BRPI0611399B1 - A process for preparing a low molecular weight gelatine hydrolyzate, the above-mentioned gelatin hydrolyzate and compositions comprising the same - Google Patents

Description

Patent Descriptive Report for "PROCESS FOR PREPARING A LOW MOLECULAR GELATIN HYDROLISATE, GELATINE HYDROLISATE, AND COMPOSITIONS UNDERSTANDING THE SAME".

FIELD OF INVENTION

This application claims priority of USSN 11 / 140,863 filed May 31, 2005, the application of which is hereby incorporated by reference in its entirety.

The present invention relates to a gelatin hydrolyzate, a process for preparing gelatin hydrolyzate, and a composition comprising gelatin hydrolyzate. More specifically, the present invention provides a low molecular weight gelatin hydrolyzate having a high primary amine content, a process for preparing gelatin hydrolyzate, and a gelatin composition including gelatin hydrolyzate. BACKGROUND OF THE INVENTION

Gelatin is made by denaturing collagen contained in materials such as pigskin, cattle skin or leather, and animal bones. Like its collagen protein, gelatin is defined by a distinctive structure comprising a unique blend of amino acids. Native collagen is a polypeptide chain-based sceroprotein comprising about 1050 amino acids. Three of these polypeptide chains together form a triple helix. Overprinting several of these triple helices produces collagen fibrils which are stabilized by crosslinking, thereby forming a three-dimensional lattice structure. This particular structure makes collagen insoluble; It is then brought in soluble form by partial hydrolysis as gelatin or gelatin hydrolyzate. The amino acid content of collagen and hence gelatin comprises one third of glycine and an additional 22% proline and 4-hydroxyproline; the remaining 45% comprises 17 different amino acids. Gelatin has a particularly high content of acidic and basic amino acids. Of the amino acids acid (glutamic acid and aspartic acid), varying amounts are present in the form of starch such as glutamine and asparagine which depend on the process conditions employed in the gelatin manufacturing process. Cysteine is completely absent; Of the sulfur-containing amino acids, methionine is the only one present.

The data for fish and poultry collagen are slightly different, but the present invention is equally applicable to fish and poultry collagen derived gelatin and / or gelatin hydrolyzate.

Gelatin can be used in a wide range of applications depending on its starting material and method of manufacture. This is because the physical and chemical behavior of gelatin is determined on the one hand by a combination of its amino acid content and the resulting spatial structure, and on the other by a myriad of conditions such as pH, ionic resistance and reactions with other molecules. For example, different types of gelatines are used in diverse applications such as food, photographic, cosmetic, and pharmaceutical.

In the pharmaceutical industry, gelatin is employed inter alia in the manufacture of hard and soft capsules. Gelatin capsules provide a convenient and efficient method for orally administering a drug because the capsules disintegrate rapidly upon exposure to stomach acid content, thereby releasing the drug into the body. While gelatin capsules provide a pharmaceutically elegant way in which to administer a drug, there is, however, a risk that the gelatin capsule may suffer from disintegration and dissolution that are the result of a process known as crosslinking. Crosslinking is believed to occur when gelatin carbonyl groups, sufficient ingredients containing carbonyl capsules, or decomposition of sufficient ingredients into carbonyl groups, react with primary amines and other nitrogenous compounds present in gelatin to form crosslinks.

[007] Crosslinking, in particular, can have dire consequences on the performance of gelatin capsules in prolonged storage and exposure to extremes of heat and humidity. Cross-linking of extensive gelatin in capsule formulations can lead to the formation of a very thin, hard and water-insoluble film, commonly referred to as a film. The film acts as a water-insoluble, rubbery layer that can restrict or prevent the release of the capsule contents.

Someone has widely reported ways to prevent focal cross-linking of gelatin capsules in products acting as carbonyl decontaminants, preventing the interaction of carbonyl groups, for example aldehyde groups, with the shell of the gelatin capsule, thereby preventing gelatin crosslinking. All of these methods generally suggest adding products to the pharmaceutical composition contained in the gelatin capsules. The addition of the amino acid glycine and citric acid in combination with gelatin hard capsule formulations has been shown to improve the dissolution profile of hard capsules (3). Addition of the amino acid glycine alone has not been shown to produce satisfactory results. But the addition of carbonyl decontaminants such as glycine, and carboxylic acids such as citrate, in the amounts necessary to reduce crosslinking in gelatin capsules is significantly cost prohibitive. Therefore, the addition of these products to gelatin is not a practical solution for reducing cross-linking in gelatin capsules.

The present invention provides a cost effective, practical means for reducing crosslinking in gelatin. Briefly, the invention encompasses a low molecular weight gelatin hydrolyzate which, when mixed with higher molecular weight gelatin, reduces gelatin cross-linking and improves dissolution properties by increasing the amounts of free glycine, other amino acids, and peptides. small in the mixed gelatin product. Advantageously, because the gelatin hydrolyzate and mixed gelatin composition of the invention have reduced cross-linking properties achieved without the addition of products such as glycine as an isolated compound mixed with citric acid, the gelatin may still be marketed as a natural product.

Among the various aspects of the invention, therefore, is a process for producing a gelatin hydrolyzate having an average molecular weight of about 100 to 2000 Da, preferably 1500 Da, and an average primary amine content. about 1.0 x 10'3 to about 1.0 x 10'2 μΜοΙ primary amine per pg of gelatin hydrolyzate. The process comprises contacting a gelatin starting material with at least one proteolytic enzyme that has endopeptidase activity to form an endopeptidase digested gelatin product. The endopeptidase digested gelatin product is then typically contacted with at least one proteolytic enzyme that has exopeptidase activity. Generally, proteolytic digestions of endopeptidase and exopeptidase proceed for a sufficient length of time and are conducted under reaction conditions to form the gelatin hydrolyzate.

Another aspect of the invention encompasses a process for preparing a gelatin hydrolyzate. The process comprises contacting a gelatin starting material with a series of at least three proteolytic enzymes that have endopeptidase activity to form an endopeptidase digested gelatin product. Typically, the three proteolytic enzymes consist of Bacillus subtilis Endopeptidase (eg Corolase® 7089), Bromelain (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 which have exopeptidase activity. Generally, the two proteolytic enzymes consist of Aspergillus oryzae Exopeptidase (eg, Validase® FPII) and Aspergillus soyee Exopeptidase (eg, Corolase® LAP).

Yet another aspect of the invention provides a gelatin hydrolyzate. The gelatin hydrolyzate will typically have an average molecular weight of about 100 to 2000 Da, preferably 1500 to 1500 Da and an average primary amine content of about 1.0 x 10-3 to 1.0 x 10 '2μΜοΙ primary amine per pg gelatin hydrolyzate. In one embodiment, the gelatin hydrolyzate is made by a process comprising contacting a gelatin starting material with a series of at least three proteolytic enzymes that have endopeptidase activity to form an endopeptidase digested gelatin product. Typically, the three proteolytic enzymes are selected from Bacillus subtilis Endopeptidase (eg Corolase® 7089), Bromelain (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 that have exopeptidase activity. Generally, the two proteolytic enzymes are selected from Aspergillus oryzae Exopeptidase (eg Validase® FPII) and Aspergillus soyee Exopeptidase (eg Corolase® LAP).

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

Other objects and aspects of the invention will be partly apparent and partly shown below.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the reduction in formaldehyde-induced crosslinking of LH-1, a calcium oxide skin gelatin, in the addition of 2 different Type BH-3 and Type LHSH gelatin hydrolysates as measured by the Vortex hardening. Type BH-3 Hydrolyzate is a low molecular weight calcium oxide skin hydrolyzate and Type LHSH is a calcium oxide skin hydrolyzate of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a novel gelatin hydrolyzate, a process for preparing gelatin hydrolyzate and gelatin compositions comprising gelatin hydrolyzate. Mixing a low molecular weight gelatin hydrolyzate and in particular the gelatin hydrolyzate of the present invention with gelatin has been found to reduce the tendency of cross-linking gelatin and to improve dissolution properties by increasing the amounts of free glycine. , other amino acids, and small peptides in the mixed gelatin product. Advantageously, the present invention provides a cost effective means for reducing gelatin crosslinking with the benefit of maintaining the original gelatin amino acid composition and without the need to add non-gelatin derived compounds as citric acid. As such, the gelatin hydrolyzate compositions of the present invention may still be marketed as natural products. /. Process for Preparing Gelatin Hydrolyzate An aspect of the present invention encompasses a process for producing a gelatin hydrolyzate having an average molecular weight of about 100 to about 2000 Da, preferably about 1500 Da and an amine content. primary amine from about 1.0 x 10'3 to about 1.0 x 10 2 μΜοΙ primary amine per pg of gelatin hydrolyzate. The process comprises contacting a gelatin starting material with at least one proteolytic enzyme that has endopeptidase activity to form an endopeptidase digested gelatin product. The endopeptidase digested gelatin product is then typically contacted with at least one proteolytic enzyme that has exopeptidase activity. Generally, proteolytic digestions of endopeptidase and exopeptidase proceed for a sufficient period of time and are administered under reaction conditions to form the gelatin hydrolyzate.

The gelatin starting material employed in the process of the invention is typically derived from collagen or collagen rich tissue available from various suitable raw materials. Collagen-rich tissues include the skin and bones of animals such as fish, poultry, pigs or cattle. Generally, there are two main types of collagen-derived gelatin, Type A and Type B, which differ in their manufacturing method. In one embodiment, the gelatin starting material is Type A, Type A gelatin with an isoionic point. from 7 to 10.0, is derived from collagen with exclusively acid pretreatment by methods generally known in the art. In an alternative embodiment, the gelatin starting material is Type B gelatin. Type B, with an isoion point of 4.8 to 5.8, is the result of an alkaline collagen pretreatment and is produced by generally known methods. known in the art.

In another alternative embodiment, the gelatin starting material is a mixture of Type A and Type B. The respective amounts of Type A and Type B gelatin may be varied greatly without detrimental effect on the properties of the gelatin hydrolyzate produced.

In principle, any of Type A and Type B gelatin may be exchanged completely or partially for enzymatically produced gelatin. However, the enzymatic process to make gelatin has not been widely used so far.

Regardless of the embodiment, the gelatin starting material will usually contain from about 80% to about 90% by weight of the protein, from about 0.1% to about 2% by weight of mineral salts (corresponding to ash content) and from about 10% to 15% by weight of water.

It is likewise considered that the physical properties of the gelatin starting material can and will vary depending upon the intended use of the gelatin hydrolyzate. The gelatin starting material will have an average molecular weight typically 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 less than about 150,000 Da.

In one embodiment, the strength resistance of the gelatin starting material will be from about 50g to about 300g, the pH will be from about 3.8 to about 7.5, the isoelectric point will be about 4.7 to 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 Type A gelatin, the strength resistance will be from about 50g to about 300g, the pH will be from about 3.8 to about 5.5, the isoelectric point will be from about 7.0 to 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 Type B gelatin, the strength resistance will be from about 50g to about 300g, the pH will be from about 5.0 to about 7.5, the isoelectric point will be from about 4.7 to 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 where gelatin hydrolyzate is employed in the manufacture of hard capsule pharmaceuticals, the gelatin starting material will have a strength resistance of about 200g to about 300g, a viscosity of about 40 to about 60 mP and a pH of about 4.5 to about 6.5. In yet another preferred embodiment where gelatin hydrolyzate is employed in the manufacture of soft shell capsule pharmaceuticals, the gelatin starting material will have a strength resistance of from about 125g to about 200g, a viscosity of about 25 to about 45 mP and a pH of about 4.5 to about 6.5.

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

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

It is considered that gelatins having varying particle sizes may be used in the invention as a starting material. For example, the gelatin particle size may range from about 0.1 mm to about 10 mm. The gelatin particle size may be larger having an average particle size of from about 0.1 to about 0.3 mm. In another embodiment, the gelatin particle size may be average having an average particle size of about 0.3 to about 0.8 mm. In yet another embodiment, the gelatin particle size may be large having an average particle size of approximately greater than about 0.8 mm. In general, the particle size of the gelatin starting material will affect the amount of time required for gelatin to dissolve in the solution. During the dilation process, the ability of the gelatin to absorb up to ten times its weight in cold water is utilized. Gelatines that have a larger particle size dilate within a few minutes, gelatines that have a medium particle size dilate within about 8 to about 12 minutes, and gelatines that have a large particle size dilate within about an hour. . Typically, low concentrated gelatin solutions, solutions having for example from about 10% to about 20% by weight gelatin, may be prepared employing all particle sizes. For highly concentrated solutions, solutions that have, for example, from about 30% to about 35% gelatin by weight, coarse particles are typically employed because they tend not to aggregate and produce fewer air bubbles when processed.

After gelatin has been brought into the solution through the dilation process and typically prior to the addition of proteolytic enzymes, the pH, temperature and oxidation state of the solution is typically adjusted to take care of the smaller amounts of residual peroxide present in the gelatin. of its manufacturing process to optimize the hydrolysis reaction, and in particular to ensure that the cysteine-containing proteolytic enzymes used in the hydrolysis reaction work at their optimal level of activity. The pH of the gelatin solution is adjusted and maintained from about 5 to about 7. In a particularly preferred embodiment, the pH of the gelatin solution is adjusted and maintained from about 6.0 to about 6.5. At this pH, the proteolytic enzymes detailed below are close to their optimal activity level. The pH of the gelatin solution may be adjusted and monitored according to methods generally known in the art. For example, to lower the pH of the gelatin solution an acid such as hydrochloric acid is typically added. Alternatively, to increase the pH of the gelatin solution a base such as sodium hydroxide is typically added. The temperature of the gelatin solution is preferably adjusted and maintained from about 40 ° to about 65 ° during the hydrolysis reaction according to methods known in the art. In a particularly preferred embodiment, the temperature of the gelatin solution is adjusted and maintained from about 50 ° C to about 60 ° C during the hydrolysis reaction. In general, temperatures above this range may disable proteolytic enzymes, while temperatures below this range tend to reduce the activity of proteolytic enzymes. Depending on the proteolytic enzyme employed in the hydrolysis reaction, the Oxidation State of the gelatin solution should typically be adjusted and kept slightly neutral on the reducing side. High levels of oxidants tend to inactivate some of the cysteine-containing proteolytic enzymes employed in the hydrolysis reaction, while low levels of reducers may serve to keep some of the proteolytic enzymes, such as papain, active until they are deactivated.

In general, the hydrolysis reaction is conducted by adding proteolytic enzymes to the gelatin solution. Various proteolytic enzymes are suitable for use in the process of the invention. In a preferred embodiment, the proteolytic enzymes will be food-grade enzymes that have endopeptidase or exopeptidase activity at a pH of from about 5 to about 7 and at a temperature of from about 40 to about 65 ° C. In a particularly preferred embodiment, proteolytic enzymes will be food grade enzymes that have exopeptidase or endopeptidase activity at a pH of about 6 to about 6.5 and at a temperature of about 50Ό to about 60Ό.

In one embodiment, the endopeptidase will be a food grade serine proteinase belonging to EC 3.4.21. In an alternative of this embodiment, serine proteinase is a chymotrypsin proteinase. In another alternative of this embodiment, serine proteinase is a subtilisin proteinase. In another embodiment, the endopeptidase will be a food grade cysteine proteinase belonging to EC 3.4.22. In yet another embodiment, the endopeptidase will be a food grade aspartic proteinase belonging to EC 3.4.23. In another embodiment, the endopeptidase will be a food grade metalloproteinase belonging to EC 3.4.24. Exemplary non-limiting examples of food grade endopeptidases which may be used in the process of the invention include Validase® AFP, Validase® FP 500, Alkaline Protease Concentrate, Validase® TSP, Enzeco® Bromelain Concentrate, Corolase® 7089, Papain 600L and Concentrate ® Sulphite Free Papain.

In another embodiment, the exopeptidase will be a food grade amino peptidase belonging to EC 3.4.11. In another embodiment, the exopeptidase will be a food grade dipeptidase belonging to EC 3.4.13. In yet another embodiment, the exopeptidase will be a food grade peptidyldi or tripeptidase belonging to EC 3.4.14. In yet another embodiment, the exopeptidase will be a food grade peptidyldipeptidase belonging to EC 3.4.15. In another embodiment, the exopeptidase will be a food grade carbine peptide peptidase belonging to EC 3.4.16. In yet another embodiment, the exopeptidase will be a food grade carboxy peptidase metal belonging to EC 3.4.17. In another embodiment, the exopeptidase will be a food grade cysteine carboxy peptidase belonging to EC 3.4.18. In yet another embodiment, the exopeptidase will be a food grade omega peptidase belonging to EC 3.4.19. An exemplary example of a food grade exopeptidase that may be used in the process of the invention includes Validase® FP II or Coolase se LAP®.

Another example of a food grade hydrolytic enzyme that may be employed in the process of the invention is Validase® FP Concentrate. Examples of other suitable proteolytic food grade enzymes are shown in Table A.

Table A

Typically, combinations of endopeptidases and exopeptidases will be employed to catalyze the hydrolysis reaction. Proteolytic enzymes are preferably selected by considering the protease activity of the enzymes and selecting the enzymes that will maximize the splitting of peptide bonds in the gelatin starting material. In a preferred embodiment, enzymes with preferred endopeptidase activity are added to the gelatin solution first to form an endopeptidase digested gelatin product. The endopeptidase digested gelatin product is then contacted with enzymes that have preferential exopeptidase activity without deactivating the endopeptidase (s). It is likewise considered that in certain embodiments enzymes having exopeptidase activity may be added before or at the same time as enzymes having endopeptidase activity.

In a preferred embodiment, 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 Sulphite Free Papain Concentrate Validase®; and the exopeptidase is Validase® FP II or Corolase® LAP. In yet another embodiment, the endopeptidase is selected from the group consisting of Corolase® 7089, Enzeco® Bromelain Concentrate, and Papain 6000L; and the exopeptidase is selected from the group consisting of Validase® FPII and Corolase® PAL. In a preferred embodiment, each proteolytic enzyme is consecutively 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 of this embodiment, each proteolytic enzyme digests the gelatin starting material for approximately about 0.5 to about 2 hours prior to addition of the subsequent proteolytic enzyme.

The amount of proteolytic enzyme added to the hydrolysis reaction may and will vary depending upon the desired degree of gelatin hydrolysis and the duration of the hydrolysis reaction. In general, about 0.025% to about 0.15% (w / w) of the proteolytic enzyme having endopeptidase activity is added and from about 0.025% to about 0.15% (w / w) of the proteolytic enzyme. which has exopeptidase activity is added for a hydrolysis reaction lasting for about 5 hours to about 24 hours. In a preferred embodiment, about 0.05% to about 0.15% (w / w) Corolase® 7089, about 0.025% to about 0.075% (w / w) Enzeco® Bromelain Concentrate, about 0.05% to about 0.15% (w / w) of Papain 6000L, about 0.025% to about 0.075% (w / w) of Validase® FPII, and about 0.05% to about 0.15% (w / w) Corolase® LAP is added to the gelatin starting material.

The hydrolysis reaction will typically proceed for up to about 24 hours. Typically, after about 24 hours the quality of gelatin hydrolyzate, in terms of color and odor, will begin to decline markedly. In another embodiment, the hydrolysis reaction will proceed from about 1 hour to about 24 hours. In yet another embodiment, the hydrolysis reaction will proceed from about 3 hours to about 15 hours. In still a more preferred embodiment, the hydrolysis reaction will proceed from about 5 hours to about 12 hours. Within this time period, highly economical process conditions and constant quality of gelatin hydrolyzate are readily achievable. To terminate the hydrolysis reaction, the hydrolyzed gelatin solution can be heated to approximately 90 ° C to deactivate proteolytic enzymes. An additional step to disable cysteine proteases may be required. If required, the addition of hydrogen peroxide or other oxidizing agent may be added, generally not exceeding 1000 ppm. The gelatin hydrolyzate may then be purified from the hydrolysis solution by any means generally known in the art, for example microfiltration.

Typically, the degree of hydrolysis (DH) of the starting gelatin material in the process of the invention is greater than about 13%. In certain embodiments, DH is from about 10% to about 20%. In other embodiments, 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 additional 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, DH is greater than about 90%. DH is the percentage of the total number of peptide bonds in the gelatin starting material that has been hydrolyzed by proteolytic enzymes. The 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 with a lower average molecular weight are more effective in preventing the crosslinking process. As a consequence, the amount of hydrolyzate in the gelatin formulation can be reduced which optimizes costs. II. Gelatin Hydrolyzate Yet another aspect of the invention encompasses a gelatin hydrolyzate made by the process of the invention. In general, the gelatin hydrolyzate, compared to the gelatin starting material, will comprise a mixture of peptides of different lengths that have an increase in amounts of free glycine, other amino acids, and small peptides. Gelatin hydrolyzate will likewise have a lower average molecular weight and higher primary amine content compared to gelatin starting material.

The gelatin hydrolyzate will typically have an average molecular weight of at least about 100 Da. In other embodiments, the gelatin hydrolyzate will typically have an average molecular weight not exceeding about 2000 Da. gelatin will have an average molecular weight of about 100 Da to about 2,000 Da. In other embodiments, the gelatin hydrolyzate will have an average molecular weight of about 700 Da to about 1800 Da. In another embodiment, the gelatin hydrolyzate will have an average molecular weight of about 700 Da to about 1500 Da. In still other embodiments, the gelatin hydrolyzate will have an average molecular weight of about 800 Da to about 1200 Da.

