U.S. patent number 3,898,129 [Application Number 05/297,461] was granted by the patent office on 1975-08-05 for enzymatically hydrolyzed composition of skin rag and derivatives thereof.
This patent grant is currently assigned to Kyowa Hakko Kogyo Kabushiki Kaisha. Invention is credited to Yasu Fujimoto, Ikuo Matsukuma, Masayuki Teranishi.
United States Patent |
3,898,129 |
Fujimoto , et al. |
August 5, 1975 |
**Please see images for:
( Certificate of Correction ) ** |
Enzymatically hydrolyzed composition of skin rag and derivatives
thereof
Abstract
A process comprising (a) enzymatically hydrolyzing skin rag with
a neutral or alkaline protease which is capable of decomposing
collagen other than collagenase, and (b) N-acylating the resultant
oligopeptide hydrolyzation products with a fatty acid type
acylating agent having from 8-22 carbon atoms to form N-acylated
oligopeptides useful as surfactants.
Inventors: |
Fujimoto; Yasu (Yokohama,
JA), Teranishi; Masayuki (Tokyo, JA),
Matsukuma; Ikuo (Tokyo, JA) |
Assignee: |
Kyowa Hakko Kogyo Kabushiki
Kaisha (Tokyo, JA)
|
Family
ID: |
13849119 |
Appl.
No.: |
05/297,461 |
Filed: |
October 13, 1972 |
Foreign Application Priority Data
|
|
|
|
|
Oct 28, 1971 [JA] |
|
|
46-10985 |
|
Current U.S.
Class: |
435/68.1;
514/773; 530/329; 530/330; 530/356; 530/842; 530/859; 510/126;
510/501 |
Current CPC
Class: |
C11D
1/32 (20130101); A61K 35/36 (20130101); A61K
38/014 (20130101); C07K 1/02 (20130101); A61K
8/65 (20130101); A61Q 19/00 (20130101); Y10S
530/859 (20130101); A61Q 5/02 (20130101); Y10S
530/842 (20130101); A61Q 9/02 (20130101) |
Current International
Class: |
A61K
35/36 (20060101); A61K 8/65 (20060101); A61K
38/01 (20060101); C11D 1/02 (20060101); A61K
8/30 (20060101); C07K 1/00 (20060101); C07K
1/02 (20060101); C11D 1/32 (20060101); C12B
001/00 () |
Field of
Search: |
;195/29,6 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Shapiro; Lionel M.
Assistant Examiner: Warden; Robert J.
Attorney, Agent or Firm: Phillips, Moore, Weissenberger,
Lempio & Strabala
Claims
What is claimed is:
1. A process for preparing a skin rag hydrolysate containing mainly
peptides having a polymerization degree of from about 4 to about 7,
comprising treating skin rag with an alkaline protease, other than
collagenase, which protease is capable of decomposing collagen, at
a pH of from about 7 to 12 and at a a temperature of from about
47.degree. to 70.degree.C for a period of from about 4 to 24 hours,
the amount of said protease being from about 0.1 to 0.5% based on
the weight of said skin rag.
2. The process of claim 1, wherein the protein element of the skin
rag is denatured prior to treatment with the protease.
Description
BRIEF SUMMARY OF INVENTION
This invention relates to (a) a process for producing enzymatically
hydrolyzed composition of skin rag by treating skin rag with a
protease, (b) a process for producing N-acyl derivatives by
subjecting the said enzymatically hydrolyzed composition to
acylation reaction by using a long chain fatty acid-type acylating
agent, and (c) the utilization of the said N-acyl derivatives as
surfactant.
DETAILED DESCRIPTION OF INVENTION
Generally, a large amount of skin rag (which denotes animal skin
rag such as e.g. hide rag, leather rag and the like) is brought
forth as waste at various leather factories. The unpleasant smell
of the skin rag itself and the smell which is generated during the
after-treatment of the rag have caused questions of air pollution
and public health to arise and for this and other reasons, the rag
has not yet been used effectively in spite of strong desire, for a
long period of time.
The collagen fiber, which is part of the main composition of skin,
has a helical structure. It is said that this collagen helix is
stable to protease other than collagenase under normal conditions.
For example, it has been reported that various enzymes, such as
pepsin, trypsin, Promnase (commercial product available from Kaken
Kagaku Kabushiki Kaisha, Japan), Proctase (commercial product
available from Meiji Seika Kabushiki Kaisha, Japan) etc., digest
only the peptide portion (less than 5% of the entire molecule)
which projects from the helix structure at the terminal of a
collagen molecule, but do not affect the helix structure itself
[Kobunshi (high molecule), Vol.19, pp. 355-361 (1970)].
