Sodium butyrate

Effects of enzymatic hydrolysis on the structural, rheological, and functional properties of mulberry leaf polysaccharide

Teng-Gen Hu a,b, Yu-Xiao Zou b,d, Er-Na Li b, Sen-Tai Liao b, Hong Wu a,*, Peng Wen c,*


Effects of enzymatic hydrolysis on the structural, rheological, and functional properties of mulberry leaf poly- saccharide (MLP) were characterized in this study. The enzymatic hydrolysis of MLP raised the carbonyl, carboxyl, and hydroxyl groups from 7.21 ± 0.86 to 10.08 ± 0.28 CO/100 Glu, 9.40 ± 0.13 to 17.55 ± 0.34 COOH/100 Glu, and 5.71 ± 0.33 to 8.14 ± 0.24 OH/100 Glu, respectively. Meanwhile, an increase in thixotropic performance and structure-recovery capacities were observed in hydrolyzed MLP, while the molecular weight, surface tension, apparent viscosity, and thermal stability were decreased. An improved antioxidant activity of MLP was also achieved after the enzymatic degradation. Moreover, the hydrolyzed MLP showed greater ability to promote the growths of Bifidobacterium bifidum, Bifidobacterium adolescentis, Lactobacillus rhamnosus, and Lacto- bacillus acidophilus and the production of acetic acid, butyric acid, and lactic acid. The results demonstrate that enzymatic modification is a useful approach for polysaccharide processing.

Keywords: Morus alba L. Enzymatic hydrolysis Characterization Antioxidant activity Prebiotic activity

1. Introduction

Mulberries (Morus alba L.) are traditional medicine and food re- sources with a long history in Asia. The polysaccharides isolated from mulberry leaves exhibit antidiabetic, anti-oxidative, immune-stimula- tory, and gut microbiota–regulatory effects (Wen et al., 2019). Mulberry leaf polysaccharides (MLPs) possess potent Fe2+-chelating capability and scavenging activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, superoxide, and 2,2′-azinobis-(3-ethyl-benzothiazolin-6-sul- fonic acid) radicals (Yuan et al., 2015). Zhao et al. (2015) reported that MLP could improve intestinal ecology by inhibiting Escherichia coli and promoting Lactobacilli and Bifidobacteria. However, owing to the critical functions of polysaccharides and their structural complexity, oligosac- charides have received increasing importance in therapeutic fields as alternatives to the polymeric structures.
Currently, acid and enzymatic hydrolysis are the main methods of preparing oligosaccharides from polysaccharides (de Moura, Macagnan, & da Silva, 2015; Yan, Wang, Liu, & Zhao, 2018). However, acid hy- drolysis often leads to more toxicants (furfural) and environmental pollution. Recently, enzymatic hydrolysis has received attention because of its significant advantages such as mild reaction conditions, good selectivity, and the production of less undesirable byproducts. Hence, enzymatic hydrolysis is being regarded as a promising method for polysaccharide modification. Enzymatic modification can reduce the viscosity and consistency index, and enhance the crystallinity and flow behavior index of polysaccharides (Mudgil, Barak, & Khatkar, 2012). In addition, in previous studies, it was found that the bioavailability and the anti-apoptotic, anti-oxidative, and immunolatory activities of poly- saccharides increased after enzymatic treatment (Kim, Kim, Kim, Lee, & Lee, 2006; Liu et al., 2021; Xu et al., 2016), which may be due to the exposure of many active groups of polysaccharides after the hydrolysis. Additionally, the unique functions of MLP, including its anti-oxidative and prebiotic activities, are related to its structural and rheological properties, such as monosaccharide composition, active groups, mo- lecular weight (Mw), viscosity, thixotropic performance, and glycosidic linkage (Yuan et al., 2015). Hence, it is important to investigate whether enzymatic hydrolysis influences the MLP bioactivity, structure, and rheological properties. Furthermore, to our knowledge, the preparation of oligosaccharides from MLP via enzymatic hydrolysis has not been previously reported. Thus, this study marks the first time oligosaccharides were enzymati- cally prepared from MLP, and the effect of enzymatic hydrolysis on the structural and rheological properties of MLP was also evaluated. Furthermore, the bioactivities associated with the structural and flow behaviors, such as the anti-oxidative and probiotic activities, were analyzed.

2. Material and methods

2.1. Materials and chemicals

Fresh mulberry (Morus alba L.) leaves purchased from Guagzhou Bosun Co. Ltd. (Guangzhou, China) were dried using a heat pump dryer (GHRH-20, Guangdong Hongke Agricultural Machinery Research and Development Co. Ltd, Guangzhou, China) at 60 ◦C and 11 kW for 12 h. Then, the dried leaves were pulverized using a crusher (BJ-2500A, Deqing Baijie Electric Appliance Co. Ltd, Huzhou, China) at 1.8 kW for 5 min. Hemicellulase (35 U/mg) was obtained from Hefei Bomei Biotechnology Co. Ltd. (Hefei, China). Moreover, DPPH, tripyridyl- triazine (TPTZ), salicylic acid, and ferrous sulfate (FeSO4) were pur- chased from Guangzhou Qiyun Biotechnology Co. Ltd. (Guangzhou, China). All other chemicals used were of analytical grade.

