EP1248534A1 - Hyperbaric pressurization of proteins and therapeutical uses of pressurized proteins thereof - Google Patents

Hyperbaric pressurization of proteins and therapeutical uses of pressurized proteins thereof

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Publication number
EP1248534A1
EP1248534A1 EP01901095A EP01901095A EP1248534A1 EP 1248534 A1 EP1248534 A1 EP 1248534A1 EP 01901095 A EP01901095 A EP 01901095A EP 01901095 A EP01901095 A EP 01901095A EP 1248534 A1 EP1248534 A1 EP 1248534A1
Authority
EP
European Patent Office
Prior art keywords
protein
proteins
group
whey
protein composition
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01901095A
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German (de)
French (fr)
Inventor
Tahereh Hosseini-Nia
Stan Kubow
Ashraf Ismail
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McGill University
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McGill University
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Publication date
Application filed by McGill University filed Critical McGill University
Publication of EP1248534A1 publication Critical patent/EP1248534A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/015—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with pressure variation, shock, acceleration or shear stress or cavitation
    • A23L3/0155—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with pressure variation, shock, acceleration or shear stress or cavitation using sub- or super-atmospheric pressures, or pressure variations transmitted by a liquid or gas
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/30—Physical treatment, e.g. electrical or magnetic means, wave energy or irradiation
    • 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 invention relates to hyperbaric pressurization of proteins and therapeutical uses of such pressurized proteins as stimulatory growth and/or antioxidant and tissue glutathione inducing agents.
  • dietary proteins such as whey to undergo digestion can greatly affect their physiological properties.
  • Increased insoluble or poorly digested protein in the upper intestine decreases the bioavailability of bioactive peptides hidden in an inactive state inside the polypeptide chain (Meisel H, Frister H, Schlimme E. Z. Ernahrungswiss 1989; 28:267- 278) .
  • TBARS plasma thiobarbituric acid reacting substances
  • GSH-promoting and antioxidant activities of whey proteins have been related to their capability to provide GSH precursors in the form of peptides, such as ⁇ -glutamylcysteine, which can readily cross cell membranes (Baruchel, S., Viau, G. Anticancer Res . 1996; 16:1095-1100).
  • ⁇ -glutamylcysteine groups has been indicated to be rare in food proteins except for whey and egg albumin (Bounous G., Gold P., 1991, Clin . Invest . Med. 14:296- 301).
  • Cow's milk allergy is believed to be due to interaction between ⁇ -lactoglobulin and other milk proteins and the immune system, which is generally IgE or IgG mediated (Pediatr 121: S16, (1992)).
  • the antigenicity of a protein molecule is a function of both its primary structure and the conformation of the molecule. Food allergens are resistant to digestion and thus can be presented to the immune system in the gut (Astwood JD et al . Nature Biotech 14:1269 (1996)). In particular, proteins with a high disulphide content are highly resistant to digestion and have been strongly implicated in the allergenic responses (Del Val G. et al., J Allergy Clin Immunol 103:690-7, (1999)).
  • Disulphide-rich whey proteins such as ⁇ -lactoglobulin are particularly resistant to both digestion due to their rigid and compact tertiary structure and conformation (Hagemeister H, Antila P. J. Anim . Physiol . Anim . Nutr. 1994; 72:86-91). These proteins apparently remain intact after their passage through the stomach (Hagemeister H, Antila P. J. Anim . Physiol . Anim . Nutr. 1994; 72:86-91).
  • ⁇ -lactoglobulin to digestive enzymes such as pepsin has been related to the simultaneous presence of two disulfide bridges and one free thiol group which give a rigid structure to this protein, allowing many exchanges with the S-S bonds (Schimdt DG, Poll JK. Neth. Milk Dairy J 1991 45:240-255).
  • Schimdt DG Poll JK. Neth. Milk Dairy J 1991 45:240-255
  • the cleavage of the S-S bonds is required to decrease the surface polarity of the molecule and to expose the hydrophobic groups to the polar environment, which could, in turn, increase the digestibility of the protein as well as decrease its allergenic
  • U.S. Patent No 5,476,677 there is disclosed a single pressure treatment of rice between 1000 atm to 9000 atm, to reduce cooking time by denaturing rice proteins. Their processing also refers to drying after with pressurization to exert the treating rice via pressure for human consumption as opposed to cooking via heating.
  • the U.S. Patent No. 5,476,677 also claim that the pressure treatment of rice induce hypoallergenic effects on the rice.
  • One aim of the present invention is to provide a specific unique pressurization technique on proteins resulting in potent stimulatory growth and antioxidant effects via increase in availability of SH group and increase in digestibility of proteins.
  • a method to induce conformational changes in proteins to enhance their susceptibility to enzymes which comprises the step of: a) exposing a protein in a solution, having a concentration of about 0.01% to about 50%, to a pressurization treatment sufficient to induce permanent conformational changes in proteins.
  • the enzymes may be digestive enzymes and when locate in vivo enhances digestibility of the protein.
  • the method in accordance with a preferred embodiment of the present invention wherein the pressurization treatment is followed by about 3-5 minutes of holding time at 400 MPa.
  • the method may further comprises a step effected prior to step a) , wherein the protein is solubilized in solution by adding a solubilization agent or by storing the protein solution at about 4°C for at least 12 hours .
  • the protein is selected from the group consisting of whey, caseins, soy, egg, peanut, legume, nut, milk, fish, meat proteins, hormones and albumin such as egg albumin and Human Serum Albumin (HAS) .
  • the protein may be solubilized in a solution selected from the group consisting of aqueous, saline, isoelectric, isotonic, and buffered solutions, nutrient-based solutions and formulas used in infant feeding and in enteral and parenteral nutrition.
  • the preferred protein concentration is of about
  • the preferred pressurization treatment is a mixture of pulse and continuous mode which is a combination of at least 2 cycles, preferably 3-6 cycles at 400 MPa and which may optionally be followed by a 5- 10 minutes of holding time, at 400 MPa.
  • the optimal number of cycles, holding time and pressure treatment however will vary according to the protein concentration of the solution and the type of application.
  • the pressurization treatment is effected at a temperature ranging between 0 and 80 °C, preferably 20°C.
  • a protein composition having enhanced in vivo susceptibility to digestive enzymes for administration to an animal which comprises between 1% (w/w) to 100% (w/w) protein prepared by the method of the present invention; and an acceptable pharmaceutical carrier between 0% (w/w) to 99% (w/w) .
  • the enhanced susceptibility of the protein composition may cause stimulatory growth effect, increased fur quality and/or calm behavior, antioxidant properties and/or tissue glutathione enhancing properties for the animal.
  • the antioxidant properties of the protein composition may cause enhanced redness of the animal meat .
  • the preferred protein composition contains 24% (w/w) protein; however, the effects can be exerted with a range of different protein concentrations .
