Isolation, Partial Characterization and Modification of

the Great Northern Bean (Phaseolus vulgaris L.) Starch

S. K. SATHE and D. K. SALUNKHE

Microscopyof starch

ABSTRACT
The yield of the Great Northern bean starch was 18.23% (bean flour
basis). The starch granule size ranged from 12 x 12 pm to 58 x 40
pm (length x width). The shape of starch granules was round to oval
to elliptical, and in some cases,concave as well. Lamellae were present on all the starch granules observed. Amylose content of the
starch was 10.2% (starch basis). Hog pancreatic cY-amylasehydrolyzed
more starch than did malt a-amylase under similar conditions. The
Great Northern bean starch had good water and oil absorption capa-

cities at room temperature(21C). The bean starchformed a stable

gel at concentrations of 7% and above (w/v). The viscoamylographic
studies of the isolated starch indicated the restricted-swelling character of the bean starch.

INTRODUCTION
contain about 60% carbohydrates including

LEGUMES
starch, reducing and nonreducing sugars, oligosaccharides
of the raffinose family, and others. Starch constitutes the

major portion of legume carbohydrates. Cerning-Beroard

and Filiatre (1976) studied the carbohydrate composition
of horsebeans, smooth and wrinkled peas, and lupine seeds.
They found that the average starch content of horsebeans,
smooth and wrinkled peas, and lupine seeds to be 41 .O,
48.0, 33.0, and 0.4%, respectively. Naivikul (1977) reported the starch content range to be 50.9-52.9% (moisture free basis) in navy bean, pinto bean, faba bean, lentil,

The purified starch was studied microscopically by employing

both light and scanning electron microscope. For light microscopic studies, starch was moistened with a drop of distilled water.
A calibrated eyepiece lens (152.5@ (calibrated against a stage
micrometer) was employed for the measurements of starch granule

size at a magnification of 344. The starch granuleswere studied

for the size, shape, hylum, and lamellae. The size measurements
were made on 25 representative granules.
Starch samples for scanning electron microscopic studies were
prepared as follows. Starch was sprinkled on an aluminum stub
(with a double-stick tape on it) and was coated with a gold-palladium alloy completely in a Polaron E 5000 Sputter Coater (U.K.)
and the specimens observed in AMR 1OOOBscanning electron microscope (Cambridge, Mass.) at 20 KV accelerating voltage and suitable

magnification(s).
Hydrolysisof starch
Starch was hydrolysed by two different ol-amylases(from Hog

pancreas,type VI-A, and from malt, type V-A; both from Sigma

and mung bean. Schoch and Maywald (1968) discussed the

difficulties encountered in the separation of horsebean

starch. They attributed these difficulties to the presence of
a highly hydrated fine fiber fraction (presumably from the
cell walls enclosing the starch granules) and high content of
insoluble proteins. Halbrook and Kurtzman (1975) studied
the water uptake by the Great Northern bean starch at
high (80-148(Z) temperatures. Recently, Lai and VarrianoMarston (1979) reported certain physicochemical characteristics of black bean starch.
The purpose of the present investigation was to isolate
the Great Northern bean starch and to study certain physicochemical properties of the unmodified and modified
starch.

RESIDUE

RESIDUE

CENTRIFUGE

RESUSPEND
IN
80% AQ. ETHANOL

F 30L 2% N&I,

24 h, 4C

1 Washed with 2L H,O, blended with

6L O.IN NaOH (1 min.1 m a Warmg
Blendor and extracted
for 48 h. 4C
10,000

I-

RPM, 30 min.

MATERIALS & METHODS

The Great Northern beans were purchased from Bean Growers
Warehouse, Filer, Idaho, and stored at 4C until experiments were
conducted. The residue obtained after preparation of the protein
concentrates on a pilot plant scale (Sathe et al., 1980) was referred
to as crude starch. All the chemicals were of reagent grade unless
mentioned otherwise. All the analyses were performed in triplicate
and means reported.

Blended

HEATING

WATER

1 min. in a Waring

Blendor

*
BATH

80C. 1 h

4h. 4-C; Discard

supernatant

Isolationof starch
The beans were ground to 20 mesh in a Fitz mill (The W.J.
Fitzpatric Co., Chicago, Ill.). Three kg of bean flour were extracted
sequentially with different solvents to yield starch. The schematic
diagram for the process is presented in Figure 1.

