# The Effect of Different Extraction Conditions on the Physical Properties, Conformation and Branching of Pectins Extracted from Cucumis melo Inodorus

^{*}

## Abstract

**:**

## 1. Introduction

## 2. Materials and Methods

^{3}factorial design. This study utilised a two-level, full factorial design for each of the three factors (pH, time and temperature), i.e., a 2

^{3}design. Therefore, there were eight individual pectin samples (A–H). Two levels (high and low) for each of the three extraction parameters (pH, temperature and time) were established, the lower level (−1) corresponding to pH 1, 60 °C and 2 h, and the upper level (+1) corresponding to pH 3, 80 °C and 4 h. Due to low yields [24], it was not possible to further analyse sample F in duplicate and this sample was only analysed in singlicate. All other samples were analysed in duplicate. Therefore, for each physical/conformational property determination, there was a minimum of 15 measurements.

#### 2.1. High-Performance Size Exclusion Chromatography Coupled to a Differential Pressure Viscometer (HPSEC-DPV)

_{n}) was then estimated from the weight–average intrinsic viscosity ([η]

_{w}) using a universal calibration [41] produced with pullulan standards of number–average molar mass from 45,000–640,000 g/mol (Sigma-Aldrich, Gillingham, UK).

#### 2.2. Estimation of Conformation

#### 2.2.1. Translational Frictional Ratio

_{0}, is a parameter which depends on conformation and molecular expansion through hydration effects [42]. It can be measured experimentally from the hydrodynamic radius and molecular weight:

#### 2.2.2. Persistence Length (L_{p}) and Mass Per Unit Length (M_{L})

_{p}of equivalent worm-like chains [44,45,46] where the persistence length is defined as the average projection length along the initial direction of the polymer chain. In the case of a theoretical perfect random coil, L

_{p}= 0, and for the equivalent extra-rigid rod, L

_{p}= ∞, although in practice limits of ~1 nm for random coils (e.g., pullulan) and 200 nm for an extra-rigid rod (e.g., xanthan) are more appropriate [44]. The mass per unit length, M

_{L}[37] is a direct measure of the degree of branching [38] and a larger value is indicative of a more branched molecule. The mass per unit length, M

_{L}of a pectin HG region is approximately 370 g/mol nm, although this will depend on the degree of methyl esterification (DM) and acetylation (DA) [39].

_{p}and mass per unit length, M

_{L}can be estimated using Multi-HYDFIT program [47] which considers data sets of intrinsic viscosity and molar mass. It then performs a minimisation procedure [47] to find the best values of M

_{L}and L

_{p}which satisfy the Bushin-Bohdanecky [44,48,49] equation (Equation (3)).

_{L}) from the composition, it was decided that, for melon pectins, we would consider the scenario where only the chain diameter was fixed at 0.8 nm [39]. The Multi-HYDFIT program then floats the variable parameters (L

_{p}and M

_{L}) in order to find a minimum of the target function [47].

_{p}/M

_{L}(nm

^{2}mol/g) which increases with increasing stiffness [51].

#### 2.2.3. Conformation Zoning (Normalised Scaling Relations)

#### 2.3. Statistical Analysis

_{p}/M

_{L}ratio, mean side chain length and mean side chain number) [54]. The polynomial Equations ((5)–(10), (13) and (14)) were used to fit the mean values of the experimental data, where X

_{1}, X

_{2}and X

_{3}correspond to pH, time and temperature, respectively [55].

## 3. Results and Discussion

#### 3.1. Intrinsic Viscosity ([η]_{w})

_{w}= 6914 − 2044X

_{1}− 2040X

_{2}−92.3X

_{3}+ 741X

_{1}X

_{2}+ 31.4X

_{1}X

_{3}+ 33.8X

_{2}X

_{3}− 12.62X

_{1}X

_{2}X

_{3}

r

^{2}= 0.87

#### 3.2. Molar Mass (M_{n})

^{5}–2.0 × 10

^{6}g/mol and the effects of different processing conditions were fitted using Equation (6).

_{n}= 4,580,501 − 4,070,497X

_{1}− 702,037X

_{2}− 60,567X

_{3}+ 892,834X

_{1}X

_{2}+ 65718X

_{1}X

_{3}+ 9077X

_{2}X

_{3}− 13,983X

_{1}X

_{2}X

_{3}

r

^{2}= 0.93

_{0}) and the persistence length (L

_{p}) and semi-quantitatively using conformation zoning.