  • An acceptable pharmaceutical carrier may be selected, without limitation, from the group consisting of exipient for oral, topical, intravenous, systemic, intraperitoneal or subcutaneous administration to the animal .
  • An oral exipient may be selected from the group consisting of food, water, juice, milk, carbonated and non-carbonated beverages, sweetened and unsweetened drinks, enteral, parenteral, infant formulas and health drinks .
  • a method to stimulate growth, increase fur quality and/or calm behavior of an animal which comprises administering an effective amount the protein composition of the present invention.
  • the protein composition of the present invention in the preparation of a medicament to stimulate growth, increase fur quality and/or calm behavior of an animal.
  • a method to induce conformational changes in proteins to reduce their allergenic properties comprises the step of: a) exposing a protein in a solution, having a concentration of about 20% to about 24%, to a pressurization treatment sufficient to induce permanent conformational changes in proteins.
  • pressurization treatment is of at least 100 MPa for at least 2 cycles.
  • the pressurization treatment is of about 400 MPa for preferably 3 cycles.
  • the method in accordance with another embodiment of • the present invention which further comprises a step effected prior to step a) , wherein the protein is solubilized in solution by adding a solubilization agent or by storing the protein solution at about 4°C for at least 2 hours.
  • the protein during the pressurization treatment is at a temperature ranging from 0 to 80°C, preferably at 20°C.
  • the following terms are defined below.
  • protein is intended to mean any food or physiological proteins.
  • food protein is intended to mean protein which is usually fed to humans and animals, including, without limitation, whey, caseins, soy, legume, egg, such as egg albumin, peanut, nut, milk, fish and meat proteins.
  • physiological protein is intended to mean any physiological proteins found in body fluids of animals, including without limitation human and animal blood proteins, such as Human Serum Albumin (HAS) , and hormones .
  • HAS Human Serum Albumin
  • body fluids is intended to mean any physiological fluid including, without limitation, saliva, plasma and blood.
  • animal is intended to mean human,, mammal, bird, amphibian, fish, reptile and insect.
  • pressurization sufficient to induce permanent conformational change in protein mainly consists in repeated cycles of pressure or equivalent treatment.
  • Fig. 1 illustrates food intake and weight gain in weanling rats following intake of native or pressurized whey proteins after 17 and 35 days;
  • Fig. 2 illustrates the feed efficiency and protein efficiency ratios in Weanling rats following intake of native or pressurized whey proteins after 17 and 35 days;
  • Fig. 3 illustrates serum levels of thiobarbituric acid reactive substances in Weanling rats following intake of native or pressurized whey proteins after 17 and 35 days;
  • Fig. 4 illustrates variations in SH/SS ratio for several pressure treatment of a 3.1% whey protein solution
  • Fig. 5 illustrates variations in SH/SS ratio for several pressure treatment of a 12.5% whey protein solution
  • Fig. 6 illustrates variations in SH/SS ratio for several pressure treatment of a 25% whey protein solution
  • Fig. 7 illustrates variations in SH/SS ratio for several pressure treatment of a 50% whey protein solution
  • Fig 8 illustrates SH and SS levels in 3.1% native whey protein solution vs 3.1% pressurized whey protein solution under different cycles and holding time;
  • Fig. 9 illustrates SH and SS levels in 12.5% native whey protein solution vs 12.5% pressurized whey protein solution under different cycles and holding time
  • Fig. 10 illustrates SH and SS levels in 25% native whey protein solution vs 25% pressurized whey protein solution under different cycles and holding time
  • Fig. 11 illustrates SH and SS levels in 50% native whey protein solution vs 50% pressurized whey protein solution under different cycles and holding time;
  • Fig. 12 illustrates the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in rat hepatic tissue after 35-day feeding trial;
  • Fig. 13 illustrates the ratio of reduces glutathione (GSH) to oxidized glutathione (GSSG) in rat hepatic tissue after 17-day feeding trial
  • Fig. 14 illustrates the reduced glutathione concentrations in rat hepatic tissue after 17-day feeding trial
  • Fig. 15 illustrates the reduced glutathione concentrations in rat hepatic tissue after 35-day feeding trial
  • Fig. 16 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 1-4) and pressurized whey for 17 days
  • Fig. 17 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 5-8) and pressurized whey for 17 days;
  • Fig. 18 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 1-5) and pressurized whey (rats # 1-3) for 35 days;
  • Fig. 19 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 6-8) and pressurized whey (rats # 4-8) for 35 days;
  • Fig. 20 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 1-4) and pressurized whey (rats # 1-5) for 17 days;
  • Fig. 21 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 5-8) and pressurized whey (rats # 6-9) for 17 days;
  • Fig. 22 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 1-5) and pressurized whey (rats # 1-4) for 35 days;
  • Fig. 23 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 6-9) and pressurized whey (rats # 5-9) for 35 days; and
  • Fig. 24 illustrates the Fourier Transform Infrared (FTIR) Deconvolution Spectroscopic Data of a native 20% whey protein isolate solution before and after exposure to different applied modes of pressure at 400 MPa.
  • FTIR Fourier Transform Infrared
  • the weight gain, glutathione inducing effect and antioxidant findings in the animals were unexpected and unique. Also, the Applicants have developed a new type of processing using pressurization in conjugation with holding time and recycle of the pressure to enable the use of lower pressures to impact upon the secondary structure protein molecules in a greater magnitude than was observed via earlier work on whey proteins using a single application of pressure at a range of high- pressure treatments up to 1200 MPa.
  • pressurization decreases the disulfide content of whey proteins while concurrently increasing the free sulfhydryl content of the proteins. This would suggest that pressurization could increase the bioavailability of sulfur amino acids towards glutathione synthesis by breaking the disulfide bonds. In this manner, pressurization of whey proteins could increase their digestibility to allow for an enhanced generation of small bioactive peptides such as ⁇ -glutamylcysteine that allow for a more rapid intracellular uptake of cysteine. This approach differs dramatically from earlier descriptions on the scientific literature and earlier patents that describe the importance of preserving whey proteins on their undenatured state in order to maintain the integrity.
  • the new pressurization approach involved a high pressure machine which could produce pressures only as high as 400 MPa but gave large volumes (2 liters) .
  • the technique allowed for a greater impact on the protein molecule's secondary structure than was observed in earlier studies using 1000-1200 MPa pressure.
  • This approach would allow pressurization to be readily used on an industrial scale whereas the pressures involved in earlier work could not be readily transferred to an industrial scale due to the enormous pressures involved (1000-1200 MPa) .
  • Protein concentrations and pressure treatment were optimized using the unique approach of: (Astwood, J.D. et al., Na ture Biotech 1996; 1269-1274) exposing the protein to recycles pressure; and (Baruchel, S., Viau, G. Anticancer Res . 1996; 16:1095-1100) holding the protein under pressure for an extended period of time to obtain the maximal changes in secondary structure in the amide I area as detected by FTIR spectroscopy .
