IT201900002415A1 - Method for the production of milk containing lipid-lowering substances - Google Patents

Description

DESCRIPTION

Attached to a patent application for INDUSTRIAL INVENTION having the title

"Method for the production of milk containing lipid-lowering substances"

FIELD OF THE INVENTION

The present invention relates to a method for the production of milk and / or a derivative thereof enriched with at least one lipid-lowering substance, preferably monacolin k. The invention also refers to milk, to a derivative thereof and to a food obtained from milk or a derivative thereof, enriched with this substance and their use for the control and / or reduction of blood cholesterol.

BACKGROUND OF THE INVENTION

Cardiovascular diseases (CVD) are the leading cause of adult mortality and morbidity in industrialized countries. Among the numerous cardiovascular risk factors, dyslipidemias, and in particular LDL cholesterolemia, certainly represent one of the factors most commonly called into question. For this reason, one of the blood parameters currently considered most important to keep under control for the prevention of ischemias, heart attacks or other cardiovascular diseases, is total cholesterolemia (TC).

A healthy adult individual produces by autonomous biosynthesis most of the necessary cholesterol (endogenous pool), while, under normal conditions, only a small part is taken daily with food (exogenous pool). Both pools are subject to regulatory mechanisms, so that, in equilibrium conditions, the quantity of cholesterol synthesized and that absorbed by food, corresponds to the cholesterol eliminated with the bile as free cholesterol and as bile acids.

The biosynthesis of cholesterol is controlled by the hepatic enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA). If the cholesterol level of a healthy individual turns out to be too low, the liver produces HMG-CoA in high quantities to raise the cholesterol level and restore it to adequate values, while if the cholesterol level of that healthy individual becomes too high, the liver inhibits or reduces the production of said coenzyme by decreasing or inhibiting the production of endogenous cholesterol.

However, the optimal level of cholesterol in the blood can increase in an uncontrolled way leading to a condition of hypercholesterolemia in the individual which, as already mentioned, represents one of the major risk factors for cardiovascular disease.

The increase in cholesterol can be caused by environmental factors (such as a diet high in saturated fat, sugar and physical inactivity) or by predisposing genetic factors or, more commonly, by a combination of both factors.

Since the distribution profiles of CVD in all industrialized countries are similar to those of the main risk factors, and since there is direct evidence of the effectiveness of reducing cholesterol levels on reducing CVD mortality, prevention strategies are now well established and are based on the combination of dietary therapies with drug therapies.

For this reason, the first line of intervention for the control of cholesterolemia and the risk of cardiovascular disease is essentially based on a healthy lifestyle, a diet that includes low fat consumption, regular and daily motor activity not less than thirty minutes as well as refraining from smoking and alcohol.

Regarding drug therapies, statins have proved to be the most widely used drugs of choice in clinical practice and their efficacy has been demonstrated in various clinical studies (Collins R. et al., "Interpretation of the evidence for the efficacy and safety of statin therapy ", Lancet 2016; 388: 2532-2561) both in terms of reduction of cholesterolemia and reduction of cardiovascular events, fatal and otherwise. Statins in fact inhibit the hepatic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), a key enzyme in the endogenous synthesis of cholesterol that catalyzes the irreversible reaction that leads to the synthesis of mevalonate starting from 3-hydroxy -3-methylglutaryl coenzyme A (HMG-CoA).

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

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

In fact, statins, in addition to blocking the production of cholesterol, also block enzymes that govern muscle activities, resulting in loss of muscle tissue, pain and numbness in the extremities, inflammation of tendons and ligaments. Additionally, dead muscle cells release myoglobin, which in turn can cause kidney problems. Other problems caused by taking statins can affect the liver (in fact, transaminases are often checked in patients who use them regularly). Among the various side effects of statins, cognitive problems as well as a general increase in blood sugar with a consequent increase in the risk of diabetes have also sometimes been noted. For this reason, in recent years, the need to find alternatives to statins and pharmacological remedies has become increasingly felt. The interest therefore turned to the identification of combinations of nutraceuticals aimed at reducing cardiovascular risk capable of acting on the levels of cholesterolemia and other circulating lipids.

An example of said combinations of nutraceuticals are food supplements containing lipid-lowering substances.

Among these, one of the most commonly used is fermented red rice. This supplement is a natural product deriving from the fermentation of rice by monascus purpureus (m.p.), which is a fungus that is able to ferment rice and other congenital substrates producing monacolin k. Monacolin k is a molecule that belongs to the statin family.

Monacolin k is of fermentation derivation and performs an action completely similar to that of synthetic lovastatin by inhibiting the same protein involved in the control of endogenous cholesterol, namely HMG-CoA reductase effectively reducing the endogenous cholesterol produced.

