Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
  • A23C1/00—Concentration, evaporation or drying
  • A23C1/06—Concentration by freezing out the water
  • A23C1/08—Freeze-drying
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/20—Dietetic milk products not covered by groups A23C9/12 - A23C9/18
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/17—Amino acids, peptides or proteins
  • A23L33/19—Dairy proteins
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23B—PRESERVATION OF FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES; CHEMICAL RIPENING OF FRUIT OR VEGETABLES
  • A23B11/00—Preservation of milk or dairy products
  • A23B11/10—Preservation of milk or milk preparations
  • A23B11/16—Preservation of milk or milk preparations by irradiation, e.g. by microwaves
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
  • A23C2210/00—Physical treatment of dairy products
  • A23C2210/15—High pressure treatment
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING OR TREATMENT THEREOF
  • A23C9/00—Milk preparations; Milk powder or milk powder preparations
  • A23C9/14—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment
  • A23C9/142—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration
  • A23C9/1422—Milk preparations; Milk powder or milk powder preparations in which the chemical composition of the milk is modified by non-chemical treatment by dialysis, reverse osmosis or ultrafiltration by ultrafiltration, microfiltration or diafiltration of milk, e.g. for separating protein and lactose; Treatment of the UF permeate
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

• the present invention relates to the treatment and prevention of asthma and allergic diseases.
• LPS bacterial lipopolysaccharide
• bovine milk like for example goat's milk, sheep's milk, soy milk, rice milk and almond milk.
• bovine milk like for example goat's milk, sheep's milk, soy milk, rice milk and almond milk.
• These sorts of milk are substitutes for people afflicted with allergies to whey proteins, as the whey proteins are not present in these milks.
• a dehydrated raw milk preparation has an effect on the prevalence of asthma and allergic diseases. This effect is obtained with a raw milk preparation wherein the whey proteins are undenatured, i.e. have not been subjected to a heat treatment. It could be shown that regular consumption of a raw milk preparation in dehydrated form comprising mainly undenatured whey proteins is inversely associated with asthma, hay fever and atopy, atopic dermatitis and food allergen sensitisation. Early exposure and daily consumption showed a stronger inverse association. More surprisingly, it was found that the microbial load of the raw milk preparation was not related to the clinical health outcome.
• the inventors could identify part of the farming lifestyle, which is responsible for the low prevalence of asthma and allergic diseases in infants and children, and they provided a preparation for use for treating and preventing asthma and allergic diseases. Furthermore, it was found that there is a correlation between raw milk consumption and the upregulation of gene expression of innate immunity receptors. Toll-like receptors 7 and 8 that are part of the innate immune system were expressed in significantly higher percentage in children that had received unboiled farm milk in their first year of life compared to controls that had received no raw milk. Therefore, it is the gist of the present invention to use a raw milk preparation in dehydrated form or an untreated raw milk preparation, particularly a raw milk preparation as defined below, for treating or preventing asthma and allergic diseases.
• the raw milk preparation is used for treating or preventing asthma.
• the raw milk preparation is particularly effective in infants and children in an age where the immune system is in development.
• the raw milk preparation according to the invention is used for treatment or prevention of asthma or allergic diseases in infants and children until completion of the 3 rd year of age.
• asthma and allergic diseases also occur in other mammalian species besides human beings, it is also contemplated to use the raw milk preparation of the present invention for the prevention or treatment of asthma and allergic diseases in mammals.
• mammals can be, without being limited to the explicitly mentioned examples, equines, felines and canines, such as horses, cats and dogs.
• the raw milk preparation of the present invention as defined and described below and in the claims can be used with advantage already before the infant is born, and, thus, the present invention provides also a raw milk preparation for use for treatment and particularly prevention of asthma or allergic diseases in infants by administration of untreated or dehydrated raw milk to a female human being, who is pregnant, nursing or preparing for a planned pregnancy.
• an untreated or dehydrated milk preparation as defined and described below is used as a nutritional supplement for a female human being during pregnancy, as a preparation for a planned pregnancy and/or during nursing of the infant or for an infant or child after birth. It has been found that administering the untreated or dehydrated raw milk preparation as defined below in the first 3, preferably at least 6 months and more preferably at least until completion of the 1 st year, more preferably until completion of the 3 rd year, has a beneficial effect for the health of the infant or child.
• raw milk when used in this specification can refer to either native raw milk (raw whole milk), or a fraction of raw milk that comprises at least a whey and fat fraction (whey-fat-preparation).
• the "raw milk preparation” of the present invention as defined below contains heat-sensitive proteins in fully or mainly native, undenatured form.
• untreated is used for a raw milk preparation that has not been subjected to any treatment that causes damage to the heat-labile milk ingredients such as the heat-labile proteins in the whey.
• a "raw milk preparation” being untreated means that the raw milk preparation of the present invention has not been subjected to heat treatment and/or mechanical treatment steps that are usually used in dairy to decontaminate, standardize or sterilize the milk and adapt the fat content.
• mechanical treatment shall refer to methods like homogenisation, which introduce mechanical strain, and thereby destroy the structure of milk components. Methods that do not or hardly modify the structure of milk components, in particular the structure of proteins or the fat globules, like filtration methods, are not comprised by the term “mechanical treatment”.
• Heat treatment method shall refer to any heating step wherein milk is heated to temperatures above 55°C for at least 20 seconds at atmospheric pressure.
• raw milk preparation in dehydrated form when used in this specification refers to a raw milk preparation as defined above which has been dehydrated using mild conditions such that the heat sensitive proteins remain in native, undenatured form.
• a preferred form of a dehydrated raw milk preparation is a freeze-dried raw milk preparation.
