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ABSTRACT

KINETICS OF THERMAL INACTIVATION OF POLYGALACTURONASE
IN SPENT CHERRY BRINES

BY

Panagiotis Evagelos Athanasopoulos

Sweet cherries, used for manufacturing into
maraschino, cocktail, or glacé fruit, are stored in calcium
bisulfite brine. Spent cherry brine is a strong pollutant
and creates serious disposal problems. If the spent brine
could be reclaimed and reused, the disposal problem would
be reduced in magnitude. The polygalacturonase enzyme may
be present in reclaimed brine and results in enzymatic
softening of the new cherry lot. The inactivation of the
enzyme by heat would provide the required safety to prevent
losses of cherries due to enzymatic softening.

Commercial sweet cherry brines were used to investi-
gate the heat resistance of purified polygalacturonase
which was diluted to a concentration of 1 mg enzyme per ml
of brine. One ml of the enzyme dilution was heated in test
tubes using a two constant-temperature bath technique
followed by cooling in an ice-water bath. The plate and cup

assay was used to estimate the enzyme activity during

Panagiotis Evagelos Athanasopoulos

inactivation determinations. Least-squares statistical
analysis was used to evaluate experimental results and
determine appropriate constants.

The inactivation of the polygalacturonase enzyme in
cherry brines was described by first-order functions. The
rate constant (k) at 70 °C was 4.93 x 10-2 sec“1 and the
decimal reduction time (D) at the same temperature was
found to be 46.8 sec. The Z value for thermal resistance
was calculated from the thermal resistance curve and found
to be 8.4 °C. The activation energy was computed from the
Arrhenius plot and had a value of 64.7 Kcal mol-l. The
effect of pH on the enzyme inactivation rate was studied
and the results indicate that the polygalacturonase enzyme
in cherry brines exhibits higher resistance in the pH 2.8 -
3.5 range. Sugar concentrations higher than 12 percent
found to have a significant protecting effect on the
enzyme. Enzyme dilution in brine with higher 802 and higher
pigment content had higher resistance to inactivation by
heat. Partially inactivated enzyme in brine was tested for
regeneration capability at room temperature and under

refrigeration conditions. Results indicated that the

inactivation of that enzyme is irreversible.

KINETICS OF THERMAL INACTIVATION OF POLYGALACTURONASE

IN SPENT CHERRY BRINES

by

Panagiotis Evagelos Athanasopoulos

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Food Science and Human Nutrition

1976

To my dear wife

Gina

ii

ACKNOWLEDGMENTS

The author wishes to express his deep gratitude
to his major professor, Dr. D. R. Heldman, Professor and
Chairman of the Agricultural Engineering Department, for
his inspiring advice, help and constant encouragement
throughout this study.

Grateful appreciation is extended to Dr. P.
Markakis, Mr. A. L. Rippen, Professors of the Department of
Food Science and Human Nutrition, to Dr. F. W. Bakker-
Arkema, Professor of the Department of Agricultural
Engineering, and to Dr. M. L. Davis, Professor of the
Department of Civil and Sanitary Engineering, who served
as members of the committee, reviewed the manuscript and
made constructive comments.

Thanks are expressed to Dr. R. F. McFeeters for
his advice and encouragement.

The author also expresses his appreciation to the
"Sweet Cherry Industry Waste Disposal Committee" for the

financial support of this project.

iii

TABLE OF CONTENTS

LIST OF TABLES . . . . . . .

LIST OF FIGURES. . . . . . .

INTRODUCTION. . . . . . . .

LITERATURE REVIEW . . . . . .

A.
B.
C.
D.
E.
F.

Structure of the Industry .
Brine Reclamation . . . .
Pectic Substances and Pectic

Enzymes.

Polygalacturonase in Brined Cherries.

Enzyme Kinetics. . . .
Heating and Cooling Methods.

METHODS AND PROCEDURES . . . .

A.
B.
C.
D.
E.
F.
G.

Heating and Cooling of the Samples
Enzyme Source and Concentration
Measurement of Polygalacturonase Activity

pH Adjustment . . . . .
Dissolved Solids Level . .
Cherry Variety . .

Regeneration Capability of the Enzyme

RESULTS AND DISCUSSION . . . .

A.

B.
C.
D.
E.
F.

Effect of Temperature. . .

1. Order of the Reaction .
2. Rate Constants. . . .

3. Activation Energy for Inactivation

Effect of pH on Inactivation Rate.
Effect of Sugar Concentration .

Effect of Cherry Variety. .

Regeneration Capability of the Enzyme

Applications. . . . . .

iv

Page
vi

vii

10
l4
16
21

23

23
26
28
33
33
34
34

35
35

35
38
42

46
53
57
59
60

SUMMARY AND CONCLUSION . . . . .

BIBLIOGRAPHY . . . . . . . .

APPENDIX A. TABLES ILLUSTRATING RAW AND CALCULATED

DATA 0 O O O O O I

APPENDIX B. CUP PLATE METHOD OF ANALYSIS FOR

POLYGALACTURONASE ENZYMES

Page
67

69

75

83

LIST OF TABLES
Page

40

Table
1. Rate constants for inactivation of poly-
galacturonase in Napoleon brine (pH = 3.0)

Data for evaluation of the activation energy
42

for heat inactivation of polygalacturonase

2.
= 3.0). o

in Napoleon brine (pH

Rate constants of polygalacturonase in-

activation in Napoleon brine at 69 °C
. . 49

3.

and different pH values

Rate constants of polygalacturonase in-

activation in Napoleon brine at different
0 O O I 49

4.
sugar concentrations . . .

Activity of polygalacturonase inactivated
. 61

in Napoleon brine by 99.12 percent.

5.
65

Contribution of heating and cooling to the

total lethality of the enzyme in brine

6.
Poly-

75

Data for standard curve construction.
galacturonase enzyme diluted in commercial

Napoleon brine at pH = 3.0

A2. Effect of temperature on zone diameter for
polygalacturonase in Napoleon brine (pH
0 O O O O O O O O 76

3.0). o o o o
3.0) . 78

Effect of temperature on polygalacturonase
inactivation in Napoleon brine (pH —

A1.

A3.
79

Effect of pH on inactivation rate of poly-
galacturonase in Napoleon brine at 69 °C.

Effect of sugar concentration on polygalac-
80

A5.
turonase in Napoleon brine at 69 °C

(pH = 3.0). . .
Effect of cherry variety on inactivation rate
= 3.0). . 81

A6.
of polygalacturonase at 69 °C (pH

A4.

vi

Figure

l.

2.

10.

11.

12.

LIST OF FIGURES

Major steps in maraschino cherry industry . .

Temperature history of 1 m1 brine in test
tube, at the slowest heating point. Curves
show heating from room temperature, 24 °C
(70, 75, and 80 °C are heating bath
temperatures). . . . . . . . . . .

Cooling curves for 1 ml of brine in test
tube from various temperatures to 0 °C . .

Effect of gel thickness on zone diameter of
1 mg/ml enzyme concentration and brine
pH = 3.0 O O O O O O O O O O O 0

Standard curve for enzyme activity estimation.

Effect of temperature on the inactivation
rate for the polygalacturonase enzyme in
Napoleon brine (pH = 3.0). . . . . . .

Normalized inactivation rate curves for poly-
galacturonase enzyme in Napoleon brine
(pH = 3.0). o o o o o o o o o o 0

Thermal resistance curve for polygalacturonase
in Napoleon brine (pH = 3.0). . . . . .

Arrhenius plot for polygalacturonase in-
activation in Napoleon brine (pH = 3.0) . .

Inactivation rate curves for polygalacturonase
enzyme in Napoleon brine at 69 °C and
different pH values. . . . . . . . .

Effect of pH on inactivation rate of poly-
galacturonase in Napoleon brine. . . . .

Effect of pH on the thermal reduction time
at 69 0C O I O O O O O O O O O 0

vii

Page

25

27

30

32

36

39

41

44

47

48

50

Ffigure Page

13. Effect of dissolved sugar concentration on
inactivation rate of polygalacturonase
in Napoleon brine at 69 °C and pH = 3.0 . . 54

14. Effect of dissolved sugars on D value at
69 oC and pH = 3.0 O O O O O O O O O 55

15. Effect of cherry variety on inactivation rate
of polygalacturonase in brine at 69 °C
and pH = 3.0 . . . . . . . . . . . 58

16. Heating and cooling profiles for lethality
calculation . . . . . . . . . . . 63

A1. Long time inactivation profile for poly-
galacturonase Napoleon brine at pH = 3.0. . 82

viii

INTRODUCTION

In the United States, the cherry brining industry
has increased since 1930 (Steele and Yang, 1960). Between
40 and 45 percent of the total sweet cherry production is
brined. In Michigan, a 75 percent of the production is
brine processed (Woodruff, 1975). In the manufacture of
maraschino and glacé cherries, the fruit is held in calcium
bisulfite brine. First, cherries are bleached to a bright
light straw color before processing.

Spent brine is normally discharged to municipal
sewage system or to local streams. Waste water from cherry
brining industry is characterized by high sulfur dioxide
content, high chemical oxygen demand, low pH, and color.
Reclamation of spent brine for re-use has been prOposed of
reducing the pollution problems created from the disposed
brine. The problems in recycling of spent brine are:

(a) The brine has reduced bleaching power which will
usually prevent the production of a bright yellow color in
the cherries during brine reuse and (b) the polygalacturo-
nase enzyme may be present in the spent brine and result in

enzymatic softening of the fruit.

A process for used brine reclamation has been
develOped at Oregon State University, (Soderquist, 1971),
which consists of the following units: (a) sand filters for
suspended solids removal, (b) activated carbon contactors
for anthocyanin and polyphenol adsorption, (c) pasteuri-
zation unit for polygalacturonase enzyme inactivation, and
(d) chemical reactor for adjustment of SO2 concentration
and pH.

The kinetics of thermal inactivation of polygalac-
turonase in spent cherry brine has not been investigated
and data required for design of the pasteurization unit of

the reclamation system are not available in current liter—

ature. This study has been conducted to evaluate the
apprOpriate design parameters.
The specific objectives of this research are:

1. To study the inactivation rates of the polygalac-
turonase enzyme as a function of (a) temperature,
(b) pH, (c) dissolved sugars, and (d) cherry
variety.

2. To investigate the regeneration capability of the
partially inactivated enzyme in brine under differ—
ent conditions.

3. To conduct mathematical analysis of experimental

data and evaluate the appropriate design parameters.

