US3178301A - Reconstitutable acid solubilized collagen - Google Patents

Description

United States Patent 3,178,301 RECONSTITUTABLE ACID SOLUBILIZED COLLAGEN Arthur Veis, Skokie, and Jerome Cohen, Chicago, IlL,

assignors, by mesne assignments, to Armour and Company, Chicago, Ill., a corporation of Delaware No Drawing. Filed Sept. 19, 1960, Ser. No. 56,646 4 Claims. (Cl. 106-124) This invention relates to collagen fibers and particularly it relates to warm acid solubilized fragments of collagen fibers which are susceptible to ready repolymerization and reprecipitation as networks of highly organized long collagen fibers, to a method for preparing them, and to a method for reconstructing collagenous fibers.

Leather, which consists of chemically modified collagen, is generally available only in form of sheets of intact animal skin. Manufacturers of leather goods cut pieces of desired shape, size and quality from the sheets of skin and discard the remainder as scrap which is disposed of in relatively uneconomical ways. Dilterences in quality, thickness and shape of natural leather sheets further add to the difiiculties and amount of scrap encountered by fabricators. It has long been recognized that it would be desirable to have methods available for dissolving collagen, the solubilized collagen being susceptible to reconstitution as uniform sheets of leather.

The treatment of collagen to dissolve it generally results in the production of material such as gelatin. Generally, gelatin is irreversibly denatured, there being no known practical method for repolymerizing or reconstituting gelatin or similar materials into the native-like fibrous structure found in leather.

A scientific fact which has been known for some time is that normally occurring collagen contains very minor amounts of a substance called tropocollagen or procollagen which may be solubilized and reconstituted into the native-like fibrous structures found in leather. However, the reconstitutable fraction of native collagen can be obtained only by a careful low temperature extraction procedure; relatively scarce and expensive collagen starting materials such as calf skins must be used; and use of mature bovine hide often results in extraction of only traces of tropocollagen.

Attempts to increase the yields of the tropocollagen or procollagen by raising the temperature of the extraction procedure have resulted only in total destruction of the material. During heated acid or alkali extraction of the collagen, the collagen is normally irreversibly depolymerized to form water soluble gelatin from which no native-like collagen fibers can be extracted.

There is need for a method of increasing the yield of the tropocollagen-like or procollagen-like materials obtained from collagen without destroying the reconstitutable character of the substances.

By the use of one aspect of our invention, we are able to extract from mature steer hide up to about by weight of soluble collagen which is amenable to reaggregation as cross-linked, highly oriented collagen fibers and which has the properties of tropocollagen.

An important object of this invention is to provide a highly elficient and simple method for solubilizing native collagen which is susceptible to reprecipitation as polymerized fibers of tropocollagen. Another object is to provide collagen material that is acid extractable at temperatures above room temperature and which subsequently displays the properties of tropocollagen. A further object is to provide a method for processing and altering collagen so as to form reconstructed collagenous structures of desired form and quality. Other objects and advantages will appear in the following description.

In accordance with this invention solubilized collagen having the properties of tropocollagen is extracted from native collagen in the presence of hydroxy acids at temperatures of from about room temperature to about 70 C.

Collagenous materials from various sources are suitable starting materials to produce the products of this invention. Examples of such materials include hides, skins, tendons, ossein and so on. Preferably mature steer hide or hide scraps are used as raw material. We may use collagen which has been subjected to various pretreatments such as dehydration, salting and the like. Optimally, the collagenous stock is dehaired, fleshed and thoroughly cleaned and washed prior to processing.

In the practice of this invention the collagen is mechanically subdivided by a variety of well-known methods, such as beating, grinding or comminuting. One satisfactory method of accomplishing this procedure is to introduce the collagen into a paper beating machine with sufliicient water and with suflicient mixing and beating to form a tree-flowing slurry. A preferred type of apparatus is the Hollander type paper beater which yields a satisfactory slurry without appreciably reducing the length of the native fibers.

Following mechanical sub-division the disaggregated collagen slurry may then be solubilized with warm acid. Preferably the fibers are first treated to remove part of the water which may have been added during the beating operation. Wringing the collagen fibers by rubber-rollers is one suitable method. Hydroxylated organic acids produce the dissolved reprecipitatable collagen which is important in this invention. Alkyl, aryl and heterocyclic hydroxylated carboxy and polycarboxy acids which may be dissolved in water to give solutions having pI-Is between about 2.5 and 4.0 are suitable reagents for this solubilization. Although we prefer to employ citric acid in the process, examples of other acids which may be used include benzilic, glycolic, glycerolphosphoric, hydroxybutyric, lactic, and the like. Acids of this type are believed to react with the collagen fibers during heating and solubilization thereby preventing protein reaction with water present, as a consequence of which the collagen fiber fragments cannot collapse. The dissolved fibers thus remain susceptible to reprecipitation in a native cross-linked polymeric pattern.

Concentration of the acid should be suflicient to give a pH of about 2.5 to 4.0 in the collagen-acid solution reaction mixture. Expressed differently, this pH will generally be obtained by employing about 0.01 molar to 1 molar acid solution, depending on the individual acid or acid mixture utilized. We prefer to use acid concentrations giving a reaction mixture pH of about 3.0 to 3.5. When reacting slurries which have been de-watered to yield wet fibers composed of about 25% protein and water, one part of collagen fibers to about one and onehalf parts by weight of 0.1 molar citric acid gives a reaction mixture with satisfactory acidity.

Throughout the solubilizing portion of our process, it

is most important that temperature of the reaction mix-V ture be maintained between about room temperature and 70 C. Best results are usually experienced when a tem perature of between about 30 C. and 60 C. is used. Poor yields of soluble reprecipitatable col-lagen fiber fragments normally result from the use of temperatures much below room temperatures. Temperatures above 70 C. should be avoided as gelatin may be formed in large quantities and we prefer not to form it in our process.

When the heated and acidified fiber mass has been properly prepared it will assume a physical state which may be described as stringy plasticity. Holding the warm acidified collagen for about 30 to 60 minutes will usually be adequate for proper solubilization. Lower acidities and Patented Apr. 13, 1965 Q as temperatures may necessitate longer times, as for example, a temperature of 30 C. at about pH 3.0 may require a reaction time of as much as 3 to 4 hours. The fiber dough is preferably agitated and blended during solubilization, a screw type blender giving good results.

The soluble portion of thefiber dough may be extracted and reprecipitated as skin-like or sheet-like structures without the presence of intact fibers, but we prefer to mix the treated fiber dough containing the dissolved collagen with wet fibers which have simply been mechanically subdivided. Preferably, the wet fibers are lightly tanned by contact with a solution of formaldehyde having a concentration of about 0.3 to 3 percent by weight. About percent fiber dough mixed with about 85 percent by weight of lightly tanned wet fibers and with an equal amount of 'water added provides an excellent mix for preparing reaggregated structures such as sheets, threads and various other forms. The wet mix may then be formed as desired and allowed to remain in a cool place for a short time. Thirty minutes at 4 C. is preferred by us but there is nothing critical about this procedure. The forms, such as sheets, may then be reaggregated into stable cross-linked structures by any of several known methods which reprecipitate the soluble tropocollagen like collagen fraction. These include heat coagulation, neutralization and salting out of the soluble matter with any of several reagents. We find good results are obtained by a combination of neutralization and salting. This is easily accomplished by placing the fiber structure in half saturated ammonium sulphate made basic with ammonium hydroxide. The resulting wet skin-like structure maybe further processed as typical native collagen.

Reprecipitated collagen materials containing the tropocollagen-like fibers of our invention may be formed into varying articles such as sheets, threads and other fabricated structure.

The tropocollagendike fibers which are produced as described above have the following properties. The fibers when viewed by means of an electron microscope exhibit the typical cross striations of native collagen, having an axial periodicity of about 600 to 650 A. The materials containing the tropocollagen-like fibers of our invention consist of interwoven networks of fibers, exhibit reversible synersis, dissolve in water at pI-Is of about 1 to 3 and are substantially insoluble in water at pHs of about 4 to 8. In addition these materials have relatively high wet tensile strength.

Our invention provides a method for extracting reprecipitatable soluble collagen from mature mammalian collagen in far greater yields than any method heretofore developed. This invention allows the use of cheaper and more readily available raw materials. Moreover, the criticality of known methods of extraction is reduced by the use of our process.

The following detailed examples are given only for the purpose of illustrating and aiding in understanding the invention, it being understood that our invention is not limited to the particular conditions and materials set forth therein.

Example I 1200 gms. of fresh steer hide corium were beaten in a paper beater with added water. The beaten collagen slurry was dewatered by wringing out the fibers between rubber rollers to give wet fibers containing protein and 75% water. 1200 gms. of the wet fibers were added to 1800 cc. of 0.1 M citric acid solutions. The reaction mixture which had a pH of 3.5, was maintained at a temperature of 50 C. for minutes while being continuously mixed in a screw type blender. 450 gms. of the acid treated fiber dough were added to 940 gms. of wet fibers, which had been de-watered, and the mixture was lightly tanned in 3% formaldehyde solution for a short period of time. To the lightly tanned wet fibers was added 450 gms. of fiber dough and 1505 cc. of water. The fiber and water mixture were blended for 30 minutes and rolled into sheets having a thickness of about .016 inch. The sheet was placed in a cold room at about 4 C. for 30 minutes after which it was immersed in half saturated ammonium sulphate which had been adjusted to pH 8.1 by the addition of ammonium hydroxide. The ammonium sulphate solution was prepared by adding 1100 cos. of water to one pound of ammonium sulphate. After four hours immersion the sheets were water-washed and chrome tanned to produce a high strength leather-like product.

Example 11 Bovine corium collagen pieces of about 1 cm. cubed were suspended in 0.1 M citric acid at room temperature. After the collagen pieces were wet through with citric acid solution, the mixture was heated with stirring to 50 C. for one hour. The majority of the material dissolved, leaving a few pieces of individual fibers remaining. The entire slurry was blended in a Waring blender to obtain uniform dispersion. This slurry was then cooled to room temperature and extruded through an orifice onto a moving belt which carried slurry through a one-half saturated solution of ammonium sulfate. pH of bath was maintained at 8.5 with ammonium hydroxide. Extruded fibers wer carried through several feet of the bath and then picked up on a second conveyor belt and transferred to a bath of cold water maintained at low ionic strength. As soon as salt was removed, fibers were picked out of the water wash and transferred to a drying tower.

Example III Beef corium which had been cleaned and cubed was suspended in 0.1 M glycolic acid at room temperature. The acidified collagen was heated to 55 C. with constant agitation and held for one hour at that temperature. The collagenous mix was centrifuged and the supernatant collected. This supernatant was poured into a pan and chilled at 4 C. for thirty minutes, after which a onehalf saturated solution of ammonium sulfate adjusted to pH 8.1 with ammonium hydroxide was layered onto the resulting chilled collagen gel. After one hour at room temperature the resulting sheet was dialyzed against tap Water to remove ammonium sulfate. A high wet strength sheet suitable for tanning resulted.

Example IV Steer hide corium was suspended in 0.15 M citric acid and heated to 50 C. for one hour. The solubilized collagen was put in a dialysis bag and dialyzed overnight against cold tap water. A rigid gel which formed in the dialysis bag air dried to form a fibrous tubular structure having high wet strength and which upon examination displayed the fine structure of native collagen.

Example V Steer hide corium was extracted by the procedure of Example 111 with 0.025 M of citric acid at 50 C. for one hour. Approximately a 1% yield of solubilized reconstitutable collagen resulted from the dilute hot acid extraction.

Example VI Steer hide was extracted with 0.1 M citric acid solution at 60 C. for thirty minutes. A 10% yield of reconstitutable collagen was recovered from the collagen thus solubilized. The reconstituted fibers exhibited typical 640 A. banding of native collagen fibers.

