3.1. Pepper Seed Flour—Nutritional and Chemical Analysis
Standard parameters such as moisture, fat, protein, and fibre content were determined in the pepper seeds,
Podravka and
Slavonka varieties (
Table 4). The results showed that there were no big differences in the contents between these two varieties. The
Podravka variety had higher initial moisture (8.80 g/100 g) and oil content (27.20 g/100 g) compared to the
Slavonka variety, with lower initial moisture (8.50 g/100 g) and oil content (26.70 g/100 g). Consequently, dry matter of
Podravka flour was 91.20 g/100 g, while for
Slavonka flour it was 91.50 g/100 g. The protein content was determined by the Dumas method. The results showed slight differences in protein content among the pepper seeds of both varieties. The protein content was lower (16.50 g/100 g) in the
Slavonka variety compared to the content found in the
Podravka variety (16.70 g/100 g). Despite the differences, seeds of both varieties can be considered as a good source of protein. El-Adaway and Taha [
7] found that
Capsicum annuum seeds were a good source of protein (24.43%) and oil (25.91%), while Azabou et al. [
6] found that
Capsicum annuum seeds had lower values of protein (18.30%) and oil (11.04%). Hot pepper seeds were also shown to be a good source of protein due to the content of 21.29 ± 0.28 g/100 g found by Zou et al. [
42]. These variations between protein and oil content occurred due to the differences in plant variety, climate, harvesting time, and ripening stage [
43]. Some reports have shown variations in the oil content of pepper seeds in the
Capsicum genus, as well as seed oil content between
Capsicum annuum varieties. According to Jarret et al. [
3], the means of the seed oil content among five cultivated species of
Capsicum genus varied from 18.26% to 28.08% and between
Capsicum annuum varieties from 21.06% to 28.08%. Matthaus et al. [
12] investigated 10 samples of
Capsicum annuum from different locations (Turkey, Italy) and found that the oil content varied in a wide range from 8.5 g/100 g to 32.6 g/100 g. Seed oil made of samples from Italy showed a higher oil content than those from Turkey. There were big differences in seed oil content between different varieties in
Capsicum annuum species, which is not in accordance with the findings in this study. A previous study by Jarret et al. [
3] showed that environment does not have a significant influence on seed oil content obtained from the same pepper variety of
Capsicum annuum. They analysed the seed oil content of seven accessions from different growing seasons and locations, and found the average difference between samples of the same pepper variety to be only 2.4%.
Regarding the results of the nutritional potential of the pepper seed flour, it was shown that they possess a high energy value of 407 kcal/100 g for the
Podravka variety and 404 kcal/100 g for the
Slavonka variety (
Table 4). The fat content of the
Podravka seed flour was higher (27.20 g/100 g) with lower values of saturated fatty acids (3.18 g/100 g) than the fats present in the flour of
Slavonka seeds (26.70 g/100 g), but with higher values of saturated fatty acids (3.89 g/100 g). Paired T-test showed significant difference in saturated fatty acid content between varieties (
p ˂ 0.05). There are no significant differences in seed oil content between the
Podravka and
Slavonka varieties, which is not in agreement with the previous study [
3,
12].
Regarding carbohydrate content, the flour of
Slavonka seeds contained 3.40 g/100 g with 3.02 g/100 g of sugars, while the flour of
Podravka seeds had lower carbohydrate content 3.20 g/100 g with 3.16 g/100 g of sugars. Embaby and Mokhtar [
10] found that the carbohydrate content in sweet pepper seeds is significantly higher than that observed in this study, with the amount of 56.28 ± 0.49% of dry weight. It can be explained by the difference in calculation due to the dietary fibre value included in the carbohydrate content.
