壳聚糖有机酸盐的制备及其抑菌性研究
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摘要
壳聚糖又名甲壳胺,是甲壳素脱乙酰的产物,是一种阳离子多糖。近年的实验证明,壳聚糖具有广谱的抗菌性能,因此已在实际生产当中得到了应用。但是壳聚糖在碱性以及中性条件下水溶性差,这在一定程度上影响了它的应用。为了改进壳聚糖的水溶性,多种壳聚糖衍生物被合成出来,但是这些合成产物中,要么对反应条件的要求过高,要么产物被加入了新的基团,而新的基团反而影响了壳聚糖的抑菌性或者影响了产物的安全性。
在本论文中,我们将1700kDa的壳聚糖进行酸降解,得到分子量为82kDa,脱乙酰度为88.7%的小分子量壳聚糖。通过将壳聚糖与有机酸盐在有机溶剂中的反应制备得到了四种固态壳聚糖有机酸盐:壳聚糖甲酸盐、壳聚糖乙酸盐、壳聚糖丙酸盐和壳聚糖乳酸盐。四种壳聚糖有机酸盐的电位滴定曲线对比壳聚糖的电位滴定曲线可以发现,突越位点只有一个。FTIR检测表明,在有机溶剂中,小分子量壳聚糖与四种有机酸(甲酸、乙酸、丙酸、乳酸)以离子键结合,在壳聚糖的氨基上形成了–NH3+.RCOO-结构,从而形成四种壳聚糖有机酸盐(CF、CA、CP、CL)。同时水溶性和稳定性实验证明,四种有机酸盐具有良好的水溶性且在一定时间内具有稳定性。相对于其它壳聚糖衍生物的制备方法,此种制备方法简单,成本较低。
抑菌实验研究了四种壳聚糖有机酸盐(CF、CA、CP、CL)对细菌( Escherichia.coli、Staphylococcus.aureus )和真菌( Candida. guilliermondii)生长的抑制作用。从之前的实验中发现,当壳聚糖有机酸盐加入到培养基中以后,会导致培养基的吸光度发生变化,因而对使用普通的浊度法进行抑菌效果检测增加了难度。为了解决这一问题,首先将MTT法与浊度法的检测效果进行了比较,证明在本实验中,MTT法能够很好的代替浊度法,而且检测结果不受培养基浊度变化的干扰。壳聚糖有机酸盐对三种微生物的最小抑制浓度因有机酸盐种类及菌种的不同而表现出一定的差异,但是随壳聚糖有机酸盐浓度的增加,四种盐对三种微生物的作用情况具有相同的变化趋势。以CA对E.coli为例:在某一较低浓度(0.05% w/v)附近,有机酸盐对菌种作用不明显;当浓度继续升高到某一点(0.08% w/v),有机酸盐对微生物的生长具有轻度的促进作用;之后随壳聚糖有机酸盐浓度的增加其抑制作用逐渐增强直到达到最小抑制浓度MIC(0.13% w/v)。电镜照片显示CA作用下的三种微生物的细胞壁和细胞膜均发生了一定的改变,由此可以推测壳聚糖有机酸盐很可能是通过对细胞表面的作用抑制了微生物的生长。同时我们还对壳聚糖有机酸盐对微生物生长抑制作用的影响因素进行了探讨,随微生物种类不同,有机酸盐会表现出不同的抑菌效果;有机酸盐中所含有机酸的量较少,故而有机酸的种类对不同有机酸盐的抑菌效果影响不大;接种的菌种浓度对有机酸的最小抑制浓度会产生影响。
在本论文中,我们还对固态壳聚糖有机酸盐作为一种新型食品防腐剂的可行性进行了探讨。将CA与高级天然防腐剂柚子提取物进行了对比:据报道,柚子提取物作为一种优良的天然防腐剂,其抗菌能力是其它防腐剂的10-100倍。实验证明,一方面,CA浓度达到0.1%(w/v)时能够达到柚子提取物所具有的抗菌效果;另一方面,壳聚糖有机酸盐的价格低于柚子提取物。所以壳聚糖有机酸盐有望成为一种质优价廉的食品、化妆品防腐剂。采用特殊处理过的蔬菜样品,对CA、CL作为食品防腐剂的实际应用进行了模仿,实验证明,这种有机酸盐在一定条件下对蔬菜加工产物(萝卜)能够达到较好的防腐效果。
Chitin is a natural polysaccharide which is usually obtained from shells of crustaceans such as crab, shrimp, and crawfish. Chitosan is a partially N-deacetylated derivative of chitin, and consists of polymeric (1→4)-linked 2-amino-2-deoxy-β-D-glucopyranose units. Due to the unique polycationic nature, chitosan and its derivatives have been proposed for various applications in biomedical, food, agricultural, biotechnological and pharmaceutical products. Chitosan has a broad spectrum of antimicrobial activities, high bactericidal rates and low toxicity toward mammalian cells, making it a potential biocide in food preserving. However, the poor solubility of chitosan at neutral and higher pH limits its applications. Some modification has been attempted. But the traditional method of modifying is to introduce some covalent bond, which is usually complex and sometimes changes some of the properties of chitosan.
