Physicochemical, Functional Properties of Edible Insects’ Chitin and Antimicrobial Properties of Derivatives Produced Through In Vitro Chitin Digestion and Fermentation

Abstract

Globally and in Kenya, there is a concerted effort to promote the consumption of edible insects. However, chitin poses a challenge to the utilization of edible insects as human food, since it is not digestible by the human gut's natural enzymes. Thus, the diverse biodiversity of edible insects offers a considerable chitin resource, prompting scientific investigations into its comprehensive benefits and potential applications. Therefore, this study aimed to characterize chitin extracted from House cricket (Acheta domesticus), field cricket (Gryllus bimaculatus) and black soldier fly larvae cocoons (Hermetica illucens) and to evaluate its potential health-promoting properties through in vitro digestion and fermentation studies. Chitin was chemically extracted and the functional groups were determined by Fourier Transform Infrared Spectroscopy (FTIR). The surface morphology of the chitin was examined using a Scanning Electron Microscope. Solubility, emulsion capacity, water holding capacity (WHC), oil binding capacity (FBC), degree of deacetylation (DDA) and purity of the extracted chitin were also determined. The extracted chitin was then in vitro digested by enzymes, followed by fermentation using ABY 10 and ABT 5 probiotic cultures. Derivatives of the in vitro digestion and fermentation process were then tested for antioxidant and antimicrobial properties. Further derivatives of in vitro digestion and fermentation were then determined as follows: fatty acid composition was determined using Gas Chromatography Mass Spectrometry (GCMS) while chitosan, chito-oligosaccharides, antimicrobial peptides and vitamins were determined using High Performance Liquid Chromatography (HPLC), Liquid Chromatography Mass Spectrometry (LCMS) and UltraViolet- Visible Spectroscopy (UV-VIS). Data analysis was done using STATA version 12. Data were subjected to one-way ANOVA to determine the differences in functional properties among the chitin samples. Further data were subjected to three-way ANOVA to determine the effects of the chitin sample, fermentation time and sample concentration on antioxidant activity, antimicrobial activity, fatty acid composition, chitosan, chito-oligosaccharides, antimicrobial peptides and vitamin content. The extracted chitin showed the characteristic functional groups i.e., O-H stretch, C=O stretch, N-H bend, CH2 ending and CH3 deformation, C-N stretch and C-O-C stretch. Chitin extracted from Gryllus bimaculatus recorded the highest values in oil absorption capacity (780.14%), emulsion capacity (65.67%) and emulsion stability (65.67%) (p<0.001). Chitin extracted from Acheta domesticus was more soluble in water than the commercial chitin. The highest level of deacetylation was reported in Hermetia illucens chitin (66.2%), while Acheta domesticus chitin had the least value (47.1%) (p<0.001). The microstructure images showed the presence of pores and fibers in all the chitin samples. The highest antioxidant activity in chitin samples fermented using ABY 10 was observed in derivatives of Gryllus bimaculatus, Acheta domesticus and Hermetia illucens chitin digested in vitro and fermented for 48 hours at a concentration of 5mg/ml (61.11%, 63.88% and 61.63%), while the least antioxidant activity was observed in the negative control (p<0.05). Derivatives obtained after in vitro digestion and fermentation of the chitin samples exhibited significantly different antimicrobial activity against Escherichia coli, Vibrio cholerae, Bacteroides fragilis, Enterobacter agglomerans, Shigella dysenteriae, Staphylococcus aureus and Bacillus cereus. The antimicrobial activity of the derivatives increased with an increase in fermentation time and sample concentration, with the highest activity being observed after 48 hours of fermentation and at a concentration of 10 mg/ml. The predominant SCFA in all the samples was acetic acid (40.86% - 64.75%), followed by propionic acid (8.76% - 17.34%), then butyric acid (7.51% - 15.33%) as well as trace levels of 4-methylvaleric acid (1.01% - 3.88%), iso- valeric acid and valeric acid. The highest values of chitosan were obtained after fermentation of Acheta domesticus chitin using ABY 10 and ABT 5 for 48 hours (2.91 g/100g and 2.90 g/100g) and fermentation of Hermetia illucens indigestible chitin using ABY 10 for 48 hours (2.90 g/100g) (p<0.05). Gryllus bimaculatus indigestible chitin fermented using ABY 10 for 48 hours and Acheta domesticus indigestible chitin fermented using ABY 10 and ABT 5 for 48 hours had the highest chito-oligosaccharides yield (9.82 mg/g, 9.07 mg/g and 9.37 mg/g). Gryllus bimaculatus indigestible chitin fermented using ABY10 for 48 hours, Acheta domesticus and Hermetia illucens indigestible chitin fermented using ABY 10 for 48 hours yielded the highest defensin like AMP (19.40 mg/100g and 18.32 mg/100g). Gryllus bimaculatus indigestible chitin fermented using ABY 10 for 48 hours yielded the highest vitamin B12 content (5.74 mg/100g). In conclusion, the edible insect’s chitin was similar to commercial chitin in regards to the FTIR spectra as well as surface morphology. ABY 10 proved to be more efficient in fermentation while Hermetia illucens indigestible chitin was more fermentable as compared to all the other samples. Regarding fermentation time 48 hours was found to be optimal in producing metabolites that had highest antimicrobial activity. The study findings collectively suggest that the development of chitin based food products could promote gut health.