Subscriber access provided by University of Newcastle, Australia Letter Nitrogen-to-protein conversion factors for three edible insects: Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens Renske H Janssen, Jean-Paul Vincken, Lambertus A.M van den Broek, Vincenzo Fogliano, and Catriona M.M Lakemond J Agric Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b00471 • Publication Date (Web): 02 Mar 2017 Downloaded from http://pubs.acs.org on March 7, 2017 Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication They are posted online prior to technical editing, formatting for publication and author proofing The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract “Just Accepted” manuscripts have been fully peer 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American Chemical Society However, no copyright claim is made to original U.S Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties Page of 16 Journal of Agricultural and Food Chemistry Janssen et al., Specific Kp to calculate protein content of insects Nitrogen-to-protein conversion factors for three edible insects: Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens Renske H Janssen1,2, Jean-Paul Vincken2, Lambertus A.M van den Broek3, Vincenzo Fogliano1, Catriona M.M Lakemond1* Food Quality and Design, Wageningen University and Research, P.O Box 17, 6700 AA Wageningen, The Netherlands AA Wageningen, The Netherlands Laboratory of Food Chemistry, Wageningen University and Research, P.O Box 17, 6700 Wageningen Food & Biobased Research, Wageningen University and Research P.O Box 17, 10 6700 AA Wageningen, The Netherlands 11 *corresponding author: Catriona M.M Lakemond, telephone +31 317 480 288, email 12 catriona.lakemond@wur.nl ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Janssen et al., Specific Kp to calculate protein content of insects 13 Abstract 14 Insects are considered as a nutritionally valuable source of alternative proteins and their 15 efficient protein extraction is a prerequisite for large scale use The protein content is usually 16 calculated from total nitrogen using the nitrogen-to-protein conversion factor (Kp) of 6.25 17 This factor overestimates the protein content, due to the presence of non-protein nitrogen in 18 insects In this paper, a specific Kp of 4.76±0.09 was calculated for larvae from Tenebrio 19 molitor, Alphitobius diaperinus and Hermetia illucens, using amino acid analysis After 20 protein extraction and purification, a Kp factor of 5.60±0.39 was found for the larvae of three 21 insect species studied We propose to adopt these Kp values for determining protein content 22 of insects to avoid overestimation of the protein content 23 Keywords 24 Protein extraction, Tenebrio molitor, Alphitobius diaperinus, Hermetia illucens, black soldier 25 fly, yellow mealworm, lesser mealworm, edible insects, amino acids, nitrogen-to-protein 26 conversion factor (Kp) ACS Paragon Plus Environment Page of 16 Page of 16 Journal of Agricultural and Food Chemistry Janssen et al., Specific Kp to calculate protein content of insects 27 Introduction 28 There is an increasing interest for alternative protein sources to feed the increasing world 29 population.1 Insects represent one of the potential sources to exploit The high protein content, 30 40-75% on dry matter basis, makes insects a promising protein alternative for both food and 31 feed.2 Their nutritional composition and ease of rearing makes insects especially interesting 32 for food and feed production when they are in the larval stage.3 To use insects as alternative 33 food protein source, efficient protein extraction is a prerequisite, as potential consumers 34 not like to recognize the insects as such 35 The protein content of different insect species in literature is mainly based on nitrogen content 36 using the nitrogen-to-protein conversion factor (Kp) of 6.25 generally used for proteins.2,4–8 37 The presence of non-protein nitrogen (NPN) in insects, e.g chitin, nucleic acids, 38 phospholipids and excretion products (e.g ammonia) in the intestinal tract, could lead to an 39 overestimation of the protein content.9,10 Finke (2007) estimated that the amount of nitrogen 40 present from chitin would not significantly increase the total amount of nitrogen.11 41 The aim of this research was to determine the specific nitrogen-to-protein conversion factor 42 (Kp) for larvae of the three insect species and their protein extracts using amino acid 43 composition data In this way an accurate protein content can be determined from analysis of 44 the nitrogen content Larvae of Tenebrio molitor (yellow mealworm), Alphitobius diaperinus 45 (lesser mealworm) and Hermetia illucens (black soldier fly) were used ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Janssen et al., Specific Kp to calculate protein content of insects 46 Materials and methods 47 Tenebrio molitor and Alphitobius diaperinus larvae were purchased from Kreca Ento-Feed 48 BV (Ermelo, The Netherlands) Hermetia illucens larvae were kindly provided by laboratory 49 of Entomology (Wageningen University, The Netherlands) Larvae were frozen with liquid 50 nitrogen and stored at -22 °C The larvae from the three