A Thesis
Entitled
Mitigation of the Tomato Lye Peeling Process
By
Bradley S. Yaniga
Submitted as partial fulfillment of the requirements for
The Master of Science in Chemical Engineering
______Advisor: Constance A. Schall
______Advisor: Sasidhar Varanasi
______Committee Member: Dong-Shik Kim
______College of Graduate Studies
The University of Toledo
May 2007 The University of Toledo
College of Engineering
I HEREBY RECOMMEND THAT THE THESIS PREPARED UNDER MY SUPERVISION BY Bradley S. Yaniga
ENTITLED Mitigation of the Tomato Lye Peeling Process
BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF Master of Science in Chemical Engineering
Thesis Advisor: Constance A. Schall Thesis Advisor: Sasidhar Varanasi
Recommendation concurred by
Committee
Committee Member: Dong Shik-Kim
On
Department Chairman: G. Glenn Lipscomb Final Examination
Dean, College of Engineering Copyright © 2007
This document is copyrighted material. Under copyright law, no parts of this document
may be reproduced without the expressed permission of the author.
______
An abstract of
Mitigation of the Tomato Lye Peeling Process
Bradley S. Yaniga
Submitted as partial fulfillment of the requirements for
The Master of Science in Chemical Engineering
The University of Toledo
May 2007
Tomato lye peeling experiments were conducted to determine whether tomato
maturity, post-harvest age, type of base, or the type of pretreatment has significant effects
upon tomato peelability. Results show that tomatoes are significantly more difficult to peel as they mature on the vine. Similarly, increasing post-harvest ages make tomatoes
more difficult to peel as well. No significant differences exist between tomato peelability
for sodium hydroxide and an equimolar mixture of ammonium and potassium hydroxide
at the same total hydroxide concentration. Thus, substituting sodium hydroxide for an
equimolar mixture of potassium and ammonium hydroxide would produce statistically
insignificant differences in tomato peeling. The best pretreatment for lye peeling was
determined to be a mixture of water and octanoic acid provided that its presence is
greater than its solubility point. Other functional groups such as aldehyde, ketones, and
alcohols proved to be ineffective as a pretreatment. Mercerization and osmotic effects
iv appear not to be mechanisms through which lye peeling occurs. Strong bases act to depolymerize pectin in the middle lamella, which separates the cuticle from the fruit.
Applying advanced statistical principles of design of experiments to tomato lye peeling would greatly increase the validity of further experiments. Future work will include additional multifactor lye peeling experiments at the laboratory scale or pilot plant scale.
v
Dedication
To my wife, Christina Yaniga, who has always been a support for me during my studies as a graduate student.
vi
Acknowledgements
First, I would like to acknowledge my advisors, Dr. Constance A. Schall and Dr.
Sasidhar Varanasi, for introducing me to the area of Food Science in Chemical
Engineering. This is a truly interesting topic, and my experience as a graduate student at the University of Toledo has been greatly enhanced through my research. They have provided much guidance and insight during the course of my research. I would also like to acknowledge Dr. Dong-Shik Kim for his involvement in my thesis committee. I would like to acknowledge the following companies for their financial contributions to the project; the Center for Innovative Food Technology (CIFT) and the Hirzel Canning
Company & Farms. Also, I would like to thank Hirzel Canning Company & Farms for providing us with tomatoes, constant temperature water baths, and the mechanical tumbler for tomato peelability experiments. Finally, I would like to acknowledge all those who helped me accomplish this project. Without these individuals, this project would not have been a success. William Hirzel, for his guidance throughout the project;
Jeff Unkford, for acquiring and organizing tomatoes for vine-ripened studies in 2005 and
2006; and Glen Gibbs for acquiring tomatoes for picking and delivering tomatoes twice a week during the 2005 and 2006 peeling seasons. In addition, credit must also be given to the undergraduate students who helped design and conduct experiments in the lab.
Jeremy Herman, for doing so much work in the lab at the start-up of the project and designing laboratory experiments; John Mehler, for his help in conducting experiments; and John Clements, for his help in conducting experiments and technical advice.
vii
Table of Contents
Abstract iv
Dedication vi
Acknowledgements vii
Table of Contents viii
List of Tables xii
List of Figures xviii
I. Introduction 1
II. Literature Review 6
The Tomato Cuticle 9
Naringenin Chalcone 12
Cutin 13
Cell Walls 14
Cellulose 17
Pectin 22
Growth & Development 26
Vine-Ripened Development 26
Post-Harvest Development 30
Tomato Peeling 33
Lye Peeling 34
Wastes from Lye Peeling 37
Steam Peeling 38
viii
Research in Lye Peeling 39
Surfactants 40
Research Goals 43
III. Experimental Procedures: Lye Peeling Protocols 45
Equipment 47
Chemical Reagents 48
Tomato Peeling 49
Tomato Selection 49
Peeling Process 50
Pretreatment 50
Pretreatment Reaction Time 51
Base Dip 51
Base Reaction Time 51
Mechanical Peeling 52
Scoring 52
Statistics 53
IV. Vine-Ripened & Post-Harvest Age Studies 54
Vine-Ripened Study 54
Post-Harvest Study 63
V. Process Parameters Controlling Lye Peeling 67
Temperature, Concentration and Time Study 68
Type of Base 76
VI. Role of Caustic in Tomato Peeling 84
ix
Chemical Reagents 85
Mercerization Studies 85
Cellulose II Standards 86
13C Solid State CP-MAS NMR 87
X-Ray Diffraction 88
VII. Pretreatment with Organic Solvents: Preliminary Studies 98
VIII. Solvent Pretreatments Used in Lye Peeling 109
Experimental Methods 110
Variation of Functional Group 111
Carboxylic Acids Above & Below Solubility 112
Effectiveness of Ketones 116
Temperature Variation with Carboxylic Acids 116
Carboxylic Acids at Room Temperature 120
IX. Conclusions 125
Age Studies 125
Base Selection 126
Solvent Studies 127
Role of Caustic in Tomato Peeling 128
X. Future Work 131
Vine-Ripened and Post-Harvest Changes 131
Solvent Studies 131
Design of Experiments 132
XI. References 134
x
XII. Appendices 143
Appendix A – Robbins Chart 143
Appendix B – Scoring of Tomatoes 144
Appendix C – Part Drawing for X-Ray Diffraction Sample Holder 147
Appendix D – X-Ray Diffraction Patterns for Cellulose 148
xi
List of Tables
Table 1. ANOVA analysis for the vine-ripened study in 2005. Tomato variety was
H9423. No pretreatment was used. NaOH concentration was 3.0 N. NaOH dip
time and reaction time were 45 and 60 seconds, respectively. NaOH temperature
was 85±2°C. 57
Table 2. ANOVA analysis for the vine-ripened study in 2006. Tomato variety was 696.
Water pretreatment was used for 60 seconds. NaOH concentration was 3.0 N.
NaOH dip time and reaction time were 60 seconds for both. Temperatures of
water pretreatment and NaOH treatment were 80±1°C. 57
Table 3. ANOVA analysis for the post-harvest study in 2006. Tomato variety was TSH-
8. Pretreatment was water Pretreatment dip time and reaction were 60 and 30
seconds, respectively. NaOH concentration was 3.0 N. NaOH dip time and
reaction time were 60 seconds for both. Temperature was 80±1°C. 64
Table 4. Time and Temperature Studies Using NaOH from 2005. Tomato variety was
H9704. No pretreatment or pretreatment reaction time was used. NaOH
concentration was 3.0 N. NaOH dip time and reaction time was 60 seconds.
69
Table 5. Experimental design for three-factor factorial experiment. Treatment totals
refers to the total sum of all six duplicated replicates. Tomato variety was TSH-8.
Water pretreatment reaction time was 30 seconds. NaOH reaction time was 60
seconds. Pretreatment and NaOH temperatures and times were the same and are
xii
listed below. Each replicate was a duplicated measurement in which the response
was the average score of the three tomatoes. 70
Table 6. Three-way ANOVA analysis for lye peeling study. Tomato variety was TSH-8.
Water pretreatment was used with a 30 second reaction time. NaOH reaction time
was 60 seconds. Temperatures and times were adjusted for both pretreatment and
caustic dips. Each replicate was a duplicated measurement in which the response
was the average score of the three tomatoes. 71
Table 7. A list of the four bases used to determine if the type of base has an affect upon
tomato peelability. Tomato variety was TSH-8. 76
Table 8. ANOVA analysis for type of base study. Tomato variety was TSH-8.
Pretreatment was water. Pretreatment dip time and reaction time were 60 and 30
seconds, respectively. Base concentration was 4.0 N. Base dip time and reaction
time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C.
77
Table 9. Comparisons of average tomato score for base study. Tukey’s value, T0.05,
was found to be 0.96. Tomato variety was TSH-8. Pretreatment was water.
Pretreatment dip time and reaction time were 60 and 30 seconds, respectively.
Base concentration was 4.0 N. Base dip time and reaction time were 60 seconds
each. Pretreatment and NaOH temperatures were 80°C. 78
Table 10. Experimental design and results for an unbalanced factorial study between
NaOH and NH4OH. An ANOVA analysis revealed no significant differences
between the type of base tested. Pretreatment was water. Pretreatment dip time
and reaction time were 60 and 30 seconds, respectively. Base dip time and
xiii
reaction time were 60 seconds each. Pretreatment and NaOH temperatures were
80°C. 81
Table 11. ANOVA analysis for effect of type of base and concentration for results in
Table 10 and Figure 20. Tomato varieties were 9423 and TSH-8. Pretreatment
was water. Pretreatment dip time and reaction time were 60 and 30 seconds,
respectively. Base dip time and reaction time were 60 seconds each.
Pretreatment and NaOH temperatures were 80°C. 82
Table 12. Experimental design for mercerization experiments 90
Table 13. Tomato lye peeling studies with solvent pretreatment and variable solvent time
and temperature. Pretreatment reaction time is 30 seconds. NaOH concentration
is 3.0 N. NaOH temperature is 85±2°C. 100
Table 14. Tomato lye peeling studies using carboxylic acids as pretreatments.
Pretreatment dip time and reaction times were 60 seconds each. NaOH dip time
and reaction times were 60 seconds. NaOH temperature was 85±2°C. 101
Table 15. Tomato lye peeling studies using an octanoic acid pretreatment followed by a
treatment with a salt solution near solubility. Tomato variety was H9423.
Pretreatment dip time and reaction time were 60 seconds each. NaOH and salt
dip time and reaction times were 60 seconds each. Octanoic acid and NaOH
temperature was 85±2°C. 101
Table 16. Tomato lye peeling studies using a pretreatment of octanoic acid mixed with
NaOH. Tomato varieties were H9423 and OX323. Pretreatment dip time and
reaction time were 30 seconds each. Pretreatment temperature was 85°C. NaOH
xiv
dip time and reaction times were 60 seconds each. NaOH temperature was 85°C.
102
Table 17. Tomato lye peeling studies using a chlorinated solvent pretreatment. Tomato
variety was OX303. Pretreatment dip time was 60 seconds and reaction time was
30 seconds. NaOH dip time and reaction times were 60 seconds each. NaOH
temperature was 85°C. 102
Table 18. Tomato lye peeling studies using dimethyl sulfoxide and 2-undecanone as
pretreatments. Pretreatment dip time was 60 seconds. Pretreatment reaction time
was 30 seconds. NaOH dip time and reaction times were 60 seconds each.
NaOH temperature was 85°C. 103
Table 19. Tomato lye peeling studies using dibutyl phthalate and tetradecane as
pretreatments. Tomato variety was H9704. Pretreatment dip time was 60
seconds. Pretreatment reaction time was 30 seconds. NaOH dip time and
reaction times were 60 seconds each. NaOH concentration was 3.00 N. NaOH
temperature was 85°C. 103
Table 20. Tomato lye peeling studies using a solvent mixture of chloroform and
hexanoic acid in the pretreatment. Tomato varieties were H9423 and OX323.
Pretreatment temperature was 80°C. Pretreatment dip time was 60 seconds.
Pretreatment reaction time was 30 seconds. NaOH dip time and reaction times
were 60 seconds each. NaOH temperature is 85°C. 103
Table 21. Physical Properties of the solvents used in lye peeling studies and their PAFA
Status. 104
xv
Table 22. Tomato peeling results for a pretreatment of neat organic solvents with an
alkyl chain length of eight carbons. 112
Table 23. Solubilities of various carboxylic acids in water at 80°C. 113
Table 24. Tomato peel scores for pretreatments with varying concentrations of octanoic
acid in water. 114
Table 25. The effect of carboxylic acids above the solubility point as a uniform layer on
top of the water. Tomato variety was TSH-8. 114
Table 26. The effect of completely dissolved carboxylic acids in water below the
solubility point. Tomato variety is TSH-8. 115
Table 27. Tomato peeling scores for the effect of alkyl chain length on the ketone
functional group. Solvents existed as neat solvents. Tomato variety was TSH-8.
116
Table 28. ANOVA analysis for the 32 experimental design (results presented in Figure
31). 117
Table 29. Multiple comparisons for all averages using Tukey’s test. Significant
differences are represented as plus (+) signs and insignificant differences are
represented as minus (-) signs. Tomato variety was TSH-8. NaOH concentration
was 2.0 N. Octanoic acid and hexanoic acid pretreatments were a uniform layer
of neat solvent on top of the water. 120
Table 30. Data from single factor experiment examining the post pretreatment effect.
Tomato variety was TSH-8. Sodium hydroxide concentration was 4.0 N. Type of
dip refers to a 1 second dip in a neat solvent. 121
xvi
Table 31. ANOVA analysis for single factor experiment examining the post pretreatment
effect of results in Table 30 and Figure 34. 122
Table 32. ANOVA analysis for the residuals of single factor experiment examining the
type of dip. Tomato variety was TSH-8. Sodium hydroxide concentration was
4.0 N. Type of dip refers to a 1 second dip in a neat solvent. 124
xvii
List of Figures
Figure 1. Fresh Market Tomato Consumption in the in the United States During the
Years 1984 to 2005. 3
Figure 2. Different Monosaccharides of Pectin. (A) Demethylated Galacturonoglycan
Monosaccharide, (B) Methylated Galacturonoglycan Monosaccharide, (C)
Galacturonic Acid Monomer. 23
Figure 3. Flow Diagram for laboratory lye peeling experiments. 46
Figure 4. Mechanical tumbler used to remove tomato peels after pretreatment and basic
dips. 47
Figure 5. (a) Average tomato peel score versus vine-ripened age for H9423 tomatoes
peeled in 3.0 N NaOH in 2005 at 85±2°C. (b) Average tomato peel score versus
vine-ripened age for 696 tomatoes peeled in 3.0 N NaOH in 2006 at 80±1°C.
Squares with error bars represent the average score and standard deviation of
three or more tomatoes. Squares without error bars represent the score of a single
tomato or the average of 2 tomatoes. A score of zero indicates no cuticle removal
whereas a score of four indicates over-peeling. 56
Figure 6. Normal probability plot of residuals for the vine-ripened study in 2005.
Tomato variety was H9423. No pretreatment was used. NaOH concentration was
3.0 N. NaOH dip time and reaction time were 45 and 60 seconds, respectively.
NaOH temperature was 85±2°C. 58
Figure 7. Plot of residuals versus average tomato scores for the vine-ripened study in
2005. Tomato variety was H9423. No pretreatment was used. NaOH
xviii
concentration was 3.0 N. NaOH dip time and reaction time were 45 and 60
seconds, respectively. NaOH temperature was 85±2°C. 59
Figure 8. Normal probability plot of residuals for the vine-ripened study in 2006.
Tomato variety was 696. Water pretreatment was used for 60 seconds. NaOH
concentration was 3.0 N. NaOH dip time and reaction time were 60 seconds for
both. Temperatures of water pretreatment and NaOH treatment were 80±1°C.
59
Figure 9. Residuals versus average scores for the vine-ripened study in 2006. Tomato
variety was 696. Water pretreatment was used for 60 seconds. NaOH
concentration was 3.0 N. NaOH dip time and reaction time were 60 seconds for
both. Temperatures of water pretreatment and NaOH treatment were 80±1°C.
60
Figure 10. Average tomato score versus post-harvest age. Each point represents the
average score of 18 tomatoes. Error bars show one standard deviation of each of
the averages. Closed circles represent average scores that are not significantly
different from the control (post-harvest age of 0 days) while open circles represent
average scores that are significantly different from the control. A score of zero
indicates no cuticle removal whereas a score of four indicates over-peeling.
Tomato variety was TSH-8. Pretreatment was water Pretreatment dip time and
reaction were 60 and 30 seconds, respectively. NaOH concentration was 3.0 N.
NaOH dip time and reaction time were 60 seconds for both. Temperature was
80±1°C. 64
xix
Figure 11. Normal probability plot of the residuals from the post-harvest experiment.
Tomato variety was TSH-8. Pretreatment was water Pretreatment dip time and
reaction were 60 and 30 seconds, respectively. NaOH concentration was 3.0 N.
NaOH dip time and reaction time were 60 seconds for both. Temperature was
80±1°C. 65
Figure 12. Average tomato scores versus residuals for the post-harvest experiment.
Tomato variety was TSH-8. Pretreatment was water Pretreatment dip time and
reaction were 60 and 30 seconds, respectively. NaOH concentration was 3.0 N.
NaOH dip time and reaction time were 60 seconds for both. Temperature was
80±1°C. 66
Figure 13. Three dimensional model graph obtained from Design Expert for lye peeling.
Sodium hydroxide concentration was 4.50 N. The score represents the extent of
cuticle removal. Time is in seconds. Temperature is in degrees Celsius. Tomato
variety was TSH-8. Water pretreatment was used with a 30 second reaction time.
NaOH reaction time was 60 seconds. Temperatures and times were adjusted for
both pretreatment and caustic dips. 72
Figure 14. Normal probability plot of the residuals for the 23 factorial design with center
points. Tomato variety was TSH-8. 73
Figure 15. Residuals versus average tomato score for each condition in the 23 factorial
design with center points. Tomato variety was TSH-8. 73
Figure 16. Residuals versus levels of temperature in the 23 factorial design with center
points. Tomato variety was TSH-8. 74
xx
Figure 17. Average score of tomatoes subjected to treatments of various bases. Error
bars represent one standard deviation. Significant differences are observed
between NaOH and NH4OH, KOH and NH4OH, and KOH and the mixture of
KOH and NH4OH. The hydroxide ion concentration was 4.0 N in each case.
Tomato variety was TSH-8. Water was used as a pretreatment for 60 seconds.
Base dip was 60 seconds. All temperatures were 80°C. 77
Figure 18. Normal probability plot of the data from the base experiment. Tomato variety
was TSH-8. Pretreatment was water. Pretreatment dip time and reaction time
were 60 and 30 seconds, respectively. Base concentration was 4.0 N. Base dip
time and reaction time were 60 seconds each. Pretreatment and NaOH
temperatures were 80°C. 79
Figure 19. Average tomato score versus residuals for the base experiment. Tomato
variety was TSH-8. Pretreatment was water. Pretreatment dip time and reaction
time were 60 and 30 seconds, respectively. Base concentration was 4.0 N. Base
dip time and reaction time were 60 seconds each. Pretreatment and NaOH
temperatures were 80°C. 80
Figure 20. Results for an unbalanced, two-factor factorial study between NaOH and an
equimolar mixture of KOH & NH4OH at three different concentrations at
80±1°C. Error bars represent standard deviations. An ANOVA analysis revealed
that no significant differences exist between any treatments. Tomato varieties
were 9423 and TSH-8. Pretreatment was water. Pretreatment dip time and
reaction time were 60 and 30 seconds, respectively. Base dip time and reaction
xxi
time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C.
81
Figure 21. Normal probability plot of the residuals for the two base experiment. Tomato
varieties were 9423 and TSH-8. Pretreatment was water. Pretreatment dip time
and reaction time were 60 and 30 seconds, respectively. Base dip time and
reaction time were 60 seconds each. Pretreatment and NaOH temperatures were
80°C. 83
Figure 22. Average tomato score versus residuals for the two base experiment. Tomato
varieties were 9423 and TSH-8. Pretreatment was water. Pretreatment dip time
and reaction time were 60 and 30 seconds, respectively. Base dip time and
reaction time were 60 seconds each. Pretreatment and NaOH temperatures were
80°C. 83
Figure 23. Solid State 13C CP-MAS NMR pattern for Cellulose I. 88
Figure 24. Solid State 13C CP-MAS NMR pattern for Cellulose II. 88
Figure 25. Powder x-ray diffraction patterns of five standard mixtures of cellulose. (a)
Pure cellulose I in the form of Avicel. (b) 25% cellulose II. (c) 50% cellulose II.
(d) 75% cellulose II. (e) Pure cellulose II. Cellulose II was obtained through
mercerization of cellulose I in 4.25 N NaOH at 65°C for 24 hours. 91
Figure 26. Characteristic ratios versus absolute peak intensities for each of the five
standard cellulose mixtures. The characteristic ratio is defined as the ratio
between the relative peak intensity at 22.5° to relative peak intensity of the
shifting peak between 19 and 20.5°. The trend-line represents the best fit for the
xxii
data. The trend-line equation and R2 value were obtained through Microsoft
Excel. 93
Figure 27. Characteristic ratios versus relative peak intensities for each of the five
standard cellulose mixtures. The characteristic ratio is defined as the ratio
between the relative peak intensity at 22.5° to relative peak intensity of the
shifting peak between 19 and 20.5°. The trend-line represents the best fit for the
data. The trend-line equation and R2 value were obtained through Microsoft
Excel. 93
Figure 28. X-ray diffraction patterns for Cellulose in 3.0 N NaOH at 100°C for 1, 2, and
5 minutes. 95
Figure 29. X-Ray diffraction patterns for Cellulose I at 100°C for extended times.
96
Figure 30. Lye peeling process used to analyze effectiveness of various organic solvents.
110
Figure 31. Average tomato scores for each of the nine conditions evaluated in the 32
Design. Tomato variety was TSH-8. Hexanoic and octanoic acid pretreatments
consisted of a layer of neat organic acid on top of the water. NaOH concentration
was 2.0 N. Error bars represent the standard deviation of five duplicated
responses. 117
Figure 32. Normal probability plot of the residuals for the 3² experiment. 118
Figure 33. Residuals versus average score for each of the nine conditions tested in the 3²
experiment. 118
xxiii
Figure 34. Average tomato peel scores for the single factor experiment with no addition
dip or a hexanoic acid or octanoic acid 1 second dip immediately following the
water pretreatment. 122
Figure 35. Normal probability plot of the residuals from the single factor experiment.
123
Figure 36. Graph of residuals versus average tomato score for the single factor
experiment. 123
Figure 37. Part drawing for x-ray diffraction sample holder. 144
Figure 38. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N NaOH at
60°C for 1, 2, and 5 minutes. 148
Figure 39. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N NaOH at
80°C for 1, 2, and 5 minutes. 148
Figure 40. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N KOH at
60°C for 1, 2, and 5 minutes. 149
Figure 41. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N KOH at
80°C for 1, 2, and 5 minutes. 149
Figure 42. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N KOH at
100°C for 1, 2, and 5 minutes. 150
Figure 43. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N NH4OH at
60°C for 1, 2, and 5 minutes. 150
Figure 44. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N NH4OH at
80°C for 1, 2, and 5 minutes. 151
xxiv
Figure 45. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N NH4OH at
100°C for 1, 2, and 5 minutes. 151
xxv
Chapter One
Introduction
Tomatoes (Lycopersicon esculentum) play a vital role in a healthy diet. Tomatoes
are extremely beneficial to human health for they are rich in folate, potassium, vitamin A
and vitamin C, carotenoids, and flavonoids (Beecher,1997). Many of the carotenoids,
such as lycopene and β-carotene, and flavonoids seem to protect men from various
cardiovascular diseases and different types of cancer (Oshima et al.,1996; Wold et al.,2004). Tomatoes are the primary source of lycopene in many peoples’ diets
(Slimestad & Verheul,2005). Lycopene is responsible for the red color present in the tomato (Heinz,2003; Sabio et al.,2003). The taste of the tomato is attributed to the various organic acids and sugars present (Slimestad & Verheul,2005).
Tomatoes exist in different varieties (i.e. cultivars). Some cultivars may grow to a total weight of 5 to 10 grams whereas other cultivars may grow to 450 grams in size (Ho
& Hewitt,1986). There has been debate whether the tomato is a fruit or a vegetable. The answer is both. It is a fruit because it produces seeds, which is not present in other vegetables such as carrots or celery. Conversely, it is a vegetable because the tomato
1 2 grows on a plant, which is not a characteristic of other fruits such as apples or peaches
(Heinz,2003).
Historically, the tomato originated from South America where it was cultivated by the Aztecs. Then, in the 1500s, the tomato was introduced to Europe by the Spanish after invading the Aztecs. While in Europe, it obtained the reputation of being an aphrodisiac. In Italian, the tomato was referred to as “poma amoris,” and in French, it was denoted as “pomme d’amour,” which means “apple of love” in both languages.
However, the tomato did not receive such success in America, Canada, and Great Britain at first because it was considered poisonous until the nineteenth century (Heinz,2003).
