Study design

We will carry out a randomized, placebo-controlled, double-blinded superiority study to evaluate the effects of a synbiotic formulation on measures of physical performance, strength and muscle health in older, independent living individuals. This clinical trial was registered with the Australian New Zealand Clinical Trials Registry (ACTRN12622000652774) in May 2022 and the study design, protocol, and informed consent procedures were approved by the Human Research Ethics Committee of Swinburne University of Technology (Ref. 20226246–9780).

Study setting

This clinical trial will take place at Swinburne University of Technology, Hawthorn campus. Dual-energy X-ray absorptiometry (DXA) evaluations will be conducted at Deakin University, Burwood campus. Recruitment commenced in November 2022 and the enrolment target is expected to be met in April 2024. The schedule of enrolment, interventions, and assessments is shown in Fig. 1.

Fig. 1

Schedule of enrolment, interventions, and assessments (adopted from SPIRIT 2013 Figure). 1include age, gender, COVID-19 history and vaccine status; 2include heart rate, and blood pressure; 3include handgrip strength, 4m gait speed, balance testing, repeat chair stands, timed up and go; 4via bioelectric impedance analysis; 5include SARC-F, Physical Activity Scale for the Elderly, Gastrointestinal Symptom Rating Scale, Constipation Assessment Scale; 6include stool sample and blood sample. IP: investigational product

Study intervention

All eligible participants will be randomized equally (1:1) into either treatment (synbiotic) or control (placebo) groups. The study treatment will consist of a multi-strain synbiotic (SYN) powder containing inulin and a combination of probiotic strains. Placebo (PLA) will consist of maltodextrin, which will match synbiotic in appearance, taste, and consistency. Both SYN and PLA sachets will be 1800 mg. The participants will be instructed to consume 1 sachet daily (orally, dissolved in water, and at the same time each day) for the entire intervention period. Both SYN and matching PLA will be provided by Lallemand Health Solutions (Montreal, QC, Canada). Enrolled participants will be asked to make no changes to their current exercise habits or usual dietary intakes and abstain from taking any dietary supplements containing prebiotics and/or probiotics during the study. A list of prohibited medications is provided in Supplementary File 1.

Study duration

Participant involvement will be approximately 22 weeks. This will consist of a screening period of no longer than 2 weeks, a 16-week intervention period, and a 4-week follow-up period. Each participant will attend a screening visit, during which eligibility will be confirmed and informed consent will be obtained. Study investigational product (IP) will be initiated within 2 weeks following successful screening (at baseline visit). Each participant will subsequently attend 3 further visits: mid-intervention at 8 weeks, an end-of-intervention visit at 16 weeks, and a follow-up visit at 20 weeks. Study endpoints will be evaluated at each visit as discussed below. The trial design is outlined in Fig. 2.

Fig. 2Study participants

Approximately 74 participants will be enrolled to receive oral synbiotic formulation or placebo (n = 37 per group), daily for 16 weeks. Community-dwelling male and female participants between 60 and 85 years of age (inclusive) will be enrolled.

EligibilityInclusion criteria

To be eligible for enrolment into the study, participants must meet all of the following:

(1)

Evidence of a personally signed and dated information consent form (ICF) document indicating that the participant has been informed of all pertinent aspects of the study.

(2)

Healthy adults* who are determined by medical history and clinical judgment of the investigator to be eligible for inclusion in the study.

*Note: Healthy participants with pre-existing stable disease, defined as disease not requiring significant change in therapy or hospitalization for worsening disease within 6 weeks before enrolment, can be included.

(3)

Male and nonchildbearing-potential female adults between 60 and 85 years of age (inclusive) at the time of enrolment (signing of the ICF).

Female participants of nonchildbearing potential must meet at least 1 of the following criteria:

Postmenopausal status, defined as follows: cessation of regular menses for at least 12 consecutive months with no pathological or physiological cause;

Have undergone a documented hysterectomy and/or bilateral oophorectomy;

Have medically confirmed ovarian failure.

All other female participants are considered to be of childbearing potential.

(4)

Willing and able to comply with scheduled visits, laboratory tests, and other study procedures.

(5)

Willing to refrain from consuming probiotic and/or prebiotic supplements as well as foods that contain naturally occurring probiotics (e.g., fermented foods with live, active cultures such as yogurt, kefir, kombucha, kimchi, curd, buttermilk) from enrolment (Screening) until the end of the study.

(6)

Able to walk 10 m.

(7)

Able to get up from a chair.

(8)

BMI between 18 and 30 (inclusive) and body weight of at least 40 kg.

Exclusion criteria

Participants with any of the following characteristics/conditions will not be included in the study:

(1)

Use of probiotics, prebiotics or antibiotics in the past 4 weeks (screened participants that are otherwise eligible may enrol into the study after a 4-week wash-out period).

(2)

Use of proton pump inhibitors in the last 3 months.

(3)

Chronic treatment with statins or other drugs with known myotoxicity.

(4)

Musculoskeletal or other disorder resulting in inability to perform physical function testing.

(5)

Presence of medical conditions causing secondary sarcopenia.

(6)

Presence of diseases or disorders that can impact muscle mass.

(7)

At risk for malnutrition (Mini Nutritional Assessment-Short Form [MNA-SF] ≤ 7).

(8)

Diagnosed gastrointestinal disease with known association with gut microbiota dysbiosis.

(9)

Lower or upper extremity fracture within the past 6 months and/or hip replacement surgery within the past 12 months.

(10)

Myocardial infarction in the past 6 months.

(11)

Coronary artery disease, peripheral vascular disease, previous stroke, or history of transient ischemic attacks.

(12)

Uncontrolled hypertension (> 160/100 mmHg).

(13)

Androgen therapy in males or estrogen therapy in females.

(14)

Kidney failure.

(15)

History of cholecystectomy.

(16)

Blindness.

(17)

Inability to read or understand English.

(18)

Unintentional weight loss of 5% or more in the last 30 days or 10% or more in the last 6 months.

(19)

Known infection with human immunodeficiency virus, hepatitis B virus, or hepatitis C virus.

(20)

Known or suspected immunodeficiency, as determined by history and/or laboratory/physical examination.

(21)

Treatment with immunosuppressive therapy, including cytotoxic agents or systemic corticosteroids.

(22)

Other acute or chronic medical or psychiatric condition that, in the judgment of the investigator, would make the subject inappropriate for entry into this study.

(23)

Any condition or abnormality that, in the judgment of the investigator, would compromise the safety of the participant or the quality of the study data.

(24)

Currently enrolled in another clinical study or having participated in another clinical study in the 30 days before the screening visit.

(25)

Milk or soy allergy.

Randomization and masking

Participants who fulfil the eligibility criteria and consent to participate will be randomly assigned (1:1) to receive either a synbiotic (intervention) or placebo (control). Randomization will be completed by an independent (unblinded) third party using a random number generator in Microsoft Excel. To perform random allocation, stratified block randomization will be used. The unblinded staff will have no interaction with study participants and will not be involved in the collection or analysis of data. They will assign each participant to one of two intervention groups (synbiotic or placebo) and allocate IP based on stratification factors consisting of gender, age, and handgrip strength (“normal” or “low”, based on EWGSOP2 criteria [8]; men: “normal” ≥ 27kg or “low” < 27 kg and women: “normal” ≥ 16kg or “low” < 16 kg). Stratification data will be collected by blinded study investigators from each participant during their screening visit and will be submitted to the unblinded staff responsible for group allocations.

Allocation concealment and masking

Intervention and placebo will be matched in appearance, taste and consistency, and will have the same packaging and weight. The unblinded staff will be provided with the code for determining which containers are treatment or control and will label all IP containers for both intervention groups. All study participants and investigative site staff involved in data collection will be blinded to treatment allocation.

Implementation

Screening, enrolment, confirming eligibility, obtaining consent, and all other data collection and analysis will be performed by investigative site staff who are blinded to treatment allocation. Randomization and treatment allocation will be performed by unblinded study staff who will have no other study involvement.

ObjectivesPrimary aim

To determine the effect of a synbiotic formulation on indicators of functional performance, balance, and muscle strength.

Secondary aim

To determine the effect of a synbiotic formulation on muscle mass and architecture, microbiota composition and diversity and their associated metabolites, and self-reported indicators of sarcopenia.

Safety aim

To assess the safety and tolerability of a synbiotic formulation in older individuals.

