Effects of Selenite and Selenate on the Growth, Nutrient Composition, Selenium Species, and In Vitro Digestibility of Mealworm Tenebrio molitor

TL;DR

This study shows that mealworms can convert toxic inorganic selenium into a bioavailable organic form, making them a potential source of healthy selenium-rich food.

Contribution

The study reveals mealworms' efficient biotransformation of inorganic selenium into organic forms, particularly selenocystine, with high bioaccessibility.

Findings

Mealworms convert inorganic selenium into organic forms, with selenocystine as the main product.

Selenate (Se6+) is more efficiently transformed and yields higher bioaccessible selenium than selenite (Se4+).

High selenium concentrations inhibit mealworm growth and fat accumulation but boost protein and polysaccharide synthesis.

Abstract

The yellow mealworm is frequently regarded as a promising, sustainable, high-quality protein-containing foodstuff that can be consumed by humans. Selenium has been demonstrated to be an essential micronutrient for humans and animals, as it plays a crucial role in promoting physical health. Nevertheless, it is important to note that inorganic selenium is toxic to organisms at elevated concentrations. Furthermore, there is a paucity of information regarding the changes in nutritional components, selenium enrichment, and the biotransformation efficiency of the worms. The present study employed two types of inorganic selenium (selenite and selenate) to address the aforementioned issues, with the purpose of selenium-enriched mealworm cultivation and utilisation. Inhibited larval biomass was observed in the presence of elevated concentrations of inorganic selenium, with both Se forms stimulating crude protein and polysaccharide synthesis. A dose-dependent Se accumulation pattern and limited bioaccumulation capacity were identified in mealworms. However, a remarkable capacity for Se biotransformation was also observed, yielding a higher proportion of organic Se in mealworms, and selenocystine was identified as the predominant compound. The in vitro gastrointestinal digestion test demonstrated a high bioaccessibility of mealworm-derived Se.

This study systematically compared the growth performance, nutrient composition, accumulation and speciation of selenium (Se), and in vitro bioaccessibility in yellow mealworm (Tenebrio molitor L.) larvae, which were reared on substrates supplemented with selenite (Se4+) and selenate (Se6+) at concentrations of 0, 5, 10, and 20 mg/kg over 28 days. The results showed that high Se concentrations (≥10 mg/kg) significantly reduced larval biomass, with Se6+ having a slightly stronger inhibitory effect than Se4+. The mealworms effectively accumulated Se in a dose- and form-dependent manner. Peak total Se concentrations were observed on day 14, after which there was a decline, suggesting the presence of potential elimination mechanisms, such as moulting. The bioaccumulation factors (BAFs) were all below 1, indicating its limited enrichment capacity for both Se4+ and Se6+. Nutrient composition was altered, with both Se forms stimulating crude protein and polysaccharide synthesis while inhibiting fat accumulation. Mineral content (Mg, Fe, Zn) was also modulated, with differences observed between the Se4+ and Se6+ treatments. Notably, mealworms exhibited a remarkable ability to biotransform inorganic Se into organic forms, with organic Se proportions exceeding 79% in all treatments. Selenate was more efficiently bio-converted, yielding a higher proportion of organic Se. In vitro gastrointestinal digestion revealed significantly higher Se bioaccessibility from Se6+-treated mealworms (up to 85.12%) than from Se4+-treated ones (up to 60.67%). Analysis of the bioaccessible fraction by Se speciation identified SeCys2 as the dominant compound (>92% of the detected species), with much lower levels of SeMet. Trace amounts of unmetabolised Se6+ were only detected in the Se6+-exposed groups. These findings highlight T. molitor as an efficient bioreactor for producing bioaccessible, organically bound Se, primarily as SeCys2, with Se6+ being the more favourable precursor for generating a high-quality, bioavailable source of Se for potential use in feed or food.

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1. Introduction

In the context of economic globalisation, there has been concomitant strengthening of public health awareness, resulting in an increasing demand for high-quality nutrition. Selenium (Se) is an essential micronutrient for humans and animals. It plays crucial roles in promoting physical health by acting as an antioxidant, aiding immune function, and regulating thyroid metabolism [1,2]. However, the supplementation of Se is limited by several factors. Firstly, Se deficiency is prevalent in most environments. Secondly, Se is unevenly distributed. Thirdly, there is a reduction in food chain transmission. Consequently, approximately 1 billion people worldwide are suffering from the health threat of Se deficiency [3,4]. It is evident that traditional Se supplements, such as inorganic sodium selenite and selenate, are associated with limitations. These limitations include narrow safety margins and lower bioavailability when compared to organic Se forms, such as selenomethionine (SeMet) and selenocystine (SeCys_2_) [5]. The transformation of inorganic Se into organic forms through biological processes represents a promising approach to enhance Se bioavailability and reduce toxicity [6]. The artificial cultivation of Se-enriched foods by adding exogenous inorganic Se fertiliser and then accumulating and biotransforming Se using the biofortification characteristics of organisms is one of the main ways to provide organic Se components with low toxicity, such as Se-containing peptides, proteins, and polysaccharides [7]. In recent decades, there has been a notable increase in the presence of foods such as corn, vegetables, fruits, and meats that are rich in organic Se in shopping malls and on people’s dining tables [6,8,9]. This development provides individuals with a high-quality and healthy source of Se, which represents a significant advancement in Se nutrition.

Notwithstanding the importance of Se nutrition, the expending global population has concomitantly engendered an augmented requirement for high-quality protein-containing foods. Conventional livestock and poultry farming is associated with a number of significant drawbacks, including elevated energy consumption, substantial land area utilisation, considerable water utilisation, and greenhouse gas emissions [10]. These drawbacks have been demonstrated to result in deleterious environmental consequences, and therefore there is an imperative for the exploration of long-term solutions.

The present study explores the nutritional significance of insects and worms, and their potential as a sustainable alternative to conventional protein sources. It is well established that these organisms are distinguished by their elevated protein, fat, and micronutrient composition. They are regarded as a cost-effective and environmentally sustainable nutritional source. The consumption of insects has been documented in the diets of some of the world’s oldest civilisations, including those in Latin America, Africa, and Asia, with an estimated global consumption of approximately two billion people [11].

Tenebrio molitor, more commonly referred to as the yellow mealworm, is an insect that has undergone the processes of decortication and metamorphosis. It has been demonstrated that the larvae stage of this species constitutes a substantial source of protein and lipid [12,13,14], and it has been identified as a potential ingredient in both animal and human diets [15,16]. In China, mealworms are consumed as a food source and incorporated into a bio-regenerative life support system in space. In this capacity, they are employed as an efficient method for the treatment of plant waste and the provision of protein for astronauts [13]. Furthermore, this alternative nutrient has garnered increased attention due to its short growth and life cycle, uncomplicated feeding operations, and minimal technical requirements. Mealworms have been found to be capable of consuming a variety of agricultural waste materials, including wheat and rice bran, tail vegetables, and kitchen garbage. These organisms have been observed to efficiently convert such waste into nutrients that meet the requirements for toxicological and microbiological safety. This conversion process renders the mealworms suitable for consumption as safe food products [17,18]. It is noteworthy that the product has been commercialised on a large scale. It is imperative that the industrial farming process of this edible insect is conducted in accordance with the prevailing energetic and environmental constraints, including the generation of an environmentally acceptable carbon footprint [19,20].

The utilisation of edible insects and worms in Se biofortification and supplementation for humans or animals has also been proposed [21]. For instance, the black soldier fly (Hermetia illucens) [22], maggot (Chrysomya megacephala) [23], earthworm (Eisenia fetida) [24], and mealworm (Tenebrio molitor) [25] have been used as Se biofortification materials and have produced encouraging results in terms of biomass, Se accumulation, and organic Se content. The ingestion of edible insects that have been enriched with Se may provide a perfect solution for the supplementation of Se and protein nutrition. Nevertheless, insects remain the most unexplored source of Se-containing functional foods and nutraceuticals [26]. The paucity of research in this field is due to factors such as the tolerance, enrichment, and organic Se transformation ability of insects, in different species and growth stages, to Se has not been systematically investigated, and the effects of inorganic Se on the growth and reproduction of insects have not been researched sufficiently.

The present study has been conducted for the purpose of investigating the possible value of mealworms as a source of bioenhancer for Se. In summary: (1) Sodium selenite- and sodium selenate-containing substrates were utilised to breed T. molitor, with a view to investigating the effects of two inorganic Se on worm growth, Se accumulation, and transformation. (2) Investigate the effects of inorganic Se supplements on the nutrient composition of mealworm. (3) Detect the organic Se contents and forms by HPLC-ICP-MS, and analyse the bioaccessibility of mealworm-derived Se using an in vitro simulated digestion test. The present study provides the data and technical basis for the cultivation, acquisition, and utilisation of Se-enriched edible insects with high nutritional quality. Furthermore, it can also provide instructions for the development of other high-protein foods containing Se.

2. Materials and Methods

2.1. Yellow Mealworm and Test Substance

Mealworm larvae, wheat bran, and rice bran utilised in the experiment were obtained from a local market in Dezhou, China. Prior to the initiation of the experimental phase, all energetic mealworms were subjected to a pre-incubation process in a two-bran (1:1 in dry weight) substance under experimental conditions for a period of three days. The mean weight of the mealworms at the commencement of the experiment was approximately 4.00 g per 100 individuals (see Table S1 for details of the initial weight of the mealworms).

The clean wheat and rice bran samples were subjected to a sieving process (4 mm), followed by oven drying, and then mixed at a ratio of 1:1 (w:w). The substance was then fortified with sodium selenite (Na_2_SeO_3_·5 H_2_O, Xiya Reagent, Linyi, Shandong, China) and sodium selenate (Na_2_SeO_4_, Sigma-Aldrich, Milwaukee, WI, USA) solution (5 mg/mL) at concentrations of 5, 10, and 20 mg Se/kg dry weight (dw), referred to as Se5, Se10, and Se20 treatments, respectively. The substance that had not undergone any treatment was designated as the control group (CK).

2.2. Experiment Process

Following to the attainment of equilibrium over a period of one week, the prepared substances were distributed into circular plastic boxes with a diameter of 20 cm at the upper extremity, 15 cm at the lower extremity, and a height of 12 cm. The substances were then utilised as sustenance (30 g per box) for mealworms. Approximately 20 g (500 individuals) was introduced to each box (three replicates for each treatment), and the experiment was maintained for 28 days in an illumination incubator at an appropriate temperature of 26 ± 2 °C, with 65% humidity and a photoperiod of 12/12 h (dim light in 150 lx/dark). Furthermore, fresh carrot was administered to mealworms on a biweekly basis (10 g each time).

Approximately 3 g of fresh mealworms was sampled at random on days 7, 14, 21, and 28. The selected worms were subjected to a 24 h depuration process, after which they were thoroughly washed using deionized water. The weight of the subjects was recorded in a fresh state (FW), and subsequently, they underwent a freeze-drying procedure for subsequent analysis.

2.3. Detection of Total and Organic Se Concentrations

The pretreatment of samples, and the subsequent determination of total Se concentrations in wheat bran, rice bran, and mealworms (in dry weight, dw), were conducted in strict accordance with the method outlined in Yue et al. [24].

The presence of organic Se in mealworms cultivated with two inorganic Se forms was detected in accordance with the method described by Peng et al. [23]. The procedure entailed the amalgamation of 1.0000 g of freeze-dried worm sample with 30 mL of ultrapure water within a flask, followed by a thorough mixing process. Subsequent to a 30 min sonication period at 37 °C, the resultant mixture was subjected to centrifugation at 3000× g for a duration of 15 min, resulting in the collection of the upper layer. Following the addition of 2.5 mL of 6 mol/L HCl and 10% (w/w) potassium ferricyanide, the water phase was collected in a centrifuge tube after a duration of 20 min. This inorganic Se-containing solution was then diluted to 25 mL with ultrapure water and determined using an inductively coupled plasma optical emission spectrometer (ICP-OES) (iCAP PRO, Thermo Fisher Scientific, Waltham, MA, USA). The organic Se concentration was calculated by the subtraction method from the total Se quantity minus the inorganic Se content. The reference standard material pork liver powder (GBW 10051) [27] was utilised and evaluated in comparison to certified values, and the Se concentrations measured in the reference material were found to be within 91–112% of the certified concentrations. The concentrations of Se in the samples were investigated using ICP-OES.

The bioaccumulation factor value of Se (BAF_Se_) is calculated using the following formula:

$$[eqn]$$

where C_1_ denotes the Se concentration in the worm, and C_2_ denotes the Se concentration in the substrates.

2.4. Nutrient Components and Elements Analysis

The contents of crude protein, crude fat, and crude polysaccharide were determined by Kjeldahl nitrogen determination, Soxhlet extraction, and the phenol-sulfuric acid method, respectively.

2.4.1. Crude Protein

Following a drying process at 105 °C for a duration of 2 h, 0.5000 g of mealworm powder was introduced into the digestion tube. Subsequently, 3.5 g of K_2_SO_4_, 0.4 g of CuSO_4_·5H_2_O, and 12 mL of H_2_SO_4_ were added to the sample and thoroughly mixed. The initial temperature setting for the cooking furnace was initially set at 150 °C, with a duration of 30 min. Subsequently, the temperature was elevated to 420 °C for a duration of 60 min. Following the cooling period, the descaling tube was installed on the Kjeldahl nitrogen analyzer (QSY-1, Hua Rui Bo Yuan GmbH, Beijing, China), and 2 drops of bromocresol green-methyl red indicator were added into a 250 mL conical bottle for distillation. The distillate was then titrated with 0.05 mM HCl. In the blank group, 0.01 g sucrose was utilised in lieu of a mealworm sample for the aforementioned determination, with each sample being replicated thrice. The calculation of crude protein percentage is derived as follows:

$$[eqn]$$

where 0.05 denotes the molar concentration of HCl, V_1_ denotes the average volume of HCl utilised for titrating the sample, V_2_ is the average volume of HCl consumed for titrating the blank solution, W is the weight of the sample (g), 14 is the relative atomic mass of nitrogen, and 6.25 is the conversion coefficient.

2.4.2. Crude Fat

Mealworm powder was subjected to a drying process at a temperature of 105 °C for 2 h, then accurately weighed (5.0000 g) and transferred into a filter paper cylinder that had been previously lined with degreasing cotton. The extraction of the crude fat from the sample was conducted using the Soxhlet extraction method. Simply, the extraction agent, petroleum ether (boiling point 30–60 °C), was added to two-thirds of the volume of the receiving bottle and heated in the electric heating jacket. The extraction agent was returned at 20 min intervals, with each sample undergoing extraction for a period of 5 h. Subsequent to extraction, the filter paper cylinder was dried at 70 °C for a constant weigh. The crude fat yield was calculated by the following formula:

$$[eqn]$$

where W_1_ is the weight of the raw material, and W_2_ is the weight of the sample after extraction.

2.4.3. Crude Polysaccharide

The dried mealworm powder was weighted (0.2000 g) and mixed with 4 mL of ultrapure water. The mixture was then extracted for 1 h at 80 °C using ultrasonic assisted at 100 Hz. The resultant supernatant was collected by means of centrifugation at 8000× g for 20 min at room temperature. This extraction process was repeated once more to incorporate the supernatants. Anhydrous ethanol was added to the upper layer (4:1, v:v) and the mixture was deposited at 4 °C overnight, then subjected to a centrifugal process at 8000× g for 30 min (4 °C). The precipitate was thoroughly dissolved in 10 mL of ultrapure water to yield a crude polysaccharide solution of mealworm.

The crude polysaccharide content of mealworm was determined by phenol–sulfuric acid assay using a UV-VIS spectrophotometer (UV-5100, Shanghai Yuanxi GmbH, Shanghai, China) at OD_490_, as described by Sangthong et al. [28], with D-(+)-glucose (BW4009) serving as the standard. The procedure entailed the incorporation of 1 mL of crude polysaccharide solution into 1 mL of a 5% phenol solution. Subsequently, 5 mL of H_2_SO_4_ was then introduced and thoroughly amalgamated. The mixture was then placed at 25 °C for 20 min. Subsequent to this, the solution was determined by UV-VIS spectrophotometer at OD_490_, and the solubility of crude polysaccharide of each treatment was calculated according to the glucose standard solution curve.

2.4.4. Mineral Elements

As outlined in Section 2.3, the presence of mineral elements, including Mg, Zn, Fe, Cu, and Mn, was detected in mealworms.

