Fat from Tenebrionidae bugs – Sterols content, fatty acid profiles, and cardiovascular risk indexes

Pol. J.Food Nutr. Sci., 2019, Vol. 69, No. 3, pp. 247–254 DOI: 10.31883/pjfns/109666 http://journal.pan.olsztyn.pl Original research article Section: Food Quality and Functionality © Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences © 2019 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/3.0/). INTRODUCTION From thenutritional point ofview, edible insects have become an increasingly discussed topic inboth theprofessional andlaic public, even incountries where their consumption isnot common [Mlček etal., 2014; Ramos-Elorduy etal., 2011; van Huis, 2016]. Inparticular, itispossible to use edible insects as avaluable alternative source ofproteins, especially inregions with alack ofconventional animal proteins [Mlček etal., 2014; Ramos-Elorduy etal., 2011; van Broekhoven etal., 2015]. Other benefi ts include higher feed conversions, low greenhouse gas emissions, better soil utilization, andtheconversion oforganic materials to valuable products. This strategy can lead to fi nancial savings andenvironmental benefi ts [Cerritos, 2009, 2011; Fontaneto etal., 2011; Mariod etal., 2011; Premalatha etal., 2011]. Insects have also agreat potential as feed, for example, inaquaculture [van Huis, 2016]. This isdue to thehigh content ofproteins andsulphur amino acids that can besuccessfully used as feed for poultry [Józefi ak etal., 2016]. One ofthemost studied species ofinsects isthemealworm [Finke, 2002, 2004; Barroso etal., 2014; Ravzanaadii etal., * Corresponding Author: Tel.: +42–057–603–3030; E-mail: [email protected] (J.Mlček) 2012; Sánches-Muros etal., 2016; Tzompa-Sosa etal., 2014; Zielińska etal., 2015]. Itisagood source ofprotein andfat. Thehighest protein content (637.0–676.5g/kg indry matter (DM)) andlowest fat content (148.8–184.0g/kg DM) was determined inadult specimen. However, larvae andpupae are more nutritionally benefi cial because oftheir better digestibility andsensory properties. For larvae, thetotal protein content isusually reported intherange from 477.6to 527.0g/kg DM andtotal fat content from 189.0to 382.9g kg DM. Sterols are key nutritional elements ofinsects. They are precursors ofsteroid hormones anddevelopment process regulators [Mondy etal., 2006]. Phytosterols, typical plant sterols, were found ininsect samples [Piironen, 2000], because insects cannot synthesize cholesterol de novo [Behmer & Nes, 2003] andhave to use plant phytosterols (β-sitosterol, campesterol, stigmasterol) to synthesize cholesterol. Cholesterol isthemost abundant sterol present ininsects. Mealworm (Tenebrio molitor) sterols contain about 17% of7-dehydrocholesterol andabout 67% ofcholesterol [Ikekawa etal., 2013]. Insects need cholesterol to synthesize vitaminD3 andsteroid hormones known as ecdysteroids. These hormones are indispensable for theindividual developmental stages oftheinstar [Nation, 2001; Klowden, 2007]. Cholesterol isfound infoods ofanimal origin, but itisalso theonly sterol thehuman body can synthetize byitself. Too high cholesterol intake causes increased levels oflow-density Fat from Tenebrionidae Bugs – Sterols Content, Fatty Acid Profi les, andCardiovascular Risk Indexes Jiří Mlček1*, Anna Adámková1, Martin Adámek2, Marie Borkovcová1, Martina Bednářová3, Ivana Knížková4 1Department ofFood Analysis andChemistry, Tomas Bata University, Zlín, Czech Republic 2Department ofMicroelectronics, Faculty ofElectrical Engineering andCommunication, Brno University ofTechnology, Czech Republic 3Department ofInformation Technology, Mendel University, Brno, Czech Republic 4Livestock Technology andManagement, Institute ofAnimal Science, Prague, Czech Republic Key words: mealworm, superworm, cholesterol, stigmasterol, β-sitosterol, cholecalciferol This work focused on analysing thecontent ofselected sterols andprofi le offatty acids ofedible insect species – mealworm (Tenebrio molitor) andsuperworm (Zophobas morio), which are expected to increase their usability inhuman nutrition. Sterols content was determined using capillary gas chromatography method. Cholesterol content was determined at 1335mg/kg indry matter (DM) for themealworm, which was less than for superworm (3224mg/kg DM). Other sterols analysed were stigmasterol andβ-sitosterol, which were once again higher insuperworm (stigmasterol – 44mg/kg DM andβ-sitosterol – 414mg/kg DM) than inmealworm (stigmasterol – 18mg/kg DM andβ-sitosterol – 171mg/kg DM). From thenutritional point ofview, theamount ofcholecalciferol isalso not negligible, which was 190μg/kg DM inmealworm and199μg/kg DM insuperworm. Atherogenic index (AI), thrombogenic index (TI), andcholesterol index (CSI) were calculated for both species andcompared with theresults ofother authors. These indexes are often considered predictors ofcardiovascular diseases. Apotential benefi t ofboth species could bethebalanced proportion ofsterols ofanimal andplant origin that could benutritionally well-accessible andlower weight ofdry matter necessary to consume to cover thedaily dose oflinoleic acid compared to dried beef. 248 J. Mlček et al. lipoprotein (LDL) andvery low-density lipoprotein (VLDL), which results inan increased risk ofthedevelopment ofthemetabolic syndrome (obesity, diabetes mellitus, cardiovascular disease) [WHO, 2004]. Thehuman body consumes about 2g ofcholesterol per day. Theoptimal intake is0.15– –0.3g per day. This amount issuffi cient because therest isproduced inthebody through endosynthesis. However, common diet means an intake of0.6–0.8g ofcholesterol. Asuitable diet can reduce cholesterol by10%, e.g. byconsumption offi - ber, antioxidants, andphytosterols [Pánek etal., 2002]. Vitamin D issynthesized inthehuman body from ergosterol and7-dehydrocholesterol byexposure to sunlight. Inthelatitudes ofthetemperate zone, sunlight does not cover these needs andvitamin D endosynthesis isinsuffi cient. For this reason, itisnecessary to supplement it. Vitamin D, along with calcitonin andparathormon hormones, controls thecalcium andphosphorus metabolism. Its defi ciency leads to rachitis inchildren, while older people develop osteopenia andconsequently osteoporosis [Pánek etal., 2002]. For this reason, therecommended daily dose ofvitamin D is5μg/day [Decree No. 225/2008Coll, 2008]. Developmental stage ofinsect isone ofthemajor factors infl uencing theamount offat andthefatty acids [Nowak etal., 2016; Finke, 2004; Adámková etal., 2016]. Other factors include gender, diet andtheenvironment [Chakravorty etal., 2011, 2014, 2016]. Fatty acid profi le description can besimplifi ed andexpressed byvarious proportional numbers, including thecholesterol index (CSI), theatherogenic index (AI), andthethrombogenic index (TI). These indexes serve often inmedicine as important predictors ofcardiovascular risks [Dobiášová, 2006]. Regarding then-3:n-6ratio offatty acids andthementioned indexes, edible insect fat may have aprotective effect on human health. One oftheobjectives ofthis study was to determine theratio ofn-3:n-6fatty acids andCSI, AI, andTI inedible insect fat andto compare them with other commodities ofanimal origin. Thecontent ofsterols inthemealworm (Tenebrio molitor) andsuperworm (Zophobas morio) bred on farms intheCzech Republic, which are fed ad libitum with conventional feed, isnot yet suffi ciently known from available literature [Sabolová etal., 2016] andso far this issue has not been thoroughly explored. Theamount ofcholesterol andphytosterols can beaffected bylong-term nutritional stress (theinsects do not have access to feed). Although insects bred infarms usually do not starve, access to feed may berestricted during longer transport or longer pre-treatment before killing. This study was carried out to defi ne initial information on thecontent ofcholesterol, phytosterols, andfatty