nitrogen to protein conversion factors for three edible insects tenebrio molitor alphitobius diaperinus and hermetia illucens

In this paper, a specific Kp of 4.76±0.09 was calculated for larvae from Tenebrio 18 molitor, Alphitobius diaperinus and Hermetia illucens, using amino acid analysis.. 22 Keywords 23

Journal of Agricultural and Food Chemistry is published by the American Chemical Society 1155 Sixteenth Street N.W., Washington, DC 20036

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

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Nitrogen-to-protein conversion factors for three edible insects: Tenebrio molitor,

1

Alphitobius diaperinus and Hermetia illucens

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Renske H Janssen1,2, Jean-Paul Vincken2, Lambertus A.M van den Broek3, Vincenzo

3

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 Laboratory of Food Chemistry, Wageningen University and Research, P.O Box 17, 6700

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AA Wageningen, The Netherlands

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3 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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catriona.lakemond@wur.nl

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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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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,

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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<0.001) than the general nitrogen factor of 6.25, which has been used up to now to

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calculate the protein content of insects.4–6,8,16 The Kp values found for insects are similar to

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those calculated for different tropical plants (Kp range 3.7-5.0)18, microalgae (Kp range

2.53-114

5.77)12,15,19, as well as different grains and legumes (Kp range 5.09-5.38).20 Higher values

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between 5.14-6.26 were found for meat, fish and egg.21

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This new Kp value gives a more accurate estimation of protein content by taking the presence

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of NPN into account This leads to lower of more than 20% the values for protein content

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compared to literature values, which are based on Kp of 6.25 So, the protein content of T

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molitor calculated in this study was 45%, which falls in the low range (45-65%) found in

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literature based on Kp of 6.25.6,22 The protein content falls out of the range for the larvae of A

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diaperinus, for which a value of 49% was found compared to literature values of 58-65%

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protein.4,16,22 Also for H illucens a lower value of 36% was found compared to the range of

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37-56% from the literature.16,23 When calculating protein content from our data using a Kp of

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6.25 the results do fall again into the ranges reported in literature

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The average Kp value of 5.60±0.24 obtained for soluble protein extracted from insects were

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significantly (P<0.001) higher compared to those for whole larvae, due to the removal of

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NPN Again, comparable Kp values among the three species were found

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Non-protein nitrogen

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The calculated Naa/Nt ratio showed the presence of 11-26% NPN in whole larvae of all three

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insect species (Table 2) Tenebrio molitor contained 12-23% NPN, which is in line with Finke

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(2002).7 The NPN of 16-26% present in H illucens is higher compared to the 2% found in

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literature, whereas the amino acid composition and content were similar.8 Beside the

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analytical procedures, differences in composition and recovery might be also caused by

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different diets fed to the insects.17

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Carbohydrates, like chitin and chitosan have glucosamine or N-acetyl glucosamine with

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nitrogen as building block During the hydrolysis conditions used, N-acetyl glucosamine was

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converted into N-glucosamine The total amount of (N-acetyl) glucosamine within polymers

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for the three insect species was 4.4-9.1% (w/w), corresponding to approximately 1/3 of the

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carbohydrates present, similar to results based on acid detergent fiber fraction for T molitor.24

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The chitin content comprised 3.0-6.8% nitrogen of the total nitrogen Apart from chitin, NPN

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might originate from nucleic acids.9 Part of the NPN can also come from inorganic nitrogen

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Examples of inorganic nitrogen are excretion products in intestinal tract of the larvae, like uric

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acid, urea and ammonia.10 This is in agreement with the removal of most NPN during dialysis

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of the protein extracts

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Protein extraction yields

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The average Kp values for the whole larvae and extracts were used to determine the protein

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content and extraction yield based on nitrogen (Table 2) Protein extraction yields between

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17.1-23.5% were calculated using the insect specific Kp factors and these were higher

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compared to those obtained with the general Kp of 6.25 (14.4-17.6%) This is due to a larger

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overestimation of the protein content within the whole larvae when the factor of 6.25 was

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used caused by NPN

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When considering insect larvae as an alternative protein source, overestimation of the protein

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content, due to the presence of NPN, should be avoided To avoid overestimation of protein

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content in insects, we propose the use of a Kp value of 4.76 for the quantification of protein

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content in whole larvae, and a Kp of 5.60 for the protein extracts derived from insects

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Abbreviations Used

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Kp: ratio of the sum of nitrogen from amino acid residue weights to total nitrogen from

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Dumas measurement

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Nt: Total nitrogen content based on Dumas measurement

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Naa: Total nitrogen from amino acid analysis

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NGlcN: Total nitrogen from glucosamine

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Acknowledgements

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Dennis Oonincx is kindly acknowledged for providing the Hermetia illucens larvae We are

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grateful to the Animal Nutrition group from Wageningen University & Research for

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performing the amino acid analysis

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