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THESlS

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This is to certify that the

thesis entitled

AN INVESTIGATION OF THE KINETICS AND
INHIBITION OF PECTINESTERASE

presented by

ANNA T.C. MAGHASI

has been accepted towards fulfillment
of the requirements for

M.S. degree in CHEMISTRY

 

 

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DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 cJCIFIC/DateDuepes-p. 15

AN INVESTIGATION OF THE KINETICS AND
INHIBITION OF PECTINESTERASE

By

Anna Tamnai Chemsito Maghasi

A THESIS

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

MASTER OF SCIENCE

Department of Chemistry

2000

ABSTRACT

AN INVESTIGATION OF THE KINETICS AND INHIBITION
OF PECTINESTERASE

By
Anna Tamnai C. Maghasi

Methanol in fruit brandy is regulated by the FDA because of its toxic properties.
To reduce the amount of methanol in fruit brandy, it is necessary to understand the
source and mode of its generation. Pectinesterase enzyme is responsible for the
deesterification of fruit pectin and generates methanol as one of the major products.
The kinetics of pectinesterase and its inactivation by heat were studied. At 70°C,
Pectinesterase was found to be inactivated significantly with an inactivation rate
constant of 2.8 x 1035".

Various inhibitors were tested for their effect on the activity of pectinesterase.
Tannic acid, polyacrylic acid, polymaleimide, citric acid and
ethylenediamminetetraacetic acid (EDTA) were tested as potential inhibitors.
Polyacrylic acid was found to reduce the activity of pectinesterase by nearly 45%.
EDTA also significantly reduced the amount of methanol generated by pectinesterase.
It is postulated that they act by providing excess carboxylate functionality thereby

shifting the equilibrium of the deesterification reaction.

Dedicated to my Parents

For their wisdom and guidance

ACKNOWLEDGEMENT

I would like to thank members of my committee: Dr. Kris Berglund, Dr. Gary
Blanchard, and Dr. Robert Ofoli for their guidance throughout my research. A special
thanks is warranted for my advisor Dr. Kris Berglund for his mentoring and guidance.

I would also like to acknowledge the assistance and friendliness of my group
members. I am especially grateful for the lunchtime discussions and debates on all kinds
of issues.

Finally, I would like to thank Mike for putting up with my endless chemistry
monologues, none of which were interesting. His support and encouragement is deeply

appreciated.

iv

TABLE OF CONTENTS

1. INTRODUCTION
1.1 Michigan Fruit Brandy Industry
1.2 Regulation of methanol
1.3 Objectives
2. LITERATURE REVIEW
2.1 Source and Nature of Pectin
2.2 Pectic Enzymes
2.3 Pectinesterase
2.4 Enzyme Kinetics
2.5 Current methods of measuring pectinesterase activity
2.6 Inhibition of Pectinesterase activity
3. MATERIALS AND METHODS
3.1 Methanol Analysis
3.1.1 Fermentation
3.1.2 Distillation
3.1.3 Analysis of Distillates.
3.1.4 Quantification of Methanol
3.2 Pectinesterase Activity
3.2.] Measurement of PE activity
3.2.2 Temperature dependence
3.2.3 Thermal Inactivation of Pectinesterase

3.3 Inhibition of Pectinesterase

10

13

15

19

19

19

19

23
25
26
26
27
27

27

3.3.1 Tannic Acid
3.3.2 Polyacrylic acid
3.3.3 Polymaleimide
3.3.4 Citric Acid
3.3.5 Ethylenediamminetetraacetic acid (EDTA)
4. RESULTS AND DISCUSSION
4.1 Composition of Fruit Brandy
4.2 Methanol in Fruit Brandy
4.2.1 Quantitative Analysis.
4.2.2 Fruit Varieties
4.3 Activity of Pectinesterase
4.3.1 Enzyme Kinetics.
4.3.2 Effect of Temperature
4.3.3 Thermal Inactivation of Pectinesterase in cherries
4.4 Inhibition of Pectinesterase
4.4.2 Effect of Tannic Acid
4.4.2 Effect of Polyacrylic Acid
4.4.2 Effect of Polymaleimide
4.4.3 Effect of EDTA
4.4.4 Effect of Citric Acid
5. SUMMARY AND CONCLUSIONS
6. RECOMMENDATIONS FOR FUTURE WORK

7. APPENDIX

8. LIST OF REFERENCES

vi

28
28
29
29
3O
31
31
31
32
37
39
39

41

45

48
50
52
55
55

58

60

63

68

Table 1. 1

Table 2.1

Table 4.1

Table 4.2

Table 4.3

LIST OF TABLES

Methanol content of Fruit Juice and Beverages 3
Classification of pectic enzymes 7
Brix values of fermented mash of different fruits. Fruit mash 31

stirred in a 200 gallon fermenter maintained at 15°C by a water

chiller. 300g Prise de mouse yeast used for each fermentation.

Amount of pectic substances in various plants 37

Inactivation rate constants for pectinesterase at various 43
temperatures. Rate constants evaluated from straight line plots
of equation (3).

vii

Figure 2.1
Figure 2.2
Figure 3.1

Figure 3.2

Figure 4.1

Figure 4.2

Figure 4.3

LIST OF FIGURES

The structure of pectin 6
Mode of action of pectinesterase 9
Schematic of a 150 L Christian Carl still 20

Typical GC chromatogram of fruit distillates The GC run conditions 24
were as follows: Oven temp: 40°C (hold for 1 minute); to 190°C

at 25°C /min (hold for 5 minutes), Injector temp: 240°C,

Detector temp: 255°C, Carrier gas: Helium at 30 cm/sec,

Injected volume: 0.5 pl.

Methanol calibration curve. Area corresponds to chromatographic 33
peak area. The GC nm conditions were as follows: Oven temp:

40°C (hold for 1 minute); to 190°C at 25°C /min (hold for 5 minutes),
Injector temp: 240°C, Detector temp: 255°C, Carrier gas:

Helium at 30 cm/sec, Injected volume: 0.5 ul. All other methanol

concentrations were determined relative to this external standard curve.

Generation of methanol during fermentation. Montmorency 34
cherries fermented at 15°C with Prise de mouse yeast. Analysis of
methanol was done every 24 hours. Samples were filtered and
centrifuged before injecting into the GC.

Methanol profile during distillation. Different distillation batches 36
from the same fermentation batch. Distillation performed with

all 3 trays engaged. Distillation outnumber corresponds to different
volumes of distillate samples collected in the course of the distillation.

Results shown are for cherry fruit brandy.

viii

Figure 4.4

Figure 4.5

Figure 4.6

Figure 4.7

Figure 4.8

Figure 4.9

Methanol profile among different fruit varieties. Fermented at 38
15°C with Prise de mouse yeast. Methanol content average of three
distillation batches for each fruit. Distillation performed with

all 3 trays engaged.

Activity of pectinesterase as a function of time. Pectin 40
concentration (4 mg/mL) and 0.432 mg/mL pectinesterase

concentration. Pectinesterase prepared in phosphate buffer pH 7.0.
Incubation at room temperature. 0.5 ul of each sample injected

into the GC after incubation for the period of time shown.

Effect of Temperature on the formation of methanol. Pectin 42
concentration: 4 mg/mL, Pectinesterase: 0.432 mg/mL. Pectinesterase
prepared in phosphate buffer pH 7.0. The samples were incubated at

the respective temperature for 5 minutes before injection into the GC.

Normalized inactivation rate curves for pectinesterase. Pectin: 44
4 mg/mL, pectinesterase: 0.432 mg/mL. Pectinesterase prepared in
phosphate buffer pH 7.0. One half ul of each sample injected into

the GC after incubation for the period of time shown. Initial methanol

concentration (Co) determined after 30 seconds of incubation.

Arrhenius plot for pectinesterase inactivation. Pectin: 4 mg/mL, 46
pectinesterase: 0.432 mg/mL. Pectinesterase prepared in phosphate
buffer pH 7.0. Activation energy was calculated from the slope to

be 5697 cal mol'l.

Thermal Inactivation of Pectinesterase in cherry juice samples. 47
Five hundred mL cherry juice heated for 5 minutes at the respective
temperature. Samples centrifuged and filtered before injection

into the GC.

ix

Figure 4.10

Figure 4.11

Figure 4.12

Figure 4.13

Figure 4.14

Effect of tannic acid on the activity of pectinesterase. #1 and #2 49
represent two separate batches of cherry juice incubated with tannic

acid. Cheery juice samples fermented with Prise de mouse yeast

in the cold room (15°C). The final pH of the samples ranged from
3.82-4.70.

Effect of polyacrylic acid on the activity of pectinesterase. 51
Pectinesterase concentration: 0.432 mg/mL, prepared in phosphate
buffer pH 7.0. One half ul of each sample injected into the GC after

incubation at room temperature for 5 minutes. Inhibition range: 30-45%.

Effect of polymaleimide on the activity of pectinesterase using 53
1% w/v sodium salt of polymaleimide. Pectinesterase concentration:
0.432 mg/mL, prepared in phosphate buffer pH 7.0. One half ul of

each sample injected into the GC after incubation at room temperature

for 5 minutes. Inhibition range: 30-40%.