2.5. In Vitro Digestibility and Se Species Evaluation

The in vitro gastrointestinal digestion model reported by Li et al. [29], with minor modification, was utilised to evaluate the bioaccessibility of mealworm-derived Se. In summary, 0.5000 g of desiccated worm power was meticulously measured and subsequently immersed in 1 mL of simulated salivary fluid (SSF, pH 7.0) for 2 min at 37 °C. Thereafter, 1 mL of simulated gastric fluid (SGF, pH 3.0) was added to the mixture and maintained for 2 h at 37 °C, with continuous stirring at 180 rpm in the dark environment. Finally, 2 mL of simulated intestinal fluid (SIF, pH 7.0) was introduced into the system and maintained at a stirring rate of 180 rpm for a further 2 h at 37 °C. Subsequently, the SSF-SGF and SSF-SGF-SIF extracts were subjected to centrifugation, and the resultant hydrolysates were filtered through 0.22 μm cellulose acetate syringe filters for the purpose of bioaccessible Se concentration detection using ICP-OES. Meanwhile, the Se species present in the SSF-SGF-SIF digests were examined by high-performance liquid chromatography (HPLC, Agilent 1260, Wilmington, DE, USA) coupled with inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700x, Santa Clara, CA, USA) (see Table S2 for details of the operating conditions of HPLC-ICP-MS).

The utilisation of five Se species standard solutions (Se^4+^, Se^6+^, SeCys_2_, MeSeCys, and SeMet, purchased from the National Institute of Metrology, Beijing, China) was undertaken with the objective of ensuring the quality of the process. In order to calculate the bioaccessibility/digestibility of Se in mealworms, the Se content of hydrolysates was divided by the total Se concentration of the mealworms.

2.6. Statistical Analysis

Statistical analyses were accomplished using SPSS (Version 24.0, IBM). Origin 2018 was utilised in the creation of the plots. The assessment of significant differences between values of the same parameters was conducted through the implementation of a one-way ANOVA. The results are presented as mean ± standard deviation, and p < 0.05 is used to designate the statistical significance between the different groups.

3. Results

3.1. Biomass of Mealworm

The fresh weight and biomass increase rate of mealworm in different treatments and sampling times is demonstrated in Figure 1. A positive correlation was observed between the duration of the culture and the increase in mealworm biomass, indicating a time-dependent effect. The maximum observed biomass was recorded as 12.63 g/100 individuals in the CK group on day 28, while the peak values in Se5, Se10, and Se20 groups were 12.17, 9.04, and 8.27 g/100 individuals for Se^4+^ treatment (Figure 1A), and 10.55, 9.28, and 8.63 g/100 individuals for Se^6+^ treatment (Figure 1C), respectively. It was evident that the mealworm biomass and the rate of biomass increase (Figure 1B,D) were both suppressed by high substrate Se addition, irrespective of the form of Se. The inhibitory effect was observed by day 14, as the mass of mealworms exposed to a high Se concentration substrate (20 mg/kg) was significantly lower than that of the low concentration of Se-treated group (CK and Se5, p < 0.05). Treatment with Se5 had no adverse effect on the rate of increase in worm biomass over the first 21 days in comparison with the CK. However, an extension of the exposure period by a further week resulted in a limitation of biomass gain, and a significant difference was observed in the Se^6+^ treatment (Se5 vs. CK).

3.2. Total Se Concentration and BAFSe

As demonstrated in Table S1, the concentrations of Se in wheat bran and rice bran, as well as the various Se-treated substrates, were examined. During the course of the experiment, the mortality rate of mealworms was less than 3% across all groups, and moulting was observed in all treatments. As can be seen in Figure 2A,C, at equivalent sampling times, worm Se concentrations were increased in proportion to the substrate Se content in all Se^4+^ and Se^6+^ treatments. The total Se concentrations in each treatment generally increased for the first 14 d and then decreased for the following 14 d. The maximum Se contents were 1.77 mg/kg (Se5, 14 d), 2.87 mg/kg (Se10, 7 d), and 4.47 mg/kg (Se20, 14 d) in Se^4+^ treatments, while the values were 1.48 mg/kg (Se5), 3.67 mg/kg (Se10), and 6.94 mg/kg (Se20) in Se^6+^ groups at 14 d. These results demonstrated that mealworms exhibited a greater propensity to accumulate Se in the Se^6+^ treatment in comparison to the Se^4+^ treatment, particularly when the Se concentration exceeded 10 mg/kg over a duration of 14 d. A notable observation was the decline in worm Se concentration beginning on day 21, which culminated in a nadir on day 28. This phenomenon may be explained by the elimination of Se through moulting, which could be an efficient method of avoiding a negative impact on itself.

The results of the BAF_Se_ analysis in different treatments are shown in Figure 2B (Se^4+^) and Figure 2D (Se^6+^). The most significant values observed in the Se^4+^ treatments were 0.35 (14 d), 0.29 (7 d), and 0.22 (14 d) for Se5, Se10, and Se20, respectively. When Se^6+^-treated was administered on day 14, the values recorded were 0.3, 0.37, and 0.35 for Se5, Se10, and Se20, respectively. It is evident that the BAF_Se_ values in both Se^4+^ and Se^6+^ groups exhibited an initial increase at 14 d, followed by a continuous decline with the passage of time, with the exception of Se10 in the Se^4+^ treatment. This phenomenon can be attributed to the rapid weight gain of mealworms coinciding with substantial Se-containing food intake during the initial phase of the experiment, while a gradual increase in biomass and Se-containing food intake occurred in the subsequent phase. The BAF_Se_ were found to be less than 1 in all treatments during the experiment. This finding indicates that mealworms possess a limited capacity for bioaccumulation of Se. Furthermore, the capacity of mealworms to bioaccumulate Se can be inhibited by prolonged exposure duration.

