WO2015022411A1

WO2015022411A1 - Dairy composition comprising hawthorn and phytosterols

Info

Publication number: WO2015022411A1
Application number: PCT/EP2014/067454
Authority: WO
Inventors: Youyou ZHAO; Junkuan Wang; Richard Shao; Jinrui DU; Yangfeng HOU; Lei GUAN; Chulin Claire LIANG
Original assignee: Nestec S.A.
Priority date: 2013-08-14
Filing date: 2014-08-14
Publication date: 2015-02-19

Abstract

The invention relates a process for manufacturing a dairy composition comprising phytosterols and hawthorn fruit composition, to such a dairy composition and its use for the treatment, alleviation or prevention of disorders associated with hypercholesterolemia and/or hyperlipidaemia.

Description

DAIRY COMPOSITION COMPRISING HAWTHORN AND PHYTOSTEROLS

TECHNICAL FIELD

The invention relates to dairy compositions, such as food and beverages, comprising functional ingredients. In particular, the functional ingredients address health benefits, such as cardiovascular health.

BACKGROUND OF THE INVENTION

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Cardiovascular diseases (CVDs) are the leading cause of death and disability globally. More people are severely affected annually by CVDs than from any other cause. From a WHO report, an estimated 17.3 million people died from CVDs in 2008, representing 30% of all global deaths. Of these deaths, an estimated 7.3 million were due to coronary heart disease and 6.2 million were due to stroke. By 2030, almost 23.6 million people will die from CVDs, mainly from heart disease and stroke. These are projected to remain the single leading causes of death.

Hawthorn and phytosterols are well known to have beneficial health effects. However, both individual ingredients have their limitations.

Hawthorn, a flowering shrub of the rose family, has an extensive history of use in cardiovascular diseases, dating back to the 1st century. Hawthorn belongs to the genus Crataegus. Hawthorn has been shown to have a promising effect on blood lipids reduction [1] [5]. Under China's Regulation hawthorn can be used as normal food or functional food. The fruits and leaves of the Crataegus pinnatifida are used as herbal medicine as mentioned in the Chinese Pharmacopoeia with daily recommended dose of 9-12 g for dry fruits (equivalent to 60-80 g fresh fruit) [6] and 3-10 g for leaves [7], respectively. However, to achieve a biologically significant effect, daily consumption of hawthorn juice frequently causes stomach discomfort due to its acidic taste.

Due to the acidity of hawthorn, it is difficult to integrate into food and beverage matrices that are pH sensitive, such as milk and dairy matrices, without effect on the texture, taste and other organoleptic properties of such matrices. In addition, the extraction of hawthorn has limited reducing effect on blood triglycerides [1], which is an important risk factor for cardiovascular disease among blood lipids.

Phytosterols are known to be able to reduce blood concentrations of total and LDL cholesterol by reducing cholesterol absorption in the gut. A daily intake of around 1.5g - 2.4 g of plant sterols (or its saturated form, stanols) is associated with an 7-10.5% reduction of LDL cholesterol [2]. The effect is usually established within the first 2-3 weeks. Studies, which covered periods of up to 85 weeks, showed that the effect could be sustained throughout that period. Based on current findings and research, the EFSA NDA Panel also concluded that foods such as yoghurts and milk, including low-fat yoghurts and cheese, margarine-type spreads, mayonnaise, salad dressing and other dairy products, were the most suitable for delivering the cholesterol-lowering effects from plant stanols and sterols to the body. For other foods, either information was lacking or they appeared to be less effective in reducing blood cholesterol levels.

However, phytosterols have unwanted limitation. For example, they inhibit alpha and beta carotene absorption. Beta carotene is an important source of Vitamin A. Vitamin A is essential for growth and development.

When administered at low dosages that avoid significant unwanted side effects or gut discomfort hawthorn or plant sterol have no statistical significant effect on blood lipids, or cholesterol reduction.

Dairy products containing milk fat are major food source of saturated fat, accounting for about 21% of total saturated fat intake in the U.S. diet. It has been postulated that the consumption of dairy productions influences the risk of CVD since a high intake of saturated fat has been linked to an increased risk of CVD. However, several meta-analysis of prospective cohort studies have shown no consistent evidence that higher intakes of milk and dairy products, regardless of milk fat levels, are associated with an increased risk of CVD, coronary heart disease (CHD), or stroke [3] [4]. In addition, low-fat and fat-free milk and milk products are recommended as part of a healthy diet to reduce the risk of CVD through the maintenance of healthy plasma lipids and lipoprotein cholesterol levels.

WO 2013/117008 Al to Nestec S.A. relates to the synergistic protection effect of hawthorn and phytosterols that allows reducing blood lipids and blood levels of LDL cholesterol statistically significantly, while keeping the individual dosages of phytosterols and hawthorn sufficiently low to avoid unwanted side effects and discomfort. This document does not describe how a hawthorn fruit composition may be prepared.

WO 2007/039040 Al to Unilever N.V. discloses a food product comprising sterols, and a hawthorn extract, where the weight ratio of sterols to the total amount of oleanolic acid and ursolic acid is from 1 : 10 to 50 : 1, and where the amount of sterols ranges from 0.1 to 20 wt%. The hawthorn extract is obtained by organic solvent extraction, for example with dichloromethane. Hawthorn in itself or hawthorn juice should be avoided due to their low concentration in active ingredients (oleanolic acid and ursolic acid). An article by the same authors discusses organic solvent extracts of hawthorn (Crataegus pinnatifida), their content triterpenic acids (oleanolic acid and ursolic acid), and the effect of such extracts on cholesterol metabolism in hamsters and in human Caco-2 cells [9].

CN 101361506 A discusses fermented milk and its method of preparation, which involves preparing a standard hawthorn extract in water, mixing it with milk and a dairy starter culture, to prepare a yogurt like product. Hawthorn is used as a lactic acid bacteria growth promoter.

CN 101243810 A discusses a yoghurt beneficial for heart-health, which comprises phytosterols or phytostanol esters.

Hence, there is a clear need in the art for natural preparations that can be safely used without side effects and that allow reducing the risk for obtaining cardiovascular disorders. It is also desirable to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative. In particular, it is desirable to provide food and beverage products which leverage the synergy between hawthorn and phytosterols. Specifically, it is desirable to provide dairy products which leverage the synergy between hawthorn and phytosterols, without compromising the properties of such dairy products, including but not limited to texture, taste, or reconstitution from powder into liquid.

SUMMARY OF THE INVENTION

To this end, a first aspect of the invention proposes a process for manufacturing a dairy composition comprising phytosterols and hawthorn fruit, said process comprising the steps of:

1) preparing a hawthorn fruit composition by: a) preparing a hawthorn fruit slurry, b) removing the hawthorn seeds from said slurry, c) homogenising said slurry, d) adjusting the pH of the homogenised slurry to a value ranging from 4 to 6, e) heat-treating said slurry, and f) optionally drying said heat-treated slurry into a hawthorn fruit powder,

2) mixing said hawthorn fruit composition with a dairy matrix, and

3) mixing phytosterols with said dairy matrix before, together, or after the mixing step 2).

Another aspect of the invention proposes a dairy composition comprising phytosterols and hawthorn fruit. Such a dairy composition is obtainable, for instance, by performing the process according to the first aspect of the invention.

Another aspect of the invention proposes a dairy comprising phytosterols and hawthorn fruit, for use in the treatment, alleviation or prevention of disorders associated with hypercholesterolemia and/or hyperlipidemia

These and other aspects, features and advantages of the invention will become more apparent to those skilled in the art from the detailed description of embodiments of the invention, in connection with the attached drawings. DETAILED DESCRIPTION OF THE INVENTION

Unless the context clearly requires otherwise, throughout the specification, the words "comprise", "comprising" and the like are to be construed in an inclusive sense, that is to say, in the sense of "including, but not limited to", as opposed to an exclusive or exhaustive sense.

As used in the specification, the singular forms "a", "an", and "the" include plura l referents unless the context clearly dictates otherwise.

Unless noted otherwise, all percentages in the specification refer to weight percent, where applicable.

Unless defined otherwise, all technical and scientific terms have and should be given the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

I n a first aspect, the invention relates to a process for manufacturing a dairy composition comprising phytosterols and hawthorn fruit. This process comprises the steps of 1) preparing a hawthorn fruit composition, 2) mixing said hawthorn fruit composition with a dairy matrix, 3) mixing phytosterols with said dairy matrix before, together, or after the mixing step 2). Preparation of a hawthorn fruit composition

In order to prepare a hawthorn fruit composition, first hawthorn fruits are crushed in water, to form a slurry. Hawthorn fruits are provided as fresh fruits or dried fruits. Preferably, whole hawthorn fruits are used, including peel, pulp and seeds. Preferably, the fruits are washed before crushing, in order to remove impurities. When dried fruits are used, they can be rehydrated before crushing. Afterwards, the fruits are cooked in water, at a temperature ranging from 80°C to 95°C, preferably from 85°C to 90°C. Cooking can be performed during 20 to 60 minutes, preferably from 30 to 45 minutes. Standard industrial cooking equipment can be used. As a matter of example, the fruit: water weight ratio is from about 1 : 1 to about 1 : 4. Preferably, cooking is done under stirring, for example using a high speed stirrer. Cooking, optionally under stirring, helps to separate the pulp from the seeds. It also inactivates fruit enzymes. Preferably, the hawthorn fruit composition is prepared from a water-based hawthorn fruit slurry.

Then the slurry is transferred to a sieve to remove the seeds from the slurry. Hawthorn pulp slurry is collected, and homogenised. Homogenisation can be performed using a colloid mill. Preferably, hawthorn pulp is milled to a particle size smaller than 700 microns, preferably smaller than 500 microns. Usually, the particle size is above a few microns, for example, above 1 micron, or above 10 microns. For instance, the particle size ranges from 1 to 700 microns, preferably from 1 to 500 microns.

Usually at this stage, the slurry has a pH below 3.5. The pH of the homogenised slurry is then adjusted to a value ranging from 4 to 6, preferably from 4.5 to 5.5. A food-grade KOH solution can be used to adjust the pH of the slurry. pH adjustment is an important step to avoid undesirable reactions between the hawthorn fruit composition and the dairy matrix, such as protein precipitation. This also ensures shelf-life of the dairy composition, as well as stability upon reconstitution in the case of dry products.