acids inTenebrioidae larvae andtheir presumed impact on human health (AI, TI andCSI). Furthermore, theimpact ofthenutritional stress (inaccessibility ofthefeed) on thecholesterol content was evaluated, as thenutritional stress has asignifi cant impact on thewelfare ofthebreed. MATERIAL ANDMETHODS Material Species used for ana lysis were mealworm larvae (Tenebrio molitor) andsuperworm larvae (Zophobas morio). Samples were purchased from breeder Radek Frýželka, Brno, Czech Republic. Insects were reared inoptimum conditions for thedevelopment ofindividual species (mealworm – 25–28°C, 60–70% relative humidity (RH); superworm – 28–30°C, 60– –70% RH) andfed with wheat bran andoat bran ad libitum till thebeginning oftheexperiment. At thebeginning oftheexperiment, two groups oflive larvae, weighing approximately 200g, were taken from abreeding. Thefi rst group was left to starve for 12h. According to theEuropean Food Safety Authority (EFSA) recommendations [EFSA, 2015], theexpiration time is12–24h. To shorten theprocessing, thelower limit was chosen. Subsequently, theinsects were killed inboiling water (100°C) anddried at 105°C.Subsequently, thesamples were homogenized andstored inahermetically sealed aseptic box with anormal atmosphere at 4–7°C until analysis. Thesecond experimental group oflarvae starved for 168h (7days) before killing, which led to nutritional stress. Furthermore, thelarvae were killed andprocessed inthesame way as thefi rst experimental group oflarvae. Wheat bran with thefollowing nutritional values (data per 100g ofproduct) were used as feed: energy 1210kJ/292kcal, fats 5.3g, ofwhich saturated fatty acids accounted for 0.88g, carbohydrates 24.9g, ofwhich sugars were at 2.2g, fi ber 40.2g andprotein 16.2g, andsalt 0.1g. Oat bran with thefollowing nutritional values (data per 100g ofproduct) were used as feed: energy 1518kJ/ 361kcal, fats 8.3g, ofwhich saturated fatty acids accounted for 1.4g, carbohydrates 45g, ofwhich sugars were at 2.0g, fi ber 12g andprotein 21.0g, andsalt 0.01g. Determination ofdry matter content ofinfrared scales Ahomogenized sample ofabout 0.5g was evenly distributed over thealuminium foil andsubsequently analysed according to ISO 1442:1997using Precisa HA 300scales with infra radiator (Precisa Gravimetrics AG, Dietikon, Schweiz). Inthis method, thetest sample isheated andtheweight loss ismeasured. Thebasis ofthemethod isthedrying ofthesample byinfrared rays. Drying was carried out at 105°C until constant weight, where thedifference oftwo consecutive weights was not less than 2mg per min. Crude protein content determination Thenitrogen andcrude protein were analysed using theKjeldahl’s method [ISO 1871:2009]. Thesamples (1g) andblank runs were mineralized at 420°C for 105min. Thedistillation was performed on Kjeltec™ 2200 (FOSS, Denmark) for 4min. Theprotein content was calculated using nitrogen-to-protein conversion factor of6.25. Fat content determination Thefat was extracted acc.to Soxhlet’s method [Soxhlet, 1879] on theGerhardt Soxtherm (C.Gerhardt GmbH & Co. KG, Germany). Thesample (5g) was placed intheextraction capsule andextracted (program selected: 70°C for 120min) with 150mL ofpetroleum ether (Ing. Petr Švec – PENTA s.r.o., Prague, Czech Republic). Theextracted sample was then dried at 103°C andrepeatedly weighed until aconstant Fat from Tenebrionidae Bugs 249 weight (difference between two subsequent weighings ofless than 10mg) was achieved. Sterols content determination Sterols infat ofthesamples were analysed andevaluated inan accredited laboratory Skúšobňa VETLAB, Ltd., Púchov, Slovakia. Determination ofsterols content was done using capillary gas chromatography method according to ES no 213/2001 [Regulation (EC) No. 2013/2001, 2001]. Cholecalciferol (vitamin D3) content determination Cholecalciferol content determination was done according to EN 12821:2009 [EN 12821:2009, 2009]. Samples were extracted with hexane. Determination ofvitamin D3content was performed using semi-preparative HPLC on normal phase followed byanalytical reverse phase HPLC.Vitamin D was detected spectrophotometrically intheUV area. When determining vitamin D3, vitamin D2was used as an inner standard. Fatty acid profi le determination Weighted portions offat (0.5g) extracted from worms were esterifi ed according to theISO 12966-2: 2011 [ISO 12966–2:2011, 2011] using 0.25mol/L ofmethanolic potassium hydroxide. Fatty acid methyl esters inthesamples were determined using gas chromatography with afl ame ionization detector (GC/FID) on GC-2010 (Shimadzu, Kyoto, Japan), using ahigh polar chromatography column HP-88 (100m × 0.25mm × 0.2μm) (Agilent Technologies, CO, Santa Clara, USA), which isintended for theidentifi cation ofcis/trans fatty acid methyl esters. Chromatographic conditions were as follows: inlet volume – 1μL; inlet temperature – 250°C; split ratio – 1:100; carrier gas – nitrogen; andtemperature program – 80°C/5min, 200°C/30min, 250°C/15min. Quantitative evaluation oftheindividual fatty acid contents inthesamples was performed using theinternal normalization method using FAME Mixture C4-C24 (Supelco Inc, Bellefonte, USA) containing 37selected fatty acid methyl esters. Thecontent ofindividual fatty acids was calculated as apercentage ofthetotal methyl ester present. Lipid nutritional quality indices evaluation For each species, theatherogenicity index (AI) andthrombogenicity index (TI) were calculated according to thefollowing formulas [Zhang etal., 2014; Kulma etal., 2016]: AI = (C12:0 + 4x C14:0 + C16:0) / (ΣMUFA + Σn-6 + Σn-3), (1) TI = (C14:0 + C16:0 + C18:0) / (0.5x ΣMUFA + 0.5x Σn-6 + 3x Σn-3 + (n-3/n-6)), (2) where: MUFA stands for Mono Unsaturated Fatty Acids. These indexes were further calculated for both animal species from thefatty acid profi les from available literature. Thecholesterol-saturated acid index (CSI) was determined inlarvae ofmealworm fed ad libitum according to thefollowing formula [Pánek, 2002]: CSI = 1.01 x ΣSFA (g/100 g) + 50 x cholesterol (g/100 g), (3) where: SFA stands for Saturated Fatty Acids. Statistical analysis Each measurement was performed 4times. All samples were from thesame batch. Thedata was analysed using Excel 2013 (Microsoft Corporation, Redmond, USA) andSTATISTICA Cz version 12 (StatSoft, Inc., Tulsa, USA). Results were expressed byaverage andstandard deviation. Comparison oftheresults was performed using aKruskal-Wallis test (α=0.05; α=0.01). RESULTS ANDDISCUSSION To determine thebasic characteristic anddescribe thematerial inmealworm (Tenebrio molitor) andsuperworm (Zophobas morio) bred intheCzech Republic, thefollowing basic components were evaluated: dry matter (DM), crude protein inDM, andfat inDM (Table 1). Further description ofthematerial isgiven inAdámková [2017]. Thecholesterol content inmealworm (Tenebrio molitor) andsuperworm (Zophobas morio) fed ad libitum andthese under nutritional stress (Table 2); andcontents ofstigmasterol, β-sitosterol, andcholecalciferol inthead libitum-fed larvae (Table 3) were analysed. Theaverage cholesterol content inthead libitum-fed mealworm larvae (Tenebrio molitor) was 1335mg/kg DM.Superworm larvae (Zophobas morio) fed ad libitum contained higher amount ofcholesterol (3224mg/kg DM). Compared with theresults ofsamples that have been subjected to nutritional stress, cholesterol levels were higher inthead libitum fed larvae (Table 2). Statistically signifi cant (p<0.01) difference was detected between theworms without nutritional TABLE 1. Composition ofmaterial from mealworm (Tenebrio molitor) andsuperworm (Zophobas morio) bred intheCzech Republic – dry matter (DM), crude protein inDM andfat inDM. Components Zophobas morio Tenebrio molitor DM (g/100g) 47.9±0.6 32.6±0.7 Crude protein (g/100g DM) 39.4±0.1 62.6±0.4 Fat (g/100g DM) 39.1±0.4 16.7±0.1 DM – dry matter. TABLE 2. Cholesterol