Salty crackers enriched with cricket (Acheta domesticus) powder: a comprehensive quality and safety assessment study

Abstract

The formation of acrylamide was investigated in salty crackers enriched with cricket powder (Acheta domesticus) since its addition increases the free asparagine content. Cricket powder additions (5, 10, 15 %) were tested on crackers prepared with white wheat flour or whole grain wheat flour, baked for 25, 35 and 45 min at 180 °C. Additionally, chloropropandiols (2- and 3-MCPD) fatty acid esters and glycidol fatty acid esters were investigated. Acrylamide increased significantly after the addition of cricket powder and a prolonged baking time, especially in crackers containing white wheat flour. Although 2-MCPD and glycidol were not affected by the addition of cricket powder, 3-MCPD increased especially in crackers baked for 45 min. Crackers enriched with 5 % cricket powder were sensorially the most preferred.

Highlights

• •Cricket powder increased acrylamide formation, especially in white wheat crackers.
• •Crackers containing 5 % cricket powder were sensorially the most preferred.
• •2-MCPD and glycidol were not affected by cricket powder addition.
• •3-MCPD increased according to the addition of cricket powder and baking time.

1. Introduction

In recent years, edible insects have become of interest in western countries as an interesting and alternative source of high-quality proteins (EFSA, 2015). Depending on insect species, protein content can reach almost 80 % (w/w) (Rumpold & Schlüter, 2013). In addition to proteins, insects are also rich in other components such as vitamins (especially group B), minerals (e.g., iron, zinc, calcium, etc.) or polyunsaturated fatty acids (van Huis et al., 2013; Nowakowski et al., 2022). A positive impact has also been shown on the human microbiome (Nowakowski et al., 2022). On the other hand, the carbohydrate content is low, which contributes to the reduction of the glycaemic index (Bas & El, 2022; Mihaly Cozmuta et al., 2023). The nutritional profile of insects has been shown to depend on many factors, e.g., their feed composition, breeding, metamorphic stage, and habit. Other benefits that can be associated with insects are their high food and feed conversion efficiency, effective reproducibility, breeding conditions, and are environmentally friendly, using less water, energy, and producing less greenhouse gas emissions (van Huis et al., 2013; Rumpold & Schlüter, 2013; Lange & Nakamura, 2021). However, it should be noted that the consumption of insects may cause allergies in sensitive consumers due to the presence of some protein allergens (de Gier & Verhoeckx, 2018), chitin, or contribute to gout in disorders of amino acid metabolism.

The production of crackers enriched with insect proteins may present a benefit to human health, as mentioned above. In January 2018 the European Food Safety Authority (EFSA) approved the Novel Food Regulation (No. 2015/2283) for the consumption and sale of insect-based food products in all EU Member States. Permitted insects for food production are Tenebrio molitor – larva – mealworm in frozen, died and powder, for Locusta migratoria – migratory loctus, grasshopper, and Acheta domesticus - house crickets.

During the baking process, many chemical reactions take place providing sensorially attractive compounds that enhance the taste, colour, and flavour of the final product (Liu et al., 2022). Along with these desirable compounds, heat-induced processing contaminants may occur (Goh et al., 2021; Pandiselvam et al., 2024).

When protein-rich insect flours are used for the production of crackers, the presence of free asparagine can serve as an important reactant of acrylamide (Bas & El, 2022). Acrylamide is classified as a probable human carcinogen (group 2A, IARC, 1994) with a genotoxic and carcinogenic effect based on animal studies (EFSA, 2015). In 2017, a Commission Regulation (EU) 2017/2158 came into force establishing mitigation measures and benchmark levels for the reduction of the presence of acrylamide in food that contributes significantly to exposure to acrylamide. When these levels are exceeded, food operators must ensure mitigation measures achieving acrylamide levels as low as reasonably achievable (ALARA) below the benchmark levels. For savoury cereal snacks, the benchmark level was set at 400 μg/kg (Commission Regulation 2017/2158).

Furthermore, the fat (especially refined) used for cracker preparation can contribute to dietary exposure to 2-monochloropropane-1,3-diol fatty acid esters (2-MCPDE), 3-monochloropropane-1,2-diol fatty acid esters (3-MCPDE) and glycidyl fatty acid esters (GE) (EFSA, 2016; Di Campi et al., 2020), compounds that are regulated especially in refined oils and fats but also in infant formulae (Commission Regulation (EU) 2023/915). These contaminants are formed primarily during the vegetable oil refining process in the deodorisation step (Pudel et al., 2011). It has already been documented that MCPDEs can also originate in cereal products during baking due to the presence of mono- and diacylglycerols or phospholipids that react with chlorine added as natrium chloride or with naturally present organochlorines (Nagy et al., 2011; Mogol et al., 2014; Birch & Bonwick, 2019; Tiong et al., 2018; Kourimsky et al., 2025). The toxic effect of 3-MCPDEs and GEs is associated with their hydrolysis in vivo by gastrointestinal lipases to free 3-MCPD (a possible human carcinogen group 2B) (IARC, 2012; EFSA (European Food Safety Authority), 2016, EFSA (European Food Safety Authority), 2018) and glycidol (a probable human carcinogen group 2 A) (IARC, 2000; EFSA, 2016). Although the toxicity of 2-MCPDEs is under investigation, still limited information is available (Oey et al., 2019).

To the best knowledge of the authors, limited studies are dealing with the investigation of cricket powder as a potential source of acrylamide formation in crackers. Furthermore, no studies on the formation of MCPDEs and GEs are available in these protein-enriched crackers. Only a few articles present the enrichment of cereal-based products by insects describing quality, safety, and sensory aspects (Osimani et al., 2018; González et al., 2019; Bartkiene et al., 2022; Mihaly Cozmuta et al., 2023). Therefore, the objective of this study was to contribute to the knowledge in a more comprehensive investigation of the impact of cricket powder (Acheta domesticus). Investigated was (i) the safety of crackers with respect to acrylamide, in addition to MCPDEs and GEs; (ii) sensory characteristics such as non-target analysis of aroma compounds and sensory evaluation; and (iii) nutritional parameters such as the content of free amino acids and fatty acids present in cricket powder, flours used for cracker preparation, and crackers that were subjected to sensory evaluation. The data obtained will contribute to the knowledge of new foods and the impact of their composition on the quality and safety of the final product.