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

For example, patent BE1020033 describes a milk-based composition for use as a hypocholesterolemic food supplement comprising a quantity of monacolin (which in this specific case is lovastatin deriving from pleurotus ostreatus) sufficient to obtain a hypocholesterolemic effect. In this patent the composition is obtained starting from pleurotus ostreatus powder, added to fermented liquid milk or to a derivative thereof.

Patent WO2005104864 instead deals with an edible oil, comprising statins produced by the fermentation of mushrooms (such as monascus fungus) and at least 90% by weight of diglycerides and / or triglycerides, which is added to a cow's milk.

The Chinese document CN103651915 instead describes a dairy product, in particular a fresh cheese, containing ingredients such as monacolin k and γ-aminobutyric acid, with the function of regulating blood fat and reducing blood pressure. In this case, a fermentation agent (lactobacillus) and a fungus (such as monascus fungus) are added to the raw, sterilized milk.

It is important to note that all these supplements described in the art are an alteration of the natural food, being produced by the addition of fermentation derivatives, exposing the product to problems related to possible chemical contamination and sophistication. These products, although not exactly drugs, are far from healthy and natural food.

In fact, the demand for so-called “clean label” products is increasingly evident, with consequent growth on the market of consumer demand for products without additives or sophistication.

Therefore, the need arises for a new method for the production of a completely natural food, without additives, which can be consumed daily, even for example by children and pregnant women, and is able to contribute to the control of blood cholesterol. , without altering the natural formulation of the food product. Another aspect to take into consideration is that the use of a lipid-lowering drug such as a statin, and / or a dietary supplement, requires that an adequate lipid-lowering diet be first suggested to the individual and vice versa. In fact, currently, the diet followed to reduce blood cholesterol is essentially a fat-free diet associated with drugs or, alternatively, with the aforementioned food supplements. Furthermore, the drug and / or food supplement cannot and should not be considered a substitute for proper nutrition or taken as a normal food having certain contraindications and methods of use strictly related to the amount of active ingredient (statins) present in it.

The present invention therefore solves the technical problem of the coupling between proper nutrition and the intake of a lipid-lowering substance for the control of blood cholesterol by providing a healthy food, namely milk and / or its derivative, naturally enriched with said substance.

Furthermore, the regular consumption of milk and / or a derivative thereof and / or the consumption of a food product comprising 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 allow to keep cholesterol levels under control, without side effects, by reducing or even completely replacing the intake of traditional drugs and / or supplements. The absence of side effects is in fact strictly connected to the method of the present invention which advantageously allows to obtain a food product in which the at least one lipid-lowering substance is preferably present in a concentration lower than that established as the limit for drugs and / or food supplements. .

SUMMARY OF THE INVENTION

The present invention relates to a method for the production of 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) feeding a dairy cow a ration of fermented cereals, with at least one fungus capable of producing at least one lipid-lowering substance, said cereal ration being partially integrated or totally substituted for the cow's feed;

(b) milking the dairy cow in the following 6-48 hours;

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

Preferably the milk and / or a derivative thereof obtained according to the method of the present invention is enriched with monacolin k. The invention also relates to a food product comprising milk and / or a derivative thereof enriched with at least one lipid-lowering substance according to the method of the invention.

The invention also relates to the use of said milk or its derivative or a food product obtained from milk or its derivative for the control and reduction of blood cholesterol.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the chromatographic profile obtained from the analysis of a milk sample (12 hours) extracted with dichloromethane or methanol as in Example 1.

Figure 2 shows the chromatographic profile obtained from the analysis of a solution with a concentration of 100 ng / ml of lovastatin.

Figure 3 shows the chromatographic profile obtained from the analysis of the two milk samples (signed 12 hours and 48 hours) extracted with dichloromethane as in Example 1.

DETAILED DESCRIPTION OF THE INVENTION

The term “milk” refers to bovine drinking milk produced by the mammary glands of dairy cows and obtained from their regular milking. The term "enriched milk" means the milk obtained according to the method of the present invention. Said enriched milk can also be defined with the terminology of "naturally enriched milk" since the at least one lipid-lowering substance taken by the cow through feeding according to the method of the present invention, is found in the milk naturally produced by the cow itself without the need to add any substance at the end of milking. This terminology is to be understood as extended also 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 "added milk" means milk that is added with a substance, such as a lipid-lowering substance, after milking.

The term "lipid-lowering substance" refers to that group of substances that lower the blood level of lipids. Examples of lipid-lowering substances are: monacolin k, monacolin j, monacolin l, dehydromonacolin k, compactin, monascin, ankaflavine.

