Title of Invention

PROCESS FOR MAKING A LOW MOLECULAR WEIGHT GELATINE HYDROLYSATE AND GELATINE HYDROLYSATE COMPOSITIONS

Abstract The present invention provides a process to make a gelatine hydrolysate, a gelatine hydrolysate, and gelatine compositions including gelatine hydrolysates. More specifically, the invention provides gelatine compositions having a reduced tendency to cross-link and improved dissolution properties.
Full Text

PROCESS FOR MAKING A LOW MOLECULAR WEIGHT GELATINE
HYDROLYSATE AND GELATINE HYDROLYSATE COMPOSITIONS
FIELD OF THE INVENTION
This application claims priority of USSN 11/140,863 filed on May 31, 2005,
which application is hereby incorporated by reference in its entirety.
The present invention generally relates to a gelatine hydrolysate, a
process for making the gelatine hydrolysate, and a composition comprising the
gelatine hydrolysate. More specifically, the present invention provides a low
molecular weight gelatine hydrolysate having a high primary amine content, to a
process for making the gelatine hydrolysate, and to a gelatine composition
including gelatine hydrolysate.
BACKGROUND OF THE INVENTION
Gelatine is manufactured by the denaturation of collagen contained in
materials such as pig skin, cattle skin or hide, and animal bones. Like its parent
protein, collagen, gelatine is defined by a distinctive structure comprising a
unique blend of amino acids. Native collagen is a scleroprotein based on a
polypeptide chain comprising approximately 1050 amino acids. Three of these
polypeptide chains come together to form a triple helix. Superimposition of
several of these triple helices produces fibrils of collagen that are stabilized by
cross-linking, hence forming a three-dimensional network structure. This
particular structure renders collagen insoluble; it is then brought into soluble
form by partial hydrolysis as gelatine or gelatine hydrolysate. The amino acid
content of collagen and hence of gelatine, is about one third glycine and a
further 22% proline and 4-hydroxyproline; the remaining 45% comprise 17
different amino acids. Gelatine has a particularly high content of acidic and
basic amino acids. Of the acidic amino acids (glutamic acid and aspartic acid),
variable amounts are present in the amido form as glutamine and asparagine
depending on the processing conditions used in the gelatine manufacturing
process. Cysteine is completely absent; of the sulphur-containing amino acids,
methionine is the only one present.

The data for poultry and fish collagen are somewhat different, but the
present invention is applicable likewise to gelatine and/or gelatine hydrolysate
derived from poultry and fish collagen.
Gelatine can be utilized in a wide array of applications depending upon its
starting material and method of manufacture. This is because the physical and
chemical behavior of gelatine is determined on one hand by a combination of its
amino acid content and the resulting spatial structure, and on another hand by a
myriad of conditions such as pH, ionic strength and reactions with other
molecules. For example, different kinds of gelatines are utilized in diverse
applications such as food, photographic, cosmetic, and pharmaceutical.
In the pharmaceutical industry, gelatine is used inter alia in the
manufacture of hard and soft capsules. Gelatine capsules provide a convenient
and efficient method to orally administer a drug because the capsules
disintegrate rapidly upon exposure to the acidic content of the stomach, thus
releasing the drug into the body. While gelatine capsules provide a
pharmaceutically elegant manner in which to administer a drug, there is,
however, a risk that the gelatine capsule may suffer from retardation of
disintegration and dissolution resulting from a process known as cross-linking.
Cross-linking is believed to occur when carbonyl groups in gelatine, carbonyl-
containing fill ingredients in capsules, or decomposition of fill ingredients into
carbonyl groups , react with primary amines and other nitrogenous compounds
present in gelatine to form cross-links.
Cross-linking, in particular, can have dire consequences on the
performance of gelatine capsules upon extended storage and exposure to
extremes of heat and humidity. Extensive gelatine cross-linking in capsule
formulations may lead to the formation of a very thin, tough and water-insoluble
film, usually referred to as a pellicle. The pellicle acts as a rubbery, water-
insoluble layer that can restrict, or prevent release of the contents of the
capsule.
One widely reported means to prevent cross-linking in gelatine capsules
focuses on products that act as carbonyl scavengers, preventing the interaction
of carbonyl groups, e.g., aldehyde groups, with the gelatine capsule shell, thus
preventing gelatine cross-linking. These methods all generally suggest adding

products to the pharmaceutical composition contained in the gelatine capsules.
For example, it has been shown that adding the amino acid glycine and citric
acid in combination to formulations encapsuled in gelatine hard capsules
improved the dissolution profile of the hard capsules (3). Addition of the amino
acid glycine alone was proven not to yield satisfactory results. But the addition
of carbonyl scavengers such as glycine, and carboxylic acids such as citrate, in
the amounts needed to reduce cross-linking in gelatine capsules is significantly
cost prohibitive. As such, adding these products to gelatine is not a practical
solution to reduce cross-linking in gelatine capsules.
SUMMARY OF THE INVENTION
The present invention provides a practical, cost-effective means to reduce
cross-linking in gelatine. Briefly, the invention encompasses a low molecular
weight gelatine hydrolysate that, when blended with higher molecular weight
gelatine, reduces the gelatine's cross-linking and improves the dissolution
properties by increasing the amounts of free glycine, other amino acids, and
small peptides in the blended gelatine product. Advantageously, because the
gelatine hydrolysate and blended gelatine composition of the invention have
reduced cross-linking properties achieved without the addition of products such
as glycine as an isolated compound admixed with citric acid, the gelatine may
still be marketed as a natural product.
Among the several aspects of the invention, therefore, is a process for
producing a gelatine hydrolysate having an average molecular weight of from
about 100 to about 2000 Da, preferably about 1500 Da, and an average primary
amine content from about 1.0 x 10"3 to about 1.0 x 10"2 )iMol of primary amine
per \ig of gelatine hydrolysate. The process comprises contacting a gelatine
starting material with at least one proteolytic enzyme having endopeptidase
activity to form an endopeptidase digested gelatine product. The endopeptidase
digested gelatine product is then typically contacted with at least one proteolytic
enzyme having exopeptidase activity. Generally, the endopeptidase and
exopeptidase proteolytic digestions proceed for a sufficient length of time and
are conducted under reaction conditions so as to form the gelatine hydrolysate.
Another aspect of the invention encompasses a process for making a
gelatine hydrolysate. The process comprises contacting a gelatine starting

material with a series of at least three proteolytic enzymes having
. endopeptidase activity to form an endopeptidase digested gelatine product.
Typically, the three proteolytic enzymes consist of Endopeptidase from Bacillus
subtilis (e.g.Corolase® 7089), Bromelain (e.g. Enzeco® Bromelain Concentrate),
and Papain (e.g.Papain 6000L). The endopeptidase digested gelatine product is
then contacted with a series of at least two proteolytic enzymes having
exopeptidase activity. Generally, the two proteolytic enzymes consist of
Exopeptidase from Aspergillus oryzae (e.g. Validase® FPU) and Exopeptidase
from Aspergillus sojae (e.g. Corolase® LAP).
Yet a further aspect of the invention provides a gelatine hydrolysate. The
gelatine hydrolysate will typically have an average molecular weight of from
about 100 to about 2000 Da, preferably about 1500 Da and an average primary
amine content from about 1.0 x 10-3 to about 1.0 x 10-2 μMol of primary amine
per μg of gelatine hydrolysate. In one embodiment, the gelatine hydrolysate is
made by a process comprising contacting a gelatine starting material with a
series of at least three proteolytic enzymes having endopeptidase activity to
form an endopeptidase digested gelatine product. Typically, the three
proteolytic enzymes are selected from Endopeptidase from Bacillus subtilis (e.g.
Corolase® 7089), Bromelain (e.g. Enzeco® Bromelain Concentrate), and Papain
(e.g. Papain 6000L). The endopeptidase digested gelatine product is then
contacted with a series of at least two proteolytic enzymes having exopeptidase
activity. Generally, the two proteolytic enzymes are selected from Exopeptidase
from Aspergillus oryzae (e.g. Validase® FPU) and Exopeptidase from Aspergillus
sojae (e.g. Corolase® LAP).
An additional aspect of the invention is directed to a gelatine composition.
The composition comprises a gelatine hydrolysate and gelatine. Typically, the
composition will comprise from about 1 % to about 20% by weight of the
gelatine hydrolysate and from about 80% to about 99% by weight of the
gelatine.
Other objects and features of the invention will be in part apparent and in
part pointed out hereinafter.

BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the reduction in formaldehyde induced cross linking of LH-1,
a limed-hide gelatine, upon the addition of 2 different gelatine hydrolysates,
Type BH-3 and Type LHSH, as measured by the Vortex Hardening procedure.
Hydrolysate Type BH-3 is a low molecular weight limed-hide hydrolysate and
Type LHSH is a limed-hide hydrolysate of the present invention.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides a novel gelatine hydrolysate, a process to
make the gelatine hydrolysate and gelatine compositions comprising the gelatine
hydrolysate. It has been discovered that blending a low molecular weight
gelatine hydrolysate and in particular, the gelatine hydrolysate of the present
invention, with gelatine, reduces the gelatine's tendency to cross-link and
improves dissolution properties by increasing the amounts of free glycine, other
amino acids, and small peptides in the blended gelatine product.
Advantageously, the present invention provides a cost effective means to reduce
gelatine cross-linking with the benefit of maintaining the original amino acid
composition of gelatine and without the need to add non-gelatine derived
compounds like citric acid. As such, the gelatine hydrolysate compositions of the
present invention can still be marketed as natural products.
J. Process for Making the Gelatine Hydrolysate
One aspect of the present invention encompasses a process to produce a
gelatine hydrolysate having an average molecular weight of from about 100 to
about 2000 Da, preferably about 1500 Da and an average primary amine
content from about 1.0 x 10-3to about 1.0 x 10-2fiMol of primary amine per μg of
gelatine hydrolysate. The process comprises contacting a gelatine starting
material with at least one proteolytic enzyme having endopeptidase activity to
form an endopeptidase digested gelatine product. The endopeptidase digested
gelatine product is then typically contacted with at least one proteolytic enzyme
having exopeptidase activity. Generally, the endopeptidase and exopeptidase

proteolytic digestions proceed for a sufficient length of time and are conducted
under reaction conditions so as to form the gelatine hydrolysate.
The gelatine starting material used in the process of the invention is
typically derived from collagen or collagen rich tissue available from several
suitable raw materials. Collagen rich tissues include the skin and bones from
animals, such as from fish, poultry, pigs or cattle. There are generally two main
types of gelatine derived from collagen, Type A and Type B that differ in their
method of manufacture. In one embodiment, the gelatine starting material is
Type A gelatine. Type A, with an isoionic point of 7 to 10.0, is derived from
collagen with exclusively acid pretreatment by methods generally known in the
art. In an alternative embodiment, the gelatine starting material is Type B
gelatine. Type B, with an isoionic point of 4.8 to 5.8, is the result of an alkaline
pretreatment of collagen and is produced by methods generally known in the
art.
In another alternative embodiment, the gelatine starting material is a
mixture of Type A and Type B. The respective amounts of Type A and Type B
gelatine may be greatly varied without detrimental effect on the properties of
the gelatine hydrolysate produced.
In principle, any one of Type A and Type B gelatine could be exchanged
completely or partially by enzymatically produced gelatine. However, the
enzymatic process for manufacturing gelatine is up to present not widely used.
Irrespective of the embodiment, the gelatine starting material will
normally contain from about 80% to about 90% by weight protein, from about
0.1% to about 2% by weight mineral salts (corresponding to the ash content)
and from about 10% to 15% by weight water.
It is also contemplated that the physical properties of the gelatine starting
material can and will vary depending upon the intended use of the gelatine
hydrolysate. The gelatine starting material will typically have an average
molecular weight of from about 50,000 Da to about 200,000 Da. In a
particularly preferred embodiment, the gelatine starting material will have an
average molecular weight of less than about 150,000 Da.
In one embodiment, the bloom strength of the gelatine starting material
will be from about 50 to about 300, the pH will be from about 3.8 to about 7.5,

the isoelectric point will be from 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 gelatine starting material is
substantially Type A gelatine, the bloom strength will be from about 50 to about
300, 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 gelatine starting material is
substantially Type B gelatine, the bloom strength will be from about 50 to about
300, 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 one preferred embodiment where the gelatine hydrolysate is used in
the manufacture of hard capsule pharmaceutical products, the gelatine starting
material will have a bloom strength from about 200 to about 300, a viscosity
from about 40 to about 60 mP and a pH from about 4.5 to about 6.5. In yet
another preferred embodiment where the gelatine hydrolysate is used in the
manufacture of soft shell capsule pharmaceutical products, the gelatine starting
material will have a bloom strength from about 125 to about 200, a viscosity
from about 25 to about 45 mP and a pH from about 4.5 to about 6.5.
In the process of the invention, the gelatine starting material is typically
mixed or dissolved in water by a process known as swelling to form a solution
comprising from about 10% to about 60% gelatine by weight. In one preferred
embodiment, the solution has from about 10% to about 50% gelatine by weight.
In a further preferred embodiment, the solution has from about 20% to
about 50% gelatine by weight. In a still more preferred embodiment, the
solution has from about 35% to about 40% gelatine by weight.
It is contemplated that gelatines having varying particle sizes may be
utilized in the invention as starting material. For example, the gelatine particle
size may vary from about 0.1 mm to about 10 mm. In one embodiment, the
gelatine particle size may be fine having an average particle size from about 0.1
to about 0.3 mm. In another embodiment, the gelatine particle size may be

medium having an average particle size of from about 0.3 to about 0.8 mm. In
still another embodiment, the gelatine particle size may be large having an
average particle size of approximately greater than about 0.8 mm. Generally
speaking, the particle size of the gelatine starting material will impact the
amount of time needed for the gelatine to dissolve in solution. During the
swelling process, the ability for gelatine to absorb up to ten times its weight in
cold water is utilized. Gelatines having a fine particle size swell within a few
minutes, gelatines having a medium particle size swell within about 8 to about
12 minutes, and gelatines having a large particle size swell within about an hour.
Typically, low concentrated gelatine solutions, solutions having for example,
from about 10% to about 20% by weight gelatine, can be prepared using all
particle sizes. For highly concentrated solutions, solutions having for example,
from about 30% to about 35% gelatine by weight, coarse particles are typically
used because they tend not to aggregate and produce fewer air bubbles when
being processed.
After the gelatine has been brought in solution through the swelling
process and typically prior to the addition of the proteolytic enzymes, the pH,
temperature and Redox State of the solution is typically adjusted to take care of
minor amounts of residual peroxide present in the gelatine from its
manufacturing process so as to optimize the hydrolysis reaction, and in
particular, to ensure that the cysteine-containing proteolytic enzymes utilized in
the hydrolysis reaction function near their optimum activity level. The pH of the
gelatine solution is adjusted and maintained at from about 5 to about 7. In a
particularly preferred embodiment, the pH of the gelatine solution is adjusted
and maintained at from about 6.0 to about 6.5. At this pH, proteolytic enzymes
detailed below are near their optimum activity level. The pH of the gelatine
solution may be adjusted and monitored according to methods generally known
in the art. For example, to decrease the pH of the gelatine solution an acid,
such as hydrochloric acid, is typically added. Alternatively, to increase the pH of
the gelatine solution a base, such as sodium hydroxide, is typically added. The
temperature of the gelatine solution is preferably adjusted and maintained from
about 40° C to about 65° C during the hydrolysis reaction in accordance with
methods known in the art. In a particularly preferred embodiment, the

temperature of the gelatine solution is adjusted and maintained from about 50°
C to about 60° C during the hydrolysis reaction. In general, temperatures above
this range may deactivate proteolytic enzymes, while temperatures below this
range tend to slow the activity of the proteolytic enzymes. Depending upon the
proteolytic enzyme used in the hydrolysis reaction, the Redox State of the
gelatine solution typically should be adjusted and maintained as neutral to
slightly on the reducing side. High levels of oxidants tend to inactivate some of
the cysteine-containing proteolytic enzymes used in the hydrolysis reaction,
while low levels of reductants 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 gelatine solution. Several proteolytic enzymes are suitable for
use in the process of the invention. In a preferred embodiment, the proteolytic
enzymes will be food grade enzymes having endopeptidase or exopeptidase
activity at a pH from about 5 to about 7 and at a temperature from about 40 °C
to about 65 °C. In a particularly preferred embodiment, the proteolytic enzymes
will be food grade enzymes having endopeptidase or exopeptidase activity at a
pH from about 6 to about 6.5 and at a temperature from about 50 °C to about
60 °C.
In one embodiment, the endopeptidase will be a food grade serine
proteinase belonging to EC 3.4.21. In one alternative of this embodiment, the
serine proteinase is a chymotrypsin proteinase. In a further alternative of this
embodiment, the 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 an additional
embodiment, the endopeptidase will be a food grade metalloproteinase
belonging to EC 3.4.24. Exemplary non-limiting examples of food grade
endopeptidases that may be utilized 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 Validase®
Papain Concentrate Sulfite Free.

