Extraction, characterization and applications of pectin from fruit wastes

Using the central composite design, the response surface methodology Minitab® was applied to the conventional heating process to optimize extraction time and temperature.. Our results sh

EXTRACTION, CHARACTERIZATION AND

APPLICATIONS OF PECTIN FROM FRUIT WASTES

Submitted in total fulfilment of the requirements for the degree of

Doctor of Philosophy

by

Dao Thi Anh Thu

Supervisors

Principal Supervisor: Dr François Malherbe

Co-Supervisor: Dr Hayden Webb

Associate Supervisor: Prof Enzo Palombo

Department of Chemistry and Biotechnology

Faculty of Science, Engineering and Technology

Swinburne University of Technology

2021 _

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Abstract

Fruit and vegetable processing operations generate large volumes of waste, often discarded or used as low-value ingredients in animal feed However, they can be a source

of chemicals, so our study investigates fruit waste as a source of pectin The diversion of waste from landfills contributes to sustainable practices, while processing of the biomass can generate value-added products The focus is yield optimisation and production of high

quality pectin from peels of white-flesh (Hylocereus undatus) and red-flesh (Hylocereus

polyrhizus) dragon fruits, and purple passion fruits (Passiflora edulis) Both conventional

and microwave-assisted heating processes were considered Using the central composite design, the response surface methodology (Minitab®) was applied to the conventional heating process to optimize extraction time and temperature For the microwave-assisted process, a three-level Box-Behnken design targeted power, pH, extraction time, and liquid:solid ratio The physicochemical properties of the pectin were assessed using a suite of analytical techniques, and compared to commercial pectin for quality, on the basis

of their degree of esterification (DE) and methylation (DM) Our results show that in conventional extraction the type of peels influences both yield and degree of esterification; microwave-assisted heating gave significantly higher yields for all types of peels The parameters giving the highest yield (18.73 %) for passion fruit peels were: extraction time -12 minutes, power - 218 W, pH - 2.9 and liquid:solid ratio - 57:1 mL/g The results also evidenced important variations in the physicochemical properties of extracted pectin with processing conditions Pectin with the highest degree of esterification was extracted from PFP by conventional heating (61.98 %); the material obtained from white-flesh DFP by microwave had the lowest (41.96 %)

The structural assessment by Fourier Transform Infrared spectroscopy evidenced that our pectin was very similar to commercially available citrus pectin The extracted pectin had a high specific surface area and was categorised as typical amorphous polymers In terms of functional properties, the pectin extracted from PFP by conventional heating showed the lowest solubility and highest emulsion capacity while the PFP pectin from microwave heating had the highest solubility, oil-holding capacity and foaming capacity The rheological properties indicated that increasing PFP pectin concentration produced solutions with enhanced viscosity The higher strength of PFP pectin gel was observed with higher calcium concentration as a crosslinking agent

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To evaluate pectin as a functional biomaterial, its use as a vector for probiotics was studied Preliminary results show that both type of peels and extraction conditions influenced the morphology of the gelatinous capsules formed, critical to their intrinsic properties To determine encapsulation efficiency, the viability of entrapped cells in simulated digestive media (salivary, gastric and intestinal fluids) was compared to that of free micro-organisms Overall, the results indicated that pectin extraction represents a viable avenue for the effective valorisation of fruit processing wastes and

microwave-assisted heating could be a significant energy saving technique for high yield extractions without compromising product quality The application of the extracted pectin as potential probiotic encapsulating material gave promising results

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Acknowledgments

I would like to express the deepest appreciation to my supervisors Dr François Malherbe, Dr Hayden Webb and Prof Enzo Palombo for the continuous support of my PhD and your patience, motivation, enthusiasm and immense knowledge Your thoughtful comments and recommendations helped me in all the time of research and writing of this thesis, without which I would have stopped my PhD a long time ago

This work would not have been possible without the financial support of the 911-Swinburne joint scholarship My sincere thanks also go to the staff of chemistry and biotechnology laboratories for your considerate guidance and suggestions to complete my questionnaire My appreciation also extends to my laboratory colleagues for your mentoring, encouragement and willingness throughout my project

Last but not least, I would like to express my sincere gratitude to my parents for supporting me spiritually throughout my life; to my husband and my daughters for helping

me survive all the stress and not letting me give up in these very intense academic years

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Declaration

I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at Swinburne or any other educational institution, except where due acknowledgement is made in the manuscript Any contribution made to the research by others, with whom I have worked at Swinburne or elsewhere, is explicitly acknowledged

in the report I also declare that the intellectual content of this report is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged

Melbourne, 17 September 2020

Dao Thi Anh Thu DaihocDaNang

List of publications

1 Dao, T.A.T., Webb, H.K and Malherbe, F (2020) Optimisation of pectin extraction from fruit peel by response surface method: conventional versus microwave-assisted

heating Food Hydrocolloids, vol 113, April 2021

2 Pectin extraction from peels of white dragon fruit (Hylocereus undatus) and red dragon fruit (Hylocereus polyrhizus) optimised by response surface methodology, 1 st