#### 3.3. Estimation of Conformation

#### 3.3.1. Translational Frictional Ratio

_{0}(Table 1) are consistent with the range of values, which have been found previously for pectins of ~7–10 [39,43] and the effects of different processing conditions were fitted using Equation (7).

_{0}= 23.15 − 4.49X

_{1}− 5.16X

_{2}− 0.228X

_{3}+ 1.87X

_{1}X

_{2}+ 0.0678X

_{1}X

_{3}+ 0.0866X

_{2}X

_{3}− 0.0325X

_{1}X

_{2}X

_{3}

r

^{2}= 0.89

#### 3.3.2. Persistence Length (L_{p}) and Mass Per Unit length (M_{L})

_{p}/M

_{L}ratio is perhaps a better indication of chain stiffness [51,61] as this mitigates against an over reliance on localized minima in the global HYDFIT analysis plot [51,61]. These values (Table 1) are again generally higher for pectins extracted at pH 1 when compared to pectins extracted at pH 3, and this is especially noticeable for pectin G, which would appear to be stiff with little or no side chains. The effects of different processing conditions were fitted using Equations (8)–(10). However, it should be noted that the fit for mass per unit length is very poor (r

^{2}= 0.51).

_{p}= 16 + 21.2X

_{1}− 14.4X

_{2}− 0.87X

_{3}+ 0.1X

_{1}X

_{2}− 0.039X

_{1}X

_{3}+ 0.584X

_{2}X

_{3}− 0.127X

_{1}X

_{2}X

_{3}

r

^{2}= 0.85

_{L}= 1104 – 23X

_{1}+ 525X

_{2}− 4.9X

_{3}– 251X

_{1}X

_{2}− 0.1X

_{1}X

_{3}− 8.7X

_{2}X

_{3}+ 4.02X

_{1}X

_{2}X

_{3}

r

^{2}= 0.51

_{p}/M

_{L}= 0.98 − 0.293X

_{1}− 0.508X

_{2}− 0.0171X

_{3}+ 0.164X

_{1}X

_{2}+ 0.00529X

_{1}X

_{3}+ 0.00893X

_{2}X

_{3}− 0.00290X

_{1}X

_{2}X

_{3}

r

^{2}= 0.69

_{p}/M

_{L}to estimate chain rigidity [51,61], where a higher value is indicative of a more rigid polymer.

#### 3.3.3. Conformation Zoning (Normalised Scaling Relations)

#### 3.4. Estimation of Branching

_{1}− 2.75X

_{2}− 0.249X

_{3}+ 2.72X

_{1}X

_{2}+ 0.123X

_{1}X

_{3}+ 0.024X

_{2}X

_{3}− 0.0258X

_{1}X

_{2}X

_{3}

r

^{2}= 0.75

_{1}+ 1437X

_{2}+ 85X

_{3}– 150X

_{1}X

_{2}+ 3.2X

_{1}X

_{3}− 25.5X

_{2}X

_{3}+ 1.2X

_{1}X

_{2}X

_{3}

r

^{2}= 0.58

## 4. Conclusions

_{p}/M

_{L}.

_{p}/M

_{L}ratio or their position in the conformational zoning plot and again demonstrates the importance of using more than one method in estimating the branching of a polysaccharide [39,43,62]. As with conformation zoning and translational frictional ratio, we can see that samples extracted at pH 1 are more rigid than those extracted at pH 3. This is consistent with results from chemical analysis which also shows clear differences between samples extracted at pH 1 and pH 3 [24]. There is also some evidence that pectins extracted at higher temperatures (C, D, G and H) have fewer side chains, which could be due to the loss of arabinan side chains [24,27]. The first and second components describe 83% of the variation in the samples. It is, therefore, clear that extraction conditions, and pH in particular, have a great influence on the number and length of side chains on the RG-I region of pectins extracted from Cucumis melo Inodorus.

