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HTHS

THE USE OF CLOSTRIDIUM PERFMNGENS»
{596K FOR ANALYZING CASE!!!

FOR ESOLEUCINE, LEUCWEJflET’HiONINE
FHENYLALANINE, AND ‘V'ALENE

“Silesia éor i‘he Dames an“ M. S.
Mi’CHfiGAN STATE COLLEGE
3979mm Eflm Gramsmifk
“€949

THE'ilS

This is to eertitg that the
thesis entitled
The Use of Clostridium Eerfringens Blfifi
for Analyzing Casein for Isoleucine, Leucine,

Methionine, lhenylalanine, and Valjne

presented an

Roberta Ellen Greensmith

has been accepted tnwarcls fulfillment

01 the requirements for

__.Ji.54_._.degrce hIJKUIJzitiLnl

  
    

lainr lvrnleswr

Date June 6; 191+?

 

In US! 0?
CLOSED” Pmmm max
FOR ANALYZING CASE]! FOR
180mm, mucm, 1313103133
W133, AID VALUE

. _ -_ 3! .. .,
Robert. 111qu Egon-nth

,, , A mm
anmttoa to the 8011001 of Giant. Studio. of menu-m
State College of Agriculture and Ap‘pnod‘”3'o”10noo
in panel {unlawfbf the ”quiz-amt.
‘ fer tho decree of
mm or game:

Depart-ant of foods on! nutrition
School of non locum”

19 49

”MOM

1110 11:}th 1131139 tovoxprosg hogepgrgeption ”-333
figment A. 01119911 for hog aggqtionl gag guidance,
phDrg W. 20.3.“?! fer his helpful: the lfilatgotgoal
39317-1! 93 the “5??! agd___t_o It.“ Ruth man: for
her inn-oat and «comment.

218322

TABLE or CONTENTS

Page
monvcrxon..................................,..... 1
REVIEW or m me.. 4
mmnmmx. mmmm.... .9
mum's mo DISCUSSION.............................. 14
SW um coucmsxons............................. 2:;
LITERATURE c1m.................................... 24
APPENDIxs........................................... 31

.O‘QQ‘O'IQVQA

wo‘s‘quo

limbo:

II

III

TABLES

. .. e. .. T310 ..,

Innatieutgrs Who £3.79 Used 1.0th
bacilgtug Arabinoaug 17-5ufor tyo
_Ang1y§13 affix-ions. Amigo Acme.

The Igoloncine.‘ Ifoucine, Methionine,
Phorwgalanigelepd Vagina Content
0.: cmzn £8. 369mm by new: ..
b.0111” Argentina 17-5 and electri-
diul'anerfz-irgono 32965, _ _‘ ‘
ll' V4991 Obtggngd encompariun or
Pergeptacea of Each Ming Acid .. h
Insured bypaptobacilgtug Arqbglgoano $
. ”1.7-5 end Cloutgiggtm Partyinzenp BPGI.
Percentggc Recoveries of Added Amino

Acids.

Page

15

18

20

MODUOT ION

MODUOTION

-. Microbiologiool nethodo for the eotinetion of
enino eeido here hooone inportent enelytioel toolo dn
m #1914 0:. mteély mum. mummy thy-9
mm..- in women; outxitéenéu 999° 1909-11310. 9
noro deteiled knowledge of enino eoid eooindletion end
netnholion. Ihgn niorobiolocioel nethodo_ ere r .
properly warned out end oerei‘nlly oontrolled, they ere
nepetle of yielding r‘eonlto. thigh oonpere‘ teyornbdy in
eoonreoy with thooe from the boot ohuioel methodo
(Snell. 1945”). a .. - .., ..,. ..

no edrento‘no _end_ linitetieno of nioro-

' hdolocioel nethodo here )een dioonoood by Snell (194§)
and P! scenimtw 89911...-(19‘37lt. A11 31" retiree:-
dre oiniler‘in prododereh yell onited to routine nee,
end donnnd only the ordinnry le‘egrntoryl ”Ellfltgt.
inoy 9’93!” opeeifiohendvnnuondelly oenoitivo.
we? mew?“ 91'. the 91‘9’6019 parrot» 0.! the mole
norwoggeretion of. the pinged“! gtermhydrolyodo ie
neeeooery, daemletevknoyrledgewod the. datinnte . ~
nutrition! the ngtdbglign. nnd'the edeptntion dyildtieo.

Lupe-«I-

of thgteot orgnniono do thenootwinpo'rtent diodd'ventege
of theoe nethodo. _ rho inhibitire effect of the _
unneturel ioonero of the nnino eoido nay introduoe
oeriono error (Prooeott, et el" 1949; romeohi. nine.
end roe, 1948; nine end For. 1945; Ion, nine. end

a“

40

I.

<.‘

IO

ID

Bollonboek. 1944).

."|' o.

_. y The leetie eoid beetorie ere the noot widely M
need grono of orceniono for niorobiologieol doterninetieno
et the preoonth tine.“ . ydotorioellyd they were enonz the
firotb 9393991.“ bo eppliod to eoou prooednreo.._ 5‘
booenoo nere woo hum of their nutritionel reouironente
then ofwow other group. Snell (1948), ettribnteo the
nidoopredd neg of tho leotio eoid beetorie to "the
emlex netnro of thedr nutritdonelhroqnironento, their
rngud netnro! end the feet that their gnonth eonld be
eeoily followed by tnrbiddnetrie neenohor by titration
of the leotio ooid produced dnrdng gronth', The
greeniono neot amenity and ere iootobeoillno r ..
erebinoouo 17-5, Lootoboeillne eeoei he-l. Leotobeoilluo
fer-enti :6, streptooooono feeoelio. end Leuoonootoo
neoenteroidoo.

nu" Iv L4 . l- ..._ I... M

._ - Other arseniono have been onggeotod for

ooooy purpooeo. “Lenoinelooo' nutento of lonroopore
ereooe here teen need for the eotinotion of lenoine ' d

by non end Bread (1944) no by Hodoon end Krueger (1947).
Door-eon $1945) oloo heo__no_od e notent of lonroopore

for the__utinetden of 'lyoine, “Tryptophene heo been
ootinetod by neono of Iberthelle typheoo T 63 by“ d .
'ooley end Sebroll (1945), hooheriohie eoli hee been
reoongnded (Roepke, Libbey. end Snell, 1944), but

deteilo of the nethod need here not yet oppoered.

F\

-3—
-- - n nierobiolecieal pnoeednro for ooeu or . ‘
ominohooidemnith Oleetridinn pontnmene. ES! hoe been
published guenggyjnoye, men. ondvatel‘l. 1348),
It ie the pngpgeo of thio etndy_to «were thin non
oethod nith o etondord nethod noino Lootoboeillue‘
erobinoeno, 17-5 byvnnolgeino ooeein for ieolenoine,
lenoino, methionine. phenylelonine, ond noline.

~\
.
H .
A
V ‘\
, .
n . .
fi\
. n
r
fl~ o
. t
m
C
. _
a\ . , .

