Effects of phosvitin phosphopeptide-Ca complex prepared by effi cient enzymatic hydrolysis on calcium absorption and bone deposition of mice

3.0 科研～小助 2025-09-01 4 4 895.8KB 10 页 1知币

侵权投诉

M.D. Zhao et al. / Food Science and Human Wellness 11 (2022) 1631-1640

1631

Effects of phosvitin phosphopeptide-Ca complex prepared by efﬁ cient

enzymatic hydrolysis on calcium absorption and bone deposition of mice

Mengdie Zhaoa, Dong Uk Ahnb, Songming Lia, Wei Liua, Shengwei Yic, Xi Huanga,*

a National Research and Development Center for Egg Processing, College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, China

b Animal Science Department, Iowa State University, Ames 50011, USA

c Chongqing Institute for Food and Drug Control, Chongqing 401121, China

A B S T R A C T

Phosvitin (PV) was treated with high-temperature, mild pressure (HTMP), and enzyme combination, and then

phosvitin phosphopeptides-calcium (PPP-Ca) complexes were prepared. The low-calcium speciﬁ c pathogen

free-Kunming (SPF-KM) mice were used to determine the effect of PPP-Ca complexes on intestinal calcium

absorption and their utilization for bone formation. The serum calcium content was the highest with the

HTMP-Enz-PPP-Ca treatment (2.19 mmol/L), and it significantly down-regulated the abnormal elevation

of serum alkaline phosphatase (AKP) caused by calcium deficiency. The low-calcium control group had

the lowest calcium deposited to the femur (80.41 mg/g) and the lowest femur bone mineral density (BMD)

(0.17 g/cm3), while HTMP-Enz-PPP-Ca significantly improved bone calcium content (94.33 mg/g) and

BMD (0.29 g/cm3). The micro-computed tomography (MCT) images showed that the femur with the

normal control, PV-Ca, and HTMP-Enz-PPP-Ca treatments had a more compact, complete, and thicker

trabecular network than the low-calcium and CaCl2 treatments. These results indicated that the organic

calcium (HTMP-Enz-PPP-Ca) promoted calcium absorption and bone deposition, and the effect of

HTMP-Enz-PPP-Ca was better than the inorganic CaCl2.

Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license

(http://creativecommons.org/licenses/by-nc-nd/4.0/).

http://doi.org/10.1016/j.fshw.2022.06.022

BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

A R T I C L E I N F O

Article history:

Received 11 July 2021

Received in revised form 12 September 2021

Accepted 5 January 2022

Available Online 1 July 2022

Keywords:

Phosvitin phosphopeptide (PPP)-Ca complex

SPF KM mice

Calcium absorption

Bone formation

* Corresponding author at: College of Food Science and Technology, Huazhong Agricultural

University, Wuhan 430070, China.

E-mail address: huangxi@mail.hzau.edu.cn (X. Huang)

Peer review under responsibility of KeAi Communications Co., Ltd.

Publishing services by Elsevier

1. Introduction

Calcium is the major divalent cation in the human body and

accounts for about 1.5%-2.2% of total body weight [1]. The

majority (99%) of calcium in the body is deposited in bones and

teeth. At the same time, calcium also exists in an ionic form in the

soft tissues, extracellular fluid, and blood, which acts not only as

an intracellular messenger for muscle contraction and relaxation,

neurotransmission, immune response, and cell proliferation but also

maintain a dynamic balance between the serum and bone calcium

content [2]. Thus, calcium deficiency can lead to metabolic bone

diseases such as rickets and osteoporosis.

Dietary intake is the only way to supplement body calcium and

is absorbed in the small intestine through the voltage-gated (active)

transcellular and the passive paracellular pathways [3]. The active

transcellular pathway is known as the main transport pathway in

the duodenum that requires the combined actions of three calcium

transport proteins: 1) the regulation of calcium influx by calcium

transport proteins (mainly transient receptor potential vanilloid type 6,

TRPV6) [4]; 2) calcium transfer mainly by the calcium-binding

proteins (calbindin-D9k) [5]; and 3) the extrusion of calcium into the

blood by the plasma membrane calcium ATPase1b (PMCA1b) [6]. In

the paracellular transport, ionized calcium diffuses through the tight

junctions into the basolateral spaces of the enterocytes and the blood.