2. Materials and methods

2.1. Chemicals

Acrylamide (CAS 79-06-1, purity 99.5 %) was purchased from Sigma-Aldrich (Buchs, Switzerland). Internal standard ¹³C₃-acrylamide (isotopic purity ≥99 %, +100 ppm hydroquinone) from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Magnesium sulphate (p.a. purity ≥98 %) was from Fluka (Tokyo, Japan). Sodium chloride (purity ≥99 %), and anhydrous sodium sulphate were from Penta (Chrudim, Czech Republic). Methanol, n-hexane, and aluminum oxide were obtained from Merck (Darmstadt, Germany). Acetonitrile, amino acids, glucose, fructose, sucrose (all purity ≥99.5 %) and formic acid (purity ≥95 %) were supplied by Sigma-Aldrich (Steinheim, Germany). Deionised water was obtained from a Millipore apparatus (Billerica, MA, USA). Standards of 2-MCPD diesters, 3-MCPD diesters, and glycidyl esters: 1,3-dipalmitoyl-2-chloropropanediol (1,3-diP-2-MCPD), 1,3-dipalmitoyl-2-chloropropanediol-d5 (1,3-diP-2-MCPD-d5), 1,2-dipalmitoyl-3-chloropropanediol (1,2-diP-3-MCPD), 1,2-dipalmitoyl-3-chloropropanediol-d5 (1,2-diP-3-MCPD-d5), glycidyl palmitate, glycidyl palmitat-d5 (all purity ≥97 %) were purchased from Toronto Research Chemicals (Ontario, Canada). Standards of fatty acids: Supelco 37 Component FAME Mix, and tetrahydrofuran (purity 99.5 %) were supplied by Sigma-Aldrich (Munich, Germany). Aminopropyl (NH₂) SPE cartridge (Enviro-Clean NAX, 500 mg, 6.0 mL) (Chromservis, Czech Republic. SPME fibres coated with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS, 50/30 μm, 1 cm) were purchased from Merck (Darmstadt, Germany). The saturated alkane mixture (C7-C30) (certified reference material, 1000 μg/mL each component in hexane) intended for the identification of retention index (RI) was obtained from Sigma-Aldrich (Steinheim, Germany).

Sulfuric acid (purity >95 %), sodium hydrogen carbonate (purity ≥99 %), sodium hydroxide, sodium chloride were from Penta (Chrudim, Czech Republic). Phenylboronic acid (purity >97.0 %) and sodium bromide (purity >99.95 %) were from Fluka Chemie (Buchs, Switzerland). The technical gases (helium 6.0, argon 5.0, and nitrogen 5.0 and 4.0) were purchased from SIAD (Prague, Czech Republic) White wheat flour and whole grain wheat flour (Triticum aestivum L.) were provided by an industrial mill company, Mlyn Perner, s.r.o. (Svijany, Czech Republic). Cricket powder (Acheta domesticus) was purchased from Grig.cz (Brno, Czech Republic). According to the producer, the cricket powder was prepared from dried adult crickets. Basic dough ingredients, including rapeseed oil and salt, were purchased from commercial suppliers.

2.2. Cracker preparation

The cracker dough was prepared using only basic ingredients: flour (200 g of white wheat flour or whole grain wheat flour), refined rapeseed oil, salt (NaCl) and distilled water. Farinograph water absorption for white wheat flour was 53.8 % and for whole grain wheat flour was 68.3 %. Crackers differed in the addition of cricket powder (Acheta domesticus): 0, 5, 10 and 15 % (w/w). The description of the recipes is summarised in Table 1.

Table 1.

Composition of cracker dough.

	R 1	R 2	R 3	R 4
Ingredients	(g)
White wheat flour	200	190	180	170
Cricket powder	–	10	20	30
Rapeseed oil	45	45	45	45
Salt	3	3	3	3
Water	56	56	56	56
	R 5	R 6	R 7	R 8
Ingredients	(g)
Whole grain wheat flour	200	190	180	170
Cricket powder	–	10	20	30
Rapeseed oil	45	45	45	45
Salt	3	3	3	3
Water	76	76	76	76

R1–R8 – corresponding to recipes 1–8.

A Kitchen Aid machine (Greenville, Ohio, USA) equipped with a 4.5 L inox bowl and a flat beater was used for mixing. The dough was prepared by first mixing flour (200 g) with 45 g of rapeseed oil. Subsequently, 3 g of diluted salt in water (56 g of water for wheat flour and 76 g of water for whole grain wheat flour) were added and properly mixed. The higher amount of water added to the dough containing whole grain wheat flour was due to the higher protein content to obtain the optimal viscosity of the dough. The viscosity of the dough was not measured as it was not the aim of the study. However, with a short mixing time, the large development of wheat gluten was avoided. This ensured a good rolling of the dough and maintaining the shape of the crackers during baking. The dough preparation was optimised to obtain a good consistency of the non-sticky dough so that it can be easily rolled out. To ensure the same properties of dough, it was necessary to strictly maintain the order of mixing of raw materials and the time regime (pre-kneading the flour with oil, final kneading with other ingredients in a constant time). To further follow the experimental procedure, the dough was rolled 2 times back and forth with an intermediate step of turning the dough sheet 90°. The dough was rolled to a thickness of 3.5 mm and manually cut to a size of 40 × 40 mm using a square rolling cutter and baked in an electric mini-oven Sencor SEO 2000BK (Japan) for 25, 35 and 45 min at 180 °C. The dough of one recipe was prepared once and from this, always 16 crackers were baked for 25, 35 and 45 min. The baking conditions were also optimised. Between each baking, we waited 10 min for the temperature equilibration. After baking, the plate of crackers was also cooled for 10 min. Each second cracker was selected and homogenised, making a pooled sample (for each time and recipe), and stored at −28 °C until analysis. The other half of the crackers was used for analysis of colour and rheological properties (these data will be used in another study).

Crackers baked with white wheat flour for 25 min (Recipe 1, without cricket powder) were assigned as ´control´, baked in a slight golden yellow colour as recommended in Acrylamide Toolbox (2019). The colour developed after such a long time due to the relatively poor recipe consisting only of basic ingredients (flour, oil, water, and salt). This basic recipe was deliberately selected with the aim of (i) eliminating flavour compounds that can originate from additional sensory enhancers and (ii) investigating how cricket powder contributes to the formation of acrylamide in crackers. Longer baking times (35 and 45 min) were selected to demonstrate the extreme of the baking conditions in terms of acrylamide formation. However, these crackers were still consumable. The repeatability of the baking procedure was verified by determining acrylamide, since acrylamide is known as a marker of thermal load in cereals. For both types of crackers (prepared from white wheat flour and whole grain wheat flour), the baking time of 35 min was selected and baked three times. The crackers were sampled as stated above, homogenised, and analysed for acrylamide content. The relative standard deviation (RSD) of these three baking repetitions was calculated. The repeatability expressed as RSD was <10 % for both types of basic (´control´) crackers. With regard to this, it was proved that baking each recipe only once is a representative way of sample preparation. The crackers are visualised in Fig. S1.

2.3. Analysis of samples

2.3.1. Determination of the quality parameters of wheat flour and cricket powder

The quality parameters of the white wheat flour (Triticum aestivum L.), the whole grain wheat flour (Triticum aestivum L.) and the cricket powder (Acheta domesticus) used for the preparation of the dough included the determination of proteins, fat, carbohydrates, fibre, ash and moisture. The total nitrogen content was determined using the Kjeldahl method according to AACC Method 46-12.01. The protein content in the cricket powder was calculated by multiplying the result by the nitrogen-to-protein conversion factor 5.0 (Ritvanen et al., 2020) and wheat flour 5.70 (AOAC, 1999). Total fibre was determined after deffating according to AOAC Method 985.29, ash content according to ICC Standard No. 104/1 and moisture content according to ICC Standard No. 110/1. The available carbohydrate content was calculated by subtracting the average protein, fat, ash, and fibre content from 100 %.

2.3.2. Reducing sugars

Ultra-high-performance liquid chromatography Acquity UHPLC™ System coupled to an evaporative light scattering detector (UHPLC-ELSD) (Waters, USA) was used for the determination of sugars. The extraction approach was carried out according to Kocadağlı et al. (2016) with a slight modification: Approximately 1 g of flour was repeatedly extracted with distilled water (10, 5, 5 mL). During each extraction, the sample was vortexed for 3 min at room temperature and centrifuged at 6000 rpm for 5 min. The three extracts were combined and filtered with a 0.2 μm PVDF (polyvinylidene fluoride) membrane filter and diluted with ethanol (1:1, v/v). For chromatographic separation, an Acquity UPLC BEH Amide column (100 × 2.1 mm; 1.7 μm) (Waters, USA) was used. The column temperature was set at 40 °C and the injection of the sample was2 μL. The mobile phase consisted of (A) 80 % (v/v) acetonitrile and (B) 30 % (v/v) acetonitrile, both containing 0.2 % (v/v) diethylamine. The elution gradient with constant flow 0.2 mL/min was as follows: 0–5 min (60 % B), 5–5.01 min (100 % A) and 5.01–10 min (100 % B). The ELSD setup was as follows: drift tube temperature 40 °C, nebuliser in cooling mode (120 °C), nitrogen pressure 40 psi and gain 500. The method was internally validated with a quantification limit of 0.1 g/100 g.

2.3.3. Free asparagine and other amino acids

Ultra-high-performance liquid chromatography Acquity U-HPLC™ system (Waters, USA) coupled to high-resolution mass spectrometry Exactive™ with an orbitrap mass analyser (Thermo Fisher Scientific, USA) was used for the determination of free amino acids. The extraction of free asparagine together with other free amino acids was carried out according to Žilić et al. (2017) with slight modifications. Approximately 0.5 g of flour or homogenised crackers were weighted in a 25 mL volumetric flask and 20 mL of deionised water was added. The suspension was sonicated in an ultrasonic bath for 10 min and the flask was topped with deionised water. The extract was filtered with a 0.2 μm PVDF (polyvinylidene fluoride) membrane filter and diluted with methanol (1:1, v/v). For chromatographic separation, an Acquity UPLC BEH amide column (100 × 2.1 mm; 1.7 μm) (Waters, USA) was used. The column temperature was set at 40 °C and the injection of the sample was 3 μL. The mobile phase consisted of (A) 100 % (v/v) water and (B) 100 % (v/v) acetonitrile, both containing 0.2 % (v/v) formic acid. The elution gradient with constant flow 0.35 mL/min was as follows: 0–5.0 min (75 % B), 5.0–7.0 min (60 % B), 7.0–7.1 (50 % B), 7.1–12.0 min (75 % B). The setup of the mass spectrometer was as follows: positive ionisation mode, scan range: 50–1000 m/z, spray voltage: 3.5 kV, capillary voltage: 60 V and capillary temperature: 280 °C. The method was internally validated with the quantification limit of 2 mg/kg.

2.3.4. Isolation of free lipids

An automatic Soxtec™ 2055 (FOSS, Denmark) was used to extract fat from both types of flour (white wheat flour and whole grain flour), cricket powder and baked crackers. The extraction approach was carried out as described by Belkova et al. (2018). Briefly, approximately 3 g of sample were mixed with anhydrous Na₂SO₄ and then extracted with petroleum ether at 135 °C. The fat content was determined gravimetrically. The extraction repeatability was 0.2 %.

2.3.5. Fatty acid profile

Gas chromatography coupled to a flame ionisation detector (GC-FID) 6890P (Hewlett – Packard, USA) was used for the fatty acid profile after their conversion to methyl esters (FAME). Approximately 10 mg of isolated fat was weighted in a vial and methylation was carried out by adding 0.5 mL of a methanolic NaOH solution (0.5 M) and heated at 80 °C for 30 min. Then 200 μL of boron trifluoride (methanol solution 14 %, v/v) was added and the mixture was heated at 80 °C for 30 min. After heating, 0.5 mL of heptane and 1 mL of a saturated sodium chloride solution were added. The upper heptane layer was transferred to a vial with an insert and analysed.

