Partial replacement of wheat flour with pumpkin seed flour in muffins offered to children

ABSTRACT

Effects of partial replacement of wheat flour with pumpkin seed flour on quality indices of muffins for children were studied. Fatty acid (FA) composition, peroxide value (PV), anisidine value (AnV) and conjugated diene (CD) and triene (CT) contents were assayed in lipids extracted from muffins and related to storage time. Both PV and AnV values increased with the increasing content of pumpkin seed flour. Regardless the pumpkin seed flour share, the content of CD was higher than of CT in muffins. As the replacement of wheat flour with pumpkin seed flour increased, the nutritional value of muffins improved. It appears appropriate to incorporate 33% of pumpkin seed flour into muffins stored for 2 weeks without special packaging conditions. More than 71% of the children evaluated muffins containing 33% of pumpkin seed flour as ‘tasty’ and ‘very tasty’. The experimental muffin formulation resulted in a novel product suitable for children’s nutrition.

RÉSUMÉ

Se estudiaron los efectos de la sustitución parcial de la harina de trigo por harina de semilla de calabaza en los índices de calidad de madalenas ofrecidas a niños. Se examinaron la composición de ácidos grasos (FA), el índice de peróxidos (PV), el índice de anisidino (AnV) y el dieno conjugado (CD) además del contenido de trieno (CT) en los lípidos extraídos de las madalenas y relacionados con el tiempo de almacenamiento. Los índices de PV y AnV se incrementaron con el aumento del contenido de harina de semilla de calabaza. A pesar de que compartían harina de semilla de calabaza, el contenido de CD fue mayor que el de CT en las madalenas. A medida que aumentaba la sustitución de la harina de trigo por harina de semilla de calabaza, mejoraban los valores nutricionales de las madalenas. Pareció apropiado incorporar un 33% de harina de semilla de calabaza en las madalenas conservadas durante 2 semanas sin ninguna condición especial de envase. Más del 71% de los niños evaluaron las madalenas que contenían un 33% de harina de semilla de calabaza como ‘sabrosas’ y ‘muy sabrosas’. La formulación experimental de las madalenas resultó ser un producto novedoso adecuado para la nutrición de los niños.

KEYWORDS:

• Pumpkin seed flour
• muffins
• lipid oxidation products
• antioxidant activityconsumer acceptance by children

PALABRAS CLAVE:

• harina de semilla decalabaza
• madalenas
• productos con oxidación lipídica
• actividad antioxidante aceptada para el consumo de niños

Introduction

The enrichment of food products has been an important tool, not only to manage or prevent specific nutritional deficiencies, but also to promote a general state of well-being in different populations and, possibly, to prevent certain chronic diseases. The identification and development of fortifying agents that would ensure high product quality and bioavailability pose some technological and scientific challenges (Revathy & Sabitha, 2013). Pastries and confectionery products contain many nutritionally controversial components and may thus serve as models for food enrichment (Onacik-Gür, Żbikowska, & Jaroszewska, 2015). Many authors (Reddy, Urooj, & Kumar, 2005; Zbikowska & Rutkowska, 2008) discussed the nutritional value of those products, focusing on the quality of fat, partial replacement of simple sugars by polysaccharides, decrease of caloric value and enrichment with bioactive compounds usually absent from such products. The authors of many reports discussed the application of a natural ingredient with a high fibre content (oat), polyphenols (sorghum, cumin, ginger, pomegranate peel), carotenoids (pumpkin powder) that were used for nutritional enrichment and for maintaining the rheological and sensory properties of confectionery products (Abdel-Samie, Wan, Huang, Kim Chung, & Xu, 2010; Chiremba, Taylor, & Duodu, 2009; Ismail, Akhtar, Riaz, & Ismail, 2014; Zbikowska & Rutkowska, 2011).

Among other ingredients, pumpkin (Cucurbita spp.) pulp and seeds have been used in the food industry (Xanthopoulou, Nomikos, Fragopoulou, & Antonopoulou, 2009). In some European countries, pumpkin seeds are used as raw material for the production of high value roasted pumpkin seed oil (Vujasinovic, Djilas, Dimic, Romanic, & Takaci, 2010), while in other countries they are regarded as an agro-industrial waste and discarded. However, in some countries they are consumed (Patel, 2013) raw or roasted, or used as an additive to bread, flakes, salads and pastries (Xanthopoulou et al., 2009) due to their beneficial phytochemical composition. Pumpkin seeds contain about 40% of fat rich in unsaturated fatty acids (FA), lecithin and antioxidants, for example carotenoids, tocopherols and phytosterols, as well as in cellulose and minerals (Makni, Fetoui, Gargouri, El Garoui, & Zeghal, 2011; Patel, 2013; Xanthopoulou et al., 2009). Moreover, pumpkin seeds are rich in exogenous amino acids (e.g. lysine, thyrosine, tryptophan, methionine) and in iron (96 ± 33 ppm), thus being recommendable to children and adolescents often prone to iron deficiency-caused anaemia (El Adawy & Taha, 2001; Patel, 2013). Ample reports have indicated health benefits resulting from the consumption of pumpkin seeds (Patel, 2013; Rezig, Chouaibia, Msaadab, & Hamdia, 2012), for example in acute schistosomiasis, a parasitic disease accompanied by fever, chills, headache, fatigue and intense gastrointestinal discomfort (Patel, 2013).

A valuable by-product in the manufacture of cold-pressed oil is pumpkin seed flour (Makni et al., 2011) that, unlike wheat, is rich in fibre (47.9% of dry mass) and, thus, enhances intestinal functions and produces the feeling of satiety that is essential in body weight control. Another interesting benefit of pumpkin seed meal is the lack of gluten, which makes it recommendable to patients with gluten intolerance (Patel, 2013). In addition, pumpkin seed flour is used in nutrition of malnourished infants in Kenya as a source of an adequate proportion of dietary energy, protein and fat (Ward & Ainsworth, 1998).

