Synergistic effect of polysaccharides, betalain pigment and phenolic compounds of red prickly pear (Opuntia stricta) in the stabilization of salami

Accepted Manuscript Synergistic effect of polysaccharides, betalain pigment and phenolic compounds of red prickly pear (Opuntia stricta) in the stabilization of salami

Nadia Kharrat, Hedia Salem, Aicha Mrabet, Fatma Aloui, Soumaya Triki, Ahmed Fendri, Youssef Gargouri PII: DOI: Reference:

S0141-8130(17)31707-5 https://doi.org/10.1016/j.ijbiomac.2018.01.025 BIOMAC 8857

To appear in: Received date: Revised date: Accepted date:

12 May 2017 4 December 2017 4 January 2018

Please cite this article as: Nadia Kharrat, Hedia Salem, Aicha Mrabet, Fatma Aloui, Soumaya Triki, Ahmed Fendri, Youssef Gargouri , Synergistic effect of polysaccharides, betalain pigment and phenolic compounds of red prickly pear (Opuntia stricta) in the stabilization of salami. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Biomac(2017), https://doi.org/ 10.1016/j.ijbiomac.2018.01.025

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ACCEPTED MANUSCRIPT Title: Synergistic effect of polysaccharides, betalain pigment and phenolic compounds of red prickly pear (Opuntia stricta) in the stabilisation of salami Authors' names: Nadia Kharrat1, Hedia Salem1, Aicha Mrabet2, Fatma Aloui1, Soumaya Triki3, Ahmed Fendri1, Youssef Gargouri1 1

Laboratory of Biochemistry and Enzymatic Engineering of Lipases, ENIS, Soukra

Food Analysis Laboratory, National Engineering School of Sfax (ENIS), Route de

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Road, BPW 1173- 3038 Sfax-Tunisia.

Laboratory of Microorganisms and Biomolecules, Center of Biotechnology of Sfax-

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Soukra, km 4.5, BP 3038 Sfax, Tunisia

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Tunisia.

* Corresponding author: Prof. Youssef Gargouri

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Laboratory of Biochemistry and Enzymatic Engineering of Lipases, ENIS, Soukra

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Road, BPW 1173- 3038 Sfax-Tunisia. Tel/ Fax: + 21674575055

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E-mail : [email protected]

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Abbreviations: PPE: Prickly pear extract, FAs: Fatty acids, FAMEs: fatty acid methyl esters, MIC: Minimum inhibitory

concentration, MBC: Minimum bactericidal concentration, WHC: water holding capacity.

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ACCEPTED MANUSCRIPT Abstract The aim of this work is to try to substitute some synthetic additives by a natural extract from red prickly pear (Opuntia stricta) which known by its richness on bioactive polysaccharides mainly consisting of galactose, rhamnose and galacturonic acid. This natural fruit has a high content of carbohydrates above 18.81 % FM. It contains also a

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high level of polyphenols 152.25 ± 0.26 µg QE/ mg PPE and flavonoids about 370.60 ±

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0.12 µg GAE/ mg of PPE. In addition, prickly pear extract (PPE) displayed a strong antioxidant and antimicrobial activities. These activities are likely due to its phenolic,

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flavonoid and carbohydrate contents. Moreover, the addition of 2.5 % of PPE, as a

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natural colorant and antimicrobial agent in salami formulation, causes a decrease in hardness and chewiness of the formulated salami. Interestingly, PPE inhibited bacterial

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growth in salami stored at 4 °C over 30 days. Sensorial analysis shows that the color, taste and texture of salami prepared with 2.5 % of PPE are markedly more appreciated

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by panellists. Our results suggest that the betalain pigment, carbohydrate and phenolic

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compounds present in PPE could be used as a natural colorant, antioxidant and

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antimicrobial agent without change of the sensory characteristics.

Keywords: Prickly pear (Opuntia stricta) extract; Antioxidant and antimicrobial

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activities; Salami; Microbial and textural analysis.

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ACCEPTED MANUSCRIPT 1.

Introduction For centuries, most of the items purchased in traditional markets have been

processed and preserved using food additives as dyes, leavening agents, stabilizers and preservatives [1]. Although several researches showed that not all additives are safe, there are many worst additives which are dangerous for health and which display side-

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effects [1-3]. A British study had established a link, in the case of children aged about 3 years,

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between the risk of hyperactivity and ingestion of foods containing dyes and synthetic

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preservatives [4-6]. The hyperactivity is reflected by an inability sit still focus as well as by impulsivity. In France, 3-5% of children suffer from hyperactivity [5;6]. Some

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additives, though allowed, are recognized as potentially carcinogenic such as coloring:

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E123, E120, E131, E142, preservatives: benzoic derivatives E210-E219 and nitrite derivatives E249-E252, with doubts for some sweeteners [5]. These dyes and

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preservatives are present in large numbers of sweets, sodas, chocolate but also in meat

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products [7].

Currently, meat products (Salami, Kafta, Sausages, Frozen meat...) occupy a large space in the market. The salami has nutritional characteristics that meet consumers’

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demand. Despite its several benefits, we cannot pass over some harmful health effects

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such as the carcinogenic effect of sodium nitrite (E250) used as preservative [8]. The synthetic preservatives can be transformed, in the presence of free amines in the human body, and produce nitrosamine molecules which are carcinogenic [9]. Moreover, the accumulation of sodium nitrite in blood prevents oxygen from binding to hemoglobin and therefore prevents its transport in the blood. In addition, the red dye (E120), used in the salami preparation, was extracted from the cochineal body Dactylopius coccus. Carmine can cause various allergies in some people who have intolerance to carminic 3

ACCEPTED MANUSCRIPT acid, such as severe anaphylactic shock, hives, asthma, urolithiasis, hyperactivity and childhood neurological diseases [10;11]. Cactus plant belongs to the Cactaceae family, originated from Mexico and was produced in North Africa in the 16th century, as underlined by the Food and Agriculture Organization [12;13]. Opuntia ficus indica is the most abundant species in Tunisia,

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which is mainly consumed as fresh fruit and as local health-food stores [14;16]. However, Opuntia stricta was found in west but it is less appreciated by the consumer,

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due to its acidic taste and the presence of a large amount of seeds.

