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Date

This is to certifg that the

thesis entitled

Exooellular Pectolytic Enzymes Broduced
by
Molds Isolated from Black Raspberries

presented bg

Lawrence Sinclair White

has been accepted towards fulfillment

of the requirements for

L degree in ________Ba°t 91'1 01 03V and
Pub 1 10 He a1 th

9]?” VCW/

Major professor

June 2, 1952.

 

 

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alliadewmlnw 44“ "in": 4.4T. i. Indians A131?“ .5: . -.. ....r<: 1.2.3.5.“! a"! shaman)”, .41fllaflr33.i.u1

‘3 Yum...

EXOCW PBCTOLYTIC ENZYMES PRODUCED BY

M3113 ISOLATED FROM BLACK RASPBERRIIS

By

Durance Sinclair write

ATEESIS

Submitted to the School of Graduate Studies of Michigan
State College of Agriculture and Applied Science ‘
in partial fulfillment of the requirements
for the degree of

MASTER OF SC MOB

Department of Bacteriology and Public Health

1952

ti“
~T' ‘.

I.
II.

III.

Iv.

V.

CONTENTS

Acknowledgments
Introduction
Review of the Literature
Experimental Studies
A. lbteriale and Methods
19 Sources of_ mterials
2, Study of Methods
3. Methods Duployed
B. Studies of Enzyme Production
1? Experiments with Citrus and Apple Pectin
2? Experiments with Black Raspberry Juice.
3;“ Develcpmerrt of Molds on Whole Black Raspberries

General Discussion of Results

Bury
Bibliography

AC KNOWLEIXELENTS

The author Iishes to express his sincere appreciation to the
folloting staff members of Michigan State College: Dr. F. W.
Fabian, Professor, Department of Bacteriology and Public Health,
for suggesting this study and for constant advice and guidance
during the investigation; Dr. E. S. Beneke, Assistant Professor,
Department of Botany, for supplying the mold cultures used; Dr.
C. L. Bedfcrd, Associate Professor, Department of Horticulture,
and Mr. W. F. Robertson, Associate Professor, Department of Horti-
culture for their interest and suggestions at various times during
this study. Grateful acknowledgment is also due to Mr. M. A.
Benarde, graduate student, for appraising the optical rotation
method as a means of determining pectic degradation and to the
Michigan Fruit Canners Inc. Benton Harbor, Michigan, for genu‘ous-

ly supplying the black raspberries used in this work.

INTRODUCTION

In some instances, arbitrary mold count tolerances by Govern-
mental agencies make little or no allowance for the types of pro-
duce, weather conditions, or the genera of molds which may be
present.

In effect, while the Howard method doubtless has shown con-
siderable merit when applied to tomatoes, its widespread use for
other food products is difficult to justify unless certain basic
fundamentals are considered. Too frequently, the term 'nold'is
deemed synonymous with "rat“ without any evidence to show that the
product or products concerned are structurally or chemically de-
graded.

It is alleged that the Howard mold count standard for black
raspberries has been set unofficially by the Government at «EC-per-
cent positive fields. This is the same as that allowed for tomato
paste, catsup and puree. Black raspberries are essentially dif-
ferent from tomatoes and it is by no means possible to predict from
one fruit the behavior of another, even under given conditions.
Producers and packers of black raspberries have sustained serious
economic losses occasioned by seizure and rerouting of produce
especially when humid conditions have prevailed during the growing
season. This has occurred in Michigan which supplies about 80-per-
cent of the U. 8. black reapberry canned pack and a substantial
proportion of the frozen pack. No evidence has been offered thus

far to show that the berries are damaged in any way when minute

amounts of molds are present.

The present study was undertaken to determine the extent of
available information concerning the degradation of black raSp-
berries after picking and to investigate experimentally, the dif-
ferences, if any, which might exist in the pectolytic properties

of several important molds isolated from black raspberries.

REVIEW OF LITERATURE

It has long been believed that pectin occurs as calcium peo-
tate in the intercollular layers of plant tissues, and that this
calcium pectate serves as a cement between the adjacent cell walls.
Loconti and Kertesz (22) presented evidence that the firmer texture
which is given to tomatoes by treating them with calcium chloride
was due to the formation of calcium pectate. The mechanics of
firming apple slices with calcium has similarly been explained by
Baker (1).

