Ptyalin action on insoluble acid-treated cellulose residues
by Eldon N Sanborn
A THESIS Submitted to the Graduate Committee in partial fulfillment of the requirements for the
degree of Master of Science in Chemistry
Montana State University
© Copyright by Eldon N Sanborn (1949)
Abstract:
Hydrochloric acid hydrolysed cellulose residues were treated with saliva amylase and the extent of this
ptyelin action upon the insoluble cellulose residue was determined by quantitatively measuring the
amount of reducing sugars produced.
A study was made of the effects of acid concentration, duration of acid hydrolysis, and the temperature
used during acid hydrolysis on the amount of cellulose residue which would be hydrolyzed by ptyalin.
Since normal cellulose is not susceptible to ptyalin hydrolysis, a theory has been proposed to explain
the action of saliva amylase on the insoluble cellulose residues. PTYALIN ACTION ON INSOLUBLE AGID-TBEATED
CELLULOSE BESIDUBS
By
ELDON N. SANBOBN
A THESIS
Submitted to the Oreduate Committee
In
p e r t l e l fu lf illm e n t o f the requirem ents
fo r the degree o f
Master o f Science in Chemistry
at
Montana S tete C ollege
Approved:
Heed o f Major Department
Examining Committee
Bozeman,
June
lontena
1949
^ *7&
h & ^
O tfi L
- 2 TABLE Og COUTEiJTS
2&.
I.
ABSTRfCT..............................................................................................................
3
I I.
IUTBOBUCTION ......................................................................................................
4
III.
HISTORICAL..........................................................................................................
5
I?.
EXPERIMENTAL.....................................................................................................
7
1.
Type o f C o llu lo se Used ......................................................................
7
2.
Treatment o f C e llu lo se by H ydrochloric Acid H yd rolysis
w ith Respect to Acid C oncentration, Temperature pad
Time .............................................................................................................
7
3.
Sslitra Treatment o f the H ydrolysis Residue
......................
9
4.
Method o f Reducing Susar D eterm ination
...............................
10
5.
Semple C a lc u la tio n s ............................................................
V.
RESULTS AND CONCLUSIONS................................................................................
1.
2.
3.
4.
VI.
13
16
R esu lts o f Concentrated H ydrochloric Acid H ydrolysis
at Room Tempereture fo r T r io u e Lengths o f Time. . . . .
16
R esu lts o f 3 N H ydrochloric Acid H ydrolysis at
B o ilin g Temperature fo r Various Lengths o f Time. . . . .
23
(A)
Ammonium Hydroxide N eu tra lised P o r tio n .............................
23
(B)
P o rtio n Dried in the Acid S ta te . . . . .................. ....
28
R esu lts o f 0 .5 N H ydrochloric Acid H ydrolysis a t
B o ilin g Temperature fo r Various Lengths o f Time. . . . .
32
C on clu sion s....................................................
33
SUMMAHI....................................................................... * ......................................
38
V II..............................................................................................................................................
V III.
LITERATURE CITED AND CONSULTED ...............................................................
40
41
100384
- 3 X.
ABSTRACT
H ydrochloric s o ld hydrolyaed c e llu lo s e resid u es were tree ted w ith
se l i v e e:ayleee end the e x te n t o f t h is p t y e lin potion upon the in so lu b le
c e llu lo s e resid u e was determined by q u a n tita tiv e ly messuring the amount o f
reducing sugars produced.
A study was made o f the e f f e c t s o f a cid co n cen tra tio n , duration o f
pcid h y d r o ly s is , end the temperature used during a cid h y d r o ly sis on the
amount o f c e llu lo s e resid u e which would be hydrolyzed by p t y e lin .
Since normal c e llu lo s e i s not s u s c e p tib le to p t y e lin h y d r o ly s is , a
theory has been proposed to e x p la in the a c tio n o f s a liv a amylase on the
in so lu b le c e llu lo s e r e s id u e s .
™
II_.
/-L —
INTRODUCTION
I t I b w e ll known th e t the p o t e n t ia lly r ic h cerbohydrete source o f
c e llu lo s e
ps
en energy food Is denied most o f the pnlmel w orld.
A few o f
the low er snim els cpn u t i l i z e c e llu lo s e d ir e c t ly as a source o f energy hut
the h igh er »nlmele are compelled to u t i l i z e I t , at le a s t in the major p e r t,
in a roundabout way.
With t h is in mind, t h is problem was conceived In the hope that some
simple procedure could be found whereby c e llu lo s e cou ld , to some e x te n t,
be made more d ir e c t ly a v a ila b le to animal l i f e .
I f a p r a c tic a l so lu tio n
i s not forthcom ing in t h i s work, i t i s hoped th at i t w i l l arouse In te r e st
in and fu r th e r work to s o lv e the many problems v is u a liz e d in the course
o f t h is study.
- 5 III.
HISTORICAL
U n til e number o f yeere ego when h sr v ee tin g o f wheat by combines
became common on email farms, th resh in g o f th is sraln l e f t many straw p il e s
on Montana farms which were e v e n tu a lly eaten by liv e s to c k , burned or decay­
ed .
Though farm anim als were known to s u b s is t on th ese straw p ile s fo r
long p eriod s w ith l i t t l e oth er supplem entation. I t was n e v e rth e less recog­
n ised th at straw was a poor food at b e s t, e s p e c ia lly fo r w in ter feed in g .
Straw, however, i s p o t e n t ia lly r ic h e r In carbohydrates than one might sus­
p ect from th ese o b se r v a tio n s.
The crude fib e r fr a c tio n is h igh (h0-$0$)
and i s n ot u t i l i s e d g r e a tly by animals.
This engendered the thought th at
p o s s ib ly t h is fr a c tio n could be r eso lv e d in to more n u tr itiv e m aterials by
chemical treatm ent and thus enable th ese animals to survive the w in ters
on ranges and farms In a much b e tte r c o n d itio n .
Experiments cen terin g
about d ilu t e acid treatm ent o f straw in stack s was in s t it u t e d by B. L.
Johnson o f the Department o f Chemistry a t Montana S ta te C ollege in 1935»
end the changes In the crude fib e r fr a c tio n and the accompanying in creases
In the reducing sugars were stu d ied .
The p resen t study o f a c id -tr e a te d c e llu lo s e resid u es grew out o f the
above mentioned fin d in g s .
The maximum decrease in crude f ib e r by prolon­
ged 0 .5 S h yd rochloric a cid h y d ro ly sis was never more then 10 p ercen t.
The in c r ea se in reducing sugars was la rg e on a percentage b a s is , only be­
cause the amount p resen t b efore treatm ent was almost n e g lig ib le .
However,
animal t e s t s were not conducted, and, th e r e fo r e , i t was not determined
whether the acid treatm ent o ffered a g r e a te r increase in n u tr itiv e value
than in d ic a te d by the changes in the amount o f crude fib e r and reducing
eu gsre.
Inpsuuch s.g p ert o f the crude f ib e r undoubtedly con eIete o f c e llu ­
lo s e es w e ll PS o f lig n in , end e s p e c ia lly lnesmich s s lig n in Is undoubtedly
combined w ith c e llu lo s e , I t wea thought acid trea ted c e llu lo s e might p o s s i­
b ly g ive r is e to In solu b le resid u es which would be su s c e p tib le to em y lo ly tic
cleavage to reducing su gars.
Bough p relim in ary t e s t s in d ica ted th at s a liv a
amylase (p ty a ltn ) d id hydrolyze the In so lu b le a d d tr e a ted c e llu lo s e
resid u es to a sm all but measurable d eg ree.
S c ie n t i f ic lit e r a t u r e from a l l over the world is abundant in the work
o f in v e s tig a to r s who have studied the p a r t ia l h y d ro ly sis o f straw end
other crude f ib e r s and th e u t i l i z a t i o n o f the in so lu b le p a r t ia lly hydro­
lyzed r e sid u e s as a f o o d s t u f f ^ ’
6, 8 .
Most o f t h is work h a s, however,
been c a rr ie d out by means o f a lk a li h y d r o ly sis and very l i t t l e , i f any,
s tu d ie s are pu blished on time use o f a c id s .
Many o f the r e s u lt s obtained by
u sin g the resid u es as food m ateria ls are only s u p e r f ic ia l, and i t appears
that no attem pt has been made to determ ine q u a n tita tiv e ly how w e ll they
could be u t i l i z e d by the animal.
Therefore the fo llo w in g study i s aimed a t
a determ ination o f the fa c to r s c o n tr o llin g the amount o f p t y e lin hydroIy z able fr a c tio n o f r e sid u e s obtained in incom plete acid h y d r o ly sis o f c e llu ­
lo s e .
- 7 jV .
JL«
EXrSPIMENTtL
Source o f C e llu lo se
Hexsjjon trend ls t o r e t o r y f i l t e r paper oenufsctured t y Schaer end
Coinpeny wee used ee the source o f c e llu lo s e fo r t h is work,
A la rg e enough
supply o f th e game trend end shipment was obtained e t the beginning o f the
research to l e s t throughout the e n tir e experim ent.
