The effects of ω-3 fatty acids and inulin addition to spelt pasta quality

LWT - Food Science and Technology xxx (2015) 1e9

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The effects of u-3 fatty acids and inulin addition to spelt pasta quality Jelena Filipovi c a, Lato Pezo b, *, Vladimir Filipovi c c, Jovana Brklja ca a, Jelena Krulj a a

University of Novi Sad, Institute for Food Technology, Bul. Cara Lazara 1, 21000 Novi Sad, Serbia University of Belgrade, Institute of General and Physical Chemistry, Studentski Trg 12e16, 11000 Beograd, Serbia c University of Novi Sad, Faculty of Technology, Bul. Cara Lazara 1, 21000 Novi Sad, Serbia b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 29 December 2014 Received in revised form 16 March 2015 Accepted 18 March 2015 Available online xxx

This paper investigates the effects of simultaneous addition of inulin (10 g/100 g of sample and 20 g/ 100 g of sample) and u-3 fatty acids content (0.2 g/100 g of sample, 0.4 g/100 g of sample and 0.6 g/100 g of sample), according to accepted experimental plan, on the technological quality of spelt pasta to obtain new products with altered nutritional properties. u-3 fatty acids content strongly influences texture properties (maximum hardness was obtained for pasta with 0.6 g/100 g of sample u-3 fatty acids content and 10 g/100 g of sample inulin HPX, the maximum of work of shear was reached for 0.6 g/100 g of sample u-3 fatty acids and without inulin HPX, while the minimum of adhesiveness, was observed for sample with 20 g/100 g of sample inulin and 0.6 g/100 g of sample u-3 fatty acids). Inulin addition showed the influence on pasta colour (maximum values for brightness and minimum for colouration were observed with 0 g/100 g of sample u-3 fatty acids and 20 g/100 g of sample inulin content). The chemometric analysis pointed at the versatile beneficial contributions of u-3 fatty acids in wholemeal spelt pasta enriched with inulin enabling the optimization of pasta dough formula. © 2015 Elsevier Ltd. All rights reserved.

Keywords: u-3 fatty acids Inulin Spelt Pasta Quality Chemical compounds studied in this article: Inulin (PubChem CID: 16219508) Trimethylsulfonium hydroxide (PubChem CID: 11105313) Methanol (PubChem CID: 887) Omega-3 fatty acid (PubChem CID: 56842239) n-hexane (PubChem CID: 8058) Etanol (PubChem CID: 702) 2-(N-Morpholino) Ethanesulfonic acid hydrate (PubChem CID: 78165) Tris(hydroxymethyl)aminomethane (PubChem CID: 6503) Dichlormethan (PubChem CID: 6344)

1. Introduction Triticum aestivum ssp. spelt is also suitable raw material for pasta production, a special type of bread and other products with altered nutritional characteristics compared to conventional wheat products. Some of spelt cultivars have very high protein content and even 30e60% higher concentration of mineral elements Fe, Zn, Cu, Mg and P compared to Triticum aestivum (Abdel-Aal, Hucl, &

* Corresponding author. Engineering Department, Institute of General and Physical Chemistry, 11000 Belgrade, Studentski Trg 12e16, Serbia. Tel.: þ381 11 3283 185. E-mail address: [email protected] (L. Pezo).

ccia et al., 2000; Esposito et al., 2005; Sosulski, 1998; Bonafa  ski et al.; 2008). Filipovi c et al., 2013; Zielin Pasta is excellent choice for incorporating some nutrient components because it is popular with consumers due to its easy handling, storage and preparation (Babuskin, Krishnan, Babu, Sivarajan, & Sukumar, 2014; Kadam & Prabhasankar, 2010; Simonato, Curioni, & Pasin, 2015). Nutritional and functional properties of pasta can be improved by using spelt as basic raw material for the production of pasta and adding some additional raw material such as u-3 fatty acids and inulin for improving functional properties (Filipovi c et al., 2013, 2014; Foschia, Peressini, Sensidoni, & Brennan, 2013; Foschia, Peressini, Sensidoni, Brennan, & Brennan, 2015a, 2015b; Kadam & Prabhasankar, 2010). Dietary fibre ingredients and u-3 fatty acids could be added into

http://dx.doi.org/10.1016/j.lwt.2015.03.082 0023-6438/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Filipovic, J., et al., The effects of u-3 fatty acids and inulin addition to spelt pasta quality, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.082

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Nomenclature: A a* ANOVA b* C Cell DW H h HSD

adhesiveness (g sec), share of red colour (), analysis of variance, share of yellow colour (), the differences in colouration (), cellulose content (g/100 g of sample), dominant wavelength (), hardness (g), difference in tone (), honestly significant difference,

