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Article

Production of Nutritional Protein Hydrolysates by Fermentation of Black Soldier Fly Larvae

by
Penghui Zhang
1,
Kelyn Seow
1,
Leo Wein
2,
Rachel Steven
3,
Rebecca J. Case
1,4,
Yulan Wang
5,* and
Patricia L. Conway
1,6,7,*
1
Singapore Centre for Environmental Life Sciences Engineering, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
2
Protenga Pte Ltd. (Singapore HQ), 3 Coleman Street, #03-24, Singapore 179804, Singapore
3
Protenga Sdn Bhd (Malaysia), No. 204, Jalan Ekoperniagaan 6, Taman Ekoperniagaan 2, Senai 81400, Malaysia
4
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore
5
Singapore Phenome Center, Lee Kong Chian School of Medicine, Nanyang Technological University, 59 Nanyang Drive, Singapore 636921, Singapore
6
School of Biological, Earth and Environmental Sciences, The University of New South Wales Sydney (UNSW), Sydney 2052, Australia
7
PC Biome Pte Ltd., 71 Nanyang Drive, Singapore 638075, Singapore
*
Authors to whom correspondence should be addressed.
Fermentation 2025, 11(9), 524; https://doi.org/10.3390/fermentation11090524
Submission received: 30 July 2025 / Revised: 28 August 2025 / Accepted: 1 September 2025 / Published: 8 September 2025
(This article belongs to the Special Issue 10th Anniversary of Fermentation: Feature Papers in the "Fermentation for Food and Beverages" Section)
Browse Figures
Versions Notes

Abstract

The black soldier fly (Hermetia illucens) has become one of the most promising alternative protein sources in the feed and food industry. The aim of this work was to utilize microbial fermentation to enhance the nutritional properties of black soldier fly larvae (BSFL) as a food ingredient for human consumption by optimizing the amino acid profile and small peptide content. Free amino acids (FAA) have a critical role in human nutrition and bioavailability. Unlike whole proteins that require enzymatic breakdown in the digestive tract, FAA are directly absorbable by the small intestine, allowing for rapid utilization in protein synthesis and metabolic functions. BSFL pastes were fermented using Lacticaseibacillus paracasei (PCB 030) or a mixed starter culture preparation, and results were compared to pea protein and BSFL pastes that were enzymatically hydrolyzed. The resultant hydrolyzed BSFL pastes were analyzed for free amino acids and small peptides. The L. paracasei PCB 030 fermented BSFL pastes yielded significantly higher amounts of free amino acids than the control or pastes fermented using a commercial starter culture (named F-LC). The increased FAA availability in fermented BSFL makes it a more efficient protein source for human consumption. The L. paracasei PCB 030 fermented pastes showed an increase in small peptides after three days fermentation; nearly 80% of normalized abundances of small peptides increased by over 100 times compared to day zero (before the fermentation started). Over 90% of these small peptides consisted of more than 50% hydrophobic amino acids, which may contribute to their antioxidant and antibacterial properties. This study provides a promising and industrially practical process for hydrolyzing BSFL protein to yield a functional protein hydrolysate with an enhanced nutritional profile.

Graphical Abstract

1. Introduction

The growth of the human population and increased global demand for food protein drives the development of sustainable and efficient protein production methods. The black soldier fly (BSF, Hermetia illucens), which has a higher protein content than conventional animal livestock, is a promising candidate for industrial rearing to supplement food production as it can bioconvert organic waste to protein with a high feed conversion efficiency, but a lower carbon footprint [1,2]. BSF contains a diversity of micro- and macro-nutrients, with high protein and fat content, as well as being rich in zinc, iron, and calcium among other micronutrients [3,4]. Because of this valuable nutritional profile, BSF is utilized as a feed ingredient, particularly in rearing fish, shrimp, chicken, swine and even pet animals [1]. This impressive nutritional profile, combined with the observed high feed conversion efficiency, fast growth rate, and ability to recycle organic matter led to the BSF being identified as a possible alternative food source for human consumption [5,6,7,8].
Despite the advantages from both sustainable and nutritional standpoints, negative perceptions associated with eating insects are a major barrier to its widespread adoption as food [9,10]. One way to overcome consumer acceptance challenges is to develop refined insect-based protein ingredients, rather than include them in their original form, so they can be readily included in familiar foods [11,12]. A strategy to refine insect-derived ingredients and improve their nutrient profile is to take advantage of liquid-solid-state fermentation [1,13] as a processing method. For example, fermentation of BSFL by lactic acid bacteria (LAB) was recently described by Luparelli et al. [14], with the aim to enhance its nutritional value. The extensive utilization of Lacticaseibacillus paracasei as a starter culture for food fermentation means it is generally recognized as safe (GRAS) by the US Food and Drug Authority (FDA) [15,16,17]. In addition, the proteolytic enzyme system of Lacticaseibacillus paracasei is well documented, including cell-envelope proteinases and multiple peptidases that hydrolyze proteins into free amino acids and peptides [18,19,20]. Enzymatic hydrolysis of BSFL has been carried out in order to produce nutritional ingredients such as amino acids and small peptides [21,22,23]. To meet the global demand for food protein, efficient utilization of high nutritional value insects, such as BSF, is required; however, most BSFL research has been focused on rearing strategies and characterization of its nutritional composition, including proteins, lipids, amino acids, and minerals. Only a few studies have explored BSFL refinement via fermentation. For example, Meng et al., fermented the BSFL with Lactobacillus crispatus and Pichia kudriavzevii in combination with neutral protease for enzymolysis [24], which mainly focused on evaluating the quality and safety of the fermented BSFL paste. Saadoun et al., fermented black soldier fly prepupae using Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum as starter cultures, and reported the in vitro antimicrobial activity of the fermented prepupae [25].
This study aimed to produce nutritional ingredients from BSF larvae (BSFL), potentially for human consumption, by hydrolyzing BSFL using a fermentation process and Lacticaseibacillus paracasei (PCB 030) as the starter culture. The strain L. paracasei PCB 030 was chosen as it was previously isolated and had been shown to cause a decrease in pH and a fermented-paste aroma, as well as proteolytic activity in preliminary screening assays (unpublished data). The L. paracasei PCB 030 showed a more favorable fermentation profile than the other tested candidates, including Lactobacillus rhamnosus, Limosilactobacillus fermentum, Lactiplantibacillus plantarum, Saccharomyces cerevisiae, and Lactobacillus acidophilus. Initially the fermentation conditions were optimized in terms of numerous parameters including the initial glucose and salt concentrations, pH, pre-treatment processes to create the BSFL paste, and the elimination of contaminating bacteria. The final product of the fermented material was assessed by monitoring the degree of hydrolysis as well as analyzing the free amino acids and small peptides in the hydrolysate. In addition, from the perspective of industrial application, we monitored dynamic changes during fermentation and established optimum fermentation conditions. This systematic investigation of procedures for hydrolyzing BSFL using L. paracasei PCB 030 provides a protocol for screening other bacteria with proteolytic properties and has established a foundation for functional black soldier fly-based ingredients for human consumption.