I t was hoped In th is
way to elim in a te as much ms p o s s ib le any v a r ia tio n s in the source o f c e l l u ­
lo s e due to d iffe r e n c e s in brands or v a r ia tio n s in the same tren d .
P relim inary t e s t s upon the f i l t e r paper gave no in d ic a tio n o f the
presence o f r e sid u a l reducing su g a rs.
P relim inary t e s t s a lso showed th at
the f i l t e r paper was not acted upon by s a liv a amylase in such a way as to
produce any reducing su gars.
2
.
Trmctrnrnnt a £ j&a C e llu lo s e Sy H ydrochloric ISlM ByArblyslS
Re.BP.gXLi
to Held C oncentration. Temuerrtnre £24 2 1 m
The treatm ent o f the c e llu lo s e p r io r to amylase a c tio n was carried out
w ith two major v a r ia tio n s in procedure, th at o f a d d con cen tration and tem­
perature.
In one c a se , the f i l t e r paper was put in concentrated h yd rochloric
acid which was kept at room temperature.
At predetermined but arb itra ry
tim es, sub-samples o f the c e llu lo s e r e sid u e , o f such a s iz e as to give
convenient q u a n titie s fo r la t e r study, were removed from the a c id .
The
excess liq u o r was drained from th ese samples and the resid u e put in the
open s i r to dry.
The approximate time from in tro d u ctio n in to the concen­
trated a c id to complete dryness was recorded fo r each sub-sam ple.
- 8 The dry residu e* were then ground end weehed w ith hot we t e r , f i r s t by
decentins; the supernetpnt liq u id e f t e r the residu e hed s e t t le d , end, f i n a l l y ,
by f i l t e r i n g w ith * g r a v ity f i l t e r .
I t wee found th et g r a v ity f l l t r e t i o n
gsve b e tt e r r e s u lt s then su ctio n f l l t r e t i o n , end in eech ceee the washing
o f the resid u e wee continued u n t il the supernatant liq u id gave a negative
t e s t fo r reducing sugars and ch lo rid e io n s.
The washed resid u e was again put in the open a ir to dry and, when
com pletely dry, p u lv er ise d as b e fo r e ,
/ f t e r t h i s , the f in e l y powdered
resid u e was preserved in la b e le d , w e ll stoppered b o t t le s fo r the f in a l
t e s t s to be d iscu ssed la t e r .
I t might be p e r tin e n t at th is p o in t to give a more d e ta ile d d isc u ssio n
o f the drying and grinding o f the sam ples, sin ce i t was found that c e r ta in
step s f a c i l i t a t e d la t e r treatm ents.
I t was found th=t o ccasion al s t ir r in g o f the residu e not only shorten­
ed the time necessary fo r drying but in the case o f the unwashed acid
residu e i t g r e a tly increased the ease w ith which the resid u e could be han­
dled during the f i r s t grinding p r o c e ss.
I t was found that in many cases
t h is acid r esid u e, when dry, formed an extrem ely hard mass which could be
handled by the grinder much more e a s il y when in sm all chunks, formed by
o c c a sio n a l s t ir r in g during the drying p r o c ess.
Since the grinder used did
not g iv e the residu e the d esired degree o f fin e n e s s , p u lv e r iz a tio n was
continued in a la rg e mortar u n t il the d esired fin e n e ss was achieved .
?re-
queat s t ir r in g o f the washed resid u es during drying was not as e s s e n tia l
sin ce in t h is case the residue did not form hard m asses, and u su a lly only
p u lv e r iz a tio n in the mortar was a l l th at was necessary to ob tain the de­
- 9 sire d degree o f fin e n e s s fo r the dry weshed resid u e.
In the second procedure, the c e llu lo s e samples were reflu x ed with the
hyd roch loric acid fo r v a rio u s p eriod s o f tim e.
Under th ese c o n d itio n s, i t
w»s found by previous t e s t s that about 3 # HCl was the h ig h e st concentra­
tio n th at could be used w ithout extreme d is c o lo r a tio n o f the residue a fte r
long p eriod s o f r e flu x in g .
On t h is b a s is , 3 H HCl wee used in th is proced­
u re.
At recorded in te r v a ls o f tim e, r e flu x in g o f the sample was stopped and
the acid residu e suspension was d iv id ed in to two eou el p a r ts .
Both p a rts
were allow ed to s e t t l e a few minutes and the supernatant liq u id was decan­
ted .
One o f the h a lv es was then n e u tr a liz e d w ith ammonium hydroxide, w hile
the oth er was l e f t a c id .
Both p a rts were then f i l t e r e d by g r a v ity f i l t r a ­
tio n and the resid u es d r ie d , ground, washed, e t c . , as b e fo r e .
Washing on
th ese samples was contin ued, however, u n t i l e l l so lu b le c o lo r in g matter
was removed to a p o in t where i t would not a ff e c t la t e r co lo rim eter t e s t s .
This r e flu x in g procedure was a lso ca rried out on a few samples u sin g
0 .5 N HCl in stea d o f 3 N HCl.
3.
S aliva Treatment o f the H ydrolysis Residue
It
wag
found by previous t r ia l runs th at room temperature was s u f f i­
c ie n t to g iv e good s a liv a amylase a c tio n , and that 2k hours was more than
s u f f i c ie n t time to complete any p o s s ib le h y d ro ly sis o f the residu e by the
amylase at t h is tem perature.
T ests a lso showed that r eg u la tio n o f the pH
was not n ecessary fo r t h is work, end th at the ch lo rid e ions in the s a liv a
were adequate fo r the a c tiv a tio n o f the am ylase.
In a l l samples d ilu tio n
was made in the amount o f one part o f s a liv a to f iv e p arts o f water by
10 volume.
D ilu tio n o f the s p llv s appeared to Introduce no dlerdvent^ see fo r
t h is work but h»d the pdventege o f d ecresein g the volume o f e e lt v s neces­
sary.
A fte r c o lle c t io n , the s a liv a was f i l t e r e d to remove eny solid, m atter
which might be p r e se n t.
The method o f preparing samples to be run and n ecessa ry c o n tr o ls is
probably b eet d escribed by the fo llo w in g model experim ent.
IlzJ tk I
0 .5 - 1 .5 g residu e + $ cc o f s a liv a f ?5 co o f EgO f Toluene
F lssk 2
(F ir s t C ontrol)
0 .5 - 1 .5 g residu e r JO cc o f HgO *• Toluene
Flask 3. ( Second C on trol)
5 cc o f s a liv a + 25 oc o f HgO f Toluene
The presence o f Toluene in each f la s k was to insure a g a in st any bac­
t e r i a l a c tio n .
Tlie f la s k s were then stoppered and allow ed to stand about
24 hours a t room temperature w ith several shakings.
At the end o f the
tim e, the m ixtures were f i l t e r e d and the f i l t r a t e was preserved in w e ll
stoppered c o n ta in er s.
The f i l t r a t e from f la s k I contained reducing sugars produced by amy­
la s e a c tio n on the resid u e plus any r e s id u a l reducing sugars in the residue
p lu s reducing fr a c tio n s which might be p resen t in the s a liv a .
Ihe q u an tity
o f reducing substances from these l a s t two sources can be determined in the
f i l t r a t e s from fla s k s 2 and J r e s p e c tiv e ly .
4.
Method 0f r.adlining Suzar D eterm ination
Except fo r minor v a r ia tio n s , the method used fo r reducing sugar d e te r ­
m inations on the f i l t r a t e i s the same as th at of Folln-Vvu^ ^ and B e n e d lc t^ ^ .
- 11In the Polin-Wu blood sugpr determ ination, the blood f i l t r p t e Ie mpde pro­
te in fr e e to elim in a te m y reducing groups due to p r o te in s , but fo r t h is
work t h is precaution i s not n ecessary sin c e pny red uction due to the pro­
te in s in the e<=live pre very sm ell end would e lso be included in the
second c o n tr o l mentioned pbove.
B r ie f ly o u tlin e d , the theory o f the Folin-Wu determ ination i s *s
fo llo w s!
the f i l t r a t e is heeted w ith en s I k e lin e copper s o lu tio n , end the
reducing augers in the f l i t r e te qu en tI t e t lv e ly reduce the cupric to the
cuprous io n .
The cuprous s e l t formed i s then perm itted to r e e c t w ith moIyb-
detephoephpte s o lu tio n .
The molyb d ete i s p e r tl e l l y reduced by the cuprous
ion to low er reduction products o f blu e c o lo r , the in t e n s it y o f which i s e
measure o f the m ount o f copper reduced to the cuprous e t e t e end, th er e fo re ,
the emount o f suger p r e se n t.
Procedure:
Two cc o f the unknown end two cc o f e stenderd glucose
s o lu tio n were tran sferred to eep erete Folin-Wu blood auger tu b es.
