Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
23~5~
ELASTIC HYDROLYZATE
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
This invention relates to the preparation of elastic hydra-
lusts.
DESCRIPTION OF THE PRIOR ART
Elastic is the major component of elastic fibers found prim
manly on connective tissue in conjunction with collagen and polyp
saccharides. Major concentrations of elastic are found in blood
vessels. Another source of elastic is in the ligaments, and more
particularly the ligamentum Nash, prominent in the necks of grazing
animals and on their hides. The ligamentum Nash is a preferred
source of elastic because of the high concentration of elastic therein.
Elastic is known to have a highly distinctive amino acid
composition. Although similar to collagen in that one-third of the
amino acid residues are Gleason, elastic is rich in praline, and in
contrast to collagen, elastic contains very little hydroxyproline,
no hydroxylysine, and is very low in polar amino acids. Elastic is
very rich in nonpolar aliphatic residues such as isoleucine~ Lawson,
valise and ala nine. Elastic, as present in mature animals, is highly
cross-linked, therefore making it very difficult to syllables. This
dense cross-link;ng is attributable to the desmosine and isodesmosine
residues which are highly functional and cross-link both intro- and
interfibrillarly. It is believed that the desmosine and ;sodesmosine
cross-linking gives the elastic fibers their elasticity. The demo-
sine residue can be represented by the structural formula:
I
T
--N--C--C
I
Shea
HEN NO
SUE
Ho SUE_ SHEA CHIHUAHUAS OH
Old Jo KIWI
N
Of ~12
(Shea
CHIC N--
11 1 1
O H H
And the isodesmosine residue can be represented by the structural
formula:
~2~3~5~
H
- -- - No N -H
Cull -(C1~2)2 (SCHICK--- I
H
I .. (SHEA --C-
34 No H
I
This highly cross-linked structure is extremely difficult to
syllables and purify, especially in the case of mature animals such
as horses, cows and the like, which are aged and thus contain elastic
which has an extremely high cross-link density.
Prior art methods of solubilizing elast;n primarily include
the use of elasticize to hydrolyze the peptize linkages to provide an
-I, acceptable product
I` Solubilized elastic has found utility in the cosmetic no
aye . However, its manufacture has been limited to
small quantities and it is not necessarily of acceptable purities
because of the enzymatic residue required for the hydrolysis
Further, partially hydrolyzed elastics have been produced;
however, the processing conditions for their production reduce the
amount of desmosine and isodesmosine amino acid residues which are
recovered in the elastic hydrolyzate. Therefore, many of the elastic
hydrolyzates lose the primary elastin-characterizing amino acids, i.e.
desmosine and isodesmosine. One particular method of producing elastic
by non enzymatic means is disclosed in "ELASTIC" by Berg et at.,
Cosmetics & Toiletries, Vol. 94, October, 1979.
~q~3~s~
Characteristically, elastic in its natural state in mature
animals, and particularly the ligamentum Nash, is present at a level
of at least 3.5 combined desmosine and isodesmosine residues/1,000
Amelia acid residues. Thus, in order to produce a hydrolyzed elastic
which retains the basic characteristics of elastic, it is necessary
to recover substantially all of the desmosine and isodesmosine residues
which are present in the starting material.
In accordance with the present invention, the method of pro
during a soluble elastic partial hydrolyzate in pure Form is provided
wherein the desmosine and isodesmosine residues are substantially
recovered.
BRIEF DESCRIPTION OF THE INVENTION
A method of preparing soluble partially hydrolyzed elastic is
provided. The method involves treating insoluble elastic with a per
oxide and subsequently partially hydrolyzing the treated elastic and
recovering substantially pure soluble partially hydrolyzed elastic.
A soluble elastic partial hydrolyzate is comprised of polyp
peptizes having at least 3.5 desmosine and isodesmosine residues/1,000
amino acid residues.
