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银杏浊汁的酶法制备及其稳定机理研究

Preparation of Ginkgo (Ginkgo Biloga L) Cloudy Juice by Enzymatic Hydrolysis and Its Stability Mechanism

【作者】 张慧

【导师】 王璋;

【作者基本信息】 江南大学 , 食品科学, 2008, 博士

【摘要】 银杏在我国分布很广,资源丰富。银杏不但具有很好的食用价值,而且还具有独特的功能性质和药用价值,深受消费者喜爱。我国银杏总产量占世界的90%左右,居世界银杏产量第一位。银杏以销售干果为主,大部分的银杏资源没有得到充分的利用,银杏深加工产品的开发能充分发挥银杏产业的资源优势,具有十分显著的社会和经济效益。银杏浊汁是以银杏种仁为原料加工而成,但是制备混浊稳定的银杏浊汁一直是生产中的难题。因此,对银杏浊汁加工技术的深入研究和对浊汁稳定机理的探讨具有重要的理论和现实意义。本文研究了复合酶法制备银杏浊汁、不同的杀菌方法处理银杏浊汁以及银杏浊汁的混浊稳定性、色泽稳定性和挥发性成分。主要研究内容如下:确定了糊化和制浆的条件。DSC测得银杏淀粉的糊化终了温度为81.904℃。通过研究银杏浆料的糊化温度和时间以及浆料的粒径分布对银杏浊汁品质的影响,确定银杏浆糊化温度和时间为90℃、10 min;银杏浆均质后更有利于酶解。通过研究不同的酶制剂对银杏浊汁品质的影响,发现使用淀粉酶单一酶制剂的效果不如淀粉酶和蛋白酶复合使用的效果,其中中温α-淀粉酶和Alcalase蛋白酶的复合可以最有效的提高银杏浊汁的悬浮稳定性和得率,改善银杏浊汁的品质。反应温度为60℃、淀粉酶用量为7.39 U/g果肉、蛋白酶用量为6.43 U/g果肉以及酶解时间为73 min。银杏浊汁保留了银杏种仁中大部分的营养物质。研究了高压脉冲电场(PEF)对银杏浊汁的杀菌效果,发现处理时间越长、电场强度越高、水浴温度越高杀灭效果越好。较佳的处理条件为:水浴温度为40℃,电场强度为30 kV/cm,处理时间为260μs,脉冲宽度为3μs,频率为303 Hz;或者水浴温度为15℃,电场强度为30 kV/cm ,处理时间为520μs,脉冲宽度为3μs,频率为303 Hz。PEF处理过程中热的影响是有限的,它和PEF杀菌具有协同作用。研究了PEF处理后银杏浊汁的货架寿命。以微生物的存活数为评价指标,在520μs,30 kV/cm和15℃的条件下,经过PEF处理后的银杏浊汁在4℃可以贮存18天,在室温下可以贮存15天。经过260μs、30 kV/cm和40℃条件下处理后的银杏浊汁,在4℃可以贮存15天,在室温下可以贮存6天。结果证明PEF处理可以延长银杏浊汁的货架寿命。确定了适合银杏浊汁的最佳杀菌方法。超高温瞬时杀菌在140℃保持5 s、10 s和15s均可使银杏浊汁达到商业无菌的要求。采用121℃高温杀菌、UHT杀菌和PEF杀菌对银杏浊汁品质的影响以及感官评定,确定适合银杏浊汁的最佳杀菌方式为超高温瞬时杀菌。