The antibacterial activity and Pickering emulsion stabilizing effect of a novel peptide, SA6, isolated from salted-fermented Penaeus vannamei

doi:10.21203/rs.3.rs-2314693/v1

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The antibacterial activity and Pickering emulsion stabilizing effect of a novel peptide, SA6, isolated from salted-fermented Penaeus vannamei

https://doi.org/10.21203/rs.3.rs-2314693/v1

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This study aimed to improve bacterial inhibition in Pickering emulsions during storage using antimicrobial peptides. A peptide (ARHQGVMVGMGQK), designated SA6, isolated from the broth of salt-fermented shrimp (Penaeus vannamei). Peptide SA6 had a minimum inhibitory concentration (MIC) of 15.6 μg/mL against Staphylococcus aureus. The mean particle size of SPI-SA6 particles (417.4 nm) was significantly smaller compared with soybean isolate protein (SPI) (463.3 nm). Moreover, the polydispersity index (PDI) decreased with increasing peptide concentration, while the particles were stabilized by hydrogen bonding. SPI-SA6 Pickering emulsions were stable for the entire storage period (7 d) and had lower creaming index and droplet size compared with SPI Pickering emulsions. Further, SPI-SA6 Pickering emulsion could effectively inhibit bacterial growth and disrupt bacterial cell membrane structure, to significantly decrease bacteria (S. aureus) numbers to 2.83 CFU/mL during storage and therefore extending the inhibition time. Collectively, peptide SA6 could stabilize Pickering emulsion while exerting antibacterial effects.

Penaeus vannamei

antimicrobial peptide

Pickering emulsion

Staphylococcus aureus

antimicrobial activity

Pickering emulsions, when stabilized by solid particles instead of traditional emulsifiers, have the advantages of anti-caking, good stability, low toxicity, and low environmental pollution, for which reason they are currently widely used in cosmetics, food, biology, and medicine (Peito et al., 2022; Sharkawy et al., 2020). Soybean isolated protein (SPI), which is mainly composed of β-accompanying soy globulin (7s) and soy globulin (11s) (Zhu et al., 2018), is often used to stabilize Pickering emulsions. Generally, SPI promotes the formation of the emulsions by reducing the water-oil interfacial tension while forming a membrane structure at the water-oil interface to avoid the aggregated flocculation of emulsions (Li et al., 2020; Huang et al., 2019). However, Pickering emulsions provide space for microbial growth while SPI provides nutrients for microbial growth. Therefore, controlling the growth of microorganisms in food-grade Pickering emulsions is critical in their application in the food industry.

Currently, two main strategies are used to improve the antibacterial properties of Pickering emulsions, i.e., the addition of chemicals or natural substances with bacteriostatic properties (e.g., chitosan or silver nanoparticles) or the of natural essential oils with bacteriostatic activity to the oil phase substances, such as encapsulating cinnamon essential oil in the emulsion (Jiang et al., 2020). However, chitosan requires chemical modification or secondary processing before use due to its poor water solubility; although silver nanoparticles have excellent bactericidal properties, their toxicity limits their application in food (Ahmed et al., 2021; Hu et al., 2022). Encapsulated essential oils release a strong odor when they leak or exert antibacterial activity, affecting food's sensory quality (Patience et al., 2018). Thus, to increase the bacterial inhibition ability of food-grade Pickering emulsions without lowering the food quality, there is the need to develop a stable and safe strategy.

In the food industry, antimicrobial peptides (AMPs) are widely used for food preservation because of their chemical stability, lack of odor, and low toxicity (Jiang et al., 2021). These peptides usually contain two or more positively charged amino acid residues and hydrophobicity between 30% and 60% (Kurpe et al., 2020). Recent studies have shown that hydrophobic proteins and SPI can mediate protein binding by forming hydrogen bonds (Wang et al., 2018). Given that negatively charged SPI and positively charged proteins can interact electrostatically to reduce the intermolecular distance, it can promote intermolecular hydrogen bond formation (Zheng et al., 2020), suggesting that AMPs could potentially bind stably to SPI to enhance the bacteriostatic properties of Pickering emulsions.

Penaeus vannamei (P. vannamei) is the most farmed shrimp species globally, from which various peptides with antimicrobial activity have been isolated from their proteomic data (Sun et al., 2021). Proteins can degrade into peptides by salting, but the potential of isolating AMPs from salted P. vannamei has received little attention. Herein, we propose a strategy to improve the antimicrobial properties of Pickering emulsions using AMPs. A peptide with antimicrobial activity was identified from the salt-fermented broth of P. vannamei and shown to bind stably to SPI. The stability of the emulsion, its antibacterial activity, as well as its inhibition mechanism were further explored.

2.1 Materials, bacteria strains, and reagents

P. vannamei were purchased from a commercial supermarket (New Huadu) in Xiamen, China, whereas nutrient broth (NB) medium, SPI, soybean oil, and agar were obtained from Lablead Biotechnology (Lablead Biotechnology Co., Beijing, China). Sodium chloride (NaCl), mass spectrometry grade trypsin, acetonitrile, and formic acid, as well as urea, propidium iodide (PI), green fluorescent nucleic acid stain (STOY-9), phosphate buffer solution (PBS), pentanediol, ethanol, acetone lead citrate, and uranyl acetate were all purchased from Solarbio (Solarbio Technology Co., Beijing, China). Staphylococcus aureus (ATCC 27217), Escherichia coli (ATCC 15224), Vibrio parahaemolyticus (ATCC 17802), and Bacillus cereus (ATCC 14579) are all bacteria strains collections in the Key Laboratory of Microbiology and Enzyme Engineering of Fujian Province, Jimei University, Xiamen, China. All bacteria were cultured in NB medium at 37°C.

2.2. Salt fermentation of P. vannamei and screening of isolated peptides with antimicrobial potential.

P. vannamei were cleaned before being placed in 20% NaCl solution for 15 days of natural fermentation. Next, the fermentation broth was collected and centrifuged at 10000 rpm (25°C, 10 min), followed by placing the supernatant in ultrafiltration units (3 kDa, Milipore) and centrifuged at 10000 rpm for 20 min (4°C). The filtrate samples were then digested with trypsin overnight at 37°C. After the samples were desalted using a small C18 column, they were injected into an ultra-performance liquid chromatograph-mass spectrometer (UPLC-MS) for analysis. The injection volume was 5.0 µL, mobile phase A was 0.1% formic acid in water, and B was acetonitrile. The gradient elution program was as follows: 0–1 min, 98% A; 1–55 min, 98% -70% A; 55–60 min, 70% -10% A. The mass spectrometry (MS) conditions were set to spray voltage: 3.0 kV; capillary temperature: 300°C. The scan mode was positive ion, primary scan: resolution 70,000, range 350–1600 m/z; secondary scan: resolution 17500. The MS data were searched against the Penaeus vannamei database in Uniprot (http://www.uniprot.org) with the Maxquant software (v1.6.5.0). The charge and hydrophobicity of the peptides were calculated using the online APD3 software (http://aps.unmc.edu/AP/).

2.3 Molecular docking prediction of SPI and AMPs interactions

The interaction between SPI and AMPs was predicted by molecular docking. Crystal structures of 7S (coded 3AUP) and 11S (coded 2D5F) proteins were obtained from the protein data bank (PDB) (http://www.rcsb.org/). The optimization of AMPs structures was performed by ChemBio Draw U1tra 14.0 software, and molecular docking was performed using Maestro software (version 11.1.012), while the docking results were visualized using Pymol software (version 1.6). The SPI was set as rigid molecules, while AMPs were set as flexible molecules whose torsion, position, and orientation were set randomly (Ao et al., 2021).

2.4 Determination of antibacterial activity

Peptide SA6 was synthesized by a commercial company (Scilight Biotechnology Co., Beijing, China) using the Fmoc solid-phase synthesis method, followed by purification to more than 99% purity and molecular weight determination using liquid chromatography-mass spectrometry (LC-MS), as previously described (Huertas et al., 2017).

The minimum inhibitory concentrations (MIC) of peptide SA6 against S. aureus, B. cereus, V. parahaemolyticus, and E. coli were determined using the plate colony counting method (Mookherjee et al., 2020). Briefly, bacteria were incubated in nutrient broth (NB) at 37°C for 12 h before diluting to 10³ CFU/mL in sterile PBS. Next, the diluted bacteria and peptide SA6 (in sterile PBS) were mixed in equal volumes and incubated at 37°C for 2 h. After 2 h incubation, 20 µL of the samples were spread on glass petri dishes and incubated at 37°C for 24 h. The MIC was defined as the peptide concentration that inhibited 80% of bacterial growth.

