Physicochemical and rheological characterization of pectin‐rich polysaccharides from Gardenia jasminoides J. Ellis flower

Abstract Gardenia (Gardenia jasminoides J. Ellis) is regarded as an edible medicine plant in China. Here, gardenia flower polysaccharide fraction (GFPF) was extracted by water at 90°C and its chemical composition, rheological properties, and antioxidant activities of GFPF were investigated. The GFPF extraction yield was 18.04 ± 1.81% (W/W) and mainly comprised neutral sugars (46.83 ± 3.14%), uronic acid (35.21 ± 0.17%), protein (1.63 ± 0.34%), and total phenol (9.49 ± 0.08 mgGAE/g). Galacturonic acid (41.05 ± 0.59%) was the main monosaccharide, and galactose, glucose, arabinose, rhamnose, xylose, mannose, and glucuronic acid were also detected in GFPF. Its degree of esterification was 32.76 ± 1.52%. FT‐IR spectra analysis showed a similar absorption pattern between GFPF and pectin from apple. The results suggested that GFPF was low methoxy pectin. Thermogravimetric analysis and zeta potential analysis indicated that the pectin was stable under high temperature and alkaline condition. Steady rheology showed that the GFPF dispersion was a shear thinned pseudoplastic fluid with high apparent viscosities at concentration above 2%. The degree of pseudoplasticity of the solutions increased with the concentrations increased and the temperatures decreased. DPPH and ABTS free radical scavenging assay indicated that GFPF had relatively high antioxidant activity. The results showed that gardenia flower was rich in pectin polysaccharides with low methoxy pectin. It had high apparent viscosities at concentration above 2% and had good antioxidant activity. The data suggested that GFPF can be a new resource of low methoxy pectin with potential application as thicker or gelling agents in food industry.


| INTRODUC TI ON
Gardenia jasminoides J. Ellis, a flowering plant belonging to the family of Rubiaceae, is widely distributed in the south of China, which is traditional regarded as an edible medicine plant in China. Its fruit contains many functional components such as crocin, geniposide, genipin, and phenolic compounds (Yin & Liu, 2018) and has traditionally been used as a folk medicine in China and many East Asian countries for thousands of years with many biological activities, such as antioxidant activities, improving insulin sensitivity and antidiabetes, anti-inflammatory activity, and antidepressant activity (Shan et al., 2017;Xiao, Li, Wang, & Ho, 2017). Besides the application in folk medicine, gardenia fruit is used as a natural food colorant and its flower is also used as edible vegetable in China .
Recently, gardenia flower was also reported to have a wide range of bioactive phytochemicals, such as iridoids and terpenoids, which may provide desirable health benefits with anticancer and antioxidant activities (Zhang et al., 2017(Zhang et al., , 2018. By contrast, the content and bioactivity of other components of flower, such as nonstarch polysaccharides (NSPs), still did not attract much attention yet.
Pectin, an structurally heterogeneous acid polysaccharides, is one of the major NSPs in plant cell wall (Marić et al., 2018). Due to its strong hydrophilic character and favorable rheological properties, pectin is widely using in food industry as a gelling agent, emulsifier, stabilizer, and food thickener (Chan, Choo, Young, & Loh, 2017). Pectin is often esterificated by methyl groups (at C-6) and/or acetyl groups (at O-2 and/or O-3) at galacturonic acid residues. According to degree of esterification (DE), the pectin is divided into two groups: low methoxy pectin (LMP, DE < 50%) and high methoxy pectin (HMP, DE > 50%) (Dranca & Oroian, 2018). The degree of esterification offers pectin with the distinct gels formation characteristics. HMP forms a gel in an acidic environment (pH < 3.5) or in the presence of a high concentration of low molecular weight cosolutes, such as sucrose (more than 55%) (Giacomazza, Bulone, San Biagio, & Lapasin, 2016). LMP can also form gel in an acidic condition (pH < 3.3). Moreover, it can form gel with a wide pH range when divalent ions such as ionic calcium are present (Han et al., 2017;Yang, Nisar, Liang, et al., 2018). LMP is more preferred in some functional foods.
Commercial pectin is mainly extracted from many cheap resources such as apple pomace (Cho et al., 2019) and citrus peels (Hosseini, Khodaiyan, & Yarmand, 2016), which is mainly HMP. The HMP can be de-esterificated by chemical or enzymatic methods to produce LMP (Buchholt, Christensen, Fallesen, Ralet, & Thibault, 2004;Wan, Chen, Huang, Liu, & Pan, 2019). However, HMP de-esterification by using chemical methods may lead to chemical waste-induced environmental damage and to degrade the pectin resulting in poor thickening or gelling properties (Yoo, Fishman, Savary, & Hotchkiss, 2003). Moreover, HMP de-esterification by enzymatic methods can increase the product price. Finding new sources of natural LMP has attracted more attention in recent years (Lu et al., 2019;Yuliarti, Chong, & Goh, 2017).
Here, we found that gardenia flower is a new resource for LMP.
The aim of this study was to determine the chemical compositions, thermal stability, rheological behaviors, and antioxidant activities of pectin extracted from gardenia. It may provide a foundation for its application in food production.