Effect of Polymaleimide on the activity of pectinesterase. 54
Concentrations of polymaleimide: 0.25, 0.5, 1% w/v. Pectinesterase
concentration: 0.432 mg/mL, prepared in phosphate buffer pH 7 .0.

One half p1 of each sample injected into the GC after incubation at

room temperature for 5 minutes.

Effect of EDTA on the activity of pectinesterase. EDTA 56
concentration: 1% w/v. Pectinesterase concentration: 0.432 mg/mL,
prepared in phosphate buffer pH 7.0. One half ul of each sample

injected into the GC after incubation at room temperature for 5

minutes. Inhibition range: 38-41%.

Figure 4.15

Figure A-l

Figure A-2

Figure A-3

Figure A-4

Effect of Citric Acid on the activity of pectinesterase. Citric acid: 57
1% w/v, pectin concentration: 4 mg/mL. Pectinesterase concentration:
0.432 mg/mL, prepared in phosphate buffer pH 7.0. Resulting pH of
samples: 3.12-3.38

GC chromatogram of fermented fruit mash samples. The GC run 64
conditions were as follows: Oven temp: 40°C (hold for 1 minute); to
190°C at 25°C /min (hold for 5 minutes), Injector temp: 240°C,

Detector temp: 255°C, Carrier gas: Helium at 30 cm/sec, Injected

volume: 0.5 ul.

GC Chromatogram for pectin-pectinesterase sample with 65
1% w/v EDTA inhibitor and without any inhibitor. Samples were
incubated for 5 minutes before analysis. The GC temperature
programming was as follows: Oven temp: 40°C to 100°C at 3°C/min (hold
for 0.5 min) to 190°C at 20°C /min.

GC chromatogram of thermally inactivated sample (90°C) and 66
sample at room temperature (25°C). Samples were incubated for 5
minutes before analysis. The GC temperature programming was as
follows: Oven temp: 40°C to 100°C at 3°C/min (hold for 0.5 min) to
190°C at 20°C /min.

GC Chromatogram of Pectin-pectinesterase sample. The GC 67
temperature programming was as follows: Oven temp: 40°C to

100°C at 3°C/min (hold for 0.5 min) to 190°C at 20°C /min. Injector temp:
240°C, Detector temp: 255°C, Carrier gas: Helium at 30 cm/sec, Injected
volume: 0.5 u].

xi

1. INTRODUCTION

1.1 Michigan Fruit Brandy Industry

The Michigan fruit industry has benefited greatly from advances made in
agricultural techniques. Consumption of the major fruits such as apples, plums and
cherries has remained the same despite the increase in production. In some cases such as
pears, consumption has even decreased. The improved productivity has led to surpluses
for most of these major fruits. Some fruit also has defects in either appearance or size
that reduce its market value significantly. These surpluses have created a need to
develop new ways to utilize the excess fruit. An alternative widely practiced in Europe
is the production of fruit brandy. In the United States, this technology is virtually non-
existent and the development of such technology will greatly benefit the fruit industry as
a whole.

Many fruit and vegetable juices contain some amount of methanol. The
methanol content of fresh juices is dependent upon the method used to extract the juice,
the type of fruit and the stage of harvesting (Kazeniac, 1970). Lund (1981) showed that
the average methanol content of fresh orange juice was 34 mg/liter, while fresh grapefruit
juice averaged 27 mg/liter in the same study. Processed juices have been reported to
contain much less methanol than their fresh counterparts. For example, Kazeniac (1970)
found that blended tomatoes had a methanol content of between 64 and 138 mg/liter

depending upon the ripeness of the tomatoes. Methanol is also found in alcoholic

beverages, especially those derived from fruit. Table 1.1 shows a summary of the
average methanol content of some beverages (Monte, 1984).

The pectin in fruits and vegetables is usually esterified by methyl groups. The
naturally occurring presence of the enzyme pectinesterase in the fruit is responsible for
deesterifying the pectin to release methanol. The activity of the enzyme gradually
increases as fruits ripen and tissue is damaged. Fruits at different stages of maturity will
contain methanol, but the activity of the pectinesterase enzyme is rather low until after

harvesting when the fruit tissue drastically softens.

 

 

 

 

 

Table 1.1 Methanol content of Fruit Juice and BeveragesI
Juice/ Beverage Methanol (mg/liter)
Orange 34
Grapefruit 27
Pear Wine 188
Cherry Wine 276

 

 

 

 

As noted in table 1.1, methanol is present in many fruit juices. This poses a
problem in the fruit brandy industry. The methanol levels that are low in fruit juices
become highly significant when fruit mash is distilled. During distillation, separation of
alcohols from the fruit mash always results in the concentration of methanol in the
product.

Use of Michigan excess fruits for brandy production therefore calls for the need to

address the methanol content of the fruit brandies. In order to develop quality products,

 

‘ Data obtained from Monte, w.c. (1984).

the amount of methanol needs to be closely monitored. Methanol contributes to good
attributes in the fruit brandy, but there are health concerns associated with methanol
toxicity. The aim of this project is therefore to quantitate the methanol content of
Michigan fruit brandies and establish ways in which this can be moderated. The main
approach used in this project is to target the pectinesterase enzyme that is responsible for

the generation of methanol and find ways to inhibit its activity.

1.2 Regulation of methanol

The United States Environmental Protection Agency recommends a minimum
acute toxicity concentration of methanol in drinking water at 3.9 parts per million
(Cleland and Kingsbury, 1977). The EPA has also set the permissible exposure limit in
air as 200 parts per million for an 8-hour time weighted average.

In the production of fruit brandy, methanol has been widely regulated in Europe
(Tanner and Brunner, 1982). In Germany, maximum methanol content in fruit brandies
varies from fruit to fruit. Cherry brandy methanol content is regulated at 400 mg/100 mL
absolute alcohol, while the maximum methanol content in brandy from Bartlett pears is
790 mg/ 100 mL absolute alcohol. In Italy the maximum methanol content in any fruit
brandy is 800 mg/ 100 mL absolute alcohol. In Austria the maximum allowed content of
methanol in spirits is 1000 mg/100 mL absolute alcohol.

The United States Food and Drug Administration (FDA) regulates the amount of
methanol that is allowed in fruit brandy and the current limit is 0.35 % v/v for both local

and imported fruit brandy. This corresponds to 700 mg/ 100 mL absolute alcohol.

1.3 Objectives

The objectives of this study are to determine the amount of methanol in fruit
brandy and find possible means of minimizing it. The approach used in this work is to
study the activity of pectinesterase, which catalyzes the formation of methanol and use
various substances to inhibit its activity. A variety of potential inhibitors are tested for

their effect on pectinesterase activity.

2. LITERATURE REVIEW

2.1 Source and Nature of Pectin

Pectic substances are carbohydrate derivatives that are widely distributed in plant
tissues, where they occupy intracellular spaces. They contribute to the adhesion between
cells and overall mechanical strength of the cell wall. Cell walls contain approximately
60% water and 40% biopolymers. Pectins make up 20-3 5% of the polymers (Jarvis,
1982). Pectins are usually classified by the procedures that are used to extract them from
cell walls. The three major classifications are: water-soluble, chelator-soluble and
protopectin that are dissolved with alkali. Either one or several of these pectin types may
exist in a given fruit tissue (Sajjaanatakul et al., 1989).

Pectin is a polysaccharide, whose main component is D-galacturonic acid, joined by
means of or-(l —>4) glycosidic linkages. Figure 2.1 shows the structure of pectin. The
galacturonic acid molecule has a carboxyl group on C5 that may be esterified with methyl
alcohol. The degree of esterification of this carboxyl group is an important factor in
characterizing pectin and has a bearing on the firmness and extent of cohesion of plant
tissues.

There are a wide range of degrees of esterification, depending on the species, tissue
and maturity (Van Buren, 1991). In general, the degree of esterification in plant tissues

can be up to 90%.

5‘
O.”

OCH,
I HO OH
O C '50 0
HO OH O
0" \
O 0 ll OCH,
C 0

Cl) '6 ‘ OCH, 0”

R

Figure 2.1 The structure of fruit pectin.

2.2 Pectic Enzymes

The Chemistry of pectic substances has been extensively reviewed by Kertesz
(1951), Dewel and Stutz (1958), and Joslyn and Dewel (1963). Enzymes that catalyze
the formation of pectin from its water insoluble precursor protopectin, are called
protopectinases. The enzymes that catalyze the degradation of pectic substances are
called “pectic enzymes.”

Depolymerization of pectin generally occurs during fruit ripening. These
enzymes also play a significant role in changes that occur after the harvest of fruits.
Pectic enzymes have been known as the cause of cloud loss in citrus juice in the food
processing industry (Pilnik and Voragen, 1993).

There are reports in the literature of various types of pectic enzymes. Some of
these are: pectate lyase, polygalacuturonase and pectinesterase (Wong, 1995). Of these,
polygalacturonase and pectinesterase have been widely studied for various reasons.

