WO2010008113A1 - Anti-cancer composition comprising a culture fluid of lactobacillus casei as an effective component - Google Patents

Anti-cancer composition comprising a culture fluid of lactobacillus casei as an effective component Download PDF

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Publication number
WO2010008113A1
WO2010008113A1 PCT/KR2008/004375 KR2008004375W WO2010008113A1 WO 2010008113 A1 WO2010008113 A1 WO 2010008113A1 KR 2008004375 W KR2008004375 W KR 2008004375W WO 2010008113 A1 WO2010008113 A1 WO 2010008113A1
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Prior art keywords
cancer
lactobacillus casei
fluid
composition
bacteria
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PCT/KR2008/004375
Other languages
French (fr)
Inventor
Myung Jun Chung
Dong Myung Kim
Hong Baek
Jung Hwa Kim
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Cell Biotech Co., Ltd.
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Application filed by Cell Biotech Co., Ltd. filed Critical Cell Biotech Co., Ltd.
Publication of WO2010008113A1 publication Critical patent/WO2010008113A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to an anti-cancer composition
  • a culture fluid of Lactobacillus casei spp. as an effective component
  • an anti-cancer composition comprising a culture fluid obtained by culturing Lactobacillus casei in a culture medium containing a carbon source, a nitrogen source, a phosphorus source, and the like and then removing the bacteria as an effective component.
  • Lactic acid bacteria are the bacteria which produce lactic acid as the major metabolic endproduct of carbohydrate fermentation. They are facultative anaerobic bacteria or obligate anaerobic bacteria that proliferate well under oxygen-deficient conditions.
  • the genera that comprise the lactic acid bacteria are Streptococcus, Lactobacillus, Leu- conostoc, Bifidobacterium and Pediococcus.
  • Streptococcui are chain-shaped ho- mofermenting bacteria and are known to inhibit saprophytes or pathogens by fermenting milk and producing lactic acid.
  • Lactobacilli are rod- shaped ho- mofermenting or heterofermenting bacteria fiequently found in the fermentation process of dairy products or vegetables.
  • Leuconostoc is a diplococcus heterofermenting bacteria mainly involved in fermentation of vegetables.
  • Bifidobacteria are obligate anaerobic bacteria which cannot survive in an oxygen-rich environment. They produce l(+)-lactic acid, which can be digested by infants, by fermenting sugars.
  • Pediococcus is a homofermenting bacteria usually occuiring in pairs or tetrads. They exist in kimchi or preserved food and are involved in fermentation of meat, such as sausage.
  • the lactic acid bacteria maintain the pH in the intestine acidic, thereby inhibiting the proliferation of harmful bacteria such as E. coli and Clostridium sp. and improving diairhea and constipation. Further, they aid in synthesis of vitamin, have anti-cancer effect, and reduce serum cholesterol. In particular, with specific proteins that can strongly bind the mucosa and epithelial cells of the intestine, the lactic acid bacteria inhibit the growth of harmful bacteria and pievent intestinal infection.
  • the lactic acid bacteria aie known to facilitate the growth of macrophages, thereby enhancing the recognition ability, antimicrobial ability and so on of the macrophages against harmful bacteria in the intestine, and promote the secretion of immune-related substances, thereby enhancing immunity
  • Lactobacilli are known to produce acidophillin, thereby inhibiting the growth of harmful bacteria, such as Shigella, Salmonella, Staphylococcus, etc., and inhibiting the proliferation of diairhea-causing bacteria and normalizing intestinal microflora, thereby stopping diairhea.
  • Lactobacillus bulgaricus Lactobacillus acidophilus
  • Streptococcus thermophilus reduced the occu ⁇ ence of DMH- induced tumors in rats (Shakelford et al., Nutr. Cancer 5:159-1647, 1983).
  • Dairy products fermented by Lactobacillus bulgaricus inhibited DMH-induced carcinogenesis in rats and DEN (diethylnitrosamine)-induced carcinogenesis of the upper respiratory tract in hamsters (Balansky et al., Cancer Lett., 147:125-137, 1999).
  • a single subcutaneous injection of peptidoglycan isolated from Bifidobacterium infantis inhibited tumors (syngeneic fibrosarcoma, Meth A in Balb/c mice)(Sekine et al., Cancer Res., 45:1300-1307, 1985).
  • Polysaccharide fractions derived from the culture medium of Lactobacillushelveticus were effective in inhibiting tumors (sarcoma 180), and glycoproteins exhibited cell toxicity against cervical cancer cells (Oda et al., Agri. Biol. Chem., 47:1623-1625, 1983; Manjunath et al., Indian Journal Exp. Biol., 27:141-145, 1989).
  • macrophage activating substances derived from BCG Bacillus Calmette-Guerin
  • BCG Bacillus Calmette-Guerin
  • Lactobacillus casei has superior anti-cancer activity. Particularly, they have found through experiments on various cancer cells that the cultuie fluid of the cells, not the cells themselves, outstandingly inhibits the proliferation of the cancer cells and, thus, has superior anticancer activity.
  • an object of the piesent invention is to provide an anti-cancer composition comprising a cultuie fluid of Lactobacillus casei as an effective component.
  • the piesent invention provides an anti-cancer composition comprising a cultuie fluid of Lactobacillus casei as an effective component.
  • the anti-cancer composition of the piesent invention comprises a cultuie fluid of
  • Lactobacillus casei spp. as an effective component.
