**Deoxynivalenol (DON) Accumulation and Nutrient Recovery in Black Soldier Fly Larvae (***Hermetia illucens***) Fed Wheat Infected with** *Fusarium* **spp.**

**Zehra Gulsunoglu 1,2, Smitha Aravind 2, Yuchen Bai 2, Lipu Wang 3, H. Randy Kutcher <sup>3</sup> and Takuji Tanaka 2,\***


Received: 22 August 2019; Accepted: 16 September 2019; Published: 19 September 2019

**Abstract:** Fusarium head blight (FHB) is one of the most significant causes of economic loss in cereal crops, resulting in a loss of \$50–300 million for Canadian agriculture. The infected grain (containing *Fusarium*-damaged kernels (FDKs)) is often both lower in quality and kernel weight, and it may be unsuitable for human and animal consumption due to mycotoxin presence. However, it still contains a considerable amount of nutrients. A method to recover the nutrients without the mycotoxins should be beneficial for the agricultural economy. In this study, our objective was to examine recovery methods of the nutrients in relation to mycotoxin accumulation in the insect. The FDKs were fermented with *Aspergillus oryzae* and/or *Lactobacillus plantarum* (solid-state fermentation (SSF)). The SSF kernels were then provided to 50 young, black soldier fly larvae (BSFL) for 12 days. Weight gain, chemical composition, and mycotoxin bioaccumulation of BSFL and spent feed were evaluated. After 12 days of insect culture, the BSFL grew 5–6 times their initial weight. While the overall weights did not significantly vary, the proteins and lipids accumulated more in SSF FDK-fed insects. During the active growth period, the larval biomass contained deoxynivalenol (DON), a mycotoxin, at detectable levels; however, by day 12, when the larvae were in the pre-pupal stage, the amount of DON in the insect biomass was nearly negligible, i.e., BSFL did not accumulate DON. Thus, we conclude that the combination of BSFL and SSF can be employed to recover DON-free nutrients from FHB-infected grain to recover value from unmarketable grain.

**Keywords:** insect culture; solid-state fermentation; mycotoxins; value-added processing; Fusarium head blight

#### **1. Introduction**

Fusarium head blight (FHB) is a fungal disease caused by several *Fusarium* spp. Wheat, barley, oats, corn, and other cereal grains can be affected by FHB, resulting in small lightweight kernels and, thus, loss of yield. *Fusarium* spp. produce various amounts and types of trichothecene mycotoxins, which are highly toxic to humans and livestock [1]. A major mycotoxin produced by *Fusarium* spp. is deoxynivalenol (DON). Toxin production occurs during disease development in the field under favorable weather conditions. Contamination of food and feedstuff with DON causes short- and long-term adverse effects on human health and livestock productivity [2]. In order to limit the mycotoxins in food and feed, regulations specify maximum allowable concentrations, which is 1 mg/kg sample in many countries. According to the regulations, products are monitored, and when mycotoxin

concentrations exceed the maximum allowable limits, products are separated from the food chain [3]. The economic loss from FHB accounts for many millions of dollars in Canada alone.

Detoxification methods are expensive, labor-intensive, inefficient, and time-consuming, and there is inadequate capacity for industrial applications. An effective way to prevent FHB in the field is to treat the flowering wheat plants with fungicides, and to develop resistant cultivars to minimize the infection of *Fusarium* spp. Fungicide, however, has limited effects on the infection, and, every year, a large number of grains are damaged by FHB. Considering that possible approaches to prevent the contamination of grain with mycotoxins are limited before harvest, alternate approaches should be considered to utilize inedible FHB-damaged kernels (FDK). In this study, we aimed to investigate if black soldier fly larvae (BSFL) can grow on FDK without any toxin accumulation in larval body.

World population is increasing, and it is predicted to reach 9.6 billion by 2050, i.e., a 2.3 billion increase in the next 30 years. Food production relies on agriculture, but the current practice in agriculture may not be sufficient to supply enough food for this population increase, without damaging Mother Earth or introducing super-high-yield crops that do not result in environmental damage. Utilization of inedible agriculture products can bypass the above concerns and can yield additional edible products from current practice agricultural production [4].

There is a considerable interest in the use of insects to recover inedible organic matter because insects can convert carbohydrates into proteins and lipids using organic wastes [5]. *Hermetia illucens* (black soldier fly) is one of the most important species, along with other insect species like *Tenebrio molitor* (yellow mealworm), *Drosophila melanogaster* (common fruit fly), *Amyelois transitella* (orange worm), *Helicoverpa zea* (corn earworm), and *Trichoplusia ni* (cabbage looper) [3]. While the regulations vary among countries and areas, the nutrient values and ease of utilization of insect nutrients draw huge interests for providing an alternative food source [6]. BSFL are considered a possibly proteinaceous animal feed or human food source because of the high accumulation of fat (29%) and protein (42%) in their body, and they do not transmit pathogenic microbes to humans and animals [7]. BSFL have high feed conversion ratios and an ability to convert various organic wastes into body mass [8,9].

Solid-state fermentation (SSF) shows great possibilities in the development of high-value products. Fungal and bacterial strains can be used in SSF, utilizing their abilities of enzyme production such as cellulase, pectinase, and xylanases. In this study, we used *Aspergillus oryzae* and *Lactobacillus plantarum* as microbial strains based on data obtained from our previous study [4]. The main objective of SSF was increasing the bioavailability of nutrients in FDK in favor of recovering them as BSFL biomass, and changing the nutrient profiles in favor of improved/efficient nutrient recovery in BSFL. Through these microbial modifications, efficiency of nutrient recovery from damaged crops should be enhanced during BSFL digestion.

The BSFL can be utilized to reduce pollution and convert low-value organic resources into a high-quality feed protein. They do not harbor diseases, and their production does not need any special equipment of facilities. BSFL are an extremely resistant species capable of dealing with demanding environmental conditions, such as drought, food shortage, or oxygen deficiency. The BSFL is already used in the waste management of some substrates such as manure, rice straw, food waste, kitchen waste, distillers' grains, rotting plant tissues, fecal sludge, animal offal, and animal manure [8]. We showed that BSFL can convert agricultural wastes into biomass with up to 95% recovery of organic matter using SSF [4,10]. Our previous research [4,10] suggests that FDK could be fed to BSFL to recover nutrients at a high rate with SSF treatment of feedstock. It is, however, unclear if the mycotoxins would be accumulated when BSFL are fed with FDK. We hypothesize that the BSFL are not affected by the mycotoxin content of wheat grain infected with *Fusarium* spp., they will not accumulate mycotoxins in their bodies, and a majority of the nutrients in the FDK will be recovered using SSF treatments. Efficient recovery of nutrients from damaged crops can be used to create a high-value product from low-value grain. In this study, the performance of BSFL in converting SSF-treated FDK into insect biomass and the accumulation of DON were investigated.

