WO2009007515A1 - Lactic acid bacteria having pro-inflammatory characteristics - Google Patents

Lactic acid bacteria having pro-inflammatory characteristics Download PDF

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Publication number
WO2009007515A1
WO2009007515A1 PCT/FI2008/050428 FI2008050428W WO2009007515A1 WO 2009007515 A1 WO2009007515 A1 WO 2009007515A1 FI 2008050428 W FI2008050428 W FI 2008050428W WO 2009007515 A1 WO2009007515 A1 WO 2009007515A1
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cremoris
dsm
composition
ssp
pia2
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PCT/FI2008/050428
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French (fr)
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Riitta Korpela
Riina Kekkonen
Anna-Maija Ahonen
Ilkka Julkunen
Minja Miettinen
Ville Veckman
Elina Kajasto
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Valio Ltd
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    • 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
    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1236—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using Leuconostoc, Pediococcus or Streptococcus sp. other than Streptococcus Thermophilus; Artificial sour buttermilk in general
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates to lactic acid bacteria such as Lacto- coccus or Leuconostoc or a mixture thereof, which induce production of Th1 type cytokines from human pheripheral blood mononuclear cells (PBMC) to impart an immunoregulatory function to the cells.
  • PBMC pheripheral blood mononuclear cells
  • the present invention relates to the use of Lactococcus or Leuconostoc or a mixture thereof in the preparation of a product for activating pro-inflammatory and Th1 type immune response.
  • the present invention relates also to a food or a nutritional product, a medical or a pharmaceutical product, and/or an animal feed, which contains said micraorganism(s).
  • Probiotic bacteria such as lactobacilli, bifidobacteria and lactococci, have been shown to modulate immunological responses both in vitro and in vivo studies, with relatively little knowledge about the mechanisms that regulate the beneficial effects of probiotic bacteria at the level of host cells of the whole organism. Furthermore, there is only limited amount of comparative data between different probiotic strains.
  • a bacterium may be referred to as a probiotic if it essentially meets the following requirements: it remains viable in the demanding conditions prevailing in the digestive tract (low pH of the stomach, acids of the digestive system, etc.); attaches to the walls of the intestine; metabolizes in the intestine; is technologically applicable (endures processing); exhibits clinically tested and reported health effects; and is safe to consume (Lee, Y-K and Salminen, S., Trends Food Sci Technol, 6 (1995) 241-245).
  • the best-documented probiotics include Lactobacillus rhamnosus GG (LGG) ATCC 53103, L johnsonii LA1 , L. casei Shirota and Bifidobacterium lactis Bb12.
  • the health-promoting effects of probiotics include for example balancing and maintenance of intestinal flora, stimulation of the immune system and anti-carcinogenic activity.
  • Immune system functions are regulated by cytokines, proteins made by cells that affect the behaviour of cells.
  • Probiotics have been found to both modulate the balance of pro- and anti-inflammatory cytokines and affect the generation of cell- and antibody-mediated immune response.
  • the immunomodulatory responses are found to be strongly strain-specific. Further, no synergistic immune response effects have been generated with bacterial combina- tions (Fujiwara et al, int Arch Allergy Immunol 2004, 135, 205-215).
  • Pro-inflammatory cytokines such as IL-1 ⁇ , IL-1 ⁇ , IL-6, TNF- ⁇ , IL-12, IL-18, IFN- ⁇ , are produced quickly and trigger the inflammation response, which is in turn followed by the production of anti-inflammatory cytokines.
  • Antiinflammatory cytokines such as IL-4, IL-10, TGF- ⁇ , IL-I Ra and IL-18BP, bal- ance the inflammation and prevent it being excessively activated.
  • Lactobacillus rhamnosus GG has been found to increase the production of pro-inflammatory TNF- ⁇ and IL-6 as well as anti-inflammatory IL -10 in human mononuclear cells (Miettinen, M. et al., Infection and Immunity 64 (12) (1996) 5403-5). Similarly, Lactobacillus casei Shirota, bifidobacteria and lactococci have been shown to be able to induce the production of proinflammatory cytokines such as TNF- ⁇ , IL-6 and IL-12.
  • EP 1 538 198 describes that certain microorganisms belonging to strain Lactococcus lactis i.e., Lactococc ⁇ s lactis subsp. cremoris C60 (FERM BP 08559) and Lactococcus lactis subsp. lactis biovar diacetylactis DRC 1 (MAFF400206), have an immunoregulatory function, which induces production of an anti-inflammatory cytokine IL-10 from mammalian dendritic or spleen cells.
  • the bacterial cultures most frequently used as starter cultures in the dairy industry in the manufacture of fermented milks comprise lactic acid bac- teria (LAB) of the Lactobacillus, Leuconostoc, Lactococcus and Streptococcus spp.
  • Bacteria from the genus Leuconostoc play an important role in the dairy industry being used in mesophilic starter cultures for the manufacture of fermented dairy products and being the dominant flavour producing strains in several cheeses.
  • the most important function of Leuconostoc spp. is their abi- lity to produce carbon dioxide (CO 2 ) and flavour compounds through lactose heterofermentation and citrate utilization. However, little is known about their physiological and biochemical capabilities and genetic properties.
  • cremoris strains are among the most useful strains for producing flavour compounds in dairy fermentations, characterized with their citrate utilization, production of diacetyl and acetoin under neutral and acidic conditions, diacetyl reductase activity, and plasmid profiles. Considerable va- riability within strains is observed. Most strains utilized citrate under neutral conditions, at about pH 6.5, without a concomitant production of diacetyl or acetoin. Furthermore, according to the current knowledge fermented dairy products such as viscous fermented milks produced with mesophilic strains(s) cannot be considered as probiotic products.
  • probiotics and their effects on inflammatory function have been extensively studied, a probiotic and a composition comprising said probi- otic with wide specific pro-inflammatory, anti-inflammatory, Th1 or Th2 type profiles together with balanced immune responses are still very welcome. Consequently, there is continued, evident need to offer the consumers probiotic compositions and products having clearly demonstrated balanced immu- noregulatory profile which are produced in a form that allows them to be con- sumed as a convenient part or a supplement, for example, of the every-day diet.
  • the present invention relates to a new probiotic composition with an efficient Th1 type cytokine profile, an antiviral activity and/or a balanced immunological response, which hence is capable of enhancing natural immunity.
  • the present invention relates to a food or nutritional product, a me- dicinal or pharmaceutical product or an animal feed product comprising said probiotic composition.
  • the present invention further relates to strains having properties that increase Th1 type cytokine responses and antiviral activity, and hence, being well suited for use both as a starter and as a health-promoting ingredi- ent.
  • the present invention relates to strains Lactococcus lactis ssp. cremoris ARH 74, DSM 18891 and/or Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 and to a bacterial starter culture and/or a probiotic composition comprising Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 and/or Lactococcus lactis ssp.
  • cremoris ARH74, DSM 18891 Further, the present invention relates to a bacterial starter culture and/or a probiotic composition consisting of Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 and/or Lactococcus lactis ssp. cremoris ARH74, DSM 18891 , as the probiotically active strain(s). The present invention further relates to the use of the strains Lactococcus lactis ssp. cremoris ARH74, DSM 18891 and/or Leuconostoc mesenteroides ssp.
  • cremoris PIA2, DSM 18892 in food, pharmaceutical or feed industry and to edible products, such as food and feed products and pharmaceuticals, which contain or which have been prepared by using said strain(s).
  • the invention further relates to the use of the probiotic composition of the invention in the preparation of a product having Th1 type immune response enhancing and antiviral characteristics.
  • the invention still further relates to the use of the probiotic composition of the invention, and strains Lactococcus lactis ssp. cremoris ARH74, DSM 18891 and/or Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 in the preparation of a product for enhancing pro-inflammatory Th 1 type immune response and/or for preventing or treating allergy and/or atopic diseases and/or viral diseases and/or infectious diseases.
  • the invention also relates to the use of the probiotic composition of the invention, and strains Lac- tococcus lactis ssp. cremoris ARH74, DSM 18891 and/or Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 for enhancing pro-inflammatory Th 1 type immune response and/or for preventing or treating allergy and/or atopic diseases and/or viral diseases and/or infectious diseases.
