【作者】 张文斌；

【导师】 张晓鸣；

【作者基本信息】 江南大学 ， 食品科学， 2009， 博士

【摘要】 脂肪酸糖酯(SFAE)是一类非离子型、可消化吸收、可生物可降解、无毒的双亲化合物,作为表面活性剂和乳化剂而被广泛应用于食品、化妆品和医药工业中。SFAE的理化性质与它的组成、取代度和化学结构有着密切的关系。与传统的化学法合成SFAE相比,酶法具有直接利用底物及反应条件温和等优点,但同样无法回避选择性不高的问题。本论文主要研究了甘露糖月桂酸酯的脂肪酶(Novozym 435)区域选择性合成和物化性质。通过甘露糖月桂酸酯间歇合成反应过程的研究,揭示了单酯及二酯的反应规律及影响区域选择性控制合成甘露糖月桂酸酯的关键因素。根据这些影响因素,建立了不同反应体系对单酯、二酯分别进行选择性合成,确定了合成甘露糖月桂酸单酯和二酯的工艺参数,研究了甘露糖月桂酸酯的理化性质,包括水溶性、表面性质及Millard反应活性。首先,对甘露糖月桂酸酯的分析检测和分离纯化方法进行了研究。确定了甘露糖月桂酸酯的高效液相色谱(HPLC)分析条件:采用Waters公司的sunfire C-18反相柱(4.6×150 mm,2.5μm),流动相采用95 %的甲醇/水(v/v),流速为1.0 ml/min,进样量为5μl。蒸发光散射检测漂移管温度45°C,喷雾管温度36°C,载气为N2,载气压力20 psi,增益为1。甘露糖月桂酸酯采用硅胶层析分离:100×600 mm硅胶层析柱(硅胶60-100目),上样量20 ml,采用梯度洗脱的方法,洗脱剂依次为乙酸乙酯/正己烷(1:1,v/v,4000 ml),乙酸乙酯/正己烷(6:4,v/v,4000 ml),乙酸乙酯/正己烷/甲醇(7:2:1,v/v/v,4000 ml),乙酸乙酯/正己烷/甲醇(4:2:4,v/v/v,3000 ml),2000 ml 100 %甲醇,流速为10 ml/min,按1管/30 min,收集洗出液,并用薄层层析TLC跟踪洗脱产物,将比移值一致的产物合并。再经过制备色谱进一步纯化:采用Waters公司sunfire C-18反相柱(19×150 mm,2.5μm)。流动相为90 %的甲醇水(v/v),流速为8.0 ml/min,进样量为200μl,蒸发光散射检测器检测。纯化的产品经过红外、质谱和核磁共振分析测定,进行结构鉴定与表征。所得产物为6-O-甘露糖月桂酸酯、1,6-di-O-甘露糖月桂酸酯、4,6-di-O-甘露糖月桂酸酯及3,6-di-O-甘露糖月桂酸酯。确定了脂肪酶催化间歇合成甘露糖月桂酸单酯和二酯合理的工艺条件,单酯的合成条件:75 mmol/L甘露糖、375 mmol/L月桂酸、15 g/L脂肪酶和10 g/L 3 ?分子筛于5 ml丙酮中50℃振荡水浴反应72 h;二酯的合成条件:75 mmol/L甘露糖、酸糖摩尔比5:1、15 g/L脂肪酶和80 g/L 3 ?分子筛于5 ml丙酮中50℃振荡水浴反应120 h。甘露糖月桂酸单酯和二酯的转化率分别为33 %和21 %。构建了循环流化床生物反应器(Circulating fluidized bed bioreactor, CFBBR)系统,对甘露糖月桂酸酯进行连续化生产,以达到对甘露糖月桂酸单酯的选择性合成。选择性合成甘露糖月桂酸二酯的较优条件:月桂酸丙酮溶液(80 mmol/L)饱和溶解甘露糖,进料速度(1.0 ml/min),加酶量(6 g),床层膨胀比(1.6), 50℃反应连续反应7.5天,产物中只有甘露糖月桂酸单酯生成,反应进行前五天,甘露糖月桂酸单酯转化率保持在30 %左右,甘露糖月桂酸单酯的平均产量为59.26 g/(d·L);反应进行至7.5天后转化率下降到25 %。建立一种由乙腈和正己烷这两种不互溶的有机相组成的两相反应体系——同时反应萃取体系(Simultaneous Reaction-Extraction system,SRE),以达到对甘露糖月桂酸二酯的选择性合成。选择性合成甘露糖月桂酸二酯的较优条件:以正己烷和乙腈组成SRE体系,相比为1:1,甘露糖的浓度为0.05 mol/L,酸醇摩尔比为4:1,脂肪酶添加量为15 mg/ml,分子筛添加量为60 mg/ml,50℃振荡水浴150 rpm反应72 h。此时甘露糖月桂酸二酯的转化率为51 %,总酯转化率为76 %。在25℃时,甘露糖月桂酸单酯在水中的溶解度要明显的高于二酯;甘露糖月桂酸单酯因取代度低于二酯,所以亲水-亲油平衡值(HLB)较高,表现出亲水性更强。通过对甘露糖月桂酸单酯和二酯的临界胶束浓度(CMC)、最大吸附量(Γmax)、表面吸附分子的最小截面积(Amin)、临界堆积参数(CPP)、胶束自由能(ΔGm)的测定和比较,表明甘露糖月桂酸二酯比单酯具有更强的胶团化倾向,更易聚集生成胶团。研究了25℃时不同浓度的甘露糖月桂酸酯的泡沫性质和乳化性质。单酯的起泡能力、乳化能力和乳化稳定性都要好于二酯,而泡沫稳定性弱于二酯。根据不同的性质差别,将单酯和二酯进行复配,通过单酯与二酯的相互作用,其配合物的乳化性能和起泡性能有了很大的提高。构建了甘露糖/甘露糖月桂酸酯-L-半胱氨酸Millard反应体系,对比了不同处理时间下不同体系的Millard反应活性。通过顶空固相微萃取和GC/MS分析,1,6-di-O-甘露糖月桂酸酯由于半缩醛羟基被月桂酰基酯化,而表现出较低的Millard反应活性。更多还原