Authors
Sathe and Salunkhe are affiliated
with the Dept.
tion & Food Sciences, Utah State Univ., Logan, UT84322.

FREEZE

DEHYDRATE

STARCH

POWDER

1
I

of NutriFig. l-Schematic
starch.

Volume

diagram

for the isolation

46 (1981kJOURNAL

of Great

OF FOOD

Northern

SCIENCE-617

bean

Chemical Co., St. Louis, MO.) according to the method described by

Decker (1977). Maltose hydrate (Grade II, Sigma Chemical Co.,
St. Louis, MO.) served as reference standard. The starch to enzyme
ratio in each case was 1:l (w/w). Liberated maltose was measured,
after inactivating the enzyme (heating in a boiling water bath for 3
min), calorimetrically (3,5dinitrosalicylic acid as color reagent)
at 540 nm in a Beccman-DBG spectrophotometer. Maltose equivalent was determinec at time intervals of 0, 15, 30, 60,90, and 120
min. The incubatiorI was, in both cases,at pH 7.0 and at a temperature of 21C.
Modifications
Acetylation-The method of Wurzburg (1964) was followed.
One hundred grams of starch were dispersed in 500 ml of distilled
water and magnetic:ally stirred for 30 min to obtain a uniform
suspension. The pH 1rf the slurry was adjusted to 8.0 with 1N NaOH.
Acetic anhydride (11.2g) was then added slowly to this slurry maintaining constant stirring and monitoring the pH between 8.0-8.4.
The reaction was al.owed to proceed for an additional 5 m m after
completion of the acetic anhydride addition. The pH of the slurry
was finally adjusted o 4.5 with 0.5N HCl and filtered through Whatman filter paper #4. The residue was then washed five times with
distilled water and freeze dehydrated.
Oxidation-A uniform slurry of starch (1OOg starch in 500 ml
distilled water) was prepared as in acetylation. Oxidation of the
purified starch wa:. accomplished by the method of Hullinger
(1964). The pH of the slurry was first adjusted to 9.0-9.5 with 3%
aqueous NaOH and log of NaOCl was added slowly (over a period
of 90 min) while rraintaining the magnetic stirring and constantly
monitoring the pH lletween 9.0-9.5. Cooling was provided (crushed
ice with NaCl) simitltaneously. The reaction was allowed to proceed for 4 hr after IJaOCl addition was completed, pH adjusted to
7.0 with 0.5N HCI and the slurry was filtered through Whatman
filter paper #4. Th: residue was washed five times with distilled
water and freeze dehydrated.
Moisture
The moisture content of the samples were determined by the
AACC method 44-15 (1962).
Proteins
Protein content of the appropriate .samples was determined
by the Kjeldahl method (N X 6.25).
Fat
The method followed was that of Schoch (1964). Starch (5g)
was hydrolyzed witlr 8N HCl solution for 1 hr on a boiling water
bath. After successb.eextraction of fat with ether and petroleum
ether, the solvents Mere evaporated on a hot water bath (80C)
and the nearly dry samples were then dried in an oven (100C)
for 20 min followel by three successiveextractions with carbon
tetrachloride (10 m. each time). The combined extracts were filtered and the solvc,nt evaporated on a hot water bath (SOC).
The beakers (previot sly weighed) containing the nearly dry samples
were dried in an oven (100C) for 30 m m and weighed. The difference was interpretet as the weight of fat. The fat content was reported on a dry weigrt basis.