Time-kill curve analysis of peptide SA6 against S. aureus was performed as described previously (Yang et al., 2020). Briefly, SA6 diluted to different concentrations (1/4×MIC − 4×MIC) was mixed with diluted S. aureus (10³ CFU/mL) and incubated at 37°C for 0, 1, 2, 3, 4, and 5 h. Next, 20 µL of samples were uniformly spread on glass petri dishes and incubated at 37°C for 24 h. Negative control samples were treated the same way but with sterile PBS in place of peptide SA6. The number of colonies was counted and recorded.

To evaluate the bacteriostatic properties of SPI Pickering and SPI-SA6 Pickering emulsions during storage, these Pickering emulsions were mixed with an equal volume of diluted S. aureus (10³ CFU/mL) and incubated at 37°C. Next, 20 µL of the samples were evenly spread onto glass petri dishes at different time points (0, 1, 2, 3, 4, and 5 d), and the Petri dishes incubated at 37°C for 24 h. The bacteria were then observed, and the number of colonies counted (Li et al., 2018).

2.5 Determination of particle size, zeta potential, and interparticle interaction of SPI-SA6 particles

The SPI-SA6 particles were prepared as previously reported (Yi et al., 2021). Briefly, SPI was dissolved in ultrapure water (1.0%, w:v) and ultrasonicated for 40 min using a JY92-IIDN ultrasonicator (Ningbo Xinzhi Biotechnology, Ningbo, China). Next, SA6 was dissolved in the SPI solution and stirred magnetically for 2 h at room temperature. The particle size and zeta-potential of SPI and SPI-SA6 were diluted 1000 times before being measured using a laser nanoparticle sizer (Brookhaven Instruments Co., Holtscille, USA) at 25°C with an equilibration time of 120 s. The pattern of interparticle interactions was determined by the relative change in the average particle size of SPI-SA6 dispersions in 6 M urea solution. To do this, SPI and SPI-SA6 were diluted to 0.1% (w:v), and the mean particle size of SPI and SPI-SA6 were analyzed using a laser nanoparticle sizer. Samples were kept at ambient temperature for 30 min before testing (Liu & Tang, 2014).

2.7 Preparation of emulsion and observation of emulsion droplets

The emulsion was prepared as previously described (Li et al., 2019). Briefly, soybean oil was added to SPI and SPI-SA6 (oil to water ratio of 7:3), and fresh emulsions obtained using a high-speed shear emulsifier (high-speed homogenizer (FA25, Fluko, Shanghai, China) operated at 12000 rpm for 2 min. The distribution patterns of SPI Pickering and SPI-SA6 Pickering emulsions were observed under an optical microscope (Motic BA240, Motic Deutschland GmbH, Wetzlar, Germany).

The stability of the SPI and SPI-SA6 emulsions was observed by the creaming index. Briefly, 10 mL of each SPI Pickering and SPI-SA6 Pickering emulsions were added into colorimetric tubes and kept at room temperature for 7 d. The appearance of the emulsions was observed and recorded daily for delamination and surface oiling (Ge et al., 2017).

2.8 Rheological Measurements

The dynamic viscoelasticity of the SPI Pickering and SPI-SA6 Pickering emulsions was measured using a rotational rheometer TA Instruments (DHR-2, TA Instruments, New Castle, USA) based on a previously described procedure (Zhang et al., 2021). The linear viscoelastic zone of the emulsions was determined by placing the appropriate amounts of SPI Pickering and SPI-SA6 Pickering emulsions on the sample table using a 40.0 mm diameter plate. The plate spacing was 1 mm, the constant shear frequency was 1 Hz, and the temperature was 25°C, with the strain scanning performed at 0.01–100% of shear strain. A constant shear strain value in the linear viscoelastic zone was selected. The frequency scan was performed at 0.1–10.0 Hz to obtain the variation curves of the elastic modulus (G′) and viscous modulus (G″).

2.9 Emulsion particle size measurement

The particle size distribution and average particle size of SPI Pickering and SPI-SA6 Pickering emulsions were measured using a laser particle size analyzer (BT-9300HT, Better, Dandong, China) according to a previously described method (Li et al., 2020). Deionized water was used as a dispersant (1.330) and a refractive index of 1.471 for the emulsions. All emulsion samples were diluted 100 times with PBS to avoid dynamic light scattering.

