Method for improving gelatin viscosity decline rate and gelatin composition

Abstract

The invention relates to a method for improving the decline rate of gelatin viscosity. The method comprises the following steps: providing a gelatin aqueous solution; combining the aqueous gelatin solution with from about 2% to about 20% of a bioactive peptide (by weight fraction of the bioactive peptide relative to the total dissolved protein content of the aqueous solution) wherein the bioactive peptide comprises a collagen peptide having an average molecular weight of from about 100 to about 5,000 Da and / or comprises an amino acid; adjusting the pH of the aqueous solution to about 3.4 to about 8.0; heating the aqueous solution to a temperature of about 60 DEG C to about 150 DEG C; and maintaining the aqueous solution at the temperature for a period of time sufficient such that the viscosity of the solution falls within the range of about 15 mP to about 25 mP when measured at a total dissolved protein concentration of 6.67% w / w, at a temperature of 60 DEG C, and at a pH of 5.3 to 5.5. The present invention also relates to a gelatin composition comprising gelatin and from about 2% to about 20% of a bioactive peptide (by weight fraction of the bioactive peptide relative to the total protein content of the gelatin composition) wherein the bioactive peptide comprises collagen peptides and / or amino acids having an average molecular weight of from about 100 to about 5,000 Da, wherein the gelatin composition has a viscosity of 15 to 17 mP and a bloom value of 55 to 171 g, or a viscosity of 17 to 19 mP and a bloom value of 73 to 189 g, or a viscosity of 19 to 21 mP and a bloom value of 91 to 205 g, or a viscosity of 21 to 23 mP and a bloom value of 109 to 220 g, or a viscosity of 23 to 25 mP and a bloom value of 124 to 233 g, when measured at a total dissolved protein concentration of 6.67% w / w, at a temperature of 60 DEG C, and at a pH of 5.3 to 5.5.

Description

Background of the Invention

[0002] Gelatin is an important component of the coatings used in the manufacture of hard capsules, soft gelatin capsules and tablets for the pharmaceutical and nutraceutical markets. Commercially produced gelatin consists of a mixture of denatured collagen molecules and partially hydrolyzed collagen molecules. There are many known types of collagen, of which type I collagen is the main source of commercial gelatin and is commonly found in connective tissues such as tendons, skin and bones. The protein chains of type I collagen consist of approximately 1014 amino acids and have a molecular weight of approximately 100 kDa. These molecules are called alpha chains. The alpha chains consist mostly of glycine, proline and hydroxyproline, with these three amino acids accounting for approximately 50% to 60% of the protein mass. The three alpha chain proteins form linear helices approximately 300 nm long, and several of these elements are bundled together to form collagen fibrils. The fibrils are chemically bound together by natural crosslinks. These natural crosslinks are produced in animals by enzymatic reactions that convert biologically selected lysine or hydroxylysine side chains into aldehyde lysine or hydroxyaldol lysine. The aldehydes of these enzymatically modified amino acids are then free to react non-enzymatically with primary amines of other lysine or hydroxylysine residues located at selective positions along the adjacent collagen.

[0003] Commercial extraction and purification of gelatin from animal tissues and bones typically involves multiple process steps, including raw material pretreatment, aqueous solution extraction, filtration, purification, concentration, drying, grinding and mixing. Details of these process steps have been taught elsewhere (see Reference A, Reference B). Acidic pretreatment produces type A gelatin, which has an isoelectric point of about 7 to 9. Alkaline pretreatment produces type B gelatin, in which glutamine and asparagine amino acids are converted to glutamic acid and aspartic acid. The isoelectric point of type B gelatin can be about 4.7 to 5.4. Depending on the intensity of the alkaline pretreatment, an intermediate isoelectric point between the isoelectric point of type A gelatin and the isoelectric point of type B gelatin can also be obtained.

[0004] Commercially available gelatin for the pharmaceutical and nutraceutical markets is typically supplied in the form of dry powders or granules with moisture contents between 9% and 12%. Actual gelatin compositions sold as individual batches are typically prepared as a mixture of several individual dried batches. These dried batches may be produced from a specific set of gelatin extracts or from material collected from a continuous gelatin extraction process at set time intervals. The mixture formulation is determined by measuring various physical and chemical properties of individual batches and then combining those batches in selected proportions to produce a final batch that meets a set of properties required for a particular application or customer specification. Properties that are typically considered in batch preparation include gel strength (e.g., Bloom's value), viscosity, viscosity breakdown, pH, optical transmittance, ash content, and specific inorganic cation and anion concentrations. The viscosity of a gelatin solution is typically related to the average molecular weight to a first order approximation ( Figure 1). However, significant changes in the average molecular weight may produce similar solution viscosities, since this physical property also depends on other chemical properties such as pH, isoelectric point, salt content, etc. Gel strength is also associated with higher average molecular weights, although the contribution of α-chain sized molecules is generally dominant, since linear gelatin molecules of this size are able to form stronger helical structures in the gelled state.

[0005] Within the gelatin manufacturing industry, there is a need to balance the physical quality and quantity of gelatin produced from a given raw material with the quality and quantity required by the market. Gelatin is typically extracted from a collagen source material by a series of extractions at successively higher temperatures. As a result, the characteristic physical properties of the processed gelatin change with each successive extraction. In order to maximize the amount of saleable gelatin produced, it is often advantageous to reduce the gel strength and / or viscosity of a particular gelatin extract to better align the production volume with the available sales volume in the market.

[0006] Gelatin undergoes both acid- and base-catalyzed hydrolysis, with the more extreme the pH, the faster the hydrolysis reaction. Higher temperatures increase the rate of hydrolysis. The pH of extraction affects the ratio of gel strength to viscosity, which can significantly affect the suitability of gelatin for a particular application. pH also greatly affects the extractability of gelatin from the raw material. Likewise, in many practical cases, reduction of gel strength and / or viscosity is limited to thermal treatment. However, thermal degradation can be quite time-consuming, often requiring many hours to achieve the desired physical properties. In addition, heating large amounts of dilute aqueous gelatin solutions requires a lot of energy and increases production costs. Introducing additional holding times into the gelatin manufacturing process is also undesirable because it limits plant capacity and requires the installation of large, costly storage vessels.

[0007] In addition, a key factor in determining the performance of different gelatin compositions in hard or soft gelatin capsule applications is the amount of high molecular weight gelatin molecules present in the gelatin batch. Large gelatin molecules are referred to in the industry as microgels and are generally defined as cross-linked gelatin chains (e.g., four or more α chains cross-linked together) with a molecular weight ≥ 400 kDa. The amount of microgels present in a gelatin composition is generally related to the solution viscosity to a first order approximation ( Figure 2 ), however, within a specified viscosity range, the amount of microgels can vary significantly. For example, for commercial batches of 6.67% / 60°C limed bovine bone gelatin with a viscosity of 40 mP, the amount of microgels present can vary by about two-fold and can have a significant impact on the performance of these gelatin compositions in hard or soft capsule applications.

[0008] It is known in the capsule industry that the higher the percentage of gelatin microgel, the higher the incidence of capsule dissolution failure. Capsule dissolution failures are due to additional chemical cross-linking reactions that may occur between gelatin molecules after capsule manufacture (see reference F). These reactions generally depend on the chemical nature of the capsule fill ingredients, although direct cross-linking between gelatin molecules is also possible under low moisture conditions. When chemical cross-linking occurs within the shell of a gelatin capsule, the molecular weight of the gelatin molecules increases. As the molecular weight and covalent connectivity within the capsule shell increases, the solubility of the shell decreases. This equates to an increase in the time required for the capsule to dissolve and release its fill. In extreme cases, the capsule may fail to open in vivo, representing a complete failure of the drug and / or nutraceutical delivery system. Gelatin capsules composed of higher molecular weight gelatin molecules are more susceptible to dissolution problems when chemical cross-linking agents are used because of the higher initial degree of cross-linking in the shell. The effect of chemical cross-linking agents on gelatin is also known to be highly pH dependent (see references C, D, E).

[0009] Common gelatin manufacturing processes known in the art result in the presence of a large amount of microgel fraction in most gelatin extracts. The specific amount varies depending on the type of raw materials, pretreatment conditions and extraction conditions. To help reduce the impact of microgels on gelatin capsules, it has been demonstrated that the addition of 1% to 20% of small peptides of about 100 to 1000 Da to the gelatin composition can reduce the effect of chemical crosslinkers on gelatin dissolution, see Reference G). This improvement occurs because when new crosslinks are formed within the capsule shell, some of them are formed with the small peptides rather than with larger gelatin molecules. This limits the potential growth of microgel molecules and helps maintain the dissolution characteristics of the capsule over time.