LITERATURE REVIEW

A. Structure of the Industry

 

The chief cherry varieties for brining in Michigan
are Napoleon, Windsor, Schmidt, and Emperor Francis.
Cherries are hand picked or mechanically harvested. Very
little damage occurs during mechanical harvest depending on
the variety and maturity (Levin et al., 1969). As maturity
advances, the machine harvested fruit decreases in quality
much more rapidly than hand picked fruit (Thienes et al.,
1969). A considerable percentage of mechanically harvested
cherries has attached stems which could create problems if
briners wished to have fruits free of stems. Thienes
(1969), reported that a reduction in cherry grade may occur
by delaying the brining for even two hours after harvesting.

To minimize the adverse effect of bruising on
quality, cherries are brined in a matter of seconds after
harvesting by placing the cherries in barrels which are
half-full of brine. These barrels are transported to the
briner plant where they are sold. Since cherry weight is
difficult to be obtained after cherries have been placed in
brine, they are sold by volume (Whitenberger, 1969). Inside

the processing plant, the cherries are kept in large tanks,

in brine, for at least 5-6 weeks for their color to be
removed.

Two general types of brine are commercially used.
Type one uses the method of gas sulfur dioxide introduced
into a suspension of calcium carbonate or calcium hydroxide.
There is no effect on texture due to the use of different
calcium salts, (Vibbert, 1976). In type two method, the
bleaching agent is sodium bisulfite (NaHSO3). Sodium
bisulfite and calcium chloride are dissolved in water and
pH is adjusted with commercial hydrochloric acid. Brined
cherries are shipped to finisher plant in plastic lined
tote bins and tank trucks. Years ago, and sometimes today,
cherries were transported in brine. Currently, considerable
quantities of brined cherries are shipped in water with
citric acid added as a preservative.

Cherries with high pigment content are not com-
pletely bleached by sulfur dioxide brines. In addition,
dark Spots (sunburn, bruising) are partially bleached.
Cherries with these dark blemishes are unacceptable for
finishing because the final product has lower market value.
A secondary bleaching is necessary to remove these dis-
colorations. A number of bleaching agents have been tested
with different degrees of success. Sodium chlorite
(NaClOz) has been reported (Beavers, 1968, and Payne, 1969)
as a successful agent leading to snow white product of firm
texture and free of off-flavors. Brined cherries are

leached in cold water to remove 802, and they are bleached

in an acidified solution of sodium chlorite. After
bleaching, sodium chloride is removed (leached in water)
and they are returned to calcium bisulfite brine.

The next step is steaming and pitting. Accordingly,
prepared cherries are soaked in running water to remove most
of the sulfur dioxide. Then, they are boiled in water
which is changed several times until the desired tenderness
is obtained and the sulfur dioxide content is below 100 ppm
(for maraschino cherries) or below 20 ppm (for fruit
salad).

Bleached cherries are ready for dyeing. They are
covered with the 30 percent sugar syrup containing dye.
Twenty-four hours later, the syrup concentration is in-
creased (by evaporating water in a vacuum pan) gradually,
to a final strength of 72-74 percent sugar. When the dye
has penetrated sufficiently, the syrup is acidified to pH
3.6-3.9 using citric acid for color fixation. Dyed cherries
are drained out of the syrup and packed.

The general steps of the brining cherries industry
are illustrated in the following flow sheet, shown in

Fig. 1.

B. Brine Reclamation

 

The sweet cherry industry is a source of pollution.
Solid wastes are produced during pitting and stemming
processes, while waste water is the most important pollutant

of this industry (Beavers et al., 1970). Sources of waste

 

HARVESTING
BRINING
SECONDARY BLEACHING

L

PITTING STEMMING
LEACHING
SYRUPING AND DYEING

J

PACKING

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 1. Major steps in maraschino cherry industry.

water are: brining, secondary bleaching, leaching, and
syruping and dyeing. A

The most important pollutants of this industry
(Soderquist, 1971) are soluble solids, which consist
principally of dextrose, acids, anthocyanins, polyphenols,
combined SO

free sulfur dioxide, 80 2, sulfate (804) and

2,
calcium (Ca++). In addition, spent brine is characterized
by low pH; effluent from dyeing consists principally of
sucrose, color, and high temperature.

Spent brine makes up the largest volume of the
effluent and it is characterized by high strength. Payne
et al. (1969) reported that spent undiluted brine may con-
tain 4,000 mg/l free 802, 1,000 mg/l sulfite, 4,000 mg/l
calcium, and a pH of 3.5. Chemical oxygen demand (COD) of
150,000 mg/l is common. Soderquist (1971) reported that
spent cherry brine is approximately 500 times stronger than
domestic wastewater.

The high sulfur content is directly related to very
high chemical oxygen demand (COD). The dissolved oxygen in
the water is depleted by microbia growth, stimulated by the
high levels of organics present, and it creates anaerobic
conditions. This anaerobiosis is conducive to the growth
of organisms, such as Desulfovibrio sp. (McKinney, 1962).
These microflora generate hydrogen sulfide and other

malodorous reduced sulfur compounds resulting in serious

odor problems.

Chemical and physical methods for brine treatment
have been described by Sapers (1975). Application of
hydrogen peroxide for odor control of sweet cherry brining
waste is under investigation (Gerrish, 1975). The waste-
waters from the brined cherry pitting process were found to
be highly treatable in an aerobic biological treatment
process (Mauldin et al., 1975).

By recycling the commercially wasted spent brine,
the volume of the waste water released from a cherry process
is significantly reduced. The spent brine contains dis-
solved pigment and has a little bleaching capacity (Beavers
et al., 1970). Pigment must be removed before re-use so
that the brine is adequate to bleach successfully a new
load of cherries. Experiments have shown that dissolved
anthocyanins and polyphenols, present in Spent brine, can
be removed by treatment with active carbon (Beavers et al.,
1970, Soderquist, 1971, Sapers, 1975). A reclamation system
for spent brine should include a sand filter to remove
suspended solids, which may escape from screens, a series
of active carbon contactors to adsorb most of the pigments
present in the brine, and an adjusting unit for full
strength brine preparation (Soderquist, 1971).

Reclaiming spent brine may not be an economical
process for finishers located at a distance from cherry
brining areas. Finishers brine may be used as a syrup
basis, after removing Ca--, 802, and concentrating it 1.78

fold by evaporation. The level of pesticides in brine have

to be taken under consideration, as well, for using spent
brine for syrup production (Sapers, 1975).

One important problem in re-using reconditioned
brine is the enzyme polygalacturonase (PG). If any portion
of the brine is contaminated with the enzyme, the entire
bulk stock of brine could become contaminated during the
reclamation process (Beavers et al., 1970). Re-using brine
contaminated with polygalacturonase could lead to enzymatic
hydrolysis of the pectic substances of the cherries
resulting in a soft unacceptable product. A safe inactiva-
tion method of the enzyme is pasteurization (Watter, 1961;
Soderquist, 1971; Sapers, 1975).

Brine reclamation and re-use would reduce sewerage
charges and lower costs of chemicals for new brine pro-
duction. Economic analysis by Soderquist (1971) has shown
that the net annual savings is a function of the plant
size. A processor handling 1,000 tons of Black Republican
cherries per year would have negative results ($574 per
year or $0.06 per ton). A larger plant handling 5,000 tons
per year would have a profit of $10,109 per year or $2.02
per ton, while production on a level of 10,000 tons per
year would result in a net annual cost savings of $27,300
per year or $3.06 per ton. The above results have been
derived under economic conditions of the year 1970. By
incorporating a pasteurization unit in the reclamation

system, the cost will be somewhat higher.

10

Preliminary work for syrup preparation from used
cherry brine, which contains about 8 percent sugars, showed
that 100 gal. brine produces 56 gal. of 15-17 percent
soluble solids syrup base. By adding 3.8-4.0 1b of sugar
per gal. of concentrate, about 80 gal. of 40 Brix syrup is
produced. The cost for that process has been estimated to

be $11.32 per 100 gal. of brine (Sapers, 1975).

C. Pectic Substances and Pectic Enzymes

 

The pectic substances, which are carbohydrate
derivatives, are widely distributed in plant tissues where
they fill the intercellular spaces (middle lamella).
Pectins are composed mostly of hydrogalacturonic acid
residues. Other carbohydrates (Joslyn and Denel, 1963),
such as arabinose, galactose, sorbose, rhamnose, are
attached to the chains of unhydrogalacturonic acid units.
The carboxyl groups in pectic polygalacturonides are either
free or partially esterified with methyl alcohol or may
form salts with various cations.

The characteristics and composition of pectic sub-
stances depend on many factors (Kertesz, 1951). These
factors include the origin of the sample, the methods used
in the preparation of the tissue, the methods of extraction
and separation, the type and extent of purification, and
the analytical procedures used in assay and characterization.

Highly esterified pectins in 65 percent sugar

solutions yield firm gels. The term PECTIN comes from that

11

property. It has been derived from the Greek "UnXTOS"
meaning to congeal or solidify. Partial hydrolysis of the
methyl esters leads to low methoxyl pectins, which form
gels with calcium ions at lower sugar concentrations. Com—
plete hydrolysis of the methyl esters leads to the pectic
acids in which there is one free carboxyl group per unit of
galacturonic acid. The jelly grade of pectin is signifi-
cantly affected by the molecular weight. Pectins with high
molecular weight, such as 180,000 yield firm gels with a
large amount of sugar (lg pectin with 220-300 9 sugar). On
the other hand, pectins with molecular weight 30,000 fail
to form jelly.

Several workers have estimated the amount of pectic
substances in cherries. Kertesz (1951) summarizes: (a) for
an unripe sample of cherries, 11.4 percent pectic substances
on a dry matter basis; (b) for a ripe sample 4.3 percent;
(c) for stone-free cherries, 0.35 percent as pectic acid on
a fresh weight basis; (d) for Morella cherries, 0.16 percent
as calcium pectate; (e) for red cherries 0.28 percent; and
(f) for white cherries 0.31 percent.

Montmorency cherries have been studied (Al-Delaimy,
1963) under different storage conditions and varying degrees
of maturity. The total pectins found to be in the range
0.304 to 0.466 percent on a fresh fruit weight basis are
expressed as unhydrouronic acid.

The chemistry of pectic substances has been exten-

sively reviewed by Kertesz (1951), Dewel and Stutz (1958),

12

and Joslyn and Dewel (1963). The enzymes which catalyze
the degradation of pectic substances are called "pectic
enzymes." Enzymes, catalyzing the formation of pectin
from its water insoluble precursor protOpectin, are called
protopectinases, but they have never been isolated (Reed,
1966).

