Study of intracellular proteinases of some bacteria
by Dharam Vir Vadehra
A thesis submitted to the Graduate Faculty in partial fulfillment of the requirements for the degree
MASTER OF SCIENCE in Dairy Manufacturing
Montana State University
© Copyright by Dharam Vir Vadehra (1962)
Abstract:
Seven organisms (Streptococcus lactis. Leuconostoc dextranicum, Lactobacillus casei, Proteus
vulgaris. Bacillus subtilis. Pseudomonas fluorescens and Staphylococcus aureus) were selected and
grown on liquid media. From these organisms the intracellular enzymes were separated and studied for
their proteolytic activity on casein and/or whey protein substrates. The proteolysis was followed by the
Folin-Ciocalteu method, with the results being expressed in micrograms of tyrosine and tryptophane.
Optimum pH for the various enzymes was found to be within the range of 6.0 to 7.0, although S. lactis
showed a second peak at pH 5.5. ' The age of the cells was found to be a factor and proteinases
production increased as the age of the cell increased up to 96 hours, Cells 144 hours old showed a
decrease in proteinase production.
The addition of gelatin and casein to the medium used to grow the organisms, increased the proteolytic
activity of the intracellular enzyme system, while the addition of Casitone (Difco) had no effect. The
absence of carbohydrates from the growth medium also increased the proteolytic activity of these
enzymes. STUDY OF INTRACELLULAR PROTEINASES OF SOME BACTERIA
by
DHARAM VIR VADEHRA
A t h e s i s s ubm itt ed t o t h e Graduate F a c u l t y in p a r t i a l
f u l f i l l m e n t o f t h e re q u ir e m e n ts f o r t h e de gre e
MASTER OF, SCIENCE
in
D a ir y Manufacturing
Approved;
Head, Major Department
gUafrmaji^ Examining Committee
D^aW, Graduate D i v i s i o n
MONTANA STATE COLLEGE
Bozeman, Montana
August, 1962
iii
'
ACKNOWLEDGMENTS
The au th o r wishes t o e x p re ss h i s s i n c e r e a p p r e c i a t i o n and thanks
t o Dr. J . C. Boyd, P r o f e s s o r o f Dairy I n d u s t r y , f o r h i s i n t e r e s t ;
h e l p f u l s u g g e s t i o n s ; p l a n n in g and w r i t i n g o f t h i s m a n u s c r i p t ; t o Dr.
R. H. McBee, Dr. W. G. W alter and Dr. N. M. Nelson o f t h e Department
o f B a c t e r i o l o g y f o r t h e i r v a l u a b l e advice and guidance in c a r r y i n g out
t h e r e s e a r c h p r o j e c t ; t o Dr. B. L, Johnson, P r o f e s s o r o f Chemistry, f o r
h i s he lp on some o f t h e chemical a s p e c t s of t h e problem.
The auth or i s
h i g h l y g r a t e f u l t o Dr. J . C. Bbyd f o r being allowed t o do e x t r a work
d ur in g t h e l a s t f i v e months which helped him out f i n a n c i a l l y .
L a s t , but by no means l e a s t , t h e a u th o r wishqs t o thank h i s p a r e n t s ,
Mr. arid Mrs. S r i Ram Vadehra f o r en couraging him to come t o t h e U. S. A.
and h e l p i n g him t o do so.
iv
TABLE CF CONTENTS
Page
VITA eeofrooe»»6eeo0oeeeoeoeeeed6e»d»*ooo»oee»edeeeo»»ooe6e#6eo
ii
ACKNOWLEDGMENTS * # d * @e_e »eodoo9a#oo6e»ooooe*o6eooo*ooopoooe*e»a i i i
LIST Cti*" TABLES
v
e 9 e 9 e e e e 0 o o o e o e f l i 9 e 9 9 e » 9 9 9 o 6 e * e 6 6 e e o » e o e e o e » o e e »
LIST OF FIGURES oooodooeoooodooooo^eftedoeoooeo'ooobeooeeeodoooo v i i i
ABSTRACT . o. »
o o o o o o 6 o 6 » o o o e o o o
INTRODUCTION
o o e o o o e e t i e e o o e e o o o o e o e o o o o o o o o e o o f l e d o e o o o t i e o o o o ,
0 0 0 0 0 0 9 0 9 9
o
I
OOOOOOOOl
3
o’ o o o o o e o e o o d o e o o o d o o d o a e o o a o o o e o b a b b o
3
REVIEW CF LITERATURE
En Zyme S
ix
t o e o o e o o o o o e e o o d o o o o o o o o o o o e o o o o
- o o e d o o o o d o e o o o o o o
4
P r o t e o l y t i c enzymes and f e r m e n t a t i o n of cheese
. 9.
6
Attempts t o a c c e l e r a t e t h e cheese r i p e n i n g . . . . . . . . . . . .
9
C o n t r i b u t i o n of b a c t e r i a t o t h e development of f l a v o r . .
10
Problem and purpose
12
Role of b a c t e r i a in r i p e n i n g of c hees e . ?
EXPERIMENTAL PROCEDURES AND RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . .
14
DISCUSSION AND CONCLUSIONS
....
40
. . c i . . . . . . 0. . . . . o o o . . 0^o'o'o.'Oo.6'o ..o. . . . . . . . . . . 0 0 0
45
LITERATURE CITED ^ . o . . . . ^ ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
47
APPENDIX . . . o ' . . . . . . . . . . . . . 0. 0. . . . o'... . . . . . . . . . . . . . . . . 0 0 . 9 0 . . . .
55
SUMMARY
V
LIST OF TABLES
Tabl e
I.
Page
Average d e c r e a s e in o p t i c a l d e n s i t y XlO of milk
( 1 :5 0 d i l u t i o n ) due to i n t r a c e l l u l a r enzyme a c t i v i t y .
C e l l s grown on medium I , Enzymes e x t r a c t e d by Toluene.
20
Average d e c r e a s e in o p t i c a l d e n s i t y XlO of milk
( 1 :5 0 d i l u t i o n ) due t o i n t r a c e l l u l a r enzyme a c t i v i t y .
Organisms grown on medium I I . Enzymes e x t r a c t e d by
toluene,
21
Decrease in o p t i c a l d e n s i t y XlO of milk (1 :2 0 d i l u t i o n )
as a r e s u l t of i n t r a c e l l u l a r enzyme a c t i v i t y . Organisms
grown on medium I . Enzymes e x t r a c t e d by t o l u e n e .
22
IF.
D ecrease in o p t i c a l d e n s i t y XlO o f milk (1: 20 d i l u t i o n )
due t o enzyme a c t i v i t y . Organisms grown on medium I I .
Enfcymes e x t r a c t e d by t o l u e n e . ;
23
V.
Decrease in o p t i c a l d e n s i t y XlO o f milk (1: 20 d i l u t i o n )
when t h e i n t r a c e l l u l a r enzymes,were e x t r a c t e d by a c e r
t o n e dry powder method, Organisms grown on medium I .
24
Summary of Table s I , I I , I I I , IF and V.
25
Micrograms o f t y r o s i n e and tr y p t o p h a n e l i b e r a t e d as th e
r e s u l t of i n t r a c e l l u l a r enzyme a c t i v i t y on c a s e i n s o l u t i o n
substrate,
,
28
II.
III.
VI.
V II.
.
F ill.
Micrograms of t y r o s i n e and t r y p t o p h a n e l i b e r a t e d as
a r e s u l t of i n t r a c e l l u l a r enzyme a c t i v i t y on whey p r o t e i n
solution.
29
IX.
Optimum pH, and micrograms o f t y r o s i n e and tr y p t o p h a n e
l i b e r a t e d a t v a r i o u s pH.
30
X.
D i f f e r e n c e s in amounts o f t y r o s i n e and tr y p t o p h a n e
l i b e r a t e d by i n t r a c e l l u l a r enzymes e x t r a c t e d from c e l l s
grown f o r 24 and 48 h o u r s .
31
XI.
D i f f e r e n c e s in t h e amounts of t y r o s i n e and tr y p t o p h a n e
l i b e r a t e d by i n t r a c e l l u l a r enzymes e x t r a c t e d from c e l l s
grown f o r 48 and 96 h o u r s ,
32
vi
Table
Page
D i f f e r e n c e s in t h e amounts o f t y r o s i n e and t r y p t ­
ophane l i b e r a t e d by i n t r a c e l l u l a r enzymes e x t r a c t e d
from c e l l s grown f o r 96 and 144 h o u r s .
23
D i f f e r e n c e s in t h e amounts of t y r o s i n e and try p t o p h an e
l i b e r a t e d by i n t r a c e l l u l a r enzymes e x t r a c t e d from c e l l s
grown f o r 24 and 144 ho u rs .
34
I n c r e a s e in amounts of t y r o s i n e and tr y p t o p h a n e l i b e r a t e d
by i n t r a c e l l u l a r enzymes due t o t h e presence o f 0.5%
g e l a t i n in t h e growth medium.
35
XV.
I n c r e a s e in t h e amounts o f t y r o s i n e and try p t o p h an e
l i b e r a t e d by i n t r a c e l l u l a r enzymes due t o t h e presence
of 0.5% c a s e i n in t h e growth medium.
36
XVI.
I n c r e a s e in t h e amounts o f t y r o s i n e and try p t o p h an e
l i b e r a t e d by i n t r a c e l l u l a r enzymes due t o t h e absence
o f c a r b o h y d r a t e s in t h e growth medium.
37
XVII.
I n c r e a s e in t h e amounts of t y r o s i n e and try p t o p h an e
l i b e r a t e d by i n t r a c e l l u l a r enzymes due t o t h e presence
o f C a s i t o r e ( D i f c o ) in th e growth medium.
38
XVIII.
C u l t u r a l c h a r a c t e r i s t i c s of t h e organisms used .
56
X II.
X III.
XIV.
XIX. ' Media I and I I used f o r growth o f c e l l s .
XX.
59
Composition o f s o l i d s u b s t r a t e s .
60
The micrograms o f t y r o s i n e and tr y p t o p h a n e l i b e r a t e d by
i n t r a c e l l u l a r enzymes from c e l l s grown f o r 24 and 48 hours
and t h e d i f f e r e n c e s .
61
XXII.
The micrograms o f t y r o s i n e and tr y p t o p h a n e l i b e r a t e d by
i n t r a c e l l u l a r enzymes from c e l l s grown fo r 48 and 96
hours piid t h e d i f f e r e n c e s .
62
xxni.
The micrograms of t y r o s i n e and try p t o p h an e l i b e r a t e d by
i n t r a c e l l u l a r enzymes from c e l l s grown fo r 96 and 144
hours and t h e d i f f e r e n c e s .
63
XXIV.
Comparative amounts (micrograms) o f t y r o s i n e and t r y p t ­
ophane l i b e r a t e d by i n t r a c e l l u l a r enzymes prepa red from
c e l l s grown on medium I and g e l a t i n r i c h medium.
64
XXV.
Comparative amounts (micrograms) o f t y r o s i n e and t r y p t ­
ophane l i b e r a t e d by i n t r a c e l l u l a r enzymes pre pa red from
c e l l s grown on medium I and c a s e i n r i c h medium.
65
XXI.
v ii
Table
Page
XXVI.
Comparative amounts (micrograms), o f t y r o s i n e and t r y p t ­
ophane l i b e r a t e d by i n t r a c e l l u l a r enzymes prepa red from
c e l l s grown on medium I and c a r b o h y d r a t e f r e e medium.
66
XXVII.
Comparative amounts (micrograms) o f t y r o s i n e and t r y p t ­
ophane l i b e r a t e d by i n t r a c e l l u l a r enzymes prepa red from
c d l l s grown on medium I and 0.5% C as it o n e r i c h medium.
67
. v iii
LIST OF FIGURES
F ig u r e
Page
1.
P e p t i d e ch ain showing N t e r m i n a l li n k a g e and C te r m i n a l
l i n k a g e and t h e p o i n t s o f a t t a c k by p r o t e o l y t i c enzymes.
2.
Sta nd a rd curve o f l i g h t absorbance v s . micrograms of
t y r o s i n e and t r y p t o p h a n e u s in g Beckman B s p e c tr o p h o to m e te r.
5
27
ix
ABSTRACT
Seven organisms ( S tr e p t o c o c c u s l a c t i s , Leuconostoc de x tr a n ic u m ,
L a c t o b a c i l l u s e a s e l , JProteus v u l g a r i s . B a c i l l u s s u b t i l i s . Pseudomonas
f l u o r e s c e n s and Sta phylococcus a u r e u s ) were s e l e c t e d and grown on l i q u i d
m e dia . From t h e s e organisms t h e i n t r a c e l l u l a r enzymes were s e p a r a te d
and s t u d i e d f o r t h e i r p r o t e o l y t i c a c t i v i t y on c a s e in an d /o r whey p r o t e i n
s u b s t r a t e s . The p r o t e o l y s i s was followed by t h e F o l i n - C i o c a l t e u method,
with t h e r e s u l t s being ex pr e sse d in micrograms of t y r o s i n e and t r y p t ­
ophane.
Optimum pH f o r t h e v a r i o u s enzymes was found t o be w i t h i n th e
range o f 6 .0 t o 7 . 0 , althoug h Si l a c t i s showed a second peak a t pH
5.5.
'
The age of t h e c e l l s was found t o be a f a c t o r and p r o t e i n a s e s
p ro d u c ti o n i n c r e a s e d as t h e age of t h e c e l l in c r e a s e d up t o 96 h o u r s ,
C e l l s 144 hours old showed a d e c r e a s e in p r o t e i n a s e p r o d u c t i o n .
The a d d i t i o n o f g e l a t i n and c a s e i n t o t h e medium used t o grow th e
o r g a n is m s , in c r e a s e d t h e p r o t e o l y t i c a c t i v i t y of t h e i n t r a c e l l u l a r en­
zyme system, w hil e t h e a d d i t i o n o f C as ito ne ( D i f c o l had no e f f e c t . The
absence o f c a r b o h y d r a t e s from t h e growth medium a l s o in c r e a s e d the
p r o t e o l y t i c a c t i v i t y , o f t h e s e enzymes.
INTRODUCTION
Fe rm e n ta ti o n s have been s t u d i e d f o r a long t i m e .
Some ferm e nta ­
t i o n s , f o r example, t h e p ro d u c ti o n of al co hol by y e a s t a r e well under­
s to o d .
Othe rs such as cheese r i p e n i n g a r e s t i l l somewhat o f a mystery,
alth ou gh e x t e n s i v e work has been done in t h e a r e a .
Q u it e a body of l i t e r a t u r e i s a v a i l a b l e on t h e r o l e played by
such t h i n g s as r e n n e t and b a c t e r i a in cheese r i p e n i n g .
The re n n e t
s t u d i e s have been c o n fi n e d l a r g e l y t o t h e f a c t o r s a f f e c t i n g t h e coag'f
u l a t i o n o f m il k , along with t h e advancement o f some t h e o r i e s as t o i t s
r o l e in t h e r i p e n i n g p r o c e s s .
The r o l e played by b a c t e r i a in t h e ferm­
e n t a t i o n of chees e has been s t u d i e d p r i m a r i l y from t h e s t a n d p o i n t of
b a c t e r i a l p o p u l a t i o n s a t v a r i o u s s t a g e s o f r i p e n i n g and t h e r e s u l t i n g
pH changes.
These f a c t o r s have been e v a l u a t e d on t h e b a s i s o f o rg a hol-
e p t i c d e t e r m i n a t i o n s of f l a v o r and body and t e x t u r e .
Body changes have
a l s o been measured by such chemical methods as d e t e r m i n a t i o n s of water
s o l u b l e n i t r o g e n , p r o t e a s e and p e p t i d e c o n t e n t s .
B a c t e r i a l enzymes have not been s t u d i e d e x t e n s i v e l y but have been
shown t o be i m p o r t a n t .
Both e x t r a c e l l u l a r enzymes s e c r e t e d i n t o th e
su rro und in g medium and i n t r a c e l l u l a r enzymes r e t a i n e d , in t h e c e l l
a p p a r e n t l y pla y some p a r t .
E x t r a c e l l u l a r enzymes have been shown to
be co nnected p r i m a r i l y with p r o t e i n breakdown.
/■";<i
Only a few i n t r a c e l l u l a r enzyme s t u d i e s have been found in the
l i t e r a t u r e revie w ed .
These s t u d i e s have been devoted c h i e f l y t o the
c h a r a c t e r i z a t i o n of p r o t e i n s p l i t t i n g enzymes and t o some o f t h e f a c t o r s
—2—
such as pH, te m p e r a t u r e and metal ion s a f f e c t i n g t h e i r r e a c t i o n .
In as much as in any f e r m e n t a t i o n t h e r e i s a c o n s t a n t tu r n o v e r
of microorganisms i . e . some a r e dead w hil e o t h e r s are a c t i v e v e g e t a t i v e
c e l l s , t h e r o l e of enzymes remaining in th e b a c t e r i a l c e l l s a f t e r they
have become i n a c t i v e i s of i n t e r e s t and pro bably im p o r ta n t i n most
f e r m e n t a t i o n , e s p e c i a l l y cheese r i p e n i n g .
The purpose o f t h i s study was t o i n v e s t i g a t e t h e i n t r a c e l l u l a r
p r o t e i n a s e s of some b a c t e r i a commonly found in milk o r s t a r t e r as well
as some common milk c o n ta m in a n ts .
The organisms s e l e c t e d which are
commonly found i n milk or s t a r t e r were ( I ) S t r e p t o c o c c u s l a c t i s . (2)
Leuconostoc dextranicum or (3) L a c t o b a c i l l u s c a s e i .
