WO2007115899A1

WO2007115899A1 - Satiety enhancing food products

Info

Publication number: WO2007115899A1
Application number: PCT/EP2007/052520
Authority: WO
Inventors: Christiaan Michael Beindorff; Cedric Yves Ghislain Lemetter; Sergey Michailovich Melnikov; Ingrid Winter
Original assignee: Hindustan Lever Ltd; Hindustan Unilever Ltd; Unilever NV
Current assignee: Hindustan Unilever Ltd; Unilever NV
Priority date: 2006-04-07
Filing date: 2007-03-16
Publication date: 2007-10-18
Anticipated expiration: 2008-10-07

Abstract

The present invention relates to an food product comprising at least 0.1 wt.% of oil bodies, said oil bodies having a volume weighted mean diameter within the range of 0.1- 100 m and comprising a lipid matrix surrounded by a layer of phospholipids embedded with structural proteins, wherein said structural proteins have been cross-linked. Furthermore the invention concerns the use of such cross-linked oil bodies in the treatment or prevention of overweight. The inventors have found that resistance of oil bodies against digestion may be improved dramatically by cross-linking the structural proteins contained therein. It is believed that the cross-linked oil bodies according to the present invention have a positive influence on appetite suppression because the oil contained in the oil bodies will reach the distal gut and thus inhibit transit through the proximal small intestine by means of the so called ileal brake mechanism.

Description

SATIETY ENHANCING FOOD PRODUCTS

TECHNICAL FIELD OF THE INVENTION

The present invention relates to satiety enhancing food products. More particularly, the present invention relates to such satiety enhancing food products containing oil bodies.

BACKGROUND OF THE INVENTION

Obesity is defined as an excess of body fat relative to lean body mass. Not only is obesity regarded as unattractive, it is also a well-established risk factor for a number of potentially life-threatening diseases such as atherosclerosis, hypertension, diabetes, stroke, pulmonary embolism, sleep apnea, and cancer. Furthermore, it complicates numerous chronic conditions such as respiratory diseases, osteoarthritis, osteoporosis, gall bladder disease, and dyslipidemias . The enormity of this problem is best reflected in the fact that death rates escalate with increasing body weight. More than 50% of all-cause mortality is attributable to obesity-related conditions once the body mass index (BMI) exceeds 30 kg/m², as seen in 35 million Americans (Lee, JAMA 268:2045-2049, 1992).

The accumulation or maintenance of body fat bears a direct relationship to caloric intake. Comprehensive treatment programs, therefore, focused on behaviour modifications to reduce caloric intake and increase physical activity using a myriad of systems. These methods have limited efficacy and are associated with extremely high recidivism rates.

Obesity has also been treated by administering specific agents, for example, anorectic agents, to obese subjects. However, anorectic agents such as dextroamphetamine, the combination of the non-amphetamine drugs phentermine and fenfluramine (Phen-Fen) , and dexfenfluramine (Redux) alone, are associated with serious side effects. Indigestible materials such as olestra (OLEAN) , mineral oil or neopentyl esters have been proposed as substitutes for dietary fat. Garcinia acid and derivatives thereof have been described as treating obesity by interfering with fatty acid synthesis. Swellable cross-linked vinyl pyridine resins have been described as appetite suppressants via the mechanism of providing non-nutritive bulk.

Surgical interventions, such as gastric partitioning procedures, jejunoileal bypass, and vagotomy, have also been developed to treat severe obesity. Although these surgical procedures are somewhat more effective in the long run, the acute risk benefit ratio has reserved these invasive procedures for morbidly obese patients. Therefore, this approach is not an alternative for the majority of overweight patients unless and until they become profoundly obese and are suffering the attendant complications. Meal replacer products have also been proposed as part of a healthy diet in order to control or reduce body weight. For example, U.S. Pat. No. 5,688,547 discloses a nutritional meal replacement composition comprising dietary fibre, protein, a cellulose gum and gel. These meal replacer products are generally products that are intended to be consumed as a single-serving food product, such as a bar, drink etc to replace one or two meals per day. The meal replacer products are designed such that on the one hand they provide a restricted caloric intake, but on the other hand they provide a healthy balance of nutritional ingredients and are convenient to incorporate into an individual's daily diet.

