**1. Introduction**

The food industry generates an enormous amount of waste in form of skins, seeds and leaves, whose disposal is a problem for the environment and expensive for companies concerned. Many residues from fruits, which are rich source of phenolic compounds, can be extracted and used by food industries as antioxidant and antimicrobial preservatives.

In this work, the antimicrobial and antioxidant capacity of several extracts from rosemary, pomegranate, and hydroxytyrosol were measured. These extracts were chosen because of their beneficial effects for human health and their notable antioxidant properties. *Rosmarinus officinalis* L. is a natural woody perennial green herb from the Mediterranean region rich in vitamins A, C, B1, B6 and B9, minerals such as Mg, Ca, Cu, Fe and Mn, as well as phenolic compounds (diterpenes and rosmarinic acid). The regular consumption of this herb has been seen to have many beneficial effects for human health [1,2] acting as a powerful antioxidant and antibacterial agen<sup>t</sup> [3]. Furthermore, pomegranate extract, from *Punica granatum* fruit, contains large quantities of phenolic compounds (ellagitannins, flavonoids, punicalagin, ellagic acid, vitamin C and minerals). For this reason, pomegranate has been used in medical applications for more than 2000 years. Among its beneficial effects for the human body [4,5], pomegranate peel extracts and other subproducts obtained from this fruit, such as juice or seeds, have shown high antioxidant and antimicrobial capacities, with a grea<sup>t</sup> scavenging power, preventing microbiological growth of different bacteria [6,7]. On the other hand, oleuropein is the main phenolic compound in olive (*Olea europaea*) tree and the precursor of hydroxytyrosol, which is also extracted from leaves and vegetation waters from the manufacture of olive oil. This phenolic phytochemical is considered the most antioxidant compound after gallic acid, whose consumption has many beneficial effects for the skin, eyes and immune system, and acting as an antimicrobial, anti-infammatory and anticancer agen<sup>t</sup> [8–10].

As these extracts act as antioxidants, antimicrobials and hence preservatives of shelf life food products, it is interesting to investigate about their action in a food matrix. In this case, fish has been chosen due to it is an important source or omega-3, particularly eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), essential fatty acids for the protection against autoimmune, inflammatory and cardiovascular diseases [11]. However, fish consumption has decreased in populations groups under 14 years old, thus manufacturation of fish products as "burgers" could be a good option to stimulate fish consumption among young people [12]. In the same way, synthetic additives are widely used to avoid the microbiological, enzymatic and oxidative degradation of these kinds of products. As substitution of synthetic additives by natural extracts is gaining in importance in the food industry [13], the use of natural extracts as preservatives in fish products could be an excellent way to produce a saludable product with high nutritional level, Clean label, and without synthetic additives. Alternatively, pomegranate has already demonstrated its antioxidant capacity after addition to chub mackerel minced muscle during frozen storage [14]. In this case, 2% pomegranate seed extract inhibited the formation of substances related with lipid oxidation regard to the control sample. Similarly, rosemary oil at 0.2%, 1% and 3% added to minced rainbow trout had a positive effect on the freshness indicators, oxidative stability, fatty acid and biogenic amine contents during refrigerated storage [15]. In addition, hydroxytyrosol has also showed an important inhibition of the formation of lipid oxidation products in foodstuffs rich in fish lipids (bulk cod liver oil, cod liver oil-in water emulsions and frozen minced horse mackerel) [16], while in a general view, hydroxytyrosol has also demonstrated to have excellent antioxidant properties in a nanostructured starch developed as active food packaging [17].

Briefly, the objective of this work was to make a comparative study of pomegranate, rosemary and hydroxytyrosol extracts measuring their antioxidant and antimicrobial capacities, as in vitro, following several methods, as antioxidants and antimicrobials in a food matrix: fish patties.

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

#### *2.1. Plant Extracts*

The plant extracts used were: Pomegranate (P), Rosemary: Rosmarinic acid extract (RA), Nutrox OS (NOS), Nutrox OVS (NOVS), Hydroxytyrosol from fruit (HYT-F), and from leaf (HYT-L). All extracts were supplied by Nutrafur-Frutarom Group (Alcantarilla, Murcia, Spain) and obtained from the corresponding dry vegetal materials using a maceration process including, as an initial step, solid-liquid extraction with different ethanol-water mixtures. Pomegranate extract (P) was obtained from dehydrated lignocellulosic materials of fruits by means of a hydro-alcoholic extraction, filtration of the vegetal material, evaporation of the ethanol and crystallisation in aqueous medium, followed by concentration and drying. De-oiled rosemary leaf was used as raw material to obtain the rosemary extracts used in this study. The water-soluble rosemary extract (RA) was obtained by extraction in aqueous medium and simple filtration of the plant material, concentration and drying. Rosemary extracts not soluble in water were obtained by extraction with acetone-water, filtration of the plant material, concentration and drying. Subsequently, the solid obtained was dissolved in different excipients to obtain the two extracts used in this study, sunflower oil (NOS) and sunflower oil

plus lecithin (NOVS). HYT-L was made by extraction in ethanol-water medium, filtration of the plant material, evaporation of the solvent and subsequent thermal treatment. Finally, the water insoluble materials were filtered and dried. While HYT-F was obtained by extraction from the dry plant material with ethanol (vegetation waters), decantation, evaporation of the solvent, drying, recrystallisation of aqueous medium, elimination of insoluble materials in water and drying. All the drying processes were carried out in a vacuum and at a reduced temperature (50–70 ◦C).

#### 2.1.1. Determination of Extract Composition (HPLC)

For the quantification of phenolics in the P extract, it was dissolved in dimethylsulfoxide (DMSO) in the ratio of 4 mg/mL; this solution was filtered through a 0.45 μm nylon membrane. The HPLC equipment used for all the extracts was a Hewlett-Packard Series HP 1100 equipped with a diode array detector. The stationary phase was a C18 LiChrospher 100 analytical column (250 × 4 mm i.d.) with a particle size of 5 μm (Merck, Darmstadt, Germany) thermostated at 30 ◦C. The flow rate was 1 mL/min and the absorbance changes were monitored at 280 nm. The mobile phases for chromatographic analysis were: (A) acetic acid/water (0.1:99.5) and (B) methanol. An initial isocratic period for 15 min with 100% (A) was run, after, a linear gradient was run from 100% (A) to 90% (A) and 10% (B) for 15 min (30 min total time), and it was maintained for 10 min (40 min total time), before reequilibrating in 10 min (50 min, total time) to initial composition. Punicalagin in P was identified and quantified by comparation of their retention times with the corresponding standard and by their UV spectra obtained with the diode array detector.

For the quantification of diterpenes in rosemary extracts (mainly carnosic acid and carnosol), for NOS and NOVS extracts, each extract was dissolved in methanol in variable ratios between 0.2 and 4 mg/mL, depending on the diterpene concentration expected for each extract; the solution was filtered through a 0.45 μm nylon membrane. The flow rate was 0.75 mL/min, and the elution was monitored at 230 nm. HPLC was used for the separation of the different diterpenes present in the rosemary extracts. The mobile phase for chromatographic analysis was an isocratic single step of acetonitrile (65%), water (35%) and phosphoric acid (0.2%) for 25 min. Diterpenes were quantified by comparing the chromatographic areas of the corresponding standards. On the other hand, for the quantification of rosmarinic acid in rosemary extracts (RA), the solid was dissolved in water at 5 mg/mL for analytical chromatography; this solution was filtered through a 0.45 μm nylon membrane. The flow rate was 1 mL/min. The absorbance changes were monitored at 340 nm.

For the quantification of phenolics in the olive (*Olea europaea*) extracts (HYT-F and HYT-L), the extract was dissolved in dimethylsulfoxide (DMSO) in the ratio of 5 mg/mL; this solution was filtered through a 0.45 μm nylon membrane. The flow rate was 1 mL/min and the absorbance changes were monitored at 280 nm. The mobile phases for chromatographic analysis were: (A) acetic acid/water (2.5:97.5) and (B) acetonitrile. A linear gradient was run from 95% (A) and 5% (B) to 75% (A) and 25% (B) for 20 min; it was then changed to 50% (A) and (B) in 20 min (40 min, total time); in 10 min it was changed to 20% (A) and 80% (B) (50 min, total time), before reequilibrating in 10 min (60 min, total time) to initial composition. Phenolic compounds in olive extracts were identified and quantified by comparation of their retention times with the corresponding standard and by their UV spectra obtained with the diode array detector [18].

Pure standards for HPLC quantification: hydroxytorosol (Code 4999S, Extrasynthése, Genay, France); carnosic acid (Sigma, Code C-0609, Madrid, Spain); carnosol (Sigma, Code C-9617, Darmstadt, Germany); rosmarinic acid (Extrasynthése, Code 4957S, Genay, France); punicalagin (Sigma, Code P-0023, Darmstadt, Germany).

#### 2.1.2. Total phenolic content (TPC)

The total phenolic content (TPC) was determined quantitatively using the Folin–Ciocalteu reagen<sup>t</sup> and gallic acid as the standard [19,20]. Each extract was diluted with water or ethanol, according to its polarity, in a 1000 ppm solution. Then, 2 mL of 2% Na2CO3 was added to 100 μL of sample and

a standard solution of gallic acid. After, 100 μL of 10:1 Folin–Ciocalteau phenol reagen<sup>t</sup> was added. Following incubation for 30 min, the absorbance was measured at 750 nm. The TPC was expressed as mg gallic acid equivalents (GAE) per g extract.