At the present time, it is almost impossible to
obtain a clear picture of the relationship between the
different kinds of pectic enzymes which have been isolated
from various micro-organisms. Kertesz (1951) discusses
two enzymes pectin-methylesterase, and pectin-polygalac-
turonase. Reed (1966) classifies the pectic enzymes into
two groups. The enzymes which Split the ester linkage
between methyl esters of the galacturonic acid molecules,
called methylesterase (ME), and that which Split the gly-
cosidic linkage between adjoining galacturonic acid mole-
cules, called polygalacturonases (PG).

The group of polygalacturonases includes enzymes
able to act within the polygalacturonic acid molecules on
a-l,4- linkages (endopolygalacturonases) and enzymes
catalyzing the step-wise splitting off of galacturonic acid
residues from the non-reducing end of the chain (Exopoly-
galacturonases).

Some enzymes act principally on methylated sub-
stances (pectins) and are called polymethylgalacturonases
(endo- and exo-) while others act on substrates containing

free carboxylic acid groups (pectic acids) and are called

13

polygalacturonases (endo— and exo-). The latter completes
the degradation of the pectins on which methylesterases
have acted.

The complexity of classification is greater than
described in the above paragraph. It has been recognized
that a nonhydrolytic type of cleavage of the a-1,4 linkage
occurs, called trans-elimination (Reed, 1966). The reaction
is enzymatically catalyzed, and the enzymes have been
called transeliminases. These enzymes are not included in
the general classification prOposed by Demain and Phaff
(1957) to help clear up the complexity problem. The most
recent classification and nomenclature is that recommended
by the International Union of Biochemistry (1973).

The activity of an enzyme can be measured by deter—
mining the substrate loss, by estimating some new compounds
or groups formed by the enzyme action, or by measuring the
changes in physical prOperties (ViSCOSity). All of these
methods have been used for polygalacturonase determination.
The techniques and the limitations of each procedure are
well described by Kertesz (1951). The interferometric
method has been proposed by Rukhlyadeva and Korchagina
(1975) for commercial pectic enzyme preparations. It is a
rapid (less than two hours) and accurate method (:2%).
Interferometer readings (m) are obtained using several
dosages of a given preparation and the enzyme activity A is
calculated in arbitrary units from the equation: A =

(.6425xm + 19.62) % (n x 1000).

14

Recently, the solid media technique has been intro-
duced in the quantitative determination of enzyme activity.
This technique, develoPed for microorganism cultivation,
has been used for detection of enzyme production by micro-
organisms (Hankin and Anagnostakis, 1975). The agar "cup-
plate" diffusion technique has been applied to the quanti-
tative determination of polygalacturonase (Dingle et al.,
1953). This test has been used by Steele and Yang (1960)
and by Beavers et a1. (1970) for polygalacturonase activity

determination in cherry brines.

D. Polygalacturonase in Brined Cherries

The polygalacturonases have been detected in higher
plants such as tomatoes (Patel et al., 1960), avocados and
pears (McCready et al., 1954). This enzyme is produced by
a number of microorganisms including bacteria yeast and
mold, such as Saccharomyces fragilis (Patel et al., 1959;
Phaff et al., 1956), ASpergillus niger (Mill et al., 1961;
Tuttobello and Mill, 1961), Aspergillus sp. (McClendon
et al., 1963).

Softening of brined cherries was reported for the
first time in 1954 (McCready et al., 1954). The damage to
cherries was Slight, and an in—depth investigation was not
initiated until 1957 when extensive softening damage
occurred in Oregon and Washington. The cherry softening
which occurred seemed to be identical to that reported in

1954. The problem was investigated and it was established

15

that the enzyme polygalacturonase was responsible for the
softening (Steele et al., 1960).

The enzyme polygalacturonase has not been detected
in cherries. Investigations on Montmorency cherries (Al-
Delaimy et al., 1966) indicated the enzyme was not present.
Severan varieties of fresh cherries tested by Steele (1960)
were free of polygalacturonase. Probably, the sources of
the enzyme are microorganisms which gain access in cherries
after damage during the ripening period, or it enters the
brines with stems and plant particles.

The activity of the enzyme in calcium bisulfite
brines does not follow a definite pattern. It has been
reported that the enzyme remained active in the brine for a
period of only 13 to 25 days (Yang et al., 1960). On the
other hand, commercial samples of brined cherries showed
enzyme activity three months after brining, but it stopped
after six months in brine (Beavers et al., 1970). Brekke
et a1. (1966) found active polygalacturonase in brine from
softened commercially brined cherries for over one year.
Cherries brined the second year in this brine produced 80ft
fruit.

Current results indicate that the degree of activity
and the period of activity of polygalacturonase in cherry
brine is not predictable. Any investigation on reclamation
of brine must include tests for the detection of poly-
galacturonase which should be conducted on each lot of

brine before it is released for reuse. No extensive study

16

of cherry polygalacturonase has been conducted, and the
inactivation of the enzyme in different brines has remained
a problem. Methods suggested for inhibition of poly-
galacturonase in cherry brines are: (a) Using alkyl aryl
sulfate (Steele et al., 1960), (b) adjusting pH in the
range from 1.0 to 1.6 (Yang et al., 1960), and (c) in-
creasing the concentration of calcium chloride in the brine
(Brekke et al., 1966). None of the mentioned methods found
commercial application, however, and the inactivation by
heat has been pr0posed as the only approach (Soderquist,

1971; Watters et al., 1961; Sapers, 1975).

E. Enzyme Kinetics

 

The magnitude of enzymatic activity is usually
proportional to the concentration of the enzyme. This
proportionality is exhibited at least during the earliest
stages of the reaction. The linear relationship between
enzyme concentration and rate of reactionn has been detected
in hydrolysis of milk fat and olive oil by purified milk
lipase (Chandan and Shahani, 1963).

In the earliest stages of an enzymatic reaction
and for very Short time intervals, the substrate concentra-
tion can be assumed constant. This is particularly true in
the case when the substrate is present in excess, and the

amount of product formed is proportional to time,

dP / dt = Ko (1)

17

where dP/dt is the rate of product formation, and K0 is a
constant. In other words the enzymatic reaction follows
zero-order kinetics. Beyond the initial phase, the
reaction slows down with time. Most enzymatic reactions
follow the kinetics of a first-order reaction described by

the equation,

dP / dt = K (S-P) (2)

where (S-P) is the concentration of substrate remaining at
any given time, t, and K is the first order reaction con-
stant.

The enzymes are proteins in nature, and they have
highly specialized biological activity. Most protein
molecules retain their biological capacity to function only
in a very narrow range of temperature and pH. Exposure of
proteins to extremes of temperature or pH results in
denaturation of the molecule. When the enzymes are heated,
they lose their ability to catalyze a chemical reaction.
The polypeptide backbone of the protein molecule is not
broken, but the polypeptide chain is unfoled and the protein
molecule loses its folded structure (Lehninger, 1970).

As the temperature is increased, two simultaneous
reactions occur (Messing, 1975): (a) The enzyme activity
increases, as observed in most chemical reactions, and
(b) The enzyme stability decreases due to thermal denatura-
tion. The net result of these two reverse effects is a

bell-shaped curve.

18

Inactivation of enzymes by heat is a very common
procedure in the food processing industry. Vegetables and
some fruits are heat treated before canning or freezing.
Cloudy juices, such as orange and tomato juice retain their
cloudiness by inactivating the pectic enzymes by heat.

Lipoxidase, inactivated by heat and by ionizing
energy, exhibit first order kinetics (Farkas and Golblith,
1962). Kinematic procedures employing D values were used
by Yamamoto et al. (1962) for kinetic studies on heat
inactivation of peroxidase in sweet corn. A first order
reaction was observed for the initial 95 to 98% reduction
of the enzyme activity.

The inactivation of an enzyme can be described by

first order kinetics,

= - RC (3)

0.10.:
('1' 0

where dC/dt is change in activity with time, k is the
reaction constant and C the activity at any time. After

intergration, equation (3),

C t
dC _
_C — kdt (4)
Co 0
the following equation is obtained,
C - —
1n ES — kt (5)

19

where Co is the initial enzyme activity and t is the heating
time. The natural log can be changed to log with base 10

and the equation (5) can be written,

 

C _ -kt _ _ kt
109 ES ' 2.303 or log C ‘ 109 CO 2.303 (6)
Equation (6) describes a straight line on log C versus t .

coordinates. The intercept is log Co and the slope is
k/2.303. Measuring the enzyme activity C, at different
times t, both the initial activity Co, and the rate con-
stant k, can be computed. The constant k is highly pH
depended. Plot of the log k versus pH resulted in a "boat
shaped" curve (Farkas and Golblith, 1962), with high k at
pHs 4 and 9 and low k at pHs 5 and 7.

Yamamoto et a1. (1962) employed techniques used in
thermobacteriology to study kinetics of enzyme inactivation
by heat. Decimal reduction time, D, is the time required
to reduce enzyme activity by 90 percent at a given temper-
ature T. This time can be computed from equation (6) for
C = 10 and Co = 100 (reduction of activity by 90 percent)

as following:

 

 

C _ 10 _ _ k ,
log CS — log I00 — 2.303 t or
= k ' I _ __ 20303
1 m t and t — D — k (7)

where t' is the time required for reduction of activity by
90 percent. Substituting k in equation (6) with 2.303 / D,

from (7),

log %3 = - % and —— = 1

-t/D
Co 0 (8)

By plotting log D values against temperature, a straight
line is obtained. This is the "thermal resistance curve."
The temperature required to reduce D value by 90 percent is
called "factor Z" which is characteristic for each enzyme.
The equation of the thermal resistance curve is,

Tz'Ti

D =D 10 z (9)

 

If the Z factor is known and the D value is known at certain
temperature T1’ the D value can be computed at any given
temperature T2.

As heating time t increases, the enzyme activity C
decreases. This activity can be very low, but it never is
zero (eq. 5). The time required to reduce the enzyme
activity to a certain desired degree at a given temperature
is called "thermal inactivation time." In establishing the
thermal death time for a certain enzyme, the following should
be taken into consideration:

(a) The curve log C versus t sometimes is not a straight
line, but a broken one. If the degree of inactivation be-
yond the break point is desired, two "D" values should be
evaluated, one for each portion of the broken curve.