However, a general problem with products intended to be used in a weight loss or weight maintenance plan, e.g. meal replacer products or low-calorie snacks, is that feelings of hunger may occur sooner than desired after consumption and/or the feeling of satiety obtained may not be as great as desired. Both of these considerations may render it difficult for the individual to adhere to the plan or it may make it and/or the products used therein less appealing to consumers.

Recognising the demand for effective and convenient satiety-inducing food products, research has been carried out to try to address the problems associated with the above approaches to controlling or reducing body weight.

One approach to addressing the aforementioned problems has been to investigate the use of satiety agents in food products in order to increase the satiety effect obtained from consuming a food product comprising the satiety agents. WO 99/02041 discloses a food composition giving a prolonged feeling of satiety and comprising a mixture of specific triglyceride oils and a food emulsifier.

Another approach to reduce the feeling of hunger which has been suggested is to use the principle of the ileal brake. The ileal brake principle itself is described by Gregg W. Van Citters in The Ileal Brake: A fifteen-year progress report, Current Gastronenterology Reports 1999, 1:404-409 and which concerns the delivery of satiety agents to parts of the gut e.g the ileum, duodenum or jejunum. However, the satiety effects obtained by the above compositions are often sub-optimal. Furthermore, many of the satiety enhancing agents proposed in the prior art can only be applied in particular types of food products and/or their use is associated with regulatory issues. Thus, there still is a need for a variety of food compositions that provide a good satiety effect for consumers, especially those wishing to control their calorie intake and/or body weight. Peng CC et al in Biotechnol. Prog. VoI 19 (2003), pages 1623-1626 describe size and stability of reconstituted sesame oil bodies. Reconstituted oil bodies are seen as carrier for hydrophobic molecules such as neutraceuticals and cosmetic lipids. No food application is described, nor any use of cross- linked oil bodies in satiety enhancement.

US2004/258803 describes the use of encapsulated satiety agents. No use of cross-linked oil bodies in satiety enhancement is disclosed.

SUMMARY OF THE INVENTION

The present inventors have unexpectedly discovered that the aforementioned need can be met by providing food products containing a significant amount of oil bodies in which the structural proteins have been cross-linked. Cross-linking of naturally derived and reconstituted oil bodies with glutaraldehyde and genipin has been described by Peng et al . (Biotechnol. Prog. 2003, 19, 16523-1626). The authors report that the cross-linking was found to improve the thermostability of the oil bodies. Oil bodies are the discrete intracellular organelles in which triacylglycerides are stored in, for instance, plant seeds. Plant oil bodies are spherical particles about 0.5-4 μm in diameter and are found in all oilseed plants (i.e. Canola, flax, sunflower, soybean and corn) . The oil-bodies consist primarily of central droplet of oil (triglycerides) that is surrounded by a coat consisting of a phospholipid monolayer and a structural protein. Oil bodies are remarkably stable both inside cells and in isolated preparations. In both situations the oil bodies occur as individual entities. Even when pressed against one another during seed desiccation or flotation centrifugation, oil bodies hardly aggregate or coalesce. The structural proteins that together with the phospholipid monolayer surround the central lipid core of the oil bodies, are believed to protect the phospholipids in the monolayer by making them inaccessible to external phospholipase A2 and phospholipase C. Structural proteins found in plant oil bodies include oleosins and caleosins .

The structural protein oleosin is a relatively small protein (15 to 26 kD) and typically contains a central stretch of about 72 hydrophobic residues that is inserted into the lipid core whereas the amphiphatic N-terminal and C-terminal are located at the surface of the oil body. Both terminal portions of the oleosin can be either short (e.g. 4 residues) or very long (e.g. 1200 residues). The central hydrophobic stretch of 72 uninterrupted hydrophobic residues is the hallmark of an oleosin. No other known protein in any organism has such a long hydrophobic stretch (Hsieh et al . , Plant Physiol. 2004 November; 136(3): 3427-3434).

