Lipids, proteins, phenolic composition, antioxidant and antibacterial activities of seeds of peanuts (Arachis hypogaea l) cultivated in Tunisia

Fatty acid composition of peanut seed oil in four varieties cultivated in Tunisia showed that linoleic (C18:2), oleic (C18:1) and palmitic (C16) acids account for more than 84% for Chounfakhi and Massriya and for more than 85% of the total fatty acids of Trabilsia and Sinya seed oil respectively. Seed oil contents were signifi cantly diff erent (P ≤ 0.05) and did not exceed 48%. The study of total phenolics revealed that Chounfakhi contained more total phenolics (2.1 mg GAE/g DW), followed by the Massriya and Sinya cultivars (1.35 mg GAE/g DW for each); Trabilsia presented the lowest total phenolic content with 1 mg GAE/g DW. Considerable antiradical ability was found, especially in the Trabilsia peanut seed cultivar (IC50 = 1550 μg/ml), the Massriya and Sinya cultivars had, respectively, 720 and 820 mg/ml IC50. In the Massriya variety the sterol fraction showed antibacterial activity against Listeria ivanovii, Listeria inocua, Pseudomonas aeruginosa, Staphylococus aureus, Enterococcus hirae and Bacillus cereus.

The aim of this study was to analyze antioxidant activity and lipid, fatty acid, protein and phenolic composition in four groundnut varieties cultivated in Tunisia, as well as to test these compounds for antibacterial activity against reference strains.

Plant material
The four groundnut varieties used in this study were Massriya, Sinya, Chounfakhi and Trabilsia. Their seeds were provided by the CRDA of Nabeul (NE Tunisia).

Determination of oil content
Oil content was determined by extracting dry material of peanut with petroleum ether using a Soxhlet apparatus (4h/42 °C) (Harwood, 1984). The extract was dried in a rotary evaporator and the oil content was determined as the diff erence in weight between a dried peanut sample before and after extraction (AOCS, 1989).

Saponifi cation and TLC analysis
The unsaponifiable fraction of lipids was determined by saponifying 5 g of oil mixed with both 200 μl α-cholestanol and an ethanolic KOH 12% solution; the mixture was heated at 60 °C for 1.30 h. The unsaponifi able matter was extracted, washed, dried over anhydrous Na 2 SO 4 and evaporated to dryness using N 2 . The unsaponifi able matter was separated into subfractions on preparative silica gel thin-layer plates (silica gel 60G F254) using one-dimensional TLC with hexane-diethyl ether (6:4, v/v) as the developing solvent. The unsaponifi able fraction diluted in chloroform was applied on the silica gel plates. After developing, the plate was sprayed with 2,7-dichlorofl uorescein and viewed under UV light.

GC-FID Fatty acids methyl ester analysis
Fatty acids were methylated using the method of Metcalfe et al. (1966) modifi ed by Lechevallier (1966). Methyl esters were analyzed with a gas chromatography-fl ame ionization detector (GC-FID) using an HP 4890 chromatograph equipped with a fl ame ionization detector (FID) on a capillary column coated with supelcowaxTM 10 (30 m length, 0.25 id, 0.2 mm fi lm thickness). Temperatures of the column, detector and injector were 200, 250 and 260 °C, respectively.

SDS-PAGE of proteins
Defatted peanut seeds (100 mg) were successively extracted with 1 ml distilled water, 1 ml 5.0 M NaCl, 1 ml absolute ethanol, and 1 ml 0.2 M phosphate buff er (pH 8.0) for the extraction of the albumin, globulin, prolamin and glutelin, respectively. Each extraction was shaken for 20 min in an Eppendorf tube and centrifuged at 10,000 g for 6 min. Protein fraction assays were performed following Bradford's method (1976). S odium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed in a discontinuous buff ered system according to the method of La emmli (1970), using 12% separating gel and 3% stacking gel.