In this paper, a new approach to prepare solid chitosan organic salts is reported. Chitosan (Mw=82kDa, DD=88.7%) was prepared first. Chitosan and organic acid was simply reacted and the chitosan organic salts (COS) were prepared: chitosan formait (CF), chiotsan acetate (CA), chitosan propionate (CP), chitosan lactate (CL). It was demonstrated by the titration curves that there was only one inflexion point, compared with two inflexion points in chitosan. It could be inferred from the FTIR that chitosan is linked by electrovalent bond other than covalent bond with the acid, so the products still keep the structure and antibacterial activity of chitosan and can be prepared easily. The solubility, titration curve, stability, IR spectrum and other basic characterizations of the products are evaluated. It is demonstrated that the chitosan organic salts are water soluble and stable in common conditions.
The antibacterial activities against E. coli (gram negative), S. aureus (gram positive) and C. guilliermondii have been investigated by MTT method. It was found that chitosan organic salts have good antibacterial acitivities. Take CA and E.coli for example, the growth of E. coli was regular at the concentration of 0.05% (w/v); when the concentration of CA reached 0.08%(w/v), the bacteria grew more than the ones with no CA; CA would inhibit the growth of E.coli at the concentration of 0.13% (w/v). It could also inhibit the growth of S.aureus at the concentration of 0.20% (w/v), and C. guilliermondii at 15ppm (w/v). It also demonstrated that COS showed stronger antibacterial activities with the increase of concentration of COS. Different kinds of microbiology also cause different antibacterial activities. The organic acid nearly has no influence on the activities. And the concentration of the bacterium suspension during incubation has effect on the MIC of COS.
The possibility of the product being used as a new kind of preservative in foods was also investigated in this paper. It is conferred that the chitosan organic acid salts will be a kind of cheap, convenient, and effective preservative in foods.
在本论文中,我们将1700kDa的壳聚糖进行酸降解,得到分子量为82kDa,脱乙酰度为88.7%的小分子量壳聚糖。通过将壳聚糖与有机酸盐在有机溶剂中的反应制备得到了四种固态壳聚糖有机酸盐:壳聚糖甲酸盐、壳聚糖乙酸盐、壳聚糖丙酸盐和壳聚糖乳酸盐。四种壳聚糖有机酸盐的电位滴定曲线对比壳聚糖的电位滴定曲线可以发现,突越位点只有一个。FTIR检测表明,在有机溶剂中,小分子量壳聚糖与四种有机酸(甲酸、乙酸、丙酸、乳酸)以离子键结合,在壳聚糖的氨基上形成了–NH3+.RCOO-结构,从而形成四种壳聚糖有机酸盐(CF、CA、CP、CL)。同时水溶性和稳定性实验证明,四种有机酸盐具有良好的水溶性且在一定时间内具有稳定性。相对于其它壳聚糖衍生物的制备方法,此种制备方法简单,成本较低。
抑菌实验研究了四种壳聚糖有机酸盐(CF、CA、CP、CL)对细菌( Escherichia.coli、Staphylococcus.aureus )和真菌( Candida. guilliermondii)生长的抑制作用。从之前的实验中发现,当壳聚糖有机酸盐加入到培养基中以后,会导致培养基的吸光度发生变化,因而对使用普通的浊度法进行抑菌效果检测增加了难度。为了解决这一问题,首先将MTT法与浊度法的检测效果进行了比较,证明在本实验中,MTT法能够很好的代替浊度法,而且检测结果不受培养基浊度变化的干扰。壳聚糖有机酸盐对三种微生物的最小抑制浓度因有机酸盐种类及菌种的不同而表现出一定的差异,但是随壳聚糖有机酸盐浓度的增加,四种盐对三种微生物的作用情况具有相同的变化趋势。以CA对E.coli为例:在某一较低浓度(0.05% w/v)附近,有机酸盐对菌种作用不明显;当浓度继续升高到某一点(0.08% w/v),有机酸盐对微生物的生长具有轻度的促进作用;之后随壳聚糖有机酸盐浓度的增加其抑制作用逐渐增强直到达到最小抑制浓度MIC(0.13% w/v)。电镜照片显示CA作用下的三种微生物的细胞壁和细胞膜均发生了一定的改变,由此可以推测壳聚糖有机酸盐很可能是通过对细胞表面的作用抑制了微生物的生长。同时我们还对壳聚糖有机酸盐对微生物生长抑制作用的影响因素进行了探讨,随微生物种类不同,有机酸盐会表现出不同的抑菌效果;有机酸盐中所含有机酸的量较少,故而有机酸的种类对不同有机酸盐的抑菌效果影响不大;接种的菌种浓度对有机酸的最小抑制浓度会产生影响。