Preferably, pH adjustment is followed by a further homogenisation of the hawthorn slurry, to a particle size below 300 microns, preferably below 200 microns. Usually, the particle size is above a few microns, for example, above 1 micron, or above 10 microns. For instance, the particle size ranges from 1 to 300 microns, preferably from 1 to 200 microns. This provides a particle size that has best stability in the dairy matrix after reconstitution, in the case of dry products. Finally, the slurry is heat-treated, preferably by UHT treatment (for instance, 20 seconds at 137°C). Optionally, the heat-treated slurry is concentrated, to provide a hawthorn fruit concentrate. Optionally, the heat-treated slurry, or the hawthorn fruit concentrate, is dried, to provide a hawthorn fruit powder. Drying can be performed conventionally by spray- drying or freeze-drying for instance.

The hawthorn fruit composition is prepared from whole hawthorn fruits from which the seeds have been removed, using water as a solvent. In other words, the hawthorn fruit composition is prepared essentially from the pulp and skin of hawthorn fruits, using water as a solvent. This is in contrast with other extraction methods which use organic solvent extraction. Indeed, it is necessary to remove all traces of organic solvent before consumption by human or animals, whereas water is a perfectly food-grade medium. Surprisingly, the hawthorn fruit compositions retain beneficial properties, contrary to what has been suggested in previous publications. The hawthorn fruit powder can be provided in a dry form, such as a powder, in a concentrated form, such as a concentrate, or in a semi- liquid form, such as a slurry or a puree.

Preparation of a dairy matrix

The dairy matrix is prepared using standard dairy technology. For instance, the dairy matrix is a dairy-based powder, a milk powder, a yogurt powder, a fermented milk product, a yogurt, a dairy-based beverage, or a drinkable yogurt. Preferably, the dairy matrix is a milk powder, such as a skim milk powder, a semi-skim milk powder, or a full-fat milk powder. Even more preferably, the dairy matrix is a skim milk powder.

Alternatively, the dairy matrix is an aqueous dairy matrix, such as a fermented milk product, for example a drinkable or spoonable yogurt.

In an embodiment, the dairy matrix is lactose-free. Lactose-free products are preferable for consumers who suffer from lactose intolerance.

Preparation of the dairy composition

The dairy composition is prepared by mixing said hawthorn fruit composition with a dairy matrix, and mixing phytosterols with said dairy matrix before, together, or after the mixing of hawthorn fruit composition. In an embodiment, the dairy composition is a dairy- based powder, a milk powder, a yogurt powder, a fermented milk product, a yogurt, a dairy- based beverage, or a drinkable yogurt.

Preferably, the hawthorn fruit composition is provided as a powder when the dairy matrix is provided also as a powder, such as milk powder. The hawthorn fruit powder and the dairy matrix powder are dry-mixed using standard equipment.

When the dairy matrix is an aqueous dairy matrix, such as yogurt or milk beverage, the hawthorn fruit composition can be added as a dry powder, as a concentrate, as a heat- treated slurry or puree.

Typical phytosterols that may be used for the purpose of the present invention may be selected from the group consisting of plant sterols, stanols, or combinations thereof. Examples are β-sitosterol, campesterol and/or stigmasterol.

Phytosterols may be provided as chemically pure compounds. As such they may be purified from plant sources or synthesized chemically. This allows a very precise dosing. It may also be preferred to provide phytosterols from natural sources or extracts thereof. As such the phytosterols may be dietary phytosterols. They may be provided as vegetable oils, nuts, cereal products, vegetables, fruit, berries or as extracts thereof.

Preferably, the dairy composition contains at least 0.5 g, 1 g, or 4 g (dry weight) hawthorn fruit composition per serving. The preparation of the hawthorn fruit composition has been described above. Hawthorn fruit composition generally represents less than half of the dry weight of the dairy composition, or less than 40% of the dry weight of the dairy composition. For instance, in a dry dairy composition, such as milk powder, the dry dairy composition comprises hawthorn fruit powder in an amount of 2-40 %wt of the dry dairy composition. Also for instance, in an aqueous dairy composition, such as liquid dairy-based beverage, yoghurt or a ready-to-drink milk-based beverage, the aqueous dairy composition comprises hawthorn fruit composition in an amount of 0.3-5 %wt of the aqueous dairy composition.

Preferably, the dairy composition contains at least 0.25 g, 0.5 g, or 0.6 g of phytosterols per serving. Preferably, the dairy composition contains up to 2.4 g, 1.8 g, or 1.2 g of phytosterols per serving. For instance, in a dry dairy composition, such as milk powder, the dry dairy composition comprises phytosterols in an amount of 1-6 %wt of the dry dairy composition. Also for instance, in an aqueous dairy composition, such as liquid dairy-based beverage, yoghurt or a ready-to-drink milk-based beverage, the aqueous dairy composition comprises phytosterols in an amount of 0.14-0.75 %wt of the aqueous dairy composition.