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It is well established that the O. ficus indica and O.stricta fruits, present many virtues like anti-inflammatory and analgesic effects [17], anti-hyperglycemia and

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hypocholesterolemic effects [18], antioxidant activity [19], anti-Leishmania activity

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[20], antibacterial effects [21], Neuroprotective and anti-ulceric effects [22;23]. These large beneficial effects can be related to the compounds present in the fruits as the

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various fatty acids of the prickly pear seeds oil and their richness in phenolic

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compounds, vitamins, carotenoids, polysaccharids and betalains [24-27]. Moreover, betalain, pigments with red color, has been specified as a new class of dietary cationized antioxidant composed of a nitrogenous core structure, betalamic acid [21;28]. Imtiyaj

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Khan showed that the betalamic acid condenses with amino compounds (cyclo- DOPA

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or its glucosyl derivatives) or amines or/and their derivatives to form a variety of betacyanins (violet) and betaxanthins (yellow), respectively [28]. Khatabi et al. showed that the red prickly pear contains betaxanthin pigments (red) in excess of the indicaxanthin (yellow) that permits to valorize human’s potential spring of genuine colorings [14;21]. Polysaccharides are polymeric carbohydrate molecules that are widely present in plants and algae. Several works have showed that bioactive polysaccharides have been 4

ACCEPTED MANUSCRIPT isolated and characterized from Opuntia species [26-29]. These polysaccharides are characterized by the presence of neutral sugar content, mainly consisting of galactose, rhamnose and galacturonic acid [26;27]. Majdoub et al. have described the presence of polysaccharides in the different parts of Opuntia ficus indica and Opuntia Litoralis (peel and pulp of prickly pear) collected in the Tunisian area [27]. Based on the sugar

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composition of the pectic polysaccharides extracted from the Opuntia ficus indica cactus fruits, authors suggested their applications as thickening additives in food

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components as well as in cosmetic and pharmaceutical preparations [26;27].

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Interestingly, cladodes powder, containing carbohydrate molecules, was used as an ingredient in breakfast cereals and milk-based drinks. Therefore, it could be used up

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to 20 % as a thickening agent in many foods such as vegetable soups and dessert gels

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[30]. Recently, cladodes powder has been used at 5% level in cookies preparation [31]. This level was optimal for improving the total phenolic content and the antioxidant

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potential of cookies without having any negative effect on the sensory properties.

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The main objective of the present work is to study the physicochemical properties of the prickly pear (Opuntia stricta) and to evaluate the antioxidant and antimicrobial activities of the PPE. Based on these in vitro results, PPE is applied as a

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natural preservative and colorant to substitute the sodium nitrite (E250) and dye (E120),

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that are commonly used in salami formulation. We investigate the influence of PPE, rich in betalain pigment, carbohydrate and phenolic compounds, on microbial growth, as well as textural, sensory and color characteristics. 2.

Material and methods

2.1. Materials and Chemical reagents Mechanically separated turkey (MST) meat was produced from turkey after meat cutting. It was obtained from local processors of charcuterie industry (Chahia, Sfax5

ACCEPTED MANUSCRIPT Tunisie). Approximate chemical composition of MST meat was 67 % water, 13 % proteins, 19 % fat and 1 % ash. Analytical grade NaCl, sodium ascorbate, Tripolyphosphate (TPP), Monosodium glutamate, Sodium nitrite (NaNO2) (E250) and cochineal or carmin (E120), which is a red colorant extracted from the cochineal body (Dactylopius coccus), were purchased from sigma and were kindly donated by a local

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industry of charcuterie. Ethanol, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), 3-(4,5dimethyl-thiazol-2-yl) -2,5-diphenyl-tetrazolium (MTT) and Folin-Ciocalteu reagents

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were purchased from Sigma chemicals (Steinheim, Germany). Gallic acid and quercetin

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were obtained from Fluka (Buchs, Switzerland). 2.2. Plant material

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The identification of the used species has been investigated with the help of a

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specialist in botany. Opuntia stricta is a low-growing plant (about 50-100 cm tall) with elliptic and elongated cladodes. The fruit are reddish-purple and covered by small

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spines. Mature prickly pear fruits (Opuntia stricta) were collected in February from the

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region of Ghraba located at 25 km in the North of Sfax (Tunisia). The fruits were immediately sorted, washed with running water several times and partially hand-peeled. The obtained whole prickly pear fruit was air-dried at ambient temperature (varying

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from 20 to 27°C for 2 weeks), was cut into fine cubes then powdered using a coffee

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grinder (Braun AG-Frankfurt/M, typ: 4 045, Germany) and stored at 4 °C until use. 2.3. Physico-chemical analysis of the prickly pear powder Moisture and dry matter were determined by oven drying the whole prickly pear fruit at 105 ◦C until a constant weight [32]. Total nitrogen was determined by the Kjeldahl method and the crude proteins were determined as previously described by Balogun and Fetuga [33]. Ash was determined by the combustion of the sample in a muffle furnace at 550 °C for 12 h [34]. The residue was used for the determination of 6

ACCEPTED MANUSCRIPT the mineral constituents (Na, Ca, Mg, Fe, K, Cu and Zn) using flame atomic adsorption spectrometry according to the procedures CEE-BIPEA [35]. Fat content was determined by using the Soxhlet extraction method using hexane as solvent [34]. Chlorophyll and carotenoid contents were determined as described previously [36]. All analysis was carried out at least in triplicates.

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2.4. Oil extraction from prickly pear seed and fatty acid composition The seeds were separated from the fresh pulp, washed several times with water,

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air-dried at ambient temperature then grinded until obtaining a fine powder. Prickly pear

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seed oil was obtained by extraction from the seed powder as described previously [37]. The prickly pear seed oil was transformed to fatty acid methyl esters (FAMEs)

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according to Maxwell and Marmer [38]. Fatty acid composition was determined by gas-

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liquid chromatography equipped with polar capillary column (Shimadzu, GC-2014, DANI/SPA type, 0.32 mm internal diameter, 50 m length and 0.25 mm film thickness,

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J&W Scientific). The operational conditions were: injector temperature 200 °C; detector

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temperature 250 °C; column temperature 165 °C for 5 min then a gradient of 5 °C/ min to 220 °C; carrier gas was nitrogen at a flow of 4.5 ml/ min. Analysis of free fatty acids

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was carried out in triplicate.

