Optimization of conditions for extraction of acid-soluble collagen from grass carp (Ctenopharyngodon idella) by response surface methodology

Grass carp is a freshwater fish species widely known by consumers over the world. Its skin has abundant collagen which possesses many healthy effects. The optimal conditions for collagen extraction were determined by response surface methodology in this work. Box–Behnken design was applied to evaluate the effects of three independent variables (acetic-acid concentration, temperature and time) on the acid-soluble collagen yield of grass carp skin. Correlation analysis of the mathematical-regression model indicated that quadratic polynomial model could be employed to optimize the extraction of acid-soluble collagen. From response surface plots, acetic-acid concentration, temperature and time exhibited interactive effects on the extraction of acid-soluble collagen. The optimal conditions to obtain the highest acid-soluble collagen yield of grass carp skin were an acetic-acid concentration of 0.54 M, a temperature of 24.7 °C and a time of 32.1 h. Under these optimal conditions, the experimental values agreed with the predicted values, using analysis of variance, indicating a high goodness of fit of the model used and the success of response surface methodology for modeling extraction of acid-soluble collagen from grass carp skin.

Industrial relevance

Grass carp is a freshwater fish species with important economic significance. The annual production of grass carp in China was 3.86 million tons in 2005 which was the highest among all the freshwater fish species (http://www.agri.ac.cn/DataIss/ZY/QT/200610/17688.html). The weight of skin accounts for approximately 9% of whole fish. It is interesting to find a way to improve its value. In this work, collagen was extracted from grass carp skin by acetic acid and a mathematical model of extraction was built by response surface methodology. The effects of acetic-acid concentration, extraction time and temperature were under investigation. The results showed that there is an optimal condition to obtain the highest collagen yield. The optimal condition was determined to be an acetic-acid concentration of 0.54 M, a temperature of 24.7 °C and a time of 32.1 h. This research is helpful to develop a new product using grass carp and to fully use this sort of fish resource.     

草鱼鱼鳞中活性胶原蛋白提取工艺及参数优化

以草鱼鱼鳞为原料,在低于蛋白变性温度的条件下提取酸溶性胶原蛋白(ASC)和酶溶性胶原蛋白(PSC),并对鱼鳞原料的前处理方法、胶原蛋白提取工艺进行优化。结果表明,在鱼鳞原料的前处理工艺中,以0.1mol/L 的Na2CO3 溶液为试剂脱除原料中杂蛋白的最佳工艺条件为室温、料液比1:40(g/mL),300r/min 搅拌处理3 次,每次6h;以0.3mol/L 的EDTA 为试剂脱除原料中矿物质杂质的最佳工艺条件为室温、料液比1:60,搅拌处理3 次,每次12h;ASC 的最佳提取工艺条件为：提取温度不高于25℃,料液比1:60,醋酸浓度0.8mol/L,提取3 次,每次24h;PSC 的最佳提取工艺条件为：胃蛋白酶用量为鱼鳞原料质量的3.0%、醋酸浓度0.8mol/L、料液比1:30、提取温度4 ℃、提取2 次,每次24h。     

草鱼鱼肠抗菌活性物质的提取

为优化草鱼鱼肠抗菌活性物质的提取条件,以草鱼鱼肠为原料,通过单因素试验和正交试验研究乙酸体积分数、料液比和超声时间对提取草鱼肠道抗菌物质的影响。试验结果表明,影响草鱼肠道抗菌物质提取的主次因素依次为乙酸体积分数＞料液比＞超声时间;草鱼肠道抗菌物质的最佳提取条件为：乙酸体积分数10%,料液比1∶3.5（g∶mL）,超声时间10 min,4 ℃浸提24 h。在此最佳提取条件下,提取液的抗菌活性较高,金黄色葡萄球菌抑菌圈直径为3.106 0 cm,大肠杆菌抑菌圈直径为2.028 4 cm。     

淡水鱼皮胶原蛋白提取工艺研究(Ⅰ)

研究了罗非鱼、鳙鱼、草鱼和鲫鱼4种淡水鱼皮胶原蛋白的提取工艺。结果表明,鱼皮用乙醚2次除脂效果显著,除脂率依次为92.4%、95.1%、66.8%和76.7%,除脂后胶原蛋白的提取率和得率都略有增加。除非胶原蛋白的工艺为:先用NaOH浸泡6h,再用NaCl浸泡6h,4种鱼皮胶原蛋白对应的提取率分别为34.4%、28.7%、31.5%和42.5%,得率依次为8.4%、8.0%、8.2%和6.1%,比其他处理相应高出0.4-2.3倍、0.04-1.6倍、0.1-0.9倍和0.07-0.5倍。乙酸的提取效果优于柠檬酸,对应提取率分别为34.8%、23.6%、33.6%、43.0%,得率依次为8.5%、6.6%、8.63%、6.19%,比柠檬酸相应高2.4-4.3倍、1.7-4.0倍。温度对罗非鱼、鳙鱼和草鱼鱼皮胶原蛋白提取影响较大,最佳提取温度都为20℃,其对应提取率依次为34.05%、27.05%、33.38%,得率分别为8.32%、7.54%、8.58%;温度对鲫鱼鱼皮胶原蛋白的提取影响较小,在520℃范围内提取率和得率分别约为42%和6%左右。     