  • the present invention relates also to a milk-based and/or milk- derived product as such having a probiotic effect selected from the group consisting of immunomodulation, Th1 type immune response enhancing activity, protection against allergies and/or atopic diseases and/or infectious diseases, anti-viral activity or as an ingredient for use in the preparation of functional food products.
  • the probiotic composition of the invention can be used as such or as a part of another product, such as a pharmaceutical or a food or a nutritional product.
  • the composition influences the immune response by increasing the amount of Th1 type cytokines, such as IL-12 and IFN- ⁇ .
  • the composition of the invention is thus useful for the prevention and treatment of intestinal disorders, allergies and cancer, for fighting against viral and/or infectious diseases and for promoting general health.
  • the probiotic and/or the probiotic composition of the invention could be used in a processed milk product such as yoghurt, curdled milk, curd, sour milk, viili, buttermilk and other sour milk products.
  • a processed milk product such as yoghurt, curdled milk, curd, sour milk, viili, buttermilk and other sour milk products.
  • other edible products such as milk, flavoured milk, beverages, ice-cream etc. are available.
  • products are also applicable as capsules, pills or tablets that allow the use as convenient part or supplement, for example, of the every-day diet.
  • Figure 1 shows the productionof TNF- ⁇ in S. pyogenes (GAS) and probiotic stimulated PBMCs with bacteria:host cell ratio of 0.2:1 , 1 :1 or 5:1 .
  • Figure 2 shows the production of IL-12 in S. pyogenes (GAS) and probiotic stimulated PBMCs with bacteria:host cell ratio of 0.2:1 , 1 :1 or 5:1.
  • Figure 3 shows the production of IFN- ⁇ in S. pyogenes (GAS) and probiotic stimulated PBMCs with bacteria:host cell ratio of 0.2:1 , 1 :1 or 5:1.
  • Figure 4 shows the production of IL-10 in S. pyogenes (GAS) and probiotic stimulated PBMCs with bacteria:host cell ratio of 0.2:1 , 1 :1 or 5:1.
  • probiotics there is a continuous need for probiotics, probiotic compositions, and products having at least one probiotic effect selected from immunomodu- lation, Th1 type immune response enhancing activity, anti-allergic activity, anti- atopic activity, anti-infectious activity and/or anti-viral activity.
  • the present invention is directed to a probiotic composition comprising at least one Leuconostoc strain which is able to activate Th1 type immunological responses in human leukocytes or a composition comprising at least one Leuconostoc and at least one Lactococcus strain which are able to activate Th1 type immunological responses in human leukocytes.
  • the present invention is further directed to a product comprising said probiotic composition.
  • the present invention is also directed to a product produced by using said composition.
  • the probiotic compositions and/or products of the present invention have an effect selected from immunomodulation, Th1 type immune response enhancing activity, anti-allergic activity, anti-atopic activity, anti-infectious activity or anti-vira! activity.
  • Leuconostoc is Leuconostoc mesenteroides ssp. cremoris.
  • Lactococcus is Lactococcus Iactis ssp. cremoris.
  • Leuconostoc mesenteroides ssp. cremoris is Leuconostoc mesenteroides ssp. cremoris PIA2, which has been deposited with the depository authority Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under accession number DSM 18892.
  • Lactococcus Iactis ssp. cremoris is Lactococcus Iactis ssp. cre- moris, ARH74, which has been deposited with the depository authority Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under accession number DSM 18891.
  • the present invention is thus directed to a probiotic composition comprising Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892.
  • the invention is directed to a probiotic composition comprising at least one Leuconostoc and Lactococcus Iactis ssp. cremoris ARH 74, DSM 18891
  • Lactococcus Iactis ssp. cremoris ARH 74 (DSM 18891) is Gram- positive, facultative anaerobe, cocci in short chains, mesophilic (with optimum temperature of about 20-25 0 C).
  • the strain ferments D-galactose, D-glucose, D-fructose, D-mannose and D-lactose (bovine origin) (API 50 CHL V5).
  • cremoris PIA2 (DSM 18892) is Gram-positive, facultative anaerobe, cocci (ovoid) in short chains, mesophilic (with optimum temperature of about 22-25X).
  • the strain ferments D-galact- ose, D-glucose and D-lactose (bovine origin) (API 50 CHL V5).
  • AMI 50 CHL V5 D-galact- ose
  • cremoris ARH74 (DSM 18891) and Leu- conostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) were found to be much stronger inducers of Th1 type cytokines than LGG. Furthermore, said strains were found to be much more potent inducers of pro-inflammatory responses (especially pro-inflammatory TNF- ⁇ ), and TM type cytokines IL-12 and IFN- ⁇ than Sfreptococcus pyogenes GAS which is a known cytokine inducer used as a positive control in the In vitro experiments.
  • the ability of the strains Lactococcus lactis ssp. cremoris ARH74 (DSM 18891) and Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) to activate pro-inflammatory and Th1 type immune response is impor- tant and useful for example in preventing and treating allergy or an autoimmune disease atopic diseases.
  • the immune response is skewed towards Th2 type immune response and therefore agents probiotic bacteria and/or their specific structural components and/or secreted compounds able to balance this situation are considered to be useful in the treatment of allergy.
  • the ability to activate pro-inflammatory and Th1 type immune response is valuable also in the treatment and prevention of mita?viral and/or infectious diseases.
  • the probiotic is and/or the probiotic composition comprises Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892).
  • Said strain induces the production of pro-inflammatory and Th1 type cytokines TNF- ⁇ , IL-12 and IFN- ⁇ , with the production increasing towards 24 h, indicating that it has excellent ability of inducing the production of pro-inflammatory and Th1 type cytokines. It is thus considered especially suitable for the purpose of the invention.
  • TNF- ⁇ production was 6.1 times higher than that of LGG.
  • IL-12 production was 34.5 times higher and IFN- ⁇ production 32.8 times higher than LGG's.
  • probiotic composition refers to orally ad- ministrable composition of metabolically active, i.e., live and/or or lyophilized, or non-viable heat-killed, irradiated or lysed probiotic bacteria.
  • the composition may contain other ingredients.
  • the probiotic composition of the invention can be administered orally as such, i.e., in the form of a tablet, capsule or powder.
  • the probiotic composition of the invention can be administered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical product.
  • the usual effective daily dose of a probiotic in humans is from 10 6 to 10 10 cfu.
  • a probiotic is optionally combined with at least one suitable prebiotic compound.
  • a prebiotic is usually a nondigestible carbohydrate such as an oli- go- or polysaccharide, or a sugar alcohol which is not degraded or absorbed in the upper digestive tract.
  • Known prebiotics used commercial products include inulin and transgalacto-oligosaccharides.
  • food product is intended to cover all consumable products that can be solid, jellied or liquid, and to cover both ready-made products and products which are produced by using the probiotic composition of the invention as a starter alone or in combination with conventional starters or other probiotics.
  • Food products can for instance be products of dairy industry or beverage industry.
  • milk-based product means any liquid or semi-solid milk or whey based product having a varying fat content.
  • the milk- based product can be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk, other sour milk products, such as viili, filling of snack bars, etc.
  • Another important group includes milk beverages, such as whey beverages, fermented milks, condensed milks, infant or baby milks; icecream; milk-containing food such as sweets.
  • Another type of product of the invention is an animal feed.
  • the probiotic composition of the invention is a fermented dairy product or it is used in the preparation of a fermented dairy product.
  • the fermentation conditions such as, starter culture(s), temperature, pH and time for the production of fermented milk products or ingredients are selected to meet the requirements of the final product.
  • suitable conditions belongs to knowledge of a person skilled in the art.
  • fermentation is usually allowed to continue until the pH is 4.2 to 4.6.
  • fermentation normally takes from 2 to 7 hours with yoghurts, up to 24 hours with sour cream or 'villi'.
  • the prabiotic composition of the invention and the starter, if any, are used in a balanced proportion to each other to produce the desired effect on pro-inflammatory and Th1 type cytokines.
  • a milk-based fermented product of the invention could be produced by using the conventional fermentation proce- dures of the dairy industry.
  • a milk-based fermented product of the invention can be soured with a chemical acidifying agent.
  • acidifying agent refers to a microbiological starter or culture, a chemical acidifying agent or mixtures thereof. Acidifying may be per- formed by fermenting with at least one product specific culture and/or by using a chemical acidifying agent, such as organic or inorganic acid, for example.