【Abstract】 Saccharide fatty acid esters (SFAEs) are nonionic, digestible, absorbable, biodegradable, and nontoxic amphiphilic derivatives, which have been widely used as surfactants and emulsifiers in food, cosmetic, and pharmaceutical industries. The physicochemical properties of SFAE depends on the fatty acid, the sugar moiety, the degree of substitution, and its chemical structure. Enzyme-catalyzed condensation is superior to the chemical synthesis because of the direct use of unmodified substrates, and moderate reaction conditions. But both of them have low regioselectivities in SFAE synthesis.In this study, regioselective synthesis of lauryl mannoses catalyzed by Novozym 435 was achieved and the physicochemical properties of lauryl mannoses were sudied. Based on the batch reactor study, the reaction rules and key factors influencing the mono- and diesters regioselective syntheses were investigated. Different reaction systems were designed and built for the selective synthesis of mono- or dilauryl mannoses, and the optimum reaction parameters for these systems were determined. The physicochemical properties of lauryl mannoses, such as water solubilities, interfacial properties and Millard activities were studied.The components of reaction solution were analyzed and separated. The reaction solution was analyzed by HPLC using a Sunfire-C18 column (4.6×250 mm, Waters, USA) eluted with methanol/water (95:5, v/v) at 1 ml/min, and detected by a Waters 2420 evaporative light scattering detector (ELSD). ELSD conditions were optimized at drift-tube temperature 45 oC; sprayer temperature 36 oC; carrier gas pressure 20 psi; and gain 1. The reaction solution was applied to a silica gel column (100 to 200 mesh, 100×600 mm) and then eluted with the mixture of n-hexane/ethyl acetate/methanol by a linear programming. The elution gradients were n-hexane/ethyl acetate (50/50, v/v, 4000 ml), n-hexane/ethyl acetate (40/60, v/v, 3000 ml), n-hexane/ethyl acetate/methanol (20/70/10, v/v, 4000 ml), n-hexane/ethyl acetate/methanol (20/40/40, v/v, 3000 ml) and 100 % methanol (2000 ml). The flow rate of the eluent was 10 ml/min, and the eluent, 1 tube/10 min, was collected and monitored by thin-layer chromatography (TLC). Fractions containing the same product were combined together. The concentrated solution was applied to a preparative HPLC with ELSD and a Sunfire-C18 column (5μm, 19×150 mm, Waters, USA) to purify the products. The methanol/water eluant (90:10, v/v), was used at the flow rate of 8.0 ml/min. The loading amount was 200μl. The purified products were identified by FT-IR, MS, 1H NMR and 13C NMR. And they were 6-O-lauryl mannose (ME), 3,6-di-O-lauryl mannose (D 3,6), 4,6-di-O-lauryl mannose (D 4,6), and 1,6-di-O-lauryl mannose (D 1,6).The batch reactor was used to synthsize lauryl mannose. The optimum parameters were achieved by single factor experiments. The optimum parameters for monolauryl mannose were as follow: mannose (75 mmol/L), the molar ratio of lauric acid to mannose 