species were freeze dried before 51 chitin, nitrogen and amino acid analysis 52 The dry matter content and ash content were determined gravimetrically by drying and 53 incinerating the samples at, respectively, 105 °C and 525 °C overnight in triplicate 54 For carbohydrate analysis, larvae were frozen and ground in liquid nitrogen The ground 55 larvae were freeze-dried and subsequently hydrolyzed and analyzed for carbohydrates 56 according to Gilbert-López et al (2015)12 with some modifications An ICS-3000 Ion 57 Chromatography HPLC system equipped with a Dionex™ CarboPac PA-1 column (2×250 58 mm) in combination with a Dionex™ CarboPac PA guard column (2×25 mm) and a pulsed 59 electrochemical detector in pulsed amperometric detection mode was used (ThermoFisher 60 Scientific, Breda, NL) A flow rate of 0.25 mL min-1 was used and the column was 61 equilibrated with H2O Elution was performed as follows: 0-35 H2O, 35-50 0-40% 62 M sodium acetate in 100 mM NaOH, 50-55 M sodium acetate in 100 mM NaOH, 55- 63 60 150 mM NaOH, 70-85 H2O Detection of the monosaccharides was possible after 64 post column addition of 0.5 M sodium hydroxide (0.15 mL min-1) Elution was performed at 65 20 °C and to discriminate between glucose and glucosamine an additional run was performed 66 at 28 °C using the same settings 67 Fat content was determined gravimetrically after petroleum ether extraction using Soxhlet in 68 duplicate.13 ACS Paragon Plus Environment Page of 16 Page of 16 Journal of Agricultural and Food Chemistry Janssen et al., Specific Kp to calculate protein content of insects 69 For protein extraction, frozen larvae were blended at °C in 0.1 M citric acid - 0.2 M 70 disodium phosphate buffer at pH in a ratio of 1:4 (w/v) using a kitchen blender (Philips, 71 Eindhoven, NL) The obtained solutions were centrifuged for 20 at 25,800 g and 15 °C 72 using a high speed centrifuge (Beckman Coulter, Woerden, NL) The supernatant was filtered 73 twice through cellulose filter paper (grade: 424, VWR, USA) and dialyzed at °C at a cut off 74 of 12–14 kDa (Medicell Membranes ltd, London, UK) Dialyzed protein extracts were 75 considered as soluble protein extract and stored at -20 °C after freeze-drying Extraction was 76 performed in duplicate 77 Amino acid composition was determined in duplicate by the ISO13903 (2005) method14, 78 adjusted for micro-scale The amide nitrogen from Asn/Gln were measured together with 79 Asp/Glu The amount of tryptophan was determined based on AOAC 988.15 Total protein 80 content was calculated from the total amino acid content 81 Nitrogen content (Nt) was determined in triplicate by the Dumas method using a Flash EA 82 1112 NC analyser (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the 83 manufacturer’s protocol Average Kp values were calculated from the ratio of the sum of 84 amino acid residue weights to Nt Kp values were statistically evaluated by analysis of 85 variance (ANOVA) with the SPSS 23 program The percentage protein nitrogen from total 86 nitrogen was determined by total amino acid nitrogen (Naa)/Nt The lower limit of this 87 percentage was calculated based on the theoretical value with 100% Asp/Glu and the upper 88 level with 100% Asn/Gln.15 ACS Paragon Plus Environment Journal of Agricultural and Food Chemistry Janssen et al., Specific Kp to calculate protein content of insects 89 Results and discussion 90 Nutritional composition of whole insects 91 The amino acid profile both from whole larvae and their protein extract contain high amounts 92 of all essential amino acids (Table 1) Overall, amino acid profiles were comparable as 93 observed before for T molitor, A diaperinus4 and H illucens.8,16 From the amino acid 94 profiles, the total nitrogen from amino acids and the accurate protein content was determined 95 (Table 2) 96 General composition data are summarized in Figure The protein values based on amino 97 acid content for T molitor and A diaperinus were lower compared to Yi et al (2013).4 A 98 diaperinus showed the highest protein content based on total amino acid content within the 99 tested species The total carbohydrate content within the three species ranged from 15-21% 100 The fat content for the three species ranged from 21-24% based on dry matter In literature, 101 fat contents between 27-49% for T molitor4,6,13, 20-22% for A diaperinus4,16 and 13-36% for 102 H illucens8,16 have been reported Differences in chemical composition were probably caused 103 by different diets.6,17 Our results show that proteins, fats, and carbohydrates accounted for 104 around 90% of the total dry matter; the remainder might come from other organic components 105 i.e phenols and nucleic acids 106 Nitrogen-to-protein conversion factors 107 In order to determine the protein content from total nitrogen content, the Kp and ratio Naa/Nt 108 were calculated (Table 2) Interestingly, comparable Kp values were found among larvae of 109 the three species with an average Kp value of 4.76±0.09, despite the fact that H illucens 110 belongs to the different order (Diptera) as T molitor and A diaperinus (which are 111 Tenebriodinae family members within the Coleoptera order) This Kp value was significantly 112 lower (P