In a further 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 still 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 an additional
embodiment, the exopeptidase will be a food grade serine-type carboxy
peptidase belonging to EC 3.4.16. In yet another embodiment, the exopeptidase
will be a food grade metallo carboxy peptidase belonging to EC 3.4.17. In an
additional embodiment, the exopeptidase will be a food grade cysteine-type
carboxy peptidase belonging to EC 3.4.18. In still an 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 utilized in the
process of the invention includes Validase® FP II or Corolase LAP®.
Another example of a food grade hydrolytic enzyme that may be used in
the process of the invention is Validase® FP Concentrate. Examples of other
suitable proteolytic food grade enzymes are shown in Table A.



Typically, combinations of endopeptidases and exopeptidases will be
used to catalyze the hydrolysis reaction. The proteolytic enzymes are
preferably selected by considering the protease activity of the enzymes and
selecting enzymes that will maximize the cleaving of peptide bonds in the
gelatine starting material. In a preferred embodiment, enzymes with
preferential endopeptidase activity are added to the gelatine solution first to
form an endopeptidase digested gelatine product. The endopeptidase digested
gelatine product is then contacted with enzymes having preferential
exopeptidase activity without deactivating the endopeptidase(s). It is also
contemplated that in certain embodiments enzymes having exopeptidase
activity may be added before or at the same time as enzymes having
endopeptidase activity.
In one preferred embodiment, the endopeptidase is selected from the
group consisting of Corolase® 7089, Validase® AFP, Validase® FP 500, Alkaline
Protease Concentrate, Validase® TSP, Enzeco® Bromelain Concentrate, Papain
6000L and Validase® Papain Concentrate Sulfite Free; 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® FPU and Corolase® LAP. In a
preferred embodiment, each proteolytic enzyme is sequentially added to the
gelatine starting material in the following order: Corolase® 7089, Enzeco®
Bromelain Concentrate, Papain 6000L, Validase® FPU and Corolase® LAP. In
one alternative of this embodiment, each proteolytic enzyme digests the
gelatine starting material for approximately 0.5 to about 2 hours before
addition of the subsequent proteolytic enzyme.
The amount of proteolytic enzyme added to the hydrolysis reaction can
and will vary depending upon the desired degree of gelatine 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 having
exopeptidase activity is added for a hydrolysis reaction, lasting for a duration of
from about 5 hours to about 24 hours. In a preferred embodiment, about
0.05% to about 0.15% (w/w) of Corolase® 7089, about 0.025% to about
0.075% (w/w) of 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® FPU, and about 0.05% to about 0.15% (w/w) of Corolase® LAP are
added to the gelatine starting material.
The hydrolysis reaction will typically proceed for up to approximately 24
hours. Typically, after about 24 hours the quality of the gelatine hydrolysate,
in terms of color and smell, will begin to noticeably diminish. 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 a still more preferred embodiment,
the hydrolysis reaction will proceed from about 5 hours to about 12 hours.
Within this time period, highly economic process conditions and constant
quality of the gelatine hydrolysate are easily achievable. To end the
hydrolysis reaction, the hydrolyzed gelatine solution may be heated to
approximately 90° C to deactivate the proteolytic enzymes. An additional step
to deactivate the cysteine proteases may be required. If so required, the
addition of hydrogen peroxide or other oxidizing agent may be added,
generally not to exceed 1000 ppm. The gelatine hydrolysate may then be
purified from the hydrolysis solution by any means generally known in the art,
e.g. microfiltration.
Typically, the degree of hydrolysis (DH) of the starting gelatine material
in the process of the invention is greater than about 13%. In certain
embodiments, the DH is from about 10% to about 20%. In other
embodiments, the DH is from about 20% to about 30%. In another
embodiment, the DH is from about 30% to about 40%. In yet another
embodiment, the DH is from about 40% to about 50%. In still another

embodiment, the DH is from about 50% to about 60%. In an additional
embodiment, the DH is from about 60% to about 70%. In yet a further
embodiment, the DH is from about 70% to about 80%. In still another
embodiment, the DH is from about 80% to about 90%. In still another
embodiment, the DH is greater than about 90%. The DH is the percentage of
the total number of peptide bonds in the gelatine starting material that have
been hydrolyzed by proteolytic enzymes. The DH may be calculated by
methods generally known in the art, such as according to the Adler-Nissen
method (19).
It has been observed that gelatine hydrolysates with a lower average
molecular weight are more effective in preventing the cross-linking process. As
a consequence, the amount of hydrolysate in the gelatine formulation may be
reduced which optimizes costs.
II. Gelatine Hydrolysate
Yet another aspect of the invention encompasses a gelatine hydrolysate
made by the process of the invention. Generally speaking, the gelatine
hydrolysate, compared to the gelatine starting material, will comprise a
mixture of peptide of different lengths having an increase in the amounts of
free glycine, other amino acids, and small peptides. The gelatine hydrolysate
will also have a lower average molecular weight and higher primary amine
content compared to the gelatine starting material.
The gelatine hydrolysate will typically have an average molecular weight
of at least about 100 Da. In other embodiments, the gelatine hydrolysate will
typically have an average molecular weight not exceeding about 2000 Da. In
some embodiments, the gelatine hydrolysate will have an average molecular
weight of from about 100 Da to about 2,000 Da. In other embodiments, the
gelatine hydrolysate will have an average molecular weight of about 700 Da to
about 1800 Da. In another embodiment, the gelatine hydrolysate will have an
average molecular weight of about 700 Da to about 1500 Da. In still other

embodiments, the gelatine hydrolysate will have an average molecular weight
of from about 800 Da to about 1200 Da.
The average molecular weight is the weight of a gelatine hydrolysate as
measured by electro-spray ionization liquid chromatography mass
spectrometry (ESI-LC/MS). For example, a gelatine hydrolysate having an
average molecular weight of approximately 1200 Da may have a molecular
weight range from about 75 Da to 8000 Da.
In general, the gelatine hydrolysate will have an average primary amine
content of not less than about 1.0 x 10 3 μMol of primary amine per μg of
gelatine hydrolysate. In another embodiment, the gelatine hydrolysate will
have an average primary amine content of not less than about 1.5 x 10 3μMol
of primary amine per μg of gelatine hydrolysate. In still another embodiment,
the gelatine hydrolysate will have an average primary amine content of not
less than about 2.0 x 10-3 μMol of primary amine per μg of gelatine
hydrolysate. In an additional embodiment, the gelatine hydrolysate will have
an average primary amine content of from about from about 1.0 x 10 -3 to
about 1.0 x 10-2 μMol of primary amine per μg of gelatine hydrolysate. The
primary amine content of the gelatine hydrolysate is measured through
derivatization and subsequent UV absorption (6-8) as illustrated in the
Examples.
The gelatine hydrolysate of the present invention comprises
polypeptides typically of up to about 75 amino acids in length, preferably up to
50 amino acids in length. In one embodiment, the average polypeptide
comprising the gelatine hydrolysate is from about 6 to about 18 amino acids in
length. In another embodiment, the average polypeptide contained in the
gelatine hydrolysate of the present invention is from about 9 to about 20
amino acids in length. The length of a polypeptide chain may be determined
indirectly by size-exclusion chromatography/high performance liquid
chromatography (SEC/HPLC).

