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a Crouch 2011 f Doctor of H ellenbosch arius Huysam Deirdre Holc riSciencesces betw
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al SciencesDeclaration
By submitting this thesis/dissertation electronically, I declare that the entirety of the work
contained therein is my own, original work, and that I have not previously in its entirety or in part
submitted it for obtaining any qualification.
Signature:
Date: December 2010
Copyright © 2011 Univesity of Stellenbosch
All rights reserved
SUMMARY
Mealiness, a soft, dry textural disorder of ‘Forelle’ pear (Pyrus communis L.), is a problem for the South African fruit export industry. Soft, dry textural disorders seem to be related to changes in cell wall breakdown. The aim of this work was, therefore, to investigate the occurrence of mealiness‐associated changes in the cell wall and elucidate the mechanism by which mealiness occurs in ‘Forelle’ pear, as well as to characterise cell wall changes occurring during normal ripening.
Mealy ‘Forelle’ tissues had significantly lower total galacturonic acids associated with the middle lamella (water‐ and CDTA‐soluble fractions). The water‐soluble pectin of mealy tissues was depolymerised at an earlier stage of ripening. The widespread disintegration of cell‐to‐cell adhesion in mealy cell walls only, suggests that the middle lamella and the plasmodesmata are more broken down. In mealy ‘Forelle’ tissues there was no indication of less broken down high molecular weight polyuronides in the CDTA fraction, normally associated with these dry, soft textures. The pectins from mealy tissues were more broken down and both mealy and non‐mealy tissue polyuronides depolymerised. Furthermore, there was a lack of light toluidine staining in the larger air spaces, which would indicate such water‐insoluble pectins. These data suggest that the formation of high molecular weight pectate gels is unlikely in mealy ‘Forelle’ pear. The slight increase in the galactose content in mealy tissues in CDTA‐ and Na2CO3‐soluble fractions and slight
decrease in the 1 M KOH glycan fraction during later stages of ripening (6+11, 9+7, 9+11; weeks at ‐0.5°C plus days at 15°C) may indicate that galactose loosely interlinked into the glycan fraction broke down sooner for mealy tissues. This didn’t increase molecular size profiles in the CDTA fraction. Arabinose content was slightly higher in the 4 M KOH fraction and slightly lower in mealy tissues of water‐ and CDTA fractions. This did not influence the molecular weight of the glycans compared to those in the non‐ mealy tissues. ‘Forelle’ data therefore seem to be more congruent with a decrease in intercellular adhesion as the mechanism by which mealiness occurs, rather than the formation of high molecular weight pectins taking up the cellular fluid.
‘Forelle’ pear water‐soluble pectin content increases with increased ripening. High amounts of water‐ soluble pectin and low amounts of Na2CO3‐soluble pectin suggests that solubilisation of
rhamnogalacturonan‐I pectins must have taken place during early ripening (at a fruit firmness of > 4.7 kg (7.9mm tip). Galactose and glucose in the pectin fraction dramatically decreased after fruit ripened to a firmness of 4.5 kg, whereafter they remained unchanged. This was also the period in which fruit softened the most and the biggest increase in pectin water‐solubility occurred. It is not known whether these events are coincidental, or linked causally. Rhamnose and arabinose extractability increased in the water fraction and xylose, fucose and mannose increased in glycan fractions with ripening. The biggest changes in
polyuronide solubilisation and depolymerisation occurred in water‐ and CDTA fractions between storage and ripening durations of 3+7 (4.7 kg) and 6+4 (2.7 kg).
OPSOMMING
Melerigheid, ʼn sagte droë tekstuur afwyking van ‘Forelle’ pere (Pyrus communis L.), is ʼn probleem vir die Suid Afrikaanse vrugte uitvoerbedryf. Sagte, droë tekstuur afwykings blyk betrekking te hê op selwandafbraak veranderinge. Die doel van die studie was dus om die melerigheid‐geassosieerde veranderinge in die selwand te ondersoek, sowel as om vas te stel wat die meganisme betrokke is by melerigheid ontwikkeling in ‘Forelle’ pere. Die selwand veranderinge gedurende normale rypwording is ook gekarakteriseer.
Melerige ‘Forelle’ weefsel het betekenisvol laer totale galakturoonsuur wat geassosieer is met die middellamella (water‐ en CDTA‐oplosbare fraksies). Die water‐oplosbare pektien van melerige weefsel was op ʼn vroeër stadium van rypwording gedepolimeriseer. Die wydverspreide disintegrasie van sel‐tot‐sel adhesie, slegs in melerige selwande, dui aan dat die middellamella en die plasmodesmata meer afgebreek is. Daar is geen indikasie van hoë molekulêre massa poliuroniedes in die CDTA fraksie van melerige ‘Forelle’ weefsel, wat gewoonlik geassosieer word met droë, sagte teksture nie. Die pektiene van melerige weefsel was meer afgebreek en melerige en nie‐melerige weefsel se poliurone was gedepolimeriseer. Daar was ook geen ligte toluïdien verkleuring in die groter intersellulêre lugruimtes nie, wat ʼn aanduiding sou wees van wateronoplosbare pektiene. Hierdie data dui dus aan dat die vorming van hoë molekulêre pektien jel in melerige ‘Forelle’ pere onwaarskynlik is. Die klein toename in galaktose inhoud in die CDTA‐ en Na2CO3‐
oplosbare fraksies en ʼn klein afname in 1 M KOH glikaan fraksie tydens latere rypheidstadiums (6+11, 9+7, 9+11; weke by ‐0.5°C plus dae by 15°C), kan beteken dat los verweefde galaktose in die glikaan fraksie vroeër afgebreek het in melerige weefsels. Die molekulêre grootte profiel is nie verander in die CDTA fraksie nie. Arabinose inhoud was bietjie hoër in die 4 M KOH fraksie en bietjie laer in melerige weefsel van die water‐ en CDTA fraksies. Die molekulêre massa van die glikane was klaarblyklik onbeïnvloed hierdeur. ‘Forelle’ data blyk dus meer saam te stem met die meganisme waar ʼn vermindering in intersellulêre adhesie ʼn rol speel in melerigheid, eerder as die meganisme waar hoë molekulêre pektien selvloeistowwe bind.
‘Forelle’ peer water‐oplosbare pektieninhoud neem toe met toenemende rypheid. Hoë vlakke water‐ oplosbare pektien en lae vlakke Na2CO3‐oplosbare pektien stel voor dat die oplossing van
rhamnogalakturonan‐I pektiene gedurende vroeë rypwording moes plaasgevind het (by ʼn fermheid van > 4.7 kg (7.9mm punt). Galaktose en glukose in die pektienfraksie het drasties verminder nadat vrugte tot ʼn fermheid van 4.5 kg ryp geword het, waarna hul onveranderd gebly het. Dit was ook die periode waarin vrugte die meeste sag geword het en die grootste toename in poliuronied wateroplosbaarheid gevind is. Dit is nie bekend of die gebeure toevallig of oorsaaklik verbind is nie. Rhamnose en arabinose
ekstraheerbaarheid het vermeerder in die water fraksies, en xylose, fukose en mannose het vermeerder in die glikaan fraksies gedurende rypwording. Die grootste verandering in oplosbaarheid en depolimerisasie het plaasgevind in die water‐ en CDTA fraksies tussen opberging en rypwordingsperiodes van 3+7 (4.7 kg) en 6+4 (2.7 kg).