RP-HPLC phenol analysis
Colorimetric quantifi cation of total phenolics was determined as described by Dewanto et al., (2002). All samples were analyzed in three replicates.
Phenolic compound analysis was carried out using a liquid chromatography RP-HPLC coupled with a UV-vis multiwavelength Waters 996 Photoperiod Array Detector: 190-400 nm. The separation was carried out in a 250×4.6-mm, LD 5-μm symmetry shield C18 reversed phase column. The mobile phase consisted of acetonitrile (solvent A) and water (80:20 v/v) with 1% formic acid (solvent B). The fl ow rate was kept at 1 ml/min. The injection volume was 20 μl and peaks were monitored at 275 nm. Peaks were identifi ed by congruent retention times compared to standards. Analyses were performed in triplicate.

DPPH radical-scavenging activity
The DPPH-quenching ability of plant extracts was measured according to Hanato et al. (1988). The ability to scavenge the DPPH radical was calculated using the following equation: (1) DPPH-scavenging eff ect (%) = [(A0 − A1)/A0]×100; where A0 is the absorbance of the control at 30 min and A1 is the absorbance of the sample at 30 min. All samples were analyzed in three replicates.

Antibacterial activity detection
The fractions of lipids and proteins were individually tested against a large panel of microorganisms including Staphylococcus aureus (ATCC 6539), Pseudomonas aeruginosa (ATCC 15442), Escherichia coli (ATCC 25922), Enterococcus hirae ATCC10541, Listeria ivanovii and L. inocua (RBL 30 and RBL 29 respectively), Bacillus subtilis and B. cereus (168 and ATCC11778 respectively). All strains were obtained from the Institut Pasteur de Tunis. The bacteriological agar was from Biokar Diagnostics (Beauvais, France). Nutrient broth (NB) was from Difco (Becton Dickinson, Le Pont de Claix, France). All the other media used in this study were manufactured by Biorad (Marnes-La Coquette, France) and Merck. Antibacterial activity is revealed by growth inhibition in the test strain, observed in solid medium. In the present study we used the well diff usion method described by Perez et al. (1990).

Statistical analysis
The data (three replicates) were statistically evaluated using the JMP SAS version 12.6 software (Statistical Analysis System). (SAS, Institute INC, Box 8000, Cary, North Carolina 27511, USA).

Physiochemical characteristics:
The average compositions of the four peanut seed varieties are shown in Table 1. It shows significantly different oil contents (P≤0.05) which were very high in Chounfakhi and Sinya (about 48%), whereas in Trabilsia and Massriya they were slightly lower (45 and 46%, respectively). These differences were probably due to the different origins of the four varieties, the environmental conditions they were subjected to and/or year eff ect. For 1000-seed weight Trabilsia exhibited the lowest mean, but this showed no eff ect on its oil content. However, diff erences between the four varieties were not statistically signifi cant (P>0.05). Similarly, no signifi cant diff erences were found in moisture. The values the of acid index were signifi cantly diff erent (P≤0.05) in these groundnut varieties. Trabilsia and Massriya showed the highest values, which shows that their oils contain a lot of free fatty acids and are therefore of low quality. This may be due to the fact that seeds used in this work were not freshly harvested or were not well stored. Chounfakhi and Sinya, however, showed acid indexes which conform to the international norms of codex alimentarius.
Total unsaponifi able content did not exceed 1% in the four groundnut varieties and varietal diff erences were not statistically (P≤0.05) signifi cant (Table 1). In the literature it was reported that unsaponifi able contents did not exceed 2% in oil seeds such as those of rapeseed and linseed (Sebei, 2005;.