In one embodiment, the gelatine hydrolysate will have an average
molecular weight from about 100 Da to about 2,000 Da, an average primary
amine content from about 1.0 x 10-3 to about 1.0 x 10-2 μMol of primary amine
per μg of gelatine hydrolysate, and an average polypeptide length of up to
about 20 amino acids. In still another embodiment, the gelatine hydrolysate
will have an average molecular weight from about 700 Da to about 1500 Da,
an average primary amine content from about 1.0 x 10-3to about 2.0 x 10 -3
μMol of primary amine per μg of gelatine hydrolysate, and an average
polypeptide length of up to about 18 amino acids. In another embodiment,
the gelatine hydrolysate will have an average molecular weight from about
800 Da to about 1200 Da, an average primary amine content from about 1.0 x
10"3 to about 2.0 x 10-3 μMol of primary amine per μg of gelatine hydrolysate,
and an average polypeptide length of from about 4 to about 18 amino acids.
III. Gelatine Compositions
Another aspect of the invention encompasses a gelatine composition
comprising a low molecular weight gelatine hydrolysate and gelatine.
Surprisingly it has been found that when a low molecular weight gelatine
hydrolysate is blended with higher molecular weight gelatine, it reduces the
gelatine's cross-linking and improves the dissolution properties by increasing
the amounts of free glycine, other amino acids, and small peptides in the
blended gelatine product, as shown in the Examples.
A number of different gelatine hydrolysates are suitable for use in the
gelatine composition. In one embodiment, the gelatine hydrolysate will be an
enzymatically-digested hydrolysate. By way of non-limiting example, the
gelatine hydrolysate of the present invention is produced via an enzymatic
hydrolysis procedure, as detailed above. In another embodiment, the gelatine
hydrolysate will be an acid digested hydrolysate. For example, acid hydrolysis
may be conducted by digesting a gelatine starting material with approximately
6 N hydrochloric acid for about 24 hours at a reaction temperature of

approximately 110° C. In yet another embodiment, the gelatine hydrolysate
will be a base digested hydrolysate. By way of non-limiting example, base
hydrolysis may be conducted by digesting a gelatine starting material with a
strong base, such as sodium hydroxide. Acid and base hydrolysis will typically
result in a hydrolysate having free amino acids. In each embodiment (i.e.,
enzymatic, acid and base hydrolysis), suitable gelatine starting materials are
detailed in section I above, which delineates structural and functional
properties for gelatine starting materials to be used in the process of the
invention.
Typically, gelatine 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 gelatine hydrolysate will have an
average molecular weight from about 700 Da to about 1500 Da. In addition,
the gelatine hydrolysate will also have an average primary amine content
ranging from about 1.0 x 10-3to about 1.0 x 10-2μMol of primary amine per μg
of gelatine hydrolysate.
In another embodiment, the average primary amine content may range
from about 1.0 x 10-3to about 2.0 x 10"VMol of primary amine per μg of
gelatine hydrolysate.
In still another embodiment, the average primary amine content may
range from about 2.0 x 10"3 to about 4.0 x 10-3 μMol of primary amine per μg
of gelatine hydrolysate. In still a further embodiment, the average primary
amine content may range from about 4.0 x 10 -3 to about 6.0 x 10 -3 μMol of
primary amine per μg of gelatine hydrolysate. In yet an additional
embodiment, the average primary amine content may range from about 6.0 x
10-3 to about 1.0 x 10-2 μMol of primary amine per μg of gelatine hydrolysate.
The gelatine hydrolysate will also generally have an average polypeptide
chain length from about 4 to about 50 amino acids. In one embodiment, the
average polypeptide comprising the gelatine hydrolysate is up to about 30
amino acids in length. In another embodiment, the average polypeptide

comprising the gelatine hydrolysate 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 gelatine hydrolysate used in the
composition will be the hydrolysate of the present invention as detailed in
section II. Examples of other exemplary gelatine hydrolysates that may be
used in the composition are delineated in Table B. Mixtures of the afore-
described gelatine hydrolysates may also be used.


The gelatine hydrolysate may be blended with several types of gelatine
having a broad range of physical and functional properties. The choice of a
particular gelatine can and will vary greatly depending upon the intended use
of the gelatine composition. Generally speaking, irrespective of the

embodiment or intended use, the gelatine is typically derived from collagen or
collagen rich tissue available from several suitable raw materials such as from
the skin and bones of animals. In one embodiment, the gelatine is Type A
gelatine. In another embodiment, the gelatine is Type B gelatine. In still
another embodiment, the gelatine is a mixture of Type A and Type B gelatine.
Again, gelatine prepared in an enzymatic process may be used to substitute
Type A and/or Type B gelatine.
The gelatine, irrespective of the embodiment, will preferably contain
from about 80% to about 90% by weight protein, from about 0.1% to about
2% by weight mineral salts (ash content) and from about 10% to 15% by
weight water.
The gelatine will typically have a high average molecular weight. In one
embodiment, the gelatine will have an average molecular weight of greater
than about 200,000 Da. In another embodiment, the gelatine will have an
average molecular weight greater than about 150,000 Da. In still another
embodiment, the gelatine will have an average molecular weight from about
100,000 Da to about 200,000 Da.
In one embodiment, the bloom strength of the gelatine will be from
about 50 to about 300, the pH will be from about 3.8 to about 7.5, the
isoelectric point will be from 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 gelatine is substantially Type A
gelatine, the bloom strength will be from about 50 to about 300, 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 gelatine is substantially Type B
gelatine, the bloom strength will be from about 50 to about 300, 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 one preferred embodiment where the gelatine composition is used in
the manufacture of hard capsule pharmaceutical products, the gelatine will
have a bloom strength from about 200 to about 300, a viscosity from about 40
to about 60 mP and a pH from about 4.5 to about 6.5.
In yet another preferred embodiment where the gelatine composition is
used in the manufacture of soft shell capsule pharmaceutical products, the
gelatine will have a bloom strength from about 125 to about 200, a viscosity
from about 25 to about 45 mP and a pH from about 4.5 to about 6.5.
The gelatine composition of the invention will generally comprise from
about 1 % to about 20% by weight of the gelatine hydrolysate and from about
80% to about 99% by weight of the gelatine. In another embodiment, the
gelatine composition will comprise from about 1 % to about 5% by weight of
the gelatine hydrolysate and from about 95% to about 99% by weight of the
gelatine. In yet another embodiment, the gelatine composition will comprise
from about 5 % to about 10% by weight of the gelatine hydrolysate and from
about 90% to about 95% by weight of the gelatine. In another embodiment,
the gelatine composition will comprise from about 10% to about 15% by
weight of the gelatine hydrolysate and from about 85% to about 90% by
weight of the gelatine. In an additional embodiment, the gelatine composition
will comprise from about 15% to about 20% by weight of the gelatine
hydrolysate and from about 80% to about 85% by weight of the gelatine. In a
typical embodiment, the gelatine composition will comprise a ratio of gelatine
hydrolysate to gelatine from about 1:4 to about 1:99 (w/w).
In a preferred embodiment, the gelatine composition will comprise the
gelatine hydrolysate of the present invention and a higher molecular weight
pharmaceutical grade gelatine. In one embodiment, the gelatine composition
will comprise from about 5% to about 10% by weight of the gelatine
hydrolysate and from about 90% to about 95% by weight of the

