sustainability
Article
Black Soldier Fly Larvae Meal as Alternative to Fish Meal for
Aquaculture Feed
Marianna Oteri 1 , Ambra Rita Di Rosa 1 , Vittorio Lo Presti 1 , Filippo Giarratana 1 , Giovanni Toscano 2 and
Biagina Chiofalo 1, *
1
2
*
Citation: Oteri, M.; Di Rosa, A.R.; Lo
Presti, V.; Giarratana, F.; Toscano, G.;
Chiofalo, B. Black Soldier Fly Larvae
Meal as Alternative to Fish Meal for
Aquaculture Feed. Sustainability 2021,
13, 5447. https://doi.org/10.3390/
su13105447
Academic Editors: Massimo Lucarini
and Just Tomàs Bayle-Sempere
Department of Veterinary Sciences, University of Messina, 98168 Messina, Italy;
marianna.oteri@unime.it (M.O.); dirosaa@unime.it (A.R.D.R.); vittorio.lopresti@unime.it (V.L.P.);
filippo.giarratana@unime.it (F.G.)
Department of Chemical, Biological, Pharmaceutical and Environmental Sciences, University of Messina,
98166 Messina, Italy; giovanni.toscano@unime.it
Correspondence: biagina.chiofalo@unime.it; Tel.: +39-0906766833
Abstract: Hermetia illucens meal (HIM) as ingredient in feed represents a way to achieve more sustainable food production. The aim was to characterize the chemical, microbiological and organoleptic
characteristics of four diets for Sparus aurata, isoenergetic and isoproteic, containing 0%, 25%, 35% and
50% of HIM in substitution of fish meal (FM). Analyses were carried out using gas chromatography
for fatty acids and amino acids, ICP-OES for minerals and liquid chromatography for aflatoxins and
following International Organization for Standardization methods for microbial flora. E-sensing
analysis of the diets was evaluated using an artificial sensory platform (E-eye, E-nose and E-tongue).
The chemical results were submitted to a one-way ANOVA while Principal Component Analysis
(PCA) of the e-sensing data was performed. No significant differences were observed for polyunsaturated fatty acids, thrombogenic and peroxidation indices among the diets. The replacement
of FM with HIM increased the content of lysine, methionine, isoleucine, leucine, threonine and
valine, while phosphorus, calcium and sodium content decreased (p < 0.01) as the percentage of
HIM increased. Lead was significantly below the maximum level set by the EU regulation. The diets
showed good hygienic and sanitary quality. The artificial senses permitted distinguishing color, odor
and taste among the diets. Data allow considering Hermetia illucens as alternative protein source in
fish nutrition.
Keywords: Hermetia illucens; aquaculture feed; fatty acids; amino acids; minerals; microbiological
quality; e-sensing profile
Received: 2 April 2021
Accepted: 11 May 2021
Published: 13 May 2021
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1. Introduction
Among the costs of the aquaculture farms, feeding represents the largest portion
(about 60%) of the economic balance; therefore, the economic success of fish production
sector is mainly linked to the use of low-cost nutritionally balanced diets [1]. Therefore, the
goal of aquaculture nutritionists, as well as fish farmers, is to obtain a very good conversion
ratio to cover the cost of feeding. Proteins with a well-balanced presence of essential and
non-essential amino acids are the most important nutrients for the maintenance, growth
and feed efficiency of fish. They are the nutrients with the highest cost, and, therefore,
their inclusion in the fish feed plays an important role in the overall feed costs. Fish meal,
characterized by a high protein content, an excellent amino acid profile, a low carbohydrate
level and a high digestibility, is considered the most important feeding source in fish
nutrition [2]. However, the significant increase in the price of fish meal, the high protein
nutritional needs of fish and the alternating availability in fish meal supply, in recent
decades, have led to the study of alternative protein sources in aquafeeds [3].
In relation to the increasing demand for protein sources in animal feeding, there is
a great interest towards unconventional sources. In recent years, industrial by-products,
Sustainability 2021, 13, 5447. https://doi.org/10.3390/su13105447
https://www.mdpi.com/journal/sustainability
Sustainability 2021, 13, 5447
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co-products, insects, seaweed ingredients [4] and ex-food or former food products have
been investigated as alternative ingredients for livestock and aquaculture feeding.
Interest in insects as feed ingredient for terrestrial and aquatic animals continues
to grow [5–8]. At present, the exploiting of insects as feed ingredients is not in direct
competition with food production. The use of insect meal as a substitute of fish meal
seems to represent interesting perspectives for limiting the environmental impact of some
production system, such as aquaculture, and for contributing to a circular economy and a
“zero waste” society [9].
Among the insect species used as unconventional protein source for fish feeding,
Hermetia illucens L. is of the most interesting source for its sustainability related to its
capacity to convert organic waste material into biomass containing proteins (40–45%) with
high biological value [10], fat (30–35%) with fatty acids of nutritional interest and ash
(11–15%) with high mineral concentrations and a high Ca/P ratio [11]. In terms of protein
quality, Hermetia illucens larvae contain a favorable essential amino acid profile closer to
fish meal than that of soybean meal [5]. The mineral profile and fatty acid composition
of the Hermetia illucens larvae were found to be influenced by the diet [12–14]. Liland
et al. [14] reported that Hermetia illucens larvae are unable to synthesize polyunsaturated
fatty acids, and, therefore, the presence of linoleic acid and alpha-linolenic acids, as well
as of eicosapentaenoic and docosahexaenoic in the larvae most likely originates from the
substrate. Furthermore, these authors observed that the presence of seaweed in the feeding
media can enrich the larvae with macro- and microelements, making the insects a good
source of minerals [14].
Nevertheless, Weththasinghe et al. [15] observed a linear decrease in protein and
lipid digestibility, protein efficiency ratio and lipid retention in extruded diets for Atlantic
salmon as the level of dietary Hermetia illucens meal increases.
The use of Hermetia illucens larvae meal (HIM) as a component of feed is a way to
achieve more sustainable food production. According to current regulation in Europe [16],
the use of HIM is mainly permitted in aquaculture. The optimal level of dietary substitution of fish meal for HIM varies considerably across studies, ranging from 25% to 100%,
probably in relation to the different quality of larvae meal, fish species and diet formulation.
Furthermore, to obtain a higher-quality fish, it is important to provide good quality and
pathogen-free feeds to the fish [17]. In this view, the extrusion method destroys undesirable
microbial flora, enzymes and anti-nutritional factors [18–20] and improves the nutritional
value of final products and the apparent absorption of minerals [21,22].