Pectate lyases degrade pectin by the mechanism of B-elimination (Rexova-Benkova and

Markovic, 1976). The degradation occurs either in a random (endo) or terminal (exo)
fashion. When endo-pectate lyase acts on pectin, the product of the degradation is a 4,5-
unsaturated galacturonosyl residue. Exo-pectate lyase is considered relatively rare
compared to endo-pectate lyase. It attacks from the reducing end of the chain and the
only product of degradation is unsaturated digalacturonic acid.

Polygalacturonase is widely studied because its activity is usually associated with
the softening of fruit. The enzyme catalyzes the hydrolytic cleavage of the O-glycosyl
bond of OL-D- (l —-)4) polygalacturonate (Burns, 1991). The pattern of degradation is
either random (endo-polygalacturonase) or terminal (em-polygalacturonase). Endo-
polygalacturonase usually prefers substrates with a low degree of esterification (Archer,
1979), typically less than 20%. Exo-polygalacturonase generally acts on deesterified
pectin (Pressey and Avants, 1977). Pressey (1986) found that within short reaction times,
random Cleavage of polygalacturonate resulted in large decreases in viscosity. Very little
change in viscosity was observed in the case of terminal cleavage. Table 2.1 shows the
main classifications of pectic enzymes.

Table 2.1 Classification of pectic enzymes

 

Enzyme Mode of action

 

Polygalacturonase (PG) Cleaves Ot-(l—)4) glycosidic bonds

 

Pectate Lyase (PEL) Cleaves non-esterified galacturonate

units via B-elimination

 

Pectinesterase (PE) Hydrolyzes the methyl ester groups of

pectin.

 

 

 

 

This study will focus on the activity of pectinesterase enzyme, which is responsible for
the generation of methanol from pectin. A literature survey of reports on pectinesterase
and the theories involved in the mode of action of pectinesterase are discussed in the next

section.

2.3 Pectinesterase

Pectinesterases have been detected in a variety of plants. Most of the fruits have
been reported to have more than one isozyme of pectinesterase. Two pectinesterase
isozymes, each having a molecular weight of 30,000 Da, were detected in banana pulp
(Hultin et al, 1966) and they both exhibited an optimum activity at pH 7.5. Orange
pectinesterase is a well-studied enzyme. Two isozymes have been reported, each having
a molecular weight of 36,200 Da. Unlike the banana pectinesterase, these isozymes have
different optimal pH. Structural studies have shown that the enzyme is a glycoprotein
formed by a single low molecular weight polypeptide (Markovic and Jorvall, 1986;
Pressey and Woods, 1992; Glover and Brady, 1994). Generally, the isoelectric points for
pectinesterase isozymes are between 7 and 11 (Alonso et al). This enzyme is also
produced by a number of micro-organisms, including bacteria yeast and mold (Fogarty
and Kelly, 1983).

Pectinesterase catalyzes the hydrolysis of the methyl esters of pectin, generating
methanol. It removes the methoxyl group by a nucleophilic attack on the ester, resulting
in an acyl-enzyme intermediate. This is then followed by deacylation, where the
intermediate is hydrolyzed to regenerate the enzyme and a carboxylic acid (Wong, 1995).

The schematic of this mechanism is shown in Figure 2.2.

i?) r.-

E—N v? 0 CH3 ——> E—N—C—O-CH3
R

E-N ------ Enzyme

0

|| 0-
R—C— —OH +E-N (.1 O "OH
4—E—N—C—OH 4— NJ
aJR E—N—C

R

Figure 2.2. Mode of action of pectinesterase.

The effect of pH on pectinesterase has also been studied. Sun and Wicker (1996)
used the fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS) to determine the
surface hydrophobicity of the enzyme. ANS is non-fluorescent in water and other polar
environments, but highly fluorescent in non-polar environments or when bound to
hydrophobic sites on proteins. Changes in protein structure that result in an increase in
hydrophobicity can be estimated by the change in fluorescence of ANS. If pH causes a
change in the hydrophobic sites of protein, the structural change is detected by ANS
fluorescence. Based on ANS fluorescence, pectinesterase molecules were found to be
more hydrophilic at neutral and alkaline pH than at acidic pH values.

Current studies of pectinesterase activity indicate that the degree of activity can be

enhanced by the presence of cations. The stimulatory effect of NaCl on pectinesterase

varies considerably. F ayyaz and coworkers (1995) found that the stimulatory effect of
NaCl was quite high for NaCl concentrations between 0.1 and 0.15 M. The enzyme had
approximately the same activity betWeen 0.15 and 0.3 M, but as the concentration of
NaCl increased the activity of the enzyme declined. Labib and El-Ashwah (1995)
reported a similar effect on mango pectinesterase by NaCl. The maximum pectinesterase
activity was at 0.1 M, and the activity decreased gradually as the concentration was
increased to 0.4 M. Calcium chloride has also been shown to have a similar effect on the
activity of pectinesterase. It is worth noting that the cations increased the activity of

pectinesterase, but were not a requirement for its activity.

2.4 Enzyme Kinetics

Enzymes are proteins by nature, and they have highly specialized biological
activity. They retain their biological capacity and function in a very narrow range of
temperature and pH. Exposure of an enzyme to extremes of temperature or pH results in
denaturation of the enzyme and loss of its ability to catalyze a chemical reaction. The
polypeptide backbone of the protein molecule is not broken, but the polypeptide chain is
unfolded and the molecule loses its structure. Local structure is key to the functioning of
an enzyme.

Enzymatic activity is generally proportional to the enzyme concentration. During
the early stages of the enzymatic reaction, the relationship between enzyme concentration
and the rate of reaction is linear. This linear relationship has been reported (Mangos and

Haas, 1997) for orange pectinesterase.

10

During the early stages of an enzymatic reaction and for very short time intervals,
the substrate can be assumed to be constant (initial rate assumption). This is especially
true in cases where the substrate is present in excess. 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,
d[S]/dt = -k[S] (1)
where
d[S]/dt = rate of substrate consumption

[S] = substrate concentration at any given time during the reaction
k = reaction rate constant
The concentration of substrate remaining at any given time, may be expressed in terms of
the initial substrate concentration and product formed in view of the mass balance

relationship:

[3] = [S]i - [M]: (2)
where,
[S]i= Initial substrate concentration
[M], = Concentration of product at any given time
Enzyme activity is affected by a variety of factors. Two major factors are

temperature and pH. As the temperature is increased, two simultaneous reactions occur.
First, the enzyme activity increases, as observed in many chemical reactions, and
secondly, the enzyme stability decreases due to thermal denaturation (Laidler, 1954).

The net result of these two effects is a bell-shaped curve.

11

In the food processing industry, inactivation of enzymes by heat is a very
common procedure. Vegetables and 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. The inactivation of an enzyme can be described by first order

kinetics. Alter integration and substituting equation (2), equation (1) can be written as:

In ([S]i - [M]t)/ [Sli = -kt (3)
where,
[S],= Initial substrate concentration
[M], = Concentration of product at any given time
t = heating time
k = inactivation rate constant
A plot of equation (3) is linear and the slope represents the inactivation rate constant, k.
The constant k is highly pH and temperature dependent. Various reports have
demonstrated that thermal inactivation rates of pectinesterase in citrus juices increased at
lower pH values (Rouse and Atkins, 1952; Atkins and Rouse, 1954).
The rates of chemical reactions generally vary with temperature according to

Arrhenius law:
k = A e('Ea’RT) (4)
where
k = reaction rate constant,
A = Arrhenius constant
E, = Activation energy

T = Absolute temperature

12

To measure the inactivation rate constant, a small range of temperature is chosen, at
which inactivation is taking place. A plot of the overall temperature effect on the enzyme
activity will assist in determining the temperature at which inactivation occurs.

Fayyaz et al (1995) studied the activity of papaya pectinesterase. They found that
papaya pectinesterase showed an optimum reaction temperature at 65°C and declines
afier 70°C. They calculated the activation energy of papaya pectinesterase to be 5690 cal
mol'1 from 20 to 60°C. Similar results have been reported for orange pectinesterase by
Komer et a] ( 1980), who determined a value of 5740 cal mol". Another study by Lee

and Wiley (1970) found an activation energy of 5800 cal mol'l for apple pectinesterase.

2.5 Current methods of measuring pectinesterase activity

Several methods have been used to monitor the activity of pectinesterase. The
analytical methods can be categorized into three, depending on changes that are used to
quantify the activity of pectinesterase. The activity may be measured by: l) the
disappearance of the substrate; 2) the formation of certain products from the substrate; 3)
changes in physical properties of the substrate mixture. In this study the second method
was used, whereby the amount of methanol produced was used as a measure of the
activity of pectinesterase (PE). Other methods that are generally spectrophotometric in
nature have been extensively reported.

Wood and Siddiqui (1971) developed a titrimetric method to measure the amount
of methanol generated from pectin. Pectin is hydrolyzed using sodium hydroxide to

generate methanol. The methanol is then oxidized to formaldehyde with potassium

13

permanganate, followed by condensation with 2,4-pentanedione to yield the colored
product 3,5-diacetyl-1,4-dihydro-2,6-dimethylpyridine. The absorbance of this colored
product is monitored by UV-VIS. The permanganate oxidation method requires the
reduction of unreacted permanganate with sodium arsenite.