stand for 30 min at room temperature (21(Z), centrifuged at 5000

X G for 30 m m and the volume of the supernatant noted. Density of
distilled water was assumedto be 1 g/ml. Results were expressedon
a dry weight basis.
Pasting properties
Starch gelatinization curves were obtained by the method of
Sandstedt and Abbott (1964). Starch (2Og, dry weight basis) was
suspended in 350 ml of distilled water in a Waring Blendor. CMC
(Cellulose Gum 7 HP, Hercules Powder Co.. Wilminaton. Del.) was
added (3.6g) with gentle stirring over 30 set to this suspension,
blended for 1 min, and poured into the amylograph bowl. The
blender was then rinsed with 100 ml distilled water and the water
was added to the amylograph bowl. The temperature of this starchCMC suspension was then raised from 25C to 95C at a rate of
1.5C/min; held at 95C for 15 min and then cooled uniformly to
50C (1.5C/min). A blank curve for CMC was prepared similarly
(with 3.6g of CMC alone) and subtracted from the starchCMC
curves.
Gelation
The method of Coffman and Garcia (1977) was employed with
slight modifications. Purified starch suspensionsof 1,3,5,7,9,11,
13, 15, 17, and 20% (w/v) were prepared in 5 ml distilled water and
the test tubes were heated in a boiling water bath for 1 hr followed
by rapid cooling under running cold tap water. The test tubes were
further cooled for 2 hr at 4C. Least gelatinization concentration
was determined as that concentration when the sample from the
inverted test tube did not fall down or slip.
Degree of substitution
The degree of substitution (D-S.) for the acetylated starch was
determined according to Wurzburg (1964). Starch (5g) was dispersed in 75% aqueous ethanol and warmed for 30 m m on a water
bath (5OC), cooled to room temperature (21C), and 25 ml of
1N NaOH added. The stoppered flasks were then allowed to stand
for 72 hr with occasional shaking at room temperature (21C).
Excess NaOH was back titrated with 1N HCl. The flasks were allowed to stand at room temperature (21C) for 2 hr and the titra-

Table

1-Physichochemical

Sample
Crude starch
Purified starch
Acetylated
starchd
Oxidized starch

data on the Great Northern

Yield
(%)

Moisture
Pd

87.50a
1 8.23a
92.00b
84.40b

3.07
2.67
3.70
4.12

a On bean flour basis

b On purified starch basis
i Dry weight basis
Degree of substitution
(D.S.)

bean starch

Protein
1%)

FatC
(%)
0.34
0.46
-

4.86
0.97
-

= 0.40

Amylose content
Amylose content of the purified starch was determined by the
procedure of Wolf et al. (1970) with slight modifications. Pure amylose (Potato, Type 111,Sigma Chemical Co., St. Louis, MO.) served
as standard. Starch lvas dissolved in 90% (v/v) dimethyl sulfoxide
and 0.2, 0.5, and 1.(1ml portions were assayedfor the amylose content. The starch concentration in 90% dimethyl sulfoxide was 100
mg/lOO ml. One ml of each of 0.005N KI03, 0.016 KI, and 0.5N
HCl were then addec to a total of 1 ml of standard/sample and final
volume (9 ml) made up with distilled water. Absorbance was read at
615 nm in Beckman DEG spectrophotometer.
Water and oil absorption
Water and oil (Crisco vegetable cooking oil, density = 0.8888
g/ml) absorption capacities of the purified starch and the modified
starches were deternined by the centrifugal method (Beuchat,
1977). One gram 0: sample was mixed with 10 ml of distilled
water/oil (Sari-whirl, mixing control, fast) for 30 set, allowed to
618-Volume

46 /1981)-JOURNAL

OF FOOD SCIENCE

Fig. 2-Light
I1 75X).

photomicrograph

of

Great

North&m

bean

starch

GREATNORTHERN

tion completed.A blank with pure starch was conductedconcurrently and the D.S. calculatedasfollows:
% Acetyl =
(ml blank - ml sample)X normality of HCl X 0.43 X 100
Weightof sample(g) dry basis
162 X % Acetyl
D.S.=
4300 - (42 X % Acetyl)

BEAN

STARCH..

(1968) have reported similar observations on legume

starches. The cell wall structure is shown in Figure 4. The
starch granules can be seen enclosed in the cell wall. The
isolation treatments did not remove completely all the cell
walls and some did survive as shown in the Figure 4.
Amylose content and starch hydrolysis
The amylose content of Great Northern bean starch was

