Compositions comprising live probiotic bacterial cultures of <i>Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus, </i>or <i>Staphylococcus</i>

Abstract

The present invention provides anallergic compositions, including food products, comprising probiotic bacterial cultures including probiotic strains of Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus, and/or Staphylococcus.

Claims

1. A live probiotic food product or a live probiotic pharmaceutical formulation comprising: (a) a substrate comprising less than 3 ppm gluten, less than 7 ppm lactose, and less than 0.05 ppm beta-globulins; and (b) live probiotic bacteria selected from the group consisting of: TABLE-US-00004 1 Streptococcus thermophilus LMG P-18383 2 Streptococcus thermophilus LMG P-18384 3 Lactobacillus pentosus LMG P-21019 4 Lactobacillus plantarum LMG P-21020 5 Lactobacillus plantarum LMG P-21021 6 Lactobacillus plantarum LMG P-21022 7 Lactobacillus plantarum LMG P-21023 8 Lactobacillus casei ssp. paracasei LMG P-21380 9 Bifidobacterium longum LMG P-21382 10 Bifidobacterium breve LMG P-21383 11 Bifidobacterium lactis LMG P-21384 12 Lactobacillus plantarum LMG P-21385 13 Lactococcus lactis ssp. lactis LMG P-21387 14 Lactococcus lactis ssp. lactis LMG P-21388 15 Lactobacillus plantarum LMG P-21389 16 Streptococcus thermophilus DSM 16506 17 Streptococcus thermophilus DSM 16507 18 Bifidobacterium longum DSM 16603 19 Bifidobacterium breve DSM 16604 20 Lactobacillus casei ssp. rhamnosus DSM 16605 21 Lactobacillus delbrueckii ssp. bulgaricus DSM 16606 22 Lactobacillus delbrueckii ssp. bulgaricus DSM 16607 23 Streptococcus thermophilus DSM 16590 24 Streptococcus thermophilus DSM 16591 25 Streptococcus thermophilus DSM 16592 26 Streptococcus thermophilus DSM 16593 27 Bifidobacterium adolescentis DSM 16594 28 Bifidobacterium adolescentis DSM 16595 29 Bifidobacterium breve DSM 16596 30 Bifidobacterium pseudocatenulatum DSM 16597 31 Bifidobacterium pseudocatenulatum DSM 16598 32 Staphylococcus xylosus DSM 17102 33 Bifidobacterium adolescentis DSM 17103 34 Lactobacillus plantarum DSM 17104 35 Streptococcus thermophilus DSM 17843 36 Streptococcus thermophilus DSM 17844 37 Streptococcus thermophilus DSM 17845 38 Lactobacillus fermentum DSM 18295 39 Lactobacillus fermentum DSM 18296 40 Lactobacillus fermentum DSM 18297 41 Lactobacillus fermentum DSM 18298 42 Lactobacillus gasseri DSM 18299 43 Lactobacillus gasseri DSM 18300 44 Lactobacillus gasseri DSM 18301 45 Lactobacillus gasseri DSM 18302 46 Bifidobacterium adolescentis DSM 18350 47 Bifidobacterium adolescentis DSM 18351 48 Bifidobacterium adolescentis DSM 18352 and 49 Bifidobacterium catenulatum DSM 18353.

2. The food product or pharmaceutical formation of claim 1, wherein the substrate further comprises a mono- or disaccharide.

3. The food product or pharmaceutical formation of claim 2, wherein the mono- or disaccharide is glucose derived from maize starch, potato starch, beet sucrose, or cane sucrose.

4. The food product or pharmaceutical formation of claim 2, wherein the mono- or disaccharide is derived from complex polysaccharide hydrolysis.

5. The food product or pharmaceutical formation of claim 1, wherein the food product or pharmaceutical formation is freeze-dried.