2.5. Preparation of crude extract of prickly pear

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The powdered whole prickly pear fruit (10 g) was extracted by maceration in 100 ml ethanol-water (70/30; v/v) at room temperature (about 25°C) over night. After that, the suspensions were filtered through a Buchner funnel. A second extraction was performed following the same conditions in order to extract the maximum of bioactive compounds. Finally, the solvent was removed under reduced pressure by rotary evaporator (Buchi Rotavapor R-200) at 50 °C. The aqueous fraction was lyophilized. The extraction yield for prickly pear extract (PPE) was calculated based on the dry 7

ACCEPTED MANUSCRIPT weight of the prickly pear powder. The resulting dry condensed extract was packed in a glass bottle and stored at 4 °C, until needed. 2.5.1. Determination of total phenolic contents The total amount of phenolic compounds was determined by the Folin-Ciocalteu reagent as described previously [39]. A volume of 0.5 ml of each extract was mixed

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with 0.5 ml of the Folin- Ciocalteu reagent. After 5 min, 0.5 ml of sodium carbonate (Na2CO3) solution (20% w/v) was added and the solution was brought up to 5 ml by

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adding distilled water. After 1 hour incubation at room temperature in darkness, the

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absorbance was measured at 760 nm against a blank. Gallic acid was used as standard at

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a concentration ranging from 31.25 µg /mL to 250µg /mL for the calibration curve. The concentrations of total phenolic compounds in the different spices extracts were

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determined as micrograms of gallic acid equivalent/mg of PPE (μg GAE/mg). The equation obtained from the standard gallic acid graph is as follows: (I)

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Absorbance = 0,006 μg gallic acid – 0.021 (R² = 0.99).

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2.5.2. Determination of total flavonoid content The method of Zhishen et al. was adapted to determine the total flavonoid

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content in PPE [40]. An aliquot of each sample (250 μl) was mixed with 1 ml of distilled water and subsequently with 150 μl of 150 mg/ml sodium nitrite solution. After

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6 min incubation, 75 μl of 100 mg/ml aluminum chloride solution was added, then the mixture was left 5 min before the addition of 1ml of 40 mg/ml sodium hydroxide (NaOH) solution. Distilled water was immediately added to the mixture until the total volume reached 2.5 ml. The absorbance was measured at 510 nm against a blank. Quercetin was used as standard at a concentration ranging from 15.63 µg /mL to 1mg /mL for the calibration curve. The total flavonoid content was expressed as micrograms

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ACCEPTED MANUSCRIPT quercetin equivalent/mg of PPE (μg QE/mg). The calibration recorded for this standard was expressed as follows: Absorbance = 0.006 μg quercetin + 0.006 (R² = 0.99).

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2.5.3. Antioxidant activity The free radical scavenging activity of sample was assessed by recording the bleaching extent of a DPPH solution from purple-colored radical, DPPH·, into the

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yellow-colored DPPH-H, according to Brand-Williams et al. [41]. An aliquot of ethanol absolute solution (500 µl) containing different concentrations (1:2 serial dilutions from

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the initial sample) of extract samples ranged from 1 to 1000 µg/ml was mixed with 500

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µl ethanol and 125 µl of freshly prepared solution of DPPH (0.02 % in 100 % absolute

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ethanol). The mixture was vortexed vigorously and incubated at room temperature in darkness for 60 min. The absorbance of the remaining DPPH radical was determined at

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517 nm against ethanol blank using a spectrophotometer. The IC50 values denote the concentration of tested extract samples, which is

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required to scavenge 50 % of DPPH free radicals. The corresponding inhibition

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percentages were calculated according to the following equation: ( Ablank  Asample ) Ablank

 100

(III)

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Radical scavenging activity (%) 

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Where Ablank is the absorbance of the control (prepared in the same manner without test compound), and Asample is the absorbance of the test compound. The values were presented as the means of triplicate analysis.

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ACCEPTED MANUSCRIPT 2.5.4. Determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) In order to determine the antibacterial activity of the PPE, various Gram-positive and Gram-negative bacteria were used: Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 27853), Salmonella enteric (ATCC 43972), Enterobacter cloacae

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Bacillus cereus (ATCC 14579) and yeast Candida albicans.

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(ATCC 13047), Staphylococcus aureus (ATCC 25923), Bacillus subtilis (ATCC 6633),

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The minimum inhibitory concentration (MIC) values, which correspond to the lowest compound concentration that completely inhibits the growth of microorganisms,

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were determined by a micro-well dilution method as previously described using 3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) [42]. The inoculum of

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each bacterium was prepared and the suspensions were adjusted to 107 CFU/mL. All the compounds were dissolved in 100 % ethanol, and then dilutions series were prepared in

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a 96-well plate, ranging from 3.125 µg/mL to 1mg/mL. Each well of the microplate

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contained 175 µL of the growth medium, 5 µL of inoculum and 20 µL of the diluted sample extract. Ethanol was used as a negative control. The plates were incubated at 37

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°C for 24 h, then 40 µL of MTT, at a final concentration of 0.5 mg/mL freshly prepared in sterile water which was added to each well and incubated for 30 min. The change to

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purple color indicated that the bacteria were biologically active. The MIC was taken where no change of MTT color was observed in the well. To determine the minimum bactericidal concentration (MBC), a liquid portion from each well that showed no change in color will be placed on solid LB and incubated at 37 °C for 24 h. The lowest concentration that yielded no growth after this sub-culturing will be taken as the MBC [42]. All experiments were made in duplicate.

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ACCEPTED MANUSCRIPT 2.6. Salami preparation Mechanically separated turkey (MST) meat was grinded in a commercial food processor (Universo, Rowenta, Germany) at the highest speed. All ingredients (salt, modified starch, etc) were added to the ground MST as powders. In the second step, cold distilled water was added to the mixture and final temperature of batters was about

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10 °C. The batters were manually stuffed in collagen reconstituted casing (30 mm

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diameter) and hand-linked to form approximately 9 cm long links. Then salami was cooked in a temperature-controlled water-bath (Haake L, Haake Buchler Instruments,

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Karlsruhe, Germany) maintained at 90 °C for 1 hour until a final internal temperature of

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74 °C was reached. A thermocouple (type-T, copper–constantan) was inserted into the centre of a link, and the time/temperature data were recorded. Finally, salami links were

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cooled immediately in an ice-water-bath and stored at 4 °C for 30 days. The substitution of the preservative (E250) and the colorant (E120) by PPE was

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carried out under several experimental conditions based on various amounts of extract.

