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This is to certify that the

thesis entitled

The Effect of Temperature and the Use of Liquefaction
Enzymes of the Congener Concentration in

Distilled Fruit Brandies
presented by

Johnny 1. Andraous

has been accepted towards fulfillment
of the requirements for

Master's Chemical Engineering

degree in

 

 

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6/01 c'JCIRC/DatoDuo.p65-p.15

THE EFFECT OF TEMPERATURE AND THE USE OF LIQUEFACT ION ENZYMES
ON THE CON GENER CONCENTRATIONIN IN DISTILLED FRUIT BRANDIES

By

Johnny I. Andraous

A THESIS

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

MASTER OF SCIENCE
Department of Chemical Engineering

2002

ABSTRACT

THE EFFECT OF TEMPERATURE AND THE USE OF LIQUEFACTION
ENZYMES ON THE CONGENER CONCENTRATION IN DISTILLED
FRUIT BRANDIES

By

Johnny 1. Andraous

The first goal of this research was to examine the effects brought about by
changing the temperature at which yeast fermentations for the production of fruit brandy
are conducted. The distillates from fruit fermented at 10, 15, and 20 °C were analyzed by
gas chromatography and the congeners of interest were analyzed. These congeners
included fusel alcohols, methanol, carbonyls, and esters. Temperatures of 15 and 20 °C
are found to be optimal for brandy yeast fermentations. Concentrations of congeners
studied were consistent with those found in the literature. The second goal was to use
liquefaction enzymes to maximize the ethanol yield. Fermentations that were performed
showed that ethanol production was not affected by the use of these enzymes, but the
methanol concentration increased dramatically above the legal limit. This is crucial
because the limit of the methanol in alcoholic beverages is strictly regulated for health
reasons. Therefore, the use of liquefaction enzymes in the treatment of mashes must be

approached with caution.

TABLE OF CONTENTS

List of TABLE S ..................................................................... v
List of FIGURES ..................................................................... vii
l . Introduction ........................................................................... 1
1.1 Michigan Fruit Brandy Industry .......................................... 1
1.2 The Distillation Process .................................................... 2
1.2.1 Mashing of the Fruit ............................................... 2
1.2.2 Fermentation of the Mash ......................................... 2
1.2.3 Distillation of the Fermentation Mash ........................... 4
1.3 Temperature Effects on the Fermentation Products .................... 5
1.3.1 Types of Yeasts ..................................................... 5
1.3.2 Temperature Change and Congener Formation ................. 8
1.3.3 Formation of Fusel Alcohols ..................................... 9
1.3.4 Formation of Esters ................................................ 11
1.3.5 Formation of Carbonyl Compounds ............................. 12
1.4 Methanol Regulation and Liquefaction Enzymes ....................... l4
(pectinesterase and polygalacturonase)
1.4.1 Methanol Regulation ............................................... 14
1.4.2 Sources and Nature of Pectin ..................................... 16
1.4.3 The Pectolytic enzymes ........................................... 18
1.4.4 Pectin Methylesterase ............................................. 20
1.4.5 The Polygalacturonase ............................................. 22
2. Materials and Methods .............................................................. 24
2.1 Materials ..................................................................... 24
2.2 Experimental Procedure .................................................... 26
2.2.1 Preparation of the Fruit and Yeast ............................... 26
2.2.2 Enzyme Preparation ................................................ 27
2.2.3 Fermentation ........................................................ 29
2.2.4 Distillation .......................................................... 29
2.2.5 Distillate Analysis .................................................. 28
2.2.6 Ethanol Calibration ................................................ 32
2.2.7 Quantification of Methanol ...................................... 33
2.2.8 Quantification of the Congeners ................................. 33
3. Results and Discussion .............................................................. 37

iii

3.1 Analysis of the Distillates Using the Calibration Curves .............. 37
3.2 Congeners Present in Distillates from Fruit Fermented at 10 °C. . . 38
3.3 Congeners Present in Distillates from Fruit Fermented at 15 °C. . . ...44
3.4 Congeners Present in Distillates from Fruit Fermented at 20 oC. . 46

3.5 Comparison of Plums, Pears, and Gala Apples at the
Three Temperatures ........................................................ 46

3.6 Fermentation with the Addition of Different Liquefaction
Enzymes ...................................................................... 53

3.7 Congeners and Ethanol Present in Distillates from Gala Apples
Fermented at 15 0C with Different Liquefaction Enzymes Added... 53

3.8 Congeners and Ethanol Present in Distillates from Bartlett Pears
Fermented at 15 °C with Different Liquefaction Enzymes Added... 54

3.9 Congeners and Ethanol Present in Distillates from Red Delicious
Apples Fermented at 15 °C with Different Liquefaction Enzymes
Added ......................................................................... 57

3.10 Congeners and Ethanol Present in Distillates from Granny Smith
Apples Fermented at 15 °C with Different Liquefaction Enzymes

Added ......................................................................... 61
Computer Simulation ................................................................ 64
4.1 ChemCAD Batch ............................................................ 64
Conclusion and Summary ........................................................... 68
Recommendation for Future Work ................................................ 70
LIST OF REFERENCES ............................................................ 71

Table 1.1

Table 1.2

Table 1.3

Table 2.1

Table 3.1

Table 3.2

Table 3.3

Table 3.4

Table 3.5

Table 3.6

Table 3.7

LIST OF TABLES

The number of aroma compounds present in alcoholic beverages.9 ......... 9

Flavor thresholds of Selected organoleptic compounds. The
range of concentration found in beer is included for reference
Purposes.lo ......................................................................... 10

Methanol content in fruit juices and beverages.12 ............................. 14

Organoleptic compounds (congeners) that were looked at and their
retention times in minutes and their boiling points ........................... 31

Analysis example from Tanner and Brunner2 that shows different
fruit with amounts of several congeners (all values are in mg/ 100 mL
A.A) ................................................................................. 39

Analysis of a cherry sample from Tanner and Brunner.2 .................... 4O

Experimentally determined concentrations of congeners and ethanol
present in distillates after fermentation at 10 °C as determined by gas
chromatography. ................................................................. 41

Sugar content and alcohol output of various raw material from
Tanner and Brunner.2 ............................................................ 43

Experimentally determined concentrations of congeners and ethanol
present in distillates after fermentation at 10 0C as determined by gas
chromatography. ................................................................. 45

Experimentally determined concentrations of congeners and ethanol
present in distillates after fermentation at 20 °C as determined by gas
chromatography. ................................................................. 47

The concentration of congeners present in distillates from plums,

pears, and gala apples that were fermented at the three

temperatures. The error is the standard deviation calculated

from the three runs for each fruit at each temperature. The

data in this table were used to plot fig 3.1, 3.2, and 3.3. The letters A,

B, and C in the table represents the value obtained from performing the
Duncan Range Test Method with an CI of 0.05 and 4 degrees of freedom.
If two values have the same letter, it signifies that the values are
Statistically similar ................................................................ 52

Table 3.8

Table 3.10

Experimentally determined concentration of congeners and ethanol
present in distillates afterfermentation at 15 0C with liquefaction enzymes
added as determined by gas chromatography. .............................. 56

Experimentally determined concentration of congeners and ethanol
present in distillates after fermentation of red delicious and granny smith
apples at 15 0C with liquefaction enzymes added as determined by gas
chromatography. ................................................................ 60

vi

Figure 1.1

Figure 1.2

Figure 1.3

Figure 1.4
Figure 1.5

Figure 1.6

Figure 1.7

Figure 1.8

Figure 2.1

Figure 2.2

Figure 2.3

Figure 2.4

Figure 3.1

Figure 3.2

Figure 3.3

LIST OF FIGURES

The Embden-Meyerhof—Parnas fermentation scheme

showing the intermediates in the production of ethanol from

glucose ............................................................................... 3
The distillation set-up used in our lab for large distillations (150L)5 ........ 5
Formation of higher alcohols from amino acids and the

corresponding keto acids. (a) Biochemical sequence: (b) relationship
between selected amino acids and the corresponding higher alcohols.l°..11
Formation of esters and medium-chain length fatty acids by yeasts"). . ..12

The formation of Vicinal diketones and 3-hydroxy-2-ketones.lo ........... 13

The structure of pectin is shown in the top of the figure and the
bottom part shows the action of pectinesterase ............................... 17

The structures and reaction catalyzed by the enzyme
pectin methylesterase ............................................................. 20

The action of endopolygalacturonase that hydrolyzes internal
glycosidic bonds of polygalacturonate .......................................... 23

The distillation apparatus used to distill the mashes. ........................ 25

A chromatogram obtained from the gas chromatograph
for distillate from gala apples ................................................... 30

Ethanol calibration curve to determine the concentration of
ethanol in the distillates .......................................................... 35

Methanol calibration curve to determine the concentration of
methanol in the distillates ........................................................ 36

Analysis of con geners from plums fermented at different
temperatures ....................................................................... 49

Analysis of congeners from Bartlett pears fermented at different
temperatures ....................................................................... 50

Analysis of congeners from gala apples fermented at different
Temperatures ...................................................................... 5 1

vii

Figure 3.4 Volume of 40 % ethanol produced and concentration of methanol
in mg/ 100 mL in 40 % ethanol solution in gala apples fermented at
15 °C with Liquefaction enzymes added. ..................................... 55

Figure 3.5 Volume of 40 % ethanol produced and concentration of methanol
in mg/ 100 mL in 40 % ethanol solution in Bartlett pears fermented
At 15 °C with liquefaction enzymes added. ................................... 58

Figure 3.6 Volume of 40 % ethanol produced and concentration of methanol
in mg/ 100 mL in 40 % ethanol solution in red delicious apples
fermented at 15 °C with liquefaction enzymes added ........................ 59

Figure 3.7 Volume of 40 % ethanol produced and concentration of methanol
in mg/ 100 mL in 40 % ethanol solution in granny smith apples
fermented at 15 °C with liquefaction enzymes added ........................ 63

Figure 4.1 Ethanol profile in the distillate from gala apples fermented at
15 °C with different liquefaction enzymes added as deterrrrined
using ChemCAD Batch simulation ............................................. 66

Figure 4.2 Methanol profile in the distillate from gala apples fermented at

15 0C with different liquefaction enzymes added as determined
using ChemCAD Batch simulation ............................................. 67

viii

Introduction

1.1 Michigan Fruit Brandy Industry

Brandy is the product obtained by the distillation of wine or fermented juice of
any fruit.l There exists many production methods that differ from country to country.
For instance, continuous stills are frequently used in the production of California brandy,
but the brandies of French Cognac and Armanac are distilled exclusively by batch pot
stills without rectification. In Michigan, the production of fruit brandy has been slow
though the need for such an outlet for fruit existed. The consumption of fruit in the state
has remained the same despite the increase in production in certain fruit such as apples,
cherries and plums. This excess fruit production has prompted the farmers and wine
distilleries’ owners to request the state to enact a new law regarding brandy production.
A major change in Michigan’s legislation in 1996 enabled Michigan wineries to sell
distilled products out of the tasting rooms directly to customers and an annual license of
$100. This change has had a dramatic impact on the industry and distillation became
economically feasible. The number of distilleries involved in the distilling of fruit spirits
increased from zero in 1996 to seven in 2000.