For instance, a serving corresponds to 10 to 25 grams of milk powder (or the volume equivalent of liquid milk or liquid dairy-based composition), or to 80 to 180 grams of yogurt. Preferably, a recommended daily intake is one, two, or three servings a day. The daily intake may depend on the age, weight and diet of the consumer. In the dairy composition, the dairy matrix is the main ingredient, meaning that it represents more than half of the weight of the composition.

For instance, a dry dairy composition, such as those mentioned above, comprises 2-40 %wt of hawthorn fruit powder, 1-6 %wt of phytosterols, and the remainder of dairy matrix, such as milk powder, yoghurt powder, or other dairy-based powder ingredients. The dry dairy composition may also comprise added vitamins and/or minerals. The dry dairy composition may also comprise sweeteners, emulsifiers, hydrocolloids, flavours, and/or colorants. The dry dairy composition may be lactose-free.

For instance, an aqueous dairy composition, such as those mentioned above, comprises 0.3-5 %wt of hawthorn fruit composition, 0.14-0.75 %wt of phytosterols, and the remainder of dairy matrix, such as milk, yoghurt, milk-based or yoghurt-based beverage, or other dairy-based ingredients. The aqueous dairy composition may also comprise added vitamins and/or minerals. The aqueous dairy composition may also comprise sweeteners, emulsifiers, hydrocolloids, flavours, and/or colorants. The aqueous dairy composition may be lactose-free.

Synergistic effect of hawthorn and phytosterols

The inventors have found in a hamster model that - when administered together - hawthorn and phytosterols act synergistically and allow reducing blood lipids and blood levels of LDL cholesterol statistically significantly, while keeping the individual dosages of phytosterols and hawthorn sufficiently low to avoid unwanted side effects and discomfort.

Without wishing to be bound by theory, the present inventors believe that this beneficial effect is due to hawthorn and phytosterols simultaneously enhancing the cholesterol excretion and reducing the cholesterol absorption. Consequently, the present invention relates in part to a dairy composition comprising phytosterols and hawthorn for use in the treatment, alleviation or prevention of disorders associated with hyperlipidaemia and/or hypercholesterolemia.

The present invention also relates to the use of phytosterols and hawthorn in the preparation of a dairy composition for treating, alleviating or preventing disorders associated with hyperlipidaemia and/or hypercholesterolemia.

Preferably, the dairy composition for use in the treatment, alleviation or prevention of disorders associated with hyperlipidaemia and/or hypercholesterolemia may be a dry dairy composition or an aqueous dairy composition, as described above.

Disorders associated with hyperlipidaemia and/or hypercholesterolemia are well known to those of skill in the art (see for example Bhatnagar D. et al., BMJ 2008;337:a993, herewith incorporated by reference). For example, hypercholesterolemia is one of the major causes of atherosclerosis or coronary heart diseases. Hyperlipidaemia is characterized by abnormally elevated levels of any or all lipids and/or lipoproteins in the blood. For example, disorders associated with hyperlipidaemia and/or hypercholesterolemia may be cardiovascular disorders.

Cardiovascular disorders are a group of disorders of the heart and blood vessels and include coronary heart disease, cerebrovascular disease, peripheral arterial disease, rheumatic heart disease, deep vein thrombosis and pulmonary embolism, etc. "Cardiovascular disorders" generally refers to any disease that affects the cardiovascular system, and in particular refers to those disorders related to atherosclerosis (arterial disease). These conditions usually have similar causes, mechanisms, and treatments.

Atherosclerosis is the build-up of fatty deposits (such as cholesterol) called plaque on the inside walls of arteries. By the time that heart problems are detected, atherosclerosis is usually quite advanced, having progressed for decades. Most commonly, the plaque suddenly ruptures which causing the formation of a thrombus that will rapidly block the blood flow, leading to death of the surrounding tissues only in 5 minutes. This catastrophic event is called an infarction. One of the most common recognized scenarios is called coronary thrombosis of a coronary artery, causing myocardial infarction (a heart attack). The same process in an artery to the brain is commonly called stroke. Another common scenario in a very advanced disease state is claudication from insufficient blood supply to the legs, typically caused by a combination of both stenosis and aneurysmal segments narrowed with clots.

The main cause of atherosclerosis is yet unknown, it is a syndrome affecting arterial blood vessels, a chronic inflammatory response in the walls of arteries, caused largely by the accumulation of macrophages and promoted by low-density lipoproteins (LDL, plasma lipoproteins that carry cholesterol and triglycerides) without adequate removal of fats and cholesterol from the macrophages by functional high density lipoproteins (HDL). It is commonly referred to as a hardening or furring of the arteries. This is caused by the formation of multiple plaques within the arteries.

Hyperlipidaemia is the most important risk factor for atherosclerosis, which is the major cause of cardiovascular disease. Hyperlipidaemias may also be classified directly into which types of lipids are elevated, that is hypercholesterolemia, hypertriglyceridemia or both in combined hyperlipidaemia. Elevated levels of Lipoprotein(a) may also be classified as a form of hyperlipidaemia.

Today, people make great efforts to keep their cholesterol levels low. Typical measures to reduce cholesterol levels are reducing dietary cholesterol intake, administration of certain medications, and rarely other treatments even including surgery. The present invention now provides a convenient dietary way to reduce cholesterol levels.