The high amounts of fibres in the amount of 41.20 g/100 g for the flour of
Podravka seeds and 42.10 g/100 g for the flour of
Slavonka seeds indicate that the pepper seeds could be beneficial for human health due to the ability of fibres to prevent colon cancer, obesity, cardiovascular problems, and diabetes [
44]. Compared with the amount of dietary fibres found in hot pepper seeds (38.76 g/100 g) by Zou et al. [
42], seeds used in this study showed higher amounts of dietary fibres and they were also the main components found in
Podravka and
Slavonka seeds. The daily requirement of dietary fibre is 21–25 g for women and 30–38 g for men, depending on their age [
45]. An investigation by Anderson et al., 2020 [
46] showed that the unweighted mean intake values for total dietary fibre were 16.7 g/day for men and 15.6 g/day for women and concluded that normal fibre intake is below the recommended levels. Therefore, one of the main strategies identified by the World Health Organization is food fortification and this is an opportunity for the food industry to apply fibre in products and develop new fibre-rich products [
47]. Dordevic et al. 2020 [
48], stated that the addition of more than 1% of bamboo fibre in fruit jams significantly affected sensory properties and caused the product to not be accepted by consumers. This value was not enough to satisfy the requirement to be labelled as the source of fibre (at least 3%), according to the [
49]. Due to the high value of dietary fibre, pepper seed flour could be an ingredient for the fortification of different products, like jams, sauces and soups, but further evaluation of their nutritional, textural, and sensory characteristics, influenced by the fortification, is needed.
The results for salt showed that the flour of
Podravka seeds contained 5.05 g/100 g of salt, while the flour of
Slavonka seeds had a higher value of 5.18 g/100 g. Ash content, when compared to the red pepper researched by Azabou et al. [
6] was lower for both of the flours;
Podravka flour contained 2.91 g/100 g, while
Slavonka flour had 2.77 g/100 g. Hot pepper seeds also showed higher ash content (4.94 ± 0.14 g/100 g) [
42], as well as sweet pepper seeds (4.88 ± 0.15 g/100 g) [
10] compared to the seeds used in this study.
In this study, vitamin C was not detected in
Podravka seed flour or in
Slavonka seed flour. However, studies have shown that ascorbic acid is present in Jalapeno peppers and its by-products, including the seeds and placenta. Sandoval-Castro et al. [
50] revealed the content of ascorbic acid ranging from 0.0394 ± 0.014 g/kg to 0.0496 ± 0.0008 g/kg. Silva et al. [
51] investigated two different species of pepper seeds, Reus long pairal and sweet Italian, and they observed that the sweet Italian pepper seeds contained a higher amount of ascorbic acid (0.2304 ± 0.0011 g/kg). Total capsaicins were not detected, which indicates that the used pepper seeds were sweet, while Jalapeno peppers and their by-products were spicy, which has been proven in the study by Sandoval-Castro et al. [
50], where the amount of total capsaicins ranged from 0.142 ± 0.123 g/kg to 0.292 ± 0.304 g/kg. The capsaicionid content can differ between the cultivars, but the maturity stage plays an important role as well. Capsaicins are developed during maturation but also can be lost due to peroxidase activity [
52].
It is well known that polyphenols contribute to the quality and the nutritional value of food, so their content, as well as their antioxidant power, were determined in this study. Polyphenols have the ability to reduce free radicals and prevent the damage they cause by providing hydrogen, quenching singlet oxygen and acting as metal chelators due to the hydroxyl groups. This study provides the confirmation of health benefits of pepper seeds due to the presence of phenolic compounds. The literature covers research on different species of pepper seeds, such as red pepper seeds, where the polyphenol content was 21.50 mg GAE g
−1 of seed extract according to Azabou et al. [
6], while Sim and Sil [
53] recorded that the polyphenol content was 29.10 ± 0.18 mg GAE g
−1 of pepper seeds. Hot peppers showed different polyphenol content when compared to red peppers. According to Gurnani et al. [
54], red chilli pepper seeds contained from 7.95 mg GAE g
−1 to 26.15 mg GAE g
−1 of phenolic compounds, while Jalapeno peppers had slightly lower values of polyphenols (10.01 ± 0.61 mg GAE g
−1 to 13.09 ± 0.98 mg GAE g
−1) [
50]. In this study,
Podravka pepper seeds contained 158.20 mg/100 g of polyphenols, while
Slavonka pepper seeds contained lower quantities (149.90 mg/100 g) (
Table 4). This can be explained by the fact that polyphenol content depends on the pepper cultivar [
55]. The results obtained by Marin et al. [
56] showed that sweet peppers had a very rich profile of polyphenols and they have observed that the maturity stage from green to red decreased the total phenolic content in peppers.