We have extensively studied the problem of the economical and
effective utilization of skin rag, and have now made the discovery
that skin rag is easily decomposed into low molecular oligopeptides
by a certain type of neutral and/or alkaline protease. We have also
discovered the interesting fact that such hydrolysis proceeds with
a very slow speed to completion, i.e., amino acid monomers.
The hydrolysis of protein is classified into three types, i.e.,
acidic hydrolysis, alkaline hydrolysis and enzymatic hydrolysis,
depending upon the hydrolyzing agent in use. The first two types of
hydrolysis have the disadvantage that the peptides and amino acids
produced are liable to be further decomposed into undesired fumins.
Furthermore, when applied to the hydrolysis of proteins into low
molecular peptides, which is a purpose of the present invention,
the first two types of hydrolysis have the disadvantage that the
hydrolysis proceeds to the production of amino acid monomers owing
to the difficulty of controlling these hydrolyzation processes.
Conventional techniques of alkaline hydrolysis of skin rag under
pressure have also the aforementioned disadvantages. Furthermore,
enzymatic hydrolysis has been conventionally used either for the
purpose of obtaining collagen with simultaneous removal of some
proteins (such as albumin, globulin and viscouse protein) which are
undesirable for the production of leather or for the purpose of
producing high molecular proteins such as gelatin.
According to the process of the present invention, it is possible
to obtain directly from skin rag, under mild conditions, a
hydrolysate containing little amino acid monomer, by using a
neutral or alkaline protease which is capable of decomposing
collagen other than collagenase.
Skin rag, which may be used as raw material for the purpose of the
present invention, includes any and all skin rag brought forth as
waste in leather manufacturing. Usually, either raw or tanned skin
rag of cow, horse, goat, pig, sheep, etc., may be used for the
process of the present invention.
The hydrolysis is carried out by immersing skin rag in water, for
example, by soaking or suspending. In this case, the amount of the
water is preferably about 4-50 times of that of the skin rag to be
immersed, although it is not necessary to specifically limit the
amount. It is difficult to carry out an enzymatic action on skin
rag without pretreatment because of its intricate structure. Thus,
it is sometimes necessary to denature the substrate prior to
decomposition. It is possible to improve the degree of
decomposition when the skin rag used in the present invention is
denatured by heating. For example, the skin rag may be suspended or
soaked in water and then subjected to heat treatment prior to
enzymatic decomposition. It is generally preferable to heat the
skin rag at a temperature of about 80.degree.C to about
100.degree.C for about 0.5 to about 5 hours. When the skin rag is
subjected to such heat treatment, the degree of decomposition may
be further improved by discarding the aqueous solution used for the
heat treatment and carrying out the enzymatic decomposition in
fresh water in which the skin rag is soaked or suspended.
Protease, which may be used for the hydrolysis, may be of animal,
plant or microorganic origin. Although either a neutral or an
alkaline protease which is capable of decomposing collagen other
than collagenase may be used, better result may be obtained by
using an alkaline protease which is capable of decomposing
collagen. It is also possible to use various types of enzyme in
combination. Typical enzymes employable are exemplified by protease
of hay bacillus, Epolozyme (commercial product available from Kyowa
Hakko Kogyo K. K., Japan), Tacynase (commercial product available
from Hakko Kogyo K. K., Japan), or Prozyme (commercial product
available from Amano Seiyaku K. K., Japan).
The conditions of the hydrolysis, of course, depend upon the enzyme
employed, and the important factors are the pH and temperature of
the reaction mixture. In all events, the pH and temperature should
be determined so that the activity of the enzyme employed may be
effectively utilized. The pH may be adjusted to a suitable value by
a suitable alkaline or acidic substance, such as sodium hydroxide,
potassium hydroxide, sodium carbonate, sodium bicarbonate,
hydrochloric acid, sulfuric acid, acetic acid, etc. It is also
possible to use a suitable buffer solution.
The hydrolysis may be carried out in the following manner.
Immediately after immersing or suspending skin rag in water or
after the said pretreatment, the reaction mixture is adjusted to
optimum temperature (e.g. 20.degree. -70.degree.C) and pH (e.g.
7-12), which may vary depending upon the types of enzyme which is
added thereto. The amount of the enzyme to be added may vary,
depending upon the activity, and is preferably within a range of
about 0.1 to about 5.0% based on the weight of the substrate. The
pH of the reaction mixture may be adjusted to optimum for the
enzyme used in the above-mentioned manner during the reaction. If
desired, a suitable antiseptic may be added. The reaction can be
carried out as long as the enzyme shows its activity and
accelerates the decomposition (usually, for example, for 4-24
hours).
