WO2018169297A1 - Tryptophanase-negative lactic bacteria, corresponding compositions and uses - Google Patents

Tryptophanase-negative lactic bacteria, corresponding compositions and uses Download PDF

Info

Publication number
WO2018169297A1
WO2018169297A1 PCT/KR2018/002969 KR2018002969W WO2018169297A1 WO 2018169297 A1 WO2018169297 A1 WO 2018169297A1 KR 2018002969 W KR2018002969 W KR 2018002969W WO 2018169297 A1 WO2018169297 A1 WO 2018169297A1
Authority
WO
WIPO (PCT)
Prior art keywords
compositions according
weight
treatment
compositions
brevis
Prior art date
Application number
PCT/KR2018/002969
Other languages
French (fr)
Inventor
Seok-Jin Kim
Original Assignee
Bioeleven Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bioeleven Co., Ltd. filed Critical Bioeleven Co., Ltd.
Priority to CN201880018611.5A priority Critical patent/CN110446497A/en
Priority to JP2019572333A priority patent/JP2020512014A/en
Publication of WO2018169297A1 publication Critical patent/WO2018169297A1/en

Links

Images

Classifications

    • 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
    • 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
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • 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
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria
    • 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
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • 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
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/122—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells for inducing tolerance or supression of immune responses