  • the probiotic composition is a milk-based product comprising Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) or Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) and a Lactococcus strain.
  • the probiotic composition is a milk-based product comprising Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) and Lactococcus lactis ssp. cremoris ARH 74 (DSM 18891).
  • the probiotic composition is a milk-based product comprising a Leuconostoc strain and Lactococcus lactis ssp. cremoris ARH 74 (DSM 18891 ).
  • the probiotic composition is a milk-based product produced by utilising Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) or Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) and a Lactococcus strain in the preparation process.
  • the probiotic composition is a milk-based product produced by utilising Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) and Lactococcus lactis ssp. cremoris ARH 74 (DSM 18891 ) in the preparation process.
  • the probiotic composition is a milk-based product produced by utilizing a Leuconostoc strain and Lactococcus lactis ssp. cremoris ARH 74 (DSM 18891 ). Furthermore, the optimum conditions for an economic, inexpensive and efficient production process for producing a fermented food etc. containing an essential amount and balanced proportion of TM type immune response and antiviral activity characteristics is provided with said strains.
  • the milk-based products described above can be used as such to achieve the desired effect.
  • Said products can also be concentrated and used as ingredients.
  • the products can also be dried and used in the form of powder or lyophilisate.
  • the products are also applicable as capsules, pifls or tablets.
  • the products can also be used in the preparation of functional food products, health and wellness edible products, or other corresponding prod- ucts. Possible forms are capsules, pills or tablets, for example, manufactured in conventional processes used in the preparation of such product for example in the pharmaceutical industry.
  • the cultures most frequently used in the manufacture of cultured milks are those comprising lactic acid bacteria.
  • the industrially most useful lac- tic acid bacteria are found among Lactococcus species, Streptococcus species, Enterococcus species, Lactobacillus species, Leuconostoc species and Pediococcus species.
  • Commonly used dairy starter culture strains of lactic acid bacteria are generally divided into mesophilic organisms having optimum growth temperatures at about 30 0 C and thermophilic organisms having opti- mum growth temperatures in the range of about 40 to about 45°C.
  • Typical organisms belonging to the mesophilic group include Lactococcus lactis subsp. lactis, Lactococcus lactis subsp.
  • cremoris Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis and Lactobacillus casei subsp. casei.
  • Thermophilic lactic acid bacterial species include as examples Streptococcus thermophilus, Enterococcus faecium, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus del- brueckii subsp. bulgaricus and Lactobacillus acidophilus.
  • the ratio of different strains is significant for the characteristics of the final fermented product. For example, in case of viili, typically 3 to 4 acid forming lactic acid bacteria strains together with one flavour forming lactic acid bacteria strain are used in the dairy industry.
  • the probiotic composition of the invention having pro-inflammatory and Th1 type characteristics can be added to a food product during its prepa- 5 ration or to a finished food product.
  • the food products in question thus contain the desired cytokines.
  • each of the food product, food material, and/or the pharmaceutical products, and the animal feed is not particularly limited.
  • suitable food and/or nutritional products are dairy products, drinks, o juices, soups or children's foods.
  • the probiotic composition of the invention is used to ferment milk or milk-based liquid or milk-derived liquid such as, whey, cheese whey, acid whey.
  • the probiotic composition and the products of the invention are pri-5 marily suitable for use for human adults and infants.
  • the positive effects of the products are also beneficial to animals, especially pets and production animals. Examples of these include dogs, cats, rabbits, horses, cows, pigs, goats, sheep and poultry.
  • PBMC peripheral blood mononuclear cells
  • Lactococcus lactis ssp. cremoris ARH74 (DSM 18891) and Leu- cx)nostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) were stored in skimmed milk at -70 c C and passaged three times before used in stimulation experiments.
  • ARH74 was grown in calciumcitrate- mediumagar and M17- medium.
  • PIA2 was grown in MRS-medium.
  • Lactobacillus rhamnosus GG (LGG) (ATCC 53103) was grown in MRS-medium.
  • bacteria were grown to logarithmic growth phase, and the number of bacterial cells was determined by counting in a Petroff-Hauser counting chamber.
  • Streptococcus pyogenes (GAS) serotype T1 M1 (IH32030) (National Public Health Institute, Helsinki, Finland) was used as a positive control. It was grown in TY-medium supplemented with 0.2% of glucose. CeH culture
  • PBMCs Human peripheral blood mononuclear cells
  • Stimulations were carried out in RPMI-1640 medium containing 10% FCS, with optimal bacteria:host cell ratio of 1 :1.
  • PBMC peripheral blood mononuclear cells
  • ELISA enzyme-linked immunoabsorbent
  • Cytokine specific ELISAs TNF- ⁇ and IL-10 were determined with antibody pairs and standards obtained from BD Pharmingen (San Diego, CA, USA) with the procedures and the conditions followed the protocol attached to the kit. IFN- ⁇ and IL-12p70 were determined with Eli-pair kits (BioSite, Taby, Sweden) in accordance with their protocol. Results
  • Results are expressed as means ⁇ SD. Cell culture supernatants from bacteria stimulated supernatants were collected and cytokine levels measured with ELISA.
  • TNF-cc production pg/ml
  • Bacteria host cell ratio of 0.2:1, 1:1 or 5:1 was used.
  • Cell culture supernatants from bacteria > stimulated supernatants were collected and cytokine levels measured with ELISA. Results are mean ⁇ SD from four different blood donors.
  • Lactococcus lactis ssp. cremoris ARH74 (DSM 18891 ) induced 7.5 times higher IL-12 production than the reference bacterium LGG (15 vs. 2 pg/ml).
  • Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) induced 34.5 times higher IL-12 production than the reference bacterium LGG (69 vs. 2 pg/ml). The results are shown in Table 2 and Figure 2.
  • Anti-inflammatory cytokine IL-10 was weakly a little bit induced by GAS, LGG and Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892). The results are shown in Table 4 and Figure 4.
  • Bacterial strains were grown, cell culture prepared and stimulation experiment carried out as described in example 1.
  • Stimulated cells were collected, washed with PBS, and lysed in guanidium isothiocyanate, followed by a centrifugation through a CsCI cushion.
  • Samples containing 10 ⁇ g total cellular RNA were size-fractionated on 1% for- maldehyde-agarose gels, transferred to Hybond-N nylon membranes and hybridized at 42 0 C in a solution containing 50% formamide, 5 x Denhardt's solution, 5 x subacute sclerosing panencephalitis and 0.5% sodium dodecyl sulphate. Ethidium bromide staining was used for control equal loading.
  • cremoris PIA2 (DSM 18892) induced IFN- ⁇ and IL-10 mRNA expression at 9 h. The strongest expressions were detected in TNF-oc and IFN- ⁇ genes when stimulated with Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892).
  • Standardised (3% fat content), homogenised, and heat-treated (95 0 C for 5 min) milk was cooled to 17-23°C.
  • Milk was inoculated with a starter culture (L-type; consisting of Lactococcus lactis biovar longi and Leuconostoc mesenteroides ssp. cremoris) (1%) together with Lactococcus lactis subs. cremoris ARH74 (DSM 18891) (0.5%) and Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) (0.5%). Milk was packed and incubated for 20 h (until pH was below 4.6). Fermented milk product was cooled to a temperature below 6°C. It has mild and slightly acidic taste, high viscosity and ropy consistence.
  • Milk for a reference fermented milk product i.e., a product without probiotic characteristics
  • a starter culture L-type (2%), consisting of Lactococcus lactis biovar longi and Leuconostoc mesenteroides ssp. cremoris.
  • Standardised (3% fat content), homogenised, and heat-treated (95°C for 5 min) milk was cooled to 17-24 0 C.
  • Milk was fermented a starter cul- ture (DL-type/L-type; consisting of Lactococcus lactis ssp. lactis, L. lactis ssp cremoris and L. lactis ssp. lactis biovar. diacetylactis) (1%) together with Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) (0.5%) in a tank for 17-24 h (until pH after cooling was below 4.3). The coagulum was broken gently, cooled to a temperature below 6 0 C. It has mild, slightly acidic and aromatic taste due to diacetyl and carbon dioxide produced from citrate metabolism of Leuconostoc.
  • Milk for a conventional fermented milk product (i.e., a product without probiotic characteristics) was inoculated with a starter culture (1.5%, DL- type, consisting of Lactococcus lactis ssp. lactis, L. lactis ssp cremoris and L. lactis ssp. lactis biovar. diacetylactis).