Two cc
o f e lk e lln e copper reegent were edded to each tube, end the tubes were
w e ll sheken.
The tubes were p ieced in b o ilin g w ster fo r s i x to e ig h t min­
u te s a ft e r which they were removed end cooled in e co ld w ster bath fo r three
m inutes.
Two cc o f phoephomoIybdIc a cid reegent were added subsequently to
each tube end the co n ten ts w e ll mixed by shaking fo r one m inute.
The so lu ­
tio n s were d ilu te d w ith w ater to the 25 cc mark on the tu b es, a ft e r which
the stoppered tubes were in verted se v e r a l tim es to in su re complete mixing,
and the con ten ts were then immediately compared c o lo r im e t r ic a lly .
For th is
work, i t was found th at two or three unknowns could be compared w ith e ch
— 12 ™
*
s tendprd in e. short enough time lc p se so th et no error wes Introduced hy
c o lo r chenges due to sten d ln g .
R esa en ts:
AIk rI Ine cooper s o l u t io n ,
ho g o f pure pnhydrous sodium carbonate
were d is s o lv e d in shout hOD cc o f w ater and tra n sferred to a I l i t e r v o lu ­
m etric f la s k .
7 .5 0 g o f ta r ta r ic acid were added and when d is s o lv e d U.5 g
o f c r y s t a llin e copper s u lfa t e ( CuSO^e SHgO) a lso added.
The con ten ts were
mixed and made up to the mark w ith w ater and stored in an amber b o t t le .
Phos^hoiaolvbdlc acid reagen t.
To 35 S o f moly b d ic acid and 5 S o f
sodium tu n g sta te were edded 200 cc o f 10$ sodium hydroxide and 200 cc o f
w ater.
The mixture was b o ile d in a. beaker fo r th ir ty m inutes, keeping the
volume approxim ately con stan t by the a d d itio n o f water from time to tim e.
The s o lu tio n was then cooled to room temperature and tran sferred to a $00
cc volum etric f la s k .
were added.
12$ cc o f concen trated phosphoric acid ( syrupy 8$$)
The con ten ts were w e ll mixed, d ilu te d to the mark w ith water
and stored in »n amber b o t t le .
Rtondard glucose s o lu tio n .
The standard glu cose s o lu tio n was pre­
pared from a stock g lu co se so lu tio n o f convenient co n cen tra tio n .
A satura­
ted b en zoic acid s o lu tio n was need as the so lv en t in a l l c a se s to prevent
any b a c t e r ia l a c tio n , and a l l glu co se s o lu tio n s were stored e ith e r in amber
b o t t le s or kept in the dark.
>11 standard so lu tio n s were compared again st
a standard glu cose s o lu tio n from the Bureau o f Standards to determine th e ir
c o n cen tra tio n ,
io r t h is work, standard co n cen tra tio n s from 0 . 020- 0.030 mg
g lu c o se /c c gave the b eet r e s u lt s .
- 135-
gmrnnle Cm lctiletlon*
C elcu lp tlon a were mede w ith the weshed a cid hydrolyzed residu e on es
near es p o s s ib le dry w eight h e a ls .
I h ls wee achieved by drying weighed
samples o f the resid u e In a vacuum oven a t about 73°C fo r about 12 hours.
The lo s s in w eight was a measure o f the water content o f the residue and
from t h is the percentage o f n o n -v o la tile m atter In the resid u e was c a lc u la ­
te d .
Samples o f the residu e to be hydrolyzed w ith amylase were not dried
w ith h e a t, in order to avoid any p o s s ib le changes In the stru ctu re o f the
resid u e due to the e lev a ted temperature and extreme d rying.
A common K le tt v is u a l colo rim eter was used to make a l l q u a n tita tiv e
reducing sugar d eterm in ation s.
With th is type o f co lo rim eter, the fo llo w ­
ing equation i s employed in the c a lc u la tio n o f mg o f sugar.
R6Zr U
x c s x v Zt x d V
d S = Cu
Where:
Rg i s the reading fo r the standard s o lu tio n .
Rq i s the reading fo r the s o lu tio n under t e s t .
Cg i s the number o f mg o f sugar in the standard s o lu tio n .
V
is the t o t a l volume o f the unknown.
v
i s the volume o f unknown used in making the co lo rim eter t e s t .
Du i s the volume to which the unknown is d ilu te d .
Dg i s the volume to which the standard i s d ilu te d .
Gu i s the mg o f sugar per t o t a l volume o f unknown.
The fo llo w in g model rep resen ts the c a lc u la tio n s in volved fo r a ty p ic a l
determ ination
- 14 .Unknownt
992.2 mg o f resid u e (9 0 .6 5 n o n -v o le tlle ) f 25 cc HgO
5 co s& live
1 st C o n tro l:
1019.8 mg o f residue (9 0 .6 $ n o n -v o la tile ) + 30 cc HgO
2nd C o n tro l;
25 cc HgO f 5 cc s a liv a
lHie t o t a l co n cen tra tio n o f g lu co se in the standard s o lu tio n used fo r
the c o lo rim eter t e s t Is 0.022 mg/co tim es the 2 cc used which g iv e s O.Ohh
mg.
The colo rim eter readings were e s fo llo w s:
Standard s o lu tio n . . . . . . . . . . .
Unknown . . . . . . .
.................. . . . .
1 5.0
5*5
1 s t c o n t r o l ..................................................11.8
2nd c o n t r o l ..................................................29 .0
C a lcu la tio n o f the t o t a l mg o f sugar (C ) in the "unknown" above
u
u sin g the equation g iv en above would he as fo llo w s:
15/ 5 .5 x .o w x 30/2 x 25/25 s JLSa MS
(A) 1
Because o f the n e c e s s ity o f c o n tr o ls , the fo llo w in g procedure must he
follow ed In ob tain in g the f in a l answer.
By c a lc u la tin g the " 1st control" end "2nd control" by the same equa­
tio n as above we ob tain :
t o t a l fo r the "1st control" i s 0 .8 4
(B) which i s the reduction due
to r e sid u a l reducing substances In the resid u e.
T otal fo r the "2nd control" i s 0 .3 4 mg (C) which Is the reduction due
to reducing substances In the s a liv a .
A ll such va lu es are la b e le d In t h is way fo r convenience.
- 15By RTiBtrpc tin g (C) from (« ) we oB tein:
1 .8 0 - .Jh - I.it6 a g
(D)
which i s the to t p l mg o f gngpr in the Hmknownw due only to em ylese a ctio n
end r e sid u a l reducing euhstsnces in the r e sid u e .
By p u ttin g the resid u e in the "unknown* end "1st control" on a dry
w eight h e e ls , we obtains
fo r the "unknown"s
fo r the "1st co n tro l" :
9 9 2 .2 x .906 = 8 9 8 .9 ma
(B)
1019.8 x .906 = 921.9 mg
(i)
D iv id in g (D) by (B) g iv e s the mg o f sugar per mg o f resid u e due only
to amylase a c tio n and r e sid u a l reducing substances in the resid u e.
1 .4 6 /8 9 8 .9 = 1,625 % 1Q~?
(S)
D ivid in g (B) by (? ) g iv e s the mg of sugar per mg o f resid u e due to
r esid u a l reducing substances in the r e sid u e .
.8 4 /9 2 3 .9 s ,,m . & IQ"3, m f a z
(H)
By su b tractin g (H) from (» ) we o b ta in the mg o f reducing sugar per
mg o f resid u e a c tu a lly due to amylase a c tio n .
1 .625 X IO"3 - .909 X IO- 3 - _tZL£ x ISl I B z lm
(I)
M u ltip lyin g ( I ) by (E) g iv e s the t o t a l mg o f sugar in the "unknown*
a c tu a lly due to amylase a c tio n .
• 716 x IO"3 x 6 9 8 .9 = .6 4 g g
(J )
or 0 .0 7 2 % con version , on a dry w eight b a s is , o f the HCl acid hydrolyzed
resid u e to reducing sugars by amylase a c tio n .
16 I*
RESULTS /ND CONCLUSIONS
JL.
R eeu lts o f Concentrated H ydrochloric Acid Hydrolyglu fo r Ysrioug
Lengths o f Time at Room Temperature.
The resid u es fo r Tables I through YII were prepared by concentrated
h yd roch loric acid h y d r o ly sis a t room tem perature.
The time the c e llu lo s e
spent In the acid s o lu tio n and the time required to dry the a cid residue
heads each t a b le .
The lon ger the c e llu lo s e was in co n ta ct w ith the acid
s o lu tio n , the more d i f f i c u l t i t became to a ir dry the la rg e samples due
to the Ir changing c o n s iste n c y .
the same r e sid u e .
about three months.
The resid u e used in Tables IV and V i s
They vary only by the time lapse between them, which i s
Table IV was the f i r s t t e s t made, o f course, and the
rath er in t e r e s t in g r e s u lt s which they show w i l l be more thoroughly taken
up l a t e r in the d is c u s sio n and c o n c lu sio n s.