conventional pasta to increase health benefits. The results showed that the dietary fibre function is the prevention of constipation, reduction of bowel transit time and reduction in the risk of colorectal cancer, lowering of blood cholesterol and regulation of blood glucose levels for diabetes management (Brennan & Cleary, 2005; Drusch & Mannino, 2009; Faivre and Bonithon-Kopp, 1999; Filipovi c & Filipovi c 2010; Filipovic, Filipovi c, & Filipovic, 2010; Filipovi c, Popov, & Filipovic, 2008; Kolanowski, 2005). Inulin, as a glucofructan, consists of a mixture of oligomers and polymers containing 2 to 60 b 2-1 linked D e fructose molecules. Inulin is not hydrolysed by the endogenous secretion of human digestive enzymes. Its ability to function as a fat or sugar replacer without adversely impacting food flavour is highlighted (Brennan & Cleary, 2005; Filipovi c, Filipovi c, Filipovi c, Popov, & Psodorov, 2012, 2014; Franck, 2002; Roberfroid, 2005; Tungland, 2000). u-6 and u-3 polyunsaturated fatty acids, also known as essential fatty acids, are not synthesised by the human body and must be provided in the diet (Verardo et al., 2009). A balanced ratio of u 6/u-3 fatty acids is desirable in reducing the risk of many diseases, including cardiovascular disease, cancer, and inflammatory and autoimmune diseases. Cereal grains are high in carbohydrates and u-6 fatty acids, but low in u-3 fatty acids and antioxidants (Simopoulos, 2002). Enrichment of food with u-3 fatty acids is regarded as a way of increasing intake of these fatty acids and improving ratio u3/u-6 in order to reduce levels of risk of ‘lifestyle’ (Kadam & Prabhasankar, 2010; Kolanowski, 2005; McManus, Howieson, & Nicholson, 2009). This paper investigates the effects of simultaneous addition of inulin (10 g/100 g of sample or 20 g/100 g of sample) and u-3 fatty acids content (0.2 g/100 g of sample, 0.4 g/100 g of sample and 0.6 g/100 g of sample) on the technological quality of spelt pasta to obtain new products with altered nutritional and functional properties.

2. Material and methods 2.1. Material For pasta production, spelt wheat “Nirvana” cultivar was grown in the year 2011 in Serbia at Ba cko Gradiste location (45.5333 N,  20.0333 E), with tested following characteristics: moisture content of 13.3 g/100 g of samples, ash, sugar, protein, cellulose, starch, lipid content (g/100 g dry matter samples) of 2.4, 1.33, 12.38, 1.27, 68.39 and 2.38, respectively (AOAC 1990). Stone milled spelt wholemeal flour with particle size ranged between 200 and 300 mm was c”-Ba cko Gradiste. Inulin HPX is a supplied by a local producer “Jefti commercial product from the root of Jerusalem artichoke, produced by “ORAFTI Active Food Ingredients”, Tienen, Belgium, with average

GC u-3 L* Lip. P PCA RSM S St SOP TDF WOS

u-3 fatty acids content (g/100 g of sample). brightness (), lipid content (g/100 g of sample), protein content (g/100 g of sample), principal component analysis, response surface methodology, sugar content (g/100 g of sample), starch content (g/100 g of sample), second order polynomial, total dietary fibre content, work of shear (g sec).

DP  23. u-3 fatty acids are also added during mixing, and were produced by Pharmanova, Obrenovac, Serbia. 2.2. Preparation of pasta Pasta was prepared using the device “La Parmigiana D45” MAC 60. The moisture of wholemeal flour with or without inulin HPX and u-3 fatty acids in a paddle mixer were adjusted to 31.5 g of water per 100 g of flour by adding water. Wholemeal spelt flour, was replaced by inulin HPX in the quantity 10 g/100 g of sample or 20 g/100 g of sample and u-3 fatty acids added in the quantity of 0.2 g/100 g of samples, 0.4 g/100 g of samples or 0.6 g/100 g of samples based on flour and Inulin HPX. Table 1 describes pasta formulation enriched with different quantities of u-3 fatty acids and inulin HPX. Hydrated wholemeal entered the extrusion screw which moved the loose dough forward and simultaneously compress it into a homogeneous plastic mass prior to extrusion through a die with 1.4 mm diameter used for spaghetti. Mixing time was 15 min (Filipovi c et al., 2013). Raw pasta (spaghetti, 20 cm long) was dried in a cabinet drier for about 12 h at controlled temperature that did not exceed 45  C and relative humidity was kept at 70% until pasta reached the moisture around 12.5 mL/100 g, followed by cooling to 25  C, 50 relative humidity for 4 h and then stored at room temperature in sealed containers. Drying conditions and air flow were stringently controlled to avoid creating a discontinuity in the moisture gradient between the interior and exterior of spaghetti. Two batches of 1.5 kg of pasta were processed for each ingredient formula. Wholemeal pasta is tested as control sample. 2.3. Pasta texture Textural properties of cooked pasta were measured with Texture Analyzer TA.HD plus (Stable Micro System, U.K.) equipped with a 5kg load cell. The preparation procedure was the same for all tested samples (duration of cooking: 10 min, the time period between cooking and testing:15 min). Hardness and adhesiveness of cooked pasta were measured using a 36 mm cylinder probe (P/36R) (instrument settings were as follows, mode: measure force in compression, pre-test speed: 2.0 mm s1; test-speed: 1 mm s1; post-test speed: 10 mm s1; strain: 75%; trigger force: 5 g). The maximum force correlates to the hardness of the sample. Adhesiveness is calculated from the negative areas of the plots. Toughness (area under the force/time curve) was measured using the WarnereBratzler shear blade (type HDP/BS) and the following settings: pre-test speed: 2 mm s1; test-speed: 2 mm s1; post-test speed: 10 mm s1; distance: 15.00 mm; trigger force: 10 g. The tests were performed on 10 replicates per batch. The two spaghetti

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Table 1 Pasta formulation with different quantities of u-3 fatty acids and quantities of inulin HPX. Samples

