Introduction

Recently, as the global demand for sustainable protein sources has been increasing, apart from conventional edible meat sources, edible insects have been suggested as an emerging food protein source (Patel et al., 2019). With the recent world trend, in Korea, an interest in edible insects has also been growing constantly, and the scale of edible insect farming and the related commercial markets has been increasing rapidly (Ghosh et al., 2017). Fifteen insect species have been legally registered as ‘livestock’ by the Ministry of Agriculture, Food and Rural Affairs in July 2020 (MAFRA, 2020). In addition, nine insect species including Allomyrina dichotoma larvae, Apis mellifera L., Bombycis corpus, Bombyx mori L., Gryllus bimaculatus, Oxya japonica Thunberg, Protaetia brevitarsis larvae, Tenebrio molitor larvae, and Zophobas atratus larvae are registered as general food ingredients in the Korea Food Code (MFDS, 2020).

The larvae of white-spotted flower chafer (P. brevitarsis) have been used as a traditional medicine to treat inflammation, hepatic disease, and breast cancer in Korea (Song et al., 2017). In practice, various physiological benefits of the P. brevitarsis larvae, such as antioxidant, antibacterial, anticancer, and antithrombotic effects, have been already proven scientifically (Lee et al., 2017; Yoon et al., 2003). With the registration of P. brevitarsis larvae as a general food ingredient, recent studies have noted that of the proximate composition of P. brevitarsis larvae varied considerably: moisture (3.99%–7.98%), protein (42.46%–57.86%), fat (7.33%–26.70%), ash (3.96%–8.45%), and carbohydrate (10.56%–23.71%) (Chung et al., 2013; Ghosh et al., 2017; Jeong et al., 2020; Kim et al., 2017, Yeo et al., 2013). Regarding the large variation in proximate composition, Choi et al. (2019) have suggested that the nutritional composition of the P. brevitarsis larvae could be affected by feeding sources, similarly to conventional livestock. Moreover, it has been reported that differences in feeding sources have a greater impact on the nutritional composition of P. brevitarsis larvae compared to the conventional livestock, since it has more short and simple digestive system (Yoon et al., 2020). Furthermore, as the whole larvae including a digestive tract are generally consumed and processed, it is known that fasting methods could be one of the most important factors affecting the nutritional value of edible insect larvae (Noh et al., 2015).

In this regard, in the Korean edible insect industry, the establishment of a standard production system has been attempted for stable production and utilization of edible insects as food ingredients with constant quality and safety. However, many edible insect farms in Korea have been producing by the rearing protocol based on the owners’ individual experiences. Thus, in order to establish a potentially applicable production system, it could be primarily necessary to compare the nutritional composition of edible insects produced by various current production systems. Until now, although there are some previous studies determining the nutritional composition of P. brevitarsis larvae (Chung et al., 2013; Ghosh et al., 2017; Jeong et al., 2020; Kim et al., 2017; Yeo et al., 2013), but little studies have been compared the nutritional composition of P. brevitarsis larvae produced from different commercial farms. Therefore, the objective of this study was to determine the major nutritional composition (proximate composition, amino acid profile, and fatty acid profile) of white-spotted flower chafer (P. brevitarsis) larvae, collected from five commercial insect farms in Korea.

Materials and Methods

Rearing information of white-spotted flower chafer larvae

Frozen whole white-spotted flower chafer (Protaetia brevitarsis, Coleoptera: Scarabaeidae) larvae, which were harvested at third instar and fasted for 3 days, were kindly provided by five large-scale commercial insect farms located in the Gyeongsang-namdo, Korea. The frozen and vacuum-packaged samples were placed in an ice cooler and transported to the laboratory. According to the manufacturers’ information, the conditions of the rearing room, such as temperature, relative humidity (RH), and lighting control, were similar for guaranteeing maximum profits as follows: average temperature of 25℃, 60% RH, and 16L:8D. However, the feeding sources for P. brevitarsis larvae in the insect farms varied as follows: Farm A, fermented oak sawdust; Farm B, fermented oak and scrub sawdust; Farm C, commercial feed (Goomlife, Gimhae, Korea); Farm D, private fermented feed (oak sawdust 50%, rice bran 5%, barley bran 5%, molasses 5%, water 25%); and Farm E, the byproduct from mushroom compost. However, the detailed feed composition, manufacturing method, and harvesting methods of the larvae were unfortunately not provided for confidentiality reasons.

