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Black Soldier Fly (Hermetia Illucens) Larvae Protein Derivatives: Potential to Promote Animal Health

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European legislation permits the inclusion of insect proteins in pet and aquaculture diets. Black soldier fly larvae (BSF) are one of the most actively produced species due to their low environmental impact and nutritional characteristics. BSF protein derivatives (proteins and protein hydrolysates) contain a substantial amount of low molecular weight peptides that are known to possess antioxidant potential. In this study, the in vitro antioxidant potential of commercial BSF proteins and protein hydrolysates was investigated for (1) radical scavenging activity, (2) myeloperoxidase activity modulation, and (3) neutrophil response modulation. Chickenmeal and fishmeal are commonly used in pet food and aquaculture formulations. Hence, both were used as industrial benchmarks during this study. The results indicate that fishmeal and chickenmeal are ineffective at suppressing the oxidative damage caused by neutrophil response and myeloperoxidase activity. Fishmeal and chickenmeal even exhibit pro-oxidant behavior in some of the models used during this study. On the other hand, it was found that BSF protein derivatives could be effective in protecting against the cellular damage resulting from neutrophil and myeloperoxidase activities. The outcomes of this study indicate that BSF protein derivatives could be potentially included in pet food and aquaculture feed formulations as health-promoting ingredients.
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animals
Article
Black Soldier Fly (Hermetia illucens) Larvae Protein
Derivatives: Potential to Promote Animal Health
Ange Mouithys-Mickalad 1, Eric Schmitt 2, Monika Dalim 2, Thierry Franck 1,
Nuria Martin Tome 2, Michel van Spankeren 2, Didier Serteyn 1and Aman Paul 2, *
1Centre of Oxygen, Research and Development—University of Liege, 4000 Liège, Belgium;
amouithys@uliege.be (A.M.-M.); t.franck@ulg.ac.be (T.F.); didier.serteyn@ulg.ac.be (D.S.)
2Protix B.V., 5107 NC Dongen, The Netherlands; eric.schmitt@protix.eu (E.S.);
monika.dalim@protix.eu (M.D.); nuria.tome@protix.eu (N.M.T.); michel.vanspankeren@protix.eu (M.v.S.)
*Correspondence: amanpaul@mail.com; Tel.: +31-162-782501
Received: 13 May 2020; Accepted: 27 May 2020; Published: 29 May 2020


Simple Summary:
In European countries, black soldier fly larvae (BSF) proteins are gaining
rapid acceptance as high value protein ingredients in pet food and aquaculture feed formulations.
BSF protein derivatives (proteins and protein hydrolysates) contain a significant share of short-chain
peptides that are known to possess antioxidant behavior. In the present study, the
in vitro
antioxidant
potential of BSF protein derivatives was analyzed using five dierent models. Chickenmeal and
fishmeal are commonly used in pet food and aquaculture feed formulations and hence were used
as industrial benchmarks. The results obtained during this study show that chickenmeal and
fishmeal oer little or no advantage in protecting animal cells against the oxidative damage resulting
from neutrophils and myeloperoxidase response. Moreover, chickenmeal and fishmeal even show
pro-oxidant responses in some of the models tested during this study. It was found that the BSF
protein derivatives used in this study could be eective in protecting the animal cells from oxidative
damage as a consequence of immune response.
Abstract:
European legislation permits the inclusion of insect proteins in pet and aquaculture
diets. Black soldier fly larvae (BSF) are one of the most actively produced species due to their low
environmental impact and nutritional characteristics. BSF protein derivatives (proteins and protein
hydrolysates) contain a substantial amount of low molecular weight peptides that are known to possess
antioxidant potential. In this study, the
in vitro
antioxidant potential of commercial BSF proteins and
protein hydrolysates was investigated for (1) radical scavenging activity, (2) myeloperoxidase activity
modulation, and (3) neutrophil response modulation. Chickenmeal and fishmeal are commonly
used in pet food and aquaculture formulations. Hence, both were used as industrial benchmarks
during this study. The results indicate that fishmeal and chickenmeal are ineective at suppressing
the oxidative damage caused by neutrophil response and myeloperoxidase activity. Fishmeal and
chickenmeal even exhibit pro-oxidant behavior in some of the models used during this study. On the
other hand, it was found that BSF protein derivatives could be eective in protecting against the
cellular damage resulting from neutrophil and myeloperoxidase activities. The outcomes of this
study indicate that BSF protein derivatives could be potentially included in pet food and aquaculture
feed formulations as health-promoting ingredients.
Keywords:
Hermetia illucens; chickenmeal; fishmeal; proteins; DPPH; ABTS; myeloperoxidase;
neutrophil response; immune response; antioxidant activity
Animals 2020,10, 941; doi:10.3390/ani10060941 www.mdpi.com/journal/animals
Animals 2020,10, 941 2 of 16
1. Introduction
Insects are commonly consumed as food in many cultures around the world [
1
3
]. In European
countries, insect proteins are gaining rapid acceptance as high value protein ingredients in animal
diets. The European Union has already approved the inclusion of insect proteins in pet food and
aquaculture feed formulations [
4
]. Chickenmeal and fishmeal are common ingredients in pet food and
aquaculture feed preparations, respectively [
5
,
6
]. Insect proteins are increasingly being viewed as an
alternative to chickenmeal and fishmeal in these markets [
4
]. Amongst all the insect being produced
on industrial scale, the black soldier fly (Hermetia illucens) larvae has gained special attention due
to its ability to grow on a wide range of organic residues and unique nutritional composition [
7
,
8
].
The nutritional suitability of black soldier fly larvae (BSF) proteins in aquaculture and pet diets is well
established [913].
Pets develop a wide range of health disorders with age. Aging can accelerate the free radical
damage in a pet’s body, which might lead to cognitive and locomotor system malfunctioning [
14
].
Similarly, oxidative stress in fish as a result of immune response could lead to compromised health [
15
].
Neutrophils (white blood cells) are responsible for the primary defense mechanism of the body.
Upon receiving the signal, neutrophils rush to the site of intrusion by pathogenic microbes. Then,
neutrophils inactivate the pathogens by phagocytosis and the release of reactive oxygen species (ROS).
The production of ROS is crucial for the host defense [
16
,
17
]. However, in the long term, excessive ROS
production by neutrophils could damage animal cells and might lead to cellular aging, cancer, reduced
immunity, etc. [
18
]. Dietary interventions that can scavenge ROS may help in reducing oxidative
damage in the animal body and resulting health conditions [14].
Some short-chain peptides and free amino acids are known to possess antioxidant activity.
These molecules can actively scavenge ROS and free radicals [
19
]. Studies on preparations obtained
from the hydrolysis of Amphiacusta annulipes,Bombyx mori,Gryllodes sigillatus,Locusta migratoria,
Schistocerca gregaria,Tenebrio molitor, and Zophobas morio proteins have indicated the strong antioxidant
potential of insect protein hydrolysates [
20
23
]. Research institutes and companies are currently
developing methods leading to the production of BSF protein hydrolysates that have superior
nutritional properties [
24
27
]. BSF proteins hydrolysates have a significant share of proteins <1000 Da.
This includes a mixture of short-chain peptides and free amino acids [
24
]. However, until now,
only a few studies have been realized to evaluate the antioxidant potential of BSF protein hydrolysates.
Firmansyah and Abduh [
27
] evaluated the DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity
of a BSF protein hydrolysate. On the other hand, Zhu et al. [
26
] evaluated the DPPH, ABTS (2,2’-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid), superoxide, and hydroxyl radical scavenging activity
of BSF protein hydrolysates. No studies have been realized to date that evaluate the antioxidant
activity of BSF protein hydrolysates using fundamental enzymatic and cellular models. Therefore,
the antioxidant potential of BSF protein hydrolysates is poorly understood on a fundamental level.
Detailed investigations on the
in vitro
antioxidant activity of BSF proteins and protein hydrolysates
may unlock new applications of these protein derivatives to improve animal health.
The current study investigates the antioxidant potential of BSF proteins and protein hydrolysates,
using (1) radical scavenging models involving DPPH and ABTS; (2) enzymatic models involving
myeloperoxidase activity; and (3) a cellular model involving neutrophil response. Chickenmeal and
fishmeal were used as industrial benchmarks in this study.