RESULTS & DISCUSSION

Composition and yield
The data on composition and yield are presented in
Table 1. The purity of the isolated starch was judged on
the basis of composition and microscopic observations.
The yield of Great Northern bean starch was 18.23% (on
bean flour basis). Naivikul and DAppolonia (1979) reported yields of 40.3, 38.3, 39.9,42.5, and 34.5% for navy
bean, pinto bean, faba bean, lentil, and mung bean starch
respectively. Schoch and Maywald (1968) obtained starch
yields of 27, 38, and 37% from navy bean, lentil, and mung
bean seeds respectively. Lineback and Ke (1975) reported
37% starch yield from horsebean flour. The differences in
yields have been attributed to the methods opted for starch
isolation (Naivikul and DAppolonia, 1979). A yield of
18.23% in the present investigation which was lower than
those of navy and pinto beans, both Phaseolus vulgaris
species may be primarily due to the method of isolation.
Granule size and microscopic appearance
The shape, size, and bifringence of starch granules are
often representative of the plant species and its maturity
(Manners, 1974). Several fiels were observed and measurements of 25 representative granule sizes were made. The
range of granule size was about 12 X 12 pm to 58 X 40
pm (length X width) which was in close agreement to the
ranges reported by Naivikul and DAppolonia for navy and
pinto bean starches (12-36 and 16-28 pm for width, 1240
and 1640 pm for length, respectively, for navy and pinto
bean starch granules). The shape of Great Northern bean
starch granules was quite varient, ranging from small
round to large oval to irregular. Some granules were concave. Similar observations have been reported by Lineback and Ke (1975) on chick pea and horsebean starches,
and by Lai and Varriano-Marston (1979) on black bean
starch.
Light microscopic observations (Fig. 2) of the starch
granules revealed the presence of hylum and lamellae. In
general, the hylum paralleled the longitudinal axis of the
starch granule; however, in case of spherical granules such
as distinction could not be made. Hylum was absent on
some granules; however, lamellae were observed in all the
granules viewed. Hylum was found to possess different
shapes and varying lengths. Similar observations have been
reported on starch grains of Phaseolus species (Dhaliwal
et al., 1964) and lima beans (Salunkhe and Pollard, 1955a,
b; Salunkhe, 1957).
Scanning electron photomicrographs are presented in
Figure 3. As can be seen from these photographs, starch
granules appear to be round, oval, and elliptical. The surfaces appeared to be smooth. The lamellae observed in the
light microscopic view (Fig. 2) were not evident in the
scanning electron microscopic observations on starch granules. This may have been due to the dehydrated state of
starch granules in scanning electron microscopy samples
versus the hydrated state in light microscouv. Some starch
granules appeared to be do-nut shaped. -Hall and Sayre
(1971), McEwen et al. (1974), and Schoch and Maywald

Fig. 3-Scanning
electron
photomicrographs
bean starch: (Al 365X; (B1 730X; (Cl 1460X.

Volume

46 (1981bJOURNAL

OF FOOD

of

Great

Northern

SCIENCE-619

10.2% (on starcll basis) which was comparable (in order)

to that reported in Amsoy 71 soybeans (15-20%) by Wilson et al. (1978). Results of starch hydrolysis employing
a-amylases are p resentedin Figures 5 and 6. Hog pancreatic
a-amylase hydrolysed 8.2% starch which was higher than
that by cw-amzla:efrom malt, in a 2-hr period at room temperature (21 C). This difference may be due to the difference in the activities of enzymes used (11 mg maltose and
3.3 mg maltose liberated per mg of enzyme in 1 min at
20C at pH 6.9 for hog and malt ol-amylase, respectively).
The low degree of, hydrolysis may be due to the starch
nature (uncookell raw starch was employed) and a relatively low temperature (21C) during incubation.

Waterandoil abrorption
The water alld oil absorption data are presented in
Table 2. Modifications did not improve both water and oil
absorption capac:ity of starch. The purified starch had oil
and water absorption capacity of about 2.9 g/g and 2.93
g/g, respectively. Halbrook and Kurtzman (1975) have
reported a water uptake of about 3.0 g/g and about 3.0
g/g at 121C and 80C, respectively. Our results of water
absorption (2.92 g/g at 21C for the purified starch) were
comparable to .:heir observation of water absorption at
8OC. The high water absorption at 21C observed in the
present investigation may have been due to the nature of

the starch and a possible contribution to water absorption

by the cell wall material(s) which was not removed completely. Comer and Fry (1978) have reported cold water
absorption of the purified pea starch to be 92-105%, and
that the water uptake was a function of temperature.

