Title of Invention	A COMPOSITION HAVING IMMUNOREGULATING ACTIVITIES COMPRISING LACTOBACILLUS PENTOSUS
Abstract	The invention provides a composition comprising novel lactic acid bacteria having immunoregulating activities. Specifically, the invention provides food, drinks or medicaments containing novel lactic acid bacteria separated from "Shibazuke," one kind of traditional Kyoto pickles, and having immunoregulating activities. The lactic acid bacteria belong to Lactobacillus pentosus and have a weak assimilating activity or no assimilating activity for glycerol.

Title of Invention

A COMPOSITION HAVING IMMUNOREGULATING ACTIVITIES COMPRISING LACTOBACILLUS PENTOSUS

Abstract

The invention provides a composition comprising novel lactic acid bacteria having immunoregulating activities. Specifically, the invention provides food, drinks or medicaments containing novel lactic acid bacteria separated from "Shibazuke," one kind of traditional Kyoto pickles, and having immunoregulating activities. The lactic acid bacteria belong to Lactobacillus pentosus and have a weak assimilating activity or no assimilating activity for glycerol.

Full Text	DESCRIPTION A COMPOSITION HAVING IMMUNOREGULATING ACTIVITIES TECHNICAL FIELD The present invention relates to a composition comprising Lactobacillus pentosus having immunoregulating activities. BACKGROUND ART Lactic acid bacteria are known to exhibit various beneficial bioactivities such as an intestine regulating activity and immunostimulating activity. Many of the Lactic acid bacteria with such beneficial bioactivities are separated from the intestinal tract or fermented dairy products obtained from animal sources. Some lactic acid bacteria from plant sources are also known to exhibit immunostimulating activities. An example of lactic acid bacteria having immunostimulating activities is found, for example, in Patent Document 1, which discloses the Lactobacillus plantarum L-137 strain. Other examples include the Lactobacillus brevis Labre strain separated from "Suguki," which is a particular kind of traditional pickles produced in Kyoto, and the Lactobacillus pentosus DA74N strain separated from "Shibazuke," another kind of Kyoto pickles (see Non-Patent Documents 1, 2). [Patent Document 1] Japanese Laid-Open Patent Publication No. 167972/1998 (Tok.ukaih.ei 10-167972; published on June 23, 1998). [Non-Patent Document 1] Screening of immune-enhancing Probiotics: Study of immune-enhancing effects of Lactobacilli strains by in vitro stimulation human peripheral blood mononuclear cells, Atsuko KISHI, Aoi KOKUBO, Kaoru AKATANI, Eriko OUGITANA, Setsuya FUJITA, Tsunataro KISHIDA, Pasken Journal 15. 21-26, 2002. [Non-Patent Document 2] The 6th Intestinal Bacteria Conference (Chonai Saikin Gakkai), May 30-31, 2002,. Abstract, Kaoru AKATANI, Atsuko KISHI, Eriko OUGITANA, Aoi KOKUBO, Setsuya FUJITA, Tsunataro KISHIDA. A wide variety of bacteria can be separated from the traditional Kyoto pickles, and it is believed that the pickles include other bacterial strains, in addition to the Labre strain, with the immunostimulating activities or other beneficial bioactivities. Among different bacterial strains separated from the pickles, Lactobacillus plantarum and Lactobacillus pentosus are most frequently separated. However, the effects of bacteria separated from the pickles have not been actively researched. In addition, since lactic acid bacteria separated from the pickles are originally contained in food, they are considered to be highly safe even if ingested by living organisms. Therefore, with the beneficial lactic acid bacteria separated from the pickles, a useful composition comprising such lactic acid bacteria can be realized. The present invention was made in view of the foregoing problem, and an object of the invention is to provide food, drinks, medicaments, or the like that are safe and containing lactic acid bacteria, separated from traditional Kyoto pickles, having beneficial bioactivities. DISCLOSURE OF INVENTION The inventors of the present invention diligently worked to solve the foregoing problems. In accomplishing the invention, the inventors investigated immunoregulating activities of 16 kinds of lactic acid bacteria separated from pickles, and conducted a detailed study of immunoregulating activities of Lactobacillus pentosus bacteria among the separated bacteria. It was found as a result that immunoregulating activities such as immunostimulating activity and anti-allergy activity were exhibited when the Lactobacillus pentosus bacteria was ingested by animals with drinking water or when a suspension of the Lactobacillus pentosus bacteria was administered to animals. Specifically, a composition according to the present invention comprises lactic acid bacteria which belong to Lactobacillus pentosus and which have a weak assimilating activity or no assimilating activity for glycerol. Preferably, the lactic acid bacteria have immunoregulating activities and are an extracellular polysaccharide-producing strain. An example of preferable lactic acid bacteria is a Lactobacillus pentosus S-PT84 strain (PERM ABP-10028). The Lactobacillus pentosus S-PT84 strain was separated from "Shibazuke" by the inventors, and was found to possess strong immunoregulating activities. The Lactobacillus pentosus S-PT84 strain is also called a DS84C strain. The Lactobacillus pentosus S-PT84 strain (Lactobacillus pentosus SAM 2336) is deposited with the deposit number PERM ABP-10028 at the International Patent Organism Depositary in the National Institute of Advanced Industrial Science and Technology (AIST). Therefore, a composition according to the present invention preferably comprises the Lactobacillus pentosus S-PT84 strain. A composition according to the present invention comprises the lactic acid bacteria and has immunoregulating activities. Further, a composition according to the present invention comprises the lactic acid bacteria and has anti-allergy activities. In addition, a composition according to the present invention may comprise lactic acid bacteria as viable cells. The composition is useful for food, drinks, and medicaments having immunoregulating activities and/or anti-allergy activities. For a fuller understanding of the nature and advantages of the invention, reference should be made to the ensuing detailed description taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS Fig. 1 is a graph representing the result of a macrophage IL-12 induction test conducted through in vitro stimulation using 16 kinds of lactic acid bacteria. Fig. 2 is a graph representing changes in serum IL-12 concentration in response to intraperitoneal administration of S-PT84. Fig. 3 is a graph representing the results of measurement on mouse serum IL-12 concentration in response to intraperitoneal administration of 16 kinds of lactic acid bacteria. Fig. 4(a) through Fig. 4(c) are flow cytometric charts representing cytokine production from splenocytes caused by S-PT84 stimulation, in which Fig. 4(a) is the result using a cxilture medium from non-stimulated splenocytes, Fig. 4(b) is the result using a culture medium from heat-killed S-PT84 cells stimulated splenocytes, and Fig. 4(c) is the result using a culture medium from concanavalin A stimulated splenocytes. Fig. 5(a) through Fig. 5(c) are flow cytometric charts representing effects of S-PT84 stimulation on CD4 + , CD8 + , and CD69+ cells, in which Fig. 5(a) is the result using non-stimulated splenocytes, Fig. 5(b) is the result using heat-killed S-PT84 cells stimulated splenocytes, and Fig. 5(c) is the result using concanavalin A stimulated splenocytes. Fig. 6 is a graph representing the result of measurement on NK activity of hepatic lymphocytes in response to intraperitoneal administration of S-PT84. Fig. 7(a) and Fig. 7(b) are flow cytometric charts representing effects of S-PT84 intraperitoneal administration on CD4 + , CD8 + , and CD69+ cells of hepatic lymphocytes, in which Fig. 7(a) is the result for the control, and Fig. 7(b) is the result with S-PT84 administration. Fig. 8 is a graph representing the result of measurement on serum IL-12 concentration in mice to which S-PT84 was orally administered. Fig. 9 is a graph representing the result of measurement of splenic NK activity of mice to which S-PT84 was orally administered. Fig. 10 is a graph representing the result of measurement of Thl/Th2 ratio in splenocytes of mice to which S-PT84 was orally administered. Fig. 11 is a graph representing changes in the weight of mice to which S-PT84 was orally administered, in response to cyclophosphamide administration. Fig. 12 is a graph representing the result of measurement of serum IL-12 concentration in mice to which S-PT84 was orally administered, in response to cyclophosphamide administration. Fig. 13 is a graph representing changes in OVA-specific IgE concentration in mice to which S-PT84 was orally administered. Fig. 14 is a graph representing the result of measurement of total IgE concentration 3 weeks after OVA administration in mice to which S-PT84 was orally administered. Fig. 15 is a graph representing NK activity reduction suppressing effects in mice to which S-PT84 was orally administered and which were placed under stress. Fig. 16 is a graph representing the results of measurement on serum IL-12 concentration in response to intraperitoneal administration of 4 kinds of lactic acid bacteria (DB22C, DS51C, DS2C, and DS84C(S-PT84)) either as heat-killed cells or viable cells. BEST MODE FOR CARRYING OUT THE INVENTION The following will describe one embodiment of the present invention. It is to be noted that the invention is not limited in any way by the following description. Lactic acid bacteria contained in a composition according to the present invention belong to Lactobacillus pentosus and have a weak assimilating activity or no assimilating activity for glycerol. A composition comprising such lactic acid bacteria is highly valuable as a composition capable of exhibiting beneficial bioactivities of the lactic acid bacteria. Among such compositions, a composition having immunoregulating activities is preferable. As used herein, the term "immunoregulating activity" refers to the function of activating a steady or dropped immune function (immunostimulating activity), or the function of suppressing an excess immune function to an appropriate level (immunosuppressing activity). In addition to or instead of these activities, the term is also used to refer to the function of achieving an optimum balance between cellular immunity and humoral immunity. Non-limiting examples of immunoregulating activities include: facilitation or suppression of cytokine production; activation of lymphocytes; enhancement of NK (natural killer) activity; improvement of Thl/Th2 balance; suppression of immune reduction; and anti-allergy activity. Further, it is preferable that lactic acid bacteria contained in a composition according to the present invention be an extracellular polysaccharide-producing strain (EPS). The property or chemical structure of EPS varies greatly from genus to genus, species to species, and strain to strain. The EPS produces capsular polysaccharides, which accumulate on the bacteria surface and is easily recognizable by Indian ink staining. The ESP-producing strain is more hydrophilic than strains that produce no ESP. This is advantageous in food applications. A representative example of lactic acid bacteria contained in a composition according to the present invention is the Lactobacillus pentosus S-PT84 strain. This bacterial strain is deposited with the deposit number PERM ABP-10028 at the International Patent Organism Depositary in the National Institute of Advanced Industrial Science and Technology (AIST). The Lactobacillus pentosus S-PT84 strain (hereinafter simply referred to as "S-PT84") will be described below. Based on the criteria noted below, the inventors of the present invention separated and selected 16 kinds of lactic acid bacteria from "Shibazuke" (4 kinds of Lactobacillus plantarum, and 12 kinds of Lactobacillus pentosus). Specifically, 16 kinds of lactic acid bacteria were selected from the plant lactic acid bacteria if ( 1 ) they were bacilli (genus Lactobacillus), (2) more than one strain with the same characteristics was separated, (3) they proliferated desirably in a culture medium, and (4) they were distinct to "Shibazuke." The 16 kinds of lactic acid bacteria were compared with respect to inducible activity of interleukin 12 (simply "IL-12" hereinafter). The result showed that the Lactobacillus pentosus S-PT84 strain yielded the highest concentration of serum IL-12 when intraperitoneally administered to mice. Detailed studies of S-PT84 immunoregulating activities led to the following findings. (1) When processed in vitro in the splenocytes prepared from mice, the S-PT84 induced the production of IFN-y (interferon y) ar*d TNF-a (tumor necrosis factor ex) of the Thl-type cytokine, and thereby increased the number of CD4 + CD69 + cells and CD8 + CD60 + cells. That is, the S-PT84 functioned to activate the helper T cells or killer T cells. (2) When intraperitoneally administered to mice, the S-PT84 enhanced the NK activity of the hepatic lymphocytes. In addition, the S-PT84 increased the number of CD8 + cells and CD8 + CD69 + cells, and thereby enhanced the cellular immunity. (3) When orally administered to mice, the S-PT84 raised the concentration of serum IL-12, increased the number of CD4 + , CD8 + , and CD3+ cells in the spleen, and thereby enhanced the NK activity of the splenocytes. As a result, the Thl/Th2 balance in the splenocytes became Thl dominant. (4) When