CN106509913A - Composition for regulating balance of intestinal flora - Google Patents
Composition for regulating balance of intestinal flora Download PDFInfo
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- CN106509913A CN106509913A CN201610939514.2A CN201610939514A CN106509913A CN 106509913 A CN106509913 A CN 106509913A CN 201610939514 A CN201610939514 A CN 201610939514A CN 106509913 A CN106509913 A CN 106509913A
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- lactobacillus
- rhamnosus
- cfu
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- 239000000203 mixture Substances 0.000 title claims abstract description 34
- 230000000968 intestinal effect Effects 0.000 title claims abstract description 30
- 230000001105 regulatory effect Effects 0.000 title claims abstract description 23
- 241000186604 Lactobacillus reuteri Species 0.000 claims abstract description 47
- 229940001882 lactobacillus reuteri Drugs 0.000 claims abstract description 47
- 241000218588 Lactobacillus rhamnosus Species 0.000 claims abstract description 43
- DLRVVLDZNNYCBX-UHFFFAOYSA-N Polydextrose Polymers OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(O)O1 DLRVVLDZNNYCBX-UHFFFAOYSA-N 0.000 claims abstract description 38
- 101100207366 Curvularia clavata TR02 gene Proteins 0.000 claims abstract description 24
- 101100207371 Curvularia clavata TR08 gene Proteins 0.000 claims abstract description 22
- 229920001202 Inulin Polymers 0.000 claims abstract description 19
- 229920001100 Polydextrose Polymers 0.000 claims abstract description 19
- 235000013325 dietary fiber Nutrition 0.000 claims abstract description 19
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 claims abstract description 19
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- 239000012467 final product Substances 0.000 claims description 7
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- 239000003814 drug Substances 0.000 claims description 4
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- 235000013351 cheese Nutrition 0.000 claims 2
- SHZGCJCMOBCMKK-UHFFFAOYSA-N D-mannomethylose Natural products CC1OC(O)C(O)C(O)C1O SHZGCJCMOBCMKK-UHFFFAOYSA-N 0.000 claims 1
- SHZGCJCMOBCMKK-JFNONXLTSA-N L-rhamnopyranose Chemical compound C[C@@H]1OC(O)[C@H](O)[C@H](O)[C@H]1O SHZGCJCMOBCMKK-JFNONXLTSA-N 0.000 claims 1
- PNNNRSAQSRJVSB-UHFFFAOYSA-N L-rhamnose Natural products CC(O)C(O)C(O)C(O)C=O PNNNRSAQSRJVSB-UHFFFAOYSA-N 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 5
- 230000036039 immunity Effects 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 2
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- 241000901050 Bifidobacterium animalis subsp. lactis Species 0.000 abstract 1
- 229940009289 bifidobacterium lactis Drugs 0.000 abstract 1
- FTSSQIKWUOOEGC-RULYVFMPSA-N fructooligosaccharide Chemical compound OC[C@H]1O[C@@](CO)(OC[C@@]2(OC[C@@]3(OC[C@@]4(OC[C@@]5(OC[C@@]6(OC[C@@]7(OC[C@@]8(OC[C@@]9(OC[C@@]%10(OC[C@@]%11(O[C@H]%12O[C@H](CO)[C@@H](O)[C@H](O)[C@H]%12O)O[C@H](CO)[C@@H](O)[C@@H]%11O)O[C@H](CO)[C@@H](O)[C@@H]%10O)O[C@H](CO)[C@@H](O)[C@@H]9O)O[C@H](CO)[C@@H](O)[C@@H]8O)O[C@H](CO)[C@@H](O)[C@@H]7O)O[C@H](CO)[C@@H](O)[C@@H]6O)O[C@H](CO)[C@@H](O)[C@@H]5O)O[C@H](CO)[C@@H](O)[C@@H]4O)O[C@H](CO)[C@@H](O)[C@@H]3O)O[C@H](CO)[C@@H](O)[C@@H]2O)[C@@H](O)[C@@H]1O FTSSQIKWUOOEGC-RULYVFMPSA-N 0.000 abstract 1
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 4
- 235000018417 cysteine Nutrition 0.000 description 4
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- AYRXSINWFIIFAE-SCLMCMATSA-N Isomaltose Natural products OC[C@H]1O[C@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)[C@@H](O)[C@@H](O)[C@@H]1O AYRXSINWFIIFAE-SCLMCMATSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
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- 210000000936 intestine Anatomy 0.000 description 3
- DLRVVLDZNNYCBX-RTPHMHGBSA-N isomaltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-RTPHMHGBSA-N 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
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- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 208000036649 Dysbacteriosis Diseases 0.000 description 2
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- 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 2
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- 239000008272 agar Substances 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- VBIXEXWLHSRNKB-UHFFFAOYSA-N ammonium oxalate Chemical compound [NH4+].[NH4+].[O-]C(=O)C([O-])=O VBIXEXWLHSRNKB-UHFFFAOYSA-N 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
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- KCIDZIIHRGYJAE-YGFYJFDDSA-L dipotassium;[(2r,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] phosphate Chemical compound [K+].[K+].OC[C@H]1O[C@H](OP([O-])([O-])=O)[C@H](O)[C@@H](O)[C@H]1O KCIDZIIHRGYJAE-YGFYJFDDSA-L 0.000 description 2
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- 229910052739 hydrogen Inorganic materials 0.000 description 2
