WO2020170117A1

WO2020170117A1 - Method for producing milk containing lipid-lowering substances

Info

Publication number: WO2020170117A1
Application number: PCT/IB2020/051322
Authority: WO
Inventors: Salvatore Valenti
Original assignee: Salvatore Valenti
Priority date: 2019-02-19
Filing date: 2020-02-18
Publication date: 2020-08-27
Also published as: IT201900002415A1

Abstract

The present invention relates to a method for producing milk and/or a derivative thereof enriched with at least one lipid-lowering substance, preferably monacolin k, comprising the steps of: (a) administering to a dairy ruminant a ration of cereals fermented with at least one fungus capable of producing the at least one lipid-lowering substance, said ration of cereals partially supplementing or totally replacing the feed of the ruminant; (b) milking the dairy ruminant in the next 6-48 hours; (c) collecting the enriched milk with the at least one lipid-lowering substance. The invention also relates to the milk, a derivative thereof (obtained after a further processing step (d)) and a food product obtained from the milk or a derivative thereof, enriched with said substance and the use thereof for controlling and/or reducing blood cholesterol.

Description

“Method for producing milk containing lipid-lowering substances”

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DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to a method for producing milk and/or a derivative thereof enriched with at least one lipid-lowering substance, preferably monacolin k. The invention also relates to the milk, a derivative thereof and a food product obtained from the milk or a derivative thereof, enriched with said substance and the use thereof for controlling and/or reducing blood cholesterol.

BACKGROUND OF THE INVENTION

Cardiovascular diseases (CVDs) are the leading causes of adult mortality and morbidity in industrialised countries. Among the numerous cardiovascular risk factors, dyslipidaemia, and in particular blood LDL cholesterol, undoubtedly represents one of the most commonly implicated factors. For this reason, one of the blood parameters that are today considered most important to keep under control to prevent ischaemia, heart attacks or other cardiovascular diseases is precisely total cholesterol (TC).

A healthy adult individual produces the majority of the cholesterol necessary {endogenous pool) by autonomous biosynthesis, whereas under normal conditions only a small part is taken in daily through the diet ( exogenous pool). Both pools are subject to regulatory mechanisms, so that, under balanced conditions, the amount of cholesterol that is synthesised and the amount absorbed from foods corresponds to the cholesterol eliminated with bile as free cholesterol and bile acids.

The biosynthesis of cholesterol is controlled by the liver enzyme 3-hydroxy-3- methylglutaryl coenzyme A (HMG-CoA). If the cholesterol level of a healthy individual is too low, the liver will produce a large quantity of HMG-CoA in order to increase the cholesterol level and restore it to adequate values, whereas if the cholesterol level of said healthy individual becomes too high, the liver will inhibit or reduce the production of said coenzyme, thereby decreasing or inhibiting the production of endogenous cholesterol. However, the optimal level of blood cholesterol can increase uncontrollably, leading to a condition of hypercholesterolaemia in the individual, which, as already said, represents one of the largest risk factors for the cardiovascular diseases.

The increase in cholesterol can be caused by environmental factors (such as, for example a diet with a high content of saturated fats, sugars and physical inactivity) or by predisposing genetic factors or, more commonly, by a combination of both factors.

Given that the distribution profiles of CVDs in all industrialised countries follow those of the main risk factors, and since there is direct evidence of the effectiveness of reducing blood cholesterol values in reducing mortality due to CVDs, the prevention strategies are by now well established and are based on a combination of dietetic therapies with pharmacological therapies.

For this reason, the first line of intervention for controlling blood cholesterol and the risk of cardiovascular diseases is based essentially on a healthy lifestyle, a diet that provides for a low fat consumption, regular motor activity of least thirty minutes on a daily basis, as well as abstention from smoking and alcohol.

With regard to pharmacological therapies, statins have shown to be the drugs of first choice, they are the most widely used in clinical practice and their effectiveness has been demonstrated in many clinical studies (Collins R. et al. , “Interpretation of the evidence for the efficacy and safety of statin therapy”, Lancet 2016; 388: 2532-2561 ), in terms of both reducing blood cholesterol and decreasing fatal and non-fatal cardiovascular events.

