TWI404781B - Process for making a low molecular weight gelatine hydrolysate and gelatine hydrolysate compositions - Google Patents

Abstract

The present invention provides a process to make a gelatine hydrolysate, a gelatine hydrolysate and gelatine compositions comprising gelatine hydrolysates. More specifically, the invention provides gelatine compositions having improved cross-linking and dissolution properties.

Description

Method for producing low molecular weight gelatin hydrolyzate and gelatin hydrolysate composition

The present application claims priority to USSN 11/140,863 filed on May 31, 2005, the entire disclosure of which is incorporated herein by reference.

The present invention generally relates to a gelatin hydrolysate, a method of making the gelatin hydrolysate, and a composition comprising the gelatin hydrolysate. More specifically, the present invention provides a low molecular weight gelatin hydrolysate having a high first amine content, a method of producing the gelatin hydrolysate, and a gelatin composition comprising a gelatin hydrolysate.

Gelatin is produced by denaturation of collagen contained in materials such as pig skin, cowhide and animal bone. Similar to its parent protein, maternal collagen, gelatin is defined by a special structure comprising a unique blend of amino acids. Native collagen is a hard protein based on a polypeptide chain comprising about 1050 amino acids. Three of these polypeptide chains aggregate to form a triple helix. Several of these triple helices are superimposed to produce fibrils of cross-linked stabilized collagen to form a three-dimensional network structure. This particular structure renders the collagen insoluble; it then achieves a soluble form by partial hydrolysis to gelatin or gelatin hydrolysate. The amino acid content of collagen and the gelatin thus produced is about one-third of glycine and another 22% of proline and 4-hydroxyproline; the remaining 45% contains 17 different amino acids. Gelatin has a particularly high content of acidic amino acids and basic amino acids. The variable acid amino acids (glutamic acid and aspartic acid) are present in the form of a guanamine such as glutamic acid and aspartic acid, depending on the processing conditions used in the gelatin manufacturing process. . Cysteine is completely absent; among the thioaminic acids, methionine is the only thiol-containing acid present.

The information on poultry collagen and fish collagen is slightly different, but the present invention is equally applicable to gelatin and/or gelatin hydrolysate derived from poultry collagen and fish collagen.

Gelatin can be used in a large number of applications depending on its starting materials and manufacturing methods. This is due to the fact that the physical and chemical properties of gelatin are determined by the combination of the amino acid content and the resulting spatial structure, and on the other hand by various conditions such as pH, ion concentration and reaction with other molecules. Decide. For example, different types of gelatin are used in different applications such as food, photography, cosmetics, and medicine.

In the pharmaceutical industry, gelatin is especially useful in the manufacture of hard and soft capsules. Gelatin capsules provide a convenient and effective means of oral administration of the drug, since the capsules rapidly disintegrate upon exposure to the acidic contents of the stomach, thereby releasing the drug into the body. While gelatin capsules provide a pharmaceutically acceptable means of administration of the drug, the presence of gelatin capsules can be subject to the risk of disintegration and dissolution delays caused by processes known as cross-linking. The carbonyl group in the gelatin, the filling component or the filling component of the carbonyl group in the capsule is decomposed into a carbonyl group, and reacts with the first amine and other nitrogen-containing compounds present in the gelatin to form a crosslink.

Cross-linking in particular can have serious adverse effects on the efficacy of gelatin capsules after prolonged storage and exposure to heat and humidity. The wide range of gelatin cross-linking in capsule formulations can result in the formation of extremely thin, tough, and water-insoluble films, commonly referred to as films. The film acts as a rubbery, water-insoluble layer that can limit or prevent the release of the contents of the capsule.

A widely reported method for preventing cross-linking in gelatin capsules focuses on acting as a carbonyl scavenger, preventing the interaction of a carbonyl group (e.g., an aldehyde group) with a gelatin capsule shell, thereby preventing gelatin cross-linked products. These methods generally suggest adding the product to the pharmaceutical composition contained in the gelatin capsule. For example, it has been shown that amino acid glycine and citric acid are added in combination to a formulation encapsulated in a hard gelatin capsule to improve the dissolution profile of the hard capsule (3). The addition of aminoglycolic acid alone did not prove to produce satisfactory results. However, the addition of a carbonyl scavenger such as glycine in an amount required to reduce cross-linking in a gelatin capsule, and a carboxylic acid such as citrate are significantly more expensive. Therefore, the addition of such products to gelatin is not a practical solution to reduce cross-linking in gelatin capsules.

The invention provides a practical and cost-effective method for reducing cross-linking in gelatin. Briefly, the present invention contemplates a low molecular weight gelatin hydrolysate which, when blended with higher molecular weight gelatin, increases the amount of free glycine, other amino acids, and small peptides in the blended gelatin product. The cross-linking of the gelatin is reduced and the solubility characteristics are improved. Advantageously, since the gelatin hydrolysate of the present invention and the blended gelatin composition have reduced cross-linking properties without the addition of a product such as glycine acid as a separation compound mixed with citric acid, gelatin can still be used. It is sold as a natural product.

Thus, in several aspects of the present invention, a method of manufacturing as having about 100 Da to about 2000 Da, preferably of about 1500 Da average molecular weight, and gelatin hydrolyzate per μg of approximately 1.0 × 10 ^{- 3} μMol to about 1.0 × 10 ^- A method of hydrolyzing a gelatin hydrolysate having an average first amine content of ² μMol of the first amine. The method comprises contacting a gelatin starting material with at least one proteolytic enzyme having endopeptidase activity to form an endopeptidase digested gelatin product. The endopeptidase-digested gelatin product is then typically contacted with at least one proteolytic enzyme having peptide chain endolytic activity. In general, proteolytic digestion of the endopeptidase and proteolytic digestion of the peptide end-endase are carried out for a sufficient period of time and under reaction conditions to form a gelatin hydrolysate.

Another aspect of the invention encompasses a method of making a gelatin hydrolysate. The method comprises contacting a gelatin starting material with a series of at least three proteolytic enzymes having endopeptidase activity to form an endopeptidase digested gelatin product. Typically, the three proteolytic enzymes are endopeptidases from Bacillus subtilis (eg Corolase) 7089), pineapple protease (eg Enzeco A mixture of a pineapple protease concentrate and papain (eg papain 6000L). The endopeptidase-digested gelatin product is then contacted with a series of at least two proteolytic enzymes having peptide chain endolytic activity. In general, the two proteolytic enzymes are derived from a peptide chain endolytic enzyme from Aspergillus oryzae (eg Validase) FPII) and from Aspergillus sojae (eg Corolase) LAP) peptide chain endolytic enzyme composition.

Yet another aspect of the invention provides a hydrolysate. The gelatin hydrolyzate will generally have from about 100 Da to about 2000 Da, preferably of about 1500 Da average molecular weight, and gelatin hydrolyzate per μg of approximately 1.0 × 10 ^- 1.0 × 10 ³ μMol about to ^- an average of ^{2 μMol} first amine First amine content. In one embodiment, the gelatin hydrolysate is comprised of a method comprising contacting a gelatin starting material with a series of at least three proteolytic enzymes having endopeptidase activity to form an endopeptidase digested gelatin product. Manufacturing. Typically, the three proteolytic enzymes are selected from an endopeptidase from Bacillus subtilis (eg, Corolase) 7089), pineapple protease (eg Enzeco Bromelain concentrate) and papain (eg papain 6000L). The endopeptidase-digested gelatin product is then contacted with a series of at least two proteolytic enzymes having peptide chain endolytic activity. In general, the two proteolytic enzymes are selected from the group consisting of peptide chain endolytic enzymes from Rice Bacteria (eg Validase) FPII) and from soy sauce (eg Corolase) LAP) peptide chain endolytic enzyme.

Another aspect of the invention is directed to a gelatin composition. The composition comprises a gelatin hydrolysate and gelatin. Typically, the composition will comprise from about 1% to about 20% by weight of the gelatin hydrolysate and from about 80% to about 99% by weight of gelatin.

Other objects and features of the present invention will be apparent in part in the description which follows.

The present invention provides a novel gelatin hydrolysate, a method of making the gelatin hydrolysate, and a gelatin composition comprising the gelatin hydrolysate. It has been found that blending low molecular weight gelatin hydrolysate and especially the gelatin hydrolysate of the present invention with gelatin reduces the amount of gelatin by increasing the amount of free glycine, other amino acids and small peptides in the blended gelatin product. Linking trends and improving solubility characteristics. Advantageously, the present invention provides a cost effective method of reducing gelatin cross-linking that has the benefit of maintaining the original amino acid composition of gelatin without the need to add non-gelatin derived compounds such as citric acid. Therefore, the gelatin hydrolysate composition of the present invention can still be sold as a natural product.