 

REVIEW OF THILIW

REVIEW or m LIME

.. The Tirot opplieetion of lootio ooid booterio
to qnontitotive eeeey nork nee node by Snell end strono
(1939) Tent!” eotinotion of riboflevin nithnl’looto- '
booillno eooei. loot of the booie geehnionoewupleyod
in ell niorobieleoieel determinetieno ted” ere derived
Tron thio eorly Vito-in eeou method. m ..

_ One of the Tiret oiorobioleoieel nethodo
for themdo‘torninotion or onino oeido utilised Looto-y
boeillno orobineono 17-5 (Bhenhen. Donn, and Ruby!
1945),,“ lneh of the pienoerwnogk‘on oninowooid eoeoy
n5. 5...; done an: Lootobeeillno nrobinoono nnd it
ie non one of fho moot oomonly need ‘- lootio ooig
booterio Tor“ ooeoy pnooednreo.” A Tootoboeillno
erobinoone oethod hoe been ehoeen oo thewetondord
method in thin etndy. The follonino dieenooion,
therefore. hoe been limited to thooo refereneeo
pertoinino to the development end one of niorebielooioel
proeednreo ntilieino Lootobooillno orobinoono end the
Oleotridinn perfrinoene nethod beino teeted.

Snell end Trioht (l94_l) nere the Tirot nee-hero
to onbenltnre Lootobooillne orebinoone onooeeofnlly on“.
o eonpletely enthetie nedinn (o oixtnre of onine ooide
oe the solo eonree ”of nitrooen). . __

The ootnol nnTno oeid requiruento oT Loote-

o—v

boeillno orobinoene hone been determined independently

-5-
by Shannon (1943). Iniken et el. (mob), Heooted (1944)
end Dunn et. cannon). Allhthenorkore ooree on the. ’
eeoentiel notnre of olntonie oeid, ieoleneiney lonoine,
tryptopheno, .- end yolino To; orenthoi’ the eroenion end
on the nonaoeentiolnetnre oi’ olyoine, Thydroryprolino.‘
39110391809 end norooline. The lotter. homer, n_e_re._\
deolored to be-ooooooery To; optinel oronth, There ie
dieeoreuoent bntnoen workere oentohnhetheruoroinine.
lyoine, phowlelonine, threonine, end tyreoine ore
eeoentiel er oeoeooory oronth foetoro. .‘
Mo Toilnre to ooreo completely hoe been ,

ottnibnten tenth inpnrity of eooo omereiel eonroee
of onino oeido (Heoeted end Iordnell. 1944), the:
oonpooition of nno'nedim (Briekoon et‘ elullueg
stokeo end Gunneoe'. 1945), and thowpoeeibility of
veriotion inutheteet- oroenien itoelf. Donn ot ol.
(1947) hoe denonetroted qnontitotive dittorenoeo inuooid
prednetion by oi: enltnreo. oT Lnotoboeillno erebineene.

_ It ie oenorelly eeennod in microbiolooioel
eeonyo thet only the I. Torn oT the onino oeido ore >1
utilized by The toot oroenieno. Initen et o1. (1943»
1943b) hove onbnitted proof thot only the_ notnrel
ieonere of iooleneine, lenoine, oln-tongenooid. lyoine.
end yeline ore ogtivefor Loefobgnlm orobinoono.
stereo end Gnnneoe (1944) hove roported thot tho

notnrolly oeonrrino enentionorpho of threonine end

r?

I‘

I"

In

.6...
nethionine ore the only onee o‘rnilnble to Loetobooillue
erobinoone. Although Heoeted (1945) found oome
oetivity for the 2D tom of leuoine, ieoleueine, ond__
noline, he concluded thot 3.11% error oonoed by the neo-
of D1. onino with no etonderde in ordinary ooeeyo would
be onell.

In niorobiolooiool eeoey procedure” totol
oronth permitted byfloi'ren oonoentrotiono of the
eeoentiel onbotenoeo io honoured rather then oonporotiye
at.» 9.1“-m'fcho .,-D°F0=n.1m§19n'. 9.! 19mm cram
otter prolonged inoubetion hovo been found tofiyi'eld”
more relioble reoulto thou doterninetieuo node beifore
growth hoe gone'toHoonplotion__(8noll.~“19455). The
ineybetionjyaigé 3.111011. '11; e110! 3h: marinara...
poooible reoponee of Loetobeoilluo_orobinoouohond H”
other- lootiowooid bootorio ie 72 119“}?! Thie. Three
dey period ie o dioodnentooe ”in the prnotiool oppli-
oetion oT Lootobooillne orebinoeue nethode. for it T ...
inpooee a limit on the number of deterninatione thet eon
be perTorned routinely end thuo inereoeee the time
neoeeoory to complete o oinen enolyeioo . m .. __

Table T sumorioee the onino oeido nhioh hn'g'e

0......-

been determined nith Lootobooilluo orebinoeue by variouo

norkero.

T9910 F .N .-
vlnneotigntpro Th9 {Into Used ..
Looto‘oooillno Arnpinogno. 17-1-5 for the

Analysis of Various Amino Aoido

In.

a v

 

Amino Acids

- Innestigntors

 

Glntnmio noid

.n

Dunni'et nl. "(1944
WY“ n1;‘(194 )
Lewis on'd Oloott 1945)
EEO, ' at al‘. . (1945
Bier; ““31. (19-4 )
Bomgort'en.‘ Mother, and Stone (1945)
Henderson ond. Snell (1948)

“H v ' -...

 

Iool eno ine

Inih‘en,‘ offal. (1943b) ..... ”
Soh’wei'gert,'1'ntmon nnd Evehjem (1945)
H'ier. ‘et"'e.l. (1945 " " “ .
Bnimgnrten, Stone”; and Burnt! (1945)
Barton Wright (1946 )

 

Leno ine

Knik’en," 'et‘ o1. (1943b) " "
Sohwei‘gert.‘et nl. (1944)
Bier, 'et‘nl. (1945) **
Bounénrten, Stone. and Burnt!
Barton Wright" (1946)
Onmi’en'nnd Dunn (1948) ' *
Henderson and Snell (1948)

(1945)

 

lethionine

Dunn; et' ‘31; (1946) ~
Earn." Jone-g" nnd‘ 3111110946) '* "
Rieoen, Sehneigert, and mvehjon (1946)

 

Phenylolnnine

- up...

Hogat’ed'(l944) '
Hendorson nnd Snell (1948)

 

TryptOphnne

Greene nnd'IBl'nok“('l944) "
Sohwsig'ert, Snnberlioh, and Elvehden (1945)
Din—1n)" et’ ol.’ (1945) " .

100193? and Bebrell“ (1945)

Henderson and Snell (194s)

 

 

Voline ‘

 

Kuik'en,""'et' al. (19431)) ~
Sohweige'rt; et"’ol. 1944
n 'linhon and Snell 1944
H er',"ot nl‘. (1945)
Barton‘Wright (1946) '
Henderoon and Snell (1948)

 

 

I!

I)

I.

I'

~8-

- The growth requirements of ClooTtrdeiun

pTerTfringeno 3261C and the mTiorobiologionl noony procedure
noingTT thiToT thotTeTriun neroT publioheg by ngydTTin 1948. do

fl.-

for no the writer knoTog.T noT 9thTer otndieo using the
Glootridinn pergt’ringenoT no on noToToTy agent hove been made.

