Soybean hawkmoth (Clanis bilineata tsingtauica) as food ingredients: a review

ABSTRACT The soybean hawkmoth, known as Clanis bilineata tsingtauica (CBT), is a traditional edible insect of East Asia. CBT larvae are an ideal nutrient source containing 18 amino acids and 19 fatty acids. The total essential amino acid contents could meet the requirement of preschool children and adults. Long chain fatty acid content, especially linolenic acid is quite high than egg or soybean. Various active components, such as oil, polysaccharide, chitosan, can change the anti-oxidative, anti-aging, anti-fatigue, hypolipidemic and anti-bacterial properties. CBT larvae are of high protein, low fat, and easily absorbed by the human body. Thus, CBT larvae could be used as an alternative food ingredient, such as whole food and individual active component resource. Edible CBT foods could meet the emerging global demand for animal protein and resolve the food security issues. This review will provide an insight into CBT nutrient composition and their potential application in functional foods.


Introduction
Insects can be a potential sustainable food source for humans owing to enormous biodiversity and high nutritional, environmental, and economic value (K. Kim et al., 2019b;Ramos-Elorduy, 2010). Around 2 billion people consume insects as a food source worldwide (Nowakowskia et al., 2021). Edible insects are gaining interest as alternative food sources for the increasing world population (Antonietta, 2020;Otero et al., 2020). The Food and Agriculture Organization of the United Nations (FAO) recognized that edible insects could play an important role in improving global food and nutrition security (Mutungi et al., 2017). In addition, FAO recommended that people can supplement their daily diet with edible insects, since they are consumed in some stage of their life cycle as food resources (van Huis et al., 2013). Currently, over one thousand identified species have historically been recorded and consumed by one third of the world's population, mostly in Asia, Latin America, and Africa, for more than two thousand years (Jiménez et al., 2020;Raheem et al., 2018). Asian people started cultivating silkworms (Bombyx mori) artificially 5200 years ago (Ghosh et al., 2017;Yi et al., 2010). Until now, 324 species of insects from 11 orders have been documented that are either edible or associated with 'Entomophagy' (Feng, Chen, et al., 2018). The most commonly consumed insects in China and other East Asian countries include caterpillars, beetles, bees, wasps, crickets, ants, termites, and flies (X. Chen et al., 2009). Although the usual approach of insect collection is from the wild or cultivated field, edible insects often fail to meet the increasing consumption demand affected by seasons and market variations. There is a growing interest in rearing insects for commercial purposes, and an industrial scale production will be required to ensure a steady supply (Raheem et al., 2018;Kim et al., 2019a). At present, Bombyx mori pupae are the only insect legally documented in the food catalogue in China (Feng, Zhao, et al., 2020;Gao et al., 2018). In addition, other insects and their by-products, including almost all edible insect species are consumed only as folk food and traded in local markets, of which soybean hawkmoth is one of them ( Figure 1). Soybean hawkmoth, Clanis bilineata tsingtauica, (CBT) belonging to the Lepidoptera, Sphingidae is an important economic insect widely distributed in East and South Asia (Sun, 2018;Wu, 2012). The CBT larvae (CBTL) are commonly called 'Doudan' and 'Bean ginseng'. Moreover, its edible stage includes the whole larval stage from the first to the fifth instar (X. Chen et al., 2009). In this review, we provide an overview of the nutrition, functional evaluation, and safety of CBTL.

Eating culture of soybean hawkmoth
CBT is an agricultural pest, mainly feeding on leguminous crops. While controlling the CBT outbreak, people innovatively converted it to a novel protein resource with great edibility (X. Chen et al., 2009;Feng, Zhao, et al., 2020). CBTL consumption has long been a traditional folkways culture, but it is difficult to find out the exact origin of this diet custom. The earliest document appeared in the book On Farming and Sericulture (C.N. Li, 1982). The cooking and oil extraction method was recorded by Song-ling Pu (1640--1715), a famous litterateur of the Qing Dynasty, in detail. In another book, The Addition to Compendium of Materia Medica, written by Xue-min Zhao (1719Zhao ( -1805, reported that CBTL were edible (Zhao, 2007). Until now, CBTL has always been favored and has become a local traditional specialty insect food in Beijing city, Jiangsu, Anhui, Henan, Shandong Province, and their surrounding regions Wu, Meng, et al., 2000). Compared to Bombyx mori and other insects, CBTL has also been highly accepted in these areas (A.J. Liu et al., 2019). CBTL provides significant economic benefits as it can be utilized continuously (Feng, Zhao, et al., 2020). China holds the largest consumer of CBTL, with a consumption rate of approximately 100, 000 tons/ year (Pan et al., 2018;Yan et al., 2020). At present, the most common processing method includes cooking the fresh larvae into delicious food using traditional Chinese cooking techniques, including stir frying, deep frying, stewing, roasting, and pan frying ( Figure 1). Besides direct consumption after cooking, CBTL can be processed into different types of common food. For instance, freeze-dried, fried, fresh meat, and canned fresh meat CBTL food are quite common. It is worth mentioning that the canned CBTL meat was developed for industrial food application (H.B. Wang et al., 2013;Wu, Lu, et al., 2017).

