THE OXIDATION OF COEN OIL BY BACTEBIA

by

JOHN ORVIN MUNDT

A THESIS

S u b m i t t e d to t h e G raduate S c h o o l o f M ic h ig a n
S t a t e C o l l e g e o f A g r i c u l t u r e and A p p l i e d
S c ie n c e in p a r t i a l f u lf ilm e n t o f the
requirem ents f o r th e degree o f

DOCTOR OF PHILOSOPHY

Department o f B a c t e r i o l o g y
1943

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Aeknowle dgement
The a u t h o r w i s h e s t o e x p r e s s h i s s i n c e r e a p p r e c ­
i a t i o n t o D r . F . W. F a b i a n f o r h i s i n s p i r a t i o n a l
encouragem ent and h is p rofound i n t e r e s t in t h i s
w o r k ; t o P r o f e s s o r C . D. B a l l f o r h i s g u i d a n c e
w i t h t h e c h e m i c a l a n a l y s e s ; a n d t o D r . C. H o p p e r t ,
who s u g g e s t e d a p o r t i o n o f t h e w o r k .

TABLE OF CONTENTS

Page
In trod u ction

1

R evie w o f t h e L i t e r a t u r e

3

Experim ental procedure

11

a . General procedure

11

b* The a b i l i t y o f b a c t e r i a to u se c o r n o i l

19

c* Comparison o f t h e o x i d a t i o n o f c o r n o i l w i t h
l i p o l y s i s o f c o r n and b u t t e r o i l s and t h e
p roduction o f o xid ase

28

d . Rate o f c o n s u m p t io n o f o x y g e n a s measured
m a n o m e tr ic a lly , the e v o l u t io n o f carbon
d i o x i d e , and t h e r e s p i r a t o r y q u o t i e n t

34

e* The e f f e c t o f lo w e r e d pH and o f sodium c h l o r ­
i d e upon b a c t e r i a l r e s p i r a t i o n

42

£.

The e f f e c t o f t h e p h o s p h a te i o n , h y d r o q u i n o n e ,
A v e n e x , and r a n c i d o i l upon t h e co n su m p tio n o f
oxygen
48

g . C hem ical d e t e r m i n a t i o n s

53

D isc u ssio n o f the r e s u lts

64

Summary and c o n c l u s i o n s

72

A pp en d ix A

74

L iterature c ite d

76

TABLES

T a b le 1 . Cmm. o f o x y g e n t a k e n up by b a c t e r i a i n t h e
Warburg i n b u f f e r e d s a l i n e s o l u t i o n , and i n
b u ffe r e d s a l i n e p lu s e m u ls if ie d corn o i l or
M/30 g l y c e r o l
T a b l e 2 . M in u te s r e q u i r e d by one m l. o f c e l l s to r e ­
duce m e t h y l e n e b lu e i n b u f f e r e d s a l i n e s o l u t i o n ,
and i n b u f f e r e d s a l i n e p l u s e m u l s i f i e d corn o i l
o r M/30 g l y c e r o l
T a b le 3 . Comparison o f t h e o x i d a t i o n o f c o r n o i l and
g l y c e r o l w i t h l i p o l y s i s o f c o r n and b u t t e r o i l s
and w i t h t h e p r o d u c t i o n o f o x i d a s e
T a b le 4# Cmm. o f o x y g en t a k e n up by b a c t e r i a i n t h e
p r e s e n c e o f c o r n o i l , t h e carbon d i o x i d e e v o l v e d
and t h e r e s p i r a t o r y q u o t i e n t
T a b le 5 . The e f f e c t o f pH o f t h e c o n t e n t s o f t h e cup
upon t h e cmm. o f o x y g e n u t i l i z e d by b a c t e r i a in
th e presence o f corn o i l
T a b le 6 . The e f f e c t o f t h e p e r c e n t a g e o f NaCl i n t h e
cup upon t h e cmm. o f oxygen u t i l i z e d by b a c t e r i a
i n t h e p r e s e n c e o f c o r n o i l a t pH 6 . 8 5
T a b l e 7 . The e f f e c t o f th e p r e s e n c e and th e a b s e n c e
o f th e p h o s p h a t e i o n upon t h e cmm. o f o x y g e n
u t i l i z e d by b a c t e r i a i n t h e p r e s e n c e o f e m u l s i ­
f i e d co r n o i l
T a b l e 8 . The e f f e c t o f hydroquin one and o f Avenex
upon th e cmm. o f oxy g en u t i l i z e d by b a c t e r i a
in the p resen ce o f e m u ls if ie d corn o i l
T a b l e 9 . The e f f e c t o f f r e s h and o f r a n c i d corn o i l
upon t h e cmm. o f oxy g en u t i l i z e d by b a c t e r i a i n
t h e Warburg ap p ar a tu s

Page

T a b le 10* The e f f e c t o f b a c t e r i a l growth i n m i n e r a l
s a l t s - f r e s h c o r n o i l medium upon ( a ) a c i d num­
ber
( b ) p e r o x i d e number ( c ) i o d i n e number
( d ) s a p o n i f i c a t i o n number ( e ) r e f r a c t i v e i n d e x
( f ) a ld e h y d e number ( g ) K r e i s t e s t and ( h )
a cid s in l i q u i d resid ue

60

T a b l e 1 1 . The ( a ) a c i d number ( b ) p e r o x i d e number
( c ) i o d i n e number ( d ) K r e i s t e s t o f o i l s from
c o r n o i l - m i n e r a l s a l t s medium; th e medium p l u s
h y d r o q u i n o n e ; t h e medium p l u s Avenex; and ea c h
o f t h e media p l u s one p er c e n t p e p t o n e , f o l l o w ­
in g growth o f A l c a l i g e n e s l i n o l y t i c u s and P s e u d o ­
monas a e r u g i n o s a
63

-1 -

INTBODUCT ION

The f a c t
has

that

b e e n known t o

teur.

It

study.

is,

bacteria

oxidize

bacteriologists

however,

t wo

s e p a ra te problem s:

the o th e r ;

Because o f
terial

the

hydrolysis

greater

ease

however,

both as

and f a c i l i t i e s

regards

lipolysis
acids

there

are

frequently

economic e f f e c t s
or lipolysis,

fo r study,

deals w ith th is

and a lso

upon f l a v o r .

It

is

the

subject.

that

the

dairym an,

s h o u l d be

interested

problem .

made o f
bacteria
ed;

this

problem .

relatively

The a b i l i t y

to o x id iz e o r u t i l i z e

b u t much o f

the

detail

of

fatty

and those

and o i l s ,

by b a c t e r i a

is

few s t u d i e s

have been

of

less

certain species

triglycerides
th e problem is

* T h e t e r m s " f a t ” a n d ’' o i l ” a r e
triglycerides.

true

liberated

making use o f f a t s

c o mmo n , a n d c o n s e q u e n t l y

is

under which

of food in d u s tr ie s

The o x i d a t i o n o f t r i g l y c e r i d e s

and the

This

operators

in t h is

of bac­

much t h e g r e a t e r

e f f e c t of the

natural

in­

exceptions.

th e mechanism and c o n d i t i o n s

occurs,

esters

and o x id atio n .

one p r o b l e m

triglycerides,

amount o f l i t e r a t u r e

tim e o f P a s ­

n eg lected phase of

lipolysis

far-reaching

of

the

a lte r a tio n of glycerol

T h e t wo a p p a r e n t l y go t o g e t h e r ;
volves

since

a relatively

The m i c r o b i o l o g i c a l

involves

fats*~ ( t r i g l y c e r i d e s )

lim ited

is

unquestion­

as y e t

in th is

of

unknown.

paper

to

-2 H ydrolysis
fats

or oxidation of

" ra n c id /1 that

tionable
notes

is,

the

flavors

and odors*

rancid

condition,

the

drolytic"

or "oxidative,"

ification

in the

given r is e

to

a relativ ely

adaptable

to

the

triglyceridesigators
study;

are

recent

but

tool,

Several

confirm ed;

a n d s o me f a c t s

have u n p le a s a n t and o b j e c ­

the

is

which de­

The t y p e o f
is

"hy­

used fre q u e n tly w ithout

evident

qual

r e a c t i o n which has

from the

context.

groundwork f o r th e a p p l i c a t i o n
th e Warburg a p p a r a t u s ,

which is

m icrobiological oxidation of

conclusions
problems

of

re n d e r the

may b e m o d i f i e d b y e i t h e r

the

study o f

frequently

The n o u n " r a n c i d i t y , "

condition

This p a p e r p r e s e n t s
of

fats

literatu re-

this

fats

are

interest

r e a c h e d by o t h e r i n v e s t ­
suggested fo r

are

presented-

future

-3 -

KEVIEW OF THE LITERATURE

In a s e r ie s
blatt
the

fur

especially
to

(1898)

reports

the

stated

unsaturated fa tty

that

various

growth

R itsert

to

(1920)

Holm, G reenbank,
essential

the

He r e ­

of

acids

triglycerides

and g l y c e r o l ,

R itsert*

(quoted

m icroorganism s,

notably

into

the organism s

rancidity

rancid

could not

in la r d

and b u t t e r -

combined a c t i v i t y o f oxygen and
In t h i s ,

a n d Emery a n d H e n l e y
and Deyscher

z i k e r a n d Hosman ( 1 9 1 7 )

groups-

introduced

bacteria.

fo r oxidation-

by b a c t e r i a ,

fats.

believed th a t

r a t h e r th an to

Salkowski

however,

in sweet

wa s d u e c h i e f l y

were

upon

(1889) and F a h rio n

were u t i l i z e d *

if

reports

of butter o il

the c o n s t it u e n t s

o i d ia ,* * and y e a s ts ,

in itiate

(1902)

by D u c l a u x a n d G r o e l g e r

growth o c cu rre d ;

lig h t,

acids

by h y d r o l y s i s ,

by J e n s e n )

fat

fatty

who f o u n d t h a t o f

lib erated

fats,

of

in the C e n tra l-

by t h e Pseudom onas an d S e r r a t i a

in b u tte r o i l ,

m olds,

published

B a k te rio lo g ie , O rla Jensen

u tilizatio n

fers

of a rtic le s

It

(1927)

Browne ( 1 8 9 9 ) ,

(1922)
stated

concurred,

th a t oxygen is

may b e s i g n i f i c a n t

failed

to

include

w hile

t h a t Hun-

bacteria

in t h e i r

* R itsertInaugural d is s e rta tio n .
Berne, S w itzerlan d ,
1899- Q u o t e d by O r l a J e n s e n , C e n t r a l b l * f . B a k t - , I I
Abt ♦, ^8:11-16, 1902* * The te r m " o i d i a " i s a l i t e r a l t r a n s l a t i o n o f t h e word
f o u n d i n t h e p h r a s e " S c h i r a m e l p i l z e , O i d i e n und H e f e n . "
c f . O rla Jensen, lo c . s i t .

-4 d is c u s s io n o f agents
Fierz-D avid
volved

(1985)

in oxidative

Schreiber

bringing

about o x id a tio n of fats*

did not b e liev e

that

that

the

(1902) s t a t e d

that

u tilization of

p re s e n c e o f oxygen*
review

that

pure

all

cases

the

discrim inate,
It
sible

of

changes

n o ted the

fat,

to

converted o le ic

He c o u l d n o t

find

carbon dioxide

J e n s e n and G -re ttie
oxidized

the

fat

fatty

cultures*
lim iting

acids

fat,
is

in

acids.

lipolytic
elaidic

are

respon­

fats

'De

bacteria
acid,

and

a c i d upon c o n t i n u e d

interm ediate

the

in­

accompanying m ani­

a c i d to

latter

and

products of

w e r e o x i d i z e d com­

and water*

( 1 9 3 7 ) o b t a i n e d two o r g a n i s m s w h i c h

of bacon.

Peppier

(1941)

and th e

stu d ied the

de-

u tilizatio n of

i n m edia o f known c o m p o s i t i o n by w a s h e d

The l e n g t h o f
factor

in a b rie f

f o l l o w e d by t h e

that bacteria

nine

com position o f pure t r i g l y c e r i d e s
pure

demanded t h e

glycerol

fatty

and the

and

in f la v o r and c h a r a c t e r i s t i c s *

o x i d a t i o n and co n clu d ed t h a t
pletely

that

u tiliz a tio n of

disappearance of the

incubation.

almond o i l

(1906) p o in te d o u t

(1907) o b t a i n e d from s o i l

which a p p a r e n t l y

in­

m icroorganism s,

component u t i l i z e d ,

complete

as

by b a c t e r i a

n i t r o g e n was n e c e s s a r y ;
first

fat

w e r e known w h i c h a t t a c k e d

the o x id a tio n of

festations

*

to

has been p roved a d e q u a te ly

for

K ruyff

fats

D r. Rahn

few b a c t e r i a

th at organic

were

rancidity.

( w i t h w h i c h h e w o r k e d ) wa s u s e l e s s
also

bacteria

in

the

the

fatty

hydrolysis

acid
of

the

c h ain proved a
e s t e r and the

-5 oxidation of
Fouts
although

the

acid*

(1940)

the

n o t e d t h a t s o me b u t t e r s a m p l e s w e r e

fatty

sam ples w ith h ig h
tio n

the

a c i d n u m b e r o f e a c h was l o w , w h i l e o t h e r
titratab le

known f o r t h e i r

acids.

Schonbrunner

bond in o l e i c
illus

a c i d wa s

L ivschitz,

are

Jensen,

interm ediate
(1936)

to

and cap-

the

of

t h e a c i d was t h e s o l e

are agreed

1902;

1906;

1928;

oxidation of

on th e

and o th e r s )
fatty

o th e r hand,

products.

Both Derx (1925)

found th a t

methyl ketones

a n d Hammer

were

evanescent

th a t of Jacobson

nut o il

reported that

appeared only

ycetes.
oxalic

e a r l i e r work is

Schenker
and o le ic

classical

work o f

the

under the

(1921),
acids,

fruity

of high m o lecu lar

ester of

using A s p e rg illu s

(1924)

(1918)

ra n c id coco­

influence of c e rta in

and g ly c e r o l,

Starkle

acids

do p r o d u c e

w eight.

is

De

H orow itz-V lassow a and

and C h a rik ,

Molds,

(Rahn,

acids

i n which i t

double

c o l l , Bac­

p ro d u cts o f the o x id a tio n of f a t t y
Among t h e

u tilize

medium.

products of the

recovered.

and Bryant

reported th at

sodium s a l t

Pigulew ski

interm ediate

seldom
such

1935;

caproic,

of bac­

f l u o r e s c e n s , a n d o t h e r unnamed

investigators

1907; O r l a

were a b le

butyric,

carbon in a s y n th e tic

A number o f
K ruyff,

of

Investiga­

species

s a t u r a t e d by E s c h e r i c h i a

m i c r o o r g a n i s m s when t h e
of

ability

(1940)

s u b t i l i s . Pseudomonas

source

and several

lipolytic

sodium an d c a lc iu m s a l t s

ry lic

that

a c i d i t y were " s w e e t . "

showed t h a t O o sp o ra l a c t i s

teria

rancid,

Phycom-

n ig e r, recovered

from o l i v e o i l .

The

s h o w s how m e t h y l k e t o n e s

may

-6 be p ro d u ced

from f a t t y

acids

beta-oxidation,

ketonic

acid

decarboxylated,

leaving

the

b o n atom t h a n
sessed.

the

Stokoe
due t o

However,
the

fatty

is

acid,

w h i c h was

correspondingly

(1921)
the

at

found t h a t

(1928)

ketones

the

of

B acteria
triglycerides

action

the

color

m oisture,

results

(1939) o b t a i n e d a c e to n e

f o u r te e n carbon atom s,

not

detrim ental

in a l l

using the

instances

may a c c r u e

products
Tracey,

as th e

(K ertesz,
Bamsey,

is

than

in m ilk of

sen,

and W hitaker

(1937).

o x i d a t i o n o f m arket milks
butterfat

the

resu lt of b a c te ria l

1934 a n d 1935;

an d Ruehe,

according

A lthough a l l

H i t t e r and

1933).

h ig h er and the b a c t e r i a l

standard grade,

to

D efinitely

content

the

and a i r .

media on w h i c h

the

that

in

h e p t y l - , and nonyl-

o c c u r r e d i n m a r k e t m ilk s o f premium g r a d e ,

sible

defects.

factors

flavors
fat

pos­

from media

chief

light,

car­

in coconut o i l s

w i t h which th e y a r e a s s o c i a t e d *

1935;

then

glaucum .

are

in d a iry

C hristen,

f o u r to

is

acid.

of o il-g ela tin

T h aler and O e is t

acids

By

precursor,

as were c e r t a i n

were

distillatio n s

mold P e n i c i l l i u m

beneficial

"perfume"

he r e p o r t e d o b t a i n i n g a m y l-,

molds had grow n.

acid

isolated

tim e he b e l i e v e d t h a t

by s t e a m

from f a t t y

its

higher f a t t y

a c t i o n o f m olds,

that

this

molds*

m ethyl keto n e w ith one l e s s

rancidity of trig ly cerid es

L ater

formed;

A number o f s u c h k e t o n e s w ere

containing

wa s

the

by th e a c t i o n o f

Oxidized
in which

content

low er,

to B o la n d ,

reactions

are

not yet

understood,

plays

a not

insignificant

Soren­

in the
it

is

role

pos­
(Rol­

-7 and and T re b lo r,
to

the

tains

The b e n e f i c i a l

growth o f b a c t e r i a

in the

1931 a ,

b).

