壳寡糖金属配合物对扇贝体内重金属镉的影响
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摘要
随着我国工业化的发展,大量的重金属以多种方式进入海洋,由此造成水环境中的重金属含量不断增加。重金属镉(Cd)是主要的海洋污染物之一,它易被生物体吸收富集,由此已对食物的安全构成了较大的隐患,尤其是水产品,其中贝类污染尤为严重。而人们通常又是以生吃或半生熟吃为主还不去内脏,使得重金属通过食物链传递到人,对人类健康形成了潜在威胁。
目前,关于提高贝类品质净化的方法主要有净水区暂养和净化工厂净化两种方法。但上述两种方法由于劳动强度大、时间长,在经济上是很难行得通;且贝类从生长区移入暂养区后,存在着污染暂养区水质的可能。贝类净化目前多数针对致病性微生物,对于受石油烃、重金属、农药及海洋毒素污染的贝类还未见有效净化处理技术,因此如何减少Cd在双壳贝类体内残留或有效脱Cd已是当务之急,这对保障我国贝类水产品的质量安全有重要意义。
本文通过对青岛几个主要的水产品批发市场一年间市售的栉孔扇贝重金属Cd的调查,结果发现栉孔扇贝多数存在重金属Cd超标现象,其中只有33.3%的样品未超过限量标准,说明如何脱除贝类Cd残留已是水产品的当务之急。进一步研究还发现,栉孔扇贝对Cd具有很强的富集能力,且内脏团中重金属Cd含量相当于闭壳肌中的20倍,其对Cd的富集与水温成正相关,栉孔扇贝个体大小对Cd富集差异较小。
由于壳寡糖(COS)具有良好的水溶性,并且含有大量的羟基(-OH)、氨基(-NH2)或酰氨基(-NHCOCH3),是一些金属离子的良好配体。因此,本文将壳寡糖与Ca、Mg、Zn、Fe及稀土元素(Ree)反应,制备了五种壳寡糖金属配合物,即COS-Ca、COS-Mg、COS-Zn、COS-Fe、COS-Ree。并对其配合条件,时间、温度、pH、金属添加量、反应液浓度及金属盐种类进行优化。采用紫外-可见光谱、红外光谱、荧光光谱对其进行表征,结果证明壳寡糖与金属原子所形成的配合物主要氨基(-NH2)和仲羟基(-OH)上N和O与金属原子形成配价键。
将制备的五种壳寡糖金属配合物应用到暴露Cd栉孔扇贝,发现配合物对栉孔扇贝体内Cd含量均有一定的清除作用。COS-Ca、COS-Mg和COS-Ree处理组Cd消除效果较好,12天内COS-Ca、COS-Mg和COS-Ree处理组栉孔扇贝体内Cd含量降低了46%、52%和47.8%。COS-Zn和COS-Fe处理组Cd脱除率达到17%、13%。壳寡糖金属配合物在脱Cd同时可以改变栉孔扇贝体内Ca、Mg及Fe的含量。其中配合物添加剂量及温度对栉孔扇贝脱Cd影响较大。按1～1.5kg栉孔扇贝、养殖海水40L计算,分别添加COS-Ca、COS-Mg、COS-Ree、COS-Zn和COS-Fe量为0.35g、0.21g、0.21～0.28g、0.21g和0.35g,其栉孔扇贝体内Cd脱除率就可达到31%、27%、39%、17%和13%。
壳寡糖金属配合物主要是降低了了栉孔扇贝肾脏及内脏团中与蛋白、小分子物质结合的重金属Cd含量。经COS-Ca、COS-Mg和COS-Ree三种壳寡糖金属配合物处理5天,相比阴性对照组栉孔扇贝肾脏中的Cd含量分别降低25.2%、36.1%和37.5%。栉孔扇贝暴露Cd经COS-Ca、COS-Mg及COS-Ree处理,影响了栉孔扇贝体内的抗氧化酶系统过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)酶活及丙二醛(MDA)含量,发挥了抗氧化作用,维持了自由基的产生与消除平衡,从生理学方面证实了壳寡糖金属配合物对栉孔扇贝体内Cd的清除机理。进而通过壳寡糖与镉金属硫蛋白(Cd-MT)的置换反应,从化学置换上证实了COS-Ca、COS-Mg及COS-Ree对栉孔扇贝体内Cd脱除的机理。
壳寡糖金属配合物不但可以有效的脱除栉孔扇贝体内Cd,而且对其他贝类紫贻贝(Mytilus edulis)、长牡蛎(Crassostrea gigas)、缢蛏(Sinonovacula constricta)、杂色蛤子(Venerupis variegata)、海湾扇贝(Argopecten irradiams)体内的Cd也有很好的脱除效果。
我国是贝类养殖大国,2007年我国贝类总产量1073.3万吨,占世界贝类生产总量的60%以上。但养殖贝类重金属污染问题较为严重,Cd含量超标现象尤为严重,已是食物安全重大隐患之一。而目前尚无有效地消除鲜活贝类重金属残留的方法,如果这一问题不能有效解决,将严重影响我国养殖贝类的可持续发展。本文所研究的多糖金属配合物对双壳贝类Cd含量的影响,是开发鲜活水产品脱重金属残留的应用基础研究内容之一,可为今后开发具有减少重金属元素残留的养殖饲料添加剂及鱼、贝类净化或暂养脱金属专用剂提供基础,为水产品安全、人类健康提供保障。
