Protection of osteogenic peptides in nanoliposomes: Stability, sustained release, bioaccessibility and influence on bioactive properties

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Food Chemistry 436 (2024) 137683

Available online 7 October 2023

Protection of osteogenic peptides in nanoliposomes: Stability, sustained

release, bioaccessibility and inuence on bioactive properties

Dongyang Zhu , Wuchao Ma , Meilian Yang , Shuzhen Cheng , Ling Zhang, Ming Du

School of Food Science and Technology, National Engineering Research Center of Seafood, Dalian Polytechnic University, Dalian 116034, Liaoning, China

ARTICLE INFO

Keywords:

Encapsulation

In vitro release

Nanoliposomes

Osteogenic peptides (atlantic cod)

Simulated digestion

ABSTRACT

This study prepared osteogenic peptides (OPs) from cod meat by hydrolysis and subsequently encapsulated them

in nanoliposomes (NLP) to enhance bioaccessibility. The characterization, stability, controlled release behavior

and bioactivity of OPs-loaded nanoliposomes (OPs-NLP) were investigated as well. The highest loading capacity

(27.32%) was achieved in NLP loaded with 6 mg/mL of OPs. The particle size, zeta potential, and encapsulation

efciency of OPs-NLP were 70.59 nm, −11.98 mV, and 75.24%, respectively. The interaction between OPs and

empty NLP was through hydrogen bonding and hydrophobic. The OPs-NLP showed the greatest stability during

storage at 4 ◦C. The in vitro release prole of OPs from OPs-NLP tted a one-level kinetic model best. The

osteogenic activity of OPs was unaffected by NLP encapsulation, and the bioaccessibility of OPs was notably

improved. These ndings suggest that OPs-NLP has the potential to be used in functional foods.

1. Introduction

The global commercial interest in functional foods is experiencing a

signicant upswing. Functional foods contain either fortifying in-

gredients, or micronutrients and natural chemicals are benecial for

health improvement and disease prevention (Domínguez Díaz, Fern´

an-

dez-Ruiz, & C´

amara, 2020). Bioactive peptides are popular ingredients

in current functional foods due to their potential to enhance physio-

logical functions and even reduce disease risks. Bioactive peptides are

reported to have antihypertensive (Ucak et al., 2021), antioxidant

(Jamnik et al., 2017; Mohammadi, Soltanzadeh, Ebrahimi, & Hamish-

ehkar, 2022), antimicrobial (Ucak et al., 2021), osteogenic (Yang et al.,

2022) activities and so on. The effectiveness of peptides hinges largely

on their capacity to maintain their activity and structural integrity until

reaching their intended target sites. However, bioactive peptides are

susceptible to enzymatic breakdown in the oral cavity and gastrointes-

tinal tract prior to entering the systemic circulation, resulting in

decreased utility (Liu, Guo, & Ma, 2022). Furthermore, several other

challenges concerning bioactive peptides need to be resolved when

being incorporated into food products, such as bitter taste, inherent

instability and interactions with the food matrix (Mor, Battula, Swar-

nalatha, Pushpadass, Naik, & Franklin, 2021). Encapsulating peptides

within carrier systems therefore becomes imperative to enhance their

stability and bioavailability.

In the food industry, the utilization of carrier matrices prominently

employed are proteins, polysaccharides, and lipids. Proteins, however,

possess limitations in acting as carriers in the encapsulation of bioactive

peptides, due to the structural heterogeneity of the encapsulated peptide

mixtures, encapsulation mechanisms involving hydrophilic or hydro-

phobic interactions appear to be difcult to realize directly using protein

carriers (Fathi, Martín, & McClements, 2014). Polysaccharides are

emerging delivery agents owing to their structural robustness, abun-

dance, and low cost. Nonetheless, extreme conditions, such as high

temperatures, can trigger interactions between polysaccharides and

peptides, resulting in intricate compounds that diminish peptide

bioactivity (Fathi et al., 2014). In contrast, lipid-based carriers, espe-

cially nanoliposomes (NLP), have gained signicant research attention

due to their high safety and non-immunological reactivity (Mazloum-

Ravasan et al., 2022). NLP is characterized by enclosing a specic vol-

ume of water within the phospholipid bilayer. This versatile structure

Abbreviations: OPs, osteogenic peptides; NLP, nanoliposomes; ENLP, empty nanoliposomes; OPs-NLP, OPs-loaded nanoliposomes; PBS, phosphate buffered saline;

EE, encapsulation efciency; LC, loading capacity; PDI, polydispersity Index; XRD, X-ray diffraction; FTIR, Fourier transform infrared; RP-HPLC, reverse-phase high-

performance liquid chromatography; TEM, transmission electron microscopy; Cryo-SEM, Cyro-scanning electron microscope; MTT, 3-(4,5-dimethyl-2-thiazolyl, -2,5-

diphenyl-2H-tetrazolium bromide; ALP, alkaline phosphatase.

