TWI243649B - Fermented and concentrated beverage derived from microorganism having disinfection/infection protection action on vancomycin-resistant enterococcus - Google Patents

Abstract

To obtain a concentrated solution of filtrate of a cultured solution prepared by cocultivation of a specific combination of plural kinds of useful microorganisms selected from Bacillus, yeast and lactic acid bacteria. Since the culture product of the microorganism thus obtained has disinfection/infection protection action at least on vancomycin-resistant Enterococcus and inhibits the growth of the Enterococcus, an infectious disease with the bacterium can be improved or prevented by drinking the concentrated solution.

Description

1243649 A7 B7 Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs 5. Description of the Invention (4) [Table 1] Probiotics used in the manufacture of concentrated concoctions 1 Bac. Bulgaricus No. 1 Bulgarian bacillus 2 // 2 // 3 Bac. Koernchen No.l 4/2 // 5 // 3 // 6 // 4 // 7 Bac. Acidophillus No.l 8 // 2 // 9 // 3 // 10 // 4 // 11 // 5 // 12 // 6 // 13 Micrococcus Lactis No.l lactococcus 14 // 2 // 15 // 3 // 16 // 4 // 17 Yeast No.l yeast 18 // 2 // 19 // 3 // 20 // 4 // a ^ --------- r --- tr --------- ^ w_w. (Please read the note on the back first Please fill in this page again for this matter) This paper size is in accordance with Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1243649 A7 __ B7 V. Description of the invention (6) [Table 3] Analysis results of concentrated feedstock Analytical method. Protein 5.6% 1 Kiyoda nitrogen method phosphorus 304mg / 100g vanadium salt molybdic acid spectrophotometry iron 1.72mg / 100g phenanthroline spectrophotometry calcium 1 65mg / l OOg potassium permanganate capacity method thiamine (Vitamin Bi) 0.03mg / l OOg 2 High Liquid Chromatography Riboflavin (Vitamin B 2) 0.1 2mg / l OOg High Speed Liquid Chromatography Total Sugar 11.9% 3 Sommer's Pressure Test Sugar Method Titration Acidity 87.8 4 pH 3.8 Glass Electrode Method ---------- (Please read the notes on the back before filling this page).--丨 · Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Note 1: Nitrogen, protein conversion number; 6.25 Note 2: In terms of thiamine hydrochloride Note 3: Glucose conversion, hydrolytic conditions; 2.3% hydrochloric acid, in a boiling bath, 2.5 hours Note 4: The number of ml of 1 n alkali titration used to neutralize the test product is' this In the concentrated concoction of the invention, in addition to amino acids other than essential amino acids such as threonine, valine, lysine, and leucine, it is possible to confirm that approximately 2 0 kinds of amino acids. In addition, the organic compounds contained in the concentrated fodder of the present invention (21 (yi \ gage A4 S) § quasi-M · home country, moderate size paper, centimeter, paper 97

HO v, LI 'X-Page printed by the Consumers' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs, Ministry of Economic Affairs, printed 1243649 A7 —_ B7 V. Description of the invention (7) Except amino acids and sugars), and separated and dissolved in the ethyl acetate layer Material and its structural formula. In addition, the extraction of organic compounds uses the liquid-liquid extraction method of the reverse extraction method, and the liquid-liquid extraction method that changes its liquid properties is used to separate the substances dissolved in the ethyl acetate layer. The method for extracting organic compounds (other than amino acids and sugars) from the concentrated feed of the present invention is shown in FIG. 1 if it is illustrated. Therefore, the substances dissolved in the ethyl acetate layer are separated into the following structural formulae and the names are as follows. Compound A (daidzein)

Compound A Compound A of the above structural formula is obtained by isolation and purification of a neutral portion, and can be defined as a glycidin obtained from a soybean isoflavone glycoside daidzein. Compound B (2,3,5-trihydroxybenzaldehyde), * ------------------ ^ WW. (Please read the note on the back first? Matters before filling in this page}

2,3,5 —trihydroxybenzaldehyde This paper is sized to the Chinese National Standard (CNS) A4 (210 X 297 mm) -10- 1243649 A7 B7 V. Description of the invention (8) Compound B of the above structural formula is medium The sexual part is obtained by isolation and purification, which can be defined as a 2 aldehyde having 3 hydroxyl groups. A novel substance not described in the literature 5-trihydroxybenzoyl compound C (2-sugar acid)