After the completion of the hydrolysis, the reaction mixture is,
for example, heated from about 80.degree. to about 100.degree.C to
inactivate the enzyme and the insoluble matter is removed
therefrom. The solution is then concentrated or dried in
conventional manner, to give the powder. It is usually preferable
to carry out the concentration and drying at a temperature below
about 60.degree.C to avoid over-heating. The resultant hydrolysate
is sometimes colored because the coloring matters which elute from
the skin rag during the decomposition adhere to the hydrolysate. If
desired, a further refined hydrolysate may be obtained by the
following treatment:
A hydrolysate solution, from which the insoluble matter is removed,
is heated at a temperature below about 60.degree.C, simultaneously
with treatment with active carbon in an amount of about 1 to about
10% weight based on the amount of the solids in the hydrolysate. It
is also possible to use a suitable decoloring resin in place of the
active carbon. The coloring matters may be removed significantly by
this treatment. When a still further purification is required, or
when fine powders of some types of active carbon contaminate the
hydrolyzed solution, coloring matters, inorganic salts, active
carbon and a part of peptides may be precipitated either by
concentrating the solution to one-tenth and adding the same to
about 10 times this amount of methanol (by volume) or by adding the
said methanol to the said concentrated solution. After removing the
precipitates, a colorless and transparent solution of peptides is
obtained. The solution may be dried in conventional manner to give
a white or slightly colored powder.
According to the present invention, a hydrolysis is easily achieved
with a yield of about 40-50% by only a single hydrolyzing
treatment. The reaction residue may be subjected to further
hydrolyzing reactions to obtain a hydrolysis yield of about 85-95%
by repeating.
The hydrolyzed composition, obtained by the process of the present
invention, contains only a small amount of amino acid monomers and
represents a mixture of various peptides. The amino acid components
of this mixture are rich in glycine, proline and hydroxyproline,
but very poor in sulfur-containing amino acids such as cystine,
cysteine, etc. Thus, it is possible to not only protect human skin
by using this hydrolysate as raw material, for example, for
cosmetics and the like, which are placed in direct contact with
human body, but also to inhibit deleterious influences upon the
human body by using this hydrolysate for various materials which
may directly or indirectly be placed in contact with human
body.
The present invention further provides a process for producing
N-acyl derivatives of the enzymatically hydrolyzed composition of
skin rag obtained in the manner stated hereinbefore by subjecting
the composition to a condensation reaction with a long chain fatty
acid-type acylating agent, and the invention further provides for
the utilization of the derivatives as surfactants which are usable
as additives to detergents, cosmetics and the like, as fully and
clearly described hereinafter.
Generally, the polymerization degree of peptides is important when
an acylated product of peptide is used as a surfactant, because it
is closely related to the stability of the final product to hard
water and its efficiency as a surfactant. Peptides having a high
polymerization degree, exhibit excellent stability to hard water
and/or acid, but exhibit the liability of the reduction of
efficiency as surfactants. Also the existence of amino acid monomer
is not preferable with respect to skin permeability. It is
preferable to use peptides having a polymerization degree of about
2 to about 7, which have desirable balance as surfactants and
desirable properties.
The hydrolyzed composition obtained by the present invention,
comprises mainly peptides having a polymerization degree of about 4
to about 7 and contains little amino acid monomers. The N-acylated
product of these peptides has properties of good wettability,
diffusing, homogenizing, washing, and surfactant, which is
advantageous for less stimulating cosmetics.
It is possible to use any type of known fatty acid-type acylating
agent having 8-22 carbon atoms as the acylating agent of the
present invention; however, it is preferable to use fatty acid
halides (especially chlorides) or fatty acid anhydrides. As the
fatty acid, it is possible to use caprylic acid, capric acid,
lauric acid, myristic acid, palmitic acid, stearic acid, arachidic
acid, behenic acid, oleic acid, palmitoleic acid, linoleic acid, or
a mixture thereof, for example, beef tallow fatty acid, beef tallow
hardened fatty acid, whale oil hardened fatty acid, beef stearic
acid, beef tallow distilled fatty acid, coconut oil fatty acid,
coconut oil decolorized fatty acid, rice bran distilled fatty acid
and so on. Especially good result can be obtained by using acid
chloride of these fatty acids.