pharmaceutical grade gelatine. In another embodiment, the gelatine
composition will comprise from about 10% to about 15% by weight of the
gelatine hydrolysate and from about 85% to about 90% by weight of the
pharmaceutical grade gelatine.
Advantageously, gelatine compositions of the present invention and of
this embodiment typically have reduced cross-linking as measured by the
vortex hardening test and viscosity test. Gelatine compositions of this
embodiment typically have a vortex hardening time of 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. Gelatine compositions of this embodiment
typically also have an average initial viscosity of from about 10 to about 15 cP
and after the addition of less than about 0.5% by weight of [2-(4-dimethyl-
carbamoyl-pyridino)-ethane-l-sulfonate] (OB1207® of H.W. Sands
Corporation) to the gelatine composition for about two hours at a reaction
temperature of about 60° C, the gelatine composition has an average viscosity
of from 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 gelatine composition of the invention. The glycine may be added to the
gelatine composition in an amount of from about 0.5% to about 5% by
weight. In a more 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 gelatine composition. The citric acid may be added in an
amount of from about 0.5% to about 5% by weight. In a more typical
embodiment, citric acid is added to the gelatine composition in an amount of
from about 0.5% to about 1.5%.
The gelatine composition of the invention may be employed in several
applications including as a food ingredient, as a cosmetic ingredient and as a
photographic ingredient. Because of the gelatine composition's reduced

tendency to cross-link and improved dissolution properties, in a preferred
embodiment, the gelatine composition is used in the manufacture of
pharmaceutical products.
In one preferred embodiment, the gelatine composition is used in the
manufacture of hard gelatine capsules. As detailed above, when the gelatine
composition is used in the manufacture of hard capsule pharmaceutical
products, the gelatine will have a bloom strength from about 200 to about
300, a viscosity from 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% by weight of the gelatine composition of the invention, approximately
65% by weight water, approximately 5% by weight of a suitable dye, and will
contain a pigment as needed. The hard gelatine capsules may be made
according to any method generally known in the art.
In yet another preferred embodiment, the gelatine composition is used
in the manufacture of soft capsule gelatine. As detailed above, when the
gelatine composition is used in the manufacture of soft shell capsule
pharmaceutical products, the gelatine will have a bloom strength from about
125 to about 200, a viscosity from about 25 to about 45 mP and a pH from
about 4.5 to about 6.5. A typical soft capsule gelatine formulation will
comprise from about 40% to about 45% by weight of the gelatine 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. The soft gelatine capsules
may be made according to any method generally known in the art. Typical
examples for plasticizers are glycerol (usually used in the form of a 85 weight
% aqueous solution) and sorbitol (usually used in the form of a 70 weight%
aqueous solution) and mixtures thereof.
All publications, patents, patent applications and other references cited
in this application are herein incorporated 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 both cationic and anionic in
character, such as a protein.
"Bloom value" is the degree of firmness of a gel measured in grams.
The bloom value is the force required for a punch of defined form and
dimension to penetrate 4 mm deep into the surface of a 6.7% by weight
gelatine solution. The bloom values of commercially available gelatines are
between 80 and 280.
"Bone chip" is chipped, degreased and dried bone from which,
subsequent to demineralisation (see maceration), gelatine is produced.
"Cross-linking" refers to the mechanism by which, e.g., a pellicle is
formed on a pharmaceutical soft capsule. Typically, cross-linking decreases
the dissolution properties of the capsule.
"Da" is an abbreviation for Dalton.
"EC" is an abbreviation for Enzyme Classification. It is typically used as
a prefix in the numerical designation of an enzyme.
"Endopeptidase" is an enzyme typically belonging within subclass EC
3.4, peptide hydrolases, that hydrolyses nonterminal peptide linkages in
oligopeptides or polypeptides and comprising any enzyme subclasses EC
3.4.21-99.
"Exopeptidase" is an enzyme of a group of peptide hydrolases within
subclass EC 3.4 that catalyzes the hydrolysis of peptide bonds adjacent to the
terminal amino or carboxyl 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 of genetically
modified organisms and is safe when consumed by an organism, such as a
human being. 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
gelatine with or without the addition of dye. They comprise an upper and
lower part; these are joined together once filling is completed.
"Instant gelatine" is powder gelatine capable of swelling in cold water.
"Microgel" is considered to be gelatine with a molecular weight greater
than 300,000 Da.
"Selenoproteins" are those proteins providing a support function within
the body. They are insoluble in water and possess a fibrous structure. These
proteins include e.g. keratin that occurs in hair and nails, the elastins and the
collagens that occur in support and connective tissue, skin, bone and cartilage.
"Soft capsules" are elastic capsule made of gelatine for filling with active
ingredient/excipient mixture. They can be produced with different wall
thicknesses and either with or without a seam.
"Split" is a gelatine raw material; mid-layer of the connective tissue of
cattle hide.
'Triple helix" is a basic structure of collagen consisting of 3 protein
chains. These often possess somewhat different amino acid sequences.
'Type A gelatine" is acid digested gelatine.
'Type B gelatine" is alkali (basic) digested gelatine.
'Type LBSH" is a limed-bone hydrolysate of the present invention
produced by proteolytic digestion of gelatine.
'Type LHSH" is a limed-hide hydrolysate of the present invention
produced by proteolytic digestion of gelatine.
As various changes could be made in the above compounds, products
and methods without departing from the scope of the invention, it is intended
that all matter contained in the above description and in the examples given
below, shall be interpreted as illustrative and not in a limiting sense.
EXAMPLES
The following examples illustrate the invention.

Example 1
The gelatine hydrolysate of the invention may be made according to the
following process. A solution containing 34% by weight gelatine (limed-bone
gelatine Type B, Bloom = 100) was made by adding 1.94 kg of water to 1.0 kg
of de-ionized processed gelatine. The gelatine was left to hydrate for 1 hour
and then placed into a 55 °C water bath to dissolve. Once completely
dissolved, the pH of the gelatine solution was adjusted to 6.0-6.5 with
aqueous sodium hydroxide. Calcium Chloride was added to the gelatine
solution in the amount of 0.037% w/w with the amount of gelatine in solution
(CDG -Commercial Dry Gelatine (having a moisture content of about 10% by
weight), all additions in this procedure were based on this amount). An
aliquot was taken and was diluted to 5% by weight in order to test the Redox
State of the solution. Peroxide testing strips (EM Science) were used to
quickly measure the amount of peroxide. If peroxide was present,
Fermcolase® 1000F (Genencor International Inc.) was added in 0.5 ml
increments. After each addition, the solution was left 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 1-hour reaction time and before the
addition of the next enzyme, a small sample was taken and the molecular
weight was analyzed. This process was repeated for each of the enzyme
additions. After a 1-hour reaction time, 0.05% w/w of Enzeco® Bromelain
Concentrate (Enzyme Development Corp.) was added and the solution was left
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 of Validase® FPU (Valley
Research) was added to the solution and was reacted for another hour. The
final enzyme addition was 0.1% w/w of Corolase® LAP (AB Enzymes). After 1
hour, the solution was heated to 90 °C to deactivate the remaining functional
enzymes. In some instances, an additional 30-40 ppm of hydrogen peroxide

was added to be certain the Papain 6000L was deactivated,. No proof of
enzymatic activity after heat deactivation was seen. A summary listing the
details of the five enzymes used during hydrolysis is given in Table 1.

The gelatine hydrolysate obtained in this example was used as Type LHSH
hydrolysate in the following examples. The average molecular weight was
determined to be about 1500 Da.

