Performing Department
Food Science & Human Nutrition
Non Technical Summary
Global food security is tenuous, pressured by a growing population, a changing climate, inequitable food distribution, and shifting demand toward more resource-intensive protein products (such as meat and dairy).Although crop production is at an all-time high, about 820 million people remain undernourished.In addition, problems of food insecurity and undernutrition are often compounded by environmental enteropathy (EE), a subclinical manifestation of intestinal inflammation and malabsorption resulting from chronic exposure to fecal pathogens. EE can contribute to impaired gut immune function, poor growth and lack of response to nutritional interventions, as well as poor oral vaccine responses.EE is prevalent in low-resource developing contexts and is a probable intermediary in childhood stunting and anemia. Interventions to ameliorate enteropathies could improve the health of millions of people. One potential solution to these issues is to increase consumption of nutrient-dense foods that can be sustainably and inexpensively produced and have the added value of promoting gut health. Edible insects offer an under-exploited and readily available source of nutrients across the globe, and our previous research suggests that edible crickets are protein-rich and may also provide benefits to gut health. We aim to build upon this research to further assess how edible crickets could alleviate intestinal inflammation and reduce EE. The overall objective of this project is to determine how whole cricket powder and isolated cricket chitin modulate gut microbiota and intestinal inflammation in a human population. We hypothesize that consumption of cricket powder, specifically cricket chitin, will support the growth of beneficial probiotic bacteria, alter the inflammatory potential of the gut microbiota, and improve intestinal barrier function and mucosal immunity.We will explore this hypothesis by completing three specific aims. The first aim examines whether Bifidobacterium and other probiotic species can assimilate cricket powder and purified cricket chitinas a sole carbon source and determineshow these substrates affect bacterial growth and viability. The second aim will assess the impact of whole cricket/cricket chitin consumption on a pilot population of individuals with irritable bowel syndrome (IBS). We selected a participant base with IBS because of their increased intestinal inflammation and permeability, which will serve as a proxy for a population with EE. Our third aim will utilize samples collected from the clinical intervention study to determine how consumption of cricket/chitin impacts intestinal barrier function. Our results will support future research on the microbiome, the use of edible insects for food, and ways to promote global food and nutrition security.
Animal Health Component
0%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Goals / Objectives
A global goal of our research is to increase the consumption of nutrient-dense foods that can be sustainably and inexpensively produced and that have the added value of promoting gut health. Edible insects offer an under-exploited and readily available source of nutrients across the globe, and our previous research suggests that edible crickets are protein-rich and may also provide benefits to gut health. We aim to build upon this previous research to further assess how edible crickets could alleviate intestinal inflammation and reduce environmental enteropathy. The overall objective of this project is to determine how whole cricket powder and isolated cricket chitin modulate gut microbiota and intestinal inflammation in a human population.We will achieve this goal by completing three specific aims. The first aim examines whether the beneficial gut microbe, Bifidobacterium, and other probiotic species can assimilate cricket powder and purified cricket chitin (and its digested derivatives) as a sole carbon source and determines how these substrates affect bacterial growth and viability. The second aim will assess the impact of whole cricket/cricket chitin consumption on a pilot population of individuals with irritable bowel syndrome (IBS). We selected a participant base with IBS because of their increased intestinal inflammation and permeability, which will serve as a proxy for a population with environmental enteropathy. Our third aim will utilize samples collected from the clinical intervention study to determine how consumption of cricket/chitin impacts intestinal barrier function using a series of in vitro experiments. We will determine how fecal waters, collected from participants during Aim 2, impact trans-epithelial electrical resistance (TEER) and migration of FITC-dextrin in Caco-2 cell monolayers. Our results will support future research on the microbiome, the use of edible insects for food, and ways to promote global food and nutrition security.