• Milk as used in the present description can be milk from any mammal, such as cattle, sheep or goat. Preferably the milk is bovine milk.
• whey proteins in the context of the present invention refers to those proteins that are present in the whey, i.e. are found in the aqueous phase of milk after precipitation of the casein fraction.
• protein as used in the present description shall comprise proteins and peptides.
• “Denaturation” is a process in which proteins lose at least their quaternary structure, or also their tertiary structure and includes processes which lead up to the loss of the secondary structure by application of some external stress or of a compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent, or by heat.
• Native stress as used above, which leads to denaturation, can be exerted by other means than mechanical stress or treatment.
• Denatured proteins in the present description are proteins that have been damaged by external stress, i.e. have lost at least part of their native structure.
• An undenatured protein in the context of the present invention displays a substantially undamaged structure and has an equivalent effect as the natural protein.
• the state of denaturation of a protein can be determined with methods known in the art such as antibody assays, electrophoretic, chromatographic or enzymatic methods.
• non-equivalent refers to a raw milk preparation that is equivalent in its effect in the treatment and prevention of asthma and allergic diseases.
• “native-equivalent raw milk preparation” shall refer to a raw milk preparation wherein the active principle, i.e. the heat-sensitive proteins are in primarily undenatured form. Primarily undenatured means that at least about 75%, preferably at least about 80%, more preferably at least about 90%, even more preferred at least about 95% and ideally at least 98% of the whey proteins are in undamaged native form.
• “Native-equivalent” or “undenatured” when used for dehydrated milk proteins refers to the state of the proteins at least after rehydration, that means that a raw milk preparation in dehydrated form of the present invention comprises at least the whey proteins after rehydration in a form that is equivalent in its effect to the native whey proteins.
• raw milk preparation when used in the following shall comprise untreated raw milk preparation, dehydrated milk preparation, and/or native-equivalent raw milk preparation, in other words any raw milk preparation having a high amount of undenatured protein.
• a significant distinction between treated milk and the raw milk preparation of the present invention is the amount of denatured whey proteins comprised in the milk and the amount of active enzymes present in the milk. Decrease of enzyme activity, particularly of lactoperoxidase and/or alkaline phosphatase activity, can be detected via methods known to the person skilled in the art.
• the level of denaturation can be assessed by a person skilled in the art by comparing the level of undenatured protein in the raw milk preparation with the level of undenatured protein in a milk preparation after a denaturing treatment.
• the distinction between denatured and undenatured protein can be assured by using, for example, an antibody that only binds to the undenatured protein displaying its native secondary and tertiary structure and/or by standardized HPLC (High pressure/performance liquid chromatography) methods.
• HPLC High pressure/performance liquid chromatography
• the existence of a native quaternary structure of proteins can be determined using methods for directly or indirectly measuring mass and/or size of the protein complex. Those methods are known to the person skilled in the art and comprise, for example, mass spectrometric immunoassay and size-exclusion chromatography.
• the denaturation process can be in part a proteolytic process.
• the resulting degradation products can be detected via methods known to the person skilled in the art.
• a raw milk preparation of the present invention contains at least about 75%, preferably at least about 80, more preferably at least about 90%, even more preferred at least about 95 and ideally at least 98 % of the whey proteins in undamaged form.
• the percentage of damage can be determined with well-known methods as disclosed above.
• “Asthma” is a common chronic disorder of the airways that is complex and characterized by variable and recurring symptoms, airflow obstruction, bronchial hyper-responsiveness, and an underlying inflammation.
• asthma Its prevalence is increasing, especially among children. Asthma comprises current and/or chronic asthma.
• Two major environmental factors have emerged as the most important in the development, persistence, and possibly severity of asthma: airborne allergens and viral respiratory infections. Allergens cause allergies. Therefore it is beneficial for the treatment or prevention of asthma to include the treatment of allergic diseases as well.
• Allergic diseases or "allergy” are used interchangeably throughout the description of this invention. Allergy is a hypersensitivity disorder of the immune system. Allergies are the result of inappropriate immune responses to normally harmless substances. Allergy symptoms vary widely, from sneezing, watery eyes, and nasal congestion of mild "hay fever” to severe rashes, swelling, and shock. Allergic diseases include without being limited to hay fever, atopy and atopic dermatitis.
• Figure 1 shows the GABRIELA (an advanced multidisciplinary study to identify the genetic and environmental causes of asthma in the European Community) study population and design.
• Figure 2 shows the milk exposure of farmers and non-farmers in Phase II and III.
• Figure 3 shows the adjusted association of reported milk exposure and asthma, atopy, hay fever and atopic dermatitis (Phase II).
• Figure 4 shows levels and percentage of detectables of all milk constituents stratified by milk type and milk's heating status.
• Figure 5 shows the adjusted association of asthma or atopy and milk's heating status, total fat content, total viable bacterial count, or whey protein levels (Phase III).
• the present invention is concerned with a raw milk preparation for use in the treatment of conditions and diseases that are caused by inappropriate immune reactions.
• a raw milk preparation particularly in dehydrated form, can be used for treating or preventing asthma, and allergic diseases, in particular in infants or children.
• an untreated raw milk preparation can be used for preventing asthma by administering a raw milk preparation to a female human during pregnancy, as a preparation for a planned pregnancy and/or during nursing of the infant.
• the inventors of the present invention found that the amount of undenatured whey protein present in a raw milk preparation according to the present invention is inversely associated with asthma, hay fever, atopy, atopic dermatitis and food allergen sensitisation. More surprisingly, it was found that the microbial load of the raw milk preparation was not related to the clinical health outcome. The beneficial effect is obtained only if an untreated or dehydrated raw milk preparation is used whereas the beneficial effect decreases or disappears when a thermally treated milk preparation or parts thereof are used. Furthermore, it has been found that the fat fraction of the raw milk preparation can also contribute to the beneficial effect.
