Phenblic Antioxidants

DAN E. PRATT,

CARMINE

of Soy Protein Hydrolyzates

DI PIETRO, WILLIAM

ABSTRACT
Soy protein hydrolyzate (SPH) was investigated to identify the
phenolic compounds responsible for antioxidant activity. Three
isoflavones-genistein, daidzein, and glycitein-were found in SPH.
These are the same isoflavones that have been identified in raw
soybeans. No isoflavone glycosides were found in SPH. The phenolic
acids found in SPH were caffeic, feruhc, pcoumaric, syringic,
vanillic, gentisic, and p-hydroxybenzoic acids. The compounds were
identified by TLC, GLC and UV spectra. The isoflavones were
confumed by their mass spectra.

INTRODUCTION
NUMEROUS
PHENOLIC
COMPOUNDS have been identified in soybeans and in soy flours and concentrates. Arai
et al. (1966) found several phenolic acids, which included
both substituted benzoic and cinnamic acids, in deffatted
soy flour. Pratt and Birac (1979) identified chlorogenic,
caffeic, ferulic, and p-coumaric
acids as primary antioxidants in soybeans and soy products. Three isoflavonesgenistein, daidzein, and glycitein-have
been identified in
soybeans and in soy flour (Naim et al., 1973). These
isoflavones are responsible for a portion of the antioxidant
acitivity attributed to soybeans and soy products (Pratt and
Birac, 1979; Pratt, 1980). The antioxidant activity of soy
flours, concentrates, and isolates have been summarized in
an excellent review (Hayes et al., 1977).
Vegetable protein
hydrolyzates .. are produced
from
several protein sources. They may be derived as the protein
hydrolyzate of a single vegetable source or as a mixture of
several vegetable sources. A number of low molecular
weight phenolic compounds have been identified in soy
protein
hydrolyzates
(SPH) by Manley and Fagerson
(1970). Bishov and Henick (1972; 1975) reported primary
and synergistic antioxidant
activity in SPH. Hayes et al.
(1977) spectulated that isoflavones and other phenolic
compounds may be found in SPH which may contribute to
their antioxidant activity. The present study was initiated
to identify phenolic compounds of SPH that contribute to
antioxidant activity.

L. PORTER, and J. WALTER

GIFFEE

developed in methanol:acetic acid:water (90:5:5). Bands were

eluted in methanol and rechromatographed on polyamide, with
ethanol:water (3:2) as the developing solvent.
Co-chromatography (TLC) in several developing systems, UV
spectral analysis GLC, and GLC-mass spectrometer were used to
identify phenolic acids and flavonoids.
W
spectral analyses were performed using a Beckman 26
spectrophotometer. Spectral analyses for flavonoids were conducted
as outlined by Mabry et al. (1970). Spectral of phenolic acids were
determined in methanol.
GLC analyses were conducted on trimethysilyl derivatives (TMS).
Isolated bands eluted in methanol were evaporated to dryness. The
residue was treated with 0.5 ml Bistrimethyl-silytrifluoroacetamide
(BSTFA). The mixtures were tightly capped and heated for 30 min
in a boiling water bath to facilitate derivatization. The gas chromatograph used was a Hewlett-Packard Model 5830A equipped with a
FID detector. TMS derivatives were separated isothermally on a 6
ft x l/8 in column of 3% S.E. 30 on 100/120 GCQ. The carrier gas
(helium) flow rate was 20 ml per minute. The column temperatures
were 240C for isoflavones and 180C or 200C for the phenolic
acids.
A Finnigan Automated GC/EI-CI Mass Spectrometer System was
used to further confirm the identity of the isoflavones. Electron
impact only was necessary due to the abundance of the parent ion.
Probe analysis was conducted on isoflavones that had been further
pruifled by TLC on polyamide plates and developed in methanol:
water (19:l). Bands were eluted in methanol and concentrated in
vacua. Samples (113 ~1) were evaporated to dryness ln a 5 ~1 vial.
The vial was placed onto the probe and heated rapidly to 300C
while in contact with the ionizer region of the mass spectrometer.
Conditions for the runs were: mass range, 20-27, 33-400 amu;
integration time, 4 msec; scan rate, 2 set/scan; filament current,
400 PA, electron energy, 70 eV; electron multiplier, 1500V; and
ionizer temperature, 33C.

Soy

Protein

Hydrolyrate
(1000 9)

(Oil

Coated)

dissolved
in 2000
"exane
extraction

EXPERIMENTAL
SOY PROTEIN HYDROLYZATE (SPH) (Vi-Zate 115 HVP powder,
oil coated) was supplied by A.E. Staley Mfg. Co. (Protein Div)
Chicago, IL. One kg of SPH was dissolved in 2L of deionizeddistilled water. The oil coating and the residual lipids were removed
by two extractions with 2L of hexane. Preliminary investigation
demonstrated that oil extraction of the aqueous dispersion was as
efficient as extraction of the dry powder. The scheme for isolating
flavonoids and phenolic acids from SPH is presented in Fig. 1.
The concentrated methanolic extract (Fig. 1) was streaked on
preparative (500~) cellulose TLC plates and developed in the upper
phase of I-butanol: acetic acid:water (5:1:4 v/v/v) (BAW). Bands
were eluted in methanol, streaked on polyamide 6 TLC plates and

Ethyl

Acetate

concentlated
to 100 ml
Phenols
adsorbed
onto Polyamide
Agitated
for 30 min

I
Methanolic

24-Volume

47 (1981)-JOURNAL

OF FOOD

SCIENCE

"1)

(25

9)

Complex
.rith
with

water
100% methanol
I
Aqueous
Phase
(discarded)

Extract

concentrste
I
Cellulose

Author
Pratt (to whom requests for reprints should be addressed) is
with the Dept. of Foods i? Nutrition,
Purdue Univ., West Lafayette,
IN 47907. Authors
Di Pietro, Porter, and Giffee are with the U.S.
Army Natick R&D Laboratories,
Natick, MA 01760.

