中国林蛙皮透明质酸提取技术的研究
摘要
透明质酸(Hyaluronic Acid,HA)是一种高分子量的直链多糖，由N-乙酰氨基葡糖和葡糖醛酸二糖单位重复交替连接形成。HA广泛的分布于动物和人体的结缔组织中，但不同组织来源的HA化学结构完全相同，只是在含量多少和相对分子量大小上存在差异，没有种属特异性。
经过多年的研究，人们对HA的结构、理化性质及生理功能已有了明确的认识。它在水溶液中的无规则卷曲状态和它的流体动力学特点赋予其重要的物理特性，即高度粘弹性、可塑性、渗透性和良好的生物相容性，作为一种可吸收、可降解的生物材料，其应用研究领域不断扩大，表现出良好的市场前景。由于HA具有保湿功能，广泛用于化妆品和口服美容保健食品中；作为粘弹性保护剂，用于眼科手术和关节疾病的治疗；作为媒介在滴眼液中广泛应用；交联的HA在粘连的预防和软组织修复等应用中获得成功；HA与其他药物反应形成的加合物对药物发挥控释作用，可达到定向和定时释放的目的。
目前，制备HA主要有两种方法，一种是从鸡冠、人脐带、动物眼球等动物组织中获得，制得的HA分子量较高，粘度好，保湿性好，但由于受原料资源的限制，不能满足市场对HA的需求；另一种是微生物发酵生产法，是从某些茵属的细菌的荚膜中提取HA，其分离、纯化工艺简单，成本低，是今后的发展方向，但国内还处于研究阶段没有成型的工艺，组织提取法仍是目前的主要生产方法。
中国林蛙是一种珍贵的两栖类药用经济动物，具有重要的保健功能，但目前市场上只有少数的林蛙油产品，林蛙资源没有得到充分利用。本试验是以雄性林蛙及摘除蛙油的雌性林蛙的皮肤为原料，采用现代生物工程技术及超滤等高新技术对原料进行加工处理，制得高纯度的HA，与将林蛙作为烹饪原料相比可增值16倍以上，因此可以大大提高林蛙产品的附加值，使我省林蛙产业的经济效益及社会效益大为提高。
本文作为吉林市科技局“林蛙高效天然保湿因子的研究”课题的部分内容，对林蛙皮HA的制备工艺进行了研究，确定了以下工艺条件。
1、确定了原料预处理方法。在低温条件下，对林蛙皮进行高速组织破碎，通过正交试验确定了最佳的破碎条件：温度为4℃，时间为3min，PH值为6.0，组织捣碎机转速为13000r／min。
2、确定了最佳提取工艺。采用复合酶法提取HA，由于作用条件温和，制得HA的活性较高，得率较高，通过正交试验确定了最佳复合酶的酶解条件：温度为45℃，
吉林农业大学硕士学位论文
中国林蛙皮透明质酸提取技术的研究
时间为sh，PH为8.5，酶用量为2%。
3、确定了最佳纯化工艺。采用链霉蛋白酶二次酶解、络合沉淀和超滤结合的方法，
对HA中间品进行纯化处理，制得了高纯度的HA精品。链霉蛋白酶最佳酶解的条件
是:温度为40℃，时间为12h，PH为8，酶量为0.25%;超滤的最佳操作条件是:压
力为0.1一0.15MPa，温度为55℃。
经本工艺分离提取出的HA，采用红外吸收光谱和紫外光谱法对其进行了定性测
定，采用咔哇(c arbazole)法和Elson-Morgall法对HA的含量及纯度进行了测定，结果表
明:本试验提取样品HA的红外图谱和紫外图谱与美国Sigma公司的标品HA的红外
图谱和紫外图谱一致，其外观呈白色粉末状，葡糖醛酸含量44.2%，氨基葡糖含量
41.5%，其摩尔比为1:1，属化妆品级及食品级。测得HA杂蛋白含量在0.25一0.35%之
间。采用shimada法对得到的HA精品进行了相对平均分子量的测定，结果为1 .6x 106。
依据《食品安全性毒理学评价程序和方法》GB 15193.1一1994的规定对林蛙皮HA
进行小鼠经口急性毒性试验、细菌致突变试验、小鼠精子崎形试验、30天喂养试验。
结果表明:产品属无毒级，无致突变性及遗传毒性，说明林蛙皮HA对人体无毒无害，
可以食用及外用。
Hyaluronic acid(HA) is a kind of high molecular weight and straight chain polyose. It is linked alternatedly and repeatedly by N-acety_glucosam ine and glucuronic acid units. HA extensively distributes in connective tissues of animals and human, but different HA that come from different tissues have the same structure. They only vary at content and relative molecular weight and have no specificity.