As a new emerging product, the brandy produced in Michigan has to be of good
quality in order for the wineries to be able to compete with European brandy products
and be profitable. Hence, extensive research work has to be done on many aspects of the
fermentation and distillation of fruits. In this work. we have two objectives: the first is
running fermentations at different temperatures (10, 15 and 20 °C) with different fruits to
look for differences in congener concentrations and ethanol yield; the second involves the

use of liquefaction enzymes as fermentation aides and study their effects on the end

products mainly ethanol and methanol. Congeners are compounds such as fusel alcohols,
aldehydes, ketones, and carrbonyls that contribute to the flavor of an alcoholic beverage.
1.2 The Distillation Process
1.2.1 Mashing of the Fruit

The first step in production of brandy requires mashing fresh fruit. The fruit has to be
washed and damaged fruit must be discarded. A general principle is that the fermentation
proceeds better and is more complete depending on the maceration of the raw fruit
material.2 This is done in several ways depending on the fruit that is being mashed.
Squeezing by hand, using a wooden pestle or rolling mills are mainly used when crushing
stone or berry fruits. Ratz mills are often used for the crushing of seed fruit.2
1.2.2 Fermentation of the Mash

After mashing the fruits, the fermentation is initiated by adding a known

amount of yeast, which is in most, cases a strain of saccharomyces cerevisiae. Fruit
fermentations usually start aerobically as there is air in the fermentation vessel, and once
that air is exhausted yeast switches to anaerobic metabolism, which produces the desired
product, ethanol. The pathway by which yeast metabolizes common sugars, such as
glucose or fructose, under anaerobic conditions is referred to as the Embden-Myerhof-
Pamas scheme. By means of this set of reactions involving phosphorylations and
dephosphorylations, oxidations and reductions, as well as isomerizations, yeast produces
as its principal end products ethanol and carbon dioxide.3 This can be seen in Figure 1.1.

The main fermentation equation is:

 

C6H|206 ) 2C2H50H + ZCOZ

Figurel.l. The Embden-Meyerhof-Pamas metabolic scheme showing the intermediates
in the production of ethanol from glucose.3

D-Glucose

Glucose-6-phosphate
Fructose-6-phosphate <— Fructose

Fructose-1 ,6-diphosphate

Di-hydroxyacetone""'> D-Glyceraldehyde-3-phosphate

phosphate
Pi DPNH

1,3-Di-phosphoglyceric acid

3-Phosphoglyceric acid

2-Phosphoglyceric acid

2-Phosphoenol pyruvate

Pyruvate

Lactate j ‘— DPNH

Acetaldehyde + C02

<— DPNH
Ethanol

The beginning of the fermentation can easily be identified by the bubbling in the
fermentation top caused by the production of C02 and a significant increase in
temperature can be noticed. The optimal fermentation temperature for fruit brandy is
between 15 and 20 °C.2 This temperature has to be kept under control throughout the
course of the fermentation. Also, the pH of the mash should be kept between the range of
2.8 and 5.2.2 Different acids or bases can be added to the mash to keep the pH in this
range, and some of these chemicals can be useful as nutrients for the yeast especially
nitrogen and phosphorous containing compounds. Outside this pH range, yeast will not
be able to ferment the sugars properly.

Refractometry is an optical measurement method used to measure the amount of
sugars still present in the fermentation medium based on the refractive index and this is
usually about 2-3g sugar/L. High performance liquid chromatography (HPLC) can be
used to monitor the amount of sugars and ethanol produced during the fermentation using
either an UV-VIS or a refractive index detector (RI). Fermentations usually last for a
period of two weeks but no significant changes occur if the fermented mash is stored
airtight for longer periods of time.

1.2.3 Distillation of the Fermentation Mash

Distillation is the technique used to separate and select by heat volatile
components from a liquid mixture.4 The distillation of fruit brandy is more complex than
a simple binary distillation between ethanol and water. For example, wine contains
approximately 300 volatile compounds. Each volatile component will distill according to
these criteria: boiling point, equilibrium relationship to water or alcohol, and the variation

of alcohol content in the vapor during the distillation.

Different opinions exist regarding the ideal form of the distillation stills and the
apparatus itself, but copper is the material used for the manufacture of the still. Copper is
a very good heat conductor, shows optimal resistance to fruit acids, and affects the
quality of brandy positively by catalytic chemistry on the surface.

The heating source can be a water or oil bath, steam, or electrical power. The
time that it is required to distill the mash depends on the amount of the mash, the rate of
heating, and the intensity of heating. Distillation proceeds as follows, the still is filled
with mash up to 65-75 % v/v and antifoaming agents are added.2 As the still is heated,
the alcohol in the mash vaporize and proceeds into the trays. Depending on the volatility
of the components vaporized, some will condense back to the still and the remainder will
proceed to the spirit tube. This tube is connected to the condenser, which completely
condenses the volatile components. The distillate is collected in three fractions: the
heads, hearts, and the tails. The hearts are kept and then diluted to produce 40 % alcohol.
The heads and tails are discarded or sometimes they are re-distilled. The hearts are then
diluted with pure water to produce 40 °/o alcohol product that is ready for consumption.
Figure 1.2 shows the distillation apparatus that is manufactured by Christian Carl in
Germany and is in use our lab for large distillations (150L). Usually for a 150L batch,
the first two liters of the distillate are the heads, the following eight or nine liters are the
hearts, and the last couple liters are the bottoms.

1.3 Temperature Effects on the Fermentation Products
1.3.1 Types of Yeasts

Temperature is undoubtedly one of the most important environmental parameters

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influencing all activities of microorganisms. It is standard practice to study yeast
metabolism at temperatures around 25 — 30 °C. even though these temperatures may not
be strictly relevant when considering the natural habitats of many species of yeasts.6
Yeast may be classified on the basis of their temperature limit for growth. The terms
“psycrophile”, “mesophile”, and “thermophile” have been used to classify
microorganisms into different thermal domains. A yeast that can grow in the temperature
range between 5 and 10 °C would be classified as psychrophilic yeast. Thermophilic
yeasts are ones that are capable of growth at temperatures at or above 50 °C. Certain
yeasts that can grow between 20 and 46 °C are also termed thermophilic. Mesophilic
yeasts are the most abundant and have temperature range for growth at or below 0 °C and
up to 48 °C. Pris de Mousse, which is mostly used in brandy fermentations, is a Sacch.
C erevisiae yeast and it is considered mesophilic.

Very few reports exist on temperature induced changes in psychrophilic and
thermophilic yeasts. It is suggested that changes in plasma-membrane composition,
especially the increase in polyunsaturated fatty-acyl residues at low temperature, is
responsible for the effects on solute transport across the membrane.7 Also, the rate of
glucose uptake at different temperatures is related to the composition of the unsaturated
fatty —acyls in all types of yeasts.8 An important trend found by many authors is that
yeasts adjust the membrane fatty-acyls composition with temperature and the lower the
growth temperature the more unsaturated the membrane fatty —acyls composition.6 This
is crucial because it will enable cell membranes at lower temperatures to remain

sufficiently fluid to allow proper functioning of metabolic processes. This temperature

adaptation by the yeast metabolic pathways will ultimately affect the composition and
amounts of the end products of these pathways.
1.3.2 Temperature Change and Congener Formation

The fermentation is an exotherrnal process wherein an increase in temperature of
the mash is observed in the beginning of the fermentation. The optimal fermentation
temperature for fruit brandy is between 15 and 20 °C and for mashes that are difficult to
ferment it is a little higher.2 It is generally known that higher fermentation temperatures
aid in making the fermentations proceed faster, but it can have an adverse effect in that
this higher temperature can promote the growth of undesired yeasts or bacteria. Some of
these microorganisms include film-forming yeast, wild yeasts, and molds. The growth of
these microorganisms contributes negatively to the aroma of the final product because of
the production of chemicals such as butanoic acid. The production of ethanol by yeast is
associated with the production of a wide variety of fermentation products, which
contribute to the final flavor of the beverage, either as organoleptic compounds, or
precursors of organoleptic compounds which are produced in subsequent maturation or
distillation processes. These organoleptic compounds are called congeners and in this
work we will be using both terms when talking about them.

Research on the aroma of the alcoholic beverages has shown that the aroma
composition of alcoholic beverages consists of several hundred distinct chemical
compounds.9 Table 1.1 shows the number and type of compounds present in alcoholic
beverages. These compounds are present in most types of alcoholic beverages but in

different concentrations. Organoleptic compounds produced by yeasts can be classified

into five categories: alcohols, esters, aldehydes and ketones, sulphur containing

compounds, and organic acids.

Table 1.1. The number of aroma compounds present in alcoholic beverages.9

 

 

Class # Observed Class # Observed
Alcohols 3 8 Hydrocarbons 41

Acids 80 Nitrogen compounds 1 l
Esters 1 l8 Sulphur compounds 18
Carbonyl comounds 41 Lactones l 1
Acetals 1 7 Sugars 4
Phenols 41 Unclassified compounds 11

 

Concentration of organoleptic compounds present are not, however, the only
parameter to be considered in evaluating the flavor of a beverage, since different
compounds have different sensory odor thresholds. Tablel .2 shows the flavor thresholds
of some organoleptic compounds. It is clear that although the higher alcohols are the
most abundant group of organoleptic compounds, they have odor thresholds ten times
higher than the esters and a thousand times higher than the carbonyl compound diacetyl.
Hence, their contribution to the overall flavor of alcoholic beverages is not the most
important on a per mass basis.