2000

Aqueous
Phase
(discarded)

Phase

Polyamide-Phenol
I
Washed
Eluted

ml Hz0
(twice
-

TLC-9

Polyamide

TLC

1
Identification

Fig. 1 -Scheme
for the separation
protein hydrolyzete.

of phenolic

compounds

from

soy

Table 4. The phenolic acids found in the hydrolyzate were

essentially the same as Arai et al. (1964) reported for
defatted soybean flour. The exceptions were chlorogenic
and salicylic acids which were not found. It is not surprising
that chlorogenic acid is not present. Due to the
severity of the acid-heat treatment in the preparation of
SPH, chlorogenic acid would be readily hydrolyzed.
From the data presented herein, it is obvious that
flavonoids and phenolic acids are able to withstand the
acid and heat treatments used in the preparation of SPH.
Certain of these compounds are both primary and synergistic antioxidants and obviously contribute to the antioxiContinued
on page 35
dant activity of SPH.

RESULTS & DISCUSSION

THIN-LAYER and gas-liquid chromatographic characteristics of isoflavones of SPH are given in Table 1. All isolated
isoflavones were present as aglycones. This is presumably
due to hydrolysis of the glycosides by the severity of the
acid-heat treatment in preparation of SPH. The isoflavonesgenistein (5,7,4-trihydroxyisoflavone),
daidzein (7,4dihydroxyisoflavone), and glycitein (7,4-dihydroxy-6 methoxyisoflavone) are the same as those of soybeans and soy
flour (Naim et al., 1973; Pratt and Birac, 1979).
The UV spectral characteristics of the isoflavones are
shown in Table 2. Spectral analysis in methanol and with
reagents to produce lathochromic wavelength shifts demonstrated that the compounds were genistein, daidzein, and
glycetein. The wavelength shifts are practically identical
with those reported by Mabry et al. (1970) and Pratt and
Birac (1979).
Mass spectral data for the isoflavones are presented in
Table 3. Spectra for two of the isolated isoflavones are
nearly identical with those of authentic genistein and
daidzein. No authentic sample of glycetein was available.
Due to similarity of data for this isoflavone with chromatographic data and UV spectral data of Naim et al. (1973)
and Pratt and Birac (1979) and mass spectral data presented,
the compound was assumed to be glycetein. In addition to
the three isoflavones shown in Table 3, the mass spectrometer analysis indicated a compound with a M.W. of 298.
This is presumably a monohydroxy-demethoxy isoflavone.
The compound was not present in sufficient quantity for
further analysis.
Spectral and chromatographic characteristics of the
phenolic acids of soy protein hydrolyzates are given in

Table d-Mass
h ydrolyza te

spectral

data for iso flaiones

Compound

of

isoflavones

of

270b
100

153
40

133
28

269
28

51
24

271
15

50
14

89
12

Daidzeina
Mass to charge
lntensityC

ratio

254b
100

153
31

253
28

123
24

51
22

255
16

152
16

128
11

Glyciteina
Mass tocharge
lntensityC

ratio

284b
100

283
61

166
31

123 69
18 15

285
15

167
11

18
11

a Isolated
compound
b Parent
ion
The
Intensities
are

soy

Acid

normalized

Ferulic

Genistein
Authentic
Isolated

1 .oo
1 .oo

Co-chromatography
4 solvent systems
(Same)

in

Co-chromatography
4 solvent systems
(Same)

in

Daidzein
Authentic
isolated

0.80
0.80

Glycitein
Isolated

1.72

a GLC analysis of TMS

Retention
time relative

derivatives
on 3% SE on GCQ
to genistein.

Table 2-UV

spectral

o-Coumaric
Syringic
Vanillic
Gentisic
p-Hydroxybenzoic

of isoflavones
hmax,

Compound

MeOHa

to

of soy protein

260,330*
260,331*

270.355
270,355

Daidzein
Authentic
Isolated

237*.
236.

247.303+
247.302

259,287.
259,285*,

Glycitein
Isolated

226*,

254,316

254,345

parent

Concentration

GLCd

moles/kg

1 .oo

3.6 x 1O-3

0.80

1.5x

0.43

4
4

0.38
0.23

4
4

0.25
0.14

TLCC

10-4

Tr
1.8 x 1O-4
1.2 x 10-S
Tr
Tr
compounds.
compounds

hydrolyzate

nm
AIC13

272.307*.
273,308.
328
326

ion.

hydrolyzate

Co-Chromatographyb

316
285
314
287
310
286
270
260
285
328
251

NaOMeb

Genistein
Authentic
Isolated

the

acids of soy protein

E hmax
are the same for isolated
and authentic
Co-chromatography
of isolated
and authentic
z Number
of chromatographic
systems
used
Retention
time
relative
to caffeic
acid

at 24OC.

characteristics

In respect

- nm

Caffeic

TLC

G LCa

soy protein

ratio

hmaxa
characteristics

from

Genisteina
Mass to charge
lntensit+

Table 4-Phenolic

Table
1-Chromatographic
pro rein h ydrolyza te

isolated

Concentration
moles/kg

372
372

240.
241,

246,301
246,301

226*,

259,310

2.0 x 10-3
l

0.6 x 1O-3

0.3 x 10-3

, Methanol

Sodium

methoxide

*Shoulder

Volume

47 (198 l)-JOURNA

L OF FOOD

SCIENCE-25