PA TRICIA A. HAMMERSCHMID T and DAN E.

PRA TT 1
Dept. of Foods & Nutrition, Purdue University, West Lafayette, IN 47907

PHENOLIC ANTIOXIDANTS O F DRIED SOYBEANS

ABSTRACT max L.) were extracted by a modified method of Pratt (1972). Dried
W:lole dried soybean seeds were investigated to determine the nature of soybeans (Corsoy variety) obtained from Purdue University Depart-
their antioxidant activity. Activity was measured by rates of carotene ment of Agronomy were soaked overnight in methanol. Each sample
bleaching in the coupled oxidation of linoleic acid and p-carotene. consisted of 408 beans and 200 ml methanol. Beans were homogenized
Methanolic extracts of the soybeans were found to possess potent for 2 m m at high speed in a Waring Blendor using the methanol in
antioxidant ability. Paper and Sephadex column chromatography gave which they were soaked. The extract was boiled 5 m m and filtered
incomplete resolution of individual components. Therefore, thin-layer through Whatman #42 filter paper and the residue was washed with an
chromatography of the whole methanolic extract rather than individual additional 150 ml hot methanol. The isoluble residue was discarded.
column fractions was used in isolating this component. Three fluores- The filtrate was evaporated in vacua at 40°C on a rotary evaporator to
cent bands were found to possess the major activity. The most potent a final volume of 12 ml. Extracts were stored at -20°C until used.
complex was further analyzed. Thin-layer chromatography was used to Paper chromatography
separate the complex into four fluorescent components. One com- Approximately 3 ml of methanolic extract were streaked an sheets
pound was determined to be responsible for the antioxidant activity of Whatman 3MM filter paper (23 cm x 57 cm). Chromatograms were
exhibited by the parent band. Further analysis of this component by developed descendingly 30 cm in the upper phase of 1-butanol:acetic
paper chromatography, chromogenic spray reagent tests, and ultraviolet acid:water (4:1:5, v/v). After drying, chromatograms were viewed
spectral analyses indicated that the antioxidant was an isoflavone under long (3660A) and short (2550A) wavelength UV radiation before
pentoside. and after fuming with ammonia. Major fluorescent bands were eluted
with 80% aqueous methanol.
INTRODUCTION Two dimensional paper chromatography was utilized to separate
antioxidant components of samples (Mabry et al., 1970). 25-50~1of
INCREASING ECONOMIC INTREST in soy proteins as meat sample were spotted on 21 cm x 21 cm sheets of Whatman 3MM filter
substitutes and extenders has led to investigations involving paper and developed 15 cm ascendingly in the first dimension in t-butyl
determinations of antioxidant activity in soybeans and soy- alcohol:acetic acid:water (3:1:1, v/v). Papers were dried overnight and
developed ascendingly in the second dimension in 15% acetic acid.
bean products. Soy protein concentrate, defatted soy flour,
Chromatograms were viewed under long and short wavelength ultra-
and fresh and dried soybean hot water extracts exhibited violet radiation before and after fuming with ammonia.
appreciable antioxidant activity (Pratt, 1972). Soy textured Ascending paper chromatography was used for comparing acid
vegetable protein also exerted a significant antioxidant effects hydrolyzed components with known sugar standards. 2-propanol:acetic
on lipids of beef slices and vegetable-beef soup (Sangor and acid:water (3:1:1, v/v ) was the developing solvent for 21 cm x 21 cm
Pratt, 1974). Isolated soy protein possessed antioxidant prop- sheets of Whatman #l filter paper spotted with 25-50 ~1 of unknown