  • whey protein isolate solutions were formulated to contain a concentration of 25-40% protein and were stored at 4°C for 12 hours before being subjected to pressure treatment. Whey protein isolates were exposed to 400 MPa for 3 cycles ' with 10 minutes of holding time at 400 MPa.
  • the samples were kept at a constant temperature of 20 °C during the pressure treatments although the method of pulse pressurization described in the present application can be carried out on proteins in solution using a wide range of temperatures (i.e. 0 to 80 °C) . Alternatively, to exert the same effect, an application of 6 cycles plus 3 minutes holding time also can be used.
  • the proteins were added as 24% protein (w/w) in semi-purified diets and fed to 21 day-old newly weaned Sprague Dawley rats. Rats were fed either pressurized and unpressurized whey protein for either 17 or 35 days. Semi-purified diets were formulated according to the NRC requirements for the rat (National Research Council (1995) 4 th rev.
  • the protein isolate solutions were formulated to contain a concentration of 25% protein.
  • a significant lowering of plasma concentrations of an index of oxidative stress, thiobarbituric acid reactive substances (TBARS) was also observed with the group of rats fed the pressurized whey protein (Fig. 3) .
  • the animals fed the pressurized whey were also observed to be calmer and had better general appearance including a thicker fur.
  • the animals fed the pressure-treated .whey were more manageable, had more linear growth and showed no increase in adipose tissue despite their increased body weight.
  • the tissues were observed to be redder in color in the pressurized treated rats, further suggesting a generalized antioxidative effect of tissues due to the preservation of hemoglobin from air oxidation.
  • Western blotting was employed for the detection of antibodies of the native and pressurized whey protein to assess the antigenicity of the both proteins in the serum of the newly weaned rats, which were fed native and mixed mode pressurized whey as dietary protein for 17 and 35 days.
  • Plasma proteins of the both group of the rats on the two diets for the two period of time were treated with sample loading buffer (SDS Reducing Buffer) at pH 6.8 and 5% ⁇ -mercaptoethanol and were heated at 95°C for 4 min.
  • the treated samples were loaded to SDS-PAGE gels in Mini-protean II Electrophoresis System, using 4-15%, 10 wells ready gel.
  • An electrode running buffer pH 8.3 containing 1% SDS was used to separate the proteins at a voltage of 110 and 62 mA current for 50-60 min for two gels. After the electrophoretic separation, the separated proteins were transferred to a nitrocellulose paper using transfer buffer at a pH of 8.3 at a voltage of 121 and 250 mA at the beginning to increase to 300 mA during 1 hour at the end of the transfer time. Immunodetection was carried out with peroxidase-conjugated affinity pure goat anti-rat IgG (H+L) from Jackson Immuno- Research (Cat . #112-035-062) in a dilution of 1:400.
  • FIG. 24 A 20% solution of the whey protein isolate was exposed to different pressure cycles (Fig. 24) .
  • Our hyperbaric-Fourier transform infrared (FTIR) deconvolution spectroscopic data show that our novel pressurization technique induced an irreversible major broadening in the Amide I area of the secondary structure of whey proteins, which is the result of a complete unfolding of the proteins (Fig. 24) .
  • Figure 24 demonstrates that, in comparison to native whey protein, the exposure of the proteins to 1 cycle of pressure at 400 MPa did not change the structure of the molecule although a reduction in the intensity of the amide I bands (1600 - 1700 cm -1 ) was observed as a function of pressure due to the reduction of volume of the molecules.
  • the present patent application shows for the first time a method by which protein structure can be irreversibly denatured, particularly in terms of disulphide bond breakage. Based on our chemical studies we have shown that pressurization alters the conformation of whey proteins such that the disulphide bonds are decreased concurrent with an increase with free sulfhydryl groups . Highly allergenic proteins such as ⁇ -lactoglobulin have a high disulphide content, which increases the resistance of the protein to digestion. The presence of disulfide bonds is also related to the allergenic properties of food proteins.

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Abstract

The present invention relates to hyperbaric pressurization of proteins and therapeutical uses of such pressurized proteins as stimulatory growth and/or antioxidant and/or tissue glutathione inducing agents. There is provided a protein composition having enhanced susceptibility to enzymes for administration to an animal, which comprises between 1% (w/w) to 100% (w/w) protein prepared by the method of the present invention; and an acceptable pharmaceutical carrier between 0% (w/w) to 99% (w/w). The present invention also relates to induce conformational changes in proteins to reduce their allergenic properties by exposing proteins to a hyperbaric treatment. There is provided a protein composition having reduced allergenic effects for administration to an animal, which comprises between 1% (w/w) to 100% (w/w) protein prepared by the method of the present invention; and an acceptable pharmaceutical carrier between 0% (w/w) to 99% (w/w).

Description

HYPERBARIC PRESSURIZATION OF PROTEINS AND THERAPEUTICAL USES OF PRESSURIZED PROTEINS THEREOF
BACKGROUND OF THE INVENTION (a) Field of the Invention
The invention relates to hyperbaric pressurization of proteins and therapeutical uses of such pressurized proteins as stimulatory growth and/or antioxidant and tissue glutathione inducing agents. (b) Description of Prior Art
The l idea that pressurization affects the secondary structure of whey proteins has been in the literature since 6-7 years ago.
The capacity of dietary proteins such as whey to undergo digestion can greatly affect their physiological properties. Increased insoluble or poorly digested protein in the upper intestine decreases the bioavailability of bioactive peptides hidden in an inactive state inside the polypeptide chain (Meisel H, Frister H, Schlimme E. Z. Ernahrungswiss 1989; 28:267- 278) .
Previous work has shown in vi tro hydrolysis of beta-lactoglobulin when this protein was exposed to pepsin, trypsin and thermolysin in the presence of hydrostatic pressure up to 300 MPa (Stapelfeldt H et al. (1996) J. Dairy Res . 63, 111-118.). This latter effect is believed to be due to the enhancement of the in vi tro action of digestive enzymes under pressurization. There is no evidence that pressurization of proteins in the absence of digestive enzymes can enhance the in vi tro or in vitro digestibility of proteins. No work to date has suggested the capability of pressurization of dietary proteins to increase the protein efficiency ratio and index of an increased efficiency of dietary protein or to stimulate in vivo growth. Studies from Applicants ' laboratory have shown that supplementation with whey protein isolate increases tissue glutathione concentrations and decreases plasma thiobarbituric acid reacting substances (TBARS) , a marker of lipid peroxidation secondary to oxidative stress, in hamsters supplemented with a whey protein isolate (Nicodermo A. et al . , FASEB J 1999; 13:A546). Others have also demonstrated an increase in circulating lymphocyte glutathione concentrations in healthy young adults, by supplementing them over 3- month period with a whey protein isolate (Lands LC et al. J Appl Physiol 1999; 87:1381-1385). The increase in GSH levels was associated with increased skeletal muscle performance, and decreased percent body fat, without weight change, suggesting muscle mass accretion. The potent GSH-promoting and antioxidant activities of whey proteins have been related to their capability to provide GSH precursors in the form of peptides, such as γ-glutamylcysteine, which can readily cross cell membranes (Baruchel, S., Viau, G. Anticancer Res . 1996; 16:1095-1100). The presence of γ- glutamylcysteine groups has been indicated to be rare in food proteins except for whey and egg albumin (Bounous G., Gold P., 1991, Clin . Invest . Med. 14:296- 301). Although previous work by Zom ara et al .