This is most often attributed to the use of pewter to prepare meals in the past—it is thought that the lead in the pewter seeped out into acidic foods, and that this phenomenon was observed exclusively in tomatoes. Though this may be plausible, the account is not likely because the overall amount of lead in pewter (Britannica,2006), rates of lead dissolution, and the nature of lead exposure (Britannica,2006) make this myth unlikely.
More likely, Britains and early Americans avoided the plant because the tomato plant
resembles the poisonous belladonna plant, which belongs to the nightshade family.
However, the tomato finally gained widespread popularity in the United States later in the
19th century and especially in the early 20th century (Britannica,2006).
There are two forms in which tomatoes arrive to the consumer—fresh market tomatoes and processed tomatoes. Fresh market tomatoes are whole tomatoes that are sent directly to the consumer from the plant without any further treatment. Processed
tomatoes are any tomato-based product in which the tomato goes through any unit
operation such as peeling or blanching. Thus, all tomato products that are not whole, 3 fresh market tomatoes are processed tomatoes. Ketchup, catsup, tomato soup, tomato sauce, diced and sliced tomatoes, and salsa are all examples of processed tomatoes.
Tomato consumption in the United States has increased overall during the last twenty years (see Figure 1). In 2005, tomatoes were the fourth most consumed whole fruit behind potatoes (42.8 pounds per capita), lettuce (22.1 pounds per capita), and onions (20.7 pounds per capita). Watermelon was the fifth most consumed fruit in 2005 with 13.0 pounds per capita consumption (USDA,2006).
21.0
20.0
19.0 an i l US vi e
h 18.0 Ci t a n t 17.0 on i i Capi pt 16.0 m
15.0 Consu 14.0 Pounds per
13.0
12.0 1980 1985 1990 1995 2000 2005 Year
Figure 1. Fresh Market Tomato Consumption in the in the United States During the Years 1984 to 2005.
In 2005, the US produced 1.97 million tons of fresh market tomatoes and 10.2 million tons of processed tomatoes. Ohio alone produced 107,250 tons and 175,000 tons of fresh market and processed tomatoes, respectively. Ohio accounts for 5.44% of all fresh market tomatoes and 1.72% of all processed tomatoes. The two states that 4 contribute most to fresh market and processed tomatoes in 2004 were Florida and
California, respectively. Florida accounts for 39.4% of all fresh market tomatoes, and
California is responsible for 94.1% of all processed tomato products in the US
(USDA,2006).
Processed tomatoes must be peeled before they are canned. Canned tomatoes are in the form of whole, half, sliced, and diced tomatoes, including salsa. An understanding of the physiology and chemistry are important so that tomato peeling can be understood at a fundamental level. Tomatoes are peeled industrially by the lye peeling process. Lye peeling involves submerging tomatoes in a 3.0 to 5.0 M NaOH solution at about 93°C for
15 to 40 seconds. The goal of this research is to determine factors that significantly affect lye peeling and changes to current lye peeling processes in order to reduce the amount of NaOH needed while producing peeled tomatoes of high quality. Listed below are the specific objectives that were met in the research.
• Significant processing parameters in lye peeling—the three major processing
parameters in lye peeling are temperature, NaOH concentration, and dip time.
A three factor, two level design with center points was conducted to determine
which parameter(s) produced the greatest changes in lye peeling (Chapter 5).
• Age Studies—effects of vine-ripened and post-harvest age were investigated
(Chapter 4). Vine-ripened age denotes the age (in days) that the tomato has
been left on the plant, and post-harvest age denotes the age (in days) that the
tomato has set off the vine.
• Preliminary Studies—initial lye peeling studies in which various organic
solvents were used are presented (Chapter 7). In these studies, tomato peeling 5 protocols were investigated and developed in 2005, with the aid of Jeremy
Herman, for the research conducted in 2006.
• Solvent studies—additional solvent studies were conducted to determine
which solvents were most effective at peeling tomatoes while reducing the
amount of NaOH required (Chapter 7).
• Mercerization studies—mercerization experiments were conducted on
cellulose I in order to ascertain whether the cellulose I to II conversion can be
accomplished during typical lye peeling processing times (Chapter 6).
Chapter Two
Literature Review
The tomato, is divided in two main sections—the mesocarp and the exocarp. The
mesocarp denotes the fruit flesh while the exocarp denotes the outer skin. This outer skin
is most commonly referred to as the cuticle. Tomato cuticles are a combination of
several layers of waxes, cutin, and a mixture of the two. These waxes are composed of
primarily long chain hydrocarbons, alcohols, and triterpenoids (Bauer et al.,2004; Bauer
et al.,2004). Waxes present anywhere within the cuticle are denoted as cuticular waxes; waxes present only on the surface are denoted as epicuticular waxes. Epicuticular waxes are present in the cuticles of all plants—including fruits and leaves (Martin &
Juniper,1970; Romberger et al.,1993). Cuticular waxes are present throughout the cuticle from the surface to the pectic layer beneath the cutin layer.
Below the outer layer of cuticular waxes lies a layer of saponafiable lipids consisting of cross-linked, long chain hydroxy fatty acids. These compounds serve to facilitate the function of living cells (Solomons & Fryhle,2002). This layer is denoted as cutin, and has also been referred to as the cuticle proper (Matas et al.,2004). The dividing line between the layer of cuticular waxes and the cutin layer is very thin. In
6 7 reality, there is a steady decrease of cuticular waxes and a steady increase of cutin
between the two layers so that some of the waxes are embedded in this cross-linked
matrix of cutin (Das & Barringer,1999). Cutin is cross-linked through polyester linkages.
Individual hydroxy fatty acid polymers within the cutin are commonly referred to as
oligomers.
Immediately below the cutin layer lies another layer in which fibrous and non-
fibrous polysaccharides are present; this layer has been denoted as the cuticular layer
(Matas et al.,2004). The cuticle proper (cutin layer) and the cuticle layer (fibrous and
non-fibrous polysaccharides) combine to form the cuticular membrane (Matas et al.,2004).
Still, below the cutin layer lays a pectin rich layer, which is located above and between the first layers of cells beneath the cuticle. Other studies have denoted these cells as hypodermal cells (Ho & Hewitt,1986), anticlinal cells, or collenchymatous cells
(Floros et al.,1987; Romberger et al.,1993). However, in this paper, they will be referred to as collenchymatous cells. The cuticle ends where these cells begin. Plant cells are different than human cells in that they have cell walls in addition to membranes that are much thicker than human cells (Romberger et al.,1993; Matas et al.,2004). Membranes of plant cells are denoted as plasmalemma, or cell plasma membranes, are composed of phospholipids or proteins. These membranes are very important to the tomato for they aid in keeping the exocarp bound to the mesocarp. Under the first layer of cells lies the hypodermis—or the outer most layer of the mesocarp. The hypodermis has also been referred to as the pericarp (Ho & Hewitt,1986). These cells can interact with other, adjacent cells through plasmodesmata, which are slender channels that allow transport 8 between cells. The cells near the surface are not spherical, but are polyhedrons because
they are in direct contact with other cells (Matas et al.,2004).
Layers of the overall cuticle—from epicuticular waxes to the first layer of cells—
do not exhibit sharp distinctions. Epicuticular waxes found on the surface steadily
decrease into the cuticle. The amount of cutin gradually increases throughout the cuticle, reaches a maximum, and continues to decrease until the hypodermis. About midway through the cuticle, the amount of pectin gradually increases. At the bottom of the cuticle, the amount of polysaccharides steadily increases until the cell wall of collenchymatous cells begin. During the red stage of maturity, there is even less of a distinction between these layers.
The pericarp consists of several layers of the cuticle, the vascular bundles in the outer parts of the mesocarp, and the parenchymatous cells in the tissues lining the locules.
Parenchymatous cells, which make up the parenchymatous tissue of the mesocarp, are similar to collenchymatous cells except that they have a thinner cell walls than collenchymatous cells. Locules are the cavities in which tomato seeds are located, and these cavities are generally filled with jelly-like tissue (Ho & Hewitt,1986).
The parenchymatous pericarp represents the mesocarpic outer boundary of the locules, and the coumella represents the side boundaries of locules further into the tomato. A tomato may have many small, or a few large, locular cavities separated by the coumella and contained by the pericarp.
Parenchymatous and collenchymatous cells differ in the strength of the membrane, but they contain many similarities. Both possess a cytoplasm, which is the jelly-like material that fills the cells, and the cytoplasm consists of the cytosol and 9 organelles. Cytosol is the volume of the cell where metabolism and biochemical
reactions take place. Organelles are particular structures that are suspended in the
cytosol, and fulfill a particular function. Some functions regulated by organelles are digestion of macromolecules (lysosome) and the conversion of ribonucleic acid (rRNA) to messenger ribonucleic acid (mRNA) (ribosomes). Vacuoles, a specific organelle, can capture food and other unwanted structures that enter into the cytoplasm; it can take up as much as 80% of the entire volume of the cytoplasm in some cases (Ho & Hewitt,1986).
Around the vacuole is a membrane referred as the tonoplast. Plastids, another organelle, are responsible for several functions such as photosynthesis, storage of compounds such as starch, and the synthesis of fatty acids and terpenes.
The Tomato Cuticle
Tomato cuticles consist of epicuticular waxes, cuticular waxes, and cutin until the first layer of collenchymatous cells. This cuticle is made up of extremely hydrophobic waxy components. The main function of the cuticle is to protect the fruit by forming a barrier from environmental conditions and keeping water in the fruit (Martin &
Juniper,1970; Luque et al.,1994; Vogg et al.,2004). It is the first barrier to penetration of agrochemicals and other solutes (Stevens & Bukovac,1987; Shafer & Bukovac,1988;
Shafer & Bukovac,1989; Bukovac et al.,1990; Luque et al.,1994). It aids in the protection from microbial attack while the tomato is in the field prior to harvesting (Das
& Barringer,1999; Fang et al.,2001; Bauer et al.,2004). As the tomato ripens, water loss through the cuticle decreases (Luque et al.,1994). When examined in plane polarized light, the cuticle is isotropic (Bukovac et al.,1971). 10 The structure of the cuticle is complex both physically and chemically. At the
surface of the tomato, epicuticular waxes are predominant. These waxes form an
amorphous layer, poor in cellulose, that consists of a mixture of alkanes, alkadienes,
alkatrienes (chain and branched), alkanols, alkenols, amyrins, sterols, and naringenin
chalcone. Analysis of relative amounts of cuticular waxes, hydrolyzable components,
and cutin are 3.93, 20.11, and 75.95% by weight, respectively at the mature green stage
of tomato ripeness; these amounts are 4.81, 22.55, and 72.64% by weight, respectively at the mature red stage. X-ray diffraction of the cuticle reveals that the degree of crystallinity is low (Luque et al.,1994).
The thickness of the cuticle cannot be exactly determined; the range of cuticle thickness lies between 1 and 15 µm. Several studies have attempted to measure the
thickness, and other studies have presented ranges for cuticle thickness. Average cuticle
thickness was found to be 4.5±0.5 µm (Norris,1974), 4-10 µm (Ho & Hewitt,1986), 0.1-
10 µm (Vogg et al.,2004), 13.0±0.9 µm (Edelmann et al.,2005), and 6.7-15.3 µm
(Petracek & Bukovac,1995). A consistent measurement cannot be determined because it
is difficult to ascertain where the cuticle exactly stops and collenchymatous cell walls
begin. In fact, some epicuticular waxes are even present in the cell walls of the tomato
(Das & Barringer,1999).
There are approximately 60 different chemical compounds that are present in
tomato cuticles (Baker et al.,1982; Bauer et al.,2004; Bauer et al.,2004). The most
prevalent compounds are henetriacontane (more commonly referred to as C31), δ-Amyrin,
and naringenin chalcone. For most tomatoes, 2-4 mg of wax covers the entire tomato.
The average amount of wax per unit area is 50 µg/cm2, and the amount of wax can 11 increase during tomato maturity from 27 to 79 µg/cm2 (Bauer et al.,2004; Bauer et al.,2004).
The long hydrocarbons give tomato cuticles polymer-like properties such as a glass transition temperature. A glass transition temperature indicates the temperature at which individual chains can easily move past one another. Below the glass transition temperature, polymer chains are locked in a particular conformation, which make the cuticle rigid. Above the glass transition temperature, however, the individual chains can move freely past one another, thus enabling the cuticle to have more rubbery properties
(McCrum et al.,1997). Differential scanning calorimetry (DSC) reveals a glass transition temperature of -30°C for the entire cuticle, and -47°C for the cutin matrix alone (Luque
& Heredia,1997). This is due to free volume. Cuticles have less free volume than the cutin matrix, and thus, do not have a glass transition temperature as low as for cutin.
Extra free volume in the cutin matrix enables cutin oligomers to be more mobile.
Therefore, lower temperatures are needed to lock all cutin oligomers into a particular conformation. Waxes present in the cuticle tend to make cutin oligomers less mobile, and so the glass transition temperature for tomato cuticles are greater than for the cutin matrix alone. Further results from DSC studies reveal that the cuticle begins to melt at 40
- 50°C. Though cuticular waxes are generally amorphous, the epicuticular waxes are more crystalline than the cuticular waxes below the surface.
Molecules of naringenin chalcone appear and cluster together as the tomato ripens. Clusters of naringenin chalcone can be observed by determining characteristic spacings in cuticles through x-ray diffraction. Characteristic spacings of tomato cuticles were determined to be 0.95 nm, and characteristic spacings of tomato cuticles when 12 naringenin chalcone had been removed was determined to be 0.97 nm. The similarities of these characteristic spacing measurements imply that they are due to the presence of naringenin chalcone. Additionally, organic acids such as benzoic and salicylic acid penetrate into the cuticle through these characteristic spacings (Jeffree,1996).
Tomato cuticles are hydrophobic. This factor can be measured by the use of contact angles. Studies show that the contact angles of water with the cuticle were 69 (±
12°), but when epicuticular waxes were removed, the contact angle increased to 86 (± 6°)
(Bukovac et al.,1971).
The tomato cuticle is a complex mixture of many chemical components that all aid in the tomato’s regulation of water loss and security from microbial attack.
Naringenin Chalcone
Naringenin chalcone is the most predominant chemical component of the cuticle
(Bauer et al.,2004; Bauer et al.,2004; Slimestad & Verheul,2005). Generally, naringenin chalcone belongs to a group of structurally similar compounds known as flavonoids (also known as polyphenol compounds). These compounds have at least two phenyl rings attached by 3 to 6 carbon atoms in a chain. These compounds are believed to aid in antioxidative activity (Slimestad & Verheul,2005), which reduces the aging of cells, induces and/ or blocks certain enzymatic reactions, and reduces the spread of certain tumor cells including leukemia (Le Gall et al.,2003).
Though the amount of naringenin chalcone increases during ripening on the vine, it decreases dramatically as it ripens off the vine (Slimestad & Verheul,2005). The amount of naringenin chalcone decreases once tomatoes enter the mature red stage of 13 development. This was evident in vine-ripened and post-harvest tomatoes. Furthermore,
naringenin chalcone decreases as temperature increases.
Cutin
The components beneath the epicuticular waxes are a structure commonly
referred to as cutin. Cutin is a mixture of high molecular weight, cross-linked, lipid
polyesters consisting of aliphatic acids, which are synthesized via enzymes in the cell
walls. It is the part of the cuticle remaining after all the wax and polysaccharides are
removed (Luque et al.,1994). In addition to cutin, there is another compound known as
cutan. Cutin is connected via ester linkages, which takes place through esterification, and
cutan is linked via ether linkages (Shishiyama et al.,1970; Jeffree,1996; Deshmukh et al.,2003).
There has been some controversy as to the predominant oligomer in cutin. At first, dihydroxyeicosanoic acid was pinpointed as the most abundant component
(Shishiyama et al.,1970), then ω,8-, ω,9-, and ω,10-dihydroxyhexadecanoic acids was identified as primary oligomers (Osman et al.,1995). Most recently, 10,16- dihydroxyhexadecanoic acid was determined to be the most abundant component in cutin
(Deshmukh et al.,2003). Results from Deshmukh et al. (2003) are significant because it has uncovered the fact that there is cross-linking at the alpha carbon next to the carbonyl carbon of the ester linkage. Previous studies have used degradation techniques to separate cutin oligomers, which destroyed cross-links around the alpha carbon. The analytical technique used to detect this oligomer was solid-state NMR, which has been used in the same context to identify individual components of lime cutin (Fang et 14 al.,2001). Other components in cutin are primary alcohols and free fatty acids, as well as
the presence of aromaticity.
Glass transition temperatures of cutin have been measured through DSC. Dry
cutin has a glass transition temperature of 23.7°C, and when the cutin has absorbed 5.5
weight-% of water, the glass transition temperature decreases to 16.3°C (Matas et
al.,2004). The lowering of the glass transition temperature has lowered the fracture
strength of the cutin. Since such a small amount of water absorption has led to a large
difference in glass transition temperature, there are dramatic implications for
physiological changes of the cutin (Matas et al.,2004). For example, a change in
humidity in the environment could have a wide ranging effect on the physiological
structure of the cutin.
Cell Walls
Immediately below the cutin layer lies collenchymatous cell walls.
Collenchymatous cells are defined as the visible section of components of the cell wall
that are deposited around the plasmalemma. The thickness of the cell wall ranges from a
fraction of a micron to several microns thick (Romberger et al.,1993). Three orthogonal
directions are used to characterize the cell wall. If all the measurements are similar in
magnitude, then the cell wall is said to be isodiametric (or cuboidal), but if one measurement is exceptionally longer than the other two, the cell wall is said to be tabular.
The overall structure of the cell wall changes with its function. Structure differs for a cell wall whose main function is to inhibit water transport versus a cell wall whose function is to readily transport solutes to adjacent cells. Cell walls that are still expanding are 15 denoted as primary cell walls, and cell walls that have stopped expanding are called
secondary cell walls (Romberger et al.,1993). Unlike human cells, plant cells expand
(Mackie,1975). One of the driving forces for cell wall expansion is through turgor pressure (Andrews et al.,2000). Primary cell walls expand during the ripening process, and as ripening continues, the secondary cell wall is formed over the primary cell wall.
Primary cell wall expansion decreases as more secondary cell wall components are deposited. Eventually, the process is complete, and the cell wall stops expanding
(Mackie,1975). Primary cell walls can be 0.1 µm thick.
Primary cell walls are important for specific functions of plant growth and control
the overall size and shape of fruit. Components in the primary cell wall are
polysaccharides and glycoproteins. The most prevalent polysaccharide is cellulose and
composes about 20% of all polysaccharides in the primary cell wall. Other
polysaccharides present are auxin; xyloglucan; xylan, which is the most prevalent
hemicellulosic component in the primary cell wall; β-glucan; homogalacturonans;
rhamnogalacturonan I and II; apiogalacturonan; arabinan; galactan; and arabinogalactan;
arabinosyl and galactosyl residues. Another component in primary cell walls is extensin, which is extremely hard to remove. It has been suggested that the isoditryosyl, a phenolic dimer, aids in the structural integrity of extensin. Primary cell walls also contain arabinogalactan proteins, which have structures similar to extensin (McNeil et al.,1984).
Cell walls are composed of two types of components—fibrous and non-fibrous component. Fibrous components are usually cellulose and are the most inert part of the cell wall that is resistant to most chemical treatments; non-fibrous components are 16 usually hemicellulose, which is β 1,4-linked polysaccharides such as glucan, mannan,
and xylan (Romberger et al.,1993). Cellulose has a high degree of crystallinity as compared to hemicellulose (Mackie,1975; Grierson & Kader,1986). Thus, the cell wall can be viewed as highly crystalline β 1,4-linked cellulose I around which non-crystalline hemicellulose is embedded (Atalla et al.,1993). Diameters of cellulose microfibrils range from 5 to 10 nm.
The cellulose in the primary and secondary cell walls aid to the overall strength of
the wall, and the orientation of cellulose microfibrils is unidirectional, which makes the
cell wall anisotropic (Bargel & Neinhuis,2005). These microfibrils link the cuticle to the
mesocarp during development (Holloway,1994). Cellulose is synthesized within terminal
complexes of the plasmalemma, but the exact mechanism is not understood. Water
accounts for 90% of the overall weight of cell walls (Frey-Wyssling,1976).
Hemicelluloses present can be bonded either covalently or through hydrogen bonds to
cellulose microfibrils. This bonding enables the cell wall to appear as one, unified matrix
(Romberger et al.,1993).
Water penetration is difficult through the microfibrils, and penetration of
compounds with molecular weights greater than 15,000 Daltons occurs extremely slowly
(Mackie,1975).
Lignin can also appear in the cell wall as incrustations, which act to increase the
stiffness of the cell wall. Most common units of lignin are coniferyl, syapyl, and
coumaryl alcohols.
The overall structure of the cell wall ensures transport of needed components
through adjacent cells, and aids in the overall size and shape of the tomato. Below this 17 layer of cells lies the first row of parenchymatous cells, whose diameters can be greater
than 500 mm (Romberger et al.,1993). Between the collecnhymatous cell walls is the
middle lamella, which, in the tomato, is primarily composed of pectin.
Cellulose
Cellulose is a naturally occurring polysaccharide in plant cell walls of higher
plants (Dinand et al.,2002), in cotton (Tanczos et al.,2000), and in sugar beets (Dinand et
al.,1999). Research has been done regarding this biopolymer for the past one hundred
years. The basic structure of cellulose has been known for years—it consists of 1,4
linked β-D-glucose units—but there are still disagreements in the exact hydrogen
bonding network of the different forms of cellulose. Primarily, the way cellulose
structure is determined is through X-ray diffraction. X-ray diffraction can identify the
placements of carbon (C) and oxygen (O) atoms, but not hydrogen (H) atoms. In
addition, solid-state 13C CP/MAS NMR have been used to determine its structure
(Nishiyama et al.,2002); and neutron diffraction has also been used as well (Langan et al.,1999; Langan,2005). Neutron diffraction is advantageous because it is able to locate
H atoms. A H bond exists when the distance between the H donor and the O acceptor is less than 2.8 Å, and the acceptor angle is greater than 110° (Langan et al.,1999).
There are five different types of cellulose—Cellulose Iα, Iβ, II, III1, and IV1. In nature, cellulose occurs either in the Iα or Iβ form. Cellulose synthesis occurs at terminal complexes, which can be viewed as small biological machines that produce either the metastable, parallel Cellulose Iα or Iβ form (Nishiyama et al.,2003). 18 Two types of H bonding can be present when speaking of cellulose—intersheet
and intrasheet H bonding. Intersheet H bonding takes place when a H atom of one chain
is interacting with an O atom with another chain. Intrasheet H bonding takes place when
a H atom interacts with an O atom in the same chain. Differences in H bonding lead to
the differences in Iα and Iβ.
Cellulose Iβ naturally exists in tunicin (Halocynthia roretzi), a small sea animal, and its purity is naturally about 90% (Nishiyama et al.,2002; Nishiyama et al.,2003). The hydroxymethyl group is in the tg conformation, and only intrasheet H bonding is present in this allomorph of cellulose (Kroon-Batenburg & Kroon,1997). Cellulose Iβ chains can
also be viewed as individual sheets that do not interact with each other and that stack on
top of one another in a parallel conformation (Langan,2005).
Cellulose Iα naturally occurs at almost complete purity (about 90%) in the fresh
water algae known as Glaucocystis. As with Cellulose Iβ, there is no intersheet H
bonding. The distance of the H bond between hydroxymethyl H atom and the in-chain O
atom of an adjacent glycosyl residue is shorter in Iα than in Iβ. There are also differences in how cellulose microfibrils are lined up in Iα and Iβ. Conversion from Iα and Iβ can be thermally induced at temperatures around 220 - 230°C. Also, at these temperatures, the distances between cellulose sheets increase by 6%. In both forms, van der Waals forces are the only intermolecular forces among sheets. Mechanisms have been proposed for this conversion. At high temperatures, cellulose Iα expands anisotropically in the stacking direction, which decrease van der Waals forces among the chains, thus allowing for chain slippage and the conversion from Iα to Iβ (Nishiyama et al.,2003). 19 Cellulose II consists of an entire H bond network that is more ordered than
Iβ (Nishiyama et al.,2002). This allomorph contains intersheet and intrasheet H bonding.
The length of the microfibrils of cellulose II are shorter and less crystalline than those of
the cellulose I forms (Kim et al.,2006). The conformation of the methoxyl group in the
center chains is tg, and gt in the origin chain (Langan et al.,2001). Cellulose II and III are
stronger than cellulose I due to the extra H bonds present. Furthermore, cellulose II and
III are oriented in the anti-parallel conformation (Dinand et al.,2002; Langan,2005).
Cellulose in plant cells are present in primary cell walls and its presence increases
throughout maturity in secondary cell walls in the collenchymatous cells directly under
the cuticle. It is synthesized in the metastable form from terminal complexes (Langan et
al.,2001), and has a degree of polymerization (DP) between 5 x 103 and 15 x 103. The
DP has been identified from 6,000 to 8,000 (Mackie,1975), and 14,000 (McNeil et al.,1984). These microfibrils extend in one direction over the cell. Around these
microfibrils exist non-crystalline hemicellulose that regulate tertiary structure of cellulose
(Atalla et al.,1993).