  • a starter culture (1.5%, DL- type, consisting of Lactococcus lactis ssp. lactis, L. lactis ssp cremoris and L. lactis ssp. lactis biovar. diacetylactis).
  • Standardised (3.5% fat content) (non-homogenised), and heat- treated (95°C for 5 min) milk was cooled to 20 0 C.
  • Milk was inoculated with a starter culture (L-type; consisting of Lactococcus lactis ssp. cremoris biovar longum and Leuconostoc mesenteroides ssp. cremoris (3%) together with Lac- tococcus lactis subs, cremoris ARH74 (DSM 18891 ) (0.5%) and Leuconostoc mesenteroides sp. cremoris PIA2 (DSM 18892) (0.5%).
  • a mould, Geotrichum candidum was added with the starter culture.
  • the inoculated milk was pack- aged, placed on the trays and transferred to a ripening tunnel.
  • the containers were incubated at 20 0 C for 18-24 h (until pH was below 4.6). Fermented milk product was cooled to a temperature below 6 0 C. It has mild and aromatic taste. The consistency is thick and slightly ropy but easily spoonable.
  • Milk for reference fermented milk product was inoculated with a starter culture (L-type, consisting of Lactococ- cus lactis ssp. cremoris biovar longum and Leuconostoc mesenteroides ssp. cremoris).
  • L-type consisting of Lactococ- cus lactis ssp. cremoris biovar longum and Leuconostoc mesenteroides ssp. cremoris

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Abstract

The invention relates to a probiotic composition containing at least one leuconostoc strain. The invention also relates to strains, Lactococcus lactis ssp. cremoris ARH74, DSM 18891 and Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892, which are capable of promoting Th1 type cytokines and antiviral activity. The probiotic compo¬sition of the invention is useful in dairy industry and as therapeutic substances in various health-promoting and functional food products and pharmaceuticals.

Description

LACTIC ACID BACTERIA HAVING PRO-INFLAMMATORY CHARACTERISTICS
FIELD OF THE INVENTION
The present invention relates to lactic acid bacteria such as Lacto- coccus or Leuconostoc or a mixture thereof, which induce production of Th1 type cytokines from human pheripheral blood mononuclear cells (PBMC) to impart an immunoregulatory function to the cells. In particular, the present invention relates to the use of Lactococcus or Leuconostoc or a mixture thereof in the preparation of a product for activating pro-inflammatory and Th1 type immune response.
The present invention relates also to a food or a nutritional product, a medical or a pharmaceutical product, and/or an animal feed, which contains said micraorganism(s).
BACKGROUND OF THE INVENTION Probiotic bacteria, such as lactobacilli, bifidobacteria and lactococci, have been shown to modulate immunological responses both in vitro and in vivo studies, with relatively little knowledge about the mechanisms that regulate the beneficial effects of probiotic bacteria at the level of host cells of the whole organism. Furthermore, there is only limited amount of comparative data between different probiotic strains.
A bacterium may be referred to as a probiotic if it essentially meets the following requirements: it remains viable in the demanding conditions prevailing in the digestive tract (low pH of the stomach, acids of the digestive system, etc.); attaches to the walls of the intestine; metabolizes in the intestine; is technologically applicable (endures processing); exhibits clinically tested and reported health effects; and is safe to consume (Lee, Y-K and Salminen, S., Trends Food Sci Technol, 6 (1995) 241-245).
The best-documented probiotics include Lactobacillus rhamnosus GG (LGG) ATCC 53103, L johnsonii LA1 , L. casei Shirota and Bifidobacterium lactis Bb12.
The health-promoting effects of probiotics include for example balancing and maintenance of intestinal flora, stimulation of the immune system and anti-carcinogenic activity.
Immune system functions are regulated by cytokines, proteins made by cells that affect the behaviour of cells. Probiotics have been found to both modulate the balance of pro- and anti-inflammatory cytokines and affect the generation of cell- and antibody-mediated immune response. The immunomodulatory responses are found to be strongly strain-specific. Further, no synergistic immune response effects have been generated with bacterial combina- tions (Fujiwara et al, int Arch Allergy Immunol 2004, 135, 205-215).
Pro-inflammatory cytokines, such as IL-1 β, IL-1α, IL-6, TNF-α, IL-12, IL-18, IFN-γ, are produced quickly and trigger the inflammation response, which is in turn followed by the production of anti-inflammatory cytokines. Antiinflammatory cytokines, such as IL-4, IL-10, TGF-β, IL-I Ra and IL-18BP, bal- ance the inflammation and prevent it being excessively activated.
Lactobacillus rhamnosus GG (LGG) has been found to increase the production of pro-inflammatory TNF-α and IL-6 as well as anti-inflammatory IL -10 in human mononuclear cells (Miettinen, M. et al., Infection and Immunity 64 (12) (1996) 5403-5). Similarly, Lactobacillus casei Shirota, bifidobacteria and lactococci have been shown to be able to induce the production of proinflammatory cytokines such as TNF-α, IL-6 and IL-12.
EP 1 538 198 describes that certain microorganisms belonging to strain Lactococcus lactis i.e., Lactococcυs lactis subsp. cremoris C60 (FERM BP 08559) and Lactococcus lactis subsp. lactis biovar diacetylactis DRC 1 (MAFF400206), have an immunoregulatory function, which induces production of an anti-inflammatory cytokine IL-10 from mammalian dendritic or spleen cells.
The bacterial cultures most frequently used as starter cultures in the dairy industry in the manufacture of fermented milks comprise lactic acid bac- teria (LAB) of the Lactobacillus, Leuconostoc, Lactococcus and Streptococcus spp. Bacteria from the genus Leuconostoc play an important role in the dairy industry being used in mesophilic starter cultures for the manufacture of fermented dairy products and being the dominant flavour producing strains in several cheeses. The most important function of Leuconostoc spp. is their abi- lity to produce carbon dioxide (CO2) and flavour compounds through lactose heterofermentation and citrate utilization. However, little is known about their physiological and biochemical capabilities and genetic properties. Leuconostoc mesenteroides ssp. cremoris strains are among the most useful strains for producing flavour compounds in dairy fermentations, characterized with their citrate utilization, production of diacetyl and acetoin under neutral and acidic conditions, diacetyl reductase activity, and plasmid profiles. Considerable va- riability within strains is observed. Most strains utilized citrate under neutral conditions, at about pH 6.5, without a concomitant production of diacetyl or acetoin. Furthermore, according to the current knowledge fermented dairy products such as viscous fermented milks produced with mesophilic strains(s) cannot be considered as probiotic products.
Major problem associated with the use of the conventional mesophilic cultures, such as lactococcal species, is bacteriophages infection which is common and has a large economic impact. Some strains that are strong producers of polysaccharides, for example exopolysaccharides, are sensitive to bacteriophages as well. Consequently, dramatic adverse effect on the texture of the final fermented product can then be seen. Bacteriophages are seldom observed with Leuconostoc species.
Although probiotics and their effects on inflammatory function have been extensively studied, a probiotic and a composition comprising said probi- otic with wide specific pro-inflammatory, anti-inflammatory, Th1 or Th2 type profiles together with balanced immune responses are still very welcome. Consequently, there is continued, evident need to offer the consumers probiotic compositions and products having clearly demonstrated balanced immu- noregulatory profile which are produced in a form that allows them to be con- sumed as a convenient part or a supplement, for example, of the every-day diet.
Further, in the pharmaceutical field there is a continuous need for compounds and/or agents which can block, control, mitigate, or prevent the formation and/or release of cytokines from cells which produce them.
BRIEF DESCRIPTION OF THE INVENTION
The present invention relates to a new probiotic composition with an efficient Th1 type cytokine profile, an antiviral activity and/or a balanced immunological response, which hence is capable of enhancing natural immunity.
The present invention relates to a food or nutritional product, a me- dicinal or pharmaceutical product or an animal feed product comprising said probiotic composition.
