Valorization of pectic polysaccharides from Gardenia jasminoides Ellis flower: Physicochemical, structural, rheological, and functional properties

2.3.1 Analysis of protein content and monosaccharide composition

The protein content was determined by an automatic Kjeldahl instrument (Kjeltec 2300, Foss, Sweden). Monosaccharide composition was analyzed by a reversed-phase HPLC system (Chen et al., 2020a,b).

2.3.2 Metal element analysis

The metal elements in pectin were determined by Inductively Coupled Plasma Mass Spectrometry (ICP-MS 720ES, Agilent, USA). A total of 100 mg of pectin was mixed with 0.5 mL of 30% hydrogen peroxide solution and 4 mL nitric acid. After storing for 15 min at room temperature, pectins were digested with a Graphite digester (SH220N, Haineng Instrument Technology Co., Ltd, Yantai, China) according to the digestion procedure. The digested samples were diluted to 10 mL with ultrapure water and analyzed with ICP-MS720ES (Agilent, USA) under the following condition: plasma flow for 15.0 L/min, auxiliary flow for 1.50 L/min, and nebulizer flow for 0.75 L/min.

2.3.3 MW distribution

The MW distribution was carried out by using an Agilent high-performance gel permeation chromatography (HPGPC) system (Agilent infinity 1260, MO, USA) equipped with a refractive index detector (RI-201H) and analyzed with PL aqua gel-OH Mixed column (7.5 × 300 mm, Agilent, USA). The sample solution (1 mg/mL) was filtered through a 0.45 μm membrane filter and injected into the column through a 100 μL loop. The mobile phase was 0.1 M NaNO₃ and 0.05% NaN₃ with a flow rate of 1 mL/min at 30 °C. A standard curve was established by calibrating the column with PEO (polyethylene oxide) standards.

2.4.1 FT-IR (Fourier transform infrared spectroscopy) and esterification analysis

The structural characteristics of extracted samples were investigated using an FT-IR spectrometer (Thermo Scientific Nicolet 10, USA) (Muñoz-Almagro et al., 2021). Sample powders were dried at 105 °C for 6 h, mixed with KBr powder (1:100, w/w), and compressed into a pellet using a hydraulic press. Next, samples were scanned 32 times at a frequency range of 4000–400 cm⁻¹ with a resolution of 4 cm⁻¹. The resultant spectra were smoothed to remove the noise. After spectral baseline correction and peak intensity normalization, the degree of methylation (DM) of pectin was calculated in accordance with the following equations (Morales-Contreras et al., 2020).

2.4.2 X-ray diffraction (XRD) analysis

The X-ray diffraction (XRD) pattern was performed by using an X-ray Cu Ka radiation diffractometer (Smartlab 9kw, Japan). The pectin powder was scanned with a diffraction angle (2θ) from 5° to 90° (0.02° angle gradient).

2.4.3 NMR (Nuclear magnetic resonance) analysis

¹H NMR spectra of samples were conducted using a Bruker 600 MHz NMR spectrometer (Bruker, Rheinstetten, Germany). Briefly, 10 mg of constant-weight sample was dissolved into 1 mL of 99.98% D₂O, and then the ¹H NMR spectrum was recorded.

2.4.4 Scanning electron microscopy (SEM) analysis

The surface morphology of the sample was observed by scanning electron microscope (Zeiss Sigma 300, UK). The sample was fixed and coated with gold under vacuum conditions. The surface morphology was photographed with an accelerating voltage of 4 kV and magnification of 20,000× and 200×.

2.5.1 Apparent viscosity

Samples were dissolved in deionized water at room temperature with magnetic stirring for 12 h. The sample solution was subjected to steady shearing at 25 °C soak time 60 s, the shear rates ranged from 0.1 s⁻¹ to 100 s⁻¹. Besides, emulsion and freeze–thaw variation on the apparent viscosity were also investigated at the same shear rate. Silicone oil (viscosity: 20mpa.s) was employed in the exposed sample edge to prevent water loss.

