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EVALUATION OF METHODS TO INCREASE FREE
PROCYANIDIN CONTENT IN THE CIDER FERMENTATION
PROCESS OF JONATHAN APPLES

presented by

Mavis Tan

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EVALUATION OF METHODS TO INCREASE FREE PROCYANIDIN
CONTENT IN THE CIDER FERMENTATION PROCESS OF JONATHAN
APPLES

By

Mavis Tan

A THESIS

Submitted to
Michigan State University
In partial fulfillment of the requirements
For the degree of

MASTER OF SCIENCE

Department of Food Science and Human Nutrition

2005

ABSTRACT
EVALUATION OF METHODS TO INCREASE FREE PROCYANIDIN
CONTENT IN THE CIDER FERMENTATION PROCESS OF JONATHAN
APPLES
By
Mavis Tan
Procyanidins impart the desired astringent mouthfeel and bitterness flavor in
fermented cider. Methods were investigated to enhance free procyanidin levels in
fermented cider made from Jonathan dessert apples during and prior to fermentation.
Colorimetric assays (i.e. BuOH/HCI and Vanillin in methanol) were found to be more
effective in analyzing procyanidins than HPLC acid — phloroglucinol method.
Cider samples were fermented using DV-lO yeast for 20 days at temperatures 13-
14 °C at O, 10, and 30% apple solids (crushed apples with no peel and core). No
significant effect of alcohol production was observed on the procyanidin content, chain
length and consequently, mean degree of polymerization (mDP). mDP of procyanidins
increased in samples containing 30% apple solids between Day 0 and S. Procyanidin
content was higher in samples containing 0% apple solids on Day 14 vs Day 5. Overall,
alcohol production, apple solids and time did not impact procyanidin content in hard

cider during yeast fermentation.

Unfermented cider samples heated at treatments of 30, 40, and 50 °C had no
significant differences in free procyanidin content. Unfermented cider samples treated
with tenfold pectolytic enzyme concentrations of industrial dosages (~1000 ppm)
significantly increased procyanidin concentrations without affecting mDP. Procyanidin

levels in cider were enhanced using high concentration pectolytic enzyme treatments.

DEDICATION

To my parents, Tan 800 J in and Seow Ah Choo for their unconditional love and words of

encouragement which will always live in me.

iii

ACKNOWLEDGEMENTS

My deepest gratitude goes to my major professor, Dr. Janice Harte for mentoring
me in my research project(s) and Dr. Pat Oriel as the research advisor in my thesis. I also
thank Dr. Kirk Dolan and Dr. Gale Strasburg, my committee members for their teachings
and support.

Special appreciation is dedicated to Dr. Maurice Bennink for the use of his lab
facilities and shared expertise in the analysis of phytochemicals, Muhammad Siddiq for
use of his pectolytic enzyme samples, Bill Chase of MSU Horticulture Farm for the apple
supplies and Juan Steibel of MSU Statistical Consulting Center for his guidance in
statistical analysis. I thank Karina Forberger, David Sterling, and Monica Fink for their
lab technical support.

This research project was generously supported by the Michigan Apple
Committee, USDA Rural Development and Uncle John’s Cider Mill.

I extend my gratitude to fellow colleagues, Norm Matella, Mitzi Ma, Shantanu
Kelkar, Darclay Popa, Maria Suparno, Kevin Yen and Jennifer Sowa for their friendship
and teamwork, which was vital to my success in graduate school. Special thanks to
Andreas Agung, Firman Priatno and Poong Se Yin, for being my confidants throughout
these years.

I thank my family-in-Christ for never giving up on me through their prayers and
words of encouragement. Finally, I praise and thank God for providing me love, comfort

and guidance throughout the trials in life.

iv

TABLE OF CONTENTS

LIST OF TABLES ................................................................................ viii
LIST OF FIGURES ................................................................................. x
INTRODUCTION ................................................................................. 1
1. LITERATURE REVIEW ....................................................................... 4
1.1 Polymeric flavan-3-ols (Procyanidin) ........................................................ 4
1.1.1 Procyanidins in apples/apple products ....................................... 7
1.1.2 Importance to industry ......................................................... 10
1.2 Analysis of procyanidins ..................................................................... 11
1.2.1 Colorimetric assays ............................................................ 12
1.2.2 Chromatography ............................................................... 14
1.3 Hard cider production ......................................................................... 16
1.3.1 Fermentation .................................................................... 17
1.3.1.1 Complexation of apple polyphenols with polysaccharides .............. 19
1.3.2 Heat treatments ................................................................. 21
1.3.3 Pectolytic enzymatic treatments .............................................. 22
2. MATERIAL AND METHODS ................................................................ 24
2.1 Plant material .................................................................................... 24
2.2 Juice preparation ................................................................................ 24
2.3 Extraction of procyanidins ........................................................................ 25
2.3.1 Centrifugation .................................................................. 25
2.3.2 Rotary evaporation ............................................................ 25

2.3.3 Solid phase extraction .......................................................... 26

2.4 Analysis of procyanidins ..................................................................... 26
2.4.1 High liquid performance chromatography .................................. 26

2.4.1.1 Sample preparation ...................................................... 26

2.4.1.2 Instrumental settings ..................................................... 27

2.4.2 Colorimetric assays ............................................................ 27

2.4.2.1 Depolymerization in Butanol/HCl (Porter’s reagent)... . .. . . . . . . . .....28

2.4.2.2 Vanillin in Methanol assay ............................................... 28

2.4.2.3. Mean degree of polymerization (mDP) ................................. 29

2.5 Fermentation .................................................................................... 29
2.6 Statistical analysis .............................................................................. 31
3. RESULTS AND DISCUSSIONS .............................................................. 32
3.1 Evaluation of analytical methods on fermented hard apple cider ........................ 32
3.2 Juice sample preparation ....................................................................... 36
3.3 Fermentation .................................................................................... 37
3.3.1 Sugar content .................................................................... 37

3.3.2 pH ................................................................................. 39

3.4. Procyanidin estimation ........................................................................ 40

3.4.1 The effect of fermentation on the concentration of procyanidin
in cider ...................................................................... 40
3.4.1.1 The effect of alcohol production by yeast fermentation on the

concentration of procyanidins in cider fermentation made from

Jonathan apples .............................................................. 41
3.4.1.2 The effect of apple solids on the concentration of procyanidins

in cider fermentation made fiom Jonathan apples ..................... 46

vi

3.4.1.3 The effect of fermentation time on the concentration of
procyanidins in cider fermentation made from Jonathan apples . ..50

3.4.2 The effect of heat on the concentration of procyanidins in cider

made from Jonathan apples ................................................ 54
3.4.3 The effect of pectolytic enzymes on the concentration of

procyanidins in cider made from Jonathan apples ...................... 59

4. CONCLUSIONS AND RECOMMENDATIONS .......................................... 65
4.1 Summary and conclusions .................................................................... 65
4.2 Recommendations for future research ...................................................... 68
APPENDIX .......................................................................................... 69
Appendix 1: Reagents ..................................................................... 69
Appendix 2: Raw data for fermentation tests .......................................... 70
Appendix 3: Raw data for heat and pectolytic enzymatic tests ...................... 75
REFERENCES ...................................................................................... 78

vii

Table
1 . l

1.2

3.4.1

3.4.2

A21

A22

A.2.3

A.2.4

A.2.5

A.2.6

A.3.l

A.3.2

A.3.3

A.3.4

A.3.5

LIST OF TABLES
Substitution patterns within procyanidin classes in reference to
Figure 1.2 (Cheynier and Fulcrand 2003)

Enzymes typically used in fruits and vegetables processing
(Richardson and Hyslop 1985)

Procyanidin terminal end unit concentrations in control and yeast
samples of Jonathan cider fermentation containing 0, 10 and 30%
apple solids on fermentation days 0, 5, 10, 14 and 20
Procyanidin extension unit concentrations in control and yeast
samples of Jonathan cider fermentation containing 0, 10 and 30%
apple solids on fermentation days 0, 5, 10, 14 and 20

RI values of Jonathan apple cider fermentation: Batch 1

RI values of Jonathan apple cider fermentation: Batch 2

pH values of Jonathan apple cider fermentation: Batch 1

pH values of Jonathan apple cider fermentation: Batch 2

Mean absorbance values of Jonathan apple cider fermentation using

Vanillin in methanol assay

Mean absorbance values of Jonathan apple cider fermentation using

BuOH/HC1 assay

Mean absorbance values of Jonathan apple cider heat treatments
using Vanillin in methanol assay

Mean absorbance values of Jonathan apple cider heat treatments using

BuOH/HCI assay

Mean absorbance values of Jonathan apple cider heat treatments using

Vanillin in methanol assay: Preliminary

Mean absorbance values of Jonathan apple cider heat treatments using

BuOH/HCl assay: Preliminary

Page

23

42

43

70
7O
71

72

74

75

75

76

76

Mean absorbance values of Jonathan pectolytic enzyme treatments using 77

Vanillin in methanol assay

viii

A.3.6 Mean absorbance values of Jonathan pectolytic enzyme treatments using 77
BuOH/HCl assay

ix

LIST OF FIGURES

Figure Page
1.1 Classification of polymeric phenolics/tannins 4
1.2 Basic C6 — C3 — C6 structure (Haard 1985) 4
1.3 Flavonoid biosynthesis (Belitz and Grosch 1987) 5
1.4 Structure of proanthocyanidins dimers (Lazarus 2003) 6
1.5 Monomeric units of procyanidins (Tsao and Yang 2003) 7
1.6 Procyanidin B2 (Tsao and Yang 2003) 8

1.7 Extension and lower terminal units of procyanidins (Kennedy and Jones 2001) 12
1.8 Acid-catalyzed cleavage of procyanidins to cyanidins (Porter and others 1986) 13

1.9 Proposed mechanism of acid cleavage of procyanidins and formation of 15
nucleophile adducts (Kennedy and Jones 2001)

1.10 Basic hard cider making steps 16
1.11 Conversion of fermentation sugars to ethanol 17
2.1 Apple samples with anthocyanins irnbedded in flesh 25
2.2 Experiment design of fermentation setup 30
2.3 Experiment design of heat treatment 30

3.1.1 HPLC chromatogram of procyanidin cleavage product from Jonathan apple 32
extracts following acid-catalysis (A) with the presence of phloroglucinol and

(B) without phloroglucinol

3.1.2 HPLC chromatogram of procyanidin cleavage products from grape seed 34
extracts following acid-catalysis (C) with the presence of phloroglucinol and
(D) without phloroglucinol.

3.3.1 Sugar content of Jonathan hard cider during fermentation 38
3.3.2 pH of Jonathan hard cider during fermentation 39
3.4.1 The effect of alcohol production on procyanidin terminal end units 41

concentrations in Jonathan cider fermentation containing 0% apple solids
on fermentation days 0, 5, 10, 14 and 20

3.4.2 The effect of alcohol production on the procyanidin extension unit 42
concentrations in Jonathan cider fermentation containing 0% apple solids
on fermentation days 0, 5, 10, 14 and 20

3.4.3 The effect of alcohol production on the mean degree of polymerization 44
of procyanidins in Jonathan cider fermentation on fermentation days

0, 5,10,14 and 20

3.4.4 Proposed behavior of procyanidins in Jonathan cider during fermentation 45
(T= terminal end units, E = Extension units): Stable or Same rate of transfer

3.4.5 The effect of apple solids on procyanidin terminal end units of Jonathan 46
cider fermentation

3.4.6 The effect of apple solids on procyanidin extension units in Jonathan 47
cider fermentation

3.4.7 The effect of apple solids on mean degree of polymerization of 48
procyanidins in Jonathan cider

3.4.8 Proposed behavior of procyanidins in Jonathan cider during 49
fermentation: Polymeric breakdown and monomeric unit elimination

3.4.9 The effect of fermentation time on procyanidin terminal end unit 50
concentration in Jonathan cider fermentation containing 0% apple solids.

3.4.10 The effect of fermentation time on the concentration of extension units 51
in Jonathan cider fermentation

3.4.11 The effect of fermentation time on the concentration of extension units 51
in Jonathan cider fermentation: Yeast samples

3.4.12 The effect of fermentation time on mean degree of polymerization 52
of procyanidins in Jonathan cider fermentation

3.5.1 Experiment design of heat treatment 54

3.5.2 The effect of pectolytic enzyme treatments on the procyanidin terminal end 56
unit concentration in Jonathan cider containing 10% apple solids

3.5.3 The effect of pectolytic enzyme treatments on the procyanidin extension unit 5 7
concentration in Jonathan cider containing 10% apple solids

xi

3.5.4

3.6.1

3.6.2

3.6.3

3.6.4

3.6.5

4.1

A21

The effect of heat on the mean degree of polymerization of procyanidins in 58
Jonathan cider containing 10% apple solids

Experiment design of pectolytic enzyme treatments 60

The effect of pectolytic enzyme treatments on procyanidin terminal end 60
unit concentration in Jonathan cider containing 10% apple solids

The effect of pectolytic enzyme treatments on the procyanidin concentration 62
of extension units in Jonathan cider containing 10% apple solids

The effect of pectolytic enzyme treatments on the mean degree of 63
polymerization of procyanidins in Jonathan cider containing 10% apple solids

Proposed behaviour of procyanidins in Jonathan cider during treatment 64
(T= terminal end units, E = Extension units): Addition of procyanidin units
with no structural change

Estimated procyanidin concentrations of extension and terminal end units
in K, Strongbow, Woodpecker, Woodchuck and MSU hard cider brands 67

Standard Curve: RI vs Sugar % 71

xii

INTRODUCTION

Fermented apple juice or hard cider was widely consumed during the early history of
the United States (U .8.) Hard cider was regarded as a safe alternative to water because
alcohol prevented bacterial contamination and it remained a popular beverage until the
late 19tlrl century. At that time, beer began to dominate in the American market with the
influx of German immigrants into the US. Beer made from grain was cheaper and easier
to produce since it did not require the development of mature fruit orchards to provide the
raw material. Beer also fermented more quickly and consistently and thus, was the more
popular option for commercial producers (Rowles 2000). The Prohibition Act dealt the
final blow to hard cider’s popularity in the 19205.

Until about 1990, there were few domestic ciders commercially available and most
imported ciders were from the United Kingdom and France (Proulx and Nichols 2003). In
1990, only 115,000 cases of hard cider were sold in the US. while by 1997, the total has
risen to 2.7 million cases (F abricant 1997; Koeppel 1998). While the hard cider accounts
for less than 0.2% of the total US. beer market in 1999, the hard cider industry is
expected to grow rapidly (Rowles 2000).

Unlike the common sweet beverage cider known as cider in the U.S., ‘hard cider’
contains alcohol and unique tastes that are produced during fermentation. The distinction
between hard cider and apple wine is usually made based on alcohol content, but there is
much overlap between the two products. Apple wine is typically above 7% alcohol
content and not carbonated while hard cider is usually below 7% and carbonated. In the
U.S., commercial hard ciders contain about 5.5% alcohol while apple wine generally has

10-12% alcohol.