  • the Lactobacillus casei used in the piesent invention may be the lactic acid bacteria generally used in the related art.
  • it may be Lactobacillus casei ATCC 334, ATCC 393, ATCC 11578, KCTC 3109, KCTC 3189, DSM 20011 or DSM L26 10633.
  • the culture fluid of Lactobacillus casei may be obtained by culturing the bacteria according to the conventional lactic acid bacteria culturing method and then removing the bacteria.
  • the culturing of the lactic acid bacteria may be cairied out using adequate medium and adequate culturing condition known to those skilled in the art. Those skilled in the art may easily modify the culturing process, depending on particularly selected bacteria.
  • Various culturing methods are disclosed in many publications [for example, James et al., Biochemical Engineering, Prentice-Hall International Editions]. The culturing methods can be classified into suspension culturing and adherent culturing depending on the mode of cell growth, and into batch culturing, fed-batch culturing and continuous culturing depending on the mode of culturing.
  • the cultuie medium should adequately satisfy the lequiiements of particular bacteria.
  • the cultuie medium includes a variety of carbon sources, nitrogen sources and trace element components.
  • the carbon source include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid.
  • carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose
  • fats such as soybean oil, sunflower oil, castor oil and coconut oil
  • fatty acids such as palmitic acid, stearic acid and linoleic acid
  • alcohols such as glycerol and ethanol
  • organic acids such as acetic acid.
  • the nitrogen source examples include organic nitrogen sources such as peptone, yeast extract, bouillon, malt extract, com steep liquor (CSL) and soybean meal, and inorganic nitrogen sources such as uiea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources may be used singly or in combination.
  • the cultuie medium may include potassium di- hydrogen phosphate, potassium phosphate dibasic, sodium dihydrogen phosphate and disodium hydrogen phosphate as phosphorus source. Further, metal salts such as magnesium sulfate or iron sulfate may be included. Besides, amino acids, vitamins, adequate piecursors, and the like may be included.
  • such compounds as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid may be added in an adequate manner to adjust pH.
  • a defoaming agent such as fatty acid polyglycol ester may be used to ieduce foaming.
  • oxygen or oxygen-containing gas e.g., air
  • the culturing temperatuie is usually from 20 0 C to 45 0C, pieferably from 25 0 C to 40 0 C. The culturing may be cairied out for 10 to 40 hours.
  • centrifugation or filtration may be cairied out to remove the bacteria from the cultuie fluid. This process may be cairied out if necessary at the discretion of those skilled in the art.
  • the removal of the bacteria refers to a substantial removal of the Lactobacillus casei through a commonly used bacteria removal method.
  • the culture fluid of the present invention may be prepared by a method comprising:
  • a culture medium comprising a carbon source selected from the group consisting of glucose, sucrose, lactose, fructose(fructose), maltose, starch, cellulose, soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, linoleic acid, glycerol, ethanol and acetic acid; a nitrogen source selected from the group consisting of peptone, yeast extract, bouillon, malt extract, com steep liquor (CSL), soybean meal, uiea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate; and a phosphorus source selected from the group consisting of potassium dihydrogen phosphate, potassium phosphate dibasic, sodium dihydrogen phosphate and disodium hydrogen phosphate, at 20 0 C to 45 0 C for 10 to 40 hours; and
  • the culture medium of the step (a) may further comprise a metal salt, an amino acid or a vitamin.
  • the culture fluid may comprise glucose, soy-peptone A3, yeast extract, K 2 HPO 4 , sodium acetate, diammonium citrate, MgSO 4 and MnSO 4 .
  • Lactobacillus casei were cultured by batch culturing (see Example 1).
  • FPLC protein components having anti-cancer activity were obtained from 3-100 kDa and 10-30 kDa fractions (see Example 2-3).
  • the protein components having anti-cancer activity obtained from the 3-100 kDa fractions were administered to gastric cancer cells (AGS), lung cancer cells (A549), breast cancer cells (MCF-7), ovarian cancer cells (SK-OV-3) and colon cancer cells (LoVo). They showed superior anti-cancer activity of at least 50% in all cancer cells, whereas they did not show cell toxicity in normal cells (see Test Example). The same experiment was cairied out for the protein components having anti-cancer activity obtained from the 10-30 kDa fractions. No cell toxicity for normal cells was observed, and anti-cancer activity was at least 60%.
  • the Lactobacillus casei or the culture fluid of the bacteria may be ef- fectively utilized as an effective component of an anti-cancer composition.
  • the cancer is selected from the group consisting of gastric cancer, lung cancer, bieast cancer, ovarian cancer and colon cancer, although not limited thereto.
  • the anti-cancer composition is a pharmaceutical composition or a food composition, although not limited thereto.
  • the pharmaceutical composition may comprise a pharmaceutically effective amount of the cultuie fluid of Lactobacillus casei singly or in combination with at least one pharmaceutically acceptable cairier, excipient or diluent.
  • the cultuie fluid of Lactobacillus casei according to the piesent invention may be included in the pharmaceutical composition in an amount of about 0.01-99 weight%.
  • the term "pharmaceutically effective amount” refers to an amount which is sufficient to treat or pievent cancer.
  • a pharmaceutically effective amount of the Lactobacillus casei or the cultuie fluid of the bacteria according to the piesent invention is from 0.1 to 100 mg/day/kg body weight, preferably from 1 to 10 mg/ day/kg body weight. IHbwever, the pharmaceutically effective amount may be varied adequately depending on the kind of cancer, severity thereof, age, body weight, physical conditions and sex of the patient, administration route, treatment period, or the like.
  • the term "pharmaceutically acceptable” means that the resulting composition is physiologically tolerable and generally does not induce allergic reactions such as gastroenteric trouble and dizziness or other similar reactions when administrated to human.
  • the pharmaceutical composition may further comprise a cairier, an excipient or a diluent.
  • Examples of the cairier, excipient or diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyirolidone, water, methyl hydroxybenzoate, propyl hy- droxybenzoate, talc, magnesium stearate and mineral oil.
  • the pharmaceutical composition may further comprise a filler, an anticoagulant, a surfactant, a wetting agent, a fragrance, an emulsifier, an antiseptic, or the like.
  • the composition of the present invention may be prepared into formulations that can provide immediate, sustained or controlled release of the effective component after administration to a mammal, according to methods known in the art.
  • the formulations may be in the form of powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterilized injection solution or sterilized powder
  • the pharmaceutical composition according to the present invention may be ad- ministered through various routes, including orally, transdermally, subcutaneously, intravenously and intramuscularly.
  • the administration dose of the effective component may be selected adequately, considering various factors including administration route, age, sex and body weight of the patient, severity of the disease, and the like.
  • composition of the piesent invention may be administered in combination with a compound proven to have the effect of pieventing or treating cancer
  • the cultuie fluid of Lactobacillus casei according to the piesent invention may be used as an effective component of a food composition for pieventing or alleviating cancer.
  • the Lactobacillus casei bacteria used in the piesent invention is food-grade and can be safely taken in without a special purification process for lemoving toxicity. Therefore, the culture fluid of Lactobacillus casei having cancer cell inhibition activity may be added to food to provide anti-cancer effect.
  • the food composition of the present invention encompasses all forms, including functional foods, nutritional supplements, health foods, food additives, and the like.
  • the food composition may be prepared into a variety of forms by the methods known in the ait.
  • the health food may be prepared by making the culture fluid of Lactobacillus casei according to the present invention in the form of tea, juice, drink, granule, capsule or powder
  • the functional food may be prepared by adding the culture fluid of Lactobacillus casei according to the present invention in drinks (including alcoholic beverages), fruits and processed foods thereof (e.g., tinned fruit, bottling, jam, marmalade, etc.), fish, meat and processed foods thereof (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, soba (buckwheat noodle), ramen (instant noodle), spaghetti, macaroni, etc.), fruit juices, drinks, cookies, yeot (Korean traditional
  • the culture fluid of Lactobacillus casei according to the present invention may be prepared into the form of powder or liquid concentrate for use as food additives.
  • the culture fluid of Lactobacillus casei according to the present invention may be included in the food composition in an amount of about from 0.01 to 90 g per 100 g of food.
  • the culture fluid of Lactobacillus casei according to the present invention has superior anti-cancer activity and can be effectively utilized as effective component of an anti-cancer composition.
  • FIG. 1 shows an FPLC iesult of the cultuie fluid according to the piesent invention
  • FIG. 2 shows an FPLC iesult of the 3-100 kDa fractions of the cultuie fluid according to the piesent invention.
  • FIG. 3 shows an FPLC iesult of the 10-30 kDa fractions of the cultuie fluid according to the piesent invention (blue line: protein (measuied as absorbance at 280 nm); brown line: conductivity; ied ellipse: fractions having anti-cancer activity).
  • Example 1 Reparation of cultuie fluid of Lactobacillus casei
  • a cultuie medium (glucose 4.0%, soy-peptone A3 1.5%, yeast extract 1.0%, K 2 HPO 4
  • Lactobacillus casei bacteria 0.1%, sodium acetate 0.1%, diammonium citrate 0.1%, MgSO 4 0.01%, MnSO 4 0.005%
  • the Lactobacillus casei bacteria wae inoculated (initial concentration: 0.5%) and cultuied at 37 0 C for about 13-14 hours. During the culturing, pH was maintained at 5.5-6.5.
  • the cultuie medium was centrifuged at 8000Dg for 20 minutes and passed through a 0.45 IM poie membrane (Millipoie, Bedford, Mass.).
  • Example 2 Isolation and purification of proteins having anti-cancer activity from cultuie fluid of Lactobacillus casei [60] ⁇ 2- 1> Isolation of proteins having anti-cancer activity from cultme fluid of Lactobacillus casei
  • the cultme fluid of Lactobacillus casei bacteria piepaied in Example 1 was pie- tieated using a cation exchange iesin (SK- 104, strong acid, Na + type), an anion ion exchange iesin (PA-412, strong base, Cl " type) and a synthetic ion exchange iesin (HP-20).
  • Ammonium sulfate was added to the pie-tieated cultuie fluid at 4 0 C for 4 hours until the final concentration was 70%, while stiiring.
  • proteins wae pie- cipitated by centrifuging at 4 0 C and 8000Dg for 20 minutes.
  • the piecipitated proteins wae isolated by dissolving with a 50 mM Tris-HCl buffer (pH 7.0).
  • Example 2-1 The proteins isolated in Example 2-1 wae concentrated using ultrafiltration membranes with fractional molecular weights of 3, 30 and 100 kDa.
  • a 250 mL ultrafiltration kit (Milipoie) was used.
  • the fraction flowing through the 100 kDa ultrafiltration membrane was collected.
  • the fraction was further flown through the 3 kDa, 10 kDa and 30 kDa ultrafiltration membranes, and the supanatants wae collected.
  • FPLC fast protein liquid chromatography, Sephadex 75
  • a pH 7.2 solution containing 0.05 M phosphate and 0.15 M sodium chloride was used as buffer and the flow rate was maintained at 0.5 mL/min.
  • Protein components having anti-cancer activity wae obtained from 3-100 kDa and 10-30 kDa fractions.
  • Test Example Anti-cancer activity of purified proteins from cultuie fluid of Lacto- bacillus casei
  • Each 900 j ⁇ of gastric cancer cells (AGS), lung cancer cells (A549), bieast cancer cells (MCF-7), ovarian cancer cells (SK-OV-3) and colon cancer cells (LoVo) was added to a 24-well plate and cultured for 24 hours (37 0 C, 5% CO 2 ). Then, after adding each 100 ⁇ of the proteins isolated and purified in Example 2, culturing was further cairied out for 24 hours (37 0C, 5% CO 2 ). Then, after adding each 100 ⁇ of MTT (0.5 mg/mL) solution, culturing was further cairied out for 4 hours (37 0 C, 5% CO 2 ).
  • cancer cell growth inhibition activity was calculated by the following equation.
  • the iesult is given in Table 1 and Table 2 below. Each fraction was collected by FPLC (Sephadex 75), at 0.5 mL/min and 3 mL/fraction.
  • Inhibition ratio (%) (1-T/QDlOO [74] (T: absorbance of test group, C: absorbance of control group) [75] Table 1 [Table 1] [Table ] Anti-cancer activity of proteins isolated from 3-100 kDa fractions (inhibition ratio, %)
  • fraction 7 As seen in Table 1, of the proteins isolated from the 3-100 kDa fractions, fraction 7 exhibited anti-cancer activity of at least 50% for all cancer cells but did not show toxicity for normal cells. And, as seen in Table 2, of the proteins isolated from the 10-30 kDa fractions, fraction 7 exhibited anti-cancer activity of at least 60% for all cancer cells but did not show toxicity for normal cells. Accordingly, it can be confirmed that the fraction 7 (5-20 kDa) of the 10-30 kDa protein concentrate fractions has anti-cancer activity.
  • the cultuie fluid of Lactobacillus casei according to the piesent invention has superior anti-cancer activity and can be used as an effective component of an anti-cancer composition.
  • the activity of the cultuie fluid of lactic acid bacteria, which has been discarded thus far, is elucidated, it holds a very promising application potential in the industries.

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Abstract

The present invention relates to an anti-cancer composition comprising a culture fluid of Lactobacillus casei spp. as an effective component, more particularly to an anti-cancer composition comprising a culture fluid, which is by culturing Lactobacillus casei in a culture medium containing a carbon source, a nitrogen source, a phosphorus source, and the like and then removing the bacteria as an effective component. The culture fluid of Lactobacillus casei according to the present invention has superior anti-cancer activity and can be used as an effective component of an anti-cancer composition. As the activity of the culture fluid of lactic acid bacteria, which has been discarded thus far, is elucidated, it holds a very promising application potential in the industries.

Description

Description
ANTI-CANCER COMPOSITION COMPRISING A CULTURE FLUID OF LACTOBACILLUS CASEI AS AN EFFECTIVE
COMPONENT
Technical Field
[1] This application claims the priority of Korean Patent Application No.
10-2008-0069407, filed on 17 July, 2008, the disclosure of which is incorporated herein by reference.
[2] The present invention relates to an anti-cancer composition comprising a culture fluid of Lactobacillus casei spp. as an effective component, more particularly to an anti-cancer composition comprising a culture fluid obtained by culturing Lactobacillus casei in a culture medium containing a carbon source, a nitrogen source, a phosphorus source, and the like and then removing the bacteria as an effective component.
[3]
Background Art
[4] Lactic acid bacteria are the bacteria which produce lactic acid as the major metabolic endproduct of carbohydrate fermentation. They are facultative anaerobic bacteria or obligate anaerobic bacteria that proliferate well under oxygen-deficient conditions. The genera that comprise the lactic acid bacteria are Streptococcus, Lactobacillus, Leu- conostoc, Bifidobacterium and Pediococcus. Streptococcui are chain-shaped ho- mofermenting bacteria and are known to inhibit saprophytes or pathogens by fermenting milk and producing lactic acid. Lactobacilli are rod- shaped ho- mofermenting or heterofermenting bacteria fiequently found in the fermentation process of dairy products or vegetables. Leuconostoc is a diplococcus heterofermenting bacteria mainly involved in fermentation of vegetables. Bifidobacteria are obligate anaerobic bacteria which cannot survive in an oxygen-rich environment. They produce l(+)-lactic acid, which can be digested by infants, by fermenting sugars. Finally, Pediococcus is a homofermenting bacteria usually occuiring in pairs or tetrads. They exist in kimchi or preserved food and are involved in fermentation of meat, such as sausage.