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

In general, the gelatin hydrolyzate will have an average primary amine content of not less than about 1.0 x 10'3 μΜοΙ primary amine per pg of gelatin hydrolyzate. In another embodiment, the gelatin hydrolyzate will have an average primary amine content of not less than about 1.5 x 10'3 μΜοΙ primary amine per pg of gelatin hydrolyzate. In yet another embodiment, the gelatin hydrolyzate will have an average primary amine content of not less than about 2.0 x 10'3 μΜοΙ primary amine per pg of gelatin hydrolyzate. In a further embodiment, the gelatin hydrolyzate will have an average primary amine content of from about 1.0 x 10 3 to about 1.0 x 10 2 μΜοΙ primary amine per pg of gelatin hydrolyzate. The primary amine content of gelatin hydrolyzate is measured by derivatization and subsequent UV absorption (6-8) as illustrated in the Examples.

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

In one embodiment, the gelatin hydrolyzate will have an average molecular weight of about 100 Da to about 2,000 Da, an average primary amine content of about 1.0 x 10 -3 to about 1.0 x 10'2 μΜοΙ primary amine per pg of gelatin hydrolyzate, and an average polypeptide length of up to about 20 amino acids. In yet another embodiment, the gelatin hydrolyzate will have an average molecular weight of about 700 Da to about 1500 Da, an average primary amine content of from about 1.0 x 10 -3 to about 2.0 x 10 '. 3 μΜοΙ primary amine per pg gelatin hydrolyzate, and an average polypeptide length of up to about 18 amino acids. In another embodiment, the gelatin hydrolyzate will have an average molecular weight of about 800 Da to about 1200 Da, an average primary amine content of about 1.0 x 10 -3 to about 2.0 x 10 -3. μΜοΙ of primary amine per pg of gelatin hydrolyzate, and an average polypeptide length of from about 4 to about 1 amino acids. III. Gelatin Compositions Another aspect of the invention encompasses a gelatin composition comprising a low molecular weight gelatin and gelatin hydrolyzate. Surprisingly, it has been found that when a low molecular weight gelatin hydrolyzate is mixed with higher molecular weight gelatin. It reduces gelatin cross-linking and improves dissolution properties by increasing the amounts of free glycine, other amino acids, and small peptides in the mixed gelatin product, as shown in the Examples.

Several different gelatin hydrolysates are suitable for use in the gelatin composition. In one embodiment, the gelatin hydrolyzate will be an enzymatically digested hydrolyzate. By way of non-limiting example, the gelatin hydrolyzate of the present invention is produced by an enzymatic hydrolysis procedure as detailed above. In another embodiment, the gelatin hydrolyzate will be an acid digested hydrolyzate. For example, acid hydrolysis may be administered by digesting a gelatin starting material with about 6 N hydrochloric acid for about 24 hours at a reaction temperature of about 110 ° C. In yet another embodiment, the gelatin hydrolyzate will be a base digested hydrolyzate. By way of non-limiting example, base hydrolysis may be administered by digesting a gelatin starting material with a strong base such as sodium hydroxide. Base and acid hydrolysis will typically result in a hydrolyzate that has free amino acids. In each embodiment (i.e., base, acid and enzymatic hydrolysis) suitable gelatin starting materials are detailed in section I above which outline structural and functional properties for gelatin starting materials to be employed in the process of the invention.

Typically, gelatin hydrolysates will have a low molecular weight. In one embodiment, the average molecular weight will be from about 400 Da to about 2000 Da. In another embodiment, the gelatin hydrolyzate will have an average molecular weight of about 700 Da to about 1500 Da. Gelatin likewise will have an average primary amine content ranging from about 1.0 x 10'3 to about 1.0 x 10'2 μΜοΙ of primary amine per μg of gelatin hydrolyzate.

In another embodiment, the average primary amine content may range from about 1.0 x 10-3 to about 2.0 x 10'3 μΜοΙ of primary amine per μg of gelatin hydrolyzate.

In yet another embodiment, the average primary amine content may range from about 2.0 x 10'3 to about 4.0 x 10'3 μΜοΙ of primary amine per μg of gelatin hydrolyzate. In yet another embodiment, the average primary amine content may range from about 4.0 x 10'3 to about 6.0 x 10'3 μΜοΙ of primary amine per μg of gelatin hydrolyzate. In still a further embodiment, the average primary amine content may range from about 6.0 x 10 -3 to about 1.0 x 10 ~ 2 μΜοΙ primary amine per μg gelatin hydrolyzate.

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

In a preferred embodiment, the gelatin hydrolyzate employed in the composition will be the hydrolyzate of the present invention as detailed in section II. Examples of other exemplary gelatin hydrolysates that may be employed in the composition are outlined in Table B. Mixtures of the gelatin hydrolysates described above may likewise be employed. Table B

Gelatin hydrolyzate can be mixed with various types of gelatin which has a wide range of physical and functional properties. The choice of a particular gelatin can and will vary greatly depending upon the intended use of the gelatin composition. In general, regardless of modality or intended use, gelatin is typically derived from collagen or collagen-rich tissue available from various suitable raw materials such as animal skin and bones. In one embodiment, gelatin is Type A gelatin. In another embodiment, gelatin is Type B gelatin. In yet another embodiment, gelatin is a mixture of Type A and Type B gelatin. Again, gelatin prepared in an enzymatic process may be be used to replace Type A and / or Type B gelatine.

Gelatin, regardless of embodiment, will preferably contain from about 80% to about 90% by weight of protein, from about 0.1% to about 2% by weight of mineral salts (ash content) and about 10% to 15% by weight of water.

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

In one embodiment, the strength resistance of the gelatin will be from about 50g to about 300g, the pH will be from about 3.8 to about 7.5, the isoelectric point will be from about 4.7 to about 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 is substantially Type A gelatin, the strength resistance will be from about 50g to about 300g, the pH will be from about 3.8 to about 5.5, 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 Type B gelatin, the strength resistance will be from about 50g to about 300g, the pH will be from about 5.0 to about 7.5, 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 an alternative embodiment when the gelatin composition is employed in the manufacture of hard capsule pharmaceuticals, the gelatin will have a strength resistance of about 200g to about 300g, a viscosity of about 40 to about 60 mP and a pH of about 4.5 to about 6.5.

In yet another preferred embodiment where the gelatin composition is employed in the manufacture of soft shell capsule pharmaceuticals, the gelatin will have a strength resistance of about 125g to about 200g, a viscosity of about 25 to about 45 mP and a pH of about 4.5 to about 6.5.

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

In a preferred embodiment, the gelatin composition will comprise the gelatin hydrolyzate of the present 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 of the gelatin hydrolyzate and from about 90% to about 95% by weight of the pharmaceutical grade gelatin. In another embodiment, the gelatin composition will comprise from about 10% to about 15% by weight of the gelatin hydrolyzate and from about 85% to about 90% by weight of the pharmaceutical grade gelatin.

Advantageously, gelatin compositions of the present invention and of this embodiment typically have reduced crosslinking as measured by the vortex hardening test and viscosity test. Gelatin compositions of this embodiment typically have a vortex hardening time of from about 200 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. Gelatin compositions of this embodiment typically similarly have an average initial viscosity of about 10 to about 15 cP and after the addition of less than about 0.5% by weight of [2- (4-dimethyl carbamoyl pyridine) ) -ethane-1-sulfonate ((OB1207® from HW Sands Corporation) to the gelatin composition for about two hours at a reaction temperature of about 60 ° C, the gelatin composition has an average viscosity of about 15 to about 50 cP The procedure for measuring viscosity is described in the examples.