Definitions

  • compositions comprising lactic bacteria thatare unable to metabolise tryptophan (tryptophanase-negative bacteria) in association with lactic bacteria inducing Interferon gamma (IFN gamma).
  • the compositions to which the invention relates can be used in the dietary, nutraceutic and pharmaceutical fields and as food supplements in all those diseases benefiting from activation of the kynurenine metabolic pathway, such as for example tolerance during pregnancy, control of chronic inflammation, protection from autoimmunity, prevention and treatment to alleviate the symptoms of autoimmune diseases, help in preventing organ rejection, and the generation of energy (adenosine triphosphate ATP), nicotinamide and related compounds (nicotinamide adenine nucleotide and nicotinamide adenine nucleotide phosphate; NAD and NADP).
  • energy adenosine triphosphate ATP
  • nicotinamide and related compounds nicotinamide adenine nucleotide and
  • Tryptophan is an essential amino acid, and is therefore one of the group of amino acids that vertebrate organisms are unable to produce and therefore have to be taken in through food.
  • tryptophan is essential for the physiological functioning of mammals, and is processed via different metabolic pathways leading to the production of biogenic amines such as serotonin, melatonin, tryptamine, a number of degradation products collectively known as kynurenines, and finally it contributes to the synthesis of nicotinamide adenine dinucleotide (NAD+), a coenzyme important in energy metabolism.
  • NAD+ nicotinamide adenine dinucleotide
  • Systemic and cellular tryptophan levels are therefore not only determined by food intake, but also and above all by the activity of the metabolic pathways converting or degrading it.
  • tryptophan is metabolised by opening the indole ring with the formation of formylkynurenine and kynurenine.
  • This process represents the initial step in the biosynthesis of nicotinic acid and nicotinamide, vitamins of the B complex, and the production of ATP ( Figure 1).
  • IDO indoleamine 2,3-dioxygenase
  • IDO immunomodulating effect of IDO
  • APC antigen-presenting cells
  • IDO has an important role as a counter-regulatory mechanism in response to inflammatory conditions to limit excessive immune activation which could be damaging to the host itself.
  • IDO in the body balances out the effects of immunity and tolerance, controlling the response of the T cells through the dendritic cells.
  • CpG ODNs oligodeoxynucleotides containing demethylated CpG sequences
  • IFN- ⁇ interferon-gamma
  • IFN- ⁇ interferon-alpha
  • cytokines Mellor et al., 2005.
  • the present invention refers to dietary, nutraceutic, pharmaceutical and food supplement compositions comprising tryptophanase-negative microorganisms selected from: lactic bacteria, bifidobacteria, streptococci and mixtures thereof, in combination with microorganisms inducing IFN gamma selected from: lactic bacteria, bifidobacteria, streptococci and mixtures thereof.
  • microorganisms selected from lactic bacteria, bifidobacteria, streptococci and mixtures thereof, selected to be without tryptophanase activity, in combination with microorganisms selected from lactic bacteria, bifidobacteria, streptococci and mixtures thereof selected for their ability to induce IFN gamma act together in compositions which when administered, orally, enterally or rectally for example, are capable of altering the intestinal flora, increasing the proportion of tryptophanase-negative bacteria, with a consequent greater availability of tryptophan to the cells of the host, in which the activation of IDO is further stimulated by the IFN gamma, the production of which is induced by IFN gamma-inducing bacteria administered in the combination to which this invention relates, as illustrated in Figure 2.
  • the kynurenine pathway is essential to the control of inflammation and the production of energy (ATP,NAD, NADH, nicotinamide).
  • the composition according to the invention is particularly effective in preventing and treating chronic inflammation at local, systemic and cerebral level, for example in the treatment of intestinal inflammatory diseases, chronic liver diseases (steato-hepatitis, chronic hepatitis), in anti-viral treatments, chronic inflammatory diseases of the central and peripheral nervous system, in autoimmune diseases, in anti-rejection treatment following transplants, in faecal transplant, in increasing tolerance of the foetus during pregnancy, in stabilising and regulating tryptophan metabolism and inflammation of the microglia in early infancy, such as for example in autism, in changes in the amygdala, hippocampus and/or frontal cortex in the elderly, which arise for various reasons in situations of tryptophan dysmetabolism, cardiovascular diseases and diabetes.
  • composition to which the invention relates is also effective in producing energy (adenosine triphosphate, ATP), nicotinamide and related compounds (nicotinamide adenine nucleotide and nicotinamide adenine nucleotide phosphate; NAD and NADP) in individuals with energy deficit or where a greater production of energy for the body is required.
  • energy adenosine triphosphate, ATP
  • nicotinamide and related compounds nicotinamide adenine nucleotide and nicotinamide adenine nucleotide phosphate; NAD and NADP
  • Preferred microorganisms according to this invention are selected from lactic bacteria, bifidobacteria and streptococci selected for the absence of the tryptophanase enzyme and the ability to induce the production of IFN- ⁇ , and mixtures thereof.
  • These may be used in the form of non-vital bacteria, for example bacteria which have been killed by heat, radiation or any other method useful for the purpose, or as live bacteria, or in both forms at the same time.
  • compositions for food, dietary, pharmaceutical, and nutraceutical use and as food supplements containing the said bacterial strains are commercially available.
  • the inventors have now found that it is possible to make use of lactic bacteria, bifidobacteria and streptococci that have already been known for some time and are used in the food industry, in a synergistic way, by selecting strains without tryptophanase enzyme together with lactic bacteria selected for the ability to induce IFN gamma to activate and potentiate the IDO enzyme and therefore the kynurenine metabolic pathway in mammals, human or animal, to which they are administered to prevent, treat and ameliorate chronic inflammatory conditions and to improve the generation of energy in the body in all cases where this is necessary.
  • FIG. 1 The TOx patway .
  • the extrahepatic human TOx patway is shown along with IDO inhibitors and inducers.
  • FIG. 2 Lactic bacteria, bifidobacteria and streptococci selected to be without tryptophanase activity, in combination with lactic probiotics selected for their ability to induce IFN gamma act together in cooperation resulting in the greater availability of tryptophan for the host's cells in which IDO activation is further stimulated by IFN gamma, the production of which is induced by IFN gamma- inducing bacteria administered in the combination to which this invention relates.
  • food composition a composition comprising at least one foodstuff to which the bacteria according to the invention may be added or with which the bacteria according to the invention may be formulated.
  • the inventors have now found that it is possible to make use of lactic bacteria, bifidobacteria and streptococci that have already been known for some time and are used in the food industry, in a synergistic way, by selecting strains without tryptophanase enzyme together with lactic bacteria selected for the ability to induce IFN gamma to activate and potentiate the IDO enzyme and therefore the kynurenine metabolic pathway in mammals, human or animal, to which they are administered to prevent, treat and ameliorate chronic inflammatory conditions and to improve the generation of energy in the body in all cases where this is necessary.
  • composition according to the invention is in fact able to modify the intestinal microflora and influence tryptophan metabolism, with positive repercussions on inflammatory conditions, helping to prevent organ rejection, including bone marrow transplants and faecal transplants, tolerance of the foetus during pregnancy, prevention and treatment of local, systemic and cerebral inflammation, and to prevent and treat autoimmune diseases, including viral diseases.
  • compositions according to the invention are particularly beneficial in early infancy for stabilising and regulating tryptophan metabolism and inflammation of the microglia, for example in autism.
  • Another preferred field of application is use for the treatment of changes in the amygdala, hippocampus and/or frontal cortex in the elderly, which come about for a variety of reasons (stays in hospitals or hospices, antibiotic treatment or treatment with drugs altering the intestinal flora, such as antiviral and antitumor drugs) in situations of tryptophan dysmetabolism.
  • composition according to the invention is potentiation of the production of ATP, NAD and NADP in mammals, humans and animals, both in energy deficit conditions and in order to improve sports performance.
  • compositions according to the present invention may be administered to humans or animals for use in the prevention and treatment of diseases characterised by chronic inflammation and other pathological conditions indicated above.
  • the compositions may be administered to individuals, humans or animals, exhibiting symptoms (for example patients suffering from inflammatory intestinal diseases), or without symptoms but with a detected potential risk of inflammation (pregnancy in individuals experiencing repeated spontaneous abortions), as well as asymptomatic individuals, for preventive purposes (for example the prevention of atherosclerosis and senile dementia), or merely to improve physical performance.
  • Ingestion of the composition according to this invention results in an increase in the number of tryptophanase-negative bacteria, that is bacteria unable to utilise tryptophan) in the intestines of the host.
  • This increases the proportion of tryptophan which is available to the IDO enzyme (indoleamine 2-3 dioxygenase); IDO is in turn potentiated in its IFN- ⁇ activity, the IFN- ⁇ being produced in greater quantity by the immunocompetent cells of the host following stimulation by the second component of the composition to which this invention relates, that is by the bacteria inducing the production of IFN- ⁇ .
  • the composition therefore makes the substrate (tryptophan) more available and also potentiates IDO through potentiating the production of IFN- ⁇ .
  • the kynurenine pathway is therefore potentiated in treated individuals.
  • the kynurenine pathway is fundamental to the control of inflammation and the production of energy (ATP,NAD, NADH, nicotinamide).
  • the inventors first selected lactic bacteria, bifidobacteria and streptococci without tryptophanase activity. Absence of tryptophanase activity in bacteria is assayed using the Kovacs reagent for indole (p-dimethyl-amino-benzaldehyde in acid solution). Indole is one of the by-products of the metabolic degradation of tryptophan. Bacteria containing the tryptophanase enzyme are capable of hydrolysing and deamining tryptophan, producing indole, pyruvic acid and ammonia. For these assays the bacterium Escherichia coli is used as a positive standard for the presence of tryptophanase activity. An example of the test is illustrated in the examples.
  • IFN- ⁇ production was measured using an ELISA kit for measuring IFN- ⁇ after healthy individuals' lymphocytes had been stimulated with the bacteria under test in culture in a 1:1 ratio. An example of the test is illustrated in the examples.
  • strains which proved to be able to induce IFN- ⁇ production of 0.2 ng/ml or more were considered worthy of selection; the best three strains inducing the production of IFN- ⁇ proved to be S. thermophilus (1.8 ng/ml), L. acidophilus (1.5 ng/ml), B. lactis (1.3 ng/ml), B. bifidum (1.3 ng/ml) and L. helveticus (1.2 ng/ml).
  • composition comprising at least one strain of bacteria without tryptophanase activity, preferably selected from S. thermophilus, L. plantarum, L. paraplantarum, L. acidophilus, L. casei, L. paracasei, L. helveticus, B. lactis, B. breve, B. Infantis, B. longum, L. lactis, L. brevis, P. Freudenreichii and at least one strain of bacteria inducing the production of IFN- ⁇ , preferably selected from S. thermophilus, L. acidophilus, L. helveticus, B. Lactis and B. bifidum, was developed.
  • strains listed in the following Table 1 are particularly preferred.
  • compositions according to the invention varies between 1.10 9 and 5.10 12 bacteria per gram.
  • the ratio between the bacteria without tryptophanase activity and the bacteria inducing the production of IFN- ⁇ lies within a range between 1:1, 1000:1 or even 1:1000, and this may be selected by those skilled in the art on the basis of daily dietary tryptophan input or level of inflammatory response by the individual being treated.