• the present invention provides a raw milk preparation in dehydrated form which comprises at least the whey fraction and the fat or cream fraction of the milk and is preferably the dehydrated form of the whole raw milk.
• the raw milk preparation of the present invention whether it is in untreated or dehydrated form comprises at least heat-sensitive or heat-labile whey proteins and the fat or cream fraction of the milk it has a beneficial effect on asthma and allergic diseases.
• the raw milk preparation that is used according to the present invention comprises the heat-sensitive whey proteins of raw milk, which are present in the whey fraction of raw milk.
• Those milk ingredients, which are heat-labile, such as the proteins of the raw milk that are found in whey are damaged by thermal and/or other external stress. It is of importance that those milk ingredients are present in native and undamaged form in the raw milk preparation used according to the present invention.
• the raw milk preparation as used according to the present invention can be referred to as native-equivalent, i.e. has an effect primarily as or ideally identically to raw milk as obtained directly from the animal. It was found that the advantageous results are obtained with a preparation wherein whey fraction and fat fraction are present, as in a whole raw milk. It is contemplated that the fat globules have a role in the treatment and the structure of these globules is maintained only if no detrimental mechanical steps are applied. The globules are destroyed by mechanical stress as is applied by homogenisation.
• the raw milk preparation of the present invention is in particular a raw milk preparation that has not been subjected to homogenisation, and has not been heated above 55° C for longer than 20 seconds at atmospheric pressure.
• a milk preparation that has been treated by heating and/or has been subjected to external stress as defined above exerted by other than thermal methods is not useful for the treatment envisaged by the present invention, as those methods lead to damage of the proteins. Thus the application of such steps shall be excluded when preparing the preparation of the present invention.
• Filtration methods like microfiltration or ultrafiltration, which are used to remove coarse particles, bacterial load and/or precipitated matter mainly do not harm the heat-labile proteins or the fat globules and can be used.
• the amount of heat-labile proteins can be determined with methods well-known in the art. Proteins that are denatured at temperatures beyond 55°C can be identified by analysing raw milk preparations and/or whey before and after heat treatment with enzymatic, electrophoretic, chromatographic or immunological methods.
• untreated when used regarding raw milk means that the milk has not been subjected to thermal or mechanical treatments that lead to damage in the protein structure as outlined above, but can have been subjected to filtration, in particular microfiltration and ultrafiltration or separation of a whey-fat-fraction provided that methods and conditions are controlled to not or hardly cause damage to the proteins or fat globules contained in the preparation.
• the raw milk preparation comprising at least the whey-fat-fraction shall be native-equivalent to raw milk.
• the beneficial effect when using the raw milk preparation of the present invention depends on the amount and frequency taken.
• the dosage depends from the preparation, from the initial composition of the milk and the composition of the preparation after processing.
• the heat-labile whey proteins possibly in combination with the fat globules are responsible for the beneficial effect of the preparation of the present invention the dosage shall be determined based on the protein content.
• a daily dosage of about 125 ml to about 250 ml of native raw milk is useful for the treatment or prevention of asthma or allergic conditions particularly in children or infants. Therefore, an amount of a raw milk preparation in dehydrated form that comprises the same amount of proteins as the raw milk preparation it has been obtained from, or in particular of whey proteins that are contained in about 125 ml to about 250 ml of raw milk or whey is a useful daily dosage for the treatment or prevention of asthma or allergic conditions.
• a daily dosage of the dehydrated raw milk preparation that corresponds in whey protein content with about 125 to about 250 ml whey is suitable. The determination of the protein content is known to the skilled person and standard methods and standard kits are available for this determination.
• the raw milk preparation of the present invention comprises 3 - 4 % proteins, of which about 20% are whey proteins. It has been found that a daily dosage of about 125 ml to about 250 ml of native raw milk or a corresponding dosage of a raw milk preparation as defined in the present application, or an amount of a freeze-dried dried powder that comprises the same amount of proteins is useful for the treatment or prevention of asthma or allergic conditions. It has been found that the dosage in the above indicated range provides enough whey proteins in undamaged form to obtain the desirable results.
• a raw milk preparation of the present invention i.e. a raw milk preparation in dehydrated form or an untreated raw milk preparation, is used for regular consumption, i.e. oral uptake, preferably for daily consumption.
• the raw milk preparation in dehydrated form as well as the untreated raw milk preparation therefore, are preferably consumed at least once a day and can be consumed more times a day, for example at every meal.
• the immune system is "trained" to differentiate between heterologue and homologue in the infant's first years. Therefore, it is preferred to regularly use the raw milk preparation in dehydrated form of the present invention for treatment of an infant or child from birth within at least 1 month, or at least three months, preferably at least until completion of the 1 st , even better of the 3 rd year of age. It has been found that regular consumption of the raw milk preparation of the present invention until the age of three could decrease substantially the susceptibility for asthma and/or allergic conditions in children.
• a raw milk preparation of the present invention i.e. a dehydrated or untreated preparation
• the raw milk preparation is used for treatment of a female human being during pregnancy, during nursing of the infant and/or in preparation for a planned pregnancy, as it has been shown, that it is beneficial to the health of the child, if the mother has consumed the raw milk preparation of the present invention during pregnancy or even before.
• regular intake of a raw milk preparation of the present invention is beneficial to the condition of human beings, in particular of pregnant females and children.