I. Method for producing gelatin hydrolysate

One aspect of the present invention encompasses a method of fabricating a sample of about 100 Da to about 2000 Da, preferably of about 1500 Da average molecular weight, and gelatin hydrolyzate per μg of approximately 1.0 × 10 ^{- 3} μMol to about 1.0 × 10 ^{- 2} μMol first A method of hydrolyzing a gelatin product having an average first amine content of an amine. The method comprises contacting a gelatin starting material with at least one proteolytic enzyme having endopeptidase activity to form an endopeptidase digested gelatin product. The endopeptidase-digested gelatin product is then typically contacted with at least one proteolytic enzyme having peptide chain endolytic activity. In general, proteolytic digestion of the endopeptidase and proteolytic digestion of the peptide end-endase are carried out for a sufficient period of time and under reaction conditions to form a gelatin hydrolysate.

The gelatin starting materials used in the methods of the present invention are typically derived from collagen or collagen-rich tissue available from several suitable materials. Collagen-rich tissues include skin and bone from animals such as fish, poultry, pigs or cattle. There are generally two main types of collagen-derived gelatin, type A and type B, which differ in their method of manufacture. In one embodiment, the gelatin starting material is Type A gelatin. Type A having an isoelectric point of 7 to 10.0 is derived from collagen under exclusive acid pretreatment by methods generally known in the art. In an alternative embodiment, the gelatin starting material is Type B gelatin. Form B, which has an isoelectric point of 4.8 to 5.8, is a result of alkaline pretreatment of collagen and is produced by a method generally known in the art.

In another alternative embodiment, the gelatin starting material is a mixture of Form A and Form B. The amount of each of Type A gelatin and Type B gelatin can vary to a large extent without detrimental effects on the properties of the gelatin hydrolysate produced.

In principle, any of type A gelatin and type B gelatin may be exchanged, in whole or in part, by enzymatically produced gelatin. However, enzymatic methods for making gelatin have not been widely used to date.

Regardless of the embodiment, the gelatin starting material will typically comprise from about 80% to about 90% protein by weight, from about 0.1% to about 2% by weight inorganic salt (corresponding to ash content) and from about 10% to 15% % by weight of water.

It is also desirable that the physical properties of the gelatin starting material can vary depending on the intended use of the gelatin hydrolysate. The gelatin starting material will typically have an average molecular weight of from about 50,000 Da to about 200,000 Da. In a particularly preferred embodiment, the gelatin starting material will have an average molecular weight of less than about 150,000 Da.

In one embodiment, the gelatin starting material will have a bloom strength of from about 50 to about 300, a pH of from about 3.8 to about 7.5, an isoelectric point of from about 4.7 to about 9.0, and a viscosity of From about 15 to about 75 mP, and the ash will be from about 0.1 to about 2.0%.

In an alternative embodiment, when the gelatin starting material is substantially Form A gelatin, the bloom intensity will be from about 50 to about 300, the pH will be from about 3.8 to about 5.5, and the isoelectric point will be about 7.0 to At about 9.0, the viscosity will be from about 15 to about 75 mP and the ash will be from about 0.1 to about 2.0%.

In an alternative embodiment, when the gelatin starting material is substantially B-type gelatin, the bloom intensity will be from about 50 to about 300, the pH will be from about 5.0 to about 7.5, and the isoelectric point will be about 4.7 to At about 5.4, the viscosity will be from about 20 to about 75 mP and the ash will be from about 0.5 to about 2.0%.

In a preferred embodiment of the gelatin hydrolysate for use in the manufacture of a hard capsule pharmaceutical product, the gelatin starting material will have a Brent strength of from about 200 to about 300, a viscosity of from about 40 to about 60 mP, and from about 4.5 to about 6.5. pH value. In a further preferred embodiment of the gelatin hydrolysate for use in the manufacture of a soft shell capsule pharmaceutical product, the gelatin starting material will have a Brent strength of from about 125 to about 200, a viscosity of from about 25 to about 45 mP, and a viscosity of from about 4.5 to about A pH of about 6.5.

In the process of the present invention, the gelatin starting material is typically mixed or dissolved in water by a process known as expansion to form a solution comprising from about 10% to about 60% by weight gelatin. In a preferred embodiment, the solution has from about 10% to about 50% by weight gelatin.

In a more preferred embodiment, the solution has from about 20% to about 50% by weight gelatin. In a more preferred embodiment, the solution has from about 35% to about 40% by weight gelatin.

It is expected that gelatin having a different particle size can be used as a starting material in the present invention. For example, the gelatin particle size can range from about 0.1 mm to about 10 mm. In one embodiment, the gelatin particle size can be a fine particle size having an average particle size of from about 0.1 to about 0.3 mm. In another embodiment, the gelatin particle size can be a medium particle size having an average particle size of from about 0.3 to about 0.8 mm. In yet another embodiment, the gelatin particle size can be a large particle size having an average particle size of greater than about 0.8 mm. In general, the particle size of the gelatin starting material will affect the amount of time required for the gelatin to dissolve in the solution. During the expansion process, gelatin is used to absorb up to ten times its weight in cold water. Gelatin having a fine particle size swells in a few minutes, gelatin having a medium particle size swells in about 8 to about 12 minutes, and gelatin having a large particle size swells in about one hour. Typically, a low concentration gelatin solution, having a solution of, for example, from about 10% to about 20% by weight gelatin, can be prepared using all particle sizes. For highly concentrated solutions, solutions having, for example, from about 30% to about 35% by weight of gelatin typically use coarse particles, since the coarse particles do not tend to aggregate and produce less bubbles as they are processed.

After the gelatin has been introduced into the solution by the expansion process and usually prior to the addition of the proteolytic enzyme, the pH, temperature and redox state of the solution are typically adjusted to treat the lesser amount of residual peroxide present in the gelatin from its manufacturing process. In order to optimize the hydrolysis reaction, and in particular to ensure that the cysteine-containing proteolytic enzyme used in the hydrolysis reaction functions close to its optimum activity level. The pH of the gelatin solution is adjusted and maintained at from about 5 to about 7. In a particularly preferred embodiment, the pH of the gelatin solution is adjusted and maintained at from about 6.0 to about 6.5. At this pH, the proteolytic enzymes detailed below are close to their optimal levels of activity. The pH of the gelatin solution can be adjusted and monitored according to methods generally known in the art. For example, an acid such as hydrochloric acid is usually added to lower the pH of the gelatin solution. Alternatively, a base such as sodium hydroxide is usually added to increase the pH of the gelatin solution. Preferably, during the hydrolysis reaction, the temperature of the gelatin solution is adjusted and maintained at a temperature of from about 40 ° C to about 65 ° C according to methods known in the art. In a particularly preferred embodiment, the temperature of the gelatin solution is adjusted and maintained between about 50 ° C and about 60 ° C during the hydrolysis reaction. In general, temperatures above this range can inactivate proteolytic enzymes, and temperatures below this range tend to slow proteolytic enzyme activity. Depending on the proteolytic enzyme used in the hydrolysis reaction, the redox state of the gelatin solution should generally be adjusted and kept neutral to slightly biased toward the reducing side. High levels of oxidizing agents tend to passivate some of the cysteine-containing proteolytic enzymes used in the hydrolysis reaction, while low levels of reducing agents can be used to keep some proteolytic enzymes such as papain active until they are inactivated.

In general, the hydrolysis reaction is carried out by adding a proteolytic enzyme to a gelatin solution. Several proteolytic enzymes are suitable for use in the methods of the invention. In a preferred embodiment, the proteolytic enzyme will be a food having endopeptidase or peptide chain endolytic activity at a pH of from about 5 to about 7 and at a temperature of from about 40 ° C to about 65 ° C. Grade enzyme. In a particularly preferred embodiment, the proteolytic enzyme will have endopeptidase or peptide chain endolytic activity at a pH of from about 6 to about 6.5 and at a temperature of from about 50 ° C to about 60 ° C. Food grade enzymes.

In one embodiment, the endopeptidase will be a food grade serine protease belonging to EC 3.4.21. In an alternative embodiment of this embodiment, the serine protease is chymotrypsin. In another alternative embodiment of this embodiment, the serine protease is a subtilisin. In another embodiment, the endopeptidase will be a food grade cysteine protease belonging to EC 3.4.22. In yet another embodiment, the endopeptidase will be a food grade aspartic protease belonging to EC 3.4.23. In another embodiment, the endopeptidase will be a food grade metalloprotease belonging to EC 3.4.24. Illustrative, non-limiting examples of food grade endopeptidase enzymes useful in the methods of the invention include Validase AFP, Validase FP 500, Alkaline Protease Concentrate, Validase TSP, Enzeco Bromelain Concentrate, Corolase 7089, papain 600L and Validase Papaya protease concentrate free sulfite.

