Can Lactobacillus Acidophilus Decrease the Risk of Postmenopausal in Women?
Phase: NA
Conditions: Bone Loss, Post Menopausal Osteoporosis, Mineralization
Sponsor: Poznan University of Life Sciences

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Can Lactobacillus acidophilus decrease the risk of postmenopausal osteoporosis 
in women? 
 
Short Description 
Background 
The current recommendations for treating bone diseases are estrogen therapy and 
pharmacological agents. However, those treatments increase the risk of cancers and are not 
prescribed for long-term use. Lactobacillus acidophilus in ovariectomized mice could inhibit 
osteoclastogenic cells and promote anti-osteoclastogenic cells. 
Aim of study 
This study aims to evaluate the effect of L. acidophilus supplementation on calcium status and 
bone densitometry in postmenopausal women in a randomized, double-blind placebo-
controlled study. 
Study design 
This study has three steps; namely, 1st step is to prepare the diet supplementation of probiotics 
(L. acidophilus 1×109 CFU). 2nd step is to perform the randomized, double-blind placebo-
controlled clinical trial. 3rd step is to analyze the effects of L. acidophilus in postmenopausal 
women. 
Methods & Analysis 
60 women of postmenopausal osteoporosis will be recruited by following inclusion and 
exclusion criteria. Participants will be randomized and divided into two groups to take a capsule 
containing probiotics and a placebo for 12 weeks. Before and after the intervention, several 
parameters will be conducted, such as the bone turnover and hormones parameter in blood 
using ELISA, the calcium mineralization in hair sample using AAS, the bone densitometry 
analysis in bone using DXA. 
 
 

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Research Design 
Study settings 
This study will be conducted within two (2) years for research activity and one (1) year for the 
publication period. There are three steps within this study; namely, 1st step is to prepare the 
sample of L. acidophilus as diet supplementation. Probiotic capsules will contain L. acidophilus 
1×109 CFU. 2nd step is to perform the randomized, double-blind placebo-controlled clinical 
trial of L. acidophilus supplementation for twelve weeks (Figure 1). 3rd step is to analyze the 
effects of L. acidophilus in postmenopausal women. 
Eligibility
(n = 60)
Enrollment
Randomization
(n = 60)
Excluded
(if any)
Allocated to
Placebo
(n = 30)
Allocated to 
Intervention
(n = 30)
Lost to follow up 
due to personal 
reasons (if any)
Lost to follow up 
due to personal 
reasons (if any)
Analyzed
(n = 30)
Analyzed
(n = 30)
Allocation
Follow up
Analysis
Patient Recruitment 
Flow Diagram
 
Figure 1. Flow diagram of patient recruitment 
Eligibility criteria (inclusion, exclusion criteria) 
The inclusion and exclusion criteria are provided in figure 2. Informed consent should be 
obtained from all patients, and the patients will be asked to notify the researchers if medical 
changes occurred during the intervention. 

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Figure 2. Inclusion and Exclusion criteria 
Interventions or exposures 
Pre-consumption
1-week
Consumption
12-week
Post Consumption
1-week
Collecting 
samples
(Baseline)
Collecting 
samples
(Endline)
Intervention Period
 
Figure 3. Diagram of the intervention period 
The intervention period during this study can be seen in figure 3. The diet intervention in this 
experiment will be performed for 12 weeks, as Ahn et al. (2019) described. Two groups will 
be designated in this study. Group 1 is the placebo group that will receive a capsule that consists 
of the excipient. The excipient will be composed of maize starch and maltodextrins (Skrypnik 
et al., 2019). Group 2 is the probiotic group that will receive a diet capsule consisting of 
Lactobacillus acidophilus with a dose of 1x109 CFU with the excipient described by Hosseini 
et al. (2019). In addition, it is essential to deliver self-administration of probiotic agents orally 
in the morning with fasting conditions to evaluate probiotic effectiveness. Subsequently, 
patients will be asked to keep the diet in refrigerated condition (2-7 °C). 
Primary endpoints 
This study will consider the change from baseline in the frequency and severity of hot flushes 
as primary endpoints. 
Secondary endpoints 
Meanwhile, the health-related quality of life, sleep, satisfaction with treatment, and cumulative 
amenorrhea and breast pain will be considered as secondary endpoints. 
Sample size calculation 
To calculate sample size, this study refers to the Soleimani et al. (2017) study. Using a formula 
suggested for clinical trials, having 25 participants in each group will be considered adequate 

