壳聚糖没食子酸衍生物酶法制备及对鲜切苹果的保鲜效果

Enzymatic synthesis of chitosan-gallic acid derivative and its preservation effect on fresh-cut apple

为提高壳聚糖水溶性和保鲜性能,该文以没食子酸为配体,漆酶为催化剂催化壳聚糖与没食子酸发生接枝反应,得到壳聚糖没食子酸衍生物,对其结构进行了初步表征,并初步探讨了其对鲜切苹果的保鲜效果。结果表明,衍生物接枝率为72.80%,溶解度达到92.77%,没食子酸中的羧基与壳聚糖分子的氨基发生了迈克尔加成反应,在壳聚糖的C2位生成了酰胺键。14℃下贮藏4 d后,10 mg/m L衍生物处理的苹果硬度损失率仅为10.23%,比空白组、壳聚糖处理组和壳聚糖没食子酸物理复合物处理组分别低20.90%(P<0.05)、16.60%(P<0.05)和15.60%(P<0.05);可溶性固形物、维生素C、谷胱甘肽和多酚质量分数分别为12.93%,1.23 mg/(100 g),6.26 mg/(100 g)和6.24 mg/(100 g),分别比空白组高16.47%(P<0.05),48.78%(P<0.05),24.92%(P<0.05)和43.75%(P<0.05);多酚氧化酶和过氧化物酶活性分别比空白组低36.73%(P<0.05)和76.94%(P<0.05);菌落总数为16.00×10~4 CFU/g,显著低于空白组、壳聚糖处理组和壳聚糖没食子酸物理复合物处理组(P<0.05)。结果表明,漆酶可催化壳聚糖与没食子酸发生接枝反应,得到的壳聚糖没食子酸衍生物对鲜切苹果具有较好的保鲜效果。

In order to broad the application scope and improve the preservation effect of chitosan（CTS） and gallic acid（GA）, CTS derivative with GA was synthesized by laccase catalysis method. The grafting rate and solubility, structural characterization and preservation effect of derivative on fresh-cut apple were investigated. Results showed that the grafting rate and solubility were respective 72.80% and 92.77%. The preliminary characterization of derivative was studied through a series of testing methods including ultraviolet spectrum, FTIR（Fourier transform infrared spectroscopy）, X-ray diffraction spectra and superconducting magnetic resonance spectrum. Ultraviolet spectrum analysis result showed that benzene ring and auxochrome-OH were introduced into CTS molecules. FTIR result showed that-NH2 of CTS molecules decreased, which reacted with GA by amid linkage and did not change the main chain structure of CTS. X-ray diffraction analysis result indicated that with the addition of GA, CTS conformation was changed, a new kind of crystal was formed gradually, the area of amorphous area increased, the order of the structure reduced, the intermolecular and intramolecular hydrogen bonding in polymer were destroyed, and the crystallinity of CTS reduced. 13C-NMR（nuclear magnetic resonance） analysis result demonstrated that CTS-GA exhibited the resonance absorption peak of C=O. 1H-NMR analysis result proved that CTS-GA exhibited the benzene ring and alcoholic hydroxyl proton peaks. All above analysis results confirmed the successful grafting of GA onto CTS. The conjugation of GA onto CTS probably occurred between amine（C-2） and carboxyl groups of GA, forming amide linkage. Effects of chitosan（CTS）, derivative of chitosan and gallic acid（CTS-GA）, physical compound of chitosan and gallic acid（CTS.GA） and deionized water（CK） treatment on fresh-cut apples＇ biochemical properties, such as hardness, soluble solid, polyphenol, vitamin C, glutathione（GSH） content, activities of polyphenol oxidase（PPO）, peroxidase（POD）, superoxide dismutase（SOD）, catalase（CAT） and total number of bacteria colony. Analytical determinations were made every day. After 4-day storage at 14 ℃, the loss of hardness of CTS-GA group was only 10.23%, which was significant lower than those of CK（20.90%）, CTS（16.60%） and CTS.GA（15.60%）. The soluble solid, vitamin C, GSH and polyphenol content of CTS-GA group were 12.93 %, 1.23 mg/100 g, 6.26 mg/100 g and 6.24 mg/100 g, which were 16.47%, 48.78%, 24.92% and 43.75% respectively higher than those of CK, while the activity of PPO and POD of CTS-GA group were 36.73% and 76.94% lower than CK treatment. And the amount of microorganism in CTS-GA group was 16.00×10~4 CFU/g, obviously lower than CK, CTS and CTS.GA groups. These results suggested appropriate CTS-GA treatment provided better maintenance of hardness, effectively reduced the loss of vitamin C, soluble solid content, polyphenol content and GSH, inhibited the PPO activities, and also maintained higher activity of SOD and CAT, and suppressed the microbial growth. Therefore CTS-GA treatment can delay the senescence rate and exert an excellent effect on fresh keeping of fresh-cut apple. The results clearly indicate that enzyme-mediated pattern of CTS and GA can provide a novel ‘green＇ pathway of preparing and broaden the application fields of CTS and GA such as food preservation industry.