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Full Research Article

Nutritional Composition and Profiling of Fatty Acids and Carotenoids in Eri Silkworm (Samia ricini Donovan) Pupae

A
Anjela Ahmed1
https://orcid.org/0000-0002-8318-4794
H
Hena Parbin1
https://orcid.org/0009-0006-1174-5456
A
Abhijit Chandra Roy2
https://orcid.org/0000-0001-9186-1399
U
Ujjal Bordoloi2
https://orcid.org/0009-0000-9878-3581
M
Moushumi Hazarika3
https://orcid.org/0000-0001-7948-3575
C
Chandan Tamuly3
https://orcid.org/0000-0001-7396-2460
B
Bhuban Chandra Chutia2,*
https://orcid.org/0000-0002-7668-5419
1Department of Zoology, Gauhati University, Guwahati-781 014, Assam, India.
2Department of Zoology, Nagaon University, Nagaon-782 001, Assam, India.
3Natural Product Chemistry Section, CSIR-North East Institute of Science and Technology, Itanagar-791 110, Arunachal Pradesh, India.

ABSTRACT

Background: Eri silkworm (Samia ricini Donovan) is conventionally cultured in Northeast India, where it is mostly raised for silk production and dietary purposes. The current study investigates the nutrient composition and carotenoid profile of Samia ricini pupae mainly to evaluate their potential as a sustainable food source.

Methods: The proximate analysis of Eri silkworm pupae including moisture, crude protein, crude fat, crude fibre, ash content were performed by following the standard methods of association of official analytical chemist (AOAC). Fatty acid and carotenoid profiling was performed through gas chromatography-mass spectrometry (GCMS) and high-performance liquid chromatography (HPLC) analysis respectively.

Result: The moisture content was 7.93%. Crude protein, lipid, carbohydrate, crude fibre and ash contents were 52.02%, 22.90%, 7.12%, 5.81 % and 4.63%, respectively. The total carotenoid content was 377.89±1.83 µg/g. GCMS analysis revealed 9, 12, 15-octadecatrienoic acid (49.09%), cholesterol (19.35%) and n-hexadecanoic acid (13.22%) as the major compounds among ten identified bioactives. HPLC identified lutein and β-carotene at 1.981 µg/g and 266 µg/g, respectively. Thus, Eri silkworm pupae possess a rich nutrient profile, suggesting their potential application as a valuable nutrient and pigment source for animal feed applications.

INTRODUCTION

Insects have historically been a vital human food source, particularly in developing countries, where entomophagy is widely practiced. They are consumed across Europe, America, Asia, Africa and Oceania, with China, Mexico, Thailand and India recognized among the leading insect-consuming countries with high edible insect diversity (Bodenheimer, 1951; DeFoliart, 1999; Nitharwal et al., 2022). Insects possess very high crude protein, essential amino acids, fats, minerals and vitamins, making them sustainable alternative feed ingredients for livestock, poultry, ruminants and aquaculture (Finke et al., 1989; FAO, 2021; Zhou et al., 2022). Insects are recognized as a productive substitute for fishmeal in animal husbandry (Hicklinz, 1962). Due to increasing fish meal cost and production constraints, several studies are conducted exploring cheap alternative protein sources to partially or entirely replace it (Sahib et al., 2025; Bordoloi et al., 2025). The larvae and pupae of Bombyx mori L., Musca domestica L., Tenebrio molitor L. and Hermetia illucens L. are utilized in this context (Khan et al., 2018; Kannan et al., 2024).
       
The native inhabitants of Northeast India utilized various insects as food and amongst them Eri silkworm (Samia ricini Donovan), which is widely distributed in the Brahmaputra valley (Peigler, 1993), is traditionally consumed by several tribal communities (Sarmah, 2011). It is an excellent source of protein and fats containing all essential amino acids and polyunsaturated fatty acids (Longvah et al., 2011; 2012). Further, B. mori pupae contain carotenoids like lutein and neoxanthin, which contribute to pigmentation and antioxidant defense (Kotake-Nara et al., 2002; Crupi et al., 2023). Most animals cannot synthesize carotenoids de novo and rely on dietary sources (Goodwin 1984). With the increasing demand for natural carotenoids source in animal feed industries (Tran Van  et al., 2025), eri silkworm pupae may serve as a potential alternative ingredient. The nutritional qualities of B. mori have been the center of numerous investigations, particularly with respect to their protein, fatty acid and carotenoid content (Chieco et al., 2019; Yeruva et al., 2023) but minimal is known about S. ricini pupae despite their traditional consumption as food in Northeast India. A few studies exist: Longvah et al., (2011) studied the nutrient composition and protein quality, while Gangopadhyay et al., (2022) reported all essential amino acids of pupae and prepupae of S. ricini. However, its in-depth carotenoid content is still unexplored. Therefore, the present study aims to evaluate the nutrient composition, including fatty acid composition and carotenoid quantification in S. ricini pupae.