3.3. Nutrient Analysis

As demonstrated in Table 1, the addition of selenite and selenate resulted in an enhancement of crude protein percentage in mealworm tissues at 28 d, and exhibited a roughly dose-dependent response. Specifically, the crude protein content of the Se10 and Se20 treatments treated with Se^4+^ increased significantly by 9.89% and 17.24%, respectively, compared to the CK group (p < 0.05). The highest value of 56.10% was observed in Se20 (Se^4+^), which was appreciably higher than the other treatments. The content of crude fat decreased gradually with increasing Se content in the substrate. A statistically significant difference (p < 0.05) was identified when the substrate Se concentration exceeded 10 mg/kg, resulting in a 42.80%, 52.98%, 24.52% and 59.20% decrease in the ratio in groups Se10 (Se^4+^), Se20 (Se^4+^), Se10 (Se^6+^), and Se20 (Se^6+^), respectively, compared to the CK group. It is interesting to note that both the supplementation of Se^4+^ and Se^6+^ led to an augmentation in the proportion of crude polysaccharide in mealworm, with all treatment groups exhibiting a significant increase in comparison to the CK group (p < 0.05), with the exception of Se5 in Se^6+^ supplementation. The study revealed that there was no discernible difference in the outcomes of the three Se^4+^ treatments, nor in those of the three Se^6+^ treatments. The crude polysaccharide proportion in the Se20 group (Se^4+^) was found to be 16.81%, which is 1.84-fold greater than the proportion observed in the CK group (9.12%). The other treatment groups exhibited levels ranging from 1.32- to 1.82-fold higher than those observed in the CK group. In conclusion, both Se^4+^ and Se^6+^ could stimulate protein and polysaccharide synthesis but inhibit crude fat production.

The quantities of five mineral elements (Mg, Zn, Fe, Cu, and Mn) in the mealworm tissues are presented in Table 1. The incorporation of Se resulted in elevated Mg concentrations, with a dose-dependent effect observed in Se^4+^ treatments. The maximum recorded value was 3031.49 mg/kg in the Se20 group, which is 27.96% greater than the CK group. However, no significant differences were detected in the Se-treated groups. In addition, the incorporation of Se resulted in a substantial augmentation of Fe content in the tissues of the worms. The highest recorded values were 72.96 mg/kg (Se^4+^) and 77.33 mg/kg (Se^6+^) in Se20 treatments, respectively. A non-significant disparity in worm Zn concentration was observed between the CK and the Se-treated groups, with concentrations ranging from 109.13 to 124.33 mg/kg. However, a significant 13.93% increase was observed in Se20 compared to Se10 treatment (Se^4+^).

In comparison with the CK group, the concentrations of Cu and Mn in the mealworm tissues were found to be reduced by the addition of Se^4+^, and the maximum inhibition rates were recorded as 26.36% and 19.68% in the Se20 group, respectively. However, when supported by Se^6+^, the concentrations of Cu were also inhibited in all groups, while the promoting effect was found for Mn. Altered concentrations of two kinds of Se have been shown to exert different effects on the accumulation of five mineral elements in mealworms, which may be attributed to the influence of Se on the metabolism of mealworms.

3.4. Organic Se Concentration

The concentrations and proportions of organic Se in mealworms treated with Se^4+^ and Se^6+^ at the end of the test were detected as presented in Figure 3. The organic Se concentration of mealworm in CK was found to be 0.20 mg/kg, equivalent to the total content, suggesting that the Se was present exclusively in organic form. As the substrate Se content was elevated, an increased level of organic Se concentration was exhibited by the three Se-treated groups. The highest values, 2.72 mg/kg (Se^4+^) and 3.87 mg/kg (Se^6+^), were observed in Se20 treatments, respectively, and significant differences (p < 0.05) were found between them. The organic Se content in the Se5 and Se10 groups treated with two Se forms ranged from 0.90 to 1.81 mg/kg, and no significant differences were observed between the two treatments with the same amount of Se supplementation in the substrate.

The organic Se percentage of mealworm in the CK group was 100%, which was higher than the values in the other three Se^4+^ treatments, i.e., 92.54%, 88.53%, and 79.72% in Se5, Se10, and Se20 treatments, respectively. Furthermore, the proportion of organic Se was also suppressed by high Se concentrations in selenate-containing substrates, with values of 100%, 96.02%, and 83.18% being recorded in Se5, Se10, and Se20 treatments, respectively. Furthermore, substantial disparities were identified among the CK, Se10, and Se20 groups when subjected to treatment by Se^4+^, and between the CK and Se20 (Se^6+^) groups. It is noteworthy that the proportion of organic Se in the worm samples exposed to Se^6+^ was higher than that exposed to Se^4+^ at equivalent Se concentrations. This finding suggests that ingested selenate may be more readily converted into organic components by mealworms than selenite. The high percentage of organic Se (>79%) indicated that the mealworm exhibited convincing transformation capacity in relation to both inorganic Se forms.

3.5. Digestibility and Species of Mealworm-Derived Se

The extraction efficiencies of mealworm-derived Se in SSF-SGF and SSF-SGF-SIF digests can be observed in Figure 4. The range of digestibility of Se in SSF-SGF was found to be ranging from 24.79% to 27.64% when the substrate was spiked with 0–20 mg/kg (Se^4+^). In a similar manner, the values in SSF-SGF ranged from 39.71% to 49.88% when worms were exposed to Se^6+^-treated substrates. The digestibility of Se from mealworms in SSF-SGF-SIF was superior to that in SSF-SGF, with values ranging from 68.38% to 85.12% for Se^6+^-treated mealworms, followed by Se^4+^-treated mealworms (53.80–60.67%), and finally CK (44.79%). The results indicate that the mealworm-derived Se obtained from selenate-treated groups is more readily absorbed and utilised by the organism, especially for Se10 treatment (Se^6+^), which exhibited the highest extraction efficiencies in both SSF-SGF and SSF-SGF-SIF. Furthermore, substantial disparities in Se digestibility were identified between SSF-SGF and SSF-SGF-SIF in Se5, Se10, Se20 (Se^4+^), and Se10 treatments (Se^6+^), suggesting that trypsin plays a pivotal role in facilitating Se release.

