Participants and Interventions
Healthy young adult women living in a hostel and eating food prepared in the hostel kitchen were recruited for the study. Individuals who had received a course of antibiotics within the last month were excluded as were those who intended to travel out of the city during the course of the feeding trial. Participants were briefed about the nature and purpose of the study, the importance of compliance with the study intervention, and the importance of maintaining a daily record of bowel movements and any abdominal symptoms. Volunteers were given a small monetary incentive to participate in the study. All participants received normal yoghurt daily for the first week of the feeding study, following which they received probiotic yoghurt daily for the next three weeks. This was again followed by regular yoghurt feeding for the next four weeks. Normal yoghurt was prepared in the diet kitchen by boiling standardized toned milk (3.0% fat & 8.5% msnf) and then cooling to 40°C, following which starter culture (YCX-11, Chr Hansen) was added at 1 unit per 10 litres of milk. The milk was distributed in 200 ml cups which were incubated at 40°C until the pH reached 4.6, and cups then transferred to a refrigerator for cooling. Probiotic yoghurt was prepared by adding Bb-12® (Batch no 2927446, Chr Hansen) at a concentration of 0.0006% to the cultured milk prepared as above. This dosage was calculated to provide approximately 109 cfu of Bifidobacterium per 200 ml serving of yoghurt. The investigator responsible for providing the diluted starter culture did not participate in yoghurt distribution or in the laboratory analyses. Bifidobacterial concentrations in yoghurt were checked by culture of diluted yoghurt (1:10 in peptone water broth, 0.9% NaCl, 0.85% peptone) and serial dilutions (10-2 to 10-10) were made and plated on Reinforced Clostridial Agar (13.5 g/250 ml, pH 6.8) (Himedia laboratories, Mumbai, India, Catalog number-M154-500G) containing mupirocin (25 μg/L of medium) (RM-6090, Himedia laboratories, Mumbai, India). Plates were incubated at 37°C in anaerobic jars for three days and colony counts calculated from the growth in serial dilutions.
Yoghurt was prepared fresh every morning and distributed at lunch time to the participants. As all participants had lunch in the hostel mess, this allowed distribution at a single point and consumption of yoghurt under supervision. The study was preceded by focus group discussions. Participants were interviewed by a social worker and a dietician. Demographic data were recorded and socioeconomic score was calculated . A 24 hour dietary recall, together with a food frequency questionnaire of commonly used foods over the past three months, was used to calculate nutrient intake using standard tables of the composition and nutritive value of Indian foods . Standard cups and spoons were used to measure meal sizes. Each participant was given a printed study diary and asked to record on a daily basis the following - stool frequency, stool consistency, abdominal pain, gaseousness, and any other symptoms that the participant noted during this period. Each participant was requested to give a fresh sample of stool before the study commenced, at the end of the first intervention period (i.e. at 4 weeks) and at the end of the second intervention period (i.e. at 8 weeks). The study protocol, incentive, and consent forms were approved by the Institutional Review Board. All participants provided written consent.
Fresh samples of stool were collected in the morning and transported immediately to the laboratory and stored at -80°C. Secretory IgA and beta-defensin 2 were assayed by ELISA (K8870 and K6500, Immunodiagnostik, Germany) following manufacturer's instructions. Stool samples weighing 80-120 mg were diluted with appropriate amounts of buffer provided in the kit to give constant dilutions, vortexed, and centrifuged at 13000 rpm for 5 minutes in 1.5 ml tubes. Supernatants were diluted 1:250 in wash buffer. Standards, controls and stool samples were simultaneously run in the ELISA and the concentration of secretory IgA and β-defensin 2 were read against a standard curve and expressed as concentration per gram wet weight of stool.