Bioactive peptides from Tenebrio molitor: physicochemical and antioxidant properties and antimicrobial capacity

Abstract

Recent research has demonstrated the increasing interest in using insects for the extraction of bioactive compounds, particularly peptides. These compounds offer a spectrum of beneficial physiological effects. The aim of this study was to standardize a methodology for obtaining bioactive peptides from Tenebrio molitor and evaluate its physicochemical characterization, antioxidant, and antimicrobial potential. Six assays were carried out to hydrolyse larvae protein, with variations in Alcalase concentration (0.04 to 0.08%) and reaction time (3 to 8 h). The results indicated that the process applied to defatted mealworm flour was effective in reducing lipids by 82.5%. Consequently, it was an observed increase of 38.4% in protein content. Additionally, an increase in glycogen content was found in defatted mealworm flour (177 µmol glucose g^-1 sample) and peptides (152.81 µmol glucose g^-1 sample). The degree of hydrolysis was higher in assays with longer hydrolysis durations (8.14 - 8.38%). The antioxidant capacity was 12 to 14% lower in assays with an incubation time of 8h. In this sense, the methodology proposed in the present study proved to be efficient in obtaining bioactive peptides from T. molitor.

Key words
functional ingredient; enzymatic hydrolysis; mealworm; edible insects

INTRODUCTION

Aquaculture is not only important for generating financial resources and jobs. It is also recognized to contribute to food security and social development in many regions and countries, such as Brazil (Chan et al. 2024, Esteban 2012).

For aquaculture becoming a competitive activity and achieve high production rates, intensification is necessary. Consequently, several factors, including overcrowding, management practices, unfavorable temperature, poor nutrition, and water quality, contribute to stress in the animals (Stanivuk et al. 2024, Awad & Awaad 2017). The immune system of fish is directly influenced by factors in the rearing environment. Disruption of the body’s homeostasis promotes a stress situation, leading physiological changes and rendering the animal susceptible to diseases (Urbinati et al. 2014). In this condition, the production of free radicals and/or reactive oxygen species (atoms or molecules highly reactive with cellular constituents) can increase, surpassing the natural antioxidant defense capacity of cells, which leads to the undesirable state of oxidative stress (Lushchak 2011). So, all these biochemical and physiological changes are capable of disturbing the immune system of fish, thereby favoring the development of diseases.

As a way of preventing and controlling pathogens, the use of antibiotics in intensive production systems has been adopted. However, these antimicrobials are responsible for several negative effects, including the development of resistance in zoonotic pathogenic bacterial strains (Acunha et al. 2023) accumulating residues in edible tissues and the environment can also compromise the effectiveness of antibiotics and weaken the immune system (Khanzadeh et al. 2024, Gufe et al. 2024, Hoseinifar et al. 2015). As a result, ecologically sound alternatives to the use of antibiotics are being sought, offering promising sustainability for disease prevention and /or control in aquaculture (Awad & Awaad 2017, Hoseinifar et al. 2015, Mohammadi et al. 2020, Yousefi et al. 2020).

In this context, bioactive compounds are considered the future of the aquaculture industry. Through preventive health management via feeding practices, aquatic animals can allocate more energy towards growth, reducing biological energy reserves required for combating diseases or stress resistance (Salomón et al. 2020).

Studies have revealed that biologically active peptides obtained from plant and animal protein sources demonstrate antioxidant, immunomodulatory, anti-inflammatory, growth-promoting, and antibacterial properties for humans (Lalani et al. 2024, Sarker 2022, Li-Chan 2015).

These functional bioactive compounds have shown satisfactory effects in the diet of aquatic animals, such as improving nutrient use efficiency by increasing the absorption of amino acids and minerals, promoting protein synthesis and digestive enzyme activity, consequently enhancing growth. This has been observed in specimens of turbot (Scophthalmus maximus) (Hao et al. 2020). In addition to benefiting fish growth, the inclusion of low molecular weight nitrogenous compounds also promotes several health benefits for the animal. For instance, the antioxidant properties of protein hydrolysates enable them to act against free radicals present in cells and minimize oxidation in macromolecules. In addition, other properties presented by protein and peptide hydrolysates include prevention of human diseases such as hypertension (Dai et al. 2013) antimicrobial, cytomodulatory, prebiotic, and hypocholesterolemic activity (Halim et al. 2016, Sarker 2022). In addition to presenting anti-inflammatory and inhibitory actions on enzymes such as lipase and angiotensin-converting enzyme (Jakubczyk et al. 2020), it also exhibits antioxidant potential (Riolo et al. 2023).

According to Samaranayaka & Li-Chan (2011), hydrolysis through the use of enzymes has been the primary method for obtaining peptides (molecules containing from 2 to 20 amino acids) from proteins. According to Quah et al. (2023), the bioactive peptide activity is influenced by the composition and sequence of amino acids. Various raw materials such as fish, milk, eggs, soy, wheat, fungi, marine macroalgae, among other protein sources, have been widely studied as potential ingredients for the production of peptides (Sarker 2022). Among the promising potential sources for obtaining peptides, insects such as Tenebrio molitor (Linnaeus 1758) deserve to be highlighted due to the abundance of this raw material, which is rich in proteins and has high levels of essential amino acids such as lysine. Considered the main limiting amino acid in agricultural by-products used in the manufacture of commercial feed, lysine plays a crucial role in ensuring optimal protein synthesis and growth in aquaculture species (Montes-Girao & Fracalossi 2006).

In aquaculture, mealworm larvae meal has already been included in the diet of various species such as gilthead seabream (Sparus aurata, Linnaeus 1758), sea bass (Dicentrarchus labrax, Linnaeus 1758), rainbow trout (Oncorhynchus mykiss, Walbaum 1792), African catfish (Clarias gariepinus, Burchell 1822), meagre (Argyrosomus regius), as a potential substitute for fish meal (Coutinho et al. 2021, Henry et al. 2015). Additionally, in diets for Nile tilapia (Oreochromis niloticus, Linnaeus 1758), grass carp (Ctenopharyngodon idellus), and mirror carp (Cyprinus carpio var. specularis), mealworm meal have been used to replace soybean meal (Li et al. 2022, 2023, Zhang et al. 2023). However, studies on bioactive peptides obtained from T. molitor are scarce.

In this context, the main aim of the present study was to apply methodologies for obtaining bioactive peptides from T. molitor and to evaluate the physicochemical properties, as well as the antioxidant and antimicrobial activities, with the intention of exploring their potential for use in fish diets.

MATERIALS AND METHODS

Cultivation and physicochemical characterization of T. molitor larvae

Tenebrio molitor larvae were cultivated at Animal Biodiversity Laboratory (Universidade Federal do Pampa, Uruguaiana City, Rio Grande do Sul state, Brazil) and fed a commercial poultry feed (Presence, Brazil) (Bordiean et al. 2022, Romero-Lorente et al. 2022) composed of ground corn, corn gluten meal, soybean meal and rice meal (proximate composition: moisture 10.64 ± 0.97, crude protein 19.96 ± 0.70%, fat 3.83 ± 0.13%, ash 10.28 ± 0.98%, acid detergent fiber 37.05 ± 1.39, and carbohydrates 18.68%). The larvae were cultivated until reaching 2 cm in size (± 80 days). T. molitor larvae were partially dried (55 °C for 48 h), ground and evaluated by proximate composition: dry matter (method 925.45b), ash (method 923.03), crude protein (method 960.52) (AOAC 1995), lipids (Bligh & Dyer 1959), and acid detergent fiber (Van Soest et al. 1991). Carbohydrates were obtained by difference using the formula: 100 - (moisture + crude protein + ash + lipids + acid detergent fiber).

Glycogen analysis

The glycogen levels of mealworm flour, defatted mealworm flour, and bioactive peptides were determined according to Bidinotto et al. (1997). Sample (50 mg) was homogenized with KOH and ethanol for hydrolysis and glycogen precipitation. The glucose was estimated using a hydrolytic method with phenol-sulfuric acid. The results were calculated based on the standard curve of glucose solution (50 to 200 μM) and expressed as μmol glucose g^-1 of sample.

Production of bioactive peptides

The larvae were ground to obtain flour. Then, the lipids were extracted with hexane (1:2, w:v) as described by Goulart et al. (2013). The defatted mealworm flour was homogenized and pre-incubated in distilled water (1:10, w:v) at 55 °C for 30 min. After pre-incubation, the following assays were conducted using Alcalase® (density 1.17 g mL^-1):

Assay 1: 0.04% enzyme for 3 h;

Assay 2: 0.08% enzyme for 3 h;

Assay 3: 0.04% enzyme for 4 h;

Assay 4: 0.08% enzyme for 4 h;

Assay 5: 0.04% enzyme for 8 h;

Assay 6: 0.08% enzyme for 8 h.

All tests were conducted at 55°C, and the pH was adjusted to 7.0 with NaOH 0.1 N. After the hydrolysis step, the enzyme was inactivated by heating (95 °C for 15 min), and the mixture was cooled to 20-25 °C in an ice bath. Hydrolyzed samples were centrifuged (9300 g/20 min) to separate the insoluble materials from soluble peptides. The supernatant was collected and stored at -20 °C until analysis. The methodology employed was described by Tang et al. (2018).

Degree of hydrolysis

After hydrolysis, 20% trichloroacetic acid (TCA) solution was added to the supernatant, and the mixture was centrifuged at 9300 g for 20 min. The nitrogen content of the fresh supernatant (10% TCA solution) was determined by the micro-Kjeldahl method, as described by Tang et al. (2018), with some modifications. The degree of hydrolysis was calculated as following:

Enzymatic hydrolysis (%) = (N (nitrogen) total of sample) / (N of 10% TCA solution) x 100

Total antioxidant capacity of bioactive peptides

The antioxidant capacity was evaluated by the Ferric Reduction Antioxidant Power (FRAP) assay as described by Pulido et al. (2000) with some modifications. Briefly, T. molitor peptides samples were diluted in distilled water (1:25 v:v). The FRAP reagent was prepared by combining 0.3 M acetate buffer, 10 mM TPTZ solution, and 20 mM ferric chloride aqueous solution (10:1:1 v:v). For the assay, 90 µL of diluted sample was transferred to test tubes, in a dark environment, followed by the addition of 270 µL of distilled water and 2.7 mL of the FRAP reagent. The tubes were mixed and incubated in a water bath at 37°C for 30 min. The absorbance was measured using a spectrophotometer at 595 nm. The results were calculated based on the standard curve of ferrous sulfate hexahydrate (500 to 2000 µM) and expressed as µmol Fe (II) equivalent g^-1 of sample.

Antimicrobial capacity of bioactive peptides

Initially, the inoculums of the following bacterial cultures were standardized: Pseudomonas sp., Staphylococcus aureus, Enterococcus faecalis, Salmonella sp., Klebsiella pneumoniae, Escherichia coli, and Proteus sp. The inoculums were spiked in PCA (Plate Count Agar) 24 h before testing. Bacterial suspensions were diluted in 10% saline solution, according to the 0.5 Mc Farland scale (approximately 1.0 x 10⁸ CFU mL^-1).

The Minimum Inhibitory Concentration (MIC) was determined using the broth microdilution method, which is defined as the lowest concentration of extract (mg mL^-1) capable of inhibiting microbial growth. A sequence of serial dilutions was prepared with the peptides from assay 5, as follows: 1:2; 1:5; 1:10; 1:50; 1:100 (v:v, peptides: Mueller Hinton broth). This assay was used due to its higher degree of hydrolysis and lower enzyme requirement. Subsequently, 50 μL of each dilution were added to 96-well microdilution plates, followed by 50 μL of bacterial inoculum. The plates were incubated at 37 ºC for 24 h. MIC values were assessed in quadruplicate (Clinical and Laboratory Standards Institute 2013).

Statistical analysis

The data were submitted to a normality test, followed by one-way analysis of variance (ANOVA). Means were compared by Tukey’s test (p<0.05).

RESULTS

Physicochemical characterization of T. molitor larvae

The physicochemical composition of the mealworm larvae used in this study is shown in Table I. The crude protein (CP) content was 45.17%, ash content was 3.22%, and fat content was 34.36%. After the fat extraction process, the CP content of mealworm flour increased by 38.4%. In contrast, the lipid content was reduced by 82.5%.

Table I
Physicochemical characterization of Tenebrio molitor larvae.

Parameter	Tenebrium flour	Defatted tenebrium flour
Crude protein (%)	45.17±1.50	62.51±3.20
Dry matter (%)	87.55±0.09	86.65±0.14
Ash (%)	3.22±0.24	4.53±0.03
Lipid (%)	34.36±0.68	6.02±1.17
Carbohydrates (%)	4.8	13.59

• Values expressed as mean ± standard deviation (n=3).

Glycogen levels in T. molitor

The glycogen levels in T. molitor larvae and bioactive peptides are shown in Figure 1. The results (μmol glucose g^-1 of sample) were numerically higher for the deffated mealworm flour (177.04) and bioactive peptides (152.81) compared to the larvae sample (144.08).

Figure 1
Glycogen levels of T. molitor larvae and bioactive peptides.

Degree of hydrolysis of bioactive peptides

Table II shows the degree of hydrolysis for each assay used for obtaining the bioactive peptides. Tests 1 to 4, with shorter hydrolysis time (3-4h), exhibited significantly lower degrees of hydrolysis compared to tests 5 and 6.

Table II
Degree of enzymatic hydrolysis of defatted Tenebrio molitor with Alcalase®.

Sample	Enzyme (%)	Incubation time (h)	Degree of hydrolysis (%)
Assay 1	0.04	3	52.68±0.15a
Assay 2	0.08	3	62.43±0.03b
Assay 3	0.04	4	63.60±0.30c
Assay 4	0.08	4	68.09±0.10d
Assay 5	0.04	8	72.13±0.13e
Assay 6	0.08	8	74.28±0.14f

• Lower case letters indicate statistical differences among tests by Tukey’s test (p<0.05).

Total antioxidant capacity of bioactive peptides

The results related to the total antioxidant capacity of the bioactive peptides measured by the Ferric Reduction Antioxidant Power – FRAP – assay, are shown in Table III. The bioactive peptides obtained in assays 1, 3, and 4 have significantly higher antioxidant potential compared to those obtained in assays 2, 5 and 6.

Table III
Total antioxidant capacity of bioactive peptides from Tenebrio molitor.

Sample	Enzyme (%)	Incubation time (h)	Antioxidant capacity (FRAP)*
Assay 1	0.04	3	276.41±6.41b
Assay 2	0.08	3	275.13±8.00b
Assay 3	0.04	4	265.15±7.00b
Assay 4	0.08	4	235.09±6.00a
Assay 5	0.04	8	237.31±7.00a
Assay 6	0.08	8	239.89±0.89a

• ]Lower case letters indicate statistical differences among tests by Tukey’s test (p<0.05). *Ferric Reduction Antioxidant Power (µmol Fe (II) equivalent/g of sample).

Antimicrobial capacity of bioactive peptides

Figures 2 and 3 demonstrate the effects of the bioactive peptides on the growth of various bacteria. As shown, the bioactive peptides did not exhibit efficacy in inhibiting the growth of the evaluated microorganisms. Bacterial growth was observed in all tested dilutions.

Figure 2
Growth of Pseudomonas sp., Staphylococcus aureus, Enterococcus faecalis, and Salmonella sp. in broth with serial dilution of T. molitor peptides.

Figure 3
Growth of Klebsiella pneumoniae, Escherichia coli, and Proteus sp. in broth with serial dilution of T. molitor peptides.

