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Metabolism of the lactic acid bacteria, particularly Bacillus dextrolacticus

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R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
MEfA30LISM OP XHs* M C f 10 . ACID BACTERIA,/
WMMlOWUmS
140I M S .BMfROLAOfXQ98
by
A r t e l Andrew A n d ersen
A fJ a o a is Su bn lfeted t o tb e S m i m t e P a e u lt y
f o r tli© B®g»© o f
DQo rtxi Of Pi.ILC10P.ET
M ajor S u b j e c t P b y e l o l o g l e e l B a c t e r io lo g y
Approved t
lo w s Stm t# C o lle g e
1041
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
UMI Number: DP12547
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UMI Microform DP12547
Copyright 2005 by ProQuest Information and Learning Company.
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R e p r o d u c e d with p e r m i s s io n of t h e co p y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
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1®th u d s U sed I n Ittferltiom & l S tu d io * * * * * *
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Hath©da {feed 1» F#waatati©ti £ipe*f»eBt*
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SUPBRIMIMfAL *, * *«,.** , *»* * . * , * * * ♦
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I m ts r itiu iia l S tea d ies* » » * » * * * « •* •* •’» *»» *•<**
24
l a c t i c A c id B ftet» v ia # « *
24
Ejqaerlmente and voeultft*******»•****.*
24
Mamas!©®* «•»***••••»• *• »* • * **. . . ***
36
Stnmary and ©©nclmsioita..** *****♦*•••*♦ ' 37
IfaaioftanM&tatlve -Laetie Acid Bacteria*.***
30
Inscription and c l a s s i f i c a t i o n of
^
Baelllma d e x t r o l a c t i m s ♦*•*..**«.**♦
30
lutpsrljiajits and in s u lts .• »**.«•*•*.»•«
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M j^saion**,
73
Snastiarj- and e s a d l t t s i o n # .*.♦.*** . 73
Fesnentetlre Metabolism of Bacillus dexter©■
*■
lactlca# in Various ’Media ••••••*****». *******
'■76
Exp®rim®nfe« and Results* «•»«**.**».**.** ***
76
Suasaary .and Conclusions*. ..*,•**.**•*.***.
106
LlT&AWm
CXIBD**..
ACE»C»E»«lifS*..** . . .
*
**..
.......
110
114
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
m 5
m*
IStROMJCflOS
Studies in b a c t e r i a l nutrition have bstaisa on mm sig­
nificance l a r g e l y -b e c a u se o f recent advances la b io c h a m ie a l
te c h n iq u e *
F orm erly th e b a c t e r i o l o g i s t ; n e v e r a s s o c i a t e d
n u t r i t i v e r e q u ir e m e n ts with. p h y s i o l o g i c a l mechanisms# be
mm Interested only la obtaining
g r o w th } sow he desire® to
know th e r e l a t i o n s h i p o f th e n u t r i t i v e constituents t o
physiological m eohaaiew s*
fitaatn % (th ia m in ) functions
in t he breakdown o f pyruvic a c id and every cell in which tills
m echanism Is fou n d m ust h ave th ia m in present*
Many of the
s u b s t a n c e s w h ich are n e c e s s a r y for cell activity a r e synthe­
sised by th e c e l l from simpler compounds but o f t e n they must
be supplied*
Hence we M v # on the mm ex trem e a u t o t r o p h lc
organism®# w h ich 'have the ability t o b u i l d all the required
o r g a n ic compound® from I n o r g a n ic m a t e r ia l* and on t h e o t h e r
extreme th e p a r a s i t i c organism s- which require a living h o s t
t o su p p ly them w ith r e q u ir e d com p lex s u b s ta n c e s *
fb n s we
see that the food requirements o f bacteria way differ widely
from om specie® t o a n o th e r *
Furbb& m ore* the r e q u ir e m e n t s ' o f
th e sew s culture lav© been shown to v a r y markedly from tim e t©
tim e*
In spite o f this#, th e general r e q u ir e m e n ts for various
c u l t u r e s do appear t o be quit® c o n s t a n t under fixed c o n d it io n s *
4 su b s ta n c e may be absolutely essential for a p a r t i c u ­
l a r culture or it may to# only b e n e f i c i a l *
In the .latter
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
♦
4
*»
c a s e th e c u lt u r e is stimulated to greater activity win® the
substance l a .added t o a aediuii la which the culture is already
growing*
sometimes th e requirement# defend on t h e objective
o f the investigator and may to#' classified as {1} requirements
for mere c o n tin u o u s growth* .(ft) requiremmts. for abundant
growth, and (31 requirements for large scale f e r m e n t a t io n .
For example, a certain lactic acid culture -may barely sur­
vive on a certain medium containing amino acids* less than
I per cent sugar being utilised, but by replacing the amino
acids w ith m a lt sprouts i t is possible t o carry out a large
scale fermentation of 10 to 15 per cent sugar*
fh® present investigation is concerned with th e n u t r i ­
t i o n a l requirements of the lactic acid bacteria, 'f h e re-'
q u ir e m e n ts for both growth and large scale lactic acid f e r ­
m e n ta tio n s (12 to 15 per cent sugar) were investigated*
fhe culture# of lactic acid bacteria employed include
four heterof© naenfc& tiv© and three hom ofexm entm tiwe species.
Among th e latter is a member of the genus Bacillus which was
isolated during the course of t h i s work.
Although it is a
sp o r e -fo r m e r * by definition it i s a homo f e r m e n t a tiv e lactic
acid form*
Da© to i t s s im p le growth requirement# and the
v ig o r o u s d e x t r o l a c t i e acid production, the organism was
carefully studied and d e s c r ib e d .
She culture was studied in
various media w it h the objective of determining the optimum
conditions for lactic acid fo r m a tio n .
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
«<* 3
**
KlVLwtf QW LITt-HATUfUi
L a e t ie a e ld *
L e e tie . a c id #
a c o l o r l e s s * od or*
l e s s , if r o p y compound was f i r s t 61aeovere& by & eh eele (17801
srd© i s o l a t e d I t * s a d i d e n t i f i e d i t s s th e a c i d of so u r 1 m ilk *
sa y ^ Iu a sa e and F s lo u s e (1833) is o la te d lias ©©aipoimd
from f e r m e n te d beet j u i e e s s d j&ade a s a n a l y t i c a l stu d y ©f
i t b u t w ere m ista k e n i n th e fo rm u la th e y a s s ig n e d . t o i t *
Many ©fciier investigators ale© erred in tis© form u la th e y
assigned t o It*- t a r t s -and Friedel tW Sl) were t be first t o
claim I t t o -be a th r e e carb on 09qr*aeid*
Many i n v e s t i g a t o r s have c o n t r i b u t e d . t o ©or k n ow led ge
o f til© p r o p e r t i e s o f l a c t i c , a e ld # w h ich may bo b r i e f l y
a®- f o l l o o a t
L a e t lc a c id # a-% dr© 3sy p r o p io a i© a c id *
(C % #ca©l*e©©S) i s a c o l o r l e s s * . o d o r l e s s # sy ru p y ecwpouaA
© e e u r r ia g n a t u r a l l y in fe r m e n t a t io n s and ts n s e le tissue.*
It
is s o l u b l e l a water* a l c o b o l and e t h e r l a a l l proportions*
I t . ©©cars la three form s*
optically inactive acid*
de.3g.fcr©** and l e r o r e t a t o r y and. the
Ml
th r e e fo rm s ©©cur Is fe r m e n ts*
tlOBS* but. o n ly t b s dextrin** f o r a is fo u n d is m u scle •m e ta b o lis e *
If tii® lev©** form- Is introduced i n t o the a n im a l b © % it la
rapidly e x c r e t e d unchanged (M eyerhof aad Lehman* 1 9 2 6 | C o rl
and C ori* 1 9 2 9 )«
The r o t a t i o n © f th e s a l t # and e s t e r s o f
l a c t l e acid Is o p p o s it e to t h a t of t h e acid*
Lactic acid
can not be distilled a t o r d in a r y p r e s s u r e but. is distilled
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
mt
Q
tm
at v a r y lo w p r e s s u r e w it h th e a i d o f s u p e r -h e a te d steam *
f t o ls m ethod h a e not
mrna
s u c c e s s f u l however* on a com m ercial
a e a le ( F r e e e o t and t a t » 1940)*'
Wmm
h e a te d at ordinary
p r e s s u r e two m o le c u le s o f l a c t i c acid l o o s e one m o le c u le of
w a te r a c t a r e c o n v e r te d t o th e a n h y d r id e *
F u r th e r b e a t in g
s p l i t s o u t a n o th e r M o le c u le of w a te r and th e l a c t l d e l e
formed*.
toaefcle a d d form ed toy b a c t e r i a *
B londeau (1 8 4 7 ) r e c o g *
allied lactic a e l d a s a p r o d u e t of f o m e n t a t i o n *
P a e te u r
.(IBS'?) m s the first t o prove that th e 'lactic acid 'was
f o m e d toy b a c t e r ia *
it was act u n t i l 1878 t h a t a t r u e
lactic -acid erg a n ia m was Isolated! M a t e r (1 8 7 8 ) o b t a in e d
Sferepfcooooeaa facts# i n pu r# c u lt u r e *
S in c e th e n many
culture#' of l a c t i c acid b a c t e r i a have 'been isolated c h i e f l y
from dairy p r o d u ct* * grains and from other plant m a t e r ia l*
-.fJae true l a c t i c acid bacteria*
An e x c e l l e n t critical
review of the l i t e r a t u r e dealing with t h e classification o f
lactic
m lA
toa«t©i*i« is gives toy K e le e n (1 8 8 6 )#
O rlA -J e n scn (Iffi)* S«slson (1956) and others us# the
'te m
* t m e lactic a c i d b a c t e r i a ” and define it a a in c lu d in g
only ncu v*ap ere~fom in g# n o a - m o t ile organiem c*
these a u th o r s
mm
A p p a r e n tly
not aware that there were other types of
organisms which produce equally ae much lactic acid as those
they included* -Spore "formation* motility or
characteristic can not exclude
m
may
other
org a n lcm from the group
if its principal end-product o f carbohydrate d i e e i m i l a t i c a
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
i s lactic s e l d with no m o m than traces of other products*
A apeve«»fonelBg o r g « t t i n f isolated by tb s author* and
d * se? ito ed as Baeillma dw cter^Laotlw m (A n d ersen sad Vertaaea*
1 9 4 0 } has b een ia e lu d e d in t h i s investigation#-
W ertonn and
A n d ersen (1 9 3 8 ) reported l&etie & © M y i e l d of 9 8 per s e n t by
this o r g e n lM u •
■ Several other investigators have isolated
spore-foMiag organisms wMoto jprodnee larg© q u a n t i t i e s of
lactic sold*
Segisat@r (1 9 1 8 ) isolated a spore-foraaer* froa
c o a g u la t e d e v a p o r a te d silk* whleh irn d e s c r ib e d and a&aed
B a e i l l u s coanulmns-*
this e r g a a tsm is
q u a n t i t i e s e f lactic acid*
kmmei
t o p rod n ee i&rg®
JEXmeter (1 9 3 0 ) isolated and
described a a p o r e H fe m ln g organism which gave a l a e t l e aeid
y i e l d of 9 1 per e e n t of t h s o r e t i e a l *
Ior©«its»*la#sowa and
N ow otelnow (1 9 3 2 ) isolated a spor®*f©rating o rg a n ism whioh.
yielded l a e t l e a e l d nearly e q u iv a le n t to th e sugar fomented*
they mused t h e i r euifcmr© 3 & c t e b a s llla a aper o g sta es* wMeii is
not i n a e e e r d a n e e with th e system of n o n s n e la tu r e a c c o r d in g
to Bergey ©t al«.< (1 9 3 9 K
fa n * Peterson and Jo-imson (1940)
Isolated two- c u l t u r e * of -spor#»for»«rs which yielded laetle
a e M upwards o f 9 3 per e o n t o f th e t h e o r e t i c a l *
to d o serl& o or identify their cultures*
f b e y failed
T i m m and no doubt
many other e p e r e * fo r a iB g organisms fora s u f f i c i e n t q u a n t i t i e s
of l a e t l e sold to
pl& ee them rightfully in tb s hoaof#ra©nta»
tire group of l a e t l e a e l d bacteria* and in :spit© of their
spore-^fonsiag e h s v a e t e r l s t i e they m m
tr u e lactic acid
organ! tan* belonging to th e g e m s B a c ll l n e *
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
s* m
n u t r i t i o n o f l a c t i c a c id bacteria
Excellent reviews
of the l i t e r a t u r e dealing o i t h bacterial nutrition are given
%
B urrow (3.996* laight (1 9 9 6 )* K o ssr and Saunders (1 9 3 8 )'
and Miller (1 9 4 0 )*
She present status o f t&o preble* with
respect to th» lactic o d d bacteria stay toe briefly stated as
fo llo w s i
©rls-Asnssn* Otto# and Snog-Xiaer (1956) s t a t s
that riboflavin and ©a® or more ©toiler activators* «**• which,
they e o a e lu d s d was pantothenic acid, art necessary for aortas!
development o f certain laetle aeld bacteria*
fhelr finding
c o n c e r n in g r i b o f l a v i n was e o n f lr a s d by Wood# Andersen and
mr'tama.
(1 9 3 ? ) who excluded pantothenic aeld as an essential
factor for th e ta e tsp o fsrsH m tstlw o laetle aeld bacteria*
Lactobacillus asiinlbQpocia#* L*
lyeopcralel* and Streptococcus
p a r a c it r o r o r u s by growing the*. in an amino acid medium la which
all constituents were knowo. except
factor*
&n
ether-soluble yeast
O r la -J e n s e n , O tte and Saog-JS.ja.er (1 9 3 8 ) state t h a t
trjptopissa© I s not essential for laetle s o l d bacteria*
food#
.Andersen s a d iCetsws flilf) state that tryptophane is neces­
s a r y for good growth of L a c t o b a c il l u s p e n t o a c e t ic u a (L 4)
(L
4 is L* Motiaeri)*
Snell* latem mwl fetorsos (1937)
found tryptopasao necessary for L a c t o b a c il l u s d e l b r u e e k l l .
Andersen* food and I s r ta s s a (1938) o b ta in e d toooa growth o f
Lac tetoaeiliaa asaajtopooss*. L* tmeSaaaarl sad L* iytoyersiei
la a ssdlws consisting of 19 purified swine acids thiamin*
riboflavin* ether extract of yeast extract* glucose* sad
inorganic salts*
tryptophan# was essential for L* baoh&eri
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
but BOt ■e s s e n t i a l for x** amnaf t:
Qg©®a.g o r L* l y c o p e r s f c i *
C y a ti m sa d t h n m l B *
mmm
e a s o n t i a l - f o r a l l tb r e e e u lt u r o a
u b l l o s e r in # and.- a o tb lo a l& e w ere am risedly s tia a l& to F y I f mat
o a o o n t la l*
. l i l l e r {1 9 8 2 ) fo u o d v lto a & a B g ' U d o m l a ) was
r e q u ir e d toy o o r t a l a o f tb e l a c t i c e a l d b a c t e r ia *
S n e ll*
S tr o n g sa d f e f e m o n (1 9 3 9 ) a b a te t lm t p a a t o t b a a ie and a l c o *
t it k le a c i d s a r e n e c e s s a r y f o r c e r t a i n o f ' fctoe i s © t i e a c i d
b a c t e r ia *
§©mfa.iiX®» w M eii
»mmm
to e x i s t l a
%1m l i t e r a t u r e
©on-
© e m ln g tto© n u t r i t i o n o f l a c t i c a c i d b a c t e r i a l o due t o a
»i»*to©F o f f c e t e r a t
X* The c u l t u r e s u s e d toy d i f f e r e n t l a e e a t i g a t e r s
tmm
not. been i d e n t i c a l ! ' tn e r e is c o n s id e r a b le v a r i a t i o n in growth,
r e q u lr e a e n t s within th e group.*
2* fh o n u t r i t i o n a l r e q u ir e n e n ta o f a © aitur© a r e n o t
o o a a ta a t#
A d a p ta tio n * v a r i a t i o n and v i t a l i t y a l l f u n c t i o n
to a l t e r th e >j m t r i t i e n a l r e q a lr e a e ii t s o f a c u ltu r e *
3* The basal n ed lo st ©f different i n v e s t i g a t o r s
been tine sa n e* . E>mm
im&
net
s u b s ta n c e s nay f u n c t i o n u n d e r one co n ­
d i t i o n and n o t a n o th e r *
Am will
be slxon®. later sea® growth,
p r o a o tlB g aatost*wa®es ©an toe r e p la c e d * wholly of i n >p o r t* toy
others# -A p a r t i c u l a r s u b s ta n c e may to®' fo u n d to exert its
i n f ] » e a e e .only in til® presence* of a b s e n c e of. c e r t a i n other
s u b s ta n c e s *
T h e r e fo r e a n e g a t iv e result may to® ©bt& lned for
an e s s e n t i a l m b a ta n c o *
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
m
30
m
4* Many of tkm materials (extracts# h a fd r e ly sa te s* ads o r h a te s # eexiee& trebe** ©to*} u s e d l a m ed ia are n o t pure sub­
stances of known e o a p o s i t i e a a a d cun mot he identically
duplicated*
5* Several methods of d e b e r ttittln g growth have been nsedi
turbidity (visual inspection or by as# of a tmrMdiaeter) *
direct cowit of ceils,# acid p ro d u ctio n # . m aes o r volum e o f
s e l l s # nitrogen c o n t e n t of culls* ssoag others*
6
* It f t dom etin® s d i f f i c u l t to interpret results as to
■whether a s u b s ta n c e i s r e q u ir e d (absolutely mocom&vy ©ran
for poor growth} or is Just beneficial (not required for
poor growth} *
Itttrition and tim lactic a c i d fermentation*
Avery
{CMunPst* 1 9 9 0 ) was the first to s u e e e a s f u l l y p ro d u ce l a c t i c
acid oa a ©oa®erelal basis (ISSl}*
At the time an a tte m p t
was o a d e .,to repine® tartrate o f baking powder with l a c t a t e ,
i n 1 9 9 4 the leather and t e x t i l e industries began using lactic
acid* ..Since then many o t h e r uses hair© been found for th e
product and to satisfy th e demands an Industry has been built*
Probably th e most. Im p ortan t p rob lem I n lactic acid pro**'
a u c t io n is t h a t o f nutrition*
Malt s p r o u t s hay# been th e most
widely, used source o f nutrient with wtmj and eklm mill becom­
in g .more im p o rta n t*
.Tatum and P e te r s o n (1 9 3 5 ) d e s c r ib e d
a process for d e x t r o l a e t l e acid production u s i n g m a lt sprouts*
Stiles and f r o e e e < 1998) reported adding malt sprouts* steep
water* and thin grain residue to blackstrap m o la s s e s *
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
■** 12. ■»
W h it t ie r s a d le g e r a (1 9 3 1 ) r e p o r te d .
& ©entiim em »
f e r m e n ta tio n
p r o s e s # m sla g wliey t o s u p p ly b o th n u t r i e n t and fe r m e n ta b le
eartoeb pd rate*
O liv a (1 9 0 6 ) t a i B u rton (1 9 6 7 ) « l« e - h av#
d eserlto ed . l a e t i © a e l d p r o c e s s e s u s in g whey*
W erknsn:a&d
Andersen. (1 9 9 8 ) r e p o r t e d u s in g soyb ean m ea l and a sp o r e *
fo rm in g o r g a n ism t o p r o d u se d e a E tr o la e tie s o ld * '
Fan# f s t e r s o u *
and Joh n son (1 9 4 0 ) r e p o r te d u s in g sp ereH T ornlng ©rg&alattaa i n
s a l t s p r o u ts H u e la s s e s *aedi»at*
A. hlg& g r a d e l a e t l e m aid i s n o t e a s i l y and e h s n p ly
p u r i f i e d from e o n p le x f e r m e n t a tio n liq u o r *
T h e r e fo r e g r o a t
e f f o r t M s b een made t o s i m p l i f y th e w i i »
and tin ts k eep
I m p a r i t i e s a t a m iniuuau
The i d e a l nediuat w ould bo a s y n t h e t ic
-osio e o n s i s t t a g o f su g a r* e a le lu m c a r b o n a te and w a l l amount#
of in o r g a n ic . s a l t s *
t h e o b s t a c l e t o “to® overcom e l i e s I n
f i n d i n g a e u lt u r o u h ie h w i l l p rod u ce a l a r g e y i e l d o f l a o t l a
a e i d i n a s im p le a e d iu a *
The b a a t o r i a l e n ltm r e s w h ich nave
M e n u s e d l a th e p a s t f o r l a c t i c a e l d p r o d u c tio n a r e s p e e l e #
o f l a e t o b a c l l l m s and a tr e g fc o e o e e u s and t h e s e a r e p a r t i c u l a r l y
fa s tid io u s *
l a n e l (1 9 9 4 ) ii la rd .
at
a l * ( 1 S 3 8 ) , .and waksman « t a l #
(1 9 9 7 ) lav© r e p o r t e d lactic a e l d p r o d u c tio n u s in g a sim p le
s y n t h e t i c medium and s p e c i e s o f E iiiz o g u s t o b r in g a b o u t th e
fe r m e n ta tio n *
The p r o s e s s l a s . c e r t a i n d is a d v a n ta g e s # feow evor,
e a o p a red t o th e b a e t e r i a l fe r m e n ta tio n *
j$Q p e r s e n t l o v e r * . fiie
p-mmm
The y i e l d I s 15 t o
©an not be c a r r ie d o u t i n
l a r g e 10*000 g a l l o n ta n k s a s ' t h e b a e t e r i a l f e m e a t a t l e n i s #
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
'feat tiui® fa r only sJsolXov pans or rotatin g mlMmlmm dnaam
l»v® boon us$&»
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
— 1 3 «*■
«A37EHIAI» ASB K R B 0 D &
C u ltu r e s
The oulturaa of Bacteria use# to th is in v estig a tio n
lnoludas
fiotarefafeaantatlve la c t ic acid B acterial
to e te -
Taaelilua MMmltapowMi (I* 2)* J** baahmgi (L 4)» B* ly eo p w a lo l
(L 5} and Streptococcus paracltrovorua {S 9)*
t l v a ' lactic act# Bacterial
IiOfflof#r»@«t&-
toeteBaclllug d a lb r a a o lt ii <L i)
ami B* fcelveticus (1 h) afotainod from ta a v ie a n Ilals# Prod­
ucts Company, sad Bacillus do&trolac-ticma (A 6).
