000 1	**
B	1	0.50	0.77	0.419 3
C	1	2.89	25.78	0.003 8	**
D	1	1.32	5.38	0.068 1
E	1	7.58	177.28	＜0.000 1	**
F	1	0.45	0.61	0.469 0
注：：P＜0.05显著；：P＜0.01极显著。 Note: : P＜0.05 significant; **: P＜0.01 extremely significant.

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表 6 最陡爬坡试验设计及结果

Table 6. Design and results of steepest ascent experiment

试验号 Test number	A/%	C/h	E	氨基酸含量 Amino acid content /(mg·g⁻¹)	多肽含量 Peptide content/ (mg·hg⁻¹)	综合评 Comprehensive score (Y)
1	0.2	4	1∶1	129.48	87.31	34.49
2	0.4	5	1∶2	223.59	168.63	62.74
3	0.6	6	1∶3	326.27	261.65	94.33
4	0.8	7	1∶4	360.43	301.25	106.38
5	1	8	1∶5	351.96	286.77	102.56

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表 7 Box-Behnken Design试验因素与水平

Table 7. Levels and factors of Box-Behnken experiment

编号 Code	因素 Factor	水平 Level
编号 Code	因素 Factor	−1	0	1
A	总加酶量 Total enzyme amount /%	0.6	0.8	1
B	总时长 Total length / h	6	7	8
C	固液比 Solid-liquid ratio	1∶3	1∶4	1∶5

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表 8 Box-Behnken Design试验设计及结果

Table 8. Box-Behnken design and experimental results

序号 Serial number	A	B	C	氨基酸含量 Amino acid content/(mg·g⁻¹)	多肽含量 Peptide content/(mg·hg⁻¹)	感官评分 Sensory score (P)	综合评分 Comprehensive score (Y)
1	−1	0	−1	321.79	264.82	46.33	67.46
2	0	0	0	351.96	281.77	83.15	89.37
3	0	1	1	358.28	286.36	69.53	83.38
4	−1	0	1	320.54	260.88	70.65	79.21
5	−1	1	0	333.88	265.63	56.38	73.39
6	0	0	0	352.55	280.92	78.18	86.86
7	0	0	0	354.02	281.96	79.65	87.78
8	0	−1	1	340.34	270.52	68.47	80.29
9	0	0	0	353.88	283.03	80.25	88.16
10	−1	−1	0	350.08	275.15	44.87	69.55
11	0	0	0	351.64	283.77	81.37	88.62
12	1	−1	0	355.25	288.32	43.77	70.45
13	1	0	1	356.28	290.63	61.28	79.47
14	0	−1	−1	344.65	265.58	50.33	71.11
15	1	1	0	365.99	292.06	62.43	80.84
16	0	1	−1	355.29	282.05	64.67	80.39
17	1	0	−1	345.37	270.93	55.05	73.96

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表 9 响应面回归模型方差分析

Table 9. ANOVA on response surface regression model

方差来源 Source of variance	平方和 Sum of square	自由度 Degree of freedom	均方 Mean square	F值 F value	P值 P value	显著性 Significance
总模型 Total model	853.19	9	94.80	88.54	＜0.000 1	**
A	28.55	1	28.55	26.66	0.001 3	**
B	88.38	1	88.38	82.54	＜0.000 1	**
C	108.26	1	108.26	101.11	＜0.000 1	**
AB	10.71	1	10.71	10.00	0.015 9	*
AC	9.73	1	9.73	9.09	0.019 5	*
BC	9.57	1	9.57	8.93	0.020 3	*
A²	355.03	1	355.03	331.58	＜0.000 1	**
B²	123.55	1	123.55	115.39	＜0.000 1	**
C²	65.67	1	65.67	61.34	0.000 1	**
残差 Residual	7.49	7	1.07
失拟项 Missing item	3.97	3	1.32	1.50	0.342 8
绝对误差 Absolute error	3.53	4	0.88
总和 Sum	860.68	16
相关系数 Correlation coefficient	0.991 3
校正决定系数 Correction decision coefficient	0.980 1
变异系数 Coefficient of variation	1.30
信噪比 Signal to noise ratio	25.528＞4
注：：P＜0.05显著；：P＜0.01极显著。 Note: : P＜0.05 significant; **: P＜0.01 extremely significant.

