PROBIOTIC COMPOSITION FOR USE AS AN ANTIOXIDANT

Abstract

This invention refers to a probiotic composition consisting of the bacteria Lactobacillus rhamnosus, Lactobacillus casei and Bifidobacterium longum, preferably of the strains L. rhamnosus CECT8361, L. casei CECT9104 and B. longum CECT7347, and its use as an antioxidant. This composition is particularly useful in the treatment and/or prevention of damage, at the molecular level, caused by oxidative stress, preferably during high intensity physical exercise.

Claims

1. A composition that consists of the bacteria Lactobacillus rhamnosus, Lactobacillus casei and Bifidobacterium longum, together with one or more food-based and/or pharmaceutically acceptable vehicles and/or excipients.

2. The composition according to claim 1, where L. rhamnosus is the strain BPL0015 deposited in the Spanish Type Culture Collection under deposit number CECT8361, L. casei is the strain BPL0004 deposited in the Spanish Type Culture Collection under deposit number CECT9104 and B. longum is the strain IATA-ES1 deposited in the Spanish Type Culture Collection under deposit number CECT7347.

3. The composition according to claim 1, where L. rhamnosus is at a concentration of 45%, L. casei is at a concentration of 45% and B. longum is at a concentration of 10%, compared to the total concentration of bacteria included in the composition.

4. The composition according to claim 1, where the total amount of bacteria in said composition is 10.sup.9 CFU.

5. The composition according to claim 1, where said composition is a pharmaceutical or food composition.

6. The composition according to claim 1, formulated for oral administration.

7. The composition according to claim 1, where said composition is presented in solid form.

8. The composition according to claim 7, where said composition is presented in capsule form.

9-13. (canceled)

14. A method for the treatment and/or prevention of oxidative stress in a subject comprising administering the composition according to claim 1 to a subject in need thereof.

15. The method according to claim 14 wherein the oxidative stress is caused by physical activity.

16. The method according to claim 14 where said composition is administered once a day.

17. The method according to claim 14 where said composition is administered for 6 weeks.

Description

DESCRIPTION OF THE FIGURES

[0059] FIG. 1. Serum malondialdehyde for each of the groups (placebo and probiotic), for each test and in the initial and final stages of each test. * p<0.05 comparison between the initial and final stage for each test.

[0060] FIG. 2. Increase in serum malondialdehyde during physical exercise tests for each group (placebo and probiotic). * p<0.05 Test comparison.

[0061] FIG. 3. Serum oxidised LDL for each of the groups (placebo and probiotic), for each test and at the initial and final stages of each test. * p<0.05 comparison between the initial and final stage for each test.

[0062] FIG. 4. Increase in serum oxidised-LDL during physical exercise tests for each group (placebo and probiotic). * p<0.05 Test comparison.

[0063] FIG. 5. 8-oxo 2′-deoxiguanosin in 24-hour urine (pg/ml) for each of the groups (placebo and probiotic), for each test and at the initial and final stages of each test. * p<0.05 comparison between the initial and final stage for each test.

[0064] FIG. 6. Increase in 8-oxo 2′-deoxiguanosin in urine for 24 hours (pg/ml) during physical exercise tests for each of the groups (placebo and probiotic). * p<0.05 Test comparison.

EXAMPLES

[0065] Below, we will show the invention using a nutrition clinical trial conducted by the inventors, which highlights the effectiveness of the composition of the invention in reducing molecular damage caused by oxidative stress during high-intensity long-duration physical exercise.

[0066] Example 1. Clinical trial to determine the effectiveness of the composition of the invention, compared to a placebo, in the reduction of oxidative stress during high-intensity long-duration physical exercise, as well as to determine the tolerance and safety of the composition.

1.1. Study Design

[0067] A randomized, placebo-controlled clinical trial was conducted with two parallel study groups based on the product consumed (experimental or placebo), double-blind and unicentric, designed to evaluate the effect of the product on the reduction of oxidative stress produced by high-intensity long-duration physical exercise.