[0010] Although the addition of small peptides to gelatin compositions is effective, it cannot fully compensate for the crosslinking activity of several important fillers on the market, such as krill oil, peppermint oil, astaxanthin, metal ions (Fe, Zn, Al), saw palmetto, chondroitin sulfate, vitamin D3, salmon oil, B complex, lutein, multivitamin cream, oregano oil, garlic oil, other herbal extracts, etc. Peroxide and aldehyde impurities in polyethylene glycol used as a plasticizer in the formation of the capsule shell can also cause significant gelatin crosslinking. Therefore, in order to further improve the performance of gelatin compositions, it is necessary to reduce the amount of microgel present.

[0011] The use of endoproteases and exoproteases for enzymatic hydrolysis of gelatin has been known for decades. These proteases include, but are not limited to, serine proteases, cysteine ​​proteases, threonine proteases, aspartic proteases, glutamic proteases, and metalloproteases. Exposing gelatin or collagen to these enzymes in large doses or for long periods of time can produce collagen hydrolysates. Collagen hydrolysates refer to gelatin or collagen that has been hydrolyzed to the extent that it can no longer form a gel, corresponding to an approximate average molecular weight of less than 10 kDa. It is known that exposing gelatin or collagen to these enzymes in low doses or for short periods of time can reduce viscosity, but has little or no effect on gel strength, see reference H). This is because the enzymes are more effective for larger native cross-linked gelatin molecules, and the products of these enzymatic hydrolysis reactions increase in the number of molecules of α-chain size. Therefore, enzymatic treatment of collagen or gelatin can be used to reduce the amount of undesirable microgels. As the enzyme dose or exposure time increases, the degree of hydrolysis becomes more extensive, resulting in a significant reduction in molecules of α-chain size, reduced gel strength, and a continuous decrease in viscosity.

[0012] Unfortunately, there are several important limitations and challenges in the practical application of enzymes for industrial gelatin pretreatment, extraction and processing. Enzyme preparations used in the gelatin industry are generally not mono-compositional (not composed of a single unique enzyme), but rather concentrated enzyme mixtures composed of several unique enzymes extracted simultaneously from various plant, animal or microbial sources. The activity of these enzyme mixtures is usually standardized with maltodextrin, which may cause undesirable turbidity or Maillard browning in gelatin batches. Enzyme mixtures sold as fine dry powders can cause strong allergic reactions after contact with trace amounts of powder, and their use in gelatin manufacturing poses significant safety issues. Enzyme mixtures supplied in liquid form usually use significant concentrations of glycerol or sorbate as stabilizers. The stabilizers glycerol and sorbate are very hygroscopic and will make the gelatin drying requirements higher. These residual stabilizers also increase the hygroscopic properties of the dried gelatin batches, thereby increasing the amount of moisture absorbed during storage or transportation. Controlling the extent of enzyme treatment is also challenging. During the manufacture of collagen peptides, enzyme dosage tolerance and process control are more tolerant because the number of enzyme susceptible sites is highly depleted when the average molecular weight is reduced to below 10 kDa. This results in a slower reaction rate in the target viscosity or molecular weight region. However, during the manufacture of high molecular weight gelatin, the requirements for enzyme dosage tolerance and process control are higher because the number of enzyme susceptible sites is still high. Therefore, the reaction rate in the target viscosity or molecular weight region is still high and is more sensitive to small changes in pH, temperature, cofactor concentration, salt concentration and molecular weight distribution of the initial gelatin composition. SUMMARY OF THE INVENTION

[0014] The present invention provides a more efficient method for producing a gelatin composition with a reduced amount of undesirable microgel components for capsule or tablet coating applications. The present invention also provides a more efficient method for reducing the viscosity of a gelatin composition extracted from a collagen-containing raw material to better match the quality and quantity requirements of the gelatin market. Specifically, the present invention provides a method for increasing the rate of decrease in gelatin viscosity, the method comprising the following steps:

[0015] Providing an aqueous gelatin solution;

[0016] combining the aqueous gelatin solution with about 2% to about 20% active peptides, the percentage being based on the weight fraction of the active peptides relative to the total dissolved protein content of the aqueous solution, wherein the active peptides comprise collagen peptides and / or amino acids having an average molecular weight of about 100 Da to about 5,000 Da;

[0017] adjusting the pH of the aqueous solution to about 3.4 to about 8.0;

[0018] heating the aqueous solution to a temperature of about 60°C to about 150°C; and

[0019] The aqueous solution is maintained at the temperature for a period of time sufficient to cause the viscosity of the solution to drop to within the range of about 15 mP to about 25 mP when measured at a total dissolved protein concentration of 6.67% w / w, at a temperature of 60°C and at a pH of 5.3 to 5.5.

[0020] The interaction of the active peptide with the gelatin composition significantly increases the rate of viscosity reduction and the rate of reduction of the microgel component and provides a more efficient manufacturing process. Another aspect of the present invention is that the active peptide can be partially or completely recovered using ultrafiltration or diafiltration.

[0021] The present invention also provides a gelatin composition that can be used to produce capsules with improved dissolution properties in the presence of a chemical crosslinking agent. In particular, the present invention provides a gelatin composition comprising gelatin and about 2% to about 20% active peptides, the percentage being based on the weight fraction of the active peptides relative to the total protein content of the gelatin composition, wherein the active peptides comprise collagen peptides and / or amino acids having an average molecular weight of about 100 Da to about 5,000 Da, wherein when measured at a total dissolved protein concentration of 6.67% w / w, at a temperature of 60° C. and at a pH of 5.3 to 5.5, the gelatin composition has

[0022] A viscosity of 15 to 17 mP and a Bloom's value of 55 to 171 g, or

[0023] A viscosity of 17 to 19 mP and a Bloom's value of 73 to 189 g, or

[0024] A viscosity of 19 to 21 mP and a Bloom's value of 91 to 205 g, or

[0025] A viscosity of 21 to 23 mP and a Bloom's value of 109 to 220 g, or

[0026] Viscosity of 23 to 25 mP and Bloom's value of 124 to 233 g.

[0027] In the present specification and claims, all percentages are by weight unless expressly stated otherwise. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 . Viscosity versus average molecular weight for several commercially available lime treated bovine bone gelatin batches. The viscosity was measured at a concentration of 6.67% w / w and a temperature of 60°C.

[0029] Figure 2 . Plot of the viscosity of several commercially available lime treated bovine bone gelatin batches as a function of the microgel fraction present therein. The viscosity was measured at a concentration of 6.67% w / w and a temperature of 60°C.

[0030] Figure 3 Schematic diagram showing different regions of a gelatin size exclusion chromatogram. The solid curve shows the chromatogram of a standard lime-treated bovine bone gelatin reference. The dashed curve shows the chromatogram of an active peptide consisting of collagen peptides with an average molecular weight of 690 Da. The thin dashed arrows indicate the elution volume cutoff boundaries between the different molecular regions as described in Example 3. Summary of the invention

[0031] Aspects of the present invention provide an improved method for reducing the amount of undesirable microgel components in gelatin compositions. The methods of the present invention can improve one or more disadvantages of gelatin performance in capsule or tablet applications by producing a gelatin composition with reduced microgel loading.

[0032] The present invention also provides a more efficient and faster method for reducing the viscosity of gelatin compositions. When the viscosity of gelatin extracted from collagen-containing raw materials is naturally higher than the viscosity requirements of the capsule or tablet market, the method of the present invention can improve one or more disadvantages of gelatin manufacturing.

[0033] One embodiment of the present invention provides a method for increasing the rate of decrease of gelatin viscosity, the method comprising the following steps:

[0034] Providing an aqueous gelatin solution;

[0035] combining the aqueous gelatin solution with about 2% to about 20% active peptides, the percentage being based on the weight fraction of the active peptides relative to the total dissolved protein content of the aqueous solution, wherein the active peptides comprise collagen peptides and / or amino acids having an average molecular weight of about 100 Da to about 5,000 Da;

[0036] adjusting the pH of the aqueous solution to about 3.4 to about 8.0;

[0037] heating the aqueous solution to a temperature of about 60°C to about 150°C; and

[0038] The aqueous solution is maintained at the temperature for a period of time sufficient to cause the viscosity of the solution to drop to within the range of about 15 mP to about 25 mP when measured at a total dissolved protein concentration of 6.67% w / w, at a temperature of 60°C and at a pH of 5.3 to 5.5.

