Fermented Foods and Bone Health: Investigating the Gut-Bone Axis in Premenopausal Vegan Women

Within-subject difference in fasting serum C-terminal telopeptide of type I collagen (CTX) concentration between the end of the fermented food intervention phase and the end of the matched control phase, measured by electrochemiluminescence immunoassay (ECLIA).

Serum procollagen type 1 N-terminal propeptide (P1NP), a marker of bone formation reflecting osteoblast activity, measured by ECLIA.

Serum alkaline phosphatase (ALP), a non-specific marker of bone formation reflecting osteoblast activity, measured by standard clinical chemistry methods.

Parathyroid hormone (PTH), an endocrine regulator of calcium homeostasis, measured by immunoassay at the University Hospital Zurich from fasting serum (ng/l).

Serum 25-hydroxyvitamin D (25(OH)D), a marker of vitamin D status, measured by immunoassay at the University Hospital Zurich (nmol/l).

Calcium-to-creatinine ratio from morning spot urine samples, reflecting renal calcium handling and dietary calcium absorption.

Phosphate-to-creatinine ratio from morning spot urine samples, reflecting renal phosphate handling.

Whole-genome metagenomic sequencing of stool sample using Illumina NextSeq 500 (150 bp paired-end). Taxonomic profiling with MetaPhlAn 4; metabolic pathway abundance with HUMAnN 3.

Targeted metabolomics of fecal SCFA concentrations from ethanol-preserved stool samples.

Fasting plasma glucose is measured using standard clinical chemistry methods.

Hemoglobin measured from fasting whole blood samples using standard clinical chemistry methods.

Serum ferritin and soluble transferrin receptor (sTfR) measured by standard methods from fasting serum or plasma.

DNA methylation-based epigenetic ageing markers: Biological age estimated from DNA methylation in EDTA whole blood using established epigenetic clock algorithms. Expressed as estimated biological age (years) and epigenetic age acceleration (difference between biological and chronological age).

CRP, a marker of systemic inflammation, measured by multiplex immunoassay from fasting serum samples.

Diet quality assessed using the VEGANScreener, a 29-item screener for vegans (Kronsteiner-Gicevic et al., Nutrients 2024). The tool includes 25 frequency-based food group questions (9 response options: never to ≥3 times/day) and 4 binary (yes/no) nutrient/supplement questions. The VEGANScreener identifies areas of dietary adequacy and inadequacy at the item level.

Unit of Measure: Frequency category per item

A study-specific structured questionnaire administered at baseline to characterize the study population. It collects data on sociodemographic background (e.g. age, education, occupation), lifestyle factors (e.g. smoking, alcohol use, sun exposure), dietary supplement use, and habitual food patterns. Data are used for descriptive analyses and as potential covariates in statistical models. No composite score is generated; individual item responses are recorded in the electronic case report form.

Physical activity is assessed using a study-specific short form of the International Physical Activity Questionnaire. The questionnaire captures the frequency and duration of walking, moderate-intensity activity, and vigorous-intensity activity over the past 7 days. Higher values indicate greater physical activity. Data are used for descriptive analyses and as potential covariates in statistical models. No composite score is generated.

Bone mineral density (BMD) at the lumbar spine, femoral neck, and total hip will be assessed by dual-energy X-ray absorptiometry (DXA).

Weight will be measured in kg

Height will be measured in m

Waist circumference will be measured in cm

Hip circumference will be measured in cm

Systolic and diastolic blood pressure will be measured

Heart rate will be measured

A study-specific structured questionnaire assessing adherence to and characteristics of the vegan diet, including duration of vegan diet (years), use of dietary supplements, and habitual food consumption patterns. Administered at each study visit to monitor changes in dietary behavior across phases. Data are used for descriptive analyses and as covariates in statistical models; no composite score is generated. Participant-reported response (categorical and continuous items)

Menstrual cycle characteristics are assessed using a standardized study-specific self-report tracking form. Menstrual cycle phase and regularity assessed via a standardized self-report tracking form completed by participants at home throughout the study. The form records cycle length (days), first day of menstrual bleeding, bleeding duration (days), and associated symptoms at each cycle. Data are used as a covariate to control for hormonal variation at the time of blood sampling. No composite score is generated.

Acceptability of the fermented food intervention and matched control products assessed using a study-specific questionnaire administered at the end of each 12-week intervention phase. 10 questionnaire evaluates palatability, ease of preparation, convenience, and willingness to continue consuming the study foods using Likert-type response options (1 = strongly disagree to 5 = strongly agree) and 3 open-end questions to assess the preference overall. Higher scores indicate greater acceptability. Results are used to characterize the feasibility of the dietary intervention.

Adherence to the fermented food intervention and control phase assessed using a study-specific weekly compliance tracking sheet. Participants self-report the number of portions consumed from each of the three study food categories (fermented or control) per week. Adherence is defined as meeting the weekly portion targets (minimum 14 total portions/week across at least 2 of 3 food categories). The proportion of compliant weeks is calculated per participant per phase.

AGP, an acute-phase protein and marker of systemic inflammation, measured by multiplex immunoassay from fasting serum samples.

IL-6, a pro-inflammatory cytokine, measured by ELISA from fasting plasma samples.

iFABP, a marker of intestinal epithelial integrity, measured by ELISA from fasting plasma samples.

Fecal pH measured on a continuous scale (range 0-14; lower values indicate a more acidic colonic environment, which is generally associated with favorable microbiota composition) from unfixed stool samples

Fecal calprotectin, a marker of intestinal inflammation, measured by ELISA from unfixed stool samples.

General health status and potential protocol deviations assessed using a study-specific structured follow-up questionnaire administered at each study visit (Weeks 4, 16, 24, and 36). The questionnaire captures changes e.g. in medication use, recent illnesses, antibiotic use, stress levels, dietary deviations, physical activity changes, and sun exposure since the last visit. Data are used to identify and document protocol deviations and potential confounders, no composite score is generated. Participant-reported response (categorical items; presence or absence of health status changes).

Dietary intake will be assessed at three timepoints using a 3-day weighed food record (two non-consecutive weekdays and one weekend day), analyzed in a dietary assessment software. Reported outcomes include total macronutrient and micronutrient intake. Data are used to characterize dietary exposure during each study phase and as covariates in statistical analyses.

Fasting serum insulin is measured using standard clinical chemistry methods.

Fasting plasma total cholesterol is measured using standard clinical chemistry methods.

Fasting plasma high-density lipoprotein (HDL) cholesterol is measured using standard clinical chemistry methods.

Fasting plasma low-density lipoprotein (LDL) cholesterol is measured using standard clinical chemistry methods.

Fasting plasma triglycerides are measured using standard clinical chemistry methods.