Unravelling the nutritional and health benefits of marketable winged termites (Macrotermes spp.) as sustainable food sources in Africa

Termites are widely distributed globally and serve as a valuable food source in many countries. However, information on the myriad nutritional benefits of processed termite products in African markets remain largely unexploited. This study evaluated the phytochemicals, fatty acids, amino acids, minerals, vitamins and proximate composition of the edible winged termites (Macrotermes spp.) from three major Counties of Kenya. A total of 9 flavonoids, 5 alkaloids, and 1 cytokinin were identified. The oil content varied from 33 to 46%, exhibiting significant levels of beneficial omega 3 fatty acids, such as methyl (9Z,12Z,15Z)-octadecatrienoate and methyl (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate, ranging from 82.7–95.1 to 6.3–8.1 µg/g, respectively, across the different regions. Four essential and cereal-limiting amino acids lysine (1.0–1.3 mg/g), methionine (0.08–0.1 mg/g), leucine (0.6–0.9 mg/g) and threonine (0.1–0.2 mg/g), were predominant. Moreover, termites had a rich profile of essential minerals, including iron (70.7–111.8 mg/100 g), zinc (4.4–16.2 mg/100 g) and calcium (33.1–53.0 mg/100 g), as well as vitamins A (2.4–6.4 mg/kg), C (0.6–1.9 mg/kg) and B12 (10.7–17.1 mg/kg). The crude protein (32.2–44.8%) and fat (41.2–49.1%) contents of termites from the various Counties was notably high. These findings demonstrated the promising nutrients potential of winged termites and advocate for their sustainable utilization in contemporary efficacious functional food applications to combat malnutrition.

dense nutritional profiles featuring high proteins, amino acids, healthy fatty acids, vitamins, phytosterols and essential minerals 13 .
Termites are considered a customary delicacy in Africa including cultures of the western Kenya.A typical colony comprises nymphs, workers, queen, and soldiers but the most commonly consumed form is the mature winged alates 14 .In Kenya, several edible termite species have been reported such as Macrotermes nigeriensis, Pseudacanthotermes militaris (Hagen), Macrotermes bellicosus (Smeathman), Macrotermes subhylanus (Rambur), Pseudacanthotermes spiniger (Sjostedt) and the others such as Neotermes spp., Coptotermes spp., and Cryptotermes spp.They plentifully emerge during the first rains marking the end of the dry periods in the early mornings, evenings or upon attraction to light at night, during which residents avidly collect them for food 15,16 .Traditionally, the residents capitalized on the nocturnal nature of the termites by setting bowls of water below light sources as traps 17 .Moreover, the villagers have mastered the art of inducing the emergence of these insects even during the dry seasons to satisfy the outstanding market demand and sustain the established enterprise 15 .Commonly embraced processing techniques for termites marketed in many communities include de-winging, blanching, sun-drying, roasting, frying, toasting and salting, or grinding into powder 9,17 .
The nutritional quality, protein digestibility and mineral bioavailability of termites have been well documented 9,15 .In many countries, consumption of termites is driven by their health and nutritional benefits, which make them highly recommendable for breastfeeding mothers and children under the age of 5 years due to richness in protein and iron 9 .Thus, they are considered as excellent candidates for alleviating malnutrition and micronutrient deficiency in many developing countries 9 .Previously, antimicrobial peptides have been isolated from Pseudocanthotermes spiniger, Reticulitermes spp.and Odontotermes formosanus Shiraki 18 .Similarly, terpenes have been extracted from Nasutitermes spp.and Prorhinotermes spp., while alcohol extracts of Macrotermes estherae Desneux and Microtermes obes Holmgren have been shown to express antimicrobial activities 18 .Despite their established use in traditional medicine for treating myriad of medical conditions, a comprehensive understanding of termites' bioactive components remain elusive 11 .Additionally, the chemical composition of termites exhibits substantial inter-and intra-species variations, influenced by factors such as species identity, environmental conditions, geographical location, feeding substrate, and the developmental life stages of the insects 16 .In light of this knowledge gap, this study aimed to investigate, document the diverse phytochemicals, dietary value and potential health benefits associated with winged termites (Macrotermes spp.) in three Counties of Western Kenya.Elucidating this information can contribute to promoting informed consumer awareness and demystifying the long-standing yet poorly understood nutritional and potentially therapeutic properties that have underpinned human utilization of termites throughout history.

Phytochemical contents
A total of 9 flavonoids, 5 alkaloids and 1 cytokinin were identified from the termites obtained from the three Counties (Table 1).Significant variations (p < 0.05) in the flavonoid contents (except luteolin), alkaloids and cytokinin were evident from the termites sourced from the different locations.The levels of flavonoids; rutin, quercetin, kaempferol, 4-hydroxybenzoic acid, naringenin, alkaloids; solanine, tomatidine, solanidine, solasodine and cytokinin; zeatin were significantly higher in samples collected in Kakamega County compared to the other two Counties.Termites sourced from Homa Bay County had 46% oil yield compared to 42% and 33% for Kakamega and Bungoma Counties.There was no significant difference in termite oil yields between Homa Bay and Kakamega Counties samples, though both were significantly higher compared to samples from Bungoma County (F 2,6 = 17.8, p = 0.003) (Fig. 1).

Amino acids profile
A total of 15 amino acids were detected, including 8 essential and 7 non-essential (Table 3).Significant variations (p > 0.05) were observed in the essential amino acids of termites collected from the three sites (Bungoma, Kakamega and Homa Bay).No significant variation was observed in the concentrations of four non-essential amino acids (cystine, glutamic acid, proline and tyrosine).Termites from Homa Bay had higher levels of histidine, isoleucine, leucine, lysine, threonine, and valine.Kakamega termites had higher levels of isoleucine, methionine, and phenylalanine.Termites from Bungoma had relatively lower levels of essential amino acids (except for methionine).Surprisingly, all the samples contained lysine, which is a critical cereal limiting amino acids.www.nature.com/scientificreports/

Mineral composition
Table 4 presents the mineral content of winged termites from the three target sites.Termites from Homa Bay County exhibited significantly higher levels (p < 0.05) of iron, zinc, magnesium, potassium, calcium, and sodium compared to other areas.Likewise, Kakamega termites showed higher concentrations for copper and manganese only.However, lower levels of all the minerals were observed for samples obtained from Bungoma.

Vitamin composition
The vitamin contents of the winged termites significantly varied p > 0.05 as indicated in Table 5. Copious amounts of vitamins A, C, B12, B1, B2, B3 and B6 were detected in termites from Homa Bay County.However, the lowest levels of all the vitamins were recorded from samples sourced from Bungoma.

Proximate composition
There were significant variations (p < 0.05) in the proximate constituents of winged termites collected from Bungoma, Kakamega and Homa Bay as illustrated in Table 6.Kakamega termites expressed higher levels of protein whereas Homa Bay termite samples recorded enhanced levels of ash, fibre, fat and carbohydrate contents.

