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

______

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.

i

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.

ii

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.

iii

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

iv

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, 1st 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, 2nd 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”, 15th International Hydrocolloids Conference, March 2020, Melbourne, Australia.

v

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 vi

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 vii

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 viii

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

ix

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 x 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

xi

Figure 5.4. FTIR spectra of pectin recovered from (a) white-flesh DFP, (b) red-flesh DFP and (c) PFP with different microwave power (50 W, 150 W and 250 W)...... 105

Figure 5.5. FTIR spectra of pectin recovered from (a) white-flesh DFP, (b) red-flesh DFP and (c) PFP with different liquid:solid ratio ...... 106

Figure 5.6. Effect of extraction time on pectin structure from PFP...... 107

Figure 5.7. SEM images of extracted pectins from different types of peel using conventional heating or microwave-assisted heating, followed by vacuum drying or freeze drying ...... 108

Figure 5.8. N2 adsorption/desorption isotherms of commercial and extracted pectins 109

Figure 5.9. XRD patterns of the commercial pectin (top) and pectin extracted from PFP by conventional heating (middle) and microwave-assisted heating (bottom)...... 111

Figure 5.10. Influence of shear rate on the shear stress of PFP pectin extracted by microwave-heating, and standard (ST) commercial citrus pectin at 25 °C. The dotted lines represent the linear fit based on the Ostwald-de Waele power-law model (Rao, 2007)...... 119

Figure 5.11. The apparent viscosity of PFP pectin solution extracted by microwave heating at 1, 3 and 5 %, measured at 25 °C...... 120

Figure 5.12. Frequency sweeps (25 °C; fixed strain at 2 %) of pectin solution (3 and 5 %) from PFP by microwave-heating...... 122

Figure 5.13. Mechanical spectra of pectin-calcium mixtures with storage (G’) and loss (G”) moduli as a function of frequency for pectin 1 %, pH 7, 0.5 M NaCl with a) 15 mM

CaCl2 and b) 12 mM CaCl2...... 123

Figure 6.1. Growth curve of L. casei in MRS broth at 37 ℃ under 100 rpm agitation...... 126

Figure 6.2. The formation mechanism of pectin gel particles by “egg-box” calcium linked junctions. Calcium cations form junctions between free acid groups on adjacent pectin chains (Lara-Espinoza et al., 2018)...... 128

Figure 6.3. Pectin beads produced with 2 % w/v of (a) commercial pectin; (b) pectin extracted from DFP; (c) pectin extracted from PFP ...... 129

xii

Figure 6.4. The distributions of pectin particle sizes (a, b, c) and freeze-dried capsules (d, e, f) loaded with probiotics, prepared using different type of pectin (2 %w/v): (a,d) commercial pectin, (b,e) DFP pectin and (c,f) PFP pectin...... 131

Figure 6.5. Structures of surface of different types of gelled beads with the same magnifications with scale bars 2 �m: (a) commercial pectin beads, (b) DFP pectin beads, and (c) PFP pectin beads...... 132

Figure 6.6. Structures of gelled beads from commercial pectin with different magnifications: (a) free cells probiotics (20 �m); (b): blank pectin beads (100 �m); (c) & (d) & (e): pectin beads loaded with probiotics (100 �m and 2 �m)...... 133

Figure 6.7. The FTIR spectra of different types of pectin capsules, presenting the changes of functional structure of pectin to blank pectin capsules and when loading with probiotic and after storage...... 134

Figure 6.8. The FTIR spectra of capsules with different calcium concentration as gelling agents (CP1: the highest [Ca2+] 20 mM to the CP4: the lowest [Ca2+] 10 mM)...... 135

Figure 6.9. Schematic representation of the effect of calcium concentration on crosslinking between galacturonic acid units with calcium ion. Monomers and short chain pectins (a) do not effectively form egg-box crosslinks at [Ca2+] 20 mM, whereas longer chain pectins (b) can form more links with comparatively less [Ca2+] 15 mM. 135

Figure 6.10. Effect of pectin concentration on viable cells by single-pectin coating. Remaining cells in free pectin solution and after capsule formation are both compared to the original culture ...... 137

Figure 6.11. Effect of double coating in comparison with single coating at the pectin concentration of 2 % to encapsulate probiotics...... 138

Figure 6.12. The FTIR spectra of double-coated pectin capsules in comparison with single-coated pectin capsules and free probiotic cells...... 138

Figure 6.13. The viability of probiotics encapsulated in different types of pectin (2 % w/v) in simulated salivary fluids...... 139

Figure 6.14. The viability of free cells and encapsulated cells incubated in simulated gastric fluid with and without pepsin...... 140

xiii

Figure 6.15. The viability of free cells and encapsulated cells incubated in simulated intestinal fluid...... 143

Figure 6.16. Release rate of encapsulated probiotics from different types of pectin in SIF ...... 145

Figure 6.17. Swelling rate of capsules in: a) SIF-no bile, b) SIF, c) SGF...... 146

Figure 6.18. The viable cells remaining in different types of pectin beads after 20 days storage at 4 °C. Error bars represent standard deviation (n = 3)...... 147

Figure 6.19. Viability of probiotic cells in capsules after freeze-drying following encapsulation with various concentrations and types of pectin...... 148

Figure 6.20. The viability of probiotics before and after heating at 63 °C for two minutes. Error bars represent standard deviation (n=3)...... 149

xiv

List of Tables

Table 2.1. Commercial pectin specifications (Dixon, 2008) ...... 8

Table 2.2. Pectin extraction from recent literature ...... 16

Table 2.3. Studies of pectin extraction from yellow passion fruit peels ...... 24

Table 3.1. The three experimental variables and their factorial design settings ...... 40

Table 3.2. Specific combinations of experimental variables tested ...... 41

Table 3.3. The levels and coded and uncoded values of two independent variables ...... 42

Table 3.4. The experimental runs following the CCD for pectin extraction by conventional heating ...... 43

Table 3.5. Independent variables and their levels from BBD at extraction 80 °C ...... 45

Table 3.6. The whole Box-Behnken Design for microwave-assisted heating with four variables X1: Microwave time (minutes); X2: Microwave power (W); X3: pH; X4: Liquid:solid ratio ...... 45

Table 4.1. Pectin yield and DE values used for the factorial design, obtained by extraction under various conditions. Values given are average values of triplicate experiments. .. 64

Table 4.2. Analysis of variance of each component of the model derived by factorial design. Bold values indicate significant results...... 64

Table 4.3. Central composite design of two variables for each type of peel and experimental results from response variables...... 68

Table 4.4. The ANOVA for testing the significance of factors based on the F and p values for extraction yield. Bold values indicate statistically significant results...... 69

Table 4.5. The ANOVA for testing the significance of factors based on the F and p values for DE of pectin. Bold values indicate statistically significant results...... 70

Table 4.6. Experimental testing of mathematical model, optimized for maximum pectin yield and degree of esterification (DE)...... 75

Table 4.7. Central composite design of experiments for pectin extraction from PFP .... 76

xv

Table 4.8. The ANOVA for testing the significance of factors based on the F and p values for extraction yield and DE from PFP. Bold values indicate statistically significant results...... 77

Table 4.9. The maximum predicted and experimental yield and DE at optimized conditions for extraction from PFP...... 80

Table 4.10. Box-Behnken Design of experiments and experimental results...... 84

Table 4.11. The ANOVA for testing the significance of factors based on the F and p-values for the yield and DE of pectin extracted by the microwave-assisted method. Only statistically significant and lack-of-fit results included...... 86

Table 4.12. Predicted and experimental yields and DE at optimized conditions ...... 94

Table 4.13. Optimum extraction conditions used for both heating methods to recover pectin for analysis ...... 95

Table 5.1. Moisture contents of pectin recovered from three types of peels...... 97

Table 5.2. The equivalent weight and methoxyl content of pectin from three types of peel using both heating methods...... 97

Table 5.3. The degree of esterification of pectin extracted at optimum conditions...... 99

Table 5.4. The D-galacturonic acid content in extracted samples...... 100

Table 5.5. The degree of amidation of extracted pectin from two heating methods ..... 101

Table 5.6. The porosity of extracted pectins analyzed with N2 adsorption/desorption porosimetry ...... 110

Table 5.7. The solubility of pectin extracted from various materials by conventional heating and microwave-assisted heating...... 113

Table 5.8. Oil-holding capacities of extracted pectin compared with commercial pectin ...... 115

Table 5.9. The foaming properties of extracted and commercial pectin ...... 115

Table 5.10. The emulsifying properties of extracted pectin and commercial pectin. ... 117

Table 6.1. Effect of pectin on the growth of L. casei probiotic ...... 126

Table 6.2. The prebiotic activity score of pectin for L. casei ...... 127

xvi

Table 6.3. Observed shapes of gelled capsules at various pectin concentration from different types of pectin...... 130

xvii

Chapter 1: Introduction

Diverting waste biomass from landfills makes significant contributions to global sustainable practices in the agrifood industry and helps transform the management of organic waste. Serious environmental problems can emerge due to these residues, such as water pollution, unpleasant odours, vegetation damage and greenhouse gas emissions, and they represent a major factor impacting the global environment (Plazzotta et al., 2020). Increasing fruit pulp productions directly result in the accumulation of the fruit peels as waste, and the weight percentage can be rather high, for example, 30-50% for mango, 40-50% for pomegranate and 30-50% for citrus (Parfitt et al., 2010). In Europe, about 8% of food waste comes from fruit and vegetable processing, the fifth-highest source of food processing waste (Fava et al., 2015). For economic reasons, landfilling is the most adopted waste management option for the disposal of fruit processing waste with high moisture content. However, it is well- documented that the landfilling of biomass leads to the formation of greenhouse gases, mostly methane, through anaerobic decomposition (Loizia et al., 2019). To mitigate such a major environmental issue, these large volumes of wastes should be considered for further processing as a source of natural products that would assist with reducing the disposal of waste, thus alleviating environmental problems. There are various approaches available to transform and valorize food processing wastes: bio-catalysis, bioconversion, clean synthesis and biofuel generation (Lin et al., 2013). In the waste generated by the agrifood industry, biopolymers are present in large amounts, especially pectin, which has an annual worldwide consumption of around 60 thousand metric tons in 2015 with a turnover value close to US$1 billion (Ciriminna et al., 2016). Applications of pectin have been reported in the food, pharmaceutical and cosmetic industries, and it costs around £4.133 million (AU$7.5M) to extract around 1,000 metric tons of pectin per annum from 50,000 metric tons of wet peels, with the product having a net commercial value of £11.8 million (AU$21.4M) (Pfaltzgraff et al., 2013). Commercial pectin is a by-product of juice production industries including apple pomace and citrus peel (Luo et al., 2019). Due to increasing market demands, alternative sources of pectin, such as red-flesh dragon fruit peel (Amid et al., 2014), yellow passion fruit peel (Canteri et al., 2012) and jackfruit waste (Begum et al., 2014) have also been explored. However, the suitability of dragon fruit and purple passion fruit peels has not been extensively evaluated.

1

The red-flesh dragon fruit (Hylocereus polyrhizus), white-flesh dragon fruit (Hylocereus undatus) and purple passion fruit (Passiflora edulis f. edulis) are widely grown and consumed in Southeast Asia. The dragon fruit produces about 30 to 45 % of peel and seeds as typical losses and waste (Cheok et al., 2018). Vietnam is one of the countries with the highest planting areas and yield of dragon fruit with 28,700 hectares yielding 520,000 tons per year over 30 provinces (Nguyen et al., 2020). During the processing of passion fruits for juice, much of the total fruit go to waste, comprising of 50%-60% as rinds and 2-12% as seeds and arils of the raw materials (Romo & Nava, 2005). The comparison of the characteristics of extracted pectin from various types of these fruit peels has not been extensively studied. These fruit peels could be utilised as alternative pectin sources to add income for the processors as well as to reduce stress on the environment. Therefore, this research is aimed at investigating the recovery, processing and characterisation of pectin from waste fruit peels and to compare its main properties with commercial products. Of the various approaches that have been investigated to transform and valorize food processing wastes into pectin instead of landfilling; the extraction process is identified as the most critical operation of the entire pectin production process. However, the large quantities of solvent used, and the extended heating periods required in conventional heating makes this approach time- and energy-consuming. Various alternative extraction processes have been shown to enhance extraction yields, such as microwave-assisted (Garcia-Garcia et al., 2019), ultrasound pulses (Cibele, et al., 2016), subcritical fluid (Wang et al., 2014) and enzyme assisted methods (Dominiak et al., 2014). The statistical and mathematical process optimisation methodologies, such as response surface methodology, have been applied to improve the microwave-heating process. However, the molecular structure and properties of the resulting pectin may be affected when different processing conditions are applied. Hence, this study also investigates the properties of pectin obtained at optimum conditions. The physicochemical, structural and functional properties of the extracted pectin are assessed using various analytical techniques. Pectin has been determined to be biodegradable, biocompatible and non-toxic, which widen their applications as a biomaterial, especially in the encapsulation of probiotics (Noreen et al., 2017). The discovery of probiotics has resulted in various significant applications to develop and improve functional foods in food processing. Williams (2010)

2

reported that Lactobacillus and Bifidobacterium species were the most commonly used probiotics, however Jurenka (2012) reported other species of Bacillus, Sporolactobacillus and Brevibacillus; endospore-forming, microaerophilic Gram-positive bacteria to also be probiotic. Probiotics are live microorganisms that support the gut and internal organs by mechanisms such as producing pathogen-inhibitory substances, challenging the pathogenic microorganisms for adhesion sites and nutrients, breaking the toxins and their receptors, and regulating host responses (Prakash et al., 2011). If insufficient amounts of probiotic are consumed, prospective health benefits are lost (Cibele, et al., 2016). To be effective to the host, it is generally recognized that the number of viable probiotics must be higher than107 cells per gram or per millilitre of final food product (Teoh et al., 2011). The severe conditions of food processing, product storage, acidity of the stomach, basic bile salts of the small intestine, hydrolytic and proteolytic enzymes in the gastrointestinal system, antimicrobials, and other gut bacteria are the main reasons for probiotic losses (Mishra, 2015). As a result, encapsulation has been developed to reduce the loss of probiotic until final release of the surviving cells in gastrointestinal regions (Zuidam & Shimoni, 2010). The microcapsules provide a proper enclosed environment and a physical barrier from external factors to improve the number of viable probiotics (Ding & Shah, 2008). The five types of micro-particles commonly used are: microcapsules, microspheres, multilayer capsules, multi-shell and multicore microspheres (Nesterenko et al., 2013). The three groups of encapsulation techniques reported by Chavarri et al. (2012) consist of physico-mechanical-, chemical- and physico-chemical approaches.. Therefore, studying pectin extraction with their properties and its novel application provides opportunities to explore alternative natural pectin resources from fruit waste as well as widen pectin markets in the area of encapsulation. The objective of this work is to investigate the effects of extraction conditions on the yield and characteristics of pectin recovered from fruit peels, by both conventional heating and microwave-assisted methods. This investigative work develops and optimizes the extraction methodology, while the understanding of the main physicochemical, structural, and functional characteristics of extracted pectin to improve its application as prebiotic and probiotic- encapsulating material provides additional objectives.

3

Chapter 2: Literature review

2.1. Valorization of food processing waste

The increasing demand for food due to the growing world population has led to higher production as well as increased losses and waste during food processing. The United Nations Food and Agriculture Organization (FAO) has reported that about 55 million metric tons of fruit and vegetable waste are produced during processing, packaging, distribution, and consumption of fruits and vegetables in the developed areas of China, India, Philippines, and the United States (FAO, 2014). According to FAO, fruit wastage in Asia, Latin America, and Europe are emerging as a major contributor to the blue water footprint (18 %) as a result of the good production and total food wastage (16 %) (FAO, 2013). The FAO has estimated that there are about 1.3 billion metric tons of lost and wasted food annually (FAO, 2014). Stenmarck (2016) reported that there were about 89 million tons of wasted food, and this could rise by 40% in the following four years in the European Union. The peeling process produces about 14 % (pineapple), 11 % (mango), 8.5 % (papaya) and 16 % (mandarin) of peels (Ayala-Zavala et al., 2010). There are also huge amounts of by-products, including peels and seeds generated by passion fruit processing (Toledo et al., 2018).

The rapid growth of fruit and vegetable losses and wastes significantly influences the environment when they decompose in landfills. There was a 14 % increase in agricultural emissions from 4.7 billion equivalent tonnes of CO2 (2001) to more than 5.3 billion tonnes (2011) (FAO, 2013). There is an emerging interest in using the bioactive molecules such as dietary fibres, phenolic compounds, pectin, lipids extracted from by-products as beneficial ingredients or pharmaceutical compounds in functional food products (de Albuquerque et al., 2019). A few compounds present in modern fruit processing waste and their emerging applications are reported in Figure 2.1. The highest proportion is platform chemicals, which could add more chemicals for food and health applications such as pectin conductive films, beverage additives, cosmetic and skincare products, tissue engineering, antioxidants, health supplements, blood sugar and diabtes management, prebiotics and dietary fibres; as well as bio oils, acids and green solvents (Banerjee et al., 2017).

4

1.7% Water Treatment 3.4% 3.4% 5.1% Biochar 1.7% 3.4% 6.8% 5.1% 5.1% Nanocellulose Sugars Platform Carbon Sequestration Chemicals 20.3% Biofuels 44.4% Platform Chemicals Bio-hydrogen

Figure 2.1. Bioactive compounds from modern fruit processing waste (data from Banerjee et al., (2017)).

Compost and charcoal derived from waste biomass are the most traditional products used as a soil additive in agriculture. Moreover, biochar, produced by high-temperature treatment of biomass in the absence of oxygen, is also used as organic fertilizer (Aziz et al., 2015). Bioactive extracts from fruit processing waste have been assessed for health benefits, especially carbohydrates from peels such as pectin, cellulose, hemicellulose, and lignin (Udenigwe & Aluko, 2012; Deng et al., 2012). A significant component that can be obtained is cellulose which has been studied for its biomedical applications in diagnostics, or as pharmaceuticals and nutraceuticals (Liu et al., 2015). Other studies have shown that biofuels and other bio-chemicals can be produced from cellulose (Pfaltzgraff et al., 2013; Singh et al., 2015; van Dyk & Pletschke, 2012), while hemicellulose and its derivatives have been studied for their benefits to gut health, as antitumor agents, for their ability control metabolic activity, and as immuno-modulators (Singh et al., 2015).

Bioactive proteins, peptides, and amino acids have been shown to possess pharmacological properties such as anti-hypertensives, anti-inflammatories, and antioxidants. Particularly high amounts of these proteins were found in carrot and apple pomace, pineapple banana, and orange peels, solid potato waste, tomato solid waste, cabbage, and cauliflower leaves (Sharma, 2016). It has also been shown elsewhere that lipids obtained from fruit seeds are effective in preventing breast cancer, and exhibit anti- asthmatic, antioxidant, and anti-obesity properties (Goula, 2013; Mandawgade & Patravale, 2008). Polyphenols from fruit waste have been studied for their antioxidant properties, preventing oxidative stress and cell damage, for example, ellagitannins (raspberry pomace), chlorogenic acid (apple pomace), tangeretin (citrus) (Deng et al.,

5

2012; Da Silva et al., 2014). Extracts from pomegranate peels were found to be efficient as food stabilizers and inhibitors of bacterial growth (Akhtar et al., 2015).

2.2. Pectin

2.2.1. Structure of pectin

Pectic substances, which include pectin as well as several forms of protopectins, pectinates, pectinic acids, pectates and pectic acids, are found in the intercellular or middle-lamellar region forming channels to transport nutrients and water (May, 1990).Pectin is a biopolymer of α-galacturonic acid, an oxidized form of D-galactose, with a variable number of methyl ester groups (Figure 2.2). The backbone of pectin is made up mostly of α-galacturonic acid with side chains of neutral sugars, mainly D- galactose, L-arabinose, and D-xylose. It also contains arabinan and galactan in its branches (Manrique & Lajolo, 2002). Pectin has various functions, including size and shapes determination, integrity control, water retention capacity, ion transport, cementing, and cell adhesion (Harris & Stone, 2009). The homopolymer, homogalacturonan (HG), is the most dominant polysaccharide (65 %) in the pectin structure. The other pectic polysaccharides are the substituted HGs rhamnogalacturonan I & II, xylogalacturonan, and arabinogalactan and arabinans. The “smooth regions” are those that have no side chains, while the “hairy regions” are those with numerous non- ionic side chains of the pectin backbone resulting from esterification of HG (Mohnen, 2008). Many factors are influencing the structure and molecular weight of pectin, including the type of tissues and plant materials (Willats et al., 2006). The composition and properties of pectin also are affected by environments, source of the plant, stage of plant growth, enzymatic and chemical reactions that occur through plant development and recovery conditions (Henk & Alphons, 2002).

6

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

2.2.2. Product specifications

Pectin is categorized as a food additive complying with the definition and specifications from different organizations and local regulations. Some of the specifications used for commercial pectin are illustrated in Table 2.1. Pectin appearance is typically creamy-white to light brown powder coming with neutral odor and taste. The most popular specifications of pectin include the degree of esterification, viscosity, pH, loss on drying, and particle size distribution. The Food Chemical Codex do not include the requirements and tests for amide substitution and the weight percent of total galacturonic acid in the pectin component, in which cases the test procedures provide for removing the sugars and soluble salts before analysis of pectin component. The European Economic Community also reported the purity specifications for amidated pectin.

Table 2.1. Commercial pectin specifications (Dixon, 2008)

Food and Food European Specifications Agriculture Chemicals Economic Organization Codex Community Loss on drying Max 12% Max 12% Max 12% Acid-insoluble ash Max 1% Max 1% Max 1% Sulfur dioxide Max 50 mg/kg Max 50 mg/kg Max 50 mg/kg Nitrogen-content, – – Max 2.5% amidated pectin

Nitrogen content Max 2.5% – Max 0.5%

Galacturonic acid Min 65% – Min 65% Degree of amidation Max 25% Max 25% Max 25%

2.2.2.1. Galacturonic acid (GalA)

The galacturonic acid content of pectin is one of the most critical parameters determining the functional characteristics of pectin and its gelling capabilities. Experimentally it is determined by hydrolyzing the polymer into monomer units via chemical or enzymatic methods, which can then be tested to determine the proportion of galacturonic acid monosaccharides. According to the US and European Union legislation,

8

the average galacturonic acid content of a polysaccharide must be higher than 65 % for it to be considered a pectic substance (Willats et al., 2006).

2.2.2.2. Degree of esterification (DE)

There are two categories of pectin following their degree of methylation, also referred to as the degree of methanol-esterification (DE). It indicates the amount of methanol present per 100 moles of galacturonic acid, or the percentage of the C-6 carboxylate groups that are esterified with methanol (-OCH3) (Dixon, 2008). Pectin is categorized as high-methoxyl pectin (HM) if the DE higher than 50 % and low-methoxyl pectin (LM) otherwise (Sriamornsak, 2003). There are many gelation mechanisms in the contexts of high-methoxyl and low-methoxyl pectin, depending on their DE, the arrangement of ester groups along the chain, the average molecular weight, the pH of the solution, the ionic strength, the relative amount of sugar and the temperature (Thakur, Singh, Handa, et al., 1997).

2.2.2.3. Degree of amidation (DA)

The DA value is the percentage of carboxyl groups that has been converted to an amide by the alkaline de-esterification process using ammonia. Amidated LM pectin can have a DA value in the range of 15-25%. This value represents the percentage of C-6 carboxylate groups that are amide groups, meaning the percentage of amidated carboxyl groups of GalA units relative to the total number of GalA units in the molecules (Vithanage et al., 2010).

2.2.2.4. Degree of acetylation (DAc)

The DAc of pectin is the percentage of galacturonic acid residues that are esterified with acetyl groups. Pectin extracted from sugar beet and potato has a high proportion of acetyl groups in HG while this number is low for pectin from apple and citrus. The acetylated pectin has emulsion-stabilizing characteristics but does not gel with Ca2+ ions (Voragen et al., 2009).

2.2.2.5. Neutral sugar composition

In pectin, there are no less than 17 different monosaccharide types that appear as branch groups on pectin molecules (Voragen et al., 2013). The presence of galactose (Gal), arabinose (Ara) and rhamnose (Rha) suggests the contribution of galactan, arabinan, and/or arabinogalactan side in the main structure of pectin molecules. The other

9

common neutral sugars in pectin fractions include xylose, mannose, glucose, and fucose. The molar ratio of Rha to Gal suggests the degree of side-chain branching (Yapo & Koffi, 2006).

2.2.2.6. Molecular weight

Pectin includes multiple chains and different MW species leading to its polydispersity. The molecular weight of pectin ranges from 50 to 150 kDa. There are three types of measuring methods used to find the molecular weight of pectin, including absolute, relative, and combined methods (Dixon, 2008). Using light scattering methods, the apparent molecular weights have been recorded as high as about 1000 kDa because of the aggregation. The high molecular weight pectin of small fractions can affect light scattering measurements but does not influence viscosity (Fishman et al., 2001; Sawayama et al., 1988). The molecular weight of extracted pectin is affected by the raw materials and the extracting conditions. Pectin aggregation depends on ionic strength, pH of the solution, and the presence of solvent additives (Sawayama et al., 1988).

2.2.2.7. Pectin aggregation

Aggregation indicates the larger apparent size of pectin molecules (Yoo et al., 2003). The size ranges of pectin aggregates could be determined by electron microscopy, membrane osmometry, end-group analysis, and high-performance size exclusion chromatography. The aggregated spherical network structures have been reported in pectin-water solution from peaches by electron microscopy method (Fishman et al., 1993). This aggregation could be broken up into linear structures by the hydrogen bond breakers, such as NaCl and glycerol.

The stronger aggregation has been reported in samples of low methoxyl-pectin based on intrinsic viscosity measurements (Yoo et al., 2006). The higher quantity of carboxyl groups in LM-pectin results in a more significant number of hydrogen bonds leading to the higher driving force. The pectin aggregation includes lateral chain aggregation of LM- pectin, which can be disassociated by high salt concentration (at pectin concentrations smaller than 0.0004 g/mL), and end-to-end chain aggregation is causing gelation (at concentrations higher than 0.0004 g/mL). In high-methoxyl pectin, the hydrogen and hydrophobic bonding are the primary forces for aggregation. At lower pH, the higher molecular weight of polygalacturonic acid leads to an increase of chain-chain aggregation (Oakenfull & Scott, 1984; Sawayama et al., 1988).

10

2.2.2.8. Pectin stability

Pectin remains stable in the pH range from 3 to 4, and are depolymerized when the extraction process is higher than 60 °C and pH lower than 5, because of β-elimination. The neutral sugars on the side chains are hydrolyzed when the ester linkages are broken at pH smaller than 3 (Albersheim et al., 1960; May, 1990). The thermal analysis indicated that the difference in degradation temperature and broader melting range depending on the difference in molecular weight and molecular arrangement (Jiang et al., 2018).

2.2.2.9. Pectin solubility

Clumps can be formed when dried pectin powder is added into the water as semi-dry particles inside highly hydrated coatings. The solubility of monovalent cation salts of polygalacturonic acids in water is high while the salts of di- and trivalent cations are less soluble or even insoluble. The water-soluble agents could be pre-mixed with dry powdered pectin to improve solubility and prevent clumping (Sriamornsak, 2003).

2.2.2.10. Gelling properties

In low DE pectin, a gel is formed in the presence of polyvalent metallic cations, such as calcium, without or with low levels of sugar as co-solute. The association of chains (junction zones) is created by calcium cross-linking of free carboxyl groups. The binding of Ca2+ to pectin involves intermolecular chelation leading to the formation of aggregates (Ralet et al., 2001). High concentrations of Ca2+ and acidity (pH < 3), together with lower temperatures, lead to an increase in gel strength (Lootens et al., 2003).

In high DE pectin, the cross-linking of homogalacturonan through hydrogen bonds and hydrophobic interactions between methoxyl groups are junction zones; it means the sugar and water could be trapped in this pectin network (Willats et al., 2006). The minimum sugar content of 55 % (typically sucrose or others co-solutes) and pH lower than 3.5 are the conditions required for high-methoxyl pectin gelling. The low pH decreases the negative charges between the pectin chains due to the carboxylic group disassociation, which reduces electrostatic and intramolecular repulsion in the pectin gel system. The sugars act as a co-solute to decrease water activity and dehydrate pectin to enhance the hydrophobic interactions between methyl ester groups (Oakenfull et al., 1984). The formation of hydrogen bonds and hydrophobic interactions in acids (to reduce electrostatic repulsions) and sugars (to reduce polymer-water interactions) causes irreversible gelling in high-methoxyl pectin (Tsoga et al., 2004).

11

2.2.2.11. Pectin rheology

The deformation and flow of matter as rheological properties of pectin suspension were important properties. The rheological behaviors of pectin solutions contribute to the shape they take in containers, the way they should be handled and processed, quality control and sensory assessment of fluid and semi-solid foods. The viscosity of pectin solutions depends on the pectin concentration and its molecular weight. The pectin solution is a Newtonian solution at lower concentrations up to 0.5% w/w but is non- Newtonian with pseudo-plastic properties at higher concentrations (Rolin, 2002). These researchers also concluded that the viscosity of pectin solution increases with a higher molecular weight of pectin

2.2.3. Industrial production of pectin

2.2.3.1. Raw materials

According to the Future Market Insights (2016), around 34,000 metric tonnes of pectin were consumed worldwide in 2016 alone, and this could potentially reach 48,735 metric tonnes in 2026 (https://www.futuremarketinsights.com/reports/pectin-market). Apple pomace and citrus peels are the most widely used industrial sources of pectin, with pectin contents around 10-15 % and 20-30 %, respectively (Srivastava & Malviya, 2011). The other developing sources include sunflower heads, beet pulp, and potato pulp (Dumitriu, 2004). Microbial activities, especially mould contamination, could lead to the development of pectinase that will accelerate the degradation process, so the starting materials are dried before storage and transported to production plants.

2.2.3.2. Chemical extraction methods

The general method employed in the industrial production of pectin is as follows. The pectin chains and other polymers in the cell walls are partially depolymerized by acid hydrolysis, using hot dilute mineral acid at pH 2, maintained at 60-100 °C for 1 to 10 hours. These polymers are dissolved following the breaking of ionic bonds. The degradation of neutral sugar chains also occurs during extraction. Higher temperatures, acidity, and time can give higher pectin yield but also decrease the polymer chain length and their functionalities. The solid residue is then removed from the hot pectin extract by filtration, centrifugation, or a combination of both methods. The pectin solution is modified from pH 3 to 4 to prevent further polymer degradation, before being vacuum concentrated to 3 – 4 % of pectin. At this step, pectin can be de-esterified or amidated as

12

an option to obtain the desired degree of esterification or degree of amidation. The pectin is then precipitated in alcohol. The precipitate is washed in ethanol to eliminate sugars, pigments, polyphenols, and other alcohol-soluble compounds before being dried and ground to raw pectin powder. The final pectin powder is sometimes blended with sugar to achieve desired functional properties in the standardization process (Dumitriu, 2004). The large volume of acid needed is one of the main disadvantages of this conventional method. The further de-polymerization and de-esterification caused by the strong acidic treatment are also significant drawbacks of this technology (Dixon, 2008)

2.2.3.3. Exploring new techniques for pectin production

(a) Enzymatic extraction

The enzymatic extraction conditions are pH from 3 to 5 at a temperature of about 50 °C. The higher pH and lower temperatures bring more advantages in terms of energy consumption and environmental effects. There are various types of enzymes used in pectin production, including the homogalacturonan- and rhamnogalacturonan-degrading enzyme group, as well as the cellulase and proteinase groups, used to degrade cell wall polymers, which can significantly affect the degree of polymerization and the release of pectin (Dixon, 2008). The enzymatic extraction method has been tested with endo- arabinase and endo-galactanase on citrus, apple, and sugar beet pulps, resulting in low pectin yield and molecular weight (Thibault et al., 1988). The proteases and cellulases have been reported in this research to obtain the high yield (12.6%) of HM pectin (DE 68%) with a high galacturonic acid content of 75% from citrus peel. Other substrates that have also been studied include chicory roots, citrus peel, and cauliflower florets and leaves (Zykwinska et al., 2009). The lower energy consumption and more accessible waste treatment are advantages of enzymatic extraction. The main disadvantages of enzymatic extraction are low yields, low molecular weights, and low content of galacturonan (Dominiak et al., 2014).

(b) Microwave-assisted extraction methods

Microwaves (from 300 MHz to 300 GHz) are non-ionizing electromagnetic waves in the lower end of the radio-frequency range the electromagnetic spectrum. There are two mechanisms transferring energy during microwave heating: dipole rotation and ionic conduction (Kubrakova & Toropchenova, 2008). The dipole rotation of molecules promotes the disruption of weak hydrogen bonds accompanied by heat release. The

13

migration of dissolved ions leads to higher penetration of the solvent into the matrix to assist with the extraction of bioactive compounds. Significant pressure is created inside the biomaterial during microwave processing, causing a modification of the physical properties of the biomaterial tissues. The matrix porosity is improved, allowing increasing penetration of the solvent into the bulk (Yeoh et al., 2008).

Solvents with high dielectric constant and dielectric loss, absorb more microwave energy and are heated faster. For thermo-labile compounds, low dielectric solvent can ensure that the temperature will remain low, to cool down the solutes after extraction into the solvent (Kaufmann & Christen, 2002). Microwave extraction can use both polar and non-polar solvents. Low-polar solvents such as benzene, chloroform, ether, and ethyl acetate are used to release less polar biological materials. Meanwhile, more polar materials should be released using more polar solvents, e.g., acetone, alcohol, water.

The amount of energy transferring to materials to be converted to heat energy is controlled by the intensity of the incident microwave (Vian et al., 2009). The microwave power influences interactions, equilibrium rates, and partition of analytes between sample and solvent. There is an increased yield of extracted compounds with increasing microwave power. High microwave power could damage the product and decrease yield by breaking down the extracted compounds. To the polar solvents, the increased rate of heating could overheat the system and cause the compounds to degrade because of the comparatively high dielectric properties (Lucchesi et al., 2007).

High temperatures increase intermolecular interactions within the solvent, leading to the faster molecular motions that improve solubility. Cell rupture due to increased pressure under higher temperatures also increase extraction efficiency. Also, at higher temperatures the viscosity of the solvent decreases, which may further improve mobility and solubility (Khajeh et al., 2010). It has also been determined that the extraction efficiency could decrease with longer extraction time. Further, in addition to the decrease in yield, product quality could also diminish with higher temperatures and extended extraction time under higher temperatures.

The microwave-assisted method is widely accepted because it is suitable for high moisture materials and is easily scaled up. The microwaves act as an external energy source affecting water molecules, which leads to structural damage due to the high moisture content of fruit waste. That could assist extraction at lower temperatures

14

(compared to hot acid extraction, e.g.), reducing the whole process to a few minutes. The strong acids also could be replaced with weak organic acids (Attard et al., 2014).

(c) Other emerging methodologies for pectin extraction

Some other novel technologies are currently used for pectin extraction (Table 2.2). Ultrasound has been applied in pectin extraction with different frequencies higher than 20 kHz. The sound wave caused the collapse of cell walls, which induced the increased solvent entrance into the cell (Misra et al., 2018). The advantages of ultrasound as non- thermal technology include low energy usage, decreased treatment time and solvent consumption, and increased safety of operation.

15

Table 2.2. Pectin extraction from recent literature

Materials Extraction method Yield (%) Galacturonic acid (%) DE (%) Reference Citrus peel Subcritical water 21.95 68.88 - Wang et al., Apple pomace 16.68 40.16 (2014) Apple peel Acid extraction (85 °C, 1 M 6.9 54.25 (tartaric acid) 54.5 (tartaric acid) Cho et al., tartaric acid/malic acid, 2 hours, 59.27 (malic acid) 64.0 (malic acid) (2019) liquid:solid ratio = 25) Blackcurrant Citric acid (liquid:solid ratio = 50, - 37.1 Mierczyńska et Peach 30 min, pH 2.5, 90 °C) 26.0 al., (2017) Raspberry 23.1 Plum 23.8 Strawberry 33.9 Carrot 16.5 Black mulberry Microwave-assisted extraction 13.23 36.94 55.07 Mosayebi et al., pomace (900 W, 18.17 min, liquid:solid (2015) ratio 15 mL/g) Cacao pod husk Nitric acid (30 min, pH 3.5, 100 10.7 (of 59.2 41 Vriesmann et °C) total sugar al., (2017) content) Cacao pod husk Citric acid (liquid:solid ratio = 25, 8.3 53.7 37.5 Muñoz-Almagro 95 min, pH 3, 95 °C) et al., (2019) Subcritical water extraction (121 10.9 59.5 39.8 °C, 103.4 bar, 30 min) Blueberry wine Hydrochloric acid (liquid:solid 1.62 65.88 51.66 Feng et al., pomace ratio = 15, 120 min, pH 1, 85 °C) (2019)

16

Materials Extraction method Yield (%) Galacturonic acid (%) DE (%) Reference Pistachio green Citric acid (liquid:solid ratio = 22.1 65 53.01 Chaharbaghi et hull 50, 30 min, pH 0.5, 90 °C) al., (2017) Carrot residues Ultrasound-enzyme assisted 27.1 97 45 Encalada et al., extraction (12.27 W/cm2, (2019) 20 kHz, 80% wave amplitude, 20 min, pH 5.2, hemicellulose: 5h-40 °C ) Gold kiwi pomace Citric acid (liquid:solid ratio = 3 3.83 82.6 (of total non- Yuliarti et al., mL/g, 60 min, pH 2.2, 50 °C) starch polysaccharides) (2015)

Water (liquid:solid ratio = 3 3.62 82.67 mL/g, 30 min, pH 3.7, 25 °C) Enzyme (Celluclast 1.5L) 4.48 84.62 Eggplant peel Ultrasonic assisted extraction 33.64 66.08 61.22 Kazemi et al., (ultra power 50W, liquid:solid (2019a) ratio = 20, 30 min, pH 1.5) Mango peel Citric acid, ultrasound-assisted 17.15 53.35 86.83 Wang et al., extraction (liquid:solid ratio = 40, (2016) 15 min, pH 2.5, 80 °C) Mango peel Ultrasound-assisted extraction 8.1 70 58 Guandalini et with nitric acid (intensity 497.4 al., (2019) W/cm2, 10 min, pH 2, 85 °C) Okra Aqueous extraction (liquid:solid 14.6 63.4 17.2 Kpodo et al., ratio = 15, 60 min, pH 6, 80 °C) (2017)

17

Materials Extraction method Yield (%) Galacturonic acid (%) DE (%) Reference Opuntia ficus- Ultrasonic-assisted extraction 18.14 68.87 41.42 Bayar et al., indica cladodes (liquid:solid ratio = 30, 70 min, (2017) pH 1.5, 70 °C) Orange waste Ohmic heating with acidified 10.69 68.24 73.30 Saberian et al., water (liquid:solid ratio = 20, pH (2018) 1.5, 90°C, voltage gradient 30 V/cm) Pomegranate peel Citric acid (liquid:solid ratio = 50, 11.34 80.95 53.09 Pereira et 120 min, pH 2.5, 88 °C) al.,(2016) Pomegranate Hot water (liquid:solid ratio = 50, 8.5 62 75 Yang et al., 120 min, pH 2.5, 86 °C) (2018) Sugar beet pulp Hydrochloric acid (liquid:solid 9.2 63.8 44.1 Guo et al., ratio = 20, 80 min, pH 1.7, 80 °C) (2016) Averrhoa bilimbi Deep eutectic solvents 14.44 - - Shafie et al., (concentration 3.74 %, 2.5 hours, (2019) 80 °C, molar ratio 1:1) Cubiu peel Boiling HNO3 (liquid:solid ratio 14.2 75 62 Colodel et al., (Solanum = 10, 120 min, pH 1.5, 80 °C) (2019) sessiliflorum D.) Ponkan (Citrus Water (liquid:solid ratio = 36, 100 25.6 84.5 85.7 Colodel et al., reticulata Blanco min, pH 1.6) (2018) cv. Ponkan)

18

Materials Extraction method Yield (%) Galacturonic acid (%) DE (%) Reference Premna Microwave pretreatment, 18.25 82.75 62.5 Lu et al., (2019) microphylla Turcz hydrochloric acid extraction leaves (liquid:solid ratio = 50, 120 min, pH 2, 90 °C) Potato pulp Ultrasound-microwave assisted 22.86 47.78 % 32.58 Yang et al., HCl extraction (liquid:solid ratio (monosaccharide (2019) = 50, 50 min, pH 2, 93 °C) composition) Orange peel Surfactant (Tween-80 8g/L), 32.8 78.1 69.8 Su et al., (2019) microwave-heating (liquid:solid ratio = 21.5, 7 min, pH 1.2, 400 W) Lime peel HCl solvent (liquid:solid ratio = 23.59 95.93 72.81 Rodsamran et 20, 60 min, 95 °C) al., (2019) Melon rinds Citric acid extraction (liquid:solid 6.54 92.97 71.98 Güzel et al., Kiwi fruit ratio = 10, 60 min, pH 1, 80 °C ) 8.03 94.75 84.72 (2019) Pomegranate peel 6.13 78.48 56.74 Apple peel 13.30 79.78 77.62 Orange peel 11.46 76.32 69.67

19

2.2.4. Applications of pectin

2.2.4.1. Food processing

The main uses of pectin in the food industry are as gelling agents, thickeners, water binders, and stabilizers (May, 1990). Bakery glazing and fillings, confectionery, dairy products, and fruit beverages are some additional applications of pectin (Dumitriu, 2004). Among the new uses, pectin is used as a filler in low-calorie food products, acidifying agents, rheological modifiers, biodegradable surfactants, emulsifiers, packaging material, dairy stabilizers and fat substitutes (Dixon, 2008). In dairy products, high-methoxyl pectin adsorbs onto casein particles due to electrostatic interactions between the positively charged casein molecules and negatively charged pectin chains in the pH range between 3.6 and 4.5 (Dixon, 2008). Therefore, pectin is widely used to prevent aggregation and precipitation of caseins as protein stabilizers.