2. Materials and Methods

2.1. Chemicals and Materials

Black soldier fly larvae (BSFL; Hermetia illucens) were provided as frozen packages by Protenga Sdn Bhd (Senai, Malaysia), which were reared on plant-based, pre-consumer, traceable agricultural by-products that comply with EU feedstock regulations. Phosphate-buffered saline (PBS, 10 mM, pH = 7.4), sodium chloride (NaCl), sodium nitrite (NaNO2), D-(+)-Glucose, and glacial acetic acid were purchased from Sigma Aldrich (Singapore). Deionized (DI) water used in this study had a resistivity of >15 MΩ·cm. The PBS, NaCl, NaNO2, glucose, and deionized water were autoclaved before use.
Liquid Chromatography Mass Spectrometry (LC-MS)-grade methanol, acetonitrile (ACN), formic acid, water, and ammonium formate reagent as well as amino acid standards including Alanine (Ala), Arginine (Arg), Asparagine (Asn), Aspartic acid, (Asp), Glutamic acid (Glu), Glutamine (Gln), Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr), and Valine (Val) were all purchased from Sigma-Aldrich (Singapore).
Lacticaseibacillus casei PCB 030 was obtained from PC Biome Pte. Ltd. (Singapore). L. paracasei PCB 030 was routinely grown using de Man, Rogosa and Sharpe (MRS) broth and agar, which were obtained from Sigma-Aldrich (Singapore) and prepared according to the manufacturer’s instructions. F-LC starter culture contained Pediococcus acidilactici, Staphylococcus xylosus, Latilactobacillus curvatus (Chr Hansen, Hørsholm, Denmark) was used according to the manufacturer’s instructions, i.e., adding 25 g of the starter culture powder per 100 kg BSFL paste.

2.2. BSFL Paste Preparation

Frozen BSFL were thawed and then washed with tap water three times and steamed for 60 min prior to being pressed to separate the exoskeleton and endoskeleton that were then mixed homogenously to form the BSFL paste. The water content of the samples was analyzed by weighing the mass before and after freezing and drying.
Overnight growth of L. paracasei PCB 030 in MRS broth was harvested by centrifugation (2000 rpm, 5 min) and washed twice with sterile PBS (pH = 7.4) solution and resuspended in the PBS prior to use. Aliquots (30 g) of BSFL paste were mixed with NaCl/NaNO2 and glucose to yield the final concentrations, 2.8 wt%/0.015 wt% and 2.5 wt%, respectively. Acetic acid (300 μL) was added to adjust the pH of the mixture, which was then homogenized by vortexing, before the addition of L. paracasei PCB 030 (5 mL; final concentration was 7.5 log CFU/g) washed suspension (the total volume of c.a. 41 mL). The BSFL paste with the same amount of PBS but no L. paracasei PCB 030 was used as the control. The mixtures were aliquoted into 15 mL falcon tubes. The lids on the tubes were tightly closed prior to incubation at 37 °C. Samples were collected at 0, 1, 2, 3, and 7 d of fermentation and all the fermentation experiments were carried out in triplicate. The F-LC fermented BSFL paste was prepared with the same conditions, except that it was dosed at 25 g per 100 kg BSFL paste; final concentration was over 11 log CFU/g.

2.3. Online pH Measurement

Online pH measurements were carried out using pH sensors (Vernier, Beaverton, OR, USA) inserted into the BSFL paste mixture. The pH sensors and 15 mL falcon tubes were sealed with parafilm and kept at 37 °C using metallic thermal beads in an Elite Dry Bath Incubator (Major Science, Saratoga, CA, USA). The pH was recorded automatically every 5 min by connecting with the data-collection interface LabQuest® 3 (Vernier, USA) during the fermentation process.