The common milk
con tamin ants s e l e c t e d were ( I ) P r o t e u s v u l g a r i s . (2) B a c i l l u s s u b t i l i s .
(3) Pseudomonas f l u o r e s c e n s and (4) Staphylococcus au r e u s .
The e f f e c t of pH of t h e s u b s t r a t e ; age of th e b a c t e r i a l c e l l ;
and composition of t h e medium used t o pro pag at e th e c e l l s on p r o t e i n a s e
p ro duc tio n a n d /o r a c t i v i t y were s t u d i e d .
REVIEW OF LITERATURE
Enzymes
Enzymes a r e p r o t e i n ? whose b i o l o g i c a l f u n c t i o n i s t h e c a t a l y s i s o f
chemical r e a c t i o n s in l i v j n g systems (34)«
E a rl y i n v e s t i g a t i o n s of en­
zymes go back t o t h e work of Buchner as c i t e d by Neilands and Stumpf (75)
who i n . 1897 o b ta in e d a c e l l f r e e p r e p a r a t i o n of y e a s t c ap a ble o f f e r -
-
j.
menting s u g a r s ,
The work of Harden and Young who demonstrated t h e r o l e
;
.
o f a he a t s t a b l e f a c t o r or doenzyme in c e r t a i n r e a c t i o n s was a noth er im.
•
-
:
.
/
'
p o r t a n t c o n t r i b u t i o n (41)„
The monograph by Harden i s a good h i s t o r i c a l
document on t h e e a r l y s t u d i e s o f enzymes ( 4 0 ) .
<
Enzymes were f i r s t c r y s t a l l i z e d in 1926 (90) and by 1956 some 75
enzymes had been so pre p a re d and t h e i r p r o p e r t i e s s t u d i e d ( 3 0 ) ? All e n -' c
:'
'y
I'
'■
.
zymes i s o l a t e d t o elate have been i d e n t i f i e d as p r o t e i n s . The monograph
• ' C' , "• ‘
1
’
o f Northop e t a l . ( 7 7 ; may be of i n t e r e s t .
Hoffmann (4 9) and Hpffmann and Thomas (50) have reviewed t h e l i t e r ■
.
■■
's
;.
a t u r e on nomenclature and c l a s s i f i c a t i o n o f enzymes. Lamanna and M a l l e t t e
•
-
•
-,.>I
C .:
.
'
“
(6 1 ) s ugg es te d among o t h e r t h i n g s a c l a s s i f i c a t i o n a c c o r d i n g ? t o ( a ) the
s i t e o f a c t i v i t y , and ( b ) th e c o n d i t i o n s governing t h e occur renc e of
1 -
"
't
enzymes, both of which a re of in terest in the present Stpdy.
•
‘
A
1
!
1
R e l a t i v e t o t h e s i t e of a c t i v i t y some enzymes may be l i m i t e d t o t h e
c o n f i n e s o f t h e c e l l and e r e c a l l e d i n t r a c e l l u l a r ^
Others a r e found on *
t h e s u r f a c e o f t h e c e l l apd a re c a l l e d e c t o c e l l u l g r , w hil e th o s e s e c r e t e d
in t h e medium s u p p o rt in g growth a re known as e x t r a c e l l u l a r .
Most orga n­
isms e x h i b i t a l l o f t h e s e t y p e s .
I n t r a c e l l u l a r enzymes and th e i n f l u e n c e of medium on enzyme produc-
-4-
a r e of p a r t i c u l a r i n t e r e s t h e r e .
Lamanna and M al le te (61) p o in t out t h a t t h e s p e c i f i c c a t a l y t i c
f u n c t i o n s o f i n t r a c e l l u l a r enzymes a re e x t e n s i v e and t h a t t h e s e enzymes
a r e r e s p o n s i b l e f o r t h e complete breakdown of t h e m a t e r i a l i n t o forms
t h a t can be u t i l i z e d by t h e c e l l s .
These s u bst a nc es a re g e n e r a l l y f i r s t
broken down by t h e e x t r a c e l l u l a r enzymes in ord e r t h a t th e y may be t r a n s ­
p o r t e d through t h e c e l l w a ll by permeases.
R e l a t i v e t o c o n d i t i o n s governing t h e occurrence o f t h e enzymes, t h e
r o l e o f a s u b s t r a t e of s t r u c t u r a l l y analogous compounds as in du c er s or
s t i m u l a t o r s f o r enzymes i s r e c og niz e d ( 7 8 ) .
The s y n t h e s i s o f a l l en ­
zymes i s g e n e t i c a l l y determined but a give n enzyme can be c o n s t i t u t i v e
in one s t r a i n o f organisms and induced in a n o t h e r .
The chemical en­
vironment of t h e p o p u l a t i o n deter mine s which enzymatic r e a c t i o n s w i l l
o c cu r , bu t only w i t h i n t h e l i m i t s s e t by t h e g e n e t i c competence of t h e
population.
S t a n i e r ( 8 9 ) , Gale (36) and Spiegelman ( 88) have reviewed
t h e l i t e r a t u r e on ad ap ti o n by b a c t e r i a .
T h e i r work and t h e symposium
on a d a p t a t i o n in microorganisms (2 6 ) may be c o n s u lt e d f o r d e t a i l s .
P r o t e o l y t i c enzymes and t h e fe r m e n t a t i o n of cheese
The enzymes which hydro lyz e p r o t e i n s were among t h e f i r s t b i o ­
lo g ical c a t a l y s t s discovered (7 0 ).
They have a l s o co n ti n u e d t o be
prominent in t h e study o f enzyme s t r u c t u r e , k i n e t i c s , a c t i v i t y and
mechanism o f enzyme a c t i o n ( 7 0 ) .
The term p r o t e a s e i s u s u a l l y employed
I
as a g e n e r a l d e s i g n a t i o n of enzymes c ap a ble of hy dro ly z in g p e p t i d e l i n k ­
a g es .
Johnson and Berger (53) p o i n t out t h a t two kinds of p r o t e a s e s e x i s t :
-5-
(1)
P e p t i d e s which a re capab le of s p l i t t i n g o f f a s i n g l e amino a cid from
one end or t h e o t h e r of t h e p e p t i d e c h a i n , and, ( 2 ) p r o t e i n a s e s which may
s p l i t a li n k a g e a t any p o s i t i o n in th e chai n y i e l d i n g p o l y p e p t i d e s .
This
phenomenon may i n f l u e n c e t h e measurement of p r o t e o l y t i c a c t i v i t y by some
methods.
For example, th e F o l i n - C i o c a l t e u method measures only th e two
amino a c i d s t y r o s i n e and t r y p t o p h a n e .
I t does not measure o t h e r p r o ­
d u c ts such as p o l y p e p t i d e s or o t h e r amino a c i d s .
F ig u r e I i l l u s t r a t e s
t h i s phenomenon.
3
O
O
R O
NH2-CH-C -NH-CH-C- NH-CH-C*
• -NH-CH-C-
R O
F ig u r e I .
R
R
4
R O
P e p ti d e chai n showing N t e r m i n a l li n k a g e and C te rm in a l
li n k a g e and t h e p o i n t s of a t t a c k by p r o t e o l y t i c enzymes.
F ig u r e I shows a p e p t i d e chain with N te rm in a l li n k a g e ( I ) and C t e r ­
minal li n k a g e ( 4 ) which a r e hydrolyzed by a p p r o p r i a t e p e p t i d a s e s and add­
i t i o n a l p e p t i d e bonds (2 and 3) which a re hydrolyzed only by p r o t e i n a s e s .
Bergman (1 6 ) and Bergman and Fruton (1 7 ) have pu blis he d d e t a i l e d accounts
of t h e c l a s s i f i c a t i o n and s p e c i f i c i t y of p r o t e o l y t i c enzymes.
Cheese r i p e n i n g i s a complex pro c e ss which has never been f u l l y ex­
p l a i n e d , a l t h o u g h , i t has been q u i t e e x t e n s i v e l y s t u d i e d by chemists and
bacteriologists.
During cheese r i p e n i n g a number o f changes t a k e p la c e and a l l major
c o n s t i t u e n t s of milk i . e . p r o t e i n , f a t and l a c t o s e undergo some changes
(9).
All t h e s e changes a r e r e s p o n s i b l e in some degree a t l e a s t f o r t h e
-6-
c h a r a c t e r i s t i c body and f l a v o r of c h ee s e.
The chemical changes r e s p o n s i b l e f o r t r a n s f o r m i n g t h e f r e s h curd
i n t o t h e f i n a l cheese a r e c a t a l y z e d by enzymes from t h r e e main sources
C33>;
( a ) r e n n e t or o t h e r enzyme p r e p a r a t i o n s , ( b ) microorganisms t h a t
grow in or on t h e s u r f a c e of ch ee se , and Ce) t h e milk i t s e l f .
Rennet i s being c o n s id e r e d in t h i s review because i t p la y s a p a r t in
t h e p r o t e i n breakdown and f l a v o r for mation of ch ee se .
A ls o, some o f i t s
p r o p e r t i e s a re common t o b a c t e r i a l enzymes and th e a c t i o n o f r e n n e t and
b a c t e r i a a r e r e l a t e d in t h a t t h e c o n d i t i o n s c r e a t e d by b a c t e r i a l growth,
p a r t i c u l a r l y pH, i n f l u e n c e t h e a c t i o n of r e n n e t ( 5 , 1 0 ) .
Als o i t i s pos ­
s i b l e , althoug h i t has not been de m onst rat ed, t h a t r e n n e t may serve some­
what t h e same f u n c t i o n as e x t r a c e l l u l a r enzymes of b a c t e r i a .
I t has
been shown c o n c l u s i v e l y t h a t re n n in i s a p r o t e a s e ( 1 8 , 8 0 ) and v a ri o u s
a u th o r s ( 1 , 1 0 , 9 5 ) have noted t h a t i n c r e a s i n g amounts Of r e n n e t cause an
i n c r e a s e in s o l u b l e n i t r o g e n o u s pro d u c ts produced.
The s u r v i v a l o f t h e p r o t e a s e o f r e n n e t during h e a t i n g in milk has
been s t u d i e d by P e l o t o l a and A u t i l a ( 8 0 ) .
Amunstad ( 5 ) concluded t h a t
t h e p r o t e a s e s of r e n n e t a r e a b le t o a c t a t a lower pH than th o s e of
b a c t e r i a e s p e c i a l l y in t h e p resen ce o f sodium c h l o r i d e and t h e r e f o r e
t h e y c o n t i n u e p r o t e o l y s i s a f t e r t h e b a c t e r i a l enzymes have ceased fu nc ­
tioning.
,The reviews by B e r r id g e ( 1 8 ) , Aschaffenburg and Ling (7) and
A sc ha ffe nbu rg and Rowland ( 8 ) should be c o n s u l t e d f o r d e t a i l e d t r e a t ­
ment of t h e s u b j e c t of t h e r e l a t i o n s h i p of r e n n e t t o cheese making.
Role of b a c t e r i a in t h e r i p e n i n g of cheese
S t r e p t o c o c c u s l a c t i s i s th e f i r s t s p e c i e s to b r i n g about an impor tant
-7-
change d ur in g t h e making and r i p e n i n g of cheese ( 5 5 , 9 6 ) .
L a c t i c ac id
th u s produced i s im po r ta nt both in t h e manufacture and t h e r i p e n i n g of
cheese.
I t makes c o n d i t i o n s f a v o r a b l e f o r t h e c u r d l i n g of milk with
r e n n e t ; t h e ex pl o si o n of whey from t h e curd and t h e f u s i o n of t h e curd
,/
p a r t i c l e s ( 3 7 ) . Ha sti ng _et _al. (4 8 ) have shown t h a t t h e organisms a re
c o n c e n t r a t e d in t h e curd a f t e r t h e c o a g u l a t i o n of t h e m il k .
Milroy (71)
' p o in te d out t h a t a c id p ro d u c ti o n a l s o f a v o r s t h e p r o t e o l y t i c a c t i o n of
rennet e x tra c t.
The r e s u l t s o f e a r l i e r workers ( 4 8 , 9 5 ) show t h a t from t h e s t a n d ­
p o i n t of numbers
l a c t i s i s im port ant in t h e e a r l y r i p e n i n g pe ri o d and
l a c t o b a c i l l i a r e dominant l a t e r .
However, r e c e n t, s t u d i e s by Engl ish
workers us in g s p e c i a l media have shown t h a t l a c t o b a c i l l i grow slowly but
s t e a d i l y from t h e be gi nn in g of r i p e n i n g p e r i o d . ( 6 5 , 7 3 , 8 5 ) .
A lle n and
Knowles ( 4 ) and Larte and Hammer (6 2) found t h a t L a c t o b a c i l l u s c a s e i
added t o p a s t u r i z e d milk made i n t o cheese produced more r a p i d and e x t e n ­
s i v e decomposition of p r o t e i n than in cheese which was, th e y claim,
devoid o f l a c t o b a c i l l i .
The l a c t o b a c i l l u s f l o r a o f Cheddar cheese i s not u s u a l l y added t o
t h e milk but a p p a r e n t l y i s d e ri v e d from milk or may a r i s e as a contami­
nant from a i r and o t h e r so urc es ( 7 4 , 8 1 ) .
L i t t l e i s known of t h e p a r t
played by 2L l a c t i s in ihaking c o n d i t i o n s fa v o r a b l e f o r t h e growth of
l a c t o b a c i l l i , however, a s s o c i a t i v e growth o f t h e s e b a c t e r i a has been
de mon strated ( 3 1 ) .
Marshall (69) showed t h e importance o f b a c t e r i a l
a s s o c i a t i o n in t h e so urin g of raw milk as e a r l y as 1903 and found t h a t
many b a c t e r i a commonly, o c c u r r i n g in milk s t i m u l a t e d t h e growth and
-8-
f e rm e nt in g a c t i v i t y of l a c t i c a c id b a c t e r i a .
Nurmikko (76) showed t h a t
d i f f e r e n t s t r a i n s of l a c t i c a c i d b a c t e r i a could grow in symbiosis in a
s y n t h e t i c medium in c a p a b le o f s u p p o rt in g t h e growth of e i t h e r s t r a i n in
pure c u l t u r e .
Dahiya and Speck (23) have more r e c e n t l y s t u d i e d t h e sym­
b i o s i s among l a c t i c s t r e p t o c o c c i .
They found t h a t a combination of
i s o l a t e s from mixed s t r a i n s of l a c t i c s t r e p t o c o c c i showed d i f f e r e n t i n t e r ­
a c t i o n when grown on m i l k .
The i n t e r a c t i o n between t h e s t r a i n s could not
be p r e d i c t e d from t h e growth r a t e o f t h e i n d i v i d u a l s i s o l a t e d .
Hansen
(3 8) showed t h a t t h e s t i m u l a t i o n of Lo_ c a s e i by SL l a c t i s or
cremoris
may pl a y a r o l e in t h e r i p e n i n g of cheese in which t h e two l a t t e r s p e c i e s
de ve lo p, d i e , d i s i n t e g r a t e and a r e followed by L. c a s e i .
The knowledge r e g a r d i n g t h e i n t r a c e l l u l a r enzymes o f t h e b a c t e r i a
concerned with cheese r i p e n i n g i s not e x t e n s i v e , though i t has been
p o in te d out t h a t th e y a r e impor tan t in cheese r i p e n i n g and f l a v o r d e v e l ­
opment ( 9 , 3 3 , 3 7 ) .
Van Der Zant and Nelson (93) r e p o r t e d t h a t a c e l l f r e e c u l t u r e med­
ium of SL l a c t i s did not show p r o t e o l y t i c a c t i v i t y , however t h e i n t r a ­
c e l l u l a r enzymes of SL l a c t i s showed a r a p i d i n c r e a s e in both s o l u b l e
n i t r o g e n and t y r o s i n e and t r y p t o p h a n e when a ct e d on c a s e i n and l a c t a l bumin.
In a n o th e r stu dy t h e same a u th o r s ( 9 4 ) have shown t h e presence
o f a h e a t l a b i l e p r o t e o l y t i c enzyme in t h e c e l l f r e e e x t r a c t s of SL l a c t i s .
They a l s o r e p o r t e d t h a t optimum a c t i v i t y a g a i n s t m ilk , c a s e i n and I a c t albumin i s near n e u t r a l i t y .
Rabin (8 4) has s t u d i e d some o f t h e f a c t o r s i n f l u e n c i n g t h e formation
of p r o t e i n a s e s in SL l i q u e f a c i e n s , such as pH, e s s e n t i a l amino a c id s and
-9-
t h e need f o r a water s o l u b l e s y n t h e t i c medium.
Bar ibo and F o s t e r ( 1 4 ) have p u b li s h e d some of t h e c h a r a c t e r i s t i c s
o f t h e i n t r a c e l l u l a r p r o t e i n a s e s o f one s t r a i n each o f
and Micrococcus f r e u d e n r e i c h i i .
c a s e i , S. I a c t i s 0
The m i c r o b i a l enzymes were compared
with t h o s e of p r o t e o l y t i c enzymes e x t r a c t e d from one ye ar old ch ee se .
They concluded t h a t t h e organisms used in t h e i r study pos se ss e d enzymes
which co uld account f o r only a p a r t o f p r o t e i n a s e s found in cheese and
t h a t enzymes from o t h e r so urc es a re n e c e s s a r y t o account f o r t h e re m a in der .
