Nutritional composition, lipid profile and stability, antioxidant activities and sensory evaluation of pasta enriched by linseed flour and linseed oil

Pasta assortments fortified with high quality foods are a modern nutritional trends. This study, explored the effects of fortification with linseed flour (LF) and linseed oil (LO) on durum wheat pasta characteristics. Wheat flour semolina was replaced with 5%, 10% and 15% of LF or 1%, 2.5% and 5% of LO. Control pasta CP (without LF or LO addition), LF-enriched pasta LFP 5%, LFP 10% and LFP 15% and LO-enriched pasta LOP 1%, LOP 2.5% and LOP 5% was compared for the proteins, fat and phenolic contents and fatty acids (FA) profile. Impact on lipid oxidation and sensory evaluation were also determined. Fortification of pasta with LF improved significantly (p < 0.05) the contents of protein, fat and phenolic compared to CP whereas the enrichment of pasta with LO resulted in a significant increase (p < 0.05) in the content of fat and a significant decrease in protein and phenolic contents. All the formulations decreased the saturated FA percent and increased the polyunsaturated FA percent with enhancement of omega-3 FA content. Antioxidant activity measured by FRAP and DPPH assays was improved after the fortification. For lipid oxidation, the replacement of semolina by LF or LO promoted an increase (p < 0.05) on TBARS values in level-dependent manner. Regarding sensory evaluation, the two types of fortification did not affect the taste; flavor and aroma of cooked pasta, but LOP 5% showed the highest score of the overall acceptability. The results recommended the possibility of producing pasta supplemented with LF or LO (even at a level of 15% and 5% respectively) as a functional food.


Introduction
Natural bioactive compounds and extracts have long been known to have health benefits [1,2] In fact, most of the bioactive compounds have antioxidant, anticarcinogenic, antiinflammatory, [3] and antimicrobial effects [4].Today, These compounds naturally found in various foods, are gaining interest in food industry due to changes in consumer habits and they may be added as natural additives in foods [5][6][7].Dietary linseeds or flaxseed is one of these foods that contain several healthful components predominantly oil (40-45%), especially ω-3 fatty acid (α-linolenic acid (ALA)), lignan (1%) especially secoisolariciresinol diglycoside (SDG) and dietary fiber (20-25%) mucilage [8].It contains also other less abundant components including the proteins like cysteine, methionine and arginine, phenolic acids, flavonoids and potassium which have health benefits.ALA is an omega-3 fatty acid and has been reported to be useful in the prevention of the cardiovascular disease by the reduction of two risk factors hypertension and high cholesterol levels [9].SDG is the most prevalent lignan in linseeds and are characterized as phenolic acid and it act as both antioxidants and phytoestrogens [10].Linseeds fiber has hypocholesterolemic effects and reduce the serum levels of Triglycerides [11].Due to the high nutritional content and health benefits, linseeds ingested in clinical trials may be administered in capsule form [12], or fortified in functional foods like yogurt [5] or supplemented other cereal based products, including bread [13,14], pasta [6] and noodles [15].However, the physicochemical properties of dried pasta manufactured with partial replacement of semolina by linseed flour and/or linseed oil is still little known.In this context, and to better explore the nutritional role of linseeds in pasta production, we investigate in the present study the optimization levels of linseeds flour (LF) and linseeds oil (LO) to improve the nutritional and sensory values of the product.Firstly, LF and LO were compared for their phenolic content and antioxidant activities.Then, for pasta production, durum wheat semolina pasta with 3 substitution levels of LF, being 5, 10, and 15 g/100 g (w/w) or 3 substitution levels of LO, being 1, 2.5, and 5 g/100 g (w/w).After pasta fabrication, we evaluated the effect of increasing levels of LF or LO in durum wheat semolina pasta by focusing on (i) nutritional and fatty acids composition, (ii) Antioxidant and anti-lipid oxidation impacts and (iii) the sensory evaluation of different cooked pasta samples.