Example 2
The following procedure was used to quantify the degree of reduction in
cross-linking for various gelatine compositions. In the control experiments,
10.0 ± 0.1 g of a gelatine was added to a 250 ml beaker, to which was added
90.0 ± 0.5 g of de-ionized water. A watch glass was placed on the beaker and
the gelatine was allowed to swell for 30-60 minutes. The swelled gelatine was
placed into a 60 + 0.1 °C water bath for 15-30 minutes or until all of the
gelatine was dissolved. A magnetic stir bar was placed into the gelatine
solution and the pH was adjusted upon a stir plate with dilute NaOH or H2SO4
to a pH of 7.00 ± 0.05 after which the magnetic stir bar was removed. The
solution was placed into a water 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 used to create 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) as the time
when the cross-linked gelatine solution collapsed upon the shaft of the 4-blade
mixer.
In experiments involving gelatine compositions containing additives, a
percentage of the gelatine was substituted with the desired additive (e.g., a
10% hydrolysate added sample contained 9.0 g of gelatine and 1.0 g of
hydrolysate). The gelatines exhibiting a longer vortex hardening time are
believed to have reduced tendencies towards formaldehyde-induced cross-
linking.
As shown in Table 2, the vortex hardening test confirms the previous
findings that the combination of glycine and citric acid can reduce the amount
of gelatine cross-linking. More importantly, the addition of glycine alone has a
dramatic effect on the vortex hardening time of this particular limed-bone
gelatine sample. The addition of citrate did not reduce cross-linking.
Curiously, the addition of 1.5% citrate promoted cross-linking in this particular

sample. These results may serve to bolster the position of glycine's role as an
aldehyde scavenger in this model system. The detrimental effects on cross-
linking experienced by one of the samples containing only citrate cannot be
readily explained.
The vortex hardening test is used herein as an analytical tool for a rapid
screening of the impact of additives to the cross-linking behavior of gelatine
compositions.

Figure 1 details the effects of adding hydrolysate Type LHSH, a limed-
hide gelatine hydrolysate obtained in a process similar to Example 1 (MW
~1200 Da), and a Type BH-3 gelatine hydrolysate (MW ~ 2200 Da) to LH-1, a
typical limed-hide gelatine with bloom of 260 g and a 6.67% viscosity of 45
mP. The term 6.67% viscosity is used as an abbreviation for a viscosity
observed with a 6.67% CDG solution in water. The results show an increase in
the vortex hardening time for both added hydrolysates. However, the
performance was better upon the addition of the hydrolysate of the present
invention, Type LHSH. Several hide gelatines exhibited this very rapid cross-
linking that was previously only seen in limed-bone gelatines with a 6.67%
viscosity near 60 mP.
Table 3 shows the vortex hardening time of several limed-hide gelatines in
relation to different properties of molecular weight. No conclusive trends

could be deduced with the exceptions of a possible correlation of an increased
percentage of microgel and viscosity with increased cross-linking and a
subsequent reduction in the vortex hardening time.

Table 4 shows the results of adding 10% of hydrolysate Type LBSH of
Example 1 (average MW = 1500), a gelatine hydrolysate Type BB-4, and
glycine to a high viscosity limed-bone gelatine with a Bloom of 240 g and
6.67% viscosity of 64 mP. This high viscosity extract exhibits similar cross-
linking properties as some limed-hide gelatines with much lower viscosity.
This gelatine showed a significant reduction in cross-linking in the presence of
all three additives. However, the Type LBSH hydrolysate increased the vortex
hardening time by nearly 30% in comparison to the Type BB-4. Glycine
showed the greatest reduction in cross-linking and subsequent increase in the
vortex hardening time as shown in Table 4.


Table 5 shows the results of an experiment trying to determine the
amount of low molecular weight hydrolysate needed to match the performance
of glycine when added to a medium viscosity limed-bone pharmaceutical
gelatine (Bloom = 244, 6.67% vis. = 47.0 mP) as measured by the vortex
hardening test. The results indicate that 4-5% of Type LBSH is needed to
match the performance of 2.5% glycine, while 5-6% Type BB-4 is needed.
Similarly, 10% Type LBSH and 11-12% Type BB-4 is needed to match the
reduction in cross-linking achieved by 5.0% glycine.


Example 3
The gelatine hardening agent OB1207® [2-(4-Dimethylcarbamoyl-
pyridino)-ethane-1-sulfonate] was acquired from H.W. Sands Corporation.
OB1207® has been touted as a replacement for formaldehyde in photographic
emulsions. Reaction 1 describes the cross-linking of OB1207® with gelatine.
The formation of amide and ester bonds between gelatine chains through the
reaction with OB1207® may closely mimic the type of cross-linking seen in
gelatine samples that have been aged and/or stressed due to exposure to
extremes of heat and humidity.


Reaction 1
In control experiments, 15.0 ± 0.1 g of gelatine (limed-bone gelatine
Type B, Bloom = 200) was added into a 250 ml flask. To this, 95.0 + 0.5 g of
de-ionized water and a magnetic stir bar was added. The gelatine was
covered with parafilm allowed to swell for 30-60 minutes. The flask was
placed into a 60 ± 1.0 °C water bath for 15-20 minutes or until all of the
gelatine was dissolved. The viscosity of this solution was measured using a
Brookfield DV-III + Rheometer at 50 RPM and 60.0 + 0.1 °C. A solution made
of 0.30 g of OB1207® dissolved in 10.0 g of de-ionized water was slowly
added to the gelatine in the flask while stirring on a stir plate. The flask was
placed back into 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 gelatine was substituted with the desired
hydrolysate (i.e., a 10% hydrolysate added sample contained 13.5 g of
gelatine and 1.5 g of hydrolysate).
The results of viscosity experiments involving a medium bloom and
viscosity gelatine (LB-1) after cross-linking with OB1207® are given in Table
6. Control A LB-1 had no hydrolysate or OB1207® added, whereas control B

LB-1 had no hydrolysate, but was cross-linked with OB1207®. After two
hours, Control B was too viscous to read on the Brookfield rheometer
indicating a very high degree of cross-linking. The samples containing
hydrolysates showed a decreased degree of cross-linking, with the best results
achieved with Type LBSH, the hydrolysate of the present invention, especially
at the 10% level.

Example 4
The following procedure was used to determine the content of primary
amines in the gelatine hydrolysate. The use of trinitrobenzenesulfonic acid
(TNBS) to measure the amount of primary amines was described by Alder-
Nissen (6). A modified version of this procedure was used to measure the
relative amounts of primary amines in gelatine hydrolysates. Reaction 2
depicts the 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 up to a weight of 200.00 ± 0.01 g using a 1% sodium
dodecyl sulfate ("SDS", Aldrich) solution (glycine solution now referred to as
G-l). Gelatine hydrolysates in the amount of 4.000 ± 0.002 g were added to
250 ml beakers and brought up to a weight of 100 ± 0.01 g using the 1% SDS
solution (hydrolysate solution now referred to as H-1). The beakers containing
the G-l and H-1 solutions were placed on a hotplate and heated to a
temperature of 80-85 °C to fully dissolve and disperse the solids. The
solutions were cooled to room temperature and then 1.00 g of G-l was added
to a 250 ml beaker and brought up to a weight of 200.00 +. 0.01 g using the
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 of G-2,
respectively. The flasks were brought up to the mark by using the 1% SDS
solution. The solution H-1 was diluted to create H-2 by adding 1.00 g of H-1
and bringing it up to a weight of 200.00 ± 0.01 g in a 250 ml beaker using the
1% SDS solution. Into a 15 ml test tube was added 2 ml of a 0.2125 M
phosphate buffer (made by adding of 0.2125 M NaH2PO4 to 0.2125 M Na2HPO4
until a pH of 8.20 ± 0.02 is reached), and 250 ^L of the G-3 standards. This
corresponds to a six-standard glycine calibration containing 0.1667, 0.1250,
0.0833, 0.0417, 0.0167, and 0.0017 (imoles of primary amines per sample,
respectively. Similarly, 250 μl of each H-2 solution was added to a 15 ml test
tube (corresponding to 50 μg of sample) along with 2 ml of the phosphate
buffer. A control sample is made by adding 250 μl of the 1% SDS solution into
a 15 ml test tube with 2 ml of buffer. A 0.1% trinitrobenzene solution was