[5] The lactic acid bacteria maintain the pH in the intestine acidic, thereby inhibiting the proliferation of harmful bacteria such as E. coli and Clostridium sp. and improving diairhea and constipation. Further, they aid in synthesis of vitamin, have anti-cancer effect, and reduce serum cholesterol. In particular, with specific proteins that can strongly bind the mucosa and epithelial cells of the intestine, the lactic acid bacteria inhibit the growth of harmful bacteria and pievent intestinal infection. Besides, the lactic acid bacteria aie known to facilitate the growth of macrophages, thereby enhancing the recognition ability, antimicrobial ability and so on of the macrophages against harmful bacteria in the intestine, and promote the secretion of immune-related substances, thereby enhancing immunity(Gabriela Perdigon et al., J. of food Protection 53:404-410, 1990; Katsumasa Sato et al., Microbiol. Immunol, 32(7):689-698, 1988). Among them, Lactobacilli are known to produce acidophillin, thereby inhibiting the growth of harmful bacteria, such as Shigella, Salmonella, Staphylococcus, etc., and inhibiting the proliferation of diairhea-causing bacteria and normalizing intestinal microflora, thereby stopping diairhea.
[6] Recently, studies on anti-cancer activity of the lactic acid bacteria are actively underway. According to these researches, Lactobacillus bulgaricus, Lactobacillus acidophilus and Streptococcus thermophilus reduced the occuπence of DMH- induced tumors in rats (Shakelford et al., Nutr. Cancer 5:159-1647, 1983). Dairy products fermented by Lactobacillus bulgaricus inhibited DMH-induced carcinogenesis in rats and DEN (diethylnitrosamine)-induced carcinogenesis of the upper respiratory tract in hamsters (Balansky et al., Cancer Lett., 147:125-137, 1999). A single subcutaneous injection of peptidoglycan isolated from Bifidobacterium infantis (ATCC 15697) inhibited tumors (syngeneic fibrosarcoma, Meth A in Balb/c mice)(Sekine et al., Cancer Res., 45:1300-1307, 1985). Polysaccharide fractions derived from the culture medium of Lactobacillushelveticus were effective in inhibiting tumors (sarcoma 180), and glycoproteins exhibited cell toxicity against cervical cancer cells (Oda et al., Agri. Biol. Chem., 47:1623-1625, 1983; Manjunath et al., Indian Journal Exp. Biol., 27:141-145, 1989). And, macrophage activating substances derived from BCG (Bacillus Calmette-Guerin) inhibited the macrophage tumor cells (Ralph et al., Nature, 249:49-51, 1974; Ralph et al., Cancer Res., 37:546-550, 1977).
[7] But, the aforesaid researches attribute the anti-cancer activity to the peptidoglycan in the cell wall (Fichera, G. A., and Giese, G., Cancer Lett., 85:93-103, 1994). Also, the cell wall component of Enterococcus faecalis is reported to be effective in inhibiting the proliferation of tumor cells (Nomoto, K., Biotherapy, 1:169-177, 1989; Park, SJ. et al., Kor. J. Appl. Microbiol. Biotechnol., 27:8-14, 1999).
[8] However, nothing is known about the anti-cancer activity of the culture fluid of lactic acid bacteria. On the contrary, as researches, developments and utilization with regard to the lactic acid bacteria aie actively underway, a large volume of cultuie fluid of lactic acid bacteria is produced and discarded. Accordingly, if the cultuie fluid of lactic acid bacteria is proven to have such activity, it holds a very promising application potential in the industries.
[9]
Disclosure of Invention Technical Problem
[10] The inventors of the present invention have elucidated that Lactobacillus casei has superior anti-cancer activity. Particularly, they have found through experiments on various cancer cells that the cultuie fluid of the cells, not the cells themselves, outstandingly inhibits the proliferation of the cancer cells and, thus, has superior anticancer activity.
[11] Accordingly, an object of the piesent invention is to provide an anti-cancer composition comprising a cultuie fluid of Lactobacillus casei as an effective component.
[12]
Technical Solution
[13] To attain the object, the piesent invention provides an anti-cancer composition comprising a cultuie fluid of Lactobacillus casei as an effective component.
[14]
[15] Hereunder, the piesent invention is described in further detail.
[16] The anti-cancer composition of the piesent invention comprises a cultuie fluid of
Lactobacillus casei spp. as an effective component.
[17] The Lactobacillus casei used in the piesent invention may be the lactic acid bacteria generally used in the related art. Preferably, it may be Lactobacillus casei ATCC 334, ATCC 393, ATCC 11578, KCTC 3109, KCTC 3189, DSM 20011 or DSM L26 10633.
[18] The culture fluid of Lactobacillus casei may be obtained by culturing the bacteria according to the conventional lactic acid bacteria culturing method and then removing the bacteria.
[19] The culturing of the lactic acid bacteria may be cairied out using adequate medium and adequate culturing condition known to those skilled in the art. Those skilled in the art may easily modify the culturing process, depending on particularly selected bacteria. Various culturing methods are disclosed in many publications [for example, James et al., Biochemical Engineering, Prentice-Hall International Editions]. The culturing methods can be classified into suspension culturing and adherent culturing depending on the mode of cell growth, and into batch culturing, fed-batch culturing and continuous culturing depending on the mode of culturing.
[20] The cultuie medium should adequately satisfy the lequiiements of particular bacteria.