orally administered to mice, the S-PT84 suppressed a weight loss caused by administration of cydophosphamide, and suppressed immune response reduction. (5) When orally administered to mice, the S-PT84 suppressed increase of ovalbumin (OVA)-specific IgE and total IgE, even when sensitized with OVA. (6) When orally administered to mice, the S-PT84 suppressed stress-induced reduction of NK activity. From these findings, the S-PT84 was confirmed to be a strain with imrnunoregulating activities. Table 1 below shows bacterial characteristics of the S-PT84. [Table 1] Cell morphology Spore Gram staining Mobility End spore Catalase reaction Growth at 15°C Growth at 5°C Sugar assimilating activity (Positive: +, Negative: -, Weakly positive: w) D-arabinose L-arabinose Ribose D -xylose L-xylose Galactose Glucose Bacillus Absent Positive Absent Absent Negative Good No growth - + + + • - + + Fructose Mannose Raffinose Mannitol Sorbitol Cellobiose Lactose Melibiose Trehalose Glycerol Xylitol + + w + + + + + + w + Lactobacillus pentosus is generally known to have a strong assimilating activity f or glycerol. However, the S-PT84 had a weak assimilating activity for glycerol, as shown in Table 1. Thus, the S-PT84 was found to be different from any other known Lactobacillus pentosus. After extracting DNA from S-PT84, a total of about 500 bp in the entire region of the IGSrRNA gene was sequenced using the Microseq Full Gene 16S rDNA kit (Applied Biosystems). The 16SrRNA gene sequence (SEQ ID NO: 1) was 100% homologous to the 16SrRNA gene sequence of Lactobacillus pentosus JCMT (D79211). From this, the S-PT84 was identified as Lactobacillus pentosus. The S-PT84 is more hydrophilic than bacteria that produce no EPS, and has essentially no adherence to a plastic surface. Further, the S-PT84 has essentially no agglomeration activity for yeasts. A composition according to the present invention comprises lactic acid bacteria which belong to Lactobacillus pentosus and which have a weak assimilating activity or no assimilating activity for glycerol. A composition according to the present invention preferably has immunoregulating activities or anti-allergy activities. It would be more advantageous if the composition exhibits both immunoregulating activities and anti-allergy activities. A composition according to the present invention is advantageously usable in the form of food, drinks, or medicaments having immunoregulating activities and/or anti-allergy activities. In other words, the composition can be suitably used as a pharmaceutical composition having immunoregulating activities and/or anti-allergy activities. The lactic acid bacteria may be contained in a composition either directly (viable or dead), or in the form of an inclusion or processed cells, for example. Viable cells may be obtained from an inclusion of lactic acid bacteria such as a broth of lactic acid bacteria. Dead cells may be obtained by subjecting viable cells to heat, UV irradiation, or a formalin treatment, for example. The viable cells or dead cells may be ground or crushed into processed cells. That is, a composition according to the present invention includes at least one of: lactic acid bacteria; an inclusion of lactic acid bacteria; and processed cells of lactic acid bacteria. Examples of lactic acid bacteria include viable cells, wet cells, and dried cells. The lactic acid bacteria inclusion may be a suspension of lactic acid bacteria, a culture medium of lactic acid bacteria (including lactic acid bacteria, supernatant, and medium itself), a broth of lactic acid bacteria (obtained by removing a solid component from the culture medium), or fermented milk of lactic acid bacteria (lactic acid bacteria beverage, sour milk, yoghurt, etc.). The processed cells of lactic acid bacteria may be, for example, ground cells, crushed cells, liquefied cells (extract, etc.), concentrated cells, paste cells, dried cells (spray-dried cells, freeze-dried cells, vacuum-dried cells, drum-dried cells), or diluted cells. The S-PT84 contained in a composition according to the present invention is separated from the fermented "Shibazuke," and as such a composition comprising the product of fermented fruits, vegetables, or cereals with S-PT84 is also suitable as one enbodyment of a composition according to the present invention. It should be noted that a composition comprising S-PT84 is safe because S-PT84 is separated from food, as described above. It is preferable that a composition according to the present invention be used as food, drinks, medicaments, or the like. More specifically, the composition is preferably used as a pharmaceutical composition having immunoregulating activities. When used as food or drink, it is preferable that the composition be provided as health food having immunoregulating activities. Further, the composition may be combined with conventional sweeteners, acidifiers, vitamins, or various other components to provide user-selective products. For example, the composition may be provided in the form of a tablet, a capsule, a health drink, a dairy product such as yoghurt or lactic acid bacteria beverage, a flavor enhancer, processed food, dessert, or confectionary. Examples of medicaments include an immunostimulant and an anti-allergic drug. The composition may be prepared into a medicament as an active component in combination with conventional auxiliaries commonly used in the field of drug preparation. Examples of such auxiliaries include: an excipient, a binder, a disintegrator, a lubricant, a fragrance, a solubilizing agent, a suspending agent, and a coating agent. The form of dosage is not particularly limited. For example, the medicament may be in the form of a tablet, a capsule, a granule, a powder, a syrup, a suppository, or an injection. The administration route of the medicament is not particularly limited either. For example, oral administration, rectal administration, and transintestinal administration are available. [Examples] [Lactic acid bacteria strains used] Four kinds of Lactobacillus plantarum and twelve kinds of Lactobacillus pentosus were separated from "Shibazuke," a kind of traditional Kyoto pickles. These bacterial strains were compared with respect to inducible activity of interleukin 12 (IL-12), in order to select strains with good Thl-type immunostimulating activity. For each strain used, Table 2 lists names of strain and species, and the presence or absence of EPS. From the result of comparison for IL-12 inducible activity, the activity of DS84C strain (S-PT84) was found to be particularly strong. As such, subsequent experiments were conducted only for S-PT84. [Table 2] Strain DW69N DW69C DS84N DS84C (S-PT84) DB30N DB30C Species Lactobacillus. pentosus Lactobacillus. pentosus Lactobacillus. pentosus Lactobacillus. pentosus Lactobacillus. pentosus Lactobacillus. pentosus Extracellular Polysaccharide (EPS) - + - + - + DA74N DA74C DS51N DS51C DB22N DB22C DS25N DS25C DS2N DS2C Lactobacillus. pentosus Lactobacillus. pentosus Lactobacillus. pentosus Lactobacillus. pentosus Lactobacillus. plantarum Lactobacillus. plantarum Lactobacillus. pentosus Lactobacillus. pentosus Lactobacillus. plantarum Lactobacillus. plantarum - + + - + - + - + [IL-12 induction by in vitro stimulation] First, 4.05% thioglycolate was intraperitoneally administered to BALB/c mice (7 weeks of age, male). After 4 days, intraperitoneal macrophages were collected with PBS, and adjusted to 2 x 106 cells/mL using RPMI medium containing 10% FBS. The culture was inoculated on a 24-well plate (0.5 ml/well). Then, heat-killed cells (10 ug/mL) of each strain were added to each well, and, after 24 hour incubation, the IL-12 concentration of the supernatant was measured. Since the active form of IL-12 is P70 combining two subunits p35 and p40, the concentration of IL-12 (p70) was measured. For the measurement of IL-12, the OptEIA mouse IL-12 ELISA kit (BD Pharmingen) was used. The results are shown in Fig. 1. As is clear from Fig. 1, the inducible activity of IL-12 varied greatly even among the strains of the same species or same parental strain. Among these strains, the activity was particularly high in DW69N, S-PT84 (DS84C), and DS25C. [IL-12 induction by in vivo stimulation] A suspension (solvent; saline solution) of heat-killed cells (500ug/0.2mL/mouse) of each strain was intraperitoneally administered to BALB/c mice (7 weeks of age, male). After 6 hours, the cervical was dislocated and the blood was collected from the heart. For the control mice, the same amount of saline solution was administered. The blood was collected 6 hours after the administration because a preliminary analysis using S-PT84 had revealed that the peak concentration of serum IL-12 occurs 6 hours after the administration of dead cells (Fig. 2). After the blood was collected, the serum was collected by centrifugation. The IL-12 concentration in the serum was measured with the OptEIA mouse IL-12 ELISA kit (BD Pharmingen). The results are shown in Fig. 3. As is clear from Fig. 3, the concentration of serum IL-12 was significantly high for samples to which S-PT84 (DS84C), DS51C, and DS25C were administered, compared with the control. Since the S-PT84 (DS84C) had the highest concentration, only this strain was used in subsequent experiments. [Effects S-PT84 on lymphocytes] Spleens were removed from BALB/c mice (7 weeks of age, male), and splenocytes were prepared according to ordinary method. The splenocytes were cultured for 24 hours in a medium containing (1 ug/ml) of heat-killed S-PT84 cells. As a control, the splenocytes were cultured alone in a medium (control). As another control, the splenocytes were also cultured in a medium with the addition of concanavalin A (2.5 ug/mL) (Con A). In order to determine the type of cytokine produced by the stimulus of the S-PT84 dead cells, the cytokine concentration in the supernatant of each medium was measured using the CBAkit (BD Pharmingen). The splenocytes were collected and labeled with the fluorescent-labeled anti-CD4 antibody (CY-CHCROME™ label, BD bioscience), anti-CD8 antibody (FITC label, Immunotech), and anti-CD69 antibody (PE label, BD bioscience), and the respective proportions of CD4-, CD8-, and CD69-positive cells were measured with the flow cytometry (Beckman Coulter). Fig. 4 depicts the result of cytokine production. Fig. 4(a) is the result for the control in which the medium was used alone, Fig. 4(b) is the result for S-PT84 in which the S-PT84 dead cells were added to the medium, and Fig. 4(c) is the result for Con A in which concanavalin A was added to the medium. As is clear from Fig. 4, the S-PT84 stimulus produced IFN-y (interferon y) and TNF-a (tumor necrosis factor a), which were not observed in the control. These cytokines were Thl-type cytokines. It was therefore believed that the S-PT84 specifically induced Thl-type cytokines. The Th2-type cytokines such as IL-4 or IL-5 were not produced at all. With the concanavalin A stimulation, another type of Thl-type cytokine, IL-2 (interleukin 2), was produced. Fig. 5 shows how the CD4 + , CD8 + , and CD69+ cells were affected. Fig. 5(a) is the result for the control in which the medium was used alone. Fig. 5(b) is the result for the S-PT84 in which the S-PT84 dead cells (1 ug/mL) were added to the medium. Fig. 5(c) is the result for Con A in which concanavalin A was added to the medium. In Fig. 5, the directions of arrows indicate increasing numbers of positive cells for each surface antigen. As is clear from Fig. 5, the helper T cells and killer T cells were activated by the S-PT84. [Changes in hepatic lymphocytes after intraperitoneal administration of S-PT84] A suspension (solvent; saline solution) of heat-killed S-PT84 cells (500ug/0.2mL/mouse) was administered intraperitoneally to C57BL/6 mice (7 weeks of age, male). After 24 hours, liver was removed, and hepatic lymphocytes were prepared by centrifugation. As a control, only the saline solution was intraperitoneally administered. The NK activity of the hepatic lymphocytes was measured by a PINK method. The PINK method is a method for calculating the cytotoxic activity of the mouse lymphocytes according to the following procedure. First, a target cell Yac-1 is labeled with 3,3'-dioctadecyloxacarbocyanine perchlorate (Dio), which is a hydrophobic fluorescent dye for labeling a membrane. Then, the nucleus of the dead cell is double stained with propidium iodide (PI), which is a membrane-impermeable nucleic acid binding fluorescent dye. The Yac-1 cells were detected with the flow cytometry, using Dio simple staining for uninjured cells, and double staining for injured cells. Further, the other hepatic cells were labeled with the fluorescent-labeled anti-CD4 antibody (CY-CHCROME™ label, BD bioscience), anti-CD8 antibody (FITC label, Immunotech), and anti-CD69 antibody (PE label, BD bioscience), and the respective proportions of CD4-, CD8-, and CD69-positive cells were measured with the flow cytometry (Beckman Coulter). Fig. 6 shows the result of NK activity measurement. In Fig. 6, the NK activity (%) indicates cellular cytotoxicity of the mouse hepatic lymphocytes against Yac-1, and E:T ratio indicates the value of the number of reacted hepatic lymphocytes versus the number of Yac-1 cells. As is clear from Fig. 6, the NK activity of the hepatic lymphocytes prepared from mice to which the S-PT84 was intraperineally administered was clearly higher than that of the control. Fig. 7 shows the results for CD4 + , CD8 + , and CD69+ cells, in which Fig. 7(a) is the result for the control, and Fig. 7(b) is the result with the administration of S-PT84. Further, in Fig. 7, the directions of arrows indicate increasing numbers of positive cells for each surface antigen. As is clear from Fig. 7, the number of CD8 + cells, as well as CD8 + CD69+ cells, clearly increased. It was therefore found that the administration of S-PT84 increases the number of the killer T cells in the liver, as well as the number of active killer T cells. [Thl/Th2 balance regulating activity by oral administration of S-PT84] Six BALB/c mice (7 weeks of age, male) were allowed to drink S-PT84 (dead cells)-containing water for a week (equivalent of 2 mg/day). As a control group, six mice with no S-PT84 were used. After one week, blood was collected from the heart, and spleen was removed. The serum was collected from the blood by centrifugation, and the IL-12 concentration in the serum was measured using the OptEIA mouse IL-12 ELISA kit (BD Biosciences). From the spleen, splenocytes were prepared by ordinary method, and the number of the CD4 + , CD8 + , and CD3+ cells in the splenic lymphocytes were counted (measurement was made with the flow cytometry, using labeled antibodies of the respective cells). Further, the NK activity was measured by the PINK method. A measurement of Thl/Th2 ratio was also carried out (2.5 ug/ml of concanavalin A was allowed to act on 5 x 106 mouse splenocytes for 24 hours, and the concentrations of resulting IL-4 and IFN-y in the supernatant were measured). The Thl/Th2 ratio was obtained by dividing the IFN-y concentration by 11-4 concentration. Fig. 8 shows the result of measurement of serum IL-12 concentration. As is clear from Fig. 8, the concentration of serum IL-12 in the S-PT84-orally administered mice was significantly higher than that of the control group (Cont). Table 3 below represents the result of measurement of CD4 + , CD8 + , and CD3+ cells. As is clear from Fig. 3, the splenic lymphocyte T subset had a tendency to increase. [Table 3] Cell (x 10" cells) Spleen CD4 + CD8 + CD3 + Control 68.3±3.9 15.1±0.7 4.6±0.4 27.6±1.6 S-PT84 128.0±42.0 36.2+11.2' 9.7+1 1.2 61.0+19.8 Ratio 1.9 2.4 2. 