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

The gelatin composition of the invention may be employed in various applications including as a food ingredient, as a cosmetic ingredient and as a photographic ingredient. Because of the reduced tendency of the reticular gelatin composition to and improved dissolution properties, in a preferred embodiment, the gelatin composition is employed in the manufacture of pharmaceuticals.

In a preferred embodiment, the gelatin composition is employed in the manufacture of hard gelatin capsules. As detailed above, when the gelatin composition is employed in the manufacture of hard capsule pharmaceuticals, the gelatin will have a strength resistance of about 200g to about 300g, a viscosity of about 40 to about 60 mP and a pH. from about 4.5 to about 6.5. A typical hard capsule formulation will comprise approximately 30 wt% of the gelatin composition of the invention, approximately 65 wt% of water, about 5 wt% of a suitable tincture, and will contain a pigment when necessary. Hard gelatin capsules may be made according to any method generally known in the art.

In yet another preferred embodiment, the gelatin composition is employed in the manufacture of soft capsule gelatin. As detailed above, when the gelatin composition is employed in the manufacture of soft shell capsule pharmaceuticals, the gelatin will have a strength resistance of about 125g to about 200g, a viscosity of about 25 to about 45 mP and a pH of about 4.5 to about 6.5. A typical soft capsule gelatin 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 plasticizer, and from about 20% to about 45% by weight of water. Soft gelatin capsules may be made according to any method generally known in the art. Typical examples for plasticizers are glycerol (usually employed as an 85 wt% aqueous solution) and sorbitol (usually employed as a 70 wt% aqueous solution) and mixtures thereof.

All publications, patents, patent applications, and other references cited in this application are incorporated herein by reference in their entirety as if each individual publication, patent, patent application, or other reference were specifically and individually indicated to be incorporated by reference. . DEFINITIONS

"Amphoteric" is a substance that can be either cationic or anionic in character, such as a protein.

"Strength Value" is the firmness of a gel measured in grams. The Force value is the force required for a definite punch and size to penetrate 4 mm deep into the surface of a 6.7 wt% gelatin solution. The strength value of commercially available gelatines is between 8üg and 280g.

"Bone splinter" is cut, degreased and dried bone from which, subsequent to demineralization (see maceration), gelatin is produced.

"Crosslinking" refers to the mechanism by which, for example, a film is formed on a soft pharmaceutical capsule. Typically, crosslinking decreases the dissolution properties of the capsule.

"Da" is an abbreviation for dalton.

"EC" is an abbreviation for Enzyme Classification. It is typically employed as a prefix in the numerical designation of an enzyme.

"Endopeptidase" is an enzyme typically belonging to the EC 3.4 subclass, peptide hydrolases, which hydrolyzes non-terminal peptide bonds to oligopeptides or polypeptides and comprises any EC 3.4.21-99 enzyme subclasses.

"Exopeptidase" is an enzyme of a group of peptide hydrolases within the EC 3.4 subclass that catalyzes the hydrolysis of peptide bonds adjacent to the carboxyl or terminal amino group of an oligopeptide or polypeptide. The group typically encompasses enzyme subclasses 3.4.11-3.4.19.

"Food Grade Enzyme" is an enzyme that is typically free from genetically modified organisms and is safe when consumed by an organism, such as a human. Typically, the enzyme and the product from which the enzyme may be derived are produced in accordance with applicable FDA guidelines.

"Hard capsules" are hollow capsules of various sizes made of pure gelatin with or without the addition of tincture. They comprise an upper and lower part; These are joined together as soon as the completion is completed.

"Instant gelatin" is gelatin powder capable of swelling in cold water.

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

"Scleroproteins" are those proteins that provide a supportive function within the body. They are insoluble in water and have a fibrous structure. These proteins include, for example, keratin occurring in hair and nails, elastins and collagen occurring in connective and supporting tissue, skin, bone and cartilage.

"Soft capsules" are elastic capsules made of gelatin to fill with active ingredient / excipient mixture. They can be produced with different wall thickness and with or without a seam.

"Division" is a gelatin raw material; half-layer of connective tissue from the skin of cattle.

"Triple helix" is a basic collagen structure consisting of 3 protein chains. These often have slightly different amino acid sequences.

"Type A gelatin" is acid digested gelatin.

"Type B gelatin" is alkali digested (basic) gelatin.

"Type LBSH" is a calcium oxide bone hydrolyzate of the present invention produced by proteolytic gelatin digestion.

"Type LHSH" is a calcium oxide skin hydrolyzate of the present invention produced by proteolytic gelatin digestion.

Since various changes may be made to the foregoing compounds, products and methods without departing from the scope of the invention, it is intended that all the subject matter contained in the foregoing description and examples given below be interpreted as illustrative and not in one form. limiting sense.

EXAMPLES

The following examples illustrate the invention.

Example 1 The gelatin hydrolyzate of the invention may be made according to the following process. A solution containing 34 wt% gelatin (Type B calcium oxide bone gelatin, Strength = 100) was made by adding 1.94 kg of water to 1.0 kg of deionized processed 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 aqueous sodium hydroxide. Calcium chloride was added to the gelatin solution in the amount of 0.037% w / w with the amount of gelatin in the solution (CDG - Commercial Dry Gelatin (having a moisture content of about 10% by weight), all additions in this procedures were based on this amount). An aliquot was taken and was diluted by 5% by weight to test the Oxidation Reduction Status of the solution. Peroxide test strips (EM Science) were employed to quickly measure the amount of peroxide. If peroxide is present, Fermcolase® 1000F (Genencor International Inc.) is added in 0.5 ml increments. After each addition, the solution was allowed to react for 30 minutes before repeating the peroxide measurement. Fermcolase® 1000F additions were repeated until the peroxide level approached zero.

Corolase® 7089 (AB Enzymes) was added to the solution in the amount of 0.1% w / w. Near the end of the reaction time of 1 hour and before the next enzyme was added, a small sample was taken and the molecular weight was analyzed. This process was repeated for each of the enzyme additions. After a reaction time of 1 hour, 0.05% w / w Enzeco® Bromelain Concentrate (Enzyme Development Corp.) was added and the solution was allowed to react for an additional hour. Liquid Papain 6000L (Valley Research) was then added 0.1% w / w. After 1 hour, 0.05% w / w Validase® FPII (Valley Research) was added to the solution and reacted for an additional hour. The final enzyme addition was 0.1% w / w Corolase® LAP (AB Enzymes). After 1 hour, the solution was heated to 90 ° C to deactivate the remaining functional enzymes. In some examples, an additional 30-40 ppm hydrogen peroxide was added to make sure Papa 6000L was deactivated. No evidence of enzymatic activity after heat deactivation has been seen. A summary listing the details of the five enzymes employed during hydrolysis is given in Table 1. Table 1 The gelatin hydrolyzate obtained in this example was employed as Type LHSH hydrolyzate in the following examples. The average molecular weight was determined to be about 1500 Da. Example 2 The following procedure was employed to quantify the degree of crosslinking reduction for various gelatin compositions.