In an attempt to improve on the technique of Wood and Siddiqui, an enzymatic
method was proposed by Klavons and Bennett (1986) using alcohol oxidase to oxidize
methanol liberated upon hydrolysis of pectin. The use of alcohol oxidase eliminated the
reduction step with sodium arsenite and offers a two-fold increase in sensitivity. The
specifity of alcohol oxidase to oxidize lower primary alcohols to aldehydes was also
utilized.

Mangos and Haas (1997) have developed an alternate procedure that also utilizes
alcohol oxidase (ADD) to oxidize methanol to formaldehyde, but subsequently employs
the use of peroxidase and hydrogen peroxide. When methanol is oxidized it produces
formaldehyde and hydrogen peroxide. They quantitated hydrogen peroxide with
peroxidase (POD) and the chromogen 2,2’~azino—bis(3-ethylbenzthiazoline-6-sulfonic
acid) (ABTS). The reaction scheme is shown below:

N OH
Pectin-COOCH3 + HzO-a—->Pectin-COOH + CH3OH

CH3OH + 02 ADD . HCHO + H202
H202 + 2 ABTS(H) LCD—+2 H20 + 2 ABTS“

The generation of ABTS radical cation, which absorbs strongly at 420 nm, is proportional
to the amount of pectinesterase in the reaction.
In the fi'uit brandy industry, presence of methanol is accompanied by relatively

large amounts of ethanol. The techniques discussed above are not selective for methanol

14

over ethanol. In fact, ethanol has been reported to interfere with the analysis of methanol
(Wood and Siddiqui, 1971 ). Measurement of the activity of pectinesterase in the brandy
industry requires a technique that is particularly selective toward methanol in the
presence of large amounts of ethanol.

Gas chromatography is a fast and accurate method to measure alcohols, both
qualitatively and quantitatively. There are specific column requirements needed to
ensure separation of methanol from ethanol. Temperature programming is utilized in gas
chromatography for fast, selective separation (Skoog et a1. 1998). Optimization of
column temperature and carrier gas flow rates is therefore necessary to ensure the
efficiency of a gas chromatographic technique.

One major advantage of the gas chromatographic technique is the elimination of
reaction steps. Since methanol can be detected directly on the column, there is no need
for any firrther reactions with the methanol as is required with the other techniques
described above. Methanol generated from the demethoxylation of pectin can therefore
be detected easily on a polyethylene glycol column without any further modification. In
this work, this approach is used to quantify the activity of pectinesterase. The versatility
of this method also makes it efficient for rapid prescreening of potential inhibitors of

pectinesterase.

2.6 Inhibition of Pectinesterase activity

The control of pectinesterase activity has been the subject of study because of its

implications in the modification of the texture of fruit and vegetables (J averi and Wicker,

15

1991). It has also been found to act as a destabilizing agent of pectin materials in fruit
and juice concentrates (Seymour et al., 1991 ).

Inhibitors decrease the rate of an enzyme-catalyzed reaction. Control of the
enzymatic activity of pectinesterase is of importance in regulating the amount of
methanol generated from the deesterification of pectin. The amount of methanol in any
food product needs to be regulated for obvious health reasons associated with the toxicity
of methanol. Several authors have attempted inhibiting the activity of pectinesterase.

Veersteg et al (197 8) were able to show that pectinesterase is inhibited by
polygalacturonic acid. Fayyaz et al (1995) also showed that papaya pectinesterase was
competitively inhibited by polygalacturonic acid. They calculated the inhibition constant
as 0.019 mg/mL. Lineweaver and Ballou (1945) suggested a cation-carboxyl
complexation of the acid was responsible for the inhibitory behavior of polygalacturonic
acid. They also suggested that at higher pH, the cations were less effective because PE
was less protonated and therefore had less of a tendency to form an inactive complex
with an anion inhibitor.

Pectinesterase has also been inactivated by low pH. Owusu-Yaw er a1 (1988)
used hydrochloric acid to lower the pH of orange juice from 3.75 to 2.0. Pectinesterase
in the juice was completely inactivated at the low pH. This method of inactivation is,
however, not applicable in the fruit brandy industry. In order to inactivate the enzyme
while fermenting the fruit mash, very low pH needs to be avoided. Very low pH results
in the inactivation of the yeast as well, and therefore pectinesterase activity is inhibited,

but this process also stalls fermentation.

16

Hall (1966) studied the effect of tannic acid on tomato pectinesterase. He was
able to obtain complete inhibition with 5 x 10'5 molar concentration of tannic acid using
0.05 per cent pectin as the substrate. Isolation of an inhibitor of pectinesterase from Kiwi
fruit has also been reported by Balestrieri et a1. (1990). The inhibitor completely
inhibited pectinesterase in the pH range between 3.5 and 7 .0. The inhibitor was tested on
a variety of pectinesterases from various sources including orange, tomato, potato,
banana and apple pectinesterases.

The most frequently used technique to inhibit the activity of pectinesterase is by
thermal inactivation of the enzyme. Nakagawa etal., (1970) completely inactivated
purified pectinesterase by heating between 65 and 90°C. Labib et al., (1995) observed
stepwise inactivation of mango pectinesterase activity, which was attributed to a possible
presence of more than one type of pectinesterase. Heat inactivated pectinesterase did not
regenerate upon frozen storage.

Ion exchange membranes have also been used to evaluate their effect on
pectinesterase-pectin complexes. Chen et al., (1998) used both anionic and cationic ion
exchangers to bind pectinesterase and pectin. They observed that at pH 7.2, anionic
exchange disks had little binding of pectinesterase, but its binding to a cationic membrane
was significantly higher at the same pH.

Interactions between pectinesterase and ion exchangers depend on several factors,
including the net surface charge on pectinesterase. Other factors include pH, ionic
strength, and the nature of the ion (Karlsson et al, 1989). One of the most important
factors that determine protein binding to ion exchange membranes is pH, since it affects

the effective charge on both the protein and the ion exchanger.

17

Theoretically, pectinesterase is positively charged at pH less than 8.0 while pectin
is negatively charged at pH values above 4.0 (Chamay et al., 1992; Lineweaver and
Ballou, 1945). Under such conditions, positively charged pectinesterase can combine
with pectin at the ionized carboxyl groups.

In understanding the effect of cations on the activity of PE, the pH of the assay
needs to be considered. At lower concentrations, cations displace pectinesterase from the
inactive pectin- pectinesterase complex and stimulate activity. At higher concentrations,
cations competitively bind carboxylic acid groups and act as competitive inhibitors of
pectinesterase activity. This competitive displacement theory is probably not the only
factor involved in binding and release of pectinesterase from pectin complexes, but is a
considerably reasonable explanation of the effect of ions on pectinesterase activity.

In this study a variety of inhibitors are tested to determine their effect on the
activity of pectinesterase. Most of the inhibitors used are known chelating agents that

should significantly bind to a positively charged protein such as pectinesterase.

18

3. MATERIALS AND METHODS

3.1 Methanol Analysis
3.1.] Fermentation

A variety of fruits were used to monitor the fermentation process, namely
cherries, pears, peaches and plums. The fruits were stored frozen and had to be thawed
before crushing. They were then crushed using a stem crusher and pumped into a 200-
gallon fermenter. 300g of yeast (Prise de Mouse) were added to 600 mL of 45°C
distilled water and left for 15 minutes to activate the yeast. The activated yeast was then
added to the fruit in the fermenter and continuously stirred. The temperature of the
fermenter was maintained at 15 °C using a water chiller. Fermentation progress was
monitored for a period of 10 days. Dissolved sugars in the broth give an indication of the
fermentation progress and this was monitored on a daily basis using a refractometer. The
pH of the broth was also monitored on a daily basis.

For monitoring the generation of methanol during fermentation, the samples were
centrifuged for 2 minutes at 1400 rpm and filtered through 0.5um filter paper to remove
all the solid particles. 0.5 uL of each sample were then injected into the gas

chromatograph.

3.1.2 Distillation

Distillation was performed using a Christian Carl still shown in figure 3.1. The
still is equipped with three trays, a partial condenser and a catalytic converter. Once the
brix of the fermented broth reached a steady value, fermentation was assumed to be

complete.

19

 

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The mash was then pumped into the still and continuously stirred for the duration
of the distillation. Cooling water was circulated through the entire system before
distillation began. Steam was used to heat the fruit mash and a pressure of 0.3 to 0.4 bar
was maintained throughout the distillation. The number of trays to be open or closed, as
well as the engaging of the “catalytic converter” varied from one distillation rim to the
other. The “catalytic converter,” is made up of a high surface area copper packing
material thought to strip cyanide out of the distillate and therefore reduce the amount of
urethane in the spirits.

The distillate started condensing at approximately 90% v/v alcohol and distillation
was continued until the alcohol level dropped to 45% v/v alcohol. The distillate collected
after this level contains a significant amount of higher alcohols and generally does not
have good taste qualities. The distillates were collected as different cuts in order to
quantify the methanol at each stage in the distillation. F our-500 mL samples were
collected to represent the heads, followed by five l-liter samples to represent the hearts
and finally 1 liter was collected as tails. The steam was then turned off, the still emptied

and cleaned for the next run.

22

3. 1.3 Analysis of Distillates.

