CN110093287A - False chainlet Bifidobacterium CCFM1045, its composition, fermented food, purposes, microbial inoculum and its bacterial preparation process - Google Patents
False chainlet Bifidobacterium CCFM1045, its composition, fermented food, purposes, microbial inoculum and its bacterial preparation process Download PDFInfo
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- CN110093287A CN110093287A CN201910205851.2A CN201910205851A CN110093287A CN 110093287 A CN110093287 A CN 110093287A CN 201910205851 A CN201910205851 A CN 201910205851A CN 110093287 A CN110093287 A CN 110093287A
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- 230000005484 gravity Effects 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 244000005709 gut microbiome Species 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 239000002471 hydroxymethylglutaryl coenzyme A reductase inhibitor Substances 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000020824 obesity Nutrition 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- AHLBNYSZXLDEJQ-FWEHEUNISA-N orlistat Chemical compound CCCCCCCCCCC[C@H](OC(=O)[C@H](CC(C)C)NC=O)C[C@@H]1OC(=O)[C@H]1CCCCCC AHLBNYSZXLDEJQ-FWEHEUNISA-N 0.000 description 1
- 229960001243 orlistat Drugs 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- 238000009928 pasteurization Methods 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 108010004621 phosphoketolase Proteins 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229960005095 pioglitazone Drugs 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 230000007420 reactivation Effects 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 230000003252 repetitive effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000000276 sedentary effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 235000013024 sodium fluoride Nutrition 0.000 description 1
- 239000011775 sodium fluoride Substances 0.000 description 1
- AGDSCTQQXMDDCV-UHFFFAOYSA-M sodium;2-iodoacetate Chemical compound [Na+].[O-]C(=O)CI AGDSCTQQXMDDCV-UHFFFAOYSA-M 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 150000001467 thiazolidinediones Chemical class 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- HFFLGKNGCAIQMO-UHFFFAOYSA-N trichloroacetaldehyde Chemical compound ClC(Cl)(Cl)C=O HFFLGKNGCAIQMO-UHFFFAOYSA-N 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 235000019163 vitamin B12 Nutrition 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
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- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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- A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
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- A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
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Abstract
The invention discloses false chainlet Bifidobacterium CCFM1045, its composition, fermented food, purposes, microbial inoculum and its bacterial preparation process, present invention vacation chainlet Bifidobacterium CCFM1045 is resistant to human intestines and stomach's environment, significantly improves the exception of metabolic syndrome mice serum liver function index ALT and AST caused by high fat diet；The oral glucose tolerance for significantly improving metabolic syndrome mouse is abnormal, reduces glucose tolerance area under the drug-time curve, reduces serum insulin level；It significantly improves metabolic syndrome mouse Triglycerides in Serum as caused by high fat diet and total cholesterol level increases；Significantly improve the raising that Proteus belongs in enteron aisle caused by high fat diet, false chainlet Bifidobacterium CCFM1045, which is used to prepare, to be alleviated metabolic syndrome, reduces Parkinson, the pathogenetic pharmaceutical composition of the diseases such as enteric infection and urinary tract, renal infection and fermented food have very extensive application prospect.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to false chainlet Bifidobacterium CCFM1045, its composition, hair
Ferment food, purposes, microbial inoculum and its bacterial preparation process.
Background technique
In recent years, with the continuous development of Chinese society economic level, public living standard constantly improves, diet knot
Carbohydrate in structure, grease specific gravity increasingly increase, and to allow people easily to form sedentary for the limitation of many operating conditions
Behavioural habits.These external environmental factors combine the inherent cause of itself to result in population of China Metabolic syndrome jointly and levy
The continuous promotion of sick rate.It is reported that between 2002-2010, the ratio of metabolic syndrome is suffered from China 20 years old or more crowd
21.3% has been risen to from 5.4%, has increased 4 times, and it is contemplated that the following data still may cumulative year after year.Therefore, seek
It is a kind of it is suitable alleviate metabolic syndrome means be very necessary.
Metabolic syndrome is syndrome made of cluster metabolic risk factors set, using insulin resistance as center ring
Section and pathologic basis, patient is high in the ratio of centric obesity (waist-to-hipratio abnormal), and often along with, high fasting blood glucose, height
Many unfavorable signs such as blood lipid (serum triglyceride and low-density lipoprotein content increase), hypertension.Metabolic syndrome patient
Intracorporal Abnormality of Glycolipid Metabolism directly enhances the occurrence risk of atherosclerotic cardiovascular disease and diabetes B.
Although metabolic syndrome and insulin resistance as the factor that clinically prediction cardiovascular disease occurs there are still it is certain not really
It is qualitative, but still there is certain reference significance, if being controlled not in time, metabolic syndrome, which is further aggravated, be may lead
Play a series of organ diseases such as fatty liver, atherosclerosis, hyperuricemia.