* Corresponding author.

E-mail address: duming@dlpu.edu.cn (M. Du).

Contents lists available at ScienceDirect

Food Chemistry

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

https://doi.org/10.1016/j.foodchem.2023.137683

Received 9 May 2023; Received in revised form 20 September 2023; Accepted 4 October 2023

Food Chemistry 436 (2024) 137683

enables the encapsulation of both lipophilic and hydrophilic substances,

guaranteeing NLP a widely explored carrier (Ajeeshkumar, Aneesh,

Raju, Suseela, Ravishankar, & Benjakul, 2021).

NLP is a promising material for food packaging (Asdagh et al., 2020;

Mehdizadeh, Shahidi, Shariatifar, Shiran, & Ghorbani-HasanSaraei,

2022) and preservation (Abdollahzadeh, Elhamirad, Shariatifar, Saei-

diasl, & Armin, 2023; Homayounpour, Jalali, Shariatifar, Amanlou, &

khanjari, 2020). It is also used in functional foods to enhance nutrient

solubility, delay release (Ebrahimi, Hamishehkar, & Amjadi, 2023) and

maintain substance activity (Amjadi et al., 2021). The encapsulation of

peptides from various sources within NLP has been documented, such as

peptides from salmon (Li, Paulson, & Gill, 2015), Spirulina platensis

(Ebrahimi, Reza Farahpour, Amjadi, Mohammadi, & Hamishehkar,

2023), milk whey (Mohan, McClements, & Udenigwe, 2016), rainbow

trout (Ramezanzade, Hosseini, & Nikkhah, 2017), oyster (Xu, Jiang, Liu,

Zhao, & Zeng, 2021), axseed (Sarabandi & Jafari, 2020), and casein

(Sarabandi, Mahoonak, Hamishehkar, Ghorbani, & Jafari, 2019). These

studies explored the physicochemical properties, morphology, stability,

release kinetics, and effects on activity of peptide-loaded NLPs. The

investigated functionality of NLP-encased peptides focuses mainly on

their antioxidant activity (Sarabandi et al., 2019), wound healing ac-

tivity, etc. (Ebrahimi, Reza Farahpour, et al., 2023). Nevertheless,

limited research is available on assessing the osteogenic activity of NLP-

encapsulated peptides from cod (Gadus morhua).

Atlantic cod (Gadus morhua) is the world’s most important and

consumed commercial sh. Our previous study found that cod peptides

had osteogenic activity (Yang et al., 2022), but digestion of the peptides

affected the activity. As a result, the study intended to investigate OPs

encapsulation in NLP and evaluate the resultant vesicle properties, in

vitro release, stability, bioaccessibility and bioactivity. This research

could provide a promising method to increase the bioaccessibility and

physiological activity of OPs to pave the way for its application in

functional foods.

2. Materials and methods

2.1. Materials and chemicals

Atlantic cod (Gadus morhua) was purchased from a Dalian local

market, Liaoning Province, China. Alkaline protease (2.4 AU-A/g) was

purchased from Novozymes (Bagsvaerd, Denmark). Lecithin, choles-

terol, and Tween-80 were purchased from Shanghai Aladdin Biochem-

ical Science and Technology Co. (Shanghai, China). PBS (phosphate

buffered saline) and MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-

2H-tetrazolium tetrazolium bromide) was purchased from Beijing

Solarbio Bio-technology Co. (Beijing, China). FBS (fetal bovine serum)

was purchased from PAN-Biotech (Adenbach, Germany).

-MEM (

modied eagle medium) was purchased from Gibco, Thermo Fisher

Scientic Co. Ltd., (Massachusetts, USA) Penicillin-streptomycin solu-

tion was purchased from VivaCell, Shanghai Longtian Biotech Co.