探讨了银杏浊汁的混浊稳定性与酶制剂作用的关系。酶制剂的作用使银杏浆中淀粉水解为糊精和低聚糖,蛋白质水解为肽。银杏浊汁的悬浮颗粒受大分子糊精或果胶柔顺分子链构象的限制而产生空间排斥效应;酶制剂的作用使蛋白质水解以及浊汁中带负电的氨基酸残基的增加,不利于蛋白质和多糖之间的相互作用,浊汁的Zeta电位增加,悬浮颗粒间的静电排斥作用增强。空间排斥效应和静电排斥作用是银杏浊汁保持混浊稳定的主要原因。研究了杀菌方法对银杏浊汁混浊稳定性的影响。杀菌方法对悬浮颗粒的Zeta电位影响不大,但对悬浮颗粒的大小影响较大,PEF、UHT、121℃杀菌后银杏浊汁悬浮颗粒的平均粒径分别为:0.925μm,1.333μm,2.467μm,混浊稳定性的效果为:PEF >UHT > 121℃高温杀菌。不同的亲水胶体对银杏浊汁的稳定性影响不同。瓜尔豆胶的增稠作用最显著,亚麻籽胶具有一定的乳化性能。在30℃、35℃和40℃贮存温度下对UHT杀菌的银杏浊汁进行加速贮存实验,预测银杏浊汁在25℃时的货架寿命为388天。通过分析银杏浊汁的汁液和沉淀物的组成,探讨了贮存过程中银杏浊汁混浊稳定的机理:一方面大分子糊精通过空间排斥效应和静电排斥作用保持银杏浊汁的混浊稳定;另一方面糊精大分子彼此交联长大,杂乱无序的缠绕在一起,易将夹杂在大分子之间的颗粒一起纠缠,聚集成较大的颗粒,在重力的作用下沉降下来。糊精、脂肪-直链淀粉络合物、果胶-蛋白或蛋白质-多酚的复合物及其细胞碎片等之间的相互作用也会使颗粒长大而发生沉降。不同的加工阶段对银杏浆非酶褐变具有不同的影响。酶反应引起的褐变主要由于美拉德反应的发生,高温杀菌引起褐变可能是由美拉德反应和焦糖化反应产生。研究了贮存过程中银杏浊汁的色泽变化。贮存温度越高,时间越长,银杏浊汁的褐变越严重。美拉德反应是银杏浊汁贮存过程中发生非酶褐变的主要原因。将贮存过程中银杏浊汁褐变指数的变化曲线与动力学方程拟合,推测贮存期间银杏浊汁的非酶褐变符合零级动力学反应。酚类化合物的氧化聚合和维生素C的氧化降解不是主要影响因素。分析了加工过程和杀菌方法对银杏浊汁挥发性成分的影响。经SPME富集,GC-MS分离与检测,从新鲜银杏种仁中测定出46种化合物,主要为萜烯类物质。从糊化后的银杏浆料中测定出30种化合物,有20种化合物与新鲜银杏种仁的挥发性物质相同。从杀菌前的银杏浊汁中测定出49种化合物,与糊化后的银杏浆料有19种物质相同。PEF杀菌后的银杏浊汁中测定出41种化合物。与杀菌前相同的挥发性成分有36种,UHT杀菌的银杏浊汁中测定出57种化合物,与杀菌前有36种相同,121℃高温杀菌的银杏浊汁中测定出53种化合物,与杀菌前有33种相同,不同杀菌方式处理后的银杏浊汁都保留了杀菌前的大部分的挥发性成分。与银杏种仁比较,PEF杀菌后的果汁保留18种化合物;UHT杀菌后的浊汁保留18种物质;121℃杀菌后的浊汁仅保留12种。加工条件对挥发性物质影响较大,但银杏浊汁仍然保留了银杏特有的香味。在银杏浊汁的加工过程中,有些加工过程改善了银杏浊汁的风味,如酶处理过程;有些加工步骤如热处理导致大量羰基化合物以及一些褐变产物糠醛、吡啶、呋喃类化合物的生成,产生蒸煮风味。

