**About the Editors**

**Man P. Huynh** is a Research Associate at the University of Missouri. His research has focused on insect nutrition and toxicology and its utilization in insect production and management via the application of multi-omics analyses and mathematical modeling. This results in the development of artificial diet formulations specialized for western and northern corn rootworm larvae that are currently utilized in research and commercial applications. His current research aims to develop a high-throughput system for mass production of corn rootworms toward accelerating discovery efforts related to novel insecticidal compounds and their related products.

**Kent S. Shelby** is a Research Entomologist stationed at the USDA Agricultural Research Service Biological Control of Insects Research Laboratory in Columbia Missouri. His research has focused on improvements to the effectiveness of biological control agents such as parasitic Hymenoptera, beneficial Heteroptera, nematodes, and viruses. His current research is a multidisciplinary effort applying multiomic approaches to studying the nutrition, toxicology, and immunity of maize pests.

**Thomas A. Coudron** is a Research Scientist (retired collaborator) with the USDA Agricultural Research Service. He has been involved in insect biochemistry and physiology for forty years. His early research focused on ways to improve the effectiveness of pathogens, parasitoids, and predators via exploiting the physiological interactions between beneficial organisms and their hosts or prey. This resulted in the discovery of new enzyme complexes and unique nonlethal and nonparalyzing venom components. His recent research has focused on insect nutrition and the application of molecular technology to advance the formulation of artificial insect diets. This has led to diet recipes that are now utilized in research and commercial applications.

### *Editorial* **Recent Advances in Insect Rearing Methodology to Promote Scientific Research and Mass Production**

**Man P. Huynh 1,2,\*, Kent S. Shelby 3,\* and Thomas A. Coudron 3,\***


The benefits obtained from our ability to produce insects have encompassed a wide array of applications, from the early stages of examining different species, to the present day of mass production for multiple purposes. Perhaps the most prominent application to date is insect management for production agriculture. Much of the considerable knowledge humans have of insect biology has been made possible by first bringing insects from the field into the laboratory, and maintaining sufficiently large colony sizes on bespoke, empirically developed diets. The ability to conduct experiments without the seasonal limitations that normally bound insect life history, to control abiotic and biotic treatments, is heavily dependent upon a nutritionally complete, inexpensive and easily produced or procured diet acceptable to the insects. Nominally, healthy insects produced in this way are more likely to respond accurately in various bioassays, to better perform the desired ecosystem services upon release, or to act as nutritional inputs for other animals. Ecosystem services provided by mass reared insects include pollination, release of predators or parasitoids for biological control and releases of sterile insects to suppress pest populations. More recently, the usage of mass-produced insects for nutrient sources such as for human consumption, animal feed, pet food, stock chemicals, or valorization of waste streams has the potential to surpass all previous applications. Of course, food sources are not the minimum requirement for successful mass rearing. Attention must be paid to innovate and optimize many other inputs including reduced labor, close observation of losses at each life stage, mating and oviposition, appropriately shaped containers, lighting, scheduling and sanitation.

The initial effort of the mass production of insects probably has its origin in scientific inquiry. The production of healthy, vigorous experimental subjects for basic and applied research purposes was an early impetus for the development of insectary diets and rearing procedures. That desire to study insects remains and has been greatly assisted by the ability to produce the specimens under natural and controlled conditions. Typical of research efforts is the progression of developing, refining and adapting supportive procedures that enable advancements. Insect rearing is a prime example.

After collection, the need to rear becomes the dominant challenge in all efforts to produce insects and that requires an understanding of environmental and nutritional needs. Along with an increased knowledge of insects came technological advances that resulted in improved methodologies and commercially available supplies. Courses, symposia and conferences in insect rearing were held and as the knowledge-based methodologies and supportive materials expanded, so did the applications of rearing insects. An industry of insect production and sales emerged and continues today, as does the need for quality assurance associated with rearing, research and production.

Numerous insects have been reared and many continue to be reared today. Applications include discovery of insect pheromones, repellents and new compounds with insecticidal properties. Some are used for monitoring purposes, including detection of

**Citation:** Huynh, M.P.; Shelby, K.S.; Coudron, T.A. Recent Advances in Insect Rearing Methodology to Promote Scientific Research and Mass Production. *Insects* **2021**, *12*, 961. https://doi.org/10.3390/ insects12110961

Received: 15 October 2021 Accepted: 19 October 2021 Published: 22 October 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

insect resistance. At the forefront of mass rearing was the emerging field of the sterile insect technique (SIT) for area-wide management of pest insects. Targeted insects included the screwworm fly, several tephritid fruit flies, the gypsy moth, pink bollworm, boll weevil, codling moth and several mosquito species. Many beneficial insects have been, and continue to be, reared for release as biological control measures against pest insects. The most notable of these may be *Trichogramma* species. The glasshouse industry has become heavily dependent on insectary reared insects for pest control. Today, we have an emerging field of insect production as biofactories, both for food and specialty substance sources. These new fields have tremendous potential.

Most agricultural applications of insect rearing require large-scale production. One of the major advancements that has enabled mass production of insects has been the substitution of alternative food sources, and in particular, artificial diets. Artificial diets allow for the mass production of insects in simpler, highly controlled, cost-effective and more convenient ways compared to rearing on native diets such as plants or natural preys. Ideal artificial diets can ultimately serve as suitable substitutes for natural foods and support the production of insects physically and behaviorally, similar to natural diets. Often, the use of artificial diets results in a more uniform and consistent production of high-quality insects for research and field applications. Consequently, the investigation of substitutes for natural food has become a common practice.

The application of new technologies is on the horizon for further refinements in insect production, such as genomics, genetic selection and engineering. Newer multi-omics technologies such as transcriptomics, nutrigenomics and nutrimetabolomics, with increased knowledge of insect microbiome contributions and statistical optimization modeling, have already enabled significant advances in diet formulation. These advances have resulted in a better understanding of the effects of the food stream ingredients on physiological and biochemical functions. Undoubtedly, this will result in improved diet formulations, higher quality control and healthier reared insects with better performance for their targeted applications.

The black soldier fly (*Hermetia illucens* L.) has a global research interest and a growing industrial application since it provides a viable option for countering the environment detriments caused by food waste and a sustainable protein source to feed the growing global population. Hopkins et al. [1] conducted a systematic literature review investigating the impacts of various foodstuffs and substrates for food waste rearing on the protein and amino acid composition of the black soldier fly larvae, finding that plant rearing substrates provide a lower protein content of the total larval mass compared to animal rearing substrates. Pliantiangtam et al. [2] investigated the growth performance, waste reduction efficiency and nutritional composition of the black soldier fly larvae reared on two plant materials (coconut endosperm and soybean curd residue), reporting a similar result of the use of plant rearing sources that yielded lower protein larvae compared with that of animal rearing materials. Furthermore, Lu et al. [3] compared the effects of nine nitrogen sources (i.e., NH4Cl, NaNO3, urea, uric acid, Gly, L-Glu, L-Glu:L-Asp (1:1, *w*/*w*), soybean flour, and fish meal) during food waste larval treatment and characterized the C/N effects on the larval development and bioconversion process. The authors found that organic nitrogen was more suitable than NH4Cl and NaNO3 as the nitrogen amendment, and an inclusion of small amounts of urea (C/N of 18:1–14:1 and 18:1–16:1) improved the waste reduction performance, and larval protein and lipid bioconversion process, respectively.

The yellow mealworm (*Tenebrio molitor* L.) is another insect species that has been considered as an alternative to fishmeal in animal feed formulations. By utilizing a nutrient self-selection approach, Morales-Ramos et al. [4] demonstrated that the optimum ratio of macro-nutrient intake of this species was 0.06:0.23:0.71 for lipid:protein:carbohydrate. Carbohydrate had positive impacts on food assimilation, food conversion and biomass gain, and several plant materials including cabbage, potato, wheat bran, rice bran (whole and defatted), corn dry distillers' grain, spent brewery dry grain, canola meal and sunflower meal were suitable macro-nutrient components in *T. molitor* diets.

Nikolouli et al. [5] evaluated inactive *Enterobacter* sp. AA26 as a protein source to potentially replace the inactive brewer's yeast, a protein ingredient in the larval diet of the spotted-wing drosophila fly (*Drosophila suzukii* (Matsumura)). This insect species is one of the most damaging insect pests of soft skinned fruits in North America and Europe and is a detrimental invasive pest in South America and Africa. The authors found that *Enterobacter* sp. AA26 provided inadequate nutrition in the larval diet compared with the inactive brewer's yeast. The replacement of *Enterobacter* sp. AA26 with the inactive brewer's yeast resulted in decreases in pupal weight, survival, fecundity and adult recovery.

Improvements in the methodology to store beneficial insects at low temperatures will facilitate biological control programs primarily relying on the mass-release of highquality bioagents to suppress agricultural pests. Lin et al. [6] characterized impacts of temperatures (4, 7, 10 and 13 ◦C) and storage durations (10, 15, 20 and 25 days) on the developmental parameters of different pupal age of *Psyttalia incisi* (Silvestri), a dominant parasitoid against *Bactrocera dorsalis* (Hendel) in fruit-producing regions of southern China. The authors reported that the emergence rate of *P. incisi* was significantly affected by storage temperature, storage duration and pupal age interval and their interactions. They further determined the optimum cold storage conditions at a temperature of 13 ◦C for 10 or 15 d for late-age pupae of this parasitoid. Separately, Lü et al. [7] demonstrated that a temperature of 13 ◦C was the cold tolerance threshold temperature and the prepupal stage was a critical developmental period for in vitro rearing of another parasitoid *Trichogramma dendrolimi* Matsumura, an important biological control agent of biological control programs.

Insect predators are also important components of biological control programs. Zou et al. [8] investigated the effects of prey species (*Drosophila melanogaster* Meigen or *Bradysia impatiens* (Johannsem)) and prey densities (6–48 preys per predator) on the performance of adult *Coenosia attenuate* Stein, a predator species native to Southern Europe effectively suppressing a wide range of agricultural pests. Their results revealed that *B. impatiens* was the better prey compared with *D. melanogaster*, and the optimal prey density for *C. attenuate* rearing was 12 to 24 adults of *B. impatiens* per predator per day.

Rearing honeybee (*Apis mellifera*) larvae in vitro is an important method for studying bee larvae diseases or the toxicity of pesticides on bees. Kim et al. [9] evaluated the emergence and deformation rates of honeybee (*Apis mellifera ligustica* Spinola) larvae reared in horizontal and vertical positions, finding that a vertical rearing method was the better approach. Compared to the horizontal rearing plates, the vertical rearing plates resulted in a twofold decrease in adult deformation rates and larger adults (11.6 mm vs. 10.8 mm).

Heat-sterilized diets are the key components of high-throughput systems for mass production of insects. Huynh et al. [10] investigated the influence of thermal exposure and lengths of thermal exposure on the quality of a commercialized diet for the western corn rootworm (*Diabrotica virgifera virgifera* LeConte), the most serious pest of maize in the United States and some parts of Europe, to further the goal of developing a diet free of antibiotics and heat-sterilized for this important pest. By using geometric and mathematical approaches, the authors demonstrated non-linear effects of thermal exposure on the performance of diet, whereas no impacts were observed on the exposure intervals evaluated. These findings will guide the continued development of sterilized rootworm diets, facilitating mass production and providing insights into the design of diets for other insects.

This unique topic has been captured in 10 articles that bring together experimental and review papers focusing on different rearing technology approaches to many facets of insect rearing for various purposes. There is every reason to believe that the rapid improvements in insect nutrition and rearing seen over the past decade will be dwarfed by the accomplishments yet to come in the next decade.

**Funding:** This editorial received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Systematic Review* **The Influence of Food Waste Rearing Substrates on Black Soldier Fly Larvae Protein Composition: A Systematic Review**

**Indee Hopkins , Lisa P. Newman, Harsharn Gill and Jessica Danaher \***

School of Science, STEM College, RMIT University, Melbourne 3083, Australia; s3744095@student.rmit.edu.au (I.H.); lisa.newman@rmit.edu.au (L.P.N.); harsharn.gill@rmit.edu.au (H.G.) **\*** Correspondence: jessica.danaher@rmit.edu.au; Tel.: +61-3-9925-6117

**Simple Summary:** The Black Soldier Fly (BSF) is a viable option for countering the environment detriments caused by food waste and can provide a sustainable protein source to feed the growing global population. This systematic literature review investigated the impacts of various foodstuffs and food waste rearing substrates on the protein and amino acid composition of BSF larvae. From the 23 articles included, BSF larvae fed 'Fish waste Sardinella aurita' for two days produced the highest total protein content at 78.8% and rearing substrates 'Fruit and vegetables' reported the lowest protein content at 12.9% of the BSF total mass. However, variation in rearing and analytical methodologies between each study potential undermines the extent to which the rearing substrates may have influenced the overall protein content of BSF larvae.