W hite

(1941)

that

according
B acillus

to Beckman

point out

and o x id iz e d

a criterion of

c o u l d be u s e d t o

(1930),

delbrueckia^ for

carbohydrate

flavor

rancidity

and p ro te in

the anaerobic
in m ateria ls

recovery of o i l s .

the

o ils

w o u l d n o t b e a l t e r e d by b a c t e r i a .
On t h e

o t h e r hand,

that

fats

are

bacteria

carbon

in Imhoff tanks

dioxide

as

copra

and w a te r ,

u tilize

to

facili­

conditions,

the

not s p a re d because o f

types of organic

shown by t h e work o f Neave a n d B u s w e ll

use o f

decom position o f

Under a n a e ro b ic

t h e i r occurrence w ith o th e r

advantage,

who r e c o m m e n d e d t h e

tate

to

(F ra z ie r and

fat.

The o x y g e n r e l a t i o n s h i p

that

t u r n main­

considered the o x id atio n -

r e d u c t io n p o t e n t i a l o f bacon f a t
of

potential

to o x i d a t i o n

may b e t r a c e d

which i n

Webb a n d H i l e m a n ( 1 9 3 7 )

a n i n c r e a s e d E^ l e a d s

i n m i l k a n d cream*

effects

product,

a low ered o x id a tio n -re d u c tio n

W hittier,
that

1937)*

m aterials

(1927),
fats,

while o t h e r

is

who f o u n d

converting

them

types of o rg an ic

m a t t e r a p p e a r to be s p a r e d .
Few i n v e s t i g a t o r s
which b a c t e r i a

bring

is

to

due

in p a rt

not been o b ta in e d

the

h ave r e p o r t e d upon t h e

about
fact

the o x id a tio n of f a t t y
that

from b a c t e r i a l

*-B a c i l l u s d e l b r u e c k i a
ed itio n ).

is

mechanism by

interm ediate
cultures

not lis te d

acids.

products

and f a t t y

in B ergey's

This

have

sub-

Manual

(fifth

strates

containing

v o lv ed in the
V lassova

t r y g l y c e r i d e s , and in p a r t

study of

and L i v s c h i t z ,

upon o l e i c

acid,

branch of physiology

1935)*

although

chem ical o x i d a t i o n ,
tion-

this

and aldehydes a re
attacks

upon th e

iodine

fat

fat

tubes

of

the

b a c t e r i a l m etabolism ,
carbon dioxide

soybean o i l
this

is

using

from o l i v e

fa t as

hundred years
heptylic,

source

O ther products,

oil

r e s u l t of purely
the

Pow ick,

on th e s u b je c t

suberic,

betw een

end pro d u cts o f

the sole

pim elic,

of

carbon,

however,

(1938) have

have

isolated

in which P u r p u r o b a c t e r i a

and a ld e h y d es were

used*

the

E ther-insoluble oxystearic

p o ssib ly because of
were

to which

wide v a r i a t i o n s

agreed that

b u t a s m a ll number o f

bacteria

the

the

acid,

and
as

r e c o v e r e d from e m u l s i f i e d

by H o ro w itz a n d L i v s c h i t z

found as the
acid,

are

and w ater-

grown-

w e ll as ketones

direct

The i n a d e q u a c y o f t h i s

Thus, P ig u le w s k i and C h a rik

r o d forms have

numbers,

uninnoculated c o n tro ls-

Many i n v e s t i g a t o r s

acid

view o f

respira­

peroxide

One o f t h e m o s t

had been added-

duplicate

ketostearic

bacterial

r e c o v e r e d from c u l t u r e s

r e a l i z e d when o n e n o t e s

been found-

in­

p r o b l e m was t h a t o f P i g u l e w s k i a n d C h a r i k

method i s

are

tim e

(Horowitz-

point of

numbers,

tim e-consum ing♦

( 1 9 3 8 ) who w e i g h e d t h e
known am ounts o f

from th e

be a p p l i e d t o

The d e t e r m i n a t i o n o f

the

The c h e m i c a l e x p e r i m e n t a t i o n

fru itfu l

cannot

to

(1935)-

In com parison,

compounds w h i c h h a v e b e e n

chem ical o x id a tio n of o le ic
s m a ll amount o f work i n w hich
reviewing the
(1923),

adipic,

literatu re

reports
azelaic,

the

of a

finding of

dihydroxystearic,

-9 b utyric,

acetic,

form ic,

pelargonic,

w e ll as h e p t y l i c ,

caproic,

aldehydes*

(1908) o b t a i n e d

Scala

oenanthylic,

idative

the

(1931),

acids,

nonic

acid,

acids.

conditions

iso-oleic

E llis

acid,

(1932),

tion.

However,

tissue

or of

to

none o f

biological

om ega-oxidation
bacterial

(1931)

treatm ent.

a c i d a t 100°
acid,

of form ic

and

in o rd er to

method o f

in th e ir

decomposi­

ma de u s e o f
studies.

living

No r e ­

theories of beta1940)

sim ulate

and

have been a p p l i e d

respiration.

the

(1941)

h a d shown t h a t

c o u l d be a d a p t e d to

(sesame o i l ) ,

corn o i l
(1)

instances

made u s e o f q u i t e m i l d

investigators

( r e v i e w e d by H a r r o w ,

Warburg a p p a r a t u s

cludes

The o x ­

C lutterbucl? and Raper (1925),

catalysts

Since Johnston and Frey

tio n of fa t

drastic

and t r a c e s

have been fo u n d i n which t h e

tio n of

correspond­

dihydroxystearic

determ ine the
these

capronic,

and from l a r d *

o f o x i d a t i o n by c h e m i c a l means

b io lo g ic al oxidation,

to

butyric,

purified oleic

as

and ep ih y d ric

w e l l as th e

recovered 9:10

a n d Smedley-MacLean and P e a rc e

cords

as

r e s u l t of very

oxidizing

w ithout a c a ta ly s t,

acetic

form ic,

from r a n c i d o l i v e o i l

d eriv ed as

Skellon

crude

butyric,

p r o d u c t s m e n t i o n e d b y P o w i c k w e r e i n s o me

compounds

C.

pelargonic,

and p e la r g o n ic

ing a ld eh y d es,

and caproic a cid s;

the p resen t

the stu d y of

a p p lic a tio n of the ap p aratu s

Thunberg te c h n iq u e ;

(3)

(2)

a correlation

the oxida­

s tu d y upon th e

b y b a c t e r i a wa s u n d e r t a k e n .

m icrobiological oxidation of o il;

the

the

This
to

u tiliza­

report

in­

a study of

results

with the

between l i p o l y s i s

and

-1 0 oxidation;
upon the
the

the

effect of

u tiliz a tio n of

respiratory

growth
as

(4)

corn o i l;

coefficient;

upon s e v e r a l o f t h e

criteria

of

made p r e v i o u s l y

various

and

fat

(5)
(6)

a determ ination of
the e f f e c t o f b a c t e r i a l

constants

chem ical c o n s t i t u t i o n .
th a t the

latter

however,

so

literature,

in the

include s e v e r a l of the

results

since
it

commonly e m p l o y e d

The s t a t e m e n t h a s b e e n

type of

proved u n sa tisfa c to ry ;
infrequently

organic m ateria ls

i n v e s t i g a t i o n has

actual
is

values

are

given

deemed a d v i s a b l e

of the p re s e n t

study.

to

-1 1 EXPERIMENTAL WORK
a.

C ultures•

General procedure

T hirty-five

cultures

These

included sp o ru latin g

ative

rods,

cultures,
P rior

to

various

rods,

m icrococci,

to g eth er w ith

b a c t e r i a were

Gram p o s i t i v e

a n d Gr am n e g

are

listed

th e y were p l a t e d o u t

during

used.

and a p la n t pathogen*

th e ir sources,

e x p e rim e n ta l work,

exam ined p e r i o d i c a l l y

of

the

course of the

below .
for purity

experim ent,

and checked m i c r o s c o p i c a l l y and p h y s i o l o g i c a l l y a t
of the
fifth

e x p erim e n tal work.

The t e r m i n o l o g y

The

the

end

to

the

conforms

e d i t i o n o f B e rg e y 's Manual.
C ultures

from th e M ichigan S t a t e C o lle g e C o l l e c t i o n :

A lcaligenes

faecalis

Serratia

marcescens
indica

Proteus

ammoniae

S erratia

Sarcina

lutea

Pseudomonas f l u o r e s c e n s

(a)

M icrococcus

aurantiacus

Pseudomonas a e r u g in o s a

M icrococcus

flavescens

Flavobacterium

M icrococcus

cinnabareus

F la v o b a c te riu m synxanthum

arborescens

Staphylococcus

aureus

Phytomonas t u m e f a c i e n s

Staphylococcus

citreus

B acillus

m esentericus

Staphylococcus

albus

B acillus

vulgatus

B acillus

mycoides

B acillus

cereus

E scherichia

coli

A ero b acter aerogenes
E scherichia

coli

var.

coramunior

-1 2 C ultures

f r o m D r . K. W. P e p p i e r ,

A lcaligenes

Kansas S t a t e

C ollege;

lipolyticus

Achrom obacter lip o ly tic u m
B acillus

s u b t i l l s , f r o m D r . W. B. S a r l e s , U n i v e r s i t y o f
Wiscons i n

C ultures

f r o m Dr- H. F .

Ps eudomonas f r a s i i

Long,

Iowa S t a t e

C ollege;

(b)

Pseudoraonas

sraveolans

Pseudomonas

fluorescens

Pseudomonas

m ephitica

(b)

Pseudomonas m ucidolans
C ultures

from D r. P. E.

Pseudomonas

putrefaciens

Pseudomonas

fragii

C ultures

isolated

laboratory,
L.

1 -

E lliker,

Purdue:

(a)
from b u t t e r

in the B a cterio lo g y

M ichigan S t a t e C o lle g e:

a Pseudomonas

(lipolytic)

L* 2 - a Gr am n e g a t i v e ,

unidentified

L.

very sh o rt

3 - a Gr a m p o s i t i v e ,

The c u l t u r e s ,
ferred

daily

for

r e f r i g e r a t e d when n o t

several

days p r i o r

rod ( l i p o l y t ic )

rod (non l i p o l y t i c )
in use,

to use as

were t r a n s ­

inoculum.

They w ere grown e i t h e r

i n b r o t h o r o n a g a r medi um o f t h e

follow ing

as u s e d by Nunheimer an d F a b i a n

com position,

(1942)

15-

B a c to P ep to n e

0.5%

Bacto

0

Beef E x tra c t

.

Bacto Y e a s t E x tr a c t

0 . 2^

Bacto P e p to n iz e d Milk

Q .Z %

(Shredded ag ar)
T h e me d i u m w a s a d j u s t e d t o
lized

in

the

autoclave

at

the

pH o f

7 .0 to

7.1,

15 p o u n d s p r e s s u r e

and s t e r i ­

f o r 20 m in­

utes .
During
bacteria

the

first

part

became a p p a r e n t t h a t

of

the

species

as

surface of

the

in

did

the h arv est

broth

the c e l l s .

in a b o ttle

many c e l l s

cultures,

sterile

the

were grown i n

activ ity of

broth,

th e e x p e r i m e n t a l work,

w e r e g r o w n o n l y o n me d i u m c o n t a i n i n g a g a r .

it

the

of

saline

quite
the

the
When

s u p p l y o f a g a r was l i m i t e d ,
the b ro th ,
It

w ithout

wa s f o u n d t h a t

sim ilar

surface of

to

many

im pairing

100 m l. o f t h e

a Ro u x f l a s k y i e l d e d a s

50 m l . o f a g a r o n t h e

same t y p e o f b o t t l e .

flat

The q u a n t i t y o f c e l l s

c o u l d be i n c r e a s e d m a t e r i a l l y by s w i r l i n g
or

flo o d in g the ag ar su rfa c e w ith

solution,

s e v e r a l hours

the

10 m l . o f

before harvesting

the

cells.
Th e b a c t e r i a w e r e g r o w n f o r 22 t o

26 h o u r s a t

p e r a t u r e w h i c h was o p t i m u m f o r t h e s p e c i e s .
moved from a g a r by w ash ing w i t h s t e r i l e
(0.85

per

c e n t sodium c h l o r i d e ) .

through c o tto n ,

th e washings

were p r e c i p i t a t e d

A ll

combined,

in a c e n trifu g e .

the

tem­

G r o w t h was r e ­

saline

solution

l i q u i d s were f i l t e r e d
and th e

bacteria

The s u p e r n a t a n t

liquid

-1 4 was

r e m o v e d by s i p h o n i n g a n d t h e

saline

solution.

tim es.

cells

C e l l s v/ere washed i n t h i s

The a m o u n t o f s a l i n e

s u s p e n s i o n wa s l i m i t e d

to

10 m l .

per b o ttle

beads,

shaken fo r fiv e

through c o tto n .
was

stored

in

the

P r i o r to
washed a i r

If not

to

m inutes,

period,

the

of

the

these

but c e lls

culture

for th ir ty

of th is

the

basis

and also

the

cause of

f o r various
and the

harvest.

suspensions

as to

at

Because

a t t e m p t s w e r e made

q u a n t i t y p e r m l. on
in Thunberg t u b e s ,
m icro-

N e i t h e r method p r o v e d s a t i s f a c t o r y ,

reasons

This

re­

I t was f o u n d b y

a s d e t e r m i n e d by t h e

v a r ie ty of organisms

10 m l .

as

uniform ity.

p a r t o f the work,

unreliable,

only sta n d a rd iz a tio n

m entioned:

m inutes,

d i f f i c u l t y was e n c o u n t e r e d w i t h

by n i t r o g e n c o n t e n t

the

the p r e p a r a tio n

(see S an d ifo rd and

o f r e d u c t io n o f methylene b lu e

K j e l d a h l method.

filtered

age were seldom u s e d .

in m aintaining

standardize

containing

c o u ld have been u sed o v er a f o u r - d a y

D uring the e a r ly
to

"restin g ” cells

heavy su s p e n s io n ,

some s p e c i e s

in the h a r v e s t.

and a g a in

commended by Q u a s t e l a n d Whetham ( 1 9 2 5 ) .

1951)

re­

t h e s u s p e n s i o n was a e r a t e d b y p a s s i n g

through

W ooldridge,

third

refrigerator.

use,

experim ent th a t

the

a bottle

used im m ediately,

fresh

fashion three

s o l u t i o n used a t

The s u s p e n s i o n was t h e n t r a n s f e r r e d
glass

suspended in

of saline
tim es

employed.

also

P late

be­

counts,

were d i s c o n t i n u e d ,

a t t e m p t e d was t h a t a l r e a d y
so lu tio n per b o ttle

p r o v e d t o be t o o h e a v y ,

in the
and the

-1 5 s u s p e n s i o n wa s s u i t a b l y
the

activity

either

o f one s p e c i e s

of b acteria

t h e Warburg s y s te m o r i n th e

quantitatively
species,
ically

d i l u t e d whe n n e c e s s a r y *

for

numbers o f b a c t e r i a

substrate

pure

Since

trig ly cerid e.

corn o i l ,

ly

because of

its

this

follow ing

containers,

is

not

of another

p e r ml* a r e

was

not

numer­

type of s tu d y

m aterial

is

is

was s e l e c t e d o n t h e
purchased lo c a l

used throughout

this

b e e n f u r n i s h e d by t h e

work.