Improve diet for the remedy measures mainly reinforcement movement that metabolic syndrome uses at present and is controlled in conjunction with drug
It treats, target is to reduce every risk factor of metabolic syndrome, and main includes the drug such as orlistat, chlorine lost weight
Ka Selin；Mitigate such as melbine, thiazolidinediones (Rosiglitazone, Pioglitazone) of insulin resistance；Reduce blood lipid
The statins such as Simvastatin of fibrate such as fenofibrate and norcholesterol etc.；The angiotensins of blood pressure lowering
Converting enzyme inhibitor class drug such as captopril etc..These drugs have for the single risk factors of items of metabolic syndrome
Good curative effect, still, there are certain side effects for prolonged administration of drugs, and stimulation one can be caused by such as taking melbine for a long time
The gastrointestinal tract of a little patients causes discomfort and may will affect absorption of the patient to vitamin B12, and Rosiglitazone can cause liver function
Energy loss injures oedema etc..
Summary of the invention
The purpose of this section is to summarize some aspects of the embodiment of the present invention and briefly introduce some preferable implementations
Example.It may do a little simplified or be omitted to avoid this is made in this section and the description of the application and the title of the invention
Partially, the purpose of abstract of description and denomination of invention is fuzzy, and this simplification or omission cannot be used for limiting model of the invention
It encloses.
In view of above-mentioned technological deficiency, the present invention is proposed.
Therefore, as one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides false small
Chain Bifidobacterium CCFM1045.
In order to solve the above technical problems, the present invention provides the following technical scheme that false chainlet Bifidobacterium CCFM1045
(Bifidobacteriumpseudocatenulatum), Guangdong Province microorganism fungus kind guarantor was preserved on 03 04th, 2019
Hiding center, address are the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building, and Guangdong Microbes Inst, deposit number is
GDMCC No:60600.
As one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides a kind of alleviation generation
Thank to the composition of syndrome.
In order to solve the above technical problems, the present invention provides the following technical scheme that a kind of combination for alleviating metabolic syndrome
Object, in which: the composition acceptable carrier including vacation chainlet Bifidobacterium CCFM1045 and its pharmaceutically；Described
The dosage form of composition includes granule, capsule, tablet, pill or oral liquid formulation；The pharmaceutically acceptable carrier
Including one of medically usually used filler, adhesive, wetting agent, disintegrating agent, lubricant, corrigent or a variety of.
As one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides a kind of fermentation food
Product.
In order to solve the above technical problems, the present invention provides the following technical scheme that a kind of fermented food, in which: the hair
Ferment food is dairy products, bean product and the fruit and vegetable product produced using the leavening containing false chainlet Bifidobacterium CCFM1045,
The fermented food includes solid-state food, food liquid, semisolid food；The leavening refers to double containing the false chainlet
The product of discrimination bacillus CCFM1045 can generate terminal food using the product with Rapid Fermentation.
A kind of preferred embodiment as the fermented food: the dairy products include milk, sour cream or cheese；
The bean product include soymilk, fermented soya bean or beans sauce；The fruit and vegetable product includes cucumber, carrot, beet, celery or circle
Chinese cabbage product.
As one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides false chainlet bifid
Application of the bacillus CCFM1045 in preparation in vivo field planting probiotics.
As one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides false chainlet bifid
Bacillus CCFM1045 is in preparation reducing blood lipid, hypoglycemic, improvement insulin resistance, alleviation hepatic injury, anti-Parkinson, anti-enteron aisle sense
Application in dye, anti-urinary tract and renal infection drug.
It is supported as vacation chainlet Bifidobacterium CCFM1045 of the present invention in preparation reducing blood lipid, hypoglycemic, improvement insulin
A kind of preferred side that is anti-, alleviating hepatic injury, anti-Parkinson, anti-enteric infection, anti-urinary tract and the application in renal infection drug
Case: the vacation chainlet Bifidobacterium CCFM1045 can improve the Indexes Abnormality of metabolic syndrome mouse, reduce metabolic syndrome
Mice serum total cholesterol (TC), liver tg (TG) content；The vacation chainlet Bifidobacterium CCFM1045 can be improved
Metabolic syndrome mouse islets element sensibility；The vacation chainlet Bifidobacterium CCFM1045 can reduce to be caused by high fat diet
Proteus Proteus abundance rising, improve the mouse intestinal flora structure disturbance that is caused by high fat diet, subtract
The generation of the diseases such as few Parkinson, enteric infection and urinary tract, renal infection.
As one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides the fermentation
Food answering in the functional food that metabolic syndrome, reduction Parkinson, enteric infection, urinary tract, renal infection are alleviated in preparation
With.