Study assessments

Data collection will include Short Physical Performance Battery (SPPB), handgrip strength (HGS), timed up and go (TUG) test, as well as body composition and muscle morphology. These outcomes will be assessed at 4 time points (baseline and weeks 8, 16, and 20). Venous blood will be collected at 4 time points by venipuncture of the median cubital vein after overnight fasting using commercial collection tubes. Two 4.5-mL samples will be taken in EDTA and two 8.5mL SST collection tubes. Samples will be rested for 30 min at room temperature followed by centrifugation at 3000 × g for 15 min at 4°C after which serum and plasma will be collected into 0.5mL aliquots and stored at − 80°C until analysis. Participants will be carefully instructed on the procedures for fecal sample collection. Participants will be given stool collection kits at each visit, to be collected at home and returned at the following visit, as described below.

Primary outcome measure: SPPB

Physical performance will be measured with the SPPB, which is a well-validated and widely used tool [36] that has excellent test–retest reliability and predictive validity [37, 38]. The SPPB measures three components (i) standing balance, (ii) 4-m habitual gait speed, and (iii) repeated chair rises. Each of the three domain scores ranges from 0 to 4 points, yielding a composite score ranging from 0 to 12 points. Higher scores indicate better function. A total score of fewer than 10 points indicates a high risk of frailty and falls [38] and a one-point change is considered clinically meaningful [39, 40]. SPPB component measures will be scored according to Table 1.

Table 1 Scoring schema for SPPB

For the balance tests, participants will be asked to stand unassisted in 3 positions:

(1)

Side-by-side stand (feet in a side-by-side position);

(2)

Semi-tandem stand (heel of one foot is beside the big toe of the other foot);

(3)

Tandem stand (toe of one foot is behind and touching the heel of the other foot).

Participants will be asked to maintain each position for 10 s. If unable, the time achieved will be recorded and the next position will be attempted.

Gait speed will be measured as the time taken to walk 4 m (between markers set at 3 and 7 m of a 10 m walking course) at the participant’s usual speed. Assistive devices will be allowed if needed. Gait speed will be calculated as meters per second (m/s) and trained staff will record time to the nearest 0.01 s. The best (fastest) of the two attempts will be included in data analysis. Gait speed is comparable to the full battery for predicting disability [38], and slower gait speeds predict higher rates of frailty and hospitalization [41]. Coleman et al. determined 0.11 m s−1 to be a clinically meaningful change [42].

For repeated chair rises, participants will be asked to rise from an armless chair, sit back down, and repeat this movement five times as quickly as possible without using their arms [43]. Participants will begin the test with their arms folded across their chest, sitting in a chair with a seat height of 43 cm. Participants will be instructed to stand up completely (defined as an upright trunk with full knee and hip extension), make firm contact when sitting and keep their arms folded across their chest. Time (in seconds) will be recorded, beginning upon the prompt, “go,” and stopping when the participant’s buttocks returned to the seat following the fifth stand [44]. No words of encouragement will be used. Chair stand time has been described as a reliable [45], useful measure of lower leg strength [43], fall risk [46], and balance control [47], and has been shown to complement measures of gait speed when screening for sarcopenia [48].

Primary outcome measure: handgrip strength

Handgrip strength will be measured using a Jamar analog hand dynamometer (Patterson Medical, Warrenville, IL) with the handle set to position II. Participants will be instructed to grip the device and squeeze as hard as possible with their dominant hand [49]. Three trials will be recorded with participants in a sitting position, elbow by their side and flexed at 90°, forearm and wrist in a neutral position, with shoulder adducted and neutrally rotated, as previously described [50]. The maximum of these values will be used for analyses [51]. Grip strength has been shown to predict future function [52] and has been negatively associated with frailty [53].

Primary outcome measure: TUG test

The TUG test measures the time (in seconds) taken to stand up from a standard chair, walk to and around a marker placed 3 m away at a comfortable pace, return to the chair and sit back down [54]. Participants will be permitted to use walking aids (if necessary) and will be instructed not to use their arms to stand up. Beginning with the participant seated with their back against the backrest of the chair, timing will commence on the command “go” and will stop when the participant returns fully seated in the back of the chair. The task will be performed twice, with the faster (shorter) time used in data analysis. TUG has been used to examine gait, agility, fall risk, balance, and dynamic (turning) movements in older adults [43, 55,56,57].

Secondary outcome measure: body composition

Dual-energy X-ray absorptiometry (DXA) will be used to measure whole and regional body composition, including lean body mass (LBM, g), appendicular lean mass (ALM, g), total and regional body fat mass (FM, %), and fat-free mass (FFM, g). Fasting whole-body scans (GE Healthcare Lunar iDXA, Madison, WI) will be acquired with participants in a supine position on the scanner table, wearing light clothing, with their limbs close to their bodies according to the manufacturer’s protocol. Segmental analyses of the whole body into arm, leg, and trunk segments will be separated with anatomical landmarks by the DXA analysis software. Appendicular skeletal muscle mass (ASMM) will be calculated as the sum of the lean soft tissue mass (LSTM) of both the right and left extremities, with the assumption that all non-bone and non-fat tissue is skeletal muscle [58]. Skeletal mass index (SMI) will be determined as the ALM divided by height squared (kg/m2) [59]. Body composition via DXA will be evaluated at baseline and week 16.

Body mass index (BMI), total body and segmental fat percent and weight, total body water percent and weight, total body and segmental muscle mass, and FFM will be estimated using bioelectric impedance analysis (BIA) with a multi-frequency body composition analyzer (Tanita MC-780U, Tokyo, JP) at 4 time points. Impedance will be analyzed in a fasting state, with participants standing barefoot on the electrode platforms, holding grips with both arms straight down at their sides.

Secondary outcome measure: muscle morphology

Muscle mass and architecture will be quantified via ultrasound examination (Sonosite iViz, Fujifilm, Tokyo, JP) of the rectus femoris (RF) and vastus intermedius (VI) at 4 time points. Participants will lie supine on an examination table, hips in a neutral position, knees in full extension, ankles at 90°, and both feet on the table, as previously described [60]. The assessed muscles will be in a relaxed state (5 min) to avoid muscle contraction-induced fluid shifts [61], and scans will be obtained before any functional testing [62]. A horizontal reference line will be drawn at 50% of the distance between the greater trochanter and the superior border of the patella of the dominant leg. These anatomical landmarks were chosen based upon guidelines proposed by the SARCopenia through UltraSound (SARCUS) working group of the European Geriatric Medicine Society [60, 63]. With adequate transmission gel applied to the transducer head, two images will be obtained at this point: (i) an image in the transverse plane with the transducer placed perpendicular to the long axis of the thigh, and (ii) an image in the sagittal plane with the transducer placed parallel to the long axis. All images obtained will be subsequently analyzed using ImageJ (NIH) software [64]. Muscle thickness (MT), echo intensity (EI), cross-sectional area (CSA), and subcutaneous tissue thickness (STT) will be measured on the transverse images; fascicle length (FL) and pennation angle (PA) will be measured on the sagittal images [65, 66]. Measurements will be taken three times, and the mean value will be recorded and included in the data analysis [67]. Low levels of muscle mass as determined by the ratio of fascicle length to muscle thickness will be factored to obtain the Ultrasound Sarcopenia Index (USI) [68].

Secondary outcome measure: fecal microbiota composition and diversity

Stool samples will be collected at 4 time points by participants at home within 72 h of each of their upcoming study appointments. Spontaneously voided feces will be collected into specimen containers, placed within provided biohazard bags, and immediately frozen until transfer to our research site, where they will be stored at − 80 °C until analysis. DNA extraction and 16S rRNA or whole-genome shotgun (WGS) sequencing will be performed to determine α- and β-diversity and taxonomic abundance [69, 70].

Secondary outcome measure: anthropometric variables

At baseline, body mass will be recorded in light clothing to the nearest 0.1 kg using a digital scale (Tanita, Tokyo, JP). Height (without shoes) will be measured using a stadiometer (SECA 213, Hamburg, Germany). BMI will be calculated as weight (kg) divided by height (m)2.

Secondary outcome measure: calf circumference

A non-elastic tape measure (SECA 201, Hamburg, Germany) will be used to measure calf circumference (CC) at the widest part of the non-dominant leg with participants in a supine position. The tape will be placed flat on the skin, taking care not to compress the sub-dermal tissue. Studies support the utility of CC as a proxy marker for measuring muscle mass [71,72,73]. CC has shown moderate to high sensitivity and specificity in predicting sarcopenia [74, 75] and predicts disability risk in older adults [76]. CC will be assessed at baseline and week 16.