.. . Thirteen nnino noido wore found to he nbeolute
TrTeaniroaTentoTT for growth on ClnntridinnT pTerfringonooT .,
“9.19.1391 ,h.1'.t.1d19°!-. 199?'u919°!_ 1°99 $119.9- ”9151,11 onino.
TtThreonTine, phmlnlnnine, tryptophnne, vnlglne! glntnnioTT
nTToid. oerTineT. oyotinenganTT tyrosine. T TTThgne nninn noido
mob. anomaly. 19.9 .nonz- 099011111919 :05ng we.” 3.119.121».
nlnninTe,TT1yo ine, TnopnTrtinTT noidT, Pmunflwfifid T hydrToTryproline.
They TwereTTnddedTTto tho '9an nTediTnn. however! hToonTnoTe
of their otinulntins effTeotTT on grothh Ton the organien.

T The amino noTTdeTTo TTIhioThT Tore eooTeTnTtTian Tour the
growth of CTootridinn partyingeno nnyTT be Tnoooyed yith
F1119 93W?! -179139-313° “57. b‘.."'”94-1f.t.h‘ - .
onnpleo tested do not contain pyridoxnnino or pyTridoxnl.

T W growth of Clootridinn poTri'rTTngonTo T T
under the oonditiono ofT the noooy proToTernre iTo obtained
in 1T6 hToTnTrTo.T T_ This Tohort ingnbntion pTeriodTwould be n
dTotiniteTTngvnTntnse in the nppliontionof the OloToTtridiun‘
perfningeno noooy aethod to routine nnnlyoio of nine

mldfle

I)

I"

I?

mum PROCEDURE

MERBCENTAL PROCEDURE

A oToneroinl Tprepnrntion of onoTeTin (Snooo;
Genernl hioohTonioane) one dried t9 Toonotnnt oeight nt
19.5 49.811901 .«nyisr-doo Ion. «39101.9? 92.000 arm.
enohT yore oToTighed into ErlennTeyer Tlnnko end 50 niTlli-
nrcers 9;. m n hydreohnrimonurs «an... m..-
1‘1"!“ 1'”? range-4 {1111 6399910019214 thoooytoohved
to; 1'1" ham 9t :5 nonhuman: (9391:»: goals...
499.5).- Whea, 90912- 394.0. ..h=rds°1!.aot°9 '05-. $1.1 tog-.04 9nd
otored in glnoo otoppTered bottleo in the refrigerntor.

Mills: W m . _L y .
The nooTToy pTroToedTure noinc Tootobnoillne

ornhinoono 17-5 one onrried out no dooorihed by
Sohoeigert, et TTTan. (1944). T TTT -- T T TTTT

.- T The Horsemen one mintnined on otnb onlturoo

of yenoTteTthrnot-dentrooe-ngnr_ Tong onhoultnreg oeekly.___h_ ..
gner tnnnei’er, .. theTonTtnreo oTeTro TinoubntedT nTtTa'I dTegTreTeo
oentignnde for 58',h9u?3.-§3d thTeTn hTerTT TinTT thn refrigerntor.
m Emula- 1’9r .5113 93”.}FP“,F8 ”Brena P!

trnnotnr to thTeTT oonpleteTTbnonTl nedTiTnn (eTeTe Appendix),

The inToonTlnnTngo inonhnted for T2T4TThTonTrToTT ntTTT3'7 dngreeTeT .
oentiTgracTTieTgTTTgknfrTiTnged. nnd the onpornntnnt dToToTandTnd.
The nTeTllToTToen-oT onnpnnded in 20 niTliliTTtero of oterile T
0.9 penoent oaninTeT nolntion. TTTTOne grop__(o,9o nillilitero)
of thiToTTnell enoponeion one need for the inoonlntion oi’

enoh noon! tube.

-10-
The anonTlTT nedinn one pTrepnrod omitting the
nnino ncid under neTeno. nod the phT one anJTuTeTted to
6.8 to 7,0 ueinTcT bronthynol blue no no exteonanT TTT
indTiontor,T -- One nilliliter on_ thieT nodal“ one Tndded
to enoh neeogT tube. TAT etnndnrdToorye of Tthe enino
neTde which one being determined one obtnTiTnedT by nddiTng

—’ ~-
' " Onon-

AppgoprinteT dilutiono of the cneein hydrolyentoe oere
mac nnd the pH ndJuoterT to 6.8 to 7,0. TThreTe -
different dilutTiono TonT eneh TteoTtTTT extrnot oere nnolyoed.
The fine} 'TroITlnnee otTTenchTTtaneT one ndJnoted to too
nillilitere oith dTietillTed onterTrTg .. T _ ..
TT TT The rnche of teet TtubeoT Toere coveTrodT Toith
Tenooth Ttoooiing nnd nuTtoclnoed for 1T5 ninnteeTT Tet 15
poundo proeToTnn'TTe. T After cooling, the tubeoTToTeTTre
inoculated noethiTonlly nnd incubnted in n onter bnth
nt :57 degreeTeT contigrnde for 72 hour-o. ,TTTT

T The 1nctie noid produced dnring grooth one _
titTiTjntTTod with 0.05 hTTeodianTthroxideT.T noing boontmTol
blue no the indicTntor. TTA otrenn of n1; one intoodueed
intoT onch tube during the titration in order to otir

the eolution.

glootridig W ne__t_h___od . - - T T
T TTTThe noony procedure for oloeTtridiun perfrinceno

.1

one follooed no outlined by Boyd. Leann, nnd Tytell (1948).

-11-

1 Stock oultureeh 91 1:110 orge111e_m were 111911119111“
_1n a_11qu1d 39911111 of 99.39111 hydrolyeaukfith ed99d 3:171:10-
ph3119,_cyst1n0.,w_9de1'11n0_. 11:35:11,}1‘19111110, 31119009; salts;
phoeph9t0hb11119r! end. d91911ec1 beef bent. .. _8er1|.1_1 ., 1
119.9919: no 310.90“ 9119e e_1r0ek_.~ “After 1:91:91», the taboo
of 0toe1l; cultur0 wer9 11191113319191.1518 degre9e 909t1gr9d9 9
for five to” _01:_hour0~__9n9. 111w. egored' 1n 111erefr1gerefior.
-- _ _89ed_ loultur9e_4_\vce£e_grownw101' inoculw 10;: th0
new prooegigre, 1119 999d 9111111129 1:19am oom1et0d of
tm119 913991: of 99.110111 p190 eelte and v1tam1n0. , 9”
hasten we_re made from 11101910411 01111111109 to thwe seed
on1turee ugptieally. ., 1110 099d _ou1tnr9e w9re then
1noube_t99 for £170 to 91: hours at 38 degrees eentisrade
0nd stored. .

0:9 911111111201- of the 09ed culture 09.9 o9ntr1-

n s A- .9 IL- on _L... r-OJ

-...-~

1u¢0d 39d the supernatept d1eoar9ed. .1 1119 9911a weye
ygehed 01111119911111.1“1-9 91 d19t1119d’ juter. 1‘110
ppe-kedn9e110 were then 20090110216.“ 1119mm 11111111119191
wafer 99d 9 d11ut1on of. one to 290 no nede__w1t11 1:91:01.
0119 6.1911 (0.05 91111111‘191‘) o1_t119_911ut09 oellpeuepenaion
n0 need for 1:119 11100111011103 91 09.911 tube 91’ n0d1un.

1 The basal med1m (090 Appendix) #1119110? 1
(1011919111; 111 the 9.31110 0o1d b01113 wasted no ”0119909.
1110 pH was edJueted to 7.1 to 7.2 u919¢vph0no1 r9d 0.0 an
91119111511 11nd1e910r.m_ five 111111111920 of £1119 919d11m
were edded to eeoh aeeey tube. To obtain a etendu'd

-12-

growth curve, graded amounts of the amino acid under

test were added tonewseries of tubes. The cascin ‘
hydrolysate samples were diluted according to estinetion
otcthe approximate concentration of the amino acid being
tested and the pH adJusted to I7.1 to 7.2. Three
different dilutions of_the hydrglyeate were analyzed.‘
his final volume of each tube as M. to lo milliliters
with distilled water. , ..

w ., Bodiun aside (9.2 milligrams) was added, and_ the
contents of each tube were thoroughly mixed. _ Ll’he tubes
wgre thenhstoppered with cotton. plugs and placed in a
hciling water. bath for_ 20 minntec. Whenhcool,” they
were in_oculated__hnd then inouhatcd in c Inter bath
at 45 degrees centigradetor a. period 9;ch hoprs.

. -. m” .1muPat109v the tab}! "12'- nmsl by“
1"??91” ”‘51 ‘99-.°°nt?nt'.??‘n'§°?r°§ 1°. “5‘?"th ”9t
tn'besi. “the dcnsity_ of grcwth yes determined with 0.
Coleman spectrophotometer using a 660 millimicron
filter.

.- -‘V n

The method of calculation of values was the

”can“

sane fcr both of the essay procedurcs. Standardcurves

o‘-.

(see hppendix)“ yerchconstructed by plcttins the concen-_ __
tration of the standhrd amino acidcgainc‘twthckmilliliters
of 0.05 N sodium hydroxide or percent transmission.

em.- tubes were tested' for similar-"light transmittance by
comparison in the Coleman spectrophotometer.

_ - '_.1_3.‘., - .. _ H.
The amount of amino acid precent 4?;FPEUEFFPl‘3 nae
detcrmincd by intcrpolation of the response of the
samples on to this standard curve.

’ , In order to determine the recovery oi’ amino
acids, known concentrations of isolencine, leucine,
methichine, phcnylalaninc. and yaline-wgrehadded to
one casein cample. this samplc gas then cuhmitted to
the same tractmcnt othgdrolysis and analysis as the

ten test samples of casein.

BEETS AID DISCUSS ION

RESULTS AND DISCUSS ION

, _.Th° everaeeflaluea Obtaiced- 1°? thatiweacineo
leuciInIe, methionine, phenylalanine, and valine content of
casein are recorded in Table II. I .

II Duplicate samples of each of three dilutions 91‘
each casein hydrolysate wIsre anaIIIyIsed with Lactohacillne
arabinosus and with Cloctridinmpcrfringens.III In micro-
??019849a1 “termination-v hows?! t1}! baofisréaénw
occasional assay tubcs Iran to grow for unhnown IreaIscne.
Therefore. inInIIvalueIs were not available ror each test
aeiraota In8999t1°n o; Fh0.éat9’°hewsd-fihet 9n03391u°
for each dilution of the hydrolysatee was available. -.
Thcn growth was obtained in the duplicatIeI tubee. there ..

was .1... aerosmeat 3n tbs Y91998- It yeehdeséyable yo
have Ia complete set ofIvaluesI so_that_ the dataIIcould be
mum renewal»- Therefpreg a. 9190???? of .
74“?! 1'39 “9'9" 399”“.“3 t9. ‘ definite 3"“‘1'9'. .919...
mm. “tam? mm the first 91' FP- 9mm»- 89992-99.
of each dilution of the casein hydrolyeates were chosen.
If the bacteria had failed to grow in the first of the

W"-V v--

duplicctetuhes, theIIIvalucIIohtainedI Tron theIIIschIohd cf
the canicatIeI tubIes wacIuIsch.II_ IIITablIeeI ch the selected
chIuIIeIe of the five amino acids may be found in the
Appendix.

I ”—4- ‘ —r - - ..._.,. ‘e . o- a, — t I. .. -74..

The Iaverage values in grams percent total

,4. a

protein obtained from the Lactobaoillus arabinosus

I,

I,

 

Table 11

.-

me Isoleucine, Leucine, lethienine.IPheny1alanine,
and Valine Content of Casein as Determined by
Lactobacillue Arabinoeue 17-5 and

V

Clostridiun Perfrinzene BPGK

 

 

 

 

 

Amino Acid b i "‘ ‘”
1.. arabinoeue 17-5 C. peri'ringens 326K

_ III percent percent
Isoleueine 5.156 ' 5.7;
Lenoine I_ 9.46 9.71
lethieniIne -- g.” 2.7e
Phenylalanine 4.84 5.04
Valine 6.17 6.73

 

 

 

 

..... .-- . _ ._.,-16- _.-- .. . .. .

method yore isoleucine. 5. 56 pcroent3 leucine. 9.46
percent3 methionine. 2.79 pcrcent3 phenylalanine, 4.84I
' percent3 and valIine._§.l7 percent. The average values
obtained from the Clostridium peri’ringencI Imethod were
isoleIucine. 5.:71I pereent3I IlIeucine._ 9.71 percIent3 methionine.
2.76 pIeIreIent3 pherlealaninc. 5.04 pIercentI3I and vcline,
6.73 percent. Differences in the average values for
individneIl IaminOI acids were prparIent.I naminaticn of the
data. however.II_rIevealed differences in the everage values
of theIten casein hydrolysates in both microbiological
nethcde. The values of ItheIIcilIutions within the hydroly-
sates also varicd. IIIIIIt was necessaryIIthdetcrmiIne ifIthe
variations between casein hydrolysatesI or the_ variations
from one dilutiIon level to another were greater than the
variation in the average valheIIe obtcined by the uIeeII of
theIIItwo micrcorghniems. The statistical device employed
to IIIeeparate and evaluate these variations for each amino acid
was the analysis of variance. . I

This statistical analysis showed that the
variation: betwcenIthIe average values of the casein
hydrolyeatee wceIInct significantIIin either theILaoto- ..
bacillus arabinosus method or the Clostridiun perfringens
metth for any ofI the five amino acids studied. IIII‘he
values obtained for the three dilutions of the oascin
hydrolysates did not differ significantly in either method

for any of the five amino acids.