K r i s t o f f e r s o n and Cole (59) r e p o r t e d t h a t p a s t u r i z a t i o n i n a c t i v a t e s
t h e milk enzymes and d e la y s cheese r i p e n i n g and f l a v o r development.
Atte mpt s t o a c c e l e r a t e cheese r i p e n i n g
Various a tt e m p ts have been made t o reduce t h e r i p e n i n g pe ri o d f o r
c h ee s e.
Hansen (39) noted t h a t t h e a d d i t i o n of 0.05% of a milk c u l t u r e of
S. I i q u e f a c i e n s or 0.5% o f a milk c u l t u r e o f an u n i d e n t i f i e d M ic ro co c c us ,
t o p a s t e u r i z e d milk improved t h e f l a v o r o f cheese made f r o m ' i t , but i t
d i d , n o t m a t e r i a l l y i n f l u e n c e t h e n it r o g e n o u s decom pos iti on.
H a r r i s and
Hammer ( 4 6 ) found t h a t out o f 34 Micrococcus c u l t u r e s , seven had an un­
d e s i r a b l e e f f e c t , 14 no e f f e c t and 13 had a d e s i r a b l e e f f e c t when in o c ­
u l a t e d i n t o p a s t e u r i z e d milk f o r cheese making.
Dahlberg and Kosikowski (24) i s o l a t e d a s t r a i n o f
faecalis that
fermented l a c t o s e r a p i d l y and th e y advocated i t s use as a s t a r t e r organ­
ism.
Another i n v e s t i g a t i o n (27) in which Su l i q u e f a c i e n s was used r e ­
s u l t e d in pronounced b i t t e r n e s s in c h e e s e .
The use o f s p e c i a l p r o t e o l y t i c enzymes ( p e p s i n , t r y p s i n ) in cheese
-10—
manufacture and t h e i r e f f e c t from t h e s t a n d p o i n t o f a c c e l e r a t e d r i p e n i n g
has been s t u d i e d by Freeman and Dahle ( 3 5 ) .
Babel J r t a l . , (12) used an
enzyme p r e p a r a t i o n from chicken p r o v e n t r i c u l i t o c o a g u l a t e milk and used
a m odif ied Edam p ro c e ss in t h e making of a new v a r i e t y of cheese ( S a v o u r e u x ) .
K r i s t o f f e r s e n has p o i n t e d out in a r e c e n t symposium on flavor^chem­
i s t r y C58) t h a t t h e work done so f a r would i n d i c a t e t h a t we can only
s h o r te n t h e r i p e n i n g p e r i o d a t t h e c o s t of f l a v o r .
C o n t r i b u t i o n o f b a c t e r i a t o t h e development of f l a v o r
Mabbitt (6 4 ) p o i n t s out t h a t t h e blown m e ta bolic pro d u c ts of d i f f e r e n t
s p e c i e s o f homofermehtive l a c t i c a c id b a c t e r i a which occur in cheese such
as s t r e p t o c o c c i , l a c t o b a c i l l i and pediotiocci are so q u a l i t a t i v e l y s i m i l a r
t h a t i t i s d i f f i c u l t on t h i s b a s i s t o suppose t h a t one s p e c i e s or s t r a i n
would be more impo rtan t t h a n t h e o t h e r .
Small d i f f e r e n c e s in minute
amounts of o d o r i f e r o u s or f l a v o r f u l s u b s ta n c e s such as d i a c e t y l which
a re produced can be i m p o r t a n t .
Most of t h e work on s t r e p t o c o c c i has been done on t h e p ro du c tio n of
acid.
The c h i e f i n t e r e s t s have been, t h e s e l e c t i o n of s u i t a b l e s t r a i n s
f o r a c i d p ro d u c ti o n ( 1 1 ) , c o n t r o l of phage i n f e c t i o n ( 4 2 , 1 0 1 ) , and o th e r
cau se s o f slowness ( 1 1 ) .
flavor.
Less work has been done on t h e i r r e l a t i o n t o
Perhaps t h e main c o n t r i b u t i o n o f s t a r t e r organism t o f l a v o r pro ­
d u c ti o n i s i n d i r e c t and a t t e n t i o n has been focused on t h e p r o t e o l y t i c
enzymes o f c e l l s ( 6 , 1 0 3 ) .
Importance o f l a c t o b a c i l l i t o f l a v o r has been
r e p o r t e d by Naylor and Sharpe ( 7 3 ) , Mabbitt and Z i e l i n s k a (67) and Johns
and Cole ( 5 2 ) .
Dacre ( 2 1 , 2 2 ) has r e p o r t e d on t h e pre s en c e o f pe diococci
in New Zealand cheese and has shown i t s p o s s i b l e r e l a t i o n t o f l a v o r ( 2 1 , 2 2 ) .
-11-
A l f o r d and F r a z i e r ( 2 , 3 ) have shown t h e r e l a t i o n s h i p of micrococci t o
flavor.
Occurrence o f Staphylococcus aureus in raw milk cheese has a l s o
^een r e p o r t e d , but i t s r e l a t i o n t o f l a v o r i s not e v id e n t C9 1 , 9 2 ) .
Though most of t h e o b s e r v a t i o n s le a d one t o b e l i e v e t h a t b a c t e r i a
a re im po r ta nt in f l a v o r , r e p o r t s t o t h e c o n t r a r y have been publ is h e d
(64,66).
I n d i v i d u a l s t u d i e s have been conducted t o determine t h e r e l a t i o n ­
sh ip between p r o t e o l y s i s and f l a v o r ( 1 3 , 2 4 , 4 3 , 4 4 , 4 7 , 5 6 , 8 7 ) , l i p o l y s i s
and f l a v o r ( 1 5 , 8 2 , 5 1 ) and carbonyl compounds and f l a v o r ( 2 8 , 2 9 , 4 5 , 6 0 ,
97,98).
Mulder, a Dutch i n v e s t i g a t o r (72) has proposed a component ba lan ce
th e o r y o f chees e f l a v o r , which in s h o r t s t a t e s t h d t c h a r a c t e r i s t i c cheese
f l a v o r i s not r e l a t e d t o a s i n g l e compound but^ m ix tu re s o f compounds
coming from t h e d e g r a d a t i o n of f a t , p r o t e i n and l a c t o s e ,
Silverman and
Kosikowski ( 86) , have confirmed t h e above view.,.
Keeny and Day (5 4) have evolved a n o th er h y p o th e s i s of cheese f l a v o r .
formation.
'
They su ggest t h a t a slow chemical . i n t e r a c t i o n over a long
"
.
*
p e r io d o f r i p e n i n g between amino a c i d s and dicarboxy compounds could
l e a d t o t h e p ro d u c ti o n of f l a v o r f u l a l d e h y d e s .
T h e i r t r i a l s of v a r io u s
compounds have i n d i c a t e d t h a t methional has a cheese l i k e f l a v o r .
Thi s
compound can p o s s i b l y be d e ri v e d as follows?,
CH3SCH2CH2CHOOH +
CH3COCOOH ------- > CHjjSCH2CH2CHO +
CH3CHGOOH +
Pyruvic a c id
NH2
Alanirid
^2
Methionine
Methional
CO2
-12-
W i t t i n g and B a t z a r (102) however, have d i s p u t e d t h e c h e e s i n e s s of
methional while Oro e t a_l, (7 9) have r e a s s e r t e d t h e importance of t h i s
compound.
Jackson and Morgan ( 5 1 ) have found 3 methyl
cheese and th ought t h a t i t c o n t r i b u t e d t o f l a v o r .
bu tu na l in Cheddar
I t i s not c e r t a i n by
which mechanism t h e s e aldehydes are formed though McCleod and Morgan ( 68)
r e p o r t t h e i r pro d u c ti o n from amino a c i d s by c e r t a i n s t r a i n s o f S l l a c t i s .
Kosikowski and Mocquot (57) s t a t e t h a t i t should be noted t h a t a l l
t h e evidenc e now s u g g e s ts t h a t t y p i c a l f l a v o r of cheese i s due t o complex
m ix tu r e s o f components and su cce ss of t a s t i n g t r i a l s w i l l depend on th e
c a r e give n t o o b t a i n t h e c o r r e c t component b a la n c e .
Problem and Purpose
In r e c e n t y e a r s t h e r e has been a renewed i n t e r e s t in r a p i d Cheddar
cheese p r o c e s s i n g , both d urin g man ufacturin g and r i p e n i n g ( 5 9 , 9 9 ) .
Thi s
has led t o t h e use of h i g h e r t e m p e r a t u r e s f o r cooking t h e curd and has
encouraged t h e use o f he a t t o l e r a n t e n t e r o c o c c i . S tr e p t o c o c c u s f a e c a l i s
as . s t a r t e r organisms ( 2 4 , 2 7 ) .
Higher c u r i n g t e m p e ra t u re s as a means of
d e c r e a s i n g t h e r i p e n i n g p e r i o d have a l s o been i n v e s t i g a t e d ( 4 , 2 7 ) .
In
most of t h e s t u d i e s t h e n a t u r a l f l a v o r of t h e cheese co uld not be pro­
duced however, and th u s t h e r e i s s t i l l a g r e a t deal of i n t e r e s t in r e ­
ducing t h e d u r a t i o n of t h e r i p e n i n g p e r i o d witho ut a f f e c t i n g t h e f l a v o r .
The purpose of t h i s study was t o e x p l o r e ways in which t h e i n t r a ­
c e l l u l a r p r o t e i n a s e s o f t h e s t a r t e r organism could be i n c r e a s e d .
If
s u c c e s s f u l , t h i s should i n c r e a s e t h e r a t e o f p r o t e i n breakdown and de ­
crease the curing time.
With t h i s end in view i t was proposed t o i n -
-In­
v e s t i g a t e t h e fo ll o w i n g f a c t o r s which might e f f e c t t h e pro d u c ti o n of
i n t r a c e l l u l a r p r o t e i n a s e s , ( a ) t h e age o f t h e c e l l „ (b ) t h e presence
of c a s e i n , g e l a t i n and C a s i t o n e in t h e growth medium and ( c ) t h e absence
of fe rm e n ta b le c a r b o h y d ra t e from t h e growth medium.
I t was a l s o pro ­
posed t o deter mine t h e optimum pH f o r t h e enzyme s u b s t r a t e r e a c t i o n s .
Some common milk con taminan ts were inc lu de d in th e study in ord er to
o b t a i n some b a s i c i n fo r m a t io n about t h e i n t r a c e l l u l a r enzymes o f t h e s e
bacteria.
EXPERIMENTAL PROCEDURE AND RESULTS
Seven c u l t u r e s o f d i f f e r e n t organisms were s e l e c t e d f o r t h i s work.
The s e l e c t i o n was based on t h e p h y s i o l o g i c a l c h a r a c t e r i s t i c s o f some
organisms commonly p r e s e n t in milk and of some common milk c o n t i m i n a n t s .
1) .
Organisms s e l e c t e d which commonly occur in milk or s t a r t e r s
were:
2) .
a.
S t r e p t o c o c c u s l a c t i s (BBlO)
bo
Leuconostoc de x tr in ic u m ( NDRL B-640)
c.
L a c t o b a c i l l u s c a s e i (U. of Indiana No. 14696)
Organisms s e l e c t e d which commonly occur in milk or milk
p ro d u c ts as co ntaminants were:
a.
P r o te u s v u l g a r i s (BB151)
b.
B a c i l l u s s u b t i l i s (ATCC 6093)
c.
Pseudomonas f l u o r e s c e n s (U. of In dia na )
d.
Sta phylococcus aureus ( BBI 37)
The c u l t u r a l c h a r a c t e r i s t i c s of above organisms have been summarized
in Table XVIII o f t h e appendix.
Growth o f c e l l s :
The c u l t u r e s were ta k e n from agar s l a n t s and were 12
hours old when i n o c u l a t e d i n t o 1000 ml o f l i q u i d medium I or I I where
they were in c uba te d a t 37 C f o r 24 h o u r s .
Medium I d i f f e r e d from med­
ium I I in t h a t t h e former had a pap aic d i g e s t o f soymeal while the
/
l a t t e r contained beef e x t r a c t .
The complete compositions of media I and
I I a r e gi ve n in Table XIX in t h e Appendix.
The B a c i l l u s s u b t i l i s c u l t u r e s
were shaken c o n ti n u o u s l y t o provid e b e t t e r a er obic c o n d i t i o n s fo r growth.
-15-
The c e l l s were h a r v e s t e d a t t h e end o f t h e i n c u b a t i o n p e r io d by
c e n t r i f u g i n g t h e c u l t u r e a t -2 C in a S e rv a l r e f r i g e r a t e d c e n t r i f u g e ,
r e v o l v i n g a t 4,000 rpm.
The mass of c e l l s so obta ine d was washed 4 or
more time s with s t e r i l e normal s a l i n e (0,9% N a d ) t o remove t h e e x t r a ­
c e l l u l a r enzymes.
The washed c e l l s were then suspended in 3 ml of
s t e r i l e normal s a l i n e s o l u t i o n and t h e suspension s t o r e d in t h e r e f r i g ­
e r a t o r f o r not more th a n 24 hours .
P r i o r t o u s e, t h e s a l i n e was de­
c a n te d , le a v i n g t h e packed c e l l s in t h e bottom of t h e t u b e .
E x t r a c t i o n of i n t r a c e l l u l a r and e c t o c e l l u l a r enzymes
The i n t r a c e l l u l a r and e c t o c e l l u l a r enzymes were e x t r a c t e d by e i t h e r
t h e t o l u e n e method or t h e aceton e dry powder method.
Toluene method;
Each c u l t u r e used h e re was grown on medium I or
I I as i n d i c a t e d above and t h e c e l l s were h a r v e s t e d and washed in the
1 :
manner p r e v i o u s l y d e s c r i b e d .
To 1.0 g o f packed c e l l s I ml o f to lu e n e
was a d d e d . f o r t h e purpose of a u t o l y z i n g t h e c e l l s .
a preservative.
I t a l s o served as
The mi xtu re was then fr o ze n f o r subsequent work.
!A c et o n e ;d ry powder method;
The enzymes e x t r a c t e d by t h i s method
were made from c e l l s grown on medium I only and were h a r v e s t e d as
i n d i c a t e d above.
One gram of packed b a c t e r i a l c e l l s was suspended in
3 ml o f normal s a l i n e ( s t e r i l e ) and mixed u n t i l a homogenous mixture
was o b t a i n e d .
Thi s mi xtu re was then added drop-wise t o 100 ml of co ld
(5 to 0 C) aceton e with v ig or ous s t i r r i n g .
A f t e r t h e e n t i r e mixture
has been added, t h e s t i r r i n g was stopped and t h e suspension allowed t o
s e t t l e f o r 15 m in u te s .
The s u p e r n a t a n t ( a c e t o n e ) was then decanted, and
-16-
t h e remainder f i l t e r e d through a Whatman #50 f i l t e r usi ng a Buchner
funnel under s u c t i o n t o enhance t h e p r o c e s s .
The e l u a t e was washed with
20 ml o f cold aceton e (5 t o 0 C).
The c e l l s as o b ta in e d above were then a s e p t i c a l l y t r a n s f e r r e d t o a
s t e r i l e watch g l a s s and d r i e d in a d e s s i c a t o r under vacuum f o r 36 h o u r s .
The d r i e d c e l l s were ground in a s t e r i l e m o rt ar and p e s t l e and t h e pow­
de r o b ta in e d suspended in 3 ml s t e r i l e normal s a l i n e .
The mixtu re was
then fr o ze n t o p r e s e r v e i t f o r f u r t h e r e xperim ent al work.
P r e p a r a t i o n and s e l e c t i o n of t h e s u b s t r a t e
Both s o l i d and l i q u i d s u b s t r a t e s were t r i e d f o r t h e purpose of
measuring p r o t e o l y t i c a c t i v i t y o f t h e enzyme mixtures?
Solid substrate?
1.
2.5% c a s e i n a g a r .
2.
Bacto stap hylo co c cu s medium No. HO.
3.
Skim milk a g a r .
4.
a.
5% skim mjlk a g a r ,
b.
2% skim milk a g a r .
D e t a i l s of t h e s e media are given in Table 5CX in t h e A p p e n d ix
The enzyme m ix tu r es p r e v i o u s l y d e s c r i b e d were thawed and a p p li e d t o
t h e 5 ml of s o l i d s u b s t r a t e in a 100 mm p e t e r i dish in t h e form of a
s t r e a k on i t s s u r f a c e .
The p l a t e s were inc uba te d f o r 24, 48, and 96
hours a t 37 C and then were examined f o r a c l e a r zone around t h e s t r e a k
as evidenc e of p r o t e o l y s i s .
None o f t h e above f o u r s u b s t r a t e s showed any evidence o f p r o t e o -
-17-
l y t i c a c t i v i t y and t h u s t h e s o l i d s u b s t r a t e s were d i s c a r d e d and l i q u i d
substrates tr ie d .
No e xact e x p l a n a t i o n as t o why t h e above s u b s t r a t e s
di d not work can be given bu t p o s s i b l y i n s u f f i c i e n t d i r e c t c o n t a c t be ­
tween t h e s u b s t r a t e ahd t h e enzyme or t h e a d s o r p t i o n of enzymes t o agar
might be t h e c a u s e .
Liquid s u b s t r a t e s :
1.
D i l u t e d skim m il k .
Fresh p a s t e u r i z e d skim milk was
d i l u t e d in t h e r a t i o of 1:20 and 1:50 with d i s t i l l e d w a t e r .
These
c o n c e n t r a t i o n s were a r b i t r a r i l y picked but were w it h in t h e range of
l i g h t t r a n s m i s s i o n used in an al y z in g t h e end p r o d u c t .