The cultuie medium includes a variety of carbon sources, nitrogen sources and trace element components. Examples of the carbon source include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. These carbon sources may be used singly or in combination. Examples of the nitrogen source include organic nitrogen sources such as peptone, yeast extract, bouillon, malt extract, com steep liquor (CSL) and soybean meal, and inorganic nitrogen sources such as uiea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. These nitrogen sources may be used singly or in combination. The cultuie medium may include potassium di- hydrogen phosphate, potassium phosphate dibasic, sodium dihydrogen phosphate and disodium hydrogen phosphate as phosphorus source. Further, metal salts such as magnesium sulfate or iron sulfate may be included. Besides, amino acids, vitamins, adequate piecursors, and the like may be included. During the culturing, such compounds as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid may be added in an adequate manner to adjust pH. Further, during the culturing, a defoaming agent such as fatty acid polyglycol ester may be used to ieduce foaming. Further, oxygen or oxygen-containing gas (e.g., air) may be injected to maintain an aerobic condition. The culturing temperatuie is usually from 20 0C to 45 0C, pieferably from 25 0C to 40 0C. The culturing may be cairied out for 10 to 40 hours.
[21] After the culturing is completed, centrifugation or filtration may be cairied out to remove the bacteria from the cultuie fluid. This process may be cairied out if necessary at the discretion of those skilled in the art. Here, the removal of the bacteria refers to a substantial removal of the Lactobacillus casei through a commonly used bacteria removal method.
[22] More specifically, the culture fluid of the present invention may be prepared by a method comprising:
[23] (a) culturing Lactobacillus casei in a culture medium comprising a carbon source selected from the group consisting of glucose, sucrose, lactose, fructose(fructose), maltose, starch, cellulose, soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, linoleic acid, glycerol, ethanol and acetic acid; a nitrogen source selected from the group consisting of peptone, yeast extract, bouillon, malt extract, com steep liquor (CSL), soybean meal, uiea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate; and a phosphorus source selected from the group consisting of potassium dihydrogen phosphate, potassium phosphate dibasic, sodium dihydrogen phosphate and disodium hydrogen phosphate, at 20 0C to 45 0C for 10 to 40 hours; and
[24] (b) lemoving the Lactobacillus casei from the culture medium of the step (a).
[25]
[26] The culture medium of the step (a) may further comprise a metal salt, an amino acid or a vitamin.
[27] More preferably, the culture fluid may comprise glucose, soy-peptone A3, yeast extract, K2HPO4, sodium acetate, diammonium citrate, MgSO4 and MnSO4.
[28] In the Example of the present invention, Lactobacillus casei were cultured by batch culturing (see Example 1).
[29] In order to investigate the components of the culture fluid of Lactobacillus casei having anti-cancer activity, the inventors of the present invention pre-tieated the culture fluid using an ion exchange resin, precipitated the protein components using ammonium sulfate, and isolated the proteins by dissolving using a buffer (see Example 2-1).
[30] Thus isolated proteins were concentrated using ultrafiltration membranes with fractional molecular weights of 3, 10, 30 and 100 kDa, into protein fractions of 3-100 kDa and 10-30 kDa (see Example 2-2).
[31] FPLC was cairied out in order to isolate proteins having anti-cancer activity from the concentrate. Protein components having anti-cancer activity were obtained from 3-100 kDa and 10-30 kDa fractions (see Example 2-3).
[32] The protein components having anti-cancer activity obtained from the 3-100 kDa fractions were administered to gastric cancer cells (AGS), lung cancer cells (A549), breast cancer cells (MCF-7), ovarian cancer cells (SK-OV-3) and colon cancer cells (LoVo). They showed superior anti-cancer activity of at least 50% in all cancer cells, whereas they did not show cell toxicity in normal cells (see Test Example). The same experiment was cairied out for the protein components having anti-cancer activity obtained from the 10-30 kDa fractions. No cell toxicity for normal cells was observed, and anti-cancer activity was at least 60%.
[33] Accordingly, the Lactobacillus casei or the culture fluid of the bacteria may be ef- fectively utilized as an effective component of an anti-cancer composition. Referably, the cancer is selected from the group consisting of gastric cancer, lung cancer, bieast cancer, ovarian cancer and colon cancer, although not limited thereto.
[34] Referably, the anti-cancer composition is a pharmaceutical composition or a food composition, although not limited thereto.
[35] The pharmaceutical composition may comprise a pharmaceutically effective amount of the cultuie fluid of Lactobacillus casei singly or in combination with at least one pharmaceutically acceptable cairier, excipient or diluent. Referably, the cultuie fluid of Lactobacillus casei according to the piesent invention may be included in the pharmaceutical composition in an amount of about 0.01-99 weight%.
[36] As used herein, the term "pharmaceutically effective amount" refers to an amount which is sufficient to treat or pievent cancer. A pharmaceutically effective amount of the Lactobacillus casei or the cultuie fluid of the bacteria according to the piesent invention is from 0.1 to 100 mg/day/kg body weight, preferably from 1 to 10 mg/ day/kg body weight. IHbwever, the pharmaceutically effective amount may be varied adequately depending on the kind of cancer, severity thereof, age, body weight, physical conditions and sex of the patient, administration route, treatment period, or the like.
[37] As used herein, the term "pharmaceutically acceptable" means that the resulting composition is physiologically tolerable and generally does not induce allergic reactions such as gastroenteric trouble and dizziness or other similar reactions when administrated to human. The pharmaceutical composition may further comprise a cairier, an excipient or a diluent. Examples of the cairier, excipient or diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyirolidone, water, methyl hydroxybenzoate, propyl hy- droxybenzoate, talc, magnesium stearate and mineral oil.