1 2.2 Fig. 9 shows the result of measurement of NK activity. In Fig. 9, the NK activity (%) indicates cytotoxicity of the mouse splenocytes against Yac-1, and E:T ratio indicates the value of the number of reacted splenocytes versus the number of Yac-1 cells. As is clear from Fig. 9, the NK activity of the splenocytes prepared from the mice to which S-PT84 was orally administered was significantly higher than that of the control group (Cont). Fig. 10 shows the result of measurement of Thl/Th2 ratio. As is clear from Fig. 10, the splenocytes prepared from mice to which the S-PT84 was orally administered had a considerably large proportion of Thl cytokine as compared with the control group (Cont). As described above, the Thl-type cytokine was induced in mice to which the S-PT84 was orally administered. As a result, the Thl/Th2 balance shifted to Thl dominant, and the NK activity increased as a result. This proved the Thl/Th2 balance adjusting activity and immunostimulating activity of the S-PT84. [Effects on change of the weight of cyclophosphamide-administered mice] Twenty BALB/c mice (7 weeks of age, male) were divided into two groups of an almost equal average weight, so as to provide an S-PT84 administered group and an S-PT84 non-administered group (control group). The S-PT84 administered group was allowed to drink S-PT84 (dead cellsj-containing water for 22 days (equivalent of 2 mg/day). After 8 days from the start of administration, 200 mg/kg of cyclophosphamide (CY) (antitumor chemotherapeutic drug, alkylating agent) was intraperitoneally administered to all individuals. A weight of each individual was measured on the 1st, 2nd, 3rd, 5th, 8th, 12th, and 15th days from the CY administration. Fig. 11 shows changes, in average body weight in the both groups. As is clear from Fig. 11, a body weight loss caused by the CY administration was suppressed in the S-PT84 administered group, as compared with the control group (Control). [Effects on IL-12 production in cyclophosphamide-administered mice] BALB/c mice (7 weeks of age, male) were divided into 5 groups: an untreated group (5 mice); an S-PT84 non-administered and CY non-administered group (10 mice); an S-PT84 non-administered and CY administered group (10 mice); an S-PT84 administered and CY non-administered group (10 mice); and an S-PT84 administered and CY administered group (11 mice). The two S-PT84 administered groups were allowed to drink S-PT84 (dead cells)-containing water for 12 days (equivalent of 2 mg/day). After 7 days from the start of administration, 200mg/kg of cyclophosphamide (CY) (antitumor chemotherapeutic drug, alkylating agent) was intraperitoneally administered to the CY administered groups. After 5 days from the CY administration, a suspension (solvent; saline solution) of heat-killed cells of S-PT84 (SOOug/ 0.2ml/mouse) was intraperitoneally administered to the mice of all groups except for the untreated group. Six hours after the administration of S-PT84 dead cells, blood was collected from the heart in all individuals, inchiding those in the untreated group. From the collected blood, the serum was collected by centrifugation, and the IL-12 concentration in the serum was measured with the OptEIA mouse IL-12 ELISA kit (BD Pharmingen). The results are shown in Fig. 12. As is clear from Fig. 12, the CY administration in the S-PT84 non-administered group reduced the IL-12 concentration in the serum by a considerable amount. On the other hand, in the S-PT84 administered group, the reduction of IL-12 concentration in the serum caused by the CY administration was suppressed significantly, and the IL-12 concentration in the serum was almost equal to that of the control group. From these results, it was proved that the S-PT84 has the ability to suppress a weight loss and immune reduction caused by CY. In other words, the immunostimulating activity of the S-PT84 was confirmed. [Analysis of anti-allergy activities] Thirty-six BALB/c mice (7 weeks of age, male) were divided into four groups: an untreated group (5 mice); a control group (10 mice); an S-PT84 group (11 mice); and a dexamethasone administered group (10 mice). The S-PT84 group was allowed to drink S-PT84 (dead cells)-containing water for 7 weeks (equivalent of 2 mg/day). To the Dex administered group, 0.5 mg/kg of S-PT84 was forcibly administered for 7 weeks by oral administration. The untreated group and control group were allowed to drink tap water for 7 weeks. Note that, the Dex is a steroid drug with anti-allergenic and anti-inflammatory activities, and was used as a positive control drug. One week and two weeks after the S-PT84 uptake or Dex administration, a mixture containing 20 ug of ovalbumin (OVA) and 2 mg of aluminum hydroxide gel was intraperitoneally administered to the mice of all groups except for the untreated group. From the second administration (0 week) to the 5th week, blood was collected every week a total of 6 times from all individuals, and an OVA-specific IgE concentration was measured. The measurement of OVA-specific IgE concentration in the serum was carried out according to the ELISA method, using a modified OptEIA mouse IgE ELISA kit (BD Pharmingen) in which OVA was coated instead of the capturing antibody. Using the blood sample from the third week, total IgE was measured. The measurement of total IgE was carried out according to the ELISA method, using the OptEIA mouse IgE ELISA kit. Fig. 13 shows changes in OVA-specific IgE concentration on the 3rd week of the experiment start. Fig. 14 shows the result of measurement on total IgE concentration. As is clear from Fig. 13, the S-PT84 group significantly suppressed the increase of OVA-specific IgE concentration as compared with the control group (control). In the Dex group, the result was not significantly different from the control group, though some suppressing effect was observed. Further, as is clear from Fig. 14, the S-PT84 group and Dex group significantly suppressed increase of total IgE concentration as compared with the control group (control). It became clear from these results that the S-PT84 had an anti-allergy activity. [Analysis of suppressing effect on stress-induced immune reduction] Nine C57BL/6 mice (6 weeks of age, male) were divided into three groups of an almost equal average weight, so as to provide a control group (3 mice), a stressed group (3 mice), and an S-PT84 administered and stressed group (3 mice). The S-PT84 administered group was allowed to drink S-PT84 (dead cellsj-containing water for 7 days (equivalent of 2 mg/day). On the eighth day of the administration, a total of 6 mice in the stressed group and the S-PT84 administered and stressed group were immersed in water, placed in a 50 ml polyethylene tube whose tip had an air vent, and restricted for 24 hours. The control group was deprived of food and water. From each animal, the spleen was removed, and splenic lymphocytes were prepared for the measurement of NK activity. Fig. 15 shows the result of measurement on NK activity. The stressed group showed a significant drop in NK activity compared with the control group. The S-PT84 administered and stressed group maintained NK activity comparable to that of the control group, i.e., the NK activity level was significantly higher than that of the stressed group. This proved that the S-PT84 had the activity of suppressing stress-induced immune reduction. [Difference in immunostimulating activity between dead cells and viable cells] The immunostimulating activities were compared between dead cells and viable cells of the lactic acid bacteria. For the comparison, the lactic acid bacteria that increased the mouse serum IL-12 concentration in response to intraperitoneal administration as shown in Fig. 3 were used. Suspensions of 4 kinds of lactic acid bacteria (DB22C, DS51C, DS2C and DS84C (S-PT84)), both in the form of heat-killed cells and viable cells, were prepared (each weighing 500 pg (2.5xl08/0.2mL/mouse)). The suspensions were intraperitoneally administered to BALB/c mice (7 weeks of age, male). After 6 hours, the cervical was dislocated and the blood was collected from the heart. As a control, a mouse to which the same amount of saline solution was administered instead of the lactic acid bacteria was used. After the blood was collected, the serum was collected by centrifugation. The IL-12 concentration in the serum was measured with the OptEIA (BD Pharmingen). The results are shown in Fig. 16. As in the case of Fig. 3, the amount of serum IL-12 was greatest for samples to which DS84C (S-PT84) was administered. As is also clear from Fig. 16, the amount of IL-12 was higher for the samples to which the viable cells of DS84C (S-PT84) were administered than for the samples to which the dead cells were administered. [Producing example 1: Tablet] An S-PT84-containing medicament (tablet) was produced according to the following procedures. A mixture containing 66.7 g of a dried pulverized powder of S-PT84, 232.0 g of lactose, and 1.3 g of magnesium stearate was punched with a single punch tableting machine, so as to produce tablets each having a diameter of 10 mm and a weight of 300 mg. [Producing example 2: Yoghurt] S-PT84 fermented milk with a 21% solid milk component was added to commercially available milk, and the mixture was allowed to stand for 3 days so as to prepare yoghurt. The resulting yoghurt had a desirable flavor. [Producing example 3: Lactic acid bacteria drinks] By using S-PT84, a lactic acid bacteria beverage was prepared with the compositions shown in Table 4. The resulting lactic acid bacteria beverage had a desirable flavor. [Table 4] Compositions S-PT84 fermented milk with 21% solid milk component Fructose glucose syrup Pectin Citric acid Flavoring agent Water Total Parts by weight 14.76 13.31 0.5 0.08 0. 15 71.2 100 The invention being thus described, it will be obvious that the same way may be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims. INDUSTRIAL APPLICABILITY A composition according to the present invention, if ingested as food or drink, or administered as medicaments, enables the immune function to be activated and thereby suppresses reduction of immune functions. Further, by adjusting the balance of immune function, adverse effects of excess immune function on the body can be suppressed. A composition according to the present invention can be implemented as health food or medicaments with immunoregulating activities. Therefore, the invention is useful is food industries and pharmaceutical industries. CLAIMS: 1. A composition comprising lactic acid bacteria which belong to Lacto bacillus pentosus, and which have a weak assimilating activity or no assimilating activity for glycerol. 2. The composition as set forth in claim 1, wherein the lactic acid bacteria have immunoregulating activities and/or anti-allergy activities. 3. The composition as set forth in claim 2, wherein the lactic acid bacteria are an extracellular polysaccharide-producing strain. 4. The composition as set forth in claim 3, wherein the lactic acid bacteria are a Lactobacillus pentosus S-PT84 strain (PERM ABP-10028). 5. The composition as set forth in any one of claims 1 through 4, wherein the composition has immunoregulating activities. 6. The composition as set forth in any one of claims 1 through 4, wherein the composition has anti-allergy activities. 7. The composition as set forth in any one of claims 1 through 6, wherein the composition contains lactic acid bacteria as viable cells. 8. The composition as set forth in any one of claims 5 through 7, wherein the composition is food, a drink, or a medicament.