Apples specifically grown and harvested for traditional European cider are
selected for their sugar, acidity and tannin contents and are not commonly available in the
US. These bittersweet and bittersharp apples, as they are called, produce a cider with an
astringent mouthfeel and somewhat bitter taste. Astringent is described as a puckery
mouth-drying attribute while bitterness is a harsh taste sensation usually detected at the
back of the tongue. These astringency and bitterness has been attributed to the
polyphenolic compounds, especially procyanidins (Lea and Arnold 1978). Procyanidins
have been found to bind with polysaccharides in the skins and structure of apples,
specifically in the cell wall (Renard and others 2001; Le Bourvellec and others 2004).
Unbound procyanidins in fermented cider impart the desired mouth feel and dry taste
through their capacity to interact strongly with salivary proteins (Murray and others
1994). This binding ability increases with the degree of polymerization (number of
flavan-3-ols units) of procyanidins (Da Silva and others 1991) while the monomeric
catechins are responsible for bitterness (Lea 1990; Peleg and others 1999). Procyanidins
measured in this research refer exclusively to unbound procyanidins (‘free’).

Various studies have documented that total polyphenol content and specifically,
procyanidin content of dessert apples are significantly lower as compared to cider apple
varieties (Guyot and others 1998). Such studies confirm that unlike the imported cider,
American hard ciders are made from sweeter, less tannic apples commonly grown in the
U. 8., produces a fruity, somewhat sweeter, less tannic flavor than the European ciders.

In addition, procyanidins in plant-based beverages such as red wine, tea, and cider
are increasingly being recognized as important in long-term health and reduction in the

risk of chronic diseases (Santos-Buelga and Scalbert 2000).

Standard cider fermentation procedures usually involve the fermentation of apple
juice or juice concentrates. The extraction and solubilization of procyanidins during
processing play an important role in regulating the phenolic pool in apple juice and cider.
Little work has been done on measuring free procyanidin levels in hard cider made from
dessert apples and investigating methods of extracting procyanidins from the flesh of the
apples into the juice.

The apples used in the present research were peeled and cored before juicing
because compounds in the peel and seeds can interfere with the analytical method used.
The apple material left after juice extraction is described as apple solids and contains
approximately 61-68% moisture content as measured in this study.

The enhancement of free procyanidin levels in hard cider made from local
Michigan dessert apples with the aim to improve its sensory characteristics may provide
an alternative method for local cider producers making quality hard cider, without
utilizing difficult to obtain rare and more expensive cider apple varieties. The goal of this
research was to develop methods to enhance free procyanidin levels in hard cider through

fermentation and various juice treatments using Michigan dessert apple varieties.

We hypothesized that mash fermentations would release procyanidins bound in
apple solids into the cider, and thus, increase the levels of free procyanidins in fermented
cider. The effects of ‘mash’ fermentations (i.e. cider fermentation with apple solids) are
investigated by determining whether alcohol production, apple solids percentage and
fermentation time influence free procyanidin levels in apple cider made from Michigan

dessert apple varieties.

1. LITERATURE REVIEW
1.1 Polymeric flavan-3-ols (Procyanidins)

Polymeric polyphenols, commonly called tannins, are found in plants and foods
of plant origin, particularly in fruits, legume seeds, cereal grains and beverages such as
wine, tea, cocoa and apple cider. Bate-Smith and Swain (1962) described tannins as water
soluble phenolic compounds having molecular weights between 500 and 3000. Besides
undergoing the usual phenolic reactions, these compounds are also reported to have

special properties such as the ability to precipitate alkaloids, gelatin and other proteins.

 

 

Polymeric phenolics/Tannins

l

 

 

 

 

 

 

L l
Hydrolysable , Condensed
(gallic, ellagic acids) (proanthocyanidins)

 

 

 

 

 

l
l l I l l

Propelargonidin Prorobinetinidin Prodelphinidin Procyanidin Profisetinidin

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.1 Classification of polymeric phenolics/tannins

Tannins are classically divided in two groups, hydrolysable and condensed as
showed in Figure 1.1. Proanthocyanidins are oligomers or polymers of flavans (2-
phenylchromanes) that are linked by C-C and occasionally C-O-C bonds and have the

typical C6—C3-C6 flavonoid skeleton (Figure 1.2).

89

, +40
321

65

Figure 1.2 Basic C6 - C3 — C6 structure (Haard 1985)

 

IV

Figure 1.3 Flavonoid biosynthesis (Belitz and Grosch 1987)

Table 1.1 Substitution patterns within procyanidin classes in reference to Figure 1.2

(Cheynier and Fulcrand 2003)

 

 

Substitution pattern
Proanthocyanidin class 3 5 7 3' 4' 5'
Propelargonidin FIavan-3-ols OH OH OH H OH H
Procyanidin OH OH OH OH OH H
Prodelphinidin OH OH OH OH OH OH
Profisetinidin 5-Deoxyflavan-3—ols OH H OH OH OH H
Prorobinetinidin OH H OH OH OH OH

 

The formation of the flavonoid structure is a result of a series of condensations

fiom hyroxycinnarnic acid to a chalcone (III) and finally, a flavanone (IV) (Figure 1.3).

These compounds are further divided into subgroups, depending on the substitution

pattern of their constitutive flavan units (Table 1.1) and named after the corresponding

red anthocyanidin (2-phenylchromenium) pigments released under acidic conditions

(Cheynier and Fulcrand 2003). These compounds have molecular weights to 20,000
(around 70 units) (Halsam and Lilley 1988).

The main proanthocyanidins encountered in foods are procyanidins, consisting of
(epi) catechin units (2-(3, 4-dihydroxylphenyl)chromane-3,5,7-triol) and prodelphinidins,
in the form of (epi)gallocatechin [2-(3,4,5-trihydroxyphenyl)chromane-3,5,7-triol].
However, the major industrial proanthocyanidins, isolated from mimosa and quebracho,
are predominantly composed of their 5-deoxy analogues, namely profisetinidins
consisting of fisetinidol [2-(3,4-dihydroxyphenyl)chromane-3,7-diol] and
prorobinetinidins, of robinetinidol [2-(3,4,5-trihydroxylphenyl)chromane-3,7-diol]

(Cheynier and Fulcrand 2003).

   

Proanthocyanidin (C4—9C8) ~ Proanthocyanidin (C4—-)C6)
R5 R3

R4

0))

 

Proanthocyanidin (C4->C8; C2-—>O—)C7)

Figure 1.4 Structure of proanthocyanidins dimers (Lazarus 2003)

While several types of units often occur within a single proanthocyanidin chain,
they can be linked by C-4—C—6 and/or C-4—C-8 bonds (a) or doubly linked, with an
additional C-2—O-C-7 or C-2—O—C-5 linkage (b) and eventually substituted (e.g.
glycosylated, galloylated) (Figure 1.4). Some plants only synthesize proanthocyanidins
based on a single constitutive unit such as epicatechin-based procyanidins in apples.
Polymeric fractions with molecular weight of approximately 55,000 (an average of 190

units) have been isolated from cider apple varieties by Guyot and others (2001).

1.1.1 Procyanidins in apples/apple products

OH

OH
~“‘$ O

i

    
 

I
0
4
OH

(+)—Catechin
OH
OH

(-)-Epicatechin

 

Figure 1.5 Monomeric units of procyanidins (Tsao and Yang 2003)
HPLC-MS analysis of an apple extract demonstrated that a complex series of
proanthocyanidin oligomers was present in whole Red Delicious apples (Lazarus and

others 2003). Oligomers through dodecamers were present and consisted entirely of the

procyanidin monomers (-) epicatechin and (+) catechin (Figure 1.5). Guyot and others
(1998) concluded that the most frequently found unit in apple or apple products was

procyanidin B2, a two-epicatechin unit with a C4—C8 interflavanic linkage (Figure 1.6).

OH

OH

 

OH

Figure 1.6 Procyanidin B2 (Tsao and Yang 2003)

Research efforts have concentrated on the procyanidin content of apples (Lees
and others 1995; Guyot and others 1997; Sanoner and others 1999; Price and others
1999), crushed apples (Foo and Lu 1999) and apple juice (Will and others 2002; Guyot
and others 2003; Van der Sluis and others 2002, 2004) rather than hard cider. Suarez and
others (1996) investigated the polyphenol content in hard cider but focused on optimizing
the analytical methodology (i.e. solid phase extraction and HPLC). To date, procyanidin
levels of hard cider made from dessert apples have not been reported in research
literature. Most studies investigate procyanidin concentrations in cider apple varieties and

utilized HPLC as their chemical analytical method.

The levels of general polyphenolic content in cider apple varieties are assumed to
be tenfold higher than in dessert apples (Lea and Drilleau 2003). The same trend was
observed specifically to procyanidins when Sanoner and others (1999) reported that cider
apples varieties (e.g. Kermerrien and Jeanne Renard) contained 1000 to 3000 mg
procyanidins per kg of fresh matter while there was 761 mg procyanidins per kg of fresh
matter in Golden Delicious apples.

The highest concentration of tannins may be found in the peel with a continual
presence of these compounds throughout the various apple tissues. Lees and others
(1995) reported the amount of 19.2 mg, 2.7 mg and 1.2 mg condensed tannin content/g
dry weight, in Golden Delicious apple peel, pulp and seed, respectively. The same trend
in McIntosh apples was observed. Specifically, Guyot and others ( 1998) found 4964 mg
procyanidins/kg fresh tissue in apple peel (epidermis zone) as opposed to 3379 mg
procyanidins/kg fresh tissue of apple flesh (parenchyma zone) by HPLC analysis. Even
when the BuOH/HCl assay resulted in lower readings as compared to HPLC (Guyot and
others 1998), the same trend was observed 1231 mg procyanidins/kg fi'esh tissue in apple
peel as opposed to 805 mg procyanidins/kg fi'esh tissue in apple flesh.

The final procyanidin content in the cider product is highly dependent on the
procyanidin content of the raw material, fresh apples. Various researchers have studied
the effect of agricultural factors such as cultivar, harvest years, weather and soil type
(Van der Sluis and others 2001) on the general polyphenol content of dessert apple
varieties while Burda and others (1990) investigated factors such as maturation and cold

storage.

1.1.2 Importance to industry

Polyphenols in the diet are becoming increasingly recognized as important in
long-term health and risk reduction of chronic disease. Various experimental studies have
investigated the effects of the consumption of food products rich in polymeric polyphenol
content such as tea, onions, apples and wine on cardiovascular diseases (CVDS) and
cancer (Santos-Buelga and Scalbert 2000). These polymeric compounds are also thought
to have antioxidant properties and are still under investigation (Santos-Buelga and
Scalbert 2000).

Five classes of phenolic compounds are present in apple fruit: (1)
hydroxycinnamic acid derivatives, (2) monomeric and oligomeric/polymeric flavan—3-ols,
(3) flavonols (quercetin derivatives), (4) dihydrochalcones (phloretin glycosides) and (5)
anthocyanidins (cyanidin glycosides) (Alonso — Salces and others 2001). Each of these
polyphenol groups plays an important role in color, aroma, formation of hazes and flavor
in cider (Alonso 2001). Polymeric flavan-3-ols (procyanidins) are major phenolic
constituents in juices and fermented beverages as they are involved in many quality
criteria such as bitterness, astringency and shelf life (Lea 1990).

In apple cider, procyanidins are recognized as principal separate contributors to
bitterness and astringency which impart the desired mouth feel and dry taste in fermented
cider (Lea and Arnold 1978). This ability is attributed to the capacity of the compound to
interact strongly with proteins and, in this case, salivary proteins (Murray and others
1994). The ability to associate increases with the degree of polymerization (number of
flavan-3-ols units) of procyanidins (Da Silva and others 1991). Lea and Arnold (1978)

also established that monomeric catechins are responsible for bitterness while the longer

10

polymeric compounds contribute to astringency. Vidal and others (2003) firrther
supported this theory through formal sensory descriptive analysis study on isolated cider
apple and grape proanthocyanidin fiactions.

Traditional ciders made from bittersweet cider apple varieties have been
distinguished by relatively high levels of bitterness and astringency caused by the
procyanidins (Lea and Drilleau 2003). Although ciderrnakers may be developing
orchards with bittersweet apple cultivars, a deficit still remains. Hard cider making in the
US. currently relies on the use of surplus dessert fruit, which has less procyanidin
content and is less suitable for cidermaking. Since processing conditions play a part in
determining the frnal procyanidin content and the final flavor profiles of the hard cider
product, this study aimed to investigate methods of processing the juice of dessert apples

varieties to increase the procyanidin concentration.

1.2 Analysis of procyanidins

Qualitative and quantitative information on the procyanidin profiles in food products
are especially lacking, due in large part to the unavailability of appropriate analytical
methodology and commercially available standards for such complex structures (Lazarus
and others 2003). While formal identification of proanthocyanidins can be achieved by
bidirnensional NMR techniques, this is restricted to pure compounds, which are
increasingly difficult to isolate as their degree of polymerization (DP) increases. This
difficulty is due to the larger number of possible isomers and smaller amounts of

individual compounds (Cheynier and Fulcrand 2003). However, with the potential for

11

health claims attached to these compounds, methods for accurate and sensitive analysis
will increasingly become more important.
1.2.1 Colorimetric assays

Historically, the analytical methods estimate procyanidins by spectrophotometric
means. Methods based on oxidizing properties such as the Folin-Ciocalteu assay have
been proposed to estimate flavanol content, but these methods lack specificity and can

only be applied to isolate tannins (Lazarus and others 2003).

4 OH
8 6| * DPn
0
01-!
Q
OH

  
 

Extension
units

 

 

Lower [HO

tenninal 00 '-‘ '-‘
Figure 1.7 Extension and lower terminal units of procyanidins (Kennedy and Jones
2001) The BuOH/HCl assay was originally created by Bate- Smith and Swain (1962)
and further developed by Porter and others (1986). This method relies on the acid-
catalyzed oxidative cleavage of interflavanic linkages between extension units (Figure

1.7) of polymeric procyanidins to produce cyanidins (Figure 1.8), which are then

measured spectrophotometrically. While this method is specific, disadvantages to this

12

method include lower reaction yield (Guyot and others 1998) and sensitivity to
interference from water (Porter and others 1986). However, Cheynier and others (2003)
have found the reaction yield to be approximately 48% and adjustments can be made
accordingly while water can be removed during solid phase extraction where extracts that
have been eluted through the cartridges are dried prior to being collected by methanol.
While the butanol-HCl assay is definitive for the polymer, it is only recommended for use
in determining polymeric polyphenols in the peel of gold or green varieties because

tissues which contain anthocyanins interfere with the colorimetric assay (Lees and others

Procyanidin a. NO 0.26::-

(polymer or oliomer) .-""""'—"—’

1995)

OH
He
1
Jon-c
O"
O:
/

 

Figure 1.8 Acid-catalyzed cleavage of procyanidins to cyanidins (Porter and others
1986)

The vanillin-HCl or dirnethylaminocinnarnaldehyde (DMCA) reactions rely on
the coupling of chemical reactions enabling them to specifically measure lower end units
of flavanol chains as seen in Figure 1.7 (Lazarus and others 2003). The vanillin assay

involves the reaction of an aromatic aldehyde, vanillin with the metasubstituted ring of

13

flavanols to yield a red adduct (Price and others 1978). Although the vanillin reaction has
been widely used to estimate condensed tannins (proanthocyanidin), there have been
disadvantages to its application. The reaction is not specific for condensed tannins, and
the monomeric unit catechin reacts to yield a red colored adduct (Price and others 1978).
A modified version of the vanillin assay using methanol (Butler and others 1982) results
in monomers present in crude plant extracts yielding color at a slower reaction rate in the

assay than polymers. Thus, less interference in the procyanidin measurement occurs.

1.2.2 Chromatography

Other alternative methods such as electrospray ionization mass spectrometry are
used to analyze a complete series of polymeric procyanidins with degrees of
polymerization up to 17 (Guyot and others 1997). The structural characterization of
proanthocyanidin fractions is performed by 13C or 1H NMR (Nuclear Magnetic
Resonance) methods. These methods provide precise information on the average degree
of polymerization, the stereochemistry of the heterocycles of the constitutive units and on
the hydroxylation pattern of the B nuclei (Guyot and others 1997). However, polymers
with molecular weight above 8000 cannot be precisely characterized according to this
procedure.

In the early 19903, various chromatographic techniques such as reversed or
normal phase liquid chromatography were developed to allow separation of oligomeric
proanthocyanidins, and acidic phenolic compounds (hydroxycinnamic acid derivatives)
from neutral compounds (flavonoids) (Jaworski and Lee 1987; Delage and others 1991;

Suarez-Valles and others 1994). While better separations have been achieved, problems

14

with procyanidins exhibiting different degrees of polymerizations of different constitutive
units remain a problem, resulting in an unresolved clump of peaks in chromatograms and
leading to underestimations (Guyot and others 1997).

Reversed phase HPLC followed by thiolysis has been developed to address this
problem in peak resolution. Concentrations of both extension and terminal units, and
consequently mDP are determined in a single reaction based on acid catalyzed
degradation in the presence of a nucleophilic agent (Figure 1.9). This is followed by
HPLC or NMR analysis of the resulting solution (Cheynier and Fulcrand 2003).
Breakage of the interflavanic C-C bond under mild acidic conditions releases the terminal
units as the corresponding flavanols and the upper and intermediate units as carbocations.
These carbocations react with the nucleophile reagent (usually phenyl-methanethiol or
phloroglucinol) to form stable adducts (e.g. benzyl tlrioethers in the presence of phenyl

methanethiol) (Cheynier and Fulcrand 2003).

Monomeric units

 

Nucleophile adducts

 

Figure 1.9 Preposed mechanism of acid cleavage of procyanidins and formation of
nucleophile adducts (Kennedy and Jones 2001)

15

Guyot and others (1997) investigated the concentrations of both extension and
terminal units, and consequently mDP in cider apple varieties through thiolysis, which
utilized benzyl thioether. Matthews and others (1997) and Gupta and Haslam (1978)
observed that benzyl mercaptan served as a better trapping agent since it resulted in
significantly higher nucleophilic adducts yields than those degraded by phloroglucinol.
However, Kennedy and Jones (2001) utilized phloroglucinol to analyze procyanidins in
grapes and found that results obtained compared favorably to those that used benzyl

mercaptan, thus, identifying a safer and less toxic alternative.

1.3 Hard cider production

 

 

 

 

 

Harvest ‘N Sweating "H Crushing "’ Pressing

l

Fermentation Filtration ‘H Packing

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 1.10 Basic hard cider making steps

The traditional steps in hard cider making are seen in Figure 1.10: harvest and
fruit selection (on the basis of cultivar and quality), sweating, crushing, pressing (or
extraction), fermentation, filtration/racking and bottling/packing (Proulx and Nichols
2003). After harvesting, the apples are left to mature for a week (i.e. sweating). In
modern plants, the apples are crushed in a grater type mill made of stainless steel. Next,
the pulp is crushed to extract the juice using a cider press. During pressing, the juice is
typically exposed to air and oxidation occurs. The polyphenol oxidase (PPO) enzyme

reacts with apple fruit tannin in the presence of air to develop soluble color. If this

16

oxidation continues further, the oxidized polyphenols (particularly procyanidins) are
tanned back into the pulp and the level of soluble polyphenols and color may be
diminished (Lea and Drilleau 2003). Mechano-hydraulically operated plate presses are
used in modern manufacturing facilities to extract the fruit juice. The freshly pressed
juice may be fermented straight away or concentrated and stored for later conversion to
cider, in which case it is extensively treated to pasteurize and remove pectin.

Apple juice concentrate (AJC) is widely used in English ciderrnaking and to a
limited extent in France cidermaking. The advantage of a 70°Brix concentrate to the
ciderrnaker is that it may be stored for months or years with relatively little deterioration
compared to fresh juice, which complements just-in-time (JIT) business practices. Hard
cider production in Europe generally utilizes AJC in a controlled fermentation process
using deliberate addition of selected yeast and malolactic bacteria, which both help to

control fermentation rates and ensure uniform product quality (Lea and Drilleau 2003).

1.3.1 Fermentation
Hard cider is a product of apple juice that has undergone two different kinds of
fermentation. The first fermentation is carried out by yeasts in anaerobic conditions,

which converts fermentable sugars to alcohol (Figure 1.11).

C6H1206 —’ 2C2H50H + 2C02
Sugar Alcohol Carbon Dioxide
(Glucose, Fructose) (Ethyl Alcohol) (Fermentation Gas)

Figure 1.11 Conversion of fermentable sugars to ethanol

Refractive Index (R1) is a measurement useful for analyzing the density of a

substance (e. g. sugar dissolved in water), based on the principle of light refraction. It is a

17

simple method of measuring the reduction of fermentable sugar. Most UK ciderrnakers
take the view that a complete ‘dry’ fermentation 10-12% alcohol in as little as two weeks
is a desirable objective (Lea and Drilleau 2003). Incomplete fermentation can be obtained
by removing the yeast halfway throughout the process, thus retaining less alcohol and
more fermentable sugars than ‘dry’ hard ciders. In the US, commercial hard ciders
usually contain about 5.5% alcohol and most are carbonated (Proulx and Nichols 2003).

The second fermentation (malo-lactic fermentation) converts L(-)-malic acid to

L(+)—lactic acid and carbon dioxide and is carried out by lactic acid bacteria present in
the apple juice. The malo-lactic fermentation can occur concurrently with the yeast
fermentation but more often it is delayed until a few months later. Most modern

commercial ciderrnakers regard this fermentation as a nuisance and do not encourage it
(Lea and Drilleau 2003).

Since the 19803, specific cultured yeasts have been used in cider making.
Inoculum mixes of active dried wine yeasts of S. uvarum and S. bayanus are widely used
as the former provides a speedy start while the latter copes better with fermentations to
dryness since some yeast types are not tolerant to high alcohol concentrations (Lea and
Drilleau 2003). The vitality and viability of cultured cider yeasts under high stress
conditions (i.e. higher alcohol concentrations and different types of alcohol) have
recently been investigated (Seward and others 1996).

Prior to fermentation, S02 is added to the juice to suppress or kill wild yeasts and
most bacteria. This step allows the added yeast to multiply and dominate the fermentation
(Lea and Drilleau 2003). The effectiveness of SO; is pH dependent since it is only the

dissociated form that has antimicrobial properties (Lea and others 2003). Therefore, cider

18

fermentation is best executed at pH <3.8. The amount of added sulfite (e.g. potassium
metabisulfite) ranges from 50-70 ppm for most commercial wines.

Fermenting cider in the presence of fruit solids (mash) is common within the
wine-making industry. The intentions are to release polyphenolic compounds such as
anthocyanins to enhance wine flavor and color from the crushed skins of grapes. Mash
fermentation of apple fruit was informally investigated by Padilla-Zakour and others
(2003) who reported that polyphenol content doubled in mash fermentation as compared

to juice fermentations.

1.3.1.1 Complexation of apple polyphenols with polysaccharides

Studies have reported that the transfer, of procyanidins from apple hit to juice
during processing is hindered by the complexation of apple polyphenols to
polysaccharides. Haslam and Lilley (1988) were among the first investigators to suggest
that higher molecular weight procyanidins become bound to soluble polysaccarides such
as pectin (which dramatically increases as fi'uit ripens) and therefore, are unavailable to
exert the astringent mouthfeel desired in hard cider. Tsao and Yang (2003) report
significant differences in the concentration of procyanidins in Red Delicious apple tissues
and juice as follows: 1655, 342, 6 pg flavan-3-ols per g fresh weight, in apple peel, fresh
and juice, respectively.

Plant cell walls consist of complex, porous polysaccharidic material. In fruit and
vegetables, the cell wall consists of three interpenetrating but not interconnected
networks: a cellulose/xyloglucan framework (>50% dry weight) embedded in a pectin

matrix (24 - 40% dw) and locked into shape by glycoproteins (~1% dw) (Carpita and

19

Gibeaut 1993). In intact plant tissues, cell walls, polyphenols and polyphenoloxidase are
present and react with each other during processing when the cell walls are ruptured. In
apples, this results in formation of apple solids, where cell walls and (oxidized)
polyphenols form a single material (Renard and others 2001). Two mechanisms are
thought to be jointly responsible: adsorption of native and oxidized polyphenols to the
cell wall matrix and formation of covalent bonds between quinines and cell wall
polymers.

The adsorption was thought to be mediated by H-bonding and hydrophobic
interactions, the latter being favored by the existence of hydrophobic cavities and
crevasses such as the internal cavity of cyclodextrins (Renard and others 2001).
Interactions between suspended cell walls and polyphenol extracts were investigated by
quantitating residue polyphenols by HPLC. It was observed that hydroxycinnamic acids
and (-) epicatechin did not bind to cell walls and that binding between apple cell walls
and procyanidins depended on the concentration and the molecular weight of the
procyanidins. Le Bourvellec and others (2004) conducted a similar experiment by
bringing into contact a solution of procyanidins and a suspension of apple cell wall
material. The amount of procyanidins bound to the cell wall increased with the degree of
polymerization (DP), the percentage of galloylation (% gall), the proportion of
(+)catechin and increasing ionic strength. Since the bindings decreased with increasing
temperature, the bonds between procyanidins and apple cell wall material were concluded
to be weak energy bonds such as hydrogen bonds and hydrophobic interactions.

The possibility of improving the flavonoid content of apple juice by adjustments

in production and processing methods is easier and faster to implement than changes in

20

the raw material itself (i.e. cultivar, storage, harvest time, maturity) (Van der Sluis and
others 2004). Since the processing steps of apple juice production are similar to hard
cider production, methods of improving procyanidin content in apple juice may be
applicable to cider making production.

Van der Sluis and others (2004) suggested that flavonoid content in juice made
from Elstar, Golden Delicious and Jonagold apple varieties and its antioxidant activity
may be improved by extracting flavonoids from the pulp using alcohol. Complex
formation between procyanidins and apple cell wall material was decreased by ethanol
production (Le Bourvellec and others 2004). However, there are no studies relating the
effect of natural alcohol production (sugar fermentation) on the release of procyanidins

from apple solids.

1.3.2 Heat treatments

A study by Lea and Tirnberlake (1978) found that warm anaerobic incubation of
apple [solids] for four hours before extraction increases the level of organoleptically
significant procyanidins in juice by 50%. The corresponding increase in bitterness and
astringency were highly significant. This study utilized total phenolic colorimetric assays
and did not offer specific information on procyanidins.

High temperatures are known to inactivate the enzyme polyphenol oxidase and
therefore, help to retain polyphenolic content. Will and others (2002) also evaluated the
influence of mash temperature on total polyphenol content using another colorimetric
method, the Folin Ciocalteu reagent assay (FCR) and HPLC. A nearly linear increase of

phenolic substance was found between ambient and 50 °C heating after 1.5 hr (Will and

21

others 2002). (-) Epicatechin and procyanidin B2 did not appear on the chromatograms
until after incubation of dessert apple solids at temperatures of 30 and 40 °C,
respectively. Diffusion extraction methods employed at HTST (High Temperature Short
Time) conditions at 55, 63, 67, and 73 °C at 90 seconds increased the procyanidin
content of Red Delicious, McIntosh and Spartan apple juice two to three times higher

(Spanos and others 1996)

1.3.3 Pectolytic enzymatic treatments

Approximately 75% of the solid matter of plants is carbohydrate and generally
consists of simple sugars, polysaccharides, pectic substances and ligrrin (Haard 1985). In
plants, carbohydrates are localized in the cell wall and intracellularly in plastic, vacuoles,
or the cytoplasm. The cell wall is primarily consists of cellulose, hemicellulose, pectins
and lignin. The relative proportion and contents of these constituents vary considerably
among species, with maturity at harvest, and with elapsed time after harvest.

Cellulose is largely insoluble and indigestible by human beings while
hemicelluloses are a heterogeneous group of polysaccharides that contain numerous kinds
of hexose and pentose sugars and in some cases, residues of uronic acids (Haard 1985).
Pectin consists of al,4-linked galacturonic acid residues esterified to varying degrees
with methanol.

Industrial enzymatic treatment of the apple solids before pressing is often applied
to increase juice yields. Pectolytic enzymes are used to increase the pressability by
degrading pectins in the cell walls where the polysaccharides are solubilized and

depolyrnerized (Dongowski and others 2002). Pectolytic, cellulolytic and hemicelluloytic

22

enzymes (Table 1.2) present in commercial enzyme preparation are used in fruit and

vegetable technology.

Table 1.2 Enzymes typically used in fruits and vegetables processing (Richardson
and Hyslop 1985)

 

 

 

 

 

Enzyme Sources Conversions catalde
Pectinase 1. Aspergillus niger, var. Pectin methylesterase
(polygalacturonase, 2. Rhizopus oryzae, var. demethylates pectin,
pectin methylesterase polygalacturonase

and pectate lyase) hydrolyzes B-D-l,4-
galacturonide.

Cellulose 1. Aspergillus nige, var. Cellulose + H2O -) B-

2. T richoderma reesei, var. dextrins (B-l, 4-glucan
bonds)

Hemicellulase Aspergillus niger, var. Hemicellulase + H2O -) B-
dextrins (B-q, 4-glucan bonds
of hum - locust bean, guar,
etc).