[0068] The selected subjects were healthy caucasian male subjects aged 18 to 45, selected from the general population, performing aerobic physical exercise 2 to 4 times a week. Excluded from the study were those subjects with a history of any chronic disease, especially digestive tract diseases, who had undergone abdominal surgery in the three months prior to the study, with a history of bronchial asthma or chronic obstructive pulmonary disease, reactive respiratory tract disease such as bronchial asthma, sinus bradycardia, second or third degree atrioventricular block, manifest heart failure or cardiogenic shock, a history of allergic hypersensitivity or poor tolerance to any component of the products under study, participation in another clinical trial in the three months prior to the study, subjects diagnosed and/or being treated for high blood pressure, smokers (>10 cigarettes a day), subjects with body mass index greater than 35 kg/m2 (BMI>30), subjects with a history of drug or alcohol abuse or of other substances or other factors limiting their ability to cooperate during the study.

[0069] As a control, a placebo with identical organoleptic characteristics and the same visual aspect as the trial product was selected.

[0070] The characteristics of the study product were the following: [0071] Pharmaceutical form: capsules, both the trial product and the placebo. [0072] Content: in no case did the excipients modify the pharmacokinetics or pharmacodynamics of the active substances, they were added for technological reasons only. [0073] Route of administration: oral [0074] Posology: 1 capsule/day. [0075] Dose regimen: 6 weeks.

[0076] The composition of the invention consisted of a mixture of: [0077] L. rhamnosus BPL0015 (CECT8361) (45%) [0078] L. casei BPL0004 (CECT9104) (45%), and [0079] B. longum IATA-ES1 (CECT7347) (10%)

[0080] The final product contained 10.sup.9 CFU/capsule.

1.2. Study Protocol

[0081] In order to demonstrate the proposed objectives, study subjects were subjected to an oxidative stress model consisting of the performance of high-intensity long-duration physical activity (90 minutes). This increased the oxidative stress of the study subjects and showed the efficacy of the product (composition of the invention) compared to the placebo in terms of improving the oxidative status of the subjects. The proposed oxidative model consisted of a preliminary test and two non-maximal stress tests of high and constant intensity, which will henceforth be called test 1 and test 2. Each one is described below. [0082] Preliminary test: the aim of this test was to be able to calculate individually the intensity at which the subjects (cyclists) should perform the subsequent physical activity, i.e., tests 1 and 2. During the realization of this test the subjects did not ingest either of the products used in the study (probiotic or placebo). This test was carried out on a bicycle roller with electromagnetic resistance (Technogym Spin Trainer) on which the bicycle is placed with a starting load that simulates a speed of 12 km/h with a load increase of 2 Km/h each minute, maintaining a constant slope of 2%. Cyclists employed a free style. In order to calculate the intensity of subsequent physical activity, subjects underwent ergo-spirometric and electrocardiographic monitoring. Thus, previously, the subject was prepared for respiratory gases analysis (breath-by-breath, open circuit, gas analyser brand Jaeger Oxicom Pro) while undertaking the test. The main variable evaluated during the performance of this test was the maximum/peak consumption of absolute and relative oxygen (VO.sub.2 max) which is the maximum volume of oxygen measured in ml/min or ml/Kg×min detected in the test or maximum value of that variable after which it does not increase, even if effort intensity is increased. Then, after a 7-day wash-out period of the oxidative stress generated in the preliminary test, the following test was performed. [0083] 1st stress test (test 1): one week after the first test, the study subjects performed the following, which consisted of high-intensity physical activity of 90 minutes duration. The study subject performed a constant intensity stress test on a bicycle roller with electromagnetic resistance, on which the subject's bicycle was placed. The maximum load maintained was equivalent to a heart rate corresponding to 75% of the subject's maximum oxygen consumption calculated in the preliminary test and a constant slope of 2% was maintained. The aim of this test was to cause high oxidative stress in the subject, so that the antioxidant effect of the trial product and placebo could be evaluated. During the test, the study subjects did not consume any of the products, only water ad libitum. Once the subjects had performed test 1, the 6-week period of product consumption (probiotic or placebo) began. [0084] 2nd stress test (test 2): after the 6-week product consumption period, the study subjects performed test 2, which consisted of the same high intensity physical activity performed in test 1.

[0085] Before and after tests 1 and 2, study subjects underwent blood extraction and a 24-hour urine collection. Blood samples were taken half an hour before the subjects performed test 1 and test 2 and half an hour after each test. Likewise, the day before and after each test, 24-hour urine collection was performed. After measuring the total volume of urine excreted within 24 hours, a sample of 9 ml was extracted and frozen at −80° C. in three different cryovials until further analysis.

