Preparation, characterization and stability of nanoliposomes loaded with peptides from defatted walnut (Juglans regia L.) meal

Abstract

This study aimed to encapsulate walnut peptides with different molecular weights (crude peptides, 5–10 kDa and < 5 kDa) within nanoliposomes. The peptides with molecular weight (MW) of 5–10 kDa (F2) was chosen as a representative sample to indicate the formation mechanism of nanoliposomes using scanning electron microscopy (SEM) and transmission electron microscope (TEM). The storage and simulated digestion experiment were carried out to evaluate the protective effect of nanoliposomes loading walnut peptides. Our results indicated that the amino acid composition was affected by peptide MW, and F2 exhibited the highest content of hydrophobic amino acids content. The MW of peptides also affected the distribution of the peptide of nanoliposomes, resulting in changes in particle size, ζ-potential, and encapsulation efficiency. The SEM exhibited that a high concentration of nanoliposomes might result in phospholipid fusion and larger particle diameters. The TEM showed individual nanoliposomes had spherical, smooth and full vesicle structures. The nanoliposomes could improve the stability of walnut peptides during storage. The maximum peptides retention after in vitro digestion was 61.6%, indicating a better sustained release in gastric digestion. The present study suggested that nanoliposomes can offer adequate protection to the walnut peptides during storage and digestion.

Introduction

Walnut (Juglans regia L.) is the most widespread nut and economically cultivated throughout Eastern Asia, Southern Europe, Western South and Northern Africa (Li et al. 2020). China is a major planting country of walnut and has highly abundant germplasm resources. In 2019, the walnut production in China was almost 2.52 million metric tons (FAOSTAT 2019). Walnut can be administered as food ingredients or consumed directly or oil. However, after extracting oil, the defatted walnut meal providing considerable amounts of proteins (61–66%) is often used as animal feed due to the lack of processing technology (Mao and Hua 2014). Greater attentions have been paid to the peptides derived from defatted walnut meal due to its healthy and functional components like anti-inflammatory, antihypertensive, antimicrobial and antioxidant activities (Wang et al. 2021; Li et al. 2020). Fang et al. (2020) prepared walnut peptides and chelated them with calcium ions, maintaining antioxidant activity of peptides after gastrointestinal digestion. Researcher has identified novel ACE inhibitory peptides sequence YVPH-WNL from defatted walnut meal protein hydrolysates (Wang et al. 2015). Jin et al. (2020) found that the limited trypsin hydrolysis could enhance the functional properties of dehulled walnut protein. However, the degradation of peptides occurred during storage and in vitro digestion, limiting its application in food processing (Wang et al. 2021). Thus, it is essential to prepare a carrier system with high delivery and absorption capabilities, which could enhance the physicochemical stability and bioavailability of walnut peptides.

Nanoliposomes are closed spherical vesicles formed by phospholipid and cholesterol with particle sizes ranging from 10 to several hundred nm (Liu et al. 2020). They can serve as nanocarriers to encapsulate and deliver hydrophilic, hydrophobic and amphipathic ingredients (Sarabandi et al. 2019b). Many studies have reported that the encapsulation of peptides in nanoliposomes could enhance stability, efficacy and bioactivity (Sarabandi et al. 2019b; Ramezanzade et al. 2017; Mosquera et al. 2014). Wang et al. (2021) reported that walnut peptides maintained their antibacterial and antioxidant activity after being loaded into proliposomes. Mazloomi et al. (2020) found that nanoliposomes could maintain the stability of orange seed protein hydrolysates after storage at 4 °C for 28 days.

To the best of our knowledge, the encapsulation of walnut peptides with different MW into nanoliposomes has not been explored. Besides, the effect of walnut peptides MW on the characteristics of nanoliposomes such as ζ-potential, EE has not been examined, which is the gap that our research could help to fill, therefore to improve the availability of walnut peptides. The main objective of this study is to understand the protective effect of nanoliposomes on walnut peptides. The characteristics and structural properties of loaded nanoliposomes are evaluated using a particle size analyzer, microscope, scanning electron microscope (SEM) and transmission electron microscope (TEM). In addition, the effect of storage conditions on the nanoliposomes’ stability and the release rate of peptides from nanoliposomes during in vitro digestion are determined. This work aims to provide a scientific basis for enhancing the walnut peptide’s stability during storage and in vitro digestion, and this work might be helpful for practical application in the food industry.

