ITMI20100325A1 - BACTERIA PROBIOTICI HAVING ANTIOXIDANT ACTIVITY AND THEIR RELATIVE USE. - Google Patents
BACTERIA PROBIOTICI HAVING ANTIOXIDANT ACTIVITY AND THEIR RELATIVE USE. Download PDFInfo
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- ITMI20100325A1 ITMI20100325A1 IT000325A ITMI20100325A ITMI20100325A1 IT MI20100325 A1 ITMI20100325 A1 IT MI20100325A1 IT 000325 A IT000325 A IT 000325A IT MI20100325 A ITMI20100325 A IT MI20100325A IT MI20100325 A1 ITMI20100325 A1 IT MI20100325A1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
Description
DESCRIZIONE "Batteri probiotici aventi attività antiossidante e loro relativo impiego". DESCRIPTION "Probiotic bacteria with antioxidant activity and their relative use".
La presente invenzione si riferisce a una composizione avente attività antiossidante. Inoltre, la presente invenzione si riferisce a batteri probiotici aventi attività antiossidante e al loro relativo impiego. The present invention relates to a composition having antioxidant activity. Furthermore, the present invention relates to probiotic bacteria having antioxidant activity and their relative use.
È noto che tutte le forme di vita mantengono un ambiente riducente entro le proprie cellule. Un'eventuale alterazione di questo stato redox può causare effetti tossici per la produzione, e il successivo accumulo, di perossidi e radicali liberi. E'solo il loro eccesso a essere implicato nello stress ossidativo che sembra essere associato a molte patologie umane, quali: aterosclerosi, ipertensione arteriosa, morbo di Parkinson, morbo di Alzheimer, diabete mellito, colite, artrite reumatoide. All life forms are known to maintain a reducing environment within their cells. Any alteration of this redox state can cause toxic effects for the production, and subsequent accumulation, of peroxides and free radicals. It is only their excess that is implicated in oxidative stress which seems to be associated with many human pathologies, such as: atherosclerosis, arterial hypertension, Parkinson's disease, Alzheimer's disease, diabetes mellitus, colitis, rheumatoid arthritis.
In condizioni fisiologiche, esiste un equilibrio tra i livelli dei radicali liberi prodotti durante un normale metabolismo cellulare e i livelli degli antiossidanti endogeni, capace di proteggere i tessuti dal danno ossidativo. La rottura di questo equilibrio, sia a causa di un aumento della produzione dei radicali, sia a causa di una diminuzione dei livelli di antiossidanti, produce come conseguenza l'instaurarsi di alterazioni a carico della struttura e della funzione delle nostre cellule. La cellula possiede fisiologicamente la capacità di svolgere azione antiossidante e, quindi, protettiva nei confronti dei radicali liberi grazie alla presenza di specifici meccanismi di difesa, sia di natura enzimatica che non enzimatica. Under physiological conditions, there is a balance between the levels of free radicals produced during normal cellular metabolism and the levels of endogenous antioxidants, capable of protecting tissues from oxidative damage. The disruption of this balance, both due to an increase in the production of radicals, and due to a decrease in the levels of antioxidants, produces as a consequence the establishment of alterations in the structure and function of our cells. The cell physiologically possesses the ability to carry out an antioxidant action and, therefore, protective against free radicals thanks to the presence of specific defense mechanisms, both of an enzymatic and non-enzymatic nature.
Anche se le nostre cellule possiedono una loro propria capacità difensiva, molti fattori della nostra vita quotidiana concorrono a diminuirla . Even if our cells have their own defensive capacity, many factors of our daily life contribute to diminish it.
E' noto che, ad esempio, il fumo di tabacco, l'assunzione di alcolici e farmaci nonché un'esposizione eccessiva non controllata alle radiazioni ionizzanti possono contribuire a ridurre la capacità di difesa delle cellule. It is known that, for example, tobacco smoke, alcohol and drug intake as well as uncontrolled excessive exposure to ionizing radiation can contribute to reducing the defense capacity of cells.
Inoltre, i ritmi della vita di tutti i giorni uniti a una dieta non bilanciata e poco ricca in frutta, verdura e pesce certamente non consentono di integrare il nostro organismo di adeguate quantità 'di vitamine, minerali ed oligoelementi ad elevata azione antiossidante . Furthermore, the rhythms of everyday life combined with an unbalanced diet that is not very rich in fruit, vegetables and fish certainly do not allow our body to be supplemented with adequate quantities of vitamins, minerals and trace elements with a high antioxidant action.
Pertanto, si rende necessario integrare una dieta alimentare con una quantità di specifiche sostanze antiossidanti che sono in grado di svolgere realmente un'attività antiossidante all'interno dell'organismo umano. Therefore, it is necessary to integrate a food diet with a quantity of specific antioxidant substances that are able to really carry out an antioxidant activity within the human organism.
In particolare, si rende necessario poter disporre di una composizione che e' in grado, una volta introdotta nell'organismo, di integrare la quantità di sostanze antiossidanti, normalmente presenti nell'organismo, in modo da contribuire alla riduzione dello stress ossidativo. In particular, it is necessary to have a composition that is able, once introduced into the organism, to integrate the quantity of antioxidant substances, normally present in the organism, in order to contribute to the reduction of oxidative stress.
Infine, si rende necessario poter disporre di una composizione che e' in grado efficacemente di mantenere elevate le difese antiossidanti dopo che uno stress ossidativo e'stato indotto da fattori esterni come ad esempio dopo l'assunzione di farmaci. Finally, it is necessary to have a composition that is able to effectively maintain high antioxidant defenses after oxidative stress has been induced by external factors such as after taking drugs.
Pertanto, forma oggetto della presente invenzione una composizione avente le caratteristiche riportate nell'unita rivendicazione. Therefore, the subject of the present invention is a composition having the characteristics reported in the attached claim.
Inoltre, forma un altro oggetto della presente invenzione l'uso di detta composizione, come riportato nell'unita rivendicazione. Furthermore, another object of the present invention is the use of said composition, as reported in the accompanying claim.
Altre realizzazioni preferite della presente invenzione sono descritte nel prosieguo della descrizione e saranno rivendicate nelle unite rivendicazioni dipendenti. Other preferred embodiments of the present invention are described in the rest of the description and will be claimed in the attached dependent claims.
La figura 1 si riferisce ad un istogramma che riporta i valori di attività antiossidante totale (TAA) rispetto a: Figure 1 refers to a histogram that shows the values of total antioxidant activity (TAA) with respect to:
- C (campione di controllo in condizioni basali), - C (control sample in baseline conditions),
- T2 (campione di controllo trattato con i batteri probiotici della presente invenzione alla dose di 10<8>/die in condizioni basali), - T2 (control sample treated with the probiotic bacteria of the present invention at a dose of 10 <8> / day in basal conditions),
- C+DOX (campione di controllo trattato con Doxorubicina), - C + DOX (control sample treated with Doxorubicin),
- T1+D0X (campione trattato con i batteri probiotici della presente invenzione alla dose di 10<9>/die e Doxorubicina), - T1 + D0X (sample treated with the probiotic bacteria of the present invention at a dose of 10 <9> / day and Doxorubicin),
- T2+D0X (campione trattato con i batteri probiotici della presente invenzione alla dose di 10<8>/die e Doxorubicina), e - T2 + D0X (sample treated with the probiotic bacteria of the present invention at a dose of 10 <8> / day and Doxorubicin), and
- T3+D0X (campione trattato con i batteri probiotici della presente invenzione alla dose di 10<7>/die e Doxorubicina). - T3 + D0X (sample treated with the probiotic bacteria of the present invention at a dose of 10 <7> / day and Doxorubicin).
La figura 2 si riferisce ad un istogramma che riporta i valori di concentrazione di Glutatione piasmatico (la forma antiossidante del Glutatione à ̈ riferita al Glutatione ridotto GSH), come valutazione della capacità antiossidante, rispetto a: Figure 2 refers to a histogram showing the concentration values of piasmatic Glutathione (the antioxidant form of Glutathione refers to reduced Glutathione GSH), as an evaluation of the antioxidant capacity, with respect to:
- C (campione di controllo in condizioni basali), - C (control sample in baseline conditions),
- T2 (campione di controllo trattato con i batteri probiotici della presente invenzione alla dose di 10<8>/die in condizioni basali), - T2 (control sample treated with the probiotic bacteria of the present invention at a dose of 10 <8> / day in basal conditions),
- C+DOX (campione trattato con Doxorubicina), - C + DOX (sample treated with Doxorubicin),
- T1+D0X (campione trattato con i batteri probiotici della presente invenzione alla dose di 10<9>/die e Doxorubicina), - T1 + D0X (sample treated with the probiotic bacteria of the present invention at a dose of 10 <9> / day and Doxorubicin),
- T2+D0X (campione trattato con i batteri probiotici della presente invenzione alla dose di 10<8>/die e Doxorubicina), e - T2 + D0X (sample treated with the probiotic bacteria of the present invention at a dose of 10 <8> / day and Doxorubicin), and
- T3+D0X (campione trattato con i batteri probiotici della presente invenzione alla dose di 10<7>/die e Doxorubicina). - T3 + D0X (sample treated with the probiotic bacteria of the present invention at a dose of 10 <7> / day and Doxorubicin).