 

 

 

 

Lea and Timberlake (1978) recommended that warm anaerobic incubation in the
presence of a pectolytic enzyme may help increase the yield of procyanidins from apple
solids. Enzymatic mash maceration of the Brettacher apple variety (European) carried out
under periodic stirring at 20 i 3 °C for 1 hr resulted in reduced concentrations of
procyanidin BZ and (-) epicatechin without the addition of ascorbic acid (Mihalev and
others 2004). This reduction was attributed to oxidative browning reactions. Spanos and
others (1990) report that enzymatic clarification caused some procyanidin degradation.
However, Will and others (2000) reported that apple solids liquefaction with pectolytic
enzymes increased (-) epicatechin and procyanidin B2 concentration in apple juice
containing apple solids as temperatures rise to 30 and 40 °C, respectively. More recently,
Renard and others (2001) found that procyanidins may inhibit pectolytic enzymatic

degradation of apple cell walls.

23

2. MATERIALS AND METHODS
2.1 Plant material

Apples fruits (Malus domestica) from the Jonathan variety were harvested from
the MSU Horticultural Farm or obtained from commercial stores. The Jonathan variety
was used as it is one of the dessert apple varieties that is abundant in the State of
Michigan, thus making the variety an appropriate choice for the development of value-
added apple products. Additionally, the Jonathan apple was selected for the sweet tart
flavor profile produced in the fermented product afier preliminary fermentation tests
involving various dessert apples such as McIntosh, Northern Spy and Winesap. The
Jonathan apples were stored in the MSU Food Science Pilot Plant cooler in the fall
season (September to November) at 8 °C until further analysis within 3 months. Apples
of similar sizes were pooled together and bruised sections of the apples were discarded
during preparation.

Since the anthocyanidins in the red skin of the Jonathan variety can interfere with
the BuOH/HCl assay (Lees and others 1995), the apples were skinned and cored before
the juice was extracted. Apples which had anthocyanidin ‘veins’ extending from the
epidermis up to the apple core within the flesh (Figure 2.1) were rejected and not

included in the experiments.

2.2 Juice Preparation

The apple juice samples were prepared using Acme 5001 White Supreme
JuiceratorTM Juice Extractor (Waring Products, Tonington, CT, USA). The apple pieces
were cored, peeled by hand and juice was collected as a single batch. The apple solids

were added into the juice samples at 0, 10 and 30% (w/w).

24

 

2.3 Extraction of procyanidins

2.3.1 Centrifugation

The apple juice samples were centrifuged using an Allegra 6R Centrifuge Rotor
Model GH3.8 with swinging buckets (Beckman, Fullerton, CA, USA) to separate the
juice from the solids at 3000 rpm (~ 1411 g) for 15 min at 10 °C. The supematants were
decanted.
2.3.2 Rotary Evaporation

For the fermented cider samples, alcohol was removed by evaporation by vacuum
(Escribano-Bailon and others 1992) to minimize procyanidin loss during extraction since
procyanidins dissolve in ethanol. The method of evaporation was adapted from Suarez
and others (2003). Rotary evaporation was achieved by using Laborota 4002 digital
(Heidolph Instruments, Cinnaminson, NJ, USA) at 60 rpm. Ten ml of supernatant was
transferred into 250 ml round flask Pyrex bottles and evaporated to dryness at 35 °C for
20 minutes. Ten ml of purified MilliQ water was reintroduced into the flask to dissolve

the dried residue.

25

2.3.3 Solid Phase Extraction

Each C13 Sep-Pak cartridge (Vac 6 cc, Waters, Milford, MA, USA) was activated
by eluting 2 ml of acidified methanol (0.01%, v/v), followed by 2 ml of acidified water
(0.01 % hydrochloric acid, v/v). The redissolved sample was distributed into the cartridge
and eluted with 5 ml of acidified water to remove sugar and other high polarity

compounds. The extracts were recovered with 5 ml of acidic methanol.

2.4 Analysis of procyanidins
2.4.1 High liquid performance chromatography

2.4.1.1 Sample preparation

25 ml of cider samples were concentrated into 5 ml using Sep Pak C13 cartridges by
eluting 5 ml of water and 5 ml of extract was recovered with 2 ml of methanol. 1 ml of
extract was added to the reaction.

Grape seed extract preparation was adapted from Escribano-Bailon and others (2003)
by homogenizing 5 g of grape seeds in 15 ml of methanol. The sample was centrifuged at
12000 rpm for 15 min. 4 m1 of extract was dried and used in the reaction.

Both samples were treated with acid-cleavage in the presence of phloroglucinol using
the method as adapted from Kennedy and others (2001). Each sample was reacted with 5
ml of 0.1 N HCl in methanol containing 50 g/L phloroglucinol and 10 g/L ascorbic acid
for 20 min at 50 °C in a water bath shaker. The reaction was stopped by addition of five
volumes of 40 mM sodium acetate and cooled at 4°C for 10 minutes. The samples were

filtered using 0.45 pm Millipore syringe filters prior to analysis by HPLC.

26

2.4.1.2 Instrumental Settings

The HPLC apparatus consist of a 717 plus autosampler, 996 Photodiode Array
Detector and Milleniurn 32 Manager System (Waters, Milford, MA, USA). The column
used was a Waters Atlantis dC18 RP column (particle size 5 ,um, 150 x 4.6 mm),
protected by a Waters Guard Sentry column. The method utilized a binary gradient with
mobile phase containing 1% aqueous formic acid (Mobile Phase A) and pure MeOH
(Mobile Phase B).

The sample injection volume was 20 11L while the elution conditions were as
follows: 1.0 mL/min, a linear gradient from 20 to 30% B in 10 min, 30 to 60% B in 10
min. The column was then washed and reequilibrated with 20% B for 40 mins before the
next injection. Standard peaks of (+) catechin and (-) epicatechin obtained from Sigma-

Aldrich (St. Louis, MO, USA) were monitored at 280 nm at ambient column temperature.

2.4.2 Colorimetric assays
Calculations of concentrations were based on the Beer Larnbert’s Law (Equation
1), where concentrations units were expressed on a molar basis.
(1) A2 = b (8)2 c
where:

A = absorbance at wavelength 7L (measured)
a = molar absorptivity at wavelength 1(M".cm") (obtained from literature)
b = path length of the cell (cm)

c = concentration (M or moles/liter) (calculated)

27

2.4.2.1 Depolymerization in Butanol-HCI (Porter’s reagent)

Each of the extract samples of 0.5 ml were dissolved into 2.5 m1 of a solution of
butanol-12 N HCl (95:5), v/v, and 0.2 ml of (NH4)Fe(SO4)2, 12 H20 (2%, w/v in 2 N
HCl). The glass test tubes were sealed with a Teflon-lined screw cap, mixed thoroughly
and heated for 30 min in a water bath at 95 °C. The solutions were cooled immediately in
a water bath at ambient temperature and the absorbance was measured at 550 nm using a
DU 520 General Purpose UVN is spectrophotometer (Beckman, Fullerton, CA, USA).

The molar concentration of released cyanidins (equal to procyanidin upper and
extension units) was calculated using the molar absorptivity of cyanidin (35,000)
estimated by Cheynier and others (2003). Calibration with known solutions of
procyanidin standards as reported showed that the reaction yield as 48% (Scalbert 1992).
Taking this reaction yield into account, adjustments to the raw measured values obtained

in this research were made to reflect 100% yields in the final estimated values.

2.4.2.2 Vanillin in Methanol assay

The vanillin method used in this study was adapted by Butler and others (1982).
Extract samples of 0.5 ml were dissolved in 2.5 ml of a 1:1 solution, (8% HCl in
methanol: 1% vanillin in methanol) (w/vl) prepared immediately before use. The
absorbance is measured at 510 nm after 20 min of incubation.

The reaction in the vanillin assay theoretically reacts specifically with
proanthocyanidin end groups. The molar absorptivity (a) was determined to be 25,200

(Cheynier and others 2001).

28

2.4.2.3 Mean Degree of Polymerization (mDP)
The mDP of procyanidins was calculated from Equation 2 as obtained from
Cheynier and others (2001).

(2) mDP = ([upper and extension units]M + [end units]M)/ [end units]M

2.5 Fermentation

The juice samples were prepared as described in Section 2.2 and distributed into 200
ml French-capped bottles and treated with 0.053g potassium metabisulfite per L of
sample for 24 hr. The addition of 802 to cider was intended to suppress or kill non-
Saccharomyces yeast and most bacteria to allow the fermentation to proceed with a more
homogenous and benign microflora and to reduce the chances of a secondary infection
(Lea and Drilleau 2003). The amount of potassium metabisulfite was used at 53 ppm,

which is within the 50-70 ppm range typical for most commercial wines.

The commercial yeast DV10 (Lallemand Inc, Rexdale, Ontario) is a Saccharomyces
cerevisiae bayanus strain. It was chosen for this study because it is a clean rapid
fermenter that retains flavor, is tolerant to alcohol levels (15%) and has the ability to
ferment under stressful conditions (i.e. low pH, high total S02, relatively low nitrogen
content, and low temperature). The yeast was added to the samples at 0.3 g/L and the

samples were stored at 12-14 °C. Day 0 indicates the day yeast was first added.

29

 

 

Apple Juice

 

 

 

 

 

 

 

 

 

 

 

 

r {r v
0% solids 10% solids 30% solids
l 1 l l T 1
Control Yeast Control Yeast Control Yeast

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5 Each sample (n=3) sampled every 5 days 3
1 Day 0, 5, 10, 15, 20.... :

 

 

 

Figure 2.2 Experiment design of fermentation setup

Refractive index (RI), pH and procyanidin levels in the hard cider samples were
measured every three days (Figure 2.2). The RI values were measured using a S-28 Hand
Refractometer (Atago, Japan) while the pH values were obtained using a pH meter 440
with “3 in 1” Combo W/RJ electrode (Corning, NY, USA). The analysis was complete
once most of the fermentable sugar in the samples had been converted into alcohol or
until ‘dry’ when RI values were relatively consistent.

Alcohol content could not be analyzed using the alcohol dehydrogenase
enzymatic assay kit (Sigma-Aldrich, St. Louis, MO, USA), which was no longer
commercially available. Alcohol % values obtained by HPLC means resulted in values
that were unreasonably higher than expected. Therefore, the potential alcohol could only
be roughly estimated by calculating RI differences from the beginning to the end of
fermentation. The RI values obtained from the cider fermentations were converted to
sugar content by making a standard curve between RI and glucose concentrations (g/ml)
(Appendix 2). After adjusting the values for residual solids, the amount of potential

alcohol was estimated to be 4.0%.

30

Procyanidin concentrations were measured using the colorimetric methods described
in Section 2.4.2. There were three variables in the fermentation data. ‘v’ referred to the
concentration values calculated from the Vanillin in methanol assay, ‘p’ referred to
concentration values calculated from the BuOH assay while ‘t’ referred to the calculated

mean degree of polymerization calculated from Equation 2.

2.6 Statistical Analysis

The three variables (v, p and t) were analyzed using a model corresponding to a
split-plot design in blocks with repeated measures, where mash was the main plot factor
and treatment was the sub plot factor and the blocks were represented by batches. For the
repeated measures (times within mash and treatments) several covariance structures were
compared and a heterogeneous compound symmetry model was selected as the best fit.
After analysis of variance, single or main effect contrasts were obtained. The model was
fitted using Proc Mixed of SAS (Littell and others 1996). The p S 0.05 was used as the
level of significance.

Images in this thesis are presented in color.

31

3. RESULTS AND DISCUSSIONS
3.1 Evaluation of analytical methods on fermented hard cider
Guyot and others (2001) reported that HPLC analysis of polyphenol extracts
result in a single large clump (marked by ‘X’). The proposed heat-acid treatments in the
presence of phloroglucinol on the samples extracts prior to HPLC are expected to resolve

the clump into single peaks consisting of (-) epicatechin and phloroglucinol adducts.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1...... .. - ._......_____._.._.- -
r phloroglucinol F
a
0.04% / 1 (-) epicatechin l
l
g 003% j
l
o 02 ‘
.1 r b
l
0011
l
0.00 i
-r...,.....__.,....r__.,....,., its, ,_, ,,._, .T ,, _,,_- ,__,._,.,_,.._,__,__1
5.00 10.00 15.00 20.00 25.00 30.00
0.05 —1 -.- l" __._._-___ ._.__._. ,...._._
l B
l a ,3
0.04 '1 ‘0 ‘ , _
l i! l (-) epicatechin ,9
it 1 / i
A003 .. -.r l 3'
D I ll . :i | t?
$ ‘ ' r ' l
i ll ‘3 f." r '
8 om g , ,. . m ’l A!
: i l '1: 'v/ ”£1.13 10,”) i:
g 0 0] i , ,fl? 3 -’- ‘81” I CLARK!
< . i i)‘ “ I" e :‘K f V. A}: if X MVM~..A
l '-"\,,/If\~/~ ‘2’ ‘ " w\"“l
0-00 f
l

 

' ""3756" " 'iofoé‘" 'Ts‘Tdc‘i "" 20766 ””3667 ”36.50
Time (min)
Figure 3.1.1 HPLC chromatogram of procyanidin cleavage product from Jonathan
apple extracts following acid-catalysis (A) in the presence of phloroglucinol (B)

without phloroglucinol

32

In this study, phloroglucinol and (-)-epicatechin standards eluted in peaks at
retention times 2.42 and 16.4 min respectively. However, the ‘X’ polyphenol clump as
seen in literature did not resolve into individual peaks when the reaction was carried out
with and without phloroglucinol (Figure 3.1.1). Peaks ‘a’ and ‘1’ were expected to be
ascorbic acid and (+)-catechin, respectively, based on literature (Kennedy and Jones
2001). Peak ‘b’ was not identified as a phloroglucinol adduct based on the order of
elution observed in the findings of Kennedy and Jones (2001) and remained an
unidentified product of the acid cleavage reaction.

The same method was repeated to a known high source of procyanidins as
described in Appendix 1. HPLC chromatograms of crushed grape seeds extracted
subjected to heat-acid treatments in the presence of phloroglucinol showed that the clump
successfully resolved into individual peaks, where phloroglucinol and (-) epicatechin

peaks were identified at 3.38 and 16.34 min, respectively (Figure 3.1.2).

33

 

' W156 " W ”70766 W W i "5106"" 50166 """""2s’;oa "' ”36:00

.. .—.--... __ .,....M.. . -_. - ---- . ... a
. ...—..r ,-,.._. .7 whxu- W.- ..._.. m..—

1 (-) epicatechin

0.105; \ l

_.
z...“

p
o’

,. r.-. -
l" /

.°
C
a.