2.2. MLP degradation

2.2.1. Extraction of polysaccharides

The MLP was extracted using a modified version of a previously re- ported method (Ma et al., 2018b). To remove lipids in mulberry leaf, per gram of powder was refluxed with 4 mL of petroleum ether at 50 ◦C for 24 h. After filtration, the residue was dried at 60 ◦C, and then the mulberry leaf powder was extracted with water at a ratio of 1:30 (w/v) at 80 ◦C for 4 h. The water extract was concentrated using a rotary evaporator (EYELA N-1100, Tokyo Rikakikai Co. Ltd, Tokyo, Japan) at 50 ◦C under vacuum and precipitated with four volumes of absolute ethanol at 4 ◦C overnight. The precipitate was collected via centrifu- gation at 10,000 × g for 10 min; then, the precipitate was dissolved with water and deproteinized using the Sevage method (Sevag, Lackman, & Smolens, 1938). Briefly, the polysaccharide solution (50 mg/mL) was mixed with the Sevage reagent (1-butanol/chloroform, v/v = 1:4) at a ratio of 4:1 (v/v). The mixture was sufficiently shaken for 30 min and then centrifuged at 10,000 × g for 5 min. The aqueous phase separated from the supernatant was added to a quarter of its volume of the Sevage reagent. The process was repeated until the solution presented no ab- sorption peak on a UV spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) at 250–280 nm. The deproteinization solution was further freeze- dried to obtain the MLP (the MLP yield was 17.23 ± 2.49%).

2.2.2. Enzymatic hydrolysis of MLP

The enzymatic hydrolysis of MLP was optimized to obtain the largest degradation under the conditions: 20 mg/mL MLP, 60 U per milliliter of reaction solution of hemicellulase, pH 4.7, reaction time of 8 h, and temperature of 55 ◦C (the hydrolysis optimization is not shown here). The hydrolysate was placed in a boiling water bath for 5 min to termi- nate the reaction and then centrifuged at 10,000 × g for 10 min to remove hemicellulase. Afterward, 10 mL of the supernatant was placed software (Xevo TQ-S Micro, Waters, Milford, MA, USA) according to a previous report but with minor modification (Cao et al., 2019). Briefly, 10 mg of the sample was added to 5 mL trifluoroacetic acid (TFA, 2 M) for hydrolysis for 2 h at 100 ◦C under nitrogen atmosphere. The excess TFA was removed through the addition of methanol and evaporated by nitrogen flow. Then, the hydrolysate was dissolved with 1 mL NaOH (0.3 M). Afterward, the hydrolysate solution (400 μL) was labeled by the addition of 0.5 M 1-phenyl-3-methyl-5-pyrazolone (PMP, 400 μL) and incubated at 70 ◦C for 2 h. The resultant solution was adjusted to pH 7.0 using 0.3 M HCl (400 μL) and then concentrated up to dryness by ni- trogen. The residue was dissolved in 1.0 mL deionized water, and the excess PMP was extracted by an equal volume of chloroform; the extraction procedure was conducted thrice. The aqueous layer was filtered through a 0.45 μm membrane before the HPLC-MS analysis.
The HPLC was performed on an Agilent EC-C18 column (2.1 mm × 50 mm, 2.7 μm), with a mobile phase composed of 20 mM ammonium acetate buffer solution (pH 7.0) and acetonitrile at a ratio of 83:17 (v/v), under a flow rate of 0.4 mL/min and temperature of 30 ◦C. The injection volume was 20 μL, and the detection wavelength was set at 245 nm. The conditions of mass spectrometry analysis under single-ion monitoring were as follows: a positive-ion model of electrospray ion source (ESI) with a spray voltage of 2.0 kV and cone voltage of 30 V; desolvation temperature, 500 ◦C; ion-source temperature, 150 ◦C; nebulizer gas, nitrogen at a flow rate of 1000 L/h; scan range, 170–800 m/z.

2.4. Mw determination

The Mw change in MLP and MLO was characterized using a gel permeation chromatography (GPC) system equipped with a differential refractive index detector and three tandem columns (Acquity APC AQ 450, Acquity APC AQ 125, and Acquity APC AQ 45, Waters Corp., Milford, MA, USA). First, 10 mg of MLP or MLO was dissolved in 1 mL of deionized water. Then, 20 μL of the sample solution was injected and eluted by water at the flow rate of 0.5 mL/min at 25 ◦C. The data were collected and analyzed using Empower 3 software (Waters Corp., Mil- ford, CT, USA). DXT180 (Mw, 180 Da), DXT504 (504 Da), DXT1K (1200 Da), DXT3K (3650 Da), DXT12K (1200 Da), and DXT72K (72700 Da) were used as molecular-mass markers to establish the standard curve. The purities of these standards were over 99% and were provided by the National Institute of Metrology (Beijing, China).