erties in both aqueous-meat and aqueous-lard systems (Hollar, samples and known sugar solutions. Papers were developed to approx-
1974). imately 15 cm and sprayed with appropriate reagents when dry.
Presence of compounds exhibiting antioxidant ability has Sephadex column chromatography
been noted in soybeans. A rather unusual flavonoid, Sephadex column chromatography was employed to separate
6,7,4’-trihydroxyisoflavone, isolated from fermented soybeans methanolic extract components (Somers, 1966). A column bed 1.5 cm
(tempeh) was reported to have active antioxidant ability. x 15 cm was prepared from Sephadex G-25 Fine after allowing the gel
Preliminary conclusions indicated either the possibility of de to swell overnight in an excess of 60 parts acidified methanol (3 ml
novo sj;nthesis of the isoflavone during fermentation, or concentrated HCl to 1L methanol) and 40 parts water. Three ml of
presence of the compound in an inactive state in unfermented concentrated extract were added to the top of the column and the
beans (Gyorgy et al., 1964). On further investigation (Ikehata aforementioned solvent used for elution. A Buchler Fraction Collector
et al., 1968) 6,7,4’-trihydroxyisoflavone was potent in Alpha 200 was used to fractionate the sample. A volume of 40 drops of
sample per fraction was collected. Fractions were stored as -20°C.
aqueous solutions but ineffective in preventing autoxidation of
soybean oil or soybean powder. The authors speculated that Thin-layer chromatography
this ineffectiveness may be due to the compound’s insolubility Thin-layer chromatography was used to obtain a better separation
in oil and difficulty to disperse in powder. of components of the methanolic extract. A volume of 350 ~1 of con-
At least nine phenolic acids including syringic, vanillic, centrated methanolic extract were streaked on each of 8-10 pre-coated
ferulic, gentisic, salicylic, p-coumaric and p-hydroxybenzoic silica get TLC plates which had been activated for 15 m m at 100°C.
Plates were developed ascendingly for 15 cm in the upper phase of
acids have been identified in defatted soybean flour. Two
ethyl acetate:formic acid:water (10:2:3, v/v). The solvent was equili-
isomers of chlorogenic acid were also present and were brated 20 min to allow the layers to separate.
thought to be isochlorogenic and chlorogenic in a ratio of After drying, bands were located by viewing under long and short
approximately 1: 10 (Arai et al., 1966). Such hydroxycinnamic wavelength UV radiation and scraped from the plate. The silica gel
acid derivatives have potent antioxidant activity (Pratt, 1976). residues which contained the separated compounds were soaked in
The present study was designed to investigate antioxidants in excess methanol (Spectral grade) for 30 mm, filtered using Whatman
whole dried soybeans. #42 filter paper, and evaporated to near dryness under in vacua at 45°C
in a rotary evaporator. The residue was redissolved in 1.0 ml methanol
MATERIALS & METHODS (Spectral grade) and filtered through glass wool to remove final traces
Preparation of extracts of silica gel.
The 1.0 ml filtrate was used to streak on two TLC plates (500 ~1 per
Methanol-soluble components of whole dried soybeans (Glycine
plate) activated as described previously. Plates were developed
ascendingly in 1-butanol:acetic acid:water (4:1:1, v/v) for 15 cm. A
blank plate was also run in this solvent. Bands of intrest were located
OOZZ-1147/78/0002-0556$02.25/O by using a UV light and were eluted by scraping as previously described.
0 1978 Institute of Food Technologists An area equal in size to the bands of intrest was eluted from the blank
plate in the same manner.

