Analysis of Microbial Load and Diversity in Crickets (Gryllus Bimaculatus and Scapsipedus icipe) used as a source of protein for food

Abstract

Crickets are drawing interest as sustainable source of protein for food and feed worldwide. However, there is little information on microbial quality of edible crickets thus the need for a profound insight into their safety. The objective of the study was to determine the microbial load of two edible cricket species Scapsipedus icipe and Gryllus bimaculatus and to evaluate the efficiency of different processing methods (boiling, sundrying, freeze-drying, snap freezing and deep frying) in reducing microbial counts. The wild-caught crickets were obtained from Nguruman (Kajiado County) and Mbita (Homa Bay County) while the farmed crickets were reared at Animal Rearing and Containment Unit at the International Centre of Insect Physiology and Ecology (icipe). The cricket samples were screened for microbiota using culture-dependent method. Fifteen adult crickets were pooled together and homogenized in Phosphate-buffered Saline (PBS). The solution was used for mycological and bacterial isolation and analysis. Bacterial microbiota was isolated using Nutrient agar (NA) and MacConkey agar (MCA). Fungal microbiota was isolated using Sabouraud Dextrose Agar (SDA) and Potato Dextrose Agar (PDA). The isolates were characterized morphologically and through sequencing of bacterial 16S SSU rRNA genes and fungal internal transcribed spacer (ITS) rRNA gene. Most of the bacterial isolates (70 %) on NA had characteristics typical of members of Bacillus spp. while on MCA, most bacteria (90 %) had characteristics suggestive of members of E. coli. Majority (80 %) of the fungal isolates on PDA had characteristics similar to those of Aspergillus spp. while most isolates (80 %) on SDA were members of Trichoderma spp. Microbial counts of fresh cricket samples were generally high, with bacterial population ranging from 2.5 × 104 - 3.8 x 104 CFU /g fresh weight. The fungal populations ranged from 2.0 × 104 – 3.0 × 104 CFU /g fresh weight. Upon processing the microbial counts reduced considerably with bacterial counts ranging from 0.1 x 102 – 0.8 x 102 CFU /g dry weight (boiled) and 0.7 x 103 – 2.2 x 103 CFU /g dry weight (sun dried). The fungal counts ranged from 0.7 x 102 - 0.9 x 102 CFU /g dry weight (boiled) and 0.9 x 103- 1.7 x103 CFU /g dry weight (sun-dried). Freeze-dried and deep fried samples had no microbial counts. The diversity of bacteria and fungi species in wildcaught crickets was considerably high compared to the lab-reared crickets with most isolates belonging to species pathogenic to humans. Phylogenetic analysis revealed that most bacterial isolates from the wild-caught crickets related to members of Bacillus spp. (57 %), Staphylococcus spp. (43 %) and E. coli (14 %). Fungal isolates related to Aspergillus spp. (57 %) and Penicillium roseopurpureum (29 %). From the farmed crickets, most of the bacterial isolates related to E. coli (60 %), Enterobacter (40 %) and Lactococcus garvieae (20 %). The fungal isolates related to Trichoderma asperellum (75 %), Aspergillus spp. (25 %) and Tetrapisispora fleetii (12.5 %). From the cricket samples processed by boiling and sun drying, the microbial diversity was very low with only two bacterial isolates related to Rickettsiella grylli (42 %) and Wolbachia spp. (29 %), while the two fungal isolates related to members of Aspergillus spp. (67 %) and Trichoderma asperellum (33 %). Thirteen potentially novel bacterial and fungal isolates from wild and reared crickets had no close matches from gene bank and need further investigation. This study shows that crickets harbor diverse microbial communities some of which are potentially pathogenic. Deep-frying, freeze drying and snap freezing completely eliminated bacterial and fungal contaminants thus minimizing microbial risks in crickets meant for food.