2018 Volume 24 Issue 6 Pages 1093-1100
The aim of this study was to improve the nutritional properties of pasta by adding red grape marc (RGM), which is rich in polyphenols and low in digestible carbohydrates, to increase phenolic antioxidants and decrease the glycemic load/energy intake. Our data showed that the spaghetti enriched with RGM (particle size ≤ 125 µm) and transglutaminase (RGM/TG) was sensory acceptable, had a higher amount of phenolic compounds and antioxidant activity than control spaghetti. Cooking did not affect these nutritional parameters. The bioaccessible fraction from RGM/TG-spaghetti showed a higher amount of polyphenols including anthocyanins, as well as antioxidant activity but a lower amount of glucose in an in vitro digestion model. Thus, enrichment of spaghetti with RGM can increase the beneficial nutritional properties by increasing bioaccessibility of compounds with antioxidant activity and reducing the glycemic load.
Natural plant bioactive compounds are of growing interest for food applications due to their potential effects on human health (Schieber et al., 2001). It is widely known that fruit and vegetables are important factors of healthy and balanced diet. They have beneficial effects on human health, being rich in substances including vitamins, minerals, fiber, and phytochemicals, such as polyphenols (phenolic acids, flavonoids, etc.). The latter are powerful antioxidants that can protect the body by means of various mechanisms against a number of degenerative diseases, such as cardiovascular diseases, neurodegenerative disorders, some types of cancer, and diabetes (Dziki et al., 2014; Gawlik-Dziki et al., 2015). Although public institutions advise consumers to increase fruit and vegetable intake, consumers fail to eat the recommended daily amount (400 g per day) by the World Health Organization. Therefore, the incorporation of fruit and vegetable compounds into foods eaten daily is a way to increase the consumption of beneficial substances and to reduce the amount of quickly digestible carbohydrates (glycemic index/load), and subsequently to reduce the risk of many diseases.
In this context, the by-products of winemaking, in particular red grape marc (RGM) is taken into account as an economic and easily available raw material rich in polyphenols (phenolic acids, flavonoids like anthocyanins), fiber and low in digestible carbohydrates. Every year the wine industry generates millions of tons of solid waste (Gonzalez-Centeno et al., 2012). This so-called waste can be used as a high-quality ingredient in food production, reducing environmental impact and disposal costs. In the food industry, these by-products can be used as high-value ingredients to replace synthetic additives, which are rejected by consumers, who favor foods with natural ingredients that are able to reduce the risk of various degenerative diseases (Schieber et al., 2001).
Different foods in literature have been enriched with bioactive compounds to increase the phenolic content and the antioxidant activity in these products, employing various food matrices (cereal, pseudo-cereals, spices, legumes) and vegetable by-products. However, these substances were often added in low concentrations to avoid altering the sensory properties. Özvural and Vural (2011) incorporated grape seed flour (GSF) into frankfurters (meat sausages) at various concentrations up to 5% and investigated the effects on physical, nutritional, and sensory characteristics of the product. They observed that the incorporation of GSF in frankfurter formulations improved the nutritional quality but also significantly affected all sensory parameters. Similar results were obtained by García et al., (2009), who added different amounts of dry tomato peel (0–6%) to raw and cooked hamburgers and noted that taste and overall quality were significantly modified. In fact, the challenge for researchers is to find a compromise between the nutritional and sensory aspect.
Furthermore, more importance is given to the nutritional quality characterization underestimating the bioaccessibility of added phytochemicals in food products. Sant'Anna et al., (2014) studied the nutritional, sensory and cooking properties of fettucine enriched with grape marc powder at different concentrations, without investigating the bioaccessibility of bioactive compounds. Other authors (Karaaslan et al., 2011; Peng et al., 2010; Tseng and Zhao, 2013) studied the nutritional aspect of an enriched common product (yogurt, bread, pasta) and found that adding plant matter to these food products led to an increase in the total polyphenol amount and antioxidant activity. However, there were no data on the bioavailability or bioaccessibility of polyphenols.
