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thesis entitled 7
Effects of Processing Factors on the
Quality Characteristics of Soaked and
Processed Navy Beans

presented by

Bih Jyu Lee

has been accepted towards fulfillment
of the requirements for

M.S. , Food Science
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EFFECTS OF PROCESSING FACTORS ON THE QUALITY
CHARACTERISTICS OF SOAKED AND
PROCESSED NAVY BEANS

By

Bih Jyu Lee

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

1979

ABSTRACT
EFFECTS OF PROCESSING FACTORS ON THE QUALITY

CHARACTERISTICS OF SOAKED AND
PROCESSED NAVY BEANS

By
Bih Jyu Lee

2 2

Effects of (1) the divalent ions, Ca+ and Mg+

(2) the additive, sodium hexametaphosphate (NaHMP). and

2 and NaHMP on qualities of

(3) the combination of Ca+
soaked and processed beans were studied.
Effects of selected enzymes, a-amylase, gluco-
amylase, pectinase, cellulase and protease on qualities
of soaked beans were performed by soaking beans with
and without seedcoats at 20, 40, 60 and 80°C for 5 hours.
Activities of the enzymes in reducing whole ground bean
flour viscosities were carried out using the Brabender
Visco-Amlegraph with temperature programmed from 45-95°C.
The divalent ions decreased drained weight and
firmed the beans. NaHMP aCcelerated water uptake, softened
soaked beans and increased scluble solids leaching. The
combination treatment decreased drained weight and

+2 effect on shear resistance.

showed no Ca
The enzymes were ineffective on water uptake and

shear resistance. The temperature and seedcoat affected

Bih Jyu Lee

these two characteristics. a-Amylase and pectinase
reduced the viscosity; while glucoamylase, cellulase

and protease had no effects.

ACKNOWLEDGMENTS

The author expresses her sincere appreciation
to Dr. M. A. Uebersax for providing an excellent
working atmosphere and for his guidance throughout
this study and assistance in manuscript preparation.

Gratitude is expressed to members of the
committee: Dr. P. Markakis, Dr. J. Cash and Dr. CSS.
Hosfield for their advice and thoroughness in reviewing
this manuscript.

Grateful acknowledgment is due to Dr. M. Zabik
for the use of her amylograph; and to Dr. P. Washko
for the use of atomic absorption spectrophotometer.

The greatest appreciation is given to the
author's countrymen, the people of Taiwan, Republic of
China, for their financial support. Without their
support, this study would be impossible.

The Michigan Bean Commission is acknowledged for
contributing indirect financial support for this research.

The author also expresses many thanks to her
friends and co-workers who have offered suggestions

and lots of help throughout this study.

ii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . . .
MATERIALS AND METHODS . . . . . . . . . . . .
Raw Material . . . . . . . . . . . . . .
Soak Water and Process Brine . . . . . .
Study Design . . . . . . . . . . . . . .
Soaking and Processing Procedure . . . .

Enzyme Soak and Gelatinization Studies .
Analytical Methods . . . . . . . . . . .
RESULTS AND DISCUSSION . . . . . . . . . . . .

Effects of Divalent ions. Ca+2 and Mg+zon

Quality Characteristics of Soaked and
Processed Navy Beans . . . . . . . . .
Effect? of NaHMP and Its Combination with
Ca+ on the Quality Characteristics of

Soaked and Processed Navy Beans . . .

Effects of Selected Enzyme Treatments . .
SWRY O O O O O O O O O O O O O O O I O I O
me RESEARCH O O O O O O I O I 0 O O l O O

REF‘ERENCES I O O O O O O O I O O O O O O I O 0

iii

Page
iv

vi

12
12
12
13
16
17
18
24

24

31
4o
52
56
57

TABLE OF CONTENTS

LIST OF TA B138 O O O O O O O O O O O O O O O O
LET OF FIGIRB O O O O O O O O O O O O O O O
INI'RODUCT ION O O O O O O O O O O O O O O O O O

MATERIALS AND METHODS . . . . . . . . . . . .
Raw Material . . . . . . . . . . . . . .
Soak Water and Process Brine . . . . . .
Study Design . . . . . . . . . . . . . .
Soaking and Processing Procedure . . . .
Enzyme Soak and Gelatinization Studies .
Analytical Methods . . . . . . . . . . .
RESULTS AND DISCUSSION . . . . . . . . . . . .

Effects of Divalent ions, Ca+2 and Mg+zon

Quality Characteristics of Soaked and
Processed Navy Beans . . . . . . . . .
Effect? of NaHMP and Its Combination with
Ca+ on the Quality Characteristics of

Soaked and Processed Navy Beans . . .

Effects of Selected Enzyme Treatments . .
SWY O O O O O O O O O O O O O O O O O O O
FURTI'IER RESEARCH O O O O O O O O O O O O O O O

REF‘ERENCES O O O O O O O O O O O O O O O O O O

iii

Page
iv

vi

12
12
12
13
16
17
18
24

24

31
4O
52
56
57

LIST OF TABLES

Table Page
1 Source and activity of selected enzymes . . 14
2 Levels of NaHMP and combination of Ca+2

and NaHMP in soak water and process
brine O O O O O O O O O O O O O O O O O 1 5

3 Concentration and incubated pH of selected
enzyme 8 O O O O O O O O O O O O O O O O O 1 6

4 Mean values of calcium ion level effects
on the quality characteristics of
soaked and processed beans . . . . . . . 25

5 Statistical summary of calcium ion level
effects O O O O O O O O O O O O O O O O O 26

6 Drained weight and shear resistance
regression on processed bean calcium
content . . . . . . . . . . . . . . . . . 27

7 Mean values of magnesium ion level effects
on the quality characteristics of soaked
and processed beans . . . . . . . . . . . 29

8 Statistical summary of magnesium ion
level effects . . . . . . . . . . . . . . 30
9 Mean values of NaHMP level effects on the

quality characteristics of soaked and
processed beans . . . . . . . . . . . . . 32

10 Statistical summary of NaHMP level effects . 34
11 Selected quality characteristics of pro-

cessed bean regression on NaHMP levels . 35
12 Mean values of combination of Ca+2 and

NaHMP effects on the quality charac-
teristics of soaked and processed beans . 37

13 Statistical summary of combination of Ca+2
and NW effeCtS O O O O O O O O O O O O 38

iv

14

15

16

17

18

Page

Mean values of water uptake (g) of beans
soaked with and without seedcoats in
selected enzyme solutions for 5 hours
at different temperatures . . . . . . . . 41

Mean values of shear resistance (lbs/50g)
of beans soaked with and without seed-
coats in selected enzyme solutions for
5 hours at different temperatures . . . . 42

Statistical summary of soaked studies with
selected enzymes . . . . . . . . . . . . 43

Mean values of viscosity of bean flour
gelatinized in selected enzyme solutions. 48

Statistical summary of selected enzyme
gelatinization studies . . . . . . . . . 50

LIST OF FIGURES

Figure Page
1 Enzyme soak studies -- The main factor
effects on water uptake of soaked
beans O O O O O O O O O O O O O O O O O 44
2 Enzyme soak studies -- The main factor

effects on shear resistance of soaked
beans . . . . . . . . . . . . . . . . . 45

3 Effects of selected enzymes on the
viscosity of whole ground bean flour . 49
4 Bean flour viscosity regression on pH . . 51

vi

INTRODUCTION

Edible dry beans are an important source of
protein, having nutritional values higher than the
cereal grains (Anonymous, 1975) but lower than the
animal proteins. However, supplemented by a relatively
small amount of methionine, the legumes may provide
the most efficient and economical medium for the
conversion of nitrogen into nutritional human food
(Rockland et al., 1974). In addition, they also
provide calories, minerals and vitamins to the diet
(de Moraes and Angelucci, 1971: Rockland et al., 1977b).

