Degradation of polysaccharides from Lycium barbarum L. leaves improves bioaccessibility and gastrointestinal transport of endogenous minerals

Journal Pre-proof Degradation of polysaccharides from Lycium barbarum L. leaves improves bioaccessibility and gastrointestinal transport of endogenous minerals

Mengze Wang, Jie Wang, Lili Fu, Majida Al-Wraikat, Shenzhu Lin, Pengpeng Lu, Lewen Shan, Junfeng Fan, Bolin Zhang PII:

S0141-8130(19)38037-7

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.11.243

Reference:

BIOMAC 14024

To appear in:

International Journal of Biological Macromolecules

Received date:

5 October 2019

Revised date:

27 November 2019

Accepted date:

30 November 2019

Please cite this article as: M. Wang, J. Wang, L. Fu, et al., Degradation of polysaccharides from Lycium barbarum L. leaves improves bioaccessibility and gastrointestinal transport of endogenous minerals, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2019.11.243

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© 2018 Published by Elsevier.

Journal Pre-proof Degradation of polysaccharides from Lycium barbarum L. leaves improves bioaccessibility and gastrointestinal transport of endogenous minerals

Mengze Wang 1, Jie Wang 1, Lili Fu, Majida Al-Wraikat, Shenzhu Lin, Pengpeng Lu, Lewen

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Shan, Junfeng Fan *, Bolin Zhang

These authors contributed equally to this work.

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Beijing Forestry University, Beijing 100083, China

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Department of Food Science and Engineering, College of Biological Sciences and Technology,

*Correspondence: Junfeng Fan, Associate Professor, Department of Food Science and

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Engineering, College of Bioscience and Biotechnology, Beijing Forestry University, P.O.112,

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35Qinghua East Road, Haidian District, Beijing, 100083, China. Phone: (8610)6233-6700, Fax:

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(8610) 6233-8221, E-mail: [email protected]

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Journal Pre-proof

Abstract Acidic heteropolysaccharide (LP) from Lycium barbarum L. leaves has compact globular structure which wrapped abundant endogenous minerals inside by ionic interactions with uronic acid. This study investigated the efficacy of chemical degradation of LP on the bioaccessibility and transport of endogenous minerals in simulated gastrointestinal fluids. Results showed that

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the degradation using vitamin C and hydrogen peroxide mildly decreased LP molecular weight

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from 162.0 kDa to 94.3 kDa, and the structure of degraded LP (LPD) was converted to loose coil.

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After the simulated intestinal digestion, the accessibility of Ca, Fe, Zn, and Mg in LPD increased

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by119%, 52%, 103% and 112.5% compared with the intact LP, respectively, and in particular, the uptake rate increased by 15.8%, 8.1%, 23.4% and 21.6% for Ca, Fe, Zn, and Mg, respectively.

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These results demonstrated that the chemical degradation is a helpful strategy to improve the

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uptake of endogenous minerals wrapped in polysaccharide.

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bioavailability

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Keywords: Calcium uptake; NMR; bioaccessibility; Caco-2 cell; simulated digestion;

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Journal Pre-proof 1. Introduction Minerals, including iron, calcium, zinc and magnesium, are necessary for normal human functioning, particularly as cofactors in metabolism and immune functions, and they ameliorate the risk of certain diseases, including cancer (Corbo & Lam, 2013; Romualdo et al., 2016; Vatanparast et al., 2009). The World Health Organization (WHO) and the United Nations Food and Agriculture Organization (UNFAO) often refer to deficiency in calcium, iron, zinc,

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magnesium and other mineral elements in the diet as “hidden hunger”. It is a growing problem

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that affects nearly 2 billion people in the world whose diet does not provide them with the

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vitamins and minerals they need (Muthayya et al., 2013). Surveys and statistical estimates have

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shown that the worldwide prevalence of calcium deficiency currently ranges from 27-87% among adults, iron deficiency affects 46% of 5-14 year olds, and zinc deficiency is present in 17-

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20% of people (Corbo & Lam, 2013; Romualdo et al., 2016; Vatanparast et al., 2009), while

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the prevalence of marginal magnesium deficit is 15%–20% of the population (Durlach, 1989).

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Therefore, mineral supplements are clearly needed to treat and prevent mineral deficiencies. Recently, due to its very high content of nutrients and bioactive substances, Lycium

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barbarum L. leaves (LL) have attracted considerable attention. Studies on LL found that the essential components involved are minerals, polyphenols and polysaccharides (Yang et al., 2009). The contents of calcium in dried LL is as high as 5.9 g/100g, 51 times higher than that of L. barbarum berries and 10 times of green tea; and the content of iron is 89 mg/100g, 10 times as high as that of L.barbarum berries. The content of zinc is 2.9 mg/100g and the content of magnesium is 1.1 g/100g, double and 10 times higher than that of L.barbarum berries, respectively. Lycium barbarum L. leaves polysaccharide (LP) was composed of six monosaccharides, i.e., arabinose, rhamnose, ribose, xylose, galactose and glucose, with ribose 3

Journal Pre-proof and glucose the most abundant (Ren et al., 2017). LP has various biological activities and its content (4.3%) is comparable to that of common vegetables (Ren, L., 2017). LP has many functions, including lipid lowering, anti-inflammation, anti-radiation, anti-oxidation, anticoagulation, and regulating blood sugar (Bosscher et al., 2001; Liu et al., 2012). Interestingly, mineral extract from LL was found to contain much polysaccharide, and these minerals interacted with polysaccharide through ionic bonds (Yang et al., 2009). The presence of

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polysaccharide in the mineral extract enhanced the solubility of minerals. Nevertheless, the

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absorption rate of these minerals is low in Caco-2 model (Ren et al., 2017), which might be due

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to LP’s high molecular weight (Mw) and conformation nature. Researches showed that LP has

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high Mw of 25×104 Da and generally globular conformation (Zhang et al., 2019).