The Se speciation in SSF-SGF-SIF digests is illustrated in Figure 5. As can be discerned, SeCys_2_ and SeMet were the only observations made in Se5 and Se10 treatments when treated by Se^4+^. Furthermore, an additional unknown Se species (U) was identified in the Se20 group. In contrast, MeSeCys, Se^4+^, and Se^6+^ were not detected in the Se^4+^-treated groups. The content and proportion of SeCys_2_ in three Se^4+^ groups were in the range of 1.06–3.19 mg/kg and 94.14–97.42%, respectively, which were evidently higher than the content (0.028–0.17 mg/kg) and proportion (2.58–5.03%) of SeMet (Table 2). It is acknowledged that U merely accounted for 0.83% (0.028 mg/kg) of the total Se form detected in the Se20 group. Concurrently, SeCys_2_ and SeMet were identified in three Se^6+^-treated groups; however, Se^6+^ was also detected, albeit with comparatively low signal intensity. The content of SeCys_2_ in Se5, Se10, and Se20 treatments was 0.53, 1.23, and 2.34 mg/kg, respectively, accounting for more than 92.68% of the total detected Se form. The Se^6+^ content detected ranged from 0.0014 mg/kg to 0.019 mg/kg, constituting 0.13–0.75% of the total Se form detected. Conversely, CK samples demonstrated no discernible Se.

4. Discussion

4.1. Effects of Inorganic Se on Mealworm Growth

The results of the present study demonstrated a decline in both the biomass of mealworms (T. molitor) and its increase rate with elevated levels of Se in the substrate. The findings of this study indicate that both of high doses of Se^4+^ and Se^6+^ can inhibit the growth of mealworms, exhibiting a duration- and dose-dependent response. It is well established that high doses of Se, even in organic species [30], are toxic. This can lead to a series of biosafety problems, such as growth inhibition [31], reproduction obstruction [32], metabolic disorders [33,34], and genotoxicity [35]. In contrast, sodium selenite at a concentration of 50 mg/kg was found to promote growth and metabolism in the maggots, while a concentration of 70 mg/kg exhibited a toxic effect and resulted in a reduction in their biomass [23]. Similarly, the fresh weight of earthworm was stimulated by 0.3 and 10 mg/kg of Se^4+^, whereas it was significantly inhibited at higher concentrations of 30 and 70 mg/kg [33]. The results demonstrate that the benefits of Se manifest at comparatively low concentrations, while the occurrence of biotoxicity is anticipated at elevated Se doses. The toxicity may be attributed to oxidative stress, resulting from the production of reactive oxygen species (ROS), or non-specific integration of Se-containing amino acids into peptides or proteins, thereby disrupting their physiological functions [36].

An increase in the biomass of mealworms was observed when exposed to a medium treated with Se^4+^ in comparison to those exposed to Se^6+^ at equivalent concentrations and durations. This phenomenon can be attributed to the heightened toxicity of selenate. There is ample evidence that metals in high concentrations have been shown to exert deleterious effects on various biological processes, including, but not limited to, growth, metamorphosis, and endocrine function [37]. An investigation was conducted into the effects of selenate and selenite on the mortality and reproduction of Enchytraeus albidus, and the results showed that the median lethal concentration (LC_50_) of selenate was 5.69 mg/kg dw and 22.5 mg/kg for selenite, while the median effective concentration (EC_50_) of selenate was 0.41 mg/kg and 7.30 mg/kg for selenite [38]. These results indicate that selenate is more toxic than selenite. Nevertheless, it has been demonstrated that selenite exhibits a higher degree of toxicity towards Tetrahymena thermophila in comparison to selenate, which is substantiated by the calculated LC_50_ values of 27.65 μM for Se^4+^ and 56.88 mM for Se^6+^ [39]. The toxicity of selenate and selenite is generally reversed in aquatic and soil environments [40], and the underlying reasons for this phenomenon require further investigation.

4.2. Se Accumulation Analysis in Mealworms

The concentration of mealworm Se increased with inorganic Se content in the medium at the same exposure stage, thus demonstrating a dose effect. This phenomenon can be explained by the observation that elevated Se levels result in increased ingestion of Se by organisms during the feeding process. However, an exceptional circumstance for mealworm Se accumulation was observed, especially after 14 days. This was characterised by a decrease in total Se concentration and BAF_Se_ values in worm tissues, with prolonged exposure time in all Se^4+^- and Se^6+^-treated groups. This outcome is in contrast with the majority of extant literature, which suggests that organisms are capable of absorbing a greater quantity of chemicals over time [41,42,43].

In general terms, the absorption of nutrients or toxins by invertebrates occurs primarily through two mechanisms: ingestion and cutaneous absorption. However, observations have been made that mealworms primarily consume chemicals through ingestion, as their skeletal carapace can significantly impede the permeation of chemicals into the body through surface contact. The expulsion of substances that are either useless or harmful to health is achieved by organisms through the process of excretion. However, the rate of chemical absorption of by organisms in a polluting medium is generally greater than the rate of excretion [44]. This phenomenon leads to an enhancement in the rate of net accumulation over time. In the case of mealworms, which are known for their moulting habits, the ingested chemicals may be expelled from the body along with the shed skin [45], thereby reducing the accumulation of hazardous substances. It is hypothesised that this may be one of the factors contributing to the observed decrease in Se accumulation in mealworms in the latter stages of the experiment. Furthermore, the mealworm exhibited a decline in biomass growth rate during the latter stage, indicative of a reduction in feeding rate. This, in turn, led to a decrease in Se absorption.

