A Large Scale Method for Preparation of Plant Thylakoids for Use in Body Weight Regulation

Abstract

A method for preparation of thylakoids from plant leaves on a large scale is described. The method involves: 1) disruption of the cells with a blender followed by filtration to remove large cell debris and non disrupted cells. 2) precipitation of the thylakoids by adjusting the pH to the isoelectric point, pH 4.7. 3) a washing step by dilution of the precipitate in water followed by precipitation at the same pH. 4) concentration of the precipitate by freeze- thawing or freeze -drying to get the final product. The product is characterized, with respect to protein composition, by SDS-PAGE and mass-spectroscopy, the content of carotenoids, particularly the xanthophylls violaxanthin, antheraxanthin, and zeaxanthin. The thylakoid preparation has about the same capacity to inhibit pancreatic lipase/colipase activity as thylakoids prepared by standard laboratory methods using sucrose in the medium and centrifugation. In a study with mice, it was found that, when the thylakoids were added to the food over 32 days, they significantly reduced the body weight gain and the percentage body fat. The large scale method described here allows studies on the effect of thylakoids in appetite regulation on experimental animals in a longer lasting time and also on humans.

Feeding spinach thylakoids to rats modulates the gut microbiota, decreases food intake and affects the insulin response

“…The experimental diet was composed of standard rat chow (R 36; Lantmännen) enriched with either only rapeseed oil (control diet) or a thylakoid–oil suspension (thylakoid diet). Thylakoid membranes were extracted and purified from fresh baby-spinach leaves as described before ( 14 , 16 ) . The thylakoid diet was prepared by mixing 4 g thylakoid powder (corresponding to 132 mg chlorophyll), 5 g rapeseed oil and 10 g water by using an Ultraturax mixer.…”

Post harvest improvement of zeaxanthin content of vegetables

“…Violaxanthin de-epoxidase is located in the lumen of thylakoids and the enzyme becomes active when the pH drops in the lumen due the light driven photosynthetic reaction (Szilayi et al, 2008;Hager, 1969;Bratt et al, 1995). We have recently shown that thylakoids isolated at low pH (4.7) contain more than 5-fold higher amount of zeaxanthin compared to thylakoids isolated at high pH (7) (Emek et al, 2010).…”

Chloroplast thylakoids reduce glucose uptake and decrease intestinal macromolecular permeability

“…The slurry was filtered through four layers of 20 mm monodur polyester mesh, and centrifuged (2000 g; 15 min; 48C). The centrifugation step was repeated four times; the pellet was re-suspended in the homogenising buffer the first time, blast media (5 mM-MgCl 2 ) for osmotic lysis the second time and washing media (10 mM-tricine-5 mM-MgCl 2 -300 mMsucrose; pH 7·4) the final two times (13) . As a final step, the pellet was dispersed in a glass/Teflon potter Elvehjem homogeniser until a homogeneous slurry of thylakoid membranes was obtained.…”

“…Thylakoid membranes were prepared from fresh baby spinach leaves (13) by mixing the leaves with homogenising buffer (50 mM-phosphate buffer (pH 7·4)-5 mM-MgCl 2 -300 mM-sucrose) for 5 min until a homogeneous slurry was obtained. The slurry was filtered through four layers of 20 mm monodur polyester mesh, and centrifuged (2000 g; 15 min; 48C).…”