(Zommara M et al . (1996) Nutr. Res . 16, 293-302;
Zommara M. et al . (1998) Biosc. Biotech . Biochem . 62,
710-717) has shown that the antioxidative effect of the whey proteins is increased by bacterial fermentation, no previous work has demonstrated that pressurization of whey proteins enhances their antioxidative properties .
Cow's milk allergy is believed to be due to interaction between β-lactoglobulin and other milk proteins and the immune system, which is generally IgE or IgG mediated (Pediatr 121: S16, (1992)). The antigenicity of a protein molecule is a function of both its primary structure and the conformation of the molecule. Food allergens are resistant to digestion and thus can be presented to the immune system in the gut (Astwood JD et al . Nature Biotech 14:1269 (1996)). In particular, proteins with a high disulphide content are highly resistant to digestion and have been strongly implicated in the allergenic responses (Del Val G. et al., J Allergy Clin Immunol 103:690-7, (1999)). Disulphide-rich whey proteins such as β-lactoglobulin are particularly resistant to both digestion due to their rigid and compact tertiary structure and conformation (Hagemeister H, Antila P. J. Anim . Physiol . Anim . Nutr. 1994; 72:86-91). These proteins apparently remain intact after their passage through the stomach (Hagemeister H, Antila P. J. Anim . Physiol . Anim . Nutr. 1994; 72:86-91). The resistance of β-lactoglobulin to digestive enzymes such as pepsin has been related to the simultaneous presence of two disulfide bridges and one free thiol group which give a rigid structure to this protein, allowing many exchanges with the S-S bonds (Schimdt DG, Poll JK. Neth. Milk Dairy J 1991 45:240-255). The cleavage of the S-S bonds is required to decrease the surface polarity of the molecule and to expose the hydrophobic groups to the polar environment, which could, in turn, increase the digestibility of the protein as well as decrease its allergenic
In U.S. Patent No 5,476,677 there is disclosed a single pressure treatment of rice between 1000 atm to 9000 atm, to reduce cooking time by denaturing rice proteins. Their processing also refers to drying after with pressurization to exert the treating rice via pressure for human consumption as opposed to cooking via heating. The U.S. Patent No. 5,476,677 also claim that the pressure treatment of rice induce hypoallergenic effects on the rice.
However, the Applicants' own molecular spectroscopy published study on whey proteins (Hosseininia T. et al. J. Agric. Food Chem . 1999;47:4537-4542) has shown that a single application of pressure up to 1200 MPa leads to no irreversible changes in protein conformation.
It would be highly desirable to be provided with a specific unique pressurization technique on proteins resulting in potent stimulatory growth, antioxidant and tissue glutathione inducing effects as well as hypoallergenic effects.
SUMMARY OF THE INVENTION
One aim of the present invention is to provide a specific unique pressurization technique on proteins resulting in potent stimulatory growth and antioxidant effects via increase in availability of SH group and increase in digestibility of proteins.
In accordance with the present invention there is provided a method to induce conformational changes in proteins to enhance their susceptibility to enzymes, which comprises the step of: a) exposing a protein in a solution, having a concentration of about 0.01% to about 50%, to a pressurization treatment sufficient to induce permanent conformational changes in proteins. The enzymes may be digestive enzymes and when locate in vivo enhances digestibility of the protein.
The method in accordance with a preferred embodiment of the present invention, wherein the pressurization treatment is of at least 100 MPa for at least 2 cycles.
The method in accordance with a preferred embodiment of the present invention, wherein the pressurization treatment is of about 400 MPa for 3 to 6 cycles.
The method in accordance with a preferred embodiment of the present invention, wherein the pressurization treatment is followed by about 3-5 minutes of holding time at 400 MPa. The method in accordance with a preferred embodiment of the present invention, wherein the enzymes are digestive enzymes.
In accordance with another embodiment of the present invention, the method may further comprises a step effected prior to step a) , wherein the protein is solubilized in solution by adding a solubilization agent or by storing the protein solution at about 4°C for at least 12 hours .
The method in accordance with a preferred embodiment of the present invention, wherein the protein is selected from the group consisting of whey, caseins, soy, egg, peanut, legume, nut, milk, fish, meat proteins, hormones and albumin such as egg albumin and Human Serum Albumin (HAS) . The protein may be solubilized in a solution selected from the group consisting of aqueous, saline, isoelectric, isotonic, and buffered solutions, nutrient-based solutions and formulas used in infant feeding and in enteral and parenteral nutrition. The preferred protein concentration is of about
12.5-40%, which will differ according to the type of application. The preferred pressurization treatment is a mixture of pulse and continuous mode which is a combination of at least 2 cycles, preferably 3-6 cycles at 400 MPa and which may optionally be followed by a 5- 10 minutes of holding time, at 400 MPa. The optimal number of cycles, holding time and pressure treatment however will vary according to the protein concentration of the solution and the type of application. The pressurization treatment is effected at a temperature ranging between 0 and 80 °C, preferably 20°C.
In accordance with another embodiment of the present invention, there is provided a protein composition having enhanced in vivo susceptibility to digestive enzymes for administration to an animal, which comprises between 1% (w/w) to 100% (w/w) protein prepared by the method of the present invention; and an acceptable pharmaceutical carrier between 0% (w/w) to 99% (w/w) . The enhanced susceptibility of the protein composition may cause stimulatory growth effect, increased fur quality and/or calm behavior, antioxidant properties and/or tissue glutathione enhancing properties for the animal. The antioxidant properties of the protein composition may cause enhanced redness of the animal meat .
The preferred protein composition contains 24% (w/w) protein; however, the effects can be exerted with a range of different protein concentrations .
An acceptable pharmaceutical carrier may be selected, without limitation, from the group consisting of exipient for oral, topical, intravenous, systemic, intraperitoneal or subcutaneous administration to the animal .
An oral exipient may be selected from the group consisting of food, water, juice, milk, carbonated and non-carbonated beverages, sweetened and unsweetened drinks, enteral, parenteral, infant formulas and health drinks .
In accordance with another embodiment of the present invention, there is provided a method to stimulate growth, increase fur quality and/or calm behavior of an animal, which comprises administering an effective amount the protein composition of the present invention.
In accordance with another embodiment of the present invention, there is provided the use of the protein composition of the present invention in the preparation of a medicament to stimulate growth, increase fur quality and/or calm behavior of an animal.