The present invention further relates to strains having properties that increase Th1 type cytokine responses and antiviral activity, and hence, being well suited for use both as a starter and as a health-promoting ingredi- ent. In addition, the present invention relates to strains Lactococcus lactis ssp. cremoris ARH 74, DSM 18891 and/or Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 and to a bacterial starter culture and/or a probiotic composition comprising Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 and/or Lactococcus lactis ssp. cremoris ARH74, DSM 18891. Further, the present invention relates to a bacterial starter culture and/or a probiotic composition consisting of Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 and/or Lactococcus lactis ssp. cremoris ARH74, DSM 18891 , as the probiotically active strain(s). The present invention further relates to the use of the strains Lactococcus lactis ssp. cremoris ARH74, DSM 18891 and/or Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 in food, pharmaceutical or feed industry and to edible products, such as food and feed products and pharmaceuticals, which contain or which have been prepared by using said strain(s). The invention further relates to the use of the probiotic composition of the invention in the preparation of a product having Th1 type immune response enhancing and antiviral characteristics.
The invention still further relates to the use of the probiotic composition of the invention, and strains Lactococcus lactis ssp. cremoris ARH74, DSM 18891 and/or Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 in the preparation of a product for enhancing pro-inflammatory Th 1 type immune response and/or for preventing or treating allergy and/or atopic diseases and/or viral diseases and/or infectious diseases. The invention also relates to the use of the probiotic composition of the invention, and strains Lac- tococcus lactis ssp. cremoris ARH74, DSM 18891 and/or Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 for enhancing pro-inflammatory Th 1 type immune response and/or for preventing or treating allergy and/or atopic diseases and/or viral diseases and/or infectious diseases.
The present invention relates also to a milk-based and/or milk- derived product as such having a probiotic effect selected from the group consisting of immunomodulation, Th1 type immune response enhancing activity, protection against allergies and/or atopic diseases and/or infectious diseases, anti-viral activity or as an ingredient for use in the preparation of functional food products. The probiotic composition of the invention can be used as such or as a part of another product, such as a pharmaceutical or a food or a nutritional product. The composition influences the immune response by increasing the amount of Th1 type cytokines, such as IL-12 and IFN-γ. The composition of the invention is thus useful for the prevention and treatment of intestinal disorders, allergies and cancer, for fighting against viral and/or infectious diseases and for promoting general health.
Furthermore, the probiotic and/or the probiotic composition of the invention could be used in a processed milk product such as yoghurt, curdled milk, curd, sour milk, viili, buttermilk and other sour milk products. According to the invention other edible products such as milk, flavoured milk, beverages, ice-cream etc. are available. In accordance with the present invention, products are also applicable as capsules, pills or tablets that allow the use as convenient part or supplement, for example, of the every-day diet.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the productionof TNF-α in S. pyogenes (GAS) and probiotic stimulated PBMCs with bacteria:host cell ratio of 0.2:1 , 1 :1 or 5:1 .
Figure 2 shows the production of IL-12 in S. pyogenes (GAS) and probiotic stimulated PBMCs with bacteria:host cell ratio of 0.2:1 , 1 :1 or 5:1. Figure 3 shows the production of IFN-γ in S. pyogenes (GAS) and probiotic stimulated PBMCs with bacteria:host cell ratio of 0.2:1 , 1 :1 or 5:1.
Figure 4 shows the production of IL-10 in S. pyogenes (GAS) and probiotic stimulated PBMCs with bacteria:host cell ratio of 0.2:1 , 1 :1 or 5:1.
DETAILED DESCRIPTION OF THE INVENTION There is a continuous need for probiotics, probiotic compositions, and products having at feast one probiotic effect selected from immunomodu- lation, Th1 type immune response enhancing activity, anti-allergic activity, anti- atopic activity, anti-infectious activity and/or anti-viral activity.
Now it has been surprisingly found that a Leuconostoc strain, Leu- conostoc mesenteroides ssp. cremoris PIA2, DSM 18892, is able to activate Th1 type immunological responses in human leukocytes.
Further, it has been surprisingly found that a Lactococcus strain, Lactococcus lactis ssp. cremoris ARH 74, DSM 18891 , is able to activate Th1 type immunological responses in human leukocytes. Accordingly, the present invention is directed to a probiotic composition comprising at least one Leuconostoc strain which is able to activate Th1 type immunological responses in human leukocytes or a composition comprising at least one Leuconostoc and at least one Lactococcus strain which are able to activate Th1 type immunological responses in human leukocytes.
The present invention is further directed to a product comprising said probiotic composition. The present invention is also directed to a product produced by using said composition. The probiotic compositions and/or products of the present invention have an effect selected from immunomodulation, Th1 type immune response enhancing activity, anti-allergic activity, anti-atopic activity, anti-infectious activity or anti-vira! activity.
In one embodiment of the invention, Leuconostoc is Leuconostoc mesenteroides ssp. cremoris. In another embodiment of the invention, Lactococcus is Lactococcus Iactis ssp. cremoris. In a further embodiment of the in- vention, Leuconostoc mesenteroides ssp. cremoris is Leuconostoc mesenteroides ssp. cremoris PIA2, which has been deposited with the depository authority Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under accession number DSM 18892. In a still further embodiment of the invention Lactococcus Iactis ssp. cremoris is Lactococcus Iactis ssp. cre- moris, ARH74, which has been deposited with the depository authority Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) under accession number DSM 18891.
In one embodiment, the present invention is thus directed to a probiotic composition comprising Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892. In another embodiment, the invention is directed to a probiotic composition comprising at least one Leuconostoc and Lactococcus Iactis ssp. cremoris ARH 74, DSM 18891
Lactococcus Iactis ssp. cremoris ARH 74 (DSM 18891) is Gram- positive, facultative anaerobe, cocci in short chains, mesophilic (with optimum temperature of about 20-250C). The strain ferments D-galactose, D-glucose, D-fructose, D-mannose and D-lactose (bovine origin) (API 50 CHL V5).
Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) is Gram-positive, facultative anaerobe, cocci (ovoid) in short chains, mesophilic (with optimum temperature of about 22-25X). The strain ferments D-galact- ose, D-glucose and D-lactose (bovine origin) (API 50 CHL V5). In the present invention it has surprisingly been found that said strains are able to activate Th1 type immunological responses in human leukocytes more efficiently than Lactobacillus rhamnosus GG (LGG), which in previous studies was found to induce strong pro-inflammatory and Th1 type responses. Lactococcus lactis ssp. cremoris ARH74 (DSM 18891) and Leu- conostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) were found to be much stronger inducers of Th1 type cytokines than LGG. Furthermore, said strains were found to be much more potent inducers of pro-inflammatory responses (especially pro-inflammatory TNF-α), and TM type cytokines IL-12 and IFN-γ than Sfreptococcus pyogenes GAS which is a known cytokine inducer used as a positive control in the In vitro experiments.
The ability of the strains Lactococcus lactis ssp. cremoris ARH74 (DSM 18891) and Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) to activate pro-inflammatory and Th1 type immune response, is impor- tant and useful for example in preventing and treating allergy or an autoimmune disease atopic diseases. In allergy, the immune response is skewed towards Th2 type immune response and therefore agents probiotic bacteria and/or their specific structural components and/or secreted compounds able to balance this situation are considered to be useful in the treatment of allergy. The ability to activate pro-inflammatory and Th1 type immune response is valuable also in the treatment and prevention of mita?viral and/or infectious diseases.
In one embodiment of the present invention, the probiotic is and/or the probiotic composition comprises Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892). Said strain induces the production of pro-inflammatory and Th1 type cytokines TNF-α, IL-12 and IFN-γ, with the production increasing towards 24 h, indicating that it has excellent ability of inducing the production of pro-inflammatory and Th1 type cytokines. It is thus considered especially suitable for the purpose of the invention. TNF-α production was 6.1 times higher than that of LGG. Respectively, IL-12 production was 34.5 times higher and IFN-γ production 32.8 times higher than LGG's.
In the present invention "probiotic composition" refers to orally ad- ministrable composition of metabolically active, i.e., live and/or or lyophilized, or non-viable heat-killed, irradiated or lysed probiotic bacteria. The composition may contain other ingredients. The probiotic composition of the invention can be administered orally as such, i.e., in the form of a tablet, capsule or powder. In addition, the probiotic composition of the invention can be administered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical product.
The usual effective daily dose of a probiotic in humans is from 106to 1010 cfu.
A probiotic is optionally combined with at least one suitable prebiotic compound. A prebiotic is usually a nondigestible carbohydrate such as an oli- go- or polysaccharide, or a sugar alcohol which is not degraded or absorbed in the upper digestive tract. Known prebiotics used commercial products include inulin and transgalacto-oligosaccharides.
The term "food product" is intended to cover all consumable products that can be solid, jellied or liquid, and to cover both ready-made products and products which are produced by using the probiotic composition of the invention as a starter alone or in combination with conventional starters or other probiotics. Food products can for instance be products of dairy industry or beverage industry.
In the present invention, "milk-based product" means any liquid or semi-solid milk or whey based product having a varying fat content. The milk- based product can be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk, other sour milk products, such as viili, filling of snack bars, etc. Another important group includes milk beverages, such as whey beverages, fermented milks, condensed milks, infant or baby milks; icecream; milk-containing food such as sweets. Another type of product of the invention is an animal feed.
In the preparation of milk-based products of the invention, conven- tional heat treatment methods such as pasteurization (heating for example at about 72°C for at least 15 seconds), ESL treatment (heating for example at about 1300C for 1 to 2 seconds), UHT treatment (heating for example at about 138°C for 2 to 4 seconds) or high temperature pasteurization (heating at 95°C for 5 minutes), are employed. In one embodiment of the invention, the probiotic composition of the invention is a fermented dairy product or it is used in the preparation of a fermented dairy product.
The fermentation conditions such as, starter culture(s), temperature, pH and time for the production of fermented milk products or ingredients are selected to meet the requirements of the final product. The selection of suitable conditions belongs to knowledge of a person skilled in the art.
The fermentation is usually allowed to continue until the pH is 4.2 to 4.6. In case of fermented milk products, fermentation normally takes from 2 to 7 hours with yoghurts, up to 24 hours with sour cream or 'villi'.
The prabiotic composition of the invention and the starter, if any, are used in a balanced proportion to each other to produce the desired effect on pro-inflammatory and Th1 type cytokines. A milk-based fermented product of the invention could be produced by using the conventional fermentation proce- dures of the dairy industry.
Alternatively, a milk-based fermented product of the invention can be soured with a chemical acidifying agent.
The term "acidifying agent" refers to a microbiological starter or culture, a chemical acidifying agent or mixtures thereof. Acidifying may be per- formed by fermenting with at least one product specific culture and/or by using a chemical acidifying agent, such as organic or inorganic acid, for example.
In one embodiment of the invention, the probiotic composition is a milk-based product comprising Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) or Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) and a Lactococcus strain. In another embodiment of the invention, the probiotic composition is a milk-based product comprising Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) and Lactococcus lactis ssp. cremoris ARH 74 (DSM 18891). In a further embodiment of the invention, the probiotic composition is a milk-based product comprising a Leuconostoc strain and Lactococcus lactis ssp. cremoris ARH 74 (DSM 18891 ).
In another embodiment of the invention, the probiotic composition is a milk-based product produced by utilising Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) or Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) and a Lactococcus strain in the preparation process. In an- other embodiment of the invention, the probiotic composition is a milk-based product produced by utilising Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) and Lactococcus lactis ssp. cremoris ARH 74 (DSM 18891 ) in the preparation process. In a further embodiment of the invention, the probiotic composition is a milk-based product produced by utilizing a Leuconostoc strain and Lactococcus lactis ssp. cremoris ARH 74 (DSM 18891 ). Furthermore, the optimum conditions for an economic, inexpensive and efficient production process for producing a fermented food etc. containing an essential amount and balanced proportion of TM type immune response and antiviral activity characteristics is provided with said strains.
The milk-based products described above can be used as such to achieve the desired effect. Said products can also be concentrated and used as ingredients. Further, the products can also be dried and used in the form of powder or lyophilisate. The products are also applicable as capsules, pifls or tablets. The products can also be used in the preparation of functional food products, health and wellness edible products, or other corresponding prod- ucts. Possible forms are capsules, pills or tablets, for example, manufactured in conventional processes used in the preparation of such product for example in the pharmaceutical industry.
The cultures most frequently used in the manufacture of cultured milks are those comprising lactic acid bacteria. The industrially most useful lac- tic acid bacteria are found among Lactococcus species, Streptococcus species, Enterococcus species, Lactobacillus species, Leuconostoc species and Pediococcus species. Commonly used dairy starter culture strains of lactic acid bacteria are generally divided into mesophilic organisms having optimum growth temperatures at about 300C and thermophilic organisms having opti- mum growth temperatures in the range of about 40 to about 45°C. Typical organisms belonging to the mesophilic group include Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, Leuconostoc mesenteroides subsp. cremoris, Pediococcus pentosaceus, Lactococcus lactis subsp. lactis biovar. diacetylactis and Lactobacillus casei subsp. casei. Thermophilic lactic acid bacterial species include as examples Streptococcus thermophilus, Enterococcus faecium, Lactobacillus lactis, Lactobacillus helveticus, Lactobacillus del- brueckii subsp. bulgaricus and Lactobacillus acidophilus.
However, even today the exact composition or the ratio of the starter cultures based on the strain(s) level is not always known. Furthermore, the ratio of different strains is significant for the characteristics of the final fermented product. For example, in case of viili, typically 3 to 4 acid forming lactic acid bacteria strains together with one flavour forming lactic acid bacteria strain are used in the dairy industry.
The probiotic composition of the invention having pro-inflammatory and Th1 type characteristics can be added to a food product during its prepa- 5 ration or to a finished food product. The food products in question thus contain the desired cytokines.
The form of each of the food product, food material, and/or the pharmaceutical products, and the animal feed is not particularly limited. Examples of suitable food and/or nutritional products are dairy products, drinks, o juices, soups or children's foods.
In one embodiment of the invention the probiotic composition of the invention is used to ferment milk or milk-based liquid or milk-derived liquid such as, whey, cheese whey, acid whey.
The probiotic composition and the products of the invention are pri-5 marily suitable for use for human adults and infants. The positive effects of the products are also beneficial to animals, especially pets and production animals. Examples of these include dogs, cats, rabbits, horses, cows, pigs, goats, sheep and poultry.
The following examples illustrate the present invention. The exam-0 pies are not to be construed to limit the claims in any manner whatsoever.
Example 1. In vitro immunomodulatory potential of strains
Cytokine gene expression patterns in human peripheral blood mononuclear cells (PBMC) in response to stimulation with different probiotic bacterial strains were analyzed. Bacterial strains
Lactococcus lactis ssp. cremoris ARH74 (DSM 18891) and Leu- cx)nostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) were stored in skimmed milk at -70cC and passaged three times before used in stimulation experiments. ARH74 was grown in calciumcitrate- mediumagar and M17- medium. PIA2 was grown in MRS-medium. Lactobacillus rhamnosus GG (LGG) (ATCC 53103) was grown in MRS-medium. For stimulation experiments bacteria were grown to logarithmic growth phase, and the number of bacterial cells was determined by counting in a Petroff-Hauser counting chamber.
Streptococcus pyogenes (GAS) serotype T1 M1 (IH32030) (National Public Health Institute, Helsinki, Finland) was used as a positive control. It was grown in TY-medium supplemented with 0.2% of glucose. CeH culture
Human peripheral blood mononuclear cells (PBMCs) were isolated by a density gradient centrifugation over Ficoll-paque gradient (Amersham- Pharmacia Biotech, Uppsala, Sweden) as described by Pirhonen et al. (J. Im- munol. 162 (1999) 7322-7329).
Stimulation experiments
Stimulations were carried out in RPMI-1640 medium containing 10% FCS, with optimal bacteria:host cell ratio of 1 :1. To characterize cytokine production pattern induced by probiotic bacteria, PBMC were stimulated with probiotics and cell culture supernatants were collected at different time points after stimulation. Supernatants were stored at -200C and used for cytokine quantification by enzyme-linked immunoabsorbent (ELISA) as described by Miettinen et al., Infect Immunol 66 (1998) 6058-6062.
Cytokine specific ELISAs TNF-α and IL-10 were determined with antibody pairs and standards obtained from BD Pharmingen (San Diego, CA, USA) with the procedures and the conditions followed the protocol attached to the kit. IFN-γ and IL-12p70 were determined with Eli-pair kits (BioSite, Taby, Sweden) in accordance with their protocol. Results
Results are expressed as means ± SD. Cell culture supernatants from bacteria stimulated supernatants were collected and cytokine levels measured with ELISA.
