Fabrication of snapper fish scales protein hydrolysate-calcium complex and the promotion in calcium cellular uptake

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Fabrication of snapper fish scales protein hydrolysate-calcium complex and the promotion in calcium cellular uptake Yanlan Lina, Xixi Caia, Xiaoping Wua, Shengnan Linb, Shaoyun Wanga, a b

⁎

College of Biological Science and Technology, Fuzhou University, Fuzhou 350108, China School of Food Science, Washington State University, Pullman, WA 99164, USA

A R T I C LE I N FO

A B S T R A C T

Keywords: Fish scales protein hydrolysate Calcium binding Cellular uptake Dietary inhibition factor TRPV6

It has become a hot topic to find natural food substances which can effectively promote calcium absorption in human body. Given this, protein hydrolysate with calcium-binding capacity was prepared from fish scales (FSPH) and its promotion in calcium cellular uptake was investigated in this study. The binding of calcium to the amino nitrogen atoms and the carbonyl oxygen atoms of FSPH led to the structural folding and aggregation. The calcium cellular uptake efficiency in Caco-2 cells population model was significantly increased in the form of FSPH-calcium complex. Furthermore, FSPH-Ca could effectively resist the inhibitory effect of dietary inhibitors on calcium cellular uptake. The mRNA expression of transient receptor potential vanilloid 6 (TRPV6) in Caco-2 cells was remarkably elevated with FSPH-Ca treatment. The results improved the understanding of the interaction mode of protein hydrolysate and calcium, and provided a basis for developing FSPH-Ca as functional foods to improve calcium absorption.

1. Introduction Calcium, a crucial mineral macroelement in the human body (Sun, Jin, Li, Yin, & Lin, 2017), is mostly stored in the bones to maintain their structure and function (Mine, Shahidi, Mine, & Shahidi, 2006) and also involves the activation of many signaling pathways (Protiva et al., 2016). Calcium deficiency bring about imbalance of calcium homeostasis, leading to metabolic bone disease, colon cancer, hypertension, kidney stones and other diseases (Power et al., 1999). Ionized calcium is always the major calcium supplement for humans, however, the calcium absorption can be easily affected by some molecules present in food (Lee & Song, 2009). The intestinal absorption of calcium significantly decrease because of the precipitation of calcium ions with dietary inhibition factors such as phytate, oxalate, phosphate, tannin (Sun et al., 2016). Therefore, maintaining the good solubility of calcium ions is the basis for the improvement of the bioavailability. More recently, how to improve the intestinal absorption of calcium has become a hot topic. There has been a great deal of interest in finding natural food substances or their products that can effectively promote calcium absorption (Lee & Song, 2009; Liu, Wang, Wang, &

Chen, 2013). Prebiotics (Garcia-Vieyra, Del Real, & Lopez, 2014; Holloway et al., 2007) and bioactive peptides (Choi, Jung, Choi, Kim, & Ha, 2005; Liu et al., 2013) are two known substances with good effects on calcium uptake. Non-digestible prebiotics are fermented in the intestines, producing short-chain fatty acids (SCFAs) to improve mineral solubility (Krupa-Kozak, Markiewicz, Lamparski, & Juskiewicz, 2017). Calcium-binding peptides, which exhibit great potentials in promoting calcium absorption, have been isolated and characterized in recent years (Hou et al., 2015; Huang et al., 2015; Sun et al., 2017; Zhao et al., 2015). Casein phosphopeptide (Liu et al., 2018) (CPP) and phosvitin phosphopeptide (PPP) (Jiang & Mine, 2001) can improve the calcium uptake through binding calcium to their phosphorylated groups. Some peptides lacking phosphorylated groups also play good roles in calcium absorption by binding calcium to amino groups and carboxyl groups. It has been reported that calcium chelating peptides fabricated from desalted duck egg white have a good effect on promoting intestinal calcium absorption in phytic acid-induced osteoporosis rat model (Hou et al., 2015; Hou, Liu, Kolba, Guo, & He, 2017; Hou, Liu, Shi, Ma, & He, 2017; Zhao, Hu, et al., 2014). Besides, calcium chelating peptides from whey protein (Zhao, Huang, Cai, et al., 2014), Schizochytrium sp.

