Effect of Fermented Milk Containing Lactobacillus Casei Strain Shirota in Sarcopenia Elderly

The Effect of Fermented Milk Containing Lactobacillus Casei Strain Shirota on Sarcopenia in Elderly Taiwanese: Interactions With the Nutrients Utilization, Diversity of Gut Microbiota, Microbiota-derived Metabolites and Muscle Loss

Sarcopenia, which refers to the progressive loss of skeletal muscle mass and strength, shares many characteristics with other disease states typically associated with risks of falling and fracture, including osteoporosis, frailty, and obesity. Sarcopenia often is linked to an increase in connective tissue, muscle steatosis, impaired muscle metabolism, an increase in inflammatory markers, an increased stiffness of myofibers, slower kinetics in establishing myosin-actin crossing bridges, increased oxidative stress, mitochondria dysfunction, hormonal imbalance, and decreased capillary flow. In addition to the Lactobacillus casei strain, Shirota is a well-known probiotic strain that has been approved and is generally recognized as safe by the United States Food and Drug Administration. L. casei strain Shirota (LCS) has been suggested to confer health benefits. Investigators found that LCS decelerated age-related muscle loss via ensuring mitochondrial function in Senescence-Accelerated Mouse Prone 8 (SAMP8) mice. Investigators also found that 3-Indolepropionic Acid (IPA) is a microbiota-derived metabolite from a healthy intestinal gut. IPA is also a protective factor for the progression of chronic kidney disease in the Chinese population. This clinical trial focuses on the effect of fermented milk containing LCS on sarcopenia in elderly Taiwanese individuals. Investigators focus on the topic of the interaction of LCS with the diversity of the gut microbiota, microbiota-derived metabolites, and protein utilization. The proposal will have four research goals. First, investigators try to investigate whether long term supplementation LCS could restructure the gut microbiota composition and gut microbial metabolites to against Aging related -Gut Dysbiosis in elderly. Second, investigators also try to link the LCS play an important role on muscle loss and alternation of gut microbiota composition. Third, investigators try to study the LCS effect of muscle deterioration with aging, and highlight the two underpinning mechanisms (ROS and protein utilization) regulating declines in muscle mass and function. Fourth, Since IPA is important Microbiota-derived metabolites in health gut, investigators try to investigate whether LCS could play an important role on modulation of IPA production in GI. Fifth, investigators hope that investigators can through gut microbiota composition found some selective microbiota clusters perform positively correlation with IPA.

Study Overview

• Status: Completed
• Conditions: Inflammation; Sarcopenia; Aging
• Intervention / Treatment: Dietary supplement: Yakult 300 LIGHT Fermented Milk

Detailed Description

1. Subject Enrollment The definition of sarcopenia is based on the algorithm of Asian Working Group for Sarcopenia (AWGS).

1. Muscle mass: measured by using Inbody S10 and standardized by height, shown by skeletal muscle index [SMI=appendicular muscle mass (kg)/height (m2)]. Low muscle mass was defined as:

   • Men: SMI <7.0 kg/m2
   • Women: SMI <5.7 kg/m2

2. Handgrip strength: measured by electronic hand grip dynamometer. Low handgrip strength was defined as:

   • Men: <28 kg
   • Women: <18 kg

3. Limb strength: measured by Time for 5 times for chair stand method. Low limb strength was defined as:

   • Time for 5 times for chair stand: ≧12s Sarcopenia was defined by (1) and one of (2), or (3)

     2. Study intervention Test beverages, Yakult 300 LIGHT Fermented Milk containing LcS, were provided by Yakult Co., Ltd. (Taipei, Taiwan). The beverages were distributed and stored at temperatures ranging from 0-10°C. The LcS-fermented milk contained LcS at more than 3x10^10 CFU per 100-ml bottle.

The subjects would take 2 bottles per day (104 kcal/day). Study intervention should be taken twice daily at approximately the same time. All milks would be sent to participant weekly (14 bottles/week).

3. Study assessment

3-1 Anthropometric measurement and Body Composition Assessment Anthropometric measurement data was detected height, weight, waist circumference, arm muscle circumference, hip circumference, and calf circumference using measure tape and weight scale.

Body composition was detected using bioelectrical impedance analysis (BIA) as a non-invasive test instrument.

3-2 BIA Subjects stand on met without shoes and socks. Arms do not touch the trunk part of body, and are spread naturally to a 15 degree angle away from trunk. The hand electrodes are marked THUMB for the thumb and MIDDLE for the middle finger. The foot electrodes should be positioned between examinee's anklebone and heel. Electric current was supplied from the electrodes on the tips of the heels of both feet and the fingertips of both hands.

3-3 Muscle strength Grip strength measured in standard conditions with a well-studied model of a handheld dynamometer with reference populations can be a reliable surrogate for more complicated measures of muscle strength in the lower arms or legs. The measurement uses electronic hand grip dynamometer. Low handgrip strength is defined in Men: <28 kg ; Women: <18kg. Limb strength measured by Time for 5 times for chair stand. Subjects sat and stood for five times, and calculated time by timer. Low limb strength is defined in time for Time for 5 times for chair stand: ≧12s.

3-4 Physical performance Physical performance was assessed by using 6-meter usual gait speed, TUG, and SPPB.

3-5 Gait speed Participants were instructed to walk from a standing start at a pace that was normal and comfortable for them or to walk as fast as participants could until participants reached the end of the marked path. A trained examiner walked behind the participant and stopped timing when the participant's foot contacted the floor at the end of the walking course. Participants were provided rest breaks as needed throughout the testing session. Low Physical performance is defined < 1 m/s.

3-6 TUG Participants were instructed to stand up from the chair, walk 3 meters forward and go back to sit on the chair. Low Physical performance is defined ≧ 20s.

3-7 SPPB Participants were followed to SPPB to do the test. Low Physical performance is defined ≦ 9 scores.

3-8 Gut Microbiota Composition and Microbiota-derived Metabolite Analysis

3-8-1 Fecal sample collection and Bacterial genomic DNA isolation Fresh fecal samples were collected from individual participant. Participants were instructed to place toilet paper at the front of the toilet bowl before defecation to prevent fecal contamination with urine or water. They were asked to defecate in the opposite direction of their usual practice, ensuring the feces were deposited onto the toilet paper. Using the collection stick provided in the sampling kit, participants collected fecal samples and transferred them into the provided tubes. One of the tubes contained a preservation solution, which required thorough mixing after sample collection. Both tubes were then stored in a freezer.

The genomic DNA was extracted by using the QIAamp DNA Stool Mini kit according to the manufacturer's protocol. DNA concentrations were measured by using NanoDrop2000. The fecal samples were stored at -80°C refrigerator.

3-8-2 16S rRNA amplicon and data analysis The V1-V9 region of the 16S rRNA gene by PCR were referred to the Illumina 16S Metagenomic Sequencing Library Preparation protocol. The amplicons were paired and sequenced on an Illumina Miseq 2000 platform according to manufacturer's protocol. The data was analyzed with Quantitative Insights into Microbial Ecology 2 (QIIME2). The UPARSE algorithm was used to align with identical sequences and determine the representative operational taxonomic unit (OTU) sequences. For sequences with 97% similarity, they were clustered as same OTU. Alpha diversity analysis (Shannon index) was performed using QIIME while beta diversity analysis was analyzed using weighted principal coordinate analysis (PCoA). The online Galaxy workflow framework was used to determine the linear discriminant analysis (LDA) effect size LEfSe).

3-8-3 Fecal Short Chain Fatty Acids (SCFAs), IPA and TMAO Determination The fecal samples were diluted in 0.5% phosphoric acid and homogenized. After centrifuge, the supernatant was mixed with 4-ethylpentanoic acid in ethyl acetate, and kept the supernatant at -20°C.

The untargeted metabolomics was performed using HPLC coupled with a Q-Exactive Orbitrap Plus mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) as previously described[42, 43]. Liquid chromatography was performed on HPLC system using a BEH C18 column (1.7 μM, 2.1 mm x 100 mm, Waters Corporation). The injection volume was 2 μL with a flow rate of 0.3 ml/min and the column temperature was maintained at 35°C. HPLC linear gradient conditions were: 0-0.5 min 1 % B, 0.5-2.5 min from 1 % B to 10 % B, 2.5-3.5 min 65 % B, 3.5-5.0 min from 35 % B to 100 % B, and 5.0-9 min 1 % B [solvent system A: water/formic acid (100:0.1, vol/vol); B: acetonitrile/formic acid (100:0.1, vol/vol)]. The capillary temperature and voltage (+ and -) were maintained at 275 °C and 3,600 and 3,200 V, respectively. The samples for quality control (QC) were prepared by mixing all the samples. Data were acquired by Thermo Data Analysis software Compound Discoverer TM2.0. The raw intensities were transformed and normalized. For multiple peaks mapped to the same metabolites, the average intensity value was used. The matrix was then subjected to principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) using the R ropls package (version 1.21.0) to obtain differential metabolites between groups.