Secondary outcome measure: dietary assessment

Habitual dietary intake will be recorded by participants using 3-day dietary intakes for the days leading up to each of the 4 study appointments. Dietary intake data will be analyzed to discern energy intake, and macro- and micro-nutrients. Participants will also complete the Australian Eating Survey [77] food frequency questionnaire at baseline only and before randomization to establish pre-intervention dietary patterns.

Secondary outcome measure: SARC-F

SARC-F is a recently developed rapid screening tool for sarcopenia [78]. It is a self-reported questionnaire consisting of 5 components: Strength, Assistance with walking, Rising from a chair, Climbing stairs and Falls, respectively. Each of the components is scored from 0 to 2 points, yielding a composite score ranging from 0 to 10 points. Lower scores indicate better function. A total score of ≥ 4 points is indicative of sarcopenia. A recent meta-analysis [79] reported that SARC-F has excellent specificity in screening for sarcopenia, however, due to its low to moderate sensitivity [80], it may detect only severe cases [8]. Participants will complete SARC-F questionnaires at each study visit.

Safety outcome measures

Serum and plasma samples will be analyzed by an external commercial pathology facility to determine changes (outside normal variation) in clinical chemistry (Comprehensive Metabolic Panel [CMP]) and clinical hematology markers (Complete Blood Count [CBC]) elicited after baseline and through week 16. Adverse events (AEs) and serious adverse events (SAEs) will be assessed and recorded from baseline through week 20.

Hypotheses and study endpointsPrimary aims

We hypothesize 16 weeks of ingesting a synbiotic preparation compared to placebo will result in statistically significant improvements (within-group and between-group) in:

(1)

4-m gait speed

(2)

Standing balance

(3)

Repeated chair rises

(4)

Timed up and go

(5)

Handgrip strength

Secondary aims

We hypothesize 16 weeks of ingesting a synbiotic preparation compared to placebo will result in statistically significant improvements (within-group and between-group) in:

(6)

Muscle mass and body composition (LBM, ALM, total and regional body FM and FFM)

(7)

Muscle quality (CSA, MT, EI, FL, and PA of Rectus femoris and Vastus intermedius muscles)

(8)

Fecal microbiota composition and diversity

(9)

Untargeted fecal and serum metabolomics

Safety aims

We hypothesize synbiotic administration will be safe and well-tolerated as defined by the absence of:

(10)

Changes (outside normal variation) in CBC elicited after baseline and through week 16

(11)

Changes (outside normal variation) in CMP elicited after baseline and through week 16

(12)

AEs elicited after baseline and through week 20

(13)

SAEs until study completion

Statistical analyses and power

This estimated sample size for the main outcome measures of physical performance was based on an assumed correlation of 0.7 between the pre- and post-intervention outcome measures, and an effect size of Cohen’s d = 1.2 for gait speed based on data from Román et al. [29] that examined the effects of a multi-strain probiotic on gait speed and the TUG test in a study cohort of 35 (17 and 18 in treatment and placebo groups, respectively). To have a power of 80% and a significance level of 5%, our study aims to enrol an estimated sample size of 74 participants (37 per group), taking into account a conservative ~ 30% dropout rate.

The study hypotheses will be addressed using a hierarchical linear model analysis to test for significant differences between the two comparative groups (intervention and placebo) over time. A primary intention to treat (ITT) analysis will be conducted on all the participants who complete the trial. Additionally, a secondary analysis will be carried out to include only participants who consumed ≥ 90% of their supplement over the course of the intervention period. Between-group comparisons will be made using linear models and statistical significance set at p < 0.05. Where there are significant group xtime interactions, planned contrasts will compare changes from baseline under each intervention. Similar tests will be performed for the secondary hypotheses, using appropriate transformations if these measures exhibit non-normal distributions across time points. This analysis will allow for the control of any baseline group differences and to test for moderation effects in terms of these variables over time. Analysis of microbial community composition will be conducted using Phyloseq and vegan package in R version 3.6.1 as per protocol.