Raw, dry legumes contain a variety of undesirable
components, most of which are eliminated during soaking
and cooking. The preparation of dry beans generally
involves hydration in water to a moisture content of
between 53 and 57%, followed by extended cooking. The
wide spread utilization of dry legumes depends on proper
handling, storage, and processing (Johnson, 1965;
Thompson et al., 1962).

Information on the chemical composition of specific
tissues,the structure of cells, and the localization of
chemical constituents in the cells of the mature navy
bean (Phaseolus vulgaris L.) seed is a prerequisite for

providing aui explanation of physical and chemical changes
1

in bean tissue which occur during mechanical, thermal,
chemical and enzymatic treatments.

According to the report of Powrie et al. (1960),
the seedcoats, cotyledons and embryonic axes constitute
7.7%, 90.5% and 1.8% respectively of the dry weight of
mature navy beans. The two catyledons are the most
important components of the seeds by weight and volume.
On dry weight basis, the cotyledons contain 27.5% protein,
1.65% ether extractables and 3.5% ash. Moreover, the
starch content of the cotyledons is 39.3%. The protein
content of the seedcoat is 4.8%, while the outer portions
of the seedcoat contain 0.4% ether extractable wax-like
material. The ash content of the seedcoat is 8.44%.

The hypocotyl and epicotyl constitute the axis of the
bean embryo; the embryonic axes contain 45.6% protein,
3.11% ether extractables and 3.58% ash.

The textural characteristics and nutritive value
of processed seeds presumably are influenced to a large
extent by the size and shape of cells, dimensions of
cell walls, and the localization of chemical constituents
in the cotyledons. The outermost layer of the cotyledon
consists of epidermal cells. The inner and outer
epidermal cells have been designated as those along the
flat and curved surfaces of the cotyledon. From a three
dimensional viewpoint, the inner epidermal cells are
elongated cubes with the long axes in the transverse
direction, while the outer epidermal cells are somewhat

cubical. The cell contents of the epidermis appear

granular, containing protein but no starch. The layer

of cells inward from the outer epidermis is the hypodermis
with cells larger than the outer epidermal cells and
elliptical in shape. The cell content appears to be a
granular matrix containing tyrosine.

Parenchyma cells and vascular bundles constitute
the remaining tissue of cotyledon. The parenchyma cells
near the outer hypodermis are somewhat smaller than the
remainder of the parenchyma cells. The thick walls in
the longitudinal section of parenchyma cells are re-
sponsible for the rigidity of the soaked beans. In each
parenchyma cell , starch granules are imbedded in a
matrix composed chiefly of tyrosine and cysteine containing
proteins. With breakdown of starch granules, the intact,
rigid materials possess cavities which vary in size.

Each cavity is envelOped by proteinaceous matter. The
shapes of the liberated granules vary from round to
elliptical and possess smooth surfaces.

The epidermal and hypodermal cells possess no
secondary walls. However, in the parenchyma cells, the
secondary walls are observed to be very thick compared to
the primary walls and contain numerous small cavities
called simple pits. Each pit is opposite another one
in the adjacent secondary wall. The presence of pits
in the secondary wall facilitates the diffusion of water
into the protoplasm during the soaking period.

Dry beans have been traditionally soaked over-

night (12-16 hours) in cold water. This initial soaking

treatment ensures uniform expansion and tenderization
during subsequent thermal processing, increases product
yield, and improves cleaning of the beans. Several
methods have been proposed to improve soaking procedures
by accelerating water uptake and tenderizing dry

beans. These include heat treatment (Gloyer, 1928;
Morris et al., 1950; Dawson et al., 1952), vacuum
treatment (Hoff and Nelson, 1965), the use of additives
(Reeve, 1947; Rockland, 1963: Rockland and Karl, 1966:
Rockland and Metzler, 1967), and irradiation (Bedford,
1971). Several factors have been shown to influence
water uptake. These include the age and composition of
dry beans, storage conditions, moisture contents and
production condition factors (Bedford, 1971). Pectic
substances, hemicellulose and protein are functional
components in absorbing water in plants (Ott and Ball,
1943). Smith and Nash (1961) concluded that the seedcoat
is the principle factor controlling the rate of water
uptake in soy beans. Hamad and Powers (1965) found rates
of water imbibition were inversely related to the pectic
content of dry peas and beans. Uebersax (1972) reported
water uptake values decreased with high temperature and
high humidity storage. Burr and Kon (1967) reported
decrease in water absorption rate for sanilac beans with in-
creased bean moisture content and storage time.
Nordstrom and Sistrunk (1979) reported low original
moisture levels before soaking resulted in high hydration

ratios. Acidity of soak water reduced the rate of water

uptake (Snyder, 1936). Luh et al. (1975) postulated

that at low pH, starch and protein swelling are inhibited,
causing a reduction in hygroscopicity. Cholinesterase
activity, phosphorous content and phytin content were
suggested to have some influence on the imbibition and
tenderness of rehydrated dry beans (Andus, 1955; Fowler,
1956, 1957). The effect of soak water additives have
also been studied. Such additives include sodium
bicarbonate (Greenwood, 1935), hexametaphosphate (HMP),
sulfite, oxalic acid, ammonium oxalate and hydrochloric
acid (Reeve, 1947), and ethylene diamine tetracetic

acid (EDTA) (Elbert, 1961: Luh et al., 1975). In general,
results of these studies have shown that HMP increased
the rate of imbibition, by serving as a chelating agent
for calcium ion, solubilizing the bean cotyledon proteins
as well as aiding in dissociating metal-salt-protein
complexes which could interfere with water absorption
(Holmquist et al., 1948; Rockland, 1963). EDTA and the
mixture of alkaline carbonates increased water uptake
apparently by softening the seedcoat. Sodium chloride
may or may not increase water uptake depending on the
quality of dry beans (Hoff and Nelson, 1965).

Most commercial canners use some type of heat
treatment during soaking. Mattson (1946) stressed that
heat was needed to promote precipitation of calcium and
magnesium by phytin. According to Mattson, precipitation
prevented divalent ions from interacting with pectin to

form tough, pectin-metal ion complexes. Morris and

Seifert (1961) reported, however, that heat inactivated
the enzymes phytase and pectinesterase. If these
enzymes were allowed to act, phytin would be dephos-
phorylated and pectin would be demethylated causing a
shift of divalent ions to form the tough pectin-metal
ion complexes (Matz, 1962).

Cooking of dry beans is necessary to develop an
acceptable flavor and texture, to destroy toxic material
and to maximize the nutrient availability of bean
protein (Kakade and Evans, 1963). The cooking rate for
intact dry lima beans was estimated with an automatic
recording Lee-Kramer shear press (Binder and Rockland,
1964). The shear peak is related to the shearing
strength of the sample or cohesiveness as defined by
Szczesniak (1963). Burr et al. (1968) studied the effect
of storage temperature and time and moisture content on
the cooking rate of dry beans. They concluded that the
cooking quality of beans with a moisture content of 10%
or less is not greatly affected by storage temperature
and time. At moisture levels above 10%, bean cookability
deteriorated in good correlation with increased storage
temperature and time. Bean processors often find that
the heat process required to sterilized canned beans is
insufficient to make them tender (Bigelow and Fitzgerald, 1927).
Several solutions have been proposed to resolve the hard
to cook phenomenon of dry beans. Such approaches have

included the development of precooked hydrated beans

(Esselen and Davis, 1942: Feldberg et al., 1956), quick
cooking dry beans (Rockland and Metzler, 1967), and heat
treated beans (Molina et al., 1976). The method used

to prepare quick-cooking beans consists of (1) loosening
the seedcoats by vacuum infiltration in a solution
containing NaCl, Na5P3010, NaHCO3 and NaZCO3‘ (2)

soaking the beans in these same salt solutions; (3)
rinsing; and (4) drying, cooking or freezing the beans
depending on their ultimate utilization. The resulting
product cooks in 15 minutes or less. Rockland and

Jones (1974) indicated that this process did not affect
the structure and appearance of the beans when compared
with untreated samples. Rockland et al. (1977a) reported
that the purified, free, dispersed lima bean starch
granules deformed and gelatinized in pure water within
the range of about 71-79°C. In a dilute mixed salt
solution which was employed to produce quick cooking
beans, the starch dispersions gelatinized within the
temperature range of about 79-850C. The increase in
extracellular gelatinization temperature of lima bean
starch granules in the mixed salt solution was consistent
with the effect of salt solution on intracellular bean
starch gelatinization observed by Hahn et al. (1977).
Varriano-Marston and De Omana (1979) studied the effect
of sodium salt on the chemical composition and morphology
of quick cooking black beans. By means of atomic

absorption and flame emission spectrophotometry, light

and scanning electron microscopy, and x-ray analysis,
these researchers reported that the amount of sodium

in the soak water did not significantly affect the amount
of water absorbed by the beans: the pH was the critical
determinant. However, the sodium salts affect the
mineral content as well as the amount of pectic substances
solubilized from the beans during the soaking and pro-
cessing periods. These researchers suggested that the
mechanism for the quick cooking of black beans presoaked
in sodium salt solutions prior to cooking was an ion
exchange and chelation mechanism. Ions which stabilized
the structure of intercellular pectic substances were
replaced by sodium ions or were removed by chelation.

The result was a solubilization of pectic substances
during soaking and cooking. The cooking process was
thought to further facilitate the dissociation of inter-
cellular cement by disruption of hydrogen bonding. The
similarities between the presoaking treatments of black beans
in Na5P3O10 and combined salt solutions with respect
to mineral losses, pectic substances solubilized, and
cell separation, indicated that the NaSPBOIO treatment
may be a satisfactory alternative in the production of
quick cooking black beans.

Color is another factor affecting the quality of
canned dry beans. Addition of calcium chloride or citric
acid to brine during canning improved the color of dry
lima beans (Luh et al., 1975). The beans treated with

calcium chloride had higher Rd values than the

control samples. Acidification of the brine with citric
acid at 0.25% and 0.50% levels decreased the formation
of gray color compounds. The lowering of pH of the
brine improved color by increasing dissociation of
metallic copper sulfite complexes.

The use of enzymes to improve the quality of
canned beans and peas has been studied by Powers et al.
(1961). Amylolytic and pectolytic enzymes were applied
to reduce the gelling of canned black eyed peas and
pinto beans. Insufficient catalytic activity made
commercial use of the enzymes impractical. However
enough hydrolytic activity did result to establish that
both starch and pectin fractions were involved in gelling
in canned peas and beans. d-Amylase (d-l,4-glucan
4-glucanohydrolase, EC 3.2.1.1) is an endosplitting
enzyme which hydrolyzes the a-1,4-glycosidic bonds of
the substrate in a random fashion. Calcium ion is
necessary to keep the enzyme active and stable. The pH-
reaction rate profile for the a-amylase is a bell shaped
curve with the optimum activity varying from pH 4.8‘r7.0
depending upon the enzyme sources. Glucoamylase (d-l,
4-glucan glucohydrase, EC 3.2.1.3) is an exosplitting
enzyme and removes successive glucose units from the
non-reducing ends of the substrate chains. The action
of the enzyme on a chain decreases markedly when an a-l,
6 linkage is encountered as in amlepectin and glycogen.
The enzyme also hydrolyzes a-l,6 and a-1,3 linkages
but more slowly than d-l,4 linkages are hydrolyzed. The

10

optimum pH is from4.0 to4.4 at 25°C. The pectic enzymes
are generally classified as pectinesterases, poly-
galacturonases and pectate lyases dependent on the mode
of degradation of the pectic substances. Pectinesterase
(pectin pectyl-hydrolase EC 3.1.1.11) removes methoxyl
groups from methylated pectic substances (pectin) and
therefore belongs to the subdivision of enzymes which
hydrolyze carboxylic acid esters. The polygalacturonases
(poly-d-l,4-galacturonide glycanohydrolase, EC 3.2.1.15)
hydrolyze glycosidic linkages in pectic substances by

the use of water. The pH optima for most of the poly-
methylgalacturonase and polygalacturonases are in the
range of 4.5 to 6.0. The pectate lyases (poly-d-1,4-D-
galacturonide lyases, EC 4.2.99.3) split the glycosidic
bond by trans elimination of hydrogen from the C-4 and
C-5 positions of the aglycone portion of the substrate.

A requirement for calcium ions is a characteristic of
the trans-eliminases. The enzymes which hydrolyze cellu-
lose and its derivatives can be divided into three groups
(King and Vessal, 1969). C1, an enzyme whose action

is not well defined, is required for hydrolysis of
highly oriented solid cellulose by the B-l,4 glucanases
and has been referred to as "hydrogen bondase." There

is no evidence that it hydrolyzes glycosidic bonds. The
8-1,4 glucanases are of two types, the exo-B-1,4 and

the endo B-l,4 glucanases. The B-glucosidases act most spe-

cifically on the small molecular products. The

11

B-glucosidases and exo-B-1,4 glucanases have in common
the substrates cellobiose to cellohexaose. The B-
glucosidase will hydrolyze cellobiose much more rapidly
than cellohexaose while the reverse is true for the exo-
B-l,4 glucanases. Homogeneous Mygothecium vegrucaria
cellulase has a molecular weight of 63,000; a pH optimum
of 5.5 to 6.0. The enzyme is inhibited by heavy metal
ions, by sulfhydryl reagents, by oxidizing and reducing
agents, and by glucoses. The proteolytic enzymes of
fungi have usually been classified on the basis of their
pH optima. The group named "acid protease" indicates
that the pH optima of the enzymes are at a low pH and
does not indicate the groups involved in the active site.
A large number of microbial proteases belong in this
group (Whitaker, 1972; Reed, 1975).

The purpose of this study was (1) to evaluate
the firming effects of calcium and magnesium ions during
soaking and processing of dry navy beans, (2) to evaluate
the effect of NaHMP and combination of NaHMP and calcium
ions on the quality of canned dry navy beans, and (3)
to study selected enzymatic soak treatments on the
softening of whole beans with and without seedcoats, as
well as these enzymatic effects on the viscosity of whole

ground bean flour during gelatinization.

MATERIALS AND METHODS

Raw Material

Samples of the 1977 harvest of Michigan-grown navy
beans were obtained from B and W Cooperative, Breckenridge,
MI. All beans were held at 20°C (68°F) until soaked and
processed or milled into whole flour. The initial
moisture content was approximately 13.6%. Whole beans
and bean flour were used in the soak water and process
brine additive studies and gelatinization experiments,
respectively. Enzyme treatment soak studies were per-

formed on whole beans with and without seedcoats.