[38] The pharmaceutical composition may further comprise a filler, an anticoagulant, a surfactant, a wetting agent, a fragrance, an emulsifier, an antiseptic, or the like. Further, the composition of the present invention may be prepared into formulations that can provide immediate, sustained or controlled release of the effective component after administration to a mammal, according to methods known in the art. The formulations may be in the form of powder, granule, tablet, emulsion, syrup, aerosol, soft or hard gelatin capsule, sterilized injection solution or sterilized powder
[39] The pharmaceutical composition according to the present invention may be ad- ministered through various routes, including orally, transdermally, subcutaneously, intravenously and intramuscularly. The administration dose of the effective component may be selected adequately, considering various factors including administration route, age, sex and body weight of the patient, severity of the disease, and the like.
[40] The pharmaceutical composition of the piesent invention may be administered in combination with a compound proven to have the effect of pieventing or treating cancer
[41] Further, the cultuie fluid of Lactobacillus casei according to the piesent invention may be used as an effective component of a food composition for pieventing or alleviating cancer.
[42] The Lactobacillus casei bacteria used in the piesent invention is food-grade and can be safely taken in without a special purification process for lemoving toxicity. Therefore, the culture fluid of Lactobacillus casei having cancer cell inhibition activity may be added to food to provide anti-cancer effect.
[43] The food composition of the present invention encompasses all forms, including functional foods, nutritional supplements, health foods, food additives, and the like. The food composition may be prepared into a variety of forms by the methods known in the ait. For example, the health food may be prepared by making the culture fluid of Lactobacillus casei according to the present invention in the form of tea, juice, drink, granule, capsule or powder And, the functional food may be prepared by adding the culture fluid of Lactobacillus casei according to the present invention in drinks (including alcoholic beverages), fruits and processed foods thereof (e.g., tinned fruit, bottling, jam, marmalade, etc.), fish, meat and processed foods thereof (e.g., ham, sausage, corned beef, etc.), breads and noodles (e.g., udon, soba (buckwheat noodle), ramen (instant noodle), spaghetti, macaroni, etc.), fruit juices, drinks, cookies, yeot (Korean traditional taffy), dairy products (e.g., butter, cheese, etc.), edible vegetable fats and oils, margarines, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauce, etc.), and the like.
[44] Further, the culture fluid of Lactobacillus casei according to the present invention may be prepared into the form of powder or liquid concentrate for use as food additives. Preferably, the culture fluid of Lactobacillus casei according to the present invention may be included in the food composition in an amount of about from 0.01 to 90 g per 100 g of food.
Advantageous Effects
[45] The culture fluid of Lactobacillus casei according to the present invention has superior anti-cancer activity and can be effectively utilized as effective component of an anti-cancer composition. As the activity of the cultme fluid of lactic acid bacteria, which has been discarded thus far, is elucidated, it holds a very promising application potential in the industries.
[46]
Brief Description of the Drawings
[47] The above and other objects, featuies and other advantages of the piesent invention will be moie clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
[48] Fig. 1 shows an FPLC iesult of the cultuie fluid according to the piesent invention;
[49] Fig. 2 shows an FPLC iesult of the 3-100 kDa fractions of the cultuie fluid according to the piesent invention; and
[50] Fig. 3 shows an FPLC iesult of the 10-30 kDa fractions of the cultuie fluid according to the piesent invention (blue line: protein (measuied as absorbance at 280 nm); brown line: conductivity; ied ellipse: fractions having anti-cancer activity).
[51]
Mode for the Invention
[52] The following examples further illustrate piesent invention.
[53] Fbwever, the following examples aie not intended to limit the scope of the piesent invention.
[54]
[55] Example 1 : Reparation of cultuie fluid of Lactobacillus casei
[56] A cultuie medium (glucose 4.0%, soy-peptone A3 1.5%, yeast extract 1.0%, K 2HPO4
0.1%, sodium acetate 0.1%, diammonium citrate 0.1%, MgSO4 0.01%, MnSO4 0.005%) for culturing Lactobacillus casei bacteria (DSM L26 10633) wae piepaied. The Lactobacillus casei bacteria wae inoculated (initial concentration: 0.5%) and cultuied at 37 0C for about 13-14 hours. During the culturing, pH was maintained at 5.5-6.5.
[57] In order to obtain the cultuie fluid only by lemoving the Lactobacillus casei bacteria, the cultuie medium was centrifuged at 8000Dg for 20 minutes and passed through a 0.45 IM poie membrane (Millipoie, Bedford, Mass.).
[58]
[59] Example 2: Isolation and purification of proteins having anti-cancer activity from cultuie fluid of Lactobacillus casei [60] <2- 1> Isolation of proteins having anti-cancer activity from cultme fluid of Lactobacillus casei
[61] The cultme fluid of Lactobacillus casei bacteria piepaied in Example 1 was pie- tieated using a cation exchange iesin (SK- 104, strong acid, Na + type), an anion ion exchange iesin (PA-412, strong base, Cl " type) and a synthetic ion exchange iesin (HP-20). Ammonium sulfate was added to the pie-tieated cultuie fluid at 4 0C for 4 hours until the final concentration was 70%, while stiiring. Then, proteins wae pie- cipitated by centrifuging at 4 0C and 8000Dg for 20 minutes. The piecipitated proteins wae isolated by dissolving with a 50 mM Tris-HCl buffer (pH 7.0).
[62]
[63] <2-2> Concentration of proteins having anti-cancer activity
[64]
[65] The proteins isolated in Example 2-1 wae concentrated using ultrafiltration membranes with fractional molecular weights of 3, 30 and 100 kDa. A 250 mL ultrafiltration kit (Milipoie) was used. First, the fraction flowing through the 100 kDa ultrafiltration membrane was collected. The fraction was further flown through the 3 kDa, 10 kDa and 30 kDa ultrafiltration membranes, and the supanatants wae collected.