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Currently, several methods are being used to improve the functional bioactivities of plantderived polysaccharides, including sulfated modification, carboxymethylated modification and

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enzymatic hydrolysis. One in particular, using hydrogen peroxide (H2O2) degradation attracted

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much attention to modify polysaccharides due to its high efficiency, mild reaction conditions, and minimal damage to sugar structures (Hou et al., 2012). These methods however have been

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focused on improving the antioxidant and antiplatelet aggregation bioactivities of polysaccharides (Tang et al. 2014). Little has been done to understand LP endogenous minerals functions, the absorption of LP endogenous minerals and the conformational changes of mineral rich polysaccharides in the gastrointestinal and their effects on mineral absorption/uptake. In vitro simulated digestion method comprises a simulation of the gastrointestinal digestion and is usually used together with Caco-2 cell model to evaluate the bioaccessibility of nutrients; And Caco-2 is a widely accepted model to simulate the functional and morphological properties of human intestinal enterocytes (Glahn, 2009). The bioaccessibility and absorption of minerals 4

Journal Pre-proof from LP depends not only on their total mineral content but also on other factors, such as Mw and molecular conformation. The objective of this study is to evaluate whether the chemical degradation using H2O2 could enhance the uptake rate of minerals in the mineral extract containing polysaccharides from LL. Therefore, we first extracted and characterized the crude LP (LPC), and then degraded LPC using vitamin C and H2O2 to produce degraded LP (LPD). And the uptake efficiency of minerals in LPC and LPD was performed using the simulated

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digestion / Caco-2 cell model. To clarify the action mechanism of degraded polysaccharide on

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mineral absorption, the structural conformation of LP and LPD was also analyzed.

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2. Materials and Methods

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2.1 Materials

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LL was harvested in Beijing, China in August 2017. The leaves were washed, dried at 60°C for 12 h, grinded into a fine powder (60 mesh), and stored at room temperature until use. Caco-2

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cells were purchased from Peking Union Medical College Hospital (Beijing, China). Minimum

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essential medium (MEM) and heat inactivated fetal bovine serum (FBS) were purchased from

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Gibco Chemical Co. (Grand Island, NY, USA). Pepsin from porcine gastric mucosa (≥500 U/mg), trypsin and pancreatin from porcine pancreas, were purchased from Sigma Chemicals (Beijing, China). Monosaccharaides standards, uronic acid standard, bovine serum albumin and trifluoracetic acid (TFA) were purchased from Sigma (St. Louis, MO, USA). Alpha-amylase was purchased from Aoboxing Biotechnology (Beijing, China). Vitamin C (Vc), H2O2, and other materials and regents were of analytical grade. 2.2 LP preparation

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Journal Pre-proof LP was prepared according to a previously described method (Lin et al., 2019). Briefly, LL powder (50 g) was dissolved in distilled water (1000 ml) and incubated for 1 h at 121°C, filtered and the filtrate was concentrated using a rotary evaporator at 70°C. The concentrated solution was precipitated overnight using 4 volumes of ethanol (99%, v/v). After repeatedly centrifuged and lyophilized, LP was further deproteined using the Sevage reagent (chloroform: butanol = 4:1, v/v), and then decolorized with AD-8 macroporous resin. The resulting LP was purified using a

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DEAE-cellulose 52 column (2.6×60 cm) pre-equilibrated with distilled water. The column was

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eluted by stepwise elution with deionized water and NaCl solutions (0.05, 0.1, 0.2, and 0.4 M

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NaCl) at a flow rate of 1 ml/min. The polysaccharide content was determined using the phenol-

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sulfuric-acid method at 490 nm (Rover et al., 2013). The fraction eluted by 0.1M NaCl had the highest polysaccharide content, so it was pooled, dialyzed, concentrated, repeatedly lyophilized,

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and used as LPC further in this study.

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LPD was obtained using the degradation method described by (Zhang et al., 2013). Briefly, 1

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g of LP solution in 100 ml distilled water was heated up to 30°C; H2O2 and Vc were then added at final concentrations of 50 mM. After magnetic stirring at room temperature for 2 h, the

LPD.

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reactant was dialyzed, precipitated with alcohol (80%, v/v), and repeatedly lyophilized to obtain

Both LPs’ protein and total sugar contents were determined using Lowry’s method (Budny et al., 2016) and phenol sulfuric acid method (Rover et al., 2013), respectively. The content of uronic acid was determined by the sulfuric acid carbazole according to the method described by (Gao et al., 2005). The content of phytic acid was assayed using the method previously reported (Liu et al., 2019). Ash and moisture contents were determined following the method of the Association of Analytical Chemists (AOAC, 2012). 6

Journal Pre-proof 2.3 Monosaccharide composition analysis For each LP, 10 mg were dissolved in 2 ml TFA and hydrolyzed at 120°C for 2 h. TFA was removed by repeatedly adding methanol (2 ml) after drying using a rotary evaporator at 70°C. Finally, monosaccharaides were analyzed according to the method described in (Liu et al., 2012) using gas chromatograph (GC, GCMSQP 2010; Shimadzu, Tokyo, Japan) equipped with an Rtx5 column and flame ionization detector (FID). The molar ratios of monosaccharaides were

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obtained by calculating their molar concentration based on the peak areas and concentrations of

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standard monosaccharaides (Han et al., 2013).