Experimental design and sample preparation

The experimental design of this study was a completely randomized block design with three independent replications. The collected P. brevitarsis larvae from each farm were separated randomly into three groups (approximately 120 g per group) as a block. The assigned larvae samples were weighed, placed in an aluminum dish, and hot-air dried at 55±1℃ for 12 h. The dried samples were re-weighed to determine the drying yield and ground using a food blender (HMF3800SS, Hanil Electric, Seoul, Korea). The obtained powder was filtered through a 100-mesh sieve, and the filtrate was vacuum-packaged in a polyamide/polyethylene bag and stored at –20℃ until further analysis.

Analysis of P. brevitarsis larvae

Drying yield

The drying yield of P. brevitarsis larvae samples was calculated as follows:

Drying yield (%) = [(Wb – Wa) / Wb] × 100

Where W_b = Weight of sample before the drying process (g), and W_a = Weight of sample after the drying process (g).

Proximate composition

The proximate composition of dried P. brevitarsis larvae was determined according to the standard methods of the Association of Official Analytical Chemists (AOAC, 2006). Moisture content (oven air-drying method, 950.46B), fat content (Soxhlet method, 960.69), and ash content (muffle furnace method, 920.153) were expressed as g/100 g of dried sample. The protein content of dried larval samples was determined by the Dumas method (N × 6.25) using a nitrogen analyzer (Rapid N Cube, Elementar Analysen systeme GmbH, Hanau, Germany).

Amino acid profile

Total amino acids in the P. brevitarsis larvae samples were determined by the method of AOAC (1998) with some modification as described by Jo et al. (2018). One gram of the sample was hydrolyzed in 15 mL of 6 N HCl at 110℃ for 24 h. The hydrolyzed samples were filtered using glass wool, and the filtrate was concentrated using a vacuum rotary evaporator at 55℃. After removal of the solvent, 10 mL of 0.2 N sodium citrate buffer was added, and the diluted sample was filtered with a 0.45 μm syringe filter before analysis.

Free amino acids were determined following the method of Jo et al. (2018) with modification by the instruction of an amino acid analyzer. Five grams of each sample was homogenized with 25 mL of distilled water for 1 min and it was filled up to 50 mL with distilled water. The homogenate was centrifuged at 7, 000×g for 10 min (4℃), and the supernatant was mixed with 12% trichloroacetic acid (TCA) in the same volume ratio (1:1, v/v). After approximately 1 h, the mixture was centrifuged at 7, 000×g for 20 min. To remove TCA and lipid components in the supernatant, hexane was added to the mixture at a 1:1 ratio (v/v). The mixture was centrifuged again at 8, 960×g for 10 min. The water phase was collected from the bottom and filtered through a 0.2 μm syringe filter. Hydrolyzed amino acids and free amino acids were analyzed with a Biochrom 30 plus amino acid analyzer (Biochrom, Cambridge, UK) using ninhydrin as the color reactant and a single ion-exchange resin column. The detection wavelength was 440 nm (proline) or 570 nm (all other amino acids), and an external standard was used to calculate the concentration of each amino acid. The results are reported as μg/g dry matter.