[0039] The reaction mixture is then preferably cooled to below 60°C to inactivate it.

[0040] In a preferred embodiment, the aqueous gelatin solution is combined with about 9% to about 13% active peptide, the percentage being based on the weight fraction of the active peptide relative to the total dissolved protein content of the aqueous solution. The total dissolved protein content of the aqueous solution can be determined according to the Kjeldahl method.

[0041] The pH of the combined aqueous solutions is preferably adjusted to about 4.5 to about 5.5.

[0042] Preferably, the aqueous solution is heated to a temperature of about 90°C to about 100°C after pH adjustment.

[0043] In a preferred embodiment, the aqueous solution is maintained at the temperature for a period of time sufficient to cause the viscosity of the gelatin to drop to a range of about 17 mP to about 22 mP when measured at a gelatin concentration of 6.67% w / w, at a temperature of 60°C and at a pH of 5.3 to 5.5.

[0044] The period of time sufficient to achieve the target viscosity reduction depends on various parameters, including the temperature at which the solution is maintained. In preferred embodiments, the aqueous solution is maintained at the temperature for less than about 24 hours, less than about 10 hours, or less than about 5 hours.

[0045] Various aspects of the present invention provide a gelatin aqueous solution comprising type A gelatin, type B gelatin or a combination thereof. In various aspects of the present invention, the gelatin is derived from cattle bones, cattle hides, pig bones, pig skins, fish skins, chicken bones, jellyfish or a combination thereof. In other aspects of the present invention, the gelatin solution may be derived from a single or multiple gelatin extracts or production batches. In another embodiment of the present invention, the gelatin solution may be made by dissolving dry gelatin powder or granules. In another embodiment of the present invention, the gelatin solution may consist of dry mixed gelatin powders or dry mixed granules. In another embodiment of the present invention, the gelatin solution consists of a variety of dry mixed gelatin powders or a variety of dry mixed gelatin granules.

[0046] In one embodiment of the present invention, the active peptide is a collagen peptide having an average molecular weight of about 100Da to about 5,000Da. In various aspects of the present invention, the average molecular weight of the collagen peptide can be about 100Da to about 1,000Da, about 1,000Da to about 3,000Da, about 3,000Da to about 5,000Da, or a range defined by any two of the above values. In a preferred embodiment of the present invention, the average molecular weight of the collagen peptide is about 400Da to about 800Da.

[0047] In a preferred embodiment of the present invention, the collagen peptide is preferably obtained by enzymatic hydrolysis of type A gelatin, type B gelatin, collagen, animal tissue or bone containing collagen, or a combination thereof.

[0048] In another embodiment of the present invention, the active peptides further comprise peptides derived from hydrolyzed soy, casein, whey or pea protein, or a combination thereof, wherein the average molecular weight of the peptides is from about 100 Da to about 5,000 Da.

[0049] In another preferred embodiment of the present invention, the active peptide comprises amino acids as a supplement or replacement of the collagen peptide. In particular, the amino acids are selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, diglycine, histidine, isoleucine, lysine, hydroxylysine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine or a combination thereof. The active peptide may also comprise diglycine.

[0050] In a preferred embodiment of the present invention, the active peptide is added to the aqueous gelatin solution in the form of a dry solid and dissolved. In another embodiment of the present invention, the active peptide is dissolved to form an aqueous active peptide solution, and then wet mixed into the aqueous gelatin solution.

[0051] The active peptide used in the method of the present invention is combined with a starting gelatin solution, wherein about 2% to about 20% active peptide (in terms of the weight fraction of the active peptide relative to the total dissolved protein content of the aqueous solution) is used to produce a pre-reaction mixture. In various aspects of the present invention, the collagen peptide is used as an active peptide and is present in an amount of 9% to 13% of the total dissolved protein content of the pre-reaction mixture. For example, the amount of the collagen peptide added can be 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20% of the total dissolved protein content of the reaction mixture, or a range defined by any two of the above values.

[0052] In a preferred aspect of the invention, the pH of the initial aqueous gelatin solution is adjusted by passing the solution through a series of cation and anion exchange resins. In another aspect of the invention, the pH of the initial aqueous gelatin solution is adjusted by adding hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, acetic acid, sodium dihydrogen phosphate, potassium dihydrogen phosphate, or a chemical mixture comprising any combination of the above compounds. In various aspects of the invention, the initial pH of the initial aqueous gelatin solution is adjusted by adding sodium hydroxide, calcium hydroxide, magnesium hydroxide, potassium hydroxide, dicalcium phosphate, dicalcium phosphate dihydrate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, calcium citrate, sodium citrate, calcium acetate, sodium acetate, or a chemical mixture comprising any combination of the above compounds.

[0053] In the method of the present invention, the pH of the pre-reaction mixture is adjusted to about 3.4 to about 8.0, preferably in the range of about 4.5 to about 5.5. In various aspects of the present invention, the pH of the pre-reaction mixture is adjusted by adding hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, citric acid, acetic acid, sodium dihydrogen phosphate, potassium dihydrogen phosphate, or by a chemical mixture comprising any combination of the above compounds. In various aspects of the present invention, the pH of the pre-reaction mixture is adjusted by adding sodium hydroxide, calcium hydroxide, magnesium hydroxide, potassium hydroxide, dicalcium phosphate, dicalcium phosphate dihydrate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, calcium citrate, sodium citrate, calcium acetate, sodium acetate, or by a chemical mixture comprising any combination of the above compounds.

[0054] In the method of the present invention, the reactive mixture is heated to about 60°C to about 150°C, and preferably maintained for up to 24 hours. In a preferred aspect of the present invention, the reaction mixture is maintained between about 90°C and about 100°C, and maintained for ≤4 hours. In another embodiment of the present invention, the reaction mixture is maintained between about 80°C and about 90°C for ≤8 hours. In another embodiment of the present invention, the reaction mixture is maintained between about 100°C and about 110°C for ≤2 hours. In another embodiment of the present invention, the reaction mixture is maintained between about 110°C and about 120°C for ≤60 minutes. In another embodiment of the present invention, the reaction mixture is maintained between about 120°C and about 130°C for ≤30 minutes. In another embodiment of the present invention, the reaction mixture is maintained between about 130°C and 140°C for ≤15 minutes. In another embodiment of the present invention, the reaction mixture is maintained between about 140°C and about 150°C for ≤7 minutes.

[0055] According to another preferred aspect of the present invention, the method further comprises the step of at least partially removing the active peptide from the reaction mixture after heat treatment using ultrafiltration, diafiltration or a combination thereof. In another aspect of the present invention, the at least partially removed active peptide can be recovered from the ultrafiltration or diafiltration permeate and concentrated by nanofiltration, reverse osmosis, evaporation or a combination thereof. The recovered active peptide can then be reused in the method of the present invention.

[0056] According to another aspect of the present invention, there is provided a gelatin composition that can be used to produce capsules with improved dissolution properties in the presence of a chemical cross-linking agent.

[0057] One embodiment of the present invention provides a gelatin composition comprising gelatin and about 2% to about 20% active peptides (measured as the weight fraction of the active peptides relative to the total protein content of the gelatin composition), wherein the active peptides comprise collagen peptides and / or amino acids having an average molecular weight of about 100 Da to about 5,000 Da, wherein when measured at a total dissolved protein concentration of 6.67% w / w, at a temperature of 60° C. and at a pH of 5.3 to 5.5, the gelatin composition has

[0058] A viscosity of 15 to 17 mP and a Bloom's value of 55 to 171 g, or

[0059] A viscosity of 17 to 19 mP and a Bloom's value of 73 to 189 g, or

[0060] A viscosity of 19 to 21 mP and a Bloom's value of 91 to 205 g, or

[0061] A viscosity of 21 to 23 mP and a Bloom's value of 109 to 220 g, or

[0062] Viscosity of 23 to 25 mP and Bloom's value of 124 to 233 g.

[0063] In a preferred embodiment of the present invention, the gelatin composition comprises a microgel fraction accounting for about 0.8% or less of the total protein content of the gelatin composition.

