Silkworm protein candy: An approach towards nutritional waste management and sustainable development

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Despite being a rich source of high-quality protein and essential amino acids, the consumption of Eri silkworm ( Samia ricini ) pupae and pre-pupae is largely restricted to tribal communities in Northeast India due to cultural taboos surrounding entomophagy. Among the six known strains of Eri silkworm, the yellow-spotted strain exhibits the highest protein content in its pre-pupal stage (66.58%), with lysine and arginine present in substantial amounts, making it a valuable dietary protein source for growth and development. Assam, the third-largest producer of raw Eri silk in India, generates approximately 4,910 metric tons of Eri cocoons annually, each containing a pupa. However, due to societal resistance, a significant proportion of these nutrient-rich pupae are discarded as waste. Addressing this issue, the present study explores the development of a novel protein candy formulated with Eri pupa protein (20 g), palm jaggery (80 g, low glycaemic index), garcinia-soaked water, and ginger leaves. Sensory evaluation revealed high consumer acceptability, with an overall score of 8.4. Furthermore, allergenicity and toxicity assessments in albino mice confirmed the safety of Eri pupa protein for human consumption. The current study highlights the potential of Eri pupa as a sustainable protein source, offering a promising approach to sericulture waste management and food security. The development of such protein-rich functional foods could facilitate broader acceptance of edible insects, drive entrepreneurship, and contribute to nutritional sustainability in the region. Eri-silkworm pupae insect protein candy waste management functional food entrepreneurship Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction The global human population has been increasing at an unprecedented rate, reaching 8.1 billion in 2024 from an estimated 2.5 billion in 1950, according to a 1998 United Nations (UN) study. This surge has equated to nearly 1 billion people being added every decade since 2010. Projections suggest that the population will reach 9.7 billion by 2050 and nearly 10.4 billion by the mid-2080s (UN article: Peace, Dignity, and Equality on a Healthy Planet ). This rapid growth presents numerous challenges, particularly in managing the availability of essential resources such as food and shelter. One of the most pressing concerns is the strain on the planet’s limited resources, especially arable land. According to the Food and Agriculture Organization (FAO), global agricultural land covers approximately 5 billion hectares—around 38% of the Earth's total land area. However, further expansion of agricultural land could lead to severe ecological imbalances, as converting natural ecosystems into farmland has historically been a major driver of greenhouse gas emissions, primarily due to biomass loss and depletion of carbon stores both above and below the ground [ 1 ]. Traditional food production systems, including staple crops like wheat and livestock farming, face mounting challenges such as land scarcity, water shortages, declining soil fertility, and increased greenhouse gas emissions. Given these constraints, there is an urgent need to explore alternative food sources and sustainable nutrition strategies to ensure food security for the growing global population. Entomophagy, or the practice of consuming insects as food, has been a part of human civilization for centuries, particularly in Asia, Africa, and Latin America [ 2 ]. Globally, approximately 2,000 insect species across 18 different orders are consumed [ 3 ]. In India, insect consumption is particularly prevalent among tribal communities in the northeastern region, where they are considered a delicacy [ 4 ]. Insects are an exceptionally nutritious source of protein, containing all nine essential amino acids [ 5 ]. They are also rich in important vitamins and minerals, such as vitamin B12, which is crucial for brain function, as well as iron, calcium, and zinc [ 6 ]. Compared to traditional protein sources, insects offer several advantages: for example, crickets contain more protein per gram than beef [ 7 ]. While conventional sources like chicken and pork provide the same essential amino acids, they may lack the diverse range of vitamins and minerals found in insects. Beyond nutritional benefits, insect consumption also has significant environmental and economic advantages. Insect farming requires considerably fewer resources, such as land, water, and feed, generating only a fraction of the greenhouse gas emissions associated with traditional livestock farming. Additionally, insect farming is cost-effective, making it a more affordable protein source for consumers. Insects also exhibit high adaptability, thriving in diverse environments, which enhances their potential as a sustainable alternative to conventional livestock production [ 8 ]. Silkworms are primarily reared for their valuable silk, but the sericulture industry also produces several by-products, including pupae, cocoon shells, and silkworm droppings, which can provide additional income for farmers. Silkworm pupae, a major by-product, are often discarded or used as animal feed and fertilizer. However, there is growing interest in their potential as a nutrient-rich food source, particularly in Asia, where they are traditionally consumed as snacks or added to various dishes [ 9 ]. Silkworm pupae are high in protein, comprising 50–60% of their dry weight, and contain essential amino acids like lysine and methionine, often lacking in plant-based diets [ 10 ]. They are also rich in essential fatty acids, vitamins, and minerals such as zinc, iron, and calcium, with an energy density comparable to chicken and beef [ 11 ]. Additionally, studies suggest that silkworm pupae may improve cholesterol levels, glucose regulation, and lipid metabolism [ 12 ], and their antioxidant properties have been linked to anticancer and anti-inflammatory effects [ 13 ]. Despite their nutritional benefits, global acceptance remains low, especially in Western countries, due to cultural stigmas. A study comparing consumer preferences in China and Germany found that Germans favored processed insect products like cricket flour, while the Chinese preferred whole insects [ 14 ]. To increase acceptance, processing silkworm pupae into more palatable and familiar forms could help integrate this sustainable protein source into mainstream diets. Samia ricini , commonly known as the Eri silkworm, is a multivoltine silk-producing insect found throughout the year in Assam and Northeast India. It belongs to the order Lepidoptera and the family Saturniidae. The species undergoes complete metamorphosis with distinct life stages: egg, larva, pupa, cocoon, and adult. Based on larval body color and markings, six distinct strains of S. ricini have been identified: Greenish Blue Plain (GBP), Yellow Plain (YP), Greenish Blue Spotted (GBS), Yellow Spotted (YS), Greenish Blue Zebra (GBZ), and Yellow Zebra (YZ) [ 15 ]. Among its life stages, the pre-pupa (just after cocoon spinning) and pupa are edible and widely consumed by tribal communities in Assam and Northeast India. However, non-tribal populations generally avoid Eri pupae due to social stigma and cultural taboos. Assam, the third-largest producer of raw Eri silk in India, produces approximately 7,364 metric tons (MT) of Eri cocoons annually, resulting in a significant quantity of Eri pupae. Despite published research highlighting its high nutritional value, particularly its rich protein content [ 16 ], much of the pupae remain underutilized and are discarded as sericulture waste, contributing to environmental hazards. Given that Assam’s tribal population accounts for only 12.4% of the total, a vast amount of nutrient-rich Eri pupae is wasted. To address this issue, the present study aims to identify the most protein-rich strain and life stage of S. ricini and develop a pupa-based candy to enhance its acceptance among the wider population. 2. Materials and Methods 2.1 Collection and indoor rearing of samples Fifth instar larvae of six distinct strains of Samia ricini were collected based on body coloration and markings from the Germplasm Conservation Centre, Chenijan, CMER&TI, Lahdoigarh, Jorhat, Assam. To obtain pure lines, each strain was reared for three successive generations. Eggs were surface-disinfected using a 2% formalin solution and thoroughly rinsed with distilled water. Upon hatching, larvae were initially fed tender leaves of Manihot esculenta (cassava), and subsequent rearing was conducted following the standardized protocol [ 17 ]. To minimize variability in nutrient composition due to environmental fluctuations, all samples for biochemical analysis were collected from a single crop reared during March–April (temperature: 25 ± 2°C; relative humidity: 80 ± 5%) at the Tangabari rearing site, Boko, Kamrup, Assam. 2.2 Sample Preparation The pre-pupal stage of the Eri silkworm ( Samia ricini ) was collected within four days of cocoon formation. Typically, the pupal stage inside the cocoon spans approximately 14 days; hence, pupae were harvested on the 10th day post-cocooning for sample preparation. Both pre-pupal and pupal specimens were carefully extracted from the cocoons, lyophilized at − 80°C to eliminate residual moisture, and subsequently ground into a fine powder following the protocol described by [ 18 ]. The resulting powder was wrapped in aluminium foil and stored in airtight zip-lock bags under appropriate conditions for further analyses. 2.3 Protein Estimation and Amino Acid Profiling Crude protein content of both the pre-pupal and pupal stages of six different insect strains was determined using the Kjeldahl method [ 19 ]. The yellow-spotted pre-pupa exhibited the highest crude protein content and was therefore selected for subsequent analyses. Amino acid profiling of the selected sample was performed using an amino acid analyser (Biochrom 30+). 2.4 Extraction of Protein Defatting of the selected sample was carried out based on the principle of “like dissolves like,” wherein non-polar compounds such as lipids were solubilized using a non-polar solvent, food-grade hexane. This selective solubilization effectively removed the lipid fraction while preserving polar constituents. Subsequently, protein extraction was performed using ultrasonication, a technique that facilitates the disruption of cellular structures and enhances the release of intracellular proteins into a polar solvent, such as ethanol. 2.5. Protein Estimation and Amino Acid Analysis of Extracted Protein Extracted protein was estimated by the Kjeldahl method, and amino acid analysis was done by an amino acid analyzer. 2.6 Candy preparation A total of 80g of palm jaggery was melted over low heat in a stainless-steel pan. Subsequently, 20 grams of protein powder, extracted from the selected yellow spotted pre-pupa strain, was incorporated into the melted jaggery to form a homogenous, thick slurry. To enhance organoleptic properties, a few drops of vanilla essence were added. Additionally, 10 mL of Garcinia-infused water (prepared by soaking 10 g of Garcinia in 100 mL distilled water for 12 hours) and 2 g of dried ginger ( Zingiber officinale ) leaves were introduced into the mixture. The entire formulation was continuously stirred and boiled until complete caramelization was achieved, as indicated by a characteristic change in color and texture. The hot mixture was then transferred into molds of various shapes and allowed to cool at refrigeration temperature (4 ± 1°C) until firm consistency was attained. 