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thesis entitled

Peotolytlo Enzymes of Garlic

presented by

Robert Walt of M1 sekaw

has been accepted towards fulfillment
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LLdegree inwagy Ind
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Major professor

June 3, 1952

 

 

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DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

PECTOLYTIC ENZYMES OF GARLIC

BY

.Robert Walter gieekow

A THESIS

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 SCIENCE
Department of Bacteriology and Public Health

Year 1952

1 m 122: " :3

/0' IV“ 5“ {'3‘}

TABLE OF CONTENTS

INTRODUCTION . . . . . . . . . . . . o . . . . . .
PRESENT INVESTIGATION. . . . . . . . . . . . . . .
LITERATURE REVIEW. . . . . . . . . . . . . . . . .
EXPERIMENTAL METHODS . . . . . . e . . . . . . . .
Preparation of Material . . . . . . . . . . . .
Qualitative Test for Pectinesterase . . . . . .
Quantitative Test for Pectinesterase. . e . . .

Effect of Various Concentrations of Salt on the
Activity of Garlic Pectinesterase . . . e e .

Effect of pH on Garlic Pectinesterase Activity.
Heat Inactivation of Garlic Pectinesterase. . o

Pectinesterase Activity of Dehydrated Powdered
Garlic and Dehydrated Garlic Flakes . . . . .

Test for a Polygalacturonase-like Enzyme in Garlic.

Decomposed Garlic as a Source of a Polygalacturonase-

like Enzyme e e e o e o e e e e e e e e e o 0
DISCUSSION 0 e o e e e e e o e e e e e e o e e o 0
SUMMARY 0 e e o o o o o e e o e o o e e e e o o e 0

LITERATURE CITED 0 o o e o o o o o e e o e e e e e

Page

11
11
lLI
15

21
22

23

2S
27

29
35
38
39

ACKNOWLEDGEMENT

The writer wishes to eXpress his sincere appreciation
to Dr. F. W. Fabian, Professor of Bacteriology and Public
Health, for his constant interest and encouragement, and
without whose help this thesis would not have been possible.
The writer also wishes to thank Dr. E. S. Beneke, Assistant
Professor of Botany and Plant Pathology, for his assistance
in identifying the molds. My thanks are also extended to
San Rosen and Bob Wheaton for their helpful assistance,
and Roland Fulde for his fine photography, and finally to
the Water Department of Flint, Michigan, for kindly per-
mitting me the use of their laboratory facilities in

carrying out certain phases of this work.

INTRODUCTION

Fabian and Wenzel (l9h5) investigated the influence
of garlic on the softening of genuine Kosher dill pickles.
In their work they used fresh, fresh moldy, fresh chOpped,
dehydrated, and sterilized garlic. Garlic vinegar was
also used. The garlic used was added in the normal amount
and in from 5 to 10 times the normal amount of garlic.
Their results showed that the lots of pickles which con-
tained no garlic, sterilized garlic, or dehydrated garlic
kept the best and showed the least amount of softening.
They found the highest percentage of spoilage and the
greatest rate of softening in the lots of pickles to which
10 times the normal amount of fresh garlic cloves had been
added, and also in those lots which contained garlic known
to be heavily contaminated with molds and bacteria.

It was significant in their investigation that those
lots of pickles which contained garlic and toluene to
prevent microbial growth became slippery after 6 weeks,
while another lot that contained toluene but no garlic

showed no softening at all.

PRESENT INVESTIGATION

Due to the fact that garlic in above normal amounts
in.Kosher dills and also garlic in pickles free from
microbial growth caused softening, it was decided to in-
vestigate the possibility that:

Normal sound garlic may contain pectolytic enzymes E23 33,
and to study the role of molds found on garlic in softening
Kosher dill pickles.

REVIEW OF LITERATURE

The commercial preparation of overnight genuine Kosher
dill pickles has been described by Schucart (l9u2) in the
following manner: "Cucumbers, usually not exceeding 1,000
size, are washed and packed in barrels with several layers
of dill weed, as in packing genuine dill pickles. One to
1% pounds of garlic and mixed spices are added; and the
stock covered with a salt brine of 22 degree to 25 degree
salometer. In some cases a quart of 100 grain vinegar is
added. The barrels are closed and sent to cold storage
where deepite the cold temperature, 36 to 38 degrees
Fahrenheit, curing at a slow rate continues, and if kept
too long (6 to 9 months) spoilage may be heavy. After the
intake season is past, barrels are withdrawn from storage
and delivered to the trade."

Greater losses due to softening occur in genuine dill
pickles than in salt stock. Fabian and Johnson (1938)
attribute this to the lower salt concentration of the
genuine dills. In some years as high as 50 percent of the
genuine dill pickles packed by certain salters have Spoiled.
Spoilage not only occurs in the fermented cucumbers but
also occurs in the finished product after it has been
bottled and marketed.

The Spoilage of pickles is progressive (Fabian and

Johnson (1938) and Fabian, Bryan, and Etchells (1932).
The first indication of Spoilage appears on the skin of the
pickles and is known as a "slip". Because of Spoilage, the
skin of the pickle is slippery and easily removed. Pro-
gressively, the deeper cells of the pickle become involved
causing the whole pickle to become soft. Cucumbers in
which spoilage has progressed to this stage are known as
"mushy" cucumbers. .

Fabian, Bryan, and Etchells (1932) studied the morpho-
logical changes from normally cured pickles to slippery
and very soft pickles. A microscOpic study showed that
normally cured pickles showed decided plasmolysis but the
cells were intact and appeared normal. Slippery pickles
when examined showed that the pectic material in the lamella
of the epidermal and parenchymatous cells was no longer
evident. Pickles in the advanced stage of decomposition
(mushy pickles) showed marked morphological differences.
Practically all the cell walls of the epidermal and paren-
chymatous cells had disappeared.