## Author Contributions

## Funding

## Conflicts of Interest

## References

- Harding, S.E.; Tombs, M.; Adams, G.; Smestad Paulsen, B.; Inngjerdingen, K.; Barsett, H. An Introduction to Polysaccharide Biotechnology; CRC Press: Boca Raton, FL, USA, 2017. [Google Scholar] [CrossRef]
- Willats, W.G.T.; McCartney, L.; Mackie, W.; Knox, J.P. Pectin: Cell biology and prospects for functional analysis. Plant Mol. Biol.
**2001**, 47, 9–27. [Google Scholar] [CrossRef] - Ridley, B.L.; O’Neill, M.A.; Mohnen, D. Pectins: Structure, biosynthesis, and oligogalacturonide-related signaling. Phytochemistry
**2001**, 57, 929–967. [Google Scholar] [CrossRef] - Houben, K.; Jolie, R.P.; Fraeye, I.; Van Loey, A.M.; Hendrickx, M.E. Comparative study of the cell wall composition of broccoli, carrot, and tomato: Structural characterization of the extractable pectins and hemicelluloses. Carbohydr. Res.
**2011**, 346, 1105–1111. [Google Scholar] [CrossRef] - Zhang, C.; Zhu, X.; Zhang, F.; Yang, X.; Ni, L.; Zhang, W.; Liu, Z.; Zhang, Y. Improving viscosity and gelling properties of leaf pectin by comparing five pectin extraction methods using green tea leaf as a model material. Food Hydrocoll.
**2020**, 98, 105246. [Google Scholar] [CrossRef] - Pi, F.; Liu, Z.; Guo, X.; Guo, X.; Meng, H. Chicory root pulp pectin as an emulsifier as compared to sugar beet pectin. Part 1: Influence of structure, concentration, counterion concentration. Food Hydrocoll.
**2019**, 89, 792–801. [Google Scholar] [CrossRef] - Fracasso, A.F.; Perussello, C.A.; Carpiné, D.; Petkowicz, C.L.d.O.; Haminiuk, C.W.I. Chemical modification of citrus pectin: Structural, physical and rheologial implications. Int. J. Biol. Macromol.
**2018**, 109, 784–792. [Google Scholar] [CrossRef] [PubMed] - Schmidt, U.S.; Koch, L.; Rentschler, C.; Kurz, T.; Endreß, H.U.; Schuchmann, H.P. Effect of Molecular Weight Reduction, Acetylation and Esterification on the Emulsification Properties of Citrus Pectin. Food Biophys.
**2015**, 10, 217–227. [Google Scholar] [CrossRef] - Cui, J.; Zhao, C.; Zhao, S.; Tian, G.; Wang, F.; Li, C.; Wang, F.; Zheng, J. Alkali + cellulase-extracted citrus pectins exhibit compact conformation and good fermentation properties. Food Hydrocoll.
**2020**, 106079. [Google Scholar] [CrossRef] - Nergard, C.S.; Kiyohara, H.; Reynolds, J.C.; Thomas-Oates, J.E.; Matsumoto, T.; Yamada, H.; Michaelsen, T.E.; Diallo, D.; Paulsen, B.S. Structure-immunomodulating activity relationships of a pectic arabinogalactan from Vernonia kotschyana Sch. Bip. ex Walp. Carbohydr. Res.
**2005**, 340, 1789–1801. [Google Scholar] [CrossRef] [PubMed] - Georgiev, Y.N.; Paulsen, B.S.; Kiyohara, H.; Ciz, M.; Ognyanov, M.H.; Vasicek, O.; Rise, F.; Denev, P.N.; Lojek, A.; Batsalova, T.G.; et al. Tilia tomentosa pectins exhibit dual mode of action on phagocytes as β-glucuronic acid monomers are abundant in their rhamnogalacturonans I. Carbohydr. Polym.
**2017**, 175, 178–191. [Google Scholar] [CrossRef] - Huang, C.; Yao, R.; Zhu, Z.; Pang, D.; Cao, X.; Feng, B.; Paulsen, B.S.; Li, L.; Yin, Z.; Chen, X.; et al. A pectic polysaccharide from water decoction of Xinjiang Lycium barbarum fruit protects against intestinal endoplasmic reticulum stress. Int. J. Biol. Macromol.
**2019**, 130, 508–514. [Google Scholar] [CrossRef] - Zou, Y.F.; Zhang, Y.Y.; Fu, Y.P.; Inngjerdingen, K.T.; Paulsen, B.S.; Feng, B.; Zhu, Z.K.; Li, L.X.; Jia, R.Y.; Huang, C.; et al. A polysaccharide isolated from codonopsis pilosula with immunomodulation effects both in vitro and in vivo. Molecules
**2019**, 24, 3632. [Google Scholar] [CrossRef] [PubMed][Green Version] - Zou, Y.F.; Zhang, Y.Y.; Paulsen, B.S.; Rise, F.; Chen, Z.L.; Jia, R.Y.; Li, L.X.; Song, X.; Feng, B.; Tang, H.Q.; et al. Structural features of pectic polysaccharides from stems of two species of Radix Codonopsis and their antioxidant activities. Int. J. Biol. Macromol.
