BIOTROPIA Vol. 28 No. 1,2021: 64 - 73 DOI: 10.11598/btb.2021.28.1.1082 

OMEGA-3 CONTENT OF BLACK SOLDIER FLY PREPUPA 
(Hefmetia illucens) FED WITH MARINE FISH OFFAL AND 

TOFU DREG 

RAMADHAS1 EIG4 PUTRA'>Z*, YENYEN FATMALASARI', IDA I(INASIH3, 
AGUS DANA PERMANA', MIA ROSMIATIl AND ELISA NURMA RIANA2 

'School ofLife Sciences and Technology, Institut Teknologi Bandzmg, Jalan Ganesha 10, Bandung 40132, Indonesia 
'Dtpan%ent o f  Biolog, Institut Teknologi Sumatera, Jalan Temsan Rjacudu, Lmpzlng 35365, Indonesia 

'Dtpar;tment ofBiology, Uniuersitas Islam Negeri Sunan Gunung Djati Bandzmg, Cibim, Bandung 40615, Indonesia 

Received 10 June 2018/Accepted 9 December 2019 

ABSTRACT 

One of the most promising materials with great potential as a nutrition source of animal feed is the black 
soldier fly prepupae (BSFP) (Hemetia illucens) which is fed with organic wastes. This study was designed to 
observe the accumulation of omega-3 from the organic wastes of marine fish offal and tofu dregs converted as 
the biomass of the harvested BSFP. A total of 150 g food consisted of marine fish offal and tofu dreg with 
different proportions of fish offa1:tofu dreg at 10:90, 25:75, 50:50, and 100% tofu dregs were fed to black soldier 
fly larvae (BSFL) for 21 days. At the end of the feeding period, all BSFL were harvested, weighed, and then 
analyzed for omega-3 fatty acids content using Gas Cromatography with Flame Ionization Detector (GC-FID). 
Results showed that BSFL reared with 25% marine fish offal produced the highest biomass (8.1 g/50 larvae) with 
the least development time (19 days). The total omega-3 recorded from the application of OO/o, 10°/o, 25%, and 
50% of marine fish offal was 0.02%, 0.87%, 2.16% and 2.61% in 100 g of dry weight, respectively. These results 
showed the possibility of producing specific nutrients from BSFP biomass converted from organic wastes, 
thereby providing baseline knowledge for further formulation of specific animal feed from BSFP. 

Keywords: black soldier fly, fish offal, omega-3, tofu dregs 

INTRODUCTION 

Organic waste is the most dominant waste 
produced in Indonesia (Zulfikar & Chaerul 
2010). Most of these end up as piles of wastes as 
the collection and waste management efforts 
could only cover about 50 - 70% of wastes 
produced by the population (Cointreau 1994; 
Rushbrook & Pugh 1999). Common methods 
applied to manage these wastes include the 
costly sanitary landfill and composting which 
produce compost of low economic value, 
thereby making both methods less desirable in 
low income regions (Cointreau 1994). 

Another alternative in managing organic 
waste is its conversion into useful biomass using 
saprophages through the natural decomposition 

*Corresponding author, email: ramadhani@sith.itb.ac.id 

process (Beard & Sands 1973; Boushy 1991; 
Barnard et  al. 1998; Ndegwa & Thompson 2001; 
Ramos-Elorduy et  aL 2002; Barry 2004; Diener et 
al. 2009; Manurung et  al. 2016; Supriyatna e t  al. 
2016). Latest developments include the 
increasingly becoming popular trend of using 
insect larvae as organic wastes decomposting 
agents, in producing a protein- and lipid-rich 
biomass (Diener et  al. 2009). Among these, the 
Black Soldier Flies Larvae (BSFL) (Hermetid 
i h c e n s )  has been considered to be the best 
canddate due to their great ability to convert 
various types of organic wastes into high 
protein-rich biomass which could be high 
quality sources of protein and lipid for animal 
feed, as well as, potential sources of biodiesel 
(Newton et  al. 1977; Newton et  al. 2005a, b; St- 
Hilaire et  al. 2007a; Myers et  al. 2008; 
Rachmawati et  al. 2010; Li et  al. 201 1; Muin et  al. 



Improvement of Omega-3 content of black soldier fly Prepua (Hemetin illzcen~) . . ... - Eka Putra et  al. 

2017; Renna et al. 2017; Spranghers e t  al. 2017; 
IGnasih et al. 201 8). 

Other benefits of using H. illzcens as organic 
wastes conversion agent, especially in tropical 
regions like Indonesia, are: 1. the species is 
adapted to tropic and warm subtropical region 
(Tomberlin et al. 2002); 2. able to suppress the 
population of house flies (Musca domestica) 
through predation and competition for nutrition 
(Furman e t  al. 1959; Newton et al. 2005a, b); 3. 
able to reduce pathogens like Escberichia cob and 
Salmonella entericd (Erickson et al. 2004); 4. has 
short development time (about 2 to 3 weeks) 
(Newby 1997); 5. adult flies do not eat fresh 
material and females do not oviposit their eggs 
directly on food source of larvae which made 
them less likely to tranfer disease (Leclercq 
1997); and 6. adult flies are not attracted to 
human habitats or food (van Huis et a/. 2013). 
Moreover, ths  insect has digestive system rich 
with amylase, lipase, protease, a-galactosidase, P- 
galactosidase, a-mannosidase, and a-fucosidase 
allowing them to digest almost any organic 
materials (Jam et al. 2011). Like other insects, 
their digestive system also allows them to 
accumulate certain organic molecules (St-Hilaire 
et al. 2007b). 
All of these benefits can be harnessed to 

solve one of the main obstacles of food security 
in Indonesia, particularly protein insufficiency, 
or the limited availability of protein rich yet 
affordable feed stock. Among organic wastes 
produced in large volumes in Indonesia, wastes 
with relatively unchanged organic matter 
properties during its production are tofu dreg 
and marine fish offal. About 731,501.5 ton of 
tofu dreg is produced annually (Bisnis UI(M 
2009), while 25 - 30% of marine fish catch 
ended up as wastes (ICI<P 2016). Valuable 
organic wastes such as tofu dreg is rich in 
carbohydrate and protein (Li et al. 2012; Li e t  al. 
2013), while the marine fish tissues are rich in 
beneficial unsaturated fatty acids (Sahena e t  al. 
2009). 

The nutritional content of BSFL biomass 
highly depends on quantity and quality of the 
feeding material (Nguyen e t  al. 2015; Oonincx et 
al. 2015). Moreover, the fat content showed 
more variation (6.6 to 39.2%) than protein (37.0 
to 62.7%) (Barragan-Fonseca e t  al. 2017) 
suggesting the strong effect of the larval dietary 
nutrient to protein and fat content. One main 

purpose of BSFL production is to manufacture 
an alternative source of feed material for several 
livestock, such as poultry (Leiber et al. 2017; 
Ssepuuya et aL 2017) and cultured fish species 
(Webster et al. 2015; Stadtlander et al. 2017). A 
growing number of studies showed the 
possibility of modifying the fatty acid profile of 
BSFL, improving the fat nutrient value (i.e., 
healthier fats such as omega-3) of local livestock 
product through manipulation of feed for better 
valuation of the product (Stoneham et al. 2018). 

The lipid content of insects is largely 
dependent on their diet (Stanley-Samuelson & 
Dadd 1983) and many of them have natural 
long-chain unsaturated fatty acids in their 
biomass (Thompson 1973). These omega-3 
content of BSFL could be enhanced by allowing 
them to feed on fish waste from rainbow trout 
processing plant (St-Hilare et al. 2007b). Unlike 
rainbow trout, most marine tropical fishes have 
low omega-3 content. In h s  study, the 
combination of both marine fish offal and tofu 
dreg were utilized as feeding material of BSFL 
to enhanced the omega-3 content of its biomass. 
The main objective of this study is to produce 
BSFL biomass with high omega-3 content at 
laboratory scale as a basis for large scale 
production of an Omega-3 rich alternative 
source of livestock feed converted from organic 
wastes. 

MATERIALS AND METHODS 

Animal Specimen 

The larvae of the black solder fly were 
obtained from the eggs that were purchased 
from BSF farm in Sumedang, West Java. All 
eggs were kept in the substance made of 
commercial chicken feed (60% moisture) and 
kept at a constant temperature (28 "C, 70% RH) 
in a container (50 x 25 x 10 cm) at the 
Laboratory of Enviromental Toxicology, School 
of Lfe  Sciences and Technology, Bandung, 
Indonesia. 

Animal Treatment 

The marine fish offal originally from local 
fish market and tofu dreg, produced as waste of 
the local tofu industry of Sumedang, West Java, 



BIOTROPIA Vol. 28 No. 1,2021 

Table 1 Proximate analysis of the feeding material 

Proportion of fish offal to Protein Lipid Carbohydrate 
tofu dreg ("10) ("10) ("10) 

0% 19.59 6.35 6.07 

were used as the growth medium in ths  study. 
All materials were washed with chlorine-free 
water. Four combinations of "marine fish offal: 
tofu dreg", at 10:90; 50:50; 25:75, and 100% tofu 
dreg, were prepared as food for the Black 
Soldier Flies larvae and kept frozen for 24 hours 
before treatment. The proximate analysis was 
conducted on all feed (Table 1). 

Fifty individuals of two-day old larvae were 
placed inside plastic cups (with three replicates 
for each treatment) and fed every 3 days with 
150 mg (wet weight, 60% moisture content) of 
the food prepared according to the treatment. 
The larvae were initially placed onto the 
prepared and defrosted food inside the plastic 
cup. Larval weight and weight of remaining food 
were measured every 3 days until 40% of all 
larvae metamorphed into prepupae when 
feedmg was also stopped (Tomberlin et a/. 2002). 
All prepupae were removed daily from each 
container and weighed, then placed in plastic 
container and prepared for extraction. 
All experiments were conducted at room 

temperature of 30 - 32 "C, relative humidity of 
65 - 85%, and 12 hour photoperiod. 

Sample Preparation for Fatty Acid Analysis 

Extraction 

All prepupae were cleaned by fresh water and 
dried at 60 OC for 12 hours. About 0.15 g of 
dried sample was ground then mixed with 3 mL 
solvent (2 part Chloroform and 1 part 
methanol). The mixture was then homogenized 
for 30 min, centrifuged at 5,400 rpm at 4 "C for 
5 to 15 min and the supernatant was then 
collected. Homogenization and centrifugation 
processes were repeated 3 times. The collected 
supernatant was then evaporized to remove the 
organic solvent. 

The crude lipid sample was mixed with 4 mL 
NaOH 0.5 M with methanol solvent. About 

0.024O/o Butylated Hydroxytoluene (BHT) was 
added, and then the solution was agitated for 8 
hours at 55 "C. 

Samples obtained after the esterification 
process were added with 300 mL methanol and 
1 - 2.5% HzS04 (v/v) in MeOH, and then 
agitated for 1 hour. The n-Hexane and NaCl 
(1 : 1) solution was added to the samples and 
then centrifugated for 10 min (4,000 g) at room 
temperature. NaZS04 was then added in order to 
remove the excess water from the samples, and 
the supernatant was collected and kept in a 
separate container. 

GC-FID Analysis for Omega 3 Content 

The Omega 3 content analysis was conducted 
using Gas Chromatography-Flame Ionzation 
Detection (GC-FID). GC Column used was 
DB-5 30 m x 0.25 mm ID x 0.25 pm with Nz 
and Hz as the carrier gases. The applied injector 
temperature was 200 "C and the detector 
temperature was 300 "C. The oven temperature 
which started at 140 "C was increased to 270 "C, 
at a rate of 4 "C/min, and maintained for 7.5 
min. The fatty acid was quantified with external 
standard method based on the formula: 

As x Ces Cs = - 
Aes 

where: 
Cs = Sample concentration 
As = Sample area 
Aes = External standard area 
Ces = External standard concentration 

Data Analysis 

The final weight of harvested prepupae were 
compared across all diet composition using 
analysis of variance, with significance level set at 
P < 0.05, followed by the Tukey-IGamer HSD 
test when significant differences were detected. 
All analyses were done using SPSS version 22.0. 



Improvement of Omega-3 content of black soldier fly Prepua (Hemzetia iLLucens) . . ... - Eka Putra et al. 

RESULTS AND DISCUSSION component has altered the intake of other 

The black soldier fly larvae were able to 
recycle a proportion of fish waste which consists 
of high protein and fat. The 10% of fish offal 
fed to the larvae has slightly improved the 
weight of harvested prepupae. However, the 
ad&tion of more fish offal at 50% showed 
negative correlation to harvested weight, 
although not significant Table 2). 

The high mortality of larvae when fed with 
higher fish offal concentration was probably 
caused by: 1. lack of balance in the protein: 
carbohydrate combination of the feeding 
material. Both of these macromolecules are very 
important for growth, reproduction and survival 
of insects (Agulla e t  al. 2013). Proximate analysis 
showed the protein : carbohydrate level of 
28.54 : 1 (Table 1). Studies showed that high 
protein content of the feed material produced 
high mortality rate in BSFL population 
(Cammack & Tomberlin 2017; Barragan- 
Fonseca e t  al. 2019; Danieli e t  al. 2019); 2. the 
production of juice through natural 
decomposition of the fish offal. This juice 
increased the moisture of feeding material, 
reduced the amount of available Oz required for 
growth of insect larvae, and induced the molting 
process (Harrison e t  al. 2006; Iaok & Harrison 
2009; Harrison & Haddad 2011; Callier & 
Nijhout 201 1). 

Since BSFL were subjected to specific diet 
restrictions, the consumption of one &et 

components. Since larval weight is highly 
associated with carbohydrate content (Le Gall & 
Behmer 201 4; Barragan-Fonseca 201 9) this 
probably explains the presence of heavier larvae 
at 25% fish offal. 

Larvae fed with 25% and 50% fish offal 
required less development time to metamorph 
into the harvested prepupae (Table 1). The 
shorter development time of BSFL reared on 
high protein diet in the current study agrees with 
other results reported by Oonincx e t  aL (2015b) 
and Cammack & Tomberlin (2017). The 
development time of larvae in this study was 
even lower than those of other studies 
(Fatchurochim e t  al. 1989; Myers e t  al. 2008; 
Diener 2009; Nguyen e t  al. 2013; Oonincx e t  al. 
2015a, b; St-Hilaire e t  al. 2007b; Cammack & 

Tomberlin 2017). This is probably related to the 
higher rearing temperature used in the current 
study. However, this result indicated the 
potential applicability of this method in tropical 
region like Indonesia. 

The growth pattern showing weight gain was 
stopped at dfferent times among the groups. 
Larvae at groups of 25% and 50% fish offal, 
stopped gaining weight at lbth day while those of 
groups 0% and 10% fish offal at 19th day 
(Fig. 1). The peak weights among groups were 
similar indicating that variations in the food 
composition did not affect the critical weight for 
prepupae development. 

Table 2 Final weight and development time of harvested prepupae 

Proportion of fish offal: Fresh weight per 50 prepupae Development time Dry weight per 50 prepupae 
tofu dreg* (Yo) (8, (day) k) 

Note: * = Percent combination of fish offal: tofu dreg 



BIOTROPIA Vol. 28 No. 1,2021 

7 10 13 16 19 

Days 
0% 010% 25% m50% 

Figure 1 Changes in the weight of black soldier fly larvae fed with various combination of fish offal and tofu dreg 
Notes: Data on 50% fish offal are not shown as more than 40% of larvae already metamorphosed into 

prepupae; * = significant at P < 0.05. 

Most of the fatty acid content of BSFP 
biomass consisted of saturated fatty acid (Fig. 2). 
Similar results of highly saturated fatty acid 
content of black soldier fly prepupae were also 
obtained by other studies (St-Hilarie 2007b; 
Finke 2013; Ramos-Bueno e t  al. 2016; Surendra 
e t  al. 2016; Renna e t  al. 2017; Schiavone e t  al. 
2017; Spranghers e t  aL. 2017). The feeding 
material rich in carbohydrate but low in protein 
produced more crude fat (Barragan-Fonseca 
2019) which is related to lipogenic activity and 
incorporation of lipid as body reserve (Nestel & 
Nemny-Lavy 2008). 

The higher saturated fatty acid content in 
10°/o fish offal group indicated the possibhty of 
BSFP converting the unsaturated fatty acid into 
saturated fatty acid while hgher fat content of 
the 50% fish offal group maybe responsible for 
the higher content of unsaturated fatty acid 
(Danieli e t  al. 201 9).  

Further analysis on each component of fatty 
acid showed high content of C12:O (Lauric Acid) 
(Table 3). This conformed with other studies on 

the bioconversion of organic wastes by black 
soldier fly larvae (St-Hilarie 2007b; Sealey e t  al. 
201 1; Leong e t  al. 201 5; Spranghers e t  a/. 2017). 
The increase of lauric acid content associated 
with the decreasing amount of tofu dreg 
indicated the possible accumulation of lauric 
acid from fish offal as tropical fish tissues are 
rich in saturated fatty acid (Gopakumar & Nair 
1972). The high content of this fatty acid might 
be related to the antibacterial function of insect 
larvae (Urbanek e t  al. 2012). 

Richness in C12 : 0 found in BSF favor its 
inclusion in poultry feed since ths  fatty acid is 
better absorbed and metabolized than the long 
chain fatty acids (LCFA) for nutrition and for 
antibacterial activities (Skrivanova e t  al. 2006; 
Kim & Rhee 2016). However, hgher saturated 
fatty acid content of the harvested prepupae 
could also increase the saturated fatty acid 
content of meat when it was applied as feed, a 
condition which is undesirable for modern 
consumers who prefer healthier meat and meat 
products (Schiavone e t  al. 2017). 



Improvement of Omega-3 content of black soldier fly Prepua (Hemzetia illucens) . . . .. - Eka Putra e t  al. 

0% fish offal 10% fish offal 25% fish offal 50% fish offal 

6 

- 5 
X - - 
2 4 
.w 
LC 

3 
0 

Figure 2 Proportion of saturatted and unsaturatted fatty acid of harvested prepupae (% dry matter basis) 

Table 3 Fatty acid compostion of black soldier fly prepupe (% dry matter basis) 

5.61 

Diet combination 
Lipid acid 

0% fish offal 10% fish offal 25% fish offal 50% fish offal 

2.59 

P 

8 2 e 
1 

0 
0 

C lo:o 
C 12:o 
C 140  
C 16:l 
C 16:O 
C 18:O 
C 18:3n-3; ALA 
C 20:5 
C 20:5n-3; EPA 
C 22:6n-3: DHA 

Unsaturated Fatty Acid 

4.23 

2.6 

Total Omega-3 0.02 0.87 2.16 2.61 

1.69 1.57 

Omega-3 consists of three main lipid acids, 
ALA, EPA, and DHA. The amount of fish offal 
added to the diet was positively correlated with 
the amount of these molecules inside prepupae 
biomass. In general, the total amount of EPA 
and DHA of prepupae in this study was higher 
than simdar study by St-Hillaire e t  al. (2007b) 
(Fig. 3). Low concentration of Omega 3 in 
prepupae might be related to the low Omega 3 
content of tropical fish offal (Osman e t  al. 2001; 
Mohanty e t  al. 201 6;  Alhazzaa e t  al. 201 8). 

Combining fish wastes with other types of 
wastes could change the molecular properties of 
the biomass produced. Higher nutritional value 
of tofu dreg compared to cow manure which 
has much lower nutritional value might explain 
the difference of the results between this study 
and that of St-Hdlaire e t  al. (2007b). 

This study showed that fatty acids content of 
the black soldier fly prepupae could be increased 
and manipulated in order to produce desirable 
"healthy" fatty acids such as ALA, EPA, and 
DHA by feeding the larvae with more tropical 
marine fish waste (Barroso e t  al. 2017; Danielli et  
al. 2019). Although the application of this 

1.07 

method as an Omega-3 source for human 
consumption would still require further studies, 
ths  method is already applicable in the 
production of a high-quality animal-grade 
foodstuff that is a suitable replacement for fish 
meal, fish oil, or vegetable oils in animal diets 
through the recycling of organic wastes. 
Furthermore, the application of Omega-3 rich 
feed material may change the fatty acid profile 
of the meat into a much healthier meat that is 
rich with unsaturated fatty acid (Saito e t  al. 1996; 
Li e t  al. 2016). 

The fatty acid composition of BSF lipid 
highly depends on the rearing substrate (Makkar 
e t  al. 2014; Oonincx e t  al. 2015b; Leong e t  al. 
2016). Therefore, further research is suggested 
to assess how much fatty acid can be improved 
through variation in substrate composition due 
to differences in fatty acid requirement for 
growth rocher  2010). When the fatty acid 
profile of BSF wdl be equal to the animal diets 
that contained fish meal/oil it will reduce the 
pressures on global marine fisheries and will also 
maintain environmental health (Tomberlin e t  aL. 
2015). 

- - 



BIOTROPIA Vol. 28 No. 1,2021 

Tofu dreg 10% fish offal 25% fish offal 50% fish offal 
and 90% tofu and 75% tofu and 50% tofu 

dreg dreg dreg 

c. 3.5 - 3.5 X - OALA H EPA I3 OHA Total X - 
E 3 E 
P .- - 

2.5 
P - -- 2.5 

m - 
4 d  m 
2 2 2 - - 

1.5 1.5 
M M 

z 1 1 

0.5 
m 

0.5 
0 
P n. 

Cow manure 10% fish offal 25% fish offal 50% fish offal 
and 90% cow and 75% cow and 50% cow 

manure manure manure 

OALA HEPA BDHA .Total 

II$ F n  * 

o n  - 
Figure 3 Percentage of a-linolenic acid (ALA), eicosapentaenoic acid @PA), and docosahexaenoic acid @HA) on a 

total dry matter basis for prepupae feed diets containing different proportions of fish offal and tofu dreg (left) 
and results of Sheppard et al. (2007) (right) 
Note: Error bars indicate the pooled SD. 

CONCLUSION 

The study results showed the possible 
applicability of a three-fronged solution (from 
basically a waste material), to three major human 
challenges, namely: 1. producing a high quality 
animal feed material from tropical marine fish 
wastes; 2. reducing the voluminous pile of 
organic wastes at the national level; and 3. 
producing a "healthier" meat for human 
consumption. The application of fish offal as 
feedstuff for black solder fly larvae has also 
improved the total Omega 3 (EPA, DHA, and 
ALA) content of the harvested prepupae. 

ACKNOWLEDGEMENTS 

This study was partly and equally supported 
by the research grant from Riset Inovasi ITB 
2019 received by the first author and Indofood 
Riset Nugraha received by the second author. 

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