#### **2. Material and Methods**

#### *2.1. Materials*

BSFL were purchased from Worm Lady (McGregor, ON, Canada). All chemicals used in this study were commercially available ACS grade and were purchased from Fisher Scientific (Ottawa, ON) and VWR International (Edmonton, AB). *Aspergillus oryzae* NRRL 32657 (*Ao*) and *Lactobacillus plantarum* NRRL B4496 (*Lp*) were obtained from the ARS Culture Collection (USDA, Peoria, IL, USA).

#### *2.2. Solid-State Fermentation*

The initial DON concentration of FDK was 0.63 ± 0.20 μg/g dry matter (dm). The FDKs were soaked in water for 18 h at 21 ◦C to obtain softer kernel for fermentation. The soaked kernels were shred using a household coffee mill (Cuisinart DBM-8, Woodbridge, ON, Canada). Seed cultures of *Ao* and *Lp* were prepared by inoculating Potato Dextrose (PD) and De Man, Rogosa, and Sharpe (MRS) broths. The seed culture of *Ao* was prepared by inoculating 250 mL of PD broth in an Erlenmeyer flask with two loopfuls of spores, followed by 72-h incubation at 30 ◦C on a rotary shaker (150 rpm). The seed culture of *Lp* was prepared by inoculating 1 mL of fully grown preculture in 250 mL of MRS broth in an Erlenmeyer flask and incubating for 24 h at 37 ◦C on a rotary shaker (150 rpm). After incubation, fungal and bacterial seed cultures were collected by centrifugation at 6000 rpm for 10 min at 10 ◦C (Sorvall, RC28S, Manasquan, NJ, US). The biomass obtained was re-suspended in 1/10 of the original volume of sterile distilled water.

Approximately 58 g (34 g in dry weight) of shredded kernels were weighed into each glass jar and, from the seed cultures, 1.5 mL of *Lp*, 2 mL of *Ao*, and 3.5 mL of a combination of these two strains (*Lp* + *Ao*) were inoculated into the crushed kernels. The moisture content was adjusted to 55% (*w*/*w*) with sterile water. The control sample was prepared without initial seed culture inoculation. The samples were fermented at 30 ◦C for four days, and the moisture content was kept constant by adding sterile water and mixing once per day under aseptic conditions.

#### *2.3. BSFL Digestion*

Fifty BSFL (second instar) were introduced to each SSF FDK sample in the glass jars that had perforated lids to allow moisture and gas transfer during BSFL digestion. Twelve jars were prepared under the same conditions for each SSF FDK sample. The jars were kept at 30 ◦C for 12 days. Water was added by weighing the sample jars and mixing each day under aseptic conditions.

#### *2.4. BSFL Separation after Digestion*

After interval days (0, 4, 8, and 12 days), the larvae were separated from the residual feed using forceps. The larvae were rinsed with water to remove residual substrate from their surface and dried on paper towel; then, their wet weight was determined. Then, the larvae were frozen at −20 ◦C for further analysis. The rinsed-off feed residues were placed back in the feed bed to avoid errors in spent feed analyses.

#### *2.5. Larval Weight Gain Determination and Survival Rate of Larvae*

For survival analysis, the number of larvae was counted at 0, 4, 8, and 12 days (initially, exactly 50 larvae). To monitor larval growth, the dry weight of each sample was determined. Larval volume was calculated by multiplying the length, width, and thickness of 10 individual larvae before and after digestion.

#### *2.6. Proximate Analysis of BSFL and Spent Feed*

Proximate analysis was performed on the BSFL and spent feed before and after larval digestion of the fermented FDK. The dry weight of the samples was measured after drying at 105 ◦C for 24 h to a constant weight according to AOAC Method #930.15 [11]. Dried samples were used for ash, crude protein, and crude fat analyses. The ash content of the larval biomass and spent feed was determined by the gravimetric method as described in AOAC Method #942.05 [11]. Samples were carbonized using a hot plate in the fume hood and, after carbonization, the crucibles were incinerated in a muffle furnace at 550 ◦C overnight. Crude protein content was measured by the micro-Kjeldahl method as described in AOAC Method #960.52 [11] with slight modification. Conversion factors of 6.25 and 5.70 were used to calculate total protein of the larval biomass and spent feed, respectively. The crude fat analysis was determined according to the Goldfisch method as described in AACC method #30-20.01 [12]. Samples were weighed onto Whatman filter paper (No.1) at 0.5 g for larval biomass and 1 g for spent feed and placed in the Goldfisch apparatus (Labconco Corporation, Kansas City, MO, US). Petroleum ether was used as the extraction solvent, and the extraction process lasted 6 h. Crude fat was determined as the weight of fats in the extract after removal of the solvent. The carbohydrate contents of BSFL and spent feed were determined by subtracting the lipid, protein, and ash contents from the total weight.

#### *2.7. Mycotoxin Analysis*

Larval biomass and spent feed were finely ground and extracted for mycotoxin analysis to determine the concentration of DON accumulated. Larval samples and spent feed were extracted according to the method developed by Dr. L. Wang, at the Cereal and Flax Pathology program at the University of Saskatchewan (personal communication). Finely ground wheat grain (2 g) and a larval sample (0.1 g) were mixed with acetonitrile/water (84:16, *v*/*v*) with a ratio of 1:4 *w*/*v* and extracted on a rotary shaker for 2 h at room temperature (250 rpm). The extract was diluted 1:10 with 5 mM ammonium acetate and syringe-filtered. Thirty microliters of this filtrate was injected into the LC–MS/MS.

The LC–MS/MS conditions were developed on a high-performance liquid chromatography system (Agilent 1260 Infinity Quaternary; Agilent Technologies, Mississauga, ON, CA) coupled to an AB Sciex 4000 hybrid triple quadrupole linear ion trap (4000 QTrap) mass spectrometer (Concord, ON, CA) equipped with a TurboionsprayTM interface. Applied Biosystems/MDS Sciex Analyst software (Version 1.6.2, AB Sciex, Foster City, CA, US) was used for system control and quantification. The mobile phase consisted of a mixture of solvent A (5 mM ammonium acetate in water) and solvent B (5 mM ammonium acetate in methanol). Samples were stored in the auto sampler at 4 ◦C and a 30-μL injection volume with a 3-s flush port wash (to minimize carryover) was used to introduce the sample into the column.

Chromatographic separation was obtained at a flow rate of 300 μL/min through an Agilent ZORBAX Eclipse XDB C18 column (4.6 × 100 mm, 1.8 μm) equipped with an Eclipse XDB C18 (4.6 mm, 1.8 μm) guard column maintained at 30 ◦C in a column heater. Multiple reaction monitoring (MRM) with electrospray was used to monitor DON with the transitions of *m*/*z* 355.0 to *m*/*z* 296.1 and *m*/*z* 355.0 to *m*/*z* 264.9 as quantifier and qualifier ions, respectively.