2. Materials and Methods
2.1. Reagents
All the reagents were of analytical grade. Dimethyl sulfoxide, methanol, ethanol,
calcium chloride, potassium chloride, sodium chloride, hydrogen peroxide, and Tween-20 were
purchased from Merck (VWR, Leuven, Belgium). Sodium nitrite, bovine serum albumin,
phorbol 12-myristate 13-acetate, and Percoll
TM
were purchased from Sigma (Bornem, Belgium).
Animals 2020,10, 941 3 of 16
Aqueous extracts and solutions were made in Milli-Q water obtained using Milli-Q water
system (Millipore, Bedford, MA, USA). Bicinchoninic acid and copper (II) sulfate solutions were
purchased from Sigma (Steinheim, Germany). Whatman filter paper grade 4 (270 mm) was
purchased from Amersham (Buckinghamshire, UK). A Sterlip 30 mL disposable vacuum filter
system was purchased from Millipore (Bedford, MA, USA). 2,2-Diphenyl-1-picrylhydrazyl and
2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) were purchased from Aldrich (Darmstadt, Germany).
8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)dione (L-012) was purchased from Wako
Chemicals (Neuss, Germany).
2.2. Raw Materials
Chickenmeal (CM) and fishmeal (FM) were purchased from an online webshop in September
2019. The chemical composition of both ingredients as declared by the supplier is indicated in Table 1.
Table 1. Chemical composition of chickenmeal and fishmeal (as in basis, provided by supplier).
Nutrients Chickenmeal Fishmeal
Moisture (g/kg) 60.0 100.0
Crude protein (g/kg) 700.0 710.0
Crude fat (g/kg) 120.0 120.0
Added antioxidant No Yes (E324 *)
Form Powder
* E324: Ethoxyquin.
BSF meat (BSF-P), BSF hydrolyzed meat (BSF-HP), and BSF aqueous protein hydrolysate (BSF-APH)
were provided by Protix B.V. (Dongen, The Netherlands) in October 2019. According to the supplier,
(1) BSF-P was pasteurized minced meat that was supplied frozen at
20
C (brand name: PureeX
TM
).
BSF-P is partially defatted and dried to produce BSF proteinmeal (brand name: ProteinX
TM
). (2) BSF-HP
was enzymatically hydrolyzed and pasteurized minced meat, which was also supplied frozen at
20
C (brand name: PureeX
proTM
). (3) BSF-APH was the hydrolysate of water-soluble BSF proteins
(brand name: ProteinAX
proTM
). The details about each hydrolysis step (type of enzyme and hydrolysis
conditions) employed for the production of BSF-HP and BSF-APH were not disclosed by the supplier.
It was also indicated that BSF-APH has high solubility in water (>95%). The chemical composition of
all three ingredients as declared by the supplier is indicated in Table 2.
Table 2.
Chemical composition of black soldier fly larvae (BSF) protein derivatives (as in basis, provided
by supplier).
Nutrients BSF-P 1BSF-HP 2BSF-APH 3
Moisture (g/kg) 700.0 a700.0 a55 a
Crude protein (g/kg) 120 a120 a455 a
Crude fat (g/kg) 122.5 a122.5 a35 a
Added antioxidant No No No
% of total proteins
<1000 Da >6>24 >98
Form Frozen minced meat Powder
1
BSF-P: PureeX
TM
;
2
BSF-HP: PureeX
proTM
;
3
BSF-APH: ProteinAX
proTM
;
a
Mean values based on the range
proposed by supplier.
Water-soluble extracts were prepared for CM, FM, BSF-P, and BSF-HP. These products (100 g each)
were dissolved with six times volumes of Milli-Q water based on their respective dry matter contents
(e.g., BSF-P had dry matter content of 33.3% and was diluted 200 mL Milli-Q water) and stirred for
2 h on a magnetic stirrer. Post centrifugation (1000
×
gfor 30 min at 4
C), the top fat layer was
removed, and the supernatant was filtered using a Whatman filter (Grade 4). The centrifugation
Animals 2020,10, 941 4 of 16
and filtration steps were repeated to remove all non-soluble residues. Finally, the supernatant was
filtered using a Sterlip filter (50 mL, 0.22
µ
m) and freeze dried over a period of two days to obtain
respective water-soluble extract powders. BSF-APH was used directly because it has water solubility
>95%. All four water-soluble extract and BSF-APH powders were stored in a desiccator (at 18
C) until
further use.
2.3. Protein Quantification
The protein content of the four water-soluble extracts and BSF-APH powder was analyzed using
bicinchoninic acid (BCA) protein assay [
28
]. The calibration curve was obtained using bovine serum
albumin (BSA) as standard at concentrations of 0, 0.125, 0.25, 0.5, and 1 mg/mL. Stock solutions of
3 mg/mL water-soluble extracts and BSF-APH were used for analysis. A test solution was made by
dissolving 4900
µ
L BCA (49/50) and 100
µ
L copper (II) sulfate (1/50). Sample stock solutions (10
µ
L)
and test solution (200
µ
L) were added in wells of a 96-well plate. This plate was incubated at 37
C
for 30 min, and absorbance was measured at 450 nm using a Multiscan Ascent (Fisher Scientific,
Asse, Belgium).
2.4. DPPH Assay
DPPH radical scavenging activity was analyzed according to the protocol of
Brand-Willams et al. [
29
], with some modifications. DPPH test solution was made by dissolving
10.5 mg of DPPH in 40 mL of ethanol. Test solution was made fresh and stored in the dark until
further use. DPPH working solution was made by diluting the test solution with 10 times ethanol
(to obtain absorbance of 0.6 to 0.8 at 517 nm). DPPH working solution (1920
µ
L) was mixed with 20
µ
L
of the sample dilutions (four water-soluble extracts and BSF-APH in Milli-Q water) to obtain a final
concentration of 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/mL. The decrease in absorbance after 30 min of
incubation in the dark was recorded at 510 nm using an HP 8453 UV-vis spectrophotometer (Agilent
Technologies, Waldbronn, Germany). Instead of sample dilutions, only Milli-Q water was used in case
of control.
2.5. ABTS Assay
ABTS cation radical scavenging activity was analyzed according to the protocol of Arnao et al. [
30
],
with some modifications. ABTS test solution was made by dissolving 7.0 mmol/L ABTS and 2.45 mmol/L
potassium persulfate in Milli-Q water. The test solution was kept overnight in the dark at room
temperature. ABTS working solution was made by diluting with methanol to obtain the absorbance
between 0.7 and 0.8 at 734 nm. ABTS working solution (1920
µ
L) was mixed with 20
µ
L of samples
dilutions (four water-soluble extracts and BSF-APH in Milli-Q water) to obtain final concentrations
of 0.0125, 0.025, 0.05, 0.1 and 0.2 mg/mL. The decrease in absorbance after 30 min of incubation in
dark was recorded at 734 nm using an HP 8453 UV-vis spectrophotometer (Agilent Technologies,
Waldbronn, Germany). Instead of sample dilutions, only Milli-Q water was used in case of the control.
2.6. Myeloperoxidase (MPO) Activity Using Specific Immunological Extraction Followed by Enzymatic
Detection (SIEFED) Assay
SIEFED assay is a licensed method developed by Franck et al. [
31
] for the specific detection of
animal origin MPO. MPO solution was made by diluting human MPO in 20 mM of phosphate buer
saline (at pH 7.4), 5 g/L BSA, and 0.1% Tween-20. Sample dilutions at final concentrations of 0.0125,
0.025, 0.05, 0.1, and 0.2 mg/mL were incubated for 10 min (at 37
C) with MPO solution at a final
concentration of 25 ng/mL. After incubation, the mixtures were loaded into the wells of a 96-well
microtiter plate coated with rabbit polyclonal antibodies (3
µ
L/mL) against equine MPO and incubated
for 2 h at 37
C in darkness. After washing up the wells, the activity of the enzymes captured by the
antibodies was measured by adding hydrogen peroxide (10
µ
M), NO
2
(10 mM) and Amplex
TM
Red
(40
µ
M). The oxidation of Amplex
TM
Red into the fluorescent adduct resorufin was monitored for
Animals 2020,10, 941 5 of 16
30 min at 37
C with Fluoroskan Ascent (Fisher Scientific, Asse, Belgium). Instead of sample dilutions
only Milli-Q water was used in case of control.
