# 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

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**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