Effects Of Proteins On Haze Formation Of Ready-to-Drink Tea

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.