made by adding 170 ± 2 μl of a 1 M TNBS solution (Sigma) into a 50 ml
volumetric flask and brought up to the mark with de-ionized water and
immediately covered with aluminum foil as TNBS is light sensitive.
The following steps were all conducted in a photographic dark room. To
the test tubes, 2 ml of the 1% TNBS solution was added. The test tubes were
then vortexed (Fisher Scientific Vortex Genie 2) for 5 seconds. The samples
were then placed into a 50.0 ± 0.1 °C water bath for 30 minutes. The
samples were then vortexed for an additional 5 seconds and placed back into
the water bath for 30 minutes. The samples were removed from the water
bath and 4 ml of 0.100 N HCI solution was added to terminate the TNBS
reaction. The solutions were vortexed for 5 seconds and allowed to cool for 10
minutes (longer cooling may lead to turbidity because of the SDS). The
absorbance of the each sample was read at 340 nm (Beckman DU-7
Spectrophotometer) against a water blank. The amounts of primary amines in
the samples were calculated by using an absorbance-based linear regression
calculation of the glycine standards.
Derivatization of primary amines with o-phthaldialdehyde (OPA) to
measure proteolysis in milk proteins was described by Church et al (7).
Nielsen et al (8) used OPA to measure the degree of hydrolysis in other food
proteins, including that of gelatine. An advantage of the Nielsen method is the
substitution of the more environmentally friendly dithiothreitol (DTT -
Cleland's Reagent) for β-mercaptoethanol as the sulfur-containing reducing
agent. This procedure is adapted from the work of Nielson et al. Reaction 3
depicts 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 SDS to a 200 ml volumetric flask.
Deionized water in the amount of approximately 150 ml was added to and the
solution was stirred until completely dissolved. OPA (Aldrich) in the amount of
160 mg 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 entire solution was brought up to
volume with deionized water. Glycine standards were created by adding 50
mg of glycine to a 500 ml volumetric flask and filling to the mark with
deionized water. Dilutions were made by adding 100, 75, 50, 25, and 5 ml of
the glycine solution to 100 ml volumetric flasks and filling to the mark with
deionized water creating 5 glycine standards. Gelatine hydrolysate samples
were prepared by adding 0.500 g of hydrolysate to a 100 ml volumetric flask
and adding deionized water to the mark. To another 100 ml volumetric flask,
10 ml of the hydrolysate solution was added and filled to the mark with
deionized water. To a 15 ml test tube, 3.0 ml of the OPA reagent solution was
added followed by 400 μL of either a glycine standard (resulting in 40, 30, 20,
10, and 2 μg of glycine) or gelatine hydrolysate sample (200 μg of
hydrolysate). A control sample using 400 μL of water was also used to
measure the absorbance of OPA alone. The sample was vortexed for 5
seconds. Absorbance was read exactly 2 minutes after the addition of sample
against a water blank. Deviation from the 2-minute requirement significantly
impacts absorbance. Each sample or standard was then tested in two minute
intervals. The amounts of primary amines in the samples were calculated by
using an absorbance-based linear regression calculation of the glycine
standards.
Table 7 and Table 8 show the results of TNBS and OPA derivatization of
primary amines in 6 gelatine hydrolysates, a first-extract gelatine, 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 HCI
hydrolyzed sample (6N HCI for 24 hours @ 110°C). The TNBS and OPA
derived molecular weights are the inverse of the amount of primary amines
per sample amount. The TNBS and OPA derived molecular weights are
considered only to be qualitative, the real significance being the measured
amount of primary amines in each of the samples. The primary amine derived
molecular weights do not take into account the double derivatization of lysine
and hydroxylysine, nor does it take into account the fact that secondary
amines are not derivatized by either derivatizing agent. However, when
assuming these factors are relatively constant for all gelatine hydrolysates, the
primary amine derived molecular weight is a useful means of comparing the
relative degrees of hydrolysis of amongst different types of gelatine
hydrolysates. Type LBSH and Type LHSH, limed-bone and limed-hide
hydrolysates according to the present invention, showed an average increase
of nearly 30-130% in primary amines over other enzymatically digested
hydrolysates. The average molecular weight as measured by SEC/HPLC
methodology is also given. Note that the molecular weights for lysine and the
glycine trimer are far from the known molecular weight values. The TNBS and
OPA derived molecular weights are also very similar to the expected results of
the HCI hydrolyzed gelatine sample, whereas the HPLC/SEC data is almost 5
times this amount. This demonstrates the relative inaccuracy of low molecular
weight SEC/HPLC methodology generally used to measure the molecular
weight of gelatine hydrolysates. TNBS and OPA derived molecular weights are
not considered for gelatine. The complexities of the gelatine macromolecule
inhibit an accurate depiction of molecular weight using this simplified model.
The OPA derivatization proves to be a much more reliable means for
measuring primary amine content in comparison to derivatization with TNBS.
Table 7



REFERENCES
All references cited in the preceding text of the patent application or in
the following reference list, to the extent that they provide exemplary,
procedural, or other details supplementary to those set forth herein, are
specifically incorporated by reference to the same extent as if each individual
publication or patent application was specifically and individually indicated to
be incorporated by reference.
1. Ofner, CM., 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". Pharmaceutical Technology. Apr. 2002; 36-58.
3. Adesunloye, T.A., Stach, P.E., "Effect of Glycine/Citric Acid on the
Dissolution Stability of Hard Gelatin Capsules". Drug Dev. Ind. Pharm.,
1998, 24(6), 493-500.
4. Rama Rao, K.V., Pakhale, S.P., Singh, S., "A film Approach for the
Stabilization of Gelatin Preparations Against Cross-Linking".
Pharmaceutical Technology. Apr. 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 Food
Protein Hydrolysates 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, Aug. 1948, 70(8): 2673-2684.
11. Ward, A. G., Courts, A., The Science and Technology of Gelatin.
Academic Press Inc.1977, pp. 231.
12. Davis, P., Tabor, B., "Kinetic Study of the Crosslinking of Gelatin by
Formaldehyde and Glyoxal". J. Polym. Sci. Part A., 1963, 1: 799-815.
13. Albert, K., Peters, B., Bayer, E., Treiber, U., Zwilling, M., "Crosslinking
of Gelatin with Formaldehyde; a 13C NMR Study". Z. Naturforsch.,
1986, 41b: 351-358
14. Gold, T.B., et al., "Studies on the Influence of pH and Pancreatin on :3C-
Formaldehyde-Induced 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. Apr. 1999;
45(l):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 its Relevance to Their in 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., Aug. 1996, 271(32): 19338-
19345.
19. "Enzymic Hydrolysis of Food Proteins"; Elsvier Applied Science
Publishers Ltd. (1986), page 122.

WE CLAIM:
1. A process for making a gelatine hydrolysate, the process comprising:
(a) contacting a gelatine starting material with series of at least three different
proteolytic enzyme having endopeptidase activity to form an endopeptidase
digested gelatine product, the three proteolytic enzymes being selected from the
group consisting of Endopeptidase from Bacillus subtillis, Bromelain, and Papain;
and
(b) contacting the endopeptidase digested gelatine product with a series of at least
two different proteolytic enzymes having exopeptidase activity, the two
proteolytic enzymes being selected from the group consisting of Exopeptidase
from Aspergillus oryzae and Exopeptidase from Aspergillus sojae,
wherein the endopeptidase and exopeptidase proteolytic digestions form the
gelatine hydrolysate, the gelatine hydrolysate having an average primary amine
content from 1.0x10" to 1.0x10" μMol of primary amine per |Jg of gelatine
hydrolysate.
2. The process as claimed in claim 1, wherein the proteolytic enzymes having
endopeptidase activity which are contacted with the gelatine starting material
have a concentration of about 0.025% to about 0.15% (w/w) and the proteolytic
enzymes having exopeptidase activity which are contacted with the endopeptidase
digested gelatine product have a concentration of about 0.025% to about
0.15%(w/w).
3. The process as claimed in claim 1, wherein each proteolytic enzyme is sequentially
added to the gelatine starting material in the following order: Endopeptidase from
Bacillus subtilis, Bromelain, Papain, Exopeptidase from Aspergillus oryzae and
Exopeptidase from Aspergillus sojae; and wherein each proteolytic enzyme digests
the gelatine starting material for approximately 0.5 to about 2 hours before addition of
the subsequent proteolytic enzyme.