5:1, lipase (15 g/L), and 3 ? molecular sieves (10 g/L) in 5 ml acetone at 50°C for 72 h; the optimum parameters for dilauryl mannoses: mannose (75 mmol/L), the molar ratio of lauric acid to mannose 5:1, lipase (15 g/L), and 3 ? molecular sieves (80 g/L) in 5 ml acetone at 50°C for 120 h. Highest equilibrium conversions were 33 % and 21 % for monoester and diesters, respectively.A novel Circulating fluidized bed bioreactor (CFBBR) was employed for continuous and selective synthesis of monlauryl mannoses by lipase-catalyzed condensation of D-mannose and lauric acid. The highest equilibrium conversion of monoesters of 30 % (the average yield of monolauryl mannose was 59.26 g/(d·L)) for first 5 days and 25 % for after 2 days with no diester detected all the time were achieved. And the optimum parameters were as follow: lauric acid (80 mmol/L), saturated mannose, feed rate (1.0 ml/min), lipase (6 g), expansion rate of the bed (1.6) at 50 oC for 7.5 days.A novel biphasic system, simultaneous reaction-extraction system (SRE), consisting of two immiscible organic solvents, acetonitrile and n-hexane, was employed for the selective synthesis of dilauryl mannoses by lipase-catalyzed condensation of D-mannose and lauric acid. The highest equilibrium conversion of diesters of 51 % and the total conversion of lauryl mannoses of 76 % were achieved at the n-hexane/acetonitrile ratio of 1:1, the molar ratio of lauric acid to mannose of 4:1, 60 g/L molecular sieves and 5 g/L lipase at 50 oC and 150 rpm for 72 hour in 15 ml SRE system.At 25 oC, monolauryl mannose was higher in the solubility and lower in hydrophile-lipophile balance (HLB) value than dilauryl mannnoses. The paremeters, critical micelle concentration (CMC),γCMC, maximal surface excess (Γmax), minimum area per molecule (Amin), critical packing parameter (CPP) and the Gibbs free energy of adsorption (ΔGm) showed that dilauryl mannoses would favor the formation of micelles.The foamabilities, foam stabilities, emulsifying activities and emulsion stabilities of different concentrations of lauryl mannoses at 25 oC were compared. Monolauryl mannose was better in foamabilities, emulsifying activities, emulsion stabilities. The foaming property and emulsifying property would be better while mixing monoester and diester together.Maillard reaction activities of mannose with L-cysteine and lauryl mannoses with L-cysteine were evaluated by headspace solid phase microextraction (HS-SPME) method combined with GC/MS. 1,6-di-O-lauryl mannose exhibited the lowest maillard reaction activity, compared with 6-O-lauryl mannose, 3,6-di-O-lauryl mannose and 4,6-di-O-lauryl mannose.更多还原