Pectin acts as a thickener, a water-binder and a stabilizer in diverse varieties of yogurts, and help minimising the color migration from fruits into the product. In the bakery industry, pectin has various roles in maintaining moisture as well as increasing the volume, flexibility, and softness of bread. The retrogradation (type of recrystallization) of starch in frozen dough could be prevented by using pectin. The amount of dough is also maintained during freezing by adding pectin (Brejnholt, 2009).

Pectin has a variety of applications in mayonnaise, dressing, ketchup, protein foams, and beverages as a stabilizer and a thickener. It also has been studied as a coating material to decrease the absorption of oil when frying (Min et al., 2010). It has been reported that pectin positively affects the human gastrointestinal tract because of its gelling and water- holding abilities. Pectin has also been successfully studied in the lowering of glucose absorption, decreasing gut transit (Roberfroid, 1993).

2.2.4.2. Emerging applications of pectin as a bioactive polysaccharide

It has been shown that the pain of many cancers has been decreased by low molecular weight pectin (Azémar et al., 2007). Small pectin fragments have been studied as an anti- cancer agent that binds to and inhibits the activities of the pro-metastatic protein galectin- 3 (Maxwell et al., 2012). Pectin has also been investigated as a drug carrier to encapsulate active molecules; remaining intact during transit to the colon through the gut intestinal system, and only releasing the drug in the colon because of its non-digestible property in the upper gastrointestinal tract (Desai, 2006; Liu et al., 2003). 20

Pectin has been studied for use as a decontaminating agent because it can bind to positively-charged heavy metal ions such as lead and mercury in the gastrointestinal system and respiratory organs of poisoned patients (Endress, 1991). It is reported that, following the ingestion of modified citrus pectin, there was a decrease in the level of lead in the blood, accompanied by an increase in urinary excretion in Chinese children intoxicated with lead. Rhamnogalacturonan II was found to play a critical role as a heavy metal chelating agent in the body (Zhao et al., 2008)

It is reported that the adhesion of pathogenic bacteria such as E. coli to intestinal uroepithelial cells can be prevented by homogalacturonan-rich structure, with dimers and trimers being the most effective agents (Guggenbichler et al., 1997). The effects of fractionated oligo- and polysaccharides of thermally modified citrus pectin (MCP) on cancer cell behavior were found to depend on their molecular size (Prado et al., 2019). The high amounts of type I arabinogalactans (AGI), less-esterified homogalacturonan (HG) oligomers, and low numbers of rhamnogalacturonan (RG-I) may lead to anticancer properties.

2.2.5. Exploring new sources for pectin production

Currently, commercial pectin is mostly obtained from citrus peels and apple pomace, however, the food and fruit processing industries around the world are generating considerable amounts of waste that may otherwise be used as potential sources for pectin, such as sunflower head residues (11.60%) (Iglesias & Lozano, 2004) and peach pomace (9.68%) (Faravash & Ashtiani, 2008). Other emerging plant sources have been investigated as pectin-rich sources including blackcurrant (Mierczyńska et al., 2017), black mulberry pomace, strawberry and raspberry (Mosayebi et al., 2015), cacao pod husks (Vriesmann et al., 2017), carrot (Mierczyńska et al., 2017), golf kiwifruit pomace (Yuliarti et al., 2015), mango peel (Wang et al., 2016), okra (Kpodo et al., 2017), pistachio green hull (Chaharbaghi et al., 2017), plum (Mierczyńska et al., 2017) and pomegranate peel (Yang et al., 2018).

2.2.5.1. Dragon fruit peels (DFP)

(a) Rationale

Dragon fruit, known as Hylocereus spp., have been gaining attention because of their economic and nutrition values (Wybraniec & Mizrahi, 2002). There are various species of dragon fruits with different colored flesh, including white (H. undatus), red (H. 21

costaricensis and H. polyrhizus) and yellow (H. megalanthus, also known as Selenicereus megalanthus) (Mizrahi et al., 2010). It is a climbing vine cactus species of the family Cactaceae with small black seeds and red peel with green scales (except for H. megalanthus, which is yellow). The dragon fruit is composed of about 30 to 45 % peel and seeds by mass, which is typically lost and discarded as waste (Cheok et al., 2018).

(b) Current research

Pectin yield from dragon fruit peel obtained previously is slightly different between authors due to the variety of extraction methodology and materials. The highest reported yield of high methoxyl pectin (26.38 %) with DE 63.74 % was achieved at 73 °C, 67 min at pH 2.03 and ratio of sample to citric acid 1:4 (w/v), using red-flesh dragon fruit peel (RDFP) as the feedstock (Muhammad et al., 2014).

The average molecular weight of the pectin in this research was 0.88 × 105 Da, consisting predominantly of galacturonic acid and other neutral sugars such as mannose, rhamnose, galactose, glucose, xylose and arabinose (Muhammad et al., 2014). However, a low methoxyl pectin with DE 47.88% was obtained at conditions of pH 2.37 in 64.67 minutes, with the highest yield of 12.56% (Liew et al., 2015). Low-methoxyl pectin with high antioxidant activity was extracted using 80 °C water with a yield ranging from 16.20 % to 20.34 % (Zaidel et al., 2017). In comparison, this number was just 15.4% when using deionized water and 15% by hydrochloric acid extraction (Pinang, 2011).

In another study, a yield of 14.86 % was achieved at pH 3.5, after 60 min extraction at 75 °C while the highest DE of pectin was obtained from 120 min extraction at pH 4.0 (Woo et al., 2010). Other novel extraction methods have been studied for pectin recovery from red-flesh dragon fruit peel. The combination of mild ultrasound and stirring in citric acid (250 rpm at 70 °C), for 120 minutes at pH of 1.5 and peel:solvent ratio of 1:10 (w/v) obtained the highest pectin yield of 42.5 % from red-flesh DFP in comparison with deionized water, acetic acid and other mineral acids (H2SO4, HCl, HNO3) (Zaid et al., 2016a, 2016b).

One of the more popular alternative methods for pectin extraction has been via microwave. Microwave-assisted extraction was conducted to extract 7.5% pectin at a temperature of 45 °C in 20 min at 400W (Thirugnanasambandham et al., 2014). Rahmati el al. (2015) extracted the highest pectin yield of 18.53 % at pH 2.07 in 65 s with solid- liquid ratio 1: 66.57 (w/v) at 800 W microwave power. A maximum yield of 23.11 % was 22

achieved after heating for 10 minutes at 600 W, which resulted in a lower viscosity of extracted pectin (Tongkham et al., 2017). The obtained results show that pectin yield from DFP is comparable with apple pomace (10 – 15%) and citrus peel (20 – 30%) (May, 1990) and higher than the pectin yield from other food waste sources such as sunflower head residues (11.60%) (Iglesias et al., 2004), peach pomace (9.68%) (Faravash & Ashtiani, 2008), cocoa husks (Mollea et al., 2008).

2.2.5.2. Passion fruit peels (PFP)

(a) Rationale

Passion fruit peel is gaining more attention as a waste product that can be used as feedstock for pectin recovery. There are two main edible passion fruit species of family Passifloraceae and genus Passiflora L. including the purple passion fruit (P. edulis Sims) and the yellow passion fruit (P. edulis f. flavicarpa Degener) with a slightly sour taste (Yapo et al., 2006). The waste generated in juice processing of passion fruit could be around 75 % of raw materials, including 90% of peel (Arvanitoyannis & Varzakas, 2008).

(b) Current research

The portion of the peel is about 85% of the total utilization of passion fruit waste, while this number from the seed is just 17 % (Cheok et al., 2018). Passion fruit peel is gaining more attention as a waste product that can be used as feedstock for pectin extraction. Some authors have studied pectin extraction from yellow PFP; the results of those studies are presented in Table 2.3.

23

Table 2.3. Studies of pectin extraction from yellow passion fruit peels

Extraction conditions Pectin characteristics References 50mM Nitric acid 5 min, 80 °C Yield: 164 to 249 g/kg; GalA: 73%; GalA/Rha molar ratio:16; Canteri et al., (2012) total neutral sugars: 52 mg/g including rhamnose, fucose, arabinose, xylose, mannose, galactose, glucose; DE 75%; Molecular weight: about 166 kDa pH 2, 98.7 °C, liquid:solid ratio = 30 Yield: 14.8g/100g; methoxyl content of 9.6g/100g; GalA Kulkarni et al., (2010) (mL/g), 60 min heating time 88.2g/100g; Jelly grade of 200 75 min, pH 2, solvent: citric acid, pH influenced yield while DE was affected by extraction time; Liew et al., (2014) Yield 14.6%, DE 54.78% 60 min, 0.086% w/v citric acid Lower citric acid concentration gave higher DE (78.59%) Pinheiro et al., (2008) 90 min, 80 °C, liquid:solid ratio = 25 GalA 64-78%; DE 52-73%; molecular weight 70-95 kDa Yapo (2009b) (mL/g) with fresh lemon juice or pure Lemon juice gave higher pectin yield and quality citric acid, pH 2.4 or1.6 Moderate electric field High DE, galacturonic acid 65% Oliveira et al., (2015) Ultrasound methods Yield 12.67%, GalA 66.65%, DE 60.36% Cibele et al., (2016) Pressurized-solvent method Yield 36%, 157 mg luteolin/100g, 244 isoorientin/100g, Cibele et al., (2016) Antioxidant content: 466 mg/100g pH 1.34, 65 °C, 2 hours, Solvent: Yield: 29%, methoxyl content 9.88%, acetyl value 1.9%, uronic Simmaky et al., (2014) hydrochloric acid acid 89.6%, DE 71.44%, equivalent weight 810.85 kDa, gel grade 110, rapid set. Enzyme/Citric acid extraction Yield 7.12-7.16%, DE 71.02-85.45% Liew et al., (2016) Pectin had pseudoplastic liquid behavior

24

Extraction conditions Pectin characteristics References Pressurized liquid extraction (50 bar, 100 Yield: 27.67 %, DE: 89.32 %, High purity Moia et al., (2019) °C, 0.5 mL/min) Enzymatic extraction (fungal enzyme GalA Yield: 26 %, GalA acid: 85 %, DE: 68 % Vasco-Correa et al., (2017) protopectinase 30 U/ml, pH 3, 37 °C, 4 hours ) Ultrasound-assisted (0.75 M citric acid, 90 Yield: 54.7 %, DE:18 % Santos et al., (2017) min, 27 °C) 75 °C, 60 minutes, liquid:solid ratio = 25 Citric acid solvent: higher DE and molecular weight, better gelling Yapo (2009a) mL/g but lowest yield Nitric and sulfuric acids solvents: low DE but higher yield Microwave-assisted heating (628W, 9min) Yield: 13% with high DE (nitric acid), 12.9% with high DE Seixas et al., (2014) (acetic acid) , 18.2% with low DE (tartaric acid) Increasing yield with higher exposure time and power Temperature 80 °C, pH 1, 10 min DM 45.94%, GalA 68.7%, acetyl value 0.3% Kliemann et al., (2009)

Boiling 80% ethanol, liquid:solid ratio = 4 Pectin fractions: water-extracted pectin, chelating agent-extracted Yapo et al., (2006) mL/g, 45 minutes pectin, acid-extracted pectin, yield 13.4%, low DM, degree of acetylation: from 1.2 to 1.9, neutral sugar: Glu (30.8%) and Xyl (12.3%), Protein residues from 1.4% to 5.1%

25

2.3. Probiotics

The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) state that probiotics is considered as live microorganisms that support and improve health benefits to the consumers (FAO/WHO, 2001). If the probiotic quantity consumed is insufficient, they will not confer the potential wellbeing to the human host (Araya et al., 2002). The probiotic number must be >107 viable cells per gram or per milliliter of a product (Ishibashi & Shimamura, 1993).

The genera of Lactobacillus and Bifidobacterium were widely categorized as probiotics, which are Gram-positive producing lactic acid. The genera of Bacillus, Sporolactobacillus, and Brevibacillus have also been reported as probiotic (Jurenka, 2012). The lactic acid bacteria (LAB) have been considered as the most important probiotic microorganisms in the food industry.

2.3.1. Probiotics in the human gastrointestinal tract and health benefits

Probiotics support the health benefits of the gut and internal organs by the production of inhibitory substances, competing with pathogens for adhesion sites and food, degrading toxins and their receptors, and regulating host responses (Prakash et al., 2011). Probiotics were also found to have positive effects as treatments for inflammatory diseases of the gastrointestinal tract, e.g., in combatting Helicobacter pylori infection (de Vrese & Schrezenmeir, 2008). Williams (2010) stated that probiotics could produce lactic acid, acetic acid, and propionic acid to decrease the intestinal pH to prevent infection. Additionally, it is claimed that probiotics reduce constipation (Ouwehand et al., 2002). De Vrese & Schrezenmeir (2008) also state that probiotics reduce the concentration of cancer-promoting enzymes. For example, one study in Japan showed that the intake of lactobacilli resulted in the reduction of the risk of bladder cancer (Ohashi et al., 2002).

Probiotics have been linked to numerous additional health benefits, such as reduction of the expression of biomarkers responsible for colonic cancer, inhibition of diarrhea in children, prevention of pouchitis, reduction of functional abnormalities, reduction of the symptoms of lactose intolerance (Aureli et al., 2011). Probiotics also have been shown to support the treatment of obesity, diabetes, fatty liver diseases (Tonucci et al., 2017) and cancer (Leslie, 2015). Recommendations for probiotics as nutritional supplements to increase the body’s immunity have been reported by other researchers (Markowiak & Śliżewska, 2017; Vandenplas et al., 2013)

26

However, the severe conditions of processing and shelf life, low pH of the stomach, bile salts in the small intestine, hydrolytic enzymes and proteases, and other competitive resident bacteria in the gut system are the main reasons to kill the probiotics (Ding & Shah, 2007). The peristaltic action of the intestine, heat, and presence of protease in intestinal mucus are also killing factors (Sarao & Arora, 2017; Lahtinen, 2012).

2.3.2. Prebiotics

Prebiotics are dietary components that improve the host health by making specific changes in the bacteria composition and activities, which includes fibers as inulin, inulin- type fructans, and (trans-) galacto-oligosaccharides (Gibson et al., 2004). Prebiotic has to be non-digestible to the host but fermentable by the intestinal microorganisms to selectively stimulate microbial growth (Schrezenmeir & de Vrese, 2001). The most popular prebiotics are fructo-oligosaccharides (FOS), galacto-oligosaccharides (GOS), human milk oligosaccharides (HMO), lactulose, and inulin (Míguez et al., 2016).

Pectin has potential as a prebiotic, as it is known to stimulate growth and activity of probiotics, inhibit pathogenic bacteria and affect bowel cancer cells apoptosis (Hotchkiss et al., 2003). These authors also reported that the oligosaccharide derived from low- methoxyl pectin has higher prebiotic properties compared with the high methoxyl. Hydrolysis of pectin polymer is important in its prebiotic application, as pectin-derived oligosaccharides are the molecules that can be utilized by native intestinal microbes (Olano-Martin et al., 2002). The purified homogalacturonides and rhamnogalacturonides from sugar beet pectin have been studied on Bacteroidetes and Firmicutes to determine the effects of the structure of prebiotic on the biological activity of the phyla (Holck et al., 2011). Pectin as encapsulating material could protect Lactobacillus plantarum in alginate-coated chitosan microcapsules from in vitro acidic conditions (Pop et al., 2016). Pectin oligosaccharide fractions from citrus peel were investigated for their role as new- generation prebiotics. The highest prebiotic activity score was 0.41 for Lactobacillus casei and 0.92 for Bifidobacterium bifidum by hydrogen peroxide-degraded pectin (Zhang et al., 2018). Apple and sugar beet pectin were determined to promote the growth of Firmicutes species Eubacterium eligens and Faecalobacterium prausnitzii (Chung et al., 2017). The prebiotic ability of passionfruit by-products was determined on ten different probiotic strains (Vieira et al., 2017). The passionfruit by-product was the most efficient substrate to stimulate the growth of bacterial populations.

27

The prebiotic potential of citrus pectin was also studied using the dynamic gastrointestinal simulator, including five regions simulating the stomach, small intestine, the ascending-, transverse- and descending colon (Ferreira-Lazarte et al., 2019). The chronic feeding with pectin resulted in greater amounts of short-chain fatty acids, especially acetate, propionate and butyrate in the end-products of indigestible carbohydrates metabolism. This group also determined the growth of Bifidobacterium spp., Bacteroides spp., and Faecalobacterium prausnitzii when citrus pectin was fed in all colon sections. In another study, 0.4 % pectin was added to broth after 48 hours and 60 hours to measure its effect on the growth of probiotics (Chatterjee et al., 2016). The optical density showed that L. casei had the highest growth in media with pectin extracted from tomato (2.4 OD at 660 nm). The titrable acidity of milk products cultured with different probiotics and pectin were from 0.52 to 0.57 %. B. bifidum and L. acidophiles were also studied in the media with pectin derived from fruit waste. The extracted pectin influenced the growth of the tested probiotics in a range of pH from 2 to 8, simulating the conditions in the gastrointestinal tract (Manuel, 2014). Similar results were obtained in other studies with of Bifidobacterium and Lactobacillus spp. (Wicker et al., 2014)

2.4. Encapsulation

Encapsulation is one of many developed methods to improve probiotic survival during manufacturing and transit through the human gut. The micro-particles with diameters of a few nm to a few mm can be formed by microencapsulation, including microcapsules, microspheres, multilayer capsules, multi-shell, and multicore microspheres (Figure 2.4) (Nesterenko et al., 2013). Microcapsules are described as a single core with a surrounding layer; microspheres with the cores mixing in a continuous wall material network; multilayer capsules with a single core surrounded by two or more layer of wall materials; multi-shell and multicore microsphere with the multicore dispersed in complex matrix network and surrounded by multilayers.

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

28

Encapsulation techniques are categorized in three groups (Chavarri et al., 2012):

1- physical-mechanical: spray drying, extrusion dripping, emulsification, centrifugal extrusion, freeze-drying, co-crystallization, centrifugal suspension separation, supercritical fluids, ultrasonic atomizer and pan coating; 2- chemical: molecular inclusion, interfacial polymerization, suspension, dispersion, emulsion polymerization, and 3- physico-chemical: coacervation -phase separation, ionotropic gelation and layer-by-layer assembly.

The popular materials for encapsulation come from many sources, for example, seaweed (carrageenan, alginate), plants (starch, gum arabic), bacteria (xanthan, gellan) and animal proteins (Rokka & Rantamäki, 2010). Choosing the proper encapsulation materials is affected by the objective of the encapsulation. Each type of material has different advantages and disadvantages.

2.4.1. Encapsulation of microbial cells

When encapsulated, the probiotic is protected against the harsh environment in the stomach until the surviving bacteria are released in the gastrointestinal tract (Zuidam et al., 2010). A microenvironment is created for bacteria to survive and then be released at a suitable place to colonize the tract. A proper anaerobic environment and the physical barrier of the microcapsules could protect the sensitive probiotics (Ding et al., 2008). It was reported that the L. casei ATCC 290 and L. casei ATCC 292 survived better among the Lactobacilli strains in an acidic condition (Sarkar, 2010). The release of encapsulated ingredients can happen when the temperature, moisture, pH, pressure, shearing forces, and/or enzymatic activities change. The encapsulated ingredients need to be free at the desired site at the desired time to perform their function (Pothakamury & Barbosa- Cánovas, 1995).

2.4.2. Pectin as emerging materials for encapsulation

2.4.2.1. Advantages of pectin as encapsulating materials

Biodegradable polymer materials bring some advantages to probiotic encapsulation, including their cryo- and osmo-protective properties. Higher protection is provided with dried microcapsules. The coating helps to postpone the cell release by its slow dissolution, and production can be set up easily on a lab-scale (Chavarri et al., 2012). The

29

biocompatibility, biodegradability, pH sensitivity, non-toxicity and low cost of pectin has stimulated much attention in its use as encapsulation material. Pectin can also adhere well to the large intestinal mucosa, further increasing its popularity for encapsulation (Alborzi et al., 2014).

Pectin hydrogels are unaffected by gastric and intestinal enzymes but can be rapidly fermented by colonic bacteria into oligomers, which results in the release of the probiotics it contains (Ashford et al., 1993; Cabrera et al., 2011). The effect of simulated digestion conditions on citrus pectin was determined using the dynamic gastrointestinal simulator (Ferreira-Lazarte et al., 2019). During the chronic feeding time, commercial pectin was mixed to the nutritive feeding medium and then added to the simulated stomach regions. The pectin remained unchanged after passing through the stomach section while the intact pectin decreased slightly in the small intestine compartment. However, there was a large decrease after the large intestine section.

Among the applications of pectin in microencapsulation, probiotic encapsulation is an emerging field. Pectin has been studied as an encapsulating material to achieve better protection of other contents. Sugar beet pectin has been used to trap fish oil due to its acetyl and ferulic acid groups and high protein (Drusch, 2007). The drug metronidazole was entrapped in calcium pectinate beads, which decreased the drug loss by around 10- 20 % compared to the theoretical metronidazole content (Pawar et al., 2008). The combination of apple pectin and octenyl succinic anhydride starch has been investigated as wall components to encapsulate �-tocopherol acetate by spray drying (Miceli-Garcia, 2014). Multinuclear microcapsules composed of gelatin and high-methoxyl pectin (ratio of 3:1 at pH 4.23) were found to carry cinnamaldehyde with high efficiency (89.2 %) (Muhoza et al., 2019).

2.4.2.2. Single pectin encapsulation to protect probiotics

The pectin beads with Lactobacillus plantarum and Bifidobacterium longum by extrusion supported the survival of probiotic cells in fruit juice for six weeks with the final number of live cells higher than 107 colony-forming units per ml (CFU/mL). The B. longum cells coated with pectin with size from 2.5 mm to 2.7 mm even had a higher survival rate than cells coated with alginate (Nualkaekul et al., 2013). Other research showed a higher number of surviving bacteria of L. rhamnosus and L. acidophilus in low pH when encapsulated in pectin beads compared to non-encapsulated cells (Gebara et al.,

30

2013). Pectin with low DE can also protect L. plantarum and L. fermentum in vitro, with an encapsulating efficiency of 99.99%. The encapsulation also enhanced the stability of freeze-dried microbeads under ambient temperature (Sun et al., 2017).

The hydrogel system of pectin, glucose, and calcium chloride was formed successfully to protect L. rhamnosus GG in storage at ambient temperatures and through the simulated gut tract. Encapsulation also improved the p40 production as a protein derivative relevant to probiotics’ benefits to intestinal homeostasis. (Li et al., 2016). Similar capsules composed of iron-crosslinked pectin were determined to protect L. plantarum in simulated saliva, gastric, and intestinal conditions. The capsules were obtained by ionotropic gelation in FeSO4 and then freeze-dried to store for 60 days. The diameter of the beads was 1-2 mm, and they were smooth and spherical and released iron and pectin in the guts (Ghibaudo et al., 2017). The solid oil/water emulsions were developed by sugar beet pectin to protect L. salivarius during storage, pasteurization, and digestions, which may due to the calcium cross-linking sugar beet pectin on emulsion droplets (Zhang et al., 2016).

Yeast cells have been used as probiotics by protecting them in pectin beads through extrusion and freeze-drying (Tanangteerapong, 2014). The morphology, internal structure, water activity of the calcium pectinate beads, and the viability of yeast were determined. The results showed that the spherical beads were around 2.3 mm in diameter with a porous internal structure and many voids before freeze-drying. The shape of the beads became irregular with a slightly rough surface, and some yeast cells began to appear on the surface after freeze-drying. Freeze-drying was also reported to produce lower water activity of cell beads than air-drying. The viability of yeast cells in beads with 5% of pectin was measured by fluorescence microscopy, showing a high live cell number. The 10 9 highest viability of cells was recorded at 1.5 % CaCl2 with 1.7 × 10 ± 3 × 10 CFU/g bead with 99% of encapsulation efficiency. Flow cytometric dot plots showed that using lower CaCl2 concentration produced a higher number of healthy cells compared to injured cells. The flow cytometric results also showed that the number of healthy cells was greater than the injured cell after 12-hour freeze-drying (Tanangteerapong, 2014).

Single-component pectin has been applied as a protectant to protect B. bifidum BB- 12 during spray drying (Salar-Behzadi et al., 2013). The data indicated that pectin concentrations of 10% or 50% w/w could protect the cells more effectively than

31

maltodextrin or skimmed milk at the same concentration. There was no significant difference in the protective effects between pectin gum arabic and gelatin during spray drying. However, during the one-month storage of dried cells, pectin showed significantly lower protective impact than gum arabic (Salar-Behzadi et al., 2013).

2.4.2.3. Combination of pectin with other polymers as encapsulating material

The disadvantages of pectin are its high porosity, weak binding behavior to the crosslinking agent (Ca2+), low sphericity of the produced capsules, flowability, and low mechanical strength that decrease the protective effect for probiotic. Unmodified pectin is highly soluble in water, enhancing its degradation and limiting its application in specific circumstances. Research on filler agents has been conducted to increase microencapsulation yield and quality of bio-capsules. Pectin alone is not always able to protect cells effectively; therefore, there is an increasing trend in using a mixture of pectin with different types of bio-compounds.

(a) Pectin and rice bran

Pectin-rice brans mixture can improve the viability of the L plantarum in acidic and bile conditions (Chotiko & Sathivel, 2016a). A higher concentration of rice bran improved the viscosity of the pectin solution and developed a mesh-like network in the calcium-pectin beads. This network slowed down the penetration of acid and bile into the capsules by longer diffusion path length. In another study, a mixture of pectin, rice bran, and Hi-maize starch coated with whey-protein can protect the L. plantarum in freeze- drying and under simulated gastrointestinal conditions (Chotiko & Sathivel, 2016b).

(b) Pectin and protein

Many researchers have investigated complex coacervation strategies for encapsulation of probiotics with pectin, frequently in conjunction with dairy proteins. Combination of the two does usually lead to increased protection of the cells in spray drying and simulated gastric conditions as well as 120-day storage at various temperatures (Oliveira et al., 2007), in bile (Gerez et al., 2012), and in yogurt products (Ribeiro et al., 2014; Shoji et al., 2013). However, when the two are applied as sequential layers, the protein layer generally does not improve cell survival through the gastrointestinal tract in comparison to capsules formed of pectin only (Gebara et al., 2013). The layer of whey-

32

protein covered the capsules by the interaction between protonated amine groups of whey protein and negatively charged carboxylic groups of pectin.

Combinations of whey protein and pectin were confirmed as effective secondary coatings in the protection of L. rhamnosus (Doherty et al., 2012) through simulated digestion. The soy protein isolate (SPI) was combined with HM pectin (7:1 v/v) to protect Lactobacillus delbrueckii in the small intestine (Sun et al., 2014). In another study, multiple lipid-protein-pectin layers exhibiting high stability were utilized to improve the viability of L. salivarius with 90 % efficiency. Pectin was working as a secondary emulsion to thicken the interface and prevent agglomeration (Zhang et al., 2015).

Gelatin, a partially hydrolyzed form of collagen, is also an emerging coating material that forms complexes through electrostatic interactions with pectin at pH lower than 4.7 (isoelectric point). The double gelatin-coated pectin mixture maintained the highest survival rate of L. plantarum and B. longum (108 CFU/mL in pomegranate juice and 106 CFU/mL in cranberry juice) after six weeks at 4 °C, while the free cells die within one week. This could be due to the stability of the polyelectrolyte complex, which was confirmed by the turbidity from pH 2 to 7 (Nualkaekul et al., 2013).

Combinations of pectin with gum Arabic, soy protein, whey protein, gelatin, and soy milk were compared for their ability to protect L. acidophilus during spray drying. The number of surviving cells carried by pectin-whey protein, pectin-gum Arabic-soymilk, pectin-gum Arabic-whey protein decreased by less than 1 log cycle, while all other tested combinations decreased by 1–2 log units. During storage at 5°C, the gum arabic/soymilk/pectin performed best, maintaining between 105 and 106 CFU/g after 14 weeks (Mosilhey, 2003).

(c) Pectin and starch

The system of pectin (0.5 % w/w) and starch (1.5 % w/w) hydrogel prepared by extrusion also improved the viability of L. plantarum in simulated gastric fluid (from 10.01 to 6.94 log CFU/g) and bile salt 2 % (from 9.98 to 7.09 log CFU/g) conditions for 2 hours while free cells were dead (Dafe et al., 2017). Gelatinized high amylose starch and pectin have been used in coatings and micro-particles and shown to be stable in simulated environments of the gastrointestinal system (Desai, 2006). Pectin-fructo- oligosaccharide-maltodextrin combination was found to protect Lactobacillus and

33

Bifidobacterium from the acidic environment of juice and drying conditions as well as during storage (Kalita et al., 2018).

(d) Pectin and alginate

Alginate is the most widely used agent in encapsulation; however, Ca-alginate has been reported significantly sensitive to calcium chelators and other cations (Ivanova et al., 2002). The microencapsulation by alginate did not effectively protect bifidobacteria in simulated gastric juice; however, the microencapsulated probiotics can survive better in milk with 2 % fat (Hansen et al., 2002; Smidsrød & Skjåk-Bræk, 1990). In a study of poultry probiotic cells, pectin based beads were more resistant and more mechanically stable than alginate beads (Voo et al., 2011). The modified citrus pectin with alginate has shown potential as a supplement to delivery Lactobacillus acidophilus in the simulated gastrointestinal tract of mice with induced colon tumors (Odun-Ayo et al., 2016, 2017). The was 82.7 % survival of cells after exposure to gastric juice for 3 hours in vitro, and an increase of 10.2 % of fecal lactobacilli numbers after 28 days in vivo.

The L. casei was significantly protected in sodium alginate-amidated LM pectin complexes (30-102 µm) in 1:4 and 1:6 ratios by extrusion in the simulated gastric juice (Sandoval-Castilla et al., 2010). The combination of 1:6 provided the best performance in yogurt during storage and gastrointestinal passage. The HM pectin combined with sodium alginate also was reported as an effective cryo-protectant protective agent during freeze-drying (Denkova et al., 2014). This hydrogel combination enriched with nutrients showed a higher protective effect than single alginate or pectin beads. FTIR confirmed the interactions between the two polysaccharides. Bekhit (2016) also reported the best combination was alginate and pectin with a ratio of 3:1 enriched with M17 broth and L. lactis was in an exponential growth phase.

Both L. plantarum X2 and L. paracasei RN5 retained high numbers of surviving cells after storage for nine months in a freeze-dried matrix with sodium alginate and high-ester pectin (Denkova et al., 2014). In another study, the loss of L. plantarum was lowest for 4 %w/v low-methoxyl citric pectin, followed by a mixture of 2 %w/v sodium alginate and 2 %w/v pectin during refrigerated storage. However, these treatments could not protect bacteria in the simulated gastric medium due to the reduced pore size (Brinques & Ayub, 2011).

34

In more recent research, the co-microencapsulation by alginate-pectin-gelatin (ratio 1.06 %:0.55 %:0.39 %) was determined to protect the L. plantarum with the survival rate of 88.66 % by entrapping them inside the matrix, as observed via scanning electron microscopy (Vaziri et al., 2018). A matrix of sodium alginate and citric pectin was used to microencapsulate L. plantarum by electrospraying with only 2.9 and 2.7 log CFU/mL reduction in simulated gut fluids and intestinal conditions respectively (Coghetto et al., 2016). There was a significantly higher survival rate for immobilized cells (about 9 log CFU/mL) in comparison with free cells (only 1 log CFU/mL) after refrigeration storage for 21 days.

(e) Pectin with chitosan

Chitosan is a cationic polymer that can form polyelectrolyte complexes with negatively charged pectin due to their opposite charges, which was confirmed by high turbidity due to the great affinity of both polymers for each other (Nualkaekul et al., 2013). The composition of 60% pectin and 40% chitin ensured the highest live cells through mimicked gastric (97.7%) and intestinal conditions (95.8%), while in free bacteria the survival percentages were only 76.2% and 73.4% in these conditions (Khorasani & Shojaosadati, 2017a).

In work by Kakimov et al, L. casei was protected by pectin capsules obtained by the extrusion method, and then the capsules were covered with chitosan by mixing to produce 2-3 mm capsules without changing the sensory properties (Kakimov et al., 2017). The system (bacteria in 3 %w/v amidated pectin into 0.15 M CaCl2) also was determined to improve the viability of cells under simulated gastric and intestinal fluid and release the cells after 1 hour in intestinal fluid (Bepeyeva et al., 2017).

Layer-by-layer self-assembly by sequential adsorption of oppositely charged biopolymer nanoparticles has been investigated as a probiotic encapsulation method. Several polyelectrolyte layers were coated on cell surfaces by sequential incubation in aqueous polycations and polyanions. The first deposition was of a polyelectrolyte, including low methoxyl pectin, chitosan, lysine, and diethylamine, followed by negatively charged nanoparticles of sulfated low methoxyl pectin. The modified cell surfaces with a negative layer enhance the stability of the capsules by enhancing the adhesion of nanoparticles. The nanoparticles deposited outside also improved the

35

viability in the simulated intestine fluid than a single coating with only one polyelectrolyte (Usmiati et al., 2017)

(f) Pectin and nanocellulose

A bio-nanocomposite system composed of bacterial nanocellulose and pectin was developed to protect Bacillus coagulans (Khorasani & Shojaosadati, 2016). The pectin- only system was not to be able to improve probiotics cell survival, but by combining with nanocellulose the highest viability was achieved (up to 99.95 %) during thermal drying and 95.18 % in gastrointestinal fluids (Khorasani & Shojaosadati, 2017c).

Studies have shown that composite of pectin with non-starch nanofibers improved the prebiotic score and gastrointestinal resistance of the biocomposites (Khorasani & Shojaosadati, 2017b). Pectin dissolution and swelling under GI fluids were slowed because of the incorporation of insoluble nanofibers. The highest viability of probiotic through artificial gastric (97.7 %) and intestinal conditions (95.8 %) was recorded with the composition of 60 % pectin and 40 % chitin, while for free bacteria, the survival proportions were only 76.2 % and 73.4 % in the same conditions.

(g) Combination with other materials

The combination of potassium alginate, potato resistant starch, pectin and heat protecting agents as encapsulants for L. plantarum was tested during spray drying, storage and incubation in gastrointestinal environments (Muhammad et al., 2017). During storage at 25 °C for 42 days and in simulated gastrointestinal conditions, pectin performed worse than potassium alginate when combined with whey protein isolate and mannose. Samples with pectin had the lowest moisture content and were least hygroscopic, which negatively affected the shelf life of spray-dried products and survival of probiotics during storage and under the simulated GI conditions. A mixture of alginate, pectin and whey proteins produced by extrusion enhanced the viability of B. bifidum during freeze-drying and under low pH of the simulated gastric conditions (Martin-Dejardin et al., 2013).

The formed gel capsules of the alginate-pectin mixture were coated with the chitosan layer by ionotropic gelation using the extrusion technique to protect L. casei PM01 (Foroutan et al., 2017). The efficiency of encapsulation of these capsules was 72.7 %, which were lower than alginate-tragacanth gum or alginate-gum Arabic mixtures. This may be due to the higher viscosity of the solutions, and so the cells adhered to the internal surfaces of tools and containers. The survival of microencapsulated cells after 48 hours

36

was also higher than free cells after adding to the MRS broth. The encapsulation also helped to improve the acid resistance of cells at pH 2 and 2.5 for 3 hours.

Whey protein and pectin were used to develop as encapsulating materials by extrusion and ionic gelation methods to enhance the survival of B. longum in simulated gastric juice and bile salts. The formulation of 2.5% whey protein concentrate and 1.5% pectin resulted in the highest encapsulation efficiency, and improved size and textural properties. This bead formulation also showed higher tolerance to bile salts compared to uncoated cells. The surviving cells were more than 107 CFU/g, and the cells only started to be released from beads after 28 days of storage at 4 °C (Yasmin et al., 2018)

The multilayer capsules of alginate/poly-L-lysine/pectin/poly-L-lysine/alginate were developed for oral delivery of L. reuteri cells through simulated gastric and intestinal fluids. The results show that these multilayer microcapsules were stable with no damage in simulated GI conditions for 12 hours at different pH values from 1 to 9. Pectin supported the acid stability and strength of microcapsules by connecting layers of poly-

L-lysine (Ouyang et al., 2004).

Pectin-based bio-nanocomposites were investigated for the development of a prebiotic, gastrointestinal-resistant matrix to support the viability of Bacillus coagulans. The optimum composition of pectin, nano-chitin and nano-lignocellulose (50:25:25% w/w) was found to increase the survivability of probiotic cells at 4 and 25 °C during 5- week storage in juice (68%) and simulated GI conditions (65 %) compared to before exposures to treatments (Khorasani et al., 2017b).

2.4.2.4. Disadvantages of pectin as an encapsulating material

Pectin capsules are usually large and highly porous resulting in a sandy texture, slow cell release rate, and low mechanical stability. These drawbacks are significant disadvantages for pectin to be applied in the encapsulation process. Using novel encapsulation methods or adding fillers and co-protective agents are emerging solutions to enable the commercial application of pectin as an encapsulating material.

The conventional delivery system, such as extrusion, spray drying creates biopolymer beads with millimeter dimensions, leading to texture defects in food products and a slow-release rate (Zhang et al., 2016). Pectin is highly porous, so fillers or co- protective agents such as starch, maltodextrin, or wheat dextrin soluble fibers need to be added to protect probiotics during freeze-drying (Dafe et al., 2017; Chotiko et al., 2016a).

37

Weak binding to cross-linking agents compared to alginate, e.g., also results in softer beads and fewer applications of pectin in encapsulation (Fang et al., 2008). The mechanical stability of the pectin beads could be improved by adding a mixture of polymers, using a lower degree of carboxylation pectin, or applying higher pectin concentration (Nualkaekul et al., 2013).

Encapsulation should enhance not only survival but also functionality by protecting the specific bioactive molecules and effector molecules in the cell wall of bacteria (Chen et al., 2017). The probiotic cells also need to be released in the targeted area to achieve full functionality and health effects by contacting the receptor molecules of the specific immune cells (Bauer et al., 2009); however, there has been little research on these issues.

Previous studies have reported that hydrogel particles from pectin can protect probiotic cells overtime under wet conditions and when exposed to simulated gastro- intestinal conditions. However, there has been little research on the dried pectin-based hydrogels in the preservation of probiotics, which may add to the potential commercial applications of probiotics in food products.

38

Chapter 3: Materials and methodology

3.1. Ingredients and chemicals

The fresh peels were obtained from the Bramston Beach farm, Queensland (Australia), following the processing of fresh fruits. All the chemical reagents were of analytical grade and purchased from Merck, Sigma (Australia). The bacteria used in this study were Lactobacillus casei ATCC334 and Escherichia coli K12 10788, obtained from the Department of Chemistry and Biotechnology at Swinburne University of Technology.

3.2. Preparation of raw materials from fruit peels

Whole peels were blanched in distilled water at 100 °C for 3 minutes and rapidly cooled in iced water to inactivate the enzymes. The blanched peel was dried at 50 °C in a vacuum oven (SuGold, China) until constant weight and was ground to produce peel powders subsequently sieved through a 40-mesh screen. The peel powders were then packed in airtight bags and stored at -24 °C to be eventually used as raw materials in the various experiments (Yapo, 2009a).

3.3. Response surface methodology (RSM)

Response surface methodology has been used to investigate the response variables and develop a model to optimize the response (Meyer & Krueger, 2001). RSM explores the relationship between response variables (properties, qualities, etc.) and several explanatory factors (processing parameters, extraction conditions, etc.) with curvatures in the response surface plot serving to identify optimum conditions, i.e., maxima or minima. The Minitab® 15 software (USA) was applied to design experimental runs and process the data.

3.3.1. Factorial Design

A factorial design approach was adopted to explore the critical processing factors that affect the pectin extraction from the two types of DFP (Figure 3.1). The significance of main effects and interaction effects could be estimated, and the presence of cubic or quadratic effects were checked by factorial design (Hanrahan & Lu, 2006). Three factors, i.e., the type of peel, the extraction temperature, and the extraction time, were tested, and the experimental settings for each variable are summarized in Table 3.1 below.