Materials and methods

Materials

Commercial walnuts (Juglans regia L.) were purchased from the Walnut Industry Base (Huize, Yunnan, China). Alcalase (≥ 2 × 10⁵ units/g), cellulase (≥ 3 × 10⁵ units/g), pectinase (≥ 3 × 10⁵ units/g) and porcine bile salt were purchased from Hebei Hongtao Biological Engineering Co, Ltd. (Handan, Hebei, China). α-Amylase (≥ 3.7 × 10⁴ units/g), pepsin (≥ 3.0 × 10⁶ units/g) and pancreatin (≥ 2.5 × 10⁵ units/g) were purchased from Beijing Solarbio Science & Technology Co, Ltd. (Beijing, China). Trichloromethane, petroleum ether, Tween 80, lecithin (phosphatidylcholine > 90%) and cholesterol (analytical grade) were purchased from Sinopharm Chemical Reagent Co, Ltd. (Shanghai, China). The deionized water was prepared using a Milli-Q water purification system (Millipore, Belford, MA, USA). The other reagents used in this study were at least analytical grade.

Preparation of defatted walnut meal hydrolysate

Walnut oil was removed using a hydraulic oil press (T32, Hinging Hydraulic Machinery Factory, Heze, Shandong, China) and the resulting meal was passed through a 40-mesh sieve. This meal was degreased with petroleum ether (1:3) for 24 h and air dried. The defatted walnut meal was mixed with distilled water (20% w/v) and subjected to enzymatic hydrolysis using a mixture of enzymes (cellulase, α-amylase and pectinase at a ratio of 5:2:3), with the enzymes: meal ratio of 1:1000 (w/v) using the optimal conditions for enzymatic activity (35 °C) for 3 h. After adjusting to pH 8.0 using 1 mol/L NaOH, the mixture was hydrolyzed using alcalase for 4 h at 50 °C, with the enzyme: meal ratio of 1:500 (w/v). The hydrolysates were heated in boiling water for 10 min to inactivate the enzymes, cooled to room temperature (25 ± 2 °C) and adjusted to pH 7.0. The supernatant was collected after centrifugation at 10,000×g for 15 min at 4 °C (HC-2518R, USTC Zonkla Scientific Instruments Co, Ltd, Hefei, Anhui, China), followed by microfiltration through 0.45 μm filters (Tianjin Jinteng Experimental Equipment Co, Ltd, Tianjin, China). The resulting hydrolysate was stored at 4 °C for a maximum of 2 weeks.

The degree of hydrolysis (DH) was determined using pH–stat method and estimated as follows:

$$\text{DH}(\%)=\frac{C\times V}{\alpha \times M_p\times h_{\mathit{ot}}}\times 100$$

where C is the concentration of NaOH (mol/L), V is the consumption of NaOH (mL), h_ot is the total number of peptide bonds in the protein substrate which was assumed to be 7.35 mmol/g in walnuts (Sun et al. 2019). M_p is the mass (g) of the protein and α represents the average degree of dissociation of the α-amino acid groups and expressed as:

$$\mathrm{\alpha }=\frac{{10}^{pH-pK}}{1+{10}^{pH-pK}}$$

where pH and pK are values at which the hydrolysis was done.

Separation of peptides fractions

The hydrolysate was fractionated using a series of ultrafiltration membranes with nominal MW cut-off membranes of 5 and 10 kDa (Vivaflow 50, Sartorius AG, Gottingen, Germany). Hydrolysates of < 5 (F1) and 5–10 kDa (F2) were obtained. The hydrolysate before ultrafiltration was also collected (F3). The fractions were lyophilized (SCIENTZ-12 N, Ningbo Xinzhi Biological Technology Co, Ltd, Ningbo, Zhejiang, China) and stored at − 20 °C for a maximum of 8 weeks.

Analysis of amino acid (AA) composition

The AA composition of walnut peptides with different MW was determined according to Mosquera et al. (2014) with slight modifications. The samples were mixed with distilled water, followed by hydrolyzed with 6 mol/L HCl (containing 0.1% phenol) in vacuum-sealed glass tubes at 110 °C for 24 h. After hydrolysis, the mixture was again vacuum-dried, dissolved in 0.2 mol/L sodium citrate loading buffer (pH 2.2) and injected onto a Biochrom 30 amino acid analyzer (Biochrom Ltd., Cambridge, UK). AA composition was reported as relative percentage of each AA to the total.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

The electrophoretic profile of peptides with different MW was determined according to Lamsal et al. (2007) with slight modifications. A 4% stacking and 12% separating gel was prepared. Samples were mixed with loading buffer containing β-mercaptoethanol (5 mg/mL). The solution was heated in a boiling water bath for 5 min (HH-8, Changzhou Yitong Analytical Instrument Manufacturing Co., Ltd, Changzhou, Jiangsu, China), and then centrifugated at 1000×g for 10 min (mySPIN™ 12, Thermo Fisher Scientific, Shanghai, China). A mixture of proteins (10–170 kDa, TransGen Biotech, Beijing, China) was used as an MW marker. Electrophoresis was run on an electrophoresis apparatus (Mini-Protean Tetra, Bio-Rad, Hercules, CA, USA). After electrophoresis, the gel was stained in Coomassie Blue R-250 for 4 h and then destained in methyl acetate until visible.

Determination of intrinsic fluorescence

The intrinsic fluorescence of peptides fractions was determined using a fluorescence spectrophotometer (F-7000, Hitachi High-Technologies Corp, Tokyo, Japan). Peptides solutions (0.1 mg/mL) were prepared with 10 mM PBS (pH 7.4). Then 2 mM ANS (160 μL) in distilled water was added into 2 mL of peptides solution and kept in the dark for 10 min, followed by measurements with a 290 nm excitation, 5 nm slit width, 200 nm/min scanning speed, 20 ms scanning interval, and a 300–400 nm scanning range.

Preparation of nanoliposomes encapsulated with walnut peptides

Nanoliposomes were prepared using a thin-film hydration method according to Sarabandi et al. (2019b) with slight modifications. Lecithin (0.09 g), cholesterol (0.17 g) and Tween-80 (10 μL) were dissolved in chloroform (10 mL) and evaporated as a thin film in a rotary evaporator (R-25, Buchi, Shanghai, China) at 60 rpm at 60 °C for 30 min. The round bottomed flask was kept in a desiccator for 12 h at room temperature to evaporate any remaining organic solvent. Then the lipid film was rehydrated with 10 mL of PBS (pH 7.4) containing 2 mg/mL lyophilized peptides. The resulting solution was stirred in the rotary evaporator at 60 °C for 60 min to form primary liposomes. Later, sonication was done using an ultrasonic probe (BioSafer 650-92, Nanjing Saifei Biological Technology Co, Ltd, Nanjing, Jiangsu, China) with nominal frequency of 20 kHz at 500 W power and 30 cycles for 10 min (0.2 s on and 0.2 s off). The nanoliposomes were stored at 4 °C for a maximum of 3 weeks. The empty nanoliposomes were L-0, those with < 5 kDa peptides were L-F1, with 5–10 kDa peptides were L-F2, and with crude peptides were L-F3.

Analysis of particle size, ζ-potential and encapsulation efficiency (EE)

The average particle size, polydispersity index (PDI) and ζ-potential of nanoliposomes solutions were measured using a dynamic light scattering (DLS) system (ZC90, Malvern Instruments, Malvern, UK) at a 90° angle at 25 °C. Samples were dissolved in deionized water to form 1 mg/mL solutions and appropriate amounts placed in the polystyrene cell and results obtained from the software with the instrument. The experiments were done in triplicate and average values were reported.

The EE was determined using an ultrafiltration centrifugation method according to Sarabandi et al. (2019b). Each sample (4 mL) was transferred to an ultra-centrifugal tube (molecular weight cut-off = 30 kDa, Millipore, Beijing Solarbio) and centrifuged (H/T16MM, Hunan Herexi Instrument & Equipment Co, Ltd, Changsha, Hunan. China) for 30 min at 4500×g. The concentration of free peptides passing through the filter was determined using a Folin–Ciocalteu method (Karadirek et al. 2016). The EE was estimated as follows:

$$\text{EE}(\%)=\frac{\text{Total}\text{peptides}\text{content - Free}\text{peptides}\text{content}}{\text{Total}\text{peptides}\text{content}}\times 100$$

Effect of storage conditions and freeze–thaw stress on stability and EE

The effect of the storage conditions and freeze–thaw stress on the particle size and EE of the nanoliposomes were evaluated according to the method described by Li et al. (2015) with slight modifications. For storage stability, each sample (4 mL) was stored at 4 °C and room temperature for 21 days. The changes in particle size and EE were evaluated in accordance with the methods described above. For freeze–thaw stability, the frozen samples (with and without 3% (v/v) food-grade glycerol) were placed at − 20 °C for 4 days, followed by thawing at room temperature for 15 min and Vortexing for 10 min. Samples without glycerol were used as the control group.

Morphological characteristics

The microstructure of nanoliposomes was observed using an optical microscope (XSP-104, Ningbo Zhanjiang Optical Instrument Co, Ltd, Ningbo, Zhejiang, China). One drop of nanoliposomes solution, one drop of 0.06% Triton (to dissolve the phospholipid membrane) and two drops of Coomassie Blue (to react with the peptides) were spread on the glass slide and allowed to stand for several min to stain prior to observing.

The morphology of nanoliposomes was also observed using a scanning electron microscope (SEM) (Gemini SEM 300, Carl Zeiss, Oberkochen, Germany). One drop of diluted sample (50-fold) was placed on an aluminum plate using two-sided adhesive tape and dried at room temperature. Samples were coated with a thin layer of gold (10 nm) using a desk sputter coater DST1 system with a magnetron cathode under vacuum. The coated samples were placed under the microscope at 10 kV with different magnification of 2200 and 6000, and the morphological properties were observed (Sarabandi et al. 2019a).