[0064] The gelatin composition preferably comprises from about 9% to about 13% active peptide (as a weight fraction of the active peptide relative to the total protein content of the gelatin composition).

[0065] The pH of the gelatin composition of the present invention is preferably 5.3 to 6.0.

[0066] In a preferred embodiment of the present invention, the collagen peptide is derived from type A gelatin, type B gelatin, collagen, animal tissue containing collagen, animal bones containing collagen, or a combination thereof.

[0067] In another preferred embodiment of the present invention, the collagen peptide is enzymatically hydrolyzed collagen peptide.

[0068] The average molecular weight of the collagen peptide is preferably about 400 to 800 Da.

[0069] In another aspect of the present invention, the active peptides further comprise peptides derived from hydrolyzed soy, casein, whey or pea protein, or a combination thereof, and wherein the average molecular weight of the peptides is from about 100 to about 5,000 Da.

[0070] In another preferred embodiment of the present invention, the amino acid is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, lysine, hydroxylysine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine or a combination thereof, and / or wherein the active peptide comprises diglycine.

[0071] The gelatin in the gelatin composition of the present invention may be obtained from cattle bones, cattle hides, pig bones, pig skins, fish skins, chicken bones, jellyfish or a combination thereof.

[0072] In another aspect of the present invention, the gelatin is Type A gelatin, Type B gelatin, or a combination thereof.

[0073] Preferably, the gel composition is in the form of granules or powder obtained by drying and grinding.

[0074] In a preferred embodiment of the gelatin composition of the present invention, the gelatin composition comprises gelatin obtained by the method according to the present invention.

[0075] Further advantages and preferred embodiments of the gelatin composition of the invention have already been described in conjunction with the method of the invention.

[0076] Another aspect of the present invention relates to the use of the gelatin composition of the present invention for the manufacture of gelatin capsules. Preferably, the gelatin capsules are hard capsules, soft capsules or microcapsules. As mentioned above, such capsules have improved dissolution properties in the presence of a chemical crosslinking agent.

[0077] The present invention is further described by the following examples, which should not be construed as limiting the scope of the present invention in any way. DETAILED DESCRIPTION

[0078] Example

[0079] Example 1

[0080] This example illustrates how to prepare an active peptide consisting of a collagen peptide composition. An approximately 35% w / w solution was prepared by dissolving 1000 g of dried porcine skin gelatin (Bloom's value 260 g, 6.67% / 60°C viscosity 37.0 mP) in 1500 g of water. The pH of the solution was adjusted to between 5.0 and 5.1 with sulfuric acid and the temperature was maintained between 58°C and 62°C. Approximately 0.08 g of sodium metabisulfite and 17.5 g of Enzyme Development Corporation were added to the mixture. T200 is added to the solution. The mixture is maintained at 60°C for 24 to 36 hours. The solution is combined with 1 mL of approximately 30% hydrogen peroxide, heated to 95°C, and maintained at 95°C for 120 minutes. The solution is then cooled to room temperature and filtered using diatomaceous earth. Size exclusion chromatography analysis determined the average molecular weight of the collagen peptide to be 590 Da. The method can be equally applicable to type A or type B gelatin. Those skilled in the art will recognize that the average molecular weight can be changed by changing the reaction time, reaction pH, reaction temperature, enzyme concentration, or the type of enzyme used.

[0081] Example 2

[0082] This example demonstrates the ability of the active peptide to increase the rate of viscosity reduction and that the efficacy of the active peptide increases as its average molecular weight decreases.

[0083] Dried lime-treated bovine bone gelatin (Bloom's value of 273 g, 6.67% / 60°C viscosity of 45.9 mP) was dissolved in water at 55°C to 60°C to produce a 20% w / w gelatin aqueous solution. The gelatin solution was then combined with 11% active peptides consisting of a collagen peptide composition produced from lime-treated bovine bone gelatin. The pH of the solution was adjusted to 5.4 and then placed in an 85°C water bath (covered with a watch glass) for 6 hours. The solution was allowed to cool to 55°C to 60°C. The above process was repeated 3 times using collagen peptide compositions of different average molecular weights. The solution was then diluted to approximately 10%, the pH readjusted to 5.4 with sulfuric acid or sodium hydroxide (if necessary), and then the solution was further diluted to 6.67%, and its viscosity was measured at 60°C using a gravity pipette.

[0084] A second series of control 20% w / w aqueous gelatin solutions was prepared using the same initial batch of dried gelatin. The pH of each solution was adjusted to 5.4 and placed (covered) in an 85°C water bath for 6 hours. The solution was then allowed to cool to 55°C to 60°C. In order to compare the viscosity of the control example with the inventive example, the control solution was then combined with 11% active peptide, which consisted of the same collagen peptide composition used in the examples of the method of the present invention. The solution was then diluted to approximately 10% w / w, the pH readjusted to 5.4 with sulfuric acid or sodium hydroxide (if necessary), and the solution was then further diluted to 6.67% w / w and its viscosity was measured at 60°C using a gravity pipette.

[0085] Table A illustrates that the process of the present invention produces a greater degree of gelatin viscosity reduction given a fixed reaction time. The viscosity was measured at 6.67% w / w and 60°C. The presence of the active peptide significantly increased the rate of viscosity reduction compared to the control in which the same active peptide was not added until after heat treatment at 85°C. The viscosity reduction was greatest when the collagen peptide composition had an active peptide with an average molecular weight of about 650 Da.

[0086] Table A

[0087]

[0088] Example 3

[0089] This example illustrates the ability of the active peptide to increase the viscosity reduction rate and microgel reduction of type B gelatin, and the efficacy of the active peptide is pH-dependent.

[0090] Dried lime treated bovine bone gelatin (Bloom's value of 280 g, 6.67% / 60°C viscosity of 41.9 mP) was dissolved in water at 55°C to 60°C to produce a 20% w / w gelatin aqueous solution. The solution was then divided into 2 parts. The first part of this initial gelatin solution was used to demonstrate the method of the present invention. This part was combined with 11% active peptides, which consisted of a dried collagen peptide composition, produced from lime treated bovine bone gelatin, and had an average molecular weight of 650 Da. Once the dried collagen peptides were completely dissolved, the pH of the solution was adjusted to one of the target set points given below using sulfuric acid or sodium hydroxide. The solution (covered) was then placed in an 85°C water bath. Samples of the gelatin solution were extracted at different time points throughout the incubation period for analysis. Once removed from the 85°C solution, the time point samples were cooled to between 55°C and 60°C. The different time point samples were then diluted to a concentration of approximately 10% w / w. The pH of the solution at each specific time point was adjusted to 5.4 using sulfuric acid or sodium hydroxide. The time point samples were then further diluted to 6.67% w / w and their viscosities were measured using a gravity pipette at 60° C. These measured viscosities for the methods of the present invention are provided in Table B, reported in mP.

[0091] The second portion of the initial aqueous gelatin solution was used as a control example and was processed together with the inventive example. The pH of this portion was adjusted to the same set point as the inventive part using sulfuric acid or sodium hydroxide. The solution (covered) was then placed in the same 85°C water bath used in the inventive method part. During the entire incubation period, samples of the illuminated gelatin solution were extracted for analysis at different time points. Once removed from the 85°C solution, the time point samples were cooled to between 55°C and 60°C. In order to compare the inventive method and the control example, the same dry collagen peptide composition used in the inventive method was added to produce a solution containing an active peptide concentration of 11%, and the required amount was calculated by the measured mass of the time point sample and its concentration. Once added, the dried collagen peptide was allowed to completely dissolve. The different time point samples were then diluted to a concentration of about 10% w / w. The pH of the solution at each specific time point was then adjusted to 5.4 using sulfuric acid or sodium hydroxide. The sample was then further diluted to 6.67% w / w and its viscosity was measured at 60°C using a gravity pipette. These measured viscosities for the comparative examples are provided in Table C, reported in mP.

[0092] The pH set points used were 6.6, 5.8, 5.4, 5.0, 4.6, 4.4, 4.0 or 3.6.

[0093] Table B

[0094]

[0095] Table C

[0096]

[0097] It was found that the viscosity drop under each condition could be well described by the following empirical formula:

[0098]

[0099] Where η is the gelatin viscosity at 6.67% CDG and 60°C, t is the incubation time, and x0, x1, and x2 are least squares fit parameters. Using standard regression techniques available in the art, a set of x0, x1, and x2 can be determined for each pH and allow interpolation of the time required to reach the target viscosity.