Sodium hydroxide (NaOH) has been known to convert the cotton-like cellulose I
to the silk-like cellulose II (Kim et al.,2006); this process is known as mercerization.
Mercerization is an irreversible transition (Kolpak & Blackwell,1976). On the
macroscopic level, the original microfibrils of cellulose I lose their crystalline order, and
take on the structure of a particulate precipitate, which appear as a traditional structure of
a polymer in the coiled state. This process begins at 10 weight-% NaOH and is complete
at 12 weight-%. If the microfibrils are more tightly packed, then a higher NaOH
concentration is needed to bring about the change from I to II. Mercerized cellulose 20 glycosyl residues attain the chair conformation, and it consists of a P2 monoclinic point
group (Langan et al.,2001). Not only can NaOH mercerize cellulose I, it is also a
swelling agent (Dinand et al.,2002). Another swelling agent is liquid ammonia, which
can covert cellulose I to III, but this conversion is reversible (Tanczos et al.,2000).
Mercerization has been studied by other authors as well (Dinand et al.,1996; Dinand et al.,1999).
Alkylammonium hydroxides have been used in mercerization studies.
Particularly, these compounds are quaternary ammonium hydroxides with the general
+ — formula R4N OH . Quaternary ammonium hydroxides were first studied in the 1920s
and 1930s, but their cost at the time did not prove to be a viable application in the
industry. Previous work indicated that the quaternary ammonium hydroxides behaved
similar to other strong bases such as NaOH, LiOH, and KOH (Dehnert & Konig,1925),
which implies that the hydroxyl group is key in mercerizing cellulose. From the 1930s to
the 1950s, the dissolving effects of tetraalkylammonium hydroxides such as
tetraethylammonium hydroxide (TEAH) (Tanczos et al.,2000) were studied. It is
hypothesized that the hydroxyl group of TEAH binds to cellulose and the
tetraalkylammonium group works to swell the cellulose sheets in the stacking direction
(Pasteka,1984). In 2000, tetramethylammonium hydroxide (TMAH) was compared to
NaOH with respect to mercerization. It was found that cotton absorbed twice as much
NaOH than TMAH, which is due to the bulkiness of the TMAH. However, in order to achieve 10% conversion from I to II, 2.8 M TMAH was required whereas 3.3 M NaOH was required. This implies that TMAH is more effective than NaOH because of the bulky cation that works to separate the cellulose sheets (Tanczos et al.,2000). This also 21 sheds light on the mechanism for mercerization. Perhaps the Na+ cation works to swell the cellulose just as the cation in TMAH (Dinand et al.,2002). TMAH has more
applications than just mercerization, for it can depolymerize the cutin as well (Deshmukh
et al.,2003).
Cellulose I structure Valonia cell walls have been studied under varying NaOH
treatments. Electron microscopy results show that the arrangement of fine cellulose
microfibrils in Valonia cell walls lose the ordered arrangement in increasing exposure
times to an 8 N NaOH solution. After 1 minute, much of the original structure could still
be detected; after 5 minutes, much of the structure had been disrupted; after 10 minutes,
only the basic structure of the original cellulose can be detected; and after 30 minutes, the
original structure can no longer be noticed (Kim et al.,2006).
Naturally occurring cellulose in Valonia consists of assemblies (bundles) of
cellulose I microfibrils that are all parallel, and there are assemblies/ bundles that are
adjacent to other bundles that are anti-parallel, but all the cellulose sheets in each bundle
are aligned in the parallel orientation. When mercerized, the parallel sheets expand in the
packing direction, which enables them to “mix” with the other bundles that are anti-
parallel. After sufficient time is allowed for the individual sheets to mix, the cellulose II
form is attained (Kim et al.,2006).
Mercerization (cellulose I to II conversion) can be brought about in organic
solvents. The solvents studied were water, primary and secondary alcohols, DMSO, and
xylene. It was found that mercerization could be completed with reduced NaOH
concentrations in an ethanol system (Mansikkamaki et al.,2005).
22 Pectin
Pectin is a polysaccharide around plant cell walls and is the predominant component of the middle lamella—it makes up approximately one third of the dry weight of plant cell walls (Morris et al.,2000). Pectin possesses a negative charge, which enables it to bind easily with calcium and other divalent cations. A main function of pectin is to add strength to the overall cuticle by forming interactions between the cell wall and cuticle components. In tomatoes, pectin is galacturonic acid joined by α-D (1,4) glycosidic bonds. Generally, pectin denotes any galacturonoglycan that is water soluble
(BeMiller,1986). Pectin is not crystalline, but it does possess structure, and its structure is very important in the ontogeny of the cell wall (Jeffree,1996). Other sugar units, such as rhamnose, arabinose, and galactose can attach to the pectin backbone in a random arrangement. This random arrangement of side chains produces regions that have branches and other regions that do not have any branches. From a molecular point of view, the presence or lack of presence of side branches produce “hairy” and “smooth” regions of pectin (BeMiller,1986).
The amount of pectin is high in immature as well as mature fruit tissues
(Pressey,1986; Jeffree,1996). The main function of pectin is to cement adjacent faces of cell walls together (Constenla & Lozano,2003). Salts produced from pectins are denoted as pectinates, which are water soluble as well.
23
Figure 2. Different Monosaccharides of Pectin. (A) Demethylated Galacturonoglycan Monosaccharide, (B) Methylated Galacturonoglycan Monosaccharide, (C) Galacturonic Acid Monomer.
The physical properties of a pectin solution can be affected in many ways.
Solubility, viscosity, degree of methylation—DM (or degree of esterification—DE) and pH are all functions of the molecular weight. Additionally, the addition of a monovalent cation to a pectin solution will decrease the viscosity of a pectin solution. However, the addition of a di- or trivalent cation to a pectin solution will increase the viscosity through gelation. De-esterification of a pectin solution take place at a pH of 4; the rate of de- esterification increases as the pH increases. The pectin chain can be broken down by acid catalyzed hydrolysis, thus making the degree of polymerization about 25. When a pectin solution (pH between 5 and 6) is heated, the chain can break through beta elimination reactions (BeMiller,1986).
Several enzymes can degrade pectins. Pectinesterase can remove the methyl ester group through hydrolysis. This enzyme attacks entire segments of a pectin chain at once instead of randomly attacking units throughout the polymer chain. Lyases and transeliminases work to depolymerize the pectin chain through beta elimination reactions, which are similar to base catalyzed depolymerization. Polygalacturonases also 24 depolymerize the pectin chain through hydrolysis, but this attacks the glycosidic bonds that join the monosaccharide units together (BeMiller,1986).
Pectins dictate how large the fruit grows and its size. They are also the first polysaccharide present in the primary cell wall during development when the fruit is primarily water. During development, enzymes transfer galactose, galacturonic acid, and arabinose to pectin polymers; their activity increases as the fruit continues to ripen. Once the pectin polymer is formed, they are transported to the primary cell walls. At maturity, pectin undergoes degradation from polygalacturonase (Northcote,1986). Though polygalacturonase is present during the entire life of the tomato, its activity increases 600 fold at maturity as compared to the earlier green stage of development (Pressey,1986).
The carbonyl carbon of a unit of pectin can be methylated or hydrated. All pectin has a variable degree of esterification (DE), which produces the ester group on the monosaccharide. Pectins can be classified as high methoxyl (HM) if the DE is 50% or above, or low methoxyl (LM) if the DE is below 50%. Degree of esterification is defined as a percentage of the number of methyl groups per the total number of monosaccharide units (BeMiller,1986). Commercially, HM pectins are obtained naturally from citrus peel and apple pomace (Morris et al.,2002) and LM pectins are obtained from the de- esterification of HM pectins. Over 50% of all pectin production is for producing jams and jellies (Beach et al.,1986). Kinetic studies have been performed in order to characterize the demethylation process (Constenla & Lozano,2003).
Pectins can also form gels in the presence of a basic solution or a highly ionic solution. Gels form when the pectin chains wrap around solutes. However, this wrapping cannot be too great, or a precipitate would form. Typically, gels are non- 25 Newtonian fluids with pseudoplastic behavior; in fact, the viscosity of some gels might
become so great that gravity-induced fluid flow will completely stop. Gelation, or the process of forming a gel, is favored at high pH. HM pectins gel more slowly than LM pectins due to the presence of the methyl group on the pectin (BeMiller,1986; Wehr et al.,2004).
Research has investigated how sodium hydroxide affects the gelation of pectin.
Gelation does not form below a sodium hydroxide concentration (NaOH) of 0.3 N.
Gelation can also be induced by lyotropic effects. However, inducing gelation of pectins
through lyotropic effects occurs only in LM pectins. If the pectin is highly methoxylated,
then NaOH is needed to induce gelation. For a LM pectin whose DE is 0%, the pectin
can gel in the presence of 0.75 M NaCl or KCl, but when the degree of methylation is
greater than 49%, the pectin can not be gelled by lyotropic effects. Furthermore, the ratio
of the sodium cation (Na+) to galacturonic acid was found to be 0.6, which indicates that
Na+ binds weakly to pectin. Pectins form gels easily with divalent cations such as
Calcium (Ca2+), but these results show that pectins can gel in the presence of an alkali
base due to an electronic charge neutralization and ionic strength effects (Wehr et
al.,2004).
Previous studies have shown that the average molecular weight of HM pectins
decreases through β-elimination as the temperature increases (Morris et al.,2002). The
average molecular weight decreases as the DE decreases; however, the DE, in and of
itself, does not affect the average molecular weight (Morris et al.,2000). These results
suggest that further increases in temperature and/or concentration of an alkali base would 26 further decrease the average molecular weight, and thus, pectin’s ability to function as
glue that binds the cuticle to the fruit.
Growth & Development
Vine-Ripened Development
Many changes take place within the first week after anthesis—or the day the
tomato first buds on the vine. Small vacuoles in individual cells combine to form large
vacuoles, and cells in the tomato divide and multiply (Ho & Hewitt,1986). After these
cells have divided, they no longer multiply, but expand. Some cells can increase as much
as tenfold during development (McNeil et al.,1984). During this first week, the plastids
contain starch, the pericarp contains between 8 and 30 layers of cells, cytoplasm is only a
peripheral layer in the cell, and the placenta accumulates in the locular cavities which
become the jelly-like material later in development. However, during the first week, the
placenta in the locular cavities is quite firm. After the second week following anthesis,
organelles, plasmalemma, and tonoplasts are formed (Ho & Hewitt,1986).
The overall weight of the tomato can dramatically change through its development. The tomato undergoes three main stages of development. In the first stage—2 to 3 weeks after anthesis—the weight increases by only 10%, and the amount of dry matter increases between 30 and 150 milligrams per day. The next stage—3 to 5 weeks—is characterized by rapid growth; the rate of growth reaches a maximum during this period between 20 and 25 days after anthesis. During these initial two stages, the 27 tomato possesses a green color due to the amount of chlorophyll present (Wold et al.,2004). Then, in the final two weeks, the tomato experiences slow growth with respect to its weight, but many metabolic changes take place during this period (Bargel &
Neinhuis,2005). It is during this final stage that the tomato turns from its original green
color, to yellow, to orange, and finally, to red (Ho & Hewitt,1986). It is during this final
stage that various carotenoids such as lycopene, α-, β-, γ-, δ-, and ζ-carotene appear via the HMG-CoA reductase pathway originating with acetyl CoA (Grierson & Kader,1986).
Lycopene and the other carotenoids are continuously synthesized in tomatoes
during ripening; with vine ripened tomatoes, the total lycopene content was 13.20
milligrams per 100 grams of fresh weight and with postharvest ripened tomatoes, the total
lycopene content was 17.62 milligrams per 100 grams of fresh weight (Slimestad &
Verheul,2005). As the tomato ripens, ethylene is also produced (Ridge & Osborne,1970;
Grierson & Kader,1986; Andrews,1995). Ethylene is a compound emitted by all climacteric fruits. Climacteric fruits are defined as fruits that can exchange gases with the environment. It is not known whether respiration induces ethylene production or vice versa. Ethylene is produced and regulated via the ACC synthase activity (Grierson &
Kader,1986).
Throughout development, the pericarp and locules increase in size. The pericarp
increases in size due to auxin activity, but the locules increase in size due to seed
production. The final size of the tomato is affected by the size of the locules, and thus, the number of total seeds inside the fruit (Ho & Hewitt,1986). During the green stage of the tomato, parenchymatous cells have diameters between 300 and 500 µm, and a definite difference can be noted between the middle lamella and cell walls. Chloroplasts, which 28 are particular organelles, are still present in the cytoplasm. The chloroplast contains
chlorophyll and is responsible for photosynthesis in the tomato. DNA, RNA, and
ribosomes are synthesized in this organelle. The chloroplasts are replaced by
chromoplasts during the mature stage. Ripening, by and large, is a process that
eventually leads to cell death, but organelles and plasmodesmata continue to remain alive
during this stage (Grierson & Kader,1986).
Peroxidase is thought to activate wall-thickening reactions. These wall-
thickening reactions have implications on the overall mechanical strength of the tomato
cuticle. Molecular weights of these enzymes were found to be 58 kDa in immature
tomatoes, and an average of 48.3 kDa in mature tomatoes (Andrews et al.,2000). Fruit
growth was believed to be primarily due to turgor pressure, which is defined as differential elasticities between inner and outer tissue layers of a fruit (Thompson et al.,1998). Measurements of turgor pressure as well as peroxidase activity have been taken in order to better understand the ripening process of the tomato (Thompson et al.,1998). Results show that cell wall-bound peroxidase activity increases sharply between 40 and 45 days after anthesis, which is the final stage of development in which fruit growth ceases (Ho & Hewitt,1986; Thompson et al.,1998). Furthermore, it is important to note that peroxidase activity is located entirely in the cell wall of the fruit— not free peroxidase. Free peroxidase is present even in immature fruit, but is not located and active within the cell walls (Andrews et al.,2000). Peroxidase that is not located in the cell walls is ineffective (Andrews et al.,2002). Turgor pressure is present during all stages of development—from anthesis to maturity. Thus, turgor pressure does not exclusively control fruit growth. Therefore, the regulation of fruit growth is determined 29 almost entirely by the cuticle and peroxidase activity (Thompson et al.,1998; Andrews et
al.,2002).
Mechanical changes in the cuticle have also been monitored. Tensile properties
have been determined for the cutin matrix and the layer underneath the cutin, also known
as the fruit skin. Results found that the layer under cutin was stronger than cutin because
the fruit skin contains the highly crystalline cellulose. Cuticular membranes possess
phenolic components at maturity, which occupy free volume in the cutin matrix, which
reduce the rigidity of the membrane (Bargel & Neinhuis,2005). Peroxidase is not
responsible for aromatic deposition in the cuticle, but it could be responsible for forming
cross-links in the cell walls (Andrews et al.,2002). Hydration also effects the mechanical
strength of the cuticle as well (Edelmann et al.,2005). The main contributor to the
structural integrity of the cuticle is through non-polar interactions between cuticle components and cellulose in the secondary collenchymatous cell walls (Bargel &
Neinhuis,2005).
Experiments reveal that the strain at failure of the cuticular membrane and fruit skin decrease as maturity increases. For mature red tomatoes, the strain at failure is 4%, which agrees with another study that identified the range of failure for the cuticular membrane between 3 and 5% (Petracek & Bukovac,1995). Cell wall degrading enzymes play an important role in the strength of the cuticle. At maturity, the activity of cell wall degrading enzymes increases (Grierson & Kader,1986; Edelmann et al.,2005).
Cuticle thickness also increases as maturity increases, which is reflected by the amount of cuticular and epicuticular waxes present (Baker et al.,1982; Bauer et al.,2004). 30 Also, changes in cutin are observed through development. The cutin structure becomes more rigid during development (Benitez et al.,2004).
Post-Harvest Development
Most work conducted in post-harvest tomatoes is aimed at increasing shelf life or the nutritive aspects of tomatoes by altering temperature and/or chemical treatment.
Post-harvest tomato quality (Ketelaere et al.,2003), and common diseases that affect post- harvest tomatoes (Cooper et al.,1998) have been investigated. Several metrics are used to characterize post-harvest tomatoes; these include the amount of lycopene, β-carotene, or ascorbic acid (antioxidant capacity), total phenolic content, weight loss, titratable acid content, the total amount of soluble solids, flesh firmness, and skin firmness.
Antioxidant capacity has been studied in both vine and post-harvest ripened tomatoes. Studies show that antioxidant capacity continually increases for both vine and post-harvest ripened tomatoes throughout development until over ripening is observed.
However, the total antioxidant capacity in post-harvest ripened tomatoes was greater than in vine ripened tomatoes (Giovanelli et al.,1999; Giovanelli et al.,2001; Wold et al.,2004). As a general rule, the increase of ascorbic acid in post-harvest tomatoes is dependent upon when the tomato is picked (Hooda et al.,1994).
Temperature and time can effect antioxidant capacity as well. Results show that the amount of lycopene is significantly less for tomatoes stored at room temperature than at 5°C (Javanmardi & Kubota,2006), and highest amounts of lycopene were observed when post-harvest tomatoes were stored at 20°C rather than at 30° or 35°C. This is due 31 to the reaction kinetics of the series reactions which produce lycopene and then β- carotene (Hamauzu et al.,1998).
Research has been conducted regarding the other measured quantities for post-
harvest tomatoes. The total acidity decreases for post-harvest tomatoes over time for
post-harvest and vine ripened tomatoes (Hooda et al.,1994). However, there is no
significant difference in total acidity, or vitamin C content, between post-harvest and vine
ripened tomatoes at any given time (Wold et al.,2004). Weight loss in post-harvest
tomatoes decreases with decreasing temperature (Garcia et al.,1995; Javanmardi &
Kubota,2006). Temperature does not affect the total soluble solids (TSS) in post-harvest
tomatoes (Garcia et al.,1995). However, the total amount of TSS increases over time for
post-harvest tomatoes (Hooda et al.,1994). Therefore, the healthiest tomatoes were those
that were picked at the mature green stage and allowed to ripen at room temperature or a few degrees cooler.
Chemically treating tomatoes is another way to lengthen shelf life or to produce
high quality tomatoes. Calcium chloride (CaCl2) and heat were used as treatments for post-harvest tomatoes to determine the effect upon cuticle firmness, flesh firmness, TSS, and titratable acids. The calcium ion has benefits for the tomato such as maintaining the function of membranes, binding to pectin in the middle lamella to strengthen cell walls, and regulating protein phosphorylation during the immature and mature green stage of development. High skin firmness was measured in tomatoes stored at 8°C, and low firmness was measure for those stored at 20°C. Significant weight loss was observed in
tomatoes treated with CaCl2. The TSS was greater in tomatoes treated with CaCl2 than those without the treatment. There was no significant difference in titratable acids 32
between tomatoes with and without a CaCl2 treatment, but the total titratable acids were affected by temperature. Higher temperature treatments produce tomatoes with higher pH (Garcia et al.,1995). No information on how CaCl2 affects antioxidant capacity was investigated.
Enzymatic activity of tomatoes was shown to be temperature dependent. At a temperature of 45°C, enzymatic activity significantly decreased, but activity was the same for both room temperature and cold temperatures (Boukobza & Taylor,2002).
Placing tomatoes in an oxygen deficient environment to either speed up or inhibit post-harvest ripening is a technique that has been studied. The chemical component displacing oxygen determines whether ripening is accelerated or reduced. Tomatoes that were placed ethylene, 2,4,6-trichlorophenoxyacetic acid, and ammonium thiocyanate sped up ripening. Ethylene possessed the capability to speed up ripening without
adversely affecting the taste (Kader et al.,1978). 2,4,6-Trichlorophenoxyacetic acid, and ammonium thiocyanate reduced the ripening time between one and four days
(Hartman,1959). Carbon dioxide has been used to enhance shelf-life of post-harvest tomatoes (Islam et al.,1995). Carbon dioxide has also been shown to decrease ethylene production in post-harvest tomatoes (Zamponi et al.,1990). Other attempts to lengthen
the shelf-life of tomatoes is through the use of gamma irradiation (El-Sayed,1978),
treatment with 1-methylcyclopropene (Opiyo & Tie-Jin,2005; Ergun et al.,2006),
polymeric films to ensure good quality (Kawada,1982), and exposing tomatoes to ethanol
vapor (Hong et al.,1995; Yanuriati et al.,1996). Furthermore, ethanol vapors can inhibit
ripening when tomatoes are picked at the mature green, breaker, and light red stage of
development without affecting taste in a negative way (Saltveit & Sharaf,1992). 33 Increasing the shelf-life of post-harvest tomatoes is not only beneficial to
suppliers, but the consumer as well. Excess amounts of antioxidants produced in post-
harvest tomatoes show this. The healthiest tomatoes are obtained when they are allowed
ripen off the vine at temperatures slightly less than room temperature.
Tomato Peeling
Two methods are commonly used to peel tomatoes commercially—lye and steam
peeling. Lye peeling involves submerging tomatoes in a 3.0 to 5.0 N solution of NaOH for a short period of time (i.e. 15 to 40 seconds) at about 90°C. After submerging tomatoes, the cuticle can easily be removed either with rubber discs or with jets of water in a rotating cylinder. Steam peeling places tomatoes in a chamber with low pressure steam between 24 – 27 psig (~127 °C) between 15 and 45 seconds. Once the chamber reaches saturation pressure, it is opened and the pressure instantly is brought back to atmospheric. The instant temperature and pressure differential created by flashing the chamber to atmospheric pressure is enough to rupture collenchymatous cells directly under the cuticle (Barringer,2004). In the home, tomatoes are peeled using boiling water.
Hot water peeling and steam peeling are similar since heat is used to enhance peeling.
However, hot water peeling is not as effective as lye or steam peeling at the industrial level. Studies show that peeling tomatoes using boiling water reduces manganese content by about 32% (Dugo et al.,2005). Though the different commercial peeling operations are effective methods of removing the cuticle, it often reduces the nutritive content compared to fresh market tomatoes (Saldana et al.,1978). A critical analysis of lye, steam, and infrared peeling was made in order to determine which method was most 34 effective and most economical. Lye peeling was found to be the most efficient and
economical method (Schulte,1965).
Three other less well known methods that are used to peel tomatoes are calcium
chloride (CaCl2), infrared peeling, and liquid nitrogen peeling. Calcium chloride peeling involves heating a 42 weight-% solution of CaCl2 to its boiling point (121.1 °C) and submerging tomatoes in this solution for a given amount of time (Stephens et al.,1973).
Infrared peeling involves placing the tomato in the presence of infrared radiation, which rapidly raises the temperature so that the cuticle can be removed. Liquid nitrogen peeling has also been used to peel tomatoes as well, but is not used commercially. The first step involves submerging the tomato in liquid nitrogen and then submerging it into warm water; the warm water acts to activate cell wall degrading enzymes more quickly in order to cleave the cuticle from the mesocarp.
Lye Peeling
Lye peeling is the most widely used peeling method in the US Midwest. This method originated in the US in the early 20th century. Before lye peeling, tomatoes were peeled with a knife, which led to significant labor costs and fruit losses. It is believed that lye peeling had its first application to the peeling of hominy. At that time, dilute lye solutions were generated by dissolving the leachings of wood ashes in water. Once the solution was made, it was brought up to a boil, and corn was added and left in the solution until the cuticles of the kernels could be removed by water. The first patent for lye peeling appeared in 1901 applied to the drying of prunes. The purpose was not to remove the cuticle of the prune, but to score it so that the prune could be dried easily. 35 Later, lye peeling was applied to the peeling of peaches. Shortly thereafter, lye peeling was applied to peeling tomatoes (Cruess,1958).
Lye peeling consists of many steps. Tomatoes are first washed to remove dirt and
dust from the fields. Second, they are sorted and graded for product quality; rotten,
discolored, and immature tomatoes are discarded during this step. Next, tomatoes are
submerged in the lye solution for 15 to 40 seconds. Lye concentrations are continuously
adjusted in order to peel all the tomatoes by adding small amounts of 50 weight-% NaOH to a peeling tank. Tomatoes most resistant to lye peeling govern the overall
concentration of NaOH to ensure all are peeled. Concentrations are measured by
electrical conductivity (Cruess,1958). After peeling, the tomatoes are sent through a device known as a tumbler, which mechanically removes tomato cuticles with rotating rubber discs or water jets. Overpeeled, underpeeled, or unhealthy tomatoes missed in the first sorting are discarded in this step before being sent to canning. Finally, those tomatoes that pass the first and second sorting are sent to be transformed into the appropriate form—dices, slices, halves, etc. (Barringer,2004).
Scanning electron microscopy (SEM) analysis is an analytical technique used to study cuticles under varying treatments in lye peeling. Temperatures in lye peeling are not as high as in steam peeling, but are higher than the glass transition temperature of the cuticular components (Eckl & Gruler,1980; Luque & Heredia,1997). This enables better diffusion of components through the cuticle to collenchymatous cell walls. As lye treatments became more severe, the outlines of the cell walls, observed using SEM analysis, became more prevalent through cuticular waxes, indicating cuticular wax dissolution (Floros et al.,1987). Furthermore, it is believed that the hydroxide ion aids in 36 the depolymerization of the cutin matrix since potassium hydroxide (KOH) has been
found to depolymerize this matrix (Holloway,1982). With respect to the cell wall and
middle lamella, the hydroxide ion is believed to remove the hemicellulosic components
while leaving the cellulose in an empty matrix (Floros et al.,1987). Studies show that
KOH in the presence of chelating agents can solubilize the hemicelluloses in the cell wall
(McNeil et al.,1984). These degrading effects—depolymerization and solubilizing hemicellulosic components—aid in destroying collenchymatous cells, thus removing the cuticle from the mesocarp. SEM Analysis also reveals that parenchymatous cells are destroyed much more rapidly than collenchymatous cells.
