高压脉冲电场水解牛骨胶原蛋白及蛋白复合钙的研究
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摘要
本文以骨粉为主要原料,以高压脉冲电场处理设备为工具,配合化学分析,光谱分析,动物试验等手段,对高压脉冲电场技术水解牛骨蛋白以及蛋白复合钙的结构性质进行系列研究。在胃蛋白酶作用下,通过二次旋转组合试验设计优化出酶解牛骨胶原蛋白的最佳参数。在酸性条件下,首先通过正交试验确定制备牛骨蛋白复合钙的最佳参数并依据傅里叶变换红外光谱及X射线晶体衍射等手段对牛骨蛋白复合钙的结构进行了探究;其次进行了牛骨钙-蛋白复合饮料的研制和稳定性研究,确定了最佳稳定性条件。最后,通过动物试验模型,对蛋白钙和蛋白饮料的持钙性进行了研究。
本文是长春市科技成果转化百亿增值工程项目(2007CZ14)“高电压脉冲电场下快速酶解牛骨胶原蛋白关键技术研究与产品开发”的组成部分。在动物骨产品开发效率较低的今天,本文不仅为骨产品的开发提供了参考,而且将高压脉冲电场技术的应用领域继续扩大,具有现实意义。
Animals body contains about 20% -30% bones, bone is a very high nutritional value of processing by-products, contains a lot of protein, fat, minerals and other nutrients. Bone powder contains 12.0% -35.0% protein, which is the highest content of collagen. Bone protein is a more full price of the soluble protein, with a high biological value.It belongs to the high quality protein. The unique quality of collagen, making it in many foods and nutrition as a function of material composition, other alternative materials with unparalleled advantages. In addition, bone contains a large number of minerals. And the calcium and phosphorus ratio is appropriate.The calcium and collagen cross-linking makes it easier to absorb calcium, so calcium is a worthwhile use of the calcium salt.
China is a major livestock breeding and fishing country. But in the long time, because of the not enough understanding of the bones and backward processing technology, a large number of animal food processing by-products have not been effective development and utilization, which not only affects the enterprise economy improving efficiency, but also a waste of resources and environmental pollution. Therefore, to improve the utilization of animal bone and the development of bone food are not only nourishing and healthy, but also increase the added-value of products, waste to treasure, to reduce environmental pollution, increase economic efficiency, while opening a new food resources for nutrition related subjects development.
PEF (pulsed electric fields) has been recognized as one of the most advanced sterilization techniques and in recent years has been gradually applied to extraction of bioactive substances and to separate the field. In this paper, the research aims at extract the bone protein by means of enzyme under the PEF. And research on bone protein-calcium structure and texture. And the development of a bovine bone-protein complex beverages. Through animal experiments , researching on the effect of calcium supplementation of products for two types of calcium. Not only for PEF technology in the field of protein will provide a reference, but also for the comprehensive utilization of bone has opened up a valuable road.
The specific content and the results of research are as follows:
1. Study on bone protein hydrolysis by PEF technology
(1) Through the single factor experiment, the paper separately studied the effect of electric field intensity, pulse number, liquid/solid ratio, enzyme content on the extraction yield of collagen.
When the liquid/solid ratio was 10:1, the enzyme content was 3%, the flow velocity was 18ml/min and the pulsed number was 8, the electric field intensity of a significant impact on protein extraction. So the better electric field intensity is 30kV/cm for extraction of bone protein by PEF.
When the liquid/solid ratio was 10:1, the enzyme content was 3%, the flow velocity was 18ml/min and the electric field intensity is 30kV/cm, the pulsed number of a significant impact on protein extraction. So the better pulsed number is 8 for extraction of bone protein by PEF.
When the enzyme content was 3%, the pulsed number was 8, the flow velocity was 18ml/min and the electric field intensity is 30kV/cm, the liquid/solid ratio of a significant impact on protein extraction. So the better liquid/solid ratio is 10:1 for extraction of bone protein by PEF.
When the liquid/solid ratio was 10:1, the pulsed number was 8, the flow velocity was 18ml/min and the electric field intensity is 30kV/cm, the enzyme content of a significant impact on protein extraction. So the better enzyme content is 3% for extraction of bone protein by PEF.