[0100] Table D shows the interpolated times required to achieve three different target viscosities using the inventive method and the control examples. Compared to the control examples using only heat treatment, the viscosity of the samples using the inventive method dropped 13% to 138% faster.

[0101] Table D

[0102]

[0103] A sample of each solution was then further diluted to approximately 0.1%. These samples were temporarily stored in a refrigerator at 4°C to 8°C until all time points for a particular pH reduction run were collected. The retained samples were then analyzed using size exclusion chromatography.

[0104] Size exclusion chromatography utilizes a mobile phase consisting of 8.67mM sodium dodecyl sulfate, 10mM sodium phosphate, 100mM sodium sulfate, and pH is adjusted to 5.2 using NaOH. The column used is TOSOH TSK-GEL G4000SWxl (7.8mm×30mm). The flow rate is 0.578mL / min, the injection volume is 50μL, and each run time is 30 minutes. The chromatogram is determined by measuring the absorbance at 214nm. Each chromatogram is then standardized. The standardized chromatogram is divided into 5 different regions, and the mass fraction of the protein in each region is determined by numerical integration (trapezoidal method). The 5 different regions are defined as microgel region, high molecular weight (HMW) region, chain region, sub-alpha chain region, and low molecular weight (LMW) region. Figure 3 A graphical representation of these molecular weight regions overlaid on the resulting chromatogram is provided.

[0105] Using a reference standard gelatin with a well-defined α peak, the center of the α peak of each chromatographic sample set was determined (in mL of eluted solution). The lower and upper limits of the α chain region are defined as ±0.35 mL from the center of the easily identifiable chromatographic peak. The microgel peak in the reference standard gelatin is also well defined. The microgel region is set to the amount of material eluted before the first minimum after the microgel peak. All materials eluted after the microgel region and before the α region are classified as the HMW region. A second reference solution containing only the collagen peptide composition used was also tested. The starting position of the collagen peptide peak is also clearly defined in the chromatogram (usually around 11.9 to 12.1 mL). All materials eluted after the starting point of the collagen peptide peak are classified within the LMW region. Materials eluted between the end of the α chain region and the starting point of the LMW region are classified as sub-α regions.

[0106] Table E provides the molecular weight distributions produced by the process of the present invention at pH 3.6 and illustrates the increased rate of microgel reduction compared to the control example.

[0107] Table F provides the molecular weight distributions produced by the process of the present invention at pH 4.0 and illustrates the increased rate of microgel reduction compared to the control example.

[0108] Table G provides the molecular weight distribution produced by the process of the present invention at pH 4.4 and illustrates the increased rate of microgel reduction compared to the control example.

[0109] Table H provides the molecular weight distributions produced by the process of the present invention at pH 4.6 and illustrates the increased rate of microgel reduction compared to the control example.

[0110] Table I provides the molecular weight distribution produced by the process of the present invention at pH 5.0 and illustrates the increased rate of microgel reduction compared to the control example.

[0111] Table J provides the molecular weight distribution produced by the process of the present invention at pH 5.4 and illustrates the increased rate of microgel reduction compared to the control example.

[0112] Table K provides the molecular weight distributions produced by the process of the present invention at pH 5.8 and illustrates the increased rate of microgel reduction compared to the control example.

[0113] Table L provides the molecular weight distribution produced by the process of the present invention at pH 6.6 and illustrates the increased rate of microgel reduction compared to the control example.

[0114] Table E

[0115]

[0116] Table F

[0117]

[0118] Table G

[0119]

[0120] Table H

[0121]

[0122] Table I

[0123]

[0124] Table J

[0125]

[0126] Table K

[0127]

[0128] Table L

[0129]

[0130] Examination of size exclusion chromatography revealed that the process of the present invention reduced the microgel and HMW fractions of gelatin at a significantly higher rate than heat treatment alone. Using a process pH of 4.4 and a 2.0 hour reaction time, the microgel content was reduced from 2.6% to 0.2% using the present invention, while the heat process only reduced the microgel content from 2.6% to 0.5%. Using a process pH of 5.0 and a 3.0 hour reaction time, the microgel content was reduced from 2.4% to 0.2% using the present invention, while the heat process only reduced the microgel content from 2.4% to 1.0%. Using a process pH of 5.8 and a 3.0 hour reaction time, the microgel content was reduced from 2.4% to 0.7% using the present invention, while the heat process only reduced the microgel content from 2.4% to 1.5%. Using a process pH of 6.6 and a 6.0 hour reaction time, the microgel content was reduced from 2.5% to 0.3% using the present invention, while the heat process only reduced the microgel content from 2.5% to 0.7%.

[0131] Example 4

[0132] This example demonstrates the ability of the active peptide to increase the viscosity decline rate and microgel reduction rate of type A gelatin.

[0133] Dried acid-treated pigskin gelatin (Bloom's value of 275 g, 6.67% / 60°C viscosity of 41.8 mP) was dissolved in water at 55°C to 60°C to produce a 20% w / w gelatin aqueous solution. The solution was then divided into two parts. The first part of this initial gelatin solution was used to illustrate the method of the present invention. This part was combined with 11% active peptides, which consisted of a dried collagen peptide composition, produced from acid-treated pigskin gelatin, and had an average molecular weight of 590 Da. Once the dried collagen peptides were completely dissolved, the pH of the solution was adjusted to 5.4 using sodium hydroxide. The solution (covered) was then placed in an 85°C water bath. Samples of the gelatin solution were extracted at different time points throughout the incubation period for analysis. Once removed from the 85°C solution, the time point samples were cooled to between 55°C and 60°C. The solution was then diluted to approximately 10% w / w and the pH was readjusted to 5.4 units using sulfuric acid or sodium hydroxide (if necessary). The samples at different time points were then further diluted to 6.67% w / w and their viscosities were measured using a gravity pipette at 60° C. The measured viscosities at 0, 1, 2, 3, 6, 22 and 23 hours were measured to be 24.95, 31.11, 28.99, 27.15, 23.04, 12.53 and 11.44 mP, respectively.

[0134] The second portion of the initial aqueous gelatin solution was used as a control example and was processed together with the inventive example. The pH of this portion was adjusted to 5.4 using sodium hydroxide. The solution (covered) was then placed in the same 85°C water bath used in the inventive method section. During the entire incubation period, samples of the illuminated gelatin solution were extracted for analysis at different time points. Once removed from the 85°C solution, the time point samples were cooled to between 55°C and 60°C. In order to compare the inventive method and the control example, the same dry collagen peptide composition used in the inventive method was added to produce a solution containing an active peptide concentration of 11%, and the required amount was calculated by the measured mass of the time point sample and its gelatin concentration. Once added, the dried collagen peptides were allowed to dissolve completely. The solution was then diluted to about 10% w / w and the pH was readjusted to 5.4 units with sulfuric acid or sodium hydroxide (if necessary). The different time point samples were then further diluted to 6.67% w / w and their viscosity was measured at 60°C using a gravity pipette. The measured viscosities at 0, 1, 2, 3, 6, 22 and 23 hours were measured to be 34.95, 32.00, 31.41, 30.20, 25.59, 17.08 and 15.01 mP, respectively.

[0135] Using the empirical relationship described in Example 3, Formula 1, it was calculated that the time required to reach a target viscosity of 25 mP was 4.3 hours when using the method of the present invention and 7.4 hours when using the comparative example. It was calculated that the time required to reach a target viscosity of 20 mP was 8.4 hours when using the method of the present invention and 14.7 hours when using the comparative example. It was calculated that the time required to reach a target viscosity of 15 mP was 15.9 hours when using the method of the present invention and 27.6 hours when using the comparative example.

[0136] A sample of each solution was then further diluted to about 0.1%. These samples were temporarily stored in a refrigerator at 4°C to 8°C until all time points for a particular pH reduction run were collected. The retained samples were then analyzed using size exclusion chromatography. The size exclusion chromatography used was the same as described in Example 3. Table M provides the molecular weight distribution produced by the method of the present invention at pH 5.4 and illustrates that the rate of microgel reduction was increased compared to the control example.