Kertess (19) presented an extensive review of the literature
relative to the chemical and physical properties of pectic sub-
stances. He stated that although pectic substances may serve a
wide variety of unknown purposes in plants, the contribution of the
insoluble pectic constituents in the middle lamella of plants to
rigidity, is one of the most important known functions and that
softening and maceration of the tissues occur when the poetic sub-
stances are transformed. Pectin materials in a state of transition
can be found in cell sap and Kertesz suggested that this solubility
my be governed at least partly by the degree of demethylation.
This hypothesis was substantiated by' the work of Leinbach, Seegmiller

and Wilbur (21) which showed that dissolved pectinic substances of

a high degree of methylation were present in the cell sap of straw-
berries and raspberries.

When overripening of fruit or vegetables, excessive heating,
fungal or bacterial rotting occurs, it may be shown that cementing
pectic material between adjacent cell walls is removed. During the
ripening of apples, Carre (8) showed that the amount of pectic sub-
stances decreased and that the parenchymous cells became loosened
or completely detached from one another. Personius and Sharp (25)
demonstrated that during the cooking of potatoes, there is an
extensive decrease in cell wall adhesion by a weakening of the inter-
cellular substances. Fabian and Johnson (11) demonstrated the
development of enzymatic activity by Bacillus mesenterigus fuscus
which produced decomposition of pectic materials in the tissues of
cucumbers. Kertess (19) stated that the pectolytic enzymes produced
by microorganisms are responsible for most plant diseases related to
the destruction of pectic constituents. This same author also
suggested (18) based on the work of Robertson 3}; El. (30) that de-
polymerisation and hydrolysis of pectin as well as other saccharides
may be caused by hydrogen peroxide in the presence of ascorbic acid.
However, Phaff (27) indicated that hydrogen peroxide has not been
conclusively demonstrated in plant tissues.

In general, the transformations which pectin substances undergo,
aside from those noted during heating, have been attributed to the
activity of pectic enzymes.

Despite the complex macromolecules of pectin substances which
are thought to be ohainlike combinations of anhydrogalacturonic acid

units possessing carboxyl groups distinguishing them from the poly-

saccharides, there are only two known pectic enzymes, pectin-
methylesterase (P. 11., pectinesterase, pectase) and pectin-poly-
galacturonase (P. G., pectinase, pectolase) (19).

Poctin-methylesterase has been characterized as the enzyme
responsible for destruction of pectinic acid by hydrolysis of the
methyl ester groups in pectinic acid and pectin. Pectin-methyles-
terase is commonly found in the root, stems, leaves and ruit of
many higher plants and without making any claims to completeness
Kertes'z (19) listed eighteen sources of this enzyme. In plant
tissues, pectin-methylestera-se, according to Phaff (27), occurs
more or less free from pectin-pclygalacturcnase.

Kertess (19) made a general statement that the activity of
pectin-methylesterase of plant origin increases as the pH is ele-
vated from 4.0 to 7.0. Above pH 7.0, however, the situation is
confused and the addition of salt causes a drift in the pH optimum.
WColloch (24) showed that heat inactivation of pectin-methyles-
terase of plant origin occurred at 30° C.

Pectin-methylesterase, according to Phaff (27) always appeared
present in fungal or bacterial preparations when pectin-polygalao-
turonase was noted and Jansen and MacDonnel (16) found that little
glycosidio hydrolysis occurs without demethylation of the pectin in
commercial fungal pectinases (a group designation for an enzyme
complex composed of at least one or more pectic enzymes in addition
to pectin-pclygalacturonase). Fungal peotin-methylesterase from a
comercial pectinase was active in a salt free medium at pH 4.0 and
had a well defined optimum at about 4.5 - 5.0. With salts, the

activity of the enzyme was increased, especially near the pH optimum

-5-

but there was not an increase in the pH value at which the action
occurred.

Calesnick, Hills and Willaman (6) reported that complete
inactivation of fungal pectase occurred at pH 3.5 when extracts
were heated for 30 minutes at 62° C. Calesnick 912.1. (6) showed
that fungal pectase with which they had experimented, was quite
stable and that there was no loss in the activity of the enzyme
at pH 6.25 when it was stored for 2 weeks at 23° C.

Pectin-polygalacturonase has been considered responsible for
the destruction of pectinic acid by hydrolysis of the 1,4 glycosidic
linkages cf the polygalacturonic acid skeleton of pectic acid or
pectinic acids with the formation of polygalacturonic acids of
smller molecular size and of (mono) galacturonic acid. Kertesz
(19) listed germinating barley as the only proven source of this
enzyme in plants. However, many microorganisms have been reported
to produce pectinvpolygalacturonase and commercial preparations
which include this enzyme are widely used. Fish and Dustman (14)
found that fungal pectin-polygalacturonase had a pH optimum in the
neighborhood of 3.5 but other workers (17) have found it in the pH
range of Si". Among the reasons suggested by Kertesz (19) to explain
this wide variation, is that many workers used unpurified pectinase
preparations and that simultaneous action of the two enzymes pectin-
methylesterase and pectin-polygalacturcnase must have been common.
In addition, he explained that the deesterifying enzyme changing
pectinic acid to pectic acid might also have had a profound effect
on the pH optimum reported. Furthermore, the same author stated (19)

that the pH optimum for fungal pectin-pclygalacturonase under most

conditions is in the range of pH 3.5 - 4.0. Phaff (26) in studies with
Penicillin]: chrysoggnum proved that complete inactivation of the
pectin-polygalacturonase occurred at 80° C in ten minutes. '

Undoubtedly, one of the most important considerations in the
production of fungal pectic enzymes is the medium. Manon (23)
presented data which indicates that the nutrients present influence
to e considerable extent the precise behavior of the enzymes of any
particular fungus. He suggested that the enzyme is the same in ‘all
instances but that certain of its properties are greatly influenced
by the absorption of the substances from the medium. Fernando (13)
showed that the optimum activity of Botrytis cinerea- on potato
decoction media was at pH 6.5 but that substances which caused a
drift to the alkaline side, such as peptone, yielded very active
enzyme solutions while those which caused an acid pH drift (am-
monium chloride, nitrate and sulfate) produced good growth but
low enzymatic activity.

The literature concerning fresh raspberries is particularly
meager. Rendle (29) was concerned with the development of mushiness
of raspberries during their transportation to canning factories. He
felt that raspberries are, perhaps, the most difficult of all fruits
to keep intact and to transport or handle. He proved that changes
which took place following ripening occurred even faster after the
fruit had been picked. Although Rendle did not report the extrac-
tion of an enzyme in this work, he showed that changes in pectic
constituents were arrested when the berries were heated. He concluded
that the character and degee of enzymatic activity in raspberries is

considerably more marked than in other fruits. When he employed the

 

Ctrre and Haynes method (7) for an estimation of pectin as calcium
pectate, he showed that the pectin content of berries decreased from
0.92 to 0.34-percent when storage was for six days at 18 to 24° C.
In this study he also reported the pH value of canned raspberries
as 5.3.

Kertesz (19) noted that it is surprising that the percentage
of pectic constituents in strawberries does not decrease when this
fruit becomes overripe and he concluded that the pectic consti-
tuents of strawberries are more stable than those of raspberries.

Lee (20) presented the following data showing that the‘blaok
raspberry, Rubus occidentalis L. respireszat a consistently higher
rate than the red raspberry or the purple variety.

Milligrams-002 evolved per kg
fresh weight per hour at

Variety 60°F 40°F 32°F
Cumberland (black) 100.? 38.5 19.9
D. S. D.‘L 322 (purple) 87.5 36.9 25.1
Iathem (red) 82.2 30.7 17.5

Eb also reported that the approximate composition of red and black

raspberries was as follows:

Total Acid
Carbo- as
fitter Protein Fat Ash hydrate Fibre Sugars citric
% % % % % % % %
Red 83.4 1.1 0.6 0.47 14.4 2.8 7.2 1.54
3130]! 80e7 1.5 1.6 1e65 1506 305 7e9 1016

In general, berry fruits such as raSpberries, blackberries and

dewberries are subject to attack‘by the same molds (15). These include

Betgytis sp., Rhizopgs sp., Penicillium sp., Phyllostocinia sp.,
Garpgggna sp., Elsinoe veneta sp., and Cladosporium sp. Probably,
the most common.and widely spread rot of strawberries is Botrytis
and it has been a major concern of raspberry growers as well. It
develops and spreads throughout the whole fruit and causes the
berry to dry up and become tough and leathery. It was further
stated that Botrytis is a good example of a mold which.may be
overlooked in the early stages on raspberries. When the aerial
mycelium.of a berry is moistened, the mycelium.seems to disappear.
In referring to Botrytis as the cause of spoilage of stone fruit,
it was stated that the organism gains entrance to the fruit through
breaks in the skin but occasionally it has been found to penetrate
sound epidermis.

Dodge and Wilcox (9) reported that any delay beyond a few hours
which increases the time interval between picking and sale to a
consumer, affords an opportunity for the development of fruit rots
and.mclds affecting raspberries. These authors also added that
raspberries which.are soft from.being overripe or from.rainy
weather are quickly attacked by certain mold fungi which cause decay
and'that in hot or wet weather, picking may be required at least
every other day.

