Glycosylated peptide-calcium chelate: Characterization, calcium absorption promotion and prebiotic effect

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Food Chemistry 403 (2023) 134335

Available online 21 September 2022

Glycosylated peptide-calcium chelate: Characterization, calcium absorption

promotion and prebiotic effect

Xiaoping Wu

, Fangfang Wang

, Xixi Cai

, Shaoyun Wang

College of Chemical Engineering, Fuzhou University, Fuzhou 350108, China

College of Biological Science and Technology, Fuzhou University, Fuzhou 350108, China

ARTICLE INFO

Keywords:

Glycosylated peptides-calcium chelate

Characterization

Calcium absorption

Prebiotic effect

Gut microbiota

ABSTRACT

Finding functional preparations that could improve the bioavailability of calcium is one of the keys to solving

calcium deciency. In this study, glycosylated peptides-calcium chelate with calcium absorption promoting

activity, named XOS-CSPHs-Ca-MR, was prepared from Crimson Sapper scales protein hydrolysates (CSPHs) and

xylooligosaccharides (XOS) via Maillard reaction. Results showed that amino nitrogen, carboxyl oxygen, and

carbonyl oxygen atom were the primary calcium chelating sites. Remarkably, XOS-CSPHs-Ca-MR exhibited good

calcium phosphate crystallization inhibitory activity, gastrointestinal stability, and could promote calcium

transport efciency in the Caco-2 cell monolayer. In vitro fermentation results showed that XOS-CSPHs-Ca-MR

improved the gut microbiota structure of calcium-decient mice. Its prebiotic effect was achieved by

increasing the number of benecial bacteria, boosting the production of short-chain fatty acids, and improving

the colonization ability of microbiota. Therefore, this study could lay a foundation for the study of glycosylated

peptide-calcium chelate as a novel calcium supplement with prebiotic effect.

1. Introduction

Calcium is the most abundant mineral element in the human body,

accounting for 1.5 %-2.2 % of human body weight (Sun et al., 2020). It is

involved in a variety of physiological functions, such as improving the

metabolic level in cells (Sun et al., 2016), promoting the growth of bone

(Zhao et al., 2014), and maintaining the stability of heart function (Wu

et al., 2019). Previous report showed that more than 95 % of adult

residents in China have insufcient calcium intake (Huang et al., 2021),

which will lead to diseases like osteoporosis (Cai et al., 2015; Sun et al.,

2020), colon cancer (Zhao et al., 2014), rickets (Walters, Esfandi, &

Tsopmo, 2018), and hypertension (Wu et al., 2019). Besides, since

planted-based food is a major component of the Oriental dietary pattern,

phytic acid, oxalic acid, phosphoric acid, and other components in food

are easy to precipitate the ingested calcium in the intestine so that cal-

cium ions cannot be absorbed by the human body, resulting in a further

decline in the bioavailability of calcium (Sun et al., 2016). Therefore, the

key to solving human calcium deciency is to explore functional prep-

arations that could effectively ameliorate calcium bioavailability for

nutritional intervention (Petersen et al., 2009).

Food-derived peptide-calcium chelate is a novel type of calcium

supplement that can interact with the cell membrane, open calcium

channels, and promote calcium absorption (Sun et al., 2016). Recent

studies have shown that peptides with calcium chelating activity

extracted from pig bone collagen (Wu et al., 2019), Snapper sh scales

(Lin et al., 2020), and sheep bone collagen (Wang et al., 2020) can

promote calcium absorption and improve the bioavailability of calcium.

Chen et al. (2017) found that the calcium bioavailability and bone

strength were signicantly increased in calcium-decient mice after

feeding tilapia skin calcium-chelating peptide complex. Zhao et al.

(2014) and Wang et al. (2018) also obtained similar results in the cal-

cium absorption and bone metabolism of collagen peptide-calcium

chelate in calcium-deciency model rats. These results suggest that

peptide-calcium chelate benets calcium absorption and has better

Abbreviations: AP, apical; BL, basal; COS, chitosan oligosaccharide; CPP, casein phosphopeptides; CSPHs, Crimson Snapper scales protein hydrolysates; DAPI, 4′,

6-diamino-2-phenylindole; DMEM, dulbecco’s modied eagle medium; FBS, fetal bovine serum; FISH, uorescence in situ hybridization; FSPH-Ca, sh scale protein

hydrolysate-calcium complex; FTIR, Fourier transform infrared spectroscopy; HBSS, D-hanks’ balanced salt solution; MRS, de Man, Rogosa, and Sharpe; OPA, ortho-

phthalaldehyde; SCFAs, short-chain fatty acids; TEER, transmembrane electrical resistance; XOS, xylooligosaccharides; XRD, X-ray diffraction; [Ca

]

, intracellular

calcium concentration.

