Biological Effects of the Consumption of Limosilactobacillus Fermentum CRL 1446 - CONICET in Volunteers With Cardiovascular Risk Factors
Phase: NA
Conditions: Disbiosis, Obesity, Overweight (BMI > 25), Hypertension (Without Type 2 Diabetes Mellitus), Diabetes (DM), Smoke, Sedentarity, Cholesterol (Total and HDL), Triglycerides High, Stress (Psychology)
Sponsor: PINER Pastor Manfredi Winery

 
 
 
 
 
 
 
 
 
 
 
Biological Effects of the 
Consumption of 
Limosilactobacillus 
fermentum CRL 1446 
CONICET in Volunteers with 
Cardiovascular Risk Factors. 
 
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NCT number 
 

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Biological Effects of the Consumption of Limosilactobacillus fermentum CRL 
1446 CONICET in Volunteers with Cardiovascular Risk Factors. 
1. Introduction The advancement of biomedical research has revealed the key 
influence of the intestinal microbiota on human health, especially regarding 
cardiovascular risk factors (CVRFs). In Argentina, results from the Fourth National 
Survey of Risk Factors show that the increase in overweight and obesity rose from 
49% in 2005 to 61.6% in 2018, representing a 2% increase per year. Overweight, 
obesity, dyslipidemia, arterial hypertension, insulin resistance, and chronic 
inflammation show a close correlation with imbalances in the intestinal 
microbiota, increasing the risk of cardiovascular diseases. Given the global 
increase in these factors, the search for innovative strategies complementary to 
conventional treatments is essential, and nutritional intervention with probiotics 
emerges as a promising alternative for modulating the intestinal microbiota's 
composition and function. Intestinal dysbiosis is characterized by a reduction in 
bacterial diversity, an increase in pro-inflammatory bacteria, and a decrease in 
beneficial short-chain fatty acid-producing bacteria. 
Unlike other probiotic strains, Limosilactobacillus fermentum CRL 1446 exhibits a 
singular combination of functional mechanisms supporting its potential benefit 
in the health of people with CVRFs. Administration of this feruloyl esterase-
producing strain has been shown to improve metabolic and oxidative parameters 
in preclinical trials. It stands out for its capacity to modulate the intestinal 
microbiota composition by producing beneficial metabolites, such as short-chain 
fatty acids, which contribute to improving the intestinal barrier integrity and 
reducing bacterial translocation. This strain can also positively influence lipid 
metabolism by regulating key enzymes involved in lipid synthesis and degradation. 
Its immunomodulatory action is also relevant: preclinical trials have observed that 
L. fermentum CRL 1446 promotes an anti-inflammatory profile by stimulating the 
production of regulatory cytokines and decreasing pro-inflammatory mediators. 
The consumption of L. fermentum CRL1446 emerges as a promising strategy for 
the modulation of key factors in the metabolic health of people with CVRFs. This 
probiotic approach not only represents an innovative complement to conventional 
therapies but also opens new avenues for the prevention and personalized 
management of complex metabolic disorders. 
2. Research Question Could the consumption of L. fermentum CRL1446 induce 
improvements in the metabolic health of adult volunteers with cardiovascular risk 
factors, and thus, prevent or mitigate non-communicable chronic diseases? 
 

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3. Working Hypothesis It is expected to demonstrate that those volunteers who 
consume L. fermentum CRL 1446 will have significant improvements in their 
metabolic health compared to those who do not consume it. 
4. Objectives 4.1  
General Objective  
4.1.1. We will measure the effectiveness of the L.l fermentum CRL-1446 strain vs 
placebo in two phases of acute and chronic intervention, in volunteers with 
cardiovascular risk factors. Time Frame: 150 days (acute) 390 days (chronic). 
4.2 Specific Objectives:  
4.2.1 We will measure the gastrointestinal symptomatology, health effects, 
tolerance, side effects, and adverse effects of two phases of acute and chronic 
intervention with the L. fermentum CRL-1446 strain vs placebo in 80 volunteers of 
both sexes with cardiovascular risk factors using the GSRS scale. Time Frame: 150 
days (acute) 390 days (chronic). 
4.2.2 We will measure postprandial glucose absorption levels (mg/dl) with 
continuous glucose monitoring in 30 volunteers with cardiovascular risk factors, 
before and after consumption of 1 capsule/day of Ll fermemtum CRL 1446 vs 
placebo and before and after consumption of 2 capsules/day Ll fermemtum CRL 
1446 vs placebo. Time Frame: 150 days 
4.2.3 We will measure the change in body composition using impedance analysis 
(in kg of muscle mass and fat mass) in 30 volunteers with cardiovascular risk 
factors, before and after consuming 1 capsule/day of Ll fermentum CRL 1446 vs 
placebo and before and after consuming 2 capsules/day of L. fermentum CRL-
1446, vs placebo. Time Frame: 150 days. 
4.2.4 We will measure postprandial glucose absorption levels (mg/dl) with 
continuous glucose monitoring in 50 volunteers with Cardiovascular Risk Factors, 
before and after consuming 1 capsule/day of Ll fermemtum CRL 1446 vs placebo 
and before and after consuming 2 capsules/day of L. fermentum CRL1446 vs 
placebo. Time Frame: 390 days. 
4.2.5 We will measure the change in body composition using impedance analysis 
(in kg of muscle mass and fat mass) in 50 volunteers with cardiovascular risk 
factors, before and after consuming 1 capsule/day of Ll fermemtum CRL 1446 vs 
placebo and before and after consuming 2 capsules/day of L. fermentum CRL-
1446, vs placebo. Time Frame: 390 days. 