Nutrition and functional evaluation
CBTL is rich in nutrition, especially protein, edible oil, and other functional components showing various activities (Table 1).

Protein and amino acids
The protein content in CBTL dry matter is around 63.7% −65.50% (w/w) which is much higher than soybean (40%) or eggs (13%) (Tian & Zhang, 2012a;Xia, Wu, et al., 2012). The crude fat content is 23.68% (w/w) in CBTL (Wu, Meng, et al., 2000). The ratio of protein to crude fat (P/G) in CBTL is 2.96; thus, CBTL is considered as a high-protein insect instead of a high-fat insect. CBTL contains 18 kinds of amino acids, excluding two nonessential amino acids, asparagine, and glutamine. Of them, eight are essential amino acids (lysine, threonine, methionine, valine, tryptophan, phenylalanine, leucine, and isoleucine) with a dry weight of 224.73 g·kg −1 (Xia, Wu, et al., 2012). According to the amino acid scoring (AAS) pattern recommended by FAO/ WHO/UNU in 1985, lysine is the first limiting amino acid of CBTL, having an AAS of 53.27. Threonine is the second limiting amino acid with an AAS of 54.71 (Table 3). Total essential amino acids (including histidine) in CBTL is 343.11 mg/g protein, which is close to the total essential amino acid content 460 (408-558) mg/g recommended by FAO/WHO/ UNU for baby, more than the total amino acids content 339 mg/g for preschool children (2-5 years old), 241 mg/g for preschool children (10-12 years old), and 127 mg/g for adults (Food and Agricultural Organization, World Health Organization, United Nations University [FAO/WHO/UNU], 1985).
In animal experiments, Xia, Wu, et al. (2012) fed three groups of rats with casein, CBTL protein, and no protein diet for 10 days, respectively. The nutritional parameters measured for the group fed with the CBTL protein diet showed a positive nitrogen balance of 1.37, net protein retention of 2.9, and true digestibility of 95.8%, which were comparable to those measured for the group fed with the casein diet. The results suggested that CBTL protein may be a suitable alternative dietary protein source for humans.

Oil
It was reported that CBTL contained 19 kinds of fatty acids (8 saturated fatty acids, 3 monounsaturated fatty acids, 5 polyunsaturated fatty acids, and 3 odd chain fatty acids) (Sun, 2018). The oil proportion in fresh and dried CBTL is 5% and 23.68% (w/w), respectively. Moreover, the content of unsaturated fatty acids was higher than saturated fatty acids. C 16 ~ C 18 fatty acids share more than 97% fatty acids. Short chain contents, medium chain, and super long chain fatty acids are less than long chain fatty acids (Table 4). The essential unsaturated fatty acid content accounts for 64.17% of the total fatty acid (Wu, Meng, et al., 2000). More importantly, CBTL contained linoleic acid and α-linolenic acid, which are essential fatty acids that cannot be synthesized in the human body and can only be provided through food. The content of linoleic acid (C 18:2 ) was 6.29 g/100 g fatty acid. Linolenic acid (C 18:3 ) was found to be with the highest content among essential unsaturated fatty acids (36.53--46.91 g/100 g fatty acid). Total essential fatty acid and linolenic acid (C 18:3 ) contents is much higher than that in egg or soybean, while other fatty acids contents are less advantageous compared with egg or soybean Tian & Zhang, 2012b;Wu, Meng, et al., 2000;Yang, 2018). Briefly, long chain fatty acids content, especially linolenic acid (C 18:3 ) in CBTL oil, is quite high. CBTL oil is a potential alternative to edible oil.

