Lanternfish (Benthosema pterotum) Hydrolysates: Dual Action on Immune Regulation

Abstract

Background:

Lanternfish (Benthosema pterotum), an abundant deep-sea resource with a unique protein profile, holds significant potential as a source of bioactive peptides. This study was designed to investigate and characterize the immunomodulatory properties of protein hydrolysates derived from lanternfish, prepared using Alcalase (BPHA) and Flavourzyme (BPHF), on the RAW 264.7 murine macrophage cell line.

Methods:

Lanternfish protein was enzymatically hydrolyzed, and the resulting peptides were fractionated to isolate those with a molecular weight below 3 kDa. RAW 264.7 macrophages were treated with B. pterotum hydrolyzed using Alcalase enzymes (BPHA) and B. pterotum hydrolyzed using Flavourzyme enzymes (BPHF) at concentrations ranging from 100 to 500 µg/mL. The cytotoxicity of the hydrolysates was evaluated using an 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. To assess immunomodulatory activity, the gene expression levels of pro-inflammatory mediators, including Inducible nitric oxide synthase (iNOS), Tumor necrosis factor alpha (TNF-α), Interleukin-1 beta (IL-1β), and Interleukin-6 (IL-6), were quantified by quantitative real-time PCR in both resting macrophages and in cells stimulated with lipopolysaccharide (LPS) to induce an inflammatory state.

Results:

The MTT assay confirmed that both BPHA and BPHF were noncytotoxic across all tested concentrations. In resting (nonstimulated) macrophages, both hydrolysates significantly upregulated the expression of iNOS, TNF-α, IL-1β, and IL-6 in a dose-dependent manner, demonstrating a clear immunostimulatory effect. In contrast, when macrophages were exposed to an inflammatory challenge with LPS, the hydrolysates exhibited potent anti-inflammatory activity by significantly downregulating the expression of these same pro-inflammatory genes.

Conclusions:

Protein hydrolysates derived from lanternfish display a notable dual immunomodulatory capability. They function as immunostimulants in a normal physiological state and act as anti-inflammatory agents during an inflammatory response. These findings underscore the potential of lanternfish hydrolysates as a valuable functional ingredient for applications in nutraceuticals and functional foods aimed at balancing and modulating the immune system.

Keywords

cytokine immunomodulatory inflammation lanternfish

Introduction

Natural products and mineral supplements have been widely explored for their therapeutic potential. Recent investigations have demonstrated the efficacy of plant-mediated biosynthesis of nanoparticles¹ and herbal extracts, such as Artemisia dracunculus,² in developing novel anticancer and antibacterial agents. Furthermore, the therapeutic potential of inorganic agents, including zinc oxide nanoparticles³ and selenium combinations,⁴ has been highlighted for their ability to induce oxidative stress and inhibit tumor growth in colon and prostate cancer models. In parallel, the oceans have long been recognized as a source of valuable nutrients, teeming with life that offers a diverse array of health benefits. Marine organisms, from fish and algae to invertebrates, are rich in essential components like proteins, lipids, polysaccharides, and minerals, contributing to their high nutritional value and making them an attractive focus for the development of functional foods and nutraceuticals.^5,6 Among these components, marine-derived proteins and peptides have garnered particular attention for their potent bioactive properties, including antioxidant, anti-inflammatory, antimicrobial, and immunomodulatory activities. These properties play a crucial role in promoting human health and preventing chronic diseases, further highlighting the potential of marine resources in the development of innovative health solutions.^7,8

The unique conditions of marine ecosystems, characterized by high pressure, low temperature, and high salinity, have driven the evolution of a vast diversity of bioactive compounds.^9,10 This diversity offers enormous potential for discovering novel compounds with specific health-promoting properties, particularly as global interest in sustainable and natural health solutions grows. In this context, marine products, and specifically fish protein hydrolysates, stand out as a promising resource for innovative bioactive compounds with potential applications in functional foods and nutraceuticals.^11,12 In fact, the exploration of marine-derived bioactive compounds has gained significant momentum, particularly focusing on their potential health benefits. Among these, protein hydrolysates from marine organisms have emerged as promising candidates for developing functional foods and therapeutic agents due to their diverse bioactivities, including antioxidant, antihypertensive, and immunomodulatory effects.^13,14