The term "feed" refers to the dry fraction of feed to be integrated to complete the daily rations of green and / or dried forage typically fed to the dairy cow and which includes sorghum, maize, broad beans, broad beans, wheat, soy, beet , alfalfa, rice, barley, oats.

The term "food grade ingredient" means an ingredient that is used in the subsequent processing of milk and / or a derivative thereof 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, fat etc.

By “cholesterolemia” we mean the concentration of cholesterol in the plasma. Since cholesterol, due to its insolubility in water, circulates exclusively incorporated in plasma lipoproteins, cholesterol is divided into total cholesterol (TC), LDL (low density lipoproteins) cholesterol, non-HDL cholesterol (which includes 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, while HDL cholesterol is recognized as having a protective role. Non-HDL cholesterol includes very low density lipoproteins (VLDL), intermediate density lipoproteins (LDL), and LDL. According to the guidelines of the World Health Organization (WHO), the classification of lipoprotein values for adulthood (mg / dl) is as follows:

The following are the reference values for individuals of pediatric age 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 terms "statins" refer to statins commonly used in drug therapy in clinical practice. Examples of this type of statin 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 synonyms perfectly interchangeable, being these molecules with identical chemical structure.

The terms "fermented rice" and "fermented red rice" are used as synonyms that are perfectly interchangeable as once fermented, the rice takes on its characteristic red color and can therefore 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. By “derivative of milk” we mean a product deriving from milk, obtained according to the most varied procedures, such as ricotta, fresh or aged cheese or yoghurt.

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

The method of the present invention provides a first step (a) where a dairy cow is given a ration of fermented cereals, with at least one mushroom capable of producing at least one lipid-lowering substance.

The cow is fed once a day and the total amount of feed administered is between 20 and 50 kg / per day, preferably between 30 and 40 kg / per day.

The ration of fermented cereals is partially integrated or totally substituted for the feed comprising sorghum, maize, broad beans, broad beans, wheat, soybeans, sunflower seeds, beets, alfalfa, rice, barley, oats and a combination thereof, so that the cow a balanced diet rich in cereals is followed.

Said ration of fermented cereals is added to the feed in a quantity ranging from 1 to 40 kg, preferably from 1 to 10 kg.

In an embodiment of the invention 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 cow. The cereals that will be fermented and which will therefore constitute the ration of fermented cereals partially integrated or totally substituted for the cow's feed, are selected from the group consisting of: rice, barley, oats, maize, soy, wheat, sorghum and a combination thereof , preferably rice.

These cereals are fermented with at least one mushroom chosen from the group consisting of the monascus species, for example: monascus pilosus, monascus purpureus, monascus ruber, monascus albidus, monascus argentinensis, monascus aurantiacus, monascus eremophilus, monascus floridanus, monascus fuligin kaus, monascus monascus lunispora, monascus pallens, monascus paxii, monascus pubigeras, monascus sanguineus, monascus kaoliang, monascus sanguineus, monascus barkeri, monascus floridanus, monascus lunisporas, monascus pallens; or from aspergillus terreus.

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

Fermentation is carried out using techniques known in the sector, 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 sieve. The collected cereals are then sterilized for 20 minutes at 120 ° C and, after cooling, are inoculated with a suitable suspension previously prepared of at least one mushroom according to the present invention, grown for 10 days at 30 ° C. The fermented cereals thus obtained are then administered as they are in the diet of the dairy cow according to the methods and quantities previously described.

The ration of fermented cereals is chosen from a ration of rice, barley, oats, maize, soy, wheat and a mixture thereof.

Preferably, the fermented grain ration is 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.

Preferably said lipid-lowering substance is selected from the group consisting of: monacolin k, monacolin j, monacolin l, dehydromonacolin k, compactin, monascin, ankaflavine.

In a preferred embodiment the lipid-lowering substance is monacolin k, preferably present in the ration of fermented cereals in a concentration lower 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 carried out using as a ration of fermented cereals an amount comprised between 1 and 9 kg of red rice fermented with monascus purpureus mushroom having a title in monacolin k comprised between 1.5 and 5% by weight.

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

The Applicant has in fact surprisingly discovered that with the present method the at least one lipid-lowering substance taken by the cow with the feed naturally passes into the milk produced thus obtaining a naturally enriched food.

For example, in the case of the preferred embodiment in which the at least one lipid-lowering substance is monacolin k (m.k.), the presence of monacolin k in the excreted milk both after 12 than after 48 hours. This result confirmed the hypothesis that part of the monacolin k is excreted by the cow in the milk in its unaltered form and not totally eliminated by the fecal or urinary route as was previously assumed.

