Allergic diseases present a large problem of public health, and morbidity to allergy seems to be increasing. Food allergies are usually the first manifestation of allergy ; they are commonest in children under 3 years but are not rare for adults. After precedence of food allergy another allergic disease can later be developed.

It is increasingly evident that allergic diseases are related to lifestyle and should, in theory, be preventable. Air pollution, consumption of manipulated and processed foods, use of various chemicals, stress and low physical activity contribute to the decreasing resistance of human to disease. Much evidence supports the fact that the human immune system poorly tolerates the dramatic changes in lifestyle that have occurred, particularly in food habits over the past 100 years.

In the immune system of humans and other mammals, a specific class of antibodies, known as immunoglobulin E (IgE) and produced by B lymphocytes, mediates the so-called type I hypersensitivity. Hypersensitive reaction is a form of atopic response which develops very quickly, within seconds or minutes of exposure of the subject to exogenous macromolecules (e. g., proteins, glycoproteins), also known as antigens or allergens. The allergens can be airborne in the environment and/or present in food products and drugs. For example, food products commonly causing reactions in children are : milk, eggs, peanuts, soy, wheat, tree-nuts, fish and shellfish. For adults, the most common reaction causing food products are peanuts, tree-nuts, shellfish and fruits.

In non-allergic subjects, there is very little IgE, but in allergic subjects the concentration of IgE is very much higher. This elevated amount of IgE mediates type I hypersensitivity by priming mast cells abundant in the skin, lymphoid organs, eye membranes, nose, mouth, respiratory tract and intestines. Mast cells have surface IgE receptors which bind the elevated concentrations of IgE present in allergy-suffering subjects. When a bound IgE is subsequently contacted by the appropriate allergen, the mast cell is caused to degranulate and release various substances such as histamine

into the surrounding tissue. The release of these substances is responsible for clinical symptoms such as contraction of smooth muscle in the airway or the intestine, dilation of small blood vessels and the increase in their permeability to water and plasma proteins, secretion of thick sticky mucus, and stimulation of nerve endings in the skin.

These clinical symptoms are manifested by acute reactions, such as hives and anaphylaxis, and/or chronic diseases, such as asthma, atopic rhinitis, atopic dermatitis, chronic sinusitis and gastrointestinal disorders.

Type I hypersensitivity is, at best, a nuisance to the sufferer; at worst it can present very serious problems and can in rare extreme cases even result in death. Over the past decades, efforts have been made to find ways to effectively treat sufferers.

Existing strategies to treat allergic diseases are of limited utility, consisting of avoidance of allergens, amelioration of an ongoing allergic reaction with therapeutic compounds, and partial desensitization of the atopic individual to an identified allergen.

Theoretically, the avoidance of the allergen could have been the most efficient approach but it is practically very difficult and usually impossible to achieve. In many cases, despite a strict diet, eliminating all suspected offending foods, the subject remains symptomatic. Moreover, strict diets oftentimes cause nutritional problems in particular to subjects which are allergic to several basic foods.

Treatments by the use of therapeutic compounds are directed at blocking the acute inflammatory cascade that is responsible for allergic reactions. These compounds include anti-histamines, decongestants, ß2 agonists and corticosteroids which act on events downstream of IgE in the allergic cascade, making them palliative remedies which address the allergic symptoms. In some cases, this approach is useful, but it is mainly directed at alleviating the symptoms of allergy rather than dealing with its causes. Oftentimes, the remedy provides only a partial relief for short terms.

Moreover, there are disadvantages in the use of certain drugs. For example, the use of certain drugs can be accompanied by adverse side effects.

Desensitization or allergen immunotherapy is a treatment in which the subject is injected by the offending allergen itself aiming to gradually reduce the tendency of the treated subject to develop type I hypersensitivity upon normal exposure to the allergen. Practically, the treatment involves the injection of the allergen into the subject over a relatively long period of time, e. g., one year or even more. Initially, the

doses used are very small. In the absence of contra-indications the doses are increased rapidly to high levels which are necessary if the treatment is to be effective.

There are several major problems in the desensitization treatment.

First, the allergens involved are difficult to identify, and the identification process itself oftentimes results in adverse reactions.

Second, it is necessary for the subject to have injections very frequently, e. g., every two or three days in the beginning of the treatment, and every one to three weeks towards the end of treatment. It is recognized that such treatment is both time- consuming and disruptive of the subject's normal routine, hence generally undesirable.

Moreover, the complexity of this treatment is considerably high because the doses of allergen administered have to be carefully and accurately monitored and controlled.

A third problem is the element of risk to the subject. Although the initial doses of the allergen are very small and precautions are routinely taken to watch for any allergic response, local or systemic allergic reactions (such as hives, asthma and faint) can occur. Risk factor further increases with the increment of the doses and period between injections. In exceptional cases systemic allergic reactions occurring during desensitization treatment can cause death.

Fourth, the desensitization treatment is typically useful in inhalant allergen- derived sensitivity and allergic reactions due to insect stings, while for many food allergies, no effective desensitization treatment has been reported.

Attempts have been made in the past to overcome or avoid these problems. To reduce the frequency of injection, preparations have been administered which release the allergen slowly over a period of time. These have not been very successful, for example, because once administered, no control can be exercised over the amount of allergen released into the blood.

Attempts have also been made to modify an allergen chemically such that, whilst its immunogenicity is unchanged, its allergenicity is substantially reduced.

Some limited success has been achieved with this approach, but it has certain disadvantages'of its own. As each allergen has to be modified individually in accordance with its particular chemical structure, there is no satisfactory universally applicable technique for modifying allergens for a desensitization treatment.