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- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 2
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- -1 phosphoric acid hydrogen Chemical class 0.000 description 2
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- 239000000843 powder Substances 0.000 description 2
- 239000006041 probiotic Substances 0.000 description 2
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- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 2
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- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
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- 241000588921 Enterobacteriaceae Species 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical class OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
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- 235000021307 Triticum Nutrition 0.000 description 1
- 241000287411 Turdidae Species 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
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- 229940095731 candida albicans Drugs 0.000 description 1
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- 208000017580 chronic wasting disease Diseases 0.000 description 1
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Classifications
-
- 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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- 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
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
-
- 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
- A23V2400/11—Lactobacillus
- A23V2400/173—Reuteri
-
- 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
- A23V2400/11—Lactobacillus
- A23V2400/175—Rhamnosus
-
- 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
- A23V2400/51—Bifidobacterium
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a composition for regulating balance of intestinal flora. The composition comprises 5-15 parts of dietary fibers, 2-10 parts of inulin, 0.5-1.5 parts of polydextrose, 1-5 parts of isomaltooligosacharide, 0.5-1.5 parts of fructo-oligosaccharide, 3000-4000 million CFU of lactobacillus paracasei, 3000-4000 millon CFU of lactobacillus reuteri (Lactobacillus reuteri) TR02, 1800-2600 million CFU of lactobacillus rhamnosus(L.rhamnosus)TR08 and 1400-2200 million CFU of bifidobacterium lactis. The invention further provides a preparation method and an application of the composition. The composition is simple in preparation technology, wide in raw material sources, low in cost and convenient to use, the immunity of the human bodies can be greatly enhanced, and the effect for treating and regulating the balance of the intestinal flora is good.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of composition of regulating intestinal canal colony balance.
Background technology
The alternate balance of gut flora is affected by interaction of physiology, food, medicine and enterobacteriaceae of host etc., various
Factor can all destroy the alternate balance of bacterium, cause enteron aisle dominant from the dominant harmful bacteria that turns to of beneficial bacterium, so as to cause many
Intestines problem.Therefore, increase the quantity of intestinal beneficial bacterium, reduce harmful bacteria quantity, be the key institute for ensureing stomach health operating
.
Flora imbalance refers to that the ratio in the normal flora of matrix a part between each bacterial classification occurs to change by a relatively large margin and exceed
The state of normal range (NR), therefore the illness for producing, become dysbacteriosis or flora replaces disease.Causing during flora imbalance double more
Infection or superinfection, i.e., in the treatment of primary infection, there occurs the infection of another kind of new pathogenic flora.Flora imbalance send out
Practical antibiotic and chronic wasting disease etc. are more common in life.It is after clinically widely applying broad-spectrum antibiotic for a long time, most of quick
Sense bacterium and normal flora are suppressed or kill, but drug-fast bacteria then obtains survival advantage and amount reproduction causes a disease, such as resistant S
Staphylococcus causes diarrhoea, septicemia, and the candida albicans bacterium insensitive to antibiotic causes thrush, enteron aisle and anus just to fire
Lamp.
At present, the mode of gut flora is adjusted mainly with probiotic supplemented type and quantity, probiotic supplemented desired nutritional
Material, suppression are killed harmful intestinal tract bacteria, strengthen body immunity to improve macrophage to the side such as phagocytosis of harmful bacteria
Formula with reach gut flora balance purpose.However, existing adjustment gut flora effect of drugs is not good.
The content of the invention
Technical problem：In order to solve the defect of prior art, the invention provides a kind of group of regulating intestinal canal colony balance
Compound.