For separation, a capillary column SP-2560 (100 m × 0.25 mm i.d., film thickness 0.20 μm) was used with constant flow of helium (1.1 mL/min) as carrier gas. An aliquot of 1 μL of sample was injected with a split ratio of 100:1 at 250 °C. The GC oven temperature programme was maintained at 140 °C (5 min); then increased at a rate of 4 °C/min to 240 °C (15 min) with a total GC run time of 45 min. The detector was set at 260 °C. For the identification of FAME, a Supelco 37 Component FAME Mix (10 mg/mL) was used. The method presents an accredited method, according to EN ISO/IEC 17025:2018, with a quantification limit of 0.1 % of the FID profile containing 37 FAMEs.

2.3.6. Acrylamide

Ultra-high performance liquid chromatography Acquity UPLC™ (Waters, USA) coupled to tandem mass spectrometry XEVO TQ-S (Waters, USA) was employed for the determination of acrylamide. The sample preparation using the modified QuEChERS technique was carried out as described by Belkova et al. (2018). Briefly, 2 g of sample was first defatted with 10 mL of hexane. Subsequently, 10 mL of deionised water was added with an internal standard ¹³C₃-acrylamide (200 μL, 10 μg/mL) and shaken for 1 min. The sample was then centrifuged at 10 °C for 7 min at 10,000 rpm. The water extract containing acrylamide was transferred to acetonitrile containing a salt mixture (MgSO₄ and NaCl). The sample was shaken intensively for 1 min, centrifuged at 10 °C for 5 min at 10,000 rpm. The acetonitrile extract was transferred to another salt mixture (Al₂O₃ and MgSO₄) to remove the remaining matrix co-extracts that could interfere with acrylamide. The extract was transferred to a 2 mL vial, evaporated with a gentle stream of nitrogen, and reconstituted with water. The sample was then subjected to analysis. Chromatographic separation was performed on column Atlantic T3 (150 × 3 mm, 3 μm) (Waters, USA). The column temperature was set at 35 °C and the sample injection 7.5 μL. The mobile phase consisted of (A) 100 % (v/v) water and (B) 100 % (v/v) acetonitrile. The elution was isocratic with a constant flow of 0.3 mL/min. The mass spectrometric detection setting was as follows: capillary voltage was 3 kV, nitrogen desolvation gas flow was 800 L/h at 350 °C, nitrogen cone gas flow was 150 L/h, source temperature 120 °C and cone voltage 21 V. Data processing was performed with MassLynx 4.1 (Waters, USA). The method represents an accredited method according to EN ISO/IEC 17025:2018, with a quantification limit of 10 μg/kg.

2.3.7. 2-MCPD, 3-MCPD and glycidol

Gas chromatography System 7890 A (Agilent Technologies, USA) coupled to a triple quadrupole 7000 B (Agilent Technologies, USA) was used for the determination of MCPD esters and glycidyl esters. The analytical approach was performed according to the AOCS Official Method Cd 29a-13 (2013) with slight modifications. The oil sample (100 ± 10 mg) was weighted in a 4 mL vial and 40 μL of the isotopically labelled standards 1,3-dipalmitoyl-2-MCPD-d5 and 1,2-dipalmitoyl-3-MCPD-d5 (both concentrations 50 μg/mL), and 50 μL of glycidyl-palmitate-d5 (concentration 45 μg/mL) was added. To remove monoacylglycerols, an analytical approach described by Zelinkova et al. (2017) was used. First, the sample was dissolved in 500 μL of a mixture of hexane:ethylacetate (85:15, v/v) and purified using an aminopropyl SPE cartridge (Enviro-Clean NAX, 500 mg, 6.0 mL). The SPE cartridge was first conditioned with 2 mL of hexane:ethylacetate (85:15, v/v). Subsequently, the sample was loaded into the cartridge and the target analytes were eluted with 10 ml of hexane:ethylacetate (85:15, v/v). The eluent was evaporated using a vacuum rotary evaporator with a water bath (40 °C). The residue was then dissolved with 2 mL. Glycidyl esters were converted to 3-monobromopropanediol (3-MBPD) monoesters in an acid aqueous solution of sodium bromide (0.3 mL, c = 3 mg/mL, sulfuric acid 5 %, v/v). Consequently, the mixture was shaken and incubated for 15 min at 50 °C. The reaction was stopped by adding 3 mL of an aqueous solution of sodium hydrogen carbonate (0.6 %, v/v). To extract the 3-MBPD esters, 2 mL of hexane was added and vigorously shaken. The upper layer was transferred to an empty flask and evaporated using a rotary vacuum evaporator to dryness with a 40 °C water bath. The residue was dissolved in 1 mL of tetrahydrofuran. The sample was incubated for 16 h at 40 °C after adding 1.8 mL of sulfuric acid in methanol (1.8 %, v/v). Hydrolysis was stopped by neutralisation with 0.5 mL of a saturated solution of sodium bicarbonate (9 %, v/v). The organic layer was evaporated using a vacuum rotary evaporator (water bath temperature 40 °C). The residue was dissolved by adding 2 mL of sodium sulphate (20 %, w/v), and the fatty acid methyl esters were discarded twice by 2 mL of hexane. A saturated solution of phenylboronic acid (250 μl) was added to the mixture and incubated for 5 min in an ultrasonic bath (room temperature). The phenylboronic acid derivatives of MCPD and 3-MBPD were extracted with 0.4 μL of hexane.

Chromatographic analysis was performed on a system consisting of a pre-column HP5-MS (0.5 m × 0.25 mm × 0.25 μm) and a separation column HP5-MS (15 m × 0.25 mm; 0.25 μm) (Agilent Technologies, USA) with a constant flow of helium (1.7 mL/min) as carrier gas. An aliquot of 2 μL of sample was injected in the solvent-vent injection mode at 50 °C (1.5 min), then increased to a rate of 600 °C/min to 300 °C (2 min), 10 °C/min to 280 °C. The detection was performed after electron impact (EI) ionisation in MRM mode. The GC oven temperature programme was held at 50 °C (2.5 min); then increased at a rate of 20 °C/min to 100 °C; 5 °C/min to 130 °C, 60 °C/min to 280 °C. A post-run with backflash set at 300 °C and helium flow 8.56 mL/min was used for 3 min. The total GC run time was 16.5 min. The following transmissions of ion fragments are summarised in Table S1.

Data processing was performed with MassHunter Version B.04.00 (Agilent Technologies, USA). The sum of 3-MCPD and 3-MCPD fatty acid esters was expressed as 3-MCPD, and glycidyl fatty acid esters expressed as glycidol (Commission Regulation (EU) 2023/915). The quantification limit for 2-MCPD and 3-MCPD was 60 μg/kg and 150 μg/kg for glycidol. The method presents an accredited method according to EN ISO/IEC 17025:2018.

2.3.8. Analysis of aroma compounds

Head-Space Solid Phase Micro Extraction (HS-SPME) coupled to gas chromatography mass spectrometry with a quadrupole and time-of-flight analyser (GC-Q-TOF) (Agilent Technologies, USA) was used to analyse the profile of volatiles in baked crackers.

Approxamitely 1 g of homogenised crackers were weighed in 10 mL head-space vials (Supelco, USA) and 2 mL of saturated sodium chloride was added. The analysis was carried out on a HP Innowax column (30 m × 0.25 mm; 0.25 μm film thickness) (Agilent Technologies, USA) with constant helium flow (1.0 mL/min) as carrier gas. The volatiles were absorbed by a Solid Phase Microextraction (SPME) fibre coated with divinylbenzene/ Carboxen/Polydimethylsiloxan phase (DVB/CAR/PDMS 50/30 μm, Supelco, USA). The fibre was incubated and extracted at 40 °C for 15 min and 15 min, respectively. The desorption time was 4 min at 240 °C. The GC injector was operated in a pulsed splitless mode. The mass spectrometer was operated in the EI mode with an ion source temperature of 230 °C. An acquisition rate of 5 spec/s and a mass range m/z 40–550 were used. The acquisition speed was 5 spectres/s and the mass analyser resolution was set >12,500 (FWHM).

2.3.8.1. Processing of data and chemometric analysis

Data obtained by SPME-GC-HRMS were transferred to SureMass format, which was subjected to peak detection, deconvolution, and library search (NIST17.L). The data processing steps were performed in MassHunter Unknowns Analysis (Version B.10.1) (Agilent Technologies, USA). Before chemometric analysis performed in SIMCA 13.0.3 (Umetrics, Sweden), the data were subjected to sum normalisation (the absolute intensity of each ion was divided by the sum of the intensities of all the ions detected in each sample) (Belkova et al., 2018). Pareto scaling was used as a data pre-processing method prior to unsupervised principal component analysis (PCA) and supervised partial least squares discriminant analysis (PLS-DA). Significant volatile compounds that contributed to group clustering were selected on the basis of their VIP (Variable Importance in Projection) score in the PLS-DA model. Compounds with a VIP score greater than 1.0 were considered important. The NIST 17 mass spectral library (NIST, USA) was used for the identification of significant compounds with a match factor ≥ 750 and relevant Kovats retention indices (RI) relative to n-alkanes (C7–C30). Identity confirmation carried out comparing the exact mass of the detected ions (mass error < 5 ppm) and the isotopic pattern.

2.3.9. Sensory analysis

The sensory evaluation of the crackers was carried out according to the international standard ISO 8589:2008. Two sets of crackers containing white wheat flour or whole grain wheat flour (each enriched with cricket flour: 0, 5, 10 and 15 %) baked for 25 min at 180 °C were evaluated by 14 trained panellists who were meticulously selected, trained, and monitored in accordance with the international standard ISO 8586:2015, ISO 5496:2009, and ISO 3972:2013. The samples were served according to the international standard ISO 6658:2005 The profile test was performed in accordance with ISO 13299:2018. A 100-point unstructured scale was used for the evaluation of 13 descriptors: overall appearance, intensity of smell, pleasantness of smell, fragility, hardness, texture pleasantness, overall taste pleasantness, fatty taste intensity, pleasantness of fatty taste, salty taste intensity, pleasantness of salty taste, bitter taste intensity, off-flavour intensity. The crackers were served randomly. Finally, a preference test was performed. RedJade software (RedJade Sensory Solutions, LLC, Martinez, CA, USA) was used for data collection and statistical processing. For statistical evaluation, one-way analysis of variance (ANOVA) followed by Duncan's post-hoc test (p < 0.05) was used.

3. Results and discussion

3.1. Consideration behind the study design

To increase the nutritional value of popular fast-food snacks, such as cereal-based crackers, cricket powder presents an interesting source of the essential components mentioned above. However, since free asparagine was identified as the limiting factor for acrylamide formation in heat processed grain-based food products (Capuano & Fogliano, 2011; Curtis et al., 2009; Curtis et al., 2010), it should be taken into account that other sources, such as cricket powder, can increase the free asparagine content and thus pose a potential for the formation of acrylamide. To clarify this hypothesis, the impact of free asparagine present in cricket powder was investigated as a potential source of acrylamide formation in high-protein crackers. In addition to acrylamide, the data were supplemented with results of other processing contaminants, such as MCPD and glycidol.

3.2. Quality parameters of wheat flours and cricket power

The protein content of both the wheat flours and the cricket powder corresponded to soft wheat flour suitable for the preparation of cracker dough that meets the criteria for the protein content <10.5 g/100 g. The low protein content (containing weak gluten) is more suitable for cracker preparation, since better rheological properties are achieved compared to high-protein wheat flour (e.g., larger spread of cookies) (Misra & Tiwari, 2014). Although the protein content of cricket powder is higher than wheat flour, it is the properties of gluten that forms a dough. The higher fat content in whole grain wheat flour is associated with the presence of germ and coating layers (testa, pericarp, and aleurone layer) (Prabhasankar & Rao, 1999). The main components of cricket powder were proteins, fat, and fibre (composed predominantly of chitin). The quality parameters are summarised in Table 2. It should be noted that the nutritional composition of cricket powder can differ among others, as it depends on factors including, e.g., its substrate, farming conditions, species, habit, and processing before food production (van Huis et al., 2013; Rumpold & Schlüter, 2013; Lange & Nakamura, 2021).