【Abstract】 Ginkgo, which widely distributed in China, has been consumed as not only a nice food, but also an herbal medication. The yield of ginkgo in China is around 90 percent of the total yield in world, presents the place of number one. Most of ginkgo is only sold as dried fruits, not fully used at present. Developing some new products from ginkgo could take advantage of ginkgo resource and promotes both social and economic benefits. Ginkgo cloudy juice was one of the main products made from ginkgo seeds. However, it is very difficult to make the juice stable. Therefore, study of processing technology and stable mechanism of ginkgo cloudy juice has the deep significant both in theory and reality.This paper studied in the method of processing ginkgo cloudy juice by multiple enzymes, and the effect of different sterilized methods for ginkgo cloudy juice and their cloud stability, color stability and flavor.The conditions of gelatinization and processing of ginkgo soup were studied, the gelatinization temperature determined by DSC was 81.904℃. Then the research of the relationship between gelatinization temperature and time and particle distribution for quality of ginkgo cloudy juice was carried out, results showed that the gelatinization temperature and time were 90℃and 10 min, respectively. Homogeneous ginkgo soup was benefited for enzymatic hydrolysis.The influences of different enzymes for quality of ginkgo cloudy juice were investigated. The combination of amylase and proteinase had a desirable effect on the quality of ginkgo cloudy juice. An appropriate enzymatic formulation containing both mild temperaturesα- amylase and Alcalase proteinase could increase the juice yield and suspension stability, and improve quality of ginkgo cloudy juice.The optimal parameters of enzymatic reaction for ginkgo cloudy juice was determined. The single experiment and RSM experiment were designed to optimize hydrolysis conditions of middle temperatureα- amylase and Alcalase proteinase. The optimal processing conditions were obtained as follows: the ratio of raw and water was 1:5, initial pH was 7.0, reaction temperature was 60℃, amylase usage was 7.39 U /g pulp, Alcalase usage was 6.43 U/g pulp and hydrolysis time was 73 min. Components analysis indicated that ginkgo cloudy juice retained most of the nutritional components in fresh ginkgo kernel.Effects of inactivate microorganism in cloudy ginkgo juice by PEF treatment were investigated. The result showed that the higher the treatment time and electric fields strength, the greater microbial destruction. The optimal conditions were showed as follows: pulse duration of 3μs, electric field strength of 30kV/cm, treatment time of 520μs and the temperature of water bath of 15?C or 260μs at 40?C. PEF combination with mild thermal treatments enhanced microbial inactivation effectiveness. Heat, which is generated during the PEF processing, has a synergistic effect with PEF treatment.The shelf time of ginkgo cloudy juice by PEF treatment was studied. Survival numbers of microorganism was selected as evaluative index. The microbial shelf life of ginkgo juice by PEF treated at 520μs, 30kV/cm and 4?C was 18 days, 15 days at room temperature. The shelf life of ginkgo juice at 260μs, 30kV/cm and 4?C was 15 days, 6 days at room temperature. The results showed PEF processing could extend the shelf life of ginkgo cloudy juice.The sterilized method which suitable to ginkgo cloudy juice was determined. Ginkgo cloudy juice was treated by UHT at 140℃and kept for 5s, 10s and 15s, respectively. Ginkgo cloudy juice by UHT treatment reached commercially sterile requirements. The effects of 121℃treatment, UHT and PEF treatment for quality of ginkgo cloudy juice and sensory evaluation were investigated, and the best sterilized methods was decided as UHT treatment. The effects of enzymatic treatment on suspension stability of ginkgo cloudy juice were investigated. Starch molecules were treated byα- amylase to become dextrin and oligosaccharide, and protein treated by proteinase become peptides. Due to the effects of pliancy molecular chain of macromolecules dextrin or pectin, the steric repulsive effects were produced among suspended particles in ginkgo cloudy juice. Hydrolysis of protein by enzyme treatment and increasing of negative amino acid residues were not benefit to the attraction between protein and polysaccharide. And the increased zeta-potential of ginkgo cloudy juice also enhanced the exclusive effects of suspended particles. Therefore, the electrostatic repulsive effects were strengthened among suspended particles. Cloud stability of ginkgo cloudy juice mainly depended on the steric and electrostatic repulsive effects among suspended particles.Effects of ginkgo cloudy juice stability by sterilized methods were studied, and the effect of Zeta potential of suspended particle by sterilized methods was not significant. But sterilized methods could effects the particle size. The average particle size of suspended particle in ginkgo cloudy juice by PEF, UHT or 121℃treatment was 0.925μm, 1.333μm and 2.467μm, respectively. Comparison of cloud stability was PEF >UHT >121℃.Different hydrocolloids had different effects on cloud stability of ginkgo cloudy juice. Guar gum was the most thickening effect, and flaxseed gum possessed a little emulsification. By accelerated storage tests at 30, 35 and 40℃for ginkgo cloudy juice by UHT treatment, suspension stability was selected as evaluative index, the activation energies of the suspension stability was 70.44 kJ/mol. The shelf life of ginkgo cloudy juice was 388 days at 25℃.According to the results of the components of ginkgo cloudy juice and sedimentation, the mechanism of cloud stability of ginkgo cloudy juice was discussed. On one side, cloud stability of ginkgo cloudy juice mainly depended on the steric and electrostatic repulsive effects among suspended particles including macromolecular substances and suspended pulp substances. On the other side, during storage some macromolecular of dextrin occurred cross-linking with each other. Those macromolecular and some particles formed sedimentation due to the action of gravity. Macromolecular material, sugar-protein or protein-polyphenol compounds, fat-amylose complex and cell fragments were aggregated together and grown up to form more sediments.Different processing stage had different effects on non-enzymatic browning of ginkgo cloudy juice. Browning during enzymatic reaction was brought by Maillard reaction. Browning during high temperature sterilization may be caused by Maillard reaction and caramel reaction.Changes of color of ginkgo cloudy juice during storage were investigated. The result showed that the higher storage temperature or the longer storage time, the more seriously non-enzymatic browning. Maillard reaction was the most important cause resulting in non- enzymatic browning and non-enzymatic browning kinetics was preferred to zero order during ginkgo cloudy juice storage. Oxidation of polyphenols and Vitamin C were not main reason of browning.The volatile compounds of ginkgo cloudy juice were concentrated by SPME, separated and identified by GC-MS. 46 kinds of compounds were identified in volatile fraction of fresh ginkgo kernels, and the main components were terpene. 30, 49, 41, 57 and 53 kinds of compounds were identified in volatile fraction of gelatinized soup and those not sterilized, PEF treated, UHT treated and 121℃treated ginkgo cloudy juice. Volatile fraction of ginkgo cloudy juice by PEF, UHT and 121℃treatment compared with fresh ginkgo kernels retained 18, 18 and 12 kinds, respectively. The results indicated processing conditions had significant effect for volatile components, but ginkgo cloudy juice retained the main aroma components of fresh gingko kernels after processing. Some processing (such as enzymatic treatment) could improve flavor of ginkgo cloudy juice, however, another processing (such as heat treatment) made many of cooking aroma such as furfural, pyridine and furan.

  • 【网络出版投稿人】 江南大学
  • 【网络出版年期】2009年 03期
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