Materials
Linseeds and linseeds oil (LO) extracted by supercritical CO2 were purchased from society of.
NATURLINA PRECIEUX (Bullarigeéa, 8100 Jendouba, Tunisia).Pure durum wheat Semolina was obtained from Society of pasta Warda, Sousse, Tunisia.All chemicals reagents were of analytical grade, and were acquired from Sigma Aldrich.

Linseeds flour (LF) preparation
LF were sundried until constant weight.Then the sundried seeds were finely ground using a Moulinex grinder (Moulinex LM242027, 450 watts, France) at high speed for 3 min with particle size of 0.5 mm and stored immediately in polypropylene package at 4 °C until used.

Pasta making
Pasta flour was prepared by mixing durum wheat semolina (SE) and LF at level of 5%, 10% and 15% (w/w) of SE or by mixing SE and LO at levels of 1%, 2.5% and 5% of SE.For each formulation, pasta flour was manually mixed with tap water (flour: water, 1:0.4,w/w) 5 min to obtain a homogeneous dough.Pasta samples were referred to as CP, control pasta without supplementation, LFP 5%, LFP 10% and LFP 15%, respectively pasta enriched with 5%, 10% and 15% of LF and LOP 1%, LOP 2.5% and LOP 5%, respectively pasta enriched with 5%, 10% and 15% of LO.The dough was fabricated using laboratory scale extruder (TR 70 INOX version, Lineapasta equipment) equipped with bronze dies (Diameter 70 mm) and the vat capacity is 2.5 kg.Fresh pasta (ziti shape) was dried at 50 °C for 14 h in a hot air drier.Finally, the pasta samples were covered by the polypropylene package and were kept at room temperature for the next analysis.

Phytochemicals screening
Total Phenolic Content (TPC) of raw materials (LF and LO) and different pasta samples was quantified according to the method of Montedoro et al. [16] and was expressed as mg of gallic acid equivalent per g of dried weight (mg GAE/g DW).Fat (method 30-10.01)and protein (method 46-10.01)contents of pasta samples were determined in triplicates according to American Association of Cereal Chemists, 2000 [17].

Fatty acids profile
The dried pasta samples were ground and then oils was extracted with hexane using a Soxhlet system.For total fatty acid analysis, extracted oils were converted to fatty acid methyl esters (FAMEs) and injected on a Hewlett-Packard gas chromatograph (Hewlett-Packard, Palo Alto, CA) according the procedure described by Mekni et al. [18].Then, FAMEs were identified by comparing their relative and absolute retention times to those of authentic fatty acid standards analyzed under the same conditions and were quantified according to their percentage area in the lipid fraction.

Antioxidant activity
The DPPH method was used to determine antioxidant activity of samples using 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical as a reagent according to Braca et al. [19].The ferric reducing antioxidant potential (FRAP) was determined according to the method described by Oyaizu et al. [20].In each assay, the absorbance was measured by LAMBDA 365 UV-Vis Spectrophotometer and results were expressed in terms of inhibition percent.

Lipid oxidation
Lipid oxidation was determined using the protocol of the thiobarbituric acid-reactive substances (TBARS) proposed by Yin et al. [21].Five grams of each sample was mixed with 22.5 mL of 11% trichloroacetic acid for 1 min in an Ultra-Turrax (IKA T25 digital) followed by 1 min in an ice bath, and another 1 min of homogenization at equal conditions.The homogenate was filtered using Whatman paper N˚1, and 1 mL of 20 mM TBA was added to 1 mL of the filtrate following incubation in dark conditions for 20 h at 25˚C.TBARS analyze was performed in triplicate.The absorbance value at 532 nm was read using a UV-1800 spectrophotometer (Shimadzu, Kyoto, Japan) and the results were expressed as TBARS number.