As one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides a kind of containing vacation
The microbial inoculum of chainlet Bifidobacterium CCFM1045.
In order to solve the above technical problems, the present invention provides the following technical scheme that a kind of contain false chainlet Bifidobacterium
The microbial inoculum of CCFM1045, in which: the microbial inoculum is the powder for being dried to obtain the bacterium solution containing false chainlet Bifidobacterium CCFM1045
Agent, containing greater than 106The false chainlet Bifidobacterium CCFM1045 of the activity of CFU/g.
As one aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides preparation containing vacation
The method of the microbial inoculum of chainlet Bifidobacterium CCFM1045.
In order to solve the above technical problems, the present invention provides the following technical scheme that preparation contains false chainlet Bifidobacterium
The method of the microbial inoculum of CCFM1045 comprising,
The preparation of culture medium: the culture medium of false chainlet Bifidobacterium is modified MRS culture medium, the modified MRS culture medium
For MRS culture medium+0.05%L- cysteine hydrochloride, adjusting its pH is 6.8 ± 0.2, obtains culture medium；
Protectant preparation: it is mixed with to obtain containing 100g/L-150g/L defatted milk using water and protective agent raw material
Powder, 100g/L-150g/L maltodextrin, 140g/L-160g/L trehalose, the freeze drying protectant that surplus is water；
False chainlet Bifidobacterium CCFM1045 strain is inoculated with according to the inoculum concentration of the 2-4% in terms of the quality of the culture medium
In culture medium after to the 15-25min that sterilizes under the conditions of 119-123 DEG C, then cultivated under 35-39 DEG C of anaerobic condition of temperature
24-48h is that 6.8-7.2 phosphate buffer cleans 2-4 times with pH, is resuspended with the protective agent, makes bacterium is dense to reach
1010CFU/mL；Then, suspension preculture 50-70min under 37 DEG C of anaerobic conditions of temperature is allowed, then at -15~-20 DEG C
Pre-freeze 8-14h finally carries out vacuum freeze drying and obtains the fermenting agent.
Beneficial effects of the present invention: vacation chainlet Bifidobacterium CCFM1045 provided by the invention is resistant to human intestines and stomach's ring
Border significantly improves the exception of metabolic syndrome mice serum liver function index ALT and AST caused by high fat diet；It significantly improves
The oral glucose tolerance of metabolic syndrome mouse is abnormal, reduces glucose tolerance area under the drug-time curve, reduces serum insulin water
It is flat；It significantly improves metabolic syndrome mouse Triglycerides in Serum as caused by high fat diet and total cholesterol level increases；
Significantly improve the raising that Proteus belongs in enteron aisle caused by high fat diet.The false chainlet Bifidobacterium CCFM1045 is used for
Preparation alleviates metabolic syndrome, reduces Parkinson, the pathogenetic pharmaceutical composition of the diseases such as enteric infection and urinary tract, renal infection
With fermented food, there is very extensive application prospect.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, making required in being described below to embodiment
Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, right
For those of ordinary skill in the art, without any creative labor, it can also be obtained according to these attached drawings
Other attached drawings.Wherein:
Fig. 1 is the colonial morphology of false chainlet Bifidobacterium CCFM1045；
Fig. 2 is the shadow that false chainlet Bifidobacterium CCFM1045 belongs to abundance to Proteus in metabolic syndrome mouse intestinal
It rings；
Fig. 3 is influence of the false chainlet Bifidobacterium CCFM1045 to metabolic syndrome mouse fasting blood glucose level；
Fig. 4 is influence of the false chainlet Bifidobacterium CCFM1045 to the oral glucose tolerance of metabolic syndrome mouse；
Fig. 5 is false chainlet Bifidobacterium CCFM1045 to oral glucose tolerance area under the drug-time curve (AUCglucose)；
Fig. 6 is influence of the false chainlet Bifidobacterium CCFM1045 to mice serum insulin content；
Fig. 7 is shadow of the false chainlet Bifidobacterium CCFM1045 to metabolic syndrome mice serum total cholesterol (TC) level
It rings；
Fig. 8 is shadow of the false chainlet Bifidobacterium CCFM1045 to metabolic syndrome mouse liver triglycerides (TG) level
It rings；
Fig. 9 is false chainlet Bifidobacterium CCFM1045 to glutamic-oxalacetic transaminease (AST) and paddy in metabolic syndrome mice serum
The influence of pyruvic transaminase (ALT) level.Note: a, b, c indicate it is different letter representated by group all exist significant difference (P <
0.05)。
Specific embodiment
In order to make the foregoing objectives, features and advantages of the present invention clearer and more comprehensible, right combined with specific embodiments below
A specific embodiment of the invention is described in detail.