Each of the different cuts was diluted with distilled water in order to obtain an
alcohol content of 40 % v/v. This is the minimum alcohol content that meets the
definition of a fruit brandy. One mL of each cut was sampled into vials for analysis.

Analysis was performed by gas chromatography using a Shimadzu GC-17A gas
chromatograph equipped with a flame ionization detector (FUD) and auto sampler. A
Stabilwax® column from Restek Corporation was used for the separation of the alcohols.
The stationary phase of this column is polyethylene glycol and is particularly good for the
separation of alcohols. The run conditions on the GC were as follows:

Column: 30m, 0.32 ID, 0.5 um Stabilwax®
0.5 uL split injection
Oven temp: 40°C (hold for 1 minute); to 190°C at 25°C /min.

190°C (hold for 5 minutes)

Injector temp: 240°C

Detector temp: 255°C

Carrier gas: Helium at 30 cm/sec
Split ratio: 65:1

Total GC run time: 65 minutes

The high final column temperature was used to ensure that most of the other
higher boiling alcohols were removed from the column at the end of the run. Very small
amounts of methanol relative to ethanol are present in the sample. A typical gas
chromatogram is shown in figure 3.2. The major component of fruit brandy is ethanol

and this is reflected on the chromatogram. The other major components detected are

23

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methanol, acetaldehyde, isopropanol and isoamylalcohol. The compound of major
interest in this work was methanol. As is evident in the chromatogram detection of

methanol was achieved with this temperature programming on the column.

3.1.4 Quantification of Methanol

In order to quantify the amount of methanol in the distillates, a calibration curve
was generated. Calibration samples were made using methanol, ethanol and water
mixtures in order to duplicate the matrix system in the distillate samples. The overall
ethanol content of all samples was 40% and the methanol concentration was varied from
0 to 1% v/v. Each calibration sample was injected into the GC and the methanol
quantified.

A calibration curve was set up using the chromatographic peak areas. Analysis
of the chromatographic data was done using Class-VP software from Shimadzu.
Quantification of methanol in all other samples was determined relative to this external

standard calibration curve.

25

3.2 Pectinesterase Activity
3. 2.1 Measurement of PE activity

Purified commercial pectinesterase (EC 3.1.1.11) enzyme and apple pectin
purchased from Sigma Chemical Company were used in this investigation. The
manufacturer specified pectinesterase activity as 130 units per mg. One unit of
pectinesterase is defined as the amount of enzyme that will liberate one mole of
methanol per minute. Concentration of 4 mg/mL of pectin was selected due to solubility
limitations. This was the maximum amount of pectin that could easily dissolve in water.
Pectinesterase was used at a concentration ranging from 0.1 mg/mL to 0.4 mg/mL. A
stock solution (21.6 mg/mL) of the enzyme was diluted with phosphate buffer (pH 7.0),
the enzyme’s optimum pH, to give the final concentrations used. Pectin substrate (0.5
ml.) was incubated with 0.5 mL of pectinesterase for a period of time and the methanol
released quantitated. All incubations were done at room temperature. The run conditions
on the GC were similar to those used for analysis of distillates, except for the oven
temperature and GC run time which were:

Oven temp: 40°C to 100°C at 3°C /min. (hold for 0.5 minute)

to 190°C at 20°C/min.

Total GC run time: 25 minutes

26

3. 2. 2 Temperature Dependence

To test the stability of the enzyme at various temperatures, a fixed incubation time
of 5 minutes was chosen. The temperature ranged from 20°C to 90°C. The enzyme was
heated in a water bath and maintained at the respective temperature for five minutes
before being added to the substrate. An enzyme to substrate ratio of 1:1 was incubated
for five minutes, and then 0.5 uL of each sample was injected into the GC. The amount

of methanol in each sample was quantitated.

3. 2. 3 Thermal Inactivation of Pectinesterase

Thermostability of pectinesterase was tested by heating in the temperature range
between 60 and 75°C. For cherry juice samples, 10 mL samples were heated to 30°C,
55°C, 70°C and 90°C. The samples were kept in water baths at the respective
temperatures for 5 minutes before sampling. They were then centrifuged and filtered

before being introduced to the gas chromatograph.

3.3 Inhibition of Pectinesterase

Once the activity of pectinesterase was established, the next step was to study
potential inhibitors for this enzyme. Several potential inhibitors were identified based on
previous literature reports or similarity in functional groups that the enzyme targets in
pectin. The specific inhibitors tested included tannic acid, polyacrylic acid,

polymaleimide, ethylenediamminetetraacetic acid (EDTA) and citric acid.

27

3. 3. I T annic Acid

Tannic acid was purchased from Sigma Chemical Company. This was used on
cherry fruit juice samples instead of commercial pectin. The appropriate amount of
tannic acid powder was added to 500 mL of cherry juice samples to make a final
concentration ranging from 0 to 1 mM. Yeast was also added to each sample to monitor
the effect of tannic acid on fermentation. The sample without any tannic acid served as a
control. Every 24 hrs, 2 mL samples were centrifuged and filtered with a 0.45 pm filter
paper to remove any particles that might clog the GC column. Brix measurements were
then taken and methanol content for each sample determined by GC. These
measurements were taken until the fermentation ceased and samples were kept in the cold

room (15°C) during the fermentation period.

3. 3.2 Polyacrylic acid

Polyacrylic acid with a molecular weight of approximately 2100 was purchased
from Sigma Chemical Company. For this study, commercial apple pectin and purified
pectinesterase were used as the substrate and enzyme, respectively. The sodium salt of
polyacrylic acid was tested as an inhibitor at a concentration of 1 % w/v. The same
inhibitor concentration was used over a range of pectin solutions (1- 4 mg/mL) in order to
understand the dependence of the inhibition process on substrate concentration. A 1:11]
mixture of pectinesterase, pectin and polyacrylic acid was incubated for 5 minutes at
room temperature. One half uL of each sample was injected into the GC to quantitate

methanol. Control samples for each pectin concentration, without polyacrylic acid were

28

also incubated for 5 minutes before injection. A comparison for each pair gave an

indication of the extent of inhibition.

3. 3.3 Polymaleimide

Polymaleimide was selected as a potential inhibitor because it possesses
carboxylate functional groups that are a likely target for pectinesterase. For preliminary
studies, 1% w/v sodium polymaleimide solution was prepared. The polymaleimide used
was synthesized in our laboratory. A 1:12] mixture of pectin, pectinesterase and
polymaleimide was incubated for 5 minutes. Various pectin concentrations ranging from
1 to 4 mg/mL were used and similar control samples without polymaleimide were also
incubated for 5 minutes at room temperature. The extent of inhibition was determined as
the difference in methanol concentrations between the samples with and without
polymaleimide.

Since the 1% w/v solution showed promising results, lower concentrations of
polymaleimide were tested. Solutions of 0.25% and 0.5% w/v were tested in a similar

manner as the 1% w/v polymaleimide inhibitor.

3. 3.4 Citric Acid

Citric acid has both low pH and carboxyl groups. These are desirable qualities for
an inhibitor of pectinesterase and therefore its inhibitory effect was tested. Commercial
pectin and pectinesterase were used for this study. One percent w/v citric acid was used
with pectin concentration of 4 mg/mL. Increasing volumes (0.1 to 0.5 mL) of 1% w/v

citric acid were added to the pectin-pectinesterase mixture. The mixtures of pectin,

29

pectinesterase and citric acid were each incubated for 5 minutes at room temperature.

One half uL of each sample was injected into the GC to quantitate methanol.

3. 3.5 Ethylenediamminetetraacetic Acid (EDT A)

EDTA is a strong chelating agent and its ability to inhibit the activity of
pectinesterase was tested. Pectin substrate concentrations used ranged from 1 mg/mL to
4 mg/mL and pectinesterase concentration was 0.432 mg/mL. 1% w/v solutions of di-
sodium EDTA were incubated for 5 minutes at room temperature with PE and pectin.
The total incubated volume was 1.5 mL consisting of 0.5 mL of each constituent.
Control samples were prepared in a similar manner except that the inhibitor was not
added to the reaction mixture. One half [L of each sample was injected into the GC to

quantitate the methanol produced.

30

4. RESULTS AND DISCUSSION

4.1 Composition of Fruit Brandy

In order to monitor the progress of fermentation, the brix measurements were
taken every 24 hrs for a period of days. The pH of the fermented mash was also taken in
order to keep track of any changes that might affect the functioning of the yeast. As is
evident in Table 4.1 the fermentation was virtually complete in 9 days. The remaining

dissolved sugar is sorbitol, which is not digested by yeast.

Table 4.1 Brix values of fermented mash of different fruits. Fruit mash stirred
in a 200 gallon fermenter maintained at 15°C by a water chiller. 300g

Prise de mouse yeast used for each fermentation.