2.5. Measurements of carbonyl, carboxyl, and hydroxyl contents

2.5.1. Carbonyl content

The carbonyl content was determined according to a previous method (Qin et al., 2021). Briefly, the sample (100 mg) was dissolved in 5 mL of deionized water, and then the solution pH was adjusted to 3.2 using 0.1 mol/L HCl. Subsequently, the sample solution was mixed with 15 mL of hydroxylamine chloride solution (50 mg/mL), which was prepared by dissolving hydroxylamine chloride in 0.1 mol/L NaOH so- lution. After the mixture was incubated for 4 h at 38 ◦C, it was titrated to pH 3.2 using 0.1 mol/L HCl. The control was carried out by using deionized water to replace the sample solution. The carbonyl content was expressed as the number of carbonyl groups per 100 units of glucose (CO/100 Glu) and calculated using the following formula: into a dialysis bag (200 Da, 44 mm × 100 mm, 28 mm) and dialyzed against 2.0 L of deionized water at 4 ◦C for 48 h to remove monosaccharides. The retention solution was collected and freeze-dried to obtain mulberry leaf oligosaccharide (MLO).

2.3. Monosaccharide composition of MLP and MLO

The sample was analyzed via high-performance liquid chromatog- raphy–mass spectrometry (HPLC-MS) controlled using MassLynx where V0 and V1 are the HCl volume consumed by the control and the sample, respectively; c is the HCl molar concentration; M is the molar mass of carbonyl group; and m is the sample mass.

2.5.2. Carboxyl content

The carboxyl content was detected using a previously reported method but with minor modification (Qin et al., 2021). First, 100 mg of the sample was dissolved in hot deionized water (5 mL, 90 ◦C). Then, the solution was titrated to pH 8.2 using 0.01 mol/L NaOH. The blank control was performed by using deionized water to replace the sample solution. The carboxyl content was determined by calculating the number of carboxyl groups every per 100 glucose units (COOH/100 Glu) using the following formula: Thecarboxylcontent(COOH/100Glu) = (V1 — V0 ) × c × M × 100 where V0 and V1 are the NaOH volumes consumed by the blank control and the sample, respectively; c is the molar concentration of NaOH; M is the molar mass of the carboxyl group; and m is the sample mass.

2.5.3. Hydroxyl content

The hydroxyl content was analyzed through the method described by Celikbag and Via (2016). First, phthalic anhydride–pyridine–imidazole solution was prepared by dissolving 74 g of phthalic anhydride into 500 mL of pyridine, and then 12 g of imidazole was added to the solution. After the system was incubated for 24 h, 25 mL of the obtained phthalic anhydride–pyridine–imidazole solution was mixed with 20 mg of MLP (or MLO), and the mixture was then placed in a boiling-water bath for 30 min. Subsequently, 10 mL boiling water was added into the mixture, which was then cooled to 25 ◦C. Finally, 2 mL phenolphthalein (10 mg/ mL) was added, and the above solution was titrated with 1 mol/L NaOH; the volume of NaOH consumed was recorded when the color changed to peach. The blank control was performed by using deionized water to replace the sample solution. The hydroxyl content (OH/100 Glu) was calculated using the following formula: Thehydroxylcontent(OH/100Glu) = (V0 — V1 ) × c × M × 100 where V0 and V1 are the NaOH volumes consumed by the blank control and the sample, respectively; c is the molar concentration of NaOH; M is the molar mass of the hydroxyl group; and m is the sample mass.

2.6. Surface tension measurements

The surface tension was determined using a DCAT 25 tensiometer (DataPhysics Instruments, Stuttgart, Germany) through the Wilhelmy Plate method (Zamani & Razavi, 2021). First, 30 mL of MLP or MLO (20 mg/mL) was added to the glass container, and the mixture was kept stationary for 30 min to achieve equilibrium before the test. The test was performed at 25 ◦C and repeated thrice.

2.7. Characterization of MLP and MLO surface morphologies

The surface morphologies of MLP and MLO were analyzed according to a previously described method (Tanaka, Tameike, Ishikawa, & Fur- uya, 2008). The sample powder was coated with Pt and then observed using a scanning electron microscope (Hitachi, Japan) at 100 kV.

2.8. Thermal stability analysis

The thermal properties of the MLP and MLO were characterized via thermogravimetric analysis (TGA, TA Instruments, New Castle, DE, USA) and differential scanning calorimetry (DSC, NETZSCH, Selb, Ger- many) using the method reported by Mohammed, Mahdi, Ahmed, Ma, and Wang (2020) with some modifications. Specifically, 5–9 mg sample was placed on the sample stage or in an aluminum pan. Then, the TGA test was performed under nitrogen atmosphere at a heating rate of 10 ◦C/min from 25 ◦C to 800 ◦C. The DSC was performed as follows: First, the temperature was increased from 0 ◦C to 200 ◦C at 5 ◦C/min and maintained at 200 ◦C for 5 min. Then, the temperature was cooled to 0 ◦C at the same rate. Then, the sample was rewarmed to 300 ◦C at 10 ◦C/min, maintained at this temperature for 5 min, and then cooled to 0 ◦C.

2.9. Fourier-transform infrared spectrometry analysis

The Fourier-transform infrared (FTIR) spectroscopy (Bruker, Ger- many) analysis was performed under the wavenumber range of 4000–400 cm—1. The sample to be analyzed (2 mg) was mixed with 250 mg of potassium bromide (KBr) and pressed into a thin slice according to the method by Jouraiphy et al. (2008).