In the following description, numerous specific details are set forth in order to facilitate a full understanding of the present invention, but the present invention may be used also
To be implemented using other than the one described here other way, those skilled in the art can be without prejudice in the present invention
Similar popularization is done in the case where culvert, therefore the present invention is not limited by the specific embodiments disclosed below.
Secondly, " one embodiment " or " embodiment " referred to herein, which refers to, may be included at least one realization of the present invention
A particular feature, structure, or characteristic in mode." in one embodiment " that different places occur in the present specification is not equal
Refer to the same embodiment, nor the individual or selective embodiment mutually exclusive with other embodiments.
The vacation chainlet Bifidobacterium CCFM1045 has following biological characteristics:
(1) thallus feature: it is in Gram-positive, does not form spore, no motion of bacterium.
(2) colony characteristics: apparent bacterium colony is formed within Anaerobic culturel 36 hours, diameter is between 0.5-2mm, positive form
Circle, side form are in overshooting shape, and neat in edge, milky is opaque, and surface wettability is smooth, not chromogenesis, referring to attached
Fig. 1.
(3) it growth characteristics: under conditions of 37 DEG C of constant-temperatureanaerobic anaerobics, cultivates and reaches within about 24 hours pair in mMRS culture medium
Number latter stage.
(4) there is preferable tolerance to simulation gastro-intestinal Fluid；
(5) it can significantly improve the exception of serum liver function index caused by high fat diet；
(6) oral glucose tolerance that can significantly improve metabolic syndrome mouse is abnormal；
(7) alleviate oral glucose tolerance area under the drug-time curve to increase；
(8) alleviate serum insulin content to increase；
(9) Triglycerides in Serum and total cholesterol level is adjusted, it is made to be reduced to normal level,
(10) raising that Proteus belongs in the enteron aisle as caused by high fat diet can significantly be restored；
The extracting method of this bacterial strain are as follows:
(1) separation screening of lactic acid bacteria
(1) 108 years old women old man fresh excreta of the 1g from Henan Boai County Changshou village is taken.It is coated on after gradient dilution
MMRS solid medium is placed in lower 37 DEG C of anaerobic environment and cultivates 72 hours.
(2) colonial morphology, picking colony scribing line purifying are observed and recorded.
(3) in mMRS fluid nutrient medium, 37 DEG C are cultivated 48 hours, and gained bacterium colony carries out Gram's staining, record bacterium colony
Form.
(4) Gram negative bacterial strain and gram-positive cocci in reject bacterium colony is selected to obtain Gram-positive bar
Bacterium.
(5) after catalase analysis, reject catalase-positive organism strain retains catalase negative strain.
(2) Preliminary Identification of Bifidobacterium: fructose-6-phosphate salt phosphoketolase measuring method
(l) lactic acid bacteria that step (1) is screened is cultivated for 24 hours in liquid mMRS culture solution, then 1mL is taken to train
It supports object 8000rpm and is centrifuged 2min；
(2) with the 0.05M KH of the pH 6.5 containing 0.05% (mass percent) cysteine2PO4Solution washes twice；
(3) it is resuspended in the above-mentioned phosphate-buffered that 200uL is added to 0.25% (mass percent) Triton X-100
Liquid；
(4) addition 50uL concentration is the mixed liquor of 6mg/mL sodium fluoride and 10mg/mL sodium iodoacetate and 50uL concentration is
The fructose-6-phosphate of 80mg/mL, 37 DEG C of incubation 1h；
(5) the light amine of hydrochloric acid that addition 300uL concentration is 0.139g/mL, pH 6.5, and in being placed at room temperature for 10 min；
(6) trichloroacetic acid and 4M HCI of 200uL15% (mass percent) are added respectively；
(7) addition 200uL contains the 0.1M HCI of 5% (mass percent) ferric trichloride, if system rapidly go to it is red
Color, as F6PPK are positive, can tentatively conclude it for Bifidobacterium.