556-JOURNAL OF FOOD SCIENCE-Volume43 11978)

 PHENOLIC ANTIOXIDANTS OF DRIED SOYBEANS. ..

Antioxidant activity evaporated to dryness in a water bath. The residue was redissolved in
Antioxidant activity was determined by measuring the coupled methanol (Spectral grade). The lower layer (water) was evaporated to
oxidation of carotene and linoleic acid. Approximately 1.0 mg near dryness on a rotary evaporator and the residue redissolved in
p-carotene was dissolved in 10 ml of chloroform. One (1.0) ml of the 0.5 ml water. The water phase was tested for sugars by paper chrom-
carotene-chloroform solution was pipetted into a boiling flask which atography.
contained 20 mg purified linoleic acid and 200 mg Tween 40. After
removal of the chloroform on a rotary evaporator at 5O”C, 50 ml of RESULTS & DISCUSSION
oxygenated distilled water were added to the flask with vigorous
swirling. Five ml aliquots of this emulsion were placed in spectrophoto- PAPER CHROMATOGRAPHY of methanol extracts devel-
meter tubes which contained 0.2 ml of the antioxidant solution being oped in the upper phase of I-butanol:acetic acid:water (4: l:S,
tested. Samples were read aganist a blank containing the emulsion v/v) provided four distinct fluorscent bands under long wave-
minus the carotene. Readings at 470 nm were taken immediately after length UV radiation. These bands corresponded to Rf values of
addition of the emulsion to the antioxidant solution. Tubes were stop- 0.21 (fluorescent light purple), 0.44 (fluorescent bright blue),
pered and placed in a water bath at 50°C. Readings were taken at 15 0.56 (fluorescent green), and 0.62 (fluorescent blue-green). All
min intervals for 105 min. Controls consisted of 0.2 ml methanol or
bands became brighter with exposure to ammonia fumes. The
solutions eluted from blank TLC plates as appropriate, in place of the
antioxidant test solution. latter three bands gave a positive test with carotene spray indi-
cating antioxidant activity. The activity was due to phenolic
Carotene spray compounds as judged by a positive reaction with ferric
A carotene spray solution was used for detecting antioxidants on chloride-potassium ferricyanide spray. However, only the blue-
thin-layer and paper chromatogams (Philip, 1974). 6 mg p-carotene
-green band (Rf, 0.63) appeared to be a single compound. UV
were dissolved in 30 ml chloroform. Two drops of linoleic acid (puri-
fied) and 60 ml ethanol were added to carotene-chloroform solution.
spectral curves and chromatography on thin-layer plates and
TLC plates ot filter paper on which 25-50 ~1 of antioxidant test solu- paper in several solvents indicated that this compound was
tions had been applied were sprayed with this solution. Plates or papers chlorogenic acid. Further chromatographic analysis of the
were exposed to daylight until background color was bleached (usually other two antioxidant bands indicated they were complex
within 3 hr). Spots in which yellow color persisted were judged to have mixtures.
antioxidant activity with intensity of color related to amount of Several investigators (Somers, 1966; Johnston et al., 1968;
activity. Woof and Pierce, 1967) have reported successful separations of
Determination of phenol concentration flavonoid compounds on Sephadex columns, even though
Concentrations of phenols present in fractions from Sephadex Sephadex tends to absorb aromatic compounds particularly
column separations were estimated using the FolinCiocalteau reagent phenols. Preparation of the gel and elution in aqueous alcohol
(Bray and Thorpe, 1954). The procedure consisted of combining 0.1 ml eliminated adsorptive effects of Sephadex (Somers, 1966).
test solution with 2.0 ml 2% Na,CO,. After 2 min 0.1 ml of 50% Sephadex column chromatography was, therefore, used as a
FolinCiocalteau reagent was added and the mixture was incubated 30 means of separating components of the extracts.
min at room temperature. Absorbance was then measured at 750 nm
using a Beckman spectrophotometer Model 25. Samples were run After separation of 21 ml methanolic extract (7 separations
aganist a blank consisting of 0.1 ml acidified methanol/water solvent, of 3 ml each) which were at a concentration of 0.3 ml ex-
2.0 ml 2% Na,CO,, and 0.1 ml 50% FolinCiocalteau reagent. tract/g bean, the fractions were tested for phenol content.
Standards of chlorogenic acid were used for determining total phenol Fractions 31 through 50 contained the greatest phenolic con-
content of fractions. centration. These fractions were tested individually for
Acid hydrolysis of antioxidant solutions antioxidant activity whereas fractions 26 through 30, and 51
Antioxidant components of the sample were hydrolyzed as reported through 55 were combined and tested. Spectrophotometric
by (Mabry et al., 1970). One (1.0) ml of antioxidant solution was determination of carotene was used for measuring antioxidant
placed in a pear-shaped flask and evaporated to dryness on a rotary ability.
evaporator at 45°C. One drop methanol (Spectral grade) was added to Result of antioxidant tests for the most active fractions
the flask to redissolve the residue and 1.5 ml of 6% HCl were added. from Sephadex column chromatography are shown in Figure
The solution was transferred to a small vial. After capping, the vial was 1. Major antioxidant activity was centered in fractions 31
heated in a boiling water bath for 45 min. Two (2.0) ml diethyl ether through 34 although all fractions exhibited some degree of
were added with shaking. The upper layer (ether) was removed and activity. The antioxidant activity of fraction 31 (most active
fraction) was essentially the same as propyl gallate. Fractions
31 through 34 reamined visably more yellow than controls in
the testing system for at least 5 days.
Several fluorescent compounds appeared more prominent
in fractions possessing antioxidant activity than in other frac-
tions. These compounds were eluted from TLC plates and
tested for antioxidant activity. Two compounds of six tested
appeared to possess antioxidant ability. These compounds
corresponded to Rf values of 0.00 (fluorescent blue-purple)
and approximately 0.67 (fluorescent blue-purple) in ethyl
acetate:formic acid:water (10:2:3 v/v/v). The band at Rf 0.67
had the greatest activity and was selected for further study.
Paper chromatography and Sephadex column chromatog-
raphy did not give adequate resolutions of compounds and
only a small amount of compound could be obtained from
each column. Therefore, the antioxidant band was isolated
directly from preparative thin-layer plates of the methanolic
.05 - whole bean extract rather than individual column fractions,
I I I I I I I using the ethyl acetate:formic acid:water solvent. Excellent
OO 15 30 45 60 75 so 105 resolution of the whole extract was obtained in this solvent
TIME (min.) with approximately 10 fluorescent bands present. Three
fluorescent bands with Rf values of 0.54, 0.67 and 0.74 having
Fig. 7-Antioxidant activities of fractions eluted from sephadex strong antioxidant activity (Fig. 2) were eluted. Characteristics
column. of the bands tested are given in Table 1.