(b) The regeneration phenomenon has been observed in
some enzymes. Peroxidase was regenerated in samples of peas
that had been blanched just sufficiently to destroy the

original peroxidase activity (Pinsent, 1962). The

21

regeneration occurred after a few months in peas stored at
-18 °C, or within a few hours in thawed peas held at room

temperature. The maximum regeneration observed was 4 percent.

F. Heating and Cooling Methods

 

A number of methods have been developed for
bacteria spores destruction studies. Stumbo (1965)
describes the most common methods. Techniques, employed
for bacteria spores studies, may be used for studying other
biological products in their original form or after proper
modification. A thermoresistometer, for example, designed
for bacteria Spores study has been properly modified for
studying the destruction of vitamins by heat (Mulley et al.,
1975). Techniques for heating can be devided into two
categories. These employing temperatures higher than the
boiling point of the water (oil baths, steam heating) and
those using a constant temperature water bath, which are
more common in enzymes studies.

The constant temperature water bath technique can
be described as follows: A small amount of liquid (.5-2 ml),
in which the substance to be studied has been dissolved, is
put in small diameter glass tubes. A number of test tubes
are placed into the constant temperature bath, and samples
are removed at scheduled time intervals, and then cooled
in ice-water.

The constant temperature bath gives good results

when the heat resistance of the substance under study is

22

relatively high. In such cases, the heating time is
relatively long allowing the liquid inside the tube to
reach the bath temperature. Agitation is sometimes employed
to assure as much uniform heating as possible. When short
times have to be employed for heat treatment, two problems
arise. First, the temperature of the liquid increases
exponentially and, as the heating time is short, it never
reaches the bath temperature. Samples removed from the
bath at different times have been heated to different
temperatures which are different from that of the constant
temperature bath. In addition, the pH may change with
temperature and this change might have an effect on the

heat resistance of the substance under study. This is

usually corrected by using buffer solutions.

METHODS AN D PROCEDURES

A. Heating and Cooling of the Samples

 

To determine the inactivation effect of heat on
polygalacturonase in cherry brine some alternative heating
methods required evaluation. To select a method for heat
treatment of the samples some preliminary tests were run.
It was established that time less than 60 seconds was
required to inactivate the enzyme by more than 90 percent
at 70 °C. Since the temperatures required for studying the
kinetics of inactivation of the enzyme polygalacturonase in
cherry brines were well below the boiling point of water,
the constant temperature bath technique was selected.

To overcome the limitations of the water bath, described
previously, the method was modified and two constant tem-
perature water baths were employed, one having a higher
temperature than the other. The higher temperature bath
will be called "heating bath" while the other, having a
lower temperature, will be referred to as "holding bath."

The temperature history of the liquid in the test
tube was studied in preliminary heating and cooling tests.
Test tubes, 8 mm in diameter and 20 mm in length, were

used. A thermocouple was placed in a tube, containing

23

24

1 ml brine, at a distance of about 1/3 of the brine height
from the bottom (slowest heating point). The sample was
heated in the heating bath at different temperatures.

After conducting an appropriate number of tests, the heating
curves were constructed (Fig. 2). Using these curves, the
time required for the heated sample to reach a desirable
temperature could be estimated as follows:

a. The heating and holding bath temperatures are
measured.

b. The temperature difference between the two baths is
calculated.

c. The temperature difference line is drawn (dotted
lines in Fig. 2).

d. The intercept of dotted and solid line is projected
on the time axis, and the heating time is directly
read.

Example for heating time estimation. Measured temperatures:
heating bath 75 °C, holding bath 69 °C. The temperature
difference is T = 75 - 69 = 6 °C. The intercept of 6 °C
(dotted line) and 75 °C (solid line) is projected on time
axis (Fig. 2) and the heating time is estimated 34.5
seconds.

The sample in the test tubes was heated in the
heating bath for a predetermined time, and then it was
transferred from the heating bath to the holding bath. A
thermocouple temperature recorder was used to check whether

the estimated heating time was the correct one. AS was

80

75

70

°C

65

60

55

Brine Temperature,

50

45

40

Fig.

25

 

 

    

 

 

 

l I r T I
I I
’ I .I
I I
I.
. I
I “ I
80
. I . '
I I | l
: I I I
: I I I I
| I l
70°C I '
_ l I I I I
12 10 8 6 4
AT, °C
- AT = temperature
difference between
heating and holding
bath.
L
I J l 1 L l
10 15 20 25 30 35 40
Time. SEC
2. Temperature history of 1 ml brine in test tube,

at the slowest heating point. Curves show heating
from room temperature, 24 °C (70, 75, and 80 °C
are heating bath temperatures).

26

indicated previously, the temperature was measured at the
slowest heating point. Tubes were removed at 10 or 15
seconds intervals and placed in an ice-water bath for
cooling. Since the heating and cooling were not instan-
taneous, both contributed to the enzyme lethality.

A number of tests were run to determine the cooling
rates of the sample. To study the temperature history
during the cooling process, the thermocouples were placed
near the top of the liquid at the location of the slowest
cooling point. Fig. 3 illustrates the shape of the

cooling curves from different temperatures to 0 °C (32 °F).

B. Enzyme Source and Concentration

 

Purified commercial polygalacturonase (Pectinase),1

from Aspergillus niger, was used in this investigation.
Concentration of 1 mg/ml of brine was selected. Concentra-
tion of 1 mg/ml of polygalacturonase may soften the cherries
extensively within 12 days (Steele and Yang, 1960). Beavers
et a1. (1970) reported failure of pure polygalacturonase

(1 mg/ml) to completely break down the texture of the
brined cherries. The failure was not due to the enzyme
concentration but to the absence of pectinesterases which
promote the polygalacturonase activity. Pectinesterases
have been detected in cherries (Al-Delaimy, 1963). The

proper amount of the purified enzyme was dissolved in

 

lPectinase is the trade name for polygalacturonase
supplied by SIGMA Chemical Company, St. Louis, MO.

27

 

 

 

 

 

 

I T
O
O
a?
H
s
4.)
m
H
é
o
a
m
c
-H
s...
m

I 1

24

Time, sec
Fig. 3. Cooling curves for 1 m1 of brine in test tubes

from various temperatures to 0 °C.

 

28

commercial brine so as to obtain an enzyme concentration of
1 mg enzyme per m1 of brine. One ml of that solution was
placed into each test tube, and heat treated as previously

described.

C. Measurement of Polygalacturonase Activity

 

The "cup-plate" technique develOped by Dingle et al.
(1953) was used. The exact assay for the enzyme activity
is deScribed in the Appendix B. Dingle et a1. (1953)
studied the effect of pH of the gel on the diameter of
zones produced by polygalacturonase. They suggested that
the pH be adjusted to 4 i 0.1 by using 0.1 N sodium
hydroxide. The substrate used in this experiment was found
to have the appropriate pH, and no adjustment was necessary.

The ingredients necessary for the gel formation
were blended and the mixture was transferred to 100 m1
Erlenmeyer flasks. The flasks with the substrate were
sterilized in an autoclave at 15 psig. (250 °F). Sterili-
zation time of 20 minutes at 15 psig. has been suggested
by Steele et a1. (1960). It was found that sterilization
for 20 minutes resulted to a soft gel, difficult to be
handled for cup formation and for develOpment of the clear
zone. The probable reason for the soft gel was the partial
hydrolysing of the agar. Preliminary tests were run to
establish the proper sterilization time. It was found that
sterilization for 5 minutes resulted in desirable firm gel
formation. That sterilization time was employed for all

the tests.

29

The next step in the procedure involved 15 ml of
the liquid substrate that were transferred to 15x85 mm Petri
dishes and allowed to solidify. The amount of the liquid
in each dish has a significant effect on the diameter of
the clear zone. The effect of the gel thickness in the
plate on the zone diameter is illustrated in Fig. 4. Very
small thickness (5 ml per dish) resulted in a non clear
zone, while, very high thickness resulted in smaller
diameter. A thickness of 2.1 mm (or 15 ml per dish) was
found to give well defined and large diameter clear zone.

It must be noted, however, the larger the diameter the
greater the accuracy.

Small plastic collars (9 mm diameter) were inbedded
in the media to provide cups into which brine, containing
enzyme, was placed. Using a device, similar to eye drOpper,
two drops of the sample were placed in each cup, each drop
was approximately 0.065 ml and then, the plate was incubated
for 20 hours at 36 °C (96.8 °F). The enzyme action on the
substrate in the incubated Petri dishes was not visible.
The clear zone was developed by flooding with 2 N hydroch-
loric acid for about 5 minutes. The development was com-
pleted by rinsing with distilled water. The activity of
the enzyme was indicated by the formation of a clear zone.
The diameter of the formed zone is a function of the enzyme
concentration. Dingle et a1. (1953) showed that the plot
of the enzyme concentration (on log scale) vs the zone

diameter is a straight line. Some other factors, such as

30

Volume of Gel Per Plate, cm3

 

 

 

 

5 10 15 20 25
‘\
32" d
31 P 2
3: '- -I
Q)
4.)
g 30_ .
-H
D
(D ,. .-
C:
O
N
29 ' .
28 b
‘.
l j 1 I l l I l l
.l 1.4 2.1 2.8 3.5

Gel Thickness, mm

Fig. 4. Effect of gel thickness on zone diameter of 1 mg/l
ml enzyme concentration and brine pH = 3.

31

the gel firmness and the incubation temperature and time,
may have an effect on the zone diameter.

A number of enzyme dilutions in brine, ranging from
10 to .001 mg/ml, were prepared and six dishes were
inoculated with each dilution. After incubation and
development, the diameter of the clear zone was measured
and the average of the six diameters were plotted against
the concentrations of the enzyme on semi log paper. The
plot (Fig. 5) was used as a reference curve to convert the
zone diameters obtained from the tests to enzyme concentra—
tion.

It was noted that the same enzyme dilution,
incubated in Petri dishes containing solid media from
different batches, might result in clear zones with a
different diameter. This is probably caused by some
unidentified factors which affected the gel firmness, and
in turn, the diffusion rate of the enzyme. For the results
to be comparable, the following procedure was followed:

a. The diameter of the clear zone (average of three
plates) of the control sample was determined for
each substrate batch.

b. From the intercept of the observed diameter line
and the 100% activity line, a curve was drawn
parallel to the standard curve (example curve in

Fig. 5).

32

 

100
4.)
C!
0)
8
m 10
Q:
C
O
"-1
4.)
(U
34
JJ
£2
0)
U
C.
O
U
(D
E
if
,3 1.0
Cd

Standard Curve

-------- Example Curve

 

 

111111 1 JLLLll

anll

L1

 

 

Zone Diameter, mm

Fig. 5. Standard curve for enzyme activity estimation.