1.3. Study Variables Analyzed

[0086] All variables were analysed at baseline and after 6 weeks of uninterrupted consumption of the product.

1.3.1. Variables of Oxidative Damage Caused by High-Intensity Long-Duration Physical Exercise.

[0087] Both aerobic and anaerobic physical exercise lead to an increase in the production of free radicals. Certain levels of these oxidizing compounds have positive effects on the body's immune functions, tissue replacement and cell resistance, and even on muscle contraction and adaptation to systematic exercise. However, physical exercise can trigger an imbalance between free radical production and antioxidant defense mechanisms in the organism, leading to different types of molecular damage, evidenced via different biological markers of molecular damage on lipids, proteins and DNA. In this study, the subjects were subjected to a source of oxidative stress (test 1 and test 2) in order to evaluate the antioxidant effect of the probiotic versus a placebo, i.e., the ability to slow down oxidative damage caused by high-intensity long-duration physical exercise that can exceed antioxidant defence mechanisms.

Oxidative Damage to Lipids

[0088] Serum malondialdehyde analysis. Serum malondialdehyde was analyzed with the MDA oxLDL ELISA (MDA (Malondialdehyde) ELISA KIT ELABSCIENCE Houston, Tex. (USA)). This analysis was made of the serum obtained from the blood extractions performed half an hour before and after each of the stress tests performed. [0089] Oxidized LDL analysis. Serum LDL in its oxidized form was quantified using the MDA oxLDL ELISA (Human OxLDL (Oxidized Low Density Lipoprotein) ELISA KIT ELABSCIENCE Houston, Tex. (USA)). This analysis was made of the serum obtained from the blood extractions performed half an hour before and after each of the stress tests performed.

Oxidative Damage to DNA

[0090] Analysis of 8-oxo 2-deoxiguanosin in 24-hour urine. The 8-oxo-2-deoxiguanosin in 24-hour urine was analyzed using the DNA/RNA Oxidative Damage EIA Kit (80HdG (8-Hydroxideoxyguanosine) ELISA KIT ELABSCIENCE Houston, Tex. (USA)). This analysis was made in 24-hour urine samples collected before and after each stress test.

1.3.2. Safety Variables.

[0091] Biochemical blood profile was analyzed to determine GOT, GPT, GGT, LDH enzyme values, and of bilirubin to assess liver function, and of biomolecules such as urea and creatinine to evaluate renal function. A blood count was also performed to evaluate the red, white and platelet cells. Blood samples were obtained twice during the study, at baseline and the end.

[0092] Adverse events were also registered and evaluated.

1.4. Statistical Analysis

[0093] A descriptive analysis (mean and standard deviation) was made of all the variables under study, both at baseline for each of them and their development. This analysis was conducted for the total group of subjects participating in the study.

[0094] The homogeneity of the population at baseline with respect to demographic variables, medical history and other clinical parameters was also analyzed. For quantitative variables, comparisons of t-Student were made between the two study groups. The qualitative variables were analyzed by means of a homogeneity test based on Chi-square distribution when made possible by the expected values and otherwise by means of an exact Fisher test.

[0095] In order to analyse the inter-group differences (experimental and control) for the trends in the different variables, an analysis of variance was performed for repeated measurements with two intra-target factors (test: before consumption and after 8 weeks of consumption and time: before and after each test) and an inter-target factor (product: experimental product and placebo product). In this way, differences were established in each of the variables analysed, taking into account these factors. Tukey or Bonferroni tests were run for the post-hoc analysis. Comparisons were made for those significant effects with the option of assuming or not equal variances.

[0096] In the set of statistical tests the level of significance used was 0.05. The statistical analysis was carried out with SPSS 21.0. software.

1.5. Results

[0097] The study was started by 45 subjects, one of whom was excluded before the first test.

[0098] The remaining 44 subjects were randomized into the two study groups. During the study, one subject in the placebo group was withdrawn for not attending follow-up visits. Therefore, 43 subjects were analyzed: 22 who consumed the probiotic product and 21 who consumed the placebo product.

[0099] In the group that consumed the probiotic product, the average age was 25.3±7.2 years, while in the placebo group, the average age was 27.1±8.4 years.