The ability of strains to induce cytokine responses was strongly strain dependent. Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) was the best inducer of pro-inflammatory and Th1 type cytokines TNF- α, IL-12 and IFN-γ, with the production increasing towards 24 h, indicating being extremely excellent in the ability of the inducing the production of proinflammatory and Th1 type cytokines. Lactococcus lactis ssp. cremoris ARH74 (DSM 18891 ) induced 1.3 times higher TNF-α production than the positive control Streptococcus pyogenes (GAS) (3346 vs. 2520 pg/ml) and 3.3 times higher than the reference bacterium Lactobacillus rhamnosus GG (LGG) (3346 vs. 1009 pg/ml). Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) induced 2.5 times higher TNF-α production than the positive control GAS (6176 vs. 2520 pg/ml) and 6.1 times higher than the reference bacterium LGG (6176 vs. 1009 pg/ml). The results are shown in Table 1 and Figure 1.
Table 1. Dose-dependent TNF-cc production (pg/ml). Bacteria: host cell ratio of 0.2:1, 1:1 or 5:1 was used. Cell culture supernatants from bacteria > stimulated supernatants were collected and cytokine levels measured with ELISA. Results are mean ± SD from four different blood donors.
Table 1. TNF-α production (pg/ml)
0.2:1 1:1 5:1
Mean ±SD Mean ± SD Mean ± SD control 0 0 0
GAS 2683 ± 1101 2520 ± 825 1211 ± 659
LGG 814 + 576 1009 ±413 471 ± 191
ARH74 2460 + 1206 3346 ±1698 2050 ± 529
PIA2 5496 + 2618 6176 ±2877 3989 ± 1215
Lactococcus lactis ssp. cremoris ARH74 (DSM 18891 ) induced 7.5 times higher IL-12 production than the reference bacterium LGG (15 vs. 2 pg/ml). Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) induced 34.5 times higher IL-12 production than the reference bacterium LGG (69 vs. 2 pg/ml). The results are shown in Table 2 and Figure 2.
Table 2. Dose-dependent IL-12 production (pg/ml). Bacteria:host cell ratio of 0.2:1, 1:1 or 5:1 was used. Cell culture supernatants from bacteria stimulated supernatants were collected and cytokine levels measured with ELISA. Results are mean ± SD from four different blood donors.
Table 2. IL-12 production (pg/rnl).
0.2:1 1:1 5:1
Mean ± SD Mean ± SD Mean ±SD control 0 0 0
GAS 63.32 ± 30.9 77 ± 26 9 ± 17
LGG 11.98 ± 8.9 2 ± 4 0
ARH74 18.73 ± 8.7 15 ± 7 0
PIA2 53.02± 21.6 69 ± 52 30 ± 25 Lactococcus lactis ssp. cremoris ARH74 (DSM 18891) induced 17.3 times higher IFN-γ production than the reference bacterium LGG (1384 vs. 80 pg/ml). Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) induced 32.8 times higher IFN-γ production than the reference bacterium LGG (2624 vs. 80 pg/ml). The results are shown in Table 3 and Figure 3.
Table 3. Dose-dependent IFN-γ production (pg/ml). Bacteria: host cell ratio of 0.2:1, 1:1 or 5:1 was used. Cell culture supematants from bacteria stimulated supematants were collected and cytokine levels measured with ELISA. Results are mean ± SD from four different blood donors.
Table 3. IFN-gamma production (pg/ml).
0.2:1 1 :1 5:1
Mean ± SD Mean ± SD Mean ± SD control 0 0 0
GAS 9805 ± 9145 1132417096 511 ± 376
LGG 526 ± 554 80 ± 93 0
ARH74 12291 1035 13841 1523 61 ±44
PIA2 4090 ± 3573 2624 ± 2163 432 + 638
Anti-inflammatory cytokine IL-10 was weakly a little bit induced by GAS, LGG and Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892). The results are shown in Table 4 and Figure 4.
Compared with Lactococcus lactis ssp. cremoris ARH74 (DSM 18891), Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) was more potent inducer of all pro-inflammatory and Th1 type cytokines.
Table 4. Dose-dependent I FN-γ production (pg/ml). Bacteria: host cell ratio of 0.2:1, 1:1 or 5:1 was used. Cell culture supernatants from bacteria stimulated supernatants were collected and cytokine levels measured with ELISA. Results are mean ± SD from four different blood donors.
Table 4. IL-1 Q production (pg/ml).
0.2:1 1:1 5:1
Mean ± SD Mean ± SD Mean ± SD control 0 0 0
GAS 62 ± 125 187 + 216 0
LGG 0 0 52 ± 105
ARH74 0 0 0
PIA2 201 ± 258 78 ± 156 0
Example 2. mRNA expression of cytokines
The best inducers of pro- and anti-inflammatory cytokines were selected for the mRNA expression experiment.
Bacterial strains were grown, cell culture prepared and stimulation experiment carried out as described in example 1.
RNA isolation and Northern Blotting
Stimulated cells were collected, washed with PBS, and lysed in guanidium isothiocyanate, followed by a centrifugation through a CsCI cushion. Samples containing 10 μg total cellular RNA were size-fractionated on 1% for- maldehyde-agarose gels, transferred to Hybond-N nylon membranes and hybridized at 420C in a solution containing 50% formamide, 5 x Denhardt's solution, 5 x subacute sclerosing panencephalitis and 0.5% sodium dodecyl sulphate. Ethidium bromide staining was used for control equal loading.
Results The kinetics of TNF-α mRNA expression was fast and it was clearly detectable already at 3 h after bacterial stimulation. However, p40, I FN-γ and IL-10 genes were induced with clearly slower kinetics and the expression of these mRNAs was detectable starting from 9 h after stimulation. LGG enhanced TNF-α mRNA expression already at 3 h. p40 and IL-10 mRNA expres- sion was detectable at 9 h, but I FN-γ mRNA expression was seen at 24 h.
Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) induced IFN-γ and IL-10 mRNA expression at 9 h. The strongest expressions were detected in TNF-oc and IFN-γ genes when stimulated with Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892).
Example 3. Fermented milk product
Standardised (3% fat content), homogenised, and heat-treated (950C for 5 min) milk was cooled to 17-23°C. Milk was inoculated with a starter culture (L-type; consisting of Lactococcus lactis biovar longi and Leuconostoc mesenteroides ssp. cremoris) (1%) together with Lactococcus lactis subs. cremoris ARH74 (DSM 18891) (0.5%) and Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) (0.5%). Milk was packed and incubated for 20 h (until pH was below 4.6). Fermented milk product was cooled to a temperature below 6°C. It has mild and slightly acidic taste, high viscosity and ropy consistence.
Milk for a reference fermented milk product (i.e., a product without probiotic characteristics) was inoculated with a starter culture (L-type) (2%), consisting of Lactococcus lactis biovar longi and Leuconostoc mesenteroides ssp. cremoris.
Example 4. Fermented milk product
Standardised (3% fat content), homogenised, and heat-treated (95°C for 5 min) milk was cooled to 17-240C. Milk was fermented a starter cul- ture (DL-type/L-type; consisting of Lactococcus lactis ssp. lactis, L. lactis ssp cremoris and L. lactis ssp. lactis biovar. diacetylactis) (1%) together with Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) (0.5%) in a tank for 17-24 h (until pH after cooling was below 4.3). The coagulum was broken gently, cooled to a temperature below 60C. It has mild, slightly acidic and aromatic taste due to diacetyl and carbon dioxide produced from citrate metabolism of Leuconostoc.
Milk for a conventional fermented milk product (i.e., a product without probiotic characteristics) was inoculated with a starter culture (1.5%, DL- type, consisting of Lactococcus lactis ssp. lactis, L. lactis ssp cremoris and L. lactis ssp. lactis biovar. diacetylactis).
Example 5. Fermented milk product
Standardised (3.5% fat content) (non-homogenised), and heat- treated (95°C for 5 min) milk was cooled to 200C. Milk was inoculated with a starter culture (L-type; consisting of Lactococcus lactis ssp. cremoris biovar longum and Leuconostoc mesenteroides ssp. cremoris (3%) together with Lac- tococcus lactis subs, cremoris ARH74 (DSM 18891 ) (0.5%) and Leuconostoc mesenteroides ssp. cremoris PIA2 (DSM 18892) (0.5%). A mould, Geotrichum candidum was added with the starter culture. The inoculated milk was pack- aged, placed on the trays and transferred to a ripening tunnel. The containers were incubated at 200C for 18-24 h (until pH was below 4.6). Fermented milk product was cooled to a temperature below 60C. It has mild and aromatic taste. The consistency is thick and slightly ropy but easily spoonable.