Fabian and Johnson (1932) isolated an organism, Bacillus
mesentericus fuscus which was capable of causing "slips"
in 6 to 12 hours and "mushy" pickles in 12 to 2k hours. By
using a stain, ruthenium red, the complete absence of the
pectic materials was noted. Sections of pickles that had
been extracted with.ammonium oxalate to remove the pectic

substances also showed absence of pectic materials. As

their chemical analysis showed the same amount of pectic
substances present in normal and spoiled pickles, they
conclude that the bacterial enzyme produced by Bacillus

mesentericus fuscgg is a protOpectinase and possibly a

 

pectase, but not a pectinase. They add, however, that this
does not preclude the possibility of a pectinase being
formed if given sufficient time.

They found the bacterial enzymes dissolving pectic
substances in pickles was inhibited by 2 percent salt and
1.1 percent acetic acid or 0.7 percent lactic acid. It
was also found that acids and heat were found to increase
the susceptibility of the pickles to softening, either
immediately as by cooking, or over a long period of time
with a weak acid.

Fabian and Faville (1949) isolated and identified a
mold, Oospora lactis which was the cause of two cases of
pickle spoilage. In one case the pickles had been allowed
to freshen too long allowing the organism to initiate growth,
while in the other case the mold was isolated from dirty
barrels which had been used to pack processed dills. For
enzyme production the molds were grown in dextrose broth
for 7 days. Freshened salt stock was then added to the
broth and layered with toluene to prevent microbial growth.
The molds isolated from both samples were able to produce
slippery pickles in 6 to 8 hours and "cheesy" pickles in
10 to 12 hours.

Jones (1909) studied the enzyme pectinase produced by

Bacillus carotovorus and certain other soft rot organisms.
Various experiments were undertaken by him to determine the
cultural conditions necessary for Optimum enzyme formation
having in mind the conclusions of previous investigators
that enzyme production in certain cases is a starvation
phenomenon. On the contrary, he noted that there seems to
be a perfect correlation between the rate and the vigor of
the growth of the organism and the amount of enzyme develOp-
ed. He also studied the effect of heat, acids, alkalies,
and long storing of the enzyme. The enzyme produced by

B. carotovorus was capable of softening carrots very quickly

 

by dissolving the middle lamella leaving the cells free.
Pittman and Cruess (1929) studied the hydrolysis of
pectin by various micrOOrganisms. Their method consisted
of growing the different organisms in apple juice to which
one percent of commercial powdered apple pectin was added.
The bottles were stored for nearly four months and at the
end of such time were tested for pectin content, viscosity,
and jellying power. One phase of the experiment was
designed to determine the protective effect of dextrose on

hydrolysis of pectin in solution by Penicillium glaucum.

 

Their results indicate that as long as such a sugar is
present, P, glaucum will not attack the pectins as rapidly

as it will in the absence of sugar; and that when the supply
of sugar is exhausted, the rate of pectin hydrolysis material-
ly increased. Of the microorganisms tested, two molds, P.

glaucum and a Pythium Sp. exerted the greatest hydrolytic

action. The former gave a more rapid hydrolysis of pectin
in media of pH 6 and pH 5 than in pH 3.

Pectin is described as the viscous colloidal sub-
stance extracted from plant tissue. It belongs to the
group of substances known as hemi-celluloses found through-
out the cell walls of plant life. Botanically, the pectins
are identified largely with the middle lamella of the cell
walls. Softening action of pickles has been considered to
be due to the result of action by pectin splitting enzymes
on the pectin composing the middle lamella of cucumber
tissue.

Phaff and Joslyn (19h?) list two principle types of
pectic enzymes: pectinesterase (syn. pectase, pectin-meth-
oxylase, pectin.methylesterase), which catalyzes the de-
esterification of pectin by removal of the methoxyl groups,
and polygalacturonase (syn. polygalacturonidase, pectinase,
pectolase, pectin polygalacturonase), which catalyzes the
glycosidic hydrolysis of pectin or pectic acid.

In regards to protOpectinase which Davison and Willa-
man (1927) included in their three categories of pectic
enzymes, which include the two Just mentioned, viz. pectin-
ase and pectase, Phaff and Joslyn state: "Pectin has never
been prepared as a product of the hydrolysis of protOpectin
by protOpectinase, probably owing to the presence of PG
(polygalacturonase) and PE (pectinesterase), which decompose
soluble pectin as formed. More critical work is needed,

however, to prove with certainty the existence or non-

existence of such an enzyme."

ProtOpectinase as defined by Davison and Willaman (1927)
is that enzyme which converts protOpectin of the cell wall
into soluble pectin and also attacks the intercellular sub-
stance with resultant maceration of the tissue.

Recently Kertesz (1951) states that the complex macro-
molecules of pectic substances would seem to offer a number
of possibilities for enzyme action, and that in Spite of
this there are only two enzymes which are definitely known;
pectin methylesterase, and pectin polygalacturonase. He
further adds that a protOpectinase might also exist, but
that so far this enzyme has been demonstrated only by its
action on plant tissues and a chemical definition of the
reaction is impossible until the nature of protOpectin
itself is clarified. As to the recently discovered pectic
acid depolymerase in tomatoes, Kertesz (l9h8) is in doubt
that it is an enzyme in the active sense of the word; but
it appears now that the loss of pectinic substances in
processed tomato products occurs as a result of the
cOOperative action of tomato pectinesterase and the pectic
acid depolymerase.

Jansen and MacDonnel (19h5) studied the influence of
the methoxyl content of pectic substances on the action of
polygalacturonase. In their work they freed the glycosidase
from pectinesterase by treatment with acid. The action of

the glycosidase was then compared with such substrates as

pectin, alkali-prepared and enzyme prepared pectinic and
pectic acids, and methyl glycoside of polygalacturonic
methyl ester. They found that de-esterification must
occur before polygalacturonase can act on pectic substances.
As Etchells and Bell (1951) state in their paper,
"considerable importance is then placed on the part pectin-
esterase may play in the destruction of the pectin of
cucumbers in relation to salt stock softening. The presence
of the esterase alone would not necessarily be a causative
agent, but together with the glycosidase enzyme system,
softening of salt stock could readily take place."
Calesnick, Hills, and Willaman (1950) studied the
prOperties of a commercial fungal pectase preparation.
They found the fungal pectase differs from the higher
plant pectases in pH relationship, in its response to
mono- and divalent cations, and in its thermal behavior.
The fungal pectase had activity from pH 2 to 6.5, whereas
the pectases of tomatoes, alfalfa, orange and tobacco are
inactive below pH h. Optimum activity was reported to be
at pH 5. Optimum salt concentration varied with pH, with
the pectase being more sensitive to calcium than to sodium
ions. The fungal pectase is also more sensitive to tempera-
ture than tomato pectase with complete inactivation at
62° C, whereas, tomato pectase is only 50 percent inacti-
vated at 62° C.
Pectinesterase occurs in the roots, leaves, and fruits

of higher plants and is also produced by a number of micro-

10

organisms. In higher plants it is more or less free of
polygalacturonase according to Phaff and Joslyn (1947).

