tsps wageingensctdemic.com/doipdL03920(IFF2014 008 -Tusséay, September 26,2017 8:30:17 AM -IP Address 80 42.77.83, Journal of insects as Food and Feed, 2016: (4): 249-259 Influence of different growing substrates and processing on the nutrient composition of black soldier fly larvae destined for animal feed M. Tschirner’ and A. Simon Humboldt-Universtat zu Berlin, Faculty of Life Sciences, Albrecht Daniel Thaer Institute of Agriculture and Hortiulture, Invalidenstr 42, 10115 Berlin, Germany; tschirnermartingngmal.comt Received: 30 june 2014 / Accepted: 27 March 2015 © 2015 Wageningen Academic Publishers RESEARCH ARTICLE Abstract Because of the ongoing depletion of fish stocks and thus continuously rising prices for fish meal, substitution, possibilities are in demand, However, since alternate protein sources from plants are mostly of lower protein quality, feedstuff produced from the black soldier fly (BSE) larvae, Hermetia illucens, are considered promising alternatives. Hence, this study investigated the influence of different growing substrates on the crude nutrient and. mineral composition of BSF larvae, The three tested substrates were a mixture of midlings (control group), dried distillers’ grains with solubles (protein group), and dried sugar beet pulp (fibre group). After a 15 day growing, period, the total larvae yield of the control, protein and fibre groups amounted to 2.58, 0.93 and 0.43 kg wet mass, respectively, the crude protein content reached 37.2, 44.6 and 52.3% of dry matter (DM), respectively. Despite a relatively low methionine content of the BSF meal when compared to fish meal, it showed a good overall protein quality. Furthermore, the BSF meal contained about 50% less P than fish meal. Moreover, an accumulation of, undesirable substances like heavy metals may occur and should be monitored during production. Because a lower fat content of BSF larvae would simplify further processing, in a second trial larvae yielded from the control group were pressed using 12 different conditions: pressures of 250 or 450 bar and temperatures of 50 or 60°C, applied over 10, 20 or 30 min, The best fat reduction from 30.8 to 16.6% of DM was achieved by pressing at 250 bar, 50°C and 30 min. In conclusion, the total yield of BSF larvae and their ingredients were highly influenced by the used growing substrates. Generally, a BSF meal could crucially contribute to substitute fish meal in animal nutrition, Keywords: aquaculture, Hermetia illucens, larvae, poultry feeding, protein sources 1. Introduction higher prices, they ae also influenced by the ongoing trend to.use pelagic fish directly for human consumption and not From the onset of the 20" century, the usage of fish real in livestock alimentation has been spread globally Nowadays, very large amounts of fish meal are used as feed components, especially in aquacultare and poultry farming. In 2012, 14% (2.17310 t) ofthe globally caught fish were used fr non-food products, mainly (about 75%) for the production of fish meal and fish il (FAO, 2014). The global demand for these product is stil increasing, thus their world market prices are also rising. Within the Inst 10 years, the price for ish meal on the world market raised by about 250% to USS 1,764 per metric ton (The ‘World Bank, 2014) According tothe FAO (2014), not only the continuous overfishing ofthe global fish stocks led to mainly forthe production of fish meal. Additionally, the aggravation of fish quotas and the improvement of feed fisheries regulations have also played a part in the price escalation (FAO, 2014) Although possibilities for fish meal substitution in animal nutrition exist, they are mostly of plant origin and therefore of lower protein digestibility and less favourable amino acd patterns. Because ofthis conflict, animal nutritionists are eager to find novel protein sources for feeding of non-ramsinants with high protein quality and digesublty Therefore, the use of Hermetiaillucens (black soldier fly; BSF) larvae as eedstulf has been investigated by several esearch groups, eg, for chicken (Hale, 973), pigs (Newton etal, 1977), channel catfish and blue tilapia | 252-4588 anne, DOV 10.39204NFF20"4 0008 249tip wageningenacsdsmic.com/doipdL03920(IFF2014 008 -Tussday, September 26,2017 8:30:17 AM-IP Address 80 42.77.83 M Techimer and A. Simon (Bondari and Sheppard, 1987), broiler chicken (Elwert et «al, 2010) and turbot (Krdcke etal, 2012). Mostly positive results were achieved and itwas also proved that BSF larvae ‘meal can provide an amino acd pattern comparable to fish meal (Elwert et al, 2010). Furthermore, BSF larvae could be used for recycling of various agricultural by-products like coffee pulp (Lardé, 1990), manure (Newton etal, 2005) and palm kernel meal (Hem ef al, 2008) or organic waste materials like market waste, municipal organic waste or dewatered faecal sludge (Diener et al, 2011). However, Tomberlin etal. (2002) observed that the development and various life-history traits of BSF are highly dependent onthe rowing substrates used. Therefor, the