Absorbanee (AU)
E

.°
0
N

500 {oj'oa' 15.00 "‘2oioo"““as;dd“ "30:00
mania)

Figure 3.1.2 HPLC chromatogram of procyanidin cleavage products from grape

seed extracts following acid-catalysis (C) with the presence of phloroglucinol and
(D) with phloroglucinol

Peaks (a) and (b) may be traces of phloroglucinol or unidentified products of the
acid cleavage while peaks (1) are identified as (+) catechin. Peak (2) should be the
phloroglucinol adduct, which agrees with results found by Kennedy and others (2001).
The unresolved clump and peak heights in Figure 3.1.2 are relatively higher than that
found in Figure 3.1.1, indicating that grape seed extracts contain significantly higher
procyanidin content than apple cider extracts made fi'om dessert varieties.

When unsatisfactory results were obtained with the HPLC methodology,
colorimetric assays were investigated for alternative methods of estimating procyanidin

content in cider samples made from Jonathan dessert apple varieties. Colorimetric means

34

such as BuOI-I/HCl and Vanillin (methanol) assays resulted in sufficient color reaction
when tested on polyphenol extracts of Jonathan hard cider. Using (-) epicatechin as a
standard on the Vanillin assay, the concentrations of procyanidins were estimated to
range from 0.093 to 4.28 g procyanidin/ml juice (Appendix 1). While overestimations
might have occurred (the Vanillin assay was subjected to interferences with procyanidin
monomeric units), the concentration values obtained from the cider samples of this study
were comparable to values obtained from juices measured in literature, which ranged
from 1.2 to 2.4 g/L (Guyot and others 2001; 2002).

It was concluded that despite multiple attempts, the heat-acid treatment prior to
HPLC analysis failed to cleave procyanidins in sample extracts into monomeric units,
thus resulting in absence of phloroglucinol adducts as seen in the HPLC chromatograms.
Among possible explanations of this outcome include the sensitivity of this reaction to
the presence of water, where the addition of water reduced the formation of phologlucinol
adducts (Kennedy and Jones 2001) and the unstability of phloroglucinol adducts. Some
factors which could have reduced the degradation yields might also be the presence of
phenolic or nonphenolic impurities and the existence of degradation resistance bonds
within the polymer units (Matthews and others 1997).

Nevertheless, using colorimetric means, the calculation of mean degree of
polymerization using the concentrations obtained from molar absorptivity of cyanidin and
procyanidins resulted in mDP ranges of 2-7, which agreed with the results of Guyot and
others (2001). Therefore, the colorimetric assays were selected for procyanidin analysis

in this study.

35

3.2 Juice sample preparation

The addition of apple solids to juice samples posed some sample preparation
challenges. First, the apple solids hinder mixing, which was necessary for ensuring
homogeneity of fermentation and collecting samples for analysis. Preliminary
experiments resulted in airlocks being blown off the bottle tops when apple material was
pushed up by the C02 gas pressure generated fi'om the fermenting yeast. The size
reduction of fermentation bottle from 1 L to 200 ml helped to increase sample size (n)

and allowed the whole bottles to be mixed by hand without opening.

Secondly, microbial contamination in the samples was increased as apple solids
were increased. In this study, the following microbial contarninations were observed: 1)
microbial growth on control samples within 10-14 days at 13-14 °C, and 2) microbial
growth in samples with yeast added after 20 — 25 days of fermentation. The first
observation was expected since there was an insufficient amount of yeast to dominate
grth in the controls and introduction of microbes from the outside environment during
sampling allowed undesirable microbial growth. The second observation was made when
an acidic aroma similar to vinegar was detected in the hard cider samples. This aroma
was considered a defect and an undesirable flavor in hard cider production that was
thought to be produced by the growth of acetic acid bacteria. Such contamination most
likely occurred when sampling was performed after the completion of the yeast
fermentation when the yeast population declines and allows other microbes to proliferate.
The equipment, whole apples and bottles used in sample preparation were sanitized using
domestic bleach (Ultra Clorox) at 200 ppm for 1 to 2 min to reduce microbial

contamination.

36

Additionally, the separation of apple solids from the juice was difficult to achieve
despite centrifugation efforts, and juice samples tended to be cloudy. Therefore, samples
of 0% solids were not completely devoid of any apple solid particles. The percentage of
apple solids of samples was defined as the weight percentage of apple solids deliberately
added back, rather than the total apple solid content of the juice.

In this study, enzymatic oxidation was a factor that was difficult to control. While the
enzyme polyphenol oxidase was responsible for producing the yellow and browning
pigments that impart the coloring effect welcomed in apple cider, oxidation played a role
in reducing procyanidin levels in cider (Guyot and others 1999, 2003). While the addition
of potassium metabisulfite at 53 ppm may help to minimize this reaction, oxidation may
have reduced the procyanidin concentration values obtained in this study due to the lack

of practical options in controlling this reaction.

3.3 Fermentation
3.3.1 Sugar content

The refractive index value (RI) was used to monitor the fermentation rate as the
concentration of fermentable sugars decrease during fermentation. RI values may vary
between cider batches. This variation was expected as the measurement was non-specific
and any dissolved solute such as soluble pectin within the cider sample matrix may
produce interferences. A sample that had higher RI value does not necessarily mean that

it contains more sugars.

37

The RI values obtained in hard cider made from Jonathan apples, which were
chosen for this study, ranged from 10.8 to 11.0. This value was converted to sugar

percentages (glucose) and adjusted according to a standard curve (Appendix 1).

 

 

 

 

 

 

 

 

 

 

 

 

I _, _ , V____ l
1 10.0 i + 0% solids C i l
...80.._ 2:, __f,l+10%solidsCr.
:3 +30% solidsC :l

E 6'0' °"<>"0% solidsY 1‘

.\° 40 10%solidsY ll
lg” . LA 30% solidsY I;
l C = Control i
l 0'0 ' ’ T Y = Yeast l
l o 5 10 15 20 ,
i Time (d) I

 

 

Figure 3.3.1 Sugar content of Jonathan hard cider during fermentation (n = 3, p
<0.001)

In this study, yeast fermentation for hard cider production was achieved within 20
days based in the reduction of RI values (Figure 3.3.1). Most large UK ciderrnakers
prefer fermentations to be completed within 14 days (Lea and Drilleau 2003) while
Johansen (2000) reported that English cider-making generally lasts for 1 to 4 weeks.
Some variations in the fermentation rates were observed between batches, in which one
batch fermented a few days more slowly than the other. The rate of fermentation varied
according to external parameters (yeast type, yeast concentration and temperature) and
internal parameters (pH, nitrogen content). Cider contains significantly less
(approximately 1/8‘“) free amino nitrogen than do grape musts and beer worts (Lea and

Drilleau 2003). Variations in fermentation rates between batches may be due to external

38

factors such as yeast viability. The capability of yeast to live, develop, or germinate
(viability) influences fermentation rates. The lower the viable yeast cells, the slower the

fermentation rates become.

3.3.2 pH

i...“ ___._ -_ _ _- , - - _ 1#__._,-_. .__._Li ____- if“,

 

F _-_ _ .

l—-o—0%solicsc
3.25 -— -—--—— ._ «ea—— —~ , Muz'
' i —l—10%solidsCi

i + 30% solids c

 

 

---0'--0%solidsY

 

 

. ---D---10%solidsY .

 

 

 

 

 

 

 

 

 

3.10 -—-—— —— — -————————- ~*— — — we
f---A---30%solidsY5
3.05 ~+~ , - ——~~~ ~ v ’—-————-———— u
C=Control l
.1 3'00 T T r T Y=Yeast I
i 0 5 T111110“) 15 20 l
L________ _, - A_ _ _-u__-____

 

Figure 3.3.2 pH of Jonathan hard cider during fermentation (n = 3, p <0.001)

Throughout the fermentation studies, the pH range of hard cider made from
Jonathan apples ranged from 3.13 to 3.25 :l: 0.01 to 0.03 (Figure 3.3.2). The pH levels of
the Jonathan hard cider increased throughout yeast fermentation, which agreed with
Carnpo and others (2003) who observed the same trend in large batch fermentations of
cider made from cider cultivars within 15 fermentation days. While the slight rise in pH
could be contributed to malo-lactic fermentations (which decreases the acidity), this
fermentation would usually occur after the yeast fermentation had completed and had

been inhibited by low pH (Lea and Drilleau 2003). One factor that could have influenced

39

this pH rise may include biochemical reactions within the cider that have yet to be

identified and investigated.

3.4 Procyanidin estimation
3.4.1 The effect of fermentation on the concentration of procyanidins in cider

Each effect was evaluated on the following dependent variables: 1) the
concentration of procyanidin terminal end units 2) the concentration of procyanidin
extension units and 3) degree of polymerization.

There were three factors that were evaluated during fermentation; alcohol
production, percentage of apple solids and time. Two batches (each n =3) were combined
for statistical analysis and samples contained apple solids ranging from 0 to 30%. There
were missing data points among the control samples due to microbial contamination,
which were denoted with an asterisk (*). The missing samples included the following:

i) Samples containing 0% apple solids: control, day 20
ii) Samples containing 10 and 30% apple solids: control, day 15 and 20

Data points marked with ‘0 o A’ indicate mean values (n = 3) calculated only

from samples with yeast.

Due to the complications of the experiment design and time constraints,
replications for the fermentation experiments were limited to two and are referred as
batches. Data sets from preliminary batches were combined to reduce errors by

increasing sample size.

40

3.4.1.1 The effect of alcohol production by yeast fermentation on the concentration
of procyanidins in cider fermentation made from Jonathan apples

 

 

 

 

 

 

 

 

 

 

 

 

 

 

9.0 ‘T # i

I: 8.0 ~ 1
o ,, ‘___., 1
E 7.0 * -~—— 1;
‘5 ’5: AContr010% AS f.
o g 6.0 - |
S a ,
§ '5 5'0 AYeast 0% AS if
i E 4.0 ~~ : 2
= c * . . ti
1’ E 3-0 *— e—v—* “Vi _-*_- . Missmgdata .
5 3° '1 .1
'23 v- 2.0 +-—-———*«m____m_w , »——-—-.. - w ‘
.E V
g 1.0 . .--.- _____
E" 0.0 . .

' '5 5 15 25

’ Time (d)

 

 

Figure 3.4.1 The effect of alcohol production on procyanidin terminal end units

concentrations in Jonathan cider fermentation containing 0% apple solids on
fermentation days 0, 5, l0, l4 and 20 (n = 3, p <0.001, half error bars)

There were no significant differences in the concentrations of procyanidins (as

 

 

 

measured by terminal end units) between control and yeast samples containing 0% apple
solids (Figure 3.4.1). Both control and yeast samples had similar concentration trends
between Day 0 and Day 14. No comparisons were made on Day 20 since control samples

were discarded due to microbial contamination after Day 14.

Similarly, no significant differences between procyanidin concentrations
(measured as terminal end units) of control and yeast samples containing 10 and 30%
apple solids were observed (Table 3.4.1). The amounts of alcohol produced at the end of
yeast fermentation may not be sufficient enough to either, 1) reduce the binding effect
between procyanidins and apple solids, or 2) release significant amounts of procyanidins

from apple solids through alcohol extraction.

41

Table 3.4.1 Procyanidin terminal end unit concentrations in control and yeast
samples of Jonathan cider fermentation containing 0, 10 and 30% apple solids on
fermentation days 0, 5, 10, 14 and 20 (n = 3, p <0.001)

 

Concentration (10" moles/ml juice)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Apple
solids% 0 10 30
Time ((1) C Y C Y C Y
6.204 6.500 4.410 4.497 0.941 0.809
0 (:l:l .04) (il .04) ($1.04) (21:1 .04) (21:1 .04) (i1 .04)
6.227 5.973 4.272 4.263 0.387 0.613
5 (11 .00) (:hl .00) (2H .00) (i1 .00) (:tl .00) (i1 .00)
6.563 6.228 4.513 3.549 0.384 0.556
10 ($1.03) (i103) ($1.04) (£1.03) (£1.06) (21:1.03)
7.177 6.528 3.910 0.549
14 ($1.07) ($1.03) N/A ($1.03) N/A (£1.03)
5.311 3.449 0.455
20 N/A ($1.01) N/A ($1.01) N/A (21:1 .01)
[*vh _ if _ — A V ‘fi 7 “ _)_
1 50 »
t a 45 4r~#- "— — - —— -~ .
E 40 ~ é -——~—— ----—-———— i
s .3 if) “5 __ “" E é” ‘ §“ *. "EBEaiQASE
Q 4).. - - fl... - —— —-—-—-—fl : |
E :l' * H
8 a 25 2.. W _ ___ _ ’ O YeaSt 0% AS i i
:2 E 20 1 _ f_g ___ __ g 7 i * Missing data ; L
a 515* ~~~~~ g~ ~4~4 ?
.2 7.: I
a a 10 A -— —* -2 .
o .
a: w 4 ~— ——~—— i
H \
. E
-5 0 5 10 15 20 25 1
Time ((1) '

 

 

 

Figure 3.4.2 The effect of alcohol production on the procyanidin extension unit
concentrations in Jonathan cider fermentation containing 0% apple solids on
fermentation days 0, 5, 10, 14 and 20 (n = 3, p <0.001)

Similarly, no significant effect of alcohol production on the concentration of

procyanidin extension units was observed (Figure 3.4.2). A similar pattern was also

observed in samples containing 10% and 30% apple solids.

42

 

Table 3.4.2 Procyanidin extension unit concentrations in control and yeast samples
of Jonathan cider fermentation containing 0, 10 and 30% apple solids on
fermentation days 0, 5, 10, 14 and 20 (n = 3, p <0.001)

 

Concentration (10" moles/ml juice)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Apple
solids% 10 30
Time (dL C Y C Y C Y
34.96 33.07 30.47 24.45 9.36 8.05
0 ($7.04) ($7.04) ($7.04) ($7 .04) ($7.04) ($7.04)
36.68 35.85 24.73 23.25 4.39 6.57
5 ($5.93) ($5.93) ($5.93) ($5.93) ($5.93) ($5.93)
29.32 30.72 18.79 20.28 1.88 4.06
10 (£6.21) ($6.21) ($6.28) ($6.21) ($6.55) ($6.21)
31.13 31.78 19.27 4.10
14 ($6.10) ($5.83) N/A ($5.99) N/A ($5.99)
33.38 18.22 4.39
20 N/A ($6.12) N/A ($5.99) N/A ($6.00)

 

The results indicated that alcohol produced during fermentation may not be

sufficient to release procyanidin units fiom the apple solids. Additionally, it was most

likely that there were no significant structural changes i.e. oligomer to polymer and vice

versa occurring within the procyanidin units (Lea and Timberlake 1978).

43

 

 

‘ --$-. **-H—“‘"fl J
3m 1.5 fix}? __ fi} __ __*._ _ . 40 Com‘ 1
‘ a 0 Yeast i

, 1

 

 

 

 

 

 

 

 

 

i 1 T'-‘ ‘ _ "—"_' “"‘— ‘ ‘ ' #01 j * Missing data. i
‘ 0.5 «w 4 --._- _ _ 1
g 0 . L . , .