Hence, the dairy composition of the present invention may be used for lowering the plasma total cholesterol level, in particular the plasma level of LDL cholesterol. A low LDL cholesterol concentration reduces the risk of cardiovascular disorders.

This effect is even more pronounced if a low LDL level is combined with a high HDL cholesterol level. Increasing HDL cholesterol levels can be achieved by weight loss and/or by exercise.

Hence it is preferred if the composition of the present invention is administered in a caloric restriction regimen and/or to subject who exercise aerobically regularly.

Typical cardiovascular disorders that can be prevented, ameliorated or treated with the composition of the present invention may be selected from the group consisting of atherosclerosis, myocardial infarction, coronary heart disease, stroke, peripheral vascular diseases or combinations thereof. Additionally or alternatively the dairy compositions of the present invention may also be for use in treating or preventing high blood triglyceride levels and/or disorders linked thereto.

One major field of public concern is heart health. With the effects that the present invention demonstrates for the compositions described herein, the compositions of the present invention may be for use in promoting heart health and/or in treating, alleviating or preventing an impaired heart health.

In therapeutic applications, dairy compositions according to the invention are administered in an amount sufficient to at least partially cure or arrest the symptoms of a disease and/or its complications. An amount adequate to accomplish this is defined as "a therapeutically effective dose". Amounts effective for this purpose will depend on a number of factors known to those of skill in the art such as the severity of the disease and the weight and general state of the patient.

In prophylactic applications, dairy compositions according to the invention are administered to a patient susceptible to or otherwise at risk of a particular disease in an amount that is sufficient to at least partially reduce the risk of developing a disease. Such an amount is defined to be "a prophylactic effective dose". Again, the precise amounts depend on a number of patient specific factors such as the patient's state of health and weight.

The compositions of the present invention are effective following a dose-response curve. They may be administered in a therapeutically effective dose or a prophylactic effective dose. Skilled artisans will be able to determine such dosages appropriately. For example, the composition of the present invention may comprise at least 7 mg hawthorn per kg body weight per day and at least 15 mg phytosterols per kg body weight per day. Alternatively, the composition of the present invention may comprise at least 14 mg hawthorn per kg body weight per day and at least 50 mg phytosterols per kg body weight per day. Further alternatively, the composition of the present invention may comprise at least 42 mg hawthorn per kg body weight per day and at least 100 mg phytosterols per kg body weight per day.

To avoid unwanted side effects such as off-tastes or inhibitions of vitamin A absorptions, the dosages should be kept at a reasonably low level. For example, the dosages of hawthorn may not exceed 2.5 g per kg body weight per day [6] and the dosage of phytosterols may not exceed 40 mg per kg body weight per day [8]. Hawthorn may be provided in any form suitable for administration to humans or animals. Preferably, hawthorn may be provided as fresh fruit, dried fruit, fruit puree, fruit powder, or fruit concentrate. Most preferably, hawthorn fruit is provided as a hawthorn fruit composition as described above.

The composition may be any composition that is suitable for human or animal consumption. As such the dairy composition may be selected from the group consisting of a food product, a drink, a functional food, a nutraceutical, a food additive, a nutritional formula, and a pet food product.

As disorders associated with hyperlipidemia and/or hypercholesterolemia typically occur with ageing, the composition of the present invention may be to be administered to adults or the elderly.

For the purpose of the present invention a subject shall be considered as "elderly" if it has surpassed the first half of its average expected lifespan in its country of origin, preferably, if it has surpassed the first two thirds of the average expected lifespan in its country of origin, more preferably if it has surpassed the first three quarters of the average expected lifespan in its country of origin, most preferred if it has surpassed the first four fifths of the average expected lifespan in its country of origin.

For example, the composition of the present invention may be to be administered to people above the age of 45, 50, 55, 60, 65, 70, 75 or 80 years.

As disorders associated with hyperlipidemia and/or hypercholesterolemia not only occur in humans but also in animals the composition of the present invention may be to be administered to humans or animals, for example companion animals such as cats or dogs.

Based on guidelines provided by the American Heart Association, NIH and NCEP for fasting HDL levels and risk for heart disease a level of less than 40 mg/dL HDL cholesterol for men and less than 50 mg/dL HDL cholesterol for women causes a heightened risk for heart disease. An HDL cholesterol level of 60-50 mg/dL for women and of 60-40 mg/dL for men is considered normal.

Consequently, the composition of the present invention may be to be administered to people with a fasting HDL cholesterol level of below 60 mg/dL, of below 50 mg/dL or of below 40 mg/dL.

The American Heart Association, NIH, and NCEP have also provided a set of guidelines for fasting LDL-Cholesterol levels and risk for heart disease. A fasting LDL-Cholesterol level of 100 to 129 mg/dL corresponds to a near optimal LDL level, corresponding to higher rates for developing symptomatic cardiovascular disease events.

A fasting LDL-Cholesterol level of 130 to 159 mg/dL corresponds to a borderline high LDL level, corresponding to higher rates for developing symptomatic cardiovascular disease events.

A fasting LDL-Cholesterol level of 160 to 199 mg/dL corresponds to a high LDL level, corresponding to much higher rates for developing symptomatic cardiovascular disease events.

