Composition for the treatment of dysbiosis of the intestinal microbiota

Abstract

A pharmaceutical composition comprising at least a probiotic and at least a carotene, for the treatment of dysbiosis of the intestinal microbiota, is disclosed. The association of these ingredients allowed to obtain a clear synergistic effect.

Claims

1. A pharmaceutical daily unitary dose for treating dysbiosis of intestinal microbiota, said pharmaceutical unitary dose comprising at least a probiotic and at least a carotene, wherein said at least a probiotic is a microorganism belonging to the species Bacillus coagulans, Bacillus clausii, Lactobacillus reuteri, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Bifidobacterium longum, or a combination thereof in an amount of at least 1×10.sup.8CFU and not higher than 5×10.sup.9CFU and said at least a carotene is in an amount of from 1 mg to no more than 30 mg.

2. The pharmaceutical unitary dose of claim 1, wherein said at least a probiotic is a microorganism belonging to the species Bacillus Coagulans, Lactobacillus Plantarum, Lactobacillus Rhamnosus, or a combination thereof.

3. The pharmaceutical unitary dose of claim 1, wherein said at least a probiotic is in an amount of at least 1×10.sup.9CFU.

4. The pharmaceutical unitary dose of claim 1, wherein said at least a probiotic is in an amount of 1.2×10.sup.9CFU to 5×10.sup.9CFU.

5. The pharmaceutical unitary dose of claim 1, wherein said at least a carotene is α-carotene, β-carotene, γ-carotene, δ-carotene, ε-carotene, lycopene, or a mixture thereof.

6. The pharmaceutical unitary dose of claim 1, wherein said at least a carotene is in an amount of from 1 mg to no more than 25 mg.

7. The pharmaceutical unitary dose of claim 1, wherein said at least a probiotic is in an amount of 1.5×10.sup.9CFU to 4×10.sup.9CFU and said at least a carotene is in an amount of from 1 mg to no more than 20 mg.

8. The pharmaceutical unitary dose of claim 1 further comprising at least a xanthophyll in an amount of from 0.1 mg to 1 mg.

9. The pharmaceutical composition of claim 8, wherein said at least a xanthophyll is astaxanthin, lutein, zeaxanthin, violaxanthin or a mixture thereof.

10. The pharmaceutical unitary dose of claim 1, wherein said at least a carotene is βcarotene.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The characteristics and the advantages of the present invention will become apparent from the following detailed description, the working examples provided for illustrative purposes and the accompanying figures, wherein:

(2) FIG. 1 shows the results of the probiotic incubation with beta-carotene, in terms of abscisic acid production, as per Example 1;

(3) FIG. 2 shows the results of the probiotic incubation with different carotenoids, in terms of abscisic acid production, as per Example 1;

(4) FIG. 3 shows the results of the probiotic incubation with a carotene and a xanthophyll, in terms of abscisic acid production, as per Example 1;

(5) FIG. 4 shows of the in vivo experimentation on healthy volunteers, as per Example 1.

DETAILED DESCRIPTION OF THE INVENTION

(6) The subject of the invention therefore is a pharmaceutical unitary dose comprising at least a probiotic and at least a carotene, wherein said at least a probiotic is in an amount of at least 1×10.sup.8CFU and said at least a carotene is in an amount not higher than 30 mg.

(7) Preferably, said pharmaceutical unitary dose is a pharmaceutical daily unitary dose.

(8) For the purposes of the present invention, said at least a probiotic is a microorganism belonging to the species Bacillus Coagulans, Bacillus Clausii, Lactobacillus Reuteri, Lactobacillus Plantarum, Lactobacillus Rhamnosus, Lactobacillus Casei, Bifidobacterium Longum, or a combination thereof.

(9) Preferably, said at least a probiotic is a microorganism belonging to the species Bacillus Coagulans, Lactobacillus Plantarum, Lactobacillus Rhamnosus, or a combination thereof.

(10) Preferably, said at least a probiotic is in an amount of at least 1×10.sup.9CFU.

(11) Preferably, said at least a probiotic is in an amount not higher than 5×10.sup.9CFU.