3-9 Blood Analyses

3-9-1 Blood Sampling Blood samples were collected from the subjects at pre-test and post-test. Whole blood were placed in collection tubes containing K2EDTA and then stored at 4°C until analysis. For plasma preparation, blood samples were placed in collection tubes containing heparin and centrifuged at 3000 g for 10 minutes then took supernatant. For serum preparation, blood samples were placed in collection tubes and then centrifuged at 3000 g for 10 minutes then took supernatant. The plasma and serum were stored at -70°C until analysis.

All biochemical parameters were performed one day before the start of the intervention and at the end of intervention. Albumin, Prealbumin, Cholesterol, High HDL-C, LDL-C, Triglyceride, TSH, Free T4, hsCRP, HbA1c (%), fasting glucose, fasting insulin, HOMA-IR, ALT, AST, creatinine, eGFR, WBC, RBC, HGB, HCT, MCHC, platelet and RDW-CV were determined after fasting for 8 hrs.

3-9-2 Metabolic Parameters All of the metabolic measurements were performed one day prior to the start of the intervention and at the end of intervention. CBC, TSH, Free T4, Vit-D, Total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, fasting glucose, fasting insulin, blood urea, nitrogen, plasma prealbumin, albumin, ALT, AST, HbA1c, HS-CRP, γ-GT, and creatinine were measured after fasting for 8 hrs.

3-9-3 Inflammatory Cytokines Analysis The inflammation-associated serum cytokines TNF-α, TGF-β, IL-6, IL-17, and IL-10 were analyzed using colorimetric kits (Elabscience, China). The procedures followed the kit instructions and were measured using an ELISA reader (Bio Tek, PowerWave XS2, City, State, USA).

3-9-4 Anti-oxidant marker analysis Many anti-oxidant enzymes in human bodies, like Superoxide dismutase (SOD) (Elabscience, China), Glutathione peroxidase (GPx) (biovision, City, State, USA), and Catalase (CAT) (biovision, City, State, USA). Enzymes in the serum were evaluated for the activity by ELISA kit. The procedures followed the kit instructions and were measured by the ELISA reader (Bio Tek, PowerWave XS2, City, State, USA).

3-9-5 Trace elements Approximately 1 mL of each whole blood sample was microwave digested with 3 mL of 65 % nitric acid (Ultrapure Reagent, J.T. Baker). Subsequently, investigators washed the residuals in microwave tubes with 2 % nitric acid and then filtered the digested fluids with 0.22 μm filter. The total filtered solutions were stored in 15 mL centrifuge tubes. The levels of Pb, Cd, As, Hg, Mn, Al, Tl, V, Na, K, Mg, Ca, Fe, Zn, Se were determined using inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7800).

3-9-6 Immune function analysis Human peripheral bloods were separated from whole blood by using PluriMate (#44-91002-11). After separation, Fc receptors in PBMC were blocked by Fc blocker (Biolegend #422302), stained with monoclonal antibodies (CD3-EV450, CD4-AF488, CD8a-AF647, CD25- PE/Cyanine7, (Elabscience Biotechnology Inc.)), and analyzed by the Invitrogen™ Attune™ NxT acoustic focusing cytometer (Thermo Fisher Scientific).

3-10 Questionnaire

3-10-1 Basic information The researchers with IRB certificate helped subjects to fill their information they said in the basic personal information, including age, disease history, etc.

3-10-2 24-hour dietary recall 24-hour dietary recall is to recall past 24 hours food intake and records food items, cooking methods, portion sizes, and calorie calculation.

3-10-3 International physical activity questionnaire (iPAQ) iPAQ is used to estimate the types of intensity of physical activity and sitting time.

3-10-4 Food frequency questionnaire Food frequency questionnaire is used to estimate long-term dietary conditions.

3-10-5 Taiwan lifestyle assessment Lifestyle questionnaire is a thirteen-item questionnaire which is reported by patients themselves.

3-10-6 Patient Assessment of Constipation symptom (PAC-SYM) PAC-SYM is used to measure specific symptoms of patients with constipation, was developed in parallel with a complementary instrument to comprehensively measure disease QoL outcomes (PAC-QOL). These instruments can be used separately or together.

3-10-7 SARC-F questionnaire SARC-F is a five-item questionnaire, which is reported by patients themselves to screen for sarcopenia. The reflection is based on the patient's perception of his own strength, ability to walk, stand up from the chair, climb stairs and fall experience. More than 4 points may be at risk of sarcopenia.