In a l l the fo llo w in g ta b le s where they are used, a l l unknowns and
c o n tr o ls were prepared according to the fo llo w in g standard procedure and
nom enclature.
Since th ese step s fo r each ta b le are ju s t a r e p e titio n o f
those b e fo r e , i t i s not deemed n ecessary to repeat t h is introd uctory part
fo r each s e t o f ta b le s .
"Unknown" ( I and 2) - residu e -f 25 cc H2O + 5 cc s a liv a
" 1st control" (3 and U) - resid u e + 3U cc H2O
"2nd control" (5 and 6 ) - 2$ cc HgO f 5 cc saliva.
The t o t a l amount o f sugar used in the standard was O.Ohh mg.
TABLE I.
Tlae In Cone. Acid S o lu tion - 3 Hour8.
Drying Tlae - 3 Lays
T otal
Sugar
In mg
Olucose
:
No.
$ NonV o la tile
Residue
Dry Wt.
Residue
Bg
Standard.
Color.
S e ttin g
C olor.
Reading
:
:
:
i
w t.
Residue
Bg
I
916.2
95-9
8 7 8 .6
10.0
2 7 .4
.24
.24
2
960.8
95-9
9 2 1 .4
10.0
2 8 .5
•23
-.23
3
8 2 2 .6
95-9
788.9
10 .0
0 .0 0
4
8 3 1 .6
9 5 .9
7 97.5
10.0
0 .0 0
5
10.0
0 .0 0
6
10.0
0 .0 0
$
S
;
:
t
%
I
:
i
i
:
i
:
:
Time in Cone. Acid S o lu tio n - 18 Hours.
Wt.
Residue
mg
NonV o la tile
Residue
Dry Wt.
Residue
Bg
Standard
Color.
S e ttin g
C olor.
Reeding
I
786.9
95-2
7 49.1
15.0
12 .0
2
995-9
9 5 .2
948.1
15.0
14.0
.71
3
1043.2
95-2
993.1
15-0
19.0
.52
k
1042.3
95-2
992.3
15.0
2 6 .2
.38
5
15.0
1 6 .5
.60
6
14.0
2 0 .0
.40
No.
T otal
Sugar
in mg
Olucose
I
;
:
i
%
Conv e r sio n
.02?
:
.024________ :
$
0.0 0 ev e. mg suger/mg :
r e s . not due to pmylaee:
a ctio n
!
0 .0 mg ev e. sugar/30 cc:
sample due to s e llv a
:
Sugar by
Amylese
coav. In
og Olucose
Conv e rsio n
:
:
:
i
N egative r e s u lt s , there-:
fore not s ig n if ic a n t
:
.453 i ICT^ av e. mg
sugar/mg r e s . not due
to amylase a c tio n
I
t
J___
:
:
i
:
Drying Time - 5 Dsya
a
TABLE I I .
Sugar by
Amylaee
cony, in
mg Olueose
•55 Bg ave. t o t a l
sugar/30 cc sample
due to s a liv a surer
:
:
;
♦
:
:
:
!SABLE I I I .
t
I
t
i_
I
I
Time In Cone. Acid S o lu tio n - ZZ Hours.
Dry in/? Tima - §, Dpvs
Ho.
Wt.
Residue
mg
^ NonV o lp ttle
ResIdue
Dry Mt.
Residue
=g
Stendsrd
Color.
S e ttin g
I
1175.7
9 5 .7
1125.1
15.0
11 .0
.90
2
1154.6
9 5 .7
1105.0
15.0
14 .2
•70
:
3
1101.6
9 5 .7
1054.2
15.0
3 1 .5
• 31
:
4
1171.8
9 5 .7
1121.4
15.0
3 9 .0
.25
*:
:
!
5
15.0
26 .5
.37
6
15.0
10 .0
.33
TABLE IT.
Time j a Cone
C olor.
Resding
Acid S o lu tio n - 42 Hours.
T otal
Sugar
in mg
Qlurto SR
Sugar T$y
Amylase
cony, in
mg Gluqose
£
Coxvv ersio n
t
t
:
$
I
.26
.023
!
$
.0 7
.01
,
:
.258 x 10” ' ave. mg
:
sugar/mg r e s . not due ;
to antsrlase a ctio n
•
•35 ag ave. t o t a l
sugar/3 0 ce sample
:
:
Drylnz Time - 12 pfiyg
:
I
Mo.
Mt.
Residue
mg
NonT o le t ile
Residue
Dry Mt.
Residue
mg
Standard
Color.
S e ttin g
C olor.
Reeding
:
I
992.2
9 0 .6
8 9 8 .9
15.0
5 .5
1.8 0
.60
.067
j
i
2
1240.4
9 0 .6
1123.8
15.0
4 .5
2.2 0
. .78
.069
t
i
3
1019.8
9 0 .6
923.9
15.0
11.8
.8 4
:
4
1130.1
9 0 .6
1023.9
15.0
9 .5
1 .0 4
!
t
:
5
15.0
29 .8
•33
6
15.0
29.0
.1 4
T otal
Sugar
in tag
Sugar by
A aylese
conv. in
$
Conv ersio n
:
:
:
.962 X 10 3 ave. tag
;
mi»»r/mg r e s . not due •
to amylase a ctio n
•
.34 iug ave. to ta l
sugar/3 0 cc sample
due to s a liv a suzer
s
j
IT*BUS Y.
S
I
t
•
:
i
;
i
t
i
t
I
Hours.
Drying Time - JZ Dwys
w t.
B esIdue
mg
i SonY o lr t ile
Residue
Dry Wt.
Residue
Standard
C olor.
S e ttin g
C olor.
Reeding
I
9 1 9.5
9 6 .4
8 8 6 .4
20 .0
5*5
2 .4 0
.0 4
.005
:
2
955*3
9 6 .4
9 2 0 .9
20 .0
5*0
2 .6 4
.20
.022
i
3
1223.9
9 6 .4
1179.8
20.0
5*5
2.40
U
1124.1
9 6 .4
1083.6
20.0
5*5
2.40
5
10.0
13*0
6
. . 10, 0-.....
14 .2
So.
TABU T I.
$
I
i
gjme In Cone. Acid S o lu tio n -
Tlmg i a Cons
Jsid S o lu tio n - 48 Hours.
T otal
Sugar
in ag
Oluso Eft
Sngar by
Amylase
conv. in
mar O lusose
i
Conv e r sio n
:
:
;
2.11 x 10"3 J5Te. rag j
sugar/mg ro e. not due:
to. amylase a c tio n
.49 mg a r e . t o t a l
su gar/30 cc sample
____ i42___ due to s a liv a sugar
*51
:
:
;
Drying Time - JLi Pays
Ho.
Wt.
Begldue
mg
,6 SonV o la tile
Residue
Dry Wt.
Residue
Standard
Color.
S e ttin g
Color.
Reading
T otal
Sugar
in mg
01 uso Ra
I
1002.3
9 6 .2
964.2
15*0
16.5
.60
2
997.9
9 6 .2
9 60.0
15.0
19.5
*51
3
1026.4
96 .2
9 8 7 .4
5*0
23*5
.1 4
4
1092.7
9 6 .2
1051.2
15.0
3 4 .5
.29
5
15.0
33*2
.30
6
1=5.0
3 2 . <5
.31
Sugar by
%
Aaylase
Conconr. in
v ersio n
mg Olusnsa
t
i
:
t
.09
.009
:
negative r e s u lt s ,
:
th erefo re not sig n I . :
.209 x 10” ^ a re. mg ;
sugor/rag r e s . not due:
to amylase antion
r
.31 mg s v e . t o t a l
su gar/30 cc sample
due to s a liv a sugar
:
:
:
Table V II.
Time 1& Gone. Acid S o lu tio n - ^
S o u rs.
Drying Time - 2 0 . Devs
:
I
Ho.
Wt.
Residue
mg
$ NonT o le t lle
Residue
Dry Wt.
Residue
Stenderd
Color.
S e ttin g
:
t
:
i
J
I
!
:
:
t
!
I
1018.5
9 6 .3
980.8
15.0
1 5 .5
.64
2
1028.1
9 6 .3
990.1
15.0
13 .5
.73
3
1091.8
9 6 .3
1051.4
15 .0
2 6 .0
.38
4
1022.9
9 6 .3
985.1
15.0
2 9 .0
.34
5
15.0
2 5 .5
•39
6
K .O
C olor.
Reeding
- J L L J ____
T otel
Suger
in mg
Glucose
.01
Soger by
Amyleee
conv. in
mg Slncnsn
$
Conv e r sio n
:
:
I
;
n eg a tiv e r e s u lt s .
t
th erefo re not e tg n i. :
.03
.003
:
:
•353 x IO- 3 e v e. mg :
suger/mg r e s . not due:
to fimvlese pctlor^
:
:
.35 mg e v e. t o t e l
S
suger/30 cc sample
i
..due to s e I I vs surer JL
- 21 For c lp r lf lc p t lo n ?nd to f a c i l i t a t e comparison o f the r e s u lt s In
Tables I through FIT, the uercentc^e conversions fo r each ta b le have been
compiled, in Table F i l l below .