DETAILED DESCRIPTION OF THE_ INVENTION
The elastin-containing material may be obtained from a variety
of sources well known to those skilled in the art. Preferably, the
source of elastic is natural insoluble elastic from bovine hide or
the bovine ligamentum Nash. Although the hides themselves may be
used as a source of elastic, it is preferred that the bovine ligament
tug Nash be used since it has about 80 percent by weight elastic
therein.
When the ligament Nash are used as a source of elastic,
just as are the hides, the collagen constituents must be removed
therefrom. In order to remove the collagen constituents therefrom,
the collagen is solubilized by the use of an acid solution containing
citric acid, tartaric acid, weak hydrochloric acid or the like. The
acid should be sufficiently weak so that it does not hydrolyze the
elastic, but must be sufficiently strong so as to syllables the collagen.
~LZ'~3~ Z
Preferably, the acid should be sufficient to provide a pi to the
aqueous solution of 3.5 to 4.5. In addition to the acid hydrolysis
of the collagen, organic peroxides may be used in the same solution
in order to initiate the destabilization of the desmosine and is odes-
cosine cross-linkages. The treatment of the ligaments with the
aqueous acid solution is for about 12 to 18 hours at room tempera-
lure with agitation. Subsequent to the acid treatment, the liquid
is drained and the ligaments washed with cold water. The ligaments
are then autoclave at 2.2 to 3 atmospheres pressure for about 2 to
8 hours. After autoclavation, the ligaments are then treated with a
base such as sodium hydroxide or potassium hydroxide in order to
hydrolyze the elastic cross-linkages, along with sodium sulfate at
a level of 1 to 1.5 molar in water to prevent overselling of the
elastic fibers. Sufficient base should be provided to establish a
pi of 10 to 12. The ligaments are immersed in the solution for about
12 to 18 hours, removed from the solution, and again washed. The
ligaments are then neutralized with dilute acid, preferably a mineral
acid such as hydrochloric acid or sulfuric acid. After neutralize-
lion, the ligaments are washed with cold water. The ligaments are
then placed in an appropriate vessel and shredded in the presence of
an acidic solution at a pi of 2.2 to 3.2, and after shredding, the
shredded ligaments are boiled in water for 3 to 5 hours After boiling,
no ligament strands should be visible and there is an apparent home-
generous solution. Subsequent to boiling, the solution is allowed to
cool and it is filtered. The pi ox the solution is adjusted with
dilute sodium hydroxide to a pi of about 6. To the solution having
the adjusted pi is added 0.1 to 0.2 percent of an organic or inorganic
peroxide or a combination thereof, along with a filtering aid, such
as diatomaceous earth or the like and/or charcoal. The solution is
then boiled for 1 to 5 hours and filtered. Additional peroxide treat-
mints may be provided depending on the extent of cross-linking of the
elastic starting material. The pi of the elastic solution is adjusted
to about 5 to 6 and the solution is filtered. Stabilizers may be added
to prevent degradation of the elastic partial hydrolyzate upon storage.
~23~
--6--
The peroxides useful in destabilizing the desmosine and
isodesmosine bonds are the organic and inorganic peroxides, and most
preferably, the inorganic peroxides. Exemplary of inorganic per-
oxides are hydrogen peroxide and ammonium per sulfate. Ammonium per-
sulfate is represented by the structural formula:
O O
N OWE O O S O N H
11 -.
.--
Organic peroxides may be used and are selected on the bass
of decomposition half-life in the presence or absence of a suitable
accelerator. Typical organic peroxides are tertiary-but~l hydropower-
oxide, bouncily peroxide, laurel peroxide, dicumene hydroperoxide and
: 10 the like. Combinations of various peroxides may be used in treating
the insoluble elastic. Preferably, the peroxide is present at a
level of about 0.025 to 0.5 percent by weight based on the weight of
the insoluble elastic, and more preferably, about 0.1 to 0.25 percent
by weight based on the weight of the insoluble elastic. It has been
I found that hydrogen peroxide and ammonium per sulfate mixed together
in approximately equal quantities are useful in treating the insoluble
elastic. It is believed that the peroxide destabilizes the desmosine
and isodesmosine cross-linkages, which is required to partially hydra-
lyre and syllables the elastic. Preferably, the insoluble elastic
is treated with the peroxide in the presence of water at above room
temperature and for the final hydrolysis under refluxing conditions.