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while considering a type I error (a) of 0.05 and type II error (b) of 0.20 (power ¼ 80%), with 
1.61 as the SD and 1.30 as the mean distinction of CTX for markers of bone resorption and 
BSAP for marker of bone formation as the key variable. By assuming five dropouts in each 
group, the final sample size will be 30 participants in each group. 
Methods assignment of interventions 
Randomization will be performed using a computerized random number generator, which 
generated the random sequence based on simple randomization. All subjects will be identified 
by a trial identification number and assigned a treatment code. The researchers and participants 
will be blinded to the treatment code until the end of the study when data will be collected and 
evaluated. The groups will be decided after the completion of the data analysis. 
Variable assessment 
All information is containing 3 days dietary records, body composition measurement, and 
fasting blood samples will be collected at the beginning and end of the study. DIETA 6.0 and 
5D computer program by Instytut Żywności i Żywienia, Poland will be used for analyzing 3 
days averages of macronutrients and micronutrient intakes. A validated questionnaire obtains 
personal and demographic information. Anthropometric measurements will be measured using 
body composition analysis. 
Biomarkers of bone turnover and hormones 
This study will analyze the biomarkers of bone turnover and hormones as described by 
Villareal et al. (2016). Briefly, venous blood samples will be obtained after fasting overnight 
and analyzed in the laboratory. Enzyme-linked immunosorbent assay (ELISA) will be used to 
measure C-terminal telopeptide of type I collagen (CTX) and tartrate-resistant acid phosphatase 
isoform-5b (TRAP5b) as markers of bone resorption and bone-specific alkaline phosphatase 
(BSAP) as a marker of bone formation. Radioimmunoassay will be used to measure N-terminal 
propeptide of type I procollagen (PINP) as an additional marker of bone formation. ELISA is 
also used to measure high-molecular-weight adiponectin and insulin-like growth factor-1. 
Chemiluminescent immunoassays will be used to measure parathyroid hormone (PTH) and 
cortisol. 
Hair sample 
A hair strand of 1 cm of length is collected from the occipital region. The sample will be taken 
by a member of the study team shortly after the hair had been washed with a shampoo not 
containing functional components. Each woman is instructed on how to wash her hair. 
Moreover, it will be clearly explained to patients that reliable results could be obtained only by 
complying with this procedure. Hair spray or hair dye is forbidden during the study (Skrypnik 
et al., 2019). 
Calcium analysis 
The calcium contents of the hair and serum will be determined after digestion in 65% (w/w) 
spectra pure HNO3 using a Microwave Digestion System. Upon digestion and dilution with 
deionized water, the concentrations in the mineral solutions are measured using flame atomic 
absorption spectrometry (Skrypnik et al., 2019). 
 

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Bone densitometry analysis 
This analysis will be conducted in the accredited laboratory in Poznan, Poland. Subsequently, 
the subjects are asked to undergo bone mineral densitometry using a DEXA scan. The scan 
will be performed on a Hologic DXA scan machine. 
Products used in the research 
1. 
A capsule consists of Maize starch and maltodextrins for the Placebo Group. 
2. 
A capsule contains Lactobacillus acidophilus with a dose of 1x109 CFU for the 
Probiotic Group. 
Ascertainment Feasibility 
The study will be conducted in the Department of Obesity and Metabolic Disorders Treatment 
and Clinical Dietetics, Karol Marcinkowski University of Medical Sciences (PUMS), Poznan, 
Poland. This place receives patients who meet the inclusion criteria described above. 
Techniques and actual available equipment 
The study will be registered to the Ethics Committee of Poznan Authority before the study 
started and followed by sending documents to the Nutricia Foundation Committee in 
September 2021. Practically, the activity will be held in the Hospital of PUMS under specialist 
doctor and expertise healthcare worker control. Meanwhile, sample measurements will be 
analysed at Poznan University of Life Sciences, and DXA measurement will also be held in 
the Department of Human Nutrition and Dietetics, Poznan University of Life Sciences, Poland. 
 
 

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