WO2013001862A1 - 新規乳酸菌及びこれを用いるサイレージ又は発酵飼料の調製方法 - Google Patents
新規乳酸菌及びこれを用いるサイレージ又は発酵飼料の調製方法 Download PDFInfo
- Publication number
- WO2013001862A1 WO2013001862A1 PCT/JP2012/056991 JP2012056991W WO2013001862A1 WO 2013001862 A1 WO2013001862 A1 WO 2013001862A1 JP 2012056991 W JP2012056991 W JP 2012056991W WO 2013001862 A1 WO2013001862 A1 WO 2013001862A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- lactic acid
- silage
- nisin
- preparation
- acid bacterium
- Prior art date
Links
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 200
- 241000894006 Bacteria Species 0.000 title claims abstract description 106
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 100
- 239000004310 lactic acid Substances 0.000 title claims abstract description 100
- 239000004460 silage Substances 0.000 title claims abstract description 66
- 238000000034 method Methods 0.000 title claims abstract description 19
- 108010053775 Nisin Proteins 0.000 claims abstract description 82
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 claims abstract description 82
- 239000004309 nisin Substances 0.000 claims abstract description 82
- 235000010297 nisin Nutrition 0.000 claims abstract description 82
- 238000002360 preparation method Methods 0.000 claims abstract description 55
- 244000025254 Cannabis sativa Species 0.000 claims abstract description 47
- 230000000813 microbial effect Effects 0.000 claims abstract description 22
- 150000002016 disaccharides Chemical class 0.000 claims abstract description 21
- 150000002772 monosaccharides Chemical class 0.000 claims abstract description 21
- 239000006228 supernatant Substances 0.000 claims abstract description 18
- 239000002609 medium Substances 0.000 claims description 42
- 235000014897 Streptococcus lactis Nutrition 0.000 claims description 14
- 239000002253 acid Substances 0.000 claims description 10
- 238000009631 Broth culture Methods 0.000 claims description 7
- 239000001963 growth medium Substances 0.000 claims description 7
- 241000186605 Lactobacillus paracasei Species 0.000 claims description 3
- 241000194035 Lactococcus lactis Species 0.000 claims 2
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Chemical compound CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 abstract description 50
- 238000000855 fermentation Methods 0.000 abstract description 35
- 230000004151 fermentation Effects 0.000 abstract description 35
- 230000000694 effects Effects 0.000 abstract description 31
- 239000000463 material Substances 0.000 abstract description 15
- 150000001720 carbohydrates Chemical class 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 244000057717 Streptococcus lactis Species 0.000 description 12
- 235000013305 food Nutrition 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
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- 239000012228 culture supernatant Substances 0.000 description 7
- 229920001817 Agar Polymers 0.000 description 6
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- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 6
- 239000008272 agar Substances 0.000 description 6
- 239000006872 mrs medium Substances 0.000 description 6
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- 240000004296 Lolium perenne Species 0.000 description 5
- 239000006227 byproduct Substances 0.000 description 5
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- -1 dehydrobutyrin Chemical compound 0.000 description 4
- 244000005700 microbiome Species 0.000 description 4
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- 244000199885 Lactobacillus bulgaricus Species 0.000 description 3
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 3
- 240000006024 Lactobacillus plantarum Species 0.000 description 3
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 3
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 3
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- 240000007298 Megathyrsus maximus Species 0.000 description 3
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- 235000007164 Oryza sativa Nutrition 0.000 description 3
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- 239000007788 liquid Substances 0.000 description 3
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- 240000007241 Agrostis stolonifera Species 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 241000194031 Enterococcus faecium Species 0.000 description 2
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- QWCKQJZIFLGMSD-UHFFFAOYSA-N alpha-aminobutyric acid Chemical compound CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 description 2
- LFYJSSARVMHQJB-QIXNEVBVSA-N bakuchiol Chemical compound CC(C)=CCC[C@@](C)(C=C)\C=C\C1=CC=C(O)C=C1 LFYJSSARVMHQJB-QIXNEVBVSA-N 0.000 description 2
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- NSGOABPZARPCFM-ZZJOKYKRSA-N (2R)-2-amino-3-[(2R)-2-amino-2-carboxyethyl]sulfanylbutanoic acid Chemical compound OC(=O)[C@@H](N)C(C)SC[C@H](N)C(O)=O NSGOABPZARPCFM-ZZJOKYKRSA-N 0.000 description 1
- NSGOABPZARPCFM-UHFFFAOYSA-N (2S,3S,2'R)-beta-Methyllanthionine Natural products OC(=O)C(N)C(C)SCC(N)C(O)=O NSGOABPZARPCFM-UHFFFAOYSA-N 0.000 description 1
- 235000007319 Avena orientalis Nutrition 0.000 description 1
- 241000209763 Avena sativa Species 0.000 description 1
- 235000007558 Avena sp Nutrition 0.000 description 1
- 235000016068 Berberis vulgaris Nutrition 0.000 description 1
- 241000335053 Beta vulgaris Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 240000007124 Brassica oleracea Species 0.000 description 1