■
.Mvthoda Uaed in Wutrtttonal Studies
I n the nutritional atudiva ife« cultures war© Inoculated
Into a Banal medium e e n v l v t l a g o f g la e o o e 1*0 per cent,
|i a 4 )gS.04 0*5 per eont* ©©dims aoot&t© 0*0 -per. cent* and
Speakman *s 11921) inorganic v o lts (KBgPOg Q«OS& per eont*
% 8 P % 0*025 per m & t $ MgSa^TBgO 0*01 per sent* S&CX 0*005
per ©eat, FaSO^TSgO 0*005 par oont "and InSO^’iHgO §*006
per aa&t) to vblefe alaa woro aid## on* or nog* 'of the fo l*
Xovingi
v at o r, aloobol* o r othov ex tra cts of n a lt sprouts,
water extract o f glu ten , "SovdMrg proeipffcafc®* or * filt» o to *
of alcetiol ex tra ct of malt aprov&a* rffeoflavin., th lou la
or the othor ex tract o f a c id ifie d yeast extract
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
14 •
( D l f e o ) * h y d r o ly s e d e a e e ia * hydrolysed gelatin fund la y d r ely a ed
album in* s is i» e a jlgs ( g ly c in e # a la n in e * v a lin e # le u c in e # i s o la u e in e # th r e o n in e # s e r i n e # c y s t i n e * m e tM e a in e # a s p a r t i c
a c id # g la t a m le a e ld # tey p to p b a a e* p r a lin e # H y d r o x y p r o lin e ,
h i s t i d i n e * a r g in in e # l y s i n e # t y r o s i n e and p h e n y la la n in e ) #
i n d o le B a c e t i c # a s c o r b ic # tia to g ly o o li© # p a n t© th e s is *
n l e o t l n l e # and p ia e X ie w i g s , b i e t i n * i* * la n £ s a # u r a e i i *
inositol, adarsiia (v it a m in
and y e a a t e x t r a c t *
lU e t e m b a s a l medium may b e d e f in e d a s
mm
in c a p a b le
of s u p p o r tin g norm al growth# and to etk leb a d d l t tonal e a b e ta n e e a
are ad d ed for th e p u rp o se of d e te r m in in g t h e i r ambrltiv®
effect.* S e v e r a l e a c h media; n e e d in this l & r e e t i g e t l e a are
l i s t e d a s f o llo w s *
B an al I s
aimc m s
1 *0
.Aswoiilias s u l f a t e
0 * 5 p e r © eat
Sodium a c e t a t e
0
<* # p e r s e n t
per cent
3peakmanf e in o r g a n ic salts
Basal XI*
d lu e o a e
1 *0
s u lfa te
par cent
0*3 per cent
■Sodium a c e t a t e
0< * # p e r c e n t
Spealtaasa1® in o r g a n ie salt#
H y d r o ly sed c a s e i n
i0-*X§
per seat
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
*
* ill
B a s a l 111 i.
S lu ces®
1*0 p e r © eat
Ammonium s a i f a t s
■ ■0*3 p e r .c e n t
Sodium a s s i s t #
0*0
p e r ©@»i
Ifestaaaa*® i n c i s a l © m a lt*
fiilaai« per 10 ml* medium 0*l>mtcrouram
. Jttaer e x t r a c t o f 0 »S gram
©f a c i d i f i e d y e a s t e x ­
t r a c t p e r 10 m l. medium
$rypt©p&aam
0*01
p e r .©eat
B asal i ¥ t
o la e e e e
1*0 p a r © eat
ftmoolixm sulfate
0-*3 per seat
Sodium a c o t a t o
0*0 per c a n t
S p # a fe » a * s l a o r g a a l e s a l t s
M r a l a per
10
ml* m adia*
R ib o f la v in p or> 10 m l*
mo&iam
0*1
m lerogram
§*0 m lo v o g m a a
&th®r e x t r a c t o f 0*3 gram
o f a c i d i f i e d y e a s t e x tr a c t
per 10 ml* medium
S ftttl f i
aitt©@se
1*0 per ©eat
jjaaioaiM m u lfa to
0*0 p e r s e n t
Bedlam a c e t a t e
0
*§ p e r ©eat
Sp eak aaa** in o r g a n ic s a l t s
Thiam in per 10 si* aadium 0 * 1 mievogrmm
B l b o f l a v i a p e r 10 ml*
medium
5*0
a lo r e g r s a s
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
■«*
i s .«*■
Sther e x t r a c t of 0*3 gram
© f a c i d i f i e d y e a s t ex­
tract per 10 m l* jacdluii
pm
Hydrolysed M N l a
0*1$
tryptophane
0*01 per cent
fatter extract of malb JggJggfef*
e©nt
100 g v u a s a l t sprouts
were places la 900 atl* distilled water «ad kept la -refrig­
erator at 10° C* for St hours-*
fli# salt sprouts
©mb and w ashed with a- little a d d it io n a l, water*
mm
filtered
fh© extract
m s adjusted to pH #.*8 with aodlas Hydroxide- and Orem fehpscl
b lu e Indicator# and wad#
up
to a $ per cent solution*.
Alcohol ex tra ct o f .s a l t sproata*
id© grata# o f s a l t
sprouts »i»® extracted' witc ethyl- n ltch o! ttS per ©eatj u n til
the alcohol m s co a p letely eolorloaa*
Sosldnal alcohol m s
vo&ovod by ©mporafeiea and replaced with s t s »
adjusted to
pH 6*8# and nude up to. a 60 per cent s o l u t i o n (#00 »!*}*
6 per cent so lu tio n m
a lso ut&de up*
l-thyl ether extract of malt sprouts*
sprouts
mm
placed in
A
WO
# § grans s a l t
ml* ethyl -ether* and l # f t stoppered
for St hours at room teaper-atar®-*
the
sprouts
fey filtration and. washed with 100 s l « ether*
m m mmom4.
fh# ether m s
replaced toy water*.the- material adjusted to pH 6*8* -and aade
up to 5 per cent -solution*
the water extract of ^lutem* 800- grams o-f gluten'were
placed, in 800- m l* distilled water and held at 60° C-* for §8
hours- -and then held in a refrigerator several -days* the
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
17 material was filtered and sad© op t o S p e r o e a t s o l u t i o n
after b e in g adjusted, t o pH 6*8*
Ether extract o f tie a l c o h o l extract, of malt s p r o u t s *
■itpwiii iiMmunnmiinniiniiii
WniniHiwmiij .inh: nwmiignMii U i
W*wi»uu'
* » ■ »■»»w<:.. i' b* w imh « ii' b
h
t mjw
w
i
m
auBKwu.j
.WttrtW)* i '"u«w
iGO sal* of the show s a l c o h o l extract were awl© acid to eong©
red. aad extracted with ether for 30 hours*. the ether waa
rtsrawsd fro® the extracted. Material .sad replaced Of water*,
the alcohol, extract* the ether soluble fractioa ©f th e alcohol
extract and the ether issolubl© f r s e t i o n of the alcohol ex­
tract were •*«& m i # tip to §5 per c e n t solution with pH 6*8
(1 ail* representing the material la 0*2$ gras of salt sprouts).
^ e u b e r a p r e c i p i t a t e " and “f i lt r a t e ® o f the a l c o h o l -extract
of s a l t sprouta*. 75 ml* of t h e alcohol extract ©f malt
s p r o u t# i s f s f r a c t i o n a t e d a c c o r d in g to lemtoerg and E@rto
(1918) with sodiwti oarbenats
cipitate
worn
mA
aereurlc acetate*' The pre­
suspended l a 40© a l * water and treated with
hydrogen sulfide for § h ou rs*
fise M ercu ric sulfide was r e -
sowed toy ©eatrifmgatioa, sad t b s supernatant liquid a s mad®
up to a 10' per cent solution at pH 6,8 (10 *1* r e p r e s e n t e d
1 gran iaa.lt sprouts)- sad designated "Ssuberg precipitate®*
She filtrate was acidified with acetic acid and. treated
with hydrogen sulfide and the n e n m r l e sulfide r s a o r s d toy
e s n t r t f t t g a t lo n *
-fh© su p e r n a ta n t liquid m ® made t o a 10
p e r ' e e n t s o l u t i o n at pi! 8*8 aad designated " lo u b e r g filtrate®,
the ether extract o f acidified y e a s t extract (life©) was
prepared as described toy Wood* fatea and P a te r s o n (1937),
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
. 1 8 «*
t h e h y d r o ly a e d p r o te i& e w ere p r e p a r e d toy a d d in g 9 6
ml*
© f distilled water. and 8 4 *dU c o n c e n tr a te d s u lfu r t© a c i d t©
I S g r a s s o f Slug p r o te in . and e a te e la v lm g th e m ix tu r e a t 15:.
pound*. f o r ti& honra*
fit© h y d r o ly a a te * were n e u t r a l i s e d w ith
t o t m a tu rated ■toarlmn h y d r o x id e s o l u t i o n J u s t t© t h e p o i n t
where a© «©r® p r e e i p f t a t e foamed*
w&m add ed
.4 te a a p o o n fh l o f c h a r c o a l.
and t h e n o t o r i a l w as f i l t e r e d and washed w it h *
l i t t l e w ater*
t o t a l o o l i d a I s th e f i l t r a t e were d e te s s d n a d
by ewajMarating. 10 m l# to d r y n e s s and in©
was made up
So 1*5 p e r s e n t s o l u t i o n o f h y d r o ly s e d p r o te in s * . a f t e r
a d j u s t in g t l m pS t o S*8 w it h sodium h y d r o x id e ©r h y d r o c h lo r ic
a e l d a s r e q u ir e d *
flie aatlae a o ld a were efetai& ed A w
th e U n iv e r s i t y ©f
I l l i n o i s and Kastman Kodak Company*
T h ese a a p e r in a a ta n a r e e a r r la d o a t l a 8 o r 10 ad* o f
medium la tu b e s*
«ii»» & si...*. a e r o u sed * r a a u lt a laare. been
e o a v e r te d t o t h e 1 0 . ml* toasls*.
e o n t r a l was subtracted.
$ 1 O r a tio n o f a a io o e u la t a d
Til# n o d io n mde s t e r i l i s e d at 18
pounds steam pressure f o r 1 5 m in u te s l a all aaaea m ales©
© therw is© in d ic a t e d *
Z nooula were- p r e p a r e d e i t h e r
by
c e n t r i f u g i n g th e © e l l s from th e medium i n w h ich th e y w ere
gr©wa -and r d i n g
them i n a s e q u a l volum e o f w ater* o r
u s i n g t h e e e l l e d i r e c t l y fro® th e w d iiH i i n w hioh th e y were
grown i f i t was' a s y n t h e t i c medium and n o t c o n t a in in g th e
t e a t su tostan ee*
©as d rop o f th e e e l l s u s p e n s io n n e e u s e d f o r
e a c h tu b e* u n l e s s o t h e r w is e in d ic a t e d *
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
«* 1 0
*»
laetle acid c u lt u r e s * 1.* » o n i t o k e n s *
k*
M S S S M l*
k*
-X y e o p T s l e l *
-mm
in c u b a te d a t 30° € , « a d
H» parse Uroyorua at roots temperature*
(& d) and L , h e lr e fe lb a a
Lactobacillus
b ) « ? e in c u b a t e d at
3? o r 40° C» and B* d s a t r o l a c t i c u a at if u n l e s s otherwise
indicated*
A f t e r 5 t o 6 days. incubation* growth m s measured
by t i t r a t i n g t h e
m i4 - io r m d
w it h s ta o d a r d m d tu a 'h y d r o x id e
using brca th ym ol tola# i n d ic a t o r *
'In some e x p e r im e a ts* where
a d d s& o d n etten ms-low* t u r b i d i t y m i u s e d as a m easure of
growth*
Wbon e x p e r im e n t* were carried through, more than one
transfer the transfer was sad© on the t& iv d ’o r -f o u r t h day of
in o u b a tio n .*
Methods Psed In Fermentation 3h£periB©nt#
I M s pdas© o f th e i n v e s t i g a t i o n w as concerned w it h lactic
acid f o r r o n t a t i o a s of IS t o 15 p©r cent sugar solutions a t i l i s *
in© various materials to su ^ly th e nutritional requirements
of Use culture B a c i l l u s d s a t g o ia e fc le u a »
ix v r e s t ig a t e d as a nutritive material*
Soyb ean m eal was
She e s s e n t i a l con­
stituents of' th e medium# their optimum e c a c e a b r a tlo x u i and
o t h e r ©ptimam conditions w ere determined,
nutrient m a t e r i a ls
mm
A number ©f other
tested "in the soybean'-, basal medium*
feast extract and i t s fractions
mm
also i vc -s tig a te d *
Bie o p t i mm s e n d i t i o a a f o r this type o f fermentation
«ay be defined as those which produce the most rapid fomentation
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
•
IQ : *
with high yield and c o m p lete uti.il.gabion of IS to IS per c e n t
su g a r.
Numerous experimental fe r m e n t a t io n s w##© carried out
t o d e te r m in e th e e s s e n t i a l c o n s t i t u e n t s of th e sodium * t h e i r
o p t i o n concentration* and o t h e r accessary conditions*
The
c o u r s e of the f e r m e n t a tio n s was follow## toy I n i t i a l and
p e r i o d c sugar d e te r m in a t io n s and l a some e s s e * by further
analysis at the completion of
the
f©rasatatton*
Aaalytloal methods.
G lu co se m s d e te r m in e d toy © it h c r th e m ethod o f S t i l e s #
f e t e r s e n and Fred (19$6) or Munson and Walker (1906)«
latter method proved more a c c u r a te .and reliable*
The
The samples
• e r e c l a r i f i e d by c e n t r i f u g a t i o n before d e te r m in a tio n s were
made*
lactic a c i d was d eterm in ed * after ethyl ether e x t r a c t io n *
toy the m ethod of F ried eaam a and Qraeeer- (19.35)*
l it h iu m lactate emit was used as a sta n d a rd *
A p u re
The type of
lactic acid was d e te r m in e d toy p r e p a r in g the sine salt*
deterainlfig
the
water o f c r y s t a l l i s a t i o n and observing th e
rotation of a 4 per cent solution in a p o la r is o o p e *
The
zinc salt e a e prepared from acid extracted from th e f e r m ented m edian, with ethyl ether* -She acid m s heated with
excess., sine eartobnate and filtered*'
The filtrate m s con­
centrated# cooled '.and the ■resulting crop o f crystals was
filtered out# washed with a l i t t l e
l e e water and dried at
room temperature to a c o n s t a n t weight*
Sine# any in a c t iv e '
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
-
S I —
&mm
the
acid was active* it w i tuuaeee weary t o ta k e mere than
the
salt precipitates before
the active and in all
first crop o f crystals*
Volatile acid m s determined toy acidifying a 100 si*
sa m p le o f fermentation l i q u o r w ith s u l f u r l e acid to ©eng©
red* and stea m d i a t i l l i n ^ , ■ailowiag th e volum e to e o n e e n tr a t e
to 00 &1» and c o l l e c t i n g 600 si*
I M t was mads alkaline*
concentrated t o BO ml** acidified and 200 al* stes® distilled*
r e flo a te d to remove
KaOH*
0 Qg
and an a l i q u o t titrated with standard
the partition m ethod of Oabarn* ‘
food aad W©rkai»n (1903)
was used to determine the acids*
Acetic aad f o n a l e acids I n
the volatile acid distillate were also d e ter m in e d qualita­
tively by the ferric c h l o r i d e m ethod of lorris {1924) and
the silver nitrate r e d u c tio n respectively*.
B u c e in t c acid was d e ter m in e d by the silver salt method*
A c ety ia a g fc h jle a r b iu o l m s d e te r m in e d according to St&hly
and Workman (1930)*
2.*3-iatylen© glycol was d e te r m in e d according to Srock»n
and Veskmam (1933)*
Sthyl alcohol was d e te r m in e d according, to the method
described .by iilo fc e la o a and workman (1938)»
.Fractionation o f yeast extract by extraction*
Sther extract mi acidified yeast extract ( l l f e o ) *
200
grams ©f yeast extract (Bifeo) mere sad® Into a heavy past©
by stirring In 8§ si* % 3 0 g (1 part acid t o 3 parts w a te r )*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
« E g •■
■Flaa ter ■of f&ris was s t i r r e d Into tit© past® u n t i l a f r i a b l e
texture resulted*. the m a t e r ia l was broken up u n t i l it
p a s s e d through a s i e v e o f i* S « • ; feel©®*;
fti@
wmt e r i a l
me
extracted eontismouiiiy w it h 86 per c e n t e t h e r f o r 5 day#*-,
fts# e t tor and eolabl® m a t e r ia l were p ou red o f f t h e .material
wJaioii tod p r e c i p i t a t e d out o f th e e t h e r e x tr a c t* '
fart of th e
e t h e r was distilled from th e extract and used in e a a h ln g the
r e s id u e * the washings
process was repeated*-
m m added'to
86
th e extract a g a in * ’, fliia
m l* water w ere added t o th e e t h e r
extract and th e e t tor removed* i:t to extract m s .neutralized.
(16 ml* 1? I haQH required) and made up to too ml** meklag
It equal to 100 per cent on th e feasia- of the original yeast
extract*
Ac®tea® extract of yeast extract*
After the ether
e x t r a c t i o n th e ether was ■completely removed fro® the yeast
e x tr a e t< » p la a te r of P a r le ..material by aeration and the material
m s then extracted for #4- hours with ace tea® (rapidly}* ■ ftm
ace ton©- m s drained .fro® the a p p a r a tu s and air passed through
to remove residual a e e t o n e completely* " A e eta a e was removed
from the- -extracted, material by ■distillation and any remaining
by >p la n in g a .jet of th e air la th e finals,
extract 160 ml*- of water
mem
f© -the a e e t e a e
ad d ed f th e e x t r a c t m i
mu*
t r a i l s e d requiring 4 m l. If 1 SaOli, and them nad® up t o 8 0 0 ml*
(100 per cent}*
Alcohol extract*
After the acetone extraction the
material was e x t r a c t e d for 60 hoars (slowly} with 96 per e e a t
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
** 3S
a lc o h o l*
At
43
h o u rs fed® f l a s k
( e x t r a c t e d m a t e r ia l) m s
reaetred and a n o th e r f l a s k w ith f r e s h a l e o h o l conn® eted t o
t h e a p p a ra tu s*
A f t e r t h e e x t r a c t i o n th e a l e o h o l m i d r a in e d
from th e a p p a r a tu s in t o - -a d i s t i l l i n g f l a s k and t h e a l c o h o l
l a r g e l y r sa o v o d by d i s t i l l a t i o n #
th e v e a a ln tie v and a l s o t h a t
I n t h e f i r s t f l a s k ta k e n o f f a t 48 h ou rs* was v e a o r o d by a
J e t o f a i r a t room te n p o r o ta r e *
She M a te r ia l was ta k e n up
l a w a ter# a e u t r & liw d (I S n l« 17 1 ImOl r e q u ir e d } and made
up fee 400 ml* ( wO p e r © e a t)* W ater e x t r a c t *
A f t e r th e e th e r # a c e to n e and a l c o h o l
e x t r a c t i o n s th e r e s id u e was sh ak en w ith m ater and c e n t r i f u g e d
r e p e a t e d ly #
The s u p e r n a ta n t l i q u i d ana e e a s e a t r a t e d u n d er a
jet of air and drying r a f l e e t o r # neutralised with 9 ml*
17 I HaOH and made up t o 1000 »1* (2 0 per c e n t ) «
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
nutritional Studies
Sfetsgttfsttmatatlva lactic Acid B a c t e r ia
Experiments and r e s u l t s .
fable I a b m m tba effect @.f adding imlifi aleoholt and
ether extra©ta of malt sprouts to tbs 'basal I » d i u m ©a tbs
growth of J>* aaannitopoeua Cl 8i ana L, baejme.ri (I» 4)#
fh®
extracts m e ® added in tin*®® concoatratloas, 0*1# 1*0 and
8*0 ml* of tbs -B for ©eat solutions* but only tbe ©pilaus
is given la tbs table*
f&bl# I*
Effect of various extracts of salt sprouts on
g v o v th of b s t s r e f s r a s n t a t l m lactic, acid bacteria*
C o m p o sitio n o f medium
a
-a
i
i
i
a lT “tm5B i a c t a f i r
1 0 m l. laedtua
8
9
*■
l
a.
*
1 4
B asal i #
0 .0
0*0
0*0
B asal- I 4- -%o e x tr a c t '
S *8
10*0
10*9
B a sa l 1 4 a lc a b o l e x tr a c t
0 .9
0*4
1 .8
B a sa l 1 4 e th e r e x tr a c t
0 .0
0*1
0*0
i 9 * 3 * p a r a e lfc r o v o r u s* 1 S * 1* m aim itopoau## 1 4 • 1*
bucm ^erlT""' -------~
------ - * ------# B a s a l I * 1*0 p e r mmt g lu e© so # 0 * 3 p e r © eat
per cent NaCMke and speakm an** in o r g a n ic salts..
