1. Introduction
Interest in the human consumption of edible insects (entomophagy) in Western countries is increasing and more and more insect-based food products are being marketed [
1]. Since January 2018, insects have been considered as a novel food in Europe, as stated in EU regulations 2015/2283 [
2]. The use of insects in both feed and food formulations is increasingly being recognized as a novel way to improve feed and food security [
3,
4,
5]. Edible insects have a high content of protein, essential amino acids, minerals, and vitamins [
6,
7,
8,
9]. Their final larval stage (prepupae) has a high content of protein and fat. Prepupae can contain up to 47% of crude protein and 35% of ether extract (on a dry matter basis), depending on the rearing substrate [
10,
11,
12,
13,
14].
Insects can, thus, be used as either food or feed source and, thus, contribute to solving global food problems [
15]. Compared to standard farming, insect rearing requires less energy and very little land. It could potentially result in a lower environmental footprint and, thus, have a beneficial effect on the environment [
15,
16,
17].
To support safety evaluations and risk assessments, as well as to establish specific microbial criteria for edible insects by the European Food Safety Authority (EFSA), additional quantitative data on their microbial quality are needed [
18,
19]. As highlighted by the EFSA, microbiological data are scarce [
20]. In fact, few studies contain data on the intrinsic properties of pH and water activity (a
w) in the diet, despite them having an important impact on the growth and survival of pathogenic microorganisms. These factors, thus, need to be taken into account when considering insects as either feed or as part of the food matrix [
21].
Farmed insects are considered by the current EU regulation as ‘farmed animals’ and, thus, they have to be fed using safe substrates [
22]. Reared insects have to be safe not only for animals, but also for human consumption; however, like any other farmed animals, the substrates (feed) used to grow insects can be contaminated by pathogens [
23].
Previous studies reported that black soldier flies (BSFs) act on microbial contamination by modifying it in the substrate and in the larvae [
24,
25,
26,
27]. However, currently, there are no microbiological challenge tests available to verify the fate of foodborne pathogens in larvae grown on a substrate contaminated from the egg to the prepupal stage.
This study aims to assess the level of contamination of BSF larvae grown on a substrate highly contaminated by foodborne pathogens in order to give useful results for a risk assessment conducted to evaluate the food and feed safety if a substrate accidentally contaminated by pathogens is used in the breeding of this important source of protein for food and feed.
In the present study, two substrates were contaminated with either Salmonella enterica serotype Typhimurium or Listeria monocytogenes in two independent challenge tests and a reduction in pathogen load in the larvae and substrate was investigated. The results of this study provide useful information aimed at improving H. illucens rearing practices. In addition, the results could be used in future microbiological risk assessments on BSF larvae.
4. Discussion
It is now internationally accepted by scientists and economists that the world population will increase to 9.6 billion by 2050. Current food production cannot support such a large number of people and it is estimated that this will need to almost double [
32]. Insects can be an important alternative source of food production for humans and farmed animals, and using insects for feed and food is an attractive option. Insects have high nutritional value, a high reproduction rate and high feed conversion efficiency, thus, making them ideal for agricultural purposes. In addition, compared to other farmed animals, insects seem to be more “respectful” of the environment [
29,
30].
Based on current legislation, animals in the EU must be fed only with safe feed (Commission Regulation (EC) No. 68/20136, Regulation (EC) No. 178/2002, and Regulation (EC) No. 767/2009), and the Regulation (EC) No. 1069/20097, which considers insects as “farmed animals”, does not allow the use of certain substrates (e.g., manure, catering waste or former foodstuffs containing meat and fish) for their feeding [
20].
The black soldier fly (BSF),
Hermetia illucens, Diptera of the family Stratiomyidae, thrives on an immense variety of organic substrates, which is why it is suitable for small-scale waste management purposes using substrates, such as manure [
33]. These biological characteristics make the species an ideal candidate to support a market based on the circular economy, as described in [
34].
This study involves the first challenge tests to study the inactivation potential and kinetic parameters of BSF larvae reared on substrates contaminated with Salmonella enterica Typhimurium and Listeria monocytogenes. In addition, for what we believe is the first time, results are reported here on L. monocytogenes, which provide important information for risk managers in view of the ubiquity of this telluric bacterium in all environments.
One limitation of this study is that larvae at 2 days of age weigh approximately 0.01 g (dry weight) and one day after hatching, they weigh approximately half this, thus, making microbiological analysis difficult. For this reason, the concentration of pathogens was only measured starting on day three after hatching. However, given that the stage of development of commercial interest is that of the prepupa, which is reached, in our conditions of study, after about 17 days, this limitation seems acceptable.
Another limitation is that our experimental plan was aimed at studying the inactivation capacity of a process against a foodborne pathogen (ISO 20976-2: challenge tests to study inactivation potential and kinetic parameters; draft under development), without demonstrating the possibility that low contamination of the substrate could magnify itself in the larvae during the production period (ISO 20976-1:2019: challenge tests to study growth potential, lag time and maximum growth rate).
Despite this limitation, the results of this study show that the BSF can be reared on a substrate contaminated with Salmonella and L. monocytogenes, obtaining a reduction in the microbial load of both pathogens. However, the reduction observed was incomplete. The slow rate of pathogen decay, observed both in Salmonella and L. monocytogenes experiments, needs to be carefully evaluated. In fact, it could represent a source of concern regarding the potential presence of a subpopulation of more resistant (persistent) bacteria. Moreover, the reduction rate observed in both pathogens in comparison to larval developmental time (D of about 7 days, measured between 3- and 17-days Vs 20 days mean developmental time) will allow a maximum reduction of about two decimal points in the pathogen load during the farming time.
Erickson et al. [
24] evaluated the effects of BSF larvae on cow, hog, or chicken manure inoculated with 10
7 CFU/g of
Salmonella and
E. coli O157:H7. They obtained a greater reduction in the contamination of both pathogens than in the control inoculated without the larvae, but starting with larvae already grown (10–11 days old). Lalander et al. [
25] observed a strong reduction in the concentration of
Salmonella in organic waste (pig manure mixed with dog food) in a continuous flow fly larvae reactor, but did not evaluate the contamination of the larvae.
These literature results, even with the differences underlined, can be considered as in agreement with our findings for
Salmonella. Regarding
L. monocytogenes experiments, the microbial load from day 3 until the end of the experiment was different from that of
Salmonella.
L. monocyotogenes, which was significantly more concentrated, although the load of pathogens in the growth substrates on day 1 was not different, with one log difference on day 13 and about two log at the end of the experiments (
Table 5).
This difference in the pathogen dynamics may reflect the diversity of their bacterial cell walls and, in general, the intrinsic greater resistance to environmental stresses of
L. monocytogenes compared to
Salmonella. A study conducted by Choi et al. [
35] observed antimicrobial activity of larval extracts on Gram-negative bacteria, but no or very little on Gram-positive bacteria. These antimicrobial agents derived from the larvae may be among the substances that are produced in the larval body for their survival and, thus, they may have an antimicrobial action in the gut of larvae when pathogens are ingested.
On the other hand, the results reported by Park et al. [
24] seem to confirm that, when induced by a septic needle,
H. illucens larvae produce an immune response that has a greater antibacterial activity, even against Gram-positive bacteria, such as methicillin-resistant
Staphylococcus aureus. The larvae in our study came into contact with a high concentration of
L. monocytogenes after hatching. It is, thus, possible that a pathogen infection in the larvae may have actually stimulated an immune response against the pathogen with a similar mechanism.
The pathogens were present both in the substrate in which the larvae grew and in the control without larvae (
Table 2 and
Table 4). The effect on the substrate can always be discussed assuming a direct action of the larvae, as also previously reported [
25,
26]. However, it is worth noting that the decay of pathogens was significantly faster in the larvae than in the growth substrate. This is even more relevant considering a feed conversion ratio for the larvae of between 1.4 and 2.6, in relation to the quantity of proteins and fats contained in the diet of BSF larvae [
31]. In theory, the concentration should be approximately twice as high in the larvae as in the substrate, providing the larvae had a direct action on the decay of pathogens, but several, not completely known, factors may influence the contamination of larvae grown on a substrate contaminated by foodborne pathogens. On the whole, the results of this study show that larvae grown on a substrate contaminated by foodborne pathogens have a lower pathogen load than the substrate; the quantification of this difference allows one to predict the final contamination of larvae depending on the initial contamination of the substrate, therefore, suggesting the possibility of classifying the different growth substrates to be used to breed BSF larvae for safety implications [
36,
37], making this breeding more attractive to the market.
However, the pathogens also decayed in the control where no larvae were added. This decay is attributable to a decrease in water activity, which, after 10 days, fell below 0.95 and then fell below approximately 0.70 at the end of the experiment (
Table 6). This can lead to stress and, therefore, a reduction in the number of microorganisms for both pathogens. However, this reduction was slower than that observed in the larvae, both for
Salmonella and especially for
L. monocytogenes, which again, demonstrated a greater resistance to environmental stresses on this occasion.
The pH remained fairly constant around neutral values in the controls, while in the D diet, the action exerted by the larvae involved an increase of about 1 pH point compared to the G diet (
Table 6). It has been hypothesized that the antimicrobial compounds present in
H. illucens larvae are either less stable or less active at lower pH values [
38]. In our experiments, the differences in pH of the two tested diets did not seem to interfere with the ability of larvae to reduce the pathogen load. In fact, the diet was never found to be a significant parameter in the ANOVA test in the two challenge tests (
p> 0.05).
5. Conclusions
Insects in current European legislation fall within the category of “novel food ingredients”, and, therefore, for them to be placed on the market, microbiological food safety concerns need to be addressed. A study conducted in Belgium found that the microbiological load of untreated insects always exceeds the food safety criteria for fresh minced meat [
37] and blanching treatment manages to reduce this contamination in different ways between species and treatments (time–temperature dependent) without, however, eliminating it [
39,
40]. The average reduction obtained after blanching treatment in the total aerobic count was around 3 to 4 logCFU/g, depending on insect species, while the total microbial load of fresh insects was 7–8 log CFU/g [
31].
The results of our study confirm that BSF larvae can grow in contaminated substrates, thus, reducing
Salmonella and
L. monocytogenes contamination through their direct action on the growth substrate. This is particularly evident in comparison with controls, where
Salmonella and
L. monocytogenes contamination decreased at a significantly slower rate (
p < 0.01). As long as the contamination level is reasonably low, the BSF larvae could, thus, be bred on contaminated substrates. In any case, we confirmed that at the end of the farming period, when larvae grow on a highly contaminated substrate, the results were also contaminated. As reported in the study by Lalander et al., which focused exclusively on
Salmonella [
25], this underlines the importance of post-production treatment before their use as food or feed.
The feeding activity of larvae modified the physical (pH and aw) and microbiological characteristics of the substrate, and this modification could be influenced by the diet’s components. In the present study, during their developmental stage, the larvae reduced the concentration of Salmonella and L. monocytogenes faster than the natural decay of pathogens observed in the controls.
Considering these observations and the results of this study, a risk assessment study would be possible to evaluate different possible growth substrates at different levels of microbial contamination. The aim would be to calculate the probability that residual contamination by Salmonella and L. monocytogenes can remain in the larvae after farming and blanching treatment.
The antimicrobial properties of larvae are not yet completely understood; however, at least against Salmonella, our results confirm the interesting abilities of BSF larvae that could be exploited for food and feed production. If, following a rigorous risk assessment, also based on the results of this study, the possibility of using substrates potentially contaminated by Salmonella and/or Listeria monocytogenes should emerge, without this entailing unacceptable risks for the consumer (even clearly following post-production treatments aimed at further improving safety), this would make this breeding more attractive in a market that must increasingly turn towards a circular economy.