Having described our invention what we claim is:

1. The method of preparing reconstitutable solubilized collagen, comprising extracting a slurry of separated native collagen fibers in an aqueous solution of an organic acid at a temperature about 30 C., but not over 70 C. to solubilize the collagen in said fibers, said organic acid having a concentration within the range from .01 to 1 molar and being selected from the group consisting of citric acid, glycolic acid, lactic acid, hydroxybutyric acid, benzilic acid, and glycerol phosphoric acid, said extraction being carried out and completed in a period of less than four hours, thereby obtaining an enhanced proportion of solubilized collagen in reconstitutable form, and subsequently reconstituting said solubilized collagen to fibrous collagen having the cross-striations of native collagen.

2. The method of preparing reconstitutable solubilized collagen, comprising extracting a slurry of separated native collagen fibers in an aqueous solution of an organic acid at a temperature of around 50 to 55 C. to solubilize the collagen in said fibers, said aqueous solution having a pH of from 2.5 to 4.0 and said organic acid being selected from the group consisting of citric acid, glycolic acid, lactic acid, hydroxybutyric acid, benzilic acid, and glycerol phosphoric acid, said extraction being carried out and completed in a period of not over one hour, thereby obtaining an enhanced proportion of solubilized collagen in reconstitutable form, and subsequently reconstituting said solubilized collagen to fibrous collagen having the crossstriations of native collagen.

3. The method of preparing reconstitutable solubilized collagen, comprising extracting a slurry of separated native collagen fibers in an aqueous solution of citric acid having a pH of from 2.5 to 4.0 and being at a temperature above 30 C. but not over 70 C. to solubilize the collagen in said fibers, said extraction being carried out and completed in a period of less than four hours, thereby obtaining an enhanced proportion of solubilized collagen in reconstitutable form, and subsequently reconstituting said solubilized collagen to fibrous collagen having the cross-striations of native collagen.

4. The method of preparing reconstitutable solubilized collagen comprising, extracting a slurry of separated References Cited by the Examiner UNITED STATES PATENTS 2,521,704 9/50 Evans et al 18-54 2,897,044 7/59 Wormell 18-54 2,934,446 4/60 Highberger et al 106-155 2,934,447 4/ Highberger et al 106155 3,071,483 1/63 Tung Tu 106155 3,073,702 1/63 Keil et al. 106155 FOREIGN PATENTS 606,427 8/48 Great Britain.

OTHER REFERENCES Progress in Leather Science 1920-1945, British Leather Manufacturers Research Association, London, England, 1948, pp. 78-80.

Gustavson: The Chemistry and Reactivity of Collagen, Academic Press Inc., New York, 1956, pp. 73-77.

OFlaherty et al.: Technology of Leather, vol. 1, pp. 151-162 (1956) (p. 155 relied upon).

MORRIS LIEBMAN, Primary Examiner.

CHARLES B. PARKER, MORRIS O. WOLK, LESLIE H. GASTON, Examiners.

Claims (1)

1. THE METHOD OF PREPARING RECONSTITUTABLE SOLUBILIZED COLLAGEN, COMPRISING EXTRACTING A SLURRY OF SEPARATED NATIVE COLLAGEN FIBERS IN AN AQUEOUS SOLUTION OF AN ORGANIC ACID AT A TEMPERATURE ABOUT 30*C., BUT NOT OVER 70*C. TO SOLUBILIZE THE COLLAGEN IN SAID FIBERS, SAID ORGANIC ACID HAVING A CONCENTRATION WITHIN THE RANGE FROM .01 TO 1 MOLAR AND BEING SELECTED FROM THE GROUP CONSISTING OF CITRIC ACID, GLYCOLIC ACID, LACTIC ACID, HYDROXYBUTYRIC ACID, BENZILIC ACID, AND GLYCEROL PHOSPHORIC ACID, SAID EXTRATION BEING CARRIED OUT AND COMPLETED IN A PERIOD OF LESS THAN FOUR HOURS, THEREBY OBTAINING AN ENHANCED PROPORTION OF SOLUBILIZED COLLAGEN IN RECONSTITUTABLE FORM, AND SUBSEQUENTLY RECONSTITUTING SAID SOLUBILIZED COLLAGEN TO FIBROUS COLLAGEN HAVING THE CROSS-STRIATIONS OF NATIVE COLLAGEN.