Technical scheme：A kind of composition of regulating intestinal canal colony balance that the present invention is provided, including dietary fiber 5-15 parts,
Inulin 2-10 parts, polydextrose 0.5-1.5 parts, oligoisomaltose 1-5 parts, FOS 0.5-1.5 parts, lactobacillus paracasei
Hundred million CFU of 30-40, lactobacillus reuteri（Lactobacillus reuteri) hundred million CFU of TR02 30-40, Lactobacillus rhamnosus
(L.rhamnosus) hundred million CFU of TR08 18-26, newborn hundred million CFU of Bacillus bifidus 14-22.
Preferably, including dietary fiber 8-10 parts, inulin 5-7 parts, polydextrose 0.8-1.2 parts, oligoisomaltose
2-4 parts, FOS 0.8-1.2 parts, hundred million CFU of lactobacillus paracasei 33-37, lactobacillus reuteri
（Lactobacillusreuteri) hundred million CFU of TR02 33-37, Lactobacillus rhamnosus (L.rhamnosus) TR08 20-24 hundred million
CFU, newborn hundred million CFU of Bacillus bifidus 16-20.
As it is another kind of preferably, including 9 parts of dietary fiber, 6 parts of inulin, 1 part of polydextrose, 3 parts of oligoisomaltose are low
1 part of Fructooligosaccharides, 3,500,000,000 CFU of lactobacillus paracasei, lactobacillus reuteri（Lactobacillus reuteri) hundred million CFU of TR0235,
2,200,000,000 CFU of Lactobacillus rhamnosus (L.rhamnosus) TR08,1,800,000,000 CFU of newborn Bacillus bifidus.
Present invention also offers the preparation method of the composition of above-mentioned regulating intestinal canal colony balance, comprises the following steps：Point
Another name takes dietary fiber, inulin, polydextrose, oligoisomaltose, FOS, mixes；Add lactobacillus paracasei, sieve
Yi Shi lactobacillus（Lactobacillus reuteri) TR02, Lactobacillus rhamnosus (L.rhamnosus) TR08, newborn bifid breast
Bacillus, mixes, obtains final product.
Present invention also offers application of the above-mentioned composition in treatment regulating intestinal canal colony balance medicine is prepared.
Beneficial effect：The composition preparation process is simple of present invention offer, raw material sources are extensive, with low cost, user
Just, body immunity is greatly improved, treatment regulating intestinal canal colony balance effect is good.
Description of the drawings
Fig. 1 is lactobacillus reuteri（Lactobacillus reuteri）The bacterium colony figure of TR02.
Fig. 2 is lactobacillus reuteri（Lactobacillus reuteri）The bacterium colony enlarged drawing of TR02.
Fig. 3 is Lactobacillus rhamnosus（Lactobacillus rhamnosus）The bacterium colony figure of TR08.
Fig. 4 is Lactobacillus rhamnosus（Lactobacillus rhamnosus）The bacterium colony enlarged drawing of TR08.
Specific embodiment
In the present invention：
Lactobacillus reuteri（Lactobacillus reuteri) TR02, its Classification And Nomenclature is lactobacillus reuteri
（Lactobacillusreuteri）TR02, in China typical culture collection center（Abbreviation CCTCC）Preservation, depositary institution ground
Location：Wuhan City, Hubei Province Wuchang District Wuhan University, postcode is 430072, and its deposit number is：CCTCC
No.M2016546, preservation date are on October 10th, 2016.
The separation of bacterial strain
1st, bacterial classification and culture medium and condition of culture
（1）Bacterium source
From certain military region health soldier's excrement of China.
（2）Culture medium
Plating medium：1000mL distilled water, peptone 10g, glucose 20g, dusty yeast 9g, sodium acetate 5g, phosphoric acid hydrogen
Dipotassium 2g, Triammonium citrate 2g, cysteine 0.5g, magnesium sulfate 0.2g, manganese sulfate 0.05g, tween 1ml, agar powder 15-20g,
pH5.5-6.5。
2nd, the separation and identification of bacterial strain
（1）The separation of bacterial strain
Proper amount of fresh excrement is taken in glove box after sterilized process in advance and is placed in the aseptic examination for being loaded with aseptic culture medium
Guan Zhong, fully shaking are vortexed and mix；
The excrement solution for mixing is vortexed as stoste with above-mentioned fully shaking, ten times of gradient dilutions is done to suitable gradient, is taken 100
μ l are spread evenly across on MRS+0.05% cysteine solid plate culture mediums, are subsequently taken out and are placed in anaerobic jar, 37 DEG C of anaerobism trainings
Support overnight.
On next day visible flat board, length has the bacterium colony of form different sizes, special with reference to lactobacillus reuteri colonies typical form
Levy, some representational bacterium colonies of picking make two generations line purification process, flat board anaerobism activates 1 day, obtains culture, subsequently do bacterium
Strain identification.
（2）The identification of bacterial strain
Gram's staining Morphological Identification：
A, smear：Sterilized slide is taken on experimental bench, is marked with marking pen on slide, be easy to observation, with
Afterwards slide is inverted, and then 10 μ l SPSSs is drawn with pipettor and is uniformly coated at mark, is chosen with liquid-transfering gun then
Take pure culture to be uniformly dissolved in physiological saline, coated face is unsuitable excessive.
B, drying：Sample is faced upwards, the both sides of hand-held slide one end, carefully on alcolhol burner, eminence adds slightly
Heat, makes moisture evaporation, but be sure not it is long against flame or heat time, in case sample is baked withered and is deformed.
C, fixation：It is fixed usually to utilize high temperature, one end of hand-held slide, sample upwards, at the alcolhol burner flame as early as possible
Back and forth through 2-3 time, altogether about 2-3 second kinds, and touch skin with the heating of the slide back side frequently are unconsciously scalded excessively and are advisable（Do not surpass
Cross 60 DEG C）, place after cold, dyeed.
D, just dye：Ammonium oxalate crystal violet 1-2 drops are added dropwise on smear film, are made dyeing liquor cover smear, is dyeed about 1min.
E, washing：Tilting slide, is rinsed with shallow bid current under water tap, until the water under washing in colourless is
Only.
F, mordant dyeing：About 300ul iodine solutions are drawn with 100-1000ul liquid-transfering guns to drop on smear film, is covered dyeing liquor and is applied
Piece, dyes about 1min.
G, washing：Tilting slide, is rinsed with shallow bid current under water tap, until the water under washing in colourless is
Only.
H, decolouring：Tilting slide, is added dropwise 95% ethanol decolorization, not showing purple to the ethanol for flowing out, about takes
20-30S, is washed immediately.
I, redye：Husky of common dye dye liquor 1-2 drops are added dropwise on smear film, are made dyeing liquor cover smear, is dyeed about 1min.
J, washing：Tilting slide, is rinsed with shallow bid current under water tap, until the water under washing in colourless is
Only.
K, drying, observation：Water droplet is sopped up with blotting paper, treats that sample slice is done under rearmounted microscope, use low power sem observation, found
Immersion oil being dripped after purpose thing on slide, the form and color of bacterium being observed with oil mirror, purple is gram-positive bacteria, red
Color is Gram-negative bacteria.
As a result show：The bacterial strain is gram-positive bacteria.
The measure of 16S rRNA sequences：
The PCR primers that use of amplification of 16S rRNA genes be general bacterium primer 8F (5 '-
CACGGATCCAGAGTTTGAT (C/T) (A/C) TGGCTCAG-3 ') and 1510R (5 '-GTGAAGCTTAGGG (C/T)
TACCTTGTTACGACTT-3 ') product entrusts qualified third party laboratory to carry out sanger sequencings.Using BLAST algorithm
Sequence and 16S rRNA genes are compared in GenBank databases, adjacent tree is set up using 4.0 programs of MEGA.
BLAST is analyzed（As shown in SEQ ID No.1）Show that TR02 bacterium are sufficiently close to lactobacillus reuteri（99% relationship
Property）.
Lactobacillus rhamnosus (L.rhamnosus) TR08 Lactobacillus rhamnosus, its Classification And Nomenclature are Lactobacillus rhamnosus
（Lactobacillus rhamnosus）TR08, in China typical culture collection center（Abbreviation CCTCC）Preservation, preservation list
Bit address：Wuhan City, Hubei Province Wuchang District Wuhan University, postcode is 430072, and its deposit number is：CCTCC NO.M
2016548, preservation date is on October 10th, 2016.
The separation of bacterial strain
1st, bacterial classification and culture medium and condition of culture
（1）Bacterium source
From certain military region health soldier's excrement of China
（2）Culture medium
Plating medium：1000mL distilled water, peptone 10g, glucose 20g, dusty yeast 9g, sodium acetate 5g, phosphoric acid hydrogen
Dipotassium 2g, Triammonium citrate 2g, cysteine 0.5g, magnesium sulfate 0.2g, manganese sulfate 0.05g, tween 1ml, agar powder 15-20g,
pH5.5-6.5。
2nd, the separation and identification of bacterial strain
（1）The separation of bacterial strain
Proper amount of fresh excrement is taken in glove box after sterilized process in advance and is placed in the aseptic examination for being loaded with aseptic culture medium
Guan Zhong, fully shaking are vortexed and mix；
The excrement solution for mixing is vortexed as stoste with above-mentioned fully shaking, ten times of gradient dilutions is done to suitable gradient, is taken 100
μ l are spread evenly across on MRS+0.05% cysteine solid plate culture mediums, are subsequently taken out and are placed in anaerobic jar, 37 DEG C of anaerobism trainings
Support overnight.