3-10-8 Mini nutrition assessment (MNA) MNA is used to screen out high-risk groups of malnutrition. The maximum score obtained by MNA is 30 points, between 17-23.5 represents the risk of malnutrition, less than 17 points is malnutrition.

• Study Type: Interventional
• Enrollment (Actual): 132
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Taiwan
  • Taipei, Taiwan, 110
    • Taipei Medical University Hospital
  • Taipei, Taiwan, 110
    • Taipei Medical University
  • Taipei, Taiwan
    • Wanfang Hospital

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: 65 years to 85 years (Older Adult)
• Accepts Healthy Volunteers: Yes

Description

Sarcopenia without treatment

Inclusion Criteria:

1. Age between 65 and 85 years
2. No use of hormonal replacement therapy (women)
3. No hospital admissions within the last 3 months
4. Sarcopenia criteria:

(1) Muscle mass: measured by using InbodyS10 and standardized by height, shown by skeletal muscle index [SMI=appendicular muscle mass (kg)/height (m2)]. Low muscle mass was defined as:

• Men: SMI <7.0 kg/m2
• Women: SMI <5.4 kg/m2 (2) Handgrip strength: measured by electronic hand grip dynamometer. Low handgrip strength was defined as:
• Men: <28 kg
• Women: <18 kg (3) Limb strength: measured by Time for 5 times for chair stand method. Low limb strength was defined as:
• Time for 5 times for chair stand: ≧12s Sarcopenia was defined by (1), (2) or (3) Sarcopenia plus Lactobacillus casei strain Shirota (LcS)

Inclusion Criteria:

1. Age between 65 and 85 years
2. No use of hormonal replacement therapy (women)
3. No hospital admissions within the last 3 months
4. Sarcopenia criteria:

(1) Muscle mass: measured by using InbodyS10 and standardized by height, shown by skeletal muscle index [SMI=appendicular muscle mass (kg)/height (m2)]. Low muscle mass was defined as:

• Men: SMI <7.0 kg/m2
• Women: SMI <5.4 kg/m2 (2) Handgrip strength: measured by electronic hand grip dynamometer. Low handgrip strength was defined as:
• Men: <28 kg
• Women: <18 kg (3) Limb strength: measured by Time for 5 times for chair stand method. Low limb strength was defined as:
• Time for 5 times for chair stand: ≧12s Sarcopenia was defined by (1), (2) or (3)

Exclusion Criteria of Three groups:

1. Active cancer: currently receiving cancer treatment or have received cancer treatment within the last 3 months
2. Weight change ≥ 5% or weight change ≥ 5 kg within the past 3 months
3. BMI > 35 kg/m2
4. Disease requiring chronic use of prescription corticosteroids
5. History of ischemic or hemorrhage stroke
6. Unstable or uncontrollable hypertension (>180/110 mmHg)
7. Doing hemodialysis or peritoneal dialysis within the last 3 months
8. Participation in a structured physical exercise training program within the past 2 year; previous use of creatinine supplementation; use of drugs that can affect bone metabolism (e.g., glucocorticoids, bisphosphonates, vitamin D or calcium).
9. Antibiotics were used in the past 3 months.
10. Products of probiotic were used in the past 2 weeks.
11. Living abroad for one month in the past 3 months
12. Hyperthyroidism without medication therapy
13. Allergic to milk

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Treatment
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: None (Open Label)

Number of Arms

3

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Sarcopenia plus LcS group(LcS); LcS group must conform to inclusion criteria of LcS group and exclusion criteria. NS group need to intervene Yakult light 300 supplementation. NS group need to finish pre test and post test.	Dietary supplement: Yakult 300 LIGHT Fermented Milk; 2 bottles of fermented Milk containing Lactobacillus casei Strain Shirota per day, Intervention for 12 weeks.
No Intervention: Sarcopenia group(SC); SC group must conform to inclusion criteria of SC group and exclusion criteria. NS group dosen't need to intervene Yakult light 300 supplementation. NS group need to finish pre test and post test.
No Intervention: non-sarcopenia group(NS); NS group must conform to inclusion criteria of NS group and exclusion criteria. NS group dosen't need to intervene Yakult light 300 supplementation. NS group only need to finish the post test.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Impact of LCS on the Change of Gut Microbiota	The V1-V9 region of the 16S rRNA gene was amplified by PCR following the Illumina 16S Metagenomic Sequencing Library Preparation protocol and sequenced on an Illumina MiSeq 2000 platform. Data analysis used QIIME2 with UPARSE for OTU clustering (97% similarity), Shannon index for alpha diversity, weighted PCoA for beta diversity, and LEfSe via Galaxy for differential analysis. In sarcopenia patients, Synergistaceae and Oscillibacter are elevated while Bifidobacterium is reduced. Post-intervention improvement (decreased Synergistaceae/Oscillibacter, increased Bifidobacterium) alleviates dysbiosis, lowers inflammation, and enhances gut-muscle axis function. This shift boosts SCFA production, muscle protein synthesis, grip strength, and mass while reducing pathogen-induced insulin resistance for metabolic recovery and symptom relief.	12 week

