**Concetta Maria Messina 1, Raimondo Gaglio 2, Maria Morghese 1, Marco Tolone 2, Rosaria Arena 1, Giancarlo Moschetti 2, Andrea Santulli 1, Nicola Francesca 2,\* and Luca Settanni <sup>2</sup>**


Received: 23 July 2019; Accepted: 6 September 2019; Published: 9 September 2019

**Abstract:** Microbiological, nutritional and bioactive properties of edible powders obtained from *Acheta domesticus* (house cricket) and *Tenebrio molitor* (mealworm) were investigated. Except for the enterobacteria, viable bacteria were at a higher concentration in mealworm flour. The diversity evaluation carried out using MiSeq Illumina that mainly identified *Citrobacter* and *Enterobacteriaceae* in mealworm powder and members of the *Porphyromonadaceae* family in house cricket powder. Enterococci were identified and characterized for their safety characteristics in terms of the absence of antibiotic resistance and virulence. Both powders represent a good source of proteins and lipids. The fatty acid profile of mealworm powder was characterized by the predominance of the monounsaturated fatty acids and house cricket powder by saturated fatty acids. The enzymatic hydrolysis produced the best results in terms of percentage of degree of hydrolysis with the enzyme Alcalase, and these data were confirmed by SDS-PAGE electrophoresis. Furthermore, the results showed that the protein hydrolysate of these powders produces a significant antioxidant power.

**Keywords:** Alcalase; insect powders; *Acheta domesticus*; *Tenebrio molitor*; *Enterococcus*; antioxidant activity

#### **1. Introduction**

Insect consumption occurs almost worldwide, and this practice would represent a potential solution to food shortages and famine [1,2]. The nutritional relevance of insects is mostly represented by their high digestible protein content [3]. Compared to conventional livestock, their breeding systems are characterized by fewer environmental issues, including lower water consumption [4], greenhouse gases and ammonia generation [5].

Insect consumption in Western countries is still limited [6,7]. First of all, because of the unpleasant perception that the majority of consumers have towards such foods, which are not considered as conventional [8]. The consumption of edible insects is also hampered by regulations regarding hygiene and safety issues. Furthermore, religious concerns should also be considered in future. In areas of Asia, Africa and South America, where insects are eaten daily, they are commonly collected from natural environments [9]. Thus, the microbiological load implications of these foods might be relevant. Durst et al. [10] reported some cases of botulism and other foodborne illnesses due to the consumption of insects stored in Africa. The major risks derive from the ingestion of the gastrointestinal tract of the insects [11]. However, several commercial insect farms are keeping the growth of edible insects under controlled hygiene conditions.

InWestern countries, insects are often consumed as flour added to some traditional food ingredients in several formulations. Insect powders were mixed with maize flour to produce tortillas [12] and emulsion sausages [13]. Commonly used species is the mealworm *Tenebrio molitor* (*T. molitor*) [14], but house cricket *Acheta domesticus* (*A. domesticus*) also showed a high potential for the enrichment of food products, including fermented ones [11]. *T. molitor* and *A. domesticus* and their powders have been studied for microbiological aspects showing a consistent presence of *Enterobacteriaceae* family [11,14–17], techno-functionality, chemical and nutritional composition showing high quantity of crude proteins, micronutrients, B-group vitamins and low crude fat [14,18,19].

Currently, there is a growing interest in the applications of food proteins and peptides in the form of functional foods or nutraceuticals as alternatives ingredients to conventional treatments. Enzymatic modification of proteins is useful to improve functionality [20]. Some peptides obtained from dietary proteins using enzymatic hydrolysis have been demonstrated to be antioxidant, antimicrobial, antidiabetic, antihypertensive, antithrombotic and immunomodulating [21,22].

Edible insects are viable sources of bioactive peptides owing to their high protein content and sustainable production [20,23]. A multidisciplinary approach consisting of chemical/nutritional, biochemical and microbiological investigations has been applied to characterize mealworm and house cricket powders.

#### **2. Materials and Methods**

#### *2.1. Raw Materials and Microbiological Analyses*

The powders analyzed were prepared from *T. molitor* and *A. domesticus* insects (two samples for each species) and were provided by Kreca Ento-Food (Harderwijk, Gheldria, The Netherlands). As reported in labels, both insect powders are important sources of proteins, since they contain all the nine essential amino acids. Furthermore, both powders have a high digestibility, possess a low carbohydrate profile and are free of preservatives, antibiotics, hormones and pesticides. The powders were kept under refrigeration in the dark as suggested by the supplier. To evaluate the changes of the chemical/nutritional, biochemical and microbiological characteristics of mealworm and cricket powder during storage, the powders were analyzed after 12 months of refrigerated storage (4 ◦C).

The insect powders were subjected to decimal serial dilution in Ringer's solution (Sigma-Aldrich, Milan, Italy). The first dilution was obtained using a Stomacher (BagMixer® 400, Interscience, Saint Nom la Bretèche, Yvelines, France) at the highest speed (260 rpm) for 2 min. Cell suspensions were measured using plate count for the enumeration of the following microbial groups: total mesophilic microorganisms (TMM) on plate count agar (PCA) incubated aerobically at 30 ◦C for 72 h; mesophilic lactic acid bacteria (LAB) rods and cocci on de Man-Rogosa-Sharpe (MRS) and M17 agar, incubated anaerobically at 30 ◦C for 48 h; enterococci on kanamycin esculin azide (KAA) agar incubated aerobically at 37 ◦C for 24 h; members of the *Enterobacteriaceae* family on violet red bile glucose agar (VRGBA) incubated aerobically at 37 ◦C for 24 h; coagulase-positive staphylococci (CPS) on Baird-Parker (BP) agar supplemented with rabbit plasma fibrinogen (RPF), incubated aerobically at 37 ◦C for 48 h; pseudomonads on *Pseudomonas* agar base (PAB) supplemented with cephaloridine sodium fusidate cetrimide (CFC), incubated aerobically at 25 ◦C for 48 h; yeasts and moulds on malt agar (MA) with chloramphenicol (0.1 g/L), incubated aerobically for 48 h and 7 days, respectively, at 28 ◦C; spore-forming aerobic bacteria were investigated after heating of cell suspensions at 85 ◦C for 15 min and then spread plated on nutrient agar (NA) before aerobic incubation at 32 ◦C for 48 h. Anaerobiosis occurred in hermetically sealed jars with the AnaeroGen AN25 system (Oxoid, Milan, Italy). All media were purchased from Oxoid. Microbiological counts were carried out in triplicates.

#### *2.2. V3-V4 Amplification and Illumina Data Analysis*

To maximize the effective length of the MiSeq's 300PE sequencing reads, the region encompassing the V3 and V4 hypervariable regions of the 16S rRNA gene (approximately 469 bp) was targeted for sequencing. Genomic DNA was extracted from insect powder samples using a QIAamp DNA Mini Kit (Qiagen, Hilden, Düsseldorf, Germany) and diluted to 5 ng/μl in 10 mM Tris pH 8.5 as indicated using the Illumina protocol 16S Metagenomic Sequencing Library Preparation, 15044223 Rev. B. Briefly, to amplify and sequence the V3-V4 hypervariable region of the 16S rRNA gene, primers were designed that had overhang adapter sequences that must be appended to the primer pair sequences for compatibility using the Illumina index (San Diego, CA, USA) and sequencing adapters. The libraries were sequenced using the MiSeq Reagent Kit v3, 600 Cycles Sequencing kit (MS-102-3003) on the MiSeq System (Illumina).

Sequences obtained from Illumina Sequencing were processed using the QIIME2 software package version 2018.4 [24]. The reads were assigned to each sample according to the unique index; pairs of reads from the original DNA fragments were first merged using an import tool implemented in QIIME2. Quality check and trimming were done to trim sequences where the Phred quality score was < 20 using the DADA2 a R packages [25] wrapped in QIIME2. The Phred quality score is a measure of the quality of the identification of the nucleobases generated by automated DNA sequencing. Moreover, to remove chimeras from the Illumina sequenced FASTQ files the "consensus" method implemented in DADA2 was used. For taxa comparisons, we used the QIIME2 q2-feature-classifier plugin and the Naïve Bayes classifier that was trained on the Greengenes 13.8 database with a 99% Operational Taxonomic Units (OTUs) full-length sequences. QIIME2 taxa barplot command and ggplot2 were used for visualization of the taxonomic composition of the samples. Alpha diversity analysis was done with the q2-diversity plugin in QIIME2. In particular, Chao1 [26] metric that is a nonparametric abundance-based estimator of species richness and observed OTU were used to study diversity within each sample. Finally, to compare the relative abundance of microbial communities between the two samples, a Kruskal-Wallis test was done [27].

#### *2.3. Phenotypic and Genotypic Characterization of LAB*

Some colonies of presumptive LAB (Gram-positive, determined using the Gregersen KOH method [28], and catalase negative, determined by addition of fresh colonies from the agar media to 5%, *w*/*v*, H2O2) from the highest plated dilutions of the microbial cells on MRS, M17 and KAA agar were collected for all different morphologies recognized considering color, shape, edge, and surface (smooth or jagged). Gram-positive and catalase-negative cultures were purified through successive sub-culturing in the same media used for plate counts. All cultures were characterized for their cell morphology determined using an optical microscope at 100 × (Zeiss, Oberkochen, Stuttgart, Germany), growth at 15 and 45 ◦C, metabolism type, testing the ability to produce CO2 from glucose and growth in the presence of a mixture of pentose carbohydrates (xylose, arabinose and ribose; 8 g/L each) in place of glucose [29]. The coccus-shaped isolates were finally tested for their growth at pH 9.2 and in the presence of 6.5% (*w*/*v*) NaCl.

Genomic DNA from the PCR assay was prepared using the InstaGene Matrix kit (Bio-Rad, Hercules, CA, USA) as described by the manufacturer. Cells were harvested from insect flour isolated cultures grown overnight in MRS or M17 broths at 30 ◦C, and genomic DNAs were extracted using the Instagene Matrix kit (Bio-Rad), as described by the manufacturer. Crude cell extracts were used as templates for the polymerase chain reaction (PCR).

Strain differentiation was done using random amplification of polymorphic DNA (RAPD)-PCR analysis using the single primers M13, AB111, and AB106 as previously described by Gaglio et al. [30] using a T1 Thermocycler (Biometra, Göttingen, Germany) to generate amplicons. The software package Gelcompare II Version 6.5 (Applied Maths, Sint-Martens-Latem, East Flanders, Belgium) was used to analyze the LAB profiles.

Gene sequencing of 16S rRNA was using as reported by Weisburg et al. (1991) with the primers rD1 (5 -AAGGAGGTGATCCAGCC-3 ) and fD1 (5 -AGAGTTTGATCCTGGCTCAG-3 ) for LAB identification at the species level. DNA fragments of about 1600 bp were purified and sequenced at Eurofins Genomics (Milan, Italy). The sequences obtained were compared with those available in the EzTaxon-e database (http://eztaxon-e.ezbiocloud.net/) with the sequences of the type strains only and the GenBank/EMBL/DDBJ (http://www.ncbi.nlm.nih.gov). The unequivocal identification of the *Enterococcus faecium* was further verified using the sodA gene-based multiplex PCR described by Jackson et al. [31] using the primers FM1 (5 -GAAAAAACAATAGAAGAATTAT-3 ) and FM2 (5 -TGCTTTTTTGAATTCTTCTTTA-3 ).

PCR mixture (22.5 μL total volume) included 20 μL of master mix and 2.5 μL of whole-cell template. The PCR program applied for all primers comprised 30 cycles of denaturation for 4 min at 95 ◦C, annealing for 1 min at 55 ◦C, and elongation for 1 min at 72 ◦C. Amplification was followed by a final extension at 72 ◦C for 7 min. The amplifications were performed using a T1 Thermocycler.

(Biometra) and the amplicons were separated by electrophoresis on a 2% (*w*/*v*) agarose gel (Gibco BRL, Cergy Pontoise, Val-d'Oise, France), stained with SYBR® Safe DNA gel stain (Molecular Probes, Eugene, OR, USA), and subsequently visualized by UV transillumination.

#### *2.4. Safety Aspects of Dominant Insect Powder LAB*

The antimicrobial susceptibility of enterococci was evaluated through the standard disk diffusion method of Kirbye-Bauer according to the Clinical and Laboratory Standards Institute guidelines [32] on Mueller Hinton Agar (Oxoid) incubated at 37 ◦C for 18 h. The following antimicrobials were tested: penicillin—10 units, ampicillin—10 μg, vancomycin—30 μg, erythromycin—15 μg, tetracycline—30 μg, ciprofloxacin—5 μg, levofloxacin—5 μg, chloramphenicol—30 μg, quinupristin-dalfopristin—15 μg, linezolid—30 μg, high-level gentamicin—120 μg and high-level streptomycin—300 μg. All antimicrobial compounds are commonly used for the treatment of human and animal infections. *Enterococcus faecalis* ATCC 29212 was used as the quality control strain for performing antimicrobial testing. All antimicrobial compounds were purchased from Oxoid.

The phenotypic assay of gelatinase production by *Enterococcus* strains was done on a plate containing gelatin agar as described by [33]. The gelatinase production was classified as positive when a clear zone of hydrolysis was detected around the colonies. The production of haemolytic activity was determined by streaking the bacterial cultures onto Columbia blood agar supplemented with 5% (*v*/*v*) horse blood (Becton Dickinson, Franklin Lakes, NJ, USA). Plates were incubated at 37 ◦C for 24–48 h with anaerobic conditions, after which the plates were examined for haemolysis. The hemolytic reactions were classified as total or β-hemolysis (clear zone of hydrolysis around the colonies), partial or α-hemolysis (green halo around the colonies) and absent or γ-hemolysis.

#### *2.5. Proximate Composition*

The proximate composition was measured as follows: moisture and ash content using the AOAC method [34], total nitrogen using the Kjeldahl method [35]; crude protein (P) and chitin (Q) content were determined applying the following equation used by Díaz-Rojas et al. [36]:

$$P = ((\text{Nt} \times \text{Cq} + \text{K} - 100) \times \text{Cp}) (\text{Cq} - \text{Cp}) \tag{1}$$

$$\mathbf{Q} = ((\text{Nt} \times \text{Cp} + \text{K} - 100) \times \text{Cq}) / (\text{Cp} - \text{Cq}) \tag{2}$$

where Nt was the total nitrogen content. K was the sum of total lipid, moisture and ash. Cp and Cq were conversion coefficients that relate the mass fraction of nitrogen with protein and chitin. The protein content of different insect species in the literature is mainly based on nitrogen content using the nitrogen to protein conversion factor (Cp) of 6.25 [37,38] while the value of Cq is 14.5 [36].

The total fatty acid (FA) methyl esters were determined from the total lipid [37] of insect powders according to Lepage and Roy [38] and analyzed using the conditions described by Messina et al. [39] employing a Perkin Elmer (Waltham, MA, USA) autosystem XL instrument equipped using a silica capillary column (30 m × 0.32 mm, df 0.25 μm, Omegawax 320, Supelco, Bellefonte, PA, USA). Individual FAME were measured by comparison of known standards (mix of PUFA 1, PUFA 2 and PUFA 3 mixed oil, Supelco).

Caloric content was measured as total energy content (kcal/100 g) using an isoperibolic oxygen bomb calorimeter (model 6200, Parr Instrument Co., Moline, IL, USA).

#### *2.6. Enzymatic hydrolysis*

The samples were subjected to enzymatic hydrolysis in distilled water (1:1 *w*/*v*), using three different proteases (peptidases) (Protamex, Flavourzyme and Alcalase, Sigma-Aldrich). The hydrolysis reaction was performed according to Messina et al. [40] at 60 ◦C keeping the pH at 8.0 with the addition of NaOH 5M. These conditions are optimal for enzymatic activity [40]. The degree of hydrolysis (DH%) of each enzyme was determined directly every 15 min for 195 min, and applying the equation used by Dumay et al. [41]. The enzymatic activity was stopped at 90 ◦C for 5 min and the samples were centrifuged at 7142 g force for 15 min at 4 ◦C. The supernatants were lyophilized for further determinations and stored at 4 ◦C [42].

#### *2.7. Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)*

The hydrolysates were separated using SDS-PAGE (SDS-PAGE, Bio-Rad). The concentration of total proteins in all samples (powder and hydrolysates) done using the Lowry et al. [43] method, using BSA as the standard assuming it was 100% pure. Aliquots of 100 μg of protein, diluted with Laemmli buffer (1970) (Sigma-Aldrich) and denaturated for 5 min at 90 ◦C, were loaded on a gradient polyacrylamide minigel (4–15%) (Bio-Rad) and subjected to electrophoresis at 20 mA for about 2 h. A mix of standards proteins, having relative molecular mass varying between 250 and 14 kDa (Bio-Rad) was run simultaneously into the gel. After the electrophoretic run, the gel was stained with a reagent which uses the reference protocol of staining with Coomassie Blue (GelCode Blue Stain Reagent, Pierce, Rockford, IL, USA). The image of the gel was acquired and elaborated using the software Image Lab 4.1 (Bio-Rad).

#### *2.8. DPPH Radical Scavenging Activity*

The total antioxidant power of the hydrolysates obtained at the end of the enzymatic processes for both insect powders was measured using the DPPH assay [44]. DPPH (1.1-diphenyl-2-picryhydrazyl, Sigma-Aldrich) is a stable free radical widely used in the detection of scavenging activity of hydrolysates for screening antioxidant compounds. Different aliquots of the sample were taken and the volume was made to 1.0 mL with ethanol. The reaction was started by the addition of 1.0 mL of 200 μM DPPH solution in 96% ethanol. The reaction mixture was kept at ambient temperature (25 ◦C) for 30 min and the absorbance was measured at 517 nm. Gallic acid (Sigma-Aldrich) was used as a positive control. The scavenging activity was determined using the following equation by Manuguerra et al. [45].

$$\text{Scavenging activity (\%)} = \left[1 - \text{(Absorbance sample/Absorbance control)}\right] \times 100\tag{3}$$