(3) molecular biology identification of Bifidobacterium
(l) single bacterium genome is extracted and (is carried out according to TIANamp Bacteria DNA kit operating procedure)
A. lactic acid bacteria overnight incubation step (2) screened takes bacteria suspension 1mL in 1.5mL centrifuge tube, and 10,
000rpm (~11,500 × g) is centrifuged 1min, as far as possible exhaustion supernatant；
B. 180 μ L buffer (20mM Tris, pH 8.0 are added into bacterial sediment；2mM Na2-EDTA；1.2%
Triton；Final concentration of 20mg/mL lysozyme (lysozyme must be dissolved in buffer with lysozyme dry powder and be prepared,
Otherwise will lead to bacteriolyze kinase inactive)), 37 DEG C of processing 30min or more；
C. 20 μ L Proteinase K solution are added into pipe, mix；
D. be added 220 μ L buffer GB, vibrate 15sec, 70 DEG C of placements 10min, solution strain it is limpid, brief centrifugation with
Remove the droplet of cap wall；
E. plus 220 μ L dehydrated alcohols, sufficiently oscillation mixes 15sec, at this time it is possible that flocculent deposit, brief centrifugation
To remove the droplet of cap wall；
F., previous step acquired solution and flocculent deposit be all added in an adsorption column CB3 (adsorption column is put into collecting pipe
In), 12,000rpm (~13,400 × g) are centrifuged 30sec, outwell waste liquid, adsorption column CB3 is put into collecting pipe；
G. 500 μ L buffer GD (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,
12,000rpm (~13,400 × g) are centrifuged 30sec, outwell waste liquid, adsorption column CB3 is put into collecting pipe；
H. 600 μ L rinsing liquid PW (please first check whether before use and dehydrated alcohol has been added) are added into adsorption column CB3,
12,000rpm (~13,400 × g) are centrifuged 30sec, outwell waste liquid, adsorption column CB3 is put into collecting pipe；Repetitive operation is primary；
I. adsorption column CB3 is put back in collecting pipe, 12,000rpm (~13,400 × g) are centrifuged 2min, outwell waste liquid.It will
Adsorption column CB3, which is placed in, to be placed at room temperature for several minutes, thoroughly to dry rinsing liquid remaining in adsorbent material；
J. adsorption column CB3 is transferred in a clean centrifuge tube, 50-200 is vacantly added dropwise to the intermediate position of adsorbed film
μ L elution buffer TE, is placed at room temperature for 2-5min, and 12,000rpm (~13,400 × g) are centrifuged 2min, by solution be collected into from
In heart pipe.
(2)16S rDNA PCR
A. bacterial 16 S rDNA 50uLPCR reaction system:
10 × Taq buffer, 5uL；DNTP, 5uL；27F, 0.5uL；1492R, 0.5uL；Taq enzyme, 0.5uL；Template,
0.5uL；DdH2O, 38uL.
B.PCR condition:
95℃5min；95℃10s；55℃30s；72℃30s；step2-4 30×；72℃5min；12℃ 2min；
(3) 1% Ago-Gel is prepared, is later mixed PCR product with 10000 × loading buffer, applied sample amount
2uL, 120V run 30min, then carry out gel imaging；
(4) PCR product of 16S rDNA is subjected to sequencing analysis, obtained sequence results is existed using BLAST
It scans for comparing with similitude in GeneBank, chooses the lactic acid bacteria that sequencing result is accredited as false chainlet Bifidobacterium, ﹣ 80
DEG C preservation is spare.
Embodiment 1: false chainlet Bifidobacterium CCFM1045 has good tolerance to simulation gastro-intestinal Fluid
The false chainlet Bifidobacterium CCFM1045 of freezen protective is inoculated in mMRS culture medium (MRS culture medium+0.05%
Cysteine hydrochloride) in, in 37 DEG C of Anaerobic culturel 48h of temperature, then after mMRS culture solution secondary culture 2~3 times, take 1mL
The culture solution of false chainlet Bifidobacterium CCFM1045 (contains 1% pepsin, pH=with 2.5 artificial simulation gastric juices of 9.0mL pH
2.5 mMRS culture medium) mixing, and the Anaerobic culturel at 37 DEG C, it is sampled respectively in 0h, 0.5h, 1h and 2h, with mMRS fine jade
The casting culture of rouge culture medium carries out plate count, measures viable count and calculates its survival rate.
Survival rate be viable count logarithm in the culture solution in sampling in 0h when viable count logarithm it
Than being indicated with %.Take the culture solution of 1mL vacation chainlet Bifidobacterium CCFM1045 that 9mL artificial simulation intestinal juice is added (containing 0.3% N
Cholate, 1% trypsase, pH=8.0 mMRS culture medium) in, the Anaerobic culturel at 37 DEG C, respectively in 0h, 0.5h, 1h and
It is sampled when 2h, carries out plate count with the casting culture of mMRS agar medium, measure viable count and calculate its survival rate.
Survival rate be viable count logarithm in the culture solution in sampling in 0h when the ratio between viable count logarithm, with % table
Show.Experimental result is as shown in Table 1 and Table 2.The result shows that false chainlet Bifidobacterium CCFM1045 to artificial gastro-intestinal Fluid have compared with
Good tolerance.
Tolerance of the 1 vacation chainlet Bifidobacterium CCFM1045 of table in artificial simulation gastric juices
Tolerance of the 2 vacation chainlet Bifidobacterium CCFM1045 of table in artificial simulation intestinal juice
Embodiment 2: false chainlet Bifidobacterium CCFM1045 has no toxic side effect to C57BL/6JBL/6J mouse
False chainlet Bifidobacterium CCFM1045 thallus is resuspended in 3% sucrose solution, be made concentration be 3.0 ×
108The bacteria suspension of CFU/mL.Health male C57BL/6JBL/6J mouse 6 of weight 20g or so is taken, is adapted to environment one week
Afterwards, it is primary that the concentration bacteria suspension stomach-filling is given daily, is observed one week, record death and body weights.