Volume 43 11978kJOURNAL OF FOOD SCIENCE-557

 Table 1 -Chromatographic characteristics of bands separated from Ascending thin-layer chromatography was used to ascertain
whole extracts by TLC the complexity of the three eluted bands. Solvent systems
included chloroform:methanol (9:1, v/v); I-butanol:acetic
uv (long) acid:water (4: 1: 1, v/v); benzene:acetic acid:methanol(45:5:8,
Rfa Visible color fluorescence
v/v); and 1-butanol:water (9:1, v/v). The band corresponding
to Rf 0.74 possessed the greatest antioxidant activity and
0.54 orange-pink yellow-pink
resolved into four components in the 1-butanol:acetic acid:
0.67 slight yellow blue
water solvent shown in Table 2. The component at Rf 0.27
0.74 slight yellow blue-green
was responsible for antioxidant activity associated with the
a Rf values in ethyl acetate:formic acid:water solvent parent band. (Fig. 3).
Two dimensional paper chromatography gave Rf values of
0.42 (t-butyl alcohol:acetic acid:water, 3:1:1, v/v) and 0.94
(15% acetic acid. Prelimary interpretation of the developed
Table 2-Chromatographic characteristics of components of the Rf chromatogram suggested the compound was an isoflavone
0. 74a band 7-0-diglycoside or a flavonol mono or diglycoside. Characteris-
uv (long) UV (short)
tics of the spot color in UV radiation before and after
Visible color fluorescence fluorescence
ammonia fuming also suggested an isoflavone or flavonol-type
Rfb
structure (Mabry et al., 1970).
0.27 slight yellow blue-green - Further characterization of the antioxidant compound was
0.58 - - dark purple attempted through use of various chromogenic reagents. Re-
0.71 - light purple - sults of spray tests are indicated in Table 3. Positive color
0.76 - - dark purple reaction with DPNA indicated a phenolic compound with free
hydroxyl groups in ortho or para positions (Swain, 1953). The
a Rf in ethyl acetate:formic acid:water solvent red-pink color produced with FeC13 also indicated a phenolic
b Rf values of 0.74 band rechromatographed in n-butanol:acetic compound in that phenols with two or more neighboring
acid:water solvent
hydroxyl groups react to give Vanillin-p-toluenesulphonic acid
designated a possible flavonoid-type substance (Roux and
Maihs, 1960). Also suggesting a compound containing phenolic
Table 3-Reactions of isolated antioxidant with chromogenic
groups was the change in both visible color and fluorescence
reagents
observed after spraying with NazC03. These characteristic
Spray reagent Visible color produced
changes are due to ionization of phenolic hydroxyl groups
(Swain, 1969). Production of a nonfading brown spot after
Diazotized p-nitroaniline (DPNA) 1. orange-brown exposure to iodine vapor suggested the compound was a
2. red-brown with overspray glycoside (Bailey, 1969). As seen by these reactions with
2% Ferric chloride (FeCI, 1 red pink chromogenic reagents, the antioxidant was determined to be a
Vanillin-p-toluenesulphonic red (slight) phenolic compound of the flavonoid type.
acid (Van-pts) UV spectual analyses of the antioxidant in methanol
Iodine vapor exposure (I, ) non-fading yellow-brown yielded major peaks at 279 nm and 242 nm with a shoulder a
20% Na, CO, 322 nm. The shoulder at 322 nm also exhibited a
Visible UV (long) bathochromic shift to 330 nm. The shift indicated a flavonoid
Before spraying slight pink blue-green compound which lacked a free 7 hydroxyl, which may be due
After spraying brown green to a sugar at that position (Mabry et al., 1970). No spectrum
shift occurred upon addition of AlC13 or AlCls/HCl. This indi-

o Rf 0.27
n Rf 0.50
O Rf 0.71
A Rf 0.76

75 90 105 OO
I I
I5
I
30
I I I
75
I
90
I
105
TME Kin.)
Fig. 2-Antioxidant activities of three componenrs resolved by thin- Fig. J-Antioxidant activities of components resolved by thin-layer
layer chromatography (ethyl acetate:formic acid:water solvent). chromatography (I-butanol:acetic acid:water solvent).

558-JOURNAL OF FOOD SCIENCE-Volume 43 (1978)

 PHENOLIC ANTIOXIDANTS OF DRIED SOYBEANS. ..

cated the lack of 6,7 or 7,8 dihydroxylation of the a-ring of Bray, H.G. and Thorpe, W.V. 1954. Analysis of phenolic compounds of
interest in metabolism. In “Methods in Biochemical Analysis.” Ed.
flavonoid compounds. This did not rule out the possibility of GIich. D.
3’, 4’-dihydroxylation of the P-ring since there is a little Gyargy. R., Murata, K. and Ikehata. H. 1964. Antioxidants isolated
from fermented soybeans (Tempeh). Nature 203: 870.
conjugation of the P-ring with the major chromophore with Heimann. W. and Reiff. F. 1953. The relationshiu between chemical
which Al& reacts (Mabry et al., 1970). Based on the position constitution and antioxidant activity of flavor&s. Fette and Seifen
of Band II (279 nm) of the spectral analyses and on 55: 451.
Hollar, N.S. 1974. A study of lipid antioxidation of soy protein iso-
chromatographic analyses, the antioxidant appeared to be an lates. M.S. thesis, Purdue University. W. Lafayette, IN.
isoflavone or insoflavone derivative. Ikehata, H., Wakaizumi, M. and Murata. K. 1968. Antioxidant and
antihemolytic activity of a new isoflavone, “Factor 2” isolated from
Acid hydrolysis was used to determine whether the antioxi- Tempeh. Agr. Biol. Chem. 32: 740.
dant was a glycoside. Upon evaporation of the water layer Johnston, K.M., Stern, D.J. and Waiss, A.C. Jr. 1968. Separation of
after hydrolysis, a white precipitate appeared which then flavonoid compounds of Sephadex LH-20. J. Chromatog. 33: 539.
Lea, C.H. and Swoboda, P.A.T. 1956. On the antioxidant activity of
redissolved in additional water.. One dimensional paper the flavonols, gossypetin and quercetagetin. Chem. and Ind. 1426.
chromatography in 2-propanol:acetic acid:water (3: 1: 1, v/v) Mabry, T.J., Markhan, K.R. and Thomas, M.B. 1970. “The Systematic
Identification of Flavonoids.” Springer-Verlag, Berlin.
of unhydrolyzed antioxidant and the hydrolyzed antioxidant Mehta, A.C. and Seshadri, T.R. 1959. Flavonoids as antioxidants. J. Sci.
(water layer) was performed. Spraying of developed chromato- Ind. Res. 18B: 24.
Naim, M.. Gesttner, B., Kirson. I.. Birk, Y. and Bondi. A. 1973. A new
grams with aniline oxalate suggested the unknown sugar was a isoflavone from soya beans. Phytochemistry. 12: 169.
pentose. Philip, F. 1974. An investigation of the antioxidants in a textured
Antioxidant ability of flavonoid compounds has been well vegetable protien product from soy flour. Ph.D. thesis, Purdue Uni-
versity, W. Lafayette. IN.
established in literature (Heimann and Reiff, 1953; Simpson Pratt, D.E. 1972. Water soluble antioxidant activity in soybeans. J.
and Uri, 1956; Lea and Swoboda, 1956; Mehta and Seshadri, Food Sci 37: 322.
Pratt, D.E. 1976. Role of flavones and related compounds in retarding
1959; Pratt and Watts, 1964). Results suggested the lipid-oxidative fhavor changes in foods. In “Phenolic. Sulfur and