An attractive greenish colour of pumpkin seed flour and its nutty taste challenge the food technologists to create new products of a high nutritional value. Pumpkin seed flour has been used as an additive to pancakes, salty and sweet breads, soups, sauces, etc. and, above all, as an additive to wheat meal in pastries, rendering them an original, unique taste (Patel, 2013). All those specificities of pumpkin seed flour make it a potentially valuable additive to children-oriented confectionery. The aim of this study was, thus, to assess the effect of partial replacement of wheat flour with pumpkin seed flour on quality indices of muffins offered to children.

Materials and methods

Preparation of experimental muffins

Muffins were prepared in four variants (0–3) differing in the degree of wheat flour (300 g) substitution with pumpkin seed flour (Szarłat, Poland) that ranged from 0 to 50% (by weight). Pumpkin seed flour was obtained from Cucurbita pepo L. seeds containing (as declared by the producer): proteins 579 g/kg, fat 85 g/kg and monosaccharides 76 g/kg. The following proportions of ingredients were used to prepare the muffins: sugar powder (100 g), milk (3.2% of fat, 120 mL), butter (90 g), baking powder (6 g) and beaten whole eggs (76 g). Butter was whipped with sugar powder, the mixture was aerated for 10 min, then milk and eggs were added and blended for 2 min. At last, flour and baking powder were added and mixed. The resulting batter was poured into moulds lined with baking paper and baked for 20 min at 180ºC in a convection oven with forced air circulation (Electrolux AR 85, Italy), and cooled at room temperature. The muffins were stored in polyethylene bags at 10 ± 1ºC for 4 weeks.

Analysis of pumpkin seed flour

Before analyses, pumpkin seed flour was stored in original containers in a cooled magazine at 8–10°C, according to the manufacturer’s recommendation. All analyses were performed from fresh pumpkin seed flour immediately after the container had been opened. The pumpkin seed flour (Szarłat, Poland) was determined for: fat content, FA composition, content of oxidation products, total phenolic content (TPC) and antioxidant activity.

Fat content was determined with the Soxhlet’s method using hexane as an extracting solvent. The hydroperoxide value (PV) was determined in extracted fat according to ISO 3960:2012 standard titration method (ISO 3960: 2012, 2012). The results were expressed as meq O/kg of fat. The content of secondary products of lipid oxidation was determined spectrophotometrically using Specord 40 (Analytic Jena, Germany) as the anisidine value (AnV) according to ISO 6885:2008 standard method (ISO 6885:2008, 2008) at λ = 350 nm. FA composition was determined as follows: FA methyl esters (FAME) were prepared by transmethylation of fat samples using a mixture of concentrated H₂SO₄ (95%) and methanol according to AOCS (2000). The analysis of FAME profile of fat extracted from pumpkin seed flour was conducted by gas chromatography (GC) using an HP-Agilent 6890N equipped with flame ionisation detector (FID), a split/splitless injector, operated with a split ratio of 1:50 and a capillary column with a stationary phase of high polarity (100 m × 0.25 mm I.D., film thickness 0.1 μm; Rtx 2330 Restek). Oven temperature was initially 120°C for 40 min, then it was ramped to 155°C at 1.5°/min and held for 50 min, then it was ramped again at 2°/min to 210°C and held for 35 min. Injector and detector temperatures were maintained at 250°C, helium was used as the carried gas at a flow rate of 0.9 mL/min (Rutkowska, Adamska, & Bialek, 2012). FAs were identified with the use of Supelco 37 standard No: 47885-U (Sigma Aldrich).

Antioxidant activity by DPPH scavenging and TPC were determined in ethanol extracts of pumpkin seed flour using ethanol/water (80/20, v/v). The extract was separated by filtering through a filter paper (POCH, S.A., Poland), and the procedure was repeated twice with 50 mL of the solvent. TPC was determined spectrophotometrically (Specord 40, Analytik Jena AG, Germany) by using the Folin-Ciocalteu’s reagent (Singleton & Rossi, 1965). Gallic acid was used as a reference and the results were expressed as gallic acid equivalents (GAE; mg GAE/100 g of extract). Then, 0.1 mL of the ethanol extract of muffin was diluted with 7.9 mL of pure water. The Folin-Ciocalteu’s reagent (0.5 mL) and a sodium carbonate solution (1.5 mL; 20 g/100 mL) were added and thoroughly mixed. The mixture was allowed to stand for 120 min with intermittent shaking, and absorbance was measured at 725 nm. Radical scavenging effect of the ethanol extract of pumpkin seed flour on 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH) was determined according to the modified method of Sánchez-Moreno, Larrauri, and Saura-Calixto (1998). An aliquot (1.0 mL) of the DPPH solution was diluted in 2.9 mL of methanol, and 0.1 mL of the ethanol extract of flour was added. The shaken mixture was left in the dark, then the absorbance was measured at 515 nm against blank (mixture without extract). The antioxidant activity of the extract was obtained from the following formula:

where A_DPPH – absorbance of blank sample, A_t – absorbance of the analysed sample.

Analysis of muffins

Determination of fat content

Fat was extracted from muffins (fresh and stored) with the Weibull-Stoldt method that including acid hydrolysis (37% HCl, w/w) of lipids bound to carbohydrates and proteins followed by hexane extraction. Weighted portions of muffin were powdered in a mortar, then hot water was added and the mixture was treated with hydrochloric acid to hydrolyse proteins and carbohydrates contained in the product. The fat was extracted with hexane.

Determination of fat oxidation

The fat extracted from muffins was analysed for lipid oxidation by determining primary products expressed by the PV and secondary products expressed by the AnV. Additionally, the contents of conjugated dienes (CD) and trienes (CT) were assayed according to Pegg (2001) at λ = 233 nm and 268 nm using the following equations:

C – concentrations (mol/L); A – absorbance; E – molar absorption of the linoleic acid peroxide (2.525 × 10^–4 M/cm); l – light path of the quartz cell (1 cm); W – sample mass (g); 2.5 × 10⁴ – amount of isooctane used to solubilise oil sample. The results were expressed in μmol/g fat.

Analysis of fatty acid composition

FA composition of fat extracted from muffins was measured at 0, 2 and 4 weeks of storage. Description of the method is presented above. Peak areas were corrected by the response factors for FAME responses of FID, and area% of FAME was appropriately converted to weight% of FA. A butter reference standard (CRM 164; Community Bureau of Reference, Belgium) was used to determine the recovery rates and correction factors for individual FAs in fat extracted from muffins. In addition, Supelco 37 standard No: 47885-U (Sigma Aldrich) was used for identification of unsaturated FAs. The contents of individual FAs were expressed as g/100 g FA.

Calculation of nutritional value

The nutritional value of the experimental muffins was calculated based on literature data: ‘Food Composition and Nutritional Value Tables’ (Kunachowicz, Nadolna, Przygoda, & Iwanow, 2005).

Sensory evaluation

A scoring test was used to determine the sensory characteristics of muffins. Sensory analyses were done after cooling the baked muffins. Ten well-trained panellists (four men and six women; 27–46 years of age) with experience in assessing bakery products participated in the study. They scored each attribute on a 10 cm analogue scale. The panellists were requested to evaluate samples three times in the following order: intensity – aroma (buttery and off-flavours), taste (buttery, sweet, off-taste), acceptability – colour, texture and overall acceptability. Muffins were delivered in coded plastic containers in a random order. Every panellist was provided with spring water to rinse mouth between samples. The results from the analogous scale were converted to numerical values (from 0 to 10 units).

Consumer acceptance evaluation by children

The muffins rated by the expert panellists as the best ones were offered to children aged 7–12 years (n = 248) as the potential consumers. All children participating in the study submitted their parental consents. Testing was done in classrooms of two schools located in the Mazovian Province in the morning hours. All children underwent a short training prior to the rating by using a five-point mimic hedonic scale (Barylko-Pikielna & Matuszewska, 2014). Children expressed the degree of liking of the product by ticking the facial expression corresponding to their preferences. Hedonic ratings were converted into numeric scores ranging from 1 (very tasteless) to 5 (very tasty). The results were averaged and the acceptance index was expressed in percentages, with score 5 being equal to 100%.

Data analysis

All chemical analyses were conducted in triplicate. The results (means ± SD) were subjected to a regression analysis or to the analysis of variance (ANOVA) followed by Scheffe’s post-hoc test, the level of P < 0.05 being considered significant.

Results and discussion

Quality of pumpkin seed flour

Pumpkin seed flour used in this study was produced from the cold-pressed cake of Cucurbita pepo L. and contained 8.5% of valuable fat rich in unsaturated FA and characterised by a high antioxidant potential resulting from many polyphenolic compounds (Table 1). The predominant saturated fatty acid (SFA) of the lipid fraction was palmitic acid (C16:0) whose content was 11.54 g/100 g FA. Regarding unsaturated FAs, pumpkin seed flour was rich in linoleic acid (c-9 c-12 C18:2; 58.10 g/100 g FA). Lipids extracted from pumpkin seed flour contained low levels of primary and secondary oxidation products: 2.89 meq O/kg of fat and 2.41 units, respectively.

Table 1. Quality of pumpkin seed flour [mean ± SD].

Tabla 1. Calidad de la harina de semilla de calabaza [promedio ± SD].

Component	Value
Fat [g/100 g product]	8.5 ± 0.75
Total SFA [g/100 g FA]:	17.30 ± 0.85
C16:0	11.54 ± 0.77
Total MUFA [g/100 g FA]:	23.24 ± 0.64
C18:1 9c	22.31 ± 0.53
Total PUFA [g/100 g FA]:	58.69 ± 0.49
C18:2 9c 12c	58.10 ± 0.38
PV [meqO2/kg fat]	2.89 ± 0.31
AnV	2.41 ± 0.28
AV [mg KOH/g of fat]	1.05 ± 0.19
*DPPH [%]	64 ± 0.03
Total phenolic content	17.60 ± 0.96
[mg GAE/100 ml extract]

Note: *DPPH −1,1-diphenyl-2-picrylhydrazyl radical; GAE – gallic acid equivalent.

Nota: *DPPH – radical 1,1-difenílico-2-picrilhidrazil; GAE – equivalente de ácido gálico.

The antioxidant capacity measured by DPPH reduction of pumpkin seed flour was high (64%) and comparable with results obtained by Andjelkovic, Van Camp, Trawka, and Verhé (2010) for pumpkin seed oil. The DPPH radical scavenging activity of pumpkin seed flour used in our experiment was much higher than that reported by Nawirska-Olszańska, Kita, Biesiada, Sokół-Łętowska, and Kucharska (2013) for pumpkin seed oils from 12 different pumpkin cultivars. The TPC was 17.6 mg GAE/100 mL of the extract.

Nutritional analysis of muffins

The application of the high resolution GC enabled determination of the contents of 32 FAs in fat extracted from muffins (Table 2). Short-chain saturated FAs (SCSFA) were represented by C4:0 – C12:0 acids; in general increasing the content of pumpkin seed flour caused a decrease of SCSFAs in the muffins. In addition, partial replacement of wheat flour with pumpkin seed flour induced a decrease in the content of the main long-chain saturated FAs (LCSFA) in fat extracted from the muffins: C14:0 – myristic, C16:0 – palmitic and C18:0 – stearic FA. The highest content of pumpkin seed flour caused a 44% decrease of C14:0 content, a 26% decrease of C16:0 content and a 26% decrease of C18:0 content. It may be considered as an advantage due to the confirmed atherogenic properties of LCSFA, especially of myristic and palmitic FAs (Zock, Vries, & Katan, 1994). Cookies and confectionery products are one of the major sources of SFAs in the European diet (Zbikowska, Rutkowska, & Kowalska, 2015). The consumption of a mixture of pumpkin with flax seeds resulted in a significant decrease in lipid parameters, which indicated their anti-atherogenic potential (Barakat & Mahmoud, 2011).