1.3.3 Formation of Fusel Alcohols

F usel alcohols are alcohols with more than two carbons. The most important are
l-propanol, 2-methy-1-propanol, l-butanol, 2-methyl-1-butanol, and 3-methyl-l-butanol.
Most of these can be derived from the carbon skeletons of common amino acids.
Formation of these fusel alcohols is thought to be independent of the raw materials used
in the mash because the formation of these longer chain alcohols can occur in whiskeys,
tequila, and gin. These fusel alcohols can be produced either from amino acids or from

sugars. Figure 1.3 shows the steps involved in producing some of these alcohols. The

 

 

 

 

 

Flavor Threshold Range of Concentration
Alcohols (ppm) in beer (ppm)
ethanol 1400
l-propanol 800 7.5-13.8
2-propanol 1500 02-24
l-butanol 450
2-methylpropanol 200 86-566
2-butanol 16
2-methylbutanol (optically active 65 70-23
amyl alcohol)
3-methylbutanol (iso-amyl alcohol) 70 27-122
2-phenethanol 125 50-27
Acids
acetic 175 150-280
propionic 150 5
butyric 2.2 0.6-3.3
iso-butyric 30 0.7-3.3
caproic 8 2.2-5.8
caprylic 13 3.3-8.2
capric 10 O. l-2.0
phenylacetic 2.5 0.93
lactic (D+L) 400 28-400
Esters
ethyl acetate 33 82-476
n-butyl acetate 7.5 0.23
iso-butyl acetate 1.6 0.03—0.25
phenylethyl acetate 3.8 0. 1-1.17
ethyl butyrate 0.4 0.09
ethyl caproate 0.23 __
ethyl caprylate 0.9 0.08-0.01
ethyl caprate 1.5
ethyl lactate 250 0.1
Aldehydes and Ketones
acetaldehyde 25 2.5-24.4
iso-butyraldehyde l 0-0.024
acetone 200 1
pyruvate 300 10-220
diacetyl 0. 15 0.5-2.0
Sulphur compounds
dimethyl sulphide 50 0-0. 144

 

 

Table 1.2. Flavor thresholds of selected organoleptic compounds. The range of
concentration found in beer is included for references purposes.10

10

 

effect of the temperature is not always consistent, but several reports of research indicate

that the maximum production of higher alcohols occurs at intermediate temperatures of

about 24 °C. A study done on the fermentation of grape juice shows that different

alcohols are affected differently when temperature is changed between 10 and 32.2 OC.ll

(a)

(b)

NH2
chzcoou
R1CO.COOH
TRANSAMINATION
<:::;R1CH2.COOH
RCO.CO0H
DECARBOXYLATION
cho + £02
<:::;NADH + H+ .
+ DEHYDROGENASE
NAD
RCHZOH

VALINE 6—) a-KETO ISOVALERATE ———) rsoaurmor.

LEUCINE <—> a-KETO ISOCAPROATE ——> 3-METHYL BUTANOL

(ISOAMYL ALCOHOL)

rsoreucme (—-—§ o-KETO 3-METHYLVALERATE ———> Z-HETHYL BUTANOL

(OPTICALLY ACTIVE
AMYL ALCOHOL)

PHENYL ALANINE é———) PHENYL Pvauvrc ACID ——> 2-PHENYLETHANOL

a-KETO BUTYRATE ———————9 u-PROPANOL

Figure 1.3. Formation of higher alcohols from amino acids and the corresponding keto
acids. (a) Biochemical sequence: (b) relationship between selected amino acids and the
corresponding higher alcohols.I0

1.3.4 Formation of Esters

Esters are numerically the largest group of organoleptic compounds found in

11

alcoholic beverages.'0 Most common esters are produced by the yeast during the
fermentation stage in addition to the esters formed during distillation and storage. Figure
1.4 shows the formation of esters and medium-chain length fatty acids by yeasts. Since
ethanol is the most abundant alcohol, the ethyl esters are the most abundant followed by
isoamyl and propyl esters. Acetate is the most abundant acid formed by yeast during

fermentation, so acetate esters of ethanol and higher alcohols are the most abundant.

 

 

 

C12 I2 c2
H20 ETOII 520 ETOH
COASH CCOASH COASH COASH

Figure 1.4. Formation of esters and medium-chain length fatty acids by yeasts.'0

Temperature doesn’t have an effect on the ester formation, but as mentioned
earlier, it will affect the level of unsaturated fatty acids in the membrane and the
fermentation medium and this affects the amount of esters formed. Esters also formed
during aging where higher alcohols and ethanol are converted to the corresponding esters.
1.3.5 Formation of Carbonyl Compounds

The carbonyl compounds consist of aliphatic and aromatic aldehydes and ketones.
Aldehydes are synthesized by yeasts as intermediates in the formation of alcohols

through the decarboxylation of keto acids. Acetaldehyde is quantitatively the most

RCO.COOH
KETO ACID

+ ACTIVE ALDEHYDE

CH3

C0

1
R - C - COOH
0H

ACETOHYDROXY ACID

DECARBOXYLATION OXIDATIVE DECARBOXYLATION

RCH(0H).CO.CH3é;' Renucrron Rco.co.CH3
IYnRoxv—Z-KETONE VICINAL DIKETONE

 

Figure 1.5. The formation of Vicinal diketones and 3-hydroxy-2-ketones.l0

B

compound in this group. Aldehydes are intermediates in the biosynthesis of
alcohols; most of the parameters that influence the production and amount of the latter
also influence the aldehyde forrnatio. Ketones are also intermediates in the biopathway
for higher alcohol formation. They are also involved in the formation of Vicinal and 3-
hydro-2-ketones as seen in Figure 1.5.

Studies to determine the parameters that affect ketone formation were focused on
determining the factors that influence acetohydroxy acid production. Increase
temperature could cause a decrease in acetohydroxy formation and hence a decrease in
the concentration of the Vicinal diketones and 2,3-pentadione. '0
1.4 Methanol Regulation and Liquefaction Enzymes ( Pectinesterase and

Polygalacturonase )

1.4.1 Methanol Regulation

Many types of 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. The average methanol content of fresh
orange juice is 34mg/L, while fresh grapefruit juice contains 27 mg/L.'2 Table1.3 shows

the amount of methanol present in some juices.

 

 

 

 

 

Juice/Beverage Methanol (mg/L)
Orange 34
Grapefruit 27
Pear Wine 188
Cherry Wine 276

 

 

 

 

Table 1.3. Methanol content in fruit juices and beverages'2

As mentioned earlier, methanol is present in fruit and is a problem for the fruit
brandy industry. The methanol levels that are low in fruit juices become much more
significant when the fruit mash is distilled. During distillation, separation of alcohols
from the fruit mash always results in the concentration of methanol in the product.
Excessive methanol present in the human body can have severe effects on many organs
and in some cases could lead to the person’s death. The level of methanol in many
products is tightly regulated for that reason. The United States Enviromental Protection
Agency recommends a minimum acute toxicity concentration of methanol in drinking
water at 3.9 parts per million.12 The EPA has also set the permissible exposure limit in
air to 200 parts per million for an 8-hour time weighted average.

This regulation extends to the alcoholic beverages and especially for brandies
because they are made from fruit. In Europe, the production of fruit brandy has had
many guidelines and regulations for many years.2 For example, in Germany the cherry
brandy methanol content is set at 400 mg/ 100mL absolute alcohol while for the brandy
from Bartlett pears at 790 mg/ 100mL absolute alcohol.2 In Austria, the maximum
allowed content of methanol in brandy is 1000 mg/ 100mL absolute alcohol.2 In the
United States of America, the bureau of Alcohol, Tobacco, and Firearms has set the
permissible amount of methanol in brandy to 0.35 % v/v. This corresponds to 700
mg/ 1 OOmL absolute alcohol.

The source of methanol is the pectin present in the fruit. Pectin is esterified by
methyl groups and the naturally occurring presence of the enzyme pectinesterase and

other liquefaction enzymes in fruit is responsible for deesterifying the pectin to release

methanol. The activity of these enzymes increases as the fruit ripens and the tissue is
damaged.2
1.4.2 Sources and Nature of Pectin

Pectic substances are carbohydrate derivatives that are widely distributed in plant
tissues where they occupy intracelluar space. The pectic substances consist of a number
of compounds, depending on the degree and type of enzymatic action. The parent
compound in the intact immature tissue is protopectin, an insoluble substance located
primarily in the middle lamella that serves as the glue to hold cells together and in the
cell walls. '3 Pectin (polymethylgalacturonate) is the soluble polymeric material in which
at least 75% of the carboxyl groups of the galacturonate units are esterified with
methanol. Pectic acid (polygalacturonic acid) is the soluble polymeric material in which
all the methoxyl groups are removed from the galacturonate units. Pectinic acids contain
>0 and <75% methylated galacturonate units. Cell walls contain approximately 60%
water and 40% biopolymers. Pectins make up 20-3 5% of the polymers.'4 Figure 1.6
shows the structure of pectin.

Pectin is polysaccharide whose main component is D-galacturonic acid, joined by
means of 0t - (1 ——>4) glycosidic linkages. The galcturonic acid molecule has a carboxyl
group on C 5 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 is a wide range for the degree

of esterification and this depends on the species, tissue and maturity.'5

16

Stucture of Pectin

 

 

OCH3
HO _‘O OH /R
OH O OH 0
fl % o
O C CH C
“\OCHa HO I \OCH3
0 . Pectin O
R
Mode of Action of Pectinesterase
E—N Represents the enzyme
0
/
o H \H
O -
I 4|
E—N C—O—CHa ——> E—N —C—-O—-CH3
a
CH3OH
0
II I o
R—C—OH i I
<— E—N—C—OH‘-— E—N—
R H
OH-

Figure 1.6. The structure of pectin is shown in the top of the figure and the bottom
part shows the action of pectinesterase.

l7

1.4.3 The Pectolytic Enzymes

The pectolytic enzymes include one esterase (pectin methylesterase); six
polygalagturonases and four lyases.l6 Pectolytic enzymes are widely distributed in higher
plants and microorganisms. They are not found in higher animals, but are found in some
protozoa, nematodes, and insects. Enzymes that catalyze the formation of pectin from its
water insoluble precursor 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. ' 7

Microbial pectolytic enzymes are known to play key roles in plant pathogenicity
and in much of fruit and vegetable spoilage involving rotting. Pectolytic enzymes are
commercially important in a number of industrial processes including retting of flax and
other vegetable fibers, extraction, clarification, and depectinization of fruit juices,
extraction of vegetable oils and maceration of fruits and vegetables to give unicellular
foods.18 The use of these enzymes has become indispensable for the fruit and vegetable
technology and for the production of wines in many places, yet it is not clear how
suitable they are for the treatment of mashes in distilleries.2 The use of enzymes in the in
the treatment of mashes improves the capability to pump mashes in the fermentation
phase because the insoluble pectin is also decomposed. The advantages of a fast
liquefaction of the mash are apparent. When the mash is treated with the liquefying

enzymes, the viscosity of the mash drops sharply and it becomes much easier to pump it

and in a much shorter period of time. Also, the speed of the fermentation is much higher
and this is very important because we can obtain the same yield of alcohol in a shorter
period of time.

