3.1. Proximate Composition
The FD materials had a moisture content of 4.55%, 4.91% and 4.24%, for the green, brown and purple-colored morphs, respectively. The final water content for the green and brown OD samples was 4.33% and 4.50%, respectively.
The purple sample was not included in the statistical analyses, as the sample was too small to obtain significant amounts for both drying methods; as such, only FD purple samples were analyzed.
The changes in moisture content, crude fat, crude protein, chitin fibers and ash are depicted in
Table 3. In general, neither drying method seems to have had any significant effect on the proximate composition of the grasshoppers.
We found that apart from the purple morph, in which the protein, fat and ash content differed, there was little variation (
p > 0.99) in the green and brown grasshoppers across both drying methods. These observations are in agreement with Kinyuru et al. [
13], who reported similar values and found no difference in protein content and only slight variations in other parameters. The overall variation could also have arisen from the insects’ diets since they were captured at different locations and pooled together. Since they were all caught in the adult stage, the stage of development could not have accounted for such variation, but the sex and reproductive state of insects have been shown to affect nutrient composition [
28].
The results for energy content, ash and chitin presented here are consistent with the findings of Rumpold and Schluter [
33], who in a review compiled the proximate composition of 51 orthopterans (see
Table 3) from the literature.
The moisture content for the insect morphs has been reported in other studies to vary between 66% and 71% [
13]. In our case, we obtained average water contents of 53.72 g/100 g fresh weight before drying. Use of different drying times and temperatures would yield different values of moisture for the same species. Adult insects tend to have less moisture than their nymph counterparts, and this trend was evident, as all samples evaluated were adults and had moisture contents lower than expected for grasshoppers [
11]. The low moisture content of the fresh grasshoppers when compared with more conventional meat, such as chicken and beef, would imply a higher content of dry matter and, by extension, this means that
R. differens constitutes a denser source of nutrients, since a relatively larger portion of its weight is represented by nutrients than other animal food sources. The insect samples were dried to moisture contents between 4.0% and 4.5%, a range carefully chosen to extend the shelf storability of the products, as well as to optimize fat extraction using the Soxhlet procedure.
Fat is known to exude with moisture evaporation during oven drying, which increases the effect of lipid and fatty acid losses. This phenomenon has been observed with OD fish in comparison to other preservation methods, as outlined by Chukwu [
20]. However, the drying method had no significant effect on the crude fat content in our case. The absence of fat reduction during oven drying in our study is explained by the lower temperature used (being 60 °C, compared to 110 °C) in the preparation of dried tilapia fish [
20].
Overall,
R. differens showed higher crude fat contents (35.5%) than most orthopterans (13.41%), although this was compensated by lower average protein content (47.75 % for OD and 46.41% for FD) in comparison to several other insects of this order (61.32%) [
33]. In a recent review, data compiled by Aman et al. [
11] reveal that
R. differens has the highest fat and lowest protein content that so far has been reported in the order of orthoptera. The high lipid content of the mean value of
R. differens morphs (35.50%) accounts for the insects’ palatability when fried or roasted, as mentioned by other authors [
34]. This value is lower than the 46.2–48.2% obtained by other researchers for the same species [
13].
The lipid contents were higher than those of chicken, fish and unprocessed milk, but similar to that of raw chicken egg [
35]. When compared to beef or fish, these insects had high lipid contents and are therefore also good energy sources. Indeed, lipids are necessary for food because they increase palatability and retain the flavor of food, as well as enhancing vitamin A, D, E and K levels [
36,
37].
The higher fat content of the FD purple morph compared to the other color morphs explains why the purple grasshoppers are anecdotally considered to be more delicious.
The protein content of most insect species is very high, with many ranging from 60%–85% [
2,
38]. Orthopterans (crickets, grasshoppers and locusts) averaged 60% protein content, the highest among all edible insect orders compared to the isopteran (termites) with just 35% [
33]. The average protein content of
R. differens was found to be 46.41–47.7%, in agreement with similar studies on this species (43.1–44.3%) [
13].
