Insight into the chemical and nutritional fat profile of Tenebrio molitor larvae reared on different Agri-food by-products

Insight into the chemical and nutritional fat profile of Tenebrio molitor larvae reared on different Agri-food by-products M. Lourdes Morales a , M. Pilar Segura-Borrego a,* , Jos´ e Raúl Aguilera-Vel´ azquez b , Raquel M. Callej´ on a , Daniel Guti´ errez-Praena c , Cristina Ubeda a a ´ Area de Nutrici´ on y Bromatología, Dpto. Nutrici´ on y Bromatología, Toxicología y Medicina Legal, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain b Dpto. Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain c ´ Area of Toxicology, Dpto. Nutrici´ on y Bromatología, Toxicología y Medicina Legal, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain ARTICLE INFO Keywords: Fatty acid Alperujo Olive leaf Mushroom Health-promoting index ABSTRACT Insects are capable of feeding with a variety of substrates, being possible obtaining products rich in protein using by-products from agri-food industry. Hence, the aim of this work was evaluating the effect of different diets based on three agri-food industry by-products of Tenebrio molitor larvae’s total fat contents and fatty acid profiles. Stabilized Alperujo (ALP), Pleorotus ostreatus spent substrate (POS) and olive leaf flour (OLF) were used mixed with wheat bran, control diet, at different percentages (20–100 %). All substrates studied modified the parameter analysed respect to the control diet. OLF subtract led to the greatest effects on the total fat content at percentage higher than 40 %. Respect to fatty acid profile, the most relevant changes were the increases of total MUFA content in samples from all T. molitor larvae fed with ALP and in most of those fed with POS, due to the specific increases of oleic acid content. On the contrary, total PUFA content decreased in most cases except for OLF substrate when the addition percentages were lower than 100 %. Moreover, OLF substrate enhanced the presence of ω -3. Finally, according to European regulations on nutritional claims, this fat fraction could be labelled as “source of ω -3”, “high in monounsaturated fat” and “high in unsaturated fats”. We can therefore conclude that feeding T. molitor with these agri-food by-products, especially from olive oil industry, enhanced the fatty acids profile of their fat fraction. 1. Introduction Tenebrio molitor larvae have been authorized as human food by the European Food Safety Authority (EFSA) (European Commission, 2021). These insects are a great source of emergent proteins considering that their contents may range between 48.8 and 68.9 % of dry weight and may provide essential amino acids, highlighting the content of leucine, valine, or isoleucine (Mu˜ noz-Seijas et al., 2024). Thus, their inclusion in the human diet can help reducing the greenhouse effect and the water deficits, thanks to the decrease of the meat protein production and due to their low water consumption. The development cycle of T. molitor consists of 4 distinct life stages: egg, larva, pupa and adult. The cycle begins with the laying of eggs by the female. The eggs hatch in 4 days at temperatures of 26–30 ◦C. The larval stage has an average duration of 112 to 203 days, depending on growth conditions. The pupal stage lasts between 6 and 20 days, after which adults emerge as white and soft beetles, gradually hardening and darkening their exoskeleton. Mating and egg-laying begin approximately 3 days after emergence, having the adults an average lifespan of 32 to 62 days (Ribeiro et al., 2018). T. molitor have showed the ability to ingest different kind of diet. Therefore, various rearing substrates have been studied, including vegetables discarded due to quality standards (L´ opez-G´ amez et al., 2024), as well as corn stover, soybean meal, and distillers’ grain byproducts (Zhang et al., 2019), among other. They are even capable of degrading different types of plastics (Brandon et al., 2018; Tsochatzis et al., 2021). Hence, the production in a high scale mode for human nutrition (food human supply) could enable to produce “food rich in protein” and the agri-food wastes exploitation simultaneously. On one hand, olive oil production generates large quantities of wastes, including leaves, alperujo, wash water, among others. Alperujo is a mixture of vegetation waters or alpechines; solid parts of the olive, such as the * Corresponding author. E-mail address: [email protected] (M.P. Segura-Borrego). Contents lists available at ScienceDirect Food Research International journal homepage: www.elsevier.com/locate/foodres https://doi.org/10.1016/j.foodres.2025.116223 Received 19 November 2024; Received in revised form 6 February 2025; Accepted 11 March 2025 Food Research International 209 (2025) 116223 Available online 19 March 2025 0963-9969/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/bync-nd/4.0/ ). stone, mesocarp and skin; and fatty remains, produced during oil extraction. This waste is highly polluting, so a way is being sought to take advantage of them and reduce their accumulation. This problem affect especially to Spain, as it is the world’s leading country in terms of olive oil production and the amount of land devoted to olive cultivation. Moreover, according to FAO (FAO (Food and Agriculture Organization of the United Nations, 2025), global mushroom production in 2023 was 50.01 million tons. In cultivation of fungi it is produced as waste the exhausted substrate. Thus, in parallel with the increase in mushroom production for human consumption, the quantity of this waste has also augmented. Therefore, if these residues could be used to feed T. molitor it would be a good alternative to reduce them. Thus, within the framework of the circular economy, the use of by-products or wastes from the agrifood sector as a substrate for the diet of T. molitor is a promising option. On the other hand, the substrates used in the rearing of T. molitor are important since changes were observed in proximal composition of larvae. Thus, the use of vegetable waste (L´ opez-G´ amez et al., 2024), corn stover, soybean meal, and distillers’ grains by products (Zhang et al., 2019) or Moringa oleifera leaves (Kotsou et al., 2023) produced, in most cases, significant increases of total protein contents of larvae, as well as decreases in ash content compared to the control. Moreover, insect fat content and fatty acid profile are affected by the rearing substrate (Pinotti et al., 2019). In general, T. molitor larvae can have total fat contents that can range between 19.12 and 50.15 % of dry weight (Huang et al., 2025). The most abundant fatty acids in the larvae of these insects are oleic acid, followed by linoleic acid and palmitic acid. If the fatty acids are grouped according to the number of unsaturations, in descending order, the contents of monounsaturated fatty acids (MUFA) are the highest, followed by polyunsaturated (PUFA) and, finally saturated (SFA) (dos Santos Aguilar, 2021). The amount and the type of fats (saturated, monounsaturated or polyunsaturated) that human ingest have a relevant effect on their health. Besides, the intake of high total SFA levels, and concretely myristic, palmitic, and stearic acids, are linked to an increased cancer risk (Mei et al., 2024). Scientific evidence has led to the World Health Organization (WHO) to update its guidance on total fat and saturated fat consumption. Hence, WHO reaffirms that adults should limit total fat intake to 30 % of total energy intake or less with a primarily composition of unsaturated fatty acids, with no more than 10 % of total energy intake coming from saturated fatty acids (WHO, 2023). Moreover, PUFA ingest recommendations should range 6–11 % of total energy intake (FAO (Food and Agriculture Organization of the United Nations), 2010). Consequently, it is not only important to explore new protein sources but also to ensure they offer a healthier fat profile, as is the case with insect-based proteins, which are known for their high nutritional value. Thus, the aims of this work were to study the effect of different agri-food by-products as rearing substrate on the fat content and fatty acid profile of T. molitor larvae assessing the potential relevance of each of the fats obtained in different “health statements”. For this purpose, T. molitor’s diets based on three kinds of agri-food by-products at different percentages were studied. 2. Materials and methods 2.1. Chemicals Hexane and methanol were supplied by Merck (Darmstadt, Germany), and hydrochloric acid 37 % v/v was supplied by PanReac (Barcelona, Spain). Two internal standards were used nonadecanoic acid from Sigma-Aldrich (Merck, Darmstadt, Germany) to test the transesterification reaction (IST) and 2-phenylethanol provided by Merck (Darmstadt, Germany) as IS of injection. To quantification were used the following standards commercial mixture: 37 component FAME mix (CRM47885) and FAME mix C8-C24 (CRM-18918) from Supelco (Merck, Darmstadt, Germany). 2.2. Collection and treatment of feeding substrates Three different types of substrates based on agri-food by-products has been used in this study: stabilized alperujo (ALP), mushroom (Pleorotus ostreatus) spent substrate (POS), and olive leafs flour (OLF). Mushroom (Pleorotus ostreatus) spent substrate was provided by Setacor company (Villafranca de C´ ordoba, C´ ordoba, Spain) and did not undergo any pretreatment. Alperujo and olive leafs were provided by the Agricultural Cooperative “Nuestra Se˜ nora de los ´ Angeles” (Montellano, Sevilla, Spain). To obtain olive leaves flour (OLF), the leaves were pulverized. The alperujo, a wet by-product from the extraction of oil, was obtained in December 2022. To stabilize the material, fresh alperujo was subjected to a drying process at 80 ◦C for approximately 43 ±4 h. This procedure was conducted using an air-drying oven (Memmert UF260). Following the drying step, the dry alperujo was finely ground using a Retsch SM 100 mill fitted with a 750 μ m sieve in order to make it available for insects. 2.3. Feeding trials T. molitor larvae were fed with 3 different types of substrates based on agri-food by-products: stabilized Alperujo, mushroom (Pleorotus ostreatus) spent substrate, and olive leaves flour . These by-products were mixture with wheat bran at different percentages (20, 40, 60, 80 and 100 %) and the control diet was 100 % of wheat bran. Three assays of each percentage and substrate were carried out. For logistical reasons, initial feeding assays with ALP and POS were conducted, followed by feeding assays with the OLF substrate. Three assays were performed for each percentage and substrate, and each round of tests included a control group. 2.4. Processing of larvae When 10 % of the individuals had pupated, the rearing assays were concluded, and the larvae were separated from the rearing substrates and fasted for 48 h. After that, larvae were killed by submerging them in hot water at 50 ◦C for 2 min (thermostatic water bath Raypa, BAD-2, Terrasa, Spain). Then, larvae were dried in an oven (Memmert model UF260, Germany) at 40 ◦C for 3 days, milled in a semi-industrial mill Restch model GM 300 (Haan, Germany), and stored in plastic bags with Zip lock (hermetic seal) at −20 ◦C until analysis. 2.5. Determination of total fat content and fatty acid composition The total content of fat was determined by Soxhlet method based on AOAC (2012). Ten grams of samples were extracted with hexane for 2 h. The fat fraction of samples was storage at −20 ◦C. Fatty acids were determined turning them into their corresponding methyl esters and analysing them by GC–MS. An acid catalysed transesterification was performed (Osimani et al., 2017). For it, 100 μ L of nonadecanoic acid (4.06 mg/mL) (IST) and 2 mL of HCl 3 N in methanol were added to 200 mg of T. molitor larvae fat. The mixture was put into an oven at 70 ◦C, for 2 h. Then, let it cool at room temperature and 1 mL of distilled H 2 O and 2 mL of hexane were added, the mixture was centrifuged at 3000 rpm during 3 min to make easier the separation of different phase. The organic layer was transferred to a volumetric flask, made up to 2 mL with hexane and storage in a vial at −20 ◦C until analysis. An Agilent 6890 GC system coupled to an Agilent 5975 inert quadrupole mass spectrometer (Agilent, Santa Clara, CA, US) was used for analysis. The volume of sample used was 148 μ L that joint to 2 μ L of IS (2-phenylethanol, 6.06 mg/mL hexane) were put into a vial micro-insert and 1 μ L were injected in split mode 10:1 at 250 ◦C. Gas carrier used was He at 0.8 mL/min. Fatty acid separation was carried out using the DBFastFAME column of 20 m ×180 μ m i.d. x 0.2 μ m of film thickness (Agilent, Santa Clara, CA, US). The oven temperature program was: M.L. Morales et al. Food Research International 209 (2025) 116223 2 70 ◦C for 0.5 min, then it was increased 20 ◦C/min to 110 ◦C, 10 ◦C/min to 171 ◦C, 3 ◦C/min 185 ◦C hold during 1 min, 0.5 ◦C/min to 190 ◦C, 7 ◦C/min to 204 ◦C during 2 min, 25 ◦C/min to 240 ◦C during 2 min (adapted from Tossavainen et al., 2017). Detection was performed on selected ion monitoring (SIM) mode at 70 eV. The ions monitored were: 55, 67,74, 79, 91, 107, 108, 122, 143, 227, 236, 241, 250, 255, 264, 273, 312, 320, 322, 340, 350, 368, and 382. The MS quadrupole, source, and transfer line temperatures were 150 ◦C, 230 ◦C and 250 ◦C, respectively. For compounds identification, spectra and retention times of real standards were used, for that, a mixture of standards was injected in full scan mode (29–400 m/z). Methyl esters of fatty acids (FAMEs) were quantified building regression lines with 6 levels of concentration (Table S1, supplementary material). 