2.4. Viable Counts

Viable counts of bacteria in the fermented BSFL paste mixture were determined using the standard plate count method. Briefly, after the sample collection at different time points, 0.5 mL of samples were added to 4.5 mL sterile PBS and then vortexed for 30 s prior to 10-fold serial dilutions in PBS. Aliquots of 5 μL of appropriate dilutions were plated in triplicate on MRS agar plates and incubated at 37 °C for 48 h after drying. Colonies were counted and analyzed on a log plot of colony forming units (CFU) per mL.

2.5. L. paracasei PCB 030 Fermented BSFL Paste Morphology

FESEM (Hitachi High-Tech, Singapore) was used to study the morphology of L. paracasei PCB 030 fermented BSFL paste at 3 d. The sample was fixed in 2.5% glutaraldehyde at 4 °C overnight followed by dehydration in a graded concentration ethanol series (25–100%) and dried at room temperature for 24 h. The dried samples were then coated with platinum before imaging.

2.6. Degree of Hydrolysis

The degree of hydrolysis (DH) was determined as the ratio of 10% trichloroacetic acid (TCA) soluble nitrogen to total nitrogen in the sample before fermentation according to the method of Hoyle and Merritt (1994) with some modification [26]. Briefly, 0.9 mL samples were added to 0.9 mL of 20% TCA and the mixture was vortexed before being stored in a fridge at 4 °C for 30 min. The mixture was then centrifuged at 10,000 rpm at 4 °C for 10 min before filtering. Aliquots (0.5 mL) of the filtrate were added into 19.5 mL Deionized H2O for nitrogen measurement using a total organic carbon analyzer (TNM-L, Shimadzu, Kyoto, Japan). To measure the total nitrogen content, 100 mg un-fermented and dried BSFL samples were completely hydrolyzed in 5 mL of 6 N HCl at 110 °C for 24 h [27]. After the hydrolysis with HCl, the suspension was filtrated before nitrogen analysis.
The DH was calculated as:
DH = (TCA soluble N)/(Total N) × 100%

2.7. Quantification of Free Amino Acids

After fermentation, collected samples (1.0 mL) were mixed with 4.0 mL methanol to extract the free amino acids by vortexing vigorously prior to being centrifuged at 10,000 rpm for 10 min. The supernatant was collected (250 µL), completely dried by a vacuum concentrator system with a −84 °C cold trap then reconstituted in 1000 µL of 80%/20% acetonitrile/H2O. Aliquots (150 µL) of the solution were syringe filtered (0.22 μm) before liquid chromatography-mass spectrometry (LC-MS) analysis. The calculation of the free amino acids (in mg/kg) was based on the dry mass of the BSFL paste.
LC-MS analysis was performed following the protocol by Andrea et al. [28], with some modification; a Waters UPLC BEH Amide column (Waters Corporation, Milford, MA, USA) (1.7 µm, 2.1 × 100 mm) was used in the LC-MS. The mobile phase was composed of 30% acetonitrile + 70% H2O + 0.1% formic acid + 10 mM ammonium formate (eluent A) and 95% acetonitrile + 5% H2O + 0.1% formic acid + 10 mM ammonium formate (eluent B). The flow rate was set at 0.5 mL/min, with an injection volume of 5 µL, column temperature 45 °C and sample temperature 4 °C. Detection was performed using a Waters Xevo TQ-S (Milford, MA, USA) mass spectrometer and the parameters settings were as follows: the ESI source was in positive ionization mode, capillary voltage 3.0 kV, source temperature 150 °C, desolvation temperature 500 °C, cone gas flow 150 L/h, and desolvation gas flow 1000 L/h. Calibration was performed with standard amino acid solutions in the range of 10 nM to 10,000 nM.

2.8. Small Peptides Analysis by LC-MS/MS

Fermented BSFL samples (1.0 mL) were mixed with 4.0 mL methanol to extract the small peptides by vortexing for 30 s and centrifugation at 10,000 rpm for 10 min. Supernatant was collected (250 µL) and completely dried before being reconstituted with 1000 µL of 0.1% formic acid. A 150 µL sample was filtered before injection into LC-MS/MS.
LC-MS/MS was performed by Singapore Phenome Centre (SPC, Singapore) at NTU using an ACQUITY ultra-performance liquid chromatography and quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) system. Furthermore, 20 µL samples were injected into a Phenomenex bioZen XB-C18 column (Phenomenex Inc., Torrance, CA, USA) (1.7 μm, 2.1 × 150 mm) which was operated at 60 °C and the flow rate was 0.2 mL/min. The total run time was 100 min with mobile phase A (water + 0.1% formic acid) and mobile phase B (acetonitrile + 0.1% formic acid). The gradient during the run time window was 98% A + 2% B from 0 to 55 min, 60% A + 40% B from 55 to 85 min, 5% A + 95% B from 85 to 96 min, 98% A + 2% B from 96 to 100 min. The separated small peptides were injected into the Xevo G2-XS QTOF (Waters, MA, USA) with the standard ESI source in positive mode, with 3.0 kV capillary voltage, 150 °C source temperature, 350 °C desolvation temperature, 25 L/h cone gas flow, and 800 L/h desolvation gas flow. MS spectra had a m/z range of 50–2000 Da with a scan time of 0.2 s. After acquisition, the MS spectra were uploaded into Progenesis QI v3.0 software for data pre-processing with a Hermetia illucens database (UniProt). Since no calibration with peptide standards was conducted, this method provides relative abundance data based on ion intensity rather than absolute quantification for the small peptide concentrations.

2.9. Fat Content Measurement

Fat content was measured with a HCI Hydrolysis System (ANKOM) and Extractor (ANKOM). Briefly, samples were completely dried before being added into filter bags containing diatomaceous earth. The filter bags were then sealed and put into a hydrolysis vessel with 500 mL of 3N HCl at 90 °C for 60 min. The filter bags were rinsed with sufficient water and dried at 110 °C for 3 h to completely remove the HCl residue. Hexane was used to extract the fat content at 90 °C for 60 min. The fat content was calculated based on the mass change after the whole process and drying.

2.10. Ash Content Measurement

The freeze-dried samples were put into crucible and then placed in a muffle furnace and burned at 550 °C for 0.5 h until ash was formed. The ash content was calculated by weighing the mass before and after the burning process.

2.11. Statistical Analysis

All the results are presented as the mean ± standard error from triplicate samples. Data was analyzed using a one-way ANOVA test, with p < 0.05 considered statistically significant.

3. Results and Discussion

3.1. BSFL Pre-Processing and Fermentation Condition

The pre-treatment and fermentation of BSFL paste is illustrated in Figure 1. The washing and steaming of BSFL was performed sequentially to remove solid residues (such as soil, stone, or wood, etc.) and contaminating bacteria. For better efficiency of fermentation and industrial application, the BSFL were pressed using a mechanical oil press and the endoskeleton and exoskeleton parts of BSFL were separated. Figure S1 shows the fat and ash contents of BSFL of the endoskeleton, exoskeleton and the mixtures. The ash content of the endoskeleton and exoskeleton, 11.6 wt% and 8.8 wt%, respectively, were similar (p = 0.008, two-sample t-test). However, the fat content of the endoskeleton (32.9 wt%) was much higher than that of the exoskeleton (7.2 wt%) (p = 0.0004, two-sample t-test), resulting in an overall fat content in larval paste of 26.4 wt%. These results are consistent with previously reported values [29,30]. The mixed endoskeleton and exoskeleton BSFL paste was then subjected to fermentation. Glucose was added to accelerate the fermentation process as the BSFL paste contains limited fermentable sugar [31,32]. After glucose and salt were added, the pH was adjusted, and the mixture was inoculated with L. paracasei PCB 030 (initial cell density was ~7.5 Log CFU/mL), the BSFL paste was fermented, and samples were collected on specific days. During the fermentation process, vigorous bubbling was observed (Figure S2b), presumably CO2 produced during the fermentation, which resulted in a fermented BSFL protein hydrolysate with a porous powder form after freeze drying (Figure S2c).

3.2. Phenotypic Characterizations of the Fermentation Process

It is essential for L. paracasei PCB 030 (or other fermenting organisms) to produce fast acidification to inhibit growth of pathogens and spoilage microbes [33,34,35]. The pH changes in the BSFL paste during the fermentation period is shown in Figure 2a. Upon inoculation of L. paracasei PCB 030, the pH declined from c.a. 6.7 to c.a. 5.0 within 16 h and stayed constant at pH5 for the remainder of the experiment. However, the pH of the control sample was c.a. 6.5 over 65 h which indicates that there was no fermentation and hence no contamination by fermenting bacteria after the pre-treatment of the BSFL. The rapid decrease in pH after inoculated with the L. paracasei PCB 030 would benefit the fermentation process. In addition, the pH value dropped to 5.0 at 1 d, this is regarded as an essential threshold from a food safety perspective [32]. Lactobacilli selective MRS agar was used to enumerate the viable L. paracasei PCB 030 during fermentation. The initial L. paracasei PCB 030 loading was 7.4 Log CFU/mL (Figure 2b) while the initial viable count was not detectable (below 2.6 Log CFU/mL) for the control (Figure S3a,b), which implies again that the steaming pre-treatment was effective to eliminate the contaminating bacteria. The L. paracasei PCB 030 concentration rapidly increased to 9.2 from 7.4 Log CFU/mL after one day of incubation, the doubling time was calculated to be 4.04 h. The viable count dropped down to 8.1, 7.2, and 7.3 Log CFU/mL at D2, D3, and D7, respectively. The maximum viable count of 9.2 Log CFU/mL during the fermentation was very close to the work reported by Borremans et al. [36]. The volume of the BSFL paste expanded and formed a sponge-like paste during the fermentation process (Figure S3c), which may be explained by the production of carbon dioxide as a by-product of the carbohydrate metabolism of L. paracasei PCB 030 [37]. This observation is consistent with the pH drop as shown in Figure 2a. During fermentation, rod-shaped L. paracasei PCB 030 cells were also observed on the surface of the BSFL paste using a field emission scanning electron microscope, which suggested an interaction between the inoculum and BSFL protein (Figure 2c), resulting in the production of BSFL protein hydrolysate.