39

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

Table 3.1. The three experimental variables and their factorial design settings

Level Independent variable -1 0 1 Extraction time (min) 43 75 107 Extraction temperature (°C) 60 70 80 100% red-flesh 100% white-flesh Type of peels - DFP DFP

The dependent responses were the yield and the degree of esterification (DE) of the crude pectin. A preliminary study was performed to choose the value of experimental conditions. The extreme settings for extraction time and temperature were tested in every possible combination, in addition to the central values for both variables were tested for each type of peel, making a total of ten sets of extraction conditions, as shown in Table 3.2.

40

Table 3.2. Specific combinations of experimental variables tested

Extraction Time Extraction Type of DFP (minutes) Temperature (℃) 107 80 White flesh 75 70 Red flesh 43 60 Red flesh 107 80 Red flesh 43 80 White flesh 107 60 Red flesh 43 60 White flesh 107 60 White flesh 43 80 Red flesh 75 70 White flesh

3.3.2. Central Composite Design (CCD)

The central composite design was applied to model the curvature by corner points, axial points, and center points allow the development of a second-order polynomial model (Figure 3.2) (Morris, 2000). The center point measurements were conducted five times to balance the precision at the edge of the design relative to the middle. The axial experimental points located at a distance α from the center, which is a function of the number of independent factors and the properties of the applied design. The experiments were run randomly to avoid biased results.

41

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

In conventional heating, the influences of the pertinent factors, including processing temperature (X1) and processing time (X2), on the processing parameters, the yield of pectin, and degree of esterification, were investigated using 13 experimental points for each type of peel. Five levels of each variable were used in this study (–α, –1, 0, 1, +α), as summarized in Table 3.3.

Table 3.3. The levels and coded and uncoded values of two independent variables

Independent variables Levels Uncoded Coded –α –1 0 +1 +α Dragon fruit peel

Extraction temperature (°C) X1 56 60 70 80 84

Extraction time (min) X2 30 43 75 107 120 Passion fruit peel

Extraction temperature (°C) X1 48 60 90 120 132

Extraction time (min) X2 30 43 75 107 120

42

The 13 experiments listed in Table 3.4 below included five center point measurements (coded 0, 0), four axial points (coded ±α, 0 and 0, ±α) and four corner points (coded ±1, ±1).

Table 3.4. The experimental runs following the CCD for pectin extraction by conventional heating

White-flesh/Red-flesh Passion fruit peel Standard Dragon fruit peel order Temperature Temperature Time (min) Time (min) (℃) (℃) 1 -1 (60) -1 (43) -1 (60) -1 (43) 2 -1 (60) 1 (107) -1 (60) 1 (107) 3 1 (80) -1 (43) 1 (120) -1 (43) 4 1 (80) 1 (107) 1 (120) 1 (107) 5 0 (70) -α (30) 0 (90) -α (30) 6 0 (70) α (120) 0 (90) α (120) 7 -α (56) 0 (75) -α (48) 0 (75) 8 α (84) 0 (75) α (132) 0 (75) 9 0 (70) 0 (75) 0 (90) 0 (75) 10 0 (70) 0 (75) 0 (90) 0 (75) 11 0 (70) 0 (75) 0 (90) 0 (75) 12 0 (70) 0 (75) 0 (90) 0 (75) 13 0 (70) 0 (75) 0 (90) 0 (75)

A second-order polynomial regression equation was fitted to the obtained data, following the general model of Equation 3.1. Post optimization, the derived models were verified in predicting the optimum response of yield and degree of esterification. The actual extraction was conducted with the optimum conditions to verify the efficacy of the model at these conditions.

2 2 � = �0 + �1�1+�2�2 + �11�1 +�22�2 + �12�1�2 Eq. 3.1

43

Where Y is predicted response (Yp: pectin yield; Yd: DE) and β are regression coefficients

(β0-a constant, β1 β2 - linear coefficients, β11 β22- quadratic coefficients, β12- interaction coefficients), and X is the independent variable.

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

A Box-Behnken design was applied for the microwave extraction method due to the greater number of independent variables, as illustrated in Figure 3.3 (Box & Behnken, 1960).

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

The BBD for four independent processing factors (extraction time, microwave power, pH, and solid: liquid ratio) requires fewer experimental runs than an equivalent CCD because BBD does not include corner points of the cubic region. The BBD also has only three levels (-1, 0, +1) of each variable compared to the five-factor levels of CCD. The number of experimental points was calculated using Equation 3.2 (Ferreira et al., 2007):

� = 2�(� − 1) + �� Eq. 3.2

Where N is the total of experimental points; k is the number of independent variables; Cp is the number of center points.

44

The independent variables and their settings for BBD for the experiments involving microwave-heating extraction of pectin are presented in Table 3.5. The whole design, including all experimental points for each type of peel, is shown in Table 3.6.

Table 3.5. Independent variables and their levels from BBD at extraction 80 °C

Independent Variables /Factors Level Uncoded Coded –1 0 +1

Extraction time (mins) X1 5 10 15

Microwave power (W) X2 50 150 250

pH X3 2 3 4

Liquid:solid ratio (mL/g) X4 30 50 70

Table 3.6. The whole Box-Behnken Design for microwave-assisted heating with four variables X1: Microwave time (minutes); X2: Microwave power (W); X3: pH; X4: Liquid:solid ratio

Standard Variable levels

order X1 X2 X3 X4 1 -1 (5) -1 (50) 0 (3) 0 (50) 2 -1 (5) 1 (250) 0 (3) 0 (50) 3 1 (15) -1 (50) 0 (3) 0 (50) 4 1 (15) 1 (250) 0 (3) 0 (50) 5 0 (10) 0 (150) -1 (2) -1 (70) 6 0 (10) 0 (150) -1 (2) 1 (30) 7 0 (10) 0 (150) 1 (4) -1 (70) 8 0 (10) 0 (150) 1 (4) 1 (30) 9 -1 (5) 0 (150) 0 (3) -1 (70) 10 -1 (5) 0 (150) 0 (3) 1 (30) 11 1 (15) 0 (150) 0 (3) -1 (70) 12 1 (15) 0 (150) 0 (3) 1 (30) 13 0 (10) -1 (50) -1 (2) 0 (50) 14 0 (10) -1 (50) 1 (4) 0 (50)

45

Standard Variable levels

order X1 X2 X3 X4 15 0 (10) 1 (250) -1 (2) 0 (50) 16 0 (10) 1 (250) 1 (4) 0 (50) 17 -1 (5) 0 (150) -1 (2) 0 (50) 18 -1 (5) 0 (150) 1 (4) 0 (50) 19 1 (15) 0 (150) -1 (2) 0 (50) 20 1 (15) 0 (150) 1 (4) 0 (50) 21 0 (10) -1 (50) 0 (3) -1 (70) 22 0 (10) -1 (50) 0 (3) 1 (30) 23 0 (10) 1 (250) 0 (3) -1 (70) 24 0 (10) 1 (250) 0 (3) 1 (30) 25 0 (10) 0 (150) 0 (3) 0 (50) 26 0 (10) 0 (150) 0 (3) 0 (50) 27 0 (10) 0 (150) 0 (3) 0 (50)

3.4. Conventional heating extraction

Peel materials were mixed with distilled water in a 1:50 (w/v) ratio, and the pH was adjusted to 2 with citric acid. Citric acid is a weak organic acid known to be effective for pectin extraction. It is also relatively environmentally friendly. The suspension formed was heated at different temperatures for various times to extract pectin. The pectin released in the solution was then separated from the residual biomass by centrifugation (10,000 × g, 25 °C) followed by vacuum filtration to remove peel residues.

The pH of the solution was then adjusted to 3.5 by adding 0.1 M NaOH, and cooled down to 4 °C using an ice bath. Two volumes of 96% ethanol were then added with mild stirring, and the mixture left overnight at room temperature to allow maximum pectin precipitation. The coagulated pectin was collected and rinsed with absolute ethanol. The raw pectin was dehydrated at 50 °C in a vacuum oven until reaching a constant weight and stored at room temperature (Rodsamran et al., 2019).

46

3.5. Microwave-assisted extraction

Microwave-assisted extractions were conducted in a Microwave Synthesis System (CEM, USA, Frequency 50/60 Hz, and maximum microwave power of 300 W) using a closed vessel to generate pressurized conditions. Peels were dispersed in distilled water at the desired pH, as described above, before loading into the reaction vessel without stirring. When it exceed the set up temperature, the system will cool down by gas. The holding time will start when the desired temperature is stable. The ramp time was affected by the input microwave power. After irradiation, the solutions were treated similarly to the conventional heating method.

3.6. Pectin characterization

The molecular structure and properties of the resulting pectin could be different when different processing conditions are applied. Therefore, the properties of pectin obtained at optimum conditions from different materials were studied. The physicochemical, structural, and functional properties of the extracted pectin were assessed using a suite of analytical techniques.

3.6.1. Pectin yield

The yields in pectin of both conventional heating and microwave-assisted heating were calculated using Equation 3:3

� The yield of pectin (%) = 0 × 100 � Eq. 3.3

Where �0: dry weight of purified pectin, and �: dry weight of raw material

All the tests for each type of peel were performed in triplicate, and the average value was used to calculate the pectin yield.

3.6.2. Equivalent weight

A mass of 0.5 g of extracted pectin and 5 mL of ethanol were dissolved in 100 mL of distilled water along with 1 g of NaCl. The mixture was stirred until complete dissolution and then titrated with 0.1 M NaOH. The neutralized solution was then used to determine the methoxy content (Ranganna, 2004). The equivalent weight was evaluated according to Equation 3.4:

1000 × pectin powder (g) Equivalent weight = NaOH concentration (N) × NaOH volume (mL) Eq. 3.4

47

3.6.3. Methoxy content

A volume of 25 mL of 0.25 M NaOH was added to the final solution from the above (Section 3.6.2) experiment of equivalent weight, and the mixture was kept at room temperature for 30 minutes. Then, 25 mL of 0.25 M HCl was added to the mixture and titrated again to pH 7.5 using 0.1 M NaOH (Ranganna, 2004). The methoxy content of pectin was determined using Equation 3.5 below:

NaOH concentration (N) × NaOH volume (mL) × 3.1 Methoxy content (%) = Pectin powder (g) Eq. 3.5

3.6.4. Moisture

One gram of extracted pectin was weighed and dried in a vacuum oven at 105 °C until constant weight. The residue was left to cool down to room temperature in a desiccator before weighing (AOAC, 1990). The moisture was determined as the percentage of the weight lost compared to the initial weight of the sample. All moisture tests were triplicated, and the average results are presented.

3.6.5. Solubility

The solubility of pectin was determined gravimetrically at pH 2, 4, 6, 8, and 10

(Monsoor & Proctor, 2001). An initial mass of 0.5 g (S1) of pectin was suspended in 100 mL of distilled water. The pH of the final mixture was adjusted by either 0.1 M HCl or 0.1 M NaOH. The mixture was kept in the incubated shaker (OM11, Ratex, Australia) at room temperature for 24 hours. After centrifugation (2700×g, 15 mins) (Eppendorf 5702, Germany), the insoluble pectin was carefully collected and then dried at 105 °C until constant weight and recorded as S2. The solubility was determined using Equation 3.6.

� −� Solubility (%) = 1 2 × 100 Eq. 3.6 �1

3.6.6. The total carbohydrate contents

The total carbohydrate contents were determined by the phenol sulfuric acid method (Dubois et al., 1956). Five microliters of 80 % aqueous phenol were added to the glucose/pectin solution and vortexed. Then 5 mL of 95 % sulfuric acid was quickly added to the solution. The absorbance of the solution was determined at 490 nm by UV-VIS spectroscopy (Shimadzu UV-2600, United States). The calibration curves were produced from ᴅ-(+)-glucose standard solutions at concentrations in the range of 0.0062-0.05 mg/mL.

48

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

The DE and DA of extracted pectin were determined using a slight variation of the titration method with (Bochek et al., 2001; Kirk et al., 1983; FAO/WHO, 2009). A mass of 0.5 g of dried pectin was wetted with ethanol (2 mL) and dispersed in 100 mL of distilled water for 2 hours at 40 °C.

The resulting solution was titrated against 0.1 M NaOH to determine the amount of free carboxylic acid groups, which was recorded as the initial titer (V1). To the neutralized solution, 20 mL of 0.5 M NaOH was added and the mixture stirred at room temperature for 2 hours to hydrolyze the side ester groups. Subsequently, 20 mL of 0.5 M HCl was added to neutralize the remaining NaOH, and the excess HCl was titrated against 0.1 M

NaOH. The total titration volume (V2) was recorded.

Then 20 mL of 10 % NaOH was added to the solution before distillation. The receiving flask contained 20 mL of 0.1 M HCl with about 80-120 mL of distillate. The resulting solution was titrated against 0.1 M NaOH with S as the titration volume. A 20 mL aliquot of 0.1 M HCl was titrated as a blank sample and the required volume of 0.1

M NaOH was recorded as B. The amide titer was the difference (B-S) recorded as V3.

The DE was calculated using Equation 3.7 below:

� �� (%) = ( 2 ) × 100 Eq. 3.7 �1+�2

The degree of amidation, reported as the percentage of total carboxyl groups, was determined by following Equation 3.8:

� �� (%) = ( 3 ) × 100 Eq. 3.8 �1+�2+�3

3.6.8. The content in galacturonic acid

The percentage of galacturonic acid (GalA) was measured by a colorimetric technique known as the sulfamate/meta-hydroxy diphenyl method (Adetunji, 2016). The sulfuric acid hydrolysis of pectic polysaccharides was followed by a colorimetric assay involving reaction with m-hydroxy diphenyl and spectrophotometric absorbance at 525 nm using a UV-VIS spectrophotometer (Shimadzu UV-2600, United States). The calibration curves were produced from ᴅ-(+)-galacturonic acid standard solutions at concentrations between 100–1500 μM.

49

3.6.9. Surface morphology analysis

The samples of pectin were mounted onto specimen stubs and gold-coated to increase the electrical conductivity. The morphological features of the pectin were imaged by a field-emission scanning electron microscope (FESEM; ZEISS SUPRA 40 VP) with an accelerating voltage of 5kV at various magnifications.

3.6.10. Fourier Transform infrared spectroscopy

Fourier Transform Infrared (FTIR) analyses were used to assess the degrees of esterification of pectin. Prior to analysis, pectin samples were kept overnight in a desiccator. The pectin was scanned using a Thermo Scientific™ Nicolet™ iS5 FTIR spectrophotometer with a universal ATR accessory, in absorbance mode over the spectral range 400 – 4000 cm-1 with a spectral resolution of 4 cm-1 and averaged over 128 scans.

3.6.11. Rheological properties

The influences of pectin on the rheological behavior of pectin solutions were determined at a concentration of 1 %, 3 % and 5 % (w/v). The pectin solution was made with distilled water and stored at 4 °C for 24 hours before use. For the rheological behavior of pectin gels, solutions of 1 % (w/v) in various calcium chloride concentrations at pH 7 were prepared with 1 M HCl and stored at 4 °C overnight for each batch.

Rheological properties were studied by a controlled-stress rheometer with a plate- plate geometry (Gigli et al., 2009). The plate diameter was 40 mm, the temperature was maintained at 25 ± 1 °C, and the plate gap was set at 1 mm for all experiments. The data of flow behaviors were fitted with a power-law model. To determine the linear viscoelasticity region, the stress sweeps were run at a constant frequency of 0.1 Hz. For oscillation experiments, the frequency sweeps were studied in this linear viscoelasticity region at a strain of 2 %. All experiments were performed in triplicate.

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

The textural properties, e.g., specific surface area, pore volume, and pore size, were measured using the BET method on a TriStar II Plus analyzer. Prior to adsorption experiments, the samples were degassed on the Smart VacPrepTM. The samples’ surface areas were determined using the multipoint method. The BET total pore volume and average pore diameter were determined via the adsorption isotherms using the Barrett- Joyner-Halenda (BJH) model.

50

3.6.13. X-ray diffraction (XRD)

The XRD analyses of the different types of pectin were carried out on a Bruker D8 Advance X-ray diffractometer. The different types of pectin were scanned between 5° to 90° (2�) at the voltage of 40 kV, current of 40 mA, step size of 0.02° (2�) and a counting time of 1 s (Hosseini et al., 2019)

3.6.14. Oil-holding capacity

The capacity of different types of pectin to absorb oil was investigated by a method adapted from the literature (Bayar et al., 2016). One gram of pectin was weighed in a centrifuge tube before adding 10 mL of sunflower oil (density: 0.919 g/mL). The mixture was then stirred for 5 hours and centrifuged at room temperature for 20 minutes at 5000 × g. The residue was drained by angling the centrifuge tube on a filter paper for 3 minutes. The oil-holding capacity was determined as the amount of oil held per unit of weight of pectin.

3.6.15. Emulsifying properties

The emulsifying properties of different types of pectin were determined by a method adapted from the literature (Sila et al., 2014). Two milliliters of sunflower oil was added to the 5 mL of pectin suspensions (2 % and 4 %w/w). The mixture was then homogenized by vortex for 1 minute. To calculate the emulsifying stability, the suspension was held for 30 minutes at room temperature and then centrifuged for 10 minutes at 4700 × g. Similarly, the emulsifying stability was measured after storing for one day at 4 °C and room temperature (25 °C). The emulsifying capacity and stability were calculated by following equations 3.9 and 3.10.

The emulsion volume = × 100 The emulsion capacity (%) The total volume Eq. 3.9

The final volume after incubation = × 100 The emulsion stability (%) The initial emulsion volume Eq. 3.10

3.6.16. Foaming properties

The foaming properties of extracted pectin were determined by a methodology adapted from the literature (Sila et al., 2014). Two grams of the sample was added in distilled water (100 mL), and the volume was recorded as V1. The solution was then homogenized for 1 minute by vortexing. The total volume of the stirring solution was then measured in a 250 mL graduated cylinder (V2). The foaming capacity corresponds

51

to the increasing percentage of volume after whipping. The foaming stability was described as the remaining foaming volume after standing for 30 and 60 minutes (Vt). The measurements were repeated three times, and the foaming properties were calculated following Equation 3.11 and 3.12:

� −� The foaming capacity (%) = 2 1 × 100 Eq. 3.11 �1

� −� The foaming stability (%) = � 1 × 100 Eq. 3.12 �1

3.7. Prebiotic score

3.7.1. Growth of probiotics in the presence of pectin

Lactobacillus casei ATCC334 was grown and maintained in MRS broth culture (BD Diagnostic 288130). The influence of pectin on the growth of L. casei was carried out by supplementing MRS broth with extracted pectin (0.5 %) from each type of peel. Probiotics were grown in these media at 37 °C for 48 hours. The effects of pectin on probiotic growth were assessed at 48 hours by optical density measurement at 660 nm and compared to growth in the absence of pectin. All experiments were done in triplicate.

3.7.2. Prebiotic activity score

The prebiotic score is a quantitative score which estimates the selective growth stimulation on lactobacilli and bifidobacteria of prebiotic materials (Huebner et al., 2007). The prebiotic scores for extracted pectin were calculated as the difference between the ratio of probiotic growth (in log CFU/mL) on pectin to growth on glucose, and the same ratio for enteric bacteria (Equation 3.13).

Probiotic growth on pectin Enteric growth on pectin Prebiotic activity score = − Probiotic growth on glucose Enteric growth on glucose Eq. 3.13

Where growth is measured in log CFU/mL, over a constant specified time interval, e.g., 24 or 48 hours.

A positive prebiotic activity score indicates that the sample selectively supports the growth of the probiotic bacteria over other bacteria present in the gastrointestinal system. The probiotic strain used here was L. casei (see above for growth conditions), while the enteric strain was E. coli grown and maintained in Luria-Bertani broth.

The L. casei cells were incubated at 37 °C for 17 hours under low oxygen exposure, while E. coli cells were incubated in a similar time and temperature settings but under

52

aerobic conditions. A small amount of the activated cultures of bacteria (1% v/v) was added to two different media including 0.5 % w/v glucose or 0.5 % w/v pectin. The cultures were then incubated at 37 °C for 48 hours under low oxygen conditions for L. casei and aerobic conditions for E. coli culture. Cell numbers at t = 0 and 48 hours were counted in triplicates as described above and used as input data for calculating prebiotic scores.

3.8. Microencapsulation

3.8.1. Preparation of cell culture

The single colonies were harvested from streaking L. casei stock culture on MRS plates, which were inoculated into 100 mL of sterilized MRS broth and incubated for 17 hours at 37 °C under shaking at 150 rpm. The L. casei was reactivated twice in MRS broth at 37 °C for 17 hours in shaking incubator with 200 rpm agitation. The cell pellets were collected by centrifugation (4000 rpm, 4 °C, 10 minutes). Only the cell suspension was harvested and washed three times by sterilized peptone water (0.2 %w/v). The final cell paste was stored at 4 °C for further experiments (Chen et al., 2017).

3.8.2. Bacterial enumeration method

The 1 mL of harvested L. casei was serially diluted with saline solution (0.89 % NaCl), and 100 �L of each dilution was plated on MRS agar by spread plate technique. The plates were then incubated at 37 °C for 48 hours. The number of colonies formed was then recorded as colony-forming units (CFUs) and expressed in log CFU/mL.

3.8.3. The growth curve of L. casei cells

The growth curves of free and encapsulated cells were generated by counting viable cells. Two hundred milliliters of the MRS broth were prepared to grow bulk culture at 37 °C, with agitation at 150 rpm for 25 hours. Culture samples were collected at 2, 6, 18, 22 and 25 hours. Microencapsulated probiotic cells were released before incubation by the stomacher machine.

3.8.4. Encapsulation process

3.8.4.1. Single pectin coating

One milliliter of cell pellets was inoculated into 9 mL of pectin solutions at different concentrations (0.5, 1 and 1.5 %) and stirred continuously for 1 hour. The concentration

53

of viable cells in this mixture before encapsulation was determined as described above. Using a syringe pump fitted with a 27G needle, the mixtures were extruded into the sterile

1 % CaCl2 solution at 10:1 v/v (mixture: hardening solution).

The capsules formed were kept in CaCl2 solution for 1 hour before filtration and subsequently rinsed with sterilized distilled water. The wet capsules were then stored at 4 °C in a 0.1 % sterilized peptone solution. One portion was retained as “gelled capsules” which were used to determine the viable cells in simulated gastrointestinal conditions. The remaining capsules were freeze-dried and stored at room temperature in a desiccator. The microencapsulation technique is summarized and illustrated in Figure 3.4.

Figure 3.4. A flow chart of the microencapsulation process

54

3.8.4.2. Double pectin coating

The single coated capsules were added to a 10 mL solution of 1 % w/v pectin and stirred for 10 minutes at 80 rpm. Then the capsules were collected and processed in the hardening solution as previously. The double-coated capsules were then harvested and washed with sterilized distilled water before storage at 4 °C.

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

3.8.5.1. Moisture

The gelled capsules were accurately weighed before being dried in a vacuum oven for two hours at 60 ± 2 °С and a pressure of 50 mmHg. All samples were cooled in desiccators before being reweighed. The samples were transferred to the vacuum oven for a further hour and repeatedly reweighed until the variation in weight was lower than 4 %. The moisture content was calculated and expressed as a percentage (AOAC, 2008). All measurements were done in triplicate.

3.8.5.2. Particle size, shape analysis and distribution

The bead size was determined using a Nikon Microscope Eclipse with an image analysis software (NIS Elements). The sphericity factor (SF), as defined by Equation 3.14, is commonly used to quantify tear-shape particles. Beads with SF > 0.5 are defined as spherical particles (Chan et al., 2011).

� −� Sphericity factor (SF) = ��� ��� Eq. 3.14 ����+����

Where ���� is the largest diameter and ���� is the smallest, perpendicular to ����.

3.8.5.3. Fourier Transform Infrared Spectroscopy (FTIR)

Both wet and freeze-dried capsules were examined by FTIR spectroscopy. The samples were placed in a desiccator overnight before being analyzed. The pectin was analyzed using ATR in absorbance mode over the spectral range 400 – 4000 cm-1 at spectral resolution 4 cm-1, averaged over 128 scans to obtain a high signal-to-noise ratio.

55

3.8.5.4. Scanning Electron Microscopy (SEM)

SEM was used to explore the surface topography of probiotic-loaded beads. The dried beads were transferred onto an aluminum specimen stub for SEM and then gold coated. Images were captured at various magnifications. The dried cell colony was also observed under the same conditions.

3.8.5.5. Swelling test

The gelled beads were dehydrated at 40 °C for 24 h and then weighed (Wd). Each dried sample was incubated separately in 10 mL of three different solutions: (i) phosphate-buffered saline (PBS) with pH maintained at 7.4, (ii) PBS with bovine bile (3 g/L) or (iii) simulated gastric fluid (SGF) at 37 °C under gentle shaking.

Samples were carefully withdrawn from the incubation solutions at intervals of 0.5, 1, 1.5, and 2 hours. The samples were rinsed with distilled water, collected by filtration, and weighed (Wt). The swelling ratio was calculated as following Equation 3.15 (Munarin et al., 2012):

� −� Swelling ratio (%w/w) = � � × 100 Eq. 3.15 ��

3.8.6. Viability of probiotic through microencapsulation

3.8.6.1. Viability of probiotic after microencapsulation

A mass of 0.1 g of encapsulated L. casei was dissolved in 0.9 mL of modified phosphate buffer under continuous agitation for 1 hour at room temperature (150 rpm) and until the gelled beads were dissolved completely. The serial dilutions of dissolved beads and free cells (for control) were prepared, and the viable cells were counted by spread plate methods on MRS agar.

The inoculated plates were incubated for 48 hours at 37 °C in a low oxygen environment. The plating was performed in triplicate for each treatment. The viability of L. casei was evaluated both in the pectin mixture before encapsulation and after encapsulation through the pour plate method.

56

The encapsulation efficiency of each capsule formulation was calculated according to Equation 3.16:

����� ������ ����� ����� ������������� � � EE% = = � � Eq. 3.16 ����� ������ ����� ������ ������������� �0�

EE: encapsulation efficiency

NE (CFU/g): the number of cells released from 1 g of the capsules

ME (g): the mass of capsules obtained after encapsulation

N0 (CFU/mL): the number of free cells in 1 mL of cell mass added to the pectin solution to be extruded for the encapsulation process

V (mL): the total volume of cell suspended to the pectin solution

3.8.6.2. Viability of probiotic after freeze-drying

The 0.1 g of freeze-dried gelled beads or free cells suspension was dissolved in 0.9 mL of buffer under continuous agitation at room temperature (150 rpm for 1 hour). The serial dilutions of dissolved beads and free cells without capsule (for control) were prepared, and the viable cells were counted by spread plating on MRS agar as described above in section 3.8.6.1. The total viable cells in freeze-dried capsules were determined according to Equation 3.17:

Cell viability = ��� × ��� Eq. 3.17

NFD (CFU/g): the quantity of surviving bacteria cells in 1 g of freeze-dried beads

MFD (g): the total weight of freeze-dried beads.

3.8.6.3. Viability of probiotic in different simulated gastrointestinal conditions

(a) Viability of probiotic in simulated mouth conditions

The simulated saliva fluid (SSG) was used to determine the survivability of free and encapsulated probiotic cells. The simulated saliva fluid included 8 g/L NaCl, 0.19 g/L

KH2PO4, 2.38 g/L Na2HPO4 and was modified to pH 6.8. The α-amylase (EC 3.2.1.1) was added to the solution to achieve 200 U of enzyme activity. The 1 g of gelled capsules were added to 9 mL of pre-warmed SSG at 37 °C and incubated for 5 minutes in shaking incubator at 37 °C and 100 rpm. The viability of the probiotics was determined as described above.

57

For free cells, 1 mL of cell mass was added to 9 mL of SSG and processed similarly prior to counting viable cells. At each time point, 100 �L of the samples were withdrawn to quantify viable cells as described above.

(b) Viability of L. casei in simulated gastric fluid (SGF)

The mimic gastric fluid was prepared following the US pharmacopeia (The United States Pharmacopeia, 2010), which includes 0.2 %w/v NaCl and the pH maintained at 2.0 by 0.2 M HCl. To observe any degradation effect of the enzymes on the capsules, a solution of 3 g/L of pepsin was added to 100 mL of gastric fluid. The wet or freeze-dried capsules (1 g) or the free cells (1 mL) were added to 9 mL of pre-warmed SGF at 37 °C and incubated at 37 °C under orbital agitation at 100 rpm. The capsules were taken out after 30, 60 and 120 minutes to account for viable cells.

In detail, the capsules were removed from the solution, rinsed with distilled water and redissolved in peptone water using a stomacher to break the beads and release encapsulated probiotic. The number of live cells was evaluated as described above. The viability of probiotic cells was expressed as viable cells (CFU/g for encapsulated cells, CFU/mL for free cells). The results were recorded for both samples: with and without pepsin.

(c) Viability of L. casei in the presence of bovine bile

The free (1 mL) or encapsulated cells (1g) were separately added to 9 mL of bovine bile solution (3 g/L). The mixture was then incubated at 37 °C under orbital agitation at 100 rpm. Viable cell counts after 1, 2, and 3 hours were determined as described previously.

(d) Viability of probiotic at high pH

A mass of 6.8 g of KH2PO4 was dissolved in 250 mL of distilled water and 77 mL of 0.2 M NaOH solution was made up to 1 L with distilled water and was used as a buffer solution. The pH of this solution was fixed to 7.4. One gram of wet or freeze-dried capsules was added to 9 mL of pre-warmed sterilized SIF at 37 °C and kept in an orbital mixer incubator at 37 °C and 100 rpm. Samples were collected at 1, 2, and 3 hours for viable cells. For the free cells, 1 mL of cell mass was added to 9 mL of SIF to count the viable cells.

58

(e) The release profile of probiotic in simulated intestinal fluid (SIF)

Bile powder was added to 100 mL of the above buffer to a final concentration of 3 g/L, which was considered as the simulated intestinal fluid. The pH of the mixture was maintained at 6.8 by 0.1 M NaOH and 0.1 M HCl. The survival rates of free and microencapsulated cells were determined as described previously in section, sampling 100 �L at each time point.

(f) Storage stability test of encapsulated probiotic in wet capsules at 4 ℃

The viability of cells in wet beads was determined after 5-, 10-, 15- and 20-days during storage at 4 °C to investigate the shelf life of encapsulated cells. The wet capsules were dissolved in sodium citrate with a ratio of 1:9 (w/v) to release entrapped live cells, which was followed by vortexing for complete dissolution. Cell viability was evaluated as described previously. The survival rates of encapsulated cells under storage conditions were determined using Equation 3.18:

� Survival rate = 1 Eq. 3.18 �2

N1 (CFU/g): the quantity of surviving cells of wet gelled beads after storage

N2 (CFU/g): the quantity of surviving cells in 1 g of wet gelled beads before storage

3.8.6.4. Viability of probiotic after heat treatment

The protective actions of pectin as an encapsulating material were explored by heating free cells and entrapped cells at the pasteurization temperature of 63 °C. One gram of each sample was exposed to 63 °C for 60 s and 120 s in a drying oven, and an untreated sample was used as a control for each type of pectin material. The survival rate (%) after treatment was determined by the spread plate technique of serial dilutions on MRS agar plates (37 °C, 48 hours). For microencapsulated probiotics, the cells were released from the gelled capsules before enumeration as described above in section 3.8.6.1.

3.9. Statistical analysis

Analysis-of-variance (ANOVA) tests were performed using Minitab®. The significant differences were examined using post-hoc multiple comparisons tests, including Tukey’s test. All the tests were performed at a significance level of 0.05.

59

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

In the waste generated by the agro-food industry, pectins are present in large amounts. Commercial pectin is a by-product of juice production of apple pomace and citrus peel. The dragon fruit and passion fruit peels have been studied in this chapter as alternative sources for pectin recovery to fulfil the increasing market demands. However, the large quantities of solvent used and extended heating periods required in conventional extraction methods makes this approach time- and energy-consuming. The further de- polymerization and de-esterification caused by the strong acidic treatment are also major drawbacks of this technology. The microwave-assisted method is widely accepted because it is suitable for high moisture materials and is easily scaled up. Therefore, this chapter is aimed to investigate and optimize of the pectin recovery from three types of peel by both conventional and microwave-assisted heating. The statistical and mathematical process optimization methodologies such as response surface methodology have been applied to improve these techniques.

4.1. Optimization of pectin extraction by conventional heating

Many processing parameters can affect the pectin extraction and the properties of the extracted pectin. To explore the optimal extraction conditions of the conventional heating method, three factors, i.e., extraction time, temperature, and the type of peel, were considered as the major variables. The effects of these factors on the yield and degree of esterification (DE) of pectin was first determined by single-factor experiments.

4.1.1. Effects of extraction time on pectin yield and DE

The peel powder was suspended in distilled water at a ratio of 1:50 w/v (4 g in 200 mL), the pH was modified by citric acid to pH 2, and the suspension was heated at 75 ℃ by hot plate with magnetic stirrer with condenser for various times to investigate the effects on pectin yield (Figure 4.1) and the DE (Figure 4.2). As seen from these figures, longer extraction from 50 minutes to 110 minutes gave a higher yield of pectin. It appears that the highest pectin yield can be achieved by setting the extraction time to 110 minutes for both red-flesh DFP and white-flesh DFP. It has been suggested that lower pH and

60

prolonging time could extract more pectin for red-flesh DFP (Liew et al., 2015). The DE value of DFP pectin remains virtually the same over the time intervals tested; however, the DE of pectin extracted from PFP decreased with longer extraction times beyond 90 minutes. Similar results were previously reported for DFP, showing that DE did not decrease significantly with longer extraction time (Liew et al., 2015). However, extended time at elevated temperature could partially degrade pectin by depolymerization of the galacturonan chains (Albersheim et al., 1960).

25

20

15 white-flesh DFP

10 red-flesh DFP Purple PFP Yield of pectin (%) pectin of Yield 5

0 0 20 40 60 80 100 120 140 Time (minutes)

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

80

70

60

50

40 white-flesh DFP

30 red-flesh DFP purple PFP 20

Degree (%) Degree of esterification 10

0 0 20 40 60 80 100 120 140 Time (minutes)

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

61

4.1.2. Effects of extraction temperature on pectin yield and DE

The effects of processing time on pectin yield and DE are determined by extracting peel powder at various temperatures for 80 minutes. It was observed that raising temperature could expedite yield in all three types of peel up to temperatures of 90 °C (Figure 4.3). The surface tension and viscosity of the matrix were decreased at the higher temperature, which enhances the diffusivity of the solvent into the materials. The pectin yield from dragon fruit in other research also increased with increasing temperature from 30 °C to 70 °C (Zaid et al., 2016a). However, the DE was found to increase here when the extraction temperature decreased (Figure 4.4). Because the slope of the line for white- flesh DFP is steeper, it is concluded that the temperature has a greater effect on white- flesh DFP than red-flesh DFP. The methyl ester group in pectin can be hydrolyzed to a carboxyl acid and methanol due to high temperature leading to lower DE (Dore, 1926).

20 18 16 14 12 10 white-flesh DFP 8 red-flesh DFP 6 purple PFP Yield of pectin (%) pectin of Yield 4 2 0 0 20 40 60 80 100 120 Temperature (°C )

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

62

80

70

60

50

40 white-flesh DFP

30 red-flesh DFP Purple PFP 20

Degree (%) esterification of Degree 10

0 0 20 40 60 80 100 120 Temperature (°C)

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

4.1.3. Factorial design for two types of dragon fruit peels

4.1.3.1. Analysis of Variance (ANOVA)

Due to the discrepancy in the available literature to refer to between dragon fruit and passionfruit peels, the two are presented separately. The results of all experimental runs and ANOVA for both dragon fruit are presented in Table 4.1 and Table 4.2. The ANOVA tables determine the significance of single effects based on the F and P values to evaluate the main effects. The analysis was done using a confidence level of 95%. The two main factors (extraction temperature and types of peel) have a significant effect on yield and DE (p<0.05), which is shown by the value F (1356.68) and F (716.97) respectively. Meanwhile, only one interaction (temperature and peel type) factor significantly affected the pectin DE. The ANOVA determined that the extraction time has no significant effect on extraction yield or DE within the range tested. Previous work has also reported that extraction time did not significantly influence pectin yield, but found that longer extraction could lead to increased DE (Woo et al., 2010).

63

Table 4.1. Pectin yield and DE values used for the factorial design, obtained by extraction under various conditions. Values given are average values of triplicate experiments.

Results Experimental Extraction Extraction Types of run time (min) temp. (°C) peel Pectin yield DE (%) (%) 1 107 80 White flesh 12.16 57.16 2 75 70 Red flesh 11.93 61.02 3 43 60 Red flesh 9.25 60.51 4 107 80 Red flesh 14.64 56.79 5 43 80 White flesh 10.02 57.76 6 107 60 Red flesh 9.63 63.35 7 43 60 White flesh 5.59 74.07 8 107 60 White flesh 7.09 72.93 9 43 80 Red flesh 13.00 57.16 10 75 70 White flesh 8.73 66.63

Table 4.2. Analysis of variance of each component of the model derived by factorial design. Bold values indicate significant results.

Yield DE Source F-value p-value F-value p -value Overall Model 279.26 0.046 678.61 0.030 Linear 734.61 0.027 1478.13 0.019 Time 130.18 0.056 0.94 0.510 Temperature 1356.68 0.017 3164.73 0.011 Types 716.97 0.024 1268.72 0.018 2-way interactions 9.15 0.237 309.01 0.042 Time*Temperature 14.55 0.163 12.86 0.173 Time*Types 10.76 0.188 31.91 0.112 Temperature*Types 2.15 0.381 882.26 0.021 3-way interactions 1.48 0.483 25.39 0.125 Time*Temperature*Types 1.48 0.483 25.39 0.125

64

4.1.3.2. Effects of extraction conditions on pectin yield and DE

The normal plot for standardized effects of extraction yield and DE is illustrated below to assess the magnitude and the statistical impact of individual variables and interaction effects (Figure 4.5). In the normal plot of yield, the temperature and type of peel were the only significant variables, and temperature displayed higher positive significant effects than the type of peel. Both of these variables also significantly affected the DE of extracted pectin, as did the interaction effect between the two.

Percent yield Percent

ercent DE ercent P

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.

The main effects plot for extraction yield and DE plots are presented in Figure 4.6. The means of yield and DE (each point) at each level of each factor are plotted and

65

connected with a line for comparison. The plots indicate that longer extraction at higher temperatures gave a higher pectin yield. The DE plot shows that the DE value remains virtually the same as time changes, while the DE increases when extraction temperature decreases. The red-flesh DFP gave a higher yield of pectin but lower DE compared to the white-flesh DFP. Because the interaction between temperature and types of peels is significant to DE, the interaction plot was examined more closely in Figure 4.7. In the interaction plots for DE, the temperature*type interaction lines are not parallel, indicating that there is an interaction between these factors, i.e., the effects of temperature on DE is dependent upon the type of peel. There was a greater difference in the pectin extracted from each peel at lower temperatures. However, at a higher temperature, DE was lower and not different between two types of peel. Because the slope of the line for white DFP is steeper, it is concluded that the temperature has a greater effect on white DFP than red DFP.

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

These results are in agreement with the literature. DE values from red DFP achieved in other studies on DFP are of the following order: < 50% (Tang et al., 2011); 36.54 – 57.81% (Liew et al., 2015) and 63.74% (Tang et al., 2011). Liew et al. (2015) reported that the difference in the DE is due to the pectin degradation by longer extraction time and that low concentration of citric acid gives higher DE values. They also suggested different extraction methods could result in different DE trend.

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

When the average of the center points falls above or below the fitted lines in the main effect and interaction graphs, the curvature on the response surface should be significant. Adding axial points in the Central Composite Design (CCD) to a previously performed factorial design allows for modelling of the curvature. The CCD also developed a fitted quadratic model describing the effect of all linear, interaction, and squared interactions of all independent variables on the responses. Table 4.3 presents all experimental results from CCD for each type of peel.

67

Table 4.3. Central composite design of two variables for each type of peel and experimental results from response variables

Extraction Extraction Red-flesh DFP White-flesh DFP Run Time Temperature Pectin yield DE Pectin yield DE (min) (℃) (%w/w) (%) (%w/w) (%) 1 43 60 13.06 60.44 5.81 72.19 2 107 60 9.58 63.38 7.74 71.93 3 43 80 13.06 58.03 10.00 59.15 4 107 80 14.45 54.01 12.19 57.01 5 30 70 10.56 58.64 6.82 69.91 6 120 70 11.25 58.32 8.47 69.10 7 75 56 8.99 62.47 6.44 72.71 8 75 84 15.12 53.50 13.11 52.38 9 75 70 12.86 59.38 7.69 75.19 10 75 70 12.49 60.99 8.29 68.82 11 75 70 11.98 56.28 7.70 72.00 12 75 70 12.38 57.46 7.76 76.13 13 75 70 12.49 59.31 8.47 59.87

4.1.4.1. Experimental data, model fitting and statistical analysis

The ANOVA (Tables 4.4 and 4.5) of the CCD data was carried out to evaluate the model's significance and suitability. The p-value of the developed model was less than

0.05, indicating that this model adequately fits the data (FWDFP=65.61, FRDFP=122.87).