TABLS F i l l .
Co mulled. R esu lts o f Tables X through FII
$
t
:
t
l
I
;
t
:
Table
No.
Time in
Acid S o lu tio n
Hre.
Drying
Time
Days
Unknown
I
3
3
I
2
II
18
5
I
2
t
:
I
!
!
S
t
I
$
I
I
$
t
I
I
I II
22
8
I
2
.023
.01
IF
UZ
12
I
2
. 06?
.069
F
42
12
I
2
.005
.022
FI
48
15
I
2
.009
n eg a tiv e V=Iue
FII
66
20
I
2
n eg a tiv e value
.003
Conversion
.02?
.025
n eg a tiv e value
n eg a tiv e value
l
:
I
:
:
:
Z
:
i
t
!
t
!
i
t
S
:
t
I
t
i
t
:
t
t
D is c u ss io n :
B arely during the course o f th ese end la t e r d eterm in ation s was i t pos­
s ib le to ob tain r e la t iv e l y c lo se r e s u lt s fo r d u p lica te sam ples.
Therefore,
only trends in r e s u lt s could be compered w ith any s a t is f a c t io n .
P o ssib ly
more experim ental study and refinem ent o f technique could elim in a te these
d is c r e p a n c ie s, but the accomplishment o f t h is was beyond the time e llo te d
fo r t h is paper.
22 From Tsble V III i t csn be seen th at h2 hours ( Tsble IV) o f sold hydro­
l y s i s under th ese co n d itio n s sppeers to have given s resid u e w ith the high­
e s t percentage o f conversion to reducing sugars by p t y s lln a c tio n .
Above
end below t h is time o f acid h y d r o ly sis (w ith the exception o f 18 hours where
the value was zero end Table V which is on the same resid u e as in IV as ex­
p lain ed above), there is a general decrease in percentage conversion w ith
d ecrea sin g and in c r ea sin g tim es o f acid h y d r o ly s is .
#t the time o f preparation o f th ese r e sid u e s, each was washed u n t il
I t gave both a n eg a tiv e Folln-Wu reducing sugar and ch lo rid e t e s t .
However,
when determ inations were run on th ese resid u es a month to h5 days la t e r ,
each, w ith the ex cep tio n o f the 3 hour r e sid u e , contained r esid u a l reducing
sugars, which becomes evid en t in the "1st control" (se e t a b le s ) .
A c lo s e r in s p e c tio n o f Tables IV and V, which, as mentioned above, are
determ inations on the same resid u e, w ith IV f i r s t , and V about three months
la t e r , r ev e a ls some p e c u l i a r i t i e s .
Bie f i r s t determ ination gave a much
h ig h er conversion to reducing sugars than did the second.
There wee e ls e
a marked increase in the r e sid u a l reducing sugars in the " 1st control"
which had increased from 0.096# fo r the f i r s t determ ination to about 0 .2 1 #
fo r the second.
Along w ith th ese changes, there was a decrease in the
m oisture content o f the residue ( about 9 .4 # m oisture fo r th e f i r s t and
about 3 .6 » m oisture fo r the second d eterm in a tio n ), even though the resid u e
was kept in a t ig h t ly clo sed co n ta in er, as were a l l the r e s id u e s .
I f fu rth er work should prove the v a lid it y o f these apparent changes,
t h is might prove to be an in t e r e s t in g problem fo r fu rth er study.
— 23 **
Z*
R esu lts Qf 3 N H ydrochloric Acid HydroLvsiB fo r Various Lengths o f Time
a t B o ilin g Temperature
(A)
Amaonlua Hydroxide n e u tr a liz e d p o rtio n
The resid u es fo r Tables IX through XIII were prepared by 3 N hydro­
c h lo r ic acid h y d r o ly sis e t b o ilin g temperature end the time o f h y d ro ly sis
in each case heads each ta b le .
As mentioned e a r li e r , the r e sid u e s from the
3 H h yd roch loric acid h y d r o ly sis were d ivid ed in to two equal p a r ts , one o f
which was n e u tr a lise d w ith HHjjOH s o lu tio n b efo re the f i r s t dlying w hile the
o th er h a lf was l e f t to dry in the acid s t a t e .
The resid u es in the fo llo w ­
ing Tables IX through X III were n e u tr a liz e d b efo re the f i r s t d ry in g .
A ll unknowns and c o n tr o ls were prepared according to the standard pro­
cedure and nomenclature as o u tlin e d p r e v io u sly .
TABLE IX.
!
I
:
:
:
:
t
!
:
$
w t.
Residue
-fl MonV o le tlle
Residue
Dry Mt.
Residue
Stenderd
C olor.
S e ttin g
I
679.2
9 8 .0
6 6 5 .6
1 5 .0
2 6 .0
&
S
:
Duration o f I N Acid H ydrolysis - X Hour
2
8 3 5 .2
9 8 .0
8 18.5
15*0
20 .0
.50
3
1 0 1 7 .0
9 8 .0
996.7
1 5 .0
0.0 0
4
7 1 2 .6
9 8 .0
698.4
1 5 .0
0.0 0
No.
5
i-------- 6—
TABLE X.
C olor.
Reeding
1 5 .0
19.0
14.0
20.0
T o tel
Suger
in mg
Glucose
Suger by
Amylese
conv. in
me- Glucose
Conv e rsio n
n eg a tiv e r e s u lt s ,
th e r e fo r e , not
sia rn tficen t
:
:
•
t
0 .0 0 eve. mg sugsr/mg:
r e s . not due to emy- :
Iese a c tio n
•
.5 1 rag ev e. to t e l
Bugsr / 30 cc sample
__ c50___ due to s a liv a su»ar
.5 2
:
:
•
Duration o f 3 E Acid H ydrolysis - % Hours
i
:
t
I
:
:
$
:
No.
Mt.
Residue
mg
t NonV o la tile
Residue
Dry Mt.
Residue
Standard
Color.
S e ttin g
C olor.
Reading
I
797.8
9 7 .4
777.1
10.0
2 3 .O
.29
.01
.001
2
1055.0
9 7 .4
1027.6
10 .0
2 1 .5
.31
.03
.003
i
3
1144.1
9 7 .4
1114.4
10 .0
0.0 0
1
4
1165.6
9 7 .4
1135.3
10.0
0.0 0
j
t
5
T otal
Sugar
in mg
Glucose
i
J________6-----
:
:
:
:
10.0
10.0
22 .0
„ - /Llt.Q___ ....
.30
Sugar by
Amylase
conv. in
mg Glucose
%
Conv ersio n
0 .0 0 ave. mg sugar/
mg r e s . not due to
amylase a ctio n
.28 mg ave. t o t a l
sugsr/3 0 cc sample
due to s a liv a susrar
J
I
t
I
:
:
*
:
t
I
t
i
M
P-
TABLE X I.
:
t
i
:
t
:
i
t
•
:
j
In rp tio n c>£ 3. E. Acid HydroIvcifi - 3. Hourg
No.
Wt.
Residue
ag
$ NonV o la tile
Re6idue
Diy Wt.
Residue
Standard
Color.
S e ttin g
I
8 1 1 .U
97-9
7 9 4 .4
1 0.0
3 0 .0
2
8 0 6 .6
9 7 .9
789.7
10.0
2 9 .0
3
968.0
9 7 .9
947.7
10.0
h
9 5 0 .4
9 7 .9
930.4
10.0
C olor.
Reading
5
10.0
2 7 .0
6
10.0
3 0 .0
T otal
Sugar
in mg
Olucose
Sugar "by
Amyiase
conv. in
hl» Olucose
%
Conv e rsio n
1
t
:
i
t
.22
ne ;et ive r e s u l t s ,
i
th erefo re. not a Ign l- :
f leant
.23
:
t
0 .0 0
0 .0 0 mg av<1. sugar/mg ;
r e s . not due to amy- ;
0 .0 0
Iase a c tio n
:
i
.2 4
.2 3 mg pYe. t o t a l
i
BUgF.r/30 Ct sample due:
i
__ iZZ— . to sa liv a su^rar
I
M
Vn
TABLE X II.
Duration o f I N Acid H ydrolysis - £ Hours
Dry Wt.
Residue
Standard
Color.
S e ttin g
I
1 1 6 5 .2
9 7 .6
1137.2
10 .0
2 2 .0
.30
:
2
1093-6
9 7 .6
1067.4
10.0
3 1 .0
.21
t
$
:
3
1357.0
9 7 .6
1324.4
10.0
0 .0 0
4
1185.2
9 7 .6
1159.7
10 .0
0 .0 0
:
$
:
5
10 .0
3 2.0
.21
6
___ 10 .0
3 4.0
.19
j
!
J
C olor.