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
0*0
,glI **
Tii® results shew feat th e wetter extra©t eontains tim
n e c e s s a r y ©laments ©f n u t r i t i o n for t h e s e c u l t u r e s w h ile
tiie o t h e r e x t r a c t s a r e lacking in so*© r e e p e e t #
fh© acid
p r o d u c tio n in t h e 'c a s e ©f th e water extract is p r o b a b ly th e
maximum amount tolerated lay organisms in t h i s medium*
T a b le IX -shows the effect of s-.p ,-la m en tin g t h e media
shorn In T a b le 1 with h y d r o ly s e d e a s e i n 0*1$ per ©©at* The
results t o g e t h e r w it h those in fable I I n d ic a t e that hydrolym *
®d c a s e i n and th e alcohol extract both, su p p ly sow beneficial
nutrients of a com plem entary n a tu r e *
Table XI*
e f f e c t o f su p p le m e n tin g the basal medium and
e x t r a c t s with h y d r o ly s e d c a s e i n ©a g ro w th o f
h e fc e r o fe r m e n ta tlv e lactic acid bacteria*
%
I ,,
Oompositlon of medium
ml* 0*05 I a c i d p e r
1 0 m i• medium
s
1 s t
Cultures
* t 4
* I» 2
0.1
0.0
0*4
Basal 1 % Q ©xfcraefe 4- nyd*
casein
10*4
10*4
10.6
Basal 1 + alcohol extract +
hyd* -casein
0*7
11.4
18*6
Basal 1 ♦ ether extract +
hyd* cas«ia
0*3
e.3
1.0
Basal I* + hyd. casein
3 9 * 3 * p a r a c it r o r orua * I*
bucime
Z
* 1* m a a n lto p o eu a » t 4 » 1 ,
*
*
*
*
~
* B a s a l I * 1 * 0 p e r c e n t g lu c o s e # 0 * 3 p e r c o a t (l®^}g.S0^# 0*6
p e r c e n t ImOAo and Bpeakmarx1a in o r g a n ic s a l t s .
R e p r o d u c e d with p e r m i s s io n of t h e co p y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e rm is s io n .
i n th e n e x t e sp erlm s& t th e n o d i* c o n s i s t e d o f ti»© b a s a l
i p l u s h y d ro i., *j»d ©as®in t o e h le h m m add ed fed® various
fractions or « © a l c o h o l e x t r a c t of m a lt s p r o u t s deserltoed
above under m ethods*
three e o u c e a t v a t le iis *
©a malt s p r o u t s ) *
Each of fed# fractions was u s e d in
0*0, 0 * 5 , nab 0*05 pur e#nt (b a s e d
fh® r e s u l t s are given ia f a b l e
Hit-
R e p r o d u c e d with p e r m i s s io n of t h e cop y rig h t o w n e r. F u r th e r re p ro d u c tio n p rohib ited w ith o u t p e r m is s io n .
m
S a b le 111,
*
Lffect of varioms fractions of th e alcoaol extract
of malt sprouts on growth of beterofamentatiw
lactic acid bacteria,
s ml. 0*05 JSi acid per
s
10 ml* medium
T~"
m 9 * L i * 1 4
Composition of medium
l
jp#l
i
*9*7
•1
45*5
*
*1*0
Basal 11*‘ (eontrol}
Basal 11 4 ale# ext# .2*0$
Basal 11 4 ale* ext* 0.5$
0*0
■
U.5
11*4
18*8 ■
7*5
8*5
1.5
8.6
Basal II 4 ether sol* ale. ext* 2.0$
0*6
■2.2
Basal 11 4 ether
0.6'
1.8
0*3
1*7
10.1
12.4
6*6
8,1
1,0.
2*3
10*0
14.5
5*5
7,2
1*0
2.4
0*7
2.0
0.8
1,7
1.0
.1*6
Basal 11 ♦ ale* ext* o.o5/6
Basal XI 4 ether
Basal 11 4 ether
Basal II 4 ether
10*3
*
sol* ale* ext# 0*S$ .*■0*3
I
-sol* ale. ext* 0*05$ SO*5
#:
insol* ale* ext* 2*0$18,7
*
insol* ale* ext* 0.6,>53.5
Basal 11 4 ether insol* ale* ext* 0*05/^0.?
wBasal 11 4 Seuberg ppt* 2.G$
J©*8
£
Basal 11 4 8*aberg ppt* 0„S$
.*8*8
|
Basal II 4 lumberg, ppt« 0*G©$
*0*7
s
Basal II 4 Seuber^ filtrate 2*0$
10*7
f
Basal IX + leaders filtrate 0*5^
*0.5
S
■Basal 11 4 Heaters filtrate 0*05$
10*3
8
9 * ||* p a r a c lt r o v o r u a » L
b u e lm e r i r ~ * * ~ ,*,~
— ——
2
* t , aannlto p o e n a » I> 4 * X.
—
-
—
a&sal II * 3U0 per cent glucese* 0.5 per cent (£84)0304#
0*6 per cent M&QAe# &peak»an*s inorganic salts and 0*15
per cent hydrolyzed casein*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
** JUS '**■
fa b le
111
.shews t h e e t h e r s o l u b l e and th e le u b e r g f i l t r a t e "
f r a c t i o n s were q u it e i n e f f e c t i v e i n s t im u la t in g g ro w th of a l l
t h r e e c u l t u r e s w h ile th e e t h e r I n s o l u b l e and, th e ^ e u b e r g
p r e c i p i t a t e " fr & e tle n a w ere v e r y e f f e e t l v e #
files© f r a c t i o n s
c o n t a in t h e satin© a e ld s * p e p t id e s and p r o t e in s *
l a th e n e x t e x p e rim en t h y d r o ly s e d cru d e c a s e i n wag
compared w ith h y d r o ly s e d p u r i f i e d p r o t e i n s ( c a s e in * g e l a t i n
albwaaea} t o d e te r m in e w h eth er the- cru d e o s s e i n m s d i f f e r e n t
from th e p u r i f i e d p r o t e i n s i n s t im u la t in g Orow th*
S in e #
tr y p to p h a n e i s . d e s t r o y e d and a c i d h y d r o l y s is o f p r o te in * i t
was added t o e a c h tu b e a s . mas a l s o th e e t h e r e x t r a c t o f
y e a s t e x t r a c t and th ia m in *
|»* b u e h n e r i (l> 4 ) was em ployed
i n t h i s e x p e r im e n t and I t was c a r r ie d th ro u g h th r e e tr a n s f e r s *
The r e s u l t s
fa b le I f ,
mm
shown i n f a b l e I f *
i i f f e c t o f r e p l a c i n g ' h y d r o ly s e d cru d e c a s e i n w ith
h y d r o ly z e d p u r i f i e d p r o t e i n s on grow th o f 1 ,
buchnter 1 *
""
«e. “T i l .
C o m p o sitio n o f medium
1
1
1
1s
t
0
10
,USE fi a c £ 3 ^ # r
ml* a e d iu a
T ra n sfe rs
* 2 nd
* 3 rd
0 .5
0 .8
0*0
byd* ©rude c a s e i n
8 .5
12*8
12*0
B a s a l I I I + hyd* Merck c a s s i a
7*0
6 ,8
6 .8
B a s e l I I I *■ hyd* Cm c a s e i n a t e
6
*a
6 .5
5 ,7
B a s a l 1X1 + hyd* album en
0 ,1
8*6
7*5
B a s a l 111 + h y d . g e l a t i n
i,s
2 ,6
2*1
B asal I H *
B a sa l
11% *
B a s a l I I I * 1 , 0 p e r c e n t g lu c o s e * 0 * 3 p e r c e n t (£ £ 4 ) 2 8 0 4 *
0 , 6 p e r c e n t haOAc and 8 j>®ale»an*s I n o r g a n ic s a l t s ; 0 ,1
s i e r o g r a a th ia m in , e t h e r e x t r a c t o f 0 . 3 ^raai y e a s t e x t r a c t
p e r 1 0 ml* and 0 , 0 1 p e r eem t try p to p h a n e *
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
<* 8 9
-
The ether extract ©f .yeast e x t r a c t pins th ia m in plus
tr y p to p h a n e d i d not fully r e p la c e the alcohol.extract o f
m a lt sprouts used In the previous e x p e rim en t*
It n s evident
that th e cru d e cassia c o n ta in e d toms factor t h a t the purified
p r o t e i n s d i d not*
It was a l s o evident that th e h y d r o ly s e d
gelatin compared w ith the other proteins- is deficient in
sos© growth p ro m o tin g su b s ta n c e *
-Qelmfein is laosa to be
deficient in c e r t a i n amino a c id s #
The p u rp o se of"the n e x t e x p e r im e n t (fable V) m s t o
determine w h ich am ino a c id s* w h en added to- h y d r o ly s e d gelatin
w ould s t im u la t e Orov/th*.
C u ltu r e I* 4 w&a used*
w ere 0 * 0 1 p e r c e n t c o n c e n tr a tio n *
Amin© acids
'Tubes number l
# -8
were ran through few© t r a n s f e r s before tubes 4* 5 and
set up*
and 3
6
w ere
Cys-tlae- m s added t o number 3 third transfer and
t h e r e was an im m ed iate r e s p o n s e *
Tubes 4* 5 .and
6
show th e
addition o f t y r o s in e # h i s t i d i n e -and p h e n y la la n in e to hydro**
l y z e d gelatin p r o d u c e s a© s t im u la t io n *
Tube
8
shews other
amino a c i d .a d d it io n s a r e b e n e f i c i a l *
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
T a b le ¥»
a f fe c t; o f a d d in g amino acids to h y d r o ly z e d gelatin
medium ©is the growth of L* buehneri,
i
:
Tab# . C o m p o sitio n o f medium
J*e, i
Basal H I 4** + iiyd* gelatin *
v a lin e + ty r o s in e
’"IJTSS’TS a c i d p e r
i__ 10 mi* medium
I'
T r a n s fe r s
lis t
1
2nd t
3rd
r ..
4*0
4*4
Basal III + hyd* g e l a t i n ♦
valine *► tyre sin® + m eth ion in e +
c y s t i n e ♦ h i s t i d i n e + phenyl& +
l& n in e
8*8
8*1
8 .3
3
Basal H I * hyd* gelatin +
ty r o s in e
3,1
2.6
* 7 ,0
4
Basal H I + hyd* g e l a t i n •*■
c je tin e
7 ,6
7*7
7 .3
5
Basal I I I ♦ hyd* gelatin *
c y s t i n e *f t y r o s i n e
7 .0
7*8
7 .3
6
Basal I I I + byd* gelatin +
cystine + tyrosine +•
p h e n y la la n in e
6 ,9
7*6
7*3.
1
2
0 .1
# Cystine added
** Basal III s 1*0 par cent glucose* 0.3 per cent (H%5gS©4*
0*6 per cent M&GAc, Speaka*an*s inorganic salts* 0*1
talcro^ram th ia m in per 10 ml., e t h e r e x t r a c t o f 0.3 gram
yeast extract per 10 ml. and 0.01 per cent tryptophane*
fable IV indicates that the hydrolysed crude casein
was. better than the purified ©as®la hydrolysate*
difference was the-
as
fills
to be dm® to riboflavin which had
been more completely rem oved from the purified protein*
The effect of a d d in g riboflavin is shewn la Table ¥1*
Con­
centrations of 0*5, 8 *0 , 10*0 and 20 miorograas per 10 ml*
R e p r o d u c e d with p e r m i s s io n of t h e co p y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e rm is s io n .
*■» S i *“
medium
given*
mm
wX&vo&mm}
tested bit only the optima*# CIO
Tim
experiment
ma
is
carried through eight serial
transfers* only 4 are shewn,
fable VI*
Effect of riboflavin on acid production in a
Hydrolyzed p u r i f i e d caseia medium*
1
Cul**'*
tur©; C o m p o sitio n o f medium
tal* 0.05 1 acid per 10
s
ml • medium
Transfer#
* ls t
2nd i 3rd $ 4t&
.1
S 9
h 2
la 4
Sasal H I * - hyd, purified ■
casein
2*6
0*5
0.6
0*3
8ssal H I «* fayd* purified
casein * Bg
5*0
4*7
4.4
4.8
Basal H I * byd* p u r i f i e d
©a seta
4*9
1*0
8.3
1*0
Basal III - hyd* purified
casein » §g
7*5
5*2
5,5
5,5
Basal III «* iiyd* purified
easels
9*0
5*6
5*7
5*7
Basal 111 - toyd* purified .
easeIn • Bg
9.0
6*9
6.8
8.5
Basal III - hyd* purified
e a s e In
5*4
5*1
8 .1
1*8
Basal III » hyd* purified
easein - Bg
4.8
3.4
5*9
5*1
1 §
8 S *
p a r a e l t r o v o m a .8 » i
2
•
i ,
m a a m lfc o p o e u a * L 4
*
1,
buchneri*"t»'I « I* Ijooperalel*
Bg s riboflavin
* B a sa l III * 1*0 per c o a t .glucose# 0*5 per cent (HS^ijgSOg, 0*6
pew m n t 1 &OA©# Speakm&n*# in o r g a n ic s a l t s # 0 * 1 microgr&m
th ia m in per 10 m l*,' e t h e r extract of 0 * 5 gram yeast mxt r a c t
per 10 ml* and 0,01 per cent, tryptophan#-.*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
»
Riboflavin p rod u ced th e greatest
p&raeltr o v o r u e CS 9} en d
J»*
effect,
oa culture S.
maanltoyeeua {I* B)g with least
effect on-&* b u e h a e r l (1* 4 ) but still rather s i g n i f i c a n t *
The medium a p p ea red to be somewhat d e f i c i e n t for J## l y c o p e r a i d
{h
5 ) a lth o u g h the addition o f f l a v i n did skew a narked atlmuia**
t lo n *
The in f lu e n c e o f th ia m in # e t h e r extmet o f acidified
yeast extract, tr y p to p h a n e .and riboflavin on growth measured
by acid production is shown in Table V II*
m edia were t e s t e d ;
Six different
CD th e substances just listed were a l l
present in th e medium# (Sj riboflavin omitted# (3) thiamin
omitted# (4 ) th ia m in and riboflavin o m itte d # (5) ether extract
of yeast extract o m itte d and Cfi) try p to p h a n e e m itte d *
serial transfers are shown.
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
Three
35 ~
T a b le V II*
E f f e c t o f th ia m in # r ib e fl& v in # e t b e r e x t r a c t of
y e a s t e x t r a c t and try p to p h a n e on a c i d p r o d u c tio n
by f t e t e r o f e r a e n t a t i v s l a c t i c a c id b a c te r ia * .
|♦
**
C o m p o sitio n o f m edia
1
1:»
•*
1
5 * 6 ! T f acTcT per
10 m l. medium
STrans- ■s
Cultures
: fer
* S 9 * X* 8 i L 4 f h S
I
t
.1
b asal x A
H y d ro ly sed casein.
Tryptophan©
E th e r extract of yeast ext*
T niam in
R ib o f la v in
1st
2*3
4*7
9,1
4.4
2nd
:4*9
4*9
7,8
4.9
3rd
5*2
4*7
8 .6
4*4
1st
2*3
4,4
9*4
5*4
2nd
2,1
3.9
9*6
3 .6
3rd
1*8
5*9
10*4
5,4
1s
1,8
5,6
6 .0
2.6
gad
1*8
5,8
7 .0
3.1
3rd
1*3
5*6
7.8
2,9
1st
6.0
0*8
7.0
1*8
End
0*0
2*6
6,5
0.6
3rd
0.0
0 .5
7*3
0.0
Basal 1
Hydrolysed casein
Tryptophan©
Thiamin.
H lb o f la v in
1st
2 .1
2 .1
5.4
3,4
2nd
0.6
0*5
0*5
0*5
3 rd
0*0
0 .0
0,0
0*0
Basal 1
le t
2*6
6.0
6*8
5*6
2nd
1 .3
5,5
3.8
4.2
Basal I
u y d r o l yz e d 'casein T rypt op.u&n©
O th er extract of yeast ext-*
Thiamin
'a#g a l I.
Hydrolysed casein
T ryptophane
E th e r extract o f yeast ext*
H ib o f la v in
Basal 1
Hydrolysed c a s e i n
Tryptophan©
E th e r e x tr a c ts o f yeast ext*
.
t
Hydrolysed c a s e i n
sther e x t r a c t o f yeast e x t*
Thiamin
Eiboflavln
—————r*r*—
bacl»«.glTm^ ' S ~ * ^ T ,Xycopeg8lcX
1,8 6.2
1*8
5*5
m&rml t opoeuaT^ i r T ^ S I
3rd
***
* Basal I a 1*0 per cent g lu c o s e # 0,3 per cent (HH^gSOg* 0*6
per cent HaQAc and Sp©akman*s in o r g a n ic salts*
R e p r o d u c e d with p e r m i s s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e rm is s io n .
** n# »
These r e s a l t a indicate that the yeast f a c t o r was
e s s e n t i a l for a l l o f th e c u l t u r e s tasted*
tryptophan© was
e s s e n t i a l for good g ro w th ©f S t r e p t o c o c c u s p a r a c itrovorua
i s 9} mid Lf b u c h a e r l | h 4.) hut was not essential f o r J»*
insrm ltopoeua Cl*
2)
or I* l y c o p e r s l c l
(1
§ )*
and th ia m in s t im u la t e d growth o f © ultares
loth r i b o f l a v i n
L 2-* L
5 and S 9
but neither was e s s e n t i a l in th e p r e s e n c e of the other
a lth o u g h some s t i m u l a t i o n s *er©
a icwnj
when b o th eompounds
were o m itte d * .growth is c o m p le t e ly stopped*
The same I* 4
culture willcii in a previous e x p e r im e n t was s t im u la t e d some*
■what toy flavin showed no response to flavin in this @xp©rim ent*
Thiam in also was net e s s e n t i a l for I 4# 'tout was pro­
bably beneficial# sine© it. showed a alight stimulation*
With a hotter u n d e r s ta n d in g o f the req u irem en ts- of
these cultures when grown in a hydrolysed casein medium# an
e x p e r im e n t was set up t o
d e te r m in e whether the hydrolysed
casein can'be replaced by amino- acids#
B ia t e e a amino acids
w ere u sed i n th e f o l lo w in g c o n c e n t r a t io n s e x p r e s s e d a s p e r
c e n t:
glycine o.oi, alanine
th r e o n in e
0
0
*0 1 # v a l i n e
0
*0 1 # l e u c i n e 0*01#
*0 1 # serin# 0*01# cystine 0*005# m e th io n in e 0*01#
.aspartic 0*01# g lu ta m a e 0*G2§# t y r o s i n e 0*005# p h e n y la la n in e
0*01# tryptophan©
0
*0 1 # p r o l in e 0*01, h y d r o x y p r o lin s
h is t id in ® 0*01# a r g in in e v . i# and l y s i n e 0*01*
0
*0 1 #
The results
are given in fable ¥111 which also shows t h e effect ©f
adding p a n to th e n ic acid to the am ino acid medium *
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
- 35 Table fill, Effect of replacing hydrolycod casein by amino
acids and by amino acids and pantothenic acid
on acid production by heterofexmentative lactic
acid bacteria*
I ml* 0,05 U acid
i per 10 aim medium
4'
%friasfers
$ let
2 nd
3rd
I
t
Cul­
tu r e iCompoalfcion of aedis
■*
5 .7
7*3
7.0
7.8
8*1
7,8
8*6
8*2
7.5
9*9
9*4
7.5
Basal if ■m 19 aaino a c ld e
11*7
1 2 .0
10.1
Basal If m* 19 sals© acids - p a n t o fchenio
1 1 .7
12.7
8 .6
8*2
5*5
5*9
5*2
5*2
3 .9
4*9
5.5
4*4
iasal IV®1 hyd* easels
19 amino acids
Basal If
L 2
Basal.if
t h e n ie
***■
19 am ino acids - p a n to -
Basal If m- hyd* c a s e i n
L4
hyd* casein
Bae&l IV
L
Basal If am- 19 am ino a ei.d e
5
Basal If
m
19 amino acids
-
panto*
tiienic
I» 1
*
L*
m a n n i t o p o e n a # I* 4 » 1 .
b u c m ie x l*
I* 5 *
Jk* X y c o g e r a l e i
* Basal If « 1*0 per cent glucose $ 0*3 per cent (Mi^gSG,^, 0*i
per cent SaGAc and Bpeakman's inorganic salts# 0*1 microgram thiamin# 5 mierograma riboflavin and ether extract of
0*3 grant yeast extract per 10 ml*
The results ahow the amin© acid medium was better than
th e h y d r o ly s e d casein laedino. for cultures JO* m m m i t o p m m ih 2)
and £*-. b a c lm e r l {h 4) and e q u a l l y as good in the case of
' Jk* X y o o p e r a lc l (L S).