Statins in fact inhibit the liver enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), the key enzyme in the endogenous synthesis of cholesterol, which catalyses the irreversible reaction leading to the synthesis of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA).

It is through this mechanism of action that statins are able to significantly lower the blood cholesterol values.

Although statins are effective in preventing cardiovascular diseases and their success has been unmatched, the use thereof is associated with a variety of side effects such as muscle weakness, myopathies and, in rare cases, fatal rhabdomyolysis (Venturi et al. , Br J Pharmacol., 2018; 175(6): 938-952).

In fact, in addition to blocking the production of cholesterol, statins also block enzymes that preside over muscle activities, with a consequent loss of muscle tissue, pain and numbness of the extremities and tendon and ligament inflammations. In addition, the dead muscle cells release myoglobin, which in turn may cause kidney problems. Other problems caused by the intake of statins can affect the liver (in fact, transaminase is often checked in patients who use them regularly). Among the various side effects of statins, cognitive problems have also sometimes been detected, as well as a general increase in glycaemia with a consequent increase in the risk of diabetes.

For this reason, in recent years there has been an increasingly felt need to find alternatives to statins and pharmacological remedies. The interest has thus focused on the identification of combinations of nutraceuticals aimed at reducing cardiovascular risk and capable of acting on the levels of blood cholesterol and other circulating lipids.

An example of said nutraceutical combinations are dietary supplements containing lipid-lowering substances.

Among the latter, one of the most commonly used is fermented red rice. This supplement is a natural product derived from the fermentation of rice by monascus purpureus (m.p.), a fungus that is capable of fermenting rice and other congeneric substrates producing monacolin k. Monacolin k is a molecule belonging to the statin family.

Monacolin k is of fermentative derivation and performs an action wholly analogous to that of synthetic lovastatin, as it inhibits the same protein involved in the control of endogenous cholesterol, namely, HMG-CoA reductase, thereby effectively reducing the endogenous cholesterol produced.

Among the dietary supplements containing lipid-lowering substances, milk-based supplements with added monacolin k or other lipid-lowering substances obtained by fermentative methods have recently been introduced into the market.

For example, patent BE1020033 discloses a milk-based composition for use as a hypocholesterolaemic food supplement composition comprising an amount of monacolin (which in this specific case is derived from pleurotus ostreatus) sufficient to obtain a hypocholesterolaemic effect. In said patent, the composition is obtained from a powder of pleurotus ostreatus added to fermented liquid milk or a derivative thereof.

Patent W02005104864, by contrast, relates to an edible oil, comprising statins produced by fermentation of fungi (such as, for example, monascus fungus) and at least 90% by weight of diglycerides and/or triglycerides, which is added to cow’s milk.

The Chinese document CN103651915, on the other hand, discloses a dairy product, in particular a fresh cheese, containing ingredients such as monacolin k and y-aminobutyric acid, with a function of regulating blood fats and reducing blood pressure. In this case, a fermentation agent ( lactobacillus ) and a fungus (such as, for example monascus fungus) are added to sterilised raw milk. It is important to note that all of these supplements described in the art are an alteration of a natural food product, being produced by adding fermentative derivatives, which exposes the product to problems related to possible chemical contaminations and adulteration. Such products, though not exactly drugs, are far from being a healthy and natural food product.

In fact, there is an increasingly evident demand for so-called “clean label’ products, with a consequent growth in the market of consumer demand for products free of additives and adulterations.

Thus, there arises a need for a new method for producing a completely natural, additive-free food that can be consumed on a daily basis, also, for example, by children and pregnant women, and is capable of contributing to the control of blood cholesterol, without altering the natural formulation of the food product.