| Materials and chemicals
Materials: G. Jasminoides J. Ellis flowers were harvested from Zhejiang Province in June 2016. After dried with hot air at 60°C for 24 hr, the flowers were milled using a blender (YB-1500A), sieved (40 mesh pass), and stored in plastic bags in 4°C before extraction.

| GFPF extraction
To remove pigments, lipids, peptides, and other low molecular weight compounds, the milled gardenia flower was washed twice with 85% ethanol overnight at room temperature. The resulting residues were dried at room temperature. To extract GFPF, the dried biomass was mixed with distilled water at a ratio of 1:40(w/v) at 90°C with stirring for 2 hr. The mixture was centrifuged at 5,000 g for 20 min. After extraction twice, the supernatants were combined and concentrated by rotary evaporator to 1/2 of original volume at 60°C. The GFPF was precipitated by adding three volumes of ethanol and kept at 4°C overnight. The precipitate was separated by vacuum filtration and dehydrated with ethanol. The resulting GFPF was dried at room temperature in a fume hood. The extract was repeated three times, and the yield was calculated according to the dried biomass obtained after treating the milled sample with 85% ethanol.
where A was the weight of GFPF from gardenia and B was the weight of powder.

| Proximate analysis
Neutral sugar content of the GFPF was determined by the phenolsulfuric acid assay using D-glucose as a standard (Rover, Johnston, Lamsal, & Brown, 2013). Uronic acid content was determined by a sulfamate/m-hydroxy-diphenyl assay using galacturonic acid as a standard (Blumenkrantz & Asboe-Hansen, 1973). Protein content was determined by Bradford method using BSA as a standard (Bradford, 1976).

| Monosaccharide composition of GFPF
Monosaccharide composition of GFPF was analyzed by reversedphase HPLC using PMP as a precolumn derivatization reagent as previously described by (Dai et al., 2010) with some modification.
Briefly, the GFPF was hydrolyzed with 4 M TFA at 110°C for 4 hr.

| Measurement of the degree of esterification
The DE of GFPF was determined by titration method as described by (Trujillo-Ramírez et al., 2018). Briefly, 0.1 g GFPF was dissolved into 50 ml distilled water. After adding 3 droplets of phenolphthalein to the solution, it was titrated using sodium hydroxide (0.1 M) until the solution turned pink and the titration volume was recorded as V1.
And then, 5 ml sodium hydroxide (0.5 M) was mixed in the solution and kept at room temperature for 20 min. Then, 10 ml hydrochloric acid (0.5 M) was added, and the sample was shaken violently until the pink color vanished. The resultant solution was titrated by using 0.1 M sodium hydroxide until the solution turned pink, and the volume was recorded as V2. The DE was calculated using the formula:

| FT-IR analysis
The structural characteristics of GFPF were investigated using infrared spectroscopy. The extracted pectin was analyzed by FT-IR (IR Prestige-21, Shimadzu, Japan) using wavelength range from 500 cm −1 to 4,000 cm −1 on KBr disks with a 90:10 KBr/pectin ratio.
Pectin from apple (Sigma-Aldrich, NO. 93854) was used as reference.

| Zeta potential measurement
The aqueous dispersion of GFPF (1 mg/ml) was adjusted by 0.1 M NaOH or HCl to pH 2.0-11.0. The zeta potential was analyzed by using high-resolution zeta potential analyzer (Brookhaven ZetaPALS) at 25°C. Each sample was measured three times, and all measurements were carried out at 25°C. to determine the molecular weights of the samples.

| Thermogravimetric analysis
The thermal properties of GFPF were evaluated by Thermo Gravimetric Analyzer (*STA 409C, NETZSCH, Germany). The samples (5-10 mg) were placed in alumina crucibles and flowed through nitrogen gas at a rate of 20 ml/min. The temperature increased from 25 to 600°C with heating rate 10°C/min. An empty alumina crucible was used as reference.

