Our previous work on the use of probiotics improved the clinical symptoms in irritable bowel syndrome with constipation-predominant. This study would like to explore further use of probiotics in the immune system of the patients with irritable bowel syndrome.

This is a case control study involving participants who fulfilled the Rome III criteria for constipation-predominant irritable bowel syndrome. The patients were recruited from the Gastroenterology clinic at Universiti Kebangsaan Malaysia Medical Centre, Kuala Lumpur. A total of 165 participants were recruited based on probability (power) of 0.8 and Type I error was set at 0.05.

The investigators included healthy participants above 18 years old. Those less than 18 years old; constipation due to other medical illnesses such as diabetes mellitus, inflammatory bowel disease, hypothyroidism, colorectal cancer, neurological diseases, major depression, lactose intolerance, pregnancy, breastfeeding women and must not be on chronic opioids or anti- depressants were excluded. Participants must not consume any antibiotics, probiotic, prebiotic, symbiotic and/or laxatives less than two weeks before recruitment.

Participants were required to consume three bottles of 125 ml cultured milk drink daily for 30 days. Each bottle contains 10^9 cfu Lactobacillus acidophilus LA-5 and Lactobacillus paracasei L. CASEI-01. In each 100 ml unit, it contains 4.06 g of fructose, 4.14 g of glucose, 0.29 g of sucrose, less than 0.1 g of maltose and 1.35 g of lactose. Cold chain was maintained below 10°C during storage, transportation and distribution. Prior to consumption of the cultured milk drinks, fecal and blood samples from the participants were obtained to determine their baseline fecal pH and serum cytokines level respectively. Each of the participants answered a food frequency questionnaire to assess their dietary profile (data are not presented in this paper) and the Garrigues Questionnaire to evaluate their clinical symptoms and physical activity. The same parameters were taken following 30 days of cultured milk drinks' consumption.

Inclusion Criteria:

• Do not fulfil the Rome III criteria for irritable bowel syndrome

Exclusion Criteria:

• constipation due to other medical illnesses such as diabetes mellitus, inflammatory bowel disease, hypothyroidism, colorectal cancer, neurological diseases, major depression, lactose intolerance, pregnancy, breastfeeding women and must not be on chronic opioids or anti- depressants. Participants must not consume any antibiotics, probiotic, prebiotic, symbiotic and/or laxatives less than two weeks before recruitment.

The investigators are bound to the Institutional Review Board Universiti Kebangsaan Malaysia Medical Research Ethics Committee rules and regulations where recruited patients identity and data are kept confidential and will only be allowed to access by the research team.