Seven m i l l i ­
l i t e r s o f t h i s d i l u t e d milk medium were d i s p e r s e d in screw cap tubes
and s t e r i l i z e d in an a u t o c l a v e f o r 20 minutes a t 15 l b s p r e s s u r e and
120 C.
2.
Casein s o l u t i o n .
Thi s s u b s t r a t e was pre pa red by d i s ­
s o lv in g 1.5 g of c a s e i n (Merck) in d i s t i l l e d w a te r .
Heat and N/10
NaOH were used t o a id in g e t t i n g t h e c a s e i n i n t o s o l u t i o n ; d i s t i l l e d
w ater whs added! tti make t h e volume t o 250 ml.
s o l u t i o n was 9 . 5 .
The i n i t i a l pH of the
Thi s was a d j u s t e d t o pH 7 . 0 by adding 10% t a r t a r i c
acid.
Tw e nt y-f iv e m i l l i t e r s of t h e above s o l u t i o n were d i l ­
ute d t o 100 ml with d i s t i l l e d water which gave a c o n c e n t r a t i o n iof
1.5 mg of c a s e i n per ml.
Thi s s o l u t i o n was dis pensed in 10 ml qua nt­
i t i e s in screw cap tu b e s and au to cl av e d f o r 20 mi nute s.
3.
Whey p r o t e i n s o l u t i o n .
T h i s s o l u t i o n was pre pa re d by
p r e c i p i t a t i n g t h e c a s e i n a t i t s i s o e l e c t r i c p o i n t from p a s t e u r i z e d
-18-
skim milk with l a c t i c a c i d .
The c a s e i n was spun down in a c e n t r i f u g e
and 10 ml o f t h e s u p e r n a t a n t l i q u i d was ta ken up in 90 ml o f d i s t i l l e d
water.
T hi s gave a c o n c e n t r a t i o n o f about 0 . 5 7 mg of whey p r o t e i n in
1.0 ml o f t h e s o l u t i o n .
The f i n a l pH of t h i s s o l u t i o n was a d j u s t e d t o
pH 7 . 0 by t h e a d d i t i o n o f N/20 NaOH.
General proce dur e f o r h a n d li n g s u b s t r a t e s
To a l l t h e above s u b s t r a t e s 0 . 2 ml o f Agromycin* s o l u t i o n was added
f o r ev ery 10 ml p o r t i o n .
0 . 3 3 g of
T hi s a n t i b i o t i c s o l u t i o n was made by suspending
Agromycin in 50 ml o f s t e r i l e phosphate b u f f e r (pH 7.0>.
m ix tu r e was shaken v i g o r o u s l y and allowq<J t o stand f o r 2 h o u r s .
c l e a r s u p e r n a t a n t l i q u i d was then u s e d .
The
The
The Agromycin was added t o d e s ­
t r o y any l i v i n g c e l l s p r e s e n t in t h e enzyme m ix tu r e.
Measurement of p r o t e o l y t i c a c t i v i t y
The fo ll o w i n g methods f o r t h e measurement of p r o t e o l y t i c a c t i v i t y
were t r i e d :
1.
V i s c o s i t y measurement of g e l a t i n .
2.
Formal t i t r a t i o n .
3.
Light a b s o r p t i o n .
4.
F o l i n - C ioc alteuo
Methods (L) and ( 2 ) were dropped a f t e r very p r e l i m i n a r y t r i a l s as
n e i t h e r methods gave r e p r o d u c i b l e r e s u l t s .
(3)
Light a b s o r p t i o n method:
Seven m i l l i l i t e r s o f 1:20 and 1:50
$
Agromycin 100 i s t h e t r a d e name of t h e Chas. P f i z e r Co. product which
has t h e fo ll o w i n g com pos it ion : Streptomycin 15%, O x y t e t r a c y c l i n e 1.5%,
T o t a l i n e r t i n g r e d i e n t s 83.5%.
-19-
d i l u t i o n o f skim milk p r e v i o u s l y d e s c r i b e d were used.
O ne -te nth m i l l i ­
l i t e r o f enzyme mi xtu re was added t o each 7 ml o f t h e s e s u b s t r a t e s and
t h e t u b e s were in c uba te d a t 37 C f o r v a ry in g l e n g t h s of t i m e .
The o p t ­
i c a l d e n s i t y was determined by a Lumentron P h o t o e l e c t r i c Colorimeter*
a t r e g u l a r i n t e r v a l s t o fo ll o w th e p r o t e i n breakdown.
The t u b e s were
i n v e r t e d two times t o mix and suspend t h e e n t i r e c o n t e n t s u nifo rm ly ,
before taking the readings.
I n v e r t i n g of t h e tu be s was a l s o done once
every 12 hours t o expose t h e maximum s u b s t r a t e s u r f a c e .
O p t i c a l d e n s i t y rou gh ly means t h e amount of l i g h t t h a t w i l l be
absorbed by a s o l u t i o n .
As the p r o t e o l y t i c a c t i o n of t h e enzyme mix­
t u r e s p r o g r e s s e d and c a s e i n broke i n t o si m p le r compounds, t h e l i g h t
absorbed by t h e s o l u t i o n d e c r e a s e d .
Thus a f a l l in o p t i c a l d e n s i t y was
a measure o f p r o t e o l y t i c a c t i v i t y , though i t did not g iv e a q u a n t i t a t i v e
measurement.
The method was used however t o e v a l u a t e t h e e f f e c t of
!
media used f b r t h e growth o f t h e org a n is m s ; t h e c o n c e n t r a t i o n of sub­
s t r a t e and methods of p r e p a r i n g th e enzyme m i x t u r e s .
C4)
F o l i n - C i o c a i t e u method;
T his method measures t h e amount of
f r e e t y r o s i n e and tr y p t o p h a n e in a s d l u t i o n .■ Q u a n t i t a t i v e e s t i m a t i o n s
o f t h e s e twb amino pci ds can be made u s in g a s ta nda rd c u r v e .
D e t a i l s of
t h e proce dur e w i l l be d i s c u s s e d l a t e r .
T hi s method was used t o determine t h e e f f e c t of pH of t h e s u b s t r a t e
on t h e enzyme a c t i v i t y , a l s o to determine t h e e f f e c t o f t h e age of th e
b a c t e r i a l c e l l and t h e e f f e c t of modified media on t h e p r o d u c ti o n and /or
.
$
The o p t i c a l d e n s i t y s c a l e in t h i s in s tr u m e n t re ad s from O t o 20.
-20-
a c t i v i t y of t h e i n t r a c e l l u l a r enzymes.
E f f e c t o f medium on p r o t e o l y t i c a c t i v i t y
Table I shows th e d e c r e a s e in o p t i c a l d e n s i t y XlO ( i n c r e a s e in
p r o t e i n breakdown) when t h e enzymes from c e l l s grown on medium I were
e x t r a c t e d from t h e b a c t e r i a l c e l l s by t h e to l u e n e method and r e a c t e d
with skim milk (1 :5 0 d i l u t i o n ) .
Table I
Average d e c r e a s e in o p t i c a l d e n s i t y XlO of milk (1 :5 0 d i l u t i o n ) due to
i n t r a c e l l u l a r enzyme a c t i v i t y . C e l l s grown on medium I .
_____ Enzymes e x t r a c t e d by t o l u e n e . ________________________
Organism
0 hr.
Enzyme s u b s t r a t e r e a c t i o n time
Difference
24 h r . 48 h r . 96 h r . 144 h r . 192 hr . between
0 & 192 h r .
Blank (no
enzyme added)
4 .7
4 .5
4.4
4 .2
4.1
4.2
0.50
I.
S. I a c t i s
5.2
4.8
4 .7
4 .6
4 .5
4 .5
0.70
2.
L. dextranicum 6.4
6. 0
5.9
5 .7
5 .7
5 .7
0 .7 0
3.
P. v u l g a r i s
6. 0
5 .8
5 .6
5 .4
5.2
5.2
0.80
4.
B. s u b t i l i s
5 .6
5.1
4 .7
4 .0
3 .7
3.6
2. 00
5.
P. f l u o r e s c e n s 5 .8
5 .0
4.8
4.2
3 .9
3 .9
1.90
4.4
4 .0
3 .6
3.4
3 .3
1.70
6 = Si aureus
5.0
I t w i l l be n o ti c e d t h a t the i n i t i a l o p t i c a l d e n s i t y i s not th e same
f o r t h e v a r i o u s o rg a nis m s .
This i s p o s s i b l y due t o s l i g h t d i f f e r e n c e s in
t h e p a r t i c l e s i z e s of v a r i o u s enzyme p r e p a r a t i o n s .
I t w i l l a ls o be seen
t h a t t h e blank showed a s l i g h t d e c r e a s e in o p t i c a l d e n s i t y .
This can
p o s s i b l y be a t t r i b u t e d t o minor d e n a t u r a t i o n changes which might be t a k i n g
pl a ce du rin g i n c u b a t i o n .
-21-
These r e s u l t s show th e change in th e blank between O and 192 hours
of r e a c t i o n time t o be 0 . 5 and t h e changes due to enzymes from organisms
1.2 and 3 t o be only s l i g h t l y g r e a t e r than t h e b la nk.
The enzymes from
organisms 4 ,5 and 6 show a c o n s i d e r a b l y g r e a t e r breakdown with d i f f e r ­
ences of 2.0 0 , 1.90 and 1.70 r e s p e c t i v e l y .
Table I I shows th e average d e c r e a s e s in o p t i c a l d e n s i t y which were
o b ta in e d when th e enzymes mixtu res were made from c e l l s grown on medium
I I and e x t r a c t e d by t o l u e n e .
Here again th e p r o t e o l y t i c a c t i v i t y of
enzymes from organisms 4 , 5 and 6 was c o n s i d e r a b l y g r e a t e r than organisms
1.2 and 3.
Table I I
Average decrea se in o p t i c a l d e n s i t y XlO of milk (1 :5 0 d i l u t i o n ) due to
i n t r a c e l l u l a r enzyme a c t i v i t y . Organisms grown on medium
_________I I . Enzymes e x t r a c t e d by t o l u e n e method._________________________
Organism
Enzyme s u b s t r a t e r e a c t i o n time_____________D if f e r e n c e
0 h r . 24 h r . 48 h r . 96 h r . 144 h r . 192 hr,, between
0 & 192 hr
Blank (no
enzyme added)
4.10
3.90
3.80
3.70
3.60
3.60
0.50
I.
S. I a c t i s
4.80
4.50
4.50
4.30
4.20
4.20
0.60
2.
L. dextranicum 4.60
4.50
4.30
4.10
4.00
3.90
0.70
3.
P. v u l g a r i s
4.50
4.30
4.00
3.80
3.60
3.60
0.90
4.
B. s u b t i l i s
4.20
3.50
3.10
2.60
2.30
2.30
1.90
5.
P. f l u o r e s e n s
4.80
3.50
3.60
3.40
3.10
3.10
1.70
6.
So aureus
4.40
3.80
3.40
3.10
2.90
2.80
1.60
On comparing th e v a lu e s in t a b l e I and I I i t can be t e n t a t i v e l y
concluded t h a t t h e enzymes from organ isms grown on medium I had s l i g h t l y
-22-
hi g h e r p r o t e o l y t i c a c t i v i t y than th o s e grown on medium I I as f o r ex­
ample in B. s u b t i l i s t h e d i f f e r e n c e between 0 and 192 hour was 1.90
when grown on medium I I and 2.00 when grown on medium I .
E f f e c t o f s u b s t r a t e c o n c e n t r a t i o n on p r o t e o l y t i c a c t i v i t y
Tabl e I I I shows th e d e c r e a s e in o p t i c a l d e n s i t y on 1:20 d i l u t i o n
of milk.
I t w i l l be n o t i c e d by comparing t h e s e data with Table I t h a t
t h e d i f f e r e n c e between 0 hour and 192 hour a re much g r e a t e r when the
d i l u t i o n was 1:20 as compared t o 1:50 d i l u t i o n .
from 0 . 3 0 in
l a c t i s to 1:40 in
The i n c r e a s e s range
aureus.
Table I I I
Decrease in o p t i c a l d e n s i t y of milk ( 1 :2 0 d i l u t i o n ) as a r e s u l t of i n t r a ­
c e l l u l a r enzyme a c t i v i t y . Organisms grown on medium I .
_________ Enzyme e x t r a c t e d by t o l u e n e . _______________________________________
Organisms
I.
Si l a c t i s
9.0
8.4
8.2
8. 2
8. 1
8.0
1.00
2.
Li dextranicum 8 . 0
8. 2
8.0
8. 0
7 .8
7 .7
1.10
3.
Pi v u l g a r i s
9 .0
8 .5
8.4
8. 1
7 .8
7 .7
1.30
4.
iL s u b t i l i s
9.0
6. 8
6 ,4
6. 0
5 .8
5.7
3.30
5.
Pi f lu o re s c e n s
03
Ul
Blank
Enzyme s u b s t r a t e r e a c t i o n time____________D i ff e re n c e
24 h r . 48 h r . 96 h r . 144 h r . 192 hr . between
0 & 192 hr
8, 1
8 .5
8.2
8.0
8.0
8.0
0.50
0 hr.
7 .8
7 .0
6. 8
6.3
6.1
2.40
6.
Si aureus
8.7
7 .2
6 .4
6. 0
5 .8
5 .6
3.10
Tabl e IV giv e s th e d e cr ea se in o p t i c a l d e n s i t y when t h e enzymes
were r e a c t e d with th e s u b s t r a t e ( 1:20 d i l u t i o n of skim milk) but the c e l l s
in t h i s experiment were grown on medium I I i n s t e a d of medium I .
The r e ­
s u l t s when compared to Table I I I show th e va lu es to be s l i g h t l y lower in
-23-
a l l but one organism.
The val ue fo r
l a c t i s is s l i g h t l y but probably
not s i g n i f i c a n t l y h i g h e r .
From t h e previo us experiments i t can be concluded t h a t growth of t h e
c u l t u r e s on medium I and t h e use of 1:20 d i l u t i o n of skim milk as an
enzyme s u b s t r a t e gave t h e b e s t r e s u l t s .
I t appears t h a t s u b s t r a t e con­
c e n t r a t i o n was a f a c t o r in th e lower v a lu es found when 1:50 d i l u t i o n of
skim milk was used.
Table IV
Decrease in o p t i c a l d e n s i t y XlO of milk ( 1 :2 0 d i l u t i o n ) due to i n t r a ­
c e l l u l a r enzyme a c t i v i t y . Organisms grown on medium
__________I I . Enzymes e x t r a c t e d by t o l u e n e method.______________
Enzyme s u b s t r a t e r e a c t i o n time_____________D if f e r e n c e
() h r . 24 h r . 48 h r . 9<) hr. 144 h r . 192 hr., between
0 & 192 h r .
7 .8
7 .6
7 .6
7 .6
0.4
8.0
8.0
Organism
Blank
I.
S. l a c t i s
8.0
7 .4
7.2
6.9
6 .9
6.9
1.10
2.
L. dextranicum 8 . 5
8.3
8. 0
7.6
7 .6
7.4
1.10
3.
P. v u l q a r i s
8 .4
8.0
7 .6
7 .4
7.2
7.1
1.30
4.
B. s u b t i l i s
8.0
7.2
7 .4
6. 0
5.2
5 .0
3.00
5.
P. f l u o r e s c e n s 8 .0
7 .4
6.0
6.5
6.0
5 .7
2.30
6.
S. aureus
8 .5
7.5
6 .5
6. 1
5 .8
5 .5
3.00
Table V shows the d e c r e a s e in o p t i c a l d e n s i t y when th e enzyme mixt u r e s were prepa red by usi ng acetone dry powder method i n s t e a d of to l u e n e
method.
The r e s u l t s show an i n c r e a s e in th e d i f f e r e n c e in o p t i c a l d e n s i ­
t y between 0 and 192 hours as compared t o Table I I I which can only be
a t t r i b u t e d to t h i s method o f p r e p a r a t i o n of enzymes.
The i n c r e a s e s
-24-
v a r i e d from ( . 1 0 to .30) ex cept fo r S. I a c t i s which gave th e same r e s u l t s .
Table V
Decrease in o p t i c a l d e n s i t y of milk (1 :2 0 d i l u t i o n when t h e enzymes
were e x t r a c t e d by acetone dry powder method. Organ__________isms grown on medium I,___________________________________________
Blank
Enzyme s u b s t r a t e Reaction Time____________D i f f e r e n c e
2<I h r . 48 h r . 96 h r . 144 hr . 192 h r . between
0 & 192 h r .
7
.8
7
.5
7
.5
7
.7
7
.5
0.50
8. 0
I.
S. I a c t i s
0.5
8.0
7 .6
7 .4
7 .4
7 .5
1.00
2.
L. dextranicum 8 . 8
8.3
8. 0
7 .8
7 .7
7 .6
1.20
3.
P. v u l g a r i s
8 .5
7 .8
7 .5
7 .4
7.3
7.1
1.40
4.
B. s u b t i l i s
9 .0
7.1
6 .5
6. 0
5 .7
5 .5
3.50
5.
P. f l u o r e s c e n s 8 .7
7 .8
7.2
6 .5
6. 2
6.1
2.60
6.
S. aureus
8 .5
7 .7
6 .5
6. 0
5.4
5.1
3.40
Organism
0 hr.