The addition of pantothenic acid pro­
duced ao stimulation in the a m in o 'a c id medium*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
* 36 •
DtaeusslOB*
It
have
wsxat
bma
be remembered t h a t any c o n c lu s io n s drawn her#
derived under special conditions and may n o t toe
exp©fttod to bold if the c o n d it io n s are altered*
it is dif­
ficult. if not I m p o s s ib le even with. th e .greatest of care t o
d u p l i c a t e t h i s type of mortg for bacterial cultures do n e t
restsin c o n s ta n t*
V a r ia tio n # adaptation and the vitality of
t he culture p r o b a b ly f u n c t i o n in altering the properties of
any culture.*
R ib o f la v in h a s been d e m o n stra te d t o stimulate
cultures when vitality i s low but soon loses its effect
(Wood# A n d ersen sad
"mwkmmig
1 » 3 6 }*
Serial transfers are
n e c e s s a r y a t tine® to deplete the culture o f carry over
material but h a s a disadvantage la that the culture .may
th ro u g h adaptation o r v a r i a t i o n be altered during the process*
Wood# Andersen and Wmwka&a, (1938) c i t e a good exam ple o f
adaptation or *tr a in in g ® as the phenomenon has been called*
A propionic acid culture (49 1) was grown on a. tMamin-free
medium for twelve t r a n s f e r s *
Growth as measured by acid
production p r a c t i c a l l y ceased during th a first six t r a n s f e r s #
then steadily increased on e a c h succeeding; transfer t o above
that at the b e g in n in g *
QrXa-Jensen. e t a l*
(193#) claim t h a t thiamin and
tr y p to p a n e are n o t iaportsnt l a the nutrition, o f the lactic
acid bacteria*
fa© r e s u l t s p r e s e n t e d above show that t i l l s
dots not hold true for a l l lactic acid bacteria* the con­
clusions eased on this Investigation should not be applied to
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
- m
other cultures* #v»n of the amm genus 'out m&y be very help­
f u l in f u r t h e r study o f t h e s e and o t h e r c l o s e l y r e l a t e d
c u ltu r es-*
Summary sad conclusions*
B u t r i t i o a a l .s tu d ie s o f f o u r c u l t u r e s o f n e te r o f e r m e n ta t i v e l a c t i c a c id bacteria* J»:* maanltoaoeua (1 2 )* J», d n eiaaerl
IL 45# J» ly e o p s r s ic l {I* 5) and Streptococcus paracltrovorus
(S 9 )> e e r e im d er ta k e n to d e te r m in e t h e -r e q u ir e m e n ts o f this
group* The four cultures v e r y c o n s id e r a b le # b u t probably are
representative of t be group.
Malt sprouts are a r i c h source
of food m a t e r ia l for t h i s group o f bacteria*
Of water#
alcohol* and ether extracts of m a lt sprouts only the water
e x t r a c t contains the essential m a t e r ia l for growth of these
cultures is a protein-free and aiai.no acid-free medium*
The
alcohol extract contains the essential m a t e r ia l when added
t o a basal medium c o n t a in in g hydrolysed c a s e I n .
The e t h e r
extract o f either the m a lt sprouts or the alcohol extract is
not effective*
The Bieaberg precipitate1
* o f the alcohol
extract is effective while th e "Jieubarg f i l t r a t e * is not*
In an attempt t o replace the alcohol extract o f salt
sprouts and h y d r o ly s e d casein ether extract of acidified
yeast extract* thiamin* r i b o f l a v i n * p a n t o th e n ic acid and
nineteen, am ino acids have been investigated*
In a basal
medium containing hydrolyzed casein the ether soluble factor
o f acidified yeast extract is essential f o r all four c u l t u r e s ;
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
- m
tr y p to p h a n e is essential for good growth, of <§*- para c l tr o y o r u s
i s 9) and J»* buchneri Cl# 4) bat not essential for I* w m ~
nttepocii# (1 2 y taiA.hf lyeepegalel Cl#- ©)f riboflavin and thiaaim
s t im u la t e d g ro w th of'
cut tares
1* m a n n itop oeu a# J** l y c o p e r s i c i
and S# paraeltr o v o r a g b u t n e i t h e r i s essential in the presence
of th e other# a lth o u g h s © » stimulation is shown; when thiamin
and r i b o f l a v i n a r e o m it te d from th e medium I*# a a o h it o p o e u s #
h* 3L -jcopersioj« -and S* g a r a c it r o y o g u s a r e completely stopped
while j** tenoiaaeri Is not s u b s t a n t i a l l y affected*
In all oases .growth m
measured toy acid production is
c o n s id e r a b ly r ed u c ed when the alcohol extract of m a lt sprouts
is r e p la c e d by the e t h e r soluble factor of acidified yeast
extract# thiamin* r i b o f l a v i n and tr y p to p h a n e *
H y d ro ly sed casein is completely replaced toy amino acids*
In the amino acid medium pantothenic acid shows no stim ulation*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
• 59 **
S©»©f©»@Htativ© £ * e t l« ' Acid B & eteria
H e s io fe n e & ta tlv e l a c t i c l e M
b a c t e r i a a r e th o s e b a c te r ia ,
which. p rod u ce o n ly t r a c e s o f p r o d u c ts e t h e r th an l a c t i c a c i d
i n th e d i a s i i a i i a t i o a o f c e r t a i n «arfeohydvat»**
Seam h a v e
thom ght th e group t o c o n s i s t o f o n ly members o f th e g e a a #
L a c to ta o iX ltte t
o th e r * h a v e in c lu d e d w m b t n o f th e g e n u s
S t r e p t o c o c c u s , aoao o f tftileh haw# b e e a shown t o c o n v e r t a s
mue& a s 98 p e r ©e&fc o f t h e su g a r fe r m e n te d t o l a c t i c a e l d f
w h ile s t i l l o t h e r e include* e v e r y t h la g f i t t i n g th e o r i g i n a l
d e fin itio n *
S t r i n g th e o o o r e e 'o f t h i s i n v e s t i g a t i o n an
a e r o h le # a p o r e -fo r a ilh g # s t o t l l e * o a t a l a e e p o s i t i v e -and n i t r a t e
r e d u c in g o rg a n ism was i s o l a t e d w h ich c o n v e r t s a s amoh a # 98
p e r c e n t o f th e f o m e n t e d su g a r t o l a c t i c .*©14*
It is a
hoM O fem eatafelve l a c t i c a e id o r g a n i s s fey d e f i n i t i o n , a lth o u g h
i t d i f f e r s fr© a i e t c t o f e a o l ll u s . l a th e p r o p e r t i e s | u s t l i s t e d #
t a g orga&lem was e a r e f a l l y e tu d le d * d e s c r ib e d and named
B a c i l l u s d e x tr o l& o tle m s (A n d ersen asui werkmaa# 1 9 4 0 }# and was
in c lu d e d i s t h i s .stu d y w ith two o t h e r fc e N e fe iM e a ta tiv e l a c t i c
a c i d c u lt u r e s # L a e to b & e ilia a d e lf e r a e c k li ( 1 d ) , and L a c to ­
b a c i l l u s h e lv e fc lo u s il# h }»
ju e a o r lp tic w and, c l a s s i f i c a t i o n o f l a a l l l a a d a a t r o lw e t ic a a .
isolation-* the organism was Isolated fro® a flask of
medium eompeaed of .salt sprouts., gltteose aad calcium
carbonate» which had been heated for 88 minutes at 10 pounds
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
- 40 •
stems p r e s s u r e *
the flank was serving as as u n in o c u la t e d
control la mm expertm m t determining the effect of various
temperatures ©a tbe rate of ftaNMmtatle&*v the flask mas taken,
M r s e tly
4?® C*
from the autoclave and placed l a an Incubator a t
r
Xt
was o b s e r v e d ©a th e s e e o a d day t h a t a v ig o r o u s
f e r m e n t a t io n was l a progress*,
A transfer mis aa'i# to a tu b s
o f sterile siediuai for su b se q u e n t study and t&e fe r m e n ta tio n
was allowed t o ©ontlon e*
to analysis of the fermented l i q u o r
showed that th e sugar fer m e n te d ( 7 * 1 ,grams per 100 at* I had
boon converted t o lactic aeid*
t h e sin© salt was p rep a red *
fit® water of c r y s t a l l i s a t i o n of 13*15 per c e n t and the s p e c iH c
optical rotation o f 7*8 stowed th e acid to to the deatfero f o m *
M ic r o s c o p ic a l e jsa m ia e tio n of the culture i n d ic a t e d that a
single m o r p h o lo g ic a l type was p r e s e n t*
plated and replated on g l u e o s e agar*
single type appeared*
The c u l t n r e was
©aaly e o lp m ie s of a
fell I s o l a t e d c o l o n i e s tore picked
inf© a medium containing m a lt s p r o u ts * g lu c o s e ana e s lc io m
c a r b o n a te *
A study o f the resulting c u l t u r e s show ed them to
to identical to the o r i g i n a l c u ltu r e * indicating t h e latter
also m s a par© culture*
The following s tu d y m s made on one
of th e c u l t u r e s obtained from a well i s o l a t e d colony*
the
parity of the ©ulta.ro has b een m a in ta in e d throughout this
work*
Lxcept for the variation described tolow th e c u lt u r e
has rem ain ed s t a b le *
Morphology ana -O s s in in
1**111g reactions*,
..■r-1.1.r.T->irtiir.||1
iwrmimfnilttwig^ I
ilnHr
M m w m kd:
<i t**>n**n*inHi'i■run.mjHit***(*
.<
~ —— —-T - . —
MtW||
the e l s e # shape
*
*
and s t a i n i n g e b s r a e t e r i s t i s s o f t h i s o r g a a is a v a r y c o n s id e r a b ly
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
■«►
. 4 1 «*►
'
d©p.©iidl»g upon t h e medium* t h e te o p e r a tu ? * # th e pi# ©xygm
sn d bo d o u b t o t h e r f a s t e r * *
grown l a
&s a lt
ih® a th e o r g a n is e i s
s p r e t t t S T g lu e o s w e a le iu * e a r b o n s te o s d iu a a t
47® C*# tb e s e l l s
-mm
sewwaat larger and leas u n ifo r m i n
sis® than when g r o w on a n u t r i e n t agar slant#
th e rods rapidly ;b#o©*e trait negative#
On unit agar
She last few
e r g a n ls a s r e v e l a l a g ©rata positive grew.Into long e s l l e *
many
mmiklug
o b se rv e d
5 0 sleroas o r longer*
m lo n g a s
so n * ©ells have been
160 a& erons .( F la t s
I# f i g u r e l l #
If
e a l e t u a c a r b o n a te Is ad d ed to the n s l t -agar# th e c e l l s remain
Sr&ai p o s i t i v e and u n ifo r m l a s is © #
l a sjmtJasfele m ed ia « M d i
/
have b©«n d e v e lo p e d for this e r g a n la a # the- ©ells are w l i t f
an d do n o t r e t a i n th e s t a i n by dram 1# a o th o d a s w e ll a s o # H s
grown on n u t r i e n t agar#
?©ry Irregularly shaped reds h a v e
been fo u n d la aerated m a lt sgroats-glaeos© aedloa*
Grown ma m n u t r i e n t agar slant a e r o b i c a l l y at 47® G* for
.14 hours# the .rods ar e fro® ©*§ t© 0*® microns In width and
■5 t© 6 m icr o n s la length*
longer sells o cc u r r a t h e r fr e q u e n tly *
Th® sells o oetir singly# la'pairs and' short chains*
the
organism is a o t l l o by means of p e r lt r le t u m e f l a g e l l a #
One#
two- or three f l a g s l l a ar# freqm@at.ly found but as many as
s e v e n have b een o b s e r v e d ©a a single © e ll*
method m
l^ifsoa1® (1958)
used for s t a i n i n g t b e f l a g e l l a (Plat# 1# f i g u r e 3)
Sporalafcloo. © oeure best la n u t r i e n t a g a r slant c u lt u r e s #
fh®
s p o r e s a r e oval# s u b t e m d n e l to te r m in a l -and t h e r e l a some
s w e l l i n g ' a s a p o r u la t io a p r o g r e s s e s *
2 o m r f a aw tbod {Committee#
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
** '^y3 ***
S., A* B *.# 193-9) has b een found s a t i s f a c t o r y f o r staining to e
spores (flat© 1# f i g u r e 3)«
ftm ends o f tb * rods are rounded*
Oram*a s t a i n Is r e t a i n e d Cfl&fee 1# f i g u r e 4 ) t a d u n d er some
c o n d i t i o n s th e © e l l s s t a i n u n e v e n ly # p a r t i c u l a r l y n o te d a t
th e t i n e t b s M i l t a r e becom ing o n u s 'n e g a t iv e *
V a r ia t io n *.
Wien B a c i l l u s d a x t r o l a o t ie a a w as i s o l a t e d
i t p ro d u ced o n ly sm ooth c o lo n ie s * mad f o r th r e e y e a r s n o
rou gh v a r ia n t * s e r e Observed*.
W all# g ro w in g th e c u lt u r e
l a s f m t a s t i « * » l a © m eld medio®# I t o a s o b se r v e d a f t a r
mmj
tr a & sfa r a t b a t t h e a o l t o r a d id n o t c o n s i s t o f th e s n a i l*
u n ifo rm ©©11s u s u a l l y s i n g l y ms p r e v io u s ly # tout ivare l a r g e r
mad u s u a l l y s e v e r a l © e l l s a t t a c h e d t o g e t h e r t o form a c h a in
u s u a l l y b a n t w here th e © e l l s jo in e d *
Tim
c u lt u r e was iaaaadl*»
a t e l y p l a t e d ©a m y e a s t a s t v a s t » g l u e e a e Ma £ a r medium eon *
s l a t i n g ©f y e a s t e x t r a c t 0 * 8 p e r c e n t# g lu c o s e 0*1 p a r c e n t*
I n o r g a n ic s a l t s and 1*© p e r c e n t agar*.
types#- a sm ooth mad a rough*
C o lo n ie s were o f two
A c u l t u r e was talsea from a w e l l
i s o l a t e d ro u g h c o lo n y .ana r e s e r v e d f o r su b se q u e n t stu d y *
She red iU B in which tbs rough form was f i r s t observed
c o n s i s t e d of g lu c o s e # inorganic salts# tiiiiwia# -and f i v e sain© a c i d s i
glutamic acid.
c y s t i n e # aetiilonlae# a r g in in e * threonine' and
At the time the rough fora was found in the
mala# acid a s d iu a * o t b a r e x p e r ia e a fc s o n simplified toe A la
were in progress# on# e c m t a in is g 13 a i e r o g r s s s of hydrolysed
casein p e r ml. and a n o th e r o e n t a l a ia g 4 a t e r e g r a n s o f y e a s t
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
*► 4JJ- «'
o x t r a o t -f«r ml* la p la o o of to® sain® a e ld o *
these organism#
move p l a t e d ta b o n l y sm ooth o o l o a l o s d e v e lo p e d *
th® sto o ls
©alter®# o f fen* org«ai» war# tested t a t a o rough utmimjs
w@r® found*
titm
It appears 'th a t th e rough strata d o v o lo p o d in
sain© meld modioau
The ©altar® baft tenia o a v r lo d earn*
tif»«MsXf on to® main® m eld medium f m f e w months* having
teen t r a a s f o r r o d o v o s y 1 t o 4 day#* toe® to® rough ©Jmraeteri s t i c was first o b serv ed *
fla® rou^h p r o p e r ty eeema t o b© a
s t a b l e e b s r s s t s r i s t l e of to® c u l t u r e on to® yeast s x t r s s t *
g iu e o o o -o tg o r «®41w»* for after f transfers #® this msdimm
to® rough strata y i e l d e d onlj rough ©©leal##*
Whoa grow®
on yeast e x t r s e t '- g l u e o s e agar- tb o rough and smooth s t r a i n s
sesm to- be a lik e morphologically -oxoopt that to® s e l l s l a
to® ro u h e u lt u r o tend t o restate s t t s e b e d #
la actual s c a n t s
mad® on stained mamma taken from omlter®# l a f o a w m t ia g
aalt spromts-glmeos# aiestfttia* to© average somber of e-ells per
segm en t of to® rough strain- -was greater than tm iae t h a t o f
the smooth*
Tte latter a v e r a g e d 1*4. s e l l s per segm en t toil®
toe f o i w r awragad 4*0.
fh@ rough strain finite?® la malt o p v o u to ^ sla o e o o medium
in t o o l a s t e n s e s ha# y i e l d e d a s m a ll proportion of sm ooth
colonies*
f b e smooth ty p o o e c u r r la g in the rough culture
appear to be toe same as to®
original m neottu
There is no
significant difference in fe r m e n ta tio n rates f o r to® smooth
and rough e u l tares la 1© per cent g l u c o s e median*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
<* 4 4
Cultural etoemoterlafclea* Aerobic growth ©a m n utrien t
agar slsm t i s moderate# filiform * opaque* s lig h t ly ra ised ,
g listen in g * emootb* bufeyrous; under transm itted lig h t fell©
in tern a l structure appears to be fin e ly grmsttlar* mad the
appeamaae o f th* sedlun i s moianged*
to the agar, fell© growth i© luxuriant*
I f glucose i s added
Axiaembimlly there
i s a© growth ©a n u trien t agar although them Is. modamt#
growth on glueoaa n utrient a_ar*
In a a a g a r stab# gmwfca occurs only ia the upper p a r t
sad i s b est a t the aurfaae*
I f glnoose Is present* growth
extends b© the bottom o f the stab*
Colonies ©a a autrieafe agar p lat# am circular* e n tir e ,
ooavex# granular and 1 to 3 mm* la d isaster*
Subsurface
o o lo & ie s 'a re w a l l .
Xaoabeted a t 48° C* a g e la tin tab© slow ly b#eem@a turbid
with « sesafey surface growth*
I f glucose I s added to the
mdimm* growth i s n tth better*
S o la tia I s net liftu lfied *
S utrien t broth ia©#ml*t©€ .and incubated a t 4 f° 0 * quletely
t»eosi®s turbid -with a Moderate amouat o f sediment*
Timm i s
no growth in lo s e r *a c itr a te aadiuau
P h y s i o l o g i c a l a & a m a ta r la t l a a *
Blmlww.
g ro w th t e a p e m ^
tur© o f th e o rg a n !sm i s n e a r tO®.C*f th e o p t l e e l tem p er a tu r e
ra n g # i s fro ®
I s n e a r US® C*
4S fee 5 3 ° C»j fclm
musSwm
g ro w th tem p er a tu r e
A r lg e r o a a f « » e » t s t i © » w i l l eoatin u © i a
u n i t sp ro u ts« g lm © e se medium a s h ig h a s gS® c*« w h ile a t
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
«* 4 f §
61*f#
Gp
th e fermentation gets. under m y r a p id ly but c o n -
ttones only 18 to 84 tours*
the ■o r g a n ism i s a f & e u l t a t i v s a e r o b e #
fh e effect -of
ail* in fermentablcas w i l l be discussed u n d er fe r m e n t a t iv e
metabolism#.
litmus isilte is rapidly reduced and coagulated* I n o c u la t e d
with 0*5 ml* of Inoculum from malt spreubs-giucea® medium,
10 al* 11 teas m ilk is e o a g a la t a d and reduced in 9 t o 18 tours*
As th e culture ages a red band form a at the top-*
form ed*
Ho gas i s
If c a lc iu m c a r b o n s te i s added th e litarns rem a in s
reduced '.and the curd i s partially d ig e s t e d *
After 48 tours Incubation to nitrate broth the nitrite
test is p o s i t i v e *
I M s nitrate reduction has been checked
repeatedly*
Cabalas® i s formed*
hydrogen sulphide Is not produced In lead acetate agar
or iron citrate agar*
Indole is not form ed ia tj^ptophane b r o th .
The V o g ea -P ro sk a u er test is positive indicating the
organism produces acetyls# feiiyfearbtool*
Bisslaii&feion of carbohydrates m s determined in th e
f o l lo w in g aodiutt*
f e a s t e x tr a c t (B ifc o )
a*0 grams
H*0A««'3H^8
6*0 grams
KrigPOg
0*85 grams
KgftfO*
'
0*E§ grams
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
%SC*4
0*10
great#
CaClg
0*10
.grass#
laCl
0*01
grass
FeS04
0*01
g r ts#
ltoS 04
0*01 grass#
Carbohydrate
10*0
g rass
M30Q ail*
i)istilled water
fti© e& rtohyd r& tes were s t e r i l i s e d s e p a r a t e l y i n 10
pmr
cent
solution at 10 pounds et#a» pressure for 10 mi&ute* and
then
1
ml* of each was aaued aseptic&lly to tabes eon twining
9 ml. of ttm sterile aediwsu
(green to brown thymol bine)*
the pfi was approximately 7.0
fhe inoculum was prepared by-
growing the culture in the medium described above w it h glucose
as the carbohydrate*
®ta« cells were ©eatrlfnged and re-
suspended in an equal veiaiB® of sterile water*
was added to ©sen tab©*
One tenth ml*
All tabes were ia duplicate#
They
w ere imombated at 4 7 ° C»* on© tu b e for 48 hours and the other
for 72 hours and then titrated witu standard NaOH to neutrality
with
bmm
thymol b lu e indicator*
fhe results are presented
in fable IX*. .©» last eolaa® giro results of the *xg»srim»&t
r e p e a t e d several souths later*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
„ 47 -
Table IX#.