• an untreated raw milk preparation or a raw milk preparation in dehydrated form is used as nutritional supplement for a female adult during pregnancy, in preparation for a planned pregnancy and/or during nursing of an infant or as nutritional supplement for an infant or child in the first time of its life, such as within the first months after birth or from birth until completion of the 1 st , or preferably until the 3 rd year of age.
• the raw milk preparation of the present invention is derived from native raw milk and can be a preparation based on native raw milk or based on at least the whey and fat fraction of raw milk as defined above and it is either the untreated preparation or the preparation in dehydrated form.
• the preparation can be prepared by methods known in the art. In particular any method that does not damage the heat-labile milk ingredients, such as the proteins of the whey fraction, can be used.
• another embodiment of the present invention is a method of producing a raw milk preparation for use according to the present invention, wherein the method comprises removal of undesired parts from raw milk obtained by milking an animal without damaging heat-labile milk ingredients, such as the proteins in the whey fraction.
• the method comprises filtration of raw milk obtained from an animal, such that a substantial amount of the whey protein fraction, such as more than about 75%, remains undenatured and wherein any heating of the milk above 55°C for longer than 20 seconds is excluded, thereby obtaining a native- equivalent or untreated raw milk preparation.
• a method for producing a raw milk preparation in dehydrated form comprises providing a raw milk, optionally fractionating the filtered raw milk, and dehydration of the raw milk or a raw milk fraction which comprises at least the whey and fat fraction of native raw milk.
• a method for producing a raw milk preparation in dehydrated form comprises at least removal of undesired parts of raw milk, optionally fractionating the filtered raw milk, and dehydration of the raw milk or a raw milk fraction which comprises at least the whey and fat fraction of native raw milk.
• Milk and whey are complex systems comprising a multiplicity of components in addition to whey proteins although mostly in minute amounts.
• Milk is an emulsion or colloid of butterfat globules within a water-based fluid. Each fat globule is surrounded by a membrane consisting of phospholipids and proteins; these emulsifiers keep the individual globules from joining together into noticeable grains of butterfat and also protect the globules from the fat-digesting activity of enzymes found in the fluid portion of the milk.
• Milk contains more than 40 proteins. Milk composition changes based on the origin of the milk, the feed of the milk animal, the race of the animal, the time in the year, and further conditions. In general cow's milk contains approximately 30 to 35 g/L (3-3.5%) of cow's milk proteins (CMPs), which can be divided into 2 main classes: Caseins (about 80%) and Whey proteins (about 20%). It has been found that the proteins, and in particular the heat-labile proteins, that are present in the whey in undamaged form are essential for the use for preventing and/or treating asthma and allergic conditions.
• CMPs cow's milk proteins
• the raw milk preparation of the present invention at least comprises the whey and fat fraction of raw milk in undenatured form and preferably is native raw milk in dehydrated form.
• Whey proteins can be separated from milk caseins by precipitation using proteases like chymosin or by acidification of the milk to pH 4.6, and thereby forming the coagulum (curd).
• the whey proteins remain soluble in the milk serum.
• the raw milk preparation of the present invention comprises at least the milk serum.
• ⁇ -lactoglobulin is the most abundant protein in whey, accounting for 50% of total protein in the whey fraction. It has no homologous counterpart in human milk.
• a-lactalbumin is a monomeric globular calcium-binding protein representing about 25% of whey proteins. It is a regulatory component of the enzymatic system of galactosyl transferase responsible in mammary secretory cells for the synthesis of lactose.
• Bovine serum albumin accounts for around 5% of the total whey proteins. BSA is physically and immunologically very similar to human blood serum albumin. Its main role is the transport, metabolism and distribution of ligands and the protection from free radicals. Lactoferrin is a protein of mammary origin and is a milk-specific iron-binding glycoprotein of the transferrin family. It can be found in the milk of most species at levels lower than 1 %. Lactoferrin is known to be an immunomodulatory substance. Although it is present in very low concentrations in cow's milk, it has been shown to be a strong milk allergen.
• the Immunoglobulin (Ig) fraction which includes IgG and IgE, accounts for about 1 % of total milk protein and 6% of whey protein. Data on the potential allergenicity of bovine immunoglobulins are very limited. However, some studies propose IgG as another milk allergen due to the observation that IgE from cow's milk allergy patients specifically binds bovine IgG.
• the proteose-peptone fraction represents about 1 .1 % of the total milk protein. It is a heat-stable and acid-soluble protein fraction of milk with important functional properties. This milk component is derived mainly from the proteolysis of D-casein, and the enzymatic activity of plasmin can over time increase its concentration in milk. Methods of milk processing
• Milk is usually processed before it is available for sale to consumers. Usually such processes involve heat-treatment and homogenisation of milk, both treatment steps that change components in the milk.
• One of the main goals of conventional heat- treatment of milk is to inactivate any pathogenic microorganisms while at the same time extending its shelf-life by reducing the microbial loads of spoilage bacteria as well as enzymatic activity.
• a disadvantage of the heat treatment is degradation of the product's quality attributes and nutritional properties due to the intense heat treatment and subsequent destruction of proteins and vitamins, including undesirable changes to other characteristics such as flavour and colour.
• the commonly used processes of heat treatment are not suitable for the raw milk preparation of the present invention because the most valuable part of the preparation is damaged thereby.
• Any alternative method can be used as long as the heat- labile milk ingredients, such as the whey proteins, and their structure are not or hardly changed or altered.
• Microfiltration is widely used for the removal of microorganisms and somatic cells. It is particularly adapted to the removal of bacteria from skimmed milk, as the size of the microorganisms is in the same range as fat globules.
• membrane equipment has been adopted throughout the dairy processing chain, including milk reception, cheese-making, whey protein concentration, fractionation of protein and effluent treatment.
• 40% of the food applications of membrane processing are developed in the dairy industry, with applications and equipment serving as references for the industries treating other food liquids.
• Skimmed milk microfiltration makes it possible to decrease the microbial load of milk while maintaining the organoleptic quality of milk due to low heat treatment (35-55°C).
• the decimal reduction of bacteria ranged from 2-3 log with first ceramic membranes and reaches 3-4 log with the currently used 1 .4 ⁇ membranes. This means that microfiltered milk contains between 10 and 50 cfu/ml.
• the observed decimal reduction in spore counts is high: retention from 99.1 to 99.99% (2-4 log) for both aerobic and anaerobic spores or even higher reductions for specific spores such as Bacillus cereus (> 4.5 log).
• Decimal reduction of pathogenic bacteria Listeria monocytogenes, Brucella abortus, Salmonella typhimurium and Mycobacterium tuberculosis
• the raw milk obtained after the microfiltration step can be used as untreated raw milk preparation of the present invention as the heat-labile milk ingredients are not altered by this step and, thus, this preparation is native equivalent.
• a raw milk obtained after the microfiltration step is subjected to a dehydration process.
• certified raw milk can be subjected directly to the dehydration process.
• Dehydration processes are well-known in the art and the skilled artisan can find the optimal process conditions to be used in a specific case.
• a suitable method is gentle and not or hardly damaging to the ingredients of the raw milk.
• a preferred method is freeze drying.
• milk powder is produced by using spray drying steps which are suitable to obtain dry milk products. However, spray drying processes cannot be used for preparing the preparations of the present invention.
• Freeze drying methods are known in the art and are commonly used for dehydrating sensitive or heat-labile materials. For example it is well-known to use freeze drying methods in the pharmaceutical field and those methods are also useful for obtaining the dehydrated raw milk preparation of the present invention.
• Freeze drying methods comprise at least two steps: a) cooling the material below its triple point and b) drying or removing water respectively; and can optionally comprise as step c) secondary drying.
• a specific form of freeze drying is lyophilization which is also suitable as dehydrating process for obtaining the preparation of the present invention.
• the cooling or freezing step can be carried out by using standard devices like mechanical refrigerators, dry ice, liquid nitrogen or other means.
• freeze drying is done in a freeze drying machine.
• temperature cycling can also be used.
• the raw milk is rapidly frozen below its triple point or eutectic point.
• the freezing step is succeeded by a primary drying step.
• the pressure is lowered and enough heat is supplied to the material for the water to sublime.
• the amount of heat necessary can be calculated using the sublimating molecules' latent heat of sublimation.
• Pressure is lowered usually by application of a partial vacuum and is controlled during the process.
• the sublimated water is condensed in a condensator and is withdrawn continuously. If necessary a third step can be carried out for secondary drying wherein the temperature is raised such that residual water can sublime and be withdrawn.
• raw milk By using the freeze drying method raw milk, optionally after filtration, can be obtained in dehydrated form. By using mild conditions the components of the raw milk remain nearly undamaged such that the obtained product is native-equivalent.
• the raw milk preparation in dehydrated form can be stored for some time and it can be administered in this form or can be rehydrated and then be administered.
• High Pressure (HP) processing of food products generally involves the treatment of products at a pressure in the range 100- 1000 MPa.
• HP-induced changes in milk fat have not been widely studied so far. It seems, however, that milk fat globules appear to remain intact under pressure, with no substantial changes in globule size being observed.
• Increased solubility of salts under pressure is highlighted particularly by the increased solubility of micellar calcium phosphate. Structural modifications of proteins under pressure have been extensively studied. Under pressure, casein micelles are disrupted, probably resulting from solubilisation of micellar calcium phosphate.
• whey proteins The globular structure of whey proteins makes them particularly susceptible to pressure-induced denaturation.
• ⁇ -lactoglobulin is the least stable to pressure and tends to unfold at pressures > 100 MPa whereas alpha-lactalbumin is considerably more stable with denaturation observed at pressure > 400 MPa.
• the reactive free sulphydryl-group of ⁇ -lactoglobulin becomes exposed and can subsequently undergo irreversible sulphydryl-disulphide interchange reactions with other surrounding molecules.
• immunoglobulins that are particularly sensitive to heat-denaturation are considerably more stable to HP processing, with c.a. 90% of colostral IgG surviving treatment at 500 MPa for 5 min.
• Pulsed Electric Field is another technique that consists of applying very short pulses (micro to milliseconds) at an electric field intensity of 10-80 kV/cm, applied to a food product held between two electrodes inside a chamber, usually at room temperature. Bacteria inactivation is achieved by changing the cell membrane permeability and is called electroporation, and is either reversible (cell membrane discharge) or irreversible (cell membrane breakdown or lysis). Depending on the experimental conditions and the microbial target selected, PEF has been shown to considerably reduce bacteria content of milk with reduction of up to 5 logs or even a total disappearance of cultivable bacteria.
• Irradiation is related to the propagation of energy from the electromagnetic spectrum. Radio and television waves, microwaves, and ultraviolet and gamma rays are some of the examples of radiation included in the spectrum.
• Radio and television waves, microwaves, and ultraviolet and gamma rays are some of the examples of radiation included in the spectrum.
• a cascade of secondary electrons with enough kinetic energy generates ionisation of atoms and molecules and formation of free radicals.
• chemical species are formed from the radiolysis of water. Irradiation can change some of the chemical and physical properties of milk, a result of denaturation, degradation, polymerisation, and rearrangement of proteins, vitamins and carotenoids, as well as hydroperoxide production.
• results showed that solubility of proteins was reduced after irradiation treatment, which could be a result of crosslinking of protein chains; however, ⁇ -lactoglobulin antigenicity was not reduced by the treatment.
• Micronutrients such as minerals are not altered in a significant way in irradiated food or can even be more available.
• irradiation can be considered as long as conditions and wave lengths are used that do not or hardly alter the structure of the whey proteins and fat globules. Any change of the structure can be determined by methods known in the art, such as methods as outlined above.
• Ohmic heating consists in the passage of an alternating electrical current through a food sample (one that has electrical conductivity), wherein the electrical resistance of the food material generates heat.
• the heat instantly generated inside the food is proportional to the square of the current induced, the electrical conductivity, and the type of food being heated. Because the generation of heat and its distribution throughout the material are unusually fast, the food retains its flavour and particulate integrity better than when using conventional thermal treatment.
• this processing step can be considered as long as conditions and heating time are such that the structure of the whey proteins and fat globules is not altered. Any change of the structure can be determined by methods known in the art, such as methods as outlined above. For the present invention only those processing steps that do not denature proteins, in particular heat-labile proteins, can be used and those steps have to be differentiated from those steps that damage proteins and, therefore, cannot be used.
• Homogenisation is a mechanical method, wherein the milk is forced through small holes at high pressure. This breaks up the fat globules in order to spread them evenly throughout the milk and prevent separation of a cream layer. This process basically results in milk of uniform composition or consistency and palatability without removing or adding any constituents. Homogenisation increases the whiteness of milk because the greater numbers of fat globules scatter the light more effectively. Homogenized milk is not within the definition of raw milk and cannot be used in the present invention.
• a filtration process is a gentle mechanical step that can be used to obtain the raw milk preparation of the present invention.
• milk or milk components are forced through ceramic microfilters, for example, to remove microorganisms.
• this processing does not or hardly modify the milk and the proteins respectively, and, therefore, can be used.
• the basic process for pasteurisation of whole milk involves heating the milk to a temperature of no less than 71 .7°C for a minimum of 15 seconds (max 25 seconds). This process is known as High Temperature Short Time (HTST) or flash pasteurization. Following heating, the milk is cooled rapidly to below 6°C using chilled water on the opposite side of the plate. This process also extends the keeping quality of the milk.
• HTST High Temperature Short Time
• Sterilisation is a more severe form of heat treatment, which destroys nearly all the microorganisms present, including the vegetative cells and endospores.
• the milk is pre-heated to around 50 C, then homogenised, after which it is poured into glass bottles which are closed with an airtight seal.
• a gentle method of sterilisation can be achieved by direct sterilisation via steam injection, which is mainly used for sensitive products which have to be treated with short heating-holding times and high temperatures, or by indirect sterilisation, wherein the heat is transferred via heat exchangers.
• the indirect sterilisation is a gentle method, as the product to be sterilized does not come into direct contact with the heating medium, which can be steam, hot water or electric heat. Nevertheless, it can alter the structure of heat-labile milk ingredients, such as the whey proteins, and, therefore, is not suitable as processing step for preparing a raw milk preparation of the present invention.
• the heating medium which can be steam, hot water or electric heat.
• heat-labile milk ingredients such as the whey proteins
• the sterilisation process results in a change of taste and colour and also slightly reduces the nutritional value of the milk, particularly the B group vitamins and vitamin C, depending on the specific sterilization technology applied.
• UHT or ultra-heat treated milk is a form of milk that has been heated to a temperature of at least 135°C for a very short time period in order to kill any harmful microorganisms including endospores, which may be present in the milk.
• the milk is then packaged into sterile containers.
• UHT milks have a longer shelf life as a result of the higher temperatures to which they are heated and the packaging used to store them.
• raw milk preparation of the present invention In contrast to the above mentioned commonly used methods of heat treatment and homogenisation, an alternative approach is used for the raw milk preparation of the present invention. In other words, methods that do not harm the proteins and fat globules are used.
• raw milk is used that has been obtained by avoiding microbial contamination.
• Raw milk of high quality and low microbial load can be obtained, as is well-known in the art, by conforming to the safety guidelines for primary and secondary food production and consumption without conducting thermal treatment of the milk by otherwise ensuring that the milk does not contain a microorganism load which exceeds safety levels for food consumption.
• a preferred form of raw milk that is used for the present invention is so-called certified raw milk, which is raw milk that has been treated only by filtration.
• Certified raw milk is obtained from cows certified as healthy. It is unpasteurized milk with a bacteria count below a specified standard.
• Certified raw milk can be obtained from suppliers known to the person skilled in the art. In Germany for example, certified raw milk (Vorzugsmilch) can be obtained from approximately 80 dairy farms listed by the Bundes vein der Vorzugsmilchermaschinemaschine und shortcutver proer von Cincinnati & Milch arean. Similar organisations can be found world wide. A listing can be found on http://www.realmilk.com/where-other.html.
• the present invention is further outlined and explained with reference to the examples that follow without being restricted to the embodiments outlined therein. In particular, the beneficial effects that can be obtained by using the preparation of the present invention regularly are demonstrated.
• Example 1 Example 1
• Serum immunoglobulin E levels against inhalant and food allergens were measured by fluorescence immunoassay.
• Atopy was defined as positive test results for specific IgE antibodies against D. pteronyssinus, cat, birch (cut-off 0.7 kU/L), or against a grass mix (cut-off 0.35 kU/L).
• Food allergy was defined as a positive fx5 test (fish, cow's milk, hen's egg, peanut, soybean, and wheat flour).
• ISAAC ISAAC The Lancet 1998; 351 :1225-312.
• Childhood asthma was defined as either wheeze in the past 12 months, asthma inhaler use ever, or a doctor's diagnosis of asthma at least once or whez bronchitis more than once.
• Current asthma was defined as childhood asthma and wheeze in the past 12 months.
• Hay fever required occurrence of nasal symptoms with itchy or watery eyes in the past 12 months or a doctor's diagnosis of hay fever ever.
• Atopic dermatitis was defined as a doctor's diagnosis ever.
• Cow milk exposure assessed by questionnaire The phase II comprehensive questionnaire gave information about the child's farm related exposures. Cow milk consumption was determined by asking whether the child consumed milk purchased at a shop (shop milk) or directly from a farm (farm milk) and whether farm milk was boiled or skimmed. Heating status of shop milk was not assessed. The parents had to indicate the life period of milk exposure from pregnancy to school age and the corresponding amounts of milk consumption.
• phase III trained field workers collected cow's milk that was consumed at the participants' homes on the day of the field visit. Parents were instructed to prepare the milk as they usually did and filled out standardized milk documentation sheets. All samples were analyzed by lab staff blinded to the milk type and the health and exposure status.
• Heating status of milk samples was defined by residual activity of the milk indigenous enzymes alkaline phosphatase (ALP) and lactoperoxidase (LPO) according to EC council directive 92/46/EC.
• ALP alkaline phosphatase
• LPO lactoperoxidase
• the measurements and the milk type allowed to categorize the samples as i) high heat treated shop milk (at least 85°C), ii) pasteurized shop milk (not heated over 85°C), iii) heated farm milk (at least 72°C), and iv) raw farm milk (not heated above 72°C). As 85% of the heat treated farm milk samples were heated above 85°C, all heated farm milk samples were combined for analysis. The total fat content and whey protein levels were determined for all available phase III samples.
• the total viable bacterial count (TVC) was assessed in all 800 milk samples and 222 samples were selected for advanced microbiological analyses using stratified random sampling (strata based on milk type, heating status, and fat content).
• the following microbiological groups were determined by selective plate count methods: Pseudomonades, Enterobacteriaceae, Micrococci plus Staphylococci, Lactobacilli, Yeast plus Mold, Bacilli plus endospores, psych rotrophic bacteria, and human pathogens.
• phase III data were used to explore associations between objectively assessed heating status of milk or measured milk components and asthma and atopy. These regression models were adjusted for the same set of confounders as the phase II data.
• Milk type and heating status were categorized into four groups with high heated shop milk as the reference category. To take into account the distribution of milk constituents with high proportions of non-detects (total viable bacterial count, lactoferrin, total IgG and bovine serum albumin), samples within the detection range were split at the median representing low and high levels while non-detects were used as reference group.
• Milk constituents that were measurable in all samples were divided into tertiles with the lowest tertile as a reference group to test for linearity of the association with health outcomes, ⁇ -lactalbumin and ⁇ -lactoglobulin were subsequently entered as continuous variables into the regression models.
• a factor analysis with continuous variables and varimax rotation extraction of eigenvalues of 1 .5 or higher was used to evaluate whether the different milk constituents could be separated into different factors.
• Results from weighted logistic regression models were expressed as adjusted odds ratios (aOR) with corresponding 95% confidence intervals (95%-CI).
• Figure 6 gives the results of the advanced microbiological analyses. In few shop milk and heated farm milk samples microorganisms could be detected (below 15% for all groups except micrococci and staphylococci (25%)). In many raw farm milk samples, micrococci and staphylococci (85.2%), lactobacilli (94.1 %), bacilli and bacterial endospores (63.4%), and psych rotrophic bacteria (58.4%) could be detected. Pathogenic Listeria innocua and Listeria ivanovii strains were found in only three unboiled farm milk samples.
• PASTURE is a large prospective birth cohort study conducted in rural areas in Austria, Finland, France, Germany and Switzerland.
• the study team contacted 2871 women of which 1772 (61 .7%) were identified as eligible for participation (Figure 1 ).
• Potential participating families were contacted in the third trimester of pregnancy. Exclusion criteria were living on a farm without livestock, maternal age below 18 years, premature delivery, genetic disease in the offspring, no telephone connection and insufficient knowledge of the country's language. Women living on a farm where livestock was held or whose partners actively run a farm were considered farming women. A subset of the population living in close neighborhood in likewise rural environments not occupationally involved in farming activities were selected as controls.
• Extensive questionnaires were administered by interview to the mother of the child within the third trimester of pregnancy and two and twelve months after birth of the study child. Questions were based on previously published studies 17"20 and designed to assess respiratory and other health issues of the mother, agricultural exposures and potential confounders such as active and passive smoking, parental education and family size. In addition to the extensive questionnaires the mothers kept a weekly diary from month 3 to year 1 of the child's life to record among others the introduction of a variety of food items. Relevant pregnancy variables were farming (living on a farm vs.
• maternal farm work mother working on a farm during pregnancy
• contact to stable/barn stay in stable/barn during pregnancy at least 15 min per week in one trimester
• contact to number of livestock horse, cow, pig, poultry: 0, 1 -2 or 3- 4
• maternal history of asthma or hay fever did not affect smoking during pregnancy (in any trimester) and farm milk consumption during pregnancy (never, only boiled farm milk or any unboiled farm milk).
• Variables during first year were farming (child living on a farm during first year of life), regular visit to farm, regular stay in stable/barn (child stayed in stable/barn at least 15 min per week), smoking during lactation, duration of breastfeeding (never, below or equal to 3 months, 3 to 6 months, more than 6 months) duration of exclusive breastfeeding (never, below or equal to 3 months or more than 3 months) and child's farm milk consumption (never, only boiled farm milk or any unboiled farm milk during year 1 ).