Fabiaan§_§g, (12) investigated the factors responsible for high
mold counts which were found to occur on'black raspberries which had
been grown in the State of Michigan. These workers found that no
tissue breakdown of the berries was noted, even when the mold count,
as determined by the Howard method, was 80-percent. Visible mold

was rarely found and when present it indicated that the berries had

 

 

not been freshly picked. Despite prompt picking and delivery to the
canning factories, mold counts by the Howard method during a humid
season sometimes reached 40-percent. As a result packers had to
drastically reduce the season's output of canned and frozen berries.

In the same study, it was also reported that the molds most
cannonly found in black raspberries were, in decreasing order of
incidence, Alternaria humicola, Cladosporium sp., Pullularia sp.,
Fusarium sp., Botrxtis cinerea and Penicillium sp.

Organoleptic tests on berries which showed a high mold count
failed to indicate that the quality of the fruit had been affected
in any my. Beneke (4) calculated on the basis of the 56-percent
positive Howard mold count that if lOO-percent positive fields had
been obtained, the volume of mold present would have amounted to
only 0.00162-percent of the total puree volume.

- Fabian gt 3.1. (12) suggested that the present Howard mold
count standard for black raspberries is unjustly severe and par-

ticularly so when berries are grown under hot and humid conditions.

STUDY OF MATERIALS

The work reported in this investigation was conducted during the
season when fresh fruit was unavailable. Therefore, it was important
that the berries resemble as much "as possible their original condition
and especially contain pectin which would serve as a carbon source
for microorganisms.

Initially, it was believed that frozen berries would be satis-
factory; but tests on berries which had been stored since the pre-

Vious season's pack showed that the pectin content of these berries.

as determined by their ability to form a gel, was very small or
negligible.

Canned black raspberries which were available on the local
market were packed in sugar sirup; and although tests showed that
they'were capable of showing good gelation, Pitman and Cruess (28)
observed in a series of experiments with Penicillium.glaucum that
when sugar was present in the medium, it was invariably utilized
before the pectin.was attacked. ‘Accordingly, it was decided not
to use berries packed in this manner.

The black raspberries which.were finally chosen for the studies
were obtained from a Michigan processor and had.been packed in.water
for use in bakeries and restaurants. .All cans had the same code
number and the berries appeared sound.

Sample portions of the berries from several cans were crushed
and tested using a Beclcnan glass electrode pH meter. L pH value
of 5.85 was noted for all samples. .Alcohol precipitation.tests

showed that a substantial amount of pectin.was present.

STUDY OF METHODS

Phaff and Jcslyn (27) list five methods for the measurement
of polygalacturonase activity: increase in reducing substances
.measured as aldoses; decrease in alcohol precipitate; decrease in
optical rotation; drOp in viscosity of the pectin solution; and
decrease in calcium pectate. For the purpose of this investigation
all of these methods had decided limitations, many of which were
similar to those noted by Bell 21; _a_._l_. (2) during a study of methods
for‘werk on cucumbers. .All methods with the exception of the drop

in.viscosity of pectin solution depend on the development of a

-11-

strong glycosidic hydrolysis which might require several hours and
the use of a large amount of substrate.

Measurement of changes in. optical rotation was employed by
Ehrlich (10) in a study of enzymatic activity which occurred in
pectin solutions. It was stated (19) that the specific rotation of
pectinic and pectic acid solutions is constant in the pH range of
3 to 6.5 and that the optical rotation decreases during enzymic
hydrolysis.

Trial experiments were conducted using a Schmidt and Hench
polarimeter and a sodium light source, but it was observed that this
method 1nd several disadvantages and was unsuitable for the present
study. It was necessary to clarify the samples to be tested and
readings, when obtained, were of insufficient accuracy to show cor-
relation. Probably, this was due to the concentration of the pectin
solutions, which approximated 1-percent, used in this work. Another
obvious difficulty with this method, had it been used, would have
been the influence of arabans on observed readings (19). The arabans
which often accompanies pectic substances in plants is strongly
levorotatory while the pectinic substances are dextrorotatory and even
the presence of minute amounts of the arabans has a considerable effect

on the optical rotation of pectic substances.

METHODS EMPLOYED
The molds which had been most prevalent on black raspberries
during the 1950 season were obtained. These were Alternaria humi-
c_ol_;, Cladosporium sp., Pullularia sp., Fusarium sp., Botrfiis

Oinerea and Penicillium‘sp. Subcultures were made periodically during

 

the study. Potato dextrose agar (Difco), pH 5.6, was used as a solid

medium unless otherwise specified.

As required for inoculations, the molds were grown on large
slants prepared by the addition of 200 m1 of agar to one-liter
dilution bottles. After 2 weeks incubation at 22° to 25° c, 50 m1
of sterile distilled water was added to each bottle and spore sus-
pensions prepared by gently rubbing the agar surfaces with an inocu-
lating needle. The suspensions were removed, placed in screw capped
bottles and thoroughly shaken.