Global Conference on Health, Agriculture and Environmental Sciences, June 2018,

Melbourne, Australia

3 Valorisation of waste industrial biomass: optimisation of pectin extraction from fruit

peels, 2 nd International Conference on Agriculture, Food and Biotechnology (ICAFB 2019), January 2019, National University of Singapore, Singapore

4 Chemical and functional properties of pectin extracted from Passiflora edulis f edulis (purple passion fruit) peel by microwave-assisted heating”, 15 th International Hydrocolloids Conference, March 2020, Melbourne, Australia

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Table of contents

Abstract i

Acknowledgments iii

Declaration iv

List of publications v

Table of contents vi

List of Figures x

List of Tables xv

Chapter 1: Introduction 1

Chapter 2: Literature review 4

2.1 Valorization of food processing waste 4

2.2 Pectin 6

2.2.1 Structure of pectin 6

2.2.2 Product specifications 8

2.2.3 Industrial production of pectin 12

2.2.4 Applications of pectin 20

2.2.5 Exploring new sources for pectin production 21

2.3 Probiotics 26

2.3.1 Probiotics in the human gastrointestinal tract and health benefits 26

2.3.2 Prebiotics 27

2.4 Encapsulation 28

2.4.1 Encapsulation of microbial cells 29

2.4.2 Pectin as emerging materials for encapsulation 29

Chapter 3: Materials and methodology 39

3.1 Ingredients and chemicals 39

3.2 Preparation of raw materials from fruit peels 39

3.3 Response surface methodology (RSM) 39

3.3.1 Factorial Design 39

3.3.2 Central Composite Design (CCD) 41

3.3.3 Box-Behnken Design (BBD) for four independent variables 44

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3.4 Conventional heating extraction 46

3.5 Microwave-assisted extraction 47

3.6 Pectin characterization 47

3.6.1 Pectin yield 47

3.6.2 Equivalent weight 47

3.6.3 Methoxy content 48

3.6.4 Moisture 48

3.6.5 Solubility 48

3.6.6 The total carbohydrate contents 48

3.6.7 Degree of esterification (DE) and degree of amidation (DA) 49

3.6.8 The content in galacturonic acid 49

3.6.9 Surface morphology analysis 50

3.6.10 Fourier Transform infrared spectroscopy 50

3.6.11 Rheological properties 50

3.6.12 Brunauer-Emmett-Teller (BET) nitrogen adsorption 50

3.6.13 X-ray diffraction (XRD) 51

3.6.14 Oil-holding capacity 51

3.6.15 Emulsifying properties 51

3.6.16 Foaming properties 51

3.7 Prebiotic score 52

3.7.1 Growth of probiotics in the presence of pectin 52

3.7.2 Prebiotic activity score 52

3.8 Microencapsulation 53

3.8.1 Preparation of cell culture 53

3.8.2 Bacterial enumeration method 53

3.8.3 The growth curve of L casei cells 53

3.8.4 Encapsulation process 53

3.8.5 Analysis of the gelled capsules and freeze-dried capsules 55

3.8.6 Viability of probiotic through microencapsulation 56

3.9 Statistical analysis 59

Chapter 4: Optimization of pectin extraction by conventional heating and microwave-assisted heating 60

4.1 Optimization of pectin extraction by conventional heating 60

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4.1.1 Effects of extraction time on pectin yield and DE 60

4.1.2 Effects of extraction temperature on pectin yield and DE 62

4.1.3 Factorial design for two types of dragon fruit peels 63

4.1.4 Optimization of pectin extraction by conventional heating from dragon fruit peels by a fitted quadratic model 67

4.1.5 Optimization of pectin extraction by conventional heating from passion fruit peels by a fitted quadratic model 75

4.1.6 Conclusion 80

4.2 Optimization of pectin extraction by microwave-assisted method by fitted quadratic model 81

4.2.1 Effects of microwave power on pectin yield 81

4.2.2 Effects of processing time on pectin yield 82

4.2.3 Effects of pH on pectin yield 82

4.2.4 Experimental data, model fitting and statistical analysis 83

4.2.5 Analysis of interaction plots and response surface plots 88

4.2.6 Validation of optimum conditions of pectin extraction by microwave-assisted method from the DFP and PFP 94

4.3 Conclusion and comparison with conventional heating 94

Chapter 5: Properties of extracted pectin 96

5.1 Physicochemical properties 96

5.1.1 Moisture content 96

5.1.2 Equivalent weight (Eq W) and methoxyl content 97

5.1.3 Degree of esterification (DE) 99

5.1.4 The total carbohydrate content and the content of galacturonic acid 99

5.1.5 Degree of amidation (DA) 101

5.1.6 Fourier Transform Infrared spectroscopy 101

5.1.7 Scanning Electron Microscopy (SEM) 107

5.1.8 BET surface area 108

5.1.9 X-ray diffraction (XRD) 110

5.2 Functional properties of pectin 112

5.2.1 Solubility 112

5.2.2 Oil-holding capacity (OHC) 114

5.2.3 Foaming properties 115

5.2.4 Emulsifying properties 116

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5.2.5 Rheological properties 118