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2.4 Fourier transform infrared spectroscopy (FTIR), Nuclear magnetic resonance (NMR)

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spectra and scanning electron microscopy (SEM)

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To scan FTIR spectra, samples were loaded after the instrument (Bruker Vertex 70, Bruker Optics, Ettlingen, Germany) was preheated and stabilized, and the scanning was performed at a

H and 13C NMR spectra of both LPs were obtained with an NMR spectrometer (Bruker

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range of 4000-400 cm-1 with a resolution of 4 cm-1 and scanning times of 70.

reported in ppm.

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Avance 500) using a sample of 20 mg/ml LP in D2O (0.5 ml, 99.9%). Chemical shifts are

Polysaccharide surface morphology was observed using a JSM-6480A scanning electron microscope (JEOL, Tokyo, Japan). 2.5 Mw and Conformation Mw and structure conformation of both LPs were determined by high performance gel permeation chromatography (HPGPC) equipped with both a refractive index detector (RID) and a multi-angle laser scattering detector (MALSD) (Lin et al., 2019). For each LP, 1 mg was 7

Journal Pre-proof dissolved in 1 ml deionized water, and then filtered using 0.45 μm and 0.22 μm syringe filters for aqueous solutions. 20 μl of the sample were loaded and the solution (pH 7.0) containing NaH2PO4 (0.05 M) and NaNO3 (0.15 M) was used as the mobile phase. The flow rate was set to 0.5 ml/min and the column temperature was 35°C. A calibration curve was produced using Dextran T-series standards (Dextran T1000, T500, T70, T40, T10, and T5) of different molecular weights. The Mw of both LPs were estimated with reference to the calibration curve using the

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RID (35°C). Simultaneously, the gyration radius (Rg) of LP molecules was determined using

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HPGPC equipment and MALSD (35°C).

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2.6 In vitro simulated digestion and the calculation of bioaccessibility

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The in vitro simulated digestion procedure was performed with some modifications according to

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the method illustrated by Faria (2018). The procedures include three solutions to simulate the digestion three phases: simulated saliva fluid (SSF) to simulate the oral phase, followed by

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simulated gastric fluid (SGF) to simulate the gastric phase and simulated intestinal fluid (SIF) to

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simulate the intestinal digestion phase.

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For each LP, 2.5g was mixed with 1.75 ml of SSF, 12.5 μl of 0.3 M CaCl2, 0.25 ml of αamylase at 1500 U/ml, and 487 μl of distilled water. The mixture (5 ml) was incubated for 2 min at 37°C and then mixed with 3.75 ml of SGF, 2.5 μl of 0.3 M CaCl2 ,0.8 ml of pepsin (25000 U/ml), 100 μl of 1M HCl and 347.5 μl of distilled water. After incubation for 2 h in a water bath at 37°C, the gastric mixture (10 ml) was mixed with 5.5 ml of SIF, 2.5 ml of pancreatin (800 U/ml), 1.25 ml of bile solution (160 mM), 20 μl of 0.3 M CaCl2, 75 μl of 1M NaOH and 0.65 ml of distilled water. After 4 h of incubation at 37°C, The intestinal mixture (20 ml) was then cooled in an ice bath then centrifuged at 5000×g at 4°C for 10 min, finally frozen at -20 °C. During the previous procedures, pH test was performed for each phase to correct the mixtures pH, results 8

Journal Pre-proof were 1.9, 6.2 for the gastric, and the intestinal phase, respectively, which agrees with the reference method (Faria et al., 2018). After the simulated gastrointestinal digestion, the samples were centrifuged at 5,000g for 10 min at 4°C to separate the soluble bioaccessible fraction from the residual fraction. Supernatants from the bioaccessible fraction were sampled to determine mineral using the inductively coupled

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plasma-atomic emission spectrometry (ICP-AES; iCAP6300, Thermo Scientific, MA, USA)

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based on the method of Faria et al. (2018). Bioaccessibility was calculated as follows:

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Bioaccessibility (%) = [mineral content (mg/100 g) in the gastric or intestinal fraction / total

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2.7 Cell culture and transport assay

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mineral content (mg/100 g)] × 100

Transport assay was performed following the same study adopted previously for the in vitro

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digestion (Faria, 2018), with some modification depending on the availability of materials. In brief, Caco-2 cells between passages 20 and 50 were routinely maintained in MEM

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supplemented with 20% FBS, 1% non-essential amino acids solution (NEAA), 1 ml glutamine

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and 0.5 ml penicillin-streptomycin. Cells were incubated at 37°C, 5% CO2 atmosphere. Medium was changed every two days and cells were subcultured every 5 days. Caco-2 cells were seeded in a density of 5×104 cells/cm2 in six-well Transwell plates and the transport experiments were conducted 22 days after seeding. Transepithelial electric resistance (TEER) was determined, cells with a TEER value > 1000 Ω cm2 were only considered to the transport. Cytotoxicity assay was detected by the MTT method (Zhang et al., 2015) and concentrations of both LPs that were safe for Caco-2 cell growth were selected accordingly.

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Journal Pre-proof Transport assay were performed to determine the gastric and intestinal uptake for LPC and LPD mixtures. For both LPs, 2.5 ml of each mixture was diluted in a transport medium (pH 7.2; 1 mM MgSO4, 5 mM glucose, 130 mM NaCl, 50 mM HEPES, and 10 mM KCl) then added to the Caco-2 cells. Cell cultures were incubated for 3 h at 37°C, 5% CO2 with 95% relative humidity. Samples were then collected from both the apical compartment (AP) and the basolateral compartment (BL). Cells were washed 3 times with PBS and detached using trypsin-

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EDTA solution, and the calcium, iron, zinc and magnesium contents were determined using the

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ICP-AES. Retention, transport and uptake were calculated as follows:

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Retention (%) = (minerals in cells – minerals in AP) / mineral intake

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Transport (%) = (minerals in BL – minerals in transport filter) / mineral intake

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2.8 Statistical analysis

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Uptake (%) = Transport (%) + Retention (%)

3. Results

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presented as mean± SD.

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Data were analyzed using SPSS 11.0 (SPSS Inc., Chicago, IL, USA) software, and were

3.1 Proximate compositions and characteristics of LPC Five major fractions of LP obtained after purification using DEAE-cellulose-52 column isolation are shown in Figure 1A, in which the fraction eluted with 0.1 M NaCl showed the highest polysaccharide content (79.0 ± 6.8%), therefore was used as LPC. The proximate composition and minerals content of LPC is summarized in Table 1 and 2, respectively. The moisture and protein content of LPC was 3.9 ± 0.2% and 2.5 ± 0.1%, respectively. The ash content was 15.0 ± 10

Journal Pre-proof 0.3%, implying that LPC had the potential to be a mineral source for human nutrition. Further analysis demonstrated that LPC had high levels of calcium (10.8 ± 1.1 mg/g), iron (2.29 ± 0.32 mg/g ), zinc (0.41 ± 0.06 mg/g ) and magnisuim (3.96 ± 0.22 mg/g), which is consistent with our prevous studies (Ren et al., 2017; Zhang et al., 2019). The uronic acid content of LPC was 13.7 ± 1.7%, which is less than that (40.6 ± 5.8%) reported in a previous study (Ren et al., 2017), while no phytic acid was detected. These results further indicate that LPC binds abundant minerals

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with uronic acids. HPGPC analysis showed that LPC had high Mw of 162.0 ± 1.2 kDa, which is

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similar to that (171.8 kDa) reported in a previous document (Ren et al., 2017).

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GC analysis showed that LPC composed of six monosaccharaides namely arabinose,

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rhamnose, ribose, xylose, galactose, and glucose at ratios of 1.43, 1.30, 2.53, 1.00, 1.86, and 2.48,

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respectively (Fig. 1B). This result is consistent with our previous study (Ren et al., 2017), while differs from that of LP eluted with 0.4 M NaCl, in which mannose and glucuronic acid were

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detected rather than arabinose and rhamnose (Zhang et al., 2019). Overall, the carbohydrate

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composition of LPC is similar to that of pectin polysaccharides; however, in LPC ribose content

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is higher (Stojilkovic et al., 1994).

FTIR spectrum of LPC showed a typical absorption pattern of sugar (Fig. 2A), which further proved that LPC contained high amount of polysaccharide. The major wide absorption peak at 3435 cm−1 was caused by the stretch vibration of O-H and N-H bonds. The minor peak at 2925 cm−1 indicate C-H stretching of –CH2–, and the peaks at 1641 cm−1 were caused by C=O stretching of –NHCOCH3– in polysaccharides (Wang et al., 2017). The peaks at 1413 cm−1 were assigned to –COO– bonds, signifying the presence of uronic acids, which was consistent with the findings of uronic acid content determination (Guo et al., 2015). The absorptions at 1085 and

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Journal Pre-proof 1050 cm−1 were attributed to the asymmetric C-O-C glycosidic rings, indicating the presence of pyranose (Liu et al., 2012). The 1H and 13C NMR spectra of LPC are represented in Figure 3A. The 1H NMR spectrum of LPC showed six anomeric proton signals at δ 4.28, 4.41, 4.90, 4.97, 5.12, and 5.36 ppm. The anomeric proton signals from δ 4.28 to 4.97 ppm were attributed to the β-configurations, while δ 5.12 and 5.36 ppm were accredited to α-configurations. 13C NMR chemical shifts of LPC ranged

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from δ 60 to 110 ppm, which represents a typical distribution of NMR signals in polysaccharides.

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LPC showed six major signals at δ 94.29, 96.72, 100.72, 103.12, 107.38, and 109.28 ppm for six

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anomeric carbons (Fig. 3B). Signals of 13C NMR at δ 90–110 ppm confirmed the presence of

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both α- and β-configurations, and the spectrum has a strong signal at δ 175.79 ppm, indicating

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the presence of uronic acids. The analysis of 1H and 13C NMR indicated that LPC had six kinds of monosaccharide, which agrees with the results of FTIR and the GC monosaccharide analysis.