Kertesz (1951) states that it is doubtful that poly-
galacturonase occurs in higher plants along with pectin-
esterase. The enzyme pectinesterase occurs in such diverse
plants as alfalfa, lilac leaves, molds, malt, tobacco stems
and leaves, and the fruits of cherry, tomato, and eggplant.
It is not abundant in most plants, however. Some of the
better sources are alfalfa, the citrus fruits flavedo and
albedo, tobacco stems and leaves, tomatoes, white clover,
and pea vines.

Garlic, to our knowledge, has not been mentioned in

the literature as a source of pectolytic enzymes.

EXPERIMENTAL METHODS
Preparation of Material

There are several methods used for pectinesterase
determination. Kertesz (1951) lists the following:

1) determining the change in ester content, 2) determining
the increase in free carboxyl groups, and 3) estimating
the amount of methanol liberated.

Our determination was based on the increase in free
carboxyl groups, by titrating with 0.1 or 0.5 N NaOH. The
procedure followed closely the work of Hills and Mottern
(19h?) and Bell and Etchells (1951).

Figure 1 shows the apparatus used in determining the
pectinesterase in garlic. The reaction was carried out
in a water bath at a constant 30° C temperature. The water
was heated with two heating elements thermostatically
controlled and kept in agitation by means of a stirrer
attached to an air driven motor. The reaction mixture
preper, contained in a 600 ml beaker, was also agitated.
The temperature in the beakerVIas also maintained at
30° i 0.50 C. The pH of the reaction.mixture was taken
with a Beckman pH meter equipped with extension leads.

A 10 ml microburette was used to add 0.1 or 0.5 N NaOH as
needed.

The enzyme extract was prepared in the following

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manner: Clusters of garlic were broken into individual
cloves which were examined very carefully for any signs

of microbial decomposition. Only those cloves that were
sound and normal in appearance were used. They were placed
in a quart mason jar and held in a deep freeze unit at a
temperature of -20° C. When ready to use, the cloves were
allowed to partially thaw. Because of the low moisture
content of garlic (oh-66 percent) enough distilled water
was added to the cloves to secure a consistency necessary
for prOper maceration of the garlic tissue. Approximately
500 g of garlic cloves and 270 ml of distilled water were
blended in a Waring blender for 20 minutes. Enough NaCl
was added to give a 2.h percent concentration to the
macerate. The pH of the garlic-distilled water macerate
was 5.6.

According to Kertesz (1951) pectinesterase can be
easily desorbed from water insoluble cellular tissues by
the use of fairly strong salt solutions; by raising the
pH of the tissue macerate above 5; or by using a combination
of these two conditions.

After thoroughly blending the macerate was squeezed
by hand through several layers of cheesecloth and then
filtered through coarse filter paper using a Buchner filter
flask. The extract was layered with toluene and stored

at 7° C until used.

lu

Qualitative Test for Pectinesterase

The following qualitative test for pectinesterase in
garlic based on the observations of Etchells gt a; (19h?)
was run as follows. Thirty ml of a 3 percent pectinI
solution was placed into each of two 50 ml Erlenmeyer flasks.
The pectin was buffered at pH u with NaOH and potassium
acid phthalate buffer. To flask No. l was added 5 ml of
the above garlic extract, and to flask No. 2 was added
5 ml of garlic extract that had been inactivated by heating
to 80° C for 15 minutes. Five drops of toluene was placed
in each flask; they were tightly stOppered, and placed in

a 300 C water bath. Table I shows the results.

TABLE I

QUALITATIVE TEST FOR PECTINESTERASE IN GARLIC

 

— w a
H—

 

 

Flask number Time in hours

12 any 36 us

 

 

_.- __ __.. .‘—'-

l + + + ++

2 - - - _

 

d”!- —w-‘ t

- = absence of gel; + = gel; ++ 2 solid gel

The results in Table I show that in flask No. l a gel
was observed whichyindicated that the enzyme was inactivated.
In flask No. 2 no gel occurred which indicated that the
enzyme was inactivated. Therefore, pectinesterase is

present in garlic.

 

*Pectin obtained from California Fruit Growers Exchange.
Labeled was "Special Pectin for Enzyme Testing" No. hu7-U-7.

15

Quantitative Test for Pectinesterase

The quantitative test for pectinesterase in garlic
was run according to the following procedure: Two hundred ml
of a 1 percent pectin solution, 5 ml of 0.2 M sodium oxalate
solution and sufficient 2 M NaCl to give a final concen-
tration of 0.15 M in the reaction mixture were added to
a 600 ml beaker. The above solutions were made up to a
final volume of 500 ml by the addition of distilled water
and 25, no, and 50 m1 quantities of the garlic extract
reapectively. The garlic extract was not added to the
mixture, however, until all the other solutions had been
heated to 30° c in a 600 m1 beaker. The beaker was then
placed in a water bath at 30° c and 25, no, and 50 ml of
the garlic extract reapectively added. The pH was then
adjusted to 7.5 with 0.1 N NaOH for the 25 and no m1
quantities of garlic extract and with 0.5 N NaOH for the
50 ml sample. They were maintained at this pH and temper-
ature until the reaction was completed.