- 235000003899 Brassica oleracea var acephala Nutrition 0.000 description 1
- 235000012905 Brassica oleracea var viridis Nutrition 0.000 description 1
- 108010059892 Cellulase Proteins 0.000 description 1
- UQBOJOOOTLPNST-UHFFFAOYSA-N Dehydroalanine Chemical compound NC(=C)C(O)=O UQBOJOOOTLPNST-UHFFFAOYSA-N 0.000 description 1
- 241000234645 Festuca pratensis Species 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 240000005979 Hordeum vulgare Species 0.000 description 1
- 235000007340 Hordeum vulgare Nutrition 0.000 description 1
- DWPCPZJAHOETAG-IMJSIDKUSA-N L-lanthionine Chemical compound OC(=O)[C@@H](N)CSC[C@H](N)C(O)=O DWPCPZJAHOETAG-IMJSIDKUSA-N 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241000194036 Lactococcus Species 0.000 description 1
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- 239000004677 Nylon Substances 0.000 description 1
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- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 235000014676 Phragmites communis Nutrition 0.000 description 1
- 241000209056 Secale Species 0.000 description 1
- 241000287219 Serinus canaria Species 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000015503 Sorghum bicolor subsp. drummondii Nutrition 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000194054 Streptococcus uberis Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
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- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 229940069780 barley extract Drugs 0.000 description 1
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- 235000013405 beer Nutrition 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- 239000012153 distilled water Substances 0.000 description 1
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- 230000003993 interaction Effects 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- DWPCPZJAHOETAG-UHFFFAOYSA-N meso-lanthionine Natural products OC(=O)C(N)CSCC(N)C(O)=O DWPCPZJAHOETAG-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
- A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/21—Streptococcus, lactococcus
- A23V2400/231—Lactis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
Definitions
- the present invention relates to a lactic acid bacterium capable of suppressing butyric acid fermentation, a microbial preparation containing the lactic acid bacterium, and a method for preparing silage or fermented feed using the preparation.
- a problem called poor fermentation often occurs, but this is a problem that the growth of butyric acid bacteria predominates over lactic acid bacteria in the silage, resulting in a high butyric acid content in the silage.
- Silage with a high butyric acid concentration becomes a problem that not only reduces the palatability of cattle and reduces the amount of food intake, but also leads to diseases such as ketosis.
- Non-Patent Document 1 As a condition that the lactic acid bacteria added to the silage have an effect, the minimum amount of soluble saccharide in the material grass is 5.0 to 6.0% in dry matter (1.0 to 3.0% in terms of the original) or more It is said that it is necessary (Non-Patent Document 1). On the other hand, because there are many pastures and forage crops with low sugar content, the lactic acid bacteria for silage that have been reported so far have not been fully effective, but are often fermented with butyric acid.
- lactic acid bacteria are known to produce bacteriocin which is an antibacterial protein by fermentation or culture.
- nisin consists of 34 amino acids produced by some strains of Lactococcus lactis, a kind of lactic acid bacteria, and contains in its molecule lanthionine, 3-methyllanthionine, dehydroalanine, dehydrobutyrin, 2-aminobutyric acid. It is an antibacterial protein containing.
- nisin A, nisin Z, and nisin Q There are several analogs of nisin A, nisin Z, and nisin Q. In recent years, Nisin U produced by Streptococcus uberis and Nisin F produced by Lactococcus lactis have been reported.
- Nisin has a broad antibacterial spectrum centering on Gram-positive bacteria including butyric acid bacteria, and may be added to silage to suppress butyric acid bacteria.
- Nisin A which is one of its analogs, has been widely used as a food preservative for cheese and canned vegetables all over the world.
- nisin when adding nisin directly to a food, a process of producing high concentration nisin by culturing lactic acid bacteria, extracting, purifying, and drying the nisin is necessary, which is generally costly.
- nisin exhibits an immediate antibacterial action, the effect is difficult to sustain, and the effect may be reduced by interaction with the component to be added.
- Non-Patent Documents 2 to 5 attempts to add nisin-producing lactic acid bacteria to foods such as cheese, sauerkraut, bean miso, and rice miso (Non-Patent Documents 2 to 5) are being studied, but grass, feed crops, food production by-products, etc. that are used as silage ingredients Is not used practically. The reason for this is that these silage raw materials have fewer nutrients, including sugar, which is essential for the growth of lactic acid bacteria, and there is no sterilization process as in the case of adding to food materials. Therefore, it is conceivable that a sufficient effect cannot be obtained even by adding nisin-producing lactic acid bacteria that are effective in food materials.