 

 

 

 

 

 

 

 

 

 

 

Day Plum Peach Cherry
1 13.0 10.3 13.7
2 12.6 9.7 13.4
3 11.1 9.2 13.3
4 8.8 9.0 12.6
5 6.8 7.1 10.5
6 6.0 4.7 6.6
7 6.1 4.4 6,4
8 6.2 4.5 6.3
9 6.2 4.4 6.0
10 6.2 4.4 6.0

 

 

 

 

 

 

31

4.2 Methanol in Fruit Brandy
4. 2. 1 Quantitative Analysis.

The amount of methanol in fruit distillates is regulated by the FDA at 0.35% v/v
in the drinkable product (40% v/v ethanol), so the initial step in this study was to quantify
the methanol content of the fruit brandy. In order to do the quantitative studies, a
methanol calibration curve was generated. The calibration curve that was used to
quantitate methanol throughout this study is shown in figure. 4.1. The linear plot has a
correlation coefficient of 0.9987 and the lower detection limit of 0.003% v/v corresponds
to 0.023 ug/mL.

Methanol generated during fermentation was monitored (figure. 4.2). The plot
shows a very small increase in methanol during fermentation. This implies that most of
the methanol present in the final distillate is already in the fruit mash even before the
onset of fermentation. Addition of yeast does not favorably affect the activity of
pectinesterase.

A typical batch brandy distillation has three main components based on the
product recovered: the heads, hearts and tails. Heads, the first 2000 mL recovered from
the 150 L Christian Carl still, constitute the low boiling components such as acetaldehyde
and methanol. The typical alcohol content for this cut is above 80% v/v. The hearts,
which is the main drinkable product and comes out next, consists mainly of ethanol and
ranges between 55% v/v to 80% v/v alcohol content. This is typically diluted to at least
40 % v/v to meet the legal definition of a fruit brandy. The tails are rich in higher boiling

components such as fusel oils, which are alcohols with more than two carbons and

32

80000

Area

Figure 4.1.

 

 

R2 = 0.9937
y = 60750x

 

 

 

I I I I I

0 0.2 0.4 0.6 0.8 l 1.2
Methanol (°/o v/v)

Methanol calibration curve. Area corresponds to chromatographic
peak area. The GC run conditions were as follows: Oven temp: 40°C
(hold for 1 minute); to 190°C at 2.5°C lmin (hold for 5 minutes),
Injector temp: 240°C, Detector temp: 255°C, Carrier gas: Helium at
30 cm/sec, Injected volume: 0.5 uL. All other methanol
concentrations were determined relative to this external standard

curve.

33

 

0.029

 

 

 

g 0.026 . i I i
.3: .
'5
E
63
5'.
° ’ i
2 0.023, f

0002 f I l l l

0 2 4 6 8 10 12

Figure 4.2. Generation of methanol during fermentation. Montmorency cherries
fermented at 15°C with Prise de mouse yeast. Analysis of methanol
was done every 24 hours. Samples were filtered and centrifuged

before injecting into the GC.

typically contain less than 5 5% alcohol. This cut is usually either discarded or re-

distilled to improve its quality attributes.

Methanol is a low boiling alcohol (65°C) relative to ethanol and would naturally
distill mostly in the heads and hearts cuts. A typical methanol profile is shown in figure.
4.3. It is evident that the highest level of methanol is present in the heads cut. Surprising
is the fact that the methanol in the tails cut is slightly higher than that in the hearts.
Methanol content of distillates from the same fermentation batch also varied

The major component of fruit brandy is ethanol. The yeast used in fermentation
converts glucose into ethanol and carbon dioxide. The conversion is a 1:2 ratio such that
for every 1 mole of glucose, 2 moles of ethanol are generated. Acetaldehyde is present in
fruit brandy and is generated from the oxidation of ethanol with catalysts such as copper.
The distillation still is made out of copper, and this copper surface catalyzes the
production of acetaldehyde during distillation. Cracked pits are thought to increase the
amount of urethane and benzaldehyde in fruit brandy. Benzaldehyde is a positive flavor
compound, but urethane is a carcinogen. Benzaldehyde is produced through the

hydrolysis of amygdalin, which is present in most stone fruits.

35

 

 

 

 

 

 

 

 

0.6
+ batch #l
-I- batch #2
0.5 - + batch #3
E
>
e\°
‘O’ 0.4 J
E
a
in:
8
2
\
0, - ‘U
0.2 I I I r
0 2 4 6 8 10
D'utillation cut number
Figure 4.3. Methanol profile during distillation. Different distillation batches

from the same fermentation batch. Distillation performed with all 3
trays engaged. Distillation cut number corresponds to different
volumes of distillate samples collected in the course of the distillation.

Results shown are for cherry fruit brandy.

36

4. 2.2 Fruit Varieties

Methanol in fruit brandy is generated from the fi'uit pectin. It has been reported
that different fruits have different amounts of pectic substances. Table 2.1 shows an

example of the total pectic substances in different plants.

Table 4.2 Amount of Pectic substances in various plants.2

 

 

 

 

 

Fruit Pectic substances (%)
Apple 14.9

Cherries l 1.4
Beets 13.2

Orange 16.8

 

 

 

 

After distillation, each cut was sampled for methanol. The results of methanol
analysis for various fruit distillates are shown in figure 4.4. The results are plotted for an
average of three distillation batches for each fruit. The amount of methanol in the
distillates varied from fruit to fruit, but generally the highest amount of methanol was
found in plums on an average basis. The amount of methanol varied from batch to batch
even within the same fruit type. The methanol content also varied with distillation

conditions such as the number of trays closed and use of the catalytic converter.

 

2 Data obtained from The Pectic Substances by L]. Kertesz.

37

 

Methanol (% WV)
9
u

9
N

 

 

+ Cherry
+ Plum
+ Peach

 
   

 

 

 

 

 

0. 1 ‘—
0 I T I I
0 2 4 6 8 10
Distillation cut number
Figure 4.4. Methanol profile among different fruit varieties. Fermented at 15°C

with Prise de mouse yeast. Methanol content average of three
distillation batches for each fruit. Distillation performed with all 3
trays engaged.

4.3 Activity of Pectinesterase

4. 3.] Enzyme Kinetics.

The magnitude of enzymatic activity is usually proportional to the concentration
of the enzyme. This proportionality is well exhibited during the earliest stages of the
reaction. The linear relationship between enzyme concentration and rate of reaction is
common in many enzyme-catalyzed reactions.

During the early stages of an enzymatic reaction, and for very short time intervals,
the substrate concentration can be assumed constant. This is particularly true in cases
where the substrate is present in excess, and the amount of product formed is proportional
to reaction time. In order to understand the kinetics of pectinesterase, the substrate-
enzyme reaction was monitored over a period of one hour. Figure 4.5 shows the activity
of pectinesterase with time. From this figure, it is evident that the reaction is linear for
the first 20 minutes and then it significantly slows down after 40 minutes. The reaction
seems to get to completion after releasing 0.049% v/v methanol.

The concentration of substrate used in this study was 4 mg/mL pectin, and the
maximum methanol released was 0.387 mg/mL. On this basis, the concentration of
pectin-bound methanol is 0.096 mg/mg pectin, corresponding to 9.6% methoxy content in
the pectin. The citrus pectin used had a methoxy content of 10% w/w as specified by
manufacturer. The incomplete demethoxylation of pectin by pectinesterase is in

agreement with results from other laboratories (Mangos, 1997), which have shown that

39

 

  

Methanol (°/o v/v)
P
c
u

0.02 -

 

 

 

0.01 4
0 T U r I 1
0 20 40 60 80 100

Time (minutes)

Figure 4.5. Activity of pectinesterase as a function of time. Pectin concentration
(4 mg/mL) and 0.432 mg/mL pectinesterase concentration.
Pectinesterase prepared in phosphate buffer pH 7.0. Incubation at
room temperature. 0.5 uL of each sample injected into the GC after

incubation for the period of time shown.

pectinesterase from higher plants demethoxylates blocks of methoxylated
polygalacturonic acids and ester groups next to free carboxyl groups. Fungal
pectinesterase on the other hand is capable of demethoxylating methyl esters that are not
present in blocks. The results obtained here are similar to those obtained using a
spectrophotometric assay that gave a methoxy content of 9.56% for citrus pectin and

orange pectinesterase.

4. 3.2 Eflect of Temperature

Pectinesterase activity was measured at different temperatures to establish the
therrnostability of the enzyme. Four mg/mL citrus pectin was used as the substrate and
the assay was carried out at pH 7.0, reported in the literature as the optimum pH for
pectinesterase. The temperature range was from 20°C to 90°C, the broad range where
pectinesterase is activated and subsequently denatured. Each sample was incubated for a
period of 5 minutes at the respective temperature.

Figure 4.6 shows the results for the effect of temperature on the formation of
methanol. The region below 50°C is where the enzyme is activated in accordance with
normal enzyme behavior. Beyond 5 5°C, the enzyme is denatured and the activity
declines dramatically. Heat-inactivation of the chemical reaction occurs according to the
Arrhenius law.

To determine the order of the inactivation reaction, the measured methanol values

for the inactivation region were plotted versus time. Under the experimental conditions

41

 

 

 

 

0.025
0.02 ~
3
>
5 0.015 —
'3
1:
a
:5
g 0.0] ~
0.005 ~
0 fl 1 I T
0 20 40 60 80 100
Temperature (°C)
Figure 4.6 Effect of Temperature on the formation of methanol. Pectin

concentration: 4 mg/mL, Pectinesterase: 0.432 mg/mL. Pectinesterase
prepared in phosphate buffer pH 7.0. The samples were incubated at
the respective temperature for 5 minutes before injection into the GC.