Pastingpropertiesandgelation
The amylograms are the plots for the corrected viscosity
(Fig. 7). The data are summarized in Table 3. Peak heights
were not reported as the amylograms did not have distinct
peaks. With the exception of the oxidized starch, all other
samples followed similar patterns. The change in viscosity
after holding for 15 min at 95C was rather slow, except
for oxidized starch in which case it decreased sharply during the cooling cycle. The gelatinization temperature range
(65.5-68.5(Z) of the purified Great Northera bean starch
was comparable to those of faba bean (66 Cd and lentil
(68C) (Naivikul, 1977); garbanzo bean (65-71 C), smooth
pea (65-69(Z), red kidney bean (64-68OC), and mung
bean (63-69C) (Biliaderis et al., 1979); and black bean
(63.8-76C) (Lai and Varriano-Marston, 1979) starches.
The trend of the purified starch curve was characteristic
of restricted swelling type starches. The viscosity behavior
of the oxidized starch was characteristic of hypochlorite
oxidized starches which show a greater degree of fluidity.

Table 2-Water

andoilabsorption

by the Great Northern

Water absorbed
Sk

Sample
Purified starch
Acetylated
starch
Oxidized starch

Table 3-Amylogram

Sample
Crude starch
Purified starch
Acetylated
starch
Oxidized starch

Fig. 4-Scanning

Fig. 5-Light
ofA.

620-Volume

eh&on

photomicrograph

photllmicrograph

of a-amylase

of the cell wail,

(hog pancreas)

1460X.

attack

bean starch
Oil absorbed
Sk

2.93
2.68
2.60

2.94
1.88
2.26

Data of the Great Northern

bean starch

Gelatinization
temp range
(C)

15 min

62.587.0
65.5-68.5
61.0-64.0
65.5-68.5

425
295
355
40

50C htb
(BU)

325
445
475
Ii

a Viscosity
of the corrected
starch curveO(in Brabender
Units)
the end of 15 min period of holding at 95 C.
b Viscosity
at 50C (In Brabender Units) during the cooling cycle.

on starch granule.

(A) 0 hr; IBJ 2 hr. The magnification

at

of B is 2.5 times that

I

41; (198lkJOURNAL

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GREAT

The tendency to set back on cooling is minimized in oxidized starches due to the presence of functional groups that
block the association tendencies of the starch chains
(Scallet and Sowell, 1967). The gelation studies indicated
that purified starch could yield stable gels at concentrations
of 7% or above (w/v).

O-0

Hog d-Amylare

- . . . . . . . .

M&d-Amyline

30

45

60

75

90

105

120

TIME (min.)

Fig. 6-Starch

hydrolysis

Fig. 7-Gelatinization
bean starch.

-*

by or-am ylases.

curves

(corrected

for CMC)

of Great Northern

NORTHERN

BEAN

STARCH..