In accordance with another embodiment of the present invention, there is provided a method to induce conformational changes in proteins to reduce their allergenic properties, which comprises the step of: a) exposing a protein in a solution, having a concentration of about 20% to about 24%, to a pressurization treatment sufficient to induce permanent conformational changes in proteins.
The method in accordance with another embodiment of the present invention, wherein the pressurization treatment is of at least 100 MPa for at least 2 cycles.
The method in accordance with another embodiment of the present invention, wherein the pressurization treatment is of about 400 MPa for preferably 3 cycles. The method in accordance with another embodiment of the present invention, wherein the pressurization treatment is followed by 10 minutes of holding time. ' The method in accordance with another embodiment of • the present invention, which further comprises a step effected prior to step a) , wherein the protein is solubilized in solution by adding a solubilization agent or by storing the protein solution at about 4°C for at least 2 hours.
The method in accordance with another embodiment of the present invention, wherein the protein during the pressurization treatment is at a temperature ranging from 0 to 80°C, preferably at 20°C. For the purpose of the present invention the following terms are defined below.
The term "protein" is intended to mean any food or physiological proteins. The term "food protein" is intended to mean protein which is usually fed to humans and animals, including, without limitation, whey, caseins, soy, legume, egg, such as egg albumin, peanut, nut, milk, fish and meat proteins. The term "physiological protein" is intended to mean any physiological proteins found in body fluids of animals, including without limitation human and animal blood proteins, such as Human Serum Albumin (HAS) , and hormones .
The term "body fluids" is intended to mean any physiological fluid including, without limitation, saliva, plasma and blood.
The term "animal" is intended to mean human,, mammal, bird, amphibian, fish, reptile and insect.
The expression "pressurization sufficient to induce permanent conformational change in protein" mainly consists in repeated cycles of pressure or equivalent treatment.
BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates food intake and weight gain in weanling rats following intake of native or pressurized whey proteins after 17 and 35 days;
Fig. 2 illustrates the feed efficiency and protein efficiency ratios in Weanling rats following intake of native or pressurized whey proteins after 17 and 35 days;
Fig. 3 illustrates serum levels of thiobarbituric acid reactive substances in Weanling rats following intake of native or pressurized whey proteins after 17 and 35 days;
Fig. 4 illustrates variations in SH/SS ratio for several pressure treatment of a 3.1% whey protein solution;
Fig. 5 illustrates variations in SH/SS ratio for several pressure treatment of a 12.5% whey protein solution;
Fig. 6 illustrates variations in SH/SS ratio for several pressure treatment of a 25% whey protein solution; Fig. 7 illustrates variations in SH/SS ratio for several pressure treatment of a 50% whey protein solution;
Fig 8 illustrates SH and SS levels in 3.1% native whey protein solution vs 3.1% pressurized whey protein solution under different cycles and holding time;
Fig. 9 illustrates SH and SS levels in 12.5% native whey protein solution vs 12.5% pressurized whey protein solution under different cycles and holding time; Fig. 10 illustrates SH and SS levels in 25% native whey protein solution vs 25% pressurized whey protein solution under different cycles and holding time; Fig. 11 illustrates SH and SS levels in 50% native whey protein solution vs 50% pressurized whey protein solution under different cycles and holding time;
Fig. 12 illustrates the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in rat hepatic tissue after 35-day feeding trial;
Fig. 13 illustrates the ratio of reduces glutathione (GSH) to oxidized glutathione (GSSG) in rat hepatic tissue after 17-day feeding trial; Fig. 14 illustrates the reduced glutathione concentrations in rat hepatic tissue after 17-day feeding trial;
Fig. 15 illustrates the reduced glutathione concentrations in rat hepatic tissue after 35-day feeding trial;
Fig. 16 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 1-4) and pressurized whey for 17 days; Fig. 17 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 5-8) and pressurized whey for 17 days;
Fig. 18 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 1-5) and pressurized whey (rats # 1-3) for 35 days;
Fig. 19 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 6-8) and pressurized whey (rats # 4-8) for 35 days;
Fig. 20 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 1-4) and pressurized whey (rats # 1-5) for 17 days;
Fig. 21 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 5-8) and pressurized whey (rats # 6-9) for 17 days;
Fig. 22 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 1-5) and pressurized whey (rats # 1-4) for 35 days; Fig. 23 illustrates the Western Blot Analysis of IgG-Mediated Antigenicity of Plasma proteins from rats fed native whey (rats # 6-9) and pressurized whey (rats # 5-9) for 35 days; and
Fig. 24 illustrates the Fourier Transform Infrared (FTIR) Deconvolution Spectroscopic Data of a native 20% whey protein isolate solution before and after exposure to different applied modes of pressure at 400 MPa.
DETAILED DESCRIPTION OF THE INVENTION
In accordance with the present invention, there is provided a specific unique pressurization technique on proteins resulting in potent stimulatory growth and antioxidant effects.
The weight gain, glutathione inducing effect and antioxidant findings in the animals were unexpected and unique. Also, the Applicants have developed a new type of processing using pressurization in conjugation with holding time and recycle of the pressure to enable the use of lower pressures to impact upon the secondary structure protein molecules in a greater magnitude than was observed via earlier work on whey proteins using a single application of pressure at a range of high- pressure treatments up to 1200 MPa.
The Applicants' recent chemical studies have indicated for the first time that pressurization decreases the disulfide content of whey proteins while concurrently increasing the free sulfhydryl content of the proteins. This would suggest that pressurization could increase the bioavailability of sulfur amino acids towards glutathione synthesis by breaking the disulfide bonds. In this manner, pressurization of whey proteins could increase their digestibility to allow for an enhanced generation of small bioactive peptides such as γ-glutamylcysteine that allow for a more rapid intracellular uptake of cysteine. This approach differs dramatically from earlier descriptions on the scientific literature and earlier patents that describe the importance of preserving whey proteins on their undenatured state in order to maintain the integrity. On this basis, earlier patents have emphasized the need of maintaining whey proteins in their undenatured state in order for these proteins to exert significant GSH inducing effects in tissues. In this regard, the present application showing patent tissue glutathione-inducing properties in pressurized whey as compared to native whey proteins was unexpected. The present application has shown that pressurization of native, undenatured whey proteins denatured these proteins and disrupted their disulfide bonds. This pressure treatment also showed a dramatic enhancement of the total glutathione content of tissues including an increase in the concentrations of the reduced form of glutathione, which is the form of glutathione that is the biologically active antioxidant . The new pressurization approach involved a high pressure machine which could produce pressures only as high as 400 MPa but gave large volumes (2 liters) . The technique allowed for a greater impact on the protein molecule's secondary structure than was observed in earlier studies using 1000-1200 MPa pressure. This approach would allow pressurization to be readily used on an industrial scale whereas the pressures involved in earlier work could not be readily transferred to an industrial scale due to the enormous pressures involved (1000-1200 MPa) .