(2) In order to achieve the purpose of precise optimization, a quadratic general rotary unitized design was arranged near the best parameter which was fixed by orthogonal test, and the Mathematics model was established. The regression equation was:
For the level of 0.01, deleting inapparent item, the simple regression equation is: Through the regression equation to determine the impact of the rate of protein extraction followed the order of primary and secondary factors were liquid/solid ratio> enzyme content> pulsed number> electric field intensity.
(3) Through the experiment analyse, the best combination parameters were fixed exactly: pulse number of 10, electric field intensity of 30kV/cm, liquid/solid ratio is 12.5:1, enzyme content is 3.5%, at this point extraction rate of 46.51%.
2. Study on preparation for bone protein-calcium and its structure and texture
(1) Studied bone protein-calcium preparation methods, through the orthogonal test to determine bone protein-calcium complex the best extraction conditions.
The best condition of hydrolysis bone calcium by PEF: water/bone powder ratio is 10:1, bone powder/mixed acid is 5:3, citric acid/malic acid is 1:1.5, electric field intensity is 20kV/cm, the flow velocity was 18ml/min, pulsed number is 12.
The best condition of preparation for bone protein-calcium under high-temperature: temperature is 115℃, heating time is 25min, and the order of primary and secondary factors are time>temperature.
(2) Analyse the structure of bone protein-calcium by FTIR and results as follows: Characteristic peaks at 1435 cm-1 caused by C-N stretching vibration. They belong to collagen peptide amino acid side chain groups; Medium intensity characteristic bands at 602 cm-1、670 cm-1、887 cm-1 caused by N-H-plane rocking vibration; All these show that the original collagen matrix of bone has been partially hydrolyzed to form acid residues of collagen and small peptide; Phosphate characteristic peaks at 2301 cm-1 Show that in the original hydroxyapatite meal to be biodegradable, phosphate free form, may be related to the formation of calcium phosphate; In addition, Amino stretching vibration and the amide I band shows the movement of collagen peptides and amino and carboxyl group are involved in calcium coordination; Characteristic peaks at 5354cm-1 Strengthen may caused by Ca-N stretching vibration; At 1387 cm-1-1078 cm-1, there are lots of Strengthen band, but can’t find its ascription, may cause by other kind of complex calcium.
(3) Analyse the structure of bone protein-calcium by XRD. The bone protein-calcium is make up of CaPO3(OH)·2H2O, Ca(H2PO4)·2H2O and Ca3(PO3)6·10H2O.
3. Study on preparation for bovine bone-protein complex beverages.
(1) The flow chart of bovine bone-protein complex beverages as follows:
Material→Homogenize→Prepare→Second-homogenize→Perfusion→Sterilization→Finished Product
(2) Study on stability of the bovine bone-protein complex beverages and results as follows:
The higher homogeneous pressure, the better the stability of beverage.The better first homogeneous pressure is 30Mpa, the second is 40Mpa.
Emulsifier choose 0.2% GMS, Stabilizer choose PGA and CMC-Na and concentration ratio is 3:1.
(3) By orthogonal test to determine the best formula additives as follows: GMS0.3%, PGA0.3%, CMC-Na0.1%, Sodium tripolyphosphate0.3%.At this point, system is the most stable. The order of primary and secondary factors are PGA> Sodium tripolyphosphate> CMC-Na>GMS.
4. Animal experiments
(1) Mice were tested 6 weeks to monitor the weight of mice in the early subjects in the comparison group and experimental group of mice had no significant differences in body weight, the comparison group and experimental groups of mice both the overall weight increase, with the subjects time, significant inter-group changes. And beverage> powder> comparison> blank.
(2) The apparent calcium absorption rate measurement results show that the first week of each group of apparent calcium absorption rate is highest, after five weeks in each group between the apparent absorption of calcium were no significant differences between that period of absorption in the young mice calcium effect more visible.
(3) On serum calcium and serum phosphorus measurement results show that the compound of calcium and bone protein osteocalcin cattle - drinks can increase the protein complex in serum calcium, phosphorus content, indicating that increased calcium absorption, but the difference was not significant.
(4) Determination of ALP results show that the experimental group and the control group serum ALP activity was significantly higher than blank group. And the experimental group was higher than the control group. That they both increase the ALP activity, can significantly promote the absorption of bone metabolism.
