Antifungal application of biosynthesized selenium nanoparticles with pomegranate peels and nanochitosan as edible coatings for citrus green mold protection | Journal of Nanobiotechnology

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The chitosan was successfully generated in current study; the attained chitosan physiognomies suggested its successful extraction, as chitosan should have ≥ 70% DD, which indicated effectual deacetylation of chitin substrate [31, 37, 41].

The FTIR analysis indicated the most effectual bonds/groups in screened molecules. For the NCTspectrum (Fig. 1-NCT), it had the main characteristic bands of the typical bands of natural chitosan [41, 42]. The band around 3426 cm−1 indicated the main locations for TPP interactions with chitosan [37]. The bands appeared at 2919 and 2874 cm−1 are indicatives to C–H symmetric/asymmetric stretching, which are typical bands for polysaccharides. The following detected bands are distinctive to NCT biochemical bonding: ~ 1655 cm−1 (stretched C = O of amide I); 1321 cm−1 (vibrated C–N stretching); 1411 and 1358 cm−1 (CH2 bending and CH3 symmetrical deformations); 1153 cm−1 (bridge of C–O–C asymmetric stretching); 1066 and 1025 cm−1 (C–O stretching) [43,44,45]. The appeared peaks at 1153 and 1066 cm−1 indicated the C–O overlapping and formation of NCT after interaction of PO4 and NH4 groups in NCTmolecules; and also the peak at 1196 cm−1 that is corresponding to stretched P = O pond, validated NCTsynthesis following TPP interaction [37, 43].

The designated biochemical bonds in PPE spectrum included the bands at 3347 cm−1 [bonded –NH and –OH groups of carboxylic acid (CA), gallic acid, tannic acid and ellagic acid] [17, 46]; 2977 cm−1 [stretched C–H vibration of methyl and methoxy groups and stretched vibration of –CH3/ –CH2 groups of CA]; 2888 cm−1 [vibrated C‒H stretching of alkyl];1723 cm−1 [N–H bonds of carboxylic and amides groups]; 1601 cm−1 [stretched C = C vibration of aromatic rings and vibrated N–H of amines]; 1361 cm−1 [C–O stretching in acid groups]; 1439 cm−1 [aromatic rings]; 1182 cm−1 [–OH deformation and C–O stretching of primary alcohols]; 1042 cm−1 [–OH deformation and C–O stretching of tertiary alcohols]; and at 879 cm−1 [aromatic ring vibration] [21, 45,46,47].

The combined PPE/SeNPs spectral analysis indicated the most responsible groups in PPE for the biosynthesis of SeNPs. The PPE band at 3426 cm−1 shifted to 3482 cm−1 in PPE/SeNPs spectrum, indicating Se interaction with N–H and O–H groups, whereas the C–H band (at 2888 cm−1 in PPE spectrum) mostly disappeared in PPE/SeNPs spectrum, indicating its roles in SeNPs conjugation/reduction [22]. Also, the bands in PPE spectrum at 1723 cm−1 (N–H of amides and CA groups) and at 1601 cm−1 (aromatic rings C = C) were remarkably shifted, as indicator of their roles in SeNPs synthesis/reduction [23, 46]. The beak at 1439 cm−1 (aromatic rings in PPE spectrum) shifted to 1378 cm−1 in PPE/SeNPs spectrum. Furthermore, the emergence of multiple notable bands at 1603 cm−1 and in the range of 756–812 cm−1 in PPE/SeNPs spectrum clearly indicated the formation of novel bonds and vibrated bending (mainly of Se–O) after interactions of Se ions with PPE biomolecules [24, 48]. These detectable bands in PPE/SeNPs spectrum strongly validated the PPE potentiality for conjugating, reducing and stabilizing SeNPs; the reduction/stabilizing of SeNPs forms are predominantly depending on the biomolecules’ nature and their stabilization capability that enable Se ions interaction with them [24, 46, 49]. Thus, PPE could be advocated as valued stabilizer/reducer for SeNPs biosynthesis.

FTIR analyses are useful to assess whether the conjugation of PPE/SeNPs with NCT is physical or chemical entrapment; if minimal or no deviations from parental compounds FTIR spectra were observed, the physical entrapment is expected, whereas spectral bands’ shift or varied intensities indicate probable chemical interactions between molecules [20]. As many peaks in NCT/PPE/SeNPs spectrum were shifted and varied from their parental compounds (NCT and PPE/SeNPs), along with the identical peaks that were detected from both agents, the spectral comparison could strongly indicates both biochemical and physical interactions during PPE/SeNPs entrapment within NCT [20, 44].

The NCT high capability of capping SeNPs and formation of highly stable nanocomposites with minute Ps were demonstrated formerly [27, 42]. Generally, NPs with elevated ζ potentials (≥ + 30 mV or ≤  − 30 mV) display high stability and dispersity degrees due to electrostatic repulsion between particles [50, 51].