33

c. The dotted curve was considered as a standard
curve for all the tests for which the same sub-

strate was used.

D. pH Adjustment

 

The pH of the commercial brines at the time of
preparation is about 2.8. The pH increases during the
storage of the brines reaching about 3.8 by the end of the
period. The resistance of the enzyme to inactivation by
heat was determined at four different pH levels to represent-
the normal commercial pH range. The pH of the commercial
Napoleon brine tested was 3.0. Samples were adjusted to
pH = 2.5 by using hydrochloric acid, and to pH = 3.5, and

4.0 by using sodium hydroxide.

E. Dissolved Solids Level

After the cherries have been cured and equilibrated,
the brine will contain soluble solids of 6 to 12 percent
(Beavers et al., 1970). The soluble solids consist
principally of sugars, pigments, acids, and other soluble
constituents diffused from cherries. Napoleon brine was
found to have a dissolved solids concentration 9 percent
(measured with ABBE refractometer). Corn syrup was used to
increase the dissolved solids concentration to 12 and
14 percent. Spent brine contains the same as corn syrup

sugars, namely fructose and glucose (Sapers, 1975).

34

Dissolved solids concentration of 14 percent is the
highest concentration which might be expected in the brine

after several years of re—use.

F. Cherry Variety

 

Commercial brines from two cherry varieties were
studied. Windsor brine is characterized by high SO2 and
high pigment concentration. Napoleon brine contains less

pigment and about one-half as much SO2 as Windsor brine.

G. Regeneration Capability of the Enzyme

 

Commerical Napolean brine which had been heat
treated to inactivate the enzyme was stored under room
conditions and in refrigerator at 4 °C. The enzyme
activity was determined at selected time intervals for 82

days.

RESULTS AND DISCUSS ION

A. Effect of Temperature

 

1. Order of the Reaction

 

To determine the order of enzyme inactivation
reaction, the measured percent activities were plotted
versus time in Fig. 6. Under the experimental conditions,
data fit a straight line plot on semi-log coordinates and
were described by a first order function. Fig. 6 illustrates
the effect of the temperature on the rate of enzyme in-
activation. The straight lines can be described by
equation (6). It must be noted that the higher the tem-
perature the higher the rate constant which results in
steeper curves. The time scale in Fig. 6 corresponds to
the heating time in the holding bath. The intercept of the
curves with the percent activity indicates the enzyme
activity at the time when the sample was transferred from
the heating bath to the holding bath. As initial activity
Co, in equation (6), is considered the activity described
by the intercept. Thus any factor affecting the value of
the intercept does not have any effect on the slope of

the inactivation curves. The activity value determined

35

36

100

 

I I l l

Holding Temperature

 

A 68 °c
0 69 °C
0 70 °C
v71 °c
072 °c

 

 

 

O
4) V
c:
0)
O
:3
m 10- .
5‘
~r-l
>
-r-I
1.)
O
:4:
(D
a O
N \
f: ‘ x
O s
\
\
s
\
O
\
\
V \
l. ‘ \ .
\ \
\ \
\ \
O ‘ ‘
1 l l I
0 20 40 60 80

Time, sec.

Fig. 6. Effect of temperature on the inactivation rate
for the polygalacturonase enzyme in Napolean
brine (pH = 3.0).

37

by the intercept is always less than the initial activity

due to the partial inactivation occurring during heating

and cooling. The value of the intercept is affected by the

following:

a.

The effect of the heat on the enzyme activity,
described by the curves, correSponds to the heating
in the holding bath. Some inactivation occurs
during heating in the heating bath and during
cooling. The contribution of heating and cooling
to the total lethality is not indicated in Fig. 6.
The temperature of the sample was measured at the
Slowest heating point and since the sample was not
agitated, all the other points are at higher tem-
peratures. The thin layer of the brine in contact
with the tube walls would be heated more than all
the other points. This nonuniform temperature
distribution contributes to depletion of the inter-
cept.

The other factor which may have a significant
effect on the intercept value is the method used
for the enzyme activity measurement. As it has
been described above, a standard curve was used to
convert the zone diameter to enzyme activity. In
each test, a line parallel to the standard was
drawn based on the zone diameter average of three
control dishes, as described in the previous

chapter. A small error in the diameter measurement

38

results in significant movement of the intercept.
By changing the control diameter by one milimeter
(from 29.5 to 30.5) the activity indicated by the
intercept decreases from 33 to about 18 percent.
Factor (b) may be controlled closely. By employing
agitation, more uniform heating of the samples should be
expected, and the temperature measured will be very close
to the average sample temperature. The factor (c) was
difficult to control and could be a deficiency of the

method.

2. Rate Constants

 

Equation (5) can be rewritten as follows:

c = Co e ‘kt (10)

The concentration indicated by the intercept may be con-
sidered as initial concentration Co. The inactivation
rate constants were computed from equation (10) by using
the exponential regression program in Wang Computer. The
enzyme activities percent at different times and tempera-
tures are summarized in Table A3. Fig. 7 illustrates the
normalized inactivation curves for three temperatures.
Table 1 shows the rate constants as they were evaluated by
using Wang Computer (2200 series). Computed values of the
rate constant are Similar to that found in thermal in-

activation of polygalacturonase in cucumber brine. The

39

V T

 

 

 

 

 

1.0 Holding Temperature ‘
C) 69 °C
E] 70 °C
8 '3 ~
° ’ A 72 °C
‘
O
o 06'- d
o
\.
U
I O
.4 .
A
.2- ' 0
I
‘ h .0.-
0.0 1 1 1 l 1 l
0 10 20 30 40 50 60
Time, sec.
Fig. 7. Normalized inactivation rate curves for

polygalacturonase enzyme in Napoleon brine
(pH = 3.0) .

40

Table 1.--Rate constants for inactivation of polygalacturo-
nase in Napoleon brine (pH = 3.0).

 

 

 

Temperature Correl.l D k x 102.
(°C) Coeff. (sec) (sec-1)
68 .9995 84.9 2.712
69 .9969 67.59 3.407
70 .9994 46.76 4.925
71 .9988 36.71 6.274
72 .9934 29.26 7.870

1

For C/Co versus time (Table A3).

rate constant in the latter case found to be equal to
7.7x10-2sec at 79.5 °c (Chavana, 1976).

Fig. 7 indicates that the experimental points fit
well to the ideal curves which describe the inactivation
process. The curves illustrated in Fig. 7 have been
obtained by using least squares fit-power curve. Thus the
inactivation proceSs follows first order kinetics. This
may be supported by the high correlation coefficients shown
in Table 1. The D values in Table 1 computed from k
values (eq. 7).

The D values were plotted versus temperature on
semilog coordinates. The straight line obtained and shown
in Fig. 8 is called the "thermal resistance curve." Using
Fig. 8, the decimal reduction time can be estimated at
different holding bath temperatures. As the temperature

increases, the D becomes smaller and smaller. The

Sec.

D Value,

41

 

 

 

 

 

 

I I I
100 »- ..
Z
10 - -
L l l L
66 68 7o 72 74

Temperature,

°C

76

Fig. 8. Thermal resistance curve for polygalacturonase

in Napoleon brine (pH = 3.0).

42

Table 2.--Data for evaluation of the activation energy for
heat inactivation of polygalacturonase in
Napoleon brine (pH = 3.0).

 

 

k x 102 T Tab (l/Tab)x103
(sec-1) (°C) (°k) (°k’1)
2.712 68 341 2.9325
3.407 69 342 2.924
4.925 70 343 2.915
6.274 71 344 2.907
7.870 72 345 2.899

 

temperature change required for changing D by 90 percent

is called "Z" value and it was found to be 8.4 °C (15.12 °F).
The Z value is characteristic for each micro-

organism, nutrient or enzyme and the Z value can be used

to compute the decimal reduction time (D) at any desired

temperature. The thermal resistance curve (Fig. 8) can be

described by the equation:

log D

 

= log D2 - (11)

l

O]:

T -T

=131o21

I’1 2 z (12)

 

if Z has been evaluated and D is known at a certain tem-
perature, the decimal reduction time can be computed at any

temperature.

43

3. Activation Energy
for Inactivation

 

 

Another way to express the influence of temperature
on rate constant, and in turns on decimal reducation time,

is the well-known Arrhenius equation:

(-Ea/RTab)

k = A e (13)

where k is reaction rate constant, A is a constant, Ea is
the activation energy, Tab is the absolute temperature, and
R is the gas constant. Equation (13) can be written as

follows:
log k = log A - Ea/(2.303)RTab (14)

Equation (14) describes a straight line known as the
"Arrhenius plot." The Arrhenius plot for polygalacturonase
inactivation in Napoleon cherry brine at pH = 3.0 is
illustrated in Fig. 9. The constant A is evaluated from
the intercept, while the activation energy is computed from
the slope of the curve. The slope is equal to Ea/(2.303)R.
The rates of chemical reactions generally increase
with increasing temperature. Increasing in temperature
results in increasing of the collision frequency and, for a
certain reactant concentration, an increasing of reaction
rate. It must be noted that only a small proportion of the
collisions occur with the required energy to promote the
reaction. This minimum energy required for a successful

reaction is called activation energy. Equation (13)

44

 

 

 

 

' I l I
.1 I 7
o
m
m
A:
.01 - .
4* 1‘ I L
.00289 .0029 .00291 .00292 .00293 . .00294
l/Tab, °K_l

Fig. 9. Arrhenius plot for polygalacturanase inactivation
in Napoleon brine CpH = 3.0).

45

formulated empirically by Arrhenius is generally used to
correlate the rate constant, the temperature, and the
activation energy. Enzyme catalyzed reactions exhibit
behavior similar to chemical reactions up to a point (Segel,
1975), and equation (13) can be used to describe the effect
of temperature on the reaction rate.

To explain the magnitude of the activation energy
for inactivation of an enzyme, the structure of the enzyme
must be taken into consideration. Enzymes are complicated
protein molecules and their catalytic properties are associ-
ated to their highly ordered tertiary structure. The
tertiary structure of the enzymes is maintained primarily
by noncovalent bonds which generally are weak bonds.
Hirschelder (Gould, 1959) suggested that the energy of
activation is proportional to the strength of the bond that
must be broken during the rate determining step of a
reaction.