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3.2 Effect of degradation on LPC’s characteristics

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LPD was obtained using the degradation method described previously. After degradation, the

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Mw decreased from 162.0 kDa in LPC to 94.3 kDa in LPD, while the uronic acid content slightly increased to 19.2% in LPD, indicating significant changes in LP molecular structure caused by degradation using H2O2 and Vc. According to Fry (1998), hydroxyl radicals originating from the reaction system of H2O2 and Vc react with hydrogen atoms of polysaccharides to break glycosidic links. The protein and total sugar content of LPD was 2.7 ± 0.4% and 77.2 ± 7.9% (Table 1), respectively, which was almost same to those in LPC, suggesting that the degradation had no significant effect on the proximate composition of LPC. Interestingly, the content of Ca and Zn 12

Journal Pre-proof in LPD was significantly higher than that in LPC, with values at 11.5 mg/g and 0.72 mg/g, respectively. However, Fe content significantly decreased from 2.29 to 1.5 mg/g (p < 0.05) and Mg slightly reduced from 3.9 to 3.6 mg/g (Table 2). These results indicated that minerals content had been severely affected by degradation. Previous studies reported that minerals such as calcium linked with polysaccharide through ionic interactions and LP binds abundant calcium with uronic acid (Ren et al., 2017; Zhang et al., 2019). Degradation allowed more ions to set free

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from such interactions, some were available for measurement while others might been lost due to

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oxidation or even weak bonds explaining the significant difference in some mineral contents

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between LPC and LPD.

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Monosaccharaides composition analysis of LPD showed that degradation had no significant

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effect on LPC composition, and LPD was similarly composed of six monosaccharaides: arabinose, rhamnose, ribose, xylose, galactose and glucose (Fig. 1C). This result differed from

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that of LPC at molar ratios of 1.26, 1.01, 2.42, 1.00, 1.89, and 2.2, respectively.

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The infrared spectrum of LPD is shown in Fig. 2A. The main peaks of LPD were almost the

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same as those of LPC, suggesting that no major functional group transformations caused by degradation. 1H and 13C spectra of LPD are shown in Fig. 3C and Fig. 3D. It could be seen that the spectra of LPD fairly resembled LPC. The anomeric proton signals of LPD were found at δ5.28, 5.15, 4.99, 4.92, 4.44, and 4.30; and the anomeric carbon signals of LPD were at δ109.18, 107.37, 103.21, 100.71, 96.71, and 94.29. Obviously, the signals of the anomeric region of LPD were almost identical to those of LPC, which demonstrated LPC and LPD had similar glycosidic linkage patterns. Furthermore, the signals at δ174.74 showed that LPD contained uronic acid (Hirsh, 1991). In summary, H2O2 and Vc degradation did not change the main structure of polysaccharides, and it was consistent with the FTIR and NMR results above. 13

Journal Pre-proof 3.3 Degradation effect on LPC’s conformation & morphology The Rg of LPC and LPD, shown in Fig.4, were determined to understand the degradation effect on LP chain conformation. The Rg and the Rg vs. Mw curve slope of LPC was 45.2 ± 4.1 and 0.32 nm, respectively, suggesting that LPC has a compact globule conformation in a thermodynamically good solvent. Furthermore, these results indicate that LPC is a compact polysaccharide that binds high amount of minerals by interacting with carboxyl groups. However,

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the Rg and the Rg vs. Mw curve slope of LPD increased to 53.4 ± 3.7 and 0.52 nm, respectively,

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indicating that LPD has an irregularly loose stretched chain coil conformation (Zeng, 2014).

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The surface morphology of LPC and LPD were considerably different, as shown by the SEM

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images results presented in Fig. 5. LPC had a rough, porous appearance, with clearly visible

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lumps of aggregates including polysaccharide particles, while LPD had a silky-smooth, relatively uniform appearance. Previous study reported that the extraction, purification, and preparation

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conditions affect polysaccharides structure and surface morphology (Fry, 1998). The changed

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degradation.

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surface morphology implied that the polysaccharide molecules had a significant transform after

3.4 FTIR spectra of LPC and LPD after the simulated gastrointestinal digestion FTIR spectra of the gastric and intestinal mixtures were analyzed to understand the action mechanism of the digestion procedures on the structural conformation of LPC and LPD (Fig. 2B). The 4 mixtures share almost the same main peaks (Fig. 2B) as those of LPC and LPD before the gastrointestinal phases as shown in Fig. 2A, suggesting that no major functional group transformations occurred during digestion. Previous study on polysaccharide from the seeds of P. asiatica L. reported in (Hu et al., 2012) found that there was no free monosaccharide released throughout the whole in vitro digestion time period, and the gastrointestinal digestion did not 14

Journal Pre-proof change the monosaccharide composition. However, it could be noticed that both LP’s intestinal mixture has higher transmittance than the corresponding gastric mixture, while no significant changes occurred in the gastric phase. This observation is coincided with the starch changes in the gastrointestinal tract (Yongliang, et al., 2014). 3.5 Gastrointestinal bioaccessibility

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The bioaccessibility fraction is an indication for the amount of a substance that is potentially available for the absorption. In this study, an overall increase of the bioaccessible fractions (%)

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of the investigated minerals could be noticed with the sequential digestion procedure from