Table 2. Mean contents (±SD) of fatty acids in muffins (g /100 g FA) at 0, 2 and 4 weeks of storage.

Tabla 2. Contenidos promedio (±SD) de ácidos grasos en las madalenas (g /100 g FA) a 0, 2 y 4 semanas de almacenamiento.

FA	Control			I (17%)				II (33%)				III (50%)
	0 w	2 w	4 w	0 w	2 w	4 w		0 w	2 w	4 w		0 w	2 w	4 w
C4:0	4.92 ± 0.05	4.89 ± 0.30	4.91 ± 0.01	4.42 ± 0.76^a	4.22 ± 1.13^a	4.06 ± 0.20^b	*	4.09 ± 0.13^a	3.73 ± 0.13^b	4.06 ± 0.10^a	*	3.61 ± 0.05^a	3.58 ± 0.05^a	3.81 ± 0.01^b	*
C6:0	2.69 ± 0.50	2.71 ± 0.59	2.65 ± 0.12	2.63 ± 0.31	2.71 ± 0.33	2.76 ± 0.20		2.44 ± 0.24^a	2.94 ± 0.18^a	3.40 ± 0.15^b	*	2.61 ± 0.05^a	2.88 ± 0.02^b	2.99 ± 0.05^b
C8:0	1.50 ± 0.09	1.43 ± 0.18	1.55 ± 0.20	1.28 ± 0.35	1.27 ± 0.25	1.23 ± 0.17	*	1.19 ± 0.19	1.09 ± 0.15	1.20 ± 0.16	*	1.04 ± 0.09	1.04 ± 0.11	1.08 ± 0.16	*
C10:0	3.22 ± 0.10	3.31 ± 0.45	3.35 ± 0.25	2.68 ± 0.38	2.69 ± 0.43	2.61 ± 0.36	*	2.47 ± 0.24	2.34 ± 0.26	2.50 ± 0.10	*	2.21 ± 0.10	2.25 ± 0.08	2.26 ± 0.10	*
C12:0	3.60 ± 0.30	3.64 ± 0.37	3.59 ± 0.25	2.97 ± 0.28	3.04 ± 0.34	2.96 ± 0.27	*	2.83 ± 0.11	2.65 ± 0.25	2.76 ± 0.20	*	2.44 ± 0.36	2.52 ± 0.37	2.48 ± 0.30	*
SCSFA	15.93 ± 0.91	15.98 ± 0.57	16.05 ± 0.33	13.48 ± 1.87	13.93 ± 1.65	13.61 ± 0.89	*	13.02 ± 0.62	12.75 ± 0.99	13.92 ± 0.68	*	11.90 ± 0.31^a	12.26 ± 0.30^a	12.61 ± 0.28^b	*
aC12:0	0.09 ± 0.03	0.09 ± 0.03	0.08 ± 0.06	0.08 ± 0.07	0.08 ± 0.07	0.08 ± 0.04		0.08 ± 0.05	0.07 ± 0.01	0.07 ± 0.04		0.07 ± 0.03	0.07 ± 0.06	0.06 ± 0.12
iC12:0	0.10 ± 0.05	0.10 ± 0.02	0.10 ± 0.02	0.07 ± 0.03	0.07 ± 0.02	0.07 ± 0.03		0.07 ± 0.05	0.06 ± 0.03	0.07 ± 0.05		0.06 ± 0.03	0.06 ± 0.02	0.07 ± 0.05
iC13:0	0.14 ± 0.05	0.14 ± 0.05	0.14 ± 0.03	0.11 ± 0.05	0.11 ± 0.04	0.11 ± 0.05		0.09 ± 0.04	0.09 ± 0.02	0.10 ± 0.04	*	0.09 ± 0.03	0.09 ± 0.02	0.09 ± 0.03	*
iC15:0	0.27 ± 0.03	0.27 ± 0.08	0.26 ± 0.02	0.22 ± 0.03	0.22 ± 0.03	0.23 ± 0.01		0.20 ± 0.02	0.19 ± 0.01	0.20 ± 0.03	*	0.18 ± 0.01	0.19 ± 0.02	0.18 ± 0.03	*
aC15:0	0.48 ± 0.04	0.47 ± 0.05	0.49 ± 0.01	0.38 ± 0.02	0.39 ± 0.04	0.39 ± 0.01	*	0.37 ± 0.01	0.34 ± 0.01	0.35 ± 0.01	*	0.31 ± 0.01	0.33 ±0.03	0.33 ± 0.01	*
iC16:0	0.16 ± 0.08	0.16 ± 0.04	0.15 ± 0.03	0.26 ± 0.03	0.26 ± 0.03	0.27 ± 0.03	*	0.25 ± 0.01	0.23 ± 0.02	0.24 ± 0.02	*	0.21 ± 0.06	0.22 ± 0.02	0.23 ± 0.02
aC17:0	0.44 ± 0.18	0.43 ± 0.10	0.44 ± 0.10	0.31 ± 0.02	0.32 ± 0.05	0.34 ± 0.06	*	0.28 ± 0.03	0.30 ± 0.08	0.30 ± 0.05	*	0.25 ± 0.03	0.27 ± 0.02	0.26 ± 0.05	*
C11:0	0.34 ± 0.04	0.33 ± 0.06	0.31 ± 0.02	0.04 ± 0.01	0.04 ± 0.02	0.04 ± 0.03	*	0.04 ± 0.01	0.03 ± 0.02	0.04 ± 0.03	*	0.03 ± 0.01	0.03 ± 0.02	0.04 ± 0.03	*
C13:0	0.11 ± 0.01	0.11 ± 0.03	0.10 ± 0.01	0.08 ± 0.03	0.09 ± 0.02	0.08 ± 0.04		0.07 ± 0.05	0.07 ± 0.01	0.08 ± 0.02		0.07 ± 0.03	0.07 ± 0.02	0.07 ± 0.02
C15:0	1.10 ± 0.08	1.12 ± 0.07	1.14 ± 0.03	0.90 ± 0.13^a	0.94 ± 0.09^b	0.96 ± 0.10^b		0.85 ± 0.04	0.83 ± 0.08	0.83 ± 0.01	*	0.76 ± 0.06	0.79 ± 0.02	0.77 ± 0.01	*
C17:0	0.54 ± 0.01	0.55 ± 0.03	0.54 ± 0.01	0.48 ± 0.05	0.49 ± 0.03	0.50 ± 0.08		0.46 ± 0.14	0.45 ± 0.12	0.45 ± 0.11	*	0.40 ± 0.03	0.43 ± 0.01	0.41 ± 0.02	*
C21:0	0.07 ± 0.01	0.06 ± 0.04	0.07 ± 0.03	0.05 ± 0.01	0.07 ± 0.05	0.06 ± 0.02		0.05 ± 0.02	0.05 ± 0.02	0.06 ± 0.01		0.05 ± 0.02	0.05 ± 0.01	0.05 ± 0.01
OBCFA	3.83 ± 0.21	3.83 ± 0.35	3.82 ± 0.15	2.98 ± 0.75^a	3.07 ± 0.52^b	3.13 ± 0.26^b	*	2.81 ± 0.18^a	2.71 ± 0.31^b	2.79 ± 0.20^a	*	2.48 ± 0.04^a	2.60 ± 0.05^b	2.56 ± 0.05^b	*
C14:0	11.22 ± 1.73	11.18 ± 1.95	11.25 ± 1.55	9.34 ± 1.03^a	9.57 ± 0.94^b	9.59 ± 1.00^b	*	8.77 ± 1.09^a	8.45 ± 1.02^b	8.68 ± 0.91^a	*	7.76 ± 0.86^a	8.07 ± 0.91^b	7.86 ± 0.90^a	*