REFERENCES
AACC 1962. Approved
Methods of the AACC, 7th ed. American
Association
of Cereal Chemists. St. Paul. Minn.
Beuchat,
L.R. 1977. Functional
and electrophoretic
characteristics
of succinylated
peanut flour proteins. J. Agr. Food Chem. 25: 258.
Billaderls,
C.G., Grant, D.R.. and Vase. J.R. 1979. Molecular
weight
distributions
of legume starches by gel chromatography.
Cereal
Chem. 56: 475.
Cerning-Beroard.
J. and Flliatre,
A. 1976. A comparison
of the carbohydrate
composition
of legume seeds: Horsebeans.
peas, and
lupines. Cereal Chem. 53: 968.
Coffman,
C.W. and Garcia, V.V. 1977. Functional
properties
and
amino acid content of a protein isolate from mung bean flour. J.
Food Technol. (U.K.) 12: 473.
Comer, F.W. and Fry M.K. 1978. Purification,
modification,
and
wooerties
of air-clas;fied
nea starch. Cereal Chem. 55: 818.
Decker. L.A. 1977. Worth&ton
Enzyme Manual, p. 173. Worthington Biochemical
Corporation,
Freehold, N.J.
Dhaliwal.
AS.. Pollard. L.H.. and Salunkhe.
D.K. 1964. Biosynthesis of starch &ins ln.cotyledons
of Phaseblus species. Amer. Sot.
Hort. Sci. 85-361.
Halbrook,
W.U. and Kurtzman,
R.H. Jr. 1975. Water uptake of bean
and other starches
at high temperatures
and pressures.
Cereal
Chem. 52: 156.
Hall. D.M. and Sayre, J.G. 1971. A scanning electron microscope
study of starches. 3. Miscellaneous
starches. Textile Res. J. 41: 880.
Hullinger.
C.H. 1964. Hypochlorite
oxidized starch. In Methods
in
Carbohydrate
Chemistry:
Starch,
Vol. 4, Ed. Whistler,
R.L.,
p. 313. Academic Press, New York.
Lai, C.C. and Varrlano-Marston,
E. 1979. Studies on the characteristics of black bean starch. J. Food Sci. 44: 528.
Lineback.
D.R. and Ke, C.H. 1975. Starches and low molecular
weight carbohydrates
from chick pea and horsebean flours. Cereal
Chem. 52: 334.
Manners,
D.J. 1974. The structure
and metabolism
of starch. In
Essays in Biochemistry,
Vol. 10, p. 37. Ed. Campbell, P.N. and
Dickens, F.. Academic
Press, New York.
McEwen.
T.J., McDonald,
B.E.. and Bushuk. W. 1974. Faba bean
(Vicia faba minor).
Physical, chemical,
and nutritional
properties.
Unpublished
report,
The Fourth
International
Food Congress,
Madrid, Spain.
Naivikul,
0. 1977. The carbohydrates
present in flour obtained
from various types of legumes. Ph.D. thesis, North Dakota State
University,
Fargo, N.D.
Naivikul.
0. and DAppolonia.
B.L. 1979. Carbohydrates
of legume
flours compared with wheat flour. 2. Starch. Cereal Chem. 56: 24.
Salunkhe,
D.K.
1957. Histological
and histochemical
changes in
gamma-irradiated
lima beans, Phaseolus lunatus. Nature 179: 585.
Salunkhe.
D.K. and Pollard, L.H. 1955a. A rapid and simple method
to determine
the maturity
and quality of lima beans. Food Technol. 9: 45.
Salunkhe,
D.K. and Pollard, L.H. 195513. Further studies on microscopic examination
of starch grains in relation to maturity
of lima
beans. Food Technol. 9: 521.
Sandstedt,
R.M. and Abbott,
R.C. 1964. A comparison
of methods
for studying
the course of starch gelatinization.
Cereal Sci. Today
9: 13.
Sathe, S.K., Ponte, J.G. Jr., Rangnekar,
P.D., and Salunkhe, D.K.
1980. Effects of addition
of Great Northern
bean (Phaseolus vulgaris L.) flour and protein concentrates
on rheological
properties
of dough and baking quality
of bread. Cereal Cbem. (In press).
Scallet, B.L. and Sowell, E.A. 1967. Production
and use of hypochlorite
oxidized
starches. In Starch
Chemistry
and Technology.
Industrial
Aspects, Vol. 2, p. 243. Ed. Whistler, R.L. and Paschall.
E.F.. Academic Press, New York.
Schoch, T.J. 1964. Fatty substances in starch. Determination
and
removal.
In Methods
in Carbohydrate
Chemistry:
Starch,
Vol. 4, p. 60. Ed Whistler, R.L. Academic Press, New York.
Schoch, T.J. and Maywald,
E.C. 1968. Preparation
and properties
of various legume starches. Cereal Chem. 45: 564.
Wilson,
L.A..
Birmingham,
V.A.. Moon, D.P., and Snyder,
H.E.
1978. Isolation
and characterization
of starch from mature soybeans. Cereal Chem. 55: 661.
Wolf, M.J., Melvin, E.H.. Garcia, W.J., Dimler.
R.J., and Kwolek,
W.F. 1970. Amylose
determination
in dimethyl
sulfoxide
extracts
of maize. Cereal Chem. 47: 437.
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Academic Press, New York.
MS received 6/26/80; revised E/16/80; accepted E/24/80.
Presented at the 40th Annual Meeting of the Institute
of Food
Technologists,
New Orleans, La., June E-11.1980.
Contribution
No. 2578 from the Utah Agriculture
Experiment
Station and a contribution
of Western Regional Project W-150.
We thank Professor J.G. Ponte Jr. and Mr. P.D. Rangnekar.
Dept.
of Grain Science & Industry,
Kansas State Univ., Manhattan,
KS
66506, for their help in viscoamylographic
studies.

Volume

46 /1981)-JOURNAL

OF FOOD

SCIENCE-621