Extraction, characterization and applications of pectin from fruit wastes

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 cD ho D Supervisors Co-Supervisor: Dr Hayden Webb Associate Supervisor: Prof Enzo Palombo g an aN Principal Supervisor: Dr Franỗois Malherbe 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 D 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 ho of their degree of esterification (DE) and methylation (DM) Our results show that in cD conventional extraction the type of peels influences both yield and degree of esterification; microwave-assisted heating gave significantly higher yields for all types of aN 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 an The results also evidenced important variations in the physicochemical properties of g 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 microwaveassisted 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 g an aN cD ho D ii Acknowledgments I would like to express the deepest appreciation to my supervisors Dr Franỗois 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 911Swinburne 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 D 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 g an aN cD ho 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 ho D Dao Thi Anh Thu g an aN cD iv List of publications 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 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 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 Chemical and functional properties of pectin extracted from Passiflora edulis f D edulis (purple passion fruit) peel by microwave-assisted heating”, 15th International g an aN cD ho 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 Introduction Chapter 2: Literature review D Chapter 1: Valorization of food processing waste 2.2 Pectin ho 2.1 Structure of pectin 2.2.2 Product specifications 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 an aN Probiotics 26 g 2.3 cD 2.2.1 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 D 3.6.10 Fourier Transform infrared spectroscopy 50 3.6.11 Rheological properties 50 ho 3.6.12 Brunauer-Emmett-Teller (BET) nitrogen adsorption 50 cD 3.6.13 X-ray diffraction (XRD) 51 3.6.14 Oil-holding capacity 51 aN 3.6.15 Emulsifying properties 51 3.6.16 Foaming properties 51 an 3.7 Prebiotic score 52 g 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 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 ho D 4.2.1 4.3 Conclusion and comparison with conventional heating 94 Properties of extracted pectin 96 aN Chapter 5: Physicochemical properties 96 an 5.1 cD 4.2.6 Validation of optimum conditions of pectin extraction by microwaveassisted method from the DFP and PFP 94 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 g 5.1.1 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 5.3 Rheological properties 118 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 D 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 °C 147 cD ho 6.5.1 Freeze-drying capsules loaded with probiotic cells 147 6.7 Heat tolerance 148 6.8 Conclusion 150 g Chapter 7: an aN 6.6 Conclusion 151 Bibliography 156 ix List of Figures Figure 2.1 Bioactive compounds from modern fruit processing waste (data from Banerjee et al., (2017)) 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) 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 D experiments for each type of peel 40 ho Figure 3.2 Central composite design with two independent factors (time, temperature) including four corner-, five center- and four axial- experiments (Morris, 2000) 42 cD Figure 3.3 The Box-Behnken Design for four variables including 24 experimental points aN for each type of peel 44 an Figure 3.4 A flow chart of the microencapsulation process 54 g 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 D ho Figure 4.15 Effects of pH on yield 83 Figure 4.16 Interaction plot of microwave power and processing time on pectin yield cD from white-flesh DFP 89 Figure 4.17 Surface plots of extraction yield showing significant square terms of aN extraction time and microwave power (curvature) from a) red-flesh DFP; b) white-flesh an DFP; c) purple PFP (pH and liquid:solid ratio 50) 90 g 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 D Figure 5.10 Influence of shear rate on the shear stress of PFP pectin extracted by ho 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) cD 119 aN Figure 5.11 The apparent viscosity of PFP pectin solution extracted by microwave heating at 1, and %, measured at 25 °C 120 an Figure 5.12 Frequency sweeps (25 °C; fixed strain at %) of pectin solution (3 and g %) 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 %, 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 % 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 𝜇m: (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 𝜇m); (b): blank pectin beads (100 𝜇m); (c) & (d) & (e): pectin beads loaded with probiotics (100 𝜇m and 𝜇m) 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 D and after storage 134 Figure 6.8 The FTIR spectra of capsules with different calcium concentration as gelling ho agents (CP1: the highest [Ca2+] 20 mM to the CP4: the lowest [Ca2+] 10 mM) 135 cD Figure 6.9 Schematic representation of the effect of calcium concentration on crosslinking between galacturonic acid units with calcium ion Monomers and short aN chain pectins (a) not effectively form egg-box crosslinks at [Ca2+] 20 mM, whereas an longer chain pectins (b) can form more links with comparatively less [Ca2+] 15 mM 135 g 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 % 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 °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 g an aN cD ho D xiv List of Tables Table 2.1 Commercial pectin specifications (Dixon, 2008) 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 D 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 ho variables X1: Microwave time (minutes); X2: Microwave power (W); X3: pH; X4: cD Liquid:solid ratio 45 Table 4.1 Pectin yield and DE values used for the factorial design, obtained by extraction aN under various conditions Values given are average values of triplicate experiments 64 an Table 4.2 Analysis of variance of each component of the model derived by factorial g 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 D pectin for analysis 95 ho 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 cD using both heating methods 97 aN Table 5.3 The degree of esterification of pectin extracted at optimum conditions 99 an Table 5.4 The D-galacturonic acid content in extracted samples 100 g 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 g an aN cD ho D xvii ... 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... 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. .. chart of the microencapsulation process 54 g 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