When ammonium per sulfate and hydrogen peroxide are used in about
equal quantities as the peroxide constituents, the elastic is treated
in the presence thereof under above ambient temperature conditions
and below reflex at 12 to 18 hours and under reflex for 3 to 5 hours,
and more preferably, 3 to 4 hours.
LO 3 2 So
Although the peroxide destabilizes the desmosine and is-
desmosine cross-linkages, it is necessary to further treat with acid
to perform the complete hydrolysis. Most preferably, the acidic
solution is composed of water and a mineral acid such as hydrochloric
ac;cl, sulfuric acid or the like. However, weaker acids such as
acetic acid may also be used but are not preferred. In order to
hydrolyze the elastic it it necessary to heat the elastic in the
presence of the acid, preferably at reflex and at atmospheric pros-
sure. In the case of a solution of 3 to 10 percent hydrochloric
acid, and preferably 5 to 7 percent hydrochloric acid, refluxing
treatment is between 2 to 8 hours, and more preferably, 3 to 5 hours.
The substantially pure partially hydrolyzed elastic in act
cordons with the above procedure has an average molecular weight of
8,000 to 15,000, with a substantial number of hydrolyzate molecules
having a molecular weight between 500 and 1,000. Thus, the water
soluble partial hydrolyzate has a molecular weight range of 500 to
15,000. It has been found upon analysis that the procedure according
to the invention recovers substantially all of the desmosine and
isodesmosine amino acid residues from the elastic, thus maintaining
elastin-like characteristics to the hydrolyzate. Further, it has
been found that the soluble elastic partial hydrolyzate in accordance
with the invention has at least 3.5 desmosine and isodesmosine residues/
1,000 amino acid residues. In the case where some polypeptides are
extracted during the hydrolysis procedure, it has been found that the
desmosine and isodesmosine remain so that higher levels than 3.5
residues/1,000 residues and up to 6 residues/1,000 residues are pro-
sent in the elastic partial hydrolyzate, evidencing the fact that all
the desmosine and isodesmosine residues present in the starting material
are recovered in the hydrolyzate.
The dissolved partially hydrolyzed elastic may be further
purified by treatment with activated carbon or the like. Further, if
the solution is hazy it may be further treated with hydrogen peroxide
or a similar peroxide to make the solution transparent. Further
~L~2~3~'3~
stabilizers and antioxidant may be added to impart prolonged shelf
life to the elastic solution. Typical stabilizers are sorbic acid,
sodium bonniest and the like. The hydrolyzate is soluble in water
and is parboil as a solution of up to 40 percent by weight at am-
blent temperature.
If a non aqueous solution of the partially hydrolyzed elastic
is desired, the water may be removed from the elastic solution by
evaporation or the like, and it may be redissolved in propylene guy-
got, dipropylene glycol or the like.
The invention is further illustrated by reverence to the lot-
lowing examples.
EXAMPLE 1
In an appropriately sized vessel were charged 100 pounds of
fresh beef neck ligaments (ligamentum Nash). water at 85C was
added until the ligaments were fully immersed. One thousand grams
of citric cold, 50 grams of ammonium per sulfate, and 500 milliliters
of 30 percent hydrogen peroxide were charged to the vessel with
agitation. The agitation was continued for 18 hours without the
addition of heat thereto. The ammonium per sulfate and hydrogen per-
oxide began the attack on the desmosine and isodesmoslne cross-
linkages while the citric acid solubilized any collagen or other
pretenses impurities in the ligaments. After agitation for 18
hours, the liquid and fat were drained from the vessel and the lima-
mints were washed with cold water for approximately 1 hour, or until
all the loose fat was removed. The ligaments were placed in an auto-
crave at 40 pi (2.8 atmospheres) for about 6 hours. After auto-
elevation, any excess liquid and fat were drained from the autoclave
and the ligaments were washed in cold water for about 1 hour. For
each 7.5 pounds of ligaments the following solution was added thereto:
10 liters of water, 2.2 pounds of sodium hydroxide and 3 pounds of
sodium sulfate. The pi of the solution was 10 to 12. The ligalnents
were immersed in this solution and allowed to stand for 18 hours. The
solution was drained from the ligaments and the ligaments were washed
with cold water for approximately 1 hour. The ligaments were charged
to an appropriately sized stainless steel vessel and water was added
thereto until fully immersed. Two liters of 37 percent Hal were added
Lucy
to the Bessel, agitated until homogeneous, and the ligaments were
allowed to stand in the solution at ambient temperature overnight.