C16:0	33.60 ± 1.81	33.64 ± 1.77	33.59 ± 1.62	29.56 ± 0.94^a	29.91 ± 0.89^b	30.20 ± 0.90^b	*	28.74 ± 0.03^a	27.77 ± 0.01^b	28.40 ± 0.01^a	*	26.62 ± 0.09^a	27.11 ± 0.13^b	27.45 ± 0.10^c	*
C18:0	9.95 ± 0.79	9.90 ± 0.93	9.89 ± 0.85	8.74 ± 1.76^a	8.91 ± 1.61^b	8.96 ± 1.71^b	*	8.42 ± 1.02^a	8.47 ± 1.11^a	8.70 ± 1.04^b	*	7.85 ± 0.93^a	7.95 ± 0.84^b	8.13 ± 0.96^b	*
C22:0	0.09 ± 0.05	0.09 ± 0.01	0.09 ± 0.04	0.07 ± 0.02	0.07 ± 0.01	0.07 ± 0.02		0.07 ± 0.01	0.07 ± 0.02	0.08 ± 0.03		0.07 ± 0.01	0.07 ± 0.02	0.07 ± 0.01
LCSFA	54.86 ± 2.74	54.81 ± 2.45	54.82 ± 2.52	47.71 ± 2.47^a	48.46 ± 2.36^b	48.82 ± 2.39^c	*	45.99 ± 2.05^a	44.76 ± 2.12^b	45.86 ± 2.16^a	*	42.30 ± 1.99^a	43.19 ± 1.91^b	43.51 ± 2.03^c	*
C14:1	1.08 ± 0.30	1.11 ± 0.16	1.09 ± 0.22	0.86 ± 0.11	0.90 ± 0.14	0.90 ± 0.10		0.82 ± 0.13	0.79 ± 0.19	0.81 ± 0.11	*	0.74 ± 0.13	0.77 ± 0.15	0.74 ± 0.20	*
C15:1	0.29 ± 0.04	0.31 ± 0.06	0.30 ± 0.02	0.23 ± 0.03	0.23 ± 0.08	0.24 ± 0.09		0.21 ± 0.10	0.20 ± 0.12	0.21 ± 0.13	*	0.19 ± 0.09	0.19 ± 0.11	0.19 ± 0.09	*
C16:1 n-7	1.52 ± 0.37	1.52 ± 0.34	1.51 ± 0.35	1.48 ± 0.24	1.52 ± 0.27	1.54 ± 0.31		1.40 ± 0.19	1.37 ± 0.21	1.38 ± 0.18	*	1.34 ± 0.24	1.38 ± 0.23	1.36 ± 0.21	*
C17:1	0.30 ± 0.11	0.30 ± 0.07	0.29 ± 0.09	0.24 ± 0.06	0.24 ± 0.02	0.25 ± 0.04		0.24 ± 0.05	0.23 ± 0.03	0.22 ± 0.07	*	0.21 ± 0.04	0.22 ± 0.08	0.20 ± 0.03	*
VA	0.94 ± 0.21	0.92 ± 0.22	0.93 ± 0.19	0.53 ± 0.21^a	0.52 ± 0.16^a	0.58 ± 0.18^b	*	0.49 ± 0.11^a	0.55 ± 0.11^b	0.50 ± 0.08^a	*	0.44 ± 0.11	0.44 ± 0.15	0.44 ± 0.11	*
C18:1 9c	19.49 ± 2.06	19.47 ± 2.01	19.44 ± 2.08	20.27 ± 2.55	20.22 ± 2.46	20.21 ± 2.53	*	21.16 ± 1.83^a	20.95 ± 1.91^a	20.65 ± 2.01^b	*	21.66 ± 2.02^a	21.45 ± 1.96^b	21.38 ± 2.11^b	*
C18:1 11c	0.47 ± 0.08	0.48 ± 0.06	0.48 ± 0.07	0.56 ± 0.17	0.57 ± 0.21	0.58 ± 0.14	*	0.56 ± 0.13	0.57 ± 0.20	0.56 ± 0.19	*	0.60 ± 0.23	0.55 ± 0.25	0.57 ± 0.20	*
MUFA	24.08 ± 2.09	24.11 ± 2.12	24.04 ± 2.05	24.18 ± 1.33	24.20 ± 1.80	24.30 ± 1.13		24.88 ± 2.09^a	24.66 ± 2.02^b	24.33 ± 2.12^c	*	25.18 ± 2.15^a	25.00 ± 2.03^b	24.88 ± 2.05^c	*
LA	1.36 ± 0.03	1.35 ± 0.08	1.31 ± 0.01	7.53 ± 0.35^a	7.25 ± 0.27^b	7.09 ± 0.34^c	*	11.52 ± 1.12^a	11.30 ± 1.62^b	11.21 ± 1.01^b	*	14.75 ± 1.26^a	14.15 ± 1.08^b	13.32 ± 1.11^c	*
GLA	0.15 ± 0.04	0.15 ± 0.06	0.16 ± 0.02	0.16 ± 0.07	0.16 ± 0.08	0.16 ± 0.03		0.19 ± 0.04	0.18 ± 0.05	0.17 ± 0.05		0.19 ± 0.11	0.17 ± 0.10	0.16 ± 0.09
ALA	0.41 ± 0.06	0.38 ± 0.07	0.36 ± 0.00	0.49 ± 0.06	0.47 ± 0.08	0.47 ± 0.02		0.50 ± 0.06	0.49 ± 0.09	0.49 ± 0.11		0.52 ± 0.13	0.47 ± 0.18	0.47 ± 0.14
CLA	0.33 ± 0.12	0.33 ± 0.08	0.32 ± 0.11	0.26 ± 0.04	0.26 ± 0.07	0.25 ± 0.09		0.25 ± 0.07	0.25 ± 0.01	0.24 ± 0.02		0.22 ± 0.09	0.23 ± 0.4	0.20 ± 0.11	*
C20:3 n-3	0.12 ± 0.02	0.12 ± 0.02	0.13 ± 0.03	0.18 ± 0.03^a	0.25 ± 0.02^b	0.20 ± 0.05^a		0.14 ± 0.03^a	0.18 ± 0.01^a	0.28 ± 0.02^b		0.13 ± 0.08	0.14 ± 0.01	0.15 ± 0.04
PUFA	2.37 ± 0.17	2.33 ± 0.28	2.28 ± 0.14	8.62 ± 0.15^a	8.39 ± 0.11^b	8.17 ± 0.11^c	*	12.59 ± 0.28^a	12.40 ± 0.21^b	12.39 ± 0.31^b	*	15.81 ± 0.42^a	15.16 ± 0.38^b	14.30 ± 0.41^c	*
SFA	74.13 ± 1.29	74.15 ± 1.12	74.13 ± 1.00	65.79 ± 1.70	65.08 ± 1.51	65.17 ± 1.18		61.45 ± 0.95	59.88 ± 1.14	62.21 ± 1.01		56.37 ± 0.78	57.73 ± 0.75	58.35 ± 0.79
UFA	26.94 ± 1.13	26.91 ± 1.20	26.94 ± 1.10	33.18 ± 0.74	32.98 ± 0.96	32.86 ± 0.62		37.85 ± 1.19	37.40 ± 1.12	37.07 ± 1.22		41.17 ± 1.29	40.50 ± 1.21	39.51 ± 1.23
UFA/SFA	0.36	0.36	0.36	0.50	0.51	0.50		0.62	0.62	0.60		0.73	0.70	0.68