In another embodiment, the peptide chain endolytic enzyme will be a food grade aminopeptidase belonging to EC 3.4.11. In another embodiment, the peptide chain endolytic enzyme will be a food grade dipeptidase belonging to EC 3.4.13. In yet another embodiment, the peptide chain endolytic enzyme will be a food grade peptidyl dipeptidase or peptidyl tripeptidase belonging to EC 3.4.14. In yet another embodiment, the peptide chain endolytic enzyme will be a food grade peptidyl dipeptidase belonging to EC 3.4.15. In another embodiment, the peptide chain endolytic enzyme will be a food grade serine acid type carboxypeptidase belonging to EC 3.4.16. In yet another embodiment, the peptide chain endolytic enzyme will be a food grade metal carboxypeptidase belonging to EC 3.4.17. In another embodiment, the peptide chain endolytic enzyme will be a food grade cysteine type carboxypeptidase belonging to EC 3.4.18. In yet another embodiment, the peptide chain endolytic enzyme will be a food grade omega peptidase belonging to EC 3.4.19. Illustrative examples of food grade peptide chain endolytic enzymes that can be used in the methods of the invention include Validase FP II or Corolase LAP .

Another example of a food grade hydrolase that can be used in the method of the invention is Validase FP concentrate. Examples of other suitable food grade proteolytic enzymes are shown in Table A.

Typically, a combination of an endopeptidase and a peptide chain endolytic enzyme will be used to catalyze the hydrolysis reaction. The proteolytic enzyme is preferably selected by considering the protease activity of the enzymes and selecting an enzyme that will maximize peptide bond cleavage in the gelatin starting material. In a preferred embodiment, an enzyme having better endopeptidase activity is first added to the gelatin solution to form an endopeptidase digested gelatin product. The endopeptidase-digested gelatin product is then contacted with an enzyme having better peptide chain endolytic activity without inactivating the endopeptidase. It is also contemplated that in certain embodiments, an enzyme having peptide chain endolytic activity can be added prior to or concurrent with an enzyme having endopeptidase activity.

In a preferred embodiment, the endopeptidase is selected from the group consisting of: Corolase 7089, Validase AFP, Validase FP 500, Alkaline Protease Concentrate, Validase TSP, Enzeco Bromelain concentrate, papain 6000L and Validase Papaya protease concentrate free sulfite; and peptide chain endolytic enzyme is Validase FP II or Corolase LAP. In yet another embodiment, the endopeptidase is selected from the group consisting of Corolase 7089, Enzeco a group consisting of a pineapple protease concentrate and a papain 6000L; and the peptide chain endolytic enzyme is selected from the group consisting of Validase FPII and Corolase A group of LAPs. In a preferred embodiment, each proteolytic enzyme is continuously added to the gelatin starting material in the following order: Corolase 7089, Enzeco Bromelain concentrate, papain 6000L, Validase FPII and Corolase LAP. In an alternative embodiment of this embodiment, each proteolytic enzyme digests the gelatin starting material for about 0.5 to about 2 hours, followed by the addition of a subsequent proteolytic enzyme.

The amount of proteolytic enzyme added to the hydrolysis reaction can vary depending on the degree of hydrolysis of the gelatin to be used and the duration of the hydrolysis reaction. In general, for a hydrolysis reaction lasting from about 5 hours to about 24 hours, from about 0.025% to about 0.15% (w/w) of proteolytic enzyme having endopeptidase activity is added and about 0.025% is added to About 0.15% (w/w) of proteolytic enzymes having peptide chain endolytic activity. In a preferred embodiment, from about 0.05% to about 0.15% (w/w) of Corolase 7089, about 0.025% to about 0.075% (w/w) of Enzeco A pineapple protease concentrate, from about 0.05% to about 0.15% (w/w) papain 6000L, from about 0.025% to about 0.075% (w/w) of Validase FPII and about 0.05% to about 0.15% (w/w) of Corolase LAP is added to the gelatin starting material.

The hydrolysis reaction will usually proceed for up to about 24 hours. Generally, after about 24 hours, depending on the color and odor, the quality of the gelatin hydrolysate will begin to decrease significantly. In another embodiment, the hydrolysis reaction will proceed for from about 1 hour to about 24 hours. In yet another embodiment, the hydrolysis reaction will proceed for from about 3 hours to about 15 hours. In a more preferred embodiment, the hydrolysis reaction will proceed for from about 5 hours to about 12 hours. During this time period, highly economical process conditions and constant quality gelatin hydrolysate can be easily achieved. To complete the hydrolysis reaction, the hydrolyzed gelatin solution can be heated to about 90 ° C to inactivate the proteolytic enzymes. Additional steps may be required to inactivate the cysteine protease. If so, the addition of hydrogen peroxide or other oxidizing agents can be added, typically not exceeding 1000 ppm. The gelatin hydrolysate can then be purified from the hydrolysis solution by any method generally known in the art, such as microfiltration.

Generally, the degree of hydrolysis (DH) of the gelatin starting material in the process of the invention is greater than about 13%. In certain embodiments, the DH is from about 10% to about 20%. In other embodiments, the DH is from about 20% to about 30%. In another embodiment, the DH is from about 30% to about 40%. In yet another embodiment, the DH is from about 40% to about 50%. In yet another embodiment, the DH is from about 50% to about 60%. In another embodiment, the DH is from about 60% to about 70%. In yet another embodiment, the DH is from about 70% to about 80%. In yet another embodiment, the DH is from about 80% to about 90%. In yet another embodiment, the DH is greater than about 90%. DH is the percentage of the total number of peptide bonds in the gelatin starting material that have been hydrolyzed by proteolytic enzymes. DH can be calculated by methods generally known in the art, such as according to the Adler-Nissen method (19).

It has been observed that gelatin hydrolysates having a lower average molecular weight are more effective in preventing the crosslinking process. Thus, the amount of hydrolysate in the gelatin formulation can be reduced, which optimizes cost.

II. Gelatin hydrolysate

Yet another aspect of the invention encompasses a gelatin hydrolysate produced by the method of the invention. In general, the gelatin hydrolysate will comprise a mixture of peptides of different lengths in comparison to the gelatin starting material, wherein the peptides of different lengths are increased in amount of free glycine, other amino acids, and small peptides. The gelatin hydrolysate will also have a lower average molecular weight and a higher first amine content than the gelatin starting material.

The gelatin hydrolysate typically has an average molecular weight of at least about 100 Da. In other embodiments, the gelatin hydrolysate will typically have an average molecular weight of no more than about 2000 Da. In some embodiments, the gelatin hydrolysate will have an average molecular weight of from about 100 Da to about 2,000 Da. In other embodiments, the gelatin hydrolysate will have an average molecular weight of from about 700 Da to about 1800 Da. In another embodiment, the gelatin hydrolysate will have an average molecular weight of from about 700 Da to about 1500 Da. In other embodiments, the gelatin hydrolysate will have an average molecular weight of from about 800 Da to about 1200 Da.

The average molecular weight is the weight of the gelatin hydrolysate as measured by electrospray ionization liquid chromatography mass spectrometry (ESI-LC/MS). For example, a gelatin hydrolysate having an average molecular weight of about 1200 Da can have a molecular weight in the range of from about 75 Da to 8000 Da.

Generally, gelatin hydrolyzate having not less than about 1.0 × 10 per μg gelatin hydrolyzate ^{- 3 μMol} first average amine content of the first amine. In another embodiment, gelatin hydrolyzate having not less than about 1.5 × 10 per μg gelatin hydrolyzate ^{- 3 μMol} first average amine content of the first amine. In yet another embodiment, gelatin hydrolyzate having not less than about 2.0 × 10 per μg gelatin hydrolyzate ^{- 3 μMol} first average amine content of the first amine. In another embodiment, the gelatine hydrolyzate will have about 1.0 × 10 per μg gelatin hydrolyzate ^--3 to about 1.0 × 10 ^{- 2} μMol first average amine content of the first amine. The first amine content of the gelatin hydrolysate was measured by derivatization as described in the Examples and subsequent UV absorption (6-8).