I. l-percent solution of special pectin.t in a sodium citrate-citric
acid buffer solution at pH 4.0 was prepared for demonstrating enzymatic
activity by the loss in viscosity method. The buffer solution was
prepared according to the method given by Bell 9311‘. (2) and con-
sisted of weighing out 2 gm each of sodium citrate and citric acid
and dissolving in 100 m1 of distilled water. The solution was heated
over a Bunsen burner until the temperature reached 60°C and then
placed in a Waring blendor. The pectin then was added slowly in a
sprinkling manner to the liquid which was simultaneously agitated
with a glass rod. To insure uniformity, the suspension was stirred
in the blendor for a period of 1 minute and then was passed through
two thicknesses of cheesecloth.) The solution was allowed to stand for
several hours before use to offset any increase in apparent viscosity
(19).

Changes in the viscosity of test pectin solutions have been

 

e .
Designated 447-U-7. Obtained from the California Fruit Growers Ex-

change. Bhnufacturer's specifications showed the following percentage
analysis: moisture, 7.7; ash 1.2; methoxyl 10.6; galacturonic acid

87e3e

widely used to measure pectolytic enzyme activity. It was reported
by Jansen and MaoDonnell (16) that a 50-percent change in the vis-
cosity of a l-percent solution occurred with only a 2-percent breakage
of the glyoosidic bonds. Phaff (26) used this method in studies with
Mllium chrysogenum, Bell 33; g._l_. (2) in determining softening of
pickles and Bell (3) in studying cucumbers. Kertesz (19) pointed out
that the decrease in viscosity is a very sensitive indicator of even
traces of pectin-polygalacturonase action. This author further
stated that it is unlikely that at the pH of the reaction mixtures,
demethorylation would have any significant effect on the viscosity as
compared to the viscosity drop produced by pectin-polygalacturonase.

It is interesting to note that a l—percent hydrolysis of the
polygalacturonic chains causes approximately a SIS-percent reduction
in the viscosity, while a l-percent demethoxylation causes a slight
clunge. Therefore, viscosity is not governed by the methylester
content but rather by the average molecular size of the pectinic acid.

Bell 9}; 9.3;. (2) added heated and unheated extracts to S-percent
test pectin solutions and incubated the mixtures at 30°C for 6 days.
Relative dropping tines were then determined by allowing the solutions
to flow through a 20 m1 pipette.

Phaff (26) added heated and unheated extracts to 1.5-percent test
pectin solutions and then incubated the mixtures at 25°C for 30
minutes. Dropping times were measured employing Ostwald viscometers.

The procedure used in the present investigation to measure the
loss in viscosity was essentially a combination of the methods em-
ployed by Phaff and by Bell. In each instance, to determine pecto-

lytic activity 2 m1 of the unheated extract was mixed with 20 m1 of

1".

- 14 -

the test pectin solution. This mixture was incubated at 30°C in a
thermostatically controlled water bath for 45 minutes. At the end of
this time, 5 ml amounts were placed in each of 2 Ostwald viscometers.
These were then placed in a water bath and dropping times measured.
Each time a determination.was made, the initial viscosity of the
pectin solutions and a heated extract from the same source served as
a centre. To obtain a heated extract, a sample was placed in a water
bath and kept at 80°C for 10 minutes. The inactivated extract was
then added to the pectin solution and the sane procedure followed as
above. Decrease in viscosity was expressed as a percentage of the
viscosity of the test pectin solution and the heated extract
according to the formula Ali—2 x 100. L represents the viscosity of
the heated enzyme mixture and B the viscosity of unheated enzyme
mixture.

The incubation period was longer than the 30 minutes employed
by Phaff (26) to permit time for adequate cleaning of the Ostwald
viscometer. Acetone, alcohol and hot water rinses as were used by
Bell g§_gg, (2) did not prove satisfactory for cleaning the Ostwald
viscometers and gels usually formed which could not be removed from
the small capillaries. ‘A satisfactory procedure, developed during
the early part of this work, consisted of flushing hot chromato-
sulfuric acid solution through the Ostwald viscometers, followed by
four hot water rinses and finally by a distilled water rinse. The
viscometers were then dried by filtered air under pressure. The

same two Ostwald visoometers were used in all tests reported.