[0137] Examination of size exclusion chromatography revealed that the process of the present invention reduced the microgel and HMW fractions of gelatin at a significantly higher rate than heat treatment alone. Using a process pH of 5.4 and a reaction time of 3.0 hours, the microgel content was reduced from 2.5% to 0.6% using the present invention, while the heat process only reduced the microgel content from 2.4% to 1.6%. After a reaction time of 6.0 hours, the microgel content was 0.3% using the present invention, while the heat process only reduced the microgel content to 1.1%.

[0138] Table M

[0139]

[0140] Example 5

[0141] This example illustrates the ability of the active peptide to increase the viscosity reduction rate and microgel reduction rate of type A gelatin when glycine is used as the active peptide.

[0142] Dried acid-treated pig skin gelatin (Bloom value 275g, 6.67% / 60°C viscosity 41.8mP) was dissolved in water at 55°C to 60°C to produce a 20% w / w gelatin aqueous solution. The solution was then divided into 2 parts. The first part of this initial gelatin solution was used to illustrate the method of the present invention. This part was combined with 11% active peptides, which consisted of dried glycine. Once the dried collagen peptides were completely dissolved, the pH of the solution was adjusted to 5.4 using sodium hydroxide. The solution (covered) was then placed in an 85°C water bath. Samples of the gelatin solution were extracted at different time points throughout the incubation period for analysis. Once removed from the 85°C solution, the time point samples were cooled to between 55°C and 60°C. The solution was then diluted to approximately 10% w / w and the pH was readjusted to 5.4 units using sulfuric acid or sodium hydroxide (if necessary). The samples at different time points were then further diluted to 6.67% w / w and their viscosities were measured using a gravity pipette at 60° C. The measured viscosities at 0, 1, 2, 3, 5, 23 and 28 hours were measured to be 35.53, 32.89, 30.20, 27.46, 24.00, 12.17 and 9.59 mP, respectively.

[0143] A second portion of the initial aqueous gelatin solution was used as a control example and was processed together with the inventive example. The pH of this portion was adjusted to 5.4 using sodium hydroxide. The solution (covered) was then placed in the same 85°C water bath used in the inventive method section. Samples of the illuminated gelatin solution were extracted at different time points throughout the incubation period for analysis. Once removed from the 85°C solution, the time point samples were cooled to between 55°C and 60°C. In order to compare the inventive method and the control example, the same dry glycine used in the inventive method was added to produce a solution containing an active peptide concentration of 11%, and the required amount was calculated from the measured mass of the time point sample and its gelatin concentration. Once added, the dry glycine was allowed to dissolve completely. The solution was then diluted to about 10% w / w and the pH was readjusted to 5.4 units with sulfuric acid or sodium hydroxide (if necessary). The different time point samples were then further diluted to 6.67% w / w and their viscosity was measured at 60°C using a gravity pipette. The measured viscosities at 0, 1, 2, 3, 5, 23 and 28 hours were measured to be 36.40, 34.07, 33.48, 31.11, 16.48 and 13.95 mP respectively.

[0144] Using the empirical relationship described in Example 3, Formula 1, it was calculated that the time required to reach a target viscosity of 25 mP was 4.6 hours when using the method of the present invention and 8.8 hours when using the comparative example. It was calculated that the time required to reach a target viscosity of 20 mP was 8.5 hours when using the method of the present invention and 15.1 hours when using the comparative example. It was calculated that the time required to reach a target viscosity of 15 mP was 15.0 hours when using the method of the present invention and 25.8 hours when using the comparative example.

[0145] Samples of each solution were then further diluted to about 0.1%. These samples were temporarily stored in a refrigerator at 4°C to 8°C until all time points for a particular pH reduction run were collected. The retained samples were then analyzed using size exclusion chromatography. The size exclusion chromatography used was the same as described in Example 3. Table N provides the molecular weight distribution produced by the method of the present invention at pH 5.4 and illustrates that the rate of microgel reduction was increased compared to the control example.

[0146] Examination of size exclusion chromatography revealed that the process of the present invention reduced the microgel and HMW fractions of gelatin at a significantly higher rate than heat treatment alone. Using a process pH of 5.4 and a reaction time of 3.0 hours, the microgel content was reduced from 4.3% to 1.1% using the present invention, while the heat process only reduced the microgel content from 4.1% to 2.4%. After a reaction time of 5.0 hours, the microgel content was 0.5% using the present invention, while the heat process only reduced the microgel content to 1.8%.

[0147] Table N

[0148]

[0149] Example 6

[0150] This example illustrates the partial removal of active peptides after they have been used to reduce the viscosity and microgel concentration of a gelatin composition. A gelatin composition derived from lime-treated bovine bone feedstock was produced according to the procedure described in Example 3. The viscosity of the gelatin composition measured at 6.67% and 60°C was 17.8 mP. The gelatin composition was dissolved in water to form a 5% w / w gelatin aqueous solution and maintained at a temperature of 58°C. The solution was passed through a cross-flow ultrafiltration chamber (manufactured by Sartorius Corporation) using a 5,000 Da membrane cutoff. 200) recirculation. The permeate solution was collected. The recirculation continued for about 35 minutes. The concentration of the aqueous gelatin solution was increased to about 15.4% w / w. Samples of the original 5% w / w aqueous gelatin solution, the concentrated gelatin solution and the collected permeate solution were analyzed by size exclusion chromatography. The microgel region, HMW region, α region, sub-α region and LMW region of the starting gelatin composition accounted for 0.2%, 12.1%, 12.8%, 60.4% and 14.5%, respectively. The microgel region, HMW region, α region, sub-α region and LMW region of the concentrated gelatin solution accounted for 0.2%, 12.9%, 13.7%, 64.2% and 9.0%, respectively. The microgel region, HMW region, α region, sub-α region and LMW region of the ultrafiltration permeate of the gelatin composition accounted for 0.0%, 0.0%, 0.0%, 0.1% and 99.9%, respectively. The amount of LMW present in the concentrated gelatin solution was reduced by 38%, while the chromatogram describing the higher molecular weight region remained consistent with the chromatogram of the original gelatin solution. The permeate solution was composed of active peptides used in the process of preparing the gelatin composition. This example shows that the amount of active peptides present in the gelatin composition can be reduced. The active peptides in the permeate can be further concentrated. The collected permeate containing active peptides can also be reused to extract gelatin from collagen-containing raw materials.

[0151] Example 7

[0152] This example demonstrates the ability of varying amounts of active peptides to increase the viscosity reduction rate and microgel reduction rate of type A gelatin.

[0153] Dried acid-pretreated bovine bone gelatin (Bloom value of 288 g, 6.67% / 60°C viscosity of 40.4 mP) was dissolved in water at 55°C to 60°C to produce a 20% w / w gelatin aqueous solution. The solution was then divided into 2 parts. The first part of this initial gelatin solution was used to illustrate the method of the present invention. The parts were combined to produce a set point concentration of active peptides, which consisted of a dried collagen peptide composition, produced from lime-treated bovine bone gelatin, and had an average molecular weight of 650 Da. Once the dried collagen peptides were completely dissolved, the pH of the solution was adjusted to 5.0 using sulfuric acid. The solution (covered) was then placed in an 85°C water bath. During the entire incubation period, samples of the gelatin solution were extracted at different time points for analysis. Once removed from the 85°C solution, the time point samples were cooled to between 55°C and 60°C. The different time point samples were then diluted to a concentration of approximately 10% w / w. The pH of the solution at each specific time point was adjusted to 5.4 using sodium hydroxide. The time point samples were then further diluted to 6.67% w / w and their viscosities were measured using a gravity pipette at 60° C. These measured viscosities for the methods of the present invention are provided in Table O, reported in mP.

[0154] The second portion of the initial aqueous gelatin solution was used as a control example and was processed together with the inventive example. The pH of this portion was also adjusted to 5.0 with sulfuric acid. The solution (covered) was then placed in the same 85°C water bath used in the inventive method section. During the entire incubation period, samples of the illuminated gelatin solution were extracted for analysis at different time points. Once removed from the 85°C solution, the time point samples were cooled to between 55°C and 60°C. In order to compare the inventive method and the control example, the same dry collagen peptide composition used in the inventive method was added to produce a solution containing the same corresponding set point concentration of active peptides as used during the inventive method, and the required amount was calculated by the measured mass of the time point sample and its concentration. Once added, the dried collagen peptide was allowed to completely dissolve. The different time point samples were then diluted to a concentration of about 10% w / w. The pH of the solution at each specific time point was then adjusted to 5.4 using sodium hydroxide. The sample was then further diluted to 6.67% w / w and its viscosity was measured at 60°C using a gravity pipette. These measured viscosities for the comparative examples are provided in Table P, reported in mP.