Fish have strongly developed chemosensory and chemical signaling systems due to
their living in an aquatic environment. The olfactory and gustatory systems comprise the
main chemosensory pathways [23]. The combination of artificial senses (Electronic Nose,
an Electronic Tongue and an Electronic Eye) proves to be a powerful tool to distinguish
different organoleptic profiles related to the different chemical compositions of aquaculture
feeds [24].
To further characterize the feasibility of HIM as a unconventional protein source in
aquafeeds, the aim of this study was to characterize the chemical, microbiological and
mineral composition of diets for Spaurus aurata L. containing HIM as a partial replacement
for fish meal (FM). The effects of inclusion of HIM on the organoleptic characteristics of the
diets were also investigated with the aim of providing detailed sensory information on the
HIM-containing fish meal useful for feed industry.
2. Materials and Methods
Four experimental diets were formulated to satisfy the nutritional needs of Sparus
aurata. The diets were isoenergetic (about 22 MJ/kg gross energy), isonitrogenous (about
43 g/100 g, as fed) and isolipidic (about 19 g/100 g, as fed). A basal diet (HIM0) with fish
meal (FM), as exclusive protein source of animal origin, was prepared. FM was partially
replaced with defatted Hermetia illucens meal at 25%, 35% and 50% (as fed basis) in the
other three diets (HIM25%, HIM35%, HIM50%) corresponding to the inclusion levels of
Sustainability 2021, 13, 5447
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0%, 7.9%, 11% and 15.7% of HIM, respectively. The other components of the formulas were
adapted to obtain diets with the same energetic content.
Diets were prepared by SPAROS Lda (Olhao, Portugal); all dietary ingredients were
ground, mixed and extruded using die with 4 mm diameter; the oils were added using a
vacuum coating technology. The ingredients of the diets and the proximate composition of
the diets (HIM0, HIM25%, HIM35% and HIM50%) are reported in Table 1.
Table 1. Diet ingredients and proximate composition of the experimental diets.
Ingredients, % as fed
Fish meal
Hermetia illucens meal
Soy protein concentrate
Wheat gluten
Corn gluten
Soybean meal 48
Rapeseed meal
Wheat meal
Whole peas
Fish oil
Rapeseed oil
Vitamin and mineral premix
Vitamin C35
Vitamin E50
Antioxidant
Sodium propionate
MCP, monocalcium phosphate
L-Lysine
L-Tryptophan
DL-Methionine
L-Taurine
Chemical composition, % as fed
Dry matter
Crude protein
Crude fat
Crude fiber
Ash
NFE *
HIM0
HIM25%
HIM35%
HIM50%
25.00
0
5.00
5.00
5.00
15.00
5.00
17.45
4.00
5.00
10.00
1.00
0.03
0.02
0.30
0.10
1.50
0.30
0.10
0.20
18.75
7.90
5.00
5.00
5.00
15.00
5.00
15.17
4.00
5.00
9.80
1.00
0.03
0.02
0.30
0.10
2.20
0.35
0.03
0.15
0.20
16.25
11.00
5.00
5.00
5.00
15.00
5.00
14.21
4.00
5.00
9.80
1.00
0.03
0.02
0.30
0.10
2.50
0.37
0.04
0.18
0.20
12.50
15.70
5.00
5.00
5.00
15.00
5.00
12.88
4.00
5.00
9.80
1.00
0.03
0.02
0.30
0.10
2.80
0.40
0.05
0.22
0.20
92.33
42.7
18.6
2.3
9.3
19.43
92.78
42.7
18.6
2.2
9.3
19.98
92.90
42.7
18.6
2.2
9.4
20.00
92.64
42.7
18.7
2.1
9.3
19.84
HIM0, fish meal; HIM25%, HIM35% and HIM50%, Hermetia illucens meal at 25%, 35% and 50% substitution rate
of fish meal, respectively. * Nitrogen-free extract, NFE (%) = 100 − (%Crude Protein + %Crude fat + %Crude fiber
+ %Ash).
2.1. Fatty Acid Analysis
Triplicate feed samples were analyzed for the fatty acid composition. Each sample
(ca. 2.5 g) added with sodium sulfate (1 g) was pounded manually. The lipids were
extracted for 6 h with petroleum ether by a Soxtec™ 8000 Extraction system (FOSS, Padua,
Italy). The fatty acid methyl esters (FAMEs) were produced from aliquots of lipids. In
detail, sulfuric acid–methanol (1:9, v/v) reagent (2 mL) was added to the extracted lipid
samples, and they were then heated at 100 ◦ C for 1 h [25]. FAMEs were analyzed by a
Trace 1310 chromatograph (Thermo Fisher Scientific, Milan, Italy) equipped with a flame
ionization detector (FID) and a fused silica capillary column (30 m × 0.25 mm I.D., 0.25 µm
film thickness) (Omegawax 250; Supelco, Bellefonte, PA, USA) maintained at 100 ◦ C for
5 min, from 100 to 240 ◦ C at 4 ◦ C/min and final isotherm of 240 ◦ C (20 min). Injector and
detector temperatures were 250 ◦ C. Injection volume and split ratio were 0.5 µL and 1:50,
respectively. The carrier gas was helium at a flow rate of 1 mL/min. Data acquisition
was carried out by a Chromeleon Software (Thermo Fisher Scientific, Milan, Italy). The
identification of individual compounds was carried out by comparing their retention times
Sustainability 2021, 13, 5447
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with those of standards (mix 37 FAMEs, Supelco, Inc., Bellefonte, PA, USA). The results
were expressed as g/100 g of the total fatty acids identified.
