Protein quality of edible insects in the view of current assessment methods

Abstract

Protein quality refers to the ability of dietary protein sources to supply indispensable amino acids (IAA) to meet the requirements of the target organism in either feed or food. Methods for quantifying protein quality are based on measures for either digestibility or growth. For insect protein quality, the evidence is growing though still a limited number of studies are conducted. Applying appropriate methods is critical to ensure the best future applications of insect protein for animal feed and direct human consumption. For evaluating the protein quality of insects for humans using DIAAS method, the generalized nitrogen-to-protein conversion factor of 6.25 is the recommended standard for quantifying crude protein. This factor results in an underestimation of protein quality due to the nature of the chitin non-protein nitrogen in insects. Thus, the evaluation should consider IAA instead of crude protein. Tenebrio molitor (Yellow mealworm) and Hermetia illucens (Black soldier fly) are the most studied species. Across studies, T. molitor has the highest crude protein digestibility, while the effect on animal growth performance is similar. Both insect species are suitable to be used in animal feed. The few studies investigating edible insect protein quality for humans indicate that among the species relevant for insect farming, cricket species may have slightly better protein quality than mealworm species, though inconclusive till more data is available. Over the past decade, following the rise in global awareness of the potential of edible insects, their nutritional properties have been studied, though still in infancy compared to established feed and food sources. The nutritional composition among more than 2,000 insect species recorded to be utilized for animal feed or direct human consumption differs widely in nutritional content, though sharing that protein is a significant component (van Huis et al., 2021). Protein and amino acid (AA) content and composition differ between species, developmental stages (pupae, larva, and adult) (Jonas-Levi and Martinez, 2017); location and season of the collection; the processing of samples, etc (Churchward-Venne et al., 2017; Janssen et al., 2019). Following the great diversity of edible insects, the AA profile of insects shows equal variability hence, it also challenges establishing a clear picture of the protein quality. Overall, insects are considered a valuable source of alternative protein for feed and food, with the potential for sustainable mass production. To fully release these potentials, insect protein needs to be characterized for quality to ensure the best future applications. This review aims to provide an overview of suitable methods to evaluate protein quality and, secondly, provide a concise review of the available information on insect protein quality. Protein quality refers to the ability of dietary protein sources to supply IAAs to meet the requirements of the target organism, in either feed or food (Fact box 1). Hence, commonly used principles of expressing protein quality are based on the ability of a protein source to supply sufficient IAA for a specific target group. Over a century, different methods have been developed to quantify and classify protein quality. The methods build on two principles, either as evaluation of the contents and digestibility of IAA or measured as the physiological impact, such as the growth of the target organism. For humans, IAA requirements are defined for different age groups. Except for breast milk for infants, no single dietary protein fully matches the requirements (FAO, 2013). While animals and humans share the physiological need to rely on a dietary supply of indispensable AA (IAA), the protein quality of a protein source is more commonly used to describe the quality of human food than the quality of animal feed. In evaluating the nutritional adequacy of feed for monogastric livestock, the IAA concentrations, the IAA ileal digestibility, and the endogenous IAA losses of the protein sources are taken into account when formulating composite diets fulfilling the minimum nutritional requirements (Stein et al., 2007). In contrast, for humans, it is, in most cases, impossible and not desirable for healthy individuals to control dietary habits. Therefore, the concept of protein quality in single foods is used to facilitate guidance on protein sources in diverse diets based on consumers' preferences. Because of the wider diversity of foods that humans consume, the Food and Agriculture Organization (FAO) has facilitated the recommendation of specific standards based on scientific criteria for evaluating comparable measures for protein quality. Assessment of the digestibility of the protein sources is used as an indicator to which extent the AA can be hydrolyzed by gastrointestinal enzymes and be available for absorption in a target organism. (Stein et al., 2007). Evaluation of protein digestibility can be carried out either in-vivo or in-vitro. For the in-vivo digestibility methods, the point of sampling for digesta, i.e., either at the ileum or after the passage of the total gastrointestinal tract in the feces, has a significant impact on the protein quality measurements (Stein et al., 2007). Total tract digestibility – also termed as “apparent total tract digestibility” (ATTD) – is the simplest and longest-standing method of measuring fecal nitrogen (N) recovery relative to intake, assuming AAs are absorbed throughout the digestive tract (Stein et al., 2007). However, in the large intestine of monogastric animals and humans, no significant AA absorption takes place, and N may be absorbed as ammonia and excreted in the urine (van der Wielen et al., 2017). Also, the microbiota in the large intestine catabolizes and synthesizes AA, making fecal digestibility not truly represent the protein source (Hendriks et al., 2012). To adjust for this, ATTD can be corrected for endogenous losses using an N-free diet but remain to account for N or AAs absorbed in the large intestine. Ileal digestibility has been developed and standardized by the swine and poultry industries for feed formulation and adapted to improve human protein quality measures. The apparent ileal digestibility (AID) – overcomes the limitations of ATTD, but the measure is more complicated by requiring surgically fitted T-cannula to tap ileal digesta (Stein et al., 2007). Also, the ileal digesta is not exclusively of dietary origin, and basal and diet-specific endogenous AA losses add some noise to this method. The “true ileal digestibility” (TID) method overcomes these limitations by adding protein-free or well-defined AA diets, along with tracers or markers, to follow the transition in the tract. The TID is also called “standardized ileal digestibility” (SID) when no diet-specific endogenous AA losses exist. TID is considered the most accurate estimation of digestibility. However, it can also overestimate bioavailability for some diets, for example, due to the Maillard reaction (Jensen et al., 2019) between AAs and carbohydrates, not affecting the digestibility, but reducing the bioavailability of AAs. Laboratory-based in-vitro digestibility methods that mimic digestive processes using proteolytic enzymes are widely used as a simple, less costly alternative to in-vivo digestibility (Egger et al., 2017). Although in-vitro digestion methods are being further developed into dynamic models of digestion (Egger et al., 2017), these methods can only partially mimic the actual gastrointestinal peristalsis conditions. Several insect species evaluated in in-vitro systems have been reviewed by Rodríguez-Rodríguez et al. (2022). This review concluded that there appeared to be great differences in in-vitro digestibility between insect species, but also found methodological issues calling for standardized in vitro protocols to secure comparable results between studies. With few exceptions of human studies, in-vivo methods are carried out in animals assessing the protein quality in relation to animal nutritional requirements or used as an animal model for protein quality in relation to human requirements. The different in-vivo methods and their principles are based on either assessing growth or IAA digestibility or absorbed IAA (Osborne et al., 1919; FAO/WHO/UNU, 1991; WHO/FAO/UNU, 2007; FAO, 2013; Devi et al., 2018), as shown in Table 1. In-vivo methods for determining protein quality of food Abbreviations: TP = Test Protein; RP = Reference Protein; AA = amino acids; N = Nitrogen; IAA = indispensable amino acid; IAAlim= first limiting indispensable amino acid. In-vivo methods for determining protein quality of food Abbreviations: TP = Test Protein; RP = Reference Protein; AA = amino acids; N = Nitrogen; IAA = indispensable amino acid; IAAlim= first limiting indispensable amino acid. Several methods are based on the assessment of the growth of animals, determined for feeding a diet containing a test protein and compared to a reference protein. The “protein efficiency ratio” (PER) and “NPR” (NPR) are widely used growth-based methods for protein quality evaluation. The PER in growing rats developed by Osborne et al. (1919) was the first method adopted for assessing the protein quality of human food. The PER method calculates the efficiency of dietary crude protein (CP) for body weight gain with casein as a reference protein. The PER ranks protein sources against a single