Another aspect to be taken into consideration is that the use of a lipid-lowering drug such as a statin, and/or a dietary supplement, requires that a suitable lipid lowering diet is first recommended to the individual and vice versa. In fact, at present, the diet followed for the purpose of reducing blood cholesterol is essentially a fat-free diet associated with drugs or, alternatively, with the above- mentioned dietary supplements. Furthermore, the drug and/or dietary supplement cannot and must not be considered a replacement for a correct diet, nor taken like a normal food product, as it has certain contraindications and methods of use that are closely connected to the amount of the active ingredient (statins) present inside it.

The present invention thus resolves the technical problem of coupling between a correct diet and the intake of a lipid-lowering substance for the control of blood cholesterol by providing a healthy food product, namely, milk and/or a derivative thereof, naturally enriched with said substance.

Furthermore, the regular consumption of the milk and/or a derivative thereof and/or the consumption of a food product comprising the milk and/or a derivative thereof obtained according to the method of the present invention, together with appropriate physical activity and a healthy diet, can advantageously enable the blood cholesterol levels to be kept under control, without side effects, reducing or even completely replacing the intake of drugs and/or traditional supplements. The absence of side effects is in fact closely connected to the method of the present invention, which advantageously enables a food product to be to obtained wherein the at least one lipid-lowering substance is preferably present in a lower concentration than is established as the limit for drugs and/or food supplements.

SUMMARY OF THE INVENTION

The present invention relates to a method for producing milk and/or a derivative thereof enriched with at least one lipid-lowering substance.

The method of the present invention comprises the steps of:

(a) administering to a dairy ruminant a ration of cereals fermented with at least one fungus capable of producing the at least one lipid-lowering substance, said ration of cereals partially supplementing or totally replacing the feed of the ruminant;

(b) milking the dairy ruminant in the next 6-48 hours;

(c) collecting the milk enriched with the at least one lipid-lowering substance.

In one embodiment, the milk enriched with the at least one lipid-lowering substance is consumed as such, possibly after partial or total skimming, or is further processed to yield a derivative enriched with the at least one lipid-lowering substance (step (d)).

The milk and/or a derivative thereof obtained according to the method of the present invention is preferably enriched with monacolin k. The invention also relates to a food product comprising the milk and/or a derivative thereof enriched with at least one lipid-lowering substance according to the method of the invention. The invention further relates to the use of said milk or a derivative thereof or a food product obtained from the milk or a derivative thereof for controlling and reducing blood cholesterol.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the chromatographic profile obtained by analysing a sample of milk (12 hours) obtained from a dairy cow extracted with dichloromethane or methanol as per Example 1.

Figure 2 shows the chromatographic profile obtained by analysing a solution of lovastatin with a concentration of 100 ng/ml. Figure 3 shows the chromatographic profile obtained by analysing the two milk samples (marked as 12 hours and 48 hours) extracted with dichloromethane as per Example 1.

DETAILED DESCRIPTION OF THE INVENTION

The term“milk” refers to drinking milk produced by the mammary glands of dairy ruminants and obtained by regular milking thereof.

The term“enriched milk” means the milk obtained according to the method of the present invention. Said enriched milk can also be defined by the term“naturally enriched milk”, since the at least one lipid-lowering substance taken in by the ruminant through the diet according to the method of the present invention is found in the milk naturally produced by the ruminant itself, without there being a need to add any substance at the end of milking. This terminology is to be understood as also extending to the milk derivative obtained according to the method of the present invention and to the food product comprising said milk and/or a derivative thereof.

The term “supplemented milk” means a milk that is supplemented with a substance, such as, for example a lipid-lowering substance, after milking.

The term“lipid-lowering substance” means the group of substances that lower the blood lipid concentration. Examples of lipid-lowering substances are: monacolin k, monacolin j, monacolin I, dehydromonacolin k, compactin, monascin and ankaflavin.

The term “feed” refers to dry fraction of feed administered as a supplement to complete the daily rations of green forage and/or the dried feed typically administered to dairy ruminants and which comprises sorghum, corn, field beans, wheat, soybean, sugar beet, alfalfa, rice, barley and oats.

The term “food-grade ingredient” means an ingredient that is used in the subsequent processing of the milk and/or a derivative thereof in order to obtain a food product comprising said milk and/or a derivative thereof. Food-grade ingredients are for example: flour, sugar, salt, eggs, butter, oil, fats etc.