MATERIALS AND METHODS

The experiment was conducted during the period of December 2022 to August 2024 at Department of Zoology, Nagaon University (Upgraded from Nowgong College (Autonomous), Nagaon. HPLC and GC-MS analysis were outsourced at CSIR-North East Institute of Science and Technology, Itanagar Branch, Arunachal Pradesh.
 
Sample preparation
 
S. ricini pupae (7 days old matured) grown exclusively on Castor (Ricinus communis L.) leaves were collected shortly after the cocoon formation from Eri-Rearing Unit of Integrated Eri Cum Ornamental Fish Rearing laboratory of Zoology Department, Nagaon University, Nagaon (Assam), India. Fresh pupae were cleansed with deionized water and spread on a filter paper. After an hour, they were homogenized, moved into stainless steel trays and dried at 60 C using hot air oven (Optics Technology, an ISO 9001:2008 certified) (Longvah et al., 2011). Dried pupae were then finely ground into powder for further analysis. A portion of dried powdered pupae samples were collected for the proximate analysis while the remaining portion was used for fatty acid profiling by Gas Chromatography- Mass Spectrometry (GC-MS), total carotenoid analysis and specific carotenoids (lutein and β-carotene) quantification through high performance liquid chromatography (HPLC).
 
Proximate analysis
 
The moisture and fiber content was determined by Association of the Official Analytical Chemists (AOAC, 2005) methods 934.01 and 962.09 respectively. The crude protein content (Nitrogen x 6.25) was estimated by Kjeldahl method (984.13) of AOAC (1984). Crude fat was estimated by Soxhlet Extraction method (AOAC, 1990). For carbohydrate estimation, difference method (100-sum of moisture, protein, fat, crude fibre and ash) was applied (Longvah et al., 2011). Crude ash was determined by the standard 942.05 method (AOAC, 1984).
 
Fatty acid profiling through GC-MS
 
For fatty acid profiling through GC-MS analysis, extraction and fatty acid methyl esters (FAME) preparation was done by following Yeruva et al., (2023) with minor modifications, where 1.0 g sample was homogenized with chloroform: methanol (3:1, v/v) and filtered, with extraction repeated four times. The pooled extract was separated, dried over anhydrous sodium sulfate, adjusted to 15 mL and evaporated to dryness in hot air oven (Optics Technology, an ISO 9001:2008 certified) at 40°C. The residue was fitted to reflux condenser and 1 mL of methanol was introduced through it and gently heated at 40°C-50°C for 10 min. Subsequently, 5 mL heptane was added through the condenser and allowed to cool. The flask was removed; 2 mL saturated NaCl was added, mixed thoroughly and the contents were transferred to test tubes. The upper heptane layer containing FAMEs were collected, passed through sodium sulfate, syringe-filtered and analyzed by GC-MS. GC-MS analysis was carried out using Shimadzu Nexis GC-2030 GC system coupled with Shimadzu GCMS-QP2020NX and AOC-20s U auto sampler, equipped with capillary column SH-Rtx-5MS (30 m x 0.25 mm; Crosson 5% diphenyl/ 95% dimethylpolysiloxane) with an auto injector AOC-20i plus. The carrier g as was  helium  (>99.999 per cent) with a linear flow rate of 0.69 mL/min and pressure of 43.6 kPa. 1.0 μL extract was injected into the GC-MS. Split less injection modes were used for the chemical profiling. The oven temperature was initially 100°C and was held for 4 min. Then it was set to rise at 3°C/min, till 220°C and was held for 4 min and then was raised till 260°C with ramp of 5°C/min. The ion source temperature was 220°C; interface temperature 260°C and solvent cut time of 2 min. and a mass range of 50 m/z to 450 m/z. The peaks were identified by comparing with those spectra available in National Institute of Standards and Technology-2007 (NIST-2007) spectral libraries.
 
Total carotenoid analysis (TCA) and carotenoid profiling through HPLC
 
For TCA, 6 g of dried pupa sample was mixed with 20 mL of methanol using a glass mortar and pestle, filtered using Whatman no. 42 filter paper (pore size: 2.5 µm). The extraction was repeated 3-4 times with fresh solvent to obtain sufficient extract. The pooled extracts were then concentrated to 5 mL using a rotary evaporator under vacuum (≤55°C) and stored in -20°C for further analysis (entire process was conducted under low light conditions). Extraction and calculation were performed following Machmudah and Goto (2013) with slight modification. The extract was diluted 100 fold and optical density was measured at 450 nm using Cary 60 UV-Vis Spectrophotometer (Model No: G6860A, Agilent Technologies) and calculated according to the formula:
 
Where,
V= Volume of extract (mL).
 = Average absorption coefficient for β-carotene =2500 L/mol.cm).
W= Weight of the sample (g).
A= Absorbance at 450 nm.
       