present stu aimed at the question, what influence the different composed substrates have on the crude nutrient and mineral contents of BSE larvae, Furthermore, the high fat content of BSE larvae meal impairs simple processing in the fed industry, especialy ding grinding and pelleting (Briggs et a, 1999), Because a reduced fat content would make the market entry of BSF larvae meal easier, a compression trial was also conducted to explore possibilities ofa mechanical fat reduction method ofthe lrvae 2. Materials and methods Substrate trial The influence of three substrates, differing in nutrient composition, on the larvae nutrient content was explored. ‘The young larvae used were provided by Hermetia Baruth GmbH (Baruth/Mark, Germany). They were produced for this trial according to Sheppard et al. (2002) with the exception that they were reared for 8 days on commercial ‘turkey chicken feed (Table 1) to ensure an optimum larvae development for the first week after hatching. In order to ensure a uniform size and mass ofthe young larvae used in the experiment, they were sieved with a mesh diameter of 1.2 mm and only those that managed to pass through ‘Table 1, Contonts of crude nutrients of the young larvae substrate the sieve by themselves were used. The mean individual larva weight was determined by weighing 20 replications of 20 larvae with a precision scale PCE-BT 200 (PCE Instruments, Meschede, Germany), The larvae transferred, to the trial containers (open plastic boxes, 60%40x30 cm) hhad an average size of 4,140.9 mm and a body weight of 2220.3 mg per larva, The tested substrates were all by-products of plant processing and could therefore be considered as main components fora commercial BSF mass production. Furthermore, the substrates were chosen to reflect different main nutrients (carbohydeates, protein and, fibre) to investigate their effect on optimal larvae growth. The three growing substrates were (Table 1) (1) a mixture ‘of midalings from a feed mill, containing a wide spectrum of cereal processing leftovers like broken pellets, spilled grains and grinding dust (control group, referred to.as abalanced. substrate); (2) dried distillers’ grains with solubles (DDGS) ade from barley, corn, wheat and sugar syrups (protein group); and (3) dried sugar beet pulp (SBP; fibre group). The trial was conducted with seven replications per substrate with two tril containers forming one replication, hence 14 tral boxes per group. Each replication contained approximately 16,000 larvae calculated with the help of the mean individual larvae weight that was determined as explained above. The aimed dry matter (DM) content of substrates for a viscous consistency was about 25 to 28%, which depended on the water holding capacity ofthe substrates. The reduction of the DM content was achieved byanaddition of water (Table 2). Nevertheless, inthe fibre group, the SBP absorbed the water completely and could not be considered as viscous but as moist solid substrate. The larvae were transferred tothe trial containers by spreading, them carefully aver the substrate surface. For the entire experiment, which lasted 15 days, the tral containers were laced randomly in a shelf and no further substrates or nutrients were added, The trial parameters are presented in Table 2. To document the climatic conditions in the ‘and the tested growing substrates, Young lavae sted substrates substrate ooo Mixture of middlings Dried dieters’ grains with Dried sugar best pulp (contrat group) solubles (poten group) (fbr group) ry mati (4) a0 183 930 s10 Crude ash (a % DM) 64 104 58 a3 Crude fe (a % DM) at 55 18 158 ‘Crude poten % DM) 238 20 312 85 Eber extrac (n% DM) 70 58 a4 uN No exvacives in %OM) 587 585 488 685 * DM = dy matt 250 Journal of Insects as Food and Feed 1(4)tip wageningenacsdsmic.com/doipdL03920(IFF2014 008 -Tussday, September 26,2017 8:30:17 AM-IP Address 80 42.77.83 Table 2 Initial rial parameters ofthe different groups ( [Nutrient composition of black sold Nl larvae ‘Til groups, Control ‘Subavate mass hg) 607 ‘mount ads HO () 1399 Tal substrate volume () 20 Dry attr content) a7 “Wiallarvae mass (9) 4s Mean invdul lara weight (rg) 218 Teal larvae ount 6000 tial room, a data-logger Volteraft” DL-121TH (Conrad Electronic SE, Hirschau, Germany) with temperature and relative air humidity sensors was used, The larvae were harvested inthe fifth or sixth instas, when their development stuttered and the consistency of the substrates was dry enough to enable an unproblematic sieving process (5 mm mesh diameter) to separate larvae and substrate residues. A sample of 250 g larvae ‘was taken from each replication and frozen at -23 °C for subsequent analyses. Additionally six replications of 10 larvae were weighed with a precision scale PCE-BT 200 (PCE Instruments) to determine the mean individual larva ‘weight. Furthermore, the total larvae yield per replication ‘yas determined by sing a scale model DE 6KID (Kern & Sohn GmbH, Balingen, Germany) Compression trial ‘The second trial aimed at the reduction of the larvae fat content with different press parameters. The pressing device was a fully automated tincture press, model HP- 5MT (Fischer Maschinenfabrik GmbH, Neuss, Germany) where pressure, temperature and duration could be set individually It was intended to reduce the fat content to less than 15% in DM to approximate the fat content of fish ‘meal (max. 12% in DM). Deep frozen larvae of the control group from the substrate trial were chopped immediately prior to the compression phase with a common kitchen shredding machine model Home Culture Mini Chopper (Gastroback GmbH, Hollenstedt, Germany). The trial included 12 variations (Table 3) each with a threefold replication. The pressure of 250 bar (variations A to F) simulates procedures commonly used in industrial press devices; the pressure of 450 bar, the highest possible pressure inside the compression chamber, was also tested (variations G to L}. Furthermore, the temperature during the pressing process was risen to 50 and 60°C, respectively, tocenhance the fluidity of the fat while minimising the risk of protein denaturation. The diferent exposure times (10, Protein Fibre 538 528 1409 1393 1948 1920 258 250 348 a8 218 218 16,000, 18000 Table 3, Compression trial set up. Variation Pressure_—Temperature Exposure ime (bay co) (in) 250 50 250 50 250 50 20 0 250 80 250 6 450 50 50 50 6 6 @ a8 exc zanmooe> eusssesesess gas 20and 30 min) were chosen to investigate which duration {s sufficient without losing efficiency. Laboratory analyses ‘The proximate and mineral analyses were performed according to common methods for crude nutrient analysis as described by VDLUFA (2013; DM, method 3.1; crude ash (CA), method 8.1; crude protein (CP), method 4.1.2, calculated by Nx6.25; ether extract (EE), method 5.1; ‘minerals, method 10.8.2). The crude fibre (CF) contents was analysed according to method AOCS’ approved procedure BA 62-05 (ANKOM, 2008). The residual organic fraction was calculated as DM — CF - CP - EE, and the N free extractives as DM - CA- CF - CP EE. The amino acid composition was analysed according to method EG 98/64 1 257/16 (EC, 1998). Due to financial constraints, the amino acid composition was only analysed for the larvae of the control group. Journal of Insects as Food and Feed 1/4) 21tip wageningenacsdsmic.com/doipdL03920(IFF2014 008 -Tussday, September 26,2017 8:30:17 AM-IP Address 80 42.77.83 M Techimer and A, Simon Calculations and statistical analyses The recorded parameters for the substrate trial were total larvae yield (kg wet mass; WM), individual larvae weight (mg), the post-trial total larvae count and the residual substrate mass (Kg). Furthermore, the survival rate (8) was caleulated by dividing the post-trial by the initial larvae count, The substrate reduction (%) was estimated by che difference between residual and initial substrate mass and dividing it by the initial substrate mass, The substrate consumption (kg DM/kg WM) is the ratio of the DM content of the growing substrate in relation to the WM of yielded larvae, All statistical analyses were done with IBM SPSS software (IBM Corp, Armonk, NY, USA). The results ofthe substrate tral and the associated laboratory analyses were tested with a one-way ANOVA for statistical significance («=0.05) followed by a Tukey's post-test to clarify which means were significantly diferent from each other: For the results of the compression tral, a three-way ANOVA was used to test the influence of various trial parameters on the crude nutrient content and possible interactions, Subsequently, a one-way ANOVA was also performed followed by a Tukey’ test to examine the significant differences between the various experimental variations, 3. Results Substrate trial The highest total larvae yield of 2.58 ke per replication ‘was observed for the contral group (<0.05; Table 4). The larvae yield ofthe protein and fibre groups was remarkably. lower and amounted to only 36 and 17% of the control respectively. Although in the control group the individual larva weight was significantly higher than for all other groups, the difference to the fibre group was especially large: the individual larva weight of this group amounted to only 12% of the larvae ofthe control group. The total larvae count was highest for the fibre group and lowest for the protein group. Ia the latter group the significantly lowest survival rate (21.7%) was also observed and the substrate reduction was only about 2/3 ofthe other groups (37.8%) (On the other hand, the highest substrate reduction (60.5%, significant to the protein group) and the highest survival rate (78.3%) were achieved forthe ibre group. Furthermore, the substrate consumption per kg larvae mass was very different between groups. Again, the lowest (most desirable) value of 2.1 kg DMikg WM was observed for the control group and the highest for the fibre group (12.3 kg/kg) Moreover, inthis group no further larvae development could be observed already after 7 days of treatment, A. progressing strong acrid smell was noticed inthe wil room during