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gastric to the intestinal phases (Fig. 5C). The highest accessible mineral was Mg, with a fraction

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increased from 26.30% to 31.84% in LPC, and from 46.38% to 68.18% in LPD gastric and

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intestinal, respectively. For Fe, which was the lowest accessible mineral, the fraction undergone gastric and intestinal digestion increased from 20.52% to 23.08% and from 26.15% to 35.29%

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for LPC and LPD, respectively. It was noticeable that the mineral loss derived by the degradation

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from LPC to LPD using H2O2 and Vitamin C were not taken into consideration when the bioaccessibility of Mg and Fe were calculated. The accessibility of Ca in LPC increased from

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27.85% to 33.22%, while in LPD from 36.67% to 53.09%, for gastric to intestinal fluid, respectively. While, Zn in LPC increased from 27.70% in gastric fluid to 32.20% in intestinal fluid, and that of LPD increased from 49.40% to 65.10%, respectively. Clearly, LPD gastric and intestinal fractions were higher than that of their corresponding LPC ones for the investigated minerals, and the bioaccessibility percentage almost doubled for Mg and Zn between gastric and intestinal mixtures in both LPC and LPD. Compared to LPC in the intestinal fluid, the accessibility of Ca, Fe, Zn, and Mg in the intestinal digestion increased by 60%, 53%, 102% and 114%, respectively. 15

Journal Pre-proof As far we know, no studies on the bioaccessibility of LP minerals have been published yet. A recent study by Faria (2018) on the bioaccessibility of home cooked and canned beans demonstrated that the bioaccessibility of Mg varied from 16–32% to 38–66%; while For Fe, Zn and Ca results varied from 5–16% to 7-47%, 31–44% to 37–56%, and 13–37% to 23–50%, respectively, which are in good agreement with our results. Furthermore, this study also

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suggested that degradation increases mineral bioaccessibility. 3.6 Caco-2 cell absorption

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The intake, retention, transport and uptake values of minerals in LPC and LPD after

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gastrointestinal digestion were determined through incubation with Caco-2 cells (Table 3).

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Overall, an increase was observed in the absorption with the sequential digestion procedures for

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minerals, and absorption for the intestinal minerals was higher than that for the gastric minerals after 3 h incubation, regardless the type of LP. For example, the uptake of Ca in LPC increased

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dramatically from 17.8 to 38.5%, while in LPD increased from 31.4 to 44.6%. Previous studies

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on LP minerals reported low absorption in Caco-2 cells for Ca, Fe and Zn in the crude status in Caco-2 cells (Ren et al., 2017). The absorption values for LP were 2.7–3.9% for calcium, 4.3–5.9%

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for iron, and 2.7–3.8% for zinc, which is much lower than that reported in this study. Interestingly, minerals absorption in LPD was significantly higher than that in LPC in both gastric and intestinal mixtures in this study. The uptake increased by 15.8% for Ca, 8.1% for Fe, 23.4% for Zn and 21.6% for Mg from LPC compared to LPD at the end of gastrointestinal digestion, indicating that degradation significantly enhanced the bioaccessibility of LP minerals. 4. Discussion

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Journal Pre-proof Plant polysaccharide generally contains abundant minerals, but the absorption of these endogenous minerals is problematic due to the disadvantage of polysaccharide structure. This is the first study to demonstrate that the degradation using H2O2 and Vc remarkably affected the composition, characteristics and structural conformation of polysaccharide from L. barbarum L. leaves. In particular, the degradation significantly increased the bioaccessibility and the absorption of endogenous calcium, iron, zinc and magnesium included in the polysaccharide

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using the in vitro digestion /Caco-2 cell model.

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Although leaves polysaccharides have abundant endogenous minerals, the polysaccharide is

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a double-edged sword for the mineral absorption. On one hand, the polysaccharide could keep

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minerals from precipitation in the gastrointestinal tract by interacting with minerals through

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weak ion bonds (Jung et al., 2006; Yin et al. 2015); On the other hand, the polysaccharide could inhibit the absorption of minerals in the tract by wrapping them in the globular structure, as

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evidenced in our previous study in which the endogenous minerals had low absorption rate when

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incubating with Caco-2 cell (Ren et al., 2017). However, when the polysaccharide was chemically modified by Vc and H2O2, high bioaccessibility and absorption of endogenous

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minerals were observed even only after the gastric digestion, and the final uptake of minerals (including Ca, Fe, Zn and Mg) in the degraded polysaccharide increased by 8.1%-23.4% at the end of the gastrointestinal digestion tested with Caco-2 cells, which is significantly higher than those previously reported (p < 0.05) (Ren et al., 2017). These findings indicated that degradation with Vc and H2O2 could be utilized as an efficient pathway to enhance the bioaccessibility of endogenous minerals wrapped in polysaccharide. The mechanism underlying the degradation of polysaccharide by Vc and H2O2 involves in the breakdown of glycosidic links caused by hydroxyl radicals originating from the reaction 17

Journal Pre-proof system of H2O2 and Vc, these hydroxyl radicals could react with hydrogen atoms of polysaccharides (Fry, 1998). In this study, this chemical degradation of polysaccharide decreased the Mw of polysaccharide from 162.0 kDa to 94.3 kDa, which is a limited degradation of polysaccharide. Therefore, this chemical degradation by Vc and H2O2 is a mild and easycontroled compared with the degradation caused by TFA. The moderate degradation of polysaccharide might eliminate the stereo obstacle of high molecular polysaccharide to facilitate

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the absorption of endogenous minerals, which was therefore considered to be one of the

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explanations for high mineral absorption after the degradation of polysaccharide in this study. As

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stated above, the intact polysaccharide in this study presented a large Mw, and more compact

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spherical structure, which caused minerals to be encrypted inside and hard to be absorped. However, after the degradation, the polysaccharide molecules become small and loose, the ions

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are easily exposed to the external environment, and the uptake was thereby increased. But we

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also noticed that the percentage of uronic acid found in LPD (19.2 ± 0.7%) is higher than in LPC (13.7 ± 1.7%) according to Table 1. It is usually thought that the more uronic acid is found

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within the LP structure, the more carboxyl groups are available for bonding with minerals, thus,

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a less quantity of these minerals are ready to be absorbed. However, it was reported that the binding between carboxyl groups of uronic acids and minerals was weak and reversible, in particular, it may prevent metal cations from precipitating in the alkaline intestinal environment, thereby increasing their bioaccessibility (Jung et al., 2006; Yin et al., 2012). Therefore, the change in structural conformation of polysaccharide was also thought to be a key factor to the improvement of mineral absorption in this study. Mineral deficiency often takes place among the elderly and children. The Recommended Dietary Allowance (RDA) value for calcium, iron, zinc and magnesium is 1000, 18, 11 and 420 18

Journal Pre-proof mg/day, respectively (Kruger, 2016). In this study, ash accounted for about 15% of LP and endogenous calcium, iron, zinc and magnesium are also abundant in LP, implying LP could be a mineral source for human health. According to the absorption rate based on our Caco-2 results, 50 grams of LPC/LPD intake could meet 20.8%/25.7%, 149.5%/110%,56.6%/122.8% and 28.5%/30.8% of RDA value for calcium, iron, zinc and magnesium, respectively. Obviously, LP is a rich mineral source with high bioaccessibility, and in particular, the degraded polysaccharide

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is more efficient than the intact one to provide minerals for human health. However, this

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conclusion is summarized only based on the observation of the simulated fluids and cell

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experiments. Further study needs to be conducted to figure out the practical uptake in animal

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tract.

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5. Conclusions

This study demonstrated that the physical configuration of the polysaccharides from LL strongly

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affects endogenous minerals digestive bioaccessibility. LP is a non-fast fermented

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heteropolysaccharide extracted from L.barbarum leaves, and it was abundant with mineral elements, especially calcium, iron, zinc and magnesium. This study found that the degradation

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using Vc and H2O2 not only decreased the Mw of LP but also changed the structural conformation from compact sphere to loose coil, thereby greatly enhanced the bioaccessibility and in vitro uptake of endogenous calcium, iron, zinc and magnesium in the simulated gastrointestinal fluids. These results from this study provide a new perspective on how to improve enhance the absorption of minerals in the nutrient supplements with high Mw polysaccharide as carrier in food industry; Meanwhile, this study will also promote further research on the structure-activity relationships of LP which could be utilized as an excellent

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Journal Pre-proof source of polysaccharide as a minerals supplement. However, further study needs to be done to observe the uptake of these minerals in the degraded polysaccharide using animal model. Acknowledgements We extend special thanks to the Key Research and Development Program of Ningxia Hui Autonomous Region (the East-West China Science and Technology Cooperation Project:

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Bioactive activities of minerals and polysaccharides from Lycium barbarum L. leaves), and the Beijing Forestry University training program for undergraduates (Grant no. X201910022073 and

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X201910022074).

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digestion on the structural, morphologic characteristics and fermentability of raw banana

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starch. Journal of Food and Nutrition Research, 2, 731–737. Zeng, H. L., Zhang, Y., Liu, J., & Zheng, B. D. (2014). Molar mass distribution and chain conformation of polysaccharides from Fortunella margarita (Lour.) Swingle. Chinese Journal of Structural Chemistry. 33(8), 1245–1252. Zhang, Z., Wang, X., Mo, X., & Qi, H. (2013). Degradation and the antioxidant activity of polysaccharide from Enteromorpha linza. Carbohydrate polymers, 92(2), 2084–2087.

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Journal Pre-proof Zhang, Y., Zhang, H., Wang, F., Yang, D., Ding, K., & Fan, J. (2015). The ethanol extract of Eucommia ulmoides Oliv. Leaves inhibits disaccharidase and glucose transport in Caco-2 cells. Journal of Ethnopharmacology, 163, 99–105. Zhang, B., Wang, M., Wang, C., Yu, T., Wu, Q., Li, Y., ... & Zhang, B. (2019). Endogenous calcium attenuates the immunomodulatory activity of a polysaccharide from Lycium

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barbarum L. leaves by altering the global molecular conformation. International journal of

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Figure legends Fig. 1 - (A) Elution curve of polysaccharide fractions obtained using a DEAE cellulose-52 column eluted with water and NaCl solution; Gas chromatogram of (B) LPC and (C) LPD monosaccharaides.

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Fig.2 - FTIR spectra of (A) LPC, LPD and (B) LPC (black) and LPD (Red) after the simulated

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gastric (solid) and intestinal (dashed) digestion.