1.5.1. Serum Malondialdehyde Analysis.

[0100] Descriptive statistics are presented in the following table:

TABLE-US-00001 TABLE 1 Statistical levels of malondialdehyde in serum (ng/ml) (mean, standard error, mean difference, P1 level of statistical significance for the difference between levels before and after each test and P2 level of significance for the difference in the increase of the levels for the placebo and probiotic). Mean Mean S. E. Dif. P1 P2 Placebo Test 1 Before 347.4 84.8 143.7 .094 .975 Afterwards 491.1 145.3 Test 2 Before 312.9 64.3 141.5 .149 Afterwards 454.4 113.3 Probiotic Test 1 Before 433.2 82.9 254.2 .003 .005 Afterwards 687.4 142.0 Test 2 Before 358.0 62.9 46.6 .623 Afterwards 404.6 110.7

[0101] The following was obtained in the comparative analysis: [0102] Comparison of the values of the variable in the initial state. There are no significant differences when comparing the values of this variable at the initial stage, so it can be said that the groups were homogeneous for this variable at the initial stage of each test. [0103] Placebo group. During the first test results show a non-significant increase (P<0.094) in serum malondialdehyde levels, secondary to the damage generated by high-intensity long-duration physical exercise. On performing the second test, after ingestion of the placebo product, physical exercise produced the same increase in the levels of this parameter as in test 1 (p<0.149). Therefore, one cannot affirm that the consumption of the placebo altered the trends in this variable during the realization of the stress tests (FIG. 1). [0104] Experimental group. There is a significant increase (P<0.001) in serum malondialdehyde levels during the first test. On performing the second test, following the intake of the probiotic product, physical exercise resulted in a much lower increase in this parameter than in test 1 (p<0.623). When comparing the changes in this parameter in test 1 with those obtained in test 2, significant differences were observed (p<0.005), i.e., in the second test, the subjects who consumed the probiotic product showed a lower increase in malondialdehyde than during the first test (FIG. 1). Therefore, one can state that the consumption of the probiotic product changed the trends in this variable during the stress tests.

[0105] On comparing the trends between the two groups (FIG. 2), significant differences (p=0.047) were observed, i.e., one can state that the trends for both products differ, so it can be concluded that the 6-week intake of the probiotic product produces significant improvements compared to the placebo for this variable.

1.5.2.—Oxidized LDL Analysis.

[0106] Descriptive statistics are presented in the following table:

TABLE-US-00002 TABLE 2 Statistical levels of oxidized LDL in serum (ng/ml) (mean, standard error, mean difference, P1 -level of statistical significance for the difference between the levels before and after each test and P2 level of significance for the difference in the increase of the levels for the placebo and probiotic). Mean S.E. Mean dif. P1 P2 Placebo Test 1 Before 740.3 61.5 159.3 .000 .536 Afterwards 899.6 64.1 Test 2 Before 777.8 69.2 196.6 .001 Afterwards 974.4 78.9 Probiotic Test 1 Before 646.2 60.1 162.8 .000 .042 Afterwards 809.0 62.6 Test 2 Before 772.9 67.6 40.4 .467 Afterwards 813.3 77.1

[0107] The following was obtained in the comparative analysis: [0108] Comparison of the values of the variable in the initial state. No significant differences were observed when comparing the values of this variable at the initial stage, so it can be said that the groups were homogeneous for this variable at the initial stage of each test. [0109] Placebo group. During the first test, a significant increase (P<0.001) in serum oxidized LDL levels secondary to the damage caused by high-intensity long-duration physical exercise was observed. On performing the second test, after ingestion of the placebo product, physical exercise produced the same increase in the levels of this parameter as in test 1 (p<0.001) (FIG. 3). Therefore, it cannot be said that placebo consumption changed the trends in this variable during stress tests. [0110] Experimental group. A significant increase (P<0.001) in serum oxidised LDL levels was observed during the first test. On performing the second test, following the intake of the probiotic product, physical exercise produced a much lower increase in this parameter than in test 1 (p<0.467) (FIG. 3). Comparison of the changes in this parameter in test 1 with those obtained in test 2 showed significant differences (p<0.042), i.e., in the second test, the subjects who consumed the probiotic product showed a lower increase in oxidized LDL than during the first test. Therefore, one can state that the consumption of the probiotic product changed the trends in this variable during the physical tests.