Carotenoid extraction for HPLC analysis followed the same procedure used for TCA. HPLC equipped with a photodiode array (PDA) detector (Model: LC-20 AP, Shimadzu, Japan) was used for carotenoid profiling. Analytical standards of high purity lutein (Product No. 07168-1 mg) and β-carotene (Lot No. BCBH8452V) were procured from Sigma Aldrich. A reversed-phase C18 column was used for chromatographic separation. The mobile phase for lutein consisted of Solvent A: acetonitrile and methanol (75:20, v: v) containing 15 mM ammonium acetate and Solvent B: pure methanol. The elution was performed with 95% Solvent B and 5% Solvent A at a flow rate of 0.8 mL /min. The injection volume was 20 µL and detection was performed at 445 nm using a UV-visible PDA detector.  The mobile phase for β-carotene comprised of methanol and acetonitrile (75:30 v: v), with 3.2 g/L ammonium acetate. Elution was performed using 100% of this mobile phase under isocratic conditions at 1.5 mL/min flow rate. Detection was performed at 460 nm. The mobile phase and chromatographic conditions for lutein and β-carotene were adopted from Sun and Zhan (2021) and Brauer (2014) respectively. Calibration curves were constructed for lutein and β-carotene by plotting the peak areas against known standard concentrations and used to quantify their concentrations in pupae samples.
 
Statistical analysis
 
For proximate analysis and TCA, all values were performed in triplicate (n=3) and results were expressed as mean± standard deviation (SD). Data were analyzed using Microsoft Excel. GC-MS and HPLC analyses were performed on a single representative sample for compound profiling purpose; hence results are presented descriptively.

RESULTS AND DISCUSSION

Proximate composition
 
Table 1 shows the proximate composition of S. ricini pupae in comparison with fish and soybean meal based on literature. The observed moisture content was lower than the value reported by Longvah et al., (2011) for S. ricini pupae. The observed protein level was 52.02% which was greater than the protein content of spent silkworm pupae (about 48.7%) reported earlier by Rao (1994) but considerably lower than the 54% that Longvah et al., (2011) reported. With high protein content, Eri pupae compare favorably with conventional fish meal and clearly surpass soybean meal. The observed carbohydrate content was relatively higher than the 3.45% reported for eri pupae by Longvah et al., (2011) and 4.7% for B. mori pupae by Yeruva et al., (2023). Further, the carbohydrate content is higher than in fish meal, while lower than in soybean meals. Insects, typically have higher protein and fat content than carbohydrate (Zhou et al., 2022). The fat contents were moderately high, exceeding the lipid levels of fish meal and soybean meal. The observed ash content was very much similar to 4% reported for Antheraea pernyi G. pupae by Zhou and Han (2006). The fibre content falls between the values reported by Kashyap et al., (2023) for non-deoiled (3.5%) and deoiled (6.8%) eri pupae.

Table 1: Proximate composition of S. ricini pupae compared to Fish and Soybean meal based on literature.


 
Fatty acid profiling
 
Ten significant compounds were detected through GC-MS analysis, predominantly sterols, fatty acids and their methyl esters (Table 2). The chromatogram of the FAMEs obtained through GC-MS analysis is presented in Fig 1. The major fatty acid identified was 9, 12, 15 octadecatrienoic acid (Z, Z, Z)-commonly known as α-linolenic acid (ALA), comprising (49.09 %), of the total fatty acids, indicating a rich percentage of polyunsaturated fatty acids (PUFAs). A remarkable level of cholesterol (19.35%), an important sterol of animal origin, was also detected. In addition, N-hexadecanoic acid (palmitic acid) constituted 13.22% representing a common saturated fatty acid. The presence of methyl esters of linolenic, linoleic, stearic and palmitic acids as minor ingredients suggested a rich profile of essential fatty acids. Similar results were reported by Mishra et al., (2025); representing that Eri pupal oil is rich in unsaturated fatty acids, including omega-3 fatty acids. Payne et al., (2016) noted high levels of monounsaturated and polyunsaturated fatty acids in silkworm pupae, while Rodríguez-Ortiz  et al. (2024) reported palmitic acid (23.61%) as the major saturated fatty acids, oleic acid (31.45%) as the main monounsaturated fatty acids and ALA as the primary polyunsaturated fatty acid (29.90%) in B. mori pupae. Javali et al., (2015) reported that silkworm pupae contain 60% to 70% of n-3, n-6 and n-9 fatty acids, supporting their utilization in food, pharmaceutical and medicinal applications.

​

Table 2: Fatty acid composition of S. ricini Pupae obtained through GC-MS analysis.



Fig 1: GC chromatogram of fatty acid profile from Samia ricini pupae.


 
Total carotenoid analysis and carotenoid profiling through HPLC
 
The total carotenoid content of Samia ricini pupae was 377.89±1.83 µg g-1 (Table 3) which is much greater than the values reported by Cheico et al., (2019) for Bombyx mori pupae, including White Polyhybrid Strain (WPS: 17.1 µg/g) and Golden Nistari Strain (GNS: 35.0 µg/g).