2.7. Myeloperoxidase (MPO) Activity Using Classical Measurement
MPO solution was prepared as mentioned in Section 2.6. Sample dilutions at final concentrations
of 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/mL were incubated for 10 min (at 37
C) with MPO solution at a
final concentration of 25 ng/mL. After incubation, the mixture (100
µ
L) was immediately transferred
into a 96-well microtiter plate. This was followed by the addition of 10
µ
L NO
2
(10 mM) and
100
µ
L of Amplex
TM
Red and hydrogen peroxide mixture (at concentrations mentioned in Section 2.6).
The oxidation of Amplex
TM
Red into the fluorescent adduct resorufin was monitored for 30 min at
37
C with Fluoroskan Ascent (Fisher Scientific, Asse, Belgium) immediately after addition of the
revelation mixture. Instead of sample dilutions, only Milli-Q water was used in case of control.
2.8. Cellular Antioxidant Activity
Preparation of the neutrophil and phorbol 12-myristate 13-acetate (PMA) solutions were made
according to Paul et al. [
17
]. The neutrophil response modulation activity of samples was analyzed using
the protocol of Tsumbu et al. [
16
]. Neutrophil suspension (1 million cells/143
µ
L PBS) was loaded in wells
of a 96-well microtiter plate and incubated for 10 min (at 37
C in the dark) with phosphate buer saline
solution of samples at final concentrations of 0.0125, 0.025, 0.05, 0.1, and 0.2 mg/mL. After incubation,
25
µ
L calcium chloride (10
µ
M) and 20
µ
L L-012 (100
µ
M) was added in wells. The neutrophils were
activated with 10
µ
L PMA (16
µ
M) immediately before monitoring the chemiluminesence response of
neutrophils during 30 min at 37
C using Fluoroskan Ascent (Fisher Scientific, Asse, Belgium). Instead
of sample dilutions, only phosphate buer saline was used in case of control.
2.9. Statistical Analyses
All the analyses were performed in triplicate. For protein quantification, the equation of a
fitted line and R-square value were calculated using linear regression. The relationships between
concentration and inhibition obtained for antioxidant assays were non-monotonic in nature. To address
this, the locally estimated scatterpot smoothing (LOESS) regression technique was used to model the
relationship [
32
]. Models were fitted using the R statistical software [
33
]. These models require a
span parameter that defines the smoothing sensitivity of the local regressions. By visual inspection, a
span parameter value of 0.4 was found to be suitable for all concentration and inhibition relationship
curves. Concentrations with a predicted inhibition percentage of interest, such as IC
50
(concentration at
which 50% inhibition is reached), were found using the fitted models in combination with a numerical
search routine.
3. Results
3.1. Protein Quantification
The calibration curve resulted in the following parameters: (1) equation of line: y =0.3314x +
0.1503 (where x is the concentration of proteins); and (2) R-squared value: 0.9989. The optical density
of samples and relative concentration of proteins (calculated using equation of line) are mentioned in
Table 3. BSF-P extract solution (3 mg/mL) exhibits the highest and BSF-HP solution exhibits the lowest
protein concentrations amongst the tested solutions using bicinchoninic acid assay.
Animals 2020,10, 941 6 of 16
Table 3. Protein quantification using bicinchoninic acid assay.
Product Product Used for Testing in all
the Assays Mean Optical Density Protein Concentration
(mg/mL)
BSF-P 1Water-soluble extract 0.486 1.013
BSF-HP 2Water-soluble extract 0.365 0.648
BSF-APH 3
Product as provided by supplier
0.383 0.702
FM 4Water-soluble extract 0.425 0.829
CM 5Water-soluble extract 0.481 0.998
1BSF-P: PureeXTM;2BSF-HP: PureeXproTM ;3BSF-APH: ProteinAXproTM;4FM: Fishmeal; 5CM: Chickenmeal.
3.2. DPPH Assay
The DPPH radical scavenging activity of all five samples after 30 min of incubation is indicated
in Figure 1. The plot shows the measured values as well as fitted curves obtained from LOESS.
CM exhibited pro-oxidant behavior at all tested concentrations. Whereas FM exhibited pro-oxidant
behavior at four out of five tested concentrations. It was not possible to calculate IC
50
for samples,
because the samples either exhibited pro-oxidant activity or 50% inhibition was not achieved during
the assay (see Table 4). The E
max
(maximum inhibition achieved during the assay) of all the samples
are also indicated in Table 5and are in the following order: BSF-HP >BSF-APH >BSF-P >FM.
Animals 2020, 10, x 6 of 17
3.2. DPPH Assay
The DPPH radical scavenging activity of all five samples after 30 min of incubation is indicated
in Figure 1. The plot shows the measured values as well as fitted curves obtained from LOESS. CM
exhibited pro-oxidant behavior at all tested concentrations. Whereas FM exhibited pro-oxidant
behavior at four out of five tested concentrations. It was not possible to calculate IC
50
for samples,
because the samples either exhibited pro-oxidant activity or 50% inhibition was not achieved during
the assay. The E
max
(maximum inhibition achieved during the assay) of all the samples are also
indicated in Table 5 and are in the following order: BSF-HP > BSF-APH > BSF-P > FM.
Figure 1. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of PureeX
TM
(BSF-P),
PureeX
proTM
(BSF-HP), ProteinAX
proTM
(BSF-APH), Chickenmeal (CM), and Fishmeal (FM) (n = 3).
Table 4. Antioxidant activity IC
50
(mg/mL) of samples obtained using different assays.
Assay BSF-P
1
BSF-HP
2
BSF-APH
3
FM
4
CM
5
DPPH NE
c
NE
c
NE
c
NE
c
PO
d
ABTS 0.04 0.05 0.03 0.11 0.09
MPO
a
SIEFED NE
c
0.14 0.18 PO
d
PO
d
MPO
a
Classical 0.10 0.09 0.05 PO
d
PO
d
CAA
b
0.15 0.15 NE
c
NE
c
NE
c
1
BSF-P: PureeX
TM
;
2
BSF-HP: PureeX
proTM
;
3
BSF-APH: ProteinAX
proTM
;
4
FM: Fishmeal;
5
CM:
Chickenmeal;
a
MPO: Myeloperoxidase;
b
CAA: Cellular antioxidant activity using neutrophil model;
c
NE: Not estimated because 50% inhibition was not achieved in tested concentrations;
d
PO: Not
estimated because sample exhibited pro-oxidant activity on tested concentrations.
Table 5. Antioxidant activity E
max
(% inhibition) of samples obtained using different assays. ABTS:
2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid.
Assay Parameter BSF-P
1
BSF-HP
2
BSF-APH
3
FM
4
CM
5
DPPH Emax (%) 14.52 48.09 16.26 0.75 PO
c
C* (mg/mL) 0.20 0.20 0.20 0.03 -
ABTS Emax (%) 89.33 76.32 90.81 70.40 69.39
C* (mg/mL) 0.20 0.20 0.20 0.20 0.20
MPO
a
SIEFED Emax (%) 36.23 77.58 53.08 PO
c
PO
c
C* (mg/mL) 0.20 0.20 0.20 - -
MPO
a
Classical Emax (%) 89.66 83.82 90.86 PO
c
PO
c
C* (mg/mL) 0.20 0.20 0.20 - -
CAA
b
Emax (%) 59.57 59.64 36.62 21.81 5.08
C* (mg/mL) 0.20 0.20 0.20 0.05 0.20
Figure 1.
DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity of PureeX
TM
(BSF-P),
PureeXproTM (BSF-HP), ProteinAXproTM (BSF-APH), Chickenmeal (CM), and Fishmeal (FM) (n =3).
Table 4. Antioxidant activity IC50 (mg/mL) of samples obtained using dierent assays.
Assay BSF-P 1BSF-HP 2BSF-APH 3FM 4CM 5
DPPH NE cNE cNE cNE cPO d
ABTS 0.04 0.05 0.03 0.11 0.09
MPO aSIEFED NE c0.14 0.18 PO dPO d
MPO aClassical 0.10 0.09 0.05 PO dPO d
CAA b0.15 0.15 NE cNE cNE c
1
BSF-P: PureeX
TM
;
2
BSF-HP: PureeX
proTM
;
3
BSF-APH: ProteinAX
proTM
;
4
FM: Fishmeal;
5
CM: Chickenmeal;
a
MPO: Myeloperoxidase;
b
CAA: Cellular antioxidant activity using neutrophil model;
c
NE: Not estimated because
50% inhibition was not achieved in tested concentrations;
d
PO: Not estimated because sample exhibited pro-oxidant
activity on tested concentrations.
Animals 2020,10, 941 7 of 16
Table 5.
Antioxidant activity E
max
(% inhibition) of samples obtained using dierent assays. ABTS:
2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid.
Assay Parameter BSF-P 1BSF-HP 2BSF-APH 3FM 4CM 5
DPPH Emax (%) 14.52 48.09 16.26 0.75 PO c
C * (mg/mL) 0.20 0.20 0.20 0.03 -
ABTS Emax (%) 89.33 76.32 90.81 70.40 69.39
C * (mg/mL) 0.20 0.20 0.20 0.20 0.20
MPO aSIEFED Emax (%) 36.23 77.58 53.08 PO cPO c
C * (mg/mL) 0.20 0.20 0.20 - -
MPO aClassical Emax (%) 89.66 83.82 90.86 PO cPO c
C * (mg/mL) 0.20 0.20 0.20 - -
CAA bEmax (%) 59.57 59.64 36.62 21.81 5.08
C * (mg/mL) 0.20 0.20 0.20 0.05 0.20
* C: Concentration at which E
max
is achieved;
1
BSF-P: PureeX
TM
;
2
BSF-HP: PureeX
proTM
;
3
BSF-APH: ProteinAX
proTM
;
4
FM: Fishmeal;
5
CM: Chickenmeal;
a
MPO: Myeloperoxidase;
b
CAA: Cellular antioxidant activity using neutrophil
model; cPO: Not estimated because sample exhibited pro-oxidant activity on tested concentrations.
3.3. ABTS Assay
The ABTS cation radical scavenging activity of samples after 30 min of incubation is shown in
Figure 2(measured values as well as fitted curves obtained from LOESS). All the samples exhibited
a similar inhibition pattern i.e., the percentage of inhibition increased as a function of increasing
concentration. The IC
50
of samples are mentioned in Table 4and are in the following order: FM >CM
>BSF-HP >BSF-P >BSF-APH. Lower IC
50
reflects a higher ABTS cation radical scavenging activity.
The E
max
(maximum inhibition achieved during the assay) of all the samples are indicated in Table 5
and are in the following order: BSF-APH >BSF-P >BSF-HP >FM >CM.
Animals 2020, 10, x 7 of 17
*C: Concentration at which E
max
is achieved;
1
BSF-P: PureeX
TM
;
2
BSF-HP: PureeX
proTM
;
3
BSF-APH:
ProteinAX
proTM
;
4
FM: Fishmeal;
5
CM: Chickenmeal;
a
MPO: Myeloperoxidase;
b
CAA: Cellular
antioxidant activity using neutrophil model;
C
PO: Not estimated because sample exhibited pro-
oxidant activity on tested concentrations.
3.3. ABTS Assay
The ABTS cation radical scavenging activity of samples after 30 min of incubation is shown in
Figure 2 (measured values as well as fitted curves obtained from LOESS). All the samples exhibited
a similar inhibition pattern i.e., the percentage of inhibition increased as a function of increasing
concentration. The IC
50
of samples are mentioned in Table 4 and are in the following order: FM > CM >
BSF-HP > BSF-P > BSF-APH. Lower IC
50
reflects a higher ABTS cation radical scavenging activity. The
E
max
(maximum inhibition achieved during the assay) of all the samples are indicated in Table 5 and
are in the following order: BSF-APH > BSF-P > BSF-HP > FM > CM.
Figure 2. ABTS cation radical scavenging activity of PureeX
TM
(BSF-P), PureeX
proTM
(BSF-HP),
ProteinAX
proTM
(BSF-APH), chickenmeal (CM), and fishmeal (FM) (n = 3).
3.4. Myeloperoxidase (MPO) Activity using Specific Immunological Extraction Followed by Enzymatic
Detection (SIEFED) Assay
The MPO response modulation activity of samples obtained using SIEFED assay is shown in
Figure 3 (measured values as well as fitted curves obtained from LOESS). BSF-HP exhibited strong
inhibition behavior, with >75% inhibition at 0.20 mg/mL concentration. The IC
50
of samples are
mentioned in Table 4 and are in the following order: BSF-APH > BSF-HP. The E
max
of samples are
shown in Table 5, and they are in the following order: BSF-HP > BSF-APH > BSF-P. FM and CM show
pro-oxidant behavior at all tested concentrations. On the other hand, E
max
for BSF-P was < 50%.
Figure 2.
ABTS cation radical scavenging activity of PureeX
TM
(BSF-P), PureeX
proTM
(BSF-HP),
ProteinAXproTM (BSF-APH), chickenmeal (CM), and fishmeal (FM) (n =3).
3.4. Myeloperoxidase (MPO) Activity Using Specific Immunological Extraction Followed by Enzymatic
Detection (SIEFED) Assay
The MPO response modulation activity of samples obtained using SIEFED assay is shown in
Figure 3(measured values as well as fitted curves obtained from LOESS). BSF-HP exhibited strong
inhibition behavior, with >75% inhibition at 0.20 mg/mL concentration. The IC
50
of samples are
Animals 2020,10, 941 8 of 16
mentioned in Table 4and are in the following order: BSF-APH >BSF-HP. The E
max
of samples are
shown in Table 5, and they are in the following order: BSF-HP >BSF-APH >BSF-P. FM and CM show
pro-oxidant behavior at all tested concentrations. On the other hand, Emax for BSF-P was <50%.
Figure 3.
MPO response modulation activity of PureeX
TM
(BSF-P), PureeX
proTM
(BSF-HP),
ProteinAX
proTM
(BSF-APH), chickenmeal (CM), and fishmeal (FM) using specific immunological
extraction followed by enzymatic detection (SIEFED) assay (n =3).
3.5. Myeloperoxidase (MPO) Activity Using Classical Assay
The MPO response modulation activity of samples obtained using classical assay is indicated
in Figure 4(measured values as well as fitted curves obtained from LOESS). CM and FM exhibited
pro-oxidant behavior at all tested concentrations. The E
max
of all the samples tested are indicated in
Table 5. BSF-APH, BSF-P, and BSF-HP exhibited E
max
>75%. The IC
50
of samples are mentioned in
Table 4and are in the following order: BSF-P >BSF-HP >BSF-APH.
Animals 2020, 10, x 8 of 17
Figure 3. MPO response modulation activity of PureeX
TM
(BSF-P), PureeX
proTM
(BSF-HP),
ProteinAX
proTM
(BSF-APH), chickenmeal (CM), and fishmeal (FM) using specific immunological
extraction followed by enzymatic detection (SIEFED) assay (n = 3).
3.5. Myeloperoxidase (MPO) Activity using Classical Assay
The MPO response modulation activity of samples obtained using classical assay is indicated in
Figure 4 (measured values as well as fitted curves obtained from LOESS). CM and FM exhibited pro-
oxidant behavior at all tested concentrations. The E
max
of all the samples tested are indicated in Table
5. BSF-APH, BSF-P, and BSF-HP exhibited E
max
> 75%. The IC
50
of samples are mentioned in Table 4
and are in the following order: BSF-P > BSF-HP > BSF-APH.
Figure 4. MPO response modulation activity of PureeX
TM
(BSF-P), PureeX
proTM
(BSF-HP),
ProteinAX
proTM
(BSF-APH), chickenmeal (CM), and fishmeal (FM) using classical measurement (n=3).
3.6. Cellular Antioxidant Activity
The neutrophil response modulation activity (measured values as well as fitted curves obtained
from LOESS) and E
max
of the samples are shown in Figure 5 and Table 5, respectively. All the tested
samples exhibited E
max
> 0%. BSF-APH, FM, and CM exhibited E
max
< 40%. CM exhibited pro-oxidant
behavior at 3 out of 5 tested concentrations. The IC
50
of samples are mentioned in Table 4. BSF-P and
BSF-HP have the same numerical IC
50
values.
Figure 4.
MPO response modulation activity of PureeX
TM
(BSF-P), PureeX
proTM
(BSF-HP),
ProteinAX
proTM
(BSF-APH), chickenmeal (CM), and fishmeal (FM) using classical measurement
(n =3).
Animals 2020,10, 941 9 of 16
3.6. Cellular Antioxidant Activity
The neutrophil response modulation activity (measured values as well as fitted curves obtained
from LOESS) and E
max
of the samples are shown in Figure 5and Table 5, respectively. All the tested
samples exhibited E
max
>0%. BSF-APH, FM, and CM exhibited E
max
<40%. CM exhibited pro-oxidant
behavior at 3 out of 5 tested concentrations. The IC
50
of samples are mentioned in Table 4. BSF-P and
BSF-HP have the same numerical IC50 values.
Animals 2020, 10, x 9 of 17
Figure 5. Neutrophil response modulation activity of PureeX
TM
(BSF-P), PureeX
proTM
(BSF-HP),
ProteinAX
proTM
(BSF-APH), chickenmeal (CM) and fishmeal (FM) (n = 3).
Figure 5.
Neutrophil response modulation activity of PureeX
TM
(BSF-P), PureeX
proTM
(BSF-HP),
ProteinAXproTM (BSF-APH), chickenmeal (CM) and fishmeal (FM) (n =3).
4. Discussion
4.1. Protein Quantification
The protein concentration of BSF-APH and four water-soluble extracts estimated using
bicinchoninic acid assay are displayed in Table 3. For BSF-APH, 3 mg/mL solution resulted in
protein concentration of 0.702 mg/mL, which translates into 0.235 mg proteins per gram of BSF-APH
(or 23.5% proteins). According to the supplier, the average protein content of BSF-APH is 45.5%
(see Table 2, analyzed using the Dumas method). Dierences in protein content arise due to the
method of analysis. Bicinchoninic acid assay is based on the detection of bonds specific to Cys, Trp,
and Tyr. On the other hand, Dumas assay is based on the estimation of total organic nitrogen [
34
].
Therefore, protein content estimated using the Dumas method is always higher than that estimated
using bichinchoninic acid assay. However, comparing the two protein estimation methods is not the
goal of this study. Considering the amino acid pattern of black soldier fly proteins, FM and CM [
35
,
36
],
it could be hypothesized that the protein content of four water-soluble extracts are in the following
order: BSF-P >CM >FM >45.5% >BSF-HP.
4.2. DPPH Radical Scavenging Activity
DPPH and ABTS assays are commonly used to analyze the antioxidant potential of food and
feed products [
17
]. DPPH radical scavenging activity represents the ability of a sample to donate
hydrogen atoms (referred as hydrogen atom transfer) or electrons (referred as single electron transfer)
to stabilize free radicals [
29
]. DPPH assay IC
50
and E
max
for all tested samples are mentioned in
Tables 4and 5, respectively. Post 30 min of incubation, all the tested samples exhibit E
max
<50%
(with BSF-HP exhibiting highest E
max
). According to the supplier, BSF-HP is manufactured by the
controlled hydrolysis of black soldier fly proteins and contains at least 24% of proteins <1000 Da
(see Table 2). On the other hand, BSF-P and BSF-APH contain at least 6% and 98% proteins <1000 Da.
Animals 2020,10, 941 10 of 16
The authors were not able to find any representative literature for the molecular weight distribution
of FM and CM. However, according to the literature, FM and CM contain 2.2% and 1.1% free amino
acid (of total proteins), respectively [
37
], which translates into FM and CM containing at least 2.2%
and 1.1% proteins <1000 Da, respectively. Zou et al. [
38
] indicated that the capacity of proteinaceous
materials to scavenge free radicals depends on the protein molecular weight distribution. Proteins
with low molecular weight peptides could scavenge free radicals more eciently. However, this does
not explain the fact that BSF-APH contains a higher amount of proteins <1000 Da and still exhibits a
lower inhibition of DPPH free radicals. The free radical scavenging activity of proteinaceous molecules
is also influenced by the following. (1) Amino acid composition: hydrophobic amino acids (for e.g.,
Tyr, Phe, Pro, Ala, His and Leu) have superior radical scavenging activity in comparison to hydrophilic
amino acids (2) Amino acid sequence: Peptides with an amphiphilic nature could enhance the radical
scavenging activity of a sample [
38
40
]. Chemical analyses have indicated that Tyr exhibits antioxidant
behavior via the hydrogen atom transfer mechanism. On the other hand, amino acids such as Cys, Trp,
and His exhibit antioxidant behavior via the single electron transfer mechanism [41].
FM and CM exhibit pro-oxidant behavior at most of the tested concentrations (see Figure 1).
This behavior mainly arises from the thermal processing. For both FM and CM, thermal processing
commonly involves heating the raw product at high temperatures for 15 to 20 min [
42
,
43
]. In Norway,
during fishmeal production, wild caught fish are subjected to heating at temperatures
70
C for
time
20 min in order to achieve 100 log
10
reductions of Enterobacteriaceae and Salmonella counts [
44
].
Such strict thermal processing conditions may result in the oxidation of fats and proteins. Fishmeal
contains lipids rich in polyunsaturated fatty acids that are more susceptible to thermal oxidation [
45
].
Antioxidants are commonly added in fishmeal to prevent the oxidation of polyunsaturated fatty acids
(also visible in Table 1). The heat-induced oxidation of amino acids leads to the development of a wide
range of oxidation products [
45
,
46
]. The pro-oxidant behavior of amino acid oxidation by-products is
already known. They can result in a wide range of health conditions in animal body [
47
]. According to
the supplier, all the black soldier fly protein derivatives used in this study were thermally processed at
temperatures <100
C for time <1.5 min. The supplier also indicated that these thermal processing
time–temperature combinations were adopted to ensure minimum damage to nutrients (proteins and
fat) and the adequate inactivation of pathogenic microbiota. This implies that the pro-oxidant behavior
of FM and CM arises mainly due to the stringent production method.
In a recent study [
27
], researchers made BSF protein hydrolysate using a bromelain enzyme.
Bromelain-derived protein hydrolysate was also tested for DPPH radical scavenging activity,
which resulted in the IC
50
of 8.4 mg/mL. The DPPH radical scavenging activity of this bromelain-derived
protein hydrolysate was much lower when compared to the activity of products such as BSF-HP
(IC
50
0.18 mg/mL after 60 min of incubation). The higher activity of BSF-HP used in this study could
arise from compositional attributes (as previously discussed in this section) and the quality of the
raw material itself. Protix is reportedly producing insect proteins in GMP+and SecureFeed certified
facilities, under HACCP conditions [7].
4.3. ABTS Cation Radical Scavening Activity
ABTS cation radical scavenging denotes the ability of a sample to donate electron and stabilize
free radicals [
17
]. The ABTS assay IC
50
of all samples are indicated in Table 4. They are in the following
order: FM >CM >BSF-HP >BSF-P >BSF-APH. The higher the IC
50
, the lower the antioxidant activity.
In this assay, even FM and CM exhibit antioxidant activity. It appears that FM and CM extracts may be
ecient where free radical(s) could be stabilized using a single electron transfer mechanism. However,
they still exhibit lower scavenging activity in comparison to BSF derivatives.
BSF-APH has at least 98% proteins <1000 Da (the lowest protein molecular weight amongst all
tested samples) and exhibited the lowest ABTS IC
50
. The dependence of radical scavenging activity on
protein molecular weight is already explained in Section 4.2. According to the supplier, BSF-P and
BSF-HP have the same amino acid composition. However, due to protein hydrolysis (hydrolysis details
Animals 2020,10, 941 11 of 16
were not disclosed by the manufacturer), the amount of proteins <1000 Da is higher in BSF-HP than in
BSF-P. It is therefore somewhat surprising that the BSF-P IC
50
value was slightly lower in comparison
to BSF-HP. This could be explained by the mechanism of hydrolysis. Enzymatic hydrolysis is achieved
through exo- and endopeptidase. Exopeptidase cleaves the terminal peptide bond; on the other hand,
endopeptidase cleaves the non-terminal peptide bond [
48
]. In both cases, the sequence of amino acids
is altered. The radical scavenging ability of the resulting peptides via single electron transfer is also
dependent on the amphiphilic nature of proteinaceous molecules [
38
]. It is possible that the peptides
in BSF-HP are less amphiphilic in nature, which results in the lower ABTS cation radical scavenging
activity of BSF-HP compared to BSF-P.
Zhu et al. [
26
] developed BSF protein hydrolysate using a wide range of commercial enzymes.
The hydrolysates were further fractionated into the following groups using ultrafiltration: group 1
(<3000 Da), group 2 (3000 to 10,000 Da), and group 3 (>10,000 Da). The activity of these hydrolyzed
fractions was also investigated for ABTS cation radical scavenging activity. Ascorbic acid was used
as the reference molecule in this study. Interestingly, the best performing fraction and ascorbic acid
were able to inhibit 85.7% and 92.1% of ABTS cation radicals at 0.05 mg/mL concentration, respectively.
In our study, BSF-P and BSF-APH exhibit ABTS cation radical scavenging E
max
of 89% and 91%,
respectively (at 0.2 mg/mL). This indicates that fractioning BSF-P and BSF-APH could result in fractions
that may have very strong antioxidant potential.
4.4. Neutrophil Response Modulation Activity
The strong free radical scavenging activities of BSF derivatives are evident from Sections 4.2
and 4.3. Furthermore, all the samples were also tested for neutrophil response modulation activity.
Neutrophils are white blood cells present in the animal body (including humans, pets, fishes, poultry,
and swine). They are involved in the primary defense against pathogens [
16
]. When pathogenic
microbes enter the animal body, neutrophils rush to the site of infestation and initiate defense via
the mechanism indicated in Figure 6. During degranulation, neutrophils release a wide range of
oxidative enzymes including myeloperoxidase, which results in the activation of nicotinamide adenine
dinucleotide phosphate (NADPH) oxidase. NADPH oxidase is responsible for the production of
superoxide anion and by-products (e.g., hydrogen peroxide) [
16
,
17
]. Superoxide anion can further
react with the nitric oxide radical to produce peroxynitrite. This process also generates a hydroxyl
radical (by the reaction of hydrogen peroxide with metal ion) [
49
,
50
]. This battery of oxidative reactions
is crucial to the defense of the host animal. However, these ROS generated during host defense can
react with enzymes, proteins, lipids, etc., of body cells and result in the development of various health
conditions (for e.g., cellular aging, cancer, etc.) [
18
]. The neutrophil assay conducted in this research
determines the ability of proteinaceous molecules to scavenge ROS produced as a result of neutrophil
activity. PMA was used to activate protein kinase C present in neutrophils, which results in the
production of NADPH oxidase responsible for catalyzing ROS production. ROS production in the
system is coupled with lucigenin-amplified chemiluminescence. The ability of a proteinaceous sample
to scavenge ROS (particularly superoxide anion) is marked by a decreased chemiluminescence [51].
To the author’s knowledge, this is the first analysis of the
in vitro
neutrophil response modulation
activity of BSF derivatives. CM exhibited pro-oxidant behavior at 3 out of 5 tested concentrations
and had an E
max
of only 5% at 0.2 mg/mL (see Figure 5and Table 5). CM is commonly used in pet
food preparations [
52
]. However, the outcomes of the current study indicate that CM inclusion oers
little or no benefits relating to scavenging the ROS produced by activated neutrophils. Moreover,
CM inclusion could even result in inflammatory damage to host cells. The repetitive inflammatory
damage of canine or feline cells could translate into conditions such as accelerated aging, slow cognitive
function, etc. [14].
Animals 2020,10, 941 12 of 16
Animals 2020, 10, x 12 of 17
production of superoxide anion and by-products (e.g., hydrogen peroxide) [16,17]. Superoxide anion
can further react with the nitric oxide radical to produce peroxynitrite. This process also generates a
hydroxyl radical (by the reaction of hydrogen peroxide with metal ion) [49,50]. This battery of
oxidative reactions is crucial to the defense of the host animal. However, these ROS generated during
host defense can react with enzymes, proteins, lipids, etc., of body cells and result in the development
of various health conditions (for e.g., cellular aging, cancer, etc.) [18]. The neutrophil assay conducted
in this research determines the ability of proteinaceous molecules to scavenge ROS produced as a
result of neutrophil activity. PMA was used to activate protein kinase C present in neutrophils, which
results in the production of NADPH oxidase responsible for catalyzing ROS production. ROS
production in the system is coupled with lucigenin-amplified chemiluminescence. The ability of a
proteinaceous sample to scavenge ROS (particularly superoxide anion) is marked by a decreas.
Figure 6. Defense mechanism of neutrophils. Source: Adapted from Tsumbu et al. [16], Paul et al. [17],
and Perobelli et al. [44].
To the author’s knowledge, this is the first analysis of the in vitro neutrophil response
modulation activity of BSF derivatives. CM exhibited pro-oxidant behavior at 3 out of 5 tested
concentrations and had an E
max
of only 5% at 0.2 mg/mL (see Figure 5 and Table 5). CM is commonly
used in pet food preparations [52]. However, the outcomes of the current study indicate that CM
inclusion offers little or no benefits relating to scavenging the ROS produced by activated neutrophils.
Moreover, CM inclusion could even result in inflammatory damage to host cells. The repetitive
inflammatory damage of canine or feline cells could translate into conditions such as accelerated
aging, slow cognitive function, etc. [14].
On the other hand, FM exhibits mild antioxidant behavior in this assay, with an E
max
of 22% (see
Table 5). At 0.2 mg/mL, FM exhibits inhibition of 5%. Aquaculture rearing media (i.e., water) offer a
continuous buffer of pathogenic bacteria. Therefore, aquaculture organisms are at the constant risk
of pathogenic bacterial invasions [53]. This results in a wide range of health conditions, including
reduced immunity, aging, etc. [15]. Our research highlights the inadequacy of FM to suppress the
inflammatory damage from repetitive neutrophil activity. This often translates into incremental cost
occurring as a result of antibiotics and nutritional supplement usage. BSF-P used in the study is also
the raw material to produce BSF proteinmeal. EU legislations already permit the use of insect
proteinmeal in aquaculture diets. BSF-P exhibits E
max
and IC
50
of 59.57% and 0.15 mg/mL, respectively
(see Table 5). Additionally, the supplier uses low drying temperature to convert BSF-P into BSF
proteinmeal. This implies that BSF proteinmeal will have activity similar to BSF-P. Therefore, BSF-P-
Figure 6.
Defense mechanism of neutrophils. Source: Adapted from Tsumbu et al. [
16
], Paul et al. [
17
],
and Perobelli et al. [44].
On the other hand, FM exhibits mild antioxidant behavior in this assay, with an E
max
of 22%
(see Table 5). At 0.2 mg/mL, FM exhibits inhibition of 5%. Aquaculture rearing media (i.e., water) oer
a continuous buer of pathogenic bacteria. Therefore, aquaculture organisms are at the constant risk of
pathogenic bacterial invasions [
53
]. This results in a wide range of health conditions, including reduced
immunity, aging, etc. [
15
]. Our research highlights the inadequacy of FM to suppress the inflammatory
damage from repetitive neutrophil activity. This often translates into incremental cost occurring as a
result of antibiotics and nutritional supplement usage. BSF-P used in the study is also the raw material
to produce BSF proteinmeal. EU legislations already permit the use of insect proteinmeal in aquaculture
diets. BSF-P exhibits E
max
and IC
50
of 59.57% and 0.15 mg/mL, respectively (see Table 5). Additionally,
the supplier uses low drying temperature to convert BSF-P into BSF proteinmeal. This implies that
BSF proteinmeal will have activity similar to BSF-P. Therefore, BSF-P-derived proteinmeal could also
be eective in preventing the inflammatory damage resulting from neutrophil activity in the fish
body. Moreover, BSF-HP also exhibits neutrophil response modulation activity comparable to BSF-P
(see Table 4).
Therefore, it is possible that BSF derivatives (particularly BSF-P and BS-HP used in this study)
could oer a natural and sustainable solution to suppress oxidative damage resulting from pathogenic
invasion. The use of these ingredients may even help the aquaculture industry to improve the immune
health of fishes.
4.5. MPO Response Modulation Activity (SIEFED and Classical Assay)
The general mechanism of neutrophil response is indicated in Figure 6. The neutrophil extracellular
trap contains several molecules required to inactivate pathogenic microbes. The MPO enzyme present
in neutrophil extracellular trap can produce hypochlorous acid from hydrogen peroxide and chloride
ion. Additionally, MPO is capable of oxidizing tyrosine into the tyrosyl free radical. Both products of
MPO oxidation (hypochlorous acid and tyrosyl free radical) are crucial to inactivate pathogens. Again,
the repetitive interaction of these molecules with animal cells results in inflammatory damage [
16
,
31
].
In an animal body, MPO-Fe (III) (active form) reacts with hydrogen peroxide to form oxoferryl
π
cation
radical (CpI form). CpI form converts back into MPO-Fe (III) coupled with chloride ion, transforming
into hypochlorous acid. However, in the present experiment, back reduction of the Cp I form to
Animals 2020,10, 941 13 of 16
MPO-Fe (III) was achieved in 2 stages. First, there was the reduction of CpI to MPO-Fe (IV) =O
via electron transfer through nitrite ions. Then, electron provisioning was done (via Amplex
TM
Red
oxidation to resorufin reaction), which converts MPO-Fe (IV) =O to MPO-Fe(III) form [
17
,
31
,
51
].
Proteinaceous molecules could prevent the oxidative damage resulting from MPO either by directly
reacting with the CpI form and terminating the halogenation or by donating hydrogen (hydrogen atom
transfer) to ROS produced as a consequence of MPO activity [
16
]. The MPO response modulation
activity was analyzed using the classical and SIEFED assay. The classical assay measures the ability of
a sample to complex with CpI form and stabilize ROS. Whereas in SIEFED assay, MPO is bound to
rabbit polyclonal antibodies (and the rest of the compounds are washed away), so it purely measures
the ability of samples to complex with the CpI form [31].
As with neutrophil response modulation activity, the MPO response modulation activity of BSF
derivatives is also being reported for the first time. FM and CM exhibit pro-oxidant behavior in both
the assays (see Figures 3and 4). The presence of oxidative reaction products in FM and CM (because
of the production process) that are capable of initiating pro-oxidative response have been already
discussed in Section 4.2. Detailed
in vitro
investigations realized during this study indicate that the
inclusion of FM and CM in animal diets may result in inflammatory damage.
In the classical assay, BSF derivatives exhibit strong antioxidant potential, with IC
50
in following
order: BSF-P >BSF-HP >BSF-APH. BSF-APH show strong antioxidant potential in the classical
assay (see Table 4), whereas, for SIEFED assay, IC
50
were in the following order: BSF-APH >BSF-HP.
In the SIEFED assay, BSF-P did not reach 50% inhibition (even at the highest concentration tested).
Thus, while BSF-P and BSF-APH are more eective in stabilizing ROS, BSF-HP has higher ecacy in
complexing with the CpI form of MPO. These observations indicate that BSF derivatives could be used
in pet food and aquaculture formulations to eectively suppress inflammatory damages resulting from
MPO activity.
Free amino acids are directly absorbed from the animal intestine [
54
]. Whereas, the intestinal
absorption of peptides takes place by one of the following mechanisms: (1) transfer and uptake
of di- and tri-peptides by PepT1; (2) paracellular transport of water-soluble and low molecular
weight peptides via intercellular junctions; and (3) uptake of short and intermediate peptides by
transcytosis [
55
], indicating that the water-soluble extracts used in this study will pass the intestinal
membrane with minimum alterations. Due to this reason, BSF-APH, with high water solubility (>95%)
and the majority of proteins below 1000 Da (>98%), could be a very interesting candidate for inclusion
in pet and fish diets to promote animal health. In the future, it could be of interest to analyze the eect
of feed processing treatments on the in vitro bioactivity of BSF protein derivatives.
BSF protein derivatives used in this study oer an antioxidative advantage over FM and CM.
However, the animal body is a complex system with several biochemical processes taking place
simultaneously. Additionally, several processes interact with each other, resulting in an adapted
response [
56
]. It is possible that BSF protein derivatives show an altered response in the animal body.
Therefore, in the future, it could also be interesting to investigate the activity of BSF protein derivatives
using in vivo animal feeding trials.
5. Conclusions
In this study, the
in vitro
antioxidant activity of commercial black soldier fly proteins and protein
hydrolysates was analyzed using radical scavenging models (DPPH and ABTS assays), enzymatic
models involving myeloperoxidases activity modulation (classical and SIEFED assays), and a cellular
model involving neutrophil response modulation. Commercial fishmeal and chickenmeal were used as
industrial benchmarks. The outcomes of the present study reveal that fishmeal and chickenmeal oer
little to no advantage in terms of suppressing the oxidative damage occurring as a result of neutrophil
response and myeloperoxidase activity. Moreover, fishmeal and chickenmeal also exhibit pro-oxidant
behavior in some of the models used in this study. Results indicate that black soldier fly proteins and
protein hydrolysate could be eective in protecting against the cellular damage resulting from host
Animals 2020,10, 941 14 of 16
neutrophil and myeloperoxidase response. Therefore, the black soldier fly derivatives used in this
study show advantages over chickenmeal and fishmeal for inclusion in pet food and aquaculture feed
formulations. In the future, it could be interesting to validate the fundamental
in vitro
knowledge
developed during this study using in vivo animal models.
Author Contributions:
Conceptualization, A.M.-M. and T.F.; methodology, A.M.-M.; formal analysis, A.M.-M.;
investigation, A.M.-M.; writing—original draft preparation, A.M.-M., A.P., E.S.; writing—review and editing,
M.D., N.M.T., M.v.S., M.D. and D.S.; supervision, A.M.-M. and T.F. All authors have read and agreed to the
published version of the manuscript.
Funding: This research received no external funding.
Acknowledgments:
Authors are thankful to Roel Boersma and Tarique Arsiwalla from Protix B.V. to provide
valuable inputs regarding the animal nutrition market. The authors are grateful to the guest editors of this journal
special issue for acknowledging the scientific quality of this research and exempting the publication fee.
Conflicts of Interest:
This research was sponsored by Protix. The sponsor is an industrial scale producer of black
soldier fly larvae proteins. The sponsor had no role in the design of the study; in the collection; analyses; or
interpretation of the data. However, the sponsor of this study does have a role in the writing of the manuscript.
The main motivation behind this manuscript is to inform the audience about the health promoting potential of
black soldier fly larvae protein derivatives.
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... However, BSF fats have received little attention from pet food producers till now. These fats contain bioactive fatty components (Mouithys-Mickalad et al., 2020;Veldkamp et al., 2022) and have a carbon footprint lower than fats with comparable fatty acid profile such as palm kernel oil (Agri-footprint®, n. d.; Smetana, 2020;Smetana et al., 2019). These properties make BSF fats interesting candidates for inclusion in pet food formulations. ...
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Black soldier fly (BSF) larvae derived ingredients are being considered as sustainable alternatives to conventional animal derived ingredients in pet food formulations. In Europe, legislations permit the use of BSF fat in pet food formulations. However, BSF fat has received very little attention from pet food producers till now. This article examines literature regarding fatty acid profile and some minor components reported in BSF fat. Literature on digestibility, antimicrobial activity, intestinal immunity, and brain health improvement potential has also been carefully reviewed. Finally, some examples of pet food brands using BSF fat commercially were mentioned, and compilation of consumer review scores from popular review websites have also been illustrated. Literature reviewed in this article indicate that BSF fat is highly digestible and has interesting health benefits, thus supporting its use as functional pet food ingredient. This article also identifies the knowledge gap in the current literature. It is essential to fill this knowledge gap to facilitate the wider use of BSF fat in canine and feline diets.
... material/referenced in article. [31,32,33,34,35,36,37,38,39,40,41,42,43,44,45] Table 4. Advantages and disadvantages of fish feed alternativesoil. ...
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As the population grows, demand for food increases. Fish is considered to be one of the most efficient sources of protein. But as demand increases, we need to think about the efficient and sustainable fish feed. There is a need to replace existing feed ingredients such as fishmeal and fish oil with more sustainable sources of protein and oil. In 1990, fish feed consisted mainly of fishmeal and fish oil, but today’s fish feed is dominated by vegetable protein and vegetable oil. Comparing the advantages and disadvantages of the alternatives is concluded that previously used fish feed ingredients such as fishmeal and fish oil are not the most efficient, sustainable, and economically viable resources. The comparison shows why the composition of fish feed has shifted from 1990 to 2020 towards the use of plant resources in fish feed, as plant resources are more efficient, sustainable, and economically viable.
... Up to 80% of waste mass can be reduced and turned into larvae biomass with a biomass conversion rate (BCR) of 20% based on dry matter (Diener et al., 2011). Due to its high fat and protein content amongst other properties, BSFL have shown to be an ideal source for animal feed and pet food (Chia et al., 2019;Mouithys-Mickalad et al., 2020). BSFL can be processed to various products, such as dried larvae, protein meal, oil, biodiesel, chitin, or used directly as unprocessed fresh larvae (Joly and Nikiema, 2019). ...
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Black soldier fly larvae (BSFL) waste processing has proven to be a promising approach, which can be applied in centralised or decentralised settings. Financial considerations however still remain undisclosed given business secrecy. Building on the experiences of a facility and operational setup in Indonesia, this study conducted an in-depth cost analysis using methods such as time motion studies, activity based costing and a mass flow balance to evaluate business models. Thereafter followed an analysis of different scenarios on how to integrate the approach into the solid waste management system of Surabaya, Indonesia assuming different degrees of decentralisation with market rates and prices from 2020. Results show that a centralised plant, managing all steps of the BSFL process (Scenario 1), achieves the highest net present value (NPV), mainly through the economy of scale. When BSFL waste treatment is conducted using a decentralised approach, close to the waste sources, and the nursery and post-processing of larvae remains at a central location (Scenario 3), other benefits besides the still positive NPV can be shown. Such a scenario for instance would reduce the spatial footprint of the central facility, create jobs within the communities and reduce the transport costs and total costs for the municipality. Another scenario (Scenario 2) hypothesises a decentralised waste treatment as well as post-processing of larvae at the same decentralised location increasing storability and the value of products. Although this achieves higher NPV values compared to Scenario 3, it requires larger scale units to achieve financial viability. Coordination and facilitation by local government can further strengthen a network of decentralised BSFL treatment plants, as the city authorities could focus on operating a centralised nursery (Scenario 2), supplying young larvae at low cost or free of charge to increase the financial viability of the decentralised plants treating the waste.
... The black soldier fly (BSF, Hermetia illucens, Stratiomyidae) is one such insect species for which mass-rearing is currently undergoing fast worldwide growth [9][10][11] and from which no viruses have so far been described. Most research on BSFs focuses on rearing optimization and application as a prominent source of proteins for the food and feed industry [9,10,12,13], as well as in biotechnology [12][13][14][15][16][17]. BSFs appear particularly robust and resistant to diseases. ...
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Black soldier flies (BSFs, Hermetia illucens) are becoming a prominent research model encouraged by the insect as food and feed and waste bioconversion industries. Insect mass-rearing facilities are at risk from the spread of viruses, but so far, none have been described in BSFs. To fill this knowledge gap, a bioinformatic approach was undertaken to discover viruses specifically associated with BSFs. First, BSF genomes were screened for the presence of endogenous viral elements (EVEs). This led to the discovery and mapping of seven orthologous EVEs integrated into three BSF genomes originating from five viral families. Secondly, a virus discovery pipeline was used to screen BSF transcriptomes. This led to detecting a new exogenous totivirus that we named hermetia illucens totivirus 1 (HiTV1). Phylogenetic analyses showed this virus belongs to a clade of insect-specific totiviruses and is closely related to the largest EVE located on chromosome 1 of the BSF genome. Lastly, this EVE was found to express a small transcript in some BSFs infected by HiTV1. Altogether, this data mining study showed that far from being unscathed from viruses, BSFs bear traces of past interactions with several viral families and of present interactions with the exogenous HiTV1.
... Also in gilthead sea bream, several genes related to the antioxidant defence were down-regulated in fish fed macroalgae supplemented diets, being this finding attributed to the fact that the diets containing compounds with antioxidant properties have reduced requirements for host antioxidant enzymes (Magnoni et al., 2017). Besides, the NoPAP SANA diet contained higher amounts of black soldier meal and bacterial by-products, which have already been described to possess antioxidant activity (Medvedkova et al., 2009;Mouithys-Mickalad et al., 2020). ...
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The exponential growth of the aquaculture sector requires the development of sustainable aquafeeds with less dependence on marine products. The maximized replacement of fish meal (FM) and fish oil (FO) with plant ingredients has been extensively studied in the economically important species gilthead sea bream (Sparus aurata). Recently, major progress has been done with other alternative raw materials, though some non-pathological inflammatory response persisted with feed formulations that increased the circularity of resource utilization. In the present study, we evaluated the effects on growth performance, gene expression, intestinal microbiota and disease resistance of a FM-free diet (NoPAP SANA), based on plant ingredients, aquaculture by-products, algae oil, insect meal and bacterial fermentation biomasses as main dietary oil and protein sources, and supplemented with a commercially available health-promoting feed additive (SANACORE®GM). Juveniles of 21 g initial body weight were fed control or NoPAP SANA diets for 34 days, and head kidney, liver and posterior intestine were collected for gene expression analyses using customized PCR-arrays. Each tissue-specific PCR covered 96 genes in total and included markers of growth performance, lipid and energy metabolism, antioxidant defence, immune system, and intestinal function and integrity. From the same fish, the adherent bacteria of the posterior intestine were studied by Illumina sequencing of the V3-V4 region of the 16S rRNA. The remaining fish were challenged with the intestinal parasite Enteromyxum leei for 78 days and sampled for parasite diagnosis. Both control and NoPAP SANA fish grew efficiently considering gilthead sea bream standards. Before parasite challenge, the NoPAP SANA group showed differential expression of 17, 2 and 4 genes in liver, head kidney and posterior intestine, respectively. The intestinal bacterial composition showed no major differences in diversity or at the phylum level. However, 29 abundant OTUs significantly changed with the diet. From these, 10 OTUs were significantly correlated with differentially expressed genes in the different target tissues. Inferred metagenome analyses revealed that the altered microbiota with NoPAP SANA diet could account for changes in 15 metabolic pathways. The intensity and prevalence of infection after the parasite challenge did not significantly vary between dietary treatments, and infected fish from both groups showed similar disease outcome. Altogether, these results indicate that the NoPAP SANA diet promoted optimal growth and a healthy condition in gilthead sea bream without affecting susceptibility against the tested intestinal parasite, as often observed with alternative diets following current industry formulations.
... Black soldier fly (BSF) larvae have been declared to be the most auspicious insect aimed at biomass production as an anticipated waste recycler . Pupa and larval stages of BSF could serve as a mainstream of valuable nutrients for livestock, fisheries, pets, lab animals, and certainly for humans (Mouithys-Mickalad et al., 2020). Subsequently, the BSF larvae-centred foodstuffs can substitute the conventional ones and turn out to be a noteworthy protein substitute in the forthcoming years (Joly and Nikiema, 2019). ...
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The use of insect-derived products in animal nutrition attracts great attention and shows a great opportunity for meeting the increasing feed raw material demand. Not only the protein fraction is of interest, but also fat and bioactive compounds are present in insects. In the aquaculture sector, the interest in the use of insects as feed pushed the European Union to allow, in 2017, the inclusion of insect-derived protein from seven insect species in aquafeeds. After a brief introduction, this chapter reviews and reports a synthesis of the available literature about the dietary use of insect protein, fat and bioactive compounds in freshwater and marine fish species with a special focus on fish performance and health. In the end, the chapter also highlights the challenges to be solved to allow the real inclusion of these products in aquafeeds.
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A feeding trial was conducted to test the growth potential, nutritional utilization, liver health and fillet sensory parameters of sea-water Atlantic salmon (Salmo salar L.) fed diets with increasing substitution of fish meal with insect meal. The insect meal was produced from black soldier fly larvae (Hermetia illucens, L.). Triplicate sea-cages of salmon were fed one of four isonitrogenous and isolipidic diets for 16 weeks. The control diet (IM0) contained 100 g kg⁻¹ fish meal, which was replaced up to 100% with insect meal (33% (IM33), 66% (IM66) and 100% (IM100)), corresponding to dietary insect meal inclusion levels at 50 g kg⁻¹, 100 g kg⁻¹ and 150 g kg⁻¹, respectively. Replacing the dietary fish meal with insect meal did not affect the apparent digestibility coefficients (ADC) of protein, lipid, amino acids and fatty acids, or the digestive enzyme activities. Feed intake, daily growth increase, and feed conversion ratio were also unaffected by the inclusion of insect meal in the diets. Whole body protein, lipid and amino acid composition were not affected by dietary substitution of fish meal with insect meal, while the whole body fatty acid composition generally reflected that of the diets. Liver lipid accumulation was not affected by replacing the fishmeal with insect meal, as assessed by both histological examinations and chemical analyses. The sensory testing of the fillet revealed only small changes in the fillet sensory quality. In general, this study showed that a total replacement of fish meal with black soldier fly larvae meal in the diets of sea-water Atlantic salmon was possible without negative effects on growth performance, feed utilization, nutrient digestibility, liver traits or the sensory qualities of the fillet.
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