Compound C 2 -sugar acid The compound C of the above structural formula is obtained by isolation and purification of an acidic moiety, and can be defined as a 2-sugar acid. Compound D (4-hydroxy-3-methoxybenzoic acid)

Compound D

0H

〇CH3 COOH 4 -hydroxyl 3 -methoxybenzoic acid II —Γ I 11-- I --- ^ -----order--------- wew. (Please read the precautions on the back first (Please fill in this page again) The compound D printed by the above-mentioned structural formula printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs is obtained by isolating and purifying the acidic part, which can be defined as vanillic acid. This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm) -11-1243649 A7 B7 V. Description of the invention (9 Compound (E) (4-hydroxy-3,5-dimethylbenzoic acid)

Compound E 0H h3〇〇, / ^^ 〇ch3

COOH 4 -hydroxy-3 -dimethylbenzoic acid Compound E of the above structural formula is obtained by isolating and purifying the acidic part, and can be defined as 4 -hydroxy-3,5-dimethylbenzoic acid. The present compound F is a known substance and it is described in the literature that it is extracted from plants. ---- * 1 —-----—— (Please read the precautions on the back before filling this page) Compound (F) (2-methyl-4-Cyclo-4H-D Biran-4- ketone)

Compound F 〇 Order H0, printed by H3c / (2-methyl-4-hydroxy-4H-pyran-4-one) in the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economy 2. The purified product can be defined as 2-methyl-1, 4-hydroxy, 4H-D, bis-4-one. Among them, it was confirmed that the concentrated tincture of the present invention has a bactericidal and infection-preventing effect on vancomycin-resistant enterococci. The test methods, etc. are explained in detail with the data. [Test method for vancomycin-resistant enterococci (VRE) (of which the size of the paper is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) -12- 1243649 A7 B7 Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs Printed 5. Description of the invention (10))] The susceptibility test plate of the concentrated condiment according to the present invention is added at an arbitrary concentration, and the bacterial solution of vancomycin-resistant enterococcus (VRE) is smeared and cultured. The concentration was determined as the minimum development-prevention concentration against vancomycin-resistant enterococci (VRE). Here, the susceptibility measurement plate, bacterial solution, and application / culture are described below. (Sensitivity measurement plate) The sterilized condensed material of the present invention was sequentially diluted twice with sterilized pure water, and prepared to be diluted twice. Secondly, the sterilized and dissolved susceptibility measurement medium [Heart Infusion Agar (DIFCO LABORATORIES INCORPORATED)] maintained at 50 to 60 ° C is added with an arbitrary amount of the concentrated condiment according to the present invention and the aforementioned diluted series solution, and mixed well. Then, each was poured into a petri dish to solidify it. This is a plate for susceptibility measurement of this test. 0 (preparation of inoculum bacterial solution) Subcultured test bacteria [E. n. Occ. Faecium NCTC 12204 (VRE)] was inoculated to the growth medium [Heart Infusion Agar (DIFCO LABORATORIES INCORPORATED)], after incubating at 35 ° C for 18 to 20 hours, dilute with the aforementioned bacterial growth medium to a bacterial count of about 106 CFU / m1. The size of liquid paper is applicable to China National Standard (CNS) A4 (210 X 297 mm) 13- -------------- r --- tr --------- ^ (Please read the precautions on the back before filling out this page) 1243649 A7 ____B7 V. Description of the invention (11) (Smearing and cultivating) Use a nickel-chromium heat-resistant alloy coil (inner diameter about 1 in m) on the plate for sensitivity measurement Smear the inoculum for inoculation with a line of about 1 to 2 c HL, and incubate at 3 5 ° C for 18 to 20 hours. This is called smearing and culturing in this test. After this incubation, the minimum concentration that prevented development was determined. The results are shown in Table 4. [Table 4], --------— (Please read the note on the back? Matters before filling out this page) The minimum development prevention concentration (VRE) of vancomycin-resistant enterococci of the concentrated feed of the present invention (M IC) Test bacteria M IC (W / V%) VRE 2.5 Order printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Printed in Table 4 It can be confirmed clearly that the use of the concentrated feed of the present invention can prevent vancomycin-resistant enterococci Grow, at least have a bactericidal effect on vancomycin-resistant enterococci. And when there is no bactericidal effect on vancomycin-resistant enterococci, its minimum development prevention concentration (w / V%) is set to 100, and it is known that the concentrated tincture of the invention does have a bactericidal effect on vancomycin-resistant enterococci Test method for vancomycin-resistant enterococcus (VRE) (2 of which are concentrated condensate of the present invention and a culture medium as a control group 10) The paper size is applicable to the Chinese National Standard (CNS) A4 specification (21 × 297 mm)- 14- Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 1243649 A7 V. Description of the invention (12) Pass the sterilized L-shaped tube and add 0 · iml to prepare 1080 CFu 'm 1 of vancomycin-resistant enterococcus (V r E) solution was shaken at room temperature and then sampled over time (0, 60 minutes, 120 minutes, and 24 hours) to calculate the number of bacteria. The results are shown in Table 5. In addition, for this test strain, enterococcus K u 1 8 56 was used. [Table 5] The bactericidal effect of Kul 856 strain on the intestinal bacteria was tested under the effect of the test substance name and the number of three bacteria (CFU / ml) 0 60 minutes 1 2 0 minutes 24 