The determination of antioxidant power (AP) is performed by ESR spectroscopy and it is based on the 1,1-diphenyl-2-picryl-hydrazil (DPPH) method, with the major difference that both the antioxidant capacity and the antioxidant activity are used to qualify an antioxidant [
34].
The reaction time
tr is a measure of the reactivity of the specific antioxidant and the characteristic weight
wc reflects the capacity of the antioxidant. Both parameters are used to calculate AP [
34]. The results for antioxidant power determined with the DPPH radical assay showed a correlation with the concentration of phenolic compounds. Apart from phenolic compounds, vitamin E and unsaturated fatty acids can also influence antioxidant capacity [
14]. Antioxidant power for
Podravka pepper seed flour was significantly higher (107 AU), compared to the
Slavonka pepper seed flour (70 AU), which is in accordance with the polyphenol content found in these two flours. This is in correlation with lower reaction time (
tr) of
Podravka seed flour (1.01 min) compared to the
Slavonka seed flour (1.10 min). The shorter the reaction time, the lower the oxidation state of polyphenols and the higher their antioxidative power.
A similar observation was presented in the study by Sim and Sil [
53], where they have suggested that phenolic compounds were responsible for the antioxidant activities of red pepper pericarp and red pepper seeds. Silva et al. [
51] observed that antioxidant activity was concentration-dependent for both of the analysed seeds, ‘‘sweet Italian’’ and ‘‘Reus long parial”. According to Sousa et al. [
57], the antioxidant potential of pepper seeds is the consequence of the protection of lipids stored in seeds, as well as ensuring their viability in the presence of high oxygen concentrations during the germination process. Chouaibi et al. [
14] revealed that the applied extraction method can have a significant influence on the oxidative stability of the oil. The oils produced by SC-CO
2 extraction and cold pressing had better stability than the oil extracted by Soxhlet, considering the content of phenolic compounds. Statistical analysis of instrumental data obtained by the Paired T-test showed significant difference in saturated fatty acid content, sugars, and antioxidant power (
p < 0.05). The Podravka variety (P1) has higher content of fatty acids, sugars, and higher antioxidant power than the Slavonka variety (P2). Differences in other measured parameters are not significant.