The structural properties were observed using a transmission electron microscope (TEM) (Libra 200, Carl Zeiss) with a negative staining method. Equal volumes of diluted sample (30-fold) and 2% ammonium molybdate solution were mixed and kept for 3 min at room temperature. A drop of suspension was placed on a Formvar-carbon coated grid (Guangzhou Jingying Scientific Instrument Co, Ltd, Guangzhou, Guangdong, China) for 5 min and air dried at room temperature. The samples were visualized at 200 kV, 40,000 × and 400,000 × magnification (Ramezanzade et al. 2017).

In vitro gastrointestinal digestion

In vitro digestion of free peptides and peptides-loaded nanoliposomes was carried out according to the method described by Liu et al. (2013) with slight modifications. In brief, the sample (5 mL) was mixed with simulated gastric fluid (SGF, pH 1.5) consisting of 0.032 mg/mL porcine pepsin or simulated intestinal fluid (SIF, pH 7.4) consisting of 0.032 mg/mL pancreatin and 0.02 mg/mL bile salt at a ratio of 1: 3 (v: v), then the mixture was transferred to a shaking incubator (SHA-BA, Jintan Ronghua Instrument Manufacturing Co, Ltd, Jintan, Jiangsu, China) at 95 rpm for 2 h. Each sample (4 mL) from SGF and SIF digestion was taken for release study.

Statistical analysis

All the experiments were done in 3 replications, and the results were expressed as mean ± standard deviation (SD). Statistical analysis of the data was carried out using the Statistical Package for the Social Sciences, version 19.0 (SPSS Inc, Chicago, IL, USA). One-way analysis of variance (ANOVA) and the Duncan’s test were used to determine differences between means. The level of P < 0.05 was set for significance.

Results and discussion

Amino acid (AA) composition

The DH of defatted walnut meal hydrolysates was 10.3%. The AA composition of the walnut peptides with different MW is shown in Table 1. All the peptides fractions were rich in Asp, Glu, Leu and Arg, which were consistent with Wang et al. (2020). The hydrophobic amino acids (HAA) in F1, F2 and F3 accounted for 40.6 ± 0.06%, 40.8 ± 0.1% and 40.3 ± 0.07%, respectively, indicating that the AA composition of the peptides was related to MW. Furthermore, the contents of HAA were highest in the F2 compared to F1, F3 and higher than the 22.8–26.4% reported for walnut protein hydrolysate (Wang et al. 2020). The AA composition affected the angiotensin I-converting enzyme (ACE) inhibitory activity. The peptides with low MW could be used as preferred ACE inhibitors, which could be attributed to that low-MW peptides contained HAA on the C-terminal and preferably bond with catalytic sites of ACE (Jia et al. 2010).

Table 1.

The amino acid (AA) composition of peptides with different MW

Amino acid	Content (%)
	F1	F2	F3
Asp	8.01 ± 0.03	8.42 ± 0.1	8.54 ± 0.06
Thr	3.53 ± 0.05	3.38 ± 0.03	3.33 ± 0.05
Ser	5.34 ± 0.1	5.04 ± 0.05	5.24 ± 0.03
Glu	19.3 ± 0.1	19.8 ± 0.06	18.6 ± 0.04
Gly	4.76 ± 0.02	4.60 ± 0.04	5.13 ± 0.03
Ala	4.11 ± 0.04	4.22 ± 0.1	4.09 ± 0.02
Cys	0.72 ± 0.05	0.51 ± 0.03	0.35 ± 0.1
Val	5.50 ± 0.1	5.35 ± 0.08	4.60 ± 0.06
Met	3.85 ± 0.05	3.67 ± 0.1	3.86 ± 0.1
Ile	5.24 ± 0.07	5.16 ± 0.1	4.73 ± 0.04
Leu	7.28 ± 0.05	7.31 ± 0.04	6.90 ± 0.01
Tyr	3.51 ± 0.07	3.72 ± 0.07	4.07 ± 0.06
Phe	3.74 ± 0.04	3.50 ± 0.05	4.66 ± 0.05
His	3.22 ± 0.06	3.19 ± 0.08	2.95 ± 0.03
Lys	3.61 ± 0.1	3.50 ± 0.1	3.19 ± 0.1
Arg	11.5 ± 0.1	11.20 ± 0.1	12.8 ± 0.08
Pro	6.78 ± 0.1	7.43 ± 0.05	6.96 ± 0.1
HAA	40.6 ± 0.06	40.8 ± 0.1	40.3 ± 0.07
Total	100 ± 0.08	100 ± 0.1	100 ± 0.07

F1: peptides with MW of < 5 kDa; F2: peptides with MW of 5–10 kDa; F3: crude peptides; HAA: hydrophobic amino acids—Ala, Cys, Val, Met, Ile, Leu, Tyr, Phe and Pro