DEDICATION
This thesis is dedicated to my husband Ian, for his unfaltering support at work and at home, positive criticism, patience and love during this study. To my two boys, Matthew and Karl, for allowing me to do the work that I love, for always waiting for me with a smile and unconditional love, and for allowing me to experience the world again for the first time through their eyes.
ACKNOWLEDGEMENTS I gratefully acknowledge the following institutions and individuals: The Deciduous Fruit Producers’ Trust / Hortgro Services for funding my research. Molteno Farm in Elgin for providing the fruit for the study. The Stellenbosch University and the Department of Horticultural Sciences for letting me further my studies and career whilst being employed and supporting me during my Ph.D. studies.
Prof. Marius Huysamer, my supervisor, for technical advice, positive criticism, patience throughout my studies, but also for the opportunities he created to broaden my knowledge in the field.
Dr. Deirdre Holcroft, my co‐supervisor, for her encouragement throughout the study and for her infectious love of postharvest physiology and technology as a field.
Prof. Karen Theron, the head of the Horticultural Department, for advice and assistance with the statistical analysis, for her support throughout the study, critical thinking and for her attempt, especially in the beginning of the study, not only to provide the necessary equipment but also the necessary support personnel for these type of laboratory oriented Horticultural studies. Prof. John Labavitch, Dr. Carl Greve, Dr. David Brummell and Dr. Ann Powell for letting me work in their labs for three months, teaching me techniques, and for making my stay in UC Davis truly memorable. Mrs. Nicole Windell, for her commitment and hard work in helping me with extractions and size exclusion chromatography. Dr. Elisabeth Rohwer for a critical scientific foundation, her intellectual and technical contributions.
Mrs. Susan Agenbach for technical assistance, ordering of chemicals, upkeep of the ‐80°C freezers, for organising the moving of the labs from the A.I. Perold Building to the Lombardi Building whilst the project continued and for making the lab a friendly and safe place for all. Shantel, Elveresha and Cecelia for helping me with maturity and mealiness evaluations. Erik van Papendorp, from Hortgro Services, for industry statistics used in the introduction. Prof. Vernon and Kay Singleton, my American grandparents, for making me feel so welcome during my stay at UC Davis. I still miss you.
Doris Gallemore, for providing me with a loving home in UC Davis. The smartest, most interesting and youngest 74 year old woman I have met.
Veerle van Linden, Karin Struijs, David Brummell, Valeriano Dal Cin and Mary Kalamaki, my international lab friends that made life at UC Davis very special and fun, and supported me throughout my studies. Prof. Paul Knox for providing me with complementary antibodies to test on ‘Forelle’ pears.
Dr. Rob Smith, Maritza Kruger and Ben Loos for letting me work in the histochemistry lab at the Department of Physiology and for helping me with the techniques. These will come in handy in future studies.
Dr. Mohammed Jaffer, of the Electron Microscopy Unit at University of Cape Town, for teaching me embedding, cutting and microscopy techniques and for letting me prepare and observe my samples in their facility.
My colleagues, friends, and in particular my fellow Ph.D. students (Mariana Jooste, Paul Cronjé, Michael Schmeisser, Simeon Hengari and Esmé Louw) at the Department of Horticultural Sciences for their support throughout my studies and for making the Department a great place to work. My M.Sc. students Tarryn de Beer, Joanna Majoni and Patricia Carmichael for their patience during the last few months of writing up. Carin Pienaar and Dianah Daniels (administrative staff) and Gustav Lötze and Tikkie Kerwel (technical staff) at the Department of Horticultural Sciences, for assistance throughout the study.
My friends throughout South Africa, Heather and David Good, Tanelle Schutte, Julie Vosloo, Anke Aschenborn, Pastor Dieter Reinstorf, Aby Louw, Nikoleen van der Spuy, Heleen Destroo, Carmen and Ernst Eggers, who have always been a great support during this study, for looking after the kids, and some even bringing our family meals whilst I was finishing writing up. Your support has touched me in such a way that I hope to be able to do the same for you and other people in future. Desmond and Pat Crouch, my late parents in law, who received me as their own, supported me throughout
my study and provided me with a family in the Western Cape.
My Nelspruit, Cape Town and Durban family: Leon, Odette and Emilia Martin, David Crouch and Ariane Spitaels, and Neil and Tanza Crouch for always being there for our family during the study and for your academic insights.
Opa, Jos* und Oma Elli Eggers, dass ihr mir immer während meinem Studium unterstützt habt.
Vielen Dank Papa Erlo* und Mama Monika Martin, dass ihr mich beigebracht habt mit Fleiβ and die Arbeit zu gehen, zu beten, alles zu hinterfragen im Versuch die Funktion der Natur zu ergründen und niemals auf Träume auf zu geben. Vor allem aber dass ihr mir Beistand leistetet und in mir geglaubt habt.
John 1:3
TABLE OF CONTENTS DECLARATION i SUMMARY ii OPSOMMING iii DEDICATION v ACKNOWLEDGEMENTS vi TABLE OF CONTENTS viii LIST OF FIGURES xi LIST OF TABLES xiii GENERAL INTRODUCTION AND OBJECTIVES 1 CHAPTER 1: LITERATURE REVIEW 7 THE ROLE OF CELL WALLS IN DRY TEXTURAL DISORDERS WITH SPECIAL REFERENCE TO PECTIN AND MEALINESS 1.1 Introduction 7 1.1.1 Definitions of texture 7 1.1.2 Textural properties 8 1.1.3 Textural properties pertaining to dry textural disorders 8 1.2 Factors influencing textural changes in ripening fruit and other fresh products 9 1.2.1 Factors influencing juiciness 9 1.2.2 Turgor 9 1.2.3 Cell‐to‐cell adhesion 10 1.3 Factors influencing the perception of juiciness 11 1.3.1 The force with which juice is released 11 1.3.2 Cell size and cell wall thickness 11 1.3.3 Cell wall, cytoplasm, and the vacuole ratio 12 1.3.4 Sensory juiciness scale of products in relation to their characteristics 12 1.4 The role of pectin in gelling and dry textures 13 1.4.1 Pectin composition and functions 13 1.4.2 Homogalacturonan 14 1.4.2.1 Middle lamella 14 1.4.2.2 Ca2+‐ cross‐linking 14 1.4.2.3 Degree and distribution of methyl groups 15 1.4.2.4 The influence of cell wall pH 15 1.4.2.5 The influence of cation levels 16 1.4.2.6 The influence of ionic strength 16 1.4.2.7 Homogalacturonan’s involvement in woolliness of peach and nectarine 17 1.4.2.8 In vitro gelling of high methoxy pectins 19 1.4.2.9 Gelling properties of cations other than Ca2+ 19 1.4.2.10 Cu2+ involvement in non‐enzymatic breakdown of the cell wall 20 1.4.3 Rhamnogalacturonan‐I 22 1.4.3.1 Structure 22 1.4.3.2 Linkages 22
