Tech Bull. Fac. Agr . Kagawa Univ..
POLYSACCHARIDES OF SOYBEAN HULLSTeiiti
(Laboratory of Agricultural Products Technology and Laborator y of Biological Chemistry)
Extensive studies have been made i n this university by KAWAMURA and co-workers on the soybean carbohydrates('-')
.Main attention of these studies was focussed on t h e pol ysacchar ides of soybean cotyldons
WHISTLER and SAARNIO(*) have shown that soybean hulls contained a galactomannan soluble in cold water. They suggested that the soybean hull qalactomannan possessed a structure similar to guaran and other galactomannans from the seeds of leguminous plants. ASPINALL and W H Y ~ E ( ' ) isolated two galactornannans from soybean hulls by extracting soybean hulls with water a t room temperature and a t 60° C.
In this report , soybean hull polysaccharides were fractionated by successive extraction with water, 0.5 % ammonium oxalate, and 5 % sodium hydroxide and the separated polysaccharides were examined for their component sugars by paper chromatography after hydrolyzing with sulfuric acid. Further, the hot- water -soluble polysaccharide was hydrolyzed with Taka-diastase and the formed oligosaccharides were examined for their component sugars and it was presumed that xylose in the h o t . w a t e ~ -soluble polysaccharide was not a mere contamination but an actual component of galactomannan.
1.Fractionation of Soybean Hull kolysaccharides by Successive Extraction 1 1. Sample
The defatted soybean flake used as the raw material was donated by Nippon Koyu K. K. through the courtesy of Mr. Torao Sakakihara, Director of the Mizushima Factory, Kura- shiki, Okayama-ken
.This flake contained
10% moisture and 1 5 % crude oil''').
1.2. Fractzonatzon of Plysaccharzdes
The defatted soybean flakes were pulverized and sieved to collect seed huils. The contamination of hypocotyls was removed by the use of controlled current of air. Then the purified hulls were subjected to the fractionation of polysaccharides according t o a scheme given in Fig 1.
1.3. Detectzon o f Component Sugars b y Paper Chromatography
Each of the polysaccharides ( 0 . 2 g ) was hydrolyzed with 2 N H2SOc in a sealed glass tube by heating in a boiling water bath for
4hrs. The hydrolyzate was neutralized with BaCOs and the formed precipitate was removed by centrifugation and filtration through Toyo No. 5 C filter paper. The filtrate was concentrated to dryness under reduced pressure below 40°C and the residue was taken up in I ml water to be examined by paper
Vol 17, No. 2 (1 966) 11 1 chromatography
.Two. dimsnsional paper chromatography was carried out with phenol-
water (4 : 1) and n-butanol-pyridine-water
(6: 4 : 3) as the solvent systems and 0.3 %
p-anisidine.HC1 in water -saturated $2-butanol as the spraying reagent. The results are shown in Table I.
The hot -HaO-soluble polysaccharide A gave xylose, mannose, galactose,and galacturonic Soybean hulls (MOg)
Extd with I 1 H 2 0 70',
S o l Infol
Concd to v /4 Add 2 vols EtOH P u t
,s!l Dissolve in I 1 H a O Insol
Extd with I I 0 5% (NH,),C,O,. 70" 4 h r s 4 times S p l Inspl Coned to v /4 Add 2 "01% EtOH
-4Add 500 ml Fehltng $01" I P p t Dzssolve I" I I
'[Add '/4 E m r---
.o 5% \NH,),c,o, -sol E ~ , O pelysaeeharide A
ID r y E t O R P p t Aar dry Uot H,O-sol polysaeeharlde A polysaecharide B H C I - E t O H ( I r 9 ) E t O H E t , O Dry
Extd with 5% NaOH. room t e m p , 24 h r s , 3 times I Insol
'i'Made pH 4 5 w ~ t h ( 2 OK) H o t - H 2 0 - s o l polysaeeharxde B ( 0 %)
IAeOH, Ice box, 24 h r s P u t
'[Add 3 w l s EtOH ~ b t Dissolve ~n 200 rnl 5% NaOH S o l P u t
1100 ml Fehll:Lsoln P u t EtOH EtOH ( 0 4 s ) HCI-EtOH (1:Q) 5% NaOH-sol EtOH polyracchar>de A Et.0 5% NaOH sol polysaeehar>dc B F i g . 1 . Fractionation of polysaccharides ( 0 48)
