摘要
2-苯乙醇(2-苯基乙醇)是最重要的香味物质之一,具有玫瑰特有的香气,广泛应用于食品、糖果、饮料、香料、化妆品等行业。随着人们越来越关心健康与和营养,崇尚“绿色”、“天然”,美国和欧盟等纷纷对化学食品添加剂的使用进行了严格的限制规定,天然2-苯乙醇的需求量越来越大。从玫瑰花中提取的天然2-苯乙醇价格昂贵,生产量少,成本高,原料受多方因素限制,不能满足人们日益增长的需求。利用微生物转化法生产天然2-苯乙醇具有周期短、成本价格低、可获得天然等同产品等优点,是具有发展前途的替代方法之一。
本论文以酵母细胞微生物转化L-苯丙氨酸生成2-苯乙醇为进行了研究,主要研究内容包括:摇瓶发酵培养条件的优化;酿酒酵母的细胞固定化及其反复分批生物转化;两相体系生物转化L-苯丙氨酸生成2-苯乙醇。
首先从酿酒酵母、马克斯克鲁维酵母和中国克鲁维酵母这三种被报道用于转化L-苯丙氨酸生成2-苯乙醇的最多的菌种中筛选出转化能力最强的酿酒酵母作为后面的实验菌种。研究了酿酒酵母转化L-苯丙氨酸生成2-苯乙醇的液体发酵的营养条件,确定了蔗糖为该转化菌株的最佳碳源,最佳液体培养基条件(g/L):蔗糖40,L-苯丙氨酸7,磷酸氢二钠0.5,YNB 1.8。蔗糖和L-苯丙氨酸对转化发酵的影响较大,其次为磷酸氢二钠和YNB。由于培养基成分YNB的添加使得发酵培养基中硫酸镁含量达到要求,继续添加反而会使2-苯乙醇产量降低,因此本实验发酵培养基中不添加硫酸镁。实验得出了微生物转化2-苯乙醇生产2-苯乙醇适宜的摇瓶条件为:种子培养时间为24h,接种量6%,优化培养基初始pH为4.0,装液量为50mL/250mL,在30℃时于转速100r/min的往复式摇床上培养,在此条件下发酵48h后,发酵液中2-苯乙醇含量可达到2.0g/L,比优化前(1.5g/L)提高了33.33%。
分别用角叉胶、海藻酸钠、明胶这3种不同载体进行包埋法固定化细胞的试验。经比较,就强度和2-苯乙醇的生成而言,采用海藻酸钙包埋法均优于其它包埋法,且该操作条件简单,容易控制固定化胶珠小球的大小,而明胶凝固后需要切块。因此,选用海藻酸钠作为载体,进行固定化细胞的条件实验。然后采用海藻酸钙包埋法对酿酒酵母细胞进行了固定化研究,确定固定化条件为海藻酸钠4.0%,细胞量20%,硬化用Ca2+浓度0.1mol/L。通过对固定化细胞性质的研究发现,其最佳反应pH 3.5,比游离细胞(pH4.0)偏移了0.5,最佳反应温度30℃,最佳反应时间48h,与游离细胞一致。游离细胞和固定化细胞在各pH下都较稳定。固定化细胞保藏的酶活半衰期超过10d,在贮存前5d内,固定化细胞的转化能力没有明显下降,其贮存稳定性较好。而游离细胞贮存稳定性较差,其转化能力随贮存时间直线下降,其酶活半衰期不足5d。对固定化细胞的反复分批生物转化进行了研究,以48h作为一个周期,固定化细胞使用的半衰期约为5次,比游离细胞多2次。
为了解决2-苯乙醇的产物抑制问题,对两相体系微生物转化L-苯丙氨酸生成2-苯乙醇进行了研究。对庚烷、辛烷、壬烷和油酸等这集中常见的有机溶剂进行了比较。结果表明,油酸不但具有合适的底物和产物的分配,而且生物相容性好,其它的几种溶剂都不能同时具备这两个条件。然后研究了水/油酸两相体系微生物转化L-苯丙氨酸生成2-苯乙醇的条件,有机溶剂油酸与水的相体积比为1:1为宜,最佳温度为30℃,最佳pH为4.0,最佳装液量为20mL/250mL。在优化反应条件下,48h后,水/有机溶剂两相体系中酿酒酵母转化L-苯丙氨酸生成2-苯乙醇的浓度可达3.0g/L,比水相体系2.0g/L高50%。
As one of the most important components of natural flavors, 2-phenylethanol is widely used in foods, beverages and perfumes industries. Natural 2-phenylethanol extracted from rose features both a high price and a limited supply. The increasing concern for health nutrition and the limit of chemistry food additive in United states and European Union stimulate a worldwide demand for natural 2-phenylethanol. Biotransformation is an alternative or equivalent technology to get natural 2-phenylethanol. Natural L-phenylalanine is available and economical substrate for the 2-phenylethanol production. Therefore, the bioconversion of L-phenylalanine to 2-phenylethanol becomes more and more attractive.
The paper focused on the biotransformation of L-phenylalanine to 2-phenylethanol by microorganisms. The optimization of fermentation with free and immobilized cells is studied. Then the production of 2-phenylalcohol from L-phenylalanine by microbial conversion in aqueous/organic solvent biphasic system was studied.
Saccharomyces cerevisiae, Kluyveromyces marxianus and Kluyveromyces sinensis were reported to be three of the best strains which could produced more 2-phenylethanol than others. The results showed that Saccharomyces cerevisiae was the best strain which had bigger capacity to transform L-phenylalanine to 2-phenylethanol among the three stains tested. The conditions of liquid fermentation of 2-phenylethanol were studied. It showed that the effect of single factors, such as carbon source, nitrogen source, growth factors and inorganic salts on the production of 2-phenylethanol. Through orthogonal test, the optimum medium composition had been obtained. It was 40g/L sucrose, 7g/L L-phenylalanine, 0.5g/L Na2HPO4, 1.8 g/L Yeast Nitrogen Base( without amino Acid and Ammonium Sulfate). The optimum shaking culture condition was as follows: 24 hours strain age, 6% inoculation quality, initial pH 4.0, 50mL medium in 250mL shaking flask, 30℃, 100r/min shaking speed, 48hours incubation. Under this condition, productivity of 2-phenylethanol was 2.0g/L, which increased about 33.3% than that in original medium.
The cells of Saccharomyces cerevisiae were entrapped into calcium alginate. The immobilized conditions were alginate 4.0%, cells 20%, Ca2+ 0.1mol/L .The optimum pH for free and immobilized cells were 4.0 and 3.5, respectively. The optimum reaction temperature for free and immobilized cells was 30℃. The pH stabilities of free cells and immobilized cells were good at all pH levels. For storage stability, the half-life of free cells was less than 5 days and the half-life of immobilized cells was more than 10 days.
Microbial conversion of L-phenylalanine to 2-phenylalcohol with yeast was performed in an aqueous/organic solvent biphasic system. Saccharomyces cerevisiae had more potential to transform L-phenylalanine to 2-phenylalcohol than Kluyveromyces marxianus. The results of the solvent screening indicated that oleic acid was the best solvent which can extract 2-phenylalcohol efficiently. The optimum values of volume ratio of organic phase to aqueous phase, reaction temperature, pH and liquid volume were 1:1, 30℃, 4.0 and 20mL/250mL, respectively. Under above optimum conditions, the content of 2-phenylethanol reached 3.0g/L 48h later, which increased about 50% than that in monophasic aqueous system.
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