Purification, Characterization, Prebiotic Preparations and Antioxidant Activity of Oligosaccharides from Mulberries

A water-soluble oligosaccharide termed EMOS-1a was prepared by enzymatic hydrolysis of polysaccharides purified from mulberries by column chromatography. The chemical structure of the purified fraction was investigated by ultraviolet spectroscopy, Fourier-transform infrared spectroscopy, and gas chromatography–mass spectrometry, which indicated that galactose was the main constituent of EMOS-1a. Chemical analyses showed that the uronic acid and sulfate content of EMOS-1a were 5.6% and 8.35%, respectively, while gel permeation chromatography showed that EMOS-1a had an average molecular weight of 987 Da. The antioxidant activities of EMOS-1a were next investigated, and EMOS-1a exhibited concentration-dependent 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, Trolox equivalent antioxidant capacity, and ferric reducing antioxidant power. The level of proliferation of Lactobacillus rhamnosus reached 1420 ± 16% when 4% (w/v) EMOS-1a was added, where the number of colonies in MRS (de Man, Rogosa, and Sharpe) medium with no added oligosaccharide was defined as 100% proliferation. These results indicate that the oligosaccharide EMOS-1a could be used as a natural antioxidant in prebiotic preparations.


Introduction
Mulberry (Morus nigra L.) is widely cultivated in Asia, Africa, Europe, and America [1], and China has a long history of planting this tree [2]. In China, mulberries and other plants are used as a traditional medicine for treating fever, sore throat, anemia, and liver damage [3][4][5]. The therapeutic benefit of mulberries may be attributable to a variety of active ingredients, including polysaccharides, polyphenols, anthocyanins, and flavonoids [6][7][8].
Mulberry polysaccharides are macromolecular active substances with antioxidant, anti-fatigue, and immune-enhancing properties [9,10]. Prior research has indicated that the components obtained following deproteinization, decolorization, and desalting of mulberry polysaccharides have hypoglycemic activity in vitro [11]. In addition, mulberry polysaccharides have been shown to

Characterization of EMOS-1a
As a homogeneous oligosaccharide, EMOS-1a was chosen for further study. Chemical analysis showed that the uronic acid and sulfate content of EMOS-1a were 5.6% and 8.35%, respectively. The oligosaccharide had no UV absorption at 260 or 280 nm, indicating that EMOS-1a contained no protein or nucleotides ( Figure 2A).
Polysaccharides with a triple-helix structure form a complex with the dye, Congo red [24]. Compared with Congo red, the maximum absorption wavelength of the complex shows a bathochromic shift, and the product is purple-red in color. When the added NaOH concentration exceeds a certain value, the maximum absorption wavelength drops sharply [25]. Structural assessment of EMOS-1a is shown in Figure 2B. The maximum absorbance of the complex formed by EMOS-1a and Congo red decreased only slightly with increasing NaOH concentration. This indicated that EMOS-1a did not have a triple-helix structure.