[0155] The active peptide concentration set points used were 3%, 5%, 7%, 9% and 11%.

[0156] Table O

[0157]

[0158] Table P

[0159]

[0160] Using the empirical relationship described in Example 3, Equation 1, the time table Q required to reach the target viscosity shows the interpolated time required to obtain 3 different target viscosities using the inventive method and the comparative example. The viscosity of the samples using the inventive method dropped 23% to 108% faster than the comparative example using only heat treatment.

[0161] Table Q

[0162]

[0163] Then a sample of each solution was further diluted to about 0.1%. These samples were temporarily stored in a refrigerator at 4°C to 8°C until all time points for a specific reduction run were collected. The retained samples were then analyzed using size exclusion chromatography. The size exclusion chromatography used was the same as described in Example 3.

[0164] Table R provides the molecular weight distribution produced by the process of the present invention at an active peptide concentration of 3% and illustrates the increased rate of microgel degradation compared to the control example.

[0165] Table S provides the molecular weight distribution produced by the method of the present invention at an active peptide concentration of 5% and illustrates the increased rate of microgel degradation compared to the control example.

[0166] Table T provides the molecular weight distribution produced by the method of the present invention at an active peptide concentration of 7% and illustrates the increased rate of microgel degradation compared to the control example.

[0167] Table U provides the molecular weight distribution produced by the method of the present invention at an active peptide concentration of 9% and illustrates the increased rate of microgel degradation compared to the control example.

[0168] Table V provides the molecular weight distribution produced by the method of the present invention at an active peptide concentration of 11% and illustrates the increased rate of microgel degradation compared to the control example.

[0169] Table R

[0170]

[0171] Table S

[0172]

[0173] Table T

[0174]

[0175] Table U

[0176]

[0177] Table V

[0178]

[0179] Examination of size exclusion chromatography revealed that the process of the present invention reduced the microgel and HMW fractions of gelatin at a significantly higher rate than heat treatment alone. Using 3% active peptide and 5.0 hours reaction time, the microgel content was reduced from 8.5% to 2.7% using the present invention, while the heat treatment only reduced the microgel content from 8.5% to 3.7%. Using 5% active peptide and 6.0 hours reaction time, the microgel content was reduced from 6.0% to 0.9% using the present invention, while the heat treatment only reduced the microgel content from 5.3% to 1.9%. Using 7% active peptide and 6.0 hours reaction time, the microgel content was reduced from 5.7% to 0.9% using the present invention, while the heat treatment only reduced the microgel content from 5.1% to 1.8%. Using 9% active peptide and 6.0 hours reaction time, the microgel content was reduced from 5.7% to 0.8% using the present invention, while the heat treatment only reduced the microgel content from 5.5% to 2.3%. Using 11% active peptide and 6.0 hours reaction time, the present invention reduced the microgel content from 5.2% to 0.7%, while the thermal process only reduced the microgel content from 5.5% to 2.2%.

[0180] Example 8

[0181] This example illustrates the gel strength of the gelatin compositions of the present invention.

[0182] Dried lime-treated bovine bone gelatin (Bloom's value of 251 g, 6.67% / 60°C viscosity of 42.6 mP) was dissolved in water at 55°C to 60°C to produce a 20% w / w gelatin aqueous solution. The gelatin solution was then combined with 2% and 20% active peptides consisting of a collagen peptide composition produced from lime-treated bovine bone gelatin. The pH of the solution was adjusted to 5.0 with sulfuric acid and then placed in an 85°C water bath (covered with a watch glass) for several hours. The solution was allowed to cool to 55°C to 60°C. The pH of each solution was then readjusted to 5.4 with sodium hydroxide. The solution was then diluted to 12.5% ​​or 6.67% w / w and its viscosity was measured at 60°C using a gravity pipette. The Bloom's value (gel strength) of the 6.67% concentrated solution was also measured. Data on the viscosity and gel strength of the example gelatin compositions of the present invention are provided in Table W.

[0183] Table W

[0184]

[0185] Example 9

[0186] This example illustrates the improved crosslinking resistance of the present invention. All gelatin compositions used in this example are made from lime-treated bovine bone gelatin. 12.5% ​​gelatin solutions were prepared by dissolving 10.0 g of different gelatin compositions into 70.0 g of distilled water. 8.00 grams of each gelatin solution was placed in a 25 mL beaker with a magnetic stirring bar. Each beaker was covered and placed in a 40°C water bath. To initiate crosslinking, 2 mL of 0.5% glutaraldehyde solution was added. After 10 seconds, the sample was loaded onto a rheometer equipped with a 60 mm plate-to-plate fixture and a steam trap. The dynamics were analyzed at 40°C, γ = 0.1 and a frequency of 1 Hz by linear oscillatory shear. The changes in the storage modulus G' and loss modulus G" of the gelatin solution were monitored. The initial crosslinking rate was determined by the initial linear viscosity increase at the start of the reaction. The time required to form a crosslinked network covering the entire sample is determined by the time when G' is equal to G". The results of different gelatin compositions are provided in Table X.

[0187] The standard composition used represents a common soft gelatin composition in the industry. The composition has a high initial crosslinking rate and a short network formation time.

[0188] The Gelita RXL composition represents the prior art described in Reference G and contains about 8% to 9% low molecular weight collagen peptides. The gelatin composition has improved initial cross-linking rate and network formation time when compared to the standard gelatin composition described above.

[0189] The present invention contains about 11% to 12% low molecular weight collagen peptides, which significantly improves the initial cross-linking rate, which is 36 times slower than the rate of the GelitaRXL combination. The network formation time is greater than the maximum experimental time, thus exceeding 3600 seconds. This is a significant improvement compared to the network formation times of 301 seconds and 602 seconds for the standard gelatin composition and the Gelita RXL composition, respectively.

[0190] Table X

[0191]

[0192] Example 10

[0193] This embodiment illustrates that the present invention can be used to make soft capsules, and the present invention provides a gelatin composition having excellent soft capsule dissolution properties in the presence of a known composition that can chemically crosslink gelatin. Four kinds of gelatin masses were prepared using different gelatin compositions. The details of each gelatin composition and the gelatin mass formulation used are provided in Table Y. All gelatins studied are from the cattle bone raw material of lime treatment. The gelatin mass formulation was selected to obtain a similar gelatin mass viscosity and a gelatin to plasticizer ratio of 2.

[0194] Table Y

[0195]

[0196] All capsules used the same filler components. The filler components were PEG-400 [97%], glycerol [3%], and brilliant blue dye [0.5%]. Soft gelatin capsules were made using a 7.5 oval die roller running at 2.5 rpm. The capsules were drum dried for 30 minutes and then dried for 1 week at about 22°C and 40% to 50% relative humidity.

[0197] The capsules were stored in sealed bottles at 40°C and contained a mixture of medium chain triglycerides (97.5%) and peppermint oil (2.5%), which is a known crosslinking agent for gelatin capsules.

[0198] After 8 weeks, the capsule is taken out from the storage solution. Then its dissolution performance is measured by immersing the soft capsule in simulated gastric juice (USP paddle method No. 2, 50rpm). Shell dissolution and filler release are monitored simultaneously under 225nm (gelatin shell detection) and 635nm brilliant blue dye (filler release). Six capsules are repeated under each test condition. The average degree of capsule shell dissolution over time is provided in Figure 4. The average amount of the filler release of the soft capsule is provided in Figure 5 over time.

[0199] The experiment found that the standard gelatin composition had the worst dissolution performance, with a significant delay in the release of the filler, indicating that the concentration of peppermint oil used in this example was sufficient to strongly crosslink the gelatin shell. The low viscosity mixture had the second worst dissolution characteristics. The GelitaRXL composition performed better than the low viscosity mixture, with a significantly reduced release time of the filler, indicating that the Gelita RXL gelatin composition was more resistant to the crosslinking effects of peppermint oil. The present invention showed little to no delay in shell dissolution or filler release, and showed excellent soft gelatin dissolution performance.