The findings that weight gain, fur quality, and redder color of tissues were affected so dramatically were unexpected.
To our knowledge, the animal trial described in the present patent application is the first evidence that the consumption of a pressurized protein exerts growth stimulatory and antioxidative effects. Based on our hyperbaric-Fourier transform infrared spectroscopic (FTIR) studies, we suggest that pressurization alters the conformation of whey proteins such that the intramolecular disulfide bonds are converted into intermolecular disulfide bonds. Due to the more accessible exposure of intermolecular disulfide bonds to digestive enzymes, this would increase the bioavailability of sulfur amino acids and thereby decrease oxidative stress since these amino acids are precursors of cellular antioxidants such as glutathione. Moreover, the unique findings by the applicants that the disulfide bonds are broken by the pressure treatment would also decrease the resistance of proteins to digestion enzymes (Astwood, J.D. et al., Nature Biotech 1996; 1269-1274) . The increased bioavailability of sulfur amino acids could also be responsible for the growth stimulatory effects as well as the improved fur quality and docile behavior of the animals. The mechanisms of these effects, however, are not completely elucidated. The 50% increase in plasma protein concentrations of rats fed pressurized whey for 35 days is consistent with a growth hormone stimulatory effect. Methods
Protein concentrations and pressure treatment were optimized using the unique approach of: (Astwood, J.D. et al., Na ture Biotech 1996; 1269-1274) exposing the protein to recycles pressure; and (Baruchel, S., Viau, G. Anticancer Res . 1996; 16:1095-1100) holding the protein under pressure for an extended period of time to obtain the maximal changes in secondary structure in the amide I area as detected by FTIR spectroscopy . Accordingly, whey protein isolate solutions were formulated to contain a concentration of 25-40% protein and were stored at 4°C for 12 hours before being subjected to pressure treatment. Whey protein isolates were exposed to 400 MPa for 3 cycles ' with 10 minutes of holding time at 400 MPa. The samples were kept at a constant temperature of 20 °C during the pressure treatments although the method of pulse pressurization described in the present application can be carried out on proteins in solution using a wide range of temperatures (i.e. 0 to 80 °C) . Alternatively, to exert the same effect, an application of 6 cycles plus 3 minutes holding time also can be used. After pressurization, the proteins were added as 24% protein (w/w) in semi-purified diets and fed to 21 day-old newly weaned Sprague Dawley rats. Rats were fed either pressurized and unpressurized whey protein for either 17 or 35 days. Semi-purified diets were formulated according to the NRC requirements for the rat (National Research Council (1995) 4th rev. edn., Na tional Academy of Sciencesr Washington, D.C.). The diet was made fresh every day, access to water and food was freely available for the control and experimental groups. The light/dark cycle was 12 hours/day. Body weight and food intake was monitored every other day.
In another embodiment of the invention, the protein isolate solutions were formulated to contain a concentration of 25% protein. Results No differences in food intake were observed between rats fed native or pressurized whey protein- based diets (Fig. 1) . A significant increase, however, was observed in weight gain, feed efficiency and the protein efficiency ratio in the rats fed the pressurized whey vs. the unpressurized whey-fed rats (Figs. 1 and 2). A significant lowering of plasma concentrations of an index of oxidative stress, thiobarbituric acid reactive substances (TBARS) , was also observed with the group of rats fed the pressurized whey protein (Fig. 3) . The animals fed the pressurized whey were also observed to be calmer and had better general appearance including a thicker fur. The animals fed the pressure-treated .whey were more manageable, had more linear growth and showed no increase in adipose tissue despite their increased body weight. The tissues were observed to be redder in color in the pressurized treated rats, further suggesting a generalized antioxidative effect of tissues due to the preservation of hemoglobin from air oxidation.
A significant (p< 0.05) increase 45% in hepatic concentrations of reduced glutathione was observed in the rats fed the pressurized whey vs. the unpressurized whey-fed rats (Figs. 8-11). A significant (p< 0.05) increase in the reduced glutathione to oxidized glutathione ratio was also observed with the group of rats fed the pressurized whey protein (Figs. 12-13). To the Applicants' knowledge, the animal trial described in this patent application is the first description that the consumption of a pressurized protein exerts tissue glutathione inducing effects. Based on the Applicants' chemical studies, however, the Applicants suggest that pressurization alters the conformation of whey proteins such that the disulfide bonds are converted into free sulhydryl groups. This would increase the bioavailability of sulfur amino acids towards glutathione synthesis and thereby decrease oxidative stress since these amino acids are precursors to glutathione. The mechanisms of these effects, however, are not completely elucidated. The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
EXAMPLE I
Hypoallergenic Effects of Hyperbaric Pressurization of
Whey Proteins
Methods
Western blotting was employed for the detection of antibodies of the native and pressurized whey protein to assess the antigenicity of the both proteins in the serum of the newly weaned rats, which were fed native and mixed mode pressurized whey as dietary protein for 17 and 35 days. Plasma proteins of the both group of the rats on the two diets for the two period of time were treated with sample loading buffer (SDS Reducing Buffer) at pH 6.8 and 5% β-mercaptoethanol and were heated at 95°C for 4 min. The treated samples were loaded to SDS-PAGE gels in Mini-protean II Electrophoresis System, using 4-15%, 10 wells ready gel. An electrode running buffer (pH 8.3) containing 1% SDS was used to separate the proteins at a voltage of 110 and 62 mA current for 50-60 min for two gels. After the electrophoretic separation, the separated proteins were transferred to a nitrocellulose paper using transfer buffer at a pH of 8.3 at a voltage of 121 and 250 mA at the beginning to increase to 300 mA during 1 hour at the end of the transfer time. Immunodetection was carried out with peroxidase-conjugated affinity pure goat anti-rat IgG (H+L) from Jackson Immuno- Research (Cat . #112-035-062) in a dilution of 1:400. To carry out the detection of IgE on blots, the peroxidase-conjugated IgG conjugated fraction of polycolonal goat antiserum to rat IgE, Fc (GARA/GE (Fc)/po) in a dilution of 1:200 was used. For color development an immuno-chemical kit employing horse- radish-peroxidase substrate (Bio-Rad) was used. To quantify the antigenicity of the proteins in each band of the blots after color development the blots were scanned and analyzed using "Quantity One" software (Bio-Rad, Mississauga, Ontario) employing the trace quantity option to perform a rough estimation of the antigenicity of the different fractions of whey protein in serum samples.
Results
Our work indicates that our novel technique of pressurization inhibits the antigenicity of whey proteins, which is directly related to their allergenic effects. Western blot analysis of the rat serum showed a major diminishment or complete absence of IgG and IgE bands corresponding to extracts of the major milk proteins, especially of bovine serum albumin and β- lactoglobulin. (Fig. 16-23). As noted in Tables 1-4, there was a significant inhibition of the antigenicity of β-lactoglobulin, bovine serum albumin, α- lactalbumin, lactoferrin and casein. This latter finding was seen when the pressurized whey was fed for either 17 days or 35 days; however, a greater inhibition of antigenicity was observed in the 35 day- fed rats. This result corresponded with a greater induction of tissue GSH and a greater inhibition of lipid peroxidation in rats fed pressurized whey for 35 days vs. 17 days.
