Antibacterial activity and chemical characterization of almond (Prunus dulcis L.) peel extract

Abstract In this study, almond skin of Prunus dulcis L. variety Casteltermini was characterised for its chemical composition and for the inhibitory activity towards food associated microorganisms belonging to pathogenic (Salmonella Enteritidis, Staphylococcus aureus, Escherichia coli and Listeria monocytgenes), spoilage (Pseudomonas poae and Brochotrix thermospacta) and useful (Levilactobacillus brevis and Saccharomyces cerevisiae) groups. The n-hexane extract of P. dulcis skin, investigated by GC–MS, evidenced linoleic, palmitic, and oleic acids as the main abundant compounds. Staphylococcus aureus ATCC 33862 growth was strongly limited by n-hexane extract and the minimum inhibition concentration (MIC) was 25.5 mg/mL. These results highlight the potential of almond by-products as food bio-preservatives as well as pharmaceutical products. Graphical Abstract


Introduction
The circular economy, based mainly on the reuse of waste and leftovers from the food industries, aims both to reduce the volume of food waste, but also to enhance the resources that can be extracted from these by-products (Lin et al. 2013). On this point, the management of by-products deriving from the world of fruit processing is fundamental. In fact, these products are still particularly rich in antioxidant (mainly polyphenols) (Campos et al. 2020; Barbaccia et al. 2021) that, after extraction, can be used to functionalised various foods (Gaglio et al. 2021;Garcia-Perez et al. 2021;Rodr ıguez et al. 2021).
One of the recent advancements in the valorisation of fruit by-products is related to the extraction of antimicrobial compounds. Nowadays, consumers are conscious of the negative health implications of chemical preservatives and are pushing food industries towards the use of natural preservatives. Plants, herbs, and fruits show antimicrobial properties mainly thanks to their essential oils (EOs) (Oussalah et al. 2007;Said et al. 2016;Settanni et al. 2014), but also aqueous (Miceli et al. 2014;Sadeghian et al. 2011;Fattouch et al. 2008) and oil extracts (Salih 2006) exert inhibitory effects against undesired microorganisms. Generally, the antimicrobial efficacy of plants is due to diverse compounds such as phenolics, tannins, and alkaloids (Cowan 1999). Plant extracts can be used as food preservatives because they are referred to as GRAS (Generally Recognised As Safe) (Viuda-Martos et al. 2008) and, thanks to their safety, they have been used for this purpose since ancient times (Ullah et al. 2016). Plant extracts are still very important for processing safe and stable foods because foodborne illnesses still pose a threat even in industrialized countries, even though hygiene standards have steadily improved in recent decades (Newell et al. 2010). The major microbial agents responsible for human diseases caused by food ingestion are bacteria; over 90% of food-poisoning illnesses are due to Staphylococcus, Salmonella, Clostridium, Campylobacter, Listeria, Vibrio, Bacillus, and Escherichia coli (Fung et al. 2018).
The almond tree is a widespread species worldwide (Sottile et al. 2020). The edible part of the fruit is the kernel used as ingredient in many desserts, ice cream, cake, pastry and drink, like almond milk (Amorello et al. 2016). However, a considerable amount of waste is obtained from processing the whole fruit. A part of them is represented by the seed skin (tegument) that is physically removed to obtain peeled almonds for food processing.
Based on the above considerations, in order to acquire information on the characteristics of almond peel for its reuse in food production, the objectives of this study were: (i) to evaluate the inhibitory effect of n-hexane extract against food pathogens and spoilage bacteria and, because of their use during food fermentation, also against pro-technological lactic acid bacteria (LAB) and yeasts to verify their harmlessness against the fermenting agents necessary to transform raw materials into final products; and (ii) to determine their chemical composition.

Antibacterial activity of almond peel extract
The inhibitory activity of almond n-hexane extract was tested at a concentration of 102 mg/mL (in hexane). Interestingly, Levilactobacillus brevis and Saccharomyces cerevisiae was not inhibited by the extract tested, indicating its harmless activity against pro-technological microorganisms generally used as starter cultures in food fermentation (Kandasamy et al. 2018). No inhibition activity was observed against B. thermosphacta commonly associated with meat spoilage (Gribble et al. 2014) and all Pseudomonas poae commonly involved in the alteration of several foods of animal and plant origin (Meng et al. 2017;Miceli et al. 2019). Regarding pathogenic bacteria, the inhibitory activity of the extracts was tested against different bacteria generally used as food safety criteria (Listeria monocytogenes ATCC19114 and Salmonella Enteritidis ATCC13076) and process hygiene criteria (E. coli ATCC25922 and Staphylococcus aureus ATCC33862) following the indication of European Commission (EC) Regulation No 2073/2005(Commission Regulation 2005. The most active extract was the n-hexane that showed a defining inhibitory activity (12 mm width of the clear area) only against S. aureus ATCC 33862 (Figure 1). An hypothesis to explain the antimicrobial activity of n-hexane extract of Prunus dulcis skin against St. aureus involves the presence of fatty acids in this by-products. However, it is well known the antibacterial activity of fatty acids such as linoleic acid, palmitoleic acid, oleic acid and their esters against both Gram-negative and Gram-positive bacteria (Huang et al. 2010). In order to retrieve the Minimum Inhibitory Concentration (MIC) of the oil extract, it was serially diluted in hexane and further tested only against S. aureus ATCC 33862. The inhibitory activity was registered until the second dilution (1:4). Thus, MIC was estimated at 25.5 mg/mL of dried extract.
S. aureus is a relevant food-borne pathogen. Its harmfulness is mainly due to the ability to produce staphylococcal enterotoxins (Hennekinne et al. 2010). Food poisoning by staphylococcal enterotoxins is one of the most common causes of food-borne outbreaks (Denayer et al. 2017). Generally, a staphylococcal food poisoning occurs after ingestion of foods contaminated with S. aureus improperly handled and kept at uncontrolled temperatures (Argud ın et al. 2010). Although the activity of n-hexane extracts from spice plants against pathogenic bacteria is known (Gul and Bakht 2015;Hasan et al. 2012), the information about these extracts from nut fruit peel are quite limited. However, n-hexane extracts from fruits' peels of Calophyllum inophyllum were able to inhibit St. aureus ATCC 6538 (Cheah and Heng 2009).

Almond skin n-hexane extract GC-MS analysis
The GC-MS analysis identified the presence of 11 fatty acids (Table 1). The most abundant compound was linoleic acid (63.19%), followed by palmitic acid (16.65%), and oleic acid (8.41%). The almond skin also contained interesting levels of stearic (4.10%) and palmitoleic acids (3.40%). All these compounds are commonly present in almond kernels cultivated in Sicily, Spain and California (Amorello et al. 2016).
Previously, only Saura-Calixto et al. (1985) had analysed the fatty acids profile of a mixture of almond skin from almond cultivar growing in Palma de Mallorca (Spain). Oleic (47.13%), linoleic (40.34%), and palmitic acids (7.53%) were found as the most abundant compounds. Significant differences were found in the essential fatty acid linoleic acid content, which was 1.6-fold higher in the Sicilian almond cv.

Conclusions
Almond skin like all unmanaged agro-wastes and food by-products creates environmental concerns and represents a disposal cost for producers. In this work, almond skin was considered a potential source of natural antibacterial compounds for further use in bio-preservation. With this in mind, nhexane extract was tested against the main food-borne pathogenic bacteria, and showed the ability to inhibit St. aureus ATCC 33862 with a MIC of 25.5 mg/mL of dried extract. This extract was then characterized by GC-MS analysis that identified linoleic acid as a major compound. From an application perspective, almond skin n-hexane extract represents an environmentalfriendly alternative to the use of chemical preservatives used to combat one of the major microbiological concerns for the food industry.

Disclosure statement
No potential conflict of interest was reported by the authors.