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4.2.6 We will measure the modification of oxidative stress biomarkers in 50 
volunteers with cardiovascular risk factors, before and after consuming 1 
capsule/day vs placebo and before and after consuming 2 capsules/day of L. 
fermentum CRL1446, vs placebo. Time frame 390 days. 
4.2.7 We will measure the modification of antioxidant defenses in 50 volunteers 
with cardiovascular risk factors, before and after consuming 1 capsule/day vs 
placebo and before and after consuming 2 capsules/day of L. fermentum 
CRL1446, vs placebo. Time frame 390 days.  
4.2.8 We will measure the modification of DNA damage biomarkers in 50 
volunteers with cardiovascular risk factors, before and after consumption of 1 
capsule/day vs placebo and before and after consumption of 2 capsules/day of L. 
fermentum CRL1446, vs placebo. Time frame 390 days. 
4.2.9 We will measure the modification of chronic inflammation biomarkers in 50 
volunteers with cardiovascular risk factors, before and after consuming 1 
capsule/day vs placebo and before and after consuming 2 capsules/day of L. 
fermentum CRL1446, vs placebo. Time frame 390 days. 
5. Materials and Methods Eighty volunteers of both sexes, aged 40 to 65, residing 
in Buenos Aires City and receiving medical attention for CVRFs, will be invited to 
participate. 
5.2 Design The research design will be a 2-branch randomized, double-blind, 
placebo-controlled, and crossover trial. 
Design Development:  
5.2.1 Acute Phase will involve a nutritional intervention in 30 volunteers with 
CVRFs. The intervention includes 30-day periods of 1 capsule/day of L. fermentum 
CRL1446 or placebo, followed by a 15-day microbiota washout period, and then 
group crossover. This sequence is repeated with a dosage of 2 capsules/day of L. 
fermentum CRL1446 or Placebo. A total of 8 sets of analyses will be performed. 
5.2.2 Chronic Phase will involve a nutritional intervention in 50 volunteers with 
CVRFs. The intervention includes 90-day periods of 1 capsule/day of L. fermentum 
CRL1446 or Placebo, followed by a 15-day washout period, and then group 
crossover. This sequence is repeated with a dosage of 2 capsules/day of L. 
fermentum CRL1446 or Placebo. In total, there will be 8 sets of analyses. 
5.3. Capsule Content The content of L. fermentum CRL1446 per product 
capsule is 2.20E+9 CFU (colony-forming units). Placebo capsules lack L. 
fermentum CRL1446 but maintain the same composition, size, color, and shape as 
the product capsules. The study is double-blind, meaning neither the responsible 

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researcher nor the patient volunteer will know which type of capsule is being 
ingested. 
5.3.1 Posology: Capsules must be swallowed whole with water. For the one-
capsule-per-day phase, they are taken at breakfast, and for the two-capsules-per-
day phase, they are taken at breakfast and dinner. Storage conditions: Keep the 
closed jar refrigerated. 
5.4. SAFETY  
5.4.1. Bacterial Translocation Assays (Indicator of acute toxicity): The strain L. 
fermentum CRL1446 did not induce bacterial translocation at concentrations of 
10E+7, 10E+8, and 10E+9 CFU/mL in the two assayed mouse strains. Histological 
analysis showed no significant modifications in the intestinal structure of the 
treated animals. 
5.4.2. Complementary Tests to Ensure Strain Safety: 
• Antibiotic Resistance: No Minimum Inhibitory Concentration (MIC) values 
superior to the EFSA-recommended cut-off were observed for the tested 
antibiotics (ampicillin, vancomycin, clindamycin, gentamicin, chloramphenicol, 
streptomycin, tetracycline, erythromycin). PCR analysis showed no resistance 
genes in the CRL1446 strain. 
• Hemolytic Activity: No presence of or -hemolysis was observed for the 
CRL1446 strain. 
5.5 Inclusion Criteria: Men and women aged 40 to 65 years with cardiovascular 
risk factors. Eligible volunteers could have one or more of the following 
conditions: 
1. Blood glucose > 100 mg/dl or pharmacological treatment for high blood glucose 
levels. 
2. HDL Cholesterol < 40 mg/dl in men or < 50 mg/dl in women, or pharmacological 
treatment for low HDL cholesterol levels. 
3. Blood triglycerides > 150 mg/dl or pharmacological treatment for high 
triglyceride levels. 
4. Waist circumference > 102 cm for men or > 88 cm for women. 
5. Blood pressure > 130/85 mmHg, or pharmacological treatment for high blood 
pressure. 
6. Sedentary lifestyle. 
7. Smoking habit. 

