COMPOSITION OF LACTIC BACTERIA FOR USE IN THE TREATMENT OF INFECTIONS DUE TO PROPIONIBACTERIUM ACNES AND IN PARTICULAR FOR ACNE

Abstract

The present invention relates to selected strains of lactic bacteria for use in the treatment of infections caused by pathogenic bacteria belonging to the species Propionibacterium acnes, in particular for use in the preventive and/or curative treatment of dermatitis in general, seborrheic dermatitis, rosacea, eczema and acne. In addition, the present invention relates to a composition which comprises a mixture comprising or, alternatively, consisting of selected strains of lactic bacteria for use in the treatment of infections caused by pathogenic bacteria belonging to the species Propionibacterium acnes, in particular for use in the preventive and/or curative treatment of dermatitis in general, seborrheic dermatitis, rosacea, eczema and acne. The composition comprising said mixture of selected strains of lactic bacteria is formulated in a pharmaceutical form for oral use (novel food, supplement product or medical device or pharmaceutical composition) in the form of powder, dispersible powder, granulate, packets or stick, solution, spray solution, syrup, tablet, capsule, pill or lozenge; or for topical use (composition for medical device or pharmaceutical composition) in the form of cream, ointment, pomade, gel, hydrogel, dispersion, emulsion or spray to be used as such for skin applications, or for transdermal use even in the form of a band aid.

Claims

1-14. (canceled)

15. A composition comprising cells of the strain Lactobacillus salivarius LS03 (ID 1382) (deposited at the DSMZ as DSM 22776).

16. The composition of claim 15, wherein the composition is formulated as a cream, ointment or gel for topical use.

17. The composition of claim 15, wherein the composition is a transdermal patch.

18. The composition of claim 15, wherein the composition comprises live bacterial cells.

19. The composition of claim 15, wherein the composition comprises dead bacterial cells.

20. The composition of claim 15, further comprising: cells of the strain Lactobacillus delbrueckii ssp. delbrueckii LDD01 (deposited at the DSMZ as DSM 22106), cells of the strain Lactobacillus reuteri LRE04 (deposited at the DSMZ as DSM 23880), and/or cells of the strain Bifidobacterium pseudolongum ssp. globosum BPS01 (deposited at the DSMZ as DSM 26456).

21. The composition of claim 20, wherein the strain of Lactobacillus salivarius LS03 and the strain of Lactobacillus delbrueckii ssp. delbrueckii LDD01 are, respectively, in a weight ratio of about 1:1, or 2:1, or 3:1, or 4:1, or 5:1, or 1:2.

22. The composition of claim 20, wherein the strain of Lactobacillus salivarius LS03 and the strain of Lactobacillus reuteri LRE04 are, respectively, in a weight ratio of about 1:1, or 2:1, or 3:1, or 4:1, or 5:1, or 1:2.

23. The composition of claim 15, wherein said composition further comprises ammonium lactate.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG. 1 shows the results of a first soaked-disk inhibition test in which Lactobacillus salivarius LS01 (1797 S” and “1797 C”), DLV8 (“1813 C” and “1813 S”), and LS03 (“1382 C” and “1382 S”), and Lactobacillus rhamnosus LR06 (“1697 C” and “1697 S”), LR04 (“1132 C” and “1132 S”) and LR05 (“1602 C” and “1602 S”) were assayed for their ability to inhibit the growth of Propionibacterium acnes ATCC 11827.

[0034] FIG. 2 shows the results of a second soaked-disk inhibition test in which Lactobacillus reuteri strains LRE 01 (“1775”), LRE 02 (“1774”), LRE 03 (“1777”), LRE 04 (“1779”) and LRE 05 (“1807”) and Streptococcus thermophilus strains YO 2 (“Yo2”), YO 3 (“Yo3”), YO 4 (“Yo4”), YO 5 (“Yo5”), YO 6 (“Yo6”) and YO 8 (“Yo8”) were assayed for their ability to inhibit the growth of Propionibacterium acnes ATCC 11827.