Paracellular calcium absorption mainly occurs in the jejunum and

ileum, especially when the dietary calcium levels are high [7].

Food Science and Human Wellness

Contents lists available at ScienceDirect

journal homepage: http://www.keaipublishing.com/en/journals/food-science-and-human-wellness

1632

M.D. Zhao et al. / Food Science and Human Wellness 11 (2022) 1631-1640

Because calcium is only absorbed in its ionic form, it should

be solubilized or released from its sources. However, some of the

solubilized calcium can form insoluble complexes with minerals

or other dietary constituents such as oxalic acid and phytate in the

alkaline pH of the small intestine, resulting in inadequate calcium

absorption and utilization [8]. Recent research showed that casein

phosphopeptides (CPPs) could bind calcium ions to form soluble

peptide-calcium complexes, promote calcium absorption, and

improve calcium accumulation in bones [9]. The CPPs produced

from caseinpromoted calcium uptake in the Caco-2 cells by

up-regulating the expression of TRPV6, a key calcium-transport protein

in the duodenum, and increased serum Ca2+ levels, femur length,

and femur calcium in a Sprague-Dawley rat model by up-regulating

the expression of TRPV6 [10]. Zhang et al. [11] reported that the

phosphorylation of functional proteins or polypeptides is vital because

the phosphate group’s multiple negative charges play an essential role

in the binding of divalent metal ions.

Phosvitin (PV), a natural phosphoprotein in egg yolk, is the

most phosphorylated protein in nature. PV accounts for 8%–11%

of the egg yolk protein and consists of 217 amino acids, of which

128 amino acid residues (123 serines, 4 threonines, and 1 tyrosine)

can be phosphorylated [12,13]. Almost all the serine and threonine

residues in PV are phosphorylated, and many groups of serine

residues are arranged in clusters of 15 consecutive residues [13].

Although all the commercial phosphopeptides are currently prepared

using casein, PV has a much higher phosphorylation level and

is a much better source for the preparation of phosphopeptide.

However, the preparation of phosvitin phosphopeptides (PPP) using

PV is exceptionally challenging because PV has extreme negative

charges that block the access of proteases to the cleavage site [13].

Recently, our group developed a high temperature and mild pressure

(HTMP, 121 °C at 0.1 MPa) pretreatment to improve the enzymatic

hydrolysis of PV without losing phosphate groups in the PV. The

result showed that HTMP pretreatment alone produced 310 peptides,

but the HTMP and subsequent enzyme treatments further improved

PV hydrolysis and produced up to 605 phosphopeptides from

PV [12]. Because PPP would have similar structural characteristics

to the CPPs, it is assumed that the PPP also would promote calcium

absorption in animals.

The objectives of this study were to determine the effects of the

dietary PPP-Ca complex on intestinal calcium absorption and to

elucidate the mechanisms involved in the use of absorbed calcium in

bone formation using a low-calcium speciﬁc pathogen free-Kunming

(SPF-KM) mice model.

2. Materials and methods

2.1 Materials

PV was prepared using the method of Lee et al. [14]. A standard

feed for mice (AIN93) was purchased from Trophic Animal

Feed High-tech Co., Ltd. (Jiangsu, China), trypsin (E.C.3.4.4.4,

15 500 U/mg protein) and thermolysin from Bacillus thermoproteolyticus

rokko (Thermoase PC10F, E.C. 3.4.24.27, 113 U/mg protein) were

obtained from American Enzyme Co., Ltd. (Elgin, IL, USA), and

serum calcium, phosphorus, and alkaline phosphatase (AKP) kit were

from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

2.2 Preparation of PPP from phosvitin

The PPP were prepared following the method of Huang et al. [12]