(12) More preferably, said at least a probiotic is in an amount of 1.2×10.sup.9CFU to 5×10.sup.9CFU, more preferably 1.5×10.sup.9CFU to 4×10.sup.9CFU.

(13) Said at least a carotene is a carotenoid containing no oxygen, being α-carotene, β-carotene, γ-carotene, δ-carotene, ε-carotene, lycopene, lycopersene, phytofluene, hexahydrolycopene, torulene, zeacarotene, or a mixture thereof.

(14) Preferably, said at least a carotene is α-carotene, β-carotene, γ-carotene, δ-carotene, ε-carotene, lycopene, or a mixture thereof.

(15) In preferred embodiment, said at least a carotene is β-carotene.

(16) Preferably, and said at least a carotene is in an amount not higher than 25 mg.

(17) More preferably, said at least a carotene is in an amount not higher than 20 mg, more preferably not higher than 10 mg.

(18) In preferred embodiments, said at least a carotene is in an amount of 3-7 mg.

(19) In preferred embodiments, the pharmaceutical composition comprises liposomes carrying at least a carotene.

(20) Preferably, said liposomes comprise phospholipids selected from natural phospholipids, phosphatidic acid, phosphatidylcholine, phosphatidylglycerol, phosphatidyl-ethanolamine, phosphatidylserine, PEG-phospholipid, or mixtures thereof.

(21) More preferably, said liposomes comprise phospholipids selected from soybean phospholipid, egg phospholipid, sunflower phospholipids, egg phosphatidylcholine, soy phosphatidylcholine, hydrogenated soy phosphatidylcholine, phosphatidylcholine sunflower, sphingomyelin, didecanoyl phosphatidylcholine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleyl phosphatidylcholine, palmitoyl-oleyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, dierucoyl phosphatidylcholine, didecanoyl phosphatidylglycerol, dilauroyl phosphatidylglycerol, dimyristoyl phosphatidylglycerol, dipalmitoyl phosphatidylglycerol, distearoyl phosphatidylglycerol, dioleyl phosphatidyl glycerol, palmitoyl-oleyl phosphatidylglycerol, dilinoleoyl phosphatidylglycerol, dierucoyl phosphatidylglycerol, didecanoyl phosphatidylethanol-amine, dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidyl-ethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleyl phosphatidylethanolamine, palmitoyl-oleyl phosphatidylethanolamine, dilinoleoyl phosphatidylethanolamine, dierucoyl phosphatidylethanolamine, dilauroyl phosphatidyl-serine, dimyristoyl phosphatidylserine, dipalmitoyl phosphatidylserine, distearoyl phosphatidylserine, dioleyl phosphatidylserine, dilinoleoyl phosphatidylserine, or a mixture thereof.

(22) In preferred embodiments, said liposomes comprise lecithin, which is a mixture of glycerophospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid.

(23) Preferably, said at least a probiotic is in an amount of 1×10.sup.8CFU to 5×10.sup.9CFU and said at least a carotene is in an amount not higher than 25 mg.

(24) Preferably, said at least a probiotic is in an amount of at least 1×10.sup.9CFU and said at least a carotene is in an amount not higher than 25 mg, more preferably said at least a probiotic is in an amount of 1.2×10.sup.9CFU to 5×10.sup.9CFU and said at least a carotene is in an amount not higher than 20 mg.

(25) In particularly preferred embodiments, the pharmaceutical unitary dose comprises at least a probiotic belonging to the species Bacillus Coagulans, Lactobacillus Plantarum, Lactobacillus Rhamnosus, or a combination thereof, in an amount of 1×10.sup.8CFU to 5×10.sup.9CFU and β-carotene in an amount not higher than 10 mg.

(26) In a particularly preferred embodiment, the pharmaceutical unitary dose comprises at least a probiotic belonging to the species Bacillus Coagulans, Lactobacillus Plantarum, Lactobacillus Rhamnosus, or a combination thereof, in an amount of 1.2×10.sup.9CFU to 2×10.sup.9CFU and β-carotene in an amount of 3-7 mg.

(27) In a further aspect, the present invention concerns the use of the pharmaceutical unitary dose for the treatment of dysbiosis of the intestinal microbiota, and pathologies ascribable thereto, wherein said at least a probiotic in the presence of at least a carotene produces a therapeutically effective amount of abscisic acid.