The key structural features of caleosins are an N-terminal region with a single Ca²⁺- binding EF hand domain, a central hydrophobic region able to form a single bilayer span, and a C- terminal region with several putative protein kinase phosphorylation sites (D.J. Murphy, LIPID ASSOCIATED PROTEINS IN PLANTS, 16^th Int. Plant Lipid Symposium, Budapest, June 2004, pages 1-8). Caleosins lack an N-terminal signal peptide, but do have a central, hydrophobic region of typically more than 30 residues. Like oleosins, caleosins contain a proline-rich region with the potential to form a "proline know" motif. In vitro studies under simulated human GI tract conditions conducted by the inventors showed that non-cross-linked oil bodies are largely stable under gastric conditions and that degradation starts upon addition of intestinal enzymes. Consequently, even if the stability of oil bodies during digestion is higher than that of oil droplets contained in simple oil-in-water emulsions, the are not sufficiently stable to survive until the distal part of the intestine (ileum) . The inventors have found that resistance of oil bodies against digestion may be improved dramatically by cross-linking the structural proteins contained therein. Although the inventors do not wish to be bound by theory, it is believed that the cross-linked oil bodies according to the present invention have a positive influence on appetite suppression because the oil contained in the oil bodies will reach the distal gut and thus inhibit transit through the proximal small intestine by means of the so called ileal brake mechanism. The term "ileal brake" refers to a physiological state of the lower small intestine that slows down the normal digestive process. Specifically, it is an endocrine-induced intestinal change in which the contents (e.g., nutrients) of the lower small intestine (ileum) influence upper gastrointestinal activity such as by decreasing gastric and pancreatic secretions and preventing gastric emptying.

DETAILED DESCRIPTION OF THE INVENTION

Accordingly, one aspect of the invention relates to an food product comprising at least 0.1 wt . % of oil bodies, said oil bodies having a volume weighted mean diameter within the range of 0.1-100 μm and comprising a lipid matrix surrounded by a layer of phospholipids embedded with structural proteins, wherein said structural proteins have been cross-linked. The oil bodies according to the present invention will be referred hereinafter as "cross-linked oil bodies" or "the oil bodies".

The aforementioned lipid matrix represents the core of the oil body and essentially consists of lipids. According to a preferred embodiment, the lipid matrix contains at least 80 wt. %, more preferably at least 90 wt.%, even more preferably at least 95 wt.% and most preferably at least 98 wt.% of glycerides selected from the group consisting of triglycerides, diglycerides, monoglycerides, phosphoglycerides and combinations thereof. In a preferred embodiment, the glycerides are selected from the group consisting of triglycerides and diglycerides. Most preferably, the glyceride is triglyceride oil. Besides glycerides of various structures and fatty acid chain length the lipid matrix may also contain other minor components that are dissolved in said matrix.

Examples of lipids that may be present in the lipid matrix include: triglycerides of various structures and fatty acid chain lengths and other minor components that are dissolved in said matrix.

In order to achieve a significant resistance against intestinal digestion, it is preferred that at least 5% of the structural proteins in the oil bodies have been cross-linked. Even more preferably, at least 20% of the structural protein is cross-linked, most preferably at least 40% is cross-linked. It should be understood that the term "cross-linking" as used herein refers to the introduction of a covalently bonded link between two different protein molecules or between different parts of the same protein molecule. The degree of cross-linking as referred to herein is defined as the average number of amino acid residues per structural protein molecule that is conjugated with (covalently bound to) a cross-linking agent. Analytical methods suitable for determining the degree of cross-linking can be found in the relevant scientific literature. Different methods may be applied to determine the degree of cross-linking, depending on the nature of the cross-linking agent. A suitable method for determining the degree of cross-linking is the amino acid analysis described in US 2005/0130261, which is incorporated herein by reference.

Another methodology for determining the degree of cross- linking makes use of LC-MS/MS. Sample preparation includes hydrolysis of the proteinaceous material, e.g. by treatment with 6 mol/L hydrochloric acid at 110 ⁰C for 24 hours. The protein hydrolysate so obtained will essentially consist of single amino acids and amino acids that are linked together by a cross-linking agent. The latter components can easily be identified on the basis of their relatively high molecular mass .