Tables II, III, and IV show the results of using 25,
no and 50 m1 of the garlic extract in a quantitative
determination of the pectinesterase of garlic.

Figure 2 shows the de-esterification curves obtained
for the different quantities of extract used.

"The number of milliequivalents of ester bonds hydro-
lyzed per minute per unit volume or weight of enzyme material
at pH 7.5, 30° C, and with 0.15 M sodium chloride when

acting on a 0.u percent pectin solution is designated as a

16

TABLE II

TIME AND AMOUNT OF 0.1 N NaOH REQUIRED TO COMPLETE
REACTION WHEN 25 ML OF THE GARLIC PECTINESTERASE
EXTRACT WAS USED

 

 

 

 

 

 

Ml of 0.1 N Ml of 0.1 N
Minutes NaOH used Minutes NaOH used
0 0.00 86 0.95
2 0.00 88 1.00
8 8.20 32 1.1g
. 0.
10 1.18 9 0.60
1 1.0a 100 0.65
1 0.39 102 0.60
18 0.90 10h 0.70
20 0.50 106 0.76
22 0.90 108 0.66
an 0.75 110 0.63
26 0.70 112 0.60
28 1.18 11 0.75
30 0.53 11 0.65
32 1.16 118 0.65
3 0.58 120 0.53
3 0.80 122 0.6a
38 0.87 12 0.73
10 1.03 12 0.55
12 0.60 128 0.55
kg 0.90 130 0.67
n 0.60 132 0.58
ea 1.05 13 0.58
50 0.30 13 0.57
52 0.87 138 0
5 0.68 180 0.80
fig 8'3? 1&2 S‘fif
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62 1.05 1&8 0.06
2 82% 15° 0.3“"
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. 0
'4. 8'25 137 0.95
33 3'3; 123 . 0:10
8H 0.85 177 0.39

17
TABLE II CONTINUED

“‘40‘

M1 or 00]. N

 

 

Ml of 0.1 N
Minut°s NaOH used Minut°s NaOH used
0 0 199 0.0?
182 0.85 0 0
0 0 204 0.05
190 0.11- 0 0
0 0 209 0.01
195 0.22 0 0
0 0 212 0.00

 

Total ml of
0.1 N NaOH used 56.36

 

O = no readings taken

TABLE III

18

TIME AND AMOUNT OF 0.1 N NaOH REQUIRED TO COMPLETE

EXTRACT WAS USED

 

 

 

 

Ml of 0.1 N
Minutes NaOH used
Trial 1 Trial 2

0 0.00 0.01
3 1.15 1.10
6 1075 1075
9 1.65 1.12
12 1.36 1. 3
15 1.18 1.15
18 1.56 1.37
21 1.77 1.85
21 1.82 1.35
27 1.29 1.55
30 1.27 1.55
33 1.65 1.12
36 1.70 1. 3
39 1.60 1.56
12 1.28 1.19
15 1. 6 1.58
18 1. 3 1.17
-51 1.69 1.38
51 1.30 1.35

57 1.59 0
60 1.59 3.17
63 1.32 1.55
66 1.58 1.05
69 1.97 1.37
72 0.15 1.38
75 1.15 1.90
78 1.55 1.60
81 1.37 1.12
81 1.18 1.58

0 = no readings taken

Results of Two Trials

REACTION WHEN 10 ML OF THE GARLIC PECTINESTERASE

 

 

 

 

M1 of 0.1 N
Minutes NaOH used
Trial 1 Trial 2
87 0087 0096
90 1.19 1.17
93 1.29 1.29
96 1.05 1.15
99‘ 1.18 1.15
102 0.81 1.10
105 0.87 1.00
108 0.91 0.75
111 1.02 0.90
111 0.73 0.65
117 0.37 0.77
120 0.10 0.63
123 00”? 0035
126 0.33 0.60
129 0.30 0.35
132 0.38 0.31
135 O°u7 0021
138 0.23 0.35
111 0.20 0.25
111 0.08 0.15
117 0.06 0.15
150 0.12 0.10
153 0.20 0.15
156 0.13 0.10
159 0.09 0.05
162 0.01 0.02
165 0.05 0.00
168 0.00
Total ml
or 001 N

NaOH used 56.80

56.29

19
TABLE IV
TIME AND AMOUNT OF 0.5 N NaOH REQUIRED TO COMPLETE
REACTION NHEN 50 ML OF THE GARLIC PECTINESTEEASE
EXTRACT WAS USED

Results of Two Trials

 

___

“—.-

 

 

 

 

 

 

 

 

 

 

Ml of 0.5 N . Ml of 0.5 N
Minutes NaOH used Minutes HaOH used
Trial 1 Trial 2 Trial 1 Trial 2

0 0.00 0.00 72 0.35 0.27

3 0.15 0.19 75 0.26 0.29

6 0.27 0.27 78 0.30 0.25

9 0.38 0.37 81 0.21 0.22
12 0.33 0.28 81 0.22 0.39
15 0.33 0.39 87 0.30 0.19
18 0.11 0.30 90 0.25 0.26
21 0.28 0.3% 93 0.22 0.21
27 0.32 0.35 99 0.17 0.19
30 0.31 0.30 102 0.19 0.15
33 0.13 0.30 105 0.15 0.18
36 0.31 0.36 108 0.07 0.11
39 0.35 0.31 111 0.13 0.13
12 0.28 0.33 111 0.01 0.12
15 0.39 0.33 117 0.07 0.16
18 0.35 0.35 120 0.08 0.08
51 0.25 0.27 123 0.01 0.01
51 0.39 0.31 126 0 0.07
57 0.30 0.31 129 0 0.00
60 0.26 0.27 132 0.10 0.01
63 0.35 0.35 135 0.00 0.01
66 0.35 0.35 138 0.00 0.00
69 0.30 0.26 Total ml _1

of 0.5 N

NaOH used 11.19 11.35

 

0 = no readings taken

20

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21

unit of pectinesterase" by Bell 33 21 (1950). They eXplain
that this unit is very similar to that described by Line-
weaver and Ballou (1915) with symbol PE, and Hills and
Mottern (1917) with symbol K.