Despite their many advantages in the fruit industry, the use of these enzymes
could pose a problem for the brandy industry. Some researchers say that the methanol
content of the mashes treated with enzymes does not change compared to the ones which
are not treated because the formation of methanol takes place through
pectimethylesterase which is already present in the fruit.2 This result could vary
considerably from fruit to fruit depending on the amount of enzyme present and the type
of treatment applied. No major changes in the alcohol amount produced or in the sensory
properties of the distillates were noticed.2

Yet, in the production of fruit brandy a slight increase in the production of
methanol could have a negative impact on the brandy because the level of methanol is
tightly regulated in alcoholic beverages. So, the production of brandy low in methanol
content is highly desirable. This study will focus mainly on two types of enzymes that
were provided by the German company Erbsloh that specializes in making products for
the brandy industry and also for the production of enzymes for different industries. The
first one is F ructozym M that is a pectinase and is commercially available, and the other
three are polygalacturonases that are not commercially available. Ethanol, methanol and
other compounds will be looked at as they are produced and compared. The next two

sections will explain in detail the enzymatic action of these two types.

19

1.4.4 Pectin Methylesterase

Pectin methylesterase is synonymous with pectinesterase, pectase, pectin
methoxylase, pectin demethoxylase, and pectolipase. Pectin methylesterases have been
detected in a variety of plants. Most of the fruits have been reported to have more than
one isozyme of pectin methylesterase. Orange pectin methylesterase is a well-studied

enzyme.

I
020. H+
+CH30H

 

Pectin methylesterase

Polymcthylgalacturonate (pectin) + H20 WW polygalacturonate + methanol + H“ (I)

Figure 1.7. The strcuture and reaction catalyzed by the enzyme pectin methylesterase.l5

Two isozymes have been reported, each having a molecular weight of 36,200 Da. The

two isozymes have different optimal pH values unlike the isozymes found in banana pulp
that both exhibited optimal activity at the same pH of 7.5. Structural studies have shown
that the enzyme is a glycoprotein formed by a single low molecular weight polypeptide.l9

Generally, the isoelectric point for this enzyme is between 7 and 11.20

20

Pectin methylesterase acts upon pectin to remove the methoxyl groups from the 6-
carboxyl group of the galacturonate unit by hydrolysis as shown in equation 1. The
enzyme is a carboxylic acid esterase, belonging to the hydrolase group of enzymes.
The products of the reaction are: (l) a further deesterified pectin which eventually
becomes a pectinic acid and finally a pectic acid; (2) methanol; (3) a H+ from the
ionization of the newly formed carboxyl group.

The effect of pH on pectin methylesterase has also been studied. Some
researchers used the fluorescent probe 8-anilinonaphtalene-l-sulfonate (ANS) to
determine the surface hydrOphobicity of the enzyme.21 ANS is non-fluorescent in water
and other polar enviroments, but highly fluorescent in non-polar enviroments or when
bound to hydrophobic sites on proteins. Changes in protein structure that result in an
increase in hydrophobicty can be estimated by the change in fluorescence of ANS. If pH
causes change in the hydrophobic sites of the protein, the structural change is detected by
ANS fluorescence. Based on this technique, pectin methylesterase molecules were found
to be more hydrophilic at neutral and alkaline pH than at acidic pH values. Current
studies of the enzyme activity indicate that the degree of activity can be enhanced by the
presence of cations. The stimulatory effect of NaCl on pectin methylesterase varies
considerably. 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. Another study reported similar effect on mango pectin methylesterase by
Nale22 The maximum pectin methylesterase activity was at 0.1 M, and the activity

decreased gradually as the concentration was increased to 0.4 M. Calcium chloride has

21

also been shown to have a similar effect on the activity of the enzyme. It is worth noting
that the cations increased the activity of the enzyme, but were not a requirement for its
activity.

The distribution of methoxyl groups along the pectin chain is also important for
pectin methylesterase. While the enzyme attacks completely methylated pectin slowly,
the rate of deesterification increases to a maximumat 50% methylation.l6 Pectin, partially
deesterified previously with pectin methylesterase, results in a slower rate than the
equivalent degree of deesterification by alkali treatment. Some reports show that the
optimum reaction temperature for this enzyme to be 65 °C and it declines after 70 °C.
1.4.5 The Polygalagturonase Enzyme

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 or - D — (1—9) polygalagturonate.23 The pattern of degradation is either random
(endopolygalacturonase) or terminal (exopolygalacturonase). Endopolygalagturonase
usually prefers substrates with a low degree of esterification, typically less than 20%.2|
Exopolygalacturonase generally acts on deesterified pectin.22 It was found that within
short reaction times, random cleavage of polygalacturonate resulted in large decrease in
viscosity. Very little change in viscosity was observed in the case of terminal cleavage.

Figure 1.7 shows the action of the polygalacturonase enzyme.

22

Polygalacturonases

 

d
Polygalacturonate + nHZO £13,me "mm—a oligogalacturonatec + galacturonate

Figure 1.7. The action of endopolgalacturonase that hydrolyzes internal glycosidic bonds of
polygalacturonate.

23

Materials and Methods

2.1 Materials

Several types of fruit were obtained from for the fermentations. The fruit selected
was fresh and had no signs of damage. The types of fruit chosen were: cherries,
California peaches, plums, apples ( gala, jonathon, red delicious, and granny smith),
Bartlett pears. Fermentations were conducted in 800 mL Fleakers® obtained from Dow
Coming. The rubber tops on the Fleakers® were fitted with a U-shaped tubes that
functioned as Air Locks. Ordinary lab glassware was used for all tasks need. The mash
was placed in a 1000 mL round bottom flask before distillation. Enzymes used in the
fermentation were four types.
1. Spirizm FM is a liquid, concentrated pectolytic enzyme preparation
2. VP 0956/2 is a purified liquid polygalacturonase from Aspergillus niger, activity 3-

fold higher than Spirizm FM. Good activity up to pH 5.

3. VP 0996/2 is a liquid polygalacturonase from Aspergillus niger, activity 2-fold higher

than Spirizm FM and good activity even up to pH of 6.

4. VP 0996/9 is a powdered polygalacturonase from Rhizopus oryzae, activity 8-fold

higher than Spirizm FM and good activity up to pH 5.5.

A simple distillation apparatus was used and duplicates were generally performed.
Figure 2.1 shows the simple distillation apparatus. The round bottom flasks were
halfway immersed in silicone oil. Heating was applied using a hotplate/stirrer
manufactured by Dow Corning (model PC-420).

The fermentations were performed in temperature controlled chambers that were

bought from Sears (model: Galaxy # 253.19501992, 9ft3). To keep precise temperature

24

Thermometer

|.{m:m

,, Inlet Adapter

1

 

 

 

@ Three-way
F e Condenser
' dapter Vacuum Adapter
Afi~ Open
c... ~ .rfl‘
Clamp Hg! 4 i m,”
—._ ‘Water Outlet Water Inlet Clamp I'gg‘.’
1000 ml. Flask 50 ml. Flask
\. . .
1 Silicon Oil BathJ Ice Water Bath

 

 

Figure 2.1. The distillation apparatus used to distill the mashes.

25

control, the electrical cord from the freezer was connected to a CN77000 controller from
MicroomegaTM. The parameters of the controller were set to achieve temperature control
at the desired temperature and deviation no more than 1 °C.

The distillates were stored in 100-mL plastic vials. One mL from each sample was
put in Gas Chromatography sample vials (model: C 4010-88) from VWR Scientific.
Samples were run on a Shimadzu GC 17A Gas Chromatograph with a Stabilwax®
column from Restek Corporation. Helium, from BOC Gases, was the carrier gas and
hydrogen and oxygen tanks were used for the flame. Chemicals used for the preparation
of standards and also ethanol and methanol were bought from Sigma Aldrich Co. HPLC
water was used for all dilutions.

2.2 Experimental Procedure
2.2.1 Preparation of the Fruit and the Yeast

The fruit was washed with water and all stems, leaves, and/or vines were removed
from each piece of fruit. Fruit, except cherries, were cut with a pairing knife and then put
into a five-gallon plastic bucket. The fruit pieces were then crushed with a wooden
pestle. After the F leakers were cleaned thoroughly with water and soap, they were
placed on a mass balance and it was tared. Seven hundred grams of fruit that was mashed
was scooped into the Fleakers®. This was done in triplicate for each fruit. The Fleakers®
were then quickly capped with their tops until it was time to add the yeast. The same
procedure was followed at each fermentation temperature 10, 15, and 20 °C.

Three tenth of a gram of yeast was weighed. This amount is a little higher than
the 0.28 grams that is recommended for thick and viscous mashes.2 This larger value was

chosen because the fermentations were not stirred during the course of the fermentation

26

so excess yeast helped insure good initiation of the fermentation. The yeast was placed in
50 mL beakers filled with 20 mL of distilled water at 40 °C. The beaker was placed in a
water bath at 40 °C for 15 minutes to ensure complete swelling of the yeast. The
suspension was added to the Fleaker® as evenly as possible and the contents were stirred
for 5 minutes to ensure mixing of the yeast and the fruit pieces. The F leaker® was
capped with its top and placed in the chest freezer. The procedure was repeated for each
fermentation at each temperature.
2.2.2 Enzyme Preparation

Liquefaction enzymes were added in some experiments and the solution was
prepared separately. The recommended dosage for berries and stone fruit is 20 —— SOmL/
Kg of mash at 15 °C.23 The amount used in our fermentations was 0.35 grams/700 grams
of mash for each enzyme (we used a relatively high value because our fermentations were
not stirred). The enzymes and 3.15 mL of distilled water were combined to form a 10%
solution. The procedure was repeated with each of the enzyme types except for the
0996/9. In this case, we weighed 0.35 grams because the enzyme was in powder form.
The liquefaction enzyme solution was evenly added to its respective Fleaker®. The
contents were kept at room temperature for 30 minutes. The yeast preparation was added
and the F leaker® placed in the chest freezer at a temperature of 15 °C. Controls were
run which had no enzyme added to them and were done in triplicates. One Fleaker® of
each enzyme was prepared except for the gala apples experiment where two sets were

prepared.

27

2.2.3 Fermentation

Fermentations were run anareobically, which requires minimum exposure to
oxygen. After the mash, yeast, and enzyme were added, the rubber cap with its air lock
was firmly put on the Fleaker®. This air lock allowed the release of CO; that was
generated from the fermentation. The pH of the fermentation was measured daily in each
Fleaker® using pH paper and was in the range of 3.0 to 4.5. Refractive index
measurements were taken. The temperature of the Fleaker® in the chest freezer was
maintained at the specified temperature using a MicroOmega controller. The On/Off
controller received power from a wall- mounted outlet. The controller was placed
adjacent to the chest freezer. The thermocouple was connected to the On/Off controller
and the temperature probe was placed in a beaker filled with water inside the freezer.
This was done to prevent fluctuations in the thermocouple temperature and to model the
situation in the Fleaker® as much as possible. The chest freezer received power from the
controller, and hence the controller controlled the power input to the chest freezer. If the
temperature inside the freezer was below the set point, the On/Off controller would not
allow power to enter the chest freezer. Hence, the compressor will not be able to run and
the temperature inside rises. If the temperature inside rose above the set point, the
On/Off controller would allow power to enter the chest freezer and the compressors will
work to bring the temperature down. This kept an excellent temperature control on the
fermentation in the Fleakers® and a deviation of only a tenth of a degree Celsius was

achieved. Fermentations were run for two weeks.