Table VI i s a summary of t h e pre vio us four t a b l e s and shows t h a t
1:20 d i l u t i o n o f skim milk as a s u b s t r a t e gave g r e a t e r d i f f e r e n c e s
(column I and 3) than t h e 1:50 d i l u t i o n skim milk s u b s t r a t e (column 2
and 4 ) .
On comparing th e v a lu es in column I with th o s e in column 3 i t
w i l l be seen t h a t medium I gave s l i g h t l y g r e a t e r d i f f e r e n c e s as compared
t o medium I I ( a t l e a s t fo r organisms 4 ,5 and 6) .
The d i f f e r e n c e s in
column 5 a r e c o m par at iv el y hig he r than t h o s e in column I which is a t t r ­
ib u t e d t o t h e aceton e dry powder method o f e x t r a c t i o n o f enzymes.
As medium I and t h e aceton e dry powder method of e x t r a c t i o n of en ­
zymes appeared t o giv e t h e hig he r v a lu es i n d i c a t i n g g r e a t e r p r o t e o l y s i s ,
they were used in t h e remaining p a r t o f t h e study.
-2 5 -
Table VI
Summary o f t h e r e s u l t s shown in Table I, I I , I I I , IV, and V.
D i f f e r e n c e s in o p t i c a l d e n s i t y between 0 and 192 hours
Method o f e x t r a c t i o n of enzymes
Toluene method
Acetone dry powder
Medium I
Medium I I
Medium I
O i l . 1:20 Di I . I :50 O i l . 1:20 O i l . 1:50 D i l . I :20
Col. I
Col .5
Col. 2
Col .4
Col. 3
Organism
I.
S. I a c t i s
1.00
.70
1.10
.60
1.00
2.
L. dextranicum
1.10
.70
1.10
.70
1.20
3.
P
vulgaris
1.30
.80
1.30
.90
1.40
4.
B. s u b t i l i s
3.30
2.00
3.00
1.90
3.50
5.
P, f l u o r e s c e n s
2.40
1.90
2.30
1.70
2.60
6.
S. aureus
3 10
1.70
3.00
1.60
3.40
The F o l i n - C i o c a l t e u method
Since t h e o p t i c a l d e n s i t y method gave only comparative r e s u l t s and
no q u a n t i t a t i v e e s t i m a t i o n o f p r o t e o l y s i s could be obta in e d on t h i s
b a s i s , t h e F o l i n - C i o c a l t e u method was used s u b s e q u e n t l y .
Thi s method
as given by Lowry e t a_^, (63) has been suggested by Cowgill and Pardee
(20) and Davis and Smith (25) f o r th e assay of p r o t e o l y t i c enzymes.
The fo ll ow in g r e a g e n t s were used.
Reagent A:
Thi s was prepared
by adding 1.0 ml of 2.7% sodium potassium t a r t r a t e (NaKC^HjO^.dHgO) and
I ml of 1% copper s u l p h a t e (CuSO^SH2O) to 100 ml of 2.0% sodium c a r b o ­
nate s o lu tio n .
Reagent B:
This phosphomolybdotungstic a c i d r e a gen t
was made acc ord ing t o t h e d i r e c t i o n s of F o l i n and C i o c a l t e u ( 3 2 ) .
-2 6 -
Pr ocedure:
One m i l l i l i t e r o f well mixed enzyme r e a c t e d s u b s t r a t e
p r e v i o u s l y d e s c r i b e d was p i p e t t e d a s e p t i c a l l y i n t o a IO ml c e n t r i f u g e
tube.
Three m i l l i l i t e r s o f 5.0% t r i c h l o r a c e t i c were added to s top the
enzyme a c t i v i t y and t o a c t as a p r o t e i n p r e c i p i t a n t .
then c e n t r i f u g e d a t 2,000 rpm f o r 10 m in u te s .
These tu b e s were
Two-tenths m i l l i l i t e r
o f t h e s u p e r n a t a n t were t r a n s f e r r e d t o a n o th er tube and 0 . 8 ml 0 . 5 N
NaOH added to make t h e volume to 1.0 ml.
Five m i l l i l i t e r s of Reagent
A were then added t o t h e above mixed, and allowed t o s ta n d f o r 15
minutes a t rpqm t e m p e r a t u r e .
F i v e - t e n t h s m i l l i l i t e r o f Reagent B were
added and t h e tu b e s shaken immediately.
s ta n d u n d i s t u r b e d f o r one hour.
Thi s mi xtu re was allowed to
At t h e end of t h i s r e a c t i o n time l i g h t
a b s o r p t i o n was determined by a Beckman B s pec tro pho to m et e r a t 750 Mu
wave l e n g t h .
The ug o f t y r o s i n e and t r y p t o p h a n e were then determined
by t h e a i d of a s t a n d a r d c u r v e .
P r e p a r a t i o n ^of t h e s t a n d a r d curve f o r t h e F o l i n - C i o c a l t e u method:
S i x t y - t h r e e m i l l i g r a m s of t y r o s i n e and 17 mg of tr y p t o p h a n e were
d i s s o l v e d in 100 ml of a l k a l i n e d i s t i l l e d w a te r .
This p r o p o r t i o n of
t h e two amino a c id s i s t h e same as found in whole c a s e i n .
One m i l l i ­
l i t e r o f t h e above s o l u t i o n was f u r t h e r d i l u t e d to 100 ml with d i s t i l l e d
water.
This s o l u t i o n gave a c o n c e n t r a t i o n of 8 .0 ug pe r ml.
From t h i s
0, 0 . 1 , 0 . 2 , 0 . 4 , 0 . 6 , 0 . 8 , 1.0 ml p o r t i o n s were then t r a n s f e r r e d in t o
numbered t u b e s .
F i v e - t e n t h s normal NaOH were added i n t o each t o make
up t h e volume t o 1.0 ml.
Thi s gave c o n c e n t r a t i o n s of 0 , 0 . 8 , 1.6, 3 .2 ,
4 . 8 , 6 . 4 and 8 . 0 micrograms of t y r o s i n e and tr y p t o p h a n e .
The F o l i n -
I
-2 7 -
C i o c a l t e u r e a c t i o n was then run as d e s c r i b e d p r e v i o u s l y and micrograms
of t h e s e amino a c id s were then p l o t t e d a g a i n s t l i g h t absorbance.
I
3.2
4,8
6,4
'
Micrograms of t y r o s i n e and try pt ophan e
F i g . 2.
Sta ndard curve of l i g h t absorbance vs. micrograms of t y r o s i n e and
try p t o p h an e us in g Beckman B spec tro phom et er.
P r o t e o l y t i c a c t i v i t y usi ng F o l i n - C i o c a l t e u method
All o f t h e seven organisms were grown on medium I f o r 24 hours at
37 C.
The c e l l s were h a r v e s t e d , washed and t h e enzyme m ix tu r e s prepared
by t h e aceton e dry powder method, p r e v i o u s l y d e s c r i b e d .
Ten m i l l i l i t e r s
o f c a s e i n or whey p r o t e i n s o l u t i o n ( a l r e a d y d e s c ri b e d ) were used as sub­
strates.
Two-tenths m i l l i l i t e r of Agromycin s o l u t i o n and 0 .1 ml of
-2 8 -
enzyme mi xtu re were added t o th e s u b s t r a t e s and th e mi xtu res inc ubated
a t 37 C f o r 144 ho u rs .
One m i l l i l i t e r samples were drawn a t 24, 48,
96 and 144 hours and assayed by F o l i n - C i o c a l t e u method.
ob ta in e d a r e summarized in Table VII and V I I I .
The r e s u l t s
Table VII shows t h a t
as t h e r e a c t i o n time in c r e a s e d th e amount o f t y r o s i n e and tr ypt op han e
lib e ra te d also increased.
The blank showed an i n c r e a s e of 0.2 0 ug
while t h e v a r i o u s enzymes mi xtu re l i b e r a t e d from 1.20 ug to 2=40 ug
of t y r o s i n e and t r y p t o p h a n e .
One f i n d s from t h e s e r e s u l t s t h a t the
p a t t e r n o f p r o t e o l y t i c a c t i v i t y i s much t h e same as determined by th e
o p t i c a l d e n s i t y method.
Organism 2 gave th e lowest va lu e with the
Table VII
Micrograms of t y r o s i n e and try p t o p h an e l i b e r a t e d as th e r e s u l t of i n t r a ­
c e l l u l a r enzyme a c t i v i t y on c a s e i n s o l u t i o n s u b s t r a t e .
_______ Enzyme s u b s t r a t e r e a c t i o n time______
Organisms________________ 24 h r . ______ 48 h r ._______96 hr. _______144 hr.
Micrograms o f t y r o s i n e and try p t o p h an e
I . S. I a c t i s
1.35
1.70
1.80
1.90
2.
L. dextranicum
1.25
1.35
1.40
1,40
3.
L. c a s e i
1 .45
1,80
2.00
2.10
4.
P. v u l g a r i s
1.30
1.70
1.90
2.00
5.
B. s u b t i l i s
1.50
2.10
2.75
3.00
6.
P. f l u o r e s c e n s
1.50
2.20
2.80
3.10
7.
S
1,40
2.15
2.70
2.95
0.7 5
0.90
0.90
0.90
aureus
Blank
l i b e r a t i o n of 1.40 micrograms while I , 3 and 4 followed next with valu es
o f 1.9 0, 2.1 0 and 2 . 0 micrograms.
Organisms 5 ,6 and 7 had co mp arati vely
h ig h e r v a lu e s of 3 . 0 , 3.1 and 2 . 9 micrograms r e s p e c t i v e l y .
Thi s t a b l e
-2 9 -
r e p r e s e n t s th e b a s i c v a lu e s and s h a l l be used fo r comparison in the
remaining p a r t o f t h i s s t u d y .
Table V III shows the micrograms of t y r o s i n e and try p t o p h an e l i b e r ­
a ted from whey p r o t e i n s o l u t i o n , i n d i c a t i n g t h a t t h e enzymes can act
upon t h i s type of p r o t e i n a l s o .
I t w i l l be seen t h a t t h e p a t t e r n amongst
d i f f e r e n t organisms i s a l i t t l e d i f f e r e n t as compared to Table VII, i . e .
organism 2 and 4 show t h e lowest va lu es of 1.20 u g , followed by organTable VIII
Micrograms of t y r o s i n e and try p t o p h an e l i b e r a t e d as a r e s u l t of i n t r a c e l l u l a r enzyme a c t i v i t y on whey p r o t e i n s u b s t r a t e .
Organisms
24 h r .
Enzyme s u b s t r a t e r e a c t i o n time
48 h r .
96 hr.
144 hr.
I.
S. I a c t i s
1.10
1.15
1.20
1.25
2.
L. dextranicum
1.00
1.10
1.20
1.20
3.
L. e a s e l
1.20
1.30
1.35
1.40
4.
P, v u l g a r i s
1.15
1.20
1.20
1.20
5.
R. s u b t i l i s
1,30
1.40
1.40
1.50
6o
P
fluorescens
1,35
1.40
1.40
1.40
7.
S
aureus
1,20
1.20
1.30
1,30
0.50
0 .6 0
0.60
0.60
Blank
ism I and 7 with 1.25 and 1.30 ug.
Organism 3,5 and 6 a r e t h e higher
group with 1,40, 1.50 and 1.60 ug of t y r o s i n e and t r y p t o p h a n e .
While on
t h e c a s e i n s u b s t r a t e organism 2 had t h e lowest val ue of 1.4 ug , organisms
5 , 6 and 7 were th e h i g h e s t with 3 .0 , 3.1 and 2.90 ug o f t y r o s i n e and t r y ptophane.
-3 0 -
E f f e c t of pH on p r o t e o l y t i c a c t i v i t y
Whey s o l u t i o n was p r e p a r e d , s t e r i l i z e d and cooled as p r e v i o u s l y d e s ­
cribed.
Measured q u a n t i t i e s of s t e r i l e t a r t a r i c a cid were added a s e p t i -
c a l l y to d i f f e r e n t p o r t i o n s to bri n g th e pH t o 4 . 0 , 4 . 5 , 5 . 0 , 5 . 5 , 6 . 0 ,
6 . 5 and 7 .0 ( t h e q u a n t i t i e s of a cid t o be added were determined in a sep­
ara te experiment.
The pH were r e c h e c k e d ) .
One t e n t h m i l l i l i t e r of enz­
yme m ix tu r e s were added t o each 10 ml of s u b s t r a t e and tu be s incubated a t
37 C fo r 15 day s.
One m i l l i l i t e r p o r t i o n of th e h y d r o ly z a te was ana­
lyzed by F o l i n - C i o c a l t e u method.
Tabl e IX
Optimum pH and the micrograms of t y r o s i n e and tr ypt op han e l i b e r a t e d at
________ v a r i o u s pH.__________________________________________________________
e Micrograms of t r y o s i n e and try pt ophan e
Organisms___________ Optimum pH___________________ l i b e r a t e d ________________
____________________ pH___________________
5.5
4.5
6 .5
5.0
7.0
6 .0
4.0
I.
S. I a c t i s
1.30
1.20
1.15
1.30
1.10
1.00
1.00
2.
L. dextranicum
6 .0
1.10
1.10
1.20
0.90
0.9 0
0.90
0.80
3.
L. c a s e i
6.5
1.40
1.50
1.30
1.20
1.20
1.00
0.90
4.
P. v u l g a r i s
e e 7.0
1.30
1.10
1.00
0.8 0
0.80
0.70
0.70
5.
B. s u b t i l i s
6.5
1.55
1.60
1.40
1.20
1.00
0.90
0.80
6.
P. f l u o r e s c e n s
6.5
1.45
1.50
1.40
1.30
1.00
0.90
0.8 0
7.
S. aureus
ee7 .0
1.45
1.30
1.20
0.90
0.90
0.80
0.8 0
0.6 5
0.60
0.65
0.60
0.6 0
0.60
0.6 0
Blank
♦
ee
**7 .0 and 5 .5
The v a lu e s given a re aftefr 15 days o f r e a c t i o n ti m e .
These va lu es may not be optimum as no tu be s were pre pa red a t pH value
above 7 .0 because t h e pH during chees e r i p e n i n g i s never a l k a l i n e .
Tabl e IX giv e s th e r e s u l t s and shows t h a t th e optimum pH fo r v a ri o u s
enzymes was in t h e range of pH 6 .0 to 7 . 0 .
I t w i l l be n o t i c e d t h a t t h e r e
-3 1 -
was s t i l l some enzyme a c t i v i t y a t lower pH r a n g e s .
The d i f f e r e n c e b e ­
tween t h e amounts l i b e r a t e d at optimum pH and pH 4 .0 were .30, .40, .60,
.60, .60, .90, and .65 micrograms fo r organisms I , 2, 3, 4, 5, 6 , and 7,
respectively.
E f f e c t of age of t h e c u l t u r e on p r o t e i n breakdown
The s e l e c t e d organism were grown on medium I a t 37 C f o r 24, 48, 96,
and 144 h o u r s .
The c e l l s were h a r v e s t e d , washed and t h e enzyme mixture
pre pa red by t h e acetone dry powder method as p r e v i o u s l y d e s c r i b e d .
The
enzyme mixtu re was then r e a c t e d on a s t e r i l e c a s e i n s o l u t i o n ( a l r e a d y d e s ­
c r i b e d ) at 37 C.
One m i l l i l i t e r p o r t i o n s were taken a t r e g u l a r i n t e r v a l s
Table X
D i f f e r e n c e * in t h e amounts of t r y o s i n e and tr ypt op han e l i b e r a t e d by i n t r a c e l l u l a r enzymes e x t r a c t e d from c e l l s grown f o r 24 and 48 ho urs .
I. s_
I a c t is
Enzyme
24 h r .
Micrograms
+ .05
2. L
dextranicum
+ .05
+.35
+ .45
+ .60
casci
+ .15
+.40
+.40
+ .40
4. IL
5. FL
vulgaris
+ .00
+ .20
+.30
+.30
subtilis
+.30
+ .40
+.35
+ .30
6. Lu
7. s_
fluorescens
+ .20
+.10
+ .20
+ .20
aureus
+ .20
+.25
+.20
+.25
Organism
3.
L-
s u b s t r a t e r e a c t i o n time
48 h r .
144 hr'.
96 h r .
of t y r o s i n e and tr ypt op han e
+ .30
+.40
+ .50
♦ D i f f e r e n c e s have been c a l c u l a t e d on t h e b a s i s of th e same r e a c t i o n ti m e ,
and F o l i n - C i o c a l t e u r e a c t i o n run .
Tables X, XI, and XII shows th e r e s u l t s
o b ta in e d from t h e s e e x p e r im e n t s.
Table X shows t h a t a l l 48 hours old c u l t u r e s showed an i n c r e a s e in
th e amounts of t y r o s i n e and try p t o p h an e l i b e r a t e d as compared to 24 hour
-3 2 -
old c u l t u r e s .
However, t h e i n c r e a s e v a r i e d from organism to organism.
D e t a i l s o f th e a c t u a l amounts of t y r o s i n e and try p t o p h an e l i b e r a t e d are
given in Table XXI in t h e Appendix.
Table XI giv e s th e d i f f e r e n c e s in t h e amounts of t y r o s i n e and t r y p o phane l i b e r a t e d between 48 and 96 hours old c u l t u r e s .
All the va lu es are
p o s i t i v e , i n d i c a t i n g an i n c r e a s e in t h e amounts of f r e e t y r o s i n e and
tryptophane.
On comparing t h e s e v a lu es with Table X i t w i l l be seen t h a t
enzymes from organisms I , 2, 3, and 4 showed l e s s e r i n c r e a s e while orga n­
isms 5 and 6 showed a g r e a t e r i n c r e a s e .
sim ilar.
The va lu es f o r organism 7 were
These r e s u l t s i n d i c a t e t h a t enzymes from organisms I , 2, 3, and
4 gain t h e maximum r a t e o f a c t i v i t y between 24 and 48 hours while enzymes
Table XI
D i f f e r e n c e s in t h e amounts of t y r o s i n e and try p t o p h an e l i b e r a t e d by i n t r a _______ c e l l u l a r enzymes e x t r a c t e d from c e l l s grown f o r 48 and 96 hours.
Organisms
I.
lactis
Enzyme s u b s t r a t e r e a c t i o n time
24 h r .
48 h r .
96 h r .
144 h r .
Micrograms of t y r o s i n e and tr ypto pha ne
+ .20
+ .15
+ .20
+ .20
2.
.Ll dextranicum
+ .20
+.20
+ .25
+ .10
3.
L l casei
+ .10
+ .20
+.20
+ .20
4.
fL vulgaris
+ .05
+ .10
+.10
+ .00
5.
EL s u b t i l i s
+ .20
+.30
+ .35
+ .50
6 < P_ f l u o r e s c e n s
+ .40
+.30
+ .25
+.70
7.
+.20
+.30
+.05
+ .25
L l aureus
from organisms 5 and 6 o b ta in e d th e maximum r a t e of p r o t e o l y t i c a c t i v i t y
between 48 and 96 hours of age.
The a c t u a l amounts o f t y r o s i n e and t r y p ­
tophane l i b e r a t e d a re given in Table XXII in the Appendix.
-3 3 -
Table XII r e p r e s e n t s t h e d i f f e r e n c e s in t h e amounts o f t y r o s i n e and
t r y p t o p h a n e l i b e r a t e d from 96 and 144 hours c u l t u r e .
All the val ues are
n e g a t i v e i n d i c a t i n g t h a t enzymes obta in e d from c e l l s grown a t 144 hours
have l e s s e r p r o t e o l y t i c a c t i v i t y as compared to c e l l s grown f o r 96 ho urs .
For d e t a i l s of t h e a c t u a l amounts o f t y r o s i n e and tr ypto pha ne l i b e r a t e d
see Table XXIII in th e Appendix.
Table XII
D i f f e r e n c e s in t h e amounts of t y r o s i n e and tr ypt op han e l i b e r a t e d by i n t r a c e l l u l a r enzymes e x t r a c t e d from c e l l s grown f o r 96 and 144 hours.
Organism
Enzyme s u b s t r a t e r e a c t i o n time
24 h r .
48 h r .
96 h r .
144 h r .
Micrograms of t y r o s i n e and try p t o p h an e l i b e r a t e d
-.30
-.25
-.30
-.40
I.
S. l a c t i s
2.
L. dextranicum
-.30
-.10
-.20
- . 20
3.
L. c a s e i
-.30
- .65
- . 65
-.70
4.
P. v u l g a r i s
-.05
-.10
—«,20
-.30
5.
B. s u b t i l i s
-.30
-.40
-.65
-.80
6.
P. f l u o r e s c e n s
-.60
-.40
—o50
-.95
7.
S. aureus
-.40
-.60
-.35
-.60
Tabl e X I I I shows t h e d i f f e r e n c e in t h e amounts of t y r o s i n e and
tr y p t o p h a n e l i b e r a t e d by 24 and 144 hours old c u l t u r e s .
The p o s i t i v e
f i g u r e s i n d i c a t e t h a t 144 hour old c u l t u r e s had hig h e r v a l u e s while
neg­
a t i v e f i g u r e s i n d i c a t e t h a t 24 hour c u l t u r e had hig he r v a lu e s by the amounts i n d i c a t e d .
Others r e p r e s e n t no change.
From Table s X, XI, XII and X I I I i t can be concluded t h a t th e p r o t e o ­
l y t i c a c t i v i t y in a l l t h e organisms i n c r e a s e d up to 96 hours of age,
though t h e i n c r e a s e s v a r i e d from one organism to a n o t h e r .
The p r o t e o l y t i c
-3 4 -
a c t i v i t y d e c l i n e d when t h e c e l l s were 144 hours o l d .
Only t h r e e organisms
( I , 2 and 3) had hig h e r v a lu es at 144 hours as compared t o 24 h o u r s .
In
organisms 6 and 7 t h e v a lu es were lower a t 144 hours as compared to 24
ho ur s, while organisms 4 and 5 showed no change.
Table X II I
D i f f e r e n c e s in th e amounts of t y r o s i n e and tr ypt op han e l i b e r a t e d by i n t r a _______ c e l l u l a r enzymes e x t r a c t e d from c e l l s grown f o r 24 and 144 hours.
Si l a c t i s
2.
L . dextranicum
+ .05
+ .45
+ .45
+ .50
3.
Li c a s e i
+ .05
+.05
+.05
+.10
4.
Pi v u l g a r i s
.00
§
.00
.00
5.
Bz s u b t i l is
+.20
+.30
.00
.00
6.
Pi f l u o r e s c e n s
.00
.00
05
-.15
7.
Si aureus
-.10
-.10
§
I.
Enzyme s u b s t r a t e r e a c t i o n time
144 h r .
24 h r .
48 h r .
96 h r .
Micrograms of t y r o s i n e and try pto ph an e
+ .05
+.20
+.30
+ .30
Organism
-.05
—
e
E f f e c t of g e l a t i n in t h e growth medium on p r o t e o l y t i c a c t i v i t y
Medium I with .50% g e l a t i n added was used for t h e c u l t i v a t i o n of t h e
organisms and t h e enzyme mi xtu res were prepa red in t h e us ua l manner.
The
enzyme mi xtu re from each organism was r e a c t e d with t h e c a s e i n s u b s t r a t e
p r e v i o u s l y d e s c r i b e d and t h e F o l i n - C i o c a l t e u r e a c t i o n s was performed a t
i n t e r v a l s on 1.0 ml samples.
The r e s u l t s obt a in e d have been summarized in Table XIV.
The e n t r i e s
under each column of t h i s t a b l e r e p r e s e n t micrograms of t y r o s i n e and t r y p ­
tophane l i b e r a t e d in ex ce s s as compared to medium I (Table VII). The i n ­
c r e a s e i s a t t r i b u t e d t o t h e presence o f g e l a t i n in th e growth medium.
It
-3 5 —
w i l l be n o t i c e d t h a t ex cept f o r organisms 4 and 7 the i n c r e a s e s were not
very g r e a t , ra nging from .10 to .30 micrograms.
isms 4 and 7 were .90 and .85
The i n c r e a s e s fo r organ­
respectively.
For d e t a i l v a lu es f o r each medium a t every r e a c t i o n time see Table
XXIV in the Appendix.
Table XIV
I n c re a s e in t h e amount of t y r o s i n e and try p t o p h an e l i b e r a t e d by i n t r a ­
c e l l u l a r enzymes due t o t h e pres enc e of 0.5% g e l a t i n in
t h e growth medium.________________________________________
Organisms
I.
lactis
Enzyme s u b s t r a t e r e a c t i o n time
48 h r .
96 h r .
144 h r .
24 h r .
In c r e a s e in micrograms of t y r o s i n e and try pto ph an e
.10
.20
.05
.20
2.
U dextranicum
.05
.15
.15
.20
3,
L . casei
.05
.10
.10
.10
4,
P, vulqaris
.60
.70
.70
.90
5,
s u b t i l is
.45
.25
.20
.30
6.
fluorescens
.25
.55
.05
.10
.70
.55
.80
.85
7.
s _ aureus
E f f e c t of c a s e i n in th e c u l t u r e media on p r o t e o l y t i c a c t i v i t y
Five grams of c a s e i n ( Merck) were d i s s o l v e d in a l k a l ire d i s t i l l e d
w a t e r , and made up to 200 ml.
F in a l pH o f t h e medium was a d j u s t e d to
pH 7. 0 and t h e s o l u t i o n a u t o c l a v e d .
Eig ht hundred m i l l i l i t e r s of medium
I were mixed a s e p t i c a l l y with th e above.
The f i n a l pH o f t h e combined
medium was 7 . 1 .
C u l t u r e s of Pr ote us v u l g a r i s . Pseudomonas f l u o r e s c e n s , S ta ph ylo c ­
occus aureus and B a c i l l u s s u b t i l i s were grown on t h e above medium,
Cul­
t u r e s of S t r e p t o c o c c u s l a c t i s , Leuconostoc dextranicum and L a c t o b a c i l l u s
-3 6 —
c a s e i were grown on t h e above medium plu s 0.2% calcium c a r b o n a t e .
Cal­
cium c a r b o n a t e served as a b u f f e r f o r th e a c id produced by t h e above c u l ­
tures.
All c u l t u r e s were grown f o r 24 hours a t 37 C.
C e l l s were h a r v e s t e d a t the end o f i n c u b a t io n pe ri o d and NaOH s o l u ­
t i o n was used to d i s s o l v e t h e c a s e i n in i n s t a n c e s where i t p r e c i p i t a t e d .
Excess calciu m c a r b o n a te was removed by t h e use of an a c i d .
The enzyme
m ix tu r es were prepa red as usual and r e a c t e d with th e c a s e i n s u b s t r a t e .
F o l i n - C i o c a l t e u r e a c t i o n was performed to follow th e p r o t e o l y t i c changes.
The r e s u l t s have been summarized in Tabl e XV.
In Table XV t h e f i g u r e s r e p r e s e n t t h e micrograms of excess t y r o s i n e
and t r y p t o p h a n e l i b e r a t e d by c e l l s when grown with added c a s e i n as com­
pared to t h e medium I (T abl e V I I ) .
On comparing t h e s e v a lu es with th o s e
Table XV
I n c r e a s e in t y r o s i n e and try p t o p h an e l i b e r a t e d by i n t r a c e l l u l a r enzymes
due t o t h e pres enc e of .05% c a s e i n in th e growth medium.
Organisms
Enzyme s u b s t r a t e r e a c t i o n time
24 h r .
48 h r .
96 h r .
144 h r .
I n c r e a s e in micrograms of t y r o s i n e and try pt ophan e
0.4 5
1.30
0.90
1.50
I.
Si l a c t i s
2.
Li dextranicum
0.6 0
1.05
1.60
1.60
3.
L« c a s e i
1.15
0.40
1.60
1.80
4.
E\ v u l g a r i s
0.4 0
0.9 0
0.9 5
1.10
5.
Bi s u b t i l i s
2.50
2.40
1.95
2.30
6.
Pi f l u o r e s c e n s
2.70
2.60
2.1 0
2.1 0
7.
Si aureus
2.40
2.05
2 .1 0
2.15
in Table XIV i t w i l l be seen t h a t d i f f e r e n c e s obta ine d by t h e a d d i t i o n of
c a s e i n t o t h e growth medium were much h i g h e r than th os e with g e l a t i n .
In
-3 7 -
ca se of g e l a t i n th e i n c r e a s e s ranged from .10 to .90 ug while in case of
c a s e i n t h e i n c r e a s e ranged from 1.10 to 2.30 ug.
For d e t a i l s of t h e v a lu e s obta in e d f o r each medium see Table XXV in
th e Appendix.
E f f e c t of c a r b o h y d ra t e f r e e medium on p r o t e o l y t i c a c t i v i t y
To stu dy t h e e f f e c t of c a r b o h y d r a t e s on t h e pro duc tio n of enzymes
t h e fo ll o w i n g medium was used fo r growth o f b a c t e r i a :
Trypticase
Polypeptone
Yeast e x t r a c t
NaCl
Potassium hydrogen
phosphate ( d i b a s i c )
D i s t i l l e d water
17.0
5.0
2.0
5 .0
g
g
g
g
2.5 g
1000
ml
The enzymes were prepared and r e a c t e d with t h e c a s e i n s u b s t r a t e and
assayed by t h e F o l i n - C i o c a l t e u r e a c t i o n .
The r e s u l t s a re shown in Table
XVI.
Table XVI
I n c r e a s e in t h e t y r o s i n e and try p t o p h an e l i b e r a t e d by i n t r a c e l l u l a r en_______ zymes due t o th e absence of c a r b o h y d ra t e in th e growth medium.
Organisms
I.
lactis
Enzyme s u b s t r a t e r e a c t i o n time
24 h r .
48 h r .
96 h r .
144 h r .
I n c r e a s e in micrograms of t y r o s i n e and trypto phane
.35
.70
.85
.80
2.
L . dextranicum
.30
.40
.60
.70
3.
L . casei
.35
.60
.65
.70
4.
P^ v u l g a r i s
.20
.25
.30
.40
5.
s u b t i l is
.40
.50
.85
1.25
6.
F\ f l u o r e s c e n s
.90
1.00
1.10
1.40
.70
.65
.70
.60
7.
aureus
-3 8 -
In Table XVI t h e f i g u r e s r e p r e s e n t t h e micrograms o f excess t y r o s i n e
and tr y p t o p h a n e l i b e r a t e d by enzymes from c e l l s when grown on a c arbo­
h y d ra te f r e e medium as compared to th e b a s i c medium (Table V I I ) .
On com­
p a r in g t h e above r e s u l t s t o Table XIV, XV, i t w i l l be n o t i c e d t h a t t h e s e
i n c r e a s e s a r e hig h e r than th o s e shown in Table XIV ( e f f e c t of g e l a t i n )
except in S. a ure us ; however t h e s e i n c r e a s e s a r e c o m p a r i t i v e l y much
s m a l le r than t h o s e in Tabl e XV ( e f f e c t o f c a s e i n ) .
For d e t a i l s of v a lu es obt a in e d from each medium see Table XXVI in
th e Appendix.
E f f e c t o f C as ito ne (D if co )* in t h e growth medium on p r o t e o l y t i c a c t i v i t y
To study th e e f f e c t o f C asi to ne in t h e medium on p r o t e o l y t i c a c t i v i t y
t h e c u l t u r e s were grown f o r 24 hours a t 37 C on medium I plus 0.5% of
C a s it o n e ( D if c o , vit am in f r e e ) .
The enzyme mixtu res were pre pared
Table XVII
I n c r e a s e in t h e t y r o s i n e and try p t o p h an e l i b e r a t e d by i n t r a c e l l u l a r enzmes due t o th e pres enc e o f 0.5% C asi to ne (Difco) in th e
growth medium.
Organism
I.
Iactis
Enzyme s u b s t r a t e r e a c t i o n time
144 h r .
24 h r .
48 h r .
96 h r .
In c r e a s e in micrograms of t y r o s i n e and try pt ophan e
.05
.05
.10
.00
2.
L l dextranicum
.00
.05
.00
.00
3.
Ll c a s e i
.05
.05
.00
.00
4.
Pl v u l g a r i s
.00
.05
.00
.00
5.
Bl s u b t i l is
.05
.05
.10
.10
6.
Pl f l u o r e s c e n s
.00
.00
.00
.05
7.
S l aureus
.00
.15
.15
.15
* Pancreatic d igest of casein .
-3 9 -
In Table XVII t h e f i g u r e s r e p r e s e n t t h e excess micrograms o f t y r o s i n e
and tr y p t o p h a n e l i b e r a t e d by c e l l s when grown on added C as it o n e as com­
pared to t h e b a s i c medium (T abl e V I I ) .
I t can be concluded t h a t Casit one
di d not a f f e c t t h e p r o t e o l y t i c a c t i v i t y in any a p p r e c i a b l e amounts.
Re­
t a i l s o f t h e v a lu e s o b ta in e d a re shown in Table XXVII o f t h e Appendix.
On comparing Table s XIV, XV, XVI, and XVII i t can be concluded t h a t
0.5% c a s e i n in th e growth medium showed maximum i n c r e a s e over a b a s ic
medium in t h e p r o t e o l y t i c a c t i v i t y o f t h e enzymes and t h e car b o h y d ra t e
f r e e medium followed n e x t .
The pres enc e o f 0.5% g e l a t i n in t h e growth
medium in c r e a s e d t h e amounts of t y r o s i n e and tr ypt op han e l i b e r a t e d but
except f o r P. v u l g a r i s and S. aureus
nigicant.
t h e q u a n t i t i e s were not very s i g -
In c a s e of 0.5% C as ito ne in t h e growth medium, t h e i n c r e a s e s
do not appear t o be s i g n i f i c a n t .
f' :
i' '
DISCUSSION AND CONCLUSIONS
The f i r s t p a r t o f t h i s study was concerned with t h e d e t e r m i n a t i o n o f
p r o t e o l y t i c a c t i v i t y o f t h e i n t r a c e l l u l a r enzymes o f t h e organisms s e l e -
,/I':
cted.
I t was found t h a t t h e enzymes from a l l t h e organisms had some a.pt.-r
i v i t y though i t v a r i e d g r e a t l y from organism t o organism e . g . S t r e p t o ­
coccus l a c t i s co uld l i b e r a t e only 1.90 ug o f t y r o s i n e and tr ypt op han e
w hi le Pseudomonas f l u o r e s c e n s l i b e r a t e d 3.10 ug.
As f a r as l a c t i c a c i d organisms a r e concerned i t r e a f f i r m s t h e be­
l i e f t h a t s t a r t e r organisms may be r e s p o n s i b l e f o r a t l e a s t p a r t of t h e
p r o t e i n breakdown d uri ng chees e r i p e n i n g .
In c o t t a g e chees e l a c t i c a c id
b a c t e r i a (Su_ l a c t i s and Leuconostoc dex tra nic um ) a r e always p r e s e n t and
Pseudomonas. Pr o te u s and B a c i l l u s s p e c i e s may be encoun tere d as contam­
inants.
Thi s l a t t e r group could b r i n g about r a t h e r e x t e n s i v e p r o t e i n
breakdown a f t e r a u t o l y s i s .
Thi s may be one of t h e caus es o f b i t t e r anvd"
o t h e r o f f f l a v o r in low count c o t t a g e c h e e s e , s i n c e some peptones have
been r e p o r t e d t o have b i t t e r t a s t e .
Chromatographic s t q d i e s o f t h e hydro-
l y z a t e should be done t o i d e n t i f y t h e peptones in ord er t o confirm t h e
p o s s i b i l i t y o f o f f f l a v o r s being due t o t h e s e s u b s t a n c e s .
Raw milk Cheddar c hees e is re c o g n iz e d as r i p e n i n g f a s t e r than pa s ­
t e u r i z e d milk c h ee s e.
Most of t h e s e l e c t e d organisms a r e p r e s e n t in raw
milk in s m a l le r or l a r g e r numbers,
During p a s t e u r i z a t i o n t h e organism's
a re d e s t r o y e d and p r o t e i n a s e s would pro ba bl y be i n a c t i v a t e d .
On th e o t h e r
hand in raw milk c h ee s e, t h e b a c t e r i a u s u a l l y ge t a chance t o grow b e f o r e
th e y a r e i n a c t i v a t e d by an unf a v o ra b le pH ( 6 . 8 to 4.3 ) th u s t h e i r number
would be h i g h e r .
This stu dy has shown t h e s e b a c t e r i a l c e l l s on death and
autolysis will lib e ra te pro tein ases.
The a c t i o n of t h e s e enzymes can in
p a r t be t h e cause o f f a s t e r c u r i n g , as t h e pH s t u d i e s o f b a c t e r i a l pro-
-4 1 -
t e i n a s e s showed, some a c t i v i t y in t h e lower pH ra n g e .
Both whey p r o t e i n s o l u t i o n and c a s e i n s o l u t i o n s u b s t r a t e were a t t ­
acked by t h e s e enzymes.
A comparison between t h e v a lu e s o b ta in e d cannot
be made s i n c e t h e r e was a d i f f e r e n c e in t h e c o n c e n t r a t i o n o f t h e two.
The study o f t h e e f f e c t of pH o f t h e s u b s t r a t e on t h e enzyme a c t ­
i v i t y r e v e a l e d t h a t most o f t h e enzymes had t h e i r optimum pH in the v i c ­
i n i t y o f 6 ,0 and 7 , 0 , though
a n o th er a t pH 5 . 5 .
l a c t i s had two p e a k s , one a t pH 7 ,0 and
These v a lu es f o r
stu dy o f Baribo and F o s t e r ,(14).
l a c t i s a r e in accordance with t h e
f l u o r e s c e n s , Liacobacillus c a s e i and
B. s u b t i l i s showed an optimum pH a t 6 . 5
Staphylococcus au reus and P.
v u l g a r i s showed t h e maximum a c t i v i t y a t pH 7 .0 ( t h i s may not be optimum
s in c e no pH h i g h e r than 7 . 0 were s t u d i e d ) .
I t was a l s o observed t h a t t h e
organisms s t i l l had some a c t i v i t y a t a lower pH ( 4 . 5 - 5 . 0 ) .
The e f f e c t o f t h e age of th e c e l l s on t h e i n t r a c e l l u l a r p r o t e i n a s e s
was a l s o i n v e s t i g a t e d and i t was found t h a t as th e c e l l s grew o l d e r , up
t o 96 hours o f age, t h e enzyme a c t i v i t y i n c r e a s e d .
In c e l l s 144 hours old
t h e p r o t e o l y t i c a c t i v i t y o f ttye enzymes d e creased in a l l c a s e s .
(
i
-
"
-
.
'
I-.-
I t was
-
:
observed t h a t in 144 hour old c e l l s Sj_ l a c t i s , L. dextranicum and Ll
■'-i
■
•
■ -r
j
c a s e i s t i l l had h i g h e r p r o t e o l y t i c a c t i v i t y than 24 hour old c e l l s .
P.
f l u o r e S c e n s and S. aureus showed a d e c r e a s e in v a lu es as compared to 24
hour old c e l l s , w hil e P. v u l g a r i s and B. s u b t i l i s had s i m i l a r va lu es a t
24 and 144 h o u r s .
The e f f e c t of age could probably be e xpla in ed from t h e f a c t t h a t
c ar bo hy dra s e s a r e t h e f i r s t enzymes t o be formed and p r o t e i n a s e s b u i l d
up l a t e r .
As to why t h e enzyme a c t i v i t y f e l l a f t e r a c e r t a i n peak one
:
i
-4 2 -
e x p l a n a t i o n t h a t could be given i s t h a t old au to ly z ed c e l l s might ser ve
as a s u b s t r a t e f o r some o t h e r typ e o f enzymes.
I t f o ll o w s , based on t h e
r e s u l t s o f t h i s stu dy t h a t old c u l t u r e s might be o f more v a lu e as an i n ­
oculum than t h e young c u l t u r e s i f f a s t p r o t e i n breakdown i s d e s i r e d .
How­
e v e r , s i n c e t h e c u l t u r e s a r e g e n e r a l l y added p r i m a r i l y f o r t h e purpose of
a c id p r o d u c t i o n and i t i s d e s i r e d t o have maximum a c id pro d u c ti o n in min­
imum ti m e , old c u l t u r e s would be d is a dva nt a geo us under t h e s e c ir c um st an c es
Some o f t h e en vironm ental f a c t o r s which could enhance t h e p r o t e o ­
l y t i c a c t i v i t y o f i n t r a c e l l u l a r p r o t e i n a s e s were a l s o s t u d i e d .
Gelatin
0.5% was added in t h e growth medium and t h e enzymes p re pa re d from such
c e l l s had a h i g h e r p r o t e o l y t i c a c t i v i t y t h a n th o s e grown in t h e absence
of g e la t in .
The i n c r e a s e s v a r i e d from .10 t o .90 ug of t y r o s i n e and t r y ­
ptophane f o r t h e d i f f e r e n t o rg a n is m s .
Thi s was a l i t t l e s u r p r i s i n g be­
cause none o f t h e organisms s e l e c t e d showed any l i q u e f a c t i o n o f g e l a t i n
when grown on i t .
Perhaps g e l a t i n c o n t a i n e d some o t h e r p r o t e i n in min­
u t e q u a n t i t i e s which might have s t i m u l a t e d t h e p ro du c tio n of p r o t e i n a s e s .
Casein 0.5% was a l s o added to t h e growth medium.
The, eftjsymes e x t r a c -
ed frdm t h e c e l l s grown.on such a medium showed t h e l a r g e s t i p p r e a s e in
p r o t e o l y t i c a c t i v i t y o f any substa nc e s t u d i e d .
1.10 ug t o 2.30 ug of t y r o s i n e and t r y p t o p h a n e .
The i n c r e a s e v a r i e d from
In Sjl l a c t i s , L. d e x t r a -
nicum and L. c a s e i two f a c t o r s i . e . c a s e i n and calcium c a r b o n a te were
involved so t h a t t h e i n c r e a s e s o f 1,50, 1.60. and 1.80 ug were probably
due t o t h e i r combined a f f e c t .
I t appea rs t h a t th e pre s e n c e of a sub­
s t r a t e i n c r e a s e s t h e p ro d u c ti o n of p r o t e i n a s e s .
The pre s e n c e o f 0.5% C as it o n e (Difco) gave no a p p r e c i a b l y i n c r e a s e .
The maximum i n c r e a s e was .15 ug o f t y r o s i n e and tr y p t o p h a n e in 5L aureus
-4 3 -
enzymes o Thi s o b s e r v a t i o n seems t o i n d i c a t e t h a t u n d ig e st e d p r o t e i n i n ­
c r e a s e s t h e for mation o f p r o t e i n a s e s in t h e organisms s t u d i e d .
Absence o f fe rm e n ta b le c a r b o h y d ra t e in t h e growing medium a l s o af f e c t s t h e p r o t e o l y t i c a c t i v i t y o f t h e i n t r a c e l l u l a r enzymes.
c r e a s e s o f t y r o s i n e and t r y p t o p h a n e were .85 ug in
The i n ­
l a c t i s , .70 ug in
L. dextranicum and Lll c a s e i , .40 ug in Pl v u l g a r i s , 1.25 ug in Bl s u b t i l i s ,
1.40 ug in Pl f l u o r e s c e n s and .60 in Sl a u r e u s .
be given f o r t h e above r e s u l t s .
No e x a c t e x p l a n a t i o n can
In t h e l a c t i c a cid b a c t e r i a , however^
t h e a d d i t i o n o f c a r b o h y d r a t e in t h e growing medium might le a d t o th e i n ­
h i b i t i o n o f deaminases p ro d u c t i o n , pH might be a f a c t o r h e r e .
But t h i s
does not e x p l a i n t h e r e s u l t s obta in e d with non-ferm enting b a c t e r i a .
In c o n c lu s io n i t can be s a i d t h a t pH s t u d i e s showed t h a t optimum f o r
enzyme a c t i v i t y f o r v a r i o u s organisms was in t h e v i c i n i t y pH 6 .0 to 7 , 0 .
Study o f t h e e f f e c t of age o f c e l l s r e v e a l e d an i n c r e a s e in p r o t e o l y t i c
a c t i v i t y t i l l t h e c e l l s were 96 hours o ld , growing them f o r an oth er 48
hours however showed an a p p r e c i a b l e d e c r e a s e .
The pre s en c e of g e l a t i n
and c a s e i n and t h e absence of c a r b o h y d ra t e in th e growth medium in c re a s e d
t h e p r o t e i n a s e s while C a s it o n e did not have an a p p r e c i a b l e e f f e c t ,
- Suggested f u r t h e r work
Combination of more than one f a c t o r should be t r i e d to see the com­
bined e f f e c t on t h e p r o d u c ti o n o f p r o t e i n a s e s , e . g . absence of carbohy­
d r a t e and a d d i t i o n o f c a s e i n ; age of t h e c e l l s might be v a r i e d f o r each
combination.
S e v e ra l such combinations can be t r i e d .
An impo rtan t thing, t o v e r i f y would be t o see whether t h i s in c re as e d
amount of p r o t e i n a s e r e s u l t s in o f f f l a v o r s or speeds up development of
desirable fla v o r.
A d d it io n s of th e a ce ton e dry powders from v a ri o u s
-4 4 -
organisms t o t h e milk used f o r making ex per im ent al chees e would be one
approach.
Comparisons between t h e p r o t e i n a s e pro d u c ti o n of t h e c e l l s on t h e s e
s p e c i a l media and t h e c e l l s o c c u r r i n g in normal cheese would be i n t e r e s t
in g, and a study t o de ter mi ne t h e e f f e c t of te m p e ra t u re on t h e a c t i v i t y
o f t h e s e enzymes may be o f val ue in e x p l a i n i n g t h e i n f l u e n c e o f p a s t ­
e u r i z a t i o n on t h e r a t e of chees e r i p e n i n g .
SUMMARY
Seven c u l t u r e s s e l e c t e d on t h e b a s i s o f t h e i r common occurrence in
milk or s t a r t e r s and con tam ina nts of milk and milk pro d u c ts were used
for t h i s study.
The organisms were S t r e p t o c o c c u s l a c t i s , Leuconostoc
d e x tr a n ic u m . L a c t o b a c i l l u s
c a s e i „ Pr ot eu s v u l g a r i s „ B a c i l l u s s u b t i l i s „
Pseudomonas f l u o r e s c e n s and Sta phylococcus
aureus.
Each organism was
grown on medium I a n d /o r medium I I f o r 24 hours a t 37 C.
At t h e end of
t h e i n c u b a t i o n p e r io d t h e c e l l s were h a r v e s t e d , washed fo ur times arid
t h e enzyme m ix tu r es p re p a re d by to l u e n e and acetone dry powder
The p r o t e o l y t i c a c t i v i t y o f t h e enzymes was
method.
determined by t h e o p t i c a l
d e n s i t y method us in g a d i l u t e d skim milk s u b s t r a t e .
C e l l s grown on medium I and enzymes e x t r a c t e d by a ce ton e dry pow­
der method gave t h e b e s t r e s u l t s „ The F o l i n - C i o c a l t e u method was ad­
apt ed f o r f o l l o w i n g t h e p r o t e o l y s i s in t h e l a t t e r p a r t o f t h e work s i n c e
t h i s method could g iv e a q u a n t i t a t i v e d e t e r m i n a t i o n of p r o t e o l y t i c a c t i ­
v i t y e x p re ss e d in terms o f th e amounts o f t y r o s i n e and tr y p t o p h a n e l i b ­
erated.
Casein ( 1 . 5 mg/ml> s o l u t i o n and whey p r o t e i n s o l u t i o n ( . 5 7 mg/ml)
were used as s u b s t r a t e s .
S t u d i e s of pH were done on whey p r o t e i n s o l ­
u t i o n w hil e t h e remaining p a r t o f t h e study was done u s in g c a s e i n s o l ­
u t i o n as a s u b s t r a t e .
I t was found t h a t optimum pH f o r v a r i o u s enzymes
f e l l in t h e v i c i n i t y 6 . 0 t o 7 .0 though
l a c t i s showed a second peak
a t pH 5 . 5 .
The e f f e c t o f age on i n t r a c e l l u l a r p r o t e i n a s e s was determined by
growing c e l l s on medium I f o r 24, 48, 96 and 144 h o u r s .
Enzymes were
p re pa re d a t t h e end o f each i n c u b a t io n p e r i o d and r e a c t e d with th e
casein s u b s tr a te .
Amounts o f t y r o s i n e and try p t o p h an e l i b e r a t e d were
—4 6 —
compared and i t was found t h a t Se I a c t i s e Le dextranicum and Le c a s e i
showed a maximum r a t e o f i n c r e a s e d uri ng t h e 24 and 48 hour p e r i o d .t h o u g h
t h e i n c r e a s e , c o n t i n u e d up t o 96 ho urs ,
. s u b t i l l s and F. -fluorescems
showed a maximum r a t e of i n c r e a s e durin g t h e 48 and 96 hour p e r i o d ,
while Se au reus gave a very s i m i l a r i n c r e a s e d urin g t h e 24 t o 48 hour
p e r i o d and t h e 48 t o 96 hour p e r i o d ,
c r e a s e beyond 48 hours p e r i o d .
Pi. vu l g a r i s d id not show any i n ­
However, a l l th e organisms showed an
a p p r e c i a b l e d e c r e a s e when t h e c e l l s were 144 hours old .
The organisms were a l s o grown on medium I w it h 0,5% a d d i t i o n a l
amounts of g e l a t i n , c a s e i n and C a s i t o n e ( D if c o ),
f o r 24 hours and enzymes p re pa re d and ass a yed .
The c e l l s were grown
I t was found t h a t g e l ­
a t i n and c a s e i n both i n c r e a s e d t h e p r o t e i n a s e s but. t h e i n c r e a s e i n
case of c a s e i n was much g r e a t e r ,
C a s i t o n e on t h e o t h e r hand d id not
b r in g about any a p p r e c i a b l e change.
D e l e t i o n of c a r b o h y d r a t e s from
medium I a l s o i n c r e a s e d t h e p r o t e o l y t i c a c t i v i t y of t h e enzymes.
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APPENDIX
56TABLE X V I I I
CULTURAL CHARACTERISTICS OF THE ORGANISMS USED
S t r e p t o c o c c u s I a c t i s BBlO
Shape, s i z e , arrangement,
and r e l a t i o n t o Og
-
Ovoid, 0.70u in dia meter C a / v ) ;
in p a i r s or s h o r t c h a i n s , Aerbic
Staining c h a r a c te r is tic s
m otility
-
Gram p o s i t i v e
non-motile
Agar c o l o n i e s
-
S m a ll , l e s s th a n I mm s l i g h t l y
mucoid, S u b - s u r f a c e c o l o n i e s ,
lenticular
Carbohydrates
fermentation
.
-
Acid from:
glu c o se , ma lto s e,
l act os e, s u c r o s e , no acid from
r a f f i n o s e ' and s o r b i t o l
Litmus milk
-
A c i d i f i e d and c u r d l e d . Litmus
reduced b e f o r e c u r d l i n g . No.,,
digestion.
Remarks
-
Growth was l u x u r i a n t .
j
Leuconostoc dextranicum NRRL B-640
Shape, s i z e , a r r a n g e m e n t,
and r e l a t i o n t o Og
S p h e r i c a l 0 . 86u in dia me ter in •
s h o r t c h a in s m o s tl y . Aerobic
Staining c h a r a c te r is tic s
m otility
Gram p o s i t i v e .
Non-motile.
Agar c o l o n i e s
S m a ll , gray, c i r c u l a r , s l i g h t l y
raised, e n tire .
!
Carbohydrates
f e r m e n ta ti o n
Acid from: g l u c o s e , f r u c t o s e ,
g a l a c t o s e , m a l t o s e , s u cr o se .
Litmus milk
S l i g h t a c i d , No c o a g u l a t i o n .
No r e d u c t i o n of l i t m u s .
■??
Remarks
!
Growth f a i r , in e nric hed
medium.
-5 7 -
L a c t o b a c i l l u s e a s e l - -Lh o f Indi an a 14696
Shape, s i z e , arrangement,
and r e l a t i o n t o Og
-
Long r o d s , o c c u r r i n g in ch ai ns
some s i n g l e c e l l s s e e n . Mieroaerophillic
Staining c h a r a c te ris tic
M otility
Gram p o s i t i v e .