1.4.3.3 Role of rhamnogalacturonan‐I in texture 23 1.5 Mealiness disorders 25 1.5.1 Peach 25 1.5.2 Pear 26 1.5.3 Plum 28 1.5.4 Apple 29 1.5.5 Tomato 30 1.6 Conclusion 31 1.7 References 32 CHAPTER 2: COMPARITIVE CELL WALL COMPOSITION OF MEALY AND NON‐MEALY 'FORELLE' PEAR 48 Abstract 48 2.1 Introduction 49 2.2 Materials and Methods 50 2.2.1 Fruit material 50 2.2.2 Maturity and quality indices 50 2.2.3 Isolation of cell wall 51 2.2.4 Total uronic acid and neutral sugar measurements 51 2.2.5 Cell wall fractionation 51 2.2.6 Gas chromatographic analysis of neutral sugars 52 2.2.7 Data Analysis 53 2.3 Results and discussion 53 2.3.1 Maturity indices, extractable juice content and cell wall yield 53 2.3.2 Neutral sugars and uronic acids after sequential extraction. 53 2.3.3 Total cell wall neutral sugar composition 54 2.3.4 Pectin‐ and glycan fraction neutral sugar composition 55 2.4 Conclusion 58 2.5 References 59 CHAPTER 3: CELL WALL COMPOSITIONAL DIFFERENCES BETWEEN MEALY AND NON‐MEALY 'FORELLE' PEAR DURING RIPENING. 69 Abstract 69 3.1 Introduction 71 3.2 Materials and method 72 3.2.1 Fruit material 72 3.2.2 Maturity and quality indices 72 3.2.3 Isolation of cell wall 73 3.2.4 Total uronic acid and neutral sugar measurements 73 3.2.5 Cell wall fractionation 74 3.2.6 Gas chromatograpic analysis of neutral sugar 75 3.2.7 Size exclusion chromatography 75 3.2.8 Microscopic preparation 76 3.2.9 Data analysis 76 3.3 Results and discussion 76 3.3.1 Ripening, mealiness and cell wall yield 76 3.3.2 Neutral sugar and uronic acid contents of the total cell wall and cell wall residues after 4 M KOH extraction. 77 3.3.3 Cell wall fractionation 78 3.3.4 Changes in neutral sugar composition during ripening 80 3.3.4.1 Total cell wall neutral sugar composition 81 3.3.4.2 Pectin fraction neutral sugar composition 81
3.3.4.3 Glycan fraction neutral sugar composition 84 3.3.4.4 Mealy ‘Forelle’ cell wall neutral sugar compositional differences 86 3.3.5 Size exclusion chromatography 89 3.3.5.1 Size exclusion chromatography of water soluble uronic acids 89 3.3.5.2 Size exclusion chromatography of water soluble neutral sugars 90 3.3.5.3 Size exclusion chromatography of CDTA soluble uronic acids 91 3.3.5.4 Size exclusion chromatography of Na2CO3 soluble uronic acids 92 3.3.5.5 Size exclusion chromatography of 1 M KOH soluble neutral sugars 92 3.3.5.6 Size exclusion chromatography of 4 M KOH soluble neutral sugars 93 3.3.6 Light Microscopy 95 3.4 Conclusion 99 3.5 References 102 GENERAL DISCUSSION AND CONCLUSIONS 142
LIST OF FIGURES
GENERAL INTRODUCTION AND OBJECTIVES
Figure 1: Total area of blushed‐pear cultivars in South Africa from 2000 to 2009. ……….……….………6 Figure 2: Blushed‐pear cultivars (12.5 kg carton equivalents) exported from 2003 to
2010………6
CHAPTER 2
Figure 1: Sequential extraction protocol used for the preparation and isolation of ‘Forelle’ pear fruit cell wall fractions………64 Figure 2: Percentage total neutral sugars (glucose equivalents) and total uronic acids (galacturonic acid
equivalents) after sequential extraction (Ext)……….….…65 Figure 3: Neutral sugar composition of total cell walls before (A) and after DMSO treatment (B)………66 Figure 4: Neutral sugar composition of the de‐starched alcohol insoluble residue after sequential extraction
with H2O (A), CDTA (B) and Na2CO3 (C)………..…67
Figure 5: Neutral sugar composition of the de‐starched alcohol insoluble residue after further sequential extraction with 1 M KOH (A) and 4 M KOH (B)………..…..68
CHAPTER 3
Figure 1: Sequential extraction protocol used for the preparation and isolation of ‘Forelle’ pear fruit cell wall fractions………..…..…109 Figure 2: Percentage total uronic acids after 3, 6 or 9 weeks of storage at ‐0.5°C and ripening for 4, 7 or 11 days at 15°C of mealy (M) and non‐mealy (N) tissues…….………...115 Figure 3: Water soluble uronic acid content as a function of flesh firmness of non‐mealy ‘Forelle’ pears after 3, 6 or 9 weeks of storage at ‐0.5°C and ripening at 15°C for 4, 7, or 11 days……….116 Figure 4: Percentage total neutral sugars (glucose equivalents) after 3, 6 or 9 weeks of storage at ‐0.5°C and ripening for 4, 7 or 11 days at 15°C of mealy (M) and non‐mealy (N) tissues……….…………117 Figure 5: Percentage total neutral sugars (glucose equivalents) after 3, 6 or 9 weeks of storage at ‐0.5°C and ripening for 4, 7 or 11 days at 15°C………..……….118 Figure 6: Percentage total neutral sugars (glucose equivalents) after 3, 6 or 9 weeks of storage at ‐0.5°C and ripening for 4, 7 or 11 days at 15°C of mealy (M) and non‐mealy (N) tissues. Material was sequentially extracted with water, CDTA, Na2CO3, 1 M‐ and 4 M KOH and data were statistically
pooled……….………….…….118 Figure 7: Neutral sugar composition (mol%) of ‘Forelle’ pear crude cell wall (CW) (A) and after sequential
Figure 8: Neutral sugar composition (mol%) after sequential extraction with Na2CO3 (A), 1 M KOH (B), 4 M KOH (C) of mealy (M) and non‐mealy (N) fruit……….………..120 Figure 9: Neutral sugar composition (mol%) of the cell wall residue after sequential extraction of mealy (M) and non‐mealy (N) fruit. ……….……..121 Figure 10: Arabinose content (mol%) of sequential extracts……….122 Figure 11: Xylose content (mol%) of sequential extracts. ………..123 Figure 12: Galactose content (mol%) of sequential extracts………..124 Figure 13: Rhamnose content (mol%) of sequential extracts……….125 Figure 14: Fucose content (mol%) of sequential extracts………..………..126 Figure 15: Glucose content (mol%) of sequential extracts………...127 Figure 16: Mannose content (mol%) of sequential extracts………..……….128 Figure 17: Rhamnose content (mol%) of pooled sequential extracts………129 Figure 18: Rhamnose content (mol%) after sequential extraction of fruit stored for 3, 6 or 9 weeks at ‐0.5°C and ripened for 4, 7 or 11 days at 15°C (combined) and mealy (M) and non‐mealy (N) tissues…….130
Figure 19: Arabinose to galactose ratio of the H2O‐, CDTA‐, Na2CO3‐, 1 M KOH‐, 4 M KOH fractions and the CW residue after sequential extraction of mealy (M) and non‐mealy (N) fruit………..……….130