“Blood cholesterol” means the concentration of cholesterol in the plasma. Since cholesterol, because of its insolubility in water, circulates exclusively incorporated in plasma lipoproteins, blood cholesterol is distinguished into total cholesterol (TC), LDL cholesterol (low density lipoproteins), non-HDL cholesterol (which includes the cholesterol transported by all plasma lipoproteins with the exception of HDL) and HDL cholesterol (high density lipoproteins). High levels of the first three parameters are considered harmful to health, where HDL cholesterol is recognised to have a protective role. Non-HDL cholesterol includes VLDL (very low density lipoproteins), IDL (intermediate density lipoproteins) and LDL. According to the guidelines of the World Health Organization (WHO), the classification of the lipoprotein values for adults (mg/dl) is the following:

Below are the reference values for children and adolescents according to the Journal of the American College of Cardiology (2018) (doi: https://doi.Org/10.1016/j.jacc.2018.11.003. Grundy SM, et al. 2018 “Cholesterol Clinical Practice Guidelines”):

The term“statins” refers to statins commonly used in pharmacological therapies in clinical practice. Examples of this type of statins are the following drugs: atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin etc.

The terms monacolin k (m.k.) and lovastatin are used as perfectly interchangeable synonyms, these molecules having an identical chemical structure.

The terms “fermented rice” and “fermented red rice” are used as perfectly interchangeable synonyms, since once it is fermented, rice takes on the characteristic red colour and can thus be identified as“fermented red rice”.

For the purposes of the present invention, the terms“food” and“food product” are used as perfectly interchangeable synonyms.

“Milk derivative” means a product derived from milk, obtained according to a whole variety of processes, such as, for example, ricotta, fresh cheese, aged cheese or yogurt.

The term “dairy ruminant” means a mammal belonging to the sub-order Ruminantia, capable of producing milk.

For the purposes of the present invention, the dairy ruminants referred to as“cow”, “goat” and“sheep” are thus perfectly interchangeable synonyms of “dairy cow”, “dairy goat” and“dairy sheep”.

The method of the present invention comprises a first step (a) wherein a ration of cereals fermented with at least one fungus capable of producing at least one lipid lowering substance is administered to a dairy ruminant. In one embodiment, said dairy ruminant is selected from the group consisting of: cows, goats and sheep.

According to a particularly preferred embodiment, said dairy ruminant is a dairy cow.

The dairy ruminant is preferably fed once a day.

According to one embodiment of the invention, wherein said dairy ruminant is a goat, the total amount of feed administered is comprised between 1 and 5 kg/pro die, preferably between 2 and 4 kg/pro-die.

According to one embodiment of the invention, wherein said dairy ruminant is a sheep, the total amount of feed administered is comprised between 1 and 5 kg/pro-die, preferably between 2 and 4 kg/pro-die.

According to a particularly preferred embodiment of the invention, wherein said dairy ruminant is a cow, the total amount of feed administered is comprised between 20 and 50 kg/pro-die, preferably between 30 and 40 kg/pro-die.

The ration of fermented cereals partially supplements or totally replaces the feed comprising sorghum, corn, field beans, wheat, soybean, sunflower seeds, sugar beet, alfalfa, rice, barley, oats and a combination thereof, so that the dairy ruminant is made to follow a balanced diet rich in cereals.

According to one embodiment of the invention, wherein said dairy ruminant is a goat, said ration of fermented cereals is added to the feed in an amount comprised between 0.2 and 2 kg, preferably between 0.5 and 1.5 kg.

According to one embodiment of the invention, wherein said dairy ruminant is a sheep, said ration of fermented cereals is added to the feed in an amount comprised between 0.2 and 2 kg, preferably between 0.5 and 1.5 kg.

According to a particularly preferred embodiment of the invention, wherein said dairy ruminant is a cow, said ration of fermented cereals is added to the feed in an amount comprised between 1 and 40 kg, preferably between 1 and 10 kg.