Soak Water and Process Brine

Chemicals used in soak water and process brine
additive: studies were analytical reagent grade CaClz,
MgClz.6H20 (Fisher Scientific Company) and sodium hexa-
metaphosphate (NaHMP)(Monsanto Company). All soak water was
prepared in one gallon polyethylene containers and held
for approximately 16 hours prior to use.

Five types of enzymes were used in the enzyme soak
and gelatinization studies: d-amylase, glucoamylase,

pectinase, cellulase and protease. The source and activity

12

13

of each enzyme are listed in Table 1. The enzyme
solutions were prepared in distilled water adjusted with
hydrochloric acid or sodium hydroxide to the specified
stable pH. Each enzyme solution and its corresponding

control solution contained 50 ppm calcium ion.

Study Design

Divalent ions, Ca+2 and Mg+2 treatments

Soak water and process brine cation levels were
0, 50, and 100 ppm.

Soakigg -- Beans were soaked in each level of
divalent ions. Water uptake and shear resistance of
soaked beans were determined. Ash and calcium content
determination were conducted on final soak water and
beans soaked in different calcium ion levels.

Processing -- Soaked beans were processed in all
possible level combinations of soak water and process brine.
Drained weight, Hunter Lab color and shear resistance
of processed beans were determined. Ash and calcium
content determinations were conducted on final process
brine and beans processed in different calcium ion levels.

NaHMP and combination of calcium ion and NaHMP

treatments

The soak water and process brine additive levels

are listed in Table 2.

14

 

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Table 2. Levels of NaHMP and combination of Ca+2 and

NaHMP in soak water and process brine

 

 

 

Additives Lavels
NaHMP (%) 0, 0.25, 0.50, 0.75, 1.0, 1.5
Ca+2(ppm)+NaHMP (%) 0+0, 50+o, 100+o. 0+1.o, 50+1.o
100+1.0
Seeking -- Beans were soaked in each level of

soak water additives. The dependent measures included
water uptake, shear resistance of soaked beans, as well
as total nitrogen (%) in soaked beans and final soak
water .

Egoceesing -- Soaked beans were processed in the
same level of brine additive as that of soak water.
The dependent measures included drained weight, Hunter Lab
color, and shear resistance of processed beans, as well
as total nitrogen (%) in processed beans and final

process brine.

Selecteg enzyme treatments
Soeki g -- Soak studies with 5 selected enzymes

were conducted on whole beans with and without seedcoats.
The enzyme concentration andincubation.pH are listed in
Table 3. The water uptake and shear resistance of soaked
beans were determined on enzyme treated and control

samples.

16

Table 3. Concentration and incubation pH of selected
enzymes

 

 

Enzyme Type Concentration pH

a-Amylase 127 unit/ml 6.9

Glucoamylase 0.22 unit/ml 4.5

Pectinase 0.1 mg/ml, 3.5

Cellulase 0.1 mg/ml 6.5

Protease 0.1 mg/ml 3.5
Gelatinization -- Whole bean flour was used in

 

this experiment. Concentration and incubation pH of each
enzyme were identical to that in Table 3 except for d-
amylase. The concentration of 12.7 unit/ml was used.

The activity of each enzyme in reducing the viscosity

of bean flour during gelatinization was measured by a

Visco-Amylograph.

Seekieg and Processing Procedege

One hundred grams of beans were soaked in solutions
containing various levels of divalent ions, NaHMP or
combination of calcium ion and NaHMP shown in the study
design. One part of dry beans was immersed in four
parts of soaking water in a 600 ml beaker. Beans were
initially soaked 30 min at 20°C (68°F) and then were
transferred to a steam kettle with water maintained at

87.8°C (190°F) for an additional 30 minute soak. The

17

temperature variation within the beaker was i 1°C. Soaked
beans of 100 g dry solids were drained and filled into
303x406 enameled cans, covered with 93.3°C (200°F) brine,
double sealed and retorted at 115.6°C (240°F) for 45
minutes. Cans were cooled to 32.2°C (90°F) to 37.7°C
(100°F) in cold running water and air dried. Cans were
stored at 20°C (68°F) for at least two months to ensure
complete equilibration prior to analyses of chemical and

quality characteristics.

Eezyme Soek and Gelatinization Studies

Soak Studies

Whole beans with and without seedcoats were used
in these studies. Beans were previously soaked in
distilled water for 30 minutes to facilitate the removal
of seedcoats. Fourty-five grams each of whole beans
with seedcoats intact and with seedcoats removed were
placed into beakers containing 180 ml of enzyme solu-
tions or control solutions. The beans were soaked and
incubated in the solutions for 5 hours at 20°C, 40°C,
60°C, and 80°C in a temperature controlled water bath.
After soaking, the beans were drained on an 8 inch No. 8
mesh sieve, and water uptake and shear resistance were

measured.

18

Gelatinization
The Brabender Visco-Amylograph (No. 888, Type Vavi,

Volt 110, Amp 5, Cy. 60, C. W. Brabender Instruments,
Inc., South Hackensack, N.J.) was used in this experi-
ment. The Visco-Amylograph peak (viscosity) was obtained
using the procedure of AACC (1962) with the modification
that 15.03% (w/v) of bean flour was used and the tem-
perature programmed from 45°C to 95°C at the rate of
1.5°C/minute. These modifications were required due

to the consistency and gelling characteristics of the

bean flour.

Analytical Methods

Moistege

Initial moisture content of dry beans were
measured by a Motomco Moisture Meter (Model 919, Motomco
Inc., Clark, N.J.) in conjunction with calibrated moisture
chart no. B-5. All measurements were obtained following
the procedure recommended by the manufacturer. Moisture
contents of soaked and processed beans or bean flour were
determined according to the AOAC (1975) air dried oven
method. Twenty grams of well mixed soaked or processed
bean flour were placed in a tared aluminum moisture dish.
The dish and its contents were dried to a constant weight
(overnight) in a forced convection oven, then transferred
to a CaSO4 desiccator and weighed soon after reaching
room temperature. Residues were determined as total

solids and loss in weight as moisture content.

19

Age,

Ash content was determined according to AOAC
(1975) on beans soaked and processed in calcium ion
solutions and on the final soak water and brine.
Approximately 500 mg of well mixed dry samples were
weighed into previously ashed and tared Coors crucibles.
Samples were pre-ashed over a Fisher burner, incinerated
in a muffle furnance set at 525°C until light gray ash
was obtained (overnight). Residues were cooled over
CaSO4 in desiccators and weighed soon after reaching
room temperature. Ash content was determined using the

following formula:

Ash (%) = ASh w x 100
Sample Weight (g)
Calcium content

Calcium content was determined on soaked beans,

 

soak water, processed beans and brine using Perkin Elmer
Model 303 Atomic Absorption Spectrophotometer (Norwalk,
Connecticut) at wave-length 211 nm. Ashed samples were
digested with 6N nitric acid for about 45 minutes, then
diluted to 200 ml with 1N nitric acid. Lanthanum chloride
(0.1% w/v) was added in sample solutions to avoid inter-
ference caused by phosphate, sulfate, silicon or alumin.
Standard calcium (Sargent Welsh) for mineral analysis was
diluted with 1N nitric acid to the linear working range

of the instrument (Sug/ml Ca+2). All the acid diluted
standards contained equal levels of lanthanum. Data

were reported on a dry weight basis.