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参考文献(21)

[1]	孙颖, 张莉莉, 张玉玉, 等. 3种酶解液中游离氨基酸的分析 [J]. 精细化工, 2018, 35(6):1004−1008, 1014. SUN Y, ZHANG L L, ZHANG Y Y, et al. Analysis of free amino acids in three kinds of enzymatic hydrolysates [J]. Fine Chemicals, 2018, 35(6): 1004−1008, 1014.(in Chinese)
[2]	何蓉蓉, 李怡芳, 李维熙, 等. 鸡汤健康功能的研究现状 [J]. 食品与生物技术学报, 2012, 31(1):17−27. doi: 10.3969/j.issn.1673-1689.2012.01.003 HE R R, LI Y F, LI W X, et al. Review on health functions of chicken broths [J]. Journal of Food Science and Biotechnology, 2012, 31(1): 17−27.(in Chinese) doi: 10.3969/j.issn.1673-1689.2012.01.003
[3]	ZHUANG M Z, LIN L Z, ZHAO M M, et al. Sequence, taste and umami-enhancing effect of the peptides separated from soy sauce [J]. Food Chemistry, 2016, 206: 174−181. doi: 10.1016/j.foodchem.2016.03.058
[4]	SONG H L, XIA L J. Aroma extract dilution analysis of a beef flavouring prepared from flavour precursors and enzymatically hydrolysed beef [J]. Flavour and Fragrance Journal, 2008, 23(3): 185−193. doi: 10.1002/ffj.1873
[5]	陈海涛, 徐晓兰, 张宁, 等. 鸡肉酶解工艺对热反应鸡肉香精香气的影响 [J]. 食品科学, 2013, 34(9):150−154. doi: 10.7506/spkx1002-6630-201309031 CHEN H T, XU X L, ZHANG N, et al. Optimal chicken enzymatic hydrolysate for the preparation of chicken flavoring [J]. Food Science, 2013, 34(9): 150−154.(in Chinese) doi: 10.7506/spkx1002-6630-201309031
[6]	张永生, 耿伟涛, 江方, 等. 水解度对热反应鸡肉香精呈味的影响 [J]. 食品科技, 2017, 42(12):270−275. ZHANG Y S, GENG W T, JIANG F, et al. Effect of degree of hydrolysis on the taste characteristic of chicken flavor [J]. Food Science and Technology, 2017, 42(12): 270−275.(in Chinese)
[7]	肖作兵, 吴旻玲, 牛云蔚. 双指标响应面优化鸡肉酶解工艺 [J]. 中国食品学报, 2016, 16(11):121−128. XIAO Z B, WU M L, NIU Y W. Optimization of enzymatic hydrolysis process by dual indicators response surface analysis [J]. Journal of Chinese Institute of Food Science and Technology, 2016, 16(11): 121−128.(in Chinese)
[8]	邱燕翔, 陆志鸿, 许守庆, 等. 鸡肉蛋白酶解工艺及水解产物成分研究 [J]. 食品科技, 2017, 42(3):284−287. QIU Y X, LU Z H, XU S Q, et al. Optimum technical conditions of chicken hydrolysis and composition of hydrolysate [J]. Food Science and Technology, 2017, 42(3): 284−287.(in Chinese)
[9]	侯佰慧, 夏杨毅, 周涛, 等. 鸡肉酶解液制备鸡汤热反应过程中呈味物质的变化 [J]. 食品科学, 2017, 38(14):175−180. doi: 10.7506/spkx1002-6630-201714027 HOU B H, XIA Y Y, ZHOU T, et al. Change of taste components in thermal reaction model system containing chicken enzymatic hydrolysate with reaction time [J]. Food Science, 2017, 38(14): 175−180.(in Chinese) doi: 10.7506/spkx1002-6630-201714027