**Abstract:** The Black Soldier Fly (BSF) offers the potential to address two global challenges; the environmental detriments of food waste and the rising demand for protein. Food waste digested by BSF larvae can be converted into biomass, which may then be utilized for the development of value-added products including new food sources for human and animal consumption. A systematic literature search was conducted to identify studies investigating the influence of food waste rearing substrates on BSF larvae protein composition. Of 1712 articles identified, 23 articles were selected for inclusion. Based on the results of this review, BSF larvae reared on 'Fish waste *Sardinella aurita*' for two days reported the highest total protein content at 78.8% and BSF larvae reared on various formulations of 'Fruit and vegetable' reported the lowest protein content at 12.9%. This review is the first to examine the influence of food waste on the protein composition of BSF larvae. Major differences in larval rearing conditions and methods utilized to perform nutritional analyses, potentially influenced the reported protein composition of the BSF larvae. While this review has highlighted the role BSF larvae in food waste management and alternative protein development, their application in nutrition is still in its infancy.

**Keywords:** alternative protein; amino acid; Black Soldier fly; food waste; insect protein; macronutrients

#### **1. Introduction**

With the predicted expansion of the global population expected to reach 8.5 billion by 2030 and 9.7 billion by 2050 [1], we have a vital need to develop and provide a safe and sustainable food system. With an annual increase of 83 million people worldwide, it is estimated that a 70% increase in food production will be required to meet demand, resulting in increased competition for arable land and natural resources such as energy and water [2]. However, our agricultural sectors' ability for growth, particularly in the production of sufficient and quality protein from traditional sources, is constrained by a deficiency of these key resources and presents as a serious challenge. In addition to our current food production system having been deemed as unsustainable from a growth perspective, it is also linked to adverse environmental implications, such as greenhouse gas emissions (GHG) and soil depletion [2].

**Citation:** Hopkins, I.; Newman, L.P.; Gill, H.; Danaher, J. The Influence of Food Waste Rearing Substrates on Black Soldier Fly Larvae Protein Composition: A Systematic Review. *Insects* **2021**, *12*, 608. https:// doi.org/10.3390/insects12070608

Academic Editors: Man P. Huynh, Kent S. Shelby and Thomas A. Coudron

Received: 26 April 2021 Accepted: 29 June 2021 Published: 4 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

A simultaneous global issue, despite being paradoxical to our food production and sector growth problem, is that much of the food produced is wasted. Whilst quantification of the extent of the problem is difficult due to lack of consistency in definitions and evaluation methods, it is estimated that as much as one-third of food produced for human consumption is wasted globally each year [3,4]. Food waste negatively impacts the environment in several ways including the net loss of finite resources such as land, water and fuel consumed during food production and distribution. In addition, foodstuffs discarded into landfill are also a contributor to GHG emissions (namely methane), making food waste a growing contributor to climate change [5,6]. It is estimated that food waste is contributing 4.4 gigatons of carbon dioxide (CO2) emissions into the atmosphere annually [6]. If put into context against national rankings, food waste would be the third-highest contributor of total GHG emissions after that of the United States and China [6]. At present, alternative treatment methods for food waste include incineration, fodder, anaerobic digestion and aerobic composting [7]. However, these methods are either not without their own environmental concerns or unable to be used in isolation to satisfy environmental needs in the long-term.

Another viable alternative to food waste treatment is Conversion of Organic Refuse by Saprophagous (CORS) technologies which use decomposer insects such as the Diptera *Hermetia illucens* L., also known as the Black Soldier Fly (BSF) larvae to manage organic waste. Deteriorated fruits and vegetables, municipal waste, crop waste, and industrial food-processing waste can be quickly and effectively digested by the BSF larvae [8–11]. This process of bioconversion diverts organic food waste from landfill to the production of biomass which can later be utilised for the development of value-added products. The BSF larvae have shown promising results in the production of biodiesel [12], fish feed [13] and have the potential to be used as an alternative source of protein for livestock [14]. The farming of mini livestock (i.e., insects) offers many benefits including high feed conversion ratios, and lower resource inputs in terms of energy, land and water requirements when compared to the farming requirements of traditional livestock (i.e., cattle) [15]. BSF larvae are also safe for human consumption [16], thus offering an opportunity to produce a new sustainable protein source for the growing global population.

Due to the variable nature of the nutritional composition of BSF larvae, the effect of various rearing substrates on BSF development needs to be investigated in further depth. This information can assist in the establishment of efficient growing and production practices in the future. To date, research on the nutritional protein value of BSF larvae fed food waste as the primary rearing substrate has not been synthesised. Thus, the purpose of this systematic literature review is to synthesise and investigate the influence that rearing substrates comprising of foodstuffs or food waste products have on the nutrient composition of BSF larvae.

#### **2. Materials and Methods**

#### *2.1. Literature Search Strategy*

The current review was performed in adherence to PRISMA-P (Preferred Reporting Items for Systematic review and Meta-Analysis Protocols) guidelines [17]. Scopus, Food Science and Technology Abstracts (FSTA), Web of Science, PubMed, Scifinder and ScienceDirect databases were used to search for articles investigating food waste and the nutritional composition of BSF larvae. The search was limited to articles published between 1 January 2000 and 30 October 2020.

Search terms were pilot tested before commencing the final search to ensure appropriate articles were identified. The final search included keywords searched within three categories (using 'OR') and then combined (using 'AND'). One category searched for articles reporting data on the insect of interest (i.e., Black Soldier Fly, or *Hermetia illucens*). The second category searched for articles reporting on rearing substrates (i.e., food waste, or diets). The third category searched for articles reporting on nutritional outcomes (i.e., protein, amino acids).

#### *2.2. Inclusion and Exclusion Criteria*

To be included in the review, articles were required to meet the following criteria: (i) be published as an original research study, with full-text availability in English; (ii) reporting on BSF in the larval or pre-pupae life stage; (iii) including a by-product of food production, foodstuffs or food waste as the rearing substrate for BSF larvae; (iv) reporting an assessment of protein composition of the BSF larvae.

The exclusion criteria included: (i) opinion articles, reviews, narrative reviews and concept papers; (ii) abstract and conference proceedings where a full-text published article could not be obtained; (iii) studies published in a non-English language; (iv) data previously reported elsewhere; (v) studies rearing BSF larvae on alternative waste products not safe for human consumption (i.e., manure); (vi) studies rearing BSF larvae on foodstuffs or food waste products with the inclusion of microbial assistance (i.e., fermentation).

#### *2.3. Study Selection*

Articles detected using the search strategy were collated using EndNote. Two reviewers (IH and JD) screened all articles based on titles and abstracts initially, and then by a full-text review. Articles that did not meet the eligibility criteria were excluded. For any articles where it was unclear whether the eligibility criteria were met, full-text articles were obtained, screened and resolved by discussion between three reviewers (IH, LPN and JD) until a consensus was reached.

#### *2.4. Data Extraction and Synthesis*

The data extraction method was pilot tested using an article retrieved whilst piloting search terms and refined accordingly. Data from the eligible articles were extracted using a Microsoft Excel spreadsheet, with the following study attributes recorded: year of publication, rearing substrates nutritional and physical characteristics, abiotic factors (including temperature, humidity, and light availability), BSF larvae rearing duration study methodology, statistical analysis, key results and author's conclusions. Data extraction was performed by one reviewer (IH) and verified by secondary reviewers (LPN and JD). In cases of disagreement, a discussion was held until consensus was reached. Extracted data were unable to be combined in a meta-analysis as the studies were not sufficiently homogenous in terms of design and comparator.

#### *2.5. Risk of Bias Assessment*

Risk of bias assessment of all included articles was independently undertaken by two reviewers (IH and JD) using the SYRCLE risk of bias tool [18]. The assessment tool considered (i) whether the allocation sequence was adequately generated and applied; (ii) were the groups similar at baseline; (iii) was allocation adequately concealed; (iv) were the animals randomly housed during the experiment; (v) were caregivers and investigators blinded from the knowledge of which intervention each group received; (vi) were larvae selected at random for outcome assessment; (vii) was the outcome assessor blinded; (viii) was incomplete outcome data adequately addressed; (ix) was the study free of selective outcome reporting; (x) was the study apparently free of other issues that could result in high risk of bias. A 'yes' response indicated a low risk of bias, a 'no' response indicated a high risk of bias, and an 'unclear' response indicated that insufficient details had been reported to allow for a valid assessment of the risk of bias [18]. Any discrepancies in the risk of bias assessment were reviewed by a third reviewer (LPN) and resolved by discussion and consensus between the three reviewers.

#### **3. Results**

#### *3.1. Search Results*

A total of 1712 records were identified through database searches (n = 1051 duplicates), of which 98 were accepted for full-text review after screening by titles and abstracts (Figure 1). Of these, 76 articles were excluded due to the following reasons: BSF larvae was

not the primary species investigated (n = 2), rearing substrate did not meet inclusion criteria (n = 17), rearing substrate included added supplementation with microbial assistance or fermentation (n = 2), measurable outcome did not include a relevant nutritional assessment of BSF larvae (n = 33), full-text was not available or was a conference proceeding (n = 10), article was not published in a scientific journal (n = 3) or was not a primary research study (i.e., review article, concept paper or patent application) (n = 9).

**Figure 1.** Flow diagram summarising the screening process.

#### *3.2. Rearing Substrates of Black Soldier Fly Larvae*

Of the 23 articles included in the current review, 16 articles reported on rearing substrates that contained grain-based ingredients [19–35]. Fifteen articles reported on rearing substrates that contained fruit and vegetable ingredients [19–21,23,25,29–33,35–39]. Six articles reported on rearing substrates that contained animal-based ingredients [24,25,28,39–41], four articles reported on rearing substrates that contained a generic food or kitchen waste description, with no further details regarding included ingredients [24,38,39,41], and one article reported on a rearing substrate that contained seaweed as an ingredient [26].

The rearing duration of the BSF larvae in the experimental trials varied with a reported range between one day [40] and 52 days [37]. Details on larvae feeding frequency were provided by 19 articles, as shown in Table 1 [19–22,24–29,31–34,36–40]. Of these the feeding frequency ranged from a singular feed at the beginning of the experiment [19,35], to a set feeding schedule throughout the experimental trial including daily [26,34,36], weekly [32], and specific days [22,23,25,27,32,33], and ad libitum feeding approaches [21,29,30,37,39,40]. The feed ration provided to BSF larvae was reported by 15 articles [19,20,22–30,33,36,38,39] and varied considerably from 12.2 mg per larvae [23] to 1,530 mg per larvae [24].

**Table 1.** Rearing substrates (RS) and rearing conditions of the BSF larvae.



**Table 1.** *Cont.*

Surendra [41] RS 1: Food waste (uncharacterised) ------- ≈, approximately; g, grams; mg, milligrams; C, Celsius. Nguyen et al. [42] article data used to support Nguyen et al. [39] reporting of rearing conditions. Second or third stage instar implies ≈ 15 days of age [43]. Neonate implies <5 days of age [44].

#### *3.3. Rearing Abiotic Conditions of Black Soldier Fly Larvae*

Abiotic conditions of the 23 articles included in the current review are shown in Table 1. Of these, ten articles included details regarding duration of light and dark exposure hours of BSF larvae [19–21,25–27,29,30,34,36,37]. Five articles reporting a duration of 12 h of light and 12 h of darkness [20,21,27,30,37], two articles reporting 16 h of light and eight hours of darkness [19,36], one article reporting 24 h of light [29], one article reporting 24 h darkness [25] and one article reporting 14 h of light and ten hours of darkness [34]. Twenty articles included in this review provided data on the relative humidity of the BSF larvae rearing environment [19–40], with ranges varying from 40.0% relative humidity [27] to 75.6% relative humidity [23].

Twenty-two articles included information regarding the temperature of the rearing environment [19–40], with a range of 24.5 ◦C [27] to 32.5 ◦C [31]. The age of larvae at the beginning of the experimental trials was reported in 21 articles [19–29,31–40], with BSF larvae ages ranging from eggs which were directly inoculated onto the rearing substrate [22,36] to BSF larvae aged 14 days before being introduced to the substrate [25]. The rearing duration of BSF larvae was reported in 20 articles [19–21,23–30,32–40], with rearing duration ranging from eight days [26,29] to 52 days [37].

#### *3.4. Macronutrient Composition of Rearing Substrates*

Twenty of the 23 articles included in this review provided details on the total protein composition of the rearing substrate provided to the BSF larvae (Table 2) [19–41]. Eleven articles reported details of the rearing substrate total lipid content [19,24–30,33,34,37,39]. Thirteen articles additionally reported details of the rearing substrate total carbohydrate content [19–30,33,34,37,39].


**Table 2.** Macronutrient Composition of Rearing Substrates (RS).

**Table 2.** *Cont.*


Results presented as a percentage. \* Indicative of original article presenting data as g/100 g of rearing substrate. <sup>ˆ</sup> indicative of original article presenting data as g/kg of rearing substrate. <sup>~</sup> indicative of original article nutrient data acquired from database or literature. Dashes used to indicate where data is unreported in the original article. Chia et al. [45] article data used to support Chia et al. [22] reporting of rearing substrate composition. DM dry matter, FW fresh weight.

There were substantial differences in the macronutrient composition of the rearing substrate provided, with an average total composition of 17.5% total protein, 7.2% total lipid and 27.3% total carbohydrate, when reported on a dry matter basis. Rearing substrate 'Apple' showed the lowest total protein content of 0.4% of dry matter [19]. The highest total protein content of a rearing substrate was reported for 'Fish waste *S. aurita*', with 72.7% protein dry matter [40]. Rearing substrates 'Apple' [19] and 'Bread waste' [28] both showed the lowest total lipid content of 0.0% of dry matter. The highest total lipid content in a substrate was reported for 'Poultry waste', with 42.9% lipid dry matter [25]. Rearing substrates 'Melon', 'Tomato' [19] and 'Fish waste *O. mykiss*' [28] all showed the lowest total carbohydrate content of 0.0% of dry matter. The highest total carbohydrate content of a rearing substrate was reported for 'Bread', with 78.6% carbohydrate dry matter [24].

Only one article reported rearing substrate macronutrient composition on a fresh weight basis, finding the highest protein and lipid content in substrate 'Vegetable—lettuce, string green beans, cabbage (ratio of 3.3:3.3:3.3)' (2.0% and 0.2% of fresh weight, respectively) and the highest substrate carbohydrate content in substrate 'Fruit—apple, pear, orange (ratio of 3.3:3.3:3.3)' (8.9% of fresh weight) [37].

#### *3.5. Amino Acid Composition of Rearing Substrates*

Two articles reported the amino acid content of the rearing substrate provided to the BSF larvae (Supplementary Table S1) [26,33]. Results collected indicated that glutamate was the most abundant amino acid found, accounting for 25.9% of total protein dry matter in 'Brown algae *A. nodosum*' [26]. No other substrate composition included this amino acid in isolation.

Cysteine and tryptophan were the least abundant amino acids reported in the rearing substrate with 'Restaurant waste—potato, rice, pasta, vegetable (ratio unspecified)' showing the presence of both amino acids at 0.2% of total protein dry matter [33].

#### *3.6. Macronutrient Composition of Black Soldier Fly Larvae Reared on Food Waste*

As per the inclusion criteria, all articles included in this review provided details on the protein composition of BSF larvae reared on food waste substrates. Of these, 22 articles reported BSF larvae protein composition on a dry matter basis, with one article reporting on a fresh weight basis (Table 3).


**Table 3.** Macronutrient Composition of Black Solider Fly Larvae Reared on Food Waste.


**Table 3.** *Cont.*

RS 4: Bread, fish *O. mykiss,* (8.5:1.5) 44.8 - - - - -


**Table 3.** *Cont.*

Results presented as a percentage of total BSF larvae biomass unless indicated. Liland et al. [26] presented as total sum of amino acids. ≈ approximate figure; \* indicative of original article presenting data as g/100g BSF larvae biomass, <sup>ˆ</sup> indicative of original article presenting data as g/kg BSF larvae biomass. Dashes used to indicate where data is unreported in the original article, DM dry matter, FW fresh weight.

> There was an average of 31.2% total protein when reported on a dry matter basis. Rearing substrate 'Exotic fruit, pineapple, kiwi, apple, melon (ratio of 2:2:2:2:2)' for 26.7 days, 'Exotic fruit, kiwi (ratio of 5:5)' for 25.3 days [19] and 'Fruit and vegetable (uncharacterised)' for an unspecified number of days [39], equally resulted in the lowest total protein content of 12.9% of BSF larvae dry matter. The highest BSF larvae total protein content was reported for larvae reared for two days on 'Fish waste *S. aurita*' with 78.8% total protein BSF larvae dry matter [40]. This was followed by 'Fish waste *S. aurita*' reared for one and four days and resulting in 77.4% and 75.4% total protein of BSF larvae dry matter, respectively [40].

> Twelve articles reported additional details of the BSF larvae total lipid content on a dry matter basis [19,20,22,24,26,27,29–31,36,37,39,41], and two articles reported details of the BSF larvae total carbohydrate content, on a dry matter basis [39,41]. One article reported additional details of the BSF larvae total lipid content of a fresh weight basis [37].

> Rearing substrates 'Fruit and vegetables (uncharacterised)' reared for an unspecified number of days [39], 'Pomace, all-year mix (ratio of 5:5)' reared for 26 days and 'Pineapple' reared for 33.7 days [19] resulted in the lowest reported total lipid content of 2.2%, 4.9%

and 5.0% total lipid of BSF larvae dry matter, respectively. The highest BSF larvae total lipid content was reported for larvae reared for 14 days on 'Bread', resulting in 57.8% total lipid BSF larvae dry matter [24]. This was followed by 'Spent barley, brewer's yeast' reared for an unspecified number of days [22] and 'Fish *O. mykiss*, wheat (ratio of 5:1)' reared for 14 days [24] (49.0% and 46.7% total lipid of BSF larvae dry matter, respectively).

Of the two articles reporting on BSF larvae total carbohydrate content (a combined total of three rearing substrates), 'Fish waste (uncharacterised)' larvae reared for 12 days produced the highest reported result of 12.7% total carbohydrate BSF larvae dry matter [40]. This [40] and 'Fruit and vegetable (uncharacterised)' reared for an unspecified number of days [39] (12.3% and 8.4% total carbohydrate of BSF larvae dry matter, respectively).

The one article reporting BSF larvae nutritional composition on a fresh weight basis showed the highest protein in BSF larvae reared on 'Fruit and vegetable mix (ratio of 1:1)' for 36.7 days (17.6% of fresh weight) and highest lipid content in BSF larvae reared on 'Fruit—apple, pear, orange (ratio of 3.3:3.3:3.3)' for 52 days (21.0% of fresh weight) [37].

#### *3.7. Essential Amino Acid Composition of Black Soldier Fly Larvae Reared on Food Waste*

Seven articles included in this review provided details on the essential amino acid composition of BSF larvae reared on different substrates. Of these, six articles reported BSF larvae essential amino acid composition on a dry matter basis [26,32,33,35,36,38], with one article reporting on a wet weight basis (Table 4) [41].



Results presented as a percentage of the BSF larvae total protein content unless indicated. Liland et al. [26] presented as percentage of total sum of amino acids. <sup>ˆ</sup> indicative of original article presenting data as g/kg of the BSF larvae total protein content, <sup>+</sup> indicative of original article presenting data as mg/g of the total BSF larvae total protein. Dashes used to indicate where data is unreported in the original article. WW wet weight.

> Histidine—Histidine comprised 2.5% of BSF larvae total protein content when reported on a dry matter basis. Rearing BSF larvae on 'Kitchen waste—potato peelings, carrot, rice, bread debris (ratio unspecified)' for an unspecified number of days, resulted in the lowest

histidine content of 0.3% of the total larval protein content [32]. The highest BSF larvae histidine content was reported for BSF larvae reared for 15 days on 'Carbohydrate—wheat middlings' with 3.3% of BSF larvae total protein content [35].

Isoleucine—Isoleucine comprised 3.8% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Brewery by-product spent grain' reared for an unspecified number of days, resulted in the lowest reported isoleucine content of 0.2% of BSF larvae total protein content [32]. The highest BSF larvae isoleucine content was reported for BSF larvae reared for 43–47 days on 'Fruit and vegetable mix—lettuce, apple, potato (5:3:2)' with 4.3% of BSF larvae total protein content [38].

Leucine—Leucine was the most abundant essential amino acid and comprised 6.3% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Kitchen waste—potato peelings, carrot, rice, bread debris (ratio unspecified)' reared for an unspecified number of days, resulted in the lowest reported leucine content of 0.3% of BSF larvae total protein content [32]. The highest BSF larvae leucine content was reported for BSF larvae reared for eight days on 'Wheat, brown algae *A. nodosum* (4:6)' with 6.9% of BSF larvae total protein content [26].

Lysine—Lysine comprised 5.6% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrates 'Kitchen waste—potato peelings, carrot, rice, bread debris (ratio unspecified)' and 'Brewery by-product spent grain' reared for an unspecified number of days, both resulting in the lowest reported lysine content of 0.5% of BSF larvae total protein content [32]. The highest BSF larvae lysine content was reported for BSF larvae reared for 19 days on 'Food waste (uncharacterised)' with 8.3% of BSF larvae total protein content [38].

Methionine—Methionine comprised 1.5% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrates 'Brewery by-product spent grain' reared for an unspecified number of days [32] and 'Restaurant waste—potato, rice, pasta, vegetables (ratio unspecified)' reared for 19 days [33] both resulting in the lowest reported methionine content of 0.7% of BSF larvae total protein content. The highest BSF larvae methionine content was reported for BSF larvae reared for 45 days on 'Fruit and vegetable mix—zucchini, apple, potato, green beans, carrot, pepper, orange, celery, kiwi, plum, eggplant (unspecified ratio)' [36] and 'Food waste (uncharacterised)' reared for 19 days, both resulting in 1.8% methionine of BSF larvae total protein content [38].

Phenylalanine—Phenylalanine comprised of 3.5% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Brewery by-product spent grain' reared for an unspecified number of days, resulted in the lowest reported phenylalanine content of 0.2% of BSF larvae total protein content [32]. The highest BSF larvae phenylalanine content was reported for BSF larvae reared for eight days on 'Wheat, brown algae *A. nodosum* (8:2)' with 4.3% of BSF larvae total protein content [26].

Threonine—Threonine comprised 3.8% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Restaurant waste—potato, rice, pasta, vegetables (ratio unspecified)' reared for 19 days, resulted in the lowest reported threonine content of 1.6% of BSF larvae total protein content [33]. The highest BSF larvae threonine content was reported for BSF larvae reared for eight days on 'Wheat, brown algae *A. nodosum* (8:2)' and 'Wheat, brown algae *A. nodosum* (4:6)' both resulting in 4.1% threonine of BSF larvae total protein content [26].

Tryptophan—Tryptophan was the least abundant essential amino acid and comprised 1.1% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Fruit and vegetable mix—zucchini, apple, potato, green beans, carrot, pepper, orange, celery, kiwi, plum, eggplant (unspecified ratio)' reared for 45 days, resulted in the lowest reported tryptophan content of 0.4% of BSF larvae total protein content [36]. The highest BSF larvae tryptophan content was reported for BSF larvae reared for 19 days on 'Food waste (uncharacterised)' and BSF larvae reared for 42–47 days on 'Fruit and vegetable mix—lettuce, apple, potato (5:3:2)' both resulting in 1.4% tryptophan of BSF larvae total protein content [38].

Valine—Valine comprised 5.5% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Kitchen waste—potato peelings, carrot, rice, bread debris (ratio unspecified)' reared for an unspecified number of days, resulted in the lowest valine content of 0.1% of BSF larvae total protein content [32]. The highest BSF larvae valine content was reported for BSF larvae reared for eight days on 'Wheat, brown algae *Ascophyllum nodosum* (9:1, 8:2 and 7:3)' all resulting in 6.0% valine of BSF larvae total protein content [26].

The one article reporting BSF larvae essential amino acid composition on a wet weight basis showed the least and most abundant essential amino acid to be methionine (0.9% of BSF larvae total protein content) and valine (2.4% of BSF larvae total protein content), respectively, when reared on 'Food waste (uncharacterised)' for an unspecified number of days [41].

#### *3.8. Non-Essential Amino Acid Composition of Black Soldier Fly Larvae Reared on Food Waste*

Seven articles included in this review provided details on the non-essential amino acid composition of BSF larvae reared on different substrates. Of these, six articles reported BSF larvae non-essential amino acid composition on a dry matter basis [26,32,33,35,36,38], with one article reporting on a wet weight basis (Table 5) [41].


**Table 5.** Non-Essential Amino Acid Profile of Black Soldier Fly Larvae Reared on Food Waste.

Results presented as a percentage of the BSF larvae total protein content unless indicated. Liland et al. [26] presented as percentage of total sum of amino acids. <sup>ˆ</sup> indicative of original article presenting data as g/kg of the BSF larvae total protein content, <sup>+</sup> indicative of original article presenting data as mg/g of the total BSF larvae total protein. Dashes used to indicate where data is unreported in the original article. WW wet weight.

> Alanine—Alanine comprised 6.2% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Restaurant waste—potato, rice, pasta, vegetable (ratio unspecified)' reared for 19 days, resulted in the lowest reported alanine content of 2.8% of BSF larvae total protein content [33]. The highest BSF larvae alanine content was reported

for BSF larvae reared for 15 days on 'Carbohydrate—wheat middlings with 7.8% of BSF larvae total protein content [35].

Arginine—Arginine comprised 4.3% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Brewery by-product spent grain' reared for an unspecified number of days, resulted in the lowest reported arginine content of 0.3% of BSF larvae total protein content [32]. The highest BSF larvae arginine content was reported for BSF larvae reared for eight days on 'Brown algae *A. nodosum*' with 6.5% of BSF larvae total protein content [26].

Aspartate—Aspartate comprised 8.1% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Restaurant waste—potato, rice, pasta, vegetable (ratio unspecified)' reared for 19 days, resulted in the lowest reported aspartate content of 3.7% of BSF larvae total protein content [33]. The highest BSF larvae aspartate content was reported for BSF larvae reared for eight days on 'Wheat' with 9.4% of BSF larvae total protein content [26].

Cysteine—Cysteine comprised 0.5% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Fruit and vegetable mix—zucchini, apple, potato, green beans, carrot, pepper, orange, celery, kiwi, plum, eggplant (unspecified ratio)' reared for 45 days, resulted in the lowest reported cysteine content of 0.05% of BSF larvae total protein content [36]. The highest BSF larvae cysteine content was reported for BSF larvae reared for 15 days on 'Carbohydrate—wheat middlings with 0.9% of BSF larvae total protein content [35].

Glutamate—Of the one article (two rearing substrates) reporting BSF larvae glutamate composition, there was an average of 9.7% glutamate of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Fruit and vegetable mix—lettuce, apple, potato (5:3:2)' reared for 42–47 days, resulted in the lowest reported glutamate content of 9.5% of BSF larvae total protein content [38]. The highest BSF larvae glutamate content was reported for BSF larvae reared for 19 days on 'Food waste (uncharacterised)' with 9.8% of BSF larvae total protein content [38].

Glutamic acid—Glutamic acid was the most abundant non-essential amino acid and comprised 9.8% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Brewery by-product spent grain' reared for an unspecified number of days, resulted in the lowest reported glutamic acid content of 0.3% of BSF larvae total protein content [32]. The highest BSF larvae glutamic acid content was reported for BSF larvae reared for eight days on 'Wheat, brown algae *A. nodosum* (4:6)' with 12.8% of BSF larvae total protein content [26].

Glutamine—One article (two rearing substrates) reporting BSF larvae glutamine composition, there was an average of 0.4% glutamine of BSF larvae total protein content when reported on a dry matter basis and was the least abundant non-essential amino acid. Rearing substrate 'Brewery by-product spent grain' for an unspecified number of days, resulted in the lowest reported glutamine content of 0.0% of BSF larvae total protein content. The highest BSF larvae glutamine content was reported for BSF larvae reared for an unspecified number of days on 'Kitchen waste—potato peelings, carrot, rice, bread debris (ratio unspecified)' with 0.8% of BSF larvae total protein content [32].

Glycine—Glycine comprised 4.8% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Fruit and vegetable mix—zucchini, apple, potato, green beans, carrot, pepper, orange, celery, kiwi, plum, eggplant (unspecified ratio)' reared for 45 days, resulted in the lowest reported glycine content of 2.3% of BSF larvae total protein content [36]. The highest BSF larvae glycine content was reported for BSF larvae reared for 15 days on 'Carbohydrate—wheat middlings with 5.6% of BSF larvae total protein content [35].

Proline—Proline comprised 4.6% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Brewery by-product spent grain' reared for an unspecified number of days, resulted in the lowest reported proline content of 0.2% of BSF larvae total protein content [32]. The highest BSF larvae proline content was reported for BSF larvae reared for 15 days on 'Carbohydrate—wheat middlings with 6.2% of BSF larvae total protein content [35].

Serine—Serine comprised 4.1% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Restaurant waste—potato, rice, pasta, vegetable (ratio unspecified)' reared for 19 days, resulted in the lowest reported serine content of 1.6% of BSF larvae total protein content [33]. The highest BSF larvae serine content was reported for BSF larvae reared for eight days on 'Wheat, brown algae *A. nodosum* (4:6)' with 4.9% of BSF larvae total protein content [26].

Tyrosine—Tyrosine comprised 4.1% of BSF larvae total protein content when reported on a dry matter basis. Rearing substrate 'Brewery by-product spent grain' reared for an unspecified number of days, resulted in the lowest reported tyrosine content of 0.3% of BSF larvae total protein content [32]. The highest BSF larvae tyrosine content was reported for BSF larvae reared for 19 days on 'Food waste (uncharacterised)' with 6.0% of BSF larvae total protein content [38].

The one article reporting BSF larvae non-essential amino acid composition on a wet weight basis showed the least and most abundant non-essential amino acid to be cysteine (1.1% of BSF larvae total protein content) and alanine (2.7% of BSF larvae total protein content), respectively, when reared on 'Food waste (uncharacterised)' for an unspecified number of days [41].

#### **4. Risk of Bias Assessment**

Risk of Bias (ROB) assessment was performed using the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) risk of bias tool [18], with the exclusion of items 5 and 7 (Supplementary Table S2). These items were removed from assessment following the instruction of the (SYRCLE) risk of bias tool to adapt the list to the specific needs of the review [18], with 'blinding of the caregiver' and 'blinding of the assessors' deemed by the authors as unlikely to influence the potential bias of the articles reviewed. This ROB assessment performed in this review highlights a potential widespread nature of poor method reporting and lack of intent to reduce the risk of bias in BSF larvae rearing investigations. All articles included in this review were missing information to various degrees, with all showing potential for selection bias due to the absence of methodological information reporting on allocation concealment or sequencing generation and as such, being at risk of a significant level of bias.

#### **5. Discussion**

BSF larvae offer a feasible and cost-effective solution to two growing global challenges: food waste management and the rising global demand for sustainable protein. For this decomposer insect to be utilised in the treatment of food waste, and then to be effectively implemented and accepted into the food supply, it is essential to further our knowledge regarding the influence of various food waste streams on the nutritional composition of the BSF larvae.

Dietary protein is an essential macronutrient in BSF larval development and is necessary for supporting adequate protein and lipid accumulation in the fat cells of the BSF [46]. The results of this review indicate the total protein content of the BSF larvae can be substantially influenced by the substrate protein content of which it is reared on, with BSF larvae reared for up to 28.7 days on various combinations of low protein 'Fruit and vegetable' substrates producing the least abundant source of BSF larval protein (12.9% total protein) [19,39]. In comparison, BSF larvae reared for two days on a high protein substrate 'Fish waste *S. aurita*' produced the most abundant source of BSF larval protein (78.8% total protein) [40].

Interestingly, BSF larvae reared on 'Fish waste *S. aurita*' for longer than two days displayed a steady decline in larval total protein content, suggesting that two days of rearing on this high protein food waste substrate would be the optimal condition to generate high protein BSF larvae [40]. Variation in the protein composition of the BSF

larvae throughout their lifespan has been supported by others using non-food waste rearing substrates (Chicken feed), in which total protein has been reported to range between 34% and 42% during larval stages and between 31% and 46% [47–50]. The articles collated in this review began experimental feeding procedures at various stages in the BSF larvae life cycle, from placing eggs directly onto the rearing diets [22,36] to waiting until the BSF larvae were aged up to 14 days before introducing them to food waste substrates [25]. Furthermore, some studies reported different harvest stages, including the sight of first prepupae [30], when 40% of BSF larvae had reached prepupae [36,37] and when 100% of BSF larvae had reached prepupae [38] (Supplementary Table S3). As such, the composition of total protein in the BSF larvae reported across different studies may be compounded by factors such as age and harvest stage, rather than a representation of the type of food waste provisions.

The articles included in this review also reported widespread difference in the quantity of feed rations provided to the BSF larvae, with a range from 12.2 mg per larvae [23] to 1530 mg per larvae [24]. Insufficient feed rations have been shown to impair biomass production and influence BSF larvae protein content [48]. Whilst the optimal feeding ration of both chicken feed and fecal sludge has been determined for optimising BSF larvae biomass and nutritional content, the literature on the ideal food waste provision is limited [51]. This is likely due to the variations and inconsistencies in food waste products from a macronutrient perspective. It is plausible that the BSF larvae in the articles included in this review, may have been provided with less than adequate feed rations and as a result their protein composition was influenced by lack of sustenance as opposed to the macronutrient content of the rearing substrate provided.

In addition to rearing conditions influencing the composition of total protein in the BSF larvae, different data analyses methods used by different studies may have impacted on the ability to accurately compare the efficacy of differing food waste substrates. The total protein content of BSF larvae is most commonly determined from the total elemental nitrogen content using the common Kjeldahl method and the standard protein conversion factor 6.25 [52]. However, due to the additional non-protein nitrogen found in the insects' chitin, it is possible to over-estimate total protein content and as such a factor of 4.67 has been proposed as a more accurate representation of total protein content in insects [52]. When presenting the proximate composition of BSF larvae, Ewald et al. [24] included results of both conversion factors highlighting the differences between both factors. BSF larvae reared on 'Bread' for 14 days, with protein determination calculated with a conversion factor of 6.25, indicated the substrate to result in a total protein content of 39.2% of BSF larvae dry matter [24]. Yet, when the same data were reanalysed using a conversion factor of 4.67, a total protein content of 29.8% of BSF larvae dry matter was indicated; a substantial difference of 9.4% in the total protein content reported in BSF larvae [24]. Spranghers et al. [33] also included chitin corrected values when observing the influence of 'Restaurant waste—potato, rice, pasta, vegetable (ratio unspecified)' on the nutritional composition of the BSF, finding a similar decrease of 9.0% in BSF larvae total protein content when compared to data not taking chitin into consideration [33]. It is possible to include chitin corrected value, as this is obtained by analysing the nitrogen content of the chitin fraction and subtracting this from total nitrogen content, yet the reporting of the conversion factors was absent in many articles included in this review [20,21,23,36,37,39], as was the reporting of chitin correction adjustments [19–22,24–28,30–32,34,35,37–41], which may hinder the ability to draw comparisons between the total protein content of BSF larvae reared on various food waste substrates included in this review.

The BSF larvae's amino acid profile has been shown to be suitable for use as pet food [47] and animal feed [41]. To date, only a few studies have examined the relationship between the substrate amino acid composition and that of the BSF larvae [25,32]. When observing the impact of food waste substrates on the amino acid profiles of BSF larvae, of the two articles included in this review, glutamic acid was reported to be the most abundant non-essential amino acid (when reared on 'Wheat, brown algae *A. nodosum* (4:6)'

for eight days) [26] and leucine the most abundant essential amino acid (when reared on 'Wheat, brown algae *A. nodosum* (4:6)' for eight days) [26]. This was consistent with the glutamic acid and leucine content also being reported as most prevalent in the food waste rearing substrates provided to BSF larvae [26,33]. Aside from both glutamic acid and leucine, there was a great variation in the amino acid content of substrates used in different studies (Supplementary Table S1.). Despite this, the BSF larvae only exhibited minimal differences in amino acid content (±20%) within each study [33,38]. This would indicate that the amino acid content of the BSF larvae has a limited opportunity for manipulation when reared on food waste products regardless of the amino acid content of the rearing substrate provided. With only two articles providing amino acid content of both BSF larvae and the rearing substrate, there is limited information available to draw a solid conclusion on the role of rearing substrates in the influence of the amino acid content on BSF larvae. This makes further studies essential to developing a clearer understand of this relationship.

The choice of processing methods has been shown to influence the amino acid content of the BSF larvae, including the culling and drying method used in preparing the BSF larvae [53]. Articles included in this review reported various drying techniques including freezing [23,34] and freeze-drying in liquid nitrogen [29]. Huang et al. [53] reported that conventional drying (60.0 ◦C) of BSF larvae produced a higher digestible indispensable amino acid score when compared with microwave drying of BSF larvae. Others have reported that culling BSF larvae by a method of freezing resulted in a reduction in amino acids cysteine and lysine content [54]. Both Liland et al. [26] and Spranghers et al. [33] reporting freezing as their method of culling BSF larvae, there is an increased likelihood of the amino acid content values of the BSF larvae being inaccurately reported.

In addition to rearing substrate, various abiotic factors can affect the development of BSF larvae and may explain the variability of the nutritional content of BSF larvae in the studies included in this review. Abiotic factors that may influence BSF larvae development include larval density [55], larval handling [42], substrate moisture content or pH level [56], however, these factors were not extracted from articles and taken into consideration in this current review.

#### **6. Conclusions and Future Directions**

The results of this review on the influence of food waste products on BSF larvae protein content infer that the total protein content of food waste products used as a rearing substrate is likely to result in producing BSF larvae with a similar total protein and amino acid content. However, due to the variation in methodologies applied within each study and absence of BSF larvae nutritional composition at the commencement of the studies, there is a reduced confidence in the extent to which these various food waste substrates may have influenced the total protein content of BSF larvae. The standardisation of methodologies in BSF larvae resource conversion studies have been proposed by others and adherence to a standard methodology may increase confidence in future studies [44].

The transformation and nutrient recovery prospects of using BSF larvae as a food waste management system and as a sustainable protein source are promising. However, further research is required regarding the influence of various food waste streams on the protein composition outcomes of the BSF larvae, as well as a greater understanding of the potential influence of anti-nutritive elements on the nutritional profiles of BSF larvae. Further research exploring these factors will improve the successful introduction of BSF larvae as a novel feed and/or food.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/insects12070608/s1, Supplementary Table S1, Amino Acid Profile of Rearing Substrates. Supplementary Table S2, Risk of bias assessment. Supplementary Table S3, Life history traits of Black Soldier Fly Larvae Reared on Food Waste.

**Author Contributions:** All authors contributed to the development of the research protocol (I.H., L.P.N., H.G. and J.D.). Titles and abstracts collected from the search were screened by two investigators (I.H. and J.D.). In circumstances where it was not clear whether an article met the eligibility criteria, full-text articles were obtained, screened and resolved by discussion and consensus between three investigators (I.H., L.P.N., H.G. and J.D.). All authors provided intellectual input to the final manuscript (I.H., L.P.N., H.G. and J.D.). All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available in The Influence of Food Waste Rearing Substrates on Black Soldier Fly Larvae Protein Composition: A Systematic Review.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