Several factors that inhibit lye peeling are the hydrophobic cuticle, the pectin in
the middle lamella, and the hemicellulosic components in the cell wall. If these
substances are present in large quantities, then lye peeling is severly reduced.
Optimization studies for lye peeling suggest the best condition for lye peeling is
submerging tomatoes in a 9 weight-% NaOH solution at 80°C for two minutes. This
operating condition allows for adequate peel removal with minimal fruit loss (Floros et al.,1987).
Sodium hydroxide diffusivity coefficients across the cuticle were evaluated for various NaOH concentrations and temperatures. Results show that the diffusivity coefficient is constant above NaOH concentration of 2 M, which agrees with Fick’s Law of Diffusion, and follows an Arrhenius-type behavior with respect to temperature. At temperatures greater than 50°C, diffusivity coefficients could be measured, but below
30°C, diffusion was virtually nonexistent. It was found that diffusivity coefficients were around 1.2 x 10-8 cm2/s (Floros & Chinnan,1989; Floros & Chinnan,1990). 37
Wastes from Lye Peeling
There is much waste produced from this process—wastewater and waste peels.
The wastewater must be neutralized before being sent to municipal sewage plants. The
peels have excessive amounts of sodium due to the high concentrations of NaOH, but
they also have lycopene as well as proteins. Tomato peels were used as chicken feed in order to transfer the lycopene from the peels to the egg yolk in order to make it more nutritious. However, results show that only about 0.1% of the lycopene was actually transmitted to the egg yolks (Knoblich et al.,2005).
Tomato peels could also be used for biomass. They contain large amounts of carbon, nitrogen, and oxygen, and very small amounts of sulfur. Tomato peels, tomato seeds, and a mixture of seeds and peels have heating values of 22.1, 24.3, and 22.2
MJ/kg, respectively. This exceeds that of other biomass residues such as grass, corn cob, or hazelnut shells. Though this might sound promising, there are processing problems.
Biomass tends to degrade relatively quickly, which means that the biomass must quickly be converted into fuel shortly after the residue is produced. Tomato processors in the mid-western US generate tomato waste about five weeks of the year. Thus, in order to
convert the tomato peels, seeds, and seeds and peels into biofuels, the process must
operate only when tomatoes are being peeled (Mangut et al.,2006).
38 Steam Peeling
Two scenarios were studied to ascertain the mechanism(s) of single and multistage tomato steam peeling. Single stage steam peeling involved placing tomatoes in a chamber, then adding steam. This increases the temperature and pressure for a given period of time. Multistage peeling involved alternating the pressure and temperature from high to low in a chamber for a number of cycles. The alternating cycles of high and low pressure and temperature aided in cuticle removal. In the single stage process, scanning electron microscopy (SEM) analysis uncovered cytoplasm damage with increasing time. At 15 seconds, there was some cytoplasm damage, but it was located in the collenchymatous cells. After 24 seconds, this damage was noted in cells in both the exocarp and mesocarp; and at 45 seconds, there was extensive cytoplasm coagulation in the exocarp and mesocarp as well as collenchymatous cell wall degradation that led to cell rupture in the exocarp. For the multistage process, cellular damage was located primarily in the exocarp (Floros & Chinnan,1988).
Higher magnifications revealed that there are no distinctions between the cytoplasm, cell wall, and middle lamella before steam treatment. With increasingly harsh steam treatments, these components began to separate. Previous studies reveal that the hemicelluloses in the cell wall can be broken down in the presence of high temperature
(Floros & Chinnan,1988).
Studies in steam peeling enable a mechanism to be proposed. When tomatoes are in the chamber and steam is introduced, the temperature and pressure inside the chamber can increase, which increases the temperature and pressure of collenchymatous cells.
During this initial increase, no cellular damage is observed because pressures and 39 temperatures inside and outside tomatoes are the same. When the chamber is opened and
the pressure instantly decreases , there is extensive cell damage due to the large pressure
differential between the collenchymatous cells and surroundings. This pressure differential enabled collenchymatous cell walls to be easily ruptured (Floros &
Chinnan,1988).
A mathematical analysis was performed on steam peeling using Fourier’s Law of
Heat Transfer for single stage and multistage steam peeling. This analysis shows that the damaging effects of multistage steam peeling can be localized entirely in the exocarp.
Furthermore, a mathematical model has been applied to lye peeling using Fick’s Law of
Mass Transfer, which states that lye peeling is theoretically more effective as a multistage
process, which was presented at Society of Agricultural Research in 1986 (Floros &
Chinnan,1988).
Research in Lye Peeling
Much research in the area of lye peeling has been aimed at mitigating the lye
peeling process in order to make it more efficient. A previous study analyzed over 50
organic solvents including carboxylic acids, esters, phosphate and fluoro containing
compounds, chloroform, and surfactants. Of all these compounds, the best pretreatment
to lye peeling was a 1% (by mass) C6 to C8 aqueous, fatty acid mixture, with the most
effective fatty acid being octanoic acid. The next best pretreatment was a 0.1% (by mass)
solution of trimethyl nonanol (Neumann et al.,1978).
Another study subjected tomatoes to a pretreatment of various organic solvents
such as hexane, butane, butanol, THF, chloroform, ethyl acetate, dioxane, acetonitrile, 40 isopropanol, butanone, and water. Chloroform was found to be the most effective solvent
as a pretreatment to lye peeling as it was able to reduce the thickness of the cuticle by
more than half from 16.5 to 6.7 µm (Das & Barringer,1999). Chloroform is effective because it can extract surface epicuticular waxes from the tomato. Previous work has shown that chloroform can extract approximately 5.8% of the dry weight of cuticular waxes (Bukovac et al.,1971). Chlorinated solvents are used in food production today, as in the production of decaffeinated coffee. Also, chilled methylene chloride has been
proposed as a replacement for NaOH, which is similar to liquid nitrogen peeling (Valle-
Riestra,1974). However, chlorinated solvents can often be detrimental to human health.
In addition to solvent pre-treatments, substitutive chemicals for NaOH have been proposed such as potassium hydroxide (KOH). Potassium hydroxide was found to be more effective than NaOH; in fact, in order to achieve the same peeling results with
KOH, only two-thirds of the concentration of NaOH is needed (Das & Barringer,2006).
A theory for KOH’s greater effectiveness is due to its stronger alkalinity. The pKa of
NaOH is 14.77 whereas the pKa of KOH is 16 (Albert & Serjeant,1971). One may expect
calcium hydroxide (Ca(OH)2) to be another good substitute for its pKa is 12.9 (Albert &
Serjeant,1971) but its low solubility in water limits its utility (Das & Barringer,2006).
Surfactants
Surfactants were proposed as pretreatments to lye peeling, but not all surfactants
are effective (Neumann et al.,1978). In agrochemicals, surfactants often aid in
transporting/ penetrating an active ingredient through a plant or fruit cuticle (Fader &
Bukovac,1997). Surfactant studies are aimed at discovering surfactant—cuticle—active 41 ingredient interactions. These studies have investigated surfactants in a purely
agricultural context. Penetration through cuticles is also denoted as foliar penetration.
Foliar penetration through leaves of Phaseolus vulgaris has been studied
extensively (Sargent & Blackman,1962; Sargent & Blackman,1963; Sargent &
Blackman,1969; Sargent & Blackman,1969; Sargent et al.,1969; Sargent &
Blackman,1970; Sargent & Blackman,1970). However, results from studies in leaves of
Phaseolus vulgaris cannot be directly applied to tomato cuticles (Bukovac et al.,1971).
Benzaldenine is an active ingredient, whoe penetration through tomato cuticles
has been studied. Results show that the surfactant Triton X-100 increases absorption of
benzyladenine into the cuticle 1.5 – 3.0 fold (Petracek et al.,1998). The cuticle has a
large impact on inhibiting the penetration of agrochemicals or surfactants (Baker,1980;
Bukovac et al.,1990). More recently, studies have investigated penetration of 2-(1-
Naphthyl)acetic acid (NAA) through tomato cuticles in order to more fully characterize
foliar penetration (Shafer & Bukovac,1989; Knoche & Bukovac,2000; Knoche &
Bukovac,2001).
Surfactants contribute to the reduction of the elasticity of the tomato cuticle,
which causes it to fracture more easily (Petracek & Bukovac,1995). This phenomenon is
surprising because one might intuitively assume that surfactants would have the same
effects as water. It is known that surfactants aid in cuticle permeability (Petracek &
Bukovac,1995) and permeability means greater diffusion capabilities due to formation of free volume (Rogers & Sternberg,1971).
Chlorination of a surfactant aids in the penetration of the active ingredient. As the chlorination of phenoxyacetic acid increases, penetration of the active ingredient 42 increases 2 to 11 fold through the cuticle. However, chlorination of benzoic acid reduces
the penetration through the cuticle (Bukovac et al.,1971). The results for phenoxyacetic acid agree with previous studies where lipid solubility increased. In contrast, chlorination of benzoic acid lowers the pKa value of its carboxylic acid group (Sargent et al.,1969).
Lowering the pKa increases dissocation and penetration since organic acid penetration
into stems and leaves is maximized in the undissociated state (Bukovac et al.,1971).
Octylphenol (OP) is another chemical that has been used to study foliar
adsorption because it is a highly active compound with the number of ethoxy (EO)
groups attached to OP affecting penetration (Stevens & Bukovac,1987; Stevens &
Bukovac,1987; Shafer & Bukovac,1988; Shafer & Bukovac,1989; Shafer et al.,1989;
Shafer & Bukovac,1991; Fader & Bukovac,1997).
Foliar absorption was monitored with different surfactants in which the number of
EO groups attached to OP varied. Maximum absorption was observed when n was 5 or
7.5 (Shafer & Bukovac,1991). If the number of EO groups exceeded 9.5 or was lower
than 5, absorption was almost negligible (Shafer & Bukovac,1988). These results agree with other studies, which also pinpoint the optimum value for n to be 5 or 7.5 (Shafer &
Bukovac,1989; Bukovac et al.,1990).
Critical micelle concentration (CMC) also plays a role in the penetration of the
active ingredient. As the concentration of OP + 9.5 EO increases below the CMC,
penetration increases, but as the concentration increases above the CMC, penetration is
severely limited (Shafer & Bukovac,1989). These results point to micelle solubilization.
Micelle solubilization is defined as solubilizing the active ingredient in the micelles
formed by the surfactant. This phenomena decreases the driving force for penetration 43 because it decreases the bulk concentration of the active ingredient (Shafer &
Bukovac,1991; Heredia & Bukovac,1992).
Common industrial surfactants are the Triton X series of surfactants. All these
surfactants are octylphenols (OP) with a different number of ethoxy (EO) groups attached
to the octylphenol group. Thus, the general formula for a Triton X series surfactant is
OP+nEO.
With respect to lye peeling, surfactants may solubilize epicuticular waxes through micellular solubilization or they may improve penetration of NaOH through the cuticle so that it can interact with pectin and collenchymatous cell walls. Another hypothesized mechanism is that surfactants may increase penetration through the rearrangement of the cross-linking in the cutin layer (Kannan,1969).
Research Goals
In this study, the primary focus was placed on tomato peeling using solvent pretreatments. Initial preliminary studies served to find an effective solvent as a pretreatment to lye peeling. Solvents were selected through the use of a Robbins Chart
(Robbins,1980; Frank et al.,1999), which displays classes of solutes and solvents and predicts whether interactions between solutes and solvents are favorable or not (see
Appendix A). If the solute-solvent interaction obeys ideal solution behavior, then the interaction is assigned “0.” If the interaction is repulsive in reality, then it is assigned a plus sign. Similarly, if the interaction is attractive, then it is assigned a minus sign. Thus, the Robbins Chart was used to identify functional groups in solvents that may display attractive interactions with solute functional groups found in tomato cuticles. Results 44 from preliminary studies revealed that octanoic acid was an effective pretreatment to lye
peeling, which agreed with previous studies (Neumann et al.,1978). Compounds that had eight carbons and alternative functional groups were screened.
In addition to favorable solute-solvent interactions, other parameters such as cost and safety of the solvent were considered. To quantify the safety of the solvent, only chemicals Generally Regarded as Safe (GRAS) were selected. Solvents found on the US
FDA (United States Food and Drug Administration) database of EAFUS (Everything
Added to Food in the United States) were chosen (FDA,2006).
Mercerization studies on the conversion from cellulose I to II were studied to determine if this is a possible transition in lye peeling. During mercerization, the structure swells (Tanczos et al.,2000) as it is altered from cellulose I to cellulose II
(Langan,2005). This swelling could foster increased NaOH diffusion into collenchymatous cells. Thus, studies of cellulose structure before and after treatments with NaOH and other caustic solutions, such as ammonium hydroxide or KOH, were performed in an attempt examine the concentration and temperature dependency of the cellulose transition. Cellulose I to II transition can be observed through x-ray diffraction
(XRD) measurements.
Completing these objectives may lead to a better understanding of the mechanisms for lye peeling, and identify alternative peeling aids.
Chapter Three
Experimental Procedures: Lye Peeling Protocols
It was the goal of the researcher to match the industrial scale lye peeling process in the laboratory. The various parts of the lye peeling process were inspection, a pretreatment dip, a pretreatment reaction time, a basic dip, a basic reaction time, tumbling, and scoring. Inspection involves assessing tomatoes for damage or disease.
All tomatoes that are damaged or diseased were discarded. Pretreatment and basic dips involved submerging tomatoes in a pretreatment (i.e. water or an organic solvent) or basic solution (i.e. NaOH or KOH). The pretreatment and basic reaction times enabled a chemical (pretreatment or base) to act upon the tomato cuticle for a given amount of time.
Tumbling is the mechanical process used to remove the peels. Tomatoes are placed in a cone-shaped device with water and allowed to rotate; the friction between the cone’s walls and cuticle provides for cuticle removal. After tumbling, tomatoes are scored based upon how much of the cuticle has been removed. A numerical value between 0 and 4 is given. If there is no cuticle removal, the tomato is given a score of 0, and if the tomato has been overpeeled, then it receives a score of 4. Each part of the lye peeling process is described in more detail later in this chapter. However, data from preliminary studies
45 46 (Chapter 7) deviated slightly in that tomato scores ranged from 0 to 3. Completely peeled and overpeeled tomatoes both received a score of 3.
Tomatoes Brought from Field
Washing / Inspection— damaged or diseased Damaged Tomatoes Discarded tomatoes were discarded
Good Tomatoes—used for lye peeling experiments
Pretreatment Dip, 80-85°C for 60 seconds a
Pretreatment Reaction Time for 30 seconds a
Basic Treatment (NaOH, KOH, NH4OH), 80-85°C for 60 seconds
Basic Reaction Time for 60 seconds
Cuticle Removal by Mechanical Tumbler
Tomatoes are Scored
Figure 3. Flow Diagram for laboratory lye peeling experiments. a – This step was not included in some preliminary 2005 experiments except when a pure organic solvent was used as a pretreatment.
47 Equipment
Solutions for lye peeling were heated in stainless-steel, 2000 mL beakers using constant temperature water baths (Precision Scientific Co., model #6606). Stainless steel mesh cylindrical baskets, 11.1 cm in diameter and 14.0 cm deep (Pronto Products, model
#980734) were modified by the addition of a stainless steel flip top lid to hold tomatoes submerged in the lye solutions. The tumbler, a hard coating panning machine, consisted of a stainless steel cone, 30.48 cm diameter at the base with an opening of 15.24 cm at the opposite end and 58 cm in length. This cone was mounted on a base in the horizontal position with a handle attached, which allowed it to be spun manually (see Figure 2). Six cylindrical stainless-steel bars 0.64 cm in diameter and 27.3 cm in length were welded on the interior of the cone equidistant from each other to provide a source of abrasion.
Figure 4. Mechanical tumbler used to remove tomato peels after pretreatment and basic dips.
48 Chemical Reagents
All NaOH solutions were prepared in the lab from solid NaOH pellets (Fisher
Scientific, #S318-5). The dissolution of NaOH in water is a very exothermic process
(enthalpy of dissolution is 42.59 kJ/gmol (Perry et al.,1997)). Solutions were cooled to room temperature for final preparation in volumetric flasks. Similarly, potassium hydroxide (KOH) solutions were prepared from KOH pellets (Fisher Scientific, #P250).
Ammonium hydroxide (NH4OH) was prepared from a 14.1 N solution (Fisher Scientific,
#A669s-212). Sodium chloride (Fisher Scientific, #S271) was used to prepare solutions
of salt near its solubility point.
Organic solvents sources and purities that were used in tomato peeling studies
were as follows: chloroform, (Acros Organics, stabilized, 99+%, #15821), 2-
chloropropionic acid (Acros Organics, 95%, #29576), cyclohexanol, (Acros Organics,
98%, #14768), decanoic acid (Sigma-Aldrich, 96%, D165-3), 1-decene (Sigma Aldrich,
94%, #D-180-7), dibutyl phthalate (Acros Organics, 99%, #16660), dichloroethane
(Acros Organics, 99.8%, #11336), dichloromethane (Acros Organics, ACS reagent,
99.5%, #40692), dimethyl sulfoxide (Fisher Scientific, >99%, #D128), hexanoic acid
(Acros Organics, 98%, #12070), octanoic acid (Acros Organics, 99%, #12939), 1-octanol
(Acros Organics, 98%, #15063), tetradecane (Acros Organics, 99%, #17413), and 2-
undecanone (Arcos Organics, 97.5%, #29938).
49
Tomato Peeling
Roma tomatoes of cultivars H9704, H9423, 611, OX323, 696, 9423, 9601, and
TSH-8 were used for testing. Tomatoes were grown throughout northwest Ohio between
August 2005 and October 2005, and August 2006 and October 2006.
Tomato Selection
Tomatoes delivered from the fields were sorted and rinsed free of dirt and debris.
The tomatoes were rinsed with cool tap water and placed into plastic trays where they were allowed to drip and air dry. During this process, tomatoes were removed from the trays individually and checked visually for physical imperfections. An imperfection was defined as any observable deformity or discoloration making the tomato unappealing to the researcher performing the sort; this includes but is not limited to brown or black patches, insect holes, or an unusually small tomato when compared to the rest of the day’s harvest. Other imperfections included unusually soft patches. Tomatoes with such imperfections were rejected. Tomatoes passing the inspections were placed in an open top plastic container and stored at room temperature until they were used in the peeling process.
50 Peeling Process
Pretreatment
Most tomato peelability tests were performed using a pretreatment prior to a base
(or lye) pretreatment. The pretreatment solutions consisted of either of water, organic
solvent, or a mixture of the two. Pretreatment solutions were placed in 2000 mL stainless
steel beakers which in turn were placed in constant temperature water baths. When water
was the pretreatment, approximately 1700 mL was placed in the beaker. The amount of
solvent in the pretreatment beaker was governed by the necessity that the tomatoes be
completely submerged in the solvent. If the density of the solvent was more than that of
the tomatoes, which would enable the tomatoes to float to the top, then approximately
1600 to 1800 mL of solvent was required, and only 1000 to 1200 mL was required if the
reverse was true.
Most of the preliminary testing was done at the highest feasible solvent
temperature and a narrow range of dip times. Physical properties such as the flash point and boiling point were parameters considered in determining suitable pretreatment temperatures. Lower pretreatment temperatures were used for those solvents that had
boiling/flash points below 100oC (e.g. chloroform, dichloroethane) to ensure safety and minimize the loss of solvent due to evaporation. Pretreatment dip times were set to 60 seconds. During heating, aluminum foil was placed over the top of the beakers to reduce evaporative losses of solvent.
51 Pretreatment Reaction Time
After pretreatment, tomatoes were held for 30 seconds with the basket out of the
solution before they were placed in the aqueous basic solution. This allowed for the
removal of the majority of any residual organic solvent or water that remained on the
tomatoes and basket. Excess water would dilute the NaOH solution over time, and there
were concerns between organic solvent and NaOH interactions.
Base Dip
After the pretreatment dip and reaction time, tomatoes were placed in a basic
solution. Most often, the base that was used was NaOH. Approximately 1700 mL of the
NaOH solution was used in the stainless-steel beakers for each dip.
Evaporation of water in NaOH solutions was also a concern. Thus, aluminum foil
was placed over the top of all solutions to eliminate evaporation during heating and
throughout the experiment. In the case of NaOH evaporation, the vapor consists of water while all the NaOH remains in solution. This meant that the initial level of NaOH solution was maintained by addition of water as needed. Most basic dip times were 60 seconds.
Base Reaction Time
The base reaction time is similar to the pretreatment reaction time discussed
earlier. A film of base (i.e. NaOH solution) is on the tomato after the basket is pulled out
of solution, and therefore that time between the removal from the base and the insertion 52 into the mechanical peeler was maintained at a constant 60 seconds (reaction time), closely replicating the reaction times in industry.
Mechanical Peeling
Once tomatoes were subjected to the pretreatment dip, pretreatment reaction time, base dip, and base reaction time, they were mechanically peeled using a laboratory scale tumbler. Approximately 500 mL of DI water was added to the peeler. The tumbler was rotated 30-40 times at about 1.5 revolutions per second by hand to provide a source of abrasion in order to remove the cuticles.
The number of revolutions for cuticle removal was determined through experimentation. Loose peels would occasionally remain with few rotations (10-25), while a large number of rotations (80-100) would damage the fruit and remove peels that had not been loosened through the lye treatment. The 30 – 40 revolutions were intermediate between these two.
Scoring
After the tomatoes were removed from the tumbler, they were given a score based on how much of the cuticle had been removed. The amount of cuticle removal was assessed by visual inspection. Scores were assigned as follows (see Appendix B for more detail):
0 – No cuticle removal
1 – Less than half of the cuticle was removed 53 2 – More than half of the cuticle was removed
3 – Complete cuticle removal
4 – Complete cuticle removal with overpeeling, which was evidenced by fruit
damage and/or the appearance of vascular bundles in the fruit
Statistics
Data were analyzed using balanced and unbalanced one-way, two-way, or three-
way Analysis of Variance (ANOVA). Blocking was used whenever tomato lye peeling
experiments were carried out over several days to eliminate the day-to-day variability
among tomatoes. Provided that the ANOVA analysis revealed significant differences due to a given factor, Tukey’s test was used to make multiple comparisons of treatment averages. Significant changes were defined either as having a P-value less than or equal to 0.05 or when the differences in averages were greater than Tukey’s value when the confidence level, α, was set to 0.05 (Montgomery,2005). Calculations were performed using Design Expert 7.0 (Stat-Ease, Inc.) and Microsoft Excel 2003 (Microsoft
Corporation). All graphs were generated by Microsoft Excel, Design Expert, or Origin
7.0 (OriginLab Corporation).
Chapter Four
Vine-Ripened & Post-Harvest Age Studies
Vine-Ripened Study
Roma tomatoes of the cultivar H9423 and 696, which were grown in 2005 and
2006, respectively, were used for the vine-ripened study. To quantify the age of the tomato on the vine, one individual marked each tomato with the date on which it turned red. Tomatoes were checked daily. For the vine-ripened study in 2005, all tomatoes that entered the mature red stage of development between July and September were picked on
September 6, and were peeled on September 8, 2005. In the vine-ripened study in 2006, all tomatoes that entered the mature red stage of development between August and
October were picked on October 12, and peeled on October 13, 2006.
Peeling conditions varied slightly between 2005 and 2006. For the vine-ripened study in 2005, three tomatoes were placed in mesh baskets and submerged in 3.0 N
NaOH at 85±2°C for 60 seconds, lifted out of the solution and held for a 45 second reaction time. The three tomatoes with 500 mL of de-ionized water were placed in the tumbler, and scored.
54 55 For the vine-ripened study in 2006, three tomatoes were placed in mesh baskets and submerged first in de-ionized water for 60 seconds and then in 3.0 N NaOH for 60 seconds with a 30 second break in between. Solution temperatures were 80±1°C. After the NaOH dip and a 60 second reaction time, tomatoes were placed in the tumbler and scored.
Vine ripened tomato age was defined as the difference between the date the tomato turned red and the date it was picked. The range of available ages in 2005 was from 9 to 42 days; and in 2006, the range was from 0 to 57 days. Peeling scores are summarized in Figures 5a and 5b where the average score for each day is plotted against the vine-ripened age of the tomato.
The tomato peel score appears to decrease as the age on the vine increases, indicating that tomatoes are more difficult to peel as they mature. As the vine-ripened age of the tomato increases, the average score decreases. As tomatoes age on the vine, cuticular components increase in mass as the tomato matures (Baker et al.,1982; Bauer et al.,2004). With a build-up of cuticular components, the barrier for lye peeling thicker.
