Luria agar

Grasshopper samples were taken from maize fields in Coronango, Puebla, Mexico. The geographical coordinates are the parallels 19^° 06′36″ and 19^° 10′42″ of north latitude and the meridians 98^° 14′54″ and 98^° 19′40″ of western longitude to 2,180 m above sea level. The recollection took place in September 2020 for early grasshopper (EG) and November 2020 for adult grasshopper (AG). Grasshoppers were collected and transported alive, and later, they were cleaned, not purged, and washed with distilled water, and frozen at −80^°C. The samples were freeze-dried, blended (NutriBullet NBR-0601), and stored at room temperature until use.
Folin–Ciocalteu reagent, gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH•), 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS•+), (S)-6-methoxy-2,5,7,8-tetramethylchromane-2-carboxylic acid trolox (Sigma Aldrich), serine endoprotease from Bacillus licheniformis 2.4L E.C.3.4.21.14 (Sigma Aldrich), Luria Agar, Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, Enterobacter aerogenes ATCC 13048 Salmonella sp., and Pseudomonas aeruginosa ATCC 77853 were used in this study. Samples of EG, AG, early grasshopper extract (EGE), adult grasshopper extract (AGE), early grasshopper hydrolysate (EGH), adult grasshopper hydrolysate (AGH), and hydrolyzed fractions were tested in this research.

Lactobacillus rhamnosus GG (LGG; ATCC 53103) was obtained from the Valio culture collection (Valio Ltd., Helsinki, Finland). Bifidobacterium bifidum (DSMZ 20456), E. coli RY13, and E. coli K12 DH5α were available internally. Prior to experiments, LGG (ATCC 53103) and B. bifidum (DSMZ 20456) were cultured at 37°C on Man-Rogosa-Sharep agar (Lactobacilli MRS Broth, Difco, France) supplemented with L-cysteine (Sigma Aldrich, Germany) for 24–48 h under anaerobic conditions. Liquid cultures of LGG and B. bifidum were then prepared from single colonies using MRS broth (Lactobacilli MRS Broth, Difco, France) supplemented with L-cysteine. The E. coli strains were cultured at 37°C on Luria agar (Sigma Aldrich, United States) overnight and colonies were picked for liquid culture in Luria-broth (Sigma Aldrich, USA). Heat treatment of the bacterial strains was performed by heating at 65°C for 0.5 h. The success of heat-killing was confirmed by plating the heat-killed bacteria on MRS or Luria agar overnight at 37°C.