(28) As it will be demonstrated in the following Examples, firstly, it has been surprisingly found that the probiotic of the invention is able to produce abscisic acid (ABA), which, as explained above, improves the metabolic parameters that are altered under conditions of dysbiosis and pathologies ascribable thereto. In this regard, the present invention also concerns a probiotic for the endogenous production of abscisic acid for use in the treatment of dysbiosis of the intestinal microbiota, and pathologies ascribable thereto.

(29) Secondly, it was unexpectedly found that a probiotic in the presence of a carotene and at the indicated amounts, produces abscisic acid at significantly higher levels than those observed for the probiotic alone at the same conditions. The results reported below clearly show that an unexpected synergistic effect is obtained.

(30) It is known that high concentrations of glucose stimulate β-pancreatic cells to release abscisic acid, which in turn induces the release of insulin from the same. In addition, the plasma concentration of abscisic acid is increased in humans after a surfeit of glucose, indicating that abscisic acid is in effect an endogenous human hormone. It has been experimentally demonstrated that, even at extremely low doses, abscisic acid has an ameliorating effect on glycaemic control without however increasing insulin secretion and therefore without representing a potential harm to the population of pancreatic beta-cells, in contrast to current therapeutic strategies.

(31) The effect observed by the above-described pharmaceutical unitary dose is particularly advantageous, since it allows the disadvantages of the prior art to be solved, including those related to conventional hypoglycaemic therapies, which cause an undesired increase in insulin secretion. This effect is also unexpected, given that abscisic acid induces β-pancreatic cells in vitro to release insulin after administration of high concentrations of glucose.

(32) As discussed in the State of the Art, perturbation of the gut microbiota composition results in higher intestinal permeability, allowing bacterial endotoxins to enter the bloodstream; this triggers a systemic low-grade inflammation, which in turn leads to insulin-resistance, hyperglycemia, dyslipidemia and concurs to body fat accumulation (obesity). The results reported in the Examples below indicate that the daily intake of above-described pharmaceutical unitary dose significantly ameliorates most of the metabolic markers altered in a condition of dysbiosis and causing T2D and metabolic syndrome. In particular, the significant reduction of the hs-CRP, a marker of low-grade systemic inflammation, suggests a beneficial action of the probiotic combined with a carotene in lowering gut permeability. As a consequence, a significant improvement of total cholesterol, HDL, LDL, TG, BMI and waist circumference are observed. Moreover, the production of abscisic acid by the probiotic combined with a carotene allows the autocrine stimulation of the probiotic proliferation, thus contributing to its colonization of the gut. The production of abscisic acid by the probiotic combined with a carotene also allows the already reported beneficial systemic effect of abscisic acid on glycemia regulation. Indeed, intake of the probiotic combined with a carotene increases basal plasma ABA 2.3-fold in all subjects, thus reaching concentrations in the range of activity having beneficial effects on hyperglycemia.

(33) Among the pathologies ascribable to dysbiosis of the intestinal microbiota, the following can be mentioned: insulin-resistance, inflammatory bowel disease, irritable bowel syndrome (IBS), and coeliac disease, allergy, asthma, non-insulin-dependent diabetes (NIDDM), syndrome X, obesity, polycystic ovarian disorder (PCOS), hair-AN syndrome, AIDS wasting, intra-uterine growth failure, post-natal growth failure, Prader-Willi syndrome, type 2 diabetes, diabetic complications, hyperglycaemia, dyslipidaemia, metabolic syndrome, hypertension, cardiovascular disease.

(34) In a further aspect, the present invention concerns abscisic acid for use in the treatment of dysbiosis of the intestinal microbiota, and pathologies ascribable thereto.

(35) In another aspect, the present invention concerns a pharmaceutical composition comprising at least a probiotic, at least a carotene, and at least a xanthophyll.

(36) Said at least a xanthophyll is a carotenoid containing oxygen.

