【作者】 张辉；

【导师】 郑晓冬； 冯凤琴；

【作者基本信息】 浙江大学 ， 食品科学， 2009， 博士

【摘要】 微乳液是一种由一定配比的表面活性剂、助表面活性剂、水和油自发形成的各向同性、外观透明或半透明、热力学稳定的分散体系,其液滴粒径约为1～100nm。虽然微乳化技术在药物、化妆品、三次采油等领域已经有了广泛的应用,但在食品领域的研究还处于起步阶段。自1990年第199届美国化学协会论坛开始,食品微乳化逐渐引起研究者的重视,特别是作为生物活性物质的载运体系,已经成为近几年的研究热点。本论文利用微乳化技术,构建了以月桂酸单甘油酯为油相的食品级微乳体系,测定了其对典型食品腐败菌的抑菌作用,并揭示了食品级微乳液的抑菌机理。主要研究结果如下:(1)利用拟三元相图,研究了各成分对以月桂酸单甘油酯为油相的食品级微乳体系相行为的影响。结果表明,只由水、油、单一表面活性剂很难形成食品级微乳液,但是当加入短链醇、多元醇、有机酸和盐时能形成食品级微乳体系。短链醇、短链酸、多元醇能大大提高微乳体系中油相的增溶量。有机盐特别是苯甲酸钠,作为助水溶物,也能提高油相的增溶量。吐温20,作为吐温系列中亲水性最强的表面活性剂,对油相的增溶量也是最大的。但是,蔗糖酯、辛癸酸甘油酯和卵磷脂形成微乳液的能力很差。通过对粘度与pH值的测定结果表明,随着水相的稀释,微乳体系会在某一点从W/O型转变为O/W型,证实了所构建的以月桂酸单甘油酯为油相的食品级微乳体系为U型微乳体系。(2)构建了三种不同的食品级U型微乳体系,即月桂酸单甘油酯-乙醇-吐温20-乳酸钠-水体系、月桂酸单甘油酯-乙醇-丙二醇-吐温20-苯甲酸钠-水体系、月桂酸单甘油酯-乙醇-吐温80-山梨酸钾-水体系,成功制备了三个微乳液配方,以抑菌率为指标,分别测试其对枯草芽孢杆菌(Bacillus subtilis)、嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia)、黑曲霉(Aspergillus niger)和意大利青霉(Penicillium italicum)的抑菌效果。结果表明,以月桂酸单甘油酯为油相的微乳体系能够增溶不同抗菌盐类物质,对食品腐败菌(革兰氏阳性、革兰氏阴性细菌以及霉菌)具有很好的抑制效果。在所得微乳液的抑菌效果中,月桂酸单甘油酯起主要抑菌作用,其次为所增溶的抗菌盐类物质。证实了增溶至少两种食品防腐剂于微乳体系的可行性,并初步验证了该体系对食品腐败菌的抑菌作用。(3)以月桂酸单甘油酯为油相,丙酸为助乳化剂,苯甲酸钠为盐、吐温80为表面活性剂构建U型食品级微乳体系,并测试该微乳体系配方的广谱抑菌作用,结果表明:微乳液对金黄色葡萄球菌(Staphylococcus aureus)、大肠杆菌(Escherichiacoli)、枯草芽孢杆菌的最低杀菌浓度分别是500μL/L、250μL/L、250μL/L。杀菌动力学曲线表明,在最低杀菌浓度下,微乳液在5 min内能杀死90%以上的细菌。微乳液对大肠杆菌的抑菌作用要强于金黄色葡萄球菌和枯草芽孢杆菌,因为在60 min后金黄色葡萄球菌和枯草芽孢杆菌细菌数下降4个数量级左右,而大肠杆菌则被全部杀死。微乳液对黑曲霉和意大利青霉的最低杀菌浓度分别是500μL/L和1000μL/L。杀菌动力学曲线表明,在最低杀菌浓度下,微乳液能在15 min和30 min内杀死99%以上的黑曲霉孢子和意大利青霉孢子。微乳液对黑曲霉的抑菌作用要强于意大利青霉,因为120 min后黑曲霉孢子数下降5个数量级左右,而意大利青霉孢子数则下降3个数量级左右。(4)以大肠杆菌和黑曲霉为代表,通过细胞表面疏水性试验和细胞膜通透性试验及超微结构观察,探讨了食品级微乳液对细菌和霉菌的抑菌作用机理。结果表明:细胞表面疏水性试验和细胞膜通透性试验与微乳液对大肠杆菌的杀菌动力学曲线的趋势是相一致的。这说明微乳液可作用于细菌细胞膜,使得细胞表面疏水性下降和细胞膜通透性增加,导致了细菌的死亡。细胞膜通透性试验以及采用光学和扫描电子显微镜观察真菌微观结构的变化,与微乳液对黑曲霉在液体和固体培养基中的杀菌结果也相一致,说明微乳液对真菌生长具有良好的抑制效果,对真菌孢子也有很好的杀灭作用,其原因在于微乳液引起真菌细胞壁和生物膜的结构与功能的破坏。更多还原