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Results of the preliminary sensorial assay (data not shown) allowed us to maintain two amounts of extract (1.0 % w/w and 2.5 % w/w) which were necessary to improve the

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color and the texture of the salami. All formulations were prepared with the same common ingredients: 60 % MST, 29 % water (ice- and cold-water), 8 % modified starch

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(E-1422, Sigma Chemical CO., St Louis, MO), 2 % NaCl, 0.5 % TPP and 0.045 % ascorbic acid. Different levels were studied by adding 1.0 % and 2.5 % of PPE to the novel formulation. In addition a standard sample was prepared in parallel which contained dye carmine or Cochineal (E120) and 0.8 % (E250). The process was replicated twice.

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ACCEPTED MANUSCRIPT 2.6.1. Salami water holding capacity and water activity To measure the stability of formulated salami during storage, the water holding capacity (WHC) was measured after 7, 21 and 30 storage days at 4 °C. About 10 g of each salami sample was centrifuged for 30 min at 9000 rpm at 4 °C. The water holding capacity (WHC) was calculated as a percentage and bound water as described

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previously [43]. WHC (%) = (Wac / Wbc) × 100

(IV)

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Where Wac is the weight of salami after centrifugation and Wbc is the weight of salami

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before centrifugation.

The water activity (aw) of the different formulated salami was determined using

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a Novasina (Aw SPRINT TH-500), Swiss mode, Novasina). All measurements were

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done on triplicate different samples per type of product. 2.6.2. Microbial analysis

The microbiological quality of the salami samples was evaluated by

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determining the total mésophills bacteria count, Clostridiums perfringens count, Staphylococcus aureus count, Salmonella count and yeast and molds count during the storage at 4 °C for 7 and then for 30 days. Ten grams of salami sample were randomly

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picked up and homogenized in the Stomacher 400 (Seward, England) with 90 ml of

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sterile peptone water for 60 s. Appropriate dilutions of samples were prepared in sterile peptone water blank and repeated in duplicate on different growth media. Total mesophilic bacteria counts were determined using Plate Count Agar (PCA, Merck, Darmstadt, Germany) after incubation at 30 °C for 48 h according to the guidelines of CSN ISO 4833:2003). The enumeration of microorganisms was performed by horizontal method. 1 ml of serial dilutions of rinse fluid (1:100–105) was poured onto Petri dishes. The quantification of yeast and moisture was performed on PDA medium 12

ACCEPTED MANUSCRIPT at 25 ± 1 °C for 3 to 5 days, in accordance with the guidelines of NF ISO 7954. However, the determination of the Staphylococcus aureus and the Clostridiums perfringens counts were carried out on Baird-Parker (BP) and TSN medium at 37 °C for 24 h, respectively and in accordance with the guidelines of ISO 6888- 1,2 (2005). The Salmonella account was also performed on sodium selenite at 37 °C for 24 h (ISO

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9579). All analyses were performed in duplicate. 2.6.3. Texture measurement

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A texturometer (Texture Analyser, TA Plus, LLOYD instruments, England) was

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used to perform the texture profile analysis (TPA) of salami samples. All instrumental

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texture analyses were done on samples stored at least for 24 h at 4 °C. For every formulation, two repeated measurements were taken for each replicate and mean values

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are reported. Slices of salami (2 cm diameter and 2 cm thick) were cut from the centre of the links and compressed twice to 50 % of their original height between flat plates

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and a cylindrical probe (1 cm2 in diameter). Force–time curves were recorded at a

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crosshead speed of 20 mm/min [44]. In these experiments Hardness (N), Springiness (mm), Cohesiveness, Gumminess (N) and Chewiness (N/mm) were evaluated using the

of product.

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computer software. All measurements were done on triplicate different samples per type

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2.6.4. Sensory analysis

Sensory analysis was evaluated by 40 panelists, who were experienced in sensory evaluation of foods, but received no specific training relevant to this product. Evaluations were performed in individual booths, prepared as described in accordance with ISO6658:2005. Panelists were asked to indicate how much they liked or disliked each product on a 5-point hedonic scale (5 = like extremely; 1 = dislike extremely) according to color, odor, taste and texture characteristics. Slices of salami with 2 cm 13

ACCEPTED MANUSCRIPT thick were presented in polystyrene plates to the panelists with three-digit codes and in random order for evaluation. Sensory tests were conducted in an appropriately designed and lighted room to ensure that the testing conditions are stable from one test to another. A mean score was estimated for each product using Design Expert 7.0.0 software (StatEase, USA).

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2.6.5. Color analysis Salami was sliced with a thickness of 1 cm and put in petri dishes before

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measuring. Samples were measured using a handheld tristimulus colorimeter (Minolta

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chroma Meter CR-300, CIE, 1976) and a CIE standard illuminant C to determine CIE

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color space coordinates, L*, a* and b* values. L* value indicates the lightness, 0-100 representing dark to light, a* value gives the degree of the green–red color, with a

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higher positive a* value indicating more red. The b* value indicates the degree of the blue–yellow color, with a higher positive b* value indicating more yellow. The

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colorimeter was calibrated against a standard white plate before each color

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measurement. All measurements were performed in triplicate. 2.7. Statistical analyses

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Analysis and samples treatment were repeated at least three times. Means and standard deviations were calculated with Microsoft Windows Excel 2003. SPSS

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(version 20.0, IBM, USA) for Windows software was used to verify significant differences between treatments and means by Duncun’s test at p < 0.05. 3. Results and Discussion 3.1. Physicochemical composition The determination of the physico-chemical composition of the prickly pear is an important step to take into account in order to exploit it in the salami formulation.

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ACCEPTED MANUSCRIPT Table 1 presents the chemical composition of the prickly pear (Opuntia stricta). According to the obtained results, these fruits were characterized by high moisture (78.45 %), low amount of lipid (0.82 %) and protein (1.29 %). Prickly pears contain a high content of carbohydrates in fresh matter basis above 18.81 % and 8.33 % of reducing sugar. This may be due to their vegetable nature, the pectocellulose wall in cell

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plants is composed of a polysaccharides complex such as cellulose and lignin. Previous work has showed that the lignin content in a dry weight basis of prickly pear peel is low

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(2.4 wt.%), and the main constituents are polysaccharides (66.1 wt.%), including

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cellulose (27 wt.%) [45].