The concentration of monacolin k found in the milk collected and produced according to the method of the present invention (step (c)) is lower than 10 mg / l, preferably between 0.01 mg / l and 10 mg / l, preferably between 0.02 mg / l and 8 mg / l, more preferably between 0.01 mg / l and 4 mg / l, more preferably between 0.01 mg / l and 3 mg / l, more preferably between 0.02 mg / l and 3 mg / l, even more preferably between 0.01 mg / l and 0.5 mg / l.

Said concentration of active substance in the final product can be increased by administering the same type of feed to the cow daily as in step (a) but increasing the percentage of fermented cereal ration, for example by increasing the percentage of fermented red rice present in the totality of the feed administered. .

This leads to a higher blood concentration of active substance and consequently increases the concentration of at least one lipid-lowering substance in milk and / or a derivative thereof. However, milk and / or its derivative, in order to be classified as food by the various authorities that deal with the regulation of food and pharmaceutical products (such as the European Food Safety Authority (EFSA) or the Food and Drug Administration American (FDA)), and in order to be approved for consumption (for example also by pregnant women and / or children), it must include at least one lipid-lowering substance in very low concentration, in order to avoid the onset of the typical side effects of traditional lipid-lowering drugs such as statins and / or 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 give a solid or semi-solid milk derivative such as 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 milk is between 0.01 mg / l and 10 mg / l, preferably between 0.02 mg / l and 8 mg / l, more preferably between 0.01 mg / l and 4 mg / l, more preferably between 0.01 mg / l and 3 mg / l, more preferably between 0.02 mg / l and 3 mg / l, more preferably between 0.01 mg / l and 0.5 mg / l L.

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 derivative of milk, produced in step (d), is 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 depending on 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 milk in an amount ranging from 0.01 mg / l to 10 mg / l, preferably from 0.02 mg / l to 8 mg / l, more preferably between 0.01 mg / l and 4 mg / l, more preferably between 0.01 mg / l and 3 mg / l, more preferably between 0.02 mg / l and 3 mg / l, even more preferably between 0, 01 mg / l and 0.5 mg / l.

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 derivative of milk, produced in step (d), in an amount ranging from 0.15 mg / kg to 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 depending on the type of derivative.

Therefore, 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 for the control and / or reduction of blood cholesterol, together with suitable physical activity and an appropriate lifestyle.

In fact, not only said milk and / or its derivative is naturally enriched with at least one lipid-lowering substance that inhibits the activity of HMG-CoA reductase in the biogenesis of endogenous cholesterol, but also contains an amount of exogenous cholesterol including, for example, between 0.03 mg / ml, in the case of skimmed milk and 0.11 mg / ml for whole milk, cholesterol, which is essential in the diet of an adult individual. In fact, the enzyme responsible for controlling the production of endogenous cholesterol is regulated by negative feedback; that is, if cholesterol is taken with the diet, the enzyme stops the production of endogenous cholesterol while, if cholesterol is not taken through the diet (for example in the case of a completely free or very low cholesterol diet), it the same enzyme induces a hepatic biosynthesis of cholesterol in order to make up for the lack of exogenous cholesterol.

The invention also relates to a food product comprising milk and / or a derivative thereof obtained from the subsequent processing of 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 milk and / or a derivative thereof according to the invention with one or more further ingredients allows to obtain a food product enriched with at least one lipid-lowering substance, such as, for example, a snack, a cake, a snack, etc. enriched with at least one lipid-lowering substance.

Since the activity of HMG-CoA-reductase is also modulated by the glycemic control hormones, i.e. glucagon and insulin, the combination of the food product of the present invention with a suitable diet, for example a diet with a reduced carbohydrate intake and a constant and not intense motor activity, allows to produce a decrease in blood cholesterol.

Advantageously, the regular consumption of milk and / or a derivative thereof or of a food product obtained from milk and / or a derivative thereof according to the invention therefore allows to keep cholesterol levels under control, without side effects, reducing or even completely replacing the intake of traditional drugs and / or supplements.

EXAMPLES

Example 1: Determination of monacolin k in two milk samples

Chromatographic analysis

The analysis was carried out with a chromatographic instrumentation from Varian (Varian Inc.) managed by the Varian MS Workstation System Control Vers.6.9 software 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 with the following conditions:

- injection volume 5 l,

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

- duration of the chromatographic run 7 minutes,

- flow 400 l / min.

The molecule of interest was eluted with a retention time of 3.5 min. The following gradient was used for the chromatographic analysis.