[0035] FIG. 3 shows the results of a third soaked-disk inhibition test in which Lactobacillus salivarius LS03 (“1382”) and Lactobacillus rhamnosus T12 (“T12”) were assayed for their ability to inhibit the growth of Propionibacterium acnes ATCC 11827.

[0036] FIG. 4 is a bar chart showing the results of an IL-8 inhibition assay in which various strans were tested for their ability to inhibit the release of the chemokine IL-8 from peripheral blood mononuclear cells.

[0037] The Applicant tested 70 strains of bacteria for assessing their ability in modulating the activity of Th1 and Th2 lymphocytes. From a first screening, 22 of the 70 tested strains were found to have a remarkable anti-inflammatory activity (pro-Th2 strains). These 22 strains of bacteria were further studied for assessing their capability in inhibiting the release of chemokine IL-8. Chemokine IL-8 is often associated with inflammation and is increased by oxidative stress, thus resulting a key parameter for the localized inflammation: for this reason it was identified as the main proinflammatory mediator in acne.

[0038] The study showed that all the 22 strains of probiotic bacteria (Table 1) are able to significantly inhibit the release of IL-8 produced by PHA-activated PBMCs from 10 to 25%, such as BPS01 (DSM 26456) and LS04 (DSM 24618). In particular, 3 strains of probiotic bacteria: LDD01 (DSM 22106), LRE04 (DSM 23880) and LS03 (DSM 22776) are able to reducing the production of IL-8 leading to a reduction of 36%, 44% and 30%, respectively. Thus, the use of these probiotic strains, having a so remarkable action in the inhibition of IL-8, is obvious to be of primary relevance for limiting the proinflammatory activity of the chemokine at the site of inflammation and infection by Propionibacterium acnes.

[0039] It is an object of the present invention a strain of bacteria belonging to the species Lactobacillus salivarius for use in the preventive and/or curative treatment of infections and/or inflammations caused by pathogenic bacteria belonging to the species Propionibacterium acnes; said strain of bacteria belonging to the species Lactobacillus salivarius is isolated and identified as Lactobacillus salivarius LS04-DSM 24618 deposited on 2 Mar. 2011 at DSMZ by Probiotical SpA (Italy).

[0040] It is an object of the present invention a strain of bacteria belonging to the species Lactobacillus delbrueckii ssp. delbrueckii for use in the preventive and/or curative treatment of infections and/or inflammations caused by pathogenic bacteria belonging to the species Propionibacterium acnes; said strain of bacteria belonging to the species Lactobacillus delbrueckii ssp. delbrueckii is isolated and identified as Lactobacillus delbrueckii ssp. delbrueckii LDD01 (DSMZ 20074)-DSM 22106 deposited on 10 Dec. 2008 at DSMZ by Probiotical SpA (Italy).

[0041] It is an object of the present invention a strain of bacteria belonging to the species Lactobacillus reuteri for use in the preventive and/or curative treatment of infections and/or inflammations caused by pathogenic bacteria belonging to the species Propionibacterium acnes; said strain of bacteria belonging to the species Lactobacillus reuteri is isolated and identified as Lactobacillus reuteri LRE04-DSM 23880 deposited on 5 Aug. 2010 at DSMZ by Probiotical SpA (Italy).

[0042] It is an object of the present invention a strain of bacteria belonging to the species Bifidobacterium pseudolongum ssp. globosum for use in the preventive and/or curative treatment of infections and/or inflammations caused by pathogenic bacteria belonging to the species Propionibacterium acnes; said strain of bacteria belonging to the species Bifidobacterium pseudolongum ssp. globosum is isolated and identified as Bifidobacterium pseudolongum ssp. globosum BPS01-DSM 26456 deposited on 2 Oct. 2012 at DSMZ by Probiotical SpA (Italy).