- Patent Document 1 lactic acid bacteria that produce nisin at a high concentration in a synthetic medium and nisin production in a fermented barley extract medium have been reported (Patent Document 1, Non-Patent Document 6). Even if a high concentration of nisin can be produced in the medium, there is no prior literature showing that a high concentration of nisin is produced in silage materials with different nutrient sources.
- Patent Document 2 a silage preparation method has been reported in which Lactococcus lactis RO50 strain producing bacteriocin is added to the silage material grass (Patent Document 2). In some cases, the effect is unstable, and it has not been clarified whether bacteriocin is produced in silage (Non-patent Documents 7 and 8). Patent Document 3 also reports a silage preparation method in which a nisin-producing lactic acid bacterium and a nisin-resistant lactic acid bacterium are added, and 200 IU / g nisin activity is detected in the silage, but other lactic acid bacteria that do not produce nisin.
- the object of the present invention is to select a lactic acid bacterium having the ability to produce nisin at a high concentration even under silage fermentation conditions, where butyric acid fermentation is likely to proceed, and adding a microbial preparation containing the lactic acid bacterium having this ability to the silage for high quality It is to provide a technique for stably preparing silage and fermented feed.
- the present inventor has prepared a grass broth culture medium with very low monosaccharide and disaccharide contents without adding any saccharides, and cultivated lactic acid bacteria using this Then, a strain producing nisin was screened.
- the screening succeeded in selecting lactic acid bacteria that exert a stronger nisin activity than the known nisin-producing strains in the grass broth medium, thereby completing the present invention.
- the present invention relates to the following [1] to [11].
- a lactic acid bacterium that produces 40 IU or more of nisin per mL of supernatant in a grass broth medium having a total content of monosaccharide and disaccharide of 0.1 to 0.4% by mass.
- [3] The lactic acid bacterium according to [1] or [2], which produces 100 IU or more of nisin per mL of supernatant in a grass broth medium containing a total content of monosaccharide and disaccharide of 0.1 to 0.4 mass%.
- the present invention can provide a lactic acid bacterium that produces nisin even in materials having a low sugar content, which is an actual silage fermentation condition, among many dairy farmers suffering from butyric acid fermentation of silage and fermented feed.
- a microbial preparation that suppresses butyric acid fermentation and enables high-quality lactic acid fermentation can be provided.
- the present invention makes it possible to produce silage or fermented feed that is highly safe and has good livestock foraging.
- the nisin-producing lactic acid bacterium of the present invention is nisin in a grass broth medium having a total content of monosaccharides and disaccharides of 1.0% by mass or less, preferably 0.7% by mass or less, more preferably 0.4% by mass or less. It is obtained by selecting lactic acid bacteria that produce
- the grass used in the grass broth medium used for selection of nisin-producing lactic acid bacteria in the present invention is not particularly limited as long as it is a grass used for silage or fermented feed.
- alfalfa, clover, timothy, orchard grass Reed canary grass, buckwheat, facility ryegrass, perennial ryegrass, tall fescue, meadow fescue, festrorium, kentucky bluegrass, red top, guineagrass, rosegrass, napiergrass etc.
- grass it may be used in the state of freshly cut grass, but it may be refrigerated or frozen, or it may be used in the form of dry powder obtained by pulverizing dried and freeze-dried grass. .
- Rusan pellets and hay cubes distributed as feed may be used.
- a medium using only a leachate obtained by leaching these grasses with hot water as a nutrient source is used as a grass broth medium.
- the temperature of the hot water is preferably 50 to 100 ° C., more preferably 70 to 100 ° C.
- the time for leaching the grass in hot water is preferably 10 to 180 minutes, more preferably 30 to 120 minutes.
- the leachate is preferably used after filtering to remove solids, if necessary.
- the total content of monosaccharides and disaccharides in the grass broth medium is 1.0% by mass or less, preferably 0.7% by mass or less, more preferably It is desirable to adjust the dilution to 0.4% by mass or less with water.
- the minimum of the total content of preferable monosaccharide and disaccharide is 0.1 mass%, More preferably, it is 0.2 mass%.
- the culture conditions for selection of the nisin-producing lactic acid bacteria of the present invention may be any conditions that allow normal lactic acid bacteria to grow. For example, 0.1 to 1.0% by mass inoculated in a grass broth culture medium at 25 to 35 ° C.
- the stationary culture may be performed for 8 to 24 hours.