The gelatin hydrolysate of the present invention comprises a polypeptide of generally up to about 75 amino acids, preferably up to 50 amino acids. In one embodiment, the gelatin hydrolysate comprising the polypeptide has an average length of from about 6 to about 18 amino acids. In another embodiment, the polypeptide contained in the gelatin hydrolysate of the present invention has an average length of from about 9 to about 20 amino acids. The length of the polypeptide chain can be determined indirectly by size exclusion chromatography/high performance liquid chromatography (SEC/HPLC).

In one embodiment, the gelatine hydrolyzate will have from about 100 Da to about 2,000 Da average molecular weight of about 1.0 × 10 per μg gelatin hydrolyzate ^--3 to about 1.0 × 10 ^{- 2} μMol first average amine content of the first amine And an average polypeptide length of up to about 20 amino acids. In yet another embodiment, the gelatine hydrolyzate will have from about 700 Da to about 1500 Da average molecular weight of about 1.0 × 10 per μg gelatin hydrolyzate ^--3 to about 2.0 × 10 ^{- 3 μMol} a first average of the first amine-amine The content, and the average polypeptide length of up to about 18 amino acids. In another embodiment, the gelatine hydrolyzate will have from about 800 Da to about 1200 Da average molecular weight of, per μg gelatin hydrolyzate of about 1.0 × 10 ^{- 3} to about 2.0 × 10 ^{- 3} μMol a first average of the first amine-amine The content, and the average polypeptide length of from about 4 to about 18 amino acids.

III. Gelatin composition

Another aspect of the invention encompasses a gelatin composition comprising a low molecular weight gelatin hydrolysate and gelatin. Surprisingly, it has been found that when low molecular weight gelatin hydrolysate is blended with higher molecular weight gelatin as shown in the examples, it increases free glycine, other amino acids and small peptides in the blended gelatin product. Amount to reduce cross-linking of the gelatin and improve dissolution characteristics.

Many different gelatin hydrolysates are suitable for use in gelatin compositions. In one embodiment, the gelatin hydrolysate will be an enzymatically digested hydrolysate. By way of non-limiting example, the gelatin hydrolysate of the present invention is made via an enzymatic hydrolysis procedure as detailed above. In another embodiment, the gelatin hydrolysate will be an acid digested hydrolysate. For example, acid hydrolysis can be carried out by digesting the gelatin starting material with about 6 N hydrochloric acid at a reaction temperature of about 110 ° C for about 24 hours. In yet another embodiment, the gelatin hydrolysate will be an alkali digested hydrolysate. By way of non-limiting example, base hydrolysis can be carried out by digesting the gelatin starting material with a strong base such as sodium hydroxide. Acid hydrolysis and base hydrolysis will generally result in a hydrolysis product having a free amino acid. In each of the examples (i.e., enzymatic hydrolysis, acid hydrolysis, and base hydrolysis), suitable gelatin starting materials are detailed in Section I above, which describes the initiation of gelatin to be used in the method of the present invention. The structural and functional properties of the substance.

Typically, the gelatin hydrolysate will have a low molecular weight. In one embodiment, the average molecular weight will range from about 400 Da to about 2000 Da. In another embodiment, the gelatin hydrolysate will have an average molecular weight of from about 700 Da to about 1500 Da. Further, gelatin hydrolyzate will also have a gelatin hydrolyzate per μg of approximately 1.0 × 10 ^{- 3} μMol to about 1.0 × 10 ^- a first average amine content in the range of ^{2 μMol} the first amine.

In another embodiment, the first average content of amine per μg gelatin hydrolyzate of about 1.0 × 10 ^{- 3 μMol} to about 2.0 × 10 ^- within a first range of amine ^{3 μMol.}

In yet another embodiment, the average content of amine per μg first gelatin hydrolyzate at about 2.0 × 10 ^- within a first range of amine ^{3 μMol - 3 μMol} to about 4.0 × 10. In another embodiment, the first average content of amine per μg gelatin hydrolyzate of about 4.0 × 10 ^{- 3 μMol} to about 6.0 × 10 ^- within a first range of amine ^{3 μMol.} In a further embodiment, the first average content of amine per μg gelatin hydrolyzate of about 6.0 × 10 ^{- 3} μMol to about 1.0 × 10 ^{- 2 μMol} the range of the first amine.

The gelatin hydrolysate will also generally have an average polypeptide chain length of from about 4 to about 50 amino acids. In one embodiment, the gelatin hydrolysate comprising the polypeptide has an average length of up to about 30 amino acids. In another embodiment, the gelatin hydrolysate comprising the polypeptide has an average length of from about 9 to about 20 amino acids. The average molecular weight, average first amine content, and average polypeptide chain length are determined as detailed in Section II.

In a preferred embodiment, the gelatin hydrolysate used in the composition will be the hydrolysate of the invention as detailed in Section II. Examples of other exemplary gelatin hydrolysates that can be used in the compositions are described in Table B. Mixtures of the aforementioned gelatin hydrolysates may also be used.

Gelatin hydrolysates can be blended with several types of gelatin having a wide range of physical and functional properties. The choice of a particular gelatin can and will vary to a large extent depending on the intended use of the gelatin composition. In general, gelatin is typically derived from collagen or collagen-rich tissue available from several suitable materials, such as the skin and bone of an animal, regardless of the embodiment or desired use. In one embodiment, the gelatin is Type A gelatin. In another embodiment, the gelatin is Type B gelatin. In yet another embodiment, the gelatin is a mixture of Type A gelatin and Type B gelatin. Further, gelatin prepared by an enzymatic method can be used in place of type A gelatin and/or type B gelatin.

Regardless of the embodiment, gelatin preferably will comprise from about 80% to about 90% protein by weight, from about 0.1% to about 2% by weight inorganic salt (ash content) and from about 10% to 15% by weight water .

Gelatin will generally have a high average molecular weight. In one embodiment, the gelatin will have an average molecular weight greater than about 200,000 Da. In another embodiment, the gelatin will have an average molecular weight greater than about 150,000 Da. In yet another embodiment, the gelatin will have an average molecular weight of from about 100,000 Da to about 200,000 Da.

In one embodiment, the gelatin of the gelatin will have a strength of from about 50 to about 300, a pH of from about 3.8 to about 7.5, an isoelectric point of from about 4.7 to about 9.0, and a viscosity of from about 15 to about 75 mP. And the ash will be from about 0.1 to about 2.0%.

In an alternative embodiment, when the gelatin is substantially Type A gelatin, the bloom intensity will be from about 50 to about 300, the pH will be from about 3.8 to about 5.5, and the isoelectric point will be from about 7.0 to about 10.0. The viscosity will be from about 15 to about 75 mP and the ash will be from about 0.1 to about 2.0%.

In an alternative embodiment, when the gelatin is substantially B-type gelatin, the blubber strength will be from about 50 to about 300, the pH will be from about 5.0 to about 7.5, and the isoelectric point will be from about 4.8 to about 5.8. The viscosity will be from about 20 to about 75 mP and the ash will be from about 0.5 to about 2.0%.

In a preferred embodiment of the gelatin composition for use in the manufacture of a hard capsule pharmaceutical product, the gelatin will have a Brent strength of from about 200 to about 300, a viscosity of from about 40 to about 60 mP, and a pH of from about 4.5 to about 6.5. .

In still another preferred embodiment of the gelatin composition for use in the manufacture of a soft shell capsule pharmaceutical product, the gelatin will have a Brent strength of from about 125 to about 200, a viscosity of from about 25 to about 45 mP, and from about 4.5 to about 6.5. pH value.

The gelatin compositions of the present invention will generally comprise from about 1% to about 20% by weight of the gelatin hydrolysate and from about 80% to about 99% by weight of gelatin. In another embodiment, the gelatin composition will comprise from about 1% to about 5% by weight of the gelatin hydrolysate and from about 95% to about 99% by weight of the gelatin. In yet another embodiment, the gelatin composition will comprise from about 5% to about 10% by weight gelatin hydrolysate and from about 90% to about 95% by weight gelatin. In another embodiment, the gelatin composition will comprise from about 10% to about 15% by weight gelatin hydrolysate and from about 85% to about 90% by weight gelatin. In another embodiment, the gelatin composition will comprise from about 15% to about 20% by weight gelatin hydrolysate and from about 80% to 85% by weight gelatin. In a typical embodiment, the gelatin composition will comprise a gelatin hydrolysate and gelatin in a ratio of from about 1:4 to about 1:99 (w/w).

In a preferred embodiment, the gelatin composition will comprise a gelatin hydrolysate of the invention and a higher molecular weight pharmaceutical grade gelatin. In one embodiment, the gelatin composition will comprise from about 5% to about 10% by weight gelatin hydrolysate and from about 90% to about 95% by weight pharmaceutical grade gelatin. In another embodiment, the gelatin composition will comprise from about 10% to about 15% by weight gelatin hydrolysate and from about 85% to about 90% by weight pharmaceutical grade gelatin.

Advantageously, the gelatin compositions of the present invention and the gelatin compositions of this embodiment typically have reduced cross-linking as measured by a vortex hardening test and a viscosity test. The gelatin composition of this embodiment typically has a vortex hardening time of from about 200 seconds to about 300 seconds. In another embodiment, the vortex hardening time is greater than about 300 seconds. The procedure for determining the vortex hardening time is described in the examples. The gelatin composition of this embodiment typically also has an average initial viscosity of from about 10 cP to about 15 cP and will have less than about 0.5% by weight of [2-(4-dimethyl-amine-formamidine) at a reaction temperature of about 60 °C. Benzyl-pyridyl)-ethane-1-sulfonate] (OB1207 of HWSands Corporation) After about 2 hours of addition to the gelatin composition, the gelatin composition has an average viscosity of from about 15 cP to about 50 cP. The procedure for measuring viscosity is described in the examples.

In one embodiment, the glycine acid can be added to the gelatin composition of the present invention as a separate compound. Glycine may be added to the gelatin composition in an amount from about 0.5% to about 5% by weight. In a more exemplary embodiment, the amount of glycine will range from about 1.5% to about 2.5% by weight. In yet another embodiment, citric acid can be added to the gelatin composition. The citric acid may be added in an amount of from about 0.5% by weight to about 5% by weight. In a more exemplary embodiment, the citric acid is added to the gelatin composition in an amount from about 0.5% to about 1.5%.

The gelatin compositions of the present invention are useful in a number of applications, including as a food ingredient, as a cosmetic ingredient, and as a photographic component. Because of the reduced tendency of the gelatin composition to crosslink and the improved solubility characteristics, in a preferred embodiment, the gelatin composition is used in the manufacture of a pharmaceutical product.

In a preferred embodiment, the gelatin composition is used to make hard gelatin capsules. As detailed above, when the gelatin composition is used in the manufacture of a hard capsule pharmaceutical product, the gelatin will have a Brent strength of from about 200 to about 300, a viscosity of from about 40 mP to about 60 mP, and a pH of from about 4.5 to about 6.5. value. A typical hard capsule formulation will comprise about 30% by weight of the gelatin composition of the invention, about 65% by weight water, about 5% by weight of a suitable dye, and if desired, will contain a pigment. Hard gelatin capsules can be made according to any method generally known in the art.

In yet another preferred embodiment, the gelatin composition is used to make soft gelatin capsules. As detailed above, when the gelatin composition is used in the manufacture of a soft shell capsule pharmaceutical product, the gelatin will have a Brent strength of from about 125 to about 200, a viscosity of from about 25 to about 45 mP, and a pH of from about 4.5 to about 6.5. value. A typical soft gelatin capsule formulation will comprise from about 40% to about 45% by weight of the gelatin composition of the invention, from about 15% to about 35% by weight of a plasticizer, and from about 20% to about 45% by weight of water. . Soft gelatin capsules can be made according to any method generally known in the art. Typical examples of plasticizers are glycerin (usually used in the form of an 85% by weight aqueous solution) and sorbitol (usually used in the form of a 70% by weight aqueous solution) and mixtures thereof.

All publications, patents, patent applications, and other references cited in this application are hereby expressly incorporated by reference herein in the extent of Patent applications or other references are incorporated by reference in their entirety.

definition

"Amphoteric" is a substance that exhibits cationic and anionic characteristics, such as proteins.

The "bloom value" is the strength of the gel measured in grams. The Buren value is the force required for the punch of the specified form and size to penetrate 4 mm deep into the surface of the 6.7% by weight gelatin solution. Commercially available gelatin has a Buren value between 80 and 280.

"Bone fragments" are sliced, degreased and dried bones from which gelatin is produced after demineralization (see maceration).

"Crosslinking" refers to, for example, the mechanism by which a film is formed on a pharmaceutical soft capsule. Generally, cross-linking reduces the solubility characteristics of the capsule.

"Da" is an abbreviation for Dalton.

"EC" is an abbreviation for Enzyme Classification. It is usually used as a prefix for the numerical name of the enzyme.

"Endopeptidase" is an enzyme generally belonging to the subclass EC 3.4, ie a peptide hydrolase, which hydrolyzes a non-terminal peptide bond in an oligopeptide or polypeptide and comprises any enzyme subclass EC 3.4.21-99.

"Peptide chain endolytic enzyme" is a group of peptidase enzymes in subclass EC 3.4 that catalyze the hydrolysis of peptide bonds adjacent to the terminal amine or carboxyl group of the oligopeptide or polypeptide. This group usually covers the enzyme subclasses 3.4.1-1.4.19.

A "food grade enzyme" is an enzyme that is generally a genetically modified organism and is safe when consumed by a living organism such as a human. Generally, enzymes and products from which enzymes can be derived are manufactured in accordance with applicable FDA guidelines.

"Hard capsules" are hollow capsules of various sizes made from pure gelatin with or without the addition of a dye. It consists of the upper part and the lower part; once the filling is completed, these parts are joined together.

"Instant gelatin" is a powdered gelatin that can be expanded in cold water.

"Microgel" is considered to be a gelatin having a molecular weight greater than 300,000 Da.

"Hard protein" is a protein that provides a supporting function in the body. It is insoluble in water and has a fibrous structure. Such proteins include, for example, keratin present in hair and nails, elastin and collagen present in supporting tissues and connective tissues (skin, bone and cartilage).

A "soft capsule" is an elastic capsule made of gelatin for filling an active ingredient/excipient mixture. It can be made with different wall thicknesses with or without seams.

"Split" is a gelatin material; the middle layer of cowhide connective tissue.

The "triple helix" is the basic structure of collagen consisting of three protein chains. These often have slightly different amino acid sequences.

"Type A gelatin" is an acid-digested gelatin.

"B type gelatin" is an alkali-digested gelatin.

The "LBSH type" is a lime bone hydrolyzate of the present invention produced by proteolytic digestion of gelatin.

The "LHSH type" is a lime peel hydrolyzate of the present invention produced by proteolytic digestion of gelatin.

Since the above-mentioned compounds, products and methods can be variously changed without departing from the scope of the invention, all the contents contained in the above description and the examples given below should be construed as illustrative and non-limiting.

Instance

The following examples illustrate the invention.

Example 1

The gelatin hydrolyzate of the present invention can be produced according to the following method. A solution containing 34% by weight of gelatin (Type B lime bone gelatin, Buren value = 100) was prepared by adding 1.94 kg of water to 1.0 kg of deionized gelatin. The gelatin was allowed to hydrate for 1 hour and then placed in a 55 ° C water bath to dissolve. Once completely dissolved, the pH of the gelatin solution was adjusted to 6.0-6.5 with an aqueous solution of sodium hydroxide. Calcium chloride is added to the gelatin in an amount of 0.037% w/w based on the amount of gelatin in the solution (CDG-commercial dry gelatin (having a moisture content of about 10% by weight), all additions in this procedure) In solution. An aliquot was taken and diluted to 5% by weight to test the redox state of the solution. The amount of peroxide was quickly measured using a peroxide test strip (EM Science). If the peroxide is present, add Fermcolase in 0.5 ml increments 1000F (Genencor International Inc.). After each addition, the solution was allowed to react for 30 minutes, after which the peroxide measurement was repeated. Repeated addition of Fermcolase 1000F until the peroxide content is close to zero.

Corolase in 0.1% w/w 7089 (AB Enzymes) was added to the solution. At the end of the 1 hour reaction time and before the next addition of the enzyme, an aliquot was taken and the molecular weight was analyzed. This process is repeated each time the enzyme is added. After 1 hour reaction time, add 0.05% w/w of Enzeco The pineapple protease concentrate (Enzyme Development Corp.) was allowed to react for an additional hour. Then 0.1% w/w of liquid papain 6000L (Valley Research) was added. After 1 hour, 0.05% w/w of Validase FPII (Valley Research) was added to the solution and allowed to react for an additional hour. The last enzyme was added as 0.1% w/w of Corolase LAP (AB Enzymes). After 1 hour, the solution was heated to 90 °C to inactivate the remaining functional enzymes. In some cases, an additional 30-40 ppm of hydrogen peroxide was added to ensure that the papain 6000L was inactivated. No signs of enzyme activity after heat deactivation were found. A summary table listing the details of the five enzymes used during the hydrolysis is given in Table 1.

