Chemical composition, fatty acid profile, antioxidant content, and microbiological loads of lesser mealworm, mealworm, and superworm larvae

Abstract

In the last decade, great attention was placed on insects to increase feed-food production without negatively affecting the environment. In this research study we compare three different insect species (Alphitobius diaperinus, Tenebrio molitor, and Zophobas morio - lesser mealworm, mealworm, and superworm), fed the same substrate, on the chemical composition, fatty acid (FA) profile, antioxidant content, and microbiological loads. Superworm larvae show higher dry matter (38.06%) and ether extract (17.65%) contents followed by mealworm and lesser mealworm larvae (32.77%, 27.11% and 13.86%, 10.78%, respectively). No differences were detected in the crude protein and ash content. Superworm larvae showed the highest content of SFA (41.43% vs 39.16% and 28.52% of total FA, in lesser mealworm and mealworm, respectively), while mealworm larvae were the richest in MUFA (40.61% vs 34.38% and 32.98% of total FA, in super and lesser mealworm, respectively) and PUFA (29.05% vs 22.29% and 21.21% of total FA in super and lesser mealworm, respectively). Anyhow, in all the larvae the three more representative fatty acids were oleic acid, linoleic acid, and palmitic acid. The least beneficial ratio of n-6/n-3 was detected in Alphitobius diaperinus larvae (53.84), while Zophobas morio larvae showed the healthier ratio (16.04). The antioxidant contents determination revealed a linkage to the fatty acids content. Low differences were determined in microbiological loads of the larvae. The characteristics of the insects revealed the great potential of these three species highlighting the capacities to respond to different requests derived from the food and feed sectors.

Highlights

• Superworm larvae showed the highest content of SFA

• Mealworms larvae were the richest in PUFA

• The fat-soluble vitamin content followed the fatty acids content

Keywords:

• Sustainability
• entomophagy
• alphitobius diaperinus
• tenebrio molitor
• zophobas morio

Introduction

Estimation of the Food and Agricultural Organisation of the United Nations prospect that in 2050 there will be approximately nine billion people in the world. Even if in the last decades food production has increased, it is urgent and necessary to change the current systems of production of feed and food focusing not only on the efficiency and enhancement of production but also looking to new resources (Cadinu et al. 2020). The insects could represent a real opportunity to respond to these issues representing a high nutritional feed and food with low environmental impact (van Huis et al. 2013). Indeed, if compared to conventional livestock, insects have valuable advantages in terms of environmental sustainability, low greenhouse gas emissions, soil utilisation, and low water consumption (van Huis et al. 2013; van Huis and Oonincx 2017; Halloran et al. 2018). Another advantage of insect farming is the possibility of feeding them with food by-products and former foodstuff, agricultural side-streams following the concept of circular economy (Cappellozza et al. 2019; Rumbos et al. 2021; Moruzzo et al. 2021a). Therefore, insects are considered a marketable solution to reduce food waste, thanks to their ability to bioconversion (Ojha et al. 2020). For these reasons, insects gained a high interest for the production of feed and food, increasing the importance of research both in the public and private sectors also inducing changes in EU legislation (Mancini et al. 2022, 2022b). Thanks to the regulation of the European Commission 2021/1372 of 17 August 2021, the use of seven insect species processed animal proteins (PAP) was allowed in the EU as feed for poultry and swine, while previously these insects’ PAP were authorised only in aquaculture feeds by the Regulation of the Commission 2017/893. Recently, the list of authorised insects was updated to 8 species with the addition of Bombyx mori (silkworm, Regulation of the Commission 2021/1925). One of the most studied species is the Tenebrio molitor (Linnaeus, 1758; Coleoptera: Tenebrionidae) known as mealworm; the interest in this insect has increased in recent years because it is rich in protein, with an amino acid profile comparable to fish and soy meals (Azagoh et al. 2016). The nutritional value of Tenebrio molitor larvae could be modified using different substrates, such as former foodstuff, showing high plasticity (Mancini et al. 2019b). Other two insect species belonging to the family of the Tenebrionidae family showed interesting features: Alphitobius diaperinus (Panzer, 1797) known commonly as lesser mealworm, and Zophobas morio (Fabricius, 1776) known as superworms. Furthermore, mealworms and lesser mealworms could be employed in the European Union for the production of PAPs. Also, the topic of insects as food is growing in Europe and worldwide. Thanks to the Regulation of the Commission 2283/2015 new food resources, called novel food, could be evaluated and marketed in the EU. At this moment, six novel food requests dealing with insects have been positively evaluated by EFSA (European Food Safety Authority). Two of the six authorised requests deal with mealworms (Turck et al. 2021a, 2021b) while one takes into consideration the employment of lesser mealworm larvae (Turck et al. 2022). More novel food applications for insects are still pending (Mancini et al. 2022a, 2022b). In contrast, Zophobas morio larvae were not listed in the species authorised as PAP nor novel food applications were presented until today about this insect. From a nutritional point of view, these insects represent an excellent source of protein. Yi et al. (2013) showed that Tenebrio molitor, Zophobas morio, and Alphitobius diaperinus have a protein content equal to 19.1%, 20.7%, and 20.6% on live weight basis, respectively. In another study, it was observed that Tenebrio molitor and Alphitobius diaperinus showed higher values of protein content, respectively 63 and 60% on dry matter (DM), than Zophobas morio (39% on DM) (Adámková et al. 2016). On the other hand, superworms were richer in lipids (39% on DM) than mealworms (17% on DM) and lesser mealworms (29% on DM) (Adámková et al. 2016). Unfortunately, these research studies did not pair the diet of the insects. Harsányi et al. (2020) fed Tenebrio molitor and Zophobas morio with organic waste materials, highlighting that feeding nutrient-poor diets resulted in a lower protein and a higher fat concentration in the larvae. Insects’ lipids (fats) also represent an interesting ingredient to be used in the formulation of feed and food. For instance, mealworms’ fatty acid profile revealed to be very interesting in relation to its high contents in oleic, α-linolenic, and palmitic acid (Mattioli et al. 2021) and it could be suitable as table oil and as food-feed ingredient (Tzompa-Sosa et al. 2019; Son et al. 2020; Tzompa-Sosa et al. 2021).

Literature data highlight a great plasticity of insects, both in terms of capacity to bio-convert a wide range of substrates but also in terms of their susceptibility to the chemical composition of the diet. Moreover, meanwhile Tenebrio molitor larvae have been extensively studied (Oonincx and de Boer 2012; van Broekhoven et al. 2015; Dreassi et al. 2017; Caparros Megido et al. 2018; Wynants et al. 2019; Zhang et al. 2019; Mancini et al. 2019a; 2021; Mattioli et al. 2021; Moruzzo et al. 2021b; Mancini et al. 2022a, 2022b), the availability of chemical characterisation of Alphitobius diaperinus and Zophobas morio larvae are still very low and not comprehensive. In addition, a lack of data is nowadays present about the different responses of Alphitobius diaperinus, Tenebrio molitor, and Zophobas morio species under the same rearing conditions. Therefore, the main aim of this study was to compare these three insect species (Alphitobius diaperinus, Tenebrio molitor, and Zophobas morio) larvae reared in the same environmental conditions and fed the same substrates. Larvae were then analysed for the determination of their nutritional-safety values as proximate composition, fatty acid profile, antioxidant content, and microbiological parameters.

Materials and methods

Experimental diet

All the insects were fed with the same diet composed of a mix (1:1) of bread (B) and brewery spent grains (SG). Bread was collected from a market shop as daily unsold remains (leftovers), while brewery spent grains were collected from a local beer producer (Birrificio del Forte, Pietrasanta, Italy). Both the ingredients were frozen at −20 °C after collecting them. To reduce excessive humidity spent grains and bread were dried in an oven at 90 °C (spent grains were previously thawed for 18 h at 4 °C). After drying, both were finely ground. Proximate compositions of bread, brewery spent grain, and their mix are reported in Table 1.

Table 1. Proximate composition of bread (B), brewery spent grain (SG), and their mix (1:1) (B-SG).

Item	B	SG	B-SG
Dry matter (%)	97.47	96.83	97.08
Ether extract (%)	0.54	3.84	2.20
Crude protein^a (%)	11.08	18.03	13.79
Ash (%)	2.73	3.05	2.40

^a Nitrogen to protein conversion factor of 5.70 for (B) and of 5.83 for (SG).

Insect rearing and sampling

Alphitobius diaperinus, Tenebrio molitor, and Zophobas morio were reared at the Department of Veterinary Sciences of the University of Pisa (Italy) under a laboratory scale production. The insects were reared in a closed environment with a temperature of 25 °C and relative humidity 50-60%. The adults of the three species were placed in plastic containers (39 × 28 × 14 cm) containing the substrate and were left freely mating and laying the eggs. After two weeks adults were removed from the containers where the larvae hatched and grew. Three containers per species were used, for a total of nine experimental units. The substrate was added when needed (ad libitum) and moisture was provided twice a week adding potato slices.

The mealworms and lesser mealworms were harvested when the first pupa was observed (larvae weight of 149.79 ± 7.79 mg and 30.16 ± 1.56 mg, respectively), while superworms were collected at the final larvae stage when no more growth was showed (larvae weight of 1.03 ± 0.08 g). Larvae were fasted for 48 h in sterile plastic containers with a plastic web on the base in order to separate faeces and to avoid faecal contact, then they were killed by freezing at −18 °C (for the microbiological analyses the larvae were frozen for only 1 h). Proximate composition, fatty acids profile, and antioxidant content were performed in triplicate per experimental unit.

Proximate composition of larvae

Before being analysed, the larvae were ground in a blender. The dry matter (DM) content was determined by drying in an oven at 105 °C to constant weight. The ether exact content was quantified by the Soxhlet method using petroleum ether as the solvent. The ash content was determined by incineration in a muffle furnace at 550 °C. The crude protein content was determined by the Kjeldahl method, two nitrogen-to-protein conversion factors were used for insects as 6.25 established for meat proteins and as 4.76 which was suggested by Janssen et al. (2017) for insect specimens. The samples were analysed according to the AOAC (1995) methods (protocol numbers 930.15, 934.01, 976.06, 920.39, and 942.05, respectively for dry matter, organic matter, crude protein, ether extract, and ash).

Fatty acids profile

The fatty acid composition of larvae and substrates was determined by gas chromatography. Total lipids were extracted from 10 g of each homogenised sample, according to Folch et al. (1957), and esterified according to Christie (1982). One mL of each solution containing fatty acid esters was transferred into vials for the gas-chromatographic analysis. The separation of fatty acid esters (FAME) was performed using a Varian gas-chromatograph (CP-3800) equipped with a flame ionisation detector (FID) and an Agilent capillary column (100 m x 0.25 mm, CPS Analitica, Milan, Italy) coated with a DB-Wax stationary phase (film thickness of 0.25 µm). Injector and detector temperatures were set at 270 °C and 300 °C respectively. The carrier gas was helium at a flow rate of 0.6 mL/min. The oven temperature was programmed as follows: from 40 °C (1-min hold) to 163 °C (10-min hold) at 2 °C/min ramp, to 180 °C (7-min hold) at 1.5 °C/min, to 187 °C (2-min hold) at 2 °C/min, and to 230 °C (25-min hold) at 3 °C/min. Single fatty acid methyl esters were identified by comparing their retention time with the retention time of commercially available FAME standard mixture (FAME mix Supelco 2560, Sigma-Aldrich, Germany). C21:0 methyl ester (CAS number 2363-71-5; Merck H5149, Germany), eluted under the same conditions of the samples, was used as internal standard (1 mg/100 µL of added solution). The area of each peak was used to calculate the fatty acid proportion and the relative data were expressed as percentages of total fatty acids for qualitative analysis. For the quantitative analysis (mg/100g of larvae), the calculation method reported by Vahmani et al. (2017) was applied. Total saturated fatty acids (SFA), total monounsaturated fatty acids (MUFA), total polyunsaturated fatty acids (PUFA), total n-6 and n-3, as well as n-6/n-3 ratios, were also calculated.

Antioxidant content

The tocotrienol (α, γ) and tocopherol (α, γ and δ) contents of the larvae were quantified according to Zaspel and Csallany (1983). Briefly, 3 g of larvae were mixed with 1 mL of water and 4 mL of ethanol solution of 0.06% BHT. The mixture was saponified with KOH (60%) at 70 °C for 30 min and extracted with hexane/ethyl acetate (9/1, v/v). Two mL of supernatant was transferred into a glass tube, dried under N₂, and re-suspended in 200 μL of acetonitrile. The pellet was re-extracted two times. A 50 μl volume of the filtrate was then injected into the HPLC/FD (pump model PerkinElmer series 200, equipped with an autosampler system, model AS 950–10, Jasco, Tokyo, Japan) on a Sinergy Hydro-RP column (4 μm, 4.6 × 100 mm; Phenomenex, Bologna, Italy). The flow rate was set at 2 mL/min. All tocopherols and tocotrienols were identified using an FD detector (model Jasco, FP-1525 - excitation and emission wavelengths of 295 and 328 nm, respectively) and quantified using external calibration curves prepared with increasing amounts (from 0.001 µg/mL to 0.1 mg/mL) of pure standard solutions (Sigma-Aldrich, Bornem, Belgium) in ethanol. The total concentration (tocols, μg/g) was also reported.

The thiols group was detected according to the method of Mattioli et al. (2018), through the 5,5′ dithio-bis-2-dinitrobenzoic acid (DTNB) assay. Briefly, the pellets from trichloroacetic acid (TCA) were mixed with the DTNB 20 µM solution and incubated for 30 min at room temperature. After centrifugation at 10000 x g, the supernatant was read at a spectrophotometer (412 nm wavelength) with an extinction coefficient of 13,600 1/M*cm and expressed as µmoL SH – group per g.

Microbiological analyses

Microbiological analyses were performed on both non-starved and starved (48h) larvae to quantify the effect of fasting. One gram of Alphitobius diaperinus, 2 grams of Tenebrio molitor, and about 5 grams of Zophobas morio larvae were weighted in sterile Stomacher bags and diluted (1:10) with a sterile saline solution. The mixture was homogenised for 60 s in a stomacher (Stomacher® 400 Circulator, VWR International Sr, Milan, Italy). Ten-fold serial dilution series were performed and plated on different media. Plate count agar was employed for the quantification of the total viable aerobic count (incubated at 30 °C for 72h) and aerobic bacterial endospores after heating the 10:1 dilution at 80 °C for 10 min and performing ten-fold serial dilutions (incubated at 30 °C for 72h); de Man–Rogosa–Sharpe agar (MRS) was employed to count lactic acid bacteria (37 °C for 48h in anaerobic conditions); Chloramphenicol Yeast Glucose Agar (YGCA) was used to count yeasts and moulds (30 °C for 24h); violet red bile glucose agar (VRBGA) was used to enumerate Enterobacteriaceae (incubation at 37 °C for 24h); Tryptone Bile X-Glucuronide medium (TBX) was employed to quantify Escherichia coli (42 °C for 24h); Agar Sale Mannitol (MSA) was used for the quantification of Staphylococcus spp. (37 °C for 24h); Kanamicina Aesculina Azide Agar (K) was used to quantify the Enterococcus spp. (42 °C for 24h); Bacillus Cereus MYP agar was employed for the evaluation of the presence of presumptive Bacillus cereus (37 °C for 24h). The detection of Listeria monocytogenes and Salmonella spp. in 25 g was assessed according to ISO 11290 and ISO 6579, respectively. All culture media and supplements were purchased from ThermoFisher Scientific, (Milan, Italy) except for Bacillus Cereus MYP agar, which was purchased from Biolife (Milan, Italy). Microbial counts were expressed in log colony-forming unit (CFU)/g as the mean of three replicates.

Statistical analysis

The data obtained from the proximal composition, the fatty acids profile, the antioxidant content, and the microbial determinations were statistically analysed using a one-way ANOVA regarding the different insect species (Alphitobius diaperinus, Tenebrio molitor, and Zophobas morio) as the main factor. The effect of starvation (starved and unstarved) was tested against each different microbiological quantification by a Student’s t-test. Statistical significance was set at 0.05 and differences were assessed using Tukey’s test. The R free statistical software was used (R Core Team 2015). In order to compare the different chemical characteristics of the three insect species reared on the same substrate a principal components analysis (PCA) was performed via Stata Statistical Software (StataCorp 2015) on proximate composition (crude protein and ether extract, expressed on DM basis), main fatty acids classes (SFA, MUFA, PUFA n-3 and n-6) and antioxidant contents (tocols and thiols) of the larvae; all the data were mean centred and scaled to a unit standard deviation before analysis.

Results and discussion

Proximate composition

Proximate composition of the different larvae reared on the same substrate are reported in Table 2. The results show some differences among the examined insect species. The percentage of DM was significantly higher in larvae of Zophobas morio, followed by the one of Tenebrio molitor and Alphitobius diaperinus.

Table 2. Proximate composition of different larvae reared on the same substrate (bread:brewery spent grain, 1:1).

Item	Alphitobius diaperinus	Tenebrio molitor	Zophobas morio	RMSE	p-value
Dry matter (%)	27.11^c	32.77^b	38.06^a	0.648	<0.001
Ether extract (%)	10.78^c	13.86^b	17.65^a	0.794	<0.001
Crude protein* (%)	14.40	14.33	13.82	0.842	0.669
Crude protein^§ (%)	18.91	18.82	18.15	1.106	0.669
Ash (%)	0.71	0.81	0.84	0.163	0.611

* Conversion factors of 4.76 for crude protein.

^§ Conversion factors of 6.25 for crude protein.

Means in the same row with no common superscripts (a–c) differ significantly (p < 0.05).

RMSE: Root Mean Square Error.

These data are consistent with those reported in the literature about the percentages of DM per insect species. In detail, our data of Tenebrio molitor larvae are in line with those of Mancini et al. (2019b) which reported respectively a DM of 33.33% and 32.62% for larvae reared on substrates made only by brewery spent grains or bread. The results on DM for Tenebrio molitor are also in line with those of Yi et al. (2013) (36.5%) who reported having purchased the samples from a supplier that fed the larvae with a mix of wheat, wheat bran, oats, soy, rye, corn, carrot, and beer yeast. Contrarily, Ghaly and Alkoaik (2009), reported higher DM content, of 41.9%, in Tenebrio molitor larvae purchased from a pet shop and fed with wheat germs and oatmeal. The DM value of Zophobas morio is in line with those of Soares Araújo et al. (2019) and Yi et al. (2013) which reported values respectively of 35.42% and 40.1%. Contrarily, the DM value of Alphitobius diaperinus is lower than that identified by Yi et al. (2013) (35.5%). All these variations could be related both to the feeding substrates or to the larval stages, not secondary also the environmental conditions of the rearing farm could affect the DM of the larvae (e.g. the relative humidity of the farming).

The values relating to the ether extract expressed on the fresh basis showed the same trend as the one of DM (Table 2); while a little variation was showed for Tenebrio molitor larvae when the ether extract was expressed on DM, indeed, this species did not show differences against the other two species (Figure 1).

Figure 1. Proximate composition on dry matter of Alphitobius diaperinus, Tenebrio molitor, and Zophobas morio larvae reared on the same substrate (bread:brewery spent grain, 1:1).

Bars with no common superscripts (a–b) differ significantly (p < 0.05) between insect species.

In general, the ether extract values showed by our samples were higher than those of Yi et al. (2013), according to which the lipid content, expressed as a percentage of fresh sample, were 8.5%, 9.9%, and 16.0%, respectively for Alphitobius diaperinus, Tenebrio molitor, and Zophobas morio. Similarly, the data reported by Adámková et al. (2016) of lipids (expressing the values on the dry matter content) of the three insect species were lower than those detected in our samples with the exception of Zophobas morio for which the results seem comparable (29.0% for Alphitobius diaperinus, 17.0% for Tenebrio molitor, and 39.0% for Zophobas morio). The authors reported that the larvae were purchased in the ultimate or penultimate instars from a private company that fed the insects with a mixture of plant material (carrots, cabbage, Chinese cabbage, tomatoes, and potatoes). Alphitobius diaperinus ether extract is lower to those reported by Soetemans et al. (2020) ranged from 14 to 28% on DM for larvae fed with rice bran, rapeseed meal, corn gluten feed, wheat middlings, brewery spent grain, and carrots.

About Tenebrio molitor larvae, the ether extract content was comparable to the one reported by Mancini et al. (2019b) of 14.82% for larvae reared on a substrate consisting exclusively of bread, while it was higher than the one of the larvae fed brewery spent grain only (6.46%). Larvae fed wheat germs and oatmeal showed similar results, varying from 12.0 to 12.5% on the fresh sample, on the other hand, the data expressed on DM showed to be lower (ranged between 29.83 and 31.17% on DM) than those determined in our samples (Ghaly and Alkoaik 2009). The Zophobas morio ether extract expressed on the dry matter is in line with the results obtained by Rumpold and Schlüter (2013) (40.80-42.04%), by Soares Araújo et al. (2019) (43.64%) and by Harsányi et al. (2020) (42.04-46.30%).

Data about crude protein, expressed on fresh basis, did not show any significant differences between insects (Table 2). On the other hand, when crude proteins were expressed on DM statistical differences were highlighted (Figure 1). Zophobas morio larvae showed significantly lower values than other insects (14.40, 14.33 and 13.82; 18.91, 18.82 and 18.15, respectively for Alphitobius diaperinus, Tenebrio molitor, and Zophobas morio larvae with a conversion factor of 4.76 and 6.25, Figure 1). The proteins expressed on the fresh sample are comparable to those reported by Yi et al. (2013) (20.6% for Alphitobius diaperinus, 19.1% for Tenebrio molitor, and 20.7% for Zophobas morio). In 2016 Adámková et al. reported percentages of protein content of about 60% for Alphitobius diaperinus, 63% for Tenebrio molitor, and 39% for Zophobas morio. About the protein content expressed on DM Alphitobius diaperinus showed values lower than reported by Soetemans et al. (2020) of 37-49% on DM. Comparing these data to the ones of our samples, we can highlight a similar range for Tenebrio molitor, while higher values were obtained for the other two species.

Furthermore, according to Mancini et al. (2019b), the protein content of Tenebrio molitor larvae reared on a 100% brewery spent grain substrate was similar to the larvae fed the mix B:SG. On the contrary, the data of the larvae fed only bread leftovers reported a slightly lower value (10.73%; Mancini et al. 2019b). Another study on Tenebrio molitor, conducted by Ghaly and Alkoaik (2009), showed values of proteins between 24.3 and 27.6% on the fresh basis and between 63.31 and 68.87% on the DM; both the data expressions revealed higher values than our data (Table 2 and Figure 1), probably due to their high protein substrate employed (wheat germs and oatmeal). Variation in proteins content in relation to the substrate used is also reported by Harsányi et al. (2020). Larvae of Tenebrio molitor fed chicken feed or a mix of it with vegetable waste or garden waste (ratio 1:9) showed protein contents of 42.30-47.18% on DM, while larvae fed a mix with cattle or horse manures showed lower protein contents of 38.92% and 37.90%, respectively. The protein percentage on DM for Zophobas morio, using a conversion factor of 6.25, is in line with the values reported by other research studies (Rumpold and Schlüter 2013; Adámková et al. 2016; Soares Araújo et al. 2019). These data are not in agreement with those reported by Oonincx and Dierenfeld (2012) which reported crude protein value on DM of 68.05% for larvae purchased by a supplier that used wheat bran as substrate. No differences are observed in the percentage of ash (both expressed as % on fresh or dry matter, Table 2 and Figure 1).

Fatty acids profile and antioxidant content

The fatty acids profile of the larvae was reported in Table 3. Interesting was the proportion of the main fatty acid classes: Zophobas morio larvae showed the highest SFA percentage, followed by Alphitobius diaperinus and Tenebrio molitor (41.33, 28.52 and 39.16%, respectively), whereas MUFA and PUFA exhibited a different trend with higher values in the larvae of Tenebrio molitor, followed by Zophobas morio and Alphitobius diaperinus. However, the fatty acids proportion was mainly affected by lipid proportion, indeed considered the quantitative fatty acids profile (Table S1), the SFA and MUFA followed the trend: Zophobas morio > Tenebrio molitor > Alphitobius diaperinus, whereas PUFA exhibited similar values in Zophobas morio and Tenebrio molitor and lower ones in Alphitobius diaperinus. The most representative SFA in Zophobas morio was palmitic acid (C16:0; 30.32%, corresponding to 3381.25 mg/100g) and it is in line with what reported by Kulma et al. (2020) where the palmitic acid percentage ranged from 25.60% to 28.34% based on the days of age (from 60 to 120). Regarding Tenebrio molitor larvae the main MUFA was represented by oleic acid (C18:1cis n-9, OL; 38.41%), whereas concerning the PUFA, was linoleic acid (C18:2cis n-6, LA; 28.06%). Similar results have been reported by Paul et al. (2017) who found in Tenebrio molitor a 35.83% and 22.83% of OL and LA, respectively. However, a different trend was found considering the mg of FA (Table S1), where OL and LA were the most abundant in Zophobas morio larvae.

Table 3. Fatty acids profile (% of total fatty acids) of larvae fed the same substrate (bread:brewery spent grain, 1:1).

	Alphitobius diaperinus	Tenebrio molitor	Zophobas morio	RMSE	p-value
C12:0	0.34^b	0.24^c	0.46^a	0.135	<0.001
C14:0	1.57^b	3.73^a	1.87^b	0.444	<0.001
C14:1cis9	0.03^b	0.13^a	0.06^b	0.098	<0.001
C16:0	26.68^b	18.79^c	30.32^a	0.973	<0.001
C16:1cis9	1.06^b	1.80^a	0.67^c	0.310	<0.001
C18:0	10.57^a	5.77^c	8.77^b	0.627	<0.001
C18:1cis9, OL	31.41^c	38.41^a	33.49^b	0.778	<0.001
C18:2 n-6, LA	20.27^b	28.06^a	20.78^b	0.842	<0.001
C20:0	0.32^b	0.27^b	0.61^a	0.175	<0.001
C20:1	0.48^a	0.28^b	0.20^b	0.163	<0.001
C18:3 n-3, α-ALA	0.38^c	0.90^b	1.38^a	0.306	<0.001
C20:4n-6, AA	0.55^a	0.07^c	0.10^b	0.207	<0.001
C22:5n-3, DPA	0.02^a	0.01^b	0.02^a	0.045	<0.001
C22:6n-3, DHA	0.002^b	0.01^a	0.002^b	0.029	0.005
Tot	93.35^b	98.18^a	98.11^a	0.699	<0.001
Others	6.65^a	1.82^b	1.88^b	0.699	<0.001
SFA	39.16^b	28.52^c	41.43^a	1.052	<0.001
MUFA	32.98^c	40.61^a	34.38^b	0.821	<0.001
PUFA	21.21^c	29.05^a	22.29^b	0.833	<0.001
n-6	20.81^b	28.13^a	20.88^b	0.827	<0.001
n-3	0.40^c	0.92^b	1.41^a	0.306	<0.001
n-6/n-3	53.84^a	30.77^b	16.04^c	1.810	<0.001

Means in the same row with no common superscripts (a–c) differ significantly (p < 0.05).

RMSE: Root Mean Square Error.

On the contrary, the α-linolenic acid (C18:3 n-3, ALA) was higher in Zophobas morio larvae, followed by Tenebrio molitor and Alphitobius diaperinus considering both the FA proportion (1.38%, 0.90% and 0.38%, respectively) and amount (154.321, 66.41 and 16.14 mg/100g, respectively). Conversely, a percentage of ALA similar to what recorded in Zophobas morio larvae was found in Alphitobius diaperinus when the basal diet was constituted by 2.5% of ALA (Oonincx et al. 2020). Similar ALA values (0.89%) were reported in Tenebrio molitor larvae fed only brewery spent grains (Mattioli et al. 2021). The PUFA trend also affected the total n-6 and n-3 fatty acids proportion. In this context the n-6/n-3 ratio was very different between insects (53.84, 30.77 and 16.04 in larvae of Alphitobius diaperinus, Tenebrio molitor and Zophobas morio respectively) and reached the best value in Zophobas morio larvae. Such a trend was mainly due to the ALA content of the latter species because the long-chain fatty acids showed a negligible proportion (less than 0.02%). It is reported that insect is not able to efficiently elongate LA and ALA into their long-chain derivatives, thus they store what take-up by the growth substrate (Mattioli et al. 2021; Rossi et al. 2022).

However, our data confirmed that every species showed a different PUFA metabolism (Twining et al. 2021), i.e. the Zophobas morio resulted in a higher n-3 proportion than the other species, mainly due to the ALA %. In agreement with this statement, the data herein reported also suggested an unusual n-3 long chain-PUFA metabolism in Zophobas morio. Indeed, a metabolic intermediate such as DPA exhibited higher values than other species, but controversy the final products, DHA, was not the highest. Further studies on metabolic activity are necessary to investigate this aspect in the lesser and superworms. However, such trend was also partially related to the higher lipid content of Zophobas morio than other species. The SFA concentration resulted in almost double respect to that of PUFA (SFA/PUFA ratio was 1.88 in lesser and 1.85 in superworms; data not shown), whereas Tenebrio molitor showed an equal concentration (2099.38 and 2137.73 mg/100g of larvae for SFA and PUFA, respectively) therefore from fatty acids profile viewpoint, seems that mealworm was better balanced.

The antioxidant contents were reported in Table 4. Only γ- and α-tocopherols showed significantly different concentrations in the studied insects. A higher total quantity of tocols was recorded in Zophobas morio than in other (0.97 vs 0.27 and 0.17 µg/g), probably due to the higher lipid concentration; indeed, the vitamin E is a fat-soluble vitamin and thus it is mainly stored in lipid tissue or cell membranes (Steenbock and Nelson 1923). However, vitamin E concentration was in line with what reported on larvae of Tenebrio molitor reared in bread and brewery spent grains but lower with respect to what was previously found in Tenebrio molitor larvae fed different substrates (100% cookies, 50% bread+ 50% cookies and 50% spent grains + 50% cookies) (Mattioli et al. 2021). The thiols also showed a higher concentration in larvae of Zophobas morio, but it did not differ from Alphitobius diaperinus (2.80 and 2.45 µmol SH-group/g, respectively), whereas the Tenebrio molitor larvae, showed a lower quantity (1.97 µmol SH-group/g), suggesting a lower antioxidants enzyme activity in this insect. Thiol-containing compounds are essential components of maintaining the oxidation–reduction homeostasis in cells and tissues (Sokolovskiǐ 1988). However, studies of glutathione-enzymes in insects are scarce and mainly deal only with the physico-chemical properties of the isolated enzymes and their participation in the detoxication of xenobiotics (Francis et al. 2002), such as non-enzymatic antioxidants, as SH-containing compounds, were studied predominantly in vertebrate animals (Sokolovskiǐ 1988; Mattioli et al. 2018; 2020).

Table 4. Antioxidant content of larvae fed the same substrate (bread:brewery spent grain, 1:1).

	Alphitobius diaperinus	Tenebrio molitor	Zophobas morio	RMSE	p-value
γ-Tocotrienol (µg/g)	0.02	0.03	0.03	0.082	0.307
α-Tocotrienol (µg/g)	0.01	0.01	0.01	0.012	0.060
d-Tocopherol (µg/g)	0.00	0.01	0.01	0.040	0.074
γ-Tocopherol (µg/g)	0.01^b	0.01^b	0.09^a	0.100	<0.001
α-Tocopherol (µg/g)	0.13^b	0.21^b	0.83^a	0.289	<0.001
Tocols (µg/g)	0.17^b	0.27^b	0.97^a	0.295	<0.001
Thiols (µmol SH-group/g)	2.45^ab	1.97^b	2.80^a	0.554	0.015

Means in the same row with no common superscripts (a–c) differ significantly (p < 0.05).

RMSE: Root Mean Square Error.

Microbiological analyses

Results obtained from the microbiological analyses are reported in Table 5. Escherichia coli, Bacillus cereus, Listeria spp., Salmonella spp., and yeasts and moulds loads were not detected in all the samples. The absence of pathogens such as Bacillus cereus, Listeria spp., Salmonella spp., as well as Escherichia coli is in accordance with several other research studies (Grabowski and Klein 2017a; Vandeweyer et al. 2017a; Mancini et al. 2019a). Differently, yeasts and moulds loads have been detected by Grabowski and Klein (2017a) in Tenebrio molitor and Zophobas morio larvae (5.2 and 1.7 log CFU/g, respectively) and also by Vandeweyer et al. (2017a, 2017b) in Tenebrio molitor larvae (3.5 − 6.0 log CFU/g). These variations in yeast and moulds loads could be ascribed to the rearing systems and to the substrates employed. Alphitobius diaperinus larvae showed total viable aerobic count, Enterobacteriaceae and lactic acid bacteria loads comparable to those reported by Wynants et al. (2018). The total viable aerobic count, the Enterobacteriaceae, and the staphylococci loads of Tenebrio molitor larvae were similar to those of Stoops et al. (2016), Grabowski and Klein (2017b) Mancini et al. (2019b) and Costa et al. (2020). Instead, the total viable aerobic count in unstarved and starved Tenebrio molitor larvae was lower with respect to what it showed by Caparros Megido et al. (2018) (8.5 log CFU/g). On the other hand, the values of lactic acid bacteria in both starved and unstarved Tenebrio molitor larvae were lower than those reported by Stoops et al. (2016) and Vandeweyer et al. (2017a), and at the same time higher than those of Mancini et al. (2019b). About spore forming bacteria on Tenebrio molitor unstarved larvae the load was lower than that observed in Costa et al. (2020) (4.1 log CFU/g) while was similar to those reported by Klunder et al. (2012) in fresh Tenebrio molitor larvae (2.15 log CFU/g). Little variations in total viable aerobic counts of superworms larvae was revealed comparing the results to another research study conducted in the same laboratory using the same rearing technique and diet (variation of 0.39 log CFU/g) (Cacchiarelli et al. 2022). These variations in a general quantification as the total viable aerobic counts could be related to little differences in the daily operations or due to changes in the management of the larvae. Other variations in Zophobas morio larvae were observed in comparison with Grabowski and Klein (2017a) as lower total viable aerobic count and staphylococci load were revealed. Among insect species have highlighted significant differences regarding the lactic acid bacteria counts, both in the case of unstarved and 48h starved larvae, as well as for staphylococci loads in starved larvae. Zophobas morio unstarved larvae showed higher lactic acid bacteria count than both other insect species, while, on 48h starved larvae specimens it can be observed that loads of lactic acid bacteria were lower in lesser mealworms than mealworms and superworms. The staphylococci determination showed higher loads in starved larvae of Zophobas morio than of Tenebrio molitor, but both showed no significant differences with the ones of Alphitobius diaperinus. No statistical differences were reported for the starvation effect, as was also shown by Wynants et al. (2018) for lesser mealworm, while Mancini et al. (2019a) (2019b); reported in mealworm larvae variations in the microbial loads in relation also with the diets.

Table 5. Microbiological loads of larvae (unstarved and starved) fed the same substrate (bread:brewery spent grain, 1:1), expressed in log CFU/g.

Item	Alphitobius diaperinus	Tenebrio molitor	Zophobas morio	RMSE	p-value
Unstarved
Total viable aerobic counts	7.72	7.38	7.23	0.292	0.188
Spore-forming bacteria	1.82	2.15	2.16	0.382	0.496
Staphylococcus spp.	5.70	5.38	5.69	0.707	0.826
Enterococci	5.81	5.56	6.07	0.476	0.477
Lactic acid bacteria	6.33^b	6.55^b	7.10^a	0.210	0.010
Enterobacteriaceae	8.17	6.86	7.47	0.665	0.133
Starved, 48h
Total viable aerobic counts	7.14	7.28	7.58	0.865	0.082
Spore-forming bacteria	3.11	4.05	2.90	0.421	0.327
Staphylococcus spp.	5.44^ab	4.67^b	5.78^a	0.309	0.012
Enterococci	6.08	5.54	5.58	0.737	0.300
Lactic acid bacteria	5.46^b	6.80^a	6.93^a	0.490	0.019
Enterobacteriaceae	7.47	6.69	7.42	0.733	0.398
Effect of starvation, P-value
Total viable aerobic counts	0.329	0.775	0.930
Spore-forming bacteria	0.473	0.317	0.790
Staphylococcus spp.	0.382	0.880	0.373
Enterococci	0.286	0.168	0.708
Lactic acid bacteria	0.623	0.976	0.185
Enterobacteriaceae	0.096	0.330	0.569

Means in the same row with no common superscripts (a–b) differ significantly (p < 0.05).

RMSE: Root Mean Square Error.

Principal components analysis (PCA)

Many studies have demonstrated the effectiveness of multivariate analysis not only in genetic classification but also for studying the trend of many variables within experimental groups (Mancini et al. 2018; Mattioli et al. 2019) (Figure 2). PCA analysis summarised what was previously affirmed regarding the different responses of studied chemical compounds in the insect species. The first principal component (Dimension 1) mainly discriminated Alphitobius diaperinus specie by Tenebrio molitor and Zophobas morio explaining 50.9% of variability. Instead, the second principal component (Dimension 2) discriminated the three insect species (explaining 31.7% of variability). Based on the observation distribution, the Tenebrio molitor larvae were mainly described by n-6 PUFA and MUFA, whereas the Alphitobius diaperinus ones by protein content. Interestingly resulted that the larvae of Zophobas morio showed a localisation mainly characterised by n-3 PUFA, lipid, and tocols. Thiols and SFA did not show an insect species-related trend. Such characterisation suggests that every species, having a different chemical-compounds metabolism, could be used a different source as feed or food: i.e. the Tenebrio molitor larvae as a source of MUFA and n-6 PUFA, the Alphitobius diaperinus larvae as a protein source and the Zophobas morio larvae as lipid, n-3 PUFA, and tocols source. The latter features are of great relevance if it is considered the wide word deficit of n-3 PUFA (Cartoni Mancinelli et al. 2022). In this context, the superworms could be retained as a very important lipid source for animal and/or human nutrition (if approved by EFSA) unlike the usual protein source, which, up to now, are considered the main species of insects (Tenebrio molitor and Hermetia illucens) (Liceaga et al. 2022). Further studies would be interesting to test the possibility of modifying the SFA/PUFA ratio in superworms by increasing the PUFA content through different growth substrates (e.g. linseed-based by-products), considering the high n-3 proportion found in this species.

Figure 2. Principal component analysis (PCA) of insect larvae. The total explained variance is 82.6% (the first dimension explains 50.9% of variability, the second dimension explains 31.7% of variability).

Conclusions

The insect species considered in the present study (Alphitobius diaperinus, Tenebrio molitor, and Zophobas morio) showed interesting features from a nutritional point of view; in particular, lesser mealworm and mealworm are distinguished by a high protein content (especially Alphitobius diaperinus), while superworm shows a high rate of lipid content; moreover a higher proportion of PUFA from n-3 series were recorded respect to the other fatty acids, such as a higher concentration of fat-soluble vitamins (tocols). However, a higher quantity of SFA was recorded. Compared to the most common animal protein sources, such as eggs and meats, Alphitobius diaperinus and Tenebrio molitor represent good protein sources, leading to their use as both food and feed alternatives. Noteworthy, although containing a lower percentage of proteins, also Zophobas morio larvae are still comparable to egg and chicken meat. From a microbiological point of view, in the studied larvae no harmful bacteria or foodborne pathogens were detected. Differences in chemical-compounds metabolism shown by the three species reared on the same substrate highlight the insects’ potential to respond to different questions derived from the food and feed sectors. Results point out the high plasticity of the studied insect species and the potential to tailor the final outcomes in relation to the required characteristics need in the final products, also in relation to their utilisation as food or feed. This characteristic plays a key role in the insects’ sector as rearing and also processing steps can meet the marketing strategies and requirements unleashing the insect potential.

Disclosure statement

No potential conflict of interest was reported by the authors.

Ethical approval

The author confirm that the study has been conducted in an ethical and responsible manner.

Data availability statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Additional information

Funding

This work is supported by the Università di Pisa under the ‘PRA – Progetti di Ricerca di Ateneo’; (Institutional Research Grants) - Project no. PRA_2020-2021_12 ‘Produzione di Insetti come Feed e Food—PIFF’.