28

2.2.4 Distillation

The contents of the F leaker® were placed in a 1000 mL round bottom flask and a
small amount of an anti-foaming reagent was added. The flask was half immersed in
silicone oil bath placed on a hot plate. Figure 2.1 shows the distillation apparatus. A
silicone oil bath was used because it is possible to heat it over 100 °C without the oil
boiling. The silicone oil bath used in these experiments typically reached temperatures of
around 115 °C. Distillates started vaporizing at about 70 °C and the distillate was
collected in a 50 mL round bottom flask and later transferred to a plastic or glass vial
depending on the volume. At 99 °C, the distillation was stopped to prevent excess water
from boiling over. After the distillation was complete, the apparatus was disassembled
and each part washed with water and soap and rinsed and placed in an oven for drying.

2.2.5 Distillate Analysis

One mL from each sample was placed in a GC vial and was run on the gas
chromatograph within 48 hours after distilling to prevent evaporation of highly volatile
samples and any additional aging from occurring in the samples. The GC that was used
for analysis was a Shimadzu GC-17A equipped with a flame ionization detector (FID)
and auto sampler. A Stabilwax® column from Restek Corporation was used for all the
experiments. This column had a stationary phase made of polyethylene glycol. The run
conditions of the GC were as follows:

Column : 30m, 0.32 ID, 0.5 pm Stabilwax®

0.5 uL split injection
Oven temp: 40 °C (hold for 1 minute ) to 190 °C at 2.5 °C/min

190 °C (hold for 5 minutes)

29

 

0.06:

0.05

004*

3110 A

0.03I

0.02

 

0.01

 

 

 

 

 

 

65
0 Time (min)

Figure 2.2. A chromatogram obtained from the gas chromatograph for
distillate from gala apples.

30

 

Compound Boiling Point (°C) Retention Time (minutes)

 

Acetaldehyde 21 1.61
Acetone 56.5 1 .95
Ethyl Forrnate 54.3 2.02
Ethyl Acetate 77 2.54
2-Methyl-2-Propanol 82 2.68
Methanol 64.7 2.83
Ethanol 78.3 3.23
2-Methyl-2-Butanol 101.9 4.47
2-Butanol 94 4.77
l-Propanol 97 5.14
2-Methyl-1-Propanol 107.7 7.41
l-Butanol 118 8.55
3-Methyl-l-Butanol 132 10.98
2-Methyl-1-Butanol 128 11.65
1-Pentanol 138 12.83
l-Hexanol 156.5 17.68
l-Heptanol 176 22.64
Furaldehyde 161.7 23.1
Benzaldehyde 179 25.63
l-Octanol 194.5 27.55
Benzyl Alcohol 205 41.68

 

 

 

Table 2.1. Organoleptic compounds (congeners) that were looked at and their retention
times in minutes and their boiling points.

31

Injector temp: 240 °C

Detector temp: 255 °C
Carrier gas: helium at 30 cm/sec
Spilt ratio: 65: l

Toatl 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. A typical gas
chromatogram is shown in Figure 2.2.

The initial step to determine the components present in our chromatogram was to
run standards of chemicals and figuring their retention times. Retention times in
triplicate were obtained for the following compounds: acetaldehyde, acetone, ethyl
formate, ethyl acetate, 2-methyl-2-propanol, methanol, ethanol, 2-methyl-2-butanol, 2-
butanol, l-propanol, 2-methyl-l-propanol, 1-butanol, 3-methyl-1-butanol, 2-methyl-l-
butanol, l-pentanol, l-hexanol, l-heptanol, furaldehyde, bennzaldehyde, l-octanol, and
benzyl alcohol. Retention times were run in triplicate and averaged to increase the
accuracy of the analysis. This qualitatively identifies peaks present in the chromatogram.
Usually, compounds that low boiling points will elute first from the column followed by
compounds with higher boiling points, but there are exceptions to this general rule.

2.2.6 Ethanol Calibration

An ethanol Calibration curve was generated. The following concentrations of
ethanol were prepared using 100% ethanol and HPLC water: 25, 27, 30, 33, 35, 38, 40,
43, 45, 47, 50, 53, 55, 57, and 60%. Each of these concentrations (%v/v) was run five

times using the same conditions as mentioned earlier. From the chromatograms obtained,

32

the peak areas were integrated and averaged. Plotting the areas versus the % ethanol we
obtain a calibration curve as seen in Figure 2.3. The equation of this curve is then used to
calculate all the other concentrations of other compounds.
2.2.7 Quantification of Methanol

The amount of methanol produced in these experiments needed to be calculated as
accurately as possible. A calibration curve was constructed by plotting concentration of
methanol in mg/ 1 00 mL in 40 % ethanol solution versus the areas under the peak
obtained from the gas chromatograph. The concentrations chosen were: 79, 118, 158,
197, 237, 276, 316, 355, 395, 474, and 513 mg/IOO mL in 40 % ethanol solution. Each
was run five times and the average area was used in the plot. Figure 2.4 shows the
methanol calibration curve along with error at each point.
2.2.8 Quantification of the Congeners

The quantity of these congeners were calculated using the ethanol calibration
curve, Figure 2.3. First the fraction of each chemical including ethanol was calculated by
dividing the compound integrated area by the total area. The ethanol concentration in our
sample was calculated by using the area of the ethanol peak and putting it in the
calibration curve equation. This gave us a value in WV units. Using ethanol density, we
converted this value to one that has units of g/mL. Then using this concentration of
ethanol and the fraction of it in the samples, we calculated the concentration of the other
compounds by equal ratios. In this procedure, we estimated the density of the
compounds to be very close to that of ethanol and that ethanol acted as an internal
standard. The values were then converted into units of mg/ 100 mL in 40 % ethanol

solution. The methanol concentration was calculated using the methanol calibration

33

curve Figure 2.4. Using the methanol density, this v/v concentration was converted into
mg/ 100 mL in 40 % ethanol solution. This procedure for calculating the methanol
concentration was repeated for all the samples. The conversion into the unit
mass/volume in 40 % ethanol solution was to make it easier to compare between samples
and also because the brandy that is sold is usually at 40 % ethanol. The volume of the
distillate from each fermentation was recorded. Using this volume and the % ethanol in
that sample, we calculated the volume of 40% ethanol produced in mL. This value was

needed to calculate the yield of pure alcohol produced from each fermentation.

34

Area of the Ethanol Peak from the GC

3.E+07

2.E+07

Figure 2.3. Ethanol Calibration Curve to Determine the Concentration

of Ethanol in the Distillates

y = 230203x + 6E+06
R2 = 0.9974

2.E+07 “r

1.E+07

5.E+06

0.E+(X) ‘ -

 

40 60
Percent Ethanol (v/v)

35

80

100

Area of the Methanol Peak frrn GC

Figure 2.4. Methanol Calibration Curve to Determine the Concentration

2.50E+05

2.00E+05

1.50E+05

1.00E+05 '

5.00E+04

0.00E+00

of Methanol in the Distillates

 

 

y = 410.76x
R2 = 0.9952

 

 

100 200 300 400 500 600
Concentration of Methanol in mg/ 100 mL in 40 % EtOH Soln.

36

Results and Discussion

3.1 Analysis of the Distillates Using the Calibration Curves

One of the two main objectives of this study was to analyze the products of the
distillation in order to quantify organoleptic compounds present and compare their
concentrations at 10, 15 and 20°C. The flavor of an alcoholic beverage depends on a
large part on the types of these organoleptic compounds present and their concentration.
Organoleptic compounds, also called congeners, include a very large number of
compounds. Table 1.1 shows the distribution of the compounds among different
chemical categories3 .

This huge number of compounds makes it impractical to look at each single
compound quantitatively in one study due to many reasons. In the current work, 21
compounds were analyzed in the distillates. These compounds were chosen becausethey
include the more important compounds reported in previous studies, and their
concentration is high enough for gas chromatography detectionz’ 3‘ 9' '0 Some of these
chemicals were not detected gas chromatography due to their low concentration or
absence. Also, some additional peaks were present in the chromatogram were not
identified. The compounds chosen and their respective retention times and boiling points
can be seen in Table 2.1. The retention times were used so we can qualitatively identify
them in our chromatograms, and the standards for these chemicals were ran under the
same conditions as our samples from the distillates. Usually, lower boiling compounds
elute first in gas chromatography, but that is not always the case when separating

alcohols as seen in Table 2.1. This is due to the interaction between the ethanol, water,

and the compound. The interaction of the compound in ethanol and water also plays a

37

role. The compound that interacts more with ethanol will have a shorter retention time
because it is vaporized with ethanol.
3.2 Congeners Present in Distillates from Fruit Fermented at 10 °C

The congeners were put in four categories: fusel alcohols, methanol, carbonyls,
and esters. The volume of 40 % ethanol produced from each fruit was also determined.
The main firsel alcohols present were: 2-methyl-2-butanol, l-propanol, 2-
methyl-l-propanol, l-butanol, 3-methyl-l-butanol, 2-methyl-l-butanol, and 1-hexanol.
Large concentration of fusel alcohols are in general a negative flavor component as they
have a damp cloth smell and impart a bad taste to the alcoholic beverages. They also can
impart bitter harsh aftertaste. Sometimes, compounds that exhibit bad taste or aroma
when present alone, could have a more pleasant aroma when in a mixture. Though, as
can be seen from Table 1.2, fusel alcohols have a high threshold value and need to be in
large concentrations to cause a change in the flavor of the product. The most abundant
fusel alcohols were l-propanol and 3-methyl-l-butanol. As seen from Tables 3.1 and
3.2, 1-propanol and 3-methyl-l-butanol are the most abundant fusel alcohols. So, it is a
very good approximation when comparing our values to those in Tables 3.1 and 3.2.
Tanner and Brunner2 have done extensive work on fruit spirits, and it is desirable to
compare the experimental values with theirs. For the fusel alcohols, the range of
concentrations measured ranges from 832.5 mg/ 100 mL for Bartlett pears to 2245
mg/100 mL in granny smith apples, Table 3.3. The values in our tables are in mg/ 100
mL in 40 % ethanol solution, but were converted to mg/ 100 mL in absolute alcohol
(A.A.) to compare them. Tanner and Brunner2 reported 304 mg/ 100 mL for the pears and

468 mg/ 100 mL for apples, but did not specify what kind of apples were studied. Our

38

 

 

 

 

Compound Kirsch Zwetschgen Bartlett pear Marc Brandy Apple
(Cherries) (Plums)
Methanol 417 931 1546 1560 58 503
Ethyl acetate 350 280 200 393 61 99
1-Propanol 888 138 69 41 52 59
2-Butanol 28 0 28 3 l 0
2-Methyl-1- 42 50 43 98 108 106
Propanol
l-Butanol 0 5 19 0 1 0
2-Methyl-1-Butanol 12 28 34 57 50
3-Methyl-l-Butanol 80 13 1 l l 1 l 14 2 14 303
Acetaldehyde 5 15 10 72 7 13
Total acid
(acetic acid) 100 33 90 45 40 55

 

 

Table 3.1. Analysis sample from Tanner and BrunnerI that shows different fruit with

concentrations of several congeners (all values are in mg/ 100 mL AA.)