Figure 20: Arabinose (Ara) plus Galactose (Gal) to Rhamnose (Rha) ratio for water‐, CDTA‐ and Na2CO3‐ soluble pectins of non‐mealy fruit……….………...131 Figure 21: Galactose content (mol%) of the CDTA fraction of fruit stored for 3, 6 or 9 weeks at ‐0.5°C and ripened for 4, 7 or 11 days at 15°C, for mealy (M) and non‐mealy (N) tissues……….………..132 Figure 22: Galactose content (mol%) of the 1 M KOH fraction of fruit stored for 3, 6 or 9 weeks at ‐0.5°C and ripened for 4, 7 or 11 days at 15°C (RIP), for mealy (M) and non‐mealy (N) tissues……….….132 Figure 23: Mannose content (mol%) of the crude cell wall (CW) of fruit stored for 3, 6 or 9 weeks at ‐0.5°C and ripened for 4, 7 or 11 days at 15°C, for mealy (M) and non‐mealy (N) tissues……….……133 Figure 24: Size exclusion chromatography uronic acid profiles as separated on a Sepharose CL‐2B column for the water fraction of mealy and non‐mealy tissues……….………….134 Figure 25: Size exclusion chromatography neutral sugar profiles as separated on a Sepharose CL‐2B column for the water fraction of mealy and non‐mealy tissues……….………….135 Figure 26: Size exclusion chromatography profiles as separated on a Sepharose CL‐2B column for the CDTA fraction of mealy and non‐mealy tissues………136
Figure 27: Size exclusion chromatography profiles as separated on a Sepharose CL‐2B column for the Na2CO3 fraction of mealy and non‐mealy tissues……….…………137
Figure 28: Size exclusion chromatography profiles as separated on a Sepharose CL‐6B column for the 1 M KOH fraction of mealy and non‐mealy tissues……….………138
Figure 29: Size exclusion chromatography profiles as separated on a Sepharose CL‐6B column for the 4 M KOH fraction of mealy and non‐mealy tissues……….…………..139
Figure 30: Light micrographs of transverse sections through tissues of ‘Forelle’ pear (A) vascular bundle (v) surrounded by parenchyma cells and (B) a sclereid cluster (s) with associated radial parenchyma cells (rp) of slightly mealy tissues (4.0 kg)……….140 Figure 31: Light micrographs of transverse sections through firm, non‐ripe tissues of ‘Forelle’ pear (A) stored for 10 weeks at ‐0.5°C (6.3 kg) and (B) 12 weeks at ‐0.5°C (6.1 kg)………..……….140 Figure 32: Light micrographs of transverse sections through tissues of ‘Forelle’ pear that were ripe, soft and juicy (A and C) or mealy (B and D) after they were stored for 10 weeks at ‐0.5°C and ripened for 7 (A and B; 3.1 and 2.6 kg, respectively) or 11 days (C and D; 2.3 and 2.2 kg, respectively) at 15°C….…141 LIST OF TABLES CHAPTER 2 Table 1: Maturity and quality indices of mealy and non‐mealy ‘Forelle’ pears, stored for 6 weeks at ‐0.5°C and ripened for 4 days at 15°C……….…………65 CHAPTER 3
Table 1: Maturity indices and mealiness incidence for ‘Forelle’ pear stored at ‐0.5°C and ripened at 15°C. ……….…110 Table 2: Percentage alcohol insoluble residue (AIR) and firmness, measured for ‘Forelle’ pears after cold storage for up to 21 weeks at ‐0.5°C……….……111 Table 3: Juice content and fruit firmness after 3, 6 or 9 weeks of storage at ‐0.5°C and ripening for 4, 7 or 11 days at 15°C for mealy (M) and non‐mealy (N) tissues………...111 Table 4: Hue angle and alcohol insoluble residue for ‘Forelle’ pear stored at ‐0.5°C for 3, 6 and 9 weeks plus ripened for 4, 7 or 11 days at 15°C (indicated as 3+7, 6+4, 6+11, 9+7, 9+11)……….…112 Table 5: Percentage total neutral sugars (glucose equivalents) and uronic acids (galacturonic acid
equivalents) of total cell walls (AIR) plus cell wall residues after sequential extraction. Presented means are averaged pooled data of AIR and the CW residue after fruit storage for 3, 6 or 9 weeks at ‐0.5°C plus ripening for 4, 7 or 11 days at 15°C. Data presented are for mealy (M) and non‐mealy (N) tissues………..…..112 Table 6: Percentage total neutral sugars (glucose equivalents) and uronic acids (galacturonic acid equivalents) of total cell walls (AIR) and cell wall residues after sequential extraction. Data presented are for mealy and non‐mealy tissues. ……….….113 Table 7: Percentage total neutral sugars (glucose equivalents) of cell wall residues after sequential
extraction. Data presented are for mealy (M) or non‐mealy (N) tissues. ……….……113 Table 8: Percentage total neutral sugars (glucose equivalents) and uronic acids (galacturonic acid
Table 9: Pr>F for neutral sugar composition (mol%) after sequential extraction of mealy (M) and non‐mealy (N) tissues (data in Fig. 7, 8 and 9 analysed as one data set)………..………121 Table 10: Pr>F for neutral sugar composition (mol%) after sequential extraction of fruit stored for 3, 6 or 9 weeks at ‐0.5°C and ripened for 4, 7 or 11 days at 15°C of mealy (M) and non‐mealy (N) tissues (each fraction in Fig. 7, 8 and 9 was analysed separately)……….………..121 Table 11: Fruit firmness after 3, 6 or 9 weeks of storage at ‐0.5°C and ripening for 4, 7 or 11 days at 15°C as
main effect (mealy and non‐mealy fruit combined)………..129 Table 12: Rhamnose content (mol %) of mealy and non‐mealy cell wall (CW) tissues, H2O‐, CDTA‐and
1 M KOH fractions and the cell wall residue after sequential extraction (CW Residue)……….….129 Table 13: Arabinose and galactose values used for arabinose to galactose ratio (Fig. 19) for the water‐,
CDTA‐, and Na2CO3 fractions after 9 weeks of storage and 7 days of ripening………..130 Table 14: Arabinose‐, galactose‐, xylose‐ and mannose content (mol %) of mealy and non‐mealy fractions. Each fraction was statistically evaluated separately……….…………131 Table 15: Arabinose (Ara) plus Galactose (Gal) to Rhamnose (Rha) ratio for mealy and non‐mealy tissues of the 4 M KOH fraction………..131 Table 16: Fucose content (mol %) of mealy and non‐mealy water‐, Na2CO3‐ and 1 M KOH fractions and the cell wall (CW) residue after sequential extraction………..132
GENERAL INTRODUCTION AND OBJECTIVES Forelle (Pyrus communis L.) is South Africa’s most important blushed pear cultivar. The blushed pear season begins with ‘Rosemarie’, followed by ‘Flamingo’ and then ‘Forelle’. ‘Flamingo’, however, is prone to internal breakdown and ‘Rosemarie’ has a poor blush development. Both these cultivars bear poorly and when crop load is increased fruit are too small for fresh fruit export purposes. ‘Forelle’ tree plantings and the number of cartons exported have, therefore, increased annually (Fig. 1 and 2, respectively), whereas the corresponding figures for ‘Rosemarie’ and ‘Flamingo’ are much smaller in comparison and have decreased (Hortgro Services/Deciduous Fruit Producers’ Trust, 2000 to 2009; Perishable Products Export Control Board, 2003 to 2009; van Papendorp, 2010).