In Tabella 1 sono riportati, a titolo esemplificativo, un gruppo di microrganismi che trovano valida applicazione nel contesto della presente invenzione. Table 1 shows, by way of example, a group of microorganisms which find valid application in the context of the present invention.
La Richiedente ha condotto un'intensa attività di ricerca a seguito della quale ha trovato che i ceppi di batteri appartenenti a una specie scelta dal gruppo comprendente Lactobacillus acidophilus , Lactobacillus brevis, e Bifidobacterium lactis mostrano un'interessante attività antiossidante grazie alla quale e' possibile utilizzare i ceppi selezionati in una composizione per uso come medicamento per ridurre lo stress ossidativo. The Applicant has carried out an intense research activity as a result of which it has found that the strains of bacteria belonging to a species chosen from the group including Lactobacillus acidophilus, Lactobacillus brevis, and Bifidobacterium lactis show an interesting antioxidant activity thanks to which it is possible use the selected strains in a composition for use as a medicament to reduce oxidative stress.
Vantaggiosamente, la composizione della presente invenzione trova impiego nei casi in cui lo stress ossidativo e' indotto a seguito dell'assunzione di farmaci, da parte di un soggetto che si trova sotto cure mediche. Advantageously, the composition of the present invention is used in cases in which oxidative stress is induced following the assumption of drugs by a subject who is under medical treatment.
In una realizzazione preferita, la composizione della presente invenzione può comprendere una miscela di ceppi che contiene uno o più ceppi di batteri appartenenti alla specie Lactobacillus acidophilus, ad esempio due o tre ceppi; uno o più ceppi di batteri appartenenti alla specie Lactobacillus brevis, ad esempio due o tre ceppi; e uno o più ceppi di batteri appartenenti alla specie Bifidobacterium lactis, ad esempio due o tre ceppi. In a preferred embodiment, the composition of the present invention can comprise a mixture of strains containing one or more strains of bacteria belonging to the Lactobacillus acidophilus species, for example two or three strains; one or more strains of bacteria belonging to the Lactobacillus brevis species, for example two or three strains; and one or more strains of bacteria belonging to the species Bifidobacterium lactis, for example two or three strains.
In una realizzazione preferita, la composizione della presente invenzione comprende una miscela contenente almeno un ceppo scelto dal gruppo comprendente: In a preferred embodiment, the composition of the present invention comprises a mixture containing at least one strain selected from the group comprising:
- Bifidobacterium lactis BS 05 (ID 1666) depositato da Probiotical SpA, Novara (Italia) il 13.10.2009 presso il DSMZ in Germania e avente numero di deposito DSM 23032, e/o - Bifidobacterium lactis BS 05 (ID 1666) filed by Probiotical SpA, Novara (Italy) on 13.10.2009 at the DSMZ in Germany and having filing number DSM 23032, and / or
- Lactobacillus acidophilus LA 06 (ID 1683) depositato da Probiotical SpA, Novara (Italia) il 13.10.2009 presso il DSMZ in Germania e avente numero di deposito DSM 23033, e/o - Lactobacillus acidophilus LA 06 (ID 1683) filed by Probiotical SpA, Novara (Italy) on 13.10.2009 at the DSMZ in Germany and having the filing number DSM 23033, and / or
- Lactobacillus brevis LBR01 (ID 1685) depositato da Probiotical SpA, Novara (Italia) il 13.10.2009 presso il DSMZ in Germania e avente numero di deposito DSM 23034. - Lactobacillus brevis LBR01 (ID 1685) deposited by Probiotical SpA, Novara (Italy) on 13.10.2009 at the DSMZ in Germany and having DSM filing number 23034.
Vantaggiosamente, la miscela di ceppi di batteri e' costituita da Bifidobacterium lactis BS 05 (ID 1666) DSM 23032, Lactobacillus acidophilus LA 06 (ID 1683) DSM 23033 e Lactobacillus brevis LBROl (ID 1685) DSM 23034. Advantageously, the mixture of bacterial strains consists of Bifidobacterium lactis BS 05 (ID 1666) DSM 23032, Lactobacillus acidophilus LA 06 (ID 1683) DSM 23033 and Lactobacillus brevis LBROl (ID 1685) DSM 23034.
I ceppi di batteri, nel contesto della presente invenzione, possono essere in forma di batteri vivi o batteri morti o loro componenti cellulari, estratti cellulari e/o batteri inattivati, lisati o permeabilizzati. The strains of bacteria, in the context of the present invention, can be in the form of live bacteria or dead bacteria or their cellular components, cell extracts and / or inactivated, lysed or permeabilized bacteria.
La composizione della presente invenzione può essere una composizione alimentare, ad esempio una composizione simbiotica, oppure un integratore o una composizione farmaceutica. The composition of the present invention can be a food composition, for example a symbiotic composition, or a supplement or a pharmaceutical composition.
In una realizzazione preferita, la composizione può inoltre comprendere uno o più ceppi dì batteri tra quelli riportati in Tabella 2. In a preferred embodiment, the composition can also comprise one or more strains of bacteria among those reported in Table 2.
Preferibilmente, la composizione può inoltre comprendere da uno a sei ceppi, ancora più preferibilmente da uno a tre ceppi scelti tra quelli riportati in Tabella 2. Preferably, the composition can furthermore comprise from one to six strains, even more preferably from one to three strains selected from those reported in Table 2.
Ceppi particolarmente preferiti sono scelti tra quelli riportati in Tabella 2, identificati con il numero presente nella colonna a sinistra: N.<0>5, N.° 20, N.° 42, N.° 49, N.° 80, N.° 81, N.° 92, N.° 93, N.° 99, N.° 100, e N.° 101. Particularly preferred strains are selected from those reported in Table 2, identified with the number in the left column: N. <0> 5, N. ° 20, N. ° 42, N. ° 49, N. ° 80, N . ° 81, No. 92, No. 93, No. 99, No. 100, and No. 101.
I ceppi N.° 99, N.° 100, e N.° 101 sono particolarmente preferiti in quanto dotati di una spiccata attività antiossidante. Strains No. 99, No. 100, and No. 101 are particularly preferred as they have a marked antioxidant activity.
I ceppi N.° 5 e N.° 20 sono dotati tra l'altro anche di proprietà anti-infiammatorie . Strains No. 5 and No. 20 are also endowed with anti-inflammatory properties.
I ceppi N.° 42, N.° 80 e N.° 81 sono anche in grado di combattere infezioni, ad esempio, infezioni intestinali da lieviti, includendo la Candida. Strains No. 42, No. 80 and No. 81 are also capable of fighting infections, for example, intestinal yeast infections, including Candida.
II ceppo N.° 49 Ã ̈ in grado anche di produrre folati Strain No. 49 is also capable of producing folate
nell'intestino. in the intestine.
I ceppi N.° 5, N.° 92 e N.° 93 sono anche in grado di antagonizzare E. coli intestinale. Strains No. 5, No. 92 and No. 93 are also capable of antagonizing intestinal E. coli.
Tutti i ceppi descritti e/o rivendicati nella presente domanda di brevetto sono stati depositati in accordo al Trattato di Budapest e sono accessibili al pubblico su richiesta alla competente Autorità di Deposito. All the strains described and / or claimed in this patent application have been filed in accordance with the Budapest Treaty and are accessible to the public upon request to the competent Filing Authority.
Vantaggiosamente, la composizione della presente invenzione può comprendere elementi o sostanze ad attività antiossidante quali ad esempio selenio, zinco, magnesio, manganese, glutatione, superossidodismutasi (SOD), vitamina C, vitamina E, beta-carotene, carotenoidi, riboflavina, taurina, L-carnosina, astaxantina, licopene, olio di semi di pomodoro, quercetina, tirosolo, resveratrolo, idrossitirosolo, oleuropeina, luteina, spirulina, capsaicina, propoli, ginseng, ginkgo biloba, coenzima Q10, acido alfa-lipoico, acidi grassi ω-3 insaturi quali, ad esempio, acido docosaesaenoico (DHA) e acido eicosapentaenoico (EPA), estratto di bacche quali ad esempio estratto di mirtillo, cranberry, ribes, uva bianca, estratto di thà ̈ verde, cactus, carciofo, papaya, melone, mela, luppolo, camelia, trifoglio rosso, sambuco, rosmarino, cacao, foglie di olivo, corteccia di pino, ostrica e altri estratti vegetali contenenti polifenoli in una quantità in peso superiore all'1%. Advantageously, the composition of the present invention can comprise elements or substances with antioxidant activity such as for example selenium, zinc, magnesium, manganese, glutathione, superoxide dismutase (SOD), vitamin C, vitamin E, beta-carotene, carotenoids, riboflavin, taurine, L -carnosine, astaxanthin, lycopene, tomato seed oil, quercetin, tyrosol, resveratrol, hydroxytyrosol, oleuropein, lutein, spirulina, capsaicin, propolis, ginseng, ginkgo biloba, coenzyme Q10, alpha-lipoic acid, fatty acids Ï ‰ -3 unsaturated such as, for example, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), berry extract such as bilberry, cranberry, currant, white grape extract, green tea extract, cactus, artichoke, papaya, melon, apple , hops, camellia, red clover, elderberry, rosemary, cocoa, olive leaves, pine bark, oyster and other plant extracts containing polyphenols in a quantity over 1% by weight.
Vantaggiosamente, detti estratti vegetali possono essere stati previamente sottoposti ad almeno una fase di fermentazione. In una realizzazione preferita, della presente invenzione, trova impiego una papaya fermentata. La papaya fermentata viene prodotta ed estratta dai frutti di papaya e da alcune erbe tropicali in seguito alla fermentazione da parte di lieviti (preferibilmente Saccharomyces) e batteri. La papaya fermentata aumenta l'attività dei ceppi oggetto della presente invenzione, oltre che per l'attività antiossidante, anche per la riduzione dei radicali liberi, l'inibizione della perossidasi lipidica, il rinforzo del sistema immunitario, le proprietà alcalinizzanti, la chelazione di metalli di transizione sia in sistemi sperimentali in vitro che in vivo con conseguente azione antisettica nei confronti di microrganismi responsabili di infezioni intestinali. Advantageously, said plant extracts may have previously been subjected to at least one fermentation step. In a preferred embodiment of the present invention, a fermented papaya is employed. Fermented papaya is produced and extracted from papaya fruits and some tropical herbs following fermentation by yeasts (preferably Saccharomyces) and bacteria. Fermented papaya increases the activity of the strains object of the present invention, as well as for the antioxidant activity, also for the reduction of free radicals, the inhibition of lipid peroxidase, the strengthening of the immune system, the alkalizing properties, the chelation of transition metals both in in vitro and in vivo experimental systems with consequent antiseptic action against microorganisms responsible for intestinal infections.
Detti elementi o sostanze ad attività antiossidante di cui sopra sono addizionati in una quantità in peso compresa da 0,0001% a 30% rispetto al peso della composizione finale, in funzione della concentrazione delle sostanze ad attività antiossidante e/o della dose giornaliera raccomandata (RDA), ove definita. The above elements or substances with antioxidant activity are added in a quantity by weight ranging from 0.0001% to 30% with respect to the weight of the final composition, depending on the concentration of the substances with antioxidant activity and / or the recommended daily dose ( RDA), where defined.
Il selenio può essere presente in forma di sodio seleniato, sodio selenito, selenito acido di sodio, nonché in forma di microrganismi, ad esempio lieviti, arricchiti in selenio, in una quantità in peso compresa da 0,0005% a 0,005% rispetto al peso della composizione finale, in ogni caso sufficiente ad apportare un quantitativo di selenio preferibilmente compreso da 10 pg a 150 pg. Selenium can be present in the form of sodium selenate, sodium selenite, sodium acid selenite, as well as in the form of microorganisms, for example yeasts, enriched in selenium, in an amount by weight ranging from 0.0005% to 0.005% with respect to weight of the final composition, in any case sufficient to add a quantity of selenium preferably comprised from 10 pg to 150 pg.
In una realizzazione preferita, la composizione della presente invenzione comprende inoltre uno o più ceppi di batteri in grado di internalizzare il selenio. In a preferred embodiment, the composition of the present invention further comprises one or more strains of bacteria capable of internalizing selenium.
Trovano, in particolare, applicazione i ceppi depositati dalla Società BIOMAN S.r.l., Via Alfieri 18, 10100 Torino, quali: In particular, the strains deposited by the Company BIOMAN S.r.l., Via Alfieri 18, 10100 Turin, are applied, such as:
Lactobacillus buchneri LB26BM, depositato in data 05/04/2004 presso la DSMZ e avente numero di deposito DSM 16341; Lactobacillus buchneri LB26BM, filed on 05/04/2004 at the DSMZ and having DSM filing number 16341;
Lactobacillus ferintoshensis LB6BM, depositato in data 17/01/2004 presso la DSMZ e avente numero di deposito DSM 16144; Lactobacillus ferintoshensis LB6BM, filed on 01/17/2004 with the DSMZ and having filing number DSM 16144;
Lactobacillus reuteri LB2BM, depositato in data 17/01/2004 presso la DSMZ e avente numero di deposito DSM 16143, in associazione con i ceppi ad attività antiossidante della presente invenzione. Lactobacillus reuteri LB2BM, filed on 17/01/2004 at the DSMZ and having filing number DSM 16143, in association with the antioxidant activity strains of the present invention.
Detti ceppi, infatti, sono in grado di accumulare all'interno della cellula elevati quantitativi di selenio, soprattutto in forma organica, se cresciuti in presenza di un'opportuna fonte di selenio nel terreno di coltura. These strains, in fact, are able to accumulate high quantities of selenium inside the cell, especially in organic form, if grown in the presence of an appropriate source of selenium in the culture medium.
Il glutatione à ̈ un forte antiossidante, sicuramente uno dei più importanti tra quelli che 1 "organismo à ̈ in grado di produrre. Glutathione is a strong antioxidant, certainly one of the most important among those that the body is able to produce.
Rilevante à ̈ la sua azione sia contro i radicali liberi o molecole come perossido di idrogeno, nitriti, nitrati, benzoati e altre. Svolge un"importante azione nel globulo rosso, proteggendo tali cellule da pericoli ossidativi che causerebbero l'emolisi. In particolare, la forma antiossidante à ̈ riferita al glutatione ridotto (o GSH). Relevant is its action both against free radicals or molecules such as hydrogen peroxide, nitrites, nitrates, benzoates and others. It plays an important role in the red blood cell, protecting these cells from oxidative dangers that would cause hemolysis. In particular, the antioxidant form refers to reduced glutathione (or GSH).
In una realizzazione preferita, la composizione comprende il glutatione in forma ridotta e il selenio in una quantita'in peso compresa da 0,5% al 10%, rispetto al peso della composizione finale . In a preferred embodiment, the composition comprises glutathione in reduced form and selenium in an amount by weight ranging from 0.5% to 10%, with respect to the weight of the final composition.
Vantaggiosamente, poiché il glutatione può essere parzialmente inattivato se assunto per via orale, la composizione può comprendere 1'amminoacido solforato cisteina e/o N-acetilcisteina e/o loro miscele. Advantageously, since glutathione can be partially inactivated if taken orally, the composition can comprise the sulfur amino acid cysteine and / or N-acetylcysteine and / or their mixtures.
In una realizzazione preferita, trova impiego l'olio di semi di pomodoro, in quanto particolarmente ricco di licopene, carotenoide con spiccata attività antiossidante, in associazione con i ceppi antiossidanti della presente invenzione. In a preferred embodiment, tomato seed oil is used, as it is particularly rich in lycopene, a carotenoid with a marked antioxidant activity, in association with the antioxidant strains of the present invention.
Nella composizione della presente invenzione, la miscela di ceppi di batteri e' presente in una quantità compresa da 0,5 a 20% in peso, rispetto al peso totale della composizione, preferibilmente da 2,5 a 8%. In the composition of the present invention, the mixture of strains of bacteria is present in an amount ranging from 0.5 to 20% by weight, with respect to the total weight of the composition, preferably from 2.5 to 8%.
In una realizzazione preferita, la composizione può inoltre comprendere almeno una fibra prebiotica e/o carboidrati ad azione bifidogena quali ad esempio inulina, frutto-oligosaccaridi (FOS), gaiatto- e trans-galatto-oligosaccaridi (GOS e TOS), In a preferred embodiment, the composition can further comprise at least one prebiotic fiber and / or carbohydrates with bifidogenic action such as for example inulin, fructo-oligosaccharides (FOS), gaiatto- and trans-galacto-oligosaccharides (GOS and TOS),
gluco-oligosaccaridi (GOSa), xilo-oligosaccaridi (XOS), chitosan-oligosaccaridi (COS), soia-oligosaccaridi (SOS), isomalto-oligosaccaridi (IMOS), amido resistente, pectine, psyllium, arabino-galattani, glucomannani, galattomannani, xilani, lattosaccarosio , lattulosio, lattatolo e vari altri tipi di gomme, fibra di acacia, di carruba, di avena, di bamboo, fibre di agrumi e in generale, fibre contenenti una porzione solubile ed una insolubile, in rapporto variabile tra di loro. gluco-oligosaccharides (GOSa), xylo-oligosaccharides (XOS), chitosan-oligosaccharides (COS), soy-oligosaccharides (SOS), isomalt-oligosaccharides (IMOS), resistant starch, pectins, psyllium, arabino-galactans, glucomannans, galactomannans, xylans, lactosaccharose, lactulose, lactatol and various other types of gums, acacia, carob, oat, bamboo fibers, citrus fibers and in general, fibers containing a soluble and an insoluble portion, in variable ratio between them.
In una realizzazione preferita dell'invenzione, la composizione comprende almeno una fibra prebiotica scelta tra quelle sopra enzionate e/o loro opportune miscele in qualunque percentuale relativa . In a preferred embodiment of the invention, the composition comprises at least one prebiotic fiber selected from those mentioned above and / or suitable mixtures thereof in any relative percentage.
La quantità delle fibre prebiotiche e/o dei carboidrati ad azione bifidogena, se presenti nella composizione, risulta compresa da 0 a 60% in peso, preferibilmente dal 5 al 45% ed ancora più preferibilmente dal 10 al 30%, rispetto al peso totale della composizione. In questo caso la composizione o integratore ha attività simbiotica e proprietà funzionali. The quantity of prebiotic fibers and / or carbohydrates with bifidogenic action, if present in the composition, ranges from 0 to 60% by weight, preferably from 5 to 45% and even more preferably from 10 to 30%, with respect to the total weight of the composition. In this case the composition or supplement has symbiotic activity and functional properties.