Table V gives the calculations of pectinesterase units

for the different quantities of enzyme extract used.

TABLE V

CALCULATION OF PE UNITS

Amount of Minutes of No. of milliequivalents PE units

 

 

extract reaction of hydrolyzed ester bonds x 100
25 10 1.56 0.156
10 39 2.00 0.128
10 39 1.96 0.126
50 39 2.26 0.116
50 39 2.22 0.111

 

Effect of Various Concentrations of Salt on
the Activity of Garlic Pectinesterase

The effect of NaCl on garlic pectinesterase activity
was determined at concentrations of 0.15 M (0.88 percent),
0.50 M (3.0 percent), and 0.63 M (3.7 percent), using 200 ml
of the reaction mixture. The NaCl added was dissolved in
the distilled water that was added to the reaction mixture
to bring the volume to 200 ml. In calculating the salt used
the 2.1 percent that was originally in the garlic extract

was taken into consideration. The reaction was carried out

22

for a 30 minute period. The total amount of 0.1 N NaOH
consumed in the 30 minute period was used to determine the
activity of the enzyme. The percentages of salt used is
typical of the range of salt used in Kosher dills. Table VI

shows the activity found at the different salt levels.

TABLE VI

ACTIVITY OF GARLIC PECTINESTERASE AT
VARIOUS SALT CONCENTRATIONS

 

 

M1 of 0.1 N Percent relative

 

EXp. No. Salt concentration NaOH used activity
1 0.15 M (0.88%) 6.11 100
2 0.50 M (3.0%) 1.11 72
3 0.63 M (3.7%) 3.15 56

 

This indicates that pectinesterase present in the garlic
would still have an activity of 56 to 72 percent in the
range of the salt concentrations found .in Kosher dill

piCklOSe
Effect of pH on Garlic Pectinesterase Activity

The effect of pH on garlic pectinesterase activity
was determined at pH levels of 7.5, 6.0, 5.0 and 1.0.
One-tenth N HCl was used in adjusting the pH. Two hundred
ml of the reaction mixture was used. Sodium chloride
concentration was maintained at 0.15 M, and the reaction
was carried out for a 30 minute period. The total amount

of 0.1 N NaOH consumed in the 30 minute period was used to

23

to determine the activity of the enzyme. Table VII shows

the activity found at the various pH levels.

TABLE VII

ACTIVITY OF GARLIC PECTINESTERASE
AT DIFFERENT pH LEVELS

 

 

 

Ml of 0.1 N Percent relative
Efig: PH NaOH used activity
1 7.5 9.89 100
2 6.0 7.22 73
3 5o0 3.17 35
1 1.0 0.36 3.6

 

 

"-..-'- J—"u- A.—...—o- ‘_ -—— ~~-—.-.——----¢-._‘-‘.-—.-——— “fl...“ ‘4

As the pH is decreased from 7.5, the activity of the

garlic pectinesterase was diminished.
Heat Inactivation of Garlic Pectinesterase

The effect of heat on garlic pectinesterase was studied
because of the wide use of fresh garlic cloves in making
certain types of pickles such as genuine, processed, and
fresh pasteurized dill pickles.

The temperature used to inactivate the enzyme was 720 C
which is equivalent to 165? F, the temperature used to
pasteurize genuine, processed, and fresh dill pickles.
Genuine and processed dill pickles may or may not be pas-
teurized depending upon the equipment available and the

manufacturer but of course it is necessary to pasteurize

21

the fresh dills;otherwise they would Spoil immediately.
The method was similar to that used by Kertesz (1938)
in his studies on heat inactivation of tomato pectin-
methoxylase (pectinesterase). The test is as follows:
A 1 percent pectin solution was made to which was added
15 drOps of methyl red indicator to every 100 ml of pectin
solution. The pH of the pectin solution was adjusted to 6.2.
with 0.1 N NaOH. Twenty-five ml of the 1 percent pectin
solution and 5 drOps of toluene were placed into 125 ml
flasks. Five ml quantities of the garlic pectinesterase
extract was placed in 20 ml pyrex test tubes. The test
tubes were placed in a rack and immersed in a 720 C water
bath to inactivate the enzyme. The tubes were allowed to
remain in the bath for various lengths of time (Table VIII)
after which the contents of each tube were emptied in the
flasks containing the 1 percent pectin solution, mixed
thoroughly and incubated at 300 C for 21 hours. At the
end of this time the pH was taken. If the enzyme was
inactivated, the pH would not be lowered and the indicator
would still have its yellow appearance. If the enzyme was
not inactivated the pH would be lowered with the formation
of a red color due to action of the enzyme on the pectin.
The results of this experiment are given in Table VIII.
Since 5 ml of the garlic pectinesterase was used, it
was not possible to heat this quantity to 720 C immediately.
A thermometer suspended a short distance from the bottom of

the tubes gave the temperature of the extract in the tubes.

25

TABLE VIII

EFFECT OF HEAT ON GARLIC PECTINESTERASE

 

-“-—.

 

 

 

Time in Temperature of Activity pH of pectin‘
water bath tubes in bath, 0C after 21 hrs.
30 sec. 15 + 3.95
15 sec. 51 + 3.97
1 min. 60 + 1.00
2 min. 70 + 1.55
3 min. 72 + 5.72
1 min. 72 + 5.62
5 min. 72 - 6.31
7 min. 72 - 6.32
10 min. 72 - 6.10
12 min. 72 - 6.10

 

+ = red color; - 2 yellow color
The enzyme was not inactivated until a temperature of 72° 0
had been reached at the end of 5 minutes.

Pectinesterase Activity of Dehydrated Powdered Garlic

and Dehydrated Garlic Flakes

Commercial powdered garlic and garlic flakes were tested
for the presence of pectinesterase. The powdered garlic
was packed in 1950 and the garlic flakes in 1951.

Sixty-gram quantities of each product, 210 ml of dis-
tilled water and 2 ml of toluene were placed in flasks which
were tightly stoppered and allowed to stand overnight in

the refrigerator at 70 C. This mixture was then macerated

26

in the Waring blendor with 2 percent NaCl and filtered
through cheesecloth and coarse filter paper. The activity
was quantitatively tested by the method previously described
except 20 ml quantities of the garlic extract were used.

