CN110468070A - Lactobacillus rhamnosus CCFM1060, its fermented food and bacterial preparation process - Google Patents
Lactobacillus rhamnosus CCFM1060, its fermented food and bacterial preparation process Download PDFInfo
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- CN110468070A CN110468070A CN201910765009.4A CN201910765009A CN110468070A CN 110468070 A CN110468070 A CN 110468070A CN 201910765009 A CN201910765009 A CN 201910765009A CN 110468070 A CN110468070 A CN 110468070A
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- XPFJYKARVSSRHE-UHFFFAOYSA-K trisodium;2-hydroxypropane-1,2,3-tricarboxylate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].[Na+].[Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O XPFJYKARVSSRHE-UHFFFAOYSA-K 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- 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.
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
- A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L19/00—Products from fruits or vegetables; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/065—Microorganisms
-
- 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
-
- 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
- A23V2400/11—Lactobacillus
- A23V2400/175—Rhamnosus
-
- 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
- C12R2001/225—Lactobacillus
Abstract
The invention discloses Lactobacillus rhamnosus CCFM1060, its fermented food and bacterial preparation process, significantly reduce the abundance raising that the Proteus of High cholesterol diet induction high in fat belongs to, and reduce the diseases such as urinary obstruction, enteric bacteria migration and enteritis and occur；Significantly improve the disorders of lipid metabolism of NAFLD patient；Significantly improve the insulin resistance of NAFLD mouse；The raising of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminease in serum can be adjusted back；The concentration for significantly reducing low density lipoprotein cholesterol in serum, reduces the risk of cardiovascular disease；The SOD that can be significantly improved in NAFLD patient's liver is horizontal；It can significantly improve liver inflammation；It can significantly improve the damage of NAFLD patient's liver organization；It can significantly improve the expression of the Nrf2 gene of fatty liver cell；The ability of vitro Adsorption perfluoro caprylic acid is stronger.
Description
Technical field
The invention belongs to functional microbial technical fields, and in particular to Lactobacillus rhamnosus CCFM1060, its fermentation food
Product and bacterial preparation process.
Background technique
In recent years, expanding economy causes our people's living-pattern preservation, activity to reduce, and fat ratio is obvious
Increase, diabetes and metabolic syndrome (Metabolic Syndrome) obtain illness rate and significantly increase.International diabetic combinations
Meeting (IDF) indicates that 2017, the whole world had people of 4.25 hundred million ages greater than 19 years old with diabetes, if maintaining this trend, about
After 30 years, diabetic's number will be up to 6.93 hundred million people.Therefore, control diabetes have become extremely urgent thing.
Type-II diabetes are with fasting blood-glucose raising, high-density lipoprotein cholesterol reduction and triglycerides raising, pancreas islet
The performances such as element resistance exist simultaneously, and are extremely a variety of danger on pathological change basis with glycometabolism, lipid metaboli and protein metabolism
Factor aggregation, inspires the clinical syndrome of the various cardiovascular and cerebrovascular disease occurrence and development such as atherosclerosis.Due to two type glycosurias
Disease is the pathological state of a variety of Metabolite abnormal aggregations, and cluster generation is related with insulin resistance, has become painstaking effort at present
The hot spot that pipe disease and liver diseases research field are paid close attention to jointly.In addition, type-II diabetes are along with the disorderly of intestinal microecology
Disorderly, while being also closely related with psychotic disorders such as depression and anxiety.In addition some researches show that occupational exposure is in the worker of PFOA
Type II diabetes mortality risk increase, therefore in daily life PFOA exposure, be the mankind's type II diabetes morbidity it is potential
Risk.
It is mostly at present to surround to alleviate hypoinsulinism and insulin resistance two for the drug therapy of type-II diabetes
Aspect, melbine, thiazolidinediones including mitigating insulin resistance；Sulfonylureas, the Rosiglitazone of good control blood glucose
Deng.These drugs have certain therapeutic effect, but with aggravation, dosage increase, drug drug interaction, drug poison
Side effect etc. also dramatically increases, and alimentary canal is caused adverse reaction occur, and shows certain liver renal toxicity, such as takes two for a long time
First biguanides can cause the gastrointestinal tract for stimulating some patients to cause discomfort and may will affect absorption of the patient to vitamin B12,
Rosiglitazone can cause liver dysfunction and oedema etc..
Non-alcohol fatty liver (non-alcoholic fatty liver disease, NAFLD) is liver cell
Steatosis caused by middle fat excess accumulation, is a kind of symptom of liver metabolism disorder, not plus intervenes and can gradually be evolved into
Non-alcoholic fatty type hepatitis (NASH) and then deterioration are the high liver fibrosis of the death rate, cirrhosis, liver cancer.In recent years, with
Improvement of living standard, more and more high-fat, high-energy diet lay out dining table, and the amount of exercise of people is not therewith
Increase, so that being increased year by year by the incidence of the metabolic syndrome of representative of non-alcohol fatty liver.Due to its disease
Reason mechanism is not perfect, and the drug having no specifically for NAFLD is sold in the market, and therapeutic modality can only be from diet and life style
On improved, while being aided with the drug for reducing blood lipid or blood glucose to reach better effect.Some researches show that, occupational exposure in
The worker of PFOA because fatty liver mortality risk increase, therefore in daily life PFOA exposure, be human liver disease morbidity
Potential risk.
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 our department is made in this section and the description of the application and the title of the invention
Point, the purpose of abstract of description and denomination of invention it is fuzzy, and this simplification or omit and cannot be used for limiting the scope of the invention.
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 rhamnose
Lactobacillus CCFM1060, deposit number are GDMCC No:60705.
As another aspect of the present invention, the present invention overcomes the deficiencies in the prior art, provides and prepares rhamnose
The method of lactobacillus CCFM1060 microbial inoculum.
In order to solve the above technical problems, the present invention provides the following technical scheme that preparation Lactobacillus rhamnosus CCFM1060
The method of microbial inoculum comprising,
The preparation of strain cultures；
The protectant preparation of bacterial strain；
Inoculated and cultured, freeze-drying.
A kind of preferred embodiment of method as preparation Lactobacillus rhamnosus CCFM1060 microbial inoculum of the present invention: described
Strain cultures, are modified MRS culture medium, and formula is tryptone 10g, beef extract 10g, yeast powder 5g, glucose
20g, sodium acetate 5g, diammonium hydrogen citrate 2g, dipotassium hydrogen phosphate 2g, epsom salt 0.5g, Tween-80 1mL, a hydration sulphur
Sour manganese 0.25g, cysteine hydrochloride 0.5g, water 1000mL；Adjusting its pH is 6.5 ± 0.2.
A kind of preferred embodiment of method as preparation Lactobacillus rhamnosus CCFM1060 microbial inoculum of the present invention: described
Bacterial strain protective agent, formula be 100g/L~150g/L skimmed milk power, 100g/L~150g/L maltodextrin, 140g/L~
160g/L trehalose, surplus are water, and freeze-drying obtains freeze drying protectant.
A kind of preferred embodiment of method as preparation Lactobacillus rhamnosus CCFM1060 microbial inoculum of the present invention: described
Bacterial strain protective agent, formula be 120g/L skimmed milk power, 120g/L maltodextrin, 150g/L trehalose, surplus are water, is lyophilized,
Obtain freeze drying protectant.
A kind of preferred embodiment of method as preparation Lactobacillus rhamnosus CCFM1060 microbial inoculum of the present invention: described
Inoculated and cultured, freeze-drying, including, Lactobacillus rhamnosus CCFM1060 is inoculated under the conditions of 119~123 DEG C with 2% inoculum concentration
In the strain cultures after 15~25min of sterilizing, 24~48h is cultivated under 35~39 DEG C of anaerobic conditions of temperature, is with pH
6.8~7.2 phosphate buffers clean 2~4 times, are resuspended with the bacterial strain protective agent, make bacterium is dense to reach 1010CFU/mL；Then,
Allow the bacterial strain re-suspension liquid 50~70min of preculture under 37 DEG C of anaerobic conditions of temperature, then in -15~-20 DEG C of 8~14h of pre-freeze, it
Vacuum freeze drying afterwards.
A kind of preferred embodiment of method as preparation Lactobacillus rhamnosus CCFM1060 microbial inoculum of the present invention: sandlwood
Sugared lactobacillus CCFM1060 is inoculated into culture medium under the conditions of 121 DEG C after sterilizing 20min with 2% inoculum concentration, in temperature
It is cultivated under 37 DEG C of anaerobic conditions for 24 hours, is that 6.8 phosphate buffers clean 2~4 times with pH, is resuspended with the protective agent, makes bacterium
It is dense to reach 1010CFU/mL；Then, suspension preculture 60min under 37 DEG C of anaerobic conditions of temperature is allowed, then in -15 DEG C of pre-freezes
12h, later vacuum freeze drying.
