Authenticity Evaluation of Insect Protein-Containing Food Products

Significance of the topic

Insect-derived proteins are emerging as sustainable alternative food ingredients, with Acheta domesticus (house cricket) gaining commercial traction. Reliable analytical approaches are required to verify species identity, ensure label compliance, and monitor potential allergen risks in highly processed matrices. LC/MS-based proteomics provides sequence-level specificity that can discriminate taxa and detect species‑unique peptides even after food processing.

Objectives and study overview

This application note presents a validated LC/MS workflow to authenticate cricket-derived ingredients in commercial protein bars. The aims were to (1) extract and profile proteins from processed bars, (2) identify species-specific peptide markers for Acheta domesticus, (3) confirm declared plant and animal ingredients, and (4) evaluate specificity using a non‑insect control product.

Methodology

Sample preparation and extraction:

• Commercial protein bars (five labelled as containing A. domesticus and one negative control) were cryogenically ground in liquid nitrogen.
• Delipidation was performed by three successive hexane washes to remove lipids and improve protein recovery.
• Proteins were solubilized in chaotropic buffer (7 M urea, 2 M thiourea, 50 mM DTT, 100 mM Tris-HCl pH 8), clarified by centrifugation, and quantified by Bradford assay.

Enzymatic digestion and cleanup:

• Aliquots corresponding to ~1 mg total protein were reduced (10 mM DTT, 60 °C, 30 min) and alkylated (30 mM IAA, 37 °C, 1 h).
• Samples were diluted and digested overnight with sequencing-grade trypsin (1:50 w/w) at 37 °C.
• Digests were acid-quenched, cleaned by solid-phase extraction, dried, and reconstituted in 2% acetonitrile/0.1% formic acid prior to LC/MS.

LC/MS acquisition and data processing:

• LC: Agilent 1290 Infinity III Bio LC with AdvanceBio Peptide Mapping C18 column (2.1 × 150 mm, 2.7 µm), column at 60 °C, flow 0.3 mL/min, 90-min acetonitrile gradient; injection equivalent to 250 µg peptide (40 µL).
• MS: Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Jet Stream ESI in positive mode. MS mass range m/z 300–3,200 (2 spectra/s); DDA MS/MS m/z 50–3,200 (8 spectra/s); isolation width m/z 4; collision energy applied by formula (4 × (m/z))/100 – 4.8 V; up to 8 precursors/cycle with active exclusion after 2 spectra (release after 1 min).
• Data analysis: database searches against UniProtKB reference proteome, an A. domesticus proteome, and a contaminant database; search parameters included trypsin specificity (≤2 missed cleavages), precursor tolerance 20 ppm, fragment tolerance 0.05 Da, fixed carbamidomethylation (Cys), variable oxidation (Met).
• Peptide-centric taxonomic mapping used Unipept to assign unique peptides to taxa and to flag nonspecific peptides shared between taxa.

Instrumental setup

The principal instrumentation and key parameters were:

• Agilent 1290 Infinity III Bio High-Speed Pump and Multisampler with integrated thermostat.
• Agilent AdvanceBio Peptide Mapping C18 column (2.1 × 150 mm, 2.7 µm).
• Agilent 1290 Infinity III Multicolumn Thermostat with Quick Connect Bio Heat Exchanger.
• Agilent 6545XT AdvanceBio LC/Q-TOF with Dual Jet Stream ESI source; drying gas 8 L/min at 320 °C; sheath gas 11 L/min at 350 °C; capillary 3,500 V; nozzle 1,000 V; fragmentor 175 V.

Main results and discussion

Identification performance and overall profiling:

• Across triplicate injections, an average of ~78,700 MS/MS spectra per sample were acquired, with an average peptide identification rate ~11%—typical for complex processed food matrices.
• Chromatographic and MS profiles varied across product formulations, reflecting different ingredient compositions (proteins from soy, rice, whey, nuts, cereals, etc.).

Taxonomic mapping and species-level assignment:

• Unipept mapping assigned many unique peptides to plant taxa consistent with declared ingredients (e.g., Glycine max for soybean, Prunus dulcis for almond, Arachis hypogaea for peanut, Avena sativa for oat, Fagopyrum esculentum for buckwheat).
• Between 25 and 41 peptides per sample mapped to Arthropoda in the cricket-containing bars; 10–12 peptides mapped to the Gryllinae subfamily.
• Five peptides were found to be unique to Acheta domesticus and were consistently detected in all cricket-containing bars but absent in the negative control, supporting high specificity.

Species-specific peptide markers and proteins identified:

• Species-unique peptides originated from three proteins: Apolipophorin-III and two Tropomyosin isoforms (Tropomyosin 1 and 2).
• Two peptides called out as particularly robust markers were LQEASEAADEAQKR (from Tropomyosin 1) and ALQTAEGEIAALNR (from Tropomyosin 2).
• MS/MS fragment ion series (b/y ions) confirmed peptide sequences, and extracted ion chromatogram (EIC) peak areas showed consistent detection across replicates.

Analytical implications and semi-quantitative potential:

• EIC peak areas for A. domesticus-specific peptides correlated qualitatively with the declared proportion of cricket powder in the different bars, suggesting potential for semi‑quantitative assessment under controlled calibration.
• The absence of insect peptides in the negative control verifies method specificity and low false‑positive risk when using curated databases and peptide-centric taxonomy.

Allergen considerations:

• Tropomyosins are well-known pan‑allergens in invertebrates with thermal stability and cross-reactivity to shellfish allergens; Apolipophorin-III has also been associated with allergenic potential in insects.
• The identified species-specific peptides (and their parent proteins) are therefore relevant both for authenticity testing and for allergen surveillance in novel food products.

Benefits and practical applications

The described LC/MS workflow provides:

• High confidence species-level authentication for insect-derived ingredients in processed foods.
• Simultaneous verification of multiple declared plant and animal ingredients from complex matrices.
• A path toward targeted monitoring: species-unique peptides identified here can be converted into focused, higher-throughput assays (MRM/PRM) for routine screening or quantitation.
• Support for regulatory compliance, label transparency, and allergen risk management.

Future trends and potential applications

Several developments can extend this approach:

• Transitioning species‑unique peptide markers into targeted LC‑MS/MS (MRM/PRM) assays to improve sensitivity and throughput for routine QC and regulatory testing.
• Developing quantitative calibration curves using isotopically labelled peptide standards to convert semi‑quantitative observations into absolute measurements of insect content.
• Expanding curated proteome databases for edible insect species to improve coverage and reduce ambiguous assignments.
• Integrating proteomics with orthogonal methods (DNA-based tests, immunoassays) to strengthen multi-layered authenticity and allergen assessment strategies.

Conclusion

This application demonstrates that a workstream combining robust sample preparation, high-performance LC separation, high-resolution DDA Q‑TOF acquisition, and peptide-centric taxonomic mapping can reliably authenticate Acheta domesticus in commercial protein bars. Five species-unique peptides from Apolipophorin-III and Tropomyosins provided consistent, specific markers for A. domesticus and can underpin targeted assays for authenticity and allergen monitoring. The method also confirmed declared plant ingredients and offers semi‑quantitative capability correlated to declared cricket content.

References

1. Giampieri F, Álvarez Suárez JM, Machì M, Cianciosi D, Navarro Hortal MD, Battino M. Edible insects: A Novel Nutritious, Functional, and Safe Food Alternative. Food Front. 2022;3(3):358–365.
2. Pilco Romero G, Chisaguano Tonato AM, Herrera Fontana ME, et al. House Cricket (Acheta domesticus): a Review Based on Its Nutritional Composition, Quality, and Potential Uses in the Food Industry. Trends Food Sci. Technol. 2023;142:104226.
3. Siddiqui SA, Zhao T, Fitriani A, et al. Acheta domesticus (House Cricket) as Human Foods—an Approval of the European Commission—A Systematic Review. Food Front. 2024;5(2):435–473.
4. Farkas VI, Máté M, Takács K, Jánosi A. The House Cricket (Acheta domesticus Linnaeus) in Food Industry: Farming, Technological Challenges, and Sustainability Considerations. Appl. Sci. 2025;15(17):9494.
5. Mesuere B, Debyser G, Aerts M, et al. The Unipept Metaproteomics Analysis Pipeline. Proteomics. 2015;15(8):1437–1442.
6. Hashiguchi T, Hashiguchi M, Tanaka H, et al. Comparative Analysis of Seed Proteome of Glycine max and Glycine soja. Crop Sci. 2020;60(3):1530–1540.
7. De Marchi L, Wangorsch A, Zoccatelli G. Allergens from Edible Insects: Cross Reactivity and Effects of Processing. Curr. Allergy Asthma Rep. 2021;21(5):35.
8. Lopata AL, Kleine Tebbe J, Kamath SD. Allergens and Molecular Diagnostics of Shellfish Allergy. Allergo J. Int. 2016;25:210–218.
9. Palmer LK, Marsh JT, Lu M, et al. Shellfish tropomyosin IgE Cross Reactivity Differs Among Edible Insect Species. Mol. Nutr. Food Res. 2020;64:e1900923.
10. Karnaneedi S, Johnston EB, Bose U, et al. The Allergen Profile of Two Edible Insect Species—Acheta domesticus and Hermetia illucens. Mol. Nutr. Food Res. 2024;68:2300811.
11. Barre A, Pichereaux C, Simplicien M, et al. A Proteomic and Bioinformatic Based Identification of Specific Allergens from Edible Insects: Probes for Future Detection as Food Ingredients. Foods. 2021;10(2):280.