In a particularly preferred embodiment of the invention said ration of fermented cereals constitutes from 10 to 40% by weight, preferably from 15 al 35% by weight of the total amount of feed administered to the dairy ruminant.

The cereals that will be fermented and that will thus form the ration of fermented cereals partially supplementing or totally replacing the feed of the dairy ruminant, are selected from the group consisting of: rice, barley, oats, corn, soybean, wheat, sorghum and a combination thereof, preferably rice.

Said cereals are fermented with at least one fungus selected from the group consisting of the species monascus, for example: monascus pilosus, monascus purpureus, monascus ruber, monascus albidus, monascus argentinensis, monascus aurantiacus, monascus eremophilus, monascus floridanus, monascus fuliginosus, monascus kaoliang, monascus lunispora, monascus pallens, monascus paxii, monascus pubigeras, monascus sanguineus, monascus kaoliang, monascus sanguineus, monascus barkeri, monascus floridanus, monascus lunisporas, monascus pallens; or aspergillus terreus;

or the species penicillium, for example penicillium citrinum.

In a preferred embodiment the fungus used is monascus purpureus, which is capable of producing the lipid-lowering substance monacolin k.

The fermentation is conducted by means of techniques that are known in the art, suitably adapted according to production needs.

The cereals listed above are covered with distilled water. After 8 hours, the water is removed by means of a strainer. The cereals collected are then sterilised for 20 minutes at 120 °C and, after cooling, they are inoculated with a suitable suspension, prepared beforehand, of the at least one fungus according to the present invention, cultured for 10 days at 30 °C. The fermented cereals thus obtained are then administered as such in the diet of the dairy ruminant according to the previously described methods and amounts.

The ration of fermented cereals is selected from a ration of rice, barley, oats, corn, soybean, wheat and a mixture thereof.

The ration of fermented cereals is preferably fermented rice.

The at least one lipid-lowering substance is present in said ration of fermented cereals in a concentration of less than 10% by weight, preferably between 1.5 % and 5% by weight.

Said lipid-lowering substance is preferably selected in the group consisting of: monacolin k, monacolin j, monacolin I, dehydromonacolin k, compactin, monascin and ankaflavin. In a preferred embodiment, the lipid-lowering substance is monacolin k, preferably present in the ration of fermented cereals in concentration of less than 10% by weight, preferably between 1.5 and 5% by weight.

In an even more preferred embodiment, step (a) of the method according to the present invention is conducted using, as a ration of fermented cereals, an amount comprised between 1 and 9 kg of red rice fermented with the fungus monascus purpureus and having a monacolin k content comprised between 1.5 and 5% by weight.

The natural enrichment of the milk produced by the ruminant (step (b)) in the next 6-48 hours with the at least one lipid-lowering substance depends on the amount of the ration of fermented cereal and the concentration of said lipid-lowering substance in said ration administered to the ruminant in step (a).

The Applicant has in fact surprisingly discovered that, with the present method, the at least one lipid-lowering substance taken in by the ruminant through the diet passes naturally into the milk produced, and a naturally enriched food product is thus obtained.

For example, in the case of the preferred embodiment wherein the at least one lipid-lowering substance is monacolin k (m.k.), the experimentation conducted and reported in the Examples section showed the presence of monacolin k in the milk excreted after both 12 and 48 hours. This result confirmed the hypothesis that part of the monacolin k is excreted by the ruminant in the milk in unaltered form and it is not totally eliminated through faeces or urine as has been supposed until today. The concentration of monacolin k found in the milk collected and produced according to the method of the present invention (step (c)) is less than 10 mg/I, preferably comprised between 0.01 mg/I and 10 mg/I, preferably between 0.02 mg/I and 8 mg/I, more preferably between 0.01 mg/I and 4 mg/I, more preferably between 0.01 mg/I and 3 mg/I, more preferably between 0.02 mg/I and 3 mg/I, even more preferably between 0.01 mg/I and 0.5 mg/I.