[0200] References

[0201] AAG Ward and A. Courts, "The Science and Technology of Gelatin", Food Science and Technology: A series of monographs, 1977, Academic Press, New York. ISBN: 0 12 735050 0

[0202] BR Schrieber and H. Gareis, "Gelatine Handbook: Theory and Industrial Practice", 2007, Wiley-VCH GmbH, KGA, Weinheim. ISBN: 978-3-527-31548-2

[0203] CP Davis and BE Tabor, "Kinetic Study of the Crosslinking of Gelatin by Formaldehyde and Glyoxal", 1963, Journal of Polymer Science: Part A, 1, pp. 799-815.

[0204] DF Moll, H. Rosenkranz and W. Himmelmann, "The Structure of Gelatin Crosslinked with Formaldehyde", 1974, The Journal of Photographic Science, 22:6, pp. 255-261.

[0205] E. Ian D. Robinson, "Rate of Crosslinking of Gelatin in Aqueous Solution", 1964, Journal of Applied Polymer Science, 8, pp. 1903-1918.

[0206] FGA Digenis and TB Gold, VP Shah, "Cross-linking of gelatin capsules and its relevance to their in vitro-invivo performance", 1994, J. Pharm Sci., 83 (7) pp. 915-921.

[0207] G. U.S. Patent 7,485,323 B2, John M. Dolphin, Tom Keenan, Jason D. Russell, Wilfried Babel, "Process for making a low molecular weight gelatine hydrolysate and gelatine hydrolysate compositions", filed May 31, 2005.

[0208] H. US Patent 6,080,843, Robert F. Rainville, Anne G. Rowlands, Deborah J. Burrows. Peter Noble, "Gelatin and Method of Manufacture", filed on November 3, 1998.

Claims

1. A method for increasing the rate of decrease of gelatin viscosity, the method comprising the following steps: Providing an aqueous gelatin solution; combining the aqueous gelatin solution with about 2% to about 20% active peptides, the percentage being based on the weight fraction of the active peptides relative to the total dissolved protein content of the aqueous solution, wherein the active peptides comprise collagen peptides having an average molecular weight of about 100 Da to about 5,000 Da and / or comprise amino acids; adjusting the pH of the aqueous solution to about 3.4 to about 8.0; heating the aqueous solution to a temperature of about 60°C to about 150°C; and The aqueous solution is maintained at the temperature for a period of time sufficient to cause the viscosity of the solution to drop to within the range of about 15 mP to about 25 mP when measured at a total dissolved protein concentration of 6.67% w / w, at a temperature of 60°C and at a pH of 5.3 to 5.

5.

2. The method of claim 1, wherein the aqueous gelatin solution is combined with about 9% to about 13% active peptide, the percentages being based on the weight fraction of the active peptide relative to the total protein content of the gelatin composition.

3. The method of claim 1 or 2, wherein the pH of the aqueous solution is adjusted to about 4.5 to about 5.

5.

4. The method according to any one of claims 1 to 3, wherein the aqueous solution is heated to a temperature of about 90°C to about 100°C.

5. The method of any one of claims 1 to 4, wherein the aqueous solution is maintained at the temperature for a period of time sufficient to cause the viscosity of the solution to drop to within the range of about 17 mP to about 22 mP when measured at a total dissolved protein concentration of 6.67% w / w, at a temperature of 60°C and at a pH of 5.3 to 5.

5.

6. The method of any one of claims 1 to 5, wherein the aqueous solution is maintained at the temperature for less than about 24 hours, less than about 10 hours, or less than about 5 hours.

7. The method according to any one of claims 1 to 6, wherein the collagen peptide is derived from type A gelatin, type B gelatin, collagen, animal tissue containing collagen, animal bone containing collagen, or a combination thereof. The method according to claim 7 , wherein the collagen peptide is an enzymatically hydrolyzed collagen peptide.

9. The method according to any one of claims 1 to 8, wherein the average molecular weight of the collagen peptide is about 400 Da to 800 Da.

10. The method according to any one of claims 1 to 9, wherein the active peptides further comprise peptides derived from hydrolyzed soy, casein, whey or pea protein, or a combination thereof, and wherein the average molecular weight of the peptides is from about 100 Da to about 5,000 Da.

11. The method according to any one of claims 1 to 10, wherein the amino acid is selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, lysine, hydroxylysine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine or a combination thereof, and / or wherein the active peptide comprises diglycine.

12. The method according to any one of claims 1 to 11, wherein the gelatin is obtained from cattle bones, cattle hides, pig bones, pig skins, fish skins, chicken bones, jellyfish, or a combination thereof.

13. The method of claim 12, wherein the gelatin is type A gelatin, type B gelatin, or a combination thereof.

14. The method according to any one of claims 1 to 13, wherein the concentration of the aqueous gelatin solution is about 5% to about 10% w / w.

15. The method according to any one of claims 1 to 14, wherein the concentration of the aqueous gelatin solution is about 17% to about 25% w / w.

16. The method of any one of claims 1 to 15, wherein the concentration of the aqueous gelatin solution is about 30% to about 44% w / w.

17. The method according to any one of claims 1 to 16, further comprising the step of at least partially removing the active peptide by ultrafiltration, diafiltration or a combination thereof after the aqueous solution has reached a viscosity of about 15 mP to about 25 mP.

18. The method according to any one of claims 1 to 17, further comprising the step of recovering the at least partially removed active peptides by concentration using nanofiltration, reverse osmosis, evaporation or a combination thereof.

19. A gelatin composition comprising gelatin and about 2% to about 20% active peptides, the percentage being based on the weight fraction of the active peptides relative to the total protein content of the gelatin composition, wherein the active peptides comprise collagen peptides having an average molecular weight of about 100 Da to about 5,000 Da and / or comprise amino acids, wherein when measured at a total dissolved protein concentration of 6.67% w / w, at a temperature of 60° C. and at a pH of 5.3 to 5.5, the gelatin composition has A viscosity of 15 to 17 mP and a Bloom's value of 55 to 171 g, or A viscosity of 17 to 19 mP and a Bloom's value of 73 to 189 g, or A viscosity of 19 to 21 mP and a Bloom's value of 91 to 205 g, or A viscosity of 21 to 23 mP and a Bloom's value of 109 to 220 g, or Viscosity of 23 to 25 mP and Bloom's value of 124 to 233 g.

20. The gelatin composition of claim 19, wherein the gelatin composition comprises a microgel fraction of about 0.8% or less relative to the total protein content of the gelatin composition.

21. The gelatin composition of claim 19 or 20, wherein the gelatin composition comprises from about 9% to about 13% active peptides, the percentages being based on the weight fraction of the active peptides relative to the total protein content of the gelatin composition.

22. A gelatin composition according to any one of claims 19 to 21, wherein the composition has a pH of 5.3 to 6.

0.

23. The gelatin composition according to any one of claims 19 to 22, wherein the collagen peptides are derived from type A gelatin, type B gelatin, collagen, animal tissue containing collagen, animal bones containing collagen, or a combination thereof.

24. The gelatin composition according to any one of claims 19 to 23, wherein the collagen peptide is an enzymatically hydrolyzed collagen peptide.

25. The gelatin composition according to any one of claims 19 to 24, wherein the average molecular weight of the collagen peptides is about 400 Da to 800 Da.

26. The gelatin composition according to any one of claims 19 to 25, wherein the active peptides further comprise peptides derived from hydrolyzed soy, casein, whey or pea protein, or a combination thereof, and wherein the average molecular weight of the peptides is from about 100 Da to about 5,000 Da.

27. A gelatin composition according to any one of claims 19 to 26, wherein the amino acids are selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, lysine, hydroxylysine, methionine, phenylalanine, proline, hydroxyproline, serine, threonine, tryptophan, tyrosine, valine or a combination thereof, and / or wherein the active peptide comprises diglycine.

28. The gelatin composition of any one of claims 19 to 27, wherein the gelatin is obtained from cattle bones, cattle hides, pig bones, pig skins, fish skins, chicken bones, jellyfish, or a combination thereof.

29. The gelatin composition according to any one of claims 19 to 28, wherein the gelatin is type A gelatin, type B gelatin or a combination thereof.

30. The gelatin composition according to any one of claims 19 to 29, wherein the gel composition is in the form of granules or powder obtained by drying and grinding.

31. A gelatin composition according to any one of claims 19 to 30, comprising gelatin obtained by a process according to any one of claims 1 to 18.

32. Use of the gelatin composition according to any one of claims 19 to 31 for manufacturing gelatin capsules, wherein the gelatin capsules are preferably hard capsules, soft capsules or microcapsules.

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