Fatty acid profile

To analyze the fatty acid composition in P. brevitarsis larvae, fatty acid methyl ester (FAME) was synthesized according to the method of O’Fallon et al. (2007) with some modifications. Briefly, 1 g of the dried larvae powder was weighed into a test tube with a screw cap, and 6.3 mL of absolute methanol and 0.7 mL of 10 N KOH were added. For permeating, dissolving, and hydrolyzing the sample, the tubes were heated in a 55℃ water bath for 1.5 h with thorough shaking every 20 min. After cooling in cold water, 0.58 mL of 24 N H₂SO₄ was added to the test tubes and mixed by inversion. Heating and cooling were carried out as described above. Three milliliters of hexane were mixed by vortexing, and the hexane layer was separated. The upper hexane layer containing the FAME was placed into a glass vial and kept at –20℃ until further analysis. FAME analysis was performed using an HP 6890N GC-FID (Hewlett-Packard, Wilmington, DE, USA) equipped with a Supelco^TM SP-2560 capillary column (100 m×0.25 mm×0.20 μm) (Sigma-Aldrich, St. Louis, MO, USA). One microliter of sample solution was injected into the column and He was used as the carrier gas. The gas flow rate was 1 mL/min, and the oven temperature was held at 140℃ for 5 min, then increased to 240℃ at a rate of 3℃/min, and the temperature was maintained at 240℃ for 10 min. The temperatures of the injector and detector were set at 260℃. Detected FAMEs were identified by comparing the retention times of peaks with those of the standards 37 component FAME mixture (Supelco, Bellefonte, PA, USA), which were analyzed under the same conditions mentioned above.

Statistical analysis

One-way ANOVA was conducted to analyze the collected data using the SPSS program (SPSS, Chicago, IL, USA). Duncan’s multiple range test was performed to compare significant differences among means (p<0.05).

Results and Discussion

Drying yield and proximate composition

The drying yield and proximate composition of P. brevitarsis larvae produced from commercial insect farms in Korea are shown in Table 1. The obtained data varied considerably depending on the insect farms (p<0.001). The drying yield ranged from 14.12% to 27.28%, and the highest yield was observed for the larvae produced from Farm D and E (p<0.05). Drying yield is one of the important processing factor directly affecting the profit of the seller, when edible insects are processed as pills and powder. Before harvesting, edible insect larvae are generally fasted for 3–4 days to remove residues in the intestine for better color and flavor (Kwon et al., 2013). According to Noh et al. (2015), fasting for 4 days before harvesting caused 27% weight loss in P. brevitarsis larvae. To our knowledge, in some cases, fasting with water immersion is carried out to promote defecation and minimize weight loss. Thus, the evaporation of absorbed water during drying process could greatly reduce the drying yield in the larvae fasted with water. If this speculation is valid, there would be similar moisture content in dried samples, despite the large variation on drying yield.

Table 1. Drying yield and proximate composition of white-spotted flower chafer (Protaetia brevitarsis) larvae produced from commercial insect farms in Korea

All values are presented as mean±SD of triplicate (n=3).

¹⁾ Farm A, Protaetia brevitarsis larvae fed with oak only; Farm B, Protaetia brevitarsis larvae fed with oak and scrub; Farm C, Protaetia brevitarsis larvae fed with commercial feed; Farm D, Protaetia brevitarsis larvae fed with private fermented feed; Farm E, Protaetia brevitarsis larvae fed with by-product from mushroom compost.

^a–e Means with different superscripts indicate significant difference within a row (p<0.05).

The difference in moisture content between the highest and lowest values (5.14–7.38 g/100 g) was approximately 2.24 g/100 g (p<0.05), which seemed to be relatively smaller than the difference in drying yield. The protein and lipid contents of P. brevitarsis larvae were greatly affected by production farms (p<0.001), in which changes in the relative content of lipids and proteins were observed. The larvae produced from Farm A, B, and D presented higher protein content, but lower lipid content compared to Farm C and E (p<0.05). The lowest ash content was found in larvae from Farm C and E (p<0.05).

In general, the large variation observed in the proximate composition of edible insects is mainly related to differences in developmental stages, feeding source, origin, and analytical methods (Rumpold et al., 2013). According to Oonincx et al. (2015), supplementation with a low-protein and high-fat diet decreased the protein content of yellow mealworm larvae but increased total fatty acid content. Moreover, they found no difference in the fatty acid profile of yellow mealworm larvae fed with different diets, despite evident differences in total fatty acid content (Oonincx et al., 2015). In this study, the larvae produced from Farm C and E were fed with commercial feed and the byproduct of mushroom compost, respectively. Thus, it seems that the feeding sources used in Farm C and E might have more digestible nutrients, particularly lipid compounds and/or their precursors, when compared to the other feeding sources used in Farm A, B, and D. As a result, the increased lipid content in P. brevitarsis larvae might cause a relative decrease in protein and ash contents. From the current perspective that edible insect has been primarily focused as an alternative protein source, our results indicate that supplementation of oak only, oak plus scrub, or private fermented feed used in Farm A, B, and D, respectively, could be beneficial in producing the P. brevitarsis larvae with high-protein and low-fat contents.