In the control experiments, 10.0 + 0.1 g of a gelatin was added to a 250 ml beaker to which 90.0 + 0.5 g of deionized water was added. A watch glass was placed in the beaker and the gelatin was allowed to dilate for 30 - 60 minutes. The swollen gelatin was placed in a 60 + 0.1 ° C water bath for 15-30 minutes or until all gelatin was dissolved. A magnetic stir bar was placed in the gelatin solution and the pH was adjusted on a stir plate with NaOH or H2 SO4 diluted to a pH of 7.00 ± 0.05 after which the magnetic stir bar was removed. The solution was placed in a 40 ± 0.1 ° C water bath for 15-60 minutes to cool. A digital stirring motor (Heidolph Brinkman 2102) equipped with a 4-blade mixer was employed to create the vortex at 750 + 10 RPM. Immediately, 20 ± 0.5 ml of a pH 7 phosphate buffered 10% formalin solution (Fisher Scientific) was added. The vortex hardening time was recorded (in seconds) at the time when the crosslinked gelatin solution collapsed on the 4-blade mixer shaft.

In experiments involving gelatin compositions containing additives, a percentage of gelatin was replaced with the desired additive (for example, a sample added to the 10% hydrolyzate contained 9.0 g of gelatin and 1.0 g of hydrolyzate). ). Gelatins exhibiting a longer vortex hardening time are believed to have reduced trends for formaldehyde induced crosslinking.

As shown in Table 2, the vortex hardening test confirms previous results that the combination of glycine and citric acid can reduce the amount of gelatin crosslinking. More importantly, the addition of glycine alone has a dramatic effect on the vortex hardening time of this particular calcium oxide bone gelatin sample. Addition of citrate did not reduce crosslinking.

Interestingly, the addition of 1.5% citrate promoted crosslinking in this particular sample. These results may serve to support the position of glycine's role as an aldehyde decontaminant in this model system. The detrimental effects on crosslinking experienced by one of the citrate-only samples could not be easily explained. [0099] The vortex hardening test is employed here as an analytical tool for a rapid assessment of the impact of additives on the crosslinking behavior of gelatin compositions.

Table 2 [Figure 1] shows the effects of the LHSH type liposuction, a calcium oxide skin gelatin hydrolyzate obtained in a process similar to Example 1 (PM ~ 1200 Da), and a Type BH gelatin hydrolyzate. -3 (PM - 2200 Da) in LH-1, a typical calcium oxide skin gelatin with strength of 260 g and a viscosity of 6.67% 45 mP. The term 6.67% viscosity is used as an abbreviation for a viscosity observed with a 6.67% CDG solution in water. Results show an increase in vortex hardening time for both added hydrolysates. However, performance was better in addition of the hydrolyzate of the present invention, Type LHSH. Several skin gelatins exhibited this very fast crosslinking than was previously seen only in calcium oxide bone gelatins with a 6.67% viscosity close to 60 mP.

Table 3 shows the vortex hardening time of various calcium oxide skin gelatins with respect to different molecular weight properties. No conclusive trend could be deduced with the exception of a possible correlation of an increased percentage of microgel and viscosity with an increased crosslinking and a subsequent reduction in the vortex hardening time.

Table 3 shows the results of the addition of 10% Type LBSH hydrolyzate from Example 1 (average MW = 1500), a Type BB 4 gelatin hydrolyzate, and glycine in a calcium oxide bone gelatin. high viscosity with a force of 240 g and 6.67% viscosity of 64 mP. This high viscosity extract exhibits similar crosslinking properties as some much lower viscosity calcium oxide bone gelatin. This gelatin showed a significant reduction in crosslinking in the presence of all three additives. However, Type LBSH hydrolyzate increased vortex hardening time by almost 30% compared to Type BB-4. Glycine showed the largest reduction in crosslinking and subsequent increase in vortex hardening time as shown in Table 4.

Table 4 shows that the results are of an attempt to determine the amount of low molecular weight hydrolyzate needing to equalize glycine performance when added to a pharmaceutical medium calcium oxide bone gelatin (Strength = 244.6). , 67% vis. = 47.0 mP) as measured by the vortex hardening test. Results indicate that 4-5% Type LBSH is required to equalize 2.5% glycine performance, while 5-6% Type BB-4 is required. Similarly, 10% Type LBSH and 11-12% Type BB-4 is required to equalize the reduction in crosslinking achieved by 5.0% glycine.

Table 5 Example 3 The OB1207® Gelatin Curing Agent [2- {4-Dimethylcarbamyl-pyridine) -etho-1-sulfonate] was purchased. from HW Sands Corporation. OB1207® was observed as a formaldehyde substitution in photographic emulsions. Reaction 1 describes the crosslinking of OB1207® with gelatin. The formation of amide and ester bonds between gelatin chains by reaction with OB1207® can rigidly mimic the type of crosslinking seen in gelatin samples that have been aged and / or enhanced due to exposure to extremes of heat and humidity.

Reaction 1 In control experiments, 15.0 ± 0.1 g of gelatin (Type B calcium oxide bone gelatin, Strength = 200} was added in a 250 ml vial. To this, 95.0 + 0.5 g of deionized water and a magnetic stir bar were added.The gelatin was coated with parapellicle and allowed to swell for 30-60 minutes.The vial was placed in a 60 + 1.0Ό water bath for 15- 20 minutes or until all gelatin was dissolved, The viscosity of this solution was measured using a Brookfield DV-III + Rheometer at 50 RPM and 60.0 + 0.113. A solution made of 0.30 g of OB1207® dissolved in 10, 0 g of deionized water was slowly added to the gelatin in the vial while stirring on a stir plate.The vial was placed back in the water bath. The viscosity of the solution was measured 2 hours later. In experiments containing the addition of hydrolysates, a percentage of the control gelatin was replaced with the desired rolled (ie, a sample added to the 10% hydrolyzate contained 13.5 g of gelatin and 1.5 g of hydrolyzate).

The results of viscosity experiments involving medium strength and viscosity gelatin (LB-1) after crosslinking with OB1207® are determined in Table 6. Control A LB-1 had no hydrolyzate or OB1207® added, as control B LB-1 was not hydrolyzed, but was crosslinked with OB1207®. After two hours, Control B was very viscous reading on the Brookfield rheometer indicating a very high degree of crosslinking.

Samples containing hydrolysates showed a decreased degree of crosslinking, with the best results obtained with Type LBSH, the hydrolyzate of the present invention, especially at the 10% level. Example 4 The following procedure was employed to determine the primary amine content in the gelatin hydrolyzate. The use of trinitrobenzenesulfonic acid (TNBS) to measure the amount of primary amines has been described by Alder-Nissen (6). A modified version of this procedure was employed to measure relative amounts of primary amines in gelatin hydrolysates. Reaction 2 describes derivatization of a primary amine with TNBS.