‘ -5 0 5 10 15 20 25

I Time ((1)

_____~_. 1

 

 

 

Figure 3.4.3 The effect of alcohol production on the mean degree of polymerization
of procyanidins in Jonathan cider fermentation on fermentation days 0, 5, 10, 14
and 20 (n = 3, p <0.001) '

There was no significant effect of alcohol production on the mDP of procyanidins
of the control and yeast samples (Figure 3.4.3). The same trend was observed in all
samples of varying apple solid percentages (0-30%). This observation was expected since

the mDP value was calculated from the concentration of terminal end units and extension

units.

 

Occurrence 1: System stable

EIEEISE EESE
EIEEIBE BEBE
T T T T T ——> T T T
mDP=3 mDP=3
Occurrence 2: Same rate of transfer

E E
EEZEEEE E(£)E
TT‘TT‘T

mDP=3
mDP=3

 

amt-n
~1an

 

 

Figure 3.4.4 Proposed behavior of procyanidins in Jonathan cider during
fermentation (T= terminal end units, E = Extension units): Stable or same transfer rate

No alteration in the concentration of procyanidin units was detected during

fermentation. If there were any changes in procyanidins being released from or absorbed

in to the solids, the transfer rate of procyanidins into and out of the juice system remained

consistent and didn’t affect the mDP of the samples (Figure 3.4.4). Therefore, it was

concluded that yeast fermentation has the potential to retain the mDP of procyanidins

during fermentation and does not increase mDP values.

45

3.4.1.2 The effect of apple solids on the concentration of procyanidins in cider
fermentation made from Jonathan apples

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 9 W _ T a
8 *u .4 $__,____ figm— I
5 ;
== 7 , ~ -—~— -~—— :3. M— l 2
E a .............. : --A-- Apple Solids i .
3‘6 "”7?“ 1 * Ti “"7 (0%)
g 55 ” A "9" Apple Solids
-§ g4 (10%)
E ,8 3 r - - O- - Apple solids
'3 32. _____ .-__£3°°4L- $2
.5
E 1 1
fl :
0 r T . E
-5 0 5 10 15 20 25 i
|
Time (day) i

L._

 

 

 

Figure 3.4.5 The effect of apple solids on procyanidin terminal end units of
Jonathan cider fermentation (n = 3, p <0.001, half error bars for 30% apple solids
samples)

Statistically, there were no significant differences in the concentrations of
procyanidins between samples of varying apple solids % (Figure 3.4.5). However, since
the statistical model was conservative, significant differences were not detected, such as
in this case, where p = 0.085 when mash was compared over time. Samples of 0% and
10% apple solids had higher levels of procyanidin concentrations than samples of 30%
apple solids, but it was not significantly high enough. This would have agreed with
Renard and others (2003) who reported that the more apple solids were added, more
binding of procyanidins with apple flesh occurred. However, it was possible that the
procyanidin - cell walls interactions that occurred in samples consisting of 0% apple
solids were sufficiently established and the addition of more apple solids did not make a

significant difference in the procyanidin content starting from Day 0 onwards.

46

 

 

 

 

(10" moles/ml juice)

 

Extension units concentration

 

 

5!

4 i
1

 

 

 

 

 

 

 

Time (d)

 

1

~--<>---- Yeast 10%AS

A Control 0% AS j

 

A ----- Yeast0%AS }

0 Control 10% AS

0 Control 30% AS
0- - - Yeast 30% AS

 

 

 

Figure 3.4.6 The effect of apple solids on procyanidin extension units of Jonathan

cider fermentation (n = 3, p <0.001, half error bars)

Similarly, there were no significant differences observed between the

concentrations of extension units in apple solid percentages (Figure 3.4.6).

47

 

[210% apple solids;
I 10% apple solids
I 30% apple solids

 

 

 

 

Time (d)

 

 

Figure 3.4.7 The effect of apple solids on mean degree of polymerization of
procyanidins in Jonathan cider fermentation (n = 3, p <0.001)

Samples containing 30% apple solids had significantly higher mDP values on Day
0 and 5 compared to samples with 0 and 10% apple solids (Figure 3.4.7). This finding
was unexpected since the concentration of terminal end units and extension units
demonstrated no significant differences on an individual basis. However, the combination
of the concentration values to calculate mDP may result in some significant differences
especially if the extension unit concentration increased while the terminal end unit

concentration decreased or remained constant.

The presence of additional apple solids may have contributed to the presence of
longer chained procyanidins for the first two sampling days. On Day 10, the differences
were no longer observed as the mDP of samples containing 30% apple solids dropped.
This observation may be due to the decrease in procyanidin extension unit concentrations
as seen on Day 10 (Figure 3.4.6) while procyanidin terminal end units remained

relatively constant. It could be possible that procyanidin units broke down into

48

monomeric units during fermentation on Day 10, when the cider samples were
undergoing yeast fermentation, possibly at its optimum. Investigations to determine the
potential role of yeast breaking down procyanidin units have not been reported in
research literature. These monomeric units may have then been eliminated from the juice
system by either being absorbed into the apple solids or were oxidized, thus reducing or

maintaining the procyanidin concentration measured as terminal end units (Figure 3.4.8).

 

Occurrence: Procyanidin breakdown and monomeric unit elimination

E E (free monomeric
g 11:: I; E E E E E E E units)
T T T T T + E E E
T T T T T . (absorbed into
_ apple cell wall
mDP “ 3 mDP = 2.4 or oxidized)

 

 

 

Figure 3.4.8 Proposed behavior of procyanidins in Jonathan cider during
fermentation: Polymeric breakdown and monomeric unit elimination

49

3.4.1.3 The effect of fermentation time on the concentration of procyanidins in cider
fermentation made from Jonathan apples

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

9
ab bc i
g 8 ab a 5
gm * if .
o
3 6 e - —— —— 1 . — a
8 :3 5 -._ ,. ! 00%apple i1
T . 5!
.2 E sohds .
= g 4 v v , ———— i_____ _
E E 3 .
TE: 72 + $___ ____
E 2 _ _ $A$$,, _ - .. __._._____________ _ _ ______
. 0
IF 1 I T
-5 0 5 10 15 20
Time ((1)

 

1

Figure 3.4.9 The effect of fermentation time on procyanidin terminal end unit

concentration in Jonathan cider fermentation containing 0% apple solids (n = 3, p
<0.001)

Significant differences in the concentrations of procyanidin terminal end units
were only observed in samples containing 0% apple solids between Day 5 and Day 14,
where Day 14 was significantly higher (Figure 3.4.9). The presence of apple solids may
have hindered the effects of fermentation on procyanidins and therefore, no significant
differences throughout time were observed in samples of higher apple solids content

(10% and 30%) when treatment was held constant.

The concentration of procyanidin terminal end units may have increased due to
the degradation of apple cell wall materials over time or other side reactions of yeast
fermentation (apart from alcohol production), which released procyanidin units into the

juice system.

50

7
3x
i
\
l
I
.l
l
J

 

h)

N
LII

20

 

(104 moles/ml juice)
CZ

 

Extension units concentration

 

 

10
5 i
o
o 5 10
Time ((1)

 

Figure 3.4.10 The effect of fermentation time on the concentration of extension units
in Jonathan cider fermentation (n = 3, p <0.001)

Procyanidin extension unit concentrations were significantly lower on Day 10
when the mash and treatment factor remained constant (Figure 3.4.10). Since data from
the control samples was limited due to microbial contamination after Day 10, the effect

of time was further investigated for the yeast samples.

1

Extension units concentration
(10 ‘ moles/ml julce)

 

 

J

‘4‘
1
l
l
i
J
1

Figure 3.4.11 The effect of fermentation time on the concentration of extension
units in Jonathan cider fermentation: Yeast samples (n = 3, p <0.001)

51

A similar pattern was observed in the yeast samples where the concentration of
extension units decreased on Day 10 onwards (Figure 3.4.11). Since the concentration of
extensions units remained consistently lower after Day 10 (when fermentation was
ongoing at its optimum), it is possible that yeast fermentation may be influencing the
structure of procyanidins by shortening the chain lengths through some mechanism,

which has yet to be investigated.

 

mDP

 

 

 

 

 

 

 

Time ((0

 

L ,
Figure 3.4.12 The effect of fermentation time on mean degree of polymerization of
procyanidins in Jonathan cider fermentation (n = 3, p <0.001)

There were significant differences in mDP values between both control and yeast

 

 

samples over time (Figure 3.4.12). Control samples at Day 10 had significantly lower
mDP than those at Day 0 and 5. The mDP may have decreased as more procyanidins
were bound to apple solids as time increased which agreed with the observation made by

Le Bourvellec and others (2004) regarding the complexation of procyanidins with apple

52

cell walls. For the fermented yeast samples, there were variations in the mDP levels
between days and no consistent trend was observed. It may be due to a combination of
factors that both increase and decrease procyanidin content (whether terminal end or

extension unit-wise) simultaneously during fermentation.

Overall, it was observed that alcohol production, apple solids and time did not
increase procyanidin content, chain length and consequently, mDP during yeast
fermentation. These results indicated that ‘mash fermentations’ practiced in the wine
industry may not yield the same procyanidin boost expectations in hard cider mash
fermentations made from dessert apple varieties. The oxidation effect on procyanidins
was a major factor that served as an obstacle towards the success of the project. For

example, sample exposure to air during preparation and analysis were unavoidable.

53

3.4.2 The effect of heat on the concentration of procyanidins in cider made from
Jonathan apples

Since fermentation did not significantly increase free procyanidin content in hard
cider, heat and pectolytic enzymatic treatments were considered as alternative options to
support the research hypothesis.

The plant material used was similar to Methods and Material (Section 2.1).
Similarly, the apple juice samples were prepared using Acme 5001 White Supreme
J uiceratorTM Juice Extractor (Waring Products, Torrington, CT, USA). The apple pieces
were cored, peeled by hand, and juice was collected as a single batch. All samples had
10% apple solids (w/w) added back to the cider and distributed into 50 ml disposable

centrifuge bottles in triplicates.

The apple juice samples were centrifuged using an Allegra 6R Centrifuge Rotor
Model GH3.8 with swinging buckets (Beckman, Fullerton, CA, USA) to separate the
juice from the solids at 3000 rpm (~ 1411 g) for 15 min at 10 °C. The supernatants were
decanted. Procyanidin concentrations were measured using the colorimetric methods
described in Methods and Material (Section 2.4.2).

The samples were held in water baths of the following temperature profiles, 40,

50, and 60 °C for 90 min (Figure 3.5.1).

 

 

Apple Juice (10% solids)

I
E l l I

Control 40°C 50°C 60°C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3.5.1 Experiment design of heat treatment

54

The data was analyzed using a one-way factorial analysis and Tukey’s HSD. The
p S 0.001 was used as the level of significance.

Elevated temperatures of 40°C and above are known to inactivate polyphenol
oxidase, the enzyme responsible for browning. Procyanidins have been reported to be
heat stable up to boiling temperatures (Renard and others 2004).

Significant differences observed between experiment batches were thought to be
due to the variances between batches of raw apple material since the apples used in this
objective were not obtained from the same source (local grocery store vs MSU
Horticulture farm). While the samples were harvested in the same Fall season, variations
in procyanidin content we potentially present in each batch or bag since growing
conditions (i.e. climate, soil type, and orchard location) may differ. Additionally, the
batches of apples may further differ in their cold storage conditions since each batch was
studied on different days. While there was no literature on the behavior of phenolic
compounds in Jonathan apples, studies reported that (-) epicatechin concentrations in
dessert apple varieties remained constant during storage (Burda and others 1990) and the
concentration of (+) catechins in apples had a tendency to decrease during storage
(Kolesnik and others 1977).

Since the phenolic content of apples influences the oxidation rate in cider
samples, it is possible that batches of apples with significantly lower procyanidin
concentrations either had reduced procyanidin content within the apples or experienced

different levels of oxidation during sample preparation for each batch.

55

 

 

 

 

 

 

 

 

fi_xi_fi____ A___m_ -‘x__ni_ x-—
10
c
9 a a a
5 8 w i e
E g 7
i 512. 6 b b b b .w. a
j g-g iUBatchl
» a 2 5 i
‘ .. c llBatchZ
1 g. 4 + + W
i .3 E 3
1 E 2 i
i 1 r
i 0 _
Control 40°C 50°C 60°C
Treatment

 

 

 

Figure 3.5.2 The effect of heat treatments on the procyanidin terminal end unit
concentration in Jonathan cider containing 10% apple solids (n = 3, p <0.001)

 

There were no significant differences observed between all samples in Batch 2
while in Batch 1, samples treated at 60°C were significantly higher in procyanidin
concentrations than the other samples of lower temperature profiles as measured by
terminal end units (Figure 3.5.2). Heat may have weakened the hydrogen bonds that
bound procyanidins with polysaccharides, and increased diffusion of procyanidin from
the apple solids into the cider significantly in Sample 60°C of Batch 2.

However, many problems were encountered in this experiment. In preliminary
tests (Appendix 3, Table A.3.3 and A.3.4), error values in the data obtained were as high
as 30% and no conclusions could be made. Additionally, repeats of this experiment with
other batches yielded varying results and no definite trend could be concluded by
comparing significant differences obtained between Batch 1, 2, and 3 (Appendix 3, Table

A.3.l and A.3.2). This inconsistency may be due to the variances between batches that

56

were discussed previously. Lea and Timberlake (1978) found that the addition of S02 was
vital in successfully increasing organoleptically procyanidins by 50% in warm anaerobic
incubation of Dabinett apple [solids] before juice extraction, since 802 helped to control
oxidation. However, the concentrations used by Lea and Timberlake were 200 ppm 802,
which is 4 times higher than the amount used in this study and was not feasible for the
industry use, especially with the regulatory pressures on the fermented beverage industry

in regards to sulfites.

 

25

 

20

‘ Erazé’ch'r }
{Bash}.

 

1

Extension units concentration
(10'4 moles/ml juice)

 

 

 

 

 

 

 

 

 

} Control 40°C 50°C 60°C

L Treatment

 

 

 

 

Figure 3.5.3 The effect of heat treatments on the procyanidin extension unit
concentration in Jonathan cider containing 10% apple solids (n = 3, p <0.001)

No significant differences were observed between the concentrations of
procyanidin extension units based on different temperature profiles (Figure 3.5.3), which
was consistent among the two batches. This observation indicated that procyanidin chain
length was not affected by temperature. An explanation may be that the concentration of

whole procyanidin units released from apple solids did not significantly change. This

57

agreed with the previous observation of terminal end concentration. Another explanation
may be that the phenomenon of oligomers changing to polymers did not occur during
heat treatment. Procyanidins had been reported to be heat stable up to boiling
temperatures (Renard 2004) and this may be why procyanidin content did not decrease

after heat treatments.