20

Wete; uptake

The water uptake of soaked beans was measured by
weight gain during the soak period. Beakers containing
soaked beans were cooled under running tap water for 5
minutes prior to draining. The soaked beans were emptied
onto an eight inch diameter, U.S. standard No. 8 sieve
at a 30 degree incline and drained for 2 minutes. Beans
plus screen were weighed. Soak water uptake was deter-
mined as total weight of soaked beans. The soaked beans
treated withtflueenzymes were emptied onto the screen,

and the water uptake was determined without cooling.

Dragged weight

Processed beans were emptied onto an eight inch
diameter, U.S. standard No. 8 screen and distributed
evenly. The screen and contents were rinsed in 20°C
(68°F) water for about 15 seconds to remove the gelling
material. Beans were immersed and screen agitated in a
slow swirling motion. The screen was drained at an
angle for 2 minutes. Beans and tared screen were weighed
for determination of washed drained weight. Canned beans
treated with calcium or magnesium were emptied onto the
screen, and the drained weight was determined without

rinsing.

Color

Objectively reflectance color measures were deter-

mined by a Hunter Lab Model D25 Color and Color Difference

21

Meter (Hunter Associates, Fairfax, Virginia). The instru-
ment was standardizedusing a standard white tile (L=94.5,
aL=-0.6, bL=+0.4). Processed beans of 100 g were placed
in an optically pure glass sample dish and covered to
shield interferring light from activating photo cells.
Coordinate values (L, :aL, ibL) were recorded for each

replicate and total color differences from the standard

 

(AE) were calculated by AB: j(AL) 2+(AaL) 2+( AbL)Z.

Shear resistance

The Lee-Kramer Recording Shear Press, Model TR-l
(Food Technology Coorporation, Reston, Virginia) was used
for all shear resistance measurements. The 3,000 pound
test ring and No. C-15 standard shear compression cell
were used. The rate of shear compression blade travel
was standardized to 0.52 cm/sec. A sample size of 50 g
of soaked or 100 g of processed beans was placed in the
cell, evenly distributed, and sheared. The entire cell

was cleaned and rinsed between each measurement.

Soaked beans -- Shear resistance was measured
directly after the soak period using range 50. Shear
resistance was reported as peak pound force/50 g of soaked
beans.

Proceseed beans -- Shear resistance was measured
on rinsed or non-rinsed processed beans using range 5.
Shear resistance was reported as peak pound force/100 g

of processed beans.

22

Total nitrogen

Analyses were conducted on beans soaked and pro-
cessed in different levels of NaHMP and the combination
of calcium ion and NaHMP. The semi-micro Kjeldahl method
for determination of total nitrogen was used according
to AACC (1962). A zooe3oo mg dry sample was digested
with concentrated HZSO4 (12N) as oxidant with the addi-
tion of [(2804 to elevate the temperature in the presence
of aselenium catalyst. The clear sample solution, neu-
tralized with concentrated NaOH (10N), was steam dis-
tilled into 10 ml of H3BO3--indicator solution until
the final volume 40 ml was collected. The distillate
was titrated with 0.1774N H2304. The total nitrogen,
reported as % was calculated by the formula:

N (%) ={[Eq. wt of N (14)xml of H2804 used x Normality
of H2804] x 100} /Sample wt in mg

Statistical Analyeis

All dependent measures for the soaking and proces-
sing studies to determine the effect of soak water and
process brine additives were analyzed using one way or
two way ANOVA, fixed effects with replications (Neter
and Wasserman, 1974). The selected enzyme soaking studies
were analyzed using multiple factor (enzyme type,
temperature, seedcoat, and group) ANOVA, fixed effects
with replications. The Tukey method was utilized for
multiple pairwise comparisons of means (Tukey, 1953).

Like letters were used to denote no significant

23

differences among means. Linear regressions were cal-
culated for selected dependent measures in soak water
and process brine additive studies using the method of
least squares. All correlations were calculated as the
Pearson Product-Moment correlation coefficient (Sokal
and Rohlf,1969). Coefficients of variation (CV) were
calculated to express the standard deviation for each

measure as a percent of the mean (Little and Hills, 1972).

RESULTS AND DISCUSSION

Effeets e: Divalent IonsI Ca+2 and Mg+2 on the Quality

Characteristics of Soaked and Processed Nayy Beans

Calcium ion

The mean values of calcium ion level effects on
processed navy beans are reported in Table 4. The
summary of statistical analyses of these data is presented

in Table 5.

Seaked beans -- The calcium ion levels did not
significantly (P=0.0S) affect water uptake during soaking.
The shear resistance of soaked beans increased signifi-
cantly (P=0.01) as the soak water calcium ion levels
increased. Results of analyses by atomic absorption
spectrophotometry showed significant differences (P=0.01)
in calcium content in the final soak water, but no
significant (P=0.05) difference in calcium content in
soaked beans among soak water calcium ion levels. This
indicated limited calcium ions diffused into the beans
from the soak water during the soaking peroid. However,
this amount of calcium was sufficient enough to result

in significant firming of soaked beans.

24

 

 

 

 

 

 

 

 

 

 

 

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He.ez Hm.me No.ee ee.Ne ee.~ me.ee om.me ee.eem o ooz
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mammm madam ”was Ac+ qm+ A AwwmmewWWH .93 czaum Hmumz
m we m 1, Hmmnm codamhm xmom
E A OH\ w... O HOUCSI NEQQVN +wU
mmhzmmmz
mason oommmOOHm
m~.m ee.e ee.e eo.e Axv>o
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e ee.ez e em.ee e m.~em e ee.m- o
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Mm I.r p AEQQV
moHSmmoz N+mo

 

mcmom noxmom

 

mo woaumaumuomumzo zuaamnd mnu co muommmm Ho>oa Goa ESHQHmo mo mosam> coo:

mason commooOHQ can omxmom

.e meeee

26

Table 5. Statistical summary of calcium ion level effects

 

I r -

 

Measures Source of variation D.F. M.S.
Seekeg beees
Water uptake Treatment 2 262.37
Error 0 85.35
Shear resistance Treatment 2 4452.89**
Error 3 318.74
Soak water Ca+2 Treatment 2 34.17%
Error 3 3.33
Soaked beans Ca+2 Treatment 2 3.74x10'2
Error 3 7.48x10"1
Processed beans
Drained wt. Treatment 8 1715.81**
Error 9 307.60
Shear resistance Treatment 8 546.46**
Error 9 29.51
Hunter L Treatment 8 3.65x10"1
Error 9 5.69x10'1
Hunter +aL Treatment 8 1.94x10'%
Error 9 1.25x10'
Hunters-bL Treatment 8 2.66x10'1*
Error 9 9.63x10‘2
41: Treatment 8 5.07x1o-1
Error 9 5.19x10'1
Brine Ca+2 Treatment 8 3.69**
Error 9 4.24x10'1
+2 **
Beans Ca Treatment 8 44.18
Error 9 6.84

 

**denotes significant F, P=0.01

*denotes significant F, P=0,05

27

Processed beans -- The ANOVA showed significant
(P50.01) differences in drained weight, shear resistance,
and processed bean and brine calcium contents among
_ different calcium ion levels. The plots of drained weight
and shear resistance on the calcium content of processed
beans indicated apparent linear relationships. Therefore,
linear regression analyses were performed to evaluate
these relationships. Table 6 contains the linear re-
gression equations and simple correlation coefficients for
drained weight and shear resistance. These significant
(P20.01) correlation coefficients indicated that the
drained weight and shear resistance of processed beans
were associated with the calcium content of processed
beans. The calcium content of processed beans directly
increased by the addition of calcium ions to soak water
and process brine. These results were in agreement with
the theory that pectin and calcium ion retarded water
uptake and increased firmness through the formation of
tough pectin-metal complexes (Matz, 1962).

Table 6. Drained weight and shear resistance regression
on processed bean calcium content

 

 

 

 

 

Dependent measures Regression rCaIZ SErCa+2
Drained weight Y=705.26-18.07X -o.9065** 0.1595
Shear resistance =-107.23+10.66X 0.9477** 0.1206

 

J;
A

*denotes significant r, P=0.01

28

Luh et a1 (1975) reported that addition of calcium
chloride affected the Rd values of processed dry lima
beans. However the color measurements Hunter L,a,b and.AE
were not significantly (P=0.05) affected by calcium ion
in this experiment. These may be due to the different

genotypic pigments between the seedcoats of lima and navy beans.

Magnesium ion

The mean values of magnesium ion level effects
are presented in Table 7. The summary of statistical

analyses of these data is reported in Table 8.

Soaked beans -- The non-significant (P=0.05)
difference in water uptake and the significant (P=0.01)
difference in shear resistance of soaked beans among
soak water magnesium ion levels were consistent with
calcium ion treatment.

Processed beans -- Drained weight and shear re-
sistance of processed beans were significantly (P=0.01)
affected by soak water and process brine magnesium ion
levels. As the magnesium ion levels increased, the
drained weight decreased, but the shear resistance
increased. The color measurements, Hunter Luawb were
significantly (P=0.05) affected by magnesium ion levels.
Hunter L decreased, Hunter +aL and +bL increased as the soak

water and process brine magnesium ion levels increased.

 

 

 

 

 

 

29

 

 

a slew

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mason commoOOHa can nuance mo
moaumauouowumco auaamsv can so muoommo Ho>oa Goa Ejamocmma no mosam> com: .5 macaw

Table 8. Statistical summary of magnesium ion level effects

30

 

 

Measures Source of variation D.F. M.S.
Seekeg beans
Water uptake Treatment 2 458.07
Error 30 163.74
Shear resistance Treatment 2 8278.13**
Error 3 234.75
Proceseed beans
Drained wt. Treatment 8 1564.64**
Error 9 198.06
Shear resistance Treatment 8 393.65%”
Error’ 9 20.16
Hunter L Treatment 8 1.45*
Error 9 3.78x10'1
Hunter +aL Treatment 8 2.14x10'1*
Error 9 4.21x10'2
Hunter +bL Treatment 8 6.20x10'1**
Error 9 8.89x10'2
A E Treatment 8 0 . 79*
Error 9 1.90x10'1

 

**denotes significant F, P=0.01

*denotes significant F, P=0.05

31

Effect on NaHMP and IES Cemelneglen Eiih Qe+2 on ene

Qualigy Cheyacteristics of Soaked and Processed Nayy Beans

NaHMP

 

The mean values of NaHMP level effects on processed
beans are reported in Table 9. The summary of statistical

analyses of these data is presented in Table 10.

Seekeg beans -- The water uptake increased, the
shear resistance decreased as the soak water NaHMP levels
increased. These results are in agreement with previous

findings (Holmquist et al., 1948: Hoff and Nelson, 1965).

 

The regression equations as well as correlation coef-
ficients of water uptake and shear resistance of soaked
beans are presented in Table 11. The water uptake was
directly correlated with NaHMP levels; the shear resistance
was negatively correlated with NaHMP levels. The ANOVA
showed significant (P=0.01) differences in total nitrogen
(%) in soak water; but no difference in the soaked beans
among the NaHMP levels. As the NaHMP levels increased, the
total nitrogen (%) in soak water increased on the fresh
weight basis but decreased on the dry weight basis. This
suggested that soluble solids, in addition to total
nitrogen compounds, leached out during soaking, supporting
the research of Varriano-Marston and De Omana (1979). These
researchers reported "Pectic substances were leached

out from beans during the soaking period. The greatest

losses occurred when beans were soaked in either the

32

 

 

 

 

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Ii

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33

 

 

 

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N>o m.~ o.~ mn.o om.c m~.o o choose:
Axv mzmoz

 

 

.A.o.ueoov a manna

Table 10. Statistical summary of NaHMP level

 

34

effects

 

Measure M.S.
Soaked beans
water uptake Treatment 5 397.40*"
Error 42 65.22
Shear resistance Treatment 5 16149.60K*
Error 6 624.19
Soak water N(%) _4**
Fresh wt. basis Treatment 5 5.49x10 5
Error 6 4.18x10'
Dry wt. basis Treatment 5 1.45**
Error 6 7.31x10'
Soaked beans N(%)
Fresh wt. basis Treatment 5 1.27x10:%
Error 6 1.24x10
Dry wt. basis Treatment 5 9.24x10:3
Error 6 5.55x10
Progessed beans
Drained wt. Treatment 5 272.68
Error 6 122.72
Shear resistance Treatment 5 53.33
Error 6 14.22
Hunter L Treatment 5 2.81
Error 6 1.39
Hunter a Treatment 5 1.92:"
Error 6 4.94x10'
Hunter b Treatment 5 0.71*
Error 6 1.61x1o-1
A E Treatment 5 2 . 22
1 Error 6 1.20
Brine N(%)
Fresh wt. basis Treatment 5 9.20x10:g""
Error 6 9.30x10 yy
Dry wt. basis Treatment 5 3.98x10"1
Error 6 6.80x10‘3
Beans N(%)
Fresh wt. basis Treatment 5 6.16x10'3
Error 6 1.64x10'
Dry wt. basis Treatment 5 5.89x10"3
Error 6 3.00x10'3

 

**denotes significant F, P
*denotes significant F, P

=0
=0

01
05

35

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Ho.onm .H unmoamacwam monocoocc

‘-‘ ‘C‘

 

 

 

 

 

 

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36

NaSPBOIO solution or the combined salts solutions.
Considerably less pectic substance was solubilized when
beans were soaked in tap water."

Egoceseeg beans -- The drained weight and shear
resistance of processed beans were not significantly
(P=0.05) affected by soak water and process brine NaHMP
levels. This could possibly be explained by the fact that
the retorting masked the effects of NaHMP on these two
dependent measures. As the concentration of NaHMP
increased, the Hunter +aL decreased dramatically and Hunter
+bL tended to increase, which implied that in the higher
levels of NaHMP, beans became less red and more yellow.
The regression equations and correlation coefficients
are also reported in Table 11. The Hunter L and A.E
were not significant (P=0.05) affected by soak water
and process brine NaHMP levels.

The total nitrogen analyses in processed brine
and beans were similar to those in final soak water and
soaked beans. The data reported on dry weight basis more

clearly demonstrate these effects.

Combination of calcium ion and NaHMP
The mean values of combination of calcium‘ion and
NaHMP effects are reported in Table 12. The summary of

statistical analyses of these data is reported in Table 13.