The amount of the acylating agent may vary, depending upon the use
of the acylated product as surfactant. It is most preferable to use
the agent in the equivalence to the terminal amino acid group or
not exceeding the equivalence, but the amount may be more than
equivalence. However, the use of acylating agent in excessive
amouht is not preferable because it may cause the introduction of
large amounts of unreacted acid chloride and hydrolyzed fatty
acid.
The condensation reaction of a hydrolyzed composition with an
acylating agent may be carried out by dissolving the hydrolyzed
composition in water and adding the acylating agent thereto with a
vigorous agitation in a conventional manner, simultaneously with
dropping alkali to keep the reaction system in an alkaline state.
It is desirable to drop the acylating agent gradually, avoiding
rapid dropping. It is preferable to keep the reaction system in an
alkaline state during the dropping. However, an excessively
alkaline state accelerates the hydrolysis of acid chloride,
therefore, it is most preferable to keep an alkaline state of about
pH 9.0 to about pH 12.5.
The reaction is completed by heating the reaction mixture to about
50.degree. to 60.degree.C, and yields N-acyl derivatives of the
hydrolyzed composition. As the reaction mixture contains unreacted
fatty acid chloride, hydrolyzed fatty acid and various salts, oily
material rises to the surface when the reaction mixture is
acidified with a suitable acid (e.g. sulfuric acid) after the
completion of the reaction. To this mixture an organic solvent in a
suitable amount (conveniently 0.5 to 2 times by volume) is added
with shaking, and allowed to stand until the mixture separates into
three layers, i.e. water, oil and organic solvent. As the organic
solvent, it is possible to use any organic solvent which is
inactive to the product, barely soluble or insoluble in water and
capable of dissolving fatty acid. However, certain organic solvents
cause emulsification and make it difficult to separate the mixture
into three layers. As the solvent for good separation, it is
preferable to use halogenized hydrocarbons (chloroform, carbon
tetrachloride, ethylene dichloride, tetrachloroethane,
trichloroethylene, perchloroethylene, etc.). The organic solvent
layer contains fatty acid and its chloride; while the aqueous layer
contains salts. After removing the organic solvent and aqueous
layers, the oil layer is adjusted to a neutral or an alkaline state
to give the alkali salt of the N-acyl derivative.
As the product obtained by the present invention is a water-soluble
oil having an adhering property and excellent qualities of
solubilizing, emulsifying, dispersing and foaming, it may be used
for various purposes as a surfactant, for example, dispersing
agent, emulsifying agent, textile treating agent, solubilizing
agent and foaming agent. The present acylated derivative consists
of peptide compounds and is therefore substantially harmless to the
human body, so that it may be advantageously added to cosmetics,
home detergents and the like.
The process of the present invention is further described in the
following examples. In place of the animal skin rag used in the
examples, both raw and tanned hide rag and leather rag of various
other animals may be used in a similar manner. It is not necessary
to limit the size of skin rag, and a large piece of skin may be
used, though it is advantageous to cut it into a suitable size for
shortening the time of hydrolysis. Also, it is possible to carry
out N-acylation by using the above-mentioned various fatty acid
chlorides.
Hitherto only collagenase has been known as enzyme which is capable
of decomposing collagen. It is now possible according to the
present invention to provide other enzymes than collagenase which
are capable of decomposing collagen by treating skin rag with a
neutral or alkaline protease specified hereinbefore.
EXAMPLE 1
Chrome-tanned leather rag of cow (400 g) was suspended in water (8
1) and heated in boiling water for 1 hour. After cooling, the
resultant mixture was adjusted to a pH 7.5 with 2N caustic soda and
heated up to 45.degree.C. To the mixture Tacynase (2 g) (commercial
product available from Kyowa Hakko Kogyo K.K., Japan) was added,
and the reaction was carried out for 20 hours. After the completion
of the reaction, the reaction mixture was heated at 80.degree.C for
15 minutes to inactivate the enzyme and was then weakly acidified
with 2N HCl. The insoluble matter was removed from the reaction
solution to give a filtrate which was then concentrated to
one-third volume at a temperature below 60.degree.C under reduced
pressure, and was freeze-dried to give 158 g of slightly colored
powder. The product was highly hygroscopic and easily soluble in
water.