Io d in e Value
F re e F a t t y Acids

-10 to

-20°

113 t o

128

maximum

05%

S a p o n i f i c a t i o n Value

188 to 193

U n sap o n ifiab le M atter

1.3

S a t u r a t e d F a t ty Acids

10 to

12

U n s a t u r a t e d F a t t y Acids

88 t o

90

S pecific G ravity at

15°

0 .9 2 1 3 to 0.9268

R efractive

15.5°

1.475 to

Index a t

** Th e l e t t e r

is

to

i n c lu d e d as A ppendix A

The

m anufactur­

and Chemical C o n s ta n ts

Point

a

rather scarce,

R e f i n i n g Company. **

Physical
Solidifying

this

B a l l . Mazola o i l ,

in fo rm a tio n has

th e Corn P r o d u c ts

for

uniform ity,

o f P r o f e s s o r C . D.

in sm all

ers,

Thunberg tu b e s ,

in

equal.

C o r n o i l . The i d e a l

advice

in a menstruum,

comparable w ith the a c t i v i t y

the

Therefore,

1.7

1.477

-1 6 Smoke P o i n t

400° to 4 5 0 ° F .

Flash Point

620° t o 6 4 0 ° F .

Burning P o in t

694°F.

A cetyl

7.5 to

Value

9.0

Compos i t i o n
Palm itic
S tearic

This

Acid

1*1%

Acid

3.5

A ra c h id ic Acid

0.4

L ig n o ceric Acid

0.2

O le ic Acid

45.5

L i n o l e i c Acid

40.9

analysis

corresponds

quite

closely

to

t h a t g i v e n by

Baughman a n d J a m i e s o n (1 9 2 1 ) *

The c o m m u n i c a t i o n f r o m t h e

m anufacturers

states

HWe h a v e no i n f o r m a t i o n o n t h e

nature

pigments

of the

dications
tain
has

in the

a trace

of

further:

literature
sterols;

that

in

Emulsions

99 m l .
tim es.

its

corn o i l

it

distilled

probably

is

stable

a b s e n t . 11

to bo th tim e and a u to corn o i l and

w a te r through a hand hom ogenizer t h r e e

The m a j o r i t y o f

f o u r te e n m icrons

may c o n ­

i n Mazola

these are

w e r e p r e p a r e d by p a s s i n g o n e m l . o f

of

in­

We h a v e no i n f o r m a t i o n

b u t we b e l i e v e

o f corn o i l ,

There a re

presence

at present

the U n sap o n ifiab le *

on hydrocarbon c o n te n t,

claving,

refined

however,

not been wholly confirm ed;

contained

to

in re fin e d corn o i l .

the

f a t globules

in diam eter,

m e a s u r e d from f i v e

w ith a very

few l a r g e r ,

-1 7 a n d many s m a l l e r *
During th e

first

sodium a l g i n a t e
ize

the

wa s a d d e d t o

em ulsion;

realized

that

p a rt of the

use o f

it

the

study,

d i s t i l l e d w ater to

Therefore,

ium h y d r o x i d e

were added to

No d i f f i c u l t y

wa s

to o x id iz e
t wo

it

according

the

to

Dixon ( 1 9 3 4 ).
at

35.2° C .,

the w ater b efo re

This

number o f

hom ogenization.

The t h e r m o - r e g u l a t o r o f t h e
length of

standardized

15 m i n u t e s
Gases were

to

cm.,

100 p e r m i n u t e .

60 m i n u t e s ,
perm it

by

b a t h wa s s e t

t h e s t r o k e was t h r e e

wa s 96 t o

a r y t i m e o f o p e r a t i o n wa s

equilibrium .

Maryland*

mercury w eight method, as o u t l i n e d

revolutions

v a l o f 12 t o

a n d show

i n s t r u m e n t wa s m a n u f a c t u r e d

th e manometers and cups were

the

was

d r o p s o f N /1 0 s o d ­

b y t h e A m e r i c a n I n s t r u m e n t Company o f B a l t i m o r e ,
of

stab il­

encountered w ith cream ing.

The Warburg A p p a r a t u s ♦

The c o n t e n t s

1:10,000

wa s d i s c o n t i n u e d w h e n i t

some b a c t e r i a w ere a b l e

a consum ption of oxygen.

one m l. o f

and th e

The c u s t o m ­

p r e c e d e d by a n i n t e r ­

tem peratures

to

c a l c u l a t e d a c c o rd in g to

come t o
the

form­

plus

0.2

u l a e g iv e n by D ixon.
Liquids

added to

m l . o f 20 p e r
determ ining
ic

acid

e a c h cup t o t a l l e d

c e n t sodium h y d r o x i d e

carbon dioxide,

(by volume)

in the

of filte r

p a p e r was a d d e d t o

dioxide.

The

successively

(by w e i g h t ) ,

0 .2 ml. o f
central

or,

when

four per cent sulphur­

receptacle.

facilitate

cups were c l e a n e d a f t e r
w ith hot w ater,

3.6 m l.,

A sm all

a b so rp tio n of carbon

each d e te r m in a tio n

95 % e t h y l

strip

alcohol,

diethyl

-1 8 -

ether,

and again rin se d

before

further

is

used

in the

use.
cups•

w ith very hot w a te r;

This p ro c e d u re

is

then d rain ed

r e c o m m e n d e d wh e n o i l

-1 9 b.

The a b i l i t y

Many s p e c i e s
fats

and o i ls ;

compounds
self

is

chiefly
one

less

to

of

of b a c te ria

the a b i l i t y

represents

preferred

of b acteria

and C o llin s

bacteria

lip olytic
This

applicab ility
u tilizatio n
of

the

sole

to

of bacteria

(1927)
of

the

results

dem onstrated
nine

strains

o n ly one h y d r o ly z e d
determ ine

the

to a study o f the

as w ell

would u t i l i z e

as

d e te rm in in g which

corn o i l

as th e

The q u a n t i t y o f o x y g e n u t i l i z e d w a s d e t e r ­

m ined f o r one ml. o f c e l l s
pH 7 . 0 a n d s a l i n e ,
saline,

buffer,

of bacteria

it,

attack

check the

of saline
om ission o f

same q u a n t i t y o f

and one ml. o f e i t h e r
G lycerol

w h ich to

in the p resen ce o f b u ffe r a t

and f o r th e

o r M /3 0 g l y c e r o l -

the

assume t h a t

because o f

was d e s i g n e d t o

in g e n e ra l,

it­

with o th e r o i l s ,

to

found t h a t ,

butterfat,

corn o i l

these

source of energy.

E xperim ental.

tity

is not s a fe

t h e Warburg a p p a r a t u s

of oils

species

(1933)

u tilize

many w o r k e r s h a v e

activity

Va n d e r W a l l e

experim ent

of

since

w i l l be h y d r o l y z e d ,

fact,

corn o i l .

It

to

S pecifically,

lipolytic

anim al o r i g i n .

o b ta in e d w ith a n o th e r.

of

o f the organisms

a n unknown f a c t o r ,

investigate

use c o rn o i l

a r e k n o wn w h i c h h y d r o l y z e

w e ll known.

type o f e s t e r

this

to

is

and i t

activity

c e l ls w ith

em ulsified corn o il

i n c l u d e d b e c a u s e many s p e c i e s
offers

of the

a good s u b s t r a t e w ith

suspensions.

s o l u t i o n u s e d wa s a d j u s t e d t o
carbon-containing

liquids,

The q u a n ­

compensate f o r

so t h a t

the

final

-2 0 volume o f

liq u id w ithin

experim ents,
bacteria

was 3*8 m l .

group of

shown i n T able
R esults *

ure

1 or

given

is

other

determ ine

th e g l y c e r o l would ta k e
experim ent.

The r e s u l t s

of bacteria

are

1.
to assume t h a t

in succeeding
an average of

representative

ism-

It

is

or

to

e x p e r i m e n t s w i t h 35 c u l t u r e s

turn,

technique,

manometers,

c o n d itio n s of the

I t w o u ld be f a l s e

in Table

and in a l l

S i m i l a r a d j u s t m e n t s w e r e made w h e n

e m u lsifie d corn o i l

up o x y g e n u n d e r t h e
this

cup i n t h i s ,

were o m i t t e d from t h e

w hether the

of

the

also

tables

duplicate

of several

of

less

absolute.

given

Each f i g ­

determ inations,

determ inations

d o u b tfu l whether,

a difference

are

the f ig u re s

and in

fo r the organ­

co n sid erin g m anipulative

than te n p e r cen t

is

signif­

icant .
I n s p e c tio n o f the
used,

ten per

B acillus

show t h e

f r o m an i n c r e a s e
255 p e r

cent

is

cent or less

present.
increase,

of

varied,

f o r Pseudomonas

Apart
the

members o f t h e

agar,

it

fo r B acillus

fluorescens

is

of

carbon.

It

is

s u b t i l i s » to

a_. To j u d g e
species

from

on min-

d o u b t f u l w h e t h e r many o f

t h e s e would grow i n d e f i n i t e l y w i t h c o rn o i l
source

genus

60 m i n u t e p e r i o d ,

s m a l l a m o u n t o f g r o w t h o f many o f t h e

eral-salts-corn oil

from t h o s e show­

increase.

during the

17 p e r c e n t

35 c u l t u r e s

in the q u a n t i t y o f oxygen

low est per cent

The p e r c e n t a g e

the

shows t h a t o f t h e

27 d e m o n s t r a t e a n i n c r e a s e

t a k e n u p whe n c o r n o i l
ing

table

probable

that

as

the o i l

the so le
is

oxidized

-2 1 -

t o f u r n i s h e n e r g y , b u t i s n o t u sed f o r growth*
ever,

T h i s , how­

i s s p e c u l a t i o n , and i s n o t a c o n c l u s i o n to be drawn

from t h e d ata p r e s e n t e d *
P r o t e u s ammoniae. members o f t h e g e n e r a S t a p h y l o c o c c u s
and E s c h e r i c h i a , F l a v o b a c t e r i u m syn xanth um , and t h e u n i d e n ­
tified

c o c c u s f a i l e d t o show an i n c r e a s e i n the u t i l i z a t i o n

o f oxygen in the p resence o f corn o i l *

They d i d , h ow ever,

o x id iz e glycerol*
N e i t h e r c o r n o i l n o r g l y c e r o l , i n the a b s e n c e o f b ac­
teria ,

a b s o r b e d o x y g e n under t h e c o n d i t i o n s o f t h e e x p e r i ­

m en t.
Equally i n t e r e s t i n g

i s the stu d y o f the a b i l i t y o f

t h e s e m ic r o o r g a n i s m s to d e h y d r o g e n a t e corn o i l ,
m e t h y l e n e b l u e as t h e h y d r o g e n a c c e p t o r .

using

T h is s t u d y i s

c a r r i e d o u t by means o f Thunberg t u b e s *
E x p e r i m e n t a l . The Thunberg t u b e c o n t a i n e d one m l. o f phos­
p h a t e b u f f e r a t pH 7 * 3 5 , one m l. o f s a l i n e s o l u t i o n , one
m l . o f 1 : 5 , 0 0 0 m e th y le n e b l u e

( f i n a l c o n c e n t r a t i o n o f the

d ye was 1 : 2 5 , 0 0 0 ) , on e m l. o f c e l l s ,

and one m l. o f e i t h e r

on e p e r c e n t e m u l s i f i e d c o r n o i l o r o f M/30 g l y c e r o l .

A d d i­

t i o n a l s a l i n e s o l u t i o n r e p l a c e d t h e co r n o i l and g l y c e r o l
in the co n tro l tu b e s .

The c e l l s were p l a c e d i n t h e s i d e -

arm and t h e tu b e was e v a c u a t e d w i t h w a te r s u c t i o n f o r 150
seconds.

The p r e s s u r e , c h e c k e d w i t h a vacuum g a u g e , was r e ­

d uced c o n s i s t e n t l y by 640 mm.

A f t e r e v a c u a t i o n , t h e tube

was p l a c e d in a w a t e r b a th a t 3 5 . 2 ° C .

When t h e c o n t e n t s

-8 2 -

T a b le 1
Cram* o f o x y g e n t a k e n up by b a c t e r i a in Warburg i n b u f f e r e d
s a l i n e s o l u t i o n , and i n b u f f e r e d s a l i n e p l u s e m u l s i f i e d c o r n
o i l o r M/30 g l y c e r o l

C ulture

C ontrol

Corn o i l Per c e n t Glycer<
increase

A lcaligen es fa e c a lis

20*6

42.7

107

49.2

A lca lig en es lip o lv tic u s

35.3

82.7

134

74.2

P r o t e u s ammonias

24 .2

22.4

7

199.0

Sarcina lu te a

68.9

205.5

198

87.8

S t a p h y l o c o c c u s au reus

46.7

35.9

-29

2 58.9

S taphylococcus c itr e u s

13 .2

13.7

-3

27.5

S ta p h y lo co ccu s albus

10.0

11.0

10

26.5

M icrococcus au ran tia cu s

78.3

2 1 7 .0

177

181.0

M icrococcus f la v e s c e n s

92.8

415.0

325

189.0

M icrococcus cinnabareus

21.2

31.5

48

27.6

E sch erich ia c o l i

20.1

21.4

6

102.4

E s c h e r i c h i a c o l i v a r . com.

29.5

30.5

3

208.7

A erobacter aerogenes

37.5

52.9

41

26.4

S e r r a t i a marcescens

34.3

74.2

116

186.2

S err a tia in d ica

46 . 4

125.7

149

143.9

A eh rom obacter l y p o l y t i c u m

36.5

68.6

88

148.0

F lavobacterium a r b o r e s c •

81.5

1 1 5 .8

42

83 .3

F l a v o b a c t e r i u m synxanthum

34.2

2 6 .S

-21

50.6

Phytomonas t u m e f a c i e n s

32.7

44.5

36

68 . 6

Pseudomonas f l u o r e s c e n s a

108.3

3 8 4 .5

255

218.1

Pseudomonas f l u o r e s c e n s b

133.0

238.3

154

298 .0

-2 3 Table
C ulture

1 (Continued)
C ontrol

Corn o i l

Per cent G lycerol
i n c r e a s e __________

72.9

2 2 1 .4

204

1 37.6

1 34.4

242.4

80

353.4

Ps eudornonas

aeruginosa

Pseudomonas

putrefaciens

P s eudornonas

fragii

a

34.9

77.5

119

51.2

P s eudornonas

fragii

b

99.0

174.3

76

250.8

Pseudomonas

graveolans

52.7

121.5

131

92.3

Pseudoraonas

m ephitica

35.9

76.4

113

126.0

Pseudomonas

muc i d o I a n s

35.0

85.5

144

72.8

21.1

24.7

17

24.3

B acillus

su b tilis

B acillus

cereus

166.3

197.9

19

2 20.3

B acillus

m esentericus

111.3

129.4

16

128.3

B acillus

vulgatus

34.2

42.7

24

34.8

88.0

258.6

194

137.3

L •2

65.2

137.2

110

127.1

L. 3

10.3

5 .1

-50

48.3

L.

1

C ontrol

0

0

0

-24tiad r e a c h e d
the

tube,

this

and th e

b l u e was n o t e d *
is

tim e o f

it

by b a c t e r i a

Table

were

d e c o lo r a tio n o f the methylene
experim ent,

than the

investigated,
as

corn o il*

27 w ere a b l e

ism s:

Sarcina

to

lutea,

table

R esults

to

layed reductions
and the

glycerol*

alone

35

dehydrogenate

dehydrogenate g ly c e ro l*
M icrococcus

o f methylene

Four o rg a n ­

c in n a b a r e u s « Micrococcus

blue

showed g r e a t l y

in the presence of

de­
the

l a t t e r organism

also

a p p e a r e d i n h i b i t e d by

No e x p l a n a t i o n o f

this

is apparent;

settled

did the

the

d e c o lo r a tio n of m ethylene b lu e ,

a u r a n t i a c u s » a n d Ps eudornonas m u c i d o l a n s

as

are given

shows t h a t o f

e i g h t were a b l e

shown by t h e

w hile

cocci

glycerol

was b e l i e v e d t o be a t t a c k e d more

Exam ination o f t h i s

corn o i l,

o il,

tipped into

2*

R esults *
cultures

the c e l l s

As i n t h e p r e v i o u s

in clu d ed because

readily
in

tem perature,

to

th e bottom of the

members o f t h e

p r o b a b ly does n o t

tube

fairly

genus B a c i l l u s » b u t

serve

to

explain

the

th e m icrorapidly,
this

fact

delayed de­

coloration.
The p r e s e n c e o f c o r n o i l
of

r e d u c tio n o f m ethylene

23 o f the

cultures*

blue

h a s no

influence

in the

Two s p e c i e s ,

tim e o f

time

in the case o f the m a jo rity ,
Escherichia

c o m m u n i o r a n d P s e u d o m o n a s m e p h i t i c a , do n o t
difference

upon t h e

re d u c tio n to

c o li var.

s how s u f f i c i e n t

be c o n s i d e r e d p o s i t i v e .