**2020**, 159, 704–713. [Google Scholar] [CrossRef] [PubMed] - Williams, M.A.K.; Cucheval, A.; Ström, A.; Ralet, M.C. Electrophoretic behavior of copolymeric galacturonans including comments on the information content of the intermolecular charge distribution. Biomacromolecules
**2009**, 10, 1523–1531. [Google Scholar] [CrossRef] [PubMed] - Sun, L.; Ropartz, D.; Cui, L.; Shi, H.; Ralet, M.C.; Zhou, Y. Structural characterization of rhamnogalacturonan domains from Panax ginseng C. A. Meyer. Carbohydr. Polym.
**2019**, 203, 119–127. [Google Scholar] [CrossRef] - Voragen, A.G.J.; Coenen, G.-J.; Verhoef, R.P.; Schols, H.A. Pectin, a versatile polysaccharide present in plant cell walls. Struct. Chem.
**2009**, 20, 263. [Google Scholar] [CrossRef][Green Version] - Axelos, M.A.V.; Thibault, J.F. Influence of the substituents of the carboxyl groups and of the rhamnose content on the solution properties and flexibility of pectins. Int. J. Biol. Macromol.
**1991**, 13, 77–82. [Google Scholar] [CrossRef] - Seixas, F.L.; Fukuda, D.L.; Turbiani, F.R.B.; Garcia, P.S.; Petkowicz, C.L.D.O.; Jagadevan, S.; Gimenes, M.L. Extraction of pectin from passion fruit peel (Passiflora edulis f.flavicarpa) by microwave-induced heating. Food Hydrocoll.
**2014**, 38, 186–192. [Google Scholar] [CrossRef] - Adetunji, L.R.; Adekunle, A.; Orsat, V.; Raghavan, V. Advances in the pectin production process using novel extraction techniques: A review. Food Hydrocoll.
**2017**, 62, 239–250. [Google Scholar] [CrossRef] - Pagán, J.; Ibarz, A. Extraction and rheological properties of pectin from fresh peach pomace. J. Food Eng.
**1999**, 39, 193–201. [Google Scholar] [CrossRef] - Pagán, J.; Ibarz, A.; Llorca, M.; Coll, L. Quality of industrial pectin extracted from peach pomace at different pH and temperatures. J. Sci. Food Agric.
**1999**, 79, 1038–1042. [Google Scholar] [CrossRef] - Pagán, J.; Ibarz, A.; Llorca, M.; Pagán, A.; Barbosa-Cánovas, G.V. Extraction and characterization of pectin from stored peach pomace. Food Res. Int.
**2001**, 34, 605–612. [Google Scholar] [CrossRef] - Denman, L.J.; Morris, G.A. An experimental design approach to the chemical characterisation of pectin polysaccharides extracted from Cucumis melo Inodorus. Carbohydr. Polym.
**2015**, 117, 364–369. [Google Scholar] [CrossRef] [PubMed] - Kliemann, E.; De Simas, K.N.; Amante, E.R.; Prudêncio, E.S.; Teófilo, R.F.; Ferreira, M.M.C.; Amboni, R.D.M.C. Optimisation of pectin acid extraction from passion fruit peel (Passiflora edulis flavicarpa) using response surface methodology. Int. J. Food Sci. Technol.
**2009**, 44, 476–483. [Google Scholar] [CrossRef] - Kumar, M.; Chauhan, A.K.R.; Kumar, S.; Kumar, A.; Malik, S. Design and evaluation of pectin based metrics for transdermal patches of meloxicam. J. Pharm. Res. Health Care
**2010**, 2, 244–247. [Google Scholar] - Levigne, S.; Ralet, M.C.; Thibault, J.F. Characterisation of pectins extracted from fresh sugar beet under different conditions using an experimental design. Carbohydr. Polym.
**2002**, 49, 145–153. [Google Scholar] [CrossRef] - Samavati, V. Polysaccharide extraction from Abelmoschus esculentus: Optimization by response surface methodology. Carbohydr. Polym.
**2013**, 95, 588–597. [Google Scholar] [CrossRef] - Samavati, V. Central composite rotatable design for investigation of microwave-assisted extraction of okra pod hydrocolloid. Int. J. Biol. Macromol.
**2013**, 61, 142–149. [Google Scholar] [CrossRef] - Sudhakar, D.V.; Maini, S.B. Isolation and characterization of mango peel pectins. J. Food Process. Preserv.
**2000**, 24, 209–227. [Google Scholar] [CrossRef] - Ayora-Talavera, T.; Ramos-Chan, C.; Covarrubias-Cárdenas, A.; Sánchez-Contreras, A.; García-Cruz, U.; Pacheco, L.N. Evaluation of Pectin Extraction Conditions and Polyphenol Profile from Citrus x lantifolia Waste: Potential Application as Functional Ingredients. Agriculture
**2017**, 7, 28. [Google Scholar] [CrossRef][Green Version] - Yapo, B.M.; Robert, C.; Etienne, I.; Wathelet, B.; Paquot, M. Effect of extraction conditions on the yield, purity and surface properties of sugar beet pulp pectin extracts. Food Chem.
**2007**, 100, 1356–1364. [Google Scholar] [CrossRef] - May, C.D. Industrial pectins: Sources, production and applications. Carbohydr. Polym.
**1990**, 12, 79–99. [Google Scholar] [CrossRef] - Happi Emaga, T.; Ronkart, S.N.; Robert, C.; Wathelet, B.; Paquot, M. Characterisation of pectins extracted from banana peels (Musa AAA) under different conditions using an experimental design. Food Chem.
**2008**, 108, 463–471. [Google Scholar] [CrossRef] [PubMed] - Mao, G.; Wu, D.; Wei, C.; Tao, W.; Ye, X.; Linhardt, R.J.; Orfila, C.; Chen, S. Reconsidering conventional and innovative methods for pectin extraction from fruit and vegetable waste: Targeting rhamnogalacturonan I. Trends Food Sci. Technol.
**2019**, 94, 65–78. [Google Scholar] [CrossRef] - Wu, D.; Zheng, J.; Mao, G.; Hu, W.; Ye, X.; Linhardt, R.J.; Chen, S. Rethinking the impact of RG-I mainly from fruits and vegetables on dietary health. Crit. Rev. Food Sci. Nutr.
**2019**, 1–23. [Google Scholar] [CrossRef] - Harding, S.E.; Berth, G.; Ball, A.; Mitchell, J.R.; de la Torre, J.G. The molecular weight distribution and conformation of citrus pectins in solution studied by hydrodynamics. Carbohydr. Polym.
**1991**, 16, 1–15. [Google Scholar] [CrossRef][Green Version] - Stokke, B.T.; Smidsrød, O.; Elgsaeter, A. Electron microscopy of native xanthan and xanthan exposed to low ionic strength. Biopolymers
**1989**, 28, 617–637. [Google Scholar] [CrossRef] - Morris, G.A.; de al Torre, J.C.; Ortega, A.; Castile, J.; Smith, A.; Harding, S.E. Molecular flexibility of citrus pectins by combined sedimentation and viscosity analysis. Food Hydrocoll.
**2008**, 22, 1435–1442. [Google Scholar] [CrossRef][Green Version] - Theisen, A.; Johann, C.; Deacon, M.P.; Harding, S.E. Refractive Increment Data-Book for Polymer and Biomolecular Scientists; Nottingham University Press: Nottingham, UK, 2000. [Google Scholar]
- Grubisic, Z.; Rempp, P.; Benoit, H. A universal calibration for gel permeation chromatography. J. Polym. Sci. Part B Polym. Phys.
**1996**, 34, 1707–1713. [Google Scholar] [CrossRef] - Tanford, C. Physical Chemistry of Macromolecules; John Willey and Sons Inc.: Hoboken, NJ, USA, 1961. [Google Scholar]
- Morris, G.A.; Foster, T.J.; Harding, S.E. The effect of the degree of esterification on the hydrodynamic properties of citrus pectin. Food Hydrocoll.
**2000**, 14, 227–235. [Google Scholar] [CrossRef] - Harding, S.E. The intrinsic viscosity of biological macromolecules. Progress in measurement, interpretation and application to structure in dilute solution. Prog. Biophys. Mol. Biol.
**1997**, 68, 207–262. [Google Scholar] [CrossRef] - Kratky, O.; Porod, G. Röntgenuntersuchung gelöster Fadenmoleküle. Recl. Trav. Chim. Pays-Bas
**1949**, 68, 1106–1122. [Google Scholar] [CrossRef] - Pavlov, G.M.; Korneeva, E.V.; Harding, S.E.; Vichoreva, G.A. Dilute solution properties of carboxymethylchitins in high ionic-strength solvent. Polymer
**1998**, 39, 6951–6961. [Google Scholar] [CrossRef] - Ortega, A.; García de la Torre, J. Equivalent radii and ratios of radii from solution properties as indicators of macromolecular conformation, shape, and flexibility. Biomacromolecules
**2007**, 8, 2464–2475. [Google Scholar] [CrossRef] - Bushin, S.V.; Tsvetkov, V.N.; Lysenko, E.B.; Emel’yanov, V.N. Conformational properties and rigidity of molecules of ladder polyphenylsiloxane in solutions according the data of sedimentation-diffusion analysis and viscometry. Vysokomol. Soedin. Ser. A
**1981**, 23, 2494–2503. [Google Scholar] - Bohdanecky, M. New Method for Estimating the Parameters of the Wormlike Chain Model from the Intrinsic Viscosity of Stiff-Chain Polymers. Macromolecules
**1983**, 16, 1483–1492. [Google Scholar] [CrossRef] - Ralet, M.C.; Crépeau, M.J.; Lefèbvre, J.; Mouille, G.; Höfte, H.; Thibault, J.F. Reduced number of homogalacturonan domains in pectins of an Arabidopsis mutant enhances the flexibility of the polymer. Biomacromolecules
**2008**, 9, 1454–1460. [Google Scholar] [CrossRef] - Morris, G.A.; Ralet, M.C.; Bonnin, E.; Thibault, J.F.; Harding, S.E. Physical characterisation of the rhamnogalacturonan and homogalacturonan fractions of sugar beet (Beta vulgaris) pectin. Carbohydr. Polym.
**2010**, 82, 1161–1167. [Google Scholar] [CrossRef][Green Version] - Pavlov, G.M.; Harding, S.E.; Rowe, A.J. Normalized scaling relations as a natural classification of linear macromolecules according to size. In Progress in Colloid and Polymer Science; Springer: Berlin, Germany, 1999; Volume 113, pp. 76–80. [Google Scholar]
- Pavlov, G.M.; Rowe, A.J.; Harding, S.E. Conformation zoning of large molecules using the analytical ultracentrifuge. TrAC Trends Anal. Chem.