1 2 3 4 5 6 7 8 9 10 11 12

Pasta composition Quantity of u-3 fatty acid g/100 g of sample

Quantity of inulin HPX g replacing 100 g of sample

0 0.2 0.4 0.6 0 0.2 0.4 0.6 0 0.2 0.4 0.6

0 0 0 0 10 10 10 10 20 20 20 20

strands were held close together and positioned centrally under the probe during testing. 2.4. Pasta colour Pasta colour attributes were measured instrumentally using a Chroma meter (CR-400, Konica, Minolta, Tokyo, Japan) tri-stimulus colorimeter. The results were expressed in terms of L*- brightness (from 0 (black) to 100 (white)), a*: greenness/redness (from a* (green) to þa* (red)), b*: blueness/yellowness (from b* (blue) to þ b* (yellow)), C: differences in colouration, h e differences in tone, DW: dominant wavelength as per CIELab system. The measurements were observed under constant lighting conditions, at 28  C, using a white control (L* ¼ 98.76, a* ¼ 0.04, b* ¼ 2.01) (Babuskin et al., 2014). 2.5. Basic chemical analyses Basic chemical analyses (protein, starch, lipid, sugar, ash, cellulose and moisture) of wholemeal spelt flour and pasta were determined according to the official methods of AOAC, 1990. Total dietary fibre was determined according to methods from AOAC Method_1991. 2.6. GCeMS analysis Fatty acids content was determined by using gas chromatography with mass spectrometry (Agilent Technologies, Palo Alto, CA, USA). Each sample was homogenized and further treated in the following manner. A 12 mL cuvette for centrifugation was used for pouring 0.5 g of flour with the precision of 0.01 g. The cuvette was additionally filled with 5 mL of n-hexane and stirred on Vortex for 2 min, after which the mixture was centrifugated at 536.64 g for 5 min. After this 3 mL of clear supernatant was poured into a 10 mL glass and left to steam up at the ambient temperature. From the oily residue an amount of 10 mL was taken, reconstituted to 500 mL of dichlormethan and additionally added 70 mL of transesterification reagent: TMSH (Trimethylsulfonium hydroxide, 0.2 mol/L in methanol, MachereyeNagel). With such a transesterification reaction fatty acids from acilglycerol esterify to methyl-esters. All the testing was conducted on a gas-chromatography system. The GCeMS analyses were performed on an Agilent Technologies 7890 instrument coupled with MSD 5975 equipment (Agilent Technologies, Palo Alto, CA, USA) operating in EI mode at 70 eV. A DP-5 MS column (30 m  0.25 mm  25 mm) was used. The temperature program was: 50e130  C at 30  C/min and 130e30  C at 10  C/min. The injector temperature was 250  C. The flow rate of the carrier

gas (helium) was 0.8 mL/min. A split ratio of 1:50 was used for the injection of 1 ml of the solutions (Vuji c et al., 2012).

2.7. Statistical analyses Simple regression models, in the form of second order polynomial models (SOP), developed using Response Surface Method  ology (RSM) (Brlek et al., 2013; Koprivica, Pezo, Cur ci c, Levi c, &  Suput, 2014; Larrosa et al., 2013) were proposed for calculation of textural properties (hardness e H, adhesiveness e A, work of shear e e WOS), colour attributes (brightness e L*, share of red colour e a*, share of yellow colour e b*, the differences in colouration e C, difference in tone e h, dominant wavelength e DW) and chemical properties (protein content e P, starch content e St, cellulose content e Cell, sugar content e S, lipid content e Lip., GC u-3 e u-3 fatty acids content and TDF e total dietary fibre content), as a function of u-3 fatty acids content and inulin content. Measuring results were collected using 3  4experimental design plan, with 12 runs (1 block) were used, according to Response surface methodology (Misljenovi c et al., 2012; Pezo,  Suput, Levi c, Cvetkovi c, & Kovacevi c, 2014). It was used to design tests for textural properties, colour attributes and chemical properties, considering two factors: u-3 fatty acids content (0 g/100 g of sample, 0.2 g/100 g of sample, 0.4 g/100 g of sample and 0.6 g/100 g of sample), and inulin content (0 g/100 g of sample, 10 g/100 g of sample and 20 g/100 g of sample). The following second order polynomial (SOP) model was fitted to the experimental data. Sixteen models of the following form were developed to relate 16 responses (Y) and two process vari cic et al., 2014; Cvetkovi ables (X) (Cur c et al., 2015):

Yk ¼ bk0 þ

2 X i¼1

bki $Xi þ

2 X

bkii $Xi2 þ bk12 $X1 $X2 ; k ¼ 1  16;

(1)

i¼1

where: bk0, bki, bkii, bk12 are constant regression coefficients; Yk e either specific textural property (H, A, WOS) or colour attributes (L*, a*, b*, C, h, DW) or chemical property (P, St, Cell, S, Lip., GC u-3, TDF); X1 e u-3 fatty acids content; X2 e inulin content. Descriptive statistical analyses for all obtained results were expressed as the mean ± standard deviation (SD). Analysis of variance (ANOVA) was utilized to show relations between applied assays, while the following post-hoc Tukey's HSD test were evaluated for comparison of nutritive data, textural properties and colour attributes for different formulations of pasta. Principal component analysis (PCA), used as the pattern recognition technique, was applied within assay descriptors to characterize and differentiate various analysed samples. The evaluations

Please cite this article in press as: Filipovic, J., et al., The effects of u-3 fatty acids and inulin addition to spelt pasta quality, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.082

