Characterization and evaluation of antioxidant and antimicrobial capacity of prepared liquid smoke-loaded chitosan nanoparticles

Highlights

• Liquid smoke-loaded Chitosan nanoparticles were prepared by ionic gelation.

• FT-IR, SEM, DLS and zeta potential were used to evaluate the nanoparticles.

• The nanoparticles exhibited 40.3% loading efficiency and 21.6% loading capacity.

• The nanoparticles function as a controlled release system of liquid smoke.

• The nanoparticles exhibited antioxidant and antimicrobial activity in vitro.

Abstract

The combined application of chitosan (CS) and liquid smoke (LS) leads to the development of a suitable material with synergistic properties and promising potential for use as an efficient preservative for the conservation of food products. The present work investigates the development and characterization of liquid smoke-loaded chitosan nanoparticles (LS-CS/NPs) with the optimization of the preparation conditions using response surface methodology and the analysis of the antioxidant and antibacterial properties of the nanoparticles as well as the controlled release of the liquid smoke. The nanoparticles were prepared by the ionic gelation method and were characterized by DLS, zeta potential, FT-IR, SEM, and EDS. The analysis of antioxidant activity of LS-CS/NPs was performed by the free radicals scavenging method (DPPH and ABTS) while the study of the total phenolic content of the particles was conducted via the application of the Folin-Ciocalteau method with some modifications. The analysis of antimicrobial activity of the LS-CS/NPs was performed using the minimum inhibitory concentration method involving the growth of Gram-positive (S. aureus and B. cereus) and Gram-negative (E. coli and Salmonella spp) bacteria. The analysis of the controlled release of liquid smoke was conducted at 4 °C using the Korsmeyer-Peppas model. The results obtained showed that the nanoparticles prepared using CS and LS in the ratio 1.5: 0.583, respectively (F′2), which had an average size of 205 nm and zeta potential of 45 mV, exhibited good antioxidant (%DPPH: 92.7% and %ABTS: 93%) and antibacterial activities, with an efficient, gradual controlled release of liquid smoke (release rate = 0.042 min⁻¹).

Introduction

The use of natural preservatives has currently become one of the most attractive mechanisms for food preservation. One of such natural preservatives is liquid smoke (LS); this additive, which is usually applied in protein foods, acts as a coloring and flavoring agent. In addition, liquid smoke has also been found to have important antibacterial and antioxidant properties (Simon et al., 2005). These properties are derived from various organic compounds present in liquid smoke; among the organic compounds present in liquid smoke include carboxylic acids, phenols, ketones aldehydes, terpenes, and alcohols (Kailaku et al., 2017; Soares et al., 2016). Dien et al. (2019) tested the application of different concentrations of liquid smoke, which varied between 0, 0.4, 0.8, 1.0, and 1.2%, on Katsuwonus pelamis L. meat. The findings of their study showed that the immersion of the fillet of Katsuwonus pelamis L. meat in 1% (v/v) solution of liquid smoke for 20 min reduced the microbial load from 4.1 × 10³ CFU g⁻¹ to 7.4 × 10² CFU g⁻¹. In addition, the application of the liquid smoke solution on Katsuwonus pelamis L. meat also inhibited the growth of some pathogenic bacteria up to 2 days (at the temperature of 30 °C) after the treatment. Dien et al. (2019) also observed a decrease in histamine concentration as time increased for the samples dipped in 1% liquid smoke solution compared to the increase in histamine concentration with time observed for the control samples – samples without the liquid smoke.

Nanotechnology allows one to design, synthesize and apply additives including preservatives, antioxidants, antimicrobial, and antifungal additives in different food matrices as a way of preserving foods and their original characteristics (Chaudhry et al., 2010). Nanoencapsulation of bioactive compounds has been proven to be a viable and efficient approach to increasing the physical stability of active substances, protecting them from interactions with food ingredients and enhancing their bioactivities (Saloko et al., 2014). The use of LS as a bioactive compound requires protection against deterioration and the nanoencapsulation technique has been found to be an effective alternative mechanism for ensuring this protection.

The encapsulation technique involves the use of small drops of liquids, solids, or gaseous particles covered with a porous polymeric network containing an active substance. According to Lupo et al. (2012), the technique enables one to create a transitory barrier, which delays chemical reactions between the active ingredient and the medium, thus increasing the useful life of the medium. Apart from that, the application of the encapsulation technique promotes a gradual release of liquid smoke and facilitates the manipulation of the applied material through the modification of the physical state of the active compound.

