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Insect farming is a potential way to meet the growing global demand for protein and is a new activity in the Western world where many questions remain regarding product quality and safety. Insects can play an important role in the circular economy by converting biowaste into valuable biomass. About half of the feed substrate for mealworms comes from wet feed. This can be obtained from biowaste, making insect farming more sustainable. This article reports on the nutritional composition of mealworms (Tenebrio molitor) fed with organic supplements from by-products. These include unsold vegetables, potato slices, fermented chicory roots and garden leaves. It is assessed by analyzing the proximate composition, fatty acid profile, mineral and heavy metal content. Mealworms fed potato slices had a double fat content and an increase in saturated and monounsaturated fatty acids. The use of fermented chicory root increases the mineral content and accumulates heavy metals. In addition, the absorption of minerals by the mealworm is selective, as only calcium, iron and manganese concentrations are increased. The addition of vegetable mixtures or garden leaves to the diet will not significantly change the nutritional profile. In conclusion, the by-product stream was successfully converted into a protein-rich biomass, the nutrient content and bioavailability of which influenced the composition of the mealworms.
The growing human population is expected to reach 9.7 billion by 20501,2 putting pressure on our food production to cope with the high demand for food. It is estimated that food demand will increase by 70-80% between 2012 and 20503,4,5. The natural resources used in current food production are being depleted, threatening our ecosystems and food supplies. In addition, large amounts of biomass are wasted in relation to food production and consumption. It is estimated that by 2050, the annual global waste volume will reach 27 billion tonnes, most of which is bio-waste6,7,8. In response to these challenges, innovative solutions, food alternatives and sustainable development of agriculture and food systems have been proposed9,10,11. One such approach is to use organic residues to produce raw materials such as edible insects as sustainable sources of food and feed12,13. Insect farming produces lower greenhouse gas and ammonia emissions, requires less water than traditional protein sources, and can be produced in vertical farming systems, requiring less space14,15,16,17,18,19. Studies have shown that insects are able to convert low-value biowaste into valuable protein-rich biomass with dry matter contents of up to 70%20,21,22. Furthermore, low-value biomass is currently used for energy production, landfill or recycling and therefore does not compete with the current food and feed sector23,24,25,26. The mealworm (T. molitor)27 is considered one of the most promising species for large-scale food and feed production. Both larvae and adults feed on a variety of materials such as grain products, animal waste, vegetables, fruits, etc. 28,29. In Western societies, T. molitor is bred in captivity on a small scale, mainly as feed for domestic animals such as birds or reptiles. Currently, their potential in food and feed production is receiving more attention30,31,32. For example, T. molitor has been approved with a new food profile, including use in frozen, dried and powdered forms (Regulation (EU) No 258/97 and Regulation (EU) 2015/2283) 33. However, large-scale production of insects for food and feed is still a relatively new concept in Western countries. The industry faces challenges such as knowledge gaps regarding optimal diets and production, nutritional quality of the final product, and safety issues such as toxic build-up and microbial hazards. Unlike traditional livestock farming, insect farming does not have a similar historical track record17,24,25,34.
Although many studies have been conducted on the nutritional value of mealworms, the factors affecting their nutritional value have not yet been fully understood. Previous studies have shown that the diet of insects may have some effect on its composition, but no clear pattern was found. In addition, these studies focused on the protein and lipid components of mealworms, but had limited effects on the mineral components21,22,32,35,36,37,38,39,40. More research is needed to understand the mineral absorption capacity. A recent study concluded that mealworm larvae fed radish had slightly elevated concentrations of certain minerals. However, these results are limited to the substrate tested, and further industrial trials are needed41. The accumulation of heavy metals (Cd, Pb, Ni, As, Hg) in mealworms has been reported to be significantly correlated with the metal content of the matrix. Although the concentrations of metals found in the diet in animal feed are below legal limits42, arsenic has also been found to bioaccumulate in mealworm larvae, whereas cadmium and lead do not bioaccumulate43. Understanding the effects of diet on the nutritional composition of mealworms is critical to their safe use in food and feed.