DISCUSSION

Physicochemical characterization of mealworm flour and defatted mealworm flour demonstrated that the chemical process used to extract fat was efficient, leading to a concentration of the protein content. In a review by Hong et al. (2020), it was demonstrated that T. molitor larvae contain between 47.0 and 60.2% crude protein and 22.87 to 37.70% fat. It is suggested that these values may vary depending on the insect diet. The levels observed in our study are similar to those reported by Hong et al. (2020), which confirms the hypothesis that these larvae are excellent sources of protein and fat, as highlighted by previous studies.

Regarding the composition of defatted mealworm flour, the values found for crude protein (62.51%) are similar to those observed by Tran et al. (2022) (71.1%) and Basto et al. (2023) (71.0%). Furthermore, it is important to highlight that these values are comparable to those of fish meal (67.5%) and higher than levels of soybean meal (on average 49.4%) (Hong et al. 2020). As a result, it is suggested that defatted mealworm flour is a highly sustainable source of protein and an alternative to conventional protein ingredients used in aquaculture, such as fish and soybean meal. Similar values were observed for dry matter and ash contents in raw and defatted flour, demonstrating that the process of removing fat does not affect the mineral content of the ingredient.

Related to glycogen concentration, samples of bioactive peptides showed higher values than T. molitor larvae. In insects, glycogen is associated with a non-reducing disaccharide composed of two glucose units known as trehalose. This compound is found in the hemolymph of insects and serves as a source of energy, particularly during muscle contraction, such as during flight. The synthesis of trehalose in insects occurs in the fat body, where it is also stored for utilization by the hemolymph when required (Lopes & Villela 1972). Some authors suggest that trehalose protects cells during stressful conditions, such as high temperatures, osmotic shock, ethanol toxicity, and dehydration (Alcarde & Basso 1997).

Alcarde & Basso (1997) observed an increase in the trehalose content of yeasts with the application of thermal treatment (increase in temperature). These results corroborate with our findings, as higher levels of glycogen were observed in bioactive peptides that underwent heat treatment at the end of the hydrolysis process to inactivate the enzyme. Furthermore, it is suggested that trehalose may interfere with the drying process of peptides obtained from T. molitor, as observed in our study. Initially, we attempted to use lyophilized samples. However, through this drying method (results not included), we did not obtain viable samples suitable for study or use in fish feed, due to its viscous and adherent appearance. This behavior can be attributed to the high levels of glycogen (trehalose) present in both the raw samples and peptides. Thus, we prioritized studying the wet sample instead of the dry one, which will consequently facilitate the homogenization of the peptides in fish feed.

In the present study, the hydrolysis time and the concentration of Alcalase enzyme influenced the degree of hydrolysis. Consistent with our findings, Centenaro et al. (2009) also observed an increase in the degree of hydrolysis with higher concentrations of Alcalase. According to Kristinsson & Rasco (2000), the increase in protease concentration is associated with a corresponding increase in the degree of hydrolysis. However, the authors highlighted that the greater the amount of enzyme used, the higher the cost of obtaining the peptides. The degree of hydrolysis in defatted mealworm flour, using Alcalase, ranged from 52.68 ± 0.15% to 74.28 ± 0.14%, higher than those values found for protein hydrolysis in other products previously studied, such as chicken leg (18.62 to 38.79%), chicken breast (20.93% to 57.42%) (Schmidt & Salas-Mellado 2009), and croaker (12.2 ±0.11 to 43.7 ±0.33%) (Centenaro et al. 2009). Despite this, the study of obtaining bioactive peptides from insects is relatively recent (Rivero-Pino et al. 2020). Further research is necessary to comprehend the biology of mealworm and, consequently, efficiently explore and optimize the production of peptides.