(Shanghai, China). Articial gastric uid (phosphate, pancreatic en-

zymes) and articial intestinal uid (sodium chloride, dilute acid,

pepsin) were purchased from Shanghai Yuan Ye, (Shanghai, China).

Methanol and chloroform were purchased from Damao Chemical Re-

agent Factory, (Tianjin, China). Acetonitrile and triuoroacetic acid

were purchased from Sigma-Aldrich Trading Co. Ltd (Shanghai, China).

The alkaline phosphatase (ALP) assay kit and ALP coloring kit were

purchased from Shanghai Beyotime Biotech Co. (Shanghai, China).

2.2. Preparation of OPs

The cod skin was removed from the thawed boneless cod. Then, it

was rinsed with tap water to remove contaminants and cut into pieces.

Subsequently, 200 g of cod meat was blended with 800 g of deionized

water and homogenized, followed by enzymatic digestion (0.03%

alkaline protease) at pH 8/55 ◦C for 2 h. The resultant hydrolysates were

boiled at 100 ◦C for approximately 10 min to deactivate the enzyme and

lyophilized. The obtained powder was redissolved in deionized water

and ltered to harvest peptides with molecular weight ranging from 200

Da to 10 KDa. The residual liquid was freeze-dried to yield the nal OPs

product.

2.3. Characterization of OPs

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-

PAGE) was conducted following established procedures (Xu et al.,

2021). OPs were diluted with loading buffer to achieve a nal protein

concentration of 2 mg/mL and subjected to heating at 100 ◦C for 10 min.

The gel was subsequently stained for 2 h, followed by overnight

destaining. The gel was scanned using a bioimaging system (Bio-Rad,

USA).

According to the method of Xu et al. (2021), size exclusion chro-

matography was employed to analyze the molecular weight distribution

of OPs. A high-performance liquid chromatography (HPLC) system

(Agilent 1260 Innity, Agilent, USA) with a TSK gel G2000SWXL col-

umn (Tosoh Corp., Tokyo, Japan) was used. Prior to analysis, OPs were

ltered through a 220 nm lter. The elution was carried out using 45%

(v/v) acetonitrile in water containing 0.1% triuoroacetic acid at a ow

rate of 0.5 mL/min, under monitoring conducted at 214 nm.

2.4. Preparation of OPs-NLP

Lecithin (90 mg), cholesterol (10 mg) and Tween-80 (20 mg) were

dissolved in ethanol (10 mL) for 20 min and evaporated in a round-

bottom ask using a rotary evaporator (SY-2000, Shanghai Yarong

Biochemical Instrument Factory, China) at 60 ◦C with an agitation rate

of 70 rpm until a thin-lm coating was formed. The ask was placed in a

desiccator for 16 h to complete the evaporation of residual solvent. 10

mL of peptide solutions in different concentrations were added to the

lm to form liposomes via hydrating. The suspension was vortexed

(VORTEX3, IKA, German) at 60 ◦C and ambient temperature for 2 min

respectively. The above process was repeated three times, followed by

sonication using an ultrasonic cell pulverizer (SCIENT2-IID, SCIENTZ,

China) at a frequency of 300 W to form OPs-NLP.

2.5. Characterization of OPs-NLP

2.5.1. Average size, polydispersity index (PDI), and zeta potential

The average size and PDI were determined using a dynamic light

scattering instrument (LS Instruments, Switzerland). The zeta potential

was determined using the Litesizer 500. Measurements were taken when

the transmittance of nanoliposome suspensions was higher than 85%.

2.5.2. Encapsulation efciency (EE) and loading capacity (LC)

The experiments were carried out according to the reported methods

(Du et al., 2020; Sarabandi et al., 2020). The NLP loaded with 1 mg/mL

peptide were transferred to an ultra-centrifugal lter membrane (mo-

lecular weight cutoff =10 kDa, Millipore, USA) and centrifuged at 4000

r/min for 10 min. The concentration of unembedded OPs was calculated

from a calibration curve at the absorbance value of 220 nm using a

UV–Vis spectrophotometer. The EE and LC of OPs-NLP were calculated

according to the following equations:

EE (%) = Amount of encapsulated OPs (mg)

Amount of total OPs (mg)∗100

LC (%) = Amount of encapsulated OPs (mg)

Amount of total nanoliposomes (mg)∗100

2.5.3. Fluorescence spectroscopy

The approach reported by Yang et al. (2022) was used herein with

slight modications. The peptide concentration of both OPs and OPs-

D. Zhu et al.

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