The gelatin hydrolyzate obtained in this example was used as an LHSH type hydrolyzate in the following examples. The average molecular weight was determined to be about 1500 Da.

Example 2

The following procedure was used to quantify the extent of cross-linking reduction in various gelatin compositions. In a control experiment, 10.0 ± 0.1 g of gelatin was added to a 250 ml beaker to which 90.0 ± 0.5 g of deionized water was added. The watch glass was placed on the beaker and the gelatin was allowed to swell for 30-60 minutes. The inflated gelatin is placed in a 60 ± 0.1 ° C water bath for 15-30 minutes or until the gelatin is completely dissolved. The magnetic stir bar was placed in a gelatin solution and the pH was adjusted to a pH of 7.00 ± 0.05 with dilute NaOH or H ₂ SO ₄ on a stirrer, followed by removal of the magnetic stir bar. The solution was placed in a water bath of 40 ± 0.1 ° C for 15 to 60 minutes for cooling. A 750 ± 10 RPM vortex was produced using a digital agitator motor (Heidolph Brinkman 2102) equipped with a 4-blade mixer. Immediately add 20 ± 0.5 ml of pH 7 phosphate buffered 10% formalin solution (Fisher Scientific). The vortex hardening time was recorded (in seconds) as the time for the cross-linked gelatin solution to collapse through the axis of the 4-blade mixer.

In an experiment involving a gelatin composition containing an additive, the percentage of gelatin to be replaced with the desired additive (for example, a sample containing 10% hydrolyzate contained 9.0 g of gelatin and 1.0 g of hydrolyzate). The gelatin with a longer vortex hardening time has a lower formaldehyde-induced cross-linking tendency.

As shown in Table 2, the vortex hardening test confirmed the previous finding that the combination of glycine and citric acid reduced the amount of gelatin cross-linking. More importantly, the addition of glycine alone has a significant effect on the hardening time of the vortex of a particular lime bone gelatin sample. The addition of citrate did not reduce cross-linking.

Curiously, the addition of 1.5% citrate promotes cross-linking in this particular sample. These results can be used to consolidate the location of glycine in the role of the aldehyde scavenger in this model system. The deleterious effects of cross-linking by one of the citrate-only samples are not easily explained.

The vortex hardening test is used herein as an analytical tool for the rapid screening of the effects of additives on the crosslinking behavior of gelatin compositions.

Figure 1 details the addition of an LHSH-type hydrolysate (a lime-skin gelatin hydrolysate obtained in a manner similar to that of Example 1) (MW about 1200 Da) and a BH-3 gelatin hydrolysate (MW about 2200 Da) to have a 260 g The effect of the Buren value and the 6.67% viscosity of 45 mP in LH-1 (typical lime skin gelatin). The term 6.67% viscosity is used as an abbreviation for viscosity observed with 6.67% CDG aqueous solution. The results show that the vortex hardening time of both hydrolysates added increases. However, the efficiency after adding the LHSH type hydrolyzate of the present invention is more excellent. Several types of skin gelatin exhibited this extremely rapid cross-linking, a phenomenon previously seen only in lime bone gelatin with a viscosity of 6.67% near 60 mP.

Table 3 shows the vortex hardening times of several lime skin gelatins involving different molecular weight characteristics. In addition to the fact that the percentage of microgels and the increase in viscosity may be related to the increase in cross-linking and subsequent reduction in vortex hardening time, a decisive trend cannot be inferred.

Table 4 shows that 10% of the LBSH type hydrolysate of Example 1 (average MW = 1500), the BB-4 type gelatin hydrolyzate, and glycine were added to have a Buren value of 240 g and a 6.67% viscosity of 64 mP. The result of viscosity in lime bone gelatin. This high viscosity extract exhibits cross-linking properties similar to some of the lower viscosity lime skin gelatin. This gelatin showed a significant reduction in cross-linking in the presence of all three additives. However, the LBSH type hydrolysate increased the vortex hardening time by nearly 30% compared to the BB-4 type. As shown in Table 4, glycine exhibited a reduction in maximum cross-linking and an increase in the subsequent vortex hardening time.

Table 5 shows the attempt to determine the equivalent of glycine acid as measured by the vortex hardening test when added to medium viscosity lime bone medical gelatin (Blenn = 244, 6.67% viscosity = 47.0 mP). The result of the experiment of the amount of low molecular weight hydrolyzate. These results show that 4-5% LBSH type is required for the efficacy of 2.5% glycine, while 5-6% BB-4 type is required. Similarly, a 10% LBSH type and a 11-12% BB-4 type are required to achieve a reduction in cross-linking achieved by 5.0% glycine.

Example 3

Gelatin hardener OB1207 [2-(4-Dimethylaminocarbamimido-pyridyl)-ethane-1-sulfonate] was obtained from HWS ands Corporation. OB1207 It has been sold as an alternative to formaldehyde in photographic latex. Reaction 1 describes OB1207 Cross-linking with gelatin. Gelatin chain between and via OB1207 The reaction to form the guanamine and ester linkages approximates the type of crosslinking found in gelatin samples that have been aged and/or inhibited by exposure to heat and humidity.

In a control experiment, 15.0 ± 0.1 g of gelatin (type B lime bone gelatin, Buren value = 200) was added to a 250 ml flask. 95.0 ± 0.5 g of deionized water and a magnetic stir bar were added to the flask. The gelatin was covered with a parafilm and allowed to swell for 30-60 minutes. The flask was placed in a 60 ± 1.0 ° C water bath for 15-20 minutes or until the gelatin was completely dissolved. The viscosity of this solution was measured using a Brookfield DV-III+ rheometer at 50 RPM and 60.0 ± 0.1 °C. While stirring on the stirrer, it will be made up of 0.30 g OB1207 The solution prepared by dissolving in 10.0 g of deionized water was slowly added to the gelatin in the flask. The flask was placed back in the water bath. The viscosity of the solution was measured after 2 hours. In the experiment containing the added hydrolyzate, the percentage of the lighting gel was replaced by the desired hydrolyzate (i.e., the sample to which 10% of the hydrolyzate was added contained 13.5 g of gelatin and 1.5 g of hydrolyzate).

Table 6 shows the medium-bulen value and medium viscosity gelatin (LB-1) in relation to OB1207 The results of the viscosity test after cross-linking. Control A LB-1 did not add hydrolysate or OB1207 While the control B LB-1 has no hydrolysate, but with OB1207 Cross-linking. After 2 hours, Control B was too viscous to read on a Brookfield rheometer indicating a very high degree of crosslinking. The sample containing the hydrolyzate exhibited a decrease in the degree of crosslinking, wherein the LBSH-type hydrolyzate of the present invention achieved better results especially at a 10% content.

Example 4

The following procedure is used to determine the amount of the first amine in the gelatin hydrolysate. The amount of the first amine measured using trinitrobenzenesulfonic acid (TNBS) is described by Alder-Nissen (6). A modified form of this procedure is used to measure the relative amount of the first amine in the gelatin hydrolysate. Reaction 2 describes the derivatization of the first amine with TNBS.

Add 2.000 ± 0.002 g of glycine (Acros) to a 250 ml beaker and use 1% sodium dodecyl sulfate ("SDS", Aldrich) solution to achieve a weight of 200.00 ± 0.01 g (now known as G-1 glycine solution). A gelatin hydrolysate of 4.000 ± 0.002 g amount was added to a 250 ml beaker and made to a weight of 100 ± 0.01 g using a 1% SDS solution (now referred to as a hydrolyzate solution of H-1). The beaker containing the G-1 and H-1 solutions was placed on a hot plate and heated to a temperature of 80-85 ° C to completely dissolve and disperse the solid. The solution was allowed to cool to room temperature and then 1.00 g of G-1 was added to a 250 ml beaker and brought to a weight of 200.00 ± 0.01 g (G-2) using a 1% SDS solution. A dilution of G-2 (G-3 standard) was prepared by adding 50 ml, 37.5 ml, 25 ml, 12.5 ml, 5 ml and 0.5 ml G-2, respectively, in a 50 ml volumetric flask. The vial was labeled by using a 1% SDS solution. Solution H-1 was diluted to give H-2 by adding 1.00 g of H-1 in a 250 ml beaker and using a 1% SDS solution to a weight of 200.00 ± 0.01 g. Add 2 ml of 0.2125 M phosphate buffer in a 15 ml tube (prepared by adding 0.2125 M NaH ₂ PO ₄ to 0.2125 M Na ₂ HPO ₄ until a pH of 8.20 ± 0.02 is reached), and 250 μl G- 3 standards. This corresponds to 6 parts of standard glycine calibrator containing 0.1667 micromoles, 0.1250 micromoles, 0.0833 micromoles, 0.0417 micromoles, 0.0167 micromoles, and 0.0017 micromoles of first amine per sample, respectively. Similarly, 250 μl of each H-2 solution was added to a 15 ml tube (corresponding to 50 μg of sample) along with 2 ml of phosphate buffer. A control sample was prepared by adding 250 μl of 1% SDS solution to 2 ml of buffer to a 15 ml tube. 0.1% trinitrobenzene solution was prepared by adding 170±2 μl of 1 M TNBS solution (Sigma) to a 50 ml volumetric flask and labeling it with deionized water, and since TNBS is photosensitive, Immediately cover it with aluminum foil.