Non-motile.
Agar c o l o n i e s
Poor growth on s u r f a c e .
Pin p o i n t c o l o n i e s .
Carbohydrates
f e r m e n ta ti o n
Acid from: g l u c o s e , f r u c t o s e ,
mannose, g a l a c t o s e , s u c r o s e .
Litmus milk
Acid and c o a g u l a t i o n . Litmus
reduc ed, p a r t i a l d i g e s t i o n .
Remarks
Growth was abundant on en­
r i c h e d medium.
Pr o te u s v u l g a r i s BB 151
Shape, s i z e , arrangement,
and r e l a t i o n t o 02
S t r i g h t r o d s , .75 x 2 .0 u
o c c u r r i n g in c h a i n s . Aerobic
Staining c h a r a c te ris tic
M otility
Gram n e g a t i v e .
M otil e .
Agar c o l o n i e s
Ameboid, opaque, gray.
Car bohydrates
fermentation
Acid and gas from g lu c ose ,
f r u c t o s e , g a l a c t o s e , m a lt o s e .
No a c id or gas from l a c t o s e .
Litmus milk
S l i g h t a c i d , becoming markedly
a l k a l i n e , quick p e p t o n i z a t i o n .
-58-
B a c i l l u s s u b t i l i s ATCC 6093
Shape, s i z e , a rr a n g e m e n t,
and r e l a t i o n to Og
Rods, .7 x 2 .5 u g e n e r a l l y
s i n g l y , not e n c a p s u l a t e d .
Spores .70 u in d ia m e te r .
Aerobic
Staining c h a r a c te r is tic
M otility
Gram p o s i t i v e . Uniformly s t a ­
in e d.
M otil e .
Agar c o l o n i e s
Rough, opaque, d u l l s p re a d in g ,
s l i g h t l y gra y .
•i
Carbohydrates
fermentation
Acid but no gas from g lu c o s e ,
su crose and m a n n it o l. No a c id
from l a c t o s e .
Litmus milk
Slow p e p t o n i z a t i o n , becoming
alkaline.
Remarks
On bro th i t gave wrinkled
rough f i l m .
Pseudomonas f l u o r e s c e n s U, of Indiana
Shape, s i z e , a rr a n g e m e n t,
and r e l a t i o n t o Og
Rods, 0 . 4 x 1.5 u o c cu r ri ng
s i n g l y and in p a i r s . A e r o b i c .
Staining c h a ra c te ris tic
M otility
Gram n e g a t i v e .
Mot ile .
Agar c o l o n i e s
Comparatively l a r g e , slimy,
r e d d i s h l a y e r becoming re d d i s h
gra y .
Carbohydrates
fermentation
Acid from g l u c o s e .
Litmus milk
No c o a g u l a t i o n becoming a l k a ­
line.
Remarks
On b r o th t u r b i d f l o c c u l e n t and
g r e e n is h p e l l i c l e and gray
sediment.
-5 9Sta phylococcus aureus BB 137
Shape, s i z e , a r r a n g e m e n t ,
and r e l a t i o n t o Og
S p h e r i c a l , 1.0 u in dia m e te r ,
o c c u r r i n g in i r r e g u l a r clumps.
Some s h o r t c h a i n s a l s o s e e n .
Aerobic.
Staining c h a r a c te ris tic
M otility
Gram p o s i t i v e .
Non-motile.
Agar c o l o n i e s
C i r c u l a r , smooth, white e n t i r e .
Carbo hyd rates f e r m e n t a t i o n
Acid from: g l u c o s e , l a c t o s e ,
s u c r o s e , m a n n i t o l . No a cid
from r a f f i n o s e , s a l i c i n .
Litmus milk
Acid c o a g u l a t i o n .
Remarks
Coagulase p o s i t i v e
4
•7
,
X,
-60T a b l e XIX
Media I and I I used f o r growth of c e l l s
Medium I
T r y p t i c a s e Soy Broth BBL
Dex trose
Yeast e x t r a c t
D i s t i l l e d water
PH
30.0
5 .0
2 .0
1000.0
7 .2
g
g
g
ml
3 .0
5.0
7.5
2.0
2.5
1000.0
7.1
g
g
g
g
g
ml
Medium I I
Bacto Beef e x t r a c t
Bacto Tryptone
Dex trose
Yeast E x t r a c t ( D if c o )
Potassium hydrogen phosphate ( D i b a s i c )
D i s t i l l e d water
PH
-61T a b l e XX
Composition of s o l i d s u b s t r a t e s
1.
Casein a g a r :
Casein
Agar (Difco)
D i s t i l l e d wa ter
pH
2.5 g
1.5 g
100.0 ml
7 .0
2.
Bacto Sta phylococcus Medium HO (d e h y d ra te d ) S t o n e ' s
re a ctio n for the detection of liq u e fa ctio n of g e la tin
was perfo rm ed .
3.
Milk ag ar :
Bacto Agar
Skim Milk
pH
4.
1.5 g
100.0 ml
7 .0
D i l u t e d milk a g a r :
a)
Skim Milk
Agar (Difco)
D i s t i l l e d water
pH
5 ml
1.5 g
I OO-O ml
7.0
b)
Skim Milk
Agar (Difco)
D i s t i l l e d water
pH
2 ml
1.0 g
100.0 ml
7 .0
T a b l e XXI
The m icr o g ra m s o f t y r o s i n e and t r y p t o p h a n e
2 4 and 48 h o u r s and t h e d i f f e r e n c e s .
lib erated
by
in tra cellu la r
enzymes
from c e l l s
grown
for
Enzyme s u b s t r a t e r e a c t i o n time
Organisms
________ 24 hours_____________ 48 hours_______________ 96 hours_______________ 144 hours
I
II
D iff.
I
I
II
D iff.
D iff.
II
Diff.
I
II
Sl I a c t i s
1.35
1.40
+ .05
1.70
2.00
+.30
1.80
2.2 0
+ .40
1.90
2.40
+ .50
Ll dextranicum
1.25
1.30
+.05
1.35
1.70
+.35
1.40
1.85
+ .45
1.40
2.00
+.60
Ll c a s e i
1.45
1.60
+. 15
1.80
2 .20
+ .40
2 .0
2.40
+ .40
2.10
2.50
+.40
Pl v u l g a r i s
1.30
1.30
+.00
1.70
1.90
+ .20
1.90
2.2 0
+ .30
2.00
2.30
+ .30
Bl s u b t i l i s
1.50
1.80
+ .30
2.1 0
2.50
+ .40
2.75
3.10
+ .35
3.00
3.30
+.30
Pl f l u o r e s c e n s
1.50
1.70
+.20
2 .2 0
2.30
+ .10
2.85
3.00
+ .15
3.10
3.30
+.20
S. aureus
1.40
1.60
+ .20
2.15
2.40
+ .25
2.70
2.90
+ .20
2.95
3.20
+ .25
I - cells
II - c e lls
grown
grown
for
for
24 h o u r s.
48 h o u r s .
T a b l e XXII
T h e m ic r o g r a m s o f t y r o s i n e and t r y p t o p h a n e
4 8 and 9 6 h o u r s and t h e d i f f e r e n c e s .
lib erated
by
in tra cellu la r
enzymes
from c e l l s
grown
for
Enzyme s u b s t r a t e r e a c t i o n time
Organisms
I
24 hours
II
D iff.
I
48 hours
II
D iff.
I
96 hours
II
D iff.
I
144 hours
II
Di f f .
5L l a c t i s
1.40
1.60
+.20
2.0 0
2.15
+ .15
2.2 0
2.40
+ .20
2.40
2.60
+ .20
L l dextranicum
1.30
1.50
+.20
1.70
1.90
+.20
1.85
2 .1 0
+ .25
2.00
2.10
+ .10
Lll c a s e i
1.60
1.70
+.10
2 .2 0
2.40
+ .20
2.40
2.60
+.20
2.50
2.70
+.20
Pl v u l g a r i s
1.30
1.35
+ .05
1.90
1.80
+.10
2.2 0
2.1 0
+.10
2.30
2.30
.00
Bl s u b t i l i s
1.80
2.00
+.20
2.50
2.80
+ .30
3.10
3.45
+ .35
3.30
3.80
+ .50
Pl f l u o r e s c e n s
1.70
2 .1 0
+.40
2.30
2.60
+ .30
3.00
3.25
+ .25
3.30
3.90
+.60
S l aureus
1.60
1.80
+.20
2.40
2.70
+ .30
2.90
2.95
+ .05
3.20
3.45
+.25
I II -
cells
cells
grown
grown
for
for
48 hours
96 h ours
T a b le XXIII
The m icr o g ra m s o f t y r o s i n e and t r y p t o p h a n e
9 6 and 144 h o u r s and t h e d i f f e r e n c e s .
lib era ted
by
in tracellu lar
enzymes
from c e l l s
grown
for
Enzyme s u b s t r a t e r e a c t i o n time
Organisms
_______ 24 hours_______________ 48 hours_______________ 96 hours______________ 144 hours
D iff.
I
II
D iff.
D iff.
I
II
I
II
Di ff.
I
II
1.30
-.30
2.15
1.90
-.25
2.40
2.1 0
-.30
2.60
2.00
- .6 0
1.50
1.20
-.30
1.90
1.80
-.1 0
2.10
1.90
- .2 0
2 .10
1.90
-.2 0
Li c a s e i
1.70
1.40
-.30
2.40
1.75
-.65
2.60
1.95
-.65
2.70
2.00
-.70
F\ v u l g a r i s
1.35
1.30
-.05
1.80
1.70
-.1 0
2.10
1.90
- .2 0
2.30
2.00
- .3 0
Bi s u b t i l i s
2.00
1.70
-.30
2.80
2.40
-.40
3.45
2.80
-.65
3.80
2.0 0
-.8 0
Pi f l u o r e s c e n s
2.10
1.50
- . 60
2.60
2.2 0
-.40
3.25
2.75
-.50
3.90
2.95
-. 9 5
Si aureus
1.80
1.40
-.40
2.70
2.1 0
-.60
2.95
2.60
-.35
3.45
2.80
- . 65
dextranicum
I - cells
II - c e lls
grown
grown
for
for
96 hours
144 h ours
-6 4-
1.60
S_ l a c t i s
T a b l e XXIV
C o m p a r a t i v e a m o u n ts ( m i c r o g r a m s ) o f t y r o s i n e and t r y p t o p h a n e l i b e r a t e d
p r e p a r e d from c e l l s grow n on medium I and g e l a t i n r i c h medium*.
by
in tra cellu la r
enzymes
Enzyme s u b s t r a t e r e a c t i o n time
Organisms
I
24 hours
II
D iff.
48 hours
II
D iff.
I
96 hours
D iff.
II
I
I
144 hours
II
D iff
1.30
1.40
+.10
1.70
1.90
+ .20
1.80
2.0 0
+ .20
1.90
2 .00
+.10
L. dextranicum
1.25
1.30
+.05
1.35
1.50
+. 15
1.40
1.55
+.15
1.40
1.60
+.20
L. c a s e i
1.45
1.40
-.05
1.80
1.70
- .1 0
2.00
2.0 0
.00
2.10
2 .20
+.10
P. v u l g a r i s
1.30
1.90
+.60
1.70
1.40
-.30
1.90
2.60
+ .70
2.00
2.90
+.90
B. s u b t i l i s
1.50
1.95
+.45
2 .1 0
2.60
+.50
2.75
2.95
+ .20
3.00
3.30
+.30
P. f l u o r e s c e n s
1.50
1.75
+.25
2 .2 0
2.40
+ .20
2.80
2.85
+ .05
3.10
3.20
+.10
S. aureus
1.40
2 .1 0
+.70
2.15
2.70
+.55
2.70
3.50
+ .80
2.95
3.90
+ .95
I II* -
cells
cells
g r o w n on b a s i c
grow n on b a s i c
medium I f o r 2 4 h o u r s
medium I - 0.5% g e l a t i n
for
24 hours
-65
S. I a c t i s
T a b l e XXV
C o m p a r a t i v e a m o u n ts ( m i c r o g r a m s ) o f t y r o s i n e and t r y p t o p h a n e l i b e r a t e d
p r e p a r e d from c e l l s gro wn on medium I and c a s e i n r i c h m e d iu m * .
by
in tra cellu la r
enzymes
Enzyme s u b s t r a t e r e a c t i o n time
Organisms
I
24 hours
II
D iff.
I
48 hours
II
D if f .
I
96 hours
II
D iff.
I
144 hours
II
D iff.
1.80
+0.45
1.70
2.60
+0.90
1.80
3.10
+1.30
1.90
3.40
+1.50
L l dextranicum 1.25
1.85
+0.60
1.35
2.40
+1.05
1.40
3.00
+1.60
1.40
3.00
+1.60
Lr c a s e i
1.45
1.60
+0.15
1.80
3.20
+2.40
2 .0
3.60
+1.60
2.10
3.90
+1.80
Pr v u l g a r i s
1.30
1.70
+0.40
1.70
2 .6
+0.90
1.90
2.85
+0.95
2.00
3.10
+1.10
Bjl s u b t i l i s
1.50
4.0
+2.5
2.1 0
4 .5
+2.40
2.75
4.70
+1.95
3.00
5.30
+2.30
Pr f l u o r e s c e n s 1.50
4.2
+2.7
2 .2 0
4.8
+2.60
2.80
4.90
+2.10
3.10
5.20
+2.10
1.40
3.8
+2.4
2.15
4.2
+2.05
2.70
4.80
+2.10
2.95
5.10
+2.15
Sr aureus
I - The organisms were grown on medium I fo r 24 hours.
I I * - The organisms were grown on medium I plus 0.5% c a s e i n fo r 24 hours.
-66
1.30
lactis
T a b l e XXVI
C o m p a r a tiv e am ounts ( m i c r o g r a m s ) t y r o s i n e and t r y p t o p h a n e l i b e r a t e d
p r e p a r e d f r o m c e l l s g r o w n on m e d i u m I a n d c a r b o h y d r a t e f r e e m e d i u m .
by
in tracellu lar
enzymes
Enzyme s u b s t r a t e r e a c t i o n time
Organisms
I
24 hours
II
D iff.
I
48 hours
II
D if f .
I
96 hours
II
D iff.
I
144 hours
II
D iff.
1.30
1.70
+0.35
1.70
2.40
+0.70
1.80
2.60
+0.80
1.90
2.75
40.85
L. dextranicum 1.25
1.55
+0.30
1.35
1.75
+0.40
1.40
2.0 0
40.60
1.40
2.1 0
+0.70
L. c a s e i
1.45
1.80
+0.35
1.80
2.40
+0.60
2.00
2.65
40.65
2.10
2.80
40.70
P. v u l g a r i s
1.30
1.50
40.20
1.70
1.95
+0.25
1.90
2 .2 0
40.30
2.0 0
2.40
40.40
B. s u b t i l i s
1.50
1.90
+0.40
2 .1 0
2.60
+0.50
2.75
3.60
+0.85
3.00
4.25
+1.25
P. f l u o r e s c e n s 1.50
2.40
+0.90
2 .2 0
3.20
+1.00
2.80
3.90
+1.10
3.10
4.50
+1.40
1.40
2 .1 0
+0.70
2.15
2.80
+0.65
2.70
3.40
40.70
2.95
3.55
40.60
S. I a c t i s
S. aureus
I - The organisms grown on medium I f o r 24 hours.
I I - The organisms grown on c a r b o h y d ra t e f r e e medium f o r 24 h o u r s .
T a b l e XXVII
C o m p a r a t i v e a m o u n ts ( m i c r o g r a m s ) o f t y r o s i n e and t r y p t o p h a n e l i b e r a t e d
p r e p a r e d f r o m c e l l s g r o w n on m e d i u m I a n d c a s e i n r i c h m e d i u m * .
by
in tra cellu la r
enzymes
Enzyme s u b s t r a t e r e a c t i o n time
Organisms
_______ 24 hours_______________ 48 hours_______________ 96 hours______________ 144 hours
D iff.
I
II
I
II
D iff.
D if f .
I
II
I
II
Dif f.
JL I a c t i s
1.30
1.35
+ .00
1.70
1.75
+.05
1.80
1.85
+ .05
1.90
2.00
+.10
Lll dextranicum
1.25
1.25
.00
1.35
1.30
-.05
1.40
1.40
.00
1.40
1.40
.00
Lll c a s e i
1.45
1.40
m
q
I
1.80
1.85
+.05
2 . OU
2 .0 0
00
2 .10
2 10
00
f\ v u l g a r i s
1.30
1.30
.00
1.70
1.60
-.1 0
1.90
1.90
.00
2 .00
2.00
.00
EL s u b t i l i s
1.50
1.55
+.05
2 .1 0
2.20
+.10
2.75
2.85
+ .10
3.00
3.10
+ .10
1.50
1.50
.00
2 .2 0
2.20
.00
2.80
2.80
.00
3.10
3.15
+ .05
1.40
1.40
.00
2.15
2.00
+. 15
2.70
2.85
+ .15
2.95
3.10
+.15
j\
fluorescens
S. aureus
I - cells
II* - c e l l s
g r o w n on m e d i u m I f o r 2 4 h o u r s
g r o w n on m e d i u m I p l u s 0 . 5 % C a s i t o n e
for
24 hours
MONTANA STATC I.. i n . . ____
3 1762 10020815 4
N378
V142
cop. 2
Vadehra, D. V.
S tu d y o f th e i n t r a c e l l u l a r
- C-