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of ANOVA and PCA analyses of the obtained results were performed using StatSoft Statistica 10.0 (StatSoft Inc. 2010; USA)® software. 3. Results and discussion 3.1. Spelt pasta texture The textural quality of cooked pasta is considered as a critical characteristic in evaluating the overall quality of pasta according to Tudorica et al. (2002). The textural characteristics of pasta play an essential role in determining the final acceptance by consumers. Spelt pasta textrure with u-3 fatty acids and inulin HPX are presented in Table 2. The highest value for hardness (2676.36 g) was observed for sample 8, while the lowest hardness value was noticed for sample 9. Statistically significant differences between hardness were noticed and they correspond to a few groups of samples, as shown in Table 2. It seems that the hardness of samples is generally decreased with inulin HPX that is consistent with Tudorica et al. (2002) and Filipovic et al. (2014) and increased with u-3 fatty acids addition. Addition of u-3 fatty acids (0.2, 0.4 and 0.6 g/100 g of samples) results in a higher value of hardness as compared to the pasta without u-3 fatty acids, probably due to strengthening of intra and inter molecular bonds in the dough, also noticed by Kadam and Prabhasankar (2010). Inulin HPX (10 and 20 g/100 g of samples) incorporated into the structure of pasta dough reduces , pasta hardness due to diluting gluten as confirmed by Tudorica Kuri, and Brennan (2002), Aravind, Sissons, Egan, Fellows (2012), Aravind, Sissons, Fellows, Blazek, Gilbert (2012), Mastromatteo, Iannetti, Civica, Sepielli, and Del Nobile (2012) and Wojtowicz and Moscicki (2014). There were also significant differences in work of shear for different pasta formulations as shown in Table 2.Work of shear was generally increased with addition of u-3 fatty acids due to above mentioned beneficial interaction with protein and starch. Work of shear generally decreased with the inulin HPX addition (Table 2), the highest value of 152.10 g sec is observed for sample 4 (0.6 g u-3 fatty acids per 100 g of samples without inulin), while the lowest value of 96.89 g sec is observed in sample 9 (20 g inulin HPX per 100 g of samples without u-3 fatty acids). These data are consistent with values of hardness confirming that u-3 fatty acids stabilize protein-starch matrix while inulin HPX supplements are made weaker protein-starch matrix in pasta dough (Brennan et al., 2004; Brunel et al., 2010; Mastromatteo  et al., 2002; Wojtowicz et al., 2012; Filipovic et al., 2014; Tudorica & Moscicki, 2014). The adhesiveness of pasta generally decreased with increasing of inulin HPX share and u-3 fatty acids addition (Table 2). The maximum adhesiveness (20.80 g sec) was observed in control

sample, while the minimum adhesiveness of 9.54 g sec, was observed in control sample 12 (20 g inulin HPX per 100 g of samples and 0.6 g/100 g u-3 fatty acids). Statistically significant differences between various samples were observed, as shown in Table 2. These data indicate a positive influence of the interaction between protein, starch, fibre and fatty acids on adhesiveness. Pasta colour attributes are very important in creating sensory expectations of consumers, which could affect their perception and acceptance of the product (Carrera et al., 2007; Foshia et al., 2015a; Fu et al., 2013). Statistically different values in various colour coordinates were observed for different pasta formulations, Table 3. Statistically significant differences between all pasta samples were found for L* coordinate (brightness) due to its inulin HPX content which contributed to the increase of the brightness value L* also confirmed by Foshia et al.(2013), Filipovi c et al. (2014), Foshia et al. (2015a, 2015b). The highest L* (79.36) was observed for sample 9, while the lowest L* value (76.33) was noticed for sample 2. While u-3 fatty acids contributes to decrease of pasta brightness L*, which leads to the formation of darker pasta, similar results as was observed by Gajula, Alavi, Adhikari, and Herald (2008), Babuskin et al. (2014). The share of red colour (a*) coordinate is found to have statistically significant difference among all samples. Due to bran colour the highest value of a* was found in control sample, while the lowest measured value was noticed for sample 9 where 20% of wholemeal is replaced with inulin HPX. The yellowness parameter (b*) also showed to be statistically different between all samples (p < 0.05). The addition of u-3 fatty acids in high concentrations influence the increase of yellow (b*), thus positively contributing to favourable yellow colour of pasta. These data are also confirmed by Foshia et al. (2015a). Statistically significant differences in colouration (C) were observed in all samples except for samples 2 and 10. The highest C was measured in sample 8 and the lowest C for sample 9. u-3 fatty acids contribute to increased colouration (C) and that lead to a more intensive colouration of pasta, as it was experienced with yellowness (Table 3). The difference in tone was statistically significant for most of the samples. The maximum value of h was observed in sample 12 and the lowest value of h was found in control sample. Values of dominant wavelength ranged between 578.89 (for control sample) and 577.78 for sample 12, thus indicating that inulin HPX and u-3 fatty acids were not affecting the dominant wavelength. On the whole pasta colour attributes are influenced by u-3 fatty acids and inulin and may contribute to consumer choice. Pasta is a complex multi component system consisting of biomacromolecules such as proteins, carbohydrates and lipids. Pasta is a source of carbohydrates (70e74%) and due to its low nutritional properties, in paricular to low glycemical index (Table 4) has great

Table 2 Texture attributes of pasta with u-3 fatty acid and inulin. Samplesa

Hardness (g)