Notes: Values with different superscripts differ significantly (P < 0.05) regarding the storage time.

Values with asterisk differ significantly (P < 0.05) from the control sample regarding pumpkin seed flour content,

SCSFA – short-chain saturated fatty acids, OBCFA – odd- and branched-chain fatty acids, LCSFA – long-chain saturated fatty acids, MUFA – monounsaturated fatty acids, PUFA – polyunsaturated fatty acids, VA – vaccenic acid, C18:1 11t, LA – linoleic acid, C18:2 9c 12c, GLA – gamma linolenic acid, C18:3 n-6, ALA – α-linolenic acid, C18:3 n-3, CLA – conjugated linoleic acid, C18:2 9c 11t.

Notas: Los valores con diferentes superíndices difieren significativamente (P < 0,05) en relación al tiempo de almacenamiento.

Los valores con asterisco difieren significativamente (P < 0,05) de la muestra control en relación al contenido de harina de semilla de calabaza,

SCSFA – ácidos grasos saturados de cadena corta, OBCFA – ácidos grasos saturados de cadena impar y ramificada, LCSFA – ácidos grasos saturados de cadena larga, MUFA – ácidos grasos monoinsaturados, PUFA – ácidos grasos poliinsaturados, VA – ácido vaccénico, C18:1 11t, LA – ácido linoleico, C18:2 9c 12c, GLA – ácido linolénico gamma, C18:3 n-6, ALA – α- ácido linolénico, C18:3 n-3, CLA – ácido linolénico conjugado, C18:2 9c 11t.

