3.1. Growth Performances and Proximate Compositions
Proximate compositions of the larvae reared on the different substrates are reported in Table 2
Dry matter, ether extract, and crude protein contents were affected by the diet. Dry matter of the SG-C larvae was the highest, followed by that of the C and B-C diets. From these data, it would seem that the presence of the cookies induced a decrease of humidity in the larval body, as the larvae fed with spent grains and bread alone (SG and B, respectively) showed the lowest values of dry matter. Cookies also played a major role in the lipid content of the larvae. Diets C and B-C showed the highest contents, followed by B. Spent grains contained low amounts of lipids (3.29%), and consequently larvae fed with SG and SG-C showed lower values compared to those fed other diets.
The highest crude protein contents were shown by diets which included spent grains (SG and SG-C) with minor differences. This value was lower in diets with bread (B and B-C). In this case, the low amount of crude proteins of the cookies (6.55%) negatively affected the chemical composition of the larvae with a consequent lowest body protein content.
As the dry matter analyses showed to be significantly affected by the diet, the proximate composition was also reported as % of dry matter (Table 2
). Expressing the data as % of dry matter, the ether extract content showed that B, C, and B-C had the highest lipid contents, followed by SG-C and SG, respectively. Thus, the effect of the cookie fat content was confirmed but the bread diet (B) also played a key role.
Crude protein contents, as % of dry matter, showed only minor differences in the statistical analyses if compared to the expression as % of fresh sample. The conversion factors of 6.25 or 4.76, as well as the expression as % of fresh sample or % of dry matter, drastically affected the final numerical expression; nevertheless, as no standardization is currently used in the insect research field, we choose to report all the data expressions.
Carbohydrates were higher in larvae fed SG and SG-C diets than those fed the other diets. Interestingly, SG had the lowest amount of carbohydrates within the employed substrates. Particularly, moisture and lipids content seemed to affect the calculations of carbohydrates in larvae reared in SG and SG-C.
Chemical compositions of mealworm confirm the data already published [7
] and in particular that body composition showed a considerable plasticity in relation to the diet [7
]. Notably, in our trial, mealworms fed cookies (C) showed a high amount of lipids (50% on DM base) mostly related to the detriment of the protein content. This data could be interesting as edible insects are almost exclusively recommended as a protein source even though they could contain a high amount of fats.
The larvae weights are reported in Figure 1
; larvae fed SG and SG-C reached the harvesting day approximately after 5 to 6.5 months. After one year of rearing, larvae fed diets with lower content of protein, C, B, and B-C did not reach the pupae stage; therefore, in order to not exceed in the rearing time, their growth was stopped. Larvae fed diets C, B, and B-C reached the final weights of 87, 95, and 112.5 mg, respectively. In linear regression and second-order polynomial quadratic equations (Figure 1
), the coefficient of determination indicates that the models explain a high percentage of the variability (R2 between 0.94 and 0.99). Between linear regression and second-order polynomial quadratic equation, the second seems to better fit the larval growth rate.
The FCR (calculated on fresh basis), the ECI, and the N-ECI were severely affected by the dietary treatments. Dietary efficiency of mealworm larvae was strictly related to the chemical composition of the substrate; indeed, larvae fed SG and SG-C showed the lowest values of FCR, 2.22 and 2.76 respectively, confirming the extremely positive potential of this species to convert feed into body weight. On the other hand, larvae fed B, C, and the mix B-C showed FCR values about 8.86, 7.31, and 4.02, which are similar to those of conventional production animals (4.0 for pork and 8.8 for cereal beef; [9
]). The variability in FCR values was related to the protein contents of the diets; indeed, SG and SG-C showed the highest amounts of protein. A large variability of the FCR value in relation to the protein content was also reported by Oonincx et al. [7
] and van Broekhoven et al. [10
] in mealworms fed different diets (from 4.1 to 19.1 and from 2.62 to 6.05, respectively).
The ECI values were higher for SG and SG-C diets (15.85 and 13.94, respectively) than for B, C, and B-C ones (3.79, 4.87, and 8.92, respectively). These data are comparable with those reported by Oonincx et al. [7
] and van Broekhoven et al. [10
]. As reported before for FCR, the ECI values were affected by the protein content of the diets, which confirms that mealworm efficiency could be modelled through the rearing substrate’s chemical composition.
Nitrogen was more efficiently converted than the other diet components in almost all diets, as in the SG, SG-C, C, and B-C diets, the N-ECIs were higher than the ECI (59.11, 71.75, 13.83, and 14.05, respectively). The diet exclusively composed of bread (B) showed the lowest value of N-ECI (2.77), which is lower than the ECI value. This means that mealworm fed only bread did not convert N of the substrate efficiently into body mass [7
]. Indeed, larvae fed only cookies (C) reached approximately the same amount of crude protein content (Table 2
) even though the substrate showed to contain half the protein (6.55% in C vs. 11.15% in B, Table 1
). The high N-ECI showed by SG and SG-C larvae represents a starting point to reach a relevant benefit of insects over conventional production animals [25
3.2. Microbiological Analyses
Microbial determinations of starved and un-starved larvae are reported in Table 3
In the un-starved larvae, staphylococci, yeast-molds, and bacterial endospores were significantly different in relation to the diet. Staphylococci and yeast-molds loads were higher in larvae fed B, C, and B-C, followed by SG-C and SG. Minor differences in the staphylococci amount was highlighted between SG and SG-C in relation to the presence of the cookies. Bacterial endospores were absent in SG, and their presence was related to B and C diets. Indeed, feeding the larvae with a mix of SG and C induced an increase of these bacteria. The highest number of bacterial endospores was detected in the B-C diet, followed by B and C substrates.
Microbial analyses highlighted the total absence of Escherichia coli
and Bacillus cereus
, as well as the absence in 25 g of Listeria monocytogenes
spp. Other research studies reported the absence of these bacteria in edible insects reared for human consumption [26
In starved larvae, diets affected only yeast-molds and bacterial endospores, showing a similar trend of that reported for the un-starved larvae with minor differences. Starvation was not effective in larvae fed SG and SG-C diets, while in larvae fed B, C, and B-C it partially affected the microbial flora. Total viable aerobic counts and lactic acid bacteria were not affected by the starvation. Staphylococci and yeast-molds amounts were significantly decreased in B, C, and B-C fed larvae. Starvation in larvae fed C induced a significant decrease in the Enterobacteriaceae amount, as well as in bacterial endospores of larvae fed C and B-C.
Studying larvae fed wheat bran supplemented with carrots, Wynants et al. [30
] reported that fasting for 24 or 48 h, both with and without fecal contact of the larvae, did not significantly affect total viable aerobic bacteria, Enterobacteriaceae, aerobic bacterial endospores, psychrotrophic aerobic bacteria, and yeast and mold amounts.
Contrarily, Mancini et al. [31
] reported that a starvation treatment for 24 h affected the bacterial endospores amount in mealworm larvae reared on wheat bran supplemented with potato slices as a water source without influencing the other microorganisms.
Moreover, starvation also resulted effective in the reduction of Salmonella enterica
and Listeria monocytogenes
in mealworm larvae reared in artificially contaminated substrates [32
]. Thus, the effect of the starvation process seems to vary in relation to the diet as well as the response of different tested bacteria.
3.3. Antioxidant Status
Antioxidant capacity of mealworm larvae is reported in Table 4
No significant differences were found among the experimental diets. However, the C diet seems to slightly improve the antioxidant capacity of the larvae with respect to the other groups (as highlighted by ABTS and FRAP values, even if no statistical significances were determined). Such a trend is justified also by the significantly higher concentration of tocopherols (mainly due to the α-isoform amount) found in the cookies-supplemented groups (C, SG-C, and B-C larvae). Nevertheless, vitamin E being a fat-soluble vitamin, it is not surprising that its concentration was higher in the mealworms with a higher fat content (ether extract values). To the best of our knowledge, this is the first evaluation of the antioxidant capacity of T. molitor larvae.
The antioxidant capacity of mealworms could be very interesting in order to enhance dietary antioxidant ingestion; however, as no thermal process was applied to the samples (cooking or drying), enzymes present in the larvae body may have altered the final outcomes. Few published data reported the antioxidant concentration of insects: Finke [15
] summarized that the vitamin E contents ranged from 3.3–24.0 mg/kg for mealworms, 5.3–9.1 mg/kg for superworms (Zophobas morio
), and 8.6–69.2 mg/kg for waxworms (Galleria mellonella
). In the present study, the total vitamin E concentration of mealworms ranged from 0.51 to 5.28 mg/kg, close to the lower values reported in the literature. However, a higher concentration (>10 mg/kg) of vitamin E is reported to be common in the wild-caught insects with respect to the reared ones [34
3.4. Principal Component Analysis
A principal component analysis of proximate composition, microbial loads, growth performance, antioxidant capacity, and antioxidant compounds was performed in order to detect the principal components that better describe the modifications highlighted (Figure 2
Eigenvalues, eigenvectors, and cumulative % of the first three principal components are reported in Table 5
. The first two principal components (PC1: 41.07% and PC2: 17.91%) well differentiate the samples in relation to the diet.
Positives PC1 eigenvectors collocate crude proteins on the right side of the biplot, near to SG and SG-C samples, as well as the growth performance. This relation highlights that diets rich in proteins (SG and SG-C) could promote growth and increase the protein content of the larvae. On the other hand, negative PC1 eigenvectors collocated ether extract content on the left side of the biplot, in contraposition with the protein content and the growth performances. The presence of the cookies in B-C and SG-C diets induced a shift on the left side of the plotted samples and then evidence a worsening of the growth performances and a higher deposition of fats.
Almost all the parameters that showed a statistical significance were plotted in the upper right or the lower left squares (both eigenvectors, positives or negatives) except for dry matter and tocopherol γ- and α-isoforms, which were plotted in the upper left square highlighting that another undetermined effect plays a role. As PC2 differentiates the antioxidant capacity and the tocopherol γ- and α-isoforms in the upper part of the plot and the microbial loads in the lower part, it could possible to hypothesize that a higher content of antioxidants played a role against microbial loads.
The observations reported above suggest that PC1 and PC2 were both a part of the diet effect related to the chemical composition of the substrates.
A residual percentage of variance (13.01%) was expressed also by PC3; this principal component was mostly related to (as absolute values) the antioxidant capacity and tocopherols δ- and α-isoforms.