-. -,.- .-17' I...
I The TI values which were calculated from the
data to test therarIiatIion ch ItheII values obtained by
the use of the turn IoIrganIismsI Iare presented in Table
III. 1'ng isoleucine. leucine. and methionine. the
variaticm of values obtained from the Lactobacillus ._
erabinosus and from the ClostridimnIperfrIinIgens methods
were not significant3 but the values for phenylalanine
and valine dIidI differ significantly. I
- These results indicate that the Lactobacillus .
arabinosuIs method and the Clostridimn perfringens method:
may bc usIeId interchangeably under the conditions of
this experiment for the analysis of isoleucine. leucine.
and methionine. In ocnsideraItion of the fact that
Clostridium perfringens requires Ia much shorter incubation
period than Lactobacillus crabinosuIsI.I it IprIIobnbly would
be advantageous to use Clostridiun perfringens for the
routine analysis of isoleucine. leucine. and methionine.
Boyd. Logan." and TytIe11(1948) have and
Clostridium perfringens for thc Ianalyeis of the II - I
arginine. histidine. iscleucine. methionine. leucine.
phenylalanine. threonine. and tryptophane content of II
fl-laotcglobulin._ egg alquImin. and cilh fibroin. Stckes.
Gnnness. DIwyer. andcaswellIanIalyzed samples ofIItheIIIsameII
preparationIofI the proteins. using §treptococcus faecalis.
Boyd. hogan. and TytellIII(_1948) stated that "the snalytical
results obtained by these two different organisms are

essentially in agreement". There was. however. one

Table III

as .— i .-

3 Values Obtained on Comparison of PIeroentageIs of
Each Amino Acid leasured by Laotobaoillus Arabinosus 17-5
and Clostridim Perfringens BPGK

 

 

 

 

 

nine Acid F Obtained
Isoleuoine 0.46
Leueine 3.24
lethionine . 2.50

Phenylalanine 9.67“

Valine 28.06“
1.05 Beouired 4.08
".01 Required 7.51

 

 

r19-

e-
.1

exception to thin statement. TheraluIe obtained {an the
phenylalanine content of fl-lantIeglobulin by the Glostriditm
perfringenIsI method wasI 3.2 percent. ant the vIaIlngI obtained
by the Streptococcus {IaIeoalis method was 4.3 percent.
There is no experimental evidence in the present
ntudy Ito explain the significant variatiIIoIn in the values
obtained for thIeII phenylalanine and valing gontent of
Icnsgin by the usIe of the two ninrgorganisms.I It is
possible that one ofI the organisms is not respondingII
gunntitatively toI Ithe phetglalIanine nnd valine in the
oaseinIIhydrplyIsate.I . _It 1. also possibleItIhat the two
grannismsI are not responding to the same eh‘ical '
substances. I . II I
IIISIatiIsIIfactIoryI reIogINeriIes of amian acids added
to the casein sample before twdrolysiIs were obtained Ii’or
‘ both microbiologicag. mnthdesI. The percentage recoveries
of added nmino acids are presentgd in Table Iv. The DI.
form of is oleucIine.I methionine. phIerwlIalnninIeI. 9nd vIaline
and the IIIforn of IleIuoine were added to_ IaI casein sample
for tho recovery deterninations.II The percgnt regenery II
9: addngIamino Iaeid was calculated as the response of the
organisms to the 1. term of the amino ncide. III
The percent recoveries Ioi’ isoieucine. valine.
phexwlalanine. Iand'I methionine were Ilower for the glostri-
dinnIIpIerfringene methIoIdItIhIanIfor the IactobIaciIlusI arabi-
nosus method. In the case of pherylalanine and valine.

I.

.
a
t ..
.
.— I b o
.
.
l
O
- 1
s
.7 ' v .
‘ w
. , .
n
e . .-
.-
\
I

 

Table IV

.0

Percentage Recoveries of Added Amino Acids

 

 

 

Amino Acid mount " b ,
added 1.. aIrabinosus c. perfringene
.A . m. perc ent percent
Isoleucine 25.0 106 98
Leucine 50.0 102 103
Iethionine .... 12.5 105 95
Phenylalanine 12.5 104 99
Valine 25.0 100 98

 

 

 

 

 

.21-
these lower rchovIery IvaluIes yigld Ispme interesting
informationgIIinnIcg thIerII Iwould suggest that Glostridim
perfringens was not responding to the D tern of phenyl-
alanine and valine. .. It has been established that
IanItIobaoiIlIus arabinosug utilises pnly the I. fnrn of
phenylaIanine and vaIIine (Iuiken. et Ia1. 1943n.II 1943in
HegstIeId. I944). Therefore. the higher values obtained
byIItheI Olgstridiun perfringens methodIIprIr the phony}:-
algnine andI IIIvInIinIeIIIcontent ofI casein were probably not
due thIIIthe responseIof the microorganism to the 1) form
of these amino aside.

em: up CONCLUSIONS

SUMMARY AND CONCLUS IONS
, 1'91!" samples of aIIIcomnerciIal preparationof I I
pasein were analyzed fcrIIisolIeuIcine. leucine. methionine.
phenylalanine. and valIineIIby a standnrd microbiological
method using Lactobacillus arabinosus l'7-I5 Iand by aIInew
microbiological method using Clostridium perfringens
Em . ..V _ ._.. d e
The average values in grams percent of total

protein obtained with Igctnbacillun arnbinosue Il'I7-5 I
were isolgucine. 5.56Iperoent; leucine. 9.45 percent;
methioninn.I 2.79 percent: and valine. 5.1? percent.
ThIeIIvaIues obtained with glostridium perfringIIenIsI'IBIPSIII
were isoleucine. 5.?1IperoIenIt; leucine.I9."Il pIIeIrIcgnth
methionine. 2.76 percent: phemrlalanine. 5.04 percent_;II
nnd vnlIiIne. §.73Iperc_ent. The variation in the values
“on inoleuIcinIe. leucine. and nethionineIobtained by the ..
use Iof the two microorganisms was not significant. The
values of phenylalanineII and valine as determined by the
two methods did differ significantly. I II

I 0n the basis of these IrIIesIults. it was
concluded that the 010stridium perfringensIIand the
Laetobacillus arabinIosus methods msbee used inter-
changeably for the Ianalysis of the isolencine. leucine.
andIInethionine content of casein. . The significant differ-
ences inII ItIhe valuesobtained for pherylalanine and valine

by the use of the two microorganisms indicated that

1’

f3

.‘

_ ~2:-.,
further investigation is needed.

ghe most important advantage of the Clcstridium
perfringens method for the estimation of amino acids is
the short incubation period required for the minus .
growth response oi the organism. Clostridiun.perfringens
attains maximum growth in the assay;procedure in 16
hours. Lactobacillus arabinosus recuires 72 hours in
the‘assay procedure to attain naximnngrcwth. in view
of this f“‘2a}ff"u;§“59 advantageous to usewthe h h
Clostridiuanerfringens nsthod for the routine analysis

.Of isoleucine. leucine. and methionine.

1.1mm CITE

mmrm 01m

Barton 'right. I. 0. l9“ - Iicrobiolcgical Assay of
mptophane. Leueine. lsoleucine. Valine. Oystine.
lethicnine. lysine. )henrlalaaine. Histidine.
Arginine. mrecnine. ~ Analyst. 71:267-278.