These test results are listed in Table 3 below.These results indicate that feeding concentration 3.0 × 108The false chainlet of CFU/mL is double
Discrimination bacillus CCFM1045 does not cause to significantly affect to mouse, and weight is generated without significant changes, the no phenomena of mortality.Mouse appearance without
Obvious pathological symptom.
The variation and death condition of 3 mouse weight of table
Note :-: mouse is without death
Embodiment 3: false chainlet Bifidobacterium CCFM1045, which has the intestinal bacilli illness as caused by high lipid food, to be restored
Effect
Health male C57BL/6JBL/6J mouse 36 of weight 18-20g is taken, adapts to environment 1 week, is randomly divided into 6 groups:
Blank control group (NC), hyperlipidemia model control group (HFD), Simvastatin control group (SC), Rosiglitazone control group (RH), vacation
Chainlet Bifidobacterium CCFM1045 intervention group (CCFM1045), animal bifidobacteria BB12 control group (BB12), every group contains mouse
6, the dosage of stomach-filling bacteria suspension is 3.0 × 108CFU/mL is resuspended in 3% sucrose solution.Experimental animal grouping and place
Reason method is shown in Table 4:
The grouping of 4 experimental animal of table
Test latter stage collects mouse fresh excreta and freezes in -80 DEG C, extracts the macro genome in excrement, and utilized for two generations
Sequenator analyzes intestinal microflora.When off-test, mouse is deprived of food but not water 12h, and 10% hydration is injected intraperitoneally
After the anesthesia of chloral solution, Culling heart blood is aided with cervical dislocation execution.3000 × g of blood sample, it is centrifuged under the conditions of 4 DEG C
15min takes supernatant, and -80 DEG C freeze for measuring associated serum index.Partial liver is immediately placed in the physiology of pre-cooling after collecting
Blood is removed in rinsing in salt water, is put into paraformaldehyde fixed, and remainder liver is quick-frozen in liquid nitrogen and is transferred to -80 DEG C and freezes,
It is subsequent that liver homogenate is made to be used to measure index of correlation, specifically the preparation method is as follows: a certain amount of liver organization is weighed, by 1:9 ratio
Example is added physiological saline and carries out tissue grinder, and 3000r is centrifuged 10min, takes supernatant to freeze spare in -80 DEG C.
It is as shown in Figure 2 that flora analyzes experimental result.Proteus belongs in the model group stool in mice of high lipid food induction
Enteric microorganism significantly increases, and the abundance that the intake of false chainlet Bifidobacterium CCFM1045 can belong to Proteus is adjusted back, this
Show that the false chainlet Bifidobacterium CCFM1045 that the present invention screens has reduction Parkinson, enteric infection and urinary tract, renal infection
Etc. the pathogenetic function of diseases.
Embodiment 4: false chainlet Bifidobacterium CCFM1045 can reduce metabolic syndrome mouse (empty stomach) blood glucose level
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.
Experimental result is as shown in Figure 3.Model group mouse fasting blood-glucose significantly increases, stomach-filling vacation chainlet Bifidobacterium
CCFM1045 significantly reduces the fasting blood glucose level of model mice and close to blank control group.It reduces mouse fasting blood
The ability of sugar level is similar with Rosiglitazone and animal bifidobacteria BB12.
Embodiment 5: false chainlet Bifidobacterium CCFM1045 can enhance metabolic syndrome mouse glucose tolerance ability
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.Before mouse is put to death, it is deprived of food but not water 12h, abdomen
Chamber injectable dextrose monohydrate solution (2g/kg weight), measures 0,30,60,120min blood glucose situation respectively.
Experimental result is as shown in Figures 4 and 5.As seen from Figure 4, model group mouse to the tolerance of glucose compared with
Difference, after stomach-filling glucose, blood glucose value is significantly raised, and declines slow.From fig. 5, it can be seen that stomach-filling vacation chainlet Bifidobacterium
CCFM1045 significantly reduces AUCglucoseArea, and with normal group without significant difference.This just illustrates, false chainlet Bifidobacterium
CCFM1045 can significantly improve oral glucose tolerance, and function and effect are better than animal bifidobacteria BB12.These results and blood glucose
Index result is consistent, prompts false chainlet Bifidobacterium CCFM1045 that can further drop by enhancing glucose tolerance
Low blood glucose level.