  • the compositions according to this invention may be administered as a single dose or in several doses, such that the daily intake of lactic bacteria or elements deriving from lactic bacteria is preferably from 0.01 to 200 mg/kg of body weight per day, and more preferably from 0.1 to 100 mg/kg of body weight per day.
  • the bacteria inducing the production of IFN- ⁇ may be non-vital, and therefore inactivated by radiation or any other means known to those skilled in the art which do not affect their ability to induce the production of IFN- ⁇ in mammals.
  • compositions according to the present invention may be dried or lyophilised or subjected to any process affecting the liquid components of the composition.
  • the stage of removing the liquid part generally water, may conveniently be performed by lyophilisation or drying or spray drying, or using any drying process suitable for the drying of bacteria, including drying under vacuum and drying in air.
  • compositions to which this invention relates may further contain one or more components selected from tryptophan, 5-hydroxytryptamine or melatonin, and the normal excipients currently used for the preparation of food, dietary, nutraceutic and pharmaceutical compositions.
  • compositions may also be suitably added to the compositions.
  • the compositions may also be prepared in the form of food supplements (for example capsules, tablets, granulates, spreadable cream), as foodstuffs with indications for health (for example food products with specific uses for health, foodstuffs with functional indications) or as functional foodstuffs (for example, dietary products, food supplements).
  • compositions according to the invention may be produced in the usual nutraceutic and pharmaceutical forms known in the literature, such as for example tablets, confectionery, capsules, granules, beverages, lyophilisates, solutions, suspensions, emulsions, pellets, syrups, suppositoriesorpessaries, and may be prepared in the usual way by mixing the active ingredient with excipients and/or vehicles, optionally adding coadjuvants and/or dispersants; for example water may be used as diluent and other organic solvents may also be used in the form of adjuvants.
  • nutraceutic and pharmaceutical forms known in the literature, such as for example tablets, confectionery, capsules, granules, beverages, lyophilisates, solutions, suspensions, emulsions, pellets, syrups, suppositoriesorpessaries, and may be prepared in the usual way by mixing the active ingredient with excipients and/or vehicles, optionally adding coadjuvants and/or dispersants;
  • the adjuvants may for example be: water, non-toxic organic solvents such as paraffins, vegetable oils (peanut oil or sesame oil), alcohols (for example ethanol, glycerine), glycols (propylene glycol, polyethylene glycol), solid substrates such as for example natural mineral flours (kaolin, talc), synthetic mineral flours (for example silicates), sugars (for example cane sugar), emulsifiers (alkyl sulfonates or aryl sulfonates and the like), dispersants (for example maltose, lignin, methylcellulose, starch and polyvinylpyrrolidone) and lubricants (for example magnesium stearate, talc, stearic acid, sodium lauryl sulfonate) and similar materials.
  • non-toxic organic solvents such as paraffins, vegetable oils (peanut oil or sesame oil), alcohols (for example ethanol, glycerine), glycols (propy
  • the ratio between the active ingredient and the excipient preferably varies between 1:10 and 100:1.
  • compositions may be administered alone or in combination with other active ingredients of a food or dietary nature (such as proteins isolated from soya, powdered albumin, dried spirulina alga, dried eggs, pumpkin seeds, brewer's yeast) of a nutraceutic and pharmaceutical nature and may be packed in the form of kits with components prepared in predosed packed units.
  • active ingredients of a food or dietary nature such as proteins isolated from soya, powdered albumin, dried spirulina alga, dried eggs, pumpkin seeds, brewer's yeast
  • the pharmaceutical forms suitable for this purpose may also contain other additives including sodium citrate, calcium carbonate or phosphate, together with various additives such as starch, gelatine and similar products in addition to the usual excipients such as lactulose, dextrose, lactose, maltose. Compatible colourings or flavourings may be added in the case of liquid forms.
  • compositions to which the invention relates may further comprise one or more components selected from amino acids and vitamins, including drugs compatible with the bacteria used that are capable of potentiating the activity of the active ingredients present therein.
  • drugs compatible with the bacteria used that are capable of potentiating the activity of the active ingredients present therein.
  • anti-inflammatories antipyretics, antiseptics, analgesics, antirheumatics, antibacterials, hepatoprotectors, antilipaemics, antivirals and chemotherapy and antitumor drugs may for example be used.
  • food compositions formulated with addition of the selected bacteria to foodstuffs in liquid form such as fruit juices and dairy products, including all food products made using milk, or milk and cheese products including but not limited to milks, yoghurts, ice creams, cheeses, butter, cream, are regarded a particularly advantageous compositions; food compositions comprising foodstuffs rich in tryptophan, for example almonds, nuts, proteins isolated from soya, powdered albumin, powdered egg, brewer's yeast, dried spirulina alga and parmesan are also considered to be particularly advantageous.
  • tryptophan for example almonds, nuts, proteins isolated from soya, powdered albumin, powdered egg, brewer's yeast, dried spirulina alga and parmesan are also considered to be particularly advantageous.
  • compositions to which this invention relates may be used as foodstuffs for men or animals, functional foodstuffs, food supplements, drugs for animal or human use, or pharmaceutical products.
  • the quantity of lactic bacteria is not particularly limited in the situation where the composition according to the present invention is used as a foodstuff.
  • the quantity may for example be from 0.00001 to 100% by weight, preferably from 0.001 to 50 % by weight and more preferably from 0.1 to 10 % by weight, of the food.
  • the pharmaceutical, nutraceutical and nutritional compositions according to the invention can be prepared by mixing the bacterial strains and/or their enzymatic products with suitable adjuvant(s) in order to obtain the final product.
  • the food and/or dietary and/or nutraceutic and/or pharmaceutical compositions comprise (% by weight):
  • composition according to the invention comprises lyophilised lactic bacterial strains selected,in the same % as the first preferred embodiment, from:
  • compositions according to the invention comprise lyophilised lactic bacterial strains selected from(% by weight):
  • composition according to the invention comprises lyophilised lactic bacterial strains selected from (same % by weight as in the third preferred embodiment):
  • composition according to the invention comprises lyophilised lactic bacteria strains selected from (% by weight):
  • composition according to the invention comprises lyophilised lactic bacterial strains selected from(% by weight):
  • composition according to the invention comprises lyophilised lactic bacterial strains selected from(% by weight):
  • starch and/or sugars and/or drugs such as antiviral agents were added to 100%.
  • the suspension tends to freeze practically instantaneously.
  • the lyophilisation cycle is started for the purposes of obtaining a lyophilised product with powder humidity below 5% which is the condition necessary for achieving good, lasting microbiological stability of the bacterial mixture.
  • the lyophilisation conditions are the following.
  • Lyophilisation heating ramp 5°C every 2 hours up to a product temperature of -5°C.
  • the product is left for 12 hours at this temperature after which the temperature recovery ramp is resumed at a rate of 5°C every 2 hours up to a product temperature of 25°C.
  • flavours that can be used are extremely varied. The best results have been obtained with a lemon flavour and a strawberry flavour.
  • L. paracaseiand E. coli ( Figure 3) were cultured in MRS broth (de Man, Rogosa and Sharp) (18 hours at +37°C) and transplanted into 1% tryptophan broth. After incubation for 24 and 48 hours at +37°C the indole test was performed by adding 5 drops of Kovacs reagent directly to the test tube.
  • the indole test (Manual of Clinical Microbiology - ASM, 1985, page 1094) is based on the formation of a red-purple complex due to reaction between the indole and the aldehyde group of p-dimethylaminobenzaldehyde.
  • bacteria assayed by the indole test have shown that they have no tryptophanase activity and have therefore been considered useful for the present invention: S. Thermophilus, L. plantarum, L. acidophilus, L. paracasei, L. helveticus, B. lactis, B. breve, B. infantis, B. longum, L. lactis, L. brevis, P. freudenreichii.
  • Other bacteria which are negative to the indole test such as Bordetella, Haemophilus, Proteus mirabilis, Pseudomonas cannot be used for the purposes of this invention, above all because they are pathogenic or potentially pathogenic.
  • Peripheral blood from healthy donor was collected into heparinized tubes and diluted 1:2 with phosphate buffered saline (PBS).Mononuclear cells were isolated by density gradient centrifugation and diluted to 1 ⁇ 10 6 /ml in RPMI-1640 supplemented with glutamine, HEPES buffer, penicillin, streptomycin and 10% of a of foetal calf serum (Flow Laboratories). When stimulating with the bacteria to test, the bacteria were added in a 1:1 ratio to the cell suspension in the wells and the incubation time was 24 hours at 37°C, 5%CO 2 .
  • PBS phosphate buffered saline
  • Abcam's Interferon gamma (IFNG) Human ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human Interferon gamma in cell lysates and tissue lysates.
  • This assay employs an antibody specific for Human Interferon gamma coated on a 96-well plate.
  • Standards and samples are pipetted into the wells and Interferon gamma present in a sample is bound to the wells by the immobilized antibody.
  • the wells are washed and biotinylated anti-Human Interferon gamma antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells.
  • the wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Interferon gamma bound.
  • the Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
  • strains capable of inducing levels of IFN- ⁇ in excess of 0.2 ng/mlin vitrocan be used for the purposes of the present invention.
  • S. thermophilus (1.8 ng/ml)
  • L. acidophilus 1.5 ng/ml
  • B. lactis 1.3 ng/ml
  • B. bifidum 1.3 ng/ml
  • L. helveticus 1.2 ng/ml.
  • composition N° 1 Six-months composition supplementation on peripheral blood and gut cellular immune activation in chronic HIV-1 infected patients under ART.
  • CSF cerebrospinal fluid
  • Histological examination primarily included the assessment of cellular infiltrates and aggregation by scoring the amount of intra epithelial lymphocytes (I.E.Ls) (mononuclear cells, such as macrophages, lymphocytes, plasma cells, and neutrophils) at a magnification of 400X.
  • I.E.Ls intra epithelial lymphocytes
  • the number of inflammatory cells was evaluated by using a semiquantitative method, as described previously (ref. IBD Working Group of the European Society for Paediatric Gastroenterology,Hepatology and Nutrition) and results are reported as the mean for the entire specimen.
  • TUNEL terminal deoxynucleotidyl transferase-mediated digoxigenindeoxyuridine triphosphate nick-end labeling
  • Endogenous peroxidase was quenched by using 3% hydrogen peroxidase for 20 minutes and then washed in phosphate-buffered saline (PBS). After equilibration, the sections were incubated in a humidified chamber with terminal deoxynucleotidyl transferase enzyme for 1 hour at 37°C. Afterward, sections were soaked in stop-wash buffer for 30 minutes and then rinsed in PBS and, thereafter, incubated with antidigoxigenin peroxidase in a humidified chamber for 30 minutes, followed by addition of diaminobenzidine and hydrogen peroxide for 1 to 3 minutes to allow color development on apoptotic nuclei. Finally, sections were counterstained with Harris hematoxylin.
  • CSF was collected by lumbar puncture, centrifuged and cell-free supernatant samples stored in aliquots at -80°C. Twenty milliliters of whole blood were collected by venipucture in Vacutainer tubes containing ethylenediaminetetraacetic acid (BD Biosciences, San Jose, CA) at each study visit. Plasma was immediately separated by centrifugation and stored at -80°C.
  • Tryptophan Chromatographic elution was performed with a binary gradient system with sodium acetate (30 mM, pH 6.5) and acetonitrile in a ratio of 80:20 (v/v) (solvent A) and propan-2-ol with acetonitrile 50:50 (v/v) (solvent B).
  • Data analysis was performed by using a dedicated software (Millennium 32 ).
  • IELs intraepithelial lymphocytes
  • the result data is as follows.