Nutritional indices were calculated from the identified fatty acids, as proposed by
Ulbricht and Southgate [26], for atherogenic (AI) and thrombogenic (TI) indices, while the
equation proposed by Santos-Silva et al. [27] was used for the calculation of the hypocholesterolaemic/hypercholesterolaemic ratio (H/H). Indices were determined according
to the following formulas:
IA = [C12:0 + (4 × C14:0) + C16:0]/(Σn6-PUFA + Σn3-PUFA + ΣMUFA)
(1)
IT = (C14:0 + C16:0 + C18:0)/[(0.5 × ΣMUFA) + (0.5 × Σn6-PUFA) + (3 × Σn3-PUFA) + (Σn3-PUFA/Σn6-PUFA)] (2)
H/H = (C18:1n9 + C18:2n6 + C20:4n6 + C18:3n3 + C20:5n3 + C22:5n3 + C22:6n3)/(C14:0 + C16:0)]
(3)
Furthermore, the peroxidation index (PI), which expresses a measure of the peroxidation susceptibility and peroxidative lipid damage for a particular phospholipid membrane,
was calculated using the formula reported below [28]:
PI = (% dienoic × 1) + (% trienoic × 2) + (% tetraenoic × 3) + (% pentaenoic × 4) + (% hexaenoic × 5)
(4)
2.2. Amino Acid Analysis
Triplicate feed samples were analyzed for the amino acid composition. For the amino
acid analysis, protein hydrolysis and derivatization were performed prior to the separation
by GC-FID. Each sample (about 0.25 g) was hydrolyzed in 10 mL of a HCl solution (6 M) at
110 ◦ C for 24 h. During the acid hydrolysis, the asparagine and glutamine were converted
to aspartic and glutamic acids [29]; therefore, they were calculated as the sum of the aspartic
acid plus asparagine and of the glutamic acid plus glutamine. For cysteine analysis, prior
to the acid hydrolysis, a preliminary oxidation was performed for the deamination. Each
sample was treated with formic acid:hydrogen peroxide (1:20, v/v) reagent (2 mL) for
30 min at room temperature. Then, a hydrolysis step with a HCl solution (6 M) was
performed [30,31]. For the tryptophan analysis, each sample was hydrolyzed in 10 mL of a
NaOH solution (4 M) at 112 ◦ C for 16 h, and, after the hydrolysis, each sample was cooled
and neutralized with acetic acid [32]. For the chromatographic analysis, procedures for
purification, pre-column derivatization and qualitative and quantitative analyses of each
amino acid were performed using the EZ:Faast Kit (Phenomenex, Torrance, CA, USA). A
Trace 1310 chromatograph (Thermofisher, Waltham, MA, USA) was used, with a flame
ionization detector (FID) and a ZB-AAA Amino Acid column (10 m × 0.25 mm ID); the
oven temperature was programmed from 110 to 320 ◦ C at 32 ◦ C/min, with a final isotherm
of 320 ◦ C (1 min). Injector and detector temperatures were 250 and 320 ◦ C, respectively.
Injection volume and split ratio were 2.5 µL and 1:15, respectively.
2.3. Aflatoxin Analysis
The analysis of aflatoxins was carried out following the EN ISO method [33] and the
criteria suggested by European regulation [34]. For the extraction of the aflatoxins, each
sample (about 25 g) of the experimental diet was treated with 125 mL of a methanol:water
mixture (7:3, v/v) and 5 g of NaCl. The extract was filtered, diluted with water and
passed through an immunoaffinity column (Vicam) containing specific antibodies for
aflatoxins B1, B2, G1 and G2. The aflatoxins were isolated, purified and concentrated
on column and then recovered with methanol. The above procedure was performed in
triplicate on each experimental diet. Aflatoxins were measured by a RP-HPLC coupled
to a fluorescence detector (RF), using a post-column derivatization (Kobra cell system).
Chromatographic separation was performed using a Nexera LC System (Shimadzu, Milan,
Italy), equipped with a Luna column C18 250 × 4.6 mm (lenght × i.d.) and 5 µm of particle
size (Phenomenex, Torrance, CA, USA). The mobile phase for the isocratic separation was
Sustainability 2021, 13, 5447
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a mixture of water:acetonitrile:methanol (3:1:1, v/v/v) with 0.35 mL of nitric acid 4 M and
120 mg/L of potassium bromide. The RF wavelength was set at 365 nm for excitation and
435 nm for emission. The injection volume was 50 µL and the amount of aflatoxins was
calculated using an external standard method. The LC method for aflatoxin analyses has
been validated for the simultaneous chromatographic determination of total (B1, B2, G1
and G2) and B1 aflatoxins.
A calibration curve was constructed for each aflatoxin; the linearity was also tested in
the range of 0.05–22 µg/kg, providing a correlation coefficient (R2) of ≥0.9996. The Limit
Of Detection (LOD, between 0 and 0.05 µg/kg) and the Limit Of Quantification (LOQ,
between 0.05 and 0.13 µg/kg) were calculated by the signal-to-noise (S/N) ratio, which
should be greater than 3 and 10, respectively, according to the IUPAC criteria.
2.4. Mineral Element Analysis
Triplicate feed samples were analyzed for the mineral composition. Each sample was
weighed (about 0.5 g) into an acid-prewashed PTFE vessels and 7 mL of HNO3 at 65% and
a Rhenium internal standard (1 mL) were added. The mixture was digested with 1 mL
of H2 O2 at 30% using a closed-vessel microwave digestion system (Ethos 1, Milestone,
Bergamo, Italy). To validate the analytical method, a Standard Reference Material of
spinach leaves (SRM, NIST-1570a) obtained from the National Institute of Standards and
Technology (Gaithersburg, MD, USA), was digested using the same analytical method
described for the feed samples. An Avio200 ICP-OES instrument (Perkin Elmer, Waltham,
MA, USA) equipped with a vertical DualView optical system and a S10 autosampler (Perkin
Elmer, Waltham, MA, USA) was used to analyze the mineral content. Table 2 shows the
recommended analytical lines length used to perform element analyses; the Argon line at
420.069 nm was used as an internal standard. The applied operational conditions are listed
in Table 3. Data were processed using a PerkinElmer Syngistix™ for ICP software (Perkin
Elmer, Waltham, MA, USA).
Table 2. Analytical lines length (nm) utilized for analysis.
Element
nm
Element
nm
Element
nm
Cr
Cu
Fe
B
267.716
327.393
238.204
249.677
K
Mg
Mn
Ca
766.490
285.592
257.610
317.933
Se
Zn
Na
Pb
196.026
213.857
589.592
220.353
Table 3. Operational conditions of the ICP-OES.
Parameter
Radiofrequency power (W)
Plasma gas flow (L/min)
Auxiliary gas (L/min)
Nebulizer gas (L/min)
Sample uptake (mL/min)
Conditions
1500
9
0.2
0.7
1
The position of the torch was optimized prior to the analytical phase using the optical
optimization procedure of Syngistix™ ICP software with Mn analytical line. All the
quantitative measurements were made against external calibration curves constructed from
a standard solution of 0.05, 0.25 and 1 ppm of Perkin Elmer (Waltham, MA, USA) for ICP
analysis. A Milli-Q ultrapure (Merck Millipore, Merck KGaA, Darmstadt, Germany) water
system was used to produce water at 1.8 MΩ/cm for the preparation of solutions and to
dilute samples as needed. The calibration curves for all elements were established using the
calibration blank and the reagent blank, and all of them resulted with correlation coefficients
(r2 ) better than 0.999; the Detection Limits (DLs) of this procedure were determined by
Sustainability 2021, 13, 5447
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analyzing a matrix blank, which consisted of the same reagents and quantities as those
used for sample preparation.
2.5. Microbiological Analysis
Twenty-five grams of each experimental diet were homogenized with buffered peptone water (Biolife, Milano, Italy) (ratio of 1:9 w/v) by using a stomacher (400 Circulator;
International PBI s.p.a., Milano, Italy) for 60 s at 230 rpm. For each sample, the following
parameters were evaluated: (i) enumeration of the aerobic colony at 30 ◦ C [35] on Tryptic
Glucose Yeast Agar (Biolife, Milano, Italy) plates, incubated at 30 ± 1 ◦ C for 72 h; (ii) Enterobacteriaceae detection [36] and count [37] on Violet Red Bile Glucose Agar (Biolife, Milano,
Italy), incubated at 37 ± 1 ◦ C for 24 h; (iii) enumeration of coliforms [38] on Violet Red
Bile Agar (Biolife, Milano, Italy) plates, incubated at 30 ± 1 ◦ C for 24 h; (iv) yeasts and
moulds count [39] on Dichloran Glycerol Agar (DG18 Biolife, Milano, Italy), incubated
at 25 ± 1 ◦ C for 5 days; (v) detection and enumeration of Clostridium spp. [40] on Tryptose Sulfite Cycloserine Agar (Biolife, Milano, Italy), incubated at 37 ± 1 ◦ C for 24 h in
anaerobic conditions; and (vi) detection of Salmonella spp. [41] on Chromogenic Salmonella
Agar (Biolife, Milano, Italy) and Xylose Lysine Deoxycholate Agar (Biolife, Milano, Italy)
incubated both at 37 ± 1 ◦ C for 24 h. The limit of detection (LOD) was 10 CFU/g for
the count of aerobic colonies at 30 ◦ C, Enterobacteriaceae, coliforms, Clostridium spp. and
100 CFU/g for the count of yeasts and molds. Further 25 g of each experimental diet, as
previously reported, were homogenized with Listeria Fraser Broth Half Concentration
(Biolife, Milano, Italy) for the detection of the Listeria monocytogenes [42], incubated at
30 ± 1 ◦ C for 20 h, followed by a passage in Listeria Fraser Broth (Biolife, Milano, Italy)
at 37 ± 1 ◦ C for 24 h and spread both on Agar Listeria according to Ottaviani & Agosti
(ALOA® ) (Biolife, Milano, Italy) and Listeria Palcam Agar (Biolife, Milano, Italy) both
incubated at 37 ± 1 ◦ C for 24–48 h.
2.6. E-Sensing Analysis
The feed samples were analyzed using an artificial sensory platform consisting of an
E-eye, E-nose and E-tongue.
E-eye: The image was acquired with an artificial vision system (Iris visual analyzer
400, Alpha MOS, Toulouse, France) equipped with a high-resolution charge-coupled device
(CCD) camera with 16 million colors. Image acquisition was performed using a top
illumination and a white tray at the bottom to easily remove the background contribution
(threshold selection applied: R 0-145, G 0-121, B 0-109). Each sample was ground, and the
powder was placed and flattened on a plastic Petri dish (diameter 92 mm, height 7 mm).
For each sample, 15 images on 15 freshly prepared dishes were acquired. Color spectra
were calculated by selecting only the contributions greater than 1%.
E-nose: Odor analysis was performed by an electronic nose device (FOX 4000, Alpha
MOS, Toulouse, France) equipped with 18 MOS (metal-oxide semiconductor) gas sensors
and an automatic headspace sampler (HS100). For each sample, 15 replicates were prepared
by placing 2 g of freshly ground fish feed into 10 mL headspace sealed vials. All parameters
of the instrument are reported in Table 4.
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Table 4. E-nose parameters.
Acquisition
Oven
Duration 120 s
Period 1 s
Time 1080 s
Flow of the carrier gas 150 mL/min
Agitator
Speed 500 rpm
On 5 s
Of 2
Incubation time 600 s
Incubation temperature 40 ◦ C
Syringe
Flushing time 120 s
Temperature 50 ◦ C
Fill speed 500 µL/s
Injection
Volume 500 µL
Speed 500 µL/s
E-tongue: Artificial taste analysis was performed using a commercially available
electronic tongue (Astree, Alpha MOS, Toulouse, France) equipped with a set of seven
potentiometric sensors (ANS, PKS, CTS, NMS, CPS, ANS and SCS), an Ag/AgCl reference
electrode (Metrohm, Pte Ltd., Singapore), a mechanical stirrer and a 48-position autosampler. Five grams of each sample were ground and placed in 50 mL of deionized water for
15 min and centrifuged at 3000 rpm for 30 min. The solution was filtered and placed in
a 25 mL beaker for the analysis. Single sample analysis was repeated 30 times to obtain
the most stable sensor response and the last 15 measurements were considered to perform
data processing. The signal was acquired every second for 120 s and the average intensity
of the last 20 s was measured. Prior to measurement, the sensors were conditioned using
one of the samples as a standard.
2.7. Statistical Analysis
The chemical data were analyzed by a one-way ANOVA, using the XLSTAT statistical
package [43]. The percentage integration of insect meal (HIM0, HIM25%, HIM35% and
HIM50%) was used as a fixed effect. Separation of means was assessed by Tukey’s test, and
differences were significant if p < 0.05.
A Principal Component Analysis (PCA) of the sensory profile data was performed
by Alpha Soft V12.4 (Alpha-MOS, Toulouse, France) to evaluate the discrimination ability
between the four experimental diets. The effectiveness of discrimination was assessed by
evaluating the discrimination index (DI), which gives the quality of discrimination through
an indication of the surface between groups. The DI is calculated automatically by the
instrument’s software according to the following formula:
DI = 100 × [1 − [(Surface (A) + Surface (B) + Surface (C))/(Total Surface)]]
(5)
The DI reaches a maximum value of 100 when the groups are completely resolved.
3. Results
3.1. Fatty Acid Profile
The fatty acid profile of the four experimental diets is shown in Table 5. Saturated fatty
acids did not show any significant (p > 0.05) differences, with the exception of the lauric acid
(C12:0), which showed significantly (p < 0.01) higher values in the HIM35% and HIM50%
diets than those observed in the HIM0 and HIM25% diets and for the palmitic acid (C16:0)
which showed a significant (p < 0.05) higher value in the HIM0 diet than those observed
in the diets containing Hermetia illucens meal. Monounsaturated fatty acids show similar
content among the experimental diets as well as the polyunsaturated fatty acids, of both
the omega 3 and omega 6 series. Table 6 shows the fatty acid classes; the sum of saturated,
monounsaturated and polyunsaturated fatty acids; and some indices of nutritional interest,
namely the atherogenic (AI) and thrombogenic (TI) indices, the peroxidation index (PI) and
the hypocholesterolaemic/hypercholesterolaemic ratio. No significant (p > 0.05) difference
was observed among the fatty acid classes, with the exception of the saturated fatty
acids which show the highest (p < 0.05) level in the control diet (HIM0). The sum of
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the eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids was similar among the
diets. Similar values were recorded for AI, TI and PI. The H/H ratio showed significantly
(p < 0.05) higher values in the HIM35% and HI50% diets than that recorded in the HIM0
diet while the HIM25% diet showed a value of the H/H ratio similar to those of the
other diets.
Table 5. Fatty acid composition (g/100 g of fatty acid methyl esters) # of the experimental diets.
Fatty Acid
HIM0
HIM25%
HIM35%
HIM50%
SEM
p
C10:0
C12:0
C13:0
C14:0
C15:0
C16:0
C16:1
C17:0
C18:0
C18:1n9
C18:1n7
C18:2 n6
C18:3n6
C18:3n3
C20:0
C20:1n9
C20:2n6
C20:3n3
C20:4n6
C20:5n3
C22:0
C22:1n9
C22:2n6
C23:0
C24:0
C22:6n3
0.04
1.12 B
0.02
2.67
0.23
12.04 a
3.10
0.20
2.72
43.49
3.21
14.34
0.11
4.25
0.47
2.12
0.11
0.30
0.04
4.61
0.25
0.34
0.02
0.22
0.59
3.46
0.03
0.52 C
0.02
2.57
0.22
11.43 ab
3.01
0.20
2.79
44.69
3.19
14.28
0.11
4.36
0.44
2.25
0.11
0.29
0.06
4.56
0.23
0.33
0.02
0.21
0.60
3.54
0.05
1.67 A
0.01
2.55
0.20
11.19 b
2.87
0.19
2.64
43.26
3.10
14.92
0.10
4.45
0.43
1.88
0.10
0.30
0.05
4.92
0.27
0.30
0.03
0.25
0.62
3.69
0.05
1.34 AB
0.01
2.54
0.21
11.17 b
2.99
0.19
2.54
43.55
3.11
14.79
0.10
4.50
0.42
2.05
0.10
0.30
0.05
4.92
0.23
0.27
0.02
0.24
0.61
3.73
0.008
0.080
0.004
0.065
0.009
0.106
0.121
0.008
0.100
0.328
0.041
0.196
0.004
0.088
0.022
0.150
0.004
0.012
0.004
0.246
0.017
0.037
0.013
0.015
0.024
0.228
0.391
0.002
0.615
0.552
0.319
0.013
0.629
0.615
0.445
0.116
0.263
0.184
0.410
0.318
0.465
0.469
0.615
0.943
0.138
0.635
0.398
0.609
0.856
0.384
0.887
0.828
HIM0, fish meal; HIM25%, HIM35% and HIM50%, Hermetia illucens meal at 25%, 35% and 50% substitution rate
of fish meal, respectively. # The concentration of fatty acid is expressed as g/100 g, considering 100 g the sum of
the areas of all FAME identified. Mean values with different letters within the same row are significantly different,
A–C at p < 0.01 and a and b at p < 0.05.
Table 6. Fatty acid classes, nutritional indices and in the experimental diets.
SFA
MUFA
PUFA
n3-PUFA
n6-PUFA
EPA + DHA
AI
TI
PI
H/H
HIM0
HIM25%
HIM35%
HIM50%
SEM
p
20.54 a
52.25
27.21
12.61
14.60
8.07
0.30 a
0.24
59.50
4.77 b
19.23 b
53.46
27.32
12.75
14.57
8.10
0.28 b
0.23
60.00
5.10 ab
20.06 ab
51.40
28.54
13.35
15.19
8.61
0.29 ab
0.22
62.90
5.19 a
19.53 ab
51.97
28.51
13.45
15.06
8.65
0.29 ab
0.22
63.10
5.21 a
0.192
0.549
0.667
0.562
0.194
0.470
0.004
0.007
2.376
0.071
0.030
0.198
0.420
0.671
0.185
0.736
0.032
0.232
0.633
0.035
HIM0, fish meal; HIM25%, HIM35% and HIM50%, Hermetia illucens meal at 25%, 35% and 50% substitution
rate of fish meal, respectively; SFA, saturated fatty acid class; MUFA, monounsaturated fatty acid class; PUFA,
polyunsaturated fatty acid class; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid; AI, atherogenic Index;
TI, thrombogenic Index; PI, peroxidation Index; H/H, hypocholesterolaemic/hypercholesterolaemic ratio. Mean
values with different letters a and b within the same row are significantly different at p < 0.05.
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3.2. Amino Acid Profile
Table 7 reports the amino acid composition of the experimental diets. Twenty amino
acids were identified and quantified; ten of these belong to the indispensable amino
acids and ten to dispensable ones. Among the indispensable amino acids, six amino
acids (isoleucine, leucine, lysine, methionine, threonine and valine) showed significantly
(p < 0.01) higher values in the diets containing insect meal, while histidine showed significantly (p < 0.01) higher values in the HIM35% and HIM50% diets than those observed
in the HIM0 and HIM25% diets. Arginine, phenylalanine and tryptophan showed the
highest (p < 0.01) values in the control diet (HIM0). Among the dispensable amino acids,
hydroxylisine, hydroxyproline and tyrosine showed similar values among the experimental
diets. Glutamic acid plus glutamine showed significantly (p < 0.01) higher values in all
diets containing the Hermetia illucens meal, while proline and serine showed significantly
(p < 0.01) higher values in the diets in which FM has been replaced with HIM at 35% and
50%. The levels of aspartic acid plus asparagine and that of glycine were significantly
(p < 0.01) higher in the HIM25% and HIM35% diets than those observed in the HIM0 and
HIM50% diets. Cysteine showed the highest (p < 0.05) value in the HIM25% diet and
alanine the highest (p < 0.01) level in the HIM0 diet.
Table 7. Amino acid composition (g/100 g dry matter) of the experimental diets.
Indispensable amino acids
Arginine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Valine
Tryptophan
Dispensable amino acids
Hydroxylysine
Alanine
Aspartic acid + Asparagine
Cysteine
Glycine
Glutamic acid + Glutamine
Proline
Hydroxyproline
Tyrosine
Serine
HIM0
HIM25%
HIM35%
HIM50%
SEM
p
2.84 A
1.10 B
1.96 B
3.40 bB
4.66 B
0.80 B
3.06 A
1.66 B
1.79 B
0.12 A
2.33 aB
1.40 C
2.42 A
4.13 aA
5.51 A
0.94 A
2.31 C
2.09 A
2.35 A
0.05 B
2.15 bB
1.60 A
2.52 A
4.22 aA
5.59 A
0.94 A
2.84 B
2.13 A
2.40 A
0.05 B
2.21 abB
1.65 A
2.38 A
4.04 bA
5.53 A
0.94 A
2.87 B
1.96 A
2.26 A
0.05 B
0.026
0.023
0.028
0.024
0.084
0.013
0.005
0.038
0.025
0.002
<0.001
<0.001
0.001
<0.0001
0.004
0.003
<0.0001
0.003
<0.001
<0.0001
0.21
1.48 C
2.37 C
0.21 b
1.76 C
1.38 B
1.88 C
0.37
1.22
2.49 B
0.24
2.04 A
3.30 A
0.39 a
2.30 A
1.64 A
2.44 B
0.66
1.39
2.65 B
0.26
2.02 A
3.16 A
0.19 b
2.26 A
1.72 A
2.76 A
0.65
1.54
2.95 A
0.23
1.81 B
2.85 B
0.17 b
2.04 B
1.74 A
2.82 A
0.60
1.57
2.82 A
0.013
0.013
0.037
0.018
0.023
0.023
0.029
0.055
0.071
0.028
0.149
<0.0001
<0.001
0.006
<0.001
0.001
<0.0001
0.059
0.075
0.001
HIM0, fish meal; HIM25%, HIM35% and HIM50%, Hermetia illucens meal at 25%, 35% and 50% substitution rate
of fish meal, respectively. Mean values with different letters within the same row are significantly different, A–C
at p < 0.01 and a and b at p < 0.05.
3.3. Mineral Element Profile
In Table 8, the average values of minerals in the experimental diets are reported as:
macroelements (phosphorus, calcium, potassium, magnesium and sodium), whose needs
by the body are in large amounts, microelements (copper, zinc, manganese, iron, etc.),
whose needs by the body are in small amounts [44] and toxic metal (lead). The amount
of phosphorus, calcium and sodium decreased significantly (p < 0.01) by increasing the
amount of HIM to replace fish meal. The presence of insect meal over 25% as a substitute
for fish meal (HIM35% and HIM50%) resulted in significantly (p < 0.01) lower levels of
potassium, although the HIM35% and HIM50% diets showed similar (p > 0.05) values
between them. The Ca/P ratio in the four experimental diets was calculated due to the
antagonist effect of these two macroelements [45]. Significantly (p < 0.01) higher levels for
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the Ca/P ratio were observed in the HIM0 and HIM25% diets than those of the HIM25%
and HIM50% diets. Magnesium was significantly (p < 0.01) higher in the HIM25% diet than
in HIM0, HIM35% and HIM50% diets, while these diets did not show (p > 0.05) differences
among them. Iron, copper and zinc showed significantly (p < 0.01) higher levels in the
HIM35% diet than those of the HIM0, HIM25% and HIM50% diets, except the HIM25%
diet, which showed a similar content to the HIM50% diet. A significantly (p < 0.01) higher
level of manganese was observed in the HIM25% diet than those of the HIM0, HIM35%
and HIM50% diets. The increase in the level of HIM to replace fish meal resulted in a
significant (p < 0.05) increase in the amount of chromium. Boron was significantly (p < 0.01)
higher in the HIM25% and HIM35% diets compared to the other diets; lead showed the
highest (p < 0.01) value in the HIM50% diet (Table 8).
Table 8. Mineral element profile (mg/kg dry matter) of the experimental diets.
Items
Macroelements
P—Phosphorus
Ca—Calcium
K—Potassium
Mg—Magnesium
Na—Sodium
Ca/P ratio
Microelements
Fe—Iron
Zn—Zinc
Mn—Manganese
Cu—Copper
Se—Selenium
Cr—Chromium
B—Boron
Toxic metal
Pb—Lead
HIM0
HIM25%
HIM35%
HIM50%
SEM
p
11,452.63 A
19,560.64 A
11,632.62 A
2045.39 B
8081.56 A
1.71 A
10,966.03 B
18,673.81 B
11,832.99 A
7600.68 A
7575.17 B
1.70 A
10,477.43 C
16,710.51 C
10,553.62 B
2041.30 B
5664.86 C
1.60 B
9557.83 D
15,064.18 D
10,262.06 B
1990.07 B
4675.18 D
1.57 B
10.30
172.16
136.03
78.62
90.82
0.017
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.001
219.84 C
175.25 C
7.13 D
14.08 C
3.90 B
4.43 b
6.49 B
232.19 B
184.76 B
10.48 A
15.20 BC
4.63 A
5.44 ab
7.82 A
250.24 A
192.10 A
9.86 C
16.56 A
3.26 B
5.62 ab
7.72 A
229.66 BC
184.47 B
10.14 D
16.03 AB
2.84 B
6.03 a
6.60 B
2.38
1.18
0.062
0.27
0.35
0.28
0.18
<0.001
<0.0001
<0.0001
0.001
<0.001
0.019
0.001
0.76 B
0.97 B
0.78 B
1.83 A
0.44
0.001
HIM0, fish meal; HIM25%, HIM35% and HIM50%, Hermetia illucens meal at 25%, 35% and 50% substitution rate
of fish meal, respectively. Mean values with different letters within the same row are significantly different, A–D
at p < 0.01 and a and b at p < 0.05.
3.4. Mycotoxin Profile
The amounts of total aflatoxins (B1, B2, G1 and G2) and aflatoxin B1 in the four
experimental diets (HIM0, HIM25%, HIM35%, HIM50%) were below the limit of detection
(LOD: 0.05 µg/kg) in all experimental diets.
3.5. Microbiological Profile
No significant difference on the load of each microbiological parameter was observed in the experimental diets. For all diets, only a few aerobic colonies with a load
below 70 CFU/g (HIM0 = 65 CFU/g, HIM25% = 70 CFU/g; HIM35% = 50 CFU/g;
HIM50% = 60 CFU/g) was observed. The counts of Enterobacteriaceae, coliforms, Clostridium spp., yeasts and molds were always under the LOD. Enterobacteriaceae, Salmonella spp.,
L. monocytogenes and Clostridium spp. were not detected.
3.6. E-Sensing Profile
Concerning data provided by the artificial sensory platform, the first step was to
separately perform PCAs on the data from E-nose and E-tongue sensors and on the colors
of the E-eye code. The next step was to look for the most effective way to combine the
data provided by the E-eye, E-nose and E-togue in order to improve the discrimination
capability. An intermediate fusion level was adopted in this study. The sensor data with
the highest discrimination power were chosen; in particular, the data of four E-nose sensors
(LY2/G, LY2/AA, P30/1 and T40/1), three E-tongue sensors (AHS, CTS and NMS) and
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four colors extracted from the E-eye (codes 1620, 1890, 1891 and 2147) were chosen. These
datasets were reduced, due to the different data size, and a new PCA was performed and
the Discrimination Index (DI) calculated. The result is shown in Figure 1.
Figure 1. Principal component analysis map for fish feed groups (HIM0, blue; HIM25%, red; HIM35%, green; HIM50%,
violet) and loading vectors of selected variables. At the top DI = 93.
The first two principal components calculated (PC1 and PC2) explain 99.4% of the
total variance and show a DI of 93, highlighting a clear difference among the four diets, in
relation to the substitution of fish meal with HIM. The combination of sensor responses
with the highest discrimination power of the three artificial senses completely separates the
four feed groups. Moreover, the loading plot helps identify the variables accountable for
clustering of our dataset, by providing a numerical value which represents the contribution
of each original variable to the score plot. Along the PC1 axis, from left to right, the
groups are separated mainly by color codes 1890, 1891 and 2147, which are the most
distinctive for the HIM0 group. On PC1, the volatile component of the different groups is
also distinguished. Figure 1 shows the contribution of LY2/G, LY2/AA, P301 and T401.
These sensors show a clear separation between HIM35% and HIM50% diets and the HIM0
and HIM25% diets. Regarding the taste profile, the three selected sensors (AHS, CTS and
NMS) help to improve discrimination between groups. In particular, the AHS and CTS
sensors have an important contribution on the HIM0 and HIM25% diets, while the NMS
sensor mainly distinguishes the groups with higher insect meal content (HIM35% and
HIM50%).
4. Discussion
Regarding the origin of fatty acids in the larvae, some studies report the possibility
of modifying the fatty acid profile through the diet, while others the possibility of an
endogenous synthesis of fatty acids. Knowledge of the fatty acid pathway is of great
importance if HIM is used as an ingredient in animal feed. Our results are similar to the
fatty acid profile determined by Belghit et al. [9] in four experimental diets formulated
for Atlantic salmon with an increasing substitution of fish meal with HIM. St-Hilaire
et al. [13] observed a reduction of alpha-linolenic acid (C18:3n3), eicosapentaenoic acid
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(C20:5n3) and docosahexaenoic acid (C22:6n3) in fish fed a diet containing HIM. This
could represent a problem for producers and consumers of the seafood supply chain [46].
However, our results show similar content of the alpha-linolenic acid, eicosapentaenoic
acid and docosahexaenoic acid among the diets, despite the increasing addition of HIM
in the feed. The content of these fatty acids, essential for the growth and development in
fish [47] and associated with disease prevention and health promotion for humans, thanks
to the production of anti-inflammatory eicosanoids [48], could be associated with the use,
in feeding of the larvae, of a substrate containing fish offal and algae [12,14], since Hermetia
illucens is not able to synthesize PUFA [14]. In fact, only plants and marine algae possess
the enzymes necessary for the synthesis of linoleic acid (C18: 2n6) and alpha-linolenic
acid (the precursor of EPA and DHA) [49]; therefore, similar concentrations of n6-PUFA
(mainly linoleic acid) and n3-PUFA among the four diets confirm our hypothesis on the
influence of the feeding substrate of Hermetia illucens larvae [46]. Another interesting
result concerned the similar values recorded for the health lipid indices (atherogenic
and thrombogenic indices), the peroxidation index and the H/H ratio. The atherogenic
and thrombogenic indices (AI and TI) take into account, in the formulas used for their
calculation, the contribution that each fatty acid has on human health and, in particular, on
the probability of influencing the incidence of cardiovascular diseases [26]. The decrease in
PI, e.g., during feed storage, indicates the oxidative degradation of PUFA in primary and
secondary oxidation products. This process results in a loss of shelf-life, nutritional value
and feed safety, as well as reduced consumer acceptability [50]. Finally, the low H/H ratio
observed in diets containing HIM suggests some positive nutritional effects on animals.
As regards the indispensable amino acids, the substitution of fish meal with Hermetia
illucens increased the lysine (EAA) and methionine content in all the levels of inclusion
(HIM25%, HIM35% and HIM50%). In fact, Hermetia illucens meal has high levels of amino
acids such as methionine and lysine [30]. Furthermore, isoleucine, leucine, threonine and
valine showed higher levels in all experimental diets containing Hermetia illucens meal; this
fact is nutritionally interesting, as fish cannot synthesize these amino acids de novo [51].
Among the dispensable amino acids (NEAA), the inclusion of HIM at 35% and 50% levels
resulted in an increase in proline and tyrosine (NEAA), as observed by De Marco et al. [30]
and by Iaconisi et al. [51].
Minerals are present in low quantities in the feed despite having important metabolic
roles [22,44]. Mineral elements in fish are involved in many biochemical processes as
enzyme cofactors and activators, in the formation of skeletal muscles, in the transmission of
nerve impulses and in the acid–base chemical balance [44,52,53]. The low levels of calcium,
potassium and sodium observed in the formulation containing Hermetia illucens meal do
not represent a problem in the feeding of fish that can readily obtain these minerals from
the surrounding water environment [44,53]. On the contrary, the phosphorus requirement
must be satisfied through the diet as the fish cannot readily obtain it from the surrounding
aquatic environment, which contains a low amount of it [54,55]. Phosphorus and calcium
are generally combined together in the fish body, so an adequate Ca/P ratio in the diet is
crucial to ensure healthy bone formation and growth performance of fish [45,52]. In relation
to their antagonistic effect [56], the dietary Ca/P ratio has to be taken into consideration,
as an excess of Ca or P can cause mineral imbalances that can affect the absorption of
other minerals (e.g., an excess of Ca into the diet can reduce the absorption of Zn, Fe and
Mn), causing some important consequences for bone development, which can be adversely
affected when this ratio increases, causing anomalies in mineral homeostasis and bone
mass [57] or environmental contamination (excess P is excreted). In our study, the Ca/P
ratio of all diets was close to 1, as recommended for fish [58,59].
As for the microelements, such as zinc, essential for the growth and development in
fish, cofactor of many enzymes and necessary for the activity of the antioxidant enzyme
superoxide dismutase [44,60]; iron, necessary for the blood and muscle pigments [44] and
actively involved in oxidation-reduction reactions [60]; and copper, involved in enzymatic
activities and oxygen transportation [60], the highest levels were observed in the diet
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containing a quote of 35% of defatted Hermetia illucens meal. This result would seem
to highlight that the HIM35% diet is the best formula from a nutritional point of view,
considering also that fish cannot obtain adequate quantities of these microelements from
water [59,61] and that they must receive them through the diet [55].
The marine environment can convey toxic metals, naturally contained or introduced
by various human activities through seafood [62]. Fish, both freshwater and marine water,
that are at the top of the food chain, are extremely sensitive to exposure to lead (Pb),
which is a highly toxic metal [63]. Its toxicity depends on various factors such diet and
environment [63]. It seems that Sparus aurata is much more sensitive to waterborne lead
exposure than in the diet [64]. However, considering the toxicity of lead, the European
regulation [65] has set a maximum level of lead for complete animal feed of 5 ppm, which
is considerably higher than the values found in our experimental diets.
Fish feeds become rapidly colonized by environmental microbes. Furthermore, all
insects are colonized by microorganism and the insect microbiota is generally different
from microorganism in the external environment, including ingested food [66,67]. Usually,
the presence in commercial fish feeds of spoilage and pathogen bacteria such as Salmonella
spp. and Escherichia coli can be related to poor hygienic storage condition, environmental
contamination and problems in the extrusion treatment [68,69]. Our results on the microbiological profile of all diets confirm the effectiveness of the extrusion treatment in reducing
the microbial load. The low charge observed for aerobic bacteria could be related to a
secondary contamination after extrusion treatment. As recent studies have shown that
aflatoxin contamination of animal feeds is a frequent issue, the European regulation has set
maximum residue limits for total aflatoxins (B1, B2, G1 and G2) and aflatoxin B1 in animal
feed [70]. To confirm the effectiveness of extrusion treatment and the good environmental
condition during the storage, the total aflatoxins (B1, B2, G1 and G2) and aflatoxin B1 were
found to be below the limit of detection in all experimental diets [71]. This is of particular
interest because, in aquaculture, their presence in the diet can destroy the availability
of certain nutrients, such as vitamin C and thiamine, reducing the immune defense of
fish [72]. In addition, the contamination by mycotoxins of fish feed has been reported to
cause intoxications [69], tissue abnormalities or liver injury, liver tumor, decreased growth
rate and appetite [73] with a decrease of production efficiency and weight of the caught
product and an increase in medical costs [74].
The E-nose and E-tongue sensors are non-specific and partially cross-sensitive; therefore, the value of each sensor is not directly related to each other. To better understand the
data obtained by the artificial senses and improve the discrimination capability, it is necessary to optimize the analysis through a fusion process often categorized in a three-level
model which includes the distinction of low, intermediate and high fusion levels [75,76]. In
our study, the use of an intermediate fusion level helped improve the discrimination power.
First, some relevant features were extracted from each data source separately, and then they
were concatenated into a single array, which was used for multivariate classification and
regression [77]. Selected color codes and E-nose and E-tongue sensors identified differences
between groups in relation to the percentage of HIM integration. The volatile component
was mainly distinguished by LY2/G, LY2/AA, P301 and T401, which are sensors primarily
sensitive to volatile organic compounds derived from proteins and lipids such as nitrogencontaining compounds, hydrocarbons and aldehydes [78,79]. These sensors showed a clear
separation of the HIM35% and HIM50% groups from HIM0 and HIM25%, in relation to
the amino acidic content of the diets. Our observations appear interesting as the use of
alternative ingredients in fish diets, in relation to their chemical composition and their level
of inclusion, can reduce the acceptability of feeds, even if nutritionally balanced. In fact,
different chemical substances can influence fish feeding behavior by acting as attractants
through smell or taste [80]. Fish, in general, have a well-defined olfactory sensitivity to
amino acids [81–85], which help them locate and identify food. Glycine and alanine are
potent odorants that can stimulate feeding behavior by increasing food intake [86,87]. Our
results show that HIM-integrated feeds had greater amounts of glycine and alanine com-
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pared to HIM0 diet; this probably contributed to the olfactory discrimination performed by
the E-nose sensors. Regarding the taste profile, the three sensors selected mainly represent
umami taste (NMS), sourness (AHS) and saltiness (CTS). The results of PCA show that
the HIM0 and HIM25% diets have a more salty and sour taste, probably related to their
high sodium content, while the diets containing insect meal showed higher percentages
of glutamic and aspartic acids, representative of the umami taste [88], than those of the
HIM0 diet.
5. Conclusions
Data suggest that inclusion of Hermetia illucens meal positively influenced the hypocholesterolaemic/hypercholesterolaemic ratio and the content of indispensable amino acids
and microelements. The microbiological quality of all diets testifies to the good practices
of hygiene and sanitation applied during the production processes of fish feeds. The Esensing analysis permitted distinguishing color, odor and taste in the four feed groups. The
combination of sensor responses (E-eye, E-nose and E-tongue) proves to be a powerful tool
for discriminating different organoleptic profiles linked to different chemical compositions
of experimental diets.
This study represents a part of a larger investigation aimed at evaluating the suitability
of HIM addition in the Sparus aurata diet through the study of the productive performance,
the chemical and organoleptic characteristics of the fillets and the possible development of
intestinal inflammation.
Author Contributions: Conceptualization, B.C.; methodology, A.R.D.R. and B.C.; software, A.R.D.R.;
formal analysis, M.O., A.R.D.R., V.L.P., F.G. and G.T.; investigation, B.C.; data curation, A.R.D.R. and
B.C.; writing—original draft preparation, M.O., V.L.P., F.G., A.R.D.R. and B.C.; writing—review and
editing, V.L.P., A.R.D.R. and B.C.; supervision, B.C.; and funding acquisition, B.C. All authors have
read and agreed to the published version of the manuscript.
Funding: This research was funded by PO FEAMP 2014–2020 mis. 2.47 CUP J46C18000570006, project
codex 03/INA/17 Title of the project “FIFA—Feed Insects for Aquaculture”, Scientific Responsible
Biagina Chiofalo.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: The authors declare no conflict of interest.
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