On next day visible flat board, length has the bacterium colony of form different sizes, with reference to lactobacillus colonies typical morphological feature, picking
Some representational bacterium colonies make two generations line purification process, and flat board anaerobism activates 1 day, obtains culture, subsequently do identification of strains.
（2）The identification of bacterial strain
Gram's staining Morphological Identification：
A, smear：Sterilized slide is taken on experimental bench, is marked with marking pen on slide, be easy to observation, with
Afterwards slide is inverted, and then 10 μ l SPSSs is drawn with pipettor and is uniformly coated at mark, is chosen with liquid-transfering gun then
Take pure culture to be uniformly dissolved in physiological saline, coated face is unsuitable excessive.
B, drying：Sample is faced upwards, the both sides of hand-held slide one end, carefully on alcolhol burner, eminence adds slightly
Heat, makes moisture evaporation, but be sure not it is long against flame or heat time, in case sample is baked withered and is deformed.
C, fixation：It is fixed usually to utilize high temperature, one end of hand-held slide, sample upwards, at the alcolhol burner flame as early as possible
Back and forth through 2-3 time, altogether about 2-3 second kinds, and touch skin with the heating of the slide back side frequently are unconsciously scalded excessively and are advisable（Do not surpass
Cross 60 DEG C）, place after cold, dyeed.
D, just dye：Ammonium oxalate crystal violet 1-2 drops are added dropwise on smear film, are made dyeing liquor cover smear, is dyeed about 1min.
E, washing：Tilting slide, is rinsed with shallow bid current under water tap, until the water under washing in colourless is
Only.
F, mordant dyeing：About 300ul iodine solutions are drawn with 100-1000ul liquid-transfering guns to drop on smear film, is covered dyeing liquor and is applied
Piece, dyes about 1min.
G, washing：Tilting slide, is rinsed with shallow bid current under water tap, until the water under washing in colourless is
Only.
H, decolouring：Tilting slide, is added dropwise 95% ethanol decolorization, not showing purple to the ethanol for flowing out, about takes
20-30S, is washed immediately.
I, redye：Husky of common dye dye liquor 1-2 drops are added dropwise on smear film, are made dyeing liquor cover smear, is dyeed about 1min.
J, washing：Tilting slide, is rinsed with shallow bid current under water tap, until the water under washing in colourless is
Only.
K, drying, observation：Water droplet is sopped up with blotting paper, treats that sample slice is done under rearmounted microscope, use low power sem observation, found
Immersion oil being dripped after purpose thing on slide, the form and color of bacterium being observed with oil mirror, purple is gram-positive bacteria, red
Color is Gram-negative bacteria.
As a result show：The bacterial strain is gram-positive bacteria.
The measure of 16S rRNA sequences：
The PCR primers that use of amplification of 16S rRNA genes be general bacterium primer 8F (5 '-
CACGGATCCAGAGTTTGAT (C/T) (A/C) TGGCTCAG-3 ') and 1510R (5 '-GTGAAGCTTAGGG (C/T)
TACCTTGTTACGACTT-3 ') product entrusts qualified third party laboratory to carry out sanger sequencings.Using BLAST algorithm
Sequence and 16S rRNA genes are compared in GenBank databases, adjacent tree is set up using 4.0 programs of MEGA.
BLAST is analyzed（As shown in SEQ ID No.2）Show that TR02 bacterium are sufficiently close to Lactobacillus rhamnosus（99% relationship
Property）.
Below the present invention is further illustrated.
Embodiment 1
The composition of regulating intestinal canal colony balance, including 5 parts of dietary fiber, 10 parts of inulin, 0.5 part of polydextrose are oligomeric
1 part of isomaltose, 1.5 parts of FOS, 3,000,000,000 CFU of lactobacillus paracasei, lactobacillus reuteri（Lactobacillus
Reuteri 2,600,000,000 CFU of) 4,000,000,000 CFU of TR02, Lactobacillus rhamnosus (L.rhamnosus) TR08, newborn Bacillus bifidus 1,400,000,000
CFU。
Its preparation method, comprises the following steps：Weigh respectively dietary fiber, inulin, polydextrose, oligoisomaltose,
FOS, mixes；Add lactobacillus paracasei, lactobacillus reuteri（Lactobacillus reuteri) TR02, sandlwood
Sugared lactobacillus (L.rhamnosus) TR08, newborn Bacillus bifidus, mix, obtain final product.