39

 

 

 

Alcohol

Methanol

Ethyl acetate

Total ester

Aldehyde
l-Propanol
2-Butanol
2-Methy1-1-Propanol
l-Butanol
2-Methyl-1-Butanol
3-Methyl-1-Butanol
Total acid (calculated

as acetic acid)

Volatile acid
(calculated
as acetic acid)

Extract ( dry residue)
Hydrogen cyanide
Copper

Furfural

 

% Vol

mg/100 mL A.A.
mg/100 mL A.A.
myIOO mL A.A.
mg/100 mL A.A.
mg/100 mL A.A.
mg/lOO mL A.A.
mg/100 rnL A.A.
mg/100 mL A.A.
mg/100 mL A.A.
mg/100 mL A.A.
mg/lOO mL A.A.

mg/100 mL A.A.

mg/L
mg/L
mg/L

mg/100 mL a.A.

42

460

658

750

90

1252

71

63

12

88

220

180

165

45

2

 

 

Table 3.2. Analysis of a cherry sample from Tanner and Brunner.

40

2

 

Concentration of congeners and volume of ethanol

produced from the different fruits

 

 

 

mg/ 100mL in 40 % ETOH mL of 40 % ETOH
Fruit Fusel ales. Methanol Carbonyls Esters Ethanol
Plums 432 107 13.7 47.0 73.0
Peaches 372 365 21.6 9.50 24.0
Bartlett Pears 333 409 17.4 28.0 29.0
Cherries 348 222 1 1.3 29.5 27.0
Gala apples 324 180 75.4 0 22.0
Granny Smith apples 898 104 9.70 133 98.0

 

 

Table 3.3. Experimentally determined concentrations of congeners and ethanol present in
distillates after fermentation at 10 °C as determined by gas chromatography.

41

 

are higher than theirs by almost four folds. As for the methanol, it is often regarded as a
positive flavor component, but in the United States the legal permissible limit for
methanol in alcoholic beverages is 0.35 %v/v or 700 mg/ 100 mL (A.A.). So, if the
concentrations are higher than this value, it is considered that methanol would contribute
negatively since it would be over the legal limit. Tanner and Brunner2 in reported
methanol concentrations that ranged from 58 to 1546 mg/ 100 mL, Table 3.1. In the
current work, Table 3.3, values ranged from 260mg/ 100 mL in granny smith apples to
1022 mg/100 mL in Bartlett pears. All fruits except peaches and pears had values lower
than the legal limit. Tanner and Brunner2 showed a concentration of 1546 mg/100 mL for
their Bartlett pears, higher than the value reported here.

The most common carbonyls present were acetaldehyde and acetone. They are
more important than the fusel alcohols in their contribution to the flavor because of their
much lower threshold limit, Table 1.2, and are considered to be a negative flavor
component. Concentrations ranged from 24.3 mg/ 100 mL in granny smith apples to 188
mg/ 100 mL in gala apples, Table 3.3. Tanner and Brunner2 (Table 3.1) report
concentrations that range from 5 to 72 mg/ 100 mL. The current is higher than theirs for
an example, a value of 43.5 mg/ 100 mL was obtained for pears while their value was 10
mg/ 100 mL. The most abundant ester identified in the current work was ethyl acetate and
in a very few samples ethyl forrnate was present. These compounds impart a fruity taste
to the flavor and aroma of an alcoholic beverage. They also have lower threshold limits
than most other compounds. The range goes from 0 mg/ 100 mL in gala apples to 333
mg/ 100 mL in granny smith apples. These values are higher than those in Table 3.1. The

amount of pure alcohol produced was determined to measure the efficiency of the

42

 

 

 

 

 

Yield ( liters of pure alcohol
Sugar Content per 100 Kg raw material)
Raw Material Variance Mean Variance Mean
Apples 6-15 10 3-6 5
Apricots 4-14 7 3-7 4
Pears 6-14 9 3—6 5
Blackben'ies 4-7 5.5 3
Gentian roots 5-13 3-5
Windfalls (seed fruit) 2-5 2.5
Yeast deposits 2-5
Blueberries 4.5-6 5.5 3
Rasberries 4-6 5.5 3
Elderberries 4-6 5 3
Currants 4-9 red 4.5 3.5
black 6.5
Pomace 2-4 2-3
Cherries, sweet 6-18 11 4-9 6
Peaches 7-12 8 4.7
Plums 6-15 8 4-8
Quinces 4-8 2.5-4
Marc 2-4 3
Juniper berries(dried) 20 10-11
Topinambour 13-18 6-8
Grapes 9-19 14 4-10 8
Zwetschgen(plums) 8-15 10 4-8 6

 

 

 

Table 3.4. Sugar content and alcohol output of various raw material from Tanner and

Brunner.2

43

 

fermentations. Values obtained were compared to values in Table 3.4 from Tanner and
Brunnerz. Volume of ethanol produced was 4.2 L for plums, 1.4 L for peaches, 1.7 L for
Bartlett pears, 1.5 L for cherries, 3 L for gala apples, and 5.6 L for granny smith apples.
These volumes would be obtained if we used 100 kilogram of raw material. These values
are considerably lower than the values in Table 3.4. Some of them are close or lie within
the variance of values presented there.
3.3 Congeners Present in Distillates from Fruit Fermented at 15 °C

The same method of analysis was followed for interpreting the results at 15 °C.
Fusel alcohols were present in higher concentrations than at 10 °C and higher than values
in Table 3.1. The range goes from 1062 mg/ 100 mL for Bartlett pears to 2502 mg/ 100
mL in plums and the values are presented in Table 3.5. The concentration found in plums
doubled at 15 °C and also increased for the other fruits except for the granny smith apples

where it decreased slightly. All methanol concentrations were under the legal limit

except for the peaches and Bartlett pears, which is consistent with the results at 10 °C.
Carbonyls were present in the same concentrations as at 10 °C with range from 22

mg/ 100 mL for plums to 111 mg/ 100 mL for peaches and Bartlett pears. Only Bartlett
pears and peaches show a dramatic increase in their concentrations. The range here is a
little higher than Table 3.1. The esters concentrations ranged in value from 1.6 mg/ 100
mL in Bartlett pears to 315 mg/ 100 mL in plums. Most fruits had an increase in their
concentration except for the granny smith apples where it dropped slightly. The values in
this work are very comparable to those in Table 3.1 except for peaches and red delicious
apples. The pure alcohol yield was as follows: 5.1 L for plums, 1.4 L for peaches, 3.4 L

for Bartlett pears, 3.9 L for gala apples, 4.1 L for granny smith apples, and 2.6 L for red

 

 

 

 

 

Concentration of congeners and volume of ethanol
produced from the different fruits
mg/ 100mL in 40 % ETOH mL of 40% ETOH

Fruit Fusel alcs Methanol Carbonyls Esters Ethanol
Plums 1001 105 8.79 126 90.0
Peaches 287 710 44.4 59.8 25.0
Bartlett pears 425 420 44.2 0.640 59.0
Gala apples 747 81.0 15.3 50.6 69.0
Granny Smith ' 651 128 10.2 51.7 72.0
apples
Red Delicious apples 600 127 18.0 21.5 46.0

 

 

Table 3.5. Experimentally determined concentrations of congeners and ethanol present in
distillates after fermentation at 15 °C as determined by gas chromatography.

45

 

delicious apples. Some of these values are higher than those obtained at 10 °C and closer
to the values in Table 3.4.
3.4 Congeners Present in Distillates from Fruit Fermented at 20 °C

Fusel alcohols range from 1588 mg/ 100 mL for plums to 1977 mg/ 100 mL in

Bartlett pears, Table 3.6. These values are higher than those at 10 °C and close to the
values at 15 °C. The range is higher than that of Table 3.1 and for plums it is almost 5

times more. The methanol concentrations range from 178 mg/100 mL for red delicious
apples and 720 mg/ 100 mL for Bartlett pears. All values are under the legal limit except
for the pears where it is slightly higher. The carbonyls range from 26.8 mg/ 100 mL for
Bartlett pears to 380 mg/ 100 mL for red delicious apples. The concentrations range in
this work were higher than the range of values in Table 3.1. Esters range from 0 mg/ 100
mL for granny smith apples to 205 mg/ 100 mL for plums. This value for plums is close
to the one in Table 3.1, which is 280 mg/ 100 mL. There is no clear trend in values for
each fruit as a function of temperature. Production of pure alcohol was as follows: 2.9 L
for plums, 4.1 L for Bartlett pears, 7.7 L for gala apples, 5.8 L for granny smith apples,
and 6.2 L for red delicious apples. These values are mostly within the range of Table 3.4,
and apples in the present study have productions closest to the mean presented in Table
3.4 while plums and Bartlett pears have values that are closer to the lower values of the
range in Table 3.4.
3.5 Comparison of Plums, Pears, and Gala Apples at the Three Temperatures
Figures 3.1, 3.2, and 3.3 show the production of the congeners at the different
temperatures for three types of fruit: plums, Bartlett pears and gala apples. The data were

analyzed to determine which is the optimum fermentation temperature for each fruit. The

46

 

 

 

 

 

Concentration of congeners and volume of
ethanol produced from the different fruits
mg/ 100mL in 40 % ETOH soln. mL of 40 % ETOH

Fruit Fusel alcs Methanol Carbonyls Esters Ethanol
Plums 602 230 67.0 82.0 51.0
Bartlett Pears 784 288 10.7 0.560 72.0
Gala apples 727 179 22.3 22.1 134
Granny Smith 664 182 20.8 0.000 102
apples
Red Delicious apples 783 71.4 152 79.6 109

 

 

Table 3.6. Experimentally determined concentrations of congeners and ethanol present in
distillates after fermentation at 20 °C as determined by gas chromatography.

