*4.1. Subjects*

We published pamphlets describing the purpose, methods, and exclusion items of our research titled "The reference of *H. pylori* infection to absorption of mozuku fucoidan" on the Internet and recruited volunteer participants. Two hundred sixty-two Japanese people submitted applications from April 2014 to June 2016. They completed a questionnaire assessing gender, age, and mozuku consumption. We enrolled 259 volunteers who completed questionnaires and collected urine samples as planned. Subjects were divided into five age groups: 20–29, 30–39, 40–49, 50–50, and ≥60 years old.

The frequency of mozuku consumption was divided into five groups as follows: approximately 1–3 times weekly, approximately once every 2 weeks, approximately once monthly, approximately once every 2–3 months, and rarely (Table 1).

This study was conducted according to the Declaration of Helsinki. The protocol of the study was approved by the Ethics Committee of South Product Co., Ltd. (UMIN000039117). Following an explanation of the study and its aim, all subjects provided informed consent.

#### *4.2. Oral Intake of Fucoidan and Collection of Urine Samples*

Subjects refrained from marine algae and fucoidan supplementation on the day before the test and on the day of the test to avoid the effects of diet. Subjects orally consumed two fucoidan drinks (1500 mg/bottle) at 9:00 in the morning. Urine samples were extracted four times, namely, before (0) and 3, 6, and 9 h after fucoidan ingestion. Urine samples were collected by a parcel delivery service.

In this study, the subjects orally consumed 3 g of mozuku fucoidan. The drink was prepared by South Product Co., Ltd.

#### *4.3. Assay for Fucoidan Levels in Urine Samples*

Urine fucoidan levels were assayed using a sandwich ELISA method developed by our laboratory [22]. The reproducibility of the fucoidan ELISA method was as follows. The intraand inter-assay CVs for serum, plasma, and urine, using high and low concentrations of fucoidan, were in the range of 1.5–13.4%. The detection limit concentration of our ELISA was less than 1 ng/mL.

#### *4.4. Assay for Anti-H. pylori Antibody Titers in Urine*

Single-void urine samples were obtained and stored at 2–8 ◦C until use. Urinary IgG antibodies to *H. pylori* were measured using a urine-based ELISA kit (URINELISA®, Otsuka Pharmaceutical Co., Ltd.) that utilizes a VacA- and CagA-positive *H. pylori* strain isolated from a Japanese patient with gastritis as the antigen source. This ELISA-based test result was considered positive when a cutoff index of 1.0 (optical density = 0.218) or greater was obtained after measurement of the optical density according to the manufacturer's instructions [32–34].

The ΔMax fucoidan value was calculated by subtracting the basal value (before ingestion) from the highest level of urinary fucoidan following fucoidan ingestion. If the basal fucoidan level was higher than that after fucoidan ingestion, then the ΔMax fucoidan value was recorded as 0.