Full Text

DESCRIPTION
A COMPOSITION HAVING
IMMUNOREGULATING ACTIVITIES
TECHNICAL FIELD
The present invention relates to a composition comprising
Lactobacillus pentosus having immunoregulating activities.
BACKGROUND ART
Lactic acid bacteria are known to exhibit various
beneficial bioactivities such as an intestine regulating activity
and immunostimulating activity. Many of the Lactic acid
bacteria with such beneficial bioactivities are separated from
the intestinal tract or fermented dairy products obtained from
animal sources. Some lactic acid bacteria from plant sources
are also known to exhibit immunostimulating activities.
An example of lactic acid bacteria having
immunostimulating activities is found, for example, in Patent
Document 1, which discloses the Lactobacillus plantarum L-137
strain. Other examples include the Lactobacillus brevis Labre
strain separated from "Suguki," which is a particular kind of
traditional pickles produced in Kyoto, and the Lactobacillus
pentosus DA74N strain separated from "Shibazuke," another
kind of Kyoto pickles (see Non-Patent Documents 1, 2).
[Patent Document 1]
Japanese Laid-Open Patent Publication No. 167972/1998
(Tok.ukaih.ei 10-167972; published on June 23, 1998).
[Non-Patent Document 1]
Screening of immune-enhancing Probiotics: Study of
immune-enhancing effects of Lactobacilli strains by in vitro
stimulation human peripheral blood mononuclear cells, Atsuko
KISHI, Aoi KOKUBO, Kaoru AKATANI, Eriko OUGITANA,
Setsuya FUJITA, Tsunataro KISHIDA, Pasken Journal 15.
21-26, 2002.
[Non-Patent Document 2]
The 6th Intestinal Bacteria Conference (Chonai Saikin
Gakkai), May 30-31, 2002,. Abstract, Kaoru AKATANI, Atsuko
KISHI, Eriko OUGITANA, Aoi KOKUBO, Setsuya FUJITA,
Tsunataro KISHIDA.
A wide variety of bacteria can be separated from the
traditional Kyoto pickles, and it is believed that the pickles
include other bacterial strains, in addition to the Labre strain,
with the immunostimulating activities or other beneficial
bioactivities. Among different bacterial strains separated from
the pickles, Lactobacillus plantarum and Lactobacillus pentosus
are most frequently separated. However, the effects of bacteria
separated from the pickles have not been actively researched.
In addition, since lactic acid bacteria separated from the
pickles are originally contained in food, they are considered to
be highly safe even if ingested by living organisms. Therefore,
with the beneficial lactic acid bacteria separated from the
pickles, a useful composition comprising such lactic acid
bacteria can be realized.
The present invention was made in view of the foregoing
problem, and an object of the invention is to provide food,
drinks, medicaments, or the like that are safe and containing
lactic acid bacteria, separated from traditional Kyoto pickles,
having beneficial bioactivities.
DISCLOSURE OF INVENTION
The inventors of the present invention diligently worked
to solve the foregoing problems. In accomplishing the invention,
the inventors investigated immunoregulating activities of 16
kinds of lactic acid bacteria separated from pickles, and
conducted a detailed study of immunoregulating activities of
Lactobacillus pentosus bacteria among the separated bacteria.
It was found as a result that immunoregulating activities such
as immunostimulating activity and anti-allergy activity were
exhibited when the Lactobacillus pentosus bacteria was
ingested by animals with drinking water or when a suspension
of the Lactobacillus pentosus bacteria was administered to
animals.
Specifically, a composition according to the present
invention comprises lactic acid bacteria which belong to
Lactobacillus pentosus and which have a weak assimilating
activity or no assimilating activity for glycerol. Preferably, the
lactic acid bacteria have immunoregulating activities and are
an extracellular polysaccharide-producing strain.
An example of preferable lactic acid bacteria is a
Lactobacillus pentosus S-PT84 strain (PERM ABP-10028). The
Lactobacillus pentosus S-PT84 strain was separated from
"Shibazuke" by the inventors, and was found to possess strong
immunoregulating activities. The Lactobacillus pentosus S-PT84
strain is also called a DS84C strain. The Lactobacillus pentosus
S-PT84 strain (Lactobacillus pentosus SAM 2336) is deposited
with the deposit number PERM ABP-10028 at the International
Patent Organism Depositary in the National Institute of
Advanced Industrial Science and Technology (AIST). Therefore,
a composition according to the present invention preferably
comprises the Lactobacillus pentosus S-PT84 strain.
A composition according to the present invention
comprises the lactic acid bacteria and has immunoregulating
activities. Further, a composition according to the present
invention comprises the lactic acid bacteria and has
anti-allergy activities. In addition, a composition according to
the present invention may comprise lactic acid bacteria as
viable cells. The composition is useful for food, drinks, and
medicaments having immunoregulating activities and/or
anti-allergy activities.
For a fuller understanding of the nature and advantages
of the invention, reference should be made to the ensuing
detailed description taken in conjunction with the
accompanying drawings.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 is a graph representing the result of a macrophage
IL-12 induction test conducted through in vitro stimulation
using 16 kinds of lactic acid bacteria.
Fig. 2 is a graph representing changes in serum IL-12
concentration in response to intraperitoneal administration of
S-PT84.
Fig. 3 is a graph representing the results of measurement
on mouse serum IL-12 concentration in response to
intraperitoneal administration of 16 kinds of lactic acid
bacteria.
Fig. 4(a) through Fig. 4(c) are flow cytometric charts
representing cytokine production from splenocytes caused by
S-PT84 stimulation, in which Fig. 4(a) is the result using a
cxilture medium from non-stimulated splenocytes, Fig. 4(b) is
the result using a culture medium from heat-killed S-PT84
cells stimulated splenocytes, and Fig. 4(c) is the result using a
culture medium from concanavalin A stimulated splenocytes.
Fig. 5(a) through Fig. 5(c) are flow cytometric charts
representing effects of S-PT84 stimulation on CD4 + , CD8 + , and
CD69+ cells, in which Fig. 5(a) is the result using
non-stimulated splenocytes, Fig. 5(b) is the result using
heat-killed S-PT84 cells stimulated splenocytes, and Fig. 5(c) is
the result using concanavalin A stimulated splenocytes.
Fig. 6 is a graph representing the result of measurement
on NK activity of hepatic lymphocytes in response to
intraperitoneal administration of S-PT84.
Fig. 7(a) and Fig. 7(b) are flow cytometric charts
representing effects of S-PT84 intraperitoneal administration
on CD4 + , CD8 + , and CD69+ cells of hepatic lymphocytes, in
which Fig. 7(a) is the result for the control, and Fig. 7(b) is the
result with S-PT84 administration.
Fig. 8 is a graph representing the result of measurement
on serum IL-12 concentration in mice to which S-PT84 was
orally administered.
Fig. 9 is a graph representing the result of measurement
of splenic NK activity of mice to which S-PT84 was orally
administered.
Fig. 10 is a graph representing the result of measurement
of Thl/Th2 ratio in splenocytes of mice to which S-PT84 was
orally administered.
Fig. 11 is a graph representing changes in the weight of
mice to which S-PT84 was orally administered, in response to
cyclophosphamide administration.
Fig. 12 is a graph representing the result of measurement
of serum IL-12 concentration in mice to which S-PT84 was
orally administered, in response to cyclophosphamide
administration.
Fig. 13 is a graph representing changes in OVA-specific
IgE concentration in mice to which S-PT84 was orally
administered.
Fig. 14 is a graph representing the result of measurement
of total IgE concentration 3 weeks after OVA administration in
mice to which S-PT84 was orally administered.
Fig. 15 is a graph representing NK activity reduction
suppressing effects in mice to which S-PT84 was orally
administered and which were placed under stress.
Fig. 16 is a graph representing the results of
measurement on serum IL-12 concentration in response to
intraperitoneal administration of 4 kinds of lactic acid bacteria
(DB22C, DS51C, DS2C, and DS84C(S-PT84)) either as
heat-killed cells or viable cells.
BEST MODE FOR CARRYING OUT THE INVENTION
The following will describe one embodiment of the present
invention. It is to be noted that the invention is not limited in
any way by the following description.
Lactic acid bacteria contained in a composition according
to the present invention belong to Lactobacillus pentosus and
have a weak assimilating activity or no assimilating activity for
glycerol. A composition comprising such lactic acid bacteria is
highly valuable as a composition capable of exhibiting
beneficial bioactivities of the lactic acid bacteria. Among such
compositions, a composition having immunoregulating
activities is preferable. As used herein, the term
"immunoregulating activity" refers to the function of activating
a steady or dropped immune function (immunostimulating
activity), or the function of suppressing an excess immune
function to an appropriate level (immunosuppressing activity).
In addition to or instead of these activities, the term is also
used to refer to the function of achieving an optimum balance
between cellular immunity and humoral immunity.
Non-limiting examples of immunoregulating activities include:
facilitation or suppression of cytokine production; activation of
lymphocytes; enhancement of NK (natural killer) activity;
improvement of Thl/Th2 balance; suppression of immune
reduction; and anti-allergy activity.