The study presented in this paper focuses on the impact of using agricultural by-products as a wet feed source on the nutritional composition of mealworms. In addition to dry feed, wet feed should also be provided to the larvae. The wet feed source provides the necessary moisture and also serves as a nutritional supplement for mealworms, increasing growth rate and maximum body weight44,45. According to our standard mealworm rearing data in the Interreg-Valusect project, the total mealworm feed contains 57% w/w wet feed. Usually, fresh vegetables (e.g. carrots) are used as a wet feed source35,36,42,44,46. Using low-value by-products as wet feed sources will bring more sustainable and economic benefits to insect farming17. The objectives of this study were to (1) investigate the effects of using biowaste as wet feed on the nutritional composition of mealworms, (2) determine the macro- and micronutrient contents of mealworm larvae reared on mineral-rich biowaste to test the feasibility of mineral fortification, and (3) evaluate the safety of these by-products in insect farming by analyzing the presence and accumulation of heavy metals Pb, Cd and Cr. This study will provide further information on the effects of biowaste supplementation on mealworm larval diets, nutritional value and safety.
The dry matter content in the lateral flow was higher compared to the control wet nutrient agar. The dry matter content in the vegetable mixtures and garden leaves was less than 10%, whereas it was higher in potato cuttings and fermented chicory roots (13.4 and 29.9 g/100 g fresh matter, FM).
The vegetable mixture had higher crude ash, fat and protein contents and lower non-fibrous carbohydrate contents than the control feed (agar), while the amylase-treated neutral detergent fibre content was similar. The carbohydrate content of the potato slices was the highest of all the side streams and was comparable to that of the agar. Overall, its crude composition was most similar to the control feed, but was supplemented with small amounts of protein (4.9%) and crude ash (2.9%) 47,48 . The pH of potato ranges from 5 to 6, and it is worth noting that this potato side stream is more acidic (4.7). Fermented chicory root is rich in ash and is the most acidic of all the side streams. Since the roots were not cleaned, most of the ash is expected to consist of sand (silica). Garden leaves were the only alkaline product compared to the control and other side streams. It contains high levels of ash and protein and much lower carbohydrates than the control. The crude composition is closest to fermented chicory root, but the crude protein concentration is higher (15.0%), which is comparable to the protein content of the vegetable mixture. Statistical analysis of the above data showed significant differences in the crude composition and pH of the side streams.
Addition of vegetable mixtures or garden leaves to mealworm feed did not affect the biomass composition of mealworm larvae compared to the control group (Table 1). Addition of potato cuttings resulted in the most significant difference in biomass composition compared to the control group receiving mealworm larvae and other sources of wet feed. As for the protein content of mealworms, with the exception of potato cuttings, different approximate composition of side streams did not affect the protein content of larvae. Feeding potato cuttings as a source of moisture led to a two-fold increase in the fat content of larvae and a decrease in the content of protein, chitin, and non-fibrous carbohydrates. Fermented chicory root increased the ash content of mealworm larvae by one and a half times.
Mineral profiles were expressed as macromineral (Table 2) and micronutrient (Table 3) contents in wet feed and mealworm larval biomass.
In general, agricultural side streams were richer in macrominerals compared to the control group, except for potato cuttings, which had lower Mg, Na and Ca contents. Potassium concentration was high in all side streams compared to the control. Agar contains 3 mg/100 g DM K, while K concentration in the side stream ranged from 1070 to 9909 mg/100 g DM. Macromineral content in the vegetable mixture was significantly higher than in the control group, but Na content was significantly lower (88 vs. 111 mg/100 g DM). Macromineral concentration in potato cuttings was the lowest of all side streams. Macromineral content in potato cuttings was significantly lower than in other side streams and control. Except that Mg content was comparable to the control group. Although fermented chicory root did not have the highest concentration of macrominerals, the ash content of this side stream was the highest of all the side streams. This may be due to the fact that they are not purified and may contain high concentrations of silica (sand). The Na and Ca contents were comparable to those of the vegetable mixture. Fermented chicory root contained the highest concentration of Na of all the side streams. With the exception of Na, horticultural leaves had the highest concentrations of macrominerals of all the wet forages. The K concentration (9909 mg/100 g DM) was three thousand times higher than the control (3 mg/100 g DM) and 2.5 times higher than the vegetable mixture (4057 mg/100 g DM). The Ca content was the highest of all side streams (7276 mg/100 g DM), 20 times higher than the control (336 mg/100 g DM), and 14 times higher than the Ca concentration in fermented chicory roots or vegetable mixture (530 and 496 mg/100 g DM).