The reaction mixture contained 200 ml and 0.1 N NaOH was
.used in the titrations. Inactivated controls of the powdered
garlic and dehydrated garlic flakes required 0.11 and 0.13

ml respectively of 0.1 N NaOH for titration. These values
were taken into consideration when calculating the PE units
in Table IX. The reactions were allowed to proceed for

30 minutes. Results are given in Table IX.

TABLE IX

PECTINESTERASE ACTIVITY OF PONDERED DEHYDRATED GARLIC
AND GARLIC FLAKES

 

 

 

 

 

 

Amount of Minutes of N8. of milli:‘j PE units

 

Extract extract reaction equivalents of x 100
(ml) hydrolyzed
ester bonds
Powdered
garlic 20 30 0.202 0.033
Garlic
flakes 20 30 0.237 0.039

Since the work was done with fresh garlic and dehydrated
garlic powder and flakes, it was necessary to use different
amounts of water to prepare the extract. This makes com-
parison of the pectinesterase activity of the reSpective
extracts difficult. In the preparation of the fresh garlic
extract 500 g of the cloves was macerated in 270 ml of

distilled water. When 25 ml of the extract was incubated

27

for 30 minutes with the 3 percent pectin solution, a PE
unit of 0.156 was obtained, Table V.‘ In the case of the
dehydrated powder and chips 210 ml of distilled water was
used to 60 g of the powder and flakes reSpectively. When
20 ml of these extracts was incubated with the 3 percent
pectin solution for 30 minutes, PE units of 0.033 and 0.039
respectively were obtained, Table IX. The label of the
dehydrated products used stated that 1 lb of the dehydrated
product was equivalent to 1 lb of the fresh product. A
very rough calculation indicates that fresh garlic was
approximately four times more active than the dehydrated
garlic products tested. Many factors can influence this
difference in activity such as age, purity and method of

preparation of the dehydrated products.
Test for a Polygalacturonase-like Enzyme in Garlic

Recently, Bell (1951) reported the presence of a
pectolytic enzyme similar to polygalacturonase in various
parts of the cucumber fruit and plant. Bell states that
the glycosidic hydrolysis of pectin or pectic acid to
galacturonic acid would not necessarily have to be complete
for salt stock to become soft, and refers to the work of
Fabian and Johnson (1938) where it.was found that both
mushy and firm salt stock had the same pectin content when
measured as calcium pectate.

Bell 22.21 (1919) reported on the softening of cucumber

salt stock in relation to polygalacturonase activity. They

28

found that the polygalacturonase-like enzyme reacted
similarly to commercial polygalacturonase (pectinol) in
respect to temperature, pH, salt and time of incubation.
Kertesz and McColloch (1919) reported an enzyme in
ripe tomatoes that was capable of the depolymerization of
pectic acid. They refer to it as pectic acid-depolymerase.
With the findings of the above workers in mind, it
was decided to test for the presence of other pectolytic

enzymes in garlic with special reference to polygalacturonase.

 

Experimental procedure. The method used was similar
to that of Bell (1951), and Bell, Etchells and Jones (1919)
and will be described later in the paper. Briefly, it
consists of measuring the loss in viscosity of a 3 percent
pectin solution caused by the action of pectolytic enzymes
or more specifically by a polygalacturonase-like enzyme.

In order to prevent gel formation in the garlic
extracts, the pectinesterase was destroyed by adjusting
the pH of the extracts to a value of 3 and incubating at
approximately 10° C for 21 hours in a water bath. Extracts
were prepared as previously described from the fresh garlic,
powdered garlic and garlic flakes reSpectively. These
extracts were tested by adding 5 m1 of the extract and 5
drOps of toluene to 30 m1 of a 3 percent pectin solution
buffered at pH 1 to 50 ml flasks. The flasks were tightly
stoppered and incubated at 30° C for 7 days. Viscosity

readings were taken at the end of one and 7 days by means

29

of a 25 m1 volumetric pipette. The drOpping time was
measured with a stepwatch. A loss in viscosity of the
pectin solution would indicate the presence of a poly-
galacturonase-like enzyme in the garlic extract.

The formation of a gel showed that in the fresh garlic
extract the pectinesterase was not inactivated by this
method so that it was impossible to test for the poly-
galacturonase-like enzyme. 0n the other hand the dehydrated
garlic powder and flakes showed no loss in viscosity and
no gel formation which indicated the presence of little or
no pectinesterase and the absence of a polygalacturonase-
like enzyme.

To check the action of the garlic extract on pickles,
several freshened salt stock pickles were kept in the.
garlic extract for a month and showed no sign of softening.

The gel formation by fresh garlic in the above eXperi-
ment may be eXplained by Kertesz (1951), who states that
the procedure for inactivating pectinesterase must be
established for each kind of enzyme preparation.

Decomposed Garlic as a Source of a
Polygalacturonase-like Enzyme

Since dehydrated garlic did not show any evidence of
a polygalacturonase-like enzyme, it was decided to test
for this enzyme in decomposed garlic. Furthermore, de-
composed garlic has already been implicated as a cause of
softening in genuine Kosher dill pickles by Wenzel and

Fabian (1915). Therefore, it was thought that a study of

30

several of the molds found on decomposed garlic would be
of interest.

Wenzel and Fabian (1915) found Aspergilli and Pani-
cillium molds on garlic and attributed the formation of
pectolytic enzymes by these fungi as the cause of Spoilage
in genuine Kosher dill pickles.

The literature cites many fungi capable of producing
pectolytic enzymes, and certain of the genera such as

Penicillium and Aspergilli, are commercially used to produce

 

pectolytic enzymes for clarifying of fruit juices. Peni-

cillium glaucum is used to produce pectolytic enzymes sold

 

under the trade name of "Pectinol".