Increasing content of pumpkin seed flour significantly decrease the content of odd- and branched-chain FAs (OBCFA) that in turn was not affected by the time of storage. Nevertheless, the content of FAs belonging to this group in the designed muffins (3.05 g/100 g FA on average) should be emphasised because of their health-promoting properties. The main sources of these beneficial FAs in a human diet include milk, dairy products and meat from ruminants. Due to apoptosis induction, the OBCFA exhibit the anti-cancer activity, whereas the branched-chain FAs may reduce the incidence of necrotising enterocolitis in newborns as well as have a beneficial effect on proper tissue function and on the functioning and development of infant gut (Adamska, & Rutkowska, 2014).

Regarding FAs belonging to MUFA group, the only significant differences in comparison with the control group were found for the muffins containing 50% of pumpkin seed flour. Increasing the share of pumpkin flour in muffins significantly increased the content of the main MUFA – oleic acid – c-9 C18:1 (from 19.49 in control to 21.66 g/100 g FA in sample III containing 50% of pumpkin seed flour). The muffins differed significantly also in the content of the main PUFA – linoleic acid – c-9 c-12 C18:2: from 1.30 in the control sample to 14.1 g/100 g FA in sample III on average. Increasing share of pumpkin seed flour in muffins improved the FA profile by increasing UFA and decreasing SFA content as evidenced by an increasing UFA/SFA ratio (Table 2). This effect is advantageous regarding the nutritional value of muffins.

The replacement of all-purpose wheat flour with pumpkin seed flour in the formulation resulted in changes in the nutritional value of the experimental muffins (Table 3). The results indicate that in the samples with pumpkin seed flour, the carbohydrate content was lower in comparison with the control sample (27%). However, as the level of pumpkin seed flour substitution increased, the caloric value and both fat and protein content in muffins was also increased. This is due to the fact that the carbohydrate (mainly starch) rich refined wheat flour was replaced by the fat and protein rich pumpkin seed flour in muffins formulation (Zdunczyk, Minakowski, Frejnagel, & Flis, 1999). Fibre contents of the experimental muffins were found to be higher when compared to the control sample. Results showed that the higher was the substitution of pumpkin seed flour, the greater fibre content was determined (incorporation of 50% of pumpkin seed flour resulted in a two-fold increase in fibre content). This is in accordance with results of studies by Kulkarni and Joshi (2013) and by Uthumporn, Woo, Tajul, and Fazilah (2015) in which wheat flour was substituted with pumpkin powder and eggplant flour, respectively, in cookies.

Table 3. Nutritional value of experimental muffins.

Tabla 3. Valor nutricional de las madalenas experimentales.

	Control (0%)		I (17%)		II (33%)		III (50%)
	Per 1 item	Per 100 g	Per 1 item	Per 100 g	Per 1 item	Per 100 g	Per 1 item	Per 100 g
Energy [kcal]	197	341	205	354	224	388	243	421
Fat [g]	7.8	13.4	8.0	13.9	8.3	14.3	8.5	14.8
Protein [g]	3.7	6.5	6.0	10.3	8.2	14.1	10.3	17.9
Monosaccharides [g]	26.6	46.1	25.3	43.8	25.6	44.3	25.9	44.8
Dietary fibre [g]	0.7	1.2	0.9	1.5	1.1	1.9	1.3	2.3

Changes in lipid fraction of muffins

The lipid fraction of confectionery products is the most susceptible to changes in the process of baking and to storage. The principal factor is oxidation that generates various compounds responsible for a deteriorated sensory quality, as well as being potentially harmful (Esterbauer, 1993). In this study, changes in the lipid fraction of muffins stored up to 4 weeks were estimated from the content of primary and secondary lipid oxidation products both reacting with p-anisidine and with transposed double bonds –CD and CT. The content of primary and secondary oxidation products in fats extracted from the muffins depended on both the degree of wheat flour substitution with pumpkin seed flour and the time of storage. Those relations were described by multiple linear regressions (shown in graphs, Figures 1–3), where the dependent variables (y) were PV, AnV, CD or CT contents, the independent ones were the time of storage (x₁) and relative content of pumpkin seed flour (x₂). Until the third week of storage, the PV increased and the highest amount of hydroperoxides was found in the muffins containing 50% of pumpkin seed flour (11.5 meq O/kg of fat on average). Similarly, the highest content of secondary lipid oxidation products was found in the muffins containing 50% of pumpkin seed flour. The content of CD and CT increased in all types of muffins during storage and the content of CD was higher than that of CT irrespective of pumpkin seed flour content. These phenomena of gradual increase in lipid oxidation products was due to a high content sensitive to oxidation linoleic acid containing two cis double bonds (Frankel, 1980) in pumpkin seed flour (58.10 g/100 FA) and in experimental muffins (Table 2). This explains the seven-fold higher content of CD than of CT in the muffins.

Figure 1. PV values in muffins differing in pumpkin seed flour content (0–50%) and in storage time (0–4 weeks).

Figura 1. Índices de PV en las madalenas que difieren en el contenido de harina de semilla de calabaza (0–50%) y en el tiempo de almacenamiento (0–4 semanas).

Figure 2. AnV values in muffins differing in pumpkin seed flour content (0–50%) and in storage time (0–4 weeks).

Figura 2. Índices de AnV en las madalenas que difieren en el contenido de harina de semilla de calabaza (0–50%) y en el tiempo de almacenamiento (0–4 semanas).

Solid horizontal line – the acceptable AnV value (8.0). Lower equation pertains to muffins containing no pumpkin seed flour, upper equation to all other data.

Línea sólida horizontal–índice aceptable de AnV (8.0). La menor ecuación pertenece a las madalenas que no contienen harina de semilla de calabaza, la mayor ecuación pertenece al resto de valores.

Figure 3. Conjugated diene (Di; μmol/ g fat) and triene (Tri; μmol/ g fat) content in muffins differing in pumpkin seed flour content (0–50%) and in storage time (0–4 weeks).