Further, it is preferable that lactic acid bacteria
contained in a composition according to the present invention
be an extracellular polysaccharide-producing strain (EPS). The
property or chemical structure of EPS varies greatly from
genus to genus, species to species, and strain to strain. The
EPS produces capsular polysaccharides, which accumulate on
the bacteria surface and is easily recognizable by Indian ink
staining. The ESP-producing strain is more hydrophilic than
strains that produce no ESP. This is advantageous in food
applications.
A representative example of lactic acid bacteria contained
in a composition according to the present invention is the
Lactobacillus pentosus S-PT84 strain. This bacterial strain is
deposited with the deposit number PERM ABP-10028 at the
International Patent Organism Depositary in the National
Institute of Advanced Industrial Science and Technology (AIST).
The Lactobacillus pentosus S-PT84 strain (hereinafter simply
referred to as "S-PT84") will be described below.
Based on the criteria noted below, the inventors of the
present invention separated and selected 16 kinds of lactic
acid bacteria from "Shibazuke" (4 kinds of Lactobacillus
plantarum, and 12 kinds of Lactobacillus pentosus). Specifically,
16 kinds of lactic acid bacteria were selected from the plant
lactic acid bacteria if ( 1 ) they were bacilli (genus Lactobacillus),
(2) more than one strain with the same characteristics was
separated, (3) they proliferated desirably in a culture medium,
and (4) they were distinct to "Shibazuke."
The 16 kinds of lactic acid bacteria were compared with
respect to inducible activity of interleukin 12 (simply "IL-12"
hereinafter). The result showed that the Lactobacillus pentosus
S-PT84 strain yielded the highest concentration of serum IL-12
when intraperitoneally administered to mice.
Detailed studies of S-PT84 immunoregulating activities
led to the following findings.
(1) When processed in vitro in the splenocytes prepared
from mice, the S-PT84 induced the production of IFN-y
(interferon y) ar*d TNF-a (tumor necrosis factor ex) of the
Thl-type cytokine, and thereby increased the number of
CD4 + CD69 + cells and CD8 + CD60 + cells. That is, the S-PT84
functioned to activate the helper T cells or killer T cells.
(2) When intraperitoneally administered to mice, the
S-PT84 enhanced the NK activity of the hepatic lymphocytes. In
addition, the S-PT84 increased the number of CD8 + cells and
CD8 + CD69 + cells, and thereby enhanced the cellular immunity.
(3) When orally administered to mice, the S-PT84 raised
the concentration of serum IL-12, increased the number of
CD4 + , CD8 + , and CD3+ cells in the spleen, and thereby
enhanced the NK activity of the splenocytes. As a result, the
Thl/Th2 balance in the splenocytes became Thl dominant.
(4) When orally administered to mice, the S-PT84
suppressed a weight loss caused by administration of
cydophosphamide, and suppressed immune response reduction.
(5) When orally administered to mice, the S-PT84
suppressed increase of ovalbumin (OVA)-specific IgE and total
IgE, even when sensitized with OVA.
(6) When orally administered to mice, the S-PT84
suppressed stress-induced reduction of NK activity.
From these findings, the S-PT84 was confirmed to be a
strain with imrnunoregulating activities.
Table 1 below shows bacterial characteristics of the
S-PT84.
[Table 1]
Cell morphology
Spore
Gram staining
Mobility
End spore
Catalase reaction
Growth at 15°C
Growth at 5°C
Sugar assimilating activity
(Positive: +, Negative: -, Weakly positive: w)
D-arabinose
L-arabinose
Ribose
D -xylose
L-xylose
Galactose
Glucose
Bacillus
Absent
Positive
Absent
Absent
Negative
Good
No
growth
-
+
+
+
• -
+
+
Fructose
Mannose
Raffinose
Mannitol
Sorbitol
Cellobiose
Lactose
Melibiose
Trehalose
Glycerol
Xylitol
+
+
w
+
+
+
+
+
+
w
+
Lactobacillus pentosus is generally known to have a strong
assimilating activity f or glycerol. However, the S-PT84 had a
weak assimilating activity for glycerol, as shown in Table 1.
Thus, the S-PT84 was found to be different from any other
known Lactobacillus pentosus.
After extracting DNA from S-PT84, a total of about 500 bp
in the entire region of the IGSrRNA gene was sequenced using
the Microseq Full Gene 16S rDNA kit (Applied Biosystems). The
16SrRNA gene sequence (SEQ ID NO: 1) was 100% homologous
to the 16SrRNA gene sequence of Lactobacillus pentosus JCMT
(D79211). From this, the S-PT84 was identified as Lactobacillus
pentosus.
The S-PT84 is more hydrophilic than bacteria that
produce no EPS, and has essentially no adherence to a plastic
surface. Further, the S-PT84 has essentially no agglomeration
activity for yeasts.
A composition according to the present invention
comprises lactic acid bacteria which belong to Lactobacillus
pentosus and which have a weak assimilating activity or no
assimilating activity for glycerol. A composition according to
the present invention preferably has immunoregulating
activities or anti-allergy activities. It would be more
advantageous if the composition exhibits both
immunoregulating activities and anti-allergy activities.
A composition according to the present invention is
advantageously usable in the form of food, drinks, or
medicaments having immunoregulating activities and/or
anti-allergy activities. In other words, the composition can be
suitably used as a pharmaceutical composition having
immunoregulating activities and/or anti-allergy activities.
The lactic acid bacteria may be contained in a
composition either directly (viable or dead), or in the form of
an inclusion or processed cells, for example. Viable cells may
be obtained from an inclusion of lactic acid bacteria such as a
broth of lactic acid bacteria. Dead cells may be obtained by
subjecting viable cells to heat, UV irradiation, or a formalin
treatment, for example. The viable cells or dead cells may be
ground or crushed into processed cells.
That is, a composition according to the present invention
includes at least one of: lactic acid bacteria; an inclusion of
lactic acid bacteria; and processed cells of lactic acid bacteria.
Examples of lactic acid bacteria include viable cells, wet cells,
and dried cells. The lactic acid bacteria inclusion may be a
suspension of lactic acid bacteria, a culture medium of lactic
acid bacteria (including lactic acid bacteria, supernatant, and
medium itself), a broth of lactic acid bacteria (obtained by
removing a solid component from the culture medium), or
fermented milk of lactic acid bacteria (lactic acid bacteria
beverage, sour milk, yoghurt, etc.). The processed cells of
lactic acid bacteria may be, for example, ground cells, crushed
cells, liquefied cells (extract, etc.), concentrated cells, paste
cells, dried cells (spray-dried cells, freeze-dried cells,
vacuum-dried cells, drum-dried cells), or diluted cells. The
S-PT84 contained in a composition according to the present
invention is separated from the fermented "Shibazuke," and as
such a composition comprising the product of fermented fruits,
vegetables, or cereals with S-PT84 is also suitable as one
enbodyment of a composition according to the present
invention. It should be noted that a composition comprising
S-PT84 is safe because S-PT84 is separated from food, as
described above.
It is preferable that a composition according to the
present invention be used as food, drinks, medicaments, or the
like. More specifically, the composition is preferably used as a
pharmaceutical composition having immunoregulating
activities. When used as food or drink, it is preferable that the
composition be provided as health food having
immunoregulating activities. Further, the composition may be
combined with conventional sweeteners, acidifiers, vitamins, or
various other components to provide user-selective products.
For example, the composition may be provided in the form of a
tablet, a capsule, a health drink, a dairy product such as
yoghurt or lactic acid bacteria beverage, a flavor enhancer,
processed food, dessert, or confectionary.
Examples of medicaments include an immunostimulant
and an anti-allergic drug. The composition may be prepared
into a medicament as an active component in combination with
conventional auxiliaries commonly used in the field of drug
preparation. Examples of such auxiliaries include: an excipient,
a binder, a disintegrator, a lubricant, a fragrance, a
solubilizing agent, a suspending agent, and a coating agent.
The form of dosage is not particularly limited. For example, the
medicament may be in the form of a tablet, a capsule, a
granule, a powder, a syrup, a suppository, or an injection. The
administration route of the medicament is not particularly
limited either. For example, oral administration, rectal
administration, and transintestinal administration are
available.
[Examples]
[Lactic acid bacteria strains used]
Four kinds of Lactobacillus plantarum and twelve kinds of
Lactobacillus pentosus were separated from "Shibazuke," a kind
of traditional Kyoto pickles. These bacterial strains were
compared with respect to inducible activity of interleukin 12
(IL-12), in order to select strains with good Thl-type
immunostimulating activity. For each strain used, Table 2 lists
names of strain and species, and the presence or absence of
EPS. From the result of comparison for IL-12 inducible activity,
the activity of DS84C strain (S-PT84) was found to be
particularly strong. As such, subsequent experiments were
conducted only for S-PT84.
[Table 2]
Strain
DW69N
DW69C
DS84N
DS84C
(S-PT84)
DB30N
DB30C
Species
Lactobacillus. pentosus
Lactobacillus. pentosus
Lactobacillus. pentosus
Lactobacillus. pentosus
Lactobacillus. pentosus
Lactobacillus. pentosus
Extracellular
Polysaccharide
(EPS)
-
+
-
+
-
+
DA74N
DA74C
DS51N
DS51C