[0043] It is an object of the present invention a composition comprising an effective amount of a mixture, which comprises or, alternatively, consists of:

[0044] (i) at least a strain of bacteria belonging to the species Lactobacillus salivarius selected from the group comprising or, alternatively, consisting of the strain Lactobacillus salivarius LS03-DSM 22776, the strain Lactobacillus salivarius LS04-DSM 24618 and/or mixtures thereof; and/or

[0045] (ii) at least a strain of bacteria selected from the group comprising or, alternatively, consisting of the strain Lactobacillus delbrueckii ssp. delbrueckii LDD01 (DSMZ 20074)-DSM 22106, the strain Lactobacillus reuteri LRE04-DSM 23880 and/or the strain Bifidobacterium pseudolongum ssp. globosum BPS01-DSM 26456; and a physiologically acceptable carrier;

[0046] said composition being for use in the preventive and/or curative treatment of infections and/or inflammations caused by pathogenic bacteria belonging to the species Propionibacterium acnes. In an embodiment, said compositions for use of the present invention further comprise also an ammonium lactate, preferably of fermentative origin from lactic bacteria.

[0047] It is an object of the present invention a composition comprising an effective amount of a mixture, which comprises or, alternatively, consists of:

[0048] (i) at least a strain of bacteria belonging to the species Lactobacillus salivarius selected from the group comprising or, alternatively, consisting of the strain Lactobacillus salivarius LS03-DSM 22776; and/or

[0049] (ii) at least a strain of bacteria selected from the group comprising or, alternatively, consisting of the strain Lactobacillus delbrueckii ssp. delbrueckii LDD01 (DSMZ 20074)-DSM 22106 and the strain Lactobacillus reuteri LRE04-DSM 23880; and a physiologically acceptable carrier;

[0050] said composition being for use in the preventive and/or curative treatment of infections and/or inflammations caused by pathogenic bacteria belonging to the species Propionibacterium acnes. In an embodiment, said compositions for use of the present invention further comprise also an ammonium lactate, preferably of fermentative origin from lactic bacteria.

[0051] Advantageously, said mixture and, thus, said composition comprise or, alternatively, consist of: [0052] a strain Lactobacillus salivarius LS03-DSM 22776; and/or [0053] a strain Lactobacillus delbrueckii ssp. delbrueckii LDD01 (DSMZ 20074)-DSM 22106; and a physiologically acceptable carrier;

[0054] said composition being for use in the preventive and/or curative treatment of infections and/or inflammations caused by pathogenic bacteria belonging to the species Propionibacterium acnes. Said strains of bacteria are preferably in a weight ratio of about 1:1, or 2:1, or 3:1, or 4:1, or 5:1, 1:2. In an embodiment, said compositions for use of the present invention further comprise also an ammonium lactate, preferably of fermentative origin from lactic bacteria.

[0055] Advantageously, said mixture and, thus, said composition comprise or, alternatively, consist of: [0056] a strain Lactobacillus salivarius LS03-DSM 22776; and/or [0057] a strain Lactobacillus reuteri LRE04-DSM 23880; and a physiologically acceptable carrier;

[0058] said composition being for use in the preventive and/or curative treatment of infections and/or inflammations caused by pathogenic bacteria belonging to the species Propionibacterium acne. Said strains of bacteria are preferably in a weight ratio of about 1:1, or 2:1, or 3:1, or 4:1, or 5:1, 1:2. In an embodiment, said compositions for use of the present invention further comprise also an ammonium lactate, preferably of fermentative origin from lactic bacteria.

[0059] Advantageously, said mixture and, thus, said composition comprise or, alternatively, consist of: [0060] a strain Lactobacillus salivarius LS03-DSM 22776; and [0061] a strain Lactobacillus delbrueckii ssp. delbrueckii LDD01 (DSMZ 20074)-DSM 22106; and [0062] a strain Lactobacillus reuteri LRE04-DSM 23880; and a physiologically acceptable carrier;

[0063] said composition being for use in the preventive and/or curative treatment of infections and/or inflammations caused by pathogenic bacteria belonging to the species Propionibacterium acnes. Said strains of bacteria are preferably in a weight ratio of about 1:1:1, or 2:1:1, or 3:1:1, or 4:1:1, or 5:1:1, 1:2:1, or 1:1:2. In an embodiment, said compositions for use of the present invention further comprise also an ammonium lactate, preferably of fermentative origin from lactic bacteria.