The metamorphosis of mealworms from the larvaal to adult stage is associated with significant chemical changes in the organisms. For instance, elevated levels of metals have been observed in the premetamorphic amphibians both prior to and during metamorphosis, with higher metal concentrations recorded in comparison to postmetamorphic individuals [46]. Furthermore, metals are predominantly lost during metamorphosis, with larval concentrations being 2–125-fold higher than those in adult insects [47]. This shift in metal concentration patterns resulted in elevated exposure risks for predators of larvae compared to predators of adult insects. Moreover, a significant decrease in the concentrations of Fe, Mn, and Cu was observed during the growth and development of mealworms during metamorphosis. Conversely, no substantial alterations were observed in the concentrations of Mg and Zn [48]. Additionally, the accumulated Cu in the larvae of Stictochironomus histrio and Chironomus anthracinus was excreted almost completely prior to the pupal stage, and trace metals were detected in the exuviae [45]. These results provide compelling evidence for the elimination pathways of trace metals during metamorphosis. In the subsequent phase of the experiment, the mealworms will undergo metamorphosis. The complex life cycles of these organisms have the capacity to exert a substantial influence on the patterns of accumulation and depuration, thereby impacting the internal composition of elements [49]. However, further exploration is required to elucidate the specific process and mechanism by which this phenomenon occurs.

Greater Se bioaccumulation of selenate in comparison to selenite has been reported in other organisms, including E. fetida [50,51] and E. andrei earthworms [52]. The present study demonstrated that mealworms accumulated more Se when exposed to selenate than to selenite. This phenomenon may be attributed to the greater bioavailability of selenate. However, a diametrically opposed scenario has also been documented in certain aquatic vertebrates, including tadpoles [53,54] and gudgeon (Pseudorasbora parva) [55]. The differential uptake of Se forms can be ascribed to variations in transporters or the rate of passive uptake, while alterations in accumulation are presumably associated with differences in metabolic processes [56].

The nutritional Se requirement for most animals is typically between 0.1 and 0.3 mg/kg of complete feed [57]. Consequently, mealworms from the lower-dose treatment group (Se5), when incorporated into feed formulations at a modest inclusion rate (e.g., 5–10%), could contribute to meeting the animal’s dietary Se requirement. Se-enriched insects with a high Se concentration are a concentrated premix, and as such, must be diluted through blending with other feed ingredients in order to ensure that the final composite feed complies with the law.

In this study, the BAF_Se_ values of mealworms were found to be less than 1, indicating that their enrichment ability to two kinds of inorganic Se was inadequate in comparison to invertebrates with BAF_Se_ values greater than 1, such as fly maggots [23] and earthworms [24]. Furthermore, an increase in exposure duration resulted in a decline in BAF_Se_ values in mealworms. This decline was concomitant with elevated selenite concentrations in the substrate, indicating that these treatments suppressed the bioaccumulation capacity of Se in mealworms. In contrast to the findings observed in selenite treatments, the worm BAF_Se_ values exhibited no significant differences when exposed to varying concentrations of selenate in the later stage. This outcome indicates that the absorption and metabolism of the two types of inorganic Se in mealworms are distinct processes.

4.3. Effects of Se Addition on Nutrient Components

In this experiment, the stimulation of protein synthesis in mealworms was observed, with the addition of inorganic Se to the substrate. These results were comparable to those reported by Dong et al. for T. molitor [25]. Peng et al. [23] also found that the administration of a precise quantity of Se^4+^ (50 mg/kg) could considerably enhance the crude protein synthesis of maggot, in comparison to the CK group. Moreover, the employment of transcriptomic methodology has been utilised to demonstrate that dietary Se levels of 0.20 and 0.45 mg/kg significantly promote the metabolism of serine, glycine, and threonine in white shrimp (Litopenaeus vannamei) [58]. This is likely to be associated with protein synthesis. Furthermore, substantial reductions in the crude fat content of mealworms were observed in each experimental treatment when exposed to Se-containing substrates. Selenite groups exhibited a reduction from 36.05% to 16.95%, while selenate groups demonstrated a decrease to 14.71%. This result was consistent with the findings reported by Peng et al. [23] that inorganic Se addition inhibited the crude fat content in Chrysomya megacephala maggots. This phenomenon may be explained by the bioaccumulation of inorganic Se, which has been demonstrated to promote lipolysis and inhibit lipid content in organisms [59]. The content of mealworm polysaccharide increased with the addition of Se in the substrate, from 9.12% to 16.81% in selenite and 14.02% in selenate, respectively. These results indicate that Se can promote the synthesis of mealworm polysaccharides. The observed alterations in the composition of crude protein, crude polysaccharide, and crude fat in T. molitor in response to varying concentrations of inorganic Se exposure may be attributable to the stress induced by high concentrations of chemicals, which subsequently led to the re-regulation of energy and metabolic processes, as evidenced by changes in amino acid content [58].

A significant decrease in the concentrations of Fe, Mn, and Cu was observed during the growth and development of mealworms (approximately 6.8 mm to 20.4 mm in length) under conventional feeding practices, while Mg and Zn concentrations remained relatively constant [48]. In this experiment, the addition of an appropriate amount of Se was found to promote an increase in the contents of Mg, Zn, and Fe, which is consistent with the results reported by Peng et al. [23] that Se promoted the uptake of Zn and Cu by the maggots, with the highest uptake observed in the 50 mg/kg group.

It is imperative to note that essential elements, such as Mg, Fe, Zn, and Cu, are indispensable in performing various biochemical functions associated with cell metabolism and regulation. The concentrations of these elements are meticulously regulated by sophisticated homeostatic mechanisms, encompassing uptake, transport, intracellular distribution and excretion [60]. Consequently, it can be deduced that the concentrations in question are intimately connected with the main physiological cell functions. Therefore, it is possible to utilise them as a tool for obtaining information regarding the biochemical adaptations of an organism.

4.4. Se Species and Bioaccessibility in Mealworm

The biotransformation of inorganic Se into organic species within edible insects is a promising strategy for producing Se-enriched functional feeds or foods. The findings demonstrate that T. molitor possesses a remarkable capacity to convert both Se^4+^ and Se^6+^ into organic Se compounds, predominantly SeCys_2_ and SeMet.

