Does microbial composition and fermentative capacity differ between stool samples collected from male adults of different ages?

Males will be recruited from 3 age ranges: young (18-25 y), middle (40-50 y), and older (65-75 y) with n = 8 participants in each of the 3 groups. They will all be provided with the same meals (breakfast, lunch, dinner and snacks) for a 10 day period (days 1 to 10; microbial adaptation period). Lunch and dinner are being prepared by a commercial meal provider. With meals and snacks the participants will receive 2132-2230 calories, 82-94 g protein, 33-37 g fibre daily. Participants will also receive a commercial multivitamin tablet to take each day of this 10 day period containing Retinol acetate (vitamin A) 300 micrograms R.E.; Betacarotene 900 micrograms; Colecalciferol (vitamin D3) 20 micrograms; Phytomenadione (vitamin K1) 25 micrograms; Calcium pantothenate 13.6 mg; Thiamine nitrate (vitamin B1) 4.6 mg; Nicotinamide 16 mg; Riboflavin (vitamin B2) 4.6 mg; Pyridoxine hydrochloride (vitamin B6) 6.7 mg; Cyanocobalamin (vitamin B12) 21.6 micrograms; Biotin (vitamin H) 54 micrograms; Folic acid 300 micrograms; dl-alpha-tocopheryl acetate (vitamin E) 40 mg; Ascorbic acid (vitamin C) 180 mg; Calcium (294mg as calcium carbonate and 6mg as calcium hydrogen phosphate) 300 mg; Magnesium (as magnesium oxide) 84 mg; Iron (as ferrous fumarate) 6 mg; Zinc (as zinc oxide) 11 mg; Manganese (as manganese sulfate monohydrate) 5.5 mg; Chromium (as chromic chloride hexahydrate) 35 micrograms; Selenium (as sodium selenate) 55 micrograms; Copper (as cupric sulfate) 900 micrograms; Iodine (as potassium iodide) 150 micrograms). The diet has been designed by nutritionists with >20 years' experience and meet all nutritional requirements. Participants will keep a food diary during the 10 day adaptation period to confirm what was consumed. On days 1 and 11, a faecal sample will be collected from each participant for individual microbial analysis and to prepare a pooled inoculum for each age group. Samples will be self-collected using at the participant's home or at our facilities. Participants will receive detailed instructions on how to safely and hygienically collect faecal samples using a standardised protocol. All samples will be collected within a few hours and processed. The inoculum produced from the faecal samples is for use in an in vitro fermentation assay which mimics colonic functioning and requires the processing of fresh faeces to ensure viable microbes for the inoculum. The composition and function of the faecal microbiome for each age group will be determined using shotgun metagenomic sequencing. The faecal samples collected on day 1 for each participant will be compared with those collected on day 11, to determine the typical variation in fermentative capacity. The samples collected on day 11 will give information on the effect of age on fermentative capacity, as with the identical diets (days 1-10), the effect of diet should be minimised. After the pooled inocula is prepared from faecal samples collected from participants in each age group, to compare the fermentative capacity of the faecal inocula across age groups, purified fibre sources with different structures (e.g., cellulose, pectin, resistant starch) will be fermented using the pooled inoculum from each age group with the in vitro colonic fermentation assay. Organic matter (OM) disappearance and microbial metabolites (e.g., organic acids) will be measured by combining HPLC and MS-based methods. The results will be compared between age groups to determine whether there is an effect of age on the microbial action (i.e., fermentative capacity). This will inform as to whether, in the future, we need to consider age as a factor for our assay.