[0064] The compositions of the present invention are effectively applied for use in the treatment of pimples or furuncles, preferably occurring in the face, shoulders, back, and upper part of the chest.

[0065] In said mixtures, said at least a strain of bacteria, or the combination of said strains of Lactobacilli/Bifidobacteria of the present invention, is in a total amount comprised from 1×10.sup.6 to 1×10.sup.12 CFU/g of mixture; preferably, from 1×10.sup.7 to 1×10.sup.11 CFU/g of mixture; more preferably, from 1×10.sup.8 to 1×10.sup.10 CFU/g of mixture. In an embodiment, said compositions for use of the present invention further comprise also an ammonium lactate, preferably of fermentative origin from lactic bacteria, in an amount comprised from 1 to 30% by weight, relative to the weight of the composition, preferably in an amount comprised from 5 to 15% by weight, relative to the weight of the composition.

[0066] In said compositions for use, said at least a strain of bacteria, or the combination of said strains of Lactobacilli/Bifidobacteria of the present invention, is in a total amount comprised from 1×10.sup.6 to 1×10.sup.11 CFU/g of composition; preferably, from 1×10.sup.7 to 1×10.sup.10 CFU/g of composition; more preferably, from 1×10.sup.8 to 1×10.sup.9 CFU/g of composition.

[0067] Said compositions for use can further comprise also the required/suitable amounts of co-formulants, excipients, carriers, surfactants, adjuvants, preservatives, coloring agents, as needed. Said substances are suitably quanti- and qualitatively selected among those known and commonly used by the pharmaceutical technician skilled in the formulation field.

[0068] The composition of the present invention comprising said mixture of selected strains of lactic bacteria is formulated in a pharmaceutical form for oral use (novel food, supplement product or medical device or pharmaceutical composition) in the form of powder, dispersible powder, granulate, packets or stick, solution, spray solution, syrup, tablet, capsule, pill or lozenge; or for topical use (composition for medical device or pharmaceutical composition) in the form of cream, ointment, pomade, gel, hydrogel, dispersion, emulsion or spray to be used as such for skin applications, or for transdermal use also in the form of band aid.

[0069] The above-cited strains of bacteria are in the mixture of the present invention in an amount comprised from 1% to 50% by weight, preferably from 5% to 40% by weight, even more preferably from 10% to 30%, relative to the total weight of the mixture. However, said percentage depends on the kind of the pharmaceutical form intended to be used. For example, in the case of capsules, the amount of said bacteria is greater than 25%, for example greater than 35%.

[0070] In an embodiment, the composition comprises strains of bacteria in a concentration comprised from 1×10.sup.6 to 1×10.sup.11 CFU/dose, preferably from 1×10.sup.8 CFU/dose to 1×10.sup.10 CFU/dose. The dose can be comprised from 0.2 to 10 g, for example can be 0.25 g, 1 g, 3 g, 5 g or 7 g. The strains of bacteria can be present in the composition in a solid form, for example in the form of powder, dry powder, or freeze-dried powder.

[0071] All the strains described and/or claimed in the present patent application were deposited according to the Budapest Treaty.

EXPERIMENTAL PART

1. Inhibition Tests for Bacterium Propionibacterium acnes ATCC 11827

[0072] Inhibition tests for the publicly available bacterium Propionibacterium acnes ATCC 11827 were conducted.

[0073] The intense research activity made by the Applicant focused on detecting, selecting and isolating one or more strains of lactic bacteria able to inhibiting the pathogenic bacterium belonging to the species Propionibacterium acnes.

[0074] For this reason, a large set of screening tests was set-up for a number of strains of bacteria belonging to the species Lactobacillus salivarius and Lactobacillus rhamnosus. The plate inhibition technique was used.

[0075] The pathogen Propionibacterium acnes (ATCC 11827) was used, by preparing a culture broth thereof. The pathogenic bacterium was grown in BHI (Brain Hearth Infusion) medium, supplemented with 1% glucose, up to a density of about 2 of McFarland's scale, and then diluted 1:10 in saline. Such a suspension was plated on Reimbold medium.