6. The food product or pharmaceutical formation of claim 1, wherein the food product or pharmaceutical formation is prepared by a method comprising: (i) providing a fermentative substrate containing sulfur dioxide and sulfites at concentrations of not more than 10 mg/kg or 10 mg/litre expressed as SO.sub.2; (ii) adding to the fermentative substrate provided in step (i) an exogenous meat peptone or an exogenous vegetal peptone selected from the group consisting of rice, potato, maize, chestnuts, tapioca, manioca, pea, fava beans, and mixtures thereof; (iii) fermenting one or more live probiotic bacteria in the fermentative substrate of step (ii) to yield a probiotic bacterial culture containing less than 3 ppm gluten, less than 7 ppm lactose, and less than 0.05 ppm beta-lactoglobulins, and (iv) preparing a live probiotic food product or live probiotic pharmaceutical formulation comprising said probiotic bacterial culture.

7. The food product or pharmaceutical formation of claim 6, wherein the fermentative substrate comprises (x) glucose derived from maize starch, potato starch, beet sucrose, or cane sucrose, or (y) mono- and disaccharides derived from complex polysaccharide hydrolysis.

8. The food product or pharmaceutical formation of claim 6, wherein said live probiotic food product or live probiotic pharmaceutical formulation obtained in step (iv) is freeze dried.

9. The food product or pharmaceutical formation of claim 1, wherein the food product or pharmaceutical formation is prepared by a method comprising: (i) preparing a fermentative substrate comprising an exogenous meat peptone or an exogenous vegetal peptone selected from the group consisting of rice, potato, maize, chestnuts, tapioca, manioca, pea, fava beans, and mixtures thereof, (ii) subjecting the fermentative substrate to an enzymatic treatment using a proteolytic enzyme, a glycoside enzyme, or both to obtain a fermentative substrate containing sulfur dioxide and sulfites at concentrations of not more than 10 mg/kg or 10 mg/litre expressed as SO.sub.2, wherein the proteolytic enzyme is selected from the group consisting of trypsin, chymotrypsin, pancreatin, pepsin, papain, and bromelain, and wherein the glycoside enzyme is selected from the group consisting of alpha-glucosidase and beta-glucosidase, (iii) fermenting one or more live probiotic bacteria in the fermentative substrate to yield a probiotic bacterial culture containing less than 3 ppm gluten, less than 7 ppm lactose, and less than 0.05 ppm beta-lactoglobulins, and (iv) preparing a live probiotic food product or live probiotic pharmaceutical formulation comprising the probiotic bacterial culture obtained in step (iii), wherein the fermentative substrate, and the live probiotic food product or live probiotic pharmaceutical formulation are each substantially free of gluten and milk-derived allergens.

10. The food product or pharmaceutical formation of claim 9, wherein said enzymatic treatment comprises treating the fermentative substrate with (a) bromelain, and (b) beta-galactosidase.

11. The food product or pharmaceutical formation of claim 9, wherein said enzymatic treatment comprises treating the fermentative substrate with bromelain.

12. The food product or pharmaceutical formation of claim 11, wherein (x) the fermentative substrate is treated with alcalase at 45° C.-55° C., pH 7-8, for 15-60 minutes, (y) the fermentative substrate is treated with lactase at 30° C.-40° C., pH 6-7, for 2-6 hours, and (z) the fermentative substrate is treated with bromelain at 30° C.-40° C., pH 5-6, for 1-6 hours.

13. The food product or pharmaceutical formation of claim 9 wherein, following the enzymatic treatment, (x) the fermentative substrate pH is adjusted to a value suitable for culturing said probiotic bacteria; and (y) the fermentative substrate is heated to a temperature of 90° C.-145° C. for a time sufficient to inactivate enzymes used in the enzymatic treatment.

14. The food product or pharmaceutical formation of claim 9, wherein the fermentative substrate comprises (x) glucose derived from maize starch, potato starch, beet sucrose, or cane sucrose, (y) a meat peptone, and (z) a vegetal peptone selected from the group consisting of: rice, potato, maize, chestnuts, tapioca, manioca, pea, fava beans, and mixtures thereof.

15. The food product or pharmaceutical formation of claim 9, wherein said live probiotic food product or live probiotic pharmaceutical formulation obtained in step (iv) is freeze dried.