Biochemical composition:
Protein composition: The four groundnut varieties exhibited relatively low protein content (7-12%); the highest were found in Chounfakhi and Massriya (Table 1). In addition, diff erences between all varieties were statistically signifi cant (P≤0.05). In general, protein content in groundnut seeds is around 20-30% (Andersen et al., 1998). The local variety Chounfakhi can produce an appreciable quantity of protein, which encourages its cultivation in regions where it can adapt to climatic and edaphic conditions. Compared to other plant proteins, especially commercial soy protein isolate, the functional properties of peanut proteins were much poorer, which might greatly limit the application of peanut proteins in many food formulations (Liu et al., 2012). Using adequate buff ers, we isolated all protein classes in seeds of the four varieties and found that they were almost totally composed of globulins (95%), albumins, prolamins, and glutelins being with negligible contents (Table 1). No signifi cant (P≤0.05) diff erence was found in the content of each protein class between the four varieties.

SDS-PAGE of total proteins and their fractions:
This qualitative study was performed in SDS-PAGE. Figure 1 illustrates the electrophoretic profi les of total proteins in the four groundnut varieties. It does not reveal marked varietal diff erences. The major proteins belong to type A, represented with bands with molecular weight (MW) varying between 100 and 50 KDa (high MW), type B (medium MW, from 35 to 25 KDa), types C and D (MW from 15 to 12 KDa), and type E (low MW, less than 10 KDa). According to Figure 2, which represents globulin electrophoretic profi les in the four groundnut varieties, there were four major bands: Band A (proteins with MW between 50 and 35 KDa), Band B (proteins with MW around 25 KDa), Band C (proteins with medium MW between 15 and 25 KDa), and Band D (proteins with low MW (around 10 KDa).
Fatty acid composition: Fatty acid composition of peanut seed oil is given in Table 2, which shows that linoleic (C18:2), oleic (C18:1) and palmitic (C16) acids accounted for more than 84% for Chounfakhi and Massriya and for more than 85% of the total fatty acids in Trabilsia and Sinya seed oil, respectively. This study shows that the oils of Tunisian peanut varieties contained oleic and linoleic acids at relatively high levels 32. 63-39.65%, 27.16-41.38%, 30.31-41.85% and 32.12-40.06% respectively for the Chounfakhi, Trabilsia, Sinya and Massriya varieties. Saturated fatty acids (SFA) accounted for 19.93%, 26.04%, 22.03 and 20.67 of total fatty acids for Chounfakhi, Trabilsia, Sinya and Massriya, respectively. The main saturated normal chain fatty acids were myristic, palmitic, stearic, behenic and arachidic acid. The ratio of unsaturated to saturated fatty acids was 4.01 in Chounfakhi, 2.84% in Trabilsia, 3.53 in Sinya and 3.83 in Massriya. According to Ingale and Shrivastava (2011), the total fatty acid composition of peanut seeds was 10.44 and 33.51% for saturated and unsaturated fatty acids, respectively. The most abundant fatty acids of groundnut seed oil were oleic (C18:1), linoleic (C18:2) and palmitic (C16:0), which together composed about 88.35% of the total fatty acids. The most abundant saturated fatty acid in groundnut seed oil was palmitic acid (6.20 percent); the main unsaturated fatty acids were oleic acid (16.28%) and linoleic acid (16.35%).The source of variability may be genetic (plant cultivar, variety grown), seed quality (maturity, harvest-caused damage and handling/ storage conditions), oil processing variables, or accuracy of detection, lipid extraction method and quantitative techniques (Rodrigues and et al., 2011).
This preliminary study shows that peanut seed oils contain high relative percentages of linoleic acid (C18:2 ω6). Potential effects of linoleic acid on health include anticarcinogenic, antiatherogenic and antidiabetogenic modulating properties; it has also attracted interest in the scientifi c community because of its potential eff ects on body composition, reducing body fat mass and increasing lean mass (Hernandez-Dıaz et al., 2010). To enhance peanut seed oil, it was investigated as an alternative source for the production of a biodiesel fuel (Kaya and et al., 2009). The maximum oil to ester conversion was 89% and the viscosity of biodiesel oil is nearer to that of petroleum diesel; the calorifi c value is about 6% less than that of diesel.