Reaction 2 Glycine (Acros) in the amount of 2,000 ± 0.002 g was added to a 250 ml beaker and brought to a weight of 200.00 + 0.01 g employing a 1% sodium dodecyl sulfate solution ( "SDS", Aldrich) (glycine solution now referred to as G-1). Gelatin hydrolysates in the amount of 4.000 ± 0.002 g were added to the 250 ml beaker and brought to a weight of 100 + 0.01 g employing a 1% SDS solution (hydrolysate solution now referred to as H-1). The beakers containing the G-1 and H-1 solutions were placed on an electric plate and heated to a temperature of 80 - 85Ό to completely dissolve and disperse the solids. The solutions were cooled 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 using 1% SDS solution (G -2). Dilutions (G-3 standards) of G-2 were made in 50 ml volumetric flasks by adding 50, 37.5, 25, 12.5, 5, and 0.5 ml G-2, respectively. The vials were brought to the mark using 1% SDS solution. The H-1 solution was diluted to create H-2 by adding 1.00 g of H-1 and bringing to a weight of 200.00 ± 0.01 g in a 250 ml beaker employing the SDS solution at 1%. In a 15 ml test tube was added to 2 ml of a 0.2125 M phosphate buffer (made by adding 0.2125 M NaH2P04 to 0.2125 M Na2HP04 until a pH of 8.20 ± 0.02 is obtained), and 250 μΙ_ of the G-3 standards. This corresponds to a standard six glycine gauge containing 0.1667, 0.1250, 0.0833, 0.0417, 0.0167, and 0.0017 primary amine hitsioIs per sample, respectively. Similarly, 250 μΙ of each H-2 solution was added to a 15 ml test tube (corresponding to 50 ng sample) along with 2 ml phosphate buffer. A control sample is made by adding 250 μΙ of 1% SDS solution to a 15 ml test tube with 2 ml buffer. A 0.1% trinitrobenzene solution was made by adding 170 ± 2 μΙ of a 1M TNBS solution (Sigma) in a 50 ml volumetric flask and brought to the mark with deionized water and immediately coated with aluminum foil. when TNBS is light sensitive.

The following steps were all administered in a dark photographic environment. To the test tubes, 2 ml of 1% TNBS solution was added. The test tubes were then vortexed (Fisher Scientific Vortex Genie 2) for 5 seconds. The samples were then placed in a 50.0 ± 0.1 x 0 water bath for 30 minutes. The samples were then vortexed for an additional 5 seconds and placed back in the water bath for 30 minutes. Samples were removed from the water bath and 4 ml of 0.100 N HCl solution was added to terminate the TNBS reaction. Solutions were vortexed for 5 seconds and allowed to cool for 10 minutes (longer cooling may lead to cloudiness due to SDS). The absorbance of each sample was read at 340 nm (Beckman DU-7 Spectrophotometer) against a cuvette of water. Primary amine quantities in the samples were calculated using a linear regression calculation based on the absorbance of glycine standards.

Derivatization of primary amines with o-phthalialdehyde (OPA) to measure proteolysis in milk proteins has been described by Church et al. (7). Nielsen et al. (8) employed OPA to measure the degree of hydrolysis in other feed proteins, including that of gelatin. An advantage of the Nielsen method is the substitution of the most environmentally friendly dithiothreitol (DTT - Cleland Reagent) for β-mercaptoethanol as the sulfur-containing reducing agent. This procedure is adapted from the operation of Nielson et al. Reaction 3 describes the reaction of primary amines with OPA in the presence of DTT.

Reaction 3 The OPA reagent was prepared by adding 7.620 g of sodium tetraborate decahydrate (Fisher Scientific) and 200 mg of SDS in a 200 ml volumetric flask. Deionized water in the amount of approximately 150 ml was added and the solution was stirred until completely dissolved. 160 mg OPA (Aldrich) was dissolved in 4 ml of ethanol (Fisher Scientific) and quantitatively transferred to the volumetric flask using deionized water. DTT (Aldrich) in the amount of 176 mg was added and the total solution was brought to volume with deionized water. Glycine standards were created by adding 50 mg of glycine in a 500 ml volumetric flask and filling up to deionized watermark. Dilutions were made by adding 100, 75, 50, 25, and 5 ml of glycine solution in 100 ml volumetric flasks and filling to the mark with deionized water creating glycine standards. Gelatin hydrolyzate samples were prepared by adding 0.500 g of hydrolyzate is poured into a 100 ml volumetric flask and added deionized water to the mark. To another 100 ml volumetric flask, 10 ml of the hydrolyzate solution was added and filled to the deionized watermark. In a 15 ml test tube, 3.0 ml of OPA reagent solution was added followed by 400 μΐ of a glycine standard (resulting in 40, 30, 20, 10, and 2 Mg glycine) or gelatin hydrolyzate (200 Mg hydrolyzate). A control sample employing 400 μΙ_ of water was similarly employed to measure the absorbance of OPA only. The sample was vortexed for 5 seconds. The absorbance was read exactly 2 minutes after the addition of the sample against a void of water. Diverging the 2 minute requirement significantly impacts absorbance. Each sample or standard was then tested at two minute intervals. Primary amine quantities in the samples were calculated using a linear regression calculation based on the absorbance of glycine standards.

Table 7 and Table 8 show the results of TNBS and OPA derivatization of primary amines in 6 gelatin hydrolysates, a first extract gelatin, a glycine trimer, and a lysine monomer. The degree of hydrolysis is reported as the amount of primary amines divided by the number of primary amines in the hydrolyzed HCl sample (6N HCl for 24 hours @ 110Ό). Molecular weights derived from TNBS and OPA are the inverse of the amount of primary amines per sample amount. Molecular weights derived from TNBS and OPA are considered to be qualitative only, the actual significance being the measured amount of primary amines in each of the samples. Molecular weights derived from primary amine do not take into account the double derivatization of lysine and hydroxylisine, nor take into account the fact that secondary amines are not derived by any derivatizing agent. However, assuming that these factors are relatively constant for all gelatin hydrolysates, the primary amine derived molecular weight is a useful means of comparing the relative degrees of hydrolysis among different types of gelatin hydrolysates. Calcium oxide skin hydrolysates and Calcium oxide bone hydrolysates Type LBSH and Type LHSH according to the present invention showed an average increase of almost 30 - 130% in primary amines over enzymatically digested hydrolysates. The average molecular weight when measured by SEC / HPLC methodology is similarly determined. It is noted that the molecular weights for lysine and glycine trimer are far from known molecular weight values. The molecular weights derived from TNBS and OPA are similarly very similar to the expected results of the HCI hydrolyzed gelatin sample, as HPLC / SEC data is almost 5 times this amount. This generally demonstrates the relative inaccuracy of the low molecular weight SEC / HPLC methodology generally employed to measure the molecular weight of gelatin hydrolysates. Molecular weights derived from TNBS and OPA are not considered for gelatin. The complexities of the gelatin macromolecule inhibit an accurate representation of molecular weight by employing this simplified model. OPA derivatization proves to be a much safer means of measuring primary amine content compared to derivatization with TNBS.