After researched for years, HA's structure, physical, and chemical characteristics and physiological functions have been clearly found. It's nun-convolute and other fluid dynamics properties make them high visoelastic, plastic, permeable and bio-soluble, so it can be used as absorbable and decomposable biogenic materials, and its practical studying field is larger and larger, putting up favorable market prospect. Because of its moisty maintainance, it has been widely used in cosmetics , oral cosmeticologic health food, eye operations joint illness treating and in eye drops as media cross-linking. HA has been applied in precaution and soft tissues repairment and achieved great success. The adduct which is formed by reacting of HA and other medicines can control in certain time medicine's releasing.
Presently,HA is prepared in two ways, one is from animal tissues, such as cock's cumb, human umbilical cord, eye balls. This kind of HA has good adhensiveness and moist maintainance and Mr are relatively high, but the sources are not enough to meet with the requirement of the market. The other way is microbe fermentation. Extracting HA from some types of bacteria's capsule, in this method, the processes of isolation, purification are simple and the cost is low. This is the correct developing direction in future, but it is researched in our country and there is no ripened process. At present, extracting from tissues is the main producing method.
R.tempoviductus is a precious amphibious animal which used in medicine and has important health function, however, in present market, there are only few R.tempoviductus oil products, the sources are not used sufficiently In this experiment, high purity HA was produced, using male ones or female ones which removed fat as material,adopting modern bioengineering technique and UF to deal with the materials,which can increase the value to 16 times comparing with cooking materials.So the R.tempoviductus products' additional value is greatly .increased, which improve the economic and society efficiency of R.
tempoviductus plants in JiLin province.
This thesis is a part content of the item 'the study of high effective moist maintenance factor of R.tempoviductus' which belong to technique department of JiLin city. After studying on preparation of HA in skin, the following process conditions were determined.
1.Determine the method of materials' pretreatment. Fracture the R.tempoviductus's skin by mini tissue fracture at low temperature. By high orthogonal, the optimum fracturing condition was determined. Temperature is 4C, time is 3min,PH is 6.0 , the rev of tissue fracturing is 13000r/min.
2.Determine the optimum extracting process. Extract HA by compound enzyme, because of the temperate condition, the HA is relatively more active , higher yield, the optimum compound zymolysis condition temperature is 45C ,time is 5h, PH is 8.5,the amount of enzyme is 2%.
3.Determine the optimum purity process. By using twice pronase zymolysis, complex action and UF, the HA intermidiary was purified. The optimum Pronase E zymolysis condition: temperature is 40C,time is 12h ,PH is 8.0, the amount of Enzyme is 0.25%. The optimum ultrafiltration condition: pressure is 0.1 MPa, temperature is 55C.
After isolated, extracted by this process, the HA was qualitively tested through infraction spectrum and UV spectrum, it's content and purity were tested by carbazole and Elason-Morgan. The results have shown that HA extracted by this process is in accordance with that of the purity which comes from Sigma company of America. It is white powder. The content of glucuromic acid and glucosam ine are 44.2% and 41.5% res
经过多年的研究，人们对HA的结构、理化性质及生理功能已有了明确的认识。它在水溶液中的无规则卷曲状态和它的流体动力学特点赋予其重要的物理特性，即高度粘弹性、可塑性、渗透性和良好的生物相容性，作为一种可吸收、可降解的生物材料，其应用研究领域不断扩大，表现出良好的市场前景。由于HA具有保湿功能，广泛用于化妆品和口服美容保健食品中；作为粘弹性保护剂，用于眼科手术和关节疾病的治疗；作为媒介在滴眼液中广泛应用；交联的HA在粘连的预防和软组织修复等应用中获得成功；HA与其他药物反应形成的加合物对药物发挥控释作用，可达到定向和定时释放的目的。
目前，制备HA主要有两种方法，一种是从鸡冠、人脐带、动物眼球等动物组织中获得，制得的HA分子量较高，粘度好，保湿性好，但由于受原料资源的限制，不能满足市场对HA的需求；另一种是微生物发酵生产法，是从某些茵属的细菌的荚膜中提取HA，其分离、纯化工艺简单，成本低，是今后的发展方向，但国内还处于研究阶段没有成型的工艺，组织提取法仍是目前的主要生产方法。
中国林蛙是一种珍贵的两栖类药用经济动物，具有重要的保健功能，但目前市场上只有少数的林蛙油产品，林蛙资源没有得到充分利用。本试验是以雄性林蛙及摘除蛙油的雌性林蛙的皮肤为原料，采用现代生物工程技术及超滤等高新技术对原料进行加工处理，制得高纯度的HA，与将林蛙作为烹饪原料相比可增值16倍以上，因此可以大大提高林蛙产品的附加值，使我省林蛙产业的经济效益及社会效益大为提高。
本文作为吉林市科技局“林蛙高效天然保湿因子的研究”课题的部分内容，对林蛙皮HA的制备工艺进行了研究，确定了以下工艺条件。
1、确定了原料预处理方法。在低温条件下，对林蛙皮进行高速组织破碎，通过正交试验确定了最佳的破碎条件：温度为4℃，时间为3min，PH值为6.0，组织捣碎机转速为13000r／min。
2、确定了最佳提取工艺。采用复合酶法提取HA，由于作用条件温和，制得HA的活性较高，得率较高，通过正交试验确定了最佳复合酶的酶解条件：温度为45℃，
吉林农业大学硕士学位论文
中国林蛙皮透明质酸提取技术的研究
时间为sh，PH为8.5，酶用量为2%。
3、确定了最佳纯化工艺。采用链霉蛋白酶二次酶解、络合沉淀和超滤结合的方法，
