Use of Collagen in Sausage Casings

383
USE OF COLLAGEN IN SAUSAGE CASINGS*
TOSHIO TZUZUKI
Devro, Incorporated
Somerville, New Jersey
-
I n t r oduct ion -C ollagen
In animals, collagen is t h e major fibrous element of t h e e x t r a c e l l u l a r connective t i s s u e s and probably t h e most abundant p r o t e i n .
It is fuund i n skin, bone, tendon, t e e t h , blood vessels, i n t e s t i n e s
and even i n t h e eye's cornea
Because collagen e x i s t s mostly i n an insoluble fiber f o r m , e a r l y
s t u d i e s on t h i s protein consisted of x-ray d i f f r a c t i o n , electronmicroscopy and chemical analyses. More recently, w i t h t h e development
of various phys ico-chemical methods, the molecular s t r u c t u r e of
collagen has been elucidated
.
A collsgen molecule, termed tropocollagen, is i n a r i g i d rod shape
with the length of 2800 A', t h e diameter of 15 A' and the molecular
weight o 300,0001
3. This rod is made up of t h r e e chains of polypeptides',
which a r e associated with each other thrau@;h a number of
hydrogen bonds f o r s t r u c t u r a l reinforcement .5
Because of i t s r i g i d , rod s t r u c t u r e , m t e r i a l s that a r e reconstituted
f r a a solution or a dispersion of collagen have generally superior
mechanical p r o p e r t i e s . For example, t h e t e n s i l e s t r e n g t h of collagen
suture which has been reconstituted from a s o l u t i o n of s o l u b i l i z e d
collagen i s about 3 g/denier. This is stronger than suture reconstituted
from soy protein r casein and is between wool and s i l k in t e n s i l e
s t r e n g t h ranking.
8
Intestinal Casings
Traditionally, i n t e s t i n e s of sheep and hogs have been used as
edible sausage casing. After removing submucosa, these n a t u r a l i n t e s t i n e s are cleaned t o become sausage casings o r tubes of collagen f i b e r s
that make up t h e w a l l of t h e i n t e s t i n e s . The collagenous s t r u c t u r e in
t h e i n t e s t i n e w a l l has not been c l a r i f i e d a s much as that in the cornea,
but it has a laminated s t r u c t u r e w i t h each layer being a network of
randomly oriented collagen f i b e r s
.
The n a t u r a l sausage casings have demonstrated t h e i r excellent
e l a s t i c i t y , appetizing appearance f o r packaged sausages and tenucious
s t a b i l i t y against punishing cooking. A l l of these advantages are
believed t o o r i g i n a t e f r o m t h e above mentioned s t r u c t u r e that is
responsible f o r t h e mechanical s t r e n g t h and e l a s t i c i t y of i n t e s t i n e s ,
even though collagen f i b e r s themselves a r e not e l a s t i c .
* Presented
a t the 29th Annual Reciprocal Meat Conference of t h e American
Meat Science Association, 1976.
However, t h e r e a r e s e v e r a l s e r i o u s shortcomings inherent t o n a t u r a l
sausage casings. FFrst of a l l , they must undergo a thorough cleaning
and s t i l l require preservation. Furthermore, t h e i r lack of uniformity
i n thickness, color and in diameter a r e d e f i n i t e disadvantages f o r
modern, high-speed s t u f f i n g . I a s t l y , if one must depend on t h e import
t o obtain l a r g e q u a n t i t i e s of a n h l i n t e s t i n e s , it would be another
handicap
There have been only a few patented improvement^^^^^^^^^^^^^^ i n
recent years on n a t u r a l casings i n t h e area of color control, uniform
diameter, reinforcement and s h i r r i n g . These products a r e probably most
u s e f u l i n specialized sausage a p p l i c a t i o n s , but not r e a d i l y manufacturable
i n an e f f i c i e n t manner.
Reconstituted Collagen Casings
A t t e m p t s t o m k e a b e t t e r e d i b l e casing from various collagen
sources have been made. Out of t h e s e a t t e q t s , a number of manufacturing
processes and products have been invented. The majority of inventors
i n t h i s a r e a appear t o have a comwn r a w material f o r collagen--beef
hide
In general, t o manufacture r e c o n s t i t u t e d collagen casing, this
collagen r a w rnaterial is cominuted, mixed with a swelling agent t o
produce a uniform dispersion fran which a continuous tube is formed.
The tube undergoes, i f it is a wet process, s e v e r a l treatments before
it becomes s h i r r e d slugs of uniform s i z e , length and s t r e n g t h . In a
d r y process, a swollen collagen dispersion may contain a tanning agent
and a higher collagen s o l i d s . From this dispersion, a tube is extruded
i n t o a i r and is simply d r i e d .
The f i r s t important s t e p in t h e manufacture of r e c o n s t i t u t e d
collagen casings is t o prepare a uniform d i s p e r s i o n f o r tube formation.
T h i s is p a r t i c u l a r l y c r i t i c a l f o r t h e extrusion process which i s t h e
Other methods of apparently
most popular method t o form collagenous tub s
m e o r importance a r e e l e c t r o d e p o s i t i o ~ 1 , ~ 3d, i~p~ coating,l5 and coextrusion
1 17,
.
Uniform, collagen dispersions f o r tube extrusion are prepared, f o r
example, by swelling c o l h g e n f i b r i l s obtained from f r e s h corium l a y e r s
o s t e e r h i d e with l a c t i c a c i d followed by homogenization and deaeration
$,19,20.
Some collagen dispersions are made from corium l a y e r which
has undergone s h o r t liming with21 or withoute2 subsequent deliming.
Hide collagen can be modified, r i o t o t h e preparation of dispersions,
through s t r o n g a l k a l i t r e a t ~ n t H 3 9 2o~r m i l d p r o t e o l y t i c enzyme t r e a t Some inventions use mixtures of s t e e r hide collagen and
pigskin collsgen15,27 mixtures of s t e e r hide collagen and casein,28
and mixtures of beef tendon collagen and gelatin29. A l s o , t h e r e is a
group of inventions which a r e based on a l o g i c a l p r i n c i p l e of having
two d i s t i n c t components i n dispersions, i.e., h e a v i l y limed collagen
f i b e r s and enzymatically soltibilized tropocollagen s 0 l u t i o n 3 ~ , 3 1 . A
most recent patent32 describes a simple method of completely llming
collagen f i b e r s followed by a c i d soaking and water washing before a
d i s p e r s i o n is formed.
38 5
The second important consideration f o r t h e manufacture of highq u a l i t y , reconstituted collagen casings is t h e alignment of collagen
fibers or f i b r i l s within uniformly formed tubes. Unlike sane p l a s t i c
materials, collagen tube, after it is formed and e s p e c i a l l y a f t e r it
i s coagulated, cannot r e a d i l y change f i b e r or f i b r i l o r i e n t a t i o n .
Therefore, any kind of o r i e n t a t i o n t o optimize mechanical properties
of collagen casing f i l m must be done almost e n t i r e l y i n the extruder.
There have been only a few developments that have been published
To design an extruder f o r t h e manufacture
of reconstituted c o l h g e n casing, it is v i t a l t o understand t h e physicochemical nature, e s p e c i a l l y t h e rheological properties, of a given
swollen collagen dispersion. One patent33 teaches us that a dispersion
of collagen f i b r i l s swollen w i t h a d i l u t e a c i d s o l u t i o n can be subjected
t o two d i r e c t i o n a l flows which w i l l be l a t e r combined t o form crosso r i e n t a t i o n of f i b r i l s i n a casing f i l m . More simply constructed and
probably more commonly used than the afore-mentioned extruder, i s the
one In which the nozzle has two counter-rotating walls t o e f f e c t similar
cross-orientation of collagen f i b e r s and f i b r i l s .34
i n t h e area of extruders.
I n addition t o t h e diameter of an extruder's nozzle, t h e pressure
difference between inside and outside of t h e extruded tube w i l l have an
important e f f e c t on t h e c o n t r o l of tube diameter. The pressure d i f f e r ence is created by e i t h e r liquid35 or gaseous2° coa@;ulant.
After collagen tubing i s reconstituted, it i s usually subjected
t o a strength development process commonly c a l l e d " t a n n h g " or I'crosslinking" o r 'hardening." This process takes advantage of one of
collagen's unique chemical properties, i.e., i t s a f f i n i t y t o heavy
metal ions and t o aldehydes.
SimiLar t o t h e chrome tanning in t h e l e a t h e r Industry, aluminum
tanning has been popular in *e hardening of e d i b l e collagen casings36~37.
Iron can a l s o be use t o e f f e c t i v e l y c r o s s l i n k collagen, e s p e c i a l l y
that from 1-d
hide$, but it should be followed by a discoloration
s t e p f o r b e t t e r appearance39.
A number of aldehydic crosslinking methods have been successfully
The simplest of
these i s t o use dextrose40 and, with b e t t e r control, l i q u i d s m 0 k e ~ 1 , ~ 2 .
tried t o strengthen reconstituted collagen casings.
P
Pro bly t h e most e f f e c t i v e and conizollable aldehyde is g l u t a r aldehyde 3 which is a l s o used i n combinations with other reagents
On a more complicated s i d e , one process is designed t o
44,45,46,4'?.
form a r e a c t i v e aldehyde48 and another requires a f i x i n g s t e p following
aldehyde tanning49
I n t e r e s t i n g l y , t h e r e are hardening processes that do not use t h e
so-called crosslinking agent, but depend on strong aUnrli media24~50.
A l l in a l l , the tanning of reconstituted collagen casings is highly
d e s i r a b l e t o make casing products withstand punishment during sausage
mnufacture. However, if it is overdone, casings could become t o o
b r i t t l e and/or t o o tough.
Generally, p r i o r t o drying, i n a wet process collagen casings
undergo a p l a s t i c i z a t i o n s t e p where a desired amount of p l a s t i c i z e r ,
such as glycerin, i s absorbed by casings. Sometines, t h i s s t e p is
modified s o that reagents other than p l a s t i c i z e r can be taken up by
casings t o impart a d d i t i o n a l desirable properties t o the f i n a l products.
The drying method of p l a s t i c i z e d collagen tubes appears t o be r a t h e r
conventional, i.e., the tube i s i n f l a t e d and passed through dry a i r f l o w a t a temperature s u f f i c i e n t 1 high, but not t o o high t o denature
t h e collagen. One recent patent5 explains that improved performnce
of casing during cooking is obtained by c o n t r o l l i n g t h e i n f l a t i o n
pressure during drying t o a moderate l e v e l . In some cases, however,
t h e diameter control must be watched c l o s e l y w i t h t h e adjustment of
i n f l a t i o n pressure
3;
In t h e a r e a of f’unctional modification of reconstituted collagen
casings, most of them seem t o aim a t improvements of appearance, t e x t u r e ,
machinability and cooking performnce. One method5* makes use of
caramel, which is blended i n swollen collagen mass t o be extruded f o r
t h e purpose of improving s t u f f i n g responses, improving p i g t a i l l i n k
r e t e n t i o n , and improving t h e s t a b i l i t y of non-smoked areas. In order
t o minimize t h e shrinkage of collagen during sausage cooking, s e v e r a l
methods have been reported. The b a s i c idea is t o add t o t h e collagen
matrix a substance that does not s h r i n k upon heating t o cooking tern e r atures A~mmin53and albumFn-carboxymethylcellulose c o m b h t i o n s 5
were used i n a s o l u t i o n form i n which wet collagen tubes are passed.
Also, sodium a l g i n a t e s were mixed i n various proportions w i t h swollen55
o r unswollen56 collagen f i b e r s and extruded tubes therefrom a r e t r e a t e d
w i t h a s e t t i n g bath f o r alginates.
.
f
Even though most casings are p l a s t i c i z e d w i t h glycerin, it i s
highly d e s i r a b l e in many cases that casings contain a hydrophobic
material f o r further softening, lmproved appearance, lubrication, and
wet s t r e n g t h r e t e n t i o n . Hydrophobic materials, unless applied d i r e c t l y
t o the surface of casings, are emulsified i n aqueous erystems, such a s a
p l a s t i c i z e r bath, w i t h a surface a c t i v e agent and absorbed by passing
collagen tubes. Edible o i l i s used i n one method57, whereas monoglyc e r i d e s p and f a t t y acid e s t e r s of propylene glycol59 are used i n others.
Conclusions
To design an i d e a l casing f o r a given sausage application, it is
necessary t o understand the composition of meat emulsions, the mode of
stuffing and linking, t h e conditions of f u r t h e r processing, i f any, the
desired s h e l f - l i f e , and t h e methods of cooking by consumers. Any
sausage casing manufacturer would l i k e t o produce u n i v e r s a l casings
which only by changing t h e diameter, can s a t i s f y worldwide sausage
industry needs. Collagen sausage caslng technology has made tremendous
progress in the last twenty years or so, but t h e r e seems t o be a long
distance separating t h e present state of a r t and t h e universal casing.
There i s no doubt that more improved products and processes w i l l be
researched and developed a t an everaccelerating frequency during t h e
next s e v e r a l years. These w i l l have t o be b u f i t on deeper understanding
of collagen raw materials, a s w e l l as sausage technolo@;y.
387
REFERENCES
1. Gross, J., J . H. Highberger and F. 0. S c h m i t t .
Acad. S c i . U.S. 40:679.
1956. J.A.C.S.
1954. Proc. National
78:4267.
2.
Boedtkes, H. and P. Doty.
3.
Holl, C . B. and P. Doty.
4.
Ramachandra, L. N.
1963. Aspects of Protein Structure. Acad.
5.
Rich, A . and F.H.C.
Crick.
Press. New York.
6. Miyata, T.
7.
1958. J.A.C.S.
80:1269.
J. Mol. B i o l . 3:483.
1962.
1970. Kobunshi 19:359.
R i n e h a r t , C . A. and L. B. Jensen.
1960. U.S.P. 2,966,415.
8. Vaessen, H. J.
1961. U.S.P.
9. Vaessen, H. J.
1965. U.S.P. 3,214,277.
2,977,233.
10.
Vaessen, H. F.
11.
Levaco, R . M. and J. A. Dolgoff.
1974. U.S.P.
12.
Levaco, R . M. and J. A. Dolgoff.
1974. U.S.P. 3,826,853.
13
Mizuguchi, J., S. Suzuki, K. Kashiwaya, S . Kinoshita and C . Kinoshita.
1968. Jap. P. 43:15774.
1973. U.S.P.
3,744,784.
1973. Jap. P. 48-12429.
14. Miyata, T. and M. Takahashi.
1963. U.S.P.
15
K e i l , H. L. and E . F. Cavanaugh.
16
Deacon, M. J . and L. Kindleysides.
17
Schilling, E. D . and P. I. B u r c h i l l .
18. Liebermn, E .
R.
3,826,852.
1973. U.S.P.
1974.
3,073,702.
3,767,821.
Can. P . 950,749.
1964. U.S.P. 3,123,482.
19
Liebermn, E. R .
20.
Fagan, P. V.
21
Talty, R . D. and M. A . Cohly.
22.
Talty, R . D.
23
Courts, A.
24.
Cohly, M. A. and J. W
1964. U.S .P. 3,123,653.
1970. U.S.P.
3,535,125.
1968. U.S.P. 3,408,ga.
1969. U.S.P. 3,425,846.
1971. U.S.P. 3,579,358.
. Sanner .
1971. U .S .P. 3,620,775.
388
25
9
1968. U .S .P. 3,373,046.
Fagan, P. V.
1972. U.S .P. 3,681,093.
26.
Tsuzuki, T. and E. R. Lieberman.
27
Takahashi, H.,
28.
Shank, J. L.
29
Lieberman, E . R .
30
Miyata, T., T. Fujimoto and M. Yamamra.
31.
Miyata, T., T. Fujimoto, S . Baba, T . Nishihara and M. Yamamura.
1970. Jap. P 45-33738
32
Burke, N . I.
33
Lieberman, E. R
34
Miyata, T.,
35
Taylor, K. W .
1971. U.S.P.
36
S e i l e r , J. A.,
Jr.
37
S . Okabe, K . Sat0 and S . Furukawa.
50-117950.
1975. Jap. P .
1972. U .S .P. 3,695,902.
1967. U.S.P.
3,346,402.
1970. Jap. P. 45-27378.
.
1976. U.S.P.
.
3,932,677.
1965. U .S .P. 3,221,372.
T. F'ujimoto and T. Higashi.
3,630,760.
1964. U.S.P.
1970. U.S.P.
1971. Jap. P. 46-4145.
3,123,481.
3,525,628.
9
Cohly, M. A .
9
Cohly, M, A . and A. F. Turbak.
38
39
3,408,916.
40.
&Knight,
41.
Nishihara, T
T. Miyata, T. FuJimoto and Y. Nishizawa.
J a p P 46-6382.
42.
Miller, A. T .
43
Rose, H . J .
44.
Burke, N. I.
1975. U.S.P.
3,930,035.
45
Burke, N. I.
1975. U.S.P.
3,930,036.
46.
Cohly, M. A .
1970. U.S.P. 3,533,809.
47
48.
49
50
J. T.
. . .,
1964. U.S.P.
1968. U.S.P.
3,151,990.
1975. U.S .P. 3,894,158.
1968. U.S.P.
3,413,130.
1971.
389
51.
Takahashi, H.,
50-117949
52.
Tanner, A . G. and J . T. Wallace.
53.
Lieberman, E . R .
54.
&Knight,
55.
Bradshav, N. J . , C. J. Sahram and A . Courts.
56.
S . Okabe, K. Sat0 and S. Furukawa.
J. T .
3,860,728.
1964. U.S.P. 3,123,480.
1964. U.S.P. 3,123,483.
Courts, A . and N . J. Bradshaw.
57. Talty, R . D.
1975. U.S.P.
1975. Jap. P.
1970.
U.S.P.
1970.
U.S.P.
3,494,772.
3,494,773.
1969. U.S.P. 3,446,633.
58.
Cohly, M. A. and J. W
59.
Takahashi, H.,
50-121452
. Sanner.
1971. U.S .P. 3,627,542.
S . Okabe, K. Sato and S . Furuhawa.
1975. Jap. P.
* * *
Dennis Campion: Now, if a l l four of t h e speakers would please
come forward, we w i l l e n t e r t a i n questions
A 1 Pearson, Michigan State: I ' d l i k e t o ask a couple of quastions.
I t h i n k Thayne might be able t o answer t h e f i r s t one which i s r e l a t i v e
t o t h e formation of collagen and t h e n u t r i t i o n a l f a c t o r s that a r e
fnvolved. I don't believe you covered that.
T. R . Dutson:
s o r t of thing?
You're speaking of t h e co-factors necessary, t h i s
A 1 Pearson, Michigan S t a t e :
on the biosynthesis of collagen?
And where do they f i t i n t o t h e p i c t u r e
That's what I ' d l i k e t o know.
T. R . Dutson: Well, p r i m r i l y the co-factors necessary f o r
synthesis of collagen a r e similar t o those required f o r synthesis of
other p r o t e i n s . However, it h a s n ' t r e a l l y been investigated t o t h e
e x t e n t that some of t h e other proteins have. The co-factors necessary
f o r hydroxylation, i n p a r t i c u l a r , involve ascorbic a c i d as one, f e r r o u s
i r o n as another, molecular oxygen, and a-ketoglutarate. CY-ketoglutarate
is necessary and it is decarboxylated In t h e process. Ferrous iron
seems t o be necessary f o r , probably binding of t h e oxygen. Ascorbic
a c i d probably a c t s l i k e a reducing agent i n t h e sequence of hydroxylation.
I n glycosylation, t h i s i s generally a t r a n s f e r r e n c e type of r e a c t i o n .
In the literature, I found no s p e c i f i c mention of micronutrients o r cof a c t o r s t h a t are nzcessary f o r t h i s r e a c t i o n .
390
A 1 Pearson, Michigan S t a t e : Okay. The other question I ' d l i k e
t o ask d e a l s w i t h t h e type I and I11 collagen. I f you look a t t h e
composition of type I and I11 collagen, t h e y ' r e both low i n carbohydrate
m a t e r i a l as compared t o t h e other two types of collagen, that is types
I1 and IV. Do you t h i n k t h a t t h i s i s of s i g n i f i c a n c e i n regard t o t h e i r
properties?
T . R. Dutson: I t h i n k c e r t a i n l y SO. The amount of carbohydrate
has been alluded t o as being important i n both t h e f o r m t i o n of t h e
types of f i b r i l s and t h e s t r e n g t h of t h e f i b r i l s . From sme of t h e
research we've done, and i n looking a t other research, I f e e l t h a t the
amount of carbohydrate is p r t i c u l a r l y important i n t h e a s s o c i a t i o n of
t h e collagen with t h e ground substance matrix. If you have a sugar
moiety on collagen, you can s e e where t h i s would be a mechanism i n
l i n k i n g t o mucopolysaccharides of t h e ground substance, which thems e l v e s a r e then bound t o proteins of' t h e ground substance. So you
have r e a l l y s o r t of a t h r e e phase s t r u c t u r e : t h e collagen with i t s
carbohydrate moiety probably bound t o t h e mucopolysaccharide of t h e
ground substance; then t h e mucopolysaccharide of t h e ground substance
being bound t o t h e p r o t e i n moiety of t h e ground substance. Also, I
t h i n k t h e amount of carbohydrate i s important i n f i b e r f o r m t i o n ,
p a r t i c u l a r l y i n type IV collagen. The high a m u n t of carbohydrate i n
t h i s collagen type seems t o be very important i n i t s amorphous s t r u c t u r e
because it does not form t y p i c a l f i b e r s .
P. E. McClain: The carbohydrate moieties are, i n a number of
cases, associated with t h e l y s i n e s t h a t a r e a l s o involved with crosslinkin:.
Now, t h e mechanism here i s not y e t c l e a r e i t h e r , b u t t h e r e i s
a relationship.
A 1 Pearson: While you've got t h e microphone, l e t me a s k you a
question, P h i l , In your p a t t e r n s of pro-crosslinking m t e r i a l s you
spoke of histidino-hydroxydesmosine. Of course, desmosine i s an amino
a c i d that i s commonly found in t h e connective t i s s u e s of a component.
P. E. McCLain: It's found as a c r o s s l i n k i n e l a s t i c t i s s u e . This
h i s t i d i n o product, though, is a r e a l weird one. H i s t i d i n e has t o be
positioned i n t h e r i g h t s p o t . Now, t h e r e ' s a school of thought that
says, "This compound i s an a r t i f a c t produced during reduction." But,
i t ' s not t h e same as t h e one found in e l a s t i n . They've searched high
and low f o r it i n collagen because t h i s i s t h e one that w i l l t i e up
four d i f f e r e n t molecules
A 1 Pearson: One other question while I've got you, P h i l . You
mentioned t h e f a c t that n u t r i t i o n may play a r o l e i n t h e r e l a t i v e
proportion of type I and I11 collagen. I d o n ' t t h i n k you elaborated
on that. Would you mind doing so?
P o E. & C h i n : This is a r e a l e x c i t i n g new a r e a of collagen
research. They're f i n d i n g that conditions such as a t h r o s c l e r o s i s , f o r
example, may be a function of t h e type of collagen present. Arthritis
i s p r e s e n t l y thought t o be a r e s u l t of type I I I / t y p e I interchange of
some s o r t . It's a brand new area. We d o n ' t r e a l l y know w h a t t h e
s i g n u i c a n c e of it i s as y e t .
391
A1 Pearson:
are these?
You're t a l k i n g about t h e n u t r i t i o n a l aspects, w h a t
P. E. b l c c l a i n : N u t r i t i o n is a f f e c t i n g collagen d i r e c t l y . There
are s e v e r a l things that w i l l . The type of carbohydrate m i g h t . Now,
I don't know whether t h i s again goes back t o t h i s carbohydrate moiety
and hydroxylysine. But, w e fed f r u c t o s e and glucose d i e t s and t h e r e
was a very rmrked a l t e r a t i o n i n t h e ty-pe of collagen w e found.
S t a r v a t i o n or food r e s t r i c t i o n has been long recognized as influncing
longevity in experimental animals. McKay, back in t h e 30's reported
t h a t food r e s t r i c t e d rats would l i v e up t o twice as long as t h e rats
that were e a t i n g ad-libitum d i e t s . Our work i n d i c a t e s that t h e r e may
be a chemical b a s i s f o r t h i s . If food r e s t r i c t i o n i n h i b i t s crosslinking,
t h e n our arteries are going t o be more p l i a b l e . The wrinkles t h a t some
of t h e l a d i e s don't l i k e w i l l be l a t e r i n coming about.
A 1 Pearson, Michigan State:
didn 't ment ion, copper
There i s another t h i n g that Thayne
T . R . Dutson: Well, copper seems t o be important i n lysyloxidase
t h e enzyme that oxidizes t h e e p s i l o n amino group of l y s i n e . The primary
p o s i t i o n of a c t i o n of t h i s micronutrient on crosslinking i s a t t h i s point
because t h i s enzyme does r e q u i r e copper f o r a c t i o n . You can i n h i b i t t h i s
enzyme by having copper d e f i c i e n t r a t i o n s .
On t h e type I11 collagen, one t h i n g t h a t I t h i n k i s very s i g n i f i c a n t
is i n f e t a l skin; type I11 collagen is t h e r e i n higher amounts. The
collagen i n f e t a l s k i n i s very, very insoluble. Then s h o r t l y a f t e r
b i r t h , t h e r e is a s h i f t from type III collagen back t o type I collagen.
Then as P h i l showed, t h e collagen s o l u b i l i t y then increases. N o w , P h i l
showed it i n t h e muscle, but t y p i c a l research w a s o r i g i n a l l y done i n
f e t a l skin. So, t h e r e ' s a s h i f t i n t o t a l collagen s o l u b i l i t y r e l a t e d
t o a s h i f t i n collagen types. Some researchers f e e l that collagen
s o l u b i l i t y , a t l e a s t i n some cases, m i g h t be almost e n t i r e l y due t o
t h e type of collagen that i s present. Possibly, l a t e r on i n maturation,
an increase i n the amount of c r o s s l i n k s that P h i l talked about decreases
t h e s o l u b i l i t y of t h e type I collagen.
D r . Greaser, Wisconsin: Would one of you like t o coment on whether
t h e r e i s such a t h i n g as a p r o t e i n r e t i c u l i n ?
Thayne Dutson: By a p r o t e i n r e t i c u l i n , you mean t h e t r a d i t i o n a l
p r o t e i n that's supposed t o be some kind of-D r . Greaser:
In t h e textbooks, we l i s t collagen and r e t i c u l i n .
Thayne Dutson: I d o n ' t h o w . My f e e l i n g is that t h i s could be
t h e type N collagen that we see i n t h e basement membrane. My b a s i s
f o r saying t h i s , i n looking a t l o n g i t u d i n a l sections of electronmicrographs, i f you look through numerous s e c t i o n s , you very seldom ever
f i n d any d e f i n i t e s t r u c t u r a l connective t i s s u e f i b e r s r i g h t down when
you g e t right darn t o t h e endomysium. Now, occasionally you do f i n d
392
them t h e r e , but I t h i n k t h e y ' r e s t i l l ramifications of t h e periqysium.
A s you come down t o t h e a c t u a l point where one f i b e r connects onto t h e
other, about a l l you can see i s t h e plasmalemma, t h e basement membrane,
some space but very l i t t l e , then another basement membrane, and another
plasmalemma. That's my b a s i s f o r saying it could v e r y w e l l be t h e
basement membrane type collagen. Another t h h g i s that basement membrane
type collagen could be binding t h e s i l v e y s t a i n t h a t has been shown
c l a s s i c a l l y . That's where t h e r e t i c u l a r f i b e r s come from, w a s t h e
s t a i n i n g with s i l v e r . This collagen could be a s i l v e r s t a i n i n g type of
collagen, p a r t i c u l a r l y with i t s high amount of carbohydrates. A s Howard
s a i d , if you remove t h e l i p i d , which could be associated with some of
the carbohydrates, you reduce t h e s t a i n i n g . Well, maybe t h e s t a i n
could be binding j u s t t o that.
H. J. Swatland: You can d i s s e c t t h e endomysium or r e t i c u l a r
fibers and p u l l them out of t h e muscle and stand them up by themselves.
N o w , that's not a membrane you're p u l l i n g out, those a r e d i s t i n c t
f i b e r s . I agree, t h e r e ' s SOIE considerable overlap i n dimension and
s t a i n i n g properties of t h e small diameter f i b e r s . I n instances l i k e
wound healing in t h e skin, you can f i n d d i s t i n c t types of f i b e r s : t h e r e
are small ones that stay small, always s t a i n e d with s i l v e r ; t h e r e are
some collagen f i b e r s t h a t never stain with s i l v e r , and y e t g e t bigger
and bigger i n a man's wounds and form s c a r t i s s u e ; t h e r e are organs
which are held together, such as t h e spleen, by r e t i c u l a r f i b e r s . You
can f i n d r e t i c u l a r f i b e r s i n a l l s o r t s of other t i s s u e s .
Thayne Dutson: O f course, i n t h e type of graphs that you showed
of t h e f a t c e l l s , t h i s was a f a i r l y f i n e network and could be basement
membrane of t h e f a t c e l l s , a l s o . Does that follow?
H. J . Swatland: Yes, f a t c e l l s , conceivably. But t h e s i l k stocking
around t h e muscle f i b e r , that's a r e a l l y s u b s t a n t i a l s i l k stocking.
D r . Allen, Minnesota: I guess t h i s is f o r Phil or Thayne. Is
t h e r e any evidence of t h e hormonal changes i n t h e animal? I ' m t h i n k i n g
now of cycling females, f o r example, cows, i f t h i s r e s u l t s in changes
i n the connective t i s s u e s collagen that might a f f e c t tenderness. I ' m
thinking about some of t h e old cows having very tender meat. Do w e
have any evidence f o r that? Is t h e r e anything that m i g h t be t i e d up
with collagen turnover e s s e n t i a l l y ?
P. E. McClain: I know t h e r e ' s a change i n t h e uterus, but I d o n ' t
know whether t h e r e ' s any r e l a t i o n s h i p t o the muscle or not. Do you
know, Thayne?
Dr. Allen: Do you have any hormonal t h i n g s you can r e l a t e t o
collagen synthesis crosslinking?
P . E. M c C h i n : I know that cortisone a f f e c t s collagen. Now
whether collagen i t s e l f o r t h e ground substance is a l t e r e d is not
known
Dr
. Allen:
Now, is t h a t used i n crosslinking?
393
is.
P. E . McClain:
I don't t h i n k so.
I ' m sorry, I c a n ' t h e l p you.
I don't know what t h e a f f e c t
D r . Allen: While I've got t h e floor, P h i l , I ' d l i k e f o r you t o
summrize t h e e f f e c t s of mar:'nates, long t i m e , l o w temperature cooking,
s o we have in our minds exactly w h a t ' s going on here on collagen. For
example, does s a l t have any a f f e c t on t h e marinate, o r is it e n t i r e l y
t h e acid?
P. E . McClain: Yes, s a l t s could a f f e c t it. Collagen i s s a l t
soluble, e s p e c i a l l y t h e e a r l y collagen, r e c e n t l y synthesized m a t e r i s l .
Acids a r e c e r t a i n l y going t o work on t h e aldamine bonds. I t ' s long
been known that if you s t o r e collagen a t room temperature it forms
c r o s s l i n k s . Now, t h e only study that I've ever seen i s t h a t one we
C . It would be i n t e r e s t i n g t o see
did and t h i s was 100 hours a t
what happens a t higher temperatures. It may a l t e r t h e u l t i m a t e
c h a r a c t e r i s t i c s of t h e meat by t h e aging process.
D r F i e l d s , Wyoming: I j u s t wanted t o follow up about your
comment about t h e old cow, or your question about t h e old cow being
tender, a c e r t a i n percentage of t h e m . The t h i n g I thought o f , P h i 1
i n connection when you were t a l k i n g , was t h e study you quoted sharing
that f a s t i n g , even f o r 48 hours or so, could reduce t h e f3 c r o s s l i n k s
e s p e c i a l l y . Certainly, many of t h e s e cows a r e n ' t i n t o o good of
condition t o s t a r t with, and if f a s t e d a l i t t l e b i t longer might t h e s e
be t h e more tender cows? I d o n ' t know i f anyone has followed up on
t h i s . I would appreciate a comment on t h a t .
P. E. McClain: Well, t h i s f a s t i n g i s another i n t e r e s t i n g t h i n g .
I t h i n k i n t h e s e f a s t i n g animals it would be a matter of accelerated
turnover. N o w , i f t h i s were a turnover of collagen, it would be nice
t o have t h i s process working f o r us, wouldn't it? There i s a need f o r
more work i n t h i s a r e a .
D r . Wierbicki: Are t h e r e going t o be differences i n collagen
compos it ion with ages?
P. E . M c C l a i n : I t h i n k you're asking are t h e r e any changes i n
amino a c i d compos i t i o n e s p e c i a l l y hydroxyproline? I t h i n k Thayne w i l l
agree that t h e r e i s . Not a whole l o t , but young immature type collagen
is somewhat under hydroxylated
Thayne Dutson: I think, probably, a t t h e point that you're
measuring it i n a n a n i m l , say two years of age or l8 months, something
l i k e t h i s , I t h i n k t h e r e probably wouldn't be. But, i f you had a s h i f t
o r d i f f e r e n c e i n t h e type of collagen, t h e r e would be a s l i g h t amount
of d i f f e r e n c e i n hydroxyproline. But even i n those s i t u a t i o n s you f i n d
l i t t l e d i f f e r e n c e unless you go t o t h e type I1 or type IV collagen,
t h e r e ' s where you would g e t a l a r g e difference i n t h e amount of hydroxyproline. Now, in muscle t i s s u e i t ' s primarily type I and type I11 which
have about t h e sane amount of hydroxyproline i n t h e r e . I t h i n k you w i l l
f i n d very l i t t l e difference, r e a l l y , unless you're looking a t very
inmature collagen which hasn't been completely hydroxylated.
394
John Romans, I l l i n o i s : This i s f x P h i l McClain. Is crosslinking
r e l a t e d t o animal age in such a nay that we might use crosslinking t o
estimate t h e anirml's age?
P. E. k c l a i n : I t h i n k very much s o . I
very t y p i c a l p a t t e r n , e s p e c i a l l y w i t h animals
regime. I wonder w h a t t h e s e grass fed c a t t l e
feed l o t c a t t l e of t h e same age? We see t h i s
on collagen crosslinking.
think it would follow a
under t h e same n u t r i t i o n
look like as compared t o
d e f i n i t e protein e f f e c t
John Romans: How long does it t a k e t o get that data? If w e took
a sample how long would it take, two or three days, t o work t o g e t that
s l i d e you had t h e r e , or one day or two?
P. E . McClain: It depends on how much you want t o know. I ' m
p r e t t y curious a l l t h e way d a m t h e l i n e . D r . Wierbicki is using heat
l a b e l cards and i t ' s probably as good as any. It t e l l s you haw much
of that is permanent crosslink.
Dennis Campion: I t h i n k that w i l l conclude then t h e session on
Connective Tissue. I ' d l i k e t o thank you four gentlemen f o r your f i n e
work.
* * *