Time (min)% solvent A% solvent B

0.0 60 40

1.0 60 40

6.0 2 98

7.0 2 98

The analysis was conducted in positive mode in MRM to maximize the analytical response, the CID (Collision-induced dissociation) experiments were conducted in the collision cell (2nd quadrupole) using argon as a collision gas. The SRM (Single Reaction Monitoring) acquisition was conducted by selecting the precursor ion (pseudomolecular ion) in the first quadrupole and monitoring the child ions with the third quadrupole, using the following instrumental 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 analysis conditions used for the Collision-induced dissociation (CID) experiments (precursor ions, child ions, capillary voltage and collision energy) are shown in Table 1.

The choice of collision energies in the fragmentation cell and the fragments that can be used for qualitative analysis were selected by the instrument software by sending the instrument a flow of 10 µl / min of the standard lovastatin solution (100 ng / ml). At the same time as the ESI source, the flow of 300 µl / min of the mobile phase used arrived.

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

transition used for quantification

Determination of monacolin k

The determination of monacolin k was carried out after searching for the best chromatographic (for separation) and fragmentation (for subsequent identification and quantification) conditions with a triple quadrupole instrumentation from Varian. On the basis of the solubility data reported below, the extraction from the two milk samples was carried out both with dichloromethane (solvent of choice for this molecule) and with methanol (to avoid excessive extraction of fats) in order to evaluate which was the best. .

Lovastatin solubility in: mg / ml

Water 0.0004

Acetone 47

Chloroform 350

Methanol 28

Acetonitrile 28

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

Extraction of milk samples

10 ml of milk were subjected to extraction with 2 aliquots of 20 ml dichloromethane. After extraction, the samples were centrifuged and the organic fraction taken up, gathered and brought to dryness under vacuum. The residue was taken up with 1 ml of methanol so as not to carry the fat extracted from the milk, and after centrifugation the solution was analyzed by LC-MS.

Similarly 10 ml of milk were extracted with two 20 ml aliquots of methanol and after centrifugation the liquid part was combined and brought to dryness under vacuum. Also in this case the sample was taken up with 1 ml of methanol and after centrifugation and filtration subjected to chromatographic analysis.

Reference Lovastatin solution

For the setting up of the method and the subsequent quantification, a 1 mg / ml methanolic solution was used, obtained by solubilizing a 20 mg Rextat® tablet in 20 ml of methanol. Before 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 best was that with dichloromethane as shown in Figure 1.

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

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

Claims (11)

CLAIMS 1. Method for the production of milk and / or a derivative thereof comprising at least one lipid-lowering substance comprising the steps of: (a) feeding a dairy cow a fermented grain ration with at least one mushroom, said fermented grain ration being partially integrated or totally substituted for the cow's feed; (b) milking the dairy cow in the following 6-48 hours; (c) collect milk enriched with at least one lipid-lowering substance. Method according to claim 1 wherein the at least one mushroom 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 and aspergillus terreus. Method according to claim 1 or 2 wherein said ration of fermented cereals constitutes from 10 to 40% by weight, preferably from 15 to 35% by weight of the amount of total feed administered to the cow. Method according to any one of the preceding claims in which the ration of fermented cereals is selected from sorghum, maize, wheat, soy, rice, barley, oats and a combination thereof, preferably rice. 5. Method according to any one of the preceding claims in which the at least one lipid-lowering substance is present in said ration of fermented cereals in a concentration lower than 10% by weight, preferably between 1.5 and 5% by weight. 6. Method according to any one of the preceding claims in which the at least one lipid-lowering substance is selected from the group consisting of: monacolin k, monacolin j, monacolin l, dehydromonacolin k, compactin, monascin, ankaflavine, preferably monacolin k. 7. Milk obtained by the method according to any one of the preceding claims. Milk according to claim 7 wherein the amount of the at least one lipid-lowering substance is between 0.01 mg / l and 10 mg / l, preferably between 0.02 mg / l and 8 mg / l, more preferably between 0.01 mg / l and 4 mg / l, more preferably between 0.01 mg / l and 3 mg / l, more preferably between 0.02 mg / l and 3 mg / l, even more preferably between 0.01 mg / l and 0.5 mg /L. Milk derivative obtained from the processing of milk according to claim 7 or 8 wherein said derivative is ricotta, fresh cheese, mature cheese or yogurt (step (d)). 10. Food product obtained from processing milk and / or a derivative thereof according to any one of claims 7 to 9 with food grade ingredients. 11. Use of milk and / or a derivative thereof and / or a food product according to any one of claims 7 to 10 as a food for the control and / or reduction of blood cholesterol.