Inoltre, la parte interna del prodotto alimentare o integratore può comprendere anche altri ingredienti e/o componenti attivi quali vitamine, minerali, peptidi bioattivi, sostanze ad attività antiossidante, ipocolesterolemizzante , ipoglicemizzante , antiinf iammatoria, anti edulcoranti in una quantità in peso generalmente compresa da 0,001% a 20% in peso, preferibilmente da 0,01% a 5%, comunque dipendente dal tipo di componente attivo e dalla sua eventuale dose giornaliera consigliata, rispetto al peso totale della composizione. Furthermore, the internal part of the food product or supplement may also include other ingredients and / or active components such as vitamins, minerals, bioactive peptides, substances with antioxidant, hypocholesterolemic, hypoglycemic, anti-inflammatory, anti-sweeteners activity in a quantity by weight generally included by 0.001% to 20% by weight, preferably from 0.01% to 5%, in any case depending on the type of active component and its possible recommended daily dose, with respect to the total weight of the composition.
La composizione alimentare oggetto della presente invenzione, ad esempio una composizione simbiotica, oppure un integratore o una composizione farmaceutica, viene preparata secondo le tecniche e le apparecchiature note all'esperto del settore. The food composition object of the present invention, for example a symbiotic composition, or a supplement or a pharmaceutical composition, is prepared according to the techniques and equipment known to those skilled in the art.
In una realizzazione preferita, la composizione contiene batteri in una concentrazione compresa da 1x10® a lxl0-^ UFC/g di miscela, preferibilmente da 1x10® a 1x10^° UFC/g di miscela. In a preferred embodiment, the composition contains bacteria in a concentration ranging from 1x10® to 1x10- ^ CFU / g of mixture, preferably from 1x10® to 1x10 ° UFC / g of mixture.
In una realizzazione preferita, la composizione contiene batteri in una concentrazione compresa da 1x10® a lxlO<11>UFC/dose, preferibilmente da 1x10® a lxlO<1>® UFC/dose. In a preferred embodiment, the composition contains bacteria in a concentration ranging from 1x10® to lxlO <11> CFU / dose, preferably from 1x10® to lxlO <1> ® UFC / dose.
La dose può essere compresa da 0,2 a 10 g, ad esempio e'di 0,25 g, 1 g, 3 g, 5 g o 7 g. The dose can range from 0.2 to 10 g, for example it is 0.25 g, 1 g, 3 g, 5 g or 7 g.
I batteri probiotici utilizzati nella presente invenzione possono essere in forma solida, in particolare sotto forma di polvere, polvere disidratata o liofilizzata. The probiotic bacteria used in the present invention can be in solid form, in particular in the form of powder, dehydrated or lyophilized powder.
In una realizzazione preferita, la miscela di ceppi di batteri comprende almeno un ceppo scelto tra Bifidobacterium lactis BS 05 (ID 1666) DSM 23032, Lactobacillus acidophilus LA 06 (ID 1683) DSM 23033 e Lactobacillus brevis LBR01 (ID 1685) DSM 23034 in forma microincapsulata, cioà ̈ detto almeno un ceppo (oppure tutti e tre detti ceppi di batteri) viene rivestito con una composizione contenente almeno un lipide, preferibilmente di origine vegetale. I batteri microincapsulati sono poi aggiunti, con modalità operative note all'esperto del settore, ad una composizione liquida a base di olio, a dare una sospensione oleosa. In a preferred embodiment, the bacterial strain mixture comprises at least one strain selected from Bifidobacterium lactis BS 05 (ID 1666) DSM 23032, Lactobacillus acidophilus LA 06 (ID 1683) DSM 23033 and Lactobacillus brevis LBR01 (ID 1685) DSM 23034 in form microencapsulated, ie at least one strain (or all three said strains of bacteria) is coated with a composition containing at least one lipid, preferably of vegetable origin. The microencapsulated bacteria are then added, with operating methods known to the expert in the field, to an oil-based liquid composition, to give an oily suspension.
I batteri sopra citati che vengono aggiunti alla composizione liquida a base di olio possono essere sotto forma di batteri microincapsulati e/o batteri "nudi" non microincapsulati, o miscele degli stessi. The aforementioned bacteria which are added to the oil-based liquid composition can be in the form of microencapsulated bacteria and / or non-microencapsulated "naked" bacteria, or mixtures thereof.
I batteri scelti tra Bifidobacterium lactis BS 05 (ID 1666) DSM 23032, Lactobacillus acidophilus LA 06 (ID 1683) DSM 23033 e Lactobacillus brevis LBR01 (ID 1685) DSM 23034, preferibilmente in forma microincapsulata, possono essere microincapsulati mediante le comuni tecnologie note all'esperto del settore. Ad esempio, può essere impiegata una tecnica a letto fluido (ad esempio, top-spray o bottom-spray) nella quale vengono utilizzati dei materiali di rivestimento di natura lipidica. Bacteria selected from Bifidobacterium lactis BS 05 (ID 1666) DSM 23032, Lactobacillus acidophilus LA 06 (ID 1683) DSM 23033 and Lactobacillus brevis LBR01 (ID 1685) DSM 23034, preferably in microencapsulated form, can be microencapsulated using common technologies known to industry expert. For example, a fluidized bed technique (for example, top-spray or bottom-spray) can be employed in which lipid-based coating materials are used.
In una realizzazione preferita, vengono utilizzati i grassi saturi vegetali aventi un punto di fusione inferiore a 75°C, preferibilmente compreso da 45 a 65°C. In a preferred embodiment, saturated vegetable fats having a melting point lower than 75 ° C, preferably between 45 and 65 ° C, are used.
In una realizzazione preferita, si possono utilizzare grassi saturi vegetali aventi un certo grado di idrofilicita' i quali ossono essere scelti tra i mono- e di-gliceridi di acidi grassi saturi, i poligliceroli esterificati con acidi grassi saturi e gli acidi grassi saturi liberi. In a preferred embodiment, saturated vegetable fats having a certain degree of hydrophilicity can be used which can be selected from mono- and di-glycerides of saturated fatty acids, polyglycerols esterified with saturated fatty acids and free saturated fatty acids.
d esempio, si possono utilizzare il poligliceril distearato (nome commerciale Plurol Stearique WL 1009), il gliceril palmìtostearato (nome commerciale Precirol Ato 5), gli acidi grassi saturi (nome commerciale Revel C) o i grassi vegetali idrogenati di origine non laurica . For example, polyglyceryl distearate (trade name Plurol Stearique WL 1009), glyceryl palmìtostearate (trade name Precirol Ato 5), saturated fatty acids (trade name Revel C) or hydrogenated vegetable fats of non-lauric origin can be used.
In una realizzazione preferita, il rapporto in peso tra microorganismo liofilizzato e materiale di rivestimento lipidico che lo riveste à ̈ 50:50 o 40:60. In a preferred embodiment, the weight ratio of the lyophilized microorganism to the lipid coating material that coats it is 50:50 or 40:60.
In una prima realizzazione, due lipidi scelti tra un grasso di palma idrogenato (Tf=60°C) e un dipalmito-stearato di glicerolo (Tf=57-60°C) vengono sprayzzati sul liofilizzato in successione, cioà ̈ sul liofilizzato viene applicata una doppia ricopertura: la prima con il grasso di palma idrogenato e la seconda con il dipalmito-stearato di glicerolo in rapporto 3:1 tra loro. Un doppio rivestimento delle cellule garantisce una migliore impermeabilizzazione dei batteri dall'ambiente, determinando una pellicola continua senza pori di comunicazione con l'esterno. In a first embodiment, two lipids selected from a hydrogenated palm fat (Tf = 60 ° C) and a glycerol dipalmito-stearate (Tf = 57-60 ° C) are sprayed on the lyophilisate in succession, i.e. on the lyophilisate it is applied a double coating: the first with hydrogenated palm fat and the second with glycerol dipalmito-stearate in a 3: 1 ratio. A double coating of the cells guarantees a better waterproofing of bacteria from the environment, resulting in a continuous film without pores of communication with the outside.
Tuttavia, questo involucro si deve aprire a livello intestinale per rilasciare i batteri e permetterne la colonizzazione. I lipidi scelti sono infatti resistenti a pH acidi, perché il rivestimento rimanga intatto nello stomaco, ma sensibili a pH anche leggermente basici, in modo da consentire la formazione di fori nel rivestimento durante il transito intestinale. However, this envelope must open in the intestine to release the bacteria and allow them to colonize. The lipids chosen are in fact resistant to acid pH, so that the lining remains intact in the stomach, but sensitive to even slightly basic pH, so as to allow the formation of holes in the lining during intestinal transit.
In una realizzazione preferita, la composizione della presente invenzione e' una composizione a base di olio comprendente batteri lattici rivestiti come sopra citato. In a preferred embodiment, the composition of the present invention is an oil-based composition comprising coated lactic bacteria as mentioned above.
Detta composizione viene preparata secondo tecniche note all'esperto del settore. Said composition is prepared according to techniques known to those skilled in the art.