Project Methods
Aim 1: To determine whether whole cricket powder and cricket chitin and its digestive derivatives impact growth of Bifidobacterium and other probiotic bacterial species in vitro.Methodology: We will use whole cricket powder, chitin isolated from two species of edible cricket, and chitin digests obtained by acid hydrolysis and treatment with digestive enzymes, pepsin and trypsin, to explore the prebiotic effects of chitin on several Bifidobacterium species and other probiotic bacteria, such as Lactobacillus. Dr. Andrea Liceaga (Purdue University) has agreed to supply the purified chitin and digestive derivatives to use for this study. Bacterial isolates will be cultured in deMann, Rogosa, and Sharpe (MRS) broth containing 0.5g/L of cysteine and incubated under anaerobic conditions for 48 hours. Cultures will then be inoculated onto plates containing minimal MRS medium supplemented with 1.0, 0.25, or 0.10% (w/v) of the purified chitins, digested derivatives, or whole suspended cricket powder to determine whether these substances can serve as a sole source carbon substrates for the bacterial isolates. Additional tests using substrates that supported bacterial growth in the plate assays will be conducted to determine their impact on bacterial growth rate and minimum inhibitory concentration. Briefly, log phase bacterial cultures will be inoculated at 104 cells/ml into substrate-supplemented (concentrations ranging from 1.0-0.10% w/v) minimal MRS medium followed by plated cell counts at 2 hour intervals over a 24 hour timeframe.Aim 2: To determine the effects of cricket chitin consumption on gut microbiota and inflammation and immune function in a population of individuals with IBS.Methodology: In a double blind, randomized-controlled crossover trial, we will investigate the effects of consuming 25g/day whole cricket powder, 4 g/day cricket chitin, or a placebo (treatments will be provided in macronutrient matched beverages) in twelve individuals with IBS. Sample size is based on the number of individuals needed to detect a significant difference (α=0.05) in Bifidobacterium sp. between whole cricket and placebo treatments with 80% power. Primary outcome measures will include: fecal microbial analysis, markers of mucosal inflammation (such as secretory IgA and fecal calprotectin), circulating cytokines, and circulating markers of intestinal barrier disruption, such as zonulin. To account for ~ 20% expected attrition, sixteen adults (>18 years old) who have obtained a diagnosis of IBS according to the Rome IV criteriawill be recruited into the study, which will be conducted in the Food and Nutrition Clinical Research Laboratory (FNCRL) at Colorado State University after approval by the CSU Institutional Review Board. Individuals with food allergies, those who are pregnant or breastfeeding, and individuals with gastrointestinal conditions, including but not limited to bowel cancers, Inflammatory Bowel Disease (IBD), and Clostridium difficile infection, will be excluded. In addition, individuals taking probiotics and those who have used antibiotics in the two months prior to starting the study will also be excluded. The study will include three 30-day intervention periods with two interspersed 30-day washout periods between treatments. Each participant will be randomly allocated to a study cohort and be provided with a daily supplemental beverage containing 4g of cricket chitin, 25g whole cricket powder, or a placebo. Participants will provide fasting blood samples at each treatment baseline and the end of each intervention period. Whole blood will be analyzed on a Piccolo Xpress Chemistry Analyzer. We will also measure plasma C-reactive protein, zonulin, and levels of various circulating cytokines, such as TNF-a, IL-6, and IL-10. Participants will also provide six self-collected fecal samples that will be subsampled for DNA extraction and 16s sequencing, short chain fatty acid analysis, secretory IgA, and fecal calprotectin as previously described.5 In addition, the validated IBS Quality of Life Assessment will be completed at each visit to the FNCRL and participants will complete a daily stool log using the Bristol Stool Scale.Aim 3: To determine how fecal waters from individuals consuming cricket/chitin impacts epithelial cell barrier function ex vivo. Methodology: We will assess how fecal waters, collected from participants during aim 2, affect TEER and migration of FITC-dextrin in stimulated (with TNF-α) Caco-2 monolayers. Briefly, we will follow previously described methods,where Caco-2 cells will be grown to 60-70% confluency and cells from passages 20-40 will be seeded onto polycarbon inserts in Transwellplates. Seeded cultures will be allowed to differentiate for ~21 days to allow formation of monolayers. Monolayers will then be treated with collected fecal waters and TNF-α and TEER values will be determined by measuring the potential difference between the two sides of the cell monolayer using a MillicellERS meter. Migration of FITC-dextrin across Caco-2 cell monolayers will also be measured. Briefly, after washing and equilibrating cells, they will be treated at the apical compartment with collected fecal waters. After 2 hours, the cells will be treated with FITC-dextrin and media will be collected from the basolateral compartment at 30, 60, 90, 120, 180, and 240 minutes and fluorescence measurements read on a 96-well plate reader. This assay will be conducted in unchallenged monolayers as well as those incubated with fecal waters and then stimulated with a barrier disrupting agent such as TNF-a.