3.3. Hydrolysate Assessment

The profile of the free amino acids in the BSFL protein hydrolysate and the control from 0 to 7 d was examined with Ultra-Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS) and the results are presented in Figure 3. The free amino acids (AA), including essential amino acids (EAA) and non-essential amino acids (non-EAA), increased during the first day of fermentation, except leucine (Leu), methionine (Met), tryptophan (Trp), and glutamine (Gln). The rapid increase in the AAs is an indication of the high activity of the L. paracasei PCB 030 metabolism, which is consistent with the observation of pH decrease and viable count increase within one day (Figure 2a,b). Some of the EAA, e.g., valine (Val), isoleucine (Ile), lysine (Lys), and histidine (His) significantly increased 223.2%, 381.0%, 161.1%, and 171.0%, respectively. For the non-EAA, serine (Ser), aspartic acid (Asp), and glutamic acid (Glu) increased by 322.0%, 578.4%, and 287.1%, respectively, after one day of fermentation with L. paracasei PCB 030. Most of the free amino acid contents of the control remained relatively unchanged over 0–3 d of fermentation, some of the free amino acids content decreased after fermentation, such as Lys, His, Asn, Gln, etc. In addition, the amino acids contents of the control had not significantly changed in the first three days, compared to the L. paracasei PCB 030 fermented samples. It has been shown that the free amino acids of Asp and Glu contribute to the umami taste in fermented meat products [33,38]. The increase in these FAA contents after fermentation would benefit their acceptance and consumption as human food. The total free essential amino acids (TFEAA) and total free amino acids of the L. paracasei PCB 030 fermented BSFL pastes showed the same trend of change as a function of fermentation time (Figure 4a).
The degree of hydrolysis (DH) is considered as the percentage of peptide bonds of the cleaved protein [26]. As shown in Figure 4b, the DH increased from 17.3% to 23.3% after one day of fermentation with L. paracasei PCB 030 with no further increase detected after three days of fermentation, while the control retained its DH at 16.5–17.7% during the first three days of fermentation. This observation indicates that there was limited BSFL protein hydrolysis in the control and the increased DH in the presence of L. paracasei PCB 030 is due to bacteria hydrolysis. In addition, the change of the degree of hydrolysis and total free amino acids content is consistent (Figure 4a,b). A notably increased DH at 7 d in the control was observed, which likely results from (a) the residual endogenous protease activity that hydrolyzes proteins during incubation [39]; (b) prolonged incubation at 37 °C would induce protein denaturation and structural breakdown, and hence unfolded proteins would be more susceptible to hydrolysis. A limited change of the DH and TFAA at 3 d until 7 d fermentation was observed. Therefore, 3 d was considered the optimal fermentation in this study. Three days fermentation of BSFL paste using F-LC as a starter culture was also investigated as a benchmark. It was used at the dosage recommended in sausage production. The changes of free amino acids before and after fermentation were compared and are shown in Figure 4c. At 3 d fermentation with F-LC as the starter culture, 11 of the 19 free amino acids decreased by more than 50%. This decrease is likely due to the mixed culture rapidly assimilating and catabolizing extracellular amino acids to support their growth and aroma formation, while providing limited proteolysis of BSFL proteins. Staphylococcus xylosus, for example, is known to actively catabolize free amino acids, particularly branched-chain amino acids (Val, Leu, Ile), into aroma precursors through catabolism pathways [40]. Pediococcus acidilactici and Latilactobacillus curvatus also contribute to amino acid uptake for growth [41,42]. In contrast, when L. paracasei PCB 030 was used as the starter culture, only three of the nineteen free amino acids decreased by more than 50%, while six out of nineteen free amino acids increased over 100%, including Ser, Val, Ile, Asp, Glu, and His. Most of the free amino acids in the control have no significant change in their contents after three days of fermentation with the F-LC starter culture. The DH of the F-LC fermented BSFL protein hydrolysate showed a decrease from 22.7% to 20.3% (Figure S4). When comparing the results for L. paracasei PCB 030 with those obtained when F-LC was used, the FAA and DH results provided evidence that the L. paracasei PCB 030 plays an important role in hydrolyzing the BSFL protein during the fermentation process.

3.4. Small Peptides Analysis

L. paracasei PCB 030 fermented samples and control samples at 0 and 3 d were selected for the small peptides analysis using a UPLC/Q-TOF-MS system. Peptides were identified using Progenesis QI v3.0 data analysis software and Hermetia illucens database in UniProt. The relative abundance of small peptides identified is shown in Figure 5 and their detailed information is listed in Table S2. These peptides have between 6 and 29 amino acids, and their peptide ions have molecular mass from 596.32 to 3034.46 Da. The relative normalized abundances for the accession of hypothetical protein of these small peptides are shown in Figure 6 and their detailed information is listed in Table S3. ArcSinh normalized abundances were used to present the data as most of the abundances of small peptides by 3 d. L. paracasei PCB 030 fermented samples were 2–3 orders of magnitude higher than those of other samples as shown in Tables S2 and S3. The relative normalized abundances for all the small peptides are similar in both control and L. paracasei PCB 030 fermented BSFL paste at the beginning of the experiment (0 d), which is expected as there is no fermentation happening yet in the BSFL paste although inoculated with L. paracasei PCB 030. At 3 d fermentation with L. paracasei PCB 030, 86.2% of the normalized abundances of small peptides increased 10 times, and 79.3% normalized abundances of small peptides increased 100 times, compared to 0 d, the beginning of fermentation (Table S2). It is well known that a high ratio of hydrophobic amino acids presented in the peptides sequence may contribute to their antioxidant and antibacterial activity [43,44]. The hydrophobic amino acids, including glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and tryptophan (W), in the sequence of small peptides were labeled in pink/blue (Figure 6b). Over 90% of the small peptides consisted of over 50% of hydrophobic amino acids in their sequencing, as shown in Figure 6c. Based on these results, the L. paracasei PCB 030 fermented BSFL paste contained a higher content of protein hydrolysate and a high number of small peptides and hence would have promising antioxidant and antibacterial properties [45,46].