1 2 3 4 5 6 7 8 9 10 11 12

2285.52 2547.07 2592.26 2573.19 2190.00 2381.85 2425.64 2676.36 2015.59 2067.59 2047.30 2033.48

± ± ± ± ± ± ± ± ± ± ± ±

117.91abcd 311.67bcd 146.11c 93.10cd 165.48abcd 281.08abcd 180.40abcd 181.24c 84.17a 175.24abd 77.51ab 129.36ab

Adhesiveness (g sec) 20.80 18.28 17.97 17.47 13.79 13.14 13.11 12.19 13.14 10.23 9.96 9.54

± ± ± ± ± ± ± ± ± ± ± ±

2.94d 2.25cd 2.64cd 0.76bcd 1.68abc 1.29ab 0.30ab 0.87a 0.98ab 0.37a 0.58a 1.48a

Work of shear (g sec) 119.83 132.99 142.65 152.10 112.54 111.40 115.87 114.42 96.89 100.42 110.65 112.22

± ± ± ± ± ± ± ± ± ± ± ±

3.94abc 11.44bcd 11.60cd 9.97d 5.04ab 8.78ab 8.87ab 9.73ab 9.19a 4.51a 8.72ab 6.76ab

The results are presented as mean ± SD; different letter within the same column indicates significant differences (p < 0.05), according to Tukey's test, number of repetitions: n ¼ 3. a Experimental cases (samples) are detaily explained in Table 1.

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Table 3 Colour attributes of pasta with u-3 fatty acid and inulin (). Samplesa

L*

a*

1 2 3 4 5 6 7 8 9 10 11 12

76.90 76.33 77.77 76.98 78.00 77.06 77.30 77.55 79.36 78.52 78.41 78.16

± ± ± ± ± ± ± ± ± ± ± ±

0.04a 0.08d 0.11g 0.08a 0.06b 0.11a 0.06e 0.08f 0.07h 0.07c 0.04c 0.05b

2.27 2.21 1.79 1.96 2.03 2.06 1.58 1.7 1.54 1.65 1.73 1.61

b* ± ± ± ± ± ± ± ± ± ± ± ±

0.01a 0.02b 0.02e 0.01d 0.02c 0.01c 0.01h 0.02f 0.01i 0.01g 0.02f 0.00h

13.16 13.64 15.45 16.36 13.35 14.42 15.21 16.66 12.74 13.83 15.16 16.44

C ± ± ± ± ± ± ± ± ± ± ± ±

0.01k 0.01i 0.00d 0.01c 0.00j 0.00g 0.01e 0.01a 0.01l 0.00h 0.02f 0.01b

13.35 13.81 15.55 16.48 13.50 14.57 15.29 16.75 12.83 13.83 15.26 16.52

h ± ± ± ± ± ± ± ± ± ± ± ±

0.01j 0.01h 0.01d 0.01c 0.01i 0.01g 0.01e 0.01a 0.01k 0.00h 0.02f 0.01b

DW

80.19 80.80 83.38 83.16 81.32 81.87 84.09 84.16 83.12 83.13 83.49 84.41

± ± ± ± ± ± ± ± ± ± ± ±

0.02h 0.07g 0.07c 0.05d 0.06f 0.03e 0.06b 0.05b 0.06d 0.04d 0.06c 0.01a

578.89 578.74 578.05 578.13 578.59 578.46 577.85 577.85 578.09 578.1 578.02 577.78

± ± ± ± ± ± ± ± ± ± ± ±

0.00a 0.02b 0.02fg 0.02e 0.02c 0.01d 0.02h 0.02h 0.01ef 0.01e 0.02g 0.01i

The results are presented as mean ± SD; different letter within the same row indicate significant differences (p < 0.05), according to Tukey's test, number of repetitions: n ¼ 3. L* e brightness, a* e share of red colour, b* e share of yellow colour, C e the differences in colouration, h e difference in tone, DW e dominant wavelength. a Experimental cases (samples) are detaily explained in Table 1.

possibilities for the improvement of functional characteristics (Bruneel, Pareyt, Brijs, & Delcour, 2010). The addition of u-3 fatty acids and Inulin HPX to pasta improve essential fatty acids pattern and dietary fibre intake. Statistically significant differences between groups of samples were observed (Table 4). Investigated pastas were characterized by high crude protein content in the sample without inulin HPX (control sample, sample 2, sample 3 and sample 4), while the minimum (9.84 ± 0.003% d.m) was observed in control sample 12. Starch content reached maximum value (71.99 g per 100 g of dry matter of sample) in sample 4 and as expected the minimum of starch 46.08 g per 100 g of dry matter of the sample was observed in sample 9. In general, the addition of inulin HPX in high concentrations influenced the decrease of protein and starch content, because part of the flour was replaced by non protein constituent (Table 4). According to Villeneuve et al. (2013), u-3 fatty acids probably influenced protein and starch content of binding lipids through starch-lipid or protein-lipid complexing. It was also confirmed by statistically significant differences in lipid content observed for almost all pasta samples, Table 4. The maximum of lipid content (3.13 g/100 g of sample) was obtained for control sample 12 (maximum u-3 fatty acids and 20 g/100 g of sample inulin HPX), while the minimum of 2.05 g/100 g of sample was observed in sample 9. Similar content was noticed by Kindt, Lercker, Mazzaracchio, and Barbiroli (2006). As expected, addition of u-3 fatty acids affects the increase in lipid content in pasta particularly positively contributes to a better ratio of u-6/u-3 fatty acids regardless added quantity (Simopoulos, 2002). Total lipid content in pasta depends on the content of inulin HPX as a non lipid