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8. Overweight 
9. Obesity 
5.5.2 Exclusion Criteria: Patients with a history of digestive surgery, inflammatory 
bowel disease, gastroenteritis in the last 6 months, oncological patients, patients 
with rheumatic pathologies, patients with liver disorders, immunological disorders 
or deficits, steatorrhea, antibiotic use in the last 3 months, anemia of any cause, 
or pregnancy/expectation of becoming pregnant during the study. 
5.6 Biomarkers to be evaluated  
5.6.1 Clinical Biochemistry in plasma and serum: Ferritin, complete blood 
count, RDW, ultra-sensitive CRP, Glycemia, Creatinine, Albumin, Alkaline 
Phosphatase, Urea, Erythrocyte Sedimentation Rate, Total Cholesterol, LDL 
Cholesterol, HDL Cholesterol, Triglycerides, Insulin, HbA1c, C-Peptide, 
Hepatogram, Haptoglobin, Plasma Zonulin. Inflammation: Oxygen consumption 
and Chemiluminescence in polymorphonuclear cells (PMN), Interleukin 6 (IL6). 
Oxidative Stress (in plasma): Lipid oxidation (TBARS); Protein oxidation (carbonyl 
proteins). DNA Damage in plasma: 8-hydroxy (OH) deoxyguanosine. Endogenous 
Antioxidant Defenses: Reduced/oxidized glutathione in erythrocytes.  
5.6.2 Non-invasive Biomarkers: Body composition by impedanciometry, 
transdermal oxidative stress scanner, and Continuous Glucose Monitoring (basal 
and postprandial up to 4 hours after a standard breakfast), and transdermal 
Advanced Glycation End-products (AGEs) Reader scanner. 
6. Outcome Measures 
6.1 Clinical Biochemistry in plasma and serum: 
Biomarker 
Units 
Ferritin 
mg/dL 
White Blood Cells 
Cells/μL 
Lymphocyte 
% 
Hematocrit 
% 
RWD 
% Red distribution width 
Ultrasensitive – CRP 
g/dl 
Glycemia 
mg/dL 
Creatinine 
mg/dL 
Albumin 
mg/dL 
Alkaline Phosphatase  
UI 
Urea  
mg/dL 
Erythrocyte Sedimentation Rate  
mm/h 
Total Cholesterol  
mg/dL 
LDL Cholesterol  
mg/dL 
HDL Cholesterol  
mg/dL 

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Triglycerides 
mg/dL 
Insulin  
μUI/mL 
HbA1c  
% 
C-Peptide  
ng/mL 
Haptoglobin 
mg/dL 
Plasma Zonulin.  
ng/mL 
Plasma Serotonin 
ng/mL 
Cerutoplasmin 
mg/dL 
Pasma nitrites 
μM/L 
 
 
Clinical Variables 
 
BMI 
Weight (kg) / height (m)2 
Blood Pressure 
mm/Hg 
 
6.2: Inflammation: 
 Interleukin 6 (IL6). Picograms/mL 
6.3 Oxidative Stress (in plasma):  
Lipid oxidation (TBARS); μM 
Protein oxidation (carbonyl proteins).  (nmol/mg prot.) 
8-hydroxy (OH) deoxyguanosine. ng/mL 
6.4 Endogenous Antioxidant Defenses:  
Reduced/oxidized glutathione in erythrocytes. μM 
6.5 Non-invasive Biomarkers:  
Body composition by impedanciometry,  (Ultraviolet absorbance) 
transdermal oxidative stress scanner AU (Arbytrary Units)  
Continuous Glucose Monitoring (basal and postprandial up to 4 hours after a 
standard breakfast) mg/dL 
Transdermal Advanced Glycation End-products (AGEs) Reader scanner. 
(Ultraviolet absorbance) 
7. Statistical Analysis In this 2-branch crossover and double-blind design, all 
volunteers will receive both treatments at different times. The Student's t-test will 
be employed for paired comparisons of continuous parametric data, and the 
Wilcoxon Mann–Whitney test for continuous non-parametric data. ANOVA 
(parametric) or Mann Whitney (non-parametric) will be used for multiple group 
comparisons. 

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8. Digital document repository: https://zenodo.org/ 
9. Digital repository for the project and results: https://clinicaltrials.gov/ 
10. Ethical Aspects Data will be used for academic purposes, handled by 
researchers according to current regulations and based on the IRB Ethics 
Committee Guidelines, the World Medical Association's Declaration of Helsinki 
(latest version, Helsinki 2024), Resolution 1480/11 of the National Ministry of 
Health, and National Personal Data Protection Law N.º 25.326.