US20060127378A1 - Composition for promoting the proliferation of lactobacillus casei subsp. casei - Google Patents

Composition for promoting the proliferation of lactobacillus casei subsp. casei Download PDF

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US20060127378A1
US20060127378A1 US10/521,947 US52194705A US2006127378A1 US 20060127378 A1 US20060127378 A1 US 20060127378A1 US 52194705 A US52194705 A US 52194705A US 2006127378 A1 US2006127378 A1 US 2006127378A1
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casei
dextran
composition
subsp
jcm
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Kenji Yasuda
Tomohiko Ogawa
Masakatsu Hasegawa
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Meito Sangyo KK
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Meito Sangyo KK
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Assigned to MEITO SANGYO CO., LTD. reassignment MEITO SANGYO CO., LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HASEGAWA, MASAKATSU, OGAWA, TOMOHIKO, YASUDA, KENJI
Publication of US20060127378A1 publication Critical patent/US20060127378A1/en
Priority to US12/190,121 priority Critical patent/US7678777B2/en
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    • 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
    • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
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Definitions

  • the present invention relates to: a dextran-comprising composition for promoting bacterial proliferation for selectively proliferating Lactobacillus casei subsp. casei (hereinafter, abbreviated as “ L. casei subsp. casei ”) which is one species of lactic acid bacteria having a variety of biological activities; and the composition for promoting bacterial proliferation, which further comprises L. casei subsp. casei . More specifically, the present invention relates to: a pharmaceutical composition for enhancing biological activities, which comprises the composition for promoting bacterial proliferation; and a health food and feed for enhancing biological activities, which comprise the composition for promoting bacterial proliferation.
  • microorganism formulations which are referred to as probiotics and consist of useful microorganisms such as living lactic acid bacteria for the purpose of effect of controlling intestinal function, prevention of infectious diseases, immunostimulation, or the like has been attracting attraction.
  • useful microorganisms can grow and colonize in the intestine of a human being, animal, or the like. If the useful microorganisms cannot grow and colonize in the intestine, expected biological activities of the useful microorganisms cannot be obtained permanently. In order to solve such a problem, the fact is that supplying the microorganism formulation at all times is proposed and performed.
  • prebiotics oligosaccharides or the like which can be utilized by useful microorganisms in probiotics
  • symbiotics ones prepared by combining prebiotics and probiotics
  • Those are also administered to a human being, animal, or the like for the aforementioned purpose.
  • prebiotics are utilized by great many enteric bacteria other than intended bacteria, those bacteria scramble for the prebiotics in the intestine, so it is difficult to grow and colonize intended useful microorganisms selectively.
  • An object of the present invention is, after ingestion of useful microorganisms referred to as probiotics to a human being, animal, or the like, to sustain a variety of biological activities originated from the useful microorganisms in a living body by selectively growing-proliferating and colonizing the microorganisms in the intestine or by selectively growing-proliferating useful indigenous microorganisms in the intestine without supplying those useful microorganisms at all times.
  • L. casei subsp. casei is a species of bacteria capable of growing-proliferating by utilizing a dextran.
  • L. casei subsp. casei is classified and named in Approved Lists (1980).
  • the bacterium is Gram positive, indigenous to the intestine of a human being, animal, or the like, and a facultative anaerobic bacillus.
  • the bacterium forms the intestinal bacterial flora together with other bacteria.
  • the bacterium itself has biological activities, for example, an immunostimulating effect in the case of subcutaneous immunization.
  • the bacterium has been found to enhance and maintain humoral immunity and cellular immunity.
  • L. casei subsp. casei has been administered to a mouse only in the initial administration, and a dextran was then orally ingested permanently, in order to confirm whether L. casei subsp. casei can selectively grow-proliferate and colonize in the intestine of a human being, animal, or the like, and the biological activities of the bacterium can be sustained or not by oral ingestion of a dextran.
  • the inventors of the present invention have found through an in vivo test that the bacterium grows-proliferates and colonizes in the intestine by utilizing the dextran, and the biological activities of the bacterium such as humoral immunity and cellular immunity are enhanced and maintained.
  • a dextran itself is also known to have a variety of biological activities, and it has been confirmed that, in the case of administration of a dextran together with L. casei subsp. casei , a higher effect is maintained than that in the case of administration of only a dextran, and a synergistic effect is large.
  • the present invention has been accomplished as described above.
  • the gist of the present invention is as follows.
  • a composition for promoting bacterial proliferation for selectively proliferating Lactobacillus casei subsp. casei comprising a dextran.