(5) Determination of StrACP results show that the experimental group and the control group serum ALP activity was significantly lower than blank group, and beverage< powder< the control group. The results show that bone protein-calcium and bone-protein complex beverages both can reduce StrACP activities and reduce the number of osteoclasts, inhibit osteoclast activity, so that bone formation than bone resorption, increase bone mass.
(6) On mice femur measurement results show that bone protein-calcium and bone-protein complex beverages both do better in bone growth and development, suggesting that delaying the aging of the skeleton to a certain extent.
本文是长春市科技成果转化百亿增值工程项目(2007CZ14)“高电压脉冲电场下快速酶解牛骨胶原蛋白关键技术研究与产品开发”的组成部分。在动物骨产品开发效率较低的今天,本文不仅为骨产品的开发提供了参考,而且将高压脉冲电场技术的应用领域继续扩大,具有现实意义。
Animals body contains about 20% -30% bones, bone is a very high nutritional value of processing by-products, contains a lot of protein, fat, minerals and other nutrients. Bone powder contains 12.0% -35.0% protein, which is the highest content of collagen. Bone protein is a more full price of the soluble protein, with a high biological value.It belongs to the high quality protein. The unique quality of collagen, making it in many foods and nutrition as a function of material composition, other alternative materials with unparalleled advantages. In addition, bone contains a large number of minerals. And the calcium and phosphorus ratio is appropriate.The calcium and collagen cross-linking makes it easier to absorb calcium, so calcium is a worthwhile use of the calcium salt.
China is a major livestock breeding and fishing country. But in the long time, because of the not enough understanding of the bones and backward processing technology, a large number of animal food processing by-products have not been effective development and utilization, which not only affects the enterprise economy improving efficiency, but also a waste of resources and environmental pollution. Therefore, to improve the utilization of animal bone and the development of bone food are not only nourishing and healthy, but also increase the added-value of products, waste to treasure, to reduce environmental pollution, increase economic efficiency, while opening a new food resources for nutrition related subjects development.
PEF (pulsed electric fields) has been recognized as one of the most advanced sterilization techniques and in recent years has been gradually applied to extraction of bioactive substances and to separate the field. In this paper, the research aims at extract the bone protein by means of enzyme under the PEF. And research on bone protein-calcium structure and texture. And the development of a bovine bone-protein complex beverages. Through animal experiments , researching on the effect of calcium supplementation of products for two types of calcium. Not only for PEF technology in the field of protein will provide a reference, but also for the comprehensive utilization of bone has opened up a valuable road.
The specific content and the results of research are as follows:
1. Study on bone protein hydrolysis by PEF technology
(1) Through the single factor experiment, the paper separately studied the effect of electric field intensity, pulse number, liquid/solid ratio, enzyme content on the extraction yield of collagen.
When the liquid/solid ratio was 10:1, the enzyme content was 3%, the flow velocity was 18ml/min and the pulsed number was 8, the electric field intensity of a significant impact on protein extraction. So the better electric field intensity is 30kV/cm for extraction of bone protein by PEF.
When the liquid/solid ratio was 10:1, the enzyme content was 3%, the flow velocity was 18ml/min and the electric field intensity is 30kV/cm, the pulsed number of a significant impact on protein extraction. So the better pulsed number is 8 for extraction of bone protein by PEF.
When the enzyme content was 3%, the pulsed number was 8, the flow velocity was 18ml/min and the electric field intensity is 30kV/cm, the liquid/solid ratio of a significant impact on protein extraction. So the better liquid/solid ratio is 10:1 for extraction of bone protein by PEF.
When the liquid/solid ratio was 10:1, the pulsed number was 8, the flow velocity was 18ml/min and the electric field intensity is 30kV/cm, the enzyme content of a significant impact on protein extraction. So the better enzyme content is 3% for extraction of bone protein by PEF.
(2) In order to achieve the purpose of precise optimization, a quadratic general rotary unitized design was arranged near the best parameter which was fixed by orthogonal test, and the Mathematics model was established. The regression equation was:
For the level of 0.01, deleting inapparent item, the simple regression equation is: Through the regression equation to determine the impact of the rate of protein extraction followed the order of primary and secondary factors were liquid/solid ratio> enzyme content> pulsed number> electric field intensity.