An ANOVA analysis reveals that there are significant differences in the average peeling scores with respect to vine-ripened tomato age with the P-values of 0.0066 and
0.0002 for 2005 and 2006, respectively. Tables 1 and 2 show the results from the
ANOVA analysis for the vine-ripened studies in 2005 and 2006, respectively. 56
Figure 5. (a) Average tomato peel score versus vine-ripened age for H9423 tomatoes peeled in 3.0 N NaOH in 2005 at 85±2°C. (b) Average tomato peel score versus vine-ripened age for 696 tomatoes peeled in 3.0 N NaOH in 2006 at 80±1°C. Squares with error bars represent the average score and standard deviation of three or more tomatoes. Squares without error bars represent the score of a single tomato or the average of 2 tomatoes. A score of zero indicates no cuticle removal whereas a score of four indicates over-peeling.
57 Table 1. ANOVA analysis for the vine-ripened study in 2005. Tomato variety was H9423. No pretreatment was used. NaOH concentration was 3.0 N. NaOH dip time and reaction time were 45 and 60 seconds, respectively. NaOH temperature was 85±2°C. Source of Sum of Degrees of Mean Variation Squares Freedom Square F0 Value P-Value Vine-Ripened 19.94 19 1.05 2.85 0.0066 Age of Tomato Error 9.93 27 0.37 Total 29.87 46
Table 2. ANOVA analysis for the vine-ripened study in 2006. Tomato variety was 696. Water pretreatment was used for 60 seconds. NaOH concentration was 3.0 N. NaOH dip time and reaction time were 60 seconds for both. Temperatures of water pretreatment and NaOH treatment were 80±1°C. Source of Sum of Degrees of Mean Variation Squares Freedom Square F0 Value P-Value Vine-Ripened 24.53 17 1.44 3.36 0.0002 Age of Tomato Error 24.47 64 0.43 Total 52.00 81
Each tomato was treated as a single replicate in both vine-ripened experiments in
the ANOVA analysis. The ANOVA analysis separates the total variability of the data into various factors based upon which factors changed during the course of the experiment. For all ANOVA analyses, the sources of variability include each factor and all possible interactions of those factors. Additionally, the ANOVA will also include an estimate for experimental error as well as the total variability of the data. The total variability must be the sum of all other sources of variability. Since the vine-ripened study included a single factor, there were no interactions. The sum of squares quantifies the variability associated with a source of variation. The degrees of freedom represent the total number of independent elements associated with a source of variation. The mean squares are determined by taking the ratio of the sum of squares to the degrees of freedom. Mean squares represent the variance associated with a source of variation. 58
The mean squares are used to calculate the F0 value. This F0 value is simply the ratio of the mean square of a source of variation to the mean square of error. If this ratio is greater than 1.0, then the variance associated with a source of variation is greater than the
variance attributed to experimental error, which indicates that the source of variation
could be significant. Tests for significance was performed by finding the P-value
associated with the F0 value.
Assumptions of normal distribution and constant variance of the residuals in the
data were verified by visual inspection of normal probability and constant variance plots,
respectively. Figures 6, 7, 8, and 9 show normal probability and constant variance plots
for vine-ripened data in 2005 and 2006. These plots reveal that there are no violations of
ANOVA assumptions.
Figure 6. Normal probability plot of residuals for the vine-ripened study in 2005. Tomato variety was H9423. No pretreatment was used. NaOH concentration was 3.0 N. NaOH dip time and reaction time were 45 and 60 seconds, respectively. NaOH temperature was 85±2°C.
59
Figure 7. Plot of residuals versus average tomato scores for the vine-ripened study in 2005. Tomato variety was H9423. No pretreatment was used. NaOH concentration was 3.0 N. NaOH dip time and reaction time were 45 and 60 seconds, respectively. NaOH temperature was 85±2°C.
Figure 8. Normal probability plot of residuals for the vine-ripened study in 2006. Tomato variety was 696. Water pretreatment was used for 60 seconds. NaOH concentration was 3.0 N. NaOH dip time and reaction time were 60 seconds for both. Temperatures of water pretreatment and NaOH treatment were 80±1°C.
60
Figure 9. Residuals versus average scores for the vine-ripened study in 2006. Tomato variety was 696. Water pretreatment was used for 60 seconds. NaOH concentration was 3.0 N. NaOH dip time and reaction time were 60 seconds for both. Temperatures of water pretreatment and NaOH treatment were 80±1°C.
The ANOVA analysis was followed by Tukey’s multiple comparison test. Only large differences in vine-ripened age resulted in significant differences in average peel scores.
Significant differences in average peel scores were found between vine-ripened ages of day 11 and 37, 11 and 40, and 11 and 42 for the 2005 study. For the vine-ripened study in 2006, the only significant differences are between days 0 and 22, 0 and 41, and 0 and
43. Thus, for the data from 2005, tomatoes had to remain on the vine for at least four weeks after ripening (turning red in color) before tomatoes became significantly harder to peel. Similarly, for the data from 2006, tomatoes needed to remain on the vine for at least three to five weeks after ripening before significant differences in peeling were observed.
In addition to a fixed effects analysis, a random effects analysis was conducted in order to ascertain the variability between levels of tomato maturity as compared to the 61 total variance according to design of experiments principles (Montgomery,2005). Such
an analysis was called for as the levels of tomato ages could have taken many possible
values. Tomato age variability is expressed as the intraclass correlation coefficient,
which represents the percentage of the total variance that is due to changes between
tomato vine-ripened ages. A confidence interval can be generated for the coefficient.
There were two sources of variance for this study. First, there exists a variability
2 between different levels of tomato maturity, σAge . Second, there exists a variability
within a specific tomato maturity level, σ2, which is also known as the mean squared
2 MSE. The total variability, σTotal , is given by the equation below.
2 2 2 σ Total = σ Age + σ 1
The interclass coefficient is listed below.
2 σ Age 2 2 2 σ Age + σ
2 Variability for the main treatment, σAge , is calculated by equation 3.
MS − MS σ 2 = Age E 3 Age n
where, n, is the number of replicates associated with each treatment level. Since this was
an unbalanced study, the number of replicates was calculated by the following equation.
⎡ a ⎤ n 2 1 ⎢ a ∑ i ⎥ n = ⎢ n − i=1 ⎥ 4 a −1 ∑ i a ⎢ i=1 n ⎥ ⎢ ∑ i ⎥ ⎣ i=1 ⎦
The upper and lower levels of the intraclass correlation coefficient confidence interval are given in equations 5. 62 2 L σ Age U ≤ 2 2 ≤ 5 1+ L σ Age + σ 1+U where
⎛ MS ⎞ 1 ⎜ Age 1 ⎟ L = ⎜ −1⎟ 6 n ⎝ MS E F0.025,a−1,N −a ⎠ and
⎛ MS ⎞ 1 ⎜ Age 1 ⎟ U = ⎜ −1⎟ 7 n ⎝ MS E F0.975,a−1,N −a ⎠
Using equations 5 through 7, estimates for the intraclass coefficient was determined for the 2005 and 2006 vine-ripened studies. Results show that vine-ripened ages in 2005 accounted for 3.1 to 71.9% of the total variance; and vine ripened-ages in
2006 accounted for 6.1 to 62.3% of the total variance. With these values, the effect of vine-ripened age (tomato maturity) cannot be neglected.
Tomatoes pass through various stages of development from immature green, mature green, breaker, and then finally to mature red. The overall process takes about six to eight weeks after anthesis (Ho & Hewitt,1986). Changes with the cuticle involve the accumulation of cuticular waxes and the appearance of naringenin chalcone (Bauer et al.,2004). Though these vine-ripened changes take place, they do not significantly alter the peelability, unless vine-ripened tomatoes are left on the vine for a period of at least three weeks after turning red. From a processing point of view, if tomatoes continue to develop past the mature red stage on the vine for more than one month, then more NaOH would be required to peel those tomatoes.
63 Post-Harvest Study
Roma tomatoes of the cultivar TSH-8, which were grown in 2006, were used for these experiments. Tomatoes were collected from random samples and set aside for 0 to
21 days until they were peeled. Three tomatoes at a time were placed in mesh baskets and submerged first in de-ionized water for 60 seconds and then in 4.0 N NaOH for 60 seconds with a 30 second break in between. Solution temperatures were 80±1°C. After the NaOH dip and a 60 second reaction time, tomatoes were placed in the tumbler and scored.
Twelve different levels of post-harvest age were considered for this study—0, 1,
2, 3, 4, 5, 6, 7, 12, 14, 19, and 21 days. The average tomato score at a post-harvest age of
0 defined the control. Three tomatoes of the same post-harvest age were used in each dip. Thus, the average tomato score of the three tomatoes for each dip was considered as a single, duplicated replicate. Six replicates were taken for each post-harvest age listed above. All tomatoes were not dipped on the same day, which placed a restriction on the total randomization of the experiment. Thus, data collected in each day was placed into a separate block as tomatoes are subject to environmental conditions from day to day and week to week on the field. There were five separate blocks. Correspondingly, the
ANOVA analysis was slightly altered to accommodate this design (Montgomery,2005).
The ANOVA analysis revealed that there are significant changes in average scores with respect to post-harvest ages with a P-value less than 0.0001. Table 3 displays the results from the ANOVA analysis. Figure 10 clearly shows that average tomato peelability scores decrease as the post-harvest age increases.
64 Table 3. ANOVA analysis for the post-harvest study in 2006. Tomato variety was TSH-8. Pretreatment was water Pretreatment dip time and reaction were 60 and 30 seconds, respectively. NaOH concentration was 3.0 N. NaOH dip time and reaction time were 60 seconds for both. Temperature was 80±1°C. Source of Sum of Degrees of Mean Variation Squares Freedom Square F0 Value P-Value Blocks 4.83 4 1.21 Post-Harvest Age 14.22 7 2.03 9.14 < 0.0001 of Tomato Error 13.33 60 0.22 Total 32.39 71
4.00
3.50
3.00
e 2.50 or c 2.00
age S 1.50 er v
A 1.00
0.50
0.00 0 5 10 15 20 25 Post-Harvest Age, Days
Figure 10. Average tomato score versus post-harvest age. Each point represents the average score of 18 tomatoes. Error bars show one standard deviation of each of the averages. Closed circles represent average scores that are not significantly different from the control (post-harvest age of 0 days) while open circles represent average scores that are significantly different from the control. A score of zero indicates no cuticle removal whereas a score of four indicates over-peeling. Tomato variety was TSH-8. Pretreatment was water Pretreatment dip time and reaction were 60 and 30 seconds, respectively. NaOH concentration was 3.0 N. NaOH dip time and reaction time were 60 seconds for both. Temperature was 80±1°C.
Applying Tukey’s multiple comparison test to the average tomato scores enables one to determine when differences in the scores are significant from the control (defined as the average score for the post-harvest age of 0 days). All multiple comparisons were made with respect to this control. Closed circles in Figure 8 represent average scores that 65 are not significantly different from the control, and open circles represent average scores that are significantly different from the control. Figure 8 shows that significant differences begin as soon as three days off the vine. After one week, all differences in average scores were significantly different from the control.
Examining residuals from the post-harvest peeling data reveal that there are no violations of the ANOVA analysis. The residuals are normally distributed and possess constant variance. This is shown in Figures 11 and 12.
Figure 11. Normal probability plot of the residuals from the post-harvest experiment. Tomato variety was TSH-8. Pretreatment was water Pretreatment dip time and reaction were 60 and 30 seconds, respectively. NaOH concentration was 3.0 N. NaOH dip time and reaction time were 60 seconds for both. Temperature was 80±1°C.
66
Figure 12. Average tomato scores versus residuals for the post-harvest experiment. Tomato variety was TSH-8. Pretreatment was water Pretreatment dip time and reaction were 60 and 30 seconds, respectively. NaOH concentration was 3.0 N. NaOH dip time and reaction time were 60 seconds for both. Temperature was 80±1°C.
These results indicate that changes in the cuticle take place shortly after tomatoes are taken off the vine. It should be noted that tomatoes, which did not show signs of excessive fruit softening were selected for this study in order to better compare results between different post-harvest ages. Fruit softening is attributed to the degradation of polysaccharides in the cell walls and in the middle lamella (Saladie et al.,2005). Thus, tomato fruit that is firmer for long post-harvest times undergo less polysaccharide breakdown than softer fruit. Thus, polysaccharide degradation does not appear to be a plausible mechanism for the phenomena. Changes with tomato peelability are more likely explained by changes in the cuticle. Structural changes or a drying of the cuticle are possible explanations for this observation. More research is required to chart cuticular changes of post-harvest tomatoes with respect to time to ascertain the mechanism for differences in post-harvest peelability.
Chapter Five
Process Parameters Controlling Lye Peeling
Three main parameters affect tomato lye peeling: temperature, concentration, and
time. Several types of experiments were conducted to reveal which processing
parameter(s) had a significant affect upon average tomato scores. First, a three factor—
two level experiment with center points was conducted in which temperature,
concentration, and time were adjusted according to principles of design of experiments
(Montgomery,2005).
Second, experiments were conducted, which tested different types of bases.
Sodium hydroxide is the base that is currently used in industry. This is due to its low cost
as a raw material. After lye peeling, waste tomatoes and peels contain a high sodium
level, which is detrimental to the environment when this waste is used as a fertilizer.
Thus, additional bases were considered to mitigate this problem. Potassium hydroxide
has also been used in tomato peeling studies (Das & Barringer,2006). NH4OH and an
equimolar mixture of KOH and NH4OH were other bases that were considered. Several experiments were conducted in which the bases NaOH, KOH, NH4OH, and an equimolar
mixture of KOH and NH4OH were simultaneously compared.
67 68
Temperature, Concentration and Time Study
Preliminary studies from Fall 2005 show that the effectiveness of sodium
hydroxide was both time and temperature dependent, regardless of whether a solvent pre-
dip was used or not. Table 4 shows the effect of tomato time exposed to NaOH and
temperature of the caustic without a pretreatment. This initial study determines that time
and temperature both affect average tomato scores. It has been suggested that processing
time was the most important parameter in lye peeling (Floros et al.,1987). Through optimization studies, it was concluded that tomatoes were peeled best in a 9 (w/v)%
NaOH solution for 2 minutes at 80°C. Furthermore, it has been demonstrated that allowing tomatoes to remain in the 9% NaOH solution for 3 total minutes resulted in damage localized to the mesocarp (Floros et al.,1987). It was found that higher NaOH concentrations could be used, which would reduce the processing time. Conversely, lower concentrations could be used to peel tomatoes provided that the processing time was long enough. Thus, in order to evaluate the effects of a solvent pre-treatment step, the temperature and time of the lye treatment was kept constant in most lye peeling studies. 69
Table 4. Time and Temperature Studies Using NaOH from 2005. Tomato variety was H9704. No pretreatment or pretreatment reaction time was used. NaOH concentration was 3.0 N. NaOH dip time and reaction time was 60 seconds. NaOH NaOH Dip Number of Avg. Score ± Temp. (°C) Time (s) Tomatoes Std. Deviation 85 30 8 0.00 ± 0.00 85 45 4 0.00 ± 0.00 90 30 7 0.00 ± 0.00 90 45 4 0.00 ± 0.00 95 30 4 0.75 ± 0.50 95 45 12 1.75 ± 0.45
Roma tomatoes of the cultivar TSH-8, which were grown in 2006, were used in
these experiments. In order to investigate significant factors associated with lye peeling, a three-factor, two level (23) design with center points was utilized to study the effect of: temperature, NaOH concentration, and time. Temperature of the water pretreatment as well as the NaOH treatment was varied, as was NaOH concentration. The amount of time tomatoes remained in the water pretreatment and the NaOH treatment was also varied. Nine different conditions were evaluated with six replicates where each replicate was the average of three tomato scores (Montgomery,2005). The experimental design is given in Table 5. 70
Table 5. Experimental design for three-factor factorial experiment. Treatment totals refers to the total sum of all six duplicated replicates. Tomato variety was TSH-8. Water pretreatment reaction time was 30 seconds. NaOH reaction time was 60 seconds. Pretreatment and NaOH temperatures and times were the same and are listed below. Each replicate was a duplicated measurement in which the response was the average score of the three tomatoes. Treatments Average ± Conc. of Treatment Standard Temp, °C Time, s Replicates NaOH, N Totals Deviation 70 3.00 30 6 2.33 0.39 ± 0.25 90 3.00 30 6 10.00 1.67 ± 0.56 70 4.50 30 6 4.00 0.67 ± 0.30 90 4.50 30 6 11.00 1.83 ± 0.46 80 3.75 45 6 6.33 1.05 ± 0.14 70 3.00 60 6 3.33 0.55 ± 0.27 90 3.00 60 6 19.33 3.22 ± 0.62 70 4.50 60 6 5.33 0.89 ± 0.34 90 4.50 60 6 21.67 3.61 ± 0.39
The ANOVA determined that all three factors, temperature, concentration, and
time, as well as an interaction between temperature and time were significant. P-values
for temperature, time, and the time-temperature interaction were less than 0.0001, and the
P-value for the main factor, concentration, was 0.0120. Additionally, the test for
curvature was significant, which stemmed from the center points of the design. All other
interaction terms were insignificant. Table 6 gives the results from the ANOVA analysis.
71
Table 6. Three-way ANOVA analysis for lye peeling study. Tomato variety was TSH-8. Water pretreatment was used with a 30 second reaction time. NaOH reaction time was 60 seconds. Temperatures and times were adjusted for both pretreatment and caustic dips. Each replicate was a duplicated measurement in which the response was the average score of the three tomatoes. Source of Sum of Degrees of Mean Variation Squares Freedom Square F0 Value P-Value A (Temperature) 46.02 1 46.02 291.23 < 0.0001 B (Concentration) 1.02 1 1.02 6.46 0.0145 C (Time) 10.39 1 10.39 65.76 < 0.0001 AB 0.0023 1 0.0023 0.015 0.9042 AC 6.50 1 6.50 41.15 < 0.0001 BC 0.058 1 0.058 0.37 0.5481 ABC 0.021 1 0.021 0.13 0.7182 Curvature 1.61 1 1.61 10.16 0.0026 Error 7.11 45 0.16 Total 72.73 53
A graph of the model generated from the experiment is given in Figure 13. 72
Figure 13. Three dimensional model graph obtained from Design Expert for lye peeling. Sodium hydroxide concentration was 4.50 N. The score represents the extent of cuticle removal. Time is in seconds. Temperature is in degrees Celsius. Tomato variety was TSH-8. Water pretreatment was used with a 30 second reaction time. NaOH reaction time was 60 seconds. Temperatures and times were adjusted for both pretreatment and caustic dips.
Validation of the ANOVA assumptions, normal distribution of the residuals and constant variance of the residuals was made by a visual examination of the appropriate graphs.
The graphs of normal probability and constant variance are displayed in Figures 14 and
15. 73
Figure 14. Normal probability plot of the residuals for the 23 factorial design with center points. Tomato variety was TSH-8.
Figure 15. Residuals versus average tomato score for each condition in the 23 factorial design with center points. Tomato variety was TSH-8.
Two points in Figure 14 give rise to some concern regarding the assumption of constant variance. Calculation of the standardized residuals revealed that the two points 74 have values of 2.66 and -3.18. Evaluating graphs of residuals versus factor levels
revealed that the greatest difference in variances was attributed to temperature (see Figure
16).
Figure 16. Residuals versus levels of temperature in the 23 factorial design with center points. Tomato variety was TSH-8.
From Figure 16, there appears to be dispersion effects due to an increased temperature.
Thus, another three-factor ANOVA analysis was performed on the standardized residuals
of the data and no significant effect of temperature was found. Thus, the assumption of
constant variance of the residuals was validated.
Elevated temperatures and concentrations provide greater driving forces for heat and mass transport, respectively. Excessive damage to collenchymatous cells in tomatoes due to heat treatments, which lead to the separation of the cuticle from the fruit, has been observed in the context of steam peeling (Floros & Chinnan,1988). Higher temperatures 75 create a greater driving force for heat transfer into the fruit. Tomatoes subjected to an
extended dipping time of 60 seconds, at the higher temperature, provided excessive heat
transfer to tomato fruits, which overpeeled many tomatoes at this condition.
Similarly, high NaOH concentrations increased the average score for tomatoes
compared to those at lower concentrations. Sodium hydroxide diffuses through the tomato cuticular membrane according to Fick’s Law of Diffusion (Floros &
Chinnan,1989; Floros & Chinnan,1990). Higher NaOH concentrations created a greater driving force for diffusion through the cuticle, which accomplishes two major objectives inside tomato fruit. First, it can degrade the pectin in the middle lamella by breaking the
α-D (1,4) glycosidic bonds. Viscosity of pectin was measured under various alkaline conditions and have shown that increasing concentration of NaOH decreases viscosity, and thus, pectin’s molecular weight (Vollmert,1950; Krachanov,1965). Second, NaOH can lead to collenchymatous cell wall damage. Partial damage of collenchymatous cell walls were observed for NaOH treatment for one minute at 0.25 N, and the damage was excessive when the treatment time increased to two minutes. Extreme damage was observed when tomatoes were treated with a 2.25 N solution of NaOH for one minute.
Collenchymatous cell wall damage is primarily attributed to the NaOH solubilizing the hemicellulosic polysaccharides in the cell walls (Floros et al.,1987). Again, an increased dipping time provides for more NaOH to diffuse into the fruit. Exposing collenchymatous cells to NaOH for prolonged exposure times, in excess of 10 minutes, completely disrupts its structure, as was observed in the cell walls of Valonia (Kim et al.,2006). Since, treatment time, temperature, and NaOH concentration were all significant, these factors were held constant. 76
Type of Base
Different bases were selected in order to evaluate their effectiveness for tomato
peeling. Bases selected for this study were potassium hydroxide (KOH), NaOH, ammonium hydroxide (NH4OH), and an equimolar mixture of KOH and NH4OH.
Concentrations of the bases were adjusted so that the total base normality was 4.0 N.
Roma tomatoes of the cultivar TSH-8 were used for this study. Table 7 lists the four
bases and concentrations used for the study.
Table 7. A list of the four bases used to determine if the type of base has an affect upon tomato peelability. Tomato variety was TSH-8. Base 4.0 N KOH 4.0 N NaOH 4.0 N NH4OH 2.0 N KOH + 2.0 N NH4OH
Three tomatoes at a time were placed in mesh baskets and submerged first in de-
ionized water for 60 seconds and then in a NaOH solution for 60 seconds with a 30
second break in between. After the NaOH dip and a 60 second reaction time, the three
tomatoes with 500 mL of de-ionized water were placed in the tumbler, and scored.
Figure 17 shows the results from the study. Basicity increases from NH4OH (pKa
= 9.246), to NaOH (pKa = 14.77), to KOH (pKa = 16) (Albert & Serjeant,1971). This chart shows that the average score of tomatoes increases with increasing basicity, which supports the results found from a previous study (Das & Barringer,2006). Bases that have a higher pKa appear more effective at peeling. An increase in basic strength may
make it more effective at degrading the pectin in the middle lamella. An ANOVA 77 analysis (Table 8) revealed that differences in the type of base were significant with a P- value of 0.0005.
4.00
3.50
3.00 e r
o 2.50 c
e S 2.00 ag
er 1.50 v A 1.00
0.50
0.00 NH4OH KOH+NH4OH NaOH KOH
Base
Figure 17. Average score of tomatoes subjected to treatments of various bases. Error bars represent one standard deviation. Significant differences are observed between NaOH and NH4OH, KOH and NH4OH, and KOH and the mixture of KOH and NH4OH. The hydroxide ion concentration was 4.0 N in each case. Tomato variety was TSH-8. Water was used as a pretreatment for 60 seconds. Base dip was 60 seconds. All temperatures were 80°C.
Table 8. ANOVA analysis for type of base study. Tomato variety was TSH-8. Pretreatment was water. Pretreatment dip time and reaction time were 60 and 30 seconds, respectively. Base concentration was 4.0 N. Base dip time and reaction time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C. Source of Sum of Degrees of Mean Variation Squares Freedom Square F0 Value P-Value Type of Base 9.72 3 3.24 9.18 0.0005 Error 7.06 20 0.35 Total 16.77 23
78 Tukey’s test was used to compare differences in tomato peel scores between the
bases (Table 9). The significance level, α, was set to 0.05. Tukey’s value, Tα, made use of a q distribution, and was determined to be 0.96.
Table 9. Comparisons of average tomato score for base study. Tukey’s value, T0.05, was found to be 0.96. Tomato variety was TSH-8. Pretreatment was water. Pretreatment dip time and reaction time were 60 and 30 seconds, respectively. Base concentration was 4.0 N. Base dip time and reaction time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C. Difference in Base Comparison Average Score a KOH and NaOH 0.28 KOH and NH4OH/KOH Mix 1.06* KOH and NH4OH 1.61* NaOH and NH4OH/KOH Mix 0.78 NaOH and NH4OH 1.33* NH4OH/KOH Mix and NH4OH 0.55 a – A star (*) indicates a significant difference with 95% confidence.
Significant differences were found between tomato peel scores for NaOH and
NH4OH, KOH and NH4OH, and KOH and the equimolar mixture of KOH and NH4OH.
No significant difference between NaOH and KOH, NaOH and a mixture of KOH and
NH4OH, and NH4OH and a mixture of KOH and NH4OH were observed. Such results
conflict with a previous study, which stated that substituting KOH for NaOH produces
significant differences in peeling (Das & Barringer,2006).