constituent. As the share of inulin HPX is increasing lipids in pasta are decreasing. Statistically significant differences in cellulose content were observed for almost all samples, Table 4. Cellulose content was higher for pastas with 10 g/100 g of sample and 20 g/ 100 g of sample inulin HPX than for pasta with no inulin HPX content, probably due to detected the cellulose molecule in the chemical structure of inulin HPX. Statistically significant differences in sugar content were not observed between following samples: 2, 3, 4, 5, 6 and 11; 8, 10 and 12, while other samples were statistically different. Statistically significant differences in total dietary fibre content were observed between almost all samples. The addition of inulin HPX, as a dietary fibre, in high concentrations influences the increase of total dietary fibre in pasta. Total dietary fibre content achieved maximum value (16.02 g/100 g of sample) with 0.4 g/ 100 g of sample u-3 fatty acids and 20 g/100 g of sample inulin HPX content. By consuming 150 g of this pasta the intake of fibre is thus 24 g reaching daily fibre intake recommended by nutritionists (World Health Organization, 2003). Gas chromatography with mass spectrometry was used for performing a qualitative analysis of u-3 fatty acids. Fig. 1 shows chromatogram which clearly indicates differences in u-3 fatty acids content in the pasta sample with 0.4 and 0.6 g/100 g of sample u-3 fatty acids. In pasta without u-3 fatty acids (control sample), (sample 5) and (sample 9) are detected 1.62 mg/g, 1.53 mg/g 1.47 mg/g of fatty acids respectively. These are fatty acids from wholemeal spelt. The addition of u-3 fatty acids (0.2 g/100 g of sample, 0.4 g/100 g of sample and 0.6 g/100 g of sample) contributed to a significant increase of fatty acids in the pasta compared to pasta without u-3 fatty acids. It is interesting

Table 4 Nutritive composition of pasta with u-3 fatty acid (g/100 g of sample) and inulin (g/100 g). Samplesa

P

1 2 3 4 5 6 7 8 9 10 11 12

12.38 12.12 12.34 12.80 11.27 11.26 10.87 10.96 10.14 10.07 10.06 9.84

Lip. ± ± ± ± ± ± ± ± ± ± ± ±

0.04a 0.04c 0.05b 0.03a 0.03d 0.02d 0.02f 0.02e 0.02g 0.02g 0.02g 0.03h

2.38 2.84 2.86 2.93 2.31 2.75 2.99 3.12 2.05 2.24 2.91 3.13

St ± ± ± ± ± ± ± ± ± ± ± ±

0.01f 0.02d 0.01cd 0.02i 0.03g 0.02e 0.01b 0.02a 0.01i 0.02h 0.01c 0.03a

68.39 71.40 67.95 71.99 57.88 57.02 57.01 56.44 46.07 48.09 48.37 50.1

Cell ± ± ± ± ± ± ± ± ± ± ± ±

0.02c 0.04b 0.04d 0.03a 0.04e 0.02f 0.02f 0.03g 0.03k 0.04i 0.04j 0.02h

1.27 1.64 1.43 1.53 1.84 1.88 1.64 1.96 1.77 1.77 1.80 1.65

GC u-3

S ± ± ± ± ± ± ± ± ± ± ± ±

0.01c 0.01a 0.00d 0.01e 0.02f 0.01g 0.00a 0.01h 0.01b 0.01b 0.00b 0.01a

1.33 0.77 0.80 1.12 0.78 0.75 0.6 1.17 0.75 1.17 0.88 1.04

± ± ± ± ± ± ± ± ± ± ± ±

0.12a 0.04c 0.07c 0.00c 0.03c 0.04c 0.04d 0.00b 0.04cd 0.00b 0.06c 0.02b

1.62 2.86 5.57 7.25 1.53 1.83 3.07 4.37 1.98 1.47 3.82 2.04

± ± ± ± ± ± ± ± ± ± ± ±

17.37a 64.30d 62.93h 63.18i 38.23a 11.64b 40.58e 190.57g 31.43bc 62.33a 149.66f 134.40c

TDF 5.62 5.89 6.37 5.98 10.80 9.23 9.6 9.64 16.01 15.32 16.02 14.92

± ± ± ± ± ± ± ± ± ± ± ±

0.02j 0.01i 0.01g 0.02h 0.03d 0.02f 0.03e 0.02e 0.01a 0.01b 0.02a 0.01c

The results are presented as mean ± SD; different letter within the same row indicate significant differences (p < 0.05), according to Tukey's test, number of repetitions: n ¼ 3. P e protein content, Lip e lipids content, St e starch content, Cell e cellulose content, S e sugar content, GC u-3 e u-3 e fatty acids content and TDF e total dietary fibre content. a Experimental cases (samples) are detaily explained in Table 1.