鱼鳞冻热处理工艺优化及性质的研究

采用正交试验法,研究了未经前处理、酸处理、酶处理3种不同前处理方式制备草鱼鱼鳞冻的热处理优化工艺。以胶原蛋白提取率和冻力作为考察指标,确定鱼鳞冻热处理工艺最佳条件的提胶温度都为100℃,提胶时间都为2 h,除酸处理鱼鳞冻提胶料水比为1∶5外,其余均为1∶4,未前处理鱼鳞冻的提胶用水pH值为6。但由于原料中胶原蛋白含量不同,需要调整料水比以满足冻力要求。优化工艺制备的3种鱼鳞冻冻力相当,质构上都要比普通果味型果冻松软、爽滑,更有弹性。酶处理鱼鳞冻胶原蛋白浓度最高,未前处理鱼鳞冻黏度最高、质构特性最好,酸处理鱼鳞冻产量最高。胶凝温度和熔化温度表明鱼鳞冻适合在10℃以下凝冻,在20℃以下保存。     

Isolation and partial characterization of pepsin-soluble collagen from the skin of grass carp (Ctenopharyngodon idella)

Pepsin-soluble collagen was extracted from the skin of grass carp (Ctenopharyngodon idella) with a yield of 46.6%, on a dry weight basis. Electrophoretic patterns showed that the collagen contained α1 and α2 chains, similar to those of calf skin collagen. The imino acid content of the collagen from grass carp skin was much lower than those of mammalian’s collagens, as also were the transition temperature and denaturation temperature which were only 24.6 °C and 28.4 °C respectively. Peptide maps of the collagen digested by trypsin and V8 protease showed different peptide fragments from those of calf skin collagen and other fish skin collagens, suggesting differences in amino acid sequences and collagen conformation. In addition, the lyophilized collagen sponge from grass carp skin had a uniform and regular network structure, just like calf skin collagen sponge. These results suggest that grass carp skin has potential for use as a supplementary source of collagen.     

正交试验优化黄河鲤鱼鳞酶促溶性胶原蛋白提取工艺

为提高黄河鲤鱼鳞胶原蛋白提取率,在4 ℃条件下,采用胃蛋白酶和0.5 mol/L乙酸溶液对其提取工艺进行研究,在单因素试验的基础上,采用正交试验设计方法探索了提取时间、加酶量和料液比3 个因素对黄河鲤鱼鳞酶促溶性胶原蛋白（pepsin soluble collagen,PSC）提取率的影响。结果表明,黄河鲤鱼鳞PSC的最适提取工艺参数为提取时间60 h、加酶量40 kU/g和料液比1∶10（g/mL）,在此条件下,PSC提取率达到（17.7±0.7）%。所提取的产品与酸溶性胶原蛋白（acid soluble collagen,ASC）有相似的电泳性质和氨基酸组成,表明其可能与ASC具有相似的分子结构。     

响应面法优化木瓜蛋白酶提取草鱼鱼鳞胶原蛋白的研究

以草鱼鱼鳞为原料,用木瓜蛋白酶解提取草鱼鱼鳞中的胶原蛋白,并对其性质进行初步研究。在单因素试验的基础上,采用响应面法Box-Behnken试验设计,对影响提取率的酶解温度、加酶量、底物质量分数3个因素进行优化,建立并分析各因素与胶原蛋白提取率关系的数学模型。确定木瓜蛋白酶提取鱼鳞胶原蛋白的最佳条件为酶解温度50℃,加酶量3.5g/L,底物质量分数22%,经过验证胶原蛋白提取率为15.4%。得到鱼鳞胶原蛋白吸水性接近于甘油,保水性优于甘油,具有一定的乳化性和乳化稳定性。     