The DON standard (purity > 99%) was supplied by Romer Labs Inc. (Tulln, Austria), and the stock solution was prepared at a concentration of 1 μg/mL in acetonitrile and kept at −20 ◦C. Working stock solutions were made by diluting in 5 mM ammonium acetate to the level of 10-fold final working concentration for each standard and quality control (QC) point. Standard and QC samples were prepared by adding 100 μL of each working stock to 900 μL of blank sample and mixing gently. A standard curve of seven points was constructed by determining the best fit of peak area versus the analyte concentration and running a weighed 1/*x* linear regression analysis.

#### *2.8. Statistical Analysis*

Each treatment was analyzed in triplicate, and results are presented as an average with standard deviation. Statistical significance of the results was analyzed by one-way analysis of variance (ANOVA) using MINITAB (MINITAB 18, Minitab Inc., Coventry, UK). Treatment means were declared significantly different from each other using Tukey's test at *p* < 0.05.

#### **3. Results and Discussion**

#### *3.1. Growth Rate of BSFL*

The amount of feed left after periods of BSFL rearing is given in Figure 1. The BSFL consumed about 65, 45, 61, and 72 mg FDK/larva/day during the first four days for the control, *Lp*, *Ao*, and *Lp* + *Ao* groups, respectively. In the next four days, the BSFL consumed 67, 82, 76, and 84 mg FDK/larva/day for the control, *Lp*, *Ao*, and *Lp* + *Ao* groups, respectively. After eight days of consumption of FDK, BSFL growth rates decreased for all treatments. The BSFL consumed 30, 42, 21, and 10 mg FDK/larva/day during the last four days for the control, *Lp*, *Ao*, and *Lp* + *Ao* groups, respectively.

**Figure 1.** The relative amount of spent feed during black soldier fly larvae (BSFL) digestion. Bars indicate differences in nutrient composition of spent feed after BSFL digestion based on initial day. During feeding time, the residual amount was calculated by taking into account the weight of Fusarium-damaged kernels (FDKs) consumed by BSFL. Each bar was divided according to the ratio of nutrients: hatched area, ashes; black area, proteins; dotted area, carbohydrates; gray area, lipids. Each panel represents the results from (**A**) control without any inoculum (i.e., unfermented FDK), (**B**) fermented FDK with *Lactobacillus plantarum*, (**C**) fermented FDK with *Aspergillus oryzae*, and (**D**) fermented FDK with both *L. plantarum* and *A. oryzae*.

Feed consumption was relative to the growth rate of larvae. The weights and volumes of BSFL from each batch fed FDK fermented with *Lp*, *Ao*, and *Lp* + *Ao* were determined for each feeding time (Figure 2). Increases in weight were observed until day eight for all treatments. From the eighth to the twelveth day, BSFL weight did not differ for any of the treatments. Initial weight of BSFL (day zero) was 8.5 ± 0.4 mg on a dry weight basis (dwb) per larva. Larvae gained significantly more weight when fed with FDK fermented with *Lp* (58.4 ± 6.3 mg/larva). Kuttiyatveetil et al. [4] assessed SSF borage meal and flaxseed meal as the feedstuffs and reported that the highest BSFL biomass was 87.4 mg/larva. The volume of BSFL increased from 66.8 <sup>±</sup> 21.8 mm<sup>3</sup> to 352.7 <sup>±</sup> 87.9 mm<sup>3</sup> when fed FDK fermented with *Ao* at day eight, and there were no significant differences among treatments. Larval survival rates did not differ among treatments. At the end of the feeding time, 90–93% of initial larvae survived.

It is speculated that the final four days were the pupation period. At prepupa, the last larval stage, the larvae stop feeding in order to produce prothoracicotropic hormone (PPTH), which is necessary for metamorphosis. At this stage, they attain maximum size and have large protein and fat contents to sustain them through metamorphosis; they do not show significant changes in morphological characteristics at this stage [13]. Our results showed low consumption of feeds and weight gains of BSFL, indicating that the last four days can be considered as prepupal periods.

**Figure 2.** *Cont*.

**Figure 2.** Weight gain, proximate composition, and deoxynivalenol (DON) concentration of larvae during black soldier fly larvae rearing. The overall length of each bar shows the dry weight of larvae divided according to the ratio of nutrients: hatched area, ashes; gray area, lipids; dotted area, carbohydrates; black area, proteins; the lines, DON concentration in black soldier fly larvae. Each panel represents the results of larvae from (**A**) Fusarium-damaged kernels (FDK) without inoculum (i.e., unfermented FDK), (**B**) FDK with *Lactobacillus plantarum*, (**C**) fermented FDK with *Aspergillus oryzae*, and (**D**), fermented FDK with both *L. plantarum* and *A. oryzae*.

#### *3.2. The Proximate Composition of BSFL and Spent Feed*

In order to achieve high larval growth, FDK was fermented for four days using generally regarded as safe (GRAS) strains (*Ao* and/or *Lp*). The nutrient profile differences among SSF treatments at day zero indicated that unfermented FDK contained 1.5% lipid, 8.9% protein, 2.2% ash, and 87.7% carbohydrates (Figure 1). At the fourth day, SSF did not change ash and lipid contents; however, the protein and carbohydrate contents differed. The protein content of the grain increased for all fermented FDK treatments; however, the carbohydrate content decreased during SSF for all treatments. While the carbohydrate ratio decreased, its absolute amount remained the greatest in the SSF feed, and the increased protein content was speculated to benefit larval growth. There was no difference in the amount of nutrients among microorganisms tested in this study; however, based on visual observation, FDK became softer compared to control FDK for easier mastication by BSFL.

During BSFL feeding trials, nutrient compositions of spent feed changed (Figure 1). Results showed that BSFL were able to consume and digest carbohydrates (i.e., starch) as their main food sources. The BSFL consumed around 23–25% of the dry matter in SSF feeds in 12 days, i.e., the residual amounts were reduced from 34 g at day zero to 25–26 g at day 12. The protein in spent feed decreased from 4.5 ± 0.5 to 1.8 ± 0.2 g on a dwb of FDK during BSFL digestion after 12 days of digestion for all SSF treatments. Lipid content of spent feed decreased significantly for all treatments after four days of digestion, and the decrease did not change by day 12. The carbohydrate content (i.e., starch) in spent feed decreased significantly during BSFL digestion for all fermented FDK. The ash content of spent feed decreased until the 12th day of digestion for the control, *Ao*, and *Lp* + *Ao* treatments. There was no difference in the ash content of FDK fermented with *Lp*. The decrease in proximate composition of spent feed during BSFL digestion was associated with the consumption of nutrients by BSFL.