DB22N
DB22C
DS25N
DS25C
DS2N
DS2C
Lactobacillus. pentosus
Lactobacillus. pentosus
Lactobacillus. pentosus
Lactobacillus. pentosus
Lactobacillus. plantarum
Lactobacillus. plantarum
Lactobacillus. pentosus
Lactobacillus. pentosus
Lactobacillus. plantarum
Lactobacillus. plantarum
-
+
+
-
+
-
+
-
+
[IL-12 induction by in vitro stimulation]
First, 4.05% thioglycolate was intraperitoneally
administered to BALB/c mice (7 weeks of age, male). After 4
days, intraperitoneal macrophages were collected with PBS,
and adjusted to 2 x 106 cells/mL using RPMI medium
containing 10% FBS. The culture was inoculated on a 24-well
plate (0.5 ml/well). Then, heat-killed cells (10 ug/mL) of each
strain were added to each well, and, after 24 hour incubation,
the IL-12 concentration of the supernatant was measured.
Since the active form of IL-12 is P70 combining two subunits
p35 and p40, the concentration of IL-12 (p70) was measured.
For the measurement of IL-12, the OptEIA mouse IL-12 ELISA
kit (BD Pharmingen) was used.
The results are shown in Fig. 1. As is clear from Fig. 1,
the inducible activity of IL-12 varied greatly even among the
strains of the same species or same parental strain. Among
these strains, the activity was particularly high in DW69N,
S-PT84 (DS84C), and DS25C.
[IL-12 induction by in vivo stimulation]
A suspension (solvent; saline solution) of heat-killed cells
(500ug/0.2mL/mouse) of each strain was intraperitoneally
administered to BALB/c mice (7 weeks of age, male). After 6
hours, the cervical was dislocated and the blood was collected
from the heart. For the control mice, the same amount of
saline solution was administered. The blood was collected 6
hours after the administration because a preliminary analysis
using S-PT84 had revealed that the peak concentration of
serum IL-12 occurs 6 hours after the administration of dead
cells (Fig. 2). After the blood was collected, the serum was
collected by centrifugation. The IL-12 concentration in the
serum was measured with the OptEIA mouse IL-12 ELISA kit
(BD Pharmingen).
The results are shown in Fig. 3. As is clear from Fig. 3,
the concentration of serum IL-12 was significantly high for
samples to which S-PT84 (DS84C), DS51C, and DS25C were
administered, compared with the control. Since the S-PT84
(DS84C) had the highest concentration, only this strain was
used in subsequent experiments.
[Effects S-PT84 on lymphocytes]
Spleens were removed from BALB/c mice (7 weeks of age,
male), and splenocytes were prepared according to ordinary
method. The splenocytes were cultured for 24 hours in a
medium containing (1 ug/ml) of heat-killed S-PT84 cells. As a
control, the splenocytes were cultured alone in a medium
(control). As another control, the splenocytes were also
cultured in a medium with the addition of concanavalin A (2.5
ug/mL) (Con A). In order to determine the type of cytokine
produced by the stimulus of the S-PT84 dead cells, the
cytokine concentration in the supernatant of each medium was
measured using the CBAkit (BD Pharmingen). The splenocytes
were collected and labeled with the fluorescent-labeled
anti-CD4 antibody (CY-CHCROME™ label, BD bioscience),
anti-CD8 antibody (FITC label, Immunotech), and anti-CD69
antibody (PE label, BD bioscience), and the respective
proportions of CD4-, CD8-, and CD69-positive cells were
measured with the flow cytometry (Beckman Coulter).
Fig. 4 depicts the result of cytokine production. Fig. 4(a)
is the result for the control in which the medium was used
alone, Fig. 4(b) is the result for S-PT84 in which the S-PT84
dead cells were added to the medium, and Fig. 4(c) is the result
for Con A in which concanavalin A was added to the medium.
As is clear from Fig. 4, the S-PT84 stimulus produced IFN-y
(interferon y) and TNF-a (tumor necrosis factor a), which were
not observed in the control. These cytokines were Thl-type
cytokines. It was therefore believed that the S-PT84 specifically
induced Thl-type cytokines. The Th2-type cytokines such as
IL-4 or IL-5 were not produced at all. With the concanavalin A
stimulation, another type of Thl-type cytokine, IL-2
(interleukin 2), was produced.
Fig. 5 shows how the CD4 + , CD8 + , and CD69+ cells were
affected. Fig. 5(a) is the result for the control in which the
medium was used alone. Fig. 5(b) is the result for the S-PT84
in which the S-PT84 dead cells (1 ug/mL) were added to the
medium. Fig. 5(c) is the result for Con A in which concanavalin
A was added to the medium. In Fig. 5, the directions of arrows
indicate increasing numbers of positive cells for each surface
antigen. As is clear from Fig. 5, the helper T cells and killer T
cells were activated by the S-PT84.
[Changes in hepatic lymphocytes after intraperitoneal
administration of S-PT84]
A suspension (solvent; saline solution) of heat-killed
S-PT84 cells (500ug/0.2mL/mouse) was administered
intraperitoneally to C57BL/6 mice (7 weeks of age, male). After
24 hours, liver was removed, and hepatic lymphocytes were
prepared by centrifugation. As a control, only the saline
solution was intraperitoneally administered. The NK activity of
the hepatic lymphocytes was measured by a PINK method. The
PINK method is a method for calculating the cytotoxic activity
of the mouse lymphocytes according to the following procedure.
First, a target cell Yac-1 is labeled with
3,3'-dioctadecyloxacarbocyanine perchlorate (Dio), which is a
hydrophobic fluorescent dye for labeling a membrane. Then,
the nucleus of the dead cell is double stained with propidium
iodide (PI), which is a membrane-impermeable nucleic acid
binding fluorescent dye. The Yac-1 cells were detected with the
flow cytometry, using Dio simple staining for uninjured cells,
and double staining for injured cells. Further, the other
hepatic cells were labeled with the fluorescent-labeled
anti-CD4 antibody (CY-CHCROME™ label, BD bioscience),
anti-CD8 antibody (FITC label, Immunotech), and anti-CD69
antibody (PE label, BD bioscience), and the respective
proportions of CD4-, CD8-, and CD69-positive cells were
measured with the flow cytometry (Beckman Coulter).
Fig. 6 shows the result of NK activity measurement. In Fig.
6, the NK activity (%) indicates cellular cytotoxicity of the
mouse hepatic lymphocytes against Yac-1, and E:T ratio
indicates the value of the number of reacted hepatic
lymphocytes versus the number of Yac-1 cells. As is clear from
Fig. 6, the NK activity of the hepatic lymphocytes prepared
from mice to which the S-PT84 was intraperineally
administered was clearly higher than that of the control.
Fig. 7 shows the results for CD4 + , CD8 + , and CD69+ cells,
in which Fig. 7(a) is the result for the control, and Fig. 7(b) is
the result with the administration of S-PT84. Further, in Fig. 7,
the directions of arrows indicate increasing numbers of positive
cells for each surface antigen. As is clear from Fig. 7, the
number of CD8 + cells, as well as CD8 + CD69+ cells, clearly
increased. It was therefore found that the administration of
S-PT84 increases the number of the killer T cells in the liver,
as well as the number of active killer T cells.
[Thl/Th2 balance regulating activity by oral
administration of S-PT84]
Six BALB/c mice (7 weeks of age, male) were allowed to
drink S-PT84 (dead cells)-containing water for a week
(equivalent of 2 mg/day). As a control group, six mice with no
S-PT84 were used. After one week, blood was collected from the
heart, and spleen was removed. The serum was collected from
the blood by centrifugation, and the IL-12 concentration in the
serum was measured using the OptEIA mouse IL-12 ELISA kit
(BD Biosciences). From the spleen, splenocytes were prepared
by ordinary method, and the number of the CD4 + , CD8 + , and
CD3+ cells in the splenic lymphocytes were counted
(measurement was made with the flow cytometry, using labeled
antibodies of the respective cells). Further, the NK activity was
measured by the PINK method. A measurement of Thl/Th2
ratio was also carried out (2.5 ug/ml of concanavalin A was
allowed to act on 5 x 106 mouse splenocytes for 24 hours, and
the concentrations of resulting IL-4 and IFN-y in the
supernatant were measured). The Thl/Th2 ratio was obtained
by dividing the IFN-y concentration by 11-4 concentration.
Fig. 8 shows the result of measurement of serum IL-12
concentration. As is clear from Fig. 8, the concentration of
serum IL-12 in the S-PT84-orally administered mice was
significantly higher than that of the control group (Cont).
Table 3 below represents the result of measurement of
CD4 + , CD8 + , and CD3+ cells. As is clear from Fig. 3, the
splenic lymphocyte T subset had a tendency to increase.
[Table 3]
Cell (x 10"
cells)
Spleen
CD4 +
CD8 +
CD3 +
Control
68.3±3.9
15.1±0.7
4.6±0.4
27.6±1.6
S-PT84
128.0±42.0
36.2+11.2'
9.7+1 1.2
61.0+19.8
Ratio
1.9
2.4
2. 1
2.2
Fig. 9 shows the result of measurement of NK activity. In
Fig. 9, the NK activity (%) indicates cytotoxicity of the mouse
splenocytes against Yac-1, and E:T ratio indicates the value of
the number of reacted splenocytes versus the number of Yac-1
cells. As is clear from Fig. 9, the NK activity of the splenocytes
prepared from the mice to which S-PT84 was orally
administered was significantly higher than that of the control
group (Cont).
Fig. 10 shows the result of measurement of Thl/Th2 ratio.
As is clear from Fig. 10, the splenocytes prepared from mice to
which the S-PT84 was orally administered had a considerably
large proportion of Thl cytokine as compared with the control
group (Cont).
As described above, the Thl-type cytokine was induced in
mice to which the S-PT84 was orally administered. As a result,
the Thl/Th2 balance shifted to Thl dominant, and the NK
activity increased as a result. This proved the Thl/Th2 balance
adjusting activity and immunostimulating activity of the
