5.1 Surface charge and hydrodynamic size
Zeta potential is a physical characteristic of suspended particles and macromolecules and refers to the electrical potential of the nanoparticle, being influenced by its constituents (Ferreira & Nunes, 2019). The zeta potential and the size of a particle are related to its antibacterial activity, as they affect particle interaction with molecular sites present on microorganism surface (Song et al., 2021).
In the present study the formation of nanoparticles and their physicochemical characteristics were strongly influenced by the concentration of TPP, chitosan and lactoferrin (Table 3).
Table 3 – Zeta potential and nanoparticle size of lactoferrin-TPP, chitosan-TPP, lactoferrin-chitosan-TPP and pure biopolymers.
Sample
|
Zeta Potential
(mV)
|
Hydrodynamic diameter (nm)
|
Pure lactoferrin (L)
|
+27.90 ± 1.35e
|
3.77 ± 0.36d
|
Pure chitosan (C)
|
+59.93 ± 0.21a
|
4.82 ± 1.69d
|
7L: 3TPP
|
-24.27 ± 1.01i
|
8.32 ± 0.69cd
|
9L:1TPP
|
-24.00 ± 0.17i
|
9.14 ± 1.66cd
|
7C: 3TPP
|
+38.03 ± 0.15c
|
108.93 ± 11.87b
|
9C:1TPP
|
+47.03 ± 0.76b
|
93.37 ± 21.88b
|
2.5L:4.5C:3TPP
|
+4.27 ± 0.96f
|
587.00 ± 48.50a
|
3.5L:3.5C:3TPP
|
+1.02 ± 0.22g
|
641.00 ± 62.22a
|
4.5L:2.5C:3TPP
|
-2.05 ± 0.06h
|
612.00 ± 14.73a
|
3.5L:5.5C:1TPP
|
+39.30 ± 0.79c
|
81.87 ± 13.76bc
|
4.5L:4.5C:1TPP
|
+33.07 ± 0.97d
|
97.67 ± 12.50b
|
5.5L:3.5C:1TPP
|
+31.27 ± 0.25d
|
123.33 ± 18.04b
|
Means followed by different letters in the same column differ significantly from each other at the 5 % significance level.
Chitosan presented a zeta potential of +59.93 mV, this positive charge is attributed to its amino groups and degree of acetylation (DA) (de Carvalho et al., 2019). The chitosan used on the present study was high deacetylated (75 – 85 %). Traditionally, when the chitosan DA is low, a greater number of amine groups are available to be protonated (Bhardwaj et al., 2021). This polysaccharide has a pKa value of 6.3 so at pH values below the pKa the amino groups became protonated, which makes the polysaccharide chain positively charged (Benltoufa et al., 2020). The chitosan hydrodynamic diameter found (4.82 nm, Table 3) is close to that reported by Rinaudo and Domard (1989).
Lactoferrin has a relatively high isoelectric point (pI = 8.7) that makes this protein positively charged over a wide pH range (Maciel et al., 2020). The lactoferrin structure is divided into two lobes, N and C, and the regions of greatest positive charge are found at the N-terminus and in the connecting helix between the two lobes (Baker & Baker, 2009). The small hydrodynamic size of 3.77 nm found for this polypeptide (Table 3) is related to its single chain with a molecular weight of 80 kDa (Niaz et al., 2019).
Lactoferrin-TPP nanoparticles (7L:3TPP and 9L:1TPP) exhibited negative charges (-24.00 and -24.27 mV) within the concentration range evaluated. Yang and collaborators (2019) also found similar zeta potential values for TPP-zein nanoparticles. The negative charge was attributed to the introduction of negatively charged phosphate groups into protein aggregates. Multivalent ions such as sodium tripolyphosphate can decrease the pI value of proteins, and thus decrease the zeta potential (Chen & Soucie, 1986).