2.10. Rheological characteristics

2.10.1. Effect of enzymatic hydrolysis on MLP apparent viscosity and thixotropic performance

The apparent viscosity and thixotropy were characterized using an AR1500EX rheometer (TA Instruments, New Castle, DE, USA) equipped with a cone and plate geometry (60 mm diameter with a gap of 0.05 mm, angle 2). First, 2 mL of the MLP or MLO solution (20 mg/mL) was placed on the sample stage, whose temperature was set to 25 ◦C. Changes in the apparent viscosities (η) of MLP and MLO with the increase in the shear rate (γ) from 0.1 to 100 and decrease from 100 to 0.1 were detected through the method by Timilsena, Adhikari, Kasapis, and Adhikari (2015) with slight modification. The power-law model was applied to fit the flow curves of MLP and MLO using the formula: where K is the consistency index, and n is the flow-behavior index (when n < 1, the solution is a pseudoplastic or a shear-thinning fluid; when n = 1, the solution is a Newtonian fluid; when n > 1, the solution is a dilatant or shear-thickened fluid) (Li, Li, Wang, Wu, & Adhikari, 2012). Furthermore, the thixotropic performances of MLP and MLO were evaluated using a previously reported method but with some modifi- cation (Ayyash et al., 2020). Here, changes in shear stress (τ) with the increase in the shear rate from 0.1 to 100 s—1 and decrease from 100 to 0.1 s—1 were monitored.

2.10.2. Effect of temperature on the apparent viscosities of MLP and MLO

The sample (20 mg/mL, 2 mL) was placed on the sample stage and allowed to remain stable for 5 min. Changes in the apparent viscosity of the sample with the increase in the temperature from 25 ◦C to 100 ◦C were recorded at the shear rate of 100 s—1 using a slightly modified version of a previously described method (Osmałek, Froelich, Jadach, & Krakowski, 2018).

2.10.3. Effect of salt ions on the apparent viscosities of MLP and MLO

The influence of NaCl or CaCl2 concentration (1, 3, 5, 10, and 15 mg/ mL) on the apparent viscosity of the sample (20 mg/mL, 2 mL) was investigated at 25 ◦C and a shear rate of 1 s—1 according to the method by Huamani-Melendez, Mauro, and Darros-Barbosa (2021) but with some modification. The viscosity retention rate was defined as the following formula:

2.10.4. Amplitude sweep

The dynamic rheological behaviors of MLP and MLO (20 mg/mL, 2 mL) were evaluated based on the storage modulus (G’) and loss modulus (G”). The measurements were conducted through an amplitude sweep assay performed at 25 ◦C over the amplitude range of 0.1%–100% as previously reported (Ayyash et al., 2020).

2.10.5. Characterization of structure recovery property

The test program comprised three stages: stage 1: low shear rate, stage 2: high shear rate, and stage 3: low shear rate. Briefly, the apparent viscosity changes of the sample (20 mg/mL, 2 mL) were measured under shear rates of 1, 100, and 1 s—1 maintained for 120, 60, and 180 s, respectively. The structure recovery was defined as the ratio of the sample peak apparent viscosity of stage 3 to the apparent viscosity of stage 1 at the endpoint (Patel et al., 2014).

2.11. Antioxidant activity

2.11.1. DPPH radical scavenging activity

The method by Hu, Cheng, Zhang, Lou, and Zong (2015) was used to evaluate the DPPH radical scavenging activity. The MLP or MLO solu- tion (2.0 mL, 0–100 μg/mL) was mixed with 2.0 mL of DPPH-ethanol solution (0.1 mM), and then the mixture was incubated in the absence of light for 30 min. Afterward, the absorbance was tested at 517 nm. The sample blank was determined by using ethanol to replace DPPH, while the negative and positive controls were prepared by adding deionized water and ascorbic acid instead of the sample solution, respectively. The scavenging activity on DPPH radical was calculated as follows: DPPHradicalscavengingactivity(%) = (1 — A1 — A2 ) × 100, where A1, A2, and A3 are the absorbances of the sample, sample blank, and negative control, respectively.

2.11.2. Hydroxyl radical scavenging activity

The hydroxyl radical scavenging activity was determined through a previously reported method (Liu, Luo, Ye, & Zeng, 2012). The reaction mixture containing 2.0 mL MLP or MLO solution (0–800 μg/mL), 2.0 mL FeSO4 (6.0 mM), and 2.0 mL H2O2 (6.0 mM) was incubated at 25 ◦C for 10 min. Afterward, 2.0 mL salicylic acid (6.0 mM) was added to the mixture, and then it was incubated for another 30 min. Then, the absorbance at 510 nm was measured. Deionized water and ascorbic acid were used as the negative and positive controls, respectively. The hy- droxyl scavenging activity was calculated according to the following equation: Hydroxylradicalscavengingactivity(%) = (1 — A1 ) × 100 where A0 and A1 are the absorbances of the negative control and the sample, respectively.

2.11.3. Fe2+ chelating capacity

The Fe2+ chelating capacity was measured according to the method by Decker and Welch (1990). First, 0.05 mL of FeSO4 solution (2.0 mM) and 0.2 mL of ferrozine solution (5 mM) were added to the MLP or MLO solution (3.0 mL, 0.2–4.0 mg/mL), and the reaction was allowed to proceed for 10 min at 25 ◦C. Then, the absorbance at 562 nm was measured. The control was measured using 3.0 mL of deionized water instead of the sample. Ethylenediaminetetraacetic acid (EDTA) was used as the positive control. The Fe2+ chelating capacity was calculated using the following equation: Fe2+chelatingcapacity(%) = (1 — A1 ) × 100, where A0 and A1 are the absorbances of the control and the sample, respectively.