Moreover, the carbohydrate was known as the non-starch polysaccharide

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(Dietary fiber). Several works have showed that the polysaccharide composition of

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Opuntia species was characterized by the presence of neutral sugar content, mainly consisting of galactose, rhamnose and galacturonic acid [26;27]. Furthermore, we can

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see from Table 1, the presence of a high level of chlorophyll (923.48 mg/g of fresh

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matter) and carotenoids (203.68 mg/g of fresh matter). Several studies showed that pigment, betaxanthin and indicaxanthin, are the major compounds of prickly pear plant

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[21;15].

3.2. Determination of mineral element contents

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The contents of prickly pear (O.stricta) in mineral elements were determined using atomic adsorption analysis (Table 2). The main elements were Calcium, Magnesium, sodium and potassium, followed by, Iron, Zinc and Copper. The most abundant electrolytes in prickly pear are calcium (Ca2+) and magnesium (Mg2+), where the amounts are in the range of 278.23 g/kg of dry matter (DM) and 192.02 g/kg DM respectively (Table 2). These minerals play a crucial rule in the physiological structure and activity of many proteins [46]. In addition, the content of sodium (Na+) and 15

ACCEPTED MANUSCRIPT potassium (K+) are in the range of 183.79 g/kg of DM and 109.51 g/kg DM, respectively. This electrolyte constitutes the main extracellular anion balance assuming an important role in the maintenance of the acid-base balances of the extracellular medium [47]. So, prickly pear fruit seems to be a valuable source of electrolytes for human being.

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3.3. Oil extraction from prickly pear seeds and fatty acid composition Oil is an important storage form of carbon in many seeds. Based on the fact that

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the extraction at high temperature can affect the quality of seeds oil, the cold extract was

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carried out in the present study [48].

The O.stricta seeds had a relatively high oil content about 7.56 % dry matter.

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Fatty acids composition of the O.stricta seeds oils is presented in Table 3. The most

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abundant fatty acid is linoleic (~71.78 %), followed by oleic (~12.65 %), palmitic (~11.03 %) and stearic (~3.26 %) acids. These results are in line with those found in

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seeds oil of Opuntia dillenii from Marocco [49]. However, the extracted oil from red

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Algerian variety of Opuntia ficus-indica seeds showed that linoleic acid was the dominating fatty acid with 58.7%. Pantaleon et al. have found a higher level of linoleic acid (around 67%) in Opuntia boldinghii seeds oil [50]. The differences in the fatty

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acids composition in extracted seeds oils were due, probably, to the environmental

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condition or to the maturity degrees of the prickly pear fruit [51]. Opuntia stricta seeds oil contain higher amount of poly-unsaturated fats, essentially linoleic acid, which is higher when compared to some edible vegetable oils such as sunflower, soybean, sesame and olive oils (49.7%, 49.7%, 44.5% and 3.5 to 21% respectively) [52;53]. Authors showed that O. ficus-indica seeds oil plays a natural preventive role in cardiovascular diseases. They also decrease the LDL cholesterol grace to its high level of poly-unsaturated fatty acids [54;55]. 16

ACCEPTED MANUSCRIPT 3.4. Determination of total polyphenol content and antioxidant activities The phenolic compound, chlorophylls and carotenoids contribute to the food stability against oil oxidation. Similarly, several studies have confirmed the existence of a direct correlation between antioxidant activities of plant extracts and their phenolic content [56]. For this reason, the level of phenolic acid content of PPE has been

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measured according the Folin–Ciocalteu method. As we can see, a high level of polyphenols (370.60 ± 0.12 µg GAE/ mg of PPE) and Flavonoids (152.25 ± 0.26 µg

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QE/ mg PPE) has been found. Similarly, Tounsi-Saidani et al. showed a high content of

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phenolic compounds in Opuntia ficus-indica from Tunisian varieties [57]. Larrauri et al. have established a direct relation between the presence of polyphenols and the

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carbohydrate content (Dietary fiber) and indicated that the polyphenols are associated

MA

with dietary fiber [58]. Moreover, several studies showed that coats of vegetable seeds and peels of fruits (present a high dietary fiber content) contain higher amounts of polyphenols [25;59]. Interstingly, the presence of these phenolic compounds and

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carbohydrates give to the prickly pear extract an interesting antiradical activity with an IC50 concentration (50% inhibition) of 29.61 mg/ml. Our results suggest that carbohydrates and phenolic compounds are responsible for enhancing the product

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texture. In fact, O.stricta can be considered as a good candidate for natural fruit source

AC

of antioxidants. Shimada et al. suggested that reductone-associated and hydroxide groups of polysaccharides can act as electron donors and can react with free radicals to convert them to more stable products and thereby terminate radical chain reactions [60]. 3.5. Determination of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC)

17

ACCEPTED MANUSCRIPT The antibacterial activity of prickly pear extract was quantitatively assessed by determining the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Table 5 shows that O.stricta extract presents a high antibacterial activity. The red prickly pear extract (PPE) displayed an interesting antimicrobial activity, especially

PT

against bacteria Gram+. The minimum inhibitory concentration of the prickly pear extract varying between 15.62 and 62.5 µg/ml was found to be comparable to the

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sodium nitrite (E250) which is commonly used by the food industry. Table 5 further

SC

shows that the MBC values of prickly pear extract exhibits more interesting and effective antimicrobial activities than the E250 with MBC values ranged between 125

NU

and 500 µg/ml, especially on the Bacillus subtilis, Bacillus cereus and Staphylococcus

MA

aureus strains. This antimicrobial activity is likely due to the betalain pigment, carbohydrate and phenolic compounds present in Opuntia stricta extract. These findings

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are in agreement with results obtained in others studies [21]. Interestingly, Ali and El-

PT E

Mohamedy showed that an increase in betalain pigment concentration, extracted from red prickly pear, leads to increased inhibition zone of all tested microorganisms (Gram+ or Gram-) for dyed wool in comparison to undyed wool [21]. Moreover, Bargougui et

CE

al. showed that Opuntia ficus-indica extracts from cladodes and fruits have anti-