2.6 Shelf-life analysis Following conventional procedures, a shelf-life analysis of the extracted protein was conducted for 3, 6, 9, and 12 months to measure the fluctuations in protein levels in the ensuing months. 2.7 Sensory Analysis Sensory evaluation was performed for the prepared candies by a taste testing panel. The test was conducted after the experimental protocols had been approved by the Institutional Human Ethics Committee, Cotton University, Assam, India (Reference No CU/HEC/055/105/2023). The parameters for evaluation were appearance, taste, texture, color, and overall acceptability on a 9-point hedonic scale [ 20 ]: 9 = Like extremely, 8 = Like very much, 7 = Like moderately, 6 = Like slightly, 5 = Neither like nor dislike, 4 = Dislike slightly, 3 = Dislike moderately, 2 = Dislike very much, 1 = Dislike extremely. 2.8 Allergenicity study of extracted protein Eight weeks old of weight 35-40g adult males of Swiss albino mice were obtained from the Veterinary College, Khanapara, Assam. They were housed under a 12:12 hour light-dark cycle at 25 ± 1℃ and were fed on a standard pellet diet with water ad libitum. The experiments were performed after the experimental protocols had been approved by the Institutional Animal Ethics Committee, Cotton University, Assam, India (Reference No CU/AEC/0513/202/2023). All methods were carried out in accordance with relevant guidelines and regulations of the Institutional Animal Ethics Committee. The mice were divided into 3 groups, 6 mice in each group where Group I was considered as control and were provided a standard laboratory animal diet along with drinking water, Group II animals were administered 5 mg/kg Body weight peanut protein extract (PPE) and considered it as a positive control, and to Group III animals were fed 1.14 mg/kg extracted pre-pupa protein along with standard diet. After 20 minutes of sensitization, at intervals of 0,3,6,9, and 12 hours, the blood serum samples were tested for concentration of IgE, histamine, and number of eosinophils in each animal group using a hematology analyzer (CBC 360 Neo). 2.9 Toxicity study The toxicity study was carried out in 8-week-old mice weighing 35–40 g. The animals were divided into three groups, each group containing 6 animals. Group I, considered as control, were provided a standard laboratory animal diet along with drinking water, and Group II animals were given only silkworm prepupal protein powder (SPPP). After oral administration of 2000 mg/kg body weight of pre-pupa protein for 14 days and 28 days. The animals were sacrificed on the 15th and 29th days of the trial. The liver and kidneys of albino mice were obtained after they were slaughtered. The organs were immediately placed in normal saline. Organs were thoroughly cleansed with normal saline. The organs were subsequently put into glass vials containing formaldehyde fixative (4% formaldehyde). Tissues were cleaned with distilled water and stored in 70% alcohol for three days before further processing. The organs were then encased in paraffin and sectioned. The sections (3–4 µm) were deparaffinized and the slides were dehydrated, using a wash of xylene, 100% ethanol, 95% ethanol, 70% ethanol, and distilled water. After staining with hematoxylin and eosin (HE) for histopathological examination, the sections were washed under running distilled water for 10 min, dehydrated, mounted, and examined by light microscopy (Leica brightfield binocular DM750). The serum levels of Serum Glutamic Oxaloacetic Transaminase (SGOT), Serum Glutamic Pyruvic Transaminase (SGPT), Alkaline phosphatase, and creatinine were estimated by biochemical analysis (Cobas C11). 2.10 Statistical analysis Two-way ANOVA followed by Post-hoc Tukey test and t-test were performed in PAST 4.03 (2020) software. 3. Results In the present study, the crude protein content in the pre-pupa stages was found to be highest in Yellow Spotted (66.58 ± 0.35 g/100 g), followed by Yellow Plain (66.23 ± 0.31 g/100 g), Yellow Zebra (62.53 ± 0.34 g/100 g), Greenish Blue Spotted (62.22 ± 0.24 g/100 g), Greenish Blue Zebra (60.56 ± 0.32 g/100 g), and Greenish Blue Plain (43.31 ± 0.28 g/100 g). On the contrary, in the pupa stages, greater values of crude protein were recorded in Greenish Blue Zebra (64.56 ± 0.36 g/100 g), followed by Yellow Zebra (63.48 ± 0.30 g/100 g), Yellow spotted (46.49 ± 0.27 g/100 g), Greenish blue plain (40.40 ± 0.28 g/100 g), Greenish Blue Spotted (39.51 ± 0.32 g/100 g), and Yellow Plain (39.38 ± 0.33 g/100 g). However, pre-pupa of the Yellow spotted strain contained slightly higher content of protein than the pupal stage. Table 1 Values of crude protein content of the different strains of Eri silkworm in pre-pupa and pupa stages Strains of Eri Silkworm Crude protein content (g/100g or %) Pre-Pupa Stage Pupa Stage t-test Yellow plain (YP) 66.23 ± 0.31 b 39.38 ± 0.33 a p < 0.05 # Yellow spotted (YS) 66.58 ± 0.35 b 46.49 ± 0.27 c p 0.05 Greenish blue plain (GBP) 43.31 ± 0.28 g 40.40 ± 0.28 f p < 0.05 # Greenish blue spotted (GBS) 62.22 ± 0.24 h 39.51 ± 0.32 af p < 0.05 # Greenish blue zebra (GBZ) 60.56 ± 0.32 e 64.56 ± 0.36 i p < 0.05 # Values are means of three replicates ± Standard Deviation and expressed in g/100g. # Individual values differ significantly (p < 0.05) in both pre-pupa and pupa stages, having different superscripts. Essential amino acids such as Phenylalanine, Valine, Arginine, Tryptophan, Methionine, Leucine, Lysine, and Histidine were present in the crude protein extract. After extraction, the protein content was reduced to 53.71%, and thus loss of protein during extraction was 12.87%. Essential amino acid composition before and after protein extraction showed considerable changes, and threonine and leucine were completely absent after extraction. Table 2 Amino acid content before and after the extraction of protein Amino Acids Amount of Amino Acid Before Protein Extraction (g/100g) (A) Amount of Amino Acid After Protein Extraction(g/100g) (B) Phenylalanine 0.26 0.25 Valine 0.33 0.31 Arginine 0.79 0.77 Tryptophan 0.17 0.16 Methionine 0.05 0.03 Leucine 0.31 0.29 Lysine 1.44 1.42 Histidine 0.51 0.49 Followed by protein extraction, shelf-life analysis of the extracted protein for 3, 6, 9, and 12 months showed a decreasing trend of protein content from 53.71*±0.67 g to 48.81*±0.58g on the 3rd month, 42.57*±0.89 g on the 6th month, 36.34*±0.87 g on the 9th month, and 29.24*±89g on 12th month. The variations were found to be statistically significant (p ≤ 0.05). Sensory evaluation of prepared candy by testing panel revealed the overall acceptability score of the candy to be 8.7, appearance 8.5, texture 8, taste 8.5, and colour 8. 3.1 Allergenicity analysis In contrast to the elevated levels of IgE, histamine, and eosinophil counts observed in the peanut protein extract (PPE)-treated positive control group, animals administered with the extracted pre-pupa protein exhibited IgE concentrations (50–100 ng/mL), histamine levels (16.5 pmol/mL), and eosinophil percentages (1–3%) within the normal physiological range. These findings suggest that the pre-pupa protein did not elicit any significant inflammatory response in the in vivo model. 3.2 Toxicity study Histological examination of liver and kidney tissues revealed no pathological alterations in animals administered the extracted protein powder for 14 and 28 days, compared to the control group. Additionally, all recorded biochemical parameters remained within normal physiological ranges, further supporting the safety of the extracted protein powder for consumption. Table 3 Creatinine values recorded in different groups of mice on the 14th and 28th day Mice groups Creatinine (U/L) (Normal range: 0.2–0.5) 14th day 28th day Group I (controlled) 0.23 ± 0.02 a 0.27 ± 0.04 a Group II 0.44 ± 0.03 b 0.49 ± 0.06 bc Values are the mean of six replicates ± Standard deviation Different superscripts along the rows and columns differ significantly Table 4 ALP values recorded in different groups of mice on the 14th and 28th day Mice groups ALP (U/L) (Normal range: 62–230) 14th day 28th day Group I (controlled) 98.00 ± 0.05 a 98.69 ± 0.04 c Group II 206.71 ± 0.06 b 220.80 ± 0.06 d Values are the mean of six replicates ± Standard deviation Different superscripts along the rows and columns differ significantly Table 5 SGPT values recorded in different groups of mice on the 14th and 28th day Mice groups SGPT (U/L) (Normal range: 7–56) 14th day 28th day Group I (controlled) 24.21 ± 0.06 a 24.55 ± 0.04 c Group II 29.29 ± 0.07 b 29.52 ± 0.06 d Values are the mean of six replicates ± Standard deviation Different superscripts along the rows and columns differ significantly Table 6 SGOT values recorded in different groups of mice on the 14th and 28th day Mice groups SGOT (U/L) (Normal range: 8–45) 14th day 28th day Group I (controlled) 22.51 ± 0.06 a 23.31 ± 0.04 c Group II 27.16 ± 0.05 b 29.56 ± 0.05 d Values are the mean of six replicates ± Standard deviation Different superscripts along the rows and columns differ significantly 4. Discussion The incorporation of edible insects into the human diet is gaining global traction due to their high-quality proteins, beneficial lipids, essential micronutrients, and favorable sensory properties. The global edible insect protein market, valued at approximately USD 144 million in 2019, is projected to grow at a compound annual growth rate (CAGR) of 45%, reaching an estimated USD 1,366 million by 2025 [ 21 ]. Despite the persistence of food neophobia in several regions, insect-based foods have been successfully commercialized and are often perceived as delicacies. These products are available in various forms, including flours, heat-dried larvae and pupae, protein isolates, canned products, extruded snacks, confectioneries, and liquid infusions. In the past decade, over 250 insect-based products have been developed by both small- and large-scale food industries [ 22 ]. Due to their high protein content and well-balanced amino acid profiles, edible insects have garnered considerable interest as potential ingredients in nutritionally enriched products, particularly those targeted at athletic populations. These include hydrolysates, protein isolates and concentrates, energy bars, and protein-fortified beverages [ 23 ]. The convergence of innovation in insect-based food technologies, regulatory advancements, increased media attention, and wider product availability has fostered significant market growth [ 24 ]. Additionally, edible insects have demonstrated utility in value-added functional foods. For instance, powders derived from Aspergillus oryzae and Bacillus licheniformis have been employed as substrates for developing fermented liquid seasonings integrated into soy sauce production [ 23 ]. To address childhood malnutrition, extruded food products incorporating sun-dried termites, germinated amaranth, corn, and soy oil have been formulated [ 25 ]. Cricket flour has also been effectively used in cookie formulations alongside amaranth, rice, and oatmeal, yielding products with high acceptability among children [ 26 ]. With the rising global interest in incorporating edible insects into food products to enhance their nutritional value and appeal, the present study evaluates the nutritional profile of the pre-pupal and pupal stages of six distinct strains of the Eri silkworm ( Samia ricini ), traditionally consumed by various tribal communities in Northeast India. Among the strains analyzed, the pre-pupal stage of the Yellow-Spotted strain demonstrated the highest protein content, measured at 66.58 g per 100 g of dry sample, surpassing the protein content of most conventional livestock sources [ 28 ]. Further amino acid profiling revealed that the protein from Yellow-Spotted pre-pupae qualifies as a complete protein, containing all essential amino acids in substantial quantities. According to the FAO/WHO (2023) essential amino acid scoring pattern, the amino acid composition generally meets the required proportions for human nutrition, with minor deficiencies noted in threonine and leucine. Notably, the high lysine content (1.69 g/100 g protein) in Yellow-Spotted pre-pupae offers a potential nutritional advantage, particularly in complementing lysine-deficient cereal-based diets [ 27 ]. These findings affirm the potential of Eri silkworm pre-pupae, especially the Yellow-Spotted strain, as a sustainable and high-quality protein source for human consumption [ 28 ]. This study represents the first sub-acute toxicity assessment of a strain-specific silkworm variant characterized by the highest protein content among its six available strains. Throughout the experimental period, no signs of toxicity or abnormal clinical symptoms, such as seizures, vomiting, anorexia, or lethargy, were observed in the treated mice. These findings align with those of [ 29 ], who similarly reported no toxicological effects in in vivo models administered Bombyx mori . However, a statistically significant increase (p < 0.05) in serum creatinine levels was observed in Group II mice on day 14 and day 28 of the feeding period. Despite this elevation, creatinine levels remained within the established physiological reference range. This increase may be attributed to enhanced nitrogen metabolism and a consequent rise in renal filtration workload, suggesting that prolonged intake of pre-pupal powder could mildly influence renal function. Monitoring liver marker enzyme levels is essential in assessing liver function and identifying potential issues with the liver's ability to process and excrete waste. Although a significant increase (p < 0.05) in ALP, SGOT, and SGPT was observed in group II mice during the feeding period for 14 days and 28 days, the resulting values were within the reference range. The significant increase in ALP might be because of greater nitrogen metabolism and filtration workload. This finding was further supported by the absence of histopathological changes in the liver. Therefore, the observed differences and the apparent lack of any impact on these enzymes make it challenging to identify any disease condition, indicating that the consumption of the test diet does not lead to hepatotoxicity and nephrotoxicity. Increasing consumer awareness regarding the edibility of insects has been shown to enhance their willingness to pay for insect-based foods [ 30 ]. To promote acceptance, it is essential to highlight the social, practical, and contextual factors influencing food consumption. Such efforts should involve ongoing education and marketing initiatives that emphasize the potential role of edible insects in addressing contemporary and future challenges related to environmental sustainability, population growth, and land use [ 31 ]. Fermented liquid seasoning, developed using Aspergillus oryzae and Bacillus licheniformis powders as substrates, has been incorporated into soy sauce fermentation [ 32 ]. Studies have also demonstrated the fortification of approximately 90% of mealworm larvae in the formulation of minced meat-like products, preserved under modified atmospheric packaging [ 33 ]. Furthermore, silkworm pupa powder has been utilized in meat batters for the production of frankfurters, significantly reducing cooking laws while improving textural properties [ 34 ]. In efforts to combat infant malnutrition, extruded food products have been developed using sun-dried termites combined with germinated amaranth, corn, and soybean oil [ 25 ]. The incorporation of Nauphoeta cinerea flour at concentrations of 5–15% in brad formulations has been studied, with an emphasis on its physical sensory properties [ 35 ]. Additionally, cookies made from cricket flour blended with amaranth, rice, and oatmeal are well accepted by children [ 13 ]. Keeping this view in mind, the present study has developed a protein-rich candy based on silkworm pre-pupa. Additionally, since sugar has been replaced with palm jaggery, which itself has many useful health benefits like antihepatotoxic, anti-inflammatory, and anti-hyperglycemic effects [ 36 ], and can be a valuable alternative for people avoiding sugar-based products due to health risks like diabetes. Unlike sugar, which spikes blood glucose levels, palm jaggery has a lower glycemic index, making it a safer option for individuals with diabetes or those at risk of developing it. Studies have shown that palm jaggery possesses anti-hepatotoxic properties, helping detoxify the liver and protect it from oxidative damage, which is vital for overall health. It also exhibits anti-inflammatory effects, which can aid in managing chronic conditions like arthritis or autoimmune diseases. Besides, palm jaggery is rich in essential minerals like iron, calcium, magnesium, and potassium, which support bone health, regulate blood pressure, and promote overall well-being. Its mild laxative effect supports digestive health, improving digestion and preventing constipation. Moreover, because palm jaggery is a natural, unrefined sweetener, it provides a wholesome source of energy and can satisfy sweet cravings without the negative impacts of processed sugar. With its various health benefits, including boosting immunity and promoting healthy digestion, palm jaggery is not just a healthier sweetener but also a valuable addition to a balanced diet. It is an ideal option for those looking to reduce sugar intake without sacrificing taste or health benefits. By incorporating palm jaggery into daily use, individuals can enjoy a more nutritious and sustainable way to sweeten their food while supporting their overall health. Furthermore, the leaves of Garcinia pedunculata and ginger may have antihyperglycemic, antidiabetic, antioxidant, and anti-inflammatory properties [ 37 – 40 ], which can improve our general health. 5. Conclusion The findings of this study highlight the potential for developing a protein-rich functional candy using silkworm pre-pupae protein powder, offering a health-promoting and accessible dietary option, particularly in developing countries facing food insecurity due to rapid population growth. Sensory evaluation results demonstrated high acceptability across all parameters, indicating favourable organoleptic properties of the formulated candy. The replacement of refined sugar with palm jaggery further enhances the product’s nutritional profile, providing added health benefits and making it suitable for individuals seeking alternatives to conventional sugar, including those managing diabetes. This innovation is expected to promote the consumption of Eri silkworm pre-pupae and pupae, thereby increasing demand and contributing to the socio-economic empowerment of local Eri silkworm rearers. Additionally, the candy may serve as a low-cost, high-protein food source during emergencies or crises. Declarations Acknowledgement The authors are thankful to Guwahati Biotech Park, Assam for providing the instrumentation necessary for the analysis of sample. Authors S.D.: original draft preparation; writing-review & editing, experimentation; P.S.: writing-review & editing, illustration, resources; M.P.: collection of data, analysis, writing-review & editing K.A.N.: writing-review & editing; K.K.: Data interpretation and analysis; S.S.: Data interpretation and analysis; R.L.: data curation, writing & editing, A.D.: writing-review & editing; S.M.: writing-review & editing; S.D.: writing-review & editing, data interpretation; A.S.: writing-review & editing; T.K.: Supervision, methodology, original draft preparation, writing-review & editing, resources. Funding Sources The author(s) received no financial support for the research, authorship, and/or publication of this article. Data availability All data generated or analyzed during this study are included in this published article. Conflict of Interests The authors declare no conflict of interest. Ethics Statement The approval for the use of Swiss albino mice during protein treatment analysis was duly taken from the Institutional Animal Ethics Committee of Cotton University. The approval for participation of human research participants for sensory evaluation test was taken from the Institutional Human Ethics Committee of Cotton University Consent to Participate All the participants were adults (18+) and informed consent for participation in the 9-point hedonic scale sensory evaluation for protein candy developed in the current study was taken accordingly. Consent to Publish Not Applicable References FAO/WHO, 1973. Energy and Protein Requirements: Report of a Joint FAO/WHO Ad Hoc Expert Committee. Rome: FAO Nutrition Meetings Report Series No. 52. Geneva: WHO Technical Report Series No. 522. Hague: Dr. W. Junk Publishers. Doi: http://dx.doi.org/10.1007/978-94-017-6159-8. Bodenheimer, F. S. Insects as human food. In Insects as Human Food: A Chapter of the Ecology of Man. 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Current opinion in Insect Science. 2021; 48:32-36. Cho, J. H., Zhao, H. L., Kim, J. S., Kim, S. H., Chung, C. H. Characteristics of fermented seasoning sauces using Tenebrio molitor larvae. Innovative Food Science & Emerging Technologies. 2018; 45 186-195. Luna, G. C., San Martin-Gonzalez, F., Mauer, L. J., Liceaga, A. M. Cricket ( Acheta domesticus ) protein hydrolysates’ impact on the physicochemical, structural, and sensory properties of tortillas and tortilla chips. Journal of Insects as Food and Feed. 2021; 7 (1), 109-120. Varelas V., Langton, M. Forest biomass waste as a potential innovative source for rearing edible insects for food and feed- A review. Innovative Food Science & Emerging Technologies. 2017; 41: 205-209. Kinyuru J.N., Mogendi J.B., Riwa C.A., Ndung’u N.W. Edible insects—a novel source of essential nutrients for human diet: learning from traditional knowledge. Animal Front. 2015; 5:14 19. Kim T.K., Yong H.I., Kim Y.B., Kim H.W., Choi Y.S. Edible insects as a protein source: a review of public perception, processing technology, and research trends. Food Science and Animal Resources. 2019; 39: 521-540. doi:10.5851/kosfa. 2019.e53 Pasiakos, S. M., Agarwal, S., Lieberman, H. R., Fulgoni, V. L. Sources and Amounts of Animal, Dairy, and Plant Protein Intake of US Adults in 2007-2010. Nutrients. 