Bauuarten. 1.. A. ll. Esther and L. Stone”, 1945

’ Glutamie Add Content of rue sun-1.1.. 0.1-..1
Chem” 22:514-521. ‘

Bamarten. W.. L. Stone and c. S. Boruft 1945 Amino
Acid compositien .: Grain Alcohol Fermentation By-
produots. Cereal 913g" 22:311-320.. -7 _

Boyd. 1. 3.. s. A. Logan ... A. 4. 17ml 1947 'Growth
ci’: Glcstridium 'elchii (M). J. Biol. Chem. 167:
eve-sec. -_ ‘

”4.1.3. I. A. Logan and A. A. Tytell__ 1948 Growth.
Becuirements of clostridim rerfringens (Velchii) BPGI.
J. 31.1. on... inane-loss. ‘ ' \

Boyd. I. 3.. I. A. Lessn and A. A. mall 1948' Incre-
biological Procedure for Assay of Amino Acids with
clostridium Bertringens (Velchii) m. J’. Biol.
chant. 174310274035. -. W

Brickson. I. L.. L. I. Henderson. 1. Bolhjell and c. A.
Brende- 1948 Antagonist: of Aainc Acids in the
Growth of Lactic Acid Bacteria. J'. Biol. Che-1.. 176:
517-528.

II

I.

a,

w -25-
Gamien. I. I. and I. 8. Dunn 1948 A Composite Basal
Iedium- for the lierobiological Assay of Leuoine.

J. 31.1. on..." 173.1374... .. '

Doormann. A. n. 1945 ._ A Bioassc for lysine by Use
of a lutant of leurospcra. J'. Biol. Oh-.. 160395-
103. ‘ p ‘

Dunn. ll. 3.. I. I. canien. 1.. 3. Rockland, s. W
and 8. O.» Goldberg 1944 Investigations of Aainc
Acids. Peptides. and Proteins. XVII. Determination
.t 01...!“ 9.1. 1. Protein 33.31;...“ by .. m...-
biological Iethod. NJ. 31.1.“ohon.._155:591-ooo.

m. no son 3---0‘319: 8» man-menu 3.1.293...
1946“ investigations .ctflAnino Acids. Peptides. and
Proteins. magneternination of‘flothignine in
Protein Bydrolysates with Laotobacillus Permenti 36.
1. 31.1. Chem. 1633577-586. __. ‘

Dunn. I. 3.. B. P. schott. V. Prankl and L. B. Bockland
19.46-.. amuse-c.1921- »! m9 “3‘!L.?'P“§!'v and
Proteins.“ ”xx. Detemination_of Apparent Pree
l'ryptophsne in Blood by Iicrcbiological lethod.

J'. 31.1. one... 157.3e7-394. ‘ _

Dunn. I. 8.. 8. Shank-an. I. 1. Camden and H. 31...: ...,
1347 . The Amino- Acid Requirqents of twenty-three
Lactic A91. Bacteria. I. 31.1. 031.1... 19931-23.

m... I: was. 1% ran 1945 . minus erasing“:
1.”... 1.31.1.1... .gcuwth of 1.....1...111u. mu.
nosus by Aline Acids. J. Biol. Chem. 1608329-336.

[‘1

13

/)

I.

If

I!

I!

1’

-25-
1.1.3. w.',_r._n1p_. and 0. 1.3.1.1..” 191.
1.1.11.1“... .1 Bacterial or... by 3-1....11... J.
”31.1. 0110s.. 15514653-458. - ' 1 .,
“"9!!! n. 9- am 49.31»: 4953 .1 anatomic-a -
1...; ofil'ryptophane 1. Proteins and 1b.... J. 31.1.
cm... 1.6.1-3. _’ .1 ‘ -
Bac. L. 3.. l. B. Bull and R. J'. willials 1945 wBicro»
Bielcgica1_ ~Determination of Amino. Acids. 11.1....
.11. Btilisat1en .1 cup...“ 1.1. and 013.91.. by
Lactebaoillus Arabinosus. J’. Biol. chem. 159:
?7§-33°o .. .
“"3“: D- 2‘: .. 194.5 Shinspwlfési!’ 01’ 3110 1.11.1...
1.31.1.1... .na‘vum. Requiregents .1 Laetcbaoillus
_. ”binosu,17-5. .. _ J'. Biol}. men. 15117-41449.
negated. B. I. ~ 19.44 -Bm1no Acid Beouirementswog
Laotobaoillus Arabincsus 17-5. 3’. Biol. Che...
_ 152.193-200. . 1
11.5.1... 3. 11. and r. B. Bhrdwell 1944 ., o. the 3.21..
.1 Bysthetic Dir-Leucine. ”J. Biol. one... 1653162459.
Hendersog.”__L. It. and I. B. Snell 194,8 A__Unitgrm Bodium
for Determination of Amino Acids with Various :1...-
organisms. J'. Biol. on... 372815-50. 1 _
Bier. 3. 1.. O. B. Graham. B. nudes and D. K1ein 1945
liercbiological Determination of Amino Acids in Aninl
Brgtein. J. Biol. chug”. 1613705-715. h
Hodson.__A. {Bend}. 1.. Krueger -1947 Bireet r..m.1..
ctLeuoino. App1ication to Bilk and certain Other
Pcodstuffs. Arch. Biochem.. 12:455-440.

O

‘\

PK

-27-

Born. I. J.. D. B. Jones and A. B. Blum 1946 _ Iicro-
Biologicgli Determination of Iethicnino in Proteins
and roods. J. Biol. Chm. 1661521-326. -

Kobayashi. Y.. I. fling and 8. V. For 1948 Antipth
Specificity in the” 1nhibiticn of Growth 01! Escherichia
Coli by Amino Acids. J. Biol. Chen" 1741591-598.

Kuiken. K. A._. V. H._Iorman.. c. I. Lyman and 3'. Hale ..
1945a Iicrobiclogical Determination of Amino Acids.
Science. 981266. _

Mitch. K. A.. O. I. Herman. G. ll. W! 1'. Halo and L.
Blotter 1945b Iicrobiologioal Determination of
Amino Acids. I. Yaline. Leucine. Isoleucine. J’.
”Biol. Chem. 1511615-626. _ ..,

1m... J. 0.... 11. s. 01.... 1945 ‘1 Lactobacillus ..
Assay Iethod for L(+)-Glutamio Acid. 3’. Biol. Chem.
1571265-285. ' 1

Lynn. 0. I.. K. A. Kuiken. L. Blotter and P. Hale f
1945 Iicrobiologioal Deternination of AmincAcide.
n. on...“ 1.1.. J. 31.1. Chen" 157.395-4015.

I3 Iahan. J. R. and B. B. Snell}. 1944” Igorobiologioal.
Determination of Amino Acids. I. Valine and Arginine.
J. 31.1. one-1.. 152133-97. '

Prescott. J. I.. B. 8. Schweigert. c. I. Lynn and I. A.
Dziken 1949 TBe Effect- of D-rryptcphane on the
Utilisation .1111. 1;. Isoner by 3... 1.1.11. 1.11
Bacteria. J'. Biol. Ohm” 1781727-752.