Aeld paeduetloa. from
OagbQtoySgat®
*
sal* Q.ll ad d peg cent 10 ail* ae&itaai
j ecmfcgol t 48 lira, t 72 hrs . . 78 lap®.
g lu m m
0*1
4*7
4.4
4#i
lAVOlOM
.0*1
4*4
4*7
5*4
maxmoae
0.1
4*0
... 4*7
6*4
g a l aoto«*
0.1
0*1
4*0'
5*0
xyl@s®
0*1
6*a
1*2 '
1*0
. 0*1
2*0
3.1
3*8
avablaeraa
0*1
2*3
3*7
5*3
tvebftlem
0*1
4*0
4*S
4*7
ea llo b lo se
0*1
2 ,6
3*1
4*3
mellbios®
0*1
3*6
4*3
4*6
auerose
0*0
0.4
0.7
0.7
maltose
0*1
1,8
1 . 8*
4.8
la cto se
0*1
0*3
4.0
gaffln osa
0.1
1*0
1*4
0*5
a d oaltol
0*1
0*0
**0*1
- 0 .2
iyeegel
0*1
1.0
1*2
1*4-
»&»»it©l
0 .1
'"*0*1
- 0*1
- 0*2
d u lc lto l
0*1
0.0
—0 *1
so r b ito l
0*1
3*9
4*1
4.7
inositol
0.1
0*3
0 .4
0.2
*- stsl& ylgluoosids'
0 .1
3*4
3*3
3.6
d extrin
u,1
1,0
1*0
2*4
rbanaoMi
"
5*8
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
** 4 ®
fa b le IX*
**
Acid production from carbohydrates* toon ;inued)
Carbohydrate
'
i
ml* G.1S a c id per cent 10 ml*, msftltai
control i 48 hrs* « ' 72 Mrs* i 72 Jars*
emygdaXta '
oa
0.3
1*1
1,4
sallein
0.1
3 .1
5*1
4*1
in u lln
0*1
o io
0*0
0*2
starch
0 .1
0*1
0,2
la .May eases acid producttea f » w ear&cliydr&tes 1ms
been d©ter»iii@d by ms© o f indicator ©sly*
to 0*5 ml* of 0*1 1
t©**
m
la th is work 0*2
id was s u ffic ie n t to change the indlea*-
Sugar mad ether fermentable sufceteaees a f t w contain
some im purities or may Ins hydrolysed s u ffic ie n tly i s tbs
s t e r i li s i n g pvoeees to gim small amounts o f substances which
m y bo fenae&ted with sufficient acid pvo&aetlotk to fram an
In&icater and thereby gig# a fa lsa result.
It may be c©n»
eluded tmm- Sable -XX that aioiegat# tacmnts of mold are readily
formed tm m
glucose# leiuless#'m annese# galactose, rlmmnese#
erablncse, trehalose# ee lleh leee # m elihiese*. maltose# la c to s e ,
so r ih llo l# oi-mettiylglueostds aad asliclm,
Aeid
in sm aller
amounts or at a slower rate i s formed from raffia®##, glycerol,
sucres®, xylose# aay^oaliia and dextrin*
She ferm entstlea of
son® of these m y be coasldersd questionable*
So acid is
formed tm m adoaltol# mannltol# d u le lto l# inulln# starch and
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
in o sito l*
f t may fee noted timb a© &eM m s produced In the
la c to se ami gala©tea# tube# in 40 hours* however* a c©nslderabl# xraoimt. had been produced at: 7B hours*
the forma**
tton o f gas from any o f -tb*; carbohydrates. sould, a ct to© d etect­
ed by the mm o f Curiam tubes# ©r i m a g lsco s# la. Smith
tabes*.
Tim low est pH reached in glucose broth m s 4*0#*
Tim
principal, and sabstsntiall^ only produet o f glu­
cose d issim ila tio n i s dsxtrol& ettc &@M.*
la a large number’
of fermentations in several different madia* chemical analysis
bat
simmi the
©©aversion o f gtoeoa# to lactic acid t® be as
high as S-S per sent# and u su ally between 92 and 95 per cent*
ft® o p tic a l a c t iv ity o f the acid has been determined for
fermentations run la malt sprouts# in corn gluten# and la
soybean me&l glucose s#dia and in each case the type ©f acid
m s dexbro*
Sms11 amounts of 0#5-hutyXens glycol mad traces
o f acetylm ethylcarblnol* 'dl&eetyl* acetic a©id -and ethyl
alcoh ol 'have bees found as eud«produeta o f fermentation,* A
mere detailed study o f the products' o f glucose dissimilation
will
be
isfcen up in a later section*
G r o w l c e l l s are s e n s itiv e -to potassium cyanide*
The
potassium cyanide was added to tabes o f s t e r ile medium con­
sistin g . o f 0*5 per ©eat yeast extract* Sp#aliBan, s inox0anic
sa lts*
0*6
per cent sodium acetate* and
0*1
per cent glucose*
The concentrations o f potassium cyanide m m 0*0* 0*002,
0*005, 0*01,' 0*0© and 0 ,1 it*
Tim
cyanide was added after
Inoculation and tbs tubes mem Immediately stoppered tightly
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
• SO
3
o
1
KGn concentration ; 040 j0*002 M
Orowfch (tu rb id ity )J 444+«1. +44
s ++
m
Sro-wth indicated. by turbldlty m s as f o llo w * *
U* **
with ©orfas*
i
*0,01 M 10*05 M |G#1*
t
s
* «*
1 *
I
s. "■
■a* .
*
.
■ -w *
After the .tubes m m observed ead growth recorded fed® ■pvuaeiaa
blue test « s positive for the 0*00$ « tube where growth
was half normal*
the laiiioitlea Is eoueldered partial at
Q*0 0 £ 1 end ocay>Xete at 0 , 0 1 M or greater#
•IssglratelhB o f a#n.*proliferatlhi£ s e l l s # la fermentation
experiments# described later# aeration -ef the fermenting
medium was found to 'he neoe&a&ry eves though i t # function
m.9 a ct understood*
Respiration, stu d ie s were undertaken to
determine whether omjgea was
produced#
and carbon dioxide
the surprising r e s u lts o f these eaqperineat* led
to further In v estig a tio n o f the d lse lm tla tiv e power o f nonp r o life r a tin g oeXle#
GysaltS# se n sitlir lty m s a lso determined#
th ese stu d ies m m carried out -on a SUtroroft^Warburg resplro*
meter#;
The
mils were
grown' on medium ©ontainiag yeast extract*
glucose# Inorganic salts and agar*
The m i l s were washed
off the agar*- eeatrlfuged and twice reeuepended*' the last
time is phosphate buffer at pH 6*8,
due.followed*
The Method of Dixon {1934)
To eaeb 'cup m s added 0*5 ml* pfceaphete buffer#
0 * 5 -ml* of "cubatrate# 0*5.ml* ©ell suspension in side arm# ■•
0*5 ml* 1 potaaclum hydroxide in ©enter ©up of those which.
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
*& measured oxygen uptake# end water to mslcs the total eon*
tents. S*§ a&* Anaerobic conditions were obtained fey re­
placing the s i r In tbs caps with. e«tj©a dioxide*
After
equilibrium at #0® #* had been reached tbs cell suspension m s
mixed witti the eon te a ts o f the cups and reeding* so re taken
for two to five hoars#
corrections wore node for endogenous
respiration*
for * glucose eofeetrate -tbe ra te o f oxygen. eoaxuoptlon
and. rate o f ca rb o n d io x id e evolution* a e r o b i c a l l y and a n -
eerehle& lly# are presented la Flgmr© 1*
Oxygen consumption
sad carbon dioxide a volution paralleled eaeb otter closely*
ffe® respiratory quotient m s ©*§?*
Anaerobically esrbea
d ioxide evolu tion was approximately one-third as mutt as
aerobically*
ffee production acid could not be detected.*
Since s o l d m s n o t f a m e d in th e previous e x p e r t e e n t #
tb e question arose as bo whether resting s e l l s would attack
lactic acid*
R a te s o f aerobic d i s s i m i l a t i o n o f glucose#
lactate# s u o o la s t e # e i t r a t e # glycerol* glycogen, e r a b ia o s e
aad acetate are s b e e n in Figure 3* . lactate m s d i e a i a l l a t e d
more r a p i d l y th a n gluoos#* and tb e otter' ecaapouada less
rapidly*
SIyeogea was not attached# arafeiaos# only very
slowly* end e it r & t e m s attacked only after 30 m in u te s*
file
respiratory quotient for glucose was 0*78 compared with. 0*97
i n tte previous e x p e rim en t*
fto® raspiratory q u o t ie n t was lee#
than on® for ©ac& of th e o t h e r compounds except glycerol
w h ich was l*tfe*
A study ©f tiies# results .ana t h o s e o f o t h e r
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
*■ • § . . . *
1000
900
800
700
■o
Consumption
a500
COo Evolution
400
A na er obi c C 0 ~
evolution
200
00
t tg w r *
40
80
U
m u*
120
160
Minutos
200
280
e x jr g tir a p to k * «ad carbon
^tyoXaotloua.
4X m im
c a ll « ©#
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
320
*• 55 ♦
600
COg
La ct ot e
O2
C09
L a c to te
Glucose
500
Glycerol
Glucose
>400
CM
CO 2
Gl yc er ol
COp
Succinate
“ 300
©
Succinate
.2 200
C0 «
100
Acet at e
Acetate
COp
20
Figure i*
40
100
60
Citrate
20
Minutes
Hafces of dlosisiiatiom of various substance®
fey noja-s>*©Ufo**fciag cells of B*.deaetro*
M
eM ibs*
R e p r o d u c e d with p e r m i s s io n of t h e co p y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e rm is s io n .
140
- 54 •
experinexite Indieotee that the substrates ottaeked are lik e ly
broken deea te ee*bea dioxide sad «it*r»
©ysuaids sensitivity ©f feta .fasting ©ells m s d©fe®miii®4
'fey wMMRsrtag a o y g en e o o s u u p t ie a l a th e p rese& ee o f Q*ul#
©.*003,* en d 0*GOG1 H* p e t a e e l u o c y a n id e *
Krehe Cl§5§l was followed*
figure
%*
$b e t e e h a l e ©f
She r e s u lts are presented in
letpfration. m s reduced to 85 per ©out of norml
fey 0 * 0001 1* p et& aslw a e y a a id e mud eom p lefcely sto p p e d by
O.OOi it*
D iffe ren tia tio n from previously described ©rjsariams*
X&ay o f the eb a ra eteria ties of th is erganlaa* p a rticu la rly
the su g a r f e r a e n t & t ie a s and th o s e w h ich d iffe r e n tia te i t
from c l o s e l y r e la te * * e r g a a ie a a h a v e been reeh eek e& a fte r
s e v e r a l swathe*
In a l l eases the p rop erties o f th e c u lt u r e
hove reaaiaed f a ir ly eonstaut* except the rough variant
occurring la the aaiae acid mMwm. described shorn*
la &m
©r two o f the sugar fevnentatloa* there appears to hove
bees, a q u a n tita tiv e change*
1 d escrip tion o f the erganlsat was ca r efu lly ©o»par®d
with those given in Sergey*e Manual o f C@temina.tive
Baeteriology# f i f t h edition. C lift)* -and elsewhere in thelitera tu r e*
la oases vhere Sergey Indicated ©lose relation *
ship or s i a ila r lt y the o r ig in a l lite r a tu r e was cheeked*
Ccunenm and Sety I MM) Iso la ted a large ausber o f
sp©r#*fomiag organisms .and divldea tmm, in to three Oroaps
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
500
300
No
cyanide
Micro liters
of
Oo
consumed
400
20 0
0 . 0 0 0 1 M.
KCn
!00
0 . 0 0 1 M.
0
Plgur®
- l - ^ ~
uasaauu
20
40
60
Minutes
KCn
80
100
Mttmmt of ©yaald® m ©xygoa^opfcajc® of nonpyollfemfeiBg cells of B. dextrolactlcua.
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
120
mk
©a a b a s i s © f t e a i^ r a t u r e r e q u ir e m e n ts Cl) grow th a t 3 7 ° C*
but not SS# C*# IS) growth at 37° 0* aaa
growth at 36° C* 111 not at 37° C m
0*# and (3)
T m second group is the
© n % ©us concerned sad by carefulij ecaparing description* of
B* deatrolac&i eu* cad Cameron and Bsfcys* organisms# differences
m m
apparent in flagellation* growth, ia nutrient broth*
growth in nutrient agar stab* nitrate- p#d»©ti©n# sad fermenta«
tlon reactions*
IOmeter Cid30) described a sp cralatiag la c t ic aeld f anting rod*
i t d iffe r * fro® j§* daatgodLaetlena in si*##
n itr a te reduction# arablnoee# s a i l ©to* lactose# maanltol* and
starch feraanitafcleas* and i s n u tr itiv e requirements*
Borovits**las*©isa and Scwotelaew C1BS8 ) Iso la ted a
sporulatlng la c t ic acid^forming rod which they named facto**
bacllltta aaorcacnca*
th e ir d escrip tion i s so Inadequate th at
comparison i s hardly worth, while.-*
The nata® L actobacillus
fo r a apor«**fora®r i s not In agreement with any accepted
system -of aoosnelatare*
fan* Peterson and Johnson (1940) recently reported
using two species -of spore»foming bacteria for fermentative
production ©f lactic acid*.
They did not describe or classify
their organisms other than stating the one culture m i
Apparently identical with Bemeters organism* and the other
•probably on© ©f €&s#roii .and Saty*a flat sour types***
Of the organ!sms described in the literature# Bacillus
.deatrolactlcua. most closely resembles B# ooagulan# described
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
m-
S f **
fey fiasater (1 9 1 6 ) and fey Sables and Bmmrn {%J6a
T'mm
str a in s o f 1* oeaflEulaas (ausba? it s * 19#* and 100) were ©to**
feataed t m m Br* assa®#** for eostparafelve studies witfe a*
dexfegelaefeloifes and all f*J»r culture* were' subjected to exactly
fell# aaae greirfeii ee&difcl<ms tmeiaiiag w f « » l transfers before
tests m r e amde*
Several distinct differences were found ia
p a ysld ld gleal ebaraefeerisfeiee alt:ia©ttgfe a»rpto#iogieally ttot
organism®
m-m
difference#*
w r y eiatilap*
l&toie X cuaaarlses stoat© o f feb#
l i e sugars' lis t e d « ? # tit# ©aly ones feasted*
ftaty m m selec ted b&e&ua# fell# lite r a tu r e suggested fefearfe ■
Miimmmm® e x is te d
ia febet? fsmeatatioas*
Ofefear dlfferenc##
amy e x is t ia fell© ©as® of ofefear e«rtoelyd»fe«g*
JU dexfer^
la o tlc a c produced a very ieflaife# darkening a ffe c t on fefe#
laoclfeol aediiia ■•till# ill# ofe&er cultures did act*
ffurfetiisr cdsparisoas w©r« sad# fey determining fcnwafea*
felon ra tes in fefe® fellowing. aediuai
aimcos©
Soybean seal
16*0
per cen t
1.0
per cent
0*8 per eeafe
4
0*06 per cent'
,
%ilP04
0*06' per eeafe
f©304
0*006 per cent
CaC03
excess
i% k
.Ib©cu1u».i
feMrd transfer on aoyfeean a#diurn described
above and equal to f 'per- ©safe*
R e p r o d u c e d with p e r m i s s io n of t h e cop y rig h t o w n e r. F u r th e r r e p ro d u c tio n prohibited w ith o u t p e rm is s io n .
S3 *»
I
I
ifc.o
ate
33
33
*4
o
o
«
*'d43- o «i o sj
O
! •I
* «tf . m$-t
iq h ! fQH
syo
Sso
*
•I♦
♦
i
I
a o i>j
U
S Q\
•w -d *-<
•ri © 43 i
^*
5*0>«4* **
I
i
ao «
4
Mf
*3
«N
R e p r o d u c e d with p e r m i s s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e rm is s io n .
-
S9 -
CVI
oc
O
CJ
OJ
ujqoi
Jed
s uj dj s
esoonio
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
60
Incubated a t
-
Cf wife aeration and a g i t a t i o n ?
Sugar
FigUr® 4 shows fet rates o f
was d e ter m in e d pericdically*
f o s n e n t f t t ie a t o be q olto d i f f e r e n t ! &# doatfegolaefelctta bad
utilised all the sugar in IS€ ho ir* w h ile is fee c a s e of
tto© e o a g u la n a strains th e r e
100
m m M to
3 gnuas of sugar per
s&* s t i l l r e a a l a ia g at 6Q0 hoars*
tfaa o rg a n ism i s u n d o u b te d ly s u f f i c i e n t l y d i f f e r e n t
front o th er® w h ich H aw b een d e s c r ib e d s s t o w arran t a sp e c ie ®
d a a lg n a t ie n u n l e s s f e e te n d e n c y t o <. reap e rg a m le* * i n t o
fe w e r s p e c i e s beooaies much g r e a t e r *
proto&bly
0®
p ls o o d I n f e e
m as
In f e a t ©as# I t w ould
s p o o lo s a s c o a g u la a a and
an d eon® ©f th e © flie r s d i s c u s s e d ab ove*
Experim ent® and r e s u l t s *
Malt sprouts &m p r o b a b ly u s e d m ore e x t e n s i v e l y th a n
any o t h e r substance as a s o u r c e of n u t r i e n t in the l a c t i c
fe r m e n ta tio n *
they are an excellent so u r c e of growth pro­
moting material# particularly for some o f the hom oferm entatlw e lactic acid o a c t e r l a whose n u t r i t i o n a l r e q u ir e m e n ts are
exacting as compared with many other o r a n io n s*
The effect of water# alcohol# and ether extracts of
malt sprouts# on acid production by h o m o fe ra te n ta tiv e lactic
acid bacteria in a basal medium Is shown in fable XI*
The
extracts# d e s c r ib e d under methods# were added to give fo u r
c o n c e n tr a t io n s # 0.G5# G.16# 1*0 and 2*S per ©eat but only tb s
optimum is given in the table*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
m '@1 **
fable XI*
bo
effect of various extractm ©f malt sprouts on acid
.production of homofernestafciee l&etio acid bacteria*
i ml* 0*06 ii acid
i per 10 ml* medium
t
Cultures
; A & :L d tt 1
osltion of median.
Basal ■t*
Basal I * .igO extract
i-4
1
1
0*1
0.4
0.3
10.0
10*8
1 9 m2
0.6
2.9
2*9
0.0
0.0
0.0
I 4 alcohol extract
Basal I + ether
I I s B* dextrola&tleua»
I* d • L* m lbraeckii#. L 3a s L*
helveticus*
#
“
Basal I * 1*0 per eeab glucose* 0*3 per cent (Hli^gSO**
per cent HaOAc .and Spe&taan*s inorganic salts*
0*6
T m result® indicate that the water extract contains
the necessary nutritive element® for these organisms.* whereas the other extracts are lackisg la some respect *
It should
to© mentioned that j§* dextrolactieus (A 5 1 i s not nearly as
acid tolerant sus-1»* delbrueekli (I» d) and L* helroticus Cl a),
m um
I M * . - * . ! n « i i M u i w ^ i '.,-'1
■■mm
■ -■ i
r i n 1m i
| - r H r i w u iii
.m m ..
consequently# the titration values are le ss for B* dsxtrolastlcaa (A S) when growth is stopped solely by acid accumula­
tion •
The effect of supplementing the media shown In fable XI
with hydrolysed casein (0*15 per cent) Is m o*
ia fable XXI.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
m
fabi# XXX*
-
E f f e c t of «ujppl<-tu®nfcli\., trie basal mediiwtt plus
e x t r a c t s with aydrolym ed c a s e i n &n acid pro­
duction.
Coaipositioa o f medium
Basal
hyi* casein
s ml. 0*05 ~£ meld
i p e r 10 ml». medium
*
Oxi.3,
i A 5 * ^ ^
^ h
0.0
0*5
0.4
Basal 1 +• Mg® e x t* ■+ hyd* casein
9 .6
19*4
19*1
Basal I+m le* ext* + hyd. casein
d .o
13.4
15.0
Basal 1 + ether ext* + iiyd. easels
§.9
0.9
0.4
I S s B» d e x t r o l a c t i e u s » 39 & » B* d e lb ir a e c k ii* L a * jd*
h e lv e t lc u m .
-------------—
# Basal 1 » 1.0 per cent glucose# 0.3 par cent CRr% ) 2 s®4* 0*§
per m n % M&O&c and Spearman* s inorganic salts.
■ She results t o g e t h e r w it h th o o e in fa b le XI show t h a t
t h e h y d r o ly s e d casein and a l c o h o l extract.# e a ch supply some
n e c e s s a r y am srfentm o f a complememtmry stator®*
the results
also show S. dextrolactieus (A f») t o to© s t im u la t e d toy th e
e t h e r extract i n th e prem enoe of h y d r o ly s e d casein,# while
1* deltormeckll (h d) and 1. h e l v e t i cum (L h) did not mho*
any s i g n i f i c a n t r e sp o n se :.