4. Conclusions

This work demonstrates an effective method for hydrolyzing BSFL protein by fermentation with L. paracasei PCB 030. Inoculation with around 7.5 log CFU/mL L. paracasei PCB 030, with the addition of glucose and salts, and a suitable pH (6.2–6.8), results in fermentation with rapid acidification and production of gas bubbles resulting in BSFL protein hydrolysate with a porous powder form after freeze drying. After one day of fermentation, the essential amino acids, such as Val, Lys, and His, significantly increased from 152.23 ± 2.68 to 429.03 ± 3.68, 89.22 ± 1.49 to 429.15 ± 2.69, 78.54 ± 7.89 to 169.74 ± 18.60, and 517.60 ± 25.80 to 1402.45 ± 68.76 mg/kg (based on the dry BSFL paste), respectively, while the control samples had no significant change in the content of amino acids. The L. paracasei PCB 030 fermented BSFL protein hydrolysate shows a better performance than that of commercial starter cultures (F-LC) used at recommended dosage rates when compared in terms of amino acid production. Six of nineteen free amino acids increased their concentration more than 100% after fermentation with L. paracasei PCB 030, but none of the free amino acids increased in the F-LC fermented BSFL. In addition, over 79.3% of small peptides at 3 d notably increased in their relative abundances by over 10,000% for the L. paracasei PCB 030 fermented BSFL protein hydrolysate, compared to those in the extracts of samples without fermentation. Furthermore, most of these small peptides comprised over 50% of hydrophobic amino acids in their sequencing, the function of these peptides requires further investigation. Our study provides a method for efficient fermentative BSFL hydrolysis and a steppingstone for the development of sustainable, functional and nutritionally enhanced BSFL protein hydrolysate ingredients.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/fermentation11090524/s1, Figure S1: The chemical content of the BSF larvae samples. The paste refers to the homogenous mixture of the exoskeleton and endoskeleton. Results expressed as percentage (%) of total dry weight. Figure S2: Digital photos of (a) raw BSFL before steaming, (b) BSFL paste in 50 mL tubes during fermentation, and (c) freeze-dried BSFL powder after fermentation; Figure S3. Digital photos show the viable counts on MRS agar plates of (a) control and (b) PCB 030 fermented BSFL paste at the start of experiment (0 D). (c) Digital photo of the BSFL paste (c.a. 7 mL) fermented with PCB 030 (left two tubes) where the BSFL paste is sponge-like at 3 D, while the control (right two tubes) is not; Figure S4: The degree of hydrolysis of the PCB 030 and F-LC fermented BSFL paste and the control at the start of the experiment (D0) and after 3 days of fermentation. Each data point was expressed as the mean and standard error of triplicate parallel fermentations; Figure S5: (a) Kinetic OD@600 nm change of the PCB 030 in MRS broth at 37 °C for 24 h using Tecan Infinite M200 Plate Reader. (b) Microscope (Zeiss Primostar) image of PCB 030 after gram staining. Figure S6. The 2D montage of m/z vs. retention time of different peptide ions used in quantitation of normalized abundances for the samples of the control and PCB 030 fermented BSFL paste at the start of the experiment (D0) and after 3 days of fermentation; Table S1: Free essential amino acids content (mg/kg, based on the dry BSFL paste) of larvae paste inoculated with PCB 030 as the starter culture and incubated at 37 °C. Samples were collected at the start of the experiment (D0) and after 1, 2, 3, and 7 days of fermentation; Table S2: The average normalized abundances of selected peptide ions (with their corresponding amino acids sequence) used in quantitation; Table S3: The average normalized abundances of small peptides accession to their corresponding proteins. The relative quantitation using Hi-3, Hypothetical protein OS = Hermetia illucens OX = 343,691. Database: UniProt.

Author Contributions

Conceptualization: Y.W., P.L.C. and L.W.; methodology: P.Z., K.S. and R.S.; supervision: Y.W., P.L.C., R.J.C. and L.W.; writing—original draft: P.Z.; writing—review and editing: P.Z., Y.W., P.L.C., R.J.C., L.W., K.S. and R.S.; project Administration, Y.W. and P.L.C.; funding acquisition, P.L.C. and Y.W. All authors have read and agreed to the published version of the manuscript.

Funding

This Research was supported by the RIE2020/RIE2025 1st Alternative Proteins Seed Challenged under the Singapore Food Story R&D Programme 2020 (Award W20W2D0014), administered by A*STAR. The Singapore Centre for Environmental Life Sciences Engineering (SCELSE) is funded by the Ministry of Education, Singapore, the National Re-search Foundation of Singapore, Nanyang Technological University Singapore (NTU) and National University of Singapore (NUS).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the corresponding authors.