Table 7 Table 8 KbhbKblNUIAS

All references cited in the previous text of the patent application or the following reference list, as they provide exemplary procedures, or other supplementary details to those mentioned herein, are specifically incorporated by reference to the same extent as if each application patent or individual publication were specifically and individually indicated to be incorporated by reference. 1. Ofner, C.M., Zhang, Y., Jobeck, V., Bowman, B., "Crosslinking Studies in Gelatin Capsules Treated with Formaldehyde and in Capsules Exposed to Elevated Temperature and Humidity". J. Pharm. Sci., Jan. 2001, 90 (1): 79-88. 2. Singh, S., Rama Rao, K.V., Venugopal, K, Manikandan, R., "Dissolution Characteristics: A Review of the Problem, Test Methods, and Solutions." Pharmaceutics! Technology - April 2002; 36-58. 3. Adesunloye, T.A., Stach, P.E., "Effect of Glycine / Citric Acid on 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. 5. Fraenkel-Conrat, H., Olcott, H., "Reaction of Formaldehyde with Proteins. II. Participation of Guanidyl Groups and Evidence of Crosslinking." J. Am. Chem. Soc., Jan. 1946, 68 (1): 34-37. 6. Adler-Nissen, J., "Determination of the Degree of Hydrolysis of Hydrolyzed Food Protein by Trinitrobenzene Sulfonic Acid", J. Agric. Food Chem., Nov.-Dec. 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. 9. Fraenkel-Conrat, H., Cooper, M., Olcott, H., "The Reaction of Formaldehyde with Proteins." J. Am. Chem. Soc., Jun. 1945, 67 (6): 950-954. 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 lnc.1977, pp. 231. 12. Davis, P., Tabor, B., "Kinetic Study of the Crosslinking of Gelatin by Formaldehyde and Glyoxal". J. Polym. Know. Part A., 1963, 1: 799-815. 13. Albert, K., Peters, B., Bayer, E., Treiber, U., Zwilling, M., "Crosslinking of Gelatin with Formaldehyde; the 13C NMR Study." Z. Naturforsch., 1986, 41b: 351-358 14. Gold, T.B., et al., "Studies on the Influence of pH and Pancreatin on 13C-Formaldehyde-Successful Gelatin Cross-Links Using Nuclear Magnetic Resonance." Pharm. Dev. Tech., 1996, 1 (1): 21-26. 15. Matsuda, S., Iwata, H., Se, N., Ikada, Y., "Bioadhesion of Gelatin Films Crosslinked with Glutaraldehyde". J Biomed Mater. Res. April 1999; 45 (1): 20-7. 16. Jiskoot, W., et al., "Indentification of Formaldehyde-induced Modifications in Proteins: Reaction with Model Peptides". J. Bio. Chem., Feb. 2004, 279 (8): 6235-6243. 17. Digenis, G.A., Gold, T.B., Shah, V.P., "Cross-Linking of Gelatin Capsules and their Relevance to Their Vitro-in Vivo Performance." J. Pharm. Sci., July 1994, 83 (7): 915-921. 18. Nagaraj, R.H., Shipanova, I.N., Faust, F.M., "Protein Cross-Linking by the Maillard Reaction." J. Biol. Chem., August 1996, 271 (32): 19338-19345. 19. "Enzymic Hydrolysis of Food Proteins"; Elsvier Applied Science Publishers Ltd. (1986), p. 122.

Claims (31)

A process for preparing a gelatin hydrolyzate 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 proteolytic enzymes consisting of Bacillus subtilis endopeptidase, bromelain, and papain; and (b) contacting the endopeptidase digested gelatin product with a series of at least two proteolytic enzymes having exopeptidase activity, the two proteolytic enzymes consisting of Aspergillus oryzae Exopeptidase and Aspergillus soyee Exopeptidase. Process according to Claim 1, characterized in that each proteolytic enzyme is sequentially added to the gelatin starting material in the following order: Bacillus subtilis endopeptidase, Bromelain, Papain, Aspergillus oryzae Exopeptidase and Aspergillus soybeane Exopeptidase ; and wherein each proteolytic enzyme digests the gelatin starting material for 0.5 to 2 hours before addition of the subsequent proteolytic enzyme. Process according to Claim 1 or 2, characterized in that the proteolytic digestion is allowed for 5 to 12 hours. Process according to any one of claims 1 to 3, characterized in that the gelatin starting material has a strength resistance of less than 150g and a viscosity of less than 30 mP at 60 ° C and a concentration of 6.67% by weight gelatin. Process according to any one of claims 1 to 4, characterized in that an aqueous solution containing 10% to 50% (w / w) of the gelatin starting material is contacted with the proteolytic enzymes. Process according to any one of claims 1 to 5, characterized in that the gelatin starting material is a pharmaceutical grade gelatin. Process according to any one of claims 1 to 6, characterized in that the proteolytic digestions are conducted at a pH of 5 to 7 and at a temperature of from about 40 ° to about 6513 ° C. Process according to any one of claims 1 to 7, characterized in that the average molecular weight of the gelatin hydrolyzate is in the range from about 100 to about 1500 Da. Process according to claim 8, characterized in that the average molecular weight of the gelatin hydrolyzate is in the range from about 400 to about 1200 Da, more preferably from about 700 to about 1200 Da. 10. Gelatin hydrolyzate having an average molecular weight from 100 to 2000 Da and an average primary amine content of 1.0 x 10'3 to 1.0 x 10'2 pMol primary amine per pg gelatin hydrolyzate , produced by the process as defined in any one of claims 1 to 9. Gelatin hydrolyzate according to Claim 10, characterized in that the gelatin hydrolyzate has a degree of hydrolysis greater than 13%. Gelatin hydrolyzate according to Claim 10 or 11, characterized in that the average molecular weight of the gelatin hydrolyzate is in the range from 100 to 1500 Da. Gelatin hydrolyzate according to claim 12, characterized in that the average molecular weight is between 400 and 1200 Da, more preferably from 700 to 1200 Da. Gelatin hydrolyzate according to any one of claims 10 to 13, characterized in that the average polypeptide in the gelatin hydrolyzate is 4 to 18 amino acids in length. Gelatin composition, characterized in that it comprises from 1% to 20% by weight of a gelatin hydrolyzate as defined in any one of claims 10 to 14 and from 80% to 99% by weight of gelatin. Gelatin composition according to Claim 15, characterized in that the composition comprises from 5% to 10% by weight of the gelatin hydrolyzate and from 90% to 95% by weight of the gelatin. Gelatin composition according to Claim 15 or 16, characterized in that the composition comprises a ratio of gelatin hydrolyzate to gelatin of 1: 4 to 1: 99 (W / W). Gelatin composition according to any one of claims 15 to 17, characterized in that the gelatin has an average molecular weight greater than 150,000 Da. Gelatin composition according to any one of claims 15 to 17, characterized in that the gelatin has an average molecular weight of from 100,000 to 150,000. Gelatin composition according to any one of claims 15 to 19, characterized in that the gelatin is a pharmaceutical grade gelatin. Gelatin composition according to any one of claims 15 to 20, characterized in that the gelatin is Type B gelatin. Gelatin composition according to any one of claims 15 to 20, characterized in that the gelatin is Type A gelatin. Gelatin composition according to any one of claims 15 to 22, characterized in that the gelatin hydrolyzate has an average molecular weight of from 100 to 2000 Da, preferably from 100 to 1500 Da, more preferably from 400 to 1200 Da. Da, even more preferably from 700 to 1200 Da. Gelatin composition according to any one of claims 15 to 23, characterized in that it also comprises glycine. Gelatin composition according to claim 24, characterized in that the amount of glycine is from 0.5% to 5% by weight. Gelatin composition according to claim 24 or 25, characterized in that it also comprises citric acid. Gelatin composition according to claim 26, characterized in that the amount of citric acid is from 0.5% to 5% by weight. Gelatin composition according to claim 24, characterized in that the amount of glycine added is from 1.5% to 2.5% by weight and the amount of citrate added is from 0.5% to 1%. 0.5% by weight. Gelatin composition according to any one of claims 15 to 28, characterized in that the composition has a vortex hardening time from 200 to 300 seconds. Gelatin composition according to any one of claims 15 to 28, characterized in that the composition has a vortex hardening time greater than 300 seconds. Gelatin composition according to any one of claims 15 to 30, characterized in that the composition has an average viscosity from 10 to 15 cP and wherein after the addition of 0.5% by weight of [ 2- (4-dimethylcarbamoyl-pyridine) -ethane-1-sulfonate] to the composition and reaction thereof for about 2 hours at a reaction temperature of 60 * 0, the composition has an average viscosity from 15 to 50 cP .