Figu 9 illustrates the Arrhenius plot for the
enzyme polygalacturonase in commercial cherry brine, from
Napoleon cherries, having pH equal to 3.0. The lepe of

the curve, calculated with Wang Computer, was found:
lepe = Ea / (2.303) R = 14,181.8 (15)

and the activation energy:

-1

Ea = 64,668.2 Cal x mol (16)

46

The activation energy for inactivation found for
the polygalacturonase enzyme falls in the range generally
accepted for inactivation of enzymes by heat. This range
(Messing, 1975) is from 50,000 to 150,000 cal mol-l. The

relatively small Ea indicates that the enzyme molecule has

a very delicate and fragile structure (Segel, 1975).

B. Effect ofij on Inactivation Rate

 

The inactivation rate was studied at pH values of
2.5, 3.0, 3.5, and 4.0 and at the temperature of 69 °C.
Commercial brine from Napoleon cherries was used in that
experiment.

Data obtained from the tests are shown in Table A4.
The inactivation rate curves are illustrated in Fig. 10.
Using the intercept of these curves as initial concentration,
Co, the normalized curves have been constructed for two pHs
(Fig. 11). The rate constants were calculated by using
Wang Computer. Results of the calculation are summarized
in Table 3. The rate constant of the inactivation reaction
is highly pH dependent.

The effect of pH on the decimal reduction time is
not linear, as it is indicated in Fig. 12. The resistance
of the enzyme to inactivation by heat is higher at pH
values between 2.8 to 3.5 than at pH values of 2.5 and 4.0.
As it is illustrated in Fig. 12, the enzyme exhibits
more stability at pH values usually employed in commerical

brines.

47

 

 

‘d O U P

Enzyme Activity Percent

 

 

 

 

\ .
\
\\
\‘
I L l
0 20 40 60 80

Time, sec.

Fig. 10. Inactivation rate curves for polygalacturonase
enzyme in Napoleon brine at 69 °C and different
pH values.

48

 

 

 

 

I l ' Ti ' I
10 pH
0 3.0
‘ A. 4.0
.8- '
o
E: .6_ ,
U
.4-
O
.2- ‘ ‘
‘ O
0 l A 1 L 1 1
0 10 20 30 40 50 60

Time, sec.

Fig. 11. Effect of pH on inactivation rate of poly-
galacturonase in Napoleon brine at 69 °C.

49

Table 3.—-Rate constants of polygalacturonase inactivation
in Napoleon brine at 69 °C and different pH

 

 

values.
. l 2

pH Correlation k x 10 D

Coefficient (sec '1) (sec)
2.5 .9988 4.848 47.5
3.0 .9984 3.464 66.54
3.5 .9976 3.812 60.41
4.0 .999 4.947 46.55

 

1For C/Co versus time (Table A4).

Table 4.--Rate constants of polygalacturonase inactivation
in Napoleon brine at different sugar concentra-

 

 

tions.

Sugar Correlation1 k x 102 D
(8) Coefficient (sec '1) (sec)
9 .998 3.462 67
12 .997 3.299 70
14 .994 2.574 92.5

 

For C/Co versus time (Table A5).

50

 

90 . -

Commercial Range
80!-

Sec.
\1
O
I

D VALUE,
ox
o»

50

4O

 

 

 

 

pH

Fig. 12. Effect of pH on the decimal reduction time
at 69 °C.

51

The "t" test was used to determine the significance
level of the effect of pH on decimal reduction time. The
calculated values were compared with "t-statistics" from
tables and found the difference between pH 2.5 and 3.0, and
3.5 and 4.0 to be significant, while the difference of D
at pHs 3.0 and 3.5 is not significant with probability of
99 percent.

Results, similar to that obtained in the lab for
polygalacturonase, have been reported for other enzymes.
Farkas and Goldblith (1962) found that the inactivation
rate of the enzyme lipoxidase is faster at pH values 4.0
and 9.0 than at pH values between 4 and 7. The stability
to inactivation by heat of 1.0 mg/ml solutions of purified
spinach catalase was higher at pH values of 5.0 or 7.0 than
at pH 9.0 at 55 °C (Sapers and Nickerson, 1962).

The effect of pH on the enzymes activity has been
studied more extensively than the effect of pH on the
thermal inactivation rate. Probably, the mechanism involved
in the effect of pH on activity should be different than
that in inactivation by heat. Lehninger (1970) describes
the possible parameters which influence the pH activity
profiles of the enzymes. The effect of pH on the inactiva-
tion rate may be more complicated and is not well understood.

To explain the effect of pH on the inactivation
rates, the nature of the enzymes must be recalled. The
enzymes are the largest class of proteins. As proteins,

the enzymes exhibit an acid-base behavior in solution which

52

is largely determined by the relatively large number of
ionizable R groups of the various aminoacids. The contri—
bution of the terminal amino and carboxyl groups in that
behavior is very little. Due to different types of
ionizing groups, the enzyme molecule has positive or
negative charge which is pH dependent. There is always a
pH at which the enzyme molecule has no charge, and this is
called isoelectic point. At pH values other than the
isoelectic point, the molecule is charged and the electrical
potential of the molecule is determined by its nature (by
the pk of the ionizable groups) and by the pH.

The enzyme molecules in solution exhibit colloidal
behavior. Proteins, and in turn enzymes, have been
characterized as thermodynamically stable, or reversible,
colloidal systems (Kruyt, 1952). Colloidal particles carry
an electric primary charge the Sign of which can be either
positive or negative. The magnitude and the sign of the
primary charge are frequently affected by the pH. The net
electrical charge of a colloidal dispersion is zero. This
is due to accumulation of opposite charge ions (counter-
ions) near the surface of the particle resulting in the
formation of double layer at the interface between a solid
and water. The counter-ions are attracted by electrostatic
forces generated by the primary charge of the particle the
magnitude of which is affected by the pH. This attraction
results in a concentration gradient of the ions, and a

thermal energy is required to diffuse the ions away from

53

the surface of the particle. This attraction of the counter-
ions may be considered as an activation energy barrier for
inactivation which is pH affected and which must be over-
come for inactivation to occur. As a result, the activation

energy is expected to be affected by pH.

C. Effect of Sugar Concentration

 

Samples of commercial brine from Napoleon variety
were treated as it is described by Sapers (1975) for syrup
production from brine. The pH of that brine, which is
essentially free of calcium bisulfite, was adjusted to 3.0
by using a small amount of sodium hydroxide. The sugars
content of the brine was measured by the ABBE refractometer
and found 9 percent. High fructose corn syrup was used to
adjust the sugars concentration in two samples to 12 and
14 percent. The heat inactivation rates were studied in all
three sugar levels, namely 9, 12, and 14 percent.

The results of the test are summarized in Table A5.
The rate constant was calculated by using Wang Computer
and the decimal reduction time computed from equation (7).
Values of both R and D are Shown in Table 4, in which the
correlation coefficient of C/Co versus time is included.
Fig. 11 indicates that the experimental points fit well to
the curve described by equation (10). The inactivation
rates of the enzyme polygalacturonase in brines with
different sugar contain is illustrated in Fig. 13. The
effect of the sugar concentration on the decimal reduction

time is described by the curve shown in Fig. 14.

54

 

 

 

 

 

 

100 l l I I
Sugar Concentration
D 9 percent
<3 12 percent
A' 14 percent
41
c
o
o
3.4
o
m
>.
4.)
-H
:>
-H
.p
0
¢
E
N
c
LL]
\ \
s
1.0- \:\‘4
\O
l J l 1
0 20 40 60 80

Time, sec.

Fig. 13 Effect of dissolved sugar concentration on
inactivation rate of polygalacturonase in
Napoleon brine at 69 °C and pH = 3.0.

55

 

 

 

 

 

I I i i I l
I
100 " d
90 P «I
6 80- .
m
U)
c?
70- .
9::
60" 1)
50L L J 4 l l i
8 9 10 ll 12 13 14
Dissolved Sugar Percent
Fig. 14. Effect of dissolved sugars on D value at 69 °C

and pH = 3.0

56

At lower sugar concentrations the inactivation rate
is not significantly affected by the sugar content. As is
illustrated by the curve (Fig. 13), the decimal reduction
time is increased when the sugar concentration is higher
than 12 percent. This effect is significant, as it was
determined by the "t" test, and has to be taken into con-
sideration when pasteurization units are designed for brines
used more than one year because the concentration of the
sugar in such brines will be higher and higher from year to
year. An observation in Fig. 8 and 14 indicates that the
pasteurization temperature must be increased by about
1.5 °C to overcome the effect of the higher sugar con-
centration at pH = 3.0.

The effect of the sugar concentration on the
resistance of the enzyme was expected. Similar observations
have been reported in thermobacteriology. It has been
reported (Stumbo, 1965) that high sugar concentrations tend
to increase the heat resistance of microorganisms. The
sugars concentration is expected to affect the activation
energy, as well. The mechanism of the protective effect
of sugar on heat inactivation of the enzyme is not known.

A possible explanation may be that the rich in hydroxyl
groups sugars form hydrogen bonds with uncharged polar R
groups of the aminoacid residues existing in the enzyme
molecule. Additional heat energy is required to break
down these hydrogen bonds which may tend to increase the

enzyme resistance to inactivation by heat.

57

D. Effect of Cherry Variety

 

Some briners change prOportions of ingredients in
brine formulations for different varieties in attempts to
get the desired product (Brekke et al., 1966). For varieties
with higher pigments content, higher 802 concentrations are
used.

Two different commercial brines were tested to
determine the effect on the decimal reduction time. One
brine was from Napoleon and the other from Windsor variety.
The main differences of the two brines were: (a) The 802
concentration in Napoleon brine was about 7,000 ppm, while
in Windsor brine it was twice as much. (b) The CaCl2 con-
centration in Windsor brine was higher than that in
Napoleon. (c) The concentration of the pigments was higher
in Windsor brine which had red color. The pH of both
brines was adjusted to 3.0 and enzyme at a concentration of
1 mg/ml was used. The inactivation rate of the enzyme was
tested at 69 °C.

Table A6 summarizes data obtained in the laboratory.
The rate constant found to be lower in Windsor brine having
higher concentration in 802, Ca, and pigments. The cal-
culated "t" values were compared with "t-statistics" from
tables, and it was found that the effect of the variety on
the D value was significant with probability 99 percent.

The inactivation rate curves are shown in Fig. 15.
The effect of the various brines on the decimal

reduction time is not easy to explain because of the

58

 

 

 

 

100 I I I l
@2221
0 Windsor
D Napoleon
4.)
c
o
o
H
m
m
>.
4.)
‘9 1&—
-a
4.)
o
.4: O
m
E.
>.
N
c
m
13.0:-
J l l L
0 20 40 60 80
Time, sec.
Fig. 15. Effect of cherry variety on inactivation

rate of polygalacturonase in brine at
69 °C and pH = 3.0.