Abstract

The present invention refers to dietary, nutraceutic, pharmaceutical and food supplement compositions comprising tryptophanase-negative microorganisms selected from: lactic bacteria, bifidobacteria, streptococci and mixtures thereof, in combination with microorganisms inducing IFN gamma selected from: lactic bacteria, bifidobacteria, streptococci and mixtures thereof.

Description

TRYPTOPHANASE-NEGATIVE LACTIC BACTERIA, CORRESPONDING COMPOSITIONS AND USES
This invention relates to compositions comprising lactic bacteria thatare unable to metabolise tryptophan (tryptophanase-negative bacteria) in association with lactic bacteria inducing Interferon gamma (IFN gamma). The compositions to which the invention relates can be used in the dietary, nutraceutic and pharmaceutical fields and as food supplements in all those diseases benefiting from activation of the kynurenine metabolic pathway, such as for example tolerance during pregnancy, control of chronic inflammation, protection from autoimmunity, prevention and treatment to alleviate the symptoms of autoimmune diseases, help in preventing organ rejection, and the generation of energy (adenosine triphosphate ATP), nicotinamide and related compounds (nicotinamide adenine nucleotide and nicotinamide adenine nucleotide phosphate; NAD and NADP).
Tryptophan is an essential amino acid, and is therefore one of the group of amino acids that vertebrate organisms are unable to produce and therefore have to be taken in through food. In addition to being used by the body to produce proteins, tryptophan is essential for the physiological functioning of mammals, and is processed via different metabolic pathways leading to the production of biogenic amines such as serotonin, melatonin, tryptamine, a number of degradation products collectively known as kynurenines, and finally it contributes to the synthesis of nicotinamide adenine dinucleotide (NAD+), a coenzyme important in energy metabolism. Systemic and cellular tryptophan levels are therefore not only determined by food intake, but also and above all by the activity of the metabolic pathways converting or degrading it.
Within the body tryptophan is metabolised by opening the indole ring with the formation of formylkynurenine and kynurenine. This process represents the initial step in the biosynthesis of nicotinic acid and nicotinamide, vitamins of the B complex, and the production of ATP (Figure 1).
The enzyme responsible for catalysing the conversion of tryptophan into kynurenine compounds, or kynurenines, is indoleamine 2,3-dioxygenase (hereinafter referred to as IDO). This property provides IDO with the ability to inhibit the immune response, both because processing tryptophan causes this amino acid, which is necessary for the growth and proliferation of T lymphocytes, to become depleted in the microenvironment, and also because it is able to produce metabolites which can cause the apoptosis of T lymphocytes. One of the most accepted hypotheses explaining the immunomodulating effect of IDO is that tryptophan depletion consequent upon expression of the protein arrests T cells in the G1 phase of the cell cycle, impeding their clonal expansion and making them more sensitive to pro-apoptotic signals (Munn et al., 1999). An alternative hypothesis is that IDO can adversely affect the biological properties of antigen-presenting cells (APC), either diminishing their ability to present the antigen or increasing the expression of suppressive ligands (for example the CD95 ligand) or favouring the release of immunoregulatory cytokines such as IL-10 or TGF-β (Mellor and Munn, 2004).
In fact IDO has an important role as a counter-regulatory mechanism in response to inflammatory conditions to limit excessive immune activation which could be damaging to the host itself. IDO in the body balances out the effects of immunity and tolerance, controlling the response of the T cells through the dendritic cells. It has also been discovered that oligodeoxynucleotides containing demethylated CpG sequences (CpG ODNs), which are known to be powerful adjuvants, act as stimulators of IDO expression in an appreciable variety of tissues through the induction of interferon-gamma (IFN-γ), interferon-alpha (IFN-α) and other pro-inflammatory cytokines (Mellor et al., 2005). This evidence may be significant from a practical point of view in that, notwithstanding the fact that CpG ODNs are particularly strong immunostimulants, in some conditions they are capable of inducing a counter-regulatory effect mediated by IDO.
The present invention refers to dietary, nutraceutic, pharmaceutical and food supplement compositions comprising tryptophanase-negative microorganisms selected from: lactic bacteria, bifidobacteria, streptococci and mixtures thereof, in combination with microorganisms inducing IFN gamma selected from: lactic bacteria, bifidobacteria, streptococci and mixtures thereof.
It has now been found that microorganisms selected from lactic bacteria, bifidobacteria, streptococci and mixtures thereof, selected to be without tryptophanase activity, in combination with microorganisms selected from lactic bacteria, bifidobacteria, streptococci and mixtures thereof selected for their ability to induce IFN gamma act together in compositions which when administered, orally, enterally or rectally for example, are capable of altering the intestinal flora, increasing the proportion of tryptophanase-negative bacteria, with a consequent greater availability of tryptophan to the cells of the host, in which the activation of IDO is further stimulated by the IFN gamma, the production of which is induced by IFN gamma-inducing bacteria administered in the combination to which this invention relates, as illustrated in Figure 2.
The kynurenine pathway is essential to the control of inflammation and the production of energy (ATP,NAD, NADH, nicotinamide). Thus the composition according to the invention is particularly effective in preventing and treating chronic inflammation at local, systemic and cerebral level, for example in the treatment of intestinal inflammatory diseases, chronic liver diseases (steato-hepatitis, chronic hepatitis), in anti-viral treatments, chronic inflammatory diseases of the central and peripheral nervous system, in autoimmune diseases, in anti-rejection treatment following transplants, in faecal transplant, in increasing tolerance of the foetus during pregnancy, in stabilising and regulating tryptophan metabolism and inflammation of the microglia in early infancy, such as for example in autism, in changes in the amygdala, hippocampus and/or frontal cortex in the elderly, which arise for various reasons in situations of tryptophan dysmetabolism, cardiovascular diseases and diabetes. The composition to which the invention relates is also effective in producing energy (adenosine triphosphate, ATP), nicotinamide and related compounds (nicotinamide adenine nucleotide and nicotinamide adenine nucleotide phosphate; NAD and NADP) in individuals with energy deficit or where a greater production of energy for the body is required.
Preferred microorganisms according to this invention are selected from lactic bacteria, bifidobacteria and streptococci selected for the absence of the tryptophanase enzyme and the ability to induce the production of IFN-γ, and mixtures thereof. These may be used in the form of non-vital bacteria, for example bacteria which have been killed by heat, radiation or any other method useful for the purpose, or as live bacteria, or in both forms at the same time.
Another object of the invention are compositions for food, dietary, pharmaceutical, and nutraceutical use and as food supplements containing the said bacterial strains. All the bacterial strains mentioned are commercially available.
Other objects will be apparent from the following detailed description of the invention.
The inventors have now found that it is possible to make use of lactic bacteria, bifidobacteria and streptococci that have already been known for some time and are used in the food industry, in a synergistic way, by selecting strains without tryptophanase enzyme together with lactic bacteria selected for the ability to induce IFN gamma to activate and potentiate the IDO enzyme and therefore the kynurenine metabolic pathway in mammals, human or animal, to which they are administered to prevent, treat and ameliorate chronic inflammatory conditions and to improve the generation of energy in the body in all cases where this is necessary.
Figure 1: The TOx patway . The extrahepatic human TOx patway is shown along with IDO inhibitors and inducers. IDO1, indoleamine 2,3-dioxygenase 1; IDO2, indoleamine 2,3-dioxygenase 2; AFMID, arylformamidase; KMO, kynurenine 3-monooxygenase; KAT, kynurenine aminotransferase; KYNU, kynureninase; HAAO, 3-hydroxyanthranilate 3,4-dioxygenase; ACMSD, aminocarboxymuconate semialdehyde decarboxylase; QPRT, quinolate phosphoribosyltransferase. (From: F. Murray. Science Translational Medicine vol 2, issue 32, 19 May 2010)
Figure 2: Lactic bacteria, bifidobacteria and streptococci selected to be without tryptophanase activity, in combination with lactic probiotics selected for their ability to induce IFN gamma act together in cooperation resulting in the greater availability of tryptophan for the host's cells in which IDO activation is further stimulated by IFN gamma, the production of which is induced by IFN gamma- inducing bacteria administered in the combination to which this invention relates.
Figure 3: Results of the indole test with L. paracasei.
For the purposes of this invention, by "food composition" is meant a composition comprising at least one foodstuff to which the bacteria according to the invention may be added or with which the bacteria according to the invention may be formulated.
The inventors have now found that it is possible to make use of lactic bacteria, bifidobacteria and streptococci that have already been known for some time and are used in the food industry, in a synergistic way, by selecting strains without tryptophanase enzyme together with lactic bacteria selected for the ability to induce IFN gamma to activate and potentiate the IDO enzyme and therefore the kynurenine metabolic pathway in mammals, human or animal, to which they are administered to prevent, treat and ameliorate chronic inflammatory conditions and to improve the generation of energy in the body in all cases where this is necessary.
The composition according to the invention is in fact able to modify the intestinal microflora and influence tryptophan metabolism, with positive repercussions on inflammatory conditions, helping to prevent organ rejection, including bone marrow transplants and faecal transplants, tolerance of the foetus during pregnancy, prevention and treatment of local, systemic and cerebral inflammation, and to prevent and treat autoimmune diseases, including viral diseases.
The compositions according to the invention are particularly beneficial in early infancy for stabilising and regulating tryptophan metabolism and inflammation of the microglia, for example in autism. Another preferred field of application is use for the treatment of changes in the amygdala, hippocampus and/or frontal cortex in the elderly, which come about for a variety of reasons (stays in hospitals or hospices, antibiotic treatment or treatment with drugs altering the intestinal flora, such as antiviral and antitumor drugs) in situations of tryptophan dysmetabolism.
Another important effect of the composition according to the invention is potentiation of the production of ATP, NAD and NADP in mammals, humans and animals, both in energy deficit conditions and in order to improve sports performance.
The compositions according to the present invention may be administered to humans or animals for use in the prevention and treatment of diseases characterised by chronic inflammation and other pathological conditions indicated above. The compositions may be administered to individuals, humans or animals, exhibiting symptoms (for example patients suffering from inflammatory intestinal diseases), or without symptoms but with a detected potential risk of inflammation (pregnancy in individuals experiencing repeated spontaneous abortions), as well as asymptomatic individuals, for preventive purposes (for example the prevention of atherosclerosis and senile dementia), or merely to improve physical performance.
Ingestion of the composition according to this invention results in an increase in the number of tryptophanase-negative bacteria, that is bacteria unable to utilise tryptophan) in the intestines of the host. This increases the proportion of tryptophan which is available to the IDO enzyme (indoleamine 2-3 dioxygenase); IDO is in turn potentiated in its IFN-γ activity, the IFN-γ being produced in greater quantity by the immunocompetent cells of the host following stimulation by the second component of the composition to which this invention relates, that is by the bacteria inducing the production of IFN-γ. The composition therefore makes the substrate (tryptophan) more available and also potentiates IDO through potentiating the production of IFN-γ. The kynurenine pathway is therefore potentiated in treated individuals.The kynurenine pathway is fundamental to the control of inflammation and the production of energy (ATP,NAD, NADH, nicotinamide).
The inventors first selected lactic bacteria, bifidobacteria and streptococci without tryptophanase activity. Absence of tryptophanase activity in bacteria is assayed using the Kovacs reagent for indole (p-dimethyl-amino-benzaldehyde in acid solution). Indole is one of the by-products of the metabolic degradation of tryptophan. Bacteria containing the tryptophanase enzyme are capable of hydrolysing and deamining tryptophan, producing indole, pyruvic acid and ammonia. For these assays the bacterium Escherichia coli is used as a positive standard for the presence of tryptophanase activity. An example of the test is illustrated in the examples.
When assayed using the indole test the following bacteria have proved to be without tryptophanase activity, specifically: S. Thermophilus, L. plantarum, L. paraplantarum, L. acidophilus, L. casei, L. paracasei, L. helveticus, B. lactis, B. breve, B.infantis, B. longum, L. lactis, L. brevis, P. Freudenreichii.
Secondly the inventors selected lactic bacteria for their ability to induce the production of IFN-γ. IFN-γ production was measured using an ELISA kit for measuring IFN-γ after healthy individuals' lymphocytes had been stimulated with the bacteria under test in culture in a 1:1 ratio. An example of the test is illustrated in the examples.
For the purposes of this invention, only strains which proved to be able to induce IFN-γ production of 0.2 ng/ml or more were considered worthy of selection; the best three strains inducing the production of IFN-γ proved to be S. thermophilus (1.8 ng/ml), L. acidophilus (1.5 ng/ml), B. lactis (1.3 ng/ml), B. bifidum (1.3 ng/ml) and L. helveticus (1.2 ng/ml).
Therefore for the purposes of this invention a composition comprising at least one strain of bacteria without tryptophanase activity, preferably selected from S. thermophilus, L. plantarum, L. paraplantarum, L. acidophilus, L. casei, L. paracasei, L. helveticus, B. lactis, B. breve, B. Infantis, B. longum, L. lactis, L. brevis, P. Freudenreichii and at least one strain of bacteria inducing the production of IFN-γ, preferably selected from S. thermophilus, L. acidophilus, L. helveticus, B. Lactis and B. bifidum, was developed.
Particularly preferred are the strains listed in the following Table 1 to be used alone or in mixture among them.
Tryptophanase neg . strains IFN -γ producing strains
L. helveticus Lafti L10 L. helveticus R0052L. helveticus HA-128
B. bifidum BB01B. bifidum MB109 B. bifidum R0071
B. longum HA-135 B. longum Rosell-175B. longum ATCC SD5588
B. infantis R0033 B. infantis HA-116B. infantis BI02
B. breve Rosell-70 B breve HA-129B. breve ATCC SD5206
L. acidophilus HA-122L. acidophilus ATCC SD5212 L.. acidophilus R0052L. acidophilus Rosell-418L. acidophilus LBA091
L. casei ATCC SD5213 L. casei Rosell-215L. casei HA-108
L. paracasei HA-196L. paracasei ATCC SD5275 L. paracasei Lafti L26 NDL. paracasei LP01
L. plantarum P83L. plantarum P94L. plantarum D747 L. plantarum Rosell-1012L. plantarum HA-119L. plantarum ATCC SD5209
L. brevis HA-112 L. brevis LBR01L. brevis ATCC SD5214
L. salivarius HA-11L. salivarius LS01
Lactobacillus delbrueckii ssp. bulgaricus SP96 Lactobacillus delbrueckii ssp. lactis LL82 MDX
B. lactis ATCC SD5219B. lactis HA-194 B. lactis Lafti B94B. lactis ATCC SD5220B. lactis ATCC SD5219B. lactis BL086
P. freudenreichii ssp shermanii HA-182P. freudenreichii ssp freudenreichii HA-273
Streptococcus thermophilus SP4 CSStreptococcus thermophilus SP4 MDX Streptococcus thermophilus (TH-4®) Streptococcus thermophilus ATCC SD5207Streptococcus thermophilus SP4
Preferably their concentration in the compositions according to the invention varies between 1.109 and 5.1012 bacteria per gram. The ratio between the bacteria without tryptophanase activity and the bacteria inducing the production of IFN-γ lies within a range between 1:1, 1000:1 or even 1:1000, and this may be selected by those skilled in the art on the basis of daily dietary tryptophan input or level of inflammatory response by the individual being treated. The compositions according to this invention may be administered as a single dose or in several doses, such that the daily intake of lactic bacteria or elements deriving from lactic bacteria is preferably from 0.01 to 200 mg/kg of body weight per day, and more preferably from 0.1 to 100 mg/kg of body weight per day.
According to the present invention the bacteria inducing the production of IFN-γ may be non-vital, and therefore inactivated by radiation or any other means known to those skilled in the art which do not affect their ability to induce the production of IFN-γ in mammals.
The compositions according to the present invention may be dried or lyophilised or subjected to any process affecting the liquid components of the composition. The stage of removing the liquid part, generally water, may conveniently be performed by lyophilisation or drying or spray drying, or using any drying process suitable for the drying of bacteria, including drying under vacuum and drying in air.
The compositions to which this invention relates may further contain one or more components selected from tryptophan, 5-hydroxytryptamine or melatonin, and the normal excipients currently used for the preparation of food, dietary, nutraceutic and pharmaceutical compositions.
Pharmaceutically acceptable agents such as excipients, disaggregating agents, lubricants, binding agents, oxidation inhibitors, colourings, aggregation inhibitors, absorption promoters, aids to dissolution and stabilisers may also be suitably added to the compositions. The compositions may also be prepared in the form of food supplements (for example capsules, tablets, granulates, spreadable cream), as foodstuffs with indications for health (for example food products with specific uses for health, foodstuffs with functional indications) or as functional foodstuffs (for example, dietary products, food supplements).
The compositions according to the invention may be produced in the usual nutraceutic and pharmaceutical forms known in the literature, such as for example tablets, confectionery, capsules, granules, beverages, lyophilisates, solutions, suspensions, emulsions, pellets, syrups, suppositoriesorpessaries, and may be prepared in the usual way by mixing the active ingredient with excipients and/or vehicles, optionally adding coadjuvants and/or dispersants; for example water may be used as diluent and other organic solvents may also be used in the form of adjuvants.
The adjuvants may for example be: water, non-toxic organic solvents such as paraffins, vegetable oils (peanut oil or sesame oil), alcohols (for example ethanol, glycerine), glycols (propylene glycol, polyethylene glycol), solid substrates such as for example natural mineral flours (kaolin, talc), synthetic mineral flours (for example silicates), sugars (for example cane sugar), emulsifiers (alkyl sulfonates or aryl sulfonates and the like), dispersants (for example maltose, lignin, methylcellulose, starch and polyvinylpyrrolidone) and lubricants (for example magnesium stearate, talc, stearic acid, sodium lauryl sulfonate) and similar materials.
The ratio between the active ingredient and the excipient preferably varies between 1:10 and 100:1.
The compositions may be administered alone or in combination with other active ingredients of a food or dietary nature (such as proteins isolated from soya, powdered albumin, dried spirulina alga, dried eggs, pumpkin seeds, brewer's yeast) of a nutraceutic and pharmaceutical nature and may be packed in the form of kits with components prepared in predosed packed units.
Administration takes place in the normal way, preferably orally. In this case the pharmaceutical forms suitable for this purpose may also contain other additives including sodium citrate, calcium carbonate or phosphate, together with various additives such as starch, gelatine and similar products in addition to the usual excipients such as lactulose, dextrose, lactose, maltose. Compatible colourings or flavourings may be added in the case of liquid forms.
The compositions to which the invention relates may further comprise one or more components selected from amino acids and vitamins, including drugs compatible with the bacteria used that are capable of potentiating the activity of the active ingredients present therein. Of these drugs, anti-inflammatories, antipyretics, antiseptics, analgesics, antirheumatics, antibacterials, hepatoprotectors, antilipaemics, antivirals and chemotherapy and antitumor drugs may for example be used.
According to the present invention, food compositions formulated with addition of the selected bacteria to foodstuffs in liquid form such as fruit juices and dairy products, including all food products made using milk, or milk and cheese products including but not limited to milks, yoghurts, ice creams, cheeses, butter, cream, are regarded a particularly advantageous compositions; food compositions comprising foodstuffs rich in tryptophan, for example almonds, nuts, proteins isolated from soya, powdered albumin, powdered egg, brewer's yeast, dried spirulina alga and parmesan are also considered to be particularly advantageous.
The compositions to which this invention relates may be used as foodstuffs for men or animals, functional foodstuffs, food supplements, drugs for animal or human use, or pharmaceutical products.
The quantity of lactic bacteria is not particularly limited in the situation where the composition according to the present invention is used as a foodstuff. The quantity may for example be from 0.00001 to 100% by weight, preferably from 0.001 to 50 % by weight and more preferably from 0.1 to 10 % by weight, of the food.
The pharmaceutical, nutraceutical and nutritional compositions according to the invention can be prepared by mixing the bacterial strains and/or their enzymatic products with suitable adjuvant(s) in order to obtain the final product.
In a first preferred embodiment of the invention the food and/or dietary and/or nutraceutic and/or pharmaceutical compositions comprise (% by weight):
Formula 1
Streptococcus thermophilus (40 - 60%)
Bifidobacterium breve (10 - 15%)
Bifidobacterium infantis (10 -15%)
Bifidobacterium longum (10 - 15%)
L. acidophilus (2 - 5%)
L. plantarum (3 - 5%)
L. paracasei (4 - 4.9%)
L. delbrueckii subsp bulgaricus (0.1 - 1%)
optionally from 0 to 80% of an excipient or from 0 to 20% of a compatible drug.
In a second preferred embodiment the composition according to the invention comprises lyophilised lactic bacterial strains selected,in the same % as the first preferred embodiment, from:
Formula 2
Streptococcus thermophilus (40 - 60%)
Bifidobacterium breve (10 - 15%)
Bifidobacterium lactis (10 - 15%)
Bifidobacterium lactis (10 - 15%)
L. acidophilus (2 - 5%)
L. casei (3 - 5%)
L. helveticus (4 - 4.9%)
optionally from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
In a third preferred embodiment the compositions according to the invention comprise lyophilised lactic bacterial strains selected from(% by weight):
Formula 3
Streptococcus thermophilus (34 - 57%)
Bifidobacterium lactis (3 - 10%)
Bifidobacterium lactis (3 - 10%)
L. acidophilus (3 - 5%)
L. plantarum (3 - 8%)
L. casei (3 - 4.9%)
L. helveticus (0.1 - 1%)
L. brevis (5 - 50%)
optionally from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
In a fourth preferred embodiment the composition according to the invention comprises lyophilised lactic bacterial strains selected from (same % by weight as in the third preferred embodiment):
Formula 4
Streptococcus thermophilus (34 - 57%)
Bifidobacterium longum (3 - 10%)
Bifidobacterium longum (3 - 10%)
Bifidobacterium infantis (3 - 5%)
L. acidophilus (3 - 8%)
L. plantarum (3 - 4.9%)
L. paracasei (0.1 - 1%)
L. helveticus (0.1 - 1%)
L. brevis (5 - 50%)
optionally from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
In a fifth preferred embodiment the composition according to the invention comprises lyophilised lactic bacteria strains selected from (% by weight):
Formula 5
L. brevis (15 -20%)
P. shermanii (5 -20%)
from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
In a sixth preferred embodiment the composition according to the invention comprises lyophilised lactic bacterial strains selected from(% by weight):
35 - 20
Formula 6
L. brevis (25 -30%)
P. shermanii (5 - 30%)
from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
In a seventh preferred embodiment of the composition according to the invention comprises lyophilised lactic bacterial strains selected from(% by weight):
Formula 7
L. brevis (10 - 40%)
from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
This invention will now be described with reference to the following examples, which are not to be regarded as limiting its scope.
EXAMPLES
The mixtures of lactic bacteria prepared in examples 1-7 have been used to prepare the compositions of the invention.
Example 1
Strains* Amount by weight
S. thermophilus ATCC SD5207; S. thermophilus SP4; S. thermophilus SP4 CS; S. thermophilus SP4 MDX; S. thermophilus (TH-4®) 40 - 60%
B breve Rosell-70; B breve HA-129; B. breve ATCC SD5206 10 - 15%
B. infantis R0033; B. infantis HA-116; B. infantis BI02 10 -15%
B. longum HA-135; B. longum Rosell-175; B. longum ATCC SD5588 10 - 15%
L. acidophilus R0052; L acidophilus Rosell-418; L acidophilus HA-122; Lactobacillus acidophilus ATCC 2 - 5%
L plantarum Rosell-1012; L plantarum HA-119; L plantarum ATCC SD5209; L plantarum P83; L plantarum P94; L plantarum D747 3 - 5%
L paracasei Lafti L26 ND; L paracasei HA-196; L paracasei ATCC SD5275; L paracasei LP01 4 - 4.9%
L. delbrueckii ssp. bulgaricus SP96; L. delbrueckii ssp. lactis LL82 MDX 0.1 - 1%
* Each strain taken alone or in mixture
Example 2
Strains* Amount
S. thermophilus ATCC SD5207; S. thermophilus SP4; S. thermophilus SP4 CS; S. thermophilus SP4 MDX; S. thermophilus (TH-4®) 40 - 60%
B breve Rosell-70; B breve HA-129; B. breve ATCC SD5206 10 - 15%
B lactis Lafti B94; B lactis HA-194; B lactis ATCC SD5220; B lactis ATCC SD5219; B lactis BL086 20 - 30%
L. acidophilus R0052; L acidophilus Rosell-418; L acidophilus HA-122; Lactobacillus acidophilus ATCC 2 - 5%