Inflammation is a natural and essential response of the immune system to infection or injury. However, chronic inflammation can lead to the development of diseases such as rheumatoid arthritis, type 2 diabetes, cardiovascular diseases, and even cancer.¹⁵ Therefore, controlling and modulating inflammation, especially in chronic conditions, is critical for maintaining immune system balance and preventing disease. In this context, the use of natural compounds with immunoregulatory properties offers a promising approach to managing unwanted inflammation and improving immune responses.^16,17

Benthosema pterotum possesses a unique biochemical profile that distinguishes it from other mesopelagic species. While many myctophids accumulate significant amounts of indigestible wax esters, B. pterotum is devoid of these compounds. This specific compositional trait, combined with adaptations to the high-pressure deep-sea environment, results in a high-quality protein matrix. Consequently, enzymatic hydrolysis of this species is less hindered by lipid interference and is likely to release a distinct spectrum of bioactive peptides compared with other marine sources.¹⁸

Lanternfish (B. pterotum), a mesopelagic species abundant in the world’s oceans, represent an underutilized resource with substantial potential. Recent studies have highlighted the bioactive properties of protein hydrolysates derived from lanternfish, demonstrating neuroprotective effects both in vitro and in vivo.¹⁹ Despite their vast biomass and underutilized status in the marine food chain, the immunomodulatory potential of lanternfish protein hydrolysates remains underexplored. Their high protein content, rich amino acid profile, abundance, and rapid reproductive cycles position them as an environmentally friendly source of marine bioresources. Utilizing lanternfish aligns with global efforts to reduce waste and improve the sustainability of marine resources.^20,21

Protein hydrolysates, obtained through the enzymatic breakdown of proteins, are a rich source of bioactive peptides with diverse health-promoting properties. Among these, immunomodulatory peptides have gained significant attention due to their ability to fine-tune the immune system, enhancing immune responses, suppressing excessive inflammation, and promoting overall immune balance. This makes them highly relevant in the prevention and management of immune-related disorders, further emphasizing the potential of lanternfish-derived hydrolysates.^14,22

Enzymatic hydrolysis using proteases such as Alcalase and Flavourzyme is a common method to produce protein hydrolysates with desired bioactive properties. These enzymes differ in their specificity and the peptides they release, which can influence the bioactivity of the resulting hydrolysates.²³ Understanding how different enzymatic treatments affect the immunomodulatory properties of lanternfish protein hydrolysates could provide valuable insights for their application in functional foods and nutraceuticals.²⁴

This study aims to investigate the immunomodulatory effects of protein hydrolysates derived from lanternfish (B. pterotum) using Alcalase and Flavourzyme on RAW 264.7 macrophage cells. By assessing cell viability and the expression of key inflammatory mediators, we seek to elucidate the potential of these hydrolysates as natural agents for modulating immune responses.

Materials and Methods

Preparation of lanternfish protein hydrolysates

Sample collection

Lanternfish (B. pterotum) were harvested from the Makran coast. Post-harvest, the fish were cleaned to remove any surface contaminants and then minced into a uniform paste to facilitate enzymatic hydrolysis.¹⁸

Enzymatic hydrolysis

The fish were cleaned, minced, and subjected to enzymatic hydrolysis using two specific proteases: Alcalase and Flavourzyme. Each enzyme was added to the fish substrate at optimal conditions to achieve hydrolysis. The reaction was maintained for a specific duration, after which the enzymes were inactivated by heating. For Alcalase hydrolysis, the reaction was conducted at a pH of 8.5, a temperature of 58°C, and a duration of 90 minutes. For Flavourzyme hydrolysis, the reaction was conducted at a pH of 7.0, a temperature of 50°C, and a duration of 90 minutes. In both cases, the enzyme-to-substrate ratio was maintained at 4% (w/w). After the hydrolysis period, the reactions were terminated by heating the mixtures to 95°C for 10 minutes to inactivate the enzymes. The hydrolysates were then cooled to room temperature.¹⁸

Determination of degree of hydrolysis

The degree of hydrolysis (DH) was quantified using the trichloroacetic acid (TCA) soluble nitrogen method. Briefly, an aliquot of the hydrolysate was mixed with an equal volume of 20% (v/v) TCA to precipitate high-molecular-weight proteins. The mixture was incubated, centrifuged at 4,800 × g for 10 minutes, and the soluble protein content in the supernatant was measured using the Lowry method. The DH was calculated as the ratio of TCA-soluble nitrogen to the total nitrogen content of the sample.¹⁸