[10]	吴晖, 任尧, 李晓凤. 单酶和双酶分步酶解鸭蛋清蛋白的工艺 [J]. 食品与发酵工业, 2010, 36(11):93−97. WU H, REN Y, LI X F. Preparation of bioactive peptides via hydrolysis of duck egg-white protein by single/binary enzymes [J]. Food and Fermentation Industries, 2010, 36(11): 93−97.(in Chinese)
[11]	CAI M H, ZHOU X S, SUN X Q, et al. Statistical optimization of medium composition for aspergiolide A production by marine-derived fungus Aspergillus glaucus [J]. Journal of Industrial Microbiology & Biotechnology, 2009, 36(3): 381−389.
[12]	侯晓慧, 张金泽, 林静, 等. 植物源α-低聚半乳糖制备工艺条件优化 [J]. 食品与发酵工业, 2014, 40(1):119−123. HOU X H, ZHANG J Z, LIN J, et al. Response surface methodology for preparation α-galactooligosaccharide from plants by fermentation [J]. Food and Fermentation Industries, 2014, 40(1): 119−123.(in Chinese)
[13]	刘雪飞, 郭端煦, 吕淑霞, 等. 莫海威芽孢杆菌J7产抑菌物质培养基发酵优化 [J]. 中国食品学报, 2018, 18(10):109−119. LIU X F, GUO D X, LÜ S X, et al. Fermentation optimization of culture medium for antibacterial material production of Bacillus mojavensis J7 [J]. Journal of Chinese Institute of Food Science and Technology, 2018, 18(10): 109−119.(in Chinese)
[14]	中华人民共和国国家质量监督检验检疫总局, 中国国家标准化管理委员会. GB/T 8314-2013, 茶游离氨基酸总量的测定[S]. 2013.
[15]	鲁伟, 任国谱, 宋俊梅. 蛋白水解液中多肽含量的测定方法 [J]. 食品科学, 2005, 26(7):169−171. doi: 10.3321/j.issn:1002-6630.2005.07.039 LU W, REN G P, SONG J M. Determination of content of peptides in protein hydrolysates [J]. Food Science, 2005, 26(7): 169−171.(in Chinese) doi: 10.3321/j.issn:1002-6630.2005.07.039
[16]	徐向宏, 何明珠. 试验设计与Design-Expert、SPSS应用[M]. 北京: 科学出版社, 2010.
[17]	朱建平, 胡朝霞, 王艺明. 高级计量经济学导论[M]. 北京: 北京大学出版社, 2009: 73-78.
[18]	周涛. 热反应鸡汤呈味物质变化研究[D]. 重庆: 西南大学, 2016. ZHOU T. Study on the change of taste components in thermal-reaction chicken soup[D]. Chongqing: Southwest University, 2016. (in Chinese)
[19]	LIU Y P, ZHENG P, SUN Z H, et al. Economical succinic acid production from cane molasses by Actinobacillus succinogenes [J]. Bioresource Technology, 2008, 99(6): 1736−1742. doi: 10.1016/j.biortech.2007.03.044
[20]	梁昌聪, 刘磊, 张建华, 等. 响应面法优化绿色木霉H06产孢培养条件 [J]. 广东农业科学, 2014, 41(19):80−85. doi: 10.3969/j.issn.1004-874X.2014.19.019 LIANG C C, LIU L, ZHANG J H, et al. Optimization of fermentation conditions for sporulation quantity by Trichoderma viride strain H06 using response surface methodology [J]. Guangdong Agricultural Sciences, 2014, 41(19): 80−85.(in Chinese) doi: 10.3969/j.issn.1004-874X.2014.19.019
[21]	安攀宇, 赵珊, 张淼, 等. 响应面法优化鸡胸肉蛋白酶解工艺 [J]. 肉类研究, 2018, 32(11):22−29. AN P Y, ZHAO S, ZHANG M, et al. Optimization of enzymatic hydrolysis of chicken breast proteins by response surface methodology [J]. Meat Research, 2018, 32(11): 22−29.(in Chinese)