WERIIEIITS WITH CITRUS Hm APPLE PBCTIK

- 15 -

Samples of high grade citrus* and apple“I pectin were obtained.
Two media were prepared by the addition of these pectins in a l-per-
cent concentration to a.mineral base described by Phaff (26). This
substrate consisted of tap water, 0.1-percent ammonium nitrate, 0.1-
percent dihydrogen phosphate, and 0.05-percent magnesium sulfate.
The ingredients appeared likely to support the growth of the molds
with pectin as the sole source of carbon since Phaff obtained luxur-
iant growth of P. chgysogenum in this medium. Each.preparation.was
thoroughly mixed and filtered through cheesecloth to obtain even
distribution of the pectin.

The pH value of the medium containing apple pectin was 5.4.

The pH value of the medium containing citrus pectin was 6.3.

The media was dispersed in 1000 ml amounts into 2000 ml
Erlenmeyer flasks which were then plugged with cotton and sterilized.
Mold suspensions of thernaria humicola, Cladosporium.sp., Pullularia

sp., Fusarium.sp., Botrytis cinerea and Penicillium sp. were prepared

 

as described under methods. Five ml of spores representing each genus
was inoculated into an individual flask containing apple pectin
medium. A.duplicate set of flasks containing citrus pectin were also
inoculated. Incubation was at 22° to 25° c for 7 days. The flasks
were then carefully tilted and samples of the medium.in each instance
were removed aseptically for testing.

The molds showed a pronounced difference in their ability to

produce exocellular pectolytic enzymes (Tables 1 and 2).

 

t
Citrus pectin - 178 grade, R. S. California Fruit Growers Exchange,

Ontario, California.
es

Apple pectin - 170 grade, uncut, Speas and Co., Philadelphia, Pa.

Fusarium sp. and Alternaria humicola showed the greatest pro-
duction of pectolytic enzymes. Exocellular enzymatic activity pro-
duced by Penicilium sp. was appreciable in the medium containing
citrus but not in apple pectin. Botrfiis cinerea, in the medium
containing citrus pectin, demonstrated pectolytic activity similar
to that of polygalacturonase while in the medium containing apple
pectin, an increase in the viscosity of test pectin solutions was
noted. This increase in viscosity was probably due to methylesterase
production.

The variation noted in the development of the enzymes in the
two media could probably be attributed to the differences inthe

pH values of the media.

EXPERIMENTS WITH BLACK RASPBERRY JUICE

Juice was expressed from the black raspberries with a screw type
fruit press. The juice obtained was thoroughly mixed and 1000 ml
quantities were distributed into 3000 m1 Erlenmeyer flasks. Large
flasks were chosen in order to provide a broad surface area for
growth of the molds. The flasks were plugged with cotton and steril-
ised. Mold suspensions of Alternaria humicola, gadosporium sp.,
Pullularia sp., Fusarium sp., Botrjgtis cinerea and Penicillium sp.
were prepared as already described.

Five ml of mold suspension prepared from each genus was inoc-
ulated into an individual flask of juice. Incubation was at 22° to
25° c for 7 days. Unless otherwise specified, determinations for
the degree of pectolytic activity were conducted following 3 and 6

day incubation periods of all cultures of molds with the exception

 

 

 

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of Ponicillium sp. More tests were made on this species in view of
the interesting results obtained by Phaff (26) for P. chrjsogenum.

Tables 3, 4 and 5 illustrate the pectolytic activity of the
molds grown on the black raspberry juice. A comparison of these data
shows that the greates pectolytic activity was produced by Penicillium
sp. followed by Pullularia sp. and Fusarium sp. 1 progressive de-
crease in the viscosity of test pectin solutions was produced by
Penicillium sp. (Table 5). While a rather pronounced variation was
noted between some readings, it is likely that this were caused by
the use of a new solution of test pectin near the end of the ex-
periment. Botrytis cinerea produced an enzyme which consistently
increased the viscosity of test pectin solutions.

One of the most interesting observations noted was the lack of
production of pectolytic enzymes by Alternaria humicola and £319-
sporium sp.

Alternaria humicola did not develop visible growth on the surface
of the black raspberry juice during these tests. Observations made
after 10 days also failed to show surface growth. A. precipitate in
the juice was noted, however, and the deveIOpment of sub-surface
growth was confirmed by microsc0pic examination.

The degree of enzymatic activity noted for the molds in black
raspberry juice was not related to the amount of visible mold which
grew on the surface of the medium.

In some instances, readings of dropping times as determined by
viscometer 1 did not correlate with viscometer 2. as well as had been
noted in previous tests. In cases of doubtful readings, tests were

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raspberries which remained in suspension and interfered with the

viscosity readings.

DEVELOPMENT OF MOLD ON’WHOLE BLACKLRASPBERRIES

It seemed desirable to provide at least some indication of the
relative ability of molds to develop quickly on the surface of black
raspberries, although it could not be assumed that mold behavior on
canned berries would be the same as on.fresh berries.

