Drinking fermented milk containing Lactobacillus paracasei 431 (IMULUS TM ) improves immune response against H1N1 and cross-reactive H3N2 viruses after influenza vaccination: A pilot randomized triple- blinded placebo controlled trial Dunyaporn Trachootham a,⇑ , Chaowanee Chupeerach a , Siriporn Tuntipopipat a , Lilly Pathomyok b , Kobporn Boonnak c,d , Kemika Praengam a , Chadamas Promkam a , Chalat Santivarangkna a,⇑ a Institute of Nutrition, Mahidol University, Nakhon Pathom 73170, Thailand b Department of Medicine, Golden Jubilee Medical Center, Mahidol University, Nakhon Pathom 73170, Thailand c Department of Clinical Tropical Medicine, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand d Vaccine Trial Centre, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand article info Article history: Received 17 November 2016 Received in revised form 25 January 2017 Accepted 6 March 2017 Keywords: Immune response Influenza Milk Probiotic Vaccine Lactobacillus casei abstract A randomized triple-blinded placebo controlled trial was conducted in healthy adults to evaluate the effects of daily consumption of fermented probiotic milk on immune response towards influenza virus type H1N1, H3N2 and Flu-B. Study and placebo groups (n = 30 each) consumed fermented milk contain- ing 10 9 Lactobacillus casei 431 and acidified milk respectively, and the vaccination was administered two weeks thereafter. The consumption continued for another four weeks. Anti-viral response was tested by Hemagglutination Inhibition (HAI) and ELISA assays. Results showed that study group had higher sero- conversion rates for H1N1 and cross-reactive H3N2; higher HAI response (2-fold) rates for cross- reactive H3N2 virus; and increased ratios of geometric mean (study/placebo) for HAI titers and IgM of H1N1 and H3N2 viruses following the vaccination. These pilot results indicated that drinking fermented milk may be an approach to raise immune response against influenza A, especially for participants with low pre-existing immune. Clinical Trial Registry: ClinicalTrials.gov NCT02909842 Ó 2017 Elsevier Ltd. All rights reserved. 1. Introduction Upper respiratory tract infections such as influenza are the world’s most frequent reason for seeking medical care and spend- ing medical expenditure by patients (Footitt & Johnston, 2009). According to World Health Organization (WHO), influenza occurs globally with an annual attack rate of 5–10% in adults and 20– 30% in children. Globally, these annual epidemics result in approx- imately 3–5 million cases of severe illness and 250,000–500,000 deaths (WHO Fact sheet N°211, 2014). Consistently, data from Cen- ter of Disease Control, Ministry of Health of Thailand reported that the incidence of influenza in Thai people was 34.9 per 100,000 peo- ple, and most cases were found in people aged between 15 and 44 years old (Center of Disease Control, Ministry of Health of Thailand, 2016). Therefore, influenza is considered as an important medical burden of many parts of the world including Thailand. Since there are no effective anti-viral therapies for such infections, preventive strategies such as immune protection by vaccination are the most important approach to reduce the risk and severity of the diseases (Footitt & Johnston, 2009). Furthermore, vaccine challenge model has been considered one of the best tools for intervention studies involving immune response (Albers et al., 2005; Burleson & Burleson, 2007). Thus, the guideline of European Food Safety Authority (EFSA) recommends the model for clinical trial of food product with immunomodulating effects (Guidance on the scientific requirements for health claims related to gut and immune function-EFSA, 2011). Probiotics are living micro-organisms that provide health bene- fits if given at an appropriate dose (FAO/WHO Guidelines for the evaluation of probiotics in food, 2002). Many probiotics have been http://dx.doi.org/10.1016/j.jff.2017.03.016 1756-4646/Ó 2017 Elsevier Ltd. All rights reserved. Abbreviations: HAI, Hemagglutination Inhibition Assay; ELISA, Enzyme-Linked Immunosorbent Assay; IMULUS TM , Lactobacillus paracasei 431. ⇑ Corresponding authors at: Institute of Nutrition, Mahidol University, 999 Phutthamonthon Sai 4 Road, Salaya, Nakhon Pathom 73170, Thailand. E-mail addresses: dunyaporn.tra@mahidol.ac.th (D. Trachootham), chalat.san@ mahidol.ac.th (C. Santivarangkna). Journal of Functional Foods 33 (2017) 1–10 Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff