1. Preparation of High Quality Pumpkin Juice by an Enzymatic Liquefaction Technique Lan Qin, Shiying Xu and Zhang Wang School of Food Science and Technology, Southern Yangtze University, Wuxi, Jiangsu, China ABSTRACT Vegetable juices have become a focus of attention due to their nutritional value. Pumpkin is one of the principal vegetables rich in bioactive carotenoids and other functional components. Therefore using enzymatic liquefaction technique to make fresh pumpkin juice with attractive color and cloud stability is desirable. Our objective was to develop a new method for preparing fresh pumpkin juice by using a complex mixture of enzymatic preparations to liquefy the pumpkin tissue. The resulting pumpkin juice should have high quality including attractable color, cloud stability and rich nutrition. Combined enzymatic preparations containing cellulase, hemicellulase and pectinase activities were used to liquefy the pumpkin cell walls. They had a desirable effect on juice yield and juice quality. Appropriate enzymatic formulation increased the juice yield by 20% and improved color and cloud stability. The different viscosities of pumpkin purees were obtained with adding different enzymatic preparations. Ultrastructure of the pumpkin tissue showed the cell walls were broken down by enzymatic maceration. The synergistic effect of enzymes was due to cooperative enzymatic hydrolysis of pectin, cellulose and hemicellulose coexisting in the primary wall and the middle lamella of pumpkin pulp cell walls. So enzymatic liquefaction technology could not only increase the juice yield but also meliorate the color and cloud stability of cloudy pumpkin juice. INTRODUCTION In recent years, fruit and vegetable juices have become a focus of attention all over the world due to their nutritional value. Cloudy pumpkin juice is a natural, healthy and nutritional vegetable juice rich in bioactive carotenoids and other functional components. The main objective of the fruit and vegetable juice industry is to process fruits and vegetables at the lowest cost while maintaining or even improving the organoleptic quality and stability of finished product. Enzymatic liquefaction offers a number of advantages in producing fruit and vegetable juices, such as high yield and soluble solids, suspended fiber solids, better color and cloud stability. Application of exogenous enzymes leads to the degradation of cell walls in fruits and vegetables and the selective extraction of some of their components. Therefore exogenous enzymes are the tools of choice to modify fruits and vegetables, and to improve the organoleptic quality of juices and their stability. Conventional pumpkin juice processing with mechanical pressing of the pulp results in poor juice yield and destability. Different commercial enzymatic preparations were used to hydrolyze pumpkin puree and enzymatic liquefaction technique was improved the quality and stability of cloudy pumpkin juice. MATERIALS AND METHODS Materials Fresh pumpkins were purchased from the local market in Wuxi. A cellulytic preparation (JC) and five kinds of pectolytic preparations (PU, PS, CC, RP, RB) were used for the enzymatic treatment of pumpkin puree. Assay of xylanase (EC 3.2.1.8), cellulase (EC 3.2.1.4), polygalacturonases (exo-PG, EC 3.2.1.67; endo-PG, EC 3.2.1.15), pectin esterase (EC 3.1.1.11) activity Xylanase, Cellulase and exo-PG activity was measured by assaying for the xylose, glucose and galacturonic acid liberated from xylan, carboxymethyl cellulose and pectin. The liberated sugars were assayed by reaction with dinitrosalicylic acid (DNS) reagent. Endo-PG activity was assayed by decreasing of pectin viscosity. Pectin esterase activity was determined carboxyl liberated from pectin with alkali titration. Rheological measurements of pumpkin puree Rheological measurements were done in an AR1000 rheometer. A 4 cm diameter parallel plate with 1 mm gap size geometry was used. Different enzymatic preparations (PU, CC, JC and their combinations) were added to pumpkin puree and their viscosity changes with time were determined. Preparation for scanning electron microscopy Rectangular pieces were cut from the pumpkin pulp and were submersed in enzyme solution (PU, JC, PU and JC) for different times. Then they were heated to inactivate the enzymes and fixed in glutaraldehyde solution and osmium oxide, coated with a layer of gold-palladium, and observed in a scanning electron microscope. Preparation of pumpkin juice Fresh pumpkins ↓Peeling ↓Deseeding ↓Slicing ↓Macerating in sodium chloride solution ↓Washing ↓Blanching ↓Cooling ↓Grinding Pumpkin purees ↓Adding cellulytic and/or pectolytic preparations ↓Inactivating enzymatic preparations ↓Cooling ↓Centrifuging Pumpkin juices ↓Bottling in glass ↓SterilizationProducts RESULTS Tab1. The enzyme activities of different commercial enzymatic preparations All the commercial enzymatic preparations are the multi-enzyme systems comprising different kinds of enzymes. PU had higher endo-PG activity, lower exo-PG and pectin esterase activity than other pectolytic preparations. JC exhibited high xylanase, cellulase activities and low exo-PG activity. Fig1. The viscosity of pumpkin puree changed with time The viscosity of pumpkin puree did not change without enzyme treatment and with CC. When only PU, the pumpkin puree’s viscosity was reduced slowly. The viscosity with JC was descended faster. If combining JC and pectolytic preparations, they were decreased very rapidly, especially JC with PU. A C B D Fig2. The SEM micrograph of pumpkin pulp cell observed using scanning electron microscopy. A, control; B, exposed 60 min to PU; C, treated with JC for 30 min; D, treated with JC for 120 min; Exposure of pumpkin pulps to enzymatic preparations resulted in the gradual disintegration of the cell walls. The cells without enzyme maceration arrayed tightly while its cell walls also are intact with cytoplasms enveloped in it. After treating with PU, parts of cell walls were broken down and some of intracellular materials were liberated. Using JC to deal with the pumpkin pulps, the cell walls were crumbled and ruptured more and more with prolonged time. The associate activities of PU and JC were more effective. The cells of pumpkin pulps treated with PU and JC for 30 min were separated. After 60 min, the cell walls were collapsed, which would provide for a more efficient release of liquid and interior components from the cells during the subsequent extraction. DISCUSSION Enzymatic liquefaction with different enzymatic preparations acting on the pumpkin cell walls effectively increased the yield of juice, the soluble solid, beta-carotene content, pigment and so on. This was related with different enzyme activities, such as xylanase, cellulase, exo-PG and endo-PG. These enzymes can destroy the ordered structure of the cell walls. By combining pectin-degrading enzymes with cellulose- degrading enzymes, their synergistic effects were very effective, degrading the pectin, cellulose and xyloglucans of middle lamella and primary wall of pumpkin cell walls. As a result, the cells were collapsed and separated and the water and other nutritional components released from the interior of the cells. By controlling the dosage of enzymes, the pectin, cellulose and cell-substances broken down to such an extent that the viscosity decreased and cloud remained.