The statistical significance of the square terms (FWDFP=20.86, FRDFP=28.63) indicate that there is a curvature in the response surface. The linear terms, interaction terms, and quadratic terms of the yield of pectin were significant at the 5% level in both types of peel, except for the 2-way interaction model for the white-flesh DFP. The interaction terms and quadratic terms of extraction conditions did not have any effect on the DE model in either type of peel at the 5% level.

68

Table 4.4. The ANOVA for testing the significance of factors based on the F and p values for extraction yield. Bold values indicate statistically significant results.

White-flesh DFP Red-flesh DFP Source DF F-value p -value F-value p -value Model 5 65.61 0.000 122.87 0.000 Linear (Main effects) 2 143.11 0.000 276.10 0.000 Extraction Temperature 1 253.85 0.000 539.96 0.000 Extraction Time 1 32.37 0.001 12.23 0.010 Square 2 20.86 0.001 28.63 0.000 Extraction Time*Extraction Time 1 0.54 0.484 3.06 0.124 Temperature* Temperature 1 39.26 0.000 56.66 0.000 2-way interactions 1 0.11 0.755 4.88 0.063 Ext. Temperature*Ext. Time 1 0.11 0.755 4.88 0.063 Lack-of-fit 3 1.41 0.363 0.29 0.830

69

Table 4.5. The ANOVA for testing the significance of factors based on the F and p values for DE of pectin. Bold values indicate statistically significant results.

White-flesh DFP Red-flesh DFP Source DF F-value p-value F-value p-value Model 5 4.23 0.043 8.33 0.007 Linear (Main effects) 2 8.13 0.015 17.89 0.002 Extraction Temperature 1 16.19 0.005 35.64 0.001 Extraction Time 1 0.06 0.809 0.14 0.720 Square 2 2.44 0.157 0.06 0.939 Extraction Time*Extraction Time 1 0.13 0.727 0.09 0.771 Temperature* Temperature 1 4.87 0.063 0.02 0.887 2-way interactions 1 0.04 0.856 5.77 0.047 Ext. Temperature*Ext. Time 1 0.04 0.856 5.77 0.047 Lack-of-fit 3 0.02 0.997 0.120 0.943

70

Regression analyses to determine the effects of extraction time and temperature on the yield of pectin were performed by fitting second order polynomials to the experimental data obtained with equations 4.1-4.4. The high coefficients of determination (R2) for extraction yield of 97.91% (white DFP) and 98.87 % (red DFP) indicate that the developed second-order models fit the experimental data quite well. On the other hand, the smaller values of R2 for the DE models (75.14% for white DFP and 85.62 % for red DFP) suggest that the independent factors in the developed model have less relevance in describing the response variations. The significance of each coefficient in the quadratic regression models was evaluated by a t-test. Both extraction time and temperature proved to be significant linear terms (p < 0.05), meaning that these terms should remain in the yield model of red-flesh DFP, but the only quadratic term kept in the model was time. The interaction between extraction time and the temperature had no pronounced effect on yield from red-flesh DFP. In the DE model, the temperature proved to be the only significant linear term for red flesh DFP and was a quadratic term for white-flesh DFP. The interaction effect temperature*time also was a major variable in the DE model for red DFP. These models, expressed in their uncoded forms using only variables determined to be significant, are presented in Equations 4.1 – 4.4 below.

2 2 �������� ��� = −11.27 + 0.391� + 0.0564� − 0.00174� − 0.000731� + 0.000906�� Eq. 4.1

2 2 ������ℎ��� ��� = 37.41 − 1.123� + 0.0274� + 0.00953� − 0.000110� + 0.000203�� Eq. 4.2

2 2 ����� ��� = 44.3 + 0.335� + 0.363� − 0.00166� + 0.000079� − 0.00544�� Eq. 4.3

2 2 ���ℎ��� ��� = −95 + 5.24� + 0.19� − 0.0417� − 0.00067� − 0.00147�� Eq. 4.4

Where T = extraction temperature in °C and t = extraction time in minutes

4.1.4.2. Analysis of factorial plots and response surface plots

From an examination of the surface plots of pectin yield from both types of DFP peel (Figure 4.8), it appears that high pectin yield can be achieved by extracting for 50 minutes at temperatures higher than 75 °C and 80 °C for red DFP and white DFP respectively. The extraction time significantly influenced the yield of pectin extraction from DFP. The raw material needs a certain amount of time to be heated and soften its structure. However, if the extraction occurs for excessive times, the glycosidic bond and the methyl ester of the pectin could be hydrolyzed. The obtained pectin becomes pectic acid, in which galacturonic acid is free from the methyl ester group (Prakash Maran & Prakash, 2015).

71

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 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.

It was observed that increasing temperature had a positive relationship with pectin yield in both types of DFP within the range studied (60 – 80 °C). Higher temperature enhanced the acidic hydrolysis necessary for pectin extraction to increase the pectin yield. Based on ANOVA results, only the temperature has significant effects on yield at the square level for all types of peels. It has been reported that the hydrolysis of protopectin is the rate-limiting step in the extraction of pectin (Minkov et al., 1996). The outcome of the process depends on how fast the protopectin separates from the raw materials. At high temperature, the transfer of protopectin increases, which accelerates pectin extraction. Additionally, increased solvent penetration helps speed up the process (Soria et al., 2014).

72

The interaction plot (Figure 4.9) and surface plot (Figure 4.10) of time and temperature for the pectin DE from red DFP indicate that the effects of temperature on DE are dependent upon the processing time. At low temperature (< 70 °C), pectin extracted over a longer time resulted in a higher DE. However, at a higher temperature, the DE decreased after extended extraction time. From the surface plots of DE response, the highest DE (>65 %) was obtained from red-flesh DFP when the temperature of extraction was lower than 60 °C and the extraction time between 65 to 120 minutes, while for white-flesh DFP the optimum conditions are below 70 °C for 30 to 120 minutes. The DE of pectin extracted from both types of peel was higher than 50%, which indicates high methoxyl content. The ANOVA showed that the extraction time did not affect the DE. Similar results were reported that DE did not reduce significantly with longer processing time (Liew et al., 2015). However, keeping extraction in extended time at elevated temperature could degrade pectin partially because of depolymerization of galacturonan groups in pectin structure (Albersheim et al., 1960).

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.

4.1.4.3. Validation of optimum conditions for pectin extraction from DFP

The regression models developed were used to predict the optimum settings to obtain the maximum yield (Table 4.6). For white-flesh DFP, the predicted optimum conditions were 84 °C and 120 minutes to obtain a predicted pectin yield of 14.13%. Meanwhile, the optimum conditions for red-flesh DFP were 84 °C and 91 minutes to achieve a predicted yield of 15.35%. The experimental values at optimum conditions were quite close to the predicted values, validating the overall adequacy of the prediction models for yield and DE. For technical feasibility, optimum conditions were slightly modified, and the obtained pectin yield and DE were still in accord with the experimental error given (< 10 %).

74

Table 4.6. Experimental testing of mathematical model, optimized for maximum pectin yield and degree of esterification (DE).

Experimental Testing conditions Predicted responses responses Temperature Time Types Yield (%) DE (%) Yield (%) DE (%) (°C) (mins) 84 120 14.13(max) – 14.11 47.86 White 57.40 flesh 80 108 12.00 (max) 12.12 57.06 (max) 84 91 15.35 (max) – 15.07 51.33 Red 58.70 flesh 70 80 12.47 (max) 11.55 53.98 (max)

As predicted, the extraction yield from red flesh dragon fruit (15.07%) was higher than white flesh dragon fruit (14.11%). The pectin yield from dragon fruit peel obtained in previous work varies slightly between authors, e.g., 10.8 % (Jamilah et al., 2011) to 11.57% using citric acid and 13.06% using sulfuric acid (Tang et al, 2011). Liew et al. (2015) also obtained a high pectin yield of 12.56% with DE of 47.88% using an extraction time of 64.67 min at 70 °C for red-flesh DFP at pH 2.37. In other research, the highest yield reported was 26.38% with DE of 63.47% using 1% citric acid at 73 °C for 67 mins at pH 2.03 from the fresh inner layer of the peel; however, this number was only 15% when using the whole dried peel (Muhammad et al., 2014).

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

4.1.5.1. Experimental data, model fitting and statistical analysis

All the experimental runs and results conducted following the central composite design of pectin extraction from purple PFP are presented in Table 4.7, with the corresponding ANOVA results in Table 4.8. The coefficient of determination (R2) was 97.41 % for extraction yield and 89.01% for DE, meaning the regression model fits the actual data quite well. Based on ANOVA results, both the extraction temperature and time had a statistically significant effect on the yield at a linear, square, and interaction level. The DE was impacted only by the extraction time at a linear level but by both

75

conditions at the square level. The quadratic regression models for the pectin yield and DE for PFP are given in following second-order polynomials (Equations 4.5-4.6) based on coded and uncoded variables:

2 2 ����� ��� = – 20.21 + 0.4026� + 0.381� − 0.001473� − 0.001233� − 0.001799��

Eq. 4.5

2 2 �� ��� = 32.70 + 0.513� + 0.323� − 0.002759� − 0.002137� − 0.000932��

Eq. 4.6

Where T = extraction temperature in °C and t = extraction time in minutes

Table 4.7. Central composite design of experiments for pectin extraction from PFP

Time (min) Temperature (°C) Yield (% w/w) DE (%) 43 60 8.56 60.31 107 60 13.95 58.62 43 120 11.95 58.52 107 120 10.43 53.25 30 90 9.12 63.12 120 90 12.56 53.33 75 48 10.45 59.62 75 132 10.98 55.65 75 90 13.51 62.65 75 90 13.65 64.41 75 90 13.02 60.12 75 90 13.92 63.65 75 90 13.84 61.54

76

Table 4.8. The ANOVA for testing the significance of factors based on the F and p values for extraction yield and DE from PFP. Bold values indicate statistically significant results.

Yield DE Source DF F-value p -value F-value p -value Model 5 52.55 0.000 11.43 0.003 Linear (Main effects) 2 12.13 0.005 11.19 0.006 Extraction Temperature 1 6.77 0.035 0.26 0.626 Extraction Time 1 11.83 0.011 21.99 0.002 Square 2 64.29 0.000 13.18 0.004 Ext. Temperature*Ext. Temperature 1 76.17 0.000 16.75 0.005 Extraction Time*Extraction Time 1 69.16 0.000 13.00 0.009 2-way interactions 1 74.42 0.000 1.25 0.285 Ext. Temperature*Ext. Time 1 74.42 0.000 1.25 0.285 Lack-of-fit 3 1.62 0.318 0.73 0.597

77

4.1.5.2. Analysis of factorial plots and response surface plots

The interaction plots for PFP (Figure 4.11), indicates that the effect of temperature on yield is controlled by the processing time. From an examination of the surface plots (Figure 4.12), it appears that high pectin yield can be achieved by setting the extraction temperature lower than 120 °C for longer than 45 minutes. The surface plots also indicate that the highest DE (> 60 %) is obtained when the extraction temperature is between 70 °C and 90 °C and the extraction time is between 45 and 70 minutes.

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.

4.1.5.3. Validation of optimum conditions for pectin extraction from PFP

The maximum predicted and experimental yield and DE of pectin extracted at optimal conditions from PFP are presented in Table 4.9. Pectin extraction from purple PFP has not been previously reported; however, Liew et al. (2015) obtained high yield (14.24 %) with a DE 55.54 % under similar conditions (extraction time of 58.47 min at 70 °C) from yellow PFP.

79

Table 4.9. The maximum predicted and experimental yield and DE at optimized conditions for extraction from PFP. Optimized conditions Predicted responses Experimental responses Temperature Time Yield (%) DE (%) Yield (%) DE (%) (°C) (minutes) 13.53 62.67 86 74.5 13.03 54.95 (max) (max) 13.98 76 99 – 13.18 – (max) 64.41 84 57 – – 54.56 (max)

In previous studies, both yield and DE of pectin from PFP were higher when using nitric acid, i.e., the yield of 16.4-24.9 % with DE 75 % (Canteri et al., 2012). Similar results have been recorded with a yield from yellow PFP of 14.8 % using an extraction temperature of 98.7 °C for shorter extraction times (Kulkarni et al., 2010) and 14.6% yield and 54.78 % DE for 75-minute extraction (Liew et al., 2014). Using hydrochloric acid, even higher yields (29 %) and DE (71.44 %) from yellow PFP have been recorded after 2 hours of extraction at pH 1.34 and 65 ℃ (Simmaky et al., 2014). A higher DE (78.59%) was also achieved from yellow PFP using citric acid at low concentration (0.086 % w/v) (Pinheiro et al., 2008). Nitric and sulfuric acids tend to give higher pectin yields but lower DE pectin from yellow PFP, whereas citric acid solvents give lower yield but higher DE, and ultimately better gelling ability (Yapo, 2009a). The results obtained here show that pectin yield from passion fruit peel is comparable with apple pomace (10 – 15%), citrus peel (20 – 30%) (May, 1990), sunflower head residues (11.60%) (Iglesias et al., 2004), peach pomace (9.68%) (Faravash et al., 2008) and cocoa husks (Mollea et al., 2008).

4.1.6. Conclusion

The conventional extraction process resulted in yields ranging from 5.81 % to 13.11 % for the white-flesh dragon fruit peels (DFP), 8.56 % to 15.12 % for the red-flesh DFP, and 8.56 % to 13.95 % for passion fruit peels (PFP). The Response Surface Methodology has shown that under conventional heating, the extraction conditions and the type of peels have significant influences on the final yield

80

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

To explore the optimal extraction conditions of the microwave-heating method, three factors including the power of microwave irradiation, extraction time and pH, were used as the major independent variables. The effects of microwave power, extraction time, and pH on the yield of extracted pectin were first determined by single-factor experiments as preliminary results for optimization.

4.2.1. Effects of microwave power on pectin yield

Power was varied between 20W and 300W while holding other conditions constant (temperature 80 °C, total extraction time of 5 minutes, pH 2). The changes in pectin yield due to power changes are illustrated in Figure 4.13.

25

20

15 white-flesh DFP 10 red-flesh DFP purple PFP

Yield of pectin (%) pectin of Yield 5

0 0 50 100 150 200 250 Microwave power (W)

Figure 4.13. Effects of microwave power on the pectin yield.

At low power, the yield of pectin was improved with increasing microwave power up to 200 W in all three types of peel. The irradiation of microwaves accelerated the increase of temperature, which causes ruptures in the cell walls to form capillary holes (Seixas et al., 2014). The solvent then quickly penetrates these holes to extract more pectin. The more intracellular space is made due to higher microwave intensity, the more damage occurs to the plant cells. The higher microwave power would also affect the kinetics of the protopectin hydrolysis reaction, also contributing to the increase in recovered pectin. The use of higher microwave power resulted in overheating and leaking of the solution from 250 W to 300 W, and so the yield decreased significantly. Compared to the conventional method, where it took 80-90 minutes to get high yield, using

81

microwave heating, the reaction time can be decreased to only 8-10 minutes to extract a similar quantity of pectin.

4.2.2. Effects of processing time on pectin yield

Processing time using microwave heating was varied between 2 and 14 minutes while holding other conditions constant (temperature 80 °C, microwave power 200W, pH 2). The effects of extraction time on the pectin yield from all three types of peel are illustrated in Figure 4.14. Increasing the time of heating to 14 minutes significantly increased the amount of pectin extracted. More protopectin was hydrolyzed due to longer contact between the raw materials and solvent, leading to an increase in yield. However, longer irradiation time could result in the degradation of pectin molecules, which would lower the yield. For this reason the optimum time was determined to be five minutes.

25

20

15 white-flesh DFP 10 red-flesh DFP

5 purple PFP Yield of pectin (%) pectin of Yield

0 0 5 10 15 Time (minutes)

Figure 4.14. Effects of extraction time on yield

4.2.3. Effects of pH on pectin yield

The pH was varied between 1 and 4 while holding other conditions constant (temperature 80 °C, microwave power 200W, extraction time 5 minutes). The effects of pH on pectin yield are presented in Figure 4.15. From pH 1 to pH 3, the yield of pectin increases significantly before decreasing under less acidic extraction conditions at pH 4. The accelerated yield in the acidic solvent was attributed to the hydrolysis of the protopectin into soluble pectin with minimum degradation (El-Nawawi & Shehata, 1988). The different degrees of solvent acidity also affect the re-adsorption of extracted pectin to the peel matrix during the cooling step (Soria et al., 2014). Also, at higher pH above 3,

82

the pectin fractions can precipitate due to reduced molecular weight, which delays pectin release, and decreases the yield of extraction (Faravash & Ashtiani, 2007).

25

20

15 White-flesh DFP 10 Red-flesh DFP

Yield Yield pectinof (%) 5 Purple PFP

0 0 1 2 3 4 5 pH

Figure 4.15. Effects of pH on yield

4.2.4. Experimental data, model fitting and statistical analysis

All of the experimental results of pectin yield from microwave-assisted heating (Table 4.10) were processed via an ANOVA test to determine the significance of each factor and the developed quadratic model. Previous literature has indicated that the ratio of solvent to peel (liquid:solid ratio) can also affect yield and/or DE, and while it was not found to influence either on its own here, each combination of variables was tested for three different ratios, for comprehensiveness.

83

Table 4.10. Box-Behnken Design of experiments and experimental results.

Variables Responses

Red-flesh White-flesh Passionfruit DFP DFP peel

solid

pH Yield DE Yield DE Yield DE power (W) power Microwave Microwave Microwave time (mins) time Liquid: ratio (mL/g) ratio (%) (%) (%) (%) (%) (%)

5 50 3 50 7.98 21.40 7.60 59.87 13.07 8.73

5 250 3 50 13.83 25.33 10.72 45.35 19.36 54.55

15 50 3 50 12.10 28.01 11.78 43.97 15.28 87.72

15 250 3 50 13.95 32.53 4.59 47.37 19.68 86.97

10 150 2 70 11.23 11.68 7.05 45.75 5.67 57.53

10 150 2 30 3.26 11.69 3.04 50.65 9.36 54.38

10 150 4 70 5.32 23.23 5.55 40.37 6.39 60.89

10 150 4 30 6.72 47.79 5.44 33.33 4.63 22.83

5 150 3 70 4.08 28.75 7.05 35.15 9.27 68.22

5 150 3 30 8.94 23.49 8.07 30.43 16.38 18.26

15 150 3 70 6.22 34.67 12.56 36.77 10.06 55.26

15 150 3 30 13.77 44.75 4.48 47.36 17.24 17.93

10 50 2 50 3.24 15.99 3.64 42.92 6.24 57.02

10 50 4 50 4.92 27.98 5.11 49.73 5.02 54.62

10 250 2 50 6.74 24.3 6.66 45.99 9.96 17.93

10 250 4 50 7.59 23.15 5.53 30.93 7.09 14.99

84

Variables Responses

Red-flesh White-flesh Passionfruit DFP DFP peel

solid

pH Yield DE Yield DE Yield DE power (W) power Microwave Microwave Microwave time (mins) time Liquid: ratio (mL/g) ratio (%) (%) (%) (%) (%) (%)

5 150 2 50 4.20 - 3.99 54.31 11.09 4.88

5 150 4 50 3.69 22.45 3.04 31.55 5.82 56.65

15 150 2 50 6.03 - 5.61 45.99 10.25 62.86

15 150 4 50 6.38 30.77 6.61 29.61 6.74 46.43

10 50 3 70 4.23 15.96 7.96 36.83 7.13 47.75

10 50 3 30 9.83 24.35 7.50 35.73 12.15 79.66

10 250 3 70 6.82 30.64 5.96 36.66 11.34 73.38

10 250 3 30 11.66 49.53 12.56 48.56 18.10 87.07

10 150 3 50 17.13 40.06 15.62 41.47 19.03 64.32

10 150 3 50 16.82 35.25 16.72 42.92 18.26 67.14

10 150 3 50 17.79 37.22 15.00 40.54 18.57 62.98

The ANOVA showed that the p-values of the developed model for yield were below 0.05 and that the model was, therefore, a good fit for pectin yield from all three peels; however, the model of DE was only significant for the red-flesh DFP (Table 4.11). The p-values of lack-of-fit tests were greater than 0.05. The fitness of the second-order polynomial equation was also indicated by the determination of the high values of R2. The R2 values for a developed model for yield were highest in PFP, which indicated that the model explained most of the total variation (95.62 %) and only 4.38 % of the variation was unexplained, followed by the yield model for red-flesh DFP (92.13%) and white- flesh DFP (86.22 %). For the DE model, the R2 value observed for red DFP was 83.58 %.

85

Table 4.11. The ANOVA for testing the significance of factors based on the F and p-values for the yield and DE of pectin extracted by the microwave-assisted method. Only statistically significant and lack-of-fit results included.

Extraction yield DE of pectin Red-flesh DFP White-flesh DFP Purple PFP Red-flesh DFP Source F-value p-value F-value p-value F-value p-value F-value p-value Model 10.03 0.000 5.36 0.030 18.73 0.000 3.27 0.040 Linear (Main effects) 9.69 0.001 – – 16.73 0.000 3.75 0.046 Extraction Time 5.91 0.032 – – – – 5.24 0.048 Microwave power 8.00 0.015 – – 23.69 0.000 5.57 0.043 pH – – – – 9.52 0.009 – – Liquid:solid ratio 24.84 0.000 – – 33.13 0.000 – – Square 24.18 0.000 15.58 0.000 48.40 0.000 – – Extraction Time 19.74 0.001 17.68 0.001 – – – – Microwave power 17.46 0.001 13.90 0.003 8.37 0.013 – – pH 87.80 0.000 57.24 0.000 172.98 0.000 7.46 0.023 Liquid:solid ratio 34.21 0.000 16.43 0.002 35.37 0.000 - - 2-way interactions – – – – – – – – Time*power – – 5.85 0.032 – – – –

86

Extraction yield DE of pectin Red-flesh DFP White-flesh DFP Purple PFP Red-flesh DFP Source F-value p-value F-value p-value F-value p-value F-value p-value pH*liquid:solid ratio ------7.51 0.023 Lack-of-fit 16.95 0.057 6.99 0.132 19.68 0.049 8.55 0.109

87

For pectin yield from red DFP, the liquid:solid ratio had the greatest impact, followed by microwave power and processing time. For the quadratic terms, all factors have significant p-values indicating a curve in the response surface. Only time and microwave power were significant linear terms affecting the DE of pectin extracted from red-flesh DFP. The pH quadratic term and pH×(liquid:solid ratio) interaction term also affected the DE. In terms of white-flesh DFP, the only significant p values (<0.05) were those for the quadratic terms of processing time, microwave power, pH, and liquid:solid ratio.

The interaction term of time×microwave power also greatly affected the yield of pectin. The yield model of PFP was affected by the microwave power, the pH and the liquid:solid ratio at both linear and square levels. The significance of the squared terms in the model indicates there is a curvature in its response surface. The second-order models for yield and DE (Equation 4.7 – 4.10) were expressed using the designed experimental data and presented as below in terms of coded factors.

2 2 2 2 ������−�ℎ��� ��� = −113.8 − 0.1553�1 − 0.000344�2 − 6.985�3 − 0.00935�4 −

0.00516�1�2 Eq. 4.7

2 ������−��� ��� = −126.3 + 2.97�1 + 0.1475�2 + 1.509�4 − 0.1436�1 − 2 2 2 0.000338�2 − 7.574�3 − 0.01182�4 Eq. 4.8

2 2 ������−��� = −104.2 + 0.0926�2 + 53.53�3 + 1.187�4 − 0.000198�2 − 8.999�3 − 2 0.01017�4 Eq. 4.9

2 ���−��� ��� = −233 + 7.29�1 + 0.393�2 − 8.81�3 + 0.0003�1�2 Eq. 4.10

Where Y is pectin yields/DE of pectin, X1, X2, X3, X4 are extraction time (mins), microwave power (W), pH, liquid:solid ratio respectively.

4.2.5. Analysis of interaction plots and response surface plots

4.2.5.1. Effects of microwave power and processing time on pectin yield

Microwave power exhibited a greater positive impact on pectin yield from red-flesh DFP and PFP. The cell wall of the peel is loosened, expanded, and broken to release the intracellular material due to microwave radiation in a short time. The reason is that the amount of energy transferred to materials to be converted to heat energy is controlled by the incident microwave power (Ma et al., 2009). The sample container was closed, so the pressure also increased in response to the microwave energy applied. Therefore the penetration and delivery of solvent into the cell can be enhanced by the microwave

88

irradiation energy (Yan et al., 2010). However, if the microwave radiation was further boosted, the yield was reduced. The superfluous energy may result in thermal degradation of the pectin and reduce the pectin recovery (Maran et al., 2015).

The irradiation time is a significant factor that influences only the yield of pectin from red-flesh DFP. The ANOVA indicated that the extraction of pectin was enhanced in longer irradiation time. This might be the required time for the peels to be exposed to the medium so that the solvent could penetrate the material to dissolve the pectin and then diffuse out for extraction (Samavati, 2013). However, the yield was reduced when the solution was exposed to the microwave activation for longer times due to the degradation of pectin chain molecules (Zheng et al., 2011). In polar solvents overheating can occur quite easily because of the comparatively high dielectric properties, which could cause the degradation of the compounds (Lucchesi et al., 2007).

The interactive effect of microwave power and processing time exhibited a significant influence only on the yield from white-flesh DFP (Figure 4.16). The interaction plot shows that under lower microwave power and prolonging irradiation time, an enhancement of the yield is observed during the first 12 minutes, and a decrease in yield is recorded following longer irradiation time. At a higher power levels, maximum extraction is achieved during the first 8 minutes and then significantly dropped. The 3D response surface plots were graphed to visualize the relationship between the significant factors and the responses, and are presented in Figure 4.17.

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).

Other reports have highlighted that with microwave-assisted extraction, temperature becomes a significant parameter affecting the process (Thirugnanasambandham et al., 2014). The authors found that increasing temperature from 35 to 45 °C led to higher pectin yield, but the yield is lowered at temperatures higher than 45 °C. The difference is potentially due to the applied duration and other experimental parameters: the authors used microwave-assisted extraction processing between 5 and 25 mins. The same

90

conclusion was reached with temperatures in the range from 30 °C to 70 °C, with the yield decreasing following further increase in temperature (Zaid et al., 2016a). This suggests that raising temperature beyond 70 °C could expedite yield, however operating a system at such a high temperature will be more costly for practical scale-up and industrial integration.

4.2.5.2. Effects of pH and liquid:solid ratio on pectin yield

The pH is another significant factor that influenced the yield on a linear level in PFP and square levels in all types of the peel. A higher pH value within the range of 2 – 4 presented positive influences on pectin extraction (Figure 4.18). The high acidity at pH 3 enhanced the hydrolysis of protopectin into soluble pectin to be easily extracted (Prakash Maran et al., 2013). The aggregation of pectin could occur at pH higher than 3, and this slowed down the pectin release (Prakash Maran et al., 2015). In terms of the DE, only the pH×(liquid:solid ratio) interaction term (Figure 4.19) and the quadratic term of pH (Figure 4.20) had a significant effect.

91

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

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

The yield from red-flesh DFP increased significantly with increasing liquid:solid ratio, up to around 56 (mL/g), however further increasing the amount of solvent reduced the yield of pectin extracted from PFP. The solvent is required in appropriate amounts to ensure complete immersion of the peels. The solvent then can absorb microwaves causing the plant cells to swell, leading to the cell walls rupturing and releasing the pectin into the medium (Guo et al., 2001). However, excessively large volumes of solvent decreased the pectin yield, most probably related to the fact that homogeneous heat dissipation was more difficult (Eskilsson et al., 1999). Larger amounts of solvent also compete with the peel powder for the absorption of microwave energy. In microwave heating, the inadequate stirring of larger solvent volumes may also contribute to lower pectin yield (Wang & Weller, 2006).

93

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

The maximum predicted and experimental yield and DE at optimized microwave- heating conditions for the three types of peels are summarized in Table 4.12. For all three types of peel, the microwave-assisted method gave significantly higher yield in a shorter lapse of time compared with conventional heating. In all cases though, the DE of the freshly extracted pectin was lower than what is observed with materials obtained by conventional heating. Pectin extracted using the microwave method can thus be be classified as low-DE pectin.

Table 4.12. Predicted and experimental yields and DE at optimized conditions

L:S Type of Time Microwave Predicted Yield pH ratio DE (%) peel (mins) power (W) Yield (%) (%) (mL/g)

Red DFP 12 183 2.97 56 18.24 17.01 36.25

White 10 153 3.00 50 15.79 13.22 30.25 DFP PFP 12 218 2.91 57 20.00 18.73 49.13

The microwave-heating extraction was previously conducted to extract 7.5% pectin at 45 °C for 20 minutes under the power of 400W for red-flesh DFP (Thirugnanasambandham et al., 2014). In another study with red-flesh DFP, a pectin yield of 18.53% was achieved with a DE of 46.95% at pH 2.07 for 65 s with liquid:solid ratio 66.57 (mL/g) at 800W (Rahmati et al., 2015). Other extraction methods have been attempted and reported in the literature, some of which can achieve higher yields still, but at the expense of pectin quality.

4.3. Conclusion and comparison with conventional heating

The optimum conditions determined in the previous chapter are summarised in Table 4.13. The pectin extracted from passion fruit peels by microwave heating exhibited the highest yield. The optimal conditions adopted from the models gave the highest pectin yield (18.73 %) from passion fruit peel: extraction time of 12 minutes, microwave power of 218 W, pH of 2.9 and liquid:solid ratio of 57:1 mL/g. Under the same operating

94

conditions, the yield of pectin obtained by the conventional heating method was only 13.58%.

The pectin from PFP exhibited higher purity than pectin from DFP in both conventional and microwave-assisted heating.Microwave irradiations have the intrinsic property to generate high temperatures in a short amount of time and this can accelerate the intermolecular interactions and molecular motion within the solvent that supports the solubility of materials. In addition, the high temperature also increases cellular pressure, eventually rupturing the cell and improving extraction efficiency. Overall, temperature gradients will also produce variations in the viscosity of the reaction mixture which in turn will affect heat transfer (Fatemeh & Rezvantalab, 2015) and pectin mobility and solubility (Khajeh et al., 2010). The microwave-assisted method is promising because it is suitable for high moisture materials and is easy to scale up. The microwaves specifically target water, and due to the high internal moisture of fruit waste, this will lead to damage of the cellular structures. As a result, irradiation could assist extraction at lower temperatures compared with conventional heating, and the extraction process could be reduced to only minutes. The microwave-assisted extraction method also allows for strong inorganic acids to be replaced with weak organic acids from natural and renewable sources, such as citric acid (Attard et al., 2014), making the process much more sustainable and environmentally-friendly. Microwave scale-up has valuable practical potential, with relatively low maintenance fees, low energy usage, small equipment size and ease of operation (Li et al., 2012).

Table 4.13. Optimum extraction conditions used for both heating methods to recover pectin for analysis

Type of peel Extraction method and conditions White- Red-flesh Purple PFP flesh DFP DFP Conventional Temperature (°C) 84 84 76 heating Time (minutes) 120 91 99 Microwave power (W) 153 183 218 Microwave- Time (minutes) 10 12 12 assisted pH 3 3 2.9 heating Liquid:solid ratio 50 56 57

95

Chapter 5: Properties of extracted pectin

Pectin is a biopolymer of α-galacturonic acid, an oxidized form of D-galactose, with a variable number of methyl ester groups. The composition and chemical properties of pectin are affected by the type of plant and extracting conditions. It is used as an additive in various applications in the food, pharmaceutical and cosmetic industries, and the increased with increasing market demands. The extraction processes of pectin from dragon fruit and passion fruit peels by conventional and microwave-assisted heating are described in Chapter 4. A survey of the literature shows that the intrinsic properties of the extracted pectin have not been extensively investigated. The molecular structure and functional properties of the resulting pectin could be affected when different processing conditions are applied. Therefore, this chapter aims to explore the effects of extraction conditions on the DE, DA, DM, equivalent weight, methoxyl content, structural and functional properties of pectin and to compare these with standard commercial product. The materials extracted under optimum conditions were used to investigate fundamental properties and compare with commercial pectin.

5.1. Physicochemical properties

5.1.1. Moisture content

Moisture content is one of the indicators of quality for pectin and important to consider when storage is concerned. Table 5.1 summarizes the moisture contents for pectins extracted from different types of peels, both by conventional and microwave- assisted heating. As anticipated, the drying method shows a non-negligible impact on the moisture content of the extracted pectins. It is generally accepted (FAO, 2009) that the maximum moisture content should be below 12 %. The freeze-drying step produced pectins with lower moisture contents, which may offer the advantage of longer shelf lives. Lower moisture content inhibits the growth of microorganisms, some of which may produce pectinase which would affects the quality of pectin (Mohamadzadeh et al., 2010). Statistical analysis of the results indicates no significant variations in the moisture contents of pectin extracted by each heating method from different types of materials.

96

Table 5.1. Moisture contents of pectin recovered from three types of peels.

Extraction Moisture content (%) Type of peel methods Vacuum Drying Freeze Drying White-flesh DFP 4.63 ± 0.70 3.93 ± 0.56 Conventional Red-flesh DFP 4.96 ± 0.17 3.76 ± 0.29 heating Passion fruit peel 5.57 ± 1.19 2.35 ± 0.59 Microwave- White-flesh DFP 5.66 ± 0.20 3.39 ± 1.81 assisted Red-flesh DFP 5.31 ± 0.71 4.18 ± 1.30 heating Passion fruit peel 4.26 ± 0.87 2.86 ± 0.26 Citrus Pectin* 3.35 ± 0.19 *Commercial, tested as-received

5.1.2. Equivalent weight (Eq. W) and methoxyl content

Eq. W and methoxyl content are critical indicators of the gel-forming ability of pectin: a higher Eq. W or methylation increases the capacity to form gels (Vaclavik et al., 2008). Table 5.2 summarizes the Eq. W and methoxyl content of pectins extracted by both heating methods. It is noteworthy that the Eq. W of materials from microwave- assisted heating were higher than those obtained by the conventional method, this could be due to the partial degradation of pectin.

Table 5.2. The equivalent weight and methoxyl content of pectin from three types of peel using both heating methods.

Equivalent weight Methoxyl content Method Type of peel (Eq. W) (%) White-flesh DFP 938.5 ± 11.5a 7 ± 0.14cd Conventional Red-flesh DFP 993.6 ± 12.8ab 7.17 ± 0.18bcd heating Passion fruit peel 1067.1 ± 31.5b 7.85 ± 0.26ab White-flesh DFP 1297.2 ± 37.8c 6.1 ± 0.17e Microwave- Red-flesh DFP 1393.6 ± 49.8cd 6.52 ± 0.02de assisted heating Passion fruit peel 1502.6 ± 18.6d 7.24 ± 0.19bc *Citrus pectin 1403.0 ± 8.51cd 8.21 ± 0.18a *Commercial, tested as-received

97

The methoxyl content of pectin extracted from PFP by conventional heating (7.85 %) was quite close to the reference commercial pectin (8.21 %). The materials recovered from different types of peels and under various extraction conditions were categorized as low methoxyl pectins, as they were around 7 % or lower, meaning that they can form gels at lower concentrations compared to the high methoxyl equivalent (Castilo-Israel, 2015). The free hydroxyl group (-OH) from natural sugars in pectin structure could be methylated to methoxyl groups (-OCH3) during extraction.

In comparison to conventional heating, microwave heating significantly ruptures cell walls, resulting in pectin with denser structure (Su et al., 2019). During conventional heating, the wall of the container must be heated first, before it will transfer heat to the samples (Figure 5.1). This results in longer processing times, and the extracted materials are subjected to more hydrolytic reactions that directly impacts on the equivalent weight of the pectin. Moreover, in microwave-assisted extraction there are minimal temperature gradients across the sample, while in conventional heating the heat transfer is reliant on convection. There is a possibility that some parts of the mixture will be exposed to higher temperatures, which could damage and destroy thermolabile compounds. In the microwave-assisted method, the material absorbs the microwave energy directly, leading to more evenly distributed and efficient heating (Soria et al., 2014).

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

98

5.1.3. Degree of esterification (DE)

The DE also was studied using FTIR spectra analyzed by Origin software to calculate the corrected peak areas at around 1650 cm-1 (COO-asymmetric stretching) and at abour 1735 (C=O esterified) using following equation 5.1 (Faravash et al., 2008): � DE (%) = 1735 ×100 Eq 5.1 (�1735+�1650) The degree of esterification of recovered pectins were recorded in the range of 42 % to 62 %, as reported in Table 5.3. For conventional heating, the ANOVA showed that for DFP the temperature had the highest influence on the DE pectin while for PFP the most critical parameter was extraction time. In microwave-assisted heating, the DE from red DFP was significantly affected by reaction time and microwave power rather than the pH and the solvent:peel ratio, i.e. liquid:solid ratio (see Chapter 4). The highest DE pectin was obtained from PFP processed by conventional heating (61.975 %) while the pectin from white DFP by microwave had the lowest (41.955 %). When comparing all peels treated by conventional heating to batches treated by microwave, it is obvious that the latter treatment produced lower DE pectin. As a convention in this report, extracted samples with DE lower than 50 % will be categorized as low-methoxyl pectin, as per Hosseini et al. (2016). Table 5.3. The degree of esterification of pectin extracted at optimum conditions.

Extraction Experimental DE (%) Type of peel method Titration FTIR Red-flesh DFP 54.61 ± 0.93c 49.32 Conventional White-flesh DFP 57.23 ± 0.38b 48.70 heating PFP 61.98 ± 0.74a 54.59 Red-flesh DFP 45.03 ± 0.45d 36.25 Microwave- White-flesh DFP 41.96 ±0.81e 30.25 assisted heating PFP 53.27 ± 0.38c 49.13

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

The composition of pectin plays a critical role in their functional properties, and of particular importance are the overall carbohydrate content and galacturonic acid (GalA) content, specifically. A standard curve of D-glucose was constructed with a coefficient

99

of correlation of 0.9951 and regression equation y = 2.5913x + 0.0013, to calculate the total carbohydrate content. The total carbohydrate contents of extracted pectin are summarized in Table 5.4.

Table 5.4. The D-galacturonic acid content in extracted samples.

Extraction Total carbohydrate Type of peel GalA Content (%) methods content (% w/w) White-flesh DFP 69.73 ± 3.82bc 63.50 ± 2.66bc Conventional Red-flesh DFP 79.38 ± 2.18ab 69.46 ± 2.51abc heating Passion fruit peel 80.73 ± 4.64ab 78.09 ± 3.05a Microwave- White-flesh DFP 74.56 ± 1.91abc 65.20 ± 1.58bc assisted Red-flesh DFP 64.91 ± 4.09c 60.10 ± 1.14c heating Passion fruit peel 79.19 ± 3ab 71.39 ± 2.95ab Citrus Pectin 87.09 ± 4.37a 66.27 ± 4.43bc

Pectin extracted from passion fruit peels by conventional heating exhibited the highest total carbohydrate content of 80.73 % by mass. The galacturonic acid (GalA) content is generally an indicator for purity of pectin and is determined by the standard D- galacturonic. According to Food Chemical Codex (1996), for an extract to be categorized a pectin this value should not be less than 65 %. A standard curve of D-galacturonic acid was constructed with a coefficient of correlation of 0.9504 and regression equation y = 0.0006x – 0.0082. Overall, the results show that the galacturonic acid (GalA) contents of pectin extracted by microwave-assisted heating are slightly lower than conventional heating (Table 5.5). These results also indicate that in conventional extraction, extended times can cause further degradations of neutral side chains, leading to a higher fraction of galacturonic acid, which is in agreement with reported data from yellow passion fruit peels (Seixas, et al., 2014). Pectins from passion fruit and white-flesh dragon fruit peels heated by microwave had similar proportions of GalA to those resulting from conventional heating. The statistical analysis also showed no significant difference in GalA content of commercial pectin compared to white-flesh DFP or PFP processed by microwave heating. This is an important point, as microwave-assisted heating can be an effective and economical process in scaling-up, with less time required to extract pectin with high quality. Lower values of GalA suggest the presence of higher amounts of protein, starch and/or sugars,

100

as reported for pectin extracted from yellow PFP by a moderate electric field (Oliveira et al., 2015). Variations in the content of GalA can be attributed to the solvent, plant species and physiological development of the plant (Lin et al., 2016). 5.1.5. Degree of amidation (DA)

The degree of amidation has a major impact on the gelling properties and the elasticity of the low-methoxyl pectins, and the overall properties of pectin could be modified if the methoxyl groups are converted to amide groups (Lootens et al., 2003). The DA values of pectin extracted by conventional and microwave-assisted heating from three different types of peel are shown in Table 5.5.