SDS-PAGE

The SDS-PAGE profiles of peptides with different MW are shown in Fig. 1. High MW peptide and low MW peptide bands in different peptide fractions were well resolved in the SDS-PAGE, respectively. The results showed that F3 consisted of molecular bands ranging from 12 to 25 kDa. Within this range, at least four major bands of ~ 13 kDa, ~ 17 kDa, ~ 23 kDa, and ~ 25 kDa were observed, which consisted of the previously report of walnut protein hydrolysis (Jin et al. 2020). After ultrafiltration, bands with MW higher than 10 kDa disappeared, and the low MW < 10 kDa bands were found in the F1 and F2 (Lanes 1–2). The F2 showed a visible band with an MW close to 10 kDa, which indicated that F1 contained peptides with much lower MW, as expected for the membrane cut-off of the ultrafiltration process (Kimatu et al. 2017). Alcalase belongs to a family of serine protease, which could cleave the peptide bonds of the carboxyl side of Arg and Lys. The disappearance of the four major bands indicated that the acid subunit bond of high-MW peptides might contain higher contents of Arg and Lys and be more sensitive to alcalase. Besides, the bands’ changes might affect walnut peptides' functional properties (i.e. ACE inhibitory activity) (Jin et al. 2020).

Fig. 1.

SDS-PAGE profile of peptides with different MW. Lane M represents the marker proteins; Lane 1: peptides with MW of < 5 kDa; Lane 2: peptides with MW of 5–10 kDa; Lane 3: crude peptides

Intrinsic fluorescence

The intrinsic fluorescence spectroscopy of peptides with different MW is shown in Fig. 2. The results showed that the optimal excitation wavelength of F1 was 341 nm, and that of F2 and F3 was 360 and 340 nm, respectively. With the increase of peptides MW, the excitation wavelength first increased and then decreased, and the F2 showed the largest excitation wavelength. The fluorescence intensity also exhibited the same trend. Qi et al. (2006) investigated that the polar environment of aromatic amino acid residues (mainly Trp) could affect the excitation wavelength, and the fluorescence intensity was affected by Trp residues exposed on the surface of peptides chains backbone. The longer peptides chains of F2 were removed during the ultrafiltration process, and more Trp residues of shorter peptides chains were exposed to a polar environment, therefore the excitation wavelength of F2 red-shifted and fluorescence intensity increased compared to F3 (Zhu et al. 2020). The excessive hydrolysis may lead to aggregation of hydrophobic groups of F1 through hydrophobic interactions, which might restore the Trp to a non-polar environment. Therefore, the excitation wavelength of F1 blue-shifted and fluorescence intensity decreased compared to F2 (Qi et al. 2006). Similar result showed that moderate enzymatic soy protein isolates helped to improve fluorescence intensity (Qi et al. 2006).

Fig. 2.

Fluorescence emission spectra of peptides with different MW. F1: peptides with MW of < 5 kDa; F2: peptides with MW of 5–10 kDa; F3: crude peptides

Size distribution and ζ-potential

As shown in Table 2, the particle size, PDI and ζ-potential of nanoliposomes were changed significantly (P < 0.05), depending on the peptides MW. After loaded with peptides, the particle size of nanoliposomes increased from 80.1 to 83.5 nm. The encapsulation of peptides with high MW resulted to the increase in the particle size of nanoliposomes can be attributed to the changes in the arrangement of the membrane, because low MW peptides entered easily within bilayer and improve the vesicular order compared to high MW peptides (Sarabandi et al. 2019a). In similar results, an increase was reported in the size distribution of nanoliposomes after loading bioactive whey peptides (Mohan et al. 2016) and Whitemouth croaker hydrolysates (da Rosa Zavareze et al. 2014). The PDI ranges from 0 to 1, indicating that the nanoliposomes change from monodisperse to polydisperse systems (Chay et al. 2015). Loading peptides with different MW increased the PDI of nanoliposomes significantly from 0.224 to 0.279 (P < 0.05), indicating a uniform and controlled particle size distribution (da Rosa Zavareze et al. 2014). Study by Mosquera et al. (2014) reported a PDI of 0.250 for nanoliposomes loading peptides from sea bream scale. Among the other samples, L-F2 had the lowest particle size (81.1 nm) and PDI (0.239), indicating a more homogenous system.

Table 2.