2.10 Flow cytometer measurements

A flow cytometer technique was used to determine bacteria (S. aureus) survival as previously described (Osman et al., 2019). Briefly, bacteria were treated with PBS, SA6, SPI Pickering, and SPI-SA6 Pickering emulsions. The SA6 treated bacteria were incubated at 37°C for 2 h, while the other samples were incubated at 37°C for 24 h. Bacteria treated with PBS were used as the negative control. Next, the treated bacteria were suspended in PBS and washed three times before being stained with 5 µL PI (1 mg/mL) and SYTO-9 at 37°C for 20 min. Samples were analyzed using a BD FACSCANTO II flow cytometer (Becton Dickinson, San Jose, CA, USA), with excitation/emission wavelengths of 490/635 nm for PI and 480/500nm for SYTO-9.

2.11 Transmission electron microscopy (TEM) observation

Bacteria ultrastructure was observed using TEM according to a previously described method (Xia et al., 2013). Briefly, bacterial suspensions (10³ CFU/mL) were mixed with SA6, SPI, SPI Pickering, and SPI-SA6 Pickering emulsions before being incubated at 37°C for 2 h, and then centrifuged at 2700 g (25°C, 2 min). The precipitates were washed three times with PBS, fixed with 0.27 mM glutaraldehyde solution, and dehydrated with ethanol (30%-100%). Ultrathin sections were stained with lead citrate and uranyl acetate before being observed on the H-7650 TEM instrument (Hitachi, Tokyo, Japan).

2.12 Statistical analysis

All experiments were performed at least three times, and data are presented as mean ± standard deviation. One-way ANOVA and Duncan's multiple comparison statistical analysis were performed on the data using SPSS software (22.0) and GraphPad Prism software (version 8.3). Statistical significance was considered at p < 0.05.

3.1. Screening of peptides isolated from salted P. vannamei

3.1.1. Identification of peptides with antimicrobial potential

To obtain the amino acid sequences of small and medium molecular weight peptides from salted P. vannamei, mass spectrometry analysis of peptides with molecular weights less than 3000 Da was carried out using UPLC-MS. A total of 12 peptides were obtained, as shown in Table 1. Among the screened peptides, the average peptide length was 11.92, of molecular weights ranging from 1093 to 1529 Da, hydrophobicity of 20–55%, and charge from − 3 to 3.

Table 1

Predicted potential AMPs in salted *P. vannamei*
No.	Peptide sequences	MW(Da)	Hydrophobic ratio (%)	Net charge
SA1	TGSNVFDMFTQK	1373.6	33%	0
SA2	LAEASQAADESER	1375.6	38%	-3
SA3	EITALAPSTLKIK	1383.8	46%	+ 1
SA4	LTQEAVSDLER	1259.6	36%	-2
SA5	AEAATMQAKAEAQK	1446.7	50%	0
SA6	ARHQGVMVGMGQK	1397.7	38%	+ 3
SA7	KDAYVGDEAQSKR	1465.7	23%	0
SA8	MQKEITALAPSTLK	1529.8	42%	+ 1
SA9	DGEAGLQGPR	998.5	20%	-1
SA10	MLKEFADLK	1093.6	55%	0
SA11	EIVRDIKEK	1128.7	33%	0
SA12	TAEDLQAVEEK	1231.6	36%	-3

Generally, most AMPs are positively charged, and their electrostatic interactions with negatively charged lipid groups on the outer surface of bacterial cytoplasmic membranes contribute to their bacteriostatic activity (Ma et al., 2020). Similarly, hydrophobicity is a crucial characteristic of AMPs; hence, most AMPs have hydrophobicity of 30–60%, which enables them to form transient pores on the bacterial outer membrane to cause the leakage of cell contents and or allow the entry of AMPs into cells to interfere with metabolism and disrupt the bacteria, resulting in death (Wang, 2013). In addition, when the proportion of functional amino acids (glycine, lysine, arginine) in the AMPs sequence is greater than 15%, it significantly increases the activity of the AMPs (Lachowicz et al., 2020). Based on the analyses of the charge number, hydrophobicity, and functional amino acid content of the isolated peptides, samples SA3, SA6, and SA8 had the best antimicrobial potential; therefore, their interaction with SPI was further investigated.