Thirty gm amounts of the water pack berries were aseptically
removed from.the cans and placed in sterile 200 ml Erlenmeyer flasks.
Two ml of the spore suspension prepared from.each of the six mold
cultures was dropped onto the surface of the berries contained in
the six flasks respectively. The berries were then incubated at
220 to 25° C and observed intermittently for the development of
visible mold growth.

In 18 hours, moderate growth of Penicillium.sp. was noted and
barely visible growth of Botrytis cinerea and Cladosporium. At
the end of 24 hours, Fusarium, Alternaris and Pullularia were faintly
visible. By 36 hours all molds had overgrown the exposed surfaces of

the berries.

DISCUSSION
Alternaria humicola and Cladosporium sp. under the conditions
of the present investigation showed a lack of ability to produce
pectolytic enzymes in'black raspberry juice. This is particularly
interesting in view of the findings of Beneke (5) that these two molds
had accounted for approximately 70-percent of all molds detected in

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-24-

Pullularia sp., Fusarium sp., Botrytis cinerea and Penicillium sp.
all showed active pectolytic enzyme production in.black raspberry
juice. When.Alternaria humicola and Cladosporium.sp. were cultured
in.mineral media, both secreted pectolytic enzymes. 0f the 6 molds
investigated, the degree of activity exhibited by Alternaria humicola
in.mineral medium.with pectin as the sole source of carbon was
exceeded only by Fusarium sp.

Botrytis cinerea, which is known to have a pronounced degenera-
tive effect on some fruits, showed evidence of a pectinqmethyles-
terase type of action.when grown in mineral medium containing citrus
pectin. In black raspberry juice and in mineral medium.containing

apple pectin, a pectin-polygalacturonase type of enzymatic action was
noted for the same organism.

This investigation has shown that production of pectolytic
enzymes by molds is not related to the amount of visible mold. It
seems obvious that in the grading of a food product, the effect of
fungal activity on the food itself should be a primary consideration
rather than the mere demonstration that fungi are present. The
Howard.method.makes no allowance for the degradative properties of
fungi as a means of evaluating the soundness and sanitary condition
of a food product. The limitations of the Howard method become even
more apparent when it is realized how little volume actually is oc-
cupied.by molds in a foodstuff, even.when up to a loo-percent mold
count is observed.

While it is recognized that any fruit or vegetable benefits from
expeditious handling, it is unreasonable to expect that growers of

black raspberries, for example, should be put in the economically

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- 25 -

disadvantageous position whereby they must harvest within an extremely
restricted period of hours, in.the hope that their produce will meet

the present Howard.mold count standard.

SUMMARY

1. .Alternaria humicola, Cladosporium sp., Pullularia sp.,
Fusarium sp., Botrytis cinerea and Penicillium sp., which had been
isolated from'black raspberries, differed markedly in their ability
to produce pectolytic enzymes when grown in'black raspberry juice
under given conditions.

2. Alternaria humicola and Cladosporium sp. showed a lack of
ability to produce pectolytic enzymes when grown in black raspberry
juice.

3. Enzyme production was most evident in black raspberry juice
by Penicillium.sp., Pullularia sp. and Fusarium sp.

4. Botrytis cinerea when grown in black raspberry juice secreted
an enzyme which increased the viscosity of test pectin solutions.

5. .dll of the molds studied except Alternaria humicola produced
visible growth on the surface of black raspberry juice under the
conditions of the test.

6. Fusarium sp., and Alternaria humicola showed the greatest
production of pectolytic activity in apple and citrus pectin media.

7. On the surface of whole, water-packed berries, Penicillium sp.
developed macroscopic growth most quickly, followed in order by
Botrytis cinerea, Cladosporium sp., Fusarium sp., Alternaria humicola
and Pullularia sp.

8. Factors relating to Howard mold count standards were reviewed

 

 

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and evidence was presented to show that the present mold count

standard for black raspberries is unreasonable.

LITERATURE CITED

1. Baker, G. L. Explanation of the role of calcium salts in the
firming of apple slices. Fruit Products J. and Am. Food Mfg., 3, 197-
198. 1947.

2. Bell, T. A., Etchells, J. L. and Jones, I. D. Softening of
commercial cucumber salt-stock in relation to polygalacturonase activity.
Food Tech., 4 (4), 157-163. 1950.

3. Bell, T. A, Pectolytic enzyme activity in various parts of
the cucumber plant and fruit. Botanical Gaz. 113 (2), 216-221. 1951.

