JP2010241945A - Lipopolysaccharide, rice fermentation extract, and rice fermentation extract composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an excellent lipopolysaccharide having a property different from that of the conventional lipopolysaccharide of Pantoea agglomerans of wheat, to provide an extract containing the lipopolysaccharide, and to provide the application product thereof. <P>SOLUTION: The lipopolysaccharide is obtained from a Gram-negative bacterium living together with a rice plant. The lipopolysaccharide has high Limulus activity, low molecular weight of a subband, a different saccharide chain structure, different lipid A structure, different protain content, saccharide content, nucleic acid content and Limulus active substance content as compared with the conventional lipopolysaccharide of Pantoea agglomerans of wheat and has high preventive effect. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  [Technical Field] The present invention relates to a lipopolysaccharide of pantoea-related bacteria symbiotic to rice, a rice fermentation extract containing the lipopolysaccharide, and a rice fermentation extract blend.

  Lipopolysaccharide is a glycolipid that exists in the outer membrane of the cell wall of Gram-negative bacteria. It has the most trace amount of innate immunity activation effect known so far, and it is beneficial if it can be used properly. Biological action can be expected (see, for example, Patent Document 1). The basic structure of lipopolysaccharide is a substance in which a sugar chain (core polysaccharide, O antigen polysaccharide, etc.) is bound to lipid called lipid A. The biological activity of lipopolysaccharide is bound to toll-like receptor-4, which is a lipopolysaccharide receptor on the membrane surface of immune system cells, etc., and a DNA transcription factor activated through an intracellular signal transduction mechanism is transferred into the nucleus To happen. The binding between Toll-like receptor-4 and lipopolysaccharide differs depending on the structure of lipid A and sugar chains constituting the lipopolysaccharide, and thus the biological activity of lipopolysaccharide differs depending on the structure of lipopolysaccharide.

  The structure of lipopolysaccharide is known to vary depending on the microorganism from which it is derived, but the lipid A moiety is highly conserved and the sugar chain moiety is highly diverse. That is, the structure of lipid A is conserved among related species, but the sugar chain structure is different. For example, E. coli strains expressed as O-157 and O-111 are the same E. coli species, but have different sugar chain structures and are different strains. Since the lipid A moiety plays a major role in binding to Toll-like receptor-4, biological activity is considered to be greatly different if the lipid A structure is different.

  In recent years, it has been reported that the gram-negative bacterium Pantoea agglomerans lipopolysaccharide (IP-PA1 (registered trademark)) coexisting with wheat exhibits a potent innate immune stimulating effect by oral and transdermal administration. In an experiment to enhance the production of tumor necrosis factor (TNF-α) using macrophages, IP-PA1 has a 3-fold stronger macrophage activation effect than lipopolysaccharide derived from E. coli. Atopic dermatitis, gastric ulcer, viral infection, acute pain, toxoplasma infection, hyperlipidemia, diabetes, cocaine morphine poisoning, tumor has been shown to have preventive or therapeutic effects, and clinical effects include tumors, herpes, It has been shown to be effective in the treatment of multiple diseases such as pain and atopic dermatitis.

International Publication No. 2005/0300938

A. Satoh et al., "PhysiologicalProperties and Phylogenetic Affiliations of Anaerobic Bacteria Isolated from Roots of Rice Plants Cultivated on a Paddy Field", Anaerobe (2002) 8, pp.233-246

  Based on the above, if there is a novel lipopolysaccharide having a biological activity different from that of wheat pantoea agglomerans lipopolysaccharide (IP-PA1), it is considered that another preventive and therapeutic effect on various diseases can be expected. By the way, Pantoea agglomerans is symbiotic with various edible plants (wheat, rice, potato, shiitake, pear, apple, etc.), and human beings have a long experience of ingesting it. Pantoea agglomerans is known to proliferate in large quantities prior to lactic acid fermentation of fermented rye bread. In other words, Pantoea agglomerans is a gram-negative bacterium with a long dietary experience. Although food experience is required as a new food, there are few gram-negative bacteria such as lactic acid bacteria and Pantoea bacteria, and there is no report of E. coli food experience. Therefore, it is necessary to find out among Pantoea bacteria that has a different character from the lipopolysaccharide of wheat Pantoea agglomerans. In order to make the personality greatly different, it is desirable that the structure of lipid A is different. However, as described above, the structure of lipid A is highly conserved, and it is unlikely that the same species of Pantoea strains with different lipid A exist.