In fell® n e x t e x p e r im e n t various fractions of th e alcohol
extract o f 'malt sprouts i n th r e e concentrations (2*0, 0.5
and 0 * 0 5 per esntl were t e s t e d in a basal medium containing
Hydrolysed casein (basal 11}*
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T a b le XXII*
iff e e t of v a r io u s fractions o f t b s a l c o h o l
e x t r a c t of salt sprouts on growth ( a c i d pro­
duction) o f hom oferm ent& tive lactic acid
bacteria%
l mS. 0*t55"’?^aeid
t per 10 ml* medium
I
. Culture
l
A 5 * L d « b h
C o m p o sitio n o f medium
Basal. II*
5.1
0 .6
0,5
Basal II + a l e * ext* 2*0$
8 ,7
7 .5
8,7
ct sal
11
ale. ext* 0 * 3 $
9*2
8*0 ■ 8*4
Basal
11
♦ ■ale #xt* 0 * 0 5 $
8*5
0 .8
1.2
Basal 11 + ether sol* ale* ext* 2*0$
5*8
0*8
1*0
Basal 11 + ether sol* ale* ext* 0*5$
5.4
0,8
0.3
4*4
0*8
0.6
4 e t h e r insol* ale* ext* t*0$
10*4
7*4
5 ,1
Basal 11 + e t h e r iaael* ale* ext* 0*5$
0*2
1*2
8*1
Basal 11 + o t h e r iasel* ale* ext* 0 *05$
8*1
0*8
1*2
Basal 11 + Heuberg ppt* 2,0$
8*3
5*6
6,8
Basal II ♦ le u b e r g ppfc* 0,5$
8,3
1*7
4.2
Basal II 4*. Hemberg ppfc* 0*05#
8,5
0 .9
0 ,9
Basal 11 •f Houborg flit rat# .2*0$
15*7
1*0
1.0
Basal 11
10*1
0*9
0.6
6.9
0*8
0 ,5
Basal 11
Basal
U
©titer eel* ale* e x t *
0
ftemberg filtrate 0*5$
Basal .11 + Neuberg filtrate 0*05$
,*05$
A 5 «
cioxtrolactlcusj L d » 1* delbrueeittl.#
h e lv e tie u s *
~~
h
h * 1*
**
* Basal j.1 " 1*0 per cent g lu c o s e # 0 * 3 per cent (M^gSO^,
0*6
per cent laOAe and Speakaan* s inorganic salts* plus
h y d r o ly s e d casein. 0*15 per c e n t*
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
* m •
Tite results show* C D B* dextrolac-telcu-S (A 5} makes
considerable growth on the Basal II medium while B,
delbruecfcll (1 d) and -L* iielwetisugi il h) do not,
tZ)
all
three eulfcures are stimulated by the alcohol extract, trie
ether insoluble fraction, and the ®Neuberg precipitate® but
it is significant that ta eaeh case the growth
m
j,#
delbraackli {£» d) and L« helveticas (I* h) diminishes with
decreasing eoaceatra fcions of the added substances, whereas ,
the growth of B# dsxfcrplacfcicua (A S) reimias relatively eoa*
staat, (5) none of.the cultures was stimulated by the ether
soluble fraction, (4) stimulation of B* aextrolac11eua Cl 5}
by the ®Ssuberg filtrate® Is abnormally high, whereas £..
delbrueckil
(h d)
ana t* helvetlcus (1» h) make no significant
response, and (5) the highest acid production by L, del*
braeekli (1 d) ami X* helvefclctts {I# h) in any case Is less
than half that of the rater extract.(see fable XII), whereas
8* dsxtralac.ticus (A S) acid production is even higher in
mom
eases than it is on the water extract,
fhis experiment
demonstrates some important differences in the nutritive
reqmir@aen.fc®. of these cultures*
Tm
alcohol extract -of malt
sprouts is not sufficient for ..aisxXasua growth of .hueto*
bacillus (£» d and 1* ft), as shown by the water extract but
probably does contain the essential material for B* dgxtro*
lactlcus (A 5) and also lain
> fcory
substances which are not
present in the flIeuberg filtrate®.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
** O S
■«*
T a b le .XI?' shows th e e f f e c t of «aai©aiu» s u l f a t e on a c id
p r o d u c tio n In 'basal
fable XIV*
1
medium plus w a te r e x t r a c t of gluten*
Effect o f ammonium sulfate o a a c i d production by
h o n e fe r o e n ta t lw e lactic a e i d b a c t e r ia *
1 ml* 0.05 S acid
I per 1 0 ml* medium
1
Cultures
I
A 5 t L d * h h
C o m p o sitio n o f medium
.Basal X# *
%0
ext* gluten 2 * 5
$
8,2
15*1
15*5
Basal X less (Sli^igSO^ f rlgG ©xt* gluten
2.bj&
i,X '11*4'" 10*1
B a sa l I + HgO e x t . 0 l u t e n 1*0$
o.u
8*5
4*4
Basal I loss
1#0>
0,2
0.5
2.3
. . . . . . .
04
4*
HgO e x t* gluten
A § 8 8 * d e x t r o l a e t i ems» I* d s 1*. delbmeekil* I* h a I**
helyefious,
«...
—
* Basal I a 1.0 per cent glucose# 0.3 per cent
per cent BaOAc and Spe&kman *a inorganic salts*
0*#
The results i n d i c a t e t h a t these c u l t u r e s all utilise
mmmlvm
low..
s u l f a t e * p a r t i c u l a r l y if the protein n i t r o g e n l a
.
The next e x p e r im e n t (fable X?) was set up t o deteraine
whether the ether extract o f acidified yeast extract pirns
th ia m in ©an replace alcohol extract of m a lt sprouts .and also
th e effect o f riboflavin*.
Sine© t tm "Heuberg f i l t r a t e 11#
w h ich does n o t contain proteins and am ino a c id s * was s o
e f f e c t i v e f o r B* d e x t r o l a e t i o a a (A &)# h y d r o ly s e d c a s e i n
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
«* f t ® «*■
m s o m itte d fram the asdiua of t h i s culture*
tested in f o u r o©ao©ntrations 0*$#, 5*0,grams per'
10
10*0
fttboflstria was
and BO m icr o *
ml* o f medium# b u t sine© it p rod u ced a© s t im u la ­
t i o n (u n d er t h e c o n d i t i o n s o f th e e x p e r im e n t) o n ly one con­
centration is listed*
fable XV#
effect of riboflavin in the presence of th ia m in
ana ether soluble fraction o f feast extract on
acid production by hom oferm eat& tlve 'lactic acid
b a c te r ia *
t
i m l* 0*05 I a c id
i per 10 ml* medium
i
transfers
s
1st * gad
J
C u lt u r e , C o m p o sitio n ©f stediust
lb .
3.®
9,6
Basal 111 (less tr y p to p h a n e ) + % .
1
9*6
9.9
.Basal 111 * hyd* c a s e in
0*5
0*0
uas&l
0*5
0*0
B a s a l 111 ♦ hyd* casein
o*a
0*0
-Basal III + byd* casein + Bg
0*5
0 .0
B a s a l III* (less tr y p to p h a n e )
A 5
T l 'l 'i * * ,
L&
Lh
3g
ill + byd* easela + %
I
riboflavin
B a s a l 1 1 1 .* 1*0 p e r eenfe g lu c o s e * 0 * 5 p e r c e n t (l% )g S % # 0*©
per cent laO&c#'Speakm an’ a in o r g a n ic salts# th ia m in # e t h e r
extract of acidified yeast extract and try p to p h a n e*
A 5 * B« dextrolacticus# I d * 1*. de.ibruecfeil# X h * J*.
h e lv e tic u a .
“
f h e r e a u l t a indicate that 1* ■ d e x tr o la o t.le a s ©an grow on
a medium c o n t a in in g only g lu c o s e * in o r g a n ic salts* e t h e r
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
■urn. @7
e x t r a c t o f a c i d i f i e d y e a s t e x t r a c t and thiamin; r i b o f l a v i n
produced. no s t im u la t io n *
ferol r e a c h e d th e
.Sine© a c i d p r o d u c tio n i n th e eon-*
imatXMxm
t o l e r a t e d by th e c u ltu r e * any
s t i m u l a t i o n w h ich r i b o f l a v i n m ig h t h ave had c o u ld n o t be
m a n ife s te d *
$he medium ©n w h ich jg» d e x t r o l& o t io a s grew
w e l l I s d e f i c i e n t f o r ju* d e lb r u e o k if and J**. h e l v e t l c a a oven,
w it h r ib o f l a v i n * tr y p to p h a n e and h y d r o ly z e d c a s e i n added*
F u r th e r a tte m p ts t o f i a t a s u b s ta n c e w hich w ou ld s tim u la t e
th e l & c t o b a c l l l i l e d t o th e I n v e s t i g a t i o n o f i n d o l e - S - a c e t i c *
a s c o r b ic # t h l o g l y o o i l o * and p a n t o t h e n ic a c id s *
th ese, su b *
stance® , w ere added# s e p a r a t e l y and com bined# t o th e .stediuiB
u se d l a th e p r e c e d in g e x p e rim en t*
fanfcofcheai© a c i d was
u s e d i n t h r e e c o n c e n t r a t io n s } -0 *0011* 0 * 0 6 and
p e r ml* o f -medium*
1 -2
a ic r o g r a ia
A s c o r b ic a c id was s t e r i l i s e d by S e l l s
f i l t r a t i o n and added to- th e s t e r i l i z e d medium*
c o n s is te d o f
1
in o cu lu m
-mm* lo o p from m a lt s p r o u ts c u lt u r e * ’ f a b l e
XVI show s th e c a r r y o v e r i s v e r y s i g n i f i c a n t ©a th e f i r s t
t r a n s f e r b u t i s s u f f i c i e n t l y d e p le t e d ©a th e s e c o n d .o r th ir d #
t h a t g ro w th was c o m p le te ly s to p p e d i n a l l b u t two tu b e s*
One ©f t h e s e s te p p e d on t h e f o u r t h t r a n s f e r *
t h e o t h e r one#
L* h e lv e t ic u a # on th e b a s a l f media®., p l u s c o m b in a tio n { a l l
f o u r t e a t s u b s t a n c e s ) c o n tin u e d th r o u g h s i x more t r a n s f e r s
on th is , sail® medium w ith t i t r a t i o n v a lu e s a s f o l l o w s i
t r a n s f e r 3*9# 3 t h — 4*6#
6*7*
6 fch— 6*5#
4 th
7 th — 4*6# 3 t h — 6 * 8 and 9 t h —
At t h i s p o i n t th e c u l t u r e was t r a n s f e r r e d t o b a s a l IV
medium pirns 19 am ino a c i d s f o r two t r a n s f e r s *
B asal I f
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
:» U fJ «*
fable XVX*
Effect of i n d o l e - 3 - a c e t i c # ascorbic# t h i o g l y c o l i e
ana p a n t o th e n ic acids on acid p r o d u c tio n when
added, to Tbas&i IV medium#
t
t
t
ml* 0 * 0 5 S a c id
t p e r 10 ml.* medium
.
",li
1
T r a n s fe r
j
1 s t t Sad tS r d
*
#
C u lt u r e 1 C o m p o sitio n o f medium
*
1 d
B a s a l ? 4 in d o le 3 a c e t i c
3.*#
0*5
X
Bm * 1 f 4 a s c o r b ic
8*1
1*6
X
sa s & l f 4- t M o g l y e o l i e
7*8
4*7
0*5
B a sa l f + p a n t o t h e n ic 0*05 yttg*
6*8
1*0
X
2*1
0*8
X
2*1
0*3
X
9*6
2*6
X
2*8
0*5
X
3*9
0*5
X
B a sa l ¥ 4 t h i o g l y c o l i e
2*9
0*3
X
B a sa l ¥ + p a s t o t h e a le 0*03 /*g*
2*3
0*5
X
B a s a l ■¥ ♦ p a n t o th e n ic 0*5 A g»
2*9
0 .5
X
Basal 1 4 p a n t o t h e n ic 10 /eg*
3*4
0*5
X
B asal ¥
4*4
2 *6
3*9
B asal ¥
■* p a n t o t h e n ic
©*5 yug*
B a s a l f 4 p a n t o t h e n ic 10
. B a sa l
L.h
¥'*
c o m b in a tio n
B a s a l f + i n d o le 3 a c e t i c
B a sa l
V
♦ assu red ©
4
c o m b in a tio n
x * failed to grow
B&aal
V
* 1 .0 p e r cent Olu c o s e # 0*3 per c e n t Cu':i*
u ,6
per cent WaGAc# SpeaJssaii1® inorganic salts# th ia m in * r i b o ­
f l a v i n # iiy&roljm € c a s e in * try p to p h a n e# ether extract o f
yeast extract*
I d ® X* d e lb r u e c k ll# L h « Ju* helvetieus
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
69
-
c o n sists of .glucose# ammonium su lfate# inorganic sa lts#
ether ex tra ct o f a c id ifie d yeast extract# thiamin and ribo­
flavin*
The amino a cid s and concentration were as follow s*
glycine# alanine# valine# leucine# Iso leu cin e, threonine#
jnefcMonine# asp artic acid# phenylalanine# tr$ptophsn©#.prolin e # aydroxyproliae# h istid in e# angenine and ly sin e 0.01
per ©eat# serine ©#02 per cent# cystin e and tyrosine I *0d5
per cent and glutamic acid 0..02S per ©eat*
Acid production
immediately increased to 8*8 on XQth transfer# and 11*4 ml*
Q*0S I on the- 11th*
The r e su lts show hydr©ly»d casein I s
d e fic ie n t in on# m more amino sle d s which are e s s e n tia l to
the metabolism o f 1* helvefciena (I* h) * The increase In acid
production on the 9th# 10th# and 11th tran sfers p a r a lle l the
d epletion o f the t e s t substances# showing that th e ir addition
to an amino acid medium containing the yeast factor produces
no stim ulation*
Table XV shows that J* doactrolactlcue {AS} grows well
on the basal media® p lu s thiamin and the ether .extract ©f
a c id ifie d yeast extract*
The only uufcaown con stitu en t o f
th is medium Is the yeast factor*
Table XVII shows the in­
fluence o f the ether ex tract o f a c id ifie d yeast extract#
thiamin and rib o fla v in on. Orowth measured by acid production*
Six d iffe r e n t media were .testedt (I) basal X# {2} basal I
plus yeast -factor# (5.) .'basal 1 plus yeast factor plus thiamin
(4) b asal 1 plus y east fa ctor pirns riboflavin# (5) basal 1
plus yeast fa cto r plus thiamin plus .rib oflavin and (6)
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
~ 70 »
basal I plus thiamin plus riboflavin*
are
S ix serial transfer*
1Tea*
The results show that the basal 1 median will not support
growth# and that the yeast factor is i n d i sponsible# and that
either thiamin or rib o fla v in is required but notto&thf. i» each
case# where the vitamins were added separately# -growth m s
equally good-and approximately equal to that when both were
present*
fable JCfll*
Effect ©f thiamin, riboflavin and the ether
extract o f acidified yeast extract on growth o f
8* dextrolacticua as measure© by acid production*
♦
•' m l*
Composition. of medium
15*Wo 1- Soil* per TP5
Si. aSllum"’
9'
:
Transfers
5 1 st i Sad i 3rd i 4th t 5th tr 6th
Basal I*
X
Bassl 1
Ether extract of yeast, ext*
0,4
9*1
6*8
5*6
f\U *#O&
Basal I
Lther extract -of yeast -ext*
10*1
Tuiamln
8*8
9*4
10*1
8*8
10.7
9*1
9*4
8*8
10.1
8*1
10*1
9*9
9*8
10.1
8*1
9*4
11.2
X
Basal I
Ether extract o f yeast ext*
Riboflavin
.basal i
Etaor extract o f yeast ext*
Thiamin
Riboflavin
Basal I
Thiamin
Riboflavin
X
* Basal i - 1 ,0 per cent of glucose# 0*3 per cent Cl-fisJg-SOA# 0*6
per cent M&QAc and Bpeakman*s inorganic salt®,
x * fa ile d to grow*
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
m
71
—
Although th e yeast factor d o e s not con t a i n protein or
main© a olds- as shewn toy the ainhydriii reaction {food# Tatum
load Peterson# 193?) i t was replaced toy b y d ro ly w sd easein
w h ich in turn was replaced toy amino a c i d s # . N in e te e n amino
acids in. combination wmm, at f i r s t u se d bat most ©f the®
wm m -eliminated as
u n n e c e ss a r y *
Table Xflli shows the
effect of r e p l& c ls g bydroly& ed casein w ith .a m in o a c id s *
fable XVIII*
Effect of replacing hydrolysed casein by sain©
acids on acid production toy B* deytrolaofelcua*
acfSf”piF*W" 1 3 7 ”mieE*
juts,
t
t
fist i 2nd s 3rd { 4 th :| 5 t h l 6it
•.
C o m p o sitio n of medium
Basal 1* 4 tulamin 4 hyd*
c a s e in
.
1 0 .2
y*o
10*2
9*4
9«6
Basal I 4 th ia m in 4 c y s t i n e 4
a r g in in e + th r e o n in e 4
glutamic acid ■
•11*2 11*6
9>»4
7*8
6*0
8*0
1 0 ,0
® Basal I * 1*0 per cent glucose, 0*3 p e r cent CMi^lgSO^* 0*fi
, per cent NaOAc and Speakman*.® inorganic s a l t s .
The amino acids which
mem to
be r e q u ir e d , or are at least
beneficial., are cystine, arginine, glutamic acid, and th r e o ­
n in e *
O th ers may; have a b e n e f i c i a l e f f e c t *
In one series
■of experiments methionine appeared t© be necessary*
Aspartic
acid has at times shown some stimulation#
Some amino acids were ■shown t© have am inhibitory effect*
Serine added to & cystine-glutamic acid medium -completely
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
■fg *
stopped growth on. the .f i r s t tran sfer (fab le XIX)*, A second
attempt also failed*
P r o l la e also
nounced inhibitory a ffect*
appeared to Im m a pro­
Although the culture was carried
on the amino acid medium tnrou^ii numerous serial tran sfers
extending omv several months* growth was not consistently
Sob© important factor present in yeast extract* malt
good*-
sprouts and other m aterials is needed fo r the immediate and
maximum activity ©f fche culture*
Biotin* n ic o tin ic acid,
p-alaalne* pantothenic acid* riboflavin* adermiu (Bg)*
pim ellc acid* i- in o s it o l* and u r a c il show no stim ulation
when added separately and c o lle c t iv e ly to the amino acid
medium* fhe potency o f yeast ex tra ct was demonstrated In the
fact that continuous growth was maintained by serial transfer
in a medium containing only the basal medium pirns
grams of yeast, extract- (D ifco) per ml* o f medium*
2
microflais
indicates that the effective substance in the yeast extract
i s inquired only In very small concentrations* probably
severe! thousand times sm aller than the- e ffe c tiv e concen­
tration o f amino a©ids which i t can rep lace*
fa b le XIX*
Effect o f serin# on acid production by Bacillus
d ex tro la ctieu s when added to amino acid'medium*'
Composition of m d lv m ,
{ SI* TJ«yiTH acId per
10 ml. medium
fraSsfer
i
i
I
Basal
4*
ias&l
+
cystine
cystine
+
♦
glutamic acid
glutamic * serine
1
2
t
5**7
5.2
X
X
x * no growth
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
-
7«
. M so u seto n *
Throughout t o i l investigation a c l o s e s i m i l a r i t y b etw een
k*
t t s l v e t i c a s and L* d a lb r u e c k ll was apparent* and at th e seme
tim e these c u l t u r e s showed. a 0 r e a t d i s s i m i l a r i t y to B* dexfcrola c tio n e *
A l l three c u l t u r e s are very much alike in cou »
verting about Si per cent o f g lu c o s e fermented to d e x t r o lactic acid* but a t the saa© tim e their nutritional r e q u ir e *
aeats d em o n stra te a fundamental d i f f e r e n c e *
If it be assum ed
. that the m echanism s o f lactic acid fo r m a tio n are the same in
both cases# then the c o n c lu s io n may be drawn that B* dsxtsd*
factious has a much greater ability than L# d e l b r u e c k ll o r L*
h e l v e t l o u t t o s y n t h e s i s e th e coaipouads which are r e q u ir e d In
m eta b o lism *
In the com m ercial l a c t i c a c i d fe r m e n ta tio n where nutri­
tion is mn .im p ortan t problem yet t o h© a d e q u a te ly solved*
B* d e x t r o la e .t ie u a a p p e a r s to b© much preferred to the other
two c u l t u r e s w hich have been maed i n the past almost exclusive­
ly*
Summary .and c o n c l u s i o n s *
The n u t r i t i o n a l studies o f three cultures o f iaoaoferaten ta ­
t i v e lactic a c id b a c t e r ia # Lactobacillus d e i b r u e e k il CL d ) #
k*
(L hi* and Bacillus
d s x tr o la c tic u a
u n d e rta k en to determine their r e q u ir e m e n ts*
(A
5 1 were
fhe s tu d y has
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
m
74
’
sh o rn that JU dexfcrolaefci c u s is very different
from
two o r g a n ism s*
of nutrients
Malt sprouts are a r ic h m w m
for all thro© species*
©4
w it h water*
th e other
fii© active m a t e r ia l is readily e x t r a c t -
the a l c o h o l extract of m a lt sprouts added to
a basal medium of* g lu c o s e 1 per ©eat, sodium acetate 0*6,
ammonium sulfate 0*5 and Speaioaan1s in o r g a n ic salt mixture#
did n o t support growth of th e ^ l ares unless it was sup­
plemented by h y d r o ly s e d casein*
th e ether extract of m a lt
sprouts aided, to the basal medium c o n t a in in g hydrolysed
cassia# d id not support growth o f L* delbrueckll or
u s but did j§* de-xfcgolacfcte u s *
Tim
1*.
helve tSo­
last organism grew poorly
on the b a s a l medium when 'hydrolysed cassia was added to it#
while 1* d e lt r u e e f c t i ana J*.* helvefcieua did net Orow*
Ml
throe ©rgaoi-aas la the basal-hydrolysed c a s e in medium
mmm
stimulated by the ether insoluble fraction mad the *Beub©rg
p r e c i p i t a t e ” of the alcohol extract of m a lt sprouts*
fta©
•ether soluble fraction o f the alcohol extract did not
s t i m u l a t e growth o f any o f the c u lt u r e s *
T im
®leuberg f i l t r a t e ”
m s e f f e c t i v e for ft* d e x fc r o ia e tlc u a but not for t* d e l b r u e c k li
and 1* h e l v e t i e u e .