A 20% solution of the whey protein isolate was exposed to different pressure cycles (Fig. 24) . Our hyperbaric-Fourier transform infrared (FTIR) deconvolution spectroscopic data show that our novel pressurization technique induced an irreversible major broadening in the Amide I area of the secondary structure of whey proteins, which is the result of a complete unfolding of the proteins (Fig. 24) . Figure 24 demonstrates that, in comparison to native whey protein, the exposure of the proteins to 1 cycle of pressure at 400 MPa did not change the structure of the molecule although a reduction in the intensity of the amide I bands (1600 - 1700 cm-1) was observed as a function of pressure due to the reduction of volume of the molecules. On the other hand, treatment of the whey protein isolate with 3 cycles of pressure at 400 MPa or 3 cycles of pressure at 400 MPa plus 10 minutes holding time resulted in a irreversible broadening of the amide I band indicating a complete unfolding of the whey proteins. The molecules underwent a new re-arrangement forming different structures (i.e., 1620 cm-1, 1653 cm" 1, 1676 cm-1, 1682 cm-1) as compared to the bands observed in the native protein at 1626 cm-1, 1636 cm-1, 1649 cm-1 and 1693 cm-1. This results thus indicate that the applied modes of pressure of 400 MPa at 3 cycles and at 3 cycles + 10 min holding time were able to make irreversible changes in the secondary structure of whey proteins. Thus, in addition to the disruption of disulphide bonds, the greater free sulfhydryl content observed from our chemical studies are . partly the consequence of an unfolded protein structure that would expose the buried free sulfhydryl groups within the protein molecules to solvent. The findings described in the present patent application were obtained from rats fed pressurized whey proteins that were generated from the pressure treatment of a 20-24% protein solution exposed to 3 cycles of pressure of 400 MPa plus 10 min holding time. Table 1. Trace Quantity Estimate of Western Blot of IgG-Mediated Antigen Reactions in Rats Fed Native and Pressurized Whey proteins for 17 Days
P<0.05 by unpaired t-test, n=8 per group
Table 2. Trace Quantity Estimate of Western Blot of IgG-Mediated Antigen Reactions in Rats Fed Native and Pressurized Whey proteins for 35 Days
*P<0.05 by unpaired t-test, n=8 per group
Table 3. Trace Quantity Estimate of Western Blot of IgE-Mediated Antigen Reactions in Rats Fed Native and Pressurized Whey Proteins for 17 Days
*P<0.05 by unpaired t-test; ND = Not Detectable, n=8 native whey, n=9 pressurized whey
Table 4. Trace Quantity Estimate of Western Blot of IgE-Mediated Antigen Reactions in Rats Fed Native and Pressurized Whey Proteins for 35 Days
*P<0.05 by unpaired t-test, n=9 per group
Table 5. Effects of hyperbaric pressurization treatment on disulfide bonds of proteins
Discussion
Since the allergenicity is dependent on antibody recognition of specific epitopes on the protein molecule, irreversible changes in protein conformation induced by high pressure might result in a reduction of allergenicity. Pressure treatment can drastically alter protein conformation under applied hydrostatic pressures; however, irreversible changes in protein structure have not been noted under single applications of hyperbaric pressure even under high pressure conditions up to 1200 Mpa (Hosseininia T. et al. J. Agric . Food Chem . 1999;47:4537-4542).
The present patent application shows for the first time a method by which protein structure can be irreversibly denatured, particularly in terms of disulphide bond breakage. Based on our chemical studies we have shown that pressurization alters the conformation of whey proteins such that the disulphide bonds are decreased concurrent with an increase with free sulfhydryl groups . Highly allergenic proteins such as β-lactoglobulin have a high disulphide content, which increases the resistance of the protein to digestion. The presence of disulfide bonds is also related to the allergenic properties of food proteins. Hence, the decrease in disulfide bond content along with the increase in free sulfhydryls induced by our pressurization technique explain our observed findings of an increase in protein digestibility (i.e., increase in protein efficiency ratio) along with the decreased allergenic action following intake of pressurized proteins. The irreversible conformational changes induced by our novel pressurization method with respect to the unfolding of whey proteins from a globular compact shape also allow for a greater accessibility to the action of digestive enzymes in vivo. The animal trial described in the present patent application is the first description that the consumption of a protein exposed to pressure treatment alone exerts hypoallergenic effects. These findings are compatible with previous research showing breakage of protein disulphides via enzymatic thioredoxin treatment induces a hypoallergenic effect on milk proteins (Del Val G. et al., J Allergy Clin Immunol 103:690-7, (1999)).
Previous work by Hayashi et al . (1987) (Hayashi, R.et al. J. Food Sci . 1987; 52; 1107-1108) has shown high pressure treatment combined with digestive enzymatic treatment eliminated the allergenic effects of milk proteins along with an increase in their digestibility. Our present patent application differs from this previous work in that pressure treatment alone was demonstrated to induce hypoallergenic effects without the need for any additional treatments. Also, our pressurization technique differs greatly from any previous pressurization methods as we have described irreversible changes in secondary structure of the proteins along with decreases in disulfide bond content. No previous high pressure study has described such molecular changes from the treatment of pressure alone, which is derived from our novel approach of the application of low levels of pressure with specific pulse and continuous modes. This innovative method has lead to the dramatic changes in the nutritional and antigenic properties of the treated proteins, not described previously via pressure treatment.
EXAMPLE II
Effects of hyperbaric treatment on soy and peanut proteins The effect of five pulse cycles up to 400 MPa plus 5 minutes holding time was tested on free sulfhydryl levels in solutions containing 12.5% concentrations of either soy protein or peanut protein. Determination of activated sulfhydryl groups of native and pressurized soy and peanut proteins was performed with dithionitrobenzoic acid (DTNB) reagent using the method of Koka (Koka, M. et al . J. Dairy Sci . 1910 , 51, 217-219) . DTNB solution was added to native and pressurized solutions containing either soy protein isolate or peanut protein and the solutions were allowed to develop the presence of yellow color at room temperature.
Pressurized solutions of both peanuts and soy protein showed the development of a strong yellow color. The development of the deep yellow color is directly related to a major increase in exposed free sulhydryl groups generated from the breakage of disulfide bonds via the pressure treatment.