 

4 b b 7 b
3.5 7 7 7 a 7

3
2.5 , 7 7 7 Hagan!
2 IBatch2
l 1.5 7
1 1 *
i 0.5 7 .

 

4.5
1

 

 

 

 

 

 

 

 

 

‘r Control 40°C 50°C 60°C
[ Treatment

 

J

Figure 3.5.4 The effect of heat on the mean degree of polymerization of procyanidins
in Jonathan cider containing 10% apple solids (n = 3, p <0.001)

No significant differences were observed in the mDP of procyanidins between
treatments of varying temperature profiles (Figure 3.5.4). This observation was expected
since the concentration of procyanidins (measured as terminal end and extension units)
influence the mDP and both concentrations demonstrated the lack of or limited
significant changes as affected by heat treatments.

The lack of trend was most likely because higher temperatures did not provide

sufficient disintegration of the apple cell walls to release procyanidin constituents since

58

the samples contain 10% apple solids. The behavior of procyanidins was thought to be

similar to what was seen from the data in Figure 3.4.4.

3.4.3 The effect of pectolytic enzymes on the concentration of procyanidins in cider
made from Jonathan apples

The plant material used was similar to Methods and Material (Section 2.1).
Similarly, the apple juice samples were prepared using Acme 5001 White Supreme
J uiceratorTM Juice Extractor (Waring Products, Torrington, CT, USA). The apple pieces
were cored, peeled by hand and juice was collected as a single batch. All samples had
10% apple solids (w/w) added back to the cider and distributed into 50 m1 disposable

centrifuge bottles in triplicates.

The apple juice samples were centrifuged using an Allegra 6R Centrifuge Rotor
Model GH3.8 with swinging buckets (Beckman, Fullerton, CA, USA) to separate the
juice from the solids at 3000 rpm (~ 1411 g) for 15 min at 10 °C. The supematants were
decanted. Procyanidin concentrations were measured using the colorimetric methods
described in Methods and Materials (Section 2.4.2).

Commercial pectolytic enzymes, Pectinex® Ultra SP-L and Crystalzyme®
ZOOXL were used at concentrations of 0.02 m1/ml juice (Figure 3.6.1). The samples
treated with Pectinex® Ultra SP-L were placed in a water bath at 35 °C while samples
treated with Crystalzyme® ZOOXL were heated at 50 °C. Both samples were held in

water baths for 90 min.

59

 

 

| Apple Juice (10% solids) l
l

 

 

 

 

 

 

 

| Pectinex® Ultra SP-L 1 ‘ Crystalzyme® 200XLJ
1__l_I I
I—_'_]
35°C Enzyme 50°C Enzyme
(Control) (Control)

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 3.6.1 Experiment design of pectolytic enzyme treatments

The data was analyzed using a one-way factorial analysis. The p 5 0.001 was used

as the level of significance.

 

_\
O

 

Terminal end units concentration
(10'4 moles/ml Julce)

    

O—‘Nm-hUIOKICDCD

 

 

 

 

35°C Pectinex® 50°C Crystalzyme®

(Control) (1000 ppm) (Control) (1000 ppm)
Treatment

 

 

 

Figure 3.6.2 The effect of pectolytic enzyme treatments on procyanidin terminal end
unit concentration in Jonathan cider containing 10% apple solids (n = 6, p <0.001)

Samples which contained 10% apple solids were treated with commercial pectolytic
enzymes, Pectinex® and Crystalzyme® had higher concentration of procyanidins as

measured by terminal end units (Figure 3.6.2). This result agreed with results of Will and

60

others (2002) who found that the concentration of procyanidins increased with enzymatic
treatments at 40, 50 and 60°C. Enhanced cell wall degradation by the pectolytic enzymes

may have led to a higher procyanidin release.

There were no significant differences between the concentrations of procyanidin,
measured as terminal end units between samples treated with the two pectolytic enzymes.
Crystalzyme 200XL contains arabinase and cellulose while both Crystalzyme 200XL and
Pectinex Ultra Sp-L contain pectolytic and a range of hemicellolulytic activities. First, the
composition of both enzyme systems may not differ significantly from each other.
Specific information on the composition of enzyme mixes is not available from the
manufacturer. If Crystalzyme 200XL does contain arabinase and cellulase, which
Pectinex Ultra Sp-L may lack, the concentrations of these enzymes may not be sufficient
enough to cause a significant impact on procyanidin released. Different temperatures
were used in order to obtain the optimum enzymatic activity as recommended by the

manufacturers.

61

 

 

 

 

 

 

 

 

 

30
25 r
l:
.2
‘ é : +
8 .3
s a
E a ‘5 ’
.5 _E
10
.5 i:
m fl
8 v
13 5 '
pa
0 l
35°C Pectiner® 50°C Crystalzyme®
(Control) “00° ppm) (Control) “’00 W“)
L Treatment

 

 

Figure 3.6.3 The effect of pectolytic enzyme treatments on the procyanidin
concentration of extension units in Jonathan cider containing 10% apple solids (n =
6, p <0.001)

Samples treated with pectolytic enzymes increased concentrations of procyanidin
extension end units (Figure 3.6.3). While there is no literature to explain the effect of
pectolytic enzymatic treatments on the concentration of procyanidin extension units in
apple cider, the increase is mostly likely due to the release of procyanidins due to the
degradation of apple solids. The released procyanidins trap these compounds within their
polysaccharide matrix as observed by Le Bourvellec and others (2004). In this
observation, it is possible that the release of procyanidins is proportional to the measured
concentration of procyanidin extension units in apple cider. Under this assumption, it
may be further proposed that procyanidins do not undergo major changes in chain length

under the conditions of this study.

62

Since there was also no significant difference between the pectolytic enzymatic
systems, it was also concluded that the composition and firnctionality of the two enzyme
systems did not differ significantly enough to have an impact on procyanidin content in

Jonathan apple cider.

 

 

 

 

 

 

 

 

 

 

35°C Pectiner® 50°C Crystalzyme®
(Control) (1000 Ppm) (Control) (10001313111) 1

L.# Treatment/ ,

Figure 3.6.4 The effect of pectolytic enzyme treatments on the mean degree of
polymerization of procyanidins in Jonathan cider containing 10% apple solids (n =
6, p <0.001)

Pectolytic enzyme treatments did not have a significant impact on the mDP values

(Figure 3.6.4) and this observation further supports the proposed theory that the chain
length of procyanidins was not affected by pectolytic enzymes. The behavior of
procyanidins in this experiment is thought to be similar to the proposed scheme

illustrated in Figure 3.6.5.

63

 

Occurrence: Addition of procyanidin units with no structural change

EEEEE EEEEE
EEEEE EEEEE EEEEE
EEEEE TTTTT + TTTTT
T T T T T . (procyanidins released)
mDP=3 mDP=3 mDP=3

 

 

 

Figure 3.6.5 Proposed behaviour of procyanidins in Jonathan cider during
treatment (T= terminal end units, E = Extension units): Addition of procyanidin units
with no structural change

Preliminary studies with pectolytic enzymes found that there was no significant
difference in procyanidin extension units at enzyme concentrations of 100 ppm, which
were recommended by enzyme manufacturers. However in this study, when
concentrations were increased to tenfold (i.e. 1000 ppm), a significantly higher
concentration of procyanidins were obtained. The increase of enzyme concentration may
have increased the enzymatic effect on the breakdown of apple solids, thus releasing

more procyanidins into the cider.

Based on these observations, it was concluded that pectolytic enzyme treatments
were not feasible for increasing procyanidin content in apple cider made from Jonathan
apples and possibly other dessert apple varieties. The enzyme concentrations necessary to
have a significant impact are higher than the typical industry dosages and enzymatic

treatments, which utilized enzymes listed in Table 1.2 would not be economical.

64

4. CONCLUSIONS AND RECOMMENDATIONS

4.1 Summary and conclusions

The original goal of the hard cider research grant was to produce a hard cider
product that Michigan consumers would accept positively and provide an alternative
option of convert excess supply of Michigan apple varieties into value-added products.
When this research effort was initiated, it was conducted under the assumption that the
average US. consumer desires bitter and astringency sensory characteristics found in
traditional hard cider made in Europe. While the effort to increase procyanidin
consumption in fermentation processing methods was carried out, extensive marketing

research on hard cider including consumer taste panels were conducted at the same time.

As part of the effort to increase procyanidin content by investigating fermentation
methods, it was found that the methodology consisting of HPLC followed by acid
hydrolysis cleavage in the presence of phloroglucinol did not successfully estimate
procyanidin in polyphenolic extracts obtained from cider samples in this research. While
possible explanations of this outcome point towards problems encountered during the
reaction treatment, these conclusions have yet to be confirmed due to time and facility
constraints. Therefore, the analysis of procyanidins in hard cider made from Jonathan
apples was achieved using classical colorimetric methods.

Alcohol production from yeast fermentations did not increase procyanidin
concentrations and mean degree of polymerization (mDP). Yeast fermentation may not
have produced sufficient alcohol (4%) to release procyanidins from apple cell wall

materials in apple solids since Renard and others (2003) indicated a minimum 15%

65

alcohol was necessary to reduce the binding effect between procyanidins and apple

solids.

Statistical analysis of measured procyanidin concentrations indicated that the
percentage of apple solids did not have a major impact on the procyanidin concentrations
and mean degree of polymerization. This observation was mostly likely due to the
procyanidin-apple cell wall interactions, which formed quickly within cider upon contact

(Renard and others 2003).

The duration of fermentation resulted in varying significant differences of
procyanidin concentration between days in samples of 0% apple solids. It was found that
mDP levels decreased after Day 10 and it was proposed that yeast fermentation breaks
down the linkages within the procyanidin chain through unknown mechanisms. Free
monomeric units were then quickly eliminated from the juice system by either absorption

by cell wall material or oxidation.

When it was found that fermentation did not affect procyanidin content in hard cider,
the study was extended to alternative methods such as heat and pectolytic treatments.
Inconclusive data obtained from heat treatments indicated that procyanidin
concentrations in dessert apple varieties may not contain significant amounts of
procyanidins and did not influence mDP of procyanidins. Pectolytic enzyme treatments
increased the procyanidins concentrations but did not influence the procyanidin mDP at
enzyme concentrations of industry standard dosages. The usage of high enzyme
concentrations is not considered feasible for the industry especially if the procyanidin

increase is not substantially increased.

66

While the lack of success in increasing procyanidin content in hard cider made
fi'om dessert apple varieties did not provide scientific support for the original hypothesis
of this study, the application of this information ultimately depends on how the US.
consumer prefers their hard cider product. Michigan consumers may not necessarily
desire a hard cider product that is as astringent or bitter as the traditional European
product. Behe and others (unpublished) have found that consumer panelists rated hard
cider made from Jonathan and McIntosh dessert varieties positively and acceptable

overall.

Additionally, the concentrations of procyanidins in hard cider made from fi'esh
Michigan dessert apples were comparably higher than commercially available imported

hard cider brands.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

35.0 i
l d 5
30.0 —-————-—-—— -_ .. _ - ._ ___..___ l
‘ 250 7— —-——-7——7—._7 7--- 7. -- .__ . _ A}, .L _fl "___-”- W n
' o ' FTermmal End Units
'9 a . .
3.. 200 L___ ______ __ _____._ _fi__,_ ,___~,__ _fl __ fi Cl Extensron UN!!!
5 l b D
E 150 'f—_—’_— }— — ifi— — _—_____ _____ __d
If . 1
7 10.0 ~ — -. _ —4 i
l
l A . ,2 B (I
i 5.0 ——# 77 _. .
E - i
0.0 . i _l ‘ N
K Strongbow Woodchuck Woodchuck MSU f

l
t Cider Brands i

 

 

 

Figure 4.1 Estimated procyanidin concentrations of extension and terminal end
units in K, Strongbow, Woodpecker, Woodchuck and MSU hard cider brands (n =
6, p >0.001)

67

In Figure 4.1, MSU cider (i.e. hard cider made from a blend of Jonathan:
McIntosh, 1:1) recorded higher procyanidin readings than K, Strongbow, Woodchuck
and Woodpecker. This observation may be due to the following explanation. While
procyanidin concentrations may be higher in apple varieties used in Europe, these high
concentrations may have been drastically reduced during the process of converting fresh
apple juice into apple juice concentrate (AJC). This process mostly likely utilizes high
temperatures and allows more oxidation to occur, thus reducing procyanidin
concentrations. While AJC is commonly used in UK. ciderrnaking, MSU cider utilized
freshly pressed apple juice, which undergoes less processing and most likely retains more

procyanidins.

As a conclusion, hard cider made fiom fresh Michigan dessert apple varieties has
the potential of retaining satisfactory levels of procyanidins, even if the dessert apple
varieties have lower levels of procyanidins than cider apple varieties within the fruit itself
without the use of alternative ‘mash’ fermentation, heat and pectolytic enzymatic
treatments. The findings of this research will provide useful information to any future

investigations on the cider making process using dessert apples varieties.

4.2 Recommendations for future research
The following topics are recommended for future research:

1. Investigate the concentration of procyanidins for longer time durations (e. g. 3
months) during fermentation and storage to determine whether flavor development in
hard cider maturity is due to increased procyanidin content.

2. Investigate the effect of extended heat treatments (higher temperatures, longer

heating times) on the concentrations of procyanidins during cider fermentation

68

APPENDIX 1: Reagents
1) High performance liquid chromatography

Cider extract: 25 ml of cider samples was concentrated into 5 ml using Sep Pak C13
cartridges by eluting 5 ml of water and 5 ml of extract was recovered with 2 ml of
methanol. 1 ml of extract was added to the reaction.

Grape seed extract: The sample preparation was adapted from Escribano-Bailon and
others (2003) was prepared by homogenizing 5 g of grape seeds in 15 ml of methanol.
The sample was centrifuged at 12000 rpm for 15 min. 4 ml of extract was dried and used
in the reaction.

Phloroglucinol Solution: Dilute 1 ml of 10M HCl and 99 ml methanol. Dissolve 5 g
phloroglucinol and 1 g ascorbic acid into 100 ml of the acidic solution.

2) Colorimetric Assays

Extraction solutions
1% HCl in methanol: Dilute 0.01 ml of 10 N HCl into 100 ml of methanol
1% HCl in H20: Dilute 0.01 ml of 10 N HCl into 100 ml of H20

Vanillin in methanol assay
Dissolve 1 g of vanillin into 50 ml of methanol and 50 ml of 8% HCl in methanol
Add 0.5 ml of sample extract into 2.5 m1 of solution.