The proximate composition of larval biomass was determined (Figure 2). During feeding time, the ash amount of BSFL increased until day eight, when it reached the maximum level for all treatments, and then it remained constant until the 12th day. The initial ash content of BSFL was 0.5 ± 0.0 mg/larva and it reached 2.0 ± 0.0 mg/larva at day eight for FDK fermented with *Lp* + *Ao*. The carbohydrate content of BSFL was 1.7 ± 0.2 mg/larva, and, after the eight-day feeding period, the carbohydrate content increased to 14.6 ± 1.0, 8.3 ± 0.8, 11.3 ± 1.5, and 12.4 ± 1.2 mg/larva fed with FDK fermented in the control, *Lp*, *Ao*, and *Lp* + *Ao* treatments, respectively. The higher carbohydrate contents can be explained by an increase in the volume of BSFL, associated with an increase in larval skin chitin, which is produced during the growing stage of the BSFL. Kaya et al. [14] reported that chitin content increased gradually from larva to adult, and the highest chitin content was observed in adults. The carbohydrate content was higher in the control BSFL than among other treatments (*p* < 0.05). This can be explained by the reduced gain in the protein, lipid, and ash contents, i.e., concentrating of carbohydrates [10]. These results indicated that BSFL can recover the nutrients in damaged FDK and are not affected by the presence of mycotoxins.

Also, it was noticed that the sums of protein and lipid gains were 42.6 ± 4.7, 47.7 ± 3.1, 45.2 ± 0.8, and 40.4 ± 2.1 mg/larva fed with FDK fermented in the control, *Lp*, *Ao*, and *Lp* + *Ao* treatments, respectively, at day 12. At day eight, these figures were 42.3 ± 1.8, 45.2 ± 1.6, 40.1 ± 2.1, and 43.5 ± 1.3, respectively. Meanwhile the accumulation of carbohydrate at day eight (and day 12) were 14.6 ±1.0 (10.1 ± 0.3), 8.3 ± 0.8 (7.0 ± 0.6), 11.3 ± 1.5 (8.5 ± 0.1), and 11.3 ± 2.3, respectively. These results indicated that SSF grain-fed insects can achieve better protein and lipid profiles compared to unfermented grain-fed insects. It is, therefore, indicated that the SSF assisted to improve the nutrient components of the insect, while overall gain in weight in this study did not significantly vary among the feeds.

#### *3.3. Amounts of DON in Spent Feed and BSFL Biomass*

During BSFL digestion, the DON concentration in spent feed at the fourth day was the lowest compared to other days (Table 1). After four days of digestion, the concentration of DON in spent feed continuously increased until day 12. This increase in DON contents of the spent feed was comparable to the 23–25% consumption rate of nutrients by larvae. This suggested that the larvae did not assimilate DON in their body, but it was simply passed through their intestinal system. The highest DON concentration in the spent feed was for FDK fermented with *Lp*.


**Table 1.** Deoxynivalenol (DON) concentration in spent feed after black soldier fly larvae digestion.

\* Each value is expressed as the mean ± SD (*n* = 3). Statistical symbols: Means of DON concentration for each fermentation type marked with different lowercase letters (a, b, c) within a row are significantly different between days, and those followed by capital letters (A, B, C) within a column represent significant differences in DON concentration compared with fermentation type on the same day (*p* < 0.05).

There are several possibilities to explain the DON increase. Some microbial enzyme yielded during fermentation could enhance the DON levels, owing to a release of DON from kernel cell walls or other cell components [15]. Secondly, the DON may be produced during the feeding periods. Microorganism type and the proportion of each may result in an increase in mycotoxin production among microorganisms [5]. For example, the increase could also result from the increasing number of *Fusarium* spp. during fermentation time. Goral et al. [16] investigated the relationship between

concentration of *Fusarium* biomass and trichothecenes B (DON and nivalenol). They reported a stronger relationship between *Fusarium* biomass and DON content, as confirmed by FHB index. As another possibility, they might be converted from precursors. Nakagawa et al. [17] reported that deoxynivalenol-3-*O*-glucoside (D3G) has lower toxicity compared to its precursor DON, and D3G can be converted to DON in human or animal gut. Lactic acid bacteria have the capability to hydrolase the derivative D3G back to the toxic DON form. When the lactic acid bacteria ferment the feed, it might be responsible for the chemical conversion of conjugated mycotoxins and explain the high amount of DON in the spent feed [18]. While these factors can affect the results, it is unlikely appropriate to explain our results. We checked the DON amounts at each sampling time for SSF and unfermented grains. Such generations and conversions were counted in the data, regardless of their effect. We did not observe significant differences in DON amounts among the four conditions. Thus, we concluded that the differences in DON amounts between SSF and unfermented FDK were small and they did not significantly affect the contents of DON.

While DON amounts in the spent feed increased during the feeding period, BSFL biomass showed different trends in terms of DON amounts. In the growing stage, the larval biomass contained considerable DON concentration; however, there was no DON accumulation observed in BSFL after the 12-day rearing. Bosch et al. [3] evaluated the tolerance and accumulation of aflatoxin B1 (AFB1) in BSFL fed with AFB1-containing feeds to utilize the mycotoxin-contaminated crops. The BSFL did not contain detectable levels of AFB1 (<0.10 μg/kg). They suggested that the larvae rapidly excreted or metabolized the AFB1 after ingestion. Another possible reason was that part of the AFB1 could be found in bound form with proteins and left undetected. Camenzuli et al. [19] also investigated the potential of accumulation of AFB1, DON, ochratoxin A, and a mixture of mycotoxins in BSFL. None of the mycotoxins accumulated in the larval body; they were shown to excrete or metabolize the four mycotoxins present in the feed. Our results showed that BSFL growing on FDK feeds do not accumulate DON in their body. As shown in Figure 1, the larvae consumed most of the feed during the first eight days, and then consumption stopped by day 12, due to pupation. This is because they defecate their intestinal system and have an empty gut before pupation or shortly after adult emergence. The DON analysis indicated that BSFL do not assimilate DON in their body, and DON observed during the growth stage is speculated to be in the contents of their intestinal system.

#### **4. Conclusions**

The FDK used in this study had a large amount of nutrients, and BSFL converted these nutrients to insect biomass without accumulating DON in their bodies. While they consumed DON-contaminated materials, they contained DON in their body; however, the toxins were excreted from their body before they became pupae. They consumed ~2.8 g of FDK per g of BSFL body mass gained, and mainly converted starch into their proteins and lipids at a high efficiency. Thus, the BSFL can be used to separate nutrients from DON in FDK. Proximate analysis of fermented FDK showed higher protein and lipid contents, while there was no significant difference in the BSFL body mass gain among treatments. The procedure can be expanded to other mycotoxin-contaminated materials to recover valuable nutrients wasted in those contaminated materials. This waste treatment technology using BSFL may contribute to reducing the burden of animal protein shortages in the animal feed market and provide new income opportunities for small entrepreneurs in low- and middle-income countries.

**Author Contributions:** Conceptualization, T.T.; Methodology of fermentation, insect culture and proximate analyses, Z.G., S.A. and T.T.; Methodology of DON analysis, L.W. and H.R.K.; Investigation, Z.G., A.S., Y.B. and L.W.; Writing original draft preparation, Z.G.; Writing-Review & Editing, T.T.; Supervision, T.T.

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

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

#### **References**


© 2019 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 (http://creativecommons.org/licenses/by/4.0/).

### *Review* **Food Wastes as a Potential New Source for Edible Insect Mass Production for Food and Feed: A review**

#### **Vassileios Varelas**

Biodynamic Research Institute, Skillebyholm 7, 15391 Järna, Sweden; vavarelas@chem.uoa.gr or vassileios.varelas@sbfi.se

Received: 9 August 2019; Accepted: 27 August 2019; Published: 2 September 2019

**Abstract:** About one-third of the food produced annually worldwide ends up as waste. A minor part of this waste is used for biofuel and compost production, but most is landfilled, causing environmental damage. Mass production of edible insects for human food and livestock feed seems a sustainable solution to meet demand for animal-based protein, which is expected to increase due to rapid global population growth. The aim of this review was to compile up-to-date information on mass rearing of edible insects for food and feed based on food wastes. The use and the potential role of the fermentation process in edible insect mass production and the potential impact of this rearing process in achieving an environmentally friendly and sustainable food industry was also assessed. Food waste comprises a huge nutrient stock that could be valorized to feed nutritionally flexible edible insects. Artificial diets based on food by-products for black soldier fly, house fly, mealworm, and house cricket mass production have already been tested with promising results. The use of fermentation and fermentation by-products can contribute to this process and future research is proposed towards this direction. Part of the sustainability of the food sector could be based on the valorization of food waste for edible insect mass production. Further research on functional properties of reared edible insects, standardization of edible insects rearing techniques, safety control aspects, and life cycle assessments is needed for an insect-based food industry.

**Keywords:** edible insects; food wastes; insect mass production; fermentation; sustainability

#### **1. Introduction**

Entomophagy, i.e., the practice of eating insects as food, formed part of the prehistoric diet in many areas worldwide [1,2]. Over the millennia since then, it has been a regular part of the diet of many people from various cultures throughout the world [3,4]. Globally, more than two billion people, mainly in Asia, Africa, and South America, are estimated to practice entomophagy [2,4,5], with more than 2000 edible insect species being used for this purpose [6]. In Western culture, however, entomophagy is not accepted and is considered a disgusting and primitive behavior, while insects are associated with pests [7]. However, this taboo seems to be weakening in recent years, as eating habits have been changing and a new trend for insect-based products and incorporation of entomophagy into the Western diet has begun [8,9].

In the near future, demand for animal-based food protein is expected to increase by up to 70% [10] due to exponential growth in the global population, which is predicted to reach 9 billion by 2050 [3,8]. The increased food production required to meet this demand will be accompanied by further exhaustion of water, agricultural, forestry, fishery, and biodiversity resources, with negative environmental impacts [11]. When the problem of climate change is added to these concerns, then global food security becomes an even more crucial issue [12,13].

Edible insects are called the insect species which can be used for human consumption but also for livestock feed as a whole, parts of them, and/or protein, and lipid extract [11,14,15]. Edible insects seem a promising alternative solution to achieving food security in the upcoming global food crisis [16], because they provide some significant advantages for human nutrition, including high protein, amino acids, lipids, energy, and various micronutrients [17,18]. Moreover, compared with livestock, insect rearing has a lower environmental impact as multiple and various food sources can be used, greenhouse gas emissions are low, the water and space requirements are low, and the feed conversion rate is high [7,11]. In addition to serving as food and feed, insects can also contribute significantly to food sustainability through biowaste degradation and conversion into food, feed, and fertilizers [19]. Furthermore, they can help preserve biodiversity [20] and assist in plant pollination and pest control [9].

In the global food industry, around 1.3 billion tonnes of various food wastes are discarded every year [21]. The waste generated in the food industry originate mainly from primary production, food processing, wholesale and logistics, combined with retail and markets, food service, and households. For 2012, the estimated volume of food waste for the EU alone was about 88 million tonnes [22]. In the USA, almost 45 million tonnes of fresh vegetables, fruits, milk, and grain products are wasted annually [23]. According to Baiano (2014), up to 42% of total food waste is produced in households [24].

In many cases, food waste residues are difficult to utilize for the recovery of value-added products due to their biological instability, potentially pathogenic nature, high water content, rapid autoxidation, and high level of enzymatic activity [25]. On the other hand, this biomaterial comprises a huge nutrient stock [26] and could be valorized through biodegradation by various edible insect species in a mass production system [9,27,28].

The aim of this review was to compile up-to-date information on rearing edible insects for food and feed purposes using food waste as a substrate. The impact of this bioconversion system in achieving an environmentally friendly and sustainable food industry was also considered.

#### **2. Edible Insect Species Commonly Mass Produced for Food, Feed, and Other Applications**

In general, within edible insect rearing and gathering three main strategies are followed: wild harvesting (not farming), semi-domestication (outdoor farming), and farming (indoor farming) [11]. Globally, 92% of edible insect species are wild-harvested, but semi-domestication and farming can provide a food supply in a more sustainable way [3]. Farming of insects for food and feed has recently begun [7].

Regarding consumer acceptance, distribution, rearing conditions, environmental impact, food safety aspects, nutritional value, and use as a component in the diet of farmed animals, pets, and fish, the main commercial edible species harvested in the wild worldwide, but also used for industrial large-scale production, belong to six major orders: Coleoptera, Hymenoptera, Isoptera, Lepidoptera, Orthoptera, and Diptera [15,29].

The most commonly used commercial insects in mass production are mulberry silkworm, waxworm, yellow mealworm, house cricket, black soldier fly, housefly (indoor farming), palm weevil, bamboo caterpillar, weaver ant, grasshopper (outdoor farming), eri silkworm, muga silkworm, giant hornet, and termite (wild farming) [15,30]. The insects most commonly used as animal feed are black soldier fly, housefly, mealworm, beetles, locusts, grasshoppers, crickets, and silkworm [31]. Some edible insect species are also used for medical applications, e.g., *Lucilia sericata* (common green bottlefly) is used as a biological indicator of post-mortem interval (PMI), in human pathology, while its larvae are used in human medicine for healing chronic injuries that cannot be cured with conventional treatments [32]. Moreover, the allantoin secreted by the larvae is used in the treatment of osteomyelitis [30]. Other applications of edible insects include biodegradation of polystyrene in the environment using *Tenebrio molitor* mealworm [33,34], use of black soldier for municipal organic waste management [35], and the use of non-mammalian models like *Galleria mellonella* larvae, also known as waxworm, to model human diseases caused by a number of bacterial pathogens [36].

The most common commercially reared edible insects and their applications for human food and animal and fish feed, as medicines, for component extraction and as environmental treatments are listed in Table 1.


**Table 1.** Summary of the edible insect species most commonly reared for food and feed, the developmental stage at which they are used, the type of farming system, and commercial applications.