Said concentration of the active substance in the final product can be increased by administering the same type of feed as in step (a) to the ruminant daily, but increasing the percentage of the ration of fermented cereals, for example increasing the percentage of fermented red rice present in the totality of the feed administered. This leads to a higher blood concentration of the active substance and consequently increases the concentration of at least one lipid-lowering substance in the milk and/or a derivative thereof. However, the milk and/or a derivative thereof, in order to be able to be classified as a food product by the various authorities responsible for regulating food products and pharmaceuticals (such as the European Food Safety Authority (EFSA) or the U.S. Food and Drug Administration (FDA)), and to be able to be approved for consumption (for example also by pregnant women and/or children), must include at least one lipid lowering substance in a very low concentration, so as to avoid the occurrence of the side effects typical of traditional lipid-lowering drugs such as statins and/or the typical food supplements in the sector.

In one embodiment, said milk produced with the method of the present invention (steps (a) - (c)) is whole milk or partially or totally skimmed milk.

The milk obtained with the method of the invention can be further processed (step (d)) to yield a solid or semi-solid milk derivative such as, for example ricotta, fresh cheese, aged cheese or yogurt.

According to the method of the present invention the concentration of the at least one lipid-lowering substance present in the milk is comprised between 0.01 mg/I and 10 mg/I, preferably between 0.02 mg/I and 8 mg/I, more preferably between 0.01 mg/I and 4 mg/I, more preferably between 0.01 mg/I and 3 mg/I, more preferably between 0.02 mg/I and 3 mg/I, more preferably between 0.01 mg/I and 0.5 mg/I.

According to the method of the present invention, the concentration of the at least one lipid-lowering substance present in the solid or semi-solid milk derivative produced in step (d), is comprised between 0.15 mg/kg and 150 mg/kg, preferably between 0.15 mg/kg and 80 mg/kg, more preferably between 0.15 mg/kg and 45 mg/kg, even more preferably between 0.15 mg/kg and 30 mg/kg, even more preferably between 0.15 mg/kg and 15 mg/kg, according to the type of derivative.

In a preferred embodiment of the invention, the at least one lipid-lowering substance is monacolin k and is present in the milk in an amount comprised between 0.01 mg/I and 10 mg/I, preferably between 0.02 mg/I and 8 mg/I, more preferably between 0.01 mg/I and 4 mg/I, more preferably between 0.01 mg/I and 3 mg/I, more preferably between 0.02 mg/I and 3 mg/I, even more preferably between 0.01 mg/I and 0.5 mg/I.

In a preferred embodiment of the invention the at least one lipid-lowering substance is monacolin k and is present in the solid or semi-solid milk derivative produced in the step (d), in an amount comprised between 0.15 mg/kg and 150 mg/kg, preferably between 0.15 mg/kg and 80 mg/kg, more preferably between 0.15 mg/kg and 45 mg/kg, even more preferably between 0.15 mg/kg and 30 mg/kg, even more preferably between 0.15 mg/kg and 15 mg/kg according to the type of derivative.

Therefore, the milk and/or a derivative thereof obtained according to the method of the present invention is identified as a healthy, natural product which can be advantageously used in diets to control and/or reduce blood cholesterol, together with suitable physical activity and an appropriate lifestyle.

In fact, not only is said milk and/or a derivative thereof naturally enriched with at least one lipid-lowering substance which inhibits the activity of HMG-CoA reductase in the biogenesis of endogenous cholesterol, but it also contains an amount of exogenous cholesterol comprised, for example, between 0.03 mg/ml, in the case of skimmed milk and 0.11 mg/ml in the case of whole milk; this cholesterol is fundamental in the diet of an adult individual. In fact, the enzyme responsible for controlling the production of endogenous cholesterol is regulated by negative feedback ; i.e. if cholesterol is taken in through the diet, the enzyme interrupts the production of endogenous cholesterol whereas if cholesterol is not taken in through the diet (for example in the case of a diet that is completely devoid of or has a very low content of cholesterol), the same enzyme induces a liver biosynthesis of cholesterol in order to compensate for the lack of exogenous cholesterol.