Fractionation

The resulting hydrolysates were subjected to ultrafiltration to obtain peptide fractions with molecular weights below 3 kDa. This was achieved using a membrane filtration system equipped with a 3 kDa cutoff membrane. The permeates, containing the desired low-molecular-weight peptides, were collected, lyophilized, and stored at −20°C until further analysis.¹⁸

Chemical composition analysis

The proximate composition of the raw lanternfish and the lyophilized protein hydrolysates was determined using standard AOAC methods. Moisture content was determined by oven-drying at 105°C until a constant weight was achieved. Crude protein was measured using the Kjeldahl method, with a nitrogen-to-protein conversion factor of 6.25. Crude fat was assessed by Soxhlet extraction using petroleum ether as the solvent. Ash content was evaluated by incineration in a muffle furnace at 550°C for 6 hours.¹⁸

Cell culture

RAW 264.7 murine macrophage cells were obtained from the Institute Pasteur of Tehran. Cells were cultured in Dulbecco’s modified Eagle medium supplemented with 2 mM glutamine, 10% fetal bovine serum, and 1% penicillin/streptomycin. Cells were maintained at 37°C in a humidified atmosphere with 5% CO₂. Subculturing was performed when cells reached 70%–80%. Cells were then detached using trypsin-EDTA, and a cell suspension containing 5 × 10⁴ cells/mL was prepared. One hundred microliter of this suspension was seeded into each well of a 96-well plate, and the plate was incubated for 24 hours prior to treatment.^25,26

Treatment of cells with protein hydrolysates

Cells were seeded in 96-well plates at a density of 1 × 10⁵ cells/well and allowed to adhere overnight. The following day, cells were treated with varying concentrations (100, 200, 300, 400, and 500 µg/mL) of BPHA and BPHF hydrolysates. Lipopolysaccharide (LPS) at 1 µg/mL served as a positive control, and untreated cells served as the negative control. Treatments were conducted in triplicate and incubated for 48 hours.¹⁸

Cell viability assay

To assess the effects of Alcalase and Flavourzyme protein hydrolysates (BPHA and BPHF) on cell viability, an MTT assay was performed. After treatment, the cells were incubated with MTT solution for 4 hours, allowing viable cells to metabolize the MTT and form formazan crystals. The formazan crystals were then dissolved in dimethyl sulfoxide, and the absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated as a percentage of the control group, and the data were analyzed to determine the significant differences between the treatment groups.^27,28

Quantitative real-time PCR

To further investigate the effects of the Alcalase and Flavourzyme hydrolysates on RAW 264.7 cells, a real-time PCR assay was conducted to analyze the expression of β-actin, iNOS, TNF-α, IL-6, and IL-1β genes.

RNA extraction and complementary DNA synthesis

Total RNA was extracted from treated and control cells using a commercial RNA extraction kit (Yekta Tajhiz Company, Iran), following the manufacturer’s instructions. Quantitative and qualitative evaluations of extracted RNA were determined by Nano Drop and agarose gel electrophoresis, respectively. Complementary DNA synthesis from total RNA was performed using a commercial kit (Yekta Tajhiz Company, Iran)

Gene expression analysis

Gene expression was quantified using a Step One Plus Real-time PCR system (Applied Biosystems, USA) with RealQ Plus 2× Master Mix Green (Ampliqon, Denmark). The thermal cycling conditions were as follows: an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. Specific primers for each gene were used, with sequences listed in Table 1. Following amplification, a melt curve analysis was performed to confirm the specificity of the products. Relative gene expression was calculated using the 2^−ΔΔCt method, with β-actin serving as the housekeeping gene for normalization.^29,30

Table 1.

Primer Sequences

Gene	Forward	Reverse	Amplicon sizes
β-actin	5′-GGCTGTATTCCCCTCCATCG-3′	5′-CCAGTTGGTAACAATGCCATGT-3′	147 bp
iNOS	5′-GGCAGCCTGTGAGACCTTTG-3′	5′-GCATTGGAAGTGAAGCGTTTC-3′	153 bp
TNF-α	5′-AGGCACTCCCCCAAAAGATG-3′	5′-GCTACAGGCTTGTCACTCGAAT-3′	199 bp
IL-6	5′-TAGTCCTTCCTACCCCAATTTCC-3′	5′-TTGGTCCTTAGCCACTCCTTC-3′	159 bp
IL-1β	5′-GCAACTGTTCCTGAACTCAACT-3′	5′-ATCTTTTGGGGTCCGTCAACT-3′	167 bp

Statistical analysis

Data were expressed as mean ± standard deviation (SD) from at least triplicate (n = 3) independent experiments. Statistical significance was determined using one-way analysis of variance followed by post hoc comparisons. A p value of <0.05 was considered statistically significant.

Results

Physicochemical properties

The physicochemical properties and DH of the hydrolysates were characterized as previously reported.¹⁸ The DH was significantly higher in BPHA (42.35%) compared with BPHF (27.37%) (p < 0.05). Both hydrolysates exhibited high protein content, with BPHA containing 80.26% and BPHF 79.45%. The moisture content was 9.36% for BPHA and 9.75% for BPHF, while the ash content was 8.41% and 8.68%, respectively. Fat content differed significantly between the two, with BPHA at 1.36% and BPHF at 1.64% (p ≤ 0.05). All values are presented as mean ± SD from triplicate determinations (n = 3), with different superscript letters indicating significant differences (p ≤ 0.05). It is important to note that the proximate composition of the raw lanternfish (B. pterotum) was determined on a wet weight basis, while the compositions of BPHA and BPHF were determined on a dry weight basis (Table 2).

Table 2.

Degree of Hydrolysis and Proximate Composition of BPHA and BPHF Hydrolysates¹⁸

Component	BPHA (mean ± SD)	BPHF (mean ± SD)
Degree of hydrolysis (%)	42.35 ± 0.50	27.37 ± 0.30
Protein (%)	80.26 ± 0.85	79.45 ± 0.36
Moisture (%)	9.36 ± 0.04	9.75 ± 0.03
Ash (%)	8.41 ± 0.07	8.68 ± 0.04
Fat (%)	1.36 ± 0.01	1.64 ± 0.05

SD, standard deviation.

MTT assay

The MTT assay was conducted to evaluate the cytotoxicity of BPHA and BPHF hydrolysates on RAW 264.7 cells. Cells were treated with varying concentrations (100, 200, 300, 400, and 500 µg/mL) of each hydrolysate for 48 hours. Cell viability was determined by measuring absorbance at 570 nm, with results expressed as a percentage relative to the untreated control group. The results indicated that both BPHA and BPHF hydrolysates did not exhibit cytotoxic effects on RAW 264.7 cells across the tested concentrations. Cell viability remained above 90% for all treatment groups, suggesting that these hydrolysates are nontoxic to RAW 264.7 cells at concentrations up to 500 µg/mL. This finding aligns with previous studies demonstrating the safety of protein hydrolysates in similar cell lines (Fig. 1).

FIG. 1.

Effect of BPHA and BPHF hydrolysates on viability. RAW 264.7 cells were treated with varying concentrations (100, 200, 300, 400, and 500 µg/mL) of BPHA and BPHF hydrolysates for 48 hours. Cell viability was assessed using the MTT assay and expressed as a percentage relative to the untreated control group. Data are presented as mean ± standard deviation (n = 3). No significant cytotoxic effects were observed for either hydrolysate across all tested concentrations, with cell viability remaining above 90%.

Effects of BPHA and BPHF hydrolysates on iNOS expression

Figure 2 shows the effects of Alcalase (BPHA) and Flavourzyme (BPHF) on iNOS expression in RAW 264.7 cells. Panels A and C display that both enzymes increase iNOS expression in a dose-dependent manner under normal conditions, with significant increases at higher concentrations (300–500 µg/mL). Panels B and D illustrate that in LPS-stimulated inflammatory conditions, both Alcalase and Flavourzyme significantly reduce iNOS expression in a dose-dependent manner, especially at 400 and 500 µg/mL. These results suggest that both enzymes can upregulate iNOS in normal conditions and exhibit anti-inflammatory effects by downregulating iNOS during inflammation.