Particle size, polydispersity index (PDI), ζ-potential and encapsulation efficiency (EE) of nanoliposomes loading peptides with different molecular weight (2 mg/mL)

Sample	Particle size (nm)	Polydispersity index (PDI)	ζ-potential (mV)		EE (%)
			Peptide	Nanoliposome
L-0	80.1 ± 0.8c	0.224 ± 0.01c	–	− 1.96 ± 0.3c	–
L-F1	81.3 ± 0.6b	0.251 ± 0.01b	− 30.1 ± 0.2b	− 7.75 ± 0.3b	52.5 ± 2b
L-F2	81.1 ± 0.6b	0.239 ± 0.005bc	− 30.3 ± 0.1ab	− 7.86 ± 0.1b	59.3 ± 2a
L-F3	83.5 ± 0.5a	0.279 ± 0.004a	− 29.9 ± 0.2a	− 8.46 ± 0.3a	51.2 ± 3b

Different letters in each column (a, b, c) represents a significant difference between treatments (P < 0.05) and data are shown as mean ± standard deviation (n = 3)

L-0: empty nanoliposomes; L-F1: nanoliposome loaded with < 5 kDa peptides; L-F2: nanoliposome loaded with 5–10 kDa peptide; L-F3: nanoliposomes loaded with crude peptide (2 mg/mL)

As shown in Table 2, after loading peptides, the ζ-potential shifted to higher negative values, suggesting the effect of high negative charge of peptides on the final potential of nanoliposomes (Sarabandi et al. 2019a). The ζ-potential of empty nanoliposomes was − 1.96 mV, and nanoliposomes increased significantly from − 1.96 to − 8.46 mV (P < 0.05) after loading peptides. The changes in ζ-potential of peptides MW may result from the decrease of hydrophobic amino acids (Sarabandi et al. 2019b). The interaction between charged peptides and phospholipids, and the placement of peptides in membranes also changed the value of this index (da Rosa Zavareze et al. 2014). Similar studies reported that the encapsulation of orange seed protein hydrolysates (Mazloomi et al. 2020) and salmon hydrolysates (Li et al. 2015) changed the ζ-potential of nanoliposomes.

EE

As can be seen from Table 2, peptides fractions with different MW had a significant influence on the EE (P < 0.05). The result was contrary to the findings obtained by Mohan et al. (2016), which could be attributed to the structural heterogeneity caused by the enzyme types and hydrolysis time, which influenced the EE of nanoliposomes when loading peptides. For instance, no significant difference was observed in the EE of nanoliposomes loading casein hydrolysates produced with alcalase and pancreatin at 90 min, while hydrolysates produced with alcalase at 90 and 150 min showed significant differences (Sarabandi et al. 2019a). From Table 2, the L-F2 had the highest EE of 59.3%, followed by L-F1 at 52.5%, while L-F3 had the lowest EE at 51.2%. The higher EE of L-F2 could be because the F2 had better placement within the nanoliposomal structure (Sarabandi et al. 2019a). Furthermore, the lower EE of L-F1 may be due to the easy release of smaller peptides from the nanoliposomes (Sarabandi et al. 2019a), and that of L-F3 may be attributed to the hydrophobic residues of high-MW peptides being bonded to phosphatidylcholine, and the hydrophilic residues interacted with the aqueous phase. These two kinds of imbalanced forces caused the noncontinuous interfacial barrier (Chay et al. 2015). Similar result has been reported for nanoliposomes loading casein hydrolysates with EE of 46.0% (Yokota et al. 2012). Chay et al. (2015) has reported that the EE of nanoliposomes loading winged bean seeds bioactive peptides was 27.6%. The poor encapsulation of their study could be due to the weak binding between hydrophilic peptides and phosphatidylcholine.

The effect of storage conditions on physical stability and EE

The effect of peptides MW and storage conditions on the physical stability and EE was investigated. As shown in Fig. 3a, b, no significant changes in the particle size of peptides-loaded nanoliposomes occurred within 8 days, but it increased significantly as the storage time increased after 8 days. From Fig. 3c, the particle size of nanoliposomes changed from 80.7–85.8 to 97.6–103 and 103-117 nm after 20 days storage at 4 °C and 25 °C, respectively. Among the nanoliposomes stored in 4 °C, the highest and lowest changes in the particle size of nanoliposomes were L-F3 and L-F2, increasing to 103 and 97.6 nm, respectively. The minimum and maximum size of the nanoliposomes were L-F1 and L-F2 increasing to 103 and 117 nm, respectively, after storage at 25 °C. The particle size of L-F2 varied from 81.1 to 97.6 nm after storage at 4 °C, and increased significantly (P < 0.05) to 117 nm after storage at 25 °C. These changes may be attributed to two reasons: (1) The effect of loaded peptides MW on the electrostatic repulsion reduction and instability of nanoliposomes during storage (Mazloomi et al. 2020), and (2) the formation of agglomerates and bridge structure in phospholipid membranes as a result of increased particle size at ambient temperature (Sarabandi et al. 2019a). In addition, the particle size of nanoliposomes stored at 25 °C were larger than those of samples stored at 4 °C (P < 0.05), which was due to the increasing fluidity of the membrane, destruction of the ζ-potential, and the high oxidation rate of unsaturated fatty acids stored at high temperature (Sarabandi et al. 2019a).