the tral period. Despite the fact that no gas analysis was executed, the characteristic smell and the occurring. Invitation ofthe respiratory system suggest a high ammonia emission. In addition, the gas appeared more abundant in the containers ofthe protein group. Moreover, on the top layer of the fibre group substrate an excessive fungal growth was observed that covered the entire substrate at first. It decreased its covering with ongoing moisture loss of the substrate only and the majority of the larvae were found directly under the mycelium, Table 5 compiles the analysed crude nutrient content of the larvae, Because only one sample was analysed for the ‘young larvae no statistical analyses could be performed After the growing period of larvae, a significant distinction between the groups existed, The DM content of fibre group larvae was significantly lower than forthe other groups, but comparable to young larvae. The CP content ofthe control group larvae was the lowest (37.2%) and forthe fibre group the highest (52.3%, P 0.05), which was again very close to the CP content of the young larvae (57.6%). It is notable that the variation for the CA contents of larvae between groups was very large. In the protein group larvae contained “Table 4, Harvest parameters of tho substrate tral (moans # standard deviation," ‘Tia groups control Totally eg) 25880 250° Irae weigh (ng WM) 290.487208 Total ln count 0) 010857 Suna at) 55.685. Resdal subabta mss (WM) ss0s08% Substata eduction (%) se5stet Subst consumo kg DUK Wit) 21402 Protein Fibre osssostt oeseo. 064 207428 shaedte® ages 2316 yassetae aunnre Taasa0 ya1ne0 58" Betendre sras2e ense22" eae25 seaeige * Dieret supers atersindcaesgnicant dflerances (P05); OM = dry mat; mater 282 Journal of insects as Food and Feed 1/4)a 4 3 nips wageningenscademie com Nutrient composition of black solely larvae ‘Table 5, Dry matter (OM) and crude nutrient contents of black soldier fly larvae (means # standard deviation).* Young larvae DM (%) zr (Crude protin(% of DV) 578 Crude ash (4 of DM) wt ter extract (% of DM) 18 Residual organic recon (% of DM) a Contrlgroup —Protvingroup Flr group 0220.86 3028103" rrast3e 5722086 45208" s2set6r ias208 48s0.17 ngs soget1e 3354302" Set 048 1852280" ra5s2.92 2283330" * Deen supercrltirs inicat signa liferences (P0.05), only 4.8% CA, which was only 1/5 of the CA content in the fibre group, The larvae of the control and the protein, groups exhibited a high content of EE, which amounted to 30.8 and 38.6%, respectively. Compared to these groups, the EE content of the fibre group larvae was extremely low (3.4% EE), Because of the large proportion of CP and EE in the larvae of the protein group, the residual organic fraction was significantly lower than for the other groups. The analysed amino acid pattern ofthe larvae ofthe control group in comparison to literature data of BSF larvae as ‘wellas fish and soybean meal is displayed in Table 6, It can be seen that the BSF larvae had a lower content of most essential amino acids compared to fish meal. Only the histidine and valine contents were higher in BSF larvae than in both fish and soybean meal. The non-essential amino acids alanine, proline, and tyrosine were found in higher concentrations than commonly contained in fish meal and soybean meal protein. ‘The mineral contents ofthe initial substrates, the young, larvae prior to the trial and the trial group larvae are presented in Table 7. Although no statistical analysis was possible the mineral contents of substrates were obviously ‘very different. Compared to the control group, the other substrates contained less Na, Mn, Fe, Zn, Cu and more K, Furthermore, the protein and fibre group substrates contained very low amounts of Ca and P, respectively A numerical comparison revealed that the contents of control group exceeded the value of young larvae by more than 50% in regard of Ca, Mn, Fe, Co and was especially ‘Table 6. Amino acid composition of the black soldier fly (BSF, larvae of the control group in comparison fo Iiterature values for BSF larva fish moal and soybean meal.! ‘Contents of essential aminoacids (in % of crude Contents of non-essential amin acids (in % of crude protein) protein) BSF larvae BSF larvae? Fish meal? Soybean BSF larvae BSF larvae? Fish meat? Soybean (contro) mea? (contret) mea? Noone = 80808 581 742 Asparicacld «8.15 B83 oat tha Hisldne = 28271 2e2 277 Anne Te 699 63t 438 Isoluche «86S AOR BG Cyne «0B nat ost 451 Louie = 88 TY 120 rat Ghianicacld 11 867 wer 180 Lysine 52 601 782 628. Gene 58 523 6 438 Metionine 1.601702 145 Proloe 624548 458 Phonylanine 260 459 398-528 Srna 42385 3988 Tweone «492408419989 Tyrosne’. 5127.08 30 3a Valine see al 481 an * Tryptophan was not analyse. 2 seis otal, (2007), * Dogussa (1898), a= ot analysed. Journal of Insects as Food and Feed 1/4) 258tip wageningenacsdsmic.com/doipdL03920(IFF2014 008 -Tussday, September 26,2017 8:30:17 AM-IP Address 80 42.77.83 M Techimer and A, Simon high in ease of Pb (350% of the content in young larvae; Table 7). On the other hand, in the control group the contents of B, Mg, K and Na was less than 65% compared tothe young larvae, Among the test groups, the fibre group larvae showed significantly higher contents of Ca, K, Mg, Na and P (Table 7). In contrary, the protein group larvae had the lowest content of many minerals, even though these differences were not significant in all cases. Particularly ‘worth highlighting is the very low Ca level in the protein group larvae, but in the control and protein groups also the contents of P, Mg, K and Na were considerably lower than in the young larvae. The P content in all trial groups ‘was hence apparently lower than in the young larvae. Furthermore the highest levels of trace elements and heavy metals were analysed forthe control group (P<0.05) and the concentration of many of these elements were the lowest inthe larvae of the protein group, The only exception was the Cd content in the fibre group, which exceeded the concentrations of ll other groups (P0.05) In comparison to herring fish meal, the BSF larvae of the control group contained more Ca, Mg, Fe and Cu, but ess P and Mn, Compression trial Table 8 summarises the crude nutrient content of the pressed larvae (control group) after different pressing procedures in comparison to the initial contents. No significant diference inthe final EE content existed between the different trial variations except to the initial content. Furthermore, the aimed EE reduction below 15% of DM. could not be realised with the applied methods. However, the highest fat reduction was achieved at 250 bas, 50°C for 30 min witha final EE content of 16.6% of DM (variation C) The highest DM content of 51.3% was found in variation L (450 bar, 60 °C for 30 min) but was only significantly different to the variation J (450 bar, 60°C for 10 min) and, the initial content, The highest CP content of 49.3% of DM resulted with variation A (250 bar, 50°C for 10 min) but was only significantly different from variations G, Hi, and the initial content, The lowest CP content (43.9% of DM) was found in variation I (450 bar, 50°C for 30 min) A pressure of 450 bar (variations G to L) appeared to lead. to lower CP levels. Although the different trial variations showed only a, significant difference in the EE content of the BSF larvae to the initial content, significant influences of the parameters pressure and temperature were observed after a statistical evaluation over al variations using a three-way ANOVA (able), but no interactions berween the different pressing. parameters were identified. It became obvious that pressing, ata lower compression (250 bar) and at higher temperature (60°C) led to a desired lower EE content in the BSE Tan meal ‘Table 7. Mineral contents of growing substrates, young larvae and trial group larvae (means ¢ standard deviation) as well as literature data for herring fish mel (values per kg DM." Growing substrates Larvae Fish meat Contre group Protein group Fibre group Younglarae Control group Protein group Fibre group cal 1409 om 1183 2228 2052370 SaRe060! S18zeKaH 2482 Pq) ra 255 09 1951 sotso4 —aiso2s razzs0.25 1828 Mgi@) 290 ant a 573 asosaie garso seta 181 Ki) 883 mn 1981 rateo7 soamsose: sa7es05e 1172 Nag 937 356 22 18 astsoo” —oagso0s — 2805021" na? Mig) 028 096 ‘08 025 700" oj9s008 — oazsnas? 838 Fafa) 108 an 028 ase ossoos —ojoso0% — oatanas? 015 29) 029 a 4 023 oz00% —o10s00P oxzsoo” on cura) 4684 183 13 m6) M2se3Hoananss> sz7ean”? 545 Colm) 148 a 02 oat 0572030" 002400 —o.es0.02" na, No(n) 188 193 095 109 0853007 0454008 0.184019" na, cama) 0.09 9 023 036 0472008 060008 2244031" na, Poina) 080 024 oe on 2884026" 0554010 1968010" na, * Dierent superstars ndeatesgnicantdforences (P05) 2 NRC (1984) 3a. = pot analysed. 254 Journal of insects as Food and Feed 1/4)a 4 3 nips wageningenscademie com ‘Table 8, Nutient contents of differently pressed black soldier fly la [Natient composition of black soley larvae (means + standard deviation) Crude nutiont contents ofthe black soir fy produet Compression paramstors Variation Prossure Temperature Exposure DM (%)? (ay (70) time (min) rita conan 0220.86 A 25050 0 0030.16 8 25050 2 48521,15% c 250 50 0 s0set.15% D250 80 0 4852288 E 25060 » 495x150 F 25060 0 47.321 08% 6 450 50 0 47521 55% x 45050 2 4751380 1 45050 0 59.85% J 45060 0 552078 k 45060 2 74x 89% L 45060 0 Bist 62 Crude protsin Crude.ash_ Ether extract Residual organic (hofDM) ——(HofDM)—(%o DM) fraction (4 of DM) S722088 1352088 RTI T8260" 495.059 185047" 2134008 1.8208 4822100 18.8008" TAR1AT® 15.4052 4o2e14s® T8207" 18464052 1578187 4320077 —TBas081" THT 15,12058% 4392188" 1902048" 1884052 18,12003" 4easi se sTBat05 1734195" 1882070 445.201 —tB0a26p — 2224252 TT 82091" a52e1as! — te9s2.12 2124287 15,120672 4eer07s! —s52s0tTe 21x25 197227 A7ss1 93% —se5s030% —tBan0.10> 1881.1 4552090 —fe3s050% —f9sei2e 1842051" 475090 1800.17 —A7Ax1.00 182108 "Means not sharing the same superset eters are significant diferent (0.05) 2DM= dy mat, ‘Table 9, Ether extract content of pressed black soldier fly larvae (% of cry matter) in relation to pressing parameters calculated ‘overall variations (means + standard deviation)" Pressure Temperature Exposure tne 250 bar(ne12) 480 bar(net2) 50 "C(n=12) °C (2), $0 min(n=8)——20.min(n=8) 30min 8) ‘79s. zoe 988 mo.ie2ee 78si.10® 9812.26 tga21.97° ‘8312.9 " Diterentsupersatetirs win a speci parameter inca signa erences (Ps0.05), 4. Discussion The present study showed that the actual nutrient content of a BSF product depenes highly on the growing substrate used. Therefore, its feed value would also vary ‘markedly in relation to the initial substrate. While Diener etal. (2009) observed that BSF larvae are able to convert carbohydrates as wells protein rich substrates very quicky, in the presented trial the substrate of the control group composed of a mixture of middlings yielded the most promising results in regards to the total larvae yield, the individual larva weight and the substrate consumption per kg larvae yield (Table 4). The protein and the fibre group substrates resulted in significantly lower values for these trial parameters, The very low EE content ofthe fibre group larvae (3.4% of DM) indicates the occurrence of a hunger period of the larvae, since the fat content was even lower than of the young larvae, thus they had to mobilise their fat reserves. However, the individual development of the fibre group larvae could also have been restrained by the excessive fungal growth on the substrate. Wertheim et al. (2002) described the impact of mycelium growth from ‘Mucor spp., Penicillium spp. and Aspergillus spp. on the development of Drosophila melanogaster. They stated that especially the development and survival rate of the younger larvae was negatively affected by the degree of mycelium coverage. Even though the survival rate of the fibre group larvae in the present study was very high, it should be considered that the individual larva development has been suppressed. Additionally, the fungal growth can be an explanation forthe high substrate reduction of 60.5% Whether the fungal growth was an assay design problem or a dietary problem remains unclear. The lower survival rate and total larvae mass of the protein group might be a sign that the protein supply ofthe control group substrate was sufficient for the larvae development and that the additional protein ofthe protein group was disadvantage for larvae development Journal of Insects as Food and Feed 1/4) 255tipi wageningensctdsmic.com/doipdfL03920(IFF2014 0008 - Tussday, September 26,2017 8:30:17 AM-IP Address 80 $2.77. M. Techimer and A. Simon According to Eisenbeis and Wichard (2003) the elimination of nitrogen in the catabolic metabolism of insects is the ‘most important task of the excretion system. Also in insects the detoxification of proteinogenic nitrogen is a process requiring energy (Ziegler, 2003). Nevertheless, Ziegler (2003) also stated that insects can on average gain similar amounts of energy from proteins as from carbohydrates. It depends on the actual amino acid composition, how much energy they finally ean acquire since different amino acids enter the citrate cycle at different locations. The high individual larva weight of the protein group is also speaking against a lower energy supply and the very high CP content of 4.6% of DM should be considered as favourable influence of the higher protein levels in this substrate Rather, the very low survival rate of 21.7% of the protein group larvae should be considered as critical. Hence, itean be assumed that other factors than the dietary energy level acted as limiting factors fr the survival of the protein group larvae. The lower total larvae yield of the fibre an protein group could be also due toa mineral deficit, since no details about mineral requirements of BSF larvae are identified yet and it might possible that the DDGS and SBP are not a preference for this insect. Indeed, very few facts are known about the nutrient requirements of BSF larvae in general, so further research inthis area is advisable, especially with regard to an industrial production, A farther constraint for the larvae growth ofthe protein group might have been an obviously increased ammonia (NH,) concentration inside the trial boxes. Even though NH, is usually lighter than air, it attaches to the water vapour resulting in a heavy mist that descends and therefore could have stayed in the boxes for an extended period, Because the relative air humidity in the room during the trial period was between 73.8 and 75.7%, the mentioned attachment of NH, to the ‘water molecules was likely. Further trials to identify and «quantify the influence of substrate specific gas emissions are advisable to prevent danget to the production labour and negative impacts on the latvae development. Another reason for the lower survival rates in the liquid substrates ofthe control and the protein group might be an increased temperature dic to larvae movernent and ingestion. In growing substrates with high larvae density, ‘Warburton and Hallman (2002) observed temperatures up to 42 °C at an ambient temperature of 23°C. Also, Tomberlin et al. (2008) reported a negative impact of high ambient temperatures (between 30 and 36 “ larvae development. In the present trial an average room temperature of 28°C was recorded and it cannot be ruled out that very high temperatures occurred in the substrates ofthe control and protein group over certain periods. The higher survival rate ofthe fibre group larvae could also be explained withthe better aeration ofthe substrate. Lardé (2989) discovered that BSF larvae development was better in well aerated zones ofa coffee pulp substrate than in less aerated, highly viscous zones. It should therefore be examined if an additional structured fibre component would be able to loosen up an energy rich liquid growing substrate without affecting the nutzient availability of liguid substrates, However, the a survival rate of 55.6% in the control group is also far from being optimal, so the rearing process is not optimised yet and more research in this regard is necessary. Considering the control group asthe most successful group in regards to che total larvae yield and the individual larva, body weighe, it becomes clear that these larvae would be a protein rich as well as an energy rich feedstuff (37.2% CP and 30.8% EE of DM). With regard to the analysed amino acid pattern of the BSF larvae, reported data for BSF larvae are comparable (Table 6). For the larvae ofthe control group marked differences exists regarding a lower content of arginine and phenylalanine (<80% of data given Inliterature), and higher contents of histidine and glutamic acid (120%). Furthermore it can be stated that the lysine (Lys) and methionine (Met) contents of the BSF larvae was about 22 and 43% lower than in fish meal, respectively, but compared to soybean meal the Lys content was similar and the Met content about 10% higher. The threonine (Thr) content of larvae is comparable to fish meal and soybean, meal. According to Jeroch et al (2013), at the beginning. of the fattening phase of broilers the desirable proportion for Lys:Met:(Met+cysteine)‘Thr in feeds is 100:36:(56)5. Therefore, the respective ratio of 100:27:(42)-66 analysed inthe BSF larvae can be considered as favourable, despite a small Met deficiency. The protein content (37.2%), however was explicitly lower in comparison to fish meal (65%). Since the market value wll be determined by the protein content, it is expected that a BSF feedstuff will therefore obtain a lower price on the world market in comparison to fish meal when sold without further processing. Regarding the mineral content in the BSF larvae, the Ca:P ratio of none of the trial groups was favourable (Table 7) and indicates a severe deficiency ofP For fish species, the "AP ratio varies between 1:2 and 1.5:1 (Andrews et al, 1973; Sakamoto and Yone, 1973). Also for other animals like poultry, a more close CaP ratio of 25: is recommended (NRC, 1994), Therefore, a BSF product will not be able to compensate the high P contents available in fishmeal about 18 g/kg; Table 7). Furthermore, some of the mineral levels found inthe different trial group larvae might be considered problematic. This may apply on the Cu concentration in the control group larvae, since Murai etl, (1981) already determined a reduced growth rate of channel catfish for Cu concentrations higher than 15 mgikg diet. The Cu content of the controf group larvae was twice that high (34.25 mg/ kg; Table 7). A decreased growth rate for Zn levels higher than 300 mg/kg of the dict was also observed for carps by Jeng and Sun (1981). The Zn content ofthe control group larvae presented here would therefore equate 90% of that content. Regarding undesirable substances in feedstulls Cd 256 Journal of insects as Food and Feed 1/4)tipi wageningensctdsmic.com/doipdfL03920(IFF2014 0008 - Tussday, September 26,2017 8:30:17 AM-IP Address 80 $2.77. and Pb were analysed, According tothe Directive 2002/32/ EG (EC, 2002) the maximum levels for Ca and Pb are2.mg/ ‘kg and 10 mg/kg feed (88% DM), respectively. The contents of these elements in the larvae of the control group were high, but cleaely below the upper limit, Only the larvae of the fibre group, which cannot be considered as feedstulf because of hampered larvae growth, exceeded the limit for Cd by 12%. Itwas a farther remarkable observation that considerable Pb and Cd accumulation has taken place in the larvae compared to the initial substrates. Moreover, for all groups such enrichment was also detected for Ca and Mn, whereas the contents of Co and Mo decreased. Diener (2010) identified in BSF larvae bioaccumulation factors (BAB Le. content inside the organism divided by the content| of the initial substrate) for Ca and Pb of 2.46 to 2,94 and 0.66 to 0.99, respectively, depending on the contamination degree of the initial substrate. When calculating the BAF for Cd and Pb for the present study, quite different results emerge. For Cd and Pb, the mean BAEs of ll tial groups were 7.4322.03 and 2.6020.61, respectively and were therefore considerably higher than in the study of Diener (2010). To produce a safe BSF product asa livestock feed, this observation underlines the necessity ofa strict control of these undesirable substances inthe initial substrates as ‘wellas in the final BSF feedstuff Because a successful fat reduction of a BSE product will not only simplify the market entry ofthat product but also help to assure the product safety to the authorities and accelerate admission of BSF larvae feedstufs, a compression trial was conducted. In this trial, several variations were chosen to determine which pressing parameters had an effect on the various crude nutrient contents. Since this was combined with a very small sample size of only ‘three replications per variation, this experiment has to be considered as a pilot study only. Although the different pressing conditions had no significant effects on the final EE content, significant influences of the trial parameters pressure and temperature on the EE content were observed lasing a three-way ANOVA. Nevertheless, no interactions between the different pressing parameters existed. It became evident that the variations pressed with 250 bar contained significantly less EE than those done at 450 bar. Thus, a very high pressure seemed to counteract an efficent fat reduction. One explanation could be the fat storage physiology of insects. Fat, as well as other nutrients such as proteins or carbohydrates, are stored in trophocytes. These large cells contain a very iregulary shaped nucleus and different types of nutrient storage structures which can vary with the type of insect and/or the phase of life of the individual (Paes de Oliveira and Cruz-Landim, 2003). An interesting fact for the present work is that even though lipids form the major cell components in the trophocytes, the type of storage may cause problems for their extraction Thus, the triglycerides, the most frequently found lipi, are stored in the core of lipid droplets. These droplets are Nutrient composition of black solely larvae surrounded by phospholipids and a protein coat (Arrese and Soulages, 2010). With an application of heat and/or high pressure dusing the fat extraction process, this protein coat could coagulate and cause an inclusion of lipids, so that the lipid extraction would be inhibited. This would explain why the inclusion would occur toa higher degree at higher pressures (Le, 450 bar), since the temperature in the pressed medium would increase sharply with such a high pressure. Additionally, also Krdckel etal (2012) are proposing that the reduced bioavailability ofthe lipids of the [BSF meal used in thee trial might have been influenced by the degreasing process. Furthermore, Table 9 reveals that a process temperature of 60°C led to significantly higher fat reduction inthe present tial than a temperature of 50°C. Even though the differences in fat content after different exposue times were not significant, it should be tested in further studies iflonger exposure times are more effective for fat reduction. In case of a mechanical fat reduction of A BSE product during processing, it should be considered, that high pressures are contradictory. But before the results| of the reported compression trial can be used in practice, the outcomes must be verified in subsequent trials with a higher number of replications 5, Conclusions From the present tral can be concluded that the nutrient composition of growing substrates havea great influence on critical production factors like total larvae yield, individual -va body weight and nutrient composition of yielded BSF Iarvae, The later fact was proved with regard to crude nutrients, amino acid composition and mineral contents, The best production results were achieved with a mixture of middlings; the use of SBP or DDGS as substrates led to significantly more unfavourable results. The larvae harvested from the substrate of middlings showed a desirable composition, eg, for amino acid pattern, and could be a substitute for fish meal in diets fr fish and various non-ruminants. Nevertheless, the production condition must be improved to increase e.g, the survival rate of larvae However, a possible accumulation of undesirable substances like heavy metals need to be monitored, In further studies, the nutrient requirements of BSF larvae should be investigated and for further processing the conditions for reducing their fat content have to be optimised. Acknowledgements We would like to thank the brothers Heinrich and Peter Katz and the entire team of the Flermetia Baruth GmbH for the substantial assistance to conduct this study. Not only. did they provide us with the larvae necessary for ths trial but also let us use the facilities on-site and furthermore helped us with their vast experience about the mass rearing. of BSF larvae. Journal of Insects as Food and Feed 1/4) 257hip wegeuingenscademie comiopalt0 3920/2014 008 - Tuesday, September 26,2017 8:30:17 AM-IP Ades 80 2.77.83 M,Techimer and A. 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