Characterization of EMOS-1a
As a homogeneous oligosaccharide, EMOS-1a was chosen for further study. Chemical analysis showed that the uronic acid and sulfate content of EMOS-1a were 5.6% and 8.35%, respectively. The oligosaccharide had no UV absorption at 260 or 280 nm, indicating that EMOS-1a contained no protein or nucleotides ( Figure 2A). . Structural analysis of EMOS-1a (B); "Congo red" indicates that Congo red was mixed with NaOH, "Congo red + EMOS-1a" indicates that Congo red was treated with EMOS-1a before the addition of NaOH.
Molecular weight analysis by GPC is shown in Figure 3. The average molecular weight of EMOS-1a was determined to be 987 Da from the standard curve. . Structural analysis of EMOS-1a (B); "Congo red" indicates that Congo red was mixed with NaOH, "Congo red + EMOS-1a" indicates that Congo red was treated with EMOS-1a before the addition of NaOH. Polysaccharides with a triple-helix structure form a complex with the dye, Congo red [24]. Compared with Congo red, the maximum absorption wavelength of the complex shows a bathochromic shift, and the product is purple-red in color. When the added NaOH concentration exceeds a certain value, the maximum absorption wavelength drops sharply [25]. Structural assessment of EMOS-1a is shown in Figure 2B. The maximum absorbance of the complex formed by EMOS-1a and Congo red decreased only slightly with increasing NaOH concentration. This indicated that EMOS-1a did not have a triple-helix structure.
Molecular weight analysis by GPC is shown in Figure 3. The average molecular weight of EMOS-1a was determined to be 987 Da from the standard curve. Figure 2. UV spectrum of EMOS-1a (A). Structural analysis of EMOS-1a (B); "Congo red" indicates that Congo red was mixed with NaOH, "Congo red + EMOS-1a" indicates that Congo red was treated with EMOS-1a before the addition of NaOH. Molecular weight analysis by GPC is shown in Figure 3. The average molecular weight of EMOS-1a was determined to be 987 Da from the standard curve. The monosaccharide composition of EMOS-1a was measured by GC-MS analysis. GC-MS analysis of seven standard monosaccharides is shown in Figure 4A. Based on the retention time and relative percentage of each sugar ( Figure 4B), EMOS-1a was composed of galactose.  The monosaccharide composition of EMOS-1a was measured by GC-MS analysis. GC-MS analysis of seven standard monosaccharides is shown in Figure 4A. Based on the retention time and relative percentage of each sugar ( Figure 4B), EMOS-1a was composed of galactose. . Structural analysis of EMOS-1a (B); "Congo red" indicates that Congo red was mixed with NaOH, "Congo red + EMOS-1a" indicates that Congo red was treated with EMOS-1a before the addition of NaOH. Molecular weight analysis by GPC is shown in Figure 3. The average molecular weight of EMOS-1a was determined to be 987 Da from the standard curve. The monosaccharide composition of EMOS-1a was measured by GC-MS analysis. GC-MS analysis of seven standard monosaccharides is shown in Figure 4A. Based on the retention time and relative percentage of each sugar ( Figure 4B), EMOS-1a was composed of galactose.

FT-IR Analysis
The FT-IR spectrum is commonly used to determine the functional groups in organic molecules. As shown in Figure 5, the spectrum of EMOS-1a displayed a broad and strong absorption peak at 3351 cm −1 (3500-3100 cm −1 ), which could be assigned to stretching vibrations of hydroxyl (-OH) groups in the polysaccharide and the water involved in hydrogen bonding. The absorption peak around 2929 cm −1 (3000-2800 cm −1 ) was attributed to the C-H stretching vibration of the methyl groups. These absorption peaks are characteristic of polysaccharides [26]. There was no absorption peak in the range 1740-1760 cm −1 , indicating that EMOS-1a does not contain esterified carboxyl groups.
Absorption peaks were detected in the region 1700-1300 cm −1 , which indicated that the polysaccharide contained a carboxyl group. The absorption peak at 1639 cm −1 indicated that EMOS-1a contained uronic acid [27]. There was no absorption peak at 1541 cm −1 , confirming the absence of protein.
The absorption peak at 1400-1200 cm −1 was assigned to C-H bending, and the peak at 1242 cm −1 to the presence of a sulfate radical [28]. The stretching peak at 1079 cm −1 indicated the presence of a pyranose ring [29].
The FT-IR spectrum is commonly used to determine the functional groups in organic molecules. As shown in Figure 5, the spectrum of EMOS-1a displayed a broad and strong absorption peak at 3351 cm −1 (3500-3100 cm −1 ), which could be assigned to stretching vibrations of hydroxyl (-OH) groups in the polysaccharide and the water involved in hydrogen bonding. The absorption peak around 2929 cm −1 (3000-2800 cm −1 ) was attributed to the C-H stretching vibration of the methyl groups. These absorption peaks are characteristic of polysaccharides [26]. There was no absorption peak in the range 1740-1760 cm −1 , indicating that EMOS-1a does not contain esterified carboxyl groups. Absorption peaks were detected in the region 1700-1300 cm −1 , which indicated that the polysaccharide contained a carboxyl group. The absorption peak at 1639 cm −1 indicated that EMOS-1a contained uronic acid [27]. There was no absorption peak at 1541 cm −1 , confirming the absence of protein. The absorption peak at 1400-1200 cm −1 was assigned to C-H bending, and the peak at 1242 cm −1 to the presence of a sulfate radical [28]. The stretching peak at 1079 cm −1 indicated the presence of a pyranose ring [29].

