JP2002238463A - Method for producing gluten-free peptide preparation and obtained preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gluten-free peptide preparation rich in glutamine. SOLUTION: This method for producing the gluten-free peptide preparation rich in the glutamine from a gluten protein comprises a step for enzymatically hydrolyzing the gluten by using one or more kinds of proteases to provide a hydrolyzate, allowing the hydrolyzate to have acidity of pH 4-5, filtering the acidified hydrolyzate to provide the objective gluten-free peptide rich in glutamine as a filtrate. The protease is referably a neutral or a basic protease, and the optimum pH for acidification is 4.5-4.7.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a process for the preparation of a glutamine-rich and gluten-free peptide preparation and to the resulting preparation. The invention also relates to the use of the preparation in various products and to products containing the preparation.

[0002]

BACKGROUND OF THE INVENTION Gluten is derived from various cereals such as wheat, barley, rye, oats and other gluten-containing wheat varieties such as triticale, spellt and kamut. A combination of proteins found in endosperm. In wheat, gluten accounts for as much as 90% of the protein, almost 1% of the total weight of the grain.
Make up to 5%. Therefore, it is an important source of protein. However, gluten is celiac (coel
iac) disease or a genetic disease known as gluten intolerance. Signs of celiac disease can range from classic features such as diarrhea, weight loss and malnutrition to potential symptoms such as isolated nutritional deficiencies. The disease mainly affects European offspring and is less common in blacks and Asians. Affected individuals have identified that they have specific edible grain antigens (toxic amino acid sequence) found on wheat, barley, rye, oats and other gluten-containing wheat varieties such as triticale, spelled and camute wheat. ) Suffers damage to the villi (shortening and villous settling) in the basement membrane and crypt area of their intestines when feeding. Gluten found in rice and corn does not cause this intolerance. For people with celiac disease, the toxic part of the gluten molecule is the prolamin part: gliadin in wheat, secalin in rye and hordein in barley. Following a gluten-free diet, people recover from the symptoms of the disease but cannot cure it. Reintroducing gluten during a meal again causes symptoms. Glutamine is an amino acid abundant in gluten. It is not an essential amino acid but is desirable for some people, especially those recovering from surgery, gastrointestinal disorders, immune dysfunction, metabolic stress, shock or behavioral endurance sports It is. Such people would benefit from supplementation of this amino acid, for example, a glutamine-rich peptide preparation. Gluten is a very cost effective source for such glutamine-rich peptide preparations. However, the known preparations are not suitable for celiac patients as they still contain the toxic part of gliadin.

[0003]

It is an object of the present invention to provide peptide preparations which are rich in bound glutamine but are also gluten free.

[0004]

The object of the present invention is to provide a peptide preparation comprising: a) enzymatically hydrolyzing gluten using one or more proteases to obtain a hydrolyzate; Object
C) filtering the hydrolyzate to obtain a glutamine-rich gluten-free peptide as a filtrate.

[0005]

DETAILED DESCRIPTION OF THE INVENTION As used herein, the term "gluten-free" refers to an anti-.OMEGA.-gliadin-based EL.
It is intended to indicate a product that yields a value of <200 ppm when tested by ISA. An ELISA suitable for testing the gluten-free properties is the Association of
Official Analytical Chemists'(AOAC's) Official M
ethodsof Analysis, 15th Edition, 2nd, Supplement
(1991). Apparently,
The protease used can be selected from a wide range of proteases known in the art, but hydrolysis with the protease will result in a preparation that yields <200 ppm in the above ELISA. Proteases include acidic, basic or neutral proteases from bacterial, fungal, animal or plant sources. p
Basic or neutral proteases active above H6 have proven to be particularly suitable. Examples of such proteases include Proleather N (Amano),
Neutrase (NOVO), PRPMOD 192
P (Biocatalysts), Alcalase 2.4
L (NOVO), protease S (Amano), peptidase A (Amano), and peptidase R (Amano). Of these, Proleather N (Amano) and Alcalase 2.4L (NOVO) are preferred.