- nisin producing strains are generally resistant to nisin, it goes without saying that strains that grow on a nisin-added medium can be selected as the primary screening.
- an MRS medium containing nisin 100 to 1000 IU / mL can be used as a selection medium.
- selection of a nisin-producing bacterium that meets the object of the present invention can be performed by measuring the nisin concentration in the culture supernatant or measuring the nisin activity. The nisin concentration can be measured, for example, by high performance liquid chromatography.
- Nisin activity can be measured using antibacterial activity against nisin-sensitive strains of lactic acid bacteria or butyric acid bacteria as an index, and can be measured, for example, by measuring antibacterial activity against Lactobacillus bulgaricus. Nisin activity is preferably measured as nisin content.
- the nisin content of the culture supernatant can be measured by a generally known method. For example, its activity can be measured by the Agarwell method. That is, a penicillin cup (diameter 8 mm) was placed on a plate of 1.5% agar MRS medium, and 0.1% of a culture solution of Lactobacillus bulgaricus JCM1002 T strain as an indicator was mixed here. Overlay with 75% agar MRS medium.
- nisin preparation Sigma, containing 2.5% nisin
- activity value per 1 ⁇ g of nisin can be measured as 40 IU (1 IU per 1 ⁇ g of nisin preparation).
- the lactic acid bacterium of the present invention is a lactic acid bacterium that produces nisin of 40 IU or more per mL of supernatant in a grass broth medium having a total content of monosaccharide and disaccharide of 0.1 to 0.4 mass%.
- a preferred embodiment of the lactic acid bacterium of the present invention is a lactic acid bacterium that produces nisin of 100 IU or more per mL of supernatant in a grass broth medium having a total content of monosaccharide and disaccharide of 0.1 to 0.7 mass%. .
- it is a lactic acid bacterium that produces 100 IU or more of nisin per mL of supernatant in a grass broth medium having a total content of monosaccharides and disaccharides of 0.1 to 0.4% by mass.
- the total content of monosaccharides and disaccharides in the grass broth medium is more preferably 0.2 to 0.4% by mass.
- the amount of nisin produced in this medium is 40 IU or more per mL of supernatant, preferably 100 IU or more, more preferably 100 to 400 IU, and even more preferably 100 to 300 IU.
- the lactic acid bacterium of the present invention has a nisin of 40 IU or more per mL of supernatant in a grass broth medium having a total content of monosaccharide and disaccharide of 0.1 to 0.4 mass% even under a low pH condition of pH 5.
- producing nisin even under a low pH condition means that the lactic acid bacteria of the present invention exhibit an effect for a longer time.
- Lactococcus lactis SBS-0001 NITE BP-1107
- Lactococcus lactis SBS-0002 NITE BP-1108
- Lactococcus lactis SBS-0004 Lactococcus lactis SBS- Examples include Lactococcus lactis SBS-0001 (NITE BP-1107) and Lactococcus lactis SBS-0002 (NITE BP-1108).
- the lactic acid bacterium of the present invention is capable of growing and fermenting even in the presence of grass having a low sugar content, and produces nisin in large quantities. Therefore, it is useful as a component of a microbial preparation for silage preparation.
- the culture of the above lactic acid bacteria can be performed using a normal medium for lactic acid bacteria.
- the medium is not particularly limited as long as it is a medium used for cultivation of lactic acid bacteria, but for example, GYP medium (Michio Kosaki et al. (1992), lactic acid bacteria experiment manual, published by Asakura Shoten), MRS medium (de Man, JC et. al. (1960), Journal of Applied Bacteriology, Vol. 23, 130-135).
- the culture conditions are not particularly limited, but the culture can usually be performed at pH 5.0 to 7.0, 25 to 40 ° C., and 10 to 24 hours.
- the cultured lactic acid bacteria can be added to the silage or fermented feed in the form of a culture solution or a concentrated solution obtained by concentrating the cells, but these solutions may be used as frozen products.
- the cultured lactic acid bacteria may be used in the form of a powder obtained by freeze drying, spray drying, or fluidized bed drying together with an appropriate protective agent and substrate.
- the microbial preparation of the present invention can contain one or more lactic acid strains that produce nisin in the present invention. Moreover, you may contain another lactic acid bacteria separately. In particular, by containing a lactic acid bacterium having acid resistance, it is possible to provide a microorganism preparation that enables high-quality lactic acid fermentation in addition to inhibiting butyric acid fermentation. In this case, it goes without saying that other lactic acid bacteria are preferably strains with high nisin resistance.