hours The concentrated seasoning of the present invention 1.5 X 1 06 2.6 X 106 1.0 X 104 Medium below detection limit 1.2 X 1〇6 1.6 X 106 3.6 X 1〇6 106 < Saline solution 1.1 X 106 1.2 X 106 1.5 X 106 0 X 1.4 In the concentrated tincture of the present invention, for Enterococcus, it is 1.5 × 10 6 CFU / m 1 at 0 hours, and it becomes below the detectable limit after 24 hours, showing resistance to at least vancomycin. Enterococci has a bactericidal and even antibacterial effect. As a result, it was found that the concentrated condiment of the present invention has a significant bactericidal effect on vancomycin-resistant enterococci (VRE). And, as shown in Table 5, the antibacterial effect of the concentrated condensate of the present invention is not It is an immediate bactericidal effect that can be seen with antibiotics or synthetic antibacterial agents, which is a type of slow action, so it can be considered to have no side effects caused by antibiotics. This paper size applies to Chinese National Standard (CNS) A4 (210 X 297) (Mm) -15- I I ----- 11 II I --- I — Order --------- (Please read the notes on the back before filling this page) 1243649 Δ7 Α7 _____ Β7 ___ V. Description of the invention (13) [Test on vancomycin-resistant enterococci (VR E) Method (wherein 3) death] Next, the present invention is concentrated Yin materials and medium as control, continuous oral administration to the rats, vancomycin resistant enterococci (V R Ε) administered in the abdominal cavity was observed in mice. The test method is described in more detail below. (Preparation of vancomycin-resistant enterococci (VR E)) After incubating in brain heart infusion agar culture medium at 37 ° C for 24 hours, inoculate 10 mg of ring-collected bacteria as standard, and suspend it in sterilization In physiological saline, a concentration of 109 CFU / m 1 was prepared. This bacterial solution was diluted ten times to prepare 10 and 106 CFU / ml with a 10% mucin solution. (Experimental Animal) Printed by the Consumer Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs 6-week-old B A L B / c female mice (S P F) were used. The test animals were housed in polycarbonate cages (2 4 X 1 7 X 1 2 cm) for rats, each containing 5 animals. The room temperature was set to 2 3 ± 2 ° C, and the relative humidity was 5 5 ± 1 5%. Rearing room. Free ingestion of feed [solid feed for rats and rats; rab 〇 M R s t ock, Japan National Agricultural Products Corporation]. Sprinkle water (tap water) in a water bottle to make it absorb water. (Administration of test substance and vancomycin-resistant enterococcus (V r E) -16- '---------- r --- order · (Please read the precautions on the back before filling (This page) This paper size is in accordance with Chinese National Standard (CNS) A4 (210 X 297 mm). Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. 1243649 A7 _B7_____ V. Description of the invention (14) Law) After the week's preliminary rearing, use a detector to administer 0,3 m 1 of the test substance once a day for 5 consecutive days, stop the administration for 2 days (Saturday, Sunday), and perform another 2 consecutive days. Vote for. 200 mg / kg endoxan was administered to the abdominal cavity on the 5th day from the administration of the test substance. The vancomycin-resistant enterococcus (VRE) prepared as described above was administered to the abdominal cavity in an amount of 0.5 m / mouse 4 hours after the last test substance was administered. In addition, the administration state of the test substance, cancer nemesis, and vancomycin-resistant enterococcus (VRE) is shown in the graph below. The time of administration is indicated by the arrow. (Administration of test substance) ^ 1 Ί Ί j 1 Ί 123456789 days (administration of endoxan) (administration of VRE) (group division) group 1: vancomycin-resistant enterococcus (VRE) Group 2: Culture medium + vancomycin-resistant enterococcus (v RE) Brother group 3 • Concentrated congee of the present invention + vancomycin-resistant enterococcus (VRE) This paper size applies Chinese National Standard (CNS) A4 specifications (210 X 297 Public love) -17- --II ΙΓ -----· II (please read the precautions on the back before filling this page). —Line · 1243649 A7 V. Description of the Invention (15) (Observation) (Please read first Note on the back, please fill in this page again) Observe the life and death of rats every day (except Saturday and Sunday). The results of this type 5 test are shown in Table 6. [Table 6] Infection defense against Enterococcus ku 1 856 phase i Strain name (CFU / ml) Test substance (-) Concentrated condiment of basic invention E. feacalis Kul856 107 4/5 (80) 6 / 6 (100) 5/6 (83) E. feacalis Ku 1 856 106 5/5 (100) 5/5 (100) 2/5 (40) As shown in Table 6, for the administration of 1 0 7 CFU / m 1 Although one case of vancomycin-resistant enterococcus (VRE) of the concentrated concoction and the non-administrative group of the present invention showed a survival phenomenon, the administration group of the culture medium all died. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. On the other hand, for vancomycin-resistant enterococci (VRE) administered with 1 06C FU / m 1, all deaths were confirmed in the non-administration group and the culture group administration group ( (100% mortality rate), but the concentrated feed administration group of the present invention was found to have only 2 cases of deaths and a low mortality rate of 40%. As a result, the concentrated tincture of the present invention was found to have a significant infection defense effect on vancomycin-resistant enterococci (VRE). As can be seen from the above results, the concentrated tincture of the present invention shows not only bactericidal and antibacterial effects and infections against vancomycin-resistant enterococci (VRE). 18- This paper is in accordance with China National Standard (CNS) A4 (210 X 297) Centimeter) 1243649