(37) A xanthophyll can be alloxanthin, cynthiaxanthin, pectenoxanthin, β-cryptoxanthin, cryptomonaxanthin, crustaxanthin, gazaniaxanthin, HO-chlorobactene, loroxanthin, lutein, lycoxanthin, rhodopin, rhodopinol, saproxanthin, zeaxanthin, oscillaxanthin, phleixanthophyll, rhodovibrin, spheroidene, diadinoxanthin, luteoxanthin, mutatoxanthin, citroxanthin, zeaxanthin furanoxide, violaxanthin, neochrome, foliachrome, trollichrome, vaucheriaxanthin, rhodopinal, warmingone, torularhodinaldehyde, torularhodin, torularhodin methyl ester, astacene, astaxanthin, canthaxanthin, capsanthin, capsorubin, cryptocapsin, 2,2′-diketospirilloxanthin, echinenone, 3′-hydroxyechinenone, flexixanthin, 3-HO-canthaxanthin, hydroxyspheriodenone, okenone, pectenolone, phoeniconone, phoenicopterone, rubixanthone, siphonaxanthin, astacein, fucoxanthin, isofucoxanthin, physalien, siphonein, β-apo-2′-carotenal, apo-2-lycopenal, apo-6′-lycopenal, azafrinaldehyde, bixin, citranaxanthin, crocetin, crocetinsemialdehyde, crocin, hopkinsiaxanthin, methyl apo-6′-lycopenoate, paracentrone, sintaxanthin, actinioerythrin, β-carotenone, peridinin, pyrrhoxanthininol, semi-α-carotenone, semi-β-carotenone, triphasiaxanthin, eschscholtzxanthin, eschscholtzxanthone, rhodoxanthin, tangeraxanthin, nonaprenoxanthin, decaprenoxanthin, bacterioruberin or a mixture thereof.

(38) Preferably, said at least a xanthophyll is alloxanthin, cynthiaxanthin, pectenoxanthin, β-cryptoxanthin, cryptomonaxanthin, crustaxanthin, gazaniaxanthin, HO-chlorobactene, loroxanthin, lutein, lycoxanthin, rhodopin, rhodopinol, saproxanthin, astaxanthin, zeaxanthin, violaxanthin, or a mixture thereof.

(39) More preferably, said at least a xanthophyll is astaxanthin, lutein, zeaxanthin, violaxanthin, or a mixture thereof.

(40) Preferably, said at least a xanthophyll is in an amount not higher than 1 mg.

(41) In preferred embodiments, said at least a xanthophyll is in an amount of 0.05-0.5 mg.

(42) In more preferred embodiments, said at least a xanthophyll is in an amount of about 0.1 mg.

(43) In preferred embodiments, the pharmaceutical composition comprises liposomes carrying at least a carotene, at least a xanthophyll, or a combination thereof.

(44) In a further aspect, the present invention concerns the use of the pharmaceutical composition for the treatment of dysbiosis of the intestinal microbiota, and pathologies ascribable thereto, wherein said at least a probiotic in the presence of at least a carotene produces a therapeutically effective amount of abscisic acid.

(45) As it will be clear from the working Examples given below, it was observed that the presence of a xanthophyll alone significantly reduces the production of abscisic acid by the probiotic. Conversely and unexpectedly, when the probiotic is in the presence of at least a carotene and at least a xanthophyll, the production of abscisic acid by the probiotic is drastically increased. The results reported below clearly show that an unexpected synergistic effect is obtained.

(46) In an additional aspect, the present invention concerns a food supplement comprising the pharmaceutical unitary dose or the pharmaceutical composition, and suitable food ingredients.

(47) The pharmaceutical unitary dose of the invention, as well as the pharmaceutical composition and the food supplement, may further comprise pharmaceutically acceptable excipients. By “excipient” it is meant a compound or a mixture thereof suitable for the use in a pharmaceutical or food formulation. For example, an excipient for use in a pharmaceutical formulation generally should not cause an adverse reaction in a subject, nor it should significantly inhibit the efficacy of the active principles.

(48) Suitable excipients are acidifying agents, acidity correctors, anti-agglomerants, antioxidants, fillers, resistance agents, gelling agents, coating agents, modified starches, sequestering agents, thickeners, sweeteners, thinners, solvents, disaggregating agents, glidants, dyes, binders, lubricants, stabilizers, adsorbents, preservatives, wetting agents, flavors, film-forming substances, emulsifiers, wetting agents, release retardants and mixtures thereof.