Milk for reference fermented milk product (without probiotic charac- teristics) was inoculated with a starter culture (L-type, consisting of Lactococ- cus lactis ssp. cremoris biovar longum and Leuconostoc mesenteroides ssp. cremoris).
Figure imgf000019_0001
Figure imgf000020_0001
FOR INTERNATIONAL BUREAU USE ONLY -5 This form was received by the international Bureau on: -5-1 Authorized officer

Claims

I. A probiotic composition comprising Leuconostoc wherein the Leuconostoc is Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892.
2. A probiotic composition comprising Leuconostoc and Lactococ- cus wherein the Lactococcus is Lactococcus lactis ssp. cremoris ARH74, DSM 18891.
3. The composition of claim 1 wherein the composition further comprises Lactococcus.
4. The composition of claim 3, wherein the Lactococcus is Lactococcus lactis ssp. cremoris ARH74, DSM 18891.
5. The composition of claim 1 or claim 2, having a probiotic effect selected from immunomodulation, pro-inflammatory Th1 type immune response enhancing activity, anti-allergic activity, anti-atopic activity, anti-infectious activ- ity and antiviral activity.
6. The composition of any one of claims 1 to 5, wherein it is for oral administration.
7. The composition of claim 1 or claim 2, wherein the bacteria is in a form selected from a live bacterial population, a lyophilized bacterial popula- tion, a fermented dairy product and a non-viable bacterial preparation.
8. The composition of claim 7, wherein said non-viable bacterial preparation is selected from heat-killed bacteria, irradiated bacteria and lysed bacteria.
9. The composition of any one of claims 1 to 8, wherein it further comprises conventional starter microbes and/or a prebiotic.
10. Use of the composition of any one of claims 1 to 9 in food industry, pharmaceutical industry, or in the manufacture of health promoting products or natural products.
I I . Use according to claim 10, characterized in that the composition is added to a dairy product, drink, juice, soup or children's food.
12. Use of the composition of claim 1 or claim 2 for enhancing proinflammatory Th1 type immune response and/or for preventing or treating allergic, atopic, viral and/or infectious disease.
13. Use of the composition of claim 1 or claim 2 in the preparation of a product enhancing pro-inflammatory Th1 type immune response and/or for preventing or treating allergic, atopic, viral and/or infectious disease.
14. Use of Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 for enhancing pro-inflammatory TM type immune response and/or for the prevention or treatment of allergic, atopic, viral and/or infectious disease.
15. Use of Lactococcus lactis ssp. cremoris ARH74, DSM 18891 for enhancing pro-inflammatory Th1 type immune response and/or for the prevention or treatment of allergic, atopic, viral and/or infectious disease.
16. A method for protecting a subject against allergy and/or atopic disease and/or viral disease and/or infectious disease autoimmune diseases comprising the step of administering to said subject an effective amount of the probiotic composition of claim 1 or claim 2.
17. Use of a product obtained from fermenting milk by Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 for the prevention or treatment of aliergy and/or atopic disease and/or viral disease and/or infectious disease.
18. The use of claim 17 wherein the product is obtained from fer- menting milk by Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892 and Lactococcus lactis ssp. cremoris ARH74, DSM 18891 for the prevention or treatment of allergy and/or atopic disease and/or viral disease and/or infectious disease.
19. Leuconostoc mesenteroides ssp. cremoris strain Leuconostoc mesenteroides ssp. cremoris PIA2, DSM 18892.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2251020A1 (en) * 2009-05-11 2010-11-17 Nestec S.A. Short-time high temperature treatment generates microbial preparations with anti-inflammatory profiles
WO2011020780A1 (en) * 2009-08-18 2011-02-24 Nestec S.A. A nutritional composition comprising lactococcus strains and reducing allergy symptoms, especially in infants and children.
WO2012091081A1 (en) * 2010-12-28 2012-07-05 キリンホールディングス株式会社 Interferon production-inducing agent containing lactic acid bacteria
TWI509069B (en) * 2013-07-12 2015-11-21 Univ Nat Yang Ming Lactic acid bacterium having immunomodulatory and anti-allergic effects
RU2575072C2 (en) * 2009-08-18 2016-02-10 Нестек С.А. Edible composition containing strains lactococcus and reducing allergy symptoms, particularly in infants and children
US20220211773A1 (en) * 2017-11-15 2022-07-07 Evelo Biosciences, Inc. Compositions and methods for treating immune disorders using immune modulating lactococcus bacteria strains
CN115109718A (en) * 2022-05-25 2022-09-27 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） Enterococcus faecium strain and application thereof
KR20230100281A (en) * 2021-12-28 2023-07-05 한국식품연구원 Composition for preventing, improving or treating inflammatory disease comprising the Leuconostoc mesenteroides WiKim0141

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1082964A1 (en) * 1998-06-05 2001-03-14 Wakamoto Pharmaceutical Co., Ltd. Lactic acid bacterium-containing compositions, drugs and foods
JP2006028047A (en) * 2004-07-14 2006-02-02 Kikkoman Corp Interleukin 12 production promoter and method for producing the same
JP2006076961A (en) * 2004-09-10 2006-03-23 Shikoku Nyugyo Kk Immunopotentiator composition
JP2007006769A (en) * 2005-06-30 2007-01-18 Asahi Breweries Ltd New lactic acid bacterium and beverage using the same
JP2007070249A (en) * 2005-09-05 2007-03-22 Shinshu Univ Immunological function regulator, antiallergic agent, composition for immunomodulation, antiallergic composition and food containing the same
JP2007117031A (en) * 2005-10-31 2007-05-17 Nakagaki Gijutsushi Jimusho:Kk Fermented milk having interferon-gamma expression inducing action and fermented milk production spawn
WO2007063075A1 (en) * 2005-11-29 2007-06-07 Actogenix Nv Induction of mucosal tolerance to antigens

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1082964A1 (en) * 1998-06-05 2001-03-14 Wakamoto Pharmaceutical Co., Ltd. Lactic acid bacterium-containing compositions, drugs and foods
JP2006028047A (en) * 2004-07-14 2006-02-02 Kikkoman Corp Interleukin 12 production promoter and method for producing the same
JP2006076961A (en) * 2004-09-10 2006-03-23 Shikoku Nyugyo Kk Immunopotentiator composition
JP2007006769A (en) * 2005-06-30 2007-01-18 Asahi Breweries Ltd New lactic acid bacterium and beverage using the same
JP2007070249A (en) * 2005-09-05 2007-03-22 Shinshu Univ Immunological function regulator, antiallergic agent, composition for immunomodulation, antiallergic composition and food containing the same
JP2007117031A (en) * 2005-10-31 2007-05-17 Nakagaki Gijutsushi Jimusho:Kk Fermented milk having interferon-gamma expression inducing action and fermented milk production spawn
WO2007063075A1 (en) * 2005-11-29 2007-06-07 Actogenix Nv Induction of mucosal tolerance to antigens

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KEKKONEN ET AL.: "Probiotic Leuconostoc mesenteroides ssp. cremoris and Streptococcus thermophilus induce IL-12 and IFN-gamma production", WORLD JOURNAL OF GASTROENTEROLOGY, vol. 14, no. 8, 2008, pages 1192 - 1203 *
KIMOTO H. ET AL.: "New Lactococcus strain with immunomodulatory activity: enhancement of Th1-type immune response", MICROBIOLOGY AND IMMUNOLOGY, vol. 48, no. 2, 2004, pages 75 - 82 *
LATVALA ET AL.: "Probiotic Bacteria Can Induce the Maturation and Cytokine Gene Expression in Human Monocyte-Derived Dendritic Cells: 50", SCANDINAVIAN JOURNAL OF IMMUNOLOGY, vol. 65, no. 6, 2007, pages 607 *
YANG Z. ET AL.: "Separation, purification and characterisation of extracellular polysaccharides produced by slime-forming Lactococcus lactis ssp. cremoris strains", INTERNATIONAL DAIRY JOURNAL, vol. 9, no. 9, 1999, pages 631 - 638 *

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