**1997**, 16, 401–405. [Google Scholar] [CrossRef] - Gadalla, H.H.; El-Gibaly, I.; Soliman, G.M.; Mohamed, F.A.; El-Sayed, A.M. Amidated pectin/sodium carboxymethylcellulose microspheres as a new carrier for colonic drug targeting: Development and optimization by factorial design. Carbohydr. Polym.
**2016**, 153, 526–534. [Google Scholar] [CrossRef] - Teja, S.P.S.; Damodharan, N. 23 Full Factorial Model for Particle Size Optimization of Methotrexate Loaded Chitosan Nanocarriers: A Design of Experiments (DoE) Approach. BioMed Res. Int.
**2018**, 2018, 7834159. [Google Scholar] [CrossRef] [PubMed][Green Version] - Mao, Y.; Millett, R.; Lee, C.S.; Yakubov, G.; Harding, S.E.; Binner, E. Investigating the influence of pectin content and structure on its functionality in bio-flocculant extracted from okra. Carbohydr. Polym.
**2020**, 241, 116414. [Google Scholar] [CrossRef] [PubMed] - Israel, L.L.; Lellouche, E.; Kenett, R.S.; Green, O.; Michaeli, S.; Lellouche, J.P. Ce3/4+ cation-functionalized maghemite nanoparticles towards siRNA-mediated gene silencing. J. Mater. Chem. B
**2014**, 2, 6215–6225. [Google Scholar] [CrossRef] [PubMed] - Urias-Orona, V.; Rascón-Chu, A.; Lizardi-Mendoza, J.; Carvajal-Millán, E.; Gardea, A.A.; Ramírez-Wong, B. A novel pectin material: Extraction, characterization and gelling properties. Int. J. Mol. Sci.
**2010**, 11, 3686–3695. [Google Scholar] [CrossRef][Green Version] - Wang, J.H.; Luo, J.P.; Yang, X.F.; Zha, X.Q. Structural analysis of a rhamnoarabinogalactan from the stems of Dendrobium nobile Lindl. Food Chem.
**2010**, 122, 572–576. [Google Scholar] [CrossRef] - Li, D.-Q.; Jia, X.; Wei, Z.; Liu, Z.-Y. Box–Behnken experimental design for investigation of microwave-assisted extracted sugar beet pulp pectin. Carbohydr. Polym.
**2012**, 88, 342–346. [Google Scholar] [CrossRef] - Morris, G.A.; Ralet, M.C. The effect of neutral sugar distribution on the dilute solution conformation of sugar beet pectin. Carbohydr. Polym.
**2012**, 88, 1488–1491. [Google Scholar] [CrossRef][Green Version] - Morris, G.A.; Patel, T.R.; Picout, D.R.; Ross-Murphy, S.B.; Ortega, A.; Garcia de la Torre, J.; Harding, S.E. Global hydrodynamic analysis of the molecular flexibility of galactomannans. Carbohydr. Polym.
**2008**, 72, 356–360. [Google Scholar] [CrossRef][Green Version] - Paniagua, C.; Posé, S.; Morris, V.J.; Kirby, A.R.; Quesada, M.A.; Mercado, J.A. Fruit softening and pectin disassembly: An overview of nanostructural pectin modifications assessed by atomic force microscopy. Ann. Bot.
**2014**, 114, 1375–1383. [Google Scholar] [CrossRef][Green Version] - Santos, E.E.; Amaro, R.C.; Bustamante, C.C.C.; Guerra, M.H.A.; Soares, L.C.; Froes, R.E.S. Extraction of pectin from agroindustrial residue with an ecofriendly solvent: Use of FTIR and chemometrics to differentiate pectins according to degree of methyl esterification. Food Hydrocoll.
**2020**, 107, 105921. [Google Scholar] [CrossRef] - Yamada, H.; Kiyohara, H. Complement-activating polysaccharides from medicinal herbs. Immunomodul. Agents Plants
**1999**, 161–202. [Google Scholar] [CrossRef] - Kpodo, F.M.; Agbenorhevi, J.K.; Alba, K.; Oduro, I.N.; Morris, G.A.; Kontogiorgos, V. Structure-Function Relationships in Pectin Emulsification. Food Biophys.
**2018**, 13, 71–79. [Google Scholar] [CrossRef] [PubMed][Green Version] - Buergy, A.; Rolland-Sabaté, A.; Leca, A.; Renard, C.M.G.C. Pectin modifications in raw fruits alter texture of plant cell dispersions. Food Hydrocoll.
**2020**, 107, 105962. [Google Scholar] [CrossRef] - Deng, L.Z.; Pan, Z.; Zhang, Q.; Liu, Z.L.; Zhang, Y.; Meng, J.S.; Gao, Z.J.; Xiao, H.W. Effects of ripening stage on physicochemical properties, drying kinetics, pectin polysaccharides contents and nanostructure of apricots. Carbohydr. Polym.
**2019**, 222, 114980. [Google Scholar] [CrossRef]