Surprisingly, protein fragments that cause hypersensitivity in celiac patients are removed by acidifying the hydrolyzate and then filtering. These fragments are expected to precipitate and remain in the filter retentate. The pH for acidifying the hydrolyzate is 4
And 5, preferably 4.1 to 4.9, more preferably 4.3 to 4.8, most preferably 4.5 to 4.7.
And 4.6 is optimal. Hydrolysis is an essential step in the process of the present invention, since toxic fragments cannot be removed without hydrolysis.

A peptide preparation obtainable by the process according to the invention consisting of a peptide which does not induce gluten hypersensitivity symptoms in celiac patients is a further aspect of the invention. Such preparations are suitable as food additives and foods to supplement the subject with additional glutamine. Thus, the preparation is applicable for sports and clinical use, and can also be used for enteral nutrition and pet food. The peptide preparations of the present invention can further be used in products that are consumed by or administered to a subject in need of supplementation. Specific examples of such products include the usual carriers, diluents and excipients for tablets and glutamine peptide tablets comprising the peptide preparations of the invention as a source of glutamine peptide, the usual glutamine peptide tablets for beverages. Ingredients, conventional carriers, diluents and excipients for glutamine peptide liquid beverages and enteral nutrition comprising the peptide preparations of the invention as a source of glutamine peptide, and the invention as a source of glutamine peptide Glutamine comprising a peptide preparation of
Peptide enteral nutrition. The invention applies more broadly to gluten from all cereals that cause celiac disease, but it is preferred to use wheat because of its high glutamine content.

[0008]

EXAMPLES The present invention will be described in more detail with reference to the following Examples, which are for illustrative purposes only and do not limit the scope of the present invention. Example 1 Preparation of Glutamine-Rich, Gluten-Free Peptide Preparation A series of experiments were performed to illustrate critical manufacturing parameters. Deionized water was heated to 63 ° C. ± 1 ° C. to prepare a series of peptide hydrolysates. In this water, a ratio of 45% liquid potassium hydroxide, 50% liquid sodium hydroxide, and hydrated calcium hydroxide was 1: 0.78: 0.7, respectively.
0 and add the appropriate p to the protease used.
Get H. Activated wheat gluten ("VWG", Cargill B.
V., Bergen op Zoom, Netherlands) is added to this solution to prepare a 12% solids mix of solubilized gluten. Hydrolysis is performed with the desired protease described in a separate experiment below. The hydrolysis reaction is carried out for 3 hours at a temperature suitable for the protease used, usually 60 ° C. ± 2 ° C. After the hydrolysis, an acid, especially sulfuric acid, is added and stirred to the desired pH (see experimental description). HTST (hi
Heat to 116 ° C. ± 2 ° C. in gh temperature short time and stop. The solution was then cooled to 66 ° C ± 2 ° C and diatomaceous earth (Eagle-Picher Minerals Inc., Reno, NV,
USA) with a 40% body feed.
The solution is recirculated through the filter press for a minimum of 3 minutes.

[0009] The pH of the filtrate is adjusted to 6.4 to
Adjust to 6.8. After evaporating the liquid, it is dried to obtain the peptide preparation of the present invention in powder form. To test if the product is gluten free, AOAC 991.1
9 (Official Methods of Analysis, 15th Edition, 2n
d Supplement (1991)). The bound glutamine content was determined by PE Wilcox, "Determinati
on of Amide Residues by Chemical Methods ", Methods
of Enzymology 11, 63-76 (1967).
A measure of the degree of proteolysis is the AN / TN ratio. AN
Can be determined by formol titration or by J. Adler-Nissen, Enzymat
ic hydrolysis of food preoteins, ElsevierApplied S
Amino nitrogen content that can be measured according to the Science Publishers, 1986. TN is the total amino nitrogen content measured according to the Kjeldahl nitrogen measurement method. AN / TN
The higher the ratio, the higher the degree of hydrolysis of the protein preparation.