Table 5.5. The degree of amidation of extracted pectin from two heating methods

Extraction methods Type of peel Degree of amidation (%) White-flesh DFP 10.29 ± 0.08c Conventional heating Red-flesh DFP 9.31 ± 0.31cd Passion fruit 12.70 ± 0.10b White-flesh DFP 6.96 ± 0.68e Microwave-assisted heating Red-flesh DFP 8.58 ± 0.09d Passion fruit 15.06 ± 0.09a Citrus pectin 16.26 ± 0.34a

The pectin recovered from white-flesh DFP had the lowest DA with about 6.96 %, while the highest value was observed for commercial pectin (16.26 %). Dragon fruit pectin extracted by conventional heating exhibited higher DA than the one from microwave-assisted heating. Previous studies on dragon fruit peels reported values of 10.8 % when extracting with citric acid, and 12.9 % when using sulfuric acid (Tang et al., 2011). For passion fruit peels pectins, the DA results obtained for conventional heating were lower with microwave heating. The less amidated low-methoxyl pectin enhances the gelling ability due to less calcium needed for gel formation (Tang et al., 2011) The amidated pectins also reduce the sensitivity to pH of gel formation (Tang et al., 2011). 5.1.6. Fourier Transform Infrared spectroscopy

5.1.6.1. The infrared profile of pectins extracted at optimum conditions

The absorption band between 4000 and 3000 cm-1 is ascribed to -OH stretching vibration, and the broad band is caused by inter- and intra-molecular hydrogen-bonding

101

of the D-galacturonic acid unit in the backbone (Figure 5.2). Moisture absorbed both on the surface and in the bulk likely contribute here. The sharp peaks in the region 2800 to -1 3000 cm corresponds to the C-H stretching and bending vibration of CH, CH2 and CH3. It can be observed that the C-H region from 2500 to 3600 cm-1 of pectin has two moderately intense bands including the C-H stretching and bending vibrations overlapping the broader O-H band. Therefore, the O-CH3 stretching band of the methyl esters of galacturonic acid in esterified pectins (between 2950 and 2750 cm-1) is masked.

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

Absorption bands around 1735 cm-1 and 1650 cm-1 are respectively due to the carbonyl functional groups (C=O) of methyl-esterified carbonyls (-COOCH3) and carboxylate anions (COO–) stretching vibrations (Kyomugasho et al., 2015). The same authors report that the intensity of the ester carbonyl peak relative to the carboxyl peak

102

can indicate the degree of esterification. For example, it was observed that the DFP pectin had a weak COO– absorption band accompanied by a strong C=O absorption band, indicating a relatively high DE. The absorption bands in the fingerprint region of 800 to 1300 cm-1 are generally assigned to monosaccharides present in pectin. The bands between 1100 and 1200 cm-1 were ascribed to the ether R-O-R and cyclic C-C bonds. The similar fingerprint region of the reference citrus pectin indicates that the recovered material can be categorized as belonging to the pectin group (Nep et al., 2016). The effects of extraction method on the structural characteristics of pectin, as evidenced by FTIR, are reported in Figure 5.2. The main functional groups present on the polysaccharide chains of extracted pectin were confirmed by FTIR analysis. The weak absorption peak at 2951 cm-1 present in all samples could be attributed to C-H stretching vibration from the CH2 and CH3 groups (Pasandide et al., 2017). It is hypothesized here that microwave irradiation, due to the electromagnetic nature, might cause longer chains to fracture, leading to higher C-H signals as more CH2 and CH3 groups are freed and exposed.

The FTIR spectra of pectin from PFP show characteristic peaks in the region of 2361- 2336 cm-1 corresponding to the N-H stretching of the amidated groups (Kumar & Chauhan, 2010). The weaker stretch at 1735 cm-1 band and a more intense one at 1630 cm-1, resulting in a lower ratio of the peaks, were observed in all pectin samples treated by microwave. This could be a consequence of more efficient heat distribution in the reaction mixture, which would increase susceptibility to acid hydrolysis. This will increase the peak area of non-esterified carboxyl groups compared to esterify carbonyl groups, hence the lower DE in these samples. The absorbance was consistent with the degree of esterification conducted by titration method.

5.1.6.2. Effect of extraction conditions on the structure of pectins

The FTIR spectra of pectin extracted from three types of peels by microwave-assisted heating are presented to assess the difference of major functional groups at different pH (Figure 5.3). For PFP and red DFP pectin, it is observed that at higher pH the absorbance peak of the esterified carboxyl groups (around 1630-1600 cm-1) is reduced while the non- esterified carboxyl group increased, indicating decreasing DE values. Similar results have been reported for apple (Yang et al., 2018) or pomelo peels (Liew et al., 2018). An

103

opposite trend was observed here, however, for white-flesh DFP pectin. The latter showed a sharper peak assigned to the non-esterified carboxyl groups at a low pH of 2 and with the peak gradually disappearing when the pH increased to 4.

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

104

The effects of the amplitude of the energy involved in microwave irradiations on the structural properties of derived pectin are presented in Figure 5.4, where three power settings were evaluated. The spectra showed no major differences in the main features with the various powers use, implying little to no changes in the structure and properties of the pectin.

a)

b)

c)

Figure 5.4. FTIR spectra of pectin recovered from (a) white-flesh DFP, (b) red-flesh DFP and (c) PFP with different microwave power (50 W, 150 W and 250 W).

105

The FTIR spectra of the pectins extracted with varying liquid:solid ratio are reported in Figure 5.5 to evaluate the effects of this ratio on their functional structures. Pectins extracted with lower liquid:solid ratio had a lower and wider peak at around 1630 cm-1 corresponding to the vibration of free carboxylic groups (-COOH). A similar trend was observed from the red-flesh DFP at the absorptions between 1100 and 1200 cm-1, which correspond to the ether R-O-R and cyclic C-C bonds (Liu et al., 2010).

Figure 5.5. FTIR spectra of pectin recovered from (a) white-flesh DFP, (b) red-flesh DFP and (c) PFP with different liquid:solid ratio

106

The FTIR spectra of samples extracted over different extraction times from PFP are presented in Figure 5.6. All spectra showed strong absorbance at intervals of 1730-1760 cm-1 for esterified carboxylic groups and 1600-1630 cm-1 for carboxylic groups. Similar, previously published spectra for white-flesh DFP also illustrate the difference in the absorptions around 1100 and 1200 cm-1, due to the vibration of ether R-O-R and C-C ring of the backbone (Venzon et al., 2015). In both cases, the spectra of samples extracted over a shorter time had a higher absorbance in the range of 1012-1030 cm-1 because of the distension of the C-O-H group.

15 minutes

Absorbance 10minutes

5 minutes

2000 1750 1500 1250 1000 750 500 -1 Wavenumber (cm )

Figure 5.6. Effect of extraction time on pectin structure from PFP.

5.1.7. Scanning Electron Microscopy (SEM)

The microstructure and morphology features of pectins extracted by conventional heating and microwave-assisted heating are illustrated in Figure 5.7. The images of extracted pectins exhibit the pectin samples after freeze-drying and vacuum drying. For pectins extracted by conventional heating, the vacuum-dried samples presented an irregular, granular-shaped structure and rough surface with wrinkles, however, the freeze- dried samples had a flat and smooth surface. For samples from microwave-assisted heating, all freeze-dried samples showed a smoother and more compact structure than those from conventional heating. During the microwave-assisted heating, smaller bubbles appeared on the surface of pectin due to heating for a shorter time. The morphology of pectin could be affected by the structural properties, the purity, the randomness of flocculation and the drying method. The microwave-assisted heating following by freeze- drying obtained the better morphology feature of extracted pectin.

107

Figure 5.7. SEM images of extracted pectins from different types of peel using conventional heating or microwave-assisted heating, followed by vacuum drying or freeze drying

5.1.8. BET surface area

The surface and porosity of extracted pectins was determined by N2 adsorption/desorption isotherms. The representative adsorption isotherms of each type of extracted pectins are shown in Figure 5.8. The textural properties of surface area, total pore volume and average pore diameter are summarized in Table 5.7. The PFP pectin had a higher BET specific surface area, which is generally attributed to higher microporosity

108

and smaller pore diameter. The pore size of white-flesh DFP pectin was the smallest among the extracted pectins however, suggesting that the more critical factor determining surface area was overall porosity. The behavior of these pectins is typical of an aerogel exhibiting large pores and high surface area, and according to the classifications from the International Union of Pure and Applied Chemistry, they behave like macroporous substances.

Figure 5.8. N2 adsorption/desorption isotherms of commercial and extracted pectins

109

Table 5.6. The porosity of extracted pectins analyzed with N2 adsorption/desorption porosimetry

Total Type Type BET Desorption Extraction desorption of of surface average pore methods cumulative peel drying area (m2/g) width (nm) volume (cm3/g) DFP VD 208.9 0.17 5.15 Conventional VD 364.7 0.33 5.30 heating PFP FD 273.7 0.23 5.12 VD 354.5 0.33 5.30 Microwave- DFP FD 210.4 0.23 5.86 assisted VD 309.0 0.28 5.30 heating PFP FD 365.2 0.33 5.31 Citrus pectin 322.1 0.29 5.32 VD: Vacuum Drying; FD: Freeze Dying DFP: White-flesh dragon fruit peels; PFP: Passion fruit peels The microwave-assisted heating following by freeze-drying obtained the better morphology feature of extracted pectin. The high frequency of microwave fieldis converted into heat causing intensive vapor formation in the capillary-porous of the peel materials. As the result of these changes, the higher surface area were measured in most of pectin samples obtained by microwave heating.

5.1.9. X-ray diffraction (XRD)

X-ray diffractometry was used to determine the crystallinity of pectin extracted from PFP, as presented in Figure 5.9. The commercial pectin and pectin extracted from PFP exhibit similar X-ray diffraction patterns and show characteristics of amorphous materials.

110

Figure 5.9. XRD patterns of the commercial pectin (top) and pectin extracted from PFP by conventional heating (middle) and microwave-assisted heating (bottom).

The X-ray diffractogram of the commercial pectin shows one major feature at 21.1° (2�) while for the pectins extracted from fruit peels, although the shape of the broad peaks at low values of 2θ are similar, the exact positions are significantly different. Two characteristic diffraction peaks of extracted pectins are observed at 21.1° and 27.3° (2�), which are partially crystalline regions (Lutz et al., 2009). Compared with the sample prepared by microwave-assisted heating, the pectin extracted by conventional heating is seen to be more crystalline than that of the microwave-assisted material, with the presence of an extra diffraction peak at 21.3° (2�).

One possible explanation would be that the process of crystallization is affected by the temperature profiles of the system. With conventional heating, as it has been pointed out previously, heat distribution across the sample is occurring at a slower rate by convection and thermal equilibrium is reached after a longer period of time. In turn, this will allow individual components of the mixture to settle and hence achieve a more ordered structure. Also, higher localized temperature due to the heating process may create pockets of enhanced crystallinity.

111

The differences in the X-ray diffractograms reflects varying degrees of crystallinity, or long distance order. The disruption of hydrogen bonds and hydrophobic forces between pectin molecules during the hydrolysis process will initiate chains separation, hence a more disordered structure. Therefore, it could be suggested that microwave-assisted heating might slightly damage the crystallinity of pectin in comparison with conventional heating. However, the characteristic diffraction peak at 2� of 27.3° of pectin extracted by microwave heating was sharper with higher intensity than these from conventional heating (46 au and 29 au respectively). Similar results have been reported for pectin extracted from orange peels (Hosseini et al., 2016), stepped hawthorn wine (Jiang et al., 2018) and waste heads from Helianthus annus by ultrasound (Ponmurugan et al., 2017).

5.2. Functional properties of pectin

The functional behaviors and gelling capabilities of pectin are some of the most critical parameters to evaluate in order to categorize and decide pectin applications. The chemical and physical structure of pectin significantly affect its solubility. Clumps can be formed when dried pectin powder is added into the water, as semi-dry particles inside highly hydrated coatings (Sriamornsak, 2003). The solubility of pectin subsequently determines its swelling, hydration and degradation rate, which contribute to its ability to perform as an efficient encapsulating material, to be discussed in detail in the next chapter. Therefore, the correlation between the chemical and physical properties of extracted pectin and their solubilities is analyzed in the following section.

5.2.1. Solubility

The solubility of commercial and extracted pectins from different types of peels and extracting condition are shown in Table 5.8. The solubility was not significantly affected by the pH except for the PFP pectin extracted by microwave-assisted heating. In general it is acknowledged that pectin exhibits maximum solubility levels at pH higher than 4 (Vaidya et al., 2009). In basic media however, when the pH is higher than 7, the amount of negatively-charged free (COO-) groups increases, engendering greater repulsive charge forces between the macromolecules, and a weaker gel is formed per unit concentration of cationic cross-linker.

112

Table 5.7. The solubility of pectin extracted from various materials by conventional heating and microwave-assisted heating. Extraction Type of Solubility (%) at the desired pH method peels pH 5 pH 7 pH 9 W-DFP 2.87 ± 2.12ab 2.97 ± 0.07ad 3.00 ± 0.06ac Conventional R-DFP 3.17 ± 0.09ab 3.12 ± 0.06acd 3.20 ac heating PFP 2.11 ± 0.02bc 2.15 ± 0.02be 2.30 ± 0.06ad Microwave- W-DFP 3.26 ± 0.11ab 3.51 ± 0.07ab 3.59 ±0.12ab assisted R-DFP 3.31 ± 0.07ab 3.40 abc 3.46 ab heating PFP 4.20 ± 0.09a 4.36 ± 0.14a 4.33 ± 0.01a Citrus pectin 3.30 ± 0.14ab 3.37 ± 0.07abc 3.46 ±0.06ab

W-DFP: White flesh DFP; R-DFP: Red flesh DFP; PFP: Passion Fruit Peels Superscripts indicate statistically significant differences.

For the same conditions of pH, the trends observed suggest that the degree of solubility is dependent on the source of the pectin, with some influences from the nature of the treatment process involved. Although these differences are sometimes minor, the implications on the applicability of the material as a gelling agent can be crucial. For example, DFP-pectin obtained by microwave treatment exhibits similar solubility threshold to commercial pectin. The pectin extracted from passionfruit peels by conventional heating were the least soluble while the PFP-pectin isolated via microwave processing exhibited the highest solubility. The pectin extracted by microwave-assisted heating and further processed by freeze-drying had high porosity with high surface area, which may facilitate pectin particles to be more easily dissolved. According to Matveev et al. (2000), the hydrophilic groups in pectin such as hydroxyl, carboxyl and amide groups, can form hydrogen bonds with water molecules thereby enhancing solubility. The higher solubility of lower DE and amidated pectins observed in our study can be explained on this basis. Both hydrogen bonding and hydrophobic interactions between methoxyl groups in pectin structures have the potential to restrict the hydrophilic groups from being exposed to water molecules. In high DE pectins, the hydrophobic area formation parallel to the helix axis may contribute to reduce solubility (Sriamornsak, 2003). Other particle properties including the shape, size

113

distribution, surface properties and physical state also affect the water uptake. The inhomogeneous surface morphology of the particles can enhance water uptake when comparison with smooth and homogenous particles (Kastner et al., 2012). Smaller particle size, higher porosity and surface area could facilitate greater solubility of pectin (Begum et al., 2017) As observed in the SEM images, pectin particles from PFP have inhomogeneous surfaces and differ significantly to the other pectins, which enhanced its solubility. The effect of the physical state of pectin particles on their solubility can be investigated by analyzing their crystalline state. All extracted pectin exhibited a mostly amorphous structure (Figure 5.9) which tends to promote the water permeation to access the hydrophilic groups. In contrast, the compact nature of crystalline materials causes a delay in the uptake of water, as the material first transforms into a soft rubbery gel that then facilitates hydration (Panchev et al., 2010).

5.2.2. Oil-holding capacity (OHC)

The oil-holding capacity of the fibre reflects the ability of the material to retain oil, and these values for the recovered and commercial pectins are summarized in Table 5.9. The oil holding capacity will affect the applicability of pectin as an additive in high-fat food products to provide greasy sensation. The pectin obtained from PFP by microwave- assisted extraction exhibited a higher oil-holding capacity than all of the other extracted and commercial pectins. For all types of peels the pectin extracted by conventional heating had a significantly lower OHC than pectins obtained by microwave-assisted methods (p < 0.05). The oil absorption might be affected by the total charge density and the resulting hydrophilic properties (Fleury & Lahaye, 1991).

The chemical structure, thickness, the hydrophobic nature and the affinity to the oil of the polysaccharides also influences the OHC (Viuda‐Martos et al., 2010). The small amount of protein in pectin samples, which show both hydrophilic and hydrophobic nature, could support the OHC of pectin. The OHC values reported here are similar to pectin from Averrhoa bilimbi extracted using special ionic solvents (Shafie et al., 2019). This OHC is lower than that reported for pectin from the pod of Parkia speciosa (3.9 g/g) (Gan et al., 2010) but greater than from cladodes of Opuntia ficus indica (1.24 g/g) (Bayar et al., 2016).

114

Table 5.8. Oil-holding capacities of extracted pectin compared with commercial pectin Extraction methods Type of peel Oil-holding capacity (g oil/g sample) White-flesh DFP 2.61 ± 0.01cd Conventional heating Red-flesh DFP 2.80 ± 0.04bc Passion fruit 2.29 ± 0.14d White-flesh DFP 2.71 ± 0.13bc Microwave-assisted Red-flesh DFP 3.07 ± 0.18b heating Passion fruit 3.54 ± 0.03a Citrus Pectin 2.84 ± 0.04bc Superscripts indicate statistically significant differences.

5.2.3. Foaming properties

The dispersion of gas bubbles (the dispersed phase) in pectin solution (the continuous phase), described as the foaming capacity, was determined for all extracted pectins (at 2 % w/v). In food processing, a foam is a sought after structure/texture in aerated food products as it improves the consistency and sensory evaluation of final food products or palatability. The foaming capacity (FC) and foaming stability (FS) of pectins are reported in Table 5.10. The FC is described as the ability of a surface-active agent to maintain a two-phase system of air and water. Pectin extracted from PFP by microwave-assisted heating presented the highest foaming capacity but the lowest stability in comparison with commercial pectin.

Table 5.9. The foaming properties of extracted and commercial pectin Extraction Foaming Foaming stability (%) Type of peel methods capacity (%) 30 minutes 60 minutes White-flesh DFP 47.25 ± 1.06a 37.00 ± 0.71a 23.75 ± 0.35b Conventional Red-flesh DFP 39.25 ± 0.35b 31.50 ± 1.41b 27.75 ± 1.06a heating Passion fruit 33.50 ± 0.71c 25.50 ± 0.71c 19.50 ± 0.71c Microwave- White-flesh DFP 22.00 ± 0.71e 16.50 ± 1.41d 10.75 ± 1.06d assisted Red-flesh DFP 26.00 ± 0.71d 16.75 ± 1.06d 11.25 ± 0.35d heating Passion fruit 34.35 ± 1.20c 18.50 ± 0.71d 21.75 ± 1.77bc Citrus Pectin 15.75 ± 0.35f 10.25 ± 0.35e 9.75 ± 0.35d Superscripts indicate statistically significant differences.

115

As reported in the literature, the interfacial properties of the surface-active components significantly affect the foaming formation and stability of the materials (Dickinson, 2003). The presence of protein fractions in pectin samples typically leads to good interfacial and foaming properties. The PFP pectin also had higher viscosity (see section 5.4.5), which contributes to prolonging the colloidal stability and increasing the FS (25.5 % after 30 minutes and 19.5 % after 60 minutes). Pectin forms a high viscosity solution that contributes to postponing the gas bubble coalescence to increase the FS. Therefore, pectin is described as a thickener or stabilizer because of its highly hydrophilic properties (Mokni Ghribi et al., 2015). The higher foaming properties of extracted pectin (the highest FC in white-flesh DFP 47.25 %) than commercial pectin (15.75 %) could widen their applications in typical foam-type food products with aerated structure. Beside the extraction method, the DE and average molecular weight are also known to determine the FS of pectin (Bayar et al., 2017). Literature findings of FC/FS have been varied, for example, pectin from Opuntia ficus indica cladodes (FC/FS: around 55 %/75 %) (Bayar et al., 2016), eggplant peels pectin extracted by microwave-assisted heating (17.13 %/4.61 %) (Kazemi et al., 2019b) and eggplant peels pectin extracted by ultrasonication (14.33%/9.67 %) (Kazemi et al., 2019a).

The improved foaming properties of the pectins extracted here compared to the commercial pectin could widen their applications in typical foam-type food products with aerated structure.

5.2.4. Emulsifying properties

Polysaccharides with highly hydrophilic natures such as pectin are described as non- surface-active agents, but are often applied as a stabilizer for edible oil-in-water emulsions in food products. Their emulsion capacity is determined by methods such as surface activity, water-holding and thickening characteristics (Mohos, 2010). The emulsion capacity (EC) and stability (ES) of two immiscible liquids including pectin solution and oil was determined at different pectin concentration (Table 5.11). The oil emulsified and aqueous phases were obtained aft er centrifuging. Emulsion stability is facilitated by the maintenance of a polymeric protective layer at the interface of a stable emulsion network during storage and heating treatment (Wu et al., 2009).

116

Table 5.10. The emulsifying properties of extracted pectin and commercial pectin.

Extraction Emulsion Emulsion stability Type of peel methods capacity After holding After incubation White-flesh 46.97 ± 1.38c 82.21 ± 1.86ab 68.56 ± 2.22bc DFP Conventional Red-flesh 40.32 ± 0.77d 74.28 ± 2.99cd 66.35 ± 4.19bc heating DFP Passion fruit 54.75 ± 0.41a 71.41 ± 2.16d 59.26 ± 0.60c White-flesh 47.87 ± 0.57bc 78.54 ± 0.30bc 76.07 ± 0.14ab Microwave- DFP assisted Red-flesh 39.43 ± 1.15d 70.73 ± 1.03d 69.56 ± 4.64bc heating DFP Passion fruit 41.65 ± 1.16d 83.64 ± 0.80ab 85.04 ± 5.36a Citrus Pectin 50.65 ± 3.45b 86.74 ± 1.08a 85.15 ± 0.80a Superscripts indicate statistically significant differences.

The EC is inversely proportional to the pectin concentration, as an increase is observed with decreasing pectin concentrations, which could be due to the hydrophobic regions of the protein components (Perez et al., 2012). At low concentrations, the exposure of hydrophobic protein regions may be facilitated due to the repulsion between proteins and polysaccharides (Perez et al., 2012). It has been suggested that these hydrophobic regions may be hindered in accessing the oil component of the mixture as a result of the establishment of the protein-polysaccharide matrix at higher polysaccharide concentration (Ganzevles et al., 2006). The emulsion capacity of pectin from PFP obtained through conventional heating was highest (54.75 %) and comparable to the commercial product (50.65 %). However, the emulsion stability of the same material (71.41 %) was the lowest when compared with other types of pectins and commercial product (86.74 %). With regard to microwave- assisted heating, the PFP pectin was able to effectively stabilize emulsions (83.64 %) even though its emulsion capacity was lower (only 41. 65 %). The pectins extracted in our study exhibited greater EC than that of pectins from Opuntia ficus indica (35 %) (Bayar et al., 2016) and from Averrhoa bilimbi (26.67 %) (Shafie et al., 2019).

117

The pectin structure, molecular weight and galacturonic acid content are significant parameters impacting on the functional property of pectin as a thickening agent. The presence of other biomolecules such as proteins, with both hydrophilic and hydrophobic functional groups in their side-chains, could also affect the emulsion capacity and stability of pectin (Huang et al., 2001). The hydrophobic characteristics of proteins in the pectin may decrease the interfacial tension between water and oil droplets by preferentially associating with the oil droplets, while the polysaccharides orient toward the aqueous continuous phase (Dickinson, 2003). The presence of arabinose and galactose in pectin have also been determined to enhance the emulsion capacity. In terms of thermal stability, emulsions prepared from commercial pectin were more stable than those prepared from extracted pectin after incubation at high temperature. The increasing temperature may accelerate the solubility of pectin, therefore there were more interactions of pectin in oil-water phase to cause a stable emulsion. The emulsion stability increased at low temperature with higher molecular weight and galacturonic acid concentration. The structure of pectin network with side chains prevented the oil droplets coalescence. It also could generate a charged surface, which would delay the oil droplets coalescence due to the repellence of particles (Wu et al., 2009). The methyl groups and the hydrophobic nature of the acetyl groups in highly acetylated passion fruit pectin could also support the emulsion stability (Dea & Madden, 1986). The commercial pectin with better gelling properties was able to generate a more stable emulsion after heat treatment than other pectins. 5.2.5. Rheological properties

The deformation and flow of matter as rheological properties of pectin suspension were studied. The rheological behaviors of pectin solutions contribute to the shape they take in containers, the way they should be handled and processed, quality control and sensory assessment of fluid and semi-solid foods. Food products with high polymer concentration can exhibit both viscous and elastic properties (Rao, 2014). yield and better quality of extracted pectin from PFP by microwave-assisted heating; only the PFP was chosen for testing rheological properties.

5.2.5.1. Estimation of flow curves

The shear stress vs. shear rate diagrams for solutions of pectins extracted by microwave heating from PFP and commercial pectin at three different concentrations (1

118

%, 3 % and 5 %) are presented in Figure 5.11. Experimental data of flow curves were fitted to the power-law fluid model since shear stress can be correlated with the shear rate using the following equation: � = �� � Eq. 5.1 Where σ is the shear stress (Pascal); K is the flow consistency index (Pa ��); � is the shear rate (s-1) and n is the flow behavior index. The flow curves of all solutions are very well described by the models with high coefficients of determination (R2).

100 y = 0.2303x0.681 y = 0.0162x0.8311 R² = 0.9596 R² = 0.9874 10 y = 2.3055x0.5268 R² = 0.987

1 y = 0.0088x0.9738 R² = 0.9923 Shear Shear (Pa)Stress 0.1 y = 0.0139x0.899 R² = 0.9807

0.01 0.001 0.01 0.1 1 10 100 1000 10000 Shear rate (1/s)

PFP pectin 1% PFP pectin 3% PFP pectin 5% ST pectin 0.5% ST pectin 1% Power (PFP pectin 1%) Power (PFP pectin 3%) Power (PFP pectin 5%) Power (ST pectin 0.5%) Power (ST pectin 1%)

Figure 5.10. Influence of shear rate on the shear stress of PFP pectin extracted by microwave-heating, and standard (ST) commercial citrus pectin at 25 °C. The dotted lines represent the linear fit based on the Ostwald-de Waele power-law model (Rao, 2007). At low pectin concentrations, the relationship between shear stress and shear rate, was an almost linear, indicating Newtonian-like flow behavior of the pectin solutions with n approaching one. At higher concentrations, based on the n values of fluids lower than one, the samples were classified as pseudoplastic fluids. All samples were shear-thinning fluids as the viscosity decreased when the shear rate increased (Rao, 2014). The lower n coefficient for the 5% compared with the 1% solution means a higher concentration of pectin increases pseudoplasticity. The flow behavior of pectin solution from PFP is similar to pectins from dragon fruit, apple and citrus (Muhammad et al., 2014). The longer extraction time and higher temperature typical of large-scale

119

commercial productions of pectin could result in less compact pectin structure with decreasing molar mass and lower intrinsic viscosity (Fishman, 2006).

5.2.5.2. Determination of viscosity

Steady shear rate sweeps were performed to study the correlation between viscosity and pectin concentrations (Figure 5.12). The results show that for all solutions the apparent viscosity was affected by the shear rate at different concentrations.

100 PFP pectin 1%

10 y = 2.6094x-0.43 PFP pectin 3% R² = 0.9778 PFP pectin 5% 1 ST pectin 0.5%

ST pectin 1% 0.1

Apperant viscosity Apperant viscosity (Pa.s) 0.01

0.001 0.001 0.01 0.1 1 10 100 1000 10000 Shear rate (1/s) Figure 5.11. The apparent viscosity of PFP pectin solution extracted by microwave heating at 1, 3 and 5 %, measured at 25 °C.

In comparison to standard pectin at 1%, the apparent viscosity values obtained for PFP pectin were of similar order of magnitude. In a 1% solution, the viscosity of the solution dropped slowly as the shear rate increased and exhibited Newtonian fluid properties, whereas pseudoplastic flow becomes dominant at higher concentrations. At low pectin concentration, the weak interactions between pectin molecules due to homogeneous dispersion might explain this trend. Increasing linear shear stress coupled with reducing viscosity at increasing shear rates is indicative of shear-thinning behavior (Rao, 2014). These changes were because of the reorganization of pectin molecules to align with the flow movement. Therefore, the pectin intermolecular forces at greater shear rate were also weaker. At the same pectin concentration, the apparent viscosity of the microwave-extracted sample was slightly higher than that obtain by the conventional method. This might be due to the lower methoxyl content leading to smaller molecules and larger space between them.

120

At 3 % and 5 % solution, clear shear-thinning behavior can be observed, with the latter exhibiting the highest viscosity because of the development of three-dimensional networks. Higher concentrations reduce distance and enhance intermolecular interactions, and as a result more energy is required to break the pectin network (Kar, 1999). Our results are in agreement with reported literature stating the existence of three dominant phases of viscosity, depending on pectin concentration: dilute (0-1 %), semi- dilute (1-2.5%) and concentrated (2.5-3%) (Hua et al., 2015). In the food industry, high shear-thinning behaviors can impart ease of pumping and thinner consistency during swallowing of liquid food (Rao, 2014). The different macromolecular compositions of the sample cause the difference of character in the viscosities (Kontogiorgos et al., 2012). The shear-thinning behavior of the 5% suspension of passion fruit peels pectin is similar to the 1% solution of many industrial-grade thickeners such as xanthan, locust bean gum and guar gum (Montoya-Arroyo et al., 2014). There are many different parameters affect the viscosity of pectin including solubility (Srivastava et al., 2011), pectin concentration and temperature (Kar & Arslan, 1999). The parameters which cause a decline in solubility will increase viscosity, including the degree of methylation, molecular weight, counter-ions present in solution and also pH (Srivastava et al., 2011; Sriamornsak, 2003). 5.2.5.3. Oscillatory measurements-gelling properties

Strain sweep tests were performed at 0.1 Hz to determine the upper limit of the linear viscoelastic region and subsequently, the strain of 2 % was selected for further tests. Under these conditions, the deformation returns to the original structure. The elastic and viscous moduli (G’ and G”) refer to the properties of viscoelastic materials. The pectin solutions with concentrations from 3 % to 5 % were applied for frequency sweep tests (Figure 5.13). Suspensions with lower pectin concentrations had no viscoelasticity as a result of low viscosity. At sufficiently high concentrations, pectin solutions exhibit both solid and liquid characteristics simultaneously. This dynamic behavior was explored to determine the viscoelastic properties and the state of interactions of the pectin chains. The results indicated liquid-like behavior in all concentrations throughout the frequency range tested, i.e. G’ < G”.

121

100

10

1

0.1

PFP pectin 5% G'

G', G" (Pa) G" G', 0.01 PFP pectin 5% G" 0.001 PFP pectin 3% G' PFP pectin 3% G" 0.0001 0.1 1 10 100 Frequency (rad/s) Figure 5.12. Frequency sweeps (25 °C; fixed strain at 2 %) of pectin solution (3 and 5 %) from PFP by microwave-heating.

5.2.5.4. Gelling properties

The strengths of PFP pectins gel in the presence of various calcium concentrations are illustrated in Figure 5.14. The values of G’ were greater than G” throughout the tested frequency range, indicating typical gel-like behavior (G’ > G”). This means that there were enough intermolecular interactions between the polymer chains to create a weak gel network structure. The pectin gel exhibited a greater storage modulus G’ at the higher calcium concentration. The fragility of Gel 2 at lower calcium concentration might be due to a lower amount of junction zones and longer average distances between chains, which may limit the water retention (Ako, 2015).

122

a) 10000 Gel 1 G' Pa G" Pa tan_delta 100

1 0.1 1 10 100 G', G", tan_delta G", G', 0.01 Frequency (rad/s)

b) Gel 2 1000 G' Pa G" Pa 100 tan_delta

10

1

G', G", tan_delta G", G', 0.1 1 10 100 0.1 Frequency (rad/s)

Figure 5.13. Mechanical spectra of pectin-calcium mixtures with storage (G’) and loss (G”) moduli as a function of frequency for pectin 1 %, pH 7, 0.5 M NaCl with a) 15 mM

CaCl2 and b) 12 mM CaCl2.

The gel network is formed by the water immobilization in the filamentous networks of pectin macromolecules (Yapo, 2015). Therefore, the gel network is affected by the water inclusion in the system and the water retention from the gel network. The extracted pectin from PFP was low-methoxyl pectin, which forms a gel by the ionotropic gelation with divalent cations forming the “egg-box” model (Fang et al., 2008). This model is formed between Ca2+ and non-esterified galacturonic acid units through an oxygen atom in the carboxylate group in the ring, and the glycosidic bond and via the hydroxyl group of the next unit (Wellner et al., 1998). It requires a pH higher than the pKa value of pectin (3.5) for a proper number of disassociated carboxyl groups (-COO-) to form ionic junction zones with divalent cations (Kastner, 2012). The mechanical properties and stability of formed gels vary on the structure of junction zones, the structure and concentration of pectin, sugar concentration, gelling agents, temperature and pH (Einhorn-Stoll, 2017). The degree of methylation, degree of acetylation, the degree of amidation, molecular

123

weight, and the heterogeneity of pectin chains affect greatly on the gelling (BeMiller, 1986). The amidated pectin includes the acid amide groups besides the acid and ester groups at C-6, which decrease the amount of charge GalA residues. Therefore, the amidation enhances the gelling of LM pectins due to the less of needed calcium ions for the gelation. Moreover, the hydrogen bonds formed between amides groups could enhance the strength of the gel network at low pH (Lofgren, 2006). The amidated pectin has a lower charge density compared to non-amidated pectin. The lower DM increases the calcium ion binding capacity due to the higher number of sequences of non-methoxylated GalA units. In consequence, the forming gel also is stronger. The block-wise distributions of ester groups can also increase the egg-box formation and gel strength than a random distribution (Fraeye, 2010). Decreasing molecular weight on the other hand could decrease the strength of the gel network due to the lower number of Ca2+ binding sites and energy of interaction (Fraeye, 2009). 5.3. Conclusion

The extracted pectin by microwave-assisted heating from all types of peel had higher equivalent weight and similar galacturonic acid content compared to thoes from conventional heating. However, the highest DE and total carbohydrates was observed from pectin isolated from PFP by conventional heating (61.98% and 80.73 % respectively). Structural assessment by FTIR spectroscopy demonstrated that the extracted pectin was very similar to commercially available citrus pectin, and would be classified as low-methoxyl pectin. Pectin extracted by microwave had higher surface area and typical amorphous structure, and was highly soluble across a wide pH range (5-9). It also had higher oil-holding ability, foaming capacity but lower foaming andemulsifying capacity under thermal treatment. Increasing pectin concentration produced solutions with enhanced viscosity, suggesting that it may be suitable for use in a variety applications for its gelling properties.

124

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

6.1. Overview

The discovery of probiotics has resulted in many significant applications to develop and improve functional foods in modern food processing. If insufficient amounts of probiotic are consumed, they will not exhibit the prospective health benefits to the gut and internal organs, as well as the overall well-being of the host. The severe conditions of food processing, product storage, the gastrointestinal system and antimicrobials are the main reasons of the probiotic losses. Encapsulation has been developed to reduce the loss of probiotic until releasing the surviving cells in the appropriate gastrointestinal regions Pectin has been determined as a biodegradability, biocompatibility and non-toxicity, which widen their applications as biomaterial, especially in probiotic encapsulation. The pectin microcapsules provide a proper enclosed environment and a physical barrier outside to improve the number of viable probiotics. Pectin can also adhere well to the large intestinal mucosa, further increasing its popularity for encapsulation. Therefore, this chapter focuses on the study of pectin as probiotic-encapsulating material to minimize the loss of probiotic in severe conditions.

6.2. Growth curve of probiotic

The growth curve of selected probiotic were studied to determine the exponential (log) phase that will be applied to all subsequent experiments. The result for Lactobacillus casei for an incubation period of 25 hours is reported in Figure 6.1. The main characteristics are a 6-hour lag phase period followed by an 11-hour exponential phase; in all, the stationary phase was finally reached after an incubation period of 17 hours at 37 °C. All cultures were refreshed twice in MRS broth before subsequent experiments. The growth profile was very similar to those of other lactobacilli strains in milk (Lin & Young, 2000).

125

Figure 6.1. Growth curve of L. casei in MRS broth at 37 ℃ under 100 rpm agitation.

6.3. Prebiotic activity score

Prebiotics are non-digestible components that preferentially favours the growth and activities of beneficial bacteria in colon (Roberfroid, 1993) . The effect of pectin (0.5 %) from each type of peels on the growth of L. casei in MRS broth was assessed before measuring their prebiotic score (section 3.7.2). The growth rates of probiotic in the presence of different types of pectin are reported in Table 6.1.

Table 6.1. Effect of pectin on the growth of L. casei probiotic

Type of pectin OD (660 nm after 48-hour incubation) Commercial pectin 1.952 ± 0.038a White-flesh DFP 1.651 ± 0.089b Red-flesh DFP 1.982 ± 0.036a PFP 1.541 ± 0.035b Control (MRS broth without added pectin) 1.002 ± 0.083c

Values are mean of three replicates ± standard deviation. Different lowercase superscript letters within the same column indicate significantly different values at p <0.05. The major observation was that all samples favored the growth of L. casei in MRS broth, with a maximum growth of, as evidenced with an optical density of 1.982 obtained

126

with pectin from red-flesh DFP, which is in close agreement with the commercial sample. The selective stimulation of the bacterial growth and the activities of extracted pectins determined their prebiotic activity score.

Prebiotics benefit host’s health by reducing pathogen flora and increasing bifidobacteria populations in gut microflora (Mussatto & Mancilha, 2007). The prebiotic activity scores of different types of pectin for L. casei are reported in Table 6.2. After 24- hour and 48-hour incubations, the L. casei grown on MRS broth with added pectin from red-flesh DFP scored highest (0.137 and 0.096 respectively). Lower values were observed for white-flesh DFP and commercial pectin, while the lowest score was only 0.013 after 24-hour and 0.004 after 48-hour incubations with purple PFP pectin. The prebiotic activity score remained unchanged after incubation for 24 hours with commercial pectin, but it nearly doubled for the white-flesh DFP while it significantly reduced for red-flesh DFP and purple PFP.

Table 6.2. The prebiotic activity score of pectin for L. casei

Prebiotic activity score Samples 24 hours 48 hours White DFP 0.027 ± 0.011b 0.046 ± 0.009b Red DFP 0.154 ± 0.022a 0.096 ± 0.021a Purple PFP 0.013 ± 0.002b 0.004 ± 0.001c Commercial pectin 0.035 ± 0.001b 0.037 ± 0.001b

Values are mean of three replicates ± standard deviation. Different lowercase superscript letters within the same column indicate significantly different values at p <0.05. The differences in monosaccharide types, the degree of esterification and the chain structure of pectin might cause the differences in prebiotic activity score (Zhang et al., 2018) . The utilization of pectin as a carbon source was affected by the degree of esterification. The fermentation of highly methylated pectin was slower than of lower methylated pectin, which caused lower growth rate. The higher prebiotic scores were attained with lower methylated pectin due to them being more easily fermented. Therefore, the oligosaccharides derived from pectin should be better candidate prebiotics. These scores were lower than for another commercial prebiotic FOS products (Huebner et al., 2007).

127

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

The pectin gel particles were prepared by extrusion of the pectin solution into a solution of calcium ions to prevent droplet breakup. As the pectin droplets come into contact with the calcium solution, the Ca2+ cations caused them to gel (Figure 6.2). The production of Ca-pectin-probiotic beads are affected by many factors including extrusion technique, pectin properties; pectin solution properties, gelation formulation, the properties of probiotics and the post-production treatment (Lee et al., 2013). In this study, only the properties of pectin and its solution were varied to determine their effects on the dimensions and shape of the capsules.

Figure 6.2. The formation mechanism of pectin gel particles by “egg-box” calcium linked junctions. Calcium cations form junctions between free acid groups on adjacent pectin chains (Lara-Espinoza et al., 2018).

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

The beads produced had relatively homogeneous spherical shapes, were translucent, white, and light pink color when using commercial pectin, DFP pectin and PFP pectin, respectively (Figure 6.3). The beads produced from PFP pectin were least regular in shape; these different shapes may be due to different contributing patterns in the molecular chains (Zhao et al., 2018). The binding sites of PFP pectin for Ca2+ decreased with higher degree of esterification, which hinder the gel formation.

128

Figure 6.3. Pectin beads produced with 2 % w/v of (a) commercial pectin; (b) pectin extracted from DFP; (c) pectin extracted from PFP

The collecting gap, viscosity and concentration of the pectin solution are known to affect the shape that is formed (Lee et al., 2013). Lower viscosity pectin solutions produce droplets that are more flattened. The effect of pectin concentrations on the shape of microbeads is reported in Table 6.3. In this study, all types of pectin were gelled at various concentrations, starting at 0.5 % w/v. For concentrations lower than 1.5 % w/v no spherical Ca-pectin capsules could not be formed. This is in agreement with literature (Seifert & Phillips, 1997) , which the author attributed to the fact that at these low concentrations the viscous force within the droplets is lower than the drag forces upon collision with the surface of the Ca gelling solution, resulting in the disruption or deformation of the droplets. Therefore, a concentration of 2 % w/v was selected for further work to investigate the viability of probiotics.

129

Table 6.3. Observed shapes of gelled capsules at various pectin concentration from different types of pectin.