Discussion
The discovery of the inhibitive and protective properties of various therapeutic compounds in insects has generated great interest in the scientific community.The exploration of insects for human medicine and nutrition has resulted in the identification of numerous bioactive compounds in species such as edible stink bugs, grasshoppers, Amphiacusta annulipes, Zophobas morio, Locusta migratoria, Brachytrupes orientalis, black soldier fly, termites, beetles, desert locusts, caterpillars, and crickets reportedly expressing myriad of bioactive compounds including peptides, isoflavonoids, alkaloids, flavonoids, anthraquinones, tannins, phlobatannins, sterols, triterpenoids and cyanogenic glycosides [19][20][21] .
Winged termites assessed in this study are no exception, manifesting flavonoids, alkaloids and a cytokinin.Since most of these insects are herbivorous, polyphagous and detritivores, they sequestrate these compounds from plants for subsequent modification into protective metabolites 22 .The identification of the flavonoids, alkaloids and cytokinin from the termites links to their acquisition from the plant materials they feed on.
A study conducted by Ameka et al. 23 discovered that termites are most attracted to forage on the tree species; Eucalyptus and Grevillea which reportedly exhibit high quantities of flavonoids, alkaloids, tannins, saponins, resins, glycosides and phenols and higher contents of alkaloids, tannins, resins and phenols respectively.Since, the presence of such trees have extensively been documented in Western Kenya 24 , it is plausible that the termites derived the chemicals from them.The notable higher levels of the flavonoids (rutin, quercetin, kaempferol, 4-hydroxybenzoic acid, naringenin), alkaloids (solanine, tomatidine, solanidine, solasodine) and cytokinin (zeatin) in samples from Kakamega may be attributed to the rich diversity of indigenous tropical forest tree species known for high quantities of phytochemicals.The variation in the phytochemical composition of the termites from the different sites may be due to availability and distribution of the specific plant species in those sites and their concentration of such bioactive compounds, as it has been established that dietary sources directly influence the chemical composition of insects 23 .However, the species effect cannot be ruled out in this context since the termites were not specifically identified and therefore Kakamega-based termites may possess genetic adaptations condusive to a heightened sequestration of phytochemicals.This assertion warrants more investigations for scientific clarity.
Flavonoids in foods play a crucial role in human health as antioxidants.The primary sources of flavonoids for humans are fruits and vegetables 25 .These compounds offer a range of benefits, including antioxidant, antiallergic, anti-inflammatory, anti-diabetic, liver and stomach protective, antiviral, and anti-cancer properties, while alkaloids such as solanine in minute amounts have been found to be anti-cancer and anti-tumor with many others reportedly offering extensive pharmacological benefits 26,27 .Therefore, humans can revamp their therapeutic arsenals against inflammatory infections by consuming winged termites.However, further research is necessary to accurately determine the levels and compositions of flavonoids, phenolics, alkaloids, and other bioactive components in these insects.
The current study confirms that winged termites furnish appreciable levels of oil with samples from Homa Bay and Kakamega manifesting richer sources compared to the samples from Bungoma County.This disparity in oil yields is characteristic of differential environmental conditions notable in terms of dietary sources, temperature and light conditions under which the termites lived 9,28,29 .This oil content plays a critical role in the retention and absorption of flavors contributing to its high palatability, acceptability and reduced applicable oil upon frying and roasting 30 .Additional research using different processing methods and other marketable insect species is needed to confirm these findings.
The dominant fatty acids detected were methyl hexadecanoate, methyl (9Z)-octadecenoate (oleic acid) and methyl (9Z,12Z)-octadecadienoate for SFA, MUFA and PUFA respectively, which corroborates the findings from previous studies 9, [30][31][32] .Also, the concentrations of fatty acids ranged 33-40% SFA, 47-63% MUFA and 11.3-14.3%PUFA which are consistent with values 39.35% SFA, 53.07%MUFA and 7.57% PUFA reported by Fombong and Kinyuru 31 and underpins the representation in Fig. 2. The fatty acid spectra of the termites collected from the three sites displayed haphazard variation from each other in terms of quantity.This variation may be attributed to differences in developmental stage, sex, biological role, analytical method employed and environmental conditions such as diet and temperature 16,29 .For instance, it has been established that wild collected insects exhibited enhanced levels of both linolenic and linoleic acids compared to commercially reared, grain-fed insects which revealed high linoleic acid but diminished linolenic acid levels 33,34 .Further, certain dynamics in PUFA concentrations were observed when aquatic larval insects feeding on algal diets, metamorphosized into adults, exclusively feeding on terrestrial diets 29 .Insects are metabolically capable of synthesizing SFAs and MUFAs de novo however, they acquire the PUFAs directly from their dietary sources 29 , thus explaining this typical phenomenon.
Termites from the three sites are endowed with PUFAs, particularly, omega 6 methyl (9Z,12Z)-octadecadienoate, methyl (7Z,10Z)-hexadecadienoate, methyl (5Z,8Z,11Z,14Z)-eicosatetraenoate, omega-3 methyl (9Z,12Z,15Z)-octadecatrienoate (α-Linolenic acid), methyl (5Z,8Z,11Z,14Z,17Z)-eicosapentaenoate (EPA), omega-5 methyl (9Z,11E,13E)-octadecatrienoate.The detection of omega-3 fatty acid, EPA, in the present study, that was undetected in previous studies 31,32 , could possibly be reflective of the sensitivities of our extraction and analytical processes used in the various experiments.Omega-3 fatty acids, ALA and EPA have been revealed to avert conditions such as cancer, mental illness, bowel disease, high blood pressure, schizophrenia, cystic fibrosis, and Alzheimer's disease as well as treatment of skin infections and Crohn's disease 35,36 .Omega-6 linoleic acids is known to render anticarcinogenic, anti-obesity, antihypertensive and antidiabetic properties to humans 37 .The most abundant MUFA, oleic acid, has been reported to confer anti-cancer and anti-inflammatory properties, and suppression of cardiovascular diseases 38 .The termites also featured a ∑(n-6)/∑(n-3) ratio of 4.7-5.3which complies with World Health Organization (WHO) recommended daily intake of 5:1 38 .A higher index parallels a significant increase in cardiovascular diseases, inflammatory and chronic diseases.The termites are also noteworthy of UFA/SFA ratios of 0.5-0.9 that agrees with the reported ratio of 0.65 31 and align with the ratios of 0.43-0.79 in other insects 13 .These ratios imply that termites are sources of highly desirable unsaturated fatty acids, concomitant with high serum high density lipoprotein (HDL) and low risk of atherosclerotic disorders 30 .This unique property has valorized termites as rich food sources in light of their nutritional and pharmacological properties outweighing the negative perception formerly ingrained in the society 11 .
Overall, histidine, lysine and leucine were the most abundant indispensable amino acids, corroborating the observations made by Mabossy-Mobouna et al. 39 on Macrotermes bellicosus while arginine was the predominant dispensable amino acid.Notably, the termites exhibited all the essential amino acids except for tryptophan which has not been detected in previous studies done on related species 39 thereby portraying termites as complete and balanced rich food source.Histidine, lysine and leucine are critical amino acids for growth 40 whereas arginine has been reported to enhance calcium absorption 41 .The lower levels of methionine and cysteine certifies the findings of Mabossy-Mobouna et al. 39 on M. bellicosus and further underpins scientific assertions labelling the two sulfur amino acids as the limiting amino acids in most insect species 29 .Additionally, the presence of high lysine qualifies the winged termites for utilization as a complement in the enrichment of lysine-deficient cereal and low protein diets.