• RNA- stabilizing solution PreAnalytiX/Qiagen
• Quantitative real-time PCR was performed on the 7900HT Fast Real-Time PCR System using the Micro fluidic card TaqMan Array system of Applied Biosystems.
• the data presented are normalized values for the endogenous controls (18S rRNA and B2M) using the comparative (AACt) method according to the manufacturer's instructions (Applied Biosystems).
• TLR3 expression was excluded from the analyses, as the expression level was below the detection limit in most of the cord blood samples.
• the transformed data were used in linear regression models to calculate associations between mRNA expression and single nucleotide polymorphisms in innate immunity genes, exposures in pregnancy and first year of life (expressed as geometric means ratios and p-values).
• a solid food score was developed to test the influence of early introduction of a variety of solid foods on mRNA expression. Time of first food introduction was sub-divided into 4 periods (introduced in month 3-6, 7-9, 10-12 or never in first year of life). Early introduction of a food item was defined as the period when at least 25% of the children received the respective food. The association of early introduction of each solid food item with mRNA expression was then tested and if significant added to the solid food score (the final score included: yogurt, butter, vegetable, fruit, meat, nut, fish, chocolate, cereal with and without gluten).
• Multivariate models were developed as follows. Pregnancy exposures were related to mRNA expression in cord blood and exposures during year 1 were related to expression at year 1 in crude regression models. Maternal history of asthma or hay fever, sex and center were chosen as covariates apriori and were included in all multivariate analyses. Variables significantly associated with mRNA expression of two or more receptor genes or significantly associated with expression of one receptor gene and significantly associated with mRNA expression in analyses of variance were included in a final model (pregnancy model included: maternal smoking during pregnancy and farming; year 1 model included: maternal smoking during breastfeeding, education, the solid food score, and child's farm milk consumption). Heterogeneity between centers was tested by means of meta- analytical techniques. If heterogeneity was present final models were additionally adjusted for center with random effect estimate, if not, center was included as fixed effect.
• the overall association of exposures on mRNA expression was additionally calculated in multivariate analyses of variance (MANOVA, adjusting for the same covariates as the regression models). Level of significance was evaluated based on Wilks' lambda. To assess the development of mRNA expression from birth to year 1 the difference of normalized mRNA expression from cord blood and year one was calculated (diff-mRNA) and regression models were developed accordingly. Finally, interaction terms were included in the final models to test for gene-environment interactions of farm milk consumption and genetic variations of innate immune receptors on the effect on mRNA expression at year 1 . All statistical analyses were performed using STATA/SE 10.1 (STATACorp, College Station, Tex) and p-values ⁇ 0.05 were considered significant.
• TLR5 aMR, 1 .20; 95% CI: 1 .02-1 .41
• p 0.030
• TLR1 , 4, 6 and 8 were significantly associated with gene expression of the respective receptors at birth and similarly at year 1 (see online repository: Table A3-1 and A3-2).
• Maternal involvement in farming during pregnancy was the exposure associated with expression of several innate immunity receptors in cord blood whereas specific activities such as working in stables or barns or contact to livestock were not. Maternal farming might thus be an overall indicator of other activities including consumption of farm milk during pregnancy.
• 34 higher gene expression of innate immunity receptors at school age in farm compared to non-farm children has clearly been observed and might reflect continuous exposure of children growing up on a farm over several years.
• the previously reported association between prenatal farm animal exposure and innate immunity gene expression 12 was, however, not confirmed in this prospective study.
• the gene expression of individual receptors significantly up-regulated by farming varied between studies 3 likely reflecting the different microbial composition of the respective environments. Recent evidence suggests that the diversity of the microbial environment and not individual microbes are important to confer protection from asthma 5 and TLR-mediated innate response pathways are believed to be important in promoting regulatory pathways that inhibit allergic immune responses. 24
• Toll-like receptors and CD14 at birth and at year 1 were not closely correlated suggesting that environmental exposures encountered by the infant induce substantial changes in innate immunity during the first year of life.
• Food is a main source of the infant's new exposure during early age.
• Breastfeeding has recently been shown to modulate innate immunity responses during the neonatal period. 15
• infant's consumption of raw farm milk during the first year of life was the exposure most strongly up-regulating the expression of TLRs, whereas duration of breastfeeding had no significant impact.
• child's contact to stables or animals was not associated with receptor gene expression. Possibly, the intensity of exposure to stable microbes is too low as long as children do not walk around themselves in stables or barns.
• Raw farm milk is rich in (mostly) non-pathogenic microorganisms. 6,25 It is conceivable that the microorganisms ingested with raw milk influence the composition of the gut flora and stimulate expression of innate immunity receptor genes. 26 Whether increased expression of TLRs associated with raw milk consumption reflects a relevant pathway underlying allergic diseases development or whether it is merely an indicator of exposure to microbes remains open. A recent cross-sectional study found whey proteins but not the actual counts of bacteria in farm milk to be associated with less asthma in children. 6 The actual level of microorganisms in farm milk consumed at school age may, however, not be a precise indicator of the child's regular exposure to a diversity of microorganisms in raw milk which may be involved in inducing tolerance and protect from chronic inflammation. 26
• the Toll-like receptor 2 R753Q polymorphism defines a subgroup of patients with atopic dermatitis having severe phenotype. Journal of Allergy and Clinical Immunology 2004; 1 13:565-7.
• Ferris BG Epidemiology Standardization Project. American Review of Respiratory Disease 1978; 1 18:1 -120.

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