37

 

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38

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ream.¢ kkmw.H «kmo.©N «kmm.on¢ «kNH.¢NmeH H mzmmz

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A 41H

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mocmumamou oxmumn .m.n COHumHHm>

Hdmflm Hmud3

mo oohsom

 

 

neooeeo mzmoz one N

+mo Ho coaumcHnEoo no humeezm Hmoaumauwum

.mH OHQMH

39

§2ekeg_22eg§ -- NaHMP in soak water softened the
beans and accelerated water uptake. The ANOVA did not
indicate the calcium ion significantly (P=0.05) affected
the water uptake or shear resistance. No interaction
effects between the two additives on these measures were
observed. The total nitrogen (%) both on dry weight and
fresh weight bases was significantly different in final
soak water, but not significantly different in soaked
beans. No effects on total bean or soak water nitrogen
were due to calcium ions. As the levels of NaHMP increased,
total nitrogen (%) in final soak water increased on the
fresh weight basis, but decreased on the dry weight basis.

Processed beans -- The calcium ion and NaHMP
significantly (P=0.01) decreased the drained weight of
processed beans. This was consistent with the results
from separate calcium ion and NaHMP studies. The
interaction effect was significant (P=0.01). Both
calcium ion and NaHMP significantly (P=0.01) affected
shear resistance of processed beans. Calcium ion in-
creased, NaHMP decreased, and the interaction of these
two additives remained as no calcium ion effect on the
shear resistance of processed beans. Hunter +aL values
were not affected by this combination treatment. No
significant (P=0.01) interaction effects between these
two additives on any color measurements were presented.
However, NaHMP increased Hunter L values. Both

calcium ion and NaHMP increased Hunter +bL values

 

40
and decreased AB. The effects due to NaHMP were more
than those due to calcium ion. The total nitrogen (%)in
the.final process brine was not significantly (P=0.01)
affected by calcium ion or NaHMP on the fresh weight
basis, but was significantly (P20.01) affected by NaHMP
on the dry weight basis. The interaction effect on
total nitrogen (%) in processed beans was significant
(P=0.01) on the dry weight basis. However, on the fresh

weight basis, no significant differences were observed.

Effecte ef Selected Enzyme Tyeatments

Soaked beans -- The mean values of selected
enzymes on water uptake and shear resistance of beans
soaked with and without seedcoats for 5 hours at dif-
ferent temperatures are presented inTables 14 and 15,
respectively. Seedcoats were removed to facilitate
enzyme penetration into the beans. The summary
of multiple factor ANOVA is presented in Table 16. The
histograms of main factor effects on water uptake and
shear resistance of soaked beans are reported in Figures
1 and 2.

The ANOVA indicated there were no significant
differences (P=0.05) in water uptake among enzyme types
and between groups (control and treatment) in either
beans with seedcoats intact or removed. The non-
difference in accelerating water uptake due to enzyme

types implied that no changes in water uptake were due

41
1

 

 

 

Table 14. Mean values of water uptake (g) of beans soaked
with and without seedcoats in selected enzyme
solutions for 5 hours at different temperatures

Who e Beans Who e Beans
(Seedcoat Intact _ (Seedcoat Removed;
Temperature Control Treatment Control Treatment
g-Amylase at pH 6.9
20°C 56.65 55.40 67.80 65.90
40 56.90 57.45 65.25 66.03
60 59.80 59.94 61.80 65.00
80 60.95 60.75 64.50 65.20
Glueeemylaee e; pH 4.5
20 56.80 61.05 66.30 65.50
40 57.10 54.10 64.35 64.10
60 58.05 60.75 61.15 62.05
80 60.00 62.80 66.20 61.30
Pectinase at pH 3.5
20 56.65 59.70 66.80 65.20
40 57.45 57.70 64.40 63.60
60 56.35 60.80 58.05 57.00
80 60.60 64.15 64.35 63.65
Celleleee a; 2H 6,5
20 57.35 60.15 67.20 63.15
40 57.25 54.60 63.50 67.25
60 56.15 61.25 57.75 62.10
80 59.85 64.20 62.45 61.60
Protease at pH 3.5
20 57.80 60.25 68.90 61.95
40 56.85 52.75 64.15 64.05
60 55.75 59.50 59.60 59.35
80 61.80 62.70 64.35 67.65

 

 

 

1

n=2 replications: CV=4.34 %

 

42

Table 15. Mean valueslof shear resistance (lbs/50g) of beans
soaked with and without seedcoats in selected enzyme
solutions for 5 hours at different temperatures

Whole Beans Whole Beane
(Seedcoat Intact Seedcoat Removed)

Temperature Control Treatment Control Treatment

 

 

 

g-Amylase at DH 6.9

 

20°C 1113.75 1102.5 967.00 907.50
40 1087.50 1095.00 900.00 892.50
60 986.25 1050.00 900.00 952.50
80 472.50 495.00 277.50 318.75

Glucoamylase at pH 4.5

 

 

20 113.75 1095.00 903.75 892.50
40 1087.50 1091.25 911.25 885.00
60 1020.00 1046.25 930.00 930.00
80 637.50 652.50 401.25 415.50
Pectinase at pH 3.5
20 1102.50 1117.50 915.00 907.50
40 1081.50 1083.75 897.75 900.00
60 1001.25 1035.00 971.25 982.50
80 577.50 585.00 487.50 480.00
Cellulase at pH 6.5
20 1185.00 1106.25 945.00 915.00
40 1107.75 1085.25 907.50 862.50
60 1005.00 1057.50 918.75 952.50
80 652.50 652.50 427.50 446.25

Protease at pH 3.5

20 1128.75 1117.50 903.75 930.00
40 1080.00 1081.50 897.75 888.75
60 1001.25 1023.75 922.50 922.50
80 600.00 615.00 397.50 357.00

 

1n==2 replications, CV=4.48%

' F" i'hl' .a’

 

43

Table 16. Statistical summary of soak studies with
selected enzymes

 

 

 

 

 

Source of M'S'

variation D.F. Water uptake Shear resistance

Mela effects
Enzyme type 4 3.43¢¢ 8157.33::
Temperature 3 91.15;; 2510688.34":
Seedcoat 1 1012.04 1107392.01*”
Group 1 13.23 61.26

Interactions
ExT 12 8.24 8566.17;"
ExSC 4 10.98 4497.94'
TxSC 3 130.38** 33925.07**
ExG 4 3.57 344.65
TxG 3 17.94 3668.19
SCxG 1 33.49* 1476.22
ExTxSC 12 3.48 1368.66
ExTxG 12 3.59 790.81
ExSCxG 4 5.67 292.41
TxSCxG 3 25.10* 553.54
ExTxSCxG 12 6.03 415.69
Error 80 7.07 1527.69

 

**denotes significant F, P=0.01

*denotes significant F, P=0.05

44

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46
to different pHs, since beans were soaked in each enzyme

solution at its stable pH. The temperature and seedcoat
contributed significantly (P=0.01) to water uptake. The
interaction effects of temperature and seedcoat: seedcoat
and group: temperature, seedcoatand group were significant
(P=0.05). Tukey's mean separations of the main

factor effects are also included in Figure 1.

The ANOVA showed that the effects on shear
resistance of soaked beans due to seedcoat, enzyme type
and temperature were significant (P=0.01). But there
was no significant (P=0.05) difference between treated
and control samples on this measure over all the main
factors. The main factor effects and Tukey's mean
separations are presented in Figure 2. The soaked beans
with seedcoats intact possessed higher shear resistance
than those with seedcoat removed. The shear resistance
of beans soaked in a-amylase solution at pH 6.9 was
significantly different (P20.01) from other enzyme
treatments. Higher temperature soaking resulted in
softer beans. However, the relationship between tem-
perature and shear resistance of soaked beans was not
linear. Tukey's mean separations showed no differences
in shear resistance of beans soaked at 40°C and 60°C.

The beans soaked at 80°C were much softer than those
soaked at lower temperatures.

