斑点叉尾鮰鱼皮胶原的制备及其应用研究
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摘要
胶原是动物组织中的纤维蛋白,广泛分布于动物的结缔、皮肤、骨骼、软骨等组织中,起着支撑器官、保护机体的功能。胶原分子是由三条α链交织组成胶原三维空间螺旋结构,大约有27种不同类型的胶原已被确定。由于胶原独特的螺旋结构和生物学特性,已被广泛应用于生物医药材料、食品、美容、保健等众多领域。目前提取胶原主要来源于哺乳动物皮、骨等,但是由于宗教信仰及“疯牛病”等因素限制了其使用范围,而另一方面,鮰鱼作为我国加工出口的主要鱼种,2009年出口量约有1.7万吨,加工过程中产生大量的鮰鱼皮等下脚料。而鱼皮中含有胶原等大量的宝贵资源,因此,本文利用水产加工废弃物斑点叉尾鮰鱼皮为原料,研究鱼胶原的提取方法和应用,具有重要的经济效益和社会效益。
本文以斑点叉尾鮰鱼皮为原料,利用国家标准方法对其基本成分进行了测定,分析结果表明：鮰鱼皮中水分含量为68.9%、总蛋白质含量为28.25%、粗脂肪含量为2.14%,总糖含量0.3136%、灰分含量1.3%。利用分光法测定胶原中特征的氨基酸—羟脯氨酸的含量为2.19%,折合胶原含量为24.31%,约占鮰鱼皮蛋白总量的86.05%。研究结果表明鮰鱼皮含有丰富胶原,是提取胶原优质原料。
本文研究了酸法提取鮰鱼皮中的胶原,比较各种酸对胶原提取得率的影响,结果表明乙酸提取得率为24.6%。在单因素实验的基础上通过正交实验对乙酸提取胶原工艺条件进行优化,研究结果表明：在5℃下乙酸浓度1.0mol/L、固液比1：30、提取时间72h,其提取得率最高为33.79%。通过紫外(UV)和傅里叶红外光谱(FTIR)对提取胶原表征,结果表明酸溶胶原在紫外236nm有最大特征吸收峰；在3450cm-1、1652 cm-1和1263 cm-1等红外区域具有吸收峰,表明胶原中具有N-H、C=O和C-H等的伸缩振动,证实该胶原具有螺旋结构。
本文研究了酶法促溶对鮰鱼皮胶原提取得率的影响。通过对胃蛋白酶、胰蛋白酶、碱性蛋白酶和酸性蛋白酶促溶的考察,结果表明：胃蛋白酶液浸泡鮰鱼皮提取得率为26.08%。采用Box-Behnken设计对胃蛋白酶量、温度、料液比和浸提时间等四因素进行研究,得出四种因素的最佳量分别为：胃蛋酶量2.21%,温度11.66℃,料液比=1：32.55,浸泡时间42.86h；实验结果表明此条件下胶原的得率为34.41%,与实验所得值34.92%相接近。酶溶胶原的紫外和红外扫描表征结果为：酶法促溶溶胶原的最大紫外吸收特征峰在235nm,与酸溶胶原吸收峰基本相同；红外扫描结果表明N-H、C=O以及C-H伸缩振动的吸收峰分别为3450 cm-1、1637cm-1、1267cm-1,说明酶法胶原螺旋结构的存在。
本文研究了利用鮰鱼皮胶原和壳聚糖混合制备胶原／壳聚糖复合膜。单因素试验结果表明,在温度为70℃,胶原：壳聚糖=1：1,添加2ml的1%的戊二醛条件下,制备的复合膜抗张强度最大为0.82 N／mm2。通过扫描电镜分析(SEM)和红外表征结果表明,在上述条件下制备的复合膜为均匀致密,胶原和壳聚糖具有良好的相容性。膜特性研究结果表明,在60℃时该膜吸水率、溶胀比,分别达到最大为212%和1.2；透明度随温度升高总体呈下降趋势,40℃时透光率最大为68.6%。
Derived from the animals fibrous protein, collagen is the principal constituent of animals connective tissue,skin,bones,cartilage and so on,which played a supporting and protecting the body function. Collagen molecules are composed of three intertwined in the so-called collagen triple-helix, some 27 different types of collagen have been identified recently.Because of its unique functional and technological properties,collagen is widely used in biomedical materials, food, cosmetic, health care and other areas. Currently the collagen mainly comes from mammal leather and bone, but its usage scope were limited by the religious belief and "cow" factors.On the other hand, the range of fish processing waste reached 730 million tons every year, causing serious pollution of environment and resources waste. Therefore, this paper actively explore the extraction and application of collagen from the cutfish processing waste,which will create the important economic benefit and social benefit.