[0076] The Applicant started the experimental activity on a first large group of strains of bacteria belonging to the species Lactobacillus salivarius and Lactobacillus rhamnosus in order to obtain a second selected group of potential candidate strains. Next, the Applicant carried out a further selection and finally tested the following strains of bacteria, showing a remarkable activity against the target pathogenic strain:

[0077] Lactobacillus salivarius LS01 (ID 1797)-DSM 22775 deposited on 23 Jul. 2009 at DSMZ (*);

[0078] Lactobacillus salivarius DLV8 (ID 1813)-DSM 25545 deposited on 12 Jan. 2012 at DSMZ (*);

[0079] Lactobacillus salivarius LS03 (ID 1382)-DSM 22776 deposited on 23 Jul. 2009 at DSMZ (*);

[0080] Lactobacillus rhamnosus LR06 (ID 1697)-DSM 21981 deposited on 14 Nov. 2008 at DSMZ (*);

[0081] Lactobacillus rhamnosus LR04 (ID 1132)-DSM 16605 deposited on 20 Jul. 2004 at DSMZ by Anidral Srl (Italy), currently Probiotical SpA (Italy);

[0082] Lactobacillus rhamnosus LR05 (ID 1602)-DSM 19739 deposited on 27 Sep. 2007 at DSMZ by Anidral Srl (Italy), currently Probiotical SpA (Italy).

[0083] All the above (*) were deposited by Probiotical SpA (Italy).

[0084] Such bacteria were revitalized through two sequential inoculations in MRS broth, centrifuged and resuspended in fresh medium (v/v), —indicated as C. The supernatant was neutralized and filtered, —indicated as S.

[0085] The inhibition test was then carried out by the soaked-disk technique as shown in FIG. 1.

[0086] The above test immediately showed a surprising and greater inhibition ability of the strain Lactobacillus salivarius LS03 (ID 1382)-DSM 22776 deposited on 23 Jul 2009 at DSMZ against the target pathogenic strain.

[0087] Said inhibition activity is due to the ability of the strain Lactobacillus salivarius LS03 (ID 1382)-DSM 22776 deposited on 23 Jul. 2009 at DSMZ to producing bacteriocins very active against the pathogen P. acnes. It can reasonably be excluded that only an effect due to the acidity and/or the possible production of hydrogen peroxide occurs. The former reason is not contemplated since the other bacteria having similar acidifying activities had no effect on the growth of the target pathogen; the second reason is not contemplated since plate incubation occurred under anaerobiosis, a condition impeding the formation of hydrogen peroxide by lactobacilli.

[0088] Based on this result, the study was extended to other possible candidates, and the obtained result was further confirmed with the strain Lactobacillus salivarius LS03 (ID 1382)-DSM 22776 deposited on 23 Jul. 2009 at DSMZ.

[0089] Next, the following strains of bacteria were tested in a second test:

[0090] Lactobacillus reuteri LRE 01 (ID 1775)-DSM 23877 deposited on 5 Aug. 2010 at DSMZ;

[0091] Lactobacillus reuteri LRE 02 (ID 1774)-DSM 23878 deposited on 5 Aug. 2010 at DSMZ;

[0092] Lactobacillus reuteri LRE 03 (ID 1777)-DSM 23879 deposited on 5 Aug. 2010 at DSMZ;

[0093] Lactobacillus reuteri LRE 04 (ID 1779)-DSM 23880 deposited on 5 Aug. 2010 at DSMZ;

[0094] Lactobacillus reuteri LRE 05 (ID 1807)-DSM 25139 deposited on 2 Sep. 2011 at DSMZ.

[0095] All the above were deposited by Probiotical SpA (Italy).

[0096] Since in literature some Streptococci thermophilus were found to be able to produce broad-spectrum bacteriocins, we also included in the test some of the strains of bacteria belonging to the species S. thermophilus:

[0097] Streptococcus thermophilus YO 2 (1427)-DSM 16590 deposited on 20 Jul. 2004 at DSMZ;

[0098] Streptococcus thermophilus YO 3 (1286)-DSM 16591 deposited on 20 Jul. 2004 at DSMZ;

[0099] Streptococcus thermophilus YO 4 (1288)-DSM 16592 deposited on 20 Jul. 2004 at DSMZ;

[0100] Streptococcus thermophilus YO 5 (1405)-DSM 16593 deposited on 20 Jul. 2004 at DSMZ.