2.5.2 Viscoelasticity characterization

Viscoelastic properties were investigated by the frequency and temperature sweep. Firstly, vibration sweeps were performed under a strain range of 0.01–1000% at 1 Hz (25 °C) to determine the linear viscoelastic region. Thereafter, the frequency sweeps of pectin and emulsion were carried out at 25 °C in the frequency range of 0.1–100 Hz within the linear viscoelastic region (1.0% strain). Secondly, temperature sweeps were performed at a frequency of 1 Hz and strain of 1.0% with heating (20–100 °C) and subsequent cooling (100–20 °C) at a rate of 5 °C/min. Silicone oil (20 MPa.s) was employed in the exposed sample edge to prevent water loss.

2.5.3 Thixotropic property

The relationship between shear stress and shear rate can be used to determine thixotropic effects (Mierczynska et al., 2017). The apparent viscosity curves were recorded by increasing the shear rate from 1 s⁻¹ to 100 s⁻¹ and then decreasing from 100 to 1 s⁻¹. A closed changing curve was used to represent the formed thixotropy area. The closed area was calculated with the rheometer software (TA Instruments Trios v5.0).

2.6.1 Preparation of emulsion and emulsifying capability

The sample was dispersed in water (2% final concentration) and hydrated for 12 h, with 0.1% sodium benzoate as a preservative. MCT oil was absorbed with a syringe and stirred with a magnetic stirrer for 2 min (1800 rpm) to make a crude emulsion. MCT oil was added during stirring. The final concentration of MCT was 10%, and the crude emulsion was homogenized by a homogenizer for 2 min to fully integrate the oil and pectin solution, and the emulsion was completed.

The emulsion stability (ES) of samples was analyzed by a reported method (Liu et al., 2019). The emulsion was heated at 80 °C for 30 min, cooled in a water bath for 20 min, and then centrifuged at 2516g for 15 min. The emulsifying activity (EA) was assessed from the particle size, which was detected by a dynamic light scattering device (Zetasizer Nano ZS, Malvern Instruments, Malvern, England). Each emulsion was diluted 500 times with ultrapure water to avoid multiple scattering effects (Wandee et al., 2019).

2.6.2 Emulsion storage stability

The prepared emulsion was stored at 4, 25, and 60 °C for 21 d, and the photos before storage and day 21 of storage were taken with a digital camera, and the height of the layer was measured. The average particle size was measured once a week.

3.4.1 FT-IR analysis

FT-IR spectra of GFPPs in the range of 4000–500 cm⁻¹ (Fig. 1C) were analyzed further to gain an insight into structural information by functional group. The result showed that all extracted GFPPs had similar spectra. The intense and wide absorption peaks near 3400 cm⁻¹ were associated with the OH stretching, which mainly arose from the hydrogen bonding of glucopyranose hydroxyl groups (Zhu et al., 2019). The weak peak in appropriately 2900 cm⁻¹ was attributed to the aliphatic CH absorption, including the stretching and bending vibration of CH₂ and CH₃ (Zhu et al., 2020a,b). The peak at around 1739 cm⁻¹ and 1632 cm⁻¹ was due to C⚌O stretching vibration of methyl esterified carboxyl groups (C⚌O) and C⚌O stretching vibration of carboxylate ion(COO^–) (Chen et al., 2020a,b). The DM of pectin can be quantified by the peak area of corresponding functional groups (Manrique and Lajolo 2002). As a result, the calculated DMs for ACP, ALP, and HMP were 42.73, 42.40, and 46.54%, respectively, indicating that they belonged to LMP (Chen et al., 2020a,b). Commercial pectin primarily extracted from low-cost sources, such as citrus peel and apple pomace, is mainly in the form of high-methoxyl pectin (HMP). HMP typically requires a deesterification process to convert it into low-methoxyl pectin (LMP) (Wan et al., 2019). The currently obtained GFPPs belong to LMP, and their prepared methods are both cost-effective and environmentally friendly. Additionally, a particular focus was placed on the absorption bands associated with proteinaceous materials. The peak in the range of 1350–1310 cm⁻¹ was assigned to the stretching of amide III band C—N (Zhu et al., 2020a,b), and the peak at 1570–1515 cm⁻¹ was attributed to the N—H bending in the protein of amide II band (Wang et al., 2021). The result indicated that the proteinaceous materials in GFPPs might be cross-linked with pectin via covalent bonds (Yang et al., 2019). The region from 1250 to 800 cm⁻¹ was proposed to be characteristic fingerprint regions of pectin. The weak bands in the range of 1235–1258 cm⁻¹ for ACP, ALP, and HAP were due to the stretching vibration of C—O in the ester or carboxyl groups, indicating the presence of uronic acids (Ji et al., 2020). The peak at 1052 cm⁻¹ indicated the presence of Ara (Coimbra et al., 1998). Moreover, the peak at 920 cm⁻¹ was regarded as the absorption of D-glucopyranosyl, and 829 cm⁻¹ was the absorption of α-D-mannopyranose (Lin et al., 2021).