Reading
T otal
Sugar
in mg
Oluco se
•
Sugar by
Amylase
conv. in
ms? Olnen se
£
Conv ersio n
:
:
:
i
$
.001
:
— .,O l.,.
I
0.0 0 ev e. mg sugar/mg ;
r e s . not due to Siny- :
Iese fiction
!
:
.20 mg eve. t o t e l
2
sample due:
to e e l ive RU=Tar
%
.10
.009
1
0
CJ
£ NonV o la tile
Residue
No.
R
Zr
Wt.
Residue
mg
i
I
I
TA".
U ur^ticn
3.
*cld HygraLysla - J1= Laa.ra
Ho.
Wt.
Residue
mg
% HonV o le tlle
Residue
Dry Wt.
Residue
Standerd
Color.
S e ttin g
I
1096.4
9 7 .8
1072.3
1 0 .0
25 .0
.26
.0 7
. 00?
2
1143.0
9 7 .8
1117.8
10.0
2 6.0
.25
.0 6
.005
3
1130.2
9 7 .8
1105.3
1 0.0
0 .0 0
1325.8
9 7 .8
1296.6
10.0
0 .0 0
I
:
3
X III.
4
♦
I
5
3
J----- ____ 6___
C olor.
Reading
T otal
Sugar
in mg
Oluco RA
10 .0
3 4 .5
.19
___ m i ___
? 4 .0
.19
Sugar by
Amylase
conv. in
n.7 Hueoee
$
Conv e rsio n
:
:
:
;
!
:
:
0 .0 0 ave. mg soger/mg :
r e s . not due to amy- :
Iase a ctio n
.
2
.19 mg ave. t o t a l
j
sugpr/30 cc sample due:
to sa liv a RUg^r
- 27 For c la r i f ic a t io n and to f a c i l i t a t e coaperleon o f the r e s u lt s In Tab­
l e s IX through X III, the percentage conversion fo r each ta b le hee been com­
p lie d In Table XIV below .
TJBLB XIV.
Table No.
Comnlled R esu lts
Time in Acid
Solu tion -E ra.
Tables U , through XIII
IInknoyn
■ib Conversion
IX
I
I
2
n eg a tiv e
v a lu es
X
2
I
2
.001
.0 0 3
XI
3
I
2
n eg a tiv e
v a lu e s
XII
6
I
2
.009
.001
XIII
15
I
______ 2_________
.00?
.OOS_______
D is c u ss io n ;
Js cen be seen by studying Tables IX through XIII end the compiled re­
s u lt s in Table XIV above, th is p a r tic u la r treatm ent o f c e llu lo s e gave very
low r e s u lt s end the establishm ent o f »ny trends from the d if fe r e n t tim es
o f h y d r o ly sis Is alm ost u s e le s s .
I t Is e v id e n t, however, th a t th is tr e a t­
ment gave much lower end l e s s uniform r e s u lt s then the treatm ent w ith con­
cen trated hyd roch loric acid In the f i r s t s e t o f ta b le s .
None o f th ese r esid u es contained any r e sid u a l reducing sugars, as cen
be seen by r e fe r r in g to the wI s t c o n tr o l” In each ta b le .
This might pos­
s ib ly be explained by the f a c t th at th ese resid u es had a much shorter time
la p se between p reparation end a c tu a l t e s t in g then did those resid u es In
Tables I through V II,
.lost o f th ese r esid u es were te sted w ith in a week
- 28 p fte r th e ir p rep aration ,
(B)
P ortion s d ried in the s o ld atp te
The resid u es in the fo llo w in g ta b le s , XV through XIX, were prepared
» t the seme time »s those under (A) end d iffe r e d from them o n ly in that
th ese completed the f i r s t drying in the acid s t a t e .
This drying process
required about 36 hours, a ft e r which they were washed to n e g a tiv e reducing
sugar end ch lo rid e t e s t s and then prepared and te s te d by the standard pro­
cedure .
TABUS XV.
D u rp tio n o f I H Acid H y d ro ly sis - I Hour
Dry Wt.
Residue
C olor.
Reeding
mg
Stenderd
C olor.
S e ttin g
I
1024.0
9 7 .2
995-3
1 5 .0
15.0
.66
.15
.015
2
9 9 3 .4
9 7 .2
965.6
15 .0
17 .5
.57
.06
.006
3
1020.4
9 7 .2
991.8
15.0
0.0 0
U
1024.6
9 7 .2
995.9
15.0
0.0 0
w t.
Beeidue
5
6
TjtBIiE XVI.
:
:
:
i
i
;
t
:
:
No.
w t.
Residue
mg
$ Non-
V o la tile
Residue
15.0
19 .0
....... I j t-Q____
2 0 .0
T otal
Sugar
in mg
Olurosa
Sugar by
£
Amylese
Conconv. in
v e rsio n
ZM? oiucose
^ NonVole t i l e
Residue
to.
0 .0 0 ev e. mg sugar/mg
r e s . not due to pmyleee
&G%%op
.51 mg ave. t o t a l
euger/30 cc sample due
___ u52____ to s a liv a ?u»faj»
.52
I
M
SO
I
Durp t ion o f I N Acid H ydrolysis - 2 Hours
Dry Wt.
Residue
mg.
Standard
Color.
S e ttin g
C olor.
Reading
To t e l
Sugar
in mg
fll nrn a#
I
1451.3
9 7 .4
1412.6
1 0 .0
17*0
.39
2
1435.4
9 7 .4
1398.1
10.0
16.5
.40
3
1326.7
9 7 .4
1292.2
10.0
3 4 .5
.19
4
1259.2
9 7 .4
1226.5
1 0 .0
35 .0
.19
.30
Sugar by
Amylase
conv. in
»
Conv e rsio n
:
$
:
flg i'll UCflRfi
;
1
negative r e s u lt s .
th erefo re, not s ig n !-
2
t
J
_3
.151 x 10“ ave. mg
sugar/mg r e s . not due
to sa liv a a c tio n
1
$
:
j
I
;
:
5
10 .0
22 .0
2
6
10.0
2<.0
___ ^
.28 mg ave. t o t a l
I
sugar/30 cc sample due I
____ to aaIiva sugar
JL
TABLE XVII.
:
Ho.
t
:
*
:
i
$
;
:
$
:
i
I
t
:
Z
w t.
Residue
ag.
% SonV o la tile
Residue
Dry Wt.
Residue
Stenderd
C olor.
S e ttin g
C olor.
Residing
Total
Sugar
in mg
Ol uen «a
Sugar By
Amylase
cony, in
m.<y OlneegA
%
Conv ersio n
918.2
9 7 .7
8 97.1
1 0 .0
19 .5
.34
.11
.012
2
9 13.6
9 7 .7
692.6
10.0
21 .0
•31
.08
♦009
3
9 9 0 .6
9 7 .7
967.8
10.0
0 .0 0
4
1137.6
9 7 .7
1111.4
10 .0
0.00
5
10 .0
2 7 .0
.24
6
10 .0
3 0 .0
.22
t NonV o la tile
Residue
Dry Wt.
Residue
Standard
C olor.
S e ttin g
I
1118.8
9 7 .8
1094.2
10 .0
16 .0
.41
2
1157.1
9 7 .8
1131.6
10 .0
1 4.5
.46
3
1106.8
97 .8
1082.4
10.0
0 .00
4
1156.4
9 7 .8
1131.0
10.0
0.00
No.
1
w t.
1
1
:
t
0 .0 0 eve. mg suge r/mg :
r e s . not due to amylase:
a c tio n
.23 mg eve. t o t a l
sugar/30 cc sample due . 1
to e g liv e sugar
Duration o f T M Acid H ydrolysis - £ Hours
Residue
rag
i
:
:
:
l
t
t
:
:
:
I
I
TABLE XVIII.
t
i
I
:
$
$
%
:
Duration o f I N Acid H ydrolysis - 2. Hours
Colo r .
Reading
5
10 .0
3 2 .0
____ 6____
10.0
35.0
T otal
Sugar
in mg
Glucose
.21
— U.9..
Sugar by
Amylase
Conv. in
rayr Glucose
£
Conv ersio n
j
1
:
z
!
.21
.019
:
I
.26
.021
i
:
0 .0 0 eve. mg suger/mg :
r e s . not due to pray- •
Ia se a c t i o n
•
I
.20 mg rr e . t o t a l
z
sugar/30 ce sample
z
due to s a liv a susrar
t
TABLE XEjL
C olor.
Beading
Ja NoaT o lp tile
Besidue
Dry V t.
Besidue
Stpndard
Color.
S e ttin g
1134.2
9 8 .2
1113.8
1 0 .0
19 .0
•35
.16
.014
2
1134.5
9 8 .2
1114.1
10.0
2 1 .0
.31
.12
.011
3
1325.3
9 8 .2
1301.4
10.0
0.0 0
4
1146.4
9 8 .2
1125.8
10.0
0 .0 0
:
i
i
:
I
i
t
No.