-2 5 T a b le

2

M i n u t e s r e q u i r e d b y o n e ml* o f c e l l s t o r e d u c e m e t h y l e n e
b lu e i n b u f f e r e d s a l i n e s o l u t i o n , and in b u f f e r e d s a l i n e
p l u s e m u l s i f i e d c o r n o i l o r M/30 g l y c e r o l
C ulture

C ontrol

Corn o i l

Glyc

A lcali^enes

faecalis

42

42

20

A lcaligenes

lipol.yticus

53

15

16

Proteus

ammoniae

26

27

6

Sarcina

lutea

20

26

16

Staphylococcus

aureus

90

90

21

Staphylococcus

albus

60

60

35

Staphylococcus

citreus

60

60

36

M icrococcus

aurantiacus

31

90

35

M icrococcus

flavescens

29

30

5

Micro coccus

cinnabareus

27

104

14

Escherichia

coli

120

120

22

E scherichia

coli

com# 1 4 0

120

19

62

37

20

120

120

10

var*

A erobacter aerosenes
S erratia

m arcescens

S erratia

indica

21

21

12

A chrom obacter ly p o ly ticu m

36

36

27

Flavobacterium

arborescens

58

57

29

Flavobacterium

synxanthum

29

30

30

60

60

18

Phytornonas

tum efaciens

Pseudomonas

fluorescens

a

23

13

11

Pseudomonas

fluorescens

b

22

22

19

-2 6 Table
C ulture

2 (Continued)

Control

Corn o i l

G lycerol

Pseudomonas

aeruginos a

14

9

6

Pseudomonas

putrefaciens

10

7

5

P s e u d o mo n a s

fragii

a

28

18

16

P s eudornonas f r a g i i

b

12

12

7

43

27

21

Pseudomonas g ra v e o la n s
Pseudomonas

m ephitica

29

26

20

Pseudomonas

muc i d o I a n s

30

52

44

B acillus

subtilis

inf.

inf.

inf

Bac i l l u s

cereus

inf*

inf.

inf

B acillus

m egatherium

15

17

B acillus

vulgatus

inf.

in f.

15
inf

L.

1

24

12

12

L.

2

26

26

20

L.

3

60

60

6

C ontrol

inf.

inf.

i n f • - no d e c o l o r a t i o n w i t h i n t wo h o u r s #

inf

-2 7 K e n d a ll and Ishikaw a
dehydrogenate
th e y were

to

those

an

increase

in the

state

that

bacteria w ill

which th ey would a t t a c k

them f o r e n e rg y and growth*

in Table 3,

ence o f co rn o i l
from t h i s

substances

utilize

be p o in ted out

(1929)

ma ny o f t h e b a c t e r i a

c o n s u m p t i o n o f o x y g e n due t o

apparently are

substrate*

unable

to

if

As w i l l
w h i c h s how
the

pres­

remove h y d r o g e n

-28-

c . Comparison o f t h e o x i d a t i o n o f c o r n o i l w i t h l i p o l y s i s o f c o r n and b u t t e r o i l s and t h e p r o d u c ­
t io n o f oxid ase
Because of

th e number o f c u l t u r e s

work,

opportunity

teria

to o x i d i z e

ties

is

g i v e n to compare t h e

corn o i l

w ith th e ir

ab ility

lipolytic

and w ith the p ro d u c tio n of oxidase#

G arrard
latter

(1940)

of bac­

proper­

C a s t e l l and

a t t a c h e d much s i g n i f i c a n c e

to

the

property#

E xperim ental *

L ipolytic

d e te r m in e d by s t r e a k i n g
taining

nile

heavy cream ,
for

used in th is

five

plates

blue su lp h ate
as d ir e c te d

days#

d i c a t e d by t h e
globules

activity

of

t h e b a c t e r i a wa s

of tryptose

a g a r con­

and e i t h e r c o rn o i l o r

by M i l l a r d ,

H ydrolysis of the
appearance of

and in c u b a tin g

triglycerides

b lu e pigment

is

in­

in the f a t

a ro u n d th e colony#

T h e p r e s e n c e o f o x i d a s e wa s d e t e r m i n e d b y f l o o d ­
ing n u t r i e n t
w ith

the

ide,

as

a fte r streak

inoculation

dye t e t r a r a e t h y l - p h e n y l e n e d i a m i n e
done by C a s t e l l

a u t h o r s were
experim ent
tity

a g a r 48 h o u r s

able

into

to

and G a r r a r d

divide

several

the

groups,

of oxidase produced.

ence o r absence of oxidase,

hydrochlor­

(1940)#

bacteria

These

used in t h e i r

based upon th e quan­

In th is

table,

the p re s­

a s shown by th e

reaction

-2 9 to

the

ple

dye,

color

is

indicated*

in the colony

The a p p e a r a n c e o f a p u r ­

indicates

the

presence of

oxidase •
The
ism s to

in fo rm a tio n as to
oxidize

rem oving

the corn o i l

hydrogen is

are

Several

Table

A ll those

drogenate
strate*
ies
to

corn o i l

dehydrogenate*

oil

are

unable

M icrocoecus
the

to

members o f t h e

able

able

to

schitz
years

points

are able

to

a n d t h e com­

however,

w ithout

are

is not

shown i n
able to

true;

dem onstrating

Several species

dehy­

this

sub­

many s p e c ­

the a b i l i t y

which o x i d i z e

dehydrogenate glycerol*

the

These a re

m upidoIans, and

genus B a c illu s *
not

in c lu d in g the

it*

spore-form ers,
w ith o u t being

H o r o w i t z —V l a s s o v a a n d L i v —

(1935) b e li e v e d t h i s
ago, because

3*

add oxygen to

to add oxygen to corn o i l ,
hydrolyze

1 and 2.

organism s which a r e

a u r a n t i a c u s , Pseudomonas

Ten s p e c i e s ,
are

experim ents

interesting

The c o n v e r s e ,

do a d d o x y g e n ,

o f the organ­

by a d d i n g o x y g e n o r by

shown s u m m a r i l y i n T a b l e

R esults*
3*

ability

t a k e n from T a b l e s

The r e s u l t s o f t h e s e
parisons

the

to be th e case

t h e y f o u n d no g l y c e r o l

several
in t h e i r

T a b le

3

C om p arison o f t h e o x i d a t i o n o f c o r n o i l and g l y c e r o l w i t h
l i p o l y s i s o f c o r n o i l and b u t t e r f a t and w i t h t h e p ro d u c ____________________________ t i o n o f _o x i d a s e _______________________
C ulture
_______________

O x i d a t i o n Meth. b lu e L i p o l y s i s
O xidof
re du ced
of
ase
c o r n g l y c - c o r n g l y c - c o r n b u t t e r p rod uco il
erol o il
erol o i l
fat
ed _____

Ale#

faecalis

4

4

-

4

—

A le.

lipolyticus

4

4

4

4

4

—

4

?

Proteus

ammoniae

-

4

-

4

-

-

4

Sarcina

lutea

4

4

-

4

-

-

4

Staph,

aureus

-

4

-

4

4*

4

-

Staph,

citreus

-

4

-

4

-

-

4

Staph,

albus

-

4

-

4

-

-

4

M. a u r a n t i a c u s

4

+

-

-

-

-

4

M.

4

4

-

4

-

-

4

M. c i n n a b a r e u s

4

4

-

4

-

-

4

E scherichia

-

4

4

-

-

-

-

4

-

4

-

-

-

A erobacter aerog.

4

4

4

+

-

-

4

Ser* m arcescens

4

4

-

4

+

4

4

Ser.

4

4

-

4

+

4

4

E.

flavescens

coli

var.

coli
c o m.

indica

^chr.

lipolvticum

4

4

-

4

4

4

-

Flav.

arborescens

f

-

-

4

-

-

0

Flav.

svnxanthum

-

4

-

-

4

4

0

4

t

-

4

4

4

4

Phyto.

tum efaciens

-31
Table
C ulture
_______________

3 (Continued)

O xidation
Meth. b l u e
of
reduced
c o m g ly c- corn glycoil
erol o i l
erol

L ipolysis
of
corn b u t te r
oil
fat

Oxidase
produced

Ps# f l u o r e s c e n s b

4

4

-

4

4

4

4

P s • aeruginosa

4

4

•f

4

4

4

4

P s • p u trefacien s

4

4

4

4

4

4

4

P s. fra g ii a

4

4

4

4

4

4

4

Ps* f r a g i i b

t

4

-

4

4

4

4

Ps • g r a v e o l a n s

4

4

4

4

-

-

P s- m ep hitica

4

4

-

4

-

-

P s * m u c id o la n s

4

4

-

-

4

-

Bac. s u b t i l i s

4

+

-

-

Bac. cereus

4

4

-

-

-

-

Bac# megatherium

4

4

-

-

-

-

Bac# v u l g a t u s

4

-

-

-

-

-

L# 1

4

4

4

4

4

4

L# 2

4

4

-

4

4

4

L.

3

—

4

-

4

-

-

-

no

t

yes

0

not determ ined

?

questionable

-

-

-3 2 -

soybean o i l c u ltu r e o f A erobacter a e r o g e n e s , y e t
c h a n g e s o c c u r r e d i n t h e c o m p o s i t i o n o f th e s u b s t r a t e *
The

experim ents

firm

this

summarized in Table

observation*

The s p e c i e s ,

A erobacter aerogenes. a re :
cIna

3 appear to

l u t e a ; M icrococcus

con­

in a d d itio n

A lcaligenes

to

f a e c a l i s ; Sar-

a u r a n t i a c u s , Micrococcus

e s c e n s , and Micrococcua

f lav-

c in n a b a re u s; Flavobacterium

a r b o r e s c e n s : Pseudom onas g r a v e o l a n s » and Pseudomonas
m ephitica*
terest,
ing
to
are

because

this
the

Pseudomonas m u cid o lan s
it

ability

latter

h y d r o l y z e s c o r n o i l w i t h o u t show­

w ith b u tte r

fat,

or else

u s e d so r a p i d l y

change

is of p e c u lia r in ­

that

fat.

It

is

the products

either

inert

of hydrolysis

the c h a r a c t e r i s t i c

color

does n o t o ccu r*

Two o r g a n i s m s w e r e f o u n d w h i c h h y d r o l y z e d c o r n
o il

and b u t t e r

the

form er,

the

Thunberg te c h n iq u e .

lococcus
plates

fat,

either

aureus

yet
in

to o x i d i z e

t h e W arburg a p p a r a t u s o r by
These org anism s

a re Staphy­

and F la v o b ac te riu m synxanthum*

on w hich t h i s

slightly

s h o w no a b i l i t y

altered,

o f bo th organism s

hydrolysis

indicating

The

was s h o w n w e r e b u t

that

lipolytic

ab ility

i s weak.

No c o r r e l a t i o n

is

d e m o n s tra te d between th e

possession

o f o x i d a s e and t h e a b i l i t y to o x i d i z e c o r n o i l *

A ll

b a c t e r i a w h i c h add o x y g e n t o t h e o i l p o s s e s s o x i d a s e
e x c e p t A ch rom obacter l i p o l y t i c u m ; h o w e v e r , s e v e r a l
s p e c i e s do c o n t a i n t h e enzyme, w i t h o u t a d d in g oxygen*

-3 4 d* R a t e o f c o n s u m p t i o n o f o x y g e n a s m e a s u r e d manom e t r i c a l l y , th e e v o l u t i o n o f c a r h o n d i o x i d e , and t h e
resp ira to ry quotient
The f a c t
uble

that

and r e s i d u a l

would

indicate

mine

fatty

that

imal m etabolism ,
terial

some i n v e s t i g a t o r s

the

is

the

quotient,

corn o il*

m enstrua

0*71

t h e o r e t i c a l one

in

l i b e r a t e d by b a c t e r i a

The r e s p i r a t o r y

quantity of

quotient

carbon dioxide

an­

fo r bac­

T h i s e x p e r i m e n t was d e s i g n e d t o

q u a n t i t y o f o x y g e n t a k e n up a n d t h e

carbon dioxide

ing the

than

recovered in s o l­

from o i l - c o n t a i n i n g

respiratory

less

m etabolism*

the

acids

have

deter­

quantity of

in the p resen ce of
is

c a l c u l a t e d by d i v i d ­

e v o l v e d by t h e

quantity

o f oxygen tak e n up.
E xperim ental*

D uplicate,

determ ining the

rate

dioxide

evolution*

viously

described,

0 * 2 ml* o f

calculated

o f oxygen u t i l i z a t i o n

and o f

B oth manometers were s e t
e x c e p t t h a t one o f

four per cent

receptacle*

p a i r e d manometers were used i n

sulphuric

up a s p r e ­

the p a ir

acid

carbon

in the

contained
central

The q u a n t i t y o f c a r b o n d i o x i d e e v o l v e d was
by t h e

indirect

m e t h o d as d e m o n s t r a t e d by D i x o n

(1934)*
R esults *

Table

four species
monas
lutea,

4 shows t h e

of b a cteria,

cmm. o f o x y g e n t a k e n

Pseudomonas a e r u g i n o s a , P seudo­

p u tr e f a c ie n s , A lcaligenes
the

cmm. o f

up by

l i p o l y t i c u s , and S a rc in a

carbon dioxide

evolved,

and t h e

res-

-3 5 piratory
Figures
the

quotient
1 to

at

each tim e

4 show t h e

evolution of

the

i n t e r v a l o f 15 m i n u t e s #

tre n d s * o f the

gases

during

consum ption and o f

the period o f

180 to

210

m inutes *
S traight

lines

sum ption o f oxygen,

may b e d r a w n t o

a lth o u g h each s u c c e s s iv e

oxygen consum ed does not
The a v e r a g e

an average

deviation of

30.4

w ith an a v e ra g e

1.9

S traight

the

the

low,

the

rises

sible,

if

experim ents

ing

all

by A l c a l -

deviation of

c mm. , w i t h a n a v e r a g e

be draw n t o d e m o n s t r a t e

is

rapidly

the

the

l u t e a t 24.5

fo r the q u a n tity

less

than t h a t

respiratory

during

that

3 . 4 cmm. ;

c mm . , w i t h a n a v e r a g e

Mor e s i g n i f i c a n t

slow ly

the

5 5 * 3 c mm. , w i t h

by P s e u d o m o n a s p u t r e f a c i e n s ,

d ev iatio n of

cannot

early periods

depicting

in itially

period is

cmm. ;

carbon dioxide,

la te r periods.

figure

increm ent of

cmm.

lines

evolution of

during

7.7

a n d by S a r c i n a

deviation of

of

e a c h 15 m i n u t e

l i p o l y t i c u s , 24.7

3*5 cmm.;

o f con­

q u a n t i t y o f o x y g e n t a k e n up b y P s e u d o m o n a s
during

igenes

rate

e x a c tly correspond w ith the average#

aeruginosa

c mm. ,

s how t h e

course

of

at
the

is

the

quotient.
first,

oil

evolved during
curve

in each

The q u o t i e n t

and con tin u es

determ inations.