The aim of this work was to improve the nutritional properties of pasta products by adding red grape marc, a by-product in winemaking containing fiber and phenolic antioxidants. The sensory quality and the nutritional characteristics (total phenolic compounds, total anthocyanins, and antioxidant activity before and after cooking) of the enriched spaghetti were evaluated and compared to control sample, made from commercial durum wheat semolina. Furthermore, bioaccessibility of polyphenols, glucose, as well as the antioxidant activity of nutrients in the bioaccessible fraction were assessed.
2.1 Raw materials Red grape marc (RGM), made up of skins, seeds and stalks, was provided by a local company of Foggia (Southern Italy, vintage 2016). The sample was dried at 30–35°C in a dryer (SG600, Namad, Rome, Italy) for 48 h, a fine powder (≤ 500 µm) (RGM-500) was produced by a hammer mill (16/BV-Beccaria s.r.l., Cuneo, Italy) and then stored at 4°C until further utilization. Then, the flour was sieved by Sieve Shakers (Mod AS 300 Retsch) to obtain medium sized particles (≤ 125 µm) (RGM-125). The commercial semolina was purchased from Agostini mill (Montefiore dell'Aso, Ascoli Piceno, Italy).
2.2 Chemicals Folin-Ciocalteu reagent, anhydrous sodium carbonate, hydrochloric acid, and formic acid were obtained from Merck (Darmstadt, Germany); methanol and acetonitrile were purchased from VWR international (Darmstadt, Germany); ferric chloride hexahydrate was obtained from neoLab Migge GmbH (Heidelberg, Germany). Ferrous sulfate heptahydrate, the ingredients for HBSS (potassium chloride, sodium chloride, disodium hydrogen phosphate dihydrate, dipotassium hydrogen phosphate, sodium hydrogen carbonate, and calcium chloride) were obtained from Carl Roth (Karlsruhe, Germany). Gallic acid monohydrate, sodium acetate trihydrate, glacial acetic acid, 2,4,6-tripyridyl-s-triazine (TPTZ), polyphenol standards, enzymes for in vitro digestion (porcine pepsin, porcine bile acid, pancreatin, alpha-amylase from Bacillus sp.), and glucose assay kit were supplied by Sigma Aldrich (Taufkirchen, Germany). Amyloglucosidase was purchased from Megazyme (Gernsheim, Germany).
2.3 Spaghetti preparation Durum wheat semolina was mixed with water (30% w/w) in the rotary shaft mixer (Namad, Rome, Italy) at 25°C for 20 min to uniformly distribute water. Spaghetti based only on durum wheat semolina were manufactured and used as the reference sample (CTRL). To produce enriched spaghetti, the red grape marc flour was added to durum wheat semolina at a concentration of 15% (w/w) and with a different particle size (500µm; 125µm). Three red grape marc flour preparations were used: (i) RGM-500, (ii) RGM-125 and (iii) RGM-125 and 0.6% (w/w) transglutaminase (TG) to produce three spaghetti formulations: RGM-500, RGM-125, RGM/TG, respectively. TG powder was previously dissolved in water in order to ensure its solubility. In all steps, the dough was extruded with a 60VR extruder (Namad). Subsequently, the extruded pasta was dried in a dryer (SG600; Namad). The drying process conditions applied were in accordance with Padalino et al., (2013).
2.4 Sensory analysis Dried spaghetti samples were cooked in distilled water to optimal cooking time (AACC approved method 66–50) and then they were served to the panelists. The sensory test panel consisted of ten panelists, four men and six women, aged between 28 and 45 years, who were trained in developing a sensory vocabulary and in identifying particular attributes to evaluate durum wheat commercial spaghetti. For the sensory evaluation they were asked to indicate color, homogeneity, and resistance to breaking of uncooked spaghetti as well as elasticity, firmness, bulkiness, adhesiveness, color, homogeneity, odor and taste of cooked samples. For the evaluation a nine-point scale, in which 1 corresponded to “extremely unpleasant”, 9 to extremely pleasant”, and 5 to “threshold of acceptability” was used to quantify each attribute. On the basis of the above mentioned attributes, panelists were also asked to score the sensory overall quality of both cooked and uncooked samples, using the same nine-point scale (Padalino et al., 2013).
2.5 In vitro digestion of spaghetti In vitro digestion was performed according to protocol described by Gille et al., (2015) with some modifications. Briefly, 0.3 g of each cooked spaghetti sample was subjected to an in vitro digestion process that consisted of two phases: gastric and intestinal. To simulate the gastric phase, porcine pepsin (40 mg/mL of 0.1 N HCl) was added and the pH adjusted to 2.2–2.4, followed by shaking at 37°C in a water bath for 1 h. Subsequently, porcine bile acid (12 mg/mL), pancreatin (11 mg/mL), and alpha-amylase (3.3 mg/mL) were added. The pH was adjusted to 7.2–7.6, followed by a treatment with nitrogen gas and shaking at 37°C in a water bath for 2 h. After digestion, the samples were centrifuged (4000 rpm × 10 min) and filtered using 0.20 µm filters. The filtrate was used for the analysis of the bioaccessible fraction.
2.6 Extraction of bioactive compounds The uncooked and cooked spaghetti were submitted to extraction with acidified methanol (80% MeOH in H2O acidified with 1% HCl) as described by Biney and Beta (2014). Before extraction, the cooked spaghetti was dried by a freeze dryer.
2.7 Chemical Analysis
2.7.1 Determination of bioactive compounds and antioxidant activity The evaluations of total polyphenols, total anthocyanins, and antioxidant activity were performed both on the methanol extracts and on digested samples. Total polyphenol content (TPC) was expressed as mg of gallic acid equivalents (GAEs)/g dry weight (dw), and were determined by UV–VIS spectrophotometry according to the Folin-Ciocalteu method (Spinelli et al., 2015). Total anthocyanin content (TAC) was evaluated according to the spectrophotometric method described by Lee et al., (2005). It was expressed as mg malvidin 3-O-glucoside (mvd-glu) per gram of dw. The antioxidant activity (µmol Fe(II)/g dry weight) was assessed by FRAP (ferric reducing antioxidant power) assay, which was carried out according to the original study of Benzie and Strain (1996).
2.7.2 HPLC After in vitro digestion, the CTRL and RGM/ TG samples were centrifuged, filtered, and if necessary concentrated using the solid phase extraction (SPE). The samples were run on an ELITE Lachrom HPLC system equipped with a diode array detector (DAD; Hitachi L-2455; Germany). The chromatographic separation was executed with a Phenomenex Luna 3 µ, C18 (2), 150 × 4.6 mm column. The temperature of the column oven was set at 40°C. The injection volume was 60 µL and the flow rate was 0.75 mL/min. The mobile phases used consisted of 100% acetonitrile (solution A) and water containing 0.5% (v/v) formic acid (solution B). A gradient elution was employed as follows: at 0 min A = 7%; at 5 min A = 12%; at 12 min A= 17%; at 20 min A = 27%; at 29 min A = 100% for 5 min. At the end of the gradient, the column was equilibrated to the initial condition (A = 7%). Detection was carried out at three different wavelengths: 280, 360 and 520 nm. Quantitative determinations were based on the peak area of different polyphenol standards present in red grape marc: gallic acid and p-coumaric acid, quercetin, pelargonidin, malvidin 3-O-glucoside (mvd-glu), and delphinidin. Furthermore, the mvd-glu identification was confirmed by spiking, namely by adding the standard to the sample.
Unidentified polyphenols (X1, X2, X3, X4 –anthocyanins) were detected at 520 nm and recognized as anthocyanins from their UV-Vis spectrum; similar to that of malvidin 3-O-glucoside and characteristic for anthocyanins. For their quantification, the calibration curve of mvd-glu was used.
2.8 Determination of bioaccessible glucose The glucose amount available for absorption in the small intestine was evaluated using the glucose assay kit (Sigma Aldrich, Taufkirchen, Germany) after gastric phase (t = 0 min) and at the end of intestinal digestion (t = 2 h). Each time 0.5 mL of CTRL and RGM samples were mixed with 1 mL ethanol, centrifuged and filtered. Then 100 µL supernatant was added to 890 µL of HBSS and 10 µL of amyloglucosidase (3260 U/mL). The solution was incubated for 1 h at 37°C before glucose determination.
2.9 Statistical analysis All experimental data were subjected to one-way analysis of variance (ANOVA) using Tukey-Kramer's test to determinate significance differences between samples (P < 0.05). SIGMA PLOT 12.3 for Windows was used.
3.1 Sensory quality Our aim was to maximally increase the concentration of red grape marc in the spaghetti and to keep the sensory quality at least at an acceptable level at the same time. Our preliminary studies indicated that the increasing concentration of red grape marc flour affected the overall quality of the spaghetti (data not shown). Here we investigated whether a smaller particle size of red grape marc and addition of TG can improve the sensory quality and nutritional characteristics of spaghetti produced using 15% (w/w) of red grape marc. Specifically, RGM flour with particle size (≤ 125 µm) (RGM-125) without or with transglutaminase at 0.6% (w/w) level (RGM/TG) were used to try to improve the quality of pasta compared to the RGM flour with particle size ≤ 500 µm (RGM-500).
Data reported in Table 1 show that the uncooked spaghetti sample with RGM-500 flour had the lowest overall quality value due to the poor resistance to breakage and the unpleasant dark purple color. In contrast, the addition of RGM-125 flour caused a noticeable improvement of the pasta color as compared to RGM-500. Furthermore, addition of TG to RGM-125 slightly increased the overall quality score of the uncooked sample due to the increase in the break resistance value.
| a) | ||||
|---|---|---|---|---|
| Uncooked Spaghetti | ||||
| Color | Homogeneity | Break Resistance | Overall Quality | |
| CTRL | 7.30±0.21a | 7.25±0.25a | 7.20±0.21a | 7.50±0.23a |
| RGM-500 | 5.75±0.19c | 5.00±0.29c | 5.28±0.29c | 5.25 ±0.28c |
| RGM-125 | 6.28±0.25b | 6.25±0.27b | 5.50±0.29bc | 6.02±0.28b |
| RGM/TG | 6.30±0.25b | 6.30±0.27b | 5.80±0.25b | 6.20±0.25b |
| b) | ||||||||
|---|---|---|---|---|---|---|---|---|
| Cooked Spaghetti | ||||||||
| Elasticity | Firmness | Bulkiness | Adhesiveness | Color | Odor | Taste | Overall Quality | |
| CTRL | 7.50±0.21a | 7.20±0.24a | 7.22±0.20a | 7.40±0.24a | 7.52±0.29a | 7.80±0.28a | 7.70±0.27a | 7.30±0.30a |
| RGM-500 | 5.00±0.27c | 5.00±0.20c | 4.00±0.25d | 4.00±0.30d | 4.50±0.27c | 6.20±0.27b | 5.25±0.29c | 4.50±0.27d |
| RGM-125 | 5.25±0.27c | 5.25±0.28c | 5.00±0.25c | 4.96±0.30c | 6.00±0.27b | 6.30±0.27b | 6.45±0.29b | 5.25±0.27c |
| RGM/TG | 5.75±0.25b | 6.00±0.29b | 6.00±0.25b | 5.96±0.30b | 6.20±0.27b | 6.25±0.27ba | 6.50±0.25b | 6.00±0.27b |
The spaghetti samples were evaluated by 10 trained panelists (for details see the ‘ematerials and methods’ section). CTRL: control spaghetti; RGM-500: spaghetti made from red grape marc flour (15% w/w, particle size ≤ 500 µm); RGM-125: spaghetti made from 125 µm red grape marc flour (15% w/w, particle size ≤ 125 µm); RGM/TG: spaghetti made from 125 µm red grape marc flour (15% w/w) and 0.6% (w/w) transglutaminase. Different letters in each column indicate statistically significant differences (P ≤ 0.001), as determined by ANOVA followed by the Tukey-Kramer test.