content indry matter ofmealworm (Tenebrio molitor) andsuperworm (Zophobas morio) fed ad libitum andunder nutritional stress. Nutrition oflarvae Tenebrio molitor Zophobas morio Ad libitum (mg/kg) 1 335±28Ab 3 224±35Aa Nutritional stress (mg/kg) 1 124±24Bb 2 985±31Ba A,B – means with different letters inthesame column are signifi cantly different at p<0.01; a,b – means with different letters inthesame row are signifi cantly different at p<0.01 250 J. Mlček et al. stress andthese subjected to nutritional stress. Theinfl uence ofinsect species on sterol content was highly statistically signifi cant. Thelevel ofsignifi cance was found to bep<0.01. Cholesterol issupposed to beburnt up innutritional stress to preserve life functions andeventually to build up theecdysteroid needed for molting. Other available literature does not provide detailed information on cholesterol content depending on thenutritional stress andthestage oflarval development inspecies we analysed. Sabolová etal. [2016] listed anumber ofselected sterols infarm insects from Sumatra Island andthese kept under European conditions. Insuperworm from Sumatra, theauthors demonstrated sterols content at 1784.1mg/kg DM andinthese from Czech Republic at 1594.9mg/kg DM.These values are lower than inour work, but higher than those detected inthesame species byRamos- -Bueno etal. [2016], i.e. 185mg/kg DM.Different values can becaused bynutrition because cholesterol isnot synthesized de novo, but theintake isdependent on thecomposition anddose offeed. Similar factors may infl uence thecholesterol levels inmealworm, inwhich Ramos-Bueno etal. [2016] determined cholesterol content at 726mg/kg DM.Inturn, Sabolová etal. [2016] reported 669.4mg/kg DM for thesame species bred inSumatra. These values are again lower than inour work. Ekpo etal. [2009] evaluated cholesterol content infats oftermites (Macrotermes bellicosus) andcaterpillars (Imbrasia Belina). They found out that theaverage cholesterol content intheir lipid fraction was up to 3.6%. Cholesterol content inmealworm (Tenebrio molitor) detected inour work iscomparable with some other commodities ofanimal origin, e.g. lobster (1460mg/kg). Superworm (Zophobas morio) iscomparable to carp (3540mg/kg), i.e. an animal commodity considered avaluable source ofnutrients andliver (up to 3450mg/kg) [Velíšek, 2002; Venugopal & Gopakuma, 2017]. Incomparison with other commodities, thespecies had higher cholesterol levels than most ofthefoods ofanimal origin. Cholesterol content inmealworm can becompared to beef tallow, mayonnaise, andLobster (northern) anditisbetween thevalues for butter andwhole eggs [Velíšek, 2002; Venugopal & Gopakuma, 2017]. Therecommended dietary allowance for cholesterol consumption is300mg/day for adult man [EFSA, 2010]. To achieve this level, theamount ofevaluated insects that would have to beconsumed is93g for superworm and224g for mealworm. However, itisassumed that normally such aquantity isnot consumed inthedried state, as itisnot expected for alarge quantity ofeggs to beeaten each day. To fi ll theRDA (Recommended Dietary Allowances) when eating eggs, 75g ofthewhole egg (about 1.5eggs day) isneeded. On theother hand, theedible insects analysed contained phytosterols (stigmasterol andβ-sitosterol) (Table 3), which serve as cholesterol antagonists, thus balancing thesterol levels. While comparing thespecies, astatistically signifi cant (p <0.01) difference was detected for stigmasterol andβ-sitosterol. Compared with thesamples from Sumatra, which were analysed bySabolová etal. [2016], thestigmasterol content was found to beup to 4times higher. On thecontrary, Sabolová etal. [2016] detected no stigmasterol intheedible insects from theCzech Republic. Inthecase ofβ-sitosterol inthemealworm, thecontent detected inthis work was lower than that measured bySabolová etal. [2016]. β-Sitosterol content ofthesuperworm measured bySabolová etal. [2016] was lower than thecontent detected inthis work at both sites oforigin. Ingeneral, nutritional values may depend on feed andliving conditions [Ghosh etal., 2017]. E.g. Van Broekhoven etal. [2015] demonstrated afat content intherange from 32.8to 43.5%, depending on themealworm feed. Oonincx [2015] reported theinfl uence offeed on nutritional values (fat, protein, fatty acid profi les, andselected minerals) under thesame breeding conditions ofthesame species. For this reason, thesterol content can beassumed to vary depending on nutrition andliving conditions. Although common commodities ofplant origin have ahigher total phytosterol content, some commodities are comparable intheir phytosterols content to edible insects (e.g. corn oil 590mg/kg, palm oil 376-627mg/kg) [Velíšek, 2002]. For this reason, edible insects may befound comparable with commodities ofplant origin. Thecontent ofstigmasterol ranges from 0% to 20% ofall sterols invegetable oils, while that ofβ-sitosterol isup to 62% ofall sterols inthese commodities [Velíšek, 2002]. When comparing theamount ofcholecalciferol, thevalues are comparable despite thedifference inthetotal content ofsterols – 190μg/kg inmealworm and199μg/kg insuperworm. Cholecalciferol content intheanalysed samples isat least three to four times higher than that ofother commodities ofanimal origin (except for fi sh), e.g. 3μg/kg inmeat, 10-20μg/kg inbutter, and30-50μg/kg ineggs [Velíšek, 2002]. Acomparable content ofcholecalciferol with theanalysed samples isstated byVelíšek [2002] for sea fi sh (50–450μg/kg). Edible insects can therefore beagood source ofcholecalciferol andconsequently meet therecommended daily dose for this compound. Inpractice, this means consuming approximately 25g ofdried mealworm or superworm to cover therequired dose ofcholecalciferol. From thenutritional point ofview, theprofi le oflipids andthecontent ofindividual fatty acids isimportant, as pointed out bytheWHO [Zielinská etal., 2015]. Table 4shows thefatty acid profi le ofthetwo insect species, where thesuperworm has ahigher saturated fatty acid content ofthetotal fatty acid content ofmore than 13% than mealworm. This material istherefore more advantageous interms oftechnological processing. However, dried material from mealworm, which has higher MUFA andPUFA contents, ismore suitable to prevent civilization diseases. TABLE 3. Content ofselected sterols andcholecalciferol indry matter ofmealworm (Tenebrio molitor) andsuperworm (Zophobas morio) larvae fed ad libitum. Compounds Tenebrio molitor Zophobas morio Stigmasterol (mg/kg) 18±6b44±12a β-Sitosterol (mg/kg) 171±20b414±37a Vitamin D3 (cholecalciferol) (μg/kg) 190±20a199±25a a,b – means with different letters inthesame row are signifi cantly different at p<0.01. Fat from Tenebrionidae Bugs 251 Differences inthefatty acid profi le against other authors inthemealworm are, for example, inthecontent ofoleic acid, which was determined inthis work byalmost 7% lower than inRavzanaadii etal. [2012] andmore than 13% than that found byTzompa-Sosa etal. [2014]. These authors reported also alower percentage oflinoleic acid – up to 8% than inour study. Insuperworm, there are differences inthefatty acid profi le, for example, inoleic acid content, where thedifference isup to 7% andinlinoleic acid content – up to 4% against thevalue reported byFinke [2002]. Thesample was similar invalues to that ofBarroso etal. [2014]. Differences between theabove nutritional values inthis work andtheother sources may bedue to different breeding conditions. Thecholesterol-saturated acid index was 36.6for mealworm and59.5for superworm. TheCSI values for mealworm are comparable to theCSI value ofgoose andduck fat, andfor thesuperworm these values correspond to thebovine tallow [Pánek etal., 2002]. Ramos-Bueno etal. [2016] analysed fatty acid profi le andcholesterol content inseven insect species, andcalculated CSI from these