With the development of industry, the large amount of heavy metals have polluted the ocean with all kinds of ways, and the content of heavy metal in water environment have been continuously increasing. And the cadmium is one of the mainest marine pollutant, it is easy to be absorbed and accumulated by the creature, which have formed the serious potential dangers to food safety, especially aquatic product, the shellfish. However, people usually like eating the raw or undercooked, and no removal the viscera, the heavy metals invaded the body by the transport through food chain, which have formed potential threat.
Now, there are two methods to improve the shellfish cleaning, including temporary raising in clean water and cleaning in factory. Considering the great labour intensity and consuming time, the two methods were difficult to carry out, and it may pollute the water in temporary zone when the shellfish moved the temporary zone from growth zone. The shellfish purification technique is mainly against pathogenic microorganism, there are no available methods to purify the shellfish polluted by petroleum hydrocarbon, heavy metals, pesticide and marine toxin. How to decrease the Cd in shellfish is urgent matter, which have important significance to guarantee the shellfish quality safety.
The Cd content in Chlamys ferrari collected in aquatic wholesale market in Qingdao was investigated for one year, the results showed the Cd content in the majority Chlamys ferrari were higher than standard limit, only 33.3% were less than the standard, which illustrated how to remove the Cd residue in the aquatic product is urgent matter. The Chlamys ferrari have the higher enrichment ability to Cd, and the Cd content in viscera is more than in meat by 20 times. The enrichment ability is positive correlation with raising temperature, the Cd enrichment among the different size of Chlamys ferrari has no obvious difference.
Chitosan oligosaccharide(COS) has excellent water-soluble, there are some groups, such as–OH, -NH2, -NHCOCH3, which are the optimum ligand of mental ionic. Therefore, 5 kinds of complexes were prepared by the binding between COS and Ca, Mg, Zn, Fe, Ree. And the preparation condition were optimized, including time, temperature, pH, the addition level of metals, the content of reaction solution, the type of metallic salt. Using the UV, IR, Fluorescence spectra, it was proved that metal elements binded with COS through N and O.