Statistical differences were witnessed in the levels of the essential amino acids and certain non-essential amino acids thus endorsing the possible influence of different feed and ecological conditions on amino acids spectra as previously discovered in Vespa spp., Apis spp.and Rhynchophorus spp. 13,42.Moreover, common variations have also been reported between life stages of completely metamorphosized insect species 43 possibly due to dissimilarities on the life stages used.However, for majority of insects, the amino acid contents remain fairly constant amidst changes in seasons, geographical areas 42 , and diet or life cycle 29 .
The set of minerals detected in this study mirrors those previously reported in termites 42 .Significant variations of individual minerals in the termites from the three regions may be due to the type of feed, biological role and accumulation of antinutrients in the guts 29 .Specifically, since the termites were collected from different sites, with different ecological and edaphic conditions, the soil types definitely influenced the mineral profiles of the vegetation 44 which includes the decaying plant matter that termites reportedly feed on 45 , owing to their detritivorous nature in their respective localities.Nonetheless, this study reveals that the termites from Bungoma, Kakamega and Homa Bay bears appreciable amounts of Fe, Zn, Mg, K, Ca, Na, Cu and Mn.Realistically, consumption of 100 g of the termites from the three sites would contribute 3.3-5.3% of 1000 mg Ca Recommended Dietary Intake (RDI), 33.3-155.6% of 0.9 mg Cu RDI, 6.7-33.3% of 3 mg Mn RDI, 321.4-508.2% of 22 mg Fe RDI, 31.4-115.7% of 14 mg Zn RDI and 1.6-5.2% of 310 mg Mg RDI 46 .Our results discern very low Ca levels which has been consistently reported in several insects and explicable by the fact of insects lacking mineralized skeleton 29 .Regardless, Ca plays central role in blood clotting, bone and teeth development 47 .
Trace minerals such as Fe, Zn and Cu are reportedly abundant in termites marked by their potentiality to surpass the RDIs indicated above and reinforcing a tendency demonstrated in other studies 10,16 .Fe is essential in energy metabolism, revamping immunity system, cognitive development, and in physical performance 48 .Mg and Zn prevent cardiomyopathy, muscle degeneration, growth retardation, impaired spermatogenesis, immunelogic dysfunction and bleeding disorder 49 .Mg alone has been affirmed to maintain normal muscles and nerve function, keeps heart rhythm steady, support a healthy immune blood and regulate blood sugar levels 14 .Cu is involved in cellular respiration, peptide amidation, neurotransmitter biosynthesis, pigment formation and strengthening of connective tissue 47 .
Considerable levels of vitamins were detected in winged termites from the three sites.Vitamins are essential dietary components involved in crucial cellular functions but in trace amounts.Except for vitamin A, the levels of the other vitamins in this study were inconsistent with the values reported by other authors 30 .Such differences were also observed in this study where higher concentration of vitamins were depicted in termite samples from Homa Bay County compared to Bungoma and Kakamega Counties.The differences may be claimed to emanate from differences in the substrate type 50 habitat and preparation methods 51 .Our results discerned higher vitamin B12 which is in agreement with that reported in a study by Oibiokpa et al. 32 .The low levels of the other B vitamins may be attributable to their susceptibility to degradative processing techniques the termites were subjected to 51 .The B vitamins play essential coenzyme roles in the various biochemical and physiological processes 52 .Vitamin A is pivotal to the performance of the visual system, growth and development, and maintenance of epithelial cellular integrity, immune function, and reproduction.On the other hand, vitamin C is essential for growth, development and repair of cells, and involved in a series of body functions such as collagen formation, iron absorption, improved immunity system, wound healing, and the maintenance of cartilage, bones, and teeth 52 .This richness of termites in essential minerals and vitamins justifies their deserved application in combating micronutrient related undernutrition cases 10,49 by either consuming them in whole forms or incorporating them in other food sources as dietary complement 49 .
The termites from the three sites showed great variability in all the proximate components.The moisture contents ranged between 4.5 and 7.3%, which is slightly lower than that reported by Igwe et al. 30 .This may be attributed to size, maturity and location of collection 53 and maybe the drying method applied 31 .Moisture content of our products from the various sites are within acceptable storage condition, known to play a pertinent role in suppressing the buildup of microbial loads and reduce chemical deterioration of these products hence influencing their shelf-life 54 .The protein content of Kakamega-sourced termites was remarkably higher compared to that reported by Igwe et al. 30 .The dissimilarities in the protein content of insects have been accredited to differences in the composition of different feeds they forage on, the species, stage of development, sex, climatic condition, and geographical location 48 .The reported protein levels in the current study are of superior quantity to conventional foods of animals and plants origin such as beef, chicken, fish, maize and soya bean 14,47 .Therefore, it can be postulated that consumption of 100 g of the termite products would contribute to 48.8-67.9% of the protein Recommended Daily Allowance (RDA) of 0.66 g/kg adult/day 55  www.nature.com/scientificreports/insects' contribution to the RDA of 23-56% 53 .The protein content of termites has been reported to be of high quality and digestibility, therefore proved ideal for alleviation and management of food malnutrition cases 11,15 .
The noticeable variability of fat contents may be ascribed to differences in sex, season of collection, stage of development, habitat and feed either individually or in combination 56,57 .The fat was the predominant component of the termites in this study which confirms previous reports 31 .The fat acts as the main energy source, provide fatty acids profiles, harbors fat soluble compounds like fat soluble vitamins 56 and volatiles 38 .The ash contents of the termites slightly differed from the reported values of 6.2-7.2% 15 and 7.6% 30 .Ash is known to represent the minerals in a food and actively promote metabolism of organic compounds such as carbohydrates and fats 14 .Fibre contents of termites has been purported to be chiefly chitin which comprise approximately10% of whole insects 31 .
In this study, significant differences in the fibre contents were apparent with Homa Bay termite samples exhibiting the highest value.The values were however comparable to 6.2-7.2% in termites 58 .Dietary fibre is lauded for promoting peristaltic movement in the gut during digestion as well as imparting a sense of satiety that translates to reduced fat and energy intake which is ideal for weight control 53 .The chitin also serves as prebiotic known to enhance the establishment of beneficial probiotic bacteria that consequently improve gut health, confers anti-inflammatory 3 , antimicrobial, antitumor and antioxidant properties 57 .Great significant variations in carbohydrates were evident in this study.Generally, it has been revealed that insects are poor sources of carbohydrate, possibly inadequate to even satisfy the adult human requirement of 400-500 g 53 .For this reason, they have been regarded as low carbohydrate-high protein food hence recommended exemplary fit diet for control of cardiovascular diseases and weight gain 48 .Overall, the chemical integrants in the present study manifested remarkable variabilities among the termites from the three sites.Notably, the specific identification of the experimental termites was not undertaken, raising the possibility that these variabilities could stem from species disparities and potential contamination or adulteration, given the known diversity of termite species across different geographical locations 15 .Additionally, development of toolkits for rapid species identification in edible insects should be considered.Therefore, this remains the study limitation that future outlooks of similar nature should take into consideration.