AC

parasitic activity [20]. However, authors reported that ethyl acetate fruit extract and ethyl acetate cladode extract were active against Leishmania donovani with IC50 values at 70.3, 70.5 µg/mL, respectively [20]. Furthermore, previous reports showed that polysaccharides from L. japonicum and garlic straw (Allium sativum L.) possess significant broad-spectrum anti-microorganism activity [21;61-63]. Authors proposed that the polysaccharide disrupted the cell wall and cytoplasmic membrane, leading to the dissolution of the protein and leakage of essential molecules, resulting in cell death. 18

ACCEPTED MANUSCRIPT They suggested also that DNA might be decomposed into small pieces after the polysaccharide entered the cell [63]. These results can be due also to the differences in the cell envelope composition between Gram+ and Gram- bacteria, which affect the permeability of these microorganisms to carbohydrate compounds [21;61-63]. 3.6. Effects of prickly pear extract on formulated salami properties

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Different formulations of the salami have been achieved in the laboratory. In these formulations, the dye E120 and preservative E250 were substituted by prickly

RI

pear extract. Preliminary tests were made using different percentages of the prickly pear

SC

extract. The obtained results have helped us to retain defined percentages (1.0 % w/w

NU

and 2.5 % w/w) for the remaining analysis (Fig.1). The water holding capacity, the microbiological, textural and sensorial analyses were assessed to evaluate the influence

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of prickly pear extract addition on developed salami properties. 3.6.1. Effect on the water holding capacity of salami

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After incorporation of prickly pear extract, the analysis of water activities (a w)

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for prepared salami was evaluated and compared to standard salami. Results showed that values are similar (aw about 0.952 ± 0.01) to those obtained in the control, with no

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differences among the different formulated salami (data not shown). Accordingly, the addition of prickly pear extract has no effect on water activity of the formulated salami.

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However, the effect of prickly pear extract addition at different concentrations and the storage time on water holding capacity of salami formulated with mechanically separated turkey meat is shown in Fig. 2. It can be seen that WHC of the salami with prickly pear extract seems to have no or a very limited effect on the water holding capacity of meat for one month at 4 °C, while WHC increases for the standard. The increase of WHC in salami standard is probably due to the water loss during storage. Indeed, Candogan and Kolsarici explicated that with the reduction in pH, the water 19

ACCEPTED MANUSCRIPT holding capacity of proteins decreases which leads to an exudation of water outside the product [64]. So the quantity of water excluded after centrifugation will be weaker and the WHC seems to be higher. However, the WHC stability in presence of the PPE could be related to its wealth of the phenolic compounds and fibers which can prevent the decrease of the salami pH. Therefore, the water holding of proteins present in salami

PT

contributes to better prevent the exudation of water outside the salami as explained previously by Candogan and Kolsarici [64]. Our results are in line with other researches

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who showed that the addition of carrageenan to salami (especially from beef meat)

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increases its capacity to retain the water during the storage which helped maintain the water holding capacity [43;44]. However, it was showed that the incorporation of

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lyophilized Melissa officinalis extract was effective in the stabilization of a new reduced

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fat sausage formulation without noticeable texture defects [65]. One can say that the addition of prickly pear extract improves salami stability by preventing the loss of water

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which allows the juiciness of salami and the conservation of its sensorial qualities.

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3.6.2. Effect on microbiological parameters of salami The interpretation of the results was made based on the guidelines of ISO mentioned by reference to each type of microbial analysis (Table 6). However, all

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results found are satisfactory and in accordance with those found with the control (Table

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6). The determination of the microbial flora counts (total mesophilic bacteria, yeasts and molds, Staphylococcus aureus, Clostridium perfringens, Salmonella) which may contaminate the prepared salami showed their total absence as required by the norms after one month of storage at 4°C (Table 6). The O.stricta fruits, present many virtues like anti-inflammatory [17], antioxidant [19], anti-Leishmania activity [20] and antibacterial effects [21]. Our results showed that PPE exhibits interesting and effective antimicrobial activities. In fact, the microbiological stability of the formulated salami 20

ACCEPTED MANUSCRIPT may be related to the richeness of PPE with flavonoids, vitamins, carbohydrates, betalains and phenolic molecules. These highly potent antioxidant compounds may contribute individually or synergistically to increase the antimicrobial activity and to stabilize the final product. 3.6.3. Effect of prickly pear extract addition on textural parameters of salami

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Table 7 shows the evolution of textural parameters of formulated salami as a function of prickly pear extract amount added. From this Table it is clear that prickly

RI

pear extract (PPE) causes a little decrease in salami hardness for each PPE

SC

concentration. Indeed, the standard salami has a hard texture so it requires more force to be deformed. However, Table 7 shows that the addition of prickly pear extract (1.0 %

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and 2.5 %) had no effect or a slight increase on salami cohesiveness and therefore

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chewiness. It requires less effort than the control product to be chewed into a state of swallowing.

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The evolution of functional properties with PPE concentration shows that the

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springiness and gumminess decrease slightly with the increase of PPE concentration from 1.0 % to 2.5 % as compared to the control (Table 7). A significant decrease in salami springiness was observed at 2.5 %. This can be explained by considering the

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decrease in the compactness of the protein network which leads to an aerated and more

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elastic structure.

All these textural changes can be explained by the fact that the presence of PPE may have an influence on the gelling process of proteins. PPE-muscle proteins interaction causes modification in microstructure of the prepared salami which could be explained by the abundance of the prickly pear structure on soluble fiber and pectocellulosic contents [43]. We can conclude that the presence of prickly pear extract allows the retention of water by his fibers content which increase the juiciness and 21

ACCEPTED MANUSCRIPT decrease the hardness and chewiness of formulated salami. Similarly, many authors reported that dietary fibers can be integrated in meat food products, to enhance textural properties, avoid syneresis, and stabilize high-fat food and emulsions [66,67]. Several studies, focused on the effect of carrageenan on functional properties of meat products, have been conducted [43;44]. Indeed, it was showed that the addition of κ-carrageenan

PT

to salt-soluble meat protein gels increases the gel strength and the water retention capacity [68]. Moreover, many works suggest that carrageenan is present in the

RI

interstitial spaces of the protein network which leads to a decrease in the compactness of

SC

protein gel network. These results are also in line with those found by Verbeken et al. who studied the influence of κ-carrageenan on the thermal gelation of salt-soluble meat

NU

proteins [43].

