【作者】 袁超；

【导师】 金征宇；

【作者基本信息】 江南大学 ， 粮食、油脂及植物蛋白工程， 2008， 博士

【摘要】 本论文通过工艺优化制备了一系列的不同取代度和取代基分布的羟丙基-β-环糊精（HPCD）,在研究HPCD结构的基础上,进一步探讨了取代度和取代基分布对HPCD包埋能力、热稳定性和降解性能的影响,并考察了HPCD产品在虾青素包埋中的应用效果。HPCD是β-环糊精的醚化衍生物,羟丙基的引入打开了β-环糊精分子内的氢键,形成无定形混合物,水溶性大大提高,并且具有更高的安全性。因此,HPCD被认为是很有潜力的母体环糊精替代品,应用于亲油性食品添加剂和药品的包埋,提高客体的水溶性和稳定性。本文通过单因素实验和响应面分析,对HPCD的生产工艺进行了优化,得到了取代度和得率都较高的HPCD生产工艺,具体工艺条件为:反应时间16h,反应温度31℃,透析时间7.4h。此时取代度为4.02,得率61.80%。在此工艺条件下,通过调节反应底物的比例制备了A、B两组各四种不同取代度和取代基分布的HPCD样品,样品具有较高的纯度,理化性质满足下一步研究的要求。红外光谱表明,β-环糊精和HPCD都显示糖类的特征吸收峰。HPCD在2960cm^-1左右出现甲基的反对称振动吸收峰,从侧面证明了羟丙基的存在。实验室制备的HPCD和标样的红外光谱出峰位置完全一致,从而证明二者是同一种物质,且自制的HPCD有较高纯度。用改良的甲基化方法对HPCD进行了甲基化分析。对样品的总离子流图和质谱图综合分析计算后,统计出了各样品的取代基分布情况和取代度。A组样品取代以6位为主,但相对比较平均,环糊精分子两个侧面的取代基比例[DS（2+3）/DS（6）]接近1。B组取代主要发生在2位氧原子上,B组HPCD样品两个侧面的取代基比例[DS（2+3）/DS（6）]都大于3,说明取代主要在2、3位羟基端。国外对照品的结构与B组产品类似,特别是与取代度差不多的6号样品非常接近,表明本实验室制备的样品的结构和品质与国外对照品很接近。羟丙基的引入改变了β-环糊精原有的紧密齐整的结晶性分子结构,形成由多种取代形式的组分组成的无定形混合物,这有助于提高HPCD及其包合物的水溶性和溶解速度。以酚酞作为“较大球形”客体模型,甲基红作为“中等线形”客体模型,苯甲酸乙酯作为“较小”客体模型,利用它们在溶液中的吸光特性和主客体复合物形成对其吸光性的影响,设计试验,测定了它们与不同取代度和取代基分布的HPCD所形成复合物的稳定常数,以确定HPCD的包埋能力。综合考虑HPCD的取代度和取代位点以及客体分子的影响,可以看出:对于“较大的球形”分子,不论取代度大小和羟丙基如何分布,空间位阻都是主导效应,所有HPCD与客体复合物的稳定常数均低于母体β-环糊精,包埋能力低于后者。对于“中等线形”分子,HPCD的包埋能力高于母体β-环糊精,低取代度时,羟丙基以“增溶”效应为主。取代基平均分布的HPCD产品具有更高的稳定常数,特别是取代度为3.5左右的此类HPCD。对于“较小”分子,HPCD的包埋能力远高于母体β-环糊精,羟丙基的“增溶”效应特别明显。由于此类客体分子只处在HPCD空腔的大口端,取代基集中于大口端的HPCD产品具有更好的稳定常数,特别是取代度4.5左右的产品。不论对何种客体,取代度太高和太低的HPCD都达不到理想的包埋效果,取代度在3-5之间应该是比较理想的,但取代基分布则要视具体客体情况而定。本文测定了HPCD的热稳定性,结果表明:其热分解温度在300-400℃之间,差热峰值为367℃。HPCD的热稳定性优于β-环糊精,分解温度提高32℃左右。通过分析其分解动力学,得到热分解动力学参数为:活化能E_A=149.15 kJ/mol,指前因子k₀=2.19×10¹⁰min^-1。按国际标准ISO 17556（2003）对HPCD在土壤中的降解性能进行了研究。HPCD的降解率随取代度的上升而降低,取代度与降解率呈负相关。取代基分布方面,A组HPCD样品的降解率高于相同取代度的B组样品,可能是因为B组样品会造成更大的空间位阻,导致更难被好氧微生物产生的CDase催化开环。土壤性质对HPCD降解率的影响要小于取代度和取代基分布的影响,在较肥沃土壤中,HPCD的降解率要略高于沙质土壤中的降解率。在污染土壤中,β-环糊精的降解非常快,经过70天的实验,有74.7%被降解。HPCD的最终降解率是40.9%。两者的降解率都高于相同时间内在未污染土壤中的降解率。添加环糊精产品对于加速土壤有机烃类污染物的降解有一定效果,从长远看,添加不易被微生物降解的HPCD等修饰环糊精有更好的效果。虾青素是一种具有高效抗氧化活性的类胡萝卜素,不溶于水,在酸、氧、高温及光照条件下易被氧化降解。通过HPCD包埋可以提高其水溶性和稳定性,利于储运和应用。本文对HPCD/虾青素复合物进行了制备,优化了制备工艺,得到的工艺参数为:HPCD浓度0.03mol/L,主客体摩尔比60,反应温度20℃,搅拌速度1000rpm,此时预测的包合率是54.0%（±5.91%）,实际测定的包合率为51.6%,说明此工艺可靠。通过紫外光谱、红外光谱和核磁共振相互印证,确定了复合物的形成并预测其结构为虾青素的六元环部分进入HPCD空腔,六元环的重心位于HPCD内壁H-5附近。此结果与Hyperchem 8.0分子模拟软件预测的结构相符。热重-差热联用分析显示,复合物的形成将虾青素的热分解开始温度至少提高了40℃,达到290℃左右。稳定性实验显示,包埋有利于提高虾青素在环境中的稳定性,并且具有很好的缓释效果;复合物具有很好的储藏稳定性,略优于原虾青素。本文还研究了复合物的体外抗氧化活性。复合物的还原能力高于虾青素和抗坏血酸。在低浓度下清除DPPH自由基的能力远高于虾青素和抗坏血酸,但随着浓度的升高清除率下降。复合物清除羟基自由基的能力略低于虾青素但大于抗坏血酸。复合物清除超氧阴离子自由基的能力也略低于虾青素,但却比抗坏血酸高出至少一个数量级。总体来说,复合物的抗氧化能力由于包埋的影响略有降低,但在亲水环境中有效果更好。更多还原

【Abstract】 A series of hydroxypropyl-β-cyclodextrin（HPCD） samples were prepared by an optimized process.The effects of degree and distribution of substitution on the complex forming ability,thermal stability and decomposing property of HPCDs were explored based on the structure investigation of HPCDs.The application of HPCD in forming inclusion complex with astaxanthin was also studied.HPCD,a hydroxyalkyl derivative ofβ-cyclodextrin,with improved water solubility properties and may be slightly more toxicologically benign,was considered a potential alternative ofβ-cyclodextrin.HPCDs have widely applications in food,agriculture and pharmaceutical field.In this study,the monofactorial tests and response surface method were performed to optimize the preparation process of HPCD.The following optimal parameters were obtained:reaction time of 16h,reaction temperature of 31℃and dialysis time of 7.4h. The degree of substitution（DS） and recovery of the production prepared under optimal parameters respectively were 4.02 and 61.80%,respectively.Under