2007; 7: 7058–7069. https://doi.org/10.3390/nu7085322 Delgado-Andrade, C., Rufián-Henares, J. A., Morales, F. J. Lysine availability is diminished in commercial fibre-enriched breakfast cereals. Food Chemistry 2007; 100: 725-731. Qian, J. Q. Chemical constituents and exploitation of silkworm pupae. Food Industry. 1997; 5: 42–43 Rattana, S. K., Teeraporn, S., and Bunleu, C. Acute and Sub-acute Toxicities of Thai Silkworm Powder (Bombyx mori Linn.) From Three Races in Male Wistar Rats and In Vitro Antioxidant Activities. Pharmacognosy Journal. 2017; 9: 541-545. 10.5530/pj.2017.4.87. Piha, S., Pohjanheimo, T., Lähteenmäki-Uutela, A., Křečková, Z., Otterbring, T. The effects of consumer knowledge on the willingness to buy insect food: An exploratory cross-regional study in Northern and Central Europe. Food Quality and Preference. 2016; 70: 1–10. https://doi.org/10.1016/j.foodqual.2016.12.006 Evans, J., Alemu, M. H., Flore, R., Frøst, M. B., Halloran, A., Jensen, A. B., Maciel- Vergara, G., Meyer- Rochow, V. B., Münke- Svendsen, C., Olsen, S. B., Payne, C., Roos, N., Rozin, P., Tan, H. S. G., Huis, A. V., Vantomme, P., and Eilenberg, J. ‘Entomophagy’: an evolving terminology in need of review. Journal of Insects as Food and Feed. 2015; 1: 293-305. Stoops, J., Vandeweyer, D., Crauwels, S., Verreth, C., Boeckx, H., Borght, M.V.D., Claes, J., Lievens, B., Campenhout, L.V. Minced meat-like products from mealworm larvae ( Tenebrio molitor and Alphitobius diaperinus ): microbial dynamics during production and storage. Innovative Food Science & Emerging Technologies . 2017; 41 , 1–9. Park, Y., Choi, Y., Hwang, K., Kim, T., Lee, C., Shin, D., Han, S. G. Physicochemical Properties of Meat Batter Added with Edible Silkworm Pupae ( Bombyx mori ) and Transglutaminase. Korean Journal for Food Science of Animal Resources. 2017; 37: 351–359. https://doi.org/10.5851/kosfa.2017.37.3.351 Oliveira, L., Carolina, C., Mellado, M. Bread enriched with flour from the cinereous cockroach (Nauphoeta cinerea). Innovative Food Science & Emerging Technologies. 2015; 44-48. Kumar, A., Singh, S., The benefits of Indian jaggery over sugar on human health. Dietary Sugar, Salt and Fat in Human Health, Academic Press Publisher, Washington, United States. 2020 10.1016/B978-0-12-816918-6.00016-0. Ozkur M, Benlier N, Takan I, Vasileiou C, Georgakilas AG, Pavlopoulou A, Cetin Z, Saygili EI. Ginger for Healthy Ageing: A Systematic Review on Current Evidence of Its Antioxidant, Anti-Inflammatory, and Anticancer Properties. Oxid Med Cell Longev. 2022; 2022:4748447. doi: 10.1155/2022/4748447 Ali MY, Paul S, Tanvir EM, Hossen MS, Rumpa NN, Saha M, Bhoumik NC, Aminul Islam M, Hossain MS, Alam N, Gan SH, Khalil MI. Antihyperglycemic, Antidiabetic, and Antioxidant Effects of Garcinia pedunculata in Rats. Evid Based Complement Alternat Med. 2017; 2:2979760. Doi: 10.1155/2017/2979760. Mashhadi N.S., Ghiasvand R., Askari G., Hariri M., Darvishi L., Mofid M.R. Anti-oxidative and anti-inflammatory effects of ginger in health and physical activity: review of current evidence. Int J Prev Med. 2013 Apr;4(Suppl 1) Espirito Santo BLSD, Santana LF, Kato Junior WH, de Araújo FO, Bogo D, Freitas KC, Guimarães RCA, Hiane PA, Pott A, Filiú WFO, Arakaki Asato M, Figueiredo PO, Bastos PRHO. Medicinal Potential of Garcinia Species and Their Compounds. Molecules. 2020; 25(19):4513. doi: 10.3390/molecules25194513. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 22 Jun, 2025 Reviews received at journal 19 Jun, 2025 Reviewers agreed at journal 17 Jun, 2025 Reviews received at journal 16 Jun, 2025 Reviewers agreed at journal 08 Jun, 2025 Reviewers agreed at journal 03 Jun, 2025 Reviewers invited by journal 30 May, 2025 Editor assigned by journal 25 May, 2025 Submission checks completed at journal 13 May, 2025 First submitted to journal 13 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6526487","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":464760317,"identity":"eaf9cf04-7c56-476d-9c90-56b31c8fa106","order_by":0,"name":"Sweta Das","email":"","orcid":"","institution":"Cotton University","correspondingAuthor":false,"prefix":"","firstName":"Sweta","middleName":"","lastName":"Das","suffix":""},{"id":464760318,"identity":"944e6b6e-8133-4581-a32f-028b6af0635b","order_by":1,"name":"Priyanku Sarma","email":"","orcid":"","institution":"Cotton 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University","correspondingAuthor":true,"prefix":"","firstName":"Tarali","middleName":"","lastName":"Kalita","suffix":""}],"badges":[],"createdAt":"2025-04-25 07:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6526487/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6526487/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83759841,"identity":"d8106737-23d9-43d8-aa85-03051f5c00aa","added_by":"auto","created_at":"2025-06-02 09:14:24","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96549,"visible":true,"origin":"","legend":"\u003cp\u003eResults showing values of essential amino acids before and after protein extraction. Different colors of the columns in the graph indicate a comparison of essential amino acids before and after protein extraction.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6526487/v1/03861ed2154eac2994ed6778.png"},{"id":83759994,"identity":"c0496a77-8b17-495c-9aae-e6ee90f2b001","added_by":"auto","created_at":"2025-06-02 09:22:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1747237,"visible":true,"origin":"","legend":"\u003cp\u003ePreparation of Candy- (a) Collection of Pre pupa of the cocoon (b) Protein after extraction from pre pupa (c) Caramelization of pre pupa protein along with palm jaggery, garcinia and ginger leaves powder (d) Pouring of the mixture into the molds.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6526487/v1/79b6a6442dc6a60b1f7062d8.png"},{"id":83759847,"identity":"56c1a7cf-0e2b-42c6-b54f-32ed334c5ce2","added_by":"auto","created_at":"2025-06-02 09:14:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":844318,"visible":true,"origin":"","legend":"\u003cp\u003eChocolates taken out from molds (left) and packed in wrappers (right).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6526487/v1/faa0e25b9710090fb8edaf4f.png"},{"id":83759839,"identity":"64d4188a-5870-4711-8a4f-04f1026459e5","added_by":"auto","created_at":"2025-06-02 09:14:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":54600,"visible":true,"origin":"","legend":"\u003cp\u003eResults showing scores of sensory evaluations tested by a trusted panel. Different colors of the pie chart show different scores of the parameters taken for sensory evaluation.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6526487/v1/cb8465c49a62a8ef366cdee9.png"},{"id":83759853,"identity":"726b0ca1-9caa-449a-84a6-b57403eea524","added_by":"auto","created_at":"2025-06-02 09:14:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3212700,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 4. \u003c/strong\u003eH\u0026amp; E-stained Liver histological slides (40X magnification): a. Group I (Control; Cv=central vein, KC=kuffer cells, H=hepatic Cells, N=Nucleus), b. Group II on the 14\u003csup\u003eth\u003c/sup\u003e day, c. Group II on 28\u003csup\u003eth\u003c/sup\u003e day\u003c/p\u003e","description":"","filename":"44.png","url":"https://assets-eu.researchsquare.com/files/rs-6526487/v1/ef2dfcfde2a00895d96300dc.png"},{"id":83759858,"identity":"184c2293-d6e2-45b5-bf13-0a713e17ed64","added_by":"auto","created_at":"2025-06-02 09:14:24","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2923850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig 5. \u003c/strong\u003eH\u0026amp; E-stained Kidney histological slides (40X magnification): a. Group I (Control), b. Group II (SPPP only) on the 14\u003csup\u003eth\u003c/sup\u003e day, c. Group II (SPPP only) on the 28\u003csup\u003eth\u003c/sup\u003e day,\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6526487/v1/36953c0b2557ca4534e136a7.png"},{"id":83761217,"identity":"45470dd4-dc10-4f72-b8c7-539489a336e5","added_by":"auto","created_at":"2025-06-02 09:38:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15234113,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6526487/v1/82511e97-2f62-472c-bc6d-3bbb38a2b67d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Silkworm protein candy: An approach towards nutritional waste management and sustainable development","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe global human population has been increasing at an unprecedented rate, reaching 8.1\u0026nbsp;billion in 2024 from an estimated 2.5\u0026nbsp;billion in 1950, according to a 1998 United Nations (UN) study. This surge has equated to nearly 1\u0026nbsp;billion people being added every decade since 2010. Projections suggest that the population will reach 9.7\u0026nbsp;billion by 2050 and nearly 10.4\u0026nbsp;billion by the mid-2080s (UN article: \u003cem\u003ePeace, Dignity, and Equality on a Healthy Planet\u003c/em\u003e). This rapid growth presents numerous challenges, particularly in managing the availability of essential resources such as food and shelter. One of the most pressing concerns is the strain on the planet\u0026rsquo;s limited resources, especially arable land. According to the Food and Agriculture Organization (FAO), global agricultural land covers approximately 5\u0026nbsp;billion hectares\u0026mdash;around 38% of the Earth's total land area. However, further expansion of agricultural land could lead to severe ecological imbalances, as converting natural ecosystems into farmland has historically been a major driver of greenhouse gas emissions, primarily due to biomass loss and depletion of carbon stores both above and below the ground [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Traditional food production systems, including staple crops like wheat and livestock farming, face mounting challenges such as land scarcity, water shortages, declining soil fertility, and increased greenhouse gas emissions. Given these constraints, there is an urgent need to explore alternative food sources and sustainable nutrition strategies to ensure food security for the growing global population.