The NCT synthesis via TPP cross-linking was proven as effective operative protocol, employing ionic-gelation interaction; the synthesized NCT with such protocol had astonishing properties for practical employment either as plain bioactive molecules or as nanocarriers for other bioactive constituents, or bases for active ECs [27, 37].

The tiny Ps of phytosynthesized SeNPs and their remarkable dispersion indicate the advanced capability of PPE for reducing/stabilizing SeNPs. The antioxidating, radical scavenging and reducing potentialities of PPE were acknowledged, principally due to the extract contents from phenolics, tannins, alkaloids, and flavonoids (e.g., punicalagin, gallic tannins, catechins, quercetin, kaempferol, ellagic acid, catechol, castalagin,, gallocatechin, and granatin) [15, 23, 24, 48]; these precious phytocompounds could effectually play principal roles in SeNPs biosynthesis.

The general antimicrobial and specific antifungal potentialities of screened agents have been documented toward various microbial pathogens [3, 19, 36, 52]. The chitosan microbicidal actions depend mainly on its surface positive charges, which enable its attachment and interaction with microbial membranes and internal organelles, beside increase of intracellular ROS “reactive oxygen species” production, suppress cellular bioactivities and upsurge cellular membranes’ permeability [32, 33]. These actions become more forceful and effectual by transforming the biomolecule to nanoforms (e.g. NCT), because of the increased reacted surface area and tiny Ps that enable more effectual interactions and biocidal actions [30, 32]. The PPE antimicrobial activities are principally attributed to its bioactive phytoconstituents (e.g. phenolics, tannins, alkaloids, flavonoids and acids), which were previously investigated, validated and applied for controlling numerous bacterial and fungal pathogens [15, 17, 19].

The PPE mediated nanometals were also verified as potent microbicidal agents that have the synergistic actions from both PPE and synthesized nanometals, including Se, Ag, Au and Zn NPs [22, 23, 48].

For the NCT/PPE/SeNPs, which was innovatively composited in current investigation, the antifungal synergism between compositing agents (NCT, PPE and SeNPs) was clear and forceful, as evidenced from the widest ZOIs and least MFCs values; this indicates that composites ingredients could preserve their distinctive antifungal actions. Matching findings were recently reported [20], employing NCT and PPE composites as antioxidant conjugates. Additionally, the application of NCT for carrying, capping and delivering further bioactive molecules such as plant extracts, essential oils and nanometals have been reported to augment their combined actions as antimicrobial, antioxidant or even anticancerous nanocomposites [29, 43,44,45]. These former findings could verify the obtained role here of NCT to strengthen the antifungal actions of both PPE and SeNPs.

The antifungal potentialities of NCT and its parent chitosan have been proved toward numerous postharvest pathogens; the exact modes of action still vague, but it could be suggested that the positively charged NCT can attach hyphal walls, interact with fungal membranes and penetrate within these membranes to inhibit/destruct the fungal biosystems and lead to their lysis [30, 32, 34]. The PPE/SeNPs are suggested to damage microbial cells because of their combined biocidal activities. The destruction and deformation of P. digitatum hyphae was formerly observed, after treating them with PPE-related phytochemicals [13], which advocates current obtained results, in addition to SeNPs antifungal action.

The innovative nanocomposite here (NCT/PPE/SeNPs) is suggested to perform multiple actions; firstly the NCT carries/holds PPE/SeNPs to the fungal hypha and attaches/interacts with them to cause softening and partial lysis of membranes, then it could penetrate inside the hypha and the liberated PPE/SeNPs beside NCT are capable to intermingle with intracellular organelles/biosystems to suppress their vital functions, which consequently lead to fungal deformation and lysis [30, 32, 53].

The chitosan- and NCT-based ECs were recurrently validated as effectual treatments for preventing postharvest decays/losses in many agricultural crops. The main distinguished functions of these ECs, beside the antimicrobial actions, are to form barriers against fungal new infection, protect fruit from moisture loss and manage the respiration and over-ripening of coated crops [29, 30, 35, 36]. PPE was also the principal component of ECs for many fruits; the extract could eliminate microbial growth on fruit and maintain their freshness because of powerful PPE antimicrobial and antioxidant potentialities [3, 7]. Furthermore, the conjugation of chitosan and PPE in ECs of fruits and vegetables could have higher functionalities than each individual component for preserving coated crops, enlarging their shelf lives and prevent their microbial decays [18, 46]. These functions were suggested to be elevated with conjugation of NCT with PPE and their usage in ECs of fruits. NCT has higher capabilities to encase the whole fruits surface, fill their pores, deliver the accompanied molecules to fruit, and prevent them from fungal invasions and quality loss [20, 37, 45]. The combination of NCT with PPE/SeNPs is innovatively presented here to employ this nanocomposite as effectual EC for orange fruit; the biosafe nature of NCT and its elevated capping ability could provide more biosafety attributes toward the potential toxicity from SeNPs, as the embedding of nanometals into biopolymer matrix was previously proven to diminish their biotoxicity and increase their biocompatibility and safety [27, 31, 35, 42, 53].

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