with some modifications. The temperature, pressure, and time

conditions used for the HPMP pretreatment were 121 °C at

0.1 MPa for 30 min, and then HTMP pretreated PV was further

hydrolyzed using trypsin and thermolysin successively to prepare

HTMP-Enz-PPP. The same enzyme:substrate ratio (1:50, m/m) and

incubation time (8 h) were used for both enzymes. The hydrolysis

conditions for trypsin were pH 8.0 and 37 °C incubation temperature,

and those of the thermolysin were pH 8.0 and 68 °C incubation

temperature. The hydrolysis with the second enzyme (thermolysin)

was performed after inactivating the first enzyme (trypsin) at the

end of the 8 h incubation. Enzymatic digestion was arrested for both

enzymes by keeping the sample in a boiling water bath for 10 min.

The hydrolyzed solutions were lyophilized and stored in a -20 °C

freezer until use.

2.3 Preparation and characterization of PPP-Ca complexes

The PPP-Ca complexes were prepared from the HTMP-Enz-PPP

combinations. The HTMP-Enz-PPP were dissolved in deionized

water (10 mg/mL), and then CaCl2 was added with different mass

ratios (PPP:CaCl2 = 5:1–10:1). The calcium-binding rate was used to

screen the peptide calcium ratio. The factors such as pH, temperature, and

time in the chelating reaction process were obtained by a single factor

experiment with calcium-binding rate as an index (specific data are

not presented). The ﬁnal chelation method was that HTMP-Enz-PPP

and CaCl2 (at ratio of 7:1) were mixed with distilled water, first.

Then the pH of mixture was adjusted to 9.5 and it was incubated for

60 min at room temperature for calcium-binding reactions. The

absolute ethanol (9 volumes of the PPP solution) was added to the

solution after chelation, held for 3 h at room temperature to precipitate

the PPP-Ca complexes, and then centrifuged at 7 100 × g for

15 min at 4 °C. The precipitant was collected, lyophilized, and

marked as HTMP-Enz-PPP-Ca complex. The natural PV was also

used to prepare the PV-Ca complex using the same method as the

HTMP-Enz-PPP.

The degree of hydrolysis (DH) and calcium-binding rate of

PPP (HTMP-PV, HTMP-Trypsin-PPP, HTMP-Thermolysin-PPP,

HTMP-Trypsin+Thermolysin-PPP) were determined using the ninhydrin

method and the atomic absorption spectrometry [15]. Zeta potential was

used to characterize the solution’s stability and pure charge of proteins

and peptides [16]. The FTIR and ﬂuorescence spectrometry was used to

characterize the structure of PPP-Ca complexes.

2.4 Stability of PPP-Ca (HTMP-Enz-PPP-Ca) complexes

The gastrointestinal stability of the PPP-Ca complex was divided

into two stages: the PPP-Ca complex was added to simulated gastric

fluid (CZ0212, Leagene Biotechnology) and then incubated for

6 h in a water bath at 37 °C. During the incubation, an aliquot of

samples was taken out at 0.5, 1.0, 2.0, 4.0, and 6.0 h of incubation,

heated at 100 °C for 10 min to inactive pepsin, and then centrifuged.

The contents of calcium ions in the supernatants were analyzed to

determine calcium release from the PPP-Ca complexes under acidic

stomach conditions. The remaining samples at each incubation time

文档加载中……请稍候！
如果长时间未打开，您也可以点击刷新试试。

下载文档到电脑，查找使用更方便

1 知币 4人已下载

立即下载

Effects of phosvitin phosphopeptide-Ca complex prepared by effi cient enzymatic hydrolysis on calcium absorption and bone deposition of mice.pdf

共10页,预览3页

还剩页未读，继续阅读

Effects of phosvitin phosphopeptide-Ca complex prepared by effi cient enzymatic hydrolysis on calcium absorption and bone deposition of mice

相关推荐

开通VIP享超值会员特权

作者详情

相关内容

推荐作者

热门标签

举报选择: