To give the actual frame of nuts and seeds consumed in the population and to compare their nutrient profile, in this study, commercial samples of nuts and seeds are analyzed and discussed regarding their position in the diet. Reference Intakes (RIs) and Nutrient Reference Values (NRVs) that were used for the calculation of the contribution of macronutrients and minerals to daily intake, respectively, were established according to European Union regulation on the provision of food information to consumers and are considered as guidelines for consumers on approximate amounts of macronutrients, micronutrients, and energy required by an average adult person for a healthy diet (20). The expression of macronutrient content in one portion of nuts and seeds as a percent of reference intakes (% RI) for macronutrients and as a percent of nutrient reference values (% NRV) for minerals could be obtained more precise insight into the significance of certain nuts and seeds in nutrition. Namely, literature data point out that the beneficial effects of consuming nuts are dose-dependent and usually are expressed as benefits that could be achieved by consuming an extra daily portion of nuts or by increasing the frequency of their consumption. Still, there are suggestions that this dose-dependency is probably curve-shaped and that at some point, such increased consumption could result in increased intake of fats, and consequently in increased body weight, since nuts are considered high-energy-dense foods, primarily because of fats high content (2).

Furthermore, though certain FBDG-recommend nuts and seeds as a part of a healthy dietary pattern, the health effects of seeds are not as comprehensively investigated as nuts. Namely, investigations concerning the health effects of seeds alone or in combination with nuts are substantially scarce. These investigations do not refer to seeds’ oil since oils are classified as a separate group of food in the majority of guidelines and represent just one of many nutrients in seeds, which all together contribute to the beneficial effects of seeds in diet (2). Results of the meta-analysis, which included 28 randomized studies (RCTs), showed that dietary intervention with flaxseed, but not flaxseed oil, decreases total and LDL cholesterol, while another meta-analysis of 11 RCTs also showed a more significant effect of flaxseed compared to flaxseed oil on lowering of systolic and diastolic blood pressure (2, 22, 23). Moreover, because of the limited ability of some dietary assessment methods to evaluate the intake of seeds as a part of commonly used food (muesli, granola, cereals, and different types of bread) or lack of information on the nutritional compositions of seeds in nutrient databases, intake of seeds in the nutritional analysis is sometimes neglected. Thus, there are certain doubts that the presence of seeds in food partially influences health effects attributed to nuts, so it is necessary to revise the current opinion on nuts to include seeds, which would contribute significantly to planning future investigations and new dietary guidelines (2). Also, as a consequence of an insufficient number of studies concerning seeds exclusively, there are no health statements referring to seeds.

Results of our analysis of fats and fatty acid content (Table 1) are in agreement with data from the USDA National Nutrient Database (24) except for walnut with twofold lower saturated fatty acid (SFA) content (3.4 g/100 g vs 6.1 g/100 g) (Supplementary Table 1). Based on the presented data, it is clear that for some representatives of nuts and seeds, above all for those with low fat and SFA content such as walnut, almonds, hazelnuts, chia, and sunflower seeds, an additional daily portion could be recommended. For some investigated nuts, such as brazil nuts, cashew nuts, and peanuts, an additional portion could contribute to excessive intake of SFAs, especially in combination with other foodstuffs that are also a significant source of SFAs (meat, dairy products) and are necessary for a well-balanced and healthy diet.

Omega-3 fatty acids possess a wide range of potential health effects, including regulation of inflammation and antioxidant signaling pathways, thus providing cardioprotective effects (25). Long-chained omega-3 fatty acids (FAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which could be, to a certain extent, biotransformed from ALA, found only in plant-based foods, are vital for growth, cognition, and immune system function (26). Fish are the most important source of these fatty acids, but in populations that have an insufficient fish intake in their diet, there is a risk of low dietary intake of these FAs. That is why including alternative nutritional sources of omega-3 FAs, such as various seeds and walnuts in the first place, is of great importance. A study comparing the effects of consuming 30 g of raw sunflower seed and the same amount of almonds in postmenopausal women with type 2 diabetes showed that both interventions lowered total and LDL cholesterol for 3 weeks. However, intervention including sunflower seeds showed more pronounced effects on decreasing triglycerides, HDL, apoA-1, and B-100, which could be explained by higher polyunsaturated FA (PUFA) content in sunflower seeds (27). According to EU regulations on the application of nutrition and health claims, all foodstuffs containing more than 0.3 g ALA/100 g and 0.6 g ALA/100 g could be labeled as “sources” and “rich sources” of omega-3 fatty acids, respectively (28). This is in line with these definitions and based on the results of the present investigation shown in Supplementary Table 1. Regarding the content of ALA, it could be concluded that sesame and black sesame seed should be considered sources of omega-3 FA, while walnut, chia, flax, and hemp seeds could be labeled as rich sources of omega-3 FA.

Our results also highlight that combining nuts rich primarily in monounsaturated fatty acids (MUFA) with seeds that are sources of PUFAs, mainly essential fatty acids LA and ALA, could increase the proportion of PUFA and significantly fulfill recommended intake of essential FA. Moreover, combining nuts and seeds could provide a satisfactory effect on blood lipid profile because of the different mechanisms of hypocholesterolemic action, most likely due to the different profiles of MUFAs and PUFAs between them (2). The total unsaturated fatty acids/saturated fatty acids (UFA/SFA) ratio presented in Table 1 showed significant differences between investigated nuts and seeds: a higher ratio was generally observed in nuts and they ranged from 3.4 (brazil nuts) to 17.5 (walnuts). In contrast, in seeds, it ranged from 4.3 (pumpkin seed) up to 10.4 (raw sunflower seeds and flax seeds). Results obtained in our work were only partly in agreement with the results of other authors. For example, UFA/SFA ratios in our experiments for hazelnuts were 1.5-fold higher (12.5 vs 8.47), while for pumpkin seed, it was twofold lower (4.3 vs 8.6) compared with the study of Kirbaslar et al. (29). The discrepancy with literature data can be explained by the fact that the composition of nuts and seeds is determined by a large number of factors, including plant variety, degree of ripening, agricultural practice, climatic conditions as well as storage conditions (14). This UFA/SFA ratio was also used previously in some investigations (29, 30), permitting a better understanding of the fatty acid profile of nuts and seeds and also being important as a predictive factor for shelf life (29). Based on data obtained in our research, a lower UFA/SFA ratio for seeds implicates their longer shelf life under the same conditions as nuts. It was also interesting to observe significant differences between this ratio’s values concerning raw and roasted sunflower seeds, which is probably influenced by different origins of raw materials for these commercial samples.

As for phytosterol content in analyzed nuts and seeds, our results are, in some cases, considerably different compared to literature data that could be explained by other analytical procedures, sample preparation, and above all, by the different origins of the samples tested. Nevertheless, β-sitosterol was also reported as the main phytosterol in nuts and seeds by many other authors (31, 32). In samples of nuts and seeds usually consumed in the United States, Phillips et al. (32) reported the highest amount of phytosterols in sesame seeds (400–413 mg/100 g) and the lowest in brazil nuts (95 mg/100 g). In our investigation, sesame and black sesame seed were also the most important source of phytosterols (489.6–505.5 mg/100 g, Supplementary Table 2), with the lowest phytosterol content in roasted cashew nut, boiled peanut, and walnut (79.3, 106.5, and 108.4 mg/100 g, respectively). As for phytosterols in brazil nuts, a sample from our investigation contained 132.9 mg/100 g, which is higher than in the respective research from the United States. Phytosterols are well-known for their cholesterol-lowering effects through competitive mechanisms with biliary and dietary cholesterol. It was shown that daily intake of 2–3 g of phytosterols decreases LDL cholesterol by 10–15% (31), while EFSA had authorized two health claims. The first points out “the beneficial effect of 0,8 g phytosterols and stanols on maintenance of normal blood cholesterol concentrations,” while the second emphasizes the “hypocholesterolemic effect of 1.5–2.4 g phytosterol daily intake” (28). Content of phytosterols in recommended daily portion (28 g) of nuts and seeds obtained in our study (Table 2) revealed that maximal intake of sterols would still be significantly lower (ca. 0,1 g) compared with amounts necessary to acquire the abovementioned beneficial effects. Also, though phytosterol content in nuts and seeds obtained in our study is substantially lower compared to those with proven beneficial physiological effects and compared with amounts obtained from plant oils, their positive effects on health in combination with other biologically active constituents from nuts and seeds should not be neglected.

According to the USDA Guideline for Americans, one ounce of nuts and seeds (about 28 g) can meet one-third of the adult protein needs for men and two-fifth for women (5, 6), based on data presented as a percentage of recommended intake (% RI) for proteins (Table 3) it is evident that this contribution from our samples is twofold lower. Analyzed samples have somewhat lower % EAAR in total proteins (Table 3) compared to some literature data for nuts (33), with the main limiting AA being methionine and lysine, while other authors (33, 34) report lysine as the main limiting AA in nut samples from the United States and Italy, respectively.