  However, we have eagerly screened pantoea bacteria having lipopolysaccharides with different characteristics from the lipopolysaccharide (IP-PA1) of wheat pantoea agglomerans among pantoea agglomerans isolated from various edible plants, Finally, an excellent lipopolysaccharide having a lipid A structure different from IP-PA1 was found from rice pantoea agglomerans.

  The lipopolysaccharide of the present invention is obtained from a gram-negative bacterium that coexists with rice.

  In addition, the rice fermented extract of the present invention is obtained by fermenting a material derived from a Gramineae plant with a gram-negative bacterium symbiotic to rice and simultaneously culturing the gram-negative bacterium.

  Moreover, the rice fermented extract powder of the present invention is obtained from the above rice fermented extract.

  Moreover, the rice fermented extract blend of the present invention is characterized in that the rice fermented extract or the rice fermented extract powder is blended.

  Moreover, the rice fermented extract blend of the present invention is a pharmaceutical product, food, cosmetics, miscellaneous goods, feed, fertilizer, or daily necessities.

  According to the present invention, it is possible to provide an excellent lipopolysaccharide, an extract containing the lipopolysaccharide, and an application product thereof having a different character from the conventional lipopolysaccharide of wheat pantoea agglomerans.

  Hereinafter, embodiments for carrying out the present invention will be described in detail.

Purification of lipopolysaccharide from rice pantoea agglomerans cells A portion of the colony of pantoea agglomerans isolated from rice (strain A46 (see Non-Patent Document 1)) was scraped and plated on a normal agar medium. The cells were cultured overnight in a thermostatic chamber. One colony was placed in a 3 liter Sakaguchi flask containing 1 liter of sterilized Luria Broth (LB) medium and cultured overnight at 30 degrees with shaking. After culturing, the culture solution was transferred to a centrifuge tube and centrifuged (HITACHI SCR-20B) at 7000 rpm (rpm) to recover wet cells of A46 strain. Purification of lipopolysaccharide from wet cells of A46 strain was performed according to the method of Westphal et al. That is, distilled water was added to 5.4 g of wet cells to make 54 ml (wet cells 100 mg / ml). The bacteria were suspended, and the same volume of 90% phenol was added to this solution, followed by stirring at 65 to 70 degrees for 10 minutes. Thereafter, the liquid was cooled to 4 degrees and centrifuged at 10,000 rpm. Collect the upper water layer in a separate container, add the same amount of distilled water as the recovered water layer to the remaining phenol layer and intermediate layer, and stir again at 65 to 70 degrees for 10 minutes to re-extract the lipopolysaccharide. did. Thereafter, the liquid was cooled to 4 degrees and centrifuged. The second aqueous layer was combined with the first aqueous layer and dialyzed against distilled water to remove phenol. This dialysis internal solution was further treated with DNA-degrading enzyme (DNase I) (50 U / ml) and RNA-degrading enzyme (RNase A) (20 μg / ml), followed by proteolytic enzyme (proteinase K) (100 μg / ml) and phenol. Extraction was performed, and then the aqueous layer was dialyzed to remove phenol. After completion of dialysis, the dialyzed solution was collected and concentrated by ultrafiltration using Microcon YM-100 (Millipore). This concentrate was used as a purified lipopolysaccharide (LPSp46) solution of A46 rice pantoea agglomerans. This solution was freeze-dried and the dry weight was measured. The dry weight of purified lipopolysaccharide LPSp46 was determined to be 42 mg. Seiko Chemical's Endspecy was used as a kit for measuring Limulus activity that specifically reacts with lipopolysaccharide. As the standard lipopolysaccharide, a standard lipopolysaccharide from Seikagaku Corporation was used. Based on the measured Limulus activity value, a converted value as IP-PA1 was calculated.