reference, but this ranking is not proportional. The NPR is a modification of PER by adjusting for the weight loss of rats in the control group that is given a protein-free diet. The weight loss of the rat fed protein free diet is considered to be equivalent to the protein needed for maintenance. Both PER and NPR use rats and the need of Sulphur amino acids in rats is higher than in humans, and the outcomes thus can overestimate the protein quality for humans (Deglaire and Moughan, 2012). The digestibility measures reflect a protein’s enzymatic breakdown and adequacy to supply IAA by sampling either ileal digesta or feces from animals or humans. As outlined, ileal digesta is preferred over fecal digesta. For protein evaluation for food, humans are preferred, and as animal models, pigs are superior to rats because their digestibility and metabolism are more like humans (FAO, 2014). Only a few human studies have determined the ileal digestibility due to the technical, economic, and ethical barriers of invasive methods for ileal sampling. Consequently, most in-vivo data come from animal models. The common methods for evaluating protein quality using animal models are: “biological value” (BV), “net protein utilization” (NPU), “protein digestibility-corrected AA score” (PDCAAS), and the “digestible indispensable AA score” (DIAAS) method. The BV, NPU, and PDCAAS use the fecal digestibility of CP in a rat model FAO/WHO/UNU (1991), whereas DIAAS uses ileal AA digestibility in a pig model (FAO, 2014). The BV measures the proportion of absorbed N retained in the body and indicates how well the body utilizes CP, though not accounting for the digestion and the interaction with other foods. The NPU and BV are similar, except that NPU is calculated from ingested N and hence considers digestibility and is a better estimate for protein quality than BV (Hoffman and Falvo, 2004). The PDCAAS method was the first to be adopted that used an indirect measure of IAA bioavailability as apparent total tract CP digestibility (FAO/WHO/UNU, 1991) and was recommended as an alternative to the PER method. The PDCAAS is calculated by multiplying AA concentrations with ATTD of CP, providing the AA scores using the IAA requirements of a reference population. The lowest AA score is considered the first-limiting AA, defining the PDCAAS. Total tract CP digestibility can overestimate AA bioavailability of protein from ingredients with poor ileal digestibility and does not consider differences in digestibility among individual AA. Also, PDCAAS values higher than 100% are truncated to 100, implying that AAs supplied above requirements do not have additional physiological value. The truncation arbitrarily underestimates the protein quality of food sources with high protein values, such as meat and milk. The DIAAS was developed to overcome the methodological limitations of PDCAAS. The DIAAS method is based on the cannulated sampling of ileal digesta and assesses digested AAs at the point of physiological absorption. The DIAAS relies on calculating a score for each IAA based on the AA content and the concentration of each digestible IAA and expresses the proportion of the reference patterns for the age groups for which the IAA has been set. The IAA with the lowest score relative to the reference pattern for the specific age is the first limiting IAA defining the DIAAS value (FAO, 2013). Compared with PDCAAS, there is no truncation for DIAAS values, and some protein ingredients can have values greater than 100. The FAO (2013) has recommended the DIAAS method, but the lack of ileal digestibility coefficients published in the literature has limited the widespread use of DIAAS. For the evaluation of insect protein quality using DIAAS, Malla et al. (2022) is currently the only published study. Though DIAAS is recommended for evaluating food’s protein quality, PER, NPR, BV, NPU, and PDCAAS are still used until a sufficient database of ileal digestibility, and DIAAS values are available. Furthermore, DIAAS can also be determined by using dual stable isotope tracer method (FAO, 2014; Shivakumar et al., 2019). More recently, the dual-isotope tracer method has been developed to determine true ileal IAA digestibility in humans (Devi et al., 2018). For this method, an intrinsically isotope-labeled test protein must be produced by growing the source, either plants or animals, on labeled substrates; the labeled protein is fed along with a different isotope-labeled “standard” protein of known digestibility. Then, the postprandial ratio of the differently labeled AA in the blood allows for determining the proper digestion and absorption of the test protein. This method measures AA bioavailability beyond intestinal digestibility. The method requires