Still, besides protein content, an important parameter is the protein’s biological value, which is determined by EAAs content. The content of EAAs methionine is 2–4 fold lower in nut proteins compared to animal proteins, but this deficiency could be beneficial. Namely, available studies with restriction in methionine intake showed a decrease in mitochondrial production of reactive oxygen species and mitochondrial DNA mutations that at least partly could be responsible for the age-delaying effect observed in an animal model of methionine deficit diet (14). On the other hand, an animal model methionine fortified diet caused neurotoxic, including oxidative stress, inflammation, increased levels of β-amyloid peptide, and memory impairment (14, 35). Nuts and seeds are a rich source of non-essential AAs, especially arginine, asparagine, and glutamine (Supplementary Table 4). Arginine is nitric oxide (NO) precursor, which is a potent vasodilatory agent responsible for the regulation of vascular function and blood pressure (36), and for increased sensitivity in diabetic patients (37).

The low lysine/arginine ratio observed in nuts could have beneficial effects on preventing diabetes development (38). Furthermore, the lower lysine/arginine ratio in nuts and seeds’ proteins compared to animal proteins is linked to a significantly lower risk of hypercholesterolemia and atherosclerosis and, consequently, cardiovascular diseases (14). According to the obtained data (Supplementary Table 4), the lowest lysine/arginine ratio is in almonds, hazelnuts, walnut, and brazil nuts (0.20), while it is several times higher in pistachios (ca. 0.57) and cashew nut (0.45). In seeds, this ratio is ranged between 0.30 (roasted sunflower seed) and 0.56 (hemp seed).

Our study showed that nuts and seeds contain all amino acids necessary in the human diet and are unvarying considering total protein content, with somewhat higher values for EAAs in entire protein content within seeds (Supplementary Table 4). Differences among analyzed samples of nuts and seeds consider limiting AA, pointing out once again the importance of seeds within a well-balanced diet, first of all, the importance of their combining with nuts to provide adequate intake of all EAAs, especially within plant-based dietary regimes.

Mineral content in nuts and seeds per one recommended portion is expressed as a percentage of contribution to NRV (% NRV). These values are established to declare foodstuffs and as guidance level for consumers on daily intake of micronutrients for the average adult to prevent deficiencies (20), allowing the perceiving role of certain nuts and seeds in obtaining daily needs from minerals. Generally, nuts are considered one of the most important sources of magnesium in the diet; thus, the beneficial cardiometabolic effects of nuts are partially attributed to their magnesium content (39, 40). Several publications and meta-analyses showed an inverse association between magnesium intake and cardiovascular disease risk (41). Based on data from these investigations, it could be concluded that seeds are generally a more significant source of magnesium and, on average, contain twofold higher magnesium content than nuts.

Almonds, brazil nuts, and hazelnuts from our study were recognized as the richest sources of calcium, in accordance with some previous results of other authors (1, 29). The calcium content in almond samples from our study was twofold higher than the results mentioned earlier but in agreement with data from USDA Nutrient Database for Standard Reference (24). It could also be observed that seeds may be considered a more significant source of calcium than nuts (Table 4). It is also important to emphasize that in some nuts and seed samples (Supplementary Table 5) calcium content was higher than in dairy products, ranging from 120 to 800 mg/100 g, depending on the type of dairy product (42). It should be kept in mind that total mineral content is not the only parameter for defining some foodstuff as its’ rich source, but bioavailability should also be considered. That is the case with plant sources of calcium characterized by low bioavailability (42). Also, based on investigations performed by Siliburska and colleagues in which the bioaccessibility of minerals from different plant sources, including nuts, was assessed using in vitro enzyme digestion model, the highest bioaccessibility of calcium was reported in brazil nuts. It was substantially higher than other nuts analyzed (hazelnut, walnut, and cashew nut) (43). Moreda-Pineiro and colleagues, using in vitro dialyzability method to assess the bioavailability ratios of essential and toxic elements, showed that bioavailability and metal dialyzability varies among nuts and seed, which could be explained by their different chemical constituent, as well as by the negative influence of fat content (44). These authors showed that the highest bioavailability of calcium was from pistachios, while other examined minerals had better bioavailability from raw and roasted hazelnuts. On the contrary, for the majority of examined minerals, the lowest bioavailability was observed in walnut. This should be especially considered in individuals with intolerance/allergy to dairy products or on a vegan diet regime, whose only dietary sources of calcium are plant-based foodstuffs.