#### **3. Edible Insect Species That Can Utilize Food Waste as Feed and Their Nutritional Requirements in Mass Production**

To date, around 1 million insect species have been described and classified, but the actual number of insect species on Earth is estimated to be between 4 and 30 million. Jongema (2015) compiled a detailed catalogue listing 2037 edible insect species [6], but the actual number of insect species suitable for human food or animal feed applications is still unknown [3].

In recent years, low cost and effective diets, so called artificial diets, are used in lab and/or industry scale in order to rear insects for various purposes (e.g., edible insects, insects as pest predators for pest biological control etc.) [57–59]. Various artificial diets have been introduced for insect rearing, but even the most promising of these is still inferior to natural nutrient sources [60]. The insect species most widely farmed for food and feed purposes are mainly omnivores, which are able to utilize various food sources and thus show broad nutritional flexibility. For this reason, their nutritional requirements and feed rate when fed an artificial diet are difficult to determine [30,61,62]. Due to their nutritional flexibility, the use of low-value food sources can be ideal for large-scale farming of edible insects [11].

A balanced diet composed of organic by-products can be as suitable for the successful growth of mealworm species as the diets used by commercial breeders [28]. It has been reported that an organic food-based diet is critical for larval growth, mass density, and colony maintenance [63]. Recycling of low-quality, plant-derived waste and its conversion into a high-quality feed rich in energy, protein, and fat can be achieved with mealworms in a relatively short time [31]. Moreover, the omnivorous house cricket *Acheta domesticus* can be fed on a large range of organic materials, making it easy to farm in a system producing six or seven generations per year [31].

Most studies with encouraging results regarding artificial diets based on food wastes or mixtures of wastes have been carried out using edible mealworm (*Tenebrio molitor* L., Coleoptera: Tenebrionidae), black soldier fly (*Hermetia illucens*, Diptera: Stratiomyidae), housefly (*Musca domestica*, Diptera: Muscidae), and Cambodian cricket (*Teleogryllus testaceus*, Orthoptera: Gryllidae) and have used raw food material as the insect feed [28,31,60,62,64–67].

Farmed edible insects that utilize food materials and wastes during rearing are summarized in Table 2.


**Table 2.** Summary of various edible insect species reared on food wastes and their characteristics.

In general, the major macronutrients required for insect mass production are (a) carbohydrates, which serve as an energy pool but are also required for configuration of chitin (exoskeleton of arthropods) [60], (b) lipids (mainly polyunsaturated fatty acids such as linoleic and linolenic), which are the main structural components of the cell membrane, and also store and supply metabolic energy during periods of sustained demands and help conserve water in the arthropod cuticle [29,59,69], and (c) the amino acids leucine, isoleucine, valine, threonine, lysine, arginine, methionine, histidine, phenylalanine, and tryptophan, which insects cannot synthesize [70], and tyrosine, proline, serine, cysteine, glycine, aspartic acid, and glutamic acid, which insects can synthesize, but in insufficient quantities at high energy consumption [61,70]. The essential micronutrients in insect rearing are (a) sterols, which insects cannot synthesize, (b) vitamins, and (c) minerals [30].

The nutrient requirements of edible insects in mass production are summarized in Table 3.


**Table 3.** Summary of the nutrient requirements of edible insects (adapted from [30,60]).

\*: Insects able to absorb and metabolize; \*\*: Insects able to absorb but not metabolize; Pfa: Polyunsaturated fatty acids; \*\*\*: Insects unable to synthesize; \*\*\*\*: Insects able to synthesize; Ws: Water-soluble; Ls: Lipid-soluble; \*\*\*\*\*: Listed in order of importance as essential for living matter (from top down). Minerals consist of combinations of cations and anions of elements; +++: Important for insect growth; ++: Important in enzyme pathways including DNA synthesis; +: Important to a lesser extent, important role in muscular excitation.

Food industry organic wastes are produced in vast quantities and can be valorized for various purposes, e.g., as biofuels, crop fertilizers, pharmaceuticals, functional foods, etc. [25]. The largest quantities are generated by the fruit, vegetable, olive oil, fermentation, dairy, meat, and seafood industries [23]. Food waste comprises a mixture of various food residues, e.g., bread, pastry, noodles, rice, potatoes, meat, and vegetables [21].

Insects are much more efficient at converting feed to body weight than conventional livestock and can be reared on organic waste streams, transforming these into high-value food and feed [31]. The use of food wastes in rearing edible insects is a quite new and promising approach [7,11]. For this purpose, various artificial food waste-based diets covering the nutritional needs of farmed insects have been proposed, without pre-treatment of the biomaterial [28,31,67] (see also Table 2).

The chemical composition and nutritional value of various wastes that have already used in insect rearing are summarized in Table 4.




**Table 4.** *Cont*.


**Table 4.** *Cont*.

\* Data refer to natural products that have not been processed or pre-treated.

#### **4. Rearing Conditions and Insect Mass Technologies**

Wild harvesting can potentially lead to depletion of natural insect species [3]. For a sustainable insect farming industry, cost-effective rearing, harvesting, and processing technologies are required [19]. The information required for industrial-scale mass production of insects from biowaste and agricultural organic residues for food and feed purposes is not complete, but much research is being conducted in this field and recent data seem very promising [30] (see also Table 2). The need for lower cost, more environmental friendly, and sustainable nutrient resources for insect mass technologies will increase as the production level increases [30]. In this regard, food biomass waste can comprise a potential source of ingredients for artificial diets used in edible insect industrial production [7,11,54].

The artificial diets used in insect mass production vary from liquid to solid, depending mainly on (a) the nutritional needs of the insect in question in terms of macronutrients, micronutrients and minerals (see also Table 3); (b) the feeding adaptation of the insect, meaning the way that food is processed by the mouthparts before ingestion, as these are adapted to match the feeding needs. Insect species possessing sucking mouthparts are liquid feeders, those possessing biting mouthparts are solid feeders, and those that possess modified sucking mouthparts, so called piercing-sucking insects, are able to pierce the host and suck liquefied animal and/or plant tissues [30,60]; (c) the pre-manufacturing of the artificial diet. Liquid diets can be used after encapsulation using different materials (paraffin, PVC, polyethylene, polypropylene) to mimic artificial eggs, a treatment step needed for their containment and presentation [60], while liquids and slurries can be dried and concentrated so that can be dissolved in water or mixed with other ingredients. Semi-liquids are used in pellet or extruded form which can be ingested by insects with biting mouthparts and also by insects with sucking mouthparts [30]. Solids are presented as a feed mash with grinding and mixing of all raw materials, after pelleting of various raw materials or by extrusion. Solids can also be encapsulated with complex coacervation technology using proteins and polysaccharides [87].

The development of low-cost commercial diets is crucial for edible insect production at industrial scale [19]. In mass production, the mechanical equipment needed in an integrated production process, automation, mechanization, and monitoring technologies for rearing, harvesting, processing, packaging, and delivering edible insects must also be applied, in order to reduce costs and produce safe food products in large-scale quantities [5,19].