It

had been co n tin u ed long

had been o x i d iz e d

rate

evolved

to
is

is
rise

pos­

enough,

q u o t i e n t o f 0 .7 1 would have b e e n r e a c h e d ,
the

the

provid­

to c a rb o n d i o x i d e and

w ater.
* The w r i t e r i s i n d e b t e d t o M r . P i o b e r t S t o e t z e r ,
C o lle g e , f o r a s s i s t a n c e in p l o t t i n g the d a ta .

o f Hobart

-3 6 Table

4

Cmm# o f o x y g e n t a k e n u p by b a c t e r i a i n t h e p r e s e n c e o f
c o r n o i l , the carb o n d ioxide ev olved and the r e s p i r a t o r y
quotient
Ps eudornonas a e r u g i n o s a
Time i n
m inutes
0
15
30
45
60
75
90
105
120
135
150
165
180
195
210

Cmm. Og
consumed
0.0
48.4
103.5
177.7
249.7
318.3
372.2
435.2
489.2
543.1
592.7
646.7
680.5
744.6
795.1

Pseudomonas
0
15
30
45
60
75
90
105
120
135
150
165
180

Cmm. COg
evoIved
0.0
7.35
24.5
55.3
89.6
119.2
149.9
183.1
210.2
248.0
273.0
299.8
311.0
353.4
384.7

R espiratory
quotient
0.0
0.15
0.24
0.31
0.32
0.37
0.40
0.42
0.43
0.45
0.46
0.46
0.46
0.47
0.48

putrefaciens

0.0
36.0
65.2
88.8
115.3
142.8
169.8
200.1
228.3
259.8
296.9
328.4
364.4

0.0
13.4
27.1
42.3
51.0
70.0
78.7
98 . 1
113.0
131.5
152.3
170.8
193.8

0.0
0.37
0.41
0.47
0.44
0.48
0.46
0.49
0.49
0.50
0.51
0.52
0.52

-3 7 Table 4 (C ontinued)
Cmm# o f o x y g e n t a k e n up b y b a c t e r i a i n t h e p r e s e n c e o f c o r n
o i l , t h e c a r b o n d i o x i d e e v o l v e d , and t h e r e s p i r a t o r y qu o ­
tien t
Time i n
m inutes

Cmm. Og
consumed

A lcaligenes

0
15
30
45
60
75
90
105
120
135
150
165
180

R espiratory
quotient

lipolyticus

0
15
30
45
60
75
90
105
120
135
150
165
180

Sarcina

Cmm. GOg
evolved

0.0
31.0
59.8
86.3
110.7
132.8
163.8
186.0
210.3
225.8
245.7
267.9
294.5

0.0
11.8
26.2
38.3
52.1
62.8
78.2
96.4
109.4
114.0
125.9
140.3
153.5

0.0
0.38
0.43
0.44
0.47
0.47
0.47
0.51
0.52
0.51
0.51
0.52
0.52

0,0
15.5
23.0
36.5
51.6
65.1
78.5
92.0
110 •5
124.1
136.5

0.0
0.48
0.40
0.45
0.47
0.50
0.51
0.52
0.54
0.55
0.55

lutea
0.0
31.9
59.0
79.9
105.7
129.3
152.9
175.4
200.0
224.8
246.2
269 .8
293.4

F ig . 1.
dioxide

T h e cmm. o f o x y g e n c o n s u m e d a n d o f c a r b o n
p r o d u c e d by Pseudomonas a e r u g i n o s a , and th e
re sp ira to ry quotient
o x y g e n consumed
carbon dioxide produced
re sp ira to ry quotient

2.QD cmm

6 0 0 cmm

5 0 0 cmm

4 0 0 cmm

0.6

3 0 0 cmm

0.5
0.4

2 0 0 cmm

0.3
0.2

0.1

Time
0

15

30

45

60

75

i n mi n u t e
90

105

120

135

150

165

180

190 210

.350 cmm
5 0 0 cmm
3 5 0 cmm
3 0 0 cmm

0.4

150. cmm

0.3

5_0 c mm

0

15

30

45

60

75

90

105

130

135

150

165 1 8 0

Ti me i n m i n u t e s

F i g . 3.
The cmm. o f o x y g e n c o n s u m e d a n d o f c a r b o n d i o x i d e
p r o d u c e d by P s e u d o m o n a s p u t r e f a c l e n s , a nd t h e r e s p i r a t o r y
quotient
♦
*

oxygen consumed
carbon d io x id e produced
re sp ira to ry quotient

3 5 0 cmm
3 0 0 cmm
2 5 0 cmm
2.0Q cm
1 5 0 cmm

0,3

1 0 0 cmm

— __ !________i_______ I________________ ________ ________________ ________________ ________ ________

0

15

30

45

60

75
Time

90

105

120

135

150

165 1 8 0

in minutes

F ig . 3.
T h e cram, o f o x y g e n c o n s u m e d a n d o f c a r b o n d i o x i d e
p r o d u c e d by A l c a l i g e n e s l i p o l . y t i c u m , and t h e r e s p i r a t o r y
quotient

*

/

oxygen consumed
carbon dioxide produced
re s p ira to ry quotient

3 5 0 cmm
R. Q
5 0 0 cmm

0. 6

2 5 0 cmm

0.5

2 0 0 cmm

0.4

1 5 0 cmm

0.5

1 0 0 cmm
5 0 c mm

0

Q1 1

30

15

45

60

75

90

105

120

135

150

165 1 8 0

T i me i n m i n u t e s

F ig . 4.
T h e cmm. o f o x y g e n c o n s u m e d a n d o f c a r b o n d i o x i d e
p r o d u c e d by S a r c i n a l u t e a » a n d t h e r e s p i r a t o r y q u o t i e n t

+
>

o x y g e n consumed
carbon dioxide produced
resp ira to ry quotient

-4 2 e.

T h e e f f e c t o f l o w e r e d pH a n d o f s o d i u m c h l o r i d e
upon b a c t e r i a l r e s p i r a t i o n

Because o f the
f o o d economy,
ces

decom position.

gested
ation.
acids

lower the

pH,

producing

on t h e
sodium

surface

and D ahlberg

of b u tte r,
Hus s o n g

by s a l t ,

butter

c o n tin u e d to

s a me c o n d i t i o n s .

under the

reported that

to tal

N elson,
butter

type of d e t e r i o r ­
inhibitors

are

black d isc o lo r­

i n h i b i t e d by 1 . 2 5 p e r
found t h a t Pseudo­

the

count

the b a c t e r i a l

the

oxidizing

in u n s a lte d b u t t e r rose

salted

C aulfield,

growth of th e

types

and c o n t r o l

f r om, g a t h e r e d

salt­

into
quick­

and G arrard (1939)

lactic

of organisms,

and M a r tin

Washburn

d e c r e a s e wh e n b r o u g h t

C astell

could

count of

the a d d itio n o f seven per cent s a l t

inhibited

count of

is

(1933)

(1917) showed t h a t

ly

the

been sug­

b u t n o t by b u t t e r c u l t u r e .

w hile

and of

to

compre­

arom a-producing organism ,

a war m r o o m ,

ed cream

subject

the

1940) h a s

a r e d d i s h brown t o

chloride.

inhibited

stored

(see

instan­

and sodium c h l o r i d e .

monas f r a g i i , a l i p o l y t i c ,

ed,

in our

(19 4 0 ) h a s shown t h a t Pseudomonas n i g r i f i c a n s ,

an organism

be

antioxidants

prevent o r delay the o x id ativ e

W hite

cent

A number o f

food-stuffs

Among t h e m o r e common b a c t e r i a l
to

ation

and a c i d u l a t i o n of

r e v i e w o f Br o wn a n d T h u r s t o n ,
to

and o i l s

emphasis has been p la c e d in c e r t a i n

upon s a l t i n g

hensive

im portance of f a t s

to g a t h e r ­

acid b a c te ria ,

but

that

the

final

creams were s i m i l a r .

(1942) o b t a i n e d

cream to which s a l t

a better

had been added,

-4 3 the

improvement b e in g

bacteria,

yeasts,

due t o

the

in h ib itio n of c ertain

and molds.

M a n o m e t r ic a l l y , Ingram (1940)

found th a t

a sm all

q u a n t i t y o f sodium chlorid.e
of

E scherichia

coli

concentrations
tio n

not

inhibited

was 7 . 3 f o r

latter.

Fouts

E xperim ental.

wa s

chloride

upon t h e

w hile

of

the

the

itated

cells

i um i o n

is

u tilization

still

salt,

and h e a t .

of oxygen.

The t o t a l

using

but

0.6 m l.,

s o l u t i o n was i n c r e a s e d

but the

con­

3*8 m l . ,

to

1.1

desired concentration
sodium c h l o r i d e ,

suspended in phosphate b u ffe r;
present,

lipolytic

e f f e c t o f a c i d and sodium

o b ta in the f i n a l

M/ 3 0 d i p o t a s s i u m

phosphate,

acid,

To o b t a i n 0 . 0 p e r c e n t
were

acid

f o u n d t h a t Ps e u d o r n o n a s

rem ained th e same,

T h e d e s i r e d pH v a l u e s o f t h e
tratin g

cream w i t h

e m u l s i f i e d o i l was d e c r e a s e d t o

in o r d e r to
salt.

wh e n l a c t i c

but

of th e Pseudomonas gro u p were

study of the

quantity of sa lt

the

retardation,

of lip o ly s is

in to le ra n t of

o f t h e Warburg cups

amount o f

and 6 .3 f o r th e

found a d e f i n i t e

Six species

for

m l.,

form er organism,

c u l t u r e s were a d d ed to

selected

the

The pH a t o p t i m u m r e s p i r a ­

L o n g a n d Hammer ( 1 9 4 1 )

putrefaciens

tents

this.

inhibition,

acid

organism s.

the

(1940)

a complete

and la c tic

in c re a s e d the r e s p i r a t o r y r a te
%
and o f B a c i l l u s c e re u s , but t h a t l a r g e r

chlorine

buffers

ion is

the

precip­
sod­

absent.

w ere o b t a i n e d by t i ­

p h o s p h a t e w i t h M/ 30 m o n o p o t a s s i u m

the g lass

electrode

a n d a B e c k m a n pH m e t e r .

-4 4The

in itial

were
as

placed into

the

the

pH v a l u e s

result

pH o f t h e

taken a t
most

the

the

are
cup,

those

and a r e

liquid

were

in the cup,

end o f th e

bacteria

the

is

pH d e t e r m i n a t i o n s

experim ental p e rio d ,

conditions

shown i n T ab le 5,

concentrations of

pH v a l u e o f

presence of o il
d efinitely

at

6.85,

one hour#

The f i r s t

t h a n was t h e

upon r e s p i r a t i o n

and the e f f e c t s

sodium c h l o r i d e ,

The

t wo s p e c i e s ,

an a cid ity

is

using b u ffe r

servations

of others,

in

the

upon r e s p i r a ­

Pseudomonas f l u o r e s c e n s
definitely

to t h e i r

unfavorable#

w ill

effect

rem aining fo u r sp e cies

acid condition

of

o f pH i n d i c a t e d v e r y

have an adverse

a slig h tly

acids

levels

aeruginosa, quite

l o w e r e d pH#

case,

Al­

a r e shown i n T ab le 6 .

various

th a t acids

Pseudomonas

of

errors
To c h e c k

T h e cmm. o f o x y g e n t a k e n up b y t h e b a c t e r i a

tio n .

latter

added#

the various
w ith

the

o f t h e a d d i t i o n o f sodium c h l o r i d e #

R e s u l t s ♦ The e f f e c t o f a c i d i c
the

as

unadjusted fo r

i n v a r i a b l e , t h e s e were n e a r e r n e u t r a l i t y

buffer

of

of the b u ffers

inhibit bacterial

sensitive

to

undoubtedly fin d

lik in g ,

The r e s u l t s

th a t natural

are

and

but too

great

confirm th e ob­

s o u r in g and th e a d d i t i o n

respiration,

and in t h i s

the o x id a tio n o f o il*
Likew ise,

vestigators,
aeruginosa

is

sodium c h l o r i d e ,
detrim ental.

a s fo u n d by p r e v i o u s

Two s p e c i e s ,

in­

Ps eudomonas. ,

and Pseudomonas m e p h i t i c a , u t i l i z e

the g r e a t—

-45-

est

quantity

A sm all

o f oxygen in the

quantity,

stim ulating

to

brings

several of

about

the

le v e l of

atio n of

cent of

the

presence

to

endogenous

cells

per

in the

inhibition

instances

salt

of the

proves

b u t w ith the

in the

except

that

p e r c e n t sodium c h l o r i d e

amount
of Pseu­
brings

amount o f oxygen consumed below
respiration,

alone w ith o u t o il*

the

endogenous

organism s,

ion#

f r a g i i _a 0 * 4 9 6 p e r c e n t o f t h i s

In a l l

f l u o r e s c e n s , 2.5

about a decrease

the

a noticeable

o f oxygen u tiliz e d *
domonas

chlorine

0#336 p e r c e n t sodium c h l o r i d e ,

e x c e p t i o n o f Pseudomonas
salt

absence of the

lowered the

that

the

respir­

A c o n c e n t r a t i o n o f 1*5
rate

of r e s p i r a t io n

em ulsified o il nearly

respiration.

is,

to

in

a point equal

-4 6 -

Table

5

T h e e f f e c t o f pH o f t h e c o n t e n t s o f t h e c u p u p o n t h e cmm.
o f o x yg e n u t i l i z e d by b a c t e r i a i n t h e p r e s e n c e o f c o r n o i l

pH o f
buffer

Ps e u d o r a o n a s
graveomuc i d o - f r a g i i
lans
Ians

me p h i
t ica

fluorescens

aeruginosa

7 *4 0

218

248

53

56

54

50

6.85

221

233

41

40

39

47

6.21

220

202

86

62

66

60

5.60

148

191

79

54

63

51

5.00

183

97

73

36

34

45

4.53

49

97

64

21

42

—

92

35

.

22

32

Endogenous
respiration

30

-4 7 -

Table

6

The e f f e c t o f t h e p e r c e n t a g e o f NaCl i n t h e cup upon t h e
cmm. o f o x y g e n u t i l i z e d b y b a c t e r i a i n t h e p r e s e n c e o f
c o r n o i l a t pH 6 . 8 5 ( u n c o r r e c t e d )

Final
percentage
o f NaCl

fluorescens

aeruginosa

Ps e u d o m o n a s
graveomucidoIans
lens

fragii

mephi
tica

0.0%

229

211

—

49

--

85

0.236^

289

181

76

64

30

75

0. 496%'

237

76

53

27

57

0.85%

238

173

36

52

34

17

1.50%

194

100

32

34

24

17

2.50%

88

75

15

21

20

17

5 .00%

37

25

6

8

13

—

7 . 50%

--

20

10.00%

19

35

-

22

32

Endogenous
respiration

30

9
*

92

-4 8 f.

The e f f e c t o f t h e p h o s p h a t e i o n , h y d r o q u i n o n e ,
A v e n e x , and r a n c i d o i l upon t h e c o n s u m p ti o n o f
oxygen

E xperim ental.
the

Because o f

metabolism o f

the

carbohydrates

review

b y We r kma n a n d Wo o d ,

gested

that

ides

influence

be o b s e r v e d .

one o f
ing

its

duplicate

in

manometers

see the

the

u tilization of

with sa lin e

triglycer­

the

buffer of

s o l u t i o n and m easur­

R esults

are

given in

7.

is

or other

have been p ro p o sed ,

to p re v e n t o x i d a t i v e
substrates,

H y d r o q u i n o n e was
o il-soluble
This

-

ion in

1 9 4 2 - Dr* C* A. H o p p e r t s u g ­

T h i s wa s d o n e b y r e p l a c i n g

Many a n t i o x i d a n t s
w hich

phosphate

a n d amino a c i d s

th e amount o f oxygen consumed.

Table

the

ro le of the

resultant

selected,

antioxidants

was e m p l o y e d a t
oil*

w ith

changes

because

(M attill,

the ra te

Avenex r e p r e s e n t s

of lipoidal m aterial,

a lte ra tio n of flavor.
it

is one o f

1931; O l c o t t ,

10 p a r t s

one o f th e

the

better

1943).

per m illion of

favored w a te r-so l­

antioxidants

T his

p r o d u c t was e m p l o y e d i n t h e W a r b u r g c u p s a t a c o n c e n ­

one

0.07

ml. o f 0 .2 5

given

in Table
Fresh o i l

shallow

layer

meyer f l a s k

per cent,
per cent

C orbet and T rac y ,

and i n the c o n t r o l
s o l u t i o n was u s e d .

1941).

manometers
R esults

are

8.
wa s a l l o w e d t o
in a la rg e ,

and le a v in g

s e v e r a l weeks.

1941;

purpose of

uble

tratio n of

(A lcott,

of

the

At t h e

it

dry,

b e come r a n c i d by p l a c i n g a
loosely stoppered E rlen-

i n a warm room i n

end o f t h i s

light

for

time a s tr o n g l y p o s i t i v e

-4 9 Table

7

The e f f e c t o f t h e p r e s e n c e a n d t h e a b s e n c e o f t h e p h o s ­
p h a t e i o n u p o n t h e cram, o f o x y g e n u t i l i z e d b y b a c t e r i a i n
th e presence of e m u lsified corn o i l

_.