FIG. 2.

Effects of Alcalase (BPHA) and Flavourzyme (BPHF) on iNOS expression in RAW 264.7 cells. (A, C) Dose-dependent increase in iNOS expression following treatment with Alcalase and Flavourzyme under normal conditions. (B, D) Dose-dependent reduction in iNOS expression in LPS (1 µg/mL)-stimulated cells treated with increasing concentrations of Alcalase and Flavourzyme. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001 compared with the LPS group. LPS, lipopolysaccharide.

Effects of BPHA and BPHF hydrolysates on cytokine expression

Figure 3 depicts the impact of Alcalase (BPHA) and Flavourzyme (BPHF) on TNF-α expression in RAW 264.7 cells. In panels A and C, treatment with increasing concentrations of Alcalase and Flavourzyme under normal conditions leads to a significant, dose-dependent rise in TNF-α expression, with the most notable increases at 400 and 500 µg/mL. Conversely, panels B and D show that in LPS-stimulated cells, which naturally have elevated TNF-α levels due to inflammation, both enzymes effectively suppress TNF-α expression in a concentration-dependent manner, with significant reductions at higher doses. These findings indicate that Alcalase and Flavourzyme can upregulate TNF-α in noninflammatory states while demonstrating anti-inflammatory properties by lowering TNF-α production in inflammatory conditions.

FIG. 3.

Effects of Alcalase (BPHA) and Flavourzyme (BPHF) on TNF-α expression in RAW 264.7 cells. (A, C) Dose-dependent increase in TNF-α expression following treatment with Alcalase and Flavourzyme under normal conditions. (B, D) Significant, dose-dependent reduction in TNF-α expression in LPS (1 µg/mL)-stimulated cells treated with Alcalase and Flavourzyme. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with the LPS group. LPS, lipopolysaccharide.

Figure 4 demonstrates the effects of Alcalase (BPHA) and Flavourzyme (BPHF) on IL-1β expression in RAW 264.7 cells. Panels A and C show that both Alcalase and Flavourzyme significantly increase IL-1β expression in a dose-dependent manner under normal conditions, with the highest levels observed at 400 and 500 µg/mL (***p < 0.001, ****p < 0.0001). In contrast, panels B and D reveal that in LPS-stimulated cells, which naturally exhibit elevated IL-1β expression due to inflammation, both enzymes significantly reduce IL-1β expression in a concentration-dependent manner, especially at higher doses. These findings suggest that Alcalase and Flavourzyme can stimulate IL-1β production in noninflammatory conditions but exhibit anti-inflammatory properties by suppressing IL-1β expression during inflammatory responses.

FIG. 4.

Effects of Alcalase (BPHA) and Flavourzyme (BPHF) on IL-1β expression in RAW 264.7 cells. (A, C) Alcalase and Flavourzyme treatments under normal conditions significantly increased IL-1β expression in a dose-dependent manner, with the highest expression observed at 400 and 500 µg/mL. (B, D) In LPS (1 µg/mL)-stimulated cells, both Alcalase and Flavourzyme significantly reduced IL-1β expression in a concentration-dependent manner, particularly at higher doses. Data are expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with the LPS group. LPS, lipopolysaccharide.

Figure 5 illustrates the effects of Alcalase (BPHA) and Flavourzyme (BPHF) on IL-6 expression in RAW 264.7 cells. Panels A and C show that both Alcalase and Flavourzyme significantly increase IL-6 expression in a dose-dependent manner under normal conditions, with the highest levels observed at 400 and 500 µg/mL (***p < 0.001, ****p < 0.0001). Conversely, panels B and D reveal that in LPS-stimulated cells, which naturally have elevated IL-6 expression due to inflammation, both enzymes significantly decrease IL-6 expression in a concentration-dependent manner, particularly at higher doses. These results suggest that Alcalase and Flavourzyme can upregulate IL-6 under noninflammatory conditions but demonstrate anti-inflammatory effects by reducing IL-6 levels during inflammation.

FIG. 5.