Acknowledgments

We acknowledge Protenga Pte Ltd. for advice and assistance throughout the research and Protenga Sdn Bhd for providing BSF larvae for this work. We appreciate Liang Xu (Singapore Phenome Center) for developing the LC-MS protocols of testing amino acids and small peptides, Cheah Yeong Cheng (Singapore Phenome Center) for her help in testing amino acids with LC-MS, and Nur Diyana Binte Zakaria (Singapore Phenome Center) for her help in testing small peptides and conducting the data pre-processing.

Conflicts of Interest

Author Leo Wein was employed by the company Protenga Pte Ltd. Author Rachel Steven was employed by the company Protenga Sdn Bhd. Author Patricia L. Conway was employed by the company PC Biome Pte Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Schematic diagram of the pre-treatment and fermentation of the black soldier fly larvae (BSFL) and subsequent analyses. The BSFL was washed and steamed before the exoskeleton and endoskeleton parts were separated with an oil-press machine. After mixing to obtain a homogenous BSFL paste, the L. paracasei PCB 030 was inoculated as the starter culture.
Figure 1. Schematic diagram of the pre-treatment and fermentation of the black soldier fly larvae (BSFL) and subsequent analyses. The BSFL was washed and steamed before the exoskeleton and endoskeleton parts were separated with an oil-press machine. After mixing to obtain a homogenous BSFL paste, the L. paracasei PCB 030 was inoculated as the starter culture.
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Figure 2. Characterizations of the fermentation process of black soldier fly larvae (BSFL). (a) pH changes of the BSFL paste after inoculating with L. paracasei PCB 030 (Log CFU/mL is around 7.5). (b) Viable counts of the BSFL paste at the start of the experiment (0 d) and at 1, 2, 3, and 7 d of fermentation. Each data point is expressed as the mean and standard error of triplicate samples. Significant differences were determined using unpaired t-tests, with each time point compared to day 0 (D0). Results are reported as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (c) Field Emission Scanning Electron Microscope image of the L. paracasei PCB 030 fermented BSFL paste, the red arrows indicate rod-shaped cells.
Figure 2. Characterizations of the fermentation process of black soldier fly larvae (BSFL). (a) pH changes of the BSFL paste after inoculating with L. paracasei PCB 030 (Log CFU/mL is around 7.5). (b) Viable counts of the BSFL paste at the start of the experiment (0 d) and at 1, 2, 3, and 7 d of fermentation. Each data point is expressed as the mean and standard error of triplicate samples. Significant differences were determined using unpaired t-tests, with each time point compared to day 0 (D0). Results are reported as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (c) Field Emission Scanning Electron Microscope image of the L. paracasei PCB 030 fermented BSFL paste, the red arrows indicate rod-shaped cells.
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Figure 3. Free amino acids profile of black soldier fly larvae (BSFL) paste during seven days of fermentation. Essential free amino acids and non-essential amino acids (mg/kg, based on the dry BSFL paste) in the BSFL paste inoculated with L. paracasei PCB 030 (red curves) and the control (black curves). Essential amino acids include (a) valine, (b) threonine, (c) leucine, (d) isoleucine, (e) lysine, (f) methionine, (g) histidine, (h) tryptophan, and (i) phenylalanine; non-essential amino acids include (j) alanine, (k) serine, (l) proline, (m) asparagine, (n) aspartic acid, (o) glutamine, (p) glutamic acid, (q) arginine, and (r) tyrosine. Samples were collected at the start of the experiment (0 d) and at 1, 2, 3, and 7 d of fermentation. Each data point is presented as the mean and standard error of triplicate parallel fermentations. Significant differences relative to the control are reported as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Amino acids are represented as a three-letter code according to the IUPAC-IUB. Detailed data can be found in Table S1.
Figure 3. Free amino acids profile of black soldier fly larvae (BSFL) paste during seven days of fermentation. Essential free amino acids and non-essential amino acids (mg/kg, based on the dry BSFL paste) in the BSFL paste inoculated with L. paracasei PCB 030 (red curves) and the control (black curves). Essential amino acids include (a) valine, (b) threonine, (c) leucine, (d) isoleucine, (e) lysine, (f) methionine, (g) histidine, (h) tryptophan, and (i) phenylalanine; non-essential amino acids include (j) alanine, (k) serine, (l) proline, (m) asparagine, (n) aspartic acid, (o) glutamine, (p) glutamic acid, (q) arginine, and (r) tyrosine. Samples were collected at the start of the experiment (0 d) and at 1, 2, 3, and 7 d of fermentation. Each data point is presented as the mean and standard error of triplicate parallel fermentations. Significant differences relative to the control are reported as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Amino acids are represented as a three-letter code according to the IUPAC-IUB. Detailed data can be found in Table S1.
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Figure 4. Total free amino acids content and degree of hydrolysis of black soldier fly larvae (BSFL) protein hydrolysate at different fermentation days when fermented with either L. paracasei PCB 030 or F-LC commercial starter culture. (a) Total essential free amino acids (TEFAA) and total free amino acids (TFAA) content (mg/kg, based on the dry BSFL paste) in the L. paracasei PCB 030 fermented BSFL paste and the control (no added bacteria) at the start of the experiment (0 d) and at 1, 2, 3, and 7 d of fermentation. (b) The degree of hydrolysis of the L. paracasei PCB 030 fermented BSFL paste and the control at the start of the experiment (0 d) and at 1, 2, 3, and 7 d of fermentation. (c) Relative change of free amino acids content after 3 d fermentation by L. paracasei PCB 030 and F-LC. Each data point is expressed as the mean and standard error of triplicate parallel fermentations. Significant differences relative to the control are reported as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