4. Beneke, E. S. The volume occupied by fungus hyphae in rasp-
berry fruits. Mich. Agr. Exp. Sta. Quat. Bull., 53 (2), 124-126. 1950.

5. Beneke, E. S. Unpublished manuscript. 1950.

6. Calesnick, E. J., Hills, C. H. and Willaman, J. J. Properties
of a commercial fungal pectase preparation. Arch. Biochem. 29, (2),
432-440. 1950.

7. Carre, M. H.,and Haynes, D. The estimation of pectin as cal-
cium pectate and the application of this method to the determination
of the soluble pectin in apples. Biochem J.,16, 60-69. 1922.

8. Carre, M. H; An investigation of the changes which occur in
the pectic constituents of stored fruit. Biochem J., 16, 705-712. 1922.

9. Dodge, B. 0., and Wilcox, R. B. Diseases of raspberries and
blackberries, U. S. D..A., Whehington F. Bull. 1488. 1941.

10. Ehrlich, F. Pectolase. Enzymologia 3, 185-99. 1937.

(Obtained from C..A. 8565)

 

-27-

ll. Fabian, F. W., and Johnson, E. A. Experimental work on cucum-
ber fermentation I Zymological studies of the cause of soft cucumbers.
Tech. 8111., 9-16, Mich. Agr. Exp. Sta. 1938.

12. Fabian, F. We, Beneke, E. 3., Rickson, F. J., and Floate R.
lint is a reasonable mold count for black raspberries in the great lakes
region. Canner, May 5, 1951.

1:. Fernando, 11., Ann. Botany (N._s.) 1.727. 1937.

14. Fish, v. 3., and Dustman, R. s. The enzyme activities of Pectinol
A on pectin and other substances. J. Am. Chem. Soc... 67, 1155-1045. 1945.

15. Food and Mg Administration, Microanalysis of food and drug prod-

ucts. U. S. Governnent Printing Office, Washington. 171 pp. 1951.

 

16. Jansen, E. F. , and MacDonnel, L. R. Influence of methoxyl content "
of pectic substances on the action of polygalactln'onase. Arch. Biochsmc,
8, 1, 97-99. 1945.

17. Joslyn, M. A., and Phaff, H. J. Recent advances in the chemistry
of pectic substance. Wallerstein Lab. Comm. 1, (29), 39-56. 1947.

18. Kortesz, z. I. A possible non-enzymatic mechanism-cf changes

occurring in the pectic substances and other polysaccharides in living
plants. Plant Phy. 18 (2), 308-309. 1943.

19. Kertesz, Z. I. The pectic substances, ed. 1, Interscience, New
York, 1951, 628 pp.

20. Lee, F. B. in the Chemistry and Technology of Food Products, ed.
by Jacobs, M. B., Interscience Publishers, New York, (2nd ed. 1951).

21. leinbach, L. 3., seepmiller, c. G., md Wilbur, J. 3. Conposi-
tion of red raspberries, including pectin characterization. Food tech.,

5 (2), 51-54. 1951.
22. Loconti, J. D., and Kertesz, Z. I. Identification of calcium

t1

 

-28ee

pectate as the tissue-firming compound formed by treatment of tomatoes
with calcium chloride. Food. Res., 6 (5), 499-508. 1940.

23.4 Manon, x. P., v. Ann. Botany, 18?, 134. 1948.

24.’ McColloch, R. J., and Kertesz, z. I. Pectic enzymes VIII. A
comparison of fungal pectin-methylesterase with that of higher plants,
especially tomatoes. Aroh. Biochem., 19 (2), 217-229. 1947.

26. Personius, C. J., and Sharp, P. F. Adhesion of potato-tissue
cells as influenced by pectic solvents and precipitants. Food Res., 4,
229-306. 1939.

26. M, R. J. The production of exocellular pectic enzymes by
Penicillium chrysogenum 1. 0n the formation and adaptive mture of
polygalacturonase and pectinesterase. Arch. Biochem., 13 (1) , 67-81. 1946.

27. Phaff, H. J., and Jcslyn, M. L. A: newer knowledge of pectic
enzymes. Wallerstein Lab. Com. X (30), 133-148. 1947.

28. Pitman, G. 1., and Cruess, W. V. The hydrolysis of pectin by
va'ious microorganisms. Ind. Eng. Chem., 21, 1292-95. 1929.

29. Rendle, T. Observations on changes in raspberries after picking.
malyst, 58, 73. 1933.

30. Robertson, W. van 3., Ropes, M. W., and Bauer, W. Degradations
of mcins and polysaccharides by ascorbic acid and hydrogen peroxide.

Biochem. J., 35, 903-908. 1941.

 

 

 

 

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