**Figure 1.**The Pareto (

**A**), main effects (

**B**) and interaction plots (

**C**) for the effect of different extraction conditions on the intrinsic viscosity of melon pectin. In the Pareto plot (

**A**), the larger the bar the greater the influence of each parameter, and values larger than 2.306 (indicated by the dashed red line) are statistically significant. In the main effects plot (

**B**), the steeper the slope the greater the magnitude of the main effect. In the interactions plot (

**C**), the Y-axis scale is always the same for each combination of factors. When the lines are parallel, interaction effects are zero [57].

**Figure 2.**The Pareto (

**A**), main effects (

**B**) and interaction plots (

**C**) for the effect of different extraction conditions on the molar mass of melon pectin. For further explanation of the individual plots, see Figure 1.

**Figure 3.**The Pareto (

**A**), main effects (

**B**) and interaction plots (

**C**) for the effect of different extraction conditions on the translational frictional ratio (f/f

_{0}) of melon pectin. For further explanation of the individual plots, see Figure 1.

**Figure 4.**The main effects (

**A**,

**C**,

**E**) and interaction plots (

**B**,

**D**,

**F**) for the effect of different extraction conditions from left to right on the persistence length, mass per unit length and their ratio (L

_{p}/M

_{L}) for melon pectin. Pareto plots not shown for clarity. For further explanation of the individual plots, see Figure 1.

**Figure 5.**Normalised scaling plot of [η]

_{w}M

_{L}versus M

_{n}/M

_{L}(adapted from [52]) where Zone A: extra rigid rod; Zone B: rigid rod; Zone C: semi-flexible; Zone D: random coil and Zone E: globular or branched [52,53]. N. B. pectins D and E are almost overlapping. This a semi-empirical plot derived from the conformation data estimated for >80 polymers in the two articles by Pavlov, Harding and Rowe (1997, 1999) [52,53].

**Figure 6.**The main effects (

**A**,

**C**) and interaction plots (

**B**,

**D**) for the effect of different extraction conditions from left to right on the length and number of side chains for melon pectin. Pareto plots not shown for clarity. For further explanation of the individual plots, see Figure 1.

**Table 1.**Physical properties: molar mass (M

_{n}), intrinsic viscosity ([η]

_{w}), frictional ratio (f/f

_{0}), mass-per-unit length (M

_{L}), persistence length (L

_{p}) and L

_{p}/M

_{L}for pectins extracted from Cucumis melo Inodorus.