* Corresponding authors.

E-mail addresses: caixx@fzu.edu.cn (X. Cai), shywang@fzu.edu.cn (S. Wang).

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

https://doi.org/10.1016/j.foodchem.2022.134335

Received 14 July 2022; Received in revised form 3 September 2022; Accepted 16 September 2022

Food Chemistry 403 (2023) 134335

bioavailability.

Prebiotics, such as indigestible oligosaccharides, are not digested

when passing through the upper digestive tract but are fermented by the

gut microbiota and can selectively promote the metabolism and prolif-

eration of benecial bacteria in the body without stimulating harmful

bacteria with potentially pathogenic or putrid activity, thus improving

the health of the host (Chalise et al., 2019). In addition to its prebiotic

effect, it also plays an active role in promoting calcium absorption in the

human body. Preliminary studies have found that the gut microbiota can

enable the fermentation of prebiotics to produce short-chain fatty acids

(SCFAs), thus reducing the intestinal pH value and improving the sol-

ubility of minerals (Bruno-Barcena & Azcarate-Peril, 2015). SCFAs can

also stimulate cell proliferation, increase the surface area of intestinal

epithelial cells, and improve calcium absorption capacity (Mineo, Hara,

& Tomita, 2001). Moreover, SCFAs can regulate human calcium ab-

sorption through mitogen-activated protein kinase, heat shock protein

27 pathway, and increase the expression of the calcium-binding protein

(Wu et al., 2017). Therefore, the effect of prebiotics on intestinal cal-

cium absorption provides a new way to alleviate the problem of calcium

deciency.

Biological polymers such as proteins/peptides and saccharides have

been used to assemble protective sustained-release carriers to delay the

reaction rate or release rate of various bioactive compounds in vivo,

which has attracted extensive attention in recent years (Liu et al., 2017).

Previous studies have shown that casein phosphopeptides (CPP) could

produce a Maillard reaction with soluble dietary ber, which could

markedly increase the content of soluble calcium in the gastrointestinal

tract, and the delivery system could delay the degradation rate of CPP-

Ca in the gastric environment, so as to ameliorate the calcium

bioavailability (Gao et al., 2018a). Similarly, Zhao et al. (2020) reported

that the Maillard reaction product obtained from the hydrolysate of

desalted duck egg white peptide had a high calcium-binding capacity,

which effectively reversed the inhibitory effect of phytic acid on calcium

transport Caco-2 cell monolayer. Hence, we wonder whether the

prebiotic-modied peptide-calcium chelates could play a prebiotic role

in regulating the structure of gut microbiota when they play a stable role

in promoting calcium absorption.

Given this, glycosylated peptide-calcium chelate with excellent cal-

cium chelating capacity was prepared from the Crimson Sapper (Lutja-

nus erythropterus) scales protein hydrolysates (CSPHs) and

xylooligosaccharides (XOS) via Maillard reaction. Its chelating proper-

ties were analyzed by structure and properties characterization. More-

over, the Caco-2 cells model was established to explore the calcium

absorption promoting activity of glycosylated peptide-calcium chelates.

Finally, the effect of glycosylated peptide-calcium chelate on the struc-

ture of gut microbiota of calcium-decient mice was explored by a

prebiotic effect experiment in vitro. This work provides a theoretical

basis for glycosylated peptide-calcium chelates as a new calcium sup-

plement reagent.

2. Materials and methods

2.1. Materials

Crimson snapper scales were provided by Putian Haiyibai Co., ltd.

(Fujian, China). Xylooligosaccharides (XOS) with a purity of 95.0 %

were products of Shanghai Yuanye Biological Technology Co., ltd

(Shanghai, China). Bidobacterium adolescentis (GDMCC1.278), Lacto-

bacillus acidophilus (GDMCC1.208), Escherichia coli (GDMCC1.737), and

Salmonella (GDMCC1.237) were purchased from Guangdong microbial

strain Preservation Center (Guangzhou, China). Fluo-3AM was the

product of Beyotime Biotechnology Co., ltd. (Shanghai, China). All other

chemicals and reagents were of analytical grade and commercially

available.

2.2. Preparation of XOS-CSPHs-MR

The Crimson Snapper scales were immersed in deionized water (4 %,

m/V) and autoclaved for 60 min. After the pH value of the mixture was

adjusted to 6.30, the avourzyme (E:S =1:25) was added. After

hydrolysed at 50 ◦C for 9 h, the hydrolysate was positioned in boiling

water to inactivate the enzyme for 10 min. The hydrolysis degree was

18.21 %, which is calculated by formaldehyde titration (Li, Liu, & Xue,

2012). Subsequently, the mixture was centrifuged at 9,615 g for 20 min,

the supernatant was freeze-dried, and the resulting powder was Crimson

Snapper scales protein hydrolysates (CSPHs).

XOS and CSPHs powders were dissolved in deionized water with a

total concentration of 50 mg/mL, wherein the mass ratio of XOS to CSPH

was 1:0.80. Then, the pH of the solution was adjusted to 9.00 and held at

90 ◦C water bath for 2.50 h. After that, the solution was cooled to room

temperature, put into a dialysis bag (molecular weight cut off 500–1000

Da), and dialyzed at 20 ◦C for 24 h (Zhao et al., 2014). Finally, the

resulting products were lyophilized. The grafting degree of the obtained

powder was 50.29 %, calculated by the ortho-phthalaldehyde (OPA)

method (Pirestani et al., 2017) and named XOS-CSPHs-MR.

2.3. Preparation of XOS-CSPHs-Ca-MR

XOS-CSPHs-MR was dissolved in deionized water (pH 7.50) to make

a concentration of 20 mg/mL. Then solid CaCl

was added to fetch a

samples/CaCl

mixture with a mass ratio of 5:1. Subsequently, the

mixture was kept at 30 ◦C for 20 min. Anhydrous ethanol was added to

the mixture, and then the mixture was centrifuged at 9,615 g for 20 min

to obtain chelates. After the precipitate was lyophilized, the calcium-

binding rate of the obtained powder was determined to be 89.68 %,

according to the method of Zhang, Lin, & Wang (2018), and it was

designated XOS-CSPHs-Ca-MR.

2.4. Structural characterization

2.4.1. UV–vis spectroscopy analysis

XOS-CSPHs-MR and XOS-CSPHs-Ca-MR in deionized water (pH

7.00) were analyzed according to the method of Cai et al. (2015) using a

UV–vis spectrophotometer (eu2600, METTLER TOLEDO). The concen-

tration of the test samples was 100

g/mL. The UV–vis spectrum was

scanned in the wavelength range of 190–400 nm, and each sample was

carried out a triple.

2.4.2. Fluorescence spectroscopy analysis

The changes in intrinsic uorescence of XOS-CSPHs-MR and XOS-

CSPHs-Ca-MR were analyzed according to the method of Cai et al.

(2015) by a uorescence spectrometer (Fluoromax-4c-l, Horiba instru-

ment Inc, Piscataway, New Jersey, USA). XOS-CSPHs-MR and XOS-

CSPHs-Ca-MR were dissolved to 100

g/mL with deionized water (pH

7.00). The uorescence spectra were measured at 290–500 nm emission

wavelength and 280 nm excitation wavelength, and the slit was 5 nm.

2.4.3. Fourier transform infrared (FTIR) spectroscopy

The XOS-CSPHs-MR and XOS-CSPHs-Ca-MR powder (1 mg) was

mixed with dry KBr (100 mg) and then ground into a uniform powder

with an agate mortar. Then, the infrared absorption spectrum was

determined according to the method of Lin et al. (2015) using an

infrared spectrophotometer (Thermo Nicolet Co., USA). The scanning

conditions were set as follows: the spectral range was 4000–400 cm

−1

the resolution was 4 cm

−1

, and the scanning number was 64.

2.4.4. X-ray diffraction (XRD)

The crystal states of CaCl

, XOS-CSPHs-MR, and XOS-CSPHs-Ca-MR

were analyzed according to the method of Feng et al. (2022) by an

X’pert3 and Empyrean diffractometer (Panalytical, Almelo,

Netherlands). Samples were swept continuously over a 2θ range of 2–75

X. Wu et al.

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