As another 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, described in use
Lactobacillus rhamnosus CCFM1060 microbial inoculum ferments, and the microbial inoculum contains greater than 106The active Lactobacillus rhamnosus of CFU/g
CCFM1060。
A kind of preferred embodiment as fermented food of the present invention: the fermented food includes dairy products, bean product
With fruit and vegetable product.
A kind of preferred embodiment as fermented food of the present invention: the fermented food, including cucumber pear juice lactic acid
Bacteria beverage, preparation method are to squeeze the juice pears to obtain pear juice, by cucumber stripping and slicing, are put into blanching 30s in 90 DEG C of hot water, mashing,
Filtering, obtains Fresh Cucumber Juice；Pear juice, Fresh Cucumber Juice are mixed with skimmed milk powder with the mass ratio of 5:4:1, the mixing of Fresh Cucumber Juice pear juice is obtained
Lotion, 5% xylitol, packing are added after homogeneous, and the rhamnose cream bar is added in 115 DEG C of sterilizing 20min after being cooled to room temperature
Bacterium CCFM1060 microbial inoculum is with 107CFU/m L is inoculated into the Fresh Cucumber Juice pear juice mixed emulsion, ferments in 37 DEG C of constant incubators
16h。
Beneficial effects of the present invention: the Lactobacillus rhamnosus CCFM1060 that the present invention screens, to simulation gastro-intestinal Fluid have compared with
Good tolerance；The abundance for significantly reducing the Proteus category of High cholesterol diet induction high in fat increases, reduction urinary obstruction,
The diseases such as enteric bacteria migration and enteritis occur；Significantly improve the disorders of lipid metabolism of NAFLD patient；Significantly improve NAFLD
The insulin resistance of mouse；The raising of glutamic-pyruvic transaminase in serum (ALT) and glutamic-oxalacetic transaminease (AST) can be adjusted back；Significant drop
The concentration of low density lipoprotein cholesterol in low serum, reduces the risk of cardiovascular disease；NAFLD patient liver can be significantly improved
SOD in dirty is horizontal；It can significantly improve liver inflammation；It can significantly improve the damage of NAFLD patient's liver organization；It can be significant
Improve the expression of the Nrf2 gene of fatty liver cell；The ability of vitro Adsorption perfluoro caprylic acid is stronger；It can significantly improve high sugar to make
With the proliferation of lower INS-1 cell and the expression of MafA gene.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, required use in being described below to embodiment
Attached drawing be briefly described, it should be apparent that, drawings in the following description are only some embodiments of the invention, for this
For the those of ordinary skill of field, without any creative labor, it can also be obtained according to these attached drawings other
Attached drawing.Wherein:
Fig. 1 is the colonial morphology that lactic acid bacteria Lactobacillus rhamnosus CCFM1060 is used in fermentation；
Fig. 2 is influence of the Lactobacillus rhamnosus CCFM1060 to Proteus Pseudomonas in NAFLD mouse intestinal；
Fig. 3 is influence of the Lactobacillus rhamnosus CCFM1060 to NAFLD mice serum cholesterol (TC)；
Fig. 4 is influence of the Lactobacillus rhamnosus CCFM1060 to NAFLD mice serum glutamic-pyruvic transaminase (ALT)；
Fig. 5 is influence of the Lactobacillus rhamnosus CCFM1060 to NAFLD mice serum glutamic-oxalacetic transaminease (AST) level；
Fig. 6 is Lactobacillus rhamnosus CCFM1060 to NAFLD mice serum low density lipoprotein cholesterol (LDL-C) level
Influence；
Fig. 7 is influence of the Lactobacillus rhamnosus CCFM1060 to NAFLD mouse fasting blood-glucose；
Fig. 8 is influence of the Lactobacillus rhamnosus CCFM1060 to the insulin resistance of NAFLD mouse
Fig. 9 is influence of the Lactobacillus rhamnosus CCFM1060 to NAFLD mouse liver triglycerides (TG)
Figure 10 is influence of the Lactobacillus rhamnosus CCFM1060 to NAFLD mouse liver superoxide dismutase (SOD)
Figure 11 is influence of the Lactobacillus rhamnosus CCFM1060 to NAFLD mouse liver inflammation；
Figure 12 is influence of the Lactobacillus rhamnosus CCFM1060 to NAFLD mouse liver histopathology；
Figure 13 is influence of the Lactobacillus rhamnosus CCFM1060 to fatty liver cell Nrf2 gene expression；
Figure 14 is the ability of Lactobacillus rhamnosus CCFM1060 vitro Adsorption perfluoro caprylic acid；
Figure 15 is that Lactobacillus rhamnosus CCFM1060 belongs to abundance to Desulfovibrio in type-II diabetes mouse intestinal
It influences；
Figure 16 is the influence that Lactobacillus rhamnosus CCFM1060 acts on high sugar lower INS-1 cell proliferative conditions；
Figure 17 is the influence that Lactobacillus rhamnosus CCFM1060 acts on high sugar lower INS-1 cell MafA gene expression；
Note: a, b, c indicate that group representated by different letters all has 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 can be with
Implemented using other than the one described here other way, those skilled in the art can be without prejudice to intension of the present invention
In the case of do similar popularization, 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 side of the invention
A particular feature, structure, or characteristic in formula." in one embodiment " that different places occur in the present specification not refers both to
The same embodiment, nor the individual or selective embodiment mutually exclusive with other embodiments.
