Xt15 extractor

The dry matter (DM), ash and ether extract (EE) content of the larvae were evaluated in triplicate on the freeze-dried larvae by standard methods according to the Association of Official Agricultural Chemists (AOAC 934.01, AOAC 942.05 and AOCS AM 5-04, respectively) on freeze-dried and granulated larvae [35 ,36 ]. Dry matter (DM) content was determined by drying the samples into a vacuum oven at 98 °C (Isotemp Standard Lab Oven, Model 230F, Thermo Fisher Scientific, Waltham, UK) until constant. Ash content was determined by incineration (Lindberg/Blue M LGO Furnace Box, Thermo Fisher Scientific, Waltham, UK) of the dry matter at 600 °C during 2 h. The EE was determined on hydrolyzed samples (4 N hydrochloric acid, 60 min, 90 °C) using extraction with petroleum ether for 120 min at 90 °C in TX4 filters (XT15 Extractor, ANKOM Technology, New York, NY, USA). Thawed larvae were analyzed in duplicate for pH measurement after homogenizing 1 g of larvae (VDI 25, VWR, Radnor, PA, USA) for 30 s in 9 mL of distilled water [37 ]. The pH of the homogenate was then measured using a digital pH meter (AB15 pH meter, BASIC accumulator, Thermo Fisher Scientific, Waltham, MA, USA).

The longissimus dorsi was cut into 2 mm of thin slices to weight by using an aluminum box, and then freeze-dried (Tofflon Freezing Drying Systems, Shanghai, China). Lyophilized meat was subsequently crushed into powder to analyze the intramuscular fat concentration by using the Soxhlet petroleum ether extraction (XT15 Extractor, Ankom Technology Corp., Macedon, NY, USA) as described by Zhang et al. [15 (link)]. Concentrations of fatty acids were determined using classical gas chromatography (6890 Series, Agilent Technologies, Wilmington, DE, USA) as described in a previous report [16 (link)]. Moreover, methionine and cysteine were determined as methionine sulphone and cysteic acid using an amino acid analyzer (Hitachi L-8900, Tokyo, Japan) after cold performic acid oxidation overnight and hydrolyzing with 7.5 mol/L HCl at 110 °C for 24 h. Tryptophan was determined using high performance liquid chromatography (Agilent 1200 Series, Santa Clara, CA, USA) after LiOH hydrolysis for 22 h at 110 °C.

Approximately 200 g of each ingredient and meat emulsions were dried in a forced air oven at 60 °C for 12 h. Dried samples were frozen at −18 °C and then ground using a mortar and pestle to pass through a 1 mm screen. Grinding was performed in a cooling room at 5 °C. An aliquot of 10 g was sent to Dairy One forage lab (Ithaca, NY, USA) for moisture, fat, and protein analyses. Moisture content was determined by oven drying samples at 105 °C for 3 h (NFTA Method 2.2.2.5). Total fat was analyzed by acid hydrolysis followed by solvent extraction (AOAC Method 954.02). Briefly, approximately 750 mg of the sample was weighed into an XT4 filter bag (Ankom Technology Inc., Macedon, NY, USA) containing 750 mg of diatomaceous earth. The bag was sealed and hydrolyzed with 4 N hydrochloric acid for 60 min in a sealed Teflon vessel (ANKOM^HCl Hydrolysis System, Ankom Technology Inc., Macedon, NY, USA). After hydrolysis, the fat extraction was performed using an ANKOM XT15 Extractor with solvent solution (45% petroleum ether, 45% diethyl ether, and 10% ethanol at 90 °C for 60 min. Nitrogen content was analyzed by complete combustion (AOAC Method 992.15) using a carbon/nitrogen determinator (CN628, LECO Corporation, St Joseph, MI, USA). Crude protein was then obtained by multiplying nitrogen content by 6.25.

Following a feeding period of 56 days, the shrimp in each tank were subjected to 24 h fasting. The weight of the shrimp was measured and the survival rate was determined through calculations. To determine the moisture content, crude lipid, and crude protein levels, a random sample of five shrimp was chosen from a tank. The hemolymph of five shrimp per tank was collected and subsequently centrifuged at 4 °C and 3500 rpm for 15 min. After the hemolymph supernatant was obtained, it was carefully collected and immediately preserved at a temperature of −80 °C. Hepatopancreas of four shrimp were taken, washed in normal saline (0.9% NaCl solution), then transferred to liquid nitrogen, and then stored at −80 °C for subsequent analysis. The intestine and hepatopancreas were collected from four shrimp, kept in RNA later reagent (Ambion^®, ThermoFisher, Waltham, MA, USA), and stored at −80 °C.
The whole shrimps underwent a drying process in a 105 °C oven, which was carried out with the aim of assessing their moisture content. The determination of the crude protein content was carried out using the Dumas Nitrogen method with a Primacs100 analyzer (Skalar, Breda, Dutch). For the determination of the crude lipid, the XT15 extractor (Ankom, NY, USA) was utilized. Additionally, the amino acid content was analyzed in accordance with the standard GB/T 18246-2019.