APPLE JUICE CLARIFICATION: IDENTIFICATION OF PECTIN RESIDUES BY IMMUNOGOLD LABELING
Special Symposium - Innovations in Food Technology (LMC Congress)
Innovations in Food Technology - Poster Session (Food - P2)
Keywords: Apple, cloudy juice, pectin, immunogold labeling
The main purpose of the clarification treatment employed in industrial apple juice processing is to eliminate constituents responsible for the turbidity and cloudiness in freshly produced juice. Conventional clarification process includes hydrolysis of pectin with specific enzymes (pectinases), flocculation of turbidity with clarifying agents, and filtration through plate and frame or vacuum filters. Although ultrafiltration (UF) is a valid alternative for the clarification of fruit juices, this membrane technique partially substituted conventional processing. Differences in the nature of ionic charges of protein, polyphenols and the fining agents, induce flocculation and sedimentation and result in the removal of these potential haze precursors from juices. Turbidity of opalescent apple juice is provided by particulate material that remains in suspension, a complex mixture of cellulose, hemicelluloses, pectins and proteins. Particle sizes above 0.5 mm are unstable and may easily settle out. Below this range particles are retained in suspension due to their small size, mutual charge repulsion, and the protective effect of pectin. Cloud particles have been modeled to consist of negatively charged pectin wrapped around a positively charged protein-carbohydrate core. This simplified clarification model assumed that after the enzymatic breaking of pectin, the positively charged proteins result partially exposed on the particle surface, promoting flocculation. However, the mechanism of particle stabilization in cloudy juices is not completely clear yet. There are different mechanisms that affect stability in a colloid, including Van der Walls attractive and electrostatic repulsive forces, and some others interactions like steric repulsion, depletion, and hydrophobic and hydrophilic interactions. For colloidal stability, the repulsive forces must be dominant. To elucidate the clarification mechanism a complete characterization of apple juice particles, in particular the participation of pectin after enzymatic treatment, should be known. Monoclonal antibodies are excellent tools for localizing pectic components of plant cell walls. Therefore monoclonal antibodies, specific to different pectin epitopes, have been used to investigate the real surface structure of cloudy apple juice particles. Pectic polysaccharides were detected using rat monoclonal antibodies raised against polygalacturonic acid, particularly homogalacturonic acid (HG). Several mAbs to HG exist. Five monoclonal antibodies (LM5, LM7, JIM5 and JIM7) kindly provided by Dr. Knox (Leeds, UK) were selected for pectin characterization. While antibodies LM5 and LM7 recognize galactan and de-esterified polygalacturonase epitopes, respectively; antibodies JIM5 and JIM7 recognize unesterified and esterified pectin epitopes, respectively. Juice treatment was as follows: Samples collected on nickel grids were floated on a drop of OH Na saturated solution, washed carefully with distilled water, and observed in a JEOL 100CXII Transmission Electronic Microscope at 80kV. Selected grids were placed floating on drops of PBS, (10 min); NH4 Cl 50 mM, (30 min), PBS/BSA, 1% (10 min), PBS/BSA, 3% , (15 min). All steps at pH=8 in wet chamber and then incubated with the hybridoma supernatants of the monoclonal antibodies JIM5, JIM7, LM5 and LM7 diluted 1:5 in PBS/BSA (1%), for 1h. at room temperature. Grids were then (i) rinsed with PBS/BSA 3%, PBS/BSA 1%, and PBS/BSA pure (10 min each step); (ii) incubated in wet chamber with a goat anti-rat antibody (immunoglobulin G) complexed to colloidal gold (15 nm) and diluted in PBS/BSA 1% (1:70); and (iii) rinsed with PBS/BSA 3%, PBS/BSA 1%, PBS/BSA 3%, (10min) and dried overnight. Grids were finally stained with uranyl acetate (15 min) and lead citrate (10 min), dried, and observed with the TEM. Observed particles from an enzymatically treated cloudy apple juice showed galactan, de-esterified polygalacturonase, and un-esterified pectin epitopes on their surfaces. Results appeared to contradict somewhat the generally accepted theory that pectinolytic treatments completely de-polymerize the apple pectin, promoting juice clarification by particle surface charge modification. As a conclusion, these cytochemical tools explained why cloudy apple juice remained as a stable colloid despite the enzymatic treatment, behavior attributed to the low effectiveness of pectinases to completely degrade pectin attached on particles.
Presented Thursday 20, 13:30 to 14:40, in session Innovations in Food Technology - Poster Session (Food - P2).