It is important to note that the difference between NaOH and the equimolar mixture of NH4OH and KOH was found to be statistically insignificant. Thus, an equimolar mixture of NH4OH and KOH could be substituted for NaOH for tomato peeling.
A residual analysis of the data validated the ANOVA assumptions. Normal probability and residual plots revealed that the residuals were normally distributed about 79 a mean of 0 and possessed constant variance. Figures 18 and 19 show the normal probability and constant variance plots, respectively.
Figure 18. Normal probability plot of the data from the base experiment. Tomato variety was TSH-8. Pretreatment was water. Pretreatment dip time and reaction time were 60 and 30 seconds, respectively. Base concentration was 4.0 N. Base dip time and reaction time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C.
80
Figure 19. Average tomato score versus residuals for the base experiment. Tomato variety was TSH-8. Pretreatment was water. Pretreatment dip time and reaction time were 60 and 30 seconds, respectively. Base concentration was 4.0 N. Base dip time and reaction time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C.
These results agree with another previous, unbalanced, two-factor experiment where peeling scores were compared for tomatoes treated with either NaOH or an equimolar mixture of KOH and NH4OH at different concentrations. Results from an
ANOVA analysis (Table 11) illustrated that there was no significant difference between
NaOH and an equimolar mixture of KOH and NH4OH. The experimental design and
results are presented in Table 10, and shown graphically in Figure 20. 81
Table 10. Experimental design and results for an unbalanced factorial study between NaOH and NH4OH. An ANOVA analysis revealed no significant differences between the type of base tested. Pretreatment was water. Pretreatment dip time and reaction time were 60 and 30 seconds, respectively. Base dip time and reaction time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C. Treatments Average ± Treatment Type of Base Concentration of Standard Replicates Totals NaOH, N Deviation NaOH 3.25 e 5 15.33 3.07 ± 0.28 3.50 e 5 15.00 3.00 ± 0.00 4.00 f 10 26.33 2.39 ± 0.55 Equimolar 3.25 e 7 17.67 2.52 ± 0.33 mixture of KOH 3.50 e 6 16.67 2.78 ± 0.17 f & NH4OH 4.00 11 28.00 2.55 ± 0.45 e – Tomato variety is 9423. f – Tomato variety is TSH-8.
4.00 NaOH 3.50 KOH + NH4OH
3.00 e r 2.50 o c 2.00 e S
ag 1.50 er v
A 1.00
0.50
0.00 3.25 3.50 4.00 Base Concentration, N
Figure 20. Results for an unbalanced, two-factor factorial study between NaOH and an equimolar mixture of KOH & NH4OH at three different concentrations at 80±1°C. Error bars represent standard deviations. An ANOVA analysis revealed that no significant differences exist between any treatments. Tomato varieties were 9423 and TSH-8. Pretreatment was water. Pretreatment dip time and reaction time were 60 and 30 seconds, respectively. Base dip time and reaction time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C.
Corrections for the sum of squares formulas had to be made to account for the unbalanced nature of the design. Moreover, all the data was not collected in one day. 82 Each level of the three levels of hydroxide ion concentration was treated as a separate
block. The main effect of concentration was confounded with the blocks due to this
design. Consequently, only the main effect of type of base and the concentration-base
interaction could be determined. Results from the ANOVA analysis are given in Table
11 below.
Table 11. ANOVA analysis for effect of type of base and concentration for results in Table 10 and Figure 20. Tomato varieties were 9423 and TSH-8. Pretreatment was water. Pretreatment dip time and reaction time were 60 and 30 seconds, respectively. Base dip time and reaction time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C. Source of Sum of Degrees of Mean Variation Squares Freedom Square F0 Value P-Value Block 1.32 2 0.66 A (Type of Base) 0.00449 1 0.00449 0.027 0.8968 B (Concentration) g 0.00 0 - - - AB 1.02 2 0.51 3.10 0.0563 Error 6.43 39 0.16 Total 8.78 44 g – This factor is confounded with the blocks.
The ANOVA analysis revealed that there were no significant differences between the average scores when NaOH or an equimolar mixture of NH4OH and KOH was used
to peel tomatoes (P-value = 0.8968). Again, assumptions of the ANOVA analysis were validated by using normal probability and constant variance plots of the residuals (see
Figures 21 and 22).
83
Figure 21. Normal probability plot of the residuals for the two base experiment. Tomato varieties were 9423 and TSH-8. Pretreatment was water. Pretreatment dip time and reaction time were 60 and 30 seconds, respectively. Base dip time and reaction time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C.
Figure 22. Average tomato score versus residuals for the two base experiment. Tomato varieties were 9423 and TSH-8. Pretreatment was water. Pretreatment dip time and reaction time were 60 and 30 seconds, respectively. Base dip time and reaction time were 60 seconds each. Pretreatment and NaOH temperatures were 80°C.
Chapter Six
Role of Caustic in Tomato Peeling
There are four main mechanisms which can account for tomato peeling. Sodium hydroxide may be due to cuticular wax dissolution. Dissolution of cuticular waxes reduces the primary barrier to lye peeling. Cuticle dissolution has been observed in previous studies (Floros et al.,1987; Das & Barringer,1999). Second, NaOH may osmotically shock collenchymatous cells due to ionic strength alone so that they collapse, thus cleaving the cuticle from the mesocarp. Third, NaOH solubilizes hemicellulosic components in collenchymatous cell walls of cuticles (McNeil et al.,1984). Fourth,
NaOH degrades pectin beneath the cutin and between collenchymatous cells through depolymerization, which reduces its ability to function as a binding agent
(Vollmert,1950; Krachanov,1965). Lastly, NaOH could affect cellulose in cell walls through mercerization. Mercerization charts the phase transition from cellulose I to II.
Cellulose swells during this transition which would allow additional NaOH diffusion into collenchymatous cells. Therefore, cellulose I to II transition was charted under varying bases, times, and temperatures to ascertain whether mercerization was another plausible mechanism.
84 85 Cellulose is an ever-present polysaccharide in plant cell walls. In tomatoes,
cellulose appears in collenchymatous cell walls as long microfibrils. The cellulose is present as cellulose I, which possesses only intrasheet bonding (Kroon-Batenburg &
Kroon,1997; Langan,2005). In the presence of NaOH, cellulose I swells and rearranges to permanently form the cellulose II allomorph in a process commonly referred to as mercerization (Kolpak & Blackwell,1976; Kim et al.,2006). The mercerization of cellulose in collenchymatous cell walls was a proposed mechanism during lye peeling.
Thus, cellulose I (Avicel from FMC) was mercerized under 3.0 N NaOH at various temperatures and times close to tomato lye peeling processing times in order to ascertain if mercerization was indeed a mechanism for lye peeling.
Chemical Reagents
Cellulose in the form of Cellulose I was obtained from Paul Langan (Los Alamos
National Lab) and from Avicel® PH-101 (Sigma Aldrich, #11363). Cellulose II was obtained by completely mercerizing Cellulose I. Hydrochloric acid (Fisher Scientific,
#A144s-212) was used to stop mercerization experiments.
Mercerization Studies
Mercerization was discovered by John Mercer in 1850 (Dinand et al.,2002). Of the polysaccharides in the primary cell wall, cellulose is often the most prevalent (McNeil et al.,1984). Throughout plant development and maturity, the amount of cellulose in the secondary cell walls increases (Langan et al.,2001). 86 Mercerization experiments were carried out in 500 mL Erlenmeyer flasks on a
heating plate with stirring using a magnetic stirring bar. Solutions of 3.0 N NaOH, KOH,
or NH4OH were used; mercerization (conversion of cellulose I to cellulose II) was evaluated at 60, 80, and 100°C for 60, 120, and 500 second exposure times of cellulose to the basic solutions. A measured quantity of cellulose I was added to the preheated solution for the given amount of time. Hydrochloric acid to neutralize the solution was added to stop the reaction. The mixture was cooled in a cooling water bath during acid addition to prevent sample heating. The product was then filtered using a sintered glass filter and washed with at least 100 mL of DI water to separate the cellulose from the solution. Finally, the isolated crystals were placed on watch glasses and allowed to dry
for at least three days in a desiccator at room temperature.
Cellulose II Standards
Cellulose II was obtained through the mercerization of cellulose I. Cellulose I
(Avicel from FMC) was added to a 4.25 N NaOH solution at 65°C and mixed for twenty-
four hours. The molar ratio of NaOH to cellulose was always greater than 5,000 for
mercerization. Such conditions were used to ensure complete conversion. Enough
cellulose II was generated in order to make five 1.0 g cellulose standard mixes of cellulose I and II. The five cellulose standard mixes were 0%, 25%, 50%, 75%, and
100% (by mass) cellulose II.
87 13C Solid State CP-MAS NMR
Solid State 13C Cross-Polarization (CP) Magic Angle Spinning (MAS) Nuclear
Magnetic Resonance (NMR) was used to detect the purity of cellulose I and II to evaluate the standard mixes. Measurements were conducted using a 1987 Chemagnetics CMC
200-0208 solid-state NMR device. The parameters used to operate the device included a
13C frequency of 50.1648 MHz; a proton dipolar decoupling for the frequency of proton
resonance of 199.485 MHz; a field strength 200 MHz, which is about 4.7 T and degree
pulses of 90° with time pulses of 5.5 µs. The spinning speed was 5 to 5.5 kHz, contact and acquisition times were 1.5 and 26 ms, respectively. The sweep width was 399 ppm.
A recycling delay of 2.00 seconds was used. Approximately 1500 scans were taken to generate the NMR patterns. The instrument was calibrated with hexamethyl benzene with the benzene carbon referenced to 132.2 ppm.
Two characteristic peaks were used to identify the presence of cellulose I and II.
Cellulose I is characterized by a peak at 65.6 ppm, which is due to the tg conformation of
C6; and cellulose II is characterized by a peak at 107 ppm, which is due to the conformation of C1 (Dinand et al.,2002). NMR pattern of Cellulose I and II are given
below. Figure 23 shows that cellulose I was initially present, which is indicated by the
strong peak at 65.6 ppm. Similarly, Figure 24 shows that cellulose II was indeed
generated as seen by the strong peak observed at 107 ppm.
88
Figure 23. Solid State 13C CP-MAS NMR pattern for Cellulose I.
Figure 24. Solid State 13C CP-MAS NMR pattern for Cellulose II.
X-Ray Diffraction
The powder X-ray diffraction (XRD) patterns were used to identify the forms of
the product. Powder diffraction measurements were taken with a PANalytical XPert Pro
MPD. Data were collected using Cu Kα radiation (λ = 1.541874 Å) with a setting of 45 kV and 40 mA. A gonio continuous scan at a scan rate of 0.0167° per step and 10 89 sec/step from 4.0 to 45.0° in 2θ was used. The incident beam path consisted of a 0.04 radian Soller slits, a 10 mm incidence mask, a ½° divergence slit, and a ¼° anti-scatter slit. The diffracted beam path consisted of a Nickel filter, a 0.04 radian Soller slits, and a
5 mm anti-scatter slit. The detector was a real-time multiple strips X’Celerator operated in a scanning mode of 2.122° active length.
Recovered cellulose samples were ground by a mortar and pestle before being
placed in a Plexiglas sample holder, which was provided by Erwin Lorenz (Custom
Machining, Toledo, OH 43615). A part drawing for the sample holder is included in
Appendix C (Figure 37). Next, the samples were packed into the sample holder and smoothed using microscope slides (Fisher Scientific, #12-550-10) to ensure the top was flush with the sample holder. Holders were placed in a flat sample stage bracket of the diffractometer.
Five standard mixes of cellulose were prepared only after pure cellulose I and II samples were verified through 13C CP-MAS NMR. Each sample was analyzed through
powder x-ray diffraction (XRD) and is analyzed in Figure 25 below.
There are several characteristics of XRD patterns as the transition from cellulose I
to II occurs. A peak at 11° appears as the presence of cellulose II increases. The two
peaks at 15° and 16° decrease to the baseline. The intensity of the shoulder at 20.5°
increases and shifts to the left as the mixture changes from cellulose I to II, and this peak
eventually becomes greater than the peak 22.5°. The intensity of the peak at 22.5°
steadily decreases. The intensity of the peak at 34.5° in cellulose I decreases to the baseline in cellulose II. 90 Twenty-seven different mercerization conditions were studied using NaOH,
KOH, and NH4OH at different temperatures and reaction times at a concentration of 3.0
N. Since cellulose is located in the tomato, it was hypothesized that mercerization may be a mechanism through which lye peeling occurs. Mercerization of cellulose involves the swelling of cellulose microfibrils in the stacking direction of the cellulose structure.
It was suggested that NaOH can more easily diffuse through collenchymatous cell walls
when cellulose swells, which would facilitate tomato lye peeling. Thus, mercerization
studies were carried out to test this hypothesis. In order for this to be true, the conversion
from cellulose I to II would need to occur within the time that is typical of lye peeling—1
to 3 minutes. The experimental design for all the mercerization experiments is listed in
Table 12.
Table 12. Experimental design for mercerization experiments Type of Base NaOH KOH NH4OH Temp., °C Time, s Temp., °C Time, s Temp., °C Time, s 60 60 60 60 60 60 60 120 60 120 60 120 60 300 60 300 60 300 80 60 80 60 80 60 80 120 80 120 80 120 80 300 80 300 80 300 100 60 100 60 100 60 100 120 100 120 100 120 100 300 100 300 100 300
91
(e)
y
it (d) s n e t (c) In (b)
(a)
0 5 10 15 20 25 30 35 40 45 2θ
Figure 25. Powder x-ray diffraction patterns of five standard mixtures of cellulose. (a) Pure cellulose I in the form of Avicel. (b) 25% cellulose II. (c) 50% cellulose II. (d) 75% cellulose II. (e) Pure cellulose II. Cellulose II was obtained through mercerization of cellulose I in 4.25 N NaOH at 65°C for 24 hours.
Changes in peak intensities of the diffraction patterns enabled one to attempt to quantify the percent conversion from cellulose I to II. Absolute and relative peak intensities were used to determine which was better at describing percent conversion.
Absolute peak intensities were found by reading the value of intensity generated from the
PANalytic XPert Data Collector. Relative peak intensities were determined by subtracting the absolute intensity from the baseline intensity. Baselines were defined by fitting a cubic equation among the points between 4.0 and 9.0°, 30.5 and 32.0°, 37.0 and
39.0°, and 42.5 and 45.0° using Microsoft Excel. 92 The largest change in peak intensities were those observed between 19 and 23°.
Thus, the defining characteristic used to quantify cellulose conversion was the ratio of the
intensity of the peak at 22.5° to the intensity of the shifting peak between 19 and 20.5°.
This ratio was calculated each of the five standard mixes using absolute as well as
relative intensities. The graphs of percent conversion of cellulose II versus the characteristic ratio are listed below in Figures 26 and 27.
Despite efforts to carefully choose baseline intensities and proper characteristic ratios to define percent conversion of cellulose II, determining precise estimates of the
cellulose I to II conversion through x-ray diffraction was not possible. A second order polynomial and natural logarithmic correlation was the best fit for the characteristic ratios using absolute and relative intensities, respectively. Linear, power, and exponential correlations were also evaluated. The R2 value of the polynomial and natural logarithmic correlation was 0.991 and 0.927, respectively. These correlations were then used to test
against the five standard mixes.
93
100%
80%
on i s
er 60% onv C
t n
e 40% c Per
20% y = -0.47x2 + 1.8683x - 0.606 R2 = 0.9913 0% 0.00 0.50 1.00 1.50 Ratio of Absolute Intensities
Figure 26. Characteristic ratios versus absolute peak intensities for each of the five standard cellulose mixtures. The characteristic ratio is defined as the ratio between the relative peak intensity at 22.5° to relative peak intensity of the shifting peak between 19 and 20.5°. The trend-line represents the best fit for the data. The trend-line equation and R2 value were obtained through Microsoft Excel.
100%
80%
on si 60% onver C 40% cent r e P
20% y = 0.761Ln(x) + 1.0848 R2 = 0.9269
0% 0.00 0.50 1.00 1.50 Ratio of Relative Intensities
Figure 27. Characteristic ratios versus relative peak intensities for each of the five standard cellulose mixtures. The characteristic ratio is defined as the ratio between the relative peak intensity at 22.5° to relative peak intensity of the shifting peak between 19 and 20.5°. The trend-line represents the best fit for the data. The trend-line equation and R2 value were obtained through Microsoft Excel.
94 For the correlation using absolute intensities, the average percent error of
measured versus actual percent cellulose II was 6.9%; and when using relative intensities,
the average percent error was 10.8%. Additionally, both correlations often produced
negative values for percent conversion of cellulose II for experimental samples.
Therefore, quantifying cellulose I to II conversion through x-ray diffraction proved to be
imprecise.
An additional correlation was used to determine cellulose I to II conversion,
which uses absolute peak intensities at 15° (2θ) (Mansikkamaki et al.,2007). Intensities
at 15° are useful for they are present in cellulose I and steadily decrease to the baseline in
cellulose II. The conversion of cellulose II can be approximated by Equation 8.
I P = (1−α )I1 + αI 2 8
In equation 8, IP is the intensity of the sample at 15°, and I1 and I2 are intensities of pure
cellulose I and II at 15°, respectively. This correlation did not produce results that were
different than correlations presented above. However, the average percent error or
measured versus actual percent cellulose II was 1.35%. Once more, this correlation
produced negative values for percent conversion cellulose II for experimental samples.
Thus, only large cellulose I to II conversions can be measured by XRD.
Results from x-ray diffraction patterns show that almost no conversion took place
in any of the conditions outlined above in Table 12. This was primarily due to the time
limitation imposed on all mercerization experiments. There simply was not enough time
for the NaOH, KOH, or NH4OH to convert cellulose I to II. An x-ray diffraction pattern for NaOH at 100°C is given in the Figure 28. It shows that no conversion was observed 95 in NaOH at the highest temperature—even for five minutes. Similar results were observed for the other bases and temperatures used. All additional XRD patterns are presented in Appendix D (Figures 38 to 45).
y
it 5 min s n e t
In 2 min
1 min
0 5 10 15 20 25 30 35 40 45
2θ
Figure 28. X-ray diffraction patterns for Cellulose in 3.0 N NaOH at 100°C for 1, 2, and 5 minutes.
Additional mercerization experiments were conducted at longer times to examine when NaOH starts to have a significant effect. From Figure 21, it is evident that, in order to achieve significant cellulose I to II conversion, mercerization times must be mercerized longer than a half hour. Since, thirty minutes is much longer than lye peeling conditions, mercerization of cellulose in the cell walls does not appear to be a plausible mechanism for lye peeling. 96
2 hours
y 1.5 hours it s n e
t 1 hour In 30 min
15 min
0 5 10 15 20 25 30 35 40 45 2θ
Figure 29. X-Ray diffraction patterns for Cellulose I at 100°C for extended times.
Results for mercerization experiments at conditions similar to tomato peeling conditions were ineffective. A previous study has shown that complete mercerization can exist for NaOH concentrations as low as 2.5 N (Dinand et al.,2002). Most mercerization experiments are carried out at room temperature for many hours at high NaOH concentrations. Carrying out mercerization experiments at higher temperatures did not prove to be beneficial. The cellulose I to II transition required more time for complete conversion (see Figure 29). Therefore, cellulose I to II conversion in collenchymatous cell walls does not appear to be a possible mechanism for tomato lye peeling. In addition, the role of NaOH is not due to osmotic effects. This is shown in Table 15
(Chapter 7). Sodium chloride (NaCl) was used as a replacement for NaOH; however, 97 NaCl did not produce increased average tomato scores. Thus, NaOH can be attributed to the breakdown of pectin in the middle lamella.
Chapter Seven
Pretreatment with Organic Solvents: Preliminary Studies
Roma tomatoes of the varieties H9704, H9423, 611, and OX323 were used in these experiments. Initial tomato lye peeling experiments using neat organic solvent pretreatments were conducted in 2005. Tomatoes were first dipped in an organic solvent followed by a 30 second hold, and then 60 second NaOH dip at 85±2°C. After the NaOH dip and a 60 second NaOH reaction time, tomatoes were placed in a mechanical tumbler, which was rotated 40 times at about 1.5 revolutions per second, and scored. Additional information pertaining to times and temperatures of the pretreatment dip are given in each table.
During these preliminary studies, a slightly different system of scoring tomatoes was used as compared to that in Chapters 3, 4, 5, and 8. The method of scoring tomatoes was as follows:
• 0 – No cuticle removal.
• 1 – Greater than 1% but less than 50% of cuticle removal.
• 2 – Greater than or equal to 50% but less than 100% of cuticle removal.
98 99 • 3 – 100% of cuticle removal, which also includes overpeeled tomatoes.
(There was no category 4 for overpeeling as in later studies.)
All organic solvent pretreatments were compared to a control for every NaOH dip
time and temperature. The control was defined as the average and standard deviation
obtained when tomatoes were subjected to a particular NaOH dip time and temperature.
This control was compared to the average and standard deviation obtained when tomatoes
were subjected to a pretreatment dip and the same NaOH dip time and temperature.
Effective solvents were defined as increasing the average score to 3.00.
Solvent pre-dip and NaOH times were varied in a limited number of peeling
studies (Table 13). Results suggest that both the pre-dip and lye dip times have an effect upon average scores, which is supported by earlier studies (Floros et al. 1987). From
Table 13, the most important factor in peeling appears to be the selection of the solvent for lye pretreatment. Therefore, in order to reduce variability in experiments, the solvent pretreatment and NaOH dip times during were kept constant at 60 seconds for most of the initial peel studies. 100
Table 13. Tomato lye peeling studies with solvent pretreatment and variable solvent time and temperature. Pretreatment reaction time is 30 seconds. NaOH concentration is 3.0 N. NaOH temperature is 85±2°C. Solvent Solvent NaOH No. of Tomatoes Avg. Score ± Std. Deviation Temp. Dip Time Dip Time Solvent Solvent Solvent (°C) (s) (s) Control Pre-Dip Control Pretreatment a Octanoic 25 45 30 8 12 0.00 ± 0.00 2.42 ± 1.08 Acid 45b 30 30 9 6 1.78 ± 0.44 2.5 ± 0.55 45 b 30 45 9 6 1.78 ± 0.44 3.00 ± 0.00 b 45 30 60 9 6 1.78 ± 0.44 3.00 ± 0.00 Chloroform 22 b 30 60 9 9 0.22 ± 0.44 1.33 ± 0.71 22 b 60 60 9 6 0.22 ± 0.44 1.5 ± 0.84 1-Octanol 25a 45 30 8 10 0.00 ± 0.00 0.00 ± 0.00 25a 45 45 4 10 0.00 ± 0.00 0.70 ± 0.48 a – Tomato variety is H9704. b – Tomato variety is H9423.
Several classes of solvents were examined as pretreatment solvents. The solvents tested included carboxylic acids (Table 14), treatments with NaCl (Table 15) octanoic acid added directly to NaOH solutions (Table 16), chlorinated solvents (Table 17), dimethyl sulfoxide (DMSO), 2-undecanone (Table 18), dibutyl phthalate (an aromatic diether), tetradecane (Table 19), and a mixture of chloroform and hexanoic acid (Table
20). 101
Table 14. Tomato lye peeling studies using carboxylic acids as pretreatments. Pretreatment dip time and reaction times were 60 seconds each. NaOH dip time and reaction times were 60 seconds. NaOH temperature was 85±2°C. Solvent No. of Tomatoes Avg. Score ± Std. Deviation Temp. NaOH Solvent Solvent Solvent (°C) Conc., N Control Pre-Dip Control Pretreatment Hexanoic Acid a 90 0.75 12 6 1.25 ± 0.97 2.00 ± 1.10 45 0.75 6 6 0.17 ± 0.41 0.17 ± 0.41 90 1.50 9 9 0.44 ± 0.53 2.89 ± 0.33 45 1.50 6 6 0.67 ± 0.52 1.50 ± 1.05 90 2.25 9 9 1.22 ± 0.67 2.78 ± 0.67 45 2.25 6 6 0.83 ± 0.75 2.33 ± 0.82 90 3.00 9 6 1.33 ± 1.00 3.00 ± 0.00 45 3.00 6 6 1.17 ± 0.98 1.83 ± 0.98 Octanoic Acid b 85 0.75 9 6 0.67 ± 0.71 1.83 ± 0.41 65 1.25 6 6 0.33 ± 0.52 2.33 ± 0.82 85 1.50 9 9 1.89 ± 0.60 3.00 ± 0.00 85 2.25 9 3 2.44 ± 0.73 3.00 ± 0.00 85 3.00 3 3 2.33 ± 1.15 3.00 ± 0.00 Decanoic Acid 85 c 0.75 12 6 0.42 ± 0.51 2.00 ± 0.89 85 c 1.50 12 6 1.75 ± 1.06 3.00 ± 0.00 85 c 2.25 12 6 3.00 ± 0.00 3.00 ± 0.00 87 c - - 6 - 0.00 ± 0.00 85 a 0.75 12 12 0.08 ± 0.29 1.92 ± 1.31 85 a 1.50 12 12 0.33 ± 0.49 2.5 ± 1.17 85 a 2.25 12 3 0.42 ± 0.51 2.67 ± 0.58 85 a 3.00 12 3 1.08 ± 0.90 3.00 ± 0.00 a – Tomato variety - mixture of H9423 and OX323. b – Tomato variety - H9423. c – Tomato variety - 611.