The invention also relates to a food product comprising the milk and/or a derivative thereof obtained from the subsequent processing of the milk and/or a derivative thereof with other food grade ingredients such as, for example, flour, sugar, salt, eggs, butter, oil, fats, etc.

The processing of the milk and/or a derivative thereof according to the invention with one or more further ingredients makes it possible to obtain a food product enriched with at least one lipid-lowering substance, such as, for example, a snack or a cake, etc. enriched with at least one lipid-lowering substance.

Since the activity of HMG-CoA-reductase is also modulated by the hormones controlling blood sugar, i.e. glucagon and insulin, combining the food product of the present invention with an appropriate diet, for example a diet with a reduced carbohydrate intake and constant, non-intense motor activity, makes it possible to produce a decrease in blood cholesterol.

Advantageously, a regular consumption of the milk and/or a derivative thereof or a food product obtained from the milk and/or a derivative thereof according to the invention thus enables the levels of blood cholesterol to be kept under control without side effects, reducing or even completely replacing the consumption of drugs and/or traditional supplements.

EXAMPLES

Example 1 : Determination of monacolin k in two samples of milk obtained from a dairy cow

Chromatographic analysis

The analysis was conducted with a Varian chromatography instrument (Varian Inc.) managed by Varian MS Workstation System Control software Vers. 6.9 and consisting of 2 pumps (212-LC) connected to a Varian triple quadrupole detector (Mod. 320-LC) equipped with an ESI ion source.

A Polaris C18A column (50x2.0 mm) (Agilent Technologies, CA) was used for the chromatographic analysis under the following conditions:

- injection volume 5 pi,

- mobile phase consisting of 0.1 % formic acid in water (A) and acetonitrile

(B),

- chromatographic run time 7 minutes,

- flow 400 mI/min.

The molecule of interest was eluted with a retention time of 3.5

min. The following gradient was used for the chromatographic analysis.

The analysis was conducted in the positive mode using MRM to maximise the analytic response; the CID ( collision-induced dissociation) experiments were conducted in the collision cell (2nd quadrupole) using argon as the collision gas. The SRM {single reaction monitoring) acquisition was conducted by selecting the precursor ion (pseudomolecular ion) in the first quadrupole and monitoring the product ions with the third quadrupole, using the following instrument parameters: Detector: 1400 Volts; CID Gas: Argon; API Drying Gas (N2): 35.0 psi; API Drying Gas: 300.0 ° C; API Nebulizing Gas (N2): 45.0 psi; Scan mode: Centroid; Collision Cell pressure: 1.8 mTorr; API Housing Temperature: 40.0 ° C; Manifold Temperature: 40.0 ° C.

The analytic conditions used for CID (collision-induced dissociation) experiments (precursor ions, product ions, capillary voltage and collision energy) are shown in table 1.

The choice of the collision energies in the fragmentation cell and the fragments utilisable for the qualitative analysis were selected by the instrument software upon the sending of 10 mI/min of the standard solution of lovastatin (100 ng/ml) to the instrument. The flow of 300 mI/min of the mobile phased used arrived simultaneously with the ESI source.

Table 1 : Analytic conditions used in the LC-MS-MS analysis (the scanning time was 0.1 s).

^a transition used for quantification

Determination of monacolin k

The determination of monacolin k was carried out after looking for the best chromatographic conditions (for the separation) and fragmentation conditions (for subsequent identification and quantification) with a Varian triple quadrupole instrument. On the basis of the solubility data shown below, the extraction from the two milk samples was performed both with dichloromethane (solvent of choice for the molecule in question) and with methanol (to avoid an excessive fat extraction) so as to evaluate which was the best.

The final results are expressed in terms of ng/ml of milk after comparison with methanol solutions of lovastatin.

