In this study, jujube polysaccharides (JP) were extracted from
Polysaccharides are complex biopolymers comprised of monosaccharides chains and widely distributed in plants, animals, and microorganisms. As one of the most important biopolymers existing in nature, polysaccharides play diverse and important roles in many biological processes [
The application of natural polysaccharides as bioactive ingredients and food additives was limited by their complex structure and various bioactivities mechanism. Many natural polysaccharides lack useful bioactivities or exhibit weak biological activities and need to be further improved in a necessary way [
Jujube (
In order to further study and utilize the
The fruiting bodies of
Chloroacetic acid and absolute ethyl alcohol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 95% ethanol, trichloroacetic acid, concentrated hydrochloric acid (HCl), concentrated sulfuric acid, chloroacetic acid, sodium hydroxide (NaOH), disodium hydrogen phosphate, sodium dihydrogen phosphate, 1,1-diphenyl-2-picrylhydrazyl (DPPH), potassium sulfate, potassium ferricyanide, anhydrous iron chloride, salicylic acid, ferrous sulfate heptahydrate, hydrogen peroxide, chloroacetic acid, and vitamin C were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). Dextrans with different molecular weight (T-10, T-40, T-70, T-100, and T-500) were purchased from Solarbio Science & Technology company (Shanghai, China). Monosaccharide standards including arabinose (Ara), rhamnose (Rha), xylose (Xyl), mannose (Man), glucose (Glc), and galactose (Gal) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals and reagents were of analytical reagent grade. The ultrapure water was utilized from a Milli-Q water purification system (Millipore, Bedford, MA, USA).
Three bacterial strains,
The crude
The carboxymethylation of JP was performed based on the method described by Wang et al. [
The influence of the concentration of NaOH solution, the content of chloroacetic acid, and the reaction temperature on the degree of substitution (DS) of CMJP was studied. Single-factor experiments showed that the optimal NaOH solution concentration, chloroacetic acid content, and reaction temperature were 2.5 mol/L, 2%, and 60°C, respectively. On the basis of single-factor test results, response surface methodology (RSM) was used to further optimize the preparation conditions of CMJP. Three-factor three-level, Box–Behnken factorial design (BBD) was used to evaluate the combined effect of three independent variables: NaOH solution concentration, chloroacetic acid content, and reaction temperature, coded as
The adequacy and significance in the model was evaluated by analysis of variance (ANOVA) for each response. Design-Expert 11 software package (Trial Version, Stat-Ease Inc., Minneapolis, MN, USA) was used to analyze the experimental data.
According to the neutralisation titration method, 20 mg CMJP was dissolved in 20 mL NaOH (0.01 mol/L) and stirred thoroughly with a magnet stirrer for 60 min at 40°C. The solution was titrated with 0.1 mol/L HCl until the color of phenolphthalein disappeared. The DS was calculated as follows:
For water solubility, 1 mL water was added to 100 mg samples and then shaken vigorously for 12 h until the sample was completely dissolved. The precipitate was dried at 55°C and weighed. The rheological properties of each sample were evaluated on a rotational rheometer (Anton Paar RheolabQC model) in three replicates. 10 mg/mL aqueous solution of JP and CMJP in distilled water was prepared. A shear rate sweep from 0.1 to 1000 s−1 was applied, and rheological data were collected at 25°C. The relationship between apparent viscosity and shear rate was modeled with a power law equation:
The total sugar contents of JP and CMJP were determined by phenol-sulfuric acid method, using
JP and CMJP samples (10 mg) were hydrolyzed with 2 mol/L trifluoroacetic acid (TFA, 4 mL) at 110°C for 6 h. The hydrolyzed samples and standard monosaccharides were acetylated by the addition of hydroxylamine hydrochloride, pyridine, and acetic anhydride. The acetylated polysaccharide was analyzed by gas chromatography (GC) on an Agilent model 7890A instrument, equipped with a TG-5MS capillary column (30 m × 0.25 mm × 0.25
The Mw of JP and CMJP was measured using high-performance liquid chromatography (HPLC) which was undertaken on an Agilent 1200 Infinity Series LC system (Agilent Technologies, Germany) coupled with an ELSD-1260 (Agilent Technologies, UK) evaporative light detector. Samples were dissolved in ultrapure water (0.5 mg/mL), passed through a 0.22
The infrared spectra of JP and CMJP were measured by a FT-IR spectrophotometer (Spectrum 65, PerkinElmer, USA) using KBr compression method. One mg of JP or CMJP was ground with 200 mg of KBr powder (completely dried at 100°C) and pressed into a 1 mm agate mortar into a pellet with 1 mm thickness for a frequency resolution of 1 cm−1 and 32 scans between 400 and 4000 cm−1.