3.4.2 NMR and XRD analysis

¹H NMR spectrum is mainly used to reflect the configuration information of glycosidic bonds. Generally, those with a chemical shift value greater than δ 4.95 ppm are α-type glycosides, otherwise they are β-type (Zhu et al., 2020a,b). The signal intensity of methyl ester (COOCH₃) and 6-position deoxysugar methyl proton is δ 3.0–3.8 and 0.8–1.4, respectively (Yang et al., 2022a,b). As shown in Fig. 2, a strong and sharp signal at 3.75 ppm was derived from methyl groups binding to carboxyl groups of GalA, and a weak signal at 1.17/1.18 or 1.25/1.26/1.27 ppm was ascribed to the 6-position deoxysugar methyl proton (Yang et al., 2022a,b). Further, five primary signals of pectin, including 5.01 ppm (H-1), 3.63 ppm (H-2), 3.92 ppm (H-3), 4.05 ppm (H-4), and 4.38 ppm (H-5), were assigned as esterified D-GalA as reported by Yang et al. (Yang et al., 2022a,b). Notably, an extremely weak signal at 4.88 ppm (H-1) was presented, suggesting the three GFPPs might also contain a small amount of β-type glycosidic bonds. The chemical shift of H-1 of three GFPPs indicated that the anomeric hydrogen configuration of D-galacturonic acid remained intact (Wang et al., 2016a,b).

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Fig.2

¹H NMR spectra of ACP, HWP, and ALP.

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X-ray diffraction can be used to characterize the crystalline structure (crystalline or amorphous) of polysaccharides. As shown in Fig. 1D, three GFPPs had two obvious characteristic diffraction peaks at around 12.10° and 21.90°, indicating that they had a mixed structure of semi-crystalline and amorphous (Guo et al., 2021).

3.4.3 Surface morphological characteristics

A scanning electron micrograph (SEM) was applied to observe the surface morphology of three GFPPs (Fig. 3). After magnifying of 20,000× (Fig. 3A), ALP had the thinnest cross-section, but three GFPPs exhibited a curly sheet shape. Additionally, ALP showed a loose structure with more voids, while ACP pectin had a smooth surface with fewer voids (Fig. 3B, C). Generally, pectin with porous and wrinkled surfaces had better solubility than pectin with a layered structure. Meanwhile, a smoother layered structure contributed to the better gel strength of pectin, which might be related to the increase of pectin-bearing area on the smooth surface (Cui et al., 2020a,b). Smooth surfaces allow pectin to withstand more uniform forces and help pectin resist deformation (Zhang et al., 2021).

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Fig 3

SEM images of under 20,000× (A, B) and 200× (C).

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3.5.1 Apparent viscosity analysis

The apparent viscosity of different concentrations (1, 2, 4, and 5% w/v) of GFPPs was investigated under the shear rate range from 0.1 to 10 00 s⁻¹. As shown in Fig. 4A, all GFPPs showed a drop of apparent viscosity along with the shear rate increasing, and their rheological behavior exhibited a pseudo-plasticity like other pectins. With the concentration raising, the apparent viscosity increased enhanced the shear-thinning behavior of the pectin solution, which was mainly attributed to the decrease of the inter-entanglement of macromolecular chains along with the increased shear rate (Li et al., 2020). In addition, some molecules easily reoriented with the flow direction also led to a significant decrease in apparent viscosity (Zhou et al., 2021). Meanwhile, the apparent viscosity of ACP was significantly greater than that of HWP and ALP, which might be partially due to its larger MW and higher RG-I content (Chan et al., 2017, Yang et al., 2020).

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Fig. 4

Steady shear flow curves of different GFPP solutions (A), freeze–thaw temperature variation curves of ACP (B), HWP (C), and ALP (D).

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The freeze–thaw property of polysaccharides is a vital parameter for its application in cold chain storage food. The prepared GFPP solution (2%, w/v) was stored at −20 °C, 4 °C, and 25 °C, respectively, for 24 h. After equilibrium at room temperature for 6 h, the apparent viscosity was measured. As illustrated in Fig. 4B–D, the apparent viscosity of ACP was not influenced by the freeze–thaw temperature variation (Fig. 4B), while the apparent viscosity of HWP was dramatically improved after freeze–thaw temperature cycle at 4 °C and −20 °C (Fig. 4C). This observation was consistent with the results of the freeze–thaw temperature variation of a polysaccharide from Mesona chinensis the syneresis (Lin et al., 2018). By contrast, the apparent viscosity of ALP was compromised by the freeze–thaw cycle compared with that of the control (Fig. 4D). It is well known that the change of apparent viscosity reflects the interaction variation of polysaccharide solution between macromolecular-macromolecule or macromolecule-water (Pongsawatmanit and Srijunthongsiri, 2008). The low-temperature storage might promote entanglement and incomplete thawing for macromolecules, thus increasing apparent viscosity (Lin et al., 2018). On the other hand, low temperatures might also be conducive to the rearrangement of hydrophobic structures of macromolecules (Zhao et al., 2020, Xie et al., 2022), thereby weakening their interaction. The structural difference of GFPPs leads to the variation of macromolecular interactions at low temperatures, which may be the reason for the apparent rheological difference of pectin with different extractants.

3.5.2 Thixotropy property analysis

Thixotropy, as one of the critical rheological properties of a non-Newtonian fluid, is defined as a rheological phenomenon in which the apparent viscosity decreases under shear stress, followed by a gradual recovery when the stress is removed (Zhao et al., 2020). The hydrocolloids with thixotropic properties are desired for their application in some foods, such as mayonnaise, and ice cream, which need flow during processing but should stiffen afterward (Zhao et al., 2020).

Hysteresis loop can be used to investigate the thixotropy, in which symmetry of up- and down-curves can be achieved with the shear rate “upward” and “downward”, and the area enclosed by the up- and down-curve is referred to as hysteresis loop (Wei et al., 2015, Alghooneh et al., 2018). The thixotropy of GFPPs was investigated by using the hysteresis loop method (Wei et al., 2015), and the result was presented in Fig. 5. All GFPPs showed hysteresis loops at a concentration of 4% and 5% (w/v), and ACP had the largest hysteresis loop area (Fig. 5A), followed by that of ALP (Fig. 5B). By contrast, the hysteresis loop of HWP was simply presented until the concentration up to 5% (w/v), but with less area (Fig. 5C). This result indicated that GFPP solutions exhibited the thixotropic behavior in a concentration-dependent manner, which could be attributed to the disentanglement–entanglement processes and the alignment of the polymer chains with corresponding to the direction of shear (Wei et al., 2015, Alghooneh et al., 2018). Thixotropy requires a microstructure that can be reversibly altered by moderate stresses, and significant viscosity changes are induced in such a manner (Alghooneh et al., 2018). The presence of a thixotropic effect might also indicate the complex structure of the materials (Mierczynska et al., 2017). The pectin extracted from citric acid usually has a high MW and apparent viscosity (Cho et al., 2019). It was reported that the pectin-enriched fractions from peach, blackcurrant, raspberry, strawberry, plum, and carrot extracted by citric acid also displayed thixotropic properties (Mierczynska et al., 2017). Our results showed that GFPP extracted by citric acid had excellent thixotropic properties, indicating a preferential application in food processing.