After Cd exposure, the Chlamys ferrari were treated with COS complexes, it was found that the all complexes can remove the Cd in viscera of Chlamys ferrari. COS-Ca, COS-Mg, COS-Ree have significant removal ability to Cd, the Cd content can be deduced by 46%, 52% and 47.8% respectively in 12 days. The removal rate of Cd reached 17% and 13% respectively by ushing COS-Zn , COS-Fe. The complexes additive dose and raising temperature have effect on removal ability of Cd. 1~1.5kg Chlamys ferrari, exposed with 0.5mg/L Cd , were raised in 40L seawater, the removal rate of Cd were 31%, 27%, 39%, 17%, 13% respectively when treated with COS-Ca, COS-Mg, COS-Ree, COS-Zn, COS-Fe, the additive dose of complexes were 0.35g, 0.21g, 0.21～0.28g, 0.21g and 0.35g respectively.
The Cd binding with protein and small molecule substances in kidney and viscera was removed by COS complexes with metal elements. Compared with negative control group, the Cd in kidney were reduced 25.2%, 36.1%, 37.5% respectively in 5 days when treated with COS-Ca, COS-Mg and COS-Ree. The COS-Ca, COS-Mg and COS-Ree have effect on the antioxidant enzyme system of Chlamys ferrari exposed with Cd, such as CAT, POD, SOD, GSH-PX and MDA. The complexes have the ability of antioxidant, maintaining the balance between creating and scavenging the free radicals. Based on physiological, theory the mechanism complexes can remove the Cd from viscera of Chlamys ferrari was proved. The mechanism was further confirmed by the replacement reaction between the complexes and Cd-MT.
The COS complexes with metal elements also can remove Cd from other shellfish except for Chlamys ferrari, such as Mytilus edulis, Crassostrea gigas, Sinonovacula constricta, Venerupis variegate, Argopecten irradiams.
Our country has the greatest stage on shellfish cultivation. The total yield of shellfish was 10.73 million tons in 2007, account for 60% yield of worldwide raising shellfish. However, the heavy metal pollution of shellfish is very serious, especially Cd, which was the one of food safety. And there are not availably technology to remove the Cd from fresh shellfish. If the problem can be solved availably, which will have great influence to the sustainable development of shellfish farming. The complexes prepared in this paper can remove Cd from fresh shellfish, which is the one of basical study of application of developing the removal of Cd from fresh shellfish, which can provide theoretical basis for exploiting feed additive and special agent of shellfish purification to remove the Cd, providing guarantee for the aquatic product safety and human health.