Ultraviolet spectra of JP and CMJP were obtained on TU-1810 ultraviolet spectrophotometer (Purkinje General Instrument Co., Ltd., Beijing, China). The scanning range was 190–900 nm at 1 nm intervals for each sample (0.1 mg/mL, w/v in distilled water).
Scanning electron micrographs of JP and CMJP were obtained with a TESCAN VEGA3 LMH scanning electron microscope (TESCAN Co., Brno, Czech Republic). Sample powders were placed on a specimen holder with the help of double-sided adhesive tapes and coated with a thin layer of gold powder. Each sample was observed with 5,000- and 10,000-fold magnification at an accelerating potential of 10 kV during micrography. Chemical analysis was performed using an energy-dispersive X-ray spectroscopy (EDS, Model TEAM, EDAX, USA) facility.
The effect of scavenging DPPH radicals of JP and CMJP was determined by a reported method, with minor modification [
The hydroxyl radical scavenging abilities of JP and CMJP were evaluated according to the method of Duan et al. [
The effect of scavenging O2.− of JP and CMJP was measured by the method of Liu and Huang [
The reducing power of JP and CMJP was quantified by the method described by Li et al. [
The activated
Each assay of all determinations was performed in triplicate and expressed as mean ± SD (standard deviation). One-way analysis of variance (ANOVA) followed by Duncan’s multiple-range test was used for multiple comparisons by the SPSS 23.0 software package (Chicago, USA).
As shown in Figure
Effects of NaOH solution concentration (a), chloroacetic acid content (b), and reaction temperature (c) on the DS of CMJP. Data with different lowercase letters represent significant differences at
The DS of CMJP had been increasing when chloroacetic acid content increased from 1% to 2% as shown in Figure
As shown in Figure
According to the single-parameter study, we adopted NaOH solution concentration of 2, 2.5, and 3 mol/L, chloroacetic acid content of 1%, 2%, and 3%, and extraction temperature of 50, 60, and 70°C for RSM experiments.
The variation of responses (DS of CMJP) at different experimental combinations was given in Table
Analysis of variance (ANOVA) for response surface quadratic model for the optimization of carboxymethylation conditions.
Source | Sum of squares | Degree of freedom | Mean of squares | Significance | ||
---|---|---|---|---|---|---|
Model | 0.0172 | 9 | 0.0019 | 14.37 | 0.0010 | |
0.0005 | 1 | 0.0005 | 3.90 | 0.0888 | ns | |
0.0010 | 1 | 0.0010 | 7.59 | 0.0283 | ||
0.0003 | 1 | 0.0003 | 1.89 | 0.2116 | ns | |
2.500E-09 | 1 | 2.500E-09 | 0.0000 | 0.9967 | ns | |
0.0000 | 1 | 0.0000 | 0.1519 | 0.7083 | ns | |
0.0007 | 1 | 0.0007 | 5.41 | 0.0530 | ns | |
0.0002 | 1 | 0.0002 | 1.46 | 0.2655 | ns | |
0.0087 | 1 | 0.0087 | 64.91 | <0.0001 | ||
0.0048 | 1 | 0.0048 | 36.10 | 0.0005 | ||
Residual error | 0.0009 | 7 | 0.0001 | |||
Lack of fit | 0.0005 | 3 | 0.0002 | 1.34 | 0.3808 | ns |
Pure error | 0.0005 | 4 | 0.0001 | |||
Total | 0.0182 | 16 |
The
The
Three-dimensional (3D) response surface and two-dimensional (2D) contour plots (Figure
Response surface plots (a, c, e) and contour plots (b, d, f) showing the effect of NaOH concentration (mol/L,
According to software analysis, the optimal preparation conditions for CMJP were NaOH solution concentration of 2.79 mol/L, chloroacetic acid content of 2.12%, reaction temperature of 60.4°C, and DS of 0.2307. Three verification experiments were carried out in order to validate the adequacy of the model equation. Taking account of the operating convenience, the optimal preparation parameters of CMJP were determined as follows: the concentration of NaOH solution was 2.8 mol/L, the content of chloroacetic acid was 2.12%, and the reaction temperature was 60.4°C. By these parameters, the DS of CMJP was 0.2275 ± 0.0108, which is close to the maximum predicted value, indicating an excellent fit with the mathematical model.