Description

(1) An aim of the present invention is to provide a method for the preparation of culture media capable of overcoming the limits of the known art.

(2) Another aim of the present invention is to provide a methodology for the production of probiotic bacterial cultures safe to administer to all the population, also to the people affected by allergies.

(3) These and other aims, which will result apparent from the following detailed description, have been attained by the Applicant, which has improved a methodology which includes a double safety level relating to the absence of allergens in the productive processes of probiotic bacterial cultures.

(4) In particular, the Applicant has set up a production methodology in which selected anallergic fermentation substrates (anallergic raw materials) are used, capable of ensuring a proper nitrogen and carbon source to the probiotic cultures.

(5) A method for the preparation of an anallergic probiotic bacterial cultures, a composition including said culture and the use of said culture for the preparation of so-called “functional” or “nutraceutic” foods form the subject of the present invention, having the features as defined in the appended claims.

(6) In an embodiment of the invention, the strains of said bacterial culture belong to the genera: Lactobacillus, Bifidobacterium, Streptococcus, Pediococcus, Lactococcus, Propionibacterium, Bacillus, Saccharomyces, Enterococcus, Leuconostoc.

(7) Preferably, of the genus Lactobacillus, the following species have found use: L. pentosus, L. plantarum, L. casei, L. casei ssp. paracasei, L. casei ssp. rhamnosus, L. acidophilus, L. delbrueckii ssp. bulgaricus, L. fermentum, L. gasseri.

(8) Examples of used strains of said species are reported in the enclosed Table 1. Preferably, of the genus Bifidobacterium, the following species have found use: B. longum. B. breve, B. lactis, B. adolescentis and B. pseudocatenulatum.

(9) Examples of used strains of said species are reported in the enclosed Table 1. Preferably, of the genus Lactococcus the following species have found use: L. lactis and L. lactis ssp. Lactis.

(10) Examples of used strains of said species are reported in the enclosed Table 1. Preferably, of the genus Streptococcus the following species have found use: S. thermophilus.

(11) Examples of used strains of said species are reported in the enclosed Table 1.

(12) In a particularly preferred embodiment of the invention, the bacteria of said bacterial culture are selected from the group including the probiotic bacterial strains reported in the enclosed Table 1.

(13) The Applicant has found useful to select and employ particular anallergic raw materials. In particular, the Applicant has found, as a nitrogen source, peptones and/or proteinic hydrolyzates of vegetal and/or animal origin, naturally free from gluten and allergens of milky origin and, as a carbon source, glucose and/or other mono- or disaccharides derived from the hydrolysis of more complex polysaccharides typical of vegetal species naturally free from gluten and allergens of milky origin.

(14) The peptones of vegetal origin are selected from the group including: rice, potato, maize, chestnuts, tapioca, manioca, pea, broad beans and their mixtures however capable of promoting the fermentation bacterial growth, but without producing allergens, either of milky nor gluten types.

(15) In a first preferred embodiment, the method subject of the present invention foresees the use, as a nitrogen source, of one or more peptones and/or anallergic proteinic hydrolyzates and, as a carbon source, glucose and/or other mono- or disaccharides derived from hydrolysis of complex polysaccharide (anallergic raw materials).

(16) In a second preferred embodiment, the method subject of the present invention foresees a pre-treatment of the raw materials with enzymes suitable for the removal of traces, if any, of allergens deriving from cross-contamination occurred along the productive and/or distributive chain.

(17) In the context of the present invention, the culture substrate is an anallergic culture substrate of vegetal and/or animal origin, naturally free from gluten, allergens or milky origin and all the substances belonging to the list of the annex III bis of the instructions (anallergic raw materials) above mentioned. The use of anallergic raw materials above mentioned allows to obtain certifiable probiotics for the use in allergic persons, as the non-use of substances belonging to the list of the annex III bis of the aforesaid community instructions and the use of ingredients certified from the supplier as free from such substances can be assured.

(18) Then, by virtue of the fact that the absence of any chemical substances in a given sample is not scientifically demonstrable, but one can simply determine that the quantity possibly existing is lower than the detection limit of the analytical method used (even if the more sensitive and refined method known in the art was used), also the use of an enzymatic pre-treatment results to be a source of an additional guarantee.

(19) By mere way of example, some anallergic formulations of medium for the growth of probiotic bacterial cultures are reported below.

(20) The components of a culture medium must bring nitrogen sources (in this case the peptones and/or proteinic hydrolyzates), carbon sources (in this case, the glucose and/or other mono- or disaccharides derived from hydrolysis of complex polysaccharides), growth bioactivators and vitamins (in this case from yeast extract) and mineral salts.

(21) For example, a culture medium can contain: glucose preferably selected from: maize starch, potato, beet sucrose or cane sucrose; peptone preferably selected from: rice, potato, maize, chestnut, tapioca, maniocak pea, broad beans, bean or generally legumes and their mixtures; peptone preferably selected from: meat; yeast extract; mineral salts (such as, by mere way of example: acetates, carbonates, phosphates, hydrogen phosphates, chlorides, citrates, sulfates and others); builder (if necessary, such as: Tween, lecithins and other) and drinking water.

(22) One of the formulation suitable for the growth of strains of the Lactobacillus and Bifidobacterium genera could preferably include the following ingredients:

(23) TABLE-US-00001 glucose (from the sources above listed) 10-100 g/l rice peptone 10-50 g/l meat peptone 10-50 g/l yeast extract 2-20 g/l mineral salts 1-10 g/l builders 0-5 ml/l drinking water q.s. to the desired volume

(24) A preferred example of a medium for anallergic probiotic bacterial cultures could be the following:

(25) TABLE-US-00002 glucose (from maize starch) 20 g/l rice peptone 10 g/l meat peptone 10 g/l yeast extract 5 g/l sodium acetate 5 g/l citrate ammonium 2 g/l dibasic potassium phosphate 2 g/l magnesium sulfate 0.1 g/l manganese sulfate 0.05 g/l tween 80 1 ml/l drinking water q.s. to the desired volume

(26) The fermentation is carried out according to the teachings known to the skilled in the art and under the experimental conditions of common use.

(27) The Applicant has verify the presence, or not, of allergen traces on a probiotic culture grown on raw materials subject of the present invention.

(28) For example, in case of milk-derived allergens, the research by analytical way of β-lactoglobulin and lactose on the end products, with confirmed specific and sensitive methodologies (analysis with ELISA kit specific for the β-lactoglobulin of the type “Bovine β-lactoglubilins ELISA quantitation kit—Bethyl Laboratories”, with a threshold limit of 0.05 ppm and analysis with chemoenzymatic kit and UV-vis detection for the lactose of the type “Lactose/D-glucose—Boehringer Mannheim”, cod. 10986119, with a threshold limit of 7 ppm) gives a negative result and, therefore, these substances, if any, should certainly be under the detection threshold.

(29) At the same time, the gluten research carried out with the more refined and, so far, more sensitive confirmed methodology (ELISA RIDASCREEN® Gliadin kit—R-Biopharm A, Darmstadt, Germany, with a sensitivity equal to 3 ppm) allows to confirm the absence of gluten. It follows that, even if the gluten were present, its concentration should be in any case under the detection threshold, namely lower than 3 ppm.

(30) The enzymatic pre-treatment on the raw materials, to be carried out or not as a function of the requirements, is able to hydrolize milk and derivatives traces and gluten and derivatives accidentally existing in the culture medium.

(31) Such treatment imparts the highest safety standard for a use also suitable to allergic and particularly sensitive persons.

(32) This manufacturing strategy is suitable for the probiotics production with an anallergic safety degree called DSS—Double Safety System.

(33) The enzymatic pre-treatment foresees the use of at least a proteolytic enzyme and/or the use of at least a glycosidase enzyme.

(34) In the context of the present invention, the proteolytic enzyme is able to perform a proteolysis. The proteolytic enzyme is selected from the group including the proteases and/or the peptidases. The proteases and the peptidases are selected from the group including: trypsin, chymotrypsin, pancreatin, pepsin, papain and bromelain. Preferably, the proteases and the peptidases are selected between pepsin and/or bromelain. In the context of the present invention, the glycosidase enzyme is able to perform a hydrolityc cleavage of a glycoside. The glycosidase enzyme is selected from the group including: alfa-glucosidase and beta-glucosidase, alfa-galactosidase and beta-galactosidase.