V. Description of Invention (16) Defensive effect. On the other hand, no toxicity is shown in the concentrated tartar of the present invention. The test methods and test results are shown together with the data. (Toxicity test) For the concentrated feed of the present invention, the acute oral toxicity of rats was investigated using the 0ECD chemical substance toxicity test indicator (19 87) as a guideline (preparation of test solution). The concentrated feed of the present invention was diluted with pure water After 5 times, use 0 (test animal) as test water for about 1 week and 4 weeks. I c R-type male and female mice (Yamoto SLC Co., Ltd.) are prepared for rearing. After confirming that there is no abnormality in normal conditions, use it in In the test. The test animals were housed in polycarbonate cages of 5 horses each. The room temperature was set at 2 3 soil 2 ° C and the lighting time was 12 hours / day. Feed [solid feed for rats and rats; RaboMRstock, Nippon Kogyo Kogyo Co., Ltd.] and water (tap water) for free intake. (Test method) A test group and a control group were each used 10 male and female. This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm) -19- (Please read the precautions on the back before filling this page) --Line · Printed by the Consumer Cooperative of Intellectual Property Bureau of the Ministry of Economic Affairs A7 1243649 _B7 V. Description of the invention (17) (Please read the precautions on the back before filling out this page) The animals should be fasted for about 4 hours before administration. After measuring their weight, the male and female of the test group simultaneously used the dose of 4 · 4 m L / K g as the sample administration amount. The concentrated material of the present invention was forcibly administered orally by a gastric detector. For the control group, the same administration was performed using 0.8 m L of males and females using 0.7 m L of pure water. Fourteen days is the observation period, and the administration day is several days. From the second day, it is observed once a day. Body weights were measured on the 7th day and the 14th day after administration, and a t-test was used to make a significant level of 5% for comparison between groups. The animals were dissected at the end of the observation period. The test results and inspection results are shown below. (Death cases and mortality) During the observation period, neither male nor female was considered dead. Line (General condition) The males in the test group showed no abnormalities during the observation period. In the female, 2 cases were found to produce sounds due to contact stimuli within 1 hour after administration, but returned to normal within 5 hours, and there were no abnormalities thereafter. Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs. The control group showed no abnormalities during the observation period. (Change in body weight) The body weight was measured on the 7th day and the 14th day after the administration. There was no difference in weight gain between the male and female groups. The change in body weight for the male group is shown in Table 7. In addition, for the body of the female group, the Chinese national standard (CNS) A4 specification (210 X 297 mm) is applicable to the paper size. -20- ~ " 1243649 A7 —____ B7 _ V. Description of the invention (M) The heavy changes are shown in Table 8. As shown. The weights shown in Tables 7 and 8 are expressed in terms of the mean jitter L standard deviation (unit: g). The number of animals is shown in parentheses. [Table 7] Weight change > Chemical (male) administration group administration before administration (after) 7 14 Test group 30.6 + 1.1 (10) 35.5 + 1.6 (10) 37.9 ± 1.8 (10) _ _Control group "30.5 + 1.1 (1〇) 35.4 + 2.0 (10) 38.2 + 1.8 (10) [Table 8] Weight change (_) After group administration and after administration (say) 7 14 ^ Test group 26.0 soil 0.9 (10) 29.1 + 1.7 (10) 31.1 + 2.4 (10) Control group 25.9 soil 1.5 (10) 28.1 ± 2.2 (10) 31.2 ± 1.7 (10) (Please read the note on the back? Matters before (Fill in this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs (anatomical examination results) After the observation period, no abnormalities of the main internal organs were observed in the males and females of all the test animals. (Examination) A single oral administration of male and female mice using the concentrated feed of the present invention at a dose of 4 · 4 m L / K g did not result in any deaths, and no Chinese paper standard (CNS) was applied to this paper size at the time of dissection. ) A4 size (210 X 297 mm) _ 21-A7 1243649 --- B7___15. Description of the invention (20) Soymilk obtained from beans (produced by Iao) is better. In the concentrated mash according to the present invention, after the end of the culture, the bacteria are heated to kill the stomach to remove the bacteria, and the filtrate of the culture liquid in which the bacteria are separated is preferably concentrated to about 15 minutes to 0. Next, an example of the method for producing the concentrated tartar of the present invention * ° As one example, Patent No. 196 2 5 12 (Japanese Patent No. 6-95914) can be mentioned. (1) Step 1 (manufacturing of special agar culture medium) The purified natural agar is added with beef, kelp, and the broth obtained by boiling in distilled water, salt and beneficial bacteria culture liquid to produce a special agar culture medium. (2) The second step (pure culture of beneficial bacteria) Transplant various beneficial bacteria into the special agar culture medium produced in the first step, put this in a thermostat and incubate at 39 ° C for 4 8 to 50. hour. (3) Step 3 (manufacturing of special soymilk culture medium) Remove soybean fat, add distilled water and boil for 70 to 110 minutes, filter the soymilk filtrate, add salt, cotton sugar, and beneficial bacteria culture solution to obtain special soymilk Culture medium. (4) The fourth step (pure culture of special soymilk culture medium of beneficial bacteria) The various beneficial bacteria purely cultured in the second step are transplanted to the special soymilk culture medium obtained in the third step, and placed in a thermostat at 39 ° C Under training 4 8 to ----_------- (Please read the notes on the back before filling in this page) Order · Thread · Printed by the Intellectual Property Bureau of the Ministry of Economic Affairs Employee Cooperatives Paper size applicable to China Standard (CNS) A4 size (210 X 297 mm) -23-