The size values of lactoferrin-TPP nanoparticles are much smaller than other nanoparticles, due to the presence of phosphate groups in the protein aggregates which increased the amount of negative charges and intermolecular repulsion, decreasing the size of the particles (Hadidi et al., 2021; Wang et al., 2019). In addition, sodium tripolyphosphate (TPP) is a crosslinker with a much lower molecular weight (0.367 kDa) compared to lactoferrin (80 kDa) and chitosan (150 kDa) (Khoerunnisa et al., 2021; Leiva et al., 2021).
Pure chitosan had a higher zeta potential than chitosan-TPP nanoparticles (Table 3) since with the addition of TPP to chitosan there was a reduction in the surface charge due to the partial neutralization of some chitosan cationic groups by the anionic groups of the TPP (de Carvalho et al., 2019).
The zeta potential of the chitosan-TPP nanoparticles, 7C:3TPP and 9C:1TPP were +38.03 and +47.03 mV respectively. Nanoparticles with zeta potential greater than ± 30 mV are considered stable (Müller et al., 2001). As the concentration of chitosan increases in the nanoparticle, the exposure of amino groups on the surface also increases that result in higher zeta potential values and smaller particle size (Pan et al., 2020). When the particle presents are high positive charge density, the electrostatic repulsion between the particles are enhanced resulting in nanoparticles with smaller hydrodynamic radius (Duarte & Picone, 2022).
The lactoferrin-chitosan-TPP nanoparticles with higher proportions of TPP (2.5L:4.5C:3TPP, 3.5L:3.5C:3TPP and 4.5L:2.5C:3TPP) presented zeta potential values of +4.27, +1.02, -2.05 mV, respectively. The neutralization of the particle's surface charge (zeta values approaching zero) decreases the stabilizing repulsive forces between the individual particles and resulted in unstable aggregates of larger sizes (587.00, 641.00 and 612.00 nm).
The nanoparticles with lower concentrations of TPP (3.5L:5.5C:1TPP, 4.5L:4.5C:1TPP and 5.5L:3.5C:1TPP) exhibited higher zeta potentials (+39.30, +33.07 and +31.27 mV) and smaller sizes (81.87, 97.67 and 123.33 nm). The increase in surface charge is related to a greater availability of positively charged chitosan and lactoferrin amino groups on the surface of the nanoparticles. Due to the higher charge and smaller size of these nanoparticles, they present promising antimicrobial action and therefore were selected for further analyses.
5.2 Fourier transform infrared spectroscopy (FTIR)
FTIR spectroscopy is a fast, noninvasive technique that can identify different functional groups in samples through the vibrations of their molecular bonds. This technique is often used for chemical characterization of the substance of interest (Ciursă et al., 2021; Fan et al., 2021). 9C:1TPP, 4.5L:4.5C:1TPP and 3.5L:5.5C:1TPP nanoparticles with the highest zeta potentials and the smallest sizes were analyzed by FTIR.
In the chitosan spectrum, the peak at 2982 cm-1 corresponds to CH vibrations, and the characteristic peaks at 1132 and 1041 cm-1 correspond to the glycosidic ring (Mauricio-Sánchez et al., 2018; Pan et al., 2020). The 1612 cm-1 peak is attributed to chitosan amide I (Yang et al., 2017). In the infrared spectrum of TPP, the peak at 1203 cm−1 can be attributed to P=O elongation (Antoniou et al., 2015; Pan et al., 2020).
The chitosan characteristic peaks (1132 and 1041 cm-1) were also observed in the FTIR spectrum of chitosan-TPP nanoparticles (Figure 1). Furthermore, the amide I peak changed from 1612 cm-1 to 1616 cm-1 indicating the establishment of electrostatic interactions between the chitosan amino group and TPP phosphate groups (Zeng et al., 2021).
The characteristic peaks of lactoferrin were observed at 1601 cm-1 (amide I), 1480 cm-1 (amide II), 1420 and 1344 cm-1 (amide III) (Duca et al., 2018; Santos et al., 2021). In the spectra of lactoferrin-chitosan-TPP nanoparticles, amide I shifted to stronger intensities. Amide I and amide II alterations are associated with electrostatic interactions between proteins and other compounds (Luo et al., 2010; Yang et al., 2019).