It was also shown that some representatives of nuts and seeds could provide up to one-fourth of daily needs for iron in one recommended portion (i.e., raw almonds and hemp seeds, 21.4 and 30.4% NRV, respectively), but in general, iron absorption is very low from plant food, that is when the iron is present in the non-hem form. Plant food, such as nuts and seeds, contain phytates, tannins, and fibers that significantly inhibit iron absorption, causing low bioavailability (44). In any case, regular consumption of nuts and seeds, especially almonds and pumpkin seeds, could contribute to the daily need for iron in persons who follow a vegan diet regime.

Nuts and seeds could be considered important sources of dietary zinc, which could be significant when planning dietary interventions for the population with zinc deficiencies. Results of previous studies conducted on the Serbian population showed that zinc is insufficiently present in the soil, water, and food and that serum zinc levels in the Serbian population are twofold lower compared to some other examined populations (45). Kafaoglu and colleagues, using in vitro intestinal method to establish bio element concentration dissolved in gastric and intestinal solution, have shown that the highest zinc bioavailability was obtained from hazelnuts and pistachios (46). All this implies that despite lower zinc content in nuts and seeds compared to other minerals, its content could be compensated by its higher bioavailability, and nuts and seeds could be recommended as an additional source of zinc in nutrition. Almost all analyzed samples could be considered significant copper and manganese sources (except boiled almonds and pistachios).

Observed differences in mineral content between raw and thermally treated samples of the same nuts and seeds could be explained in one part with the processing of the sample. For example, the lower content of some minerals in boiled peanuts is explained by the leaching of the mineral in water during the cooking process (47). Some data suggest that roasting could decrease the bioavailability of minerals since the structure of foodstuff and nutrients is changed so that digestive enzymes’ actions, and consequently bioaccessibility of minerals, are reduced (48).

Content of dietary fibers in commercial samples of nuts and seeds is mostly in agreement with United States Nutrient Database (24), except for chia seeds characterized by substantially higher fibers content (37.7 vs. 27.3 g/100 g) and pumpkin seeds with lower fibers content (3.9 vs. 6.5 g/100 g) (Supplementary Table 6). In general, nuts are considered one of the most important sources of dietary fibers, next to wholegrain cereals, but seeds seem to be more significant. Actual recommendations on fiber intake necessary for obtaining good health and recommended by various scientific authorities range from 25 to 35 g/day, that is between 10 and 13 g of fibers per 1,000 kcal (49). Accordingly, based on obtained results (Table 5), the fiber content in nuts and seeds could be considered significant and could increase dietary fiber intake. In vitro and in vivo studies showed that non-digestible components of nuts, such as dietary fibers, act as prebiotics that could modulate the gut microbiota profile, thus aiding intestinal homeostasis. Studies on walnut, pistachios, and almonds have shown that consumption over 3–16 weeks increases beneficial intestinal bacteria, especially butyric acid-producing strains. Moreover, intestinal microbiota-positive remodeling potential was found higher for almonds compared to commercially available fructooligosaccharides with previously proven prebiotic effects (3, 50).

In recent years, many authors have explained specific positive effects of nutrients by calculating the ratio of carbohydrates/fibers (51, 52). Fontanelli and colleagues have shown that increased consumption of grains, with a total carbohydrates/fibers ratio ≤ 10:1, influences cardiometabolic risk factors (atherogenic dyslipidemia and insulin resistance) by lowering blood triacylglycerols and fasting insulin (51). In our samples, this ratio was far below 10:1, while in chia, flax, and sesame seeds, it was almost 1:1. This is confirmed by their position in the PCA plot (Figure 4B). Our research is the first to represent carbohydrates/fibers and fibers/sugars ratios in nuts and seeds. Identical explanation of grains effects should be applied during monitoring of nuts and seeds effects considering calculations on carbohydrates/fibers and fibers/sugars ratio.

PCA established the results obtained in our study, and the conclusions were revealed. PCA confirmed nuts as the primary source of oleic acid and 11-eicosenoic acid (Figure 2A) and seeds as an important source of α-linolenic acid (Figure 2B). Also, PCA revealed that nuts and seeds are sources of different amino acids (Figures 1A,B), while some nuts and seeds samples are good sources of calcium and zinc.