Table 3: Total carotenoid, lutein and â-carotene concentration (µg/g) of S. ricini pupae with chemical structures and bioactivities.


       
HPLC analysis revealed the presence of lutein and β-carotene in Samia ricini pupae extract, with concentrations of 1.981 µg/g and 266µg/g, respectively (Table 3). The chromatographic profile (Fig 2 and 3) exhibited major peaks corresponding to lutein (6.442-7.268 min) and β-carotene (25.387 min), identical to their respective standards. These findings confirm the presence and abundance of both carotenoids in S. ricini pupae. Lutein (C40H56O2) is a naturally produced fat soluble carotenoid pigment with 40 carbon atoms and a conjugated double bond structure (Table 3) for which it provides striking red hue and produce free radicals (Fuad et al., 2020). Unlike other pro vitamin A carotenoids, lutein cannot be converted to vitamin A and hence must be obtained through food or supplements (Johnson, 2004). Having numerous biological and therapeutic potentials, it is widely used in pharmaceutical, animal and fish feed industries (Fuad et al., 2020).  Some of its biological activities are presented in Table 3. Meanwhile, β-carotene, an isoprenoid, naturally produced lipid soluble compound with 40 carbon atoms and a long chain of conjugated double bonds (Table 3) which undergoes isomerization at high temperature, enhancing color. Besides pigmentation, β-carotene exhibits different biological activities; some are presented in Table 3.  Cheico et al., (2019) reported lutein (11.1 µg/g in WPS; 21.7 µg/g in GNS) and β-carotene (6.0 µg/g in WPS; 13.3 µg/g in GNS) in B. mori pupae. Kotake-Nara  et al. (2002) reported lutein (1.28 mg), neoxanthin (0.22 mg) and violaxanthin (0.07 mg) per 250 g in B. mori pupae. S. ricini pupae showed markedly higher total carotenoids compared to the commercial feeds reported by Wallat et al., (2005) where Zeiglar Tropical Fish Feed (21.70 µg/g), BioKyowa Series C-700 (27 µg/g), Arkat VibraGro (5.70 µg/g) and minnow meal (249 µg/g), with substantially higher β-carotene content, suggesting its superior potential as a natural carotenoid rich ingredient. Besen et al., (2019) observed improved survivability and color enhancement in juveniles of Carassius auratus when supplemented with lutein. Kaur et al., (2016) demonstrate that natural β-carotene supplementation (25 ppm), derived from carrot powder, significantly improves growth and pigmentation in ornamental Koi carp.

Fig 2: Chromatogram of lutein in Samia ricini pupae.



Fig 3: Chromatogram of â-carotene in Samia ricini pupae.

CONCLUSION

The present study revealed significant amounts of protein, lipids with diverse composition of fatty acids mainly PUFAs and sterols and specific carotenoids like lutein and β-carotene in S. ricini pupae which indicate their potential as an alternative substitute for animal feed and nutritional applications. Further investigations on feeding trials, bioavailability and commercial application are required.

ACKNOWLEDGEMENT

The authors sincerely thank the Department of Zoology, Nagaon University for providing the necessary requirements to conduct this research. Also acknowledge the Researchers and Scientists of Natural Product Chemistry Section, CSIR-North East Institute of Science and Technology, Itanagar, Arunachal Pradesh for analytical support. Also thankful to Dr. Hafizul Islam Pathan for proofreading the manuscript and the Assam Science Technology and Environment Council, (ASTEC), Govt. of Assam, for supporting the study.
 
Contribution of authors
 
All authors contributed to the plan of the study. Author Anjela Ahmed designed the study, performed the experiments with Hena Parbin. Abhijit Ch. Roy and Ujjal Bordoloi carried out FAME preparations for GC-MS analysis. Anjela Ahmed and Abhijit Ch. Roy wrote the first draft of the manuscript. Author Moushumi Hazarika performed carotenoid quantification through HPLC; while Chandan Tamuly analyzed the data. Bhuban Chandra Chutia contributed to the management and execution of the study and supervised the whole work. All authors read and approved the final manuscript.
 
Statements and declarations
 
We, the submitting authors declare that the research work is original and has not been published previously to any other journal.
 
Informed consent
 
As in this research no human or mammalian subjects were involved, no ethical approvals were required for the study.
 
Funding declaration
 
This work was supported by ASTEC (Assam Science Technology and Environment Council), Government of Assam, Science, Technology and Climate Change, under the Research and Development Minor Project entitled “Eri-Pupa Based Ornamental Fish Feed: Preparation, Standardization and Sustainable Utilization” (File No: ASTEC/SandT/192(204)/2022-23/1658; dated 17th September 2022). Authors Anjela Ahmed and Bhuban Ch. Chutia have received the research grant. 

CONFLICT OF INTEREST

There is no conflict of interest among the authors.

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    Full Research Article

    Nutritional Composition and Profiling of Fatty Acids and Carotenoids in Eri Silkworm (Samia ricini Donovan) Pupae

    A
    Anjela Ahmed1
    https://orcid.org/0000-0002-8318-4794
    H
    Hena Parbin1
    https://orcid.org/0009-0006-1174-5456
    A
    Abhijit Chandra Roy2
    https://orcid.org/0000-0001-9186-1399
    U
    Ujjal Bordoloi2
    https://orcid.org/0009-0000-9878-3581
    M
    Moushumi Hazarika3
    https://orcid.org/0000-0001-7948-3575
    C
    Chandan Tamuly3
    https://orcid.org/0000-0001-7396-2460
    B
    Bhuban Chandra Chutia2,*
    https://orcid.org/0000-0002-7668-5419
    1Department of Zoology, Gauhati University, Guwahati-781 014, Assam, India.
    2Department of Zoology, Nagaon University, Nagaon-782 001, Assam, India.
    3Natural Product Chemistry Section, CSIR-North East Institute of Science and Technology, Itanagar-791 110, Arunachal Pradesh, India.

    ABSTRACT

    Background: Eri silkworm (Samia ricini Donovan) is conventionally cultured in Northeast India, where it is mostly raised for silk production and dietary purposes. The current study investigates the nutrient composition and carotenoid profile of Samia ricini pupae mainly to evaluate their potential as a sustainable food source.

    Methods: The proximate analysis of Eri silkworm pupae including moisture, crude protein, crude fat, crude fibre, ash content were performed by following the standard methods of association of official analytical chemist (AOAC). Fatty acid and carotenoid profiling was performed through gas chromatography-mass spectrometry (GCMS) and high-performance liquid chromatography (HPLC) analysis respectively.

    Result: The moisture content was 7.93%. Crude protein, lipid, carbohydrate, crude fibre and ash contents were 52.02%, 22.90%, 7.12%, 5.81 % and 4.63%, respectively. The total carotenoid content was 377.89±1.83 µg/g. GCMS analysis revealed 9, 12, 15-octadecatrienoic acid (49.09%), cholesterol (19.35%) and n-hexadecanoic acid (13.22%) as the major compounds among ten identified bioactives. HPLC identified lutein and β-carotene at 1.981 µg/g and 266 µg/g, respectively. Thus, Eri silkworm pupae possess a rich nutrient profile, suggesting their potential application as a valuable nutrient and pigment source for animal feed applications.

    INTRODUCTION

    Insects have historically been a vital human food source, particularly in developing countries, where entomophagy is widely practiced. They are consumed across Europe, America, Asia, Africa and Oceania, with China, Mexico, Thailand and India recognized among the leading insect-consuming countries with high edible insect diversity (Bodenheimer, 1951; DeFoliart, 1999; Nitharwal et al., 2022). Insects possess very high crude protein, essential amino acids, fats, minerals and vitamins, making them sustainable alternative feed ingredients for livestock, poultry, ruminants and aquaculture (Finke et al., 1989; FAO, 2021; Zhou et al., 2022). Insects are recognized as a productive substitute for fishmeal in animal husbandry (Hicklinz, 1962). Due to increasing fish meal cost and production constraints, several studies are conducted exploring cheap alternative protein sources to partially or entirely replace it (Sahib et al., 2025; Bordoloi et al., 2025). The larvae and pupae of Bombyx mori L., Musca domestica L., Tenebrio molitor L. and Hermetia illucens L. are utilized in this context (Khan et al., 2018; Kannan et al., 2024).
           
    The native inhabitants of Northeast India utilized various insects as food and amongst them Eri silkworm (Samia ricini Donovan), which is widely distributed in the Brahmaputra valley (Peigler, 1993), is traditionally consumed by several tribal communities (Sarmah, 2011). It is an excellent source of protein and fats containing all essential amino acids and polyunsaturated fatty acids (Longvah et al., 2011; 2012). Further, B. mori pupae contain carotenoids like lutein and neoxanthin, which contribute to pigmentation and antioxidant defense (Kotake-Nara et al., 2002; Crupi et al., 2023). Most animals cannot synthesize carotenoids de novo and rely on dietary sources (Goodwin 1984). With the increasing demand for natural carotenoids source in animal feed industries (Tran Van  et al., 2025), eri silkworm pupae may serve as a potential alternative ingredient. The nutritional qualities of B. mori have been the center of numerous investigations, particularly with respect to their protein, fatty acid and carotenoid content (Chieco et al., 2019; Yeruva et al., 2023) but minimal is known about S. ricini pupae despite their traditional consumption as food in Northeast India. A few studies exist: Longvah et al., (2011) studied the nutrient composition and protein quality, while Gangopadhyay et al., (2022) reported all essential amino acids of pupae and prepupae of S. ricini. However, its in-depth carotenoid content is still unexplored. Therefore, the present study aims to evaluate the nutrient composition, including fatty acid composition and carotenoid quantification in S. ricini pupae.

    MATERIALS AND METHODS

    The experiment was conducted during the period of December 2022 to August 2024 at Department of Zoology, Nagaon University (Upgraded from Nowgong College (Autonomous), Nagaon. HPLC and GC-MS analysis were outsourced at CSIR-North East Institute of Science and Technology, Itanagar Branch, Arunachal Pradesh.
     
    Sample preparation
     
    S. ricini pupae (7 days old matured) grown exclusively on Castor (Ricinus communis L.) leaves were collected shortly after the cocoon formation from Eri-Rearing Unit of Integrated Eri Cum Ornamental Fish Rearing laboratory of Zoology Department, Nagaon University, Nagaon (Assam), India. Fresh pupae were cleansed with deionized water and spread on a filter paper. After an hour, they were homogenized, moved into stainless steel trays and dried at 60 C using hot air oven (Optics Technology, an ISO 9001:2008 certified) (Longvah et al., 2011). Dried pupae were then finely ground into powder for further analysis. A portion of dried powdered pupae samples were collected for the proximate analysis while the remaining portion was used for fatty acid profiling by Gas Chromatography- Mass Spectrometry (GC-MS), total carotenoid analysis and specific carotenoids (lutein and β-carotene) quantification through high performance liquid chromatography (HPLC).
     
    Proximate analysis
     
    The moisture and fiber content was determined by Association of the Official Analytical Chemists (AOAC, 2005) methods 934.01 and 962.09 respectively. The crude protein content (Nitrogen x 6.25) was estimated by Kjeldahl method (984.13) of AOAC (1984). Crude fat was estimated by Soxhlet Extraction method (AOAC, 1990). For carbohydrate estimation, difference method (100-sum of moisture, protein, fat, crude fibre and ash) was applied (Longvah et al., 2011). Crude ash was determined by the standard 942.05 method (AOAC, 1984).
     
    Fatty acid profiling through GC-MS
     
    For fatty acid profiling through GC-MS analysis, extraction and fatty acid methyl esters (FAME) preparation was done by following Yeruva et al., (2023) with minor modifications, where 1.0 g sample was homogenized with chloroform: methanol (3:1, v/v) and filtered, with extraction repeated four times. The pooled extract was separated, dried over anhydrous sodium sulfate, adjusted to 15 mL and evaporated to dryness in hot air oven (Optics Technology, an ISO 9001:2008 certified) at 40°C. The residue was fitted to reflux condenser and 1 mL of methanol was introduced through it and gently heated at 40°C-50°C for 10 min. Subsequently, 5 mL heptane was added through the condenser and allowed to cool. The flask was removed; 2 mL saturated NaCl was added, mixed thoroughly and the contents were transferred to test tubes. The upper heptane layer containing FAMEs were collected, passed through sodium sulfate, syringe-filtered and analyzed by GC-MS. GC-MS analysis was carried out using Shimadzu Nexis GC-2030 GC system coupled with Shimadzu GCMS-QP2020NX and AOC-20s U auto sampler, equipped with capillary column SH-Rtx-5MS (30 m x 0.25 mm; Crosson 5% diphenyl/ 95% dimethylpolysiloxane) with an auto injector AOC-20i plus. The carrier g as was  helium  (>99.999 per cent) with a linear flow rate of 0.69 mL/min and pressure of 43.6 kPa. 1.0 μL extract was injected into the GC-MS. Split less injection modes were used for the chemical profiling. The oven temperature was initially 100°C and was held for 4 min. Then it was set to rise at 3°C/min, till 220°C and was held for 4 min and then was raised till 260°C with ramp of 5°C/min. The ion source temperature was 220°C; interface temperature 260°C and solvent cut time of 2 min. and a mass range of 50 m/z to 450 m/z. The peaks were identified by comparing with those spectra available in National Institute of Standards and Technology-2007 (NIST-2007) spectral libraries.
     
    Total carotenoid analysis (TCA) and carotenoid profiling through HPLC
     
    For TCA, 6 g of dried pupa sample was mixed with 20 mL of methanol using a glass mortar and pestle, filtered using Whatman no. 42 filter paper (pore size: 2.5 µm). The extraction was repeated 3-4 times with fresh solvent to obtain sufficient extract. The pooled extracts were then concentrated to 5 mL using a rotary evaporator under vacuum (≤55°C) and stored in -20°C for further analysis (entire process was conducted under low light conditions). Extraction and calculation were performed following Machmudah and Goto (2013) with slight modification. The extract was diluted 100 fold and optical density was measured at 450 nm using Cary 60 UV-Vis Spectrophotometer (Model No: G6860A, Agilent Technologies) and calculated according to the formula:
     
    Where,
    V= Volume of extract (mL).
     = Average absorption coefficient for β-carotene =2500 L/mol.cm).
    W= Weight of the sample (g).
    A= Absorbance at 450 nm.
           
    Carotenoid extraction for HPLC analysis followed the same procedure used for TCA. HPLC equipped with a photodiode array (PDA) detector (Model: LC-20 AP, Shimadzu, Japan) was used for carotenoid profiling. Analytical standards of high purity lutein (Product No. 07168-1 mg) and β-carotene (Lot No. BCBH8452V) were procured from Sigma Aldrich. A reversed-phase C18 column was used for chromatographic separation. The mobile phase for lutein consisted of Solvent A: acetonitrile and methanol (75:20, v: v) containing 15 mM ammonium acetate and Solvent B: pure methanol. The elution was performed with 95% Solvent B and 5% Solvent A at a flow rate of 0.8 mL /min. The injection volume was 20 µL and detection was performed at 445 nm using a UV-visible PDA detector.  The mobile phase for β-carotene comprised of methanol and acetonitrile (75:30 v: v), with 3.2 g/L ammonium acetate. Elution was performed using 100% of this mobile phase under isocratic conditions at 1.5 mL/min flow rate. Detection was performed at 460 nm. The mobile phase and chromatographic conditions for lutein and β-carotene were adopted from Sun and Zhan (2021) and Brauer (2014) respectively. Calibration curves were constructed for lutein and β-carotene by plotting the peak areas against known standard concentrations and used to quantify their concentrations in pupae samples.
     
    Statistical analysis
     
    For proximate analysis and TCA, all values were performed in triplicate (n=3) and results were expressed as mean± standard deviation (SD). Data were analyzed using Microsoft Excel. GC-MS and HPLC analyses were performed on a single representative sample for compound profiling purpose; hence results are presented descriptively.

    RESULTS AND DISCUSSION

    Proximate composition
     
    Table 1 shows the proximate composition of S. ricini pupae in comparison with fish and soybean meal based on literature. The observed moisture content was lower than the value reported by Longvah et al., (2011) for S. ricini pupae. The observed protein level was 52.02% which was greater than the protein content of spent silkworm pupae (about 48.7%) reported earlier by Rao (1994) but considerably lower than the 54% that Longvah et al., (2011) reported. With high protein content, Eri pupae compare favorably with conventional fish meal and clearly surpass soybean meal. The observed carbohydrate content was relatively higher than the 3.45% reported for eri pupae by Longvah et al., (2011) and 4.7% for B. mori pupae by Yeruva et al., (2023). Further, the carbohydrate content is higher than in fish meal, while lower than in soybean meals. Insects, typically have higher protein and fat content than carbohydrate (Zhou et al., 2022). The fat contents were moderately high, exceeding the lipid levels of fish meal and soybean meal. The observed ash content was very much similar to 4% reported for Antheraea pernyi G. pupae by Zhou and Han (2006). The fibre content falls between the values reported by Kashyap et al., (2023) for non-deoiled (3.5%) and deoiled (6.8%) eri pupae.

    Table 1: Proximate composition of S. ricini pupae compared to Fish and Soybean meal based on literature.


     
    Fatty acid profiling
     
    Ten significant compounds were detected through GC-MS analysis, predominantly sterols, fatty acids and their methyl esters (Table 2). The chromatogram of the FAMEs obtained through GC-MS analysis is presented in Fig 1. The major fatty acid identified was 9, 12, 15 octadecatrienoic acid (Z, Z, Z)-commonly known as α-linolenic acid (ALA), comprising (49.09 %), of the total fatty acids, indicating a rich percentage of polyunsaturated fatty acids (PUFAs). A remarkable level of cholesterol (19.35%), an important sterol of animal origin, was also detected. In addition, N-hexadecanoic acid (palmitic acid) constituted 13.22% representing a common saturated fatty acid. The presence of methyl esters of linolenic, linoleic, stearic and palmitic acids as minor ingredients suggested a rich profile of essential fatty acids. Similar results were reported by Mishra et al., (2025); representing that Eri pupal oil is rich in unsaturated fatty acids, including omega-3 fatty acids. Payne et al., (2016) noted high levels of monounsaturated and polyunsaturated fatty acids in silkworm pupae, while Rodríguez-Ortiz  et al. (2024) reported palmitic acid (23.61%) as the major saturated fatty acids, oleic acid (31.45%) as the main monounsaturated fatty acids and ALA as the primary polyunsaturated fatty acid (29.90%) in B. mori pupae. Javali et al., (2015) reported that silkworm pupae contain 60% to 70% of n-3, n-6 and n-9 fatty acids, supporting their utilization in food, pharmaceutical and medicinal applications.

    ​

    Table 2: Fatty acid composition of S. ricini Pupae obtained through GC-MS analysis.



    Fig 1: GC chromatogram of fatty acid profile from Samia ricini pupae.


     
    Total carotenoid analysis and carotenoid profiling through HPLC
     
    The total carotenoid content of Samia ricini pupae was 377.89±1.83 µg g-1 (Table 3) which is much greater than the values reported by Cheico et al., (2019) for Bombyx mori pupae, including White Polyhybrid Strain (WPS: 17.1 µg/g) and Golden Nistari Strain (GNS: 35.0 µg/g).

    Table 3: Total carotenoid, lutein and â-carotene concentration (µg/g) of S. ricini pupae with chemical structures and bioactivities.


           
    HPLC analysis revealed the presence of lutein and β-carotene in Samia ricini pupae extract, with concentrations of 1.981 µg/g and 266µg/g, respectively (Table 3). The chromatographic profile (Fig 2 and 3) exhibited major peaks corresponding to lutein (6.442-7.268 min) and β-carotene (25.387 min), identical to their respective standards. These findings confirm the presence and abundance of both carotenoids in S. ricini pupae. Lutein (C40H56O2) is a naturally produced fat soluble carotenoid pigment with 40 carbon atoms and a conjugated double bond structure (Table 3) for which it provides striking red hue and produce free radicals (Fuad et al., 2020). Unlike other pro vitamin A carotenoids, lutein cannot be converted to vitamin A and hence must be obtained through food or supplements (Johnson, 2004). Having numerous biological and therapeutic potentials, it is widely used in pharmaceutical, animal and fish feed industries (Fuad et al., 2020).  Some of its biological activities are presented in Table 3. Meanwhile, β-carotene, an isoprenoid, naturally produced lipid soluble compound with 40 carbon atoms and a long chain of conjugated double bonds (Table 3) which undergoes isomerization at high temperature, enhancing color. Besides pigmentation, β-carotene exhibits different biological activities; some are presented in Table 3.  Cheico et al., (2019) reported lutein (11.1 µg/g in WPS; 21.7 µg/g in GNS) and β-carotene (6.0 µg/g in WPS; 13.3 µg/g in GNS) in B. mori pupae. Kotake-Nara  et al. (2002) reported lutein (1.28 mg), neoxanthin (0.22 mg) and violaxanthin (0.07 mg) per 250 g in B. mori pupae. S. ricini pupae showed markedly higher total carotenoids compared to the commercial feeds reported by Wallat et al., (2005) where Zeiglar Tropical Fish Feed (21.70 µg/g), BioKyowa Series C-700 (27 µg/g), Arkat VibraGro (5.70 µg/g) and minnow meal (249 µg/g), with substantially higher β-carotene content, suggesting its superior potential as a natural carotenoid rich ingredient. Besen et al., (2019) observed improved survivability and color enhancement in juveniles of Carassius auratus when supplemented with lutein. Kaur et al., (2016) demonstrate that natural β-carotene supplementation (25 ppm), derived from carrot powder, significantly improves growth and pigmentation in ornamental Koi carp.

    Fig 2: Chromatogram of lutein in Samia ricini pupae.



    Fig 3: Chromatogram of â-carotene in Samia ricini pupae.

    CONCLUSION

    The present study revealed significant amounts of protein, lipids with diverse composition of fatty acids mainly PUFAs and sterols and specific carotenoids like lutein and β-carotene in S. ricini pupae which indicate their potential as an alternative substitute for animal feed and nutritional applications. Further investigations on feeding trials, bioavailability and commercial application are required.

    ACKNOWLEDGEMENT

    The authors sincerely thank the Department of Zoology, Nagaon University for providing the necessary requirements to conduct this research. Also acknowledge the Researchers and Scientists of Natural Product Chemistry Section, CSIR-North East Institute of Science and Technology, Itanagar, Arunachal Pradesh for analytical support. Also thankful to Dr. Hafizul Islam Pathan for proofreading the manuscript and the Assam Science Technology and Environment Council, (ASTEC), Govt. of Assam, for supporting the study.
     
    Contribution of authors
     
    All authors contributed to the plan of the study. Author Anjela Ahmed designed the study, performed the experiments with Hena Parbin. Abhijit Ch. Roy and Ujjal Bordoloi carried out FAME preparations for GC-MS analysis. Anjela Ahmed and Abhijit Ch. Roy wrote the first draft of the manuscript. Author Moushumi Hazarika performed carotenoid quantification through HPLC; while Chandan Tamuly analyzed the data. Bhuban Chandra Chutia contributed to the management and execution of the study and supervised the whole work. All authors read and approved the final manuscript.
     
    Statements and declarations
     
    We, the submitting authors declare that the research work is original and has not been published previously to any other journal.
     
    Informed consent
     
    As in this research no human or mammalian subjects were involved, no ethical approvals were required for the study.
     
    Funding declaration
     
    This work was supported by ASTEC (Assam Science Technology and Environment Council), Government of Assam, Science, Technology and Climate Change, under the Research and Development Minor Project entitled “Eri-Pupa Based Ornamental Fish Feed: Preparation, Standardization and Sustainable Utilization” (File No: ASTEC/SandT/192(204)/2022-23/1658; dated 17th September 2022). Authors Anjela Ahmed and Bhuban Ch. Chutia have received the research grant. 

    CONFLICT OF INTEREST

    There is no conflict of interest among the authors.

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