- Lactobacillus plantarum Lactobacillus plantarum
- Lactobacillus casei Lactobacillus casei
- Lactobacillus rhamnosus Lactobacillus rhamnosus
- Lactobacillus salivarius Lactobacillus salivarius
- Enterococcus faecium Enterococcus faecium
- Pediococcus acidilactici Pediococcus acidilactici
- Pediococcus pentosaceus Pediococcus pentosaceus
- Lactobacillus para Zei SBS-0003 strain NITE BP-1109
- Lactobacillus rhamnosus SBT-2300 strain Lactobacillus plantarum No.
- Lactobacillus paracasei SBS-0003 strain (NITE BP- 1109).
- the above-mentioned SBS-0003 strain was deposited at the Patent Evaluation Microorganism Deposit Center (Address: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture, Japan 292-0818) (Deposit Date: Heisei) June 15, 2011).
- the content ratio of the lactic acid bacterium of the present invention and the acid-resistant lactic acid bacterium in the microbial preparation is not particularly limited, but is preferably 1: 100 to 100: 1 by mass ratio, more preferably 1: 9 to 9: 1, 3: 7 to 7: 3 is more preferable.
- an enzyme may be contained in the microorganism preparation of the present invention as necessary.
- the enzyme include cellulase and hemicellulase.
- the microorganism preparation of the present invention can be used for the preparation of various silages and fermented feeds.
- the silage and fermented feed material used for the preparation of silage and fermented feed is not particularly limited as long as it is a raw material usually used as feed, but for example, alfalfa, clover, timothy, orchardgrass as pasture and feed crops.
- Examples of food production by-products include beer lees, tofu lees, tea lees, shochu, whiskey lees, beet pulp, bacas, coffee lees, juice lees, kale lees, starch lees, and the like.
- Agricultural by-products include rice straw, wheat straw, and non-standard vegetables. It can also be added to fermentation TMR prepared by mixing food production by-products, silage, agricultural by-products, hay, concentrated feed, vitamin / mineral preparations and the like. These silage and fermented feed materials are desirably used with moisture adjusted to 40-90% by mass.
- a microbial preparation is dissolved / suspended in water or the like and sprayed on the raw material, or a powdered microbial preparation is sprayed and mixed on the raw material.
- the total number of bacteria added is 10 3 to 10 7 , preferably 10 4 to 10 6 , per gram of raw material.
- Silage and fermented feed are usually fermented under anaerobic conditions.
- the vessel for fermentation is not particularly limited as long as the anaerobic state can be maintained to some extent, for example, bunker silo, stack silo, trench silo, tower silo, underground silo, block silo, cup silo, roll bale, flexible container bag, etc. Can be mentioned. Usually, it can be fermented by leaving it to stand for 1 week or longer at an outside temperature (about 5 to 30 ° C).
- silage and fermented feed obtained using the microbial preparation of the present invention are effectively inhibited from butyric acid fermentation, so that the palatability and the amount of food intake are good and ketosis does not occur.
- Nisin activity in grass broth medium The nisin activity of the culture supernatant when each lactic acid bacterium was cultured in the grass broth medium was measured.
- the pasture broth medium was prepared as follows. 1 liter of distilled water was added to 100 g of Lusan pellets, and the mixture was boiled for 30 minutes in a boiling water bath after the liquid temperature reached 90 ° C. The residue was filtered by gauze, and the filtrate was centrifuged and filtered through filter paper. The filtrate was made up to 1 liter, dispensed into test tubes, and sterilized by autoclaving (115 ° C., 20 minutes).
- the sugar content in the grass broth medium was measured as follows. 840 ⁇ l of acetonitrile was added to 360 ⁇ l of the medium, mixed, and allowed to stand at room temperature for 15 minutes. The precipitate was removed by centrifugation (15,000 rpm, 15 minutes), and the supernatant was filtered through a 0.2 ⁇ m filter and analyzed by HPLC. The contents of the detected monosaccharide and disaccharide (xylose, glucose, fructose, sucrose) were summed to obtain the sugar content.
- Each lactic acid bacterium shown in Table 1 was precultured in an MRS liquid medium, the preculture was centrifuged, the medium was discarded, and the same amount of physiological saline as the medium was added to the cells. 1% of this cell suspension was inoculated into a grass broth culture medium and left to stand at 32 ° C. for 8 hours, and the absorbance (660 nm) of the culture solution was measured. The culture solution was centrifuged, and the nisin activity of the culture supernatant from which the cells were removed with a 0.2 ⁇ m filter was measured by the Agarwell method.
