Sinan Cem Emek

Chloroplast membranes retard fat digestion and induce satiety: effect of biological membranes on pancreatic lipase/co-lipase

Human obesity is a global epidemic, which causes a rapidly increased frequency of diabetes and cardiovascular disease. One reason for obesity is the ready availability of refined food products with high caloric density, an evolutionarily new event, which makes over-consumption of food inevitable. Fat is a food product with high caloric density. The mechanism for regulation of fat intake has therefore been studied to a great extent. Such studies have shown that, as long as fat stays in the intestine, satiety is promoted. This occurs through the fat-released peptide hormones, the best known being CCK (cholecystokinin), which is released by fatty acids. Hence, retarded fat digestion with prolonged time for delivery of fatty acids promotes satiety. Pancreatic lipase, together with its protein cofactor, co-lipase, is the main enzymatic system responsible for intestinal fat digestion. We found that biological membranes, isolated from plants, animals or bacteria, inhibit the lipase/co-lipase-catalysed hydrolysis of triacylglycerols even in the presence of bile salt. We propose that the inhibition is due to binding of lipase/co-lipase to the membranes and adsorption of the membranes to the aqueous/triacylglycerol interface, thereby hindering lipase/co-lipase from acting on its lipid substrate. We also found that chloroplast membranes (thylakoids), when added to refined food, suppressed food intake in rats, lowered blood lipids and raised the satiety hormones, CCK and enterostatin. Consequently, the mechanism for satiety seems to be retardation of fat digestion allowing the fat products to stay longer in the intestine.

Thylakoids promote release of the satiety hormone cholecystokinin while reducing insulin in healthy humans

Objective. The effects of a promising new appetite suppressor named “thylakoids” (membrane proteins derived from spinach leaves) were examined in a single meal in man. Thylakoids inhibit the lipase/colipase hydrolysis of triacylglycerols in vitro and suppress food intake, decrease body-weight gain and raise the satiety hormone cholecystokinin (CCK) in rats, but their effects in man remain unclear. The aim of this study was to investigate whether thylakoids, when added to a test meal, affect appetite regulation and blood parameters in healthy individuals. Material and methods. In an intervention crossover study, healthy individuals of normal weight (n=11) were offered a high-fat meal with and without the addition of thylakoids. Blood samples were taken 0 (prior to meal), 30, 60, 120, 180, 240, 300 and 360 min after the start of the meal. Blood samples were analysed for satiety and hunger hormones (CCK, leptin and ghrelin), insulin and blood metabolites (glucose and free fatty acids). Results. The CCK level increased, in particular between the 120 min time-point and onwards, the ghrelin level was reduced at 120 min and leptin level increased at 360 min after intake of the thylakoid-enriched meal. The insulin level was reduced, whereas glucose concentrations were unchanged. Free fatty acids were reduced between time-point 120 min and onwards after the thylakoid meal. Conclusions. The addition of thylakoids to energy-dense food promotes satiety signals and reduces insulin response during a single meal in man.

Thylakoids suppress appetite by increasing cholecystokinin resulting in lower food intake and body weight in high-fat fed mice

Thylakoids are membranes isolated from plant chloroplasts which have previously been shown to inhibit pancreatic lipase/colipase catalysed hydrolysis of fat in vitro and induce short‐term satiety in vivo. The purpose of the present study was to examine if dietary supplementation of thylakoids could affect food intake and body weight during long‐term feeding in mice. Female apolipoprotein E‐deficient mice were fed a high‐fat diet containing 41% of fat by energy with and without thylakoids for 100 days. Mice fed the thylakoid‐enriched diet had suppressed food intake, body weight gain and body fat compared with the high‐fat fed control mice. Reduced serum glucose, serum triglyceride and serum free fatty acid levels were found in the thylakoid‐treated animals. The satiety hormone cholecystokinin was elevated, suggesting this hormone mediates satiety. Leptin levels were reduced, reflecting a decreased fat mass. There was no sign of desensitization in the animals treated with thylakoids. The results suggest that thylakoids are useful to suppress appetite and body weight gain when supplemented to a high‐fat food during long‐term feeding. Copyright

A LARGE SCALE METHOD FOR PREPARATION OF PLANT THYLAKOIDS FOR USE IN BODY WEIGHT REGULATION

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.