EXAMPLE 2
A mixture of skin rags of cow and pig (500 g) was suspended in
water (10 1) and heated in a boiling water for 2 hours. After
cooling, the reection mixture was adjusted to a pH 10.0 with 2N
caustic soda and heated up to 47.degree.C. To the mixture Epolozyme
(2.5 g) (commercial product available from Kyowa Hakko Kogyo K.K.,
Japan) was added, followed by reaction for 24 hours. After the
completion of the reaction, the mixture was heated at 80.degree.C
for 30 minutes to inactivate the enzyme and was weakly acidified
with 2N HCl. The insoluble matter was removed from the reaction
mixture to give a filtrate which was treated with active carbon (5%
based on the filtrate) at 50.degree.-60.degree.C. After the carbon
treatment, the filtrate was concentrated to one-tenth volume and
the concentrate was added to about 10 times by volume of methanol
with agitation. After removing the precipitate, a colorless and
transparent solution was concentrated at a temperature below
60.degree.C under reduced pressure to evaporate the methanol almost
completely, to which fresh water was added and freeze-dried to give
183 g of almost white powder. The product was barely colored as
compared with the product obtained in Example 1. The average
molecular weight measured by analyzing the terminal group of
nitrogen was about 640. The composition of the product obtained is
as follows:
Total nitrogen 14.3% Ash 1.9%
The product was highly hygroscopic and easily soluble in water.
EXAMPLE 3
The enzymatically hydrolyzed composition obtained in a similar
manner to that described in Example 2 (100 g) was dissolved in
water (300 ml) and the pH of the mixture was adjusted to 10.0 with
10% caustic soda. Lauroyl chloride (44g) was gradually dropped into
the solution at 30.degree.C with vigorous agitation and with
simultaneous adjustment of the pH to about 10.0 with 10% caustic
soda. After dropping for 45 hours, the reaction mixture was heated
at 50.degree. -55.degree.C for 1 hour to complete the reaction. The
resultant reaction mixture was slightly white and turbid. The
mixture was then adjusted to a pH of 3.0 with 30% sulfuric acid,
whereupon creamy matter rose to the surface. An equal volume of
ethylenedichloride was added to the mixture. After agitation, the
mixture was allowed to stand to separate into three layers of
water, oil and ethylenedichloride. The oil layer was separated and
adjusted to a pH 8.0-9.0 with 25% caustic soda, whereupon a
yellowish and transparent material having a strong adhering
property was obtained. The product was powder of N-acylated product
of the hydrolyzed composition which was easily soluble in water and
had a good foaming property.
EXAMPLE 4
Process for the production of shaving cream:
(a) N-lauroyl compound of an enzymatically hydrolyzed composition
of skin rag of cow 10.0 % (b) Stearic acid 10.0 % (c) Mineral oil
2.0 % (d) Butyl stearate 5.0 % (e) Sperm head oil 5.0 % (f)
Antiseptic (methyl-p-benzoate) 0.1 % (g) Perfume a small amount (h)
Water 67.9 %
The above ingredients, except perfume, were admixed and boiled,
followed by a cooling to a temperature below 35.degree.C with slow
kneading. To the mixture, the perfume was added and well kneaded to
give shaving cream.
EXAMPLE 5
Process for the production of liquid shampoo:
(a) N-stearoyl compound of an enzymatically hydrolyzed composition
of skin rag of cow 30.0 % (b) Sodium arginate 0.9 % (c) Propylene
glycol 3.0 % (d) Antiseptic (methyl-p-benzoate) 0.1 % (e) Perfume a
suitable amount (f) Water 66.0 %
The above ingredients, except perfume, were admixed and treated in
a similar manner to that described in Example 4 to produce liquid
shampoo.
EXAMPLE 6
Process for the production of liquid cream:
(a) N-acylated product of an enzymatically hydrolyzed composition
of skin rag of pig by means of whale oil hardened fatty acid
chloride 15.0 % (b) Stearic acid 10.0 % (c) Triethanol amine 4.0 %
(d) Barium hydroxide 2.0 % (e) Antiseptic (methyl-p-benzoate) 0.1 %
(f) Water 68.9 %
According to the amounts stated above, water was added to a mixture
of the acylated product, stearic acid and antiseptic, and heated to
70.degree.-80.degree.C, to which a mixture of the barium hydroxide
and triethanol amine with a small amount of water, was added. The
resultant mixture was stirred until cooled to produce liquid
cream.
EXAMPLE 7
Process for the production of vanishing cream:
(1) Stearic acid 15.0 % (2) Cetyl Alcohol 2.5 % (A) (3) N-acylated
product of an enzymatically hydrolyzed composition of skin rag of
cow by rice bran distilled fatty acid chloride 0.5 % (1) Triethanol
amine 6.6 % (2) Glycerol 7.0 % (B) (3) Water 74.4 % (4) Perfume a
suitable amount
Component (A) and component (B) were separately prepared in
solution. Component (A) was heated to 85.degree.C and admixed with
component (B) with vigorous agitation to produce a vanishing cream
composition.
* * * * *