Total and free amino acid profiles

The total amino acid profiles of P. brevitarsis larvae produced from commercial insect farms in Korea are shown in Table 2. No difference in total amino acid content was found (p>0.05), regardless of insect farms, in which the essential and non- essential amino acid contents of P. brevitarsis larvae were 38.45%–42.75% and 57.25%–61.55%, respectively. Eight essential amino acids, including histidine (for infants), isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and valine were found in the larvae. Among them, the phenylalanine and methionine contents were greatly affected by insect farms (p=0.027 and p=0.006, respectively). In particular, the larvae produced from Farm B, which used oak plus scrub feed had higher essential amino acids (methionine) and sulfur-containing amino acid (cysteine) contents compared to those from other farms (p<0.05).

Table 2. Total amino acid profile of white-spotted flower chafer (Protaetia brevitarsis) larvae produced from commercial insect farms in Korea

All values are presented as mean±SD of triplicate (n=3).

¹⁾ Farm A, Protaetia brevitarsis larvae fed with oak only; Farm B, Protaetia brevitarsis larvae fed with oak and scrub; Farm C, Protaetia brevitarsis larvae fed with commercial feed; Farm D, Protaetia brevitarsis larvae fed with private fermented feed; Farm E, Protaetia brevitarsis larvae fed with by-product from mushroom compost.

²⁾ Indicates essential amino acids for infants.

³⁾ Indicates conditional essential amino acid for adult human.

⁴⁾ NS, non-significance (p≥0.05).

^a–c Means with different superscripts indicate significant difference within a row (p<0.05).

The obtained data for total amino acids in this study were considerably similar to the previous observation on P. brevitarsis larvae (mostly third instar), which was reported by Chung et al. (2013), Noh et al. (2015), and Yoon et al. (2020). In particular, Chung et al. (2013) suggested that P. brevitarsis larvae could be a potentially useful source of essential amino acids (methionine, threonine, valine, isoleucine, leucine, phenylalanine, histidine, and lysine) to humans. In addition, Noh et al. (2015) reported that the supplementation of rice bran during fasting could slightly increase the total amino acid content of P. brevitarsis larvae. Recently, Yoon et al. (2020) evaluated the supplementary effects of the five natural feeding sources, such as aloe, apple, banana, sweet persimmon, and sweet pumpkin, on the nutritional composition of P. brevitarsis larvae, and found that different feeding sources could change the proportion of essential amino acids, but did not affect the total amino acid content. Consequently, it is expected that the enrichment of some essential amino acids could be possible through dietary feeding control, but which might have little to no impact on the total amino acid content of P. brevitarsis larvae.

A total of 33 free amino acids, including 8 essential amino acids (histidine, isoleucine, leucine, lysine, methionine, phenylalanine, tryptophan, and valine), were detected in five larval samples from different production farms (Table 3). Except for cystathionine, the contents of all free amino acids of P. brevitarsis larvae significantly differed by insect farms. The content of essential amino acids in detected free amino acids ranged from 4, 073 to 5, 6 μg/g, in which the highest content was observed for the larvae from Farm A. Moreover, free amino acids such as phosphoserine, taurine, and γ-amino-butyric acid (GABA), which are well-known to provide physiological benefits to human health (Diana et al., 2014; Huxtable, 1992; Mcmahon and Oommen, 2008), were detected, depending on production farms.

Table 3. Free amino acid profile of white-spotted flower chafer (Protaetia brevitarsis) larvae produced from commercial insect farms in Korea

All values are presented as mean±SD of triplicate (n=3).

¹⁾ Farm A, Protaetia brevitarsis larvae fed with oak only; Farm B, Protaetia brevitarsis larvae fed with oak and scrub; Farm C, Protaetia brevitarsis larvae fed with commercial feed; Farm D, Protaetia brevitarsis larvae fed with private fermented feed; Farm E, Protaetia brevitarsis larvae fed with by-product from mushroom compost.

²⁾ Indicates essential amino acids for adult human.

³⁾ Indicate conditional essential amino acid for infants.

⁴⁾ ND, not-detected.

⁵⁾ NS, non-significance (p≥0.05).

^a–c Means with different superscripts indicate significant difference within a row (p<0.05).

Phosphoserine acts as a calcium stabilizer, which is rich in casein residues in milk proteins, and in turn contributes to improvement in calcium absorption (Mcmahon and Oommen, 2008). According to Jarboe and Mabrouk (1974), moreover, aqueous beef extract contained 1.84 mg of phosphoserine per 100 g of sample, as a form of free amino acid. In this study, it was observed that the larvae from Farm A, B, and C included 1, 001, 1, 153, and 773 μg of free phosphoserine per gram of dry matter. However, opposite results have been reported by Yoon et al. (2020), who reported no detection of free phosphoserine in P. brevitarsis larvae fed with oak-fermented sawdust plus aloe, apple, banana, sweet persimmon, or pumpkin. However, given that the free phosphoserine was detected in the larvae fed with oak (in the case of Farm A and B in this study), it could be thought that the free phosphoserine content might also be affected by other rearing conditions.

Taurine, 2-aminoethane sulfonic acid, has been well-known to have positive effects on osmoregulation, calcium modulation, antioxidation, radioprotection, and energy production in the mammalian body (Huxtable, 1992). In this study, except for the larvae from farm C, 25.29–44.11 μg of taurine per gram of dry matter was detected, which was similar to the previous finding (Yoon et al., 2020). It has been reported that beef (semitendinosus muscle) and lamb (longissimus lumborum muscle) contained 38.6 and 31.0 mg of taurine/100 g, respectively (Purchas et al., 2004). Considering that the larvae sample was analyzed as a dried form in this study, it seems that the taurine content of P. brevitarsis larvae might be lower compared to conventional meat sources.

Recently, GABA has received a great interest in the food industry, due to its various physiological effects on blood pressure control, activation of liver function, and improvement in brain function etc. (Diana et al., 2014). In this study, P. brevitarsis larvae contained 10.36–99.12 μg of GABA per gram of dry matter sample. GABA is generally found in fermented foods, since lactic acid bacteria produce glutamic acid decarboxylase for catalysis of L-glutamic acid to GABA. In this regard, the observed GABA content in white-spotted flower chafer larvae was potentially comparable to those of fermented goat’s milk (28 mg/kg; Minervini et al., 2009) and fermented pork sausage enriched with GABA through lactic acid bacteria fermentation (0.124 mg/kg; Li et al., 2009). Consequently, our results show that white-spotted flower chafer larvae are not only an excellent resource for supplying essential amino acids, but also that they could be a useful food source for supplying some free amino acids (e.g., phosphoserine, taurine, and GABA) to promote physiological activity.

Fatty acid profile

A total of 17 FAMEs were found in the larvae produced from commercial insect farms (Table 4), in which all larvae samples showed a higher proportion of unsaturated fatty acids (UFA, 76.0%–81.2%) compared to saturated fatty acids (SFA, 18.8%–24.0%). The major fatty acids contained in the white-spotted flower chafer larvae were oleic acid (C_18:1, 51.6%–59.5%), palmitic acid (C_16:0, 14.1%–19.5%), palmitoleic acid (C_16:1, 6.6%–11.9%), and linoleic acid (C_18:2, 5.4%–12.9%), and these fatty acids accounted for approximately 90% of the total fatty acids (minimum 88.1% and maximum 92.0%). This finding was in good agreement with the results from previous studies, which have reported that oleic acid is the major lipid composition of white-spotted flower chafer larvae (Chung et al., 2013; Noh et al., 2015; Yoon et al., 2020). In the previous studies, oleic acid was shown to be effective in improving cardiovascular disease and lowering cholesterol levels in the blood, a high content of oleic acid has been suggested as a nutritionally good indicator in the white-spotted flower chafer larvae (Chung et al., 2013).

Table 4. Fatty acid profile of white-spotted flower chafer (Protaetia brevitarsis) larvae produced from commercial insect farms in Korea

All values are presented as mean±SD of triplicate (n=3).

¹⁾ Farm A, Protaetia brevitarsis larvae fed with oak only; Farm B, Protaetia brevitarsis larvae fed with oak and scrub; Farm C, Protaetia brevitarsis larvae fed with commercial feed; Farm D, Protaetia brevitarsis larvae fed with private fermented feed; Farm E, Protaetia brevitarsis larvae fed with by-product from mushroom compost.

²⁾ ND, not-detected.

³⁾ NS, non-significance (p≥0.05).

^a–d Means with different superscripts indicate significant difference within a row (p<0.05).

In this study, the larvae from Farm A (oak feed), B (oak plus scrub feed), and E (mushroom byproduct feed) showed higher oleic acid content than those from Farm B and C (p<0.05). However, the contents of essential fatty acids, such as linoleic acid (C_18:2) and α-linolenic acid (C_18:3n-3), were higher in the larvae from Farm C (commercial feed) than in those from the other insect farms (p<0.05). There were no significant differences in the contents of arachidic acid (C_20:0, one of the essential fatty acids) and cis-4, 7, 10, 13, 16, 19-docosahexaenoic acid (C_22:2, DHA). Recently, Yoon et al. (2020) suggested that the fatty acid composition of white-spotted flower chafer larvae could be changed by feeding sources. In addition, Noh et al. (2015) noted that supplementation with aloe, rice bran, or pumpkin during 4 days of fasting could alter the content of oleic acid, from 62.5% to 67.1%. Thus, it could be expected that the fatty acid composition of P. brevitarsis larvae could be modified by the supplementary feed during fasting as well as basal feeding during production.

The saturated-to-unsaturated fatty acid ratio (SFA/UFA) of P. brevitarsis larvae ranged from 0.23 to 0.32. It has been well documented that a decrease in SFA/UFA positively contributes to the improvement in the nutritional value of foods (Vural and Javidipour, 2002). Based on the SFA and UFA contents previously reported by Zotte and Szendrő (2011), the SFA/UFA of pork loin, beef loin, and chicken breast was calculated as approximately 0.63, 0.86, and 0.52, respectively. In this regard, it could be presumed P. brevitarsis larvae provides better SFA/UFA values to human health compared to conventional meat sources. To the best of our knowledge, although there have been no studies on the physiological benefits of edible insect oils in the human body, some recent animal studies have found the potential benefits of insect oil intake on digestibility (Kierończyk et al., 2018) and fatty acid profiles in liver and muscle tissues (Benzertiha et al., 2019). Thus, it seems that P. brevitarsis larvae from Farm A (only oak feed), which showed higher oleic acid content and the lowest SFA/UFA value, could be the most beneficial source of lipids for human health.

Conclusion

In conclusion, this study confirmed that the white-spotted flower chafer (P. brevitarsis) larvae could be an excellent food alternative to supply high-quality protein and lipids. Moreover, phosphoserine, taurine, and GABA, which are known to be physiologically useful, were detected in the form of free amino acids. The contents of the bioactive compounds and the proximate composition were greatly affected by the farms where the larvae were produced. Although the underlying mechanisms of the different nutritional compositions have not yet been clearly understood, this study suggests that the production system of Farm A, using only oak feed, could be potentially beneficial in increasing protein content and decreasing SFA/UFA ratio in P. brevitarsis larvae.

Conflicts of Interest

The authors declare no potential conflicts of interest.

Acknowledgements

This work was supported by the research invigoration program of 2020 Gyeongnam National University of Science and Technology. In addition, this study was evidently conducted to obtain data for public interest purposes and the results cannot be used for commercial promotion of a certain farm. Therefore, the actual name of participating insect farms is not disclosed.

Author Contributions

Conceptualization: Kim IS. Data curation: Ham YK. Formal analysis: Ham YK, Song DH. Methodology: Ham YK, Kim IS. Software: Ham YK, Kim HW. Investigation: Kim IS, Kim SW. Writing - original draft: Ham YK. Writing - review & editing: Ham YK, Kim SW, Song DH, Kim HW, Kim IS.

Ethics Approval

This article does not require IRB/IACUC approval because there are no human and animal participants.