 

59

complexity of the system. The possible explanation may be
the difference in ionic strength of the brines. Ionic

strength is defined by the equation:

Mizi2 (17)

H
II
NI I—‘

Where Mi is the molarity of the ion and Zi is the charge of
the ion (regardless of Sign). An enzyme may be more stable
in one buffer of a certain pH than in another buffer with
the same pH and different ionic strength (Segel, 1975).
Dyson et a1. (1973) showed that not the kind of the cations
(monovalent or divalent) but the ionic strength alone
affected the enzyme activity.

When brines of different formulations have to be
pasteurized, the effect of the ionic strength on the
inactivation rates of the enzyme should be taken into

account in designing pasteurization units.

E. Regeneration Capability of the Enzyme

 

Napoleon brine was adjusted to pH = 3.0 and purified
polygalacturonase was dissolved in it so as to obtain
enzyme concentration 0.1 percent expressed as weight by
volume. The brine was heated so as the enzyme activity was
reduced to .88 percent of the initial activity. The heat
treated brine was divided into two lots one of which was
stored at room temperature and the other under refrigeration
conditions at 4 °C. The lots were tested for activity at

time intervals.

60

No regeneration was observed for storage time equal
to 82 days, which indicates that the enzyme inactivation is
irreversible (Table 5). In the sample stored at room
temperature a precipitate, which probably consisted from
crystals of calcium sulfate, was formed. This precipitation

was not found to have any effect on the enzyme activity.

F. Applications

 

The parameters evaluated by this work cannot be
used for design purposes before evaluating the thermal
death time (F). This parameter is the time required to
reduce the enzyme activity to a level at which it should be
unable to result in softening of the cherries packed in that
brine.

The thermal death time is difficult to evaluate
from data in current literature because different types of
enzyme preparations and different substrates have been used
by the researchers. Brekke et a1. (1966) used Pectinol A
and found that it softened cherries when 0.05 to 0.20 per-
cent of the weight of the fruit was added. This enzyme
concentration is roughly equivalent to 0.5 to 2 mg of the
enzyme per m1 of brine. Steele and Yang (1960) found that
polygalacturonase, in "cats claw" infected fruit, had
activity equivalent to 1.5 to 0.5 mg/ml of Pectinol M
solutions. Softening and disintegration of these cherries
was excessive and their appearance was similar to that
reported by McCready and McComb (1954). Purified poly-

galacturonase enzyme was added by Beavers et a1. (1970) in

lull!“

61

Table 5.--Activity of polygalacturonase inactivated in
Napoleon brine by 99.12 percent.

 

 

 

 

 

Storage Room Temperature Refrigerator
(gimg) Enzyme Enzyme1
y Mean Diam. Activity Mean Diam. Activity
(mm) (%) (mm) (%)
0 23.52 0.881 23.51 0.88
16 23.52 0.88 23.52 0.88
26 23.03 0.90 23.03 0.90
34 24.54 0.88 24.44 0.82
38 23.52 0.88 23.52 0.88
65 23.03 0.90 23.03 0.90
74 23.52 0.88 23.42 0.82
82 23.03 0.90 23.03 0.88
1Activity at time zero = 0.88 percent of the
initial.

2Control Zone diameter 3.0 mm.

3Control Zone diameter = 29.5 mm.

4Control Zone diameter 31.0 mm.

62

cherry brine in a concentration of 1 mg/ml and cherries
were packed in the brine on an one weight by weight basis.
They found that a decrease in texture accompanies the
polygalacturonase activity.

The concentration of the enzyme of the type used in
this study, which is capable to produce soft fruit, Should
be determined experimentally. Data obtained from such a
study will allow for determination of the degree of inactiva-
tion of the enzyme for safe re-use of brine. Preliminary
studies indicated that inactivation of the enzyme up to
99.3 percent follows the described kinetic mechanism
(Fig. Al). As long as the desirable degree of inactivation
has been established, the F value can be evaluated for a
certain temperature. If F value is known at one temperature
T, it can be computed at any temperature T', using the
equation:

T'-T

F = F x 10 "'z (18)

T T'

where Z has already been computed from the thermal resistance
curve (Fig. 18) and found 8.4 °C.

In a continuous pasteurization process three seg-
ments are well defined: (a) heating, (b) holding, and
(c) cooling. In such a process, the heating and cooling
of the pasteurized liquid can be considered uniform. The
contribution of the heating and cooling to the total
lethality can be calculated. The following example

illustrates the procedure to be used for the equivalent

°C

Temperature,

Fig.

75

70

65

60

55

50

16.

63

 
 

 

   

   

 

 

,
l
- I
I
I
I
I
I
. I
I
l
"-“““‘-'--r ............ 1---
I I
I I
I
I
I I
l I
I ' ‘
I I
' I
I
I I
I I
; I
I I
1 1 I 1 n 1 AV l %l 1 l
3 6 9 12 15 18 0 4 8 12
Time, sec.
Heating and cooling profiles for lethality

calculation.

64

process time determination and for sizing the holding tube
of the heat exchanger.

Problem: Tubular heat exchanger is to be used for
cherry brine determination. The flow rate is 10 gal per
min, the holding tube inside diameter is 1 inch, the thermal

death time for the enzyme is F S = 10 sec, and the Z value

7
is 8.4 °C. Assuming that the heating and cooling process
is described1 by Fig. 16, calculate the minimum holding
time at 75 °C and the length of the holding tube.
Solution:

1. The time required for heating and cooling is
divided in appropriate intervals (3 sec. for heating,
2 sec. for cooling) as it is shown in Table 6.

2. The midpoint temperature is evaluated from Fig. 16
(column 2 in Table 6).

3. The lethality for each time interval is computed by
multiplying the time intervals with the lethality
computed (column 3). Time intervals for heating
selected 3 sec. and for cooling 2 sec.

4. The total lethality effect of heating and cooling
is the sum of column 4. This is expressed as
equivalent process time at 75 °C and it is 3.824

seconds.

 

1The heating and cooling profiles may be obtained
by running a pilot plant or from theoretical calculations
(Welty, 1974).

65

Table 6.--Contribution of heating and cooling to the total
lethality of the enzyme in brine.

 

 

Midpoint Midpoint Midpoint1 Time Interval2
Time Temp. T Lethality Lethality
(see) (°C)

Heating
1.5 47.25 .0005 .0015
4.5 56.50 .0063 .0188
7.5 62.50 .0395 .0975
10.5 67.25 .1195 .3851
13.5 71.0 .334 1.0021
16.5 73.75 .70989 2.1297
Subtotal 3.635
Cooling
. 65.9 .0825 .16508
. 57.8 .00896 .0179
. 53.25 .00257 .0051
. 47.25 .0005 .0010
Subtotal .18908
Total lethality 3.824

 

Midpoint lethality = 10 Z

T-75

, where Z = 8.4 °C.

Time interval lethality = Midpoint lethality x
time interval.

66

5. The minimum holding time of the brine at 75 °C is:
F75 - 3.824 = 10 - 3.824 = 6.175 seconds. Time of
6.5 sec. may be used for holding tube sizing.

6. The brine flow rate is 10 gal per min or 10 % 7.5 =

3

1.34 ft per min.

7. The velocity of the brine in the holding tube is:

V UA where A is the cross area of the tube, so

u = v + A = (1.34 : nD2)/4 = (1.34) e (3.14 x

(1/12)2/4 245.8 ft/min = 4.1 ft/sec.

8. The length of the holding tube should be: L = Ut =
4.1 ft/sec 6.5 sec = 26.65 ft or 27 ft.
The holding tube may have coil shape and if the

diameter of each turn is 1 ft, the total number of turns

will be N = 26 % nD = 8.49 or 9 turns.

S UMMARY AND CONCLUS IONS

Kinetics of inactivation of the polygalacturonase
enzyme by heat in commercial cherry brines was studied.
Commercial purified preparation of polygalacturonase was
used at a concentration of 1 mg per ml of brine and tested
at different temperatures, pH levels, at different dis-
solved sugar concentrations, and in brines from different
cherry varieties. The regeneration capability of partial
inactivated enzyme at room temperature (26 °C) and refri-
gerator temperatures (4 °C) was studied, as well. The
agar "cup-plate" diffusion technique was used in this work
for the quantitative determination of polygalacturonase.

Laboratory data were analyzed (Wang Computer, 2200
series) and the following were obtained.

1. The inactivation of the polygalacturonase enzyme by
heat can be described by a first-order function.

2. The rate constant at 70 °C was 4.925 x 10.2 sec.-1
and the decimal reduction time at the same
temperature 46.8 sec.

3. The activation energy for enzyme inactivation in

Napoleon cherry brine at pH = 3.0 computed from

the Arrhenius plot was 64.2 Kcal-mol-l.

67

68

4. The Z values for the temperatures range investi-
gated was 8.4 °C.

5. A significant effect of pH on the decimal reduction
time was found for the 2.5, 3.0, and 4.0 pH range
while the difference between the effect of pH 3.0
and 3.5 was insignificant. The highest resistance
of the enzyme to heat inactivation was at pH = 3.0.

6. Sugar concentrations higher than 12 percent seem
to have a significant protective effect on the
enzyme.

7. Enzyme preparation in brine from the Windsor cherry
variety exhibited more resistance to inactivation
by heat than enzyme in brine from Napoleon cherries.

8. No regeneration was observed within 82 days of
storage for partially inactivated enzyme.
Additional investigation is necessary to establish

the concentration of the enzyme needed to create softening
of cherries, and in turn, the degree of inactivation
required for safe re-use of pasteurized spent cherry brine.
The most common cherry variety should be tested for
softening in various brines containing different enzyme

concentrations.

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73

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74

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APPENDICES

APPENDIX A

TABLES ILLUSTRATING RAW AND CALCULATED DATA

APPENDIX A

TABLES ILLUSTRATING RAW AND CALCULATED DATA

Table A1.--Data for standard curve construction. Polygalacturonase
enzyme was diluted in comercial Napoleon brine at pH = 3.0.

 

 

 

Enz2 Clear Zone Diameter, mm Aver. Stand.
Conc. Diam. Deviat.
(mg/m1) I II III Iv v VI (mm)

10.0 33.0 33.0 32.8 33.0 33.0 33.0 32.967 .069
5.0 32.5 32.5 31.8 32.5 32.0 32.0 32.22 .269
1.0 30.0 30.5 30.0 30.0 30.2 30.0 30.12 .1725
0.5 28.5 29.0 29.0 28.5 29.0 29.0 28.84 .2182
0.1 27.0 27.0 27.0 27.0 27.0 27.0 27.00 0.0
0.05 25.5 26.0 . . 26.0 25.9 26.0 25.8 .224
0.01 23.5 23.5 23.5 24.0 23.5 24.0 23.67 .218

0.001 20.5 20.5 20.5 20.5 20.5 20.5 20.417 .173

 

75

76

Table A2.--Effect of temperature on zone diameter for polygalacturonase
in Napoleon brine (pH = 3.0).

 

Clear Zone Diameter, mm

 

 

 

 

Time Mean
sec lst Trial 2nd Trial Zone Stand.
Diam. Dev1ation
mm
I II III I II III
68 °C1
0 28.0 28.0 27.5 28.0 28.0 28.0 27.92 .204
15 27.5 27.5 27.5 27.5 27.0 27.5 27.42 .204
30 27.0 27.0 26.5 27.0 26.5 27.0 26.83 .258
60 26.0 25.5 25.5 25.5 25.5 26.0 25.67 .258
75 25.0 25.0 25.5 25.0 25.0 25.5 25.17 .258
69 °c2
;6 28.0 28.0 27.5 28.0 27.5 28.0 27.83 .258
15 27.5 27.5 27.0 27.0 27.5 27.5 27.34 .258
30 27.0 26.5 . . 27.0 26.5 . . 26.75 .289
45 26.0 26 25.5 26.0 26.0 26.0 25.92 .204
60 25.0 25.5 25.5 25.0 25.0 25.0 25.17 .258
75 25.0 24.5 24.5 24.5 24.5 25.0 24.67 .258
70 °c3
0 28.0 28.0 28.0 28.0 28.0 28.0 28.01 .00
5 27.5 28.0 27.5 28.0 28.0 27.5 27.75 .274
15 27.0 27.0 26.75 27.0 27.0 27.0 26.95 .167
30 26.0 26.0 25.75 26.0 25.75 25.75 25.86 .233
45 25.0 25.0 25.0 25.0 25.0 25.0 25.0 .00

60 23.75 24.0 24.0 24.0 24.0 23.75 23.91 .202

Table A2.--(cont'd.).

77

 

Clear Zone Diameter, mm

 

 

 

 

Mean
Time lst Trial 2nd Trial Z?“e SFané’
sec Diam. Dev1ation
I II III I II III mm
71 °C
0 27.5 28.0 27.5 27.5 28.0 27.5 27.67 .258
10 27.0 26.5 27.0 27.0 27.0 27.0 26.92 .204
20 26.0 26.0 26.0 26.0 26.0 26.0 26.0 .00
30 25.0 25.0 25.0 25.0 25.0 25.0 25.0 .00
40 24.0 24.5 24.5 24.0 24.0 24.0 24.2 .258
50 23.5 23.5 23.0 23.5 23.5 23.5 23.42 .204
72 °C5
5 26.0 26.0 26.0 26.0 26.0 26.0 26.0 .00
15 24.5 24.5 24.5 24.5 25.0 24.5 24.6 .224
30 23 23 . . 22.5 23.0 23.0 22.9 .224
45 21.5 21.5 22.0 22.0 21.5 21.5 21.67 .258
60 21.0 21.0 20.5 20.5 21.0 21.0 20.84 .258
1Control Zone diameter = 29.5 mm.
2Control Zone diameter = 29.5 mm.
3Control Zone diameter = 29.5 mm.
4Control Zone diameter = 29.5 mm.
5Control Zone diameter = 28.5 mm.

78

Table A3.--Effect of temperature on polygalacturonase
inactivation in Napoleon brine (pH = 3.0).

 

 

 

Temp. Time % Enzyme 1
°C sec Activity C/Co
o 31.0 .968
15 22.0 .687
68 30 14.5 .453
60 6.2 .194
75 4.2 .131
6 30.0 .908
15 20.5 .620
69 30 13.8 .417
45 7.4 .224
60 4.2 .127
75 3.0 .091
o 33.5 .993
5 27.8 .824
70 15 15.8 .468
30 7.2 .213
45 -3.8 .113
60 1.78 .053
0 26 .960
10 15.5 .572
71 20 8.0 .295
30 3.85 .142
40 2.12 .078
50 1.23 .045
5 16.00 .9976
72 15 5.80 .2795
30 1.65 .0795
45 0.68 .328
1C0 is: 68 °c = 32.033, 69 °c = 33.05, °c = 33.73
71 °c = 27.087, and 72 °c = 20.75.

79

Table A4.--Effect of pH on inactivation rate of polygalacturonase in
Napoleon brine at 69 °C.

 

 

 

Time Mean Diam. Standard % Enzyme 1
pH sec mm Deviation Activity,C C/Co
0 26.917 .1725 15.0 .956
15 25.917 .1725 7.8 .497
2.5 30 25.084 .1725 3.7 .236
45 24.084 .1725 1.9 .121
60 23.167 .218 0.8 .05100
6 27.834 .258 14.5 .824
15 27.34 .258 10.2 .579
3.0 30 26.75 .289 16.6 .375
45 25.92 .204 3.6 .204
60 25.17 .258 2.05 .116
75 24.67 .258 1.39 .079
5 27.67 .218 14.0 .841
15 27.25 .231 9.5 .571
3.5 30 26.40 .183 5.38 .323
45 25.834 .218 3.4 .204
60 24.917 .1725 1.8 .108
5 26.37 .194 10.0 .8299
15 25.5 .00 5.4 .448
4.0 30 25.0 .00 2.8 .232
45 23.5 .00 1.25 .104
60 22.5 .00 0.6 .0498
75 21.625 .1936 0.31 .026
1
pH : 2.5 3.0 3.5 4.0

Co : 15.686 17.60 16.6418 12.049

80

Table A5.--Effect of sugar concentration on polygalacturonase
inactivation in Napoleon brine at 69 °C (pH = 3.0).

 

 

 

Brix Time Mean Diam. Standard Enz. Act. 1
(6) (sec) (mm) Deviation (%) C/Co
6 27.83 .258 14.5 .8239
15 27.33 .258 10.2 .5795
9 30 26.75 .289 6.6 .3750
45 25.92 .204 3.6 .2045
60 25.17 .258 2.05 .11648
75 24.61 .258 1.39 .0789
5 28 .0 16.5 .8967
15 27.34 .218 10.2 .5543
12 30 26.84 .218 7.1 .3859
45 26.17 .218 4.3 .2337
60 25.42 .173 2.5 .1359
5 27.79 .237 20 .9257
15 27.29 .237 14 .6480
25 21.08 .172 12 .5554
14 30 26.75 .235 9.5 .4397
45 26.17 .218 6.1 .2823
55 25.84 .218 4.9 .2268
60 25.83 .218 4.8 .2222
75 25.34 .218 ' 3.3 .1527
1

Co is: for 9° = 17.59, 12° = 18.4, 14° = 21.605.

81

Table A6.--Effect of cherry variety on inactivation rate of poly-
galacturonase at 69 °C (pH = 3.0).

 

 

 

 

 

Variety Time Enzyme Standard C/Co Notes
(sec) Activity,C Deviation
(%)
w 4 24.00 .00 .9123 15510:
I 5 23.00 .1725 .8743 2.9068
N 9 21.00 .204 .7983
D 15 16.00 .218 .6082 CORR.COEFF.
S 30 10.0 .224 .3820 .99697
O 45 7.2 .224 .7737
R 60 4.55 .188 .1796
N 6 14.5 .258 .8239 E2193.
A 15 10.2 .258 .5795 3.4611
P 30 6.6 .289 .3750
O 45 3.6 .204 .2045 CORR.COEFF.
L 60 2.05 .258 .11648 .9984
E 75 1.39 .258 .17898
O
N
1

Co is: Windsor = 26.307, Napoleon = 17.60

Activity Percent

100

10

1.0

0.1

Fig. A1.

82

 

 

 

 

 

Holding Temperature
0 70 °C
A 72°C
0
‘ O
O
O
A a
A o
100 200 300 400 500

Time, sec.

Long time inactivation profile for poly-
galacturonase in Napoleon brine at pH = 3.0.

APPENDIX B

CUP PLATE METHOD OF ANALYAIS FOR

POLYGALACTURONASE ENZYMES

APPENDIX B
CUP PLATE METHOD OF ANALYSIS FOR

POLYGALACTURONASE ENZYMES

The following is the cup plate method for poly-

galacturonase enzyme assay according to the method of

Dingle and others (1953) as given by Beavers et a1. (1970).

follows:

1.

One thousand.milliliters of medium is prepared as

To 500 ml distilled water in a one-quart blender
jar, add 2.500 g of polygalacturonic acid.

While blending add 75 m1 of 0.1 N sodium hydroxide.
(Check pH, which should be 4.0 i 0.1.)

Add 2.5 g ammonium oxalate and 10.0 g potassium
acid phthalate. Continue to blend until completely
dissolved.

Transfer to a 1-1iter Erlenmeyer.

Blend 20.0 g Bacto Agar into 425 m1 of distilled
water and add 0.25 g thymol.

Transfer to a second l-liter Erlenmeyer.

Steam both flasks about 30 minutes or until dis-

solved. (Agar will become clear above 97 °C.)

83

84

8. Cool contents of both flasks to 50 °C; mix in a
2-liter flask.

9. Dispense to flat bottomed petri dishes, 25 ml per
dish.

Small stainless steel collars (8 mm dia.) may be
imbedded in the media to provide cups into which a few
drops of brine can be placed. The collars (1/4 inch long)
can be cut from stainless steel tubing.

With an eye drOpper or similar device, dispense
four or five drops of test sample into a cup. Incubate
overnight at 35 °C. At the end of the incubation period,
pour 6 N hydrochloric acid over the media in the plates.
Allow to stand one hour and observe. If polygalacturonase
is present, a clear zone will appear around the cup;
the diameter will vary depended on the concentration of the
enzyme.

Polygalacturonase activity is evaluated by measuring
the diameter of the clear zone, including the diameter of
the cup. Control samples of solutions of pectin enzymes in
water or brine should be included in any series of tests.
Solutions containing 1, 0.1, and 0.1 percent of pectin
enzyme will give clear zones of different diameters around
the cup. By comparing the sample of cherry brine under
test with the enzyme solutions, one can estimate the
degree of polygalacturonase activity in turns of concentra-

tion of the pectic enzyme used.

"‘WVIIII'IIIIIII'IIIITIES