2.6. Dietary indicators Several indexes related to cardiovascular health have been calculated. Thus, the ratios between PUFA:SFA, MUFA:PUFA, ω -6: ω -3, joint to atherogenic index (AI), thrombogenic index (TI), hypocholesterolemic/ hypercholesterolemic ratio (h/H) and health-promoting index were calculated (HPI) (Chen & Liu, 2020). 2.7. Statistical analysis Analysis of variance (ANOVA) was performed to check whether the changes in the values of fatty acids and healthy indexes were statistical significant (p <0.05). For that, we have used Infostat software (FCA, Universidad Nacional de C´ ordoba, Argentina) (posthoc LSD Fisher). Pearson correlation analysis (p <0.05) was undertaken in order to ascertain the possible relationship and the direction of this, between fatty acids contents and the percentages of substrate added to T. molitor’s diet. Statsoft Statistical, version 7.0 (Statsoft, Tulsa, OK) was used for this purpose. Table 1 Total fat content and profile of fatty acids of T. molitor crushed samples fed with control and with different percentage of ALP substrates (means values of triplicate of biological assays ±SD). Samples Control-1 ALP20 ALP40 ALP60 ALP80 ALP100 Total fat content (%w/w) 21.0 ±1.0 b 22.3 ±0.6 b,B 23.3 ±2.5 b 24.0 ±1.7 b 25.0 ±1.7 b,A 30.3 ±5.9 a,A Fatty acid content (% of total fatty acids content) SFA  Butanoic acid C4:0 0.49 ±0.26 0.51 ±0.44 n.q. 0.43 ±0.40 0.48 ±0.70 n.q. Dodecanoic acid C12:0 0.359 ±0.018 a 0.305 ±0.003 b,B 0.289 ±0.015 b 0.268 ±0.035 b,c,B 0.296 ±0.033 b 0.246 ±0.011 c,B Tridecanoic acid C 13:0 0.113 ±0.002 a 0.105 ±0.001 a,B 0.101 ±0.010 a,b,B 0.089 ±0.010 b,B 0.092 ±0.010 b,B 0.052 ±0.004 c,B Myristic acid C14:0 3.10 ±0.13 a 2.76 ±0.10 b,c,B 2.82 ±0.21 a,b,c 2.61 ±0.19 c,B 2.97 ±0.24 a,b 2.60 ±0.20 c Pentadecanoic acid C 15:0 0.303 ±0.009 a 0.273 ±0.020 a,b 0.288 ±0.020 a,b 0.266 ±0.019 b,B 0.265 ±0.013 b 0.228 ±0.030 c,C Palmitic acid C16:0 17.1 ±1.6 a 13.8 ±1.9 b,c 12.1 ±1.8 c 12.7 ±1.1 b,c,B 13.9 ±0.7 b,c,B 15.5 ±2.4 a,b,A Heptadecanoic acid C17:0 0.438 ±0.014 b,c 0.471 ±0.009 a,b,A 0.512 ±0.027 a 0.449 ±0.010 b,c,B 0.409 ±0.029 c,B 0.320 ±0.070 d,B Stearic acid C18:0 2.57 ±0.20 c 2.71 ±0.15 b,c 2.82 ±0.10 a,b 2.66 ±0.10 b,c,B 2.71 ±0.0.4 b,c,B 2.97 ±0.11 a,B Arachidic acid C20:0 0.437 ±0.010 b 0.535 ±0.061 a,b 0.590 ±0.077 a 0.553 ±0.032 a 0.524 ±0.026 a,b,C 0.596 ±0.095 a,B Heneicosanoic acid C21:0 0.009 ±0.001 a n.q. b n.q. b n.q. b n.q. b,B - b,B Behenic acid C22:0 0.158 ±0.007 a,b 0.168 ±0.033 a,b 0.195 ±0.033 a 0.150 ±0.032 a,b 0.161 ±0.008 a,b,B 0.128 ±0.039 b,B Tricosanoic acid C23:0 0.013 ±0.003 b,c 0.048 ±0.042 a n.q. c,B 0.031 ±0.005 a,b n.q. c,B n.q. c,B Lignoceric acid C24:0 –n.q. n.q. n.q. n.q. – MUFA  Palmitoleic acid C16:1 ( ω -7) 1.86 ±0.20 b 1.88 ±0.07 b,B 1.86 ±0.08 b,B 1.93 ±0.12 b,C 1.99 ±0.07 b 2.80 ±0.12 a,B cis-10-Heptadecenoic acid C17:1 ( ω -7) 0.352 ±0.029 b 0.419 ±0.011ª ,B 0.415 ±0.011ª ,B 0.431 ±0.009ª ,B 0.439 ±0.007ª ,B 0.375 ±0.044 b,B Oleic acid C18:1 ( ω -9) 31.0 ±1.3 c 38.2 ±1.4 b,A 39.8 ±6.4 b 43.4 ±4.2 b,A 44.0 ±2.5 b,A 61.0 ±0.6 a,A cis-11-Eicosenoic acid C20:1 ( ω -9) 0.326 ±0.019 b 0.389 ±0.045 a,A 0.373 ±0.035 a,b 0.391 ±0.031 a 0.380 ±0.016 a,b,A 0.393 ±0.031 a PUFA  Linoleic acid C18:2 ( ω -6) 39.9 ±1.2 a 36.0 ±0.4 a,b 36.7 ±8.4 a,b 32.3 ±4.3 b,B 30.5 ±2.7 b,B 12.4 ±1.6 c,B Linolenic acid C18:3 ( ω -3) 1.3 ±0.04 a 1.26 ±0.01 a,b,A 1.04 ±0.13 c,d,B 1.13 ±0.13 b,c,B 0.90 ±0.05 d,B 0.35 ±0.04 e,B cis-11,14-Eicosadienoic acid C20:2 ( ω -6) 0.166 ±0.026ª ,b 0.182 ±0.004ª ,A 0.087 ±0.075 b 0.137 ±0.019ª ,b 0.086 ±0.075 b n.q. c,B Total contents and Fatty acid indexes SFA 25.0 ±2.1 a 21.6 ±1.3 b,c,A 19.7 ±1.7 c,B 20.2 ±1.0 b,c,B 21.8 ±0.2 b,c,B 22.7 ±2.3 a,b MUFA 33.6 ±1.1 c 40.9 ±1.4 b 42.4 ±6.5 b 46.2 ±4.3 b,A 46.8 ±2.5 b,A 64.6 ±0.7 a,A PUFA 41.4 ±1.2 a 37.5 ±0.4 a,b 37.8 ±8.2 a,b 33.6 ±4.4 b,B 31.5 ±2.5 b,B 12.7 ±1.7 c,B ω -6 40.1 ±1.2 a 36.2 ±0.4 a,b 36.8 ±8.3 a,b 32.5 ±4.3 b,B 30.6 ±2.7 b,B 12.4 ±1.6 c,B ω -3 1.3 ±0.04 a 1.26 ±0.01 a,b,A 1.04 ±0.13 c,d,B 1.13 ±0.13 b,c,B 0.90 ±0.05 d,B 0.35 ±0.04 e,B PUFA:SFA 1.66 ±0.18 a 1.74 ±0.10 a 1.94 ±0.59 a 1.66 ±0.25 a,A 1.45 ±0.13 a,B 0.57 ±0.14 b,B MUFA:PUFA 0.812 ±0.022 c 1.09 ±0.04 b,c 1.18 ±0.38 b,c 1.40 ±0.32 b,c 1.50 ±0.21 b,A 5.12 ±0.61 a,A ω -6: ω -3 30.8 ±1.1 28.8 ±0.3 B 36.3 ±13.4 28.8 ±1.0 B 34.0 ±3.8 35.2 ±4.1 A,B AI 0.398 ±0.040 a 0.32 ±0.03 b 0.30 ±0.03 b 0.294 ±0.015 b,B 0.333 ±0.022 b,A 0.34 ±0.05 a,b,A TI 0.56 ±0.06 a 0.45 ±0.05 b,c 0.42 ±0.05 c,B 0.42 ±0.03 c,B 0.473 ±0.023 a,b,c,B 0.54 ±0.08 a,b,A h/H 3.6 ±0.4 c 4.5 ±0.6 a,b,c 5.2 ±0.8 a 4.9 ±0.4 a,b,A 4.4 ±0.3 a,b,c,B 4.1 ±0.7 b,c,B HPI 2.53 ±0.24 b 3.1 ±0.3 a 3.4 ±0.4 a 3.40 ±0.17 a,A 3.01 ±0.20 a,b,B 3.0 ±0.5 a,b,B -: no peak detected; n.q.: amount unquantifiable, n.d.: amount undetectable. SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid; AI: atherogenic index; TI: thrombogenic index; h/H: hypocholesterolemic/hypercholesterolemic ratio; HPI: health-promoting index. Different lowercase letter in the same line means significant differences (p <0.05) between samples fed with the same substrate with different percentage (Control-100 %). Different capital letter means significant differences (p <0.05) between samples of larvae fed with the same percentage of the three substrates studied. M.L. Morales et al. Food Research International 209 (2025) 116223 3 3. Results and discussion 3.1. Effect of the different T. molitor diets on total fat contents and fatty acid profiles The total content of fat in the feeding substrates used in this work were different, thus, the substrate that provided more fat to the larvae diet was Alperujo (ALP) (14 % w/w), then, in the