Sensory assessment
Pasta samples were cooked in distilled water (pasta: water ratio is 1:4 gmL −1 ) to optimum cooking time (between 7-8 min) and served to the panelists.The sensory test panel consisted of eight panelists (4 females and 4 males aged 25-55 years) who were trained academic staff.Panelists judged flavor, taste, odor and overall acceptability using a 10-point hedonic scale ranging from 10-5 (like extremely) to 4-1 (dislike extremely) for each sensory characteristic.The score for each sensorial component was the average of three individuals' assessments [22].

Statistical analysis
The results were expressed as mean ± standard deviation of three independent experiments (n = 3).Analyses of variance were performed by the one-way ANOVA.Procedure and significance of differences was verified by Tukey's multiple range test (P < 0.05).ANOVA analyses were performed using the statistical package SPSS version 21 (Statistical Package for Social Science, SPSS Inc., Chicago, IL).

Total phenolic content (TPC) and antioxidant activity of linseeds flour and oil
Table 1 showed TPC of linseeds flour LF and oil LO and the IC 50 values which correspond to the amount of extract required to scavenge 50% of DPPH radicals present in the reaction mixture.LF showed higher TPC at 279.5 ± 30 mg GAE/g DW and an IC 50 value of 2.28 ± 0.04 mg/ml.The lipid faction of linseeds LO contained also a phenolic content estimated by 20 ± 0.04 mg GAE/g DW and exhibited an antioxidant activity with IC 50 value of 0.16 ± 0.01 mg/ml.Our results concerning LO are similar to that found by Nazia et al. [23] who reported that TPC of linseeds cultivars, native to Pakistan ranged from 2560 to 3286 mg gallic acid equivalent (GAE)/100 g.It has been reported that lignans are the major phenols present in linseed and also in linseeds oil but in relatively low levels [3].These molecules are known as power antioxidants and display as sequestrators of hydroxyl radicals [24].

Chemical composition of pasta samples
Table 2 reports the lipids, protein and TPC of control and supplemented pasta.LF incorporation increased slightly the protein content and highly the lipids compared to CP whereas LO addition decreased the protein content and increased highly the lipid content potentially due to the LO composition.In fact, it has been reported that Whole and ground linseed is rich in fat (42%), protein (18%) and fiber (27%) with minimal vitamin and mineral however, flax oil in its natural state is 100% fat, 53% of which is ALA, has low protein content and is devoid of carbohydrates and dietary fiber [12,25].Linseed proteins contain rich proportions of amino acids such as glutamic acid, methionine, arginine, cysteine and aspartic acid, with low amounts of lysine, threonine and tyrosine [26] and they have been associated with antifungal properties and antioxidant activities.The addition of LF and LO increased lipids contents in pasta by 8-ninefold compared to CP. Lipids are one of the high value functional ingredients in linseeds and are partly the reason why linseeds are considered to be a major health promoting agent.Food Products containing linseed and its derivatives have been proposed as the principal source of omega 3 fatty acids and as nourishing supplements for a range of dietary entities [5,6,15].Manthey et al. 2002, reported that lipid, ALA, and FFA contents declined during the extrusion process in dried spaghetti samples containing semolina fortified with 5 and 10% ground linseed.Moreover, they supposed that this decline in lipid content probably occurred during dough development in the extrusion process [6].
The fortification of pasta with LF improved the phenolic content (TPC) compared to CP.The highest value of 60.4 ± 4.2 mg GAE/g DW was recorded for the LFP 15%.However, pasta fortified with LO showed lower TPC compared to CP. Linseeds polyphenols such as SDG have multiple biological functions such as antioxidant, antiinflammatory, anti-cancer and antimicrobial activities.Nowadays there is interest in using whole linseed (flour) as functional food ingredients owing to the healthier and more sustainable properties than those of isolated linseed ingredients [27].Moreover, recent studies suggested that Linseed lignans found in grain flour can be used as a good antioxidant for improving the stability of linseed oil incorporated into functional foods [28].

Fatty acids profiles of pasta samples
The fatty acid composition of the pasta samples is presented in Table 3 and Fig. 1.A total of seventeen fatty acids were identified in fortified pasta samples.Generally, all the formulations showed significance difference (p < 0.05) in total SFA and PUFA compared to the control pasta.LF and LO fortification increased significantly the PUFA percent particularly the omega-3 fatty acids (C18:3) amount and decreased the SFA percent.
Regarding SFA, the decrease is estimated by 30% in pasta manufactured with LF 15% and LO 5% compared to CP and the palmitic acid (C16:0) was the major SFA.For MUFA, a significant decrease in oleic acid C18:1 w9 was recorded from 18 ± 0.05% in CP to 14 ± 0.18% in LFP15% and 14.44 ± 0.39% in LOP 5%.The PUFA fraction presented 66-68% of the composition of the pasta enriched by LF and LO.A remarkable decrease in linoleic acid C18:2 w6 was also showed in enriched pasta (from 53.60 ± 0.16% in CP to 39.36 ± 0.31in LFP15% and 38.53 ± 0.51% in LOP5% fortified pasta samples at highest dose.However, the percent of omega-3 fatty acid, the linolenic acid was enhanced compared to that of CP and it increased by sevenfold.These findings are consistent

Table 3 Fatty acids composition (%) of different pasta samples
Values are presented as means ± DS.CP, control pasta; LFP 5%, LFP 10% and LFP 15%: pasta prepared with 5, 10 and 15 g of linseeds flour, LOP 1%, LOP 2.5% and LOP 5%: pasta prepared with 1, 2.5 and 5 g of linseeds oil. to that found by Manthey et al., 2002 who showed that ALA content in spaghetti made from semolina fortified with ground linseed increased with increased ground linseed concentration.Whereas linoleic acid was the predominant fatty acid in lipid extracted from semolina (54.9%) followed by palmitic acid (20%) [6].Same findings were showed in bakery and dairy foods.In fact, the partial substitution of soybean oil with linseed oil in the preparation of whole-wheat linseed bread enriched with omega-3 increased the omega-3 percentage in formulations, which improved the nutritional value of wholewheat linseed rolls enriched with LO [13].the addition of linseed powder in yogurt formulation significantly decreased the saturated fatty acids (SFAs), the ratio of omega-6 to omega-3 fatty acids, and atherogenic indices of yogurts and increased the polyunsaturated fatty acids (PUFAs) [5].

Antioxidant activities
The percentage inhibition of DPPH and FRAP measured the free radical scavenging attributes of the different pasta samples and indicate its antioxidant potential.The antioxidant inhibition of pasta ranged from 8 to 50% (Fig. 2).Significant improvement in both the parameters was observed in comparison to control pasta, and maximum inhibition was recorded for LFP 15% followed by LFP 10%.Similarly, the fortification of pasta with LO improved the antioxidant activities in dose dependent manner.These results are in accordance with a previous study carried out on noodles fortified with ground linseed which showed that the in vitro antioxidant activities, measured by FRAP, DPPH and ABTS assays were increased [29].In addition, Chinese steamed bread containing barley, linseed, and barley-linseed hull extracts showed that antioxidant activity and total phenolic content was increased compared to control [30].The antioxidant activity of enriched pasta may be attributed to phenolic compounds found in linseeds such as lignan, a hydroxyl radical scavenger.These antioxidant capacity might contribute to a several health benefits, including protective effects against cardiovascular diseases, diabetes, cancer, and mental stress [31].