| Steady shear flow behavior
The GFPF was dissolved in distilled water by magnetic stirring to obtain different concentrations samples (0.5%, 1%, and 2% w/v). The samples were stored (25°C, 12 hr) for adequately hydration in distilled water before experiments. The effects of various concentrations at 25°C and temperatures (5, 25, and 45°C) at 2% w/v were on the rheological behavior of GFPF.
The power law model was employed to explain the flow behavior of pectin.
where τ represents the shear stress, γ represents the shear rate, k represents a consistency index, and n is the index of power law model.

| Linear viscoelastic region and dynamic oscillatory measurements
Strain sweeps were performed (0.01%-1000% strain, 1 HZ) to determine the extension of the linear viscoelastic region (LVR). The LVR of GFPF was conducted for latter dynamic oscillatory measurements.
The frequency sweep (0.1HZ -10HZ) was performed in strain control mode, and the selected strain was within the LVR range of all samples.

| Antioxidant activity
The antioxidant activity of GFPF was evaluated by using DPPH and ABTS assays as described by (Shao et al., 2014) with some modification. The ability to scavenge the DPPH and ABTS radicals was calculated according to the following equation: where A 0 is the absorbance value of DPPH or ABTS radical cations and A i is the absorbance value of the sample. The IC 50 value was defined as the concentration of GFPF required for reducing 50% of the DPPH or ABTS radicals.

| Statistical analysis
All the data were analyzed by Excel software, and the results were expressed as the mean and standard deviation. All the figures were plotted using Origin 8.5 software.
GFPF mainly consisted of neutral sugars (46.83 ± 3.14) and small amount of proteins (1.63 ± 0.34%). The phenol composition was also presented in GFPF with 9.49 ± 0.08 mg GAE/g. The uronic acid sugars of GFPF were 35.21 ± 0.17%, which indicated that it is a strongly acidic polysaccharide. The DE of GFPF was 32.76 ± 1.58%.
The molecular weight distribution is an important parameter related to the rheological properties of polysaccharide. As shown in GFPF. Galactose, glucose, arabinose, rhamnose, xylose, mannose, and glucuronic acid were also detected in GFPF. The result indicated that GFPF is a galacturonic acid-rich acidic polysaccharide.

| FT-IR analysis
FT-IR spectroscopy of GFPF and pectin from apple was compared by using wavelength range from 500 cm −1 to 4,000 cm −1 . As shown in Figure 2a, the strong and broad peak of GFPF was found at 3,435.04 cm −1 , which was attributed to O-H stretching vibration . The peak at 2,924.31 cm -1 was referred to C-H stretching from CH 2 groups. The peak at 1,750.18 cm -1 was contributed to C=O stretching vibration of -COOH, while the peak at 1617.74 cm -1 was the C=O asymmetric stretching vibration of the free carboxylic acid or carboxylate (Manrique & Lajolo, 2002 (Han et al., 2016). The peaks at 1,103.54 cm −1 and 1,018.22 cm −1 indicated that the GFPF contains uronic acid. The FT-IR spectra of GFPF displayed a similar general pattern and exhibited similarities of the absorption patterns to that of pectin from apple ( Figure 2b), suggesting that GFPF may be a pectin-rich polysaccharide.