Porter Reagents

i) 2% ferric ammonium sulphate in 2 N HCl in methanol

Dilute 20 ml of 10 M hydrochloric acid into 80 ml of methanol. Dissolve 2 g of ferric
ammonium sulphate in 100 ml of the acidic solution

ii) 95:5, butanol: hydrochloric acid
Dilute 5 ml of 10 M hydrochloric acid into 95 ml of butanol.

69

APPENDIX 2: Raw data for fermentation tests

Table A.2.1 RI values of Jonathan apple cider fermentation: Batch 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment % apple Time (d)
solids 0 5 10 15 20

Control 0 10.8 10.8 10.8 10.8 N/A
0 10.8 11.0 10.8 10.8 N/A

0 10.8 10.8 11.0 10.8 N/A

10 10.8 10.8 11.0 N/A N/A

10 11.0 10.8 11.0 N/A N/A

10 10.8 1 1.0 10.6 N/A N/A

30 11.0 10.8 10.8 N/A N/A

30 10.8 10.8 10.8 N/A N/A

30 10.8 11.0 10.8 N/A N/A

Yeast O 10.8 9.6 8.4 5.2 N/A
0 11.0 9.6 8.2 5.4 N/A

0 11.0 9.6 8.2 5.2 N/A

10 11.0 10.2 6.6 5.2 N/A

10 10.8 10.2 6.6 5.2 N/A

10 10.8 10.2 6.6 5.0 N/A

30 11.0 9.0 5.2 4.8 N/A

30 10.8 9.4 5.4 5.2 N/A

30 10.8 9.2 5.2 5.0 N/A

 

 

 

 

 

 

 

Table A.2.2 RI values of Jonathan apple cider fermentation: Batch 2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment % apple Time (d)
solids 0 S 10 15 20
Control 0 10.8 10.8 10.8 N/A N/A
0 10.8 11.0 10.8 N/A N/A
0 10.8 10.8 10.6 N/A N/A
10 10.8 10.8 10.6 N/A N/A
10 10.8 10.8 10.8 N/A N/A
10 10.8 10.8 N/A N/A N/A
30 11.0 11.0 N/A N/A N/A
30 10.8 10.8 N/A N/A N/A
30 11.2 10.8 N/A N/A N/A
Yeast O 10.8 9.6 8.2 6.6 5.2
0 10.8 9.6 8.4 6.8 5.4
0 10.8 9.6 8.2 6.6 5.2
10 10.8 10.2 9.2 8.2 6.0
10 10.8 10.4 9.2 9.4 6.2
10 10.8 10.2 9.0 7.8 5.8
30 11.0 10.2 8.6 6.6 5.6
30 10.8 10.4 8.4 7.8 5.2
30 10.8 10.2 8.6 7.8 5.8

 

 

 

 

 

 

 

7O

 

 

 

 

 

 

10 15 20
Sugar %

25

 

 

 

 

 

 

Figure A.2.l Standard Curve: RI vs Sugar %

Table A.2.3 H values of Jonathan apple cider fermentation: Batch 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment °/o apple Time (d)
solids 0 5 . 10 15 20
Control 0 3.14 3.11 3.13 3.16 N/A
0 3.15 3.10 3.15 3.17 N/A
0 3.14 3.12 3.15 3.16 N/A
10 3.11 3.12 3.16 N/A N/A
10 3.14 3.13 3.17 N/A N/A
10 3.11 3.09 3.15 N/A N/A
30 3.13 3.16 3.16 N/A N/A
30 3.13 3.16 3.17 N/A N/A
30 3.14 3.15 3.15 N/A N/A
Yeast O 3.14 3.08 3.15 3.20 N/A
0 3.14 3.07 3.14 3.21 N/A
0 3.14 3.07 3.17 3.23 N/A
10 3.11 3.11 3.14 3.24 N/A
10 3.11 3.10 3.14 3.22 N/A
10 3.11 3.11 3.15 3.23 N/A
30 3.14 3.11 3.14 3.24 N/A
30 3.15 3.10 3.16 3.23 N/A
30 3.15 3.08 3.17 3.26 N/A

 

 

 

 

 

 

 

71

 

Table A.2.4. pH values of Jonathan apple cider fermentation: Batch 2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment % apple Day

solids 0 5 10 15 20

Control 0 3.14 3.17 3.18 N/A N/A
0 3.14 3.15 3.19 N/A N/A

0 3.12 3.16 3.18 N/A N/A

10 3.17 3.19 3.19 N/A N/A

10 3.12 3.19 3.19 N/A N/A

10 3.15 3.19 N/A N/A N/A

30 3.15 3.19 N/A N/A N/A

30 3.15 3.20 N/A N/A N/A

30 3.14 3.19 N/A N/A N/A

Yeast O 3.11 3.16 3.18 3.18 3.21
O 3.14 3.14 3.17 3.19 3.19

0 3.15 3.14 3.19 3.18 3.19

10 3.14 3.19 3.18 3.19 3.21

10 3.15 3.19 3.19 3.17 3.21

10 3.13 3.18 3.17 3.19 3.25

30 3.17 3.19 3.21 3.18 3.26

30 3.17 3.18 3.22 3.16 3.26

30 3.17 3.19 ' 3.22 3.19 3.24

 

 

 

 

 

 

 

72

 

Table A.2.5 Mean absorbance values of Jonathan apple cider fermentation using
Vanillin in methanol assay

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Batch Treatment % apple Time ((1)
solids 0 5 10 15 20
1 Control 0 0.948 0.855 0.925 1.051 N/A
(3.4%) (6.2%) (6.5%) (8.8%)
10 0.824 0.994 1.085 N/A N/A
(4.1%) (5.6%) (2.7%)
30 0.173 0.079 0.088 N/A N/A
(14.3%) (6.3%) (11.8%)
Yeast 0 0.935 0.837 1.005 1.118 N/A
(2.1%) (6.7%) (7.7%) (8.3%)
10 0.826 0.975 0.819 0.881 N/A
(5.3%) (10.4%) (23.4%L (4.3%)
30 0.166 0.145 0.128 0.113 N/A
(34.2%) (4.4%) (14.1%) (14.3%)
2 Control 0 0.569 0.618 0.639 N/A N/A
(10.4%) (6.2%) (7.3%)
10 0.329 0.221 0.180 N/A N/A
(12.8%) (9.2%) (13.0%)
30 0.072 0.025 N/A N/A N/A
(28.7%) (9.9%)
Yeast 0 0.624 0.585 0.544 0.538 0.518
(4.2%) (6.5%) (11.7%) (5.3%) (6.5%)
10 0.343 0.228 0.184 0.216 0.169
(7.5%) (3.8%L (9.12%) (9.8%L (3.9%)
30 0.053 0.031 0.029 0.036 0.024
(45.1%) (10.4%) (3.4%) (4.3%) (31.1%)

 

 

 

 

 

Mean based on three measurements. % CV given in parenthesis

73

 

Table A.2.6 Mean absorbance values of Jonathan apple cider fermentation using

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

BuOH/HCI assay
Batch Treatment % apple Time ((1)
solids 0 5 10 15 20
1 Control 0* 0.465 0.754 0.729 0.578 N/A
(6.7%) (5.9%) (11.2%) (18.2%)
10* 0.451 0.719 0.580 N/A N/A
(6.4%) (9.3%) (4.6%)
30 0.567 0.298 0.172 N/A N/A
(13.4%) (6.4%) (13.4%)
Yeast 0* 0.458 0.702 0.624 0.573 N/A
(2.4%) (6.7%) (7.1%) (2.9%)
10* 0.428 0.670 0.593 0.480 N/A
(4.4%) (5.1%) (15.8%) (6.4%)
30 0.511 0.494 0.246 0.188 N/A
(13.0%) (4.6%) (6.1%) (13.2%)
2 Control 0* 0.741 0.650 0.456 N/A N/A
(7. 1%) (2.4%) (31.3%)
10* 0.455 0.333 0.220 N/A N/A
(8.8%) (2.0%) (8.0%)
30 0.241 0.097 N/A N/A N/A
(30.8%) (11.3%)
Yeast 0* 0.697 0.652 0.548 0.603 0.608
(7.6%) (8.5%) (3.33%) (9.0%) (7.2%)
10* 0.471 0.315 0.271 0.299 0.252
(7.7%) (2.3%) (9.2%) (5.1%) (2.3%)
30 0.195 0.121 0.105 0.139 0.088
(36.3%) (8.2%) (8.1%) (2.9%) (18.2%)

 

 

 

 

 

Mean based on three measurements. % CV given in parenthesis

* Samples subjected to 1:1 dilutions prior to measurement

74

 

APPENDIX 3: Raw data for heat and pectolytic enzymatic tests

Table A.3.1 Mean absorbance values of Jonathan apple cider heat treatments using
Vanillin in methanol assay

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Batch Rep Temperature (°C)
C 40 50 60
1 1 0.492 0.450 0.420 0.568
2 0.469 0.486 0.431 0.461
3 0.445 0.456 0.448 0.473
4 0.482 0.540 0.415 0.484
5 0.506 0.475 0.466 0.453
6 0.506 0.486 0.471 0.443
Mean 0.483 (4.8%)“ 0.482 (6.6%)“ 0.442 (5.3%)“ 0.480 (9.4%)“b
2 1 0.529 0.519 0.497 0.435
2 0.524 0.509 0.535 0.489
3 0.462 0.503 0.525 0.505
4 0.450 0.527 0.476 0.522
5 0.448 0.481 0.485 0.471
6 0.458 0.514 0.439 0.437
Mean 0.478 (7.8%)“ 0.508 (3.1%)“ 0.492 (7.1%)“ 0.476 (7.5%)“
3 1 0.671 0.651 0.633 0.755
2 0.684 0.641 0.662 0.896
3 0.717 0.704 0.759 0.713
4 0.532 0.652 0.636 0.831
5 0.725 0.76 0.687 0.664
6 0.671 0.666 0.733 0.698
Mean 0.667 (10.4%)“ 0.679 (6.6%)“ 0.685 (7.5%)“b 0.760 (11.6%)“

 

Mean based on six measurements. % CV given in parenthesis

Table A.3.2 Mean absorbance values of Jonathan apple cider heat treatments using

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

BuOH/HCI assay
Batch Rep Temperature (°C)
C 40 50 60
1 1 0.836 0.837 0.736 1.104
2 0.883 0.944 0.839 0.870
3 0.842 0.960 0.804 0.913
4 0.873 0.864 0.873 0.861
5 0.916 0.954 0.884 0.866
6 0.885 0.955 0.873 0.903
Mean 0.872 (3.4%)“ 0.919 (5.8%)“ 0.834 (6.7%)“ 0.919 (10.0%)“
2 1 0.929 0.896 0.874 0.828
2 0.877 0.834 0.847 0.863
3 0.903 0.834 0.858 0.778
4 0.955 0.849 0.811 0.897
5 0.846 0.855 0.866 0.874

 

 

 

 

75

 

 

 

 

 

 

 

 

 

 

 

 

6 0.726 0.835 0.816 0.897
Mean 0.872 (9.3%)3 0.860 (3.2%)a 0.845 (3.1%)8 0.856 (5.3%)“

3* 1 0.479 0.472 0.556 0.278

2 0.609 0.545 0.578 0.289

3 0.559 0.543 0.561 0.280

4 0.546 0.621 0.542 0.271

5 0.581 0.531 0.620 0.310

6 0.584 0.586 0.603 0.301

Mean 0.560

(8.0%)ab 0.549 (9.2%)“ 0.576 (5.1%)b 0.586 (7.8%)“

 

 

 

 

 

 

Mean based on six measurements. % CV given in parenthesis
* Samples subjected to 1:1 dilutions prior to measurement

Table A.3.3 Mean absorbance values of Jonathan apple cider heat treatments using
Vanillin in methanol assay: Preliminary

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Batch Rep Treatment (°C)
C 40 50 60
1 1 0.088 0.133 0.095 0.222
2 0.092 0.119 0.174 0.225
3 0.092 0.179 0.119 0.231
Mean 0.091 (2.6%) 0.144 (21.8%) 0.129 (31.3%) 0.226 (2.0%)
2 1 0.142 0.130 0.220 0.214
2 0.143 0.141 0.196 0.254
3 0.091 0.157 0.155 0.348
Mean 0.125 (23.7%) 0.143 (9.5%) 0.190 (17.2%) 0.272 (25.3%)

 

 

 

 

 

Mean based on three measurements. % CV given in parenthesis

Table A.3.4 Mean absorbance values of Jonathan apple cider heat treatments using
BuOH/HCl assay: Preliminary

 

 

 

 

 

 

 

 

 

 

 

 

 

Batch Rep Treatment (°C)
C 40 50 60
l 1 0.274 0.369 0.296 0.523
2 0.286 0.344 0.510 0.560
3 0.317 0.480 0.326 0.564
Mean 0.292 (7.6%) 0.398 (18.2%) 0.377 (30.7%) 0.549 (4.1%)
2 1 0.394 0.388 0.581 0.865
2 0.305 0.413 0.518 0.684
3 0.421 0.399 0.435 0.613
Mean 0.373 (16.2%) 0.400 Ql%) 0.511(14.3%) 0.721 (18.0%)

 

 

 

 

Mean based on three measurements. % CV given in parenthesis

76

 

Table A.3.5 Mean absorbance values of Jonathan pectolytic enzyme treatments
usingVanillin in methanol assay

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Batch Rep Treatment
30°C Pectinex® 50°C Crystalzyme®
1 1 0.378 0.546 0.489 0.535
2 0.416 0.553 0.483 0.531
3 0.251 0.595 0.409 0.627
Mean 0.348 (24.8%) 0.565 (4.6%) 0.460 (9.6%) 0.564 (9.6%)
2 1 0.235 0.678 0.408 0.737
2 0.409 0.733 0.457 0.650
3 0.348 0.737 0.368 0.579
Mean 0.331 (26.6%) 0.716 (4.6%) 0.411 (10.8%) 0.655 (12.0%)

 

 

 

Mean based on three measurements. % CV given in parenthesis

Table A.3.6 Mean absorbance values of Jonathan pectolytic enzyme treatments

 

 

 

 

 

 

 

 

 

 

 

 

 

using BuOH/HCl assaL
Batch Rep Treatment
35°C* Pectinex®* 50°C* Crystalzyme®*
l 1 0.534 0.669 0.549 0.274
2 0.536 0.636 0.531 0.265
3 0.402 0.678 0.501 0.250
Mean 0.490 (15.6%) 0.661 (3.3%) 0.527 (4.6%) 0.659 (3.5%)
2 1 0.525 0.783 0.458 0.229
2 0.386 0.829 0.452 0.226
3 0.353 0.665 0.43 0.215
Mean 0.421(21.6%) 0.759 (11.1%) 0.419 (3.3%) 0.714 (12.1%)

 

 

 

 

Mean based on three measurements. % CV given in parenthesis

* Samples subjected to 1:1 dilutions prior to measurement

77

 

 

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