I
3
i
i
«
:
:
:
:
J___
D ur?tlos pf 3 K Acid HydroEyRis - J 5 Hours
Y t.
Beeidue
=&
T otal
Sugar
in ag
Olueoee
5
10.0
3 4 .5
.19
6
10.0
I-LO
___i l l
Sugar by
Amylase
eonv. in
m» Crluooee
$
Conv e r sio n
:
t
:
:
}
:
:
0 .0 0 ave. mg sugar/mg :
r e s . not due to amy- $
Ie e e a c tio n
...
:
:
.19 mg a r e. t o V l '
:
sugar/3 0 cc sample
d u e to p allva suffer
:
- 32 As b e fo r e , the r e s u lts o f T ebles XV through XlX have been compiled In
Table XX below.
TABLS XjL
I
;
I
i
I
»
C om piled Re s u i t s o f T a b le s XV th r o u g h XIjf
Table No.
Time In Acid
R olutlnn-H rs.
XV
I
XVI
2
Unknown
I
2
.
I
2
I
:
XVII
3
I
2
I
!
XVIII
6
I
2
XIX
15
I
2
J__
:
Kt Conversion :
:
.015
I
i
.006
$
n eg a tiv e
;
:
v a lu es
I
.012
X
:
.009
S
:
.019
.023
:
I
.Olh
I
.011
-L
D is c u ss io n :
Drying in the acid s ta te g r e a tly increased the y ie ld from these r e s i­
dues as compared to the same duration o f h y d r o ly s is in Tables IX through
X III.
I t i s apparent, th e r e fo r e , that the resid u es were s t i l l hydrolyzed
by the concentrated hyd roch loric acid which r e su lte d during the drying.
The residu e in Table XVIII then gave the b e tt e r y ie ld .
The absence o f r e sid u a l reducing sugars, w ith the e x cep tio n of Table
XVI, can probably be explain ed again by the same reasoning th a t was given
in Part ( >) .
2,-
R esu lts o f 0 .4 S H ydrochloric Acid H y d ro ly sis at B o ilin g Temperature
The r e s u lt s from the treatm ent o f c e llu lo s e with 0 .5 S hydrochloric
a c id were not s ig n if ic a n t ly d if fe r e n t from th ose obtained on resid u es from
the 3 & h yd roch loric acid h y d r o ly sis: th e r e fo r e , i t w i l l not be necessary
- 33 *
to en ter tp b les shoving these r e s u lt s .
I t should s u ffic e to et? be th et the
con cen tration o f the meld e t th ese low co n cen tra tio n s app arently had l i t t l e
e f f e c t upon the f in a l r e s u lt s , even when the h y d ro ly sis was carried out a t
the elev a ted tem peratures employed,
h.
Conclusions
B efore proceeding fu rth er w ith t h is d is c u s s io n , i t might be w ell to
s ta te again the major purpose o f t h is works
to study the e f f e c t c f p ty a lin
h y d r o ly s is o f in so lu b le c e llu lo s e resid u es r e s u ltin g from inorganic acid
h y d r o ly s is o f c e ll u lo s e .
A study o f the preceding ta b les would seem to in­
d ic a te th at p ty a lin h y d r o ly sis o f the in so lu b le c e llu lo s e r e sid u e s does
take p la c e , although i t s amount i s rather v a r ied throughout the d iffe r e n t
r esid u es te s te d , and the t o t a l amount o f h y d r o ly sis was not as la rg e or
sp ecta cu la r as i t was hoped i t might b e.
I t i s the b e l i e f o f t h is author,
however, th at fu r th e r study and m o d ific a tio n s o f techniques employed could
g r e a tly Increase the desired o b ta in a b le r e s u lt s ,
( n)
Pacsu end H ille r r
have shown that the stru ctu re o f c e llu lo s e i s be­
lie v e d to be a l a t t i c e work o f long bets-D -glucopyrenose c h a in s, which are
held togeth er by the h em lacetal lin k a g es o f g lu c o se, c e llo b io s e , c e ll o t r i o s e ,
e tc .
They go on to s ta te th at h y d r o ly sis o f c e llu lo s e by week acids does
not hydrolyze the !,h - g lu c o s id ic bonds o f the primary chain, but rather
hyd rolyzes hem lacetal bonds which connect the primary chain m olecules to­
g e th e r , thus g iv in g r is e to secondary chain m olecu les.
This p ro cess, they
s t a t e , r e s u lt s in a lea ch in g -o u t o f the sm aller m olecules such as g lu c o se,
c e llo b io s e , e t c . , le a v in g a resid u e o f la rg e primary ch a in s.
According to
Pacsu and H ille r , the hem lacetal bonds are r e a d ily broken by week acids but
the l,h -g lu c o B ld lc bonds o f the primery m olecule chains ere extrem ely
r e s is t a n t even to h y d r o ly sis w ith h ig h ly concentrated a c id s and are, th ere­
fo r e , broken only very slow ly.
I f t h is theory o f Pscsu end H ille r Is to be accepted, then the c e llu ­
lo s e r e sid u e s, known as h y d r o c e llu lo se , which are used In t h is work, can
o n ly c o n s is t o f primary m olecular chains o f the b e ta -1 ,h -g lu c o s ld ic ty p e.
I t does not seem reason ab le, however, to assume th at a l l the bonds
In th ese h y d r o c e llu lo se chains are o f the b e ta -lin k a g e type e.s supposedly
found In normal c e llu lo s e sin c e I t Is w e ll e sta b lis h e d th a t p ty e lln Is
on ly a c tiv e on lin k a g es o f the alpha-type as found In common starch .
There Is a lso l i t t l e reaeon to doubt the alpha s p e c i f i c i t y o f the ensyme.
>11 through t h is work the In d ica tio n s were th a t there was only a c er ta in
lim ite d amount o f the given resid u e which could be attacked by p ty a lln ,
and len g th o f time o f amylase con tact w ith the residue or treatment w ith
fr e sh enzyme did not lncreaee t h is given lim ite d y ie ld .
This would fu r th e r
seem to elim in a te any p o s s i b i l i t y o f p t y e lln a c tio n on b e ta -lin k a g e s .
(o )
I t might be w e ll at t h is p o in t to in clu d e a statem ent by StarkX7/ who
Ie o f the opin ion th at the products o f the a c tio n o f p t y e lln on starch in­
clude g lu c o se , m altose, and “an array o f nonferm entable copper-reducing
p o ly sa cch a rid es" .
U n til fu rth er work has proven oth erw ise, i t is not un­
reasonable to assume th at the products o f the a c tio n o f p ty a lln on hydro­
c e llu lo s e are the same as those which Stark b e lie v e s are obtained by the
a c tio n o f p ty a lln on sta rch .
I t i s a lso b e lie v e d th at s a liv a amylase Ie
composed o f two fr a c t io n s , th at c e lle d beta-am ylase or m altogenic amylase
which a tta ck s the long elp h e-D -l,b -g lu co p y ren o ee chains o f sta r ch , and
- 35 th e t pprt c e lle d elphe-pngrlese or dextro^ enlc eiaylaee, which atteck e the
g lu c o s e , m pltose, e t c . , cro ss lln lcfg es which hold togeth er the long l , k g lu c o s id ic chains by p lp h e-h em iecetel lin k a g e s.
Keeping In mind the previous d is c u s s io n , cm attempt w i l l now be made
to d eriv e p s a tis fa c to r y exp lan a tio n as to how and where p t y s lin might
a tta ck h y d ro c e llu lo se r e sid u e s.
Even though Pscsu and H ille r have sta te d th at ! ,b - g ly c o s id ic lin k ages
ere broken only by strong a c id s , i t i s not beyond the realm o f reason to
assume th a t some o f th ese lin k a g es w i l l be broken whether the acid is
strong or weak, the g r ea ter h y d r o ly s is , o f course, being favored by the
stron ger p cid .
I t should, th e r e fo r e , not be out o f order to assume that
some o f th ese broken lin k a g es could reform, in p p rt, as a lp h a -lin k a g e s.
This would g ive an In solu b le h y d ro c e llu lo se m olecule co n ta in in g most o f
the o r ig in a l b e ta -g lu c o s ld ic lin k a g e s and one or more n lp h p -g ly co eid ic lin k ­
ages which would be la b ile to p ty a lin a c tio n .
The r e s u lt s obtained in th is work appear to conform f a i r l y w ell w ith
t h is id e a .
The resid u es o f Tables I through VII were prepared w ith con­
cen trated acid which would favor g r ea ter h y d r o ly sis o f l.h - g ly c o s id ic
lin k a g e s and in crease the p o s s i b i l i t i e s o f reform ation o f th ese lin k a g e s.
On the other hand, the resid u es in Tables IX through X III were hydrolyzed
w ith a d ilu te a c id , a co n d itio n not favorab le to breaking o f l.I t-g ly c o s ld lc
lin k a g e s , and t h is lower percentage o f h y d r o ly sis would g iv e a correspond­
ing lower percentage o f reform ation.