在本论文中,我们还对固态壳聚糖有机酸盐作为一种新型食品防腐剂的可行性进行了探讨。将CA与高级天然防腐剂柚子提取物进行了对比:据报道,柚子提取物作为一种优良的天然防腐剂,其抗菌能力是其它防腐剂的10-100倍。实验证明,一方面,CA浓度达到0.1%(w/v)时能够达到柚子提取物所具有的抗菌效果;另一方面,壳聚糖有机酸盐的价格低于柚子提取物。所以壳聚糖有机酸盐有望成为一种质优价廉的食品、化妆品防腐剂。采用特殊处理过的蔬菜样品,对CA、CL作为食品防腐剂的实际应用进行了模仿,实验证明,这种有机酸盐在一定条件下对蔬菜加工产物(萝卜)能够达到较好的防腐效果。
Chitin is a natural polysaccharide which is usually obtained from shells of crustaceans such as crab, shrimp, and crawfish. Chitosan is a partially N-deacetylated derivative of chitin, and consists of polymeric (1→4)-linked 2-amino-2-deoxy-β-D-glucopyranose units. Due to the unique polycationic nature, chitosan and its derivatives have been proposed for various applications in biomedical, food, agricultural, biotechnological and pharmaceutical products. Chitosan has a broad spectrum of antimicrobial activities, high bactericidal rates and low toxicity toward mammalian cells, making it a potential biocide in food preserving. However, the poor solubility of chitosan at neutral and higher pH limits its applications. Some modification has been attempted. But the traditional method of modifying is to introduce some covalent bond, which is usually complex and sometimes changes some of the properties of chitosan.
In this paper, a new approach to prepare solid chitosan organic salts is reported. Chitosan (Mw=82kDa, DD=88.7%) was prepared first. Chitosan and organic acid was simply reacted and the chitosan organic salts (COS) were prepared: chitosan formait (CF), chiotsan acetate (CA), chitosan propionate (CP), chitosan lactate (CL). It was demonstrated by the titration curves that there was only one inflexion point, compared with two inflexion points in chitosan. It could be inferred from the FTIR that chitosan is linked by electrovalent bond other than covalent bond with the acid, so the products still keep the structure and antibacterial activity of chitosan and can be prepared easily. The solubility, titration curve, stability, IR spectrum and other basic characterizations of the products are evaluated. It is demonstrated that the chitosan organic salts are water soluble and stable in common conditions.
The antibacterial activities against E. coli (gram negative), S. aureus (gram positive) and C. guilliermondii have been investigated by MTT method. It was found that chitosan organic salts have good antibacterial acitivities. Take CA and E.coli for example, the growth of E. coli was regular at the concentration of 0.05% (w/v); when the concentration of CA reached 0.08%(w/v), the bacteria grew more than the ones with no CA; CA would inhibit the growth of E.coli at the concentration of 0.13% (w/v). It could also inhibit the growth of S.aureus at the concentration of 0.20% (w/v), and C. guilliermondii at 15ppm (w/v). It also demonstrated that COS showed stronger antibacterial activities with the increase of concentration of COS. Different kinds of microbiology also cause different antibacterial activities. The organic acid nearly has no influence on the activities. And the concentration of the bacterium suspension during incubation has effect on the MIC of COS.
The possibility of the product being used as a new kind of preservative in foods was also investigated in this paper. It is conferred that the chitosan organic acid salts will be a kind of cheap, convenient, and effective preservative in foods.
引文
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