Type of pectin Pectin concentration (% w/v) Shape 0.5 Dissolveda 1 Undefinedb Commercial pectin 1.5 Tear/Irregular 2 Sphere 0.5 Dissolved 1 Undefined DFP pectin 1.5 Irregular 2 Ellipsoidal 0.5 Dissolved 1 Undefined PFP pectin 1.5 Irregular 2 Tear-shaped a Droplet did not gel on entering CaCl2. bDroplet shapes were non-homogeneous and varied greatly.

In the food industry, small beads sizes are desired as they lead to faster diffusion rate, resulting in higher processing yields and productivity. The diameter and size distribution of Ca-pectin-probiotic beads in wet and dried capsules measured at 2 % w/v pectin solution from different types of pectin are reported in Figure 6.4. The gelled capsules ranged from 1.87 to 2.41 mm depending on the source of the pectin, with the largest sizes observed for gelled particles produced from DFP pectin. After freeze-drying, the bacteria- loaded capsules were smaller, ranging from 1.35 to 1.88 mm in diameter.

130

Figure 6.4. The distributions of pectin particle sizes (a, b, c) and freeze-dried capsules (d, e, f) loaded with probiotics, prepared using different type of pectin (2 %w/v): (a,d) commercial pectin, (b,e) DFP pectin and (c,f) PFP pectin.

The sphericity factor (SF) of Ca-pectin probiotic beads was determined due to its significant influence on the strength of the capsules, the release of the core materials and the aesthetic quality (Lee et al., 2013) . None of the pectins produced capsules with defined shapes at low concentrations (1 % w/v), however at 2 % (w/v) commercial pectin capsules were the most spherical. The highest SF and size distributions were observed with PFP capsules.

The low DE of all samples resulted in more binding sites for Ca2+, which facilitated the gel formation and the generation of capsules (Zhao et al., 2018). The commercial pectin with a higher DE, and therefore fewer binding sites, required fewer calcium ions to fill the cavities and achieve equilibrium. The increasing chain stiffness due to low viscosity from lower DE of DFP pectin was also an important factor in delaying the gelation and morphological changes when the droplets reached the gelling bath. Amidation of the pectin could further enhance the size and mechanical strength of the

131

capsules because it supports the gel-forming ability (Thakur et al, 1997). The gel strength has been reported to increase due to formation of hydrogen bonds between amide groups. It has been reported that larger capsules has the potential to enhance the viability of encapsulated cells, however, these dimensional properties could also negatively affect the texture and sensory evaluation of the capsules (Heidebach et al., 2012).

6.4.2. Scanning Electron Microscopy (SEM)

SEM images were recorded to investigate the morphological characteristics of gel capsules. The morphologies of the gel beads were examined after drying under the same conditions for all types of gel beads (Figure 6.5).

Figure 6.5. Structures of surface of different types of gelled beads with the same magnifications with scale bars 2 �m: (a) commercial pectin beads, (b) DFP pectin beads, and (c) PFP pectin beads.

The rod-shaped bacterial cells are clearly seen on the surface. The moisture loss during drying caused shrunken beads with wrinkles on their surfaces. The control samples without probiotic cells, however, had smooth surfaces (Figure 6.6). The surface of the probiotic-loaded PFP pectin gel beads appeared rougher than those of the DFP or commercial beads suggesting that more bacteria could be exposed at the surface. Some of the probiotics present at the surface of the beads might be more susceptible to harsher environmental conditions, however, the integrity of L. casei cells was confirmed in a cross-section of the dried beads, indicating that the bacteria were entrapped within the beads (Figure 6.6e).

132

Figure 6.6. Structures of gelled beads from commercial pectin with different magnifications: (a) free cells probiotics (20 �m); (b): blank pectin beads (100 �m); (c) & (d) & (e): pectin beads loaded with probiotics (100 �m and 2 �m).

6.4.3. Chemical structure by FTIR

The variations in superficial functional structures of pectin-probiotic capsules were monitored by FTIR spectroscopy through comparison with blank pectin beads (Figure 6.7). The spectra of probiotic capsules included peaks at around 1670 cm-1 and 1220 cm-1 indicative of the amide and phosphate groups in the cell walls of the bacteria (Vaziri et al., 2018). The absorption band at 1607 cm-1 corresponded to the stretching vibration of the carboxyl group of the carboxylate ion (COO–), which decreased compared with the structure of pectin in solution. The reason for this was the electrostatic interactions between COO– and Ca2+ “egg-box” model to form beads (Wellner et al., 1998). The asymmetric stretching of carboxylate ions is delayed due to strong electrostatic interactions, which led to a significant decrease in intensity of the peak at 1607 cm-1. The stretching vibration of the C=O group around 1735 cm-1 also weakened, further indicating the binding of calcium cations to the carboxylate anions. The symmetric vibrations of the carboxylate group COO– at 1414 cm-1 remained unchanged after the ionic bonds formation (Noh et al., 2018). The FTIR spectra of cell-loaded capsules included an increasing absorption band at 3600-3100 cm-1 and 1620 cm-1 corresponding to the hydroxyl groups and carboxyl group stretching respectively.

133

Figure 6.7. The FTIR spectra of different types of pectin capsules, presenting the changes of functional structure of pectin to blank pectin capsules and when loading with probiotic and after storage.

Calcium concentration is also a critical parameter in determining the microstructure of low-methoxyl pectin gel. The difference in functional structure of pectin-Ca gels were also observed at different Ca concentrations by FTIR spectroscopy (Figure 6.8). The asymmetric stretching of carboxylate groups, centered at around 1730 cm-1 is shown to progressively decrease in intensity as the concentration of Ca2+ increases. At higher calcium concentrations more egg-box crosslinks are formed, producing tighter structures, as illustrated in Figure 6.9.

134

Figure 6.8. The FTIR spectra of capsules with different calcium concentration as gelling agents (CP1: the highest [Ca2+] 20 mM to the CP4: the lowest [Ca2+] 10 mM).

a) b)

Figure 6.9. Schematic representation of the effect of calcium concentration on crosslinking between galacturonic acid units with calcium ion. Monomers and short chain pectins (a) do not effectively form egg-box crosslinks at [Ca2+] 20 mM, whereas longer chain pectins (b) can form more links with comparatively less [Ca2+] 15 mM.

6.5. Microencapsulation efficiency

Three different types of pectin were used as starting materials to form the gel beads as a protection for the probiotic cells. The effect of pectin concentration on the total viable cells after single-layer coating is reported in Figure 6.10. In terms of decrease in the number of bacterial cells, the least effective capsules were those produced using pectin

135

from white DFP, at a concentration of 1%. These showed the highest loss of 3.15 log CFU/mL of total viable cells. There was only a 0.32 log CFU/mL reduction in bacterial population when being microencapsulated with commercial pectin at 2 % concentration. Generally though, between the different types of pectin at the same concentration, the PFP pectin had the lowest protective ability.

The survival of probiotic cells after encapsulation was improved for each type of pectin as the concentration was increased. Larger capsules with higher pectin concentration generally give better protection than those with lower concentration, by forming a better barrier to cell desiccation (Sabikhi et al., 2010). More spherical beads also support the survival rate of probiotics due to their minimal surface area. Additionally, the positive prebiotic properties of pectin can stimulate the growth and multiplication of the cells that do survive, potentially making up for those lost.

136

Figure 6.10. Effect of pectin concentration on viable cells by single-pectin coating. Remaining cells in free pectin solution and after capsule formation are both compared to the original culture

6.5.1. Double coating

Probiotics may sometimes be double-coated with encapsulation material in order to better protect them from gastrointestinal conditions. The efficiency of double coating of probiotics with pectins was also studied here (Figure 6.11). The double coating did not show significantly higher protecting ability to the probiotic survival in comparison with the single–coated samples. The double coated beads were treated with more processing steps might harm the cell wall. A slightly higher survival rate of probiotic cells in double- coated capsules was observed only for those prepared using PFP pectin. The FTIR spectrum (Figure 6.12) does not show much difference in the surface functionalization of the beads, which is probably due to the thickness of the double coat.

137

CP DFP PFP Type of pectin Figure 6.11. Effect of double coating in comparison with single coating at the pectin concentration of 2 % to encapsulate probiotics.

CP-probiotic bead (double coating)

CP-probiotic bead (single coating) Absorbance

Probiotic cells

4000 3000 2000 1000 0 -1 Wavenumber (cm ) Figure 6.12. The FTIR spectra of double-coated pectin capsules in comparison with single-coated pectin capsules and free probiotic cells.

6.5.2. Survival of encapsulated cells under simulated gastrointestinal conditions

Probiotics should remain viable throughout the gastrointestinal system until they reach their active site in the colon (Yeung et al., 2016) . For this reason, the viability of free and encapsulated cells as they pass through different conditions within the gastrointestinal tract was investigated. The viability of probiotics in different pectin

138

capsules after transiting through simulated salivary fluids was examined and illustrated in Figure 6.13.

Figure 6.13. The viability of probiotics encapsulated in different types of pectin (2 % w/v) in simulated salivary fluids.

The viability of free probiotics after incubation was unchanged and remained around

8.2 log10 CFU/mL. There were slight reductions in the number of viable cells observed in encapsulated cells, however these differences were small, and may be within experimental error. There was no significant difference between different pectin capsules when exposed to simulated saliva. Therefore, the free and protected probiotic cells were stable under simulated saliva fluids. It has been reported that saliva fluids had no significant effect on the viability of Lactobacillus salivarius Li01 when exposed for 40 minutes at 37 °C (Yao et al., 2017).

6.5.2.1. Survival in simulated gastric fluid (SGF) with and without pepsin

Free cells and probiotic-loaded capsules were compared for their survival in simulated gastric fluid with and without pepsin after 30, 60 and 120 minutes (Figure 6.14). As expected, the low pH of gastric fluid without pepsin caused a significant reduction in the viability of free L. casei (6.22 log CFU/mL reduction after 120 minutes incubation). Reductions were also observed in the number of surviving cells in capsules, however these decreases were lower, indicating that the pectin capsules helped protecting the cells.

139

Figure 6.14. The viability of free cells and encapsulated cells incubated in simulated gastric fluid with and without pepsin.

140

In the first 30 minutes, a significant reduction of viable cells was observed in capsules with extracted pectins (1.63 and 2.13 log CFU reduction for DFP and PFP pectin, respectively). The number of viable cells decreased further in the next 30 minutes and reached 3.23 and 3.92 log CFU reduction for DFP and PFP respectively. The viable cells in encapsulated beads from commercial pectin was slightly higher than that of the extracted pectins (only 0.52 and 3.09 log CFU reduction after 30- and 120-minute incubation), meaning these capsules provided better protection to probiotics cells through simulated gastric conditions. The electrostatic repulsion between pectin chains is reduced at lower pH due to the protonation of carboxyl groups, which makes the capsule tighten and leads to the difficulty of liquid to penetrate into the capsule. This suggests that the probiotic cells that do survive are those located most centrally in the core of the capsules. These results are similar to other previously reported studies (Park et al., 2010).

The addition of pepsin to the SGF made little difference to the number of surviving cells after 120 minute-incubation. In most samples, the number of surviving cells was increased slightly compared to when pepsin was absent. For free cells, there was a 1.53 log CFU reduction after the first 30-minute incubation in SGF, reaching 5.68 log CFU reduction after 120 minutes of incubation. By comparison, the number of viable cells of L. casei in gel beads showed a slight reduction after 120 minutes of SGF exposure. The log reduction values were only 3.24, 3.04 and 3.1 for commercial, DFP and PFP pectin respectively. In the presence of pepsin, the DFP pectin performed the best, while the PFP and commercial pectin capsules were slightly more sensitive to these simulated conditions.

The probiotic cells present at the surface of the pectin capsules are likely more susceptible when exposed to harsh environmental conditions. The surfaces of the probiotic-loaded PFP pectin gel beads were rougher than those of the DFP or commercial beads, which suggests that more bacteria were exposed at the surface. This may explain why the DFP pectin had higher protective ability than PFP pectin in simulated gastrointestinal conditions. This may also explain why the addition of pepsin led to slightly greater survival numbers; the adsorption of a small amount of enzyme to the bead surfaces might provide an additional barrier to the penetration of the bead by the acidic medium. This protective effect was greatest for the rough PFP pectin beads. Overall, the data indicate that the encapsulation of L. casei by pectin in gel beads could protect probiotic cells during its delivery in simulated gastric fluid.

141

6.5.2.2. In simulated intestinal fluids (SIF) with and without bovine bile

The viability of free and encapsulated cells were determined in bile (pH 6.4) and in a buffer solution (pH 7.4, no bile). The release profile in simulated intestinal fluid, pH 6.8 with bile, was also studied for the different types of capsules (Figure 6.15). At a pH of 6.4, the presence of bile as surface-active compounds may affect the integrity of the cell membrane, which in turn may cause a higher susceptibility to inactivation of probiotics. For the free cells, there was 3.68 log CFU/mL reduction after 3 hours incubation in bile solution. The viability of encapsulated cells was slightly improved by the DFP pectin (3.09 log reduction) but greatly improved by commercial pectin (only 2.54 log reduction). The presence of an intricate biopolymer network might hinder the capacity of disruptive factors including bile salts and enzymes when they reach the surface of the capsules.

142

Figure 6.15. The viability of free cells and encapsulated cells incubated in simulated intestinal fluid.

143

In buffer solution at pH 7.4, the free cells showed a 7.02 log cfu reduction after 3- hour incubation. The DFP pectin capsules (4.16 log cfu reduction) provided better protection to the probiotic than commercial pectin capsules (5.08 log cfu reduction) in higher pH environments. There were no surviving cells from PFP capsules after 3-hour incubation in either a bile solution or the higher pH solution. The gel beads might have swelled and then partly dissolved or broke down into smaller fragments, which caused the release of probiotics.

6.5.2.3. The release profile of probiotic in SIF

The encapsulated probiotics need to be released from the microcapsules to exert their beneficial effects to the host by their growth and colonization in the colon. The rate of bacterial release from the capsules was affected by the type of pectin used. Within the first 60 minutes, the release of probiotics from PFP pectin and commercial pectin capsules was faster than from with DFP pectin. The phosphate ions availability in SIF might lead to formation of calcium phosphate salts, withdrawing calcium ions from the capsules. The loss of calcium induced the decrease of crosslinking density of the matrix (Pasparakis & Bouropoulos, 2006). The decreased crosslinking of the network would cause the erosion of the capsules, releasing of probiotic cells. In the human colon, the breakdown of pectin is also aided by the endo-type pectate lyases from Bacteroides sp. and the Clostridium butyricum-Clostridium beijerinckii groups, which produce unsaturated pectic- oligosaccharides (Pasparakis & Bouropoulos, 2006).

The encapsulation of L. casei in PFP pectin did not protect the cells after 90 minutes in SIF, a result of the high solubility of PFP pectin capsules. This is likely due of the large pores in the PFP pectin capsules, resulting in the quick diffusion of acids, bile salts and bile enzymes inside the capsules (McClements, 2017). Additionally, the PFP pectin had a higher degree of esterification, which means there was fewer locations for calcium ion crosslinking to occur, and hence weaker cohesive forces overall that could cause easy disruptions of the surface (Cardoso et al., 2003). Other contributing factors include lower viscosity, molecular weight and gel strength, enhancing the swelling of these capsules and resulting in their faster destruction.

After 90 minutes, the DFP capsules showed a slower release compared with the commercial pectin capsules (Figure 6.16). The slower degradation might be due to the denser structure of the DFP pectin capsules. In the simulated gastrointestinal conditions,

144

the exchange of available K+ ions with the Ca2+ leads to formation of soluble regions in the Ca-pectin network, which make the pectin capsules become more porous (Jantrawut et al., 2013). While the PFP pectin beads were not stable in the simulated intestinal conditions and released the encapsulated probiotics early, the DFP and commercial pectin capsules exhibited better stability in these simulated conditions.

Figure 6.16. Release rate of encapsulated probiotics from different types of pectin in SIF

6.5.3. Swelling studies

The swelling behavior of different Ca-pectin-probiotic beads formed with commercial and extracted pectin were investigated under simulated gastrointestinal conditions (Figure 6.17). In simulated gastric fluids, different trends in the swelling rate were observed for each type of pectin beads. The swelling rate of PFP pectin capsules was higher than that of the commercial and DFP pectin beads in the first hour of incubation due to their larger surface area and high solubility. The larger pores of PFP pectin are less effective in retarding molecular diffusion. After 1.5 hours incubation, the swelling rate of PFP and DFP beads still increased steadily while it decreased for CP beads. The PFP beads were completely dissolved within 2 hours of incubation, while the swelling rate of the DFP beads continued to increase and the CP beads were unchanged. The swelling rates are affected by the degree of crosslinking between molecular chains by Ca2+, which results in the difference in the order and arrangement of junction zones. In simulated intestinal fluid with bovine bile, the sodium ions from bile would be

145

exchanged with the calcium ions in the capsules, decreasing crosslinking and increasing the swelling rate. Also, at higher pH, the carboxyl groups became negative charged, which resulted in increased osmotic pressure due to electrostatic repulsion along the carbon skeleton. This behavior increases the hydrophilicity of the material and leads to network expansion, which promotes liquid infiltration and higher swelling rates.

Figure 6.17. Swelling rate of capsules in: a) SIF-no bile, b) SIF, c) SGF.

146

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

The encapsulated cells in gelled beads and free cells in solution were stored at 4 °C over a period of 20 days to simulate the storage of commercial food products (Figure 6.18). For the free cells there was a significant decrease in viability from 10.66 log CFU/mL in the day “0” to 3.6 log CFU/mL after 20 days storage. By comparison, the viable cells remaining in the encapsulated probiotics decreased by only 3.51, 3.59 and 1.59 log CFU/mL over the same time period for the DFP, PFP and commercial pectin beads, respectively. A relatively large decrease in the number of surviving cells was observed in PFP pectin encapsulated samples over the first 15 days, but the survival rate became more stable the longer the experiment continued. The cells encapsulated by commercial pectin retained their viability for longer, with only 1.5 log reduction within 20 days. The cells loaded into DFP pectin capsules steadily decreased in the first 10 days and last 5 days of storage, however retained viability significantly better than free cells in solution. Therefore, all of the encapsulated cells had improved survival rates, with the commercial pectin being the most effective material.

Figure 6.18. The viable cells remaining in different types of pectin beads after 20 days storage at 4 °C. Error bars represent standard deviation (n = 3).

6.6. Freeze-drying capsules loaded with probiotic cells

After encapsulation by extrusion, the gelled beads were freeze-dried for longer storage. The effects of pectin concentration on the viability of encapsulated cells after

147

freezing and drying are presented in Figure 6.19. The formation of ice crystals during freezing typically destroys the membranes of unprotected cells, however encapsulation prior to freezing might limit ice crystal size and protect against this damage. During freeze-drying, the reduction in viable cells in the unprotected samples was particularly high; viability decreased by 8.43 log CFU/mL. This number was much improved for microencapsulated cells, with PFP pectin (2 % w/v) performing best (only 4.2 log CFU/mL reduction), followed by DFP pectin (2 % w/v) with 4.56 log CFU/mL reduction. It has been reported that cell membranes are significantly damaged by the freezing process (Santivarangkna et al., 2008). During the dehydration step, moisture removal can also negatively affect the fluidity of the membrane (Borst et al., 2000). Here, the freeze- drying process caused a great reduction in viable free cells, however the encapsulated probiotic cells sustained much less damage.

Figure 6.19. Viability of probiotic cells in capsules after freeze-drying following encapsulation with various concentrations and types of pectin.

6.7. Heat tolerance

The viability of encapsulated probiotic cells after processing at the recommended pasteurization temperature for food products, i.e. 63 °C, are reported in Figure 6.20. For the free cells, there was only a few surviving cells after one minute of heating (and none

148

after two minutes), as L. casei has poor heat stability (Gardiner et al., 2002). By comparison, the thermal stability of encapsulated cells were significantly improved (p < 0.05), with a much higher number of cells surviving heat treatment even over two minutes. The probiotic cells in the commercial pectin capsules maintained the highest survival rate (only 1.06 and 2.43 log CFU reduction after 60 s and 120 s incubation). There was no significance (p< 0.05) between the survival of cells after 120 s incubation in with DFP (3.27 log CFU reduction) and PFP pectin (4.93 log CFU reduction), indicating that both sources of pectin were equally as effective in protecting cells from heat stress.

Figure 6.20. The viability of probiotics before and after heating at 63 °C for two minutes. Error bars represent standard deviation (n=3).

Cells death is caused following denaturation of proteins and nucleic acids as well as the broken linkages between monomeric units as a result of thermal treatment (Fritzen- Freire et al., 2013). The loss of water molecules from the cells also results in the reduction of viable cells (Daemen, 1982). The physical barrier provided by capsules can support the cells against severe environmental conditions (Kailasapathy, 2002). In another study, it has been reported that the lower water diffusion rate after encapsulation improves the heat stability of the cells (Mandal et al., 2006). These results further indicate that pectin microencapsulation may offer better protection to probiotic cells against harsh processing conditions.

149

6.8. Conclusion

Regarding the use of pectin as a biomaterial to encapsulate probiotics, L. casei showed the strongest growth with materials from red-flesh DFP and commercial pectin. The regular and spherical pectin beads (1.87 mm to 2.41 mm in dimater) with transparent/translucent, white and light pink coloration were collected from commercial pectin, DFP and PFP pectin, respectively. The structural assessment by FTIR evidenced that the lower absorption band at 1607 cm-1 and 1735 cm-1 compared to the non-jellified pectin and depended strongly on the degree of esterification and the calcium concentration. There was only a 0.32 log CFU/mL reduction in bacterial population when being microencapsulated with commercial pectin at 2 % concentration as the best performance. In term of microencapsulation efficiency, the lowest reductions were observed in encapsulated cells by commercial pectin in the simulated salivary fluids, gastric fluids without pepsin and the bile solution. However, when exposed to both gastric fluids with pepsin and the high pH environment, the DFP pectin capsules proved to be the best coating material and slower cell-release compared to the PFP and commercial pectin. While the viable cells released from DFP pectin capsules grew faster than others early in the log phase, the highest growth rate was exhibited by cells from commercial pectin at the end of the log phase. During freeze-drying, the highest survival of probiotic cells was observed by encapsulating with 2 % PFP pectin, followed by 2 % DFP pectin. The highest thermal stability at 63 °C was achieved in the cells encapsulated by the commercial pectin.

150

Chapter 7: Conclusion

The conventional extraction process resulted in yields ranging from 5.81 % to 13.11 % for the white-flesh dragon fruit peels (DFP), 8.56 % to 15.12 % for the red-flesh DFP, and 8.56 % to 13.95 % for passion fruit peels (PFP). The Response Surface Methodology has shown that under conventional heating, the extraction conditions and the type of peels have significant influences on the final yield.

Microwave-assisted heating gave significantly higher yields for all types of peels, up to 17.79 % for red-flesh DFP, 15.79 % for white-flesh DFP and 19.84 % for purple PFP. Only microwave power, pH, and liquid:solid ratio had a significant independent impact on the yield of pectin for PFP. For red flesh DFP, the yield was affected by time, microwave power and liquid:solid ratio while pectin extracted from white-flesh DFP was influenced by all four factors at a square level. The optimal conditions giving the highest pectin yield (18.73 %) from passion fruit peels were: extraction time of 12 minutes, microwave power of 218 W, pH of 2.9 and liquid:solid ratio of 1:57 mL/g. The experimental values obtained under the optimal conditions correlated well with the predicted value, strongly suggesting that microwave-assisted heating has the potential to improve pectin recovery and lead to higher yields.

Regarding the degree of esterification, temperature proved to be the only significant linear term for red-flesh DFP while it is a quadratic term for white-flesh DFP. Meanwhile, in the PFP, the degree of esterification was shown to be mostly affected by extraction time at linear level but by both conditions at the square level. However, the models for DE were only relevant for the red-flesh DFP with the significant factors being time and microwave power for a linear term, pH for the quadratic term and pH×(liquid:solid ratio) for the interaction term. The optimization results indicated that these alternative sources of fruit waste gave yields very similar to what is achieved in the commercial production.

The results of the various characterizations of pectin indicated no significant variations in the moisture content of pectin obtained by both heating methods, irrespective of the types of peels. However, the equivalent weight of those from microwave-assisted heating was higher than those from the conventional method. The methoxyl content of all pectin samples was lower than 7 % and the materials are thus characterized as low- methoxyl pectin. The highest DE pectin was isolated from PFP by conventional heating

151

(61.98%) while the material extracted from white DFP by the microwave-assisted protocol had the lowest DE (41.96%).

Pectin recovered from PFP by conventional heating was also shown to have the maximum amount of total carbohydrates at 80.73%. The materials obtained from PFP and white DFP heated by microwave had similar content in galacturonic acids to those resulting from conventional heating. Pectin from white-flesh DFP exhibited the lowest degree of amidation of about 6.97%, while the highest values observed with commercial pectin were in the range of 16.26%.

The structural assessment by Fourier Transform infrared spectroscopy evidenced that pectin produced in this research was very similar to commercially available citrus pectin. The change in the main functional groups present on the polysaccharide chains of extracted pectin under effects of pH, microwave power, processing time and liquid:solid ratio and were confirmed by FTIR analysis.

The microstructure and morphology features of the different types of pectin were also investigated. The PFP had a higher BET specific surface area attributed to its higher porosity and smaller pore diameter. Meanwhile, the pore size distribution of white-flesh DFP was the narrowest among all extracted pectins. The commercial pectin and extracted pectin from PFP showed similar X-ray diffraction patterns as typical amorphous polymers, while for other samples, the conventional heating produced more crystalline pectin than from microwave-assisted heating.

In terms of functional properties, in general a greater solubility of pectin is observed at pH higher than 4. The pectin extracted from PFP by conventional heating showed the lowest solubility while the PFP pectin from microwave heating had the highest, together with high oil-holding capacity and foaming capacity. However, the foaming stability of this pectin was lower compared to commercial pectin. Furthermore, pectin extracted by conventional heating from PFP had the highest emulsion capacity but the lowest emulsion stability under thermal treatment. All pectins behaved as Newtonian fluids at low concentrations, however approach pseudoplasticity at higher concentrations. These characteristics of extracted pectin provide opportunities to widen alternative nature resources from fruit waste and their potential applications in food processing.

Regarding the use of pectin as a biomaterial to encapsulate probiotics, L. casei showed the strongest growth with materials from red-flesh DFP, with a measured OD of

152

1.982, which is of the same order of magnitude to what is observed with commercial pectin. After 24-hour and 48-hour incubations, the L. casei grown in de Man, Rogosa & Sharpe broth with added pectin from red-flesh DFP recorded the highest growths (0.137 and 0.096 respectively).

The beads prepared from different types of pectin had regular and spherical shapes with transparent/translucent, white and light pink coloration for commercial pectin, DFP and PFP pectin, respectively. The size of the gelled capsules ranged from 1.87 mm to 2.41 mm with the largest being observed in DFP gelled particles, while the PFP capsules had the highest sphericity factor and size distributions. The scanning electron microscope images showed that the surface of the probiotic-loaded PFP pectin gel beads was rougher and clearly showed more exposed bacteria than other samples.

The structural assessment by Fourier Transform Infrared spectroscopy evidenced that the pectins we isolated were very similar to commercially available citrus pectin. In the FTIR spectra of these beads, the lower absorption band at 1607 cm-1 and 1735 cm-1 compared to the non-jellified pectin was as a result of the electrostatic interactions between COO– and Ca2+ to form the gel. The most important factor affecting the intensity of IR peaks is the change in dipole moment occurring during the vibration which directly characterizes the “polarizability”: in other words how easily electrons can move. Jellification is a result of the complexation properties of calcium which will mobilize the electrons and decreases polarizability, hence peak intensity. The intensities of the absorption peaks in the fingerprint area were also affected after gelling occurred, and the extent depended strongly on the degree of esterification and the calcium concentration. It is generally recognized that the gel forming ability of low methoxy pectins is inversely proportional to the degree of methylation.

In term of microencapsulation efficiency, the highest loss of total viable cells was observed in capsules with pectin (1 %) from white DFP after encapsulation, exhibiting a decrease of 3.15 log CFU/mL. The double pectin coating did not evidence a significantly higher protecting ability to ensure probiotic survival in comparison with the single coating in either CP or DFP pectin. Only minimal reductions inviable cells were observed in encapsulated cells in the simulated salivary fluids. Under the low pH of gastric fluids, without pepsin and the bile solution, the viability of the cells from beads of commercial pectin were slightly higher than that of other extracted pectin (only 0.52 and 2.54 log CFU

153

reduction respectively). However, when exposed to both gastric fluids with pepsin and the high pH environment, the DFP pectin capsules proved to be the best coating material with only 3.1 log CFU reduction. The cell-release from PFP and commercial pectin was faster than that from the DFP pectin, and was achieved within the first 60 minutes. After 90 minutes after incubation, the PFP pectin capsules were destroyed while the DFP capsules showed a slower release compared to the commercial pectin. This could be due to the swelling rate of PFP pectin which is higher than that of the commercial and DFP pectin beads.

The release of probiotic cells from pectin capsules, and their growth rates compared with free cells were determined. While the viable cells from DFP pectin capsules grew faster than others early in the log phase, the highest growth rate was exhibited by cells from commercial pectin at the end of the log phase. During freeze-drying, the survival of probiotic cells was improved by encapsulating with 2 % PFP pectin, resulting in only 4.2 log CFU/mL reduction, followed by 2 % DFP pectin with 4.56 log CFU/mL reduction. The highest thermal stability at 63 °C was achieved in the cells encapsulated by the commercial pectin.

Overall the results achieved in this project strongly suggest that the application of microwave energy in pectin extraction has significant potential to improve pectin recovery from dragon fruit and passion fruit peels. Furthermore, the physicochemical, structural and functional properties of extracted pectin are shown to be suitable for applications targeting the development of probiotic-encapsulating materials. Pectin has been determined to be an effective coating material with the ability to protect the probiotics when exposed to the simulated gastrointestinal fluids and other harsh conditions such as freeze-drying and heating.

Limitations

A few points should be highlighted, that should be taken into account for the development of further research. The initial limitation was the sample bias due to the limited availability of the fruit peels in Australia, and seasonal variations. Due to time constraints, some variables of microwave-assisted heating could not be optimized to obtain higher extraction yield. Also to fully comprehend the gelling properties of the final materials, and to be in a position to compare with commercial and off-the-shelf products more rheological tests will be required. Accessibility to these highly specialized

154

equipment was rather limited, although we have now been able to secure an industry link to conduct full systematic studies. The effects of other extracting conditions such as gelling time and the ratio of pectin to cell suspension on encapsulation efficiency were also not studied, which suggests further room for optimization of the process.

Recommendations for future work

We have identified throughout this project a few approaches that could be improved and developed into future research topics to extend the work:

- include more types of fruit peels and other waste biomass to widen the valorization of organic waste, - explore other alternative green technologies for pectin extraction such as ultrasound, enzyme addition or a combination of technologies to obtain more effective extraction, - carry out a full analysis of pectin by extensive analytical techniques that inform attempts to scale up productions, - conduct more longer-term studies to quantify the impact of storage and food processing on the retention of pectin properties, - explore the combination of pectin with other biomaterials in probiotic encapsulation, - study the major parameters of encapsulation by extrusion to achieve highest viable cells, - consider other encapsulation methods such as emulsion and nano-encapsulation using pectin as a coating agent to produce smaller beads.

155

Bibliography

Adetunji, Lanrewaju 2016, Valorising Canadian cranberry ( V . macrocarpon ) pomace using microwave heating : recovery and physicochemical characterization of pectin, McGill University. Akhtar, Saeed, Ismail, Tariq, Fraternale, Daniele & Sestili, Piero 2015, ‘Pomegranate peel and peel extracts: Chemistry and food features’, Food Chemistry, vol. 174, pp. 417– 425. Albersheim, Peter, Neukom, Hans & Deuel, Hans 1960, ‘Splitting of pectin chain molecules in neutral solutions’, Archives of Biochemistry and Biophysics, vol. 90, no. 1, Academic Press, pp. 46–51. Alborzi, Solmaz, Lim, Loong Tak & Kakuda, Yukio 2014, ‘Release of folic acid from sodium alginate-pectin-poly(ethylene oxide) electrospun fibers under invitro conditions’, LWT - Food Science and Technology, vol. 59, no. 1, pp. 383–388. Albuquerque, Marcela de Albuquerque Cavalcanti, Levit, Romina, Beres, Carolina, Bedani, Raquel, de Moreno de LeBlanc, Alejandra, Saad, Susana Marta Isay & LeBlanc, Jean Guy 2019, ‘Tropical fruit by-products water extracts of tropical fruit by-products as sources of soluble fibres and phenolic compounds with potential antioxidant, anti-inflammatory, and functional properties’, Journal of Functional Foods, vol. 52, Elsevier, pp. 724–733. Amid, Mehrnoush, Manap, Yazid & Zohdi, Khanani 2014, ‘Purifcation and characterisation of thermo-alkaline pectinase enzyme from hylocereus polyrhizus’, European Food Research and Technology, vol. 239, no. 1, pp. 21–29. AOAC 1990, Official methods of analysis, Kenneth Helrich (ed.), 15th ed, Association of official analytical chemists, Virginia, USA. Araya, Magdalena, Morelli, Lorenzo, Reid, Gregor, Sanders, Mary Ellen, Stanton, Catherine, Pineiro, Maya & Ben Embarek, Peter 2002, Guidelines for the evaluation of probiotics in food, London Ontario. Arvanitoyannis, Ioannis S & Varzakas, Theodoros H 2008, ‘Fruit/fruit juice waste management: Treatment methods and potential uses of treated waste’, Waste Management for the food industries, vol. 2, Academic Press Amsterdam, pp. 569– 628. Ashford, Marianne, Fell, John, Attwood, David, Sharma, Harbans & Woodhead, Philip 1993, ‘An evaluation of pectin as a carrier for drug targeting to the colon’, Journal of Controlled Release, vol. 26, no. 3, pp. 213–220. Attard, Thomas M, Watterson, Baillie, Budarin, Vitaliy L, Clark, James H & Hunt, Andrew J 2014, ‘Microwave assisted extraction as an important technology for valorising orange waste’, New Journal of Chemistry, vol. 38, pp. 2278–2283. Aureli, Paolo, Capurso, Lucio, Castellazzi, Anna Maria, Clerici, Mario, Giovannini, Marcello, Morelli, Lorenzo, et al. 2011, ‘Probiotics and health: an evidence-based review’, Pharmacological research, vol. 63, no. 5, Elsevier, pp. 366–376. Ayala-Zavala, JF, Rosas-Domínguez, C, Vega-Vega, V & González-Aguilar, GA 2010, ‘Antioxidant enrichment and antimicrobial protection of fresh-cut fruits using their own byproducts: Looking for integral exploitation’, Journal of Food Science, vol.

156

75, no. 8, pp. R175–R181. Azémar, Marc, Hildenbrand, Bernd, Haering, Brigitte, Heim, Manfred E & Unger, Clemens 2007, ‘Clinical benefit in patients with advanced solid tumors treated with modified citrus pectin: A prospective pilot study’, Clinical medicine: Oncology, vol. 1, pp. 73–80. Aziz, Nur Shahidah binti Ab, Nor, Mohd Adib bin Mohd, Manaf, Shareena Fairuz binti Abdul & Hamzah, Fazlena 2015, ‘Suitability of biochar produced from biomass waste as soil amendment’, Procedia - Social and Behavioral Sciences, vol. 195, pp. 2457–2465. Banerjee, Jhumur, Singh, Ramkrishna, Vijayaraghavan, R, MacFarlane, Douglas, Patti, Antonio F & Arora, Amit 2017, ‘Bioactives from fruit processing wastes: Green approaches to valuable chemicals’, Food Chemistry, vol. 225, pp. 10–22. Bauer, Stefan, Muller, Thomas & Hamm, Svetlana 2009, ‘Pattern recognition by Toll- like receptors’, Advances in experimental medicine and biology, vol. 653, pp. 15– 34. Bayar, Nadia, Bouallegue, Tahani, Achour, Mabrouka, Kriaa, Mouna, Bougatef, Ali & Kammoun, Radhouane 2017, ‘Ultrasonic extraction of pectin from Opuntia ficus indica cladodes after mucilage removal: Optimization of experimental conditions and evaluation of chemical and functional properties.’, Food Chemistry, vol. 235, pp. 275–282. Bayar, Nadia, Kriaa, Mouna & Kammoun, Radhouane 2016, ‘Extraction and characterization of three polysaccharides extracted from Opuntia ficus indica cladodes’, International Journal of Biological Macromolecules, vol. 92, Elsevier, pp. 441–450. Begum, R, Aziz, MG, Uddin, MB & Yusof, YA 2014, ‘Characterization of jackfruit (Artocarpus heterophyllus) waste pectin as influenced by various extraction conditions’, Agriculture and Agricultural Science Procedia, vol. 2, pp. 244–251. Begum, Rokeya, Yusof, Yus Aniza, Aziz, Mohammad Gulzarul & Uddin, M Burhan 2017, ‘Structural and functional properties of pectin extracted from jackfruit (Artocarpus heterophyllus) waste: Effects of drying’, International Journal of Food Properties, vol. 20, no. 1, Taylor & Francis, pp. S190–S201, viewed . Bekhit, Mariam, Sánchez-González, Laura, Ben Messaoud, Ghazi & Desobry, Stéphane 2016, ‘Encapsulation of Lactococcus lactis subsp. lactis on alginate/pectin composite microbeads: Effect of matrix composition on bacterial survival and nisin release’, Journal of Food Engineering, vol. 180, pp. 1–9. BeMiller, James N 1986, ‘An introduction to pectins: structure and properties’, ACS symposium series-American chemical society. Bepeyeva, Aigerim, de Barros, Joao MS, Albadran, Hanady, Kakimov, Aitbek K, Kakimova, Zhaynagul Kh, Charalampopoulos, Dimitris & Khutoryanskiy, Vitaliy V. 2017, ‘Encapsulation of Lactobacillus casei into calcium pectinate-chitosan beads for enteric delivery’, Journal of Food Science, vol. 82, no. 12, pp. 2954–2959. Bochek, AM, Zabivalova, NM & Petropavlovskii, GA 2001, ‘Determination of the esterification degree of polygalacturonic acid’, Russian Journal of Applied Chemistry, vol. 74, no. 5, pp. 796–799.

157

Borst, Jan Willem, Visser, Nina V., Kouptsova, Olga & Visser, Antonie JWG 2000, ‘Oxidation of unsaturated phospholipids in membrane bilayer mixtures is accompanied by membrane fluidity changes’, Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. Box, George EP & Behnken, Donald W 1960, ‘Some new three level designs for the study of quantitative variables’, Technometrics, vol. 2, no. 4, Taylor & Francis, pp. 455– 475. Brejnholt, Sarah M 2009, ‘Pectin’, in Alan Imeson (ed.), Food Stabilisers, Thickeners and Gelling Agents, Wiley Online Library, p. 237. Brinques, Graziela Brusch & Ayub, Marco Antônio Záchia 2011, ‘Effect of microencapsulation on survival of Lactobacillus plantarum in simulated gastrointestinal conditions, refrigeration, and yogurt’, Journal of Food Engineering, vol. 103, no. 2, pp. 123–128. Cabrera, Juan Carlos, Cambier, Pierre & van Cutsem, Pierre 2011, ‘Drug encapsulation in pectin hydrogel beads- a systematic study of simulated digestion media’, International Journal of Pharmacy and Pharmaceutical Sciences, vol. 3, no. SUPPL. 5, pp. 292–299. Canteri, MHG, Scheer, AP, Ginies, C, Reich, M, Renard, CMCG & Wosiacki, G 2012, ‘Rheological and macromolecular quality of pectin extracted with nitric acid from passion fruit rind’, Journal of Food Process Engineering, vol. 35, no. 5, pp. 800– 809. Cardoso, Susana M, Coimbra, Manuel A & Lopes da Silva, JA 2003, ‘Calcium-mediated gelation of an olive pomace pectic extract’, Carbohydrate Polymers, vol. 52, no. 2, pp. 125–133. Chaharbaghi, Elnaz, Khodaiyan, Faramarz & Hosseini, Seyed Saeid 2017, ‘Optimization of pectin extraction from pistachio green hull as a new source’, Carbohydrate Polymers, vol. 173, Elsevier Ltd., pp. 107–113. Chan, Eng Seng, Wong, Sze Ling, Lee, Peh Phong, Lee, Jau Shya, Ti, Tey Beng, Zhang, Zhibing, et al. 2011, ‘Effects of starch filler on the physical properties of lyophilized calcium-alginate beads and the viability of encapsulated cells’, Carbohydrate Polymers, vol. 83, no. 1, Elsevier Ltd., pp. 225–232. Chatterjee, Emon, Manuel, Suba & Hassan, Syed Shamimul 2016, ‘Effect of fruit pectin on growth of lactic acid bacteria’, Journal of Probiotics & Health, vol. 4, no. 2, p. 147. Chavarri, Maria, Maranon, Izaskun & Carmen, Maria 2012, ‘Encapsulation technology to protect probiotic bacteria’, in Everlon Rigobelo (ed.), Probiotics, IntechOpen, London, pp. 501–540. Chen, Hai-Yan, Li, Xiang-Yi, Liu, Bing-Jie & Meng, Xiang-Hong 2017, ‘Microencapsulation of Lactobacillus bulgaricus and survival assays under simulated gastrointestinal conditions’, Journal of Functional Foods, vol. 29, Elsevier, pp. 248–255. Chen, Jun, Wang, Qi, Liu, Cheng Mei & Gong, Joshua 2017, ‘Issues deserve attention in encapsulating probiotics: Critical review of existing literature’, Critical Reviews in Food Science and Nutrition, vol. 57, no. 6, pp. 1228–1238.