【Abstract】 Microemulsions are colloidal nanodispersions of oil and water stabilized by an interfacial film of surfactant molecules,typically in conjunction with a cosurfactant. The technique has been widely used in pharmaceutics,cosmetics,enhanced oil recovery,but its application in foods hadn’t caught much attention before 199th ACS (American Chemical Society) symposium in 1990.As a potential delivery system for bioactives in food,microemulsions recently have been of increasing interest to researchers and exhibited great potential on its industrial applications.The physicochemical characterization of food-grade microemulsion systems with glycerol monolaurate as oil and the antimicrobial activities against foodborne pathogens have been studied in this paper.The main research findings are as follows:(1) The effects of short-chain alcohols,organic acids,polyols,nonionic surfactants and salts on the phase behavior of the food-grade microemulsions have been elucidated.Results showed that the food-grade microemulsions were difficult to formulate from a three-component systems based on water,oil and single surfactant, but it is possible to formulate these microemulsions by the addition of short-chain alcohols,organic acids,polyols and salts.The oil solubilization was dramatically improved in the presence of short-chain alcoho-ls,organic acids and polyols,and organic salts contributed to the improved oil solubilization as hydrotropes.Tween 20 being the most hydrophilic surfactant in Tweens solubilized the maximum oil. However,it was hard to form microemulsions in the presence of sucrose esters even when mixed with ethanol.The oil solubilization capability in these systems was clearly reflected in the phase behavior of these systems.Viscosity and pH results showed that along the dilution lines,at a certain composition the systems inverted from water-in-oil to oil-in-water microemulsions, demonstrated as U-type microemulsion systems capable of being infinitely and progressively aqueous phase diluted.(2) Three different food-grade microemulsion systems(glycerol monolaurate/ethanol/Tween 20/sodium lactate/water microemulsion system,glycerol monolaurate/ethanol/propylene glycol/Tween 20/sodium benzoate/water microemulsion system and glycerol monolaurate/ethanol/Tween 80/potassium Sorbate/water microemulsion system) were established and their antimicrobial activities against Bacillus subtilis,Stenotrophomonas maltophilia,Aspergillus niger and PeniciIlium italicum respectively were examined.Results showed that the food-grade microemulsion systems with glycerol monolaurate as oil was able to sotubilize different antimicrobial salts and exhibited effective inhibitory activities against foodborne pathogens,including Gram-positive,Gram-negative bacteria and fungi.It was found that in the antimicrobial activities of these microemulsion systems, glycerol monolaurate made a major contribution,followed by the solubilized salts. These results confirmed the feasibility of solubilizing at least two food preservatives in a microemulsion system and the antimicrobial effects on foodborne pathogens.(3) The U-type microemulsion system with glycerol monolaurate as oil, propionic acid as cosurfactant,sodium benzoate as salt and Tween 80 as surfactant was characterized and the broad-spectrum antimicrobial activities were examined.The minimum bactericidal concentrations of the microemulsion against Staphylococcus aureus,Escherichia coli and B.subtilis were 500μL/L,250/μL/L and 250μL/L,respectively.Kinetics of killing results showed that the microemulsion at minimum bacterial concentrations killed over 90%bacteria in 5 min.The antimicrobial activities of the microemulsion against E.coli were stronger than those against S.aureus and B.subtilis,since after 60 min the microemulsion caused a 4 log reduction in S.aureus and B.subtilis titre while a complete loss ofE.coli viability.The minimum fungicidal concentrations of the microemulsion against A.niger and P.italicum were 500μL/L and 1000μL/L,respectively.Kinetics of killing results showed that the microemulsion at minimum fungicidal concentrations killed over 99%A.niger and P.italicum spores in 15 min and 30 min respectively.The antimicrobial activities of the microemulsion against A.niger were stronger than those against P.italicum,since after 120 min the microemulsion caused a 5 log reduction in A.niger spores titre while a 3 log reduction in P.italicum spores titre. (4) Based on the broad-spectrum antimicrobial activities,the antimicrobial mechanisms of the microemulsion against E.coli representing foodbome bacteria and A.niger representing foodborne fungi were examined.The cell surface hydrophobicity and membrane permeability experiments were in good agreement with the kinetics of killing results,and indicated that the interaction between the antimicrobial microemulsion and bacterial membranes significantly decreases the bacterial cell hydrophobicity and induces the quick release of 260 nm absorbing materials,leading to the rapid loss of bacterial viability.The membrane permeability experiment and microstructure observation using light microscopy and scanning electron microscopy were in good agreement with the antifungal activities by agar dilution method and broth dilution method,and indicated excellent growth inhibition and sporicidal activities against A.niger,These findings suggested that the observed antimicrobial activities may be attributed to a disruption and disfunction of fungal cell walls and biological membranes.更多还原