Typically, the cross-linked oil bodies according to the present invention exhibit a degree of cross-linking of at least 0.3, preferably of at least 1.0, more preferably of at least 3.0 and most preferably of at least 5.0.

According to a particularly preferred embodiment, a significant fraction of the structural proteins in the oil bodies contain intermolecular cross-links that link these structural proteins to one or more other structural proteins within the same oil body or another oil body. Advantageously, the degree of intermolecular cross-linking is at least 0.1, more preferably at least 0.3 and most preferably at least 1.0. Here the degree of intermolecular cross-linking is defined as the average number of amino acid residues per structural protein molecule that is covalently bound to another structural protein molecule by means of a cross-linking agent.

Cross-linking of the structural protein in the oil bodies may suitably be achieved with any method known in the art, provided the cross-linked material can be considered food- grade. The structural proteins may, for instance, be cross- linked by carbonylic cross-linkers, phenolic cross-linkers and/or enzymatic cross-linkers. The term "carbonylic cross-linkers" covers cross-linking agents that form covalent bonds with protein residues through a reactive carbonyl group. Preferred carbonylic cross-linkers are ketoses, polyfunctional aldehydic or phenolic cross-linkers. A typical example of a polyfunctional aldehydic cross-linker is glutaraldehyde (pentane-1, 5-dial) . A typical example of a ketose cross-linker is dihydroxy-acetone (1, 3-dihydroxy-2- propanone) .

Typical examples of phenolic acid cross-linkers are phenolic acid cross-linkers and polyphenolic cross-linkers. Preferred phenolic acid cross-linkers include chlorogenic acid, caffeic acid, ferulic acid, coumaric acid and mixtures and derivatives thereof. Polyphenolic cross-linkers that can suitably be employed include polyphenols from plant sources such as polyphenols from grapes (e.g. anthocyanidins) , coffee, tea, grains, cocoa and chocolate, fruits and berries.

Enzymatic cross-linkers that may suitably be employed include transglutaminases and/or oxidoreductases (e.g. peroxidase, laccase, monoamine oxidase or polyphenol oxidase) . The oil bodies employed in according with the present invention may be obtained from plant sources or microbial sources. Preferably, the oil bodies are obtained from plant seeds. Suitable plant seed sources include flax, safflower, rapeseed, soybean, maize and sunflower.

The food product according to the present invention advantageously comprises 0.3-10 wt . % of the cross-linked oil bodies. Even more preferably, said food product contains 0.5-8 wt .% of said cross-linked oil bodies. The volume weighted mean diameter of the cross-linked oil bodies typically is within the range of 0.2-50 μm, preferably of 0.3-20 μm, especially within the range of 0.5-6 μm. The phospholipid layer surrounding the lipid core of the oil body usually is a monolayer.

Glycerides advantageously constitute the bulk of the cross-linked oil bodies of the present invention. Typically, the oil bodies contain at least 90 wt . % of glycerides selected from the group consisting of triglycerides, diglycerides and monoglycerides . Even more preferably the oil bodies contain at least 90 wt . % of triglyceridesl The glycerides contained in the lipid matrix preferably contain at least 20 wt . % of polyunsaturated fatty acids. According to a particularly preferred embodiment, said lipid matrix contains at least 20 wt .% of ω-3 polyunsaturated fatty acids by weight of the total amount of fatty acids contained therein.

Phospholipids are normally present in a concentration of 0.2-5 wt. %, whereas the (cross-linked) structural proteins usually represent 0.2-7 wt . % of the oil bodies. The structural protein present in the cross-linked oil bodies of the present invention preferably has a hydrophilic N- terminal portion and/or a hydrophilic C-terminal portion; and a central stretch of at least 50, preferably at least 70 uninterrupted hydrophobic residues. According to an even more preferred embodiment, the central stretch of hydrophobic residues of the structural protein is inserted into the lipid core and the amphiphatic N-terminal and/or amphiphatic C- terminal are located at the surface of the oil bodies, with positively charged residues embedded in a phospholipid monolayer and the negatively charged ones exposed to the exterior. In accordance with a particularly preferred embodiment, the structural protein contains both an amphiphatic N-terminal and an amphiphatic C-terminal with are both located at the surface of the oil bodies. More preferably, the structural protein employed in accordance with the present invention is selected from the group consisting of oleosin, caleosin or combinations thereof. Most preferably, the structural protein is oleosin.

The structural proteins present in the oil bodies of the present invention typically have a molecular size of 10-100 kD, more preferably of 12-70 kD. It is noted that these molecular sizes refer to the individual proteins, i.e. not to the aggregates formed by two or more cross-linked proteins.

The lipid matrix in the oil bodies of the present inventions was found to be surprisingly stable against lipid oxidation. Even if the oil bodies are employed in an food product that contains significant levels of pro-oxidative metals, such as iron, copper and/or zinc, oxidation rates remain surprisingly low. Thus, the food product of the present invention may suitably contain at least at least 10 mg/kg of a transition metal selected from iron, copper or zinc, especially in the form of a metal salt. Most preferably the food product contains at least 10 mg/kg of iron. The exceptional stability of the oil bodies is especially appreciated in case the oil bodies contain a significant level of polyunsaturated fatty acids, notably ω-3 polyunsaturated fatty acids.

Furthermore, the stability of the present oil bodies is particularly manifest in food products that contain at least 1 wt .% of water, especially food products that contain at least 1 wt .% of an aqueous phase containing dispersed oil bodies. Even more preferably, the present food product contains at least 5 wt. %, most preferably at least 10 wt . % of a continuous or dispersed aqueous phase containing the dispersed oil bodies.

The food product according to the present invention is advantageously selected from the group consisting of beverages (e.g. nutritional beverages and dairy drinks), meal replacers, spreads, nutrition bars, pasta products, ice cream, desserts, dairy products (e.g. yogurt, quark, cheese), dressings, sauces, soups, instant powders, fillings, dips and breakfast type cereal products (e.g. porridge). According to a particularly preferred embodiment, the food product is a beverage, meal replacer, a nutrition bar or an instant powder. The term "instant powder" refers to an essentially dry powder that prior to consumption can be reconstituted with water to produce, for instance, a beverage or a soup. The food composition of the present invention may suitably contain additional ingredients such as carbohydrates, additional proteins, additional oil, emulsifiers, herbs, spices, anti-oxidants, preservatives, flavourings, colourings, vitamins and minerals. Furthermore, the food composition may optionally comprise, in suitable amounts, one or more agents which may beneficially influence (post-prandial) energy metabolism and substrate utilisation, for example caffeine and flavonoids (including tea catechins, capsaicinoids and carnitine) . According to a preferred embodiment, the food composition contains added vitamins selected from at least one of: Vitamin A, Vitamin Bl, Vitamin B2 (riboflavin), Vitamin B3 (Niacinamide) , Vitamin B5 (d-Ca-pantothenate) , Vitamin B6, Vitamin BIl, Vitamin B12 (Cyanocobalamin) , Vitamin C (Ascorbic acid), Vitamin D, Vitamin E (Tocopherol) , Vitamin H (Biotin) , and Vitamin K.

The composition also preferably comprises added minerals selected from at least one of; calcium, magnesium, potassium, zinc, iron, cobalt, nickel, copper, iodine, manganese, molybdenum, phosphorus, selenium and chromium. Calcium is preferably present in the food compositions in amounts of from 5 to 50% of the European Commission Directive 96/8/EC of 26 Feb. 1996 on foods intended for use in energy-restricted diets for weight reduction, more preferably about 10 to 35%, most preferably 15 to 35% per serving. Any suitable calcium source may be used.

It is preferred that the food compositions comprise potassium, especially in an amount of at least 300 mg of potassium per serving of the food composition, more preferably 400-1000, most preferably 450-700 mg. Any suitable potassium source may be used.

One or more of the above-mentioned vitamins and minerals are preferably present at amounts of from 5 to 45% of the amounts given in the above European Commission Directive 96/8/EC, especially 5 to 40%, most especially 10 to 30%.

