Nutritional, functional, and allergenic properties of silkworm pupae

Abstract Edible insects are a food source that has high nutritional value. Domestic silkworm pupae are an important by‐product of sericulture and have a long history as food and feed ingredients in East Asia. Silkworm pupae are a good source of protein, lipids, minerals, and vitamins and are considered a good source of nutrients for humans. Silkworm pupae are a valuable insect source of substances used in healthcare products, medicines, food additives, and animal feed. Because silkworm pupae are being increasingly used in the human diet, potential allergic reactions to the substances they contain must be elucidated. Here, we present an overview of the benefits of silkworm pupae. First, we describe their nutritional value. Second, we report their functional properties and applications, focusing on their potential use in the food and pharmaceutical industries. Finally, we consider the current state of research regarding silkworm pupae‐induced allergies.

for sustainable animal-derived dietary protein, edible insects are of interest as a source of protein (FAO, 2013). Silkworm pupae are considered a premium source of animal protein. They represent the only insect food in the List of Novel Food Resources published by the Ministry of Health of China and are widely used in dietary supplements, medicines, and animal feed in China and Korea (Kim et al., 2008;Zhu, 2004). In China, more than 100,000 tons of fresh silkworm pupae are produced annually (Dong & Wu, 2010). In recent years, silkworm pupae are used as raw materials in the food industry because of their high nutritional value and varied biological activities.
A major concern regarding novel sources of protein, including silkworm pupae, is their potential to cause food allergy (Pali-Schöll et al., 2019). In recent years, large numbers of cases of allergic reactions to silkworm pupae have been reported (Ji et al., 2008), including serious allergies, and, even anaphylactic shock (van der Poel & Chen, 2009;Araujo et al., 2014). Thus, allergies to silkworm pupae are now the subject of extensive research worldwide.
This review describes the nutritional value of silkworm pupae, their functional properties, and potential applications. Within this framework, allergies to silkworm pupae are evaluated.