R. differens tends to contain midrange levels of protein relative to other edible insects. According to WHO/FAO [
39], the requirement for food to be labeled ‘high in protein’ is a 10 g/100 g edible portion. This limit, like for most insects, is far exceeded by
Ruspolia proteins, and this edible insect can thus be considered a good protein source.
The observed high protein and fat contents, which comprise more than 75% of the dry mass, justify the cultural perception of high nutritional value attributed to Ruspolia differens.
Insects are known to contain significant amounts of fiber, and Finke [
40] suggested that the fiber in insects is predominantly composed of chitin. Chitin is a major component of the insect cuticle, which is covalently bound to catechol compounds and sclerotin-like proteins [
30]. Chitin is present only in the insects’ exoskeleton and is expected to be present in relatively small amounts. Rumpold and Schlüter [
33] reported 9.55% as the average fiber content for grasshoppers. This fiber value (predominantly chitin) is close to the mean values (13.4% and 11.33%) for either drying method obtained in the current study. As before, no significant differences were observed between morphs, but a slight influence of drying mode was noticed. However, much lower values were reported by Kinyuru et al. [
13] for the same species.
R. differens’ high chitin content might, therefore, present potential value to both the food and pharmaceutical industries [
26].
Average ash content after oven and freeze drying was 4.66% and 4.79%, respectively, which was almost two-fold more than what has been previously reported for the same species (2.7%) [
13]. This difference could be explained by the use of different analytical methods. Higher amounts of minerals were also observed as shown in Table 5, but were nonetheless consistent with the average for 51 orthopterans compiled from the literature [
33]. Higher ash values of 8.55% and 9.36% in grasshoppers have also been mentioned in other studies [
11,
33].
Nitrogen-free extract (NFE) levels, largely representing carbohydrates (but not chitin), are usually low in insects, which explains the dearth of information on the carbohydrate content of insects [
27]. In the current study, NFE levels were low (0.7–1.99%), but were highest in the green morphs (1.39–3.97%). The average NFE values obtained for each drying method (i.e., 0.7% and 2.0% for OD and FD, respectively) were significantly lower than previously reported for most orthoptera, being approximately 13% [
33]. Ramos et al. [
41] obtained similarly low values for NFE in the house cricket,
Acheta domestica. In contrast, extremely high values of up to 63.20% have been reported for the grasshopper
Zonocerus variegatus [
42].
Due to their high fat content, the average energy content (539 and 519 Kcal/g) for this study estimated by calculation [
28] was greater than the mean reported for insects. The above statement is true for other lipid-rich insects, in particular caterpillars, palm weevil larvae and termites [
43]. The values mentioned above obtained for proximate composition in our study were more consistent with those obtained for a related Ugandan species,
Ruspolia nitidula [
18].
When data are expressed on a dry matter basis, the sum of proximates should exceed the expected level of >95 g/100 g edible portion [
44]. This interval was true for the sum of proximates calculated in the current study.
3.2. Fatty Acid Composition
The fatty acid composition of the samples examined is shown in
Table 4 below. These fatty acids are mainly stored in the insect’s fat body and comprise more than 90% of the total lipid content of the fat body [
45]. Very similar values were obtained for all fatty acids irrespective of the drying method.
Oleic (44%), palmitic (28%) and linoleic (14%) acids were the major fatty acid components of
R. differens contributing up to 86% of the total fatty acids present; a trend that was also observed by Kinyuru et al. [
13] for the same species. For FD and OD samples, respectively, the main saturated fatty acids (SFA) were palmitic (C16:0, 28.2% and 27.8%) and stearic acid (C18:0, 7.88% and 8.45%), while the most dominant unsaturated fatty acids (UFA) were oleic (C18:1, 44.3% and 44.0%) and linoleic acid (C18:2, 14.0% and 14.1%). These values were again consistent with other studies [
43,
45,
46,
47].