[0101] All the above were deposited by Anidral Srl (Italy), currently Probiotical SpA (Italy).

[0102] We carried out the screening of the bacterial resuspensions as described in the first experiment, thus obtaining the following results as shown in FIG. 2.

[0103] In this second screening of the strains, no useful results were obtained, further confirming the exceptional nature of the bacteriocin activity of the strain of the present invention LS03.

[0104] Then, a new test with the strain LS03 was conducted, in order to corroborate such an inhibition activity against the target pathogenic strain.

[0105] This second test served as a comparison with the strain Lactobacillus rhamnosus T12 (ID 1738) LMG P-25211 (WO2011/029784) as positive control, being part of a commercially available anti-acne formulation called Lichtena Norma-Acn®.

[0106] After growing the strains of bacteria, as described in the first test, the broth cultures were divided into two aliquots; a first broth culture aliquot was neutralized prior to the supernatant removal by centrifugation; whereas the other second aliquot was first centrifuged and than the supernatant only was neutralized.

[0107] In literature some bacteriocins in fact were found to adhere or not to the cell membrane of the pathogen depending on the acidic pH.

[0108] The tested samples were thus 4 for either of the two tested strains: (Lactobacillus rhamnosus T12 (ID 1738) LMG P-25211 and Lactobacillus salivarius LS03 (ID 1382)-DSM 22776). Results are shown in FIG. 3.

[0109] Again, the strain Lactobacillus salivarius LS03 (ID 1382)-DSM 22776 deposited on 23 Jul. 2009 at DSMZ exhibits a better inhibition ability than the strain Lactobacillus rhamnosus T12 LMG P-25211 (Lichtena Norma-Acn®) used as control.

[0110] The bacteriocin produced by the strain of bacteria LS03 shows adhesive abilities depending on the pH and, thus, is able to exert its inhibitory action in different contexts. The activity in an acidic environment alone would not be completely useful.

2. Assessment of the Ability of Probiotic Strains to Inhibit the IL-8 Release in PBMCs (Peripheral Blood Mononuclear Cells)

2.1 Aim

[0111] In this study 70 strains of probiotic bacteria were tested for assessing their ability in modulating the activity of lymphocytes Th1 and Th2. From a first screening of the 70 tested strains, 22 of them (Table 1) were found to have a remarkable anti-inflammatory activity (pro-Th2 strains).

[0112] These 22 strains of probiotic bacteria (Table 1) were further studied for assessing their ability in inhibiting the release of chemokine IL-8 (FIG. 4). Chemokine IL-8 is often associated with inflammation and is increased by oxidative stress, thus resulting a key parameter for the localized inflammation: for this reason it was identified as the main proinflammatory mediator in acne.

[0113] In this study the ability of selected strains of probiotic bacteria (Table 1) in inhibiting the release of IL-8 by PBMCs was analyzed. IL-8 is a typical proinflammatory chemokine overproduced in acne.