Ga

I.

:3

I)

I.

I)

.‘O

I.

'9

-23-

Biesen. I. 11.. B. 8._ Schweigert and C. A. IlvohJem 1946“
Iicrobielogioei Determination of Iethionine in Proteins
‘ .andl'oodstuffs. J. Biol. Chem" 1651547-558.

3...)... 3. 3.. 3. 1.11.11» and I. Hts-.11 . 1944 1.1.11.1.
criteriatiog. of lscheriehia Coli with Respect to Growth
“Requirements. J. Bact.. 461401-412. 1 .-

Hm. I. J. and 3. Brand 1944 Iethod for Determination
of Leucina .imuiProteinHIydrolysates and in foodstuffs.
by Use of a Ieurespcra Intent. J. Biol. 011... 1541
..1.5.1‘1759 -, __ .w

Suaberligh. I. B. and C. A. Baumann l946w n. Bffeezt
of Dietary Protein upon Amino Acid nation by Bats
and _Iioe. J. Biol. Che... 1661417-428. __

8ohweigert. B. 8.. J. I. I; Intire. C. A. nrebJem and
B. I. Strong . 1945 Direct Determination of Valine,
and Leucine in Fresh Animal l'issues. J. Biol. Chem.
135.1e3-191. _

Schweigert. B. 8. and B. B. 8nell 1947 Bierebiological
Iethods for the htination of Aline Acids. Intrition
1b.“. Ben. 161497-510. 1.

Schweigert. B. 8.. B. B. saubgrlich and C. A. 1179“!!!
1945 the Free i'ryptophane Content of Human Urine.
_8oienee. 1021275-277. 1

achwe1gert. 3. 3.. I. r. 1.1... and c. 4'. urem- 1%“
l'he Leucine. Valine. and Isoleucine Content of Ieats.

Arch. Biochem.. 61177-184.

I7

'9

1‘)

«1

u

I)

I.

I"

I)

x,

-. .. _ -29- A A. ._ . ..
Shah-an. 8.1 1943 Alino _Asiql nutrition of Bactebseillns

__Arabinesns. J. 31.1. Che... 1502505410.. __ .. k

Shannan. 3.. n. 3.8 m and 1.. B. mun” 1915 Anus-4.
ef._6,Anino Acids by Iicrobiolocioal Isthod. J. Biol.

”Ohm. 150.477-4713. _ ‘9 ., h ..

Snell. B. B. 1946 Use. oi'Iicroorgeniens for Assay .1

jute-ins. Bhysiol. Ben. 281255-268.

Snell. I. B. .- 3.946 Iierobiolpcioal Isthon in Anino

Acid Analysis. An. Iew York Acad. Science. 47:
.lol-179. - 1““

Snell. B. B. .1949. .l‘hs IicrobiolecicalwAssey of Aging
1.11.. m. Anson. n. 1.. ans! 3. 1.19.11” mm...
inrretein chemistry. Aoadenio Press In... Bnblu

“Ion York. B‘. 1.. Vol. II. .,

3.1.11.1. 1:. and 1‘. I. an... . 1939 A Iicrobiolozioal
1.... 1.: munuu. Ind. m. chem. mu. m... 11.
.. 349-35“. . . ‘ _ ,

Snell. ll. 3. and. 11..” D. night 1941 _A Iicrobio1o¢ioal
mm... 1.: the mama...“ or Iiootinic 1.14. J.
3131. chm. 139305-669: _ ..

Stokes. J. 1.. snde. Guinness 1944 “Utilisation ei’~ ._
”0 Acid: .Bncntienorghs by Leotobaeillus _ Arabinosus
anstnaotobaeillns (:asei.~ J. Biol. chem. 1541715416.

Stokes. J”. I‘L‘E“ I. 9mees .19“ _:Pyridoxanine anti
the synthesis of Aline Acids by Isotobssilli. Science.
101143-44.

It

-30-

Stokes. J. I... I. Sunnese. J. I.,__Dlyo_er snail. 9. Smell
1945 Iicrebiolocicaljethcgs for, Determigetion of"
Amino Acid... 11. traitors Bessy for the l'en Essential

. .91... 1.14.. J. 31.1,, 911.... 160135-49. ‘

'eoley. .1. 9.9114111. H. 3.52.11 1 1945 M Iiorcbig-
1._.1_._.1 1.111... {or the Determination .g- 1(1),:1mtq-
phane inNProteins and Other Couple: Substances. J.
Biol. chm. 1571141-151.

I \e-

APPMIX

 

 

Double Strength Basel -1991“ for
Lactobscillns arabinosns 17-59

 

m~mao1ns ” "
BL-Isole‘ucine
DL-Valine ‘”

I.(~ )1'cyst‘ine' ’
DL-Iethionin‘e"
Dl’r l‘ryptoiahehe
LEO-Tyrant. "
DL-Phenylalenin’e
L(+)-Glntanio acid
m-Tr’eonine
DEL-Alanine ”
L-Aspar‘agine'

I. + lysine H Cl
1. + -Arginins“‘H Cl
1’. + ~Hietidine
DD-Serine
Glycine"
L(-.)-"P’r'oline
Glucose"'“" “
Sodii’m acetate
Salts A

K 1’04

Kznfi 1’04. A
Salts B 73'
Is SO -
1?. 302.7322-
In 304.4320

”Ba (31" -
Adenine snli’ateo 2829
0mm. 1! orange
Uracil
miuine 3 Cl ~ -
Pyridoxine E‘cl' ‘ “ “
Dlwcal'o'im‘n psntothenate

4o mill igrans
‘0 I
40 II
20 II
20 II
10 II
10 II
20 II
80 II
40 "
4o "
‘0 II
‘0 II
10 II
10 I!
19 II
10 "
1o “ I
4 era-s
‘ It
109 milligrams
1oo "
‘0 I
2 I -
2 I
2 I
2 II
2 I
. 2 .‘ ‘v

100 micrograms
200

 

 

100 n
Riboflavin" 100 u
Bieotinio" “acid 100 a
p-Aninobensoio acid 29 s
Biotin" 0.2 .
30116 I014 20 I . .
tater up to 100 nillilitm

 

 

*Sanberlich and Ban-an (1946).

 

Doub1s Strength Basel Iodium for
Olostridiun peririncsne m

 

Glucose "

2.0 crane

 

 

 

Ascorb‘ic ’a’eid 50.0 aillisrsns
DEL-Alanine 100.0 '
D~Ar¢inins ‘ 50.0 I!
EDD-Aspartie acid 100.0 I!
Glycine ‘ 100.0 v
L-Glutalic"‘aeid 150.0 "
B-Histidi‘ne " 50.0 !
bdrmrolin’e 2530 '
DL-lsoleueine 50.0 "
L'L‘uom. 75.0 I
131051110" 100.0 '
m'Iethi‘o’nine 50.0 "
L—Proline" 25.0 .
Din—Phenylalanine 50.0 "

Dl.- Scrine '"" 150.0 '
DL'mooniho 599° '
B-Tryptophsne 50.0 '
L-lyrcsine 50.0 "
m-Vsline 75.0 '
Uracil“~” ' 2.5 -v.
Adenine sulfate - 5.4 ‘0 " “
Riboflavin" “ " " 100.0 micrograms
calcium D-psntothsnate “ 200.0 '
Pyridoxsaine dihydrechlorids 100.0 . ' ..
31°t1‘ 10° 9 b
It 804-7E20 40.0 .1111ng
’0 804 . 20° .
In 804’ 2.0 7'
" 01 20° ‘

-. 4 .. ‘ 9°32 " . .-
Iatsr up to 100 milliliters

 

*Boyd. Logan. end i‘ytell (1948).