In a previous study reported in the literature, Saloko et al. (2014) investigated the antioxidant and antimicrobial activities of nano-encapsulated liquid smoke using chitosan (CS) and maltodextrin (MD) as encapsulants, where the authors applied the nano-encapsulated material on fresh tuna fillets at room temperature. The addition of nanocapsules, prepared from a mixture of CS (1.5% w/v) and MD (8.5% w/v) in LS concentrations higher than 5.0% helped maintain the fish freshness for 48 h at room temperature. The results obtained from the study conducted by Saloko et al. (2014) showed that the nanocapsules exhibited a higher total phenolic content (5.46% - as opposed to 0.17% for the blank sample) and mean particle size of approximately 15 nm. The nanocapsules also presented strong DPPH free radical scavenging activity (48.12–58.97%) and the results obtained from the antibacterial test indicated higher inhibitory activity against Gram-negative bacteria (P. fluorescens and E. coli) and lower inhibitory activity against Gram-positive bacteria (B. subtilis and S. aureus). The preparation of CS nanoparticles in the study carried out by Saloko et al. (2014) was facilitated by the use of a cross-linking agent – TPP, which is a non-toxic polyanion known for its ability to cross-link with CS through electrostatic forces, resulting in the formation of ionic cross-linked networks (Rodrigues et al., 2012).

Ariestya et al. (2016) investigated the ability to maintain the quality of Oreochromis niloticus (tilapia) meat during cold storage through the addition of coconut shell liquid smoke (1.5%) microencapsulated in a matrix containing dextrin (1%, 2%, and 3%). The addition of 3% dextrin in liquid smoke led to the generation of the highest content of phenols (0.98%), indicating that the application of this amount of dextrin yields the most effective result in terms of protecting the bioactive compound in microencapsulated liquid smoke. Based on the findings of Ariestya et al. (2016), the application of 1.5% liquid smoke content in tilapia meat helped reduce the total volatile base nitrogen (TVBN) content and the total plate count (TPC), and this contributed toward increasing the shelf life of the meat. Furthermore, Ariestya et al. (2016) also showed that, after 9 days of treatment, the application of 0% and 1.5% liquid smoke contents yielded TVBN concentrations of 37.787 mg N g⁻¹ and 31.070 mg N g⁻¹, respectively, and TPC concentrations of 7.717 log CFU g^{− 1} and 6.263 log CFU g^{− 1}, respectively.

In a recent study reported in the literature, Abdel-Naeem et al. (2021) investigated the microbiological, physicochemical, and sensorial properties of smoked herring (Clupea harengus) coated with CS after storage at −18 °C for 3 months. The authors found that the chitosan-coated samples (3% and 4%) exhibited a reduction of more than 4 log₁₀ CFU g⁻¹ in aerobic plate count and a complete suppression of psychrotrophic, Enterobacteriaceae, yeasts and molds counts, which were all below detectable levels (2 log₁₀ CFU g⁻¹). Based on the application of the free radical scavenging technique, the coating of the smoked herring with chitosan helped enhance and maintain the phenolic and flavonoid compounds in the fish at suitable levels during the storage period in the refrigerator (Abdel-Naeem et al., 2021).

Other studies reported in the literature also demonstrated the use of CS as a matrix for the encapsulation of other bioactive components. Amiri and Morakabati (2017) investigated the incorporation of Satureja khuzestanica essential oil in chitosan nanoparticles where the authors obtained nanoparticles with sizes between 150 and 210 nm in diameter and zeta potential of less than −30 mV (indicating low stability). The authors applied the chitosan nanoparticles modified with Satureja khuzestanica essential oil in tomatoes in order to evaluate the antifungal activity of the material (Amiri and Morakabati, 2017). In another interesting study reported in the literature, Chanphai and Tajmir-Riahi (2018) investigated the incorporation of catechins (polyphenols) from tea in chitosan nanoparticles. Khan et al. (2018) also conducted a study on the formation of nisin-loaded chitosan nanoparticles using sodium tripolyphosphate (TPP) as a crosslinking agent where they obtained nanoparticles with an average size of 134.34 nm.

Taking into account the previous studies reported in the literature, it is essentially important to carry out a comprehensive assessment of the antioxidant and antibacterial capacity of nanoparticles loaded with bioactive compounds and to investigate the mechanism involving the controlled release of the bioactive compounds incorporated in the nanoparticles with a view to obtaining suitable materials with high loading efficiency and loading capacity which contribute toward improving shelf life of food products.

In this context, the present work aims to investigate the preparation and characterization of liquid smoke-loaded chitosan nanoparticles by the ionic gelation method and optimize the preparation conditions through the application of response surface methodology (RSM). In addition, this study also seeks to evaluate the loading efficiency and loading capacity of the liquid smoke-loaded chitosan nanoparticles as well as the antioxidant and antibacterial properties of these nanoparticles. A release test is also performed in other to study the gradual controlled release of the liquid smoke.