Materials. Unless otherwise stated, all reagents and chemicals were purchased from Sigma-Aldrich. α-amylase (A3176, ≥ 10 units/mg solid), porcine pepsin (P-7125, ≥ 400 units/mg protein), porcine pancreatin (P-1750) and porcine bile extract (B-8631) were stored as directed until use. A Milli-Q plus system (Merck Millipore, Darmstadt, Germany) was used to purify water to a minimum resistance of 18.2 MΩ cm. All other chemicals, such as solvents used, were of analytical grade. Solutions of enzymes were prepared fresh before each use. The first round of insects was sourced from a farm in the United Kingdom. Insects were received fresh, immediately frozen to kill, then oven dried at 80℃ for 36 hours, prior to grinding in a coffee grinder. All were stored at -20℃ when not in use. The second round of insects were purchased from the online platform Bug Bazaar.
Simulated gastrointestinal digestion. 1 g of each insect sample was weighed out for digestion. To each sample, 5ml of α-amylase solution (75 units/ml in MQ) was added and sample agitated on a rotating plate (200 RPM) for two minutes. Following this 5ml of MQ water was added and pH adjusted to 2 using HCl (6M). During peptic digestion, 0.3ml of pepsin solution (0.15 g/ml) was added and incubated at 37℃ for 60 mins on rotating plate (70 RPM), followed by pH to 7 with NaOH (5M) to stop peptic digestion. Afterward, 1.7 ml of pancreatin-bile solution (24 mg/ml bile extract and 4 mg/ml pancreatin) was added to start pancreatin-bile digestion and samples incubated at 37℃ for 60 mins on rotating plate (70 RPM). Following digestion, sample volumes were brought up to 15ml using MQ water and stored at -20℃ until use. Samples were not heat inactivated to avoid degradation of the insect material which would not be true to physiological reality i.e. digestion does not involve a point at which material reaches above body temperature. Trials using only non-heat-inactivated in vitro digestion fluids at varying concentrations showed these had no significant impact on cell survival or viability over 24 hours at levels utilised in this trial (supplemental data).
Caco-2 cell culture. We were using the human intestinal Caco-2 cell line as the cells form an epithelial layer which displays several morphological and functional characteristics of mature enterocyte when grown in post-confluent cultures [30,31]. Further, a strong correlation has been found between published human absorption data and uptake by Caco-2 cells, establishing this method as useful in assessing iron bioavailability [32]. Caco-2 cells (ATCC; HTB-37) were obtained at passage 21 and 29 and used in experiments until passage 68. Cells were grown in Eagle’s Minimum Essential Medium (EMEM, BioWhittaker, BE12-125F) with 10% fetal bovine serum (FBS, Gibco, 10500-064), 1% L-Glutamine (200mM, BioWhittaker, 17-605E), and 0.18% Normocin (InvivoGen, ant-nr-2) at 37℃ with 5% CO2 and 95% humidified air with medium changed every two days. The cells were passaged at 80% confluence using trypsin-EDTA (0.05%, Gibco, 25300-054).
Cell survival measures. Cells were plated in 24-well plates (90,000 cells/well, Corning CellBIND) and cultured for 24 hours or 14 days, dependent on if the experiment was with differentiated or non-differentiated cells. Cell medium was changed 24 hours prior to the start of the trial. The cells were incubated with digest dilutions for 24 hours, following which digests and medium were aspirated and cells washed twice with DPBS. Cells were lysed in 100 µl cold RIPA buffer with added PPI, collected and stored at -80℃ until analysis. Digest dilutions with which cells were treated, were made by mixing digests with cell medium already present on the cells. In order to approximate actual insect concentrations represented by these dilutions (mg/ml) on the cells, it was assumed that 100% of the insect was digested in the 15 ml of digestion fluid. Given this, it was assumed that base digest prior to dilution contained 66.67 mg of insect per ml of digestion fluid. It is acknowledged that 100% digestion of the insect samples is an overestimate, as evidenced by visible precipitate in the digests. Total cell density (surface attached) as a proxy for cell survival after treatment with the various insect digests and controls were estimated by measuring the total protein content per well by using the Pierce BCA Protein Assay Kit [33].