Embodiment 2
The composition of regulating intestinal canal colony balance, including 15 parts of dietary fiber, 2 parts of inulin, 1.5 parts of polydextrose are oligomeric
5 parts of isomaltose, 0.5 part of FOS, 4,000,000,000 CFU of lactobacillus paracasei, lactobacillus reuteri（Lactobacillus
Reuteri 1,800,000,000 CFU of) 3,000,000,000 CFU of TR02, Lactobacillus rhamnosus (L.rhamnosus) TR08, newborn Bacillus bifidus 2,200,000,000
CFU。
Its preparation method, comprises the following steps：Weigh respectively dietary fiber, inulin, polydextrose, oligoisomaltose,
FOS, mixes；Add lactobacillus paracasei, lactobacillus reuteri（Lactobacillus reuteri) TR02, sandlwood
Sugared lactobacillus (L.rhamnosus) TR08, newborn Bacillus bifidus, mix, obtain final product.
Embodiment 3
The composition of regulating intestinal canal colony balance, including 8 parts of dietary fiber, 5 parts of inulin, 1.2 parts of polydextrose are oligomeric different
4 parts of maltose, 0.8 part of FOS, 3,300,000,000 CFU of lactobacillus paracasei, lactobacillus reuteri（Lactobacillus
Reuteri 2,000,000,000 CFU of) 3,700,000,000 CFU of TR02, Lactobacillus rhamnosus (L.rhamnosus) TR08, newborn Bacillus bifidus 2,000,000,000
CFU。
Its preparation method, comprises the following steps：Weigh respectively dietary fiber, inulin, polydextrose, oligoisomaltose,
FOS, mixes；Add lactobacillus paracasei, lactobacillus reuteri（Lactobacillus reuteri) TR02, sandlwood
Sugared lactobacillus (L.rhamnosus) TR08, newborn Bacillus bifidus, mix, obtain final product.
Embodiment 4
The composition of regulating intestinal canal colony balance, including 10 parts of dietary fiber, 7 parts of inulin, 0.8 part of polydextrose are oligomeric
2 parts of isomaltose, 1.2 parts of FOS, 3,700,000,000 CFU of lactobacillus paracasei, lactobacillus reuteri（Lactobacillus
Reuteri 2,400,000,000 CFU of) 3,300,000,000 CFU of TR02, Lactobacillus rhamnosus (L.rhamnosus) TR08, newborn Bacillus bifidus 1,600,000,000
CFU。
Its preparation method, comprises the following steps：Weigh respectively dietary fiber, inulin, polydextrose, oligoisomaltose,
FOS, mixes；Add lactobacillus paracasei, lactobacillus reuteri（Lactobacillus reuteri) TR02, sandlwood
Sugared lactobacillus (L.rhamnosus) TR08, newborn Bacillus bifidus, mix, obtain final product.
Embodiment 5
The composition of regulating intestinal canal colony balance, including 9 parts of dietary fiber, 6 parts of inulin, 1 part of polydextrose, oligomeric different wheat
3 parts of bud sugar, 1 part of FOS, 3,500,000,000 CFU of lactobacillus paracasei, lactobacillus reuteri（Lactobacillus reuteri)
3,500,000,000 CFU of TR02, Lactobacillus rhamnosus (L.rhamnosus) TR08 2,200,000,000 CFU, 1,800,000,000 CFU of newborn Bacillus bifidus.
Its preparation method, comprises the following steps：Weigh respectively dietary fiber, inulin, polydextrose, oligoisomaltose,
FOS, mixes；Add lactobacillus paracasei, lactobacillus reuteri（Lactobacillus reuteri) TR02, sandlwood
Sugared lactobacillus (L.rhamnosus) TR08, newborn Bacillus bifidus, mix, obtain final product.
The pharmacodynamic evaluation of the composition of regulating intestinal canal colony balance of the present invention.
1st, adjustment effect of the composition of regulating intestinal canal colony balance of the present invention to Experimental intestinal flora imbalance
Ampicillin modeling is used, SPF-BALB/C mouse intestine dysbacteriosis disease models are prepared, the observation present invention adjusts intestines
Adjustment effect of the composition of road colony balance to Experimental intestinal flora imbalance.The results are shown in Table 1～7, with natural recovering group and
Physiological saline group compares, and function nutrition agent of the present invention has rapid and clear and definite role of correcting to Experimental intestinal flora imbalance, its
To enteron aisle main advantage flora（Bifidobacterium Bifidum, lactobacillus, long coccus）Proliferation be substantially better than the list of its each component
Solely act on.