3.1.2. Molecular docking screening

To screen the potential interaction of peptides SA3, SA6, and SA8 with SPI, their predicted docking models with 7S and 11S proteins was analyzed using the Maestro software. As shown in Table 2, the binding free energy of SA3, SA6, and SA8 with 7S were − 2.4, -5.4, and − 2.7 Kcal/mol, respectively, whereas the binding free energy with 11S were − 3.4, -7.4, and − 3.1 Kcal/ mol, respectively. The binding model of SA6 with 7S and 11S was the most stable. Detailed hydrogen bonds formed between the SA6 and SPI were shown in Fig. 1. Hydrogen bonding is the intermolecular force that plays a major role in the binding process of peptides and proteins. In this case, the hydrogen bonding energy of SA6 with 7S and 11S was − 3.7 Kcal/mol and − 3.1 Kcal/mol, respectively, which is higher than the values for SA3 (-2.4 Kcal/mol and − 3.4 Kcal/mol) and SA8 (-2.7 Kcal/mol and − 3.1 Kcal/mol).

Table 2

Docking results of potential AMPs and SPI
SPI	peptide No.	Binding Free energy Kcal/mol	H Bond energy Kcal/mol	Van Der Waalss energy Kcal/mol	Electrostatic energy Kcal/mol	Total Intermolecular energy Kcal/mol
7S	SA3	-2.4	-0.9	-35.5	-16.7	-52.2
	SA6	-5.5	-3.7	-50.1	-20.8	-70.9
	SA8	-2.7	-1.4	-40.1	-18.8	-58.8
11S	SA3	-3.4	-1.9	-26.7	-20.3	-47.0
	SA6	-7.4	-3.1	-34.2	-30.1	-64.3
	SA8	-3.1	-1.4	-28.1	-21.9	-50.0

Molecular docking is an effective method for analyzing molecular interactions, hence, it can be used to screen peptides that interact with 7S and 11S proteins. Previous results on 7S and 11S molecular docking showed that electrostatic and hydrophobic interactions are the basis of the interaction between SPI and small molecules, while hydrogen bonding is the main force that facilitates the binding (Zhang et al., 2022). As shown in Figs. 1A and B, Gly in SA6 could form hydrogen bonds with the residues of 7S or 11S. The negatively charged carboxyl group of glycine drives the electrostatic force to approach the positively charged Arg in 7S and 11S proteins, while the head of the guanidine group in Arg has a special rigid planar hydrogen bond donor that can form bidentate hydrogen bonds with the negative group to facilitate the tight binding between SA6 and SPI (Mota et al., 2018). In addition, hydrophobic interactions facilitate the formation of hydrogen bonds between SA6 and 7S and 11S proteins. Pro, Ile, and Met are the main amino acids in the hydrophobic regions of 7S and 11S proteins and are the main contributors to the hydrophobic forces (Chu et al., 2018). In peptide SA6, the Ala, Val, and Met residues promote the formation of hydrogen bonds by interacting with hydrophobic residues in 7S and 11S proteins. We, therefore, speculate that SA6 could form stable particles with SPI through hydrogen bonding.

3.2. Bacteriostatic activity of SA6

Table 3 shows the MICs of peptide SA6 against Gram-positive (S. aureus and B. cereus) and Gram-negative (V. parahaemolyticus and E. coli) bacteria. The MICs of SA6 against V. parahaemolyticus and E. coli were 125.0 µg/mL, while that against S. aureus and B. cereus were 15.6 µg/mL and 31.2 µg/mL, respectively. These results indicated that SA6 has broad-spectrum bacterial inhibition with a strong inhibitory effect on S. aureus. Further analysis of the inhibitory activity of peptide SA6 on S. aureus using time-kill curve analysis revealed that bacterial numbers decreased significantly with treatment time (Fig. 2). When the bacteria were exposed to 1 × MIC SA6 for 5 h, bacterial numbers decreased by 99%, indicating that SA6 inhibited bacteria concentration-dependently.

Table 3

Antibacterial activity of peptide SA6 against Gram-negative and Gram-positive bacteria
Peptides	MIC (µg/mL)
	Gram negative		Gram positive
	V. parahaemolyticus	E. coli	S. aureus	Bacillus cereus
ARHQGVMVGMGQK	125	125	15.60	31.25

The bacteria inhibitory effect of SA6 on Gram-positive bacteria was superior to that on Gram-negative bacteria, which could be due to differences in their cell wall composition. Moreover, the outer membrane of Gram-positive bacteria has a thick peptidoglycan layer that limits the entry of AMPs (Pasquina-Lemonche et al., 2020). However, given that peptide SA6 has an Arg residue in its sequence, which has a unique spatial geometry, and is facilitated by the amphiphilic amino acids Lys and Met, allows SA6 to disrupt the peptidoglycan layer and gets inserted into the cell wall of Gram-positive bacteria (Torcato et al., 2013). The MIC of SA6 against S. aureus (i.e., 15.6 µg/mL) was lower than that of some previously reported peptides against S. aureus, including peptides GRRRRSVQWCA (MIC = 64.0 µg/mL) and SFETRFEKMDNL (MIC = 33.5 µg/mL). Thus, SA6 has good potential for controlling foodborne pathogenic bacteria, especially S. aureus (Costa et al., 2014; Yao et al., 2016).

3.3. SPI-SA6 stability analysis

The effect of different concentrations of SA6 on the average particle size of SPI-SA6 particles is shown in Fig. 3A. The particle size of SPI-SA6 (363.97 nm and 364.34 nm) was significantly lower than that of SPI (464.34 nm) when the concentration of SA6 was 2× MIC and 4× MIC. The particle size of SA6 at a concentration of 1/4× MIC to 1× MIC was not significantly different from that of SPI. The PDI also decreased to 0.46 and 0.40 with the addition of SA6 at 2× MIC and 4× MIC (SPI was 0.62), indicating excellent particle size and dispersibility. For the Zeta potential of the SPI-SA6 particles (Fig. 3B), the zeta potential of SPI-SA6 containing 4× MIC and 2× MIC SA6 were − 30.5 and − 27.3, respectively, which were not significantly different from that of SPI (-35.2). In addition, SA6-SPI interaction analysis showed that adding urea significantly increased the particle size of SPI-SA6 (Fig. 3C), indicating the breaking of hydrogen bonds.

Small molecules can effectively improve the stability of SPI (Wang & Wang, 2015), which is consistent with our data, given the ability of peptide SA6 to reduce the average particle size and PDI values in SA6-SPI, although this phenomenon was only observed when SA6 was added at a concentration greater than or equal to 2× MIC. The compact spherical structure of natural SPI makes it easy to form a polymerized state to accumulate in solutions and destabilize the solution (Huang et al., 2020). When SA6 was added at a high concentration, small peptides were distributed on the SPI surface through hydrogen bonding, and the hydrophobic bundles on the SPI surface were shielded (Wang et al., 2022). This shielding effect reduced the aggregation of SPI and the instability of the dispersion. On the contrary, interactions between SPIs were not sufficiently shielded by a low concentration of SA6, with no significant difference between particle size and PDI. In general, the absolute value of zeta in SPI solutions increased with increasing stability (Ghribi et al., 2015). However, in this study, the zeta values were not significantly different from the control after adding a high concentration of SA6, which could be due to the cationic properties of SA6. The addition of SA6 reduced the electronegativity of the solution system rather than due to the solution instability, which is similar to previous studies on metal cation addition to SPI solution, where the zeta values decreased with increasing metal ion concentration in the solution system (Danaei et al., 2018). For the relationship between SA6 and SPI interactions, the results in Fig. 3C are consistent with that of molecular docking (Figs. 1A and B). The polarity of the environment surrounding the SPI was changed by the urea molecules, resulting in protein swelling, hydrogen bond breakage, and a significant increase in average particle size (Huang et al., 2022). Previous studies have shown that the hydrogen bonds of soy peptide nanoparticles are disrupted by urea leading to an increase in their average particle size (Zhang et al., 2018). In the current study, urea increased the average particle size of SPI-SA6 to 18.14%, a value significantly higher than that of SPI (6.36%), indicating that urea broke the hydrogen bonds between SPI-SA6.

3.4. Stability and antibacterial analysis of Pickering emulsions

3.4.1. Stability of Pickering emulsions

Microscopic examination of Pickering emulsions prepared with SA6 (Figure. 4A) revealed that the average droplet size of Pickering emulsions decreased with increasing SA6 concentration. Moreover, the droplet distribution was uniform without the appearance of flocculated droplets. The viscosity of emulsions decreased with increasing shear rate, a shear thinning behavior similar to non-Newtonian fluids (Figure. 4B). Besides, the energy storage modulus (G') and loss modulus (G'') of both Pickering emulsions increased (larger G' and G'' values) with increasing frequency compared with control (Fig. 4C). Visualization of the stability of the SPI-SA6 Pickering emulsions over a storage period of 6 days (25°C) revealed that the Pickering emulsions were stable (Fig. 4D), while that of the control group showed significant separation and flocculation after 5 d (Fig. 4E). When changes in particle size of the SPI-SA6 Pickering emulsions at the microscale level were examined (Figs. 4F and 4G), the mean particle size was smaller compared with the control during storage, which did not change significantly with storage time. In contrast, the control group had a significant rightward shift in mean particle size at 7 d. These results were consistent with the observations in Fig. 4D, indicating that SPI-SA6 improved the stability of Pickering emulsions.

Given that the addition of SA6 reduced the mean particle size of SPI-SA6 (Fig. 3) to enhance the quality of Pickering emulsions, it further indicates that SPI-SA6 improves the stability of Pickering emulsions. Generally, reducing the droplet size of Pickering emulsions to improve the structure of the surrounding dense interface is an effective way to enhance the stability of emulsions (Wu & Ma, 2016). Thus, we observed that SA6 at 2×MIC and 4×MIC concentrations significantly reduced the emulsion droplet size and enhanced the interfacial structure, possibly due to the small particle size of SPI-SA6. When the concentration of SA6 was greater than 2×MIC, the particle size and PDI of SPI-SA6 particles were smaller than in other samples. The droplet surface was entirely covered by SPI-SA6 particles, which facilitated the formation of tiny droplets. The particle size increased and self-aggregated when the SA6 concentration was less than 2×MIC due to insufficient particles to cover the oil-water interface and, therefore, an increase in droplet size. Analysis of the rheological properties of the Pickering emulsions showed that SPI-SA6 particles contributed to forming a stable emulsion network structure, thereby improving the stability of the emulsion. As shown in Fig. 4C, the elastic and viscous moduli of SPI-SA6 Pickering emulsions gradually increased, with a noticeable increase in G' compared with G'', indicating that both samples form a gel-like network structure. Both moduli were weakly dependent on frequency, typical of elastic structures formed by highly dense layers of particles (Chevalier & Bolzinger, 2013). The absence of intersections between the emulsion samples G′ and G″ suggested that the viscosity and elasticity of SPI-SA6 Pickering emulsions were not disrupted with increasing strain (Luo et al., 2019). Moreover, smaller droplets might lead to tighter buildup and reduced mobility; thus, SA6 Pickering emulsion was more stable. For the stability of the emulsions during storage, the SPI Pickering emulsion showed an increase in the creaming index after 5 d, which is consistent with previous studies that found that emulsions stabilized by SPI alone were not stable enough to prevent droplet aggregation during storage (Chevalier et al., 2013). The properties of SA6 Pickering emulsion did not change significantly during storage, probably due to the reduced buoyancy effect of droplets. Usually, during storage, the large-size droplets in the emulsion move upward, increasing the creaming index. The buoyancy effect diminished as the emulsion's particle size decreased, which retards the movement of the droplets.

3.4.2. Bacterial inhibition of Pickering emulsions

When the inhibitory effect of the Pickering emulsions on bacteria (S. aureus) was examined, SA6 and SPI-SA6 Pickering emulsions were found to significantly inhibit S. aureus by 84.8% and 74.1%, respectively, compared with control and SPI Pickering emulsion (Fig. 5A). Observation of the bacterial microstructure revealed that the cell membrane was disrupted by SA6 and SPI-SA6 Pickering emulsion, resulting in the cell surface becoming irregular or wrinkled and reduced electron density in the cytoplasm (Fig. 5B). In contrast, for bacteria cells treated with PBS control or SPI Pickering emulsion, the cell membrane structure was smooth and intact, while the electron density in the cytoplasm was high and uniform. During the storage period, the number of S. aureus colonies in the Pickering emulsion decreased from 1.9 CFU/mL to 0.3 CFU/mL and 0.8 CFU/mL at 1 d and 2 d, respectively (Fig. 5C). On the other hand, the number of S. aureus in the SPI-SA6 Pickering emulsion remained consistently low compared with control throughout the storage period, indicating that SA6 conferred antimicrobial properties to the emulsion and prolonged the effective bacterial inhibition time.