Additionally, the devising of an acceptable modified allergen is very difficult because

on the one hand such modified allergen has to be useful in the desensitization treatment, while on the other hand it cannot cause any adverse reaction in the subject.

An additional strategy for treating food allergy is by modifying the food products so as to reduce or remove food allergenicity. Existing technologies include the use of heat, treatment with extreme pH and proteolysis. The capabilities of these technologies to remove or even reduce food allergenicity are, however, far from being satisfactory. Additionally, prior art technologies are known to cause undesirable sensory changes in the food products (color, odor, taste).

There is thus a widely recognized need for, and it would be highly advantageous to have a method and apparatus for reducing allergenic activity of food products, devoid of the above limitations.

SUMMARY OF THE INVENTION According to one aspect of the present invention there is provided a method of processing food. The method comprises: applying an effective amount of shock waves to the food so as to reduce or eliminate an allergenic activity of the food.

According to further features in preferred embodiments of the invention described below, the method further comprises placing the food in a liquid medium, wherein the application of the effective amount of shock waves to the food comprises generating the shock waves in the liquid medium.

According to still further features in the described preferred embodiments the effective amount of shock waves comprises a plurality of shock wave pulses.

According to still further features in the described preferred embodiments the shock waves comprise pressure shock waves.

According to still further features in the described preferred embodiments the shock waves are generated in the liquid medium by a spark produced by an electrical discharge.

According to still further features in the described preferred embodiments the effective amount of shock waves to the food is generated by a chemical explosion.

According to still further features in the described preferred embodiments the method further comprises monitoring at least one parameter of the shock waves.

According to still further features in the described preferred embodiments the method further comprises focusing the shock waves onto the food.

According to another aspect of the present invention there is provided an article of manufacture comprising food treated for reducing or eliminating an allergenic activity of the food as compared to the same food before treatment.

According to further features in preferred embodiments of the invention described below, the treatment of the food for reducing or eliminating the allergenic activity of the food is by application of an effective amount of shock waves as described herein.

According to still further features in the described preferred embodiments the food is a raw material used in the preparation of a food product. According to still further features in the described preferred embodiments the food is a food product.

According to still further features in the described preferred embodiments the food is a raw material used in the preparation of animal feed. According to still further features in the described preferred embodiments the food is animal feed.

According to yet another aspect of the present invention there is provided a cosmetic treated for reducing or eliminating an allergenic activity of the cosmetic as compared to the same cosmetic before treatment.

According to further features in preferred embodiments of the invention described below, the treatment of the cosmetic for reducing or eliminating the allergenic activity of the cosmetic is by application of an effective amount of shock waves as described herein.

According to still further features in the described preferred embodiments the cosmetic is a raw material used in the preparation of a cosmetic product. According to still further features in the described preferred embodiments the cosmetic is a cosmetic product.

According to still further features in the described preferred embodiments the shock waves are applied through a liquid medium.

According to still another aspect of the present invention there is provided an apparatus for reducing or eliminating an allergenic activity of a substance, such as, but not limited to, food material, cosmetic material, clothing article and the like. The apparatus comprises a chamber for receiving the substance and a shock wave generating device for applying an effective amount of shock waves to the substance so as to reduce or eliminate an allergenic activity of the substance.

According to further features in preferred embodiments of the invention described below, the chamber contains a liquid medium therein, and the shock wave generating device is capable of generating the shock waves in the liquid medium.

According to still further features in the described preferred embodiments the shock wave generating device produces a spark by an electrical discharge.

According to still further features in the described preferred embodiments the shock wave generating device comprises a chemical explosive.

According to still further features in the described preferred embodiments the shock wave generating device comprises a conductive membrane.

According to still further features in the described preferred embodiments the apparatus further comprises a monitoring device for monitoring at least one parameter of the shock waves.

According to still further features in the described preferred embodiments the apparatus further comprises a focusing element for focusing the shock waves onto the substance.

According to still further features in the described preferred embodiments each shock wave pulse of the plurality of shock wave pulses has a characteristic rise time in the microsecond or sub-microsecond range. According to still further features in the described preferred embodiments each shock wave pulse of the plurality of shock wave pulses has a characteristic pulse width in the microsecond or sub-microsecond range.

According to still further features in the described preferred embodiments the shock waves are characterized by an amplitude in the Mega or Giga Pascal range.

According to still further features in the described preferred embodiments the shock waves are characterized by an amplitude in the Giga Pascal range.

According to still another aspect of the present invention there is provided a method of processing food or cosmetic having S-S bonds, the method comprising transmitting an effective amount of energy to the food or cosmetic, so as to break at least a portion of the S-S bonds, so as to reduce or eliminate an allergenic activity of the food or cosmetic.

According to still further features in the described preferred embodiments the transmission of the effective amount of energy comprises applying an effective amount of shock waves.

According to still further features in the described preferred embodiments the transmission the effective amount of energy comprises applying an effective amount of electromagnetic waves.

According to still further features in the described preferred embodiments the effective amount of energy is carried by particles.