Functional components
The functional food research directs towards ingredients, their functions and underlying mechanisms (Im et al., 2019). Active components, such as oil, polysaccharide, and chitosan could exhibit anti-oxidative, anti-aging, anti-fatigue, hypolipidemic, and anti-bacterial properties. The structure of some active components has been identified (Tian, 2016;Wu, Lu, et al., 2017), and their mechanism of action has been tested in vivo or in vitro Tian & Zhang, 2012c). The active components in CBTL and their function are summarized here (Figure 2).  (2018) Chen (1997) NR, non-reported.
When the individual active components of CBTL are separated and tested for their anti-oxidation effects independently, Sun (2018) found that CBTL extract exhibited better effect presumably due to the synergistic effects of various active components in the CBTL extract. In contrast, the protein extract of Tenebrio molitor, Ulomoides dermestoides, and Bombyx mori exhibited high antioxidant activities in some edible insects (Baek et al., 2020;Flores et al., 2020). CBTL also contains high protein content, suggesting the peptides or amino acids to be antioxidant. However, little is known about the anti-oxidation activities of protein. Thus, CBTL is a good source of functional food ingredients.

Anti-aging active components
In vivo and in intro research showed that CBTL exhibited anti-aging function, which may be related to chitosan components. Chitosan, a polymer composed primarily of β-(1 → 4)-2-amino-2-deoxy-D-glucose (D-glucosamine) monomers, is derived by deacetylation of naturally occurring biopolymer. Water-soluble chitosan of CBTL skin showed favourable anti-ageing activities on superoxide anion, showing considerable scavenging activities and hydroxyl radicals and reducing capacity, with scavenging activities increased to 87.04% (hydroxyl radical scavenging activity) and 79.81% (2,2-Diphenyl-β-picrylhydrazyl radical scavenging activity) at a concentration of 120 mg/mL (Wu, Lu, et al., 2017). Using the model of subacute aging mice induced by subcutaneous injection of D-galactose, it was found that both water extraction and 75% ethanol extraction significantly prolonged the time of loaded-swimming, enhanced the viscera index and the activity of total superoxide dismutase and glutathione peroxidase, and decreased malondialdehyde contents. CBTL exhibited anti-aging effect by improving immune and antioxidant capacity. Moreover, intragastric water-soluble chitosan of CBTL skin administration significantly increased the activities of superoxide dismutase and glutathione peroxidase and inhibited the formation of malondialdehyde in brains and sera of mice in a dose-dependent manner in D-galactose-induced-aged mouse model in vivo study (Wu, Lu, et al., 2017). Thus, anti-aging effect of CBTL results from its anti-oxidation activity. Interestingly, the anti-aging effect of an edible grasshopper (Oxya chinensis sinuosa) results from downregulated production of inflammatory cytokines (Im et al., 2019). Thus, the anti-aging mechanism of CBTL needs to be investigated at cellular and molecular levels.

Anti-fatigue active components
The anti-fatigue properties of CBTL were also studied with water and alcohol extraction (X.W. Liu et al., 2014;Xu et al., 2014). At a high CBTL dose, the swimming and climbing time of the mice was significantly prolonged, with improved moving endurance. In addition, the contents of blood urea nitrogen and blood lactic acid decreased, and the contents of hepatic glycogen increased after exercise. It was concluded that CBTL exhibited anti-fatigue functions by alleviating physical fatigue in the mouse model. This is similar to a stinkbug Aspongopus chinensis (S. Li et al., 2020). Table 4. Composition and content of fatty acids in Clanis bilineata tsingtauica larvae, egg and soybean.

Hypolipidemic active components
Chitooligosaccharide was capable of inhibiting the increase in body weight by decreasing plasma total cholesterol, triacylglycerol, and low-density lipoprotein cholesterol levels to 1.88 ± 0.46 mmol/L, 1.37 ± 0.05 mmol/L, 0.61 ± 0.02 mmol/L, respectively, and increasing high-density lipoprotein cholesterol (HDL-C) level to 0.82 ± 0.11 mmol/L in the mouse fed with high fat diets (Xia, Chen, et al., 2013). A similar phenomenon was observed in a Hercules beetle Allomyrina dichotoma (K. Kim et al., 2019b).

Anti-bacterial active components
Many insects and their products, such as Musca domestica and Dinoponera quadriceps exhibited anti-bactericidal effects (Rocha et al., 2021;Z.T. Wang et al., 2017). In CBTL, both chitooligosaccharides and original chitosan showed significant inhibitory activities against Bacillus subtilis (Bacillales: Bacillaceae). With the addition of the chitooligosaccharides, the number of colonies at 0 h was slightly lower than that at 48 h. These findings suggested that the chitooligosaccharides from CBTL skin were highly effective at inhibiting the growth of B. subtilis (Wu, 2012). In brief, CBT, unlike other common edible insects, has high protein content. CBTL contains 18 amino acids and 19 fatty acids. The total essential amino acid content can meet the demand of preschool children and adults. Compared to egg and soybean, the long chain fatty acid content of CBTL, especially linolenic acid (C 18:3 ) is quite high. Both oil and polysaccharide have excellent biological activity, and protein may also be a potential antioxidant component. It possesses different anti-aging mechanisms than edible locusts. Moreover, complex mixtures showed higher efficacy than their individual components due to synergistic effects (Im et al., 2019;Sun et al., 2018). Thus, CBTL could be used as an alternative food ingredient, such as whole food and individual active component resource.

Potential hazards and risks
Although the presence of toxic and allergic substances is important, a limited number of studies exist on the possible toxicity of edible insects (Mishynaa & Glumacb, 2021). However, all the studied edible insects in China are nontoxic (Gao et al., 2018). A previous study reported that two entomogenous fungus, 'Jiuzhou' caterpillar fungus (Cordyceps kyushuensis) and 'Taishan' caterpillar fungus (Cordyceps taishanensis) grown in CBTL are non-toxic (C. Chen et al., 2005;Guo & Li, 2000;B. Liu et al., 1984). Nonetheless, the toxicological assessment of CBTL has not been reported yet. The toxicological evaluation should be implemented according to the current version of the standard in effect, such as Regulation (EU) 2015/2283 of the European Parliament and Council on novel foods (Turck et al., 2021), 'Procedures for toxicological assessment of food' (NHCPRC, 2014) (GB 15193.1-2014) and 'Regulations on Application and Acceptance of Raw Materials for New Food' (NHCPRC, 2014). The toxicological assessment is a judgment process based on the data/results obtained from standard testing procedures. Specifically, suppose the relevant literature and component analysis did not find toxic effects of the food and had long-term consumption history without any harmful effects in substances (or raw materials for new food). In that case, it can be evaluated by acute toxicity test, genotoxicity tests, ninety days oral toxicity test, and teratogenic test (NHCPRC, 2020). According to the test results, assessors determine whether to conduct a toxicokinetics test, a reproductive toxicity test, a chronic toxicity test, and a carcinogenicity test (NHCPRC, 2014). Ji et al. (2009) reported that 18% of the reported cases of fatal anaphylaxis and anaphylactic shock to food in China were due to the ingestion of insects. However, CBTL caused only one reported case of anaphylaxis for such a long time (Steffie & Kitty, 2018). The case was about a 36 year old male who consumed CBTL and drank beer, resulting in an allergic reaction (Zhang, 2007). According to the diagnosis, it might have been related to the heterotypic protein in CBTL. This case of anaphylaxis occurred rapidly, belonging to type I allergy. In addition, attention must be paid to harmful microorganisms, parasites, toxins, heavy metals, veterinary drugs, hormones, and pesticide residues in the context of food safety (Antonietta, 2020;Mutungi et al., 2017;Schröge & Wätjen, 2019).

Prospect
The present study sought to review the research progress on nutrition, functional evaluation, and safety of a traditional edible insect CBTL. Previous studies on CBTL mainly focused on three aspects: bio-ecological characteristics related to rearing techniques and nutrition evaluation and its utilization as an edible insect. CBTL is a rich source of novel proteins, fatty acids, and active components. CBTL are excellent alternative to foods that can be used as essential supplement of protein, edible oil, and functional food ingredients. However, exploration of their characteristics and the development of more active components is still needed. Overall, CBTL consumer market still needs to be expanded, which is influenced by season and region. The total amount of CBTL resources is small, the development of cooking methods and specialty dishes is relatively slow, the processing capacity is limited, and the development degree of nutrition and health care products is not significant. Recent research focuses on the use of edible insects as food ingredients to fortify more traditional forms of food such as bread, tortilla, cookies. This trend might open up alternative ways to include them in our daily diet (Melgar-Lalanne & Alvarez, 2019). CBTL is generally considered to be safe with a long history of consumption in China. Toxicological assessment, allergens, and pollutants should be considered for the evaluation process. In addition, the effects of insect farming on the ecological environment should be evaluated. Insect farming and integrated utilization can result in great economic benefits and significant commercial development value in developing countries. Besides evaluation, process development, and utilization, there will also be a need for new regulations and legislation about the use of insects in food production in order to ensure consumer safety. Certainly, edible insect production has great potential with respect to sustainable food production for the growing population (S.K. Kim et al., 2017;van Huis & Oonincx, 2017). Edible CBT foods could meet the emerging global demand for animal protein and resolve the food security issues (Nowak et al., 2016). The insect industry is rapidly developing but still faces many challenges, which can only be encountered with the cooperation of all stakeholders (van Huis, 2020).