Patent Description:
In particular, the invention relates to a method for inactivating and the inactivation testing of xenoantigens in foods of vegetable and animal origin, in particular of the alpha-Gal epitope, in particular for whole cow's milk, for soy milk and for rice milk, and derivatives, through the use of biological activities identified in phenolic compounds, polyphenolic compounds, or derivatives thereof, including phenylpropanoids, hereinafter referred to for the sake of simplicity with the abbreviation FPF.

To date, over <NUM> types of foods have been identified as being capable of causing allergy and/or intolerance.

Of such foods, the most common are milk and its derivatives, soya, and red meat, followed by eggs, peanuts, nuts in general, crustaceans and molluscs.

Today, food allergies affect <NUM>% overall of the children and <NUM>% overall of adults living in Europe, Australasia and the USA. Recently, new cases of allergies have come to light which were triggered by the specific intake of red meat or of milk (cow's or goat's milk), which can develop into a series of events ranging from classic reactions such as hives, gastrointestinal disorders, delayed growth in children and systemic anaphylaxis, up to the possible death of the subject.

Such phenomenon is determined by the presence in the foods in question of a specific molecule called alpha-Gal.

This specific allergic form particularly concerns the US and Australian market, to the point where in those countries special surveillance teams have been set up, named respectively "Alpha-Gal National Surveillance" and "TIARA - Tick Induced Allergies Research and Awareness", dedicated to monitoring the phenomenon and offering health education on it.

<NUM>% of patients admitted for treatment following the onset of food allergies in the USA are in fact affected by the development of a form of intolerance of and/or allergy to the alpha-Gal molecule.

In Europe similar reactions have been found in Denmark (<NUM>% of the population), Spain (<NUM>% of the population), Sweden, Germany, France and Switzerland.

The alpha-Gal molecule is a determining antigenic constituted by two galactose residues bonded to each other by an alpha-glycosidic bond, and it has been found in all mammals except humans and the more advanced primates.

Such molecule is predominantly expressed on glycoproteins and on membrane glycolipids by virtue of the action of an enzyme named alpha-galactosyltransferase.

In its evolution as a consequence of natural mutations, the human race has lost the functionality of this enzyme, by developing antibodies from infancy that are directed against the alpha-Gal antigen and which constitute <NUM>% of all the immunoglobulins in circulation (IgG, IgA, IgM and IgE).

In the milk of mammals (except for humans), the alpha-Gal epitope is present because, in mammary glands, the apical cytoplasm of the secreting elements is eliminated together with the secretion product.

In this manner, in the secreted liquid there are also fragments of cellular membranes exhibiting reactive alpha-Gal xenoantigens that are capable of instigating phenomena that can range from states of intolerance up to full-blown allergic reactions.

The presence of the alpha-Gal epitope in the milk, in addition to contributing apocrine secretion, is also influenced by the phenomenon of glycosylation of proteins.

Glycosylation means a post-translational modification of a protein, which sees sugars added to the peptidic chain.

Most of the proteins that are glycosylated in eukaryote cells are destined to become membrane proteins.

Allergy to the proteins of cow's milk is a far different condition from lactose intolerance, in that it is not a difficulty in digesting owing to an enzymatic deficit, but rather it is an actual immuno-mediated reaction aimed at the protein/glucose component, which is completely absent from or different in structure to human milk, and therefore foreign to our physiology.

The elimination diet, as with the subsequent therapeutic diet, entails the complete absence of such glycoproteins of cow's milk, and indeed of all foods that contain them, even in very low amounts (this is the case with many stuffed-meat products, various kinds of biscuits, many kinds of common bread, stock cubes etc.).

To this end, highly hydrolyzed types of milk or soy milk can be administered.

Recently a major link has emerged that correlates an anomalous over- expression of anti-alpha-Gal antibodies in highly-inflammatory forms of Inflammatory Bowel Diseases (IBDs). This group of diseases comprises illnesses like colitis, ulcerous rectocolitis and Crohn's disease. Significant correlations between circulating anti-alpha-Gal antibodies and the development of inflammatory diseases have also been highlighted for diseases like rheumatoid arthritis, eosinophilic esophagitis, and irritable colon syndrome (in general all the autoinflammatory forms that cause a variation of intestinal permeability). In all these cases, the ingestion of foods containing variable amounts of alpha-Gal epitope is potentially capable of further stimulating an already-overloaded immune system, decisively contributing to aggravating the inflammatory state and favoring the chronicity of the disease.

The choice to develop an effective treatment capable of destroying the pro-allergenic properties of milk proteins/oligosaccharides is therefore urgent, indispensable and economically attractive.

The aim of the present invention is to provide a method for the inactivation and inactivation testing of the alpha-Gal epitope in cow's milk.

In particular, in this context, an object of the invention is to devise a method for the inactivation of the alpha-Gal epitope, in cow's milk.

Another object of the invention is to provide a method for inactivating the above mentioned epitopes, thus ensuring an effective clearance that can be applied to the different kinds of cow's milk that are currently on the market.

Another object of the invention is to provide a method that can be carried out with conventional devices and machines.

This aim and these and other objects which will become better evident hereinafter are achieved by a method for the inactivation and inactivation testing of the alpha-Gal epitope in cow's milk, according to claim <NUM>.

Further characteristics and advantages of the invention will become better apparent from the detailed description that follows of a preferred, but not exclusive, embodiment of the method according to the invention, the steps and outcomes of which are given, by way of non-limiting example, in the accompanying drawings, wherein:.

The term "phenolic compounds" refers to molecules characterized, at least in part thereof, by the presence of an aromatic nucleus (benzene ring) bound to one or more hydroxyl functional groups.

The above mentioned compounds include, for the purposes of nonexclusive example:.

In the present invention the terms "phenols", "polyphenols" and "phenylpropanoids" have the same meaning and can be used individually, in the form of a mixture, or to substitute for each other for the set aims.

The term "xenoantigen" refers to molecules of animal origin that can be recognized by the immune system and can induce an antibody, immune-mediated, inflammatory or allergic response in the human host organism.

In the present invention the terms "xenoantigen", "antigen", "xenogeneic antigen", "epitope" and "determining antigenic" have the same meaning, and can be used together or to substitute for each other.

With reference to the figures, a method for the inactivation and inactivation testing of the alpha-Gal epitope from cow's milk according to the present invention comprises the steps of claim <NUM>.

Therefore, such method for the inactivation and inactivation testing of alpha-Gal epitope in cow's milk, characterized in that it comprises the following steps:.

In particular, a first embodiment of such method for the inactivation of alpha-Gal epitopes in samples of cow's milk is described below in detail.

Two solutions are made up with different concentrations of phenylpropanoid, using the milk as solvent in a total volume of <NUM>. In this specific example, which obviously should be understood to be non-limiting of the invention, an extract of "Lippia Citriodora" was used, titrated at <NUM>% verbascoside.

The different concentrations used for the preparation of the milk samples are: <NUM> +/- <NUM>% and <NUM> ± <NUM>% w/v.

These solutions are left to act under moderate but constant stirring, for a total of <NUM> ± <NUM> hours at <NUM> ± <NUM>.

Samples are taken at <NUM> ± <NUM> minutes, <NUM> ± <NUM> hour and <NUM> ± <NUM> hours.

An aliquot of milk is taken from each sampling, comprised between <NUM>µl and <NUM>µl, and preferably <NUM>µl, to which a buffer is added, NasCeHsOv <NUM> at pH <NUM> ± <NUM>, until a final volume is reached comprised between <NUM>µl and <NUM>µl, and preferably a final volume of <NUM>µl.

Then a murine antibody, directed against the alpha-Gal epitope, is added (in the present example this is an IgM clone called M86), at the preferable concentration of [<NUM> :<NUM>] w/v and the whole is incubated for <NUM> ± <NUM> minutes at <NUM> ± <NUM> under constant but moderate stirring.

At the end the samples are subjected to centrifugation at <NUM>,<NUM> x g for <NUM> ± <NUM> minutes at <NUM> ± <NUM>.

During incubation with the M86 antibody, a plate with <NUM> wells is prepared with <NUM>µl per well of alpha-Gal/HS A (Human Serum Albumin) at 5µg/ml in a PBS buffer (pH <NUM> ± <NUM>).

The plate thus prepared is incubated for <NUM> ± <NUM> minutes at a temperature comprised between <NUM> - <NUM>, although it is preferable to stabilize everything at <NUM> ± <NUM>.

Then <NUM> washes are carried out with <NUM>µl per well of PBS (physiological pH) at ambient temperature.

The first wash is left to act for <NUM> minutes, the two subsequent washes for <NUM> minutes each.

The blocking is done with <NUM>µl per well of <NUM> ± <NUM>% of serum albumin in PBS, followed by covering the plate with protective film and incubation for <NUM> ± <NUM> minutes at ambient temperature, in darkness.

Subsequently <NUM> washes are performed as above.

For each individual well, <NUM>µl of supernatant, taken from the treated samples after centrifugation, are added; the samples are loaded into the plate, each type of sample occupying at least <NUM> wells per column.

<NUM>µl is loaded into the first column of the plate, taken from a batch constituted by an aliquot comprised between <NUM> and <NUM>µl of buffer (preferably a dose of <NUM>µl is used) in which the aliquot of anti-alpha-Gal antibody is dissolved at the preferable concentration of [<NUM> :<NUM>] v/v without the presence of the sample of milk.

Such sample constitutes the reference value, also called "blank" value, and corresponds to the maximum bond on the plate between the anti-alpha-Gal antibody and alpha-Gal epitopes bonded to the HSA and exposed on the bottom of the wells.

Then the plate is covered with protective film and incubated at <NUM> ± <NUM> for <NUM> ± <NUM> minutes.

Then <NUM> washes with PBS are performed as above and <NUM>µl per well is added of a solution of secondary antibody (rabbit polyclonal anti-mouse) conjugated with peroxidase enzyme in phosphate buffer at pH <NUM> ± <NUM> (the ideal solutions of such antibody have been found to be [<NUM> : <NUM>], [<NUM> :<NUM>] and [<NUM> : <NUM>] v/v, preferably the intermediate one, [<NUM> :<NUM>] v/v, was adopted).

The plate is then covered again with protective film and incubated at <NUM> ± <NUM> in darkness for <NUM> ± <NUM> minutes.

Then <NUM> washes are performed as above.

Subsequently <NUM>µl is added per well of a development solution for the peroxidase enzyme, followed by covering the plate with protective film and incubation for <NUM> ± <NUM> minutes in darkness.

Then <NUM>µl per well of the stop solution is added, which is constituted by H<NUM>S0<NUM> <NUM> and the plate is then read in a plate reader at the wavelength of <NUM>.

The test of inactivation is based on the comparison between the absorbance values of the column that constitutes the blank value (<NUM>% of antibody available) and the respective columns of the samples.

If the absorbance (Abs) detected in the samples of treated milk corresponds to the Abs detected in the first column (blank batch), then it is possible to say that the anti-alpha-Gal antibodies left to incubate with the milk have not identified antigenic structures.

The unbound antibody was not able to create the interactions with the lipoprotein components of the milk that are responsible for the formation of the immune complex.

As a consequence, the unbound antibody was not sequestered by the centrifugation process, but instead remained free and available to interact with the alpha-Gal epitope bonded to the HSA and exposed on the bottom of the wells.

<FIG> shows a graph of the treatment of samples of cow's milk (three experimental sets, n=<NUM> for each experimental set) with a vegetable extract titrated in verbascoside used at the concentration of <NUM>% at ambient temperature (RT).

The vegetable extract administered at the concentration of <NUM>% w/v has been found to be capable of inactivating <NUM> ± <NUM>% of the antigens after <NUM> minutes, <NUM> ± <NUM>% after <NUM> hour and <NUM> ± <NUM> % of the antigen after at least <NUM> hours of incubation.

<FIG> shows a graph of the treatment of samples of cow's milk (four experimental sets, n=<NUM> for each experimental set) with a vegetable extract titrated in verbascoside used at the concentration of <NUM>% at ambient temperature (RT).

The vegetable extract administered at the concentration of <NUM>% w/v is capable of inactivating <NUM>± <NUM>% of the antigens after <NUM> minutes, <NUM> ± <NUM>% of the antigens originally present after <NUM> hour of incubation, and reaches a percentage of <NUM> ± <NUM> after at least <NUM> hours.

As a consequence, it has been found that this vegetable extract titrated at <NUM>% verbascoside is effective if present in solution in amounts not lower than <NUM> ± <NUM>% w/v.

A second embodiment of the method according to the invention for the inactivation of alpha-Gal epitopes in samples of cow's milk, soy milk and rice milk is described below in detail, with the application of phenyl derivatives for the removal of the alpha-Gal epitopes in samples of full-fat cow's milk.

A solution is made up with a phenyl derivative of cinnamic acid, specifically with caffeic acid, using the milk as solvent in a total volume of <NUM>.

The preferable concentration to be used is <NUM> ± <NUM>% w/v.

The preparation is left to act under moderate but constant stirring, for a total of <NUM> ± <NUM> hours at <NUM> ± <NUM>.

An aliquot of milk is taken from each sampling, comprised between <NUM>µl and <NUM>µl, and preferably a dose of <NUM>µl, to which a buffer is added, NasCeHsOv <NUM> at pH <NUM> ± <NUM>, until a final volume is reached comprised between <NUM> and <NUM>µl, and preferably a final volume of <NUM>µl.

Then a murine antibody, directed against the alpha-Gal epitope, is added (in this specific case an IgM clone called M86), at the concentration of [<NUM> :<NUM>] v/v and is left to incubate for <NUM> ± <NUM> minutes at <NUM> ± <NUM> under constant but moderate stirring.

During incubation of the samples with the M86 antibody, a plate with <NUM> wells is prepared with <NUM>µl per well of alpha-Gal/HSA at <NUM>µg /ml in a PBS buffer (pH <NUM> ± <NUM>).

The plate is subsequently incubated for <NUM>±<NUM> minutes at a temperature comprised between <NUM> - <NUM>, although it is preferable to stabilize everything at <NUM>.

Then <NUM> washes are carried out with <NUM>µl per well of sterile PBS (physiological pH) at ambient temperature.

The blocking is done with <NUM>µl per well of <NUM> ± <NUM>% of serum albumin in PBS and incubation for <NUM> ± <NUM> minutes at ambient temperature, in darkness.

For each individual well, <NUM>µl of supernatant, taken from the treated
samples after centrifugation, are added, and the samples are loaded into the plate, each type of sample occupying at least four wells per column.

If the absorbance detected in the samples of treated milk corresponds to the absorbance detected in the first column (blank batch), it means that the antibodies left to incubate with the milk have been recovered and consequently they have not identified antigenic structures. The unbound antibody was not able to create the interactions with the components of the milk that are responsible for the formation of the immune complex, and as a consequence it was not sequestered by the centrifugation process and it was recovered through the supernatant, going on to bond with the alpha-Gal antigen which is found processed together with the HSA on the bottom of the wells.

<FIG> shows a graph of the treatment of samples of cow's milk (<NUM> experimental sets, n=<NUM> for each experimental set) with caffeic acid at the concentration of <NUM>% w/v at ambient temperature (RT).

From the comparison with the blank column, it emerges that an inactivation of <NUM> ± <NUM>% of the antigens can be achieved after <NUM> minutes, of <NUM> ± <NUM>% after at least one hour of incubation, and this threshold rises to the percentage of <NUM> ± <NUM>% after <NUM> hours of incubation.

Such treatment is optimal for a significant clearance of the treated milk.

A third embodiment of the method according to the invention for the inactivation of alpha-Gal epitopes in samples of cow's milk, soy milk and rice milk is described below in detail, with the application of phenyl derivatives for the removal of the alpha-Gal epitopes in samples of full-fat cow's milk.

Different solutions are made up with a phenyl derivative of tannin and with a phenylpropanoid, using the milk as solvent in a total volume of <NUM>. In this specific example, which obviously should be understood to be non-limiting of the invention, tannic acid and a vegetable extract of "Ajuga reptans" titrated at <NUM>% teupolioside are used, and are preferably present in solution at the concentration of <NUM> ± <NUM>% w/v.

Preferably, the tannic acid is present in a solution at a concentration of <NUM> ± <NUM>% w/v. The preparation is left to act under moderate but constant stirring, for a total of <NUM> ± <NUM> hours at <NUM> ± <NUM>.

The blocking is done with <NUM>µl per well of <NUM> ± <NUM>% of serum albumin in PBS and incubation for <NUM> ± <NUM> minutes at ambient temperature, in darkness. Subsequently <NUM> washes are performed as above.

For each individual well, <NUM>µl of supernatant, taken from the treated samples after centrifugation, are added, and the samples are loaded into the plate, each type of sample occupying at least four wells per column.

Then <NUM>µl per well of the stop solution is added, which is constituted by H<NUM>S0<NUM> <NUM> and the plate is then read in a plate reader at <NUM>.

If the absorbance detected in the samples of treated milk corresponds to the absorbance detected in the first column (blank batch), then we can say that the anti-alpha-Gal antibodies left to incubate with the milk have been recovered and consequently they have not identified antigenic structures. The unbound antibody was not able to create the interactions with the components of the milk that are responsible for the formation of the immune complex, and as a consequence it was not sequestered by the centrifugation process and it was recovered through the supernatant, going on to bond with the alpha-Gal antigen which is found processed together with the HSA on the bottom of the wells.

<FIG> shows a graph of the treatment of samples of cow's milk with a vegetable extract titrated in teupolioside and with tannic acid at the concentration of <NUM>% w/v at ambient temperature (RT).

From comparison with the blank column, it can be seen that through the use of a vegetable extract titrated in teupolioside it is possible to achieve a limited capacity for inactivation of the alpha-Gal antigen equal to <NUM> ± <NUM>% after <NUM> minutes, to <NUM> ± <NUM>% after <NUM> hour and to <NUM> ± <NUM>% after <NUM> hours of incubation. The activity of the tannic acid has been shown to be capable of inactivating <NUM> ± <NUM>% of the antigens after <NUM> minutes, <NUM> ± <NUM>% after <NUM> hour and <NUM> ± <NUM>% after <NUM> hours of incubation.

The treatment of this food with such compounds has been shown to be capable of significantly lowering the reactivity of alpha-Gal epitopes, with a marked activity with reference to tannic acid.

A fourth embodiment of the method according to the invention for the inactivation of alpha-Gal epitopes in samples of cow's milk, soy milk and rice milk is described below in detail, with the application of plant cellular extracts with high content of phenylpropanoids, for example higher than <NUM>%, and of their phenyl derivatives for the inactivation of the alpha-Gal epitopes in samples of milk substitutes based on rice and soya.

Three solutions are made up, using a milk substitute based on rice and soya as solvent, for example a substitute comprising <NUM>% rice, <NUM>% soy seeds, in a total volume of <NUM>.

In the present example, which is non-limiting of the invention, extracts of "Lippia citriodora" are used, titrated at <NUM>% verbascoside.

The concentrations used for the preparation of the milk samples are: <NUM> ± <NUM>% w/v and <NUM> ± <NUM>% w/v. In addition a solution is made up of <NUM> ± <NUM>% w/v of a phenyl derivative of cinnamic acid, or of caffeic acid.

An aliquot of milk is taken from each sampling, comprised between <NUM> and <NUM>µl, and preferably a dose of <NUM>µl, to which a buffer is added, Na<NUM>C<NUM>H<NUM> <NUM> at pH <NUM> ± <NUM>, until a final volume is reached comprised between <NUM> and <NUM>µl, and preferably a final volume of <NUM>µl.

Then a murine antibody, directed against the alpha-Gal epitope, is added, for example an IgM clone called M86, at the preferable concentration of [<NUM> :<NUM>] w/v and the whole is incubated for <NUM> ± <NUM> minutes at <NUM> ± <NUM> under constant but moderate stirring.

During incubation with the M86 antibody, a plate with <NUM> wells is prepared with <NUM>µl per well of alpha-Gal/HS A (Human Serum Albumin) at <NUM>µg/ml in a PBS buffer (pH <NUM> ± <NUM>).

The blocking is done with <NUM>µl per well of <NUM> ± <NUM>% of serum albumin in sterile PBS, followed by covering the plate with protective film and incubation for <NUM> ± <NUM> minutes at ambient temperature, in darkness.

For each individual well, <NUM>µl of supernatant, taken from the samples after centrifugation, are added.

The plate is loaded, each type of sample occupying at least <NUM> wells per column.

<NUM>µl is loaded into the first column of the plate, taken from a batch constituted by an aliquot comprised between <NUM> and <NUM>µl of buffer (preferably a dose of <NUM>µl is used) in which the aliquot of anti-alpha-Gal monoclonal antibody is dissolved at the preferable concentration of [<NUM> :<NUM>] v/v without the presence of the sample of milk.

Then <NUM> washes with PBS are performed as above and <NUM>µl per well is added of a solution of secondary antibody (for example, rabbit polyclonal anti-mouse) conjugated with peroxidase enzyme in phosphate buffer at pH <NUM> ± <NUM>; the ideal solutions of such antibody are [<NUM> : <NUM>], [<NUM> :<NUM>] and [<NUM> : <NUM>] v/v, and preferably the intermediate one, [<NUM> : <NUM>] v/v, is adopted.

The plate is covered again with protective film and incubated at <NUM> ± <NUM> in darkness for <NUM> ± <NUM> minutes.

The test of inactivation is based on the comparison between the absorbance values of column number <NUM> which constitutes the blank value (<NUM>% of antibody available) and the respective columns of the samples. If the Abs detected in the samples of treated milk corresponds to the Abs detected in the first column (blank batch), then we can say that the anti-alpha-Gal antibodies left to incubate with the milk have not identified antigenic structures.

As a consequence, it was not sequestered by the centrifugation process, but instead remained free and available to interact with the alpha-Gal epitope bonded to the HSA and exposed on the bottom of the wells.

<FIG> shows a graph of the treatment of samples of milk substitutes based on rice and soya with a vegetable extract titrated in verbascoside used at the concentration of <NUM> % and <NUM> % w/v and with caffeic acid <NUM>% w/v (n=<NUM> for each experimental set) at ambient temperature.

From comparison with the blank column, it can be seen that the treatment with a vegetable extract titrated in verbascoside and used at the concentration of <NUM> ± <NUM>% w/v has highlighted a limited capacity to inactivate <NUM> ± <NUM>% of the antigens after <NUM> minutes, <NUM> ± <NUM>% after <NUM> hour and <NUM> ± <NUM>% after <NUM> hours of incubation. The treatment with a vegetable extract titrated in verbascoside and used at the concentration of <NUM> ± <NUM>% w/v has highlighted an activity capable of inactivating <NUM> ± <NUM>% of the antigens after <NUM> minutes, <NUM> ± <NUM>% after <NUM> hour and <NUM> ± <NUM>% after <NUM> hours of incubation. The activity of the caffeic acid has been shown to be significantly more effective, and capable of inactivating <NUM> ± <NUM>% of the antigens after <NUM> minutes, <NUM> ± <NUM>% after <NUM> hour and <NUM> ± <NUM>% after <NUM> hours of incubation.

In practice it has been found that the invention, as set out in the appended set of claims, fully achieves the intended aim and objects.

In particular, with the invention a method has been devised for the inactivation and inactivation testing of the alpha-Gal epitope, in cow's milk.

Furthermore, with the invention a method has been devised for inactivating at least part of the above mentioned epitopes, thus ensuring a significant clearance that can be applied to the different kinds of cow's milk that are currently on the market.

Claim 1:
A method for the inactivation and inactivation testing of alpha-Gal epitope in cow's milk, characterized in that it comprises the following steps:
- making up a solution with cow's milk as a solvent and caffeic acid or tannic acid, as a solute, for the inactivation of at least part of the alpha-Gal epitopes from said cow's milk;
- incubating samples of said cow's milk with the addition of an antibody aimed at the alpha-Gal epitope that is present in said cow's milk;
- separating the resulting immune complex created owing to the bond between antigen and antibody;
- preparing a well plate for the E.L.I.S.A. test with coating comprising said alpha-Gal epitope,
- adding, in said wells, supernatant taken from said samples, said supernatant containing the part of anti-alpha-Gal epitope antibody that has not bonded with the alpha-Gal epitopes, a column of wells being adapted to define a reference value that corresponds to the maximum signal between antibody and epitopes,
- completing said plate with a secondary antibody conjugated with an enzyme, or other molecule, adapted to chromatically highlight any presence of anti-alpha-Gal antibody,
- reading said plate, determining the presence of anti-alpha-Gal epitope antibody that has remained free in the solutions of the samples,
- comparing the absorbance values detected in the column that defines the reference values with the values detected in the other columns of samples of the plate.