- the Agarwell method was performed as follows. A 0.75% agar MRS medium in which a penicillin cup (diameter 8 mm) is placed on a plate of 1.5% agar MRS medium and 0.1% of the culture solution of Lactobacillus bulgaricus JCM1002 T strain is mixed as an indicator bacterium here. Layered. After the agar solidified, the penicillin cup was removed and used as an antibacterial test plate. The culture supernatant after filter sterilization was added to a well opened with a penicillin cup, and after anaerobic culture at 37 ° C. for 24 hours, the diameter of the inhibition circle against the indicator bacterium was measured. A nisin preparation (Sigma, containing 2.5% nisin) was used as a standard, and the activity value per 1 ⁇ g of nisin was 40 IU (1 IU per 1 ⁇ g of nisin preparation).
- the ATCC11454 strain which is known to produce nisin A
- the JCM7638 strain which is known to produce nisin Z
- the RO50 strain which is known to produce bacteriocin
- the nisin activity was very weak although it showed the same growth (OD 660) as the SBS-0001 and SBS-0002 strains.
- the SBS-0001 and SBS-0002 strains showed very high nisin activity in a grass broth medium having a sugar content of about 0.3%.
- the SBS-0001 and SBS-0002 strains grow more in the grass broth medium having a sugar content of about 0.7% than in the grass broth medium having a sugar content of about 0.3%. Both nisin activity increased.
- the JCM7638 strain showed less than half the activity of the SBS-0001 and SBS-0002 strains.
- Silage has a low pH during the fermentation process, so it is desirable to produce nisin even at low pH in order to exert its effect for a longer time, but nisin production is known to decrease when the culture pH is lowered. Yes.
- Table 5 when the initial pH of the grass broth culture medium was set to 5.0, the nisin activity in the culture supernatant decreased overall for each strain, but the RO50 strain had a nisin activity of 0.
- the SBS-0001 and SBS-0002 strains showed nisin activity of 40 IU / mL or more.
- Silage preparation and fermentation quality Silage was prepared by adding each lactic acid bacterium to pasture, and the fermentation quality was investigated.
- Lactic acid bacteria prepared the strains shown in Table 6 as follows. Each strain was inoculated into a GYP liquid medium and cultured at 37 ° C. for 24 hours. The culture solution was centrifuged (6,500 rpm, 15 minutes), the supernatant was discarded, the remaining cells were suspended by adding 10% trehalose and 1% sodium glutamate solution, and dried with a freeze dryer.
- Silage preparation was performed as follows. Timothy, orchardgrass, reed canarygrass, shibamugi, facility ryegrass, and rye were cut at the heading stage, and alfalfa was cut at the flowering stage and chopped to a length of about 2 cm.
- alfalfa was cut at the flowering stage and chopped to a length of about 2 cm.
- the number of bacteria becomes 1.0 ⁇ 10 5 cfu per 1 g of the material grass and the number of bacteria is 5.0 ⁇ 10 4 cfu when mixed with 2 strains.
- Ingredients were added to the grass and mixed. These material grasses were packed into a 1 liter polyethylene plastic bottle or plastic pouch bag (a laminated film of nylon and polyethylene).
- the ability to produce nisin in the grass broth medium is 40 IU / mL or less in the SBT-2300 strain, which is used as a commercial lactic acid bacterium for silage, with almost no improvement in fermentation quality.
- the addition of the RO50 strain has little effect on improving fermentation quality or the effect of reducing butyric acid is weak, whereas the addition of SBS-0001, which has a high ability to produce nisin in the grass broth culture medium, clearly added butyric acid. The content was reduced and the fermentation quality of the silage was improved.
- the SBS-0003 strain is superior in lactic acid fermentation ability to the SBT-2300 strain, but the addition of the SBT-0001 strain and the SBT-0002 strain further reduced the butyric acid content.
- the SBS-0001 strain and the SBS-0002 strain are added to the SBS-0003 strain even in materials where the fermentation quality improvement effect cannot be obtained with the SBT-2300 strain or the SBS-0003 strain.
- the butyric acid content was greatly reduced, and the fermentation quality was greatly improved.