目前,关于提高贝类品质净化的方法主要有净水区暂养和净化工厂净化两种方法。但上述两种方法由于劳动强度大、时间长,在经济上是很难行得通;且贝类从生长区移入暂养区后,存在着污染暂养区水质的可能。贝类净化目前多数针对致病性微生物,对于受石油烃、重金属、农药及海洋毒素污染的贝类还未见有效净化处理技术,因此如何减少Cd在双壳贝类体内残留或有效脱Cd已是当务之急,这对保障我国贝类水产品的质量安全有重要意义。
本文通过对青岛几个主要的水产品批发市场一年间市售的栉孔扇贝重金属Cd的调查,结果发现栉孔扇贝多数存在重金属Cd超标现象,其中只有33.3%的样品未超过限量标准,说明如何脱除贝类Cd残留已是水产品的当务之急。进一步研究还发现,栉孔扇贝对Cd具有很强的富集能力,且内脏团中重金属Cd含量相当于闭壳肌中的20倍,其对Cd的富集与水温成正相关,栉孔扇贝个体大小对Cd富集差异较小。
由于壳寡糖(COS)具有良好的水溶性,并且含有大量的羟基(-OH)、氨基(-NH2)或酰氨基(-NHCOCH3),是一些金属离子的良好配体。因此,本文将壳寡糖与Ca、Mg、Zn、Fe及稀土元素(Ree)反应,制备了五种壳寡糖金属配合物,即COS-Ca、COS-Mg、COS-Zn、COS-Fe、COS-Ree。并对其配合条件,时间、温度、pH、金属添加量、反应液浓度及金属盐种类进行优化。采用紫外-可见光谱、红外光谱、荧光光谱对其进行表征,结果证明壳寡糖与金属原子所形成的配合物主要氨基(-NH2)和仲羟基(-OH)上N和O与金属原子形成配价键。
将制备的五种壳寡糖金属配合物应用到暴露Cd栉孔扇贝,发现配合物对栉孔扇贝体内Cd含量均有一定的清除作用。COS-Ca、COS-Mg和COS-Ree处理组Cd消除效果较好,12天内COS-Ca、COS-Mg和COS-Ree处理组栉孔扇贝体内Cd含量降低了46%、52%和47.8%。COS-Zn和COS-Fe处理组Cd脱除率达到17%、13%。壳寡糖金属配合物在脱Cd同时可以改变栉孔扇贝体内Ca、Mg及Fe的含量。其中配合物添加剂量及温度对栉孔扇贝脱Cd影响较大。按1～1.5kg栉孔扇贝、养殖海水40L计算,分别添加COS-Ca、COS-Mg、COS-Ree、COS-Zn和COS-Fe量为0.35g、0.21g、0.21～0.28g、0.21g和0.35g,其栉孔扇贝体内Cd脱除率就可达到31%、27%、39%、17%和13%。
壳寡糖金属配合物主要是降低了了栉孔扇贝肾脏及内脏团中与蛋白、小分子物质结合的重金属Cd含量。经COS-Ca、COS-Mg和COS-Ree三种壳寡糖金属配合物处理5天,相比阴性对照组栉孔扇贝肾脏中的Cd含量分别降低25.2%、36.1%和37.5%。栉孔扇贝暴露Cd经COS-Ca、COS-Mg及COS-Ree处理,影响了栉孔扇贝体内的抗氧化酶系统过氧化氢酶(CAT)、过氧化物酶(POD)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)酶活及丙二醛(MDA)含量,发挥了抗氧化作用,维持了自由基的产生与消除平衡,从生理学方面证实了壳寡糖金属配合物对栉孔扇贝体内Cd的清除机理。进而通过壳寡糖与镉金属硫蛋白(Cd-MT)的置换反应,从化学置换上证实了COS-Ca、COS-Mg及COS-Ree对栉孔扇贝体内Cd脱除的机理。
壳寡糖金属配合物不但可以有效的脱除栉孔扇贝体内Cd,而且对其他贝类紫贻贝(Mytilus edulis)、长牡蛎(Crassostrea gigas)、缢蛏(Sinonovacula constricta)、杂色蛤子(Venerupis variegata)、海湾扇贝(Argopecten irradiams)体内的Cd也有很好的脱除效果。
我国是贝类养殖大国,2007年我国贝类总产量1073.3万吨,占世界贝类生产总量的60%以上。但养殖贝类重金属污染问题较为严重,Cd含量超标现象尤为严重,已是食物安全重大隐患之一。而目前尚无有效地消除鲜活贝类重金属残留的方法,如果这一问题不能有效解决,将严重影响我国养殖贝类的可持续发展。本文所研究的多糖金属配合物对双壳贝类Cd含量的影响,是开发鲜活水产品脱重金属残留的应用基础研究内容之一,可为今后开发具有减少重金属元素残留的养殖饲料添加剂及鱼、贝类净化或暂养脱金属专用剂提供基础,为水产品安全、人类健康提供保障。
With the development of industry, the large amount of heavy metals have polluted the ocean with all kinds of ways, and the content of heavy metal in water environment have been continuously increasing. And the cadmium is one of the mainest marine pollutant, it is easy to be absorbed and accumulated by the creature, which have formed the serious potential dangers to food safety, especially aquatic product, the shellfish. However, people usually like eating the raw or undercooked, and no removal the viscera, the heavy metals invaded the body by the transport through food chain, which have formed potential threat.