‘Forelle’ pears, however, are prone to a dry textural disorder that occurs after storage at low temperatures plus ripening to firmness below 4 kg (39.2 N). Mealiness has been present for as long as the cultivar has been grown, but is not well understood. In a susceptible season mealiness would typically increase as firmness gradually decreases with an increase in storage time and ripening potential between 6 and 12 weeks after storage at ‐0.5°C, depending on the season and harvest maturity (Carmichael, in press; Martin, 2002). Thereafter, mealiness development decreases as the storage period lengthens prior to ripening. Cold storage of ‘Forelle’ pears in regular atmosphere can last up to 21 weeks by which time mealiness is very low during subsequent ripening (Martin, 2002). The shortest recommended storage period of 12 weeks at ‐0.5°C is, therefore, mandatory to ensure consistent and acceptable eating quality (Hurndall, 2008).
Research into factors affecting mealiness of ‘Forelle’ pears in South Africa include: effects of harvest maturity (Carmichael, in press; Martin, 2002,), climatic and ripening models (Lötze and Bergh, 2004), intermittent warming (de Vries and Hurndall, 1993), chilling injury (Martin, 2002; Martin et al., 2003), controlled‐ and regular atmosphere storage intervals (de Vries and Hurndall, 1994; de Vries and Moelich, 1995), ethylene treatments (du Toit et al., 2001; Martin, 2002), pre‐harvest temperatures above 40°C and overhead cooling (Crouch et al., 2004), and rootstocks and mineral nutrients (North and Reinten, 2007). Although, it seems as if harvest maturity plays a role after storage shorter than 12 weeks at ‐0.5°C (Carmichael, in press), none of the other factors seem to play a role or explain the development of mealiness in ‘Forelle’ pear.
Factors affecting mealiness in other pears are high seasonal heat units (Hansen, 1961) and high total heat units six weeks prior to harvest (Mellenthin and Wang, 1976). This does not seem the case in ‘Forelle’ pear (Crouch et al., 2004). Harvest at a post‐optimum maturity causes mealiness in ‘La France’ and ‘Marguerite Marillat’ pears (Murayama et al., 1998). Prolonged cold storage plays a role in the development of mealiness in ‘d’Anjou’, ‘La France’ and ‘Marguerite Marillat’ pears (Chen et al., 1983; Murayama et al.,
2002). This is unlike ‘Forelle’ pear, where prolonged cold storage reduces mealiness upon ripening (Martin et al., 2003).
Many other fruit are also known to develop mealy or dry and soft textures. Woolliness in nectarines and peaches has been described extensively and generally occurs due to long term storage at low temperature. Chilling injury affects ethylene production (Zhou et al., 2001) which in turn influences normal cell wall disassembly (Brummell et al., 2004). This in turn may cause calcium‐pectate gels to form which can take up cell fluids (Zhou et al., 2000a). More advanced disassembly of the middle lamella in affected fruit further cause enlarged intercellular air spaces or large air pockets resulting from extensive cell separation (King et al., 1989; Luza et al., 1992; von Mollendorff., 1991). This may lead to cell‐to‐cell sliding, preventing cells from breaking and releasing juice. It is, therefore, suggested that the combination of calcium‐pectate gels and cell‐to‐cell sliding are involved in the mechanism of peach mealiness development (Brummell et al. 2004). Cell wall changes were also noted in chilling injured plum with a dry textural disorder (Manganaris et al., 2008; Taylor et al., 1994), as well as in grainy kiwifruit and mealy persimmon and tomato fruit (Bauchot et al.1999; Jackman et al., 1992; Lallu, 1997; Woolf et al., 1997). Mealiness in apples has also been associated with changes in the cell wall. The mechanism of mealiness development also involves the dissolution of the middle lamella (due to senescence) which causes cells to part rather than to rupture and release juice during eating (De Smedt et al., 1998; Harker and Hallet, 1992), without the formation of pectin gels. Pear mealiness hasn’t been studied as extensively as peach or nectarine woolliness. However, pear mealiness is associated with low water‐soluble pectin, little Na2CO3 solubilisation, although
depolymerisation occurred, limited depolymerisation of glycan neutral sugars and xyloglucan, restricted degradation of cellulose and low endo‐polygalacturonase activity (Hiwasa et al., 2004; Muryama et al., 2002 and 2006). Advanced degradation of pectic substances from the middle lamella has also been reported in mealy tissues (Yamaki et al., 1983).
All soft, dry textural disorders, whether induced by chilling injury or other factors, seem to be related to a difference in cell wall breakdown when compared to juicy fruit. The aim of this work was, therefore, to investigate the occurrence of mealiness‐associated changes in the cell wall i.e. solubilisation and depolymerisation of pectins and glycans as well as cell‐to‐cell adhesion. This would explain whether the mechanism of mealiness development in ‘Forelle’ pear is possibly via the formation of pectate‐gels absorbing free juice (Ben‐Arie and Lavee, 1971; von Mollendorff and de Villiers, 1988; Taylor et al., 1993a,b and 1995; Zhou et al., 2000a,b), or via extensive cell separation, where cell‐to‐cell sliding prevents cells from breaking and releasing juice (Harker and Hallet, 1992), or a combination of both (Brummell et al., 2004).
REFERENCES:
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Ben‐Arie, R. and Lavee, S., 1971. Pectic changes occurring in Elberta peaches suffering from woolly breakdown. Phytochem. 10, 531‐538.
Brummell, D.A., Dal Cin, V., Lurie, S., Crisosto, C.H. and Labavitch, J.M., 2004. Cell wall metabolism during the development of chilling injury in cold‐stored peach fruit: association of mealiness with arrested disassembly of cell wall pectins. J. Exp. Bot. 55(405), 2041‐2052.
Carmichael, P.C., in press. Predicting optimum harvest time and eating quality of Forelle pears. Thesis presented for the degree of Master of Science (Agric) in Horticulture. Department of Horticultural Sciences, Stellenbosch University.
Chen, P.M., Mellenthin, W.M. and Borgic, D.M., 1983. Changes in ripening behavior of ‘d’Anjou’ pears (Pyrus communis L.) after cold storage. Scientia Hort. 21, 137‐146.
Crouch, E.M., Huysamer, M.H. and Holcroft, D.M., 2004. Mealiness of ‘Forelle’ pears – Quo Vadis? Acta Hort. 671, 369‐376. De Smedt, V., Pauwels, E., De Baerdemaeker, J., Nikolaï, B., 1998. Microscopic observation of mealiness in apples: a quantitative approach. Postharvest Biol. Technol. 14, 151‐158. de Vries, P.J. and Moelich, J., 1995. Shortening the required cold storage period for ‘Forelle’ pears. Unifruco Research Report 1995, 268‐271. de Vries, P.J. and Hurndall, R.F., 1994. Maturity parameters and storage regimes to obtain ‘Forelle’ pears of an acceptable eating quality. Unifruco Research report 1994, 160‐163. de Vries, P.J. and Hurndall, R.F., 1993. Maturity parameters and storage regimes to obtain ‘Forelle’ pears of an acceptable eating quality. Unifruco Research Report 1993, 95‐99. du Toit, P.G., Jacobs, G., Huysamer, M. and Holcroft, D.M., 2001. Exogenously applied ethylene reduces the cold requirement for ripening of pears (Pyrus communis L.) cv. Forelle. South African J. Plant Soil 18(4), 147‐153.