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Abstract
Description
[2]単糖および二糖の合計含有量が0.1~0.7質量%の牧草煮汁培地中で上清1mLあたり100IU以上のナイシンを生産する[1]記載の乳酸菌。
[3]単糖および二糖の合計含有量が0.1~0.4質量%の牧草煮汁培地中で上清1mLあたり100IU以上のナイシンを生産する[1]又は[2]記載の乳酸菌。
[4]ラクトコッカス・ラクティスSBS-0001株(NITE BP-1107)又はラクトコッカス・ラクティスSBS-0002株(NITE BP-1108)である[1]~[3]のいずれか1項記載の乳酸菌。
[5]単糖および二糖の合計含有量が0.1~0.4質量%の牧草煮汁培地中で上清1mLあたり40IU以上のナイシンを生産する乳酸菌を含有する、サイレージ調製用微生物製剤。
[6]乳酸菌が[2]~[4]のいずれかに記載の乳酸菌である請求項5記載のサイレージ調製用微生物製剤。
[7]さらに、耐酸性を有する乳酸菌を含有する[5]又は[6]記載のサイレージ調製用微生物製剤。
[8]耐酸性を有する乳酸菌が、ラクトバチルス・パラカゼイSBS-0003株(NITE BP-1109)である[7]記載のサイレージ調製用微生物製剤。
[9]単糖および二糖の合計含有量が0.1~0.4質量%の牧草煮汁培地中で上清1mLあたり40IU以上のナイシンを生産する乳酸菌を含有するサイレージ調製用微生物製剤を添加することを特徴とするサイレージ又は発酵飼料の調製方法。
[10]乳酸菌が、[2]~[4]のいずれか1項記載の乳酸菌である[9]記載のサイレージ又は発酵飼料の調製方法。
[11]前記サイレージ調製用微生物製剤が、さらに耐酸性を有する乳酸菌を含有するものである[9]又は[10]記載のサイレージ又は発酵飼料の調製方法。
また、本発明の乳酸菌の好ましい態様は、単糖および二糖の合計含有量が0.1~0.7質量%の牧草煮汁培地中で上清1mLあたり100IU以上のナイシンを生産する乳酸菌である。より好ましくは、単糖および二糖の合計含有量が0.1~0.4質量%の牧草煮汁培地中で上清1mLあたり100IU以上のナイシンを生産する乳酸菌である。
上記のSBS-0001株及びSBS-0002株は、(独)製品評価技術基盤機構 特許微生物寄託センター(住所:〒292-0818 日本国千葉県木更津市かずさ鎌足2-5-8)に寄託した(寄託日:平成23年06月15日)。
上記のSBS-0003株は、(独)製品評価技術基盤機構 特許微生物寄託センター(住所:〒292-0818 日本国千葉県木更津市かずさ鎌足2-5-8)に寄託した(寄託日:平成23年06月15日)。
各乳酸菌を牧草煮汁培地で培養した時の培養液上清のナイシン活性を測定した。
牧草煮汁培地は、以下のように作成した。ルーサンペレット100gに1リットルの蒸留水を加え、沸騰湯浴中で液温が90℃に到達してから30分間煮出した。ガーゼで濾して残渣を絞り、ろ液を遠心分離にかけてからろ紙でろ過した。このろ液を1リットルにメスアップして、試験管に分注し、オートクレーブ(115℃、20分)で滅菌した。
各乳酸菌を牧草に添加してサイレージを調製し、発酵品質を調査した。
乳酸菌は、表6に示した菌株を以下のように作成した。GYP液体培地に各菌株を接種し、37℃で24時間培養した。培養液を遠心(6,500rpm、15分)して、上清を捨て、残った菌体に10%トレハロース、1%グルタミン酸ナトリウム溶液を加えて懸濁し、凍結乾燥機で乾燥した。
Claims (11)
- 単糖および二糖の合計含有量が0.1~0.4質量%の牧草煮汁培地中で上清1mLあたり40IU以上のナイシンを生産する乳酸菌。
- 単糖および二糖の合計含有量が0.1~0.7質量%の牧草煮汁培地中で上清1mLあたり100IU以上のナイシンを生産する請求項1記載の乳酸菌。
- 単糖および二糖の合計含有量が0.1~0.4質量%の牧草煮汁培地中で上清1mLあたり100IU以上のナイシンを生産する請求項1又は2記載の乳酸菌。
- ラクトコッカス・ラクティスSBS-0001株(NITE BP-1107)又はラクトコッカス・ラクティスSBS-0002株(NITE BP-1108)である請求項1~3のいずれか1項記載の乳酸菌。
- 単糖および二糖の合計含有量が0.1~0.4質量%の牧草煮汁培地中で上清1mLあたり40IU以上のナイシンを生産する乳酸菌を含有する、サイレージ調製用微生物製剤。
- 乳酸菌が請求項2~4のいずれかに記載の乳酸菌である請求項5記載のサイレージ調製用微生物製剤。
- さらに、耐酸性を有する乳酸菌を含有する請求項5又は6記載のサイレージ調製用微生物製剤。
- 耐酸性を有する乳酸菌が、ラクトバチルス・パラカゼイSBS-0003株(NITE BP-1109)である請求項7記載のサイレージ調製用微生物製剤。
- 単糖および二糖の合計含有量が0.1~0.4質量%の牧草煮汁培地中で上清1mLあたり40IU以上のナイシンを生産する乳酸菌を含有するサイレージ調製用微生物製剤を添加することを特徴とするサイレージ又は発酵飼料の調製方法。
- 乳酸菌が、請求項2~4のいずれか1項記載の乳酸菌である請求項9記載のサイレージ又は発酵飼料の調製方法。
- 前記サイレージ調製用微生物製剤が、さらに耐酸性を有する乳酸菌を含有するものである請求項9又は10記載のサイレージ又は発酵飼料の調製方法。
Priority Applications (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2013522489A JP5931064B2 (ja) | 2011-06-29 | 2012-03-19 | 新規乳酸菌及びこれを用いるサイレージ又は発酵飼料の調製方法 |
DK12803868.4T DK2727994T3 (en) | 2011-06-29 | 2012-03-19 | New lactic acid bacteria and process for preparing silage or fermented feed using it |