4. The process as claimed in claim 1, wherein the proteolytic digestion are allowed to
proceed for about 5 to about 12 hours in total.
5. The process as claimed in claim 1, wherein an aqueous solution containing about
10% to about 50% (w/w) of the gelatine starting material is contacted with the
proteolytic enzymes.
6. The process as claimed in claim 1, wherein the gelatine starting material is a
pharmaceutical grade gelatine.
7. The process as claimed in claim 1, wherein the proteolytic digestions are conducted
at a pH of about 5 to about 7 and at a temperature of about 40°C. to about 65°C.
8. A gelatine composition comprising:
a) a gelatine hydrolysate as claimed in claim 1-7 having an average primary amine
content from about 1.0x10" to about 1.0x10" μmol of primary amine per |Jg of
gelatine hydrolysate, wherein the gelatine hydrolysate is a proteolytic hydrolized
gelatine; and
(b) gelatine wherein the composition comprises from about 5% to about 10% by
weight of the gelatine hydrolysate and from about 90% to about 95% by weight of the
gelatine; and
wherein the composition has an average viscosity of from about 10 to about 15 cP
and after the addition of less than about 0.5% by weight of 2-(4-Dimethylcarbamoyl-
py-ridino)-ethane-l-sulfonate to the composition for about two hours at a reaction
temperature of about 60°C, the composition has an average viscosity of from about
15 to about 50 cP.

9. The gelatine composition as claimed in claim 8, wherein the composition
comprises a ratio of gelatine hydrolysate to gelatine from about 1:4 to about 1:99
(v/w).
10. The gelatine composition as claimed in claim 8, wherein the gelatine has an
average molecular weight greater than about 150,000 Da.
11. The gelatine composition as claimed in claim 10, wherein the gelatine is
pharmaceutical grade gelatine.
12. The gelatine composition as claimed in claim 11, wherein the gelatine is Type B
gelatine.
13. The gelatine composition as claimed in claim 11, wherein the gelatine is Type A
gelatine.
14. The gelatine composition as claimed in claim 8, comprising adding glycine to the
composition.
15. The gelatine composition as claimed in claim 14, wherein the amount of glycine
added is from about 0.5% to about 5% by weight.
16. The gelatine composition as claimed in claim 14, comprising adding citric acid to
the composition.
17. The gelatine composition as claimed in claim 16, wherein the amount of citric
acid added is from about 0.5% to about 5% by weight.

18. The gelatine composition as claimed in claim 16, wherein the amount of glycine
added is from about 1.5% to about 2.5% by weight and the amount of citric acid
added is from about 0.5% to about 1.5% by weight.
19. The gelatine composition as claimed in claim 8, wherein the composition has a
vortex hardening time of from about 200 to about 300 seconds.
20. The gelatine composition as claimed in claim 8, wherein the composition has a
vortex hardening time of greater than approximately 300 seconds.



ABSTRACT


PROCESS FOR MAKING A LOW MOLECULAR WEIGHT GELATINE
HYDROLYSATE AND GELATINE HYDROLYSATE COMPOSITIONS
The present invention provides a process to make a gelatine hydrolysate, a gelatine
hydrolysate, and gelatine compositions including gelatine hydrolysates. More
specifically, the invention provides gelatine compositions having a reduced
tendency to cross-link and improved dissolution properties.

Documents:

02823-kolnp-2007-abstract.pdf

02823-kolnp-2007-claims.pdf

02823-kolnp-2007-correspondence others 1.1.pdf

02823-kolnp-2007-correspondence others.pdf

02823-kolnp-2007-description complete.pdf

02823-kolnp-2007-drawings.pdf

02823-kolnp-2007-form 1 1.1.pdf

02823-kolnp-2007-form 1.pdf

02823-kolnp-2007-form 2 1.1.pdf

02823-kolnp-2007-form 2.pdf

02823-kolnp-2007-form 3.pdf

02823-kolnp-2007-form 5.pdf

02823-kolnp-2007-international publication.pdf

02823-kolnp-2007-international search report.pdf

02823-kolnp-2007-priority document.pdf

2823-KOLNP-2007-(06-02-2013)-CORRESPONDENCE.pdf

2823-KOLNP-2007-(06-02-2013)-FORM 1.pdf

2823-KOLNP-2007-(06-02-2013)-PETITION UNDER RULE 137.pdf

2823-KOLNP-2007-(20-02-2013)-CORRESPONDENCE.pdf

2823-KOLNP-2007-(20-02-2013)-OTHERS.pdf

2823-KOLNP-2007-(21-08-2012)-ANNEXURE TO FORM 3.pdf

2823-KOLNP-2007-(21-08-2012)-CORRESPONDENCE.pdf

2823-KOLNP-2007-(22-01-2013)-ABSTRACT.pdf

2823-KOLNP-2007-(22-01-2013)-CLAIMS.pdf

2823-KOLNP-2007-(22-01-2013)-CORRESPONDENCE.pdf

2823-KOLNP-2007-(22-01-2013)-DESCRIPTION (COMPLETE).pdf

2823-KOLNP-2007-(22-01-2013)-DRAWINGS.pdf

2823-KOLNP-2007-(22-01-2013)-FORM 1.pdf

2823-KOLNP-2007-(22-01-2013)-FORM 2.pdf

2823-KOLNP-2007-(22-01-2013)-FORM 3.pdf

2823-KOLNP-2007-(22-01-2013)-OTHERS.pdf

2823-KOLNP-2007-(22-01-2013)-PETITION UNDER RULE 137.pdf

2823-KOLNP-2007-AMENDED CLAIMS.pdf

2823-KOLNP-2007-CANCELLED PAGES.pdf

2823-KOLNP-2007-CORRESPONDENCE OTHERS 1.2.pdf

2823-KOLNP-2007-CORRESPONDENCE OTHERS 1.3.pdf

2823-KOLNP-2007-CORRESPONDENCE.pdf

2823-KOLNP-2007-EXAMINATION REPORT.pdf

2823-KOLNP-2007-FORM 18-1.1.pdf

2823-kolnp-2007-form 18.pdf

2823-KOLNP-2007-FORM 26.pdf

2823-KOLNP-2007-GRANTED-ABSTRACT.pdf

2823-KOLNP-2007-GRANTED-CLAIMS.pdf

2823-KOLNP-2007-GRANTED-DESCRIPTION (COMPLETE).pdf

2823-KOLNP-2007-GRANTED-DRAWINGS.pdf

2823-KOLNP-2007-GRANTED-FORM 1.pdf

2823-KOLNP-2007-GRANTED-FORM 2.pdf

2823-KOLNP-2007-GRANTED-FORM 3.pdf

2823-KOLNP-2007-GRANTED-FORM 5.pdf

2823-KOLNP-2007-GRANTED-SPECIFICATION-COMPLETE.pdf

2823-KOLNP-2007-INTERNATIONAL PUBLICATION.pdf

2823-KOLNP-2007-INTERNATIONAL SEARCH REPORT & OTHERS.pdf

2823-KOLNP-2007-OTHERS-1.1.pdf

2823-KOLNP-2007-OTHERS.pdf

2823-KOLNP-2007-PA.pdf

2823-KOLNP-2007-PETITION UNDER RULE 137.pdf

2823-KOLNP-2007-REPLY TO EXAMINATION REPORT.pdf

abstract-02823-kolnp-2007.jpg


Patent Number 258325
Indian Patent Application Number 2823/KOLNP/2007
PG Journal Number 01/2014
Publication Date 03-Jan-2014
Grant Date 31-Dec-2013
Date of Filing 02-Aug-2007
Name of Patentee GELITA AG
Applicant Address UFERSTRASSE 7 69412 EBERBACH
Inventors:
# Inventor's Name Inventor's Address
1 BABEL, WILFRIED KLEINGEMUNDENER STR. 34/1, 69118 HEIDELBERG
2 KEENAN, TOM 31465 W. HORSES LAKE DR., SIOUX CITY 1A 51106
3 RUSSELL, JASON, D. 4230 HICKORY LANE APT. 637, SIOUX CITY 1A 51106
4 DOLPHIN, JOHN, M. 7100 CHRISTY RD., SIOUX CITY 1A 51106
PCT International Classification Number C08H 1/06
PCT International Application Number PCT/EP2006/005179
PCT International Filing date 2006-05-31
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 11/140,863 2005-05-31 U.S.A.