In pratica, una determinata quantità di olio viene introdotta in un contenitore munito di mezzi di agitazione e di riscaldamento. Successivamente, i batteri probiotici rivestiti in forma solida vengono gradualmente aggiunti, sotto agitazione, evitando la formazione di grumi e agglomerati. Terminata l'aggiunta dei batteri si mantiene la sospensione oleosa in agitazione per un tempo compreso da 1 a 30 minuti, eventualmente mediante leggero riscaldamento ad una temperatura compresa da 25 a 40°C, preferibilmente da 30 a 35°C. In practice, a certain quantity of oil is introduced into a container equipped with stirring and heating means. Subsequently, the probiotic bacteria coated in solid form are gradually added, under stirring, avoiding the formation of lumps and agglomerates. At the end of the addition of the bacteria, the oily suspension is kept under stirring for a time ranging from 1 to 30 minutes, possibly by light heating at a temperature ranging from 25 to 40 ° C, preferably from 30 to 35 ° C.
La composizione che si ottiene e' simile ad una sospensione oleosa. La composizione contiene i batteri in una quantità minore o uguale al 30% in peso, compresa da 0,05 a 20% in peso, rispetto al peso totale della composizione; preferibilmente in una quantità compresa da 0,5 a 10%; ancora più preferibilmente in una quantità compresa da 1,5 a 5% in peso, rispetto al peso totale della composizione . The resulting composition is similar to an oily suspension. The composition contains the bacteria in an amount less than or equal to 30% by weight, comprised between 0.05 and 20% by weight, with respect to the total weight of the composition; preferably in an amount ranging from 0.5 to 10%; even more preferably in an amount ranging from 1.5 to 5% by weight, with respect to the total weight of the composition.
La composizione comprende almeno un olio, commestibile e idoneo a essere somministrato a soggetti in età pediatrica, scelto dal gruppo comprendente: olio di oliva, olio di mais, olio di soia, olio di lino, olio di arachide, olio di sesamo, olio di pesce e olio di riso e altri oli di semi tra cui, in particolare, trova impiego l'olio di semi di pomodoro. The composition includes at least one oil, edible and suitable for administration to pediatric subjects, chosen from the group comprising: olive oil, corn oil, soybean oil, linseed oil, peanut oil, sesame oil, fish and rice oil and other seed oils including, in particular, tomato seed oil.
Detti oli possono essere utilizzati singolarmente o in opportune miscele tra loro, in rapporti ponderali appropriati. Said oils can be used individually or in suitable mixtures with each other, in appropriate weight ratios.
Vantaggiosamente, detti oli sono di grado biologico e, nella loro preparazione, possono prevedere una fase di raffinazione e/o una fase di pressatura a freddo. Advantageously, said oils are of biological grade and, in their preparation, they can include a refining step and / or a cold pressing step.
La composizione comprende almeno un olio in una quantità maggiore o uguale al 70% in peso, rispetto al peso totale della sospensione, preferibilmente in una quantità compresa da 75 a 95% in peso, vantaggiosamente almeno 90% in peso. The composition comprises at least one oil in an amount greater than or equal to 70% by weight, with respect to the total weight of the suspension, preferably in an amount ranging from 75 to 95% by weight, advantageously at least 90% by weight.
Vantaggiosamente, la composizione contiene solo olio di oliva oppure olio di oliva in miscela con olio di mais e/o olio di soia e/o olio di lino e/o olio di semi di pomodoro. Vantaggiosamente, l'olio di oliva e' extravergine e di grado Bio. Advantageously, the composition contains only olive oil or olive oil mixed with corn oil and / or soybean oil and / or linseed oil and / or tomato seed oil. Advantageously, the olive oil is extra virgin and Bio grade.
In una realizzazione preferita, la composizione comprende inoltre almeno un composto alimentare finemente suddiviso scelto dal gruppo comprendente silice, biossido di silicio, gel di silice, silice colloidale, silice precipitata, Syloid 244, talco, magnesio silicato, magnesio ossido, magnesio carbonato, calcio silicato, lecitina, mono- o di-gliceridi quali gliceril monostearato , gliceril monooleato, acido plurol-oleico, amido, amidi modificati, gomma Konjac, gomma xantano, gomma gellano, carragenine . In a preferred embodiment, the composition further comprises at least one finely divided food compound selected from the group comprising silica, silicon dioxide, silica gel, colloidal silica, precipitated silica, Syloid 244, talc, magnesium silicate, magnesium oxide, magnesium carbonate, calcium silicate, lecithin, mono- or di-glycerides such as glyceryl monostearate, glyceryl monooleate, plurol-oleic acid, starch, modified starches, Konjac gum, xanthan gum, gellan gum, carrageenans.
Detto materiale e' presente in una quantità compresa da 0,1 a 15% in peso, rispetto al peso totale della composizione, preferibilmente da 1 a 5% in peso, rispetto al peso totale della composizione . Said material is present in an amount ranging from 0.1 to 15% by weight, with respect to the total weight of the composition, preferably from 1 to 5% by weight, with respect to the total weight of the composition.
In questo caso il procedimento di preparazione prevede che, a una determinata quantità di olio, venga addizionato sotto agitazione il materiale alimentare finemente suddiviso, ad esempio biossido di silicio. Successivamente, l'olio contenente detto materiale viene riscaldato sotto agitazione a circa 60°C, fino a completo scioglimento . In this case, the preparation process provides that the finely divided food material, for example silicon dioxide, is added under stirring to a certain quantity of oil. Subsequently, the oil containing said material is heated under stirring to about 60 ° C, until it is completely dissolved.
Alternativamente, il biossido di silicio può essere addizionato anche a freddo, però la solubilizzazione necessita di un tempo più lungo . Alternatively, silicon dioxide can also be added when cold, but solubilization requires a longer time.
Successivamente, la composizione viene lasciata raffreddare da 60°C a temperatura ambiente. Poi, si pesa il liofilizzato che viene addizionato alla sospensione sotto agitazione, fino a completa ed omogenea dispersione. La composizione che si ottiene e" simile a una sospensione oleosa. Thereafter, the composition is allowed to cool from 60 ° C to room temperature. Then, the lyophilisate is weighed and added to the suspension under stirring, until complete and homogeneous dispersion. The resulting composition is similar to an oily suspension.
Esempi di composizioni preferite, in accordo alla presente invenzione, sono riportati in Tabella 3 (Esempi 1-4). Examples of preferred compositions, according to the present invention, are reported in Table 3 (Examples 1-4).
Gli esempi 1-4 sono riportati, a solo scopo esemplificativo e non limitativo della presente invenzione, considerano un volume di sospensione oleosa idoneo ad un periodo di trattamento pari a 30 giorni. La carica vitale riportata, espressa in miliardi di cellule vive, si riferisce, pertanto, a 30 dosi. La singola dose à ̈ in grado di apportare, al momento della fabbricazione, 1,5 miliardi/ceppo negli esempi 1 e 2, 2,5 miliardi/ceppo negli esempi 3 e 4 . Examples 1-4 are reported, for illustrative and non-limiting purposes only of the present invention, they consider a volume of oily suspension suitable for a treatment period equal to 30 days. The reported vital charge, expressed in billions of live cells, therefore refers to 30 doses. The single dose is capable of providing, at the time of manufacture, 1.5 billion / strain in examples 1 and 2, 2.5 billion / strain in examples 3 and 4.
La Richiedente ha trovato possibile l'utilizzo di volumi diversi di sospensione oleosa, ad esempio 5 mi, idonei per periodi di trattamento più brevi. The Applicant has found it possible to use different volumes of oily suspension, for example 5 ml, suitable for shorter treatment periods.
In una realizzazione preferita, la sospensione oleosa può inoltre comprendere, in una quantità compresa dallo 0,5 al 25% in peso, rispetto al peso totale della sospensione, almeno una fibra prebiotica e/o almeno un carboidrato bifidogeno scelto tra inulina, frutto-oligosaccaridi (FOS), gaiatto e trans-galattooligosaccaridi (GOS e TOS), gluco-oligosaccaridi (GOSa), xilooligosaccaridi, (XOS), chitosan-oligosaccaridi (COS), soiaoligosaccaridi (SOS), isomalto-oligosaccaridi (IMOS), maltodestrine, amido resistente, pectine, psyllium, arabinogalattani, gluco-mannani, galatto-mannani, xilani, lattosaccarosio, lattulosio, lattitolo, fibra di acacia, fibra di carruba, fibra di avena, fibra di bamboo e fibra di agrumi. In a preferred embodiment, the oily suspension can further comprise, in an amount ranging from 0.5 to 25% by weight, with respect to the total weight of the suspension, at least one prebiotic fiber and / or at least one bifidogenic carbohydrate selected from inulin, fruit oligosaccharides (FOS), gaiatto and trans-galactooligosaccharides (GOS and TOS), gluco-oligosaccharides (GOSa), xylooligosaccharides, (XOS), chitosan-oligosaccharides (COS), soyoligosaccharides (SOS), isomalt-oligosaccharides (IMOS), resistant starch, pectins, psyllium, arabinogalactans, gluco-mannans, galacto-mannans, xylans, lactosaccharose, lactulose, lactitol, acacia fiber, carob fiber, oat fiber, bamboo fiber and citrus fiber.
Le fibre prebiotiche e i carboidrati hanno una duplice funzione. La prima e' quella di svolgere una valenza prebiotica. La seconda e" quella di svolgere una valenza tecnologica come addensante e stabilizzante . Prebiotic fibers and carbohydrates have a dual function. The first is that of carrying out a prebiotic valence. The second is "that of carrying out a technological value as a thickener and stabilizer.
Vantaggiosamente, detta almeno una fibra e detto almeno un carboidrato sono scelti tra gluco-oligosaccaridi (GOSa), fruttooligosaccaridi (FOS), inulina e/o maltodestrine. La sospensione contiene ceppi di microorganismi microincapsulati con almeno un lipide avente un punto di fusione inferiore a 75 °C, preferibilmente compreso da 45 a 65°C. Advantageously, said at least one fiber and said at least one carbohydrate are selected from gluco-oligosaccharides (GOSa), fructooligosaccharides (FOS), inulin and / or maltodextrins. The suspension contains microencapsulated strains of microorganisms with at least one lipid having a melting point lower than 75 ° C, preferably between 45 and 65 ° C.
La sospensione à ̈ indicata per uso come un medicamento per il trattamento di disturbi intestinali quali ad esempio le coliche in soggetti pediatrici. The suspension is indicated for use as a medicament for the treatment of intestinal disorders such as colic in pediatric subjects.
In un'altra realizzazione preferita, la composizione oggetto della presente invenzione à ̈ formulata in bustine. La Tabella 4 riporta gli Esempi 5-8. In another preferred embodiment, the composition object of the present invention is formulated in sachets. Table 4 shows Examples 5-8.
Nell'esempio 6, il ceppo Lactobacillus buchneri LB26BM (DSM 16341) contiene 50 Î1⁄4g/g di selenio accumulato all'interno della cellula prevalentemente in forma di seleniometionina e selenio cisteina,pertanto 1 grammo à ̈ in grado di apportare il 90% della RDA di tale elemento. In example 6, the Lactobacillus buchneri LB26BM strain (DSM 16341) contains 50 Î1⁄4g / g of selenium accumulated inside the cell mainly in the form of selenomethionine and selenium cysteine, therefore 1 gram is able to provide 90% of the RDA of this element.
Nell "esempio 7, la composizione comprende 3 grammi di papaya fermentata ad azione sinergica con il ceppo B. lactis BS05 (DSM 23032) . In Example 7, the composition comprises 3 grams of fermented papaya with synergistic action with the B. lactis BS05 strain (DSM 23032).
Di seguito sono riportate le condizioni operative per coltivare i ceppi Bifidobacterium lactis BS 05 (ID 1666) DSM 23032, Lactobacillus acidophilus LA 06 (ID 1683) DSM 23033 e Lactobacillus brevis LBR01 (ID 1685) DSM 23034. Le condizioni sono valide per tutti i ceppi ad eccezione di dove diversamente indicato . Below are the operating conditions for growing Bifidobacterium lactis BS 05 (ID 1666) DSM 23032, Lactobacillus acidophilus LA 06 (ID 1683) DSM 23033 and Lactobacillus brevis LBR01 (ID 1685) DSM 23034 strains. strains except where otherwise indicated.
i) Mezzo utilizzato: TPY brodo Cys HC1 0,5 g/1 per DSM 23032, e MRS Difco ref. 288130 per DSM 23033 e DSM 23034. i) Medium used: TPY broth Cys HC1 0.5 g / 1 for DSM 23032, and MRS Difco ref. 288130 for DSM 23033 and DSM 23034.
ii) pH del mezzo prima della sterilizzazione: 7,10 per DSM 23032 e 7,00 per DSM 23033 e DSM 23034. ii) pH of the medium before sterilization: 7.10 for DSM 23032 and 7.00 for DSM 23033 and DSM 23034.
iii) Sterilizzazione: 15 minuti a 121°C. iii) Sterilization: 15 minutes at 121 ° C.
iv) pH del mezzo dopo la sterilizzazione: 6,60 per DSM 23032 e 6,50 per DSM 23033 e DSM 23034. iv) pH of the medium after sterilization: 6.60 for DSM 23032 and 6.50 for DSM 23033 and DSM 23034.
v) Relazione con l'ossigeno: specie anaerobica obbligata per DSM 23032 e anaerobica facoltativa o microaerofila per DSM 23033 e DSM 23034. v) Relationship with oxygen: obligatory anaerobic species for DSM 23032 and facultative anaerobic or microaerophilic for DSM 23033 and DSM 23034.
vi) Temperatura di incubazione: 37°C. vi) Incubation temperature: 37 ° C.
vii) Tempo di incubazione: 17 ore per DSM 23032 e 15 ore per DSM 23033 e DSM 23034. vii) Incubation time: 17 hours for DSM 23032 and 15 hours for DSM 23033 and DSM 23034.
viii) Temperatura di conservazione a breve termine: 5°C. viii) Short-term storage temperature: 5 ° C.
ix) Tempo di trasferimento: 2 giorni. ix) Transfer time: 2 days.
x) Temperatura di conservazione a lungo termine: -25°C. x) Long-term storage temperature: -25 ° C.
xi) Condizioni per testare la vitalità : crescita in TPY brodo a 37°C per una notte o sino a quando una torbidità adeguata e" raggiunta per DSM 23032 e crescita in MRS brodo a 37°C per una notte per DSM 23033 e DSM 23034. xi) Conditions for testing viability: growth in TPY broth at 37 ° C overnight or until adequate turbidity is achieved for DSM 23032 and growth in MRS broth at 37 ° C overnight for DSM 23033 and DSM 23034 .
xii) Descrizione: bastoncelli di varie forme, gram positivo, formazione di acidità non veloce, formazione di spore negativa, anaerobico, degrada il glucosio esclusivamente e in maniera caratteristica attraverso la via della fruttosio-6-fosfato xii) Description: rods of various shapes, gram positive, non-fast acidity formation, negative spore formation, anaerobic, degrades glucose exclusively and in a characteristic way through the fructose-6-phosphate pathway
fosfochetolasi per DSM 23032; bastoncelli con terminali rotondi singolarmente o in catena, crescita buona a 37°C e metabolismo omofermentante obbligato per DSM 23033, eterofermentante obbligato per DSM 23034. phosphoketolase for DSM 23032; rods with round terminals individually or in chains, good growth at 37 ° C and obligatory homofermentative metabolism for DSM 23033, obligatory heterofermentative for DSM 23034.
Parte sperimentale Experimental part
La Richiedente ha effettuato uno screening su larga scala di numerosi batteri probiotici al fine di identificare uno o più microrganismi dotati di attività antiossidante. The Applicant carried out a large-scale screening of numerous probiotic bacteria in order to identify one or more microorganisms endowed with antioxidant activity.
La prima attività di screening à ̈ stata realizzata mediante una serie di analisi in vitro. In particolare, e" stata valutata l'attività ' antiossidante totale (TAA) sia delle cellule intere che degli estratti cellulari. The first screening activity was carried out through a series of in vitro analyzes. In particular, the total antioxidant activity (TAA) of both whole cells and cell extracts was evaluated.
Per le cellule intere l'eventuale attività antiossidante à ̈ stata indagata mediante i due test: For whole cells the possible antioxidant activity was investigated by means of the two tests:
- Autossidazione dell'acido ascorbico AA% - Autoxidation of ascorbic acid AA%
- Ossidazione dell'acido linoleico LA% - Oxidation of linoleic acid LA%
Entrambi i test quantificano la capacità del ceppo batterico, usato come cellule intere, di proteggere l'acido ascorbico o l'acido linolenico dall'ossidazione. Both tests quantify the ability of the bacterial strain, used as whole cells, to protect ascorbic acid or linolenic acid from oxidation.
Nel dettaglio, la cinetica della reazione di autossidazione dell'acido ascorbico può essere determinata spettrofotometricamente registrando a 265 nm la presenza di acido deidroascorbico , offrendo quindi una misura del potere antiossidante valutato come la capacità di inibire tale reazione di autossidazione. In detail, the kinetics of the ascorbic acid auto-oxidation reaction can be determined spectrophotometrically by recording the presence of dehydroascorbic acid at 265 nm, thus offering a measure of the antioxidant power evaluated as the ability to inhibit this auto-oxidation reaction.
Il saggio dell'acido tiobarbiturico à ̈ stato utilizzato, invece, per monitorare la capacità dei ceppi batterici di inibire la perossidazione dell'acido linolenico. L'ossidazione dell'acido linolenico, infatti, determina una catena autocatalitica che porta alla formazione di diverse specie radicaliche. Uno dei prodotti di decomposizione delle specie radicaliche à ̈ la malonaldeide, che può essere utilizzata come indicatore dello stress ossidativo nel saggio dell'acido tiobarbiturico in quanto capace di reagire con quest'ultimo per formare un complesso cromogeno Instead, the thiobarbituric acid assay was used to monitor the ability of bacterial strains to inhibit linolenic acid peroxidation. The oxidation of linolenic acid, in fact, determines an autocatalytic chain which leads to the formation of different radical species. One of the decomposition products of radical species is malonaldehyde, which can be used as an indicator of oxidative stress in the thiobarbituric acid assay as it is capable of reacting with the latter to form a chromogenic complex
rosso determinabile spettrofotometricamente a 534 nm. spectrophotometrically determinable red at 534 nm.