She resid u es in Tables XV through
XIX, w h ile drying, were under co n d itio n s s im ila r to those found in the prep-
— 36 —
Fr a tio n o f the resid u es in Tables I through V II.
The in creased p ty a lin
s u s c e p tib le fr a c tio n over th at o f resid u es d ried in the n e u tr e l sta te in
Tables IX through XIII bears out the more favorab le c o n d itio n s o f concen­
tr a te d sc id .
I f the assumption th at 1 ,4 -g lu c o s id lc lin k a g es csn reform a fte r they
have been broken i s v a lid , then i t should a ls o be V alid to assume that they
could reform s s h em iacatsl lin k a g es ln ste e d o f l,h - g lu c o s id ic lin k a g es.
The reform ation could g ive p lp h s-h em iecetp ls in ste e d o f b e te -h e m le c e te ls,
and again we could have h y d r o c e llu lo se m o lecu les, part o f which would be
s u s c e p tib le to p ty a lin a c tio n .
H em lpcetele are un stable end, as sta ted by
Prcsu rnd H ille r , lin k a g es o f t h is type are r e a d ily broken by weak a c id s .
I t may be th at sm all amounts o f hyd rochloric acid ere bound to the hydroc e ll u lo s e , to complete a lo o se oxonium s tr u c tu r e , during a c id h y d ro ly sis
and i s not removed by washing which would lea v e the di*y resid u e in n poten­
t i a l l y acid s t a t e .
This bound acid In the presence o f the sm all percentage
o f m oisture p resen t in a l l the dried resid u es would favor the h y d ro ly sis o f
the reformed h e a la c e t e l lin k a g e s on long sta n d in g .
These co n d itio n s might
e x p la in the appearance a ft e r a long time o f the so lu b le sugars found in the
w e ll washed resid u es o f Tables I through V II.
This co n d itio n i s p a r tic u la r ­
l y brought out in Tables IV and V, where an increase in so lu b le sugars in
the resid u e was accompanied by a corresponding decrease in w ater content
which was presumably used up, a t le a s t in p a r t, in the h y d r o ly sis o f the
h e a la c e ta l bonds.
Whether sim ila r concepts nave a p p lic a tio n in connection
w ith starch i s , at t h is tim e, e n t ir e ly c o n je c tu r a l.
- 37 The time th st the c e llu lo s e wea in co n ta ct w ith the acid so lu tio n
appears to he an import=nt fa c to r in a l l the s e ts o f r e s id u e s , since above
and below e -jiven time the percent'5?* conversion to reducing sugars by
p t y a lln a c tio n d e c r e s se s.
The r o le that i s played by time i s , however,
not w e ll understood, th er e fo re , i t w i l l probably have to aw ait fu rther
study in t h is f i e l d fo r a s a t is f a c t o r y exp la n a tio n o f i t s in flu e n c e .
In short the a ll- o v e r r e a c tio n o f acid h y d ro ly sis o f h y d ro c e llu lo se
appears to be an equilibrium between the h y d r o ly sis o f l,b ~ g lu c o s id ic
lin k a g e s and the reform ation o f th ese lin k a g es as l,h - g lu c o s id ie lin k ages
and h em le c etsl lin k a g e s.
/!th ou gh the p ic tu re presented above might appear to be a f a ir ly s a t i s ­
fa c to r y exp lan ation , the reader should be warned that a l l th a t can be con­
cluded from t h is work i s th at acid h y d r o ly sis o f c e llu lo s e appears to g iv e
an in so lu b le resid u e which i s part t e l l y la b i le to p t y e lin a c tio n ,
fny
attem pt to e x p la in the changes which have taken p l*ce in the residue
during acid h y d r o ly sis i s s t i l l m ostly theory and much more study along
t h is lin e should be undertaken b efore th ese th e o r ie s , i f proven v a lid , can
be accepted as f a c t s .
— 30 —
II-
SUMMARY
1.
Lpboretory f i l t e r peper wee used ee the source o f c e ll u lo s e .
2.
The c e llu lo s e was hydrolyzed fo r v a rio u s len gth s o f time w ith both con­
cen trated end d ilu t e hyd roch loric a cid and a t room temperature as w e ll
ee b o ilin g tem perature.
A fter th is a cid h y d r o ly sis, the washed end
d ried c e llu lo s e resid u es were allow ed to stead w ith d ilu t e s a liv a
s o lu tio n s fo r about 24 hours, then f i l t e r e d end the f i l t r a t e te ste d
c o lo r lm e tr lc e lly fo r reducing sugars.
3.
The mein o b je c tiv e o f t h is work wes f u l f i l l e d when i t was found th at
in almost e l l c a se s the c e llu lo s e resid u e was apparently p a r t ia lly
s u s c e p tib le to p ty e lin a c tio n .
Since b e ta -lin k a g e s, as found In
normal c e ll u lo s e , are not attacked by p t y e lin , t h is s u s c e p t ib ilit y o f
the resid u e was exp lain ed by assuming th a t In acid h y d r o ly s is some o f
the 1 ,4 -g lu c o s id lc lin k a g e s o f the primary c e llu lo s e chains could be
broken and then reform In an eq uilibrium r ea c tio n , as alpha lin k a g es,
thus making p art o f the resid u e la b i le to p ty e lin a c tio n .
4.
In c e r ta in in s ta n c e s , there were in d ic a tio n s th at the w e ll washed and
d ried re dueI n g -su g a r -fre e r esid u e, upon standing fo r extended p eriod s
in stoppered b o t t l e s , underwent fu rth er changes, sin c e th ere were
found p resen t so lu b le reducing sugars, a f t e r th is tim e, where there
had been none o r ig in a lly .
This was exp lain ed as p o s s ib ly due to the
reform ation o f broken ! ,b - g lu c o e ld ic lin k a g e s as h em ia ceta l lin k a g e s,
which are e a s il y hydrolyzed by weak a c id s .
I f th ese lin k a g e s were
a lp h a-h em ip cetals, they would a lso be su sc e p tib le to p t y e lin a c tio n .
It
was then assumed th at some hyd roch loric a cid was bound to the c e llu lo s e
r e sid u e , p o s s ib ly to complete e lo o se oionium str u c tu r e.
L lbereted
hydrogen ions from t h is bound a cid p lu s the sm all emount o f m oisture
which wee p resen t in a l l dry resid u es could hydrolyse th ese h ig h ly
s u s c e p tib le h e m iecetel lin k a g e s, thus producing the reducing sugars
which appeared in the resid u es a f t e r long p erio d s o f sta n d in g .
The len g th o f time o f acid h y d r o ly sis apparently had an e f f e c t upon
the amount o f resid u e which would be hydrolyzed by p t y s lin .
t h is was brought about was, however, not w e ll understood.
no exp lan ation o f t h is phenomenon wae attem pted.
How
Therefore,
-UOV II.
/CKNOWLBDOMENT
Thle puthor would lik e to tsk e t h is op p ortunity to express h is thcnks
end g ra titu d e to Dr. B. L. Johnson, P ro fesso r o f Chemistry a t Montane State
C o lle g e , w ithout whose kind a s s is ta n c e end guidance th is work could not
have been accom plished; to a l l those who In a more minor y e t not le s s
Important way were Instrum ental in h elp in g com plete th is paper; and la s t
hut c e r ta in ly not le a s t to ay w ife , who, though w ith only a meager knowledge
o f chem istry, was my se v e r e st c r i t i c and source o f c o n so la tio n whenever
problems became d i f f i c u l t or overwhelming.
-UlVIII.
LITBRATUBE CITED AND CONSULTED
(1 )
B en ed ict, S. I . , J . B io l. Chem. 2 2 :141-59 (1931).
(2 )
Edlaf H ., H e lle d sy , T ., end N o rd feld t, S ., C. A. 3Z i35245 (1943)?
(Chea. Zentr. J , 2835-6 (1942) ) .
(3 )
F ie ld , C* H ., C, A. 3 6 :1 8 6 2 (1942); ( B r it . 519,600 (A p r il I , 1940) ) .
(4 )
F o lln , 0 . end Wu, H ., J . B io l. Chem.
(5 )
H ehst, W., C. A. 3 5 ! 56987 (1941)? (F r. 847,587 (O ct. 12. 1939) ) .
(6 )
H ess, K. end Ulmsnn, M., C. A. 35:3310^ (1 9 4 l)j(B e r . T B . 119-36
(1941) ) .
367-74 (1 9 2 0 ).
(7 )
Pacsu, E. end H ille r , L. A ., J r . , T e x tile Res, J . 2 5 :2 4 3 -4 8 , 490-97*
564-70 (1 9 4 6 ).
(8 )
Sen, K. C ., R sy , 3. C. ead T elep a tre, S. E ., C. A. 42LJ290* (1 9 4 8 )|
(Indian J . V et. S c i. 2 2 ; 2 6 > 9 6 (1942) ) .
(9 )
Stark, I . E ., J . B io l. Chea. J1B = 569-77 (19 4 2 ).
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