(49) Preferably, said excipients are starch, modified starch, cellulose, modified cellulose, microcrystalline cellulose, sodium carboxymethylcellulose, pectin, tragacanth, mannitol, dicalcium phosphate, xanthan gum, carrageenan, sodium alginate, guar gum, maltodextrin, silicon dioxide, or mixtures thereof.

(50) The composition of the present invention can be prepared by methods known in the art. In fact, for oral administration, the components may, for example, be mixed with one or more excipients, enclosed in a soft gel capsule, capsule, tablet, mini-tablet, micro-tablet, granule, micro-granule, pellets, multiparticulate, micronized particulate, powder, solution, suspension, dispersion, emulsion, gel, drop, or aerosol, preferably capsule.

(51) In a further aspect, the present invention concerns a kit comprising:

(52) i) a first formulation comprising at least a probiotic, and one or more pharmaceutically acceptable excipients;

(53) ii) a second formulation comprising at least a carotene, and one or more pharmaceutically acceptable excipients, and optionally at least a xanthophyll,

(54) iii) a leaflet comprising instructions for using the kit,

(55) for simultaneous, separate or sequential use in the treatment of dysbiosis of the intestinal microbiota, and pathologies ascribable thereto, wherein said at least a probiotic in the presence of at least a carotene produces a therapeutically effective amount of abscisic acid.

(56) The pharmaceutical unitary dose of the invention, as well as the pharmaceutical composition, the food supplement and the kit, may be administered via oral, sublingual, or buccal route, preferably, via oral route.

(57) It should be understood that all the aspects identified as preferred and advantageous for the pharmaceutical unitary dose are to be deemed as similarly preferred and advantageous also for the pharmaceutical composition, the food supplement, the kit, and uses of the same.

(58) It should be also understood that all the combinations of preferred aspects of the pharmaceutical unitary dose of the invention, as well as of the pharmaceutical composition, the food supplement, the kit and uses of the same, as above reported, are to be deemed as hereby disclosed.

(59) Below are working examples of the present invention provided for illustrative purposes.

EXAMPLES

(60) Materials

(61) Bacillus coagulans was from ATCC (Virginia, USA) and from tablets commercially available (Thorne Research, Dover, UK). The mixture of Lactobacillus plantarum and Bacillus coagulans was from ProLife, a commercially available product of Zeta Farmaceutici (Sandrigo, Vicenza, Italy); Lactobacillus rhamnousus was from Kaleidon 60, a commercially available product of Malesci (Grassina, Firenze, Italy). Beta-carotene and LB medium were purchased from Sigma (Milano, Italy); zeaxanthin was obtained from BioReagents (Montigny, France). Abscisic acid ELISA Kit was purchased from Agdia Biofords (Evry Cedex, France).

Example 1

(62) Bacterial Cell Culture

(63) Lyophilized probiotics were grown separately in LB medium in shake-flasks equipped with vented cap at 37° C. for 24 hours. After growth, each probiotic suspension was diluted with LB medium to reach approximately 0.1 O.D. at 600 nm. 3.5 ml of each diluted suspension was added with 500 μl of:

(64) i) empty liposomes,

(65) ii) liposomes containing 1 mg carotene,

(66) iii) liposomes containing 0.1 mg xanthophyll,

(67) iv) both liposomes containing carotene and xanthophyll, and

(68) incubated with shaking at 350 rpm at 37° C. for 24 hours.

(69) At the end of the incubation, each probiotic suspension was pelleted by centrifugation at 3000×g for 10 minutes. Supernatant was recovered, added with 4 vol of methanol and stored at −20° C. for 24 hours. Pellet was washed once with saline buffer, centrifuged at 3000×g for 10 minutes, resuspended in 2 ml of methanol, sonicated in ice and stored at −20° C. for 24 hours.

(70) Liposomes Preparation

(71) 4 mg of carotene and 0.4 mg of xanthophyll were dissolved each in 200 μl of chloroform by vortexing. For each liposome preparation 200 mg of lecithin were dissolved in 500 μl of chloroform. Both carotene and xanthophyll chloroform solutions were added, separately, to the chloroform lecithin solution, vortexed, and completely dried with N.sub.2 to create a thin layer on the inner wall of a glass Corex. The thin layer was detached with 2 ml of LB medium by vortexing at maximum speed and the solution obtained was finally sonicated on ice to obtain liposomes. The final concentration of carotene and xanthophyll in 500 μl of each liposome preparation used for bacteria incubation was 1 mg and 0.1 mg, respectively.

(72) To prepare empty liposomes or liposomes with carotene or liposomes with xanthophyll, were added to the chloroform lecithin solution the same volume of chloroform, i.e. 200 μl, used to dissolve carotene and xanthophyll.

(73) Detection of Abscisic Acid Produced by Bacteria

(74) Quantification of abscisic acid (ABA) was performed on both pellets and supernatants obtained as described above (see Bacterial cell culture). Briefly, after 24 hours at −20° C. in methanol, pellets and supernatants were centrifuged at 3000×g for 5 minutes. Supernatant from each centrifuged sample was recovered, lyophilized, resuspended in 500 μl of TRIS buffer provided by the ABA ELISA kit and filtered with spin-x in microfuge for 30 min at maximum speed. Measurement of ABA was performed in quadruplicate on each sample according to manufacturer's instructions of the ELISA kit.

(75) Result Discussion

(76) In FIG. 1A, it was reported the comparison between a culture of Bacillus coagulans and empty liposomes, and a culture of Bacillus coagulans and liposomes containing 1 mg beta-carotene. It is clearly shown that, in the presence of beta-carotene, the production of abscisic acid is drastically increased, i.e. about 6.6 times higher (p=0.0093).

(77) The abscisic acid produced corresponds to 0.1 μg/10(9) bacteria. Considering the bacterial intestinal content (in the colon) of 10(12) bacteria/g, the amount of abscisic acid produced in vivo, if all the bacteria per g of the intestine were Bacillus coagulans, would have been 100 ug/10(12) bacteria/g. In other words, 50 ug abscisic acid per 0.5 g of intestinal contents. Hypothetically supposing that Bacillus coagulans content of 1/100 of bacteria/g of intestinal contents, 50 g of that intestinal content would be sufficient to reach the amount of abscisic acid needed.

(78) In FIG. 1B, it was reported the comparison between a culture of Bacillus coagulans, Lactobacillus Plantarum and empty liposomes, and a culture of Bacillus coagulans, Lactobacillus Plantarum and liposomes containing 1 mg beta-carotene. It is clearly shown that, in the presence of beta-carotene, the production of abscisic acid is drastically increased, i.e. about 7.5 times higher.

(79) In FIG. 1C, it was reported the comparison between a culture of Lactobacillus Rhamnosus and empty liposomes, and a culture of Lactobacillus Rhamnosus and liposomes containing 1 mg beta-carotene. It is clearly shown that, in the presence of beta-carotene, the production of abscisic acid is drastically increased, i.e. about 20 times higher.

(80) In FIG. 2, it was reported the comparison between: a culture of Bacillus coagulans and empty liposomes, a culture of Bacillus coagulans and liposomes containing 1 mg beta-carotene, a culture of Bacillus coagulans and liposomes containing 0.1 mg zeaxanthin, a culture of Bacillus coagulans and liposomes containing 0.1 mg astaxanthin,

(81) It is clearly shown that, in the presence of beta-carotene, the production of abscisic acid is drastically increased, i.e. about 5 times higher, whereas in the presence of a xanthophyll, the production of abscisic acid is unexpectedly and significantly reduced.

(82) In FIG. 3, it was reported the comparison between: a culture of Bacillus coagulans and empty liposomes, a culture of Bacillus coagulans and liposomes containing 1 mg beta-carotene, a culture of Bacillus coagulans and liposomes containing 1 mg beta-carotene and 0.1 mg zeaxanthin.

(83) It is clearly shown that, in the presence of beta-carotene and zeaxanthin, the production of abscisic acid is drastically increased, i.e. 10 times higher when compared to the culture of Bacillus coagulans alone, and 5 times higher when compared to the culture of Bacillus coagulans in the presence of beta-carotene alone.