对HA中间品进行纯化处理，制得了高纯度的HA精品。链霉蛋白酶最佳酶解的条件
是:温度为40℃，时间为12h，PH为8，酶量为0.25%;超滤的最佳操作条件是:压
力为0.1一0.15MPa，温度为55℃。
经本工艺分离提取出的HA，采用红外吸收光谱和紫外光谱法对其进行了定性测
定，采用咔哇(c arbazole)法和Elson-Morgall法对HA的含量及纯度进行了测定，结果表
明:本试验提取样品HA的红外图谱和紫外图谱与美国Sigma公司的标品HA的红外
图谱和紫外图谱一致，其外观呈白色粉末状，葡糖醛酸含量44.2%，氨基葡糖含量
41.5%，其摩尔比为1:1，属化妆品级及食品级。测得HA杂蛋白含量在0.25一0.35%之
间。采用shimada法对得到的HA精品进行了相对平均分子量的测定，结果为1 .6x 106。
依据《食品安全性毒理学评价程序和方法》GB 15193.1一1994的规定对林蛙皮HA
进行小鼠经口急性毒性试验、细菌致突变试验、小鼠精子崎形试验、30天喂养试验。
结果表明:产品属无毒级，无致突变性及遗传毒性，说明林蛙皮HA对人体无毒无害，
可以食用及外用。
Hyaluronic acid(HA) is a kind of high molecular weight and straight chain polyose. It is linked alternatedly and repeatedly by N-acety_glucosam ine and glucuronic acid units. HA extensively distributes in connective tissues of animals and human, but different HA that come from different tissues have the same structure. They only vary at content and relative molecular weight and have no specificity.
After researched for years, HA's structure, physical, and chemical characteristics and physiological functions have been clearly found. It's nun-convolute and other fluid dynamics properties make them high visoelastic, plastic, permeable and bio-soluble, so it can be used as absorbable and decomposable biogenic materials, and its practical studying field is larger and larger, putting up favorable market prospect. Because of its moisty maintainance, it has been widely used in cosmetics , oral cosmeticologic health food, eye operations joint illness treating and in eye drops as media cross-linking. HA has been applied in precaution and soft tissues repairment and achieved great success. The adduct which is formed by reacting of HA and other medicines can control in certain time medicine's releasing.
Presently,HA is prepared in two ways, one is from animal tissues, such as cock's cumb, human umbilical cord, eye balls. This kind of HA has good adhensiveness and moist maintainance and Mr are relatively high, but the sources are not enough to meet with the requirement of the market. The other way is microbe fermentation. Extracting HA from some types of bacteria's capsule, in this method, the processes of isolation, purification are simple and the cost is low. This is the correct developing direction in future, but it is researched in our country and there is no ripened process. At present, extracting from tissues is the main producing method.
R.tempoviductus is a precious amphibious animal which used in medicine and has important health function, however, in present market, there are only few R.tempoviductus oil products, the sources are not used sufficiently In this experiment, high purity HA was produced, using male ones or female ones which removed fat as material,adopting modern bioengineering technique and UF to deal with the materials,which can increase the value to 16 times comparing with cooking materials.So the R.tempoviductus products' additional value is greatly .increased, which improve the economic and society efficiency of R.
tempoviductus plants in JiLin province.
This thesis is a part content of the item 'the study of high effective moist maintenance factor of R.tempoviductus' which belong to technique department of JiLin city. After studying on preparation of HA in skin, the following process conditions were determined.
1.Determine the method of materials' pretreatment. Fracture the R.tempoviductus's skin by mini tissue fracture at low temperature. By high orthogonal, the optimum fracturing condition was determined. Temperature is 4C, time is 3min,PH is 6.0 , the rev of tissue fracturing is 13000r/min.
2.Determine the optimum extracting process. Extract HA by compound enzyme, because of the temperate condition, the HA is relatively more active , higher yield, the optimum compound zymolysis condition temperature is 45C ,time is 5h, PH is 8.5,the amount of enzyme is 2%.
3.Determine the optimum purity process. By using twice pronase zymolysis, complex action and UF, the HA intermidiary was purified. The optimum Pronase E zymolysis condition: temperature is 40C,time is 12h ,PH is 8.0, the amount of Enzyme is 0.25%. The optimum ultrafiltration condition: pressure is 0.1 MPa, temperature is 55C.
After isolated, extracted by this process, the HA was qualitively tested through infraction spectrum and UV spectrum, it's content and purity were tested by carbazole and Elason-Morgan. The results have shown that HA extracted by this process is in accordance with that of the purity which comes from Sigma company of America. It is white powder. The content of glucuromic acid and glucosam ine are 44.2% and 41.5% res
引文
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