Method for the preparation of anallergic probiotic bacterial cultures and related use

Abstract

The aim of the present invention is a method for the preparation of anallergic probiotic bacterial cultures.

Claims

1. A method for preparing a live probiotic food product or a live probiotic pharmaceutical formulation comprising (i) providing a fermentative substrate containing sulfur dioxide and sulfites at concentrations of not more than 10 mg/kg or 10 mg/liter expressed as SO.sub.2; (ii) adding to the 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 wherein one of the one or more live probiotic bacteria is the Lactobaciillus acidophilus strain deposited at the BCCM LMG under deposit accession number LMG P21381.

2. The method of claim 1, wherein the fermentative substrate comprises (a) glucose derived from maize starch, potato starch, beet sucrose, or cane sucrose, or (b) mono- and disaccharides derived from complex polysaccharide hydrolysis.

3. The method of claim 1, wherein the fermentative substrate comprises glucose derived from maize starch, potato starch, beet sucrose, or cane sucrose.

4. The method of claim 1, wherein said live probiotic food product or live probiotic pharmaceutical formulation obtained in step (iv) is freeze dried.

5. The method of claim 1, wherein said method prepares a live probiotic pharmaceutical formulation.

6. A method for preparing a live probiotic food product or a live probiotic pharmaceutical formulation 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/liter 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, and wherein one of the one or more live probiotic bacteria is the Lactobaciillus acidophilus strain deposited at the BCCM LMG under deposit accession number LMG P21381.

7. The method of claim 6, wherein said enzymatic treatment comprises treating the fermentative substrate with (a) bromelain, and (b) beta-galactosidase.

8. The method of claim 6, wherein said enzymatic treatment comprises treating the fermentative substrate with bromelain.

9. The method of claim 8, wherein (a) the fermentative substrate is treated with alcalase at 45 C.-55 C., pH 7-8, for 15-60 minutes, (b) the fermentative substrate is treated with lactase at 30 C.-40 C., pH 6-7, for 2-6 hours, and (c) the fermentative substrate is treated with bromelain at 30 C.-40 C., pH 5-6, for 1-6 hours.

10. The method of claim 6 wherein, following the enzymatic treatment, (a) the fermentative substrate pH is adjusted to a value suitable for culturing said probiotic bacteria; and (b) 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.

11. The method of claim 6, wherein the fermentative substrate comprises (a) glucose derived from maize starch, potato starch, beet sucrose, or cane sucrose, or (b) mono- and disaccharides derived from complex polysaccharide hydrolysis.

12. The method of claim 6, wherein the fermentative substrate comprises (a) glucose derived from maize starch, potato starch, beet sucrose, or cane sucrose, (b) a meat peptone, and (c) a vegetal peptone selected from the group consisting of: rice, potato, maize, chestnuts, tapioca, manioca, pea, fava beans, and mixtures thereof.

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

14. The method of claim 6, wherein said method prepares a live probiotic pharmaceutical formulation.

Description

(1) 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.

(2) 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.

(3) 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.

(4) 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.

(5) 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.

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

(7) 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.

(8) 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.

(9) 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.

(10) 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.

(11) 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).

(12) 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.

(13) 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.

(14) 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.

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

(16) 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.

(17) For example, a culture medium can contain:

(18) glucose preferably selected from: maize starch, potato, beet sucrose or cane sucrose;

(19) peptone preferably selected from: rice, potato, maize, chestnut, tapioca, maniocak pea, broad beans, bean or generally legumes and their mixtures;

(20) peptone preferably selected from: meat;

(21) 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 -lactoglobulin and lactose on the end products, with confirmed specific and sensitive methodologies (analysis with ELISA kit specific for the -lactoglobulin of the type Bovine -lactoglubilins ELISA quantitation kitBethyl 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-glucoseBoehringer 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 DSSDouble 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 -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:

(38) alcalase, which practically hydrolyzes all the proteins and particularly those of the milk;

(39) lactase, which hydrolyzes the lactose;

(40) bromelain, which hydrolyzes the gluten.

(41) 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 -lactoglobulin, the -lactaalbumin and the caseins, allows to eliminate allergenic residuals, if any, deriving from fortuitous and unintentional cross-contaminations with milk derivatives.

(42) 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/l, corresponding to 0.001-0.020 AU/l (Anson Units per Liter).

(43) 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 7.500.20.

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

(45) 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 6.500.20 with organic acids (preferably lactic acid) by adding 250-2.000 NLU/l (Neutral Lactase Units per Liter), corresponding to 0.05-0.40 ml of an enzyme solution titrated at 5.000 NLU/g.

(46) The solution is maintained at 375 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.

(47) 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/l, 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 375 C. for a time between 1 and 6 hours.

(48) 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).

(49) 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.

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

(51) a. selection of the anallergic raw materials

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

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

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

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

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

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

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

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

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

(61) k. cooling at the inoculum temperature typical of the probiotic strain under production (372 C.).

(62) l. inoculum of the strain.

(63) m. fermentation

(64) n. separation of the biomass and crioprotection

(65) o. freeze drying.

(66) 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.

(67) 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.

(68) 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.

(69) The present invention is then useful:

(70) to the consumers, for which the transparency in the labelling is fundamental;

(71) 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.

(72) TABLE-US-00003 TABLE 1 International Depositary Accession Name Authority Number Filing Date Depositor 1 Streptococcus thermophilus BCCM LMG LMG P-18383 5 May 1998 ANIDRAL S.R.L. 2 Streptococcus thermophilus BCCM LMG LMG P-18384 5 May 1998 ANIDRAL S.R.L. 3 Lactobacillus pentosus BCCM LMG LMG P-21019 16 Oct. 2001 Laboratorio Microbiologico Grana Provolone SRL Via P. Custodi 12 I-28100 Novara (Italy) 4 Lactobacillus plantarum BCCM LMG LMG P-21020 16 Oct. 2001 Laboratorio Microbiologico Grana Provolone SRL Via P. Custodi 12 I-28100 Novara (Italy) 5 Lactobacillus plantarum BCCM LMG LMG P-21021 16 Oct. 2001 Laboratorio Microbiologico Grana Provolone SRL Via P. Custodi 12 I-28100 Novara (Italy) 6 Lactobacillus plantarum BCCM LMG LMG P-21022 16 Oct. 2001 Laboratorio Microbiologico Grana Provolone SRL Via P. Custodi 12 I-28100 Novara (Italy) 7 Lactobacillus plantarum BCCM LMG LMG P-21023 16 Oct. 2001 Laboratorio Microbiologico Grana Provolone SRL Via P. Custodi 12 I-28100 Novara (Italy) 8 Lactobacillus casei ssp. paracasei BCCM LMG LMG P-21380 31 Jan. 2002 ANIDRAL S.R.L. 9 Lactobacillus belonging to the acidophilus group BCCM LMG LMG P-21381 31 Jan. 2002 ANIDRAL S.R.L. 10 Bifidobacterium longum BCCM LMG LMG P-21382 31 Jan. 2002 ANIDRAL S.R.L. 11 Bifidobacterium breve BCCM LMG LMG P-21383 31 Jan. 2002 ANIDRAL S.R.L. 12 Bifidobacterium lactis BCCM LMG LMG P-21384 31 Jan. 2002 ANIDRAL S.R.L. 13 Lactobacillus plantarum BCCM LMG LMG P-21385 31 Jan. 2002 MOFIN S.R.L. Via P. Custodi 12 I-28100 Novara (Italy) 14 Lactococcus lactis ssp. lactis BCCM LMG LMG P-21387 15 Mar. 2002 MOFIN S.R.L. Via P. Custodi 12 I-28100 Novara (Italy) 15 Lactococcus lactis ssp. lactis BCCM LMG LMG P-21388 31 Jan. 2002 MOFIN S.R.L. Via P. Custodi 12 I-28100 Novara (Italy) 16 Lactobacillus plantarum BCCM LMG LMG P-21389 15 Mar. 2002 MOFIN S.R.L. Via P. Custodi 12 I-28100 Novara (Italy) 17 Streptococcus thermophilus DSMZ DSM 16506 18 Jun. 2004 ANIDRAL S.R.L. 18 Streptococcus thermophilus DSMZ DSM 16507 18 Jun. 2004 ANIDRAL S.R.L. 19 Bifidobacterium longum DSMZ DSM 16603 20 Jul. 2004 ANIDRAL S.R.L. 20 Bifidobacterium breve DSMZ DSM 16604 20 Jul. 2004 ANIDRAL S.R.L. 21 Lactobacillus casei ssp. rhamnosus DSMZ DSM 16605 20 Jul. 2004 ANIDRAL S.R.L. 22 Lactobacillus delbrueckii ssp. bulgaricus DSMZ DSM 16606 20 Jul. 2004 ANIDRAL S.R.L. 23 Lactobacillus delbrueckii ssp. bulgaricus DSMZ DSM 16607 20 Jul. 2004 ANIDRAL S.R.L. 24 Streptococcus thermophilus DSMZ DSM 16590 20 Jul. 2004 ANIDRAL S.R.L. 25 Streptococcus thermophilus DSMZ DSM 16591 20 Jul. 2004 ANIDRAL S.R.L. 26 Streptococcus thermophilus DSMZ DSM 16592 20 Jul. 2004 ANIDRAL S.R.L. 27 Streptococcus thermophilus DSMZ DSM 16593 20 Jul. 2004 ANIDRAL S.R.L. 28 Bifidobacterium adolescentis DSMZ DSM 16594 21 Jul. 2004 ANIDRAL S.R.L. 29 Bifidobacterium adolescentis DSMZ DSM 16595 21 Jul. 2004 ANIDRAL S.R.L. 30 Bifidobacterium breve DSMZ DSM 16596 21 Jul. 2004 ANIDRAL S.R.L. 31 Bifidobacterium pseudocatenulatum DSMZ DSM 16597 21 Jul. 2004 ANIDRAL S.R.L. 32 Bifidobacterium pseudocatenulatum DSMZ DSM 16598 21 Jul. 2004 ANIDRAL S.R.L. 33 Staphylococcus xylosus DSMZ DSM 17102 01 Feb. 2005 ANIDRAL S.R.L. 34 Bifidobacterium adolescentis DSMZ DSM 17103 01 Feb. 2005 ANIDRAL S.R.L. 35 Lactobacillus plantarum DSMZ DSM 17104 01 Feb. 2005 ANIDRAL S.R.L. 36 Streptococcus thermophilus DSMZ DSM 17843 21 Dec. 2005 ANIDRAL S.R.L. 37 Streptococcus thermophilus DSMZ DSM 17844 21 Dec. 2005 ANIDRAL S.R.L. 38 Streptococcus thermophilus DSMZ DSM 17845 21 Dec. 2005 ANIDRAL S.R.L. 39 Lactobacillus fermentum DSMZ DSM 18295 24 May 2006 ANIDRAL S.R.L. 40 Lactobacillus fermentum DSMZ DSM 18296 24 May 2006 ANIDRAL S.R.L. 41 Lactobacillus fermentum DSMZ DSM 18297 24 May 2006 ANIDRAL S.R.L. 42 Lactobacillus fermentum DSMZ DSM 18298 24 May 2006 ANIDRAL S.R.L. 43 Lactobacillus gasseri DSMZ DSM 18299 24 May 2006 ANIDRAL S.R.L. 44 Lactobacillus gasseri DSMZ DSM 18300 24 May 2006 ANIDRAL S.R.L. 45 Lactobacillus gasseri DSMZ DSM 18301 24 May 2006 ANIDRAL S.R.L. 46 Lactobacillus gasseri DSMZ DSM 18302 24 May 2006 ANIDRAL S.R.L. 47 Bifidobacterium adolescentis DSMZ DSM 18350 15 Jun. 2006 ANIDRAL S.R.L. 48 Bifidobacterium adolescentis DSMZ DSM 18351 15 Jun. 2006 ANIDRAL S.R.L. 49 Bifidobacterium adolescentis DSMZ DSM 18352 15 Jun. 2006 ANIDRAL S.R.L. 50 Bifidobacterium catenulatum DSMZ DSM 18353 15 Jun. 2006 ANIDRAL S.R.L.