Figura 3. Contenido de dieno conjugado (Di; μmol/ g de grasa) y trieno (Tri; μmol/ g de grasa) en las madalenas que difieren en el contenido de harina de semilla de calabaza (0–50%) y en el tiempo de almacenamiento (0–4 semanas).

It should be emphasised that CD and CT are harmful as they may induce morphological lesion in the liver and suppress prostaglandin synthesis (Yamasaki et al., 2000). The highest CD content in muffins was noted in those containing 50% of pumpkin seed flour and stored for 4 weeks (about 30 μmol/ g fat); yet, several fold higher amounts were reported for frying oils (sunflower) or in controlled oxidised oils (Faroosh & Moosavi, 2009; Marmesat, Morales, Velasco, Ruiz-Méndez, & Dobarganes, 2009). Thus, in our opinion, the CD content is an important criterion and ought to be included in the quality control of muffins.

Following 3 weeks of storage, decomposition of non-conjugated hydroperoxides was manifested by decreasing PV values as well as by a simultaneously increase in AnV values (Figures 1 and 2). This phenomenon is typical of the oxidative decomposition of linoleic acid, which in our study was manifested by a decreasing content during storage of muffins (Table 2).

The content of the secondary oxidation-derived carbonyl compounds may serve as an indicator of product safety (Wasowicz et al., 2004). Considering the safety of the product and the upper limit of secondary lipid oxidation products (AnV < 8), muffins containing up to 33% of pumpkin seed flour and stored in polyethylene bags at 10°C for up to 2 weeks are fully acceptable. Our results seem more encouraging than those of African researchers, who claimed that only a 10% replacement of wheat flour with pumpkin seed flour was acceptable (Atuonwu & Akobundu, 2010; Giami, Achinewhu, & Ibaakee, 2005).

It should be noted that the AnV level could have been somewhat increased due to the buttery flavour generated in the baking process and consisting mainly of 2-heptanone and nonanal (Mallia, Escher, & Schlichtherle-Cerny, 2008), as well as to the baking powder (Zbikowska & Marcinkiewicz, 2010). Such effects were reported by other authors (Atuonwu & Akobundu, 2010; Giami et al., 2005).

Sensory analysis by experts and by children

Results of the sensory assessment of muffins by expert panellists are presented in Table 4. The sensory rating differed significantly regarding pumpkin seed flour content except sweet taste. Intensity ratings of buttery flavour and taste, colour and off-taste significantly decreased with an increasing level of pumpkin seed flour in the muffins. The acceptability of texture, off-flavour and the overall acceptability rating were significantly different when the content of pumpkin seed flour was 50%.

Table 4. Sensory rating of muffins by experts (rating scale 0–10) [mean ± SD].

Tabla 4. Valoración sensorial de las madalenas realizada por los expertos (escala de valoración 0–10) [promedio ± SD].

Indices	Content of pumpkin seed flour [%]
	0%	17%	33%	50%
Buttery aroma	6.63 ± 0.50^a	4.94 ± 1.06^b	4.54 ± 0.45^b	2.89 ± 0.98^c
Off-flavour	2.23 ± 0.62^a	1.77 ± 0.83^a	1.63 ± 0.59^a	3.78 ± 0.67^b
Colour	7.31 ± 0.92^a	5.42 ± 0.71^b	5.6 ± 0.63^b	4.42 ± 0.81^c
Texture	6.50 ± 0.95^a	6.91 ± 0.62^b	6.65 ± 0.52^a,b	5.15 ± 0.79^c
Buttery taste	6.44 ± 0.51^a	5.07 ± 0.81^b	4.55 ± 0.78^b	3.84 ± 0.74^c
Sweet taste	4.83 ± 0.78	4.73 ± 0.68	4.63 ± 0.87	4.21 ± 0.61
Off-taste	0.89 ± 0.41^a	1.73 ± 0.64^b	1.65 ± 0.80^b	2.77 ± 0.64^c
Overall quality	7.34 ± 0.68^a	6.64 ± 0.57^a	6.8 ± 0.65^a	5.07 ± 0.81^b

Note: Values in rows with different superscripts differ significantly (P < 0.05).

Nota: Los valores en cada fila con diferentes superíndices difieren significativamente (P < 0,05).

Confectionery products are widely consumed by children. The majority of kids start buying confectionery products as early as below 5 years of age (Singh & Ram, 2010). This may be due to a higher exposure of young consumers to products in general that makes them more critical in their appraisals (Martı́nez-Navarrete, Camacho, Martı́nez-Lahuerta, Martı́nez-Monzó, & Fito, 2002).

Muffins containing 33%, that is the highest acceptable content of pumpkin seed flour, were forwarded to an acceptability survey by schoolchildren aged 7–12 years. The results of muffins rating by the children are shown in Figure 4. The majority of children evaluated muffins as ‘tasty’ and ‘very tasty’ (71.49%), while only 13.1% of them scored muffins as ‘very untasty’ or ‘untasty’. The average acceptability was 4.05 pts in a 5-point scale (data not shown). In this study, the average acceptability of muffins containing 33% of pumpkin seed flour was twice higher than that of muffins with 30% of defatted pumpkin seed flour measured by Nigerian researchers (Atuonwu & Akobundu, 2010).

Figure 4. Distribution of individual preferences of muffins with 33% of pumpkin seed flour.

Figura 4. Distribución de las preferencias individuales de las madalenas con 33% de harina de semilla de calabaza.

Conclusion

Substantial contents of dietary fibre and PUFAs in muffins with incorporated pumpkin seed flour are among the most essential characteristics in this research since such products will bring much advantageous impacts to the human body system. It appears appropriate to incorporate 33% of pumpkin seed flour into muffins without special packaging conditions when the storage time does not exceed two weeks. The experimental muffin formulation resulted in a novel product which is considered beneficial to human health, especially children.

Disclosure statement

No potential conflict of interest was reported by the authors.