Finally, a fasting LDL-Cholesterol level of above 200 mg/dL corresponds to a very high

LDL level, corresponding to highest increased rates of symptomatic cardiovascular disease events.

Consequently, the composition of the present invention may be to be administered to people with a fasting LDL cholesterol level of above 100 mg/dL, of above 130 mg/dL, of above 160 mg/dL or of above 200 mg/dL.

Advantageously, the composition of the present invention may be consumed briefly before, with or briefly after the consumption of a high-fat or high LDL cholesterol meal. A meal shall be considered high in fat if it contains more than 60%, more than 80% or more than 100% of the daily recommended fat intake. Similarly, a meal shall be considered high in cholesterol if it contains more than 60%, more than 80% or more than 100% of the daily recommended cholesterol intake.

As such, the composition may be to be administered in the time frame from 1 hour before to 1 hour after a high fat and/or high cholesterol meal.

Further advantages and features of the present invention are apparent from the following Examples and Figures.

Figure 1 shows that the combination treatment of hawthorn and plant sterol at the same dose significantly and synergistically reduced blood cholesterol compared to an HFD- placebo. Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison tests and unpaired student t test. MeaniSEM, n=8-12, *P<0.05, versus control; ^†P<0.05, versus HFD placebo. Figure 2 shows that the combination treatment of hawthorn and plant sterols at the same dose significantly and synergistically reduced LDL cholesterol compared to an HFD- placebo. Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison tests and unpaired student t test. MeaniSEM, n=8-12, *P<0.05, versus control; ^†P<0.05, versus HFD placebo.

Figure 3 shows that the combination treatment of hawthorn and plant sterols at the same dose significantly and synergistically caused a robust reduction in blood triglyceride in HFD hamsters. Data were analyzed by one-way ANOVA followed by Tukey's multiple comparison tests and unpaired student t test. MeaniSEM, n=8-12, *P<0.05, versus control; ^†P<0.05, versus HFD placebo.

EXAMPLES

In this study, male Golden Syrian hamsters (n=54; body weights=120~130 g) from Beijing Vital River Laboratory Animal Co., Ltd., were housed in an animal room at 23^QC with 12/12-h light-dark cycles. The hamsters were adapted to these conditions for 2 weeks, during which period they were fed a pelleted form of a cereal based rodent diet. Then, all hamsters were randomly divided into five groups (n=10~12 each group) and fed one of the five diets formulated shown in the table below. The fresh diets were given to the hamsters daily and uneaten food was discarded.

Throughout the feeding period, the animals had free access to food and drinking water. Clinical observations were routinely done, 24h food intake and body weight gain were measured weekly, and their total feces per cage were collected every week. Five diets were prepared by modifying the formulation as previous described (Chan et al., 1999). The negative control diet (Control) containing no cholesterol, was prepared by mixing the ingredients together. A high cholesterol control diet (hight-fat diet, HFD) was similarly prepared by adding 0.1% (w/w) cholesterol and 10% lard into the control diet. The other three experimental diets were prepared by adding hawthorn puree (H FD-H), phytosterol esters (HFD-PS) and hawthorn puree plus phytosterol esters (HFD-H/PS), respectively, into the HFD diet. (g/kg) Control HFD HFD-H HFD-PS HFD-H/PS

Cholesterol 0 1 1 1 1

Lard 50 150 150 150 150

Casein 242 242 242 242 242

Cornstarch 508 407 389 404 386

Sucrose 119 119 119 119 119

DL-methionine 1 1 1 1 1

Vitamin Mix AIN-76A^b 20 20 20 20 20

Mineral Mix AIN-76^a 40 40 40 40 40 gelatin 20 20 20 20 20

Hawthorn puree 18 18

(dry matter)

Phytosterol esters - - - 3 3

Total 1000 1000 1000 1000 1000 ^a Mineral mix AIN-76 provided (in g/kg of mix): calcium phosphate, dibasic, 500; sodium chloride, 74; potassium citrate-H₂0, 220; potassium sulfate, 52; magnesium oxide, 24; manganous carbonate, 3.5; ferric citrate, 6; zinc carbonate, 1.6; cupric carbonate, 0.3; potassium iodate, 0.01; sodium selenite, 0.01; chromium K sulfate.12H20, 0.55; sucrose, finely powdered, 118.03; Dyets, Bethlehem, PA, USA.

b Vitamin mix AIN-76A provided (in g/kg of mix): thiamine HCL, 0.6; pyridoxine HCL, 0.7; niacin, 3; calcium pantothenate, 1.6; folic acid, 0.2; biotin, 0.02; vitamin B12 (0.1 vitamin A palmitate (500,000 lU/g), 0.8; Vitamin D3 (400,000 lU/g), 0.25; Vitamin E acetate (500 lU/g), 10; menadione sodium bisulfite, 0.08; sucrose, finely powdered, 981.15; Dyets, Bethlehem, PA, USA.

1. Synergistic effect of plant sterol (PS) and hawthorn (H) on blood total cholesterol and LDL-cholesterol reductions.

Background: Abnormal elevation of blood cholesterol level (hypercholesterolemia) especially with the higher concentration of LDL is strongly associated with cardiovascular disease because these promote atheroma development in arteries (atherosclerosis). This disease process leads to myocardial infarction (heart attack), stroke, and peripheral vascular disease.

1.1 Blood Total Cholesterol: After 6 weeks of treatment, a high fat diet (HFD) significantly elevated the blood cholesterol by 67mg/dL (Tablel.l, HFD-placebo vs. control). Simultaneously treatment with plant sterol or hawthorn has no significant alteration on high blood cholesterol compared to HFD placebo (Figl, HFD-H, HFD-PS vs. control, *P<0.05; HFD- H, HFD-PS vs. HFD-Placebo, P>0.05). Although two individual ingredients did not show the significant protection effect per se, the combination treatment of hawthorn and plant sterol at the same dose significantly and synergistically reduced blood cholesterol compared to an HFD-placebo (Fig. 1, HFD-PS/H vs. HFD placebo, ^†P<0.05). In addition, the combination treatment showed synergistic reduction effect on high blood cholesterol versus individual treatments (Table 1.1).

Table 1.1. Blood total cholesterol (TC) reduction effect of Hawthorn (H) and Plant Sterol (PS) in high fat diet (HFD) hamster.

1.2 LDL Cholesterol: After 6 weeks of treatment, a high fat diet (HFD) significantly elevated LDL cholesterol by 27mg/dL (Tablel.2, HFD-placebo vs. control). Simultaneously treatment with plant sterol or hawthorn had no significant alteration on high LDL cholesterol compared to HFD placebo (Figl, HFD-H, HFD-PS vs. control, *P<0.05; HFD-H, HFD-PS vs. HFD- Placebo, P>0.05). Although two individual ingredients did not show the significant protection effect per se, the combination treatment of hawthorn and plant sterol at the same dose significantly and synergistically reduced LDL cholesterol compared to HFD-placebo (Fig.2, HFD-PS/H vs. HFD placebo, ^†P<0.05). In addition, the combination treatment showed a synergistic reduction effect on LDL cholesterol versus individual treatments (Table 1.2).

Table 1.2. LDL cholesterol reduction effect of Hawthorn (H) and Plant Sterol (PS) in high fat diet (HFD) hamster.

Control HFD-placebo HFD-H HFD-PS HFD-H/PS

LDL (mg/dL) 45.2±3.44 72.6±3.29* 80.8±3.11* 61.6±1.52* 60.5±1.23^† 2. Synergistic effect of plant sterol (PS) and hawthorn (H) on blood triglyceride (TG) reduction.

Background: In medicine, hypertriglyceridemia denotes high blood levels of triglycerides, the most abundant fatty molecule in most organisms. It has been associated with atherosclerosis, even in the absence of hypercholesterolemia (high cholesterol levels). It can also lead to pancreatitis in excessive concentrations (i.e. when the triglyceride concentration is greater, and often very much greater, than 1000 mg/dl or 12 mmol/l).

Blood Triglyceride: After 6 weeks of treatment, high fat diet significantly elevated the blood triglyceride by 126mg/dL (Table 2, HFD-Placebo vs. control). Plant sterol alone showed robust reduction on blood triglyceride in HFD hamsters (Fig. 3, HFD-PS vs. control, P>0.05; HFD-PS vs. HFD-Placebo, ^†P<0.05). Although it did not show the reduction effect alone, hawthorn has enhanced the blood triglyceride reduction effect of plant sterol by 8.8mg/dL.

Table 2. Blood Triglyceride (TG) reduction effect of Hawthorn (H) and Plant Sterol (PS) in high fat diet (HFD) hamster.

3. Potential mechanism of the synergistic effect of plant sterol (PS) and hawthorn (H) on cholesterol-regulated.

Background: The NPC1L1 (Niemann-Pick Cl-like protein 1) protein is found on the gastrointestinal tract epithelial cells. It appears to be crucial for cholesterol and phytosterol uptake into enterocyte. ACAT-2 (cytosolic acetyl-CoA acetyltransferase) is an enzyme responsible for the synthesis of cholesteryl esters, a part of lipoproteins, and further transportation into blood circulation.

CYP7A1 (cholesterol 7 alpha-hydroxylase, or cytochrome P450 7A1) is an enzyme involved in the synthesis of bile acids from cholesterol. It is up-regulated by LXR (liver X receptor) when cholesterol levels are high. LXRa is an isoform of LXR. LXRa and CYP7A1 are associated with the enhanced amount of neutral and bile acid production and excretion. PPARa (peroxisome proliferator-activated receptor alpha) is a transcription factor and a major regulator of lipid metabolism in the liver. Activation of PPARa promotes uptake, utilization, and catabolism of fatty acids by up-regulation of genes involved in fatty acid transport, binding, and activation, and peroxisomal and mitochondrial fatty acid β-oxidation.

Gene expression: The gene expression of intestinal and hepatic tissues was compared between hamsters who received the combination treatment of hawthorn and phytosterols, and hamsters who received the high fat diet placebo.

A significant down-regulation of NPC1L1 and ACAT-2 was observed in HFD-H/PS hamsters compared with HFD placebo (Table 3.1, HFD-H/PS vs. HFD-placebo, *P<0.05).