The present invention successfully addresses the shortcomings of the presently known configurations by providing a method and apparatus suitable for reducing allergenic activity of substances, and substances having a reduced or no allergenic activity.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings : FIG. 1 is a flowchart of a method of processing a substance, according to a preferred embodiment of the present invention;

FIG. 2 is a schematic illustration of an apparatus which can be used to reduce or eliminate allergenic activity of a substance, according to a preferred embodiment of the present invention; FIG. 3 is a is a schematic illustration of an experimental setup used for treating P-Lg protein solutions, according to a preferred embodiment of the present invention; FIG. 4 shows typical waveforms of a pressure as a function of time, as observed at a distance of 33 mm from a spark produced by an electrical discharge, according to a preferred embodiment of the present invention; FIG. 5 shows values of surface hydrophobicity of R-Lg, before (control) and after application of 1, 4,6 and 9 shock wave pulses, according to a preferred embodiment of the present invention ; FIG. 6 shows intrinsic fluorescence spectra of P-Lg after application 1, 4,6 and 9 shock wave pulses, according to a preferred embodiment of the present invention; FIG. 7 shows microdifferential scanning calorimetric thermograms of ß-Lg solution before (control) and after application of 4 and 9 shock wave pulses, according to a preferred embodiment of the present invention; FIG. 8 shows the change in the number of exposed sulfhydryll (SH) groups and the number of total SH groups, after fully unfolding by urea, per dimer of ß-Lg before (control) and after application of 1,4, 6 and 9 shock waves pulses, according to a preferred embodiment of the present invention; FIGs. 9a-b show far (Figure 9a) and near (Figure 9b) UV circular dichroism spectra of P-Lg before (control) and after application of 9 shock wave pulses, according to a preferred embodiment of the present invention; and FIG. 10 shows IgE levels (mean value standard deviation) of rats after receiving daily doses of 1 mg of protein in their diet; shown in Figure 10 are: a negative control group (purple), for which no allergen was given, a positive control group (brown), for which untreated allergen was given, and a test group (green), for which a protein treated by 6 pulses of shock waves was given, according to a preferred embodiment of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is of a method and apparatus which can be used for processing substances. Specifically, the present invention can be used to reduce or eliminate allergenic activity of various substances. The present invention is further of foods, food products, cosmetic materials, cosmetics, bath products and clothing articles treated for reducing or eliminating allergenic activity thereof.

The principles and operation of a method and apparatus according to the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and the arrangement of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Referring now to the drawings, Figure 1 is a flowchart of a method of processing a substance, according to a preferred embodiment of the present invention.

The method is suitable for processing many kinds of substances, in particular materials having the potential of causing hypersensitive reaction upon contacting external or internal tissues of humans or animals. Representative examples include, without limitation, foods, food products, cosmetic materials, cosmetics, bath products and clothing articles.

It is to be understood that selected method steps described hereinbelow are optional, hence can be executed in one embodiment and not executed in another embodiment. Additionally the method steps described hereinbelow can be executed contemporaneously or sequentially in many combinations or orders of execution.

Specifically the ordering of the flowchart of Figure 1 is not to be considered as limiting. For example, two or more method steps, appearing in the following description and/or the flowchart of Figure 1 in a particular order, can be executed in a different order (e. g., a reverse order) or substantially contemporaneously.

The method begins in step 10 and, optionally and preferably, continues to step 12 in which the substance is placed in a liquid medium. The liquid medium can be,

for example, water or any other liquid which does not damage the treated substance.

Preferably, the properties of the liquid medium are selected so as to allow propagation of shock waves through the liquid medium and efficient interaction of the shock waves with the substance. For example, the liquid medium can have an acoustic impedance which is similar to the acoustic impedance of the substance.

Whether or not step 12 is executed, the method continues to step 14 in which an effective amount of energy is applied to the substance. The energy can be transmitted in many forms, including, without limitation, shock waves, electromagnetic radiation, flow of particles and the like.

While the embodiments below are described with a particular emphasis to energy carried by shock waves, it is to be understood that more detailed reference to shock waves is not to be interpreted as limiting the scope of the invention in any way.

In the preferred embodiment in which the substance is placed in the liquid medium, an effective amount of shock waves can be generated in the liquid medium and allowed to interact with the substance.

As used herein"effective amount of shock waves"refer to duration, amplitude, energy, frequency, number of pulses and/or spatial distribution of the shock waves which is selected such that upon interaction of the shock waves with the substance, its allergenic activity is reduced or eliminated.

As known, a shock wave is generated whenever a wave generating source moves at a velocity exceeding the propagation velocity of the sound waves typical for this specific medium.

As demonstrated in the Example section that follows, the application of shock waves to the substance can significantly reduce or eliminate allergenic activity, substantially without any further modification of the substance. The advantage of using shock waves for reducing or eliminating allergenic activity is that the shock waves do not elevate the temperature of the treated substance. This is particularly advantageous for foods, where thermal treatments are known to cause undesirable sensory changes to the food. Additionally, the use of shock waves does not require any additional chemical treatment to be employed. This is advantageous, for example, in the field of cosmetics where it is desired to reduce or eliminate the potential of the cosmetic to cause hypersensitive reaction, while preserving the chemical composition of the cosmetic.

The level of allergenic activity depends, inter alia, on the ability of the cell- bound IgE antibodies to recognize and bind a site on protein or other macromolecules present in the substance. The binding sites on a protein, also known as epitopes, typically comprise about 5-15 amino acids, that the cell-bound IgE antibody recognizes and is specific to. As known, cross linking of an epitope and a specific cell-bound IgE antibody correlates with the structure of the epitopes. Once the shock waves impinge on the substance, energy carried by the wave front of the shock waves is absorbed by the substance resulting in a modification of the structural features the allergenic epitopes present in the substance.

The structure of proteins is commonly described in terms of four structural levels: primary structure, secondary structure, tertiary structure and quaternary structure. As further detailed hereinunder and demonstrated in the Example section that follows, the interaction between protein and the shock waves can result in changes in secondary and tertiary structure levels. These modifications substantially reduce the ability of the epitopes to cross-link the cell-bound IgE antibodies, making them substantially unrecognizable by the immune system.

The treated substance can be subsequently used by the consumer, substantially without developing hypersensitive reaction. Thus, the present invention successfully provides articles having reduced allergenic activity. Such articles include raw materials used in the preparation of food products, food products, raw materials used in the preparation of animal feed, animal feed, raw materials used in the preparation of cosmetics, cosmetics, raw materials used in the preparation of clothing articles, clothing articles and the like.