Now, there are two methods to improve the shellfish cleaning, including temporary raising in clean water and cleaning in factory. Considering the great labour intensity and consuming time, the two methods were difficult to carry out, and it may pollute the water in temporary zone when the shellfish moved the temporary zone from growth zone. The shellfish purification technique is mainly against pathogenic microorganism, there are no available methods to purify the shellfish polluted by petroleum hydrocarbon, heavy metals, pesticide and marine toxin. How to decrease the Cd in shellfish is urgent matter, which have important significance to guarantee the shellfish quality safety.
The Cd content in Chlamys ferrari collected in aquatic wholesale market in Qingdao was investigated for one year, the results showed the Cd content in the majority Chlamys ferrari were higher than standard limit, only 33.3% were less than the standard, which illustrated how to remove the Cd residue in the aquatic product is urgent matter. The Chlamys ferrari have the higher enrichment ability to Cd, and the Cd content in viscera is more than in meat by 20 times. The enrichment ability is positive correlation with raising temperature, the Cd enrichment among the different size of Chlamys ferrari has no obvious difference.
Chitosan oligosaccharide(COS) has excellent water-soluble, there are some groups, such as–OH, -NH2, -NHCOCH3, which are the optimum ligand of mental ionic. Therefore, 5 kinds of complexes were prepared by the binding between COS and Ca, Mg, Zn, Fe, Ree. And the preparation condition were optimized, including time, temperature, pH, the addition level of metals, the content of reaction solution, the type of metallic salt. Using the UV, IR, Fluorescence spectra, it was proved that metal elements binded with COS through N and O.
After Cd exposure, the Chlamys ferrari were treated with COS complexes, it was found that the all complexes can remove the Cd in viscera of Chlamys ferrari. COS-Ca, COS-Mg, COS-Ree have significant removal ability to Cd, the Cd content can be deduced by 46%, 52% and 47.8% respectively in 12 days. The removal rate of Cd reached 17% and 13% respectively by ushing COS-Zn , COS-Fe. The complexes additive dose and raising temperature have effect on removal ability of Cd. 1~1.5kg Chlamys ferrari, exposed with 0.5mg/L Cd , were raised in 40L seawater, the removal rate of Cd were 31%, 27%, 39%, 17%, 13% respectively when treated with COS-Ca, COS-Mg, COS-Ree, COS-Zn, COS-Fe, the additive dose of complexes were 0.35g, 0.21g, 0.21～0.28g, 0.21g and 0.35g respectively.
The Cd binding with protein and small molecule substances in kidney and viscera was removed by COS complexes with metal elements. Compared with negative control group, the Cd in kidney were reduced 25.2%, 36.1%, 37.5% respectively in 5 days when treated with COS-Ca, COS-Mg and COS-Ree. The COS-Ca, COS-Mg and COS-Ree have effect on the antioxidant enzyme system of Chlamys ferrari exposed with Cd, such as CAT, POD, SOD, GSH-PX and MDA. The complexes have the ability of antioxidant, maintaining the balance between creating and scavenging the free radicals. Based on physiological, theory the mechanism complexes can remove the Cd from viscera of Chlamys ferrari was proved. The mechanism was further confirmed by the replacement reaction between the complexes and Cd-MT.
The COS complexes with metal elements also can remove Cd from other shellfish except for Chlamys ferrari, such as Mytilus edulis, Crassostrea gigas, Sinonovacula constricta, Venerupis variegate, Argopecten irradiams.
Our country has the greatest stage on shellfish cultivation. The total yield of shellfish was 10.73 million tons in 2007, account for 60% yield of worldwide raising shellfish. However, the heavy metal pollution of shellfish is very serious, especially Cd, which was the one of food safety. And there are not availably technology to remove the Cd from fresh shellfish. If the problem can be solved availably, which will have great influence to the sustainable development of shellfish farming. The complexes prepared in this paper can remove Cd from fresh shellfish, which is the one of basical study of application of developing the removal of Cd from fresh shellfish, which can provide theoretical basis for exploiting feed additive and special agent of shellfish purification to remove the Cd, providing guarantee for the aquatic product safety and human health.
引文
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