Table 15. Tomato lye peeling studies using an octanoic acid pretreatment followed by a treatment with a salt solution near solubility. Tomato variety was H9423. Pretreatment dip time and reaction time were 60 seconds each. NaOH and salt dip time and reaction times were 60 seconds each. Octanoic acid and NaOH temperature was 85±2°C. Average ± Concentration, Number of Standard Solvent Treatment N Tomatoes Deviation Octanoic Acid a NaOH 0.75 6 1.83 ± 0.41 NaOH 1.50 9 3.00 ± 0.00 NaOH 2.25 3 3.00 ± 0.00 NaOH 3.00 3 3.00 ± 0.00 NaCl 4.11 6 0.50 ± 0.84
102
Table 16. Tomato lye peeling studies using a pretreatment of octanoic acid mixed with NaOH. Tomato varieties were H9423 and OX323. Pretreatment dip time and reaction time were 30 seconds each. Pretreatment temperature was 85°C. NaOH dip time and reaction times were 60 seconds each. NaOH temperature was 85°C. Number of Tomatoes Avg. Score ± Std. Deviation
NaOH Solvent Solvent Solvent Conc., N Control Pre-Dip Control Pretreatment Octanoic Acid a 0.50 6 6 0.00 ± 0.00 0.00 ± 0.00 1.25 6 6 0.33 ± 0.52 0.67 ± 0.52 2.00 6 6 0.67 ± 0.52 2.17 ± 0.98 Octanoic Acid b 0.50 6 6 0.00 ± 0.00 0.17 ± 0.41 1.25 6 6 0.33 ± 0.52 0.33 ± 0.52 2.00 6 6 0.67 ± 0.52 1.17 ± 0.75 a – 5 moles Octanoic Acid per 100 moles Sodium Hydroxide. b – 10 moles Octanoic Acid per 100 moles Sodium Hydroxide.
Table 17. Tomato lye peeling studies using a chlorinated solvent pretreatment. Tomato variety was OX303. Pretreatment dip time was 60 seconds and reaction time was 30 seconds. NaOH dip time and reaction times were 60 seconds each. NaOH temperature was 85°C. Solvent NaOH No. of Tomatoes Avg. Score ± Std Deviation Temp. Conc., Solvent Solvent Solvent (°C) N Control Pre-Dip Control Pretreatment Chloroform 45 0.75 12 6 1.25 ± 0.97 2.33 ± 0.82 45 0.75 6 6 0.17 ± 0.41 1.17 ± 0.41 45 1.50 9 6 0.44 ± 0.53 3.00 ± 0.00 45 1.50 6 6 0.67 ± 0.52 1.50 ± 0.84 45 2.25 6 6 0.83 ± 0.75 2.50 ± 0.55 45 2.25 9 6 1.22 ± 0.67 3.00 ± 0.00 45 3.00 6 6 1.17 ± 0.98 2.33 ± 0.52 45 3.00 9 6 1.33 ± 1.00 3.00 ± 0.00 a, b 22 3.00 9 9 0.22 ± 0.44 1.33 ± 0.71 a 22 3.00 9 6 0.22 ± 0.44 1.50 ± 0.84 Dichloroethane 50 0.75 6 9 0.00 ± 0.00 0.33 ± 0.5 50 1.50 6 9 0.00 ± 0.00 1.33 ± 0.87 50 2.25 6 9 0.00 ± 0.00 2.22 ± 0.97 50 3.00 6 9 1.17 ± 0.75 1.89 ± 0.78 2-Chloropropionic 85 0.75 9 6 0.22 ± 0.44 1.50 ± 1.05 Acid 85 1.50 9 6 0.67 ± 0.87 2.17 ± 1.17 85 2.25 9 6 0.33 ± 0.50 2.67 ± 0.82 85 3.00 9 6 1.11 ± 0.78 3.00 ± 0.00 a – Solvent dip time was 30 seconds. b – Tomato variety - H9423. 103
Table 18. Tomato lye peeling studies using dimethyl sulfoxide and 2-undecanone as pretreatments. Pretreatment dip time was 60 seconds. Pretreatment reaction time was 30 seconds. NaOH dip time and reaction times were 60 seconds each. NaOH temperature was 85°C. Solvent NaOH No. of Tomatoes Avg. Score ± Std. Deviation Temp. Conc., Solvent Solvent Solvent (°C) N Control Pre-Dip Control Pretreatment Dimethyl 55 0.05 6 9 0.17 ± 0.41 0.11 ± 0.33 Sulfoxide a 55 1.25 6 9 1.53 ± 1.37 1.89 ± 1.27 55 2.00 6 9 2.00 ± 0.89 2.69 ± 0.71 2-Undecanone b 85 0.75 9 6 0.22 ± 0.44 0.83 ± 0.41 85 1.50 9 6 0.67 ± 0.87 1.00 ± 0.63 85 2.25 9 6 0.33 ± 0.50 1.17 ± 0.75 85 3.00 9 6 1.11 ± 0.78 1.5 ± 0.55 a – Tomato variety - 611. b – Tomato variety - mixture of H9423 & OX323.
Table 19. Tomato lye peeling studies using dibutyl phthalate and tetradecane as pretreatments. Tomato variety was H9704. Pretreatment dip time was 60 seconds. Pretreatment reaction time was 30 seconds. NaOH dip time and reaction times were 60 seconds each. NaOH concentration was 3.00 N. NaOH temperature was 85°C. Solvent Solvent No. of Tomatoes Avg. Score ± Std. Deviation Temp. Dip Time Solvent Solvent Solvent (°C) (s) Control Pre-Dip Control Pretreatment Dibutyl 66 30 9 6 1.11 ± 0.60 1.33 ± 0.52 Phthalate 66 60 9 6 1.11 ± 0.60 1.83 ± 0.75 Tetradecane 69 60 9 3 1.11 ± 0.60 0.00 ± 0.00 63 60 9 3 1.11 ± 0.60 0.00 ± 0.00
Table 20. Tomato lye peeling studies using a solvent mixture of chloroform and hexanoic acid in the pretreatment. Tomato varieties were H9423 and OX323. Pretreatment temperature was 80°C. Pretreatment dip time was 60 seconds. Pretreatment reaction time was 30 seconds. NaOH dip time and reaction times were 60 seconds each. NaOH temperature is 85°C. No. of Tomatoes Avg. Score ± Std. Deviation NaOH Solvent Solvent Solvent Conc., N Control Pre-Dip Control Pretreatment 25% Chloroform, 0.75 9 9 0.33 ± 0.71 1.78 ± 0.93 75% Hexanoic Acid 1.50 9 9 0.67 ± 0.71 2.56 ± 0.73 2.25 9 6 1.33 ± 1.00 3.00 ± 0.00 3.00 9 6 1.00 ± 0.87 2.83 ± 0.41
A list of the solvents, and some physical and chemical properties are included in
Table 21 below. This table also identifies which solvents are categorized as Generally
Regarded As Safe (GRAS) chemicals for use as food additives. The United States Food 104 and Drug Administration (FDA) and the Center for Food Safety and Applied Nutrition
(CFSAN) have jointly established a program known as the Priority-based Assessment of
Food Additives (PAFA). This is an on-going program that documents chemicals that are used in the food processing industry. The database includes chemicals which are GRAS; the entire database is generally known as Everything Added to Food in the United States
(EAFUS).
Table 21. Physical Properties of the solvents used in lye peeling studies and their PAFA Status. Boiling Vapor Solubility in Point Flashpoint Pressure a, Water (g/L PAFA b Status as a Solvent (°C) (°C) 20 °C, Pa 20 °C) GRAS c Chemical Hexanoic acid 205 102 2.772 11 GRAS Chemical Octanoic acid 239 110 0.278 0.68 GRAS Chemical Decanoic acid 269 112 0.0385 insoluble GRAS Chemical Chloroform 61.7 none 20996.5 8.22 No Reported Use d Dichloroethane 83 13 24383.4 8.1 Not Listed 2-Chloropropionic Acid 186 107 141.3f miscible Not Listed 1-Octanol 194 81 6.379 0.54 GRAS Chemical Dimethyl Sulfoxide 189 95 57.1 miscible Reported Use e 2-Undecanone 231 98 132.1g insoluble GRAS Chemical Dibutyl phthalate 340 157 0.0018 0.0112 Not Listed Tetradecane 251 107 0.921 insoluble Not Listed a - Calculated using UNIFAC method on CHEMCAD b - Priority-based Assessment of Food Additives c - Generally Regarded As Safe d - No toxicology information for foods found. e - Reported use of the chemical, but there is no assigned toxicology literature search f - Vapor pressure measured at 25 °C g - Vapor pressure measured at 68.1 °C using Antoine’s Equation developed from NIST at lowest acceptable temperature
In the preliminary studies, the most effective solvents appear to be the carboxylic acids and chloroform (Tables 14, 15, 17, and 20). Solvents that marginally increased peeling scores were DMSO and dibutyl phthalate (Tables 18 and 19). All other solvent pretreatments increased average peel scores, with the exception of tetradecane (Table 19).
Tetradecane was the only solvent that reduced tomato peel scores. 105 Acids (or thiols) and multihalogenated solvents were expected to be good candidates for dissolution of compounds contain ketone functional groups (found in naringenin chalcone) (See Robbins Chart in Appendix A), generally exhibiting negative deviations from ideality. Acids are also good solvents for solutes containing alcohol functional groups (found in the triterpenoids, i.e. amyrins, and OH substituted paraffin), whereas multihalogenated solvents exhibit positive deviations from ideality. Both these solvents exhibit positive deviations from ideality for paraffin solutes (Robbins,1980;
Frank et al.,1999). Carboxylic acids and chloroform were found to be an effective solvent pretreatment and are also expected to be good solvents for ketones. These results may indicate that dissolution of the naringenin chalcone component of the cuticle may be critical in selecting solvent pretreatments for lye peeling.
Carboxylic acids were found to be the most effective solvent class for pre- treatment of lye-peeled tomatoes. Use of carboxylic acids often resulted in complete peel or overpeel of tomatoes. Hexanoic, octanoic, and decanoic acids were examined as possible carboxylic acids. All three proved to be effective pretreatment solvents.
Neumann et al. (1978) also found carboxylic acids as an effective pre-peeling aid . They found that the best pre-peeling aid was not pure solvent, but a 0.5% mixture of octanoic acid in water.
It has been proposed that micelles formed by the carboxylic acid aid by the removal of cuticular waxes, thus enabling the sodium hydroxide to diffuse into the cuticle more readily. Using a micellular solution would eliminate the need for the use of pure octanoic acid. Thus, octanoic acid was added directly to the sodium hydroxide container creating sodium octanoate, a known surfactant. Small amounts of octanoic acid were 106 added directly to the sodium hydroxide container in a molar ratio of 0.05 and 0.10 moles
octanoic acid per mole of NaOH. It was found that a molar ratio of 0.05 improved peeling, but a ratio of 0.10, through increasing peelability, was not as effective as the ratio of 0.05 (see Table 16). This may be due to the chemical reaction of octanoic acid and NaOH to produce water and sodium octanoate.
The effectiveness of octanoic acid was examined in conjunction with salt
solutions. Table 15 shows tomato peeling results for octanoic acid pretreatments
followed with caustic treatments and a salt treatment. Salt treatments were of interest as
it was proposed that tomato peeling was due to osmotic shock of collenchymatous cells.
Octanoic acid interacts with tomato cuticles in order to render NaOH more effective at
removing the cuticle. However, a NaCl treatment after an octanoic acid pretreatment was
not enough to peel tomatoes. Therefore, tomato peeling cannot be attributed to osmotic
effects due to ionic strength.
All of the chlorinated solvents tested improved the peeling scores to a large extent
(Table 16). Dichloroethane was less effective than either chloroform or 2-
chloropropionic acid. The potential usefulness of these solvents in an industrial setting is severely limited due to their poor properties such as high volatility, and toxicity
(Neumann et al.,1978).
There was little to no improvement in peeling score with the use of DMSO as a pretreatment. The same can be said of 2-undecanone. However, the numbers do not accurately convey the physical changes that occurred in the tomato when 2-undecanone was used. In many cases the tomatoes emerged from the mechanical peeler with some 107 skin still attached (as the peeling scores show), but the interior of the fruit was dissolved
to some degree. This degradation increased as the NaOH concentration increased.
The increase of peelability by using dibutyl phthalate was minimal. Dibutyl
phthalate would be a poor choice for foods because it is a teratogen. Such results were
not expected since dibutyl phthalate has two ester functional groups. Dibutyl phthalate is
different from other solvents tested because it possesses aromaticity. Thus, aromatic
compounds do not appear to increase the peelability of tomatoes.
Tetradecane was also investigated since epicuticular waxes are composed of long
chain alkanes (Bauer et al.,2004; Bauer et al.,2004) and it was expected that a long chain alkane could solubilize those waxes. However, this was the only solvent that adversely affected the tomato peeling scores. These results suggest that the tetradecane forms an added coating around the tomato cuticle, thickening the waxes already present as opposed to solubilizing them, thus making the tomato more difficult to peel. Such results clarify the barrier the cuticular waxes present to diffusion of NaOH through the cuticle.
The hexanoic acid/chloroform solution was very effective as a pretreatment. The
use of a mixture of chloroform and hexanoic acid allowed for the combination of two
solvents that had proved to be effective alone. Mixtures also allowed the use of a higher
temperature than was possible with chloroform alone. At 80°C, vapor rich in the more
volatile chloroform were formed, making it difficult to maintain the 1:3 ratio of
chloroform to hexanoic acid.
These initial solvent pretreatment studies enabled the researcher to identify
classes of solvents that were favorable to lye peeling. Carboxylic acids were superior as 108 their pretreatments resulted in cuticle removal at low NaOH concentrations. Though the chlorinated solvents proved to be effective, their use in food manufacturing is unfeasible.
Therefore, since carboxylic acids were effective pretreatments, additional studies involving carboxylic acids were conducted (see Chapter 8). Water—carboxylic acid mixtures as well as single-phase aqueous solutions of carboxylic acids were also tested.
It is of interest to determine if the alkyl chain of the carboxylic acid or the carboxylic acid function group is affecting lye peeling. Thus, additional eight-carbon solvents with different functional groups such as ketones, aldehydes, and alcohols were also tested.
Chapter Eight
Solvent Pretreatments Used in Lye Peeling
Utilizing organic solvents as pretreatments for lye peeling has been the focus of earlier research (Neumann et al.,1978; Das & Barringer,1999). Neumann et al. (1978) performed an initial solvent screening of various organic solvents and surfactants and found that carboxylic acids were superior. Results found that carboxylic acids— especially octanoic acid—were superior to the other solvents investigated. Hexanoic and decanoic acid, as well as a mixture of hexanoic, heptanoic, and octanoic acid were also effective pretreatments for tomato lye peeling. Das & Barringer (1999) conducted studies with various common industrial, organic solvents. Thus, solvents were tested to determine which part of the solvent (functional group or alkyl chain) is affecting tomato peelability. Also, the effect of chain length for two functional groups—the ketone and the carboxylic acid—was also investigated.
109 110
Experimental Methods
Roma tomato cultivars TSH-8, 9423, and 9601, which were grown in 2006, were used for this study. NaOH solutions were prepared from 0.25N to 4.25N. The following figure depicts the lye peeling process used in these experiments.
Tomatoes Brought from Field
Washing / Inspection— damaged or diseased Discarded tomatoes were discarded
Pretreatment Dip (water control or solvent), 80±1°C for 60 seconds
Pretreatment Reaction Time for 30 seconds a
NaOH Dip, 80±1°C for 60 seconds
NaOH Reaction Time for 60 seconds
Cuticle Removal by Mechanical Tumbler
Tomatoes are Scored
Figure 30. Lye peeling process used to analyze effectiveness of various organic solvents.
111
Stainless steel beakers (2000 mL) were filled with approximately 1700 mL of
water/organic solvents for pretreatment and varying NaOH solutions for peeling. These
beakers were then heated in constant temperature water baths to a temperature of 80±1°C.
Three tomatoes at a time were randomly chosen and placed in mesh baskets and first
subjected to a pretreatment in either water (control) or a solvent (or water/ solvent
mixture) for 60 seconds, removed from the pretreatment beaker and held for 30 seconds,
and then a solution of NaOH for 60 seconds. Tomatoes were held after removal from the
caustic solution for a 60 second reaction time before being placed in the tumbler and scored.
Tomatoes were first peeled with a water pretreatment control followed by a dip in
increasing NaOH concentrations until at least 15 tomatoes were completely peeled or the
concentration reached 4.25 N (17 w/v %). Tomatoes were then peeled with an organic
solvent pretreatment followed by dips in increasing NaOH concentrations until either at
least 15 were completely peeled or the NaOH concentration reached 4.25 N. For
incomplete peeling with the water and organic solvent pretreatment, the average scores at
a NaOH concentration of 4.25 N was compared. For complete peeling with a water and
organic solvent pretreatments, the difference in NaOH concentrations were compared.
Variation of Functional Group
Neumann et al. (1978) found that the most effective of fifty solvents tested were
the carboxylic acids, specifically octanoic acid—a saturated fatty acid. For comparison,
three additional eight-carbon organic solvents with saturated alkyl chains and varying 112 functional groups were investigated—1-octanol, 2-octanone, and octyl aldehyde in addition to octanoic acid. Results from these solvents give insight into the effect of the functional group on peeling. Alcohols and ketones of shorter alkyl chain length were previously studied (Das & Barringer,1999). Thus, to assess the importance of the polar and non-polar segment of octanoic acid, peeling studies on other eight-carbon solvents were performed. Furthermore, studies on hexanoic acid, decanoic acid, and 2- undecanone assessed the effect of solvent chain length on tomato peelability for the ketone and carboxylic acid functional groups.
The maximum NaOH concentration used was 4.25 N. Both the average tomato
score and the difference in NaOH concentration from controls were considered. Results
for the eight-carbon neat solvents with one functional group are listed in Table 22.
Table 22. Tomato peeling results for a pretreatment of neat organic solvents with an alkyl chain length of eight carbons. Water Pretreatment Organic Solvent Pretreatment NaOH No. of NaOH No. of Solvent Conc., N Tomatoes AVG ± SD Conc., N Tomatoes AVG ± SD Octanoic Acid a 3.25 15 3.13 ± 0.35 1.75 18 3.00 ± 0.00 2-Octanone b 4.25 18 1.89 ± 1.02 4.25 18 2.06 ± 1.16 Octyl Aldehyde b 4.25 15 2.53 ± 0.74 4.25 18 1.60 ± 1.30 1-Octanol c 4.00 15 3.00 ± 0.00 4.00 15 2.60 ± 0.74 a – Tomato Variety - 9423. b – Tomato variety - TSH-8. c – Tomato variety - 9601.
Carboxylic Acids Above & Below Solubility
Octanoic acid was the most effective solvent as it resulted in the greatest decrease
in the required NaOH concentration while keeping the average tomato score above 3.0
(completely peeled). 2-Octanone and octyl aldehyde were studied with tomatoes that
could not be completely peeled at a NaOH concentration of 4.25 N and a pretreatment of 113 water. Thus, peel scores with these solvents were compared with controls. Results
showed that both 2-octanone and octyl aldehyde were not effective as they did not increase peel scores when compared to a water pretreatment. Tomato scores for 1- octanol was examined at a NaOH concentration of 4.00 N and were determined to be ineffective as it did not enhance average tomato scores. The ketone, aldehyde, and alcohol functional groups appear to be either ineffective pretreatments or inhibit tomato
peeling. A straight chain alkane was not investigated as tetradecane proved to severely
inhibit tomato lye peeling (see Table 19, Chapter 7).
Table 23. Solubilities of various carboxylic acids in water at 80°C. Carboxylic Acid Solubility (g/kg H2O) Hexanoic Acid 10.8 a Octanoic Acid 0.68 a, 0.70 b Decanoic Acid 0.10 b a –Source: (Huq,2005) b –Source: (Khuwijitjaru et al.,2002)
Carboxylic acids are effective, but are their use warranted as neat solvents? Table
23 presents solubilities for hexanoic, octanoic, and decanoic acids. Octanoic acid/ water
mixtures were investigated above and below its solubility limit at pretreatment
temperatures (80°C). Table 24 summarizes tomato peeling results for varying octanoic
acid concentrations. All pretreatments with mixtures above the solubility point were
effective. However, the solution below the solubility point (0.607 g octanoic acid/kg
H2O) was no more effective than a water pretreatment. These results were similar to those of Neumann et al. (1978) where octanoic acid pretreatment at 4.25 N at 80°C, above the solubility limit was also found more effective than other neat solvents tested 114 (Table 22). As long as octanoic acid is present above its solubility point in water, its effectiveness is the same as that of the neat solvent.
Table 24. Tomato peel scores for pretreatments with varying concentrations of octanoic acid in water. Water Pretreatment Organic Solvent Pretreatment Octanoic Acid NaOH # of NaOH # of Concentration Conc., N Tomatoes AVG ± SD Conc., N Tomatoes AVG ± SD Neat Solvent a 3.25 15 3.13 ± 0.35 1.75 18 3.00 ± 0.00 Layer b 4.25 15 2.53 ± 0.74 2.00 15 3.13 ± 0.35 b 10.141 g/kg H2O 4.25 15 3.00 ± 0.00 2.00 21 3.00 ± 0.00 b 5.041 g/kg H2O 4.25 15 3.00 ± 0.00 2.00 15 3.00 ± 0.00 b 0.607 g/kg H2O 4.25 18 2.83 ± 0.62 4.25 15 2.87 ± 0.52 a – Tomato cultivar - TSH-8. b – Tomato cultivar - 9423.
Carboxylic acids were a class of solvents that proved to be effective at enhancing tomato scores by decreasing the required amount of NaOH needed to peel tomatoes.
Similar trends were observed when hexanoic and decanoic acids were studied (Tables 25 and 26). These acids were effective when present above their solubility points (see Table
23). When hexanoic, octanoic, and decanoic acid pretreatments were prepared as a uniform layer on top of the water for the pretreatment step, the NaOH concentration needed to completely peel tomatoes was reduced almost in half in all cases. It appears that the efficacy decreases as alkyl chain increases (Table 25). Conversely, these carboxylic acids are ineffective when they are below the solubility point (Table 26).
Table 25. The effect of carboxylic acids above the solubility point as a uniform layer on top of the water. Tomato variety was TSH-8. Water Pretreatment Organic Solvent Pretreatment NaOH # of NaOH # of Solvent Conc., N Tomatoes AVG ± SD Conc., N Tomatoes AVG ± SD Hexanoic Acid 4.25 15 2.60 ± 0.74 1.50 15 3.00 ± 0.00 Octanoic Acid 4.25 15 2.53 ± 0.74 2.00 15 3.13 ± 0.35 Decanoic Acid 4.25 15 2.60 ± 0.74 2.25 15 3.13 ± 0.35
115
Table 26. The effect of completely dissolved carboxylic acids in water below the solubility point. Tomato variety is TSH-8. Water Pretreatment Organic Solvent Pretreatment Carboxylic Acid NaOH # of NaOH # of Concentration Conc., N Tomatoes AVG ± SD Conc., N Tomatoes AVG ± SD Hexanoic Acid 4.25 18 2.39 ± 0.85 4.25 15 2.78 ± 0.65 7.028 g/kg H2O Octanoic Acid 4.25 18 2.83 ± 0.62 4.25 15 2.87 ± 0.52 0.607 g/kg H2O Decanoic Acid 4.25 18 2.83 ± 0.62 4.25 18 2.80 ± 0.56 0.100 g/kg H2O
Solubilities of the three carboxylic acids are presented in Table 26 below. Carboxylic
acid solubility decreases with increasing chain length. For alkyl chain lengths of four carbons or less, the corresponding carboxylic acids are infinitely soluble at 80°C
(Huq,2005).
Octanoic acid that came in contact with the cuticle had a large effect upon average tomato scores as it enabled NaOH to be more effective at lower concentrations. Thus, octanoic acid possesses the capability to affect the cuticle in order to facilitate diffusion of NaOH during the lye dip. Tomato cuticles develop clusters of naringenin chalcone as they ripen (Luque et al.,1994). Naringenin chalcone appears immediately before the red stage of maturity (Bauer et al.,2004). It has been reported that organic acids such as benzoic and salicylic acid can easily penetrate through these clusters in the cuticle
(Jeffree,1996). The same phenomenon for the penetration of benzoic and salicylic acid might be at work for octanoic acid.
116
Effectiveness of Ketones
The effect of chain length for both ketone and carboxylic acid functional groups was investigated. Results show that chain length in ketones has no appreciable effect on tomato peel scores. Table 27 displays peel scores for ketones of varying chain length.
Undecanone did not improve the peelability above that of octanone. One might assume that a more non-polar substance would possess a greater ability to interact with the non- polar cuticular components (Frank et al.,1999). Effectiveness of the organic solvent does not appear to be due to the non-polar segment.
