Antimicrobial activity of grape, apple and pitahaya residue extracts after carbohydrase treatment against food-related bacteria

Highlights

• Carbohydrase-assisted extraction of antimicrobial compounds from fruit residues.

• Extracts subjected to antibiofilm and anti-quorum sensing activity assays.

• The antimicrobial activity correlated with the total phenolic content of samples.

• Phenolic products inhibited the growth and biofilms of food-related bacteria.

• Extracts and phenolics suppressed the violacein production in Chromobacterium.

Abstract

In this study, antimicrobial, anti-quorum sensing and anti-biofilm forming activities of phenolic-enriched extracts produced by enzyme-assisted extraction from oven-dried and lyophilized black grape (Vitis vinifera x (Vitis labrusca x Vitis riparia)), apple (Malus domestica cv. Jonagold) and yellow pitahaya (Hylocereus megalanthus) residues were investigated. Both disk-diffusion and minimal inhibitory concentration (MIC) assays showed increased antimicrobial capacity for many extracts after the carbohydrase treatments. The lowest MIC (12.5 mg/mL) was found for black grape samples against Bacillus cereus and Bacillus subtilis, and for apple and pitahaya samples against Pseudomonas putida. The percent quorum sensing inhibition effect on Chromobacterium violaceum has varied between 9 and 36%, 15 and 24% and 6 and 23% for black grape, apple and pitahaya extracts, respectively, depending on the enzyme treatment. Pseudomonas aeruginosa, P. putida and Staphylococcus aureus biofilms were the most sensitive to the extracts, while the Salmonella enterica, Listeria monocytogenes and Escherichia coli biofilms were modulated by certain enzyme-free and treated samples. The extracts can be good candidates for the development of natural preservatives.

Introduction

Foodborne diseases frequently occur worldwide. Salmonellosis and listeriosis are well-known infections but Staphylococcus aureus and certain Escherichia coli strains can also be associated with foodborne outbreaks (Callejón et al., 2015). These bacteria can form biofilm, that is, communities enclosed in self-synthesized polymer matrices, attached to biotic or abiotic surfaces. Within the biofilm, the cells are metabolically less active than the planktonic form, therefore, they are significantly less sensitive to the action of antimicrobial agents, which is the main cause of chronic infections (Landini, Antoniani, Burgess, & Nijland, 2010). Formation of biofilms is a complex process and depends on different cellular mechanisms. One of these is the population dependent cell-to-cell communication system, namely quorum sensing, that regulates additional cellular functions such as virulence gene expression, sporulation, conjugation, exopolysaccharide production and toxin production as well (Deep, Chaudhary, & Gupta, 2011). By quorum sensing inhibitors, biofilm formation of foodborne pathogenic and spoilage bacteria could also be inhibited (Kalia, 2013).

Recently, many synthetic antimicrobials are used successfully for shelf life extension, however, the consumers' growing concern against such preservatives in foods encourages researchers to screen and enrich the natural alternatives (Gyawali & Ibrahim, 2014). Polyphenols, e.g., phenolic acids, flavan-3-ols, flavonols and tannins, are intensively studied in this regard because of their ability to suppress various microbial virulence factors including those that are regulated by the quorum sensing system (Asfour, 2018; Brackman & Coenye, 2015; Slobodníková, Fialová, Rendeková, Kováč, & Mučaji, 2016). Furthermore, they are potent free radical scavengers and metal chelators. Thus, they can inhibit the lipid peroxidation and exhibit various additional physiological activities as antioxidants and antimicrobials (Daglia, 2012). Therefore, these kinds of phytochemicals are widely used in the food preservative developments as potential antimicrobial agents against various food-related pathogens (Papuc, Goran, Predescu, Nicorescu, & Stefan, 2017).

Grapes, apple and pitahaya (also known as dragon fruit) are promising sources of phenolic compounds. Besides the usable part of the fruits, by-products generated during the fruit processing are also rich in bioactive phenolics (Dembitsky et al., 2011; Kalinowska, Bielawska, Lewandowska-Siwkiewicz, Priebe, & Lewandowski, 2014; Teixeira et al., 2014). In a recent study, we successfully produced antioxidative phenolic-enriched extracts from oven-dried and lyophilized black grape, apple and pitahaya residues by direct enzymatic treatment using a cellulolytic cocktail from the zygomycete fungus Rhizomucor miehei NRRL 5282 and a pectinase from Aspergillus niger (Zambrano et al., 2018). The substrate pretreatment (oven-drying and lyophilization) affected the phenolic yield and the antioxidant activity. Furthermore, some extracts exhibited significant increase in the concentration of 4-hydroxybenzoic acid, syringic acid, gallic acid, vanillic acid, cinnamic acid, p-coumaric acid, (+)-catechin, (−)-epicatechin, quercetin, polydatin and resveratrol compounds. Here, the potential bioactive properties, i.e. the antimicrobial and anti-biofilm forming activities against five Gram-positive and four Gram-negative foodborne pathogen and food spoilage bacteria, and the anti-quorum sensing capacity of the extracts were investigated before and after enzymatic treatments. Furthermore, pure form of the previously examined individual phenolics was also subjected to bioactivity tests to study the possible correlation between the activity changes in the extracts and the increase in the phenolic compound concentrations after the enzymatic treatments.

Although antimicrobial capacity of various grape, apple and pitahaya cultivars has been investigated (Teixeira et al., 2014; Yadav, Kumar, Kumar, & Mishra, 2015; Zhang et al., 2016), there are only scarce data about yellow pitahaya residue samples (Choo, Koh, & Ling, 2016). Furthermore, the present assay provides useful information on the less studied Othello grape and Jonagold apple variants as potential sources of antimicrobial compounds.