**1. Introduction**

In recent years, there has been an increased interest in natural antimicrobials, especially those obtained from plants. Some plant species are a rich source of natural compounds with antimicrobial properties, which are able to prevent, at some extent, the growth of foodborne pathogens, and extend the shelf life of the food [1].

Pathogens involved in foodborne diseases or food processing plant contamination are often capable to adhere and form biofilms. These structures are organized communities of bacterial cells enclosed in a self-produced polymeric matrix, composed of polysaccharides, proteins and other organic components, adhering to inert or living surfaces [2]. It is known that bacteria within biofilms are more resistant to antibiotics and other chemical agents than planktonic cells in suspension and their increased tolerance towards antimicrobial agents reduces the e ffectiveness to the treatment of biofilm-related infections [3,4]. Bacterial biofilms spread widely and play important roles in many industrial activities. In the dairy industry or other food processing industries or food-contact surfaces, biofilm formation is a potential source of contamination and can lead to serious hygiene problems and economic losses [4].

*Staphylococcus aureus* is a well-known pathogen living as biofilm in a wide variety of environments and represents a severe risk of food contamination. It is has been found frequently on surfaces of food processing plants and it is responsible for infections related to consumption of fresh and processed foods [5].

Phenolic compounds occurring in vegetable foods and medicinal plants have been extensively investigated against a wide range of microorganisms. Several studies demonstrated the antimicrobial activity of dietary polyphenols, their activity on bacterial growth being mainly related to the strain, the polyphenol structure, and the dosage assayed [6–8]. Plant extracts, rich in polyphenols, has been reported to inhibit the biofilm formation by *Staphylococcus aureus*, including methicillin-resistant *Staphylococcus aureus* MRSA, *Escherichia coli* and *Pseudomonas aeruginosa* [9,10].

*Opuntia ficus-indica* (L.) Mill. (cactus pear) is a succulent species native from America, afterwards domesticated in other countries, occupying arid and semiarid zones. The first economic importance of *Opuntia ficus-indica* relies on the production of edible fruits, consumed fresh or transformed. Cladodes (succulent stem), known as pads or nopals, are also consumed at young stage in Mexico and United States in several different food preparation, or at older stage as forage, when there is shortage of fresh forage due to droughts [11,12].

The aims of this study were to investigate the potential of polyphenolic extracts from cladodes of *Opuntia ficus-indica* to inhibit the growth of some enterobacteria and the biofilm formation by *Staphylococcus aureus*. Moreover, cladode extracts were chemically and biochemically characterized, as high performance liquid chromatography time-of-flight mass spectrometry (HPLC/MS-TOF) profile and antioxidant capacity on different in vitro and ex vivo systems.

#### **2. Materials and Methods**

#### *2.1. Chemicals and Reagents*

Reagents were purchased from various suppliers as follows: authentic standards of rutin, isoquercitrin, isorhamnetin, isorhanmetin 3-*O*-glucoside, narcissin, kaempferol (Extrasynthèse, Genay, France); FCR (Folin-Ciocalteu's reagent), gallic acid, *p*-hydroxybenzoic acid, sodium carbonate, sodium hydroxide, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-Azobis (2-methylpropionamidine) dihydrochloride (AAPH), fluorescein sodium salt and 2,7-dichlorofluorescein diacetate (DCFH-DA), as well as acetonitrile (HPLC grade), ethanol, methanol, formic acid (Sigma-Aldrich, St. Louis, MO, USA). In all experiments, Milli-Q (Merck Millipore, Darmstadt, Germany) water was used.

#### *2.2. Plant Material and Extraction*

*Opuntia ficus-indica* cladodes were collected from plants producing purple fruits ('*Rossa*' variety), in the Salento countryside (Apulia Region, Italy, N 40◦2118"; E 17◦5947").

Cladodes were collected from the same plants at two developmental stages: immature (from spring shoots, fully developed cladodes at 3 weeks of development) and mature (from fall shoots, old cladodes at around 24 weeks).

Cladodes were cleaned from spines and glochids, cut in small pieces, processed in a Waring blender with liquid nitrogen, freeze-dried using a Freezone® 2.5 model 76530 lyophilizer (Labconco Corp., Kansas City, MO, USA) for 48 h and stored at −20 ◦C. This product was defined as the dry weight (DW) of cladode. Extraction was done in triplicate from 500 mg (DW) macerated with 25 mL aqueous methanol (80%) overnight at 4 ◦C. After centrifugation (4000× *g*), the supernatant was recovered and evaporated in vacuo at 32 ◦C using a R-205 Büchi rotavapor (Büchi Labortechnik AG, Essen, Germany), then re-suspended in distilled water to a concentration of 50 mg/mL. Extracts were then filtered on a 0.45-μm CA syringe filter (Filtres Fioroni, Ingré, France) and portioned in 1-mL aliquot, stored at −20 ◦C until analysis.

#### *2.3. Identification and Quantification of Phenolic Compounds*

The identification and quantification of phenolic compounds in cladode extracts was performed in triplicate (an analysis for each of the three extractions/replicas) using an Agilent 1200 Liquid Chromatography system (Agilent Technologies, Palo Alto, CA, USA) and the chromatographic conditions and column were the same already reported by Sabella et al. [13]. The identification of phenolic compounds was confirmed by a TOF LC/MS system (Agilent 6320, Agilent Technologies, Palo Alto, CA, USA), equipped with a dual ESI interface operating in negative ion mode [13].

The identified phenolic compounds were quantified by the external standard method using a six-point calibration curve of *p*-hydroxybenzoic acid (0.5–100 mg/L), rutin (0.5–50 mg/L), isorhamnetin (0.5–50 mg/L), and narcissin (0.5–50 mg/L).

#### *2.4. In Vitro Antioxidant Activity*

The Folin–Ciocalteu reducing capacity assay, the antioxidant activity by Oxygen Radical Absorbance Capacity (ORAC) and Trolox equivalent antioxidant capacity (TEAC) were evaluated in cladode extracts, as described in [14]. A rapid microplate methodology, using a microplate reader (VictorTM X3, Perkin Elmer, Waltham, MA, USA) and 96-well plates (Costar, 96-well black round bottom plate, Corning) were used. All experiments were performed in triplicate, and two independent assays were performed for each sample.

#### *2.5. Ex Vivo Antioxidant Activity*

#### 2.5.1. Cellular Antioxidant Activity (CAA-RBC) Assay in Red Blood Cells

The antioxidant activity of *Opuntia* cladode extracts was evaluated in an ex vivo erythrocytes system as described in Frassinetti et al. [15] as well as erythrocytes preparation. Trolox was used as a standard and the fluorescence was read at 485 nm excitation and 535 nm emission by using a VictorTM X3 microplate reader. Human erythrocytes from random subjects were exposed to a peroxyl radical generator, the AAPH, following one hour pre-treatment with 500 and 1000 μg/mL of cladode extracts. Each value was express according to the formula:

$$\text{CAA unit} = 100 - (\int \text{S} \mathcal{N} \int \text{CA}) \times 100$$

where SA is the integrated area of the sample curve and CA is the integrated area of the control curve [16].

#### 2.5.2. Erythrocytes Oxidative Hemolysis

Hemolysis of human erythrocytes was induced by thermal decomposition of AAPH as previously described [15]. Briefly, erythrocytes were incubated with Trolox 500 μM (antioxidant standard) and cladode extracts at 37 ◦C for 1 h, followed by incubation with 50 mM AAPH at 37 ◦C for 4 h. The erythrocytes hemolysis was evaluated by spectrophotometric reading (λ = 540 nm) of the hemoglobin released in the supernatant, and expressed as percentage compared to the control (AAPH-treated erythrocytes).