The food compositions according to the present invention preferably have a caloric content in the range of from 50 kilocalories (kcals) to 500 kcals, more preferably 100 kcals to 400 kcals per serving. However, it will be understood that the caloric content per serving will vary according to the type of food composition. For a dairy or soy based beverage or dessert the caloric content is typically in the range of from 50 kcals to 400 kcals, more preferably 100 or 150 kcals to 350 kcals, most preferably 200 kcals to 350 kcals per serving. For a soup the calorie content is typically in the range of from 50 kcals to 350 kcals, more preferably 100 kcals to 250 kcals. These products may be consumed either to replace a meal (a meal replacer product) or as a snack product which is not intended to replace a meal.

If the food composition is a meal replacer product the caloric content per serving is typically in the range of from 150 to 350 Kcal . If the food composition is a product which is intended to be eaten as a snack product (i.e. not intended by itself to replace a whole meal) the caloric content per serving is typically in the range of from 50 to 150 Kcal. The size of a serving of the food composition will depend upon the type of composition. A serving of the food composition as referred to herein refers to the amount of the food composition that is intended to be consumed as a single portion. For beverages and soups, the typical serving size is in the range of from 100 to 500 ml, preferably 150 to 400 ml, such as 200 to 350 ml. For desserts the typical serving size is in the range of from 75 g to 300 g, preferably 100 g to 250 g, such as 125 g to 200 g. Another aspect of the present invention relates to the use of the cross-linked oil bodies as defined herein before in the treatment or prevention of overweight. Furthermore the present invention relates to the use of the cross-linked oil bodies in weight loss. In addition the use of the cross-linked oil bodies in satiety enhancement is envisioned. In accordance with a preferred embodiment, these cross-linked oil bodies are consumed in an amount of at least 100 mg, more preferably at least 300 mg and most preferably at least 500 mg per consumption event. As will be appreciated from the above, the cross-linked oil bodies are advantageously orally ingested in the form of a food product, e.g. a beverage or meal replacer such as a bar, soup, or shake. It will be understood that the present invention may suitably be employed to treat or prevent overweight in animals, such as mammals. Most preferably, the present invention is used to treat or prevent overweight in humans .

The terminology "treatment or prevention of overweight" encompasses the treatment or prevention of obesity as well as the treatment or prevention of any diseases or disorders that are directly associated with being overweight or obese. The present invention may advantageously be used to improve compliance with a pre-defined dietary plan to control, reduce or maintain body weight. Subjects will find it easier to comply with such a dietary plan if they consume the cross-linked oil bodies of the present invention as such consumption will strongly decrease the temptation to snack or over-eat. As will be understood from what has been described herein before, the cross-linked oil bodies are suitably consumed in the form of an food product such as a beverage, a nutritional bar or a meal replacer .

In accordance with a preferred embodiment, the cross- linked oil bodies are consumed during a meal (breakfast, lunch or dinner) . In this embodiment, the oil bodies are advantageously provided by a meal component or by a beverage that is consumed during the meal. The consumption of oil bodies during the meal offers the advantage that the satiety effect of the meal will be prolonged. Consumption of a composition according to the invention is intended to enhance and/or prolong the feeling of satiety for the consumer and/or extend the time interval between meals and/or reduce the amount of calories consumed in the following meal. This in turn aids the individual concerned to better adhere to a weight loss or weight control plan.

According to another preferred embodiment, the cross- linked oil bodies are consumed between 2 meals (e.g. at least 1 hour before a meal) in order to reduce food intake during the meal. In this embodiment, the cross-linked oil bodies are suitably consumed in the form of a snack or a beverage.

The food composition of the present invention may be consumed as desired. Preferably, the composition is consumed at least daily in order to provide advantageous satiety effects, more preferably it is consumed at least twice daily.

The invention is further illustrated by means of the following examples. EXAMPLES

Examples 1-6

Oil bodies obtained from different plant seeds were subjected to a cross-linking treatment with glutaric dialdehyde (pentane-1, 5-dial) . The oil bodies were obtained from commercial sources or extracted from seeds as described below. Oil bodies from the following seed sources were included in the experiment .