2.11.4. Ferric-ion reducing antioxidant power

A ferric-ion reducing antioxidant power (FRAP) working solution was prepared by mixing 0.3 M sodium acetate–acetic acid buffer (pH 3.6), 20.0 mM FeCl3, and 10.0 mM TPTZ solution at a ratio of 10:1:1 (v/ v). The sample (MLP or MLO, 125–1500 μg/mL) or the calibration so- lution (FeSO4, 0.3 mL) was mixed with 2.7 mL of FRAP working solu- tion, and then the mixture was incubated at 37 ◦C for 10 min. Then, the absorbance value at 595 nm was measured according to the method by Ribeiro et al. (2020). The FRAP values of the samples were quantified based on the linear calibration curve of FeSO4 and expressed as mM FeSO4 equivalents.

2.12. Prebiotic activity

Bifidobacterium bifidum, Bifidobacterium adolescentis, Lactobacillus rhamnosus, and Lactobacillus acidophilus (Guangdong Culture Collection Center, Guangzhou, China) were used to evaluate the prebiotic activities of MLP and MLO according to the method by Li, Xia, Nie, and Shan (2016). Galacto-oligosaccharide (GOS, Yunfu Xinjinshan Biological Technology Co., Ltd, Yunfu, China) was chosen as the positive control. The lyophilized powders of these above bacteria were dissolved in 500 μL MRS broth medium (Guangdong Huankai Biotechnology Co., Ltd, Guangzhou, China). Then, the mixture was transferred to 100 mL of sterilized MRS broth medium and then incubated at 37 ◦C for 24 h under anaerobic conditions. After the activation of these strains, 1% (v/v) inoculum (108 CFU/mL) was added to the MRS broth in which MLP, MLO, or GOS (10 mg/mL) replaced glucose as the carbon source. The cultures were inoculated at 37 ◦C under static and anaerobic conditions. The absorbance at 600 nm and the pH values were monitored between 0 and 72 h. Furthermore, the short-chain fatty acids (SCFAs) were determined through a method established in our previous study (Hu et al., 2019). The SCFAs were analyzed using a gas chromatograph (GC- 2010 Plus; Shimadzu, Kyoto, Japan) equipped with a DB-FFAP column (30 m × 0.25 μm × 0.25 μm). Afterward, 20 μL of fermentation broth after filtration using a 0.22 μm pore membrane filter was injected the column. The temperatures of both the injector and the detector were set to 240 ◦C. Nitrogen and hydrogen were used as the carrier gases at the split ratio of 1:5 at 30.0 mL/min.