Fig. 3.

The effect of storage temperature a 4 °C and b 25 °C on the particle size of nanoliposomes; the influence of peptide MW and storage temperature (4 and 25 °C) on the c particle size and d encapsulation efficiency (EE) after 20 days storage. Different uppercase and lowercase letters indicate significant differences (P < 0.05) among different samples stored with similar conditions and for each peptide-loaded nanoliposomes stored with different conditions, respectively. L-0: empty nanoliposomes; L-F1: nanoliposome loaded with < 5 kDa peptides; L-F2: Nanoliposome loaded with 5–10 kDa peptide; L-F3: nanoliposomes loaded with crude peptide fractions (2 mg/mL)

As shown in Fig. 3d, the EE of peptides-loaded nanoliposomes went to 43.3–46.2 and 38.3–41.0% after 20 days storage at 4 and 25 °C, respectively. Among different samples, the L-F2 showed the largest changes in EE during storage, which can be attributed to the difference in peptides composition and MW. The smaller peptides showed faster release rate from nanoliposomes membranes (Mazloomi et al. 2020). The nanoliposomes stored at 4 °C showed better stability, which can be attributed to that lower temperature decreased the permeability and flexibility, as well as increased the fluidity of membranes, leading to a slower release of loaded peptides from nanoliposomes (Li et al. 2015). Similar study reported that the EE of nanoliposomes loaded with flaxseed peptides decreased From About 90.0 to 56.0–63.0 and 40.0–45.0% after 8 weeks storage at 4 and 25 °C, respectively (Sarabandi et al. 2019a).

The effect of freeze–thaw stress on physical stability and EE

Figure 4a shows the changes in particle size of frozen-thawed nanoliposomes loading peptides with different MW in the absence or presence of food-grade glycerol. In the absence of glycerol, the particle size of empty nanoliposomes showed more than 3 folds increase after freeze–thaw process, and that of peptides-loaded nanoliposomes varied from 81.1 to 157 nm. The reason for these changes can be attributed to the formation of ice crystals in the sample solution, and subsequent diffusion and aggregation into the lipid membranes (Mazloomi et al. 2020). Similar findings on the instability of nanoliposomes loading flaxseed and casein hydrolysates have been reported (Sarabandi et al. 2019a, b). However, the particle size was not significantly different between glycerol-containing nanoliposomes and primary nanoliposomes after the freeze–thaw process (P ≥ 0.05), indicating that the concentration of glycerol (3%) was protective. Harrigan et al. (1990) reported that the protective ability of glycerol on nanoliposomes depended on the concentration, with too little or too much cryoprotectant resulting in an instability of nanoliposomes.

Fig. 4.

The influence of peptide MW and freeze-thawing on the a particle size and b encapsulation efficiency (EE) of nanoliposomes. Different uppercase and lowercase letters indicate significant differences (P < 0.05) among different samples stored with similar conditions and for each peptide-loaded nanoliposomes stored with different conditions, respectively. L-0: Empty nanoliposomes; L-F1: Nanoliposome loaded with < 5 kDa peptides; L-F2: Nanoliposome loaded with 5–10 kDa peptide; L-F3: Nanoliposomes loaded with crude peptide fractions (2 mg/mL)

The influence of peptides MW and freeze–thaw stress on the EE is shown in Fig. 4b. The EE of nanoliposomes varied from 59.2 to 30.4% after freeze–thaw process. The highest and lowest EE of nanoliposomes was L-F2 and L-F1, reaching 46.7 and 38.9%, respectively. This difference was due to the different magnitudes and composition of peptides. The smaller peptides may be released from nanoliposomes faster than larger ones (Sarabandi et al. 2019a). In the absence of glycerol, the peptides MW did not have significant effect on the EE of nanoliposomes (P ≥ 0.05). While the EE of nanoliposomes containing glycerol was relatively higher than those without glycerol, indicating that glycerol had protective effect during freeze-thawing process (Sarabandi et al. 2019b). The ways of adding glycerol had effect on the EE of nanoliposomes. In a study, the glycerol was added directly to the nanoliposomes-forming dispersion, and the results showed that more than 80.0% of the hydrolysates were released from nanoliposomes (Li et al. 2015).