Diarrhea of mouse symptom dynamic change before and after 1 ampicillin modeling of table
2 normal mouse of table is with model mice gut flora average ratio compared with (x ± s)
3 groups of experiment mice symptom of diarrhea recovery situations of table compare (n=100)
2nd day intestinal flora quantitative analysis results after the treatment of 4 each group experiment mice of table
2nd day intestinal flora quantitative analysis results after the treatment of 5 each group experiment mice of table
2nd day intestinal flora quantitative analysis results after the treatment of 6 each group experiment mice of table
Obtained composition in embodiment 1-5 is used for into clinical testing, 20 diarrhea patients of each embodiment, men and women are each
Half, oral three days, once a day, therapeutic effect was shown in Table 7.
Table 7
SEQUENCE LISTING
<110>Tian Yi health sciences research institute（Zhenjiang）Co., Ltd
<120>A kind of composition of regulating intestinal canal colony balance
<130> SC20161025001
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 1490
<212> DNA
<213> Lactobacillus reuteri
<220>
<221> misc_feature
<222> (1)..(1490)
<400> 1
gggtacgcgg cggtgtgcta tacatgcaag tcgtacgcac tggcccaact gattgatggt 60
gcttgcacct gattgacgat ggattaccag tgagtggcgg acgggtgagt aacacgtagg 120
taacctgccc cggagcgggg gataacattt ggaaacagat gctaataccg cataacaaca 180
aaagccacat ggcttttgtt tgaaagatgg ctttggctat cactctggga tggacctgcg 240
gtgcattagc tagttggtaa ggtaacggct taccaaggcg atgatgcata gccgagttga 300
gagactgatc ggccacaatg gaactgagac acggtccata ctcctacggg aggcagcagt 360
agggaatctt ccacaatggg cgcaagcctg atggagcaac accgcgtgag tgaagaaggg 420
tttcggctcg taaagctctg ttgttggaga agaacgtgcg tgagagtaac tgttcacgca 480
gtgacggtat ccaaccagaa agtcacggct aactacgtgc cagcagccgc ggtaatacgt 540
aggtggcaag cgttatccgg atttattggg cgtaaagcga gcgcaggcgg ttgcttaggt 600
ctgatgtgaa agccttcggc ttaaccgaag aagtgcatcg gaaaccgggc gacttgagtg 660
cagaagagga cagtggaact ccatgtgtag cggtggaatg cgtagatata tggaagaaca 720
ccagtggcga aggcggctgt ctggtctgca actgacgctg aggctcgaaa gcatgggtag 780
cgaacaggat tagataccct ggtagtccat gccgtaaacg atgagtgcta ggtgttggag 840
ggtttccgcc cttcagtgcc ggagctaacg cattaagcac tccgcctggg gagtacgacc 900
gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 960
aattcgaagc tacgcgaaga accttaccag gtcttgacat cttgcgctaa ccttagagat 1020
aaggcgttcc cttcggggac gcaatgacag gtggtgcatg gtcgtcgtca gctcgtgtcg 1080
tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg ttactagttg ccagcattaa 1140
gttgggcact ctagtgagac tgccggtgac aaaccggagg aaggtgggga cgacgtcaga 1200
tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacggta caacgagtcg 1260
caagctcgcg agagtaagct aatctcttaa agccgttctc agttcggact gtaggctgca 1320
actcgcctac acgaagtcgg aatcgctagt aatcgcggat cagcatgccg cggtgaatac 1380
gttcccgggc cttgtacaca ccgcccgtca caccatggga gtttgtaacg cccaaagtcg 1440
gtggcctaac ctttatggag ggagccgcct aaggcggaca gagactgggt 1490
<210> 2
<211> 1474
<212> DNA
<213> Lactobacillus rhamnosus
<220>
<221> misc_feature
<222> (1)..(1474)
<400> 2
cgctggcggc gtgcctaata catgcaagtc gaacgagttc tgattattga aaggtgcttg 60
catcttgatt taattttgaa cgaatggcgg aagggtgaat aacccgtggg taacctggcc 120
ttaaatgggg gataaccttt ggaaaccgaa gctaataccg cataaatccc aaaaccgcat 180
ggttcttggc tgaaagatgg ggtaaactat cccttttgga tggacccccg gcgtattaac 240
ttgttggtga ggtaacggct ccaccaggga atgataccta acccaactga aaggttgatc 300
ggccaccttg ggactgagac ccggcccaaa ctcctaccgg agggagcaat agggaatctt 360
ccccaatgga cgccagtctg aaggaacaac cccccgtggg tgaaaaaggg tttccggtcc 420