C ulture
_

Phosphate
ion p resen t

Phosphate
ion absent

C ontrol

Pseudomonas a e r u g in o s a

94.5

72.0

--------

Pseudomonas a e ru g in o sa

255.2

211.3

83.8

Sarcina

186.6

186.9

52.9

A lcaligenes
( 6 0 M)
55.2
l i p o l y t i c u s ( 1 2 0 M) 1 2 1 . 4

52.7
90.9

___ _
--------

lutea

Table 8
T h e e f f e c t o f h y d r o q u i n o n e a n d o f A v e n e x u p o n t h e cmm. o f
o x y g e n u t i l i z e d by b a c t e r i a i n t h e p r e s e n c e o f e m u l s i f i e d
c o r n o i l , a n d i n t h e p r e s e n c e o f Avenex
C ulture
Pseudomonas
Pseud,
Sarcina
A le.

aeruginosa

putrefaciens
lutea

lipolyticus

M icro,

flavescens

Corn Corn o i l Corn o i l Avenex
o i l & Hydro * & A venex

Con­
trol

339.6

326.1

405.8

445.8

79.8

--------

237.8

232.9

288.7

---------

153.9

172.0

163.2

208.5

62.0

99.5

108.1

111.4

50.7

52.4

100.2

108.0

150.9

115.4

35.4

50-

Table

9

T h e e f f e c t o f f r e s h a n d o f r a n c i d c o r n o i l u p o n t h e cmm# o f
o x y g e n u t i l i z e d by b a c t e r i a i n t h e Warburg a p p a r a t u s

C ulture

Pseudomonas a e r u g in o s a
Ps eudomonas p u t r e f a c i e n s
Sarcina

lutea

A le. lipolyticus
M icro,

flavescens

Fresh corn
oil

Bancid corn
oil

C ontrol

394.8

379.3

79.1

85.7

114.8

61.7

186.4

208.1

86.8

103.5

103.5

36.5

98.9

94.5

20.1

-51K reis
the

test

are

em ulsified
given

f r e s h corn o i l

in Table

R esults♦

in the

The

of this

amount o f

latter;
hour

the

ther,

in

of

form er organism,
is

absence

the

namely,

oil

three
at

the

of

the

a ratio

stim ulating.

was n o t o b t a i n e d ;
o x y g e n was
the

effects

lip o ly ticu s.

in each

a n d i n two h o u r s

instance,
fo r the

per cent
is

in one

noted be­
lutea

of th e experim ent had been a t t a i n ­
ion does

appear in f lu e n tia l

presence of corn o i l .
five

instances

instance,

fresh

but the

u t i l i z e d w ith
were n o t

t wo

The p r o b l e m was n o t p u r s u e d f o r -

T h e cmm. o f o x y g e n u t i l i z e d
p u t r e f a c i e n s , using

presence

at

r e s p ir a tio n of Sarcina

of ten parts

In one

cent

No d i f f e r e n c e

phosphate

in the

aeruginosa

approxim ately fiv e

in the

purpose

that

respiration

ion brin g s about a

a n d by A l c a l i g e n e s

l a t t e r organism .

its

since

phosphate

is about 20 p e r

end o f one h o u r .

In
the

organism ,

decrease

fo r the

cause

ed:

the

the

by P s e u d o m o n a s

decrease

one hour fo r the

and

R esults

q u a n t i t y o f o x y g e n consumed i n t h e

e m u ls ifie d corn o i l

dilutions

was d e t e r m i n e d .

it

9.

The a b s e n c e o f

decrease

at

T h i s o i l wa s e m u l s i f i e d a n d

q u a n t i t y o f o x y g e n t a k e n up b y b a c t e r i a w i t h

w ith

of

was o b t a i n e d .

shown,

it

was

corn o i l

the

severely

in

per m illio n proved s l i g h tl y
slightly

by o n e s p e c i e s ,

fact

hydroquinone

that

Fseudomonas

w ithout hydroquinone,
a large

antioxidant
felt.

inhibitory.

quantity of

indicates

that

The d i f f e r e n c e s

in

-5 2 the

q u a n t i t y o f oxygen consumed w ith hydro qu in on e

and absent
ence,
is

are

although

not

slig h t,

indicative

sufficiently

Avenex,
increase

less

a n d no a p p a r e n t

increase

is

Pseudomonas

putrefaciens•

all

of

source
of

of

energy,

Table 8, w ith

w ith th is

organism ,

noted

in the

Avenex i t s e l f

used in th is

figures

respiration

shown i n

column s i x o f

Rancid o i l
fresh

oil

faciens
other

b y two

species

and S a rc in a

three

apparent

apparently

species

influence

are

this

table.

is

utilized

resp ira tio n of
is

utilized

of rancidity

is

by

i n column f i v e
lip o ly ticu s;

respiration,

mo r e

which

rapidly

Pseudomonas

in magnitude
observed*

than

putre-

The v a l u e s o b t a i n e d w i t h

comparable

slight,

in the presence of

of b a c te ria ,

lutea-

is

flaves-

e x p e r i m e n t as a

A v e n e x was o n a l e v e l w i t h e n d o g e n o u s
is

lutea

exception of A lcaligenes
the

a significant

up b y P s e u d o m o n a s

of Sarcina

a s s h o w n by t h e
the

differ­

retardation,

l i p o l y t i c u s , and M ic r o c o c c u s

respiration

bacteria

or of

brought about

cmm. o f o x y g e n t a k e n

The i n c r e a s e d

This

t o be c o n s i d e r e d s i g n i f i c a n t -

on the o th e r hand,

in the

species

of stim u la tio n ,

great

aeru g in o sa , A lcaligenes
cens #

th a n ten per cent#

present

the

a n d no

-53g.
The c h a n g e s
o ils

subjected

several

in the

to

chem ical com position o f f a t s

have g iv en

Jensen

(1902)

fatty

the

H orow itz-V lassova

acids,

o il.

but his

Investigators
are

various

from t h o s e
notable

substrate

occurring
to

(Jensen,

date*

iodine

numbers,

of dairy

c o n t a i n e d t h e compounds
a n d was n o t p u r e b u t t e r

this

the

Orla

values of b u t t e r f a t

aspect of b a c te ria l

although changes

constants,"

exception

instances

discussing

agreed th a t

"fat

in

to

s p e c ie s and s p e c ie s

found in d airy p ro d u cts,

m etabolism
the

changes

and s a p o n i f i c a t i o n

in which s e v e r a l b a c t e r i a l

norm ally

and L iv s c h itz

most co m p le te r e p o r t

determ ined the

m olds had grown,

and

b a c t e r i a l a c t i o n h a v e b e e n s t u d i e d by

investigators*

(1935)

free

Chemical d e te rm in a tio n s

do o c c u r i n

c h a n g e s do n o t

differ

in s t r i c t l y

chem ical o x id a tio n .

this

a c i d n u m b e r a n d i n some

1902)

is

the

the

decrease

in s o lu b le

The

fatty

acids.
R eports
values

in the

of the

fat

seemed a d v i s a b l e
the

substrate,

fat,

literature

constants
to

study

regarding

are

these

sin ce Je n se n 's

and H orow itz-V lassova

meagre.

the

changes o f

Therefore,

it

changes w ith co rn o i l

as

w o r k was d o n e w i t h b u t t e r -

and L i v s c h i t z

had used soy bean

o il.
E xperim ental m ethods.
salts

The b a s i c

medium o f C l i f t o n ,

the

Cahen,

medi um was t h e

m ineral

a n d Mo r r o w ( 1 9 3 6 - 7 ) .

-5 4 -

Large, f l a t
3 5 ml *

bottles

fresh

when c o o l

containing

corn o i l

i n o c u l a t e d w i t h o n e ml* o f a n o t i c e a b l y

controls,

to

were

incubated

tem peratures

flat,

and the

cover the

basic

the

m ineral

hydroquinone

salts

and o f Avenex,

and 0 .2 5

in

the

to

alchohol
diethyl

all

ous and e t h e r

in the

e m p lo y e d was n o t s u f f i c i e n t

To d e t e r m i n e
10 p a r t s

thus

perm itting

the

con­

adding th is

latter

the

effects

per m illion of

o i l before

p e r cent of the

the

of

the
to

m a t e r i a l was

incubation period,

the b o ttle s

and o th e r s

grew s c a n t i l y .

of

Ten m l. o f e t h y l

t h e Ro u x b o t t l e ,

a separatory funnel,

l a y e r s were s e p a r a t e d .

r i n s e d w ith second and t h i r d
and th e aqueous

failed

an e q u a l m i x t u r e o f p e t r o l e u m and

were added to
into

were

Growth in

a l t h o u g h some o r g a n i s m s

a n d 25 m l .
ethers

were l a i d

was h e a v y ,

l i q u i d was p o u r e d

tures,

The b o t t l e s

c o n ta m in a tio n and f o r growth.

instances

grow a t

proper incuba­

aqueous menstruum.

end o f

examined f o r

together w ith

o f o r g a n i c m a t t e r upon th e

medium.

the

most

the

turbid

o n e p e r c e n t p e p t o n e wa s a d d e d t o

dissolved

At the

th ree weeks.

effects

fo rm er were

dissolved

darkness a t

and

of gases.

the o i l ,

b ottle,

The b o t t l e s ,

aqueous menstruum c o m p l e t e l y ,

To s t u d y
of

for

in

quantity of o il

an in te rc h a n g e

stants

me d i u m a n d

were p re p a re d and s t e r i l i z e d ,

s u s p e n s i o n o f washed b a c t e r i a .

tion

1 0 0 ml* o f t h i s

in

entire

and th e

The f l a s k s

portions of the

discard

the

aque­

were

e t h e r mix­

each in stan ce

e x ce p t the

last

wa s p e r m i t t e d

to

flow

into

the

new e t h e r m i x t u r e .

The c o m b i n e d e t h e r e x t r a c t was f i l t e r e d
flasks

t h r o u g h W h a t m a n N o . 43 f i l t e r

washed w ith
chips

e th e r before

and th e

w ater bath at

90 C .

S ev eral sm all b o ilin g

volatile

bination

and re d u c tio n o f p re s s u re

fatty

the o i l

are

in a

by 6 4 0 mm.

has one s e r i o u s

objection

a c i d s may b e d r i v e n o f f b y t h e

o f h e a t and red u c ed p r e s s u r e ;

procedures

in e th e r

e t h e r s w e re removed by h e a t i n g

method o f o b t a i n i n g

free,

suction

p a p ers w hich were

and one m l. o f 0 . 0 0 2 p e r c e n t h y d ro q u in o n e

were added,

This

use.

into

however,

com­

drastic

n e c e s s a r y to remove th e l a s t o f t h e

ethers,

a n d no o t h e r m e t h o d h a s o c c u r r e d w h e r e b y t h e o i l may b e
extracted,

or the

ethers

c o m p l e t e ly removed.

The m e t h o d o u t l i n e d a b o v e
Pigulew ski
ever,
the

and C harik

dried th e ir

present

m itted

to

(1928).

oil

that

employed a ls o

These i n v e s t i g a t o r s ,

by adding sodium s u l p h a t e ,

investigation

settle

is

was

and c o a le s c e ,

w hich were f i t t e d
o il

caught

and th e o i l

in c le a n ,

w ith sh o rt

was w e i g h e d b y d i f f e r e n c e

fo r the
^ * Th e

determ inations
a cid number.

hydroxide

required

listed

This
to

how­

w hile

in

r e s i d u a l w a t e r d r o p l e t s were p e r
then decanted

w h i l e wa r m t h r o u g h w a s h e d a n d d r i e d f a t - f r e e
The f i l t r a t e

by

is

filter

papers

sm all Erlenm eyer fla s k s

pipettes

and s t o p p e r s .

to o b t a i n

The

the q u a n titie s

below.
the

ml. o f normal sodium

neutralize

the

free

fatty

acids

-56-

i n 100 g r a m s o f o i l .
The p e r o x i d e
of peroxide

number.

iodide

solution.

3.

of

expressed

iodine

term ined
in

the

is

used,

Because o f th e

Th e i o d i n e

as

This

m o d i f i e d by E w e b a n k
so lu tio n of

than of a s a tu r a te d

low v a l u e s o b t a i n e d ,

a b s o rp tio n number.

This

is

results

the percentage

absorbed p e r weight of the o i l .
according

fifth

moles

wa s d e t e r m i n ­

a 50 p e r c e n t

rather

the

in m illim o le s .

to

t h e Hanus m e t h o d ,

It

was d e ­

as o u t l i n e d

e d i t i o n o f t h e O f f i c i a l Methods o f A g r i ­

c u ltu ra l A nalysis.

(1940).

^ • The s a p o n i f i c a t i o n n u m b e r .
m illigram s o f potassium
ify

e x p r e s s e d as

method (1932),

i n w h ic h one m l. o f

potassium

are

is

p e r 1000 g r a m s o f o i l *

e d by W h e e l e r s
(1942),

This

hydroxide

c o m p l e t e l y one gram o f

te r m in e d a c c o rd in g to

T h is number r e f e r s

the

to th e

r e q u i r e d to sa p o n ­

fat or o il.

T h i s wa s d e ­

method o u t l i n e d

i n t h e A. 0 .

A. C.
5# The r e f r a c t i v e
term ined w ith

index.

The

i n d e x o f r e f r a c t i o n was d e ­

t h e Abbe r e f r a c t o m e t e r a t

a tem perature

o f 20°C.
6.

The a l d e h y d e n u m b e r .
number o f ml.

This

number i s

of 0.003 normal

of o il.

The m e t h o d i s

W hite*s

recommendation

iodine

e x p r e s s e d as th e
t a k e n up p e r g r a m

t h a t o f Lea ( 1 9 3 4 ) ,
(1941).

t y wa s e n c o u n t e r e d i n o b t a i n i n g

At

first,

without
much d i f f i c u l ­

good c h e c k s ,

but

it

-5 7 was

found th a t

freshly

diluting

boiled,

cooled,

the

s o l u t i o n w i t h ICO ml* o f

distilled

w ater gave b e t t e r

resu lts•
7 • Tlie K r e i s

test»

ml * o f o i l ,

This

t e s t wa s made b y a d d i n g t o

t wo ml* o f c o n c e n t r a t e d h y d r o c h l o r i c

two
acid,

o n e ml * o f e t h e r c o n t a i n i n g

0.1 p er cent p h lo ro g lu c in -

ol,

ether.

and f o u r m l.

according
ty

to

and th e

S m ith's

of diethyl

Powick (1 9 3 3 )

presence

indicates oxidative

of epihydric

observations

(1920)

A positive

are

aldehyde,
not

test,
rancidi­

although

in complete a g re e ­

ment .
8.

The v o l a t i l e

acids *

w e r e made o n b o t h t h e
tra c tio n of
o il,

using

tion,
of

the

o il,

o f the

aqueous

shown on page 168,

R esults *

litre

of the

The r e s u l t s

given in Tables

res id u e »

several other

made.

E scherichia

grow b u t

residual

o f a few o f

10 a n d 1 1 .

shown,

slig h tly

e th e r ex­
of the

fifth

edi­

A g ric u ltu ra l Chem ists.
This

result

liquid
the

and P r o te u s

is

expressed

r e q u i r e d to
to

neutral

phenoIphthalein*

determ inations

In a d d i t i o n to

determ inations

coli

acids

and T e n t a t i v e Methods o f A n a ly s i s

t h e ml* o f n o r m a l s o d i u m h y d r o x i d e

iz e one

to

after

and on w e ig h e d q u a n t i t i e s

apparatus

the O f f i c i a l

for v o la tile

liq u id residue

the A s s o c ia tio n of O f f i c i a l

Acids
as

of

the

D eterm inations

the

are

results

and o b s e r v a t i o n s were
a mmo n i a e w e r e a b l e

and b r in g a b o u t a f a i n t l y

perceptible

-5 8 turbidity*
Sarcina

The o t h e r o r g a n i s m s

l u t e a , which u t i l i z e d

oxygen but

failed

to

any tim e ,

grow a t

included

in th is

to

r e p o r t e d grew a b u n d a n t l y *

ra th e r large

decolorize

quantities

m e t h y l e n e b l u e , wa s u n a b l e

and r e s u l t s

with th is

species

are

from b o t t l e s

h a d grown a b u n d a n t l y c r y s t a l l i z e d

in which the

in the

bacteria

refrigerator;

other o ils ,

in which b a c t e r i a

had grown b u t s l i g h t l y

not

rem ained liq u id .

A lso,

all,

oils

of the

g r o u p h a d a d e e p e r amber c o l o r t h a n d i d t h e
u le w s k i and C h a rik
but

this

(1988)

be an e t h e r - s o l u b l e

are

Th e o i l

crease

in

m oles)

and in the

o il.
to

in Table

in the o il
from t h e

the peroxide

since

Proteus
up oxygen in

flasks

number ( t h i s

aldehyde number,

is

the

medium d u r i n g

was

definitely

growth

but

three

Fig-

cells.
t h a t changes

shows an i n ­

expressed in m i l l i ­

decrease.

This

is

chem ical o x id a tio n .