Effects of Alcalase (BPHA) and Flavourzyme (BPHF) on IL-6 expression in RAW 264.7 cells. (A, C) Treatment with increasing concentrations of Alcalase and Flavourzyme under normal conditions significantly elevated IL-6 expression in a dose-dependent manner, with the highest expression at 400 and 500 µg/mL. (B, D) In LPS (1 µg/mL)-stimulated cells, both Alcalase and Flavourzyme significantly reduced IL-6 expression in a concentration-dependent manner, especially at higher doses. Data are presented as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 compared with the LPS group. LPS, lipopolysaccharide.

Discussion

This study investigates the immunomodulatory properties of protein hydrolysates derived from lanternfish (B. pterotum) treated with Alcalase (BPHA) and Flavourzyme (BPHF) enzymes. The results demonstrate that both hydrolysates exhibit promising effects in modulating the immune response, especially by regulating the expression of key inflammatory mediators in RAW 264.7 macrophage cells. A mechanistic exploration of these findings involves understanding the signaling pathways affected by these bioactive peptides and comparing the observed results with other similar studies in the field.

The bioactivity observed in this study is largely attributed to the molecular weight distribution of the hydrolysates. By subjecting the hydrolysates to ultrafiltration with a 3 kDa cutoff membrane, we enriched the fractions with low-molecular-weight peptides (<3 kDa). Short-chain peptides within this specific distribution range are known to exhibit enhanced solubility and cellular uptake compared with larger polypeptides, thereby facilitating their interaction with macrophage receptors and triggering the observed immunomodulatory signaling pathways.

The MTT assay demonstrated that both BPHA and BPHF hydrolysates maintained cell viability at all tested concentrations (100–500 µg/mL), which is an important finding as it confirms that these hydrolysates are noncytotoxic. This noncytotoxicity is crucial for the potential application of these hydrolysates as functional ingredients, as they do not induce cell death or toxicity at the concentrations used. The noncytotoxic nature of protein hydrolysates has been consistently observed in similar studies. For instance, fish protein hydrolysates have been reported to have no adverse effects on cell viability in various cell types, including immune cells,^31,32 confirming the safety of these compounds in cell-based applications.

Both BPHA and BPHF hydrolysates were shown to modulate the expression of key inflammatory cytokines such as iNOS, TNF-α, IL-6, and IL-1β in a dose-dependent manner. Specifically, BPHA treatment upregulated iNOS and TNF-α at a concentration of 400 µg/mL, while BPHF enhanced IL-6 and IL-1β expression at 300 µg/mL.

The increased expression of iNOS suggests that BPHA may promote a pro-inflammatory response, as iNOS is a critical enzyme involved in the production of nitric oxide (NO), which plays a pivotal role in inflammation and immune responses.^33,34 In a similar study by Sung et al., fish protein hydrolysates enhanced iNOS expression in macrophages, resulting in an increase in NO production and activation of the immune system.³⁵ This aligns with the notion that BPHA might activate immune responses through NO production.

The upregulation of TNF-α by BPHA is indicative of an inflammatory response, as TNF-α is a potent pro-inflammatory cytokine that plays a central role in systemic inflammation. Similar findings have been reported with other fish protein hydrolysates, where TNF-α levels were increased, suggesting that bioactive peptides derived from fish proteins can activate immune cells to release pro-inflammatory cytokines.^36,37 The mechanism likely involves the activation of the Nuclear factor-kappa B (NF-κB) signaling pathway, a key regulator of inflammation and immune responses.³⁸

This observed dose-dependent upregulation of pro-inflammatory mediators in resting RAW 264.7 macrophages suggests that BPHA and BPHF act as potent immunostimulants. This phenomenon is characterized as low-level activation or immune priming, which enhances the surveillance capacity of the innate immune system without triggering pathological systemic inflammation. Rather than inducing a chronic inflammatory state, this mild stimulatory effect prepares macrophages for a more efficient and rapid response to potential pathogen encounters. This dual-action profile stimulating immune vigilance under homeostatic conditions while suppressing hyper-inflammation during an LPS challenge is a hallmark of effective immunomodulators derived from marine sources. Such biological responses align with the trained immunity concept, where natural bioactive peptides fine-tune the immune system’s baseline activity to maintain overall host defense integrity.^39,40

On In contrast, BPHF significantly increased the expression of IL-6 and IL-1β, cytokines that are important in both innate and adaptive immune responses. IL-6 is known for its role in acute-phase reactions and as a key mediator of inflammation.⁴¹ IL-1β, similarly, is involved in the inflammatory response, influencing the activation of NF-κB and the inflammasome. The increased production of these cytokines in response to BPHF indicates that this hydrolysate might promote both local and systemic immune responses. Similar findings have been observed with other fish-derived bioactive peptides, which upregulate IL-6 and IL-1β expression.⁴²

The observed immunomodulatory effects are likely mediated through the activation of key inflammatory signaling pathways. The NF-κB pathway is a primary candidate, as it regulates the expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. Fish protein hydrolysates, including those from lanternfish, have been shown to activate NF-κB signaling, leading to the upregulation of these inflammatory mediators.^29,39 Moreover, the MAPK pathway, including JNK, ERK, and p38 MAPKs, could also be involved in regulating cytokine expression following protein hydrolysate treatment.^27,43,44 These pathways are critical in mediating immune responses and inflammation, with macrophages being key players in their activation.

Comparing these results with similar studies reveals a consistent trend in the immunomodulatory effects of marine-derived protein hydrolysates. For instance, a study by Rivera et al. demonstrated that fish protein hydrolysates promoted the production of pro-inflammatory cytokines such as TNF-α and IL-6 in RAW 264.7 macrophages.³⁶ Similarly, Liu et al. reported that hydrolysates from tilapia fish protein enhanced the expression of iNOS and TNF-α, which is consistent with the findings of this study.⁴⁵ These results collectively support the hypothesis that fish protein hydrolysates can regulate immune responses by modulating the expression of key inflammatory mediators.

However, the specific effects of BPHA and BPHF on IL-1β expression in RAW 264.7 cells appear to be more pronounced than in some other studies, where IL-1β was not significantly affected.⁴⁶ This suggests that the enzymatic treatment with Alcalase and Flavourzyme might produce specific peptides with a higher capacity to stimulate IL-1β production, indicating a unique immunomodulatory profile for lanternfish-derived hydrolysates.

The distinct immunomodulatory activity observed in BPHA and BPHF may be linked to the specific protein composition of the source material. B. pterotum has been reported to possess a total amino acid content superior to that of other deep-sea organisms, such as deep-sea lobsters and Indian white shrimp. This rich amino acid profile, evolved to sustain life in extreme mesopelagic conditions, likely facilitates the generation of unique peptide sequences upon hydrolysis that are capable of the dual regulation—stimulation and suppression—of cytokine expression seen in this study.

Finally, it is important to acknowledge a limitation of the current study. While our qRT-PCR results demonstrate significant transcriptional modulation of key inflammatory mediators, we did not directly validate the activation of the NF-κB and MAPK signaling pathways at the protein level (e.g., via Western blot analysis of p65 or p38 phosphorylation). Consequently, the involvement of these pathways, although strongly supported by the observed gene expression profiles and concurrent literature, remains to be experimentally confirmed in this specific context. Future investigations will focus on the precise mechanistic elucidation of these pathways to verify the signaling cascades triggered by B. pterotum hydrolysates.

Conclusion

In conclusion, BPHA and BPHF hydrolysates from lanternfish (B. pterotum) exhibit potent immunomodulatory effects on RAW 264.7 macrophage cells, modulating the expression of key inflammatory mediators such as iNOS, TNF-α, IL-6, and IL-1β. The activation of these pathways suggests the potential application of these hydrolysates in functional foods and nutraceuticals aimed at modulating immune responses. The findings of this study are consistent with other reports on fish protein hydrolysates and add to the growing body of evidence supporting the use of marine-derived bioactive peptides in the management of inflammation and immune-related disorders.

Authors’ Contributions

N.G.B., E.G.B., F.M., and J.G.G. contributed to the conception and design of the study. N.G.B. and E.G.B. conducted the data collection and analysis. F.M. and J.G.G. contributed to the interpretation of the results and article drafting. M.S. contributed to the preparation of lanternfish protein hydrolysates. All authors reviewed, revised, and approved the final version of the article.

Data Availability Statement

The datasets generated and/or analyzed during the current study are not publicly available but are available from the corresponding author upon reasonable request.

Footnotes

Author Disclosure Statement

The authors have no relevant financial or nonfinancial interests to disclose.

Funding Information

The authors declare that no funds, grants, or other support were received during the preparation of this article.

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