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The effect of fermentation time and Trichoderma levels on digestibility and chemical components of Black Soldier fly (Hermetia illucens) larvae

M Mulyono, V D Yunianto, N Suthama and D Sunarti

Faculty of Animal and Agricultural Sciences, Diponegoro University, Tembalang Campus, Semarang 50275, Central Java - Indonesia


The study aimed to evaluate the chemical composition and digestibility of fermented Black Soldier Fly/BSF (Hermetia illucens) larvae by Trichoderma. The study was set as a 2×4 with three replicates in a randomized factorial design with fermentation time (0 and 72 hours) and the level of Trichoderma culture (0, 2, 4 and 6%) as the first and the second factors. The fermentation time reduced crude protein (CP), ether extract (EE), in vitro dry matter digestibility, and in vivo dry matter digestibility, but increased the chitin, ash, and crude fiber (CF). The levels of Trichoderma culture increased chitin, ash, and CF, but also it decreased the EE content. Fermentation of BSF larval meal using Trichoderma reduced its digestibility; however, the addition of 2% Trichoderma without fermentation increased the digestibility of BSF larvae.

Keywords: chitin, insect, in vitro


Recently, there has been a growing interest to use insects as a feed ingredient for poultry, since the insect is rich in protein, polyunsaturated fatty acids, and antimicrobial peptides that may be beneficial for the growth and health performance of poultry (Józefiak et al 2016). There are many types of insects that can be exploited as poultry feed ingredients, one of which is black soldier fly (BSF, Hermetia illucens). The larvae of BSF contains 44% dry matter, 42% crude protein, and 35% fat, including essential amino acids and fatty acids (Sheppard et al 2002). The amino acid pattern of larvae meal of BSF is very similar to that of fishmeal (Elwert et al 2010), so it can be used to partly substitute the proportion of fishmeal in the poultry rations. However, it should be noticed that the exoskeleton larvae of BSF contain chitin polysaccharide (Henry et al 2015) of about 8.7%, which can negatively affect the digestibility and utilization of nutrients (Shiau and Yu 1999; Diener et al 2009; Marono et al 2015). Moreover, high chitin levels in insects may impose negative effects on feed intake and protein use in poultry (Longvah et al 2011). However, it is noted that reducing the nutrient utilities may compromise the growth performance of poultry (Kroeckel et al 2012). Therefore, reducing the content of chitin, and thus improving the digestibility BSF larvae, seems to be necessary when using BSF larvae as a feed ingredient for poultry.

Reducing the chitin content on organic materials can be performed by fermentation using specific microorganisms that are able to degrade chitin (Beier and Bertilsson 2013; Brzezinska et al 2014). Trichoderma is one of the fungi that can produce chitinase enzymes that can degrade chitin. Trichoderma spp., is known to produce chitinases, β-13-glucanases, and proteases (Elad et al 1982; Sandhya et al 2004). The chitinase enzyme is an enzyme capable of overhauling the chitin polymer into the monomer unit of N-acetyl glucosamine (Ulhoa and Peberdy 1991; Isahak et al 2014). Previous studies showed that fermentation of agro-industrial wastes such as brewer's dried grains, rice bran, palm kernel, and maize bran using Trichoderma increased the protein content (Iyayi and Aderolu 2004). The use of Trichoderma viridae at the level of 4% during fermentation for 48 hours could reduce chitin content in shrimp shell waste from about 25% to 3.01% (Palupi and Imsya 2011).

To the best of our knowledge, no data concerning the fermentation of BSF larvae using Trichoderma can be found in the literature. Therefore, this present study aimed to evaluate the effect of Trichoderma fermentation on the chitin content, proximate compositions, and digestibility of BSF larvae.

Materials and Methods

This research was conducted at the Laboratory of Nutrition and Feed Science Faculty of Animal and Agricultural Sciences, Diponegoro University. The BSF larvae were obtained from the laying farm in Kalisidi village, West Ungaran, Semarang, Central Java, Indonesia. The inoculum was TRICHOR-TM with T. viridae (108 CFU/g), T. harzianum (7x108 CFU/g), Trichoderma spp., (7x108 CFU/g) purchased from CV Pradipta Paramita Solo, Central Java, Indonesia.

The study was set as a 2×4 randomized factorial design with fermentation time (0 and 72 hours) and the level of Trichoderma culture (0, 2, 4, and 6%) as the first and the second factors. The experiment was performed in triplicate. The measured parameters were chitin, proximate components, in vitro dry matter digestibility, and in vivo dry matter digestibility. Fermentation was done by the solid-state method. The BSF larvae meal were put into a plastic bag and weighed for 100 g each, and then sterilized using an autoclave at 121°C for 15 minutes. After sterilization, it was inoculated with Trichoderma culture according to the doses of 2, 4, and 6%. The mixture was then added with water until the moisture content reaches approximately 50%, mixed thoroughly, and incubated for 0 and 72 hours. The rest of the BSF larval meal was not fermented and used as a control. After incubation, the larvae were harvested, weighed, and then placed in aluminum foil and subsequently dried in the oven at 50°C for 36 hours. The samples were used for the determination of chitin, proximate components and digestibility.

Data collection

The proximate components were analyzed by applying the AOAC method (AOAC 1990). The in vitro digestibility was analyzed according to the AOAC method 971.09 (AOAC 1990) with some modifications. Briefly, about 1 g of the sample is weighed in a test tube, added with 10 ml of 0.2% pepsin in 0.1N HCl, and then incubated at 40°C for 20 hours in a water bath. Then, the contents of the test tube were filtered using filter paper commonly used to weigh. The dietary dry matter was analyzed by drying the sample at 105°C until a constant weight was obtained. In vitro dry matter digestibility (DMD) is obtained through the following equation:

DMD = (DM initial)-(DM residual)/DM initial*100%

In vivo digestibility was measured using total excreta collection according to El-Husseiny et al (2007). Briefly, 48 quails of 8 weeks old with body weights of 162 ± 11.2 g were placed in 24 metabolic cages (45×45×25 cm) and fed with BSF larvae for 4 days of the collection period. The excreta were collected in a plastic tray under the cage. The excreta collection was performed for 2 days and all contaminants, such as feed particles and feathers, were discarded. The excreta were weighed and then sun-dried to obtain the air-dry weight. Dry matter of feed and excreta was determined by drying samples at 105°C until the constant weight. In vivo dry matter digestibility was calculated by the following equation:

DMD = (DM intake-DM excreta)/DM intake*100%

Chitin extraction was performed according to Ghanem et al (2003). About 1.0 g of dried sample was mixed with 12.5 ml NaOH 2.5 N, placed in an oven at 75°C for 6 hours and then filtered. The filtrate residue, called raw chitin, was dried at 105°C in an electric oven for 1 hour. After drying, the raw chitin was mixed with 10 ml of HCl 1.7 N for 6 hours and filtered. The filtrate residue was washed with 95% ethanol followed by the washing with distilled water. The filtrate residue was dried and eventually weighed as chitin. Statistical analysis Data were analyzed with the general linear model (GLM) – analysis of variance (ANOVA) procedure using the SPSS 16.0 statistical software. The differences among the treatments′ means were examined by Duncan’s multiple range test (Steel et al 1997). The level of significance was p<0.05.

Results and Discussion


There was no interaction between fermentation times with Trichoderma levels in terms of chitin (p>0.05), on the other hand, both of the fermentation time and Trichoderma culture were significant to chitin (p<0.05). The fermentation time shows that after 3h, chitin increased from 9.8 to 10.3%. The chitin content after Trichoderma culture for 2, 4 and 6h was increased by 5.2, 6.7 and 7.3% respectively. The present study is in contrast to the study of Palupi and Imsya (2011). The increased content of chitin might be due to the addition of chitin from the mycelium and cell walls of Trichoderma which consist of chitin substances (Cauchie 2002; Hamid et al 2013). The chitin has a function similar to cellulose (Arbia et al 2013).

Crude protein

Trichoderma levels did not affect CP, however, the time of fermentation decreased the CP. The fermentation time decreased the crude protein of BSF larvae presumably due to the proteolytic enzyme activity produced by Trichoderma which breaks down protein (Jayalakshmi et al 2009; Sharma 2011). However, the biosynthesis of biomass proteins was less than the protein breakdown. Trichoderma will utilize the organic matter of BSF larvae including proteins. This result was consistent with the study of Palupi and Imsya (2011), that the duration of fermentation could reduce the crude protein content.

Table 1. The chitin, proximate composition, and digestibility of fermented BSF larvae meal (% DM basis)
Chitin Ash CF CP EE In vitro
In vivo
Time (H)
0 hour (H0) 9.8b 15.3b 10.5b 40.2a 25.6a 40.6a 64.8a
72 hour (H3) 10.3a 20.5a 11.1a 39.1b 19.0b 35.6b 59.8b
SEM 0.12 0.18 0.09 0.28 0.20 0.33 0.39
p 0.027 <0.001 <0.001 0.011 <0.001 <0.001 <0.001
Trichoderma (T)
0% (T0) 9.6b 15.1c 10.5b 40.5 26.0a 38.6a 63.2ab
2% (T2) 10.1a 18.2b 10.6b 39.1 22.2b 39.1a 63.4a
4% (T4) 10.2a 18.5b 11.0a 39.3 21.3c 38.4a 61.7bc
6% (T6) 10.3a 19.8a 11.0a 39.6 19.9d 36.4b 60.9c
SEM 0.17 0.26 0.12 0.40 0.29 0.47 0.55
p 0.039 <0.001 0.009 0.118 <0.001 0.004 0.013
Interaction (HxT)
H0T0 9.7 15.1c 10.4 40.4 26.0a 38.4c 63.3b
H0T2 9.7 15.2c 10.4 40.1 26.0a 42.8a 66.7a
H0T4 9.8 15.3c 10.6 40.3 25.9a 41.3ab 65.1ab
H0T6 10.1 15.5c 10.5 40.0 25.8a 39.9bc 64.3ab
H3T0 9.4 15.2c 10.6 40.5 24.9a 38.7c 63.2b
H3T2 10.6 21.1b 10.8 38.1 18.4b 35.5d 60.2c
H3T4 10.6 21.7b 11.5 38.4 16.7c 35.4d 58.3cd
H3T6 10.5 24.0a 11.6 39.2 15.0d 32.8e 57.6d
SEM 0.24 0.36 0.18 0.57 0.41 0.66 0.78
p 0.150 <0.001 0.069 0.248 <0.001 <0.001 0.001
CF= crude fiber, CP = crude protein, EE = ether extract, DMD = dry matter digestibility,
a-d Means in a column with different superscripts differ at p<0.05
Ether extract

There was an interaction between fermentation time with Trichoderma level in terms of the EE. The EE content at the treatments of H3T2, H3T4 and H3T6 decreased by 28.7, 45.7 and 46.5%, respectively. The longer the fermentation time and the more the amount of Trichoderma inoculum reduced the content of EE. The enzyme activities of Trichoderma were in accordance with the substrate (Gusnawaty et al 2017) which produced hydrolytic lipase (Jayalakshmi et al 2009; Saba et al 2012; Toscano et al 2013). A decrease in EE content might be due to the activity of the lipase enzyme, which degraded EE and used it as energy for proliferation.

Crude fiber

Both the time of fermentation and the levels of Trichoderma culture increased the content of crude fiber. The crude fiber content in H3 increased by 6.3% compared to H0. Along with the dosage of Trichoderma, the CF in T2, T4, and T6 increased by 1.2 5.1 and 5.1%, respectively. Data from the present study are in line with the studies of Ezekiel et al (2010), Ahmed et al (2017), Sugiharto et al (2018), in which the fermentation could increase the content of crude fiber. On the other hand, it was in contrast to the studies of Ofuya and Nwanjiuba (1990), Iyayi and Losel (2001), Iyayi and Aderolu (2004). Although Trichoderma produces cellulase enzymes, the work of this enzyme is not considered optimal, since the extracellular enzyme activity of Trichoderma depends on the composition of the substrate. The increase of the CF content is thought to be due to the addition of CF from the body of the fungus. Fungus mycelium and sporangia wall mainly consist of chitin, which is a compound that has a function similar to cellulose in plant cells (Arbia et al 2013).


There was an interaction between the fermentation time and the Trichoderma levels in ash content. The ash content increased in the treatments of H3T2, H3T4, and H3T6 by 33.1%, 39.1%, and 57.5%, respectively. During fermentation, Trichoderma degraded organic matter as food to build their cells and obtained the energy needed for mycelium proliferation and growth (Ali et al 2015). As a result, it increases ash content due to the decreased organic matter after fermentation. This is presumably due to the activity of Trichoderma extracellular enzymes that degrade organic matter of BSF larval especially fat content that causes shrinkage of organic matter. The decrease in organic matter resulted in an increase in the proportion of ash content. This is in line with the studies of Oboh (2006), Ezekiel et al (2010), and Ahmed et al (2017), that fermentation increases the ash content.

In vitro and in vivo digestibility

It appears that the BSF larval meal added with 2% Trichoderma culture and incubated at 0 hours had the highest in vitro DMD and in vivo DMD. On the other hand, the lowest in vitro DMD and in vivo DMD were observed in BSF larval meal added with 2% Trichoderma culture and incubated for 72 hours. The results of this study indicate that the fermentation of BSF larvae meal with Trichoderma culture reduced the digestibility. There are several possibilities due to a decrease of organic matter and an increase in ash, crude fiber and chitin content (Table 1), which negatively affect the digestibility of dry matter. The high content of ash, crude fiber, and chitin will reduce digestibility. Increasing crude fiber will reduce the digestibility, this is due to the limited digestion by cellulase enzyme in the digestive tract.

During the fermentation process, carbon and nitrogen compounds are used as energy to multiply and produce enzymes. The activity of the enzymes produced by Trichoderma spp., (carboxylase, protease, and lipase) will break down the components in BSF larval meal according to the function of each enzyme. Trichoderma spp. is known to produce enzymes mucolytics such as β-1, 3 glucanase, β-1, 4 endo-glucanase, chitinase, and protease (Harman 2006; Elad et al 1982), lipase (Saba et al 2012). The microorganism enzymes degrade complex compounds such as proteins, carbohydrates, and fats into simpler compounds. Proteolytic activity breaks down protein into amino acids, while fat is used as an energy source for microbial growth by the lipase enzyme. However, Kelley (1977) states that the growth of Trichoderma ssp., is dependent on the availability of carbohydrates used as an energy source for growth.



The research was supported by the Doctoral Research grants by the Ministry of Research, Technology and Higher Education of the Republic of Indonesia through the Domestic Graduate Scholarship program.


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Received 1 June 2019; Accepted 22 August 2019; Published 2 October 2019

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