Moreover, up-regulation of LXRa and CYP7A1 could also contribute to increasing the cholesterol clearance in hepatocytes in HFD-H/PS hamsters, and up-regulation of PPARa in the HFD-H/PS hamsters could be associated with an increased metabolism of fatty acids (Table 3.2, HFD vs. control, *P<0.05; HFD-H/PS vs. HFD-placebo,^†P<0.05).

The action of the combination diet H/PS could lead to an activation of PPARa, which play an important role to reduce the production of triglyceride-rich VLDL, and further decrease plasma triglyceride levels.

Table 3.1. Gene expression in intestinal tissue

Table 3.2. Gene expression in liver tissue

Although preferred embodiments have been disclosed in the description with reference to specific examples, it will be recognised that the invention is not limited to the preferred embodiments. Those skilled in the art will understand that they can freely combine all features of the present invention described herein, without departing from the scope of the invention as disclosed. In particular, features described for the uses of the present invention may be applied to the composition of the present invention and vice versa. Various modifications may become apparent to those of ordinary skill in the art and may be acquired from practice of the invention.

REFERENCES

[1] Ho Walter KK, Chen ZY, Huang Y, Zhang ZS, Chang Q., Chow Moses, Liu Y, Tomlinson Brian. Cardiovascular Protective Effects of Hawthorn. Nutraceutical Beverages. December 1, 2003. 62-75

[2] SCIENTIFIC OPINION :Plant Stanols and Plant Sterols and Blood LDL-Cholesterol. The EFSA Journal (2009) 1175 : 1-9

[3] Elwood PC, Pickering JE, Givens Dl, Gallacher JE. The consumption of milk and dairy foods and the incidence of vascular disease and diabetes: an overview of the evidence. Lipids

(2010) 45 : 925-39.

[4] Soedamah-Muthu SS, Ding EL, Al-Delaimy WK, Hu FB, Engberink MF, Willett WC, Geleijnse JM. Milk and dairy consumption and incidence of cardiovascular diseases and all- cause mortality: dose-response meta-analysis of prospective cohort studies. Am J Clin Nutr

(2011) 93 : 158-71.

[5] Chen Jidi, Xue Bin, Li Keji , Shi Jingda , Krempin D , Zhu M , Garland C. The effects of an instant hawthorn beverage on lipid levels, antioxidant enzyme and immune function in hyperlipidemia patients. Chin J Prev Med (May 2002) 36 (3)

[6] Hawthorn Fruit. Pharmacopoeia of the People's Republic of China, 2005, vol. I : 91 [7] Hawthorn Leaf. Pharmacopoeia of the People's Republic of China, 2005, vol. I : 69 [8] Phytosterol (Esters) as novel ingredient in China. http://www.moh.gov.cn/mohbgt/sl0697/201003/46297.shtml

[9] Yuguang Lin, Mario A. Vermeer, Elke A. Trautwein. Triterpenic Acids Present in Hawthorn Lower Plasma Cholesterol by Inhibiting Intestinal ACAT Activity in Hamsters. Evidence-Based Complementary and Alternative Medicine (2011) Article ID 801272

Claims

1. Process for manufacturing a dairy composition comprising phytosterols and hawthorn fruit, said process comprising the steps of:

2) mixing said hawthorn fruit composition with a dairy matrix, and

2. Process according to claim 1, wherein said hawthorn fruit slurry is prepared by crushing hawthorn fruits in water, followed by cooking at a temperature ranging from 80°C to 95°C optionally under stirring.

3. Process according to claim 1 or 2, wherein said pH adjustment d) is followed by a further homogenisation of the slurry to a particle size smaller than 300 microns.

4. Process according to any one of claims 1 to 3, wherein said heat-treatment e) is a UHT treatment.

5. Process according to any one of claims 1 to 4, wherein said heat-treatment e) is followed by a concentration step of the hawthorn fruit slurry, to provide a hawthorn fruit concentrate.

6. Process according to a ny one of claims 1 to 5, wherein said drying f) is performed by spray-drying or freeze-drying.

7. Process according to any one of claims 1 to 6, wherein said hawthorn fruit composition is provided as a powder, and is dry-mixed with a dairy matrix.

8. Process according to any one of claims 1 to 7, wherein said dairy matrix is selected from dairy-based powder, milk powder, yogurt powder, fermented milk product, yogurt, dairy-based beverage, and drinkable yogurt.

9. Process according to any one of claims 1 to 8, wherein said hawthorn fruit is provided as fresh fruit, or dried fruit.

10. Dairy composition comprising phytosterols and hawthorn fruit, obtainable by a process according to any one of claims 1 to 9.

11. Dairy composition according to claim 10, containing at least at least 0.5 g, 1 g, or 4 g hawthorn fruit (dry weight) per serving.

12. Dairy composition according to claim 10 or 11, containing at least 0.25 g, 0.5 g, or 0.6 g of phytosterols per serving.

13. Dairy composition according to any one of claims 10 to 12, selected from dairy-based powder, milk powder, yogurt powder, fermented milk product, yogurt, dairy-based beverage, and drinkable yogurt.

14. Dairy composition according to any one of claims 10 to 13, for use in the treatment, alleviation or prevention of disorders associated with hypercholesterolemia and/or hyperlipidemia.

15. Dairy composition for use according to claim 14, wherein the composition is to be administered to adults or the elderly.