AU569112B2 - Crosslinked collagen products - Google Patents

Description

LARATION FORM FOR COMPANY APPLICATIONS, AUSTRALIA L Form 7,8.

Regulations 12 and 12 (2) COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952 69

DECLARATION IN SUPPORT OF AN APPLICATION FOR A PATENT

In support of the Application made by NITTA GELATIN CO., LTD. (Name of Applicant Company) of 2-55-1, Honmachi, Higashi-ku, Osaka, 541 JAPAN for a patent for an invention entitled: PROCESS FOR PRODUCING A SHAPED PRODUCT OF COLLAGEN BY SYNERESIS

I, Seiichi Nitta of the Applicant's (Full Name of Individual Declarant) address, do solemnly and sincerely declare as follows:

1. I am authorized by the applicant for the patent to make this Declaration on its behalf.

2. Yoshimasa Yoden Tsuneo Okuda Eiji Fuchigami and Toshihiro Kuwabara (Full Name of Inventor) 4-11-8, Taiho Kanan-cho Minami Kawachi-gun, Osaka, JAPAN; o 3-7-45, Habikigaoka Nishi, Habikino, Osaka, JAPAN; 1-2-6, Sakuragaoka, Kgnmki-cho, Kitakatsuragi-gun Nara, :JAPAN and 373-9, Yanagida-cho, Gose, Nara, JAPAN,respectively ndsfare the actual inventor(s) of the invention and the applicant is the assignee of the said inventor(s). for the said invention 3.The basic application(s) as defined by Section 141 of the Act, was/were made in S

JAPAN on the 29th (Country)

day of September 1983, by Nitta Gelatin Co. Ltd. (Name of Applicant for Basic Application)

4. The basic application(s) referred to in paragraph 3 of this Declaration was/were first ap- *plication(s) made in a Convention country in respect of the invention, the subject of the application.

Declared at Osaka, JAPAN this 12th (City and Country)

day of September 19 8.4

(Signature)

To: (Seiichi Nitta) The Commissioner of Patents, CANBERRA.

NOTE: Initial all Deletions and Alterations. No-witnessing and no Tegalisatioh, For a Non-Convention application delete paragraphs 3 and 4 and initial the deletion. For Multiple Priorities Incorporate details of all basic applications in paragraph 3, For application for a Patent of Addition add "of Addition" after the word "patent" wherever the word occurs, and initial each such insertion. For Joint Application by a Company and an Individual amend form so that the individual declares he is one of the applicants and is authorised by the other applicant to make this declaration upon the latter's behalf (Paragraph and that the Company is an Assignee of a part interest in the invention from him (Paragraph 2.)

GRIFFITH, HASSEL FRAZER, Box 2133, G.P.O., SYDNEY 2001. AUSTRALIA.

(12) PATENT ABRIDGEMENT (11) Document No. AU--B-33461/84 (19) AUSTRALIAN PATENT OFFICE (10) Acceptance No. 569112

(51)4 International Patent Classification C08J 005/18 A22C 013/00 A23J 003/00 C08J 005/02 (21) Application No. 33461/84 (22) Application Date 24.09.84 Priority Data (31) Number (32) Date (33) Country 58-182437 29.09.83 JP JAPAN (43) Publication Date 18.04.85 (44) Publication Date of Accepted Application 21.01.88 (71) Applicant NITTA GELATIN CO. LTD.; (72) Inventor YOSHIMASA YODEN TSUNEO OKUDA EIJI FUCHIGAMI TOSHIHIRO KUWABARA (74) Attorney or Agent GRIFFITH, HASSEL FRAZER (54) Title CROSSLINKED COLLAGEN PRODUCTS (56) Prior Art Documents 68521/81 535551 C08J 5/00, C08L 5/08, A23L 1/30 32895/84 A22C 13/00, A23J 1/10 37184/78 519348 C08J 5/18, A61L 15/01, 15/03 (57) Claim

1. In a process for producing a shaped product of collagen by a wet or electrochemical method from a pasty composition comprising a collagenous substance or a mixture thereof with another high molecular substance selected from one or more of natural fibers, synthetic fibers, protein, seaweed extracts, polysaccharides and synthetic high molecular weight substances, the improvement which comprises treating said composition with a crosslinking agent when said composition is shaped, freezing the shaped product and thawing it, the crosslinking agent being capable of reacting with the amino group in the collagen.

Form PATENTS ACT 1952

COMPLETE SPECIFICATION (ORIGINAL) FOR OFFICE USE Short Title: Int. Cl: 569112 Application Number: .3 1i If Lodged:

erplt Specification-Lodged: 0:::.Accepted: 000: Lapsed: Tis documet contains the Published: arnerldmetits made undeir se~otion 49. .i'riority:

Related Art:, Liand is coMret tor prwting. fl: *00.

9* .5 5 TO BE COMPLETED BY APPLICANT

N4me of Applicant: NITTA GELATIN CO., LTD.

:48dress of Applicant: 2-53-1, Honmachi, Higashi-ku, Osaka, 541 JAPAN

Actual Inventor: Yoshimasa Yoden, Tsuneo Okuda Eiji Puchigami and Toshihiro Kuwabara

Address for Service: GRIFFITH HASSEL PRAZER 71 YORK STREET SYDNEY, N.S.W. 2000, AUSTRALIA

Complete Specification for the invention entitled: PROCESS FOR PRODUCING A SHAPED PRODUCT OF COLLAGEN BY SYNERESIS

The following stutement Is a full description of this Invention, including the best method of performing It known to me Noti The description Is to be typed In double spacing, pica type face, In an area not exceeding 250 mm In depth and 160 mm In width, on tough white paper of good quality and It Is to be Inserted Inside this form. 14099/78-L rinted by C. ,Tito"sot4, Commonwealth Government Printer, Canberra 1459918-t.

I PROCESS FOR PRODUCING A SHAPED PRODUCT OF COLLAGEN BY SYNERESIS ,BACKGROUND OF T'IH.; INVI'NTION

1. Field of the Invention:

This invention relates to a process for producing

a shaped,product of collagen by syneresis.

2. Description of the Prior Art:

There are known various kinds of shaped products 9 consisting mainly of collagen. They are, for example,

tubular products such as sausage casings, threadlike or

stringy products'such as sutures, spongy or feltlike prod- ucts such as medical sponge, and tapes and sheets which are

usable for a wide variety of applications.

A shaped product of collagen is formed from a pasty

composition prepared by finely devided fibers of connective

S tissue containing collagen, such as the skin or tondons of

an animal, and/or solubilized collagen or by the addition a

of one or more of fibrous materials, proteins, polysaccha-

S. rides and synthetic high molecular materials to a collagenous substance. The product is obtained by a wet method such as the extrusion of the composition into a salt solution or

by an electrochemical method such as eloctrodeposition.

A wet shaped product of collagen obtained by a wet

or electrochemical method is usually very soft, as it con-

tains a large amount of water. At least 95% of the product

TA- is water. The low pressure resistance of the product brings about a lot of difficulty in its drying, especially if it is a tubular product which is dried by the air intro- duced thereinto. Moreover, a large amount of energy and a correspondingly extensive set of equipment are required for removing a large amount of water from the product.

It is an object of this invention to provide a

process which produces a shaped product of collagen having 0* improved quality. It is another object of this invention to provide

Sa process which is greatly improved in efficiency for the

production of a shaped product of collagen.

These objects are attained by a process which in-

1Z cludes applying a crosslinking agent to the composition be- ing shaped.or a shaped product, freezing the shaped;product 4.

and thawing it. The crosslinking agent may b a natural

or synthetic crosslinking agent reacting with the amino group

in collagen, for example, formaldehyde, glutaraldehyde, gly-

oxal, dialdehyde starch or a liquid smoke. The freezing of 9S the product enables its crosslinking reaction. According

to this invention, it is possible to produce by syneresis

a tough shaped product of collagen in the wet state but not

containing or absorbing much water, or in the dry state.

In the event a wet method is employed, the cross-

,4

linking agent may be added to a coagulant when the pasty

composition is shaped.' Alternatively, it is possible to

apply the crosslinking agent after coagulation by spraying

a solution thereof onto the shaped product, or dipping it in such a solution, or treating it with the fume or gas of

the solution. The product is, then, frozen at a tempera- ture of -5°C to -400 C, and thawed. In the event an elec-

trochemical method is employed, the shaped product is treated with the crosslinking agent, frozen and thawed.

According to the process of this invention, the

separation of water is promoted during the addition of the

S. crosslinking agent and the freezing of the shaped product and takes place immediately upon thawing. Although the

reason for this phenomenon still remains to be clarified,

it is probably true that the sealing of the amino group in

collagen'by the crosslinking agent lowers the hydrophilicity

of collagen and thereby promotes the separation of water therefrom, and that the separation of several tens per cent

of water from the shaped product with the progress of its

*0 freeziig brings about a reduction in the distance betweenr the cross linkages and thereby enables achievement of an

improved crosslinking effect.

