Sera pak plus

Glycogen was measured in 40 mg of hepatopancreas. Frozen tissue was homogenized with 5% of trichloroacetic acid (TCA), and centrifuged for 6 min at 5,000 × g. 100 μL of the supernatants were mixed with 500 μL ethanol 95% and incubated at 37 °C for 3 h; the mix was centrifuged for 15 min at 5,000 × g and the pellet was dissolved in 20 μL of distilled water at 100°C, 200 μL of phenol 5% and 1 mL of sulfuric acid. Absorbance was recorded at 490 nm. Concentration was reported as mg⋅g^–1, calculated from a commercial glucose standard reagent (1 mg⋅ml^–1) (Sera Pak Plus^®).
In addition, glucose, and acylglycerids concentrations in plasma were determined using a clinical diagnostic reactive kit (Sera Pak Plus^®; Bayer, Whippany, NJ, United States). Concentrations were reported as mg⋅mL^–1.
Kruskal–Wallis non-parametric analyses were used to test for statistically significant differences in each variable among healthy and infected lobsters. Statistical analysis were carried out in R (R Development Core Team, 2016 (link)).

The total starch (TS) and resistant starch (RS) contents were determined using the methodology reported by Goñi et al. [28 (link)]. For the determination of the TS, an enzymatic solution of amyloglucosidase (brand Roche, No. 102,857, Roche Diagnostics, Indianapolis, IN, USA) was used. The RS content was quantified using different enzyme solutions and incubation times as follows, 60 min incubation with pepsin, 16 h incubation with α-amylase, and 45 min incubation with amyloglucosidase. The glucose released by enzymatic digestion was quantified using the glucose oxidase/peroxidase assay (SERA-PAK^® Plus, Bayer de Mexico, SA de CV), reading the optical densities of the samples at 510 nm. The percentage of starch hydrolysis (the rate of in vitro digestion) was determined according to the methodology reported by Zamudio-Flores et al. [29 ].

Total starch (TS) content was determined under the method reported by Goñi et al. [53 (link)]. For this, 50 mg of sample was dispersed in a solution of KOH 2 M to gelatinize the starch. The mixture was left for 30 min and subsequently incubated at 60 °C for 45 min, at a pH of 4.75, with an amyloglucosidase enzyme solution (Marca Roché, No. 102 857, Roche Diagnostics, IN, USA). After that time, the glucose content released was determined by the glucose oxidase–peroxidase test (GOPOD) (SERA-PAK^® Plus, Bayer de Mexico, S.A. de C.V.). The TS content was calculated as glucose (mg) × 0.9, using potato starch as a reference. The analysis was performed in triplicate for each sample.