Cell viability estimated by the MTT assay. The cells were cultured, maintained, and seeded as stated above in 2.3. and 2.5; cells were seeded in 96-well plates (10,000 cells/well, Corning CellBIND) and cultured for 24 hours. Cell viability was measured based on metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as measured by MTT Cell Growth Kit (MTT, Merck Millipore, CT02). Dimethyl sulfoxide (DMSO) was used to dissolve MTT crystals in place of acidified isopropanol, as it was ineffective.
Expression of stress-related protein markers. The protein levels of apoptosis-related proteins were identified using the Proteome Profiler™ antibody arrays (Human Apoptosis Array Kit; R&D Systems, MN, USA), which estimates the protein levels of 35 different apoptosis-related human proteins. Pooled samples of cell lysates were loaded on the arrays at 15 µg protein/membrane. Membranes with controls, digest controls, and insect digests were processed and developed simultaneously in the same run for each trial. The chemiluminescent signal was developed using a Chemidoc XRS+ (Bio-Rad Laboratories Inc., Hercules, USA). Samples from cells exposed to tolerable and cytotoxic levels of T. molitor and G. bimaculatus were additionally sent to OLink Proteomics (Uppsala, Sweden) for analysis with their organ-damage panel which analyses 92 protein biomarkers relevant to processes involved in biological response to organ damage.
Verification trials. Verification trials were done to account for potential impact of processing or source of insects utilised. The first verification utilised insect samples heated to 105℃ for 60 minutes in non-confluent cells. The second verification examined freeze dried insects, T. molitor and Locusta migratoria, which were explicitly marketed as human food from a farm in the Netherlands in non-confluent cells.
Based on continually updating research these samples were run through a slightly modified simulated gastrointestinal digestion as follows. 1 g of each insect was weighed out. To each sample 4 ml of salt solution (140 mM NaCl and 5 mM KCl) pre-heated to 37°C was added, following which the pH was adjusted to 7 using 1 M NaHCO3. Sample volume adjusted to 6.5 ml and 500 µl of the amylase stock solution (1050 U/ml; Sigma-Aldrich A3176-1MU, 10 U/mg) was added to each sample and then incubated at 37°C with shaking (165 rpm) for 4 mins. The pH was immediately lowered to 2 using 1 M HCl, and volume of each sample adjusted to 9.5 ml with salt solution. Then 500 µl of the pepsin stock solution (40000 U/ml; Sigma-Aldrich P7012-5G, 2529 U/mg) was added to each sample and they were incubated at 37°C with shaking (165 rpm) for 60 mins. Subsequently the pH was raised to 5.5 using 1 M NaHCO3 and 2 ml of bile solution (10.635 mg/ml; Sigma-Aldrich B8631-100G, 1.68 U/mg) and 0.5 ml of pancreatin solution (7 mg/ml; Sigma-Aldrich P7545, 2.6U/mg) were added. The pH was adjusted to 7 using 1 M NaHCO3, total sample volume adjusted to 15 ml using salt solution and incubated at 37°C with shaking (165 rpm) for 60 mins. Digests were stored at -20°C until use. Cells were treated with concentrations previously identified as tolerable or cytotoxic and dilution was done in blank digestion fluids to ensure all cells were equally exposed to digestion enzymes.
Statistical analysis. Data was analysed with Microsoft Office Excel 2016, IBM SPSS Statistics 23 and Bio-Rad Image Lab 6.0.1. Data are shown as means with standard deviation or standard error in which n= number of separate trials, within trials the repeats were between 2- 4. Comparison of means was done with Student’s t-test, equal variances not assumed, two-tailed and significant differences were considered at p < 0.05.