Morphology

L-F2 showed a better physicochemical property and stability, therefore, chosen as a representative sample to indicate the morphological characteristics. As shown in Fig. 5a, the nanoliposomes were assembled in aggregates in the optical microscope, and the purple points showed the placements of peptides in the nanoliposomes (Sarabandi et al. 2019a). The SEM images of peptides-loaded nanoliposomes were revealed in Fig. 5b, c. The images revealed a dense and aggregated spherical particles. In addition, the phospholipid bilayers of nanoliposomes were seen to be fused to each other due to the moisture removed before the SEM was used. Therefore, the average particle size increased at high concentrations, which may correlate with Ostwald ripening (Reyes et al. 2021). This theory suggests that the smaller particles dissolve into the larger ones, which grow by means of this molecular diffusion. The TEM images showed the production of smooth surfaces, spherical shapes and vesicular structures (Fig. 5d, e). In addition, nanoliposomes had good dispersion in water. The particle size observed using TEM was consistent with that measured using the particle size analyzer. Similar structures were observed in the nanoliposomes loaded with orange seed protein hydrolysates (Mazloomi et al. 2020), bioactive whey peptides (Mohan et al. 2016) and winged bean seeds bioactive peptides (Chay et al. 2015).

Fig. 5.

Nanoliposome loading peptides with MW of 5–10 kDa: a optical microscope image at 400× magnification; b, c SEM images at 2200 and 6000× magnifications; d, e TEM images at 40,000× and 400,000× magnifications

In vitro digestion

Figure 6 shows the release behavior of F2 and L-F2 in SGF and SIF digestion system. The results showed that the release rate of F2 reached up to 90.0% within 30 min in gastrointestinal (GI) digestion. The L-F2 showed fast release within 30 min in SGF and SIF digestion system by around 35.2% and 66.1%, respectively, then the release rate slowed down subsequently, suggesting effective protection of nanoliposomes from acidic or alkaline environment in GI system. After intestinal digestion, around 83.3% of peptides released from nanoliposomes, however, the release of peptides was sustained and observed a total of 38.4% in SGF. At the gastric pH, the acidic environment could cause protonation on nanoliposomes' surface because negatively charged phospholipids were hydrolyzed by enzyme and become swollen, leading to the fast release of peptides from nanoliposomes in the first 30 min (Li et al. 2015). In the SIF digestion, the poor stability of nanoliposomes in the first 30 min could be due to the pancreatic enzymes and bile salts, the former hydrolyzed phospholipid and produced fatty acids. The later formed mixed micelles with fatty acids and caused synergistic reductions in interfacial tensions through electrostatic interactions (Liu et al. 2020). Similar results were reported by Li et al. (2015), who found the fast release of salmon peptides from nanoliposomes within 30 min in GI digestion, and about 40.0–90.0% of salmon peptides released after in vitro digestion.

Fig. 6.

Release rate of peptides from nanoliposomes in a simulated gastric fluid (SGF) and b simulated intestinal fluid (SIF) condition. F2: peptides with MW of 5–10 kDa; L-F2: nanoliposome loaded with 5–10 kDa peptide fraction

Conclusion

In this study, nanoliposomes loaded with peptides from the defatted walnut meal were investigated. The results showed that the AA composition was related to the peptide MW, and peptides of 5–10 kDa had the highest content of HAA. Furthermore, the particle size, PDI and ζ-potential were also affected by peptides MW. The storage conditions and peptides MW could affect the stability and EE of nanoliposomes. The nanoliposomes loading peptides with MW of 5–10 kDa were selected for morphological characteristics and in vitro digestion. The TEM showed individual nanoliposomes had spherical, smooth and full vesicle structures, and the SEM showed that a high concentration of nanoliposomes might result in phospholipid fusion. The peptides-loaded nanoliposomes showed a better-sustained release effect in the SGF system. Our results revealed that the molecular weights of peptides might be an essential indicator in preparing the nanoliposomes. Moreover, the storage stability and in vitro digestion stability of walnut peptides could be improved by nanoliposomes. This work might be helpful for preparing a stable nanoliposomal delivery system for walnut peptides and the development of the food industry in beverage processing and food nutrition.

Abbreviations

MW

Molecular weight

F1

Peptides with molecular weight of < 5 kDa

F2

Peptides with molecular weight of 5–10 kDa

F3

Crude peptides

L-0

Empty nanoliposomes

L-F1

Nanoliposomes loading peptides with molecular weight of < 5 kDa

L-F2

Nanoliposomes loading peptides with molecular weight of 5–10 kDa

L-F3

Nanoliposomes loading crude peptides

Authors contributions

ZGH Experiments, Methodology, Software, Writing-Original Draft. FSX Experiments, Validation, Visualization, Investigation. RJM Writing-Review & Editing, Data Curation. WFJ: Resources, Writing—Review & Editing, Supervision, Data Curation.

Funding

This research was supported by a Beijing Municipal Science & Technology Project (Z181100009318012) and the National Innovation Alliance of Walnut Industry (NAWI).

Availability of data and material

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Consent to participate

We are giving our consent for participation. Fengjun Wang, having a specialization in Walnut Protein Processing, will be grateful to review at least three manuscripts of JFST in future.

Consent for publication

The work was original research that has not been published previously, and is not under consideration for publication elsewhere, in whole or in part.