| N UTRITI ONAL VALUE OF S ILK WORM PUPAE
Extensive data show that silkworm pupae have a well-balanced nutrient profile, making them suitable as human food. Essentially, silkworm pupae are a good source of protein, fat, minerals, and vitamins. Compared to conventional food, they have higher content of three calorigenic nutrients (protein, fat, and carbohydrates), providing up to 230 kcal per 100 g. The values of key nutrients in silkworm pupae compared with other common foods are listed in Table 1.
The protein content of silkworm pupae is about 21.5%, which is higher than that of other typical animal products. On a dry-weight basis, the protein content of silkworm pupae has been reported to be as high as 49%-54% (Longvah et al., 2011;Nowak et al., 2016).
The proteins of silkworm pupae are considered complete proteins because of their high content of essential amino acids. In fact, silkworm contains all the amino acids required by the human body and in the appropriate proportions based on the recommendations of the FAO/World Health Organization (WHO) (Köhler et al., 2019;Ni et al., 2003;Wang et al., 2009;Yang et al., 2009;. In general, insect proteins are highly digestible (Capinera and Finke, 2008). The amino acid score and protein digestibility-corrected amino acid score of silkworm pupae are 100 and 86, respectively (Longvah et al., 2011). Furthermore, the amino acid score of proteins in silkworm pupae protein was 100 with respect to the amino acid profile of a 2-5-year-old child (FAO/WHO, 1985) (Longvah et al., 2011). Therefore, silkworm is a good source of protein that could be used as an alternative dietary source of protein for human nutrition.
TA Edible insects also have high fat content. In particular, dry silkworm pupae have a fat content of 25%-30% (Kouřimská and Adámková, 2016;Longvah et al., 2011). Silkworm pupae have high monounsaturated and polyunsaturated fatty acid contents and low saturated fat content (Payne et al., 2016). About 70%-80% of the fatty acid content of silkworm pupae is unsaturated, while about 1% is unsaponifiable matter in oil, including campesterol, β-sitosterol, and cholesterol. In particular, silkworm pupae have almost 71% αlinolenic acid content (Pereira et al., 2003;Rao, 1994;Zhao, Wang, et al., 2015). Silkworm pupae are a good source of functional fatty acids. Furthermore, the oxidative stability of their total lipid content is very high, as the synergistic effect between tocopherol and phospholipids prevents the oxidation of lipids (Kotake-Nara et al., 2002).
Some insect species, including weevil larvae and termites, have high quantities of saturated fat, however silkworm pupae do not (Payne et al., 2016). Based on WHO guidelines, limiting the dietary intake of saturated fat minimizes the risk of cardiovascular disease (World Health Organization (WHO), 2015). Silkworm pupae contain edible lipids of high quality that are used as raw materials in medicine (Shanker et al., 2006;Wang et al., 2013). For instance, after eating silkworm pupae oil for 18 weeks, rats showed a notable increase in high-density lipoprotein cholesterol levels, with significantly reduced triglyceride and total cholesterol levels (Longvah et al., 2012).
Silkworm pupae oil also regulates plasma lipid and lipoprotein levels in the serum of rats by activating apoproteins and lipid-metabolizing enzymes. Thus, it could be used to treat hyperlipidemia (Hu & Chen, 2011). Therefore, silkworm pupae represent a good source of fats for human consumption.
Edible insects are also rich in minerals, including phosphorus, calcium, potassium, sodium, iron, zinc, magnesium, copper, and manganese (Kouřimská & Adámková, 2016). Silkworm pupae are a good source of calcium and iron (Table 1) and have high potassium content (34.0 mg/g), with a low Na/K ratio (0.08), and high zinc content (36 μg/g) , all of which are important for human health and nutrition. In addition, the heavy metal content (lead, arsenic, cadmium, and mercury) (Köhler et al., 2019) of silkworm pupae is below the maximum recommended level for human consumption and use in animal feed (National Standardization Management Committee of China, 2005). The mineral content reported in the published literature is highly varied (Köhler et al., 2019;Kouřimská & Adámková, 2016;Longvah et al., 2011;Nowak et al., 2016;. These differences might be explained by mineral content varying with geographical location, seasonality, and diet, among other items. In general, edible insects contain a variety of vitamins, but have limited vitamin A (Kouřimská & Adámková, 2016). Silkworm pupae are a good source of thiamin, riboflavin, and niacin (Table 1).
They contain about 10 mg tocopherols per 100 g, which is a much higher level than that found in other common foods. Importantly, the mineral and vitamin content of farm-bred silkworm pupae can be controlled by regulating what they are fed.
However, these antinutrients are present at low levels and fall within human tolerance levels. Thus, silkworm pupae are safe for human consumption (Omotoso, 2015).
Overall, silkworm pupae are a good source of protein, lipids, minerals, and vitamins, all of which are all important components of the human diet.

| FUN C TIONAL PROPERTIE S AND APPLI C ATI ON S OF S ILK WORM PUPAE
Silkworm pupae have many functional properties that could be utilized in the food and pharmaceutical industries. In recent years, various bioactive compounds have been found in silkworm pupae that have potential functions and health benefits. For instance, silkworm proteins and hydrolyzed peptides have several functions, including improving immunity, and antitumor and antioxidant activity (Table 2). Hydrolyzed peptides of silkworm pupae have multiple physiological functions. However, methods for their extraction, purification, and structural identification need to be developed, and the mechanisms of their pharmacological actions need describing.
Current methods for preparing peptides from silkworm pupae by enzymatic hydrolysis generate poor yields. Thus, enzymatic hydrolysis must be optimized to facilitate the commercialization of this process.
In addition, after deproteinization and degreasing, silkworm pupae residues contain 2%-8% chitosan, about 4% polysaccharides, and small amounts of antibacterial peptides and other bioactive materials, which have various biological activities (Battampara et al., 2020;Luo et al., 2010;Mishraa et al., 2003;Ni et al., 1998;Wang et al., 2007;Yue et al., 2013;Zhang et al., 2000). The precise functions of chitin and polysaccharides extracted from silkworm pupae in humans need further investigation. A dimethyladenosine compound extracted from silkworm pupae could be used as a vasorelaxant to treat vasculogenic impotence in men (Ahn et al., 2008). Many natural drugs have been isolated from microbes, marine organisms, and plants; however, few drugs isolated from insects are available on the market. The silkworm has long been used in Chinese medicine (Ratcliffe et al., 2011). Scientific studies have demonstrated the health benefits of silkworm pupae, and it has been reported that eating silkworm pupae does not cause mutation or hepatotoxicity (Yang et al., 2010;. Thus, silkworm pupae represent a valuable insect source for use in medicines and other healthcare products. Although some active substances in silkworm pupae have been identified, their pharmacological actions remain unclarified.
With in-depth research, silkworm pupae have great potential for use in the pharmaceutical industry.
A study on the functional properties of silkworm pupae demonstrated their potential for use as food additives (Omotoso, 2015).
In addition to direct consumption, the protein powder of silkworm pupae has been used as a food additive in cookies, noodles, bread, and other foods. Kim et al. (2016) used flour from silkworm pupae to replace 10% lean pork as a novel protein ingredient and showed that it enhanced the hardiness and cooking yield of emulsion sausages.
One of the greatest barriers to the adoption of edible insects as a direct food is consumer acceptance (Mlcek et al., 2014). Although consumers recognize the high nutrition value of insects, they prefer to not know what they are eating in this case (Govorushko, 2019