developing an intrinsic isotope labeled food, is expensive, and cannot be routinely conducted on all food ingredients. The different methods for assessing protein quality have been applied to insects in various studies, providing results that are to some extent comparable, but also represent different principles of evaluating protein quality (Longvah et al., 2011; Yang et al., 2014; Oibiokpa et al., 2018; Poelaert et al., 2018; Jensen et al., 2019; Hermans et al.,2021; Malla et al., 2022). In the review the of published in-vivo studies. The overview of the studies reviewed on protein digestibility and quality measures by insect species methods applied is shown in Table of the protein digestibility and quality measures by insect species methods applied in the studies reviewed ileal total tract BV indispensable amino acid protein protein digestibility corrected amino acid ileal ileal digestibility. The highest value for each insect species each method. of the protein digestibility and quality measures by insect species methods applied in the studies reviewed ileal total tract BV indispensable amino acid protein protein digestibility corrected amino acid ileal ileal digestibility. The highest value for each insect species each method. has been conducted on the of insect protein on the growth performance and digestibility of pigs and poultry For digestibility, have reviewed in-vivo studies CP, and AAs in-vitro studies due to the et al. that dietary of Tenebrio molitor (Yellow mealworm) in diets of growing pigs higher digestibility and of CP and some IAAs compared to poultry and hydrolyzed The to be higher in hydrolyzed than T. molitor by the that are more also found that T. molitor better digestibility of CP and IAAs than or poultry et al., 2019). The of insect protein the digestibility of CP and AAs. of T. molitor protein’s digestibility of studies on the of insect on growth performance protein digestibility AA = amino acids; = apparent ileal ATTD = apparent total tract CP = crude IAA = indispensable amino acid; N = Nitrogen; = standardized ileal digestibility. of studies on the of insect on growth performance protein digestibility AA = amino acids; = apparent ileal ATTD = apparent total tract CP = crude IAA = indispensable amino acid; N = Nitrogen; = standardized ileal digestibility. et al. compared two insects, Hermetia illucens soldier fly) and and found of most IAAs in except for and In Malla et al. (2022) also found the of CP of species from for illucens to for Both et al. and Malla et al. (2022) that the insects with higher CP and AAs content higher digestibility of CP and AA. concentrations of chitin may also AA digestibility. contents not in these studies, and the of chitin in ileal digestibility but the of AA. For growth performance as the measure of protein quality, studies in fed different insect species compared to different control diets as shown in Table et al., et al., 2018; and 2019; et al., 2019; et al., 2022). studies of insects in the feed. found an T. molitor by to at the of and growth by feed due to and with T. molitor et al., The in growth performance between studies can be to of insect species, other feed protein and studies are needed to the value of insects in animal feed insect species in results in protein and AA digestibility the studies As for growth the in species, in of other protein and different insect the among studies. the of the insect to be such as for the growth chitin content, which may the digestibility. Also, some animals – as well as some human – may as by the of of in pigs et al., 2021). Both and fed with diets CP digestibility compared to et al., 2018; et al., 2018). et al. that CP digestibility with of insect in et al. also that fed illucens of CP than fed diets containing to pig studies, in the illucens ATTD than the T. However, der et al. higher N for illucens compared to to their high content of Sulphur amino acids and and high of The quality of insect protein in human diets must be considered in of the method on which the evaluation is The studies available on determining the protein quality of insects for humans are in Table Oibiokpa et al. used a rat model for determining the protein quality of species PER, NPR, NPU, BV, and PDCAAS. the highest protein quality among the species, as in the various protein quality The protein quality of two species, T. molitor and was determined by Poelaert et al. and Jensen et al. T. molitor and also using the rat The PDCAAS values by Poelaert et al. and Jensen et al. are higher than the DIAAS values by Malla et al. (2022) for T. that the PDCAAS values to overestimate the protein quality. protein amino acid is in PER = protein efficiency NPR = protein NPU = protein BV = PDCAAS = protein digestibility corrected amino acid DIAAS = digestible indispensable amino acid = Sulphur amino acids Poelaert et al. Jensen et al. and Malla et al. calculated protein quality of insects based on the FAO (2013) pattern of amino acid requirements for age to to and and et al. Yang et al. and Oibiokpa et al. calculated protein quality of insects based on FAO/WHO/UNU pattern of amino acid requirements for age group protein amino acid is in PER = protein efficiency NPR = protein NPU = protein BV = PDCAAS = protein digestibility corrected amino acid DIAAS = digestible indispensable amino acid = Sulphur amino acids Poelaert et al. Jensen et al. and Malla et al. calculated protein quality of insects based on the FAO (2013) pattern of amino acid requirements for age to to and and et al. Yang et al. and Oibiokpa et al. calculated protein quality of insects based on FAO/WHO/UNU pattern of amino acid requirements for age group PDCAAS of and of et al., was based on in-vitro digestibility of these insects. For the of PDCAAS, in-vitro measures used as a for in-vivo total tract digestibility, for digestibility coefficients for PDCAAS. fecal total tract N digestibility for ileal digestibility of protein and free AA is currently the in-vitro method used for PDCAAS. The in-vitro method has been (Egger et al., 2017). However, this method is to be used to determine protein quality based on DIAAS. For studies in humans, Hermans et al. the of and protein on protein and metabolism and protein in For this, used intrinsically AA and in or protein. the postprandial there no differences in the of and milk AA into the and protein between groups and milk studies are needed to this that insects can provide protein of quality to milk for human consumption. The standard measure for CP content in feed and food is based on total N and the conversion factor of This standardized measure was recommended for calculating DIAAS as in the FAO (FAO, 2013). While it is well that the true conversion factor with the in the AA for insects, the conversion is by the chitin a of This non-protein N from chitin overestimate the protein content when the standard conversion Several studies have conversion for insects, adjusting for the et al., 2017; et al., et al. established in insect species and as a standard for insects. For the protein quality methods for which the of AAs is a such as the DIAAS method, total AAs is considered to be a more relevant measure for total protein to overcome the of the protein content, hence underestimation of protein quality using of 6.25 et al., 2022). using an of instead of the DIAAS in Table by for example, T. molitor compared to score to be than and from The of standardized assessment methods of protein quality, a standardized is to comparable measures protein sources. However, for insects, the nature of the non-protein N from the chitin should be considered in the of methods for true quality evaluation. To insects in the food systems as a protein source for animal feed and for direct human and comparable assessment of the protein quality is are methods for determining protein digestibility and quality to meet the requirements of animals and humans. methods have and a scientific ileal digestibility measures as TID and for animals, and DIAAS for humans, are considered the most accurate of protein quality. However, other methods be sufficient to insects against other such as growth studies for the quality of animal feed. Hence, the which the method be utilized and available determine the The protein quality of insects must be considered in of the applied Also, the generalized of 6.25 for calculating CP needs accounting for non-protein N Across animal studies, T. molitor has higher protein digestibility and well animal growth performance and hence, is suitable to be used in animal feed. Across studies evaluating the protein quality of insects for human different species of cricket and to have slightly better protein quality compared to mealworm species molitor and though needs more data to and these species are all as alternative protein sources for human consumption. This was of the in a and by the The also from for and of Malla is a protein quality of insect protein in of and at the of has conducted to determine DIAAS values for number of insect species and potential of using insects in has in animal and feeding from and of as a scientific at of to is an at of and and of monogastric group. is on applied and of The are to in is an in human at of is on the nutritional from foods in diets in as well as in the transition to more sustainable has conducted human studies in and with on and in to food and Over the past decade, has to the on edible insects by in and consumption of insects as a protein

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