158

Cheok, Choon Yoong, Mohd Adzahan, Noranizan, Abdul Rahman, Russly, Zainal Abedin, Nur Hanani, Hussain, Norhayati, Sulaiman, Rabiha & Chong, Gun Hean 2018, ‘Current trends of tropical fruit waste utilization.’, Critical reviews in food science and nutrition, vol. 58, no. 3, pp. 335–361. Cho, Eun Hi, Jung, Ho Tak, Lee, Byung Hoo, Kim, Hyun Seok, Rhee, Jin Kyu & Yoo, Sang Ho 2019, ‘Green process development for apple-peel pectin production by organic acid extraction’, Carbohydrate Polymers, vol. 204, Elsevier, pp. 97–103. Chotiko, Arranee & Sathivel, Subramaniam 2016a, ‘Development of a combined low- methoxyl-pectin and rice-bran-extract delivery system to improve the viability of Lactobacillus plantarum under acid and bile conditions’, LWT - Food Science and Technology, vol. 66, pp. 420–427. Chotiko, Arranee & Sathivel, Subramaniam 2016b, ‘Three protective agents for pectin- rice bran capsules for encapsulating Lactobacillus plantarum’, Food Bioscience, vol. 16, Elsevier, pp. 56–65. Chung, Wing Sun Faith, Meijerink, Marjolein, Zeuner, Birgitte, Holck, Jesper, Louis, Petra, Meyer, Anne S, et al. 2017, ‘Prebiotic potential of pectin and pectic oligosaccharides to promote anti-inflammatory commensal bacteria in the human colon’, FEMS Microbiology Ecology, vol. 93, no. 11, pp. 1–9. Cibele, Freitas de Oliveira, Giordani, Diego, Lutckemier, Rafael, Gurak, Poliana Deyse, Cladera-Olivera, Florencia & Ferreira Marczak, Ligia Damasceno 2016, ‘Extraction of pectin from passion fruit peel assisted by ultrasound’, LWT - Food Science and Technology, vol. 71, Academic Press, pp. 110–115. Cibele, Freitas de Oliveira, Gurak, Poliana Deyse, Cladera-Olivera, Florencia, Marczak, Ligia Damasceno Ferreira & Karwe, Mukund 2016, ‘Combined effect of high- pressure and conventional heating on pectin extraction from passion fruit peel’, Food and Bioprocess Technology, vol. 9, no. 6, Springer, pp. 1021–1030. Coghetto, Chaline Caren, Brinques, Graziela Brusch, Siqueira, Nataly Machado, Pletsch, Jéssica, Soares, Rosane Michele Duarte & Ayub, Marco Antônio Záchia 2016, ‘Electrospraying microencapsulation of Lactobacillus plantarum enhances cell viability under refrigeration storage and simulated gastric and intestinal fluids’, Journal of Functional Foods, vol. 24, Elsevier, pp. 316–326. Collins, JK, Auty, MAE, O’Sullivan, E, Stanton, C, Ross, RP, Gardiner, GE, et al. 2002, ‘Comparative survival rates of human-derived probiotic Lactobacillus paracasei and L. salivarius strains during heat treatment and spray drying’, Applied and Environmental Microbiology, vol. 66, no. 6, pp. 2605–2612. Colodel, Cristiane & Petkowicz, Carmen Lúcia de Oliveira 2019, ‘Acid extraction and physicochemical characterization of pectin from cubiu (Solanum sessiliflorum D.) fruit peel’, Food Hydrocolloids, vol. 86, Elsevier, pp. 193–200. Colodel, Cristiane, Vriesmann, Lúcia Cristina, Teófilo, Reinaldo Francisco & de Oliveira Petkowicz, Carmen Lúcia 2018, ‘Extraction of pectin from ponkan (Citrus reticulata Blanco cv. Ponkan) peel: Optimization and structural characterization’, International Journal of Biological Macromolecules, vol. 117, Elsevier, pp. 385– 391. Daemen, ALH 1982, ‘The destruction of enzymes and bacteria during the spray-drying of milk and whey, 1: the thermoresistance of some enzymes and bacteria in milk and

159

whey with various total solids contents’, Netherlands Milk and Dairy Journal, vol. 35, no. 2, pp. 133–145. Dafe, Alireza, Etemadi, Hossein, Dilmaghani, Azita & Mahdavinia, Gholam Reza 2017, ‘Investigation of pectin/starch hydrogel as a carrier for oral delivery of probiotic bacteria’, International Journal of Biological Macromolecules, vol. 97, pp. 536– 543. Dea, ICM & Madden, JK 1986, ‘Acetylated pectic polysaccharides of sugar beet’, Topics in Catalysis, vol. 1, no. 1, Elsevier, pp. 71–88. Deng, Gui Fang, Shen, Chen, Xu, Xiang Rong, Kuang, Ru Dan, Guo, Ya Jun, Zeng, Li Shan, et al. 2012, ‘Potential of fruit wastes as natural resources of bioactive compounds’, International Journal of Molecular Sciences, vol. 13, no. 7, pp. 8308– 8323. Denkova, RS, Georgieva, L V & Denkova, ZR 2014, ‘Highly active freeze-dried probiotic concentrates with long shelf life obtained using cryoprotectants of plant origin’, Journal of Food and Packaging Science, vol. 4, pp. 66–70. Desai, Kashappa Goud H 2006, ‘Preparation and characteristics of high-amylose corn starch/pectin blend microparticles: A technical note’, AAPS PharmSciTech, vol. 6, no. 2, pp. E202–E208. Dickinson, Eric 2003, ‘Hydrocolloids at interfaces and the influence on the properties of dispersed systems’, Food Hydrocolloids, vol. 17, pp. 25–39. Ding, WK & Shah, NP 2007, ‘Acid, bile, and heat tolerance of free and microencapsulated probiotic bacteria’, Journal of Food Science, vol. 72, no. 9, pp. 446–450. Ding, WK & Shah, NP 2008, ‘Survival of free and microencapsulated probiotic bacteria in orange and apple juices’, International Food Research Journal, vol. 15, no. 2, pp. 219–232. Dixon, Daniel Wayne 2008, Characterization of commercial pectin preparations by spectroscopic and chromatographic techniques, East Tennessee State University, viewed . Doherty, SB, Auty, MA, Stanton, C, Ross, RP, Fitzgerald, GF & Brodkorb, A 2012, ‘Application of whey protein micro-bead coatings for enhanced strength and probiotic protection during fruit juice storage and gastric incubation’, Journal of Microencapsulation, vol. 29, no. 8, pp. 713–728. Dominiak, Malgorzata, Søndergaard, Karen M, Wichmann, Jesper, Vidal-Melgosa, Silvia, Willats, William GT, Meyer, Anne S & Mikkelsen, Jørn D 2014, ‘Application of enzymes for efficient extraction, modification, and development of functional properties of lime pectin’, Food Hydrocolloids, vol. 40, pp. 273–282. Dore, WH 1926, ‘The pectic substances’, Journal of Chemical Education, vol. 3, no. 5, Interscience, N.Y., p. 505. Drusch, Stephan 2007, ‘Sugar beet pectin: A novel emulsifying wall component for microencapsulation of lipophilic food ingredients by spray-drying’, Food Hydrocolloids, vol. 21, no. 7, Elsevier, pp. 1223–1228. Dubois, Michel, Gilles, KA, Hamilton, JK, Rebers, PA & Smith, Fred 1956, ‘Colorimetric method for determination of sugars and related substances’, Analytical

160

Chemistry, vol. 28, no. 3, pp. 350–356. Dumitriu, Severian 2004, Polysaccharides: structural diversity and functional versatility, Severian Dumitriu (ed.), CRC press, Boca Raton. Dyk, JS Van & Pletschke, BI 2012, ‘A review of lignocellulose bioconversion using enzymatic hydrolysis and synergistic cooperation between enzymes-Factors affecting enzymes, conversion and synergy’, Biotechnology Advances, vol. 30, no. 6, pp. 1458–1480. Einhorn-Stoll, Ulrike 2017, ‘Pectin-water interactions in foods - From powder to gel’, Food Hydrocolloids, Elsevier Ltd, pp. 1–11, viewed . El-Nawawi, Sohair A & Shehata, Fadia R 1988, ‘Effect of the extraction temperature on the quality characteristics of pectin extracted from Egyptian orange peel’, Biological Wastes, vol. 24, no. 4, pp. 307–311. Encalada, Alondra M Idrov, Pérez, Carolina D, Flores, Silvia K, Rossetti, Luciana, Fissore, Eliana N & Rojas, Ana M 2019, ‘Antioxidant pectin enriched fractions obtained from discarded carrots (Daucus carota L.) by ultrasound-enzyme assisted extraction’, Food Chemistry, vol. 289, Elsevier, pp. 453–460. Endress, HU 1991, Nonfood uses of pectin, Reginald H Walter (ed.), Academic Press, New York . Eskilsson, Cecilia Sparr, Björklund, Erland, Mathiasson, Lennart, Karlsson, Lars & Torstensson, Arne 1999, ‘Microwave-assisted extraction of felodipine tablets’, Journal of Chromatography A, vol. 840, no. 1, pp. 59–70. Fang, Yapeng, Al-Assaf, Saphwan, Phillips, Glyn O, Nishinari, Katsuyoshi, Funami, Takahiro & Williams, Peter A 2008, ‘Binding behavior of calcium to polyuronates: Comparison of pectin with alginate’, Carbohydrate Polymers, vol. 72, no. 2, pp. 334–341. FAO/WHO 2009, Compendium of food additive specificaions, Organization, Rome. FAO/WHO 2001, Probiotics in food: Health and nutritional properties and guidelines for evaluation, Food and Nutrition Paper, Cordoba, Argentina, viewed . FAO 2014, Definitional framework of food loss, Rome, viewed . FAO 2013, Food wastage footprint. Impacts on natural resources, viewed . Faravash, Ram Sanati & Ashtiani, Farzin Zokaee 2007, ‘The effect of pH, ethanol volume and acid washing time on the yield of pectin extraction from peach pomace’, International journal of food science & technology, vol. 42, no. 10, Wiley Online Library, pp. 1177–1187. Faravash, Ram Sanati & Ashtiani, Farzin Zokaee 2008, ‘The influence of acid volume, ethanol-to-extract ratio and acid-washing time on the yield of pectic substances extraction from peach pomace’, Food Hydrocolloids, vol. 22, no. 1, Elsevier, pp. 196–202. Fatemeh, Bahadori & Rezvantalab, Sima 2015, ‘Viscosity variation effects on heat

161

transfer and fluid flow through two-layered porous media’, Journal of Chemical Technology and Metallurgy, vol. 50, no. 1, pp. 35–38. Fava, Fabio, Totaro, Grazia, Diels, Ludo, Reis, Maria, Duarte, Jose, Carioca, Osvaldo Beserra, et al. 2015, ‘Biowaste biorefinery in Europe: opportunities and research & development needs’, New biotechnology, vol. 32, no. 1, pp. 100–108. Feng, Liyuan, Zhou, Yun, Ashaolu, Tolulope Joshua, Ye, Fayin & Zhao, Guohua 2019, ‘Physicochemical and rheological characterization of pectin-rich fraction from blueberry (Vaccinium ashei) wine pomace’, International Journal of Biological Macromolecules, vol. 128, Elsevier, pp. 629–637. Ferreira-Lazarte, Alvaro, Moreno, F Javier, Cueva, Carolina, Gil-Sánchez, Irene & Villamiel, Mar 2019, ‘Behaviour of citrus pectin during its gastrointestinal digestion and fermentation in a dynamic simulator (simgi®)’, Carbohydrate Polymers, vol. 207, Elsevier, pp. 382–390. Ferreira, SLC, Bruns, RE, Ferreira, HS, Matos, GD, David, JM, Brandão, GC, et al. 2007, ‘Box-Behnken design: An alternative for the optimization of analytical methods’, Analytica Chimica Acta, Elsevier, pp. 179–186. Fishman, Marshall L, Chau, Hoa K, Kolpak, Frank & Brady, James 2001, ‘Solvent effects on the molecular properties of pectins’, Journal of Agricultural and Food Chemistry, vol. 49, no. 9, pp. 4494–4501. Fishman, ML, Cooke, P, Hotchkiss, A & Damert, W 1993, ‘Progressive dissociation of pectin’, Carbohydrate Research, vol. 248, pp. 303–316. Fleury, N & Lahaye, M 1991, ‘Chemical and physico‐chemical characterisation of fibres from Laminaria digitata (kombu breton): A physiological approach’, Journal of the Science of Food and Agriculture, vol. 55, no. 3, Wiley Online Library, pp. 389–400. Foroutan, Nafiseh Sadat, Tabandeh, Fatemeh, Khodabandeh, Mahvash, Mojgani, Naheed, Maghsoudi, Amir & Moradi, Meisam 2017, ‘Isolation and identification of an indigenous probiotic Lactobacillus strain: Its encapsulation with natural branched polysaccharids to improve bacterial viability’, Applied Food Biotechnology, vol. 4, no. 3, pp. 133–142. Fritzen-Freire, Carlise B, Prudêncio, Elane S, Pinto, Stephanie S, Muñoz, Isabella B & Amboni, Renata DMC 2013, ‘Effect of microencapsulation on survival of Bifidobacterium BB-12 exposed to simulated gastrointestinal conditions and heat treatments’, LWT - Food Science and Technology, vol. 50, no. 1, pp. 39–44. Gan, Chee Yuen, Abdul Manaf, Normaliza Hj & Latiff, Aishah A 2010, ‘Physico- chemical properties of alcohol precipitate pectin-like polysaccharides from Parkia speciosa pod’, Food Hydrocolloids, vol. 24, no. 5, Elsevier Ltd, pp. 471–478. Ganzevles, Renate A, Stuart, Martien A Cohen, van Vliet, Ton & de Jongh, Harmen HJ 2006, ‘Use of polysaccharides to control protein adsorption to the air–water interface’, Food Hydrocolloids, vol. 20, no. 6, Elsevier, pp. 872–878. Gebara, Clarice, Chaves, Karina S, Ribeiro, Maria Cecília E, Souza, Flavia N, Grosso, Carlos RF & Gigante, Mirna L 2013, ‘Viability of Lactobacillus acidophilus La5 in pectin-whey protein microparticles during exposure to simulated gastrointestinal conditions’, Food Research International, vol. 51, no. 2, pp. 872–878. Gerez, CL, de Valdez, GF, Gigante, ML & Grosso, CRF 2012, ‘Whey protein coating

162

bead improves the survival of the probiotic Lactobacillus rhamnosus CRL 1505 to low pH’, Letters in Applied Microbiology, vol. 54, no. 6, pp. 552–556. Ghibaudo, Florencia, Gerbino, Esteban, Campo Dall’ Orto, Viviana & Gómez-Zavaglia, Andrea 2017, ‘Pectin-iron capsules: Novel system to stabilise and deliver lactic acid bacteria’, Journal of Functional Foods, vol. 39, Elsevier, pp. 299–305. Gibson, Glenn R, Probert, Hollie M, Loo, Jan Van, Rastall, Robert A & Roberfroid, Marcel B 2004, ‘Dietary modulation of the human colonic microbiota: updating the concept of prebiotics.’, Nutrition research reviews, vol. 17, no. 2, England, pp. 259– 275. Gigli, Jiabril, Garnier, Catherine & Piazza, Laura 2009, ‘Rheological behaviour of low- methoxyl pectin gels over an extended frequency window’, Food Hydrocolloids, vol. 23, no. 5, Elsevier, pp. 1406–1412. Goula, Athanasia M 2013, ‘Ultrasound-assisted extraction of pomegranate seed oil - Kinetic modeling’, Journal of Food Engineering, vol. 117, no. 4, pp. 492–498. Guandalini, Bruna Bárbara Valero, Rodrigues, Naira Poerner & Marczak, Ligia Damasceno Ferreira 2019, ‘Sequential extraction of phenolics and pectin from mango peel assisted by ultrasound’, Food Research International, vol. 119, Elsevier, pp. 455–461. Guggenbichler, JP, De Bettignies-Dutz, A, Meissner, P, Schellmoser, S & Jurenitsch, J 1997, ‘Acidic oligosaccharides from natural sources block adherence of Escherichia coli on uroepithelial cells’, Pharmaceutical and Pharmacological Letters, vol. 7, no. 1, Springer Science and Media, pp. 35–38. Guo, Xiaoming, Meng, Hecheng, Tang, Qiang, Pan, Runquan, Zhu, Siming & Yu, Shujuan 2016, ‘Effects of the precipitation pH on the ethanolic precipitation of sugar beet pectins’, Food Hydrocolloids, vol. 52, Elsevier Ltd, pp. 431–437. Guo, Zhenku, Jin, Qinhan, Fan, Guoqiang, Duan, Yuping, Qin, Chen & Wen, Minjie 2001, ‘Microwave-assisted extraction of effective constituents from a Chinese herbal medicine Radix puerariae’, Analytica Chimica Acta, vol. 436, no. 1, pp. 41– 47. Güzel, Melih & Akpınar, Özlem 2019, ‘Valorisation of fruit by-products: Production characterization of pectins from fruit peels’, Food and Bioproducts Processing, vol. 115, Institution of Chemical Engineers, pp. 126–133, viewed . Hanrahan, Grady & Lu, Kenneth 2006, ‘Application of factorial and response surface methodology in modern experimental design and optimization’, Critical Reviews in Analytical Chemistry, vol. 36, Taylor & Francis, pp. 141–151. Hansen, L Truelstrup, Allan-Wojtas, PM, Jin, YL & Paulson, AT 2002, ‘Survival of Ca- alginate microencapsulated Bifidobacterium spp. in milk and simulated gastrointestinal conditions’, Food Microbiology, vol. 19, no. 1, pp. 35–45. Harholt, Jesper, Suttangkakul, Anongpat & Scheller, Henrik Vibe 2010, ‘Biosynthesis of pectin’, Plant Physiology, vol. 153, no. 2, pp. 384–395. Harris, Philip J & Stone, Bruce A 2009, ‘Chemistry and Molecular Organization of Plant Cell Walls’, in Himmel Michael (ed.), Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy, Wiley-Blackwell, pp. 61–93.

163

Heidebach, Thomas, Först, Petra & Kulozik, Ulrich 2012, ‘Microencapsulation of probiotic cells for food applications’, Critical Reviews in Food Science and Nutrition, vol. 52, no. 4, pp. 291–311. Henk, A Schols & Alphons, GJ Voragen 2002, ‘The chemical structure of pectins’, in Graham B Seymour & J Paul Knox (eds), Pectins and their manipulation, CRC Press, pp. 1–30. Holck, Jesper, Lorentzen, Andrea, Vigsnæs, Louise K, Licht, Tine R, Mikkelsen, Jørn D & Meyer, Anne S 2011, ‘Feruloylated and nonferuloylated arabino-oligosaccharides from sugar beet pectin selectively stimulate the growth of Bifidobacterium spp. in human fecal in vitro fermentations’, Journal of agricultural and food chemistry, vol. 59, no. 12, pp. 6511–6519. Hosseini, Seyed Saeid, Khodaiyan, Faramarz, Kazemi, Milad & Najari, Zahra 2019, ‘Optimization and characterization of pectin extracted from sour orange peel by ultrasound assisted method’, International Journal of Biological Macromolecules, vol. 125, Elsevier, pp. 621–629. Hosseini, Seyed Saeid, Khodaiyan, Faramarz & Yarmand, Mohammad Saeid 2016, ‘Optimization of microwave assisted extraction of pectin from sour orange peel and its physicochemical properties’, Carbohydrate Polymers, vol. 140, Elsevier, pp. 59– 65, viewed . Hotchkiss, AT, Olano-Martin, Estibaliz, Grace, William E, Gibson, Glen & Rastall, Bob 2003, ‘Pectic oligosaccharides as prebiotics’, Oligosaccharides in Food and Agriculture, American Chemical Society, Washington, pp. 54–62. Hua, Xiao, Wang, Kun, Yang, Ruijin, Kang, Jiaqi & Zhang, Jianing 2015, ‘Rheological properties of natural low-methoxyl pectin extracted from sunflower head’, Food Hydrocolloids, vol. 44, pp. 122–128. Huang, X, Kakuda, Y & Cui, W 2001, ‘Hydrocolloids in emulsions: particle size distribution and interfacial activity’, Food hydrocolloids, vol. 15, no. 4–6, Elsevier, pp. 533–542. Huebner, J, Wehling, RL & Hutkins, RW 2007, ‘Functional activity of commercial prebiotics’, International Dairy Journal, vol. 17, Elsevier, pp. 770–775. Iglesias, MT & Lozano, JE 2004, ‘Extraction and characterization of sunflower pectin’, Journal of Food Engineering, vol. 62, no. 3, Elsevier, pp. 215–223. Ishibashi, N & Shimamura, S 1993, ‘Bidobacteria: Research and development in Japan’, Food Technol June, vol. 47, pp. 126–136. Ivanova, Evelina, Chipeva, Valentina, Ivanova, Iskra, Dousset, Xavier & Poncelet, Denis 2002, ‘Encapsulation of lactic acid bacteria in calcium alginate beads for bacteriocin production’, Journal of Culture Collections, vol. 3, pp. 53–58. Jamilah, B, Shu, CE, Kharidah, M, Dzulkifly, MA & Noranizan, A 2011, ‘Physico- chemical characteristics of red pitaya (Hylocereus polyrhizus) peel’, International Food Research Journal, vol. 18, pp. 279–286. Jantrawut, Pensak, Assifaoui, Ali & Chambin, Odile 2013, ‘Influence of low methoxyl pectin gel textures and in vitro release of rutin from calcium pectinate beads’, Carbohydrate Polymers, vol. 97, no. 2, pp. 335–342.

164

Jiang, Yang, Du, Jinhua, Zhang, Liguo & Li, Wenqian 2018, ‘Properties of pectin extracted from fermented and steeped hawthorn wine pomace: A comparison.’, Carbohydrate Polymers, vol. 197, Elsevier, pp. 174–182. Jurenka, Julie S 2012, ‘Bacillus coagulans: Monograph.’, Alternative medicine review, vol. 17, no. 1, pp. 76–81. Kailasapathy, Kaila 2002, ‘Microencapsulation of probiotic bacteria: technology and potential applications’, Current issues in intestinal microbiology, vol. 3, pp. 39–48. Kakimov, Aitbek, Kakimova, Zhaynagul, Mirasheva, Gulmira, Bepeyeva, Aigerim, Toleubekova, Sandugash, Jumazhanova, Madina, et al. 2017, ‘Amino acid composition of sour-milk drink with encapsulated probiotics’, Annual Research and Review in Biology, vol. 18, no. 1, Cantonment Public College, pp. 1–7. Kalita, Dipankar, Saikia, Sangeeta, Gautam, Gitanjali, Mukhopadhyay, Rupak & Mahanta, Charu Lata 2018, ‘Characteristics of synbiotic spray dried powder of litchi juice with Lactobacillus plantarum and different carrier materials’, LWT-Food Science and Technology, vol. 87, Elsevier, pp. 351–360. Kar, F & Arslan, N 1999, ‘Effect of temperature and concentration on viscosity of orange peel pectin solutions and intrinsic viscosity-molecular weight relationship’, Carbohydrate Polymers, vol. 40, pp. 277–284. Kastner, H, Einhorn-Stoll, U & Senge, B 2012, ‘Structure formation in sugar containing pectin gels – Influence of Ca2+ on the gelation of low-methoxylated pectin at acidic pH’, Food Hydrocolloids, vol. 27, no. 1, Elsevier, pp. 42–49. Kaufmann, Béatrice & Christen, Philippe 2002, ‘Recent extraction techniques for natural products: Microwave-assisted extraction and pressurised solvent extraction’, Phytochemical Analysis, vol. 13, no. 2, pp. 105–113. Kazemi, Milad, Khodaiyan, Faramarz & Hosseini, Seyed Saeid 2019a, ‘Eggplant peel as a high potential source of high methylated pectin: Ultrasonic extraction optimization and characterization’, LWT - Food Science and Technology, vol. 105, no. December 2018, Elsevier, pp. 182–189. Kazemi, Milad, Khodaiyan, Faramarz & Hosseini, Seyed Saeid 2019b, ‘Utilization of food processing wastes of eggplant as a high potential pectin source and characterization of extracted pectin’, Food Chemistry, vol. 294, Elsevier, pp. 339– 346. Khajeh, Mostafa, Akbari, Moghaddam Ali Reza & Sanchooli, Esmael 2010, ‘Application of Doehlert design in the optimization of microwave-assisted extraction for determination of zinc and copper in cereal samples using FAAS’, Food Analytical Methods, vol. 3, no. 3, pp. 133–137. Khorasani, Alireza Chackoshian & Shojaosadati, Seyed Abbas 2016, ‘Bacterial nanocellulose-pectin bionanocomposites as prebiotics against drying and gastrointestinal condition’, International Journal of Biological Macromolecules, vol. 83, Elsevier B.V., pp. 9–18. Khorasani, Alireza Chackoshian & Shojaosadati, Seyed Abbas 2017a, ‘Improvement of probiotic survival in fruit juice and under gastrointestinal conditions using pectin- nanochitin-nanolignocellulose as a novel prebiotic gastrointestinal-resistant matrix’, Applied Food Biotechnology, vol. 4, no. 3, pp. 179–191.

165

Khorasani, Alireza Chackoshian & Shojaosadati, Seyed Abbas 2017b, ‘Pectin-non-starch nanofibers biocomposites as novel gastrointestinal-resistant prebiotics’, International Journal of Biological Macromolecules, vol. 94, Elsevier, pp. 131–144. Khorasani, Alireza Chackoshian & Shojaosadati, Seyed Abbas 2017c, ‘Starch- and carboxymethylcellulose-coated bacterial nanocellulose-pectin bionanocomposite as novel protective prebiotic matrices’, Food Hydrocolloids, vol. 63, Elsevier Ltd, pp. 273–285. Kirk, James, McNair, Harold, Nielsen, S, Taylor, Stephen, Auerbach, Michael, Blumenthal, Herbert, et al. 1983, Food Chemicals Codex, Food Chemicals Codex, The National Academies Press, Washington, DC. Kliemann, Erika, De Simas, Karina Nunes, Amante, Edna R, Prudêncio, Elane Schwinden, Teófilo, Reinaldo F, Ferreira, Márcia MC & Amboni, Renata DMC 2009, ‘Optimisation of pectin acid extraction from passion fruit peel (Passiflora edulis flavicarpa) using response surface methodology’, International Journal of Food Science and Technology, vol. 44, no. 3, pp. 476–483. Kontogiorgos, V, Margelou, I, Georgiadis, N & Ritzoulis, C 2012, ‘Rheological characterization of okra pectins’, Food Hydrocolloids, vol. 29, no. 2, Elsevier Ltd, pp. 356–362. Kpodo, FM, Agbenorhevi, JK, Alba, K, Bingham, RJ, Oduro, IN, Morris, GA & Kontogiorgos, V 2017, ‘Pectin isolation and characterization from six okra genotypes’, Food Hydrocolloids, vol. 72, Elsevier Ltd, pp. 323–330. Kubrakova, I V & Toropchenova, ES 2008, ‘Microwave heating for enhancing efficiency of analytical operations’, Inorganic Materials, vol. 44, no. 14, Springer, pp. 1509– 1519. Kulkarni, SG & Vijayanand, P 2010, ‘Effect of extraction conditions on the quality characteristics of pectin from passion fruit peel (Passiflora edulis f. flavicarpa L.)’, LWT - Food Science and Technology, vol. 43, no. 7, Academic Press, pp. 1026– 1031. Kumar, Amit & Chauhan, Ghanshyam S 2010, ‘Extraction and characterization of pectin from apple pomace and its evaluation as lipase (steapsin) inhibitor’, Carbohydrate Polymers, vol. 82, no. 2, Elsevier, pp. 454–459. Kyomugasho, Clare, Christiaens, Stefanie, Shpigelman, Avi, Van Loey, Ann M & Hendrickx, Marc E 2015, ‘FT-IR spectroscopy, a reliable method for routine analysis of the degree of methylesterification of pectin in different fruit- and vegetable-based matrices’, Food Chemistry, vol. 176, Elsevier, pp. 82–90. Lahtinen, Sampo J 2012, ‘Probiotic viability – does it matter?’, Microbial Ecology in Health & Disease, vol. 23, pp. 10–14. Lara-Espinoza, Claudia, Carvajal-Millán, Elizabeth, Balandrán-Quintana, René, López- Franco, Yolanda & Rascón-Chu, Agustín 2018, ‘Pectin and pectin-based composite materials: Beyond food texture’, Molecules. Lee, BB, Ravindra, P & Chan, ES 2013, ‘Size and shape of calcium alginate beads produced by extrusion dripping’, Chemical Engineering and Technology, vol. 36, no. 10, pp. 1627–1642. Leslie, Mitch 2015, ‘Microbes aid cancer drugs’, Science, vol. 350, no. 6261, pp. 614–

166

615, viewed . Li, Ran, Zhang, Yufeng, Polk, D Brent, Tomasula, Peggy M, Yan, Fang & Liu, Lin Shu 2016, ‘Preserving viability of Lactobacillus rhamnosus GG in vitro and in vivo by a new encapsulation system’, Journal of Controlled Release, vol. 230, pp. 79–87. Li, Ying, Radoiu, Marilena, Fabiano-Tixier, Anne-Sylvie & Chemat, Farid 2012, ‘From laboratory to industry: Scale-up, quality, and safety consideration for microwave- assisted extraction’, in Farid Chemat & Giancarlo Cravotto (eds), Microwave- assisted extraction for bioactive compounds: Theory and Practice, Springer, Boston, MA, pp. 207–229. Liew, Shan Qin, Chin, Nyuk Ling & Yusof, Yus Aniza 2014, ‘Extraction and characterization of pectin from passion fruit peels’, Agriculture and Agricultural Science Procedia, vol. 2, Elsevier, pp. 231–236. Liew, Shan Qin, Chin, Nyuk Ling, Yusof, Yus Aniza & Cheok, Choon Yoong 2015, ‘Citric acid extraction of pectin from tropical fruit peels of passion fruit, dragon fruit and soursop’, Journal of Food, Agriculture and Environment, vol. 13, no. 2, pp. 45– 51. Liew, Shan Qin, Ngoh, Gek Cheng, Yusoff, Rozita & Teoh, Wen Hui 2018, ‘Acid and Deep Eutectic Solvent (DES) extraction of pectin from pomelo (Citrus grandis (L.) Osbeck) peels’, Biocatalysis and Agricultural Biotechnology, vol. 13, Elsevier, pp. 1–11. Liew, SQ, Chin, NL, Yusof, YA & Sowndhararajan, K 2016, ‘Comparison of acidic and enzymatic pectin extraction from passion fruit peels and its gel properties’, Journal of Food Process Engineering, vol. 39, pp. 501–511. Lin, Carol Sze Ki, Pfaltzgraff, Lucie A, Herrero-Davila, Lorenzo, Mubofu, Egid B, Abderrahim, Solhy, Clark, James H, et al. 2013, ‘Food waste as a valuable resource for the production of chemicals, materials and fuels. Current situation and global perspective’, Energy and Environmental Science, vol. 6, no. 2, Royal Society of Chemistry, pp. 426–464. Lin, MY & Young, CM 2000, ‘Folate levels in cultures of lactic acid bacteria’, International Dairy Journal, vol. 10, no. 5–6, pp. 409–413. Lin, Z, Fischer, J & Wicker, L 2016, ‘Intermolecular binding of blueberry pectin-rich fractions and anthocyanin’, Food Chemistry, vol. 194, Elsevier Ltd, pp. 986–993. Liu, Jun, Willför, Stefan & Xu, Chunlin 2015, ‘A review of bioactive plant polysaccharides: Biological activities, functionalization, and biomedical applications’, Bioactive Carbohydrates and Dietary Fibre, pp. 31–61. Liu, Lin Shu, Fishman, Marshall L, Kost, Joseph & Hicks, Kevin B 2003, ‘Pectin-based systems for colon-specific drug delivery via oral route’, Biomaterials, vol. 24, no. 19, pp. 3333–3343. Lootens, Didier, Capel, François, Durand, Dominique, Nicolai, Taco, Boulenguer, Patrick & Langendorff, Virginie 2003, ‘Influence of pH, Ca concentration, temperature and amidation on the gelation of low methoxyl pectin’, Food Hydrocolloids, vol. 17, Elsevier, pp. 237–244. Lu, Jike, Li, Jingjing, Jin, Ruichong, Li, Shufang, Yi, Juanjuan & Huang, Jinyong 2019, ‘Extraction and characterization of pectin from Premna microphylla Turcz leaves’,

167

International Journal of Biological Macromolecules, vol. 131, Elsevier, pp. 323– 328. Lucchesi, Marie E, Smadja, Jacqueline, Bradshaw, Steven, Louw, Willem & Chemat, Farid 2007, ‘Solvent free microwave extraction of Elletaria cardamomum L.: A multivariate study of a new technique for the extraction of essential oil’, Journal of Food Engineering, vol. 79, no. 3, pp. 1079–1086. Lutz, R, Aserin, A, Wicker, L & Garti, N 2009, ‘Structure and physical properties of pectins with block-wise distribution of carboxylic acid groups’, Food Hydrocolloids, vol. 23, Elsevier, pp. 786–794. Ma, Wenyan, Lu, Yanbin, Dai, Xiaojing, Liu, Rui, Hu, Ruilin & Pan, Yuanjiang 2009, ‘Determination of anti-tumor constitute mollugin from traditional Chinese medicine Rubia cordifolia: Comparative study of classical and microwave extraction techniques’, Separation Science and Technology, vol. 44, no. 4, pp. 995–1006. Mandal, S, Puniya, AK & Singh, K 2006, ‘Effect of alginate concentrations on survival of microencapsulated Lactobacillus casei NCDC-298’, International Dairy Journal, vol. 16, no. 10, pp. 1190–1195, viewed . Mandawgade, SD & Patravale, VandanaB 2008, ‘Formulation and evaluation of exotic fat based cosmeceuticals for skin repair’, Indian Journal of Pharmaceutical Sciences, vol. 70, no. 4, pp. 539–542. Manrique, Guillermo D & Lajolo, Franco M 2002, ‘FT-IR spectroscopy as a tool for measuring degree of methyl esterification in pectins isolated from ripening papaya fruit’, Postharvest Biology and Technology, vol. 25, no. 1, pp. 99–107. Manuel, Suba GA 2014, ‘Fruit waste pectin in enhancing the establishment of probiotic bacteria’, Journal of Nutritional Health & Food Engineering, vol. 1, no. 3, pp. 124– 126. Maran, J Prakash & Prakash, K Arun 2015, ‘Process variables influence on microwave assisted extraction of pectin from waste Carcia papaya L. peel’, International Journal of Biological Macromolecules, vol. 73, pp. 202–206. Markowiak, Paulina & Śliżewska, Katarzyna 2017, ‘Review: Effects of probiotics, prebiotics, and synbiotics on human health’, Nutrients, vol. 9, no. 9, Multidisciplinary Digital Publishing Institute, p. 1021. Martin-Dejardin, F, Ebel, B, Lemetais, G, Nguyen Thi Minh, H, Gervais, P, Cachon, R & Chambin, O 2013, ‘A way to follow the viability of encapsulated Bifidobacterium bifidum subjected to a freeze-drying process in order to target the colon: Interest of flow cytometry’, European Journal of Pharmaceutical Sciences, vol. 49, no. 2, Elsevier B.V., pp. 166–174. Matveev, Yu I, Grinberg, V Ya & Tolstoguzov, VB 2000, ‘The plasticizing effect of water on proteins, polysaccharides and their mixtures. Glassy state of biopolymers, food and seeds’, Food Hydrocolloids, vol. 14, Elsevier, pp. 425–437. Maxwell, Ellen G, Belshaw, Nigel J, Waldron, Keith W & Morris, Victor J 2012, ‘Pectin - An emerging new bioactive food polysaccharide’, Trends in Food Science and Technology, vol. 24, no. 2, Elsevier Ltd, pp. 64–73. May, Colin D 1990, ‘Industrial pectins: Sources, production and applications’,

168

Carbohydrate Polymers, vol. 12, no. 1, pp. 79–99. McClements, David Julian 2017, ‘Designing biopolymer microgels to encapsulate, protect and deliver bioactive components: Physicochemical aspects’, Advances in Colloid and Interface Science, pp. 31–59. Meyer, Ruth & Krueger, David 2001, Minitab guide to statistics, Prentice Hall PTR. Miceli-Garcia, Lucia Guadalupe 2014, Pectin from apple pomace - extraction, characterization, and utilization in encapsulating alpha-tocopherol acetate, University of Nebraska-Lincoln, University of Nebraska-Lincoln, viewed . Mierczyńska, Joanna, Cybulska, Justyna & Zdunek, Artur 2017, ‘Rheological and chemical properties of pectin enriched fractions from different sources extracted with citric acid’, Carbohydrate Polymers, vol. 156, pp. 443–451. Míguez, Beatriz, Gómez, Belén, Gullón, Patricia, Gullón, Beatriz & Alonso, José Luis 2016, ‘Pectic oligosaccharides and other emerging prebiotics’, in Venketeshwer Rao & Leticia Rao (eds), Probiotics and Prebiotics in Human Nutrition and Health, IntechOpen, London. Min, Bockki, Bae, In Young, Lee, Hyeon Gyu, Yoo, Sang Ho & Lee, Suyong 2010, ‘Utilization of pectin-enriched materials from apple pomace as a fat replacer in a model food system’, Bioresource Technology, vol. 101, no. 14, Elsevier Ltd, pp. 5414–5418. Minkov, S, Minchev, A & Paev, K 1996, ‘Modelling of the hydrolysis and extraction of apple pectin’, Journal of Food Engineering, vol. 29, no. 1, Elsevier, pp. 107–113. Mishra, Munmaya 2015, ‘Microencapsulation by spray drying’, Handbook of Encapsulation and Controlled Release, CRC Press, London. Misra, NN, Martynenko, Alex, Chemat, Farid, Paniwnyk, Larysa, Barba, Francisco J & Jambrak, Anet Režek 2018, ‘Thermodynamics, transport phenomena, and electrochemistry of external field-assisted nonthermal food technologies’, Critical reviews in food science and nutrition, vol. 58, no. 11, Taylor & Francis, pp. 1832– 1863. Mizrahi, Yosef, Nerd, Avinoam & Nobel, Park S 2010, ‘Cacti as Crops’, in Jules Janick (ed.), Horticultural Reviews, pp. 291–319. Mohamadzadeh, J, Sadeghi-Mahoonak, AR, Yaghbani, M & Aalami, M 2010, ‘Extraction of pectin from sunflower head residues of selected Iranian cultivars’, World Applied Sciences Journal, vol. 8, no. 1, IDOSI Publications, pp. 21–24. Mohnen, Debra 2008, ‘Pectin structure and biosynthesis’, Current Opinion in Plant Biology, vol. 11, no. 3, pp. 266–277. Mohos, Ferenc Á 2010, ‘Introduction to Food Colloids’, Confectionery and Chocolate Engineering, Oxford University Press, pp. 176–233. Mokni Ghribi, Abir, Sila, Assaâd, Maklouf Gafsi, Ines, Blecker, Christophe, Danthine, Sabine, Attia, Hamadi, et al. 2015, ‘Structural, functional, and ACE inhibitory properties of water-soluble polysaccharides from chickpea flours’, International Journal of Biological Macromolecules, vol. 75, Elsevier B.V., pp. 276–282, viewed .