Antioxidant Activity
Antioxidant activities of EMOS-1a were determined based on DPPH radical scavenging activity and the FRAP and ABTS tests. The DPPH radical scavenging results are shown in Figure 6A. DPPH free radicals are stable and purple in color in organic solvents (λmax = 517 nm). When an antioxidant is added, the DPPH radical is removed and the absorption at this wavelength is attenuated. The scavenging of DPPH free radicals by EMOS-1a was dependent on the EMOS-1a concentration. When the concentration of EMOS-1a was 600 μg/mL, the scavenging ability was 46.96 ± 1.68%. The scavenging effect was weaker than that of vitamin C, and the half-clearance concentration of EMOS-1a for DPPH free radicals was approximately 650 μg/mL.
The results of the ABTS test showed a similar trend ( Figure 6B). The Trolox-equivalent antioxidant capacity of EMOS-1a was positively correlated with its concentration. ABTS is oxidized to green ABTS + under the action of an oxidizing agent, and the production of ABTS + is inhibited in the presence of an antioxidant. The Trolox-equivalent antioxidant capacity of a sample can be determined by measuring the absorbance of ABTS + at 734 nm. At concentrations of 100-800 μg/mL, the Trolox-equivalent antioxidant capacity of EMOS-1a was 0.6020 ± 0.0088 to 5.8420 ± 0.1155, which is lower than that of vitamin C.
The FRAP value of EMOS-1a was concentration-dependent ( Figure 6C). In acidic conditions, an antioxidant can reduce Fe 3+ -TPTZ to produce blue Fe 2+ -TPTZ. The FRAP value in the sample can then

Antioxidant Activity
Antioxidant activities of EMOS-1a were determined based on DPPH radical scavenging activity and the FRAP and ABTS tests. The DPPH radical scavenging results are shown in Figure 6A. DPPH free radicals are stable and purple in color in organic solvents (λ max = 517 nm). When an antioxidant is added, the DPPH radical is removed and the absorption at this wavelength is attenuated. The scavenging of DPPH free radicals by EMOS-1a was dependent on the EMOS-1a concentration. When the concentration of EMOS-1a was 600 µg/mL, the scavenging ability was 46.96 ± 1.68%. The scavenging effect was weaker than that of vitamin C, and the half-clearance concentration of EMOS-1a for DPPH free radicals was approximately 650 µg/mL.
The results of the ABTS test showed a similar trend ( Figure 6B). The Trolox-equivalent antioxidant capacity of EMOS-1a was positively correlated with its concentration. ABTS is oxidized to green ABTS + under the action of an oxidizing agent, and the production of ABTS + is inhibited in the presence of an antioxidant. The Trolox-equivalent antioxidant capacity of a sample can be determined by measuring the absorbance of ABTS + at 734 nm. At concentrations of 100-800 µg/mL, the Trolox-equivalent antioxidant capacity of EMOS-1a was 0.6020 ± 0.0088 to 5.8420 ± 0.1155, which is lower than that of vitamin C.
The FRAP value of EMOS-1a was concentration-dependent ( Figure 6C). In acidic conditions, an antioxidant can reduce Fe 3+ -TPTZ to produce blue Fe 2+ -TPTZ. The FRAP value in the sample can then be obtained by measuring the absorbance of Fe 2+ -TPTZ at 593 nm. At concentrations of 100-800 µg/mL, the FRAP value of EMOS-1a was 0.0376 ± 0.0114 to 0.4627 ± 0.01015, which was lower than that of vitamin C. be obtained by measuring the absorbance of Fe 2+ -TPTZ at 593 nm. At concentrations of 100-800 μg/mL, the FRAP value of EMOS-1a was 0.0376 ± 0.0114 to 0.4627 ± 0.01015, which was lower than that of vitamin C.