The present study revealed that the samples studied were rich in polyunsaturated fatty acids (PUFA), especially linolenic and linoleic acids. Previous studies [
13] did not detect lauric, arachidonic or EPA fatty acids in their samples, as they occur in low proportions and are not present in easily quantifiable amounts. PUFA/SFA ratios lower than 0.33 are not desirable as they likely lead to atherogenesis, whereas PUFA/SFA ratios greater than 0.8 are associated with desirable levels of cholesterol and reduced risk of coronary heart diseases [
48]. With a mean PUFA/SFA ratio of 0.44,
R. differens was found to be significantly higher in SFA than PUFA, though still above the cut-off point for triggering undesirable effects.
Another health index associated with fatty acids is that of the omega-6/omega-3 fatty acid ratio (n6:n3), with a ratio of 3:1 considered optimal [
49]. The current study gave an n6:n3 ratio of 9:1 on average, which is regarded as high. Lower ratios of omega-6/omega-3 fatty acids are desirable for reducing the risk for an array of chronic diseases of high occurrence in both developed and developing countries [
48]. Previous research revealed significant variation in n6:n3 ratios, due to omega-3 fatty acid differences [
50], thus confounding comparisons with existing literature. Essential fatty acids (EFA), which include linoleic and alpha-linolenic acids, were present in appreciable quantities (15.42% and 15.60%) for FD and OD samples, respectively.
Finke [
28,
45] concluded that, for a given insect species and developmental stage, the fatty acid composition is affected by environmental factors such as temperature, light, and humidity. These influences may also explain the variations observed in this study.
3.3. Mineral Composition
Table 5 depicts the mineral composition of the
Ruspolia grasshoppers. Again, there were no significant differences (
p > 0.88) between drying methods, although high standard deviations were obtained for the average drying values of some minerals, indicating variability, which has been reported by other researchers, as well [
51,
52], and attributed to a small sample size or contamination. A trend evident in
Table 5 was the higher mineral content in FD relative to OD samples; except for Zn (14.6 and 14.2 mg/100g) and Cu (1.66 mg/100g for both). However, the morph type had a bigger influence than did the drying method (
p > 0.79). The insects were high in most macro minerals, as well as trace minerals; which is true for most edible insects [
28,
42,
53]. With the exception of sodium (Na), the mineral values obtained in this study were higher than values previously reported [
13] for this grasshopper species.
Average calcium (Ca) levels were high, being 895.7 and 1035 mg/100 g dry matter (DM) for OD and FD, respectively; i.e., values well below the recommended daily intake (RDI) for adult humans (1300 mg) [
39,
54]. According to WHO/FAO [
39], foods containing Ca levels above 240 mg/100 g edible portion are considered to be ‘high in calcium’. These results suggest this edible insect could serve as an alternative source of calcium, especially for people who are lactose intolerant or allergic to soy. This is entirely in contrast to statements made by other researchers that insects are low in Ca [
28,
45,
52,
55]. The highest value previously reported for Ca in an edible insect was 2010 mg/100 g in the housefly
Musca domestica (Linnaeus), although some orthopteran species have been found to contain high Ca values (1290 mg/100 g in
Acheta domesticus (Linnaeus)), as well [
33]. This could be attributed to the high Ca content of their gut [
41].
Potassium (K) was the next most abundant mineral (779 and 816 mg/100 g DM for OD and FD, respectively), and although levels were found to be very high compared to those reported for other edible insects [
33], they did not meet the adult RDI of 4700 mg [
54]. These values were less than half those obtained for
Zonocerus variegatus (Linnaeus) (2030 mg) [
56], which are by far the highest recorded K values amongst the grasshopper family.
The Mg content was also elevated at 145 and 161 mg/100 g DM for OD and FD, respectively, compared with previously reported data for
R. differens (33.1–33.9 mg/100g) [
13]. Comparable values for other orthopterans, such as the cricket,
Acheta domesticus, have been reported in the literature [
56]. Adult RDI for Mg lies within 220–260 mg; thus, consuming about 200 g (100 fresh grasshoppers) in a day will surpass the Mg RDI.