169

Mollea, C, Chiampo, F & Conti, R 2008, ‘Extraction and characterization of pectins from cocoa husks: A preliminary study’, Food Chemistry, vol. 107, no. 3, Elsevier, pp. 1353–1356. Monsoor, MA & Proctor, A 2001, ‘Preparation and functional properties of soy hull pectin’, Journal of the American Oil Chemists’ Society, vol. 78, no. 7, Springer, pp. 709–713. Montoya-Arroyo, Alexander, Schweiggert, Ralf M, Pineda-Castro, María Lourdes, Sramek, Martin, Kohlus, Reinhard, Carle, Reinhold & Esquivel, Patricia 2014, ‘Characterization of cell wall polysaccharides of purple pitaya (Hylocereus sp.) pericarp’, Food Hydrocolloids, vol. 35, Elsevier, pp. 557–564. Morris, Max D 2000, ‘A class of three-level experimental designs for response surface modeling’, Technometrics, vol. 42, no. 2, Taylor & Francis, pp. 111–121. Mosayebi, Vahid & Yazdi, Farideh Tabatabaei 2015, ‘Optimization of microwave assisted extraction ( MAE ) of pectin from black mulberry ( Morus nigra L .) pomace’, Journal of Food and Bioprocess Engineering, vol. 34, pp. 34–48. Mosilhey, Sameh Hassan 2003, Influence of Different Capsule Materials on the Physiological Properties of Microencapsulated Lactobacillus acidophilus, Universitäts-und Landesbibliothek Bonn. Muhammad, Kharidah, Nur, Nur Izalin, Gannasin, Sri Puvanesvari, Mohd. Adzahan, Noranizan & Bakar, Jamilah 2014, ‘High methoxyl pectin from dragon fruit (Hylocereus polyrhizus) peel’, Food Hydrocolloids, vol. 42, no. P2, Elsevier, pp. 289–297. Muhammad, Zafarullah, Ramzan, Rabia, Huo, Gui-Cheng, Tian, Hui & Bian, Xin 2017, ‘Integration of polysaccharide-thermoprotectant formulations for microencapsulation of Lactobacillus plantarum, appraisal of survivability and physico-biochemical properties during storage of spray dried powders’, Food Hydrocolloids, vol. 66, pp. 286–295. Muhoza, Bertrand, Xia, Shuqin, Cai, Jibao, Zhang, Xiaoming, Duhoranimana, Emmanuel & Su, Jiakun 2019, ‘Gelatin and pectin complex coacervates as carriers for cinnamaldehyde: Effect of pectin esterification degree on coacervate formation, and enhanced thermal stability’, Food Hydrocolloids, vol. 87, Elsevier, pp. 712–722. Munarin, Fabiola, Petrini, Paola, Tanzi, Maria C, Barbosa, Mario A & Granja, Pedro L 2012, ‘Biofunctional chemically modified pectin for cell delivery’, Soft Matter, vol. 8, no. 17, Royal Society of Chemistry, pp. 4731–4739. Muñoz-Almagro, Nerea, Valadez-Carmona, Lourdes, Mendiola, José A, Ibáñez, Elena & Villamiel, Mar 2019, ‘Structural characterisation of pectin obtained from cacao pod husk. Comparison of conventional and subcritical water extraction’, Carbohydrate Polymers, vol. 217, Elsevier, pp. 69–78. Mussatto, Solange I & Mancilha, Ismael M 2007, ‘Non-digestible oligosaccharides: a review’, Carbohydrate polymers, vol. 68, no. 3, Elsevier, pp. 587–597. Nep, EI, Sims, IM, Morris, GA, Kontogiorgos, V & Smith, AM 2016, ‘Evaluation of some important physicochemical properties of starch free grewia gum’, Food Hydrocolloids, vol. 53, Elsevier, pp. 134–140. Nesterenko, Alla, Alric, Isabelle, Silvestre, Françoise & Durrieu, Vanessa 2013,

170

‘Vegetable proteins in microencapsulation: A review of recent interventions and their effectiveness’, Industrial Crops and Products, vol. 42, pp. 469–479. Noh, Juran, Kim, Jin, Kim, Jin Sic, Chung, Young Soo, Chang, Suk Tai & Park, Juhyun 2018, ‘Microencapsulation by pectin for multi-components carriers bearing both hydrophobic and hydrophilic active agents’, Carbohydrate Polymers, vol. 182, pp. 172–179. Noreen, Aqdas, Nazli, Zill i Hum, Akram, Javeria, Rasul, Ijaz, Mansha, Asim, Yaqoob, Nazia, et al. 2017, ‘Pectins functionalized biomaterials; a new viable approach for biomedical applications: A review’, International Journal of Biological Macromolecules, vol. 101, pp. 254–272. Nualkaekul, Sawaminee, Cook, Michael T, Khutoryanskiy, Vitaliy V. & Charalampopoulos, Dimitris 2013, ‘Influence of encapsulation and coating materials on the survival of Lactobacillus plantarum and Bifidobacterium longum in fruit juices’, Food Research International, vol. 53, no. 1, pp. 304–311. Oakenfull, David & Scott, Alan 1984, ‘Hydrophobic interaction in the gelation of high methoxyl pectins’, Journal of Food Science, vol. 49, no. 4, pp. 1093–1098. Odun-Ayo, Frederick, Mellem, John & Reddy, Lalini 2016, ‘Improving the survival of probiotic in simulated conditions and azoxymethane-induced colon tumour bearing mice using modified citrus pectin-alginate microencapsulation’, African Journal of Traditional, Complementary and Alternative Medicines, vol. 13, no. 2, pp. 101–109. Odun-Ayo, Frederick, Mellem, John & Reddy, Lalini 2017, ‘The effect of modified citrus pectin-probiotic on faecal lactobacilli in Balb/c mice’, Food Science and Technology, vol. 37, no. 3, pp. 478–482. Ohashi, Yasuo, Nakai, Satoshi, Tsukamoto, Taiji, Masumori, Naoya, Akaza, Hideyuki, Miyanaga, Naoto, et al. 2002, ‘Habitual intake of lactic acid bacteria and risk reduction of bladder cancer’, Urologia Internationalis, vol. 68, no. 4, Karger Publishers, pp. 273–280. Olano-Martin, E, Gibson, GR & Rastall, RA 2002, ‘Comparison of the in vitro bifidogenic properties of pectins and pectic-oligosaccharides’, Journal of Applied Microbiology, vol. 93, no. 3, pp. 505–511. Oliveira, AC, Moretti, TS, Boschini, C, Baliero, JCC, Freitas, O & Favaro-Trindade, CS 2007, ‘Stability of microencapsulated B. lactis (BI 01) and L. acidophilus (LAC 4) by complex coacervation followed by spray drying’, Journal of Microencapsulation, vol. 24, no. 7, pp. 685–693. Oliveira, Cibele Freitas, Giordani, Diego, Gurak, Poliana Deyse, Cladera-Olivera, Florencia & Marczak, Ligia Damasceno Ferreira 2015, ‘Extraction of pectin from passion fruit peel using moderate electric field and conventional heating extraction methods’, Innovative Food Science & Emerging Technologies, vol. 29, Elsevier, pp. 201–208. Ouwehand, Arthur C, Salminen, Seppo & Isolauri, Erika 2002, ‘Probiotics: an overview of beneficial effects’, Lactic Acid Bacteria: Genetics, Metabolism and Applications, Springer, pp. 279–289. Ouyang, Wei, Chen, Hongmei, Jones, Mitchell L, Metz, Terrence, Haque, Tasima, Martoni, Christopher & Prakash, Satya 2004, ‘Artificial cell microcapsule for oral delivery of live bacterial cells for therapy: Design, preparation, and in-vitro

171

characterization’, Journal of Pharmacy and Pharmaceutical Sciences, vol. 7, no. 3, pp. 315–324. Panchev, IN, Slavov, A, Nikolova, Kr & Kovacheva, D 2010, ‘On the water-sorption properties of pectin’, Food Hydrocolloids, vol. 24, Elsevier, pp. 763–769. Parfitt, Julian, Barthel, Mark & MacNaughton, Sarah 2010, ‘Food waste within food supply chains: Quantification and potential for change to 2050’, Philosophical Transactions of the Royal Society B: Biological Sciences, vol. 365, no. 1554, pp. 3065–3081. Park, Hyun Jin, Chen, Xi Guang, Li, Xiao Yan, Cha, Dong-Su & Sun, Zhong Wu 2010, ‘Preparation of alginate/chitosan/carboxymethyl chitosan complex microcapsules and application in Lactobacillus casei ATCC 393’, Carbohydrate Polymers, vol. 83, pp. 1479–1485. Pasandide, Bahare, Khodaiyan, Faramarz, Mousavi, Zeinab E & Hosseini, Seyed Saeid 2017, ‘Optimization of aqueous pectin extraction from Citrus medica peel’, Carbohydrate Polymers, vol. 178, Elsevier, pp. 27–33. Pasparakis, George & Bouropoulos, Nikolaos 2006, ‘Swelling studies and in vitro release of verapamil from calcium alginate and calcium alginate-chitosan beads’, International Journal of Pharmaceutics, vol. 323, pp. 34–42. Pawar, Atmaram P, Gadhe, Anil R, Venkatachalam, Prabakaran, Sher, Praveen & Mahadik, Kakasaheb R 2008, ‘Effect of core and surface cross-linking on the entrapment of metronidazole in pectin beads’, Acta Pharmaceutica, vol. 58, no. 1, pp. 75–85. Pereira, Paulo Henrique F, Oliveira, Túlio Ítalo S, Rosa, Morsyleide F, Cavalcante, Fabio Lima, Moates, Graham K, Wellner, Nikolaus, et al. 2016, ‘Pectin extraction from pomegranate peels with citric acid’, International Journal of Biological Macromolecules, vol. 88, pp. 373–379. Perez, Adrián A, Sánchez, Cecilio Carrera, Patino, Juan M Rodríguez, Rubiolo, Amelia C & Santiago, Liliana G 2012, ‘Effect of enzymatic hydrolysis and polysaccharide addition on the β-lactoglobulin adsorption at the air–water interface’, Journal of food engineering, vol. 109, no. 4, Elsevier, pp. 712–720. Pfaltzgraff, Lucie A, De Bruyn, Mario, Cooper, Emma C, Budarin, Vitaly & Clark, James H 2013, ‘Food waste biomass: A resource for high-value chemicals’, Green Chemistry, vol. 15, no. 2, pp. 307–314. Pierucci, Sauro, Piazza, Laura, Moia, Thais A, Pimentel, Tatiana C, Barão, Carlos Eduardo, Feihrmann, Andresa C, et al. 2019, ‘Bioactive compounds and pectin from residues of the passion fruit processing: Extraction using green technology and characterization’, Chemical Engineering Transactions, vol. 75, pp. 157–162. Pinang, Pulau 2011, ‘Pectins from dragon fruit (Hylocereus polyrhizus) peel’, Malays. Appl. Biol., vol. 40, no. 1, pp. 19–23. Pinheiro, Eloísa Rovaris, Silva, Iolanda MDA, Gonzaga, Luciano V., Amante, Edna R, Teófilo, Reinaldo F, Ferreira, Márcia MC & Amboni, Renata DMC 2008, ‘Optimization of extraction of high-ester pectin from passion fruit peel (Passiflora edulis flavicarpa) with citric acid by using response surface methodology’, Bioresource Technology, vol. 99, no. 13, Elsevier, pp. 5561–5566.

172

Ponmurugan, K, Al-Dhabi, Naif Abdullah, Maran, J Prakash, Karthikeyan, K, Moothy, I Ganesh, Sivarajasekar, N & Manoj, J Jony Blessing 2017, ‘Ultrasound assisted pectic polysaccharide extraction and its characterization from waste heads of Helianthus annus’, Carbohydrate Polymers, vol. 173, Elsevier, pp. 707–713. Pop, Oana L, Vodnar, Dan C, Suharoschi, Ramona, Mudura, Elena & Socaciu, Carmen 2016, ‘L. plantarum ATCC 8014 entrapment with prebiotics and lucerne green juice and their behavior in simulated gastrointestinal conditions’, Journal of Food Process Engineering, vol. 39, no. 5, pp. 433–441. Pothakamury, Usha R & Barbosa-Cánovas, Gustavo V 1995, ‘Fundamental aspects of controlled release in foods’, Trends in food science & technology, vol. 6, no. 12, Elsevier, pp. 397–406. Prado, Samira Bernardino Ramos, Shiga, Tânia Misuzu, Harazono, Yosuke, Hogan, Victor A, Raz, Avraham, Carpita, Nicholas C & Fabi, João Paulo 2019, ‘Migration and proliferation of cancer cells in culture are differentially affected by molecular size of modified citrus pectin’, Carbohydrate Polymers, vol. 211, Elsevier, pp. 141– 151. Prakash Maran, J, Sivakumar, V, Thirugnanasambandham, K & Sridhar, R 2013, ‘Optimization of microwave assisted extraction of pectin from orange peel’, Carbohydrate Polymers, vol. 97, no. 2, Elsevier, pp. 703–709. Prakash, Satya, Tomaro-Duchesneau, Catherine, Saha, Shyamali & Cantor, Arielle 2011, ‘The gut microbiota and human health with an emphasis on the use of microencapsulated bacterial cells’, Journal of Biomedicine and Biotechnology, vol. 2011, pp. 1–12. Rahmati, S, Abdullah, A, Momeny, E & Kang, OL 2015, ‘Optimization studies on microwave assisted extraction of dragon fruit (Hylocereus polyrhizus) peel pectin using response surface methodology’, International Food Research Journal, vol. 22, no. 1, pp. 233–239. Ralet, Marie-Christine, Dronnet, Vincent & Thibault, Jean-François 2001, ‘Enzymatcally and chemically de-esterified lime pectins: characterisation, polyelectrolyte behaviour and calcium binding properties’, Carbohydrate Research, vol. 336, pp. 117–125. Ranganna, S 2004, Handbook of Analysis of quality control for fruit and vegetable products, Co. Ltd. New Delhi, 2nd ed, TataGrawHill, Newdelhi. Rao, M Anandha 2014, ‘Rheological behavior of food gels’, Rheology of Fluid and Semisolid Foods Principles and Applications, Springer, Boston, MA, pp. 331–390. Ribeiro, Maria Cecília E, Chaves, Karina S, Gebara, Clarice, Infante, Flávia NS, Grosso, Carlos RF & Gigante, Mirna L 2014, ‘Effect of microencapsulation of Lactobacillus acidophilus LA-5 on physicochemical, sensory and microbiological characteristics of stirred probiotic yoghurt’, Food Research International, vol. 66, Elsevier, pp. 424–431. Roberfroid, Marcel 1993, ‘Dietary fiber, inulin, and oligofructose: a review comparing their physiological effects’, Critical Reviews in Food Science & Nutrition, vol. 33, no. 2, Taylor & Francis, pp. 103–148. Rodsamran, Pattrathip & Sothornvit, Rungsinee 2019, ‘Microwave heating extraction of pectin from lime peel: Characterization and properties compared with the

173

conventional heating method’, Food Chemistry, vol. 278, Elsevier, pp. 364–372. Rokka, Susanna & Rantamäki, Pirjo 2010, ‘Protecting probiotic bacteria by microencapsulation: Challenges for industrial applications’, European Food Research and Technology, vol. 231, Springer Berlin, pp. 1–12. Rolin, Claus 2002, ‘Commercial pectin preparations’, Pectins and their manipulation, Blackwell Publishing Oxford, pp. 222–241. Romo, G & Nava, G 2005, ‘A protocol for evaluating locally-sourced alternative feed ingredients: an example using passion fruit seed meal.’, Proceedings of Alltech’s 21st Annual Symposium, Alltech UK, Lexington, pp. 43–53. Saberian, Hamed, Hamidi-Esfahani, Zohreh, Ahmadi Gavlighi, Hassan, Banakar, Ahmad & Barzegar, Mohsen 2018, ‘The potential of ohmic heating for pectin extraction from orange waste’, Journal of Food Processing and Preservation, vol. 42, no. 2, pp. 1–9. Sabikhi, Latha, Babu, R, Thompkinson, D Kapila & Kapila, Suman 2010, ‘Resistance of microencapsulated Lactobacillus acidophilus LA1 to processing treatments and simulated gut conditions’, Food and Bioprocess Technology, vol. 3, no. 4, Springer, pp. 586–593. Salar-Behzadi, Sharareh, Wu, Shengqian, Toegel, Stefan, Hofrichter, Maria, Altenburger, Iris, Unger, Frank M, et al. 2013, ‘Impact of heat treatment and spray drying on cellular properties and culturability of Bifidobacterium bifidum BB-12’, Food Research International, vol. 54, no. 1, pp. 93–101. Samavati, Vahid 2013, ‘Polysaccharide extraction from Abelmoschus esculentus: Optimization by response surface methodology’, Carbohydrate Polymers, vol. 95, no. 1, Elsevier, pp. 588–597. Sandoval-Castilla, O, Lobato-Calleros, C, García-Galindo, HS, Alvarez-Ramírez, J & Vernon-Carter, EJ 2010, ‘Textural properties of alginate-pectin beads and survivability of entrapped Lb. casei in simulated gastrointestinal conditions and in yoghurt’, Food Research International, vol. 43, no. 1, pp. 111–117. Santivarangkna, C, Kulozik, U & Foerst, P 2008, ‘Inactivation mechanisms of lactic acid starter cultures preserved by drying processes’, Journal of Applied Microbiology. Santos, Eloyza Karoline Rozendo, Azoubel, Patricia Moreira & Gouveia, Ester Ribeiro 2017, ‘Better pectin yield from passion fruit peel (Passiflora edulis f. flavicarpa): from shaker or ultrasound? A comparison’, Waste and Biomass Valorization, vol. 8, pp. 905–910. Sarao, Loveleen Kaur & Arora, M 2017, ‘Probiotics, prebiotics, and microencapsulation: A review’, Critical Reviews in Food Science and Nutrition, vol. 57, no. 2, pp. 344– 371. Sarkar, S 2010, ‘Approaches for enhancing the viability of probiotics: a review’, British Food Journal, vol. 112, no. 4, Emerald Group Publishing Limited, pp. 329–349. Sawayama, S, Kawabata, A, Nakahara, H & Kamata, T 1988, ‘A light scattering study on the effects of pH on pectin aggregation in aqueous solution’, Topics in Catalysis, vol. 2, no. 1, Elsevier Ltd., pp. 31–37. Schrezenmeir, J & de Vrese, M 2001, ‘Probiotics, prebiotics, and synbiotics-approaching a definition’, The American journal of clinical nutrition, vol. 73, United States, pp.

174

361S-364S. Secretariat, Executive & Pharmacopeia, US 2010, ‘Commentary—to USP31-NF26’, pp. 1–7. Seifert, Douglas B & Phillips, Janice A 1997, ‘Production of small, monodispersed alginate beads for cell immobilization’, Biotechnology Progress, vol. 13, no. 5, pp. 562–568. Seixas, Fernanda L, Fukuda, Deise L, Turbiani, Franciele R B, Garcia, Patrícia S, Petkowicz, Carmen L de O, Jagadevan, Sheeja & Gimenes, Marcelino L 2014, ‘Extraction of pectin from passion fruit peel (Passiflora edulis f.flavicarpa) by microwave-induced heating’, Food Hydrocolloids, vol. 38, pp. 189–192. Seixas, Fernanda L, Fukuda, Deise L, Turbiani, Franciele R.B., Garcia, Patrícia S, Petkowicz, Carmen LdO, Jagadevan, Sheeja & Gimenes, Marcelino L 2014, ‘Extraction of pectin from passion fruit peel (Passiflora edulis f.flavicarpa) by microwave-induced heating’, Food Hydrocolloids, vol. 38, Elsevier, pp. 186–192. Shafie, Muhammad Hakimin, Yusof, Rizana & Gan, Chee-Yuen 2019, ‘Deep eutectic solvents (DES) mediated extraction of pectin from Averrhoa bilimbi: Optimization and characterization studies’, Carbohydrate Polymers, vol. 216, Elsevier, pp. 303– 311, viewed 24 July, 2019, . Shafie, Muhammad Hakimin, Yusof, Rizana & Gan, Chee Yuen 2019, ‘Deep eutectic solvents (DES) mediated extraction of pectin from Averrhoa bilimbi: Optimization and characterization studies’, Carbohydrate Polymers, vol. 216, pp. 303–311. Shoji, AS, Oliveira, AC, Balieiro, JCC, Freitas, O, Thomazini, M, Heinemann, RJB, et al. 2013, ‘Viability of L. acidophilus microcapsules and their application to buffalo milk yoghurt’, Food and Bioproducts Processing, vol. 91, no. 2, pp. 83–88. Sila, Assaâd, Bayar, Nadia, Ghazala, Imen, Bougatef, Ali, Ellouz-Ghorbel, Raoudha & Ellouz-Chaabouni, Semia 2014, ‘Water-soluble polysaccharides from agro- industrial by-products: Functional and biological properties’, International Journal of Biological Macromolecules, vol. 69, Elsevier, pp. 236–243. Da Silva, Larissa Morais Ribeiro, De Figueiredo, Evania Altina Teixeira, Ricardo, Nagila Maria Pontes Silva, Vieira, Icaro Gusmao Pinto, De Figueiredo, Raimundo Wilane, Brasil, Isabella Montenegro & Gomes, Carmen L 2014, ‘Quantification of bioactive compounds in pulps and by-products of tropical fruits from Brazil’, Food Chemistry, vol. 143, pp. 398–404. Simmaky, S & Jaanaki, G 2014, ‘Extraction and characterization of pectin from yellow passion fruit Passiflora edulis f.flavicarpa L) endocarp peel’, SAITM Research Symposium on Engineering Advancements, pp. 27–29. Singh, Ramkrishna D, Banerjee, Jhumur & Arora, Amit 2015, ‘Prebiotic potential of oligosaccharides: A focus on xylan derived oligosaccharides’, Bioactive Carbohydrates and Dietary Fibre, vol. 5, no. 1, pp. 19–30. Smidsrød, Olav & Skjåk-Bræk, Gudmund 1990, ‘Alginate as immobilization matrix for cells’, Trends in Biotechnology, vol. 8, no. C, pp. 71–78. Soria, Ana Cristina, Ruiz-Aceituno, Laura, Ramos, Lourdes & Sanz, Luz Maria 2014,

175

‘Microwave-assisted extraction of polysaccharides’, in Ramawat K. & Mérillon JM. (eds), Polysaccharides, Springer, pp. 1–18. Sriamornsak, Pornsak 2003, ‘Chemistry of pectin and its pharmaceutical uses: a review’, Silpakorn University International Journal, vol. 3, no. 1–2, pp. 206–228. Srivastava, Pranati & Malviya, Rishabha 2011, ‘Sources of pectin, extraction and its applications in pharmaceutical industry - an overview’, Indian Journal of Natural Products and Resources, vol. 2, no. 1, pp. 10–18. Stenmarck, Åsa, Jensen, Carl, Quested, Tom, Moates, Graham, Cseh, Balázs, Juul, Selina, et al. 2016, Estimates of European food waste levels, FUSIONS, Stockholm. Su, Dong Lin, Li, Pei Jun, Quek, Siew Young, Huang, Zhi Qin, Yuan, Yu Jun, Li, Gao Yang & Shan, Yang 2019, ‘Efficient extraction and characterization of pectin from orange peel by a combined surfactant and microwave assisted process’, Food Chemistry, vol. 286, Elsevier, pp. 1–7. Sun, Qingshen, Wang, Fuying, Han, Dequan, Zhao, Yao, Liu, Zhidong, Lei, Hong, et al. 2014, ‘Preparation and optimization of soy protein isolate-high methoxy pectin microcapsules loaded with Lactobacillus delbrueckii’, International Journal of Food Science and Technology, vol. 49, no. 5, pp. 1287–1293. Sun, Qingshen, Zhang, Yanyan, Ma, Xiuyan, Han, Dequan, Shi, Yue, Sun, Quan, et al. 2017, ‘Preparation and in vitro characterization of Lactobacillus plantarum and Lactobacillus fermentum beads using low methoxyl pectin’, SCIREA Journal of Food, vol. 2, no. 1, pp. 1–14. Tanangteerapong, Duangkanok 2014, Understanding the physiology of probiotic yeast cells under different formulation, processing and environmental conditions, University of Birmingham. Tang, Pek Yee, Kek, Thiam Seng, Gan, Cheng Zu, Hee, Chiu Yen, Chong, Cheng Hin & Woo, Kwan Kit 2011, ‘Yield and some chemical properties of pectin extracted from the peels of dragon fruit [Hylocereus polyrhizus (Weber) Britton and Rose]’, Philippine Agricultural Scientist, vol. 94, no. 3, pp. 307–311. Tang, P. Y., Wong, CJ & Woo, KK 2011, ‘Optimization of pectin extraction from peel of dragon fruit (Hylocereus polyhizus)’, Asian Journal of Biological Sciences, vol. 4, no. 2, pp. 189–195. Teoh, Peck Lian, Mirhosseini, Hamed, Mustafa, Shuhaimi, Hussin, Anis Shobirin Meor & Manap, Mohd Yazid Abdul 2011, ‘Recent approaches in the development of encapsulated delivery systems for probiotics’, Food Biotechnology, vol. 25, no. 1, pp. 77–101. Thakur, Beli R, Singh, Rakesh K & Handa, Avtar K 1997, ‘Chemistry and Uses of Pectin - A Review’, Critical Reviews in Food Science and Nutrition, vol. 37, no. 1, pp. 47– 73, viewed . Thakur, Beli R, Singh, Rakesh K, Handa, Avtar K & Rao, MA 1997, ‘Chemistry and uses of pectin—a review’, Critical Reviews in Food Science & Nutrition, vol. 37, no. 1, pp. 47–73. Thibault, Jean François, De Dreu, René, Geraeds, Carolien CJM & Rombouts, Frank M 1988, ‘Studies on extraction of pectins from citrus peels, apple marks and sugar-beet

176

pulps with arabinanase and galactanase’, Carbohydrate Polymers, vol. 9, pp. 119– 131. Thirugnanasambandham, K, Sivakumar, V & Prakash Maran, J 2014, ‘Process optimization and analysis of microwave assisted extraction of pectin from dragon fruit peel’, Carbohydrate Polymers, vol. 112, Elsevier, pp. 622–626. Toledo, Nataly, Camargo, Adriano, Ramos, Paula, Button, David, Granato, Daniel & Canniatti-Brazaca, Solange 2018, ‘Potentials and pitfalls on the use of passion fruit by-products in drinkable yogurt: Physicochemical, technological, microbiological, and sensory aspects’, Beverages, vol. 4, no. 47, MDPI AG, pp. 1–15. Tongkham, Nudthapong, Juntasalay, Boonyawee, Lasunon, Patareeya & Sengkhamparn, Nipaporn 2017, ‘Dragon fruit peel pectin: Microwave-assisted extraction and fuzzy assessment’, Agriculture and Natural Resources, vol. 51, no. 4, Elsevier, pp. 262– 267. Tonucci, Livia Bordalo, Olbrich dos Santos, Karina Maria, Licursi de Oliveira, Leandro, Rocha Ribeiro, Sonia Machado & Duarte Martino, Hercia Stampini 2017, ‘Clinical application of probiotics in type 2 diabetes mellitus: A randomized, double-blind, placebo-controlled study’, Clinical Nutrition, vol. 36, no. 1, pp. 85–92. Tsoga, A, Richardson, R. & Morris, E. 2004, ‘Role of cosolutes in gelation of high- methoxy pectin. Part 1. Comparison of sugars and polyols’, Food Hydrocolloids, vol. 18, no. 6, pp. 907–919. Udenigwe, Chibuike C & Aluko, Rotimi E 2012, ‘Food protein-derived bioactive peptides: Production, processing, and potential health benefits’, Journal of Food Science, vol. 71, no. 1, pp. R11–R24. Usmiati, Sri, Mangunwidjaja, Djumali, Noor, Erliza, Richana, Nur & Prangdimurti, Endang 2017, ‘Chracteristics improvement of probiotic encapsulated with functional nanoparticles’, Advances in Biotechnology & Microbiology, vol. 2, no. 5, pp. 2474– 7637. Vaclavik, Vickie A, Christian, Elizabeth W & Christian, Elizabeth W 2008, Essentials of food science, Springer. Vaidya, Ankur, Jain, Aviral, Khare, Piush, Agrawal, Ram K & Jain, Sanjay K 2009, ‘Metronidazole loaded pectin microspheres for colon targeting’, Journal of pharmaceutical sciences, vol. 98, no. 11, Wiley Online Library, pp. 4229–4236. Vandenplas, Y, De Greef, E, Devreker, T, Veereman-Wauters, G & Hauser, B 2013, ‘Probiotics and prebiotics in infants and children’, Current infectious disease reports, vol. 15, no. 3, Springer, pp. 251–262. Vasco-Correa, Juliana & Zapata Zapata, Arley D 2017, ‘Enzymatic extraction of pectin from passion fruit peel (Passiflora edulis f. flavicarpa) at laboratory and bench scale’, LWT - Food Science and Technology, vol. 80, Academic Press, pp. 280–285. Vaziri, Asma Sadat, Alemzadeh, Iran, Vossoughi, Manouchehr & Khorasani, Alireza Chackoshian 2018, ‘Co-microencapsulation of Lactobacillus plantarum and DHA fatty acid in alginate-pectin-gelatin biocomposites’, Carbohydrate Polymers, vol. 199, Elsevier, pp. 266–275. Venzon, Simoni Spohr, Canteri, Maria Helene Giovanetti, Granato, Daniel, Demczuk, Bogdan, Maciel, Giselle Maria, Stafussa, Ana Paula & Haminiuk, Charles Windson

177

Isidoro 2015, ‘Physicochemical properties of modified citrus pectins extracted from orange pomace’, Journal of Food Science and Technology, vol. 52, no. 7, pp. 4102– 4112. Vian, Maryline Abert, Maingonnat, Jean Francois & Chemat, Farid 2009, ‘Clean recovery of antioxidant flavonoids from onions: Optimising solvent free microwave extraction method’, Journal of Chromatography A, vol. 1216, no. 45, Elsevier, pp. 7700–7707. Vieira, Antonio Diogo Silva, Bedani, Raquel, Albuquerque, MAC, Biscola, Vanessa & Saad, Susana Marta Isay 2017, ‘The impact of fruit and soybean by-products and amaranth on the growth of probiotic and starter microorganisms’, Food Research International, vol. 97, Elsevier Ltd, pp. 356–363. Vithanage, Chandra R, Grimson, Malcolm J, Wills, Peter R, Harrison, Paul & Smith, Bronwen G 2010, ‘Rheological and structural properties of high-methoxyl esterified, low-methoxyl esterified and low-methoxyl amidated pectin gels’, Journal of Texture Studies, vol. 41, no. 6, pp. 899–927. Viuda‐Martos, M, Fernández‐López, J & Pérez‐Álvarez, JA 2010, ‘Pomegranate and its many functional components as related to human health: a review’, Comprehensive Reviews in Food Science and Food Safety, vol. 9, no. 6, Wiley Online Library, pp. 635–654. Voo, Wan Ping, Ravindra, Pogaku, Tey, Beng Ti & Chan, Eng Seng 2011, ‘Comparison of alginate and pectin based beads for production of poultry probiotic cells’, Journal of Bioscience and Bioengineering, vol. 111, no. 3, pp. 294–299. Voragen, Alphons GJ, Coenen, Gerd Jan, Verhoef, René P & Schols, Henk A 2009, ‘Pectin, a versatile polysaccharide present in plant cell walls’, Structural Chemistry, vol. 20, no. 2, pp. 263–275. Voragen, Fons, Schols, Henk & Visser, Richard GF 2013, Advances in pectin and pectinase research, Springer. Vrese, Michael de & Schrezenmeir, J 2008, ‘Probiotics, prebiotics, and synbiotics’, in Stahl U., Donalies U.E. & Nevoigt E. (eds), Food biotechnology, Springer, Berlin, Heidelberg, pp. 1–66. Vriesmann, Lúcia Cristina & de Oliveira Petkowicz, Carmen Lúcia 2017, ‘Cacao pod husks as a source of low-methoxyl, highly acetylated pectins able to gel in acidic media’, International Journal of Biological Macromolecules, vol. 101, Elsevier B.V., pp. 146–152. Wang, Lijun & Weller, Curtis L 2006, ‘Recent advances in extraction of nutraceuticals from plants’, Trends in Food Science and Technology, vol. 17, no. 6, pp. 300–312. Wang, Miaomiao, Huang, Bohui, Fan, Chuanhui, Zhao, Kaili, Hu, Hao, Xu, Xiaoyun, et al. 2016, ‘Characterization and functional properties of mango peel pectin extracted by ultrasound assisted citric acid’, International Journal of Biological Macromolecules, vol. 91, Elsevier B.V., pp. 794–803. Wang, Xin, Chen, Quanru & Lü, Xin 2014, ‘Pectin extracted from apple pomace and citrus peel by subcritical water’, Food Hydrocolloids, vol. 38, pp. 129–137. Wellner, Nikolaus, Kačuráková, Marta, Malovı́ková, Anna, Wilson, Reginald H & Belton, Peter S 1998, ‘FT-IR study of pectate and pectinate gels formed by divalent

178

cations’, Carbohydrate Research, vol. 308, pp. 123–131. Wicker, Louise, Kim, Yookyung, Kim, Mi-Ja, Thirkield, Brittnee, Lin, Zhuangsheng & Jung, Jiyoung 2014, ‘Pectin as a bioactive polysaccharide – Extracting tailored function from less’, Food Hydrocolloids, vol. 42, Elsevier, pp. 251–259. Willats, William GT, Knox, J Paul & Mikkelsen, Jørn Dalgaard 2006, ‘Pectin: New insights into an old polymer are starting to gel’, Trends in Food Science and Technology, vol. 17, no. 3, pp. 97–104. Williams, Nancy Toedter 2010, ‘Probiotics’, American Journal of Health-System Pharmacy, vol. 67, pp. 449–458. Woo, KK, Chong, YY, Li Hiong, SK & Tang, PY 2010, ‘Pectin extraction and characterization from red dragon fruit (Hylocereus polyrhizus): A preliminary study’, Journal of Biological Sciences, vol. 7, no. 10, pp. 631–636. Wu, Y, Cui, W, Eskin, NAM & Goff, HD 2009, ‘An investigation of four commercial galactomannans on their emulsion and rheological properties’, Food Research International, vol. 42, no. 8, Elsevier, pp. 1141–1146. Wybraniec, Sławomir & Mizrahi, Yosef 2002, ‘Fruit flesh betacyanin pigments in Hylocereus cacti’, Journal of Agricultural and Food Chemistry, vol. 50, no. 21, pp. 6086–6089. Yan, Ming Ming, Liu, Wei, Fu, Yu Jie, Zu, Yuan Gang, Chen, Cai Yun & Luo, Meng 2010, ‘Optimisation of the microwave-assisted extraction process for four main astragalosides in Radix Astragali’, Food Chemistry, vol. 119, no. 4, Elsevier, pp. 1663–1670. Yang, Jin Shu, Mu, Tai Hua & Ma, Meng Mei 2019, ‘Optimization of ultrasound- microwave assisted acid extraction of pectin from potato pulp by response surface methodology and its characterization’, Food Chemistry, vol. 289, Elsevier Ltd, pp. 351–359. Yang, Xi, Nisar, Tanzeela, Hou, Yanjie, Gou, Xiaoju, Sun, Lijun & Guo, Yurong 2018, ‘Pomegranate peel pectin can be used as an effective emulsifier’, Food Hydrocolloids, vol. 85, Elsevier, pp. 30–38. Yang, Xi, Nisar, Tanzeela, Liang, Di, Hou, Yanjie, Sun, Lijun & Guo, Yurong 2018, ‘Low methoxyl pectin gelation under alkaline conditions and its rheological properties: Using NaOH as a pH regulator’, Food Hydrocolloids, vol. 79, Elsevier Ltd, pp. 560–571. Yao, Mingfei, Wu, Jiong, Li, Bo, Xiao, Hang, McClements, David Julian & Li, Lanjuan 2017, ‘Microencapsulation of Lactobacillus salivarious Li01 for enhanced storage viability and targeted delivery to gut microbiota’, Food Hydrocolloids, vol. 72, pp. 228–236. Yapo, Beda M 2009a, ‘Biochemical characteristics and gelling capacity of pectin from yellow passion fruit rind as affected by acid extractant nature’, Journal of Agricultural and Food Chemistry, vol. 57, no. 4, pp. 1572–1578. Yapo, Beda M 2009b, ‘Lemon juice improves the extractability and quality characteristics of pectin from yellow passion fruit by-product as compared with commercial citric acid extractant’, Bioresource Technology, vol. 100, no. 12, Elsevier, pp. 3147–3151.

179

Yapo, Beda Marcel & Koffi, Kouassi Leonard 2006, ‘Yellow passion fruit rind - A potential source of low-methoxyl pectin’, Journal of Agricultural and Food Chemistry, vol. 54, no. 7, pp. 2738–2744. Yasmin, Iqra, Saeed, Muhammad, Pasha, Imran & Anjum Zia, Muhammad 2018, ‘Development of whey protein concentrate-pectin-alginate based delivery system to improve survival of B. longum BL-05 in simulated gastrointestinal conditions’, Probiotics and antimicrobial proteins, vol. 11, pp. 413–426. Yeoh, S, Shi, J & Langrish, TAG 2008, ‘Comparisons between different techniques for water-based extraction of pectin from orange peels’, Desalination, vol. 218, no. 1– 3, pp. 229–237. Yeung, Timothy W, Arroyo-Maya, Izlia J, McClements, David J & Sela, David A 2016, ‘Microencapsulation of probiotics in hydrogel particles: Enhancing: Lactococcus lactis subsp. cremoris LM0230 viability using calcium alginate beads’, Food and Function, vol. 7, pp. 1797–1804. Yoo, Sang-Ho, Fishman, Marshall L, Hotchkiss, Arland T & Lee, Hyeon Gyu 2006, ‘Viscometric behavior of high-methoxy and low-methoxy pectin solutions’, Food Hydrocolloids, vol. 20, no. 1, Elsevier, pp. 62–67. Yoo, Sang Ho, Fishman, Marshall L, Savary, Brett J & Hotchkiss, Arland T 2003, ‘Monovalent salt-induced gelation of enzymatically deesterified pectin’, Journal of Agricultural and Food Chemistry, vol. 51, no. 25, pp. 7410–7417. Yuliarti, Oni, Goh, Kelvin KT, Matia-Merino, Lara, Mawson, John & Brennan, Charles 2015, ‘Extraction and characterisation of pomace pectin from gold kiwifruit (Actinidia chinensis)’, Food Chemistry, vol. 187, Elsevier, pp. 290–296. Zaid, Rubaiyi M, Zularisam, AW & Sakinah, AM Mimi 2016a, ‘Effect of process parameters on pectin extraction from dragon fruit (Hylocereus polyrhizus) peels via chemical and physical treatment’, Australian Journal of Basic and Applied Sciences, vol. 10, no. 17, pp. 69–74. Zaid, Rubaiyi M, Zularisam, AW & Sakinah, AM Mimi 2016b, ‘Ultrasound-assisted extraction and characterizations of pectins from dragon fruit (Hylocereus polyrhizus) peels in various acid solution: A Preliminary Study’, International Conference in Organic Synthesis (ICOS2016), Universiti Teknologi MARA, Sarawak, Malaysia, p. 58. Zaidel, Dayang Norulfairuz Abang, Rashid, Jamaeyah Md, Hamidon, Nurul Hazirah, Salleh, Liza Md & Kassim, Angzzas Sari Mohd 2017, ‘Extraction and characterisation of pectin from dragon fruit (hylocereus polyrhizus) peels’, Chemical Engineering Transactions, vol. 56, no. 1, pp. 805–810. Zhang, Shanshan, Hu, Haijuan, Wang, Lufeng, Liu, Fengxia & Pan, Siyi 2018, ‘Preparation and prebiotic potential of pectin oligosaccharides obtained from citrus peel pectin’, Food Chemistry, vol. 244, Elsevier, pp. 232–237. Zhang, Yun, Lin, Jun & Zhong, Qixin 2016, ‘S/O/W emulsions prepared with sugar beet pectin to enhance the viability of probiotic Lactobacillus salivarius NRRL B- 30514’, Food Hydrocolloids, vol. 52, Elsevier, pp. 804–810. Zhang, Yun, Lin, Jun & Zhong, Qixin 2015, ‘The increased viability of probiotic Lactobacillus salivarius NRRL B-30514 encapsulated in emulsions with multiple lipid-protein-pectin layers’, Food Research International, vol. 71, pp. 9–15.

180

Zhao, Shan, Zhang, Ying, Liu, Yang, Yang, Fan, Xiu, Zhilong, Ma, Xiaojun & Sun, Guangwei 2018, ‘Preparation and optimization of calcium pectate beads for cell encapsulation’, Journal of Applied Polymer Science, vol. 135, no. 2, pp. 1–9. Zhao, ZY, Liang, L, Fan, X, Yu, Z, Hotchkiss, AT, Wilk, BJ & Eliaz, I 2008, ‘The role of modified citrus pectin as an effective chelator of lead in children hospitalized with toxic lead levels’, Alternative Therapies in Health and Medicine, vol. 14, no. 4, pp. 34–39. Zheng, Xianzhe, Yin, Fangping, Liu, Chenghai & Xu, Xiangwen 2011, ‘Effect of process parameters of microwave assisted extraction (MAE) on olysaccharides yield from pumpkin’, Journal of Northeast Agricultural University, vol. 18, no. 2, pp. 79–86. Zuidam, Nicolaas Jan & Shimoni, Eyal 2010, ‘Overview of microencapsulates for use in food products or processes and methods to make them’, in N J Zuidam & Viktor Nedovic (eds), Encapsulation technologies for active food ingredients and food processing, Springer, New York, pp. 3–29. Zykwinska, Agata, Gaillard, Cédric, Boiffard, Marie Hélène, Thibault, Jean François & Bonnin, Estelle 2009, ‘“Green labelled” pectins with gelling and emulsifying properties can be extracted by enzymatic way from unexploited sources’, Food Hydrocolloids, vol. 23, no. 8, pp. 2468–2477.

181