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Impact of LCS on the Change of Sarcopenia Markers (Handgrip)	Handgrip: The measurement used electronic hand grip dynamometer. Low handgrip strength was defined as Men: <28 kg; Women: <18kg.	12 week
Impact of LCS on the Change of Sarcopenia Markers (Walk Speed)	Walk speed: The time required for participants to walk 6 meters along the line.	12 weeks
Impact of LCS on the Change of Sarcopenia Markers (Chair Stand Test & TUG Test)	Chair stand test (second): Participants sat and stood five times, and time was calculated using a timer. Low limb strength was defined time ≧12s. TUG:Participants were instructed to stand up from the chair, walk 3 meters forward and go back to sit on the chair. Low Physical performance was defined ≧ 20s.	12 week
Impact of LCS on the Change of Sarcopenia Markers (SPPB Score)	The Short Physical Performance Battery (SPPB) is a validated objective assessment tool developed by the National Institute on Aging to evaluate lower extremity function in older adults. It comprises three standardized components administered in sequence: a balance test (side-by-side, semi-tandem, and tandem stands), a 4-meter gait speed test, and a five-times chair stand test, each scored from 0-4 based on time or ability, yielding a total score of 0-12. Higher scores (10-12: minimal limitations; 4-6: moderate; 0-3: severe) indicate better physical performance and predict lower risks of mobility disability, falls, hospitalization, and mortality. The SPPB takes ~10 minutes, requires minimal equipment, and demonstrates excellent reliability for clinical and research use in geriatric populations, including sarcopenia studies. Low Physical performance was defined ≦ 9 scores	12 weeks
Impact of LCS on the Change of Inflammatory Biomarker	Serum pro-inflammatory cytokines (IL-6 and TNF-α) and anti-inflammatory cytokines (IL-10, IL-17 and TGF-β) were determined by using ELISA Kits (Elabscience Biotechnology Inc) via ELISA reader (BioTek, PowerWave XS2, City, State, USA).	12 week
Impact of LCS on the Change of Anti-oxidant Biomarker	Superoxide dismutase (SOD) (E-BC-K019, Elabscience Biotechnology Inc.), Glutathione peroxidase (GPx) (E-BC-K096, Elabscience Inc.) and Catalase (CAT) (E-BC-K031, Elabscience Biotechnology Inc.) are antioxidant enzymes in human. Enzymatic activity in serum was determined by using ELISA Kits via ELISA reader (BioTek, PowerWave XS2, City, State, USA).	12 week

Collaborators and Investigators

• Sponsor: Taipei Medical University
• Collaborators: Yakult Honsha Co., LTD
• Investigators: Study Chair: Huang Hui-Yu, PhD, Taipei Medical University

Study record dates

Study Major Dates

• Study Start (Actual): March 23, 2021
• Primary Completion (Actual): February 23, 2023
• Study Completion (Actual): February 23, 2023

Study Registration Dates

• First Submitted: June 16, 2021
• First Submitted That Met QC Criteria: July 25, 2021
• First Posted (Actual): August 2, 2021

Study Record Updates

• Last Update Posted (Actual): February 17, 2026
• Last Update Submitted That Met QC Criteria: January 28, 2026
• Last Verified: January 1, 2026

More Information

Terms related to this study

• Keywords: Inflammation; Aging; Sarcopenia; Lactobacillus casei strain Shirota
• Additional Relevant MeSH Terms: Neurologic Manifestations; Nervous System Diseases; Neuromuscular Manifestations; Pathologic Processes; Pathological Conditions, Anatomical; Muscular Atrophy; Atrophy; Pathological Conditions, Signs and Symptoms; Signs and Symptoms; Inflammation; Sarcopenia
• Other Study ID Numbers: N202010025

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No