Warburg s y s te m .
the

latter;

as com pared w i t h f r e s h

ammoniae and E s c h e r i c h i a
the

former

as a r e s u l t o f b a c t e r i a l

control

in s t r i c t l y

or

t h e p i g m e n t may

10 i n d i c a t e

The o t h e r n u m b e r s show a s l i g h t

be e x p e c t e d

the

observed pigm entation,

compound fr o m b a c t e r i a l

given

brought about

action.

also

may b e o f no s i g n i f i c a n c e ,

The v a l u e s

not

table.

The e x t r a c t e d o i l s

at

of

coli

They grew s l i g h t l y

week p e r i o d ,

not deeply

did not take

since

the

in

medi um

turbid*

This

quantity of

c o u l d b e e x p e c t e d when r e s t i n g

cells

are placed

-5 9 into

a favorable

environm ent,

of

carbon,

according

It

is

possible

red

also

during

extent

protection

ide

S a n d if o r d and W ooldridge

that

a sm all

sterilizatio n ,

that

species

to

the
is

are

offered,

if

compared w ith

the

the

No a p p a r e n t

values obtained fo r

those

of fresh o i l .

as

would expect

in each

that

be l i p o l y t i c ,

the

lines

changes
tion
ly

instance

is

a l t h o u g h one

th e a c i d number o f o i l

in which the

all

number a nd t h e

these

iodine

i n some i n s t a n c e s .

values

are q u ite

A ll values

rem aining organisms

show

The s a p o n i f i c a ­

a b s o r p t i o n number d ro p marked­

The p e r o x i d e n u m b e r s a n d t h e

alde­

l o w ; o n e may h a v e e x p e c t e d h i g h e r

f o r each of th e s e .

However,

bacteria

may b e a b l e

remove t h e o x y g e n from t h e m o l e c u l e o f o i l ,

w itz-V lassova

h a s made u s e o f

logical"

for

test

with

high,

when c o m p a r e d w i t h t h e f r e s h o i l .

hyde numbers

to

w ith

add

The a c i d num­

a b o v e name d o r g a n i s m h a d g r o w n w o u ld be l o w .
on

to

p r e v i o u s l y d e t e r m i n e d mano-

a n d h a v e b e e n shown t o

the o i l

The p e r o x ­

showed an a b i l i t y

e x c e p t i o n o f Pseudomonas g r a v e o l a n s .

ber of

these

r a t h e r high.

The r e m a i n i n g o r g a n i s m s

m etrically,

(1931).

and t h e s e organism s grew to th e

numbers a r e

em ulsified o il,

source

amount o f h y d r o l y s i s o c c u r

freed g ly cero l perm itted.

and aldehyde

oxygen to

w ithout a u t il i z a b le

oxidative

this

ab ility

rancidity.

f o r Horo-

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ISI

-6 1 The r e f r a c t i v e
taken a t

20°C .,

index of fre s h o i l ,

v a r i e d somewhat from t h e

b y Baughman a n d J a m i e s o n
m anufacturer of

the

oil

may n o t b e c o m p a r a b l e ,
15.5°C#

S ignificant

to which b a c t e r i a
the
in

m olecule a re
doubt,

1.4742 to

(1921).

value

The v a l u e

(se e Appendix A ),
since

the

latter

decreases occur

indicated,

because of the

but

the

1.4717 g i v e n

g i v e n by t h e

1.475

value

in the

have b e e n expo sed.

1.4747,

to

is

1.477,

taken at

various

Internal

oils

changes o f

interpretations

numerous p o s s i b l e

are

intram olecular

changes•
Table
n ificant

11 s h o w s t h e

determ inations

follow ing

media;

same,

plus

same

as t h e

same

(1)

10 p a r t s

results

o f f o u r o f t h e mo r e s i g ­

upon c o r n o i l

extracted

m ineral s a lts - c o r n

(2)

the

p e r m illio n of hydroquinone;

(3)

f i r s t , plus 0.25

oil;

from th e

the

p e r c e n t Avenex; (4)

the

as th e f i r s t ,

p lu s one p e r c e n t p e p to n e ; (5)

the

same

as th e s e c o n d ,

p lus one p e r cent peptone;

same

as thet h ir d ,

p l u s o n e p e r c e n t p e p t o n e . Two o r g a n ­

isms were u s e d :

A lcaligenes

lipolyticus

and (6)

the

and Pseudomonas

aeruginosa.
Th e p r o t e c t i o n o f
to

the o i l

numbers
case

is

reflected

and the

lesser

of A lcaligenes

form er v a lu e ,

a utilizable
in the
change

organic

decrease
in the

m aterial given

in the

iodine

numbers

l i p o l y t i c u s , and a d e c r e a s e

and the

relatively

high io d in e

peroxide
in the

o f the

numbers in

the

case

tone

is

K reis

o f Pseudomonas a e r u g i n o s a .
also

test-

reflected
Both the

in the

Th e p r e s e n c e o f p e p -

lowered i n te n s i t y of

antioxidants,

Avenex in

the

concentrations

used,

iceably.

The c o n c e n t r a t i o n s ,

the

hydroquinone or
protect

the o i l n o t ­

incidentally,

are

those

re­

commended f o r e a c h .
No r e f e r e n c e s
atile
oil

acids.

or

in the

a r e made t o

None w e r e
aqueous

none were formed,

or

and subsequently

lost

residue.
that

Two c o n c l u s i o n s

in the

are

possible
ethers,

d u ring the v o l a t i l i z a t i o n of the

free

were

cultures

either

of vol­

t h e y w e r e t a k e n up b y t h e

The f o r m e r p o i n t

the

determ inations

f o u n d a t any t i m e ,

solvents.
acids

the

found in th e

upon t i t r a t i o n .

seems more p r o b a b l e ,
aqueous

residue

since

o f many o f

-6 3 T ab le

11

The ( a ) a c i d n u m b er ( b ) p e r o x i d e number ( c ) i o d i n e number
( d ) K r e i s t e s t o f o i l s from c o r n o i l - m i n e r a l s a l t s medium;
t h e m e d i u m p l u s h y d r o q u i n o n e ; t h e medi um p l u s A v e n e x ; a n d
e a c h o f t h e m ed ia p l u s one p e r c e n t p e p t o n e , f o l l o w i n g g r o w t h
o f A l c a l i g e n e s l i p o l y t i c u r n and Pseudomonas a e r u g i n o s a

(a)
Fresh o il
A lcaligenes

1.83

(b)

(c)

(d)

0.19

121.76

0

8.01

115.98

++++

lipolyticum

M ineral s a l ts - c o r n

oil

17.53

M ineral s a lts -c o rn o i l
& hydroquinone

19.53

7.60

114.67

++++

M ineral s a lts - c o r n o i l
& Avenex

12.79

6.23

120.58

+++

M ineral s a lts -c o rn o i l
8c p e p t o n e
25.37

5*22

120.20

1*+ +

M ineral s a l ts - c o r n o i l
p e p t o n e 8c h y d r o q u i n o n e

23.46

2.74

M ineral s a l ts - c o r n
p e p t o n e 8c A v e n e x

70.85

2.97

Ps eudomonas
M ineral

?

+

oil
119.58

■+

119.28

++•*■•*

119.63

+++■ +
++++

aeruginosa

salts-corn

oil

114*86

M ineral s a l ts - c o r n o il
& hydroquinone
113.32
M ineral s a lts -c o rn
& Avenex

oil

7.88
7.28

84.10

6.58

119.43

M ineral s a l ts - c o r n o i l
& peptone

112.31

6.95

119.19

M ineral sa lts -c o rn o il
p e p t o n e 8c h y d r o q u i n o n e

98.98

6.81

119.28

+++

128.60

4.85

118.09

+++

M ineral s a lts - c o r n
p e p t o n e & Avenex

oil

-64DISCUSSION OF THE RESULTS
The W a r b u r g a p p a r a t u s
term ination of
and o i l s .
ly

It

the
has

ability
the

and a c c u r a t e l y ,

involved
rather

a unique

tool

of b a c te ria

whereas methods
of

fo r the

to o x i d i z e

advantage of g iv in g

long p erio d s

tedious

is

results

fats
rapid­

p r e v i o u s l y u s e d have

incubation,

f o l l o w e d by t h e

pro ced u res of e x t r a c t i o n of the o i l

chem ical a n a l y s i s .

not

d i s t i n g u i s h between chem ical and b i o l o g i c a l o x i d a t i o n ,

this

d istinction.

fat

or

that

this

conclusively
is

instrum ent w ill

no t y p e o f a n a l y s i s

The W a r b u r g a p p a r a t u s

a n unknown s t r a i n

does,

makes

however,

t h a t a given sp e c ie s of
capable of o x id iz in g a

o r an o i l .
M ention has

of

true

is oxidized;

e n a b l e one to pro v e
bacteria

is

and o f

the

once a tr ig ly c e r id e

It

de­

b e e n made p r e v i o u s l y

chem ical changes of f a t s

distinguishing

and o i l s

agency o f b a c t e r i a .

drastic

of oxidation,

methods
are

employed.

conclusion.

are

inconclusive

b e t w e e n o x i d a t i o n by s t r i c t l y

and th ro u g h th e

ducts,

th a t determ inations

In these

This

resulting

The d a t a o f t h i s
determ inations,

The o n l y
biological

c h e m i c a l means
true

unless

in p e c u l i a r pro­
paper confirm t h is

the p e rio d of

c u b a t io n has been u n ifo rm ly th r e e weeks,
investigators

is

in

in­

although o th e r

h a v e e m p l o y e d much l o n g e r p e r i o d s a t t i m e s .
a lt e r a t io n of f a ts

action

is

the

and o i l s

a l t e r a t i o n of the

due s o l e l y

to

a c i d number.

-6 5 Even th is

criterion

is

H o r o w it z - V la s s o v a and L i v s c h i t z

aware,

investigators
to

u tilize

is

undependable*

who h a v e s h o w n t h a t

a triglyceride

T heir stu d ies

s i s , is

not

ed above,

add sev eral
of

these

atus*

a fat

organisms

to
to

These s p e c ie s

l u t e a : M icrococcus
M icrococcus
Phytomonas

o r an o i l ,

investigations

species

this
take

are:

then,

reported
list,

is

has been

is

paper w ill

up o x y g e n i n t h e W a r b u r g a p p a r ­
A lcaligenes

f a e c a l i s : Sarcina

a u r a n t i a c u s ; Micrococcus

flevescens:

arborescens;

m ephitica*

dem onstrated with
fat.

may b e a d d e d t o
me mb e r s o f

probable

needed before
The

lipoly-

t u m e f a c i e n s ; Pseudomonas g r a v e o l a n s , Pseudo-

gen which th e
it

is,

of

b a s e d upon t h e a b i l i t y

c in n a b a re u s ; Flavobacterium

but not w ith b u tte r

and

that

in th is

m o n a s m u c i d o l a n s may n o t p r o p e r l y b e l o n g

B acillus

ab ility

To t h e o r g a n i s m m e n t i o n ­

monas m u c i d o l a n s , a n d Pseudomonas

lipolysis

the only

A erobacter aerogenes*

determ ining the

com pletely re lia b le *

the

are

the w rite r

a m i c r o o r g a n i s m wa s a b l e

u n c o v e r e d one s p e c i e s ,

to a t t a c k

as

w ithout previous hydrolysis*

The c u s t o m a r y m e t h o d o f
a species

As f a r

they

lipolytic

puzzling*

that
are

in th is

this
the

list;

the

fam ily take

further

list;

in corn o i l ,

S e v e r a l me mb e r s o f t h e

genus

amounts o f oxy­
up a r e

sm all,

i n v e s t i g a t i o n w i l l be

definitely

activity

Several

it

Pseudo­

included o r

excluded.

o f Pseudomonas m ucidolans

reasons

may be a d v a n c e d a s

to

-6 6 failure
fat

to

medium:

rapidly,
the

detect
the

lipolysis

products

and th is

gives

or

tective

properties

ly tic
that

that

is

of the

ab ility

organisms

differences

fo r the

coli

and W alker

coccus

lipase,
albus

found th a t

to

lost

after

this

ab ility

conditions

I t wa s p o i n t e d o u t e a r l i e r

species of bac­

hydrolyzed o liv e
found i t

some

oil,

negative

(1933)

found i t

butter fat.

re­

while
to

butter

and S taphy lo ­

Peppier

low m o l e c u l a r w e i g h t ,

of the

Staphylococcus

in lip o ly tic

i s o l a t e d Staphylococcus

of

is

found S ta p h y lo c o cc u s aureus to

hydrolyze

hydrolyzed esters

It

W ells and C o rp er (1912)

(1914)

while B erry

a freshly

lipo­

have been found l i p o ­

of several

of opinion e x ist*

Day,

the

above*

determ ination of lip a s e ,

The f o r m e r a u t h o r s

possess

not hydro­

oils*

reactions

th at E scherichia

K endall,

species

substrates.

between d i f f e r e n t

to media

listed

have n o t e d a d i f f e r e n c e

Because of the

fat.

is

found in heavy cream o f f e r a p r o ­

some o f t h e s e

investigators

ported

actually

very

d e t e c t i o n by

made i n B e r g e y * s M a n u a l t o

on o t h e r f a t t y

teria

for

utilized

action*

possible

only

no o p p o r t u n i t y

th at m aterials

No r e f e r e n c e
lytic

o rg a n is m on b u t t e r -

of h y d ro ly sis are

usual means; the b u t t e r f a t

lyzed;

the

by t h i s

five

months

experim ents

a u r e u s was l i p o l y t i c

aureus

but th at

in s to c k .

reported

(1941)

in

it

Under
this

paper,

on b u t t e r f a t

and

“67on corn o i l ,
ab ility

to

and none o f th e s e organism s

u tilize

manometers*

It

is

indicated

corn o i l

as a source of energy in the

possible

th a t Staphylococcus

w ould show an i n c r e a s e d u p ta k e o f oxygen i n
cup

if

sufficient

about hydrolysis

of a substrate

state,

relatively

few,

however,

blue.

K endall

the

distinction
Sarcina
m eter;

genating

may b e ma de

dehydrogenate
and Ishikawa

is

n o r does

is

therefore,

possible,

the o i l

is

the
as

available

to

in the

decolirize

(1929)

state

that

used as

The f a c t

i n d i c a t e d by t h e

that a

fact
in the

that
ma no­

that

g r o w i n a s y n t h e t i c me d i u m
sole

source o f carbon.

th is organism

as a source o f energy,

possibility

the

it

the

assum ption should not

volved,

corn o i l

d o e s n o t d e c o l o r i z e m e t h y l e n e b l u e by d e h y d r o “
the o i l ;

sidering

it

consumes oxygen v e ry r a p i d l y

corn o i l

This

to o x i d iz e

d ehydrogenated which a re

i n which th e

u tilize

and fo r growth.

b o th energy and grow th.

lutea
it

fo r energy,

as shown by the a b s o r p t i o n o f oxygen;

compounds a r e

source of

bring

s h o u l d b e made b e t w e e n t h e

are able

em ulsified

those

t h e Warburg

of the o i l .

A number o f o rganism s

m ethylene

aureus

tim e were g iv e n th e organism to

A pparently a d is tin c tio n
u tilizatio n

the

be made,

that

able to

but not fo r growth.

however,

w ithout con­

o t h e r f a c t o r s may b e i n ­

a c c e ss o ry growth f a c t o r s

nitrogen.

is

It

and the type o f

-6 8 Because o f o c c a s io n a l f l a v o r
foods

containing

d u c t s , due to
natural

paper,

h ib itin g

action,

and s a l t i n g ,

as

entirely,

Under th e

the

conditions

ion

processes

involving o i l .

is

shown t o

although fu r th e r

available
for

is,

contain
that

in decreasing,

of

these

be

influential

if

not in ­

processes.

experim ents,

the

in o x id ativ e

The p r o b l e m h a s n o t b e e n p u r ­

investigation

is

indicated.