Oil bodies were extracted from the seeds using the following procedure. Seeds were ground to flour using a water- cooled laboratory M20 universal mill (ex IKA, Germany) . Fifty gram of the flour was mixed with 500 ml of aqueous grinding solution (0.6M sucrose) in a Waring blender at a speed of 22,000 rpm for one minute. The extraction solution which contained the oil bodies and different solutes, such as storage proteins, was separated from the fibres and other debris using a laboratory sieve with a mesh size of 150 μm. The liquid phase was further processed in a Sorvall centrifuge, equipped with a GSA rotor, for 30 minutes at 8,000 rpm in order to isolate the oil bodies. A separation in three phases was observed. At the bottom a layer of sediment, consisting mainly of fibres and proteins was formed. In the middle an aqueous phase consisting of water and solutes was formed. The top layer consisted of a creamy phase consisting of oil bodies mixed with water and some impurities .

The cream top layer was carefully removed with a spoon and subjected to three more liquid-liquid separation steps successively using three different aqueous buffer solutions 1, 2 and 3.

Floating buffer 1 0 .2M sucrose / 1 .0M NaCl

Floating buffer 2 0 .1M sucrose / 1 .0M NaCl

Floating buffer 3 0 .2M NaCl

The isolated and commercial oil bodies were cross-linked by means of glutaric dialdehyde by means of the following method. Twenty-five gram of oil body slurry was carefully mixed with 250 ml of demineralised water in a beaker. To this mixture 100 μl (low degree of cross-linking) or 1000 μl (high degree of cross-linking) of glutaric dialdehyde solution was added. The beaker was covered with foil to prevent light from affecting the cross-linking reaction. Under constant stirring (50 rpm) at room temperature, the cross-linking reaction was performed overnight. Next, the diluted cross-linked oil body mixture was centrifuged at 5000 rpm for 30 minutes and a slurry containing the cross-linked oil bodies was recovered.

Lipolysis tests were carried out under intestinal conditions to determine the digestion resistance of the cross- linked oil bodies in comparison to non-cross-linked oil bodies. For each lipolysis assay 40 ml of incubation buffer, 1. Og of bile (ex SIGMA, USA) and 1. Og of oil cross-linked oil body slurry were mixed. The incubation buffer contained 40 mM NaCl, 2mM tris (hydroxymethyl) -aminomethane, 80 mM CaCl2 and the pH of the buffer had been adjusted to 7.0 with 0.1 M NaOH solution. The lipolysis reaction was started with the addition of 25 mg of pancreatin powder (ex SIGMA, USA) . Pancreatin contains a broad mixture of different types of enzymes, proteases and lipases being predominant. When added to oil bodies, the proteases in the pancreatin will hydrolyse the oleosins and the phospholipases will start to hydrolyse the phospholipid membrane layer. Subsequently, the oil contained in the oil bodies is hydrolysed by the lipases.

During the incubation period of 60 minutes, automatic addition of sodium hydroxide solution was performed to maintain pH at 7.0. Temperature during the lipolysis assay was kept constant at 37 ⁰C. The molar amount of sodium hydroxide added is equal to the molar amount of fatty acids formed due to triglyceride and phospholipid hydrolysis. For the calculation of the degree of hydrolysis it is assumed that pancreatic lipase hydrolyses 1 mole of triglycerides into 2 moles of fatty acids and 1 mole of monoglyceride . The results obtained form the lipolysis assay are summarised below.

Table 1: degree of hydrolysis observed in sunflower oil bodies (GDA = glutaric dialdehyde)

Table 2: degree of hydrolysis observed in canola oil bodies

Table 3: degree of hydrolysis observed in linseed oil bodies

Table 4: degree of hydrolysis observed in sesame oil bodies Please leave the sesame oil bodies out of the patent! It seems that something went wrong with the crosslinking protocol.

Table 5: degree of hydrolysis observed in pine tree oil bodies

Table 6: degree of hydrolysis observed in safflower oil bodies (Natrulon OSF)

Example 7

Oil bodies from safflower (Natrulon OSF) were also cross- linked by means of transglutaminase using the same procedure as described above, except that instead of glutaric dialdehyde solution, transglutaminase (type WM, ex Ajinomoto, Germany) was added in concentrations of 0.1 to 5 wt . % to 25 gram of oil body slurry. Furthermore, pH of the diluted oil body mixture was adjusted to 7.2 with 0. IM sodium hydroxide solution prior to cross-linking .

Table 7: degree of hydrolysis observed in safflower oil bodies (Natrulon OSF)

These results show that, as regards protecting the oil bodies against lipolysis, cross-linking of safflower oil bodies with transglutaminase was less effective than cross-linking of these same oil bodies with glutaric dialdehyde.

Claims

1. A food product comprising at least 0.1 wt . % of oil bodies, said oil bodies having a volume weighted mean diameter within the range of 0.1-100 μm and comprising a lipid matrix surrounded by a layer of phospholipids embedded with structural proteins, wherein said structural proteins have been cross-linked.

2. A food product according to claim 1 wherein the food product is a satiety enhancing food product

3. A food product according to any of the previous claims comprising at least 10 mg/kg of a transition metal selected from iron, copper or zinc.

4. A food product according to any of the previous claims wherein at least 5% of the structural proteins in the oil bodies have been cross-linked.

5. A food product according to claim 1 or 2, wherein the structural proteins have been cross-linked by carbonylic cross-linkers, phenolic cross-linkers and/or enzymatic cross-linkers.

6. A food product according to to any of the previous claims, comprising 0.3-10 wt . % of the oil bodies.

7. A food product according to any one of the preceding claims, wherein the oil bodies contain at least 90 wt . % of glycerides selected from the group consisting of triglycerides, diglycerides and monoglycerides, 0.2-5 wt . % of phospholipids and 0.2-7 wt . % of structural proteins.

8. A food product according to any one of the preceding claims, wherein the structural protein has a hydrophilic N-terminal portion and/or a hydrophilic C-terminal portion; and a central stretch of at least 50, preferably at least 70 uninterrupted hydrophobic residues.

9. A food product according to any one of the preceding claims, wherein the structural protein is selected from the group consisting of oleosin, caleosin and combinations thereof .

10. A food product according to any one of the preceding claims, said food product being selected from the group consisting of beverages, meal replacers, spreads, nutrition bars, pasta products, ice cream, desserts, dairy products, dressings, sauces, soups, instant powders, fillings, dips and breakfast type cereal products.

11. Use of oil bodies having a volume weighted mean diameter within the range of 0.1-100 μm and comprising a lipid matrix surrounded by a layer of phospholipids embedded with structural proteins, wherein said structural proteins have been cross-linked, in the treatment or prevention of overweight .

12. Use of oil bodies having a volume weighted mean diameter within the range of 0.1-100 μm and comprising a lipid matrix surrounded by a layer of phospholipids embedded with structural proteins, wherein said structural proteins have been cross-linked, in a weight-loss treatment.

13. Use of oil bodies having a volume weighted mean diameter within the range of 0.1-100 μm and comprising a lipid matrix surrounded by a layer of phospholipids embedded with structural proteins, wherein said structural proteins have been cross-linked, to enhance satiety.

14. Use of oil bodies having a volume weighted mean diameter within the range of 0.1-100 μm and comprising a lipid matrix surrounded by a layer of phospholipids embedded with structural proteins, wherein said structural proteins have been cross-linked in the manufacture of a food product for use in weight-loss treatment.

15. Use of oil bodies having a volume weighted mean diameter within the range of 0.1-100 μm and comprising a lipid matrix surrounded by a layer of phospholipids embedded with structural proteins, wherein said structural proteins have been cross-linked in the manufacture of a food product that enhances satiety.

16. Use of oil bodies having a volume weighted mean diameter within the range of 0.1-100 μm and comprising a lipid matrix surrounded by a layer of phospholipids embedded with structural proteins, wherein said structural proteins have been cross-linked in the manufacture of a food product for use in the treatment or prevention of overweight .