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3.6.4. Effect on the sensory characteristics and coloration parameters of formulated salami

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The sensory analyses of formulated salami with different concentrations of

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prickly pear extract are shown in Table 8. This Table shows that prickly pear extract amount had no significant influence on salami taste (p > 0.05). Average scores for color, odor and texture show that the panellist acceptability of the salami formulation increase

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with prickly pear extracts concentration. However, in the case of salami prepared with

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2.5 % prickly pear extract a significant increase in acceptability was obtained compared to control samples (p < 0.05). Moreover, it was found that the color of both formulation were not significantly different (p > 0.05). This result suggested that PPE could be incorporated into the product giving a color similar to that of the standard. Furthermore, sensory evaluation for salami taste and odor revealed that the salami prepared with 2.5 % prickly pear extract tended to higher score than the control. It'is well established that after oxidation the sensorial quality of the salami is well affected [63]. Interstingly, 22

ACCEPTED MANUSCRIPT Velasco and P. Williams have showed that meat quality could be improved through natural antioxidants [69]. Therefore, it was found that pigment, betanin and indicaxanthin, from prickly pear plant (Opuntia lasiacantha Pfeiffer) are eco-friendly natural colorants [21;26]. Thus, the color, odor and taste of salami prepared with 2.5 % prickly pear extract were

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very well appreciated by panellists when considering all attributes (Table 8). Interestingly, concerning the color acceptation, this result has been confirmed by

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the color measurement showed in table 9. It was found that lightness (L*) and

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yellowness (b*) of both formulation (with 1.0 % and 2.5 %) were not significantly different (p > 0.05) compared to the standard (Table 9). Lightness is related to the oil

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globule diameters of animal fat, which reflect light. However, table 9 shows that the

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addition of prickly pear extract at 1.0 % and 2.5 % had no effect or a slight increase on salami redness (a*). This result suggested that prickly pear extract could be incorporated

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into salami product with a slight change in color which depends on the prickly pear

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amount added. 4. Conclusion

In the present study, prickly pear (Opuntia stricta) extract was analyzed for its

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bioactive compounds and then valorized by its incorporation in salami production to

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substitute sodium nitrite (E250) and cochineal E120 which present side effects. Prickly pear extract showed highest antioxidant and antimicrobial potential which may be due to its richness on bioactive compounds such as betalain pigment, carbohydrates and total phenolic compounds. In fact, the results obtained showed that prickly pear extract addition ameliorate microbiological stability and allowed stability on water binding capacity of the novel formulated salami compared to the control. Furthermore, results showed that at 2.5% of substitution level, prickly pear extract could be included in a 23

ACCEPTED MANUSCRIPT salami preparation without altering its textural, sensory properties and extend its shelflife during refrigerated storage. In conclusion, PPE could be regarded as a potential health-promoting functional ingredient in meat products. Acknowledgements This study received financial support from the “Ministry of Higher Education,

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Scientific Research in Tunisia”. The authors are grateful to Pr. Sofiane Bezzine (ENIS, Sfax- Tunisia) for his generous gift of bacterial strains. The authors are indebted to

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Professor Anouar FENDRI (FSG) for English language correction.

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Corporation, (Canberra, RIRDC Publication: 2006) No: 06-132. [53] M. Nissiotis, M. Tasioula-Margari. Food Chem. 77 (2002) 371-376. [54] O.B. Ajayi, D.D. Ajayi. J. Nutr. 8 (2009) 116-118.

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ACCEPTED MANUSCRIPT [60] K. Shimada, K. Fijikawa, K. Yahara, T. Nakamura. J. Agric. Food Chem. 40 (1992) 945-948. [61] F. He, Y. Yang, G. Yang, L.J. Yu. Food Control. 21 (2010) 1257-1262. [62] X. L. Li, A. G. Zhou, Y. Han. Carbohydr. Polym. 66 (2006) 34-42. [63] F. Kallel, D. Driss, F. Bouaziz, L. Belghith, S. Zouari-Ellouzi, F. chaari, A.

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Haddar, S. Ellouz Chaabounia, R, Ghorbel. RSC Adv. 5 (2015) 6728-6741. [64] K. Candogan, N. Kolsarici. Meat Sci. 64 (2003) 199-206.

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[65] I. Berasategi, Í. Navarro-Blasco, M. Isabel Calvo, R. Yolanda Cavero, I.

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Astiasarán, D. Ansorena. Meat Sci. 96 (2014) 1185-1190.

[66] N. Ktari, S. Smaoui, I. Trabelsi, M. Nasri, R. Ben Salah. Meat Sci. 96 (2014) 521-

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[67] V. S. Eim, S. Simal, C. Rosselló, A. Femenia. Meat Sci. 80 (2008) 173-182. [68] Z. DeFreitas, J.G. Sebranek, D.G. Olson, J.M. Carr. J. Food Sci. 62 (1997) 539-

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543.

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[69] V. Velasco, P. Williams. Chil. J. Agr. Res. 71 (2011) 313-322.

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Figure captions Fig. 1. Salami prepared with various amount of PPE. (A) Salami with 1% (w/w) PPE; (B) Salami with 2.5 % (w/w) PPE; (C) Standard salami with E120 and E250. PPE = Prickly pear extract. E120 = Cochineal or carmin. E250 =Sodium nitrite. Fig. 2. Effect of PPE amount and storage time on WHC of salami. WHC: water holding

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D

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Capacity

29

ACCEPTED MANUSCRIPT

Fig. 1.

C

SC

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PT

B

A

AC

CE

PT E

D

MA

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Salami with 1% PPE

30

ACCEPTED MANUSCRIPT

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CE

PT E

D

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NU

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Fig. 2.