Abbreviations: FSPH, fish scales protein hydrolysate; CPP, Casein phosphopeptide; PPP, phosvitin phosphopeptide; SCFAs, short-chain fatty acids; BBM, brush border membrane; BLM, basolateral membranes; TRPV6, transient receptor potential vanilloid member 6; PMCA, plasma membrane calcium ATPase; FT-IR, Fourier transform infrared spectroscopy; SEM, Scanning electron microscopy; 1H NMR, 1H Nuclear magnetic resonance spectroscopy; TG-DSC, Thermogravimetry and differential scanning calorimetry; MTT, 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide ⁎ Corresponding author. E-mail address: [email protected] (S. Wang). https://doi.org/10.1016/j.jff.2019.103717 Received 20 July 2019; Received in revised form 8 November 2019; Accepted 28 November 2019 1756-4646/ © 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Yanlan Lin, et al., Journal of Functional Foods, https://doi.org/10.1016/j.jff.2019.103717

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2.3. Characterization of FSPH and FSPH-Ca

protein (Cai, Lin, & Wang, 2017; Lin, Cai, Tang, & Wang, 2015), sea cucumber (Cui et al., 2018), bovine milk proteins (Picariello et al., 2013) and wheat protein (Liu et al., 2013) were also prepared and characterized. Generally, calcium transports across the epithelial barriers of intestine by passive, concentration-dependent paracellular and active, vitamin D-dependent transcellular pathways (Hoenderop, Nilius, & Bindels, 2005). The latter is a saturable process that transports calcium from the brush border membrane (BBM) to the basolateral membranes (BLM) (Perez et al., 2008). It involves three steps: calcium ions enter cells via the transient receptor potential vanilloid member 6 (TRPV6) channel at the BBM, then are transported to BLM by simple diffusion, vesicular transport or combining with Calbindin-D9k, and are finally released from enterocytes by plasma membrane calcium ATPase (PMCA) (Diaz de Barboza, Guizzardi, & Tolosa de Talamoni, 2015; Ward & Boyd, 1980). Fish scales, as low value by-products, are often discarded directly during processing. However, fish scales contain 30–50% protein and 30–40% hydroxyapatite (Mace, Morgan, Affleck, Lister, & Kellett, 2007), which make them value-added materials to produce calcium binding peptides. In this study, fish scale protein hydrolysate (FSPH) with calcium binding capacity was derived from snapper scales by enzymatic hydrolysis. The possible binding sites and thermal stability of FSPH and FSPH-calcium complex were characterized, and the effect of FSPH-Ca on calcium cellular uptake and possible mechanism were investigated. The results suggest that FSPH-Ca could be used as functional foods to improve calcium uptake.

2.3.1. Analysis of amino acid composition The amino acid composition of FSPH was determined according to the method of Lin et al. (2015) by the High-Speed Amino Acid Analyzer L-8900 (Hitachi, Japan). FSPH was dissolved in 6 M HCl and hydrolyzed at 110 °C for 24 h before test. 2.3.2. Determination of molecular mass distribution The molecular mass distribution of FSPH was analyzed using highperformance liquid chromatography in a Waters 650E Advanced Protein Purification System (Waters Corporation, Milford, MA, USA) at the Instrumental Analysis Center of Jiangnan University, China. Sample was loaded to a TSK gel 2000 SWXL column (7.8 i.d. × 300 mm). Elution conditions: mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: 0.05% trifluoroacetic acid in acetonitrile; gradient elution: 0 to 15 min, 2% B → 30% B; 15 to 20 min, 30% B → 35% B. The flow rate was 0.5 mL/min and the detected wavelength was 214 nm (Chen, Wu, Yang, & Wang, 2017; Fang, Xu, Lin, Cai, & Wang, 2019). 2.3.3. Fluorescence spectroscopy The fluorescence spectroscopy was used to analyze the conformational changes of FSPH (25 μg/mL) resulted from binding with different concentrations of calcium ions (0, 20, 40, 60, 80, 100 μM) by a Fluoromax-4C-L fluorescence spectrophotometer (Horiba Instrument Inc, USA). Fluorescence spectra were recorded at an excitation wavelength of 280 nm and emission wavelength of 290–500 nm after incubation at 37 °C for 30 min.

2. Materials and methods 2.3.4. Fourier transform infrared spectroscopy (FT-IR) The freeze-dried powder (50 μg) of FSPH and FSPH-Ca were fully mixed with dried KBr (5 mg) for grinding in an agate mortar under the dry environment, respectively. The uniform powder was transformed into tablet by a tablet machine. Then, the infrared absorption was determined according to the method of Lin et al. (2015) using an infrared spectrophotometer (Thermo Nicolet Co., USA.).