The shock waves can be generated in more than one way.

In one embodiment, the shock wave are preferably generated by explosive evaporation of the liquid medium, caused, e. g., by a spark produced by an electrical discharge. A typical spark gap includes a pair of electrodes connected to a capacitor charged to a high voltage. When the capacitor abruptly discharges its electrical energy, streamers begin to propagate from the positive to the negative electrode.

When the streamers reach the negative electrode fast formation of a discharge channel occurs. This discharge channel is characterized by a plasma with high conductivity due to the large plasma electron temperature. The large discharge channel plasma density and temperature gradients, together with the non-elastic properties of the

liquid, lead to the formation of shock waves. In addition to the shock wave formation, the discharge plasma channel is characterized by intense light emission due to collision radiative processes inside the plasma. By changing parameters of the electrical system, (e. g., capacitance, inductance, charging voltage, etc.), the parameters of the shock waves and the radiation flux can be controlled.

In another embodiment, the shock waves are preferably generated by a chemical explosive positioned in a certain distance from the substance. When the chemical explosive explodes, gases are released at high speed generating a shock wave traveling outwardly from the explosion site to thereby impinge on the substrate.

In still another embodiment, the shock waves are preferably generated by a conductive membrane. The motion of the conductive membrane can be controlled by a magnetic field which repels the membrane. The motion of the membrane generates an impulse, which can be focused by a lens to thereby form the shock waves.

According to a preferred embodiment of the present invention the shock waves are in the form of a plurality of shock wave pulses. Preferably, each shock waves consist of a plurality of dominant pressure pulse which climb steeply to high pressure levels. Specifically, the amplitude of the pressure is in the Mega or Giga Pascal range, e. g., above 50 Mega Pascals, more preferably above 100 Mega Pascals, between about 100 Mega Pascals and about 10 Giga Pascals.

As used herein the term"about"refers to 10 %.

The characteristic time rise of the pulses is preferably in the microsecond or sub-microsecond range, e. g., preferably below 10 us, more preferably below 1 us, most preferably below 500 ns. The pulse width (typically full-width at half- maximum) of the pulses, although longer than the characteristic time rise, is preferably still in the microsecond or sub-microsecond range, preferably a few times (say, about 2-5 times) the characteristic time rise.

According to a preferred embodiment of the present invention the method proceeds to step 16 in which at least one parameter of the shock waves is monitored.

The monitoring is preferably for verifying that an effective amount of shock waves has been delivered to the substance. Parameters which can be monitored include either parameters directly characterizing the shock waves (e. g., the amplitude of the shock wave, the propagation velocity, the spatial distribution, etc.), or parameters characterizing the generation mechanism of the shock waves, hence indirectly

characterizing the shock waves. Representative examples for such indirect parameters include, without limitation, output voltage, discharge current, and magnetic field.

The method may further proceed to step 18 in which the shock waves are focused on the substance. This can be done, for example, using a focusing element, such as, but not limited to, an acoustic mirror. It is known that the condition for reflection of a shock wave upon impinging on a boundary between two media is that the speed of sound, which varies depending on the medium through which it travels, changes at an interface between the two media. It is also known that the speed of sound in larger in solids than in the liquid. Thus, when a shock wave in liquid encounters, e. g., a metal surface, most of its energy is reflected away from the surface.

According to various exemplary embodiments of the present invention the shock waves are focused using a concavely shaped solid surface (e. g., metal surface) which can be designed to reflect most of the energy of the waves to a desired direction. For example, when the concavely shaped solid surface has a shape of an ellipsoid, the shock waves can be generated in one focal point of the ellipsoid and the subspace can be positioned in another focal point of the ellipsoid. As will be appreciated by one ordinarily skilled in the art, with such configuration, the shock waves are focused onto the substance either by reflection from the surface of the ellipsoid or via direct propagation between the two focal points.

The method preferably continues to optional step 20 in which the substance is withdrawn from the liquid medium. The method ends in step 22.

According to another aspect of the present invention there is provided an apparatus 30 which can be used to reduce or eliminate allergenic activity of a substance.

Reference is. now made to Figure 2 which is a schematic illustration of apparatus 30. In its simplest configuration, apparatus 30 comprises a chamber 32, for receiving substance 34, and a shock wave generating device 36, for applying an effective amount of shock waves to substance 34 as further detailed hereinabove.

Device 36 preferably generates a plurality of shock wave pulses. According to various exemplary embodiments of the present invention chamber 32 contains a liquid medium, and device 36 generates the shock waves in the liquid medium. Device 36 can be any device known in the art which is capable of generating sufficiently strong shock waves. Representative examples include, without limitation a an electrical

discharge mechanism for producing a spark, a chemical explosive charge, and a conductive membrane as further detailed hereinabove. It is expected that during the life of this patent many relevant technologies for generating shock waves will be developed and the scope of the term shock wave generating device is intended to include all such new technologies a pi-iot-i.

According to a preferred embodiment of the present invention device 36 comprises a focusing element 38, such as, but not limited to, an acoustic lens for focusing the waves generated by device 36. Focusing element 38, or, more preferably an additional focusing element 42 can be used for focusing the shock waves onto substance 34. Elements 38 and 42 can form, for example, a concave surface characterized by two focal points whereby the shock waves are generated in a first such focal point and substance 34 is positioned in a second focal point.

Optionally and preferably, apparatus 30 further comprises a monitoring device 40 for monitoring at least one parameter of the shock waves. Monitoring device 40 can be, for example, a pressure probe which can be configured to monitor the amplitude of the wave front. In another embodiment an arrangement of pressure probes can be used such that the spatial distribution of the wave front or the propagation velocity. Other monitoring devices which are contemplated include, without limitation, voltage measuring device, current measuring device, magnetic field measuring device and temperature measuring device. Additionally, monitoring device 40 can include combination of several types of measuring devices and/or an arrangement of several units of a particular types of measuring device.

Without being bound by any theory, it is believed that the reduction in allergenic activity described herein is due to energy-induced disruption or breaking of S-S bonds formed between Cysteine residues of proteins present in the food or cosmetic.

Hence, according to another aspect of the present invention there is provided a method of processing food or cosmetic having S-S bonds. The method according to this aspect of the present invention is effected by transmitting an effective amount of energy, carried by, e. g., shock-waves, electromagnetic waves or particles, to the food or cosmetic, so as to break at least a portion of the S-S bonds, so as to reduce or eliminate an allergenic activity of the food or cosmetic. The type and amount of

energy is selected such that it will not substantially disrupt other characteristics of the food or cosmetic, such as, but not limited to, taste, smell, texture, color and the like.

In any of the above embodiments, "food"refer to any food material or food product, including, without limitation, any kind of confectionery, bread, noodle, meat product, fishmeat product, soybean product, milk product, condiment, fruit and beverage.

In any of the above embodiments, "cosmetic"refer to any raw material or cosmetic product, including, without limitation, lotion, gel, emulsion, ointment, cream, cleanser, make up cosmetic, hair cosmetic and bathing agent.

Additional objects, advantages and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following example, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following example.

EXAMPLE Reference is now made to the following example, which together with the above descriptions illustrate the invention in a non limiting fashion. hlaterials and Aletlzods Sera collected from allergic human subjects were used for i71-l'it7'0 assessment of the effect of shock wave treatment in accordance with preferred embodiments of the present invention on the allergenic activity of sesame proteins. All sera samples were received from people who exhibited positive clinical symptoms. These symptoms were shown after consumption of foods containing, sesame, milk or peanut within 2 hours after exposure to the suspected food, showed positive skin prick test (SPT), demonstrating an IgE-mediated food induced reaction. The combined IgE specific for the various allergens was assessed by determination of total IgE, using the Pharmacia CAP system.

I7l-vivo experiments were performed with Brown Norway rats which have been reported to exhibit an allergic reactions similar to those of humans.

ELISA was used for detection and quantification of specific IgE in sera formed due to exposure of Brown Norway rats to proteins.

The shock waves were applied using an experimental setup, schematically illustrated in Figure 3. Two 0.5 RF, 50 kV Maxwell low-inductance capacitors, were charged in parallel to 35 kV using a high-voltage power supply. The total energy stored in the capacitors was about 800 J or below. The capacitors were discharged via two gaseous spark gap switches having a distortion electrode to which a high-voltage pulse (up to 60 kV) was applied. A high-voltage pulse with an amplitude of about 70 k-V or below was obtained at the output.

A stainless steel electrode was placed inside a cylindrical chamber filled with de-ionized water. The electrode was electrically connected to the output of the capacitors-spark gap switches system to thereby serve as a high-voltage electrode. An additional, grounded, electrode was positioned at a variable distance from the high- voltage electrode. The output voltage was measured using an active voltage divider and the discharge current was measured using and a self-integrated Rogowsky coil.

The pulsed pressure was monitored by a four channels digitizing oscilloscope (Tektronix TDS 224) connected to three high-frequency high-pressure probes (109A PCB Piezotronics, NY) which were installed in the chamber. The amplitude of the pressure pulse, at a distance of 33 mm from the discharge gap was about 130 Mega Pascal and the rise time of the pulse was about 1 jus. The width of the pressure pulse was about 3 lls (full width at half maximum).

Sodium phosphate buffer (0. 01 M, pH 7. 0) was used to prepare P-Lg protein solutions of 0. 5, 1 and 2 mg/ml. The final concentration of ß-Lg was determined spectrophotometrically (Cam Spec M330 CAMSPEC UK) using a molar extinction coefficient at 2S0 nm of, 80= 17600 M~lcm-l [Yang et al.,"Lactoglobulin molten globule induced by high pressure", Journal of Agriculture and Food Chemistry, 2001, 49: 3236-3243]. Plastic bags (PE/PA/EVOH/PA/PE Plastophil Co., Hazorea, IL) were filled with 4 ml of the ß-Lg solutions, eliminating air bubbles and heat-sealed. Half of the bags remained as control samples and were exposed to the same temperature (4 °C) as the treated bags. A predetermined number of pulses from one to nine were applied in each test with a 10 mm inter-electrode gap and a discharge current amplitude of 30 kA. Each experiment was repeated at least 5 times.

Intrinsic and extrinsic fluorescence intensities were measured at room temperature using a Perking-Elmer LS-50B spectrophotofluorometer and a quartz cell (1 cm path length). An excitation wavelength of 295 nm and observed wavelengths of 300-400 nm, with slit width of 5 nm, were used for intrinsic tryptophan fluorescence spectra of ß-Lg.

The surface hydrophobicity (extrinsic fluorescence) of ß-Lg was determined using an ANS (1-anilinonaphthalene-8-sulfonate) solution (2. 0x10' M). The wavelengths of the excitation and emission were 390 mn and 470 nm, respectively. A quantification of the ANS binding was carried out fluorimetrically by adding 1 ml of ß-Lg solution (0. 5/1 mg/ml) to 5 ml of ANS solution. Buffer phosphate containing ANS solution was used as a blank.

The enthalpies of thermal denaturation (AH) and the temperature of the maximum of the endothermic peak (Tm) were monitored by a microdifferential scanning m calorimetric DSC (VP-DSC MicroCal, LLC Northampton, MA). The heating scan was performed from 30 °C to 110 °C, at a rate of 1 °C min~l, using sodium phosphate buffer (0.01 M, pH 7.0) as a reference. The parameters AH and Tm were extracted from the DSC signals using"Origin 50"software (MicroCal) after the subtraction of the baseline.

Ellman's reagent 5, 5'-Dithiobis (2 nitrobenzoic acid) (DTNB) was used to estimate total free and exposed sulfhydryll (SH) groups [Ellman, G. L."Tissue sulfhydryl groups", Archives of Biochemical and Biophysical, 1959, 52 : 70-77]. For total free sulfhydryll groups 10 mM DTNB solution was prepared in sodium phosphate buffer (0.01 M, pH 7.0) containing 8.0 M urea and 1 mM EDTA. One microlitre of ß-Lg was mixed with 3. 8 ml sodium phosphate buffer (0.01 M, pH 7. 0), 8. 0 M urea, 1 mM EDTA, and 200 ml DTNB solution. Fifteen minutes after mixing, the absorbance was determined at 412 nm. To estimate the number of moles of SH per mol of ß-Lg, a molar extinction coefficient of 1. 36x10 M-l cm~l was used 4 (Ellman, 1959 supra ; supra). The exposed (accessible) free SH groups in ß-Lg were similarly determined without adding urea to the buffer and DTNB solution.

Small-angle X-ray scattering (SAXS) data of 1 and 2 mg/ml of ß-Lg solutions were obtained at 20 °C, using slit-collimated Kratky camera (A. Par Co), and an Ni- filter CuKa one-dimension detector. A 1 mm thick capillary with protein solution was

used for the SAXS determinations. Data were collected in intervals of 0.1 < h < 6. 4 nrri 1. The wave vector h is defined as : h = (4 7u/k) sin (20/2), were # = 15.4 nm is the length and 2A is the scattering angle. Scattering data were corrected for slit smearing before the calculation of the sample structure parameter. The treated and control ß-Lg samples were kept at 4 °C and equilibrated to 25'C for the analysis.

Circular dichroism (CD) spectra of ß-Lg solution (2 mg/ml) near-UV and Far- UV were recorded before and after subjecting to nine shock waves by a CD spectrophotometer (Model 202 CD, Aviv Instruments, Lakewood, NJ) and analyzed with Selcon 3 and Contin software packages [Sreerema, N. and Woody, R. W.,"A self-consistent method for the analysis of protein secondary structure from circular dichroism", Analytical Biochemistry, 1993,209 : 32-44 ; Sreerema, N. and Woody, R.

W. , "Estimation of protein secondary structure from CD spectra : Comparison of CONTIN, SELCON and CDSSTR methods with an expanded reference set", Analytical Biochemistry, 2000, 282 : 252-260 ; Provencher, S. W. and Glockner, J., "Estimation of protein secondary structure from circular dichroism", Biochemistry, 1981,20 : 33-37; Van Stokkum et al.,"Estimation of protein secondary structure and error analysis from CD spectra", Analytical Biochemistry, 1990,191 : 110-118].

Spectra in the far UV and near UV ranges were collected at 25 °C using 0.1 mm and 1 cm pathlength cuvettes, respectively. Spectra were recorded in the far UV region down to at least 190 nm, so as to obtain confident information related to the conformation. The mean residue ellipticity at the wavelength k is quoted in units of deg cnr/dmol and given by: [0] v = MRWxOx/lOdc, where MRW is the mean residue weight defined as the molecular weight mass divided by the number of repeated units, 0> is the measured ellipticity, d is the cuvette path length and c is the protein concentration in units of g/ml. The mean residue ellipticity was calculated using a molecular weight of 18200 and 162 residues (Yang et al., sitpra).

Results asld DscussoBt Figure 4 shows typical waveforms of the pressure as a function of time, as observed at a distance of 33 mm from the electrodes at a voltage of Uo = 70 kV. As shown in Figure 4, the underwater discharge is characterized by the appearance of several pressure pulses. The amplitude of the first pressure pulse and the time of its

appearance depend on the amplitude of the discharge current. It was found that the first pressure pulse propagates with an ultrasound velocity of v ~ 2x 105 rats indicating a shock wave type of the pressure wave. Additionally, several pressure pulses with larger amplitude and pulse duration were observed with a large time delay with respect to the beginning of the discharge current. The pressure in the shock wave was up to 130 Mega Pascal measured at 33 mm from the inter electrode gap, and pressure values of up to 300 Mega Pascal were estimated closer to the inter electrode gap.

Figure 5 shows values surface hydrophobicity of ß-Lg, before (control) and after application of 1,4, 6 and 9 shock wave pulses, in accordance with preferred embodiments of the present invention. Treated samples were normalized to the unexposed samples, which were set as 100 % hydrophobicity. As shown in Figure 5, an increase in the number of applied pulses resulted in an increase in the extrinsic fluorescence intensity of ANS associated with ß-Lg for both 0.5 (P < 0.0001) and 1 (P-0.0001) mg/ml. An increase in surface hydrophobicity of B-Lg was observed after 9 pulses.

In order to eliminate the effect of light emitted during the application of shock waves, the samples were packed in an opaque plastic bag. Only a slight change in surface hydrophobicity was detected under these conditions. This result shows that the emission of light does not contribute significantly to the reduction of alergenicity.

Figure 6 shows intrinsic fluorescence spectra of ß-Lg after application of 1, 4, 6 and 9 shock wave pulses, in accordance with preferred embodiments of the present invention. As shown in Figure 6, an increase in the number of the applied pulses resulted in a decrease in the intrinsic fluorescence intensity of Tryptophan associated with ß-Lg. A decrease of up to 15 % in intrinsic fluorescence intensity was observed after application of 9 shock wave pulses (P < 0.01).

The fluorescence of Tryptophan at 295 nm is affected by the energy transfer from Tyrosine to Tryptophan (if the distance between them is less than 10-18 A) and also by fluorescence quenching of adjacent groups [Lakowicz, J. R. ,"Principles of fluorescence spectroscopy", New York : Plenum Press, 1983, pp. 341-381]. Trp is reported to be the major fluorophore in native ß-Lg facing into the base of the hydrophobic pocket and fully buried [Manderson et al.,"Effect of heat treatment on bovin ß-Lactoglobulin A, B and C Explored using thiol availability and fluorescence,

Journal of Agriculture and Food Chemistry, 1999,47 : 3617-3627 ; Brownlow et al., "Bovine ß-Lactoglobulin at 1.80 A resolution still an enigmatic lipocalin, Structure, 1997,5 : 481-495]. Arg124 functions for this group as a fluorescence quencher. Trp61 is reported to be relatively exposed to the solvent and is adjacent to the Cys66-Cysl60 disulfide bridge which is one of the most important fluorescence quenchers [Manderson et al., supra].

The obtained decrease of Tryptophan fluorescence can be explained by the movement of one or two Tryptophan residues closer to the quenchers as well as the movement of Tyrosine residues away, such that the energy transfer to Tryptophan decreases and the quantum yield is reduced.

Note that the decrease in quantum yield of Tryptophan occurred without change of Sm y indicating an exclusive change in the protein, substantially without a change in the polarity of the environment. Reduction of Tryptophan fluorescence of ß-Lg, subjected, according to a preferred embodiment of the present invention, to shock waves is different from the increase obtained after conventional high hydrostatic pressure or heating. This difference is explained by the movement of Tryptophan residues away from their quenchers. In addition, thermal denaturation of ß-Lg increased ma. \ which is explained by an increase in exposure of one or two Tryptophan residues [Mills, O. E. , "Effect of temperature on Tryptophan fluorescence of ß-Lactoglobulin B. , Biochemica et Biophysica Acta, 1976,434 : 324-332].

Figure 7 shows microdifferential scanning calorimetric (DSC) thermograms of ß-Lg solution before (control) and after application of 4 and 9 shock wave pulses according to a preferred embodiment of the present invention. Enthalpies of thermal denaturation (Ah) and the temperature of the endothermic peak, Tm, are summarized in Table 1, below.

Table 1 concentration 0. 5 mg/ml 1 mg/ml #H Tm #H Kcal/mol Tm Kcal/mol control 92. 735. 47a 77. 9Si0. 603 95. 37i5. 22' 76. 630. 3' 4 pulses 58.77~7.07b 78.09~0.47a 71. 744. 91 76. 82~0.23a 9 pulses 50.26~4.44b 77.87~0.64a 58.41~7.78b 77.53~0.38b

a values in the same row with different superscripts are significantly different (P<0. 05) due to treatment Endothermal effects on protein DSC thermograms reflect unfolding and loss of protein structure as a result of disruption of intramolecular hydrogen bonds [Dumay et al.,"High pressure unfolding and aggregation of ß-Lactoglobulin and the baroprotective effects of sucrose", Journal of Agriculture Food Chemistry, 1994, 42: 1861-1S68]. Exothermic aggregation was moderate, thus it did not interfere in the estimation of the thermal unfolding enthalpy. AH decreased with number of shock wave pulses. A decrease in AH of up to 45 % (P < 0.001) was observed in a 1 mg/ml ß-Lg solution. A greater decrease was observed for solutions with smaller protein concentrations. For high ß-Lg concentrations, the endothermic peak, Tm, was increased with the number of pulses (P < 0. 05). Due to low sensitivity of the instrument, this effect was not observed for the low ß-Lg concentrations. The decrease in AH can be explained by partial protein unfolding as a result of application of to shock waves thereto.

Figure 8 shows the change in the number of exposed sulfhydryll (SH) groups and the number of total SH groups (after fully unfolding by urea) per dimer of ß-Lg before (control) and application of 1,4, 6 and 9 shock waves pulses, in accordance with preferred embodiments of the present invention. The reactivity of the SH group to Ellman's reagent, which is not accessible to the SH group in the native structure, increased significantly (P < 0. 01) immediately after the first applied pulse. An increase of up to ten-fold was observed after subjecting the samples to 9 pulses. These results are similar to partial unfolding of the protein structure and the exposure of free SH groups obtained at hydrostatic pressure of 50 Mega Pascal [Moller et al.,"Thiol reactivity in pressure unfolding ß-Lactoglobulin. Antioxidative properties and thermal refolding", Journal of Agriculture and Food Chemistry, 1998, 46: 425-430; and Yang et al. slep*a]. An untreated fully unfolded (using urea) protein resulted in 2 mol SH per mol dimer. The number of total SH groups significantly (P < 0.0001) increased to 3 mole SH per mole dimer after the protein was subjected to 9 shock wave pulses. This result can be attributed to the rupture of some of the 4 S-S bonds of ß-Lg dimer.

Table 2, below presents results obtained using SAXS. Shown in Table 2 are values of the apparent radius of gyration, Rg, of 1 mg/ml and 2 mg/ml ß-Lg solutions, with Uo = 70 kV; and C = 0.5 I1F.

Table 2 1 mg/ml 2 mg/ml Control 2. 132+0 25a 2. 198+0. 24a 9 pulses 2. 398+0. 29a 2. 047+0. 103

a values in the same row with the same superscript are not significantly different (P>0.05) due to treatment As shown in Table 2, the application of the shock wave pulses in accordance with preferred embodiments of the present invention did not cause any aggregation.