  Table 1 shows the sugar content of LPSp46 and IP-PA1 and the limulus activity per real weight (the activity of inducing aggregation of horseshoe crab blood by LPS). Since the converted value of IP-PA1 based on rimlas activity was 120 mg, the dry weight was 42 mg, so the specific activity based on IP-PA1 of rimlas activity per unit weight was 2.85 (120/42) It became. That is, it was revealed that lipopolysaccharide LPSp46 has a specific activity about 3 times higher than IP-PA1.

Measurement of the molecular weight of lipopolysaccharide LPSp46 from rice pantoea agglomerans LPSp46 (5 μg / lane) and IP-PA1 (5 μg / lane) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15% concentration gel) using tricine The molecular weight of was analyzed. After electrophoresis, silver staining was performed with a sylveststein (Cat No30642-41, Nacalai Tesque, Japan) kit for the purpose of visualizing the molecules of lipopolysaccharide. The molecular weight size of each stained band was read from a molecular weight marker (Prestained Protein Marker, NEW ENGLAND BioLabs).

  The results are shown in Table 2. Lipopolysaccharide LPSp46 mainly had a molecular weight of 5000 and was equivalent to IP-PA1, but the minor band on the polymer side had a lower molecular weight than IP-PA1.

Examination of cross-reactivity of purified lipopolysaccharide LPSp46 to IP-PA1 monoclonal antibody
Since the A46 strain belongs to the genus Pantoea, there is a possibility that cross-reactivity is observed with an antibody that specifically reacts with IP-PA1. Thus, the cross-reactivity of lipopolysaccharide LPSp46 was examined using six monoclonal antibodies that specifically react with IP-PA1. Three types of lipopolysaccharides, lipopolysaccharide LPSp46, IP-PA1 and E. coli O128 lipopolysaccharide, were added to a 96-well immunoplate at a concentration of 10 μg / ml at 0.05 ml / well and left overnight at 4 ° C. Then, 0.05% polyoxyethylene (20) sorbitan monolaurate (manufactured by Wako Pure Chemical Industries, Tween 20 equivalent) in phosphate buffered saline (PBS) (pH 7.3 to 7.7, manufactured by Nissui Pharmaceutical) ) Was added 3 times with a solution (PBS-T) added, and 3% bovine serum albumin was added and left at room temperature for 1 hour to prevent non-specific adsorption of antibodies and other proteins. Then, after washing 3 times with PBS-T, 0.05 ml of a hybridoma culture supernatant containing monoclonal antibodies specifically reacting with 6 types of IP-PA1 was placed in the well and left at room temperature for 1 hour. Next, each well was washed 5 times with PBS-T, and alkaline phosphatase-conjugated anti-mouse IgG, M, A immunoglobulin antibodies (manufactured by Sigma) diluted with 1% bovine serum albumin were added to 0.05 ml / well at room temperature. And left for 1 hour. Thereafter, the plate was washed 5 times with PBS-T, and a solution of p-nitrophenyl phosphate disodium (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in a substrate buffer so as to be 1 mg / ml was added at 0.1 ml / well, Then, the reaction was stopped by adding 0.05 ml / well of 2N aqueous sodium hydroxide solution and mixing with a plate mixer, and the absorbance at 415 nm was measured with a microplate reader. The results are shown in Table 3. As in the case of the purified lipopolysaccharide of E. coli O128 in which no cross-reactivity was observed, no color development was observed, and no cross-reactivity of lipopolysaccharide LPSp46 was observed against monoclonal antibodies against six types of IP-PA1. That is, it was revealed that lipopolysaccharide LPSp46 has a sugar chain structure different from that of IP-PA1.

Measurement of immunostimulatory effect of purified lipopolysaccharide LPSp46 Nitric oxide (NO) production from cells after addition of lipopolysaccharide to RAW264.7, a cultured cell line of mouse macrophages The concentration in the culture solution was measured as an index. RAW264.7 was purchased from ATCC (No.TIB-71). As controls, E. coli O128 lipopolysaccharide and IP-PA1 were used.

RAW264.7 cells were collected from the culture flask by pipetting, and the cell concentration was adjusted to 8 × 10 ⁵ cells / ml with the culture solution (RPMI1640 medium containing 10% fetal bovine serum, kanamycin 50 μg / ml, ampicillin 60 μg / ml). did. 100 μl of the cell suspension (8 × 10 ⁴ cells / 100 μl) was transferred to each well of a 96-well flat bottom plate and used for the test 6 hours after the cells were almost attached. Lipopolysaccharide LPSp46 was adjusted to 4000 ng / ml in terms of IP-PA1 lipopolysaccharide concentration. Further, serial dilution in 5 steps was performed 10 times each. Prepare each of the diluted solutions further diluted 2-fold with the culture solution and each diluted solution further diluted 2-fold with 40 μg / ml polymyxin B-containing culture solution. It was a product. At the same time, IP-PA1 was also tested. 100 μl of each sample was added to each well of a 96-well flat bottom plate to which cells had been added in advance. The cells were cultured for 20 hours at 37 ° C. in a 5% carbon dioxide incubator, and after completion of the culture, 50 μl of the supernatant was collected in another 96-well plate. The amount of nitrous acid which is a metabolite of nitric oxide in the culture solution was measured using a Gries reagent according to a conventional method.

  Table 4 shows the measurement results. Lipopolysaccharide LPSp46 was shown to induce nitric oxide from RAW264.7 cells at concentrations of 1 ng / ml (limulus activity) and higher. Lipopolysaccharide LPSp46 (based on Limulus activity) was found to show a dose-dependent response approximately equivalent to IP-PA1 measured simultaneously.

  The antibiotic polymyxin B is a cyclic polypeptide and has a high affinity for the lipid A part of lipopolysaccharide. It is known that the bioactivity is strongly suppressed because it binds well to E. coli and IP-PA1. Therefore, lipopolysaccharide LPSp46 was treated with polymyxin B, and nitric oxide production ability was measured.

  The results are shown in Table 4. In IP-PA1, it was confirmed that the NO inducing ability was reduced to about 1/1000 by pretreatment with polymyxin B. In contrast, when lipopolysaccharide LPSp46 was pretreated with polymyxin B, only a reduction of about 1/5 was observed. From the above, it was revealed that the lipid A structure of lipopolysaccharide LPSp46 is different from IP-PA1.

a)IP-PA1は重量濃度で示した。リポ多糖LPSp46はリムラス活性より求めたIP-PA1としての換算重量で示した、b)PolyB−：ポリミキシンＢとの前処理なし、PolyB+：ポリミキシンBとの前処理あり、c)各測定値は４例の平均±標準偏差

a) IP-PA1 is shown by weight concentration. Lipopolysaccharide LPSp46 was expressed as a converted weight as IP-PA1 determined from Limulus activity, b) PolyB-: no pretreatment with polymyxin B, PolyB +: pretreatment with polymyxin B, c) each measured value was 4 Mean ± standard deviation of examples

Production of rice bran fermented extract using pantoea agglomerans of rice 5 ml of distilled water was suspended in 0.5 g of rice bran, and 0.1 ml of the supernatant was added to Luria broth agar and cultured at 37 ° C. overnight. Colonies found on the agar medium were isolated, bacteria were identified by a conventional method, rice pantoea agglomerans was isolated, suspended in 50% glycerol solution, and stored in a freezer. A part of this stock was taken up on Luria broth agar medium and allowed to stand at 37 ° C. to produce rice pantoea agglomerans independent colonies.

  50 g of rice bran was placed in a 2 liter Erlenmeyer flask and purified water was added to make a total volume of 1000 ml. This was similarly autoclaved. Each of the prepared solutions and the like was put in a 3 liter Sakaguchi flask aseptically sterilized in the amount shown in Table 1 to prepare a rice bran medium (A). To 10 ml of rice bran medium prepared with the same composition in advance, add one Pantoea agglomerans colony isolated from rice bran and gently stir at 37 ° C. overnight (12-15 hours), fermented, An inoculum for rice bran fermentation was prepared (B).