The 1600-1700 cm-1 bands were more intense on the 4.5L:4.5C:1TPP and 3.5L:5.5C:1TPP nanoparticles (Figure 1) and are related to hydrogen bonds. These bonds may occur due to the presence of several types of functional groups, such as –NH2 present on lactoferrin and chitosan, –OH of lactoferrin, chitosan and TPP, and –COOH groups of lactoferrin (Tavassoli et al., 2021).
5.3 Circular dichroism (CD)
Circular dichroism (CD) spectroscopy is a method traditionally used to examine the structures and conformational changes of proteins (Miles et al., 2021), to monitor structural changes induced by interactions with ligands, and to determine the stability of the protein under changing pH or temperature conditions (Miles & Wallace, 2006).
A negative peak at 200 nm and a smooth peak at 222 nm can be seen in the spectrum of Figure 2 indicating the protein content of disordered structures and alpha helices respectively (Miles et al., 2021). The presence of an intense positive band at 190 nm reveals the presence of β structures (Saraiva et al., 2017). Changes in the magnitude of ellipticity across the measured range indicate conformational changes in lactoferrin (Wang et al., 2017). Table 4 shows an increase in alpha helix in the presence of TPP, related to a more ordered structure. This is attributed to the electrostatic balance of the protein surface and steric hindrance effect (Jin et al., 2021).
Table 4 - Fractions of secondary structure of lactoferrin and lactoferrin - TPP nanoparticles (9L:1TPP).
|
α helix
|
β sheet
|
Turn
|
Unordered coil
|
Lactoferrin
|
0.209±0.002a
|
0.260±0.010a
|
0.193±0.009a
|
0.371±0.004a
|
9L:1TPP
|
0.327±0.017b
|
0.203±0.043a
|
0.182±0.003a
|
0.306±0.055a
|
Means followed by different letters in the same column differ significantly from each other at the 5 % significance level.
Changes in the secondary structure of lactoferrin when it interacts with TPP suggest partial unfolding of the protein (Alhalwani et al., 2019). Electrostatic interactions can contribute to protein folding (Apurva & Mazumdar, 2020). Molecular forces influence α-helix shapes and β-sheets, as they influence the length and strength of the between two atoms (Howell, 1992). Thus, there are changes in the surface of the protein and consequently in the external charges of the nanoparticles influenced by the new molecular organization (García-Otero et al., 2021).
5.4 Morphology and size
The nanoparticles that showed higher zeta potential, smaller sizes and higher antimicrobial activity (section 5.5) were chosen for morphology characterization. Figure 3 shows the morphological aspect and size of chitosan-TPP nanoparticles 9C:1TPP, and lactoferrin-chitosan-TPP nanoparticles 3.5L:5.5C:1TPP and 4.5L:4.5C:1TPP.
All samples exhibited spherical morphology and size close to the measured by DLS assays. The spherical character of nanoparticles is associated with greater bactericidal action (Divya et al., 2017) due to increased interaction with the microorganism membrane, resulting in bacterial cell disruption (Linklater et al., 2020).
5.5 Antimicrobial activity
The minimum inhibitory concentration (MIC) of lactoferrin-chitosan-TPP nanoparticles and the pure compounds in GYP broth against Staphylococcus aureus are shown in Table 5.
Table 5 – Minimum inhibitory concentration (MIC) of pure compounds and lactoferrin-TPP, chitosan-TPP and lactoferrin-chitosan-TPP nanoparticles against Staphylococcus aureus.
Sample
|
MIC (mg/mL)
|
chitosan
|
0.1053
|
lactoferrin
|
2.0000
|
TPP
|
*
|
9C:1TPP
|
0.0741
|
9L:1TPP
|
*
|
3.5L: 5.5C:1TPP
|
0.0370
|
4.5L:4.5C:1TPP
|
0.0463
|
5.5L:3.5C:1TPP
|
0.0556
|
**no inhibition at concentrations up to 8 mg/ml
Means followed by different letters in the same column differ significantly from each other at the 5 % significance level.