Gelatinization -- The experimental results reported

above indicated that the selected enzymes were inactive

47
on water uptake and shear resistance in either beans with
seedcoats intact or removed. It was questioned whether
the enzymes were able to contact their substrates in the
cotyledons. Therefore, the whole ground bean flour was
used to demonstrate the activities of selected enzymes
on the viscosity of bean flour during gelatinization.
The results are presented in Table 17 and Figure 3.
The summary of statistical analyses of these data is
reported in Table 18. Only a-amylase and pectinase
significantly (P=0.01) reduced the viscosity of bean
flour. Glucoamylase, cellulase and protease remained
inactive. It is known that the glucoamylase is inactive
in this experiment (Reed, 1975). The inactivities of
cellulase and protease may be due to their low concen-
trations and short reaction time, or due to these
substrates'limited effects on viscosity. Table 18
contains the effect of pH on the viscosity of bean flour
during gelatinization. The viscosity increased as pH
decreased. The phenomenon may be explained by the
denaturation of some proteins and suppression of hydration
of the proteins and starch in the bean flour by hydro;
chloric acid. The plot of the viscosity data indicated
a linear relationship between pH and viscosity (Figure 4).
Increased degrees of freedom would be needed to establish

the significance of a linear relationship.

48

mGOHDMOHHQmH Nu:

 

 

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mo.H o.mNoH o.omoH m.m ommououm
oc.m m.NNw m.wmw m.o ommHsHHoo
mm.H o.mmm o.omoH m.m ommcHuoom
No.m o.omm o.mom m.q mmmHhemooDHw
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>0 N unoEumouH Houucoo me make oemncm

 

was: Hoococmnm

i|ll||

 

 

 

 

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oouooHom CH ooNHGHumHow HSOHH coon mo anamooma> onmoDHm> memo: .mH chmH

49

 

 

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oHocz no muamooma> can so mosasco wouooHom mo muoommm .m madman

mm mo mm mm HO HO mu mo ma mm mm HO mm O0 O0 mm mm HO mu mo OO ma NO HO mm 00
ON OH on N.m O.N ON O.N OH on Nm ON ON ON OH on NO ON «N ON OH on NO ON «N ON OH
O

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CHE

(JTUH JSDUGQEJQ) A1IsoostA

50

Table 18. Statistical summary of selected enzyme

gelatinization studies

 

l

 

-_—___

1

 

 

Measure Source of variation D.F. M.S.
a-Amylase Treatment 78230.5**
Error 1212.5
Glucoamylase Treatment 625.0
Error 1225.0
Pectinase Treatment 11250.0**
Error 250.0
Cellulase Treatment 2881.2
Error 5335.5
Protease Treatment 25.0
Error 125.0
pH Treatment 21216.7"x
Error 675.0
** . . .
denotes Significant F, P=0.01

51

utmOHMchHw coz

 

 

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no so coammouwou anamoomH> uson comm .¢ ouswam
mm
O.m 0.0 O.m O.q Oflm

OON.

ONmN.O 0 9mm

. 1 1 me

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xMN.mO 1 Nn.omNH n %
OOOJ
Omw-
ooea
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(iron Jepueqexq) A1IsoostA

SUMMARY

Two critical factors of processed navy bean
quality and subsequent consumer acceptance are degree
of bean hydration and firmness. Addition of divalent

2 and Mg+2, to processed navy beans not only

ions, Ca+
increased their firmness but had a pronounced effect
on the drained weight. The calcium content of processed
beans maintained a negative relationship with processed
bean drained weight and a direct relationship with pro-
cessed bean firmness throughout soaking and processing.
These results were in agreement with the theory that
pectin and calcium ion retarded water uptake and increased
firmness through the formation of tough pectin-metal ion
complexes (Matz, 1962). The addition of calcium ions
into soak water and process brine did not significantly
(P=0.01) affect the color of processed beans.

The effects on drained weight and shear resistance
of processed beans by magnesium ions were similar to
those caused by calcium ions. However, bean color Hunter
L, +aL and especially Hunter +bL were affected by magnesium
ions.

Increasing levels of NaHMP increased water uptake,-

softened soaked beans and affected color Hunter +aL and +bL

52

 

53

of processed beans. No significant changes in drained
weight or shear resistance of processed beans were
observed. As the NaHMP level increased the total
nitrogen (%) in the final soak water increased on the
fresh weight basis, but decreased on the dry weight basis.
A similar phenomenon was observed in the processed
products. These results suggested that soluble solids,

in addition to nitrogen compounds, leached out during
soaking and processing of beans in different levels of
NaHMP additive.

2 and NaHMP as an

The study of combining Ca+
additive indicated that NaHMP softened the soaked beans
and accelerated water uptake. The calcium ion retarded
water uptake and increased shear resistance of soaked
beans. No interaction effects were observed. Both
calcium ion and NaHMP, as well as theirinteraction,
decreased drained weight of processed beans. The calcium
ion increased, but NaHMP decreased and the combination
treatment remained as no calcium ion effect on the shear
resistance of processed beans. Increasing Hunter +bL and
decreasing 14E values by the combination treatment were
observed. The two additives did not yield interaction
effects on any color measurements. Total nitrogen (%)
in the final soak water increased on a fresh weight basis,
but decreased on a dry weight basis due to the addition

of NaHMP to soak water. The final process brine total

nitrogen (%) decreased on a dry weight basis. The

54

2 and NaHMP on the

significant interaction effects of Ca+
processed beans total nitrogen (%) were observed, on a
dry weight basis.

The selected enzyme soaking studies indicated
that the enzyme types did not show effects on water
uptake in whole beans, as well as not showing differences
between treated and control samples. The enzymes also
remained inactive in beans with seedcoats removed under
the identical soaking conditions. The removal of seed-
coats increaSed the rate of water uptake. No difference
in shear resistance was observed between control and
treated samples over all the other main factors. Shear
resistance of beans soaked in a-amylase solution at
pH 6.9 was different from those soaked in other enzymes.
This. difference was likely due to the function of pH,
since each enzyme was incubated at its stable pH. The
large difference between soaked beans with and without
seedcoats in shear resistance peak height indicated that
seedcoats contributed a major component to the shear
resistance. The beans soaked at 80°C, either with or
without seedcoats, were much softer than those soaked at
lower temperatures.

To evaluate whether enzymes were in contact with
their substrates in cotyledons in the soaking studies
with or without seedcoat, whole ground bean flour was
used to test the activities of enzymes in reducing the
viscosity of gelatinized bean flour. Only a-amylase

and pectinase decreased the viscosity, while glucoamylase,

55

cellulase and protease remained inactive possibly due

to low concentration and short reaction time, or due

to these substrates' limited effects on viscosity. The
hydrogen ion increased the viscosity of gelatinized bean
flour. This phenomenon may be explained by the denatu-
ration of some proteins and suppression of hydration of
the proteins and starch in the bean flour with-decreasing

pH.

FURTHER RESEARCH

Use a radioactive isotope to trace the distribution

of divalent ions from soaking and processing to
confirm the relative firming action of soak and
process brine.

Analyze the pectic substances in the final soak

water and process brine for NaHMP treatment to confirm
that NaHMP softening is partly due to solubilizing
pectic substances.

Conduct morphological and chemical studies on relative
effects of magnesium and calcium ions in intercellular
firming of processed beans.

Study effects of cellulase and protease on the
viscosity of whole bean flour by increasing the
concentration and reaction time of these enzyme

treatment S .

56

 

LIST OF REFERENCES

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