(84) These results were absolutely unexpected and significant, especially in view of the fact that a xanthophyll, when present alone, reduces the production of abscisic acid of the probiotic, whereas in the concomitant presence of a carotene, a synergistic effect is clearly observed.

(85) Detection of ABA in Human Plasma

(86) Human blood samples (5 ml), drawn in heparin, were centrifuged immediately after withdrawal at 2000×g for 10 minutes. Two milliliters of plasma were immediately extracted with 4 vol methanol, stored at −20° C. for 24 hours and after centrifuged at 3000×g for 5 minutes. Supernatant was recovered, lyophilized, resuspended in 250 μl of TRIS buffer provided by the ABA ELISA kit and filtered with spin-x in microfuge for 30 min at maximum speed. Measurement of ABA was performed in quadruplicate on each sample according to manufacturer's instructions of the ELISA kit.

(87) Human Volunteers

(88) Four healthy volunteers were instructed not to change their dietary habits during the period of the study, and to take one tablet of Bacillus coagulans containing 2×10.sup.9CFU (Thorne Research, Dover, UK) and one tablet containing 7 mg of beta-carotene (Natural Point, Milano, Italy) daily, before breakfast. At the beginning of the study (day 1) and after 15 days of treatment (day 15), a blood sample was taken from each subject, after overnight fasting, and waist circumference and BMI were measured. Measurement of fasting glycemia (FBG), total, LDL and HDL cholesterol, triglycerides was performed by the clinical chemistry laboratory of the IRCCS Polyclinic Hospital San Martino-IST in Genova. Measurement of hs-CRP was performed by using a specific ELISA kit (Cayman Chemical, Michigan, USA) according to manufacturer's instructions.

(89) In Table 1 below, the mean values of the metabolic parameters of four subjects treated for 15 days with Bacillus coagulans and beta-carotene, are reported.

(90) TABLE-US-00001 TABLE 1 Metabolic markers day 1 day 15 P value Cholesterol 196 ± 17 180 ± 18  p = 0.07  HDL  65 ± 15 78 ± 16  p = 0.0002 LDL 109 ± 11 90 ± 16 p = 0.053 TG 111 ± 14 74 ± 23 p = 0.044 Cardiovascular risk  3.1 ± 0.7 2.4 ± 0.5 p = 0.019 Cardiovascular protection  3.2 ± 0.05  4.3 ± 0.06 p = 0.004 hs-CRP  4.29 ± 1.19 3.64 ± 1.20 p = 0.005 BMI 22.9 ± 4.6 22.5 ± 4.7   p = 0.0023 Waist circumference (cm) 78.8 ± 3.6 78.0 ± 3.4  p = 0.019

(91) As reported in the State of the Art, perturbation of the gut microbiota composition results in higher intestinal permeability, allowing bacterial endotoxins to enter the bloodstream; this triggers a systemic low-grade inflammation, which in turn leads to insulin-resistance, hyperglycemia, dyslipidemia and concurs to body fat accumulation (obesity). The results summarized in Table 1 indicate that the daily intake of a probiotic and carotene significantly ameliorates most of the metabolic markers altered in a condition of dysbiosis and causing T2D and metabolic syndrome. In particular, the significant reduction of the hs-CRP, a marker of low-grade systemic inflammation, suggests a beneficial action of probiotic combined with a carotene in lowering gut permeability. As a consequence, a significant improvement of total cholesterol, HDL, LDL, TG, BMI and waist circumference are observed in all subjects. Moreover, production of ABA by probiotic combined with a carotene (shown in FIGS. 1A, 2) allows the autocrine stimulation of the probiotic proliferation, contributing to its colonization of the gut. Production of ABA by probiotic combined with a carotene also allows the already reported beneficial systemic effect of ABA on glycemia regulation. Indeed, intake of probiotic combined with a carotene increased basal plasma ABA 2.3-fold in all subjects (FIG. 4), thus reaching concentrations in the range of activity having beneficial effects on hyperglycemia.

(92) In FIG. 4, it was reported the comparison between the plasma concentration of abscisic acid at the beginning of the study (t0) and after 15 days (t15). The plasma concentration increases on average and significantly by 2.3 times reaching a stable and effective value of 3.4 nM (corresponding to 4.45 μg in the blood stream).