EXperimental procedure. Several clusters of garlic

 

were placed in mason jars to which a small amount of water
was added to hasten the development of mold growth. After
one week there was considerable growth. Molds present on

O
the garlic clusters were identified as Penicillium can-

 

escens and Fusarium oxysporgm.. These were cultured on
Sabouraud dextrose agar.

The molds isolated were first grown on a pectin medium
developed by Manchester and Baier (1915) to determine their
ability to liquefy pectin. Both molds grew very well on

this medium. g. canescens liquefied the pectin readily,

 

while B. oxygporwm“ liquefied it slowly and only partially.
As production of pectolytic enzymes may be more or less

profuse according to the substrate used, these molds were

31

grown in pure culture on sterile garlic cloves. Sound,
fresh garlic cloves with the layer of skin removed were
immersed in a solution of 1:1250 Roccal for 5 minutes.
The cloves were then removed and rinsed twice in sterile
water. The cloves were aseptically placed into two sterile
mason jars and inoculated with the two molds. Several of
the cloves were placed in dextrose broth and in Sabouraud
dextrose agar to check on the sterility of the cloves. One
lot of cloves was run as a control without any inoculum.
The mason jars were placed in a dark room to keep the garlic
cloves from Sprouting. After three weeks of incubation,
the inoculated cloves were completely covered with growth
and very badly decomposed, while the uninoculated control
showed no change.
Fifteen g of each of the two lots of moldy garlic
were macerated inizhe Waring blendor in 15 ml of distilled
water. The uninoculated control and two clusters of garlic
that had been allowed to decompose naturally were treated
in the same manner. The extract from each lot was filtered
through cheesecloth and then through filter paper to remove
any particles that would interfere with the viscosity
measurements. After 5 drOps of toluene was added, the
flasks were stoppered and placed in the refrigerator at 7° C.
The methods used to test for the presence of pecto-
lytic enzymes in the extract was similar to that of Etchells
32 El (1919). The method uSed in these eXperiments was as

follows: A 3 percent pectin solution was buffered at a pH

32

of 1 with potassium acid phthalate and NaOH. It was then
filtered through cotton, layered with toluene, stOppered
tightly, and stored in the refrigerator until used.

As a known source of pectolytic enzymes a commercial
product, "Pectinol M"*, was used. One portion was in-
activated by heating for 15 minutes at 80° C and then
tested for its activity.

Thirty ml of the 3 percent pectin solution was added
to 50 ml Erlenmeyer flasks. To each flask were added 5 ml
of the prepared extracts and 5 drOps of toluene after which
the flasks were stoppered and placed in the 30° 0 water
bath. Viscosity readings were made at the end of l, 2, 1,
and 6 days.

The dropping time of the solutions was measured with
a 25 ml volumetric pipette. The pectin solution was drawn
into the pipette with a rubber suction bulb to a mark on
the pipette which was calibrated to deliver 25 ml. The end
of the pipette was cut off to give a faster delivery rate.
The time required to empty the pipette was taken with a
stOpwatch.

Since all readings were taken with the same pipette,
it was necessary to clean the pipette thoroughly after
each reading. The pipette was cleaned by flushing it with
boiling water, followed by an alcohol, and then a boiling
water rinse, and finally in an alcohol and acetone rinse.

The activity of the enzyme was measured by the loss in

viscosity of the pectin solution. The loss in viscosity

 

*"Pectinol M" obtained from Rohm and Haas Co., Phila-
delphla, Pa.

33

was calculated by comparing the loss in viscosity of the

active enzyme solution to the inactive control which should

show no loss in viscosity.

in Table X.

PECTOLYTIC ACTIVITY OF THE VARIOUS EXTRACTS
EXPRESSED AS PERCENT LOSS IN VISCOSITY

m .
Ave. drOpping time in
seconds at the end of days

Source of
enzyme

TABLE X

___-'_.

 

 

l

2

1

6

The results obtained are given

 

Percent loss
in viscosity

 

Extract from
naturally
decomposed
garlic

Inactivated

15.0

0.1% Pectinol M 18.5

0.1% Pectinol M

'E. oxysporum;
gafIic extract

‘3. canescens
garlic extract

 

Control --
uninoculated
garlic extract

Control ~-
inactivated
garlic extract

12.5

51-6

9.3

57.5

51.1

10.1

18.1
10.5

5906

8.8

58.0

(+8.7

9.1

17.6

9.1

72.3

8.5

53.5

19.9

8.9

17.1
8.5

83-5

8.5

59.1

18.5

81.6

2.2
82.0

82.1

5.0

 

The results in Table X show that the extract from the

decomposed P. canescens garlic showed a loss in viscosity

 

comparable to the 0.1 percent Pectinol M, as did also the

naturally decomposed garlic extract.

oxySpoEum. garlic extract and the uninoculated garlic

The decomposed E.

31

extract showed no loss but a gain in viscosity and after

two weeks both of these extracts showed a gel formation.

DISCUSSION

In the quantitative determination of the garlic

2 to 0.111 x 10"2

pectinesterase, values of 0.156 x 10-
'PE units per ml were obta.ned when using 25 to 50 m1 of
the garlic extract reapectively. The difference in values
may be due to the concentration of enzyme, the concentration
of substrate, or due to storage of the garlic extract. The
amount of 0.1 N NaOH or 0.5 N NaOH used in titrating when
various quantities of the garlic extract were used gave
comparable results as to the total amount of milliequivalents
of hydrolyzed ester bonds. In the case of the 25 ml quantity
of extract used 56.36 ml of 0.1 N NaOH was used, while in
the case of the 10 and 50 m1 quantities, 56.51 and 11.12 ml
of 0.1 N and 0.5 N NaOH respectively were used. This would
give values of 5.63, 5.65, and 5.63 milliequivalents of
hydrolyzed ester bonds for the 25, 10, and 50 m1 quantities
of the extracts reSpectively. Although different PE
values were obtained for the different quantities of
extract used, the total amount of hydrolyzed ester bonds
indicates the completeness of the reaction in all cases.
It was found that the normal salt range of the various
kinds of Kosher dill pickles decreased the activity of
garlic pectinesterase from 56 to 72 percent. However, at

the beginning of a genuine Kosher dill fermentation the salt

36

concentration is generally greater than the values tested
for in these eXperiments, viz. 3.0 and 3.7 percent.
Therefore, the concentration of salt at the beginning of
a genuine Kosher dill fermentation would be unfavorable
for the activity of garlic pectinesterase.