| Zeta potential analysis
As shown in Figure 3, the zeta potential value of GFPF decreased from −21.17 mV to −42.46 mV along with pH elevation from 2.0 to 11.0, which implied the presence of high fraction of hydroxyl and carboxyl radicals. This may be due to the carboxylate anion concentration increase with pH increase (Yang, Nisar, Liang, et al., 2018).
However, the zeta potential values of GFPF solution were −42.46 mV and −41.30 mV at pH 9.0 and 11.0, respectively. The results indicated that the GFPF had better stability under alkaline conditions.
The effect of different temperatures (5, 25, and 45°C) on the rheological properties was investigated using 2% GFPF. As expected, the apparent viscosity decreased with the temperature increase and with the shear rate raising (Figure 5b). The weakening of the electrostatic effect and the increasing of the thermodynamic effect were companied with temperature increase (Wu et al., 2018), which might led to the decrease of viscosity.
The steady rheological characteristics of GFPF solutions were described by fitting the shear rate and shear stress to power law equation. As illustrated in Table 2, the coefficients of determination (R 2 ) for all measured samples were above 0.93, implying that the power law model can be applied to describe the flow properties of the GFPF solutions. The consistency coefficient K increased from 0.133 to 62.683 Pa s n as GFPF concentration increases from 0.5% to 2%. The K value of GFPF was similar to a pectin from the roots of Arctium lappa L. at 2% (k = 60.657 Pa s n )  and the high methyl-esterified pectic fraction (STW-A) from tamarillo at 3% mixing with 50% sucrose (k = 43.979 Pa s n ) (Nascimento, Simas-Tosin, Iacomini, Gorin, & Cordeiro, 2016) and is more higher than that of many other pectins (Feng et al., 2019).
The flow behavior index n of all tested samples was less than 1.0 (0.19-0.86) which demonstrated the pseudoplastic (shear-thinning) nature of GFPF. The n value decreased from 0.856 to 0.205 with the concentration increasing from 0.5% to 2%, while the effect of temperature on flow behavior index (n) was negligible, implying that GFPF is stable below 45°C. Hydrocolloid with low n value has high viscosity and provides a pleasant mouthfeel (Marcotte, Taherian Hoshahili, & Ramaswamy, 2001).The results indicated that GFPF may be suitable for the application in food industry.

Strain sweep
The strain sweep tests of GFPF were carried out under different concentrations (2% and 2.5%) or different temperatures (5, 25, and 45°C), and the results are shown in Figure 6. The storage modulus (G′) and loss modulus (G′′) values of GFPF 2.5% were higher than that of 2%, indicating higher rigidity of the system at higher concentrations ( Figure 6a). Conversely, both moduli values decreased with temperature increase from 5 to 45°C (Figure 6b).

Frequency sweep
The frequency sweep test was conducted to investigate the mechanical response of GFPF gel over the frequency of 0.1 HZ-10 HZ.
The value of G' and G" of 2.5% was much higher than those of 2%, moduli almost 5 times higher than that of 2% at 0.1 HZ, indicating that the gel strength was concentration dependent (Figure 7a).
Besides, the value of G' and G" tends to converge at higher frequency and tan δ increased from 0.23 to 0.5 with frequency argument ( Figure 7b). The results indicated that the formation of a network between interchain and intrachain (Yang, Nisar, Liang, et al., 2018) and gel properties became weaker in higher frequency.
The effect of temperature on its viscoelasticity is shown in Figure 7c. The values of G′ and G″ decreased with the temperature increase. The tan δ increased with frequency argument, and the value of 5°C was lower than that of 25°C or 45°C (Figure 7d). The results demonstrated that the gel became weak with the temperature increase.

| Antioxidant activity of GFPF
To evaluate the antioxidant activity of GFPF, its DPPH or ABTS free radical scavenging activity is investigated. As illustrated in Figure 8, GFPF demonstrated the free radical scavenging capability in a concentrationdependent manner at the concentration range of 0.1-0.8 mg/ml. The DPPH free radical scavenging activity increased from 12.51% to 79.32% along with concentration from 0.1 to 0.8 mg/ml (Figure 8a). The DPPH free radical scavenging IC 50 for GFPF was 0.47 mg/ml. As for ABTS free radical, GFPF exhibited the similar dose-dependent scavenging effect as for DPPH (shown in Figure 8b). The IC 50 for ABTS free radical scavenging was 0.43 mg/ml. However, its DPPH or ABTS free radical scavenging capability was lower than that of ascorbic acid (16 μg/ml).

| CON CLUS IONS
This study demonstrated that gardenia flower is a new resource of low methoxy pectin (DE = 32.76 ± 1.52%) and the extraction yield was 18.04 ± 1.81%%. It was rich in galacturonic acid (41.05 ± 0.59%).
The flow and viscoelastic properties of GFPF were dependent on concentration and temperature. The apparent viscosity was fit with power law model (R 2 > 0.93) and showed a good pseudoplastic behavior. The dynamic viscoelastic behaviors of GFPF displayed gellike behaviors with the G′ that was higher than G′′. Furthermore, GFPF showed high antioxidant activity with low IC 50 for DPPH and ABTS free radical scavenging. The results suggest that GFPF can be a very promising source of high-quality low methoxyl pectin and for food and nonfood applications.

ACK N OWLED G EM ENT
This research was funded by the Natural Science Foundation of Zhejiang Province (LY17C200019) and the Planted Talent Plan for College Student of Zhejiang Province (2019R412040).

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.