2.1. Materials Snapper scales were kindly provided by Putian Huifeng Food Co., Ltd (Fujian, China). The commercial protease Flavourzyme (EC.3.4.21.62, 1.1 × 105 U/g) was purchased from Novozymes Biotechnology Co., Ltd. Fluo-3 AM was the product of Beyotime Biotechnology Co., Ltd (Shanghai, China). All other chemicals and reagents were of analytical grade and commercially available.

2.3.5. Scanning electron microscopy (SEM) The surface morphology of FSPH and FSPH-Ca were detected by field scanning electron microscope (Nova Nano SEM 230, FEI CZECH REPUBLIC S.R.O. Czech Republic). The lyophilized samples were fixed on an SEM sample column with conductive adhesive, gold was sprayed under high vacuum conditions, and then the morphology was observed by SEM using a 2000× magnification. (Kovacs et al., 2012).

2.2. Preparation of the FSPH and FSPH-Ca Dried snapper fish scales were grinded to powder by pulverizer at room temperature for 25 min. The fish scale powder was immersed into deionized water at a ratio of 1:30 (w/v) for high-temperature and highpressure pretreatment (121 °C, 0.2 MPa, 60 min). As shown in Fig. S1, the protein hydrolysate produced by flavourzyme possessed the highest calcium-binding capacity and DH. So we chose flavourzyme as the tool enzyme in this study. The optimum hydrolysis and calcium-binding conditions had been determined by the Box-Behnken Design beforehand. Briefly, the pH was adjusted to 7.0 prior to the addition of Flavourzyme (enzyme/substrate = 7.9%) and stirred at 50 °C. After 9 h, the enzyme was inactivated by heating at 100 °C for 10 min, and the supernatant was collected by centrifuged at 10,000 rpm for 15 min. The lyophilized hydrolysate was referred to as FSPH. Subsequently, FSPH solution (1 mg/mL) was mixed with calcium chloride at a mass ratio of 4:1 (w/w), and incubated at pH 6.5, 30 °C for 50 min. Eight times volume of absolute ethanol was introduced continuously into the mixture solution and deposited for 2 h. The FSPH-calcium complex precipitate was collected by centrifuged at 10,000 rpm, 4 °C for 10 min (Cai et al., 2017; Lin et al., 2015; Sun et al., 2016). The binding rate of calcium was determined to be 88.44% (Zhang, Lin, & Wang, 2018). The FSPH-Ca precipitate was re-dissolved in deionized water, lyophilized, and stored at −20 °C for further analysis.

2.3.6. Zeta potential The zeta potentials of FSPH and FSPH-Ca were determined using a Zetasizer Nano ZEN3600 particle size analyzer (Malvern Panalytical Ltd., Malvern, UK). The folded capillary cells containing 1 mg/mL of the sample was equilibrated at 25 °C for 2 min and then the zeta potentials were determined. 2.3.7. 1H Nuclear magnetic resonance spectroscopy (1H NMR) FSPH and FSPH-Ca (1.0 mg) were dissolved in 1 mL deionized water and the pH was adjusted to 7.0 prior to the addition of 50 μL deuterium oxide (D2O). Then, the samples were transferred into 5 mm NMR tubes for analysis using a Bruker Avance III spectrometer (Bruker Biospin, Rheinstetten, Germany). 2.3.8. Thermogravimetry and differential scanning calorimetry (TG-DSC) The thermal stabilities of FSPH and FSPH-Ca were performed by TGDSC (STA449C/6/G, Germany). An aliquot of 5 mg of the sample was sealed in the crucible and heated from 30 to 500 °C at 10 °C/min under an argon atmosphere of 30 mL/min. 2

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2.4. Calcium uptake studies in Caco-2 cells population model

Table 1 Amino Acid Compositions of FSPH.

2.4.1. Cell culture The Caco-2 cells were cultured in Dulbecco’s modified eagle medium (DMEM) containing 1% non-essential amino acids, 1% penicillin-streptomycin and 15% fetal bovine serum (FBS) at 37 °C with 5% CO2. At 80–90% confluence, the Caco-2 cells were detached with trypsin-EDTA solution and seeded at 5 × 103 cells/well onto 96-well plastic cell culture clusters. Then the cytotoxicity of FSPH and CaCl2 on Caco-2 cells was determined by MTT assay. 2.4.2. Calcium imaging Calcium imaging was performed to show calcium influx into Caco-2 cells according to the method of Sun et al. (2017). The cells were seeded at 1 × 104 cells/well onto confocal dishes and cultured with 10 mM CaCl2 (control) and FSPH-Ca with 10 mM CaCl2 and different FSPH contents (2, 4, 8 mg/mL) for 24 h, respectively. Then, Fluo-3 AM (2.5 μM) was loaded. After incubated at 37 °C in the darkness for 1 h, cells were washed three times with Hank's balanced salt solution (HBSS), and incubated another 30 min for the de-esterification of Fluo3-AM to form fluorescent Fluo-3. Fluorescence images of cells were observed by an inversion fluorescence microscope (Nikon Corporation, Tokyo, Japan) with 10× magnification.