(3) Through the experiment analyse, the best combination parameters were fixed exactly: pulse number of 10, electric field intensity of 30kV/cm, liquid/solid ratio is 12.5:1, enzyme content is 3.5%, at this point extraction rate of 46.51%.
2. Study on preparation for bone protein-calcium and its structure and texture
(1) Studied bone protein-calcium preparation methods, through the orthogonal test to determine bone protein-calcium complex the best extraction conditions.
The best condition of hydrolysis bone calcium by PEF: water/bone powder ratio is 10:1, bone powder/mixed acid is 5:3, citric acid/malic acid is 1:1.5, electric field intensity is 20kV/cm, the flow velocity was 18ml/min, pulsed number is 12.
The best condition of preparation for bone protein-calcium under high-temperature: temperature is 115℃, heating time is 25min, and the order of primary and secondary factors are time>temperature.
(2) Analyse the structure of bone protein-calcium by FTIR and results as follows: Characteristic peaks at 1435 cm-1 caused by C-N stretching vibration. They belong to collagen peptide amino acid side chain groups; Medium intensity characteristic bands at 602 cm-1、670 cm-1、887 cm-1 caused by N-H-plane rocking vibration; All these show that the original collagen matrix of bone has been partially hydrolyzed to form acid residues of collagen and small peptide; Phosphate characteristic peaks at 2301 cm-1 Show that in the original hydroxyapatite meal to be biodegradable, phosphate free form, may be related to the formation of calcium phosphate; In addition, Amino stretching vibration and the amide I band shows the movement of collagen peptides and amino and carboxyl group are involved in calcium coordination; Characteristic peaks at 5354cm-1 Strengthen may caused by Ca-N stretching vibration; At 1387 cm-1-1078 cm-1, there are lots of Strengthen band, but can’t find its ascription, may cause by other kind of complex calcium.
(3) Analyse the structure of bone protein-calcium by XRD. The bone protein-calcium is make up of CaPO3(OH)·2H2O, Ca(H2PO4)·2H2O and Ca3(PO3)6·10H2O.
3. Study on preparation for bovine bone-protein complex beverages.
(1) The flow chart of bovine bone-protein complex beverages as follows:
Material→Homogenize→Prepare→Second-homogenize→Perfusion→Sterilization→Finished Product
(2) Study on stability of the bovine bone-protein complex beverages and results as follows:
The higher homogeneous pressure, the better the stability of beverage.The better first homogeneous pressure is 30Mpa, the second is 40Mpa.
Emulsifier choose 0.2% GMS, Stabilizer choose PGA and CMC-Na and concentration ratio is 3:1.
(3) By orthogonal test to determine the best formula additives as follows: GMS0.3%, PGA0.3%, CMC-Na0.1%, Sodium tripolyphosphate0.3%.At this point, system is the most stable. The order of primary and secondary factors are PGA> Sodium tripolyphosphate> CMC-Na>GMS.
4. Animal experiments
(1) Mice were tested 6 weeks to monitor the weight of mice in the early subjects in the comparison group and experimental group of mice had no significant differences in body weight, the comparison group and experimental groups of mice both the overall weight increase, with the subjects time, significant inter-group changes. And beverage> powder> comparison> blank.
(2) The apparent calcium absorption rate measurement results show that the first week of each group of apparent calcium absorption rate is highest, after five weeks in each group between the apparent absorption of calcium were no significant differences between that period of absorption in the young mice calcium effect more visible.
(3) On serum calcium and serum phosphorus measurement results show that the compound of calcium and bone protein osteocalcin cattle - drinks can increase the protein complex in serum calcium, phosphorus content, indicating that increased calcium absorption, but the difference was not significant.
(4) Determination of ALP results show that the experimental group and the control group serum ALP activity was significantly higher than blank group. And the experimental group was higher than the control group. That they both increase the ALP activity, can significantly promote the absorption of bone metabolism.
(5) Determination of StrACP results show that the experimental group and the control group serum ALP activity was significantly lower than blank group, and beverage< powder< the control group. The results show that bone protein-calcium and bone-protein complex beverages both can reduce StrACP activities and reduce the number of osteoclasts, inhibit osteoclast activity, so that bone formation than bone resorption, increase bone mass.
(6) On mice femur measurement results show that bone protein-calcium and bone-protein complex beverages both do better in bone growth and development, suggesting that delaying the aging of the skeleton to a certain extent.
引文
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