【作者】 张希；

【导师】 冯凤琴；

【作者基本信息】 浙江大学 ， 食品科学， 2014， 博士

【摘要】 随着食品工业的发展,对防腐剂的要求也越来越高,不但要求防腐剂安全、无毒、高效,而且具有营养化、功能化的特点。脂肪酸及其衍生物以其高效、安全且功能多样化而成为筛选天然食品防腐剂和医药、兽药及农药的目标群。目前抑菌化合物的筛选主要通过大量试验进行,难免盲目性和偶然性,迫切需要定量构效关系的指导。本课题组已经研究报道了脂肪酸及其衍生物对革兰氏阳性菌金黄色葡萄球菌(Escherichia coli, E.coli)和蜡样芽孢杆菌(.Bacillus cereus, B. cereus)的定量构效关系(Quantitative structure-activity relationship. QSAR),为了更全面研究和了解脂肪酸及其衍生物抑菌活性的定量构效关系,本研究选用35种脂肪酸及其衍生物,用半数抑制浓度作为评价其抑菌活性的参数,构建针对革兰氏阴性菌肠出血性大肠杆菌O157:H7(Escherichia coli O157:H7, E. coli O157:H7)和真菌中的酵母白色念珠菌(Candida albicans, C. albicans)的QSAR模型。分析所得模型发现,立体效应和电性特征是影响脂肪酸及其衍生物抑菌活性的最主要因素,原子排布较密,空间体积在一定范围,某些位置没有大的立体位阻,羧基、酯基端电负性强,特定的电子云排布和对外电场敏感的分子倾向于具有更高的抑菌能力。研究还证实,在脂肪酸及其衍生物范围内,中链脂肪酸衍生物具有最强抑菌活性,其中的代表物为月桂酸单甘油酯(Glycerol monolaurate, GML),但是在实际应用过程中,GML存在水溶性和口感较差的缺陷,制约其后续应用发展,因此本研究采用糖基取代甘油基与中链脂肪酸合成糖单酯,以期获得抑菌性、水溶性、口感及理化性质等综合性能优良的脂肪酸衍生物。为全面比较和了解中链脂肪酸糖单酯的抑菌及其他性能,本研究首先在叔戊醇与DMSO混合溶液中,利用脂肪酶Lipozyme TLIM催化合成蔗糖月桂酸单酯,在单因素试验的基础上,利用响应面分析法,优化了蔗糖月桂酸酯的合成条件,得到的最佳合成条件为：蔗糖浓度为0.04mol/L,溶剂叔戊醇：二甲亚砜=4：1(v/v),加酶量为100mg/mL分子筛量为100mg/mL,在50℃下水浴摇动30min后,加入0.4mol/L的月桂酸乙烯酯反应4h。之后,在相似条件下分别合成、分离精制和鉴定了19种中链脂肪酸糖单酯,包括6种单糖脂肪酸酯、10种二糖脂肪酸单酯及3种三糖脂肪酸单酯。再对合成的糖单酯的抑菌活性及表面张力、临界胶束浓度(Critical micelle concentration,CMC)、起泡能力和泡沫稳定性、乳化能力和乳化稳定性等进行了测定比较,同时与市售月桂酸糖酯L-1695及GML进行了对比分析。结果发现,大部分糖酯对金黄色葡萄球菌的抑制能力均较强,而对E. coli O157:H7和白色念珠菌的抑菌能力较弱,它们的抑菌活性与链长、亲水基团、酯化程度相关。相对而言,随着脂肪酸链长增加,糖单酯对金黄色葡萄球菌和白色念珠菌的抑菌活性增强,月桂酸糖单酯的抑菌活性最强,而碳链较短的脂肪酸二糖单酯则对E.coli O157:H7的抑制率较高。单糖脂肪酸单酯的抑菌性明显优于二糖脂肪酸单酯,其中甲酯葡萄糖月桂酸单酯最佳,对三种被测菌种的最低抑制浓度(Minimum inhibitory concentration, MIC)值均为188μg/mL,三糖脂肪酸单酯则没有抑菌活性,蔗糖月桂酸二酯或多酯的抑菌活性弱于蔗糖单酯。中链脂肪酸糖单酯的表面活性、乳化性、起泡性等随着碳链长度和糖基变化而变化,综合分析发现二糖月桂酸单酯在上述性能中表现优异,且口感和水溶性明显优于GML,具有较好的开发应用价值。更多还原

【Abstract】 With the development of lfood industry, higher requirements are presented for food preservatives, which are not only safe, non-toxicand efficient, but also possess, nutritional and functional festures. Fatty acids and their derivatives have become the target group to screen natural food preservatives and pharmaceuticals, veterinary drugs and pesticides for theirefficient, safe and functional diversification. However, previous reports and experiences of researchers are mainly accumulated on abundant experiments, which would inevitably be blindness or random, therefor, it is urgent to understand the relationships between molecular structure of fatty acids and their derivatives and their antibacterial activities. Our group has reported the antibacterial quantitative structure-activity relationship (QSAR) model of fatty acid and their derivatives against Gram-positive Staphylococcus aureus and Bacillus cereus. In order to get a more comprehensive understanding of the antimicrobial quantitative structure-activity relationship of fatty acids and their derivatives,35kinds of fatty acids and their derivatives were chosen to test their IC50against Gram-negative Escherichia coli O157:H7and the typical Eufloria Candida albicans and3statistically reliable QSAR models were establish.By analyzing the obtained models, it was found that steric and electronic properties are the most related factors with antimicrobial activity, and the molecules of fatty acid derivative which possessed denser atomic distribution, space volume in certain range, no steric effect in some regions, more electronic negativity of carboxyl terminal of fatty acid chains, special electron cloud distribution and particular sensitivity to out electric fieldwere tend to display more potent antimicrobial activity. This study also confirmed that medium chain fatty acid derivatives have the strongest antimicrobial activities, especially glycerol monolaurate (GML). However, GML is deficient in water solubility and taste when applied to actual food system, which may restrict its application and development in food industry. In view of this, we use sugar instead of glycerol to sythensis sugar medium chain fatty acid monoester with medium chain fatty acid, aimed to obtain new antibacterial fatty acid derivatives with good water-solublility, taste, physical and chemicalproperties as well.In order to comprehensively analyze and compare the antimicrobial activity and physico-chemical properties of sugar medium chain fatty acid monoesters, firstly, the synthesis of monolauroyl sucrose in2-methyl-2-butanol and dimethylsulfoxide (DMSO) solution by Lipozyme TL IM, and the separation and purification of monoester have been studied. On the basis of single factor experiments, response surface methodology was used to optimize the synthesis system of sucrose laurate, the obtained conditions are: sucrose (0.04mol/L),2-methyl-2-butanol:DMSO=4:1(v/v), plus the amount of Lipozyme TL IM100mg/mL, molecular sieve amount of100mg/mL, after oscillating30min at50℃, lauric acid vinyl ester (0.4mol/L) was added tothe system, and the reaction time was4h. Then,the other18kinds of different monoesters were synthesized under the similiar conditions. Finally,19different sugar monoesters involved6monosaccharide monoesters,10kinds of disaccharide monoesters and3types of trisaccharide monoesters were synthesized and purified.Afterwards, the antimicrobial activities and surface-active properties including surface tension, critical micelle concentration (CMC), foamability, foaming stability, oil-water emulsifying ability and emulsion stability of sugar fatty acid monoesters were tested and analyzed. It was found that most monoesters showed the strongest antimicrobial activity against S. aureus. However, inhibition rate against E. coli O157:H7and C. albicans were relatively lower. Among the tested monoesters, MGL showed the best antimicrobial activity against the three tested microbes, and the minimum inhibitory concentrations (MICs) for them were all188μg/mL, and the raffinose monoesters exhibited no antimicrobial activity against the three tested cultures. The antimicrobial activities of diester and triesters were lower than monoesters. The length of fatty acid chain (hydrophobic group) and sugar groups (hydrophilic group) for sugar medium chain fatty acid monoesters both affected the surface properties and antimicrobial activities. According to the results, the MGL possessed the best antimicrobial activity; while disaccharide monoester showed better surface properties, water solubility, taste than GML, and has great potential developing and applying value.更多还原