47

 

concentrations of the congeners were compared as well as the statistical results obtained
from performing the Duncan’s Multiple Range Test on the data. Standard deviation was
also calculated for each concentration as seen in Table 3.7. The Duncan test is essentially
done to reduce the comparison-wise error rate to reduce the escalation in experiment-wise
error. The values are arranged horizontally and vertically from smallest to largest and the
difference is calculated. The difference between two values is compared to the least
significant range for two adjacent treatments which is calculated based on the variance,
significance level or, and q which is based on the degrees of freedom. or was chosen to be
0.05 and the degrees of freedom were 4. From Table 3.7, the best fermentation
temperature for plums is 15 °C. This is because there is a higher concentrtaion of fusel
alcohols and esters, very low concentration of methanol and cabonyls, and the largest
volume of ethanol. Though, it can be seen that certain values at the three temperatures
had no statistical difference and more than one temperature could have been chosen. As
for the Bartlett pears, 20 °C would be chosen by looking at the same criteria as before.
For gala apples, 15 or 20 °C could be the best temperature to run the fermentation at.
Also, these results show that there doesn’t exist a stringent temperature at which brandy
yeast fermentation should be run. As can be seen from the Duncan’s test, that many
values are statistically similar at two temperatures and also if they were different it is not
exactly clear the extent they have on the final flavor of the alcoholic beverage. These
data could be further analyzed by looking at different compounds in each category of

congener and calculating their flavor thresholds from Table 1.2.

48

Figure 3.1. Analysis of Congeners from Plums Fermented at Different Temperatures

1200 /
- ‘ ‘ . fusel alcohols
- methanol
00 - carbonyls

10°C 15 °C 20°C

 

 

 
  
  

00°

 

 

40 % Ethanol Solutjpn
o o o o
o o o o
l 1

Concentration in mg/ 100mL
N b 0‘

m

 

 

Temperature at which the fermentation was done

Concentration in mg/ 100mL

in 40 % Ethanol Solution

 

Figure 3.2. Analysis of Congeners from Bartlett Pears Fermented at Different
Temperatures

 

 

 

 

 

10 °C 15 °C 20 °C

Temperature at which the fermentation was done

50

Concentration in mg/ 100 mL

in 40 % Ethanol Solution

Figure 3.3. Analysis of Congeners from Gala Apples Fermented at
Different Temperatures

 

800* /
700

600

   

 

 

   

Fusel alcohols

Methanol

100 - Carbonyls
- Esters

10 °C 15 °C 20 °C

200

 

 

Temperature at which the fermentation was done

51

 

 

 

Concentration of congeners and the error in each

 

 

 

 

 

 

 

 

 

Fruit measurement

Plums Temperature °C
Congeneri error 10 15 20

mg/100 mL in 40% ETOH

Fusel alcs 432 $14.80 A 1001 $150 B 602 $270 A
Methanol 107 $18.6 A 105 $5.50 A 230 $78.0 B
Carbonyls 13.7 $0.850 A 8.79 $1.38 A 67.0 $47.0 B
Esters 47.0 $1.17 A 126 $33.0 B 82.0 $142 A
Bartlett pears

Fusel alcs 333 $3.90 A 424 $12.6 B 784 $157 C
Methanol 409 $31.5 B 420 $54.0 B 288 $2.30 A
Carbonyls 17.4 $1.30 A 44.2 $0.40 B 10.7 $0.250 A
Esters 27.9 $1.20 B 0.640 $ 0.20 A 0.56 $0.12 A
Gala apples

Fusel alcs 324 $32.0 A 747 $ 84.0 B 727 $184 B
Methanol 180 $11.7 B 81.0 $17.6 A 179 $4.80 B
Carbonyls 75.4 $26.0 B 15.3 $2.00 A 22.3 $13.4 A
Esters 56.1 $15.4 A 50.6 $6.90 C 22.1 $31.0 B

 

 

Table 3.7. The amount of congeners present in distillates from plums, pears and
gala apples that were fermented at the three temperatures. The error is the standard
deviation calculated from the three runs for each fruit at each temperature. The data
in this table were used to plot fig. 3.1, 3.2, and 3.3. The letters A, B and C in the
table represents the value obtained from performing the Duncan Range Test Method
with an or of 0.05 and 4 degrees of freedom. If two values have the same letter, it
signifies that they are statistically similar.

52

 

 

3.6 Fermentation with the Addition of Different Liquefaction Enzymes

The enzyme experiments were done with four different fruits: gala apples, Bartlett
pears, red delicious apples, and granny smith apples. The Same amount of each different
enzyme was added to each respective fermentation. All fermentations were run under the
same conditions and the mashes were treated with the enzymes in the same manner.

The enzymes are used because they cause rapid degradation of the pectin and thus
resulting in a quick liquefaction of mash and improved pumpability. Also, they are
recommended because they cause a faster and easier onset of fermentation and complete
fermentation of mash for an optimal alcohol yield. All fermentations were run for 14
days and noticeable change occurred in the viscosity of the mash between the enzyme
treated and the non-enzyme treated mash. There were barely any fruit pieces left in the
mash treated with the enzymes. All types of enzymes used produced the almost the same
amount of liquefaction as observed qualitatively. The temperature used for all

fermentations was 15 °C. This temperature was chosen because it is in the range

recommended by many researchers and experts in this field.2 Controls were run in each
experiment in which no enzymes were added. Congener concentrations were compared
for the different enzyme treatments, although, the main focus was the amount of ethanol
and methanol produced by each enzyme treatment.
3.7 Congeners and Ethanol Present in Distillates from Gala Apples Fermented at
15 °C with Different Liquefaction Enzymes Added
Each particular fermentation was designated by the name of the enzyme that was
used to treat the mash. The amount of fusel alcohols present range from 1257 mg / 100

mL in the fermentation with FM enzyme added to it, to 1867 mg/ 100 mL in the

53

fermentation with no enzyme added. The methanol concentration shows a dramatic
increase from the control to the enzyme treatment ranging from 182 to 1450 mg/ 100 mL
(A.A.) in the FM treatment. The enzyme treatment that produced the lowest
concentration of methanol was 0996/9. Carbonyl concentration was 38 mg/ 100 mL in the
FM, to 43 mg/ 100 mL in 0956/2 and 0996/2. Esters were only present in the control and
at a concentration of 127 mg/ 100 mL. Ethanol yield of pure alcohol was as follows: 3.9
L for the control, 4.3 L for FM, 3.9 L for 0956/2, 4.9 L for 0996/2, and 4.5 L for 0996/9.
All of these values were in the range presented in Table 3.4. The fermentations where
enzymes were added did not produce more ethanol as hypothesized. Figure 3.4 shows
the production of 40 % ethanol and the concentration of methanol in each sample and
Table 3.8 presents the concentrations of the congeners. The trend is that ethanol
production goes down slightly as enzymes were added while the methanol concentration
went up drastically.
3.8 Congeners and Ethanol Present in Distillates from Bartlett Pears Fermented at
15 °C with Different Liquefaction Enzymes Added

The same analysis was followed in the Bartlett pears experiment. Fusel alcohols
concentration was lower in the pears for all samples than their production from gala
apples. It was consistent with the concentrations present at 10 and 15 °C but lower than
the value at 20 °C. All values are presented in Table 3.9. Values ranged from 755
mg/ 100 mL in 0956/2 to 1060 mg/ 100 mL in the control. Table 3.1 shows a
concentration of 304 mg/ 100 mL for bartlett pears and it is lower than the value we

obtained. Methanol concentrations in all samples was above the legal limit and it ranged

from 896 in the control to 1400 mg/ 100 mL (A.A.) in the 0996/2. The final value of

54

Volume of 40 % Ethanol in mL

Figure 3.4. Volume of 40 % Ethanol Produced and Concentration of Methanol in Gala

Apples Fermented at 15 °C with Liquefaction Enzymes Adtbd

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100 600
90 El Ethanol I Methanol

. 500
80 «
70 .

«L . 400

60 .
50 . I 300
40 «

200
30 I
20

. 100
10 .

0 0
Control FM 0956/2 0996/2 0996/9

Type of Liquefaction Enzyme Added

55

Concentration of Methanol in mg/ 100 mL in 40 % Ethanol

 

 

 

 

 

 

 

 

 

 

Concentration of congeners and volume of ethanol

produced from fruits with liquefaction enzymes added
Fruit
Gala apples mg/ 100mL in 40 % ETOH mL of 40 % ETOH
Enzyme Fusel alcs Methanol Carbonyls Esters Ethanol
No enzyme 747 81.0 15.3 50.6 69.0
FM 503 530 15.3 0 75.0
0956/2 582 525 17.3 0 69.0
0996/2 549 508 17.5 0 87.0
0996/9 653 424 16.2 0 79.0
Ifiartlett Peard
No enzyme 424 421 44.2 0.65 59.0
FM 336 588 34.6 84.5 68.0
0956/2 302 583 32.7 82.9 71.0
0996/2 354 623 36.1 89.9 61.0

 

Table 3.8. Experimentally determined concentration of congeners and
ethanol present in distillates after fermentation at 15 °C with liquefaction

enzymes added as determined by gas chromatography.

56

 

1400 mg/ 100 mL is close to the value presented for Bartlett pears in Table 3.1 which is
1546 mg/ 100 mL. Tanner and Brunner2 did not specify if any enzyme treatment was
used for the mashes. Carbonyls concentrations ranged from 82 mg/ 100 mL in 0956/2 to
110 mg / 100 mL in the control. All samples’ concentrations are very close to each other
and far from the values in Table 3.1. Ester concentrations ranged from 1.6 mg/ 100 mL
in the control to 225 mg/ 100 mL in the FM and the 0996/2 samples. This increase is
positive because it adds to the positive component of the aroma of an alcoholic beverage.
This value is also close to the one presented in Table 3.1, which is 200 mg/ 100 mL.
Ethanol yield of pure alcohol goes as follows: 3.4 L for the control, 3.9 L for FM, 4.1 L
for 0956/2, and 3.5 L for 0996/2. All values are very similar and are inside the range of
values presented in Table 3.4. Figure 3.5 shows the ethanol yield at 40 % and the
methanol concentration of each sample, and it is clear that ethanol production stayed the
same while the methanol concentration increased.
3.9 Congeners and Ethanol Present in Red Delicious Apples Fermented at 15 °C

with Different Liquefaction Enzymes Added

Fusel alcohol concentrations ranged from 1145 mg/ 100 mL in 0996/9 to 1600 mg/ 100
mL in the control. The concentration was higher for the control than for the other
samples and was higher than the value reported in Table 3.1. Methanol concentrations
jumped from 38 in the control to 1238 mg/ 100 mL (A.A.) in 0996/9 and was higher than
700 mg/ 100 mL (A.A.) in the other samples, Table 3.9. Methanol concentration reported
in Table 3.1 for apples is 503 mg/ 100 mL and between both values determined
experimentally. Carbonyls concentrations ranged from 27.3 mg/ 100 mL in 0956/2 to 64

mg/ 100 mL. Table 3.1 presented a concentration of 13 mg/ 100 mL, lower than the

57

Volume of 40 % Ethanol in mL

1(1)