[0111] On comparing the trends between the two groups (FIG. 4), significant differences (p=0.05) were observed, i.e., the trends for both products can be said to differ, so it can be concluded that the 6-week intake of the probiotic product produces significant improvements compared to the placebo for this variable.

1.5.3. Analysis of 8-Oxo 2-Deoxiguanosin in 24-Hour Urine.

[0112] Descriptive statistics are presented in the following table:

TABLE-US-00003 TABLE 3 Statistical levels of 8-oxo 2′-deoxiguanosin in 24-hour urine (pg/ml) (mean, standard error, mean difference, P1-level of statistical significance for the difference between the levels before and after each test and P2 level of significance for the difference in the increase in levels for the placebo and probiotic). Mean S.E. Mean dif. P1 P2 Placebo Test 1 Before 10.7 2.0 12.4 .000 .620 Afterwards 23.1 3.8 Test 2 Before 11.8 2.4 11.5 .000 Afterwards 23.4 3.3 Probiotic Test 1 Before 13.3 2.0 15.7 .000 .000 Afterwards 29.0 3.7 Test 2 Before 13.6 2.4 4.8 .007 Afterwards 18.4 3.2

[0113] The following was obtained in the comparative analysis: [0114] Comparison of the values of the variable in the initial state. No significant differences were observed when comparing the values of this variable at the initial stage, so it can be said that the groups were homogeneous for this variable at the initial stage of each test. [0115] Placebo group. During the first test, a significant increase (p<0.001) in the levels of 8-oxo 2′-deoxiguanosin in 24-hour urine was observed secondary to the damage caused by high-intensity long-duration physical exercise. On performing the second test, after ingestion of the placebo product, physical exercise produced the same increase in the levels of this parameter as in test 1 (p<0.001) (FIG. 5). Therefore, it cannot be said that placebo consumption changed the trends in this variable during stress tests. [0116] Experimental group. A significant increase (15.7 pg/ml; p<0.001) in 24-hour urine 8-oxo 2′-deoxiguanosin levels was observed during the first test. On performing the second test, following the intake of the probiotic product, physical exercise resulted in a much lower increase in this parameter than in test 1, but was also significant (4.8 pg/ml; p<0.007) (FIG. 5). Comparison of the changes in this parameter in test 1 with those obtained in test 2 revealed significant differences (p<0.001), i.e. in the second test the subjects who consumed the probiotic product had a lower increase of 24-hour urine 8-oxo 2′-deoxiguanosin levels than during the first test (FIG. 5). Therefore, we can state that the consumption of the probiotic product changed the trends in this variable during the stress tests.

[0117] On comparing the trends between the two groups (FIG. 6), significant differences (p=0.001) were observed, i.e., the trends for both products can be said to differ, so it can be concluded that the 6-week intake of the probiotic product produces significant improvements compared to placebo for this variable.

[0118] In conclusion, the intake of the probiotic product of the invention for 6 weeks decreased the oxidative damage to lipids and DNA generated by high-intensity long-duration physical exercise.

[0119] High-intensity long-duration physical exercise generates oxidative damage to lipids, proteins and DNA. This can be demonstrated by analyzing different metabolites: oxidative damage to lipids causes an increase in serum malondialdehyde and an increase in serum oxidised LDL-cholesterol, and oxidative damage to DNA causes an increase in urine levels of 8-oxo 2′-deoxiguanosin. The intake of probiotic decreased the increase in serum malondialdehyde, serum oxidized chol-LDL and 8-oxo 2′-deoxiguanosin in 24-hour urine. That is to say, the results demonstrate that after ingestion of the probiotic there is a decrease in the oxidative damage to lipids and DNA caused by high-intensity long-duration physical exercise; this exercise has previously been shown to generate oxidative damage to lipids and DNA.

1.5.4. Safety Variables.

[0120] No adverse events related to the intake of the probiotic of the invention were observed in any of the study subjects. No changes were found in the blood count, liver or kidney function of the subjects evaluated. Therefore, the intake of the composition of the invention is safe.

[0121] In conclusion, the daily consumption of the probiotic of invention for 6 weeks: [0122] Decreased oxidative damage to lipids and DNA generated by high-intensity long-duration physical exercise. [0123] Improved the antioxidant status of subjects. [0124] No adverse events were observed related to its intake in any of the study subjects, no changes in liver or kidney function in the subjects evaluated, therefore it can be concluded that it is safe.