Claims (1)

1243649 A8 B8 C8 D8 Γ Ql Application for patent scope No. 89 1 1 277 No. 1 patent application Chinese application for patent scope amendment Amendment on December 18, 1991 1. A bactericidal and defense effect against vancomycin-resistant enterococcus The fermented fermented condensate is characterized by a condensed culture filtrate obtained by symbiotically cultivating five kinds of beneficial bacteria of B. californicus, granulobacteria, acidophilus, lactic acid bacteria, and yeast. 2. A fermented concentrated material having a bactericidal and anti-infective effect on vancomycin-resistant enterococci according to item 1 of the scope of patent application, which contains at least one compound A to F formed by the following structural formula. Compound A Compound B y * 丨 丨 —- page ugly (Please read the notes on the back before filling out this page) Printed by the Consumer Cooperatives of the Intellectual Property Bureau of the Ministry of Economic Affairs Compound C 5 Hydroxybenzaldehyde COOH 〇 2 -Saccharic acid This paper size applies to Chinese National Standard (CNS) A4 specification (210X297 mm) 6. Patent application scope Compound D 1243649 A8 B8 C8 D8 0H 〇ch3 COOH 4 monohydroxy-3-methoxybenzoic acid compound E 0H h3co .och3 COOH 4 monohydroxy-3,5-dimethylbenzoic acid compound F 2 —methyl-1 4-hydroxy-4 hydrazone —pyran-4 —one (Please read the precautions on the back before filling this page) > r_ Set the paper size printed by the Employees' Cooperative of the Intellectual Property Bureau of the Ministry of Economic Affairs to apply the Chinese National Standard (CNS) Α4 specification (210X297 mm) 2