Like for most edible insects, the samples evaluated in this study were also found to be high in phosphorus 652.31–685.9 mg/100 g DM for OD and FD, respectively. It has been suggested in the literature that a Ca:P ratio of 1:1 to 1:2 is acceptable in food and feed for most vertebrates [
57]. This ratio incorporates the one obtained in this study (1:1.5). Thus, the inclusion of
R. differens in feed for other animals could balance the Ca:P ratio required for most diets.
The trace minerals were also present in good amounts (
Table 5), particularly Fe and Zn, which concurs with other reports [
8,
53,
58]. Consumption of mineral-rich insects could help mitigate Fe and Zn deficiencies, which are prevalent in developing countries [
53]. That Fe was the most prominent of the trace minerals was also true for Kinyuru et al. [
13], though much higher Fe levels of between 216 and 220 mg/100 g were measured in the current study. The RDI of Fe for female adults stands between 20 and 59 mg, depending on bioavailability [
59]. When compared to the Fe levels present in a variety of red meats (1.1–3.3 mg/100 g) [
60],
R. differens is a superabundant source of Fe, although the bioavailability of Fe is not known.
Very similar zinc (Zn) concentrations (being 14.6 and 14.2 mg/100 g) were observed for the two drying methods, and these were comparable to levels reported previously [
13,
52]. The RDI of Zn for adults is between 4.9 and 7.0 mg (moderate bioavailability) [
59]; as such, either FD or OD preparation of
R. differens would provide a relatively rich source of Zn.
Another trace metal, manganese (Mn) was present, at levels ranging between 7.4 and 8.3 mg/100 g, i.e., levels above the RDI of 1.8–2.6 mg [
59].
Ruspolia was found to be a significant source of selenium in the brown morphs within 40–50 µg/100 g, which exceeds the adult RDI of 26–36 µg.
The samples investigated in this study yielded promising mineral compositions, i.e., levels surpassed those of conventional meats [
60]. The compositional difference observed between different color morphs could be ascribed to environmental factors [
40].
3.4. Amino Acid Composition
Table 6 comprises the amino acid content of the FD grasshoppers; with the amino acids commonly found in proteins being identified in these samples. During acid hydrolysis, asparagine (Asn) and glutamine (Gln) were converted into aspartic acid (Asp) and glutamic acid (Glu), respectively.
No significant difference in amino acid content was observed between the two methods as was to be expected given the similar overall protein content. With the exception of methionine and cysteine, the essential amino acids were present at concentrations that met, and surpassed, the levels recommended for humans by the WHO/FAO. Glutamic acid (+ glutamine), alanine and aspartic acid (+ asparagine) were the most abundant amino acids. Defoliart [
58] showed that insect proteins are low in methionine and cysteine. This was true in the current study, however, the opposing belief that insect proteins are high in threonine and lysine was not observed. Nonetheless, not all insects are the same in their nutritional contributions.
Protein quality and nutritional value are determined by the amino acid composition and digestibility of the protein fraction of foods [
6,
44]. Lysine and threonine are indispensable since they are not transaminated, and their deamination is irreversible. The
R. differens amino acid profile was found to be high in leucine, lysine and threonine. In the predominantly cereal-based diet common in developing nations, lysine and threonine are particularly limiting. Therefore, the inclusion of this insect species into the staple diets of these nations would be expected to significantly improve the nutritional status of their population.
Another crucial parameter for the correct determination of protein quality is the ratio of essential I and nonessential (N) amino acids. According to the FAO/WHO criteria, E/(E + N) should be about 40% with E/N = 0.6 [
54]. FAO/WHO/European food safety authority (EFSA) dietary criteria state that adults should consume 0.66 g/kg of body weight of protein, daily [
61]. In the current study, an E/N of 0.61 and E/(E + N) value of 38% indicate that the amino acid composition of
R. differens satisfies these criteria.