Table 27. Tomato peeling scores for the effect of alkyl chain length on the ketone functional group. Solvents existed as neat solvents. Tomato variety was TSH-8. Water Pretreatment Organic Solvent Pretreatment NaOH # of NaOH # of Solvent Conc., N Tomatoes AVG ± SD Conc., N Tomatoes AVG ± SD 2-Octanone 4.25 18 1.89 ± 1.02 4.25 18 2.06 ± 1.16 2-Undecanone 4.25 30 2.23 ± 0.86 4.25 15 1.73 ± 1.28
Temperature Variation with Carboxylic Acids
Because the carboxylic acids were identified as effective solvents, a factorial
experiment was conducted in which two factors, pretreatment type (water, hexanoic acid,
and octanoic acid) and temperature, were factors evaluated at three different levels.
Thus, the experiment was a 32 Design. All treatments were subjected to a NaOH
concentration of 2.0 N at each temperature. The levels of the factors and average peel
scores are displayed in Figure 31. Hexanoic acid and octanoic acid pretreatments refer to
a uniform layer of pure carboxylic acid on top of the water. Since three tomatoes were
subjected to the same condition at one time, each dip was evaluated as a duplicated 117 replicate. The response for each replicate was the average for all three tomatoes tested for that dip. The ANOVA analysis revealed that pretreatment type, temperature, and the interaction between them were significant as the P-values were 0.0002, <0.0001, and
<0.0001, respectively. Table 28 shows results from the ANOVA analysis.
4.00 Water 3.50 Hexanoic Acid
e Octanoic Acid r o
c 3.00 S o t 2.50 a m
o 2.00 e T
ag 1.50 ver
A 1.00
0.50
0.00 40 50 60 70 80 90 100 Temperature, °C
Figure 31. Average tomato scores for each of the nine conditions evaluated in the 32 Design. Tomato variety was TSH-8. Hexanoic and octanoic acid pretreatments consisted of a layer of neat organic acid on top of the water. NaOH concentration was 2.0 N. Error bars represent the standard deviation of five duplicated responses.
Table 28. ANOVA analysis for the 32 experimental design (results presented in Figure 31). Source of Sum of Degrees of Mean Variation Squares Freedom Square F0 Value P-Value Pretreatment Type 13.79 1 6.90 36.75 < 0.0001 Temperature 60.01 2 30.01 159.91 < 0.0001 Interaction 7.35 4 1.84 9.79 < 0.0001 Error 6.76 36 0.19 Total 87.91 44
118 Assumptions of constant variance and normal distribution of the residuals were validated through visual inspection of the appropriate diagnostic graphs in Figures 32 and 33.
Figure 32. Normal probability plot of the residuals for the 3² experiment.
Figure 33. Residuals versus average score for each of the nine conditions tested in the 3² experiment. 119
Table 29 displays the results from Tukey’s multiple comparison test. All
comparisons represented with a plus sign were significant whereas all comparisons
represented with minus signs were found to be insignificant. Three noteworthy
conclusions can be drawn from the results in Table 28. First, at 90°C, the pretreatment
(water of carboxylic acid) has no significant affect upon peeling. At this high
temperature, the primary mechanism for lye peeling may be due to thermal effects. A
study found that hypodermal cells can be disrupted by exposing tomatoes to steam
treatments (Floros & Chinnan,1988). Second, tomato peel scores was not significantly
different between hexanoic and octanoic acid when the temperature was 75°C. However,
both carboxylic acid pretreatments produced tomato peel scores significantly greater than
a pretreatment of water at 75°C. Third, at 50°C, the only significant difference is the average score between a pretreatment of water and octanoic acid. At these low temperatures, lye peeling is severely inhibited.
120
Table 29. Multiple comparisons for all averages using Tukey’s test. Significant differences are represented as plus (+) signs and insignificant differences are represented as minus (-) signs. Tomato variety was TSH-8. NaOH concentration was 2.0 N. Octanoic acid and hexanoic acid pretreatments were a uniform layer of neat solvent on top of the water. Pretreatment Type Water Hexanoic Acid Octanoic Acid Temperature 50°C 75°C 90°C 50°C 75°C 90°C 50°C 75°C 90°C
Water 50°C - - + - + + + + + 75°C - - + - + + + + + 90°C + + - + - - + - - Hexanoic Acid 50°C - - + - + + + + + 75°C + + - + - - + - + 90°C + + - + - - + - - Octanoic Acid 50°C + + + + + + - + + 75°C + + - + - - + - + 90°C + + - + + - + + -
Carboxylic Acids at Room Temperature
Octanoic acid and hexanoic acid proved to be effective solvents for the
pretreatment of tomatoes for lye peeling. Previous experiments have used such
pretreatments at lye peeling temperatures (80°C). Could carboxylic acids be used at
room temperature after a pretreatment with water? Thus, a single factor experiment was
conducted in order to determine the effectiveness of a 1 second dip in octanoic or
hexanoic acid at room temperature after a water pretreatment. This was tested at three
levels—no dip, hexanoic acid, and octanoic acid. This experiment represents the
industrial application in which tomatoes are sprayed with carboxylic acids after leaving a water pretreatment tank immediately prior to the caustic dip. 121 Roma tomatoes of the cultivar TSH-8 were used for this single factor experiment.
Three tomatoes at a time (a single replicate) were placed in mesh baskets and submerged
first in de-ionized water for 60 seconds, which was followed by a 1 second dip in a
carboxylic acid at 23°C (room temperature) and a 30 second reaction time, and then a 60
second dip in 4.0 N NaOH. All solution temperatures were 80±1°C. After the NaOH dip
and a 60 second reaction time, tomatoes were placed in the tumbler and scored. The data
for the experiment is represented in Table 30 and Figure 34 below with an ANOVA
analysis in Table 31.
Table 30. Data from single factor experiment examining the post pretreatment effect. Tomato variety was TSH-8. Sodium hydroxide concentration was 4.0 N. Type of dip refers to a 1 second dip in a neat solvent.
Number of Treatment Average ± Standard
Type of Dip Replicates Totals Deviation
None 5 6.67 1.33 ± 0.24
Hexanoic Acid 5 8.00 1.60 ± 0.76
Octanoic Acid 5 14.33 2.87 ± 0.30
122
4.00
3.50
3.00
e
or 2.50 c
2.00 age S ver
A 1.50
1.00
0.50
0.00 None Hexanoic Acid Octanoic Acid
Figure 34. Average tomato peel scores for the single factor experiment with no addition dip or a hexanoic acid or octanoic acid 1 second dip immediately following the water pretreatment.
Table 31. ANOVA analysis for single factor experiment examining the post pretreatment effect of results in Table 30 and Figure 34. Sum of Degrees of Mean Type of Dip Squares Freedom Square F0 Value P-Value Application Type 6.711 2 3.356 13.94 0.0007 Error 2.889 12 0.241 Total 9.600 14
Examining graphs to validate the assumptions of the ANOVA analysis proved to be problematic. From Figure 35, the residuals are normally distributed, but there may be problems with constant variance of the residuals (Figure 36).
123
Figure 35. Normal probability plot of the residuals from the single factor experiment.
Figure 36. Graph of residuals versus average tomato score for the single factor experiment.
Figure 36 shows that dispersion effects are associated with the data. By comparing the standard deviations listed in Table 30, the greatest source of variability is 124 observed when tomatoes are treated with hexanoic acid immediately after the
pretreatment. The standard deviation for this condition, 0.76, is much larger than the
other two—0.24 and 0.30. An ANOVA analysis on the residuals was performed Table
32), which indicates that there are dispersion effects associated with the data (see Table
30) (P-value = 0.0098).
Table 32. ANOVA analysis for the residuals of single factor experiment examining the type of dip. Tomato variety was TSH-8. Sodium hydroxide concentration was 4.0 N. Type of dip refers to a 1 second dip in a neat solvent. Sum of Degrees of Mean Type of Dip Squares Freedom Square F0 Value P-Value Application Type 0.547 2 0.274 6.98 0.0098 Error 0.470 12 0.039 Total 1.017 14
This violates one of the assumptions of the ANOVA, which calls into question the
validity of the results found in Table 31. Power, logarithmic (natural and base 10), and
inverse transformations of the data were carried out, but subsequent ANOVA analyses of
the transformed residuals shown that dispersion effects still exist in the data.
Though results from the ANOVA analysis cannot be directly used, useful
conclusions can still be made from this experiment. A 1 second dip in a carboxylic acid
immediately following the pretreatment dip increased the average score above that which
was observed when there was no 1 second dip. Further, studies are required to assess the
efficacy of a carboxylic acid spray in order to reduce NaOH concentrations in lye
peeling.
Chapter Nine
Conclusions
Age Studies
The ease of peeling vine-ripened tomatoes decreased as they continue to mature on the vine. However, tomatoes must continue to develop past the mature red stage for at least three to five weeks before significant changes in lye peeling are observed.
Tomatoes that remain on the vine for extended periods of time past the mature red stage are also susceptible to fruit softening and bacterial attack from the environment. Though tomatoes became significantly harder to peel as the vine ripened age increased, the impact it would have on tomato processors would be small.
For post-harvest tomatoes, tomatoes become significantly more difficult to peel as soon as three days after being picked from the plant. Post-harvest age has a greater effect upon tomato peelability than vine-ripened age. These results have implications for tomato processors. Therefore, it would be beneficial for tomato processors to harvest and peel tomatoes within several days, and ensure that tomatoes do not continue to develop on the vine for more than three to five weeks after entering the red stage of maturity. For tomatoes that develop on the vine well past the mature red stage or age off the vine for
125 126 more than three days, higher concentrations of sodium hydroxide for lye peeling, which
would result in higher raw material costs.
Base Selection
Caustic temperature, concentration, time, and a temperature-time interaction were
all significant factors associated with lye peeling. Any one of the main factors can be
altered to increase the average score of tomatoes. According to the ANOVA analysis, temperature and time had a more significant effect on the average score than did concentration (see Table 5). Thus, changes in these two factors would produce greater changes in how tomatoes are peeled in industry than changes in concentration.
Results from the base study were surprising because a multiple comparison test
revealed that no significant difference exists between NaOH and an equimolar mixture of
KOH and NH4OH. An equimolar mixture of KOH and NH4OH would be advantageous
to the tomato processor since the solution would enhance the effectiveness of tomato
waste as a fertilizer. Thus, such a mixture could be used in tomato peeling. However,
the KOH and NH4OH mixture evolves large amounts of ammonia into the atmosphere,
which can be hazardous to the environment as well as to employees.
The fact that no cellulose I to II conversion was seen for any of the mercerization
experiments is evidence that tomato lye peeling is primarily due to collenchymatous cell
disruption or pectin degradation as opposed to cell wall swelling due to conversion of
cellulose I to cellulose II.
127 Solvent Studies
Octanoic acid and hexanoic acid possess the capability to affect the cuticle in such
a way that can lead to lower concentrations of NaOH required for effective tomato peeling. No other functional group was shown to be as effective as the carboxylic acid.
Data shows that the required NaOH concentration needed to peel tomatoes can be
reduced by approximately one-half. This reduction in NaOH concentration has
substantial implications for tomato processors. If hexanoic acid or octanoic acid are
incorporated into existing canning processes, then this would result in lower raw material
costs for NaOH. Additionally, differences in lye peeling can be seen for solution
temperatures below 90°C. When caustic and pretreatment temperatures were held at
90°C, no significant changes in lye peeling were observed. When the temperature was
75°C, it did not matter if either hexanoic or octanoic acid were used as a pretreatment.
Therefore, if tomato processors choose to run their operation at temperatures greater than
90°C, pretreatment at that temperature would not be of any benefit. However, if
temperatures were close to 75°C, then a pretreatment of either carboxylic acid would be
beneficial. The increased effectiveness of the carboxylic acid pretreatments might enable
processors to use lower temperatures and achieve the same quality in tomato peeling.
The effectiveness of carboxylic acids were observed only when their presence
was above the solubility limit. Carboxylic acids may remove some cuticular waxes,
which would reduce the hydrophobicity of the cuticle and decrease cuticle thickness and
hence the distance over which NaOH would have to diffuse to cleave the cuticle from the
fruit. The carboxylic acid groups can react with ester linkages between cutin groups,
decreasing cross links in cutin. Carboxylic acids can also react with hydroxyl groups 128 found in many cuticle components enhancing their solubilization and removal. In
particular, carboxylic acids could react with phenyl groups in naringenin chalcone or
ester groups within the cuticle.
Below the solubility, carboxylic acids were no more effective than a water
pretreatment. This could be due to two reasons. First, the concentration of hexanoic,
octanoic, and decanoic acids in water is so low that mass transfer is severely inhibited.
Second, when carboxylic acids are dissolved in water, they dissociate into the hydronium
cation and the carboxylate anion. The carboxylate anion (hexanoate, octanoate, or
decanoate) has a -1 charge on the functional group. This ionic system may not be able to
interact with the hydrophobic cuticle. Thus, acids in the undissociated state may better be
able to diffuse into tomato cuticles. Previous studies suggest that penetration into leaves
and steams of plants are greatest when organic acids are in the undissociated state
(Bukovac et al.,1971). As long as carboxylic acids exist as neat solvents and not as a single-phase solution in water, it was effective.
Role of Caustic in Tomato Peeling
There are several possible mechanisms that explain how tomato lye peeling is
effective. First, lye peeling could be attributed to breakdown of collenchymatous cells due to osmotic shock. As these cells are the bridge between the exocarp and mesocarp, destroying these cells with ionic strength alone would cleave the cuticle from the fruit.
However, Table 15 (Chapter 7) shows that a highly concentrated NaCl solution is not effective at lye peeling and this mechanism alone does not appear to be adequate for tomato peeling. 129 Lye peeling could also be attributed to cellulose I to II conversion. Cellulose is
formed in the I state in nature, and can be converted to the II form by treatment with
NaOH solutions, which is known as mercerization. Studies have shown that conversion
from cellulose I to II can occur at NaOH concentrations of 2.5 N, which comparable to
tomato lye peeling concentrations (Dinand et al.,2002). During the mercerization
process, cellulose swells as it changes from cellulose I to II. It was hypothesized that
additional NaOH could diffuse into collenchymatous cells while cellulose was swelled.
However, mercerization studies show that significant cellulose I to II conversions do not
take place during typical lye peeling times and temperatures (Chapter 6).
Thus, the role of caustic in tomato peeling can be attributed to changes with
pectin in the middle lamella. Pectin’s function is to work as a binding agent that glues
the cuticle to collenchymatous cells. If pectin is affected in an adverse way, then its role
as a binding agent can be reduced; this can cleave the cuticle from the fruit. Previous
research has shown that viscosities of pectin solutions decrease with increasing NaOH
concentration and increasing temperature (Vollmert,1950; Krachanov,1965). Viscosity
reduction can be attributed to depolymerization of the pectin chain or expansion of
adjacent pectin chains. Pectin’s function can be attributed to a long backbone with
“smooth” and “hairy” regions consisting of additional sugars such as rhamnose,
arabinose, and galactose attached. Breaking α-1,4 glycosidic bonds could decrease
pectin’s ability to bind the cuticle to the fruit. Similarly, the strong base of NaOH could
neutralize the carboxylic acid groups of polygalacturonic acid (see Figure 2), which
would result in chain expansion due to electrostatic forces created by COO- groups along pectin chains. Additionally, lye peeling could also be due to interactions between NaOH 130 and ester groups in cutin. Previous work has shown that ester hydrolysis can take place
in the presence of 1 N NaOH (Samelson & Hammett,1956). The exact mechanism(s)
is(are) unknown, but this research shows that tomato lye peeling can be attributed to affecting pectin in the middle lamella. More research will have to be done in order to
assess what exactly is taking place with the pectin during tomato lye peeling.
Chapter Ten
Future Work
Vine-Ripened and Post-Harvest Changes
Much work has been done in the context of vine ripened changes of the tomato
(Andrews,1995; Thompson et al.,1998; Andrews et al.,2000; Andrews et al.,2002;
Andrews et al.,2002; Bauer et al.,2004). However, not as much work has been aimed at post-harvest changes of tomato cuticles; although a recent study charted cuticle and skin firmness after foliar treatments of calcium chloride (Garcia et al.,1995). Post-harvest studies can be conducted that charts changes of tomato cuticular integrity given various post-harvest treatments.
Solvent Studies
Extensive work has been done with respect to solvent pretreatments for lye peeling (Neumann et al.,1978; Das & Barringer,1999). Octanoic and hexanoic acid were identified as optimum pretreatments. However, all previous work has been conducted in the laboratory. Thus, future tomato research can be centered on tomato peeling at a
131 132 larger scale. This has three main implications. First, experiments can be scaled to pilot plant size in order to determine if there are any additional issues associated with the incorporation of a carboxylic acid into the lye peeling process. Second, studies of tomato fruit quality can be conducted in order to determine if the addition of a carboxylic acid positively or negatively affects fruit firmness, shelf-life, etc. Third, microscopic studies can be performed in order to ascertain the mechanism through which the carboxylic acids enhance tomato lye peeling.
Design of Experiments
In order to analyze most of the data obtained from lye peeing experiments, various forms of the ANOVA was used as most of the experiments conducted were factorial experiments. The specific form of the ANOVA analysis was dependent upon the method in which the data was collected. All statistical analyses used were based on principles of design of experiments; all analyses first assume a mathematical model for the data, and then proceeds to test one or more hypotheses. Such principles are based upon three central ideas: randomization, replication, and blocking. Randomization states that the order in which the experimental runs are conducted are completely randomized with respect to all factors, levels, and replicates. Replication is simply taking more than one measurement for each experimental condition. Blocking is a technique used to systematically block the effect of one or more factors that the experimenter believes may affect the response. Tomato lye peeling experiments is another subject that can benefit from statistical analyses. 133 Experiments conducted with respect to the identification of significant factors in lye peeling (see Table 5), the type of base (see Table 8), the 3² experiment with organic solvents (see Table 11), and the investigation into post pretreatment effects (see Table 20) all were based upon principles of design of experiments. However, experiments, which investigated the effectiveness of various organic solvents, were not based upon such principles. Issues with day to day variability of tomatoes gathered from fields can be remedied through blocking. Many experimenters like to perform an entire experiment in one day; however, attempting to conduct all tomato lye peeling experiments on all available organic solvents is not feasible. Thus, the following experimental design is proposed: a blocked factorial experiment in which the factors are pretreatment type, and
NaOH concentration, which are evaluated at two levels each. The first and second levels of pretreatment type are water and organic solvent (or water and organic solvent).
Similarly, the two levels of NaOH concentration would be 2.5 N and 4.5 N, or whatever two concentrations are appropriate to the type of tomatoes that are being used. In other words, the NaOH concentration range should not be too wide (e.g. 0.50N and 7.00N) as this may result in severe underpeeling and overpeeling. If m different organic solvent pretreatments are to be tested, then the entire experiment will last for m days with m different blocks. Placing each separate day into blocks systematically eliminates the variability associated with day-to-day differences between tomatoes. The variability due to the blocks reduces the variability due to random error in the experiment, which would lead to less ambiguous results. This design also would also enable the experimenter to perform multiple comparison tests between pretreatment type, and NaOH concentration.
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143
Appendices
Appendix A – Robbins Chart (Sources: (Robbins,1980; Frank et al.,1999)) Solvent Class 1 2 3 4 5 6 7 8 9 10 11 12
Solute Class H Donor Groups 1 Phenol 0 0 - 0 ------+ + 2 Acid, thiol 0 0 - 0 - - 0 0 0 0 + + 3 Alcohol, water - - 0 + + 0 - - + + + + 4 Active H on multihalogen paraffin 0 0 + 0 ------0 + H Acceptor Groups Ketone, amide with no H on N, 5 - - + - 0 + + + + + + + sulfone, phosphine oxide 6 Tertiary amine - - 0 - + 0 + + 0 + 0 0 7 Secondary amine - 0 - - + + 0 0 0 0 0 + Primary amine, ammonia, amide 8 - 0 - - + + 0 0 + + + + with 2H on N 9 Ether, oxide, sulfoxide - 0 + - + 0 0 + 0 + 0 + Ester, aldehyde, carbonate, phosphate, nitrate, nitrite, nitrile, 10 - 0 + - + + 0 + + 0 + + intramolecular bonding, e.g., o- nitrophenol Aromatic, olefin, halogen aromatic, multihalogen paraffin 11 + + 0 + 0 0 + 0 + 0 0 without active H, monohalogen paraffin Non-H-Bonding Groups 12 Paraffin, carbon disulfide + + + + + 0 + + + + 0 0
144
Appendix B – Scoring of Tomatoes
Upon removal from the mechanical peeler the tomatoes were scored based on
how much of the cuticle was removed. The scoring system differed between Fall 2005
and 2006, and a more defined system was adopted to eliminate ambiguities. The old
system as well as the reasons behind adopting the new system is explained here
extensively.
Under the old system the following tomato scores were given based upon how
much of the cuticle was removed. The old system was:
• 0 – No cuticle removal.
• 1 – Greater than 1% but less than 50% of cuticle removal.
• 2 – Greater than or equal to 50% but less than 100% of cuticle removal.
• 3 – 100% of cuticle removal, which also includes overpeeled tomatoes.
However, this system contained ambiguities for it did not allow one to distinguish
between treatments that were adequate and those that were damaging tomatoes.
Therefore, a new, more defined scoring system was established for all the 2006 peeling
season, which is presented below.
• 0 – A score of zero indicates that no peel was removed by the process. This category
involves a tomato that is no different after being subjected to a peeling condition than
what it was before. No changes in the cuticle, color, or firmness are observed.
• 1 – A score of one indicates that greater than 1% but less than 50% of the cuticle is
removed. This category involves all cracks, and “holes” that appears in the cuticle of
the tomato. A tomato that received this score can also be defined as a tomato with 145 any break in the integrity of the cuticle provided that the sum of the peeled areas does
not exceed 50% of the total surface area. A score of 1 also includes tomatoes in
which the cuticle seems to be loosened from the fruit, but unbroken by the
mechanical friction of the tumbler.
• 2 – This describes a tomato that has more than 50% but less than 100% of the cuticle
was removed. This category includes tomatoes that experience localized overpeeling,
but whose cuticles have not been completely removed.
• 3 – Perfectly and completely peeled tomatoes received a score of 3. When a tomato
has been perfectly peeled, the outer cuticle is removed, but the pectin and fruit
firmness remains. There are two exceptions to this. First, tomatoes with an attached
“tail.” “Tails” denote the portion of the cuticle that remains attached to the bottom of
the tomato though the rest of the cuticle has been removed. These tails are prevalent
in tomatoes bred to produce tougher cuticles. In the processing industry, these tails
may be difficult to remove, and tomatoes with tails are acceptable for canning.
Second, a score of 3 also includes those tomatoes whose entire cuticle seems to be
removed from the fruit, but it hasn’t been broken by the mechanical friction of the
tumbler. This type of tomato has been given the name of “bags” in the industry
because the fruit is encapsulated in cuticle like a bag without being attached to the
cuticle. In other words, the cuticle is physically around the tomato, but is no longer
attached to the fruit.
• 4 – In order for a tomato to receive a score of 4, it must have its cuticle completely
removed and satisfy any one or more of the following conditions. The vascular
bundles in the mesocarp are clearly visible, indicating that the fruit is severely 146 softened, broken up, dissolved away, or abraded. If the entire cuticle is removed and there is significant distortion in the firmness of the fruit or a disappearance of the fruit, then these tomatoes will receive a score of 4.
147 Appendix C – Part Drawing for X-Ray Diffraction Sample Holder
Figure 37. Part drawing for x-ray diffraction sample holder.
148 Appendix D – X-Ray Diffraction Patterns for Cellulose
y it s
n 5 min e t In
2 min
1 min
0 5 10 15 20 25 30 35 40 45
2θ
Figure 38. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N NaOH at 60°C for 1, 2, and 5 minutes.
y it s n e
t 5 min In
2 min
1 min
0 5 10 15 20 25 30 35 40 45
2θ
Figure 39. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N NaOH at 80°C for 1, 2, and 5 minutes.
149 y it s n
e 5 min t In
2 min
1 min
0 5 10 15 20 25 30 35 40 45
2θ
Figure 40. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N KOH at 60°C for 1, 2, and 5 minutes.
y it s n e
t 5 min In
2 min
1 min
0 5 10 15 20 25 30 35 40 45
2θ
Figure 41. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N KOH at 80°C for 1, 2, and 5 minutes.
150 y it s n e
t 5 min In
2 min
1 min
0 5 10 15 20 25 30 35 40 45
2θ
Figure 42. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N KOH at 100°C for 1, 2, and 5 minutes.
y t i s n
te 5 min In
2 min
1 min
0 5 10 15 20 25 30 35 40 45
2θ
Figure 43. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N NH4OH at 60°C for 1, 2, and 5 minutes.
151 y t i s n 5 min te In
2 min
1 min
0 5 10 15 20 25 30 35 40 45
2θ
Figure 44. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N NH4OH at 80°C for 1, 2, and 5 minutes.
y t i s n
te 5 min In
2 min
1 min
0 5 10 15 20 25 30 35 40 45
2θ
Figure 45. X-ray diffraction pattern of cellulose I (Avicel) subjected to 3.0 N NH4OH at 100°C for 1, 2, and 5 minutes.