  • composition for promoting bacterial proliferation according to (1), further comprising Lactobacillus casei subsp. casei.
  • composition for promoting bacterial proliferation according to (1) wherein the dextran has a molecular weight of 2,000 to 40,000,000.
  • a pharmaceutical composition comprising the composition for promoting bacterial proliferation according to any one of (1) to (3).
  • a health food comprising the composition for promoting bacterial proliferation according to any one of (1) to (3).
  • a feed comprising the composition for promoting bacterial proliferation according to any one of (1) to (3).
  • FIG. 1 is a drawing showing an effect of a dextran on the bacterial number of L. casei subsp. casei in mouse feces.
  • FIG. 2 is a drawing showing the results of enhancing humoral immunity by a dextran in the case of orally administering an antigen.
  • FIG. 3 is a drawing showing the results of enhancing cellular immunity by a dextran in the case of orally administering the antigen.
  • FIG. 4 is a drawing showing the results of enhancing humoral immunity by a dextran in the case of subcutaneously administering the antigen.
  • FIG. 5 is a drawing showing the results of enhancing cellular immunity by a dextran in the case of subcutaneously administering the antigen.
  • the composition of the present invention is a dextran-comprising composition for promoting bacterial proliferation for selectively proliferating L. casei subsp. casei and the composition for promoting bacterial proliferation, which further comprises L. casei subsp. casei .
  • Dextrans to be used in the composition of the present invention can be synthesized, prepared, or the like according to a general method that is well known to a person skilled in the art.
  • culture of L. casei subsp. casei methods of preparing and drying of bacterial cells, gene recombination, and the like can be performed according to a general method that is well known to a person skilled in the art.
  • a dextran is a polysaccharide which consists of glucose, which are mainly bonded with an ⁇ -1,6 bond.
  • the dextrans to be used in the present invention may be commercially available dextrans or dextrans obtained by culturing bacteria selected from the group consisting of genus Leuconostoc and genus Streptococcus , for example, Leuconostoc mesenteroides , under a general condition, for example, in a sucrose-containing enriched medium.
  • Each of the molecular weights of dextrans to be used in the present invention is 2,000 to 40,000,000, preferably 4,000 to 5,000,000, more preferably 4,000 to 2,000,000.
  • a dextran having a molecular weight of less than 2,000 is not preferable because bacteria other than L. casei subsp. casei may proliferate.
  • Examples of the dextrans to be used in the present invention include a dextran obtained by culturing the aforementioned bacteria without any treatment, and a dextran having an appropriate molecular weight obtained by further partial hydrolysis treatment of such a dextran.
  • L. casei subsp. casei can be used in the form of viable cells, dried cells capable of growing-proliferating, or the like.
  • L. casei subsp. casei to be used in the present invention there can be used a recombinant of L. casei subsp. casei which has at least a utilizing ability for a dextran in the composition for promoting bacterial proliferation of the present invention, in addition to a wild type.
  • the recombinant is a novel bacterium in which a new biological activity is expressed by gene recombination of L. casei subsp. casei or another novel bacterium obtained by transducing a gene which participates in a dextran-utilizing ability originated from L. casei subsp. casei to another bacterium.
  • the composition of the present invention is a dextran-comprising composition for promoting bacterial proliferation for selectively proliferating L. casei subsp. casei and the composition for promoting bacterial proliferation which further comprises L. casei subsp. casei .
  • the composition of the present invention can be prepared, administered, or the like according to a general method which is well known to a person skilled in the art except that the composition comprises a dextran or further comprises L. casei subsp. casei.
  • the composition of the present invention may be a dextran only or a mixture of a dextran and L. casei subsp. casei .
  • the composition of the present invention can be provided as a composition in the form of solid, semi-solid, or liquid containing a dextran in combination with a diluent, carrier, or the like which is pharmaceutically well known and pharmaceutically acceptable, or a composition in the form of solid, semi-solid, or liquid containing a dextran and L. casei subsp. casei .
  • the composition of the present invention includes: a form in which the dextran of the present invention and L. casei subsp. casei have already been combined; and a form in which the dextran of the present invention and L.
  • casei subsp. casei have been separately processed, and are combined when the composition is used.
  • the amount of a diluent, carrier, or the like in the composition of the present invention is appropriately varied in accordance with a purpose or usage, and is not particularly limited. In general, the amount is, for example, about 0.1 to about 99.9% by weight, preferably about 1 to about 99% by weight, more preferably about 5 to about 95% by weight based on the weight of the composition.
