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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
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Journal of Agricultural and Food Chemistry Janssen et al., Specific Kp to calculate protein content of insects
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Nitrogen-to-protein conversion factors for three edible insects: Tenebrio molitor, Alphitobius diaperinus and Hermetia illucens
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Renske H. Janssen1,2, Jean-Paul Vincken2, Lambertus A.M. van den Broek3, Vincenzo
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Fogliano1, Catriona M.M. Lakemond1*
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1
Food Quality and Design, Wageningen University and Research, P.O. Box 17, 6700
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AA Wageningen, The Netherlands
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2
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AA Wageningen, The Netherlands
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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,
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6700 AA Wageningen, The Netherlands
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*corresponding author: Catriona M.M. Lakemond, telephone +31 317 480 288, email
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[email protected] 1
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Journal of Agricultural and Food Chemistry Janssen et al., Specific Kp to calculate protein content of insects
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Abstract
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Insects are considered as a nutritionally valuable source of alternative proteins and their
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efficient protein extraction is a prerequisite for large scale use. The protein content is usually
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calculated from total nitrogen using the nitrogen-to-protein conversion factor (Kp) of 6.25.
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This factor overestimates the protein content, due to the presence of non-protein nitrogen in
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insects. In this paper, a specific Kp of 4.76±0.09 was calculated for larvae from Tenebrio
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molitor, Alphitobius diaperinus and Hermetia illucens, using amino acid analysis. After
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protein extraction and purification, a Kp factor of 5.60±0.39 was found for the larvae of three
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insect species studied. We propose to adopt these Kp values for determining protein content
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of insects to avoid overestimation of the protein content.
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Keywords
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Protein extraction, Tenebrio molitor, Alphitobius diaperinus, Hermetia illucens, black soldier
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fly, yellow mealworm, lesser mealworm, edible insects, amino acids, nitrogen-to-protein
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conversion factor (Kp)
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Journal of Agricultural and Food Chemistry Janssen et al., Specific Kp to calculate protein content of insects
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Introduction
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There is an increasing interest for alternative protein sources to feed the increasing world
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population.1 Insects represent one of the potential sources to exploit. The high protein content,
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40-75% on dry matter basis, makes insects a promising protein alternative for both food and
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feed.2 Their nutritional composition and ease of rearing makes insects especially interesting
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for food and feed production when they are in the larval stage.3 To use insects as alternative
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food protein source, efficient protein extraction is a prerequisite, as potential consumers do
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not like to recognize the insects as such.
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The protein content of different insect species in literature is mainly based on nitrogen content
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using the nitrogen-to-protein conversion factor (Kp) of 6.25 generally used for proteins.2,4–8
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The presence of non-protein nitrogen (NPN) in insects, e.g. chitin, nucleic acids,
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phospholipids and excretion products (e.g. ammonia) in the intestinal tract, could lead to an
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overestimation of the protein content.9,10 Finke (2007) estimated that the amount of nitrogen
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present from chitin would not significantly increase the total amount of nitrogen.11
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The aim of this research was to determine the specific nitrogen-to-protein conversion factor
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(Kp) for larvae of the three insect species and their protein extracts using amino acid
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composition data. In this way an accurate protein content can be determined from analysis of
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the nitrogen content. Larvae of Tenebrio molitor (yellow mealworm), Alphitobius diaperinus
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(lesser mealworm) and Hermetia illucens (black soldier fly) were used.
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Materials and methods
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Tenebrio molitor and Alphitobius diaperinus larvae were purchased from Kreca Ento-Feed
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BV (Ermelo, The Netherlands). Hermetia illucens larvae were kindly provided by laboratory
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of Entomology (Wageningen University, The Netherlands). Larvae were frozen with liquid
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nitrogen and stored at -22 °C. The larvae from the three species were freeze dried before
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chitin, nitrogen and amino acid analysis.
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The dry matter content and ash content were determined gravimetrically by drying and
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incinerating the samples at, respectively, 105 °C and 525 °C overnight in triplicate.