Bacterial Strain
Lactobacillus rhamnosus (ATCC7469) was purchased from the Guangdong Culture Collection Center. The strain was stored at −80 °C in MRS broth containing 25% (v/v) glycerol.

Preparation, Isolation, and Purification of Mulberry Oligosaccharides
Mulberry polysaccharides were prepared according to the method described by Chen et al. with some modifications [13]. Polysaccharides in the mulberry powder were extracted in a water bath at 80 °C for 4 h. The resulting extract was reduced to a quarter of its original volume by vacuum

Bacterial Strain
Lactobacillus rhamnosus (ATCC7469) was purchased from the Guangdong Culture Collection Center. The strain was stored at −80 • C in MRS broth containing 25% (v/v) glycerol.

Preparation, Isolation, and Purification of Mulberry Oligosaccharides
Mulberry polysaccharides were prepared according to the method described by Chen et al. with some modifications [13]. Polysaccharides in the mulberry powder were extracted in a water bath at 80 • C for 4 h. The resulting extract was reduced to a quarter of its original volume by vacuum filtration, followed by precipitation for 24 h with four volumes of 95% (v/v) ethanol at 4 • C. The precipitate was collected by centrifugation (9000× g for 15 min), and the pellet was dissolved in water to obtain a crude mulberry polysaccharide solution. For the production of the enzymatic hydrolysate containing the mulberry oligosaccharides (EMOS), the crude mulberry polysaccharide solution was incubated with 5% (w/v) β-mannanase at 50 • C for 4 h and then lyophilized. The polysaccharide content of the mulberry polysaccharide solution and EMOS were detected with the phenol-sulfuric acid method using d-glucose as the standard [30].
EMOS was dissolved in distilled water (10% (w/v)) and then loaded onto a DEAE-52 cellulose column (2.5 cm × 25 cm) previously equilibrated with distilled water at room temperature. The column was eluted with distilled water and a step gradient of 0.1, 0.3, and 0.5 mol/L NaCl at a flow rate of 1 mL/min. The eluates were collected with an automatic fraction collector (10 mL per tube). The elution profile detected by the phenol-sulfuric acid assay showed three main elution peaks, namely EMOS-1, EMOS-2, and EMOS-3, which were then lyophilized. Fraction EMOS-1 was selected for further fractionation because of its superior proliferation effect (see Methods 2.4). Size-exclusion chromatography on a Sephadex G-100 column (2.5 cm × 25 cm) with distilled water at a flow rate of 1 mL/min yielded a fraction that was denoted EMOS-1a, which was also then lyophilized. The uronic acid content of the EMOS fractions was estimated with the vitriol-carbazole method using d-galacturonic acid as a standard, and the sulfate content was tested with the barium chloride-gelatin method [31].

Determination of Structure
Congo red was used to determine whether EMOS-1a has a triple-helix structure, as described by Wood et al., based on a shift in the maximum absorption wavelength [32]. Briefly, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 mol/L NaOH was mixed with 2 mg/mL EMOS-1a and 80 µmol/L Congo red at a volume ratio of 2:1:1, and the mixtures were analyzed with a Shimadzu UV-1800 spectrophotometer.

Fourier-Transform Infrared (FT-IR) Spectroscopy
EMOS-1a (1 mg) was mixed with KBr powder and then pressed into pellets for analysis. The FT-IR spectrum was recorded in the range 400-4000 cm −1 on a Vertex 70 FT-IR spectrometer (Bruker Optics, Ettlingen, Germany).