(35) Advantageously, the enzymatic treatment of the raw materials forming the growth broth for the probiotics is carried out with proteases (alcalases and bromelain) and with the glycosidases.

(36) The glycosidases are selected from the group including: lactase (or (3-galactosidase). In a preferred embodiment, the pre-treatment of the raw materials foresees the use in a sequence including three enzymes: alcalase, lactase and bromelain.

(37) In a preferred embodiment, the selection of the enzymes and their sequence is the following: alcalase, which practically hydrolyzes all the proteins and particularly those of the milk; lactase, which hydrolyzes the lactose; bromelain, which hydrolyzes the gluten.

(38) The sequence shown is a function of the optimal hydrolysis pH in a gradient from basic to acid; in this way, the medium preserves the nutritional properties. The alcalase, active towards the β-lactoglobulin, the α-lactaalbumin and the caseins, allows to eliminate allergenic residuals, if any, deriving from fortuitous and unintentional cross-contaminations with milk derivatives.

(39) Such treatment foresees the addition to the raw materials dissolved in water of a quantity of enzyme varying from 0.0025 and 0.0500 g/1, corresponding to 0.001-0.020 AU/1 (Anson Units per Liter).

(40) The solution is then brought to a temperature between 45 and 55° C. for 15-60 minutes, with a pH between 7 and 8; preferably, a controlled pH of 7.50±0.20.

(41) The lactase, also known as β-galactosidase, is charged to the hydrolysis of the glycoside bond between glucose and galactose in the lactose disaccharide.

(42) The treatment with lactase is carried out following to the hydrolysis with proteins alcalase after having brought the pH of the culture broth to a value between 6 and 7; preferably, a value of 6.50±0.20 with organic acids (preferably lactic acid) by adding 250-2.000 NLU/1 (Neutral Lactase Units per Liter), corresponding to 0.05-0.40 ml of an enzyme solution titrated at 5.000 NLU/g.

(43) The solution is maintained at 37±5° C. for a varying period of 2-6 hours. Finally, the bromelain is a proteolytic enzyme naturally contained in the pineapple, capable of effectively hydrolysing the gliadin in fragments not recognized by the immune system and therefore non allergenic.

(44) The treatment is carried out by adding the fermentation medium with the enzyme to the amount of 0.005-0.010 g/l (equal to 110-220 GDU/1, Gelatin Digesting Units per Liter), after correction of the pH to values of 5.0-6.0 with organic acids (preferably lactic acid). The working temperature must be maintained at 37±5° C. for a time between 1 and 6 hours.

(45) Following to the three enzymatic treatments, it is necessary to restore the pH at the optimal value for the fermentation of the single strains (preferably with 5N NaOH in order to basify, or with acid lactic in order to acidify).

(46) Next, a heat treatment for the purification of the medium is carried out (performed at temperatures between 90 and 145° C. for times varying from few seconds to 45 minutes), which will however denature and inactivate the added enzymes, without further risks for the end product and their intended people deriving from residuals of the enzyme used.

(47) A typical industrial production design therefore foresees the following steps:

(48) a. selection of the anallergic raw materials

(49) b. dissolution of the raw materials in water

(50) c. correction of the pH and temperature to proper values for the use of the proteolytic enzyme, preferably alcalase

(51) d. addition of the enzyme and its action for the required time

(52) e. correction of the pH and the temperature to proper values for the use of the glycolytic enzyme, preferably lactase

(53) f. addition of the enzyme and its action for the required time

(54) g. correction of the pH and the temperature to proper values for the use of the proteolytic enzyme, preferably bromelain

(55) h. addition of the enzyme and its action for the required time

(56) i. correction of the pH up to values suitable for the fermentation

(57) j. purification through pasteurisation and/or sterilization of the culture medium.

(58) k. cooling at the inoculum temperature typical of the probiotic strain under production (37±2° C.).

(59) l. inoculum of the strain.

(60) m. fermentation

(61) n. separation of the biomass and crioprotection

(62) o. freeze drying.

(63) The present invention allows then to produce anallergic probiotic strains and in particular with absolute absence of allergens, more preferably of milk and gluten derivatives, with a wide safety of use for all the populations classes.

(64) Advantageously, the anallergic probiotic bacterial cultures prepared according to the teachings of the present invention can be effectively used for the preparation of pharmaceutical formulations.

(65) In view of the high number of persons allergic to the milk (3-5% of the population with an age below 2 years) and the celiac persons (1% of the total population) it is useful to try to develop probiotic bacteria which can be administered also to this classes of population.

(66) The present invention is then useful: to the consumers, for which the transparency in the labelling is fundamental; to the producers, which in this way can rely on a product with a total guarantee of its anallergic properties, therefore proposable to the whole purchasing population.

(67) TABLE-US-00003 TABLE 1 1 Streptococcus thermophilus LMG P-18383 2 Streptococcus thermophilus LMG P-18384 3 Lactobacillus pentosus LMG P-21019 4 Lactobacillus plantarum LMG P-21020 5 Lactobacillus plantarum LMG P-21021 6 Lactobacillus plantarum LMG P-21022 7 Lactobacillus plantarum LMG P-21023 8 Lactobacillus casei ssp. paracasei LMG P-21380 9 Lactobacillus acidophilus LMG P-21381 10 Bifidobacterium longum LMG P-21382 11 Bifidobacterium breve LMG P-21383 12 Bifidobacterium lactis LMG P-21384 13 Lactobacillus plantarum LMG P-21385 14 Lactococcus lactis ssp. lactis LMG P-21387 15 Lactococcus lactis ssp. lactis LMG P-21388 16 Lactobacillus plantarum LMG P-21389 17 Streptococcus thermophilus DSM 16506 18 Streptococcus thermophilus DSM 16507 19 Bifidobacterium longum DSM 16603 20 Bifidobacterium breve DSM 16604 21 Lactobacillus casei ssp. rhamnosus DSM 16605 22 Lactobacillus delbrueckii ssp. bulgaricus DSM 16606 23 Lactobacillus delbrueckii ssp. bulgaricus DSM 16607 24 Streptococcus thermophilus DSM 16590 25 Streptococcus thermophilus DSM 16591 26 Streptococcus thermophilus DSM 16592 27 Streptococcus thermophilus DSM 16593 28 Bifidobacterium adolescentis DSM 16594 29 Bifidobacterium adolescentis DSM 16595 30 Bifidobacterium breve DSM 16596 31 Bifidobacterium pseudocatenulatum DSM 16597 32 Bifidobacterium pseudocatenulatum DSM 16598 33 Staphylococcus xylosus DSM 17102 34 Bifidobacterium adolescentis DSM 17103 35 Lactobacillus plantarum DSM 17104 36 Streptococcus thermophilus DSM 17843 37 Streptococcus thermophilus DSM 17844 38 Streptococcus thermophilus DSM 17845 39 Lactobacillus fermentum DSM 18295 40 Lactobacillus fermentum DSM 18296 41 Lactobacillus fermentum DSM 18297 42 Lactobacillus fermentum DSM 18298 43 Lactobacillus gasseri DSM 18299 44 Lactobacillus gasseri DSM 18300 45 Lactobacillus gasseri DSM 18301 46 Lactobacillus gasseri DSM 18302 47 Bifidobacterium adolescentis DSM 18350 48 Bifidobacterium adolescentis DSM 18351 49 Bifidobacterium adolescentis DSM 18352 50 Bifidobacterium catenulatum DSM 18353.

(68) The enclosed Table 1 identifies strains deposited at the BCCM/LMG Bacteria Collection of Gent, Belgium and at the DSMZ—Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, Inhoffenstrasse 7 B, 38124 Braunschweig, GERMANY; the deposits are in accordance with the Budapest Treaty.