Amino acids

Content (%)

Amino acids

Content (%)

Gly Pro Glu Ala Arg Asp Lys Ser Val

21.19 13.41 9.66 8.88 7.04 4.79 3.06 2.96 2.36

Leu Thr Met Phe Tyr Ile His Cys

2.19 2.10 2.09 2.05 1.09 1.07 1.05 0.18

reverse transcriptase (RT) reaction to created cDNA by TransSprit OneStep gDNA Removal and cDNA Synthesis SuperMix (Tansgen Biotech, CO., Beijing). RT-PCR was performed in MyGo PCR systems (BI-MYGO, IT-IS Life Science Ltd, USA) after adding the primers and SYBR green I to cDNA. DNA sequences of the primers were shown in supplemental materials Table S1. The expressed efficiency and specificity of the primers were also tested.

2.5. Statistical analyses

2.4.3. Establishment of Caco-2 cells population model Caco-2 cells were the typical model for studying mineral uptake in vitro. At 80–90% confluence, the Caco-2 cells were detached with trypsin-EDTA solution and seeded at 1 × 105 cells/well onto 12-well plastic cell culture clusters for 7 days. The medium was replaced every other day.

All experiments were performed in triplicate and the experimental data was finally represented as means ± SD. T-test was carried out by SPSS 22.0 to analyze the significant differences of data. Data showed significant differences at p < 0.05.

3. Results and discussion 2.4.4. Calcium uptake studies After 7 days, the medium was discarded. Subsequently, different concentrations of CaCl2, FSPH-Ca, tannic acid/FSPH-Ca, tannic acid/ CaCl2, phytate/FSPH-Ca, phytate/CaCl2, oxalate/FSPH-Ca, oxalate/ CaCl2, phosphate/FSPH-Ca, phosphate/CaCl2, Fe2+/FSPH-Ca, Fe2+/ CaCl2 were added to the wells, respectively. FSPH-Ca with different FSPH and calcium ratios were prepared as described in 2.2 before the cellular uptake study. After incubation at 37 °C with 5% CO2 for 2 h, cells were washed three times with HBSS, and 500 μL 2.5 μM Fluo-3-AM was loaded for 60 min. Then the Caco-2 cells were washed again and incubated in HBSS for another 30 min. The cells were detached by trypsin, centrifuged at 3,000 rpm for 3 min and resuspended uniformly with HBSS. The fluorescence intensity was measured at excitation wavelength of 485 nm and emission wavelength of 525 nm by a fluorescence spectrophotometer (Fluoromax-4C-L, Horiba Instrument Inc, USA). Intracellular calcium concentrations [Ca2+]i were expressed as an increase in fluorescence intensity compared to the control (cells treated with CaCl2 with equivalent calcium amount to FSPH-Ca).

3.1. Composition analysis of FSPH The function of bioactive peptide was closely related to its amino acid composition. As the result shown in Table 1, the content of glycine (Gly) and proline (Pro) in FSPH was 21.19% and 13.41% respectively, which were in accordance with the material used. Collagen is the main protein in fish scales, which has highly repetitive Gly-Pro-X sequence. Glutamic acid (Glu, 9.66%), alanine residues (Ala, 8.88%), arginine (Arg, 7.04%), aspartic acid (Asp, 4.79%) and lysine (Lys, 3.06%), which were regarded as the primary binding sites for calcium (Lin et al., 2015), were also abundant in FSPH. In Table 2, the acidic amino acid, basic amino acid and hydrophobic amino acid accounted for 14.44%, 11.15% and 32.04%, respectively. A calcium-binding peptide Asp-GlyAsp-Asp-Gly- Glu-Ala-Gly-Lys-Ile-Gly was derived from tilapia scale (Chen et al., 2014). The calcium-binding capacity was demonstrated to be enhanced with the increase of carboxyl content (Liu et al., 2013). Moreover, the basic amino acid (Lys and Arg) also made contribution to the calcium-binding capability (Lv, Bao, Liu, Ren, & Guo, 2013).

2.4.5. The pathway of calcium uptake improved by FSPH-Ca The Caco-2 cells were seeded at 1 × 105 cells/well onto 12-well plastic cell culture clusters for 7 days. According to the result of cytotoxicity test, Caco-2 cells were pre-incubated with TRPV6 inhibitor (0–50 μΜ 2-aminoethoxydiphenyl borate, 2-APB) for 1 h. The culture medium was discarded. Continually, FSPH-Ca (4 mg/mL FSPH and 10 mM CaCl2) and 2-APB were added. The calcium uptake efficiency was determined by Fluo-3-AM probe as described in Section 2.4.4.

Table 2 Amino Acid Compositions of FSPH. Amino acid

Content (%) a

Acidic amino acid Basic amino acidb Hydrophobic amino acidc Essential amino acidd Aromatic amino acide

2.4.6. Real-Time quantitative polymerase chain reaction (RT-PCR) The Caco-2 cells were seeded at 1 × 105 cells/well onto 6-well plastic cell culture clusters for 7 days. The mRNA expression of TRPV6, calbindinD9k and PMCA1b were determined in Caco-2 cells after treatment with FSPH-Ca for 24 h. Total RNA was extracted from cells using RNA prep Pure Cell Kit (Tiangen Biotech CO., Ltd, USA) according to the manufacturer’s protocol. Afterward, RNA experienced a

a b c d e

3

14.44 11.15 32.04 14.92 3.14

Containing Glu and Asp. Containing Lys, Arg, and His. Containing Ala, Phe, Ile, Leu, Pro, Val, Try, and Met. Containing Lys, Try, Phe, Met, Thr, Ile, Leu, and Val. Containing Phe, Tyr, and Trp.

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Fig. 1. The characterization of FSPH-chelate. (A) Molecular mass distribution of FSPH. (B) Fluorescence spectra of FSPH with different CaCl2 concentrations; (C) Fourier transform infrared (FT-IR) spectra of FSPH and FSPH-Ca in the region from 4000 to 500 cm−1; (D) Zeta-potential of FSPH and FSPH-Ca; (E) 1H NMR spectral region from 3.29 to 12.69 ppm of FSPH and FSPH-Ca (the red line means FSPH, and the blue line means FSPH-Ca). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

4

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eC]O in O]CeNH2 after binding with calcium (Ye, Wu, Zhang, & Wang, 2019). These findings indicated that amino nitrogen atoms and the carbonyl oxygen atoms were the main interaction sites during the binding process of FSPH and calcium ions, with the generation of new chemical compounds. Previous work also showed that peptides with the increase of carboxyl groups possessed better effect on the calcium binding (Xiao, Mei, Jing, Yang, & Shun, 2007). The amino nitrogen atoms and the carbonyl oxygen atoms were the main calcium-binding sites in oyster protein hydrolysates (Chen et al., 2013).

3.2. Analysis of molecular mass distribution According to previous reports, the molecular mass of peptide was likely to associate with its calcium chelating capacity (Li, Jiang, Zhang, Mu, & Liu, 2008). As shown in Fig. 1A, the molecular mass of FSPH distributed < 2000 Da was 97.90%, among which, the hydrolysate with molecular mass between 180 and 500 Da accounted for 52.41%. Similar results have been reported in previous studies, showing that food-derived protein hydrolysates with smaller molecular mass have higher calcium affinity. Jin, Fu, and Ma (2011) proved that bone collagen peptide with the molecular mass less than 5000 Da had a prominent calcium chelating capacity. Protein hydrolysate prepared from wheat germ with molecular mass distributed in 180–1000 Da was also reported to possess a high calcium-binding capacity (Liu et al., 2013).

3.5. Zeta potential Zeta potential analysis was used to determine the surface charge state of protein particles due to the ionization of amino acid residue (Vanapalli & Coupland, 2000; Zhang, Zhang, Fang, & Wang, 2019). Fig. 1D manifested the isoelectric point of FSPH and FSPH-Ca were 2.70 and 2.31, respectively. The zeta potential of FSPH and FSPH-Ca ranged from 7.21 and 1.88 to −26.75 and −15.2, respectively, and the absolute value of the zeta potential of FSPH-Ca decreased dramatically compared with FSPH at the same pH conditions. The chargeability of FSPH-Ca was weaker than FSPH under the same conditions, which could be predicted that FSPH-Ca existed as a neutral structure without a double electron layer. We speculated that calcium ions were entrapped in the middle of FSPH by a coordinate bond, forming a soluble complex with improved stability.

3.3. Fluorescence spectroscopy Fluorescence spectroscopy was a convincing method for analyzing the binding of peptides and other molecules (Cai, Yang, Lin, Fu, & Wang, 2017). The structural changes of peptides could be analyzed by the change of fluorescence spectra. As revealed in Fig. 1B, the fluorescence intensity of FSPH dropped from 398 147 to 321 524 with the increase of calcium ions concentrations (20, 40, 60, 80, 100 μM). This might attribute to the formation of FSPH-calcium complex, which destroyed the conjugated double bond of FSPH and caused the substituent effect, leading to endogenous fluorescence quenching. Especially, the endogenous fluorescence decreased significantly when 20 μM calcium chloride was loaded into FSPH solution. Previous study shown that metal ions chelated with peptides could generate endogenous fluorescence quenching, resulted from the structural folding and aggregation of peptides during the chelation process (Beyer, Hoang, Appleton, & Fairlie, 2004; Zhao, Hunag, Hunag, et al., 2014). The fluorescence intensity decreased dramatically with the increasing concentrations of calcium ions, obviously reflecting the structural folding and aggregation of the FSPH (Hou et al., 2017). The conclusion was verified in the SEM analysis (Fig. 1B). This was in accordance with the previous research, proving that Zn2+ could bind with zinc-finger peptide and result in the folding of peptides (Reddi, Guzman, Breece, Tierney, & Gibney, 2007). In addition, the level of fluorescence quenching gradually weakened with the increasing calcium ions concentration, which might be due to the reduction of potential binding sites of peptide chain.

3.6. 1H Nuclear magnetic resonance spectroscopy (1H NMR) The 1H NMR was an important technique that could analyze the distribution of the electron cloud around a hydrogen nucleus. The decrease of electron cloud density and shielding effect could transfer the resonance signal peaks to a lower field region (Beyer et al., 2004). Therefore, the changes of chemical shift and intensity on the 1H NMR spectra were used to reveal the binding process of FSPH and calcium ions. As shown in Fig. 1E, the peak shifted from 0.85 ppm to the low magnetic field of 1.15 ppm after binding with calcium. The binding of FSPH and calcium affected the electron density around the hydrogen nucleus, thereby change the signal peaks of hydrogen atom spin coupling cracking resonance. The fine peaks (0.75–3.3 ppm and 6.8–8.0 ppm) decreased distinctly attributed to the aggregation and folding of calcium-binding peptides (Lin et al., 2015). 3.7. Thermogravimetry and differential scanning calorimetry (TG-DSC)

3.4. Fourier transform infrared spectroscopy (FT-IR) The thermal stability could be explored by TG-DSC analysis. Many chemical compounds changed in heat and mass during the thermal decomposition processes, and the combination of the two indicators could be used to predict changes in physical and chemical properties of substances such as evaporation, sublimation, redox, etc. The TG-DSC curves of FSPH and FSPH-Ca were shown in Fig. 2. A distinct endothermic peak was obtained at 134.7 °C on the DSC curve, but there was no significant mass loss on the corresponding TG curve, which caused by the volatilization of the adsorbed water in the FSPH and FSPH-Ca during the heating process. What’s more, the two endothermic peaks appeared at 251.8, 316.3 °C on the DSC curve of FSPH might be due to the destruction of the CeN chemical bond, along with the mass loss of 69.94%. However, this phenomenon was not observed in the DSC analysis of FSPH-Ca and the mass loss of FSPH-Ca was 54.31%. These differences indicated that FSPH-Ca had a more stable structure and thermal properties compared with FSPH.

When calcium ions bind to the organic ligand groups of the peptides, the characteristic FT-IR absorption peaks were significantly changed owing to the vibration of the coordinate bonds (Cai, Zhao, Wang, & Rao, 2015; Chen et al., 2019). Fig. 1C exhibited the FT-IR spectra of FSPH and FSPH-Ca. Displacement and intensity changes of absorption peaks could be observed after the reaction of calcium ions and FSPH. The absorption band at 3500–3100 cm−1 was assigned to the stretching vibration of the NeH and OeH bonds. After the addition of calcium, the peak shifted from 3295.41 cm−1 to 3352.30 cm−1, which was attributed to transfer of electron pairs from nitrogen atoms to calcium, forming NeCa instead of the NeH bond (Fig. 1C). The amide I bands (1600–1500 cm−1), primarily caused by the stretching vibration of the C]O bonds, was shifted from 1651.16 cm−1 to a higher wavenumber of 1655.23 cm−1 after binding with calcium ions. The peak (1402.20 cm−1) belonging to the eCOOe group was shifted to 1410.37 cm−1 in the FT-IR spectra of FSPH-Ca, along with a split of the peak. The results revealed that the amide carbonyl (C]O) and the carboxyl (eCOOe) also played an important role in the binding reaction. Furthermore, the characteristic peak at 660.53 cm−1 derived from the in-plane vibration of the O]CeN bond was shifted to 618.56 cm−1, which might be due to the increase of the electron cloud density around

3.8. Effect of FSPH on calcium cellular uptake In present study, Caco-2 cells population model was used to assess the influence of FSPH-Ca on calcium uptake by testing the calcium influx into the cytoplasm after exposure to FSPH-Ca and CaCl2. As 5

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3.9. Calcium uptake in the presence of dietary inhibition factors Calcium maintained a soluble form in the intestinal lumen was necessary for its absorption, usually in free calcium ions form or chelated with a soluble organic molecule. However, calcium from diet often failed to meet the physiological needs of the human body due to a lack of intake and the low bioavailability. The latter was mainly because of the calcium precipitation effects of dietary inhibition factors in foods such as phytic acid in grains, tannic acid in mulberry and oxalate in spinach, etc., which decreased the solubility of calcium. Besides, all divalent metal ions could be absorbed by divalent metal transport 1 (DMT1) and there was competitive inhibition. Therefore, it was meaningful to assess cellular uptake of FSPH-Ca in the presence of dietary inhibition factors such as oxalate (OA), phytate (PA), tannic acid (TA) and phosphate (PPA) as well as the competitive divalent metal ions Fe2+ (Beyer et al., 2004). As shown in Fig. 4, the inhibition effect of Fe2+ on calcium uptake was significantly attenuated with the treatment of FSPH-Ca, illustrating that FSPH-Ca promoted calcium enter Caco-2 cells primarily via specific pathways rather than DMT1 receptor. As expected, at the same concentration of calcium ions, the calcium uptake efficiency of CaCl2 group seriously decreased by 47.94%, 79.86%, 59.74%, 75.43% versus the control group after the addition of OA, PA, TA and PPA, respectively, while FSPH could prevent calcium ions from precipitating and retain 143.11%, 130.81%, 122.69%, 113.14% of calcium uptake, respectively. Phytate played a stronger role in precipitating calcium ions and decreased the calcium uptake efficiency to 20.14%. FSPH could attenuate the effect and reverse the inhibitory effect by 110.67%. These results suggested that FSPH-Ca could resist the inhibitory effect of dietary inhibitors and improve the cellular uptake of calcium, which may be due to the stronger affinity of FSPH to calcium than OA, PA, TA and PPA. 3.10. FSPH-Ca significantly increased the expression of TRPV6 in Caco-2 cells

Fig. 2. Typical TG-DSC thermograms of (A) FSPH and (B) FSPH-Ca.

It was reported that the transcellular transport of calcium in Caco-2 cells was mainly through the TRPV6 channel. Calcium ions entered into the cells by TRPV6 channel, and were brought to the basolateral membranes by binding to Calbindin D9k and released into the blood by PMCA1b (Diaz de Barboza et al., 2015). Kovacs et al. demonstrated that 2-APB inhibited the activity of TRPV6 but not inhibited TRPV5 activity in HEK293 cells using fluorescence imaging, patch clamp and radioactive tracer techniques (Kovacs et al., 2012). As shown in Fig. 5A, calcium uptake increased 60.43% at FSPH-Ca (composed of 4 mg/mL FSPH and 10 mM CaCl2) versus the blank and then significantly decreased after the addition of 2-APB, which implied that the TRPV6 channel might involve in the process of FSPH-Ca improved calcium uptake in Caco-2 cells. The products of RT-PCR are specific, and efficiency (E) values (Supplementary material Figs. S3–S6) for the four genes were as follows: β-actin 0.924; PMCA1b 1.069; Calbindin D9k, 1.043; TRPV6 1.004. As shown in Fig. 5B, the mRNA level of TRPV6, calbindin D9k and PMCA1b increased significantly after the treatment of FSPH-Ca. This result indicated that FSPH-Ca could regulate the opening and mRNA expression of TRPV6 channel, promoting the calcium entered into the Caco-2 cells. The opening of TRPV6 channel stimulated by FSPH-Ca increased the intracellular calcium. Then calcium diffusion in cytoplasm was driven by combining with calbindin D9k, simple diffusion or vesicle transmission. Finally, calcium ions could be released extracellular by PMCA1b (Fig. 6). Similarly, caseinphosphopeptides (CPPs) was reported to be a kind of mineral carriers with the ability to bind calcium ions and improve calcium absorption by TRPV6 channel (Ferraretto, Gravaghi, Fiorilli, & Tettamanti, 2003; Ferraretto, Signorile, Gravaghi, Fiorilli, & Tettamanti, 2001). CPP could regulate gene expression of TRPV6 channel (Perego, Cosentino, Fiorilli, Tettamanti, & Ferraretto, 2012) and promote the uptake of extracellular calcium in differentiated HT-29 and Caco-2 cells (Cosentino, Gravaghi

shown in the supplementary materials Fig. S2, the concentrations of FSPH and CaCl2 for subsequent experiments were determined according to the results of cytotoxicity test by MTT assay. Cell viability was > 85% after treatment with different FSPH (0–8 mg/mL) or CaCl2 (0–10 mM) concentrations. Fig. 3A exhibited the cellular uptake effect of FSPH-Ca with different FSPH and calcium ratio in Caco-2 cells population model. At the concentrations of 5 and 10 mM Ca2+, the intracellular calcium fluorescence exhibited a dose-dependent increase with the increase FSPH ratios. In the presence of FSPH-Ca with 4 and 8 mg/mL FSPH, the calcium uptake increased 52.36 and 50.60% over that of the control group (10 mM CaCl2), respectively. This result confirmed that the addition of FSPH-Ca significantly improved the calcium uptake in Caco-2 cells population model. For a given calcium concentration, the increase in calcium uptake was almost equal at the presence of 4 or 8 mg/mL FSPH, illustrating that the effect of FSPH on promoting calcium absorption was saturable. The cell fluorescence images exhibited similar changes with calcium uptake studies. As shown in Fig. 3B, the intracellular fluorescence intensity increased with the increasing concentration of FSPH, which indicated that more calcium influx into Caco-2 cells after the treatment of FSPH-Ca. To sum up, FSPH-Ca effectively promoted calcium uptake in Caco-2 cells. Similar results were also published for CPP (Cosentino, Donida et al., 2010), soybean protein hydrolysates (SPH) (Lv, Bao, Yang, Ren, & Guo, 2008) and serum protein hydrolysates (Choi, Lee, Chun, & Song, 2012), which revealed calcium-binding peptide promoted calcium uptake by acting as calcium carriers and interacting with the plasma membrane to increase calcium influx.

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Fig. 3. Effects of FSPH-Ca on calcium uptake. (A) Cellular uptake of CaCl2 and FSPH-Ca. * Statistical significance p < 0.05, compared with FSPH-Ca composed with 1 mg/mL FSPH and 5 mM calcium treated group. # Statistical significance p < 0.05, compared with FSPH-Ca composed with 1 mg/mL FSPH and 10 mM calcium treated group. (B) Intracellular calcium fluorescence images. Caco-2 cells were treated with FSPH-Ca with 10 mM calcium and 0 (a), 2 (b), 4 (c), 8 (d) mg/mL FSPH. Fluorescence analysis was performed by inversion fluorescence microscope using a 10× magnification.

Ethics statements

et al., 2010; Liu et al., 2018).

This study does not involve any the human or animal experiments. 4. Conclusion In summary, fish scales protein hydrolysate (FSPH) with calciumbinding capacity was prepared, and the amino group and the carboxyl group of FSPH were determined to be the primary binding sites for calcium to form a stable complex FSPH-Ca. FSPH-Ca could effectively promote calcium cellular uptake and resist the inhibitory effect of dietary inhibitors. Moreover, FSPH-Ca could upregulate the corresponding mRNA expression including TRPV6, calbindinD9k and PMCA1b, and thus promote the calcium cellular uptake and intracellular transport. Further research is on-going to explore the exact mechanisms of the interaction of FSPH-Ca and TRPV6. The results in this study suggest that FSPH-Ca could be developed as functional foods as calcium supplement.

Acknowledgements This work was supported by National Natural Science Foundation of China (No. 31771922).

Declaration of Competing Interest The authors declare no competing financial interests.

Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.103717. 7

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Fig. 4. Effect of FSPH-Ca on calcium uptake in the presence of dietary factors (ferrous ion (Fe2+), oxalate (OA), phytate (PA), tannic acid (TA) and phosphate (PPA)). Conditions: OA/Ca or PA/Ca or Zn/Ca = 20:1. The concentration of calcium is 10 mM. * Statistical significance p < 0.05, compared with the CaCl2 control group.

Fig. 6. The possible mechanisms of calcium-promoting cellular uptake by FSPH-Ca.

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