Sample (Extraction Conditions) | M_{n} (g/mol) | [η]_{w} (mL/g) | f/f_{0} | M_{L} (g/mol nm) | L_{p} (nm) | L_{p}/M_{L} (nm^{2} mol/g) |
---|---|---|---|---|---|---|

A (pH 1, 2 h, 60 °C) | 610,000 ^{b} | 1160 ^{a,b} | 9 ^{a,b} | 760 ^{a} | 9 ^{b} | 0.012 |

B (pH 3, 2 h, 60 °C) | 570,000 ^{b} | 770 ^{b,c} | 8 ^{a,b,c} | 660 ^{a} | 17 ^{a,b} | 0.022 |

C (pH 1, 2 h, 80 °C) | 405,000 ^{b} | 1110 ^{a,b,c} | 9 ^{a,b} | 750 ^{a} | 35 ^{a,b} | 0.048 |

D (pH 3, 2 h, 80 °C) | 580,000 ^{b} | 650 ^{b,c} | 7 ^{b,c} | 610 ^{a} | 13 ^{b} | 0.018 |

E (pH 1, 4 h, 60 °C) | 520,000 ^{b} | 790 ^{b,c} | 8 ^{a,b,c} | 475 ^{a} | 9 ^{b} | 0.018 |

F (pH 3, 4 h, 60 °C) | 2,000,000 ^{a} | 650 ^{b,c} | 7 ^{b,c} | 690 ^{a} | 5 ^{b} | 0.007 |

G (pH 1, 4 h, 80 °C) | 115,000 ^{b} | 1580 ^{a} | 10 ^{a} | 275 ^{a} | 54 ^{a} | 0.295 |

H (pH 3, 4 h, 80 °C) | 680,000 ^{b} | 360 ^{c} | 6 ^{c} | 770 ^{a} | 9 ^{b} | 0.012 |

**Table 2.**The mean length and number of side chains for pectins extracted from Cucumis melo Inodorus.

Sample | HG: RG-I | HG M _{n} (g/mol) | HG M _{L} (g/mol nm) | RG-I M _{n} (g/mol) | RG-I M _{L} (g/mol nm) | ^{a} Mean Side Chain Length | ^{a} Mean Number of Side Chains |
---|---|---|---|---|---|---|---|

A | 0.3 | 140,000 | 367 | 470,000 | 887 | 5 | 800 |

B | 0.6 | 205,000 | 368 | 370,000 | 831 | 4 | 850 |

C | 0.3 | 90,000 | 368 | 315,000 | 876 | 4 | 450 |

D | 1.1 | 300,000 | 371 | 280,000 | 1494 | 9 | 180 |

E | 0.3 | 135,000 | 363 | 390,000 | 516 | 2 | 1590 |

F | 0.5 | 725,000 | 373 | 1,300,000 | 868 | 5 | 1850 |

G | 0.7 | 45,000 | 365 | 70,000 | 472 | 2 | 260 |

H | 1.1 | 370,000 | 372 | 315,000 | 1228 | 7 | 300 |

^{a}in order to determine the degree of number and degree of branching, knowledge of the mass per unit length, HG: RG-I ratio [24], number average molecular weight, degree of methyl esterification and the average mass of side chain sugar residue are required. The monosaccharide composition and degree of methylation has been published in Tables 2 and 3 of our previous publication [24]. Furthermore, knowledge the degree of acetyl esterification can also be included if available.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## Share and Cite

**MDPI and ACS Style**

Reynolds, D.C.; Denman, L.J.; Binhamad, H.A.S.; Morris, G.A. The Effect of Different Extraction Conditions on the Physical Properties, Conformation and Branching of Pectins Extracted from *Cucumis melo* Inodorus. *Polysaccharides* **2020**, *1*, 3-20.
https://doi.org/10.3390/polysaccharides1010002

**AMA Style**

Reynolds DC, Denman LJ, Binhamad HAS, Morris GA. The Effect of Different Extraction Conditions on the Physical Properties, Conformation and Branching of Pectins Extracted from *Cucumis melo* Inodorus. *Polysaccharides*. 2020; 1(1):3-20.
https://doi.org/10.3390/polysaccharides1010002

**Chicago/Turabian Style**

Reynolds, Danielle C., Laura J. Denman, Hana A. S. Binhamad, and Gordon A. Morris. 2020. "The Effect of Different Extraction Conditions on the Physical Properties, Conformation and Branching of Pectins Extracted from *Cucumis melo* Inodorus" *Polysaccharides* 1, no. 1: 3-20.
https://doi.org/10.3390/polysaccharides1010002