Protein hydrolysis can be performed through various methods, including the use of acids, alkalis, or enzymes. Among the procedures, enzymatic hydrolysis has been widely recommended due to several advantages, such as the modification of functional, physicochemical, and sensory properties of the original proteins. In addition, enzymatic hydrolysis allows greater control over the process and enables the retention of beneficial properties in the resulting product (Schmidt & Salas-Mellado 2009). According to Fennema et al. (2018), the functional properties of protein hydrolysates are influenced by the type of enzyme used in their production. In this sense, the Alcalase derived from Bacillus licheniformis is the main commercial enzyme used and recommended for the production of protein hydrolysates due to its non-specific specificity and efficiency (Schmidt & Salas-Mellado 2009). The study carried out by Tang et al. (2018), which evaluated the enzymatic hydrolysis of T. molitor protein using different enzymes (Alcalase and Flavourzyme) and enzymatic combinations, found that the highest protein hydrolysis yield was achieved with Alcalase, ranging from 10.0% to 38.7%. Based on these findings, it is suggested that further studies could be conducted to explore the combination of different physico-enzymatic methods to enhance the degree of hydrolysis of T. molitor protein while minimizing hydrolysis time and enzyme concentration.

The total antioxidant capacity of the bioactive peptides varied among the tests applied. Higher values were found after applying shorter hydrolysis time. In contrast, a study carried out by Wu et al. (2003) reported that hydrolysates with greater antioxidant activity were obtained with longer durations of hydrolysis. The authors suggest that a longer duration of enzyme-substrate contact leads to greater antioxidant capacity, which contradicts the findings of our study. In the same point, Tang et al. (2018) also observed high antioxidant capacity of bioactive peptides obtained from T. molitor using Alcalase.

Several methodologies have been developed and applied with the aim of evaluating the antioxidant capacity of different ingredients. Due to the diversity of techniques and the absence of a universal method capable of measuring the antioxidant capacity, it is difficult to make numerical comparisons between the results obtained in different studies (Morais et al. 2013). However, when compared with cerrado fruits, our results (µM ferrous sulfate/g) were higher than those reported for Solanum lycocarpum seed (fruta-do-lobo) (167.11±0.219), Byrsonima verbascifolia pulp (murici) (148.42±0.047), and Cipocereus minensis peduncle (quiabo da lapa) (151.67±0.043) (Morais et al. 2013). Therefore, regardless of the values found in the different tests, all hydrolysates obtained in the present study demonstrated antioxidant capacity. As potential sources for inclusion in fish diets, exerting an effective protective action against oxidative processes occuring in the animal’s organism and aiding in disease prevention. Antioxidant molecules have the ability to decrease the rate of oxidation by inhibiting free radicals or removing metals that damage cells (Kabel 2014). According to Chi et al. (2014), peptides with lower average molecular weight, particularly those consisting of shorter and more active peptides, act as electron donors and react with free radicals, transforming them into more stable substances that interrupt chain reactions.

Regarding the antimicrobial capacity of the bioactive peptides, any effective action was observed to inhibit the growth of the evaluated bacteria. Our results contradict studies that emphasize the antimicrobial potential of insect peptides, including those derived from mealworm larvae (Azmiera et al. 2022). The findings presented here are relevant, especially considering that there is only one study (Chae et al. 2012) focusing on evaluating the antimicrobial capacity of peptides obtained from mealworm has been identified, as reported in the review by Azmiera et al. (2022). Despite the absence of direct in vitro inhibitory effects on pathogenic microorganisms, peptides may still confer indirect positive effects. When animals receive these additives through the diet, improvements in the immune system may occur, leading to an enhanced ability to combat pathogens.

CONCLUSIONS

The hydrolysis process applied to defatted mealworm flour, using Alcalase, exhibited effectiveness particularly with longer enzymatic treatment periods. The obtained peptides demonstrated bioactive potential, as evidenced by the high antioxidant capacity observed across all enzymatic treatments tested. Additionally, this study presents promising and innovative findings that allow for further exploration as alternative sources to be used in aquaculture in future research.

ACKNOWLEDGMENTS

The authors would like to thank the Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS) for supporting the project (protocol 21/2551-0000605-6) and provision of a scientific initiation scholarship for Jéssica Cristina Verus Villanova.

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Publication Dates

• Publication in this collection
  23 Sept 2024
• Date of issue
  2024

History

• Received
  19 Dec 2023
• Accepted
  15 June 2024