On the other hand, native protein solutions of soy and peanut proteins showed minimal development of yellow color indicating low levels of exposed free sulfhydryl groups in the native proteins. The results thereby indicate that repeated pulse cycling of pressure with holding time can result in breakage of disulfide bonds in proteins other than whey proteins. As we observed that pressure-treated disruption of disulfide bonds was related to an inhibition of the antigenic response of whey proteins, our findings thus demonstrate that repeated pulse cycling could be utilized as a means to inhibit the allergenic properties of diverse types of proteins. Also, similar to our observations with whey proteins, the increase in free sulfhydryl groups would increase the growth stimulatory and antioxidant potential of a variety of other proteins such as soy or peanut. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features herein before set forth, and as follows in the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A method to induce conformational changes in proteins to enhance their susceptibility to enzymes, which comprises the step of: a) exposing a protein in a solution, having a concentration of about 0.01% to about 50%, to a pressurization treatment sufficient to induce permanent conformational changes in proteins .
2. The method of claim 1, wherein the pressurization treatment is of at least 100 MPa for at least 2 cycles.
3. The method of claim 1, wherein the pressurization treatment is of about 400 MPa for 3 to 6 cycles .
4. The method of claim 1, wherein the pressurization treatment is followed by about 3-5 minutes of holding time at 400 MPa.
5. The method of claim 1, wherein said enzymes are digestive enzymes .
6. The method of claim 5, wherein said digestive enzymes when located in vivo enhances digestibility of said protein.
7. The method of claim 1, which further comprises a step effected prior to step a) , wherein said protein is solubilized in solution by adding a solubilization agent or by storing said protein solution at about 4°C for at least 12 hours.
8. The method of claim 1, wherein said protein is selected from the group consisting of whey, soy and peanut proteins .
9. The method of claim 1, wherein said protein is selected from the group consisting of caseins, egg, legume, nut, milk, fish, meat proteins, albumin and hormones .
10. The method of claim 1, wherein said protein is solubilized in a solution selected from the group consisting of aqueous, saline, isoelectric, isotonic, and buffered solutions, nutrient-based solutions and formulas used in infant feeding and in enteral and parenteral nutrition.
11. The method of claim 1, wherein said protein concentration is of about 12.5-40%.
12. The method of claim 11, wherein said pressurization treatment is of at least 100 MPa for at least 2 cycles.
13. The method of claim 11, wherein said pressurization treatment of about 400 MPa for 3 to 6 cycles .
14. The method of claim 11, wherein said pressurization treatment is followed by about 5-10 minutes of holding time at 400 MPa.
15. The method of claim 11, wherein said protein during said pressurization treatment is at a temperature ranging from 0 to 80°C.
16. The method of claim 15, wherein said temperature is 20°C.
17. The method of claim 11, wherein said protein is kept at 4°C.
18. A protein composition having enhanced susceptibility to enzymes for administration to an animal, which comprises between 1% (w/w) to 100% (w/w) protein prepared by the method of claim 1; and an acceptable pharmaceutical carrier between 0% (w/w) to 99% (w/w) .
19. The protein composition of claim 18, wherein said enzymes are digestive enzymes.
20. The protein composition of claim 19, wherein said enhanced susceptibility to digestive enzymes enhances digestibility of said proteins.
21. The protein composition of claim 20, wherein said enhanced digestibility causes stimulatory growth effect, increased fur quality and/or calm behavior, antioxidant properties and/or glutathione inducing effect for said animal .
22. The protein composition of claim 21, wherein said antioxidant properties causes enhanced redness of said animal meat .
23. The protein composition of claim 18, wherein said animal is selected from the group consisting of human, mammal, bird, amphibian, fish, reptile and insect .
24. The protein composition of claim 18, wherein said protein is at 24% (w/w) .
25. The protein composition of claim 18, wherein said acceptable pharmaceutical carrier is selected from the group consisting of exipient for oral, topical, intravenous, systemic, intraperitoneal or subcutaneous administration to said animal.
26. The protein composition of claim 25, wherein said oral exipient is selected from the group consisting of food, water, juice, milk, carbonated and non-carbonated beverages, sweetened and unsweetened drinks, enteral, parenteral, infant formulas and health drinks .
27. The protein composition of claim 18, wherein said protein is selected from the group consisting of whey, soy, and peanut proteins.
28. The protein composition of claim 18, wherein said protein is selected from the group consisting of caseins, egg, legume, nut, milk, fish, meat proteins, albumin and hormones .
29. A method to stimulate growth, increase fur quality and/or calm behavior of an animal, which comprises administering an effective amount the protein composition of claim 18.
30. Use of the protein composition of claim 14 in the preparation of a medicament to stimulate growth, increase fur quality, induce glutathione and/or calm behavior of an animal .
31. A method to induce conformational changes in proteins to reduce their allergenic properties, which comprises the step of: a) exposing a protein in a solution, having a concentration of about 20% to about 24%, to a pressurization treatment sufficient to induce permanent conformational changes in proteins.
32. The method of claim 31, wherein the pressurization treatment is of at least 100 MPa for at least 2 cycles.
33. The method of claim 31, wherein the pressurization treatment is of about 400 MPa for preferably 3 cycles.
34. The method of claim 31, wherein said pressurization treatment is followed by 10 minutes of holding time.
35. The method of claim 31, which further comprises a step effected prior to step a) , wherein said protein is solubilized in solution by adding a solubilization agent or by storing said protein solution at about 4°C for at least 2 hours .
36. The method of claim 31, wherein said protein is selected from the group consisting of whey, soy and peanut proteins.
37. The method of claim 31, wherein said protein is solubilized in a solution selected from the group consisting of aqueous, saline, isoelectric, isotonic and buffered solutions, nutrient-based solutions and formulas used in infant feeding and in enteral and parenteral nutrition.
38. The method of claim 31, wherein said protein during said pressurization treatment is at a temperature ranging from 0 to 80°C.
39. The method of claim 38, wherein said temperature is 20 °C.
40. The method of claim 31, wherein said protein is kept at 4°C.
41. A protein composition having reduced allergenic effects for administration to an animal, which comprises between 1% (w/w) to 100% (w/w) protein prepared by the method of claim 31; and an acceptable pharmaceutical carrier between 0% (w/w) and 99% (w/w) .
42. The protein composition of claim 41, wherein said animal is selected from the group consisting of human, mammal, bird, amphibian, fish, reptile and insect.
43. The protein composition of claim 41, wherein said acceptable pharmaceutical carrier is selected from the group consisting of exipient for oral, topical, intravenous, systemic, intraperitoneal or subcutaneous administration to said animal .
44. The protein composition of claim 43 wherein said oral exipient is selected from the group consisting of food, water, juice, milk, carbonated and non-carbonated beverages, sweetened and unsweetened drinks, enteral, parenteral, infant formulas and health drinks .
45. The protein composition of claim 41 wherein said protein is selected from the group consisting of whey, soy, and peanut proteins.
46. The protein composition of claim 41 wherein said protein is selected from the group consisting of caseins, egg, legume, nut, milk, fish and meat proteins .
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WO2005067731A1 (en) * 2004-01-20 2005-07-28 Technion Research & Development Foundation Ltd. Method and apparatus for reducing allergenic activity
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