Trial 1: efficacy examination

To explore the efficacy of intake of L. paracasei K71, we conducted a randomized, double-blind, parallel-group, placebo-controlled study at the incorporated medical institution Aisei Hospital Ueno Clinic (Tokyo, Japan), with the study being supported by funds from Kameda Seika Co., Ltd. The study protocol conformed to the principles of the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Subjects issued by the Ministry of Health, Labour and Welfare, Japan. The study was approved by the institutional review board of Aisei Hospital Ueno Clinic on June 25, 2015. The study period was from June to December 2015. This study was registered under ID No. UMIN000018423 in the UMIN Clinical Trials Registry, Japan.

Subjects aged 20–64 years old were recruited for the study. The study details were disclosed to subjects before enrolment, and the investigators obtained informed consent from each subject. The inclusion criteria were as follows: male or female, age between 20 and 64 years, and relatively low rates of salivary sIgA release in a pretrial test (subject selection was based on our preliminary clinical test, in which subjects with relatively low sIgA release rate were enrolled). The exclusion criteria were as follows: prior use of health foods or medicines with high levels of lactic acid bacteria three or more times a week; (ii) use of health foods or dietary supplements that might enhance immune function; (iii) history of allergic disease such as seasonal rhinitis, perennial allergic rhinitis, asthma, atopic dermatitis, allergic conjunctivitis, food allergy, and metal allergy; (iv) receipt of therapy (such as hyposensitization therapy) that may affect the study results; (v) dental or intraoral treatment within 1 month before the screening test or plans for such treatment; (vi) dental or intraoral trouble accompanied by bleeding; (vii) shift-work or heavy physical labor; (viii) heavy exercise, such as long-distance running; (ix) disease requiring treatment; (x) serious disease such as diabetes mellitus, liver disease, kidney disease, heart disease, or a disease that affects corticosteroid secretion, or a history of such disease; (xi) possible allergy to the test compound; (xii) subjects judged unsuitable based on screening-test values; (xiii) women who were pregnant, nursing, or planning to get pregnant or nurse during the study period; (xiv) subjects involved in other clinical trials within 1 month before giving informed consent or planning to join other clinical trials after giving informed consent; (xv) subjects judged unsuitable according to their responses to a lifestyle questionnaire; and (xvi) subjects judged unsuitable by the principle investigator. Sixty-two subjects who met the eligibility criteria were assigned sequentially based on random number tables to one of the two masked products: the L. paracasei K71-containing tablet (group A; n=31) and placebo tablet (group P; n=31). The number of subjects was calculated based on a preliminary clinical trial in which the effect of intake of L. paracasei K71 on the rate of salivary sIgA release was investigated. The allocation of subjects was performed by TTC Co., Ltd. (Tokyo, Japan), and an envelope that contained the allocation table was sealed off until breaking of the blinding. Thus the allocation information was concealed from the subjects, investigators, and researchers who recruited and assessed the participating subjects.

The subjects underwent clinical surveys in weeks 4, 8, and 12 following initial intake of the test food. The clinical survey and pretrial tests included the following: a lifestyle questionnaire (anamnesis, intake of pharmaceutical products and health foods, allergies, smoking, alcohol consumption, and life habits), medical interview (physical conditions and presence or absence of adverse events), somatometry (height, body weight, body mass index, blood pressure, and pulse), and saliva analysis (determination of sIgA concentration and rate of saliva secretion). Laboratory examinations (fasting levels of white and red blood cells, hemoglobin, hematocrit, platelets, total protein, albumin, total bilirubin, alkaline phosphatase, aspartate transaminase, alanine transaminase, lactate dehydrogenase, γ-glutamyltranspeptidase, total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, urea nitrogen, creatinine, uric acid, Na, K, Cl, and glucose) and urinalysis (protein, glucose, and occult blood) were performed in the pretrial test and in week 12. From 2 weeks before supplement intake to completion of the study, subjects were asked to log details including their test-tablet ingestion, physical condition, and use of medications.

During the trial, the principal investigator and assistants instructed the participants as follows: (i) not to change their lifestyle, including diet, alcohol consumption, and sleep; (ii) to avoid over exercising, abstemious eating, or overeating; (iii) not to change their exercise habits; (iv) not to consume health foods, dietary supplements, lactic acid bacteria beverages, lactobacillus preparations, yogurt, or kimchi (Korean pickles); (v) to avoid vaccinations (e.g., for influenza) as much as possible; (vi) to log the amount and dose of pharmaceuticals used; (vii) to take the specified dose of the test food and log the intake time and dose every day; (viii) to record a lifestyle log every day; (ix) to avoid drinking alcohol the day before testing; (x) to avoid strenuous exercise on the day before and the day of testing; (xi) to finish eating their last meal of the day by around 10 p.m. and not consume anything thereafter other than water or warm water until the end of testing the following day; (xii) to go to bed by midnight and sleep well on the day before testing; (xiii) to brush their teeth without using toothpaste or mouthwash by 1 hr before saliva analysis on the day of testing; and (xiv) to avoid smoking until the end of testing on the day of testing.

The primary outcome measure was the rate of salivary sIgA release, and the secondary outcome measure was the concentration of salivary sIgA. Subjects who consumed less than 80% of the expected dose of the provided test food, who did not keep an adequate log or whose behavior cast doubt on the reliability of their clinical data, who met the exclusion criteria after enrollment or did not follow the required restrictions, or who met justifiable reasons for exclusion were excluded from efficacy analysis. To analyze the safety of the provided test food, we examined adverse events in subjects who consumed the test food at least once and explored measurement data for subjects who completed the study schedule.