L casei Rosell-215; L casei HA-108; L casei ATCC SD5213 3 - 5%
L. Helveticus R0052; L. helveticus Lafti L10; L helveticus HA-128 4 - 4.9%
* Each strain taken alone or in mixture
Example 3
Strains* Amount
S. thermophilus ATCC SD5207; S. thermophilus SP4; S. thermophilus SP4 CS; S. thermophilus SP4 MDX; S. thermophilus (TH-4®) 34 - 57%
L brevis HA-112; L brevis LBR01; L brevis ATCC SD5214 5 - 50%
L plantarum Rosell-1012; L plantarum HA-119; L plantarum ATCC SD5209; L plantarum P83; L plantarum P94; L plantarum D747 3 - 8%
B lactis Lafti B94; B lactis HA-194; B lactis ATCC SD5220; B lactis ATCC SD5219; B lactis BL086 6 -20%
L. acidophilus R0052; L acidophilus Rosell-418; L acidophilus HA-122; Lactobacillus acidophilus ATCC 3 - 5%
L casei Rosell-215; L casei HA-108; L casei ATCC SD5213 3 - 4.9%
L. Helveticus R0052; L. helveticus Lafti L10; L helveticus HA-128 0.1 - 1%
* Each strain taken alone or in mixture
Example 4
Strains* Amount
S. thermophilus ATCC SD5207; S. thermophilus SP4; S. thermophilus SP4 CS; S. thermophilus SP4 MDX; S. thermophilus (TH-4®) 34 - 57%
B. longum HA-135; B. longum Rosell-175; B. longum ATCC SD5588 6 - 20%
B. infantis R0033; B. infantis HA-116; B. infantis BI02 3 -5%
L. acidophilus R0052; L acidophilus Rosell-418; L acidophilus HA-122; Lactobacillus acidophilus ATCC 3 - 8%
L plantarum Rosell-1012; L plantarum HA-119; L plantarum ATCC SD5209; L plantarum P83; L plantarum P94; L plantarum D747 3 - 4.9%
L paracasei Lafti L26 ND; L paracasei HA-196; L paracasei ATCC SD5275; L paracasei LP01 0.1 - 1%
L. Helveticus R0052; L. helveticus Lafti L10; L helveticus HA-128 0.1 - 1%
L brevis HA-112; L brevis LBR01; L brevis ATCC SD5214 5 - 50%
* Each strain taken alone or in mixture
Example 5
Strains* Amount
L brevis HA-112; L brevis LBR01; L brevis ATCC SD5214 15 - 20%
P. shermanii 5 - 20%
* Each strain taken alone or in mixture
Example 6
Strains* Amount
L brevis HA-112; L brevis LBR01; L brevis ATCC SD5214 25 - 30%
P. shermanii 5 - 30%
* Each strain taken alone or in mixture
Example 7
Strains* Amount
L brevis HA-112; L brevis LBR01; L brevis ATCC SD5214 10 - 40%
* L brevis HA-112 in mixture with L brevis LBR01 and/or L brevis ATCC SD5214
To the mixtures prepared in examples 1-7 starch and/or sugars and/or drugs such as antiviral agents were added to 100%.
Lyophilisation of a mixture of lactobacilli and bifidobacteria
The mixtures of lactobacilli, bifidobacteria and streptococci indicated below were prepared as follows
In a 1,000 ml beaker equipped with a magnetic stirrer, 21 g of D-maltose are dissolved in 350 ml of demineralised water suitably cooled to 5℃. A perfectly clear yellowish solution is obtained after a few minutes' stirring. Keeping the solution cooled at a temperature from 2℃ to 5℃, 45 g of the lactobacilli mixture are added under stirring. The suspension is stirred for approximately 5 minutes keeping its temperature between 2℃ and 5℃. The suspension is then poured into an AISI 316 stainless steel plate precooled to -20℃ in the freezer.
The suspension tends to freeze practically instantaneously. The lyophilisation cycle is started for the purposes of obtaining a lyophilised product with powder humidity below 5% which is the condition necessary for achieving good, lasting microbiological stability of the bacterial mixture. The lyophilisation conditions are the following.
Edward Minifast 2000 Lyophilizer
Condenser temperature:-47℃
Plate tempetrature:-45℃
Product temperature prior to starting the vacuum cycle:-40℃
Lyophilisation heating ramp: 5℃ every 2 hours up to a product temperature of -5℃. The product is left for 12 hours at this temperature after which the temperature recovery ramp is resumed at a rate of 5℃ every 2 hours up to a product temperature of 25℃.
Product unloading.
Example 4
With the mixtures of the strains described above, the following formulations were prepared:
Children's sachets containing a mixture of eight/nine strains with a dose of 150 bln CFU /sachet
Strain mixture 0.5 g
Lactose 0.352 g
Sucrose 1.5 g
Acesulfame K and/or aspartame 0.035 g
Flavour 0.100 g
Silica 0.013 g
Total sachet weight 2.5 g
Adult sachets containing a mixture of eight/nine strains with a dose of 450 bln CFU /sachet
Strain mixture 1.5 g
Maltose 4.38 g
Aspartame 0,04 g
Flavour 0.07 g
Silica 0.01 g
Total sachet weight 6.0 g
The flavours that can be used are extremely varied. The best results have been obtained with a lemon flavour and a strawberry flavour.
Selection of tryptophanase -negative bacterial strains - Indole test
The results of an experiment performed with L. paracasei are reported by way of an example.
L. paracaseiand E. coli (Figure 3) were cultured in MRS broth (de Man, Rogosa and Sharp) (18 hours at +37℃) and transplanted into 1% tryptophan broth. After incubation for 24 and 48 hours at +37℃ the indole test was performed by adding 5 drops of Kovacs reagent directly to the test tube.
The indole test (Manual of Clinical Microbiology - ASM, 1985, page 1094) is based on the formation of a red-purple complex due to reaction between the indole and the aldehyde group of p-dimethylaminobenzaldehyde.
The test, at 24 hours and 48 hours, was only positive for E. colias illustrated in Figure 3(E colion the left; L paracasei on the right).
The following bacteria assayed by the indole test have shown that they have no tryptophanase activity and have therefore been considered useful for the present invention: S. Thermophilus, L. plantarum, L. acidophilus, L. paracasei, L. helveticus, B. lactis, B. breve, B. infantis, B. longum, L. lactis, L. brevis, P. freudenreichii. Other bacteria which are negative to the indole test such as Bordetella, Haemophilus, Proteus mirabilis, Pseudomonas cannot be used for the purposes of this invention, above all because they are pathogenic or potentially pathogenic.
Selection of bacterial strains capable of inducing the production of IFN -γ
Peripheral blood from healthy donor was collected into heparinized tubes and diluted 1:2 with phosphate buffered saline (PBS).Mononuclear cells were isolated by density gradient centrifugation and diluted to 1 Х 106/ml in RPMI-1640 supplemented with glutamine, HEPES buffer, penicillin, streptomycin and 10% of a of foetal calf serum (Flow Laboratories). When stimulating with the bacteria to test, the bacteria were added in a 1:1 ratio to the cell suspension in the wells and the incubation time was 24 hours at 37℃, 5%CO2.
Interferon-γ measurements
Abcam's Interferon gamma (IFNG) Human ELISA (Enzyme-Linked Immunosorbent Assay) kit is an in vitro enzyme-linked immunosorbent assay for the quantitative measurement of Human Interferon gamma in cell lysates and tissue lysates.
This assay employs an antibody specific for Human Interferon gamma coated on a 96-well plate. Standards and samples are pipetted into the wells and Interferon gamma present in a sample is bound to the wells by the immobilized antibody. The wells are washed and biotinylated anti-Human Interferon gamma antibody is added. After washing away unbound biotinylated antibody, HRP-conjugated streptavidin is pipetted to the wells.The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Interferon gamma bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.
For the purposes of this invention strains capable of inducing levels of IFN-γ in excess of 0.2 ng/mlin vitrocan be used for the purposes of the present invention. Preferably S. thermophilus (1.8 ng/ml), L. acidophilus (1.5 ng/ml), B. lactis (1.3 ng/ml), B. bifidum (1.3 ng/ml) and L. helveticus (1.2 ng/ml).
Anti-inflammatory activity in patients suffering from AIDS
Antiretroviral Therapy (ART) has led to dramatic improvements in the lives of HIV-infected people. However, residual immune activation and inflammation, which persists despite ART, is associated with increased risk of related non-AIDS diseases (Ishizaka A. et al, 2016). Different evidences show that HIV-1 infection induces alteration in gut immune homeostasis, mucosa structure and microbial composition. We analyzed the impact of six-months composition (composition N° 1) supplementation on peripheral blood and gut cellular immune activation in chronic HIV-1 infected patients under ART.
Given the strong relationship between the damage of the intestinal epithelium, the altered GUT microbiota composition, the neurocognitive impairment and the modification of the pathway of tryptophan observed in HIV patients we evaluated before and after six months of diet probiotic supplementation:
a) the inflammatory infiltrate and the damage to the integrity of the epithelium in intestinal biopsies ;
b) the production of the IDO by GUT;
c) the levels of neopterin in cerebrospinal fluid (CSF);
d) the levels of serotonin in peripheral blood;
e) the amount of tryptophan in peripheral blood;
Patients were sampled for peripheral blood and colonscopy, before and after probiotic supplementation. Colonic washing was carried out by PEG administration 24 hours before the examination. The endoscopic procedure was performed with conscious sedation (midazolam 5mg/iv) using large cup forceps (Radial Jaw 4, Boston Scientific, Natick,Massachusetts, USA). All HIV-1 positive patients underwent a total colonoscopy and retrograde ileoscopy for at least 10 cm of distal ileum with conventional or slim scope (model CF or PCF-160 AI, Olympus Medical Europe GmbH, Hamburg, Germany). We obtained specimens (2 biopsies from each site) from the terminal ileum, cecum, ascending, transverse, and descending colon.
All the numbers evaluated for each microscopic field are calculated in 5 randomly selected field of the histological section, evaluated at 40XHPFS. All the cells immunohistochemically positive for each antigen were counted.
Histological examination primarily included the assessment of cellular infiltrates and aggregation by scoring the amount of intra epithelial lymphocytes (I.E.Ls) (mononuclear cells, such as macrophages, lymphocytes, plasma cells, and neutrophils) at a magnification of 400X.
The number of inflammatory cells was evaluated by using a semiquantitative method, as described previously (ref. IBD Working Group of the European Society for Paediatric Gastroenterology,Hepatology and Nutrition) and results are reported as the mean for the entire specimen.
For the in situ detection of epithelial and lymphocytes levels of apoptotic cells, terminal deoxynucleotidyl transferase-mediated digoxigenindeoxyuridine triphosphate nick-end labeling (TUNEL) was applied on histological sections of endoscopic ileal and colonic biopsy specimens obtained from all HIV-1 positive patients according to the modifications performed by Gavrieli et al., (Gavrieli Y et ., ). The sections were digested with proteinase K, 20 μg/mL (Sigma Chemical, St Louis, MO) for 15 minutes at room temperature and then washed in tap water. Endogenous peroxidase was quenched by using 3% hydrogen peroxidase for 20 minutes and then washed in phosphate-buffered saline (PBS). After equilibration, the sections were incubated in a humidified chamber with terminal deoxynucleotidyl transferase enzyme for 1 hour at 37℃. Afterward, sections were soaked in stop-wash buffer for 30 minutes and then rinsed in PBS and, thereafter, incubated with antidigoxigenin peroxidase in a humidified chamber for 30 minutes, followed by addition of diaminobenzidine and hydrogen peroxide for 1 to 3 minutes to allow color development on apoptotic nuclei. Finally, sections were counterstained with Harris hematoxylin.
CSF was collected by lumbar puncture, centrifuged and cell-free supernatant samples stored in aliquots at -80℃. Twenty milliliters of whole blood were collected by venipucture in Vacutainer tubes containing ethylenediaminetetraacetic acid (BD Biosciences, San Jose, CA) at each study visit. Plasma was immediately separated by centrifugation and stored at -80℃.
About the amount of Tryptophan Chromatographic elution was performed with a binary gradient system with sodium acetate (30 mM, pH 6.5) and acetonitrile in a ratio of 80:20 (v/v) (solvent A) and propan-2-ol with acetonitrile 50:50 (v/v) (solvent B). Data analysis was performed by using a dedicated software (Millennium32).
Results:
First of all we evaluated the level of IDO, which is involved in tryptophan metabolism, in GALT, measuring its amount in LPL of HIV-1 infected patients. We found in our studied population higher levels of IDO after probioic supplementation (Coefficient of variability > 100%). On the contrary the amount of tryptophan was reduced in all patients after six months of probiotic dietary integration.
In addition in order to evaluate the inflammation rate both in GUT and in CNS, we evaluated the inflammatory infiltrate in intestinal biopsies collected before and after the probiotic supplementation, focusing on intraepithelial lymphocytes (IELs) density and on the rate of enterocytes undergoing apoptosis.We found that the decline of IELs infiltrating the intestinal epithelium after the probiotic supplementation was strictly associated to a statistically significant decrease in the levels of enterocytes apoptosis index both in epithelium and intestinal crypts (p=0.04).
Similarly in all studied population we found significant decrease of the level of neopterin in the CSF and a significant increase of the levels of serotonin before (T0) and after (T6) probiotic supplementation.
In conclusion probiotics supplementation for 6 months in 6 HIV-1+ under combined antiretroviral therapy (cART) induced higher levels of IDO related to altered tryptophan metabolism.
In addition in all patients was observed after six months of probiotic supplementation a significant reduction of the general inflammation both in the GUT and in the CSF.
The result data is as follows.
Figure PCTKR2018002969-appb-T000001