Optimization of Gelatin Extraction from Silver Carp Skin

ABSTRACT:  Fish skins are a by-product of the fish processing industry that can be successfully processed into gelatin. This study was designed to optimize the extraction process to obtain the highest yield, gel strength, and viscosity for gelatin production from silver carp skin. A fractional factorial design (2 levels, resolution III, 2^9-5) was chosen to screen 9 parameters to determine the most significant ones. Those found to be significant were optimized to determine the maximum value for 3 dependent variables mentioned above. The hydroxyproline content and hydroxyproline/protein ratio of the skin were 1.7% and 6.5%, respectively. The protein content of the skin was 26%. The hydroxyproline content of the gelatin for the sample giving the highest hydroxyproline/protein ratio was 10.9%. This sample was arbitrarily called pure gelatin and the purity of the remaining samples was between 71.8% and 97%. The highest protein and gelatin recovery was 78.1% and 98.8% of the total available, respectively. The latter, gelatin recovery, is proposed to be used instead of protein yield. Four variables were determined as significant in screening and these variables were studied by a central composite rotatable design (4-factor and 5-level with 6 central points) to model the system and response surface methodology was used for optimization. The optimum extraction conditions were 50 °C for the extraction temperature, 0.1 N HCl for the acid concentration, 45 min for the acid pretreatment time, and finally 4 : 1 (v/w) for the water/skin ratio. The predicted responses for these extraction conditions were 630 g gel strength, 6.3 cP viscosity, and 80.8% gelatin recovery. The data suggest that silver carp skin gelatin is similar to those of fish gelatins currently being exploited commercially.     

鱼鳞胶原蛋白提取残渣中角蛋白的回收与表征

为充分开发鱼鳞的经济价值,增加其回收利用率,本研究以草鱼鱼鳞为原料,研究了分离鱼鳞胶原蛋白的最佳方法以获得较纯的角蛋白,并以提取过胶原蛋白的鱼鳞残渣为原料,运用单因素实验和正交试验确定角蛋白的最优提取工艺,再对提取的鱼鳞角蛋白进行表征。结果表明:1%的柠檬酸溶液中加0.60 g胃蛋白酶于4 ℃下浸泡24 h,胶原蛋白去除率最高,去除效果及结构完整性最好;提取草鱼鱼鳞角蛋白的最优工艺条件为:提取温度60 ℃,氢氧化钠浓度9%,料液比1:12.5。鱼鳞角蛋白的紫外吸收峰分别在218.2、280.4 nm处,SDS-PAGE电泳显示鱼鳞角蛋白的表观分子量约为48 kDa,红外结果显示角蛋白的二级结构为α-螺旋,氨基酸组分的特征与羊毛角蛋白基本一致。     

白鲢鱼鳞胶原蛋白提取工艺条件优化研究

利用白鲢鱼鳞为原料,采用酶法提取胶原蛋白,并利用响应面分析法对影响鱼鳞胶原蛋白提取率的关键因素的分析,得到酶法提取白鲢鱼鳞胶原蛋白的最佳工艺条件为底物浓度2.89％、加酶量0.26％、酶解温度58℃、酶解时间54min,在此条件下提取,提取液中胶原蛋白提取率可达57.84％以上.     

超声波辅助酶法提取青鱼鱼皮胶原蛋白工艺

以青鱼鱼皮为原料,考察超声波功率、超声波处理时间、加酶量、酶解温度、酶解时间、pH值对胶原蛋白提取率的影响,在单因素基础上对青鱼鱼皮胶原蛋白提取工艺进行正交试验和方差分析。结果表明,最佳提取工艺条件为:加酶量100 U/g、pH 7.5、酶解温度40℃、超声波处理时间50 min,胶原蛋白的提取率达45.3%。     

海参体壁酸溶性胶原提取及氨基酸组成分析

以海参体壁为原料,采用乙酸溶剂法提取酸溶性胶原。以酸溶性胶原得率为指标,通过均匀设计试验确定了乙酸溶剂法提取酸溶性胶原的优化条件:温度为4℃,乙酸浓度为0.5 mol/L,料液比为1∶1 000,提取时间为72 h,在此条件下酸溶性胶原的得率可达到胶原纤维原料的7.35%(质量分数)。氨基酸组成分析表明,海参体壁酸溶性胶原的甘氨酸(Gly)含量为33.6%,羟脯氨酸(Hyp)含量为7.04%。     

The influence of different acids and pepsin on the extractability of collagen from the skin of Baltic cod (Gadus morhua)

Solutions of (0.5 M) citric, lactic and acetic acids and 0.15 M HCl were used for the extraction of collagen from the whole skins of Baltic cod (Gadus morhua). The extractions were performed at a temperature of 4 °C for 24, 48 and 72 h using a solid/solution ratio of 1:6 (w/v). Of the acids used, HCl was the least effective solvent for collagen. The maximal yield of collagen extracted with citric acid was 60%. Collagen extraction with acetic or lactic acid give a maximal yield of about 90% with HCl yielding of only 18%. After enzymatic treatment of cod skin the yield of protein extracted with HCl and citric acids increased to 40% and 20%, respectively. Collagen was completely solubilized under the same conditions in acetic and lactic acids. Electrophoretic analysis of collagens extracted in HCl and citric acids with enzymatic treatment proved that the isolated protein was denaturated. The solutions of acetic and lactic acids are solvents for native collagen.     