Il lavoro di ricerca condotto sugli estratti cellulari ha previsto, invece, i seguenti test: The research work conducted on cellular extracts, however, provided for the following tests:
- Trolox<®>Equivalence Antioxidant Capacity (TEAC) % - Trolox <®> Equivalence Antioxidant Capacity (TEAC)%
- Glutatione (GSH) nmoli/mg - Glutathione (GSH) nmoles / mg
- Superossidodismutasi (SOD) U/mg - Superoxide dismutase (SOD) U / mg
Il primo test si basa sulla reazione di molecole antiossidanti con il radicale cationico ABTSâ— + (2,2'-azinobis(3-et ilbenzotiazolin- 6-sulfonato) ). The first test is based on the reaction of antioxidant molecules with the cationic radical ABTSâ— + (2,2'-azinobis (3-et ilbenzothiazolin-6-sulfonato)).
Questo radicale può essere ridotto, con conseguente perdita di assorbanza, da un antiossidante la cui capacità scavenger può essere misurata spettrofotometricamente a 734 nm. Il test viene eseguito a 37°C e i risultati vengono ottenuti dal confronto con il Trolox<®>(antiossidante sintetico, analogo idrofilo della vitamina E) , definendo così la concentrazione millimolare di una soluzione di Trolox<®>che ha capacità antiossidante equivalente a una soluzione 1 mM della sostanza sotto esame (TEAC). This radical can be reduced, resulting in loss of absorbance, by an antioxidant whose scavenger capacity can be measured spectrophotometrically at 734 nm. The test is performed at 37 ° C and the results are obtained from the comparison with Trolox <®> (synthetic antioxidant, hydrophilic analogue of vitamin E), thus defining the millimolar concentration of a solution of Trolox <®> which has equivalent antioxidant capacity to a 1 mM solution of the test substance (TEAC).
Il secondo e il terzo test misurano la concentrazione di glutatione ridotto (GSH) e di enzima superossidodismutasi (SOD) che il singolo ceppo batterico à ̈ in grado di produrre. Entrambe le molecole sono note per avere attività antiossidante. The second and third tests measure the concentration of reduced glutathione (GSH) and superoxide dismutase (SOD) enzyme that the single bacterial strain is capable of producing. Both molecules are known to have antioxidant activity.
L'attività della superossido dismutasi (SOD) viene determinata tramite metodo spettrofotometrico, sfruttando il principio di inibizione enzimatica dell'ossidazione The activity of superoxide dismutase (SOD) is determined by a spectrophotometric method, exploiting the principle of enzymatic inhibition of oxidation
dell 'epinefrina (4- [l-idrossi-2- (metil-ammino) -etil] -1,2-benzenediolo) . L'ossidazione avviene a pH alcalino ad opera dell '02 con produzione di anioni superossido (02-) epinephrine (4- [1-hydroxy-2- (methyl-amino) -ethyl] -1,2-benzenediol). Oxidation occurs at alkaline pH by 02 with the production of superoxide anions (02-)
che, accumulandosi in soluzione, promuovono la conversione dell 'epinefrina ad adrenocromo (3-idrossi-1 -metil-5 ,6-indolindione) , un composto colorato che consente, a fronte di una misura dell 'assorbanza, di seguire il decorso della reazione. which, accumulating in solution, promote the conversion of epinephrine to adrenochrome (3-hydroxy-1-methyl-5, 6-indolindione), a colored compound that allows, against an absorbance measurement, to follow the course of reaction.
La presenza di superossido dismutasi (SOD) sottrae ioni 02- e riduce la velocità di formazione e la quantità di adrenocromo. La percentuale di inibizione dell'ossidazione à ̈ funzione iperbolica della concentrazione di SOD. The presence of superoxide dismutase (SOD) subtracts 02- ions and reduces the rate of formation and the amount of adrenochrome. The percentage of oxidation inhibition is a hyperbolic function of the SOD concentration.
L'analisi quantitativa del glutatione può essere effettuata tramite metodo spettrofotometrico che si avvale della reazione enzimatica di Ellman: il gruppo sulfidrilico del glutatione (GSH) reagisce con l'acido 5,5'-ditiobis-2-nitrobenzoico (DTNB) formando un composto di colore giallo: l'acido 5-tio-2-nitrobenzoico (TNB). Contemporaneamente, il glutatione ossidato viene nuovamente ridotto dalla glutatione reduttasi, portando all'ulteriore formazione di TNB. La velocità di formazione di TNB à ̈ direttamente proporzionale alla concentrazione di GSH totale, The quantitative analysis of glutathione can be carried out by means of a spectrophotometric method that makes use of the Ellman enzymatic reaction: the sulfhydryl group of glutathione (GSH) reacts with 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) forming a compound yellow in color: 5-thio-2-nitrobenzoic acid (TNB). At the same time, oxidized glutathione is again reduced by glutathione reductase, leading to the further formation of TNB. The rate of TNB formation is directly proportional to the total GSH concentration,
espressa come nanomoli per mg di proteine presenti nell'estratto cellulare. expressed as nanomoles per mg of proteins present in the cell extract.
I risultati sono riportati in tabella 1. The results are reported in table 1.
Dalla tabella 1 si evince che i ceppi possiedono una rilevante attività antiossidante, evidenziata sia con le cellule intere che con estratti cellulari. Table 1 shows that the strains have a significant antioxidant activity, highlighted both with whole cells and with cell extracts.
Successivamente, per confermare l'attività antiossidante dei ceppi della presente invenzione anche in un modello in vivo, la Richiedente ha effettuato uno studio su animale. Subsequently, to confirm the antioxidant activity of the strains of the present invention also in an in vivo model, the Applicant carried out an animal study.
In particolare, lo studio à ̈ stato eseguito su un campione di 48 ratti adulti sani del tipo Wistar (Harlan, Milano, Italia) i quali sono stati alimentati per 7 giorni con una dieta standard. In particular, the study was performed on a sample of 48 healthy adult rats of the Wistar type (Harlan, Milan, Italy) which were fed a standard diet for 7 days.
Successivamente, i 48 ratti sono stati suddivisi nei seguenti gruppi: Subsequently, the 48 rats were divided into the following groups:
- Gruppo C: gruppo di controllo comprendente 20 ratti alimentati per 18 giorni solo con la dieta standard, priva dei probiotici. - Group C: control group comprising 20 rats fed for 18 days only with the standard diet, devoid of probiotics.
- Gruppo TI: gruppo di studio comprendente 7 ratti alimentati per 18 giorni con una dieta addizionata di una miscela di batteri contenente i ceppi Bifidobacterium lactis BS 05 (ID 1666) DSM 23032, Lactobacillus acidophilus LA 06 (ID 1683) DSM 23033 e Lactobacillus brevis LBR01 (ID 1685) DSM 23034 in un rapporto ponderale 1:1:1 (di seguito indicata con M, per brevità ) a una concentrazione di 1x10^ ufc/die. Nel dettaglio, i suddetti ceppi probiotici sono stati miscelati con il mangime dei ratti in una concentrazione tale che nel quantitativo medio consumato quotidianamente da un singolo animale fossero presenti un totale di 1x109 UFC. - Group TI: study group comprising 7 rats fed for 18 days with a diet supplemented with a mixture of bacteria containing the strains Bifidobacterium lactis BS 05 (ID 1666) DSM 23032, Lactobacillus acidophilus LA 06 (ID 1683) DSM 23033 and Lactobacillus brevis LBR01 (ID 1685) DSM 23034 in a 1: 1: 1 weight ratio (hereinafter referred to as M, for short) at a concentration of 1x10 ^ cfu / day. In detail, the aforementioned probiotic strains were mixed with the rats' feed in such a concentration that a total of 1x109 CFU were present in the average daily quantity consumed by a single animal.
- Gruppo T2: gruppo di studio comprendente 14 ratti alimentati per 18 giorni con una dieta addizionata di miscela M, come sopra descritto, a una concentrazione di lxlO<8>ufc/die. - Group T2: study group comprising 14 rats fed for 18 days with a diet supplemented with mixture M, as described above, at a concentration of lxlO <8> cfu / day.
- Gruppo T3: gruppo di studio comprendente 7 ratti alimentati per 18 giorni con una dieta addizionata di miscela M, come sopra descritto, a una concentrazione di lxlO<7>ufc/die. - Group T3: study group comprising 7 rats fed for 18 days with a diet supplemented with mixture M, as described above, at a concentration of lxlO <7> cfu / day.
Al termine dei 18 giorni i ratti del gruppo C e del gruppo T2 sono stati suddivisi in due sottogruppi, rispettivamente dì 10 e 7 ratti ciascuno: ad un sottogruppo à ̈ stata iniettata una soluzione fisiologica (Cf e T2f), mentre all'altro una soluzione di doxorubicina (in breve DOX), farmaco antineoplastico ad attività pro-ossidante che e'in grado di indurre un forte stress ossidativo, ad un dosaggio di 20 pg/g di peso corporeo (CDOX e T2DOX) . A tutti i ratti del gruppo TI e T3 à ̈ stata iniettata la stessa dose di doxorubicina (T1DOX e T3DOX). At the end of the 18 days the rats of group C and group T2 were divided into two subgroups, respectively of 10 and 7 rats each: one subgroup was injected with a physiological solution (Cf and T2f), while the other a solution of doxorubicin (DOX for short), an antineoplastic drug with pro-oxidant activity that is able to induce strong oxidative stress, at a dosage of 20 pg / g of body weight (CDOX and T2DOX). All rats in the TI and T3 group were injected with the same dose of doxorubicin (T1DOX and T3DOX).
I ratti sono stati quindi sacrificati entro 24 ore dall'iniezione di soluzione fisiologica o DOX. Campioni di plasma sono stati quindi prelevati per quantificare l'attività antiossidante totale (TAA) in vitro e i livelli di glutatione in forma ridotta (GSH) (Figure 1 e 2). The rats were then sacrificed within 24 hours of saline or DOX injection. Plasma samples were then taken to quantify total antioxidant activity (TAA) in vitro and reduced form glutathione (GSH) levels (Figures 1 and 2).
II confronto tra CDOX e T1DOX, T2DOX e T3DOX consente di valutare una possibile protezione da parte della miscela di probiotici nei confronti dello stress ossidativo indotto dalla doxorubicina, evidenziando anche una possibile relazione dose-risposta in funzione del numero di cellule vitali somministrate. The comparison between CDOX and T1DOX, T2DOX and T3DOX allows to evaluate a possible protection by the probiotic mixture against oxidative stress induced by doxorubicin, also highlighting a possible dose-response relationship as a function of the number of viable cells administered.
Il confronto tra Cf e T2f serve ad evidenziare un possibile incremento dell'attività antiossidante indotto dai ceppi probiotici in condizioni fisiologiche, in assenza quindi dello stress ossidativo indotto dalla doxorubicina. The comparison between Cf and T2f serves to highlight a possible increase in antioxidant activity induced by probiotic strains in physiological conditions, therefore in the absence of oxidative stress induced by doxorubicin.
In Fig. 1 e 2 (attività totale antiossidante, TAA e glutatione piasmatico, rispettivamente) l'effetto del trattamento con probiotici in caso di stress ossidativo (differenze tra il gruppo di controllo CDOX e i gruppi T1DOX, T2DOX e T3DOX) sono state valutate con l'analisi statistica ANOVA confrontando tutti i ratti che avevano ricevuto la DOX. Gli effetti della somministrazione di doxorubicina sono stati valutati mediante test ANOVA (usando Tukey come post test) e confrontando con il controllo basale (Cf) tutti i gruppi trattati con DOX. Le differenze tra controllo (Cf) e trattato con probiotici in condizioni basali (T2f) sono state valutate con il test del t di Student. In Fig. 1 and 2 (total antioxidant activity, TAA and piasmatic glutathione, respectively) the effect of treatment with probiotics in case of oxidative stress (differences between the CDOX control group and the T1DOX, T2DOX and T3DOX groups) were evaluated with ANOVA statistical analysis comparing all rats that received DOX. The effects of doxorubicin administration were evaluated by ANOVA test (using Tukey as post test) and by comparing all DOX treated groups with the baseline control (Cf). The differences between control (Cf) and treated with probiotics in basal conditions (T2f) were evaluated with Student's t test.
Dalla figura 1 si osserva che, in condizioni basali (C e T2) il trattamento con probiotici non modifica la TAA (confronto non significativo, n.s.). Nei ratti non trattati con probiotici, la somministrazione di DOX causa un calo significativo della TAA rispetto ai valori basali (t test p<0,001). Anche nei ratti trattati con probiotici, la somministrazione di DOX causa un calo di TAA rispetto ai valori basali (Cf), ma nei trattamenti a concentrazione maggiore di probiotici (TI e T2) il calo à ̈ minore (t test p<0,02). Nei ratti trattati con una concentrazione minore di probiotici (T3), dopo somministrazione di DOX il calo di TAA à ̈ più elevato (t test p<0,001). La differenza tra i gruppi trattati con DOX à ̈ significativa (ANOVA p<0,05). From figure 1 it is observed that, in basal conditions (C and T2) the treatment with probiotics does not modify the TAA (comparison not significant, n.s.). In rats not treated with probiotics, administration of DOX causes a significant decrease in TAA from baseline (t test p <0.001). Even in rats treated with probiotics, the administration of DOX causes a decrease in TAA compared to baseline values (Cf), but in treatments with a higher concentration of probiotics (TI and T2) the decrease is smaller (t test p <0.02 ). In rats treated with a lower concentration of probiotics (T3), after administration of DOX the decrease in TAA is higher (t test p <0.001). The difference between the groups treated with DOX is significant (ANOVA p <0.05).
In figura 2, la concentrazione di GSH à ̈ usata come valutazione della capacità antiossidante. Nello stress ossidativo, i livelli del GSH si abbassano. In figure 2, the concentration of GSH is used as an evaluation of the antioxidant capacity. In oxidative stress, GSH levels are lowered.
C vs T2: t test p>0,05 (statisticamente non significativo, n.s.) Test ANOVA confrontando C con tutti i gruppi trattati con DOX: p<0,001 . C vs T2: t test p> 0.05 (statistically not significant, n.s.) ANOVA test comparing C with all groups treated with DOX: p <0.001.
Post test (Tukey): Post test (Tukey):
C vs C+DOX p<0,001 C vs C + DOX p <0.001
C vs Tl+DOX n.s. C vs Tl + DOX n.s.
C vs T2+DOX p<0,05 C vs T2 + DOX p <0.05
C vs T3+DOX p<0,01 C vs T3 + DOX p <0.01
La figura 2 mostra che in condizioni basali non c"Ã ̈ differenza tra controlli e ratti trattati con la miscela M. Lo stress ossidativo, nei ratti di controllo, determina un forte calo della concentrazione di GSH (p<0,001). Nei ratti trattati con la miscela M alla concentrazione maggiore (Tl), l'iniezione di DOX non determina una riduzione significativa dei livelli di GSH (n.s.). Figure 2 shows that in basal conditions there is no difference between controls and rats treated with mixture M. Oxidative stress, in control rats, causes a sharp drop in GSH concentration (p <0.001). In treated rats with the mixture M at the highest concentration (Tl), the injection of DOX does not cause a significant reduction in GSH levels (n.s.).
Nei ratti trattati con una dose intermedia di miscela M si osserva una diminuzione rispetto al controllo basale, ma con significatività statistica minore (p<0,05). Nei ratti trattati con probiotico alla dose più bassa, la riduzione dei livelli di GSH rispetto al controllo con probiotico à ̈ ancora più evidente (p<0,01). La differenza tra i diversi gruppi trattati con DOX à ̈ significativa (ANOVA p<0,05). In rats treated with an intermediate dose of mix M there is a decrease compared to the baseline control, but with less statistical significance (p <0.05). In rats treated with the probiotic at the lowest dose, the reduction in GSH levels compared to the probiotic control is even more evident (p <0.01). The difference between the different groups treated with DOX is significant (ANOVA p <0.05).
Da tutti i dati sopra riportati si evidenzia che la somministrazione di una miscela dei tre ceppi probiotici (miscela M) à ̈ in grado di ridurre lo stress ossidativo indotto dall'iniezione di doxorubicina, farmaco antineoplastico ad attività pro-ossidante (vedi figura 1, confronto tra C DOX e TI DOX). From all the above data it is highlighted that the administration of a mixture of the three probiotic strains (mixture M) is able to reduce the oxidative stress induced by the injection of doxorubicin, an antineoplastic drug with pro-oxidant activity (see figure 1, comparison between C DOX and TI DOX).
Lo stress ossidativo à ̈ stato valutato sia mediante la diminuzione dell'attività antiossidante totale a livello piasmatico, che quella del glutatione in forma ridotta (GSH). Il glutatione à ̈ un'importante molecola in grado di combattere lo stress ossidativo e di neutralizzare i radicali liberi presenti nel plasma. Oxidative stress was assessed both by decreasing total antioxidant activity at the piasmatic level and by reducing glutathione in reduced form (GSH). Glutathione is an important molecule capable of fighting oxidative stress and neutralizing free radicals present in the plasma.
I confronti più interessanti sono tra il gruppo C DOX (gruppo controllo, trattato senza probiotici e con iniezione di doxorubicina dopo 18 gg) e i gruppi TI D0X/T2 DOX (gruppi trattati con probiotici, rispettivamente in ragione di 10<*>9 e 10<*>8 UFC/die, a cui à ̈ stata iniettata doxorubicina dopo 18 gg). La concentrazione più bassa di probiotici, ovvero 10<*>7 UFC/die (gruppo T3), si à ̈ rivelata di utilità più contenuta nella riduzione dello stress ossidativo indotto dalla doxorubicina. The most interesting comparisons are between the C DOX group (control group, treated without probiotics and with doxorubicin injection after 18 days) and the TI D0X / T2 DOX groups (groups treated with probiotics, respectively in the ratio of 10 <*> 9 and 10 <*> 8 CFU / day, who was injected with doxorubicin after 18 days). The lowest concentration of probiotics, that is 10 <*> 7 CFU / day (group T3), proved to be of more limited utility in the reduction of oxidative stress induced by doxorubicin.
In ogni caso, riteniamo che la concentrazione di 10<*>8 UFC/die nei ratti possa essere rappresentativa dei quantitativi abitualmente utilizzati nell'uomo (10<*>10-10<*>11 UFC/die). In any case, we believe that the concentration of 10 <*> 8 CFU / day in rats may be representative of the quantities usually used in humans (10 <*> 10-10 <*> 11 CFU / day).
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