HgmfiVdmmcflO%EhmiHorhnedaflCammaimdh/bdnrflmBaflaths
WalSTVtitlrliqrdmtimErzyrmAdtbd

 

 

«—

 

Control

 

 

 

 

 

 

 

FM 0956/2

 

UBhand IMathmd

 

 

0996/2

Type of Liquefaction Enzyme Added

58

 

 

é

' V.

g

Concentration of Methanol in mg/ 100 mL in 40 % Ethanol

§

§

§

§

Volume of Ethanol in mL

Figll'elfi Volmreof40%EhamledmedmrdCamlrafimthfl1amlinRed
DeliciousAmlesFanentedat 15 °CwithquuefactionErzyrrmAdded

 

m‘ %
'1'

 

 

8

2.9

.8

{5

 

10*

 

 

 

 

 

04—-

 

Control FM 0956/2 0996/2
Type of Liquefaction Enzyme Added

.59

DEinnol
III/Ethanol

 

 

0996/9

é

a
Concentration of Methanol in mg/ 100 mL in 40 % Ethano

I

§

 

 

 

 

 

 

 

 

 

 

 

Concentration of congeners and volume of ethanol
Fruit produced from fruits with liquefaction

enzymes added
Red Delicious apples mg/ 100mL in 40 % ETOH soln. mL of 40 % ETOH
Enzyme Fusel alcs Methanol Carbonyls Esters Ethanol
No enzyme 642 66.0 22.9 79.5 47.0
FM 463 408 11.6 38.6 84.0
0956l2 569 407 15.4 64.1 58.0
0996/2 552 398 26.6 63.5 44.0
0996/9 458 354 12.7 47.0 59.0
Ikiranny Smith apple;
No enzyme 651 145 10.2 51.7 73.0
FM 583 501 38.2 66.2 89.0
0956l2 732 583 50.9 118 64.0
0996/2 555 506 12.7 42.9 93.0
0996/9 765 488 12.3 62.0 86.0

 

Table 3.9. Experimentally determined concentration of congeners and ethanol present in
distillates after fermentation of red delicious apples and granny smith apples at 15 °C with
liquefaction enzymes added as determined by gas chromatography.

60

 

current values. Esters ranged from 96.5 mg/ 100 mL in FM to 198 mg/ 100 mL in the
control. The concentration of esters went down in the samples treated with the enzymes
and this will lower their contribution to the positive components in the aroma. Ethanol
yield of pure alcohol was: 2.7 L for the control, 4.8 L for FM, 3.3 L for 0956l2, 2.5 L for
0996/2, and 3.4 L for 0996/9. These values are within the range of Table 3.4. The
production increased in the most of the fermentations treated with enzymes, but not
greatly. Figure 3.6 shows the production of ethanol and the concentration of methanol
present in each sample.
3.10 Congeners and Ethanol Present in Granny Smith Apples Fermented at 15 °C

with Different Liquefaction Enzymes Added

Fusel alcohols concentrations ranged from 1387 mg/ 100 mL in 0996/2 to 1915
mg/ 100 mL in 0996/9 as seen in Table 3.9. This is higher than the value presented in
Table 3.1, which is 468 mg/ 100 mL. No major differences is seen between fermentations
with enzymes added and the control. Methanol concentrations show a dramatic increase
from 320 in the control to 1275 mg/ 100 mL (A.A.) in 0996/2. All methanol
concentrations for fermentations that had enzymes added to them had concentrations
above the legal limit. Carbonyl concentrations ranged from 25 mg] 100 mL in the control
to 127 mg/ 100 mL in the 0956l2. These values are higher than those values presented in
Table 3.1. Esters ranged in concentration from 107 mg/ 100 mL in 0996/2 to 295 mg/ 100
mL in 0956/2. The control had a concentration of 130 mg/ 100 mL. So, it is within the
range and no clear trend could be seen for the ester concentration. Production of pure
ethanol was: 4.2 L for the contol, 5.1 L for FM, 3.7 L for 0956l2, 5.3 L for 0996/2, and

4.9 L for 0996/9. All values are within the range presented in Table 3.4. Figure 3.7

61

shows the concentration of methanol and the production of 40 % ethanol for all samples.

No advantages are gained by treating the mash with enzymes.

62

Volume of 40 % Ethanol in m].

100

70 «

10

Hgne37.Vdrmof40%maolProdmddeaxnmaionofMalmmeny
SrithApplesFemrnted15°CwithIkiuefmimEnzynesAckled

 

 

 

 

I——I

 

 

Control

 

 

 

 

 

FM 0956l2

Type of Liquefaction Enzyme Added

63

DEthanol Ill/Ethano]

 

0996/2 0996/9

assess

Concentration of Methanol in mg/ 100 mL in 40 % Ethanol

§

 

 

Computer Simulation

4.1 ChemCAD Batch

ChemCAD Batch® is a commercial batch distillation program marketed by
Chemstations®. Common applications of this software include: modeling existing batch

column equipment, exploring alternative processes for existing products, design of new
equipment, and validation of thermodynamics using batch column equipment. In this

study, ChemCAD Batch® was used to model the distillation of a fruit mash using the

experimentally determined congener concentration. The main congener of interest is
methanol and it is useful to track the concentration of the methanol as it is distilled.
These results could aid in knowing when to make the cuts and eliminate as much of the
methanol as possible. It also can allow a preliminary assessment as to whether methanol
can be removed by distillation. The ethanol profile in the distillate will be looked at as
well.

To start the simulation, it is assumed that ethanol is present at 8% v/v in the mash
before the start of the distillation. The concentrations of the congeners at 40 % ethanol
were used to find the concentrations in 8 % v/v of ethanol. The remainder of the mash
composition was assumed to be water. All these values were entered into the software
as the pot charge. The mash was assumed to be at 25 °C and 1 atmosphere. The
column selected had four trays, a partial condenser, and a total condenser. Also, the
flow rate was set at 0.333 Liters/minute, and a reflux ratio of 1.5 was chosen as
determined experimentally.

The profile for ethanol and methanol in the distillate volumes was determined.

The distillate from gala apples was chosen including distillates that had liquefaction

enzymes added to the mash. Figure 4.1 shows the ethanol profile. It starts at 80 % v/v
and goes down to almost 29 % v/v. Usually, the first three cuts, which are called the
heads, are discarded. The hearts, which are the middle cuts, are saved and then later
diluted to produce the brandy used for consumption. After the concentration of the
ethanol drops below 40 % v/v, the collected distillate called the tails is discarded. All
distillates showed the same trend for the ethanol concentration and there was no
difference whether it was an enzyme treated mash or not. The values that were used to
obtain Figure 4.1 are very close to each other and that is why we are not able to
differentiate the five series clearly. The Methanol profile shown in Figure 4.2 shows
the methanol concentration in % v/v versus the distillate volume from gala apples. The
control which had no enzymes added to the mash had methanol concentrations much
lower than the enzyme treated mashes. All enzyme treated mashes had methanol
concentration above the legal limit of 0.35 % v/v.

The trend in the profile is the same for all distillates. The concentration starts low and
peaks in the middle and then it goes back down. So, the middle, which is the hearts,

has the highest concentration of methanol.

65

Ethanol Concentration % v/v

80

70

50

30‘

20

10 7

Figure 4.1. Ethanol Profile in the Distillate from Gala Apples Fermented at

15 0C with Different Liquefaction Enzyn‘es Added as Determined Using ChemCAD

Batch Simulation

 

 

 

 

2 4 6
Curmlative Distillate Volume (L)

66

10

 

 

0.9

0.8 7

0.7 a

0.6

0.4

Methanol Concentration % v/v

0.3

0.2

0.1

Figure 4.2. Methanol Profile in the Distillate from Gala Apples Fermented

at 15 °C with Different Liquefaction Enzymes Added as Determined Using
ChemCAD Batch Simulation

 

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Cumulative Distillate Volume (L)

67

Conclusion and Summary

This research work was undertaken to improve understanding of two key areas in the
production of brandy from fruits. The temperature at which the fermentations are done
has not been studied quantitatively in relation to the amount and type of congeners and
ethanol produced. In this research work, we compared our results with those of Tanner
and Brunner2 who have done extensive research on fruit distillation and production of
brandy. The results obtained from the distillates of the fermentations that were ran at 10,
15, and 20 °C showed relatively consistent results regarding the production of these
congeners and ethanol. The fusel alcohols at all three temperatures were within the range
that Tanner and Brunner mentioned and that was 200 - 3000 mg/ 100 mL. These values
could vary a great deal depending on the quality of the fruit used. Esters and carbonyls
were studied and they were present in higher concentrations than what was reported by
Tanner and Bruner. Yet, this increase in these values or those of the fusel alcohols are
not the only parameter to be considered when evaluating the flavor of an alcoholic
beverage. As seen in Table 1.2, different compounds have different sensory odor
thresholds. These odor thresholds and evaluation of the contribution of a compound to
the overall odor of a product are difficult areas of study. The production of ethanol was a
little higher from fermentations at 15 and 20 °C, but that was not true for all the fruit.
Methanol was below the legal limit for mostly all fruit except Bartlett pears and peaches
at all fermentation temperatures.

Liquefaction enzymes can be used in the manufacture of brandy for optimal yield
of alcohol and intensification of fruit aroma. The enzymes used in this study, are the

commercially available Spirizym FM, which is pectin methyl esterase and three

68

polygalacturonases. Our results showed that for the four fruits studied, methanol
concentrations increased dramatically when the mash was treated with any of the four
enzymes. The concentration of methanol went well above the legal limit. As for ethanol,
the production did not change and in some cases the fermentations with the enzymes in
them had lower ethanol production. The increase of methanol was expected since the
action of enzymes results in the release of methanol from the pectin and from extensive
degradation and liquefaction of the mash. There was no trend regarding the change of the
congeners’ concentrations. In some cases certain compounds were produced more but
most values were in a range that is very comparable in certain fruit to values presented by
Tanner and Brunner.2 The large increase in methanol concentration makes the use of

liquefaction enzymes a very questionable practice for fruit brandy production.

69

Recommendation for Future Work

This work presented new information that would help in producing brandy of good
quality. The organoleptic compounds that are present in alcoholic beverages are huge as
seen by Table 1.1. The compounds that were analyzed here are only a fraction of the
total number. Other detection methods such as gas chromatography/mass spectrometry
could be used to detect compounds that are present in very low concentrations, but are of
importance because they contribute significantly to the aroma of the final product. The
use of enzymes could further be studied by using other enzymes that are commercially
available in the market and study their effects. After treating the mashes with the
liquefaction enzymes, enzymes could be deactivated by heat or a chemical method to see

if methanol production could be lowered and still retain the advantages of liquefaction.

70

10.

ll.

12.

13.

14.

15.

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ERBSLOH GEISENHEIM. Getranke-Technologie Gmbh & Co. KG

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