second place the olive leaf substrate (OLF) (7.5 %) with the half content of the previous one. Wheat bran substrate used as control contained a 6 % of fat and Pleorotus ostreatus spent substrate (POS) showed the lowest content of fat (5 %). In the crushed of T. molitor larvae, total fat content ranged from 11.1 to 30 % w/w in the case of larvae fed with 100 % of OLF and in the case of 100 % of ALP substrate, respectively (Table 1–3). An observation to highlight was the increase in the total content of fat in the crushed of larvae fed with ALP directly proportional to the percentage of this substrate in the diet (r =0.91), whereas an inverse correlation was found in the case of OLF substrate percentages (r = − 0.89). Zhang et al. (2019) and Lawal et al. (2021) also observed that the total content of fat in T. molitor larvae was not correlated to the fat content of substrates. If we compare the fat content at the same percentage of each substrate, the contents were significantly higher in the case of OLF at 20 %, in ALP at 80 % and in ALP and POS at 100 %. Therefore, there was influence of the feeding substrate in the total content of fat, although this only clearly depended on the amount of fat of the diet in the case of alperujo substrate. In consequence, if the objective of breeding T. molitor was to produce oil, a good strategy would be to include a high percentage of alperujo in its diet. Due to the different effects of fatty acid profiles of food on human health, it is important to know if changes occur in the fatty acid profile and which are them. The fat of T. molitor larvae fed with the control diet was rich in PUFA, containing primarily C18:2 ω -6 (Linoleic acid) followed by C18:1 ω -9 (oleic acid) and palmitic acid as saturated fatty acid (Tables 1 and 3). A possible explanation could be that C18:2 fatty acid was the most abundant in the control substrate. This result agrees with those found by Kotsou et al. (2023), who also used wheat bran as diet control. In control samples, the total content of PUFA reached the highest significant values, followed by the MUFA total content, being also this significantly higher than total content of SFA. However, it was observed that this trend changed as the diet of the larvae was modified. In larvae fed with ALP substrate (Table 1), a clear shift in fatty acids composition was observed, the total content of MUFA increased while the total content of PUFA decreased. It can also be observed a trend to increase of the percentage of MUFA as the percentage of ALP substrate in the diet increased (r =0.90). This was primarily due to the significant increases of C18:1 ( ω -9) contents, which becomes the most abundant fatty acid in these samples. The highest statistically significant percentage of total MUFA contents was reached when the larvae were fed with a 100 % of ALP (Fig. 1). MUFAs such as C16:1(n-7), C18:1(n-9), and C20:1(n-9), present in our samples, have been associated with a decrease in the impact of obesity and related metabolic syndromes (Guerendiain et al., 2018). On the contrary, the C18:2 ( ω -6) contents decreased, being significantly lower when the diet contained percentage of alperujo higher than 40 respect to the control samples. A similar trend was observed for C18:3 ( ω -3), for this compound in most of the ALP percentage studied, except for 20 %, the contents in T. molitor crushed were significantly lower than in the control which in turn meant a decrease in the total percentage of ω -3 (Table 1). Both changes lead to a decrease of the total PUFA contents whish were inversely proportional to the percentage of alperujo used (r = − 0.86), being the larvae samples fed with the 100 % of ALP diet those which accounted the significantly lowest values of total PUFA contents (Fig. 1). Palmitic, followed by myristic and stearic acids, these two last with similar values, were, as in the control samples, the saturated fatty acids most abundant when ALP is used in the diet of T. molitor (Table 1). We did not observe relevant changes of total content of SFA, remaining Table 2 Total content and profile of fatty acids of T. molitor crushed samples fed with different percentage of POS substrate (means values of triplicate of biological assays ±SD). Samples POS20 POS40 POS60 POS80 POS100 Total fat content (%w/w) 21.0 ± 1.0 a,b,B 22.0 ± 3.5 a,b 22.0 ± 4.4 a,b 18.3 ± 1.5 b,C 25.3 ± 2.9 a,A Fatty acid content (% of total fatty acids content) SFA  Butanoic acid C4:0 0.62 ± 0.49 a,b 1.23 ± 1.37 a,b 1.43 ± 1.26 a n.q. b 0.16 ± 0.28 a,b Dodecanoic acid C12:0 0.319 ± 0.021 B 0.305 ± 0.066 0.305 ± 0.017 A,B 0.323 ± 0.008 0.372 ± 0.056 A Tridecanoic acid C 13:0 0.110 ± 0.008 a,B 0.096 ± 0.022 a,b, B 0.094 ± 0.009 a,b, B 0.098 ± 0.016 a,B 0.073 ± 0.013 b,B, * Myristic acid C14:0 2.81 ± 0.13 B 2.59 ± 0.58 2.62 ± 0.10 B 2.82 ± 0.13 3.14 ± 0.55 Pentadecanoic acid C 15:0 0.283 ± 0.026 0.318 ± 0.074 0.396 ± 0.067 A 0.356 ± 0.125 0.333 ± 0.037 B Palmitic acid C16:0 12.7 ± 2.3 b, * 15.1 ± 1.6 a,b 17.2 ± 1.8 a,A 16.2 ± 1.0 a,A 16.2 ± 1.8 a,A Heptadecanoic acid C17:0 0.413 ± 0.027 a,B 0.447 ± 0.090 a 0.420 ± 0.053 a,B 0.502 ± 0.108 a 0.284 ± 0.048 b,B, * Stearic acid C18:0 2.73 ± 0.22 2.99 ± 0.55 2.93 ± 0.16 A 2.92 ± 0.18 2.98 ± 0.23 B Arachidic acid C20:0 0.667 ± 0.131 0.663 ± 0.231 0.664 ± 0.158 0.637 ± 0.038 B 0.637 ± 0.123 B Heneicosanoic acid C21:0 n.q.* n.q.* n.q.* n.q. B, * n.q. B, * Behenic acid C22:0 0.245 ± 0.08 a 0.233 ± 0.09 a 0.174 ± 0.08 a,b 0.190 ± 0.02 a,b,B 0.110 ± 0.06 b,B Tricosanoic acid C23:0 0.019 ± 0.011 a 0.017 ± 0.004 a,A 0.012 ± 0.011 a - b,B, * n.q. b,B, * Lignoceric acid C24:0 – – n.q. n.q. – MUFA  Palmitoleic acid C16:1 ( ω -7) 1.80 ± 0.01 b,B 1.91 ± 0.15 b,B 2.18 ± 0.06 b,B 2.18 ± 0.33 b 3.51 ± 0.43 a,A, * cis-10Heptadecenoic acid C17:1 ( ω -7) 0.383 ± 0.010 b,C 0.408 ± 0.025ª ,b, B 0.463 ± 0.045ª , B, * 0.445 ± 0.051ª , B, * 0.386 ± 0.022 b,B Oleic acid C18:1 ( ω -9) 37.2 ± 3.8 b,c, A, B, * 34.6 ± 1.9 c,d 38.5 ± 3.6 b,c,A, B, * 42.8 ± 3.9 b,A, * 56.6 ± 3.7 a,A, * cis-11-Eicosenoic acid C20:1 ( ω -9) 0.362 ± 0.039 A,B 0.351 ± 0.068 0.356 ± 0.048 0.365 ± 0.006 A,B 0.366 ± 0.078 PUFA  Linoleic acid C18:2 ( ω -6) 38.2 ± 4.9 a 37.4 ± 1.7 a,b 31.2 ± 3.5 b,c,B, * 29.2 ± 4.2 c,B, * 14.6 ± 5.4 d,B, * Linolenic acid C18:3 ( ω -3) 1.03 ± 0.08 b,c, B, * 1.16 ± 0.9 a,b,B 0.93 ± 0.1 c,B, * 0.83 ± 0.21 c,B, * 0.28 ± 0.07 d,B, * cis-11,14Eicosadienoic acid C20:2 ( ω -6) 0.152 ± 0.012ª ,B 0.153 ± 0.007ª 0.166 ± 0.019ª 0.148 ± 0.031 a - b,B, * Total contents and Fatty acid indexes SFA 20.9 ± 1.3 b,A,B, * 24.0 ± 0.5 a,A 26.3 ± 0.6 a,A 24.1 ± 1.1 a,A 24.3 ± 1.8 a MUFA 39.7 ± 3.8 c, * 37.3 ± 1.9 c 41.5 ± 3.6 b,c,A, B, * 45.8 ± 4.1 b,A, * 60.8 ± 3.6 a,A, * PUFA 39.3 ± 4.8 a 38.7 ± 1.6 a,b 32.3 ± 3.6 b,c,B, * 30.1 ± 4.4 c,B, * 14.9 ± 5.3 d,B, * ω -6 38.3 ± 4.8 a 37.6 ± 1.7 a,b 31.3 ± 3.6 b,c,B, * 29.3 ± 4.2 c,B, * 14.6 ± 5.4 d,B, * ω -3 1.03 ± 0.08 b,c, B, * 1.16 ± 0.9 a,b,B 0.93 ± 0.1 c,B, * 0.83 ± 0.21 c,B, * 0.28 ± 0.07 d,B, * PUFA:SFA 1.89 ± 0.33 a 1.61 ± 0.05 a,b 1.23 ± 0.15 b,B, * 1.26 ± 0.22 b,B, * 0.63 ± 0.27 c,B, * (continued on next page) M.L. Morales et al. Food Research International 209 (2025) 116223 4 their values below the total values of MUFA and PUFA, despite the decreases of the latter, except for 100 % of ALP diet, in this case it was significantly higher than total PUFA content. Alperujo, derived from olives, was rich in C18:1 ω -9, resulting this fatty acid to be the most abundant in the substrate (Table S2, supplementary), followed by palmitic, linoleic, and stearic acids. The high C18:1 content in this substrate results in the enrichment of larvae fed with it, reaching in the ALP100 samples percentages similar to those in the substrate. Regarding the POS rearing substrate (Table 2), a similar trend was observed. The percentage of total MUFA content increased significantly in most samples compared to the control, except at 40 %, with the highest significant value being achieved in the 100 % POS diet. Moreover, a direct correlation was observed between the increase in total MUFA content and the percentage of POS in the diet (r =0.88). The lowest significant percentage of total PUFA content was also observed with the 100 % POS diet. As with the ALP rearing substrate, the most relevant changes occurred in the content of MUFA and PUFA. Thus, as in the control samples, the most abundant fatty acid was C18:2 when the diet contained 20 % and 40 % POS. However, when the percentage of POS exceeded 40 %, C18:1 became the most abundant fatty acid. This result shows the influence of the substrate included in the diet since, as occurred with ALP, POS substrate presents a high content of C18:1 and total MUFA (Table S2, supplementary). Regarding SFA, the amount was similar to that found in the crushed T. molitor fed with ALP. In most cases, total SFA content was significantly lower than MUFA and PUFA contents. However, in samples fed with 100 % POS, the decrease in C18:2 made palmitic acid the second most abundant FA after C18:1, and the total SFA content became significantly higher than PUFA content, similar to what occurred with the ALP substrate. In the case of T. molitor larvae fed with the OLF substrate (Table 3), the total PUFA contents were significantly higher than SFA and MUFA contents in all cases except with the 100 % OLF diet, in which a substantial increase in total MUFA content was observed due to the rise in C18:1 content. Conversely, the total SFA content was significantly lower in all cases, showing an inverse correlation with the percentage of OLF used in the diet (r = − 0.90). In these samples, the palmitic acid content was the lowest, while significant increases in stearic and arachidic acids were observed when the two highest percentages of OLF were consumed by larvae. Additionally, unlike with the two previous substrates, an increase in C18:3 content was observed in these samples, directly correlated with the percentage of OLF substrate (r =0.89) and statistically significant when the diet contained more than 20 % of OLF. This may be because this substrate had the highest content of C18:3 fatty acids (Table S2, supplementary). This suggests that OLF may be a promising substrate for enhancing ω -3 fatty acids levels in T. molitor larvae fat fraction. Overall, the most important change observed in our experiments was the decrease of C18:2 contents whilst C18:1 contents increased as the percentage of control substrate decreased for ALP and POS substrates. A similar trend was observed by Kotsou et al. (2023) when M. oleifera leaves were added at different rates to in the T. molitor diet. When comparing the fatty acid profile of crushed T. molitor fed with the same percentage of addition of the three different substrates studied, the highest total PUFA contents were observed for OLF at 60, 80 and 100 %. In contrast, the higher total MUFA content was found in the case of ALP and POS at 80 and 100 %; whereas the lowest total SFA content was obtained with the diets of 40, 60 and 80 % of POS. Furthermore, the highest total contents of ω -3 were achieved in the case of OLF with addition percentages between 40 and 100 %. Hence, OLF substrate could not only enhance the presence of ω -3, as mentioned above, but also the total content of PUFA in the fat fraction of these larvae. Several authors have carried out assays to compare the effect of different diet in the nutritional composition of T. molitor larvae. Lawal et al. (2021) studied concretely the effect of several seed such as flax seed, chia seed, hemp seed, and rapeseed at different percentage on FA profile, using whole-wheat meal as control diet. These diets incremented the amount of fat in the larvae. They found levels of total SFA and MUFA contents higher than our values and total PUFA values clearly lower than those obtained by us. On the contrary, when mushroom spent corn stover, highly denatured soybean meal, and spirit distillers’ grains were used as substrate to rear T. molitor larvae (Zhang et al., 2019), the values of SFA and MUFA were lower than those of our samples and the total PUFA values were higher than our ones. 3.2. Health related indexes The different results relatives to the total contents of SFA, MUFA and PUFA can make us think that both the crushes of T. molitor and their fat fraction isolate may have a different impact on the consumer’s health depending on the rearing diet used. For this reason, different dietary indicators, related to the consumer’s health, were calculated. PUFA:SFA ratio is an index that has been related with a favourable effect of dietary fat against the cardiovascular diseases (CVD) (Chen & Liu, 2020), among others. In the case of the diets with the addition of different percentage of ALP, there was no significant differences respect to the control, except for the 100 % ALP diet, being significantly lower than the control (Table 1). In the case of POS addition, the values