Claims (33)

  1. Dietary, nutraceutic, pharmaceutical and food supplement compositions comprising tryptophanase-negative microorganisms selected from: lactic bacteria, bifidobacteria, streptococci and mixtures thereof, in combination with microorganisms inducing IFN gamma selected from: lactic bacteria, bifidobacteria, streptococci and mixtures thereof.
  2. Compositions according to the preceding claim in which the microorganisms inducing IFN gamma are microorganisms able to induce a production of IFN gamma equal to or higher than 0.2 ng/ml.
  3. Compositions according to claims 1-2 for use in conditions that need or benefit or are improved from activation of the kynurenine metabolic pathway in mammals, human or animal.
  4. Compositions according any one of claims 1-3 in which the conditions are selected from: need of increased tolerance to the fetus in pregnancy, control of chronic inflammation, protection from autoimmunity, prevention and treatment to alleviate the symptoms of autoimmune diseases, help in preventing organ rejection, increased provision of energy, in particular formation of adenosine triphosphate, nicotinamide and related compounds such as nicotinamide adenine nucleotide and nicotinamide adenine nucleotide phosphate.
  5. Compositions according to any one of claims 1-4 for use in the prevention and treatment of chronic inflammation at local, systemic and cerebral level, such as in the treatment of intestinal inflammatory diseases, chronic liver diseases such as steato-hepatitis and chronic hepatitis, in chronic inflammatory diseases of the central and peripheral nervous system, in autoimmune diseases, in inflammation in anti-rejection treatment following transplant, in faecal transplant, in antiviral treatments, in treating inflammation of microglia, in the treatment of autism, in the treatment of cardiovascular diseases and diabetes.
  6. Compositions according to any one of claims 1-5 for use in the prevention and treatment of HIV-infected people.
  7. Compositions according to any one of claims 1-6 for use in stabilising and regulating tryptophan metabolism in early infancy.
  8. Compositions according to any one of claims 1-7 for use in the prevention and treatment of changes in the amygdala, hippocampus and/or frontal cortex in individuals, in particular the elderly, who are hospitalised, in hospices, receiving antibiotic treatment or treatment with drugs altering the intestinal flora, such as antiviral and antitumor drugs, and in all situations of tryptophan dysmetabolism.
  9. Compositions according to any one of claims 1-8 for use in potentiation of the production of ATP, NAD and NADP in mammals, humans and animals, both in conditions of energy deficit and to improve sporting performance or to alleviate fatigue or in conditions which require increased provision of energy for the body, in particular in conditions of physical fatigue and in sporting activities.
  10. Compositions according to any one of claims 1-9 for use in individuals suffering from repeated spontaneous abortions.
  11. Compositions according to any one of claims 1-10 for oral, enteral or rectal administration.
  12. Compositions according to any one of claims 1-11 in which the microorganisms are used in a vital or non-vital form, and mixtures thereof.
  13. Compositions according to the preceding claim in which the non-vital forms are obtained by killing with heat or radiation.
  14. Compositions according to any one of claims 12-13 in which the microorganisms are in dried or lyophilised or spray dried form, or subjected to vacuum drying and air drying processes.
  15. Compositions according to any one of claims 1-14 further comprising one or more components selected from tryptophan, 5-hydroxytryptamine, melatonin and excipients used for the preparation of food, dietary, nutraceutic and pharmaceutical compositions.
  16. Compositions according to any one of claims 1-15 further comprising pharmaceutically acceptable agents selected from: adjuvants, excipients, disaggregating agents, lubricants, binding agents, oxidation inhibitors, colourings, aggregation inhibitors, absorption promoters, dissolution aids and stabilisers.
  17. Compositions according to the preceding claim in which the adjuvants are selected from: water, non-toxic organic solvents such as paraffins, vegetable oils such as peanut oil or sesame oil, alcohols such as ethanol, glycerine, glycols such as propylene glycol and polyethylene glycol, solid substrates such as natural mineral flours such as kaolin and talc, synthetic mineral flours such as silicates, sugars such as cane sugar, emulsifiers such as alkyl sulfonates or aryl sulfonates; dispersants such as maltose, lignin, methylcellulose, starch and polyvinylpyrrolidone; lubricants such as magnesium stearate, talc, stearic acid, sodium lauryl sulfonate; lactulose, dextrose, lactose, maltose, other additives including such as sodium citrate, calcium carbonate or phosphate, together with various additives such as starch, gelatine; colourings, flavourings.
  18. Compositions according to any one of claims 1-17 for administration alone or in combination with other food nutraceutical and dietary active ingredients such as almonds, nuts, proteins isolated from soya, powdered albumin, powdered egg, brewer's yeast, dried spirulina alga, parmesan; proteins isolated from soya, albumin powder, desiccated spirulina alga, powdered egg, pumpkin seeds, brewer's yeast; amino acids and vitamins;
  19. Compositions according to any one of claims 1-18 for administration alone or in combination with other farmaceutically active compounds such as anti-inflammatories, antipyretics, antiseptics, analgesics, antirheumatics, antibacterials, hepatoprotectors, antilipaemics, antivirals, or chemotherapy and antitumor drugs.
  20. Compositions according to any one of claims 1-19 packed in the form of kits with the components prepared in predosed and packed units.
  21. Compositions according to any one of claims 1-20 which are food compositions in the form of fruit juices, dairy products including but not limited to milk, yogurt, ice cream, cheeses, butter, cream.
  22. Compositions according to the preceding claim in which the quantity of lactic bacteria, bifidobacteria, streptococci varies from 0.00001 to 100% by weight, preferably 0.001 to 50% by weight, and more preferably from 0.1 to 10% by weight of the food.
  23. Compositions according to the preceding claim in which the bacteria are selected in the following list:
    Figure PCTKR2018002969-appb-I000001
    taken alone or in mixture among them.
  24. Compositions according to any one of claims 1-23 comprising as % by weight:
    Streptococcus thermophilus, (40 - 60%)
    Bifidobacterium breve (10 - 15%)
    Bifidobacterium infantis (10 - 15%)
    Bifidobacterium longum (10 - 15%)
    L. acidophilus (2 - 5%)
    L. plantarum (3 - 5%)
    L. paracasei (4 - 4.9%)
    L. delbrueckii subsp bulgaricus (0.1 - 1%)
    optionally from 0 to 80% of an excipient or from 0 to 20% of a compatible drug.
  25. Compositions according to any one of claims 1-23 comprising as % by weight:
    Streptococcus thermophilus (40 - 60%)
    Bifidobacterium breve (10 - 15%)
    Bifidobacterium lactis (10 - 15%)
    Bifidobacterium lactis (10 - 15%)
    L. acidophilus (2 - 5%)
    L. casei (3 - 5%)
    L. helveticus (4 - 4.9%)
    optionally from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
  26. Compositions according to any one of claims 1-23 comprising as % by weight:
    Streptococcus thermophilus (34 - 57%)
    Bifidobacterium lactis (3 - 10%)
    Bifidobacterium lactis (3 - 10%)
    L. acidophilus (3 - 5%)
    L. plantarum (3 - 8%)
    L. casei (3 - 4.9%)
    L. helveticus (0.1 - 1%)
    L. brevis (5 - 50%)
    optionally from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
  27. Compositions according to any one of claims 1-23 comprising as % by weight:
    Streptococcus thermophilus (34 - 57%)
    Bifidobacterium longum (3 - 10%)
    Bifidobacterium longum (3 - 10%)
    Bifidobacterium infantis (3 - 5%)
    L. acidophilus (3 - 8%)
    L. plantarum (3 - 4.9%)
    L. paracasei (0.1 - 1%)
    L. helveticus (0.1 - 1%)
    L. brevis (5 - 50%)
    optionally from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
  28. Compositions according to any one of claims 1-23 comprising as % by weight:
    L. brevis (15 - 20%)
    P. shermanii (5 - 20%)
    from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
  29. Compositions according to any one of claims 1-23 comprising as % by weight:
    L. brevis (25 - 30%)
    P. shermanii (5 - 30%)
    from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
  30. Compositions according to any one of claims 1-23 comprising as % by weight:
    L. brevis (10 - 40%)
    from 0 to 80% of an excipient and from 0 to 20% of a compatible drug.
  31. Foodstuffs, food supplements or drugs comprising the compositions according to any one of claims 1-30.
  32. Use of compositions according to any one of claims 1-30 for the preparation of foodstuffs, drugs, nutraceutic products and food supplements.
  33. Use according to claim 32 to be administered to subjects that need or benefit or are improved from activation of the kynurenine metabolic pathway, the subjects being mammals, human or animal.
PCT/KR2018/002969 2017-03-15 2018-03-14 Tryptophanase-negative lactic bacteria, corresponding compositions and uses WO2018169297A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201880018611.5A CN110446497A (en) 2017-03-15 2018-03-14 Tryptophanase feminine gender lactic acid bacteria, correspondent composition and purposes
JP2019572333A JP2020512014A (en) 2017-03-15 2018-03-14 Tryptophan-degrading enzyme-negative lactic acid bacterium, corresponding composition and use

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
KR10-2017-0032383 2017-03-15
KR1020170032383A KR102089836B1 (en) 2017-03-15 2017-03-15 The pharmaceutical composition for treating or preventing for inflammation disease using Tryptophanase-negative bacteria and bacteria inducing Interferon gamma and method using thereof

Publications (1)

Publication Number Publication Date
WO2018169297A1 true WO2018169297A1 (en) 2018-09-20

Family

ID=63522511

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2018/002969 WO2018169297A1 (en) 2017-03-15 2018-03-14 Tryptophanase-negative lactic bacteria, corresponding compositions and uses

Country Status (4)

Country Link
JP (1) JP2020512014A (en)
KR (1) KR102089836B1 (en)
CN (1) CN110446497A (en)
WO (1) WO2018169297A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10639319B2 (en) 2011-08-29 2020-05-05 Abbott Laboratories Human milk oligosaccharides for preventing injury and/or promoting healing of the gastrointestinal tract
US11179406B2 (en) 2010-12-31 2021-11-23 Abbott Laboratories Methods for decreasing the incidence of necrotizing enterocolitis in infants, toddlers, or children using human milk oligosaccharides
US11311562B2 (en) 2010-12-31 2022-04-26 Abbott Laboratories Methods for reducing the incidence of oxidative stress using human milk oligosaccharides, vitamin c and anti-inflammatory agents
WO2022184637A1 (en) * 2021-03-01 2022-09-09 Evonik Operations Gmbh Preparations comprising probiotic strains and l-tryptophan
US11446316B2 (en) 2011-07-22 2022-09-20 Abbott Laboratories Galactooligosaccharides for preventing injury and/or promoting healing of the gastrointestinal tract

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111012804B (en) * 2019-12-16 2022-05-17 南京大学 Probiotic mineral material composite preparation and application of mineral material in preparation of medicine for inhibiting malignant tumor growth

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4459938B2 (en) * 2006-09-04 2010-04-28 オハヨー乳業株式会社 Streptococcus lactic acid bacteria that normalize immune balance and food and drink using the same
US20170067065A1 (en) * 2014-12-22 2017-03-09 Synlogic, Inc. Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9980988B2 (en) * 2002-03-13 2018-05-29 Kibow Biotech, Inc. Compositions and methods for augmenting kidney function
US7179460B2 (en) * 2002-12-05 2007-02-20 Danisco A/S Bacterial composition and its use
CN102459571B (en) * 2009-06-04 2014-01-08 香港中文大学 Anti-inflammatory bacteria
JP6052485B2 (en) * 2012-02-16 2016-12-27 国立大学法人金沢大学 Immune activated lactic acid bacteria composition and immune activated lactic acid fermented food
PL2937424T3 (en) * 2014-04-23 2017-09-29 National Yang-Ming University Lactic acid bacterium, composition containing the same and their use

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4459938B2 (en) * 2006-09-04 2010-04-28 オハヨー乳業株式会社 Streptococcus lactic acid bacteria that normalize immune balance and food and drink using the same
US20170067065A1 (en) * 2014-12-22 2017-03-09 Synlogic, Inc. Bacteria engineered to treat diseases that benefit from reduced gut inflammation and/or tightened gut mucosal barrier

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DEVLIN, A. SLOAN ET AL.: "Modulation of a circulating uremic solute via rational genetic manipulation of the gut microbiota", CELL HOST & MICROBE, vol. 20, no. 6, 2016, pages 709 - 715, XP029849087 *
LEE, DO KYUNG ET AL.: "Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content", LIPIDS IN HEALTH AND DISEASE, vol. 8, no. 21, 2009, pages 1 - 8, XP055548123 *
ROUSE, MICHAEL ET AL.: "Indoles mitigate the development of experimental autoimmune encephalomyelitis by induction of reciprocal differentiation of regulatory T cells and Thl7 cells", BRITISH JOURNAL OF PHARMACOLOGY, vol. 169, no. 6, 2013, pages 1305 - 1321, XP055548125 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11179406B2 (en) 2010-12-31 2021-11-23 Abbott Laboratories Methods for decreasing the incidence of necrotizing enterocolitis in infants, toddlers, or children using human milk oligosaccharides
US11311562B2 (en) 2010-12-31 2022-04-26 Abbott Laboratories Methods for reducing the incidence of oxidative stress using human milk oligosaccharides, vitamin c and anti-inflammatory agents
US11690859B2 (en) 2010-12-31 2023-07-04 Abbott Laboratories Methods for decreasing the incidence of necrotizing enterocolitis in infants, toddlers, or children using human milk oligosaccharides
US11446316B2 (en) 2011-07-22 2022-09-20 Abbott Laboratories Galactooligosaccharides for preventing injury and/or promoting healing of the gastrointestinal tract
US10639319B2 (en) 2011-08-29 2020-05-05 Abbott Laboratories Human milk oligosaccharides for preventing injury and/or promoting healing of the gastrointestinal tract
WO2022184637A1 (en) * 2021-03-01 2022-09-09 Evonik Operations Gmbh Preparations comprising probiotic strains and l-tryptophan

Also Published As

Publication number Publication date
KR102089836B1 (en) 2020-03-16
CN110446497A (en) 2019-11-12
KR20180105381A (en) 2018-09-28
JP2020512014A (en) 2020-04-23

Similar Documents

Publication Publication Date Title
WO2018169297A1 (en) Tryptophanase-negative lactic bacteria, corresponding compositions and uses
WO2018143678A1 (en) Novel lactic acid bacteria and use thereof
WO2019216662A1 (en) Lactobacillus paracasei strain and use thereof
WO2015122717A1 (en) Novel lactic acid bacteria having obesity inhibitory effect and use thereof
WO2021182829A1 (en) Lactobacillus rhamnosus strain having intestinal immunomodulatory function and preventive or therapeutic activity for inflammatory bowel disease
WO2016053003A1 (en) Method for preparing microbial preparation in which aglycone accumulates in cells, and microbial preparation prepared thereby
WO2019226002A1 (en) Lactobacillus crispatus kbl693 strain and use thereof
KR20100015371A (en) Use of selected lactic acid bacteria for reducing atherosclerosis
WO2020262755A1 (en) Novel probiotic composition for regulation of intestinal immunity
WO2021066549A1 (en) Lactobacillus acidophilus kbl409 strain and use thereof
WO2020080736A1 (en) Probiotic strain for ageing, muscle and bone, gut, hyperlipidemia, skin and brain
WO2017131402A1 (en) Novel human gut-derived lactic acid bacteria having immune regulatory function, and use thereof
JP7126004B2 (en) Composition and use thereof
WO2021194225A1 (en) Lactobacillus delbrueckii subsp. lactis ckdb001 strain, and composition for prevention, amelioration, or treatment of non-alcoholic fatty liver comprising same
WO2017047968A1 (en) Novel lactobacillus having various functions, and use thereof
WO2017047962A1 (en) Novel lactobacillus and composition for preventing, improving, or treating degenerative brain diseases or cognitive function disorders
WO2021221398A1 (en) Novel lactic acid bacteria having excellent immune function enhancement effect, and food composition, health functionl food composition and probiotics comprising same
WO2019103198A1 (en) Lactobacillus fermentum kbl 375 strain and use thereof
WO2016093599A1 (en) Pharmaceutical composition for prevention or treatment of metabolic disease, comprising bacteroides acidifaciens as effective ingredient
WO2023055188A1 (en) Novel probiotics and use thereof
WO2020045972A1 (en) Human body-derived lactobacillus fermentum mg4231 or lactobacillus fermentum mg4244 strain having anti-obesity activity and composition comprising same
WO2023068857A1 (en) Novel bacterial strain having anticancer activity and composition for relieving, preventing, or treating cancer using same
WO2023058801A1 (en) Composition for alleviating, preventing, or treating bowel disorder, comprising lactobacillus acidophilus kbl402 or kbl409 strain
CN113164533A (en) Probiotic supplement for metabolic health comprising lactobacillus
WO2022231178A1 (en) Novel lactobacillus fermentum atg-v5 strain, or composition for enhancing immunity, comprising same

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18767061

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2019572333

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 05.12.2019)

122 Ep: pct application non-entry in european phase

Ref document number: 18767061

Country of ref document: EP

Kind code of ref document: A1