2.13. Statistical analysis

All measurements were conducted at least in triplicates, and the results were expressed as mean ± standard deviation where feasible. The differences between the mean values were analyzed via one-way anal- ysis of variance followed by Duncan’s multiple-range test (p < 0.05 was deemed significant). All statistical analyses were performed using SPSS (SPSS-17, Chicago, IL, USA). 3. Results and discussion 3.1. Determination of monosaccharide compositions and Mw As shown in Fig. 1a–c and Table S1, mannose (Man), ribose (Rib), rhamnose (Rha), glucuronic acid (GlcA), galacturonic acid (GalA), galac- tosamine (Galac), glucose (Glu), galactose (Gal), xylose (Xyl), arabinose (Arab), and fucose (Fuc) were well separated through HPLC. Accordingly, MLP was composed of Man, Rib, Rha, Glc, Gal, Arab, GlcA and GalA, with a molar ratio of 9.96:4.90:5.20:29.51:13.23:23.37:1.00:7.22. The main monosaccharides of MLP were consistent with the findings by Ma et al. (2018b). After the enzymatic hydrolysis of MLP to MLO, the monosaccharide compositions were not significantly changed. Moreover, MLP and MLO were analyzed via GPC to characterize the change in Mw. After hemicellulase treatment, the average Mw of MLP decreased to 852.63 Da, from 51.21 kDa (Fig. 1d). Meanwhile, the homogeneity of MLO was better than that of MLP, as evidenced by MLP presenting two separate peaks and MLO presenting a single peak. Furthermore, the decrease in the Mw of a polysaccharide may affect its biological activity (Sun, Wang, Shi, & Ma, 2009). 3.2. Carbonyl, carboxyl, and hydroxyl contents, and surface tension As presented in Table S2, the carbonyl and hydroxyl contents increased remarkably (p < 0.05), which may be a result of the break- down of the glycosidic bonds of MLP after enzymatic hydrolysis. Meanwhile, the carboxyl content after enzymatic treatment (17.55 ± 0.34 COOH/100 Glu) was significantly higher than that before the treatment (9.40 ± 0.13 COOH/100 Glu) (p < 0.05), which was mainly due to the oxidation of free aldehyde and hydroxyl groups during the enzymatic modification (Rossi et al., 2016). These results indicate that the enzymatic hydrolysis produced structural changes in MLP, which is related to the modification of its functional properties. Additionally, the surface tension of MLP decreased to 37.26 ± 0.03 mN/m after hydro- lysis, from 42.18 ± 0.03 mN/m. After enzymatic hydrolysis, the MLP rigid structure was broken down into more flexible short-chain struc- tures, which increased the MLO surface activity, promoted the adsorp- tion at the interface, and accordingly reduced the surface tension (Zamani & Razavi, 2021). 3.3. Surface characterization of MLP and MLO Fig. 2a and b show the surface morphologies of MLP and MLO powders, as revealed by scanning electron microscopy (SEM) images with 2000 × magnification. The structural integrity of MLP was main- tained and was severely damaged after enzymatic treatment (Fig. 2a and b). After hydrolysis, the MLP degraded into smaller segments. The re- sults indicate that enzymatic hydrolysis could change the structures of the polysaccharides, which was similar to the findings by Ma et al. (2018a). 3.4. Thermal stability analysis The weight-loss and derivative-weight-loss curves of the MLP and MLO are presented in Fig. 2c. The curves of MLP and MLO featured three main loss steps. The initial weight losses of MLP and MLO, observed in the temperature range of 30 ◦C–200 ◦C, were connected with the loss of free and bound water (Nawrocka, Szymanska-Chargot, Mis, Wilczewska, & Markiewicz, 2017). The second step represented the decompositions of MLP and MLO, which occurred at approximately 200 ◦C–320 ◦C and were related to the sugar-chain degradation (Zhu et al., 2019). The third step represented the atomic reorganization (350 ◦C–550 ◦C) (Wen et al., 2016). The weight loss of MLO (59.43%) was higher than that of MLP (43.74%), indicating that the MLP thermal stability decreased after enzymatic hydrolysis. Moreover, the thermal properties of the MLP and MLO were also characterized via DSC (Fig. 2d), which was performed to investigate the endothermal and exothermal changes with the increase in temperature. After enzymatic treatment, the functional and structural group differ- ences between MLP and MLO affected the thermal characteristics and transition temperature. Specifically, the glass-transition temperature (Tg) and crystallization temperature (Tc) slightly decreased to 84.24 ◦C (from 94.74 ◦C) and 237.09 ◦C (from 248.84 ◦C), respectively. The glass- transition temperature (Tg) of polymers is connected with their crys- talline nature. A smaller Tg value indicates a lower degree of crystallinity and a higher degree of amorphous nature (Munir, Shahid, Anjum, & Mudgil, 2016). The higher degree of crystallinity afforded MLP more structural stability and also made it more resistant to higher tempera- tures, a finding similar to the TGA result. 3.5. FTIR analysis The FTIR spectra of MLP and MLO are shown in Fig. 2e. The wide bands in 3500–3000 cm—1 indicate hydroxyl groups (–OH) of carbohy- drates (Andrade, Nunes, & Pereira, 2015). The absorptions at 2937 and 1267 cm—1 are ascribed to the C–H antisymmetric stretching and the bending vibration of the methylene groups, respectively (Gao, Lin, Sun, & Zhao, 2017). The strong absorptions at 1568 and 1414 cm—1 might be due to the stretching vibration of the C–O group and the C–O–H of carboxylic acid (Kong et al., 2015). The bands at 1051 and 1016 cm—1 are characteristic of the C–O–C glycosidic bond and vibrations over- lapping with stretching vibrations in the side groups of the C–O–H bonds (Huang et al., 2018). In addition, the signals at 925 and 790 cm—1 were from β-type linkages (Wang, Yin, Nie, & Xie, 2018; Yang, Zhang, Jin, Jin, & Xu, 2017). After enzymatic hydrolysis, the peaks at 3004 and 2937 cm—1 of MLP were absent, while the peak of C–H (1344 cm—1) bending vibration appeared in the MLO spectrum; the C–O (1713 cm—1) stretching- vibration peak, –OH and C–O coupling strong-absorption peak (1267 cm—1), and C–C–C deformation vibration peak (456 cm—1) of MLP were weakened. These phenomena may result from the fracture of glycosidic bonds, leading to the exposure of more carbonyl/carboxyl and hydroxyl groups, which agrees with the changes in the carbonyl, carboxyl, and hydroxyl contents. 3.6. Rheological characteristics 3.6.1. Effect of enzymatic hydrolysis on MLP apparent viscosity The typical flow curves of MLP before and after hydrolysis are shown in Fig. 3a. The apparent viscosity of MLP after enzymatic hydrolysis was significantly decreased, probably due to depolymerization of MLP, and the viscosities of MLP and MLO both declined with the increase in shear rate. The static flow curves of MLP and MLO well obeyed the power-law model, with high correlation coefficients (R2) of 0.9959 and 0.9940, respectively (Fig. 3b). The flow behavior index (n) and the consistency index (K) parameters of the power-law model for MLP and MLO are summarized in Table S3. The n values of MLP and MLO are 0.0621 (n < 1) and 0.1174 (n < 1), respectively, indicating that both MLP and MLO behaved like a pseudoplastic fluid, showing shear-thinning behavior. The enzymatic treatment of MLP also resulted in a tremendous decrease in the K value; this observation is similar to the reported changes in guar gum after enzymatic hydrolysis (Mudgil et al., 2012). 