of this ratio were significant lower when percentage equal or higher than 60 were used (Table 2). However, the samples of T. molitor fed with a diet with 80 % of OLF, reached the highest significant values. In similar way that occur with ALP substrate, the only case with values significantly lower than the control was 100 % of OLF, although the ratio was not as low as with the other two substrates (Table 3). Thus, the lowest values of PUFA:SFA ratio were presented by ALP and POS at 100 %, but all showed values higher than recommended 0.4 (Simopoulos, 2003). Chen and Liu (2020) carried out a review in which included the values of several dietary or nutritional indexes related to the fatty acid content from different food protein sources. Hence, we verified that the values of the PUFA:SFA ratio of our samples were higher than those of several dairy products, many meats and similar to those of shellfish and fish. Therefore, the fatty acid profile of our T. molitor crushed provides greater cardiovascular benefits than some representative human diet protein foods, especially when the OLF substrate was used in percentage less than 100. The atherogenicity index (AI) relates the content of SFA considered pro-atherogenic and the unsaturated fatty acids described as antiTable 2 (continued) Samples POS20 POS40 POS60 POS80 POS100 MUFA:PUFA 1.03 ± 0.24 b 0.96 ± 0.09 b 1.30 ± 0.25 b 1.56 ± 0.38 b,A 4.45 ± 1.58 a,A, * ω -6: ω -3 37.6 ± 6.8 a,b,A 32.8 ± 4.2 a,b 33.7 ± 2.5 a,b,A 36.4 ± 5.6 a,b 55.9 ± 30.5 a,A, * AI 0.31 ± 0.03 b, * 0.34 ± 0.05 a,b 0.38 ± 0.03 a,A 0.367 ± 0.013 a,b, A 0.39 ± 0.06 a,A TI 0.43 ± 0.05 b, * 0.51 ± 0.04 a,b,A 0.58 ± 0.05 a,A 0.55 ± 0.03 a,A 0.58 ± 0.07 a,A h/H 4.9 ± 0.8 a, * 4.1 ± 0.5 a,b 3.5 ± 0.4 b,B 3.8 ± 0.3 b,B 3.7 ± 0.5 b,B HPI 3.3 ± 0.3 a, * 3.0 ± 0.4 a,b 2.65 ± 0.22 b,B 2.73 ± 0.10 a,b,B 2.6 ± 0.4 b,B -: no peak detected; n.q.: amount unquantifiable, n.d.: amount undetectable. SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid; AI: atherogenic index; TI: thrombogenic index; h/H: hypocholesterolemic/hypercholesterolemic ratio; HPI: health-promoting index. *Mean significant difference with control sample (values in Table 1) p <0.05. Different lowercase letter in the same line means significant differences (p < 0.05) between samples fed with the same substrate with different percentage (20–100 %). Different capital letter means significant differences (p <0.05) between samples of larvae fed with the same percentage of the three substrates studied. M.L. Morales et al. Food Research International 209 (2025) 116223 5 atherogenic (Orkusz et al., 2024). Thus, a fat with low AI is considered healthy. The values of AI in our samples ranged from 0.231 (HHO100) to 0.398 (C1) (Tables 1–3). If we compare them with other foods which are rich in proteins, our samples presented values much lower than dairy products, with values at least three times higher, lower than most of meats and similar o lower than fish (Chen & Liu, 2020). The different diets studied modified this index in different ways respect to the control diet. Thus, in the diet in which Alperujo was added, the values of this index decreased positively, except in 100 % ALP, due to the important decrease of ω -6 fatty acid contents in this sample. On the contrary, in the diet containing POS, only in the case of the lowest percentage of addition presented significantly lower values than in the control. In the case of OLF substrate, the favourable effect on health occurs with percentages equal to or greater than 60. Regarding the thrombogenicity index (TI), that reflects the predisposition or propensity of certain saturated fatty acids to form clots in blood vessels (Ulbricht & Southgate, 1991) against the antithrombogenic effect of certain unsaturated fatty acids. Therefore, this index may show the positive effect of dietary fat against CVD when its value is low. The inclusion of ALP in the diet of T. molitor was beneficial when percentage between 20 and 60 were used, while in the case of OLF substrate is necessary to go up to percentages of the order of 80 or 100. The trend of this index in T. molitor samples fed with POS is equal to AI. Our results showed that our products were again healthier than meat and dairy products (Chen & Liu, 2020). For both indexes, AI and TI, comparing the same percentage of the three different substrates in the diet, lower significant values were only observed for 80 and 100 % of OLF. The hypocholesterolemic/hypercholesterolemic (h/H) ratio was also calculated as an indicator of the cardiovascular health (CVH) promotion of fat dietary. The values determined in our study ranged from 3.54 (POS60) to 6.89 in OLF100. For this index, the values of our samples were higher than fish, meat, dairy products, and most of shellfish (Chen & Liu, 2020). Respect to the values of the control samples, the only case that showed significant higher values were ALP40 and 60; POS20; OLF80 and 100. Table 3 Total fat content and profile of fatty acids of T. molitor crushed samples fed with control and with different percentage of OLF substrates (means values of triplicate of biological assays ±SD). Samples Control-2 OLF20 OLF40 OLF60 OLF80 OLF100 Total fat content (%w/w) 26.3 ±6.6 a,b 28.5 ±1.8 a,A 23.6 ±1.4 a,b,c 19.7 ±4.3 c 21.1 ±0.4 b,c,B 11.1 ±1.4 d,B Fatty acid content (% of total fatty acids content) SFA  Butanoic acid C4:0 0.79 ±0.3 0.98 ±0.76 0.35 ±0.22 0.79 ±0.36 0.96 ±0.53 0.63 ±0.54 Dodecanoic acid C12:0 0.415 ±0.027 a 0.384 ±0.008 a,b,A 0.360 ±0.016 b,c 0.328 ±0.012 c,d,A 0.298 ±0.014 d,e 0.270 ±0.061 e,B Tridecanoic acid C 13:0 0.151 ±0.007 a 0.139 ±0.003 a,b,A 0.148 ±0.009 a,A 0.144 ±0.008 a,b,A 0.146 ±0.001 a,b,A 0.125 ±0.025 b,A Myristic acid C14:0 3.55 ±0.007 a 3.12 ±0.11 b,A 3.08 ±0.11 b 2.99 ±0.10 b,A 2.76 ±0.03 b 2.39 ±0.48 c Pentadecanoic acid C 15:0 0.290 ±0.015 d 0.286 ±0.012 d 0.312 ±0.009 c 0.314 ±0.020 c,A,B 0.369 ±0.009 b 0.433 ±0.012 a,A Palmitic acid C16:0 13.2 ±0.2 a 12.8 ±0.3 a 12.7 ±1.7 a 12.2 ±0.5 a,B 9.9 ±1.6 b,C 8.6 ±1.6 b,B Heptadecanoic acid C17:0 0.443 ±0.025 d 0.450 ±0.026 d,A,B 0.539 ±0.027 c 0.570 ±0.014 c,A 0.762 ±0.006 b,A 0.933 ±0.058 a,A Stearic acid C18:0 2.52 ±0.07 d 2.68 ±0.07 c,d 2.68 ±0.06 c,d 2.89 ±0.08 c,A,B 3.24 ±0.08 a,B 4.86 ±0.27 a,A Arachidic acid C20:0 0.593 ±0.070 c 0.614 ±0.023 c 0.549 ±0.077 c 0.622 ±0.054 c 0.796 ±0.056 b,A 1.30 ±0.12 a,A Heneicosanoic acid C21:0 n.q. c n.q. c n.q. c n.q. c 0.039 ±0.006 b,A 0.052 ±0.004 a,A