| Livestock Research for Rural Development 34 (10) 2022 | LRRD Search | LRRD Misssion | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
The study was carried out to determine the amino acid and fatty acid compositions of black soldier fly larvae (Hertemia illucens) fed by tofu by-products (TF). This was a completely randomised design with 2 treatments and 4 replicates. The larvae were fed by TF and collected at 2 times as 2 treatments according to the change of colour as dark yellow (7 days after rearing or 12 day old larvae, namely 7D) and black yellow (9 days after rearing or 14 days old larvae, namely 9D) for amino acid and fatty acid analyses. Results show that two essential amino acids histidine and valine were higher in 7D than in 9D (P<0.05) and crude fat contains low lauric acid and high linoleic acid (w-6) and α-linolenic acids (w-3). It is concluded that proteins in the larvae were rich in lysine and methionine as in fishmeal but poor in histidine and leucine. Ether extract of the larvae contains low lauric acid and high linoleic acid and α-linolenic acid. Beside protein, it seems that larvae can be used as good source of essential fatty acid. In recommendation, black soldier fly larvae fed by tofu by-product could be collected at 7-9 days after rearing at temperature 30-35°C for supplying sources of protein and essential fatty acids.
Keywords: larvae development, linoleic acid and α-linolenic acid, lauric acid
The black soldier fly (Hermetia illucens) has been studied in recent years as a source of animal and aquaculture feed, replacing protein-rich feed sources such as fishmeal and soybean meal (Barragan-Fonseca et al 2017; Cullere et al 2017; Spanghers et al 2017; Moula et al 2018). Black soldier fly is native to the Americas but is now widely reared around the World due to its ability to adapt to high temperature ranges (10-42oC), but at 30oC, it has a high growth potential (Lalander et al 2019). Black soldier fly larvae (BSFL) has a high nutritional content such as dry matter (DM) (35-45%), crude protein (37-63% as DM), crude fat (7-39%), crude fibre (7.0%), ash (14.6-28.4%) and 5278.49 kcal gross energy per kg DM (Barragan-Fonseca et al 2017; Sheppard et al 2008; Arango Gutierrez et al 2004; St-Hilaire et al 2007). Besides, Arango Gutierrez et al (2004) reported that larvae are rich in macro-minerals such as calcium (5-8% DM) and phosphorus (0.6-1.5% DM), and micro-minerals such as potassium (0.69% DM), sodium ( 0.13% DM), magnesium (Mg; 0.39% DM), iron (Fe; 0.14% DM), manganese (Mn; 246 mg/kg DM), zinc (Zn; 108 mg/kg DM) and copper (Cu; 6.0 mg/kg DM).
Cullere et al (2016) reported that the most abundant essential amino acid (EAA) were valine and leucine, whereas alanine and glutamic acid were rich in defatted BSF larvae meal. The AAs concentration of defatted BSF larvae meal differed from the full-fat BSF larvae meal presented by De Marco et al (2015): regarding EAAs, lysine, methionine, arginine and histidine contents were lower in defatted BSF larvae meal compared with those of the above-mentioned study, whereas for isoleucine, leu, phenylalanine, threonine and valine the situation was reversed.
The fatty acid (FA) composition of the BSF larvae depends on the FA composition of the diet. The lipids of larvae fed on cow manure contained 21% of lauric acid, 16% of palmitic acid, 32% of oleic acid and 0.2% of omega-3 FA, while these proportions were respectively 43%, 11%, 12% and 3% for larvae fed 50% fish offal and 50% cow manure (Makkar et al 2014). Total lipid content also increased from 21% to 30% DM. Feeding BSF larvae by cow manure and 22% fish offal within 24 h of their pupation was sufficient for a substantial enrichment in polyunsaturated fatty acid (PUFA), especially in docosahexaenoic acid and eicosapentaenoic acid (St-Hilaire et al 2007).
In Viet Nam, Tofu by-product (TF) from tofu processing is main source of agro-industrial by-products. TF has a high content of crude protein (20-25%) and crude fat (9-12%), and high crude fibre (50-65%) (Nguyen et al 2020). Recently, the use of TF to feed BSFL has been implemented in practice in some areas in the South. Nguyen Thi Bich Hao et al (2017) reported that BSFL developed well in TF substrate compared with chicken manure or mixture of chicken manure and TF. However, recent publications on chemical composition and nutritive values of BSFL fed TF are still limited, particularly amino acid and fatty acid compositions. This study, therefore, aimed at determing chemical, amino acid and fatty acid compositions of BSFL fed by Tofu by-products.
The experiment was conducted at the Research and Training Centre (RTC), Faculty of Animal Husbandry and Veterinary Medicine, University of Agriculture and Forestry (HUAF), Hue University from Feb. to May 2021.
The larvae of 5 days old were randomly arranged in a completely randomised design with 2 treatments and 4 replicates. The larvae were collected at 2 times as 2 treatments according to the change of colour as dark yellow and black yellow at day 7th (12 days old larvae, namely 7D) and 9 th (14 days old larvae, namely 9D) after larval rearing, respectively.
The larvae of 5 days old were reared in a plastic containers with a density of 2 larvae/cm2 (2,000 larvae/each box) at temperature of 30-35 oC and fed tofu by-products. They were fed daily at 100 mg/larvae (based on DM), ensuring excess feed for larvae to have maximum intake. Feed was prepared in the form of air-dry, after weighing, it was mixed with water at the ratio of 10 g of air dry feed and 17 g of water, ensuring that the substrate reaches a moisture content of about 70% suitable for the development of larvae, and was given once a day at 8:00am.
Samples of larvae were collected at day 7th and 9th after rearing for chemical analysis: DM, EE, CP and CF following AOAC (1990) procedures at the Lab of the Faculty of Animal Husbandry and Veterinary Medicine, HUAF.
Amino acid compositions were analysed by Performic Acid Oxidation with Acid Hydrolysis–Sodium Metabisulfite Method (AOAC 994.12; 1997) at the Lab of Biotechnology of the National Institute of Animal Sciences, Ha Noi. Fatty acid compositions were analysed by Gas Chromatography of Fatty Acid Methyl Esters Method (ISO 2017) at the Lab of Upscience, Ha Noi.
Data were presented in the form of the mean (M), standard error of the mean (SEM). The data were statistically processed by analysis of variance (ANOVA) by General Linear Model in Minitab v. 16.2 (2010). The difference between the mean values was determined by the Tukey method at a confidence level of 95%. Statistical model:
Yij = µ + Ti + eij
Where: µ is the average value; Ti is the effect of growth stage; eij is the experimental error.
Samples of adult larvae and pre-pupae were collected at day 7th and day 9th after rearing for amino acid and fatty acid analyses (Photo. 1A, B).
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| Photo 1. A 12 days old larvae (A) and 14 days old larvae (B) | |
Results in Table 1 on amino acid composition of 7D and 9D show no statistical difference (P<0.05) except for alanine, histidine and valine. As DM percentage, amino acids: alanine, histidine and valine concentrations are higher in 7L than those in 9L, and other amino acids are the same. However, as CP percentage, only amino acid alanine content of the 7L is higher than that of 9L, and other amino acids are not different.
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Table 1. Amino acid composition of full-fat larvae fed tofu by-products collected at day 7th and 9th after rearing (n=4) |
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|
Amino acids |
% dry matter |
Source |
% crude protein |
||||||||
|
7D |
9D |
SEM |
p |
7D |
9D |
SEM |
p |
||||
|
Alanine |
2.42a |
2.02b |
0.059 |
0.009 |
2.33 |
4.13a |
3.70b |
0.105 |
0.045 |
||
|
Arginine |
2.19 |
1.97 |
0.281 |
0.598 |
2.05 |
7.74 |
7.16 |
0.314 |
0.839 |
||
|
Aspartic acid |
4.54 |
3.92 |
0.189 |
0.081 |
3.63 |
7.74 |
7.16 |
0.313 |
0.260 |
||
|
Cystine |
0.56 |
0.66 |
0.059 |
0.290 |
- |
0.95 |
1.21 |
0.107 |
0.163 |
||
|
Glycine |
2.42 |
2.12 |
0.239 |
0.429 |
1.78 |
4.12 |
3.88 |
0.443 |
0.720 |
||
|
Glutamic acid |
6.88 |
6.07 |
0.378 |
0.202 |
4.52 |
11.73 |
11.09 |
0.675 |
0.540 |
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|
Histidine |
0.98a |
0.74b |
0.055 |
0.038 |
3.16 |
1.67 |
1.35 |
0.102 |
0.094 |
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|
Isoleucine |
2.21 |
1.63 |
0.342 |
0.301 |
1.56 |
3.77 |
3.00 |
0.608 |
0.421 |
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|
Leucine |
3.53 |
3.10 |
0.312 |
0.382 |
2.71 |
6.02 |
5.67 |
0.557 |
0.681 |
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|
Lysine |
3.63 |
3.56 |
0.096 |
0.645 |
2.32 |
6.18 |
6.51 |
0.191 |
0.289 |
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|
Methionine |
2.08 |
2.03 |
0.161 |
0.812 |
2.24 |
3.56 |
3.71 |
0.278 |
0.721 |
||
|
Phenylalanine |
1.74 |
1.59 |
0.122 |
0.428 |
1.86 |
2.97 |
2.91 |
0.212 |
0.837 |
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|
Proline |
3.11 |
2.42 |
0.342 |
0.225 |
2.19 |
5.31 |
4.43 |
0.609 |
0.361 |
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|
Serine |
2.21 |
1.72 |
0.184 |
0.133 |
1.60 |
3.77 |
3.14 |
0.321 |
0.240 |
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|
Threonine |
2.49 |
2.50 |
0.089 |
0.947 |
1.84 |
4.25 |
4.57 |
0.143 |
0.185 |
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|
Valine |
3.91a |
3.22b |
0.148 |
0.030 |
1.87 |
6.66 |
5.88 |
0.269 |
0.110 |
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|
ab
: means in the same row for each parameter with
different superscripts are significantly different (p
<0.05) |
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Recently, not very much publications on amino acid composition of the BSFL are found. In this study, protein of BSFL contain 17 amino acids as previous reports of Rawski et al (2020) and Vedkamp and Bosch (2015). The authors reported that BSFL with full-fat contained 17 amino acids. In this study, however, amino acid tryptophan could not determined. Many authors indicated that BSFL proteins contained almost essential and non-essential amino acids, and the ratio of amino acids was also affected by the substrate.
In this study, the lysine and methionine contained in the 7D and 9D 3.56-3.63% and 2.03-2.08% as DM, respectively and 6.18-6.51 g/16 g N and 3.56-3.71 g/16 g N, respectively (Table 1). Vedkamp and Bosch (2015) reported also that lysine contents of larvae and pre-pupae were 6.5 and 5.7 g/16 g N, respectively, and methionine of 1.9 and 1.7 g/16 g N. Therefore, data in this study are relevant to above previous publications.
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Table 2. Amino acid profile of black soldier fly larvae (BSFL) as comparison with fish meal (FM), soybean meal (SBM) and ideal protein (IP) (g/160 g N) |
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Amino acid |
BSFL# |
FM## |
SBM## |
IP### |
|
|
Lysine |
63.5 |
67.1 |
57.2 |
70 |
|
|
Methionine |
36.4 |
24.7 |
13.8 |
18 |
|
|
Met + Cys |
47.2 |
34.8 |
29.2 |
36 |
|
|
Threonine |
41.1 |
36.9 |
37.4 |
45 |
|
|
Tryptophan |
- |
10.2 |
14.7 |
12 |
|
|
Isoleucine |
33.9 |
34.9 |
41.0 |
40 |
|
|
Leucine |
58.5 |
66.9 |
74.7 |
80 |
|
|
Valine |
62.7 |
42.2 |
43.2 |
52 |
|
|
Histidine |
15.1 |
24.1 |
26.3 |
25 |
|
|
Phenyalanine |
29.4 |
27.5 |
43.6 |
40 |
|
|
Phe + Tyr |
- |
52.7 |
74.6 |
80 |
|
|
# Mean of larvae collected at day 7th and 9 th; ##: La Van Kinh et al 2019; ### Boisen 1997 |
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Amino acid profile of BSFL, fishmeal and soybean meal are comparable (Table 2), particularly, two common limiting amino acids lysine and methionine. In this study, lysine and methionine concentrations in BSFL are 63.5 and 36.4 g/160 g N, respectively and are not limiting amino acids, however, concentrations of histidine and leucine are two. This results are relevant to previous reports that BSLF proteins are rich in two limiting amino acids, lysine and methionine (Rawski et al 2020). The authors indicated that lysine and methionine contents in BSFL were of 68.2 g/160 g N and 21.2 g/160 g N, respectively.
Data in Table 3 show that Omega-6, Omega-9 fatty acid and PUSFA concentrations are higher in the larvae that those in pre-pupae as percentage of crude fat (P<0.05). However, Omega-3 and MUSFA concentrations as DM percentage are higher in the pre-pupae than in the larvae (P<0.05).
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Table 3. Fatty acid profile of the larvae fed Tofu by-products collected at day 7th and 9th after rearing (n=4) |
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|
Fatty acids |
% relative fat |
g per 100 g dry matter |
Sources# (% |
||||||||
|
7D |
9D |
SEM |
p |
7D |
9D |
SEM |
p |
||||
|
Capric acid |
0.30 |
0.34 |
0.028 |
0.400 |
0.05b |
0.07a |
0.004 |
0.027 |
0.3-3.2 |
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|
Lauric acid |
12.95 |
15.39 |
1.131 |
0.202 |
2.28 |
3.31 |
0.346 |
0.103 |
19.2-58.5 |
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|
Myristic acid |
2.81 |
3.32 |
0.242 |
0.209 |
0.48 |
0.69 |
0.064 |
0.075 |
3.1-8.8 |
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|
Palmitic acid |
19.69 |
19.17 |
0.370 |
0.372 |
3.31b |
3.92a |
0.146 |
0.042 |
11.2-14.8 |
||
|
Palmitoleic acid |
3.11b |
3.47a |
0.039 |
0.003 |
0.52b |
0.71a |
0.043 |
0.036 |
1.3-11.6 |
||
|
Stearic acid |
4.36a |
3.74b |
0.091 |
0.009 |
0.72 |
0.76 |
0.032 |
0.472 |
1.4-3.6 |
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|
Oleic acid |
22.69a |
20.68b |
0.394 |
0.023 |
3.72 |
4.14 |
0.164 |
0.146 |
7.2-23.7 |
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|
Linoleic acid |
29.70 |
27.57 |
0.697 |
0.096 |
4.84 |
5.47 |
0.217 |
0.109 |
5.8-22.1 |
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|
α-Linolenic acid |
1.89 |
2.04 |
0.069 |
0.201 |
0.31b |
0.40a |
0.016 |
0.017 |
0.04-2.2 |
||
|
Arachidic acid |
0.38a |
0.266b |
0.039 |
0.115 |
0.06 |
0.05 |
0.005 |
0.772 |
0.04-0.05 |
||
|
Eicosadienoic acid |
0.26b |
0.38a |
0.039 |
0.110 |
0.04b |
0.07a |
0.003 |
0.001 |
- |
||
|
Omega-3 |
1.67 |
1.80 |
0.047 |
0.116 |
0.31b |
0.40a |
0.016 |
0.017 |
- |
||
|
Omega-6 |
26.13a |
24.37b |
0.310 |
0.016 |
4.84 |
5.49 |
0.222 |
0.105 |
- |
||
|
Omega-9 |
20.67a |
19.00b |
0.388 |
0.039 |
3.84 |
4.31 |
0.165 |
0.119 |
- |
||
|
SFA## |
39.03 |
40.90 |
0.805 |
0.176 |
0.16 |
0.21 |
0.016 |
0.072 |
- |
||
|
MUSFA## |
31.27 |
30.83 |
0.585 |
0.628 |
5.82b |
7,00a |
0.265 |
0.031 |
- |
||
|
PUSFA## |
29.70a |
28.27b |
0.322 |
0.035 |
5.49 |
6.36 |
0.269 |
0.084 |
- |
||
|
Total fatty acid |
100 |
100 |
18.86b |
22.90a |
1.094 |
0.049 |
- |
||||
|
ab : means in the same row for each parameter with different superscripts are significantly different (p <0.05) # Li et al 2011; Nguyen et al 2017; Abduh et al 2020 ##SFA: Saturated fatty acids; MUSFA: monounsaturated fatty acids; PUSFA: Polyunsaturated fatty acids |
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Lauric acid has been shown to act as a prebiotic on the micro-biota of animals (Mohana Devi and Kim 2014) and as an antibiotic against gastrointestinal pathogens (Skrivanova et al 2006). In this study, lauric acid contents are not significant in the larvae and pre-pupae and range 12.95-15,39% as total fatty acid or 2.28-3.31% as DM, and these data are lower than previous publications. Rawski et al (2020) and Cullere et al (2017) reported that BSFL contained lauric acid of 21% and 23.4-49.3% as total fatty acid, respectively. Therefore, BSFL can be considered as an alternative to antibiotics which are increasingly banned for use in animal feed.
In addition, the concentrations of two essential fatty acids (linoleic and α-linolenic acid) in 7D and 9D in this study are not different (P>0.05) and are 27.57-29.7% and 1.89-2.04% as total fat, respectively. The data in this experiment are higher than previous publications (Rawski et al 2020; Sealey et al 2011; Cullere et al 2017). Rawski et al (2020) reported that BSFL contained linoleic acid of 3.8% and α-linolenic acid of 0.5% as crude fat. Sealey et al (2011) showed that two essential fatty acids were both presented in the fat of the larvae although in small concentrations (linoleic acid: 3.6-9.4% and α-linolenic: 0.1-0.8% of total fat). The authors concluded that the fatty acid content of the larvae depends on the substrate they feed on. In this study, Tofu by-products are used, or layer’s commercial feeds in other studies (Sealey et al 2011; Cullere et al 2017) or avocado or mixture of avocado and tofu by-product (Abduh et al 2020) were utilised, etc. Further, more studies on the effects of substrates feeding BSFL on their fatty acid profile are needed.
In recommendation, black soldier fly larvae fed by Tofu by-products can be harvested 7-9 days after rearing under environmental temperature of 30-35oC for protein and essential fatty acid feed sources.
The authors acknowledge the financial support of the Strong Research Group Program of Hue University (06/HĐ-ĐHH) and Scientific Research Program of Hue University (DHH2021-02-149).
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