[66]
[67] <2-3> Purification of proteins having anti-cancer activity
[68] FPLC (fast protein liquid chromatography, Sephadex 75) was cairied out in order to purify the proteins having anti-cancer activity included in the concentrate obtained in Example 2-2. Specifically, a pH 7.2 solution containing 0.05 M phosphate and 0.15 M sodium chloride was used as buffer and the flow rate was maintained at 0.5 mL/min. The sample amount was 1 mL (2-4 mg/mL) and a Sephadex 75 column (lesolution Mr = 3,000-70,000) was used. Protein components having anti-cancer activity wae obtained from 3-100 kDa and 10-30 kDa fractions.
[69]
[70] Test Example: Anti-cancer activity of purified proteins from cultuie fluid of Lacto- bacillus casei
[71] Normal kidney cells (293) wae used as control group. Each 900 jΛ of gastric cancer cells (AGS), lung cancer cells (A549), bieast cancer cells (MCF-7), ovarian cancer cells (SK-OV-3) and colon cancer cells (LoVo) was added to a 24-well plate and cultured for 24 hours (37 0C, 5% CO2). Then, after adding each 100 ≠ of the proteins isolated and purified in Example 2, culturing was further cairied out for 24 hours (37 0C, 5% CO2). Then, after adding each 100 ≠ of MTT (0.5 mg/mL) solution, culturing was further cairied out for 4 hours (37 0C, 5% CO 2). Then, the supernatant was removed using an aspirator and, the produced formazan was dissolved using 300 μJl of DMSO (dimethyl sulfoxide). Absorbance was measuied at 540 nm using an ELISA reader.
[72] From the absorbance measurement lesult, cancer cell growth inhibition activity was calculated by the following equation. The iesult is given in Table 1 and Table 2 below. Each fraction was collected by FPLC (Sephadex 75), at 0.5 mL/min and 3 mL/fraction.
[73] Inhibition ratio (%) = (1-T/QDlOO [74] (T: absorbance of test group, C: absorbance of control group) [75] Table 1 [Table 1] [Table ] Anti-cancer activity of proteins isolated from 3-100 kDa fractions (inhibition ratio, %)
Figure imgf000012_0001
[76] [77] Table 2
[Table 2]
[Table ]
Anti-cancer activity of proteins isolated from 10-30 kDa fractions (inhibition ratio, %)
Figure imgf000013_0001
[78] [79] As seen in Table 1, of the proteins isolated from the 3-100 kDa fractions, fraction 7 exhibited anti-cancer activity of at least 50% for all cancer cells but did not show toxicity for normal cells. And, as seen in Table 2, of the proteins isolated from the 10-30 kDa fractions, fraction 7 exhibited anti-cancer activity of at least 60% for all cancer cells but did not show toxicity for normal cells. Accordingly, it can be confirmed that the fraction 7 (5-20 kDa) of the 10-30 kDa protein concentrate fractions has anti-cancer activity.
[80]
Industrial Applicability [81] As described above, the cultuie fluid of Lactobacillus casei according to the piesent invention has superior anti-cancer activity and can be used as an effective component of an anti-cancer composition. As the activity of the cultuie fluid of lactic acid bacteria, which has been discarded thus far, is elucidated, it holds a very promising application potential in the industries.
[82] [83] Although the piefened embodiments of the piesent invention have been disclosed for illustrative purposes, those skilled in the art will appieciate that various modifications, additions and substitutions aie possible, without departing from the scope and spirit of the invention as disclosed in the accompanying drawings.

Claims

Claims
[1] An anti-cancer composition comprising a cultme fluid of Lactobacillus casei spp. as an effective component.
[2] The anti-cancer composition as set forth in claim 1, wherein the cancer is at least one selected from the group consisting of gastric cancer, lung cancer, breast cancer, ovarian cancer and colon cancer
[3] The anti-cancer composition as set forth in claim 1, wherein the cultuie fluid is piepaied by a method comprising:
(a) culturing Lactobacillus casei in a cultuie medium comprising a carbon source selected from the group consisting of glucose, sucrose, lactose, fructose(fructose), maltose, starch, cellulose, soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, linoleic acid, glycerol, ethanol and acetic acid; a nitrogen source selected from the group consisting of peptone, yeast extract, bouillon, malt extract, com steep liquor (CSL), soybean meal, uiea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate; and a phosphorus source selected from the group consisting of potassium dihydrogen phosphate, potassium phosphate dibasic, sodium dihydrogen phosphate and disodium hydrogen phosphate, at 20 0C to 45 0C for 10 to 40 hours; and
(b) removing the Lactobacillus casei from the culture medium of the step (a).
[4] The anti-cancer composition as set forth in claim 3, wherein the culture medium further comprises at least one compound selected from the group consisting of metal salt, amino acid and vitamin.
[5] The anti-cancer composition as set forth in claim 1, wherein the culture fluid is prepared by a method comprising:
(a) culturing Lactobacillus casei in a culture medium comprising glucose, soy- peptone A3, yeast extract, K 2HPO4, sodium acetate, diammonium citrate, MgSO4 and MnSO4, at 20 0C to 45 0C for 10 to 40 hours; and
(b) removing the Lactobacillus casei from the culture medium of the step (a).
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