The pi ox the solution was about 7. After standing for 18 hours,
the liquid was drained and the ligaments were washed with cold water
for about 1 hour.
The washed ligaments were placed in an appropriately sized
vessel and water was added thereto until fully immersed. Approxi-
mutely 2.3 liters of 37 percent Hal were added to the water. The
contents of the vessel were agitated with a high-speed rotatable
mixer for 2 to 3 hours and the ligaments were fully shredded. The
dispersion of shredded ligaments and water was placed in an appropriate
vessel and heated at reflex for about 4 hours. After 4 hours, no
ligament strands were visible. The solution was cooled to room them-
portray and filtered. The filtered solution was placed in 5 gallon
pails and adjusted to a pi of 6 with S normal aqueous sodium hydroxide.
To each 5 gallons of solution were added 100 milliliters of 30 percent
hydrogen peroxide and 60 grams of ammonium per sulfate, along with 50
grams of diatomaceous earth and 75 grams of animal charcoal as a
clarifier. The material was then placed in the appropriate vessel
and heated For 2 to 3 hours under reflex and allowed to cool. After
cooling, the material was filtered through fine filter paper and to
each 40 pounds of material were added 80 milliliters of 30 percent
hydrogen peroxide and 40 grams of ammonium per sulfate. The material
was allowed to stand overnight. After standing overnight, the ye was
adjusted to 5-5.5 with aqueous ammonium hydroxide and the solution
was adjusted to 10 percent solids with water. The material was again
filtered through fine filter paper, and to each 40 pounds of material
were added 40 grams of sorbic acid, 20 grams of sodium bonniest and
40 grams of Germ all 115 antioxidant. The elastic partial hydrolyzate
30 produced in accordance with the Example had the -following analysis:
Total nitrogen 1.5 percent
Elastic partial hydrolyzate 8.43 percent
Dry matter 12 percent
Ash 3 percent
pi 5 to 5.5
Z
-10-
Amino acid analysis of the material showed that there were
3.9 combined desmosine and isodesmosine residues/l,OOO residues of amino
acid residues.
The average molecular weight of the elastic was about 10,000,
hazing a molecular weight distribution between 500 and 20,000.