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Fig.3 - NMR spectra of (A) 1H NMR LPC, (B) 13C NMR LPC, (C) 1H NMR LPD and (D) 13C

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NMR LPD.

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Fig.4 - Plots of gyration radius by molecular mass for (A) LPC and (B) LPD, respectively.

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Fig.5 - SEM images of (A) LPC and (B) LPD; (C) Gastric and intestinal bioaccessibility of Ca, Fe, Zn and Mg in LPC and LPD, respectively. Black bars, LPC; White bars, LPD. The first two

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columns in each mineral represent the bioaccessibility of mineral in gastric fluid, and the

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remaining two columns represent in intestinal fluid.

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Journal Pre-proof Table 1 Proximate composition (dry weight) of LPC and LPD. Mw (kDa)

Total sugar (%)

Protein (%)

Uronic acid (%)

Moisture (%)

Ash (%)

LPC

162.0 ± 1.2

79.0 ± 6.8

2.5 ± 0.1

13.7 ± 1.7

3.9 ± 0.2

15.0 ± 0.3

LPD

94.3 ± 2.0

77.2 ± 7.9

2.7 ± 0.4

19.2 ± 0.7

2.3 ± 0.1

13.4 ± 0.6

All experiments were performed at least in duplicate, and analyses of all samples were run in

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triplicate. The results were presented as means of three determinations ± standard deviation (SD).

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Journal Pre-proof Table 2 Mineral content (mg/g, dry weight) of LPC and LPD. Ca

Fe

Zn

Mg

LPC

10.8 ± 1.1a

2.3 ± 0.3a

0.4 ± 0.1a

4.0 ± 0.2a

LPD

11.5 ± 0.2b

1.6 ± 0.2b

0.7 ± 0.0b

3.6 ± 0.4a

All experiments were performed at least in duplicate, and analyses of all samples were run in triplicate. The results were presented as means of three determinations ± standard deviation (SD).

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Values in the same column represented by different letters differ significantly (p < 0.05).

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Journal Pre-proof Table 3 Retention, transport and uptake percentage of Fe, Zn, Ca and Mg from LPC and LPD gastric and intestinal fluids after incubation with Caco-2 cells for 3 h. Mineral LP

Fluid

Intake (mg)

Cell retention (%)

Transport (%)

Uptake (%)

Gastric

0.19

1.30 ± 0.69a

16.50 ± 0.67a

17.80 ± 7.57a

Intestinal 0.54

14.80 ± 0.68b

23.70 ± 0.46b

38.50 ± 1.14b

Gastric

0.20

10.10 ± 0.45c

21.30 ± 0.16c

31.40 ± 0.61c

Intestinal 1.31

18.90 ± 0.41d

25.70 ± 0.30d

44.60 ± 0.71c

Gastric

3.07 ± 0.26a

5.12 ± 0.56a

8.20 ± 0.82a

6.66 ± 1.96a

23.50 ± 2.24b

LPC Ca

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0.13

Intestinal 0.39

16.90 ± 0.28a

Gastric

0.14

4.10 ± 1.82b

7.17 ± 1.50b

11.20 ± 3.32b

Intestinal 0.87

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LPC

15.0 ± 0.19c

10.40 ± 0.96b

25.40 ± 1.15b

2.60 ± 0.63a

13.30 ± 1.79a

16.0 ± 4.58a

10.10 ± 0.79a

20.20 ± 0.86a

30.40 ± 1.64b

6.00 ± 1.86b

18.20 ± 0.77b

24.20 ± 2.63b

Intestinal 1.61

14.50 ± 0.49b

22.90 ± 0.52bc 37.50 ± 1.01a

Gastric

0.18

15.20 ± 0.40a

37.90 ± 1.70a

53.10 ± 2.10a

Intestinal 0.68

16.60 ± 1.50a

43.90 ± 0.40b

60.50 ± 1.90b

Gastric

0.19

18.30 ± 1.30b

34.20 ± 0.60c

52.50 ± 1.90b

Intestinal 1.69

14.90 ± 0.20b

56.40 ± 2.90d

71.30± 3.10bc

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Fe

Gastric

0.19

LPC

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Mg

0.19

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Gastric LPD

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Intestinal 0.73 Zn

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LPD

LPC

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LPD

LPD

All experiments were performed at least in duplicate, and analyses of all samples were run in triplicate. The results were presented as means of three determinations ± standard deviation (SD). Different letters indicate significant differences between the different types of polysaccharide (LPC and LPD) in gastrointestinal (p < 0.05). Uptake (%) = cell retention + transport. 29

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Intensity (×104)

Absorbance (490 nm)

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Fig. 3 Wang et al. 33

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Fig. 5 Wang et al. 35

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Author Statement

Mengze Wang: Conceptualization, Methodology, Investigation Jie Wang: Software, Writing- Original draft preparation

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Shenzhu Lin：Data curation, Investigation

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Pengpeng Lu: Investigation

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Lewen Shan: Validation Junfeng Fan: Supervision

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Majida Al-Wraikat: Visualization, Investigation

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Lili Fu: Writing- Reviewing

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Bolin Zhang: Supervision, Editing

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Journal Pre-proof Highlights Physical structure of polysaccharide affects the release of endogenous minerals.

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Chemical degradation coverts polysaccharide structure to a loose coil.

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Degradation increases the bioaccessibility and uptake of endogenous minerals.

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37