Behenic acid C22:0 0.155 ±0.015 c 0.205 ±0.014 b,c 0.168 ±0.051 c 0.203 ±0.048 b,c 0.275 ±0.046 b,A 0.538 ±0.059 a,A Tricosanoic acid C23:0 n.q. c n.q. c n.q. c,B 0.010 ±0.017 c 0.045 ±0.005 b,A 0.082 ±0.010 a,A Lignoceric acid C24:0 n.q. n.q. n.q. 0.022 ±0.038 n.q. 0.019 ±0.033 MUFA  Palmitoleic acid C16:1 ( ω -7) 2.50 ±0.03 a 2.38 ±0.05 b,c,A 2.38 ±0.04 b,c,A 2.41 ±0.02 b,A 2.35 ±0.03 b,c 2.32 ±0.04 c,B cis-10-Heptadecenoic acid C17:1 ( ω -7) 0.547 ±0.02 d 0.562 ±0.002 d,A 0.615 ±0.023 c,d,A 0.658 ±0.012 c,A 0.870 ±0.076 b,A 1.17 ±0.10 a,A Oleic acid C18:1 ( ω -9) 33.1 ±0.8 b,c 32.7 ±1.0 c,B 33.7 ±1.0 b,c 34.9 ±1.3 b,B 33.7 ±1.1 b,c,B 46.6 ±1.3 a,B cis-11-Eicosenoic acid C20:1 ( ω -9) 0.328 ±0.027 c,d 0.317 ±0.017 d,B 0.357 ±0.008 b 0.354 ±0.006 b,c 0.356 ±0.005 b,B 0.453 ±0.013 a PUFA  Linoleic acid C18:2 ( ω -6) 40.1 ±1.0 a 41.1 ±1.9 a 40.5 ±2.5 a 39.0 ±1.3 a,A 41.4 ±2.3 a,A 27.1 ±0.9 b,A Linolenic acid C18:3 ( ω -3) 1.18 ±0.02 c 1.27 ±0.04 c,A 1.45 ±0.09 b,A 1.42 ±0.10 b,A 1.51 ±0.12 b,A 2.00 ±0.06 a,A cis-11,14-Eicosadienoic acid C20:2 ( ω -6) 0.126 ±0.006 c n.q. d,C 0.150 ±0.017 a,b 0.164 ±0.013 a 0.149 ±0.021 a,b 0.130 ±0.004 b,c,A Total contents and Fatty acid indexes SFA 22.1 ±0.1 a 21.7 ±0.9 a,b,B 20.9 ±1.6 a,b,B 21.1 ±0.3 a,b,B 19.6 ±1.1 b,C 20.2 ±2.2 a,b MUFA 36.4 ±0.9 b,c 36.0 ±1.1 c 37.0 ±0.9 b,c 38.3 ±1.3 b,B 37.3 ±1.0 c,b,B 50.6 ±1.4 a,B PUFA 41.4 ±0.9 a 42.4 ±1.9 a 42.1 ±2.4 a 40.6 ±1.2 a,A 43.1 ±2.1 a,A 29.2 ±0.9 b,A ω -6 40.2 ±1.0 a 41.1 ±1.9 a 40.6 ±2.4 a 39.2 ±1.3 a,A 41.6 ±2.2 a,A 27.2 ±0.9 b,A ω -3 1.18 ±0.02 c 1.27 ±0.04 c,A 1.45 ±0.09 b,A 1.42 ±0.10 b,A 1.51 ±0.12 b,A 2.00 ±0.06 a,A PUFA:SFA 1.87 ±0.05 b 1.96 ±0.17 a,b 2.03 ±0.28 a,b 1.93 ±0.06 a,b,A 2.21 ±0.23 a,A 1.46 ±0.21 c,A MUFA:PUFA 0.88 ±0.04 b 0.85 ±0.06 b 0.88 ±0.07 b 0.94 ±0.06 b 0.87 ±0.07 b,B 1.73 ±0.04 a,B ω -6: ω -3 34.1 ±1.3 a 32.3 ±1.2 a,b,A,B 28.2 ±3.4 b,c 27.7 ±2.9 c,B 27.8 ±3.6 c 13.57 ±0.04 d,B AI 0.36 ±0.01 a 0.33 ±0.01 a,b 0.32 ±0.01 a,b 0.31 ±0.01 b,B 0.26 ±0.03 c,B 0.23 ±0.04 c,B TI 0.46 ±0.01 a 0.44 ±0.01 a 0.43 ±0.04 a,A,B 0.42 ±0.01 a,B 0.36 ±0.04 b,C 0.35 ±0.05 b,B h/H 4.3 ±0.1 c 4.6 ±0.2 c 4.7 ±0.6 c 4.9 ±0.2 b,c,A 6.0 ±0.8 a,b,A 6.9 ±1.4 a,A HPI 2.8 ±0.0 c 3.1 ±0.1 c 3.1 ±0.2 c 3.2 ±0.1 b,c,A 3.8 ±0.4 a,b,A 4.4 ±0.8 a,A n.q.: amount unquantifiable, n.d.: amount undetectable. SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid; AI: atherogenic index; TI: thrombogenic index; h/H: hypocholesterolemic/hypercholesterolemic ratio; HPI: health-promoting index. Different lowercase letter in the same line means significant differences (p <0.05) between samples fed with the same substrate with different percentage (Control-100 %). Different capital letter means significant differences (p <0.05) between samples of larvae fed with the same percentage of the three substrates studied. M.L. Morales et al. Food Research International 209 (2025) 116223 6 Finally, we also considered the healthy-promoting index (HPI), although this index is the inverse of the AI (Chen & Liu, 2020). For that, the effect of different percentage of substrates studied provided similar results to AI. However, for this index, when we compare all studied samples, the sample OLF100 showed the highest significant values. If we compare the values of indices related with cardiovascular health obtained in this work with those obtained by other authors, we observed similar results or even better. In the case of Orkusz et al.’s study (2024), in which T. molitor from market were analysed, these turned out to be less healthy than ours, showing PUFA:SFA and h/H ratios lower and TI and AI values higher than ours. Moreover, in the case of the inclusion of vegetal wastes (L´ opez-G´ amez et al., 2024) or M. oleifera leaves (Kotsou et al., 2023) in the T. molitor’s diet, the PUFA: SFA ratios were similar to ours, but TI and AI values were higher and h/ H ratios and HPI lower than ours, showing that the diets assayed by us seem to be slightly more healthier, specially respect to the last kind of diet. Another important factor in a healthy diet is a balanced intake of omega-6 versus omega-3 fats. The desired ω -6 to ω -3 ratio seem to be between 1/1 and 5/1 (Lupette & Benning, 2020). In our case, the fat fraction of larvae fed with modified diets as well as with control diet showed high ω -6: ω -3 ratios compared to the recommended values, the lowest significant value was found in the case of the OLF100 diet. In the case of these insects, when different diet were studied, only the inclusion of a percentage of 10 % of flax or chia seed was able to adequately the values of this ratio (Francardi et al., 2017; Lawal et al., 2021). Therefore, this ratio would be easily improved with the addition of a source of ω -3. Furthermore, it has recently been suggested that ω -9 FA may be as important as ω -3 and ω -6 FAs for human health (Wang et al., 2024). The fat fractions obtained in our study were rich in ω -9 MUFAs, and their contents ranged between 33.04 and 61.42 %. Several authors have suggested that this type of FA may contribute positively to cardiovascular diseases, neurodegenerative diseases, and exert an antiinflammatory effect (Hu et al., 2021; Santa-María et al., 2023; Wahle et al., 2004). Although ω -9 MUFAs can be synthesized endogenously by the human body the main source is the diet (Wang et al., 2024). However, a specific recommended intake range has not yet been defined. Since the MU fat fraction is primary consisted of ω -9 MUFA, the ω -9 MUFA intake recommendations could be practically equated with MUFA intake. Among the different ω -9 that exist, we have found in our samples two, oleic acid and cis-11-eicosenoic acid (gondoic acid). The first, as it has been pointed above, is the most abundant fatty acid in all larvae samples fed with ALP substrate, in those fed with a percentage of POS equal or greater than 60 and in the case of OLF100 diet. Among the healthy properties attributed to this FA, we can highlight the possible contribution for improving the atherosclerotic process and plaque stability (Perdomo et al., 2015). Moreover, cis-11-eicosenoic acid seems to have effect on oxidative stress that produce short-term memory deficit (Cojocariu et al., 2020). Another healthy effect is replacing some saturated fatty acids (SFA) with ω -9 MUFAs in the diet (Maki et al., 2018). In our case, all samples presented high values of ω -9 respect to SFA contents, especially in the case of ALP substrate. Hence, in making an assessment of the results of the most relevant health related indexes presented by the samples studied, stood out the fat fraction of T. molitor fed with 80 % and 100 % of OLF. However, the weaknesses of this study should be considered which are that these health related indexes are mathematical calculations from the results derived of laboratory scale experiments and, therefore, to confirm their healthy effect, future assays in animal models could be necessary. 