S-PT84.
[Effects on change of the weight of
cyclophosphamide-administered mice]
Twenty BALB/c mice (7 weeks of age, male) were divided
into two groups of an almost equal average weight, so as to
provide an S-PT84 administered group and an S-PT84
non-administered group (control group). The S-PT84
administered group was allowed to drink S-PT84 (dead
cellsj-containing water for 22 days (equivalent of 2 mg/day).
After 8 days from the start of administration, 200 mg/kg of
cyclophosphamide (CY) (antitumor chemotherapeutic drug,
alkylating agent) was intraperitoneally administered to all
individuals. A weight of each individual was measured on the
1st, 2nd, 3rd, 5th, 8th, 12th, and 15th days from the CY
administration.
Fig. 11 shows changes, in average body weight in the both
groups. As is clear from Fig. 11, a body weight loss caused by
the CY administration was suppressed in the S-PT84
administered group, as compared with the control group
(Control).
[Effects on IL-12 production in
cyclophosphamide-administered mice]
BALB/c mice (7 weeks of age, male) were divided into 5
groups: an untreated group (5 mice); an S-PT84
non-administered and CY non-administered group (10 mice); an
S-PT84 non-administered and CY administered group (10
mice); an S-PT84 administered and CY non-administered group
(10 mice); and an S-PT84 administered and CY administered
group (11 mice). The two S-PT84 administered groups were
allowed to drink S-PT84 (dead cells)-containing water for 12
days (equivalent of 2 mg/day). After 7 days from the start of
administration, 200mg/kg of cyclophosphamide (CY) (antitumor
chemotherapeutic drug, alkylating agent) was intraperitoneally
administered to the CY administered groups. After 5 days from
the CY administration, a suspension (solvent; saline solution)
of heat-killed cells of S-PT84 (SOOug/ 0.2ml/mouse) was
intraperitoneally administered to the mice of all groups except
for the untreated group. Six hours after the administration of
S-PT84 dead cells, blood was collected from the heart in all
individuals, inchiding those in the untreated group. From the
collected blood, the serum was collected by centrifugation, and
the IL-12 concentration in the serum was measured with the
OptEIA mouse IL-12 ELISA kit (BD Pharmingen).
The results are shown in Fig. 12. As is clear from Fig. 12,
the CY administration in the S-PT84 non-administered group
reduced the IL-12 concentration in the serum by a considerable
amount. On the other hand, in the S-PT84 administered group,
the reduction of IL-12 concentration in the serum caused by
the CY administration was suppressed significantly, and the
IL-12 concentration in the serum was almost equal to that of
the control group.
From these results, it was proved that the S-PT84 has the
ability to suppress a weight loss and immune reduction caused
by CY. In other words, the immunostimulating activity of the
S-PT84 was confirmed.
[Analysis of anti-allergy activities]
Thirty-six BALB/c mice (7 weeks of age, male) were
divided into four groups: an untreated group (5 mice); a control
group (10 mice); an S-PT84 group (11 mice); and a
dexamethasone administered group (10 mice). The S-PT84
group was allowed to drink S-PT84 (dead cells)-containing
water for 7 weeks (equivalent of 2 mg/day). To the Dex
administered group, 0.5 mg/kg of S-PT84 was forcibly
administered for 7 weeks by oral administration. The untreated
group and control group were allowed to drink tap water for 7
weeks. Note that, the Dex is a steroid drug with anti-allergenic
and anti-inflammatory activities, and was used as a positive
control drug. One week and two weeks after the S-PT84 uptake
or Dex administration, a mixture containing 20 ug of
ovalbumin (OVA) and 2 mg of aluminum hydroxide gel was
intraperitoneally administered to the mice of all groups except
for the untreated group. From the second administration (0
week) to the 5th week, blood was collected every week a total of
6 times from all individuals, and an OVA-specific IgE
concentration was measured. The measurement of OVA-specific
IgE concentration in the serum was carried out according to
the ELISA method, using a modified OptEIA mouse IgE ELISA
kit (BD Pharmingen) in which OVA was coated instead of the
capturing antibody. Using the blood sample from the third
week, total IgE was measured. The measurement of total IgE
was carried out according to the ELISA method, using the
OptEIA mouse IgE ELISA kit.
Fig. 13 shows changes in OVA-specific IgE concentration
on the 3rd week of the experiment start. Fig. 14 shows the
result of measurement on total IgE concentration. As is clear
from Fig. 13, the S-PT84 group significantly suppressed the
increase of OVA-specific IgE concentration as compared with
the control group (control). In the Dex group, the result was
not significantly different from the control group, though some
suppressing effect was observed. Further, as is clear from Fig.
14, the S-PT84 group and Dex group significantly suppressed
increase of total IgE concentration as compared with the
control group (control).
It became clear from these results that the S-PT84 had an
anti-allergy activity.
[Analysis of suppressing effect on stress-induced immune
reduction]
Nine C57BL/6 mice (6 weeks of age, male) were divided
into three groups of an almost equal average weight, so as to
provide a control group (3 mice), a stressed group (3 mice), and
an S-PT84 administered and stressed group (3 mice). The
S-PT84 administered group was allowed to drink S-PT84 (dead
cellsj-containing water for 7 days (equivalent of 2 mg/day). On
the eighth day of the administration, a total of 6 mice in the
stressed group and the S-PT84 administered and stressed
group were immersed in water, placed in a 50 ml polyethylene
tube whose tip had an air vent, and restricted for 24 hours.
The control group was deprived of food and water. From each
animal, the spleen was removed, and splenic lymphocytes were
prepared for the measurement of NK activity.
Fig. 15 shows the result of measurement on NK activity.
The stressed group showed a significant drop in NK activity
compared with the control group. The S-PT84 administered and
stressed group maintained NK activity comparable to that of
the control group, i.e., the NK activity level was significantly
higher than that of the stressed group. This proved that the
S-PT84 had the activity of suppressing stress-induced immune
reduction.
[Difference in immunostimulating activity between dead
cells and viable cells]
The immunostimulating activities were compared between
dead cells and viable cells of the lactic acid bacteria. For the
comparison, the lactic acid bacteria that increased the mouse
serum IL-12 concentration in response to intraperitoneal
administration as shown in Fig. 3 were used.
Suspensions of 4 kinds of lactic acid bacteria (DB22C,
DS51C, DS2C and DS84C (S-PT84)), both in the form of
heat-killed cells and viable cells, were prepared (each weighing
500 pg (2.5xl08/0.2mL/mouse)). The suspensions were
intraperitoneally administered to BALB/c mice (7 weeks of age,
male). After 6 hours, the cervical was dislocated and the blood
was collected from the heart. As a control, a mouse to which
the same amount of saline solution was administered instead
of the lactic acid bacteria was used.
After the blood was collected, the serum was collected by
centrifugation. The IL-12 concentration in the serum was
measured with the OptEIA (BD Pharmingen). The results are
shown in Fig. 16.
As in the case of Fig. 3, the amount of serum IL-12 was
greatest for samples to which DS84C (S-PT84) was
administered. As is also clear from Fig. 16, the amount of IL-12
was higher for the samples to which the viable cells of DS84C
(S-PT84) were administered than for the samples to which the
dead cells were administered.
[Producing example 1: Tablet]
An S-PT84-containing medicament (tablet) was produced
according to the following procedures.
A mixture containing 66.7 g of a dried pulverized powder
of S-PT84, 232.0 g of lactose, and 1.3 g of magnesium stearate
was punched with a single punch tableting machine, so as to
produce tablets each having a diameter of 10 mm and a weight
of 300 mg.
[Producing example 2: Yoghurt]
S-PT84 fermented milk with a 21% solid milk component
was added to commercially available milk, and the mixture was
allowed to stand for 3 days so as to prepare yoghurt. The
resulting yoghurt had a desirable flavor.
[Producing example 3: Lactic acid bacteria drinks]
By using S-PT84, a lactic acid bacteria beverage was
prepared with the compositions shown in Table 4. The
resulting lactic acid bacteria beverage had a desirable flavor.
[Table 4]
Compositions
S-PT84 fermented milk with
21% solid milk component
Fructose glucose syrup
Pectin
Citric acid
Flavoring agent
Water
Total
Parts by weight
14.76
13.31
0.5
0.08
0. 15
71.2
100
The invention being thus described, it will be obvious that
the same way may be varied in many ways. Such variations
are not to be regarded as a departure from the spirit and scope
of the invention, and all such modifications as would be
obvious to one skilled in the art are intended to be included
within the scope of the following claims.
INDUSTRIAL APPLICABILITY
A composition according to the present invention, if
ingested as food or drink, or administered as medicaments,
enables the immune function to be activated and thereby
suppresses reduction of immune functions. Further, by
adjusting the balance of immune function, adverse effects of
excess immune function on the body can be suppressed.
A composition according to the present invention can be
implemented as health food or medicaments with
immunoregulating activities. Therefore, the invention is useful
is food industries and pharmaceutical industries.