#### **5. Nutritional Composition, Ingredient Characterization, and Food Functional Properties of Edible Insect Species**

Insect farming conditions, insect developmental stage, the artificial diet selected, and the preparation and processing methods used (e.g., frying, boiling, drying) are factors that affect the nutritional composition of the reared insects [11]. Different diets composed of various food wastes have been reported to result in differences in the nutritional value of mealworm larvae [88]. However, most previous studies provide no details about the artificial diets and conditions used for insect rearing or about the preparation and process stages [29,53,54].

To date, data required in INFOODS/EuroFIR recommendations concerning the nutritional value of most common edible insect ingredients are lacking [29]. These data refer to protein, crude proteins, crude lipids, available carbohydrates, moisture, dry matter, energy, vitamins, and minerals.

The nutrient content of some of the most commonly reared edible insects reared on food wastes, in terms of crude proteins, crude lipids, available carbohydrates, vitamins, and minerals, is summarized in Table 5.


**Table 5.** Nutritional value of the most common edible insects reared on food materials and wastes.

**Insect Species Common Name Develop-mental Stage Crude Protein (% Dry Weight) Lipids (% Dry Weight) Carbohydrates, Vitamins, Minerals etc. General Comments Reference** *Acheta domesticus* House cricket Adult 16% - - Crickets fed the solid filtrate from food waste processed at an industrial scale via enzymatic digestion were able to reach a harvestable size and achieve feed and protein efficiencies. Crickets reared on waste substrates of sufficient quality might be the most promising path for producing crickets economically [41] *Acheta domesticus* House cricket Adult 15.6% <sup>±</sup> 8.1% 4.56% <sup>±</sup> 2.15% Carbohydrates: - Minerals: Na, Fe, Zn, Ca, I Vitamins: B12, B2 Data show considerable variation within insect species [29]

**Table 5.** *Cont*.

The research field concerning characterization of food functional properties of the most common edible insects (e.g., amino acid and lipid composition, foam ability and foam stability, water absorption capacity (WAC), fat absorption capacity (FAC), protein solubility, microstructure and color, rheological properties, etc.) is quite new. Some data is available, mainly for yellow mealworm, silkworm, house cricket, and housefly [54,89–91].

The food functional properties characterized for the most commonly reared edible insects are summarized in Table 6.

**Table 6.** Ingredient characterization and food functional properties of most common edible insect species.


#### **6. Fermentation Process in Edible Insect Chain Production**

The fermentation process is applied during the edible insect production to the following stages: (a) Valorization of food waste via fermentation and then use of edible insects, especially of the black soldier fly (BSF) [94,95]. The use of pre-fermentation can be performed for the waste stabilization and the food safety increasement. Moreover, the pre-fermentation can enhance the digestibility and bioavailability of nutrients to the insect larvae as most nutrients present in agricultural residue or byproducts are found in insoluble form [94]. The solid residues produced by processing of food waste via microaerobic fermentation (MF) and by black soldier fly larvae (BSF) have been proposed as soil fertilizers for plant growth [95].

(b) Use of fermentation by-products and food wastes as ingredients of artificial diets used for edible insect production. The edible mealworm species *Tenebrio molitor* L., *Zophobas atratus* Fab. and *Alphitobius diaperinus Panzer* were grown successfully on diets composed of organic by-products originating from beer brewing, bread/cookie baking, potato processing, and bioethanol production [28]. The *Hermetia illucens* edible insect, commonly named black soldier fly (BSF), was used for the biodegradation of kitchen residues, grass, sewage sludge, and separated solid material from biogas plants [68]. House crickets (*Acheta domesticus*) have been reared on diets based on food waste processed at an industrial scale via enzymatic digestion [41].

(c) Fermentation of the produced edible insect orders to increase the product's shelf-life and minimize the microbial risks for the consumers associated with edible insect consumption [96,97]. Successful acidification and effectiveness in product's safeguarding shelf-life and safety was achieved by the control of *Enterobacteria* and bacterial spores after lactic fermentation of flour/water mixtures with 10% or 20% powdered roasted mealworm larvae [97]. Techniques such as drying, acidifying, and lactic fermentation can preserve edible insects and insect products without the use of a refrigerator [16].

#### **7. Legislation, Food Safety, and Potential Hazards Associated with the Edible Insect Food-to-Food Production Chain**

The legislation concerning edible insects for food and feed varies worldwide. Current EU legislation is quite strict, with the application of two regulations: (a) Regulation 2015/2283 (European Food Safety Authority, EFSA) refers to the use of edible insects as food. Since these were not consumed in the EU before March 1997, they were initially considered 'novel foods' [98], while in the reformed regulations they are not specifically mentioned as novel foods [99]. However, if they are intended to be sold on the EU market, they require authorization from the EFSA. (b) Regulation EU 999/2001 refers to the use of edible insects as feed [100]. According to the International Platform on Insects for Food and Feed (IPIFF), only purified insect fat and hydrolyzed insect proteins are allowed to be used as feed for livestock, while non-hydrolyzed insect proteins can currently only be used and sold as pet food and for fur animals feeding while insects derived proteins are not allowed for use in pig or poultry feed [101]. The recent EU regulation No 2017/893 authorizes the use of insect proteins originating from seven insect species: Common Housefly (*Musca domestica*), Black Soldier Fly (*Hermetia illucens*), Yellow Mealworm (*Tenebrio molitor*), Lesser Mealworm (*Alphitobius diaperinus*), House Cricket (*Acheta domesticus*), Banded Cricket (*Gryllodes sigillatus*), and Field Cricket (*Gryllus assimilis*), as feed in aquaculture [99].

Despite the strict regulatory framework, some EU countries are moving rapidly towards approval of edible insects for food and feed purposes [102]. The Netherlands tolerates the sale of edible insects included in the 'List of Edible Insects of the World' [6], while in Belgium the Agence Fédérale pour la Sécurité de la Chaîne Alimentaire (AFSCA) is carrying out a risk analysis on the sale of edible mealworms, crickets, and locusts as novel foods for the Belgian market [102,103]. In Germany, the EU regulation referring to processed animal proteins (PAPs) is interpreted such that insects PAPs are not allowed as feed (not even in aquaculture), as insects are not slaughtered, but this feed ban does not apply to live insects. Therefore a proposal has been made to Deutsche Landwirtschaftsgesellschaft (DLG) to list live insects as a direct animal feed ingredient [103]. In the United Kingdom the law is looser, allowing insects to be sold as food, but this will change relatively soon with a compulsory application procedure required for classification of insect-based food products [103]. In Switzerland, edible insects require authorization from the Federal Office of Food Safety and Veterinary Services (FFSVO) if they are intended to be sold on the open market [103], but recently FFSVO followed Belgium's policy in allowing particular insect species to be sold for food on the Swiss market [102].