Extraction of the milk samples

10 ml of milk were subjected to extraction with 2 aliquots of 20 ml dichloromethane. After the extraction, the samples were centrifuged and the organic fraction was reconstituted, reunited and vacuum dried. The residue was reconstituted with 1 ml of methanol so as not to carry along with it the fat extracted from the milk, and after centrifugation, the solution was analysed by LC-MS. Similarly, 10 ml of milk were extracted with two 20 ml aliquots of methanol and after centrifugation the liquid part was reunited and vacuum dried. In this case as well the sample was reconstituted with 1 ml of methanol and after centrifugation and filtration subjected to chromatographic analysis.

Reference solution of lovastatin

For the purpose of developing the method and the subsequent quantification, use was made of a 1 mg/ml methanol solution obtained by solubilising a 20 mg tablet of Rextat® in 20 ml of methanol. Prior to use, the solution was centrifuged and filtered to remove the excipients.

Results

The method developed made it possible to obtain a quantification limit of 10 ng/ml, which proved to be satisfactory for the determination of the analyte in the two milk samples.

Of the two extraction methods, the one with dichloromethane proved to be better, as may be seen from Figure 1.

Figures 2 and 3 respectively show the chromatograms obtained for a 100 ng/ml solution of lovastatin and for the two samples (marked as 12h and 48h) after extraction with dichloromethane.

Following extraction with dichloromethane and the subsequent chromatographic analysis, the content of lovastatin in the two samples may be estimated as 20.29 and 11.27 ng/ml respectively for the 12h and 48h samples.

Claims

1. A method for producing milk comprising at least one lipid-lowering substance, comprising the steps of:

(a) administering to a dairy ruminant a ration of cereals fermented with at least one fungus, said ration of fermented cereals partially supplementing or totally replacing the feed of the dairy ruminant;

(b) milking the dairy ruminant in the next 6-48 hours;

2. The method according to claim 1 , wherein said dairy ruminant is selected from the group consisting of: cows, goats and sheep, preferably a cow.

3. The method according to claim 1 or 2, wherein the at least one fungus is selected from the group consisting of: monascus pilosus, monascus purpureus, monascus ruber, monascus albidus, monascus argentinensis, monascus aurantiacus, monascus eremophilus, monascus floridanus, monascus fuliginosus, monascus kaoliang, monascus lunispora, monascus pallens, monascus paxii, monascus pubigeras, monascus sanguineus, monascus kaoliang, monascus sanguineus, monascus barkeri, monascus floridanus, monascus lunisporas, monascus pallens, aspergillus terreus and penicillium citrinum.

4. The method according to any one of the preceding claims, wherein said ration of fermented cereals constitutes from 10 to 40% by weight, preferably from 15 to 35% by weight, of the total amount of feed administered to the dairy ruminant.

5. The method according to any one of the preceding claims, wherein the ration of fermented cereals is selected from among sorghum, maize, wheat, soybean, rice, barley, oats and a combination thereof, preferably rice.

6. The method according to any one of the preceding claims, wherein the at least one lipid-lowering substance is present in said ration of fermented cereals in a concentration of less than 10% by weight, preferably between 1.5 and 5% by weight.

7. The method according to any one of the preceding claims, wherein the at least one lipid-lowering substance is selected from the group consisting of: monacolin k, monacolin j, monacolin I, dehydromonacolin k, compactin, monascin and ankaflavin, preferably monacolin k.

8. A milk obtained with the method according to any one of the preceding claims.

9. The milk according to claim 8, wherein the amount of the at least one lipid lowering substance is comprised between 0.01 mg/I and 10 mg/I, preferably between 0.02 mg/I and 8 mg/I, more preferably between 0.01 mg/I and 4 mg/I, more preferably between 0.01 mg/I and 3 mg/I, more preferably between 0.02 mg/I and 3 mg/I, even more preferably between 0.01 mg/I and 0.5 mg/I.

10. A milk derivative obtained by processing the milk according to claim 8 or 9, wherein said derivative is ricotta, fresh cheese, ripened cheese or yogurt (step

(d)).

11. A food product obtained by processing the milk and/or a derivative thereof according to any one of claims 8 to 10 with food grade ingredients.

12. A use of the milk and/or a derivative thereof and/or a food product according to any one of claims 8 to 11 as a food for controlling and/or reducing blood cholesterol.