3.2. Pepper Seed Oil—Chemical Analysis
The determined fatty acid profile for the oils of
Podravka and
Slavonka seeds produced by SC-CO
2 extraction, as well by cold pressing (CP), revealed the same trend among the detected fatty acids. Of the saturated fatty acids, palmitic and steric acid were determined, while oleic and linoleic acid were the main unsaturated fatty acids. Palmitic acid (C16:0) was dominant among the saturated fatty acids (SFA), with the highest amount present in the
Podravka oil produced by SC-CO
2 extraction (11.89%), followed by stearic acid (C18:0) (3.25%). It was found that linoleic acid (C18:2) was dominant in the oils produced from both seed varieties, using both of the methods. However, the cold pressed oil produced from
Slavonka seeds had the highest amount of C18:2 (77.69%), while its content in the oil produced by SC-CO
2 extraction was the lowest compared to the other obtained oils (74.01%) (
Table 5). Significant amounts of oleic acid (C18:1) were found in the cold pressed oil of
Podravka seeds (10.41%), while in other oils it was present in lower amounts; 8.35% for the oil of
Slavonka seeds produced by SC-CO
2 extraction, 8.43% for the cold pressed oil of
Slavonka seeds, and 8.84% for the oil of
Podravka seeds produced by SC-CO
2 extraction. These findings were in accordance with the study of Azabou et al. [
6], where they have found the dominance of C18:2 (70.93 ± 0.84) among the polyunsaturated fatty acids (PUFA); the dominant monounsaturated fatty acid (MUFA) was C18:1 (12.18 ± 0.22%), while C16:0 and C18:0 were the dominant SFAs (11.90 ± 0.33 and 3.54 ± 0.20). Konscek et al. [
13] reported that variety and growing season have no influence on the fatty acid composition in spice pepper seed oil. The results showed small variations in the amount of determined fatty acid C18:2 (70.79–74.31%), C18:1 (7.9–9.11%), C18:0 (3.10–3.75%), and C16:0 (11.08–12.2%). A similar conclusion was found according to Matthaus et al. [
12]; the variation in linoleic acid (C18:2) between varieties of
Capsicum annuum was small (69.5 g/100g–74.7 g/100g), as well as for the other determined fatty acids (C16:0/10.7–14.2 g/100g, C18:0/2.5–4.1 g/100g and C18:1/8.9–12.5 g/100g). Reddy and Sarojini [
9] found that chili (
Capsicum annuum) seed oil contained 16.4.g/100 g of palmitic acid, 2.2 g/100 g of stearic acid, 10.9.g/100 g of oleic acid, and 70.6 g/100 g of linoleic acid.
Embaby and Mokhtar [
10] suggested that the sweet pepper seed oil can be used as an edible cooking or salad oil due to the presence of high amounts of C18:2 (71.55%). In their study, they have obtained similar values of fatty acids to those determined in this study (C16:0/12.32%; C18:0/3.15%; C18:1/12.98%; C18:2/71.55%). These findings are in accordance with the fact that the fatty acid composition of the species within a genus is generally similar to Jarret et al. [
3].
Linoleic acid is essential in the diet, as it is incorporated into cell membranes and it is involved in the synthesis of compounds that are responsible for regulating blood pressure, as well as for inflammatory response. In addition, PUFAs are considered beneficial for health due to their ability to reduce total cholesterol and body fat [
6].
Konscek et al. [
13] have showed that 10 g of pepper seed oil as salad oil can cover 70–74% of the suggested beneficial minimum daily intake and, according to the Commission (EU) No 1924/2006 [
49] and 432/2012 [
58], the following statements can be used for oils: “Linoleic acid (LA) contributes to the maintenance of normal blood cholesterol levels. The beneficial effect is obtained with a daily intake of 10 g of LA” and “Replacing saturated fats with unsaturated fats in the diet contributes to the maintenance of normal blood cholesterol levels.”
Li et al. [
59] obtained red pepper seed oil by SC-CO
2 extraction with and without ethanol as a co-solvent. The results showed that the oil obtained by SC-CO
2 extraction without ethanol contained a lower amount of C18:2 (46.49%) compared to the one obtained with ethanol as a co-solvent (48.20%), but it was the dominant fatty acid in both of the oils. Chouaibi et al. [
14] obtained similar results for C18:2 content to those in this study. The red pepper seed cold pressed oil had lower content of C18:2 (73.65%) compared to the
Podravka and Slavonka variety seed oils used in this study (C18:2 75.37–77.69%), but the oil produced by SC-CO
2 extraction had very similar content of C18:2 (76.26%) to the
Podravka seed oil used in this study C18:2 (76.01%). Comparison with other oils such as soybean, sunflower, and peanut oils revealed that pepper seed oil had higher amounts of palmitic and linoleic acid [
60,
61,
62] and it is classified among linoleic acid-rich edible oils.
Fatty acid compositions for both varieties and both methods are in accordance with previous results for Capsicum annuum L. species as well as linoleic acid (C18:2) content. The results showed that the variety of seeds or extraction methods had no effect on fatty acid content. Two-way ANOVA with repeated measurements indicated that there are significant differences in the content of oleic fatty acid between both varieties and extraction methods (p < 0.05). There was no significant influence of the variety or the extraction method on other measured fatty acids. The results showed that the variety of seeds, as well as the extraction methods, had no effect on the most dominant linoleic acid content (C18:2) of pepper seed oil.
Hence, among the analysed tocopherols, only γ-tocopherol was detected; α-tocopherol, α-tocopherol acetate, β-tocopherol, δ-tocopherol, and α-tocotrienol were not detected in this study (
Table 6). The results showed that γ-tocopherol is the most dominant vitamin E-active compound in pepper seed oil. The highest amount of γ-tocopherol was found in the oil of
Podravka pepper seeds produced by cold pressing (80.1 mg/100 g), followed by the amount in the oil of
Slavonka pepper seeds also produced by cold pressing (65.3 mg/100g). Generally, the oil of
Podravka pepper seeds showed a higher amount of γ-tocopherol compared to the oil of
Slavonka pepper seeds. Matthaus and Őzcan [
12] analysed 10 different varieties of pepper seed oil and showed that γ-tocopherol was also dominant in all of the samples, but other tocopherols were also detected (α-tocopherol, δ-tocopherol and β-tocotrienol). The amount varied from 306.6 mg/kg (Italy, red) to 602.6 mg/kg (Anamur table, bitter) which is a lower concentration compared to the cold pressed oils obtained in this study from both seed varieties. α-tocopherol, δ-tocopherol and β-tocotrienol were present in a much lower amount (49.8 ± 0.8 mg/kg, 2.8 ± 0.5 mg/kg and 6.1 ± 0.5 mg/kg), while these were not detected in the oils obtained in this study. Moreover, they noticed that the content of vitamin E-active compounds was higher in the pepper seed oil obtained from bitter pepper seeds, when compared to those with sweet taste. In addition, Koncsek et al. [
13] did not detect β-tocopherol and δ-tocopherol in cold pressed spice pepper seed oil, while the amounts of α-tocopherol (13.51–16.41 mg/100 g) were significantly lower compared to the dominant γ-tocopherol (57.85–83.57 mg/100 g). They found a big variation in γ-tocopherol content of pepper seed oil among different varieties and harvest seasons and concluded that γ-tocopherol content in pepper seed oil was significantly affected by raw material variety and the growing season factors and their interactions. The results in this study match the previous results. There is significant variation in γ-tocopherol content between cold pressed pepper seed oil of
Podravka and
Slavonka varieties (80.1–65.3 mg/100g), as well as between
Podravka and
Slavonka pepper seed oil obtained by SC-CO
2 (44.7–16.0 mg/kg). The obtained results for γ-tocopherol content showed variations between extraction methods and varieties too. γ-tocopherol content in
Podravka and
Slavonka seed oil obtained by cold pressing were higher than γ-tocopherol content obtained by SC-CO
2 for both varieties. These values do not match those found by Chouaibi et al. [
14] for γ-tocopherol content in pepper seed oil obtained by SC-CO
2 extraction (130.56 mg/kg) and γ-tocopherol content of cold pressed pepper seed oil (113.24 mg/kg). There are significant variations in γ-tocopherol content between two extraction methods inside one variety. When it comes to vitamin E, there are significant differences and a noticeable influence of the variety and the extraction method on the content of vitamin E (tocoph. equi.).
According to the study performed by Gunstone [
60], γ-tocopherol protected the cold pressed spice pepper seed oils from autooxidation and provided oxidative stability to the oils. This was confirmed in the study by Yang et al. [
63], where it was shown that capsaicins and tocopherols protected pepper oil from thermal oxidation during frying.