Conclusion
This study sheds light on the nutritional resourcefulness of dried and marketed termite swarmers or "alates" [i.e., winged termites] in Western Kenya.The identification of various flavonoins, alkaloids, and a cytokinin in termites expands our understanding of the diverse array of phytochemicals present in these insects, thereby providing scientific validation for their historical use in local diets and therapeutic practices.This finding offers a strong foundation for further exploration of termites from a pharmacological perspective, emphasizing the importance of extending phytochemical assessments to termites of the same species across different geographical regions to elucidate influence of location on chemical composition.Furthermore, our study highlights termites as rich sources of extractable oils, abundant in monounsaturated and polyunsaturated fatty acids, notably omega-3 and omega-6, which hold promising applications in both culinary and cosmetic industries.Additionally, the high protein content and appreciable levels of essential amino acids (such as histidine, lysine, and leucine) and minerals (including zinc, iron, and calcium) present in termites underscore their potential in formulating modern foods, particularly of plant origin, to address malnutrition in the region.However, the exploitation of termites for food and other resources may be impeded by seasonal availability and their rearing may prove difficult owing to their complexed social structures.Further research directed towards rearing termites on feed-specific diets which guarantees maximum derivable chemicals and nutrients, for mass production of nutrient and phytochemical rich termites is necessary.Also, research involving comparison of the chemical composition of different edible life stages of termites consumed by other communities in Africa should be considered.