  • the dextran content reduces gradually due to bacterial utilization of the dextran, so simultaneous presence of them for a long time is preferably avoided. So, it is desirable that those be separately processed and packed, and the resultant products be mixed when they are ingested, or be separately ingested.
  • the pharmaceutical composition of the present invention may be the composition of the present invention itself without other components.
  • the composition may be one prepared in combination with a diluent, carrier, or the like, which is pharmaceutically well known and pharmaceutically acceptable, into a dosage form such as a soft capsule, hard capsule, granule, pill, powder, tablet, syrup, troche, or elixir.
  • a diluent, carrier, or the like which is suitable to each dosage form, pharmaceutically well known, and pharmaceutically acceptable may further be contained.
  • the pharmaceutical composition of the present invention can be used as a drug which aims at sustaining a variety of biological activities originated from L. casei subsp. casei in a living body by: selectively growing-proliferating and colonizing L. casei subsp. casei in the intestine of a human being, animal, or the like; or selective growth-proliferation of L. casei subsp. casei in the intestine, which is indigenous to the intestine.
  • the addition amount of the composition of the present invention may be appropriately determined with reference to the description which relates to the dose and dosage cycle of the composition of the present invention described below.
  • the health food of the present invention is one containing the composition of the present invention and a composition such as a food or beverage.
  • the health food of the present invention includes: a form in which the composition of the present invention and a composition such as a food or beverage have already been combined; and a form in which the composition of the present invention and a composition such as a food or beverage have been separately processed, and are combined when the composition is ingested.
  • a food or beverage include: confectioneries such as candy, chocolate, and biscuit; breads; noodles; beverages in the form of powder, liquid, and the like; dairy products such as yogurt.
  • yogurt can be produced using such L. casei subsp. casei , and it is desirable that a dextran be mixed when they are ingested or be individually ingested.
  • the feed of the present invention contains the composition of the present invention and formula feed of livestocks, fowls, fishes, pets, or the like.
  • the feed of the present invention includes: a form in which the composition of the present invention and a feed composition have already been combined; and a form in which the composition of the present invention and a feed composition have been separately processed, and are combined when the composition is ingested.
  • the health food or feed of the present invention can be used for sustaining a variety of biological activities originated from L. casei subsp. casei in a living body by: selective growth-proliferation and colonization of L. casei subsp. casei in the intestine of a human being, animal, or the like; or selective growth-proliferation of L. casei subsp. casei in the intestine, which is indigenous to the intestine.
  • the addition amount of the composition of the present invention may be appropriately determined with reference to the description which relates to the dose and dosage cycle of the composition of the present invention described below.
  • the dose of the composition of the present invention is, in terms of the dose of a dextran in the composition, 0.01 mg to 2 g/kg weight ⁇ day, preferably 0.1 mg to 1 g/kg weight ⁇ day, more preferably 1 mg to 100 mg/kg weight ⁇ day, and it is preferable that at least a composition comprising the dextran of the present invention be generally administered every day.
  • L. casei subsp. casei is originally a bacterium which is indigenous to the intestine of a human being, animal, or the like, and in order to induce the reliable effects of the biological activities of L. casei subsp. casei , viable cells or dried cells capable of growing-proliferating are administered at least once, preferably at an appropriate interval. In addition, in order to induce more reliable effects, those cells may be administered every day.
  • Leuconostoc mesenteroides which is one species of lactic acid bacteria, was inoculated in a medium containing sucrose as a principal material (sucrose-containing enriched medium), and cultured anaerobically at 25° C. for 24 hours. Subsequently, methanol was added to the culture supernatant, and the resultant precipitates were dissolved in distilled water. After the insolubles had been removed, methanol was added thereto again, and the resultant precipitates were redissolved in distilled water. The mixture was subjected to spray drying, to thereby yield a dextran. The dextran was further partially hydrolyzed, and the resultant products were subjected to methanol fractionation and spray drying, to thereby yield dextrans having a variety of molecular weights.
  • a variety of related enteric bacteria were inoculated in a PYF medium (Peptone yeast extract Fildes solution broth) to which a dextran having a molecular weight (number average) of 10,000 (in the following examples, a dextran having such a molecular weight was used unless otherwise defined) was added to 0.5%, and the dextran-utilizing ability was determined.
  • a dextran having a molecular weight (number average) of 10,000 in the following examples, a dextran having such a molecular weight was used unless otherwise defined
  • JCM 1134 and JCM 8129 two strains of L. casei subsp. casei (JCM 1134 and JCM 8129) were confirmed to have the dextran-utilizing ability.