The results obtained in this study demonstrate that mealworms exhibit a high degree of efficiency in the accumulation and transformation of both selenite and selenate, with organic Se proportions exceeding 79% even at the maximum supplementation level of 20 mg/kg. This finding is consistent with the results of studies conducted on other insect taxa. For instance, the black soldier fly (Hermetia illucens) larvae have demonstrated high bioconversion rates of selenite into organic forms, primarily as SeMet [61]. Similarly, earthworm Eisenia fetida, has been observed to assimilate and transform soil Se into organoselenium compounds within its tissues [24]. The compelling transformation capacity of T. molitor observed here, particularly the higher organic Se proportion from selenate compared to selenite at equivalent concentrations, suggests a potentially more efficient metabolic pathway for selenate assimilation. This phenomenon may be ascribed to variations in uptake mechanisms; selenate frequently utilises sulfate transporters and may follow assimilation pathways that efficiently lead to SeCys_2_ synthesis, whereas selenite can involve passive diffusion or other transporters and may be more prone to non-specific protein incorporation. The negligible presence of residual Se^6+^ only in the selenate-treated groups, albeit in minute quantities, serves to further substantiate the differential metabolic fate of the two inorganic forms, with selenite being more completely transformed within the organism. This observation is consistent with the findings in the field of plant research, where selenate is frequently found to be more readily absorbed via sulfate transporters and incorporated into amino acid synthesis pathways than selenite. This discrepancy may be attributed to the potential involvement of distinct uptake mechanisms and the potential for selenite to encounter greater sequestration or reduction challenges prior to assimilation [62].

The in vitro gastrointestinal digestion assay provides critical insights into the potential bioaccessibility of mealworm-derived Se. The results obtained demonstrate that Se the digestibility of Se is consistently higher in groups treated with selenate during the intestinal phase. This finding indicates that the organic Se compounds biosynthesized from selenate are more accessible to digestive proteases, resulting in higher nutritional efficacy. The substantial increase in digestibility from the gastric (SSF-SGF) to the gastro-intestinal phase (SSF-SGF-SIF), especially evident in specific treatments, highlights the essential role of trypsin and intestinal enzymes in liberating protein-bound Se amino acids, such as SeMet and SeCys_2_, for absorption, from the insect matrix. This finding is consistent with the prevailing concept that the bioaccessibility of Se from food sources is predominantly determined by the digestibility of the selenoproteins or selenoamino acids in which it is incorporated [63].

It is imperative to undertake a speciation analysis of the bioaccessible fraction to ensure safety and nutritional assessment. The predominance of SeCys_2_ and SeMet, two organic Se compounds that have been shown to possess chemopreventive properties and favourable toxicological profiles in comparison to inorganic Se, is highly advantageous [1]. SeCys_2_ is the biologically active form found in crucial selenoenzymes such as glutathione peroxidases [64,65]. The predominance of SeCys_2_ over SeMet in the present study may reflect a specific metabolic preference or pathway activity in mealworms under Se stress. Indeed, the biological toxicity of inorganic Se can be reduced by biotransformation to achieve the purpose of reducing negative effects. The absence of toxic inorganic forms (Se^4+^, Se^6+^) in the bioaccessible fraction of selenite-treated groups and their minimal presence in selenate-treated groups confirms the efficiency of biotransformation and suggests a low risk of toxicity from consuming these enriched mealworms. The emergence of an unidentified Se species (U) at the maximum selenite dosage may signify the development of particular metabolic intermediates or Se-containing compounds under elevated Se pressure. This observation necessitates further identification in subsequent studies.

5. Conclusions

This study comprehensively analysed the effects of selenite and selenate on the biomass, Se accumulation, nutrients, mealworm-derived Se species and digestibility of mealworm. The effects of selenite and selenate on mealworms can be complex, with both species having the potential to stimulate growth at moderate concentrations, while being toxic at high levels. The results demonstrated that the biomass of T. molitor larvae, as well as its increase rate, could be inhibited by elevated levels of Se^4+^ and Se^6+^ substrate, a condition that was further exacerbated by an extended cultivation time. In the present study, the time- or dose-effect characters that frequently manifested in conventional biological Se enrichment were not identified. This phenomenon may be attributable to distinctive characteristics exhibited by mealworms, such as moulting and metamorphic development. Conversely, the enrichment of Se in insects began to decline after the 14th day. This finding indicates that when the objective is to obtain Se-enriched mealworm products with a high Se concentration, it is imperative to take into account their growth and development period. Elevated levels of substrate Se (i.e., ≥10 mg/kg) have been demonstrated to induce a trade-off, resulting in the inhibition of growth biomass. Concurrently, these levels have been observed to trigger a significant metabolic shift within the insects, enhancing the levels of crude protein, crude polysaccharide, elements Mg and Fe, and concomitantly reducing fat. It has been demonstrated that the larvae of T. molitor function as an effective bioreactor, with the capacity to produce high-value, bioaccessible organic Se. Selenate has been demonstrated to be a superior precursor for generating mealworm, with a higher organic Se content and potentially greater digestibility than selenite. The final worm product is primarily composed of beneficial Se species, such as SeCys_2_ and SeMet, in a highly bioaccessible form. This finding indicates that the bioavailability of Se species that accumulate in the mealworms can be influenced by the form of Se and the overall Se concentration in the diet. The present findings provide robust evidence that Se-enriched mealworms have considerable potential as a sustainable and safe nutritional supplement or functional food ingredient to address Se deficiency. Nevertheless, further research is necessary to elucidate the mechanisms underlying these effects in greater depth, and to optimise Se supplementation in mealworm diets for a range of applications, including animal feed and human nutrition.