Figure 4. Total free amino acids content and degree of hydrolysis of black soldier fly larvae (BSFL) protein hydrolysate at different fermentation days when fermented with either L. paracasei PCB 030 or F-LC commercial starter culture. (a) Total essential free amino acids (TEFAA) and total free amino acids (TFAA) content (mg/kg, based on the dry BSFL paste) in the L. paracasei PCB 030 fermented BSFL paste and the control (no added bacteria) at the start of the experiment (0 d) and at 1, 2, 3, and 7 d of fermentation. (b) The degree of hydrolysis of the L. paracasei PCB 030 fermented BSFL paste and the control at the start of the experiment (0 d) and at 1, 2, 3, and 7 d of fermentation. (c) Relative change of free amino acids content after 3 d fermentation by L. paracasei PCB 030 and F-LC. Each data point is expressed as the mean and standard error of triplicate parallel fermentations. Significant differences relative to the control are reported as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.
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Figure 5. The amino acid sequence and their relative abundances of peptide ions in the black soldier fly larvae (BSFL) protein hydrolysate produced by fermentation with L paracasei PCB 030 for three days. Most of the small peptide ions in the L. paracasei PCB 030 3 d fermented BSFL paste show a significantly higher abundance compared to those of the control. (a) Control fermentation at day zero (D0) and day three (D3); (b) L. paracasei PCB 030 at zero and three days fermentation. * represents where there were no related peptides for the samples. Data is presented as the mean of ArcSinh normalized abundances of average normalized abundances of triplicated fermented samples with p < 0.01. Detailed information, including score, ANOVA (p), fold, m/z, and retention time, etc., can be found in Table S2.
Figure 5. The amino acid sequence and their relative abundances of peptide ions in the black soldier fly larvae (BSFL) protein hydrolysate produced by fermentation with L paracasei PCB 030 for three days. Most of the small peptide ions in the L. paracasei PCB 030 3 d fermented BSFL paste show a significantly higher abundance compared to those of the control. (a) Control fermentation at day zero (D0) and day three (D3); (b) L. paracasei PCB 030 at zero and three days fermentation. * represents where there were no related peptides for the samples. Data is presented as the mean of ArcSinh normalized abundances of average normalized abundances of triplicated fermented samples with p < 0.01. Detailed information, including score, ANOVA (p), fold, m/z, and retention time, etc., can be found in Table S2.
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Figure 6. The hypothetical protein accession number and the amino acids sequence of the detected peptide ions. The relative average normalized abundances of hypothetical proteins used in quantitation of the small peptides for the control (a) and L. paracasei PCB 030 fermented BSFL (b) paste at the start of the experiment (D0) and after three days (D3) of fermentation. Data is presented as the mean of ArcSinh normalized abundances of average normalized abundances of triplicated samples. Detailed information, including score, Anova (p), fold, and protein source description, can be found in Table S3. Database: OS = Hermetia illucens and OX = 343,691 in UniProt. (c) The amino acid sequence of the small peptides, the hydrophobic amino acids are highlighted with pink boxes and blue letters.
Figure 6. The hypothetical protein accession number and the amino acids sequence of the detected peptide ions. The relative average normalized abundances of hypothetical proteins used in quantitation of the small peptides for the control (a) and L. paracasei PCB 030 fermented BSFL (b) paste at the start of the experiment (D0) and after three days (D3) of fermentation. Data is presented as the mean of ArcSinh normalized abundances of average normalized abundances of triplicated samples. Detailed information, including score, Anova (p), fold, and protein source description, can be found in Table S3. Database: OS = Hermetia illucens and OX = 343,691 in UniProt. (c) The amino acid sequence of the small peptides, the hydrophobic amino acids are highlighted with pink boxes and blue letters.
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Zhang, P.; Seow, K.; Wein, L.; Steven, R.; Case, R.J.; Wang, Y.; Conway, P.L. Production of Nutritional Protein Hydrolysates by Fermentation of Black Soldier Fly Larvae. Fermentation 2025, 11, 524. https://doi.org/10.3390/fermentation11090524

AMA Style

Zhang P, Seow K, Wein L, Steven R, Case RJ, Wang Y, Conway PL. Production of Nutritional Protein Hydrolysates by Fermentation of Black Soldier Fly Larvae. Fermentation. 2025; 11(9):524. https://doi.org/10.3390/fermentation11090524

Chicago/Turabian Style

Zhang, Penghui, Kelyn Seow, Leo Wein, Rachel Steven, Rebecca J. Case, Yulan Wang, and Patricia L. Conway. 2025. "Production of Nutritional Protein Hydrolysates by Fermentation of Black Soldier Fly Larvae" Fermentation 11, no. 9: 524. https://doi.org/10.3390/fermentation11090524

APA Style

Zhang, P., Seow, K., Wein, L., Steven, R., Case, R. J., Wang, Y., & Conway, P. L. (2025). Production of Nutritional Protein Hydrolysates by Fermentation of Black Soldier Fly Larvae. Fermentation, 11(9), 524. https://doi.org/10.3390/fermentation11090524

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