Although there were significant differences in the macromineral composition of the diets (Table 2), no significant differences were found in the macromineral composition of mealworms raised on vegetable mixtures and control diets.
Larvae fed potato crumbs had significantly lower concentrations of all macrominerals compared to the control, with the exception of Na, which had comparable concentrations. Additionally, potato crisp feeding caused the greatest reduction in larval macromineral content compared to the other sidestreams. This is consistent with the lower ash observed in the nearby mealworm formulations. However, although P and K were significantly higher in this wet diet than the other sidestreams and the control, the larval composition did not reflect this. The low Ca and Mg concentrations found in mealworm biomass may be related to the low Ca and Mg concentrations present in the wet diet itself.
Feeding fermented chicory roots and orchard leaves resulted in significantly higher calcium levels than controls. Orchard leaves contained the highest levels of P, Mg, K and Ca of all wet diets, but this was not reflected in mealworm biomass. Na concentrations were lowest in these larvae, while Na concentrations were higher in orchard leaves than in potato cuttings. Ca content increased in larvae (66 mg/100 g DM), but Ca concentrations were not as high as those in mealworm biomass (79 mg/100 g DM) in the fermented chicory root experiments, although the Ca concentration in orchard leaf crops was 14 times higher than in chicory root.
Based on the microelement composition of the wet feeds (Table 3), the mineral composition of the vegetable mixture was similar to the control group, except that the Mn concentration was significantly lower. Concentrations of all analyzed microelements were lower in potato cuts compared to the control and other by-products. Fermented chicory root contained almost 100 times more iron, 4 times more copper, 2 times more zinc and about the same amount of manganese. The zinc and manganese content in the leaves of garden crops was significantly higher than in the control group.
No significant differences were found between the trace element contents of the larvae fed the control, vegetable mixture, and wet potato scraps diets. However, the Fe and Mn contents of the larvae fed the fermented chicory root diet were significantly different from those of the mealworms fed the control group. The increase in Fe content may be due to the hundredfold increase in the trace element concentration in the wet diet itself. However, although there was no significant difference in Mn concentrations between the fermented chicory roots and the control group, Mn levels increased in the larvae fed the fermented chicory roots. It should also be noted that the Mn concentration was higher (3-fold) in the wet leaf diet of the horticulture diet compared to the control, but there was no significant difference in the biomass composition of the mealworms. The only difference between the control and horticulture leaves was the Cu content, which was lower in the leaves.
Table 4 shows the concentrations of heavy metals found in substrates. European maximum concentrations of Pb, Cd and Cr in complete animal feeds have been converted to mg/100 g dry matter and added to Table 4 to facilitate comparison with concentrations found in side streams47.
No Pb was detected in the control wet feeds, vegetable mixtures or potato brans, while garden leaves contained 0.002 mg Pb/100 g DM and fermented chicory roots contained the highest concentration of 0.041 mg Pb/100 g DM. C concentrations in the control feeds and garden leaves were comparable (0.023 and 0.021 mg/100 g DM), while they were lower in the vegetable mixtures and potato brans (0.004 and 0.007 mg/100 g DM). Compared with the other substrates, the Cr concentration in the fermented chicory roots was significantly higher (0.135 mg/100 g DM) and six times higher than in the control feed. Cd was not detected in either the control stream or any of the side streams used.
Significantly higher levels of Pb and Cr were found in larvae fed fermented chicory roots. However, Cd was not detected in any mealworm larvae.
A qualitative analysis of the fatty acids in the crude fat was carried out to determine whether the fatty acid profile of mealworm larvae could be influenced by the different components of the lateral stream on which they were fed. The distribution of these fatty acids is shown in Table 5. The fatty acids are listed by their common name and molecular structure (designated as “Cx:y”, where x corresponds to the number of carbon atoms and y to the number of unsaturated bonds).
The fatty acid profile of mealworms fed potato shreds was significantly altered. They contained significantly higher amounts of myristic acid (C14:0), palmitic acid (C16:0), palmitoleic acid (C16:1), and oleic acid (C18:1). Concentrations of pentadecanoic acid (C15:0), linoleic acid (C18:2), and linolenic acid (C18:3) were significantly lower compared to other mealworms. Compared to other fatty acid profiles, the ratio of C18:1 to C18:2 was reversed in potato shreds. Mealworms fed horticultural leaves contained higher amounts of pentadecanoic acid (C15:0) than mealworms fed other wet diets.
Fatty acids are divided into saturated fatty acids (SFA), monounsaturated fatty acids (MUFA), and polyunsaturated fatty acids (PUFA). Table 5 shows the concentrations of these fatty acid groups. Overall, the fatty acid profiles of mealworms fed potato waste were significantly different from the control and other side streams. For each fatty acid group, mealworms fed potato chips were significantly different from all other groups. They contained more SFA and MUFA and less PUFA.
There were no significant differences between the survival rate and total yield weight of larvae bred on different substrates. The overall average survival rate was 90%, and the total average yield weight was 974 grams. Mealworms successfully process by-products as a source of wet feed. Mealworm wet feed accounts for more than half of the total feed weight (dry + wet). Replacing fresh vegetables with agricultural by-products as traditional wet feed has economic and environmental benefits for mealworm farming.
Table 1 shows that the biomass composition of mealworm larvae reared on the control diet was approximately 72% moisture, 5% ash, 19% lipid, 51% protein, 8% chitin, and 18% dry matter as non-fibrous carbohydrates. This is comparable with values reported in the literature.48,49 However, other components can be found in the literature, often depending on the analytical method used. For example, we used the Kjeldahl method to determine crude protein content with an N to P ratio of 5.33, whereas other researchers use the more widely used ratio of 6.25 for meat and feed samples.50,51
Addition of potato scraps (a carbohydrate-rich wet diet) to the diet resulted in a doubling of the fat content of mealworms. The carbohydrate content of potato would be expected to consist mainly of starch, whereas agar contains sugars (polysaccharides)47,48. This finding is in contrast to another study that found that fat content decreased when mealworms were fed a diet supplemented with steam-peeled potatoes that were low in protein (10.7%) and high in starch (49.8%)36. When olive pomace was added to the diet, the protein and carbohydrate contents of mealworms matched those of the wet diet, while the fat content remained unchanged35. In contrast, other studies have shown that the protein content of larvae reared in side streams undergoes fundamental changes, as does the fat content22,37.
Fermented chicory root significantly increased the ash content of mealworm larvae (Table 1). Research on the effects of byproducts on the ash and mineral composition of mealworm larvae is limited. Most byproduct feeding studies have focused on the fat and protein content of larvae without analyzing the ash content21,35,36,38,39. However, when the ash content of larvae fed byproducts was analyzed, an increase in ash content was found. For example, feeding mealworms garden waste increased their ash content from 3.01% to 5.30%, and adding watermelon waste to the diet increased ash content from 1.87% to 4.40%.
Although all wet food sources varied significantly in their approximate composition (Table 1), the differences in the biomass composition of mealworm larvae fed the respective wet food sources were minor. Only mealworm larvae fed potato chunks or fermented chicory root showed significant changes. One possible explanation for this result is that in addition to the chicory roots, the potato chunks were also partially fermented (pH 4.7, Table 1), making the starch/carbohydrates more digestible/available to the mealworm larvae. How mealworm larvae synthesize lipids from nutrients such as carbohydrates is of great interest and should be fully explored in future studies. A previous study on the effect of wet diet pH on mealworm larval growth concluded that no significant differences were observed when using agar blocks with wet diets over a pH range of 3 to 9. This indicates that fermented wet diets can be used for the culture of Tenebrio molitor53. Similar to Coudron et al.53, control experiments used agar blocks in the wet diets provided because they were deficient in minerals and nutrients. Their study did not examine the effect of more nutritionally diverse wet diet sources such as vegetables or potatoes on improving digestibility or bioavailability. Further studies on the effects of fermentation of wet diet sources on mealworm larvae are needed to further explore this theory.
The mineral distribution of the control mealworm biomass found in this study (Tables 2 and 3) is comparable to the range of macro- and micronutrients found in the literature48,54,55. Providing mealworms with fermented chicory root as a wet diet source maximizes their mineral content. Although most macro- and micronutrients were higher in the vegetable mixes and garden leaves (Tables 2 and 3), they did not affect the mineral content of mealworm biomass to the same extent as fermented chicory roots. One possible explanation is that the nutrients in the alkaline garden leaves are less bioavailable than those in the other, more acidic wet diets (Table 1). Previous studies fed mealworm larvae with fermented rice straw and found that they developed well in this sidestream and also showed that pre-treatment of the substrate by fermentation induced nutrient uptake. 56 The use of fermented chicory roots increased the Ca, Fe and Mn contents of mealworm biomass. Although this sidestream also contained higher concentrations of other minerals (P, Mg, K, Na, Zn and Cu), these minerals were not significantly more abundant in mealworm biomass compared to the control, indicating selectivity of mineral uptake. Increasing the content of these minerals in mealworm biomass has nutritional value for food and feed purposes. Calcium is an essential mineral that plays a critical role in neuromuscular function and many enzyme-mediated processes such as blood clotting, bone and tooth formation. 57,58 Iron deficiency is a common problem in developing countries, with children, women, and the elderly often not getting enough iron from their diets. 54 Although manganese is an essential element in the human diet and plays a central role in the functioning of many enzymes, excessive intake can be toxic. Higher manganese levels in mealworms fed fermented chicory root were not of concern and were comparable to those in chickens. 59
The concentrations of heavy metals found in the sidestream were below the European standards for complete animal feed. Heavy metal analysis of mealworm larvae showed that Pb and Cr levels were significantly higher in mealworms fed with fermented chicory root than in the control group and other substrates (Table 4). Chicory roots grow in soil and are known to absorb heavy metals, while the other sidestreams originate from controlled human food production. Mealworms fed with fermented chicory root also contained higher levels of Pb and Cr (Table 4). The calculated bioaccumulation factors (BAF) were 2.66 for Pb and 1.14 for Cr, i.e. greater than 1, indicating that mealworms have the ability to accumulate heavy metals. With regard to Pb, the EU sets a maximum Pb content of 0.10 mg per kilogram of fresh meat for human consumption61. In an evaluation of experimental data, the maximum Pb concentration found in fermented chicory root mealworms was 0.11 mg/100 g DM. When the value was recalculated to a dry matter content of 30.8% for these mealworms, the Pb content was 0.034 mg/kg fresh matter, which was below the maximum level of 0.10 mg/kg. No maximum content of Cr is specified in the European food regulations. Cr is commonly found in the environment, foodstuffs and food additives and is known to be an essential nutrient for humans in small amounts62,63,64. These analyses (Table 4) indicate that T. molitor larvae can accumulate heavy metals when heavy metals are present in the diet. However, the levels of heavy metals found in mealworm biomass in this study are considered safe for human consumption. Regular and careful monitoring is recommended when using side streams that may contain heavy metals as a wet feed source for T. molitor.
The most abundant fatty acids in the total biomass of T. molitor larvae were palmitic acid (C16:0), oleic acid (C18:1), and linoleic acid (C18:2) (Table 5), which is consistent with previous studies on T. molitor. The fatty acid spectrum results are consistent36,46,50,65. The fatty acid profile of T. molitor generally consists of five major components: oleic acid (C18:1), palmitic acid (C16:0), linoleic acid (C18:2), myristic acid (C14:0), and stearic acid (C18:0). Oleic acid is reported to be the most abundant fatty acid (30–60%) in mealworm larvae, followed by palmitic acid and linoleic acid22,35,38,39. Previous studies have shown that this fatty acid profile is influenced by mealworm larval diet, but the differences do not follow the same trends as diet38. Compared with other fatty acid profiles, the C18:1–C18:2 ratio in potato peelings is reversed. Similar results were obtained for changes in the fatty acid profile of mealworms fed steamed potato peelings36. These results indicate that although the fatty acid profile of mealworm oil may be altered, it still remains a rich source of unsaturated fatty acids.
The aim of this study was to evaluate the effect of using four different agro-industrial biowaste streams as wet feed on the composition of mealworms. The impact was assessed based on the nutritional value of the larvae. The results showed that the by-products were successfully converted into protein-rich biomass (protein content 40.7-52.3%), which can be used as a food and feed source. In addition, the study showed that using the by-products as wet feed affects the nutritional value of mealworm biomass. In particular, providing larvae with a high concentration of carbohydrates (e.g. potato cuts) increases their fat content and changes their fatty acid composition: lower content of polyunsaturated fatty acids and higher content of saturated and monounsaturated fatty acids, but not concentrations of unsaturated fatty acids. The fatty acids (monounsaturated + polyunsaturated) still dominate. The study also showed that mealworms selectively accumulate calcium, iron and manganese from side streams rich in acidic minerals. The bioavailability of minerals appears to play an important role and further studies are needed to fully understand this. Heavy metals present in the side streams may accumulate in mealworms. However, final concentrations of Pb, Cd and Cr in larval biomass were below acceptable levels, allowing these side streams to be safely used as a wet feed source.
Mealworm larvae were reared by Radius (Giel, Belgium) and Inagro (Rumbeke-Beitem, Belgium) at the Thomas More University of Applied Sciences at 27 °C and 60% relative humidity. The density of mealworms reared in a 60 x 40 cm aquarium was 4.17 worms/cm2 (10,000 mealworms). Larvae were initially fed 2.1 kg of wheat bran as dry food per rearing tank and then supplemented as needed. Use agar blocks as a control wet food treatment. Starting from week 4, start feeding side streams (also a moisture source) as wet food instead of agar ad libitum. The dry matter percentage for each side stream was pre-determined and recorded to ensure equal amounts of moisture for all insects across treatments. The food is distributed evenly throughout the terrarium. Larvae are collected when the first pupae emerge in the experimental group. Larval harvest is done using a 2 mm diameter mechanical shaker. Except for the potato diced experiment. Large portions of dried potato diced are also separated by allowing larvae to crawl through this mesh and collecting them in metal trays. Total harvest weight is determined by weighing the total harvest weight. Survival is calculated by dividing the total harvest weight by the larval weight. Larval weight is determined by selecting at least 100 larvae and dividing their total weight by the number. Collected larvae are starved for 24 h to empty their guts before analysis. Finally, larvae are screened again to separate them from the remainder. They are frozen and euthanized and stored at -18°C until analysis.
Dry feed was wheat bran (Belgian Molens Joye). Wheat bran was pre-sifted to a particle size of less than 2 mm. In addition to dry feed, mealworm larvae also need wet feed to maintain moisture and mineral supplements required by mealworms. Wet feed accounts for more than half of the total feed (dry feed + wet feed). In our experiments, agar (Brouwland, Belgium, 25 g/l) was used as a control wet feed45. As shown in Figure 1, four agricultural by-products with different nutrient contents were tested as wet feed for mealworm larvae. These by-products include (a) leaves from cucumber cultivation (Inagro, Belgium), (b) potato trimmings (Duigny, Belgium), (c) fermented chicory roots (Inagro, Belgium) and (d) unsold fruit and vegetables from auctions. (Belorta, Belgium). The side stream is chopped into pieces suitable for use as wet mealworm feed.
Agricultural by-products as wet feed for mealworms; (a) garden leaves from cucumber cultivation, (b) potato cuttings, (c) chicory roots, (d) unsold vegetables at auction and (e) agar blocks. As controls.
The composition of the feed and mealworm larvae was determined three times (n = 3). Rapid analysis, mineral composition, heavy metal content and fatty acid composition were assessed. A homogenized sample of 250 g was taken from the collected and starved larvae, dried at 60°C to constant weight, ground (IKA, Tube mill 100) and sieved through a 1 mm sieve. The dried samples were sealed in dark containers.
The dry matter content (DM) was determined by drying the samples in an oven at 105°C for 24 hours (Memmert, UF110). The percentage of dry matter was calculated based on the weight loss of the sample.
Crude ash content (CA) was determined by mass loss during combustion in a muffle furnace (Nabertherm, L9/11/SKM) at 550°C for 4 hours.
Crude fat content or diethyl ether (EE) extraction was performed with petroleum ether (b.p. 40–60 °C) using Soxhlet extraction equipment. Approximately 10 g of sample was placed in the extraction head and covered with ceramic wool to prevent sample loss. Samples were extracted overnight with 150 ml petroleum ether. The extract was cooled, the organic solvent was removed and recovered by rotary evaporation (Büchi, R-300) at 300 mbar and 50 °C. The crude lipid or ether extracts were cooled and weighed on an analytical balance.
The crude protein (CP) content was determined by analysing the nitrogen present in the sample using the Kjeldahl method BN EN ISO 5983-1 (2005). Use the appropriate N to P factors to calculate the protein content. For standard dry feed (wheat bran) use a total factor of 6.25. For side stream a factor of 4.2366 is used and for vegetable mixtures a factor of 4.3967 is used. The crude protein content of larvae was calculated using an N to P factor of 5.3351.
The fibre content included neutral detergent fibre (NDF) determination based on the Gerhardt extraction protocol (manual fibre analysis in bags, Gerhardt, Germany) and the van Soest 68 method. For NDF determination, a 1 g sample was placed in a special fibre bag (Gerhardt, ADF/NDF bag) with a glass liner. The fibre bags filled with samples were first defatted with petroleum ether (boiling point 40–60 °C) and then dried at room temperature. The defatted sample was extracted with a neutral fibre detergent solution containing heat-stable α-amylase at boiling temperature for 1.5 h. The samples were then washed three times with boiling deionized water and dried at 105 °C overnight. The dry fibre bags (containing fibre residues) were weighed using an analytical balance (Sartorius, P224-1S) and then burned in a muffle furnace (Nabertherm, L9/11/SKM) at 550°C for 4 hours. The ash was weighed again and the fibre content was calculated based on the weight loss between drying and burning of the sample.
To determine the chitin content of the larvae, we used a modified protocol based on the crude fiber analysis by van Soest 68 . A 1 g sample was placed in a special fiber bag (Gerhardt, CF Bag) and a glass seal. The samples were packed in the fiber bags, defatted in petroleum ether (c. 40–60 °C) and air-dried. The defatted sample was first extracted with an acidic solution of 0.13 M sulfuric acid at boiling temperature for 30 min. The extraction fiber bag containing the sample was washed three times with boiling deionized water and then extracted with 0.23 M potassium hydroxide solution for 2 h. The extraction fibre bag containing the sample was again rinsed three times with boiling deionized water and dried at 105°C overnight. The dry bag containing the fibre residue was weighed on an analytical balance and incinerated in a muffle furnace at 550°C for 4 hours. The ash was weighed and the fibre content was calculated based on the weight loss of the incinerated sample.
Total carbohydrate content was calculated. Non-fibrous carbohydrate (NFC) concentration in the feed was calculated using NDF analysis, and insect concentration was calculated using chitin analysis.
The pH of the matrix was determined after extraction with deionized water (1:5 v/v) according to NBN EN 15933.
Samples were prepared as described by Broeckx et al. Mineral profiles were determined using ICP-OES (Optima 4300™ DV ICP-OES, Perkin Elmer, MA, USA).
The heavy metals Cd, Cr and Pb were analyzed by graphite furnace atomic absorption spectrometry (AAS) (Thermo Scientific, ICE 3000 series, equipped with a GFS furnace autosampler). About 200 mg of sample was digested in acidic HNO3/HCl (1:3 v/v) using microwaves (CEM, MARS 5). Microwave digestion was performed at 190°C for 25 min at 600 W. Dilute the extract with ultrapure water.
Fatty acids were determined by GC-MS (Agilent Technologies, 7820A GC system with 5977 E MSD detector). According to the method of Joseph and Akman70, 20% BF3/MeOH solution was added to a methanolic KOH solution and fatty acid methyl ester (FAME) was obtained from the ether extract after esterification. Fatty acids can be identified by comparing their retention times with 37 FAME mixture standards (Chemical Lab) or by comparing their MS spectra with online libraries such as the NIST database. Qualitative analysis is performed by calculating the peak area as a percentage of the total peak area of the chromatogram.
Data analysis was performed using JMP Pro 15.1.1 software from SAS (Buckinghamshire, UK). Evaluation was performed using one-way analysis of variance with a significance level of 0.05 and Tukey HSD as a post hoc test.
The bioaccumulation factor (BAF) was calculated by dividing the concentration of heavy metals in mealworm larval biomass (DM) by the concentration in wet feed (DM) 43 . A BAF greater than 1 indicates that heavy metals bioaccumulate from wet feed in larvae.
The datasets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request.
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Post time: Dec-19-2024