骆驼掌胶原蛋白提取工艺优化与性能分析

本文以骆驼掌为原料,以提取时间、加酶量、乙酸浓度、料液比为实验因素,通过正交试验优化胃蛋白酶提取骆驼掌胶原蛋白工艺,并对所提骆驼掌胶原蛋白进行性能分析。结果表明,骆驼掌胶原蛋白的最佳提取工艺为提取时间48 h、胃蛋白酶加酶量4%、乙酸浓度0.50 mol/L、料液比1:15 g/mL,此条件下提取率为30.33%±0.19%。紫外光谱和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表明,所提胶原蛋白符合I型胶原特征,保持了胶原蛋白的三螺旋结构。与牛蹄胶原蛋白性质比较,发现骆驼掌胶原蛋白的吸湿性和保湿性优于牛蹄胶原蛋白,吸油性弱于后者。     

响应面分析法优化艾叶中绿原酸提取工艺

目的:探索艾叶中绿原酸的醇提工艺。方法:以乙醇为溶剂,在单因素实验的基础上,选取提取次数、提取温度、提取时间、料液比为自变量,绿原酸的得率为响应值,采用中心组合设计的方法,利用响应面分析方法,模拟得到二次多项式回归方程的预测模型。结果:艾叶中绿原酸醇提工艺的最佳条件为60%乙醇为提取溶剂、料液比1∶13、提取温度63℃、提取次数3次、提取时间60min,在此条件下,绿原酸的得率为0.820%。结论:通过对绿原酸提取工艺的研究,艾叶中绿原酸的提取率达94.04%。     

超声辅助酶法提取中华鳖裙边胶原蛋白及其热稳定性能

以中华鳖裙边为原料,采用单因素试验探讨加酶量、超声时间、超声功率、料液比对胶原蛋白的影响,通过Box-Behnken响应面方法建立数学模型,求得最佳酶提工艺,比较常规酶提和超声辅助酶提胶原蛋白的热稳定性能。结果表明,在提取溶剂为乙酸（0.5?mol/L）、料液比1∶20（g/mL）的条件下,采用超声酶提胶原蛋白的最佳工艺参数为加酶量0.8%、超声时间43?min、超声功率176?W,此条件下胶原蛋白得率为74.50%;差示扫描量热实验结果表明,超声酶提胶原蛋白的热稳定性显著优于常规酶提胶原蛋白。该研究可为中华鳖裙边胶原蛋白的精深加工及其应用提供一定科学依据。     

响应面优化草鱼鱼鳔酶溶胶原蛋白的提取工艺

为提高草鱼下脚料鱼鳔中胶原蛋白的提取率,采用响应面法优化胃蛋白酶提取草鱼鱼鳔胶原蛋白。探讨加酶量、提取时间和液料比3个因素对提取率的影响;在单因素试验的基础上进行响应面优化试验。结果表明：胃蛋白酶提取草鱼鱼鳔胶原蛋白的最佳工艺条件为加酶量40mg/g、提取时间48h、液料比80:1(mL/g),在此工艺条件下胶原蛋白实际提取率为17.82%,与模型预测值(18.04%)接近。方差分析显示,模型可靠,3个因素均对草鱼鱼鳔胶原蛋白的提取率影响显著。     

梅花鹿鹿筋胶原蛋白提取工艺条件优化

为应用胃蛋白酶酶解法从鹿筋中提取制备胶原蛋白。采用单因素试验和正交试验筛选出鹿筋胶原蛋白提取的最佳条件为醋酸体积分数5.5%、料液比1:7(mL/g)、浸提时间14h,胃蛋白酶与底物比1:1000(g/g)、酶解时间24h。验证实验结果表明,采用本方法提取的鹿筋胶原蛋白收率为74.56%,蛋白含量为65.54%。本实验优选所得梅花鹿鹿筋胶原蛋白提取工艺,方法简便,提取率高。     

秘鲁鱿鱼皮酸溶性胶原蛋白提取工艺优化及其特性

以秘鲁鱿鱼皮为原料,通过单因素试验及响应面优化法得到秘鲁鱿鱼皮酸溶性胶原蛋白（acid soluble collagen,ASC）最佳提取工艺,并对ASC的理化特性进行研究。结果表明：最佳提取工艺为在4 ℃条件下,冰醋 酸浓度0.47 mol/L、液料比24.5 mL/g,浸泡43 h后,ASC提取率为31.76%;紫外全波长扫描结果表明,所得ASC在 231 nm波长处有最大吸收峰,符合Ⅰ型胶原蛋白特征;凝胶电泳分析表明,提取的ASC含有α1、α2和β链;拉曼光 谱分析表明,所得ASC结构完整,三螺旋结构未受到破坏;ASC的相变温度为50.07 ℃。