3.6.2. Effect of temperature on the apparent viscosities of MLP and MLO As shown in Fig. 3c, the apparent viscosities of MLP and MLO decreased with the increase of the temperature. This trend was similar to those exhibited by other polysaccharides (Zamani & Razavi, 2021), which may because the enhancement in the motility and movement of MLP and MLO reduced the resistance to flow as the temperature increased. The weaker intermolecular bonds at higher temperatures may be another reason for this phenomenon (Karazhiyan et al., 2009). Accordingly, the cleavages of molecules after hydrolysis resulted in a decrease in the MLP apparent viscosity, indicated by the lower apparent viscosity of MLO than that of MLP at the same temperature. 3.6.3. Effect of salt ions on the apparent viscosities of MLP and MLO Fig. 3d indicates that the addition of salt ions decreased the apparent viscosities of both the MLP and MLO; for both, the apparent viscosity dropped sharply with increasing salt ion concentration up to 5 mg/mL, after which no significant variation occurred. This behavior could mean that the metal ions altered the hydrodynamic size of the solutes (Jin et al., 2014). Additionally, CaCl2 showed a greater effect than NaCl on reducing the apparent viscosities of MLP and MLO. This could mean that Ca2+ carried more charges than Na+, which enhanced the interaction with the opposite-charge ions in the solution, and the hydrated layer around the long-chain molecules lead to a more decrease in viscosity. The phenomenon agrees with the report by Xu et al. (2019). At the concentration of 15 mg/mL, the viscosity retention rates of MLO in NaCl and CaCl2 were 41.8% and 31.6%, respectively; the values were significantly decreased for MLP: 16.6% and 11.2%, respectively. The result demonstrates that MLO showed better tolerance to a high con- centration of salt ions than MLP. 3.6.4. Thixotropic performances of MLP and MLO With the increase and decrease in the shear rate, the shear stress of MLP/MLO did not coincide to form an obvious hysteresis loop (Fig. 3e), indicating that both MLP and MLO were thixotropic systems. The thixotropic behavior was evaluated by measuring the area (AUC) of the hysteresis loop (Ayyash et al., 2020). The hysteresis area of MLP (26.5 Pa/s) was higher than that of MLO (16.5 Pa/s), indicating that MLP had a better thixotropic performance. The result suggests that the structure recovery capacity was enhanced after enzymatic treatment. One expla- nation for this is that the enzyme destroyed the MLP rigid construction to a flexible short-chain structure (Zamani & Razavi, 2021). 3.6.5. Dynamic rheological properties The viscoelastic properties of MLP before and after hydrolysis were evaluated via amplitude and frequency sweeps. The storage (elastic) modulus G’ and loss (viscous) modulus G” denote the elastic and viscous properties of a viscoelastic material, respectively (Gao et al., 2015). In addition, the material showed a liquid-like behavior when G” > G’ at low frequencies, and a solid-like behavior when G”< G’ at high frequencies (Zou et al., 2017). To confirm the dominance of the solid or liquid characteristics of MLP and MLO, the parameters G’ and G” of MLP and MLO were investigated. Fig. 3f shows that the G’ and G” of MLP were significantly decreased after enzymatic treatment. Meanwhile, the moduli (G’, G”) of MLP and MLO decreased with an increase in the oscillation strain (Fig. 3f). The MLP and MLO did not show any linear viscoelastic region and hence was not subjected to frequency sweep tests. These observations suggest that the MLP network was much stronger than the MLO network. This implies that enzymatic hydrolysis of MLP decreased the moduli of MLP by destroying its sugar-chain structures. Furthermore, the time-dependent fluid behavior was investigated to characterize the structure-recovery properties of MLP and MLO. At a constant shear rate, the viscosities of MLP and MLO remained stable over time (Fig. 3g), indicating that the fluid behavior depended only on the shear rate. It can be assumed that the MLP and MLO chains were entangled in a disordered state; at a low shear rate, the polymer chains were aligned and stretched in the flow direction; however, they could return to random distribution under the influence of Brownian motion; as the shear rate further increased, the effect of shear force was stronger than that of Brownian motion, thus resulting in the gradual disintegra- tion of the entanglements of molecular chains (molecular chains ori- ented in an ordered state), and the state could not be restored; at a high shear rate, the solution viscosity did not further increase with the in- crease in shear rate when the entanglements of molecular chains were completely broken up (Patel et al., 2014). The structure recoveries of MLP and MLO were 60.4% and 85.0%, respectively. The result suggests that MLP showed better structure recovery after enzymatic treatment, which agrees with the change in the thixotropic performance. 3.7. Antioxidant activity The antioxidant activities of MLP and MLO were evaluated via DPPH radical scavenging assay, hydroxyl radical scavenging assay, Fe2+ chelating capacity assay, and FRAP assay (Fig. 4). 3.7.1. DPPH radical scavenging activity Fig. 4a shows the change in the abilities of MLP and MLO to scavenge DPPH radicals. The results show that the DPPH scavenging activities of MLP, MLO, and ascorbic acid enhanced with the increase in the sample concentration, and MLO displayed higher antioxidant activity than MLP, which was evidenced by the lower IC50 (concentration inhibiting 50% of the free radical DPPH) of MLO (20.00 mg/mL) than that of MLP (24.82 mg/mL). This phenomenon might be due to the enzymatic treat- ment–induced changes in the molecular and glycosidic linkages (Yuan et al., 2015). 3.7.2. Hydroxyl radical scavenging activity As shown in Fig. 4b, MLP, MLO, and ascorbic acid displayed hy- droxyl radical scavenging activities in a dose-dependent manner. At the concentration of 800 μg/mL, the scavenging activities of MLP, MLO, and ascorbic acid were 74.89%, 88.62%, and 99.02%, respectively. Notably, MLO showed higher scavenging activity than MLP, thus showing more protective effects on the decrease in cell membrane and DNA base sequence damages (Halliwell, Dittmar, & Orsborn, 2007). This might be explained by the enzymatic hydrolysis of MLP resulting in the exposure of more active groups. 3.7.3. Fe2+ chelating capacity In this study, the Fe2+ chelating capacities of MLP and MLO were evaluated. The Fe2+ chelating capacity of the samples was concentration-dependent under MLP/MLO concentration of less than 1 mg/mL (Fig. 4c). With a further increase in the concentration, the Fe2+ chelating capacity of the samples slightly increased. Moreover, the Fe2+ chelating capacity of MLO was superior to that of MLP, especially under the concentration of >0.6 mg/mL. The results suggest that the MLP after enzymatic treatment exhibited a stronger ability to bind iron in oxidized form to prevent lipid peroxidation and minimize oxidative damage (Niki, 2012; Djerrad, Kadik, & Djerrad, 2015; Gülçin, Berashvili, & Gepdiremen, 2005).