Experiment Experiment 1 Wheat gluten is dispersed in water. The pH is adjusted to 4.6 with sulfuric acid and the solution is filtered. Experiment 2 Wheat gluten is dispersed in water. pH is adjusted to 3.2 with sulfuric acid
Adjust to 3.4. Gluten to acidic protease II
Digest using (Amano). The enzyme is heat inactivated and the solution is filtered. Experiment 3 Wheat gluten is dispersed in caustic water. Gluten is converted to the alkaline and neutral protease Alcalase 2.4 L
(NOVO) and Proleather N (Amano), and amylase BAN240L (NOVO). After inactivation of the enzyme, the pH is adjusted to near neutral with sulfuric acid and the solution is filtered. Experiment 4 Wheat gluten is dispersed in caustic water. Gluten is converted to the alkaline and neutral protease Alcalase 2.4 L
(NOVO) and Proleather N (Amano). Adjust the pH to 3.8-4.1 with sulfuric acid. The enzyme is heat inactivated and the solution is filtered. Then, caustic pH
To neutral. Experiment 5 Wheat gluten is dispersed in caustic water. Gluten is converted to the alkaline and neutral protease Alcalase 2.4 L
(NOVO) and Proleather N (Amano). The pH is adjusted to 6.5 with sulfuric acid. The enzyme is heat inactivated and the solution is filtered. Adjust the pH to neutral with caustic. Experiment 6 Wheat gluten is dispersed in caustic water. Gluten is converted to the alkaline and neutral protease Alcalase 2.4 L
(NOVO) and Proleather N (Amano). The pH is adjusted to 4.3 with sulfuric acid. The enzyme is heat inactivated and the solution is filtered. Adjust the pH to neutral with caustic. Experiment 7 Wheat gluten is dispersed in caustic water. Gluten is converted to the alkaline and neutral protease Alcalase 2.4 L
(NOVO) and Proleather N (Amano). The pH is then adjusted to 4.5 with sulfuric acid. The enzyme is heat inactivated and the solution is filtered. Adjust the pH to neutral with caustic. Experiment 8 Wheat gluten is dispersed in caustic water. Gluten is converted to the alkaline and neutral protease Alcalase 2.4 L
(NOVO) and Proleather N (Amano). The pH is adjusted to 4.6 with sulfuric acid. The enzyme is heat inactivated and the solution is filtered. Adjust the pH to neutral with caustic. Experiment 9 Wheat gluten is dispersed in caustic water. Gluten is converted to the alkaline and neutral protease Alcalase 2.4 L
(NOVO) and Proleather N (Amano). Adjust the pH to 4.8 with sulfuric acid. The enzyme is heat inactivated and the solution is filtered. Adjust the pH to neutral with caustic.

Table 1 shows the results of the above experiment. As is evident from the examples above, hydrolysis of gluten and filtration at acidic pH are essential for gluten-free products.

[Table 1] TN = 13%

Example 2 Application of the glutamine-rich gluten-free peptide preparation of the invention Three examples of application of the preparation of the invention are given below. 1. Glutamine peptide tablets Ingredients (1) Enzymatically hydrolyzed wheat protein (granules) (Preparation of the present invention) (2) Pharmacel 102 (3) CAB-O-SIL M-5 Formulation Enzymatically hydrolyzed wheat protein ( 1) 91.1% Microcrystalline cellulose (2) 5.0% Dicalcium phosphate 2.0% Silicon dioxide (3) 0.9% Stearic acid 0.5% Magnesium stearate 0.5% Total 100% Manufacturing method Premix the powder (magnesium stearate is added at the end of the mixing). The tablets are manufactured by direct compression. Tablet performance Glutamine peptide per tablet 170 mg Tablet weight 758 mg Tablet length (ellipse) 19.04 mm Compression pressure 13.3 kN Hardness 140 N

2. Glutamine peptide liquid beverage Ingredients (1) Enzyme hydrolyzed wheat protein (preparation of the present invention) (2) Enzyme hydrolyzed whey protein (WE80BG, DMV Internat
ional) (3) Grapefruit flavor (Tastemaker 946)
048) Formulation Water (amount to be 1 L) 920.00 g Enzymatically hydrolyzed wheat protein (1) 13.21 g Enzymatically hydrolyzed whey protein (2) 13.04 g Sucrose 26.60 g Glucose 15.00 g Fructose 5.00 g Glucose. Polymer (Maltodextrin DE18) 10.00 g Malic acid 3.33 g Citric acid 0.67 g Sodium citrate 1.00 g Grapefruit flavor (3) 0.60 g Aspartame 0.10 g Acesulfame potassium 0.10 g 1000.0 mL Manufacture Method Add all ingredients to water and mix well. Acid is added last to bring the pH to 3.9. The liquid is bottled, heated at 85 ° C. for 1 minute, and cooled. Nutritional status (per 100 mL) Protein 2.09 g Glutamine peptide 0.26 g Carbohydrate 6.0 g