Chitosan presented an MIC value of 0.1053 mg/ml and lactoferrin exhibited an MIC value of 2 mg/ml. The main antimicrobial mechanism of chitosan and lactoferrin is the electrostatic interaction between positively charged amino groups and negatively charged sites in the bacterial cell, causing cell lysis (Duarte et al., 2022; Goy et al., 2016). Therefore 9L:1TPP nanoparticle did not exhibit antimicrobial activity since they were negatively charged (Table 3).
The MIC of chitosan-TPP nanoparticles was 0.0741 mg/ml, which was lower than the MIC of chitosan. These MIC values show that there is an increase in the antibacterial potential of chitosan in the form of nanoparticles, and this is due not only to the high positive charge density of chitosan but also to the nanometric size of particles that leads to great surface area in relation to their volume (Perinelli et al., 2018; Mousavi et al., 2018; Kritchenkov et al., 2019). The smaller the particle size, the greater the area of the particles in contact with the bacterial surface (Maillard & Hartemann, 2013; Sullivan et al., 2018; Castelo Branco Melo et al., 2018).
This finding was supported by the MIC values of the lactoferrin-chitosan-TPP nanoparticles, which were much smaller than those of the pure compounds and the chitosan-TPP nanoparticles. Lactoferrin contributed to this decrease in MIC and can be attributed to the bioactivity of its peptides (Nakamura et al., 2022).
The alpha-helical peptides of lactoferrin are reported to provide bactericidal activity against several bacteria (Alhalwani et al., 2019). The dichroism spectra (section 5.3.) showed that alpha helix residues increased due to lactoferrin-TPP interactions, that may also have contributed to the greater bacteriostatic action of lactoferrin-chitosan-TPP nanoparticles against S. aureus in comparison to the pure compounds. Moreover, the antimicrobial action of lactoferrin-chitosan TPP nanoparticles can be related to a better distribution of positive charges and changes in the amphipathic balance of nanoparticle surfaces. Lactoferrin alpha helices have hydrophobic residues on one face and positively charged amino acids on the opposite face that confer hydrophilic character to the structure (Chaparro et al., 2018). Positively charged lactoferrin residues interact electrostatically with the surface of negatively charged bacteria. And the hydrophobic residues of lactoferrin interact with the lipophilic portion of the membrane of the microorganism, inducing cell rupture (Wang et al., 2018). Studies show that the antibacterial activity of lactoferrin is directly linked to the joint action of hydrophobic (such as tryptophan) and hydrophilic (such as Arginine) residues (Håversen et al., 2010; Nakamura et al., 2022).
5.6 The nanoparticles in strawberry coatings
The nanoparticles with the lowest MIC (3.5L: 5.5C:1TPP) were added to the coating composition of fresh strawberries, which were evaluated over 144 h. The appearance over time of the control and coated strawberries is shown in Figure 4.
All strawberries became darker over time (Figure 4). Anthocyanins are primarily responsible for strawberry color change (Wang et al., 2020). The process of ripening and senescence of strawberries is accompanied by the degradation of chlorophyll and synthesis of anthocyanins (Li et al., 2019a).
Anthocyanins are catalyzed by complex enzymes of the phenylpropanoid and flavonoid biosynthetic pathways, and tend to accumulate in the vacuole (Wang et al., 2020). The increase in sucrose content in strawberry also promotes the biosynthesis of anthocyanins.(Li et al., 2019b).
Although all groups visually shrank after 120 hours (Figure 4), the reduction in volume was less intense for strawberries in the NP+CMC group due to the weight loss barrier promoted by the coating (Figure 5). Fruit water loss is mainly caused by transpiration or transfer of water vapor from the surface of the fruit to the surrounding air (Parreidt et al., 2019).
5.6.1 Weight loss, TA, TSS and pH
Figure 5 shows the results for fruit weight loss with the coatings. Significant differences (p < 0.05) were found between the strawberries treated with the formulations and the controls. The control group showed weight loss close to 90 % after 144 h, whilst the groups coated with CMC and with NP+CMC lost 63 % and 55 % of their weight, respectively. Coatings serve as semipermeable barriers against moisture and gases transfer and can reduce respiration rate and weight loss of fruit (Maqbool et al., 2011). The results shown in Figure 5, indicate the effectiveness of coating in delaying strawberries weight loss is related to coating formulation and was more intense in the presence of nanoparticles.