the optimized process,A, B two groups of HPCD were prepared,each had 4 types of samples with different DS and substituent distribution.The samples had higher purity and their physicochemical properties fulfilled the requirement of next step investigation.The infrared spectrums showed that bothβ-cyclodextrin and HPCDs displayed the characteristic absorption bands of saccharide,3400 cm^-1（O-H）,2930 cm^-1（C-H） and 1640 cm^-1（C=O）.HPCDs showed absorption bands at 2960 cm^-1 for methyl antisymmetric vibration,it proved the existence of hydroxypropyl group.The absorption bands of HPCDs prepared in the lab were in accordance with that of standard,therefore,they were proved to be the same substance,and the prepared HPCDs had higher purity.The improved reductive-cleavage method and methylation analysis were performed to determine the DS and distribution of substitution of HPCDs through analyzing the total ion current and mass spectrums of HPCDs.In group A,the DS value of O-6 was the highest,however,there also had many substituents at O-2 and O-3 positions.The ratio of DS（2+3） to DS（6） is close to 1. The distribution of substituents on primary（DS（6）） and secondary hydroxyl groups（DS（2+3）） was even.In group B,substituents concentrated at O-2.The DS value of secondary hydroxyl groups was about three times as much as that of primary face.The configurantion of reference was similar to that of group B HPCDs,especially to that of number 6.It was obviously that the quality and configuration of prepared HPCDs were equivalent to that of reference.The introduction of hydroxypropyl groups changed the crystalline structure ofβ-cyclodextrin, madeβ-cyclodextrin an amorphous compound containing a large mumber of chemically individual.These conduce to improve water solubility and dissolving speed of HPCDs and their inclusion complexes.A test was designed to determine the complex forming ability of HPCDs,based on the change of absorbance of the guest before and after complex forming.The choosed guests were phenolphthalein as a model for "larger spheriform" guest,methyl red as a model for "middling linear" guests and ethyl benzoate as a "smaller" model.Consider the effect of DS,distribution of substitution and guest molecules,the following conclusion should be drown:the stability constants of the complexes of HPCDs with the "larger spheriform" guests were all lower than that ofβ-cyclodextrin,no matter what DS and distribution of substitution.Steric hindrance was the dominant factor.The stability constants of the complexes of HPCDs with the "middling linear" guests were higher than that ofβ-cyclodextrin."Solubility increasing" was the major factor under low DS values.The HPCD products with substituents distributed evenly could reach higher stabilities,especially which the DS value was about 3.5.For the "smaller" guests,the complex forming ability of HPCDs was far higher than that ofβ-cyclodextrin.Hydroxypropyl group showed obvious "solubility increasing" effect.Because this type of molecules can only situate at the larger end of HPCDs,the products with substituents concentrared on the larger end had better stabilities,especially that the DS value was about 4.5.No matter what kind of guestes,the HPCDs which had too higher or lower DS values could not reach the best complexes forming result.The DS values between 3 and 5 were commended;however,the effect of substituent distrbution should be determined by the guests.The thermal stability of prepared HPCDs were determined,decomposition temperature of HPCDs was between 300 and 400℃,the peak value was about 367℃.Thermal stability of HPCDs was excelled thanβ-cyclodextrin,their decomposition temperature enhanced about 32℃.The thermal decomposition kinetics was investigated,the activation energy E_A is 149.15kJ/mol and the pre-exponential factor k₀ is 2.19×10¹⁰min^-1.The biodegradation of HPCDs in soils was studied according to ISO 17556（2003）.All HPCDs were found to be more or less biodegradable.Increasing the DS had negative effect on the biodegradation rate of HPCDs.The substitution pattern affected the biodegradation,too. The soil characteristics might also slightly affect the biodegradation of HPCDs,however,the effect was lower than that of DS and substitution pattern.The degradation rate of HPCDs in fertile soil was a little higher than that in sandy soil.The biodegradation speed ofβ-cyclodextrin in the contaminated soil was rapid,which was degradated by 74.7%at the end of the 70 days test.The final biodegradation ratio of HPCD was 40.9%.Both were higher than that obtained in the uncontaminated soils.The contamination removing ability of cyclodextrins was highly affected by their own biodegradation fate in soil.The experiments in the contaminated soil indicated that bothβ-cyclodextrin and HPCD enhanced the bioavailability of contaminants in soil.HPCDs were better because they were more endurable in soil.Astaxanthin is a high value carotenoid pigment with strong antioxidant properties.It can easily be decomposed by light and oxygen,which can cause the loss of antioxidant properties. Furthermore,the poor aqueous solubility limits its use in aqueous phase antioxidant.The inclusion of astaxanthin with HPCD should improve the solubility and stability of astaxanthin and be helpful to store,carry and application.In this study,the HPCD/astaxanthin complex was prepared.The preparation process was optimized,the optimized factors were:HPCD concentration of 0.03mol/L,molar ratio of host-guest of 60,reaction temperature of 20℃, mixture speed of 1000rpm and the forecasted inclusion ratio was 54.0%（±5.91%）.The real inclusion ratio was 51.6%under the optimized process.Therefore,the optimized process was credible.The formation of HPCD/astaxanthin complex was confirmed and the configuration was speculated by UV-Vis,IR and NMR.The configuration of HPCD/astaxanthin complex was that the hexacyclic ring end of astaxanthin entered the cavity of HPCD,and the barycenter of the hexacyclic ring located around the H-5 of HPCD cavity.The TG/DTA result indicated that the formation of complex improved the thermal decomposition start temperature by at least 40℃,reached up to 290℃.The light/thermal test showed that inclusion increased the stability of astaxanthin,and regulated the release of astaxanthin.The complex also had high storage stability.The antioxidant ability of HPCD/astaxanthin complex was investigated in vitro.The deoxidization ability of the complex was higher than that of astaxanthin and ascorbic acid.The ability of eliminating DPPH free radical of the complex was far higher than that of astaxanthin and ascorbic acid at a low concentration,however,the elimination rate decreased along with the increase of the concentration.The ability of eliminating hydroxy free radical of the complex was a little lower than that of astaxanthin but higher than that of ascorbic acid.The superoxide anion radical scavenging activity of the complex was lower than that of astaxanthin but far higher than that of ascorbic acid.In conclusion,the antioxidant ability of the complex became a little lower due to the effect of inclusion.However,it had better results in hydrophilic systems.更多还原