Similar results were obtained from gel electrophoresis (12 % and 15 % polyacrylamid). The obtained Rg values for the native protein dimer coincide with values found in the literature, and no change was observed after 9 pulses. Such unaltered value for Rg indicates that the treatment performed in accordance with preferred embodiments of the present invention changed the structure of the protein substantially without causing any aggregation. Note that application of conventional high hydrostatic pressure or heating to protein is known to cause aggregation by creating new disulfide bonds from reactive SH groups [Yang et al., sup7a ; Panick et al.,"Differences between the pressure and temperature induced denaturation and aggregation of-Lactoglobulin A, B and AB monitored by FT-IR spectroscopy and small-angle X-ray scattering", Biochemistry, 1999, 38 : 6512-6519 ; Kolakowski et al., "Effect of high pressure and low temperature on P-Lactoglobulin unfolding and aggregation", Food Hydrocolloids, 2001,15 : 215-232; Dumay et al., sllpra].

Figures 9a-b show the far (Figure 9a) and near (Figure 9b) UV CD spectra of ß-Lg before (control) and after application of 9 shock wave pulses, according to a preferred embodiment of the present invention. The spectra shown in Figure 9a reflect the structure observed after the application of the shock wave pulses. Spectral modifications were evident by a decrease in the overall intensity between 210 and 190 nm of the treated ß-Lg. The latter provide evidence for secondary structural modifications.

A quantitative analysis of the CD data showed that a fraction of protein had undergone partial unfolding. As calculated, the structure of the native protein included 44 % beta strand, which was reduced to 36 % by subjecting the samples to the shock wave pulses. In other words, up to 18% of the 9 (3 strands, naturally existing in P-Lg, were affected by the treatment. A corresponding increase in the unordered structure was observed. The single a helix, which is reported in the native protein (Dumay et al., supra ; Tanaka et al."Modification of the single unpaired sulfhydryl group of P- Lactoglobulin under high pressure and the role of intermolecular S-S exchange in the pressure denaturation (Single SH of P-Lactoglobulin and pressure denaturation)", International Journal of Biological Macromolecules, 1996, 19: 63-68].

The near UV CD spectra shown in Figure 9b exhibit a pattern of negative peaks between 250 and 300 nm due to the presence of aromatic residues: two tryptophans (Trpl9 and 61), four tyrosines (Tyr20, 42,99 and 102) and four phenyl- alanines (Phe 82, 105, 136 and 151). Although all of the above residues can contribute to the CD spectrum, tryptophan residues are often the major determinants of the near UV CD curve. Tryptophan signals are generally more intense than those of tyrosine and phenylalanine and they occur between 280 and 300 nm, whereas phenylalanine and tyrosine usually do not absorb above 270 and 290 nm, respectively [Woody, R. W. and Dunker, A. K., in G. D. Fasman, Circular Dichroism and the Conformational Analysis of Biomolecules, New York : Plenum Press, 1996, pp. 109-158]. Therefore, the negative bands at 293. 2 and 284. 4 nm can be assigned to asymmetrically perturbed tryptophans while peaks below 280 nm are likely the result of the chiral environment of phenylalanine and tyrosine residues.

As shown in Figure 9b, the near UV spectra of the control and the treated protein are identical, with the exception of the slight increase of A between 300 and 330 nm. This difference can be explained by the change in the amount of disulfide bonds. This assumption is confirmed by the results obtained by measuring the reactivity of SH groups using Ellman's reagent. These observations opposite to the effects of conventional high hydrostatic pressure or heating in which aromatic side chains become more mobile as a result of unfolding (Ikeuchi et al.,"Pressure induced denaturation of monomer ß-Lactoglobulin is partially irreversible: comparison of monomer form (highly acidic pH) with dimer form (meutral pH) ", Journal of Agriculture and Food Chemistry, 2001,49 : 4052-4059; and Yang et al., sllp1 a].

Figure 10 shows IgE levels (mean value + standard deviation) of rats after receiving daily doses of 1 mg of protein in their diet. Shown in Figure 10 are: a negative control group (purple), for which no allergen was given, a positive control group (brown), for which untreated allergen was given, and a test group (green), for which a protein treated by 6 pulses of shock waves in accordance with preferred embodiments of the present invention was given. As shown in Figure 10, the IgE levels of the negative control group and the test group are similar, indicating that the application of shock waves reduced or eliminated the allergenic activity of the protein.

No regeneration of allergenicity of the treated proteinswas observed within 14 days following treatment.

Table 3 below summaries the in vitro results for 6 and 10 shock wave pulses, in accordance with preferred embodiments of the present invention. The results in Table 3 (mean value standard deviation) represent percentage of the positive control groups.

Table 3 No. Allergenic 6 pulses (%) 10 pulses (%) Globulins (kDa*) 1 14 25-20 2 14 3 14 6. 8 ~ 1. 3 28. 6 ~ 1. 8 20-25 3.2 ~ 0.5 4.4 ~ 0. 4 4 14 5 14 25-20 35-30 6 14 7 14 8 14 9 14 - - 20-25 - - 10 25-20 35-30 11 14 - - 12 14 13 14 25-20 14 14 15 14 - - 16 14 17. 14

* : Determined by SDS-PAGE - : No allergenic activity The results shown in Table 3 indicate that in 17 out of the 17 samples, the allergenic activity was reduced or eliminated, where in 16 samples the allergenic activity was eliminated and in one sample (No. 3), the allergenic activity was reduced to about 3. 2 % of the original activity for 6 pulses and about 28. 6 % of the original activity for 10 pulses.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention.