Synthesis and fractionation of Protein Hydrolysate from Hermetia illucens L. pre-treated with Lactobacillus spp.

Synthesis and fractionation of Protein Hydrolysate from Hermetia illucens L. pre-treated with Lactobacillus spp. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis and fractionation of Protein Hydrolysate from Hermetia illucens L. pre-treated with Lactobacillus spp. M.Y. Abduh, Hana Safira, Ghassani Raisa Pramestiandini, Mohammad Adiba Syahada, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6142604/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Jul, 2025 Read the published version in Waste and Biomass Valorization → Version 1 posted 5 You are reading this latest preprint version Abstract This study aimed to determine the effects of fermentation pretreatment using Lactobacillus spp. on the protein content of black soldier fly larvae, and the protein recovery, degree of hydrolysis, and amino acid composition of protein hydrolysate from black soldier fly larvae. Solid state fermentation of black soldier fly larvae with Lactobacillus spp. carried out for 72 h at 37℃ increased the protein and fat content from 32.35 to 36.75–41.99% and 36.91 to 40.03–41.99%, respectively. Protein content in the fermented samples was extracted using an alkaline extraction method which resulted in a protein recovery of 31.41 to 35.99%. The protein extract was then hydrolyzed using bromelain enzymes for 24 h to produce protein hydrolysates with a degree of hydrolysis in the range of 47.64 to 52.61%. The protein hydrolysate was further fractionated using size-exclusion chromatography with the flow rate of 0.5 to 1.5 mL/min. The protein concentration of each fraction was determined using a UV-visible spectrophotometer and the result varied from 0.51 to 1414.69 mg/mL with the highest protein concentration obtained when the flow rate of the chromatography was set at 1 mL/min. Amino acid composition of the fractionated protein hydrolysate was determined using the ultra-high-performance liquid chromatography. The results show that the fractionated protein hydrolysates fermented with Lactobacillus spp. contained several amino acids such as glycine, and L-methionine, which was significantly different than the unfermented samples. The findings highlight that fermentation of black soldier fly larvae using Lactobacillus spp. positively influence the protein and amino acid composition of protein hydrolysate from black soldier fly larvae. Amino acid Black soldier fly larvae Lactobacillus spp. Protein hydrolysate Solid-state fermentation Figures Figure 1 Figure 2 INTRODUCTION Black soldier fly ( Hermetia illucens L.) larvae (BSFL) started to attract the world’s attention due to their ability to convert organic waste into protein and lipid-rich biomass [ 1 – 2 ]. Several studies have reported that BSFL have a relatively high protein content (40–50%) and can be utilised as an alternative source of protein hydrolysate with special biological activities that highly depend on the composition of the amino acids [ 3 – 5 ]. Firmansyah and Abduh [ 3 ] investigated the synthesis of protein hydrolysates of BSFL using bromelain enzymes with the degree of hydrolysis in the range of 14.6–39.9%. The protein hydrolysates had a molecular weight in the range of 14–25 kDa and mainly comprised of lysine (8.0%) followed by leucine (7.7%), and valine (7.2%). The synthesised protein hydrolysates had the ability to inhibit free radicals 2,2-diphenyl-2-picryl hydrazyl (DPPH) up to 2.6 ± 0.41% and IC 50 of 0.84%. Riolo et al. [ 4 ] also produced and characterised protein hydrolysates from BSFL using an enzyme mix of flavourzyme:protamex:alkalase at a ratio of 1:1:1. The molecular weight of the protein hydrolysates were in the range of 10–75 kDa and mainly comprised of alanine (13.4%), glutamic acid (12.9%) and valine (9.4%). The synthesised protein hydrolysates possessed a strong antioxidant activity by reducing intracellular reactive oxygen species in challenged cells. In another study, Ferdinand et al. [ 5 ] demonstrated that protein hydrolysates produced from BSFL, and bromelain enzymes have a very good antioxidant activity with an IC 50 value of 9.43 ± 0.84% (as determined by the DPPH method). In addition, the protein hydrolysates also had a good antibacterial activity against Vibrio cholerae with an inhibition zone diameter of 11.2 ± 3.8 mm (as determined by the agar well diffusion method). Batish et al. [ 6 ] examined the effects of protein hydrolysis using different enzymes (alcalase, papain, pepsin) on the functional properties, antioxidant activities and amino acids composition of the protein hydrolysates. The degree of hydrolysis lied in the range of 9.8–18.4% and all protein hydrolysates had the ability to scavenge DPPH radicals with alcalase hydrolysates showing a higher antioxidant activity as compared to the protein hydrolysates produced by pepsin and papain. The most dominant amino acid in the protein hydrolysates was glutamic acid (35.10-74.86 mg g -1 ). The study highlighted that enzymatic hydrolysis improved the functional properties and bioactivities of the protein hydrolysis but highly influenced by the types of enzymes. According to Luparelli et al. [ 7 ], solid-state fermentation of adult and prepupae of black soldier fly using Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum enriched the essential amino acids such as histidine and cysteine while significantly reduced other amino acids such as isoleucine, leucine, lysine, valine, glycine and glutamic acid. The results indicated lactic acid bacteria (LAB) activity on the protein fraction of the black soldier fly biomass with some proteins being utilised while new proteins being synthesized by the LAB. According to Zacharof et al. [ 8 ], Lactobacillus sp. which is an environmentally friendly LAB that can produce bacteriocins is able to modify components inherent in insects, including amino acid components. Systematic studies that investigate the effects of solid-state fermentation using Lactobacillus sp . on the protein content and composition of protein hydrolysate from BSFL are still scarce. In addition, studies that report on the fractionation of protein hydrolysate are still limited. It is expected that pre-treatment of BSFL by fermentation using Lactobacillus sp . can positively influence the protein content and composition of fractionated protein hydrolysate from BSFL. Hence, this study aimed to investigate the effects of solid-state fermentation using Lactobacillus sp . on the protein content of BSFL, protein recovery, degree of hydrolysis, and amino acid composition of protein hydrolysates from BSFL. This study also investigated the effects of different chromatographic flow rates on protein concentration of the fractionated protein hydrolysates from BSFL. MATERIAL AND METHODS Materials Dried BSFL were obtained from a local shop in West Java, Indonesia. The chemicals used in this study included n-hexane, K 2 SO 4 , CuSO 4 , concentrated H 2 SO 4 , NaOH 40%, H 3 BO 3 , methylene blue, methylene red, 0.1 N HCl, 0.25 M NaOH, 0.25 M HCl, sucrose, Coomassie Brilliant Blue G-250, phosphoric acid 85%, ethanol 95%, Na 2 HPO 4 .7H 2 O, NaH 2 PO 4 , K 2 HPO 4 , KH 2 PO 4 , formaldehyde 37%, and 0.1 N NaOH, which were obtained from the chemical warehouse of the School of Life Sciences and Technology, Institut Teknologi Bandung, Indonesia. The biological agents used in this study were L. plantarum, L. acidophilus, and L. lactis culture obtained from Microbiology Laboratory, School of Life Sciences and Technology, Institut Teknologi Bandung. Bromelain enzymes were obtained from PT Bromelain Enzyme and Sephadex G-25 gels were obtained from PT Enigma Saintia Solusindo, Indonesia. Proximate Analysis Proximate analysis was carried out to determine moisture, ash, fat and protein content of the samples. Determination of moisture content was done by thermogravimetric method at 105°C for 24 h (method 934.01) whereas determination of ash content was carried out by drying method using a furnace at 500–600°C for 6 h (method 942.06) [ 9 ]. The fat content was determined by the Soxhlet method using n-hexane solvent at 70°C (method 948.22) whereas the protein content was measured by the Kjedahl method (method 960.52) [ 9 ]. The carbohydrate content was calculated based on the difference between moisture, ash, fat, and protein content of the samples. Solid-State Fermentation of Black Soldier Fly Larvae Solid-state fermentation was carried out using Lactobacillus spp. culture maintained at 37 ℃ in Man Rogosa and Sharp (MRS) broth for 18 h. Approximately 50 g of dried BSFL that were crushed using a blender and sieved with 80 mesh (< 0.177 mm) were mixed with 50% (weight/volume) distilled water and 4% (weight basis) sucrose. The samples were then sterilised in an autoclave at 121 ℃ for 20 min and then incubated with 7% (weight/volume) Lactobacillus spp. (bacterial concentration of 10 8 CFU/mL) at 37 ℃ for 72 h. Inactivation of the samples was carried out at 60 ℃ and dried overnight at 105 ℃ [ 10 ]. Defatting of Black Soldier Fly Larvae Defatting of BSFL was carried out by Soxhlet method using n-hexane solvent at 70 ℃ for 6 h to remove the fat content in the samples. The ratio of biomass to solvent used was 1:10 (weight to volume). The extracted mixture of fat and solvent was then separated using a rotary vacuum evaporator at 60 ℃ for 2 h [ 3 ]. Extraction of Protein from Black Soldier Fly Larvae Protein was extracted from the BSFL using 0.25 M NaOH at a ratio of 1:5 (weight to volume). The mixture was incubated in a water bath shaker at 60 ℃, 400 rpm, for 60 min. After that, the extracted mixture was added with the same amount of solvent as in the previous process and incubated again in a water bath shaker at 60 ℃, 400 rpm, for 60 min. The mixture was then centrifuged at 4000 rpm for 15 min. The precipitate and supernatant were then separated using a filter paper and a glass funnel. The precipitate was then re-extracted using the same procedures for the extraction of protein. The supernatants from the second and third extractions were mixed and then neutralized to pH 7 using 0.25 M HCl prior to the determination of protein concentration using the Bradford method [ 11 – 12 ]. Determination of Protein Recovery with the Bradford Method The concentration of protein extract from the BSFL was determined using the Bradford method. A protein sample of 0.1 mL was poured into a test tube and mixed with 5 mL of Bradford reagent. The mixture was then incubated for approximately 2 min at room temperature (27 \(\:\mathbf{℃})\) . The absorbance of the solution was determined using a UV-visible spectrophotometer at a wavelength of 595 nm. The protein concentration was determined based on the bovine serum albumin standard curve prepared at concentrations of 0, 100, 250, 500, and 1000 ppm [3-Firmansyah and Abduh, 2019]. Each concentration of 0.1 mL solution was added with 5 mL of Bradford reagent and incubated for 2 min. The solution was then measured using a UV-visible spectrophotometer at a wavelength of 595 nm [ 13 ]. The protein extract concentration was then used to determine protein recovery using the following equation. $$\:\text{P}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}\:\text{r}\text{e}\text{c}\text{o}\text{v}\text{e}\text{r}\text{y}\:\left(\text{%}\right)=\:\frac{\text{P}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}\:\text{e}\text{x}\text{t}\text{r}\text{a}\text{c}\text{t}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\:\left(\frac{\text{m}\text{g}}{\text{m}\text{L}}\right)}{\text{I}\text{n}\text{i}\text{t}\text{i}\text{a}\text{l}\:\text{p}\text{r}\text{o}\text{t}\text{e}\text{i}\text{n}\:\text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n}\:\left(\frac{\text{m}\text{g}}{\text{m}\text{L}}\right)}\text{x}\:100$$ 1 Hydrolysis of Protein Extract from Black Soldier Fly Larvae The supernatant produced in the protein extraction stage was dried using a freeze-drying method whereby the samples were placed in a freeze dryer at -55 ℃ and 1500 mTorr pressure until a constant weight was obtained [ 3 ]. The freeze-dried protein extract was then dissolved in a phosphate buffer solution (pH 8) with a ratio of 1:10 (weight/volume). The solution was then mixed with 3% bromelain enzyme (enzyme activity of 1200 GDU/mg) in a 250 mL Erlenmeyer flask and incubated using a water bath shaker at 50 ℃ and 150 rpm for 24 h. The enzyme in the solution was then inactivated by incubating the solution using a water bath shaker at 90 ℃ for 10 min. The solution was then centrifuged at 4000 rpm for 15 min. The supernatant obtained was then filtered and stored in a refrigerator at 4 ℃ for further analysis [ 3 ]. Determination of Degree of Hydrolysis The degree of hydrolysis was determined based on the ratio of α-amylase and total nitrogen in the samples. Total nitrogen in the samples was determined using the Kjeldahl method while α-amylase was determined using the formol titration method. Approximately 5 mL of samples were placed into a Kjeldahl destruction flask and mixed with 7 g K 2 SO 4 ; 0.8 g CuSO 4 ; and 12 mL of concentrated H 2 SO 4 . The mixture was then destructed until the colour of the solution into blue green. The samples in the flask were then settled to cool at room temperature (27°C) before mixed with 25 mL of distilled water. The mixture was then transferred into a Kjeldahl distillation flask and then mixed with 50 mL of NaOH 40%. An Erlenmeyer flask was prepared and filled with 30 mL of H 3 BO 3 3% and 3 drops of Tashiro indicator as. The green distillate was then collected as much as possible, and the solution was then titrated with 0.1 M HCl standard solution [ 14 ]. For the formol titration method, approximately 1.5 g of protein hydrolysate samples was added to distilled water until it reached a weight of 50 g. The mixture was then mixed with 0.1 N NaOH to reach pH 7 and then mixed with approximately 10 mL of a formaldehyde solution of 37% (weight/volume) and allowed to stand for 5 min. Then, the solution was titrated using 0.1 N NaOH until the pH of the solution became 8.5 [ 15 ]. The degree of hydrolysis was determined using the following equations. $$\:\text{F}\text{r}\text{e}\text{e}\:\text{a}\text{m}\text{i}\text{n}\text{o}\:\text{a}\text{c}\text{i}\text{d}\text{s}\:\left(\text{%}\right)=\left(\text{V}\:\text{x}\:\text{C}\:\text{x}\frac{\text{0,014007}}{\text{M}}\right)\text{x}\:100$$ 2 $$\:\text{D}\text{e}\text{g}\text{r}\text{e}\text{e}\:\text{o}\text{f}\:\text{h}\text{y}\text{d}\text{r}\text{o}\text{l}\text{y}\text{s}\text{i}\text{s}\:\left(\text{%}\right)=\:\frac{\text{%}\text{F}\text{r}\text{e}\text{e}\:\text{a}\text{m}\text{i}\text{n}\text{o}\:\text{a}\text{c}\text{i}\text{d}\text{s}}{\text{%}\text{T}\text{o}\text{t}\text{a}\text{l}\:\text{N}\text{i}\text{t}\text{r}\text{o}\text{g}\text{e}\text{n}}\text{x}\:100$$ 3 Where V is the volume of NaOH used to reach pH 8.5, C is the concentration of the titration solution (0.1 N), and M is the mass of the sample. Fractionation of Protein Hydrolysate Fractionation of the protein hydrolysate samples was performed using the size-exclusion chromatography method. Approximately 3 g of Sephadex G-25 beaded gels were soaked in 18 mL of phosphate buffer solution pH 7.4 for 24 h and then inserted into a column (30 cm high and 1.5 cm diameter). Approximately 1.5 mL of samples were fed into the column followed by the injection of 160 mL of phosphate buffer solution pH 7.4 at a flow rate of 0.5 to 1.5 mL/min. Absorbance of the fractionated samples was measured using a UV-visible spectrophotometer with wavelengths of 260 nm (A 260 ), 280 nm (A 280 ), and 320 nm (A 320 ) [ 16 ]. Protein concentration in the fractionated samples can be determined using the following equation: $$\:Protein\:concentration\:\left(\frac{mg}{mL}\right)=\:\left(\left({A}_{280}-{A}_{320}\right)x\:1552\right)-\left(\left({A}_{260}-{A}_{320}\right)\:x\:\text{757,3}\right)$$ 4 Determination of Amino Acid Composition Composition of amino acids in the samples was determined at Saraswati Indo Genetech, Bogor, Indonesia using ultra high-performance liquid chromatography combined with photodiode array detector (UPLC-PDA/18-5-17/MU/SMM-SIG) and high-performance liquid chromatography combined with photodiode array detector (HPLC-PDA/18-5-63/MU/SMM-SIG). Statistical Analysis Statistical analysis in this study was carried out using Minitab version 21.1.0 by testing One-way ANOVA to determine the significance of fermentation pretreatment on several parameters and two-way ANOVA to determine the significance of chromatography flow rate on protein concentration. Test results that showed a significant value (P < 0.05) were further examined by post hoc testing using the Duncan test to determine the significance between one data and another in a group [ 17 ]. Effects of Fermentation on Protein Recovery from Black Soldier Fly Larvae RESULTS AND DISCUSSIONS Proximate Analysis of Black Soldier Fly Larvae Table 1 shows the proximate composition of the BSFL samples. The moisture content of the BSFL samples investigated in this study was lower than the reported values in the literature. The differences may be due to different drying method, temperature and time that play vital roles in determining the final moisture content of the BSFL. Nevertheless, the protein, fat, carbohydrate and ash content of the samples corresponded to the values reported in other studies. Table 1 Proximate analysis of dried black soldier fly larvae Parameter Composition (%) This study Reference Moisture 3.22 \(\:\pm\:\) 0.19 4.2 [18] – 12 [19] Ash 10.47 \(\:\pm\:\) 0.31 6.4 [18] – 10.6 [19] Protein 32.35 \(\:\pm\:\) 0.89 30 [19] – 37.9 [18] Fat 36.91 \(\:\pm\:\) 1.07 33.2 [19] – 43.6 [18] Carbohydrates 17.16 \(\:\pm\:\) 1.80 7.9 [18] – 23.8 [19] Effects of Fermentation on Protein and Lipid Content of Black Soldier Fly Larvae Figure 1 shows the effects solid-state fermentation of BSFL using Lactobacillus spp. on the protein and fat content of BSFL. In general, the fermentation increased the protein content of BSFL. Statistically, fermentation of BSFL using L. lactis did not significantly increase ( P > 0.05) the protein content of BSFL. However, fermentation of BSFL using L. plantarum and L. acidophilus significantly increased (P < 0.05) the protein content of the BSFL from 32.25 ± 0.89% (unfermented BSFL) up to 35.69 ± 0.33%. This is in accordance with the results obtained by Luparelli et al. [ 7 ] that solid-state fermentation using L. plantarum on adult black soldier fly (BSF) increased the protein content of the adult BSF up to 1.4% as compared to the control samples. The increase in protein content in the fermented samples may be attributed to the protein in bacterial cells [ 20 ] and biotransformation of soluble carbohydrates in the BSFL into bacterial proteins [ 21 ]. According to da Silva Sabo et al. [ 22 ], lactic acid bacteria have probiotic properties that can produce bacteriocins which are proteins synthesised in the ribosome and released into the extracellular medium by the lactic acid bacteria. Wang et al. [ 20 ] also demonstrated that fermentation of sea cucumber using L. plantarum increased the protein content up to 2.03% as compared to the control samples. In this study, the protein content of BSFL fermented with L. plantarum was higher than that of L. acidophilus and L. lactis . As such may be attributed to the smaller cell size of L. plantarum than L. acidophilus and L. lactis . The smaller cell size can result in higher bacterial concentration and faster growth which can lead to higher bacteriocin activity [ 23 ]. Solid-state fermentation of BSFL using Lactobacillus spp. also influenced the fat content of the BSFL. In general, the fermentation increased the fat content of BSFL. Statistically, fermentation of BSFL using L. lactis did not significantly increase ( P > 0.05) the fat content of BSFL. However, fermentation of BSFL using L. plantarum and L. acidophilus significantly increased ( P < 0.05) the fat content of the BSFL from 36.91 ± 1.07% (unfermented BSFL) to 41.99 ± 1.56%. This is in accordance with the results obtained by Luparelli et al. [ 7 ] that solid-state fermentation using L. plantarum on BSF prepupae increased the fat content of the BSF prepupae up to 2% as compared to the control samples. In another study by Cabuk et al. [ 24 ], fermented peas using L. plantarum also increased the fat content by 1%. The increase in lipid content can be influenced by the activity of lipolytic enzymes produced by the lactic acid bacteria [ 25 ]. According to Kumari et al. [ 26 ], the fermentation process may increase the fat content by forming simple fatty acids through the breakdown of large fat molecules. From Fig. 1 , it can be observed that the highest fat content was observed in the BSFL fermented with L. acidophilus followed by L. plantarum and L. lactis . These results are in accordance with the previous findings by de Marins et al. [ 27 ] that burger fermented with L. acidophillus produced a higher fat content than L. plantarum . A lower lipid content in L. plantarum -fermented samples may occur because L. plantarum is known to have a more significant effect on degrading fats than the L. acidophilus [ 28 ]. A lower fat content in the L. lactis -fermented samples may be due to limited lipolytic activity in L. lactis which allows limited formation of fatty acids by the bacteria [ 29 ]. In this study, the protein content in the BSFL samples was first extracted prior to protein hydrolysis and the results of protein recovery are shown in Table 2 . From the table, it can be observed that the protein recoveries obtained in this study are higher than the protein recovery of 27.58% reported by Smets et al. [ 30 ]. As such may be influenced by the defatting process prior to the extraction stage carried out in this study. Defatting can increase the protein recovery because it can remove confounding factors in the form of fat during the extraction process [31-Ravi et al., 2020]. Nevertheless, the protein recoveries obtained in this study were lower than the the protein recovery of 59.3% reported by Wang et al. [ 11 ] which may be due to different protein extraction methods used in both studies. In this study, protein extraction was carried out using NaOH without any addition of HCl. In contrast, Wang et al. [ 11 ] used additional 1 N HCl after extraction of protein to aid the precipitation of extracted protein from BSF powder. Previous studies have reported that the addition of HCl will trigger an increase in protein interactions followed by protein aggregation and precipitation and eventually increase the protein recovery [ 32 ]. Table 2 Protein recovery of extracted protein from black soldier fly larvae. Sample Protein recovery (%) This study Reference Control 41.60 \(\:\pm\:\) 2.36 b 27.58–59.3 [11, [ 30 ]] Fermentation - L. plantarum 35.99 \(\:\pm\:\) 1.99 a Fermentation - L. acidophilus 31.71 \(\:\pm\:\) 1.14 a Fermentation - L. lactis 31.41 \(\:\pm\:\) 4.65 a *Differences in notation (a and b) indicate significant differences in different species ( P < 0.05). Protein recoveries of all the fermented samples are significantly different ( P < 0.05) and lower as compared to the control (unfermented) samples. This may occur because lactic acid bacteria possess proteolytic enzymes that can hydrolyse protein into smaller peptides or amino acids and utilisation of essential amino acids for bacterial growth [ 33 ]. In addition, the protein content in the fermented samples is also influenced by the presence of bacteriocin which is known to show decreased stability under alkaline conditions [ 34 ]. From Table 2 , it can be observed that the protein recovery of BSFL fermented with L. plantarum was higher than L. acidophilus and L. lactis . This is most probably due to the higher proteolytic activity of L. plantarum compared to the other bacteria [ 35 ]. A higher proteolytic activity may lead to a higher protein recovery due to more peptide bonds with smaller molecular weight that can cause protein interactions with water and consequently increase the solubility [ 36 ]. Effects of Fermentation on Degree of Hydrolysis of Protein Hydrolysate from Black Soldier Fly Larvae In this study, the degree of hydrolysis was assessed to determine the amount of protein that was being hydrolysed during enzymatic hydrolysis of BSFL using bromelain enzymes and the results are shown in Table 3 . The values of degree of hydrolysis obtained in this study were slightly higher than the values reported in the literature. In this study, the protein content from the BSFL was first extracted prior to the enzymatic hydrolysis stage whereas the previous studies did not extract the protein prior to the hydrolysis stage. The stage of protein extraction helps to separate the protein fraction from chitin and increase the contact surface between enzymes and the protein [ 37 ]. From Table 2 , it can be observed that the values of degree of hydrolysis for BSFL pre-treated with solid-state fermentation using Lactobacillus spp. were not significantly different from the control ( P > 0.05). According to Le et al [ 39 ], fermentation of soy milk using L. plantarum increased the degree of hydrolysis up to 3% as compared to samples hydrolysed without the fermentation process. In another study, Rai et al. [ 40 ] also reported an increase of degree of hydrolysis when fish processing waste was pre-treated with fermentation using lactic acid bacteria compared to the control. The differences may be due to the inactivation of bacteria at higher temperatures (60 ℃) followed by drying overnight at 105 ℃ carried out in this study whereas in the literature, hydrolysis was carried out directly using alcalase enzymes after the fermentation without any bacterial inactivation. The presence of bacterial proteolytic and alcalase enzymes may work together to hydrolyse the substrate and consequently increase the degree of hydrolysis as compared to the control [ 39 ]. Table 3 Degree of hydrolysis of protein hydrolysate from black soldier fly larvae. Sample Degree of Hydrolysis (%) This study Reference Control 53.19 \(\:\pm\:\) 2.73 a 19.83 [38] – 47.4 [3] Fermentation - L. plantarum 51.52 \(\:\pm\:\) 1.06 a Fermentation - L. acidophilus 47.64 \(\:\pm\:\) 2.61 a Fermentation - L. lactis 52.61 \(\:\pm\:\) 5.94 a *a indicate no significant difference in different species ( P > 0.05). Effects of Chromatography Flow Rate on Fractionated Protein Concentration No studies have reported on the influence of chromatography flow rates on the fractionation of protein hydrolysate from BSFL. In this study, fractionation of protein hydrolysate was carried out using size-exclusion chromatography at different flow rates and the results are shown in Fig. 2 . The highest protein concentration in the control sample and fermentation using L. plantarum , L. acidophilus , and L. lactis samples were 1,578.81 \(\:\pm\:\) 52.34 mg/mL, 1,252.30 \(\:\pm\:\) 112.17 mg/mL, 1,414.69 \(\:\pm\:\) 54.98 mg/mL, and 1,331.73 \(\:\pm\:\) 66.62 mg/mL, respectively. Statistically, there were no significant differences ( P > 0.05) among control and fermented samples at all flow rates (0.5–1.5 mL/min) but the highest protein concentration was observed at a chromatography flow rate of 1 mL/min. The chromatography flow rate plays important roles in the separation of compounds in the stationary phase and mobile phase. A relatively low flow rate has better adsorption equilibrium and more efficient separation. However, a flow rate that is too low can also allow diffusion effects in the mobile phase while a flow rate that is too high can cause the compounds not having the opportunity to reach an equilibrium state due to the contact time between the mobile phase and stationary phase which is very small [ 41 ]. The results of this study suggest that a chromatography flow rate of 1mL/min is deemed sufficient to separate the protein fractions in the protein hydrolysate. Effects of Fermentation on Amino Acid Composition of Fractionated of Protein Hydrolysate from Black Soldier Fly Larvae In this study, the amino acid composition was determined for the fractionated protein hydrolysate that had the highest protein concentration in all samples and the results are presented in Table 4 . The sample with the highest protein concentration was obtained in the 5th tube with a chromatography flow rate of 1 mL/min for the control, fermentation using L. plantarum , and fermentation using L. lactis sample while for the fermentation using L. acidophilus the highest protein concentration was obtained in the 4th tube with a chromatography flow rate of 1 mL/min. From Table 4 , it can be observed that there were differences in amino acid composition between the control and fermented fractionated protein hydrolysate. For BSFL fermented with L. plantarum and L. lactis , the fractionated protein hydrolysates contained only three types of amino acids particularly L-alanine, glycine, and L-methionine but the concentrations were significantly different ( P < 0.05). For BSFL fermented with L. acidophilus , the fractionated protein hydrolysate also contained L-alanine, glycine and L-methionine but with the addition of L-glutamic acid, leucine, L-methionine, L-valine, L-proline, and L-serine that were not present in the case of fermentation using L. plantarum and L. lactis. From Table 4 , it can be noticed as well that serine was present in the fractionated protein hydrolysate fermented with L. acidophilus but not in the control. This can be attributed to the synthesis of serine by L. acidophilus through the de novo pathway as suggested by Altermann et al. [ 42 ]. These results are in accordance with the previous findings by Hadj et al. [ 43 ] that fermentation of BSF puparia using lactic acid bacteria can cause changes in the amino acid composition including a decrease in several amino acids. The decrease in several amino acid content in the fermented samples may occur due to the role of lactic acid bacteria which utilises the amino acids as nitrogen sources in the growth process [ 33 ]. In this study, the absence of several amino acids that were previously present in the unfermented (control) samples may have occurred because the amino acids are essential for the bacteria and consequently being consumed by the bacteria [ 44 ]. Table 4 Amino acid composition of fractionated protein hydrolysate from black soldier fly larvae Amino acid Content (mg/kg fraction) Control Fermentation L. plantarum Fermentation L. acidophilus Fermentation L. lactis L-Alanine 84.63 84.63 85.14 84.63 L-Arginine - - - - L-Aspartic acid 190.57 - - - Glycine 312.2 b 314.95 c 325.48 d 274.77 a L-Glutamic acid 152.43 - 152.43 - L-Histidine - - - - L-Isoleucine - - - - L-Cysteine - - - - L-Leucine 167.3 - 167.3 - L-Lysine - - - - L-Methionine 2.31 d 1.24 b 2.02 c 1.21 a L-Tryptophan - - - - L-valine 128.75 - 128.75 - L-Phenylalanine - - - - L-Proline 128.38 - 128.38 - L-Serine - - 149.74 - L-Threonine - - - - L-Tyrosine - - - - *Differences in notation (a, b, c, and d) indicate significant differences in in different species ( P < 0.05). Statistically, the amount of glycine and methionine content in the control and fermented samples were significantly different ( P 0.05) which resembles the previous findings by Patterson et al. [ 45 ]. In this study, the glycine content in the fraction of protein hydrolysate fermented with L. plantarum and L. acidophilus increased by 2.75 mg/kg and 13.28 mg/kg, respectively whereas the glycine content in the fraction of protein hydrolysate fermented with L. lactis decreased by 37.43 mg/kg fraction. The increased glycine content can be influenced by the glycolysis process that synthesises serine with the change of 3-phosphoglycerate and converts it into glycine. In addition, the Lactobacillus spp. can utilise carbohydrates in the form of glucose and convert them into pyruvic acid which then produces aspartic acid that can be synthesised into methionine and then into serine and glycine [ 46 ]. This also allows a decrease in methionine content due to the biosynthesis of serine and glycine [ 43 ]. The alanine content that was not different between the control sample and fermented sample may be due to alanine being a non-essential amino acid for bacteria so it was not the main nitrogen source utilised by bacteria for growth [ 44 ]. CONCLUSIONS In summary, solid-state fermentation of BSFL using L. plantarum, L. acidophilus, and L. lactis increased protein and fat content of the BSFL up to 3.34 and 5.08%, respectively. However, the solid-state fermentation reduced the protein recovery and degree of hydrolysis of the protein hydrolysate upon alkaline extraction and enzymatic hydrolysis using bromelain enzymes. Fractionation of the protein hydrolysate at different chromatographic flow rates slightly affected the protein concentrations which varied from 0.51 mg/mL to 1414.69 mg/mL with the highest protein concentration obtained when the flow rate of the chromatography was set at 1 mL/min. The fractionated protein hydrolysates fermented with Lactobacillus spp. contained several amino acids such as glycine, and L-methionine that were significantly different from the unfermented samples. The effects of these changes towards the bioactivity of the fractionated protein hydrolysate were not examined in this study. Potential future studies might should focus on the correlation between the amino acid composition and bioactivity of the fractionated protein hydrolysate from BSFL. Declarations Ethical approval Not applicable. Competing interests The authors declare no competing interests Funding: This work was financially supported by Institut Teknologi Bandung (Riset Internasional 2023, LPPM.PN-16-19-2023). Data availability Not applicable Author contributions MYA: funding acquisition, conceptualization, supervision, formal analysis, writing original draft, review, and editing. HS, GRP, MAS, BAS: data curation, verified analytical methods, investigation, methodology, and writing original draft. KL, NIMP, AK: conceptualization, supervision, review. References Müller A, Wolf D, Gutzeit HO (2017) The black soldier fly, Hermetia illucens–a promising source for sustainable production of proteins, lipids bioactive substances. Z. Naturforsch. C Biosci. 72: 351–363 Abduh MY, Nadia MH, Syaripudin, Manurung R, Putra RE (2018) Factors affecting the bioconversion of Philippine tung seed by black soldier fly larvae for the production of protein and oil-rich biomass. J. Asia-Pac. Entomol. 21: 836–842 Firmansyah M, Abduh MY (2019) Production of protein hydrolysate containing antioxidant activity from Hermetia illucens . Heliyon 5(6): e02005 Riolo K, Rotondo A, La Torre GL, Marino Y, Franco GA, Crupi R, Fusco R., … & Giannetto, A (2018) Cytoprotective and Antioxidant Effects of Hydrolysates from Black Soldier Fly ( Hermetia illucens ). 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Journal of Dairy Science 102(1): 124-134 Smets R, Verbinnen B, Van De Voorde I, Aerts G, Claes J, Van Der Borght M (2020) Sequential extraction and characterisation of lipids, proteins, and chitin from black soldier fly ( Hermetia illucens ) larvae, prepupae, and pupae. Waste and Biomass Valorization, 11: 6455-6466 Ravi HK, Degrou A, Costil J, Trespeuch C, Chemat F, Vian MA (2020) Larvae mediated valorization of industrial, agriculture and food wastes: Biorefinery concept through bioconversion, processes, procedures, and products. Processes 8(7): 857 Martínez-Maqueda D, Hernández-Ledesma B, Amigo L, Miralles B, Gómez-Ruiz JÁ (2013) Extraction/fractionation techniques for proteins and peptides and protein digestion. Proteomics in foods: principles and applications 21-50 Solval KM, Chouljenko A, Chotiko A, Sathivel S (2019) Growth kinetics and lactic acid production of Lactobacillus plantarum NRRL B-4496, L. acidophilus NRRL B-4495, and L. reuteri B-14171 in media containing egg white hydrolysates. LWT 105: 393-399 Song DF, Zhu MY, Gu Q (2014) Purification and characterization of plantaricin ZJ5, a new bacteriocin produced by Lactobacillus plantarum ZJ5. PLoS one 9(8): e105549 Moslehishad M, Mirdamadi S, Ehsani MR, Ezzatpanah H, Moosavi‐Movahedi AA (2013) The proteolytic activity of selected lactic acid bacteria in fermenting cow's and camel's milk and the resultant sensory characteristics of the products. International Journal of Dairy Technology 66(2): 279-285 He S, Franco C, Zhang W (2013) Functions, applications and production of protein hydrolysates from fish processing co-products (FPCP). Food Research International 50(1): 289-297 Omana DA, Xu Y, Moayedi V, Betti M (2010) Alkali-aided protein extraction from chicken dark meat: Chemical and functional properties of recovered proteins. Process Biochemistry 45(3): 375-381 Chi CF, Cao ZH, Wang B, Hu FY, Li ZR, Zhang B (2014). Antioxidant and functional properties of collagen hydrolysates from Spanish mackerel skin as influenced by average molecular weight. Molecules 19(8): 11211-11230 Le PH, Parmentier N, Le TT, Raes K (2021) Evaluation of using a combination of enzymatic hydrolysis and lactic acid fermentation for γ-aminobutyric acid production from soymilk. LWT 142: 111044. Rai AK, Jini R, Swapna HC, Sachindra NM, Bhaskar N, Baskaran V (2011) Application of native lactic acid bacteria (LAB) for fermentative recovery of lipids and proteins from fish processing wastes: bioactivities of fermentation products. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6142604","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":434854732,"identity":"041a025f-17f5-4c89-9f87-78a223b5f776","order_by":0,"name":"M.Y. Abduh","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYLCCigoGHgYJBoYDjA0gLmODBEEtZ84w8PCAtBwkWsvZNgYGkBYGiBYGBrxaDG6kP5M4OK9Oxl66gfHwxx0M8vwNzI038GvJMZM4uO0wD4/MAaDDzjAYzjjA2GxBQAub9MdtB4B+SQBqaWNg3MDA2EaEw+bUwbXYE6ElAeiwBma4lkSCWiTPvDG2OHAM6JcbiQ0Hzp6RSJ5xmIBf+I6nP7xxoKbOnn1G8uEPlTtsbPvb2x/iDTGFA3AmOOqBTmLGpx4I5BsIKBgFo2AUjIJRwAAApRFO6pUdsf4AAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-9619-6014","institution":"Institut Teknologi Bandung","correspondingAuthor":true,"prefix":"","firstName":"M.Y.","middleName":"","lastName":"Abduh","suffix":""},{"id":434854733,"identity":"676bb717-ebea-4650-96e6-d51b7cfca5e2","order_by":1,"name":"Hana Safira","email":"","orcid":"","institution":"Bandung Institute of Technology: Institut Teknologi Bandung","correspondingAuthor":false,"prefix":"","firstName":"Hana","middleName":"","lastName":"Safira","suffix":""},{"id":434854734,"identity":"a26641c2-8eaa-489e-97e7-8f2228faa6b4","order_by":2,"name":"Ghassani Raisa Pramestiandini","email":"","orcid":"","institution":"Bandung Institute of Technology: Institut Teknologi Bandung","correspondingAuthor":false,"prefix":"","firstName":"Ghassani","middleName":"Raisa","lastName":"Pramestiandini","suffix":""},{"id":434854735,"identity":"62f36879-25d4-45f7-bdc6-9365189db534","order_by":3,"name":"Mohammad Adiba Syahada","email":"","orcid":"","institution":"Bandung Institute of Technology: Institut Teknologi Bandung","correspondingAuthor":false,"prefix":"","firstName":"Mohammad","middleName":"Adiba","lastName":"Syahada","suffix":""},{"id":434854736,"identity":"dc35815e-7917-42fb-a99b-7ce52d239dee","order_by":4,"name":"Bunga Ayu Salsabila","email":"","orcid":"","institution":"Bandung Institute of Technology: Institut Teknologi Bandung","correspondingAuthor":false,"prefix":"","firstName":"Bunga","middleName":"Ayu","lastName":"Salsabila","suffix":""},{"id":434854737,"identity":"46418afd-eadd-4964-a1a9-ca367bfa3215","order_by":5,"name":"Khalilan Lambangsari","email":"","orcid":"","institution":"Bandung Institute of Technology: Institut Teknologi Bandung","correspondingAuthor":false,"prefix":"","firstName":"Khalilan","middleName":"","lastName":"Lambangsari","suffix":""},{"id":434854738,"identity":"1fd4f4ec-9fdf-467b-b6e4-1674e9cd34aa","order_by":6,"name":"Noor Illi Mohamad Puad","email":"","orcid":"","institution":"International Islamic University Malaysia","correspondingAuthor":false,"prefix":"","firstName":"Noor","middleName":"Illi Mohamad","lastName":"Puad","suffix":""},{"id":434854739,"identity":"72799c42-14aa-46a8-8883-50ac18fcce26","order_by":7,"name":"Akos Kenez","email":"","orcid":"","institution":"City University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Akos","middleName":"","lastName":"Kenez","suffix":""}],"badges":[],"createdAt":"2025-03-03 04:43:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6142604/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6142604/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12649-025-03197-0","type":"published","date":"2025-07-05T15:58:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":80530461,"identity":"aabacb61-8f2b-49c1-8b87-62c61e257bd9","added_by":"auto","created_at":"2025-04-14 10:50:07","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":129042,"visible":true,"origin":"","legend":"\u003cp\u003eProtein and lipid content of black soldier fly larvae. Differences in notation (a and b) indicate significant differences in different species (\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6142604/v1/062885ebd4e582d7483eb3cb.jpeg"},{"id":80531212,"identity":"0e6940d0-01b4-4a1c-b84a-da913f1eef74","added_by":"auto","created_at":"2025-04-14 10:58:07","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":378603,"visible":true,"origin":"","legend":"\u003cp\u003eProtein concentration of fractionated protein hydrolysate at various flow rates.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6142604/v1/7862c68356f210d5063fb120.jpeg"},{"id":86179212,"identity":"da9553e3-74ba-4dae-9751-71f9400d24be","added_by":"auto","created_at":"2025-07-07 16:17:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1611747,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6142604/v1/52a9ad79-97b4-4974-a245-19d6a009121f.pdf"}],"financialInterests":"","formattedTitle":"Synthesis and fractionation of Protein Hydrolysate from Hermetia illucens L. pre-treated with Lactobacillus spp.","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eBlack soldier fly (\u003cem\u003eHermetia illucens\u003c/em\u003e L.) larvae (BSFL) started to attract the world\u0026rsquo;s attention due to their ability to convert organic waste into protein and lipid-rich biomass [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Several studies have reported that BSFL have a relatively high protein content (40\u0026ndash;50%) and can be utilised as an alternative source of protein hydrolysate with special biological activities that highly depend on the composition of the amino acids [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFirmansyah and Abduh [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] investigated the synthesis of protein hydrolysates of BSFL using bromelain enzymes with the degree of hydrolysis in the range of 14.6\u0026ndash;39.9%. The protein hydrolysates had a molecular weight in the range of 14\u0026ndash;25 kDa and mainly comprised of lysine (8.0%) followed by leucine (7.7%), and valine (7.2%). The synthesised protein hydrolysates had the ability to inhibit free radicals 2,2-diphenyl-2-picryl hydrazyl (DPPH) up to 2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.41% and IC\u003csub\u003e50\u003c/sub\u003e of 0.84%.\u003c/p\u003e \u003cp\u003eRiolo et al. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] also produced and characterised protein hydrolysates from BSFL using an enzyme mix of flavourzyme:protamex:alkalase at a ratio of 1:1:1. The molecular weight of the protein hydrolysates were in the range of 10\u0026ndash;75 kDa and mainly comprised of alanine (13.4%), glutamic acid (12.9%) and valine (9.4%). The synthesised protein hydrolysates possessed a strong antioxidant activity by reducing intracellular reactive oxygen species in challenged cells. In another study, Ferdinand et al. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] demonstrated that protein hydrolysates produced from BSFL, and bromelain enzymes have a very good antioxidant activity with an IC\u003csub\u003e50\u003c/sub\u003e value of 9.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84% (as determined by the DPPH method). In addition, the protein hydrolysates also had a good antibacterial activity against \u003cem\u003eVibrio cholerae\u003c/em\u003e with an inhibition zone diameter of 11.2\u0026thinsp;\u0026plusmn;\u0026thinsp;3.8 mm (as determined by the agar well diffusion method).\u003c/p\u003e \u003cp\u003eBatish et al. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] examined the effects of protein hydrolysis using different enzymes (alcalase, papain, pepsin) on the functional properties, antioxidant activities and amino acids composition of the protein hydrolysates. The degree of hydrolysis lied in the range of 9.8\u0026ndash;18.4% and all protein hydrolysates had the ability to scavenge DPPH radicals with alcalase hydrolysates showing a higher antioxidant activity as compared to the protein hydrolysates produced by pepsin and papain. The most dominant amino acid in the protein hydrolysates was glutamic acid (35.10-74.86 mg g\u003csup\u003e-1\u003c/sup\u003e). The study highlighted that enzymatic hydrolysis improved the functional properties and bioactivities of the protein hydrolysis but highly influenced by the types of enzymes.\u003c/p\u003e \u003cp\u003eAccording to Luparelli et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], solid-state fermentation of adult and prepupae of black soldier fly using \u003cem\u003eLacticaseibacillus rhamnosus\u003c/em\u003e and \u003cem\u003eLactiplantibacillus plantarum\u003c/em\u003e enriched the essential amino acids such as histidine and cysteine while significantly reduced other amino acids such as isoleucine, leucine, lysine, valine, glycine and glutamic acid. The results indicated lactic acid bacteria (LAB) activity on the protein fraction of the black soldier fly biomass with some proteins being utilised while new proteins being synthesized by the LAB. According to Zacharof et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], \u003cem\u003eLactobacillus sp.\u003c/em\u003e which is an environmentally friendly LAB that can produce bacteriocins is able to modify components inherent in insects, including amino acid components.\u003c/p\u003e \u003cp\u003eSystematic studies that investigate the effects of solid-state fermentation using \u003cem\u003eLactobacillus sp\u003c/em\u003e. on the protein content and composition of protein hydrolysate from BSFL are still scarce. In addition, studies that report on the fractionation of protein hydrolysate are still limited. It is expected that pre-treatment of BSFL by fermentation using \u003cem\u003eLactobacillus sp\u003c/em\u003e. can positively influence the protein content and composition of fractionated protein hydrolysate from BSFL. Hence, this study aimed to investigate the effects of solid-state fermentation using \u003cem\u003eLactobacillus sp\u003c/em\u003e. on the protein content of BSFL, protein recovery, degree of hydrolysis, and amino acid composition of protein hydrolysates from BSFL. This study also investigated the effects of different chromatographic flow rates on protein concentration of the fractionated protein hydrolysates from BSFL.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eDried BSFL were obtained from a local shop in West Java, Indonesia. The chemicals used in this study included n-hexane, K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, CuSO\u003csub\u003e4\u003c/sub\u003e, concentrated H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e, NaOH 40%, H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e, methylene blue, methylene red, 0.1 N HCl, 0.25 M NaOH, 0.25 M HCl, sucrose, Coomassie Brilliant Blue G-250, phosphoric acid 85%, ethanol 95%, Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO, NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e, KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, formaldehyde 37%, and 0.1 N NaOH, which were obtained from the chemical warehouse of the School of Life Sciences and Technology, Institut Teknologi Bandung, Indonesia. The biological agents used in this study were \u003cem\u003eL. plantarum, L. acidophilus, and L. lactis\u003c/em\u003e culture obtained from Microbiology Laboratory, School of Life Sciences and Technology, Institut Teknologi Bandung. Bromelain enzymes were obtained from PT Bromelain Enzyme and Sephadex G-25 gels were obtained from PT Enigma Saintia Solusindo, Indonesia.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProximate Analysis\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e\u003cul\u003e \u003cli\u003e \u003cp\u003eProximate analysis was carried out to determine moisture, ash, fat and protein content of the samples. Determination of moisture content was done by thermogravimetric method at 105°C for 24 h (method 934.01) whereas determination of ash content was carried out by drying method using a furnace at 500–600°C for 6 h (method 942.06) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The fat content was determined by the Soxhlet method using n-hexane solvent at 70°C (method 948.22) whereas the protein content was measured by the Kjedahl method (method 960.52) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The carbohydrate content was calculated based on the difference between moisture, ash, fat, and protein content of the samples.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eSolid-State Fermentation of Black Soldier Fly Larvae\u003c/h3\u003e\n\u003cp\u003e \u003c/p\u003e\u003cul\u003e \u003cli\u003e \u003cp\u003eSolid-state fermentation was carried out using \u003cem\u003eLactobacillus\u003c/em\u003e spp. culture maintained at 37 ℃ in Man Rogosa and Sharp (MRS) broth for 18 h. Approximately 50 g of dried BSFL that were crushed using a blender and sieved with 80 mesh (\u0026lt; 0.177 mm) were mixed with 50% (weight/volume) distilled water and 4% (weight basis) sucrose. The samples were then sterilised in an autoclave at 121 ℃ for 20 min and then incubated with 7% (weight/volume) \u003cem\u003eLactobacillus\u003c/em\u003e spp. (bacterial concentration of 10\u003csup\u003e8\u003c/sup\u003e CFU/mL) at 37 ℃ for 72 h. Inactivation of the samples was carried out at 60 ℃ and dried overnight at 105 ℃ [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eDefatting of Black Soldier Fly Larvae\u003c/h3\u003e\n\u003cp\u003eDefatting of BSFL was carried out by Soxhlet method using n-hexane solvent at 70 ℃ for 6 h to remove the fat content in the samples. The ratio of biomass to solvent used was 1:10 (weight to volume). The extracted mixture of fat and solvent was then separated using a rotary vacuum evaporator at 60 ℃ for 2 h [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eExtraction of Protein from Black Soldier Fly Larvae\u003c/h3\u003e\n\u003cp\u003eProtein was extracted from the BSFL using 0.25 M NaOH at a ratio of 1:5 (weight to volume). The mixture was incubated in a water bath shaker at 60 ℃, 400 rpm, for 60 min. After that, the extracted mixture was added with the same amount of solvent as in the previous process and incubated again in a water bath shaker at 60 ℃, 400 rpm, for 60 min. The mixture was then centrifuged at 4000 rpm for 15 min. The precipitate and supernatant were then separated using a filter paper and a glass funnel. The precipitate was then re-extracted using the same procedures for the extraction of protein. The supernatants from the second and third extractions were mixed and then neutralized to pH 7 using 0.25 M HCl prior to the determination of protein concentration using the Bradford method [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e–\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Protein Recovery with the Bradford Method\u003c/h2\u003e \u003cp\u003eThe concentration of protein extract from the BSFL was determined using the Bradford method. A protein sample of 0.1 mL was poured into a test tube and mixed with 5 mL of Bradford reagent. The mixture was then incubated for approximately 2 min at room temperature (27 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\mathbf{℃})\\)\u003c/span\u003e\u003c/span\u003e. The absorbance of the solution was determined using a UV-visible spectrophotometer at a wavelength of 595 nm. The protein concentration was determined based on the bovine serum albumin standard curve prepared at concentrations of 0, 100, 250, 500, and 1000 ppm [3-Firmansyah and Abduh, 2019]. Each concentration of 0.1 mL solution was added with 5 mL of Bradford reagent and incubated for 2 min. The solution was then measured using a UV-visible spectrophotometer at a wavelength of 595 nm [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The protein extract concentration was then used to determine protein recovery using the following equation.\u003c/p\u003e\u003cdiv id=\"Equ1\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ1\" name=\"EquationSource\"\u003e\n$$\\:\\text{P}\\text{r}\\text{o}\\text{t}\\text{e}\\text{i}\\text{n}\\:\\text{r}\\text{e}\\text{c}\\text{o}\\text{v}\\text{e}\\text{r}\\text{y}\\:\\left(\\text{%}\\right)=\\:\\frac{\\text{P}\\text{r}\\text{o}\\text{t}\\text{e}\\text{i}\\text{n}\\:\\text{e}\\text{x}\\text{t}\\text{r}\\text{a}\\text{c}\\text{t}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\left(\\frac{\\text{m}\\text{g}}{\\text{m}\\text{L}}\\right)}{\\text{I}\\text{n}\\text{i}\\text{t}\\text{i}\\text{a}\\text{l}\\:\\text{p}\\text{r}\\text{o}\\text{t}\\text{e}\\text{i}\\text{n}\\:\\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n}\\:\\left(\\frac{\\text{m}\\text{g}}{\\text{m}\\text{L}}\\right)}\\text{x}\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e1\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHydrolysis of Protein Extract from Black Soldier Fly Larvae\u003c/h3\u003e\n\u003cp\u003eThe supernatant produced in the protein extraction stage was dried using a freeze-drying method whereby the samples were placed in a freeze dryer at -55 ℃ and 1500 mTorr pressure until a constant weight was obtained [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The freeze-dried protein extract was then dissolved in a phosphate buffer solution (pH 8) with a ratio of 1:10 (weight/volume). The solution was then mixed with 3% bromelain enzyme (enzyme activity of 1200 GDU/mg) in a 250 mL Erlenmeyer flask and incubated using a water bath shaker at 50 ℃ and 150 rpm for 24 h. The enzyme in the solution was then inactivated by incubating the solution using a water bath shaker at 90 ℃ for 10 min. The solution was then centrifuged at 4000 rpm for 15 min. The supernatant obtained was then filtered and stored in a refrigerator at 4 ℃ for further analysis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eDetermination of Degree of Hydrolysis\u003c/h3\u003e\n\u003cp\u003eThe degree of hydrolysis was determined based on the ratio of α-amylase and total nitrogen in the samples. Total nitrogen in the samples was determined using the Kjeldahl method while α-amylase was determined using the formol titration method. Approximately 5 mL of samples were placed into a Kjeldahl destruction flask and mixed with 7 g K\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e; 0.8 g CuSO\u003csub\u003e4\u003c/sub\u003e; and 12 mL of concentrated H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. The mixture was then destructed until the colour of the solution into blue green. The samples in the flask were then settled to cool at room temperature (27°C) before mixed with 25 mL of distilled water. The mixture was then transferred into a Kjeldahl distillation flask and then mixed with 50 mL of NaOH 40%. An Erlenmeyer flask was prepared and filled with 30 mL of H\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e 3% and 3 drops of Tashiro indicator as. The green distillate was then collected as much as possible, and the solution was then titrated with 0.1 M HCl standard solution [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor the formol titration method, approximately 1.5 g of protein hydrolysate samples was added to distilled water until it reached a weight of 50 g. The mixture was then mixed with 0.1 N NaOH to reach pH 7 and then mixed with approximately 10 mL of a formaldehyde solution of 37% (weight/volume) and allowed to stand for 5 min. Then, the solution was titrated using 0.1 N NaOH until the pH of the solution became 8.5 [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The degree of hydrolysis was determined using the following equations.\u003c/p\u003e\u003cdiv id=\"Equ2\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ2\" name=\"EquationSource\"\u003e\n$$\\:\\text{F}\\text{r}\\text{e}\\text{e}\\:\\text{a}\\text{m}\\text{i}\\text{n}\\text{o}\\:\\text{a}\\text{c}\\text{i}\\text{d}\\text{s}\\:\\left(\\text{%}\\right)=\\left(\\text{V}\\:\\text{x}\\:\\text{C}\\:\\text{x}\\frac{\\text{0,014007}}{\\text{M}}\\right)\\text{x}\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e2\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equ3\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ3\" name=\"EquationSource\"\u003e\n$$\\:\\text{D}\\text{e}\\text{g}\\text{r}\\text{e}\\text{e}\\:\\text{o}\\text{f}\\:\\text{h}\\text{y}\\text{d}\\text{r}\\text{o}\\text{l}\\text{y}\\text{s}\\text{i}\\text{s}\\:\\left(\\text{%}\\right)=\\:\\frac{\\text{%}\\text{F}\\text{r}\\text{e}\\text{e}\\:\\text{a}\\text{m}\\text{i}\\text{n}\\text{o}\\:\\text{a}\\text{c}\\text{i}\\text{d}\\text{s}}{\\text{%}\\text{T}\\text{o}\\text{t}\\text{a}\\text{l}\\:\\text{N}\\text{i}\\text{t}\\text{r}\\text{o}\\text{g}\\text{e}\\text{n}}\\text{x}\\:100$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e3\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e \u003cp\u003eWhere V is the volume of NaOH used to reach pH 8.5, C is the concentration of the titration solution (0.1 N), and M is the mass of the sample.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFractionation of Protein Hydrolysate\u003c/h2\u003e \u003cp\u003eFractionation of the protein hydrolysate samples was performed using the size-exclusion chromatography method. Approximately 3 g of Sephadex G-25 beaded gels were soaked in 18 mL of phosphate buffer solution pH 7.4 for 24 h and then inserted into a column (30 cm high and 1.5 cm diameter). Approximately 1.5 mL of samples were fed into the column followed by the injection of 160 mL of phosphate buffer solution pH 7.4 at a flow rate of 0.5 to 1.5 mL/min. Absorbance of the fractionated samples was measured using a UV-visible spectrophotometer with wavelengths of 260 nm (A\u003csub\u003e260\u003c/sub\u003e), 280 nm (A\u003csub\u003e280\u003c/sub\u003e), and 320 nm (A\u003csub\u003e320\u003c/sub\u003e) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Protein concentration in the fractionated samples can be determined using the following equation:\u003c/p\u003e\u003cdiv id=\"Equ4\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equ4\" name=\"EquationSource\"\u003e\n$$\\:Protein\\:concentration\\:\\left(\\frac{mg}{mL}\\right)=\\:\\left(\\left({A}_{280}-{A}_{320}\\right)x\\:1552\\right)-\\left(\\left({A}_{260}-{A}_{320}\\right)\\:x\\:\\text{757,3}\\right)$$\u003c/div\u003e\u003cdiv class=\"EquationNumber\"\u003e4\u003c/div\u003e\u003c/div\u003e\u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of Amino Acid Composition\u003c/h2\u003e \u003cp\u003eComposition of amino acids in the samples was determined at Saraswati Indo Genetech, Bogor, Indonesia using ultra high-performance liquid chromatography combined with photodiode array detector (UPLC-PDA/18-5-17/MU/SMM-SIG) and high-performance liquid chromatography combined with photodiode array detector (HPLC-PDA/18-5-63/MU/SMM-SIG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e\u003cul\u003e \u003cli\u003e \u003cp\u003eStatistical analysis in this study was carried out using Minitab version 21.1.0 by testing One-way ANOVA to determine the significance of fermentation pretreatment on several parameters and two-way ANOVA to determine the significance of chromatography flow rate on protein concentration. Test results that showed a significant value (P \u0026lt; 0.05) were further examined by post hoc testing using the Duncan test to determine the significance between one data and another in a group [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eEffects of Fermentation on Protein Recovery from Black Soldier Fly Larvae\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS AND DISCUSSIONS","content":"\u003ch2\u003eProximate Analysis of Black Soldier Fly Larvae\u003c/h2\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the proximate composition of the BSFL samples. The moisture content of the BSFL samples investigated in this study was lower than the reported values in the literature. The differences may be due to different drying method, temperature and time that play vital roles in determining the final moisture content of the BSFL. Nevertheless, the protein, fat, carbohydrate and ash content of the samples corresponded to the values reported in other studies.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProximate analysis of dried black soldier fly larvae\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eComposition (%)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoisture\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.22 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.19\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.2\u003csup\u003e[18]\u003c/sup\u003e – 12\u003csup\u003e[19]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAsh\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.47 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.31\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.4\u003csup\u003e[18]\u003c/sup\u003e – 10.6\u003csup\u003e[19]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e32.35 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 0.89\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30\u003csup\u003e[19]\u003c/sup\u003e – 37.9\u003csup\u003e[18]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFat\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.91 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 1.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e33.2\u003csup\u003e[19]\u003c/sup\u003e – 43.6\u003csup\u003e[18]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCarbohydrates\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.16 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 1.80\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.9 \u003csup\u003e[18]\u003c/sup\u003e – 23.8\u003csup\u003e[19]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003eEffects of Fermentation on Protein and Lipid Content of Black Soldier Fly Larvae\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the effects solid-state fermentation of BSFL using \u003cem\u003eLactobacillus\u003c/em\u003e spp. on the protein and fat content of BSFL. In general, the fermentation increased the protein content of BSFL. Statistically, fermentation of BSFL using \u003cem\u003eL. lactis\u003c/em\u003e did not significantly increase (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) the protein content of BSFL. However, fermentation of BSFL using L. \u003cem\u003eplantarum\u003c/em\u003e and \u003cem\u003eL. acidophilus\u003c/em\u003e significantly increased \u003cem\u003e(P\u003c/em\u003e \u0026lt; 0.05) the protein content of the BSFL from 32.25 ± 0.89% (unfermented BSFL) up to 35.69 ± 0.33%. This is in accordance with the results obtained by Luparelli et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] that solid-state fermentation using \u003cem\u003eL. plantarum\u003c/em\u003e on adult black soldier fly (BSF) increased the protein content of the adult BSF up to 1.4% as compared to the control samples.\u003c/p\u003e\u003cp\u003eThe increase in protein content in the fermented samples may be attributed to the protein in bacterial cells [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and biotransformation of soluble carbohydrates in the BSFL into bacterial proteins [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. According to da Silva Sabo et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], lactic acid bacteria have probiotic properties that can produce bacteriocins which are proteins synthesised in the ribosome and released into the extracellular medium by the lactic acid bacteria. Wang et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] also demonstrated that fermentation of sea cucumber using \u003cem\u003eL. plantarum\u003c/em\u003e increased the protein content up to 2.03% as compared to the control samples. In this study, the protein content of BSFL fermented with \u003cem\u003eL. plantarum\u003c/em\u003e was higher than that of \u003cem\u003eL. acidophilus\u003c/em\u003e and \u003cem\u003eL. lactis\u003c/em\u003e. As such may be attributed to the smaller cell size of \u003cem\u003eL. plantarum\u003c/em\u003e than \u003cem\u003eL. acidophilus\u003c/em\u003e and \u003cem\u003eL. lactis\u003c/em\u003e. The smaller cell size can result in higher bacterial concentration and faster growth which can lead to higher bacteriocin activity [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSolid-state fermentation of BSFL using \u003cem\u003eLactobacillus\u003c/em\u003e spp. also influenced the fat content of the BSFL. In general, the fermentation increased the fat content of BSFL. Statistically, fermentation of BSFL using \u003cem\u003eL. lactis\u003c/em\u003e did not significantly increase (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) the fat content of BSFL. However, fermentation of BSFL using \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eL. acidophilus\u003c/em\u003e significantly increased (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) the fat content of the BSFL from 36.91 ± 1.07% (unfermented BSFL) to 41.99 ± 1.56%. This is in accordance with the results obtained by Luparelli et al. [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] that solid-state fermentation using \u003cem\u003eL. plantarum\u003c/em\u003e on BSF prepupae increased the fat content of the BSF prepupae up to 2% as compared to the control samples. In another study by Cabuk et al. [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], fermented peas using \u003cem\u003eL. plantarum\u003c/em\u003e also increased the fat content by 1%. The increase in lipid content can be influenced by the activity of lipolytic enzymes produced by the lactic acid bacteria [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. According to Kumari et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], the fermentation process may increase the fat content by forming simple fatty acids through the breakdown of large fat molecules.\u003c/p\u003e\u003cp\u003eFrom Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, it can be observed that the highest fat content was observed in the BSFL fermented with \u003cem\u003eL. acidophilus\u003c/em\u003e followed by \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eL. lactis\u003c/em\u003e. These results are in accordance with the previous findings by de Marins et al. [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] that burger fermented with \u003cem\u003eL. acidophillus\u003c/em\u003e produced a higher fat content than \u003cem\u003eL. plantarum\u003c/em\u003e. A lower lipid content in \u003cem\u003eL. plantarum\u003c/em\u003e-fermented samples may occur because \u003cem\u003eL. plantarum\u003c/em\u003e is known to have a more significant effect on degrading fats than the \u003cem\u003eL. acidophilus\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. A lower fat content in the \u003cem\u003eL. lactis\u003c/em\u003e-fermented samples may be due to limited lipolytic activity in \u003cem\u003eL. lactis\u003c/em\u003e which allows limited formation of fatty acids by the bacteria [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn this study, the protein content in the BSFL samples was first extracted prior to protein hydrolysis and the results of protein recovery are shown in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. From the table, it can be observed that the protein recoveries obtained in this study are higher than the protein recovery of 27.58% reported by Smets et al. [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. As such may be influenced by the defatting process prior to the extraction stage carried out in this study. Defatting can increase the protein recovery because it can remove confounding factors in the form of fat during the extraction process [31-Ravi et al., 2020]. Nevertheless, the protein recoveries obtained in this study were lower than the the protein recovery of 59.3% reported by Wang et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] which may be due to different protein extraction methods used in both studies. In this study, protein extraction was carried out using NaOH without any addition of HCl. In contrast, Wang et al. [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] used additional 1 N HCl after extraction of protein to aid the precipitation of extracted protein from BSF powder. Previous studies have reported that the addition of HCl will trigger an increase in protein interactions followed by protein aggregation and precipitation and eventually increase the protein recovery [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtein recovery of extracted protein from black soldier fly larvae.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eProtein recovery (%)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e41.60 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 2.36 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e27.58–59.3 [11, [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]]\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFermentation - \u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e35.99 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 1.99 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFermentation - \u003cem\u003eL. acidophilus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.71 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 1.14 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFermentation - \u003cem\u003eL. lactis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e31.41 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 4.65 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e*Differences in notation (a and b) indicate significant differences in different species (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\u003cp\u003eProtein recoveries of all the fermented samples are significantly different (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) and lower as compared to the control (unfermented) samples. This may occur because lactic acid bacteria possess proteolytic enzymes that can hydrolyse protein into smaller peptides or amino acids and utilisation of essential amino acids for bacterial growth [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In addition, the protein content in the fermented samples is also influenced by the presence of bacteriocin which is known to show decreased stability under alkaline conditions [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. From Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, it can be observed that the protein recovery of BSFL fermented with \u003cem\u003eL. plantarum\u003c/em\u003e was higher than \u003cem\u003eL. acidophilus\u003c/em\u003e and \u003cem\u003eL. lactis\u003c/em\u003e. This is most probably due to the higher proteolytic activity of \u003cem\u003eL. plantarum\u003c/em\u003e compared to the other bacteria [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. A higher proteolytic activity may lead to a higher protein recovery due to more peptide bonds with smaller molecular weight that can cause protein interactions with water and consequently increase the solubility [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003ch2\u003eEffects of Fermentation on Degree of Hydrolysis of Protein Hydrolysate from Black Soldier Fly Larvae\u003c/h2\u003e\u003cp\u003eIn this study, the degree of hydrolysis was assessed to determine the amount of protein that was being hydrolysed during enzymatic hydrolysis of BSFL using bromelain enzymes and the results are shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. The values of degree of hydrolysis obtained in this study were slightly higher than the values reported in the literature. In this study, the protein content from the BSFL was first extracted prior to the enzymatic hydrolysis stage whereas the previous studies did not extract the protein prior to the hydrolysis stage. The stage of protein extraction helps to separate the protein fraction from chitin and increase the contact surface between enzymes and the protein [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFrom Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, it can be observed that the values of degree of hydrolysis for BSFL pre-treated with solid-state fermentation using \u003cem\u003eLactobacillus\u003c/em\u003e spp. were not significantly different from the control (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05). According to Le et al [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], fermentation of soy milk using \u003cem\u003eL. plantarum\u003c/em\u003e increased the degree of hydrolysis up to 3% as compared to samples hydrolysed without the fermentation process. In another study, Rai et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] also reported an increase of degree of hydrolysis when fish processing waste was pre-treated with fermentation using lactic acid bacteria compared to the control. The differences may be due to the inactivation of bacteria at higher temperatures (60 ℃) followed by drying overnight at 105 ℃ carried out in this study whereas in the literature, hydrolysis was carried out directly using alcalase enzymes after the fermentation without any bacterial inactivation. The presence of bacterial proteolytic and alcalase enzymes may work together to hydrolyse the substrate and consequently increase the degree of hydrolysis as compared to the control [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDegree of hydrolysis of protein hydrolysate from black soldier fly larvae.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eDegree of Hydrolysis (%)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eThis study\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e53.19 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 2.73 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e19.83\u003csup\u003e[38]\u003c/sup\u003e– 47.4\u003csup\u003e[3]\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFermentation - \u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e51.52 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 1.06 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFermentation - \u003cem\u003eL. acidophilus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e47.64 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 2.61 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFermentation - \u003cem\u003eL. lactis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e52.61 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 5.94 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e*a indicate no significant difference in different species (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05).\u003c/p\u003e\u003ch2\u003eEffects of Chromatography Flow Rate on Fractionated Protein Concentration\u003c/h2\u003e\u003cp\u003eNo studies have reported on the influence of chromatography flow rates on the fractionation of protein hydrolysate from BSFL. In this study, fractionation of protein hydrolysate was carried out using size-exclusion chromatography at different flow rates and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The highest protein concentration in the control sample and fermentation using \u003cem\u003eL. plantarum\u003c/em\u003e, \u003cem\u003eL. acidophilus\u003c/em\u003e, and L. \u003cem\u003elactis\u003c/em\u003e samples were 1,578.81 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 52.34 mg/mL, 1,252.30 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 112.17 mg/mL, 1,414.69 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 54.98 mg/mL, and 1,331.73 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 66.62 mg/mL, respectively. Statistically, there were no significant differences (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) among control and fermented samples at all flow rates (0.5–1.5 mL/min) but the highest protein concentration was observed at a chromatography flow rate of 1 mL/min. The chromatography flow rate plays important roles in the separation of compounds in the stationary phase and mobile phase. A relatively low flow rate has better adsorption equilibrium and more efficient separation. However, a flow rate that is too low can also allow diffusion effects in the mobile phase while a flow rate that is too high can cause the compounds not having the opportunity to reach an equilibrium state due to the contact time between the mobile phase and stationary phase which is very small [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The results of this study suggest that a chromatography flow rate of 1mL/min is deemed sufficient to separate the protein fractions in the protein hydrolysate.\u003c/p\u003e\u003cp\u003e \u003cb\u003eEffects of Fermentation on Amino Acid Composition of Fractionated of Protein Hydrolysate from Black Soldier Fly Larvae\u003c/b\u003e \u003c/p\u003e\u003cp\u003eIn this study, the amino acid composition was determined for the fractionated protein hydrolysate that had the highest protein concentration in all samples and the results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The sample with the highest protein concentration was obtained in the 5th tube with a chromatography flow rate of 1 mL/min for the control, fermentation using \u003cem\u003eL. plantarum\u003c/em\u003e, and fermentation using \u003cem\u003eL. lactis\u003c/em\u003e sample while for the fermentation using \u003cem\u003eL. acidophilus\u003c/em\u003e the highest protein concentration was obtained in the 4th tube with a chromatography flow rate of 1 mL/min. From Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, it can be observed that there were differences in amino acid composition between the control and fermented fractionated protein hydrolysate.\u003c/p\u003e\u003cp\u003eFor BSFL fermented with \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eL. lactis\u003c/em\u003e, the fractionated protein hydrolysates contained only three types of amino acids particularly L-alanine, glycine, and L-methionine but the concentrations were significantly different (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). For BSFL fermented with \u003cem\u003eL. acidophilus\u003c/em\u003e, the fractionated protein hydrolysate also contained L-alanine, glycine and L-methionine but with the addition of L-glutamic acid, leucine, L-methionine, L-valine, L-proline, and L-serine that were not present in the case of fermentation using \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eL. lactis.\u003c/em\u003e From Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, it can be noticed as well that serine was present in the fractionated protein hydrolysate fermented with \u003cem\u003eL. acidophilus\u003c/em\u003e but not in the control. This can be attributed to the synthesis of serine by \u003cem\u003eL. acidophilus\u003c/em\u003e through the de novo pathway as suggested by Altermann et al. [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThese results are in accordance with the previous findings by Hadj et al. [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] that fermentation of BSF puparia using lactic acid bacteria can cause changes in the amino acid composition including a decrease in several amino acids. The decrease in several amino acid content in the fermented samples may occur due to the role of lactic acid bacteria which utilises the amino acids as nitrogen sources in the growth process [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In this study, the absence of several amino acids that were previously present in the unfermented (control) samples may have occurred because the amino acids are essential for the bacteria and consequently being consumed by the bacteria [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAmino acid composition of fractionated protein hydrolysate from black soldier fly larvae\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAmino acid\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eContent (mg/kg fraction)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFermentation\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. plantarum\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFermentation\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. acidophilus\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFermentation\u003c/p\u003e \u003cp\u003e\u003cem\u003eL. lactis\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Alanine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e84.63\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e84.63\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e85.14\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e84.63\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Arginine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Aspartic acid\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e190.57\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGlycine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e312.2 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e314.95 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e325.48 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e274.77 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Glutamic acid\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e152.43\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e152.43\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Histidine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Isoleucine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Cysteine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Leucine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e167.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e167.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Lysine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Methionine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.31 \u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.24 \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.02 \u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.21 \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Tryptophan\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-valine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128.75\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e128.75\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Phenylalanine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Proline\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e128.38\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e128.38\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Serine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e149.74\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Threonine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eL-Tyrosine\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003e*Differences in notation (a, b, c, and d) indicate significant differences in in different species (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\u003cp\u003eStatistically, the amount of glycine and methionine content in the control and fermented samples were significantly different (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) while alanine was not significantly different (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) which resembles the previous findings by Patterson et al. [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In this study, the glycine content in the fraction of protein hydrolysate fermented with \u003cem\u003eL. plantarum\u003c/em\u003e and \u003cem\u003eL. acidophilus\u003c/em\u003e increased by 2.75 mg/kg and 13.28 mg/kg, respectively whereas the glycine content in the fraction of protein hydrolysate fermented with \u003cem\u003eL. lactis\u003c/em\u003e decreased by 37.43 mg/kg fraction. The increased glycine content can be influenced by the glycolysis process that synthesises serine with the change of 3-phosphoglycerate and converts it into glycine. In addition, the \u003cem\u003eLactobacillus\u003c/em\u003e spp. can utilise carbohydrates in the form of glucose and convert them into pyruvic acid which then produces aspartic acid that can be synthesised into methionine and then into serine and glycine [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. This also allows a decrease in methionine content due to the biosynthesis of serine and glycine [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The alanine content that was not different between the control sample and fermented sample may be due to alanine being a non-essential amino acid for bacteria so it was not the main nitrogen source utilised by bacteria for growth [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn summary, solid-state fermentation of BSFL using \u003cem\u003eL. plantarum, L. acidophilus, and L. lactis\u003c/em\u003e increased protein and fat content of the BSFL up to 3.34 and 5.08%, respectively. However, the solid-state fermentation reduced the protein recovery and degree of hydrolysis of the protein hydrolysate upon alkaline extraction and enzymatic hydrolysis using bromelain enzymes. Fractionation of the protein hydrolysate at different chromatographic flow rates slightly affected the protein concentrations which varied from 0.51 mg/mL to 1414.69 mg/mL with the highest protein concentration obtained when the flow rate of the chromatography was set at 1 mL/min. The fractionated protein hydrolysates fermented with \u003cem\u003eLactobacillus\u003c/em\u003e spp. contained several amino acids such as glycine, and L-methionine that were significantly different from the unfermented samples. The effects of these changes towards the bioactivity of the fractionated protein hydrolysate were not examined in this study. Potential future studies might should focus on the correlation between the amino acid composition and bioactivity of the fractionated protein hydrolysate from BSFL.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eThe authors declare no competing interests\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis work was financially supported by Institut Teknologi Bandung (Riset Internasional 2023, LPPM.PN-16-19-2023).\u003c/p\u003e \u003cp\u003eData availability Not applicable\u003c/p\u003e\u003cp\u003eAuthor contributions MYA: funding acquisition, conceptualization, supervision, formal analysis, writing original draft, review, and editing. HS, GRP, MAS, BAS: data curation, verified analytical methods, investigation, methodology, and writing original draft. KL, NIMP, AK: conceptualization, supervision, review.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eM\u0026uuml;ller A, Wolf D, Gutzeit HO (2017) The black soldier fly, Hermetia illucens\u0026ndash;a promising source for sustainable production of proteins, lipids bioactive substances. Z. Naturforsch. C Biosci. 72: 351\u0026ndash;363\u003c/li\u003e\n\u003cli\u003eAbduh MY, Nadia MH, Syaripudin, Manurung R, Putra RE (2018) Factors affecting the bioconversion of Philippine tung seed by black soldier fly larvae for the production of protein and oil-rich biomass. J. Asia-Pac. Entomol. 21: 836\u0026ndash;842\u003c/li\u003e\n\u003cli\u003eFirmansyah M, Abduh MY (2019) Production of protein hydrolysate containing antioxidant activity from \u003cem\u003eHermetia illucens\u003c/em\u003e. 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Food Research International 102: 323-332\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Amino acid, Black soldier fly larvae, Lactobacillus spp., Protein hydrolysate, Solid-state fermentation","lastPublishedDoi":"10.21203/rs.3.rs-6142604/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6142604/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study aimed to determine the effects of fermentation pretreatment using \u003cem\u003eLactobacillus\u003c/em\u003e spp. on the protein content of black soldier fly larvae, and the protein recovery, degree of hydrolysis, and amino acid composition of protein hydrolysate from black soldier fly larvae. Solid state fermentation of black soldier fly larvae with \u003cem\u003eLactobacillus\u003c/em\u003e spp. carried out for 72 h at 37℃ increased the protein and fat content from 32.35 to 36.75\u0026ndash;41.99% and 36.91 to 40.03\u0026ndash;41.99%, respectively. Protein content in the fermented samples was extracted using an alkaline extraction method which resulted in a protein recovery of 31.41 to 35.99%. The protein extract was then hydrolyzed using bromelain enzymes for 24 h to produce protein hydrolysates with a degree of hydrolysis in the range of 47.64 to 52.61%. The protein hydrolysate was further fractionated using size-exclusion chromatography with the flow rate of 0.5 to 1.5 mL/min. The protein concentration of each fraction was determined using a UV-visible spectrophotometer and the result varied from 0.51 to 1414.69 mg/mL with the highest protein concentration obtained when the flow rate of the chromatography was set at 1 mL/min. Amino acid composition of the fractionated protein hydrolysate was determined using the ultra-high-performance liquid chromatography. The results show that the fractionated protein hydrolysates fermented with \u003cem\u003eLactobacillus\u003c/em\u003e spp. contained several amino acids such as glycine, and L-methionine, which was significantly different than the unfermented samples. The findings highlight that fermentation of black soldier fly larvae using \u003cem\u003eLactobacillus\u003c/em\u003e spp. positively influence the protein and amino acid composition of protein hydrolysate from black soldier fly larvae.\u003c/p\u003e","manuscriptTitle":"Synthesis and fractionation of Protein Hydrolysate from Hermetia illucens L. pre-treated with Lactobacillus spp.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-14 10:50:02","doi":"10.21203/rs.3.rs-6142604/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-03-29T02:45:10+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-27T13:18:25+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Waste and Biomass Valorization","date":"2025-03-21T19:06:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-03T12:05:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Waste and Biomass Valorization","date":"2025-03-02T23:42:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"waste-and-biomass-valorization","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"wave","sideBox":"Learn more about [Waste and Biomass Valorization](http://link.springer.com/journal/12649)","snPcode":"12649","submissionUrl":"https://submission.nature.com/new-submission/12649/3","title":"Waste and Biomass Valorization","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c7b98950-1c21-4048-9f26-036bf960facb","owner":[],"postedDate":"April 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-07-07T16:07:53+00:00","versionOfRecord":{"articleIdentity":"rs-6142604","link":"https://doi.org/10.1007/s12649-025-03197-0","journal":{"identity":"waste-and-biomass-valorization","isVorOnly":false,"title":"Waste and Biomass Valorization"},"publishedOn":"2025-07-05 15:58:33","publishedOnDateReadable":"July 5th, 2025"},"versionCreatedAt":"2025-04-14 10:50:02","video":"","vorDoi":"10.1007/s12649-025-03197-0","vorDoiUrl":"https://doi.org/10.1007/s12649-025-03197-0","workflowStages":[]},"version":"v1","identity":"rs-6142604","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6142604","identity":"rs-6142604","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}