The following steps were all carried out in a photographic dark room. Add 2 ml of 1% TNBS solution to the tube. The tube was then vortexed (Fisher Scientific Vortex Genie 2) for 5 seconds. The sample was then placed in a 50.0 ± 0.1 ° C water bath for 30 minutes. The sample was then vortexed for another 5 seconds and placed back in the water bath for 30 minutes. The sample was removed from the water bath and 4 ml of 0.100 N HCl was added to terminate the TNBS reaction. The solution was vortexed for 5 seconds and allowed to cool for 10 minutes (longer cooling may cause turbidity due to SDS). The absorbance of each sample was read at 340 nm (Beckman DU-7 spectrophotometer) with reference to the water blank. The amount of the first amine in the sample was calculated by linear regression calculation based on absorbance using a glycine acid standard.

Derivatization of the first amine with o-phthalaldehyde (OPA) to quantify the proteolysis of milk proteins is described by Church et al. (7). Nielsen et al. (8) used OPA to measure the degree of hydrolysis of other food proteins, including the degree of hydrolysis of gelatin. An advantage of the Nielsen process is the replacement of beta-mercaptoethanol as a sulfur-containing reducing agent with a more environmentally friendly dithiothreitol (DTT-Cleland reagent). This program was adapted from a study by Nielson et al. Reaction 3 describes the reaction of the first amine with OPA in the presence of DTT.

The OPA reagent was prepared by adding 7.620 g of sodium tetraborate (Fisher Scientific) and 200 mg SDS to a 200 ml volumetric flask. Approximately 150 ml of deionized water was added and the solution was stirred until completely dissolved. A 160 mg amount of OPA (Aldrich) was dissolved in 4 ml of ethanol (Fisher Scientific) and quantitatively transferred to a measuring flask using deionized water. A 176 mg amount of DTT (Aldrich) was added and the entire solution was brought to volume with deionized water. The glycine acid standard was generated by adding 50 mg of glycine to a 500 ml volumetric flask and filling the mark with deionized water. The dilution was prepared by adding 100 ml, 75 ml, 50 ml, 25 ml and 5 ml glycine acid solution to a 100 ml volumetric flask and filling the mark with deionized water to produce 5 parts of glycine standard. Things. A gelatin hydrolysate sample was prepared by adding 0.500 g of hydrolyzate to a 100 volumetric flask and adding deionized water to the label. 10 ml of the hydrolysate solution was added to another 100 ml volumetric flask and filled with deionized water to the mark. Add 3.0 ml of OPA reagent solution to a 15 ml tube, then add 400 μL of glycine standard (40 μg, 30 μg, 20 μg, 10 μg, and 2 μg glycine) or gelatin hydrolysate (200 μg) Hydrolyzate). A control sample using 400 μL of water was also used to measure the absorbance of the individual OPA. The sample was vortexed for 5 seconds. The absorbance was read with reference to the water blank 2 minutes after the sample was added accurately. Does not meet the 2 minute requirement significantly affects the absorbance. Each sample or standard was then tested at 2 minute intervals. The amount of the first amine in the sample was calculated by linear regression calculation based on absorbance using a glycine acid standard.

Tables 7 and 8 show the results of TNBS and OPA derivatization of the first amine in 6 parts of gelatin hydrolysate, first extracted gelatin, glycine trimer and lysine monomer. The degree of hydrolysis is reported as the amount of the first amine divided by the amount of the first amine in the HCl-hydrolyzed sample (6N HCl, at 110 ° C for 24 hours). The molecular weight obtained from TNBS and OPA is the reciprocal of the amount of the first amine per sample amount. The molecular weights obtained from TNBS and OPA are only considered qualitative, and what really matters is the amount of the first amine measured in each sample. The molecular weight of the first amine does not take into account the dual derivatization of the amine acid and the hydroxyl acid, and it does not take into account the fact that the second amine is not derived from the derivatizing agent. However, when it is assumed that these factors are relatively constant for all gelatin hydrolysates, the molecular weight obtained from the first amine is a useful method for comparing the relative degrees of hydrolysis between different types of gelatin hydrolysates. According to the LBSH type and the LHSH type of the present invention, the limestone and lime peel hydrolysate exhibits an average increase of nearly 30-130% compared to the other enzymatically digested hydrolysate first amine. The average molecular weight as measured by the SEC/HPLC method is also given. Note that the molecular weight of the lysine and glycine trimer is quite different from the known molecular weight. The molecular weights obtained from TNBS and OPA are also very similar to those expected for HCl-hydrolyzed gelatin samples, and the HPLC/SEC data is almost five times this amount. This indicates the relative error of the low molecular weight SEC/HPLC method generally used to measure the molecular weight of the gelatin hydrolysate. The molecular weight obtained from TNBS and OPA is not considered for gelatin. The complexity of gelatin macromolecules limits the use of this simplified model to accurately represent molecular weight. OPA derivatization was confirmed to be a more reliable method of measuring the first amine content than TNBS derivatization.

references

All references cited in the above-mentioned text of this patent application or the following list of references are hereby expressly incorporated herein by reference inso- The subject matter is hereby incorporated by reference in its entirety in its entirety in its entirety in the in the the the the the the the the

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3. Adesunloye, T.A., Stach, P.E., "Effect of Glycine/Citric Acid on the Dissolution Stability of Hard Gelatin Capsules". Drug Dev. Ind. Pharm., 1998, 24(6), 493-500.

4. Rama Rao, K.V., Pakhale, S.P., Singh, S., "A film Approach for the Stabilization of Gelatin Preparations Against Cross-Linking". Pharmaceutical Technology. April 2003: 54-63.

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6. Adler-Nissen, J., "Determination of the Degree of Hydrolysis of Food Protein Hydrolysates by Trinitrobenzene Sulfonic Acid", J. Agric. Food Chem., November-December 1979, 27(6): 1256-62 .

7. Church, F., et al. "Spectrophotmetric Assay Using o-phthaldialdehyde for Determination of Proteolysis in Milk and Isolated Milk Proteins". J. of Dairy Sci., 1983, 66(6): 1219-1227.

8. Nielsen, P.M., Petersen, D., Dambmann, C, "Improved Method for Determining Food Protein Degree of Hydrolysis". J. of Food Sci., 2001, 66(5): 642-646.

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10. Fraenkel-Conrat, H., Olcott, H., "The Reaction of Formaldehyde with Proteins V. Cross linking between Amino and Primary Amide or Guanidyl Groups". J. Am. Chem. Soc, August 1948, 70 ( 8): 2673-2684.

11. Ward, A.G., Courts, A., The Science and Technology of Gelatin. Academic Press Inc. 1977, p. 231.

12. Davis, P., Tabor, B., "Kinetic Study of the Crosslinking of Gelatin by Formaldehyde and Glyoxal". J. Polym. Sci. Part A., 1963, 1: 799-815.

13. Albert, K., Peters, B., Bayer, E., Treiber, U., Zwilling, M., "Crosslinking of Gelatin with Formaldehyde; a ^{1 3} C NMR Study". Z. Naturforsch., 1986, 41b :351-358.

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16. Jiskoot, W. et al. "Indentification of Formaldehyde-induced Modifications in Proteins: Reaction with Model Peptides". J. Bio. Chem., February 2004, 279(8): 6235-6243.

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Figure 1 shows the reduction in formaldehyde-induced cross-linking of LH-1 (lime skin gelatin) after addition of two different gelatin hydrolysates, BH-3 and LHSH, as measured by the vortex hardening procedure. The BH-3 type hydrolyzate is a low molecular weight lime peel hydrolysate and the LHSH type is the lime peel hydrolysate of the present invention.