Based on the Ictalurus Punctatus skin as raw materials, the basic ingredients was determined by use of the national standard method,the results show:Ictalurus Punctatus skin water contented 68.9%,total protein contented 28.25%, crude fat contented 2.14%, total sugar contented 0.3136%, ash contented 1.3%. Using the spectral determination,the intrinsic collagen amino acids-hydroxyproline contented 2.19%,estimation of collagen content 24.31%, which contented about 86.05% in total cutfish skin protein. Results showed that cutfish skin is rich in collagen and can be considered as collagenous raw materials.
This paper examined the acid-extraction collagen from Ictalurus Punctatus skin and compared collagen yield of various acids,we found acetic yield was 24.6%.Based on the single factor experiment,the orthogonal experiment was adopted in collagen processing for acetic extracting optimization,the results showed that:extracting temperature=5℃,acid concentration= 1.0 mol/L, the liquid-solid ratio=1:30, extracting time=72h, the highest collagen yield was 33.79%.After the purification of ultraviolet (UV) and detecting of infrared spectroscopy (FTIR) scanning,the results show that original UV maximum absorption of acid soluble collagen in 236nm peak, accord with representative collagen absorption peaks. In the 3450 cm-1、1652 cm-1,1263 cm-1 and other aeras also have infrared absorption peaks,which confirmed the vibration of the region N-H,C=O,C-H in collagen and spiral structure of collagen.
This paper examined the protease-extraction collagen from Ictalurus Punctatus skin and compared collagen yield of pepsin, trypsin, alkali protease and acid protease,the results showed:yield of pepsin-extraction collagen was 26.08%.Using Box-Behnken design of pepsin, temperature, fluid extraction time and more than four factors, we found the best respectively of them:pepsin=2.21%, temperature=11.66℃, liquid ratio=1:32.55, extracting time=42.86h.Theoretical experimental results showed that the collagen yield was34.41%,which was similar to the actual value 34.92%. By detecting of the ultraviolet and infrared scanning,the results of protease soluble collagen showed:the maximum peaks of protease ultraviolet absorption was 235nm as similar to absorption with acid soluble, infrared scanning results showed N-H, C= O andC-H telescopic vibration absorption peaks respectively 3450 cm-1,1637cm-1 and 1267cm-1,Which explained the existence of protease spiral structure of collagen.
This paper prepared collagen/chitosan composite film based on the cutfish skin collagen and chitosan mixture. Single factor experiment results indicated:temperature=70℃, collagen:chitosan=1:1,added 1% glutaraldehyde, the maximum tensile strength of the compound film was 0.82 N/mm2By analysis of scanning electron microscope (SEM) and infrared scanning, results showed that composite film under the preparation conditions was uniform, collagen and chitosan had good compatibility. Film function research results showed:When the film in 60℃, bibulous rate and the swelling ratio reached the maximum 21.2% and 1.2%;transparency declined overall with temperature, in 40℃light transmittance reached maximum 68.6%.
本文以斑点叉尾鮰鱼皮为原料,利用国家标准方法对其基本成分进行了测定,分析结果表明：鮰鱼皮中水分含量为68.9%、总蛋白质含量为28.25%、粗脂肪含量为2.14%,总糖含量0.3136%、灰分含量1.3%。利用分光法测定胶原中特征的氨基酸—羟脯氨酸的含量为2.19%,折合胶原含量为24.31%,约占鮰鱼皮蛋白总量的86.05%。研究结果表明鮰鱼皮含有丰富胶原,是提取胶原优质原料。
本文研究了酸法提取鮰鱼皮中的胶原,比较各种酸对胶原提取得率的影响,结果表明乙酸提取得率为24.6%。在单因素实验的基础上通过正交实验对乙酸提取胶原工艺条件进行优化,研究结果表明：在5℃下乙酸浓度1.0mol/L、固液比1：30、提取时间72h,其提取得率最高为33.79%。通过紫外(UV)和傅里叶红外光谱(FTIR)对提取胶原表征,结果表明酸溶胶原在紫外236nm有最大特征吸收峰；在3450cm-1、1652 cm-1和1263 cm-1等红外区域具有吸收峰,表明胶原中具有N-H、C=O和C-H等的伸缩振动,证实该胶原具有螺旋结构。
本文研究了酶法促溶对鮰鱼皮胶原提取得率的影响。通过对胃蛋白酶、胰蛋白酶、碱性蛋白酶和酸性蛋白酶促溶的考察,结果表明：胃蛋白酶液浸泡鮰鱼皮提取得率为26.08%。采用Box-Behnken设计对胃蛋白酶量、温度、料液比和浸提时间等四因素进行研究,得出四种因素的最佳量分别为：胃蛋酶量2.21%,温度11.66℃,料液比=1：32.55,浸泡时间42.86h；实验结果表明此条件下胶原的得率为34.41%,与实验所得值34.92%相接近。酶溶胶原的紫外和红外扫描表征结果为：酶法促溶溶胶原的最大紫外吸收特征峰在235nm,与酸溶胶原吸收峰基本相同；红外扫描结果表明N-H、C=O以及C-H伸缩振动的吸收峰分别为3450 cm-1、1637cm-1、1267cm-1,说明酶法胶原螺旋结构的存在。
本文研究了利用鮰鱼皮胶原和壳聚糖混合制备胶原／壳聚糖复合膜。单因素试验结果表明,在温度为70℃,胶原：壳聚糖=1：1,添加2ml的1%的戊二醛条件下,制备的复合膜抗张强度最大为0.82 N／mm2。通过扫描电镜分析(SEM)和红外表征结果表明,在上述条件下制备的复合膜为均匀致密,胶原和壳聚糖具有良好的相容性。膜特性研究结果表明,在60℃时该膜吸水率、溶胀比,分别达到最大为212%和1.2；透明度随温度升高总体呈下降趋势,40℃时透光率最大为68.6%。