3.7.4. FRAP

The FRAP was applied to evaluate the MLP and MLO abilities to reduce ferric iron to the ferrous state in the TPTZ solution (Fig. 4d). In the range of the test concentrations, the FRAPs of MLP or MLO had a good linear relationship with the concentrations; the FRAP of MLP was increased after hydrolysis. The results show that the MLO had a stronger ferric reducing antioxidant power than MLP, which might be due to the exposure of more reductive hydroxyl group and aldehyde group termi- nals to scavenge free radicals (Fimbres-Olivarria et al., 2018). These results suggest that enzymatic hydrolysis may be an effective approach to increase the reducing power of polysaccharides by decreasing their molecular weight.

3.8. Prebiotic activity

Many bifidobacteria and lactic acid bacteria can secrete glycosyl hydrolases to catabolize non-digestible plant polysaccharides and oli- gosaccharides. In this study, B. bifidum, B. adolescentis, L. rhamnosus, and L. acidophilus were applied to investigate the MLP prebiotic activity before and after enzymatic hydrolysis. The growth curves and pH vari- ations in the culture of these bacteria are displayed in Fig. 5. Since the turbidity was proportional to the concentration of the bacterial sus- pension, the turbidity was applied to express the number of colonies (Li et al., 2016). As shown in Fig. 5, all of the four microorganisms could utilize MLP and MLO and showed rapid growth, as evidenced by the increase in the absorbance at 600 nm. Decreases in the pH of the culture were also observed, which may be associated with the utilization of prebiotic (MLP or MLO) by the four microorganisms to produce lactic acid and SCFAs via fermentation (Leijdekkers et al., 2014). As shown in Fig. 6h, the SCFA production ability of the bacteria-fermented GOS was highest, followed by those of the bacteria-fermented MLO and MLP, which agrees with the observed pH values. Specifically, these four bacteria grown in MLO had higher contents of acetic acid, butyric acid, and lactic acid than those grown in MLP, and isovaleric acid and lactic acid than those grown in GOS (Fig. 6a, c, and g). Additionally, B. bifidum, L. rhamnosus, and L. acidophilus incubated in MLO or MLP also accu- mulated propionic acid, valeric acid, isobutyric acid, and isovaleric acid (Fig. 6b and d–f). Although the B. adolescentis that fermented GOS, MLO, or MLP did not produce valeric acid and produced less isobutyric acid, it could produce more propionic acid and butyric acid than the other three bacteria, resulting in more decrease in pH value than the effects of B. bifidum, L. rhamnosus, and L. acidophilus (Fig. 6b–e). In summary, these four microorganisms displayed a much higher growth under MLO as the carbon source than under MLP. The result indicates that a higher degree of polysaccharide polymerization blocked the degradation by probiotics. Overall, the glycosidic bonds and the molecular weight of the carbohydrates affected their prebiotic activity (Li et al., 2016).

4. Conclusions

Mulberry leaf oligosaccharides were enzymatically prepared from MLP for the first time. This study demonstrated that the enzymatic hy- drolysis of MLP increased the content of carbonyl, carboxyl, and hy- droxyl groups. However, the Mw, surface tension, apparent viscosity, and thermal stability were decreased. The hydrolysis also improved the thixotropic performance and structure-recovery properties of MLP. The changes in the MLP structure and rheological behavior after enzymatic treatment may contribute to the increase in the DPPH radical scavenging activity, hydroxyl radical scavenging activity, Fe2+ chelating capacity, FRAP, and the ability to promote the growths of B. bifidum, B. adolescentis, L. rhamnosus, and L. acidophilus. The enzymatic modifi- cation of MLP is beneficial for changing its structural and rheological properties, which can affect its function. It can be concluded enzymatic modification is a promising approach for polysaccharide processing.


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