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Fig. 5

Thixotropic characteristics of ACP (A), HWP (B), and ALP (C) at different concentrations.

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3.5.3 Viscoelastic property analysis

Viscoelastic properties of GFPPs were investigated by the frequency and temperature sweep. The storage modulus (G′) and the loss modulus (G″) reflected the elastic and viscous character of viscoelastic material, respectively, whereas the damping factor (tan δ, G″/G) was used as an index to determine the predominance of solid or liquid character. As shown in Fig. 6A, the storage modulus (G′) and loss modulus (G″) of GFPPs were increased with the frequency increasing in a dose-dependent manner. Specifically, the storage modulus (G′) of ACP was always greater than the loss modulus (G″) with the damping factor (tan δ, G″/G′) values between 0.1 and 1.0, indicating its “weak gel” properties (Lin et al., 2018). As for HWP, the storage modulus (G′) was lower than that of the loss modulus (G″) in low frequency (nearly 10 Hz), the G″ and G′ cross-over could be observed, and G′ surpassed G″ at a frequency higher than appropriately 15 Hz, indicating an observed viscous characteristic of HWP (Wang et al., 2018). Although the storage modulus (G′) and loss modulus (G″) of ACP were complex, it had an observed elastic property at 0.1–2.51 Hz, a viscous property at 2.51–39.81 Hz, and an elastic property at 39.81–100 Hz. The viscoelastic property of ALP showed no regularity and was similar to a mechanical characteristic of pectin from Cerasus humilis (Zhang et al., 2021).

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Fig. 6

Viscoelastic properties of different concentrations of GFPPs in frequency sweep (A) and temperature sweep (B-D, B-ACP, C-HWP, and D-ALP), temperature blue (cooling): 90–20 °C; red (heating): 20–90 °C.

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In order to evaluate the thermo-mechanical structural characteristics of GFPPs, temperature sweeps were carried out and the results were shown in Fig. 6B–D. The G′ and G″ of all GFPPs were decreased during heating and were increased during cooling regardless of the concentration. The storage modulus (G′) of ACP was dominant in either heating or cooling process (Fig. 6B). In contrast, the storage modulus (G′) of HWP was less than the loss modulus (G″), presenting a fluidic character (Fig. 6C). As for ALP, G′ was higher than G″ in cooling and gel point was observed at two moduli of 4% ALP at 58.33 °C in the heating scan, which indicated that the system underwent phase transition and changed from elastic to adhesive characteristics (Fig. 6D). In addition, the storage modulus (G′) of 5% ALP was higher than the loss modulus (G″), and the modulus of ALP temperature change was consistent with the result of frequency sweep, which was embodied as elastic property from 0.1 to 2.51 Hz before frequency sweep. As a result, 5% ALP had better stability between 20 °C and 90 °C. For 4% or 5% ACP, its G′′ was always dominant whether heating or cooling. Numerous studies have shown that the side chains of pectin molecules further tighten their network structure through entanglement and topological constraints at high concentrations (Feng et al., 2019, Lin et al., 2021). More side chains and short chain structures in ACP (Table 2) provide stable structural support during heating and cooling.