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Fig. 1. Chromatograms of the liposoluble extract of pasta containing u-3 fatty acids at 0.4 g/100 g sample (sample 7) and 0.6 g/100 g sample (sample 8).

that pasta without Inulin HPX (control sample, sample 2, sample 3, sample 4) have higher fatty acids content than pasta with 10 g/ 100 g of sample inulin HPX (sample 5, sample 6, sample 7, sample 8) and 20 g/100 g of sample of inulin HPX (sample 9, sample10, sample 11, sample 12) and added fatty acids. The International Society for the Study of Fatty acids and Lipid's (ISSFAL) recommend dietary intake of 0.65 g/day of EPA and DHA. By consuming 100 g of wholemeal spelt pasta with 0.6 g/100 g of sample u-3 fatty acids without inulin HPX fatty acids level in pasta is higher (7.25 mg/g) than recommended by ISSFAL. Data presented in Table 4, also point that u-3 fatty acids did not decompose during pasta preparation. For example, consumption of 150 g pasta control sample1 (0.4 g/ 100 g of sample u-3 fatty acids and 20 g/100 g of sample inulin HPX) could be a good opportunity to achieve the recommended daily intake both in u-3 fatty acids and dietary fibre and improve functional properties of the diet. Pasta with incorporated u-3 fatty acids and inulin HPX in different levels significantly contributes to the improved multi functionality of food. It is worth stressing that this kind of pasta is easily prepared with favourable texture and colour.

inulin HPX showed higher dominant wavelength, share of red colour, higher adhesiveness, hardness and work of shear, increased protein and starch content, while samples located at the right side of graphic with inulin HPX content of 20 g/100 g of sample showed increased total dietary fibre content and higher brightness value. Quality results show that the first two principal components, account for 74.06% of the total variance and can be considered sufficient for data representation. The biplot diagram of spelt pasta composition, regarding u-3 fatty acids and inulin HPX content is presented on Fig. 2. The first two principal components were marked as Factor 1 and Factor 2, while the percentage of describing variance was written in the brackets. Concerning textural properties, colour attributes and chemical characteristics, adhesiveness (with 11.8% contribution based on correlation), share of red colour (10.6%), protein content (12.6%), starch content (12.3%) and total dietary fibre content (11.7%) mostly contributed to the first factor calculation, while share of yellow (17.9%), colouration (17.9%), tone (10.1%), cellulose content (11.2%) and GC measured u-3 fatty acids content (14.9%) contributed more to the second factor coordinate calculation.

3.2. PCA and standard score analysis The PCA allows a considerable reduction in a number of variables and the detection of structure in the relationship between measuring parameters and different samples of spelt pasta formulations that give complementary information (Fongaro & Kvaal, 2013; Kaiser & Rice, 1974; Otto, 1999). The full auto scaled data matrix consisting of spelt pasta with 0.2, 0.4 and 0.6 g/100 g of sample u-3 fatty acids and 10 and 20 g/100 g of sample inulin HPX content formulations were submitted to PCA. For visualizing the data trends and for the discriminating efficiency of the used descriptors a scatter plot of samples using the first two principal components (PCs) from PCA of the data matrix is obtained (Fig. 2). As can be seen, there is a neat separation of the twelve samples of spelt pasta formulations, according to textural properties, colour attributes and chemical characteristics. Samples are grouped according to u-3 fatty acids content (groups are coloured in grey tone) and inulin HPX content (tin line border). Samples located on the bottom of the graphic and the lower right corner are those with maximum added u-3 fatty acids content, with increased GC measured u-3 fatty acids and lipids content, cellulose content, increased brightness, share of yellow, colouration and tone. Samples located on the upper side of graphic (with no u-3 fatty acids) showed increased dominant wavelength and share of red. Samples located at the left side of graphic, having no

Fig. 2. Biplot diagram of spelt pasta composition, regarding u-3 fatty acids and inulin content. A e adhesiveness, a* e share of red colour, b*- share of yellow colour, C e the differences in colouration, Cell e cellulose content, DW e dominant wavelength, H e hardness, h e difference in tone, L* e brightness, Lip e lipid content, P e protein content, S e sugar content, St e starch content, TDF e total dietary fibre content, WOS e work of shear and u-3 e GC u-3 fatty acids content.

Please cite this article in press as: Filipovic, J., et al., The effects of u-3 fatty acids and inulin addition to spelt pasta quality, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.082

Inulin Inulin2 u-3  Inulin Error r2

* Significant at p < 0.05 level, ** Significant at p < 0.10 level, 95% confidence limit. dF e degrees of freedom. H e hardness, A e adhesiveness, WOS e work of shear, L* e brightness, a* e share of red colour, b*- share of yellow colour, C e the differences in colouration, h e difference in tone, DW e dominant wavelength, P e protein content, St e starch content, Cell e cellulose content, S e sugar content, Lip e lipids content, u-3 e GC u-3 fatty acids content and TDF e total dietary fibre content.

0.28 0.03 184.32* 2.36* 0.42 1.82 0.990 1.4  107* 2.7  105 8.0  106* 1.0  106 7.3  106* 6.5  106 0.823

GC u-3 Lip.

0.52* 0.20** 0.01 0.15 0.61* 0.34 0.814 0.02 0.12 0.00 0.07 0.04 0.32 0.433

S Cell

0.23* 0.00 0.02 0.00 0.45* 0.15 0.819 3.96 0.09 948.41* 10.25** 0.63 17.31 0.982

St P

0.01 0.04 11.38* 0.05 0.14** 0.23 0.981 0.78* 0.00 0.42* 0.00 0.10 0.20 0.867

DW h

11.65* 0.01 5.47* 0.06 1.32 2.56 0.878 19.35* 0.10 0.07 0.28** 0.05 0.36 0.982

C b

20.01* 0.10 0.04 0.28 0.03 0.37 0.982 68.72* 7.31 2036.28* 145.38* 630.99* 43.96 0.985

a L

0.39 0.50 5.22** 0.29 0.72 5.96 0.544

WOS

1.00 0.13 138.61* 5.31* 6.75* 2.16 0.986

A

104 103 105* 105* 104 104 H

2.58  1.05  4.20  1.64  1.91  7.49  0.894 1 1 1 1 1 6

u-3 u-32

Chemical composition

* * * * *

Colour attributes Texture attributes dF Term

Table 5 ANOVA calculation for nutritive composition of pasta from different formulations.