\u003c/p\u003e \u003cp\u003eEntomophagy, or the practice of consuming insects as food, has been a part of human civilization for centuries, particularly in Asia, Africa, and Latin America [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Globally, approximately 2,000 insect species across 18 different orders are consumed [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In India, insect consumption is particularly prevalent among tribal communities in the northeastern region, where they are considered a delicacy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Insects are an exceptionally nutritious source of protein, containing all nine essential amino acids [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. They are also rich in important vitamins and minerals, such as vitamin B12, which is crucial for brain function, as well as iron, calcium, and zinc [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Compared to traditional protein sources, insects offer several advantages: for example, crickets contain more protein per gram than beef [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. While conventional sources like chicken and pork provide the same essential amino acids, they may lack the diverse range of vitamins and minerals found in insects. Beyond nutritional benefits, insect consumption also has significant environmental and economic advantages. Insect farming requires considerably fewer resources, such as land, water, and feed, generating only a fraction of the greenhouse gas emissions associated with traditional livestock farming. Additionally, insect farming is cost-effective, making it a more affordable protein source for consumers. Insects also exhibit high adaptability, thriving in diverse environments, which enhances their potential as a sustainable alternative to conventional livestock production [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSilkworms are primarily reared for their valuable silk, but the sericulture industry also produces several by-products, including pupae, cocoon shells, and silkworm droppings, which can provide additional income for farmers. Silkworm pupae, a major by-product, are often discarded or used as animal feed and fertilizer. However, there is growing interest in their potential as a nutrient-rich food source, particularly in Asia, where they are traditionally consumed as snacks or added to various dishes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Silkworm pupae are high in protein, comprising 50\u0026ndash;60% of their dry weight, and contain essential amino acids like lysine and methionine, often lacking in plant-based diets [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. They are also rich in essential fatty acids, vitamins, and minerals such as zinc, iron, and calcium, with an energy density comparable to chicken and beef [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, studies suggest that silkworm pupae may improve cholesterol levels, glucose regulation, and lipid metabolism [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], and their antioxidant properties have been linked to anticancer and anti-inflammatory effects [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Despite their nutritional benefits, global acceptance remains low, especially in Western countries, due to cultural stigmas. A study comparing consumer preferences in China and Germany found that Germans favored processed insect products like cricket flour, while the Chinese preferred whole insects [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. To increase acceptance, processing silkworm pupae into more palatable and familiar forms could help integrate this sustainable protein source into mainstream diets.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSamia ricini\u003c/em\u003e, commonly known as the Eri silkworm, is a multivoltine silk-producing insect found throughout the year in Assam and Northeast India. It belongs to the order Lepidoptera and the family Saturniidae. The species undergoes complete metamorphosis with distinct life stages: egg, larva, pupa, cocoon, and adult. Based on larval body color and markings, six distinct strains of \u003cem\u003eS. ricini\u003c/em\u003e have been identified: Greenish Blue Plain (GBP), Yellow Plain (YP), Greenish Blue Spotted (GBS), Yellow Spotted (YS), Greenish Blue Zebra (GBZ), and Yellow Zebra (YZ) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Among its life stages, the pre-pupa (just after cocoon spinning) and pupa are edible and widely consumed by tribal communities in Assam and Northeast India. However, non-tribal populations generally avoid Eri pupae due to social stigma and cultural taboos. Assam, the third-largest producer of raw Eri silk in India, produces approximately 7,364 metric tons (MT) of Eri cocoons annually, resulting in a significant quantity of Eri pupae. Despite published research highlighting its high nutritional value, particularly its rich protein content [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], much of the pupae remain underutilized and are discarded as sericulture waste, contributing to environmental hazards. Given that Assam\u0026rsquo;s tribal population accounts for only 12.4% of the total, a vast amount of nutrient-rich Eri pupae is wasted. To address this issue, the present study aims to identify the most protein-rich strain and life stage of \u003cem\u003eS. ricini\u003c/em\u003e and develop a pupa-based candy to enhance its acceptance among the wider population.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Collection and indoor rearing of samples\u003c/h2\u003e \u003cp\u003eFifth instar larvae of six distinct strains of \u003cem\u003eSamia ricini\u003c/em\u003e were collected based on body coloration and markings from the Germplasm Conservation Centre, Chenijan, CMER\u0026amp;TI, Lahdoigarh, Jorhat, Assam. To obtain pure lines, each strain was reared for three successive generations. Eggs were surface-disinfected using a 2% formalin solution and thoroughly rinsed with distilled water. Upon hatching, larvae were initially fed tender leaves of \u003cem\u003eManihot esculenta\u003c/em\u003e (cassava), and subsequent rearing was conducted following the standardized protocol [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. To minimize variability in nutrient composition due to environmental fluctuations, all samples for biochemical analysis were collected from a single crop reared during March\u0026ndash;April (temperature: 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C; relative humidity: 80\u0026thinsp;\u0026plusmn;\u0026thinsp;5%) at the Tangabari rearing site, Boko, Kamrup, Assam.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Sample Preparation\u003c/h2\u003e \u003cp\u003eThe pre-pupal stage of the Eri silkworm (\u003cem\u003eSamia ricini\u003c/em\u003e) was collected within four days of cocoon formation. Typically, the pupal stage inside the cocoon spans approximately 14 days; hence, pupae were harvested on the 10th day post-cocooning for sample preparation. Both pre-pupal and pupal specimens were carefully extracted from the cocoons, lyophilized at \u0026minus;\u0026thinsp;80\u0026deg;C to eliminate residual moisture, and subsequently ground into a fine powder following the protocol described by [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The resulting powder was wrapped in aluminium foil and stored in airtight zip-lock bags under appropriate conditions for further analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Protein Estimation and Amino Acid Profiling\u003c/h2\u003e \u003cp\u003eCrude protein content of both the pre-pupal and pupal stages of six different insect strains was determined using the Kjeldahl method [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The yellow-spotted pre-pupa exhibited the highest crude protein content and was therefore selected for subsequent analyses. Amino acid profiling of the selected sample was performed using an amino acid analyser (Biochrom 30+).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Extraction of Protein\u003c/h2\u003e \u003cp\u003eDefatting of the selected sample was carried out based on the principle of \u0026ldquo;like dissolves like,\u0026rdquo; wherein non-polar compounds such as lipids were solubilized using a non-polar solvent, food-grade hexane. This selective solubilization effectively removed the lipid fraction while preserving polar constituents. Subsequently, protein extraction was performed using ultrasonication, a technique that facilitates the disruption of cellular structures and enhances the release of intracellular proteins into a polar solvent, such as ethanol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Protein Estimation and Amino Acid Analysis of Extracted Protein\u003c/h2\u003e \u003cp\u003eExtracted protein was estimated by the Kjeldahl method, and amino acid analysis was done by an amino acid analyzer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Candy preparation\u003c/h2\u003e \u003cp\u003eA total of 80g of palm jaggery was melted over low heat in a stainless-steel pan. Subsequently, 20 grams of protein powder, extracted from the selected yellow spotted pre-pupa strain, was incorporated into the melted jaggery to form a homogenous, thick slurry. To enhance organoleptic properties, a few drops of vanilla essence were added. Additionally, 10 mL of Garcinia-infused water (prepared by soaking 10 g of Garcinia in 100 mL distilled water for 12 hours) and 2 g of dried ginger (\u003cem\u003eZingiber officinale\u003c/em\u003e) leaves were introduced into the mixture. The entire formulation was continuously stirred and boiled until complete caramelization was achieved, as indicated by a characteristic change in color and texture. The hot mixture was then transferred into molds of various shapes and allowed to cool at refrigeration temperature (4\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) until firm consistency was attained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Shelf-life analysis\u003c/h2\u003e \u003cp\u003eFollowing conventional procedures, a shelf-life analysis of the extracted protein was conducted for 3, 6, 9, and 12 months to measure the fluctuations in protein levels in the ensuing months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Sensory Analysis\u003c/h2\u003e \u003cp\u003eSensory evaluation was performed for the prepared candies by a taste testing panel. The test was conducted after the experimental protocols had been approved by the Institutional Human Ethics Committee, Cotton University, Assam, India (Reference No CU/HEC/055/105/2023). The parameters for evaluation were appearance, taste, texture, color, and overall acceptability on a 9-point hedonic scale [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]:\u003c/p\u003e \u003cp\u003e \u003cb\u003e9\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Like extremely, \u003cb\u003e8\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Like very much, \u003cb\u003e7\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Like moderately, \u003cb\u003e6\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Like slightly, \u003cb\u003e5\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Neither like nor dislike, \u003cb\u003e4\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Dislike slightly, \u003cb\u003e3\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Dislike moderately, \u003cb\u003e2\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Dislike very much, \u003cb\u003e1\u003c/b\u003e\u0026thinsp;=\u0026thinsp;Dislike extremely.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Allergenicity study of extracted protein\u003c/h2\u003e \u003cp\u003eEight weeks old of weight 35-40g adult males of Swiss albino mice were obtained from the Veterinary College, Khanapara, Assam. They were housed under a 12:12 hour light-dark cycle at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1℃ and were fed on a standard pellet diet with water \u003cem\u003ead libitum.\u003c/em\u003e The experiments were performed after the experimental protocols had been approved by the Institutional Animal Ethics Committee, Cotton University, Assam, India (Reference No CU/AEC/0513/202/2023). All methods were carried out in accordance with relevant guidelines and regulations of the Institutional Animal Ethics Committee.\u003c/p\u003e \u003cp\u003eThe mice were divided into 3 groups, 6 mice in each group where Group I was considered as control and were provided a standard laboratory animal diet along with drinking water, Group II animals were administered 5 mg/kg Body weight peanut protein extract (PPE) and considered it as a positive control, and to Group III animals were fed 1.14 mg/kg extracted pre-pupa protein along with standard diet. After 20 minutes of sensitization, at intervals of 0,3,6,9, and 12 hours, the blood serum samples were tested for concentration of IgE, histamine, and number of eosinophils in each animal group using a hematology analyzer (CBC 360 Neo).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Toxicity study\u003c/h2\u003e \u003cp\u003eThe toxicity study was carried out in 8-week-old mice weighing 35\u0026ndash;40 g. The animals were divided into three groups, each group containing 6 animals. Group I, considered as control, were provided a standard laboratory animal diet along with drinking water, and Group II animals were given only silkworm prepupal protein powder (SPPP). After oral administration of 2000 mg/kg body weight of pre-pupa protein for 14 days and 28 days. The animals were sacrificed on the 15th and 29th days of the trial. The liver and kidneys of albino mice were obtained after they were slaughtered. The organs were immediately placed in normal saline. Organs were thoroughly cleansed with normal saline. The organs were subsequently put into glass vials containing formaldehyde fixative (4% formaldehyde). Tissues were cleaned with distilled water and stored in 70% alcohol for three days before further processing. The organs were then encased in paraffin and sectioned. The sections (3\u0026ndash;4 \u0026micro;m) were deparaffinized and the slides were dehydrated, using a wash of xylene, 100% ethanol, 95% ethanol, 70% ethanol, and distilled water. After staining with hematoxylin and eosin (HE) for histopathological examination, the sections were washed under running distilled water for 10 min, dehydrated, mounted, and examined by light microscopy (Leica brightfield binocular DM750).\u003c/p\u003e \u003cp\u003eThe serum levels of Serum Glutamic Oxaloacetic Transaminase (SGOT), Serum Glutamic Pyruvic Transaminase (SGPT), Alkaline phosphatase, and creatinine were estimated by biochemical analysis (Cobas C11).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003e Two-way ANOVA followed by Post-hoc Tukey test and t-test were performed in PAST 4.03 (2020) software.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eIn the present study, the crude protein content in the pre-pupa stages was found to be highest in Yellow Spotted (66.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35 g/100 g), followed by Yellow Plain (66.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 g/100 g), Yellow Zebra (62.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34 g/100 g), Greenish Blue Spotted (62.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24 g/100 g), Greenish Blue Zebra (60.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 g/100 g), and Greenish Blue Plain (43.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 g/100 g).\u003c/p\u003e \u003cp\u003eOn the contrary, in the pupa stages, greater values of crude protein were recorded in Greenish Blue Zebra (64.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 g/100 g), followed by Yellow Zebra (63.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 g/100 g), Yellow spotted (46.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 g/100 g), Greenish blue plain (40.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 g/100 g), Greenish Blue Spotted (39.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 g/100 g), and Yellow Plain (39.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33 g/100 g). However, pre-pupa of the Yellow spotted strain contained slightly higher content of protein than the pupal stage.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eValues of crude protein content of the different strains of Eri silkworm in pre-pupa and pupa stages\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eStrains of Eri Silkworm\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCrude protein content (g/100g or %)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003ePre-Pupa Stage Pupa Stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003et-test\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYellow plain (YP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.33\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYellow spotted (YS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e66.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.35\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e46.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eYellow zebra (YZ)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e63.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreenish blue plain (GBP)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e43.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e40.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreenish blue spotted (GBS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e62.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003csup\u003eh\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003eaf\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGreenish blue zebra (GBZ)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e64.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36\u003csup\u003ei\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003csup\u003e#\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003eValues are means of three replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Deviation and expressed in g/100g.\u003c/p\u003e \u003cp\u003e\u003csup\u003e\u003cb\u003e#\u003c/b\u003e\u003c/sup\u003eIndividual values differ significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in both pre-pupa and pupa stages, having different superscripts.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eEssential amino acids such as Phenylalanine, Valine, Arginine, Tryptophan, Methionine, Leucine, Lysine, and Histidine were present in the crude protein extract. After extraction, the protein content was reduced to 53.71%, and thus loss of protein during extraction was 12.87%. Essential amino acid composition before and after protein extraction showed considerable changes, and threonine and leucine were completely absent after extraction.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAmino acid content before and after the extraction of protein\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmino Acids\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAmount of Amino Acid Before Protein Extraction (g/100g)\u003c/p\u003e \u003cp\u003e(A)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmount of Amino Acid After Protein\u003c/p\u003e \u003cp\u003eExtraction(g/100g)\u003c/p\u003e \u003cp\u003e(B)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePhenylalanine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eValine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eArginine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTryptophan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMethionine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLeucine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLysine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.42\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHistidine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFollowed by protein extraction, shelf-life analysis of the extracted protein for 3, 6, 9, and 12 months showed a decreasing trend of protein content from 53.71*\u0026plusmn;0.67 g to 48.81*\u0026plusmn;0.58g on the 3rd month, 42.57*\u0026plusmn;0.89 g on the 6th month, 36.34*\u0026plusmn;0.87 g on the 9th month, and 29.24*\u0026plusmn;89g on 12th month. The variations were found to be statistically significant (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSensory evaluation of prepared candy by testing panel revealed the overall acceptability score of the candy to be 8.7, appearance 8.5, texture 8, taste 8.5, and colour 8.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Allergenicity analysis\u003c/h2\u003e \u003cp\u003eIn contrast to the elevated levels of IgE, histamine, and eosinophil counts observed in the peanut protein extract (PPE)-treated positive control group, animals administered with the extracted pre-pupa protein exhibited IgE concentrations (50\u0026ndash;100 ng/mL), histamine levels (16.5 pmol/mL), and eosinophil percentages (1\u0026ndash;3%) within the normal physiological range. These findings suggest that the pre-pupa protein did not elicit any significant inflammatory response in the \u003cem\u003ein vivo\u003c/em\u003e model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Toxicity study\u003c/h2\u003e \u003cp\u003eHistological examination of liver and kidney tissues revealed no pathological alterations in animals administered the extracted protein powder for 14 and 28 days, compared to the control group. Additionally, all recorded biochemical parameters remained within normal physiological ranges, further supporting the safety of the extracted protein powder for consumption.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCreatinine values recorded in different groups of mice on the 14th and 28th day\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMice\u0026nbsp;groups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eCreatinine (U/L)\u003c/p\u003e \u003cp\u003e(Normal range: 0.2\u0026ndash;0.5)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14th day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28th day\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u0026nbsp;I\u003c/p\u003e \u003cp\u003e(controlled)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u0026nbsp;II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.44\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eValues are the mean of six replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard deviation\u003c/p\u003e \u003cp\u003eDifferent superscripts along the rows and columns differ significantly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eALP values recorded in different groups of mice on the 14th and 28th day\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMice\u0026nbsp;groups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eALP (U/L)\u003c/p\u003e \u003cp\u003e(Normal range: 62\u0026ndash;230)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14th day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28th day\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u0026nbsp;I\u003c/p\u003e \u003cp\u003e(controlled)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e98.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u0026nbsp;II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e206.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e220.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eValues are the mean of six replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard deviation\u003c/p\u003e \u003cp\u003eDifferent superscripts along the rows and columns differ significantly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSGPT values recorded in different groups of mice on the 14th and 28th day\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMice\u0026nbsp;groups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSGPT (U/L)\u003c/p\u003e \u003cp\u003e(Normal range: 7\u0026ndash;56)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14th day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28th day\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u0026nbsp;I\u003c/p\u003e \u003cp\u003e(controlled)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e24.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e24.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u0026nbsp;II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e29.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eValues are the mean of six replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard deviation\u003c/p\u003e \u003cp\u003eDifferent superscripts along the rows and columns differ significantly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSGOT values recorded in different groups of mice on the 14th and 28th day\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMice\u0026nbsp;groups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eSGOT (U/L)\u003c/p\u003e \u003cp\u003e(Normal range: 8\u0026ndash;45)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14th day\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e28th day\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u0026nbsp;I\u003c/p\u003e \u003cp\u003e(controlled)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e22.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e23.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u0026nbsp;II\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e27.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e29.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e \u003cp\u003eValues are the mean of six replicates\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard deviation\u003c/p\u003e \u003cp\u003eDifferent superscripts along the rows and columns differ significantly\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe incorporation of edible insects into the human diet is gaining global traction due to their high-quality proteins, beneficial lipids, essential micronutrients, and favorable sensory properties. The global edible insect protein market, valued at approximately USD 144\u0026nbsp;million in 2019, is projected to grow at a compound annual growth rate (CAGR) of 45%, reaching an estimated USD 1,366\u0026nbsp;million by 2025 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Despite the persistence of food neophobia in several regions, insect-based foods have been successfully commercialized and are often perceived as delicacies. These products are available in various forms, including flours, heat-dried larvae and pupae, protein isolates, canned products, extruded snacks, confectioneries, and liquid infusions. In the past decade, over 250 insect-based products have been developed by both small- and large-scale food industries [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Due to their high protein content and well-balanced amino acid profiles, edible insects have garnered considerable interest as potential ingredients in nutritionally enriched products, particularly those targeted at athletic populations. These include hydrolysates, protein isolates and concentrates, energy bars, and protein-fortified beverages [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The convergence of innovation in insect-based food technologies, regulatory advancements, increased media attention, and wider product availability has fostered significant market growth [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, edible insects have demonstrated utility in value-added functional foods. For instance, powders derived from \u003cem\u003eAspergillus oryzae\u003c/em\u003e and \u003cem\u003eBacillus licheniformis\u003c/em\u003e have been employed as substrates for developing fermented liquid seasonings integrated into soy sauce production [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. To address childhood malnutrition, extruded food products incorporating sun-dried termites, germinated amaranth, corn, and soy oil have been formulated [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Cricket flour has also been effectively used in cookie formulations alongside amaranth, rice, and oatmeal, yielding products with high acceptability among children [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith the rising global interest in incorporating edible insects into food products to enhance their nutritional value and appeal, the present study evaluates the nutritional profile of the pre-pupal and pupal stages of six distinct strains of the Eri silkworm (\u003cem\u003eSamia ricini\u003c/em\u003e), traditionally consumed by various tribal communities in Northeast India. Among the strains analyzed, the pre-pupal stage of the Yellow-Spotted strain demonstrated the highest protein content, measured at 66.58 g per 100 g of dry sample, surpassing the protein content of most conventional livestock sources [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Further amino acid profiling revealed that the protein from Yellow-Spotted pre-pupae qualifies as a complete protein, containing all essential amino acids in substantial quantities. According to the FAO/WHO (2023) essential amino acid scoring pattern, the amino acid composition generally meets the required proportions for human nutrition, with minor deficiencies noted in threonine and leucine. Notably, the high lysine content (1.69 g/100 g protein) in Yellow-Spotted pre-pupae offers a potential nutritional advantage, particularly in complementing lysine-deficient cereal-based diets [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These findings affirm the potential of Eri silkworm pre-pupae, especially the Yellow-Spotted strain, as a sustainable and high-quality protein source for human consumption [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study represents the first sub-acute toxicity assessment of a strain-specific silkworm variant characterized by the highest protein content among its six available strains. Throughout the experimental period, no signs of toxicity or abnormal clinical symptoms, such as seizures, vomiting, anorexia, or lethargy, were observed in the treated mice. These findings align with those of [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], who similarly reported no toxicological effects in in vivo models administered \u003cem\u003eBombyx mori\u003c/em\u003e. However, a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in serum creatinine levels was observed in Group II mice on day 14 and day 28 of the feeding period. Despite this elevation, creatinine levels remained within the established physiological reference range. This increase may be attributed to enhanced nitrogen metabolism and a consequent rise in renal filtration workload, suggesting that prolonged intake of pre-pupal powder could mildly influence renal function.\u003c/p\u003e \u003cp\u003eMonitoring liver marker enzyme levels is essential in assessing liver function and identifying potential issues with the liver's ability to process and excrete waste. Although a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in ALP, SGOT, and SGPT was observed in group II mice during the feeding period for 14 days and 28 days, the resulting values were within the reference range. The significant increase in ALP might be because of greater nitrogen metabolism and filtration workload. This finding was further supported by the absence of histopathological changes in the liver. Therefore, the observed differences and the apparent lack of any impact on these enzymes make it challenging to identify any disease condition, indicating that the consumption of the test diet does not lead to hepatotoxicity and nephrotoxicity.\u003c/p\u003e \u003cp\u003eIncreasing consumer awareness regarding the edibility of insects has been shown to enhance their willingness to pay for insect-based foods [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. To promote acceptance, it is essential to highlight the social, practical, and contextual factors influencing food consumption. Such efforts should involve ongoing education and marketing initiatives that emphasize the potential role of edible insects in addressing contemporary and future challenges related to environmental sustainability, population growth, and land use [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Fermented liquid seasoning, developed using \u003cem\u003eAspergillus oryzae\u003c/em\u003e and \u003cem\u003eBacillus licheniformis\u003c/em\u003e powders as substrates, has been incorporated into soy sauce fermentation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Studies have also demonstrated the fortification of approximately 90% of mealworm larvae in the formulation of minced meat-like products, preserved under modified atmospheric packaging [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Furthermore, silkworm pupa powder has been utilized in meat batters for the production of frankfurters, significantly reducing cooking laws while improving textural properties [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In efforts to combat infant malnutrition, extruded food products have been developed using sun-dried termites combined with germinated amaranth, corn, and soybean oil [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The incorporation of \u003cem\u003eNauphoeta cinerea\u003c/em\u003e flour at concentrations of 5\u0026ndash;15% in brad formulations has been studied, with an emphasis on its physical sensory properties [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Additionally, cookies made from cricket flour blended with amaranth, rice, and oatmeal are well accepted by children [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Keeping this view in mind, the present study has developed a protein-rich candy based on silkworm pre-pupa. Additionally, since sugar has been replaced with palm jaggery, which itself has many useful health benefits like antihepatotoxic, anti-inflammatory, and anti-hyperglycemic effects [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and can be a valuable alternative for people avoiding sugar-based products due to health risks like diabetes. Unlike sugar, which spikes blood glucose levels, palm jaggery has a lower glycemic index, making it a safer option for individuals with diabetes or those at risk of developing it. Studies have shown that palm jaggery possesses anti-hepatotoxic properties, helping detoxify the liver and protect it from oxidative damage, which is vital for overall health. It also exhibits anti-inflammatory effects, which can aid in managing chronic conditions like arthritis or autoimmune diseases. Besides, palm jaggery is rich in essential minerals like iron, calcium, magnesium, and potassium, which support bone health, regulate blood pressure, and promote overall well-being. Its mild laxative effect supports digestive health, improving digestion and preventing constipation. Moreover, because palm jaggery is a natural, unrefined sweetener, it provides a wholesome source of energy and can satisfy sweet cravings without the negative impacts of processed sugar. With its various health benefits, including boosting immunity and promoting healthy digestion, palm jaggery is not just a healthier sweetener but also a valuable addition to a balanced diet. It is an ideal option for those looking to reduce sugar intake without sacrificing taste or health benefits. By incorporating palm jaggery into daily use, individuals can enjoy a more nutritious and sustainable way to sweeten their food while supporting their overall health. Furthermore, the leaves of \u003cem\u003eGarcinia pedunculata\u003c/em\u003e and ginger may have antihyperglycemic, antidiabetic, antioxidant, and anti-inflammatory properties [\u003cspan additionalcitationids=\"CR38 CR39\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], which can improve our general health.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThe findings of this study highlight the potential for developing a protein-rich functional candy using silkworm pre-pupae protein powder, offering a health-promoting and accessible dietary option, particularly in developing countries facing food insecurity due to rapid population growth. Sensory evaluation results demonstrated high acceptability across all parameters, indicating favourable organoleptic properties of the formulated candy. The replacement of refined sugar with palm jaggery further enhances the product\u0026rsquo;s nutritional profile, providing added health benefits and making it suitable for individuals seeking alternatives to conventional sugar, including those managing diabetes. This innovation is expected to promote the consumption of Eri silkworm pre-pupae and pupae, thereby increasing demand and contributing to the socio-economic empowerment of local Eri silkworm rearers. Additionally, the candy may serve as a low-cost, high-protein food source during emergencies or crises.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to Guwahati Biotech Park, Assam for providing the instrumentation necessary for the analysis of sample.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.D.: original draft preparation; writing-review \u0026amp; editing, experimentation; P.S.: writing-review \u0026amp; editing, illustration, resources; M.P.: collection of data, analysis, writing-review \u0026amp; editing \u0026nbsp; K.A.N.: writing-review \u0026amp; editing; K.K.: Data interpretation and analysis; S.S.: Data interpretation and analysis; R.L.: data curation, writing \u0026amp; editing, A.D.: writing-review \u0026amp; editing; S.M.: writing-review \u0026amp; editing; S.D.: writing-review \u0026amp; editing, data interpretation; A.S.: writing-review \u0026amp; editing; T.K.: Supervision, methodology, original draft preparation, writing-review \u0026amp; editing, resources. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding Sources\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author(s) received no financial support for the research, authorship, and/or publication of this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe approval for the use of Swiss albino mice during protein treatment analysis was duly taken from the Institutional Animal Ethics Committee of Cotton University. The approval for participation of human research participants for sensory evaluation test was taken from the Institutional Human Ethics Committee of Cotton University\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the participants were adults (18+) and informed consent for participation in the 9-point hedonic scale sensory evaluation for protein candy developed in the current study was taken accordingly.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFAO/WHO, 1973. Energy and Protein Requirements: Report of a Joint FAO/WHO Ad Hoc Expert Committee. Rome: FAO Nutrition Meetings Report Series No. 52. Geneva: WHO Technical Report Series No. 522. Hague: Dr. W. Junk Publishers. 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The effects of consumer knowledge on the willingness to buy insect food: An exploratory cross-regional study in Northern and Central Europe. Food Quality and Preference. 2016; 70: 1\u0026ndash;10. https://doi.org/10.1016/j.foodqual.2016.12.006\u003c/li\u003e\n\u003cli\u003eEvans, J., Alemu, M. H., Flore, R., Fr\u0026oslash;st, M. B., Halloran, A., Jensen, A. B., Maciel- Vergara, G., Meyer- Rochow, V. B., M\u0026uuml;nke- Svendsen, C., Olsen, S. B., Payne, C., Roos, N., Rozin, P., Tan, H. S. G., Huis, A. V., Vantomme, P., and Eilenberg, J. \u0026lsquo;Entomophagy\u0026rsquo;: an evolving terminology in need of review. Journal of Insects as Food and Feed. 2015; 1: 293-305.\u003c/li\u003e\n\u003cli\u003eStoops, J., Vandeweyer, D., Crauwels, S., Verreth, C., Boeckx, H., Borght, M.V.D., Claes, J., Lievens, B., Campenhout, L.V. Minced meat-like products from mealworm larvae (\u003cem\u003eTenebrio molitor \u003c/em\u003eand \u003cem\u003eAlphitobius diaperinus\u003c/em\u003e): microbial dynamics during production and storage. \u003cem\u003eInnovative Food Science \u0026amp; Emerging Technologies\u003c/em\u003e. 2017;\u003cem\u003e41\u003c/em\u003e, 1\u0026ndash;9. \u003c/li\u003e\n\u003cli\u003ePark, Y., Choi, Y., Hwang, K., Kim, T., Lee, C., Shin, D., Han, S. G. Physicochemical Properties of Meat Batter Added with Edible Silkworm Pupae (\u003cem\u003eBombyx mori\u003c/em\u003e) and Transglutaminase. Korean Journal for Food Science of Animal Resources. 2017; 37: 351\u0026ndash;359. https://doi.org/10.5851/kosfa.2017.37.3.351\u003c/li\u003e\n\u003cli\u003eOliveira, L., Carolina, C., Mellado, M. Bread enriched with flour from the cinereous cockroach (Nauphoeta cinerea). Innovative Food Science \u0026amp; Emerging Technologies. 2015; 44-48.\u003c/li\u003e\n\u003cli\u003eKumar, A., Singh, S., The benefits of Indian jaggery over sugar on human health. Dietary Sugar, Salt and Fat in Human Health, Academic Press Publisher, Washington, United States. 2020 10.1016/B978-0-12-816918-6.00016-0.\u003c/li\u003e\n\u003cli\u003eOzkur M, Benlier N, Takan I, Vasileiou C, Georgakilas AG, Pavlopoulou A, Cetin Z, Saygili EI. Ginger for Healthy Ageing: A Systematic Review on Current Evidence of Its Antioxidant, Anti-Inflammatory, and Anticancer Properties. Oxid Med Cell Longev. 2022; 2022:4748447. doi: 10.1155/2022/4748447 \u003c/li\u003e\n\u003cli\u003eAli MY, Paul S, Tanvir EM, Hossen MS, Rumpa NN, Saha M, Bhoumik NC, Aminul Islam M, Hossain MS, Alam N, Gan SH, Khalil MI. Antihyperglycemic, Antidiabetic, and Antioxidant Effects of \u003cem\u003eGarcinia pedunculata\u003c/em\u003e in Rats. Evid Based Complement Alternat Med. 2017; 2:2979760. Doi: 10.1155/2017/2979760. \u003c/li\u003e\n\u003cli\u003eMashhadi N.S., Ghiasvand R., Askari G., Hariri M., Darvishi L., Mofid M.R. Anti-oxidative and anti-inflammatory effects of ginger in health and physical activity: review of current evidence. Int J Prev Med. 2013 Apr;4(Suppl 1)\u003c/li\u003e\n\u003cli\u003eEspirito Santo BLSD, Santana LF, Kato Junior WH, de Ara\u0026uacute;jo FO, Bogo D, Freitas KC, Guimar\u0026atilde;es RCA, Hiane PA, Pott A, Fili\u0026uacute; WFO, Arakaki Asato M, Figueiredo PO, Bastos PRHO. Medicinal Potential of \u003cem\u003eGarcinia\u003c/em\u003e Species and Their Compounds. Molecules. 2020; 25(19):4513. doi: 10.3390/molecules25194513. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"discoverfood","sideBox":"Learn more about [Discover Food](https://www.springer.com/44187)","snPcode":"","submissionUrl":"","title":"Discover Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Eri-silkworm pupae, insect protein candy, waste management, functional food, entrepreneurship","lastPublishedDoi":"10.21203/rs.3.rs-6526487/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6526487/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eProtein deficiency remains a significant challenge in many developing countries, where food scarcity limits access to essential nutrients. Despite being a rich source of high-quality protein and essential amino acids, the consumption of Eri silkworm (\u003cem\u003eSamia ricini\u003c/em\u003e) pupae and pre-pupae is largely restricted to tribal communities in Northeast India due to cultural taboos surrounding entomophagy. Among the six known strains of Eri silkworm, the yellow-spotted strain exhibits the highest protein content in its pre-pupal stage (66.58%), with lysine and arginine present in substantial amounts, making it a valuable dietary protein source for growth and development. Assam, the third-largest producer of raw Eri silk in India, generates approximately 4,910 metric tons of Eri cocoons annually, each containing a pupa. However, due to societal resistance, a significant proportion of these nutrient-rich pupae are discarded as waste. Addressing this issue, the present study explores the development of a novel protein candy formulated with Eri pupa protein (20 g), palm jaggery (80 g, low glycaemic index), garcinia-soaked water, and ginger leaves. Sensory evaluation revealed high consumer acceptability, with an overall score of 8.4. Furthermore, allergenicity and toxicity assessments in albino mice confirmed the safety of Eri pupa protein for human consumption. The current study highlights the potential of Eri pupa as a sustainable protein source, offering a promising approach to sericulture waste management and food security. The development of such protein-rich functional foods could facilitate broader acceptance of edible insects, drive entrepreneurship, and contribute to nutritional sustainability in the region.\u003c/p\u003e","manuscriptTitle":"Silkworm protein candy: An approach towards nutritional waste management and sustainable development","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-02 09:14:19","doi":"10.21203/rs.3.rs-6526487/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-22T08:30:23+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-19T07:51:38+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155230622971070635667788889725940852929","date":"2025-06-17T09:43:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-16T14:36:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"215081363723608158311414436494489850426","date":"2025-06-08T10:26:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"244319210031516462635086491862641059597","date":"2025-06-04T03:09:52+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-30T13:50:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-25T14:17:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-13T08:37:21+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Food","date":"2025-05-13T08:36:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-food","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"discoverfood","sideBox":"Learn more about [Discover Food](https://www.springer.com/44187)","snPcode":"","submissionUrl":"","title":"Discover Food","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c75e63a4-c59e-48d0-bb70-50a510f7f987","owner":[],"postedDate":"June 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-12T09:38:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-02 09:14:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6526487","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6526487","identity":"rs-6526487","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}