Barrier properties of heat treated starch Pickering emulsions

HYPOTHESIS There is a recognized technological need for delivery systems encapsulating lipophilic substances in food and pharmaceutical products. Pickering emulsions can provide well-defined and highly stable systems, but may not provide good enough barrier properties. Starch granules, recently being used for Pickering stabilization, have the advantage of the ability to swell during gelatinization. Hence, this property could be used to tune and control barrier properties. EXPERIMENTS Oil-in-water Pickering emulsions stabilized by starch were subject to heat treatment at different conditions. The influence of temperature, time, and storage on emulsion drop characteristics was evaluated. In order to further evaluate the barrier properties, lipolysis using the pH-stat method was applied and the effect of starch concentration, treatment temperature, and preliminary oral conditions were also investigated. FINDINGS A better encapsulating barrier was obtained by starch swelling at the oil drop interface. This was seen as reduced lipase activity. The internal oil drop size remained intact and the starch was kept at the interface during heat treatment. The extent of swelling could be controlled by the heating conditions and had impact on the ability to prevent lipase transport through the starch barrier layer. Addition of α-amylase simulating oral digestion only had minor impact on the barrier effect.

Photocurrent Generation from Thylakoid Membranes on Osmium‐Redox‐Polymer‐Modified Electrodes

Thylakoid membranes (TMs) are uniquely suited for photosynthesis owing to their distinctive structure and composition. Substantial efforts have been directed towards use of isolated photosynthetic reaction centers (PRCs) for solar energy harvesting, however, few studies investigate the communication between whole TMs and electrode surfaces, due to their complex structure. Here we report on a promising approach to generate photosynthesis-derived bioelectricity upon illumination of TMs wired with an osmium-redox-polymer modified graphite electrode, and generate a photocurrent density of 42.4 μA cm(-2).

Chloroplast thylakoids reduce glucose uptake and decrease intestinal macromolecular permeability.

Thylakoid membranes, derived from chloroplasts, have previously been shown to retard fat digestion and lower blood glucose levels after oral intake. The purpose of the present study was to investigate the effect of thylakoid membranes on the passage of methyl-glucose, dextran and ovalbumin over rat intestine in vitro using Ussing chambers. The results show that thylakoids retard the passage of each of the test molecules in a dose-dependent way. The thylakoids appear to be attached on the mucosal surface and a mechanism is suggested that the thylakoids delay the passage of the test molecules by sterical hindrance. The present results indicate that thylakoid membranes may be useful both to control intestinal absorption of glucose and to enhance the barrier function of the intestine.

Pancreatic lipase-colipase binds strongly to the thylakoid membrane surface.

BACKGROUND Isolated thylakoid membranes, i.e. the photosynthetic membranes of green leaves, inhibit the activity of pancreatic lipase and colipase during hydrolysis of fat in vitro. This inhibition has been demonstrated to cause reduced food intake and improved hormonal and lipid profile in vivo. One of the reasons suggested for the inhibiting effect is binding of lipase-colipase to the thylakoid membrane surface. This prompted a study of the binding of lipase and colipase to thylakoids. RESULTS The results showed that lipase and colipase strongly bind to the thylakoid membrane surface. The dissociation constant was determined at 1.2 × 10⁻⁸  mol L⁻¹; binding decreased after treatment of thylakoids with pepsin/trypsin to 1.0 × 10⁻⁷ and to 0.6 × 10⁻⁷  mol L⁻¹ after treatment with pancreatic juice. Similarly, delipidation of thylakoids caused a decrease in binding, the dissociation constant being 2.0 × 10⁻⁷  mol L⁻¹. CONCLUSION The binding of pancreatic lipase-colipase to the thylakoid membrane is strong and may explain the inhibition of lipase-colipase activity by thylakoids. After treatment with proteases to mimic intestinal digestion binding is decreased, but is still high enough to explain the observed metabolic effects of thylakoids in vivo.

The effect of heat treatment of thylakoids on their ability to inhibit in vitro lipase/co-lipase activity

Thylakoids has been shown to prolong lipolysis by the inhibition of lipase/co-lipase, which makes thylakoids suitable as a functional food ingredient with satiating properties. The components of thylakoids that provide its function as a lipolysis modulator are primarily photosystems I and II, which are structurally stabilised by chlorophyll. However, chlorophyll is known to be heat sensitive yet the enzymatic inhibiting capacity after heat treatment has not been previously studied. It was hypothesised that the retained function of thylakoids after heat treatment could be correlated to the degree of degradation. Heat treatment at either 60 °C, 75 °C or 90 °C for time interval ranging from 15 s to 120 min induced a color shift from bright green to olive brown which was attributed to degradation. The ability of heat-treated thylakoids to inhibit lipolysis in vitro was also reduced. A correlation between chlorophyll a degradation and the enzymatic inhibiting capacity could be established which opens possibilities to use a spectrophotometric method to quantify the ability of thylakoids to inhibit lipase/co-lipase in a more rapid and cost effective way to complement the pH-stat method used today. With the degradation pattern investigated, it is then possible to design a thermal treatment process to ensure a microbiological safe appetite-reducing product and at the same time minimize the loss of functionality.