Impact on lipid oxidation
The replacement of semolina by LF or LO promoted an increase (p < 0.05) on TBARS values in level-dependent manner (Fig. 3).Linseeds contain high amount of polyunsaturated fatty acids (PUFA) which are more susceptible to lipid oxidation supporting our findings for the TBARS values.Waszkowiak et al., 2019 suggested that lipid oxidation in linseeds-enriched foods can be attributed to that linseed roasting caused the destruction of the membrane integrity of oil body, thus increasing the direct contact between bulk oil and oxygen [32].In these cases, the addition of some natural or synthetic antioxidants such as ascorbic acid and butylated can be an alternative solution to reduce oxidation of in linseed-enriched foods.Lipid oxidation in LOP is higher than that in LFP.This finding can be explicated by that the lipids in linseed are packed into oil bodies, which are protected by their phospholipid-protein membrane.In addition, the intrinsic antioxidants such as phenolic compounds in linseed may hinder the occurrence of lipid oxidation for Fig. 2 DPPH and FRAP assays (inhibition %) in control pasta (CP), pasta fortified with different linseeds flour levels (LFP 5%, LFP 10% and LFP 15%) and linseeds oil (LOP 1%, LOP 2.5% and LOP 5%).Results are expressed as means ± standard deviation (n = 3).Different letters indicate significance differences (p < 0.05) among pasta samples according to Tukey's test linseed-enriched products [33].Recent studies suggested that both Linseed lignan extract and SDG were good plant-based antioxidants for improving the stability of linseed oil nanoemulsions and their incorporation into functional foods and beverages [28].Our results were not consistent with those reported by Manthey et al. 2002, who showed that the levels of FFA and conjugated dienes, the two parameters of lipid oxidation, were reduced during processing and cooking of spaghetti fortified with ground linseed indicating that the triacylglycerols and ALA remained stable [6].Moreover, lipid remained relatively stable during processing and cooking of macaroni fortified with 15% of ground linseed [34].

Sensory evaluation
Generally, enrichment of a food product with bioactive components should not affect its sensory quality.In addition to its health benefits, the product should be very good in terms of taste, aroma and appearance.Results in Table 4 showed that linseed flour or oil could be successfully incorporated into pasta without adversely affecting their sensory attributes.Regarding overall acceptability, LO-enriched pasta with 5% of LO showed the highest score.However, there is no statistical difference between CP, LF-enriched pasta at different levels and LO-enriched pasta at 1% and 2.5%.Wheat bread and biscuits could be enriched by linseed fibre for 5%, but a larger addition could cause adverse sensory evaluation.In other studies, the sensory profiles of bread and other baking products with yellow and brown linseed fibre [35,36] in the amounts of 2.5% and 5.0% were also acceptable.In another study, linseed at 30-50% substitution for flour greatly enhanced the nutritional qualities of some nutrients such as Linolenic acid, fiber and folate contents without affecting the overall acceptability of bakery products like breads and muffins [37].

Conclusion
Obtained results showed that linseed flour and oil could be useful additives for durum wheat pasta production with enhanced pro-health properties.En effect, enrichment of durum wheat pasta with the addition of linseed flour and oil resulted in high increase in fat content compared with control pasta with an important enhancement

Table 1
Phenolic content and antioxidant activity of linseeds flour (LF) and Oil (LO)

Table 2
Composition of control pasta (CP) and pasta samples enriched with linseeds flour (LFP) and linseeds oil (LOP) Values are presented as means ± DS.CP, control pasta; LFP 5%, LFP 10% and LFP 15%: pasta prepared with 5, 10 and 15 g of linseeds flour, LOP 1%, LOP 2.5% and LOP 5%: pasta prepared with 1, 2.5 and 5 g of linseeds oil a,b,c,d,e Means followed by the letters within the same row indicate significant difference (p < 0.05) in Tukey test a,b,c,d Means followed by letters within the same row indicate significant difference (p < 0.05) in Tukey test

Table 4
Results of cooked pasta sensory evaluationValues are presented as means ± DS.CP, control pasta; LFP 5%, LFP 10% and LFP 15%: pasta prepared with 5, 10 and 15 g of linseeds flour, LOP 1%, LOP 2.5% and LOP 5%: pasta prepared with 1, 2.5 and 5 g of linseeds oil a,b Means followed by the letters within the same row indicate significant difference (p < 0.05) in Tukey test