Embodiment 6: false chainlet Bifidobacterium CCFM1045 can reduce metabolic syndrome mice serum insulin (INS)
It is horizontal
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.When off-test, mouse is deprived of food but not water
12h, after 10% chloraldurate solution anesthesia is injected intraperitoneally, Culling heart blood.3000 × g of blood sample, it is centrifuged under the conditions of 4 DEG C
10min takes supernatant, according to the content of insulin (INS) in the detection method measurement blood of kit.
Experimental result is as shown in Fig. 6.High fat diet induces C57BL/6J mice serum insulin as seen from Figure 6
Content significantly rises, and serum insulin content can be significantly reduced in vacation chainlet Bifidobacterium CCFM1045, to insulin
Horizontal recovery capability is significantly stronger than control drug and animal bifidobacteria BB12.
Embodiment 7: false chainlet Bifidobacterium CCFM1045 can reduce metabolic syndrome mice serum total cholesterol (TC)
It is horizontal
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.When off-test, mouse is deprived of food but not water
12h, after 10% chloraldurate solution anesthesia is injected intraperitoneally, Culling heart blood.3000 × g of blood sample, it is centrifuged under the conditions of 4 DEG C
10min takes supernatant, according to the content of total cholesterol (TC) in the detection method measurement blood of kit.
Experimental result is as shown in Fig. 7.As seen from Figure 7, high fat diet group mice serum total cholesterol level is obvious
It increases, stomach-filling vacation chainlet Bifidobacterium CCFM1045 can reduce the content of serum total cholesterol, to the extensive of total cholesterol level
Reactivation power is better than control drug and animal bifidobacteria BB12.
Embodiment 8: false chainlet Bifidobacterium CCFM1045 can reduce metabolic syndrome mouse liver triglycerides (TG)
It is horizontal
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.When off-test, mouse is deprived of food but not water
12h, after 10% chloraldurate solution anesthesia is injected intraperitoneally, Culling heart blood, cervical dislocation is put to death.- 80 DEG C of liver are taken to freeze,
A certain amount of liver organization is weighed when measurement, and physiological saline is added in 1:9 ratio and carries out tissue grinder, 3000r is centrifuged 10min, takes
Supernatant, according to the content of triglycerides (TG) in the detection method measurement liver of kit.
Experimental result is as shown in Fig. 8.It can be seen from experimental result compared with Normal group, high fat diet group is small
Mouse liver tg content significantly increases, and stomach-filling vacation chainlet Bifidobacterium CCFM1045 can reduce containing for liver tg
Amount, the horizontal abnormal rise of drug Rosiglitazone control group liver TG, and stomach-filling BB12 is without remarkable effect, false chainlet Bifidobacterium
CCFM1045 is significantly stronger than control drug and animal bifidobacteria BB12 to the recovery capability of liver tg level.
Embodiment 9: false chainlet Bifidobacterium CCFM1045 to metabolic syndrome mice serum glutamic-oxalacetic transaminease (AST) and
The influence of glutamic-pyruvic transaminase (ALT) level
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.When off-test, mouse is deprived of food but not water
12h, after 10% chloraldurate solution anesthesia is injected intraperitoneally, Culling heart blood.3000 × g of blood sample, it is centrifuged under the conditions of 4 DEG C
10min takes supernatant, according to glutamic-oxalacetic transaminease (AST) and glutamic-pyruvic transaminase (ALT) in the detection method measurement blood of kit
Content.
Experimental result is as shown in Fig. 9.It can be seen from experimental result compared with Normal group, high fat diet group is small
The content of mouse serum AST and ALT significantly increase, false chainlet Bifidobacterium CCFM1045 can reduce in mice serum AST and
The level of ALT, and BB12 is not significantly reduced effect to AST and ALT level, false chainlet Bifidobacterium CCFM1045 is to small
The recovery capability of mouse serum glutamic oxalacetic transaminase (AST) and glutamic-pyruvic transaminase (ALT) level is significantly stronger than control drug and animal is double
Discrimination bacillus BB12.
Embodiment 10: the leavening containing false chainlet Bifidobacterium CCFM1045 produces fermented fruits and vegetables juice
It squeezes the juice after selecting fresh fruit and vegetables to clean, food-grade albumen peptone is added according to the ratio of 5g/L, then carries out
Flash pasteurization is cooled to 37 DEG C at 140 DEG C of temperature after high temperature thermal sterilization 2 seconds immediately, then accesses prepared by the present invention
False chainlet Bifidobacterium CCFM1045 bacteria fermentation agent, makes its concentration reach 106CFU/mL or more, and ferment at 37 DEG C
For 24 hours, then stored refrigerated at 4 DEG C of temperature, then obtain the fruit containing false chainlet Bifidobacterium CCFM1045 viable bacteria of the invention
Vegetable beverage.
It should be noted that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although referring to preferable
Embodiment describes the invention in detail, those skilled in the art should understand that, it can be to technology of the invention
Scheme is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered in this hair
In bright scope of the claims.
Claims (10)
1. vacation chainlet Bifidobacterium CCFM1045 (Bifidobacterium pseudocatenulatum), in 03 month 2019
It is preserved within 04th Guangdong Province's Culture Collection, address is the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building, extensively
Eastern institute of microbiology, province, deposit number are GDMCC No:60600.
2. a kind of composition for alleviating metabolic syndrome, it is characterised in that: the composition includes false chainlet Bifidobacterium
CCFM1045 and its pharmaceutically acceptable carrier；The dosage form of the composition includes granule, capsule, tablet, ball
Agent or oral liquid formulation；The pharmaceutically acceptable carrier includes medically usually used filler, adhesive, wetting
One of agent, disintegrating agent, lubricant, corrigent are a variety of.
3. a kind of fermented food, it is characterised in that: the fermented food is using containing false chainlet Bifidobacterium CCFM1045
Dairy products, bean product and the fruit and vegetable product of leavening production, the fermented food includes solid-state food, food liquid, semisolid
Food；The leavening refers to containing the product of the false chainlet Bifidobacterium CCFM1045, can quickly be sent out using the product
Ferment generates the fermented food.
4. fermented food according to claim 3, it is characterised in that: the dairy products include milk, sour cream or dry
Junket；The bean product include soymilk, fermented soya bean or beans sauce；The fruit and vegetable product include cucumber, carrot, beet, celery or
Cabbage product.
5. application of the vacation chainlet Bifidobacterium CCFM1045 in preparation in vivo field planting probiotics.
6. vacation chainlet Bifidobacterium CCFM1045 is preparing reducing blood lipid, hypoglycemic, improvement insulin resistance, is alleviating hepatic injury, anti-
Application in Parkinson, anti-enteric infection, anti-urinary tract and renal infection drug.
7. application as claimed in claim 6, it is characterised in that: the vacation chainlet Bifidobacterium CCFM1045 can improve metabolism
The Indexes Abnormality of syndrome mouse reduces metabolic syndrome mice serum total cholesterol TC, liver tg TG content；It is described
False chainlet Bifidobacterium CCFM1045 can be improved metabolic syndrome mouse islets element sensibility；The vacation chainlet Bifidobacterium
CCFM1045 can reduce the rising of the abundance of Proteus Proteus as caused by high fat diet, improve by high fat diet
The mouse intestinal flora structure disturbance of initiation reduces the generation of the diseases such as Parkinson, enteric infection and urinary tract, renal infection.
8. fermented food described in claim 3 or 4 alleviates metabolic syndrome, reduction Parkinson, enteric infection, urine in preparation
Road, renal infection functional food in application.
9. a kind of microbial inoculum containing false chainlet Bifidobacterium CCFM1045, it is characterised in that: the microbial inoculum is will be containing false chainlet
The pulvis that the bacterium solution of Bifidobacterium CCFM1045 is dried to obtain, containing greater than 106The false chainlet Bifidobacterium of the activity of CFU/g
CCFM1045。
10. the method for microbial inoculum of the preparation containing false chainlet Bifidobacterium CCFM1045, it is characterised in that: including,
The preparation of culture medium: the culture medium of false chainlet Bifidobacterium is modified MRS culture medium, and the modified MRS culture medium is MRS
Culture medium+0.05%L- cysteine hydrochloride, adjusting its pH is 6.8 ± 0.2, obtains culture medium；
Protectant preparation: using water and protective agent raw material be mixed with to obtain containing 100g/L-150g/L skimmed milk power,
100g/L-150g/L maltodextrin, 140g/L-160g/L trehalose, the freeze drying protectant that surplus is water；
False chainlet Bifidobacterium CCFM1045 strain is inoculated into according to the inoculum concentration of the 2-4% in terms of the quality of the culture medium
In culture medium under the conditions of 119-123 DEG C after sterilizing 15-25min, 24- then is cultivated under 35-39 DEG C of anaerobic condition of temperature
48h is that 6.8-7.2 phosphate buffer cleans 2-4 times with pH, is resuspended with the protective agent, makes bacterium is dense to reach 1010CFU/
mL；Then, suspension preculture 50-70min under 37 DEG C of anaerobic conditions of temperature, then in -15~-20 DEG C of pre-freeze 8-14h, carry out
Vacuum freeze drying obtains the fermenting agent.
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| CN113559129A (en) * | 2021-08-18 | 2021-10-29 | 唐颐控股(深圳)有限公司 | Nano armor protection unicellular product for treating Parkinson's disease through bacteria and preparation method thereof |
| CN113559129B (en) * | 2021-08-18 | 2023-07-28 | 唐颐控股(深圳)有限公司 | Nanometer armor protection single cell product for treating parkinsonism through bacteria and preparation method thereof |
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