The activity of the garlic pectinesterase diminished
as the pH decreased from 7.5 to 1.0. Thus, in genuine
Kosher cucumber fermentation the activity of the garlic
pectinesterase would be at its greatest before the fermen-
tation actually began. Therefore, in actual conditions,
the pH at the beginning of the fermentation would be favor-
able for the activity of the enzyme while the salt concen-
tration would not.

Since pectinesterase has been found in garlic, it may
be significant in paving the way for softening of genuine
Kosher dill pickles. Jansen and MacDonnel (1915) have
demonstrated that de-esterification must occur before poly-
galacturonase can act on pectic substances. Therefore,
garlic could be responsible for the de-esterification of
pectin in genuine Kosher dill pickles.

It has been shown in this paper that moldy garlic could
be a source of polygalacturonase. The extract from the

garlic decomposed by 2. canescens gave an activity com-

 

parable to commercial Pectinol M, as did the extract from
the garlic that was allowed to decompose naturally. The

extract inoculated with the F. oxysponhn‘ showed a gain in

 

viscosity and a gel formation at the end of two weeks.

37

This could be attributed to formation of pectinesterase
'by the E. oxySporum_ mold or to the extraction of more of
the garlic pectinesterase due to the decomposition of the
garlic.

The heat inactivation tests showed that the garlic
pectinesterase was inactivated at the end of 5 minutes
under the COnditions of the experiment. Since fresh
pasteurized dills are processed at approximately 165° F
for a period of 15 to 20 minutes, the garlic pectinesterase
would doubtless be inactivated. The same would be true if
genuine and processed dills were pasteurized at this tempera-
Iture and time. Most processors of such pickles use chOpped

garlic so that the heat penetration into the garlic would

be faster in such cases.

2.

3.

1.

SUMMARY

Garlic was found to contain the enzyme pectinesterase

in amounts of 0.156 x 10"2 to 0.111 x 10"2 PE units

per ml depending on the amount of extract used.

Garlic pectinesterase showed decreased activity as the
pH and salt concentration approached those found in
different kinds of Kosher pickles.

Pectinesterase was found in dehydrated garlic products.
The powdered garlic and garlic flakes showed similar
activity but showed approximately one-fourth the activity
of the fresh garlic extract.

Polygalacturonase was not demonstrated in the dehydrated
garlic products.

Naturally decomposed garlic and garlic decomposed by

the mold, Penicilligm canesggns, showed pectolytic
activity comparable to commercial Pectinol M, while
garlic decomposed by Fusarium oxySpgrumi showed a strong

gel formation.

BIBLIOGRAPHY

Bell, T. A. (1951). Pectolytic enzyme activity in various
parts of the cucumber plant and fruit. Bot. Gaz., 113
(2), D80.

Bell, T. A., Etchells, J. L., and Jones, I. D. (1951).
Pectinesterase in the cucumber. Arch. Biochem., 3; (3).

Bell, T. A., Etchells, J. L., and Jones, I. D. (1950).
Softening of commercial cucumber salt stock in relation
to polygalacturonase activity. Food Tech., 1 (1),

157'1630

Calesnick, E. J., Hills, C. H., and Willaman, J. J. (1950).
PrOperties of a commercial fungal ectase preparation.
Arch. Biochem., 22 (2), Dec., 132- 0.

Davison, F. R., and Willaman, J. J. (1927). Biochemistry
of plant diseases. IX. Pectic enzymes. Bot. Gaz.,

83. 329-361.

Fabian, F. W., and Bryan, C. S., and Etchells, J. L. (1932).
Mich. Agr. Exp. Sta. Tech. Bull. 125.

Fabian, F.Id., and Faville, L. W. (1919). Isolation and
identification of a mold, Oospora lactis, as the
causative agent of two cases of ickle spoilage.
Fruit Products Jour., 28, 297-298.

 

Fabian, F. W., and Johnson, E. A. (1938). EXperimental
work on cucumber fermentation. Mich. Agr. EXp. Sta.
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Hills, 0. H., and Mottern, H. H. (1917). Properties of
tomato pectase. Jour. Biol. Chem., 168, 651-663.

Jansen, E. F., and MacDonnel, L. R. (1915). Influence of
methoxyl content of pectic substances on the action
of polygalacturonase. Arch. Biochem., 8 (1), Oct.,
97-111 0

Jones, L. R. (1909). Pectinase, the cytolytic enzyme
produced by Bacillus carotovorus and certain other
soft rot organisms. N.Y. Agr. EXp. Sta. (Geneva)
Tech. Bull. 11.

 

10

Kertesz, Z. I. (1939). Pectic Enzymes. III. Heat in-
activation of tomato pectin-methoxylase (pectase).
Food Research, 1 (2), 113-116.

Kertesz, Z.I. (1951). "The pectic substances". Interscience
Publisher, New York.

Lineweaver, H., and Ballou, G. A. (1915). The effect of
cations on the activity of alfalfa pectinesterase
(pectase). Arch. Biochem., 6, 373-387.

McColloch, R. J., and Kertesz, Z, I. (1919). Recent
develOpments of practical significance in the field
of pectic enzymes. Food Tech., 3, March, 91-96.

Phaff, H. J., and Joslyn, M. A. (1917). The newer know-
ledge of ectic enzymes. Wallerstein Lab. Comm.,
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Pittman, G. A., and Cruess, W. V. (1929). Hydrolysis of
pectin by various microorganisms. Ind. and Eng.
Chem., 3;, 1292-1295.

Schucart, H. S. (1912). Practical observations on the
manufacture of Kosher style dill pickles. Fruit
Products Jour., 21, 206-212.

Wenzel, F. W., and Fabian, F. W. (1915). EXperimental
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A? 16 '53 6°