| Silkworm-induced allergy
The possible allergic reactions associated with eating insects are of concern for both consumers and scientists. Safety is a vital factor in food quality, with it being important to build confidence in the consumption of edible insects. Silkworms contain important sources of allergens, including excrement, dander, and silk. These allergens can cause various allergic reactions, such as cutaneous symptoms, respiratory symptoms, cardiovascular symptoms, and, even, anaphylactic shock (Gautreau et al., 2017;Ji et al., 2008;Vovolis & Galatas, 1999). These reactions could limit the use of silkworm pupae in food and feed. Potential silkworm-induced allergies include respiratory allergy, contact allergy, and food allergy.
The silkworm is widely cultivated, and its silk is widely used.
During processing and use, silk particles and wild silk floating in Hydrolysis peptide Umami taste enhancement  the air might be inhaled, causing allergies (Häcki et al., 1982;Inoue et al., 1997). Furthermore, inhaling the scales of the silkworm moth suspended in the air might cause respiratory allergies (Blanc et al., 1999;Suzuki et al., 1995). People allergic to silkworm might have a reaction when they are in contact with silk products (contact allergy) (Borelli et al., 1999;Ohansson et al., 1985). Silkworm pupae also cause allergic reactions when consumed as food (Ji et al., 2008;Choi et al., 2010). Thus, allergens likely exist at different developmental stages of the silkworm lifecycle; however, it is not clear whether these allergens are the same protein.
Silkworm pupae are consumed raw, boiled, and fried in several Asian countries. Allergic reactions after consuming silkworm pupae include urticaria, dizziness, skin itching, and shock (Feng et al., 2018).
In China, many cases of silkworm pupae-induced allergies have been reported. Each year, at least 1,000 patients have anaphylactic reactions after eating silkworm (Ji et al., 2008). In Korea, 9.4% of patients were found to be allergic to silkworm pupae (Kim et al., 2003). In East Asia, allergic reactions to eating silkworm pupae are considered a common cause of anaphylaxis (van der Poel & Chen, 2009).
However, allergic reactions caused by silkworm pupae are no more serious than those caused by conventional foods, such as shrimp and fish (de Gier & Verhoeckx, 2018). Because silkworm pupae are being increasingly incorporated into the daily human diet, the mechanisms driving these allergic reactions need to be elucidated.

| Allergens present in silkworm pupae
To date, 26 silkworm pupae proteins have been identified as allergens (Table 3). The only protein characterized as an allergen in silkworm larvae was an arginine kinase, which cross-reacted with an arginine kinase in cockroaches Liu, Xia, et al., 2009).
However, except for this arginine kinase (officially named Bomb m 1) Liu, Xia, et al., 2009), no allergens of silkworm pupae have been officially confirmed and registered by the WHO and International Union of Immunological Societies (WHO/ IUIS) Allergen Nomenclature Sub-committee (www.aller gen.org).
Five allergen proteins belong to lipoprotein 11 family (30-kDa family proteins), while three are insect cuticle proteins ( Arginine kinase is another well-known invertebrate pan-allergen. It is an enzyme found in insects and crustaceans, belonging to the guanidino phosphotransferase family (Azzi et al., 2004). However, arginine kinase has been reported as an allergen of silkworm larvae, but not of silkworm pupae. Wang and Xu (2006) showed that the gene for arginine kinase is also expressed in pupae at a relatively low level, with expression increasing as larvae mature. Thus, studies are needed to examine the allergenicity of arginine kinase in silkworm pupae. Vitellogenin is an allergen of the honey bee (Api m 12), chum salmon (Onc k 5), and yellow jacket (Ves v 6). Profilin is a pan-allergen (McKenna et al., 2016). The homology of arginine kinases and tropomyosins from different insect species is greater than 70%, while that of other allergenic proteins ranges from 35% to 95%. This phenomenon might induce cross-reactive reactions between proteins from different insect species and from crustaceans, such as shrimp (de Gier & Verhoeckx, 2018). Thus, the allergens of some silkworm pupae have high homology and structural similarity with those from other species, with cross-reactive potential. Cross reactions between invertebrate proteins are usually attributed to homologous allergens (Marti et al., 2007). Silkworms belong to the phylum Arthropoda, which includes crustaceans. A comparative proteomic study showed that some pupae proteins are differentially expressed when comparing gender and diet of silkworm larvae (Lamberti et al., 2019). Of note, the levels of known allergens in female silkworm pupae reared on mulberry leaves were lower compared to those reared under other experimental conditions (Lamberti et al., 2019). Thus, the allergen profile of silkworm pupae could be associated with gender and diet and so could be regulated.
In recent years, there has been extensive research to minimize food allergenicity in technical food processing. However, only the 27-kDa hemolymph glycoprotein has been identified as a heatstable allergen in silkworm pupae (Jeong et al., 2016). Knowledge remains limited on how food processing affects the allergenic potential of proteins in silkworm pupae. Methods to minimize allergens in silkworm pupae need to be developed. In the food industry, only hydrolysis and fermentation have achieved a satisfactory reduction in food allergenicity (Lepski & Brockmeyer, 2013;Verhoeckx et al., 2015). These processes might have similar effects on silkworm pupae. Enzymatic hydrolysis represents a promising processing technology for exploiting the bioactive properties of silkworm pupae.
This process might also represent an effective means of minimizing allergenicity. Through understanding the structure-allergenicity relationship of allergens in silkworm pupae, effective methods to process hypoallergenic proteins could be developed. Further studies are required to characterize the immunological properties of allergens in silkworm pupae.

| PER S PEC TIVE S AND CON CLUS I ON S
The silkworm is an insect that can be produced at a large scale. After extracting the silk thread, silkworm pupae are a subproduct of the textile industry. These pupae are rich in nutrients and have high Good cooking methods could promote the consumption of insects as food (Feng et al., 2018). Furthermore, the incorporation of insects into everyday food items is a documented strategy that overcomes the challenges associated with the low acceptance of insects as food (Khatun et al., 2021;Bawa et al., 2020;Nyangena et al., 2020). For instance, consuming protein powder and hydrolyzed peptides in a food product might be more acceptable than direct consumption.

TA B L E 3 Allergens in silkworm pupae
However, developing silkworm pupae as a food source requires an effective risk assessment of potential allergenic hazards.
Evaluation of the allergies of silkworm pupae is scarce and is primarily based on case reports without the consistent use of standardized tests. Overall, 26 silkworm pupae proteins have been identified as allergens; however, their immunological properties remain poorly characterized.
Potential strong cross reactions of silkworm pupae allergens should be carefully examined. Large-scale investigations of such cross-reactions are required. The detection, labeling, and elimination of silkworm pupae allergens is needed to combat allergic reactions.
Thus, the allergens present in silkworm pupae, and the mechanisms by which they cause allergic reactions, must be fully elucidated.

ACK N OWLED G EM ENTS
This study was supported in part by the Key-Area

CO N FLI C T O F I NTE R E S T
The authors declared they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.