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For carbohydrate analysis, larvae were frozen and ground in liquid nitrogen. The ground
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larvae were freeze-dried and subsequently hydrolyzed and analyzed for carbohydrates
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according to Gilbert-López et al. (2015)12 with some modifications. An ICS-3000 Ion
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Chromatography HPLC system equipped with a Dionex™ CarboPac PA-1 column (2×250
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mm) in combination with a Dionex™ CarboPac PA guard column (2×25 mm) and a pulsed
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electrochemical detector in pulsed amperometric detection mode was used (ThermoFisher
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Scientific, Breda, NL). A flow rate of 0.25 mL min-1 was used and the column was
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equilibrated with H2O. Elution was performed as follows: 0-35 min H2O, 35-50 min 0-40% 1
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M sodium acetate in 100 mM NaOH, 50-55 min 1 M sodium acetate in 100 mM NaOH, 55-
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60 min 150 mM NaOH, 70-85 min H2O. Detection of the monosaccharides was possible after
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post column addition of 0.5 M sodium hydroxide (0.15 mL min-1). Elution was performed at
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20 °C and to discriminate between glucose and glucosamine an additional run was performed
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at 28 °C using the same settings.
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Fat content was determined gravimetrically after petroleum ether extraction using Soxhlet in
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duplicate.13
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For protein extraction, frozen larvae were blended at 4 °C in 0.1 M citric acid - 0.2 M
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disodium phosphate buffer at pH 6 in a ratio of 1:4 (w/v) using a kitchen blender (Philips,
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Eindhoven, NL). The obtained solutions were centrifuged for 20 min. at 25,800 g and 15 °C
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using a high speed centrifuge (Beckman Coulter, Woerden, NL). The supernatant was filtered
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twice through cellulose filter paper (grade: 424, VWR, USA) and dialyzed at 4 °C at a cut off
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of 12–14 kDa (Medicell Membranes ltd, London, UK). Dialyzed protein extracts were
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considered as soluble protein extract and stored at -20 °C after freeze-drying. Extraction was
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performed in duplicate.
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Amino acid composition was determined in duplicate by the ISO13903 (2005) method14,
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adjusted for micro-scale. The amide nitrogen from Asn/Gln were measured together with
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Asp/Glu. The amount of tryptophan was determined based on AOAC 988.15. Total protein
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content was calculated from the total amino acid content.
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Nitrogen content (Nt) was determined in triplicate by the Dumas method using a Flash EA
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1112 NC analyser (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the
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manufacturer’s protocol. Average Kp values were calculated from the ratio of the sum of
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amino acid residue weights to Nt. Kp values were statistically evaluated by analysis of
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variance (ANOVA) with the SPSS 23 program. The percentage protein nitrogen from total
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nitrogen was determined by total amino acid nitrogen (Naa)/Nt. The lower limit of this
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percentage was calculated based on the theoretical value with 100% Asp/Glu and the upper
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level with 100% Asn/Gln.15
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Results and discussion
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Nutritional composition of whole insects
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The amino acid profile both from whole larvae and their protein extract contain high amounts
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of all essential amino acids (Table 1). Overall, amino acid profiles were comparable as
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observed before for T. molitor, A. diaperinus4 and H. illucens.8,16 From the amino acid
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profiles, the total nitrogen from amino acids and the accurate protein content was determined
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(Table 2).
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General composition data are summarized in Figure 1. The protein values based on amino
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acid content for T. molitor and A. diaperinus were lower compared to Yi et al. (2013).4 A.
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diaperinus showed the highest protein content based on total amino acid content within the
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tested species. The total carbohydrate content within the three species ranged from 15-21%.
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The fat content for the three species ranged from 21-24% based on dry matter. In literature,
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fat contents between 27-49% for T. molitor4,6,13, 20-22% for A. diaperinus4,16 and 13-36% for
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H. illucens8,16 have been reported. Differences in chemical composition were probably caused
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by different diets.6,17 Our results show that proteins, fats, and carbohydrates accounted for
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around 90% of the total dry matter; the remainder might come from other organic components
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i.e. phenols and nucleic acids.
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Nitrogen-to-protein conversion factors
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In order to determine the protein content from total nitrogen content, the Kp and ratio Naa/Nt
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were calculated (Table 2). Interestingly, comparable Kp values were found among larvae of
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the three species with an average Kp value of 4.76±0.09, despite the fact that H. illucens
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belongs to the different order (Diptera) as T. molitor and A. diaperinus (which are
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Tenebriodinae family members within the Coleoptera order). This Kp value was significantly
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lower (P