In any event, the separation of a lot of water

makes it possible to obtain by the process of this invention

a tough shaped product of collagen in the wet state but not

containing or absorbing any appreciable amount of water, 1, and which can be used in the wet state, depending on the purpose. Although the product shaped ,by syneresis in accordance with this invention is usually in the shape of a tube, sheet, tape or string, the process is also useful for shaping a product in the form of a thin film or thread.

The process of this invention, therefore, enables the pro-

duction of shaped products which are not only excellent

in properties, but also available in a wide variety of spe- S* cial shapes. This inventi.on enables a drastic reduction

p. in the time required for coagulation and washing with water in the event a wet method is employed for shaping.

Moreover, the wide-angle X-ray diffraction of a

dry shaped,product obtained by the process of this invention

does not reveal any crystal structure, but confirms that it

has a very high affinity for coloring matter having a bulky

structure, such as Erythrosine. A shaped product obtained

by treatment with a crosslinking agent and drying without being frozen, however, has a crystal structure, and its

affinity for coloring matter having a bulky structure is low. According to this invention, it is possible to use

additionally a crosslinking agent which is not reactive with

the amino group in collagen, e.g. alum.

The invention will now be described in further detail

with reference to several examples thereof.

EXAMPLE 1

A fresh corium layer of oxhide from which the hair

had been removed was cut into 10 cm square pieces, and they were dipped for 10 days in two parts of 0.4% lime milk per part of the corium. The material thus treated was washed

by water and neutralized by hydrochloric acid. The mate- rial was, then, refined by dipping for five hours in two parts of a 1% aqueous solution of ammonium chloride per part *sees: of the corium and washing in flowing water. The refined iio material was ground by a meat grinder including a plate pro-

vided with holes having a diameter of 1 mm, whereby collagen

5 fibers were obtained.

An aqueous suspension containing the corium at a

solid concentration of 8,0% and sodium hydroxide at a con-

centration of 3.0% was prepared from 20% by weight of the collagen fibers, and kept at a temperature of 20 0 C plus or

minus 10 C for two days. This treatment solubilized the

Scollagen and formed an emulsion thereof. Hydrochloric

acid was added to the emulsion at a temperature not exceed-

o.2 0 ing 200 C to lower its pH value to 4.0 and cause the precipi- tation of a fibrous agglomerate, and the agglomerate was

dehydrated. The remainder (80% by weight) of the collagen

fibers was added to the dehydrated product. They were

swollen in an aqueous solution of citric acid having a pH

value of 3.0 at a solid corium concentration of 3.5% and

mixed together homogeneously. The homogeneous mixture was passed through a Manton Gaulin type homogenizer to form a pasty composition.

The pasty composition was extruded into a coagulat-

ing solution through an annular nozzle device comprising a

pair of dies having a nozzle spacing of 0.5 mm and a dia-

meter of 17 mm and adapted for rotation in mutually opposite

directions. The coagulating solution was a 20% saline

solution containing 1000 ppm of glutaraldehyde, having its

pH value adjusted to 9.5 by sodium hydroxide and having a i

alp. temperature of 20°C. The tube as extruded had a pH value

of 3.6, and was left in the coagulating solution for o*

minutes until its pH value rose to

After the tube had been washed by flowing water for

minutes, it was immediately placed in a freezer having a

temperature of -20 0 C, and kept frozen for five hours. The

tube had a water content of 96% and a burst strength

of 600 mm Aq/cm2 before it was frozen. When it was frozen

and thawed, its water content was 75%. This means the re-

moval of 88% of the absolute quantity of water which the

tube contained prior to freezing. The freezing and thawing S

0 of the tube increased its pressure resistance to 1500 mm Aq/ 2 cm The tube did not show any appreciable change in

weight when it had been caused-to absorb water for 60 minutes.

The wet tube was filled with sausage meat, dried at 75°C for 0C for 20 minutes to make a sau- 20 minutes and boiled at 80

sage. The sausage was cooked in a frying pan and the tube

was not broken.

SI

The process of this invention showed excellent

results in the production of not only wet tubes, but also

dry tubes. Although a tube made by the conventional

process without freezing required 20 minutes for drying,

the tube formed by the process of this invention including

freezing took only two minutes for drying. According to this invention, it was not necessary at all to employ any

cellulose or other substance for preventing the shrinkage S* of the tube upon drying. Moreover, the improved strength

"*1T of the tube obtained by freezing made it possible to realize

,ee a dry tube wall thickness of 25 microns, while it was impos-

sible to reduce the wall thickness to less than, say, microns in accordance with the prior art. This reduction

in wall thickness enabled the production of a sausage cas-

ing which was closer to a natural intestinal casing in

twisted form and taste. The dry tube of this invention

was filled with sausage meat, and could be boiled at 4.

for 20 minutes without breaking, though it had not been

dried or smoked. oe*

**020 EXAMPLE 2

*The procedures of EXAMPLE 1 were repeated for pre-

paring a pasty composition. It was extruded through a hole

having a diameter of 0.5 mm into a coagulating solution con-

taining 2% of potassium alum and 20% of salt and having a

pH value of 4.5 to form a stringy product. It was left for

solidification for 10 minutes, and washed by flowing water for 10 minutes. There was obtaiined a product having a water content of 96%, and which was low in strength and difficult to dry. It was, then, kept frozehi for 24 hours in a freezer having a temperature of -25 0 C. When the product was thawed, it had a water content of 92% and a solid salt content of The product was allowed to absorb water for 60 minutes, and showed an increase of in weight.

Another stringy product was dipped in a 1% glyoxal solution immediately (within 10 seconds) after extrusion, and left thorein* for a minute. Then, it was kept frozen

at a tempera,'-ure of -150W for 20 hours. When it was -thawed, it had a water contbent of 80% and a solid salt content of It was allowed to absorb water for 60 minutes, and showed an increase of 10% in weight. This product was e*.tough, and required for drying only a half of the time re- quirod for drying the product frozen without being treated with gltyoxal. The -treatment of the product prior to its freezing enablIed a drastic reduction in solidification time anid *9 0the omission of the washing stepo The product had, a solid 0

salt content of 40% immediately after extrusion, or when it had been allowed -to solidify for 10 seconds after extru- Sion.

EXAMPLE 3 The procedures of tXAMPIJE I were repeated for pre- paring finely divided collagen fibers. They were used to form a pasty composition having a solid corium content of

0.6% and a pH value of 3.6 and containing 0.02% of propylene glycol alginate, while not containing solubilized collagen. The pasty composition was degassed carefully, and a pair of parallel stainless steel plate electrodes having a distance of 5 cm therebetween were placed in the composition. A DC voltage of 50 V was applied across the electrodes for seconds at 20WC, and a wet layer of collagen formed thereby on the cathode was continuously drawn up to yield a shaped product in the form df a sheet having a thickness of about so 1 to 2 mm. The product had a water content of 98%, and

was soft and weak. The shaped product was dipped in a solution contain- ing 500 ppm of glutaraldehyde for a minute, and frozen at *see a temperature of -10C for 18 hours. When the product was thawed, it was a tough sheet having a thickness of about 0.1 to 0.2 mm and a water content of 80%. When it was

allowed to absorb water for 60 minutes, it showed an increase

.of 10% in weight. It was easy to dry to form a dry shaped EP product in the form of a sheet having a thickness of microns. EXAMPLE 4 The procedures of EXAMPLE 1 were repeated for pre- paring finely divided collagen fibers. Four liters of a caustic soda solution having a pa value of 11 and 2.5 liters of an aqueous solution containing 57 g of sodium polyacrylate and 15 g of a copolymer of polyvinyl methyl ether and maleic anhydride were added to 1 kg of the collagen fibers.

The fibers were dispersed carefully by a homogenizer to

prepare a pasty composition. The pasty composition was

extruded through a slit having a width of 0.5 mm into a

aluminum sulfate solution, and allowed for solidifica-

tion for 10 minutes. The solidified composition was washed

by flowing water for five minutes to yield a shaped product

S"in the form of a tape. The product had a water content of

A sausage liquid smoke was sprayed onto tlhe product for :g a minute, and it, was frozen at a temperature of -30*C for

hours. When the product was thawed, it had a water con- tent of 80%, and when it had been allowed to absorb water for 60 minutes, it showed an increase of 10% in weight.

The frozen product required for drying only one-fifth of in.. the time required for drying the unfrozen product, and

yielded a thin and tough product in the form of a tape.

EXAMPLE A pasty composition, which had been prepared in S.oo

"020 accordance with the procedures of EXAMPLE 1, was extruded to into a coagulating solution through an annular nozzle device

having a nozzle width of 0.7 mM and a diameter of 25 mm and comprising an inner member and an outer member adapted for

roLation in mutually opposite directions, The soluHton had a p value of 4.5 and contained 1.5% of potassium alum, of a sausage liquid smoke and 80% of salt. Tho extrLtded prod- q uct was allowed to solidify for eight minutes, and washed in flowing water for five minutes to yield a tubular prod- uct. It had a water content of 95% and a burst strength of 500 mm Aq/cm 2 The product was frozen at a tem- perature of -100 C for 20 hours. When it had been thawed, it had a water content of 85%. When it had been allowed to absorb water for 60 minutes, it showed an increase of in weight and had a pressure resistance of 1200 mm Aq/ cm Its drying required only one"+,ighth of the time re- quired for drying the unfrozen product. The resulting dry tooo tubular product having a wall thickness of 25 microns was filled with sausage meat, and dried at 750 C for 30 minutes. It was, then, smoked at 600 C for 45 minutes in a smoking house to yield a sausage. It had a satisfactorily smoked •:15 color, and could be fried in a large quantity of oil with- 00*• out shrinking very much.

0*@0 EXAMPLE 6 6•

The procedures of EXAMPLE 3 were repeated for form-

ing a shaped product in the shape of a sheet having a thick- "20 ness of about 1 to 2 mm from a wet layer of collagen on the oi cathode. It was smoked with cherry wood at 700 C for two

minutes, and frozen at -1l0C for 18 hours. When it had been thawed, it formed a tough sheet having a thickness of

about to 0.2 mm. It was dried to yield a sheet product

having a thickness of 20 microns.

COMPARATIVE EXAMPLE

A dry tubular product having a wall thickness of microns, which had been prepared in accordance with the

procedures of EXAMPLE 5, was filled with sausage meat to

form a sausage. It was dried at 750 C for 20 minutes and

cooked with steam at 750 C for 30 minutes. The cooked

product was dipped in a 1% solution containing equal propor-

tions of erythrosine and norbixin at 70C for a minute to

yield a vividly colored sausage.

A tubular product obtained by extrusion through an annular nozzle, solidifying for eight minutes and washing 0

for five minutes in accordance with the procedures of EXAMPLE

was dried by hot air immediately without being frozen.

It was filled with sausage meat, dried at 750C for 20 minutes and cooked with steam at 75 0 C for 30 minutes to prepare a ese. 44

6. sausage. It was dipped in a 1% solution containing ery-

throsin,- and norbixin at 70'C for a minute. it was, how-

ever, not satisfactorily colored at all, but was only yellow-

ish as a whole. The wide-angle X-ray diffraction of this 0 "002 product revealed a peak of crystallinity at 4.6 A due to the 99 0surface distance between the adjoining collagen molecules, o

and at 2.8 A due to the recurring amino acid residues. The same wide-angle X-ray diffraction of the dry

tubular product obtained in accordance with the procedures

of EXAMPLE 5 and having a wall thickness of 25 microns con-

firmed the absence of any such peak of crystallinity and the presence of an amorphous structure. This difference coin- cides with a difference in the adsorbability of bulky coloring matter, such as erythrosine.

A natural sheep intestine, which had long been used

for a sausage casing, showed visual dyeing test results

comparable to those on the product of this invention. Its

wide-angle X-ray diffraction confirmed the absence of any 0 peak of crystallinity at 4.6 A. too**

09

s 0* a

0* ha 4

SO a a S**

Claims (5)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. In a process for producing a shaped product of collagen by a wet or electrochemical method from a pasty composition comprising a collagenous substance or a mixture thereof with another high molecular substance selected from one or more of natural fibers, synthetic fibers, protein, seaweed extracts, polysaccharides and synthetic high molecular weight substances, the improvement which comprises treating said composition with a crosslinking agent when said composition is shaped, freezing the shaped product and thawing it, the crosslinking agent being capable of reacting with the amino group in the collagen.
2. 2. In a process for producing a shaped product of collagen by a wet or electrochemical method from a pasty composition comprising a collagenous substance or a mixture thereof with another high molecular substance selected from one or more of natural fibers, synthetic fibers, protein, seaweed extracts, polysaccharides and synthetic high S" molecular weight substances, the improvement which comprises *20 treating with a crosslinking agent a product shaped from said composition, freezing said shaped product and thawing it, the crosslinking agent being capable of reacting with the amino group in the collagen. a *e
3. 3. A process as set forth in claim 1 or 2, wherein **25 said composition comprises collagen fibers obtained by finely dividing connective tissue containing collagen.
4. 4. A process as set forth in claim 3, wherein said 9S**4 composition further contains solubilized collagen.
5. 5. A process of producing a shaped product of "30 collagen substantially as herein described with reference to any one of Examples 1 to 6.

   DATED this 25th day of November, 1987 NITTA GELATIN CO., LTD. By their Patent Attorneys GRIFFITH HASSEL FRAZER

   5636S/bn -14-