Insect obtainment and processing
Representative samples of sun-dried winged termite (Macrotermes spp.) (alates) were purchased from three local markets in Western Kenya namely Bungoma (0.5695° N, 34.5584° E), Kakamega (0.2827° N, 34.7519° E) and Homa Bay (0.6221° S, 34.3310° E).The samples were purchased from ten vendors in each town/market during the swarming period of the termites.From each vendor, termite samples sun-dried within 24 h, collectively weighing 2 kg were obtained.The samples were then stuffed into polyethylene sterile zip lock bags, appropriately labeled and ice-packed in cooler boxes.The samples were then transported to International Center of Insect Physiology and Ecology (icipe) laboratory, Nairobi, Kenya within 24 h after collection and immediately stored at − 80 °C awaiting further processing and analysis.

Phytochemicals analysis
Flavonoids, alkaloids and cytokinin levels in the termite samples were determined according to procedures described by Matsuura and Fett-Neto 27 with slight modifications.Termites ground into powder in liquid nitrogen (1 g) were mixed with 10 mL of 80% methanol (v/v) and vortex-shaken for 1 min and ultra-sonicated for 1 h to enhance phytochemicals extraction.The remaining residues were further re-extracted twice and later pooled together.The extracts were then centrifuged at 4200 rpm for 15 min, the supernatant filtered through a Whatman filter paper No. 32 and the filtrate analyzed by LC-MS.The chromatographic separation was achieved on Agilent 1260 Infinity HPLC system (Agilent Technologies, Palo Alto, CA) coupled to an Agilent 6120 mass detector MS with a single quadrupole analyzer (Agilent Technologies, Palo Alto, CA) using ZORBAX SB-C18, 4.6 × 250 mm, 3.5 μm column, operated at 40 °C.Mobile phases used were made up water (A) and acetonitrile (B) each with 0.01% formic acid.The following gradient system was used: (0.01 min, 5% B; 0.01-5 min, 5% B; 5-10 min, 5