CN201280031729.4A CN103781898B (zh) | 2011-06-29 | 2012-03-19 | 新型乳酸菌以及使用其的青贮饲料或发酵饲料的制备方法 |
EP12803868.4A EP2727994B1 (en) | 2011-06-29 | 2012-03-19 | Novel lactic acid bacterium and method for preparing silage or fermented feed using same |
RU2014102762/10A RU2598270C2 (ru) | 2011-06-29 | 2012-03-19 | Новая молочнокислая бактерия и способ производства с ее помощью силоса или ферментированного корма |
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JP (1) | JP5931064B2 (ja) |
CN (1) | CN103781898B (ja) |
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Cited By (5)
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WO2015041556A1 (en) | 2013-09-23 | 2015-03-26 | Ivetić Aleksandra | The method and the use of additives in feed preservation |
WO2017159483A1 (ja) | 2016-03-15 | 2017-09-21 | 雪印種苗株式会社 | サイレージ調製用乳酸菌及びサイレージ調製用添加剤 |
JP2019118303A (ja) * | 2018-01-04 | 2019-07-22 | 雪印種苗株式会社 | サイレージ調整用乳酸菌製剤 |
CN113527447A (zh) * | 2021-07-21 | 2021-10-22 | 山东元泰生物工程有限公司 | 一种乳酸链球菌素的提取方法 |
KR20230038477A (ko) | 2020-10-12 | 2023-03-20 | 도쿠리츠교세이호징 고쿠리츠코토센몬갓코키코 | 유산균 및 그 취득 방법과 유산균 함유 음식품 |
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CN104531566A (zh) * | 2014-12-11 | 2015-04-22 | 中国农业大学 | 一种青贮接种剂及其应用 |
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RU2658978C1 (ru) * | 2017-09-22 | 2018-06-26 | Владимир Львович Темнянский | Комбинированная закваска для переработки растительного сырья с получением кормового белка |
CN109662208A (zh) * | 2019-02-26 | 2019-04-23 | 天津九州大地饲料有限公司 | 一种仔猪用微生物发酵饲料的制备工艺 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2015041556A1 (en) | 2013-09-23 | 2015-03-26 | Ivetić Aleksandra | The method and the use of additives in feed preservation |
WO2017159483A1 (ja) | 2016-03-15 | 2017-09-21 | 雪印種苗株式会社 | サイレージ調製用乳酸菌及びサイレージ調製用添加剤 |
KR20180120264A (ko) | 2016-03-15 | 2018-11-05 | 유키지루시 슈뵤 가부시키가이샤 | 사일리지 조제용 유산균 및 사일리지 조제용 첨가제 |
JP2019118303A (ja) * | 2018-01-04 | 2019-07-22 | 雪印種苗株式会社 | サイレージ調整用乳酸菌製剤 |
JP7062443B2 (ja) | 2018-01-04 | 2022-05-06 | 雪印種苗株式会社 | サイレージ調製用乳酸菌製剤 |
KR20230038477A (ko) | 2020-10-12 | 2023-03-20 | 도쿠리츠교세이호징 고쿠리츠코토센몬갓코키코 | 유산균 및 그 취득 방법과 유산균 함유 음식품 |
CN113527447A (zh) * | 2021-07-21 | 2021-10-22 | 山东元泰生物工程有限公司 | 一种乳酸链球菌素的提取方法 |
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Publication number | Publication date |
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JPWO2013001862A1 (ja) | 2015-02-23 |
EP2727994A4 (en) | 2015-02-18 |
EP2727994B1 (en) | 2017-05-10 |
DK2727994T3 (en) | 2017-07-17 |
CN103781898B (zh) | 2016-03-02 |
RU2598270C2 (ru) | 2016-09-20 |
JP5931064B2 (ja) | 2016-06-08 |
EP2727994A1 (en) | 2014-05-07 |
RU2014102762A (ru) | 2015-08-10 |
CN103781898A (zh) | 2014-05-07 |
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