In the US, the legislation on edible insects is also strict and more complex. The main authorities are the Food and Drug Administration (FDA), which regulates the industry and coordinates closely with the United States Department of Agriculture (USDA), and the Animal and Plant Health Inspection Service (APHIS) [102]. Concerning food insect-based products, these must conform to the standard

practices of all other US foods, including *Salmonella* and *E. coli* testing and, as edible insects are considered food additives, they must follow FDA regulations as described in the Federal Food, Drug, and Cosmetics Act (FFDCA) for Food Additives [104]. All producers of edible insect products must also conform to all FDA manufacturing procedures, known as Good Manufacturing Practice (GMP) ([103].

In Canada, insect-based foods are considered 'novel' and the legislation is complex, as the food safety and public health standards are set by the Canadian Food Inspection Agency (CFIA), which falls under Health Canada, while novel food safety assessments are conducted under the Food Directorate [103]. In Australia and New Zealand, the food safety and hygiene standards are set by Food Standards Australia New Zealand (FSANZ), in which edible insects are classified as 'novel foods' (non-traditional foods), as in EU regulations, and require an assessment of public health and safety issues before their commercialization, unless they are prohibited from sale [102,103].

In Asia, Thailand appears to be a pioneer and one of the most progressive and innovative countries in edible insect mass production, collection, processing, transport, and marketing of cricket (with most farms being medium- or large-scale enterprises) and palm weevil larvae, but also weaver ants, bamboo caterpillars, and grasshoppers, which are collected from the wild or are harvested seasonally [38]. In China, despite its population and economic growth, mass production of edible insects has not yet been established [102].

In Africa, collaborations between African and European companies are being developed on value chain production in rearing, processing, distribution, and consumption of edible insects [103].

Industrial mass production of edible insects for food and feed is associated with the hazards involved in any food production chain, which can mainly be classified into heavy metals, mycotoxins, pesticide residues, and pathogens [16]. During the relevant processes in an insect-based food chain, the associated hazards concerning food safety are of two origins: (a) specific to the species and (b) related to rearing, processing practices, preservation, and/or transport conditions. They are classified into (a) chemical, (b) physical, (c) allergen, and (d) microbial [98,105].

The data concerning the potential hazards associated with a food-to-food production chain based on most common edible insects are summarized in Table 7.


**Table 7.** Hazards associated with food-to-food edible insect production.


**Table 7.** *Cont*.

#### **8. Edible Insect Rearing Using Food Wastes: Towards Green and Sustainable Food Waste Management**

The organic wastes generated in food industry processes are huge in volume and numerous in type [21,125]. Household food streams also comprise a significant quantity of waste that is not exploited but landfilled, causing environmental damage [126]. In recent years, food waste management has attracted much attention, as these waste products can be valorized with green technologies in a sustainable way [127,128] for the production of renewable chemicals, biomaterials, and biofuels [129].

In recent years, more and more consumers from the USA and various European countries, like Netherlands and Belgium, adopted the entomophagy trend as accepted [130,131].

The utilization of food wastes for edible insect rearing for food and feed seems a promising approach [16] and some of the most common edible insects have already been reared on food wastes with encouraging results (see Table 2). Regarding crickets reared on various food waste streams in a controlled temperature and relative humidity greenhouse, with pre-analyzed ratios of feed substrates (moisture content, total N, crude protein content, acid detergent fiber content, crude fat content, ash content) in order to assess their feed quality, the biomass accumulation was strongly influenced by the quality of the diet [41]. Regarding the rearing of three edible mealworm species (*Tenebrio molitor* L., *Zophobas atratus* Fab., and *Alphitobius diaperinus* Panzer) on food industry organic by-products, the effects of dietary composition on feed conversion efficiency and mealworm crude protein and fatty acid profile were assessed, indicating that larval protein content was not influenced by diet composition while larval fat composition was affected by the used diet to a certain extent [28]. The substitution of diets comprising of mixed grains with agro-food industry by-products can lower the cost of commercial mealworm rearing [64]. During an experimental design for rearing of black soldier flies on various food waste, the weight reduction of rested waste materials was determined, indicating the ability of the black soldier fly to degrade food and plant organic waste [68].

The effect of larval density on food utilization during mealworm *T. molitor* rearing on a determined mixture of food materials was evaluated, thus indicating that although the space considerations in insect mass rearing are important in reducing production costs, crowding larvae to save space may be counterproductive. Additionally, it was demonstrated that increasing larval density impacts negatively on the productivity resulting in a reducing efficiency of food conversion linearly, higher food expenses, and lower biomass production [63].

However, in the most up-to-date experimental trials, the artificial diets, the rearing conditions, the nutritional value of the reared edible insects on food wastes, the yield (in terms of protein, fat content, chitin, etc.), the quality, and also the cost-efficiency of each rearing technique are not determined. Additionally, in none of the referred technologies (see Table 2) is a technical and economical evaluation presented. In addition to this, the up-to-date trials have been applied with simple food mixtures of wastes which in many cases the proportion, chemical composition of the used food materials and wastes and the conditions of the feeding substrate (temperature, humidity, microbial stability, etc.) are not referred, thus resulting in a not standardized insect mass rearing method and technology. That, in the case of the valorization of household food wastes is very critical as they consist of a heterogeneous substrate of various food material [41] and the compilation of a standardized artificial diet based on this appears to be complicated. The compilation of an artificial diet based on simpler food industry mixtures of wastes (e.g., spent grain), seems easier and effective [28]. Finally, clinical trials of reared insects on food materials and wastes have not been performed in humans and animals until now.

#### **9. Conclusions**

Edible insects could provide a solution to meeting future increasing demand for animal-based protein. In addition, the sustainability of the food industry sector could be improved through the use of food wastes as new substrates or dietary components in large-scale processes rearing edible insects for human food and animal feed purposes. This bioconversion could also contribute significantly to reducing climate change and the environmental impacts of food and feed production. The first trials on feeding insects with food wastes have produced encouraging results. Prospective candidates for this purpose are the black soldier fly, which has also been tested for municipal organic waste management with very good results, mealworms, houseflies, and house crickets.

Although there are some promising experimental results on the valorization of food wastes for edible insect rearing, further research is needed on the creation of artificial diets based on food by-products for edible insect mass production, isolation, and characterization of the nutrient content of reared insects, techno-economical evaluation of used technology, food-to-food chain safety control evaluation, and life cycle assessments of farmed insect species, in order to enable establishment of a modern insect-based food industry. Additionally, the use of various fermentation by-products (e.g., yeast, bacteria, micro-algae, etc.) as potential materials for rearing edible insects, has been studied a little and not sufficiently, and further research on the combination of fermentation techniques with edible insect rearing technologies is proposed.

**Author Contributions:** Writing—Original draft, preparation, creation and presentation of the published work, V.V. **Funding:** This research received no external funding

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

#### **References**


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