possibility of an isoflavone glycoside based on chromato- Nitrogen Comnounds in Food Flavor.” Ch. 1. Ed. Charalambous. G.
and I&z, I. A& Symposium Series. No. 26.
graphic and spectral analyses. Presence of 6,7,4’-trihydroxyi- Pratt, D.E. and Watts, B.M. 1964. The antioxidant activity of vegetable
rihydroxyisoflavone in unfermented soybeans has not been extracts: 1. Flavone aglycones. J. Food Sci. 29: 27.
Reio. L. 1958. A method for the paper-chromatographic separation and
demonstrated. Other investigators also have demonstrated the identification of phenol derivatives, mould metabolites. and related
presence of isoflavones in soybeans. These included glycosides compounds of biochemical interest, using a “reference system.”
of genistein and daidzein (Walz, 193 1; Walter, 1941) and a J. Chromatog. 1: 338.
Reio, L. 1960. Supplementary data for the paper chromatographic
7,4’-dihydroxy, 6-methoxy insoflavone (Naim et al., 1973), separation and identification of phenol derivatives and related
although based on structural characteristics these compounds comaounds of biochemical interest. usine - a “reference svstem.” J.
Chromatog. 4: 458.
would not be expected to exhibit antioxidant activity. Roux. D.G. and Maihs, H.E. 1960. Selective spray reagents for the
The data reported herein indicates that soybeans contain identification and estimation of flavonoid compounds associated
with condensed tannins. J. Chromatog. 4: 65.
an isoflavone with lipid antioxidant activity. Ortho or para Sangor, M.R. and Pratt, D.E. 1974. Lipid oxidation and fatty acid
dihydroxylation of either the 01 or P-ring is essential for changes in beef combined with vegetables and textured vegetable
protein. J. Am. Dietet. Assoc. 64: 268.
antioxidant activity (Pratt, 1976). The ortho dihydroxy Simpson, T.H. and Uri, N. 1956. Hydroxyfhavones as inhibitors of the
configuration on the a-ring appears more likely; since para aerobic oxidation of unsaturated fatty acids. Chem & Ind. 956.
dihydroxy flavonoids are rare in plants and highly substituted Somers. T.C. 1966. Wine tannins-isolation of condensed flavormid
pigments by gel-filtration. Nature 209: 308.
b-rings of isoflavones are not common in soybeans. It appears Swain. T. 1953. The identification of coumarins and related com-
likely that 7,4’ dihydroxy, 6-methoxy isoflavone (Naim et al., pounds by filter-paper chromatography. Biochem. J. 53: 200.
Swain. T. 1969. Phenols and related comDounds. In “Data for Bio-
1973) may be the methoxylated parent of the isoflavone chemical Research,” Ed. Dawson, R.M.C.. Elhot, D.C., Elliot, W.H.
antioxidant. and Jones, K.M. Oxford University Press, New York.
Waiter, E.D. 1941. Genistin (an isoflavone giucoside) and its aglucone,
genistein, from soybeans. J. Am. Chem. Sot. 63: 3273.
Walz, E. 1931. Insoflavon-und-saponin-giucoside in soja hispida. Justus
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WATER BINDING BY SOY FLOURS BY WIDE LINE NMR . . . From page 555

unfrozen control although the BWC was the same for both. Miiier, B.S. and Kaslow, H.D. 1963. Determination of moisture by
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Volume 43 (1978)-JOURNAL OF FOOD SClfNCE-559