Claims (40)

A method of producing a gelatin hydrolysate having an average molecular weight of from about 100 Da to about 2000 Da and an average of from about 1.0 x 10 ^-3 μMol to about 1.0 x 10 ^-2 μMol of first amine per μg of gelatin hydrolysate a monoamine content, the method comprising: (a) contacting a gelatin starting material with at least one proteolytic enzyme having endopeptidase activity to form an endopeptidase digested gelatin product, and (b) The endopeptidase-digested gelatin product is contacted with at least one proteolytic enzyme having a peptide chain endolytic activity, wherein the endopeptidase proteolytic digestion and the peptide chain endolytic enzyme proteolytic digestion are sufficiently long The time is carried out under the reaction conditions for forming the gelatin hydrolysate. The method of claim 1, wherein the proteolytic enzyme is a food having endopeptidase or peptide chain endolytic activity at a pH of from about 5 to about 7 and at a temperature of from about 40 ° C to about 65 ° C. Grade enzyme. The requested item 1 or 2, wherein the proteolytic enzyme is selected from the group consisting of each composition: chain endopeptidase derived from Bacillus subtilis (Bacillus subtilis) of (e.g. Corolase ^® 7089), ananain (e.g. Enzeco ^® Bromelain concentrate), papain (eg papain 6000L), peptide chain endolytic enzyme from Aspergillus oryzae (eg Validase ^® FPII) and peptide chain end from Aspergillus sojae Enzymes (eg Corolase ^® LAP). The method of claim 1 or 2, wherein from about 0.025% to about 0.15% (w/w) of the proteolytic enzyme having endopeptidase activity and the gelatin starter Qualitatively contacting, and contacting from about 0.025% to about 0.15% (w/w) of the proteolytic enzyme having peptide chain endolytic activity to the endopeptidase digested gelatin product. A method of producing a gelatin hydrolysate, the method comprising: (a) contacting a gelatin starting material with a series of at least three proteolytic enzymes having endopeptidase activity to form an endopeptidase-digested gelatin product, the three kinds of proteolytic enzymes within the peptide chain by the cutting of an enzyme derived from Bacillus subtilis (e.g. Corolase ^® 7089), ananain (e.g. Enzeco ^® ananain concentrate) and papain (e.g., papain 6000L) composition; and (b The endopeptidase-digested gelatin product is contacted with a series of at least two proteolytic enzymes having a peptide chain endolytic activity, the peptide chain endolytic enzyme from rice bacterium (eg, Validase ^® FPII) and peptide chain endolytic enzymes (such as Corolase ^® LAP) from soy sauce. The method according to item 5 of the request, wherein each proteolytic enzyme is added continuously to the gelatin in the following order starting material: from the chain endopeptidase of Bacillus subtilis (e.g. Corolase ^® 7089), ananain (e.g. Enzeco ^® ananain Concentrate), papain (eg papain 6000L), peptide chain endolytic enzyme from rice bran (eg Validase ^® FPII) and peptide chain endolytic enzyme from Soybean sojae (eg Corolase ^® LAP); Wherein each proteolytic enzyme digests the gelatin starting material for about 0.5 to about 2 hours, followed by the addition of a subsequent proteolytic enzyme. The method of any one of 2, 5 or 6, wherein the protein water is The digestion is carried out for about 5 to about 12 hours. The method of any one of clauses 2, wherein the gelatin starting material has a bloom strength of less than about 150 and a bloom strength of less than about 30 mP at about 60 ° C and at a gelatin concentration of 6.67 wt%. Viscosity. The method of any of 2, 5 and 6, wherein the aqueous solution containing from about 10% to about 50% (w/w) of the gelatin starting material is contacted with the proteolytic enzymes. The method of any of 2, 5, wherein the gelatin starting material is pharmaceutical grade gelatin. The 5 or 6 of the item request, wherein from about 0.05% to about 0.15% (w / w) of Corolase ^® 7089, from about 0.025% to about 0.075% (w / w) of Enzeco ^® ananain concentrate, about 0.05 % to about 0.15% (w/w) papain 6000L, from about 0.025% to about 0.075% (w/w) of Validase ^® FPII and from about 0.05% to about 0.15% (w/w) of Corolase ^® LAP added To the gelatin starting material. The method of any of 2, 5 and 6, wherein the proteolytic digestion is carried out at a pH of from about 5 to about 7 and at a temperature of from about 40 ° C to about 65 ° C. The method of any of 2, 5 and 6, wherein the gelatin hydrolysate has an average molecular weight in the range of from about 100 Da to about 1500 Da. The method of claim 13, wherein the gelatin hydrolysate has an average molecular weight in the range of from about 400 Da to about 1200 Da, more preferably in the range of from about 700 Da to about 1200 Da. A gelatin hydrolysate having an average molecular weight of from about 100 Da to about 2000 Da and an average first amine content of from about 1.0 x 10 ^-3 μMol to about 1.0 x 10 ^-2 μMol of first amine per μg of gelatin hydrolysate. The gelatin hydrolysate of claim 15, wherein the gelatin hydrolysate has a degree of hydrolysis greater than about 13%. The gelatin hydrolysate of claim 15 or 16, wherein the gelatin hydrolysate has an average molecular weight in the range of from about 100 Da to about 1500 Da. The gelatin hydrolysate of claim 17, wherein the average molecular weight is between about 400 Da and about 1200 Da, more preferably between about 700 Da and about 1200 Da. The gelatin hydrolysate of claim 15 or 16, wherein the average polypeptide length in the gelatin hydrolysate is from about 4 to about 18 amino acids. A gelatin hydrolysate produced by the method of any one of claims 1 to 14. A gelatin composition comprising from about 1% to about 20% by weight of a gelatin hydrolysate and from about 80% to about 99% by weight of the gelatin. The gelatin composition of claim 21, wherein the composition comprises from about 5% by weight to about 10% by weight of the gelatin hydrolysate and from about 90% by weight to about 95% by weight of the gelatin. The gelatin composition of claim 21 or 22, wherein the composition comprises a gelatin hydrolysate and gelatin in a ratio of from about 1:4 to about 1:99 (w/w). The gelatin composition of claim 21 or 22, wherein the gelatin has an average molecular weight of greater than about 150,000 Da. The gelatin composition of claim 21 or 22, wherein the gelatin has about An average molecular weight of from 100,000 to about 150,000. The gelatin composition of claim 21 or 22, wherein the gelatin is a pharmaceutical grade gelatin. The gelatin composition of claim 21 or 22, wherein the gelatin is type B gelatin. The gelatin composition of claim 21 or 22, wherein the gelatin is Type A gelatin. The gelatin composition of claim 21 or 22, wherein the gelatin hydrolysate has from about 100 Da to about 2000 Da, preferably from about 100 Da to about 1500 Da, more preferably from 400 Da to about 1200 Da, more preferably from about 700 Da to The average molecular weight of about 1200 Da. The gelatin composition of claim 29, wherein the gelatin hydrolysate has an average molecular weight of between about 100 Da and about 1500 Da and from about 1.0 x 10 ^-3 μMol to about 1.0 x 10 ^-2 μMol per μg of gelatin hydrolysate. The average first amine content of the first amine. The gelatin composition of claim 21 or 22, wherein the gelatin hydrolysate is proteolytic gelatin obtained by the method of any one of claims 1 to 14 or as claimed in any one of claims 15 to 20. Gelatin hydrolysate. The gelatin composition of claim 21 or 22, wherein the gelatin hydrolysate is acid hydrolyzed gelatin. The gelatin composition of claim 21 or 22, further comprising glycine. The gelatin composition of claim 33, wherein the amount of glycine is from about 0.5% to about 5% by weight. The gelatin composition of claim 33, which further comprises citric acid. The gelatin composition of claim 35, wherein the amount of citric acid is from about 0.5% to about 5% by weight. The gelatin composition of claim 33, wherein the amount of glycine added is from about 1.5% to about 2.5% by weight and the amount of citrate added is from about 0.5% to about 1.5% by weight. The gelatin composition of claim 21 or 22, wherein the composition has a vortex hardening time of from about 200 seconds to about 300 seconds. The gelatin composition of claim 21 or 22, wherein the composition has a vortex hardening time of greater than about 300 seconds. The gelatin composition of claim 21 or 22, wherein the composition has an average viscosity of from about 10 cP to about 15 cP, and wherein about 0.5% by weight of [2-(4-dimethylaminocarbamyl)- Pyridyl)-ethane-1-sulfonate] is added to the composition and allowed to react at a reaction temperature of about 60 ° C for about 2 hours, the composition having an average viscosity of from about 15 cP to about 50 cP. .