3. Clinical Enteral Nutrition Prototype Containing Glutamine Peptide and Wheat Peptide Ingredients (1) Enzymatically hydrolyzed wheat protein (preparation of the present invention) (2) Enzymatically hydrolyzed whey protein (WE80BG, DMV Internat
ional) Formulation Water (amount to be 1 L) 720.00 g Enzyme-hydrolyzed wheat gluten (1) 40.00 g Enzyme-hydrolyzed whey (2) 35.60 g Chemically modified starch 84.00 g Maltodextrin 59.00 g Soybean oil 30.00 g MCT oil 10.00 g Potassium citrate 2.20 g Sodium citrate 1.60 g Magnesium chloride 3.20 g Calcium phosphate 2.80 g Potassium phosphate 2.00 g Sodium phosphate 1.00 g Carrageenan 0.50 g 1000.0 mL Manufacturing method Constant The minerals are dissolved in water with stirring. The premixed carbohydrate is added to this mixture. Mix 70
Heat to ° C. and hold for 10 minutes with constant stirring. To this protein is added and heated to 70 ° C. with constant stirring. Add oil to this and mix well. The mixture was then added to 400
Homogenize twice at 0 psig (276 bar).
Adjust pH and solids content appropriately. 121 ° C
Sterilize by heating in a retort for 10 minutes. Nutrition status (per 100 mL) Protein 6.0 g Glutamine peptide 1.0 g Carbohydrate 13.8 g Fat 4.0 g

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification FI theme coat ゛ (Reference) A61P 3/02 A61K 37/02 (72) Inventor Edward Allen Hunter Box 77, Hancock, New York, USA H 63 F term (reference) 4B017 LC03 LK15 LP06 4B018 LB08 LE01 MD20 ME07 MF12 4C084 AA02 AA03 AA06 MA02 MA66 NA05 ZA66 4C206 AA01 AA02 FA53 MA02 MA36 MA55 MA72 MA86 NA05 ZA66

Claims (16)

[Claims] 1. a) enzymatic hydrolysis of gluten with one or more proteases to obtain a hydrolyzate; b) acidification of the hydrolyzate to pH 4-5; c) said hydrolyzate A process for obtaining a glutamine-rich gluten-free peptide as a filtrate, comprising the steps of: preparing a glutamine-rich gluten-free peptide preparation from a gluten protein; 2. The method according to claim 1, wherein the protease is a neutral or basic protease. 3. The process according to claim 1, wherein the pH is 4.2 to 4.8. 4. The method according to claim 1, wherein the pH is 4.5 to 4.7. 5. The method according to claim 1, wherein a protease active at pH 6 or higher is used. 6. The process according to claim 1, wherein the enzyme is inactivated between steps b) and c). 7. The method according to claim 6, wherein the enzyme is inactivated by heating.
Production method as described. 8. The method of claim 1, wherein the gluten is wheat gluten.
8. The method according to any one of claims 1 to 7. 9. A peptide preparation prepared from gluten protein, which is glutamine-rich and gluten-free and can be produced by the production method according to any one of claims 1 to 8. 10. The peptide preparation according to claim 9, wherein the gluten for preparing the preparation is wheat gluten. 11. The peptide preparation according to claim 9, which is for a component of a glutamine peptide tablet. 12. The peptide preparation according to claim 9, which is used as a component of a glutamine peptide liquid beverage. 13. The peptide preparation according to claim 9, which is for an enteral nutrition component of a glutamine peptide. 14. Glutamine comprising a usual carrier, diluent and excipient for tablets and a peptide preparation according to claim 9 or 10 as a source of glutamine peptide.
Peptide tablets. 15. A normal ingredient for beverages, comprising glutamine
A glutamine peptide liquid beverage comprising a glutamine peptide comprising the peptide preparation according to claim 9 or 10 as a peptide source. 16. A common carrier, diluent and excipient for enteral nutrition and as a source of glutamine peptide.
Or a glutamine peptide enteral nutrition comprising the peptide preparation of 10 above.