Malt sprout# In iu
1ion to nutrients
appear to contain an i n h i b i t o r which is not p r e s e n t in the
"Heuberg filtrate**
In a basal medium c o n t a in in g water extract of gluten a l l
three cultures deaoastrated ability to u t i l i s e ammoniua sul­
fate particularly when the protein, nitrogen was low*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
* fs -
\
jj, d e x fcro la cfcicu s grew w e l l in. th e basal medium con­
t a i n i n g th e e t h e r extract o f acidified yeast extract p lu s
th ia m in s I** d e lh a m e o k ii and L* h e l v e t i c u e failed even though
riboflavin.# tr y to p h a a e and h y d r o ly s e d casein war# also added*
Further work on B* de.xtrol.aotioaa revealed t h a t the
ether soluble factor could be replaced by hydrolyzed casein
and this by
main© acids*
C y s tin e * arginine* m e th io n in e *
g lu t a m ic acid and threonine we r e essential or beneficial*
R ib o f la v in replaced th ia m in * o m
but not both*
or the other was necessary
Certain amino acids appear to have an I n ­
h i b i t o r y effect*
Clrowth of
1
* h e l v e t i e a s was -o b ta in ed in the- basal medium
plus e t h e r extract o f acidified yeast extract# th ia m in #
riboflavin* tr y p to p h a n e *- hydrolyzed casein* and p a n to th e n ic #
ascorbic* t h i o g l y c o l i c and l n d o l e - 5 - a c e t i c acids*
delbrueckll failed la the same medium,
L*
L* lielvetlcus after 9
transfers of poor growth* was transferred t o an amino acid
medium consisting of o&sal* th iam in * riboflavin# th e e t h e r
soluble yeast factor and 19 amino acids*
There was an im­
mediate marked stimulation*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
m f# m
F e r m e n ta tiv e Metabolism o f B a c ill u s d e x t r o l a e t i c u a
in
V a r io u s
m edia
Experiment®' and Eesulfe®
m m&l
MM*Mr
i.i. *1.
as mmm
a so u r c e of n u t r i e n t ,
Lii|ii'i,ntii.i •
.iwmi*«umic . . ii»i;;ii»«"iiiinw
n itw a
■mm m niiirwi'uiiihuhrinrttmnanmum-
The exp erim en t® war# e a r n e d o u t in la r g e Pyrex tube®#
with 130 ml* o f medium u n l e s s ot.a@rwi.se stated* b 's u a lly th e
in ocu lu m for t h e s e e x p e r im e n ts was 7*7 per c e n t (b y volume)
of a 24 h ou r ©ulfcare In a medium similar to that being
te ste d *
fii© in c u b a t io n tem p er a tu r e was 4f0 C, Calcium
c a r b o n a te was used t© keep th e acid n e u t r a l i s e d as it « «
formed*
The c a r b o n a te was kept In s u s p e n s io n either by
a#ration or by m e c h a n ic a l agitation*
Medina*
The medium w hich proved moat satisfactory when
soybean meal was used as a s o u r c e of nutrient is as followst
Soybean m eal
J S f i J L i t IQS .si*
Coro sugar
13*0
Jaaonlua salfat®
u.oO
M oaopot&ssium p h o sp h a te
0*03
JDipotassium p h o sp h a te
0*03
ferrous sulfate
0*003
CaCOg
N
8 ,0
The c o n s t it u e n t ® of the medium were sterilised as
fellowsi
the so y b ea n meal was placed in 33 per cent of th e
R e p r o d u c e d with p e r m i s s io n of t h e co p y rig h t o w n e r. F u r th e r r e p ro d u c tio n prohibited w ith o u t p e rm is s io n .
*
7 f.
water and h e a te d a t 100° C* for 75 minutesj. th e sugar w
s t e r i l i s e d ia 50 per cent ©f the water sad th e soluble salt#
ia th e e t h e r
10
per c e n t ©f th e water a t IS pounds e te e ia
for 20 m in u te * f th e c a lc iu m e a r b e n a te was s t e r i l i s e d dry.
T h ese materials w ere mixed a s e p t i e a l l y while hot# ©©©led t©
€5° C** in o c u la te d # and Im m ed ia tely placed ia the in c u b a to r *
Malt sprouts, 2 t o 5 per cent# f r e q u e n t ly meed as th e
source o f nitrogen am. Oi»owth factors in th e com m ercial pro*
'd u c tio n of lactic acid* proved s a t i s f a c t o r y h » t h a te th e
d is a d v a n ta g e o f im p a r tin g c o n s id e r a b le color to the l i q u o r
w h ich is sot e a s i l y removed i n th e refining p r o c e s s *
Soy*
b ean meal imparts relatively little c o l o r to the medium# h u t
h a s n o t b een u s e d p r e v io u s l y b e c a u se i t f a i l s t o s a t i s f y th e
growth r e q u ir e m e n ts of cultures h e r e t o f o r e used* Under the
c o n d i t i o n s w h ich a r e h ere, o u t l i n e d , how ever# so y b ea n m eal
satisfies th e r e q u ir e m e n ts of jg* dextrolaeticua»
A a e r i e s o f f e r m e n t a t io n s m e c a r r ie d o u t w it h g lu c o s e
c o n c e n t r a t io n s o f 12*5# lo*G# 17*5 and 20*0 g r a n s p e r 10© ml*
The f e r m e n t a tio n s p r o c e e d e d rapidly in a l l cases*
The ©on*
c e n t r a t i o n s o f 12*5 and 15*0 grams per 100 «!..» were c o m p le te *
ly fermented# whereas i n the ether two fe r m e n t a t io n s 15*45
and l b *81 grams of glucose per 100 ml* were fermented when
the tub® c o n t e n t s began to solidify with calcium l a c t a t e
and the e x p e r im e n t was stopped* Sugar c o n c e n t r a t io n s of 15
gram s .per
100
ml* .were u s e d i n t h e s e e x p e r im e n ts b e c a u se a t
h ig h e r c o n c e n tr a t io n s difficulties are e n c o u n te r e d in
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
tm
h a n d lists tbs calcium lactate la t h s refining process*
F u r th e r e x p e r im e n ts showed that IS gram s o f g lu c o s e « f i
f«meafc#d as rapidly w ith
1
gpwtt
o f so y b ea n m eal p e r
ml*
100
as w it h Mftof 'c o n c e n tr a tio n s *
E f f e c t of p h o sp h a te *
Hi© effect of Speakman*-* in o x ^ a n ic
salts - s o lu t io n and som e. of its c o n s t i t u e n t s is shown in
F ig u r e
8*
the p r e s e n c e of phosphate appears to be highly
effective w h ile wnganous sulfate and potassium sulfate have
no s i g n i f i c a n t effect#
The I n f lu e n c e of p h o sp h a te concentre**
fcion on the rate o f fe r m e n ta tio n is shown la Figure §* which
Indicates the o p tim a l p h o sp h a te c o n c e n t r a t io n lies between
0 .2
ash
1*0
gram per liter*
In a su b se q u e n t ex p e rim en t* i n
w h ich th e p h o sp h a te m s v a r ie d toy
0 .1
gram b etw een
and
0*2
0*7 gran per liter* the o p tim a l c o n c e n tr a tio n ' w as found to
toe 0*0 gram per l i t e r (0.5 gram adgFO^ ♦ 0*3 gram JtgMPO^J.
E f f e c t o f ammo.niuia- s u l f a t e *
The effect of ammonium
s u l f a t e on the rate of fe r m e n ta t io n m s .'determ ined toy setting
up a s e r i e s o f fe r m e n ta tio n s * in w h ich t t » c o n c e n t r a t io n s o f
a l l constituents o f th e medium w ere held constant except
ammonias sulfate w h ic h was used in c o n c e n tr a t io n * of
2
*0 * 3 .0 * and 4* 0 grams per liter*
1
*0 *.
F ig u r e 7 shows t h a t the
addition of ammoaitaia sulfate i s e s s e n t i a l for rapid f e r ­
m e n ta tio n ; one gram i a .effective and 3*0 grams appears to
to# optimum*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
a* 7 0 •
O
CM
X
to
Q .
CM
CM
•|UI 001
SUIDJ9
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
*». 8 0 •
CM
L
Q_
CM
X
O
O O
r?
o
X
CD
a .
CM
O
■|UJ 0 0 1
CD
-* 9 d
SLU D Jf
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
m
81 #
CO
CM
cn
—
c m 10
0 0 0 0
CM
o
CM
00
■| l u
ja d
s u jo jg
a s o o n ig
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
o
g f f e o t o f f e r r o u s sulfate and, o t h e r , minerala*
The
t e c h n i c a l c a lc iu m c a r b o n a te meed in t h e s e exp* r im e n ts to
a e a t r a l i s e th e sold eontain©# soate iron and m agnesium and
t r a c e s of son® oilier e le m e n ts as I m p u r it ie s *
T© d e te r m in e
whether say o f these wisr® exerting an e f f e c t * a series of
fermentations m s set up with
p la c in g th e t e c h n ic a l*
0
* f* c a lc iu m c a r b o n a te r e - .
.In e v e r y ©as® th e fe r m e n t a t io n s w ith
the C* P* m a t e r ia l was equal, tenor slightly b e t t e r than
t os© c o n t a in in g th e t e c h n i c a l ^ a d e #
ferrous s u l f a t e
0
$M© a d d i t i o n o f
*«jO6 grams per l i t e r appeared t o be .slightly
fees© f i e l d *
effect o f a e r a t i o n *. Air- m s 'bubbled th rou gh t h e s e
f e r m e n t a t io n s p r im a r ily for th e purpose o f keeping the c a l ­
cium carbonate ia s u s p e n s io n to f a c i l i t a t e the neutral!Eation
of the a c i d as it was form ed*
In e x p e r im e n t to d e te r m in e
th e in f l u e n c e o f a i r c « th e p h y sio l© j.c a l b e h a v io r -of th e
organism r e p e a le d t h a t aeration was w®wy im p o r ta n t in th e
so y b ea n m eal
mMxm*
The p a r t i c u l a r e x p e r im e n t was carried
out i n IfeQO ml.* o f medium i n 2 l i t e r flasks with continuous
m e c h a n ic a l a g i t a t i o n *
Air was p a s s e d through on# f la s k *
n i t r o g e n .g a s th r o u g h a seco n d * carb on d io x i.d e th r o u g h a
third and no gas tu r o u jh t h e f o u r t h flask* F ig u r e
8
shows
th e r e s u l t s o f th e e x p e r im e n t and i n d i c a t e s t h a t oxygen is
essential sine© in its a b s e n c e th e s u g a r was n o t ferm en ted *
The c o n s t r u c t io n of t h e a g i t a t i n g d e -r ic e p r o b a b ly allowed a
small amount o f a i r t o e a t e r th e f l a s k s when g a s was n o t
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
-8 3
-
o
o» +
CVJ
CM
'|ui 001 J*d suiojg;
etoanig
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
. m *
passing through * t o account for th e sudden drop in th e cu r v e
when nitrogen -was s h u t off by mistake*
fix© behavior of oxygen
in this fe r m e n ta tio n i s v e r y pu&zlln^ s i n c e l a c t i c a c id #
w h ich a c c o u n ts for nearly all of the su g a r ferm en ted * Is
n o t o x i d i s e d compared to g lu c o s e #
k
m ic r o s c o p ic e x a m in a tio n
o f the cells in aerated and n©n~&ef&ted fe r m e n tin g media
revealed that is the latter* th e ceils were s n a i l and i r ­
r e g u la r In si2 © while in- the aerated flasks th e y w ere much,
larger and more unifora*
By c e n t r i f u g i n g e q u a l volumes o f
the fermenting »#dius* it- w&s found t h a t th e volum e of
cells in the -aerated medium was four times that- In the nonaerated aediuat*
It should be noted t h a t these remarks apply
only when soybean., m eal is t h e s o l e source of accessory
growth factors* fh© subject o f aeration, w i l l he discussed
f u r t h e r under e x p e r im e n ts on yeast- e x tr a c t* .
S a d -p r o d u c te #
.Several fo.ur liter fermentations were
carried o u t a c c o r d in g t© th e o p tim a l c o n d i t i o n s described
above*,
fa© fe r m e n te d me-dium was analysed for lactic acid,
volatile acids* e t h y l alcohol* a e e t y lm e t h y lc a r h ln e l*
1
*3 *
butylene glycol and s u c c i n i c acid (.fa b le XX).
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
■urn-
T a b le XX*
m-
P r o d u c ts recovered p e r 100 grams o f su g a r ferm ented#
*
*
s
lactic &©14$ tolatil®; A o e ty lm e th -.j 2,3 -Bufcyleae5 EtJ3.fi alcohol
acid j c a r b in e ! f
Ol y c o l
s
s
92.25
0.43
9 3 .4 5
0*91
0 .4 ©
■0 . 2 ©
1*65
0*15
JUl-fi
93.45
95• 94*
All c o n a fc itu e u te o f medium sterilised a t IS pounds steam
p r e s s u r e for 25 m in u te s* Xa fchis fe r m e n ta tio n th e selmtel©
€& + free acid were e q u iv a le n t fc© 9 4 * 0 3 grama o f lactic.
,
s e id *
S u c e l a lc a c i d was n o t d e t e s t e d *
th e t o t a l v o l a t i l e
a c i d p o r t io n was so s m a ll t h a t q u a n t i t a t i v e d e te r m in a tio n of
its individual acids was not u n d ertak en #
however* e e r e
Qualitative tests*
o s l t l v e -fo r acetic meld and n e0a t i v e for fo r m ic
acid* Ethyl a l c o h o l aaousted t o 'but a trace# 0 * 1 5 per c e n t
of th e sugar la one fe r m e n t a tio n while i n another i t was not
detected*
2#5-Butflen@ g l y c o l m i t h e m o st Im p o rta n t product
next t o lactic, acid* Aeetylsethylcarblsol was found la s m a ll
am ounts*
Di&cefcyl was Included ia th e a c e t y lm e t h y lc a r h ln o l*
Some o f the trace s u b s t a n c e s © © earring as end-products may
arise from the soybean, m eal#
With regard t o p r o d u c ts t h i s
fem@ntat.lon differ# tm& the o r d in a r y l a c t i c acid fer m en ta ­
t io n * brought about by species of t t a e t e b a e l l l u s # in t h a t
a o e t y lm e t y lc a r b ln o l and 2 * 3 ~ b u ty le n e glycol are for»©d*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
—8# '**
Sine# volatile a d d occurred o n ly ia traces Class them
1 pm mn%}$
tb e s o l u b l e calcium plus free a d d In th a f e r ­
mented medium can to© e o a a i& ered p r a c t i c a l l y equivalent t o
tiao lactic acid form ed*
fa© soluble calcium determination
was u s e d as a eb eek on th e l e c t i o acid determination*
There
'was ©lea© rngmmamitfe*
. In m m m l
f o m e n t a t i o n s s e r i a l a n a l y s i s sticwed fcimfe
l a c t i c acid fo r m a tio n followed c l o s e l y th e u t i l i s a t i o n of
su g a r ( F l g o r * .9 }*
fiie c o n v e r s io n f a c t o r * a s b t g b a s t a t tb©
en d o f. ted© e x p e r im e n t wben I t r e a e b e d 9 3 * 9 0 p e r c e n t*
fa® type o f l a c t i c a c i d form ed I n soyb ean meal medium
was dexter©
(T a b le .XXIJ*
fable M l *
Type o f l a c t i c acid form ed in various media*
tfedlum
I
■i
d n e la c ta te
i"
HgO o f j n o t a t i o n of
solution s
f %
c r y s t a l - i ~ ”w”"‘ *™ ■
■
t
i iz a tio n
t O bserved %S p e c i f i c
Type
of
a c id
#■
9
♦
Malt sprouts
1 3*13
0*62 l e w
7*75. l e w
d e x tr o
C om gluten
1 3*06
0*€? l e w
8*57 l e w
d& xtro
Soybean, m eal
13*13
0*64 l e w
8*00 l e w
dext r o
f b e type o f acid lias also, bma d e ter m in e d i a co rn gluten
uad m a lt sprouts m ed ia a t different times and in e a c h case
t b e acid was dexter© rotatory*
the c u l t u r e appears t o be
s t a b l e as to fed® ty p e o f a d d . formed*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
• Sf *
O
3
00 O
«*HI
i t
OJ
O
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00
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to
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9 SOOn|9
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
—-88 —
The te m p er a tu r e employed la these
expertaients
(4 7 ° C«)
I s sufficiently high t o p r e v e n t most- of th e c o n ta m in a tio n
l i k e l y to ©eetir In a com m ercial p r o c ea a *
fh© fe r m e n ta tio n r e q u ir e d f o u r t o s i x d a y s . fco fer m en t
X§ gram s o f c o rn su g a r p e r 100 ml*
The r a t e ©£ su g a r u t i l i z a ­
t i o n i s m t m ark ed ly r e ta r d e d h a r in g fcne f i n a l s t a g e s *
Other .sources o f grow th .factors* S te e p water r e s id u e #
com. seal, distillers g r a i n s , m a lt sp r o u ts * hydrolysed
casein pias th ia m in and yeast extract h ave replaced the
s o y b e a n .m eal without r e d e te r m in in g optimal conditions, except
in the case ©£ y e a s t - e x t r a c t which will he ta k e n up later*
The f e r m e n t a tio n s proceeded a t v a r io u s rates*
r e s id u e * 0*6 p e r c e n t , produ ced a
mom
Steep water
r a p id f e r m e n t a tio n
than soybean m ea l while the o t h e r s were s lo w e r j all indicated
possibilities, provided optimal c o n d i t i o n s are determined*
The hydrolyzdd casein e x p e r im e n t is of greatest interest
b e c a u se It most nearly a p p ro a c h e s a s y n t h e t i c medium*
the
c o m sugar was reduced t o 18 grams per 100 ml* and 1*0
m ierogram of th ia m in per 100 ml* o f media® was added.
Hydrolysed c a s e i n mm added in 0*01* 0*1, 0*3, 0,6 and 1,0
per c e n t c o n c e n tr a t io n s *
Inoculum was grown in soybean m eal
medium, but c o n ta in e d insufficient nutrient material t e
significantly influence the f e r m e n t a t io n as indicated, (F ig u r e
10 ) when ©sly 0*01 per ©eat hydrolyzed casein i a present*
The optimal c o n c e n tr a t io n lies b etw een 0*1 .and 0*6 per c e n t*
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
m
89
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V.
3O
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CM
CM
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Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
** H Q <•*
p r o b a b ly near 0*5 sine© t h a t was
c e n t r a t i o n s em ployed*
x xumin
the best
of
the five
mm essential and
co n ­
c o u ld not
tee replaced fey riteoflavin && was t3ae case in the previous
growth factor work*
lliteoflavia p rod u ced m stimulation when
add ed t o t h e & y d ro ly sed c a s e i n M edina*
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
•
8 1 #•
Yeast extract as a e o u r c e o f nutrient*
least e x t r a c t
mm
promoting su b sta n c e #
2
shown to be a potent source of growth,
m ierogm uta per ml* being sufficient
to support c o n tin u o u s transferable growth*
Its effect was
Investigated ia 12 per c e n t sugar fe r m e n ta tio n s *
Numerous fermentations were run to determine o p tim a l
c o n d i t i o n s with 0*4, per c e n t yeast extract-supplying growfch
promoting substances*
the feraentatioaa were run in 100
ml* of medium in 850 ml*
r l nm eyer flasks*
The c o n s t i t u e n t s of th e basal medium w h ich proved m ost
satisfactory are a s followss
M ono*potassium p h o sp h a te
(tea*, per 100 ml*
laaoxiium s u l f a t e
0*20
Calcium carbonate
6*20
Glucose (99 per c e n t pure)
12*00
All c o n s t i t u e n t s of th e medium were- s t e r i l i s e d under
15 pounds steam, pressure for 18 m inutes*,
th e m on o-p otassiu m
p h o sp h a te and ammonium sulfate were sterilised separately in
10 ml* of water and added, a s e p t i c a l l y *
laoculua for each
flask consisted of 7 ml* of a 24 hou r culture in a medium
that did not interfere with th e particular..experiment! or as
was- the case i n m o st of th e fe r m e n ta tio n s * th e cell® were
centrifuged from the yeast extract Medina and r e su sp e n d e d i n
one seventh, the volum e of salt solution ( 0 ,0 5 $ X % f
0
4
* 0*8$
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
«■» §11 m
and
1
ail.* added to ©aeh fl&afc*
fit# fe r m e n t a t io n s were in c u b a te d at 47 to 50° C*
Some
were aerated slowly toy means of a small glass tube 'reaching
to tli@ b ottom of the flask*
Agitation was a c c o m p lish e d toy
a m achine (flat# II) which imparted a whirling motion to t h e
fe r m e n tin g medium thirty s e c o n d s out of ©very s e v e n minutes*
As ia th e so y b ea n m eal fe r m e n ta tio n ammonium sulfate and
mono-potsssiua phosjria&te m m found to to# very Im p ortan t#
« sen 0*4 per e e n t yeast extract m
used ammonium sulfate
was not so essential a s when fractions of yeast e x t r a c t were
used*
la 0*4 per cent yeast extract medium 0*8 grain of
amwenluB sulfate per
100
ml*' o f medium red u ced th e tim e r e *
paired to complete th e fermentation toy 40 per cent*
the
addition of p h o sp h a te was very essential for a rapid and com­
plete fermentation? c o n c e n t r a t io n s of 0*04* 0*05 and 0*06
gran m o n o-p otassiu m p h o sp h a te per 100 al* .gave about the same
result* reducing tbe fermentation time to one-half»' the
p h o sp h a te alone gave a more rapid f e r m e n t a t io n than Sp@ata»anfs
salt mixture which c o n ta in e d th e saw amount o f phosphate and
in addition other inorganic salts*'
the soybean m eal o f the fermentations described in th e
previous section was replaced toy yeast extract in the same
c o n c e n t r a t io n with rather unexpected results (Figure
1 1
)*'
The fermentation was extremely rapid* r e q u ir in g only about
o n e - t h i r d as much tim e to utilize all the sugar as the aerated
soybean, meal fe r m e n ta tio n *
Aeration is not essential*
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
<*• 9 5 . #
Iujqoi
Jsd
s
ujo j
9
esoonio
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
«
m
m
p r o d u c in g o n ly a s m a ll s t i m u l a t i o n .
A d d it io n a l f e r m e n ta t io n s
showed* however# t M t at a lower concentration o f yeast e x tr a c t#
0*4 p e r cent* aeration-Is more s i g n i f i c a n t (F ig u r e Ai}*
flier©
w ere ©aly five n©ura difference feetwees aerated sad non­
a e r a t e d fermentations at 1 per cent yeast ©xtraet* and ab ou t
40 hours d l f f s v s a s * at -0*4 per s e a t yeast extract in tb s
tim e r e q u ir e d t o completely use t b s sugar:*.