The rheological behavior in terms of dynamic viscosity and shear rate of JP and CMJP is shown in Figure
Effect of carboxymethylation modification on apparent viscosity (a) and shear stress (b) of JP and CMJP aqueous solution at 25°C.
After carboxymethylation, water solubility improved in CMJP. The content of total carbohydrate in JP (75.33 ± 0.11%) was relatively higher than that in CMJP (69.22 ± 0.32%), indicating that JP may have been hydrolyzed during the carboxymethylation process (Table
Water solubility, viscosity, and the chemical composition of polysaccharide from JP and CMJP.
Sample | Water solubility (mg/mL) | Total carbohydrate (%) | Uronic acid (%) | Monosaccharide composition (%) | |||||
---|---|---|---|---|---|---|---|---|---|
Rhamnose | Arabinose | Xylose | Mannose | Glucose | Galactose | ||||
JP | 50.8 ± 0.93 | 75.33 ± 0.11 | 39.35 ± 0.10 | 0.31 | 7.69 | 0.54 | 0.15 | 1.08 | 1 |
CMJP | 85.1 ± 1.27 | 69.22 ± 0.32 | 40.38 ± 0.15 | 0.18 | 9.09 | 0.45 | ND | 0.36 | 0.98 |
As in previous studies, the
The weight-average molecular mass increased 10.62% from 2.75 × 105 Da (JP) to 3.04 × 105 Da (CMJP) after carboxymethylation. The raise might be owing to the incorporation of carboxymethyl-containing groups into the polymer structure during the carboxymethylation process. Wang et al. [
Figure
Fourier transform infrared spectra (FT-IR) of JP and CMJP.
The UV scanning analysis of crude jujube polysaccharide, JP, and CMJP was shown in Figure
Ultraviolet and visible spectrophotometry (UV-Vis) spectra of crude JP, JP, and CMJP were recorded in the range of 190–900 nm.
Figures
Scanning electron micrographs (SEM) and energy-dispersive X-ray spectroscopy (EDS) analysis of polysaccharide. (a) 5000x; (b) 10000x; (e) JP-point and carboxymethylated polysaccharide (c) 5000x; (d) 10000x; (f) CMJP-point from
The designated points in JP (Figure
DPPH is a stable free radical which could be reduced on acceptance of hydrogen donated by the antioxidant and converted to DPPH-H (the nonradical from of DPPH) [
Scavenging effect of JP and CMJP on DPPH radicals (a), hydroxyl radicals (b), superoxide radicals (c), and reducing power (d) compared with that of vitamin C. Results were expressed as means ± SD of three parallel measurements.
Hydroxyl radical is well known as a strong oxidant which can react with most of biomolecules in living cells and resulting in severe damage or cell death [
Superoxide radical is considered as an initial free radical, easily transformed from molecular oxygen and causes other cell damaging free radicals (H2O2, hydroxyl radicals) in living systems [
The electron-donating ability of antioxidants was measured using the potassium ferricyanide reduction method. As shown in Figure
In the present study, carboxymethylated modification could reduce DPPH radicals, superoxide radicals, and power but increase hydroxyl radical scavenging activity of
Grow curves at pH 5.7 ± 0.2 of the three
Growth curves of
Effects of JP and CMJP on the proliferation of
Wang et al. [
In this study, both of JP and CMJP possessed the potential as prebiotics and CMJP showed stronger prebiotic activities than JP. The mechanisms need to be disclosed, and future study will be continued to explore the
In this study, carboxymethylated
The data used to support the study are included within the article. Raw data can be acquired from the corresponding author upon reasonable request (
Jingjing Kou is the co-first author.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
Runfang Feng and Jingjing Kou contributed equally to this work.
This research was financially supported by the Natural Science Foundation of Hebei Province (C2019204366) and the Key R&D Projects in Hebei Province (20327123D), Top Young Talents of Hebei Province.
Table S1: Box–Behnken experimental design and the results for degree of substitution (DS) of CMJP polysaccharides (