Table 2.

Characterisation of flours and cricket powder.

	White wheat flour	Whole grain wheat flour	Cricket powder
	g/100 g
Proteins	9.0	10.4	55.6
Carbohydrates	84.8	74.9	5.5
Fat	0.8	1.8	16.0
Fibre	4.9	12.0	19.3
Moisture	12.7	0.5	4.3
Ash	1.2	10.4	3.7

3.3. Free asparagine and other amino acids, reducing sugars, and acrylamide

To define the content of free asparagine, the wheat flours and cricket powder used for baking experiments were analysed. The free asparagine content in white wheat flour (72.8 ± 10.9 mg/kg) was significantly lower (p < 0.05) in comparison to its content in whole grain wheat flour (222.0 ± 33.3 mg/kg) and cricket powder (218.6 ± 32.8 mg/kg). The lower amount of asparagine and, in general, other amino acids in white wheat flour (Table S2) was mainly caused by the elimination of bran and aleurone by sieving (Capuano et al., 2009; Žilić et al., 2020). These outer layers contain beneficial health compounds such as fibre, minerals, vitamins, or phenolic compounds, and are rich in proteins, especially aleurone (Žilić et al., 2012). It should be noted that the composition of amino acids in wheat flour can differ depending on, e.g., weather and soil conditions, location, application of fungicides, grain injuries, germination, fertilisers, harvest and post-harvest conditions (Halford et al., 2012; Oddy et al., 2022; Gunathunga et al., 2024). In addition to these agronomic and agrotechnical properties, milling technology has been shown to have a significant impact on the profile of amino acids. Prabhasankar and Haridas Rao (2001) presented that a greater loss of amino acids was observed when wheat was ground in stone and plate mills compared to hammer and roller mills.

Free amino acids were determined in both types of wheat fours (white and whole grain wheat flour) and cricket powder. Furthermore, the amino acid profile of the crackers baked for 25 min (containing 0 % cricket powder - ´control´ and the maximum added amount of 15 % cricket powder) is presented since these crackers were selected to be suitable for sensory evaluation and meet the criteria for the content of acrylamide (below the benchmark level 400 μg/kg) (Commission Regulation 2017/2158) (Table S2). The cricket powder compared to the wheat flours contained a significantly higher amount (p < 0.05) of free essential and non-essential amino acids. The essential amino acids in cricket powder were 77.2 %, while in white wheat flour and whole grain wheat flour it was 44.0 and 31.4 %, respectively. In general, the total content of essential amino acids in cricket powder was more than five times higher and more than two times higher compared to white wheat flour and whole grain wheat flour, respectively. The most abundant essential amino acid in cricket powder was valine (1270.6 ± 190.6 mg/kg), similarly to white wheat flour and whole grain wheat flour, which corresponded to 504.2 ± 75.6 and 1351.2 ± 135.1 mg/kg, respectively. Regarding the non-essential amino acids in cricket powder, the most abundant amino acid was arginine (2808.4 ± 421.3 mg/kg), followed by proline (2367.0 ± 355.0 mg/kg), glycine (1314.9 ± 197.2 mg/kg), glutamic acid (1266.3 ± 189.9 mg/kg) and alanine (1204.7 ± 180.7 mg/kg). The lowest content was observed for sulphur containing amino acids methionine (102.5 ± 15.4 mg/kg) and cysteine (<2 mg/kg). In general, insect proteins were found to have low proteinogenic amino acids methionine and cysteine (Rumpold & Schlüter, 2013). It should be noted that the addition of cricket powder significantly (p < 0.05) increased the content of essential amino acids, especially crackers containing white wheat flour. With regard to this observation, cricket powder may be a promising alternative to the traditional cracker composition that improves the nutritional value of the product.

Wheat flour and cricket powder were also subjected to analysis of reducing sugars, such as glucose, fructose, and maltose, but also sucrose. Sucrose is known to be a non-reducing sugar that can involve acrylamide formation as well, especially when subjected to a thermal reaction or when hydrolysed under acid or enzymatic conditions (De Vleeschouwer et al., 2009). Although the content of all sugars investigated in cricket powder was below the quantification limit (1.2 g/kg), the main reducing sugar detected in wheat flour was the expected maltose disaccharide. Its content in white wheat flour and whole grain wheat flour was 9.3 ± 0.3 g/kg and 9.6 ± 0.3 g/kg, respectively. Sucrose was the second major sugar detected. White wheat flour contained a significantly lower amount (p < 0.05) of sucrose (2.1 ± 0.1 g/kg) in comparison to whole grain wheat flour (7.4 ± 0.2 g/kg). Glucose was quantified only in whole grain wheat flour corresponding to 2.5 ± 0.1 g/kg. Fructose was not detected (below the LOQ, <1.2 g/kg) in any wheat flour.

During the baking experiment, it was obvious that asparagine was the determinant of acrylamide formation (Fig. 1A, B). Its content decreased significantly (p < 0.05) during prolonged baking time, while acrylamide increased especially in the case of white wheat flour crackers (recipe R1–R4). In the ´control´ crackers (R1, 0 % cricket powder, baked for 25 min) acrylamide was below the quantification limit (10 μg/kg). However, after 25 min of baking with the addition of 5, 10 and 15 % cricket powder, the acrylamide increased to 37 ± 24, 47 ± 12 and 82 ± 16 μg/kg, respectively (Fig. 1A). Compared to ´control´ crackers, acrylamide increased from 7 to 16 times. A similar increasing trend in acrylamide formation was also observed when the crackers were baked for 35 min (the increase in acrylamide in the case of 15 % added cricket powder was up to 4 times). After 45 min of baking, the increase was up to 2 times compared to the ´control´ crackers baked at the same time. The addition of cricket powder (15 %) was the most pronounced in the formation of acrylamide after 35 min of baking (corresponding to 459 ± 69 μg/kg). Although acrylamide in crackers with 5 % and 10 % cricket powder was fairly below this value, which corresponded to 165 μg/kg and 175 μg/kg, respectively.

Fig. 1.

Acrylamide and asparagine in crackers enriched with cricket powder. Error bars present 95 % confidence interval. (A) white wheat flour crackers and (B) whole grain wheat flour crackers, CP (cricket powder), Recipes R1–R4 corresponding to white wheat flour crackers and recipes R5-R8 to whole grain wheat crackers.

In the case of whole grain wheat flour crackers (recipe R5-R8), the benchmark level was exceeded 1.5–2 times already after 35 min of baking in all crackers, including the ‘control’ crackers (Fig. 1B). It should be noted that the contribution of cricket powder to the formation of acrylamide was not as obvious in whole grain wheat flour crackers compared to white wheat flour crackers, especially when baked for 35 and 45 min. This can be attributed to the asparagine content that was similar in both whole grain wheat flour and cricket powder. Rufián-Henares et al. (2009) presented that flour rich in proteins and fibres is a factor that contributes to a high amount of Maillard products. The results obtained in our study indicated that cricket powder containing free asparagine can be considered as an additional source that contributes to the formation of acrylamide in crackers. Similar results were presented by Bas and El (2022). Therefore, the processing conditions of crackers enriched with cricket powder must be carefully optimised to avoid additional acrylamide formation.

3.4. Fatty acid profile

The fatty acid profile was determined for both types of flour (white wheat flour and whole grain wheat flour) and cricket powder to supplement the data with the nutritional composition. Furthermore, the free fatty acid profile in the crackers baked for 25 min (containing cricket powder 0 % - ´control´ and the maximum added amount 15 %) is presented in Table S3. The dominant fatty acids in the cricket powder were linoleic acid (27.45 %), followed by oleic acid (22.73 %) and palmitic acid (21.36 %). The profile of the main fatty acids in cricket powder was in agreement with other studies (Osimani et al., 2018). Stearic acid (9.39 %), linolenic acid (1.20 %) followed by palmitoleic acid (0.53 %) and myristic acid (0.45 %) were identified as minor. The predominant fatty acid in white wheat flour was linoleic acid (53.16 %), followed by palmitic acid (15.76 %) and oleic acid (10.80 %). These results were consistent with previously published data (Roncolini et al., 2020; Tavoletti et al., 2018). A similar composition was observed in whole grain wheat flour, containing 50.5 % linolenic acid, 13.97 % palmitic acid and 12.64 % oleic acid. Compared to cricket powder, both wheat flours were almost two times higher in linoleic acid, but more than two times lower in oleic acid. The ratio of linoleic acid (n-6) to alpha-linoleic acid (n-3) corresponded to 23:1, which was significantly higher than the optimal ratio 5:1 to maintain healthy balance (EFSA, 2010). The addition of 5, 10 and 15 % cricket powder to the dough reached the optimal 3:1 ratio of n-6/n-3 in the final product (Table S3). The free fatty acid profile is in addition to other factors that are also dependent on the insect diet (Lange & Nakamura, 2021). Based on these results, cricket powder may present a good source of desirable fatty acids. However, the shelf life of these products during storage should be taken into account, as the high content of unsaturated fatty acids makes them significantly susceptible to oxidation and therefore reduces food quality (nutritional and sensory value) but can also have adverse effects on human health (Tao, 2015). The stability of cricket powder during one-year period at room temperature was investigated by Marzoli et al. (2023). The study compared three different processes, including oven drying at 80 °C, 120 °C and lyophilization, and revealed that higher amounts of hexanal and pentanal (secondary oxidation products responsible for the rancid smell) were detected in oven dried cricket powders.

3.5. MCPD and glycidol

To our knowledge, there are no studies that describe the formation of MCPD and glycidol in crackers enriched with cricket powder. The 2-MCPD content was found to be below the quantification limit (60 μg/kg). However, a significant increase in 3-MCPD was observed in all whole grain wheat crackers baked for 45 min with additions of 5 to 15 % cricket powder. For white wheat crackers, the only significant increase was observed when baked 45 min with 15 % cricket powder (Fig. 2A).

Fig. 2.

3-MCPD (A) and glycidol (B) in crackers enriched with cricket powder. Error bars present 95 % confidence interval. CP (cricket powder), R1–R8 (recipes).

It is already known that 3-MCPD increases with prolonged baking time (Mogol et al., 2014; Goh et al., 2019) due to the presence of chlorine ions (added as sodium chloride salt) or organochlorine compounds (naturally present) that react with mono- or diacylglycerols (precursors were not measured in our study, since the presented data were only supplementary to acrylamide) present in oil or cricket powder. This will be a task for further investigation. In the case of glycidol (Fig. 2B), neither the addition of cricket powder nor the prolonged baking time have shown an increase in its content. The glycidol content in the crackers prepared with white wheat flour (recipe R1–R4) and the crackers prepared with whole grain wheat flour (recipe R5-R8) corresponded to 13–44 μg/kg and 13–34 μg/kg, respectively. Although there is no limit for these processing contaminants in crackers, oils and fats used for food preparation are regulated (Commission Regulation (EU) 2023/915). The maximum level of 3-MCPD is 1250 μg/kg and glycidol 1000 μg/kg. In our study, the content of 3-MCPD and glycidol in the rapeseed oil used for the baking experiments was fairly below the maximum allowed levels. 3-MCPD corresponded to 101 ± 30 μg/kg. Glycidol and 2-MCPD were below the quantification limit of 150 μg/kg and 60 μg/kg, respectively.

3.6. Aroma compounds

To obtain a more comprehensive view of the aroma compounds originating during the baking procedure of crackers enriched with cricket powder, the volatile profile was investigated. The recipe was prepared as simple as possible to eliminate flavour compounds that can be affected by, e.g., baking powder, sugar, or other sensory enhancers commonly used for cracker preparation. However, having them still tasty. Data obtained by SPME-GC-HRMS were subjected to multivariate analysis (Section 2.3.8.1). The most significant factor that contributed to group clustering was baking time. Clustering was not proven by adding cricket powder. Although the main compounds that contributed to the clustering of crackers baked for 25 min were shown to be alcohols and aliphatic aldehydes (especially hexanal), typical secondary lipid oxidation products originating from hydroperoxide decay (Chang et al., 2020; Chu et al., 2023). The susceptibility of crackers to oxidation was mainly due to the presence of a high amount of unsaturated fatty acids present in cricket powder, but also in wheat flour (Table S3).

For long-baked crackers (35 min and 45 min), typical compounds were mainly alkylpyrazines and furan derivatives originating mainly through the Maillard reaction (Liu et al., 2022), but also aldehydes such as hexanal, typically originating via Strecker degradation or Maillard reaction (Perez-Santaescolastica et al., 2022). Fig. 3 presents the main compounds identified in the crackers baked for 45 min. It was obvious that with the addition of cricket powder (15 %) their content increased significantly.

Fig. 3.

Chromatogram and accompanying table of compounds detected in crackers baked for 45 min. * MSD responces for base peak ×10e-5.

3.7. Sensory evaluation

For sensory evaluation, crackers (white wheat flour and whole grain wheat flour) containing (0, 5, 10 and 15 % cricket powder) baked for 25 min (Fig. 4) were selected due to the low acrylamide content (<100 μg/kg) thus meeting the criteria for the acrylamide benchmark level (400 μg/kg) assigned to crackers (Commission Regulation 2017/2158). Regarding crackers prepared with white wheat flour, the addition of cricket powder (5 %) has been shown to have a positive impact on the senses of the evaluators with the highest rates on the pleasantness of aroma, texture, overall taste, fatty and salty taste. These crackers (5 % cricket powder) were evaluated to be less pronounced in intensity of bitter taste and off-flavour intensity compared to crackers with 15 % of cricket powder (Fig. 5, Table S4). Furthermore, the increase in cricket powder content changed the colour of the crackers from grey to brownish (Fig. S1). This was evaluated as pleasant, especially in crackers prepared with white wheat flour. In general, crackers with cricket powder were evaluated to be more fragile than ´control´ crackers (0 % cricket powder). This is attributed to the chitin content, which increased with increasing amount of cricket powder (Acosta-Estrada et al., 2021). In the case of whole grain wheat flour crackers, the addition of cricket powder (5 %) was rated similarly to that of wheat flour crackers with the same level of cricket powder (5 %). Compared to ´control´ crackers (0 % cricket powder), the evaluators appreciated crackers with cricket powder more. This has been reflected in the preference test, where 64 % of the evaluators appreciated the crackers (containing white wheat flour) with 5 % cricket powder. Crackers containing white wheat flour with 0 % and 10 % cricket powder were equally appreciated by 14 % of the evaluators. In the case of crackers containing whole grain flour, the highest rate (50 % of the evaluators) appreciated the crackers with 5 % cricket powder, followed by 10 % cricket powder (36 % of the evaluators) and 15 % cricket powder (14 % of the evaluators). Surprisingly, no evaluator preferred traditional ´control´ crackers (0 % cricket powder). Regarding the preferences of the crackers, the evaluators especially highlighted the taste, texture, fragility, absence of off-flavours, and bitterness. Several studies reviewed by Amoah et al. (2023) have been conducted with the aim of improving the nutritional quality of bakery products, e.g., bread, cookies, biscuits, crackers, muffins with edible insects and their larvae. This review summarised that, except for bread, the optimal addition of insect powder, acceptable to consumers, was 5 %. This observation was consistent with our study.

Fig. 4.

Crackers sensorially evaluated, baked for 25 min, enriched with cricket powder (0, 5, 10, 15 %).

Fig. 5.

Sensory evaluation of (A) white wheat flour crackers and (B) whole grain wheat flour crackers enriched with cricket powder (0, 5, 10 and 15 %) and baked for 25 min. CP (cricket powder). ** Significant differences between the values (p < 0.05).

4. Conclusions

This study presents comprehensive information on the relationship of the addition of cricket powder to crackers and the formation of acrylamide primarily, and also MCPD and glycidol. The results obtained in this large baking experiment may be summarised as follows:

• •The addition of cricket powder increased the formation of acrylamide. This was obvious, especially in crackers containing white wheat flour. In general, acrylamide was higher in crackers prepared from whole grain wheat flour.
• •The formation of 2-MCPD and glycidol was not affected by the addition of cricket powder, even baking time. Although the addition of cricket powder to the formation of 3-MCPD was more obvious during the prolonged baking time (45 min) especially in whole grain wheat flour crackers.
• •Most of the evaluators preferred crackers containing 5 % cricket powder in both types of crackers containing white wheat flour and whole grain wheat flour. Pleasant smell, texture, overall taste, fatty and salty taste were the attributes most appreciated.
• •The baking time was the most significant factor that affected the formation of volatile sensory active compounds. In crackers baked for a shorter time, there were dominant compounds such as alcohols and aldehydes, while the longer baking time contributed to typical Maillard products, e.g., pyrazines, aldehydes, and furan derivatives.

In conclusion, crackers enriched with cricket powder can present a suitable snack that provides a valuable nutritional source of fatty acids and essential amino acids. However, the profile of these beneficial compounds is affected by many factors that should be taken into account along with the formation of processing contaminants.

CRediT authorship contribution statement

Beverly Hradecka: Writing – original draft, Validation, Project administration, Methodology, Investigation, Formal analysis, Conceptualization. Lucie Stara: Visualization, Investigation. Tomas Kourimsky: Validation, Investigation. Michal Stupak: Validation, Formal analysis. Ivan Svec: Validation, Investigation. Marcela Slukova: Writing – review & editing, Validation, Methodology, Investigation. Pavel Skrivan: Methodology. Jana Hajslova: Writing – review & editing, Resources, Funding acquisition, Conceptualization.

Ethical statement

Ethical permission in case of food sensory evaluation was not required by our university. The panellists gave their consent to sensorially assess the crackers with regard to presenting the results. The rights and privacy of all participants utilized during the research were protected.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.