' EXAMPLE 2
One hundred kilograms of collagen containing material, in-
eluding raw hide, untanned tannery wastes, limed splits and trimming
scraps were charged to a suitable vessel containing 300 liters of
water having therein 10 kilograms of calcium hydroxide, Kilograms
of sodium hydroxide and 5 kilograms of sodium chloride. The fibrous
protein counterweighing material was allowed to stand in the aqueous soul-
lion at ambient temperature for 5 days. After 5 days, the vessel was
drained and the fibrous protein containing material free of hair and
fats was neutralized to a pi of 7 by treating with an aqueous soul-
lion of 30 liters of water which contained 1.5 kilograms of ammonium
chloride and 1.5 kilograms of hydrochloric acid. The pal of the
cross section of fibrous protein containing material was about 7. The
neutralized fibrous protein containing material was washed with 300
liters of running water and deposited in 600 liters of distilled water
for 12 hours. The distilled water extracted residual salts from the
fibrous protein containing material. The distilled water was drained
and the fibrous protein containing material was charged to an auto-
crave for 8 hours at a pressure of 2 kilograms/cm2. The treatment
under heat and pressure hydrolyzed the palpated bonds within the
collagen while leaving the elastic in a cross-linked condition. The
oligopeptides formed of the hydrolyzed collagen were water soluble
and had a molecular weight between 5,000 and 20,000. After treatment
under heat and pressure, the oligopeptide solution was cooled to
about 4C. Upon cooling a small amount of fat rose to the surface of
the solution and cross-linked elastic along with other minor impurities
precipitated. The collagen oligopeptides in solution were heated and
filtered through filter paper. The filter khakis autoclave at 145C
for 4 hours to separate fats. The filter cake had 25.56 percent dry
matter, 0.7 percent ash, and 13.8 to 14 percent cross-linked elastic -
based upon the weight of the dry matter. Ten pounds of the raw elastic
~L2~23~:52
containing material (i.e. the autoclave filter cake) was washed with
300 percent by weight water at 60C for 1 hour to remove salt and other
soluble impurities. The dispersion of the cross-linked elastic in
water alas separated by filtration through a stainless steel sieve and
washed in accordance with the procedure previously described. Ten
liters of 0.5 percent by weight ammonium per sulfate and 0.5 percent
hydrogen peroxide solution was prepared and the cross-iinked elastic
containing material combined therewith. The material was then boiled
for 3-1/2 hours at 100C. After boiling the raw elastic containing
material in peroxide aqueous media was filtered and mixed with 10
liters of 6 percent hydrochloric acid solution and agitated there-
with. The resulting elastic solution was reflexed for 6 hours to
partially hydrolyze the elastic. The partially hydrolyzed solubilized
elastic was filtered through filter paper and the filtrate dried
lo under vacuum.
The dried material was redissolved in 2.5 kilograms of disk
tilled water and the pi adjusted to I using a 0.1 percent aqueous
sodium hydroxide solution.
Five pounds of the elastic solution was mixed under agitation
with 0.05 pounds of activated carbon and boiled for one hour to de-
colonize the elastic solution. The active carbon was removed by lit-
traction.
The filtered elastic solution was treated with 0.05 percent of
hydrogen peroxide based on the weight of the elastic overnight at
room temperature to remove haze from the solution and thus impart ox-
tree clarity to the solution. The solution was again filtered and the
concentration of the partially hydrolyzed elastic adjusted to 10 to
30 percent.
The final solution was stabilized to impart shelf life thereto
at a ratio of US kilograms of elastic solution to 2.5 grams sorbic
acid and 5 grams of phenip.
The elastic solution so produced is useful in eschew
cosmetic applications.
I
-12-
The elastic hydrolyzate prepared in accordance with Example 2
has substantially the same composition and the same chemical and pry-
steal properties as the elastic prepared according to Example 1.
EXAMPLE 3
Example 2 was repeated except that the dried partially hydra-
lazed soluble elastic was dissolved in 70 percent ethanol.
EXAMPLE 4
Example 2 was repeated except that the partially hydrolyzed
soluble elastic was dissolved in propylene glycol.
EXAMPLE 5
Example 2 was repeated except that the partially hydrolyzed
soluble elastic which was dried was redissolved in dipropylene glycol.
Thus, in accordance with the invention, a partial hydrolyzate
of elastic which was water soluble was prepared having from 3 to 4
desmosine and isodesmosine residues/1,000 residues and having an
average molecular weight of 8,000 to 15,000.
Although the process in accordance with the invention is spew
civically set forth in exemplary fashion based upon a starting material
of raw hide, untanned tannery wastes, limed splits, trimming scraps,
and tendons, other sources of elastic may be used, such as blood Yes-
sots, hearts, lungs and the like.
When these other materials are used as starting materials, it
is necessary to treat them in the same manner when low in elastic as
raw hides, untanned tannery wastes and the like in order to remove got-
lager, globular proteins, fats and other impurities therefrom in order
to have an elastin-rich starting material to produce the partially
hydrolyzed soluble elastic in accordance with the invention.
Although the invention has been described with reference to
specific materials and specific processes, it is only to be limited so
far as is set forth in the accompanying claims.