3.3. Health claims The health-related organizations have given various recommendations on fat intake. On one hand, according to the WHO recommendation (2023), in the diet of adults, the 30 % of the total energy intake should come from fats, with no more than 10 % of this total energy intake coming from SFA. On the other hand, European Food Safety Authority (EFSA) (2017) recommended that the total energy intake from fat should be between 20 and 35 % maximum and the percentage from SFA should be as low as possible. Moreover, the adequate intake recommended for linoleic acid is 4 % of total energy intake and 0.5 % for alfa-linolenic acid. Considering these recommendations, we will proceed Fig. 1. Percentage of total SFA, MUFA and PUFA contents in crushed of T. molitor larvae’s fat fraction fed with different substrate: Control diet (100 % of wheat bran), Stabilized Alperujo (ALP), Pleorotus ostreatus spent substrate (POS) and olive leaf flour (OLF) mixed with different percentage of wheat bran. M.L. Morales et al. Food Research International 209 (2025) 116223 7 to analyze the qualities of the fat fraction of the crushed products obtained with diets modified with waste. According to the population reference intake (PRI) of energy for adults by European Food Safety Authority (2013), this should be between 2000 and 2600 Kcal/day. Considering an average value of energy of 2300 Kcal/day, 100 g of crushed T. molitor obtained in this work would provide between 4.3 and 11.8 % of the total PRI of energy provided by the fat fraction, most of them with values around 8–9 % of the PRI of energy. Furthermore, between 0.9 and 2.7 % of this energy come from SFA, 2.2–7.6 % from MUFA and 1.3–4.7 % from PUFA, i.e. mainly MUFA and PUFA. In addition, the content linoleic acid was close to the values required to meet the recommended adequate intake requirements for this fatty acid since, most of them, provided more than 3 % of the total PRI of energy. If we focus on the extracted fat fraction and it would be marketed as food product, according to European regulations on nutritional claims allowed to be included in a label (European Commission, 2006), these could be labelled as “source of ω -3” except ALP100, POS100 and OLF20, since their contents exceed 0.3 g of α -linolenic acid in 100 g of product. Some of them, such as ALP60, ALP80, ALP100, POS80, POS100 and OLP100, also meet the requirements to be referred to as “high in monounsaturated fat” contributing to the total content of fatty acids with percentages ranging between 45.8 % and 64.6 %, more than 45 % of the required fatty acids and also representing more than 20 % of the energy value of the product. Finally, all of them can be labelled as “high in unsaturated fats” because their content (73.7–80.4 %) exceeded the required value (70 %). 4. Conclusions The use of different percentages of alperujo, mushroom spent substrate and olive leaf flour in the diet of T. molitor lavae influences both the total fat contents and the fatty acid profiles. The fatty acid profiles experienced changes, especially notable in the case of diet spiked with the ALP substrate, compared to the control diet. The most remarkable change was the increase of oleic acid, a ω -9 fatty acid, which have recently been linked to health benefits. This fact is of great relevance since, among the three substrates studied, the one with the greatest ecological interest is the reuse of Alperujo, as it is a highly productive waste and the most polluting of the three. Moreover, OLF substrate enhanced the presence of ω -3 and the total content of PUFA in the fat fraction of these larvae. Regarding the impact on the consumer’s health of the fat fraction of crushed T. molitor larvae obtained in this work, for most of health related indexes we obtained more adequate values than those of other conventional protein sources. In addition, in all cases there was an increase of the total content of unsaturated fatty acids with respect to that of saturated. In addition, high percentages of OLF substrate in the diet presented the most positive results respect to health indexes. Hence, the waste from olive oil production appeared to provide the healthiest fat fraction. Although most of crushed T. molitor obtained presented high total fat contents, it was a healthy fat fraction, rich in unsaturated fats, especially ω -9, and may be considered, in some cases, as “source of ω -3”, which would make this fraction suitable as an ingredient to improve the healthiness of food. CRediT authorship contribution statement Morales M. Lourdes: Writing – original draft, Methodology, Investigation, Formal analysis, Conceptualization. M. Pilar Segura-Borrego: Visualization, Investigation, Formal analysis. Jos´ e Raúl AguileraVel´ azquez: Resources, Investigation. Raquel M. Callej´ on: Writing – review & editing. Daniel Guti´ errez-Praena: Writing – review & editing, Project administration, Funding acquisition. Cristina Ubeda: Writing – review & editing, Project administration, Funding acquisition. Funding sources This work is part of the R +D +i project TED2021-129351 A-I00, funded by MICIU/AEI/10.13039/501100011033/ and by the “European Union NextGenerationEU/PRTR”. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment The authors wish to thank Almazara Virgen de los ´ Angeles for supplying the alperujo. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.foodres.2025.116223. Data availability Data will be made available on request. References AOAC. (2012). Official method 948.22. Fat (crude) in nuts and nut products. Gravimetric methods. In AOAC international, official methods of analysis of AOAC international (19th ed.). MD, USA: Gaithersburg. Brandon, A. M., Gao, S. H., Tian, R., Ning, D., Yang, S. S., Zhou, J., Wu, W. M., & Criddle, C. S. (2018). Biodegradation of polyethylene and plastic mixtures in mealworms (larvae of Tenebrio molitor) and effects on the gut microbiome. 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