'Table I , Component sugars of the hull polysaccharides Mannose
- - - - -
,Fucose Xylose Ar abinose
StGlucose 0 5 % ( ~ ~ 4 ) 2 ~ 2 0 4 ~ B Galactose Galacturonic acid
StF $I- it
+itt -t - t 5
'tt l - -I I - -- - - tfr
112 Tech. Bull Fac Agr Kagawa Univ..
acid i n the ~ a t i o of 1 : 1.5 : 1 : 1 together with a trace of arabinose and rhamnose. This seemed to be very similar to the galactomannan of WHISTLER and SAARNIO'~), but t h e presence of xylose and galacturonic acid was a marked difference of the two polysaccha- ~ i d e s . The 0.5 % (NH4) 2C204-soluble polysaccharide B appeared to be a typical polygalacturonic acid. The presence of pectin in the soybean hulls was reported by SASAKI and FUJI'"). The 5
%NaOH-soluble polysaccharide A seemed to be a xylan. All the polysaccharides contained comparatively large amounts of arabinose and galactose as in the case of soybean cotyldons. These two sugars were presumed to be in the forms of araban, galactan, and arabogalactan as in the case of the cotyldon hemicellul~ses.(~)
2 .Hydrolysis of t h e Mot-M20-Soluble Polysaccharide A by Taka-Diastase and Paper Chromatography of t h e Hydrolyzate
.Preparation o f Enzyme Solutzon
A commercial Taka- diastase (50 g) was dissolved in 500 ml H 2 0 . (NH4) 2SOa was added to saturation and the precipitate formed after 4 hrs. was collected by centrifugation and washed with saturated (NH4) 2S04 solution. This (NH4) 2SO4 precipitation was repeated three times and finally the precipitate was dissolved in 100 ml H 2 0 and dialyzed against deionized water for
4days. The dialyzed enzyme solution was centrifuged to remove a precipitate and filled up to 150ml with deionized water to be used as an enzyme solution. 2.2. Hydrolyszs o f the Polysacchar zde and Paper Chromatography o f the Hydro1 yzate
The polysaccharide (1.5 g) was dissolved in 150 ml H z 0 by heating. MCILVAINE buffer
at pH 4.5 (50 ml) and 150 ml of the enzyme
solution were added to the polysaccharide
solution. The mixture was covered with small Galactose
amount of toluene and incubated for 24 hrs. Galacturonic
at 3S0C. The hyd~olysis degree was 70 % as acid
shown in Fig.
Incubation T i m e (hrs ) Fig 3 Paper chromatograms of the
Fig 2 Hydrolysis of the hot-HzO-soluble enzymatic hydrolyzate of the polysaccharide by Taka-diastase hot -HzO-soluble polysaccharide
80 100 -
,5 < 0 I I I I 1 \ Unkown 2
.\ t \
-BuOH-AcOH-HzO(4: 1:2) BuOH-CsHsN-H20(6:4:3) 0 4 8 12 16 20 24 (double development) (single development)
Vol, 17, No. 2 (1 966) 11 3
After the incubation, 2 vols. of ethanol was added to precipitate the enzyme and the unhydrolyzed polysaccharide. T h e clear hydrolyzate was concentrated to dryness zn vacuo
and the residue was dissolved in 5 ml H 2 0 . One-dimensional paper chromatograms of the hydrolyzate a r e given in Fig. 3. An unknown spot 1 gave xylose and mannose in the ratio of 1 : 1 by hydrolysis with H2S04. The other unknown spot 2 gave xylose, mannose, and galactose in the ratio of 1 : 1 : 1. These results seemed t o indicate conclusively t h a t xylose was chemically combined with mannose in the polysaccharide. ASPINALL and WHYTE'') showed that the purified soybean hull galactomannan contained traces of arabinose and xylose and these trace components weIe contaminations. The findings of xylose- containing oligosaccharides from the enzymatic hydrolyzate of the polysaccharide suggest that xylose was not a mere contamination but an actual component of the hot-H20-soluble polysaccha- ride of soybean hulls.
(1) Most part of the soybean hull polysaccharides were shown to be precipitated by Fehling solution in the absence of ethanol.
( 2 ) Xylose in the hot-H20-soluble polysaccharide was proved to be an actual component
of the polysaccharide and to combine with mannose residue of the polysaccharide. (3) Soybean hulls contained a polygalactur onic acid containing ar abinose.
The authors wish to thank prof. Akira KAJI, Laboratory of Fermentation Chemistry, this University, for his kind advice. A part of expenditure was defrayed by t h e research fund donated by the Ministry of Education to T . NARASAKI (Enzymatic hydrolysis of soybean hemicelluloses, 1962) and to A. KAJI (Improvement of the utilization of agricultural and horticultural products by the use of enzymes, 1962-1963).
References (1) KAWAMURA, S . Kagawa Datgaku Nogakubu
Gakuzyutu Hokoku, 4 , 65 (1952) (2) ---- : I b z d . , 5, 1 (1953)
(3) - : Ibzd , 5, 190 (1953)
(4) -- : Nippon Nogei Kagaku Kazshi, 28, 851 (1 954)
(5) - , KOBAYASHI, T , OSHIMA, M , MINO,
M : Tech B u l l . Agr Chem Soc Japan, 19,
(61 - , NARASAKI, T Agr B i o l . Chem ,25,
(7) NARASAKI, I?
,FUJIMOIO, K : K a g a w a Dai
-gaku Nogakubu Gakuz yutu Hokoku, 16, 73 (1964)
(8) WHISTLER, R L
,SAARNIO, J : J A m . Chem. Soc , 79, 6055 (1957)
(9) ASPINAIL, G 0 , WHY IE, T N C
.Pe~sonal communication (1964).
(10) SAKAKINARA, T Pe I sonal communication
(11) SASAKI, S , FUJI, S Nzppon Nogei Kagaku Katshi, 15, 624 (1939)