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

Vegan diets are associated with lower bone mineral density and elevated fracture risk, partly due to reduced intake of calcium and zinc and higher concentrations of phytates and oxalates, which inhibit intestinal mineral absorption. Emerging evidence suggests that the gut microbiota plays a key regulatory role in bone metabolism through the gut-bone axis, primarily via production of short-chain fatty acids (SCFAs), which enhance mineral absorption, modulate immune responses, and suppress osteoclastogenesis. Fermented plant-based foods may beneficially modulate this axis by delivering live microorganisms and by reducing antinutritional factors through microbial phytase activity, thereby improving mineral bioavailability.

FERMBONE is a randomized, controlled, open-label, two-period crossover trial conducted at two sites: ETH Zürich (Switzerland) and Královské Vinohrady University Hospital (Prague, Czech Republic). Fifty premenopausal vegan women are enrolled (25 per site).

Study Design and phases:

After a screening visit, participants attend a baseline visit (week 0) to assess baseline characteristics and enter a 4-week run-in period during which fermented plant-based foods are excluded from the diet to standardize gut microbiota baseline. Participants are then randomized to one of two sequences: fermented foods in Phase I (weeks 4-16) followed by control foods in Phase II (weeks 24-36), or the reverse. The two 12-week intervention phases are separated by an 8-week washout period (weeks 16-24) during which the same dietary restrictions as in the run-in apply.

Intervention:

The fermented food intervention comprises three categories consumed daily: calcium-fortified fermented plant-based yogurt alternatives with live cultures, lacto-fermented vegetables including sauerkraut and kimchi, and Rhizopus-fermented tempeh, for a minimum combined total of 14 portions per week across at least two categories daily. The matched control condition consists of non-fermented plant-based equivalents: calcium-fortified plant-based milk, fresh raw vegetables, and cooked non-fermented legumes, matched at the group level for portion size, energy, protein, and calcium content. All study foods are commercially available and provided to participants.

Assessments:

Study visits are conducted at baseline and at the beginning and end of each intervention phase (weeks 0, 4, 16, 24, 36). At each visit, fasting venous blood samples, morning spot urine, and stool samples are obtained. Anthropometric measurements, blood pressure, standardized questionnaires, and a weighed 3-day dietary record are completed at each visit. A single DXA scan is performed during the run-in period to assess the baseline bone density.

Biological analyses:

Bone turnover markers (CTX, P1NP, ALP) and endocrine regulators (PTH, 25(OH)D) are measured from fasting serum. Further, urinary calcium, phosphate, and creatinine are measured. Gut microbiota composition is assessed by whole-genome metagenomic sequencing and fecal SCFAs are quantified by targeted metabolomics. Inflammatory and intestinal integrity markers (CRP, AGP, IL-6, iFABP), fecal pH and calprotectin, hemoglobin, and DNA methylation-based epigenetic aging markers are analyzed along with iron status markers (ferritin, soluble transferrin receptor) and metabolic parameters (glucose, insulin, lipid profile).

Participants consume three categories of fermented plant-based foods daily for 12 weeks: lacto-fermented vegetables, fermented plant-based yogurt alternatives with live cultures and calcium fortification, and Rhizopus-fermented tempeh, with a minimum combined weekly target of 14 portions.

Other: Fermented Plant-Based Foods

Participants consume non-fermented plant-based equivalents matched for portion size, energy, protein, and calcium content for 12 weeks: non-fermented calcium-fortified plant-based milk, fresh raw vegetables, and cooked non-fermented legumes.

Other: Non-Fermented Plant-Based Foods

Daily consumption of three categories of fermented plant-based foods for 12 weeks: lacto-fermented vegetables, fermented plant-based yogurt alternatives with live cultures and calcium fortification, and Rhizopus-fermented tempeh. Minimum combined weekly target: 14 portions.

Daily consumption of non-fermented plant-based equivalents for 12 weeks, matched to the intervention arm for portion size, energy, protein, and calcium content: non-fermented calcium-fortified plant-based milk, fresh raw vegetables, and cooked non-fermented legumes.

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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).

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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

Time frame: Baseline (Week 0); Week 4, Week 16, Week 24, Week 36

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

Time frame: Baseline (Week 0); Week 4, Week 16, Week 24, Week 36

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

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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.

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

Fasting plasma glucose is measured using standard clinical chemistry methods.

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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).

Time frame: Baseline (Week 0); Week 4, Week 16, Week 24, Week 36

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

Time frame: Baseline

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

Time frame: Baseline

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.

Time frame: Baseline (week 0), week 4, week 16, week 24, week 36

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.

Time frame: Baseline

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

Time frame: Baseline (week 0), week 4, week 16, week 24, week 36

Weight will be measured in kg

Time frame: Baseline (week 0), week 4, week 16, week 24, week 36

Height will be measured in m

Time frame: Baseline (week 0), week 4, week 16, week 24, week 36

Waist circumference will be measured in cm

Time frame: Baseline (week 0), week 4, week 16, week 24, week 36

Hip circumference will be measured in cm

Time frame: Baseline (week 0), week 4, week 16, week 24, week 36

Systolic and diastolic blood pressure will be measured

Time frame: Baseline (week 0), week 4, week 16, week 24, week 36

Heart rate will be measured

Time frame: Baseline (week 0), week 4, week 16, week 24, week 36

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)

Time frame: Baseline (Week 0) through Week 36 (up to 36 weeks)

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.

Time frame: Administered twice after completion of the fermented food intervention phase and the control phase (week 16 and week 36)

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.

Time frame: Week 4 through Week 16 (Phase I) and Week 24 through Week 36 (Phase II)

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.

Time frame: Baseline (Week 0); Week 4, Week 16, Week 24, Week 36

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

Time frame: Baseline (Week 0); Week 4, Week 16, Week 24, Week 36

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

Time frame: Baseline (Week 0); Week 4, Week 16, Week 24, Week 36

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

Time frame: Baseline (Week 0); Week 4, Week 16, Week 24, Week 36

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

Time frame: Baseline (Week 0); Week 4, Week 16, Week 24, Week 36

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

Time frame: Week 4, Week 16, Week 24, Week 36

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).

Time frame: Pre-baseline (Week -3 to 0), mid-Phase I (Week 11-12), mid-Phase II (Week 31-32)

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.

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

Fasting serum insulin is measured using standard clinical chemistry methods.

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

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

Time frame: Baseline (Week 0), at the beginning and end of each 12-week intervention phase (Weeks 4, 16, 24, and 36)

Fasting plasma triglycerides are measured using standard clinical chemistry methods.

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