3.6.1 Emulsifying capability and emulsifying stability

In order to evaluate the emulsifying property of GFPPs, different concentrations (1.5, 2.0, and 2.5% w/v) of samples were mixed with 10% (w/v) of MCT to prepare the emulsion, and their average particle size and emulsification stability were examined. As shown in Fig. 7A, the average particle size of all GFPP emulsions was in the range of 1–3 μm. Among them, the emulsion prepared with ALP had the smallest average particle size (appropriately 1 μm), followed by that of ACP and HWP, implying that ALP might have the strongest emulsifying capability. The emulsifying function of pectin can be influenced by its structural properties, including the extraction method used and the presence of hydrophobic groups. Pectin extracted by different methods may have variations in its structural properties, leading to differences in emulsifying ability. In the case of ALP, its high emulsifying ability can be mainly attributed to its high protein content and small-MW distribution. Wang et al. found that the lower-MW pectin after sonication showed better emulsifying properties (Wang et al., 2016a,b). Furthermore, studies have indicated that pectin polysaccharides with an MW between 100 × 10³ g mol⁻¹ and 200 × 10³ g mol⁻¹ are more favorable for emulsification and can provide long-term stability (Alba and Kontogiorgos 2017). The small MW of ALP resulted in lower interfacial tension, resulting in smaller droplets and higher emulsifying capacity. As for protein content, Funami et al. discovered that removing proteins from pectin molecules using protease resulted in a significant increase in emulsion particle size and a decline in stability, indicating the crucial role of protein in pectin's emulsification process (Funami et al., 2007). The protein attached to pectin can quickly adsorb on the surface of oil droplets and form a viscoelastic film to prevent oil droplets from focusing (Niu et al., 2022). As expected, the average particle size of emulsions also decreased with the increase in pectin concentration. The larger average particle size caused by a high concentration of pectin might be related to the higher protein content contained in the sample.

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Fig. 7

Emulsifying capability and emulsifying stability of GFPPs. A. Average particle size of emulsions; B. Emulsion stability.

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The stability of GFPP emulsions was further evaluated by incubating at 80 °C for 30 min. As shown in Fig. 7B, the emulsification stability exhibited a positively correlated with the concentration. The emulsification stability was increased from 60–70 to 90% along with the concentration from 1.5 to 2.5%, while the emulsification stability of GFPPs did not show a significant difference. Since the emulsification stability of GFPPs was about 90% at a concentration of 2%, the concentration of 2% was selected for subsequent experiments.

3.6.2 Rheological properties of emulsion

In order to gain an insight into the emulsifying mechanism of GFPPs, the apparent viscosity and viscoelasticity properties of sample emulsions were assessed at the concentration of 2%. As presented in Fig. 8A, the emulsification resulted in a significant increase in the apparent viscosity of ALP and HWP, with almost 100 times larger than pure solutions at a stress rate of 0.1 s⁻¹. In contrast, the emulsifying could only slightly increase the apparent viscosity of ACP. The strong shear stress for emulsion preparation might lead to the rearrangement of droplets, which not only magnified the steric hindrance between emulsion droplets but also increased the intermolecular friction of pectin, increasing viscosity. This result was also consistent with that of pomegranate pectin (Yang et al., 2018) and persimmon pectin (Jia et al., 2022). Usually, increasing the viscosity of the continuous phase is one of the primary methods to enhance the stability of the emulsion (Yang et al., 2018), and higher viscosity can give the emulsions better stability (Jiang et al., 2020).

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Fig. 8

Apparent viscosity for solutions and emulsions prepared with GFPPs at 2% (w/v) pectin (A). Frequency dependence of storage modulus and loss modulus for solutions and emulsions (B-HWP, C-ACP, and D-ALP).