Extraction of winged termite oil
Ground insect samples (50 g) were separately mixed with 250 mL solvent solution (dichloromethane-methanol (2:1 v/v) containing butylated hydroxyl toluene (10 mg/L) to extract the oil, as previously described 59 with some modifications.The mixture was manually shaken for 30 s, sonicated for 1 h, followed by centrifugation (1500 g, 23 °C, 5 min).The supernatant was then collected into a separating funnel mixed with 100 mL of 0.9% NaCl solution, shaken vigorously and allowed to stand until the biphasic system appeared.The upper aqueous phase was discarded.The lower phase was passed through a plug containing anhydrous sodium sulfate into a pre-weighed 500 mL rotary flasks and the solvent removed in vacuo before storing the oil sample at − 20 °C for further analysis.The following equation was used to compute the percentage (%) of the extracted oils (yield):

Determination of fatty acids profile
The fatty acid (FA) compositions of the three termites' oil (100 mg), were converted to the fatty acid methyl esters by adding 1 mL sodium methoxide in dry methanol (15 mg/mL) at 60 °C for 1 h.Thereafter, the reaction was quenched by adding 100 μL of deionized water followed by a 1 min vortexing.The resulting methyl esters were extracted using gas chromatography (GC)-grade hexane (1 mL; Sigma-Aldrich, St. Louis, MO, USA) and centrifuged at 14,000 rpm for 5 min.The supernatant was dried over anhydrous Na 2 SO 4 and analyzed (1.0 μL) by GC-MS (7890A gas chromatograph; Agilent Technologies, Inc., Santa Clara, CA, USA) coupled to a 5975 C mass selective detector (Agilent Technologies, Inc., Santa Clara, CA, USA).The GC was fitted with a (5%-phenyl)methylpolysiloxane (HP5 MS) low bleed capillary column of (30 m × 0.25 mm i.d., 0.25 µm; J&W, Folsom, CA, USA).Helium was the carrier gas, and the column flow rate was 1.25 mL/min.The GC's inlet temperature was maintained at 270 °C, while that of the transfer line was set at 280 °C.The column oven's temperature was preset to rise from 35 to 285 °C, with the former temperature being held for 5 min followed by a 10 °C increase for every minute until the temperature reached 280 °C, where it was maintained for 20.4 min.The mass selective detector was maintained at quadrupole (180 °C) and ion source (230 °C) temperatures.Electron impact (EI) mass spectra was obtained at the acceleration energy of 70 eV with fragment ions being analyzed over 40-550 m/z mass range in the full scan mode, having a solvent delay time set at 3.3 min.The extracting solvent (hexane), blank machine runs was similarly analyzed and their peaks excluded from analysis.Fatty acids were identified via their methyl esters using comparisons of their fragmentation patterns and retention times to those of known fatty acid methyl ester standards where available and their reference spectra published by library-MS databases: National Institute of Standards and Technology (NIST) 05, 08, and 11.Authentic sample of methyl octadecenoate (0.2-125 ng/μL) was subsequently analyzed under the same GC-MS conditions to obtain a linear calibration curve [y = 7E + 06x − 4E + 07; (R 2 = 0.9757)] that was used for external quantification of the fatty acid methyl esters.The data are expressed as µg/mg of detected total analyzed fatty acids.