Analysis of Monosaccharide Composition
The monosaccharide composition of EMOS-1a was analyzed by gas chromatography-mass spectrometry (GC-MS) [33] with some modifications. Briefly, EMOS-1a (10 mg) was hydrolyzed to monosaccharides using trifluoroacetic acid (2 mL, 4 mol/L) at 110 • C for 6 h. The hydrolyzed solution was evaporated to dryness, added to hydroxylamine hydrochloride (10 mg) and pyridine (1 mL) for reaction for 30 min at 90 • C, and then acetylated with acetic anhydride (1 mL) for 30 min at 90 • C. Thus, the monosaccharides were derivatized as acetylated aldononitriles. The final product was analyzed by GC-MS using an Agilent 6890 GC instrument (Agilent Technologies Co., Ltd., Colorado Springs, CO, USA) equipped with an HP-1701 column and an Agilent 5973 MS detector. Nitrogen was used as the carrier gas (1 mL/min). The temperature of the column was initially set at 100 • C, then increased to 280 • C at 10 • C/min, followed by 280 • C for 15 min. The injection temperature was 280 • C and the temperature of the mass spectrometer ion source was 230 • C. Seven monosaccharides (rhamnose, ribose, fucose, arabinose, xylose, mannose, and galactose) were converted into their acetylated derivatives as standards to identify the composition of the oligosaccharides.

DPPH Radical Scavenging Assay
The DPPH assay followed the methodology developed by Brand-Williams et al. [34] with modifications. Briefly, 3 mL EMOS-1a aqueous solutions (100, 200, 400, 600, and 800 µg/mL) and 3 mL DPPH in ethanol (0.10 mmol/L) were mixed and shaken vigorously. The mixtures were kept in the dark for 30 min and the absorbance was then measured at 517 nm. The blank comprised water instead of EMOS-1a, and vitamin C was used as the positive control. The antioxidant power of the mixtures was calculated with the equation:

ABTS [2,2 -Azino-bis(3-ethylbenzothiazoline-6-sulfonic) Acid] Assay
The ABTS radical-scavenging activity of EMOS-1a was determined with an ABTS assay kit (Beyotime Biotechnology Co., Ltd., Shanghai, China). ABTS working liquor was prepared by reacting ABTS stock solution with potassium persulfate solution at a 1:1 volume ratio. The mixture was stored at room temperature for 12-16 h in the dark before use and left to stabilize for 2 to 3 days. The concentration of the resulting blue-green ABTS radical solution was adjusted to give an absorbance of 0.7 ± 0.05 at 734 nm. A total of 200 µL of ABTS working solution and 10 µL EMOS-1a solution were mixed in 96-well plates, which were protected from light. Absorbance was measured at 734 nm after 2-6 min of incubation at room temperature. Antioxidant activity was expressed as the Trolox-equivalent antioxidant capacity of the EMOS-1a solution.

FRAP (Ferric Reducing Antioxidant Power) Assay
The antioxidant activity of EMOS-1a was determined with an antioxidant capacity assay kit and the FRAP method (Beyotime Biotechnology Co., Ltd., Shanghai, China). A working solution was prepared by mixing a TPTZ dilution, TPTZ solution, and detection buffer at a ratio of 10:1:1 (v/v), and this FRAP working solution was then kept at 37 • C. Calibration solution, blank, or sample (5 µL) were added to 180 µL of working solution. Absorbance was measured at 593 nm after 3-5 min incubation at 37 • C. The FRAP values of the samples were calculated from a linear calibration curve and expressed as mmol FeSO 4 equivalents.

Statistical Analysis
All data are presented as the mean ± standard deviation (SD) of three independent experiments. Statistical significance (p < 0.05) between treatments was analyzed by one-way analysis of variance, followed by Duncan's multiple range test. All statistical analyses were performed with SPSS software version 19.0 (SPSS Inc., Chicago, IL, USA).

Conclusions
In this study, the oligosaccharide EMOS-1a was prepared by enzymatic hydrolysis of polysaccharides purified from mulberries by DEAE-52 cellulose and Sephadex G-100 column chromatography. EMOS-1a consisted of galactose with an average molecular weight of 987 Da. In addition, the antioxidant activity of EMOS-1a was positively correlated with its concentration. EMOS-1a promoted the proliferation of L. rhamnosus; when 4% (w/v) EMOS-1a was added, the proliferation level reached 1420 ± 16%, which indicated that EMOS-1a has potential as a natural antioxidant in prebiotic foods. This research provides a basis for further studies of mulberry prebiotics. The anti-digestive properties and proliferative effect of EMOS-1a on human fecal microbiota will be investigated in future studies.