CLAIMS:
1. A composition comprising lactic acid bacteria which belong to Lacto bacillus pentosus, and which have a weak assimilating activity or no assimilating activity for glycerol.
2. The composition as set forth in claim 1, wherein the
lactic acid bacteria have immunoregulating activities and/or
anti-allergy activities.
3. The composition as set forth in claim 2, wherein the
lactic acid bacteria are an extracellular
polysaccharide-producing strain.
4. The composition as set forth in claim 3, wherein the
lactic acid bacteria are a Lactobacillus pentosus S-PT84 strain
(PERM ABP-10028).
5. The composition as set forth in any one of claims 1
through 4, wherein the composition has immunoregulating
activities.
6. The composition as set forth in any one of claims 1
through 4, wherein the composition has anti-allergy activities.
7. The composition as set forth in any one of claims 1
through 6, wherein the composition contains lactic acid
bacteria as viable cells.
8. The composition as set forth in any one of claims 5 through 7, wherein the composition is food, a drink, or a medicament.

Documents:

7718-delnp-2006-Abstract-(10-05-2013).pdf

7718-delnp-2006-abstract.pdf

7718-DELNP-2006-Assignment-(14-05-2009).pdf

7718-delnp-2006-Claims-(10-05-2013).pdf

7718-DELNP-2006-Claims.pdf

7718-delnp-2006-Correspondence Others-(10-05-2013).pdf

7718-delnp-2006-Correspondence Others-(28-05-2012).pdf

7718-delnp-2006-Correspondence Others-(31-08-2012).pdf

7718-DELNP-2006-Correspondence-others (07-04-2008).pdf

7718-DELNP-2006-Correspondence-others (23-05-2008).pdf

7718-delnp-2006-Correspondence-Others-(12-10-2012).pdf

7718-DELNP-2006-Correspondence-Others-(14-05-2009).pdf

7718-delnp-2006-correspondence-others.pdf

7718-delnp-2006-description (complete).pdf

7718-delnp-2006-drawings.pdf

7718-DELNP-2006-Form-1-(07-04-2008).pdf

7718-DELNP-2006-Form-1.pdf

7718-DELNP-2006-Form-13 (23-05-2008).pdf

7718-DELNP-2006-Form-18 (23-05-2008).pdf

7718-delnp-2006-form-2.pdf

7718-DELNP-2006-Form-3.pdf

7718-DELNP-2006-Form-5.pdf

7718-delnp-2006-form-6-(14-05-2009).pdf

7718-DELNP-2006-GPA (07-04-2008).pdf

7718-DELNP-2006-GPA-(14-05-2009).pdf

7718-delnp-2006-gpa.pdf

7718-delnp-2006-pct-237.pdf

7718-delnp-2006-pct-304.pdf

7718-delnp-2006-pct-request form.pdf

7718-delnp-2006-pct-search report.pdf

7718-delnp-2006-Petition-137-(10-05-2013).pdf

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Patent Number

258392

Indian Patent Application Number

7718/DELNP/2006

PG Journal Number

02/2014

Publication Date

10-Jan-2014

Grant Date

06-Jan-2014

Date of Filing

19-Dec-2006

Name of Patentee

SUNTORY HOLDINGS LIMITED

Applicant Address

1-40, DOJIMAHAMA 2-CHOME, KITA-KU, OSAKA-SHI, OSAKA, 5308203 (JP)

Inventors:

#	Inventor's Name	Inventor's Address
1	YUJI NONAKA	C/O SUNTORY LIMITED, RESEARCH CENTER 1-1-1, WAKAYAMADAI, SHIMAMOTO-CHO, MISHIMA-GUN, OSAKA 618-8503, JAPAN
2	TAKAYUKI IZUMO	C/O SUNTORY LIMITED, RESEARCH CENTER 1-1-1, WAKAYAMADAI, SHIMAMOTO-CHO, MISHIMA-GUN, OSAKA 618-8503, JAPAN
3	KEIKO IIDA	C/O SUNTORY LIMITED, RESEARCH CENTER ANNEX, 3-3-22, TAKAHAMA, SHIMAMOTO-CHO, MISHIMA-GUN, OSAKA 618-0012, JAPAN

PCT International Classification Number

A61K 35/74

PCT International Application Number

PCT/JP2005/006585

PCT International Filing date

2005-03-29

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	159461/2004	2004-05-28	Japan