  • no dextran-utilizing ability was observed for other lactic acid bacteria such as bacteria of the genus Lactobacillus or Bifidobacterium , and other related enteric bacteria.
  • JCM 5820 ⁇ globosum pseudolongum subsp. JCM 1205 ⁇ pseudolongum pullorum JCM 1214 ⁇ ruminantium JCM 8222 ⁇ saeculare JCM 8223 ⁇ subtile JCM 5822 ⁇ suis JCM 1269 ⁇ thermophilum JCM 1207 ⁇
  • mice 12 eight-week-old BALB/c male mice were divided into two groups of six each. Those groups were referred to as (1) control group and (2) dextran group. Over the test period from the beginning of the test, a conventional feed was given to (1), while a dextran-added conventional feed (75 mg dextran/kg conventional feed) was given to (2). Over the period of three days from the beginning of the test, L. casei subsp. casei cells were orally inoculated to both groups (10 6 cells/mouse). On 28th day, each mouse was dissected, and the feces were collected from the large intestine.
  • mice 30 eight-week-old BALB/c male mice were divided into five groups of six each. Those groups were referred to as (1) control group, (2) BSA (bovine serum albumin) group, (3) BSA+ L. casei subsp. casei group, (4) BSA+dextran group, and (5) BSA+ L. casei subsp. casei +dextran group.
  • BSA bovine serum albumin
  • BSA+dextran group BSA+ L. casei subsp. casei +dextran group.
  • a conventional feed was given to each of (1), (2), and (3), while a dextran-added conventional feed (75 mg dextran/kg conventional feed) was given to each of (4) and (5).
  • mice 30 eight-week-old BALB/c male mice were divided into five groups of six each. Those groups were referred to as (1) control group, (2) BSA group, (3) BSA+ L. casei subsp. casei group, (4) BSA+dextran group, and (5) BSA+ L. casei subsp. casei +dextran group.
  • a conventional feed was given to each of (1), (2), and (3), while a dextran-added conventional feed (75 mg dextran/kg conventional feed) was given to each of (4) and (5).
  • L. casei subsp. casei cells were orally inoculated to (3) and (5) (10 6 cells/mouse).
  • BSA was dissolved in PBS at a concentration of 10 mg/ml, and the solution was orally administered (1 mg (100 ⁇ l)/mouse) to (2) to (5).
  • BSA was orally administered (1 mg/mouse).
  • Five days after the administration BSA was dissolved in PBS, and the solution was intradermally administered to the earlobe, followed by measuring the thickness of the mouse earlobe after 24, 48, and 72 hours.
  • BSA+ L. casei subsp. casei +dextran group the thickness of the earlobe significantly increased. (See FIG. 3 )
  • mice 30 eight-week-old BALB/c male mice were divided into five groups of six each. Those groups were referred to as (1) control group, (2) BSA group, (3) BSA+ L. casei subsp. casei group, (4) BSA+dextran group, and (5) BSA+ L. casei subsp. casei +dextran group.
  • a conventional feed was given to each of (1), (2), and (3), while a dextran-added conventional feed (75 mg dextran/kg conventional feed) and the resultant feed was given to each of (4) and (5).
  • casei bacteria cells were orally inoculated to (3) and (5) (10 6 cells/mouse).
  • FIA Fluorescence-Activated adjuvant
  • PBS+FIA phosphate buffered saline
  • BSA+FIA BSA (5 mg/ml)
  • booster was performed in a manner similar to that on the third day.
  • mice 30 eight-week-old BALB/c male mice were divided into five groups of six each. Those groups were referred to as (1) control group, (2) BSA group, (3) BSA+ L. casei subsp. casei group, (4) BSA+dextran group, and (5) BSA+ L. casei subsp. casei +dextran group.
  • a conventional feed was given to each of (1), (2), and (3), while a dextran-added conventional feed (75 mg dextran/kg conventional feed) was given to each of (4) and (5).
  • L. casei subsp. casei bacteria cells were orally inoculated to (3) and (5) (10 6 cells/mouse).
  • L. casei subsp. casei a variety of biological activities originated from L. casei subsp. casei can be sustained in a living body by selectively growing-proliferating and colonizing L. casei subsp. casei in the intestine of a human being, animal, or the like or by selectively growing-proliferating L. casei subsp. casei in the intestine, without supplying L. casei subsp. casei at all times.

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