【作者】 陆建良；

【导师】 梁月荣；

【作者基本信息】 浙江大学 ， 茶学， 2008， 博士

【摘要】 饮料是重要的食品种类之一。丰富多彩的饮料产品不仅满足了消费者的各种需求,而且创造了良好的经济效益和社会效益。2007年全球饮料产量估计达到1.3万亿升,销售收入达9100亿美元。浑浊问题是目前饮料研发和生产过程中普遍面临的重要技术障碍。多酚和蛋白互作引起的浑浊是各种饮料中最重要、也是最难彻底清除的浑浊类型之一。资料显示,蛋白分子中脯氨酸或者组氨酸等氨基酸侧链通过疏水堆叠方式与多酚分子中芳香环结合,分子间的氢键进一步强化两者的互作。形成的可溶性多酚/蛋白复合物通过“多酚桥”相互团聚,并形成浑浊和沉淀。多酚/蛋白互作强度主要取决于多酚、蛋白浓度以及两者比例,环境温度、酸度、金属离子等对两者互作也有显著影响。添加明胶或单宁加速絮凝、皂土等吸附剂吸附、超滤以及酶处理是常用的饮料浑浊控制措施。虽然蛋白对茶饮料浑浊有重要影响,但由于茶饮料中蛋白含量低、分离纯化困难,有关茶汤蛋白性质及其对茶饮料浑浊影响的研究报道较少。本项目主要采用电泳技术研究茶汤蛋白的理化特性;并以EGCG/蛋白/咖啡因浑浊模型研究蛋白以及不同温度和酸度条件对茶饮料浑浊形成的影响;比较不同吸附剂对茶汤蛋白的去除效果,优选吸附剂的种类和使用剂量,为茶饮料浑浊控制奠定基础。利用24个杀青叶样品,对离心前后透过率差值△T与色差和粒度等相关指标进行了分析。结果显示,△T660nm与L值、遮光比、比表面积、D3等粒度参数具有显著或极显著的相关关系。以△T660nm为依据建立了L值和遮光比等参数的预测模型,以外部验证法对建立的模型进行了验证。证明以△T660为基础建立的模型可以有效对遮光比、L、比表面积等参数进行预测,△T660是一种可用于少量样品(＜1ml)浑浊情况评价的有效方法。电泳结果显示,茶汤经冷藏诱导后,大分子量(166kD和60kD)的蛋白倾向于进入沉淀,而小分子量蛋白(56kD和48kD等)则存在于上清液中。将EGCG添加到初步纯化的茶汤蛋白溶液中温浴并冷藏,发现60kD的蛋白先于166kD蛋白进入沉淀,166kD蛋白也容易进入沉淀;在较高环境温度时,几乎所有类型的蛋白都可与EGCG互作而从上清液中消失。可见,高温不利于有效区分浑浊活性蛋白(HAPs)与非HAPs。从HAPs定义来看,60kD和166kD蛋白是较典型的HAPs,且60kD蛋白起浑能力更强。等电电泳显示,60kD和166kD蛋白的等电点为pH9.0-pH10.5。此外,研究还发现,杀青叶冷藏后,提取液60KD和166KD的蛋白含量下降,冷藏1月以后,提取液中未检测到这两种大分子蛋白。EGCG/蛋白/咖啡因浑浊模型体系研究结果显示,在37℃温浴条件下,明胶(GEL)的起浑能力显著高于牛血清白蛋白(BSA),茶汤蛋白(PFT,从冷藏1月以上的杀青叶中萃取)介于两者之间;在80℃温浴条件下,GEL的起浑能力显著高于PFT,而BSA介于两者之间。总体而言,由冷藏1个月后杀青叶制备的PFT的起浑能力更接近BSA,说明普通茶饮料中的蛋白是非HAPs。研究还显示,蛋白类型和浓度、EGCG浓度以及体系温度对模拟体系△T660有显著影响。进一步的研究表明,热处理后蛋白结构变化是引起EGCG/蛋白体系变浑浊的最主要原因;酸度对EGCG/蛋白体系浑浊的影响主要与蛋白的等电点有关,在等电点附近EGCG/蛋白体系最浑浊,EGCG和蛋白因沉淀引起的损失最大。分析了16个不同品种杀青叶样品的△T660、色差、粒度指标以及儿茶素类等化学成分,采用主成分分析法,从27个变量中抽提出5个主成分,其累计方差达到92%。以5个主成分中特征向量绝对值较大变量对△T660、色差和粒度指标进行多元回归分析,建立了预测模型。并采用外部验证法对建立的模型进行了可靠性分析,结果显示,模型计算的△T660、L、△E、比表面积、遮光比以及粒径指标(除DAV外)均与实测值相吻合,说明通过茶叶样品的化学分析可以有效地预测茶汤的浑浊程度,茶叶化学组份不同是导致茶汤浑浊程度差异的根本原因。该结果为通过原料选择来控制茶饮料冷后浑奠定了基础。不同吸附剂对蛋白的吸附试验结果显示,硅胶对蛋白吸附效果最好,其对BSA、GEL和PFT的吸附比例分别达到99%、88%和77%。洗脱试验结果显示,柠檬酸缓冲液对蛋白的洗脱效率较低,而NaOH(0.1M)洗脱效果好。将硅胶按0.5%(w/v)的比例添加到茶汤中,可去除85%以上的茶汤蛋白,但对茶汤中儿茶素类和咖啡因等功能成分没有显著影响。经硅胶吸附处理后的茶汤热稳定显著提高,因此硅胶可作为茶饮料浑浊控制的良好吸附剂。更多还原

【Abstract】 Beverage is one of the most important foods around the world. Various beverages meet the different demands of consumers, and bring about considerable benefits economically and socially. Estimated total consumption volume and revenues of the beverages worldwide reached to 1.3 trillion liters and 910 billion USD in 2007 respectively. Haze formation is the most serious and common technical obstacles in beverage development and production. Polyphenols and proteins interaction is the key factor responsible for haze formation and overcoming the problem is quite difficult. According to the published reports, the complex of polyphenols/proteins is principally a hydrophobic stacking of the polyphenols galloyl ring against the pyrrolidine ring face of the proline residue and basic amino acids such as histidine. Secondary hydrogen bonding effects stabilization of the complex. Aggregation and precipitation occurred when "polyphenols-bridge" was established between polyphenols/protein complexes. The interaction strength of polyphenols and protein is subjected to the concentration, as well as, ratio of polyphenols and proteins. Environment temperature, acidity and metal ions concentration also affect the polyphenols/proteins interaction significantly. Flocculation by addition of gelatin or (and) tannin, absorption of protein by absorbents such as bentonite, ultrafiltration and treating with hydrolytic enzymes are usually used to minimize haze formation.Characteristics of tea liquor proteins and their effects on the haze formation are still unknown thoroughly because of their low concentration and high purification difficulty. In this present investigation, effects of the proteins on haze formation at different temperature and acidity were investigated in a model system comprising of Epigallocatechin gallate (EGCG)/protein/caffeine and the characteristics of the proteins were studied by electrophoresis. Comparison of various protein absorbents was also conducted in this paper.Liquor color difference, particle distribution and liquor transmittance difference(AT),.before and after centrifugation, were assayed in water extracts of steam -fixed leaves harvested from 24 different cultivars. The results showed thatΔT660 (transmittance difference at 660nm) was significantly correlated to "L" value (index for liquor brightness), obscuration, specific surface area and the particle index. Prediction models of the "L" value, obscuration and other index of liquor were established by usingΔT660 as independent variable. The reliability of models was tested by external validation method. AndΔT660 measurement was proved as effective method for haze analysis of liquor samples with small volume(<1ml).Electrophoresis result showed that, after quenching treatment of the tea infusion, proteins with high molecular weight (such as 166KD and 60KD) are more prone to cream formation, whilst low molecular weight proteins were stable in supernatant. EGCG was added and incubated 37℃for 1hr to enhance the precipitation of protein. It was found that, 60 KD protein was precipitated first followed by 166KD protein. These two proteins were identified as typical haze-active proteins (HAPs) in tea infusion, and 66KD protein exhibited high ability of haze formation. After high temperature (80℃) incubation and quenching induction, all the protein fragments were aggregated by EGCG and creamed down. High temperature incubation was not suitable to identify the HAPs or non-HAPs. Electrophoresis results also showed that the isoelectric point (pI) of 60KD and 166KD proteins was around pH9.0-pH10.5. Furthermore, concentration of 60KD and 166KD protein in extracts decreased with the extension of cold-storage time. After one month, these fragments could not be detected in protein extracts.In EGCG/protein/caffeine model system at 37℃, haze formation ability of gelatin(GEL) was significantly higher than bovine serum albumin (BSA), and proteins extracted from fixed tea leaves(PFT, more than 1 month cold-storage) was lied in between GEL and BSA. After 80℃incubation, the sequence of haze formation ability was GEL> PFT> BSA. In general, PFT is much similar to BSA because of their low ability of haze formation. Proteins extracted from common tea belonged to non-HAPs in this manner. Significant effects of proteins type and concentration, EGCG concentration and system temperature on the haze formation were found in model system. Furthermore, structure damage and exposing more polyphenols binding sites was the main cause for much more haze formation after heat treatments of EGCG/protein system. Haze of model system also affected by pH value, i.e. when pH value was near the protein pI much more haze was developed, and much more lose of EGCG and protein was also found.ΔT660, liquor color difference, particle index and ingredients such as catechins were investigated in 16 samples plucked from different cultivars. Five eigenvalues were extracted from 27 variables, which represented 92% cumulative variation. Eigenvectors with big absolute value were selected for regression equation establishment. Reliability of equations was estimated by another 8 validation samples.ΔT660, "L" value,ΔE (total color difference), specific surface area, and particle index (except DAV) which estimated by the equation were consistent with the "truth value" which was gotten by actual analysis. It was concluded that haze forming ability of the tea samples could be estimated effectively by chemical measurement. This result can be adopted for haze relief in the manner of raw material selection.Results showed that silica gel had the highest absorption potential comparing to the bentonite and white bole. Silica gel could effective remove the BSA (99%), GEL (88%) and PFT (77%) from the aqueous solution. Protein could be eluted from gel easily by 0.1M NaOH, but inefficient elution was obtained when using citrate buffer. More than 85% proteins were removed by addition 0.5% (w/v) silica gel into tea infusion and centrifugation. The gradients such as catechins and caffeine were affected significantly by the silica gel treatment. And heat-stability was enhanced dramatically after gel absorption. It was proved that the silica gel was a effective clarification reagent for preparation of ready-to-drink tea.更多还原