5.3 Conclusion 124

Chapter 6: Pectin as potential material for the microencapsulation of probiotics 125 6.1 Overview 125

6.2 Growth curve of probiotic 125

6.3 Prebiotic activity score 126

6.4 Examination of the gelled capsules and freeze-dried capsules 128

6.4.1 Particle shape, size distribution and sphericity factor of capsules 128

6.4.2 Scanning Electron Microscopy (SEM) 132

6.4.3 Chemical structure by FTIR 133

6.5 Microencapsulation efficiency 135

6.5.1 Double coating 137

6.5.2 Survival of encapsulated cells under simulated gastrointestinal conditions 138 6.5.3 Swelling studies 145

6.5.4 Storage stability of cells in wet capsules at 4 °C 147

6.6 Freeze-drying capsules loaded with probiotic cells 147

6.7 Heat tolerance 148

6.8 Conclusion 150

Chapter 7: Conclusion 151

Bibliography 156

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List of Figures

Figure 2.1 Bioactive compounds from modern fruit processing waste (data from

Banerjee et al., (2017)) 5

Figure 2.2 Schematic diagrams of four domain pectin structures: The HG (smooth) regions are linear galacturonic acid, an oxidized form of D-galactose, with partially methyl-esterification; the XG is an HG substituted with xylose; the side chains of RGI region including galactans, arabinans and arabinogalactans; the RGII including different types of neutral sugars Adapted from Harholt et al., (2010) 7

Figure 2.3 The micro-particles structure by encapsulation: (a) microcapsules, (b) microspheres, (c) multilayer capsules, (d) multi-shell and multicore microsphere 28

Figure 3.1.The factorial design for two variables (time, temperature) including five experiments for each type of peel 40

Figure 3.2 Central composite design with two independent factors (time, temperature) including four corner-, five center- and four axial- experiments (Morris, 2000) 42

Figure 3.3 The Box-Behnken Design for four variables including 24 experimental points for each type of peel 44

Figure 3.4 A flow chart of the microencapsulation process 54

Figure 4.1 Effect of processing time on pectin yield from three types of fruit peel 61

Figure 4.2 Effect of processing time on pectin DE from three types of fruit peel 61

Figure 4.3 Effect of processing temperature at 80-minute extraction on pectin yield from three types of fruit peel 62

Figure 4.4 Effect of processing temperature at 80-minute extraction on pectin DE 63

Figure 4.5 Normal probability plot of standardized effects plots for a) yield and b) DE The red lines indicate standardized t-statistics testing the null hypothesis 65

Figure 4.6 Main effects plots of processing parameters on: a) yield and b) DE 66

Figure 4.7 Interaction plots showing the link between temperature and type of peels for DE 67 Figure 4.8 Response surface plots showing the effects of processing time and temperature on pectin yield from (a) red DFP and (b) white DFP in conventional

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extraction methods The surface plots were created based on the regression model to illustrate the relationship between the response (pectin yield) and two variables

(processing time and temperature 72

Figure 4.9 The interaction plot (time*temperature) for DE of pectin from red DFP 73

Figure 4.10 Three-dimensional plots for the extraction conditions showing their effects on DE of pectin from (a) red DFP and (b) white DFP 74

Figure 4.11 Interaction plot for extraction yield from the PFP 78

Figure 4.12 Response surface plots demonstrating the effects of processing time and temperature on a) yield and b) DE of pectin extracted from PFP 79

Figure 4.13 Effects of microwave power on the pectin yield 81

Figure 4.14 Effects of extraction time on yield 82

Figure 4.15 Effects of pH on yield 83

Figure 4.16 Interaction plot of microwave power and processing time on pectin yield from white-flesh DFP 89

Figure 4.17 Surface plots of extraction yield showing significant square terms of extraction time and microwave power (curvature) from a) red-flesh DFP; b) white-flesh DFP; c) purple PFP (pH 3 and liquid:solid ratio 50) 90

Figure 4.18 Surface plots of extraction yield showing significant square terms of pH and liquid:solid ratio (curvature) from a) red-flesh DFP, b) white-flesh DFP, and c) purple PFP Samples were irradiated for 10 minutes at 150 W 92

Figure 4.19 Interaction plot of pH and liquid:solid ratio on DE for red-flesh DFP 93

Figure 4.20 Surface plots of pectin DE showing significant square terms of pH indicated by a curve on their response surface plot from red-flesh DFP 93

Figure 5.1 The difference of heating mechanisms for conventional heating and microwave irradiation 98

Figure 5.2 The FTIR spectra of pectins extracted by (a) conventional, and (b) microwave-assisted heating 102

Figure 5.3 FTIR spectra of pectin recovered from (a) white-flesh DFP, (b) red-flesh DFP and (c) PFP at different pH 104

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