An

su p p ly o f phosp h orus would a p p ea r d e s i r a b l e

oxidation,
relatively

bacteria

conditions.
ated

inhibitors.

and c o n firm e d by d a t a

rapid oxidation of fa tty m ateria ls.

preclude

especially

use o f sodium

biological oxidative

phosphate

sued,

that

successful

dairy pro­

acidulation,

shown by o t h e r s

are

in c e r t a i n

especially

h a v e b e e n e m p l o y e d as b a c t e r i a l

B oth p ro c e ss e s ,
this

and o i l s ,

m icrobial

souring,

chloride,

of

fats

defects

are

large

the

it

i s w e l l known t h a t b a c t e r i a

quantities

capable

A lso,

activity

since

Absence would n o t

of this

of existing

elem ent,

under q u ite

consequences of prolonged,

h a v e b e e n s h o w n i n many f i e l d s

and

adverse
deceler­

of investiga­

tion.
A ntioxidants
m aterials
fats
of

as w e ll

and o i l s .

s o me o f

are

to

employed in f o o d s t u f f s
prevent the

G ortner

these.

antioxidants

are

It

is

(1958)

chem ical a l t e r a t i o n of

has review ed the a c tio n

possible

protective

and o th e r

that

in th a t

some s o - c a l l e d

they fu rn is h

a more

-6 9 desirable

substrate

for b acterial

action,

thereby

spar­

ing tr ig l y c e r i d e s .
Hydroquinone
of

oxygen in the

quinone
three
w ith

a n d A v e n e x do n o t p r e v e n t a c o n s u m p t i o n
manometers.

stim ulates

of

the

the

rate

four species

t h e Warburg s y s te m

tects

the

corn o i l

Table 8 in d ic a te s

t h a t hydro­

o f a b s o r p t i o n o f oxygen f o r

of b a c te ria .

It

is

not p o ssib le

to d e t e r m i n e w h e t h e r Avenex p r o ­

from o x i d a t i o n ,

s i n c e Avenex i t s e l f

is

r e a d i l y o x i d i z e d by f o u r s p e c i e s

of bacteria,

it

is

m aterial.

the

sole

source of oxidizable

ism a p p a r e n t l y n o t o x i d i z i n g

it,

of

columns 5 and 6 o f Table 8,

in

this

able

instance,

w ith

the

when

The o r g a n ­

a s s h o w n by c o m p a r i s o n

is A lcaligenes

the endogenous r e s p i r a t i o n

lipolyticus;
is

compar­

r e s p i r a t i o n o b t a i n e d when A v e n e x i s

present

w ith the b a c te r ia .
L ittle

inform ation

dissim ilation
to

apply

the

of

fatty

is

available

acids.

theories of

It

beta-

ture

tract

Pearce

to

from th e

C lutterbuck

may b e p r e s u m p t u o u s

to

b a c t e r i a l m etabolism .

f o u n d no e x p e r i m e n t a l e v i d e n c e

which a f f i r m e d th e s e

o f evidence

to th e b a c t e r i a l

and o m e g a - o x i d a t i o n as r e ­

v i e w e d by G - o r t n e r a n d b y H a r r o w ,
The w r i t e r

as

the

theories,

contrary

possibilities

and Raper (1925)

(1931)

each have

in the

and th e

sm all

does n o t o f n e c e s s i t y
of

these

litera­
amount
de­

types of o x id a tio n .

and Smedley-MacLean and

advanced t h e o r ie s

as to p o s s i b l e

m ethods o f
tiated

u tilization,

but

th e s e have n o t

by p r o o f o f a b i o l o g i c a l n a t u r e .

It

is

probable

that,

more t h a n one m eth od o f
and th e

constituents

upon th e

in the case of m icroorganism s,

d issim ila tio n of

exists.

acids,

acids;
uses

uses

a n d t h a t B. b u t y r i

glycerol,

acids

the g ly c e ro l

but

brings

a s s o c ia te d with the

g ators,
Fouts

including Jensen

(1940),

action

in

the

have

the

t h a t Leuconostoc

I I * -, a l i p o l y t i c

the f a t t y

organism,

a b o u t no c h a n g e

in the

triglycerides.

O ther

shown t h a t

Peppier

bacteria

of fatty

in itially
during
that

inform ative,

low v a l u e s

the

is,

is

early

oxygen

stages
is

p o ssib ility

exists,

tion

by th e

occurs

fo u r organism s,
faciens,
of

indicate

however;

this

and

exert a selectiv e

respira­

conclusive.

the

The

processes

of oxidation are

being added to

investi­

acids.

but not
that

fatty

(1941),

The i n f o r m a t i o n g a i n e d b y d e t e r m i n i n g t h e
tory quotient

occurring

chiefly

additive;

the carbon ch ain .
is

that

removal o f hydrogen.

further

dehydrogenating

Three o f the
putre-

l i p o l y t i c u s , have proved cap a b le

corn o i l ;

No s p e c i e s o f b a c t e r i a
B e r g e y 's Manual ( f i f t h

Another

desatura­

Pseudom onas a e r u g i n o s a , Pseudomonas

and A lc alig e n es

who

unsaturated

and o x i d i z e s

(1902),

utilization

is based

and L iv s c h itz ,

saturates

w i t h no f u r t h e r a t t a c k ;

m esenteroides

triglycerides

This s ta te m e n t

fin d in g s of H orow itz-V lassova

found th a t Bacterium cloacae
fatty

been s u b s ta n ­

the

fo u rth organism ,

o f t h i s name i s
edition).

listed

in

Sarcina

-7 1 l u t e a . is

unable

to

do s o *

Th e a d d i t i o n o f o x y g e n a t
bonds appears
that

several

type

of

a reasonable

acid

site

process,

investigators

fatty

the

of the

double

in view o f th e

fact

have r e c o v e r e d th e s t e a r i c

f r o m a me di um c o n t a i n i n g o l e i c

acid

initially*
The a s s u m p t i o n
that

the

presence

a b s o r p t i o n o f o x y g e n by c e r t a i n
of corn o il

the o il*
case;
sis

It

is

however,

of

the

it

oil

the

result

It

the

theories
(1927);

of

is

in these

the

that

such is

source of the

oxidations.

decarboxylation of

arise

as

the

the

result

of double bonds,

the
analy­

that oxida­

It

carbon

could a r is e

fatty acids,

as

o r as

of

the

continued o x id a ­

in accordance with

a d v a n c e d b y P o w i c k ( 1 9 2 3 ) ; Hol m a n d G - r e e n b a n k
T sirsch

and Barben ( 1 9 2 4 ) ; -and T s irs c h

of

this

point

biological oxidations
the

to b e l i e v e

taken place.

disputable

site

C larification
the

in the

o f om ega-oxidation of the hydrolyzed a c id .

could also

tion at

paper

in the o x id a tio n of

c a n b e p r o v e d o n l y whe n c h e m i c a l

has

recovered

result of

bacteria

i n t h e W arburg cup d e m o n s t r a t e s

Sim ilarly

the

has r e s u l t e d

reasonable

t i o n of the o i l

dioxide

h a s b e e n made t h r o u g h o u t t h i s

individual

fatty

must a w a i t

of fa ts ,

acids.

(1925).

fu rth e r study of

o ils,

and above a l l ,

-7 2

SUMMARY AND CONCLUSIONS
It
able,
of

is

shown t h a t

tim e-saving

bacteria

tions

t h e Warburg a p p a r a t u s

instrum ent

to o x i d i z e

show i t

to

fats

The a b i l i t y

than

and o i l s *

to add oxygen to c o r n o i l ,

is

the a b i l i t y

These

than the

a depend­

investiga­

usual plate

study*

by t h e Warburg a p p a r a t u s ,
of bacteria

in determ ining

be more r e l i a b l e

and c u l t u r a l methods o f

is

the

is

a s shown

p o s s e s s e d b y mor e s p e c i e s

ab ility

to

remove h y d r o g e n ,

as

shown by t h e Thunberg te c h n iq u e *
A lcaligenes

f a e c a l i s . S a rc in a l u t e a , Micrococcus

a u r a n t i a c u s . M icrococcus

c in n a b a r e u s . M icrococcus

flaves-

c e n s , F la v o b a c te r i u m a r b o r e a c e n s . Phytomonas tumefac i e n s ,
Pseudom onas g r a v e o l a n s , and Pseudomonas m e p h itic a a r e
able

to o x i d i z e

organism s
previous

are

triglycerides

in a d d itio n

investigators

The r e s p i r a t o r y
teria

oxidizing

The
rise
not

in itially
slow ly

quotients of

the

low v a l u e

during

exceed three

are

this

ability*

four species of

g r e a t l y below th e

rises

abruptly,

bac­

theoretical

complete o x i d a t i o n o f f a t s

the p e rio d of

hours.

These

to A e ro b a c te r a e r o g e n e s . which

h a d shown to p o s s e s s

corn o i l

v alue proposed fo r

w ithout hydrolysis*

and o ils *

and continues

investigation,

to

which did

-7 3 -

The c h e m i c a l
by b a c t e r i a l
ges

resulting

changes of t r i g l y c e r i d e s

activity

are

from s t r i c t l y

Two a n t i o x i d a n t s ,
w a t e r - s o l u b l e Avenex,
oil
is

in the

indistinguishable

the

the

study,

employed.

as

hydroquinone

five

The l a t t e r

species

whereby f a t t y

apparently

acids

are

and

corn

compound

employed i n

this

s h o w n by t h e a m o u n t o f o x y ­

g e n consumed i n th e Warburg a p p a r a t u s .
Phosphates

chan­

non-biological oxidation.
oil-soluble

a t t a c k e d by f o u r o f t h e

about

from t h e

do n o t p r e v e n t o x i d a t i o n o f

concentrations

experim ent of

brought

enter

utilized*

into

th e mechanism

-7 4 APPENDEX A
L e t t e r from Mr. L. B. R o th e , T e c h n i c a l S e r v i c e ,
P r o d u c t s R e f i n i n g Company, A r g o , I l l i n o i s .
F e b r u a r y 17,

o f Corn

1943

Mr. J . 0 . M u n d t ,
D ept, of B iology,
Hobart C olleg e,
G e n e v a , New Y o r k
Dear S ir:
We h a v e y o u r l e t t e r o f J a n u a r y 3 0 t h r e q u e s t i n g i n f o r m a t i o n
on th e p h y s i c a l and chem ical c o n s ta n ts and co m position o f
Mazo l a .
T h is i s a h i g h l y r e f i n e d c o r n o i l p r o d u c e d from
o i l p r e s s e d from th e c o rn germ.
The i n f o r m a t i o n g i v e n b e ­
low r e p r e s e n t s t h e a v a i l a b l e d a t a on t h i s p r o d u c t .
Ph.ys i c a 1 a n d C h e m i c a l C o n s t a n t s :
Mazo l a
S o lid ify in g Point
I o d i n e Value
Free F a t t y Acids
S a p o n i f i c a t i o n Value
U nsaponifiable M atter
S a t u r a t e d F a t t y Acids
U n s a tu ra te d F a tty Acids
S p e c i f i c G r a v i t y a t 15°C
R e f r a c t i v e In d e x a t 15.5°C

-1 0 to -20°C
113 to 128
. 0 5 % maximum
188 t o 1 9 3
1 . 3 to 1 .7
10 t o 12
88 t o 90
0 .9213 to 0.9268
1 .4 7 5 to 1.477

S mo k e P o i n t
Flash Point
Burning Point
A c e t y l Value

400 - 4 5 0 ° F
620 - 640°F
694° F
7.5 - 9.0
Compos i t i o n

M a zo la i s composed o f g l y c e r i d e s
follow ing f a tty acids:

combined w ith the

S a t u r a t e d F a t t y Acids
Palm itic
Stearic
A rachidic
L ignoceric

7.7 %
3.5
0.4
0.2

-75-

Compos i t i o n C o n t 1 d .
U n s a t u r a te d F a t t y Acids
O leic
L inoleic
U nsaponifiable

45.5
40.9
1.7

M a z o l a c o n t a i n s a maxi mum o f . 0 5 % F r e e F a t t y A c i d s b u t
t h i s w i l l i n c r e a s e s l i g h t l y upon e x p o s u re to a i r and
m oisture.
T h e r e f o r e , Mazola i s packed in an a i r - t i g h t c o n ­
t a i n e r an d so f i l l e d t h a t t h e r e i s o n l y a v e r y s m a l l p o r ­
t i o n o f the o i l exposed to a i r and a p r a c t i c a l absence of
mo i s t u r e .
We h a v e no i n f o r m a t i o n o n t h e n a t u r e o f t h e p i g m e n t s i n r e ­
f in e d corn o i l .
There a r e in d ic a tio n s i n the l i t e r a t u r e
t h a t r e f i n e d c o r n o i l may c o n t a i n a t r a c e o f s t e r o l s h o w e v e r ,
i t s p r e s e n c e i n Mazola has n o t been w h o lly c o n firm e d ; i f
p r e s e n t i t probably is contained in the U n sap o n ifiab le .
We h a v e no i n f o r m a t i o n o n h y d r o c a r b o n c o n t e n t , b u t we
b eliev e th ese are absent.
I f y o u d e s i r e f u r t h e r i n f o r m a t i o n o n M a z o l a we w i l l
g l a d to g i v e i t to you i f such is a v a i l a b l e .

be

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M aster's

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F r a z i e r , W. C . , a n d W h i t t i e r , E . 0 . 1 9 3 1 a .
S t u d i e s on t h e
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t i a l of m ilk.
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The r e l a t i o n s h i p s o f a l i p o l y t i c
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I I I . The c h a n g e s i n t h e r a t e o f r e s p i r a t i o n
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-79-

------------------- 1 9 3 5 *
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C entralbl.

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’’R e s t i n g "
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M.S.B.
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The o x i d a ­
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The r a n c i d i t y
Biochem. J . ,

of coconut o i l
2 2 :80-95.

p r o d u c e d by

T h a l e r , H . , a n d G e i s t , G-.
1939.
The c h e m i s t r y o f k e t o n e
rancidity.
I . D e c o m p o s i t i o n o f f a t t y a c i d s by P e n i c illiu m glaucum .
I I . F o rm a tio n o f m ethyl k e t o n e s from
b e t a - h y d r o x y g a t t y a c i d s by P e n i c i l l i u m g l a u c u m .
B i o c h e m . Z . , 3 0 2 : 1 2 1 - 1 3 6 , 3 6 9 - 3 8 3 ; Chem. Abs t . , 3 4 :
5111, 1940.
T h u n b e r g , T.
1918.
Zur K e n n tn is d e r Einw irkung t i e r i s c h e r
Gewebe a u f M e t h y l e n b l a u .
Skand. Arch. f . P h y s io lo g ie ,
35:163-195*
T r a c e y , P . H . , R a m s e y , R. J . , a n d Rue h e , H. A.
1933.
C e r t a i n b i o l o g i c a l f a c t o r s r e l a t e d to t a l l o w i n e s s
m ilk and cream .
I I I . Agr. Exp. S t a . B ui. 389.

in

T s i r s c h , A.
1925.
A u to x id a tio n in f a t s , r e s i n s , t e r p i n e s ,
and ta n n in s .
Chem.
U m s c h a u , 3 2 _ : 2 9 - 3 1 ; Chem. A b s t . ,
JL9 : 1 5 5 7 , 1 9 2 5 .
T s i r s c h , A . , a n d B a r b e n , A.
1924.
R ancidity of f a t s .
A p o . Z t g . , 6 2 : 2 8 1 - 2 8 5 ; Chem. A b s t . , 1 8 : 2 9 7 0 , 1 9 2 4 .
V a n d e r W a l l e , N.
1927.
b a k t e r i e l l e Lipasen.
70:369-373.

U e b e r s y n t h e t i s c h e Wirkung
C en tralb l. f. B akt., II A bt.,

W e l l s , H . G. , a n d C o r p e r , H. J .
1912.
The l i p a s e o f
B a c illu s tu b erc u lo s is and o th e r b a c t e r i a .
J. Inf.
B i s . , 11:388-396.
W a s h b u r n , R. M . , a n d D a h l b e r g , A. C.
1917.
The i n f l u e n c e
o f s a l t on t h e changes t a k i n g p la c e in s t o r a g e b u t t e r .
J . D a i r y S c i . , 1_:114-126.

-8 2 -

W e b b , R. E . , a n d H i l e m a n , J . L*
1937.
The r e l a t i o n o f
o x i d a t i o n - r e d u c t i o n p o t e n t i a l of m ilk to o xidized
flavor.
J . D airy S c i . , 20:47.
W e r k m a n , C. H . , a n d Wood, H. G.
1942.
of b acteria.
B o t . R e v . , *3:1-68.
W h e e l e r , D. H.
1932.
Peroxide
autoxidative d e te rio ra tio n .
W h i t e , A. H .
butter.

On t h e

metabolism

f o r m a tio n as a measure of
O i l and Soap, 9^:89-97.

1940.
A b a c te ria l discoloration of print
S c i. A g r ., 20:658-645.

W h i t e , W. H .
1941.
Methods f o r i n v e s t i g a t i o n o f r a n c i d ­
i t y , t h e i r i n t e r r e l a t i o n , and a p p l i c a t i o n to b a c o n
fat.
C a n . J . o f R e s . , S e c . D, 1 9 : 2 7 8 - 2 9 3 .