data, which reached 27.5for mealworm and46.3for superworm. These values are lower than thevalues calculated inthesamples analysed inour study. This may bedue to different feeds administered. TheAI, TI, andn-3:n-6ratio are determined infats to evaluate their health effects. Theatherogenic index was lower inmealworm than insuperworm (Table 5). Asimilar trend was also found inthethrombogenic index, which isalmost twice as low inmealworm. TheAI, TI andn-3:n-6ratio were calculated from fatty acid profi les ofother authors to enable comparison ( Table5), TABLE 4. Fatty acid composition inmealworm (Tenebrio molitor) andsuperworm (Zophobas morio) bred intheCzech Republic (% oftotal fatty acids). Fatty acid Zophobas morio Tenebrio molitor C12:0 0.1±0.00 0.2±0.03 C14:0 1.3±0.04 3.5±0.03 C16:0 32.3±0.01 18.6±0.08 C17:0 0.9±0.04 0.3±0.02 C18:0 8.2±0.10 6.7±0.46 C20:0 0.3±0.02 0.3±0.08 SFA sum 43.0 29.7 C16:1 (cis-9) 0.6±0.05 1.4±0.07 C18:1 (cis-9) 32.4±0.01 36.9±1.53 MUFA sum 33.0 38.3 C18:2 (cis-9.12) 23.4±0.15 30.9±1.08 C18:3 (cis-9.12.15) 0.6±0.01 1.1±0.03 PUFA sum 24.0 32.0 n-3sum 0.6 1.1 n-6sum 23.4 30.9 SFA – saturated fatty acids, MUFA – monousaturated fatty acids, PUFA – polyunsaturated fatty acids TABLE 5. Atherogenicity index (AI), thrombogenicity index (TI) andn-3:n-6ratio inmealworm (Tenebrio molitor) andsuperworm (Zophobas morio) bred intheCzech Republic with values calculated from thefatty acid profi le measured byother authors (% oftotal fatty acids). Stage Origin n-3:n-6 AI TI References Zophobas morio Larvae Czech Republic 0.027 0.7 1.4 This study Larvae Spain 0.045 0.7 1.4 Ramos-Bueno etal. [2016] Larvae USA 0.033 0.6 1.3 Finke [2002] Larvae Spain 0.062 0.6 1.2 Barroso etal. [2014] Tenebrio molitor Larvae Czech Republic 0.036 0.5 0.8 This study Larvae Spain 0.05 0.4 0.5 Ramos-Bueno etal. [2016] Larvae USA 0.04 0.4 0.6 Finke [2002] Adult USA 0.029 0.4 0.7 Finke [2002] Larvae Republic ofKorea 0.045 0.4 0.5 Ravzanaadii etal. [2012] Adult Republic ofKorea 0.023 0.4 0.7 Ravzanaadii etal. [2012] Larvae Spain 0.036 0.3 0.5 Sánches-Muros etal. [2016] Larvae Spain 0.036 0.3 0.5 Barroso etal. [2014] Not specifi ed Netherlands 0.038 0.4 0.6 Tzompa-Sosa etal. [2014] Larvae Poland 0.054 0.4 0.6 Zielińska etal. [2015] 252 J. Mlček et al. as theindices themselves have not yet been presented intheavailable literature. TheAI andTI determined byother authors were lower than inour work. AI inour research was 0.7for mealworm, while itwas 0.6acc. to both Finke [2002] andBarroso etal. [2014]. For superworm, thecalculated AI ranged between 0.3and0.4 [Finke, 2002; Ravzanaadii etal., 2012; Sánchez-Muros etal., 2016; Tzompa-Sosa etal., 2014; Zielińska etal., 2015]. Our results were slightly higher (0.5). Thesituation was similar for TI.Itcalculated values ranged from 1.2to 1.3for mealworm andfrom 0.5to 0.7for superworm [Finke, 2002; Barroso etal., 2014; Ravzanaadii etal., 2012; Sánchez-Muros etal., 2016; Tzompa-Sosa etal., 2014; Zielińska etal., 2015]. Inour work, TI was again slightly higher for both analysed species. Edible insect fat can becompared to thefat ofbeef or pork or to vegetable margarine [Stajić etal., 2011]. Thethrombogenic index for superworm (1.4) can becompared with these fats again, but thethrombogenic index ofmealworm issimilar to that ofchicken meat fat. Thecalculated atherogenic index oftheprofi les reported byother authors can becompared with polyunsaturated acids ofmargarines, its value issimilar to that ofolive oil. Taking into account therisk ofcardiovascular disease, theconsumption ofthemealworm ismore favourable. Another important factor describing therisk ofthemetabolic syndrome arising from fat consumption istheevaluation oftheratio ofn-3andn-6fatty acids, which, according to WHO recommendations should be1:2to 1:6 [Mourek & Mourek, 2011; Jirák & Zeman, 2007]. Thereal n-3:n-6ratio isnormally 1:15inthediet oftheWestern civilization, as reported bySimopoulos [2002]. Theratio determined for our samples (Table 5) as well as for thevalues calculated byother authors ishigher than this. Although this ratio isnot too favorable for theconsumption ofedible insect fats, theother parameters observed inthis work balance this drawback. TheScientifi c Opinion on Dietary Reference Values for fats, including saturated fatty acids, polyunsaturated fatty acids, monounsaturated fatty acids, trans fatty acids, andcholesterol [EFSA, 2010] does not mention therecommendation for n-6:n-3ratio. According to this material, linoleic acid intake should not fall below 4% andthat oflinolenic acid below 0.5% oftotal energy intake. An average man with light work has atotal energy intake of10,000kJ andshould receive 400kJ oflinoleic acid and50kJ oflinolenic acid. Assuming an energy value of37.6812kJ/g, itispossible to calculate theamount which isnecessary to betaken inthediet, i.e. 10.6g oflinoleic acid and1.3g linolenic acid. Theamount ofdry matter from mealworms required to cover theneed for linoleic acid per day was calculated at 205g andthat need to cover for linolenic acid at 708g. Inthecase ofZophobas morio, therespective amount ofdry matter is116g for linoleic acid and554g for linolenic acid. Detected contents oflinoleic acid (5.16g/100g DM) andlinolenic acid (0.18g/100g DM) for Tenebrio molitor were lower than these determined for Zophobas morio (linoleic acid content 9.15g/100g DM, linolenic acid content 0.23g/100g DM). Inthecase ofdried meat, thecontent oflinoleic acid is1.02g/100g andthat oflinolenic acid is0.39g/100g [Huis etal., 2013]. Therefore, to receive adaily dose oflinoleic acid, up to 9times less dry matter ofsuperworm isneeded incomparison to beef. On theother hand, for linoleic acid, less dry meat isneeded than mealworm or superworm dry matter. Therefore, inorder to minimise theweight ofthedry matter as anutritional dose (for example, inaperson’s special diet inplaces without access to aregular diet), acombination ofboth commodities can berecommended inthediet. CONCLUSIONS Inthis study, cholesterol, β-sitosterol, andstigmasterol were analysed intwo edible insect species: mealworm (Tenebrio molitor) andsuperworm (Zophobas morio). When fed ad libitum, superworm had ahigher content ofsterols than mealworm. During thenutritional stress there was astatistically signifi cant drop ofthecholesterol content inboth species. This suggests that cholesterol levels could beregulated inedible insects byproper nutrition. Although material oftheanimal origin isanalysed, sterols ofplant origin (β-sitosterol, stigmasterol) have been detected. For this reason, itispossible to assume that both species could serve as asource ofcholesterol (zoosterol) andphytosterols simultaneously (two inone). Further analysis proved asignifi cant content ofcholecalciferol, which was thesame for both species, although thefat content differed. Another benefi t isthelower weight ofdry matter necessary to consume to cover thedaily dose oflinoleic acid compared to dried beef. Thelinoleic acid content ofmealworm (30.9g/100g) or superworm (23.4g/100g) iscomparable with, for example, chicken lard, pork lard or goose lard. Based on thelegalization ofedible insects as anovel food inEurope since 2018, itisnot aproblem to include this commodity inthediet both inthehidden form (dry matter for food fortifi cation) andinthevisible form as an experience food. RESEARCH FUNDING This research was supported bytheinternal grant ofTBU inZlín [No. IGA/FT/2019/004] andproject BUT inBrno [No. FEKT S-17–3934]. CONFLICTS OFINTEREST Theauthors declare no confl ict ofinterest. REFERENCES 1. Adámková, A., Kouřimská, L., Borkovcová, M., Kulma, M., Mlček, J. (2016). Nutritional values ofedible Coleoptera (Tenebrio molitor, Zophobas morio andAlphitobius diaperinus) reared intheCzech Republic. 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Published on-line: 3 July 2019.