Amino acids determination
The amino acid composition was determined as previously described 60 .Samples (100 mg) were hydrolyzed with 1.5 mL of 6N HCl at 110 ℃ for 24 h under nitrogen to completion.The hydrolysates were vacuum-evaporated to dryness at 40 °C and subsequently reconstituted in 1 mL of 0.01% formic acid-acetonitrile (95:5) mix, vortexed for 30 s, sonicated for 30 min, and then centrifuged at 14,000 rpm for 15 min.The supernatant was filtered using 0.20 μm membrane filter (Life Science, USA) and analyzed (0.2 μL) by LC-MS with conditions similar to the phytochemicals analysis section with exception of mass scan range of m/z 50-600 and gradient system (0-8 min, 10% B; 8-14 min,10-100% B; 14-19 min, 100% B; 19-21 min, 100-10% B; 21-25 min, 10% B).Mass spectrometric data, retention time, and co-injection of the hydrolysate with an authentic standard amino acid mixture were used to identify the amino acids.Amino acid standard solution (AAS 18) obtained from Sigma-Aldrich (1-105 µg/µL, Chemie GmbH, Munich, Germany) was similarly analyzed by LC-MS and used for external quantification of the amounts of each amino acid present.This was repeated three times using different batch of termite's samples.

Mineral analysis
The winged termite powder (1 g each) was incinerated to ash in a muffle furnace at 550 ℃ overnight.The resulting ashes were digested in 6N HCl and the content of the various minerals determined using atomic absorption spectrometry (Shimadzu, AA-6300, Tokyo, Japan) according to previously published methods 61 .
weight of rotary flask + oil − the weight of the rotary flask (initial weight of the sample) × 100.

Figure 1 .
Figure 1.Termites oil yield in the three study sites.Homa Bay, Kakamega and Bungoma Counties.Error bars indicate the standard error of the mean.Asterisks indicate significant difference between the means: **p < 0.01.

Figure 3 .
Figure 3. (a) Histogram showing the contribution of the 10 most important fatty acids to the discrimination of all the sites.(b) Non-metric multidimensional scaling plot (NMDS) clustering the sites based on the fatty acids that were detected in the winged termites oil using the most important fatty acids (c) Shepard plot showing the great ordination of the NMDS analysis (stress value < 0.2).

Table 1 .
Phytochemical composition (µg/g DM) of termites from three Counties in Kenya.DM dry matter.Mean values with different subscripts along each row differ significantly at p > 0.05.

Table 3 .
Amino acids composition (mg/g DM) of termites.ns not significant.Mean values with different subscripts along each row differ significantly at p > 0.05.

Table 4 .
Mineral content (% Dry matter) of winged termites' powder (mg/100 g).Mean values with different subscripts along each row significantly different at p > 0.05.

Table 5 .
Vitamin content of winged termites' powder (mg/kg).ns not significant.Mean values with different subscripts along each row differ significantly at p > 0.05.