0.11 0.00 0.36* 0.00 0.07 0.17 0.762

TDF

J. Filipovic et al. / LWT - Food Science and Technology xxx (2015) 1e9

7

3.3. ANOVA and RSM calculation The ANOVA calculation for nutritive composition of analysing pastas (Table 5) revealed the significant effects of independent variables, u-3 fatty acids and inulin HPX content. The SOP models for all variables were found to be statistically significant and the response surfaces were fitted to these models. Linear and quadratic terms for inulin HPX content were the most important for the calculation of texture attributes. These terms were statistically significant at p < 0.05 level, 95% of significant limit. Interchange term of u-3  inulin HPX content was found significant for adhesiveness and work of shear calculation, wherein the linear term of u-3 content was significant for work of shear calculation. The prediction of texture attributes, concerning u-3 fatty acids and inulin HPX content as independent variables could be achieved with good accuracy (0.894, 0.986 and 0.985, for H, A and WOS, respectively). Brightness, the share of red colour, the difference in tone and dominant wavelength are mostly influenced by inulin HPX content (linear term of inulin HPX is the most important variable for prediction of these responses). The share of red colour and the difference in tone are also influenced by u-3 fatty acids content (linear term is statistically significant for SOP models calculation, p < 0.05). The linear term of u-3 fatty acids content is the most important for colouration calculation (p < 0.05), while the quadratic term was also statistically significant at p < 0.10 level. The predictive capabilities of colour attributes are not good enough, (0.544, 0.762, 0.982, 0.982, 0.878 and 0.867, for L*, a*, b*, C, h and DW, respectively). The linear term of inulin HPX content in the SOP model was the most influential variable for protein content, starch and total dietary fibre content calculation, statistically significant at p < 0.05 level. Interchange term was important for the protein content calculation and quadratic term of inulin HPX content was influential for starch calculation (both statistically significant at p < 0.10 level). The quadratic term of inulin HPX content was influential in TDF calculation, statistically significant at p < 0.05 level. The calculation of cellulose and the lipid content were mostly influenced by interchange term, and also by the linear term of u-3 fatty acids content. 4. Conclusion Based on data resulting from the investigations of quantity of u3 fatty acids and inulin HPX influenced in spelt pasta quality it can be concluded:  u-3 fatty acids positively affect the pasta texture: increase hardness (maximum value obtained was 2676.36 g with 0.6 g/ 100 g of sample u-3 fatty acids and 10 g/100 g of sample inulin HPX), reduce adhesiveness (minimum value obtained was 9.54 g sec, with 20 g/100 g of sample inulin HPX and 0.6 g/100 g of sample u-3 fatty acids) and increase work of shear (maximum value obtained was 152.10 g sec with 0.6 g/100 g of sample u-3 fatty acids and 0 g/100 g of sample inulin HPX).  Inulin HPX showed the influence on pasta colour, maximum value of brightness 79.36 with 0 g/100 g of sample u-3 fatty acids and 20 g/100 g of sample inulin HPX content. u-3 fatty acids influence the yellow colour, with a maximum value of 16.66 with 0.6 g/100 g of sample u-3 fatty acids and 10 g/100 g of sample inulin HPX content.  Nutritive composition of pasta is dependent on u-3 fatty acids and inulin content, with maximum protein and lipid content measured with 0.6 g/100 g of sample u-fatty acids and no inulin. Starch content is reduced by the addition of inulin, while maximum dietary fibre content (16.02 g/100 g of sample) is

Please cite this article in press as: Filipovic, J., et al., The effects of u-3 fatty acids and inulin addition to spelt pasta quality, LWT - Food Science and Technology (2015), http://dx.doi.org/10.1016/j.lwt.2015.03.082

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observed with 20 g/100 g of sample inulin HPX content and 0.4 g/100 g of sample u-3 fatty acids. Daily average consumption of 100 g of pasta with only 0.6 g/ 100 g of sample u-3 fatty acids contributes to essential fatty acids intake of 0.72 g per day, that is satisfying the daily needs of u-3 fatty acids intake recommended by ISSFAL. Pasta with 0.4 g/100 g of sample u-3 fatty acids and 20 g/100 g of sample inulin HPX has the best functional properties and acceptable quality. Consumption of 150 g pasta with 0.4 g/100 g of sample u fatty acids and 20 g/100 g of sample inulin HPX could be a good opportunity to achieve the recommended daily intake in u-3 fatty acids and dietary fibre and improve functional properties of meals. PCA, as the multivariate method of analysis enabled better visualization to distinguish the samples. The RSM was used to optimize the pasta formulation, utilizing texture and colour attributes, as well as the nutritive composition of pasta in the form of responses, regarding u-3 fatty acids and the inulin content. Mathematical models for all responses were statistically significant. The chemometric analyses pointed to the versatile beneficial contributions of u-3 fatty acids in wholemeal spelt pasta enriched with inulin which enables the optimization of pasta dough formula. It contributes to the positive balance of essential matters in the human organism with modified nutritive properties as the functional food.

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