 

Milliliters of 0.05 N Ne 0H

 

6.0

11.0

3.0

2.0

1.0

 

 

 

I I I I I I I ' I I

 

0 5 10 15 20 25 30 35 40 45 50
Iflcrograms of Dir-Isoleucine

Titration Values Obtained from
Lactobacillus Arabinosus 17-5 for

Known Concentrations of DL-Ieoleucine

Milliliters of 0.05 N Na OH

6.0

5.0

Loo

3.0

2.0

1.0

 

 

 

 

 

2.

L l 4 J I l l l I
5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0
Micrograms of LpLeucine

Titration Values Obtained from
Lactobacillus Arebinosus 17-5 for

Known Concentrations of LpLeueine

Millilitera of 0.05 N Na 0H

6.0

5.0

4.0

3.0

2.0

1.0

 

 

 

 

I I l l I I l l i

 

2 A 6 8 10 12 ll. 16 18 20
Micrograna of DL—Methionine ,

Titration Values Obtained from
Lactobacillus Arabinosua 17-5 for

Known Concentrations of DL-Hethionine

Milliliters of 0.05 N Na 0H

6.0

5.0

4.0

3.0

2.0

1.0

 

 

 

LA I L I II I, I 1

 

2 3 h 5 6 7 3 9 10
Micrograms of DLmPhenylalanine
Titration Values Obtained from
Lactobacillus Arabinosus 17-5 for

Known Concentrations of DL-Phenylalanine

Milliliters of 0.05 N Na OH

6.0

5.0

4.0

3.0

2.0

1.0

 

 

 

 

I I I I I I I I

 

10 15 20 25 30 35 40 #5 50
Micrograms of’DLpValine

Titration Values Obtained from

Lactobacillus Arabinosus 17-5 for-

Knoun Concentrations of DLQValine

Percent Transmission

10

20

30

50

60

70

80

90

100

 

 

I I L I I I I I I

 

 

50 100 150 200 250 300 350 400 450 500
Lflcrograms of DL-Valine
Percent Transmissions Obtained from
Clostridium Perfringens BP6K for Known

Concentrations of DL-Valine

M

Selected”hperinent31 17.15.5351: the
Isoleueine Content of casein

I. is

 

 

 

 

 

 

 

Casein Sample ‘ ncrobiologieagjefiod
1.. arabinoeue 1 - C. Mrhzene m
percent percent _

1 5.52 5.61
5.31 5086
5.36 6.12
2 5.52 5.61
5e31 5.85
5.36 5.94
5 5.52 5.35
5.31 5.35
5.36 5.35
4 5e” 5e51
5.31 5.86
5.36 6.12
5 5.95 5.86
5.52 5.35
5036 5.35
6 5.95 5.61
5e52 5e48
5.48 5.“
5.52 5086
5.61 5.86
8 5.52 5.55
5.52 5.94
5.61 5.94
9 5.52 5035
5.52 5.86
5.61 5.86

10 5.95 6
5.95 5;§§
5.52 5.55
Arerece 5.56 5.71

 

 

 

 

Selectedlltperinental‘Vaineg for the

Leueine Content e! Casein

 

casein Banp1e

 

 

 

 

' ‘ ill "51 1 then '
13. arebineeue 17. o. yeah-155m 325:

percent percent
1 11.05 10.20
9.76 9.78
8.42 9.78
2 9.35 10.20
8.92 9.78
8.67 9.78
3 10.20 10.20
8.92 9.78
9.95 9.78
4 11.05 10.20
9.35 9078
9.95 9.75
5 9.35 10.20
8.50 9078
9.75 9.78
6 19.20 10.20
8.93 9.35
8.67 9.35
7 10.20 10.20
8.93 9.35
8.92 9.35
8 9.35 10.20
9.35 9.35
8.93 9.35
9 10.20 9.35
. 9.35 9.35
8.93 9.35
10 10.20 9 35
v 9.35 9255
8.92 “.35
Average 9.46 9.71

 

 

 

 

H

sauna kperhentai Values for the

Iethicnine Content of Casein

 

Casein Sample

 

 

 

 

 

 

1
2
3
4
5
6

2972 2.87

2.86 2e59

7 3.72 2e58

2.89 2.87

2.65 2.37

8 2.72 2.68

2.72 2. 59

2.89 2.69

9 3.06 2.58

3.72 2e87

2.95 2.69

10 3.06 2.68

2.55 2. 8‘7

2.86 2.87

AV”. 2.” 2. 75

 

 

Selected lsperi-entai‘vaiues_ggr the
Ehenylaianine Content of Casein

 

 

i

ethod

 

 

 

 

Casein Sample ‘ “ “ bA ’
1.. arabinesus 17-5 0. pefiringens 3'96!
percent percent '
1 4.53 5.10
5.10 5.10
5.10 5.10
2 5.10 5.10
4.96 5.10
4.84 5.10
5 4.25 5.10
4.96 4.97
4.84 5.10
4 4.53 4.97
5.10 4.97
4.68 4.97
5 5.67 ‘09?
4.82 4.97
4.68 4.97
6 4.53 4.97
4.96 5.10
4.59 5.10
7 4.53 4.97
4.96 4.97
5.10 5.10
8 4.53 5.10
4.82 4.97
4.76 ‘0"
9 4.53 4.97
4.68 5.10
4.68 4.97
10 5.67 5.10
4.96 4.97
_“_ 4.68 5.10
Average 4.84 5.04

 

 

 

 

‘ the

Selected uperhental Yaluee fer the

valine Content of Caaein

 

 

 

 

 

“ b c ‘ -

1.. araiinegua 17-5 c.3eflr1neene m

percent percent . '
1 6.38 7.39
6.16 6.00
6.12 6.48
8 6.38 7.39
8.16 6.00
6.25 6.48
3 6.38 7.14
6.16 7.14
6.12 7.14
4 6.38 7.39
6.16 6.40
6.12 6.00
5 6.38 7.39
6.16 6.38
6.12 6.40
6 5.95 7.39
5.95 .038
6.25 6.80
7 6.38 705.
5.95 6.38
6.99 6.40
a 6.38 7.14
6.16 6.38
6.25 a.”
9 5Q’6 7.39
6.16 6.38
5.86 6.40
10 5.95 7.14
‘016 6.”
. , ., 6.18 6.”
Average 6.17 6.73

 

 

 

 

 

ROOM USE ONLY

 

 

MICHIGAN STATE UNIVERSITY LIB

III III II I ll IIHIIIIIIIIIES
3 1293 0306] 788

4