31

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Table 1 Chemical Composition of prickly pear (Opuntia stricta). ± indicates SD from 3 individual measurements. Contents (% fresh matter FM) 78.45 ± 1.260

Dry matter

21.55 ± 1.255

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Moisture

0.75 ± 0.066

Total lipid

0.82 ± 0.130

Total nitrogen (N)

0.21 ± 0.035

Proteins (N* 6.25)

1.29 ± 0.217

SC

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Ash

18.81 ± 1.170

Carbohydrate

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Reducing sugar

337.95 ± 0.083 585.53 ± 0.211 203.68 ±1.010

AC

CE

PT E

D

Carotenoids (µg/g FM)

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Chlorophyll a (µg/g FM) Chlorophyll b (µg/g FM)

8.33 ± 0.921

32

ACCEPTED MANUSCRIPT

Table 2

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Mineral contents in prickly pear (Opuntia stricta). ± indicates SD from three individual measurements. Mineral contents Element (g/kg dry matter (DM)) 183.79 ± 0.91

Ca

278.23 ± 1.05

Mg

192.02 ± 0.12

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Na

3.42 ± 0.32

Fe

109.51 ± 1.08

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K

0.54 ± 0.13

Cu

2.07 ± 0.43

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CE

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D

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Zn

33

ACCEPTED MANUSCRIPT

Table 3

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Fatty acid compositions of prickly pear seeds oil (expressed as a percentage (% w/w) of total fatty acid content). ± indicates SD from three individual measurements. Fatty acids

Composition (%)

C16:1 ω9

Palmitoleic

C18:0

Stearic

C18:1

Oleic

C18:2 ω6

Linoleic

C18:3 ω3

Linolenic

C20:0

Arachidic

0.17 ± 0.016

C20:1 ω9

Gondoic

0.24 ± 0.023

C22:0

Behenic

D

0.11 ± 0.006

C24:0

Lignoceric

0.10 ± 0.010

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Palmitic

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11.03 ± 0.156

C16:0

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NU

SC

0.40 ± 0.012 3.26 ± 0.060

12.65 ± 0.064 71.78 ± 0.956 0.26 ± 0.007

14.67 ± 0.110

MUFA : Mono-unsaturated fatty acids

13.29 ± 0.074

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SAFA : Saturated fatty acids

72.04 ± 0.950

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PUFA : Poly-unsaturated fatty acids

34

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ACCEPTED MANUSCRIPT

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Table 4

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Phenolic and flavonoid contents in PPE expressed as (μg Gallic acid Equiv (AGE)/mg dry extract) and as (μg Quercetin Equiv (QE)/mg dry extract), respectively. DPPH free-radical scavenging activity of PPE was expressed as IC50 (μg extract/ml). ± indicates SD from three individual measurements.

Total phenolic contents (µg GAE/mg PPE)

370.60 ± 0.12 152. 25 ± 0.26 29.61 ± 0.05

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Activité anti-radicalaire IC50 (µg/ml)

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Flavonoid contents (µg QE/mg PPE)

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PPE (Opuntia stricta)

35

ACCEPTED MANUSCRIPT

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Table 5

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Minimum inhibitory concentration (MIC) and Minimum Bactericidal concentration (MBC) of prickly pear extract (PPE) and the sodium nitrite (E250) on bacteria.

Strain

Gram

SC

MIC (µg/ml) PPE

E250

PPE

E250

125

500

500

125

250

500

62.5

250

500

125

125

500

1000

125

250

500

>1000

125

500

1000

1000

250

500

> 1000

1000

500

250

> 1000

500

62.5

Bacillus subtilis

+

15.62

Staphylococcus aureus

+

62.5

Enterobacter cloacae

-

Escherichia coli

-

Salmonella enteric

-

Pseudomonas aeruginosa

-

PT E CE

AC

Candida albicans

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+

D

Bacillus cereus

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Bacteria

Yeast

MBC (µg/ml)

36

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ACCEPTED MANUSCRIPT

Table 6

Methods

Total bacteria (UFC/g)

103/g

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ISO 4833 (2003)

Standard salami

Limits

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Strains

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Microbiological analysis of salami prepared with different concentrations of prickly pear extract (PPE). Salami with Salami with 1.0 % PPE 2.5 % PPE

Storage at 4°C (Days) 7

30

7

30

7

30

0

2. 102

0

< 10

0

< 10

NF ISO 7954

104/g

0

< 102

0

< 102

0

< 102

Staphylococcus aureus (UFC/g)

ISO 6888- 1,2 (2005)

103/g

0

0

0

0

0

0

Clostridium perfringens (UFC/g)

ISO 6888- 1,2 (2005)

102/g

0

0

0

0

0

0

AC

Salmonella

CE

Yeasts and Molds

ISO 9579

Absence/25g

Absence/25g

Absence/25g Absence/25g

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Table 7

Textural parameters of different formulations of salami. ± indicates SD from 3 individual measurements. Cohesiveness

Springiness (mm)

Chewiness (Nmm)

Gumminess (N)

Standard salami

16.22 ± 3.25

0.36 ± 0.21

8.48 ± 1.60

46 ± 0.08

6.71 ± 0.79

Salami with 1.0 % w/w PPE

8.83 ± 0.94

0.35 ± 2.76

8.87 ± 0.66

39.36 ± 1.10

4.44 ± 0.53

Salami with 2.5% w/w PPE

11.31± 1.85

0.45 ± 1.09

7.76 ± 0.45

33.5 ± 0.84

4.31 ± 0.30

PT E

D

MA

Hardness (N)

AC

CE

Samples

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Table 8 Odor

Taste

Texture

Appreciation (%)

Standard salami

3.25 ± 0.64 a

3.45 ± 0.76 a

3.5 ± 0.69 a

3.3± 0.92 a

51.3

Salami with 1.0%w/w PPE

3.30 ± 0.85 a

3.5 ± 0.76 a

3.75 ± 0.86 a

3.6 ± 0.89 a,b

56.1

Salami with 2.5% w/w PPE

3.95 ± 0.66 a

4.1 ± 0.95 b

4.1 ± 0.91 a

3.9 ± 0.88 b

70.5

NU

Color

MA

Samples

SC

Sensory characteristics of “salami formulation” containing different concentrations of PPE.

AC

CE

PT E

D

a,b: Means in the same columns followed by the same letters represent no significant differences at (p≤0.05).

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SC

Table 9

NU

Coloration measurements of “salami formulation” containing different concentrations of PPE. ± indicates SD from 3 individual measurements. L*

a*

b*

Standard Salami

49.30 ± 0.65

22.45 ± 0.87

5.61 ± 0.81

Salami with 1.0% w/w PPE

49.43 ± 0.51

23.17 ± 0.62

5.75 ± 0.52

48.15 ± 0.26

25.53 ± 0.75

5.21 ± 0.45

AC

CE

PT E

D

Salami with 2.5% w/w PPE

MA

Samples

40