This study showed significant differences in γ-tocopherol content between two varieties of pepper seed oil, as well as between two extraction methods.
3.3. Pepper Seed Oil—Sensory Evaluation
Sensory profile of the samples was described by the 11 attributes (
Table 7) of taste, odour, and appearance. Sensory evaluation was performed on the pepper seed oil produced by the CP and SC-CO
2 extraction methods. Overall, the results did not show significant differences between oil samples. Both obtained pepper seed oils had dark orange-red colour, pleasant odour, and the characteristic taste of pepper. When compared to the obtained oils of both the
Podravka (P1) and
Slavonka (P2) varieties, it was noticed that the pepper seed oil produced by cold pressing (CP) had more characteristic flavour than the one produced by SC-CO
2 extraction. The sensory properties of the pepper seed oil confirmed that this oil could be applied in the food industry as salad oil or cooking oil for the preparation of spreads, soups, stews, and meat. Koncsek et al. [
13] evaluated the technological and nutritional properties of the cold pressed spice pepper seed oil and concluded that it can be used in many food industries, as cooking and salad oil, due to the high content of phytonutrients.
The sensory profile of two different oil extraction techniques on two varieties of pepper seeds (
Table 8) results in a slight difference in colour; the difference is significant between cold pressed oil samples (P1-CP) and supercritical CO
2 extraction processed samples (P1-SC-CO
2) without a big impact of variety (
p < 0.01). Samples P1-CP and P2-CP have a slightly darker, more intense orange colour than P1-SC-CO
2 and P2-SC-CO
2. Turbidity shows a difference in variety; the P1 in both extraction methods is slightly clearer than the P2 samples, but not significantly.
Odour is characteristic for red pepper, more intense in P1-CP and P2-CP than P1-SC-CO
2 and P2-SC-CO
2. Nutty odour is also mildly present; differences between varieties and extraction methods are not significant. Bitterness is very slightly present, without differences between varieties and extraction methods. Red pepper aroma is more expressed in the P1 variety, significantly between extraction methods for the P1 variety (
p < 0.05). Nutty flavour is almost equally present in both varieties and extraction methods. Spiciness is more present in the P1-CP sample, but differences are not significant. There is only a slightly present waxy taste. Viscosity is typical of oils. Mouthcoating is also present to some extent, but mildly; however, there is a difference between extraction methods for the P1 variety (
p < 0.05). A common characteristic of different seed oils is mild nutty flavour and spiciness, as shown in Aydeniz et al. [
38]; in this study, those characteristics are more strongly expressed for CP oil samples. Despite the differences in the type of pepper seed oil samples, the negative attributes like bitterness of the SC-CO
2 oil samples were similar in intensity and the waxy taste of all oil samples was less expressed, in comparison with Yılmaz et al. [
64]. Multifactor PCA analysis (
Figure 3) shows that PC1 participates with 62.23%, and PC2 24.35% in the variability of samples. The sample P1-CP is characterized by a red pepper odour and aroma, and intense colour. P2-CP is characterized by turbidity and waxiness. P1-SC-CO
2 is characterized by a nutty odour, viscosity, and bitterness. P2-SC-CO
2 is characterized by a nutty flavour and mouthcoating.
The obtained results from this study also show that oils produced by cold pressing have better acceptability (
Table 9) than samples produced by SC-CO
2. The variety of pepper seed oil Podravka (P1) has significantly better acceptability than the variety of pepper seed oil Slavonka P2 obtained by both techniques (
p < 0.05). In comparison with Yılmaz et al. [
64], hedonic test results of P1-CP showed high acceptance (8.1), which indicates a pepper oil with high potential for commercial use.
According to the sensory analysis, it can be concluded that the Podravka variety (P1) of pepper seed oil obtained by cold pressing has the most intense sensory profile, due to the darker orange colour, more intense red pepper odour and aroma, and pleasant spiciness, and it is the most acceptable according to the consumer acceptability test.