  The whole amount of (B) was added to (A) and fermented for 20 to 30 hours while stirring at 37 ° C. This rice bran fermentation solution was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 5000 rpm, 20 minutes, 4 ° C.), and the precipitate was collected. The precipitate was suspended by adding a phosphate buffer solution to a total volume of 100 ml, transferred in 33 ml portions to a 50 ml centrifuge tube, and extracted by heating in a boiling water bath for 30 minutes. After completion of the heating, the mixture was cooled to room temperature and centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 10000 rpm, 20 minutes, 20 ° C.). After centrifugation, 82 ml of pale yellow supernatant was decanted and collected in another container.

  To 80 ml of this supernatant, 8.9 ml of 5 molar sodium chloride solution was added. When 178 ml of ethanol was added thereto, white turbidity was produced. This was left overnight in a freezer (−90 ° C.), and then the liquid was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 10000 rpm, 20 minutes, 4 ° C.). The supernatant was removed to obtain a precipitate. 10 ml of 70% ethanol cooled to the precipitate was added and suspended, and then the liquid was centrifuged (Hitachi, high-speed cooling centrifuge SCR-20B, 10000 rpm, 20 minutes, 20 ° C.) to wash the precipitate. The precipitate was air-dried and dissolved in distilled water to obtain 11 ml of rice bran fermented extract solution. The weight was transferred to a 1.5 ml plastic tube that weighed 0.3 ml of the solution in advance, freeze-dried, and the weight was measured.

  Three rice bran fermented extracts were produced independently by the same method, and the amount of protein in each sample was measured using protein quantification BSA by Bradford method as a standard protein. The measurement results are shown in Table 5. For the rice bran fermented extract, the content per gram of the weight obtained by drying the extract obtained above was expressed in mg. The sugar content was measured by the phenol-sulfuric acid method using glucose as a standard sugar. The nucleic acid content was determined by measuring absorbance at 210 to 340 nm of a sample diluted 500 times, and calculating the maximum content as 50 μg per absorbance of 1 nm of DNA and the value obtained by subtracting the absorbance of 320 nm from the absorbance of 260 nm. As for the amount of Limulus active substance, Seikagaku Corporation CSE-L was used as a standard Limulus active substance. The measurement results are shown in Table 5. Each value in lipopolysaccharide IP-PA1 was shown as a control. From Table 5, it is clear that the dried rice bran fermented extract is different from the lipopolysaccharide of wheat pantoea agglomerans in terms of protein content, sugar content, nucleic acid content, and amount of limulus active substance.

Infection control effect of rice bran fermented extract Rice bran fermented extract or wheat fermented extract was diluted in water, sprayed on cultured fish feed, and dried. As a control, only water was sprayed on the feed. Each of the 20 diets with an average weight of 20 g was fed daily with 7% of the body weight for 7 days. In addition, it prepared so that lipopolysaccharide LPSp46 or IP-PA1 might be 10 microgram per kg body weight. Erythronas hydrophila was inoculated intraperitoneally with 1 × 10 ⁷ viable bacteria to the tiger moth that had been fed for 7 days. Survival of the swordfish 10 days after the infection operation was observed. In the carp without added fermented wheat extract, all carp died by the 6th day. In the group administered with the fermented wheat extract-containing feed, the survival rate was 30% on the 10th day of infection. The group administered with rice bran fermented extract showed an survival rate of 80%. Rice bran fermented extract was found to have a higher preventive effect than wheat fermented extract.

In addition, this invention is not limited to the said Example.

Claims (5)

  A lipopolysaccharide obtained from a gram-negative bacterium symbiotic to rice.   A fermented rice extract obtained by fermenting a material derived from a Gramineae plant with a gram-negative bacterium symbiotic to rice and simultaneously culturing the gram-negative bacterium.   A rice fermented extract powder obtained from the rice fermented extract according to claim 2.   A rice fermented extract blend comprising the rice fermented extract according to claim 2 or the rice fermented extract powder according to claim 3. The rice fermented extract composition according to claim 4, wherein the fermented rice extract composition is a pharmaceutical, food, cosmetics, miscellaneous goods, feed, fertilizer, or daily necessities.