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

1. Subject Enrollment The definition of sarcopenia is based on the algorithm of Asian Working Group for Sarcopenia (AWGS).
2. 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

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

• Men: <28 kg
• Women: <18 kg

1. 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)

1. 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).

1. 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.