The titratable acidity (TA), soluble solids content (TSS) and pH of strawberries during time are presented in Table 6.
Table 6. Titratable acidity (TA), soluble solids content (TSS) and pH of strawberries over time
|
Storage time (hours)
|
Control
|
CMC
|
NP
|
NP+CMC
|
TA (%)
|
0
|
0.155±0.003Aa
|
0.155±0.003Aa
|
0.155±0.003Aa
|
0.155±0.003Aa
|
|
48
|
0.141±0.001Ab
|
0.125±0.001Bb
|
0.124±0.003Bb
|
0.128±0.002Bb
|
|
144
|
0.105±0.001Ac
|
0.114±0.002Bc
|
0.106±0.001Ac
|
0.116±0.001Bc
|
TSS(%)
|
0
|
6.17±0.29Aa
|
6.17±0.29Aa
|
6.17±0.29Aa
|
6.17±0.29Aa
|
|
48
|
7.50±0.50Aa
|
6.43±0.40BCa
|
7.33±0.58ABa
|
6.23±0.25Ca
|
|
144
|
14.33±0.58Ab
|
11.93±0.40Bb
|
13.17±0.29Cb
|
10.87±0.12Db
|
pH
|
0
|
3.23±0.02Aa
|
3.23±0.02Aa
|
3.23±0.02Aa
|
3.23±0.02Aa
|
|
48
|
3.29±0.01Aa
|
3.24±0.04Aa
|
3.28±0.11Aa
|
3.25±0.05Aa
|
|
144
|
3.65±0.06Ab
|
3.49±0.01Bb
|
3.55±0.02Bb
|
3.40±0.02Cb
|
Means followed by different capital letters on the same line indicate significant differences between treatments (p <0.05).
Different lowercase letters in the same column indicate significant differences between the means over time (p < 0.05).
The TA content decreased significantly (p<0.05) for all treatments during the storage period. This decline in titratable acidity is due to a decrease in fruit citric acid (Hashemi & Jafarpour, 2020). The decrease in TA was more pronounced in uncoated (control) samples. The greatest loss of acidity in uncoated fruit is due to the use of organic acids as substrates for metabolism during storage (Díaz-Mula et al., 2012). The NP+CMC coatings can decrease the respiration rate and reduce the use of organic acids, which leads to TA retention in this group (Table 6).
Regarding the content of soluble solids (TSS), there was a gradual increase in all fruit (p<0.05) (Table 6). However, the control group presented higher TSS values after 144h (Table 6). The NP+CMC group maintained lower TSS values than the control group and the CMC and NP groups. The increase in Brix with time occurs due to the hydrolysis of starch into sugars, or anthocyanin, as the fruit ripens (Bashir & Abu-Goukh, 2003, Shankar et al., 2021). Therefore, ripening occurs less intensely in strawberries coated with NP+CMC.
Table 6 shows that the pH of fruit increased significantly over the storage time for all treatments (p<0.05). However, the fruit treated with NP+CMC exhibited a significantly lower pH in relation to the other three conditions after 144h. This indicates the NP+CMC coating decreases the degradation of organic acids, giving strawberries a controlled, low-oxygen environment (Rana et al., 2015). The pH increase is usually associated to a decrease in ascorbic acid content due to oxidation of vitamin C during ripening (Gutierrez-Molina et al., 2021).
5.6.2 Total aerobic mesophilic bacteria counts
Significant differences (p < 0.05) in total aerobic mesophilic bacteria counts were found for the fruit treated with the coatings and controls and over the days after 48 and 144 h, as shown in Figure 6. At the end of the storage period (144 h), fruit treated with NP+CMC had the lowest CFU, followed by those treated with NP. In the NP+CMC coating, the CMC promoted greater adhesion of the nanoparticles to the fruit, resulting in more intense bacteriostatic effect.