In previous studies, SPI was used as nanoparticles dispersed in emulsions with no antibacterial activity, and SPI served as a "medium" for microorganisms that led to emulsion deterioration (Chen et al., 2021). Although the bacterial inhibition of natural food-grade Pickering emulsions is usually produced by encapsulating small molecules of bacteriostatic substances, here we propose a non-encapsulation strategy to improve bacterial inhibition of emulsions using SPI-SA6 antimicrobial emulsification particles. We revealed that SPI-SA6 Pickering emulsion could effectively inhibit the growth of S. aureus. From the microstructure of bacteria, SA6 and SPI-SA6 Pickering emulsions exerted bacteriostatic properties by disrupting the bacterial cell membrane, which is the typical bacteriostatic behavior of cationic AMPs, indicating that SPI-SA6 adsorbed onto the surface of oil droplets did not affect the binding of SA6 to bacterial cell membranes (Almeida et al., 2019). In addition, we observed a significant increase in the effective bacteriostatic inhibition time of SPI-SA6 Pickering emulsions compared with SPI Pickering emulsions. Previous studies have shown that the tight buildup of solid particles at the interface of Pickering emulsions reduces the effective diffusion surface area of oil droplets, resulting in slow release (Xu et al., 2020). Our results may be due to the combination of hydrogen bonding between SA6 and SPI and adsorption between droplets to produce a slow-release behavior of SA6, thus prolonging the effective antimicrobial time of SPI-SA6 Pickering emulsions.

In this study, a peptide designated SA6, identified from salted P. vannamei, could stably bind to SPI through hydrogen bonding and effectively inhibit the growth of S. aureus. In addition, SA6 inhibited the aggregation and sedimentation of SPI through the shielding effect, reducing the particle size and PDI of SPI-SA6. Further, we successfully prepared Pickering emulsions with bacterial inhibitory properties using SPI-SA6. The effective bacterial inhibition time of Pickering emulsion was significantly extended during storage by SPI-SA6.

Acknowledgements

This study was financially supported by National Key R&D Program of China (2020YFD0900900).

Funding

The work has been funded by the National Key R&D Program of China (2020YFD0900900).

Data Availability

Data supporting the findings of this study are available upon request from the corresponding author.

Authors Contribution

Jingyi Dai: Experiments, Data analysis, Writing - original draft. Jude Juventus Aweya: Methodology, Writing - review & editing. Ritian Jin: Software. Wuyin Weng: Validation, Methodology. Rong Lin: Visualization. Yuanhong Xie: Methodology. Shen Yang: Supervision, Resources, Writing - review & editing.

Conflict of Interest

The authors declared that they have no conflicts of interest to this work.

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The antibacterial activity and Pickering emulsion stabilizing effect of a novel peptide, SA6, isolated from salted-fermented Penaeus vannamei

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Materials, bacteria strains, and reagents

2.2. Salt fermentation of P. vannamei and screening of isolated peptides with antimicrobial potential.

2.3 Molecular docking prediction of SPI and AMPs interactions

2.4 Determination of antibacterial activity

2.5 Determination of particle size, zeta potential, and interparticle interaction of SPI-SA6 particles

2.7 Preparation of emulsion and observation of emulsion droplets

2.8 Rheological Measurements

2.9 Emulsion particle size measurement

2.10 Flow cytometer measurements

2.11 Transmission electron microscopy (TEM) observation

2.12 Statistical analysis

3. Results And Discussion

3.1. Screening of peptides isolated from salted P. vannamei

3.1.1. Identification of peptides with antimicrobial potential

3.1.2. Molecular docking screening

3.2. Bacteriostatic activity of SA6

3.3. SPI-SA6 stability analysis

3.4. Stability and antibacterial analysis of Pickering emulsions

3.4.1. Stability of Pickering emulsions

3.4.2. Bacterial inhibition of Pickering emulsions

4. Conclusions

Declarations

References

Additional Declarations

Supplementary Files

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