摘要
壳聚糖是甲壳素脱乙酰化的产物，而甲壳素是地球上仅次于纤维素的第二大再生资源，亦是地球上除蛋白质外数量最大的含氮天然化合物。低聚壳聚糖是壳聚糖经水解生成的一类低聚糖，是指由2～10个单糖通过糖苷键连接而形成的直链或支链的低聚糖，具有较高的生物相溶性，容易被吸收利用。特别是分子量低于3000Da的低聚壳聚糖更展现出独特的生理活性和功能性质。但是低聚壳聚糖的制备一直以来是科研和生产的热点和难点。
本论文针对壳聚糖经脉冲电场作用及双氧水、臭氧等协同作用下的降解展开研究。在此基础上提出了脉冲电场作用下壳聚糖的降解机理，为后续的研究及产业化推广提供了基础。主要的研究内容及成果如下：
1.首次研究了脉冲电场对壳聚糖降解的影响。以脉冲电场为处理手段，研究了电场强度、处理时间、壳聚糖质量浓度和电导率等因素对壳聚糖降解的影响。结果表明，壳聚糖降解产物的平均分子量随着电场强度的增大而降低，且在高场强下降解显著。场强为33.3kV/cm时，降解率达54.2%；降解速率随时间的延长逐渐变缓，前20min和后70min的降解率分别为18.5%和8.5%；而溶液浓度和电导率的升高则不利于壳聚糖的降解。FTIR及X射线衍射分析表明：降解前后，壳聚糖的酰胺结构未发生改变，表明产物的基本化学结构没有发生变化。
2.研究了一定浓度的双氧水在不同处理时间下对壳聚糖溶液的降解影响。发现双氧水对壳聚糖的降解效果明显，而且随着处理时间的增加，其分子量逐步下降，分子量分布（Mw/Mn）也呈现出降低趋势。但不同反应时间差别不大，说明处理时间对双氧水降解壳聚糖的影响不明显。
在此研究基础上进一步研究了PEF与双氧水协同作用对壳聚糖降解的影响。研究发现，PEF和双氧水协同作用效果明显。处理60min后，PEF和双氧水的降解效率分别为25%和90.7%，而PEF和双氧水联用的降解效率达到94.8%。低分子量壳聚糖的重均分子量（Mw）随电场强度的增加而降低，分子量分布（Mw/Mn）也呈现出降低趋势。同时，通过原样和降解产物的红外光谱分析，发现其基本的化学结构并没有发生改变。
3.以臭氧为处理手段，研究了臭氧流量、处理时间和壳聚糖的质量浓度等因素对壳聚糖降解的影响。发现，随着臭氧流量的增加壳聚糖的分子量逐渐降低，但臭氧在溶液中饱和后分子量的变化比较平缓。饱和浓度臭氧流量处理下，随着处理时间的增加壳聚糖不断降解，但处理30min以后壳聚糖的分子量基本没有很大的变化。
同时，对PEF和臭氧协同降解壳聚糖进行了研究。发现PEF和臭氧降解壳聚糖的协同作用效果明显，随着处理时间的增加，分子量显著降低，40min后已达5000Da以下，为完全水溶性产物。处理30min后，PEF和臭氧的降解效率分别为20.5%、93.8%，而PEF和臭氧联用的降解效率达到98.5%。对降解产物进行了GPC、FTIR和XRD分析，验证了壳聚糖的降解效果，发现降解后壳聚糖的分子量降低、结晶区破坏，但其酰胺结构基本保持不变。
4.脉冲电场及其协同作用降解壳聚糖的机理为自由基理论。研究发现高压脉冲电场能增加溶液中羟自由基、氧自由基和氢自由基的数量，加速其作用于壳聚糖分子中的β-(1,4)糖苷键的断裂。电场单独作用，电场和双氧水、臭氧协同作用，均能增加壳聚糖溶液中的自由基浓度，从而增加壳聚糖的降解效率。同时，壳聚糖乙酸溶液中，氨基被质子化，在电场处理下，壳聚糖在溶液中的链状结构更舒展，从而增加了自由基攻击β-(1,4)糖苷键的机会，加速了壳聚糖的降解。
Chitosan is the deacetylation product of chitin, and chitin is the second largest renewableresource just after cellulose on earth, also the largest number of nitrogen-containing naturalcompounds in addition to proteins. Oligochitosan is the hydrolysate of chitosan, and it isformed from2～10monosaccharides connected by glycosidic bond, which has highdissolubility, easily absorbed. Oligochitosan shows unique physiological activity andfunctional properties, when its molecular weight is less than3000Da. Meanwhile thepreparation of oligochitosan is always difficult for researches and manufactures.
In this paper, the degradation of chitosan was investigated by the way of high voltage-pulsed electric field and the synergetic degradation with hydrogen peroxide or ozone. On thisbasis, the paper proposed the mechanism of chitosan degradation under the high-voltagepulsed electric field, and it can be referred to the researcher as a basis for following researchand industrial application. The main research contents and results are as follows:
1. This is the first time to study the impact of high-voltage pulsed electric field on thechitosan degradation. Base on processing method of the pulse electric field, this paper hasstudied the effects of chitosan degradation by some factors, such as electric field strength,processing time, chitosan concentration, electrical conductivity, and so on. The results showedthat the average molecular weight of chitosan was decreased with the increase of electric fieldstrength and significant degradation effect was obtained under higher field strength. Forexample, the degradation rate reached54.2%when the field strength was33.3kV/cm; Thedegradation rate was slowing down gradually with time extended, and the degradation rates atthe first20minutes and the last70minutes were18.5%and8.5%, respectively. The increasedconcentration and conductivity of chitosan didn’t benefit the degradation of chitosan. Theanalysis of FTIR and X-ray diffraction showed that the side chain structure of chitosan wasnot changed before and after degradation, indicating that the basic chemical structure ofproducts was not changed.
2. This paper has studied the impact of different processing time of hydrogen peroxide atthe certain concentration for the chitosan degradation. It was found that hydrogen peroxidehas significant effect on the chitosan degradation, and with increasing of processing time, themolecular weight of chitosan was decreased gradually, molecular weight distribution (Mw/Mn)was also showing a decreasing trend. However, the processing time for hydrogen peroxide onchitosan degradation was limited.
Based on the above study, this paper studied the synergy degradation of chitosancombining with PEF and hydrogen peroxide. It was found that the synergic effect of PEF andhydrogen peroxide was obvious. After treated for60minutes, the degradation rates of PEFand hydrogen peroxide was25%and90.7%respectively, meanwhile the degradation rate ofcombination with PEF and hydrogen peroxide reached94.8%. When the electric field strengthwas increasing, the weight-averge molecular weight (Mw) of low molecular weight chitosanwas decreased, and the molecular weight distribution (Mw/Mn) was also decreased. Byanalyzing the FTIR of the original and degraded chitosan, we found that the basic chemicalstructure was not changed.
3. This paper studied the impact of ozone on chitosan degradation, including the ozoneflux, processing time, concentration of chitosan. It was showed that the molecular weight ofchitosan decreasing with the increasing ozone flux until it was saturated. After that, thechitosan continued to degrade with the increasing processing time, but after30minutes,molecular weight of chitosan changed little.
Therefore, the synergy degradation of chitosan with PEF and ozone was studied. It wasfound that the synergic effect of PEF and ozone was obvious for degradation of chitosan. Asthe processing time increasing, the weight of molecule has evidently decreased. For example,after being treated for40minutes, the molecular weight was below5000Da, which wasalmost the water-soluble product. After being treated for30minutes, the degradation rates ofPEF and ozone was20.5%and93.8%, respectively, while the degradation rate of thesynergetic effect of PEF and ozone reached to98.5%. And through analyses of GPC, FTIR andXRD, it was showed that the molecular weight was decreased and the crystalline region wasdestroyed, but the structure of acylamide remained the same.
4. According to above research, a free radical theory is proposed to explain themechanism of chitosan degradation by PEF and its synergic effect. Pulsed electric filed isfound to be able to increase the number of hydroxyl free radical, oxygen free radical andhydrogen free radical in the solution, which can enhance the effect on β-1,4-glycosidic bond.The effects of PEF, hydrogen peroxide, ozone and their synergy can all increase the density offree radical, which improved the degradation rate of chitosan. Meanwhile, amino group wasprotonated in acetic acid solution, and under the condition of electric field, the chain ofchitosan was more stretched and much easier to be attacked by free radical.
引文
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