taaaactctg ttgttggaaa aaaatggtcc gcaaaataac tggtggccgc gtggccgtat 480
ccaacccgaa agccacggct aactacctgc ccagcaccgc ggtaataccg aggtggcaag 540
cgttatccgg atttattggg cgtaaagcga gcgcaagcgg gttttttagt ctgatgggaa 600
agccctcggc ttaaccgaag aagtgcatcg gaaactggga aacttgaatg cagaagaaga 660
cagtggaact ccatgtgtag cggtgaaatg cctagatata tggaagaaca ccagtggcga 720
aagccgctgt ctggtctgta actgacgctg aagctccaaa gcctgggtag cgaacaggat 780
tagataccct gggagtccat gccgtaacga tgaatgctag tggtggaagg tttccgccct 840
tcaatgccga gcttacgcaa taagcaatcc gcctggggga atacgaccgg caggttgaaa 900
cttcaaagaa tttgaccggg gccgcacaag ccggtggagc attgtggttt aattcgaagc 960
acgcgaagaa cttaccaggt cttgacatct tttgatcact tgaagatcaa gtttcccttc 1020
gggcaaatga cagtggtgca tggttgtcgt cagctccgtg tcgtgagatg ttgggttaag 1080
tcccgcaacg agcgcaaccc ttatgactag ttgccagcat ttagttgggc actctagtaa 1140
gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat gccccttatg 1200
acctgggcta cacacgtgct acaatggatg gtacaacgag ttgcgagacc gcgaggtcaa 1260
gctaatctct taaagccatt ctcagttcgg actgtaggct gcaactcgcc tacacgaagt 1320
cggaatcgct agtaatcgcg gatcagcacg ccgcggtgaa tacgttcccg ggccttgtac 1380
acaccgcccg tcacaccatg agagtttgta acacccgaag ccggtggcgt aaccctttta 1440
gggagcgagc cgtctaaggt ggacaaatga tact 1474
Claims (5)
1. a kind of composition of regulating intestinal canal colony balance, it is characterised in that：Including dietary fiber 5-15 parts, inulin 2-10 parts,
Polydextrose 0.5-1.5 parts, oligoisomaltose 1-5 parts, FOS 0.5-1.5 parts, hundred million CFU of lactobacillus paracasei 30-40,
Lactobacillus reuteri (Lactobacillus reuteri) hundred million CFU of TR02 30-40, Lactobacillus rhamnosus (L.rhamnosus)
Hundred million CFU of TR08 18-26, newborn hundred million CFU of Bacillus bifidus 14-22.
2. the composition of a kind of regulating intestinal canal colony balance according to claim 1, it is characterised in that：Including dietary fiber
8-10 parts, inulin 5-7 parts, polydextrose 0.8-1.2 parts, oligoisomaltose 2-4 parts, FOS 0.8-1.2 parts, secondary cheese
Hundred million CFU of lactobacillus 33-37, lactobacillus reuteri (Lactobacillus reuteri) hundred million CFU of TR02 33-37, rhamnose breast
Hundred million CFU of bacillus (L.rhamnosus) TR08 20-24, newborn hundred million CFU of Bacillus bifidus 16-20.
3. the composition of a kind of regulating intestinal canal colony balance according to claim 1, it is characterised in that：Including dietary fiber
9 parts, 6 parts of inulin, 1 part of polydextrose, 3 parts of oligoisomaltose, 1 part of FOS, 3,500,000,000 CFU of lactobacillus paracasei, Roy
Family name's lactobacillus (Lactobacillus reuteri) 3,500,000,000 CFU of TR02, Lactobacillus rhamnosus (L.rhamnosus) TR08 22
Hundred million CFU, 1,800,000,000 CFU of newborn Bacillus bifidus.
4. a kind of preparation method of the composition of regulating intestinal canal colony balance, it is characterised in that：Comprise the following steps：
Dietary fiber, inulin, polydextrose, oligoisomaltose, FOS are weighed respectively, are mixed；Add secondary cheese breast
Bacillus, lactobacillus reuteri (Lactobacillus reuteri) TR02, Lactobacillus rhamnosus (L.rhamnosus) TR08, breast
Bacillus bifidus, mix, obtain final product.
5. application of the composition described in any one of Claims 1-4 in treatment regulating intestinal canal colony balance medicine is prepared.
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| CN110189827A (en) * | 2019-05-24 | 2019-08-30 | 天益健康科学研究院（镇江）有限公司 | A method of obtaining intestinal flora sequencer address |
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