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The frequency sweep experiments were performed at the constant strain of 1%, which was in the linear viscoelastic region (Fig. 8B–D). The G′ and G″ cross-over points were found in HWP and ACP but in a reversed manner. Moreover, G″ of HWP was higher than that of G′ at low frequency, and the cross-over point was presented at 10 Hz and 2.51 Hz for the HWP solution and HWP emulsion, respectively (Fig. 8B). For ACP, the G″ was lower than that of G′ at low frequency, whereas the cross-over point of ACP solution and ACP emulsion was at 13.34 Hz and 1.58 Hz, respectively (Fig. 8C). The G′ of ALP solution was less than that of G″, while the G′ of ALP emulsion was higher than that of G″ (Fig. 8D). Interestingly, the ALP emulsion had a creaming-like appearance (Fig. 8D, inner), which was similar with the emulsion from watermelon rind pectin (Mendez et al., 2021) and Camellia oil-based oleo gels prepared by polyphenol-palmitate and citrus pectin (Luo et al., 2019). Emulsion gel is a viscoelastic colloid that can confine emulsified oil droplets to a biopolymer three-dimensional network.

3.6.3 Emulsion storage stability

Oil-in-water emulsion stabilized by GFPP solution (2%, wt%) at an oil fraction of 10% v/v was prepared and stored at 4 °C, 25 °C, and 60 °C for 21 days. As shown in Fig. 9 A-C, the average particle size of the GFPP emulsions gradually increased with time, and ALP always had the smallest average particle size. On the one hand, the particle size was consistent with the stability behavior of the lotion. The emulsion stored at 4 °C and 25 °C had higher kinetic stability than that stored at 60 °C. The main reason for this was that the increase in temperature not only increased the free energy of the colloidal system but also reduced the viscosity of the polymer at the water–oil interface, which led to emulsion destabilization (Santiago et al., 2018).

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Fig. 9

Droplets diameter of the GFPP emulsions on the 1st day, 7th day, 14th day, and 21st day (A, 4 °C; B, 25 °C, and C, 60 °C). Macroscopic examination of GFPP emulsions during storage at room temperature for 21 days (D). Red arrows indicate a phase separation.

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The physical stability of emulsions was inspected visually using digital photos, and photographs of the emulsions by freshly-preparation and storage for 21 days at 4 °C, 25 °C, and 60 °C were presented in Fig. 9D. All emulsions showed no demulsification or phase-separation after preparation at 4 °C and 25 °C. However, after being stored at 60 °C for 21 days, a small amount of continuous phase was precipitated from HWP and ACP, especially HWP showed obvious phase separation (red arrows in Fig. 9D). In contrast, ALP showed no demulsification or phase separation.

The emulsifying properties of pectin were affected by their structure, neutral sugar side chain, and hydrophobic groups (Ngouémazong et al., 2015, Alba and Kontogiorgos 2017, Niu et al., 2022). Three GFPPs exhibited favorable emulsifying properties, which might be explained by the following reasons: (i) The amount of RG-I region of GFPPs was in the range of 42–54%. The hairy structure of RG-I and RG-II domains drove the long main chain to be adsorbed on the oil–water interface, while the other parts swelled in the water phase. Besides, the hydrophobic effect derived from the carbon atoms drove the polymer chain adsorption, while the solvent effect from the hydroxyl group generated the polymer swelling (Neckebroeck et al., 2020). At the same time, the thick interfacial layer could provide a stronger steric hindrance and was important for the long-term stability of the emulsion (Zhang et al., 2021). (ii) The protein content of GFPPs was 1.36–3.11%. Pectin and protein might form a pectin–protein conjugate through covalent and non-covalent interactions, which improved the surface activity of pectin and made the emulsions more resistant to environmental stress (Ngouémazong et al., 2015, Niu et al., 2022). However, ALP had stronger emulsification stability than ACP or HWP, especially because it could maintain the emulsion stability at 60 °C for 21 d (Fig. 9D). It was proposed by Alba and Kontogiorgos (Alba and Kontogiorgos 2017) that pectin with protein content in the range of 3%, with a degree of acetylation greater than 10%, MW between 100 and 200 kDa and enriched in RG-I segments were more likely to perform well as an emulsifier. The MW, protein content, esterification degree, and amount of RG-I region of ALP were 192 kDa, 3.11, 42.40, and 42.20%, respectively. Therefore, the strongest emulsification capability of ALP could be partly attributed to its unique structure and chemical composition.