Regarding the cooked pasta, the RGM-500 sample again showed the lowest overall quality value as compared to the other investigated samples. In particular, the incorporation of grape marc flour negatively influenced parameters, such as elasticity, adhesiveness and bulkiness. Table 1 shows that the RGM/TG sample recorded an acceptable value (6.00) of overall quality. Specifically, this sample exhibited significantly higher sensory scores for elasticity and firmness as well as a significant decline of both adhesiveness and bulkiness, when compared to the other fortified samples (RGM-500 and RGM-125). No significant differences in color, odor and taste were observed between the RGM-125 and RGM/TG sample. Based on its sensory quality, the RGM/TG spaghetti sample was selected for the investigations reported below.
3.2 Polyphenol concentration and antioxidant activity: effect of cooking Here we investigated the nutritional quality (total polyphenol and anthocyanin concentration, antioxidant activity) of spaghetti enriched with 125 µm red grape marc powder at 15% (w/w) and containing 0.6% TG in comparison to the control spaghetti made from commercial wheat durum semolina.
Table 2 shows the total polyphenol content of uncooked and cooked spaghetti samples. The fortification of spaghetti with RGM/TG flour resulted in a statistically significant higher polyphenol concentration. The uncooked pasta contained 0.54 ± 0.08 and 2.27 ± 0.20 mg GAEs/g dw in CTRL and RGM/TG spaghetti, respectively; on the contrary, the cooked pasta had 0.28±0.02 mg GAEs/g dw in the reference sample and 2.11 ± 0.15 mg GAEs/g dw in the enriched sample. There was no statistically significant difference between raw and cooked pasta for both CTRL and RGM/TG pasta (Table 2), thus indicating no significant effect of cooking on the content of bioactive compounds.
| Uncooked spaghetti | Cooked spaghetti | |||
|---|---|---|---|---|
| CTRL | RGM/TG | CTRL | RGM/TG | |
| Total polyphenols (mg GAEs/g dw) | 0.54±0.08a | 2.27±0.20b | 0.28±0.02a | 2.11±0.15b |
| Antioxidant activity (µmol Fe(II)/g dw) | 3.13±0.33a | 54.53±7.67b | 2.75±0.05a | 56.19±9.67b |
| Total anthocyanins (mg mvd-glu/g dw) | ND | 0.095±0.008a | ND | 0.084±0.002a |
ND: not detectable. CTRL: control spaghetti; RGM/TG: spaghetti containing red grape marc flour (15% w/w; particle size ≤ 125 µm) and 0.6% (w/w) transglutaminase; GAEs: gallic acid equivalents - expressed as mg GAEs/g dry weight (dw). Antioxidant activity - expressed as µmol FeSO4* 7H2O/g dry weight (dw). Results are expressed as means ± SD, n = 3.
RGM contains a number of different bioactive polyphenols including anthocyanins. No total anthocyanins (TAC) were detected in the CTRL sample, before and after cooking. In the case of fortified spaghetti the content of anthocyanins was: 0.095 ± 0.008 and 0.084 ± 0.002 mg mvd-glu-equivalents/g dw for uncooked and cooked pasta, respectively (Table 2). Thus, cooking did not cause any significant changes in the concentration of anthocyanins.
Table 2 shows that the uncooked and cooked RGM/TG spaghetti exhibited a high antioxidant activity, 54.53±7.67 before cooking and 56.19 ± 9.67 µmol Fe(II)/g dw after cooking. No loss of antioxidant activity of either CTRL or RGM/TG was observed after cooking (Table 2). These findings are in line with the observations for the total polyphenol and anthocyanin content.
3.3 Bioaccessibility of polyphenols and glucose Table 3 shows bioaccessible total polyphenols, available for absorption in the intestine, after in vitro digestion. RGM/TG spaghetti contained a statistically significant higher amount of bioaccessible total polyphenols than CTRL spaghetti: 5.53 ± 0.61 vs. 4.16 ± 0.50 mg GAEs/g dw. The difference in the concentration of total polyphenols between samples was 1.37 mg GAEs/g which was comparable to the difference observed for cooked spaghetti samples before the in vitro digestion (Table 2).
| Cooked spaghetti | ||
|---|---|---|
| Bioaccessible fraction | CTRL | RGM/TG |
| Total polyphenols (mg GAEs/g dw) | 4.16±0.50a | 5.53±0.61a |
| Antioxidant activity (µmol Fe(II)/g dw) | 6.30±2.64a | 25.30±4.89b |
| Total anthocyanins(mg mvd-glu /g dw) | ND | 0.037±0.001 |
ND: not detectable. CTRL: control spaghetti; RGM/TG: spaghetti containing red grape marc flour (15% w/w; particle size ≤ 125 µm) and 0.6% (w/w) transglutaminase; GAEs: gallic acid equivalents - expressed as mg GAEs/g dry weight (dw). Antioxidant activity - expressed as µmol FeSO4* 7H2O/ g dry weight (dw) . Results are expressed as means ± SD, n = 3.
There were no detectable bioaccessible anthocyanins (a class of polyphenols) in the CTRL samples, assessed spectrophotometrically as well as by HPLC. Both these methods used showed similar values of total anthocyanins in RGM/TG sample: 0.037 ± 0.001 mg mvd-glu/g dw and 0.039 ± 0.005 mg mvd-glu /g dw, respectively (Table 3). The antioxidant activity of samples in the bioaccessible fraction was evaluated after in vitro digestion and the results are shown in Table 3. There was an evident difference between CTRL and RGM/TG samples. The RGM-enriched spaghetti showed a higher antioxidant activity than the control pasta; 25.3 ± 4.89 vs. 6.3 ± 2.64 and µmol Fe(II)/g dry weight (dw). This indicates that in vitro digestion of RGM/TG led to a significantly higher antioxidant activity of bioaccessible fraction than that of CTRL.
The glucose amount available for absorption in the small intestine phase was also investigated and data are shown in the Figure 1. At t=0 (directly after the gastric phase and before the small intestine phase of digestion), the bioaccessible glucose concentration was 121.7 ± 10.15 and 99.4 ± 7.13 mg Glu/g fw for CTRL and RGM/TG spaghetti, respectively. No statistically significant difference between experimental spaghetti samples (P = 0.273) was detected. But at the end of the small intestinal phase of digestion (after 2h) the enriched spaghetti showed a significantly lower concentration of bioaccessible glucose (291.4 ± 69.3 mg Glu/g fw) compared to the control spaghetti (511.5 ± 73.7 mg Glu/g fw). This is due to the fact that in the fortified sample the wheat flour starch was substituted with RGM flour, which does not contain starch or other digestible carbohydrates.
Bioaccessibl. (available for absorption) glucose during in vitro digestion of CTRL and RGM/TG spaghetti. CTRL: control spaghetti without the addition of red grape marc flour; RGM (RGM/TG): spaghetti containing red grape marc flour (15% w/w; particle size ≤ 125 µm) and 0.6% (w/w) transglutaminase; Glu: glucose; fw: fresh weight. t=0 min (before digestion of the spaghetti samples in the small intestinal phase). Results are expressed as means ± SD. aData with different letters are significantly different (P < 0.05), as determined by ANOVA followed by the Tukey-Kramer test.
Here we showed that the substitution of wheat durum semolina by 125 µm RGM flour improved the sensory characteristics of spaghetti such as color (light purple color) and taste as compared to RGM-500 (500 µm) spaghetti. This result is in agreement with previous observations showing that a spaghetti sample enriched with medium particle size tomato peel flour showed a pleasant orange color as compared to that with coarse particle size tomato peel flour (Padalino et al., (2017)). The addition of the 125 µm RGM flour also improved the taste of the cooked pasta possibly due to small fibrous sensation during mastication. This result is also in line with previous reports (e.g. Padalino et al., 2017).
The significant decline of overall quality in terms of elasticity, adhesiveness, and bulkiness recorded for RGM-500 cooked spaghetti samples might be explained by the dilution of the gluten strength by RGM leading to interrupted and weakened overall structure of pasta (Rayas-Duarte et al., 1996; Rekha et al., 2013). Conversely, the addition of RGM flour with a lower particle size (125 µm) and transglutaminase (RGM/TG) improved the sensory characteristics. Possibly, lower particle size can help to form a more stable network able to bind starch granules with untraditional flour and prevent a loss of solids during cooking, reducing pasta adhesiveness and bulkiness (Padalino et al., 2017; Rekha et al., 2013). Furthermore, transglutaminase catalyzes covalent crosslinks strengthening the gluten network which prevents texture deterioration during cooking, increases hardness, elasticity and decreases stickiness of cooked pasta (Yeoh et al., 2011).
According to our data, the fortification resulted in a reduction of dry matter in the RGM-enriched cooked spaghetti of about 6%. This was probably due to a higher solid release during cooking from the enriched sample. Semolina was substituted with red grape marc flour, which does not contain gluten. Therefore, the structure of RGM spaghetti is weaker than that of the control sample. It has been reported that gluten is critical for a strong protein network that helps to reduce loss of solids during cooking (Biney et al., 2014; Rayas-Duarte et al., 1996). Furthermore, the fiber contained in RGM can also react with the protein network and break protein-starch bonds, thereby leading to a greater release of solids in the water (Tudorica et al., 2002).
Regarding the nutritional characterization of the product, our results clearly show that the RGM enrichment enhanced the levels of total polyphenols significantly in both raw and cooked spaghetti compared to the CTRL sample (Table 2). Furthermore, cooking did not affect the concentrations of polyphenols in RGM spaghetti. RGM polyphenols are mostly in glycosidic form which are localized in cellular vacuoles and other organelles, that may protect them from leaching or decay during cooking process (Sakihama et al., 2002). Similar to the total polyphenol concentration the cooking process had no significant effect on the total anthocyanin concentration in the RGM/TG spaghetti. No anthocyanins were detected in CTRL spaghetti. This is in line with data from Sant'Anna et al., (2014).
The RGM spaghetti was characterized by a significantly higher antioxidant activity compared to the control sample estimated using the FRAP assay. Again, the cooking procedure had no effect. Fares et al., (2010) observed an even greater antioxidant capacity in the pasta samples enriched with the debranning fractions after cooking; possibly this is due to the better extraction efficiency of bound polyphenols like phenolic acids. A similar effect was observed by Turkmen et al., (2005), who studied the effect of the cooking method on total polyphenols and antioxidant activity of different vegetables.
In order to affect human health beneficially, it is important that the added phytochemicals in food products are available for absorption in the gastrointestinal tract. Therefore, the bioaccessible total polyphenol and anthocyanin content as well as the antioxidant activity were evaluated in this study. The RGM/TG-enriched pasta showed a significantly higher concentration of bioaccessible total polyphenols than the CTRL sample. Furthermore, in both experimental samples (RGM/TG and CTRL) the polyphenol concentration increased after in vitro digestion compared to cooked pasta before digestion, while maintaining a similar difference observed between cooked and non-digested RGM and CTRL samples. A possible explanation may be the differences in extraction efficiency from samples (in vitro digested versus non-digested). As Gawlik-Dziki et al., (2015) showed, the use of gastrointestinal digestive enzymes led to release of bound phenol acids as well as amino acids from wheat proteins such as cysteine, tryptophan, and tyrosine. These amino acids can react with Folin-Ciocalteu reagent and mimic the reaction of polyphenols (Abdel-Aal and Hucl, 2002; Everette et al., 2010). The same trend was also observed by Miranda et al., (2013), who compared the polyphenol amount extracted from potatoes by means of the chemical method and in vitro digestion.
We observed a significant amount of bioaccessible anthocyanins in the digested RGM/TG-samples but no anthocyanins in the CTRL spaghetti. In contrast to the total polyphenols, the bioaccessible total anthocyanins after in vitro digestion was lower than that observed in the cooked spaghetti before digestion. Probably, a part of the anthocyanins was destroyed or remained in the food matrix. Several authors found that the total polyphenols increased while the anthocyanin amount decreased during digestion, due to the instability of anthocyanins in the neutral/alkaline intestinal environmental (pH = 7.2–7.6) (McDougall et al., 2005; Podsędek et al., 2014; Tagliazucchi et al., 2010).
Regarding the antioxidant activity of the bioaccessible fraction (Table 3), the RGM-enriched pasta showed a significantly higher antioxidant activity than the CTRL sample. However, there was an increase in the activity of the CTRL sample (6.30 vs. 2.75 µmol Fe(II)/g dw) and a decrease in the RGM/TG spaghetti (25.30 vs. 56.19 µmol Fe(II)/g dw) compared to the respective cooked, non-digested samples (Table 2, 3). In the CTRL spaghetti the observed increase is probably due to the release of amino acids from wheat durum semolina proteins and liberated phenolic acids such as ferulic acid, substances characterized by antioxidant activity, during digestion by enzymes (Gawlik-Dziki et al., 2015; Miranda et al., 2013). In contrast, the antioxidant activity of the fortified sample (RGM/TG) decreased from approx. 56 to approx. 25 µmol Fe(II)/g dw. This decrease can be due to loss of active but also sensitive to in vitro digestion RGM-polyphenols and the efficiency of antioxidant extraction from the matrix. The cooked spaghetti was extracted using methanol/water/HCl. In contrast, the bioaccessible fraction was obtained using gastrointestinal enzymes, water and physiological temperature, pH. This can lead not only to different concentrations of polyphenols but maybe most importantly to a different composition of individual polyphenols in these extracts. The antioxidant activity varies enormously among polyphenols. Despite the higher total polyphenol level in the bioaccessible fraction seems that the individual polyphenols in this fraction exhibit lower antioxidant activity than those in the methanol/ water/HCl extract before in vitro digestion. Possibly, effective polyphenol antioxidants like anthocyanins are partly destroyed under physiological pH at the small intestine conditions yielding polyphenols with lower antioxidant activity.
The surge in obesity and associated diseases expresses the need for strategies to manage this problem. Reducing energy density and glycemic load can have a positive effect to prevent these conditions (Rolls, 2009). Spaghetti is rich in complex carbohydrates that are effectively digested to glucose in the small intestine. Here we showed that the enrichment of spaghetti with red grape marc caused a significant reduction of bioaccessible glucose release in the small intestine model after 2h of digestion. This effect is due to a lower amount of semolina and therefore of starch in the RGM spaghetti. Similar data were reported by Biney and Beta (2014).
Our results showed that the substitution of starch-rich semolina in spaghetti by (15% (w/w) red grape marc (RGM)) increased the total polyphenol, anthocyanin content and antioxidant activity. These important nutritional parameters were not affected by cooking. The bioaccessible fraction from RGM-spaghetti showed a higher amount of polyphenols including anthocyanins as well as antioxidant activity. An additional positive effect of the spaghetti fortification was the decrease of bioaccessible glucose Thus, enrichment of spaghetti with RGM can increase its beneficial nutritional properties by increasing bioaccessibility of compounds with antioxidant activity and reducing the amount of bioavailable glucose and glycemic load.
The authors have declared no conflicts of interest.
Acknowledgement We grateful thank Benjamin Peters and Konstanze Schelm for their excellent technical assistance during the performance of the experiments.