42

the data fit a straight line on semi-log coordinates and were described by a first order

function. Figure 4.7 illustrates the normalized inactivation rate curves for pectinesterase.

Table 4.3 Inactivation rate constants for pectinesterase at various temperatures.

Rate constants evaluated from straight line plots of equation (3).

 

 

 

 

 

Temperature (°C) Rat; i033?"
60 2.2
65 2.5
70 2.8
75 3.2

 

 

 

 

The inactivation rate constants were determined from equation (3 ). Table 4.3 shows the
rate constants as they were evaluated from the straight line plots at different temperatures.
Computed values of the rate constant are similar to that reported in the literature. It is
evident that the higher the temperature the higher the rate constant which results in
steeper curves.

Another way to express the influence of temperature on the rate constant is the

well known Arrhenius equation (4) rewritten as:

 

1.2

 

. + 60°C

1 i -I- 65°C
-a- 70°C

 

 

0.8 -

0.4 -

 

0.2 *

 

 

0 l I I I
0 100 200 300 400 500
Time (seconds)

 

Figure 4.7 Normalized inactivation rate curves for pectinesterase. Pectin: 4
mg/mL, pectinesterase: 0.432 mg/mL. Pectinesterase prepared in
phosphate buffer pH 7.0. One half 11L of each sample injected into the
GC after incubation for the period of time shown. Initial methanol

concentration (Co) determined after 30 seconds of incubation.

1n k = In A - Ea/RT (5)
where,
k = reaction rate constant,
A = Arrhenius constant
B, = Activation energy
T = Absolute temperature
The Arrhenius plot for pectinesterase inactivation at pH 7.0 is illustrated in figure 4.8.
The activation energy was calculated to be:
E, = 5697 cal mol'1
To explain the magnitude of the activation energy, the structure of the enzyme
must be taken into consideration. Enzymes are complicated protein molecules and their
catalytic properties are associated to their highly ordered tertiary structure. The tertiary
structure is maintained primarily by weak noncovalent bonds that must be broken during
the rate determining step. The activation energy found here falls in the range generally
accepted for pectinesterase. This range is from 5600 to 7644 cal mol'](Fayyaz et al,
1995). The relatively small activation energy indicates that the enzyme molecule has a

very delicate structure.

4. 3.3 Thermal Inactivation of Pectinesterase in Cherries
The amount of methanol present in the cherry juice sample was measured at different
temperatures under natural pH conditions. The samples were maintained at the specified

temperature for 5 minutes. The results (figure 4.9) show a sudden drop in the amount of

-5.8 i

-5.9 -

Lnk

-6.l -

-6.2

 

 

 

 

I I I

0.00285 0.0029 0.00295 0.003 0.00305

Figure 4.8

III

Arrhenius plot for pectinesterase inactivation. Pectin: 4 mg/mL,
pectinesterase: 0.432 mg/mL. Pectinesterase prepared in phosphate
buffer pH 7.0. Activation energy was calculated from the slope to be
5697 cal mol".

0.07

0.06

P

G

U!
r

Methanol (%v/v)

0.02 1

0.01

0 I r T

Figure 4.9

p
E

P
G
b)

a

 

d

 

 

 

0 25 50 75 100
Temperature (°C)

Thermal Inactivation of Pectinesterase in cherry juice samples. Five
hundred mL cherry juice heated for 5 minutes at the respective
temperature. Samples centrifuged and filtered before injection into
the GC.

47

methanol at 55°C. At temperatures above this, low levels of methanol are observed,
indicating that PE has been substantially inactivated. Heated samples were cooled to
room temperature and further measurements were taken, but the inactivation of PE was
irreversible therefore no change was observed in the results. These observations are in

accordance with normal enzyme inactivation behavior.

4.4 Inhibition of Pectinesterase

Studies were conducted to test the ability of different compounds to inhibit
pectinesterase. Initial studies were done using citrus pectin as the substrate that was
incubated with pectinesterase from orange peel, both purchased from Sigma Chemical
Company. The fact that theoretically pectinesterase is positively charged at pH 7.0 was
used to study its complexation with inhibitors. Most of the inhibitors tested are well
known chelating agents that should be able to bind to a positively charged protein such as

pectinesterase.

4. 4.2 Eflect of T annic Acid

Tannic acid was tested for its effect on pectinesterase using cherry juice samples
as a source of pectinesterase. The results shown in figure 4.10 indicate a loss in
pectinesterase activity for tannic acid concentration of 0.25 mM. However, an increase in
tannic acid concentration did not have an inhibitory effect on pectinesterase.
The effect of tannic acid on pectinesterase was studied by Hall (1966) and found to

inhibit pectinesterase with extremely high losses in pectinesterase activity. However, this

48

 

0.026

 

0.023 -

0.02 J

 

 

 

 

0.017 -1

0.014 -

 

Methanol (% v/v)

0.011 4

0.008 -

 

 

0.005 U I I I U
0 0.2 0.4 0.6 0.8 l 1.2

Tannic Acid (mM)

 

Figure 4.10 Effect of tannic acid on the activity of pectinesterase. #1 and #2
represent two separate batches of cherry juice incubated with tannic
acid. Cheery juice samples fermented with Prise de mouse yeast in the
cold room (15°C). The final pH of the samples ranged from 3.82-4.70.

49

study was carried out on a model system of purified commercial pectinesterase and
pectin. Results obtained in this study indicate that fruit juice samples may contain
compounds that interfere with the inhibitory effect that tannic acid has on pectinesterase,

but further studies need to be conducted to ascertain this.

4. 4.2 Eflect ofPolyacrylic Acid

The role of carboxylates in the inhibition process was investigated using
polyacrylic acid. One percent w/v solutions of the sodium salt of polyacrylic acid were
mixed with pectinesterase and pectin, and incubated for 5 minutes. Comparison was
made between methanol concentrations of samples with and without the inhibitor. Figure
4.11 shows the effect of polyacrylic acid on the reaction. It is worth noting that in all
cases some decrease in methanol generated after incubation was observed. The best
inhibition, 45%, was obtained using 4 mg/mL pectin as the substrate. This is a significant
change in pectinesterase activity and is very useful in applications where the objective is
to minimize and not eliminate methanol. In the fruit brandy industry, methanol
contributes to good flavor qualities but the amount needs to be regulated for health
reasons. Therefore, the kind of inhibition obtained with polyacrylic acid is ideal for this
kind of application.

Polyacrylic acid was selected as a potential inhibitor due to its carboxylate nature.
As discussed previously, pectinesterase has an overall positive charge at pH 7 .0, the pH
at which all the inhibition studies were conducted. Use of Polycarboxylates therefore

allows pectinesterase to complex with the inhibitor and ultimately reduces the activity of

the enzyme.

 

0.025

 

.. after inhibitor

 

 

 

0.02 4

0.015 r

0.01 —

Methanol (% v/v)

 

0.005 ~

 

 

 

0 I I
0 2 4 6
Pectin ( mg/ mL)

Figure 4.11 Effect of polyacrylic acid on the activity of pectinesterase.
Pectinesterase concentration: 0.432 mg/mL, prepared in phosphate
buffer pH 7.0. One half uL of each sample injected into the GC after
incubation at room temperature for 5 minutes. Inhibition range: 30-

45%.

51

4. 4.2 Eflect of Polymaleimide

Based on the results obtained with polyacrylic acid, it was proposed to conduct
further studies using polymaleimide, which has similar properties but also possesses the
advantage of being biodegradable.

The effect of polymaleimide on the activity of pectinesterase was studied in order
to ascertain its inhibiting capability. Preliminary experiments were carried out with 1%
w/v solutions of the sodium salt of polymaleimide. Solutions of citrus pectin,
pectinesterase and polymaleimide were incubated for a period of 5 minutes. Results for
this experiment are shown in Figure 4.12. The average inhibition was 35%, close to that
obtained with polyacrylic acid.

It was then decided to reduce the amount of polymaleimide used in the inhibition
in order to establish the minimum amount that would inhibit pectinesterase. The same
incubation reaction was carried out under lower concentrations of polymaleimide. The
results (figure 4.13) indicate that a concentration as low as 0.25% w/v of polymaleimide
is capable of inhibiting PE. This is very promising, since lower concentrations of the

inhibitor would greatly enhance its applicability.

52

 

0.025

0.02 4

0.015 ~

0.01 .

Methanol ( °/o v/v)

0.005 -

 

 

l —- before I

 

a» after inhibitor

l—_——'

 

I
r
4
l

 

 

Figure 4.12

I I T I

1 2 3 4 5
Pectin ( mg/ml)

Effect of polymaleimide on the activity of pectinesterase using 1% w/v
sodium salt of polymaleimide. Pectinesterase concentration: 0.432
mg/mL, prepared in phosphate buffer pH 7.0. One half 11L of each
sample injected into the GC after incubation at room temperature for

5 minutes. Inhibition range: 30-40%.