Derived from the animals fibrous protein, collagen is the principal constituent of animals connective tissue,skin,bones,cartilage and so on,which played a supporting and protecting the body function. Collagen molecules are composed of three intertwined in the so-called collagen triple-helix, some 27 different types of collagen have been identified recently.Because of its unique functional and technological properties,collagen is widely used in biomedical materials, food, cosmetic, health care and other areas. Currently the collagen mainly comes from mammal leather and bone, but its usage scope were limited by the religious belief and "cow" factors.On the other hand, the range of fish processing waste reached 730 million tons every year, causing serious pollution of environment and resources waste. Therefore, this paper actively explore the extraction and application of collagen from the cutfish processing waste,which will create the important economic benefit and social benefit.
Based on the Ictalurus Punctatus skin as raw materials, the basic ingredients was determined by use of the national standard method,the results show:Ictalurus Punctatus skin water contented 68.9%,total protein contented 28.25%, crude fat contented 2.14%, total sugar contented 0.3136%, ash contented 1.3%. Using the spectral determination,the intrinsic collagen amino acids-hydroxyproline contented 2.19%,estimation of collagen content 24.31%, which contented about 86.05% in total cutfish skin protein. Results showed that cutfish skin is rich in collagen and can be considered as collagenous raw materials.
This paper examined the acid-extraction collagen from Ictalurus Punctatus skin and compared collagen yield of various acids,we found acetic yield was 24.6%.Based on the single factor experiment,the orthogonal experiment was adopted in collagen processing for acetic extracting optimization,the results showed that:extracting temperature=5℃,acid concentration= 1.0 mol/L, the liquid-solid ratio=1:30, extracting time=72h, the highest collagen yield was 33.79%.After the purification of ultraviolet (UV) and detecting of infrared spectroscopy (FTIR) scanning,the results show that original UV maximum absorption of acid soluble collagen in 236nm peak, accord with representative collagen absorption peaks. In the 3450 cm-1、1652 cm-1,1263 cm-1 and other aeras also have infrared absorption peaks,which confirmed the vibration of the region N-H,C=O,C-H in collagen and spiral structure of collagen.
This paper examined the protease-extraction collagen from Ictalurus Punctatus skin and compared collagen yield of pepsin, trypsin, alkali protease and acid protease,the results showed:yield of pepsin-extraction collagen was 26.08%.Using Box-Behnken design of pepsin, temperature, fluid extraction time and more than four factors, we found the best respectively of them:pepsin=2.21%, temperature=11.66℃, liquid ratio=1:32.55, extracting time=42.86h.Theoretical experimental results showed that the collagen yield was34.41%,which was similar to the actual value 34.92%. By detecting of the ultraviolet and infrared scanning,the results of protease soluble collagen showed:the maximum peaks of protease ultraviolet absorption was 235nm as similar to absorption with acid soluble, infrared scanning results showed N-H, C= O andC-H telescopic vibration absorption peaks respectively 3450 cm-1,1637cm-1 and 1267cm-1,Which explained the existence of protease spiral structure of collagen.
This paper prepared collagen/chitosan composite film based on the cutfish skin collagen and chitosan mixture. Single factor experiment results indicated:temperature=70℃, collagen:chitosan=1:1,added 1% glutaraldehyde, the maximum tensile strength of the compound film was 0.82 N/mm2By analysis of scanning electron microscope (SEM) and infrared scanning, results showed that composite film under the preparation conditions was uniform, collagen and chitosan had good compatibility. Film function research results showed:When the film in 60℃, bibulous rate and the swelling ratio reached the maximum 21.2% and 1.2%;transparency declined overall with temperature, in 40℃light transmittance reached maximum 68.6%.
引文
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