Phenol analysis and antioxidant activities:
The total phenolics, expressed as mg GAE/g dry peanut seeds per cultivar, are presented in Table 3. Chounfakhi contained the most total  phenolics (2.1 mg GAE/g DW), followed by the Massriya and Sinya cultivars (1.35 mg GAE/g DW for each), Trabilsia presented the least total phenolic content, with 1 mg GAE/g DW. As found for total phenolic content, antioxidant activity showed diff erences between peanut seed varieties. The HPLC study of Arachis hypogea seed extract identifi ed 6 phenolic compounds in the Massriya cultivar, with rutin trihydrate and p-coumaric acid as the major compounds (6.79% and 6.35%, respectively), 7 phenolic compounds in the Sinya cultivar with p-coumaric acid as the major compound (4.03%), 8 phenolic compounds in the Chounfakhi cultivar with p-coumaric acid as the major compound (4.87%) and 5 phenolic compounds in Trabilsia cultivar with rutin trihydrate as the major compound (4.52%). The compounds identifi ed were (Table 4): caff eic acid, dihydroxyphenylacetic acid, syringic acid, p-coumaric acid, rutin trihydrate, nephtoresorinol, trans-2-dihydroxycinamic acid and dihydrate quercetin. Sales and Resurreccion (2010) reported that peanut skins contain fl avonoids such as epigallocatechin, epicatechin, catechin gallate and epicatechin gallate; proanthocyanidins including procyanidin B, A-type and B-type procyanidin dimers, trimers and tetramers; phenolic acids such as chlorogenic, coumaric, caffeic and ferulic acids; and the stilbene trans-resveratrol. Peanut kernels contain caffeic, ferulic, and coumaric acids (Sales and Resurreccion, 2009) and the stilbenes trans-resveratrol and its glucoside, transpiceid. Free p-coumaric acid, along with three tentatively identifi ed esterifi ed derivatives, was the predominant soluble polyphenolic that contributed to the antioxidant capacity of peanut kernels (Talcott et al., 2005 a).
Nuts are a good source of a wide range of bioactive compounds with health benefits. Many of the bioactive compounds that occur in nuts are associated with the oil fraction. Nuts contain tocopherols, tocotrienols, phytosterols and many different flavonoids, including isoflavones and quercetin. There is scientifi c evidence that health benefi ts are mediated by the bioactive components in the oil fraction of the nuts (Jonnala et al., 2006).

Antibacterial activity
Groundnut seed oil of the four varieties showed no antibacterial activity against all studied strains. This was not surprising, since no data were found in literature mentioning an antibacterial activity in fi xed oil. This is probably due to the fact that fixed oil is composed almost entirely of triacylglycerols (TAG) that are structurally composed of aliphatic carbon and hydrogen chains that induce no antibacterial activity. Phytosterols, minor compounds of plant oils, constitute the major fraction of their unsaponifi ables. Groundnuts showed phytosterol content varying from 900 to 3000 ppm; the most represented ones are β-sitosterol (48%), campesterol (10%), and stigmasterol (5%) (Dewanto et al., 2002). We found that sterols from the Massriya oil exhibited antibacterial activities against Listeria ivanovii, Listeria inocua, Pseudomonas aeruginosa, Staphylococus aureus, Enterococcus hirae and Bacillus cereus (Table 5). For triterpenic alcohols and carbohydrates that can be considered as terpenes, antibacterial activity was not clear. This was probably due to their low concentrations. Indeed, only few strains were inhibited by these compounds.

CONCLUSION
In summary, this study revealed that Tunisian peanut seeds are a rich source of many important components that have good nutritional attributes and appear to have a very positive eff ect on human health (polyunsaturated fatty acids (w 3 and ω 6 ), phenolic components, antioxidant biomolecules, antibacterial activity…). The high levels of fatty acids and protein content make peanuts a healthy food for human and animal nutrition. Crude peanut seed oil has a good potential as alternative fuel, which we will use in Tunisia as a source of biodiesel as perspective for our work.