TABLE-US-00001 TABLE 1 PRO-TH2 STRAINS LISTING Commercial Deposit NAME abbreviation number Deposit date Lactobacillus acidophilus LA02 DSM 6 Aug. 2008 21717 Lactobacillus delbrueckii ssp. LDD01 DSM 10 Dec. 2008 delbrueckii 22106 Lactobacillus fermentum LF09 DSM 24 May 2006 18298 Lactobacillus fermentum LF10 DSM 20 Mar. 2007 19187 Lactobacillus plantarum 476LL LP01 LMG P- 16 Oct. 2001 20 bi 21021 Lactobacillus plantarum 776/1 LP02 LMG P- 16 Oct. 2001 bi 21020 Lactobacillus reuteri LRE02 DSM 5 Aug. 2010 23878 Lactobacillus reuteri LRE04 DSM 5 Aug. 2010 23880 Lactobacillus reuteri DLLRE08 DSM 16 Feb. 2012 25684 Lactobacillus reuteri DLLRE09 DSM 16 Feb. 2012 25685 Lactobacillus reuteri DLLRE07 DSM 16 Feb. 2012 25683 Lactobacillus rahmnosus LR06 DSM 14 Nov. 2008 21981 Lactobacillus salivarius LS01 DSM 23 Jul. 2009 22775 Lactobacillus salivarius LS04 DSM 2 Mar. 2011 24618 Lactobacillus salivarius LS03 DSM 23 Jul. 2009 22776 Lactobacillus salivarius DLV1 DSM 2 Sep. 2011 25138 Lactobacillus salivarius LS05 DSM 6 Jun. 2012 26036 Lactobacillus salivarius LS02 DSM 13 Nov. 2015 32204 Bifidobacterium adolescentis BA05 DSM 15 Jun. 2006 EI-18 18352 Bifidobacterium breve BR03 DSM 20 Jul. 2004 16604 Bifidobacterium pseudolongum BPS01 DSM 2 Oct. 2012 ssp. globosum 26456 Bifidobacterium longum B1975 DSM 7 Apr. 2011 24709

2.2 Method: PBMC Stimulation

[0114] Isolating PBMCs from peripheral blood of healthy subjects according to protocol FICOLL. [0115] Pre-incubating PBMCs with PHA (phytohemagglutinin) 1 μg/ml for 1 hour. [0116] Stimulating PBMCs with the probiotic strains for 24 hours. [0117] Collecting supernatants after centrifugation at 10000 rpm for 5 minutes and storing at −80° C. until use. [0118] Assessing the IL-8 release with the E.L.I.S.A. kit (eBioscience, Human IL-8 ELISA Ready-Set-Go, 2nd generation). [0119] Analyzing the data and statistical assessing carried out with a paired t-test, data are considered significant for p<0.05 values.

2.3 Method: ELISA Protocol for Assessing IL-8

[0120] Plate coating with 1× Coating Buffer, incubating at 4° C. overnight; [0121] Washing with Wash Buffer (3×); [0122] Dispensing 1× Blocking Buffer in each well and incubating for 1 hour at room temperature; [0123] Washing with Wash buffer (1×); [0124] Dispensing the samples and standard curve and incubating for 2 hours at room temperature; [0125] Washing with Wash Buffer (5×); [0126] Dispensing in each well the Detection Antibody Buffer and incubating for 1 hour at room temperature; [0127] Washing with Wash Buffer (5×); [0128] Dispensing in each well Avidin-HRP Buffer and incubating for 30 minutes at room temperature; [0129] Washing with Wash Buffer (7×); [0130] Incubating for 15 minutes with 1×TMB Solution and interrupting with Stop Solution; [0131] Spectrophotometric reading at 450 nm.

2.4 Results

[0132] Results for the chemokine IL-8 assay after 1-hour pre-stimulation with PHA followed by 24-hour incubation with selected strains of probiotic bacteria Th2 (Table 1) and subsequent assessment with the technique ELISA are shown in FIG. 4.

[0133] In FIG. 4, the value “0” (zero) relates to the reference value of the inflammatory status. Basically, it represents the PHA-activated cells. It is the stimulus of choice for the IL-8 release (it represents the maximum production of IL-8=positive control). Whereas the value “−0.25%” represents a reduction of 25% of IL-8 release.

2.5 Conclusions

[0134] From the study, all the 22 strains of probiotic bacteria (Table 1) were found to be able to significantly inhibit the release of IL-8 produced by PHA-activated PBMCs from 10 to 25%, such as BPS01 (DSM 26456) and LS04 (DSM 24618). In particular, 3 strains of probiotic bacteria: LDD01 (DSM 22106), LRE04 (DSM 23880) and LS03 (DSM 22776) are able to reducing the IL-8 production achieving a reduction equal to 36%, 44% and 30%, respectively.

[0135] It is thus obvious that the use of these probiotic strains, with a so remarkable action in inhibiting IL-8 has a fundamental relevance for limiting the proinflammatory action of chemokine in the site of inflammation and infection by Propionibacterium acnes.