Hansen, E., 1961. Climate in relation to postharvest physiological disorders of apples and pears. Proc. Oregon. Hort. Soc. 53, 54‐58 (Cited by Rease, 1989). Harker, F.R. and Hallet, I.C., 1992. Physiological changes associated with development of mealiness of apple fruit during cool storage. HortSci. 27(12), 1291‐1294. Hiwasa, K., Nakano, R., Hashimoto, A. Matsuzaki, M., Murayama, H. Inaba, A. and Kubo, Y., 2004. European, Chinese and Japanese pear exhibit differential softening characteristics during ripening. J. Exp. Bot. 55(406), 2281‐2290.
Hortgro Services/Deciduous Fruit Producers’ Trust, 2000 to 2009. Key Deciduous Fruit Statistics 2000 to 2009, DFPT tree census. Paarl, South Africa. www.hortgro.co.za
Hurndall, R.F., 2008. ‘Forelle’ dispensation procedures for 2008 season. http://www.deciduous.co.za (Accessed 27‐3‐2010).
Jackman, R.L., Gibson, H.J. and Stanley, D.W., 1992. Effects of chilling injury on tomato fruit texture. Plant Physiol. 86, 600‐608.
King, G.A., Henderson, K.G. and Lill, R.E., 1989. Ultrastructural changes in the nectarine cell wall accompanying ripening and storage in a chilling‐resistant and chilling‐sensitive cultivar. N.Z. J. Crop Hort. Sci. 17, 337‐344. Lallu, N., 1997. Low temperature breakdown in kiwi fruit. Acta Hort. 444(2), 579‐584. Lötze, E. and Bergh, O., 2004. Summary of the quality prediction project results for 2003/04 for pome fruit. South African Fruit J. 3(5), 28‐29. Luza, J.G., van Gorsel, R., Polito, V.S. and Kader, A.A., 1992. Chilling injury in peaches: a cytochemical and ultrastructural cell wall study. J. Amer. Soc. Hort Sci. 117, 114‐118. Manganaris, G.A., Vicente, A.R., Crisosto, C.H. and Labavitch, J.M., 2008. Cell wall modifications in chilling‐ injured plum fruit (Prunus salicina). Postharvest Biol. Technol. 48, 77‐83. Martin*, E.M., Crouch, I.J. and Holcroft, D.M., 2003. Ripening and mealiness of ‘Forelle’ pears. Acta Hort. 600, 449‐452 (*Now Crouch).
Martin*, E.M., 2002. Ripening responses of ‘Forelle’ pear. Thesis presented for the degree of Master of Science (Agric) in Horticulture. Department of Horticultural Sciences, Stellenbosch University (*Now Crouch).
Mellenthin, W.M. and Wang, C.Y., 1976. Preharvest temperatures in relation to postharvest quality of ‘d’Anjou’ pears. J. Amer. Soc. Hort. Sci. 101, 302‐305.
Murayama, H., Katsumata, T., Endou, H., Fukushima, T. and Sakurai, N., 2006. Effect of storage period on the molecular mass distribution profile of pectic and hemicellulosic polysaccharides in pears. Postharvest Biol. Technol. 40, 141‐148.
Murayama, H., Katsumata, T., Horiuchi, O. and Fukushima, T., 2002. Relationship between fruit softening and cell wall polysaccharides in pears after different storage periods. Postharvest Biol. and Technol. 26, 15‐21.
Murayama, H., Takahashi, T., Honda, R. and Fukushima, T., 1998. Cell wall changes in pear softening on and off the tree. Postharvest Biol. Technol. 14, 143‐149.
North, M. and Reinten, E., 2007. Pear rootstocks and planting systems. Deciduous Fruit Producers Trust Annual Report 2007/2008, Project no. 2200009.
Perishable Products Export Control Board, 2003 to 2009. Statistics on cartons passed for export per pear cultivar. Montague Gardens, Cape Town, South Africa. www.PPECB.com. Cited by: Hortgro Services/Deciduous Fruit Producers’ Trust, 2000 to 2009.
Taylor, M.A., Rabe, E., Jacobs, G. and Dodd, M.C., 1995. Effect of harvest maturity on pectic substances, internal conductivity, soluble solids and gel breakdown in cold stored ’Songold’ plums. Postharvest Biol. and Technol. 5, 285‐294. Taylor, M.A., Rabe, E., Dodd, M.C. and Jacobs, G., 1994. Effect of storage regimes on pectolytic enzymes, pectic substances, internal conductivity and gel breakdown in cold stored ‘Songold’ plums. J. Hort. Sci. 69, 527‐534. Taylor, M.A., Jacobs, G., Rabe, E. and Dodd, M.C., 1993a. Physiological factors associated with overripeness, internal breakdown and gel breakdown in plums stored at low temperature. J. Hort. Sci. 68, 825‐ 830. Taylor, M.A., Rabe, E., Jacobs, G. and Dodd, M.C., 1993b. Physiological and anatomical changes associated with ripening in the inner and outer mesocarp of cold stored ‘Songold’ plums concomitant development of internal disorders. J. Hort. Sci. 68, 911‐918.
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von Mollendorff, L.J., 1991. Anatomical and ultrastructural study on changes in mesocarp tissue during storage and ripening of nectarine. In: Post‐Harvest factors involved in the development of woolliness in nectarines (Prunus persica). Chapter 7, 163‐179. Thesis presented for the degree of Doctor of Philosophy (Agric) in Horticulture, Department of Horticultural Science, Stellenbosch University.
von Mollendorff, L.J., and de Villiers, O.T., 1988. Role of pectolytic enzymes in the development of woolliness in peaches. J. Hort. Sci.63, 53‐58.
Woolf, A.B., MacRae, E.A., Spooner, K.J. and Redgwell, R.J., 1997. Changes to physical properties of the cell wall and polyuronides in response to heat treatment of ‘Fuyu’ persimmon that alleviate chilling injury. J. Amer. Soc. Hort. Sci. 122(5), 698‐702.
Yamaki, S. Sato Yoshihiko and Machida, Y., 1983. Degrading enzyme activities in mealy fruit and “Ishinashi” fruit of Japanese pear (Pyrus serotina Rheder var. culta Rehder). Fruit Tree Research Station contribution A‐157, 123‐134. Ministries of Agriculture, Forestry and Fisheries, Yatabe, Ibaraki, 305, Japan. Zhou, H., Dong, L., Ben‐Arie, R. and Lurie, S., 2001. The role of ethylene in the prevention of chilling injury in nectarines. J. Plant Physiol. 158, 55‐61. Zhou, H., Ben‐Arie, R. and Lurie, S., 2000a. Pectin esterase, polygalacturonase and gel formation in peach fractions. Phytochem. 55, 191‐195. Zhou, H., Sonego, L., Khalchitski, A., Ben‐Arie, R., Lers, A. and Lurie, S., 2000b. Cell wall enzymes and cell wall changes in ‘Flavortop’ nectarines: mRNA abundance, enzyme activity, and changes in pectic and neutral polymers during ripening in woolly fruit. J. Amer. Soc. Hort. Sci. 125(5), 630‐637.
Figure 1: Total area of blushed‐pear cultivars in South Africa from 2000 to 2009. (Hortgro Services/Deciduous Fruit Producers’ Trust, 2000 to 2009).