53

 

 

 
   

 

 

 

 

 

 

0.025
+ control
+ 1% PMA
“~02 ‘ ~n—0.5°/. PMA
~316- 0.25% PMA
3
a; 0.015 -
'8
1:
.2
2'5 0.01 .
2
0.005 ~
0 I l l 1
0 1 2 3 4 5
Pectin ( mg/ml)
Figure 4.13 Effect of Polymaleimide on the activity of pectinesterase.

Concentrations of polymaleimide: 0.25, 0.5, 1% w/v. Pectinesterase
concentration: 0.432 mg/mL, prepared in phosphate buffer pH 7.0.
One half uL of each sample injected into the GC after incubation at

room temperature for 5 minutes.

4. 4.3 Eflect of EDT A

Considering the performance of polyacrylate and polymaleimide, it was decided
to test the inhibiting properties of a known chelating agent. Ethylenediamminetetraacetic
acid (EDTA) was selected for this study. EDTA has six electron donating sites that make
it a very good ligand. One percent w/v solutions of the sodium salt of EDTA were
incubated with pectinesterase and pectin for a period of 5 minutes. Control samples for
each incubated one were also done in order to determine the extent of inhibition.

The results are shown in Figure 4.14. The highest inhibition was observed for 4
mg/mL pectin substrate. The overall effect of EDTA on the inhibition process is very
similar to that observed with both polyacrylic acid and polymaleimide. This suggests that
chelating effects of the inhibitors used can explain a reasonable amount of the inhibition

process.

4.4.4 Effect of Citric Acid

Citric acid has carboxylate groups that have been shown to inhibit pectinesterase
in the tests performed above. It also has a low pH that can be used to reduce the overall
pH environment of the enzyme. Different volumes of citric acid were added to the
incubation mixture and its effect monitored. The results (Figure 4.15) show that 0.5 mL
of the 1% w/v citric acid solution reduced the activity of pectinesterase to an extremely

low level.

55

 

 

 

 

 

 

 

 

 

0.03
—-Without

0.025 . ....... With EDTA
3 0.02 ,
>
.\°
'5 0.015 . f
a
e
0
2 0.01 ~

0.00s - I

0 . . . .
0 1 2 3 4 5

Pectin (mg/ml)

Figure 4.14 Effect of EDTA on the activity of pectinesterase. EDTA
concentration: 1% w/v. Pectinesterase concentration: 0.432 mg/mL,
prepared in phosphate buffer pH 7.0. One half pl of each sample
injected into the GC after incubation at room temperature for 5

minutes. Inhibition range: 38-41%.

 

 

Methanol (% v/v)
P
SE
UI

0.01 i

0.005 ~

 

 

 

0 I I I I T
0 0.1 0.2 0.3 0.4 0.5 0.6

Citric acid ( mL)

Figure 4.15 Effect of Citric Acid on the activity of pectinesterase. Citric acid: 1%
w/v, pectin concentration: 4 mg/mL. Pectinesterase concentration:
0.432 mg/mL, prepared in phosphate buffer pH 7.0. Resulting pH of
samples: 3.12-3.38

57

5. SUMMARY AND CONCLUSIONS

The amount of methanol in fruit brandies routinely exceeds the 0.35 %v/v legal limit
set by the FDA. This is particularly true for cherry brandy, which was found to have the
highest amount of methanol on average, compared to the other fruit brandies. The
highest concentration of methanol was recovered in the heads cut during the distillation
for all the different varieties of fruit brandy.

Results obtained from monitoring the amount of methanol during fermentation
showed no major increases in the methanol content of the fermented mash. This
indicates that most of the methanol is produced well before the initialization of
fermentation of the fruit.

Kinetics of the activity of pectinesterase were studied. Commercial purified
pectinesterase was used at a concentration of 0.432 mg/mL. It was tested at different
temperatures with various concentrations of pectin substrate. The inactivation rate
constant at 70°C was found to be 2.8 x 10'3 s". The activation energy for the
inactivation reaction at pH 7.0 was calculated from the Arrhenius plot to be
5697 cal mol".

Inactivation of pectinesterase enzyme in cherry juice was also studied. The enzyme
lost more than 50% of its activity at 55°C. The regeneration capability of the enzyme at
room temperature was studied as well. Further tests upon cooling of the enzyme showed
that the thermal inactivation was irreversible. Inactivation by lowering of pH to 2.0 was
successful in eliminating the activity of pectinesterase, but the yeast required for

fermentation was also destroyed, therefore this approach is not very useful.

Inhibition of pectinesterase by various compounds was studied as well. Very
promising results were obtained with the potential inhibitors tested. The inhibition was
tested using commercial purified pectinesterase and apple pectin. All the potential
inhibitors tested showed a capability to slow down the activity of pectinesterase.

As indicated by inhibition studies conducted with polymaleimide, very low
concentrations of the inhibitor are needed to reduce the activity of pectinesterase. This is
a positive attribute for fruit brandy application, since using small amounts of inhibitor
implies that the aroma and taste of fruit brandy will not be significantly affected.

All the inhibitors tested were anionic in nature. The positive results obtained for the
inhibition tests suggests that there is some enzyme-inhibitor complexation taking place.
The pH at which all the inhibitions were conducted was favorable for this kind of
complexation.

The best inhibition ability was observed with citrate, which is a well-known chelating
agent. This might explain the affinity of the inhibitor to pectinesterase but further tests

need to be conducted to ascertain this.

6. RECOMMENDATIONS FOR FUTURE
WORK

It has been established in this work that methanol in fruit brandy can exceed the
legal limits. Various fruits were used to study fermentation and distillation procedures.
To understand the general trend in methanol content among fruit brandies, differences in
fermentation as well as distillation need to be tested for any influence on the overall
methanol content in the distillates. A difference in the set up of the distillation trays on
the still will cause a change in the separation process, and this will either pre-concentrate
or dilute the amount of methanol in a particular stage of distillation.

The kinetic studies of pectinesterase were conducted using commercial pectin and
pectinesterase. This is a model system, since in practice pectin co-exists with
pectinesterase. The kinetic studies need to be conducted under this condition so as to get
a clear understanding of the mode of action of fruit pectinesterase. However, since
pectinesterase activity increases dramatically during the post harvest period when the
fruit tissue is damaged, the kinetic studies need to be done immediately before, and
during this period. This will also allow the kinetic studies to be conducted at the native
pH of the fruit pectin. The effect of all the other dissolved sugars in the fruit such as
sucrose and glucose, which have been speculated to aid in the inhibition of pectinesterase
activity, should also be ascertained.

Testing of potential inhibitors produced very promising results. Most of the
inhibitors were tested at a concentration of 1% w/v. Further reduction in concentration
for polymaleimide inhibitor showed that concentrations as low as 0.25% w/v were able to

inhibit pectinesterase by the same magnitude as the 1% w/v concentration. Lowering of

60

the concentration of the other inhibitors needs to be done in order to determine the
optimum concentration for each. The inhibition studies also need to be transferred to
fruit samples as opposed to the commercial pectin and pectinesterase already used in this
work. Kinetic studies of the inhibition process also need to be conducted to obtain the
inhibition rate constants and understand the rate of inhibition for each inhibitor.

The formation of a pectinesterase-inhibitor complex was suggested as the
mechanism by which the inhibition was taking place. However, a deep understanding
into the mechanism of inhibition was not obtained. Use of various spectroscopic
techniques will aid in getting deeper insight into the mechanism of inhibition. Infrared
Spectroscopy can be used to monitor the break up of the methyl ester bond in pectin that
generates methanol. Since there is a strong band for ester carbonyl at 1760 — 1745 cm'I
and COO" band at 1640 - 1620 cm", these can be used to monitor the deesterification
process. As methanol is generated there should be a decrease in the carbonyl ester as the
COO' band increases. These data can give supporting evidence of the inhibiting effect of
the inhibitors, and may be, show a difference in the extent of inhibition for each. The IR
studies may be conducted for the fruit samples too, to determine the effect of the fruit
juice matrix on the deesterification process. Proton Nuclear Magnetic Spectroscopy can
also be used to monitor the inhibition process. Structural information obtained from this
technique can be used to gain insight into the chemical changes taking place during the
inhibition process. Comparison of the proton shifts of the pure inhibitor and those of the
inhibitor-pectinesterase mixture should be able to give an indication of the chemical sites

involved in the inhibition process.

61

As discussed previously, the ultimate aim of this work is to reduce the methanol
content of fruit bandy. All the inhibitors tested in this study were used with commercial
pectinesterase and pectin. It is therefore necessary to test these inhibitors directly in fruit
juice samples. The fruit samples will contain other compounds that may or may not aid
in the inhibition of pectinesterase.

Ultimately, the inhibited samples need to undergo the fermentation process as
well as distillation. The methanol content of these samples will then be compared to the
uninhibited samples to ascertain the applicability of the inhibitors in the fruit brandy

industry.

62

7. APPENDIX

GAS CHROMATOGRAMS FOR VARIOUS SAMPLES

The gas chromatogram of various samples with different magnification to show
the chromatographic peak of interest. The chromatograms are for distillates, fermented
mash and the pectinesterase—pectin model system. Comparison of the inhibited and

uninhibited sample chromatogram is also shown.

63

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