From t iie s e fermentations It appears that yeast extract
contains & factor ©r factor# which permit th e fermentation
to c o n tin u e w ith o u t aeration*
further study o f yeast extract
mas made by fractionating t b s material by extraction*
the
-acidified y e a s t extract was extracted first with e th e r # then
with acetous* then with alcohol and finally with water*
fiie
fractions will fee designated as e t n e r s o lu b le # e t h e r in*
s o lu b X e -s e e te n e s o lu b le # e t h e r and a c e tea© i n s o l u b l e - a l c o h o l
s o l u b l e and ©feher* acetone .and alcohol I n s o lu b le - w a t e r
s o l u b l e » respectively*
fhes® fractions
mm
s u b s t i t u t e d c o l l e c t i v e l y and I n d iv id ­
u a l l y for th e yeast extract in the previous e x p e r im e n ts*
The
extract of on© gram of yeast extract s e e added In each e s s e *
Figure 13 shows th e coarse o f the fermentations ( a e r a t e d and
not aerated}- when the- extracts are all eombi&ed*
In Figure
13 -and Figure AS# It Is-.seen t h a t some activity has b een
lost during the 'treatm en t* particularly with the aerated
fe r m e n ta tio n s *
Figure 13 ale# Audi cates that 'the non­
a e r a t e d f o m e n t a t i o n proceeds at a b o u t the same rat# as th e
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
* 95
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* 86 *
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R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
* ©f »
aerated u n til most of til© sugar is utilised* 'but slows up
markedly thereafter*
Figaros 14* 15# 16# aad 1? show, bow the course of for*
mentation Is affected by the various fractions*
la each
ease the carve# for 1 per cent yeast extract are added to
f a c i l i t a t e comparison*
fbe ether s o l u b l e fraction (Figure 14)
appears to be deficient l a ..son s important respects and
aeration bad little or no o f f s e t *
ties (Figure
IS)
the acetone soluble fr&e*
produoed a fairly rapid fermentation when
aerated but considerably slower «r.ben not aerated*
of
the
In spite
fact that the ether and acetone extractions 'had
removed a great deal of m a t e r i a l from th e yeast extract#
the alcohol extract produced the most rapid fermentation
(F ig u r e 1 6 ) .
It most nearly approached the f e r m e n ta t io n s
produced by the combined e x t r a c t s and also t h a t of the whole
yeast extract*
Aeration .mad® a significant difference
although the non«aerated was a fairly rapid fermentation
but slowed up considerably after 60 hours*
ether* a c e t o n e and alcohol
Even after the
e x t r a c t i o n s had been made there
was sufficient material remaining ia the yeast extract
residue to support a fairly rapid fermentation (Figure 1 7 )*
Aeration increased the rat® of fermentation considerably*
these experiments demonstrate’the complexity of yeast
extract and further indicate t h a t the culture is'1
,able to
utilise a number of different substances individually or
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
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R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
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R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
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R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
*
c o lle c t iv e ly *
In no
mm
xm
**
was the hope realised that
mm
f r a c t i o n w ould p rod u ce a rapid fe r m e n t a tio n u n a f f e c t e d fey
aeration for in each e e e e aeration m o m ore effective than
wag the c a s e w ith whole yeast extract.
Sine® .th e ether s o l u b l e f r a c t i o n produ ced th e poorest
fe r m e n ta tio n * few© more fe r m e n t a t io n s
mm
set
up
with all
th e fractions preseat except the ether s o l u b l e fraction.
One was aerated# t h e o t h e r not*
The extracts of 0*4 gr&sr
yeast extract wove used in these two fermentation#.
18 shows
the m r w m
Figaro
for t h e s e f©mentations along with the
carves for two with 0*4 per c e n t whole yeast e x t r a c t *
The
o m is s io n of th e ether s o l u b l e fraction I n c r e a s e d th e rate of
fomentation when aerated but r e t a r d e d the f e r m e n t a t io n
when not a e r a te d #
In t h® nutritional experiments it m s found t h a t B*
d e x t r e l a c t i c u c would not grow on th e basal medio® pirns ■the
ether yeast factor unless thiamin m s added* a finding that
explains why th e fe r m e n ta tio n , was-poor ©a th e ether soluble
fraction.
figure IP shows th e effect of th ia m in on the rate
of fermentation in a medium confeainin*
fraction*
the ether .soluble
Thiamin show# a reaarmbl© effect .and may b@
eonaldem ed t o be essential for a satisfactory fe r m e n ta tio n *
The cells used for inoculum * a lth o u g h c e n t r i f u g e d from
sedi.ua and reeuapem ded in buffer* 'mo doubt contained a e m a il
amount of fchiaaiiii# s u f f i c i e n t for a slow f e r m e n t a t io n .
Thismln
mmrm
in yeast e x t r a c t and would be e x t r a c t e d by
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
-103 -
|U J
001 JSd
S O ID J9
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R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
»
104
■**
aeetom a* aleehaJL# or w a te r tout not by ethyl e t h e r ,
However*
I f til# s e e t o n e extraction e e n p X e ts iy rem oved til# th ia m in
them the alcohol f r a e t l e a and t h e m ater fraction c o n t a in a
sy ste m of .accessory g ro w th factor® which m akes It possible
for the fe r m e n ta tio n t o proceed by sem e o t h e r m echanism *
figure ii shows that a e r a t io n Is n o t effective in in»
c r e a t i n g th e r a t a o f f e r m e n t a tio n u n t i l th ia m in i s added*
T hiam in p r o d u ce s e o n a id e r a h ie s t i m u l a t i o n w ith o u t a e r a t io n #
fch© two t o o t h e r p ro d n ee s t i l l g r e a t e r s t im u la t io n *
P o s s io ly
oxygen acts as .a-hydrogen acceptor in the system only when
th ia m in l a added*
To th e e x t a n t t h a t h yd rogen i s d i v e r t e d
from th e pyruvic a a id * the yield of lactic a e l d is red u eed *
T h is agrees with the lactic a e l d d e t e r m in a t io n s w h ic h show
that aeration in e a c h c a se lo w e r e d t h e yield o f lactic a c id
(T a b le X&XX) *
T h is r e a so n in g , i s # how ever# c o n fu s e d by th e
such r e d u c e d l a c t i c acid yields obtained, when just the ethersoluble fraction was present and no thiaaia*
The addition
of thiamin t o the medium c o n t a in in g th e ether soluble fraction,
resulted i n i n c r e a s i n g both th e rat® of f e r m e n ta t io n and the
yield of l a c t i e acid*
It therefore appears t h a t thiamin
f u n c t io n s , in th e fo r m a tio n o f lactic acid# aerobically or
a n a e r e b le a ll? *
On© of t h e effects of aeration may ho due to its action
on inhibitory substances*
This « i d em o n stra to d by a d d in g
10 m illig r a m s of cysteln® to 100 m l, medium containing e t h e r
soluble fraction of yeast and th ia m in #
Figure SO shows, that
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
* lO i *
|iu 001
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R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
* lo t *
fable XXII*
field o f lactic acid i n aerated and n c n ^ a e r a te d
fermentations* e x p r e s s e d a s grams p e r 100 grams
o f g lu c o s e fe r m e n te d .
t field of l&etie sc M
C o m p o sitio n o f medium
,|l©t aerated • Aerated
Basal* • yeast extract (0.4£)
08*7
87*8
Basal «* combined extracts
04*8
88,8
Basal - ether soluble fraction
63*3
78.6
06*8
88.6
acetone soluble fraction
87*8.
84*2
Basal • alcohol soluble fraction
$2*8
88*0'
Basal » ether soluble fraction ** .
thiamin
B asal
# B a s a l * iHH*}gS04 0.3 graa# SHgPO. 0*03 g f m , CaC05 6 » 2
gram s and g lu c o s e 12*0 .grams p e r 100 m l.
in tUt noB»a#rat#d fermentations the cysteine had a definite
i n h i b i t o r y a c t i o n 'which was absent with aeration#
T here may
he otrier substances more ©r less i n h ib it o r y - which are similar­
ly affected by
aeration#
R e p r o d u c e d with p e r m i s s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e rm is s io n .
CM
XJ
X
OJ
. '|W00l -Od SUJDJ9
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
108 4**
Smasaary and C w c lm s ic a s
fis® optimal c o n d i t i o n s f o r lactic acid fcmabicn
by B*
Several scarce® o f
dextrciaetlcms w ere investigated*
o n tf& o a t* a i o e r a l r o q u ir o a o o to * and othosr e a v ir e n a u ffita l eo n *
diblcn® have been s tu d ie d *
soybean meal Cl gram por 100 a l* } was feu&d to s a t is f y
the nutritional rogttlroawiito of the emltnr© far a IS per
cent glaoooo fonumtatloa when 0*06 £#** phosphate* and 0*3
gram
mmmm.Ixm
addition
of
oalfoto were added p e r 100 ml. aodiutt*
th e
these inorganic eoootltoonto was vital to '
■
its© fermentation*
Aeration was f c m d to bo very ©seentiai*
fh© prooooo converted IS par oa&t glucose solution to t o x t r o Xaetle acid to four t© five toys with a yield ©f i t 'to 94
per coat*
Ottor produoto -ooeurriag la small awouato ©r
traces wens t,*S*femtyl©a© glycol, aeetylaethj&eerblJael* acetic
acid and ethyl a lc o h o l*
Steep water r e e id a e # corn meal* distill#**© grains, w a it
©promts and hydrolysed cassia pirns t h la w la were a u b e t it u t a d
in t h e
more
for soybean m e a l.
Steep water r e s id u e gave a
rapid f ©Mentation t Mm soybean a e e l *
stamms
g « v e a lo w o r fe n u e u te tio B ft*
gram pits.® 1 w ie r o g r e a t h l e n i n
par 100 mX*
of
t h e o t h o r «***
SgrdroXysed casein 0.3
fmmmmtsA
Ig greats o f a u g er
»diast in 124 h o a rs*
least extract was fo u n d t o be an extraordinary oou roo
o f nutrient*
* h ea 1 per cent yeast extract was substituted
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
** 109 ••
In the medium for soybean meal only one-third m saieh time
was r e q u ir e d t o c o m p le te th e fe r m e n ta tio n # end a e r a t i o n was
foted to have but little ©ffe©t*
At 0*4 per ©eat yeast extract
the effect o f aeration m s more significant*
feast extract was fractionated by auecessive extraction
with ©the?* acetone* alcohol and water*
Bach of the f r a c t i o n s
supplied sufficient nutrient for a j>ou f©»©ntatioa except
in the ease of the ether -soluble fraction which required the
addition of thiamin#
it*
The other fractions probably contained
Aeration ©f the fermentation m s more necessary'with
each of the fractions than with the whole yeast extract*
combined fractions g i n
Tim
a more rapid fermentation when the
ether soluble fraction was omitted*
The yield of lactic acid was somewhat lower in aerated
asdic#
The addition, of. thiamin to a sedluat containing the
other soluble fraction increased the yield of lactic a c id
from 72.5 to 88*0 per c e n t in the aerated and 8 5 * 5 to 9§*6
per cent In the aoa-aevatetiU ,Thlemln appears, to be an
essential in the formation of lactic acid*
Aeration of the fomenting sadiaai has a nullifying
effect on the inhibitory action of cysteine «uad possibly other
substances*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
—
n
o
—
klTSSATWtfa CITED
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1940.* Description of a tiextro-lactic acid forming
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"
Andersen, A* A*, I. 0. Wood and C* ft* Itrtaaa*
1938* tolas acid r@fmir#®»ats of the lactic acid
bacteria* J» Baet* 36* 80S* (Abstract}»
Sergey, D* a*, It* S* Breed*, E* 0* D. lurry and A. f* Hitchens#
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Blondeau, 0.
1847, Dee fermentations*
244-261.
i» ph&ra* efclau (3) 12,
Brockman, It* C* and C. H* Workman*
1933. Determination of 2,3-butylene glycol in fer­
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Burrows, W*
%SMm
The nutritional
<$tart* lev* Biol, 11, 408-424*
Burton, L. ?*
1937. By-products of milk.
575.
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198i* fhe examination of spoiled canned foods,
Infectious Diseases* 89, 89-105,
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Demeter, J,
1930* Sin Beitr&g sur Seantttls der for- die Sraskonservitag. (wararverearing) wiehtlgen Microorganism* Ceatr.
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R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
- 1,11
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351-355,
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Knight* 8* C * 9* (i*
1956* Bacterial nutritionf material for a ©ompar&blvs
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■ 1936. Accessory growth factors for bacteria and related
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K rebs# a . A .
1955, M etab olism of amino acids. III* Peam in& tiott ©f
amino-acids. B ioch em , 1, 2 9 * 1620-1644*
Leifson# Einar*
1938, Staining of bacterial flagella,
658. (Abstract)*
1* Baet,> §6# -
Lister# Joseph
1878. 6a the la©tie fomentation* and its bearing on
p a th o lo g y * Trans* P ath# So©* London. 29* 425-467.
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
— 112 —
Meyerhof* 0.,,and K* Loiunan*
u b er den Unfce r sc h ie d . von d - unel l-M llc h s fiu r e £&$'
und X o h le h y d ra tsy n th es© im orgaaisaaia* Sloehem *
2,,171, 421-455.. .
1926#
Atmx&£
M i& celson* 1# and C. H* wertama*
1938# I n f lu e n c e of pH on the dissimilation of g lu c o s e
by A erobaefcer In d b lo ^ en ee#
Baet*? 58# 6 7 -7 6 #
M d ller* S« F .
1938# V ita m in Bg f Ademla) .ala fu c h s fc o ff fur
M ilc h a a u r e b a k te r le n # %
% phyml e i * Cham# 254# 285-288.#
1x5H e r , K. F*
1940* M ahrsfcoffe und Wuclisstoff® del* l& Xchs& ureb& kterlea
Aagew* Chem. 5 5 # 2 0 4 - 2 0 9 .
Munson, L. s. and 1. H. Walker*
1906# The unification of reducing sugar methods*
Am* Chem.# So©* 28* 665-686#
J#
n e ls o n * «* &*
1956* .Physiology ©£ the l a c t i c acid bacteria* Un­
published R u »* thesis# library, tom, State College.
B eu b erg , C a r l and l e t m a # s E e r b ,
1912* tber e i n Fallungsaittel fur Aainos&urea*
Biochea Seife# 40* 498-512*
lorri#, «T. f*.
1924* experimental organic eheaistrf* .2nd e d .
H i l l Book Co** New Xork*
MeSmt-
Olive, T* 1.
1936* Waste lactose is raw m a t e r ia l for a new lactic
acid process* Cham* and let* lag*. 43, 480-483*
Grla-Jensen, S.
1919. The lactic acid 'bacteria* X* fieaeke fidenskab.
Selskab, Skrifter* laturvidenskab* Math* Afdel* (8),
8* 81-198.
Orla-Jensen, S.* 1. C. Otfce and .Agnete 8aog-SJaer*
1956* .Oer Vi1 su sia h e d a r f der M lle b e & a r e b a ltte r le n , C entr*
Bakt# I I , 9i, 454-447.
Oebtum, 0# I * , H. Qm Wood and C, H# Workman*
1935* Determination o f formic# acetic and propionic
acids in a mixture. lad. tog. Chem** Anal* Ed. 6#
247-250*
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
- 13.3 Pan, S. C., */, H. P e te r s o n and M arvin J, J o h n son .
1940. Acceleration oi lactic acid fermentation fey h®atlatile substances. Ind. Eng, Chem* 3 2 , 7 0 9 -7 1 4 *
P a s t e u r , Lemi|*
1 8 5 7 , Mmm&im stir l a -fe r m e n ta tio n a p p ell© l a e t i q a e *
Compt* read* mead* s e t ? 4 5 , 915-916*
Freseob, c . and 0, §* JDunn*
1940. industrial aticr©biology*
lew fork.
McGmw-Hill Book Ce»#
S a r ie s * W. B , and 1 . » • Sanaa**
1932* Observations os- Bacillus co^ulans*
23, 301-314. .
J. Baet*
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1780. On milk and its acid. The collected papers of
Carl ftilh e lm S c h e e le • Translate© by L eonard D ob b in ,
p p . 2 1 5 -2 2 0 . Q* Sell and Sons L td * , London ( 1 9 3 1 ) ,
Snell, u* l. and i. H, Peterson*
1940* arowth f a c t o r s for bacteria* X* Additional,
f a c t o r s r e q u ir e d by certain, lactic acid bacteria*. J .
B & et. 3 9 , 2 7 3 - 2 8 5 .
Snell, L. L,, P* M, Strong and 9* 1* Peterson*
1939. Crowfeh factors for bacteria. VIII. Pantothenic
and nicotinic acids as essential growth factors for
lactic and propionic acid bacteria* J, Sact* 38,
2 9 9 -3 0 8 *
.
Snell, B* &*, 1. L* T&tum and W* E* Pefceraoa*
1937. Growth factors for bacteria. III. Some nutritive
r e q u ir e m e n ts o f L a c t o b a c il l u s delbrueiiii, J, Beet* 33,
207-225*
' “
' !
~
Society of American Bacteriologist#,. Com m ittee on. Bacteriologi­
cal Tedmic.
1 9 3 9 , Manual of metnods f o r pur© c u l t u r e study of
bacteria* Biotech P u b l i c a t i o n s , Otnem, S* f*
Speakinan, H. B.
1 9 2 3 . M o le c u la r c o n f ig u r a t io n i n th e s u g a r s and a c id
production fey Bacillus p e c t la o v a r u a , 3* B i o l . Chem.
5 8 , 395-413*
“~
Sfc&hly, a. L, and e* 1* Werteman*
1236* Defeeratsatlon of a e e ty lm e t h y le a r f e in o l in fe r m e n t* tion liquors* Iowa State Coll* <1*. S e l* 10, 205-211,
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
— 114
Stiles# H. 1 * , W. H. Peterson and E . B. F re d .
1926* A rapid m ethod for the d e te r m in a tio n of su gar
in b a c t e r i a l cultures* J* B&et* 1 2 , 4 2 7 -4 5 9 .
S t i l e s , I* K. and 1. M. B ru ess*
1 9 3 8 . M u trien t r e q u ir e m e n ts o f 1* d e l b r u e k l i i n th e
l a c t i c a c i d fe r m e n ta tio n of molasses* 1* Bant*, 3$.#
149-155*
tabu®, S* L, and W* S., Peterson*
1935* F e r m e n ta tio n m ethod for production of d e x t r e lactic acid* la d * Sag* Chem, 27, 1493-1494*
Wsksaiaa, S. A . and I . J* H utching*
1957* L a c t ic a c i a p r o d u c tio n by species of
J* Am* Chem* S o c . 5 9 , 5 4 5 -5 4 7 *
Ward, G. &«, L . B. Lockwood, B* T ahenkin and J?« A* Well*
1936* S a p id fe r m e n t a tio n p r o c e s s f o r d e x t r o l a e t i e
a c id * I n d . Eng* Chem, 5 0 , 1 2 3 5 -1 2 3 5 *
Workman, C , H. and A* A* A ndersen*
1958* d -L & c tic a c i d fermentation*
J* B&&t* 55, 69-70*
Whittier, E. 0* and L* A* E ogers*
1 9 3 1 . C oxitinuous fe r m e n t a tio n i n th e p r o d u c tio n o f
lactic acid* lad* Eng. Cham*jg§* 632-534*
Wood, fi* 6v* A* A* A ndersen an d C . H. Wsricmaa*
1 9 3 7 . Growth f a c t o r s for propionic and l a c t i c acid
b a c t e r ia * F ree* Soc* laeptl* Biol, Med* jgg>, 217-219*
food, H. G*, A. A. Andersen and C* X* Werkmen,
IS IS * n u t r i t i o n o f th e p r o p io n ic a c i d b a c t e r i a *
B eet* 3 6 , 2 0 1 —214*
1.
wood, H* G*, 1* L. fatam and f, ii* Peterson*
1 S 3 7 . Growth f a c t o r s f o r b a c t e r i a , IV . An a c i d i c
e t h e r - s o l u b l e f a c t o r e s s e n t i a l f o r growth o f p r o p io n ic
s o l d b a c t e r i a . J* B& ct. 3 3 , 2 2 7 -2 4 2 *
W orts# A, and. G* Pried®!. (>
1861, Bat«rsael»ngen u b er di® iilchafbr#*
Phara. 43, 369-575*
Ann, c h e iu
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
•- 115 -
The
to jDr*
a u th o r
C*
vl9h.ee
t o e x p r e s s & i# s in c e ? * a p p r e c ia t io n
S* Wexttanan f o r til® r a ln a b le s u g g e s t io n s and helpful
c r itic is m s *
A p p r e c ia tio n is a l s o expressed t© © the? members
o f th e d ep artm en t f o r t b e t r f r i e n d l y c o o p e r a tio n d u r in g th e
mm>m
o f iM s in v e s tig a tio n *
R e p r o d u c e d with p e r m is s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e r m is s io n .
P lat© f ♦ F to ta m lera g a a p lu i ©f S* _ _ _ _ _ _ _ _
Fig©*© 1* M alt agar; 48 hours at 47° C*.| tram ©tain#
Figa** t#
FLagaUa stained by g*lfaoa*s ®©fei»d*
Figure S*
..figure 4 .
Spores stained by D o m e ? 1a metbad*
latrient agar; 24 h ou rs# 4f® &j Grain ©tain*
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
—-117
Plat© 11,
Agitating aaaMaa
R e p r o d u c e d with p e r m i s s io n of t h e cop y rig h t o w n e r. F u r th e r r e p r o d u c tio n prohibited w ith o u t p e rm is s io n .
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