Figure 2: Blushed‐pear cultivars (12.5 kg carton equivalents) exported from 2003 to 2010 (Perishable Products Export Control Board, 2003 to 2009; von Papendorp, 2010). 0 500 1000 1500 2000 2500 3000 3500 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 He ct ar e s Year Forelle Rosemarie Flamingo 0 0.5 1 1.5 2 2.5 3 3.5 2003 2004 2005 2006 2007 2008 2009 2010 Ca rt o n s ex p o rt e d (m illi o n ) Year Forelle Rosemarie Flamingo
CHAPTER 1: LITERATURE REVIEW THE ROLE OF CELL WALLS IN DRY TEXTURAL DISORDERS WITH SPECIAL REFERENCE TO PECTIN AND MEALINESS 1.1 Introduction Consumer acceptance of fresh fruit is dependent on the sensory quality or experience of a certain fruit kind and is influenced by what is expected of a known product (Manning, 2009). For most fruit the primary quality attributes are flavour and appearance together with texture (Lapsley, 1989). The importance of flavour and appearance (size, shape and colour) of fresh products is well known and has been studied extensively. Texture was only later recognized as an important factor of food quality for the consumer (De Smedt, 2000) and was first described by Matz (1962). Lammertyn et al. (2002) also notes that even though there is a subconscious awareness of texture, flavour very often overshadows texture at the conscious level. Szczesniak (2002) explains that texture of a food is taken for granted and is not distinguished as a separate and distinct characteristic. However, the texture awareness is increased when expectations are violated and unpleasant mouth sensations are experienced. In apple, texture seems to be the predominant quality attribute, together with flavour and appearance (Lapsley et al., 1992). In pear the overall preference for consumers is a sweet tasting pear with a strong flavour and a soft, melting, juicy but not mealy texture (Manning, 2009). This preference can differ with various consumers. Some consumers prefer a firm, juicy fruit (Hoehn et al., 1996). Eccher Zerbini et al. (2000) states further that texture is a critical feature of pear and due to its complex nature mechanical measurements have not been able to replace the mouth‐feel that consumers perceive. Interestingly, juiciness was one of the most frequently mentioned textural attributes in word association tests conducted in the U.S.A. (Szczesniak and Kleyn, 1963; Szczesniak, 1971) and in Japan (Yoshikawa et al., 1970).
1.1.1 Definitions of texture
There are many definitions describing various aspects and attributes of texture. Bourne (1982) describes texture as a group of physical characteristics that arise from the structural elements of food that are sensed by the mouth, are related to the deformation, disintegration, and flow of food under force, and are measured objectively by functions of mass, time and distance. Corey (1970) also gives a detailed definition, but aspects of his definition focus on the human sensory characteristics. He said that: “…the textural experience during chewing is a dynamic integration of mouth‐feel, the prior tactile responses while handling the foodstuff, and a psychic anticipatory state arising from the visible perception of the food’s overall geometry and surface features…”. Many definitions have been summarised in one generally accepted definition encompassing aspects mentioned in the above two definitions. Important aspects of
this definition are further described by Szczesniak (2002) of which one has not been mentioned clearly by the above descriptions (Bourne, 1982; Corey, 1970). This is namely that texture is a sensory property and therefore only a human being can perceive it. The texture testing instruments can only measure certain physical parameters which have to be interpreted in terms of sensory perception.
1.1.2 Textural properties
Food textural properties are multi‐dimensional and can also be divided into mechanical characteristics, geometrical characteristics and other characteristics (e.g. moisture and fat content) (Szczesniak, 1963). The European pear fruit exhibits all of these characteristics and was given as an example of the above mentioned classification by Harker et al. (1997a). The mechanical properties are related to the cell strength. The inherent grittiness is associated with geometric properties of stone cells and the juiciness changes with fruit ripening. A change of a single characteristic can interfere with the perception of the others, which points to the complex nature of texture (Harker et al., 1997a).
1.1.3 Textural properties pertaining to dry textural disorders
Various textural attributes can be precisely described in order to classify them correctly. Some of these attributes pertaining to dry textural disorders are: mealiness, woolliness, flouriness, pastiness, starchiness and leatheriness. Opposites of these characteristics would be a melting texture and juiciness. Most of these attributes are clearly defined with absent/low and extreme/high reference standards in Harker et al. (1997a). A combination of textural attributes can be useful in explaining various aspects of the texture. Harker et al. (1997a) used hardness and juiciness as combined attributes, as an example. This was used to describe the following: a soft and dry banana; hard and dry unripe fruit; hard and juicy apple and a soft and juicy peach. The complex nature of texture is associated with the diversity of attributes needed to fully describe a specific texture at a specific time.
Dry textural disorders of fruit can be classified into soft, dry tissues or hard, dry tissues. Mealiness, woolliness and pastiness fall into the soft category and are normally associated with ripened fruit that have a dry texture, even though the water content seems to be similar to non‐affected fruit (Ben‐Arie and Lavee, 1971; Zhou et al., 2000a). Expressible juice is, however, negatively affected in these soft and dry tissues (Brummell et al., 2004a, Martin, 2002). Gel breakdown and internal browning of plums are also related to dry, soft tissues (Taylor et al., 1993a, b). These disorders are often related to over maturity (apples) (Snowdon, 1990), post‐optimum harvest maturity (apple, pear, plum gel breakdown) (Mitcham and Mitchell, 2002; Spotts, 1981; Taylor et al., 1995), long cold storage durations (apple, pear) (Murayama et al., 1998; Snowdon, 1990) and chilling injury (peach, nectarine, plum, kiwifruit, tomato and melon) (Bauchot et al., 1999; Brovelli et al., 1998; Brummell et al., 2004a; Fernández‐Trujillo et al., 2008; Hartmann et al., 1988; Jackman et al., 1992; Lallu, 1997; Taylor et al., 1994; von Mollendorff and de Villiers, 1988a) in combination with pre‐optimum harvest maturity (nectarine, plum internal browning) (Kotzé et al., 1989;
von Mollendorff, 1987). It is also influenced by cultivar (Crisosto et al., 1999; De Smedt, 2000). Leatheriness is also characterized by a dry texture, but is normally associated with hard or firm and rubbery fruit (Ju and Ju., 2000; McGlasson et al., 2005) that does not soften normally during ripening. This disorder can be related to pre‐optimum harvest maturity (pear, peach) (Ju and Ju, 2000; Murayama et al., 1998) or long term cold storage (pear, peach, loquat) (Cao et al., 2010a; Brummell et al., 2004a; Wang et al., 1985). 1.2 Factors influencing textural changes in ripening fruit and other fresh products
Textures of fresh plant products are dependent on the characteristics of their living cells (Harker et al., 1997a). Such characteristics would be cell size and shape, cell wall thickness and strength (Harker et al., 1997a), cell turgor (Harker and Sutherland 1993; Ilker and Szczesniak, 1990; Szczesniak and Ilker, 1988;) and starch content (Tucker, 1993) in immature pome fruit and bananas. 1.2.1 Factors influencing juiciness Szczesniak and Ilker (1988) identified the following factors as prerequisites to juiciness: high water content, an organized cellular network with proper integrity and turgidity, cell walls mechanically weaker than the middle lamella, low viscosity and few suspended solids in the expressed liquid. Juicy fruit typically contain 80‐90% water (Szczesniak and Ilker, 1988). When fruit that are typically juicy and non‐juicy are classified according to their water content, apple, grape, honeydew melon, orange, plum, peach and strawberry fall into the typically juicy category and have a water content of between 81% and 90%. Avocados and bananas which are typically non‐juicy have a water content of 74% and 76%, respectively. How much fluid must, however, be lost for a product to be perceived as non‐juicy? This value varies considerably for various fruit types (Szczesniak and Ilker, 1988). Apples which were juicy at 85.7% were perceived non‐juicy at 51.4%. Juicy cucumbers with a water content of 92.6% were perceived non‐juicy when left with only 78% water content. Oranges on the other hand had to lose more than 60% of their water content before they were classified non‐juicy. This indicates, therefore, that different fresh plant materials could lose different amounts of water before being perceived as non‐juicy (Szczesniak and Ilker, 1988).
1.2.2 Turgor
In all cases of partial water loss in the above mentioned study there was a loss in turgor (Szczesniak and Ilker, 1988). For plant tissue to be regarded as juicy, the network within which water is held must have integrity and turgor. For example, fruit parenchyma tissue that has been processed has a similar moisture content to unprocessed tissue, but may not be regarded as juicy but rather as watery or wet (Szczesniak and Ilker, 1988). This is due to the tissue having lost its turgor. Turgor is maintained by the semi‐permeable cell membrane and the physical strength of the cell wall. This selective permeability is often lost with fruit ripening or damage (e.g. chilling injury) which contributes to textural changes (Harker and Sutherland, 1993; Shackel et al., 1991). Postharvest water loss (Saladié et al., 2007) and the accumulation of osmotic
solutes in the cell wall space could also partly contribute to the change in turgor during ripening (Almeida and Huber, 1999). In contrast, mealiness or woolliness occurs when free available juice levels decrease after extended cold storage of late season peaches and nectarines. The juice content of this fruit is similar to normally ripened fruit, but is not available for the consumer to perceive during mastication (Ben‐Arie and Lavee, 1971; Zhou et al., 2000a). When free juice percentages in ‘O’Henry’ and ‘Summer Lady’ peaches declined to 38% and 46%, respectively, it was clear that fruit were mealy (Obenland et al., 2003).
Turgor pressure in parenchyma cells, however, also generates a tension on the cell walls which tends to pull cells towards a sphere, the shape of lowest energy and stress on the walls (Jarvis et al., 2003). This in turn could cause cell separation which is seen in fruit tissues toward the end of ripening or during the development of mealiness (De Smedt, 2000). To withstand this and maintain intercellular adhesion during cell growth and development great intercellular adhesion strength is necessary at the cell corners where this tension is the greatest (Jarvis, 1998). These areas have reinforcing zones that differ from the rest of the wall and middle lamella. They carry the turgor‐imposed pressure and are the first line of defense against cell separation (Parker et al., 2001). An extensin‐2 class of hydroxyproline‐rich glycoprotein has also been found exclusively in certain instances in the tricellular junctions (Smallwood et al., 1995; Swords and Staehelin, 1993). Laurenzi et al. (2002) also found this for polyamine oxidases. These components may function together generating specific cell wall matrix properties at cell wall junctions which are currently unknown (Jarvis et al., 2003). 1.2.3 Cell‐to‐cell adhesion The manner in which fruit cells bind also plays a role, including cell‐cell adhesion (Brummell et al., 2004a; De Smedt, 2000) and packing. All fruit flesh is composed of parenchyma cells which have thin, non‐lignified walls (0.4 μm – 1.0 μm) and a large vacuole that may contain 90% of the water in the cell (Pitt, 1982). The cells are separated by the middle lamella (Knox, 1992). The strength of the cell wall versus the strength of the middle lamella largely determines the way parenchyma tissue collapses during mastication, which influences the way one perceives juiciness. If the cell wall is weaker than the middle lamella, fracture occurs across the cells. If the cell wall is stronger than the middle lamella, tissue failure will occur between cells (Szczesniak and Ilker, 1988). In apple texture the above mentioned mechanism plays a role in the perception of juiciness. In firm, juicy fruit the juice is released via breaking of the cell wall rather than the strong middle lamella. As senescence progresses, the intercellular spaces grow due to weakening of the middle lamella, resulting in low cell‐to‐cell binding. This causes tissue failure to occur in the middle lamella rather than through the cell wall, which causes no release of cell fluids upon mastication. These tissues are therefore perceived mealy (De Smedt et al., 1998; Harker and Hallet, 1992). In a peach study, however, the fracture surfaces of normally ripening and mealy tissues didn’t contain broken cells as was the case in firm apple. Instead, the cell surface of normally ripened fruit was covered in juice (Harker and Sutherland, 1993). Harker et al. (1997b) explained that when fruit ripen to a soft texture, e.g. peach, nectarine, kiwifruit,
avocado and strawberry the cells separate rather than break from each other as happens in harder fruit like apple, and they all have a characteristic juice layer on the fracture surface. The origin of the juice is unclear, but could be from the apoplast that is normally hydrated, especially during ripening when the higher membrane permeability plays a role, or from exudation of cellular content caused by the tensile pressure applied. It is therefore important to consider the relationships between anatomy and cellular construction, the mechanical and physiological properties as well as the mechanisms of cell failure in evaluating texture. 1.3 Factors influencing the perception of juiciness
Szczesniak and Ilker (1988) indicated that the common definition of juiciness: “the amount of juice released on mastication” is too simplistic since several sensations are involved in the oral perception of juiciness. The composite multi‐dimensional sensational factors were listed as: 1. the force with which the juice squirts out, 2. the amount of juice expressed on first chew and subsequent chews, 3. consistency of the liquid and 4. contrast between the liquid and the solid (or semi‐solid) phase (cellular debris). In addition factors that may further influence the sensory perception of juiciness are: initial mouth feel (wet/dry), initial mechanical properties (hardness, crispness, etc.), mechanical/geometrical properties of residue (fibrous, granular or pulpy, etc.), and the effect of saliva production (stimulating, reducing, dehydrating). As mentioned before there are many ways of describing texture (Harker et al., 1997a) and the types of juiciness (Szczesniak and Ilker, 1988). There is, however, no general consensus on how juiciness could be expressed quantitatively as one number.
1.3.1 The force with which juice is released
The force with which juice squirts out and the amount of juice released upon first chew seemed to be the most influential factors (Szczesniak and Ilker, 1988). Different tissues, however, react differently to these criteria, but, they also influence perception of juiciness of these different tissues. In pear and orange (and most other fresh products) the amount of juice decreased with successive chews. In mushrooms it increased, whereas watermelons released an almost constant amount of juice on successive chews. In mushrooms the increase can be explained by not having a defined cellularity, but rather criss‐crossing myceliar fibers which have a high structural integrity which is only broken down by successive chewing or by low temperature heating (Szczesniak and Ilker, 1988).
1.3.2 Cell size and cell wall thickness
Szczesniak and Ilker (1988) noticed that cell size increased steadily with an increase in perception of juiciness (with the exception of tomato and mushroom). Larger cells contain larger vacuoles which release more juice upon injury. Cell wall thickness decreased with increasing sensory juiciness. The cell wall polymers are more broken with ripening, decreasing their density and, therefore, their strength. Less dense (and thinner) cell walls break easier, as long as the middle lamella is still intact.