Lactobacillus rhamnosus CCFM1060 (Lactobacilus rhamnosus) was preserved in wide on 06 28th, 2019
East saves Culture Collection, and address is the compound the 59th of Xianlie Middle Road, Guangzhou City 100 5 building, building, deposit number GDMCC
No:60705.
Lactobacillus rhamnosus CCFM1060 has following biological characteristics:
(1) thallus feature: it is in Gram-positive, does not form spore, no motion of bacterium.
(2) colony characteristics: aerobic or Anaerobic culturel 36 hours form apparent bacterium colony, diameter is between 0.5-2mm, front
Form is round, and side form is in overshooting shape, and edge is uneven, creamy-white, and opaque, surface wettability is smooth, not chromogenesis, ginseng
See attached drawing 1.
(3) it growth characteristics: under conditions of 37 DEG C of constant temperature are aerobic or anaerobism, cultivate about 16 hours and reaches in mMRS culture medium
To late log phase.
(4) there is preferable tolerance to simulation gastro-intestinal Fluid；
(5) the abundance raising that the Proteus of High cholesterol diet induction high in fat belongs to is significantly reduced, urinary obstruction, intestines are reduced
The diseases such as road bacterial migration and enteritis occur；
(6) disorders of lipid metabolism of NAFLD mouse is significantly improved；
(7) insulin resistance of NAFLD mouse is significantly improved；
(8) raising of glutamic-pyruvic transaminase (ALT) and glutamic-oxalacetic transaminease (AST) in the serum of NAFLD mouse can be adjusted back；
(9) concentration for significantly reducing low density lipoprotein cholesterol in the serum of NAFLD mouse, reduces cardiovascular disease
Risk；
(10) SOD that can be significantly improved in NAFLD mouse liver is horizontal；
(11) it can significantly improve the liver inflammation of NAFLD mouse；
(12) it can significantly improve NAFLD mouse liver tissue damage；
(13) it can significantly improve the expression of the Nrf2 gene of fatty liver cell.
(14) ability of vitro Adsorption perfluoro caprylic acid is stronger.
(15) it can significantly improve high sugar and act on the lower proliferation of INS-1 cell and the expression of MafA gene.
The preparation method of this bacterial strain are as follows:
(1) separation screening of lactic acid bacteria:
(l) fresh excreta of the 1g from healthy population is taken.Sample is being contained into 35 DEG C of enrichments in sorbierite GM17 culture medium
12h；
(2) it will be coated on after enriched sample progress gradient dilution and be added to the 0.02% GM17 solid plate for smelling cresol-purple
On, cultivate 24~48h；
(3) single colonie chosen discoloration circle obviously, and meet lactic acid Zoopagales grown form carries out plate streaking purifying, sieve
Lactic acid bacteria is isolated in choosing；
(4) above-mentioned single colonie is incubated to cultivate in liquid GM17 culture solution and carries out Gram's staining afterwards for 24 hours, it is blue to choose leather
Family name's positive bacteria carries out follow-up test.
(2) the fermentation Preliminary Identification of lactic acid bacteria: molten calcium circle measuring method
(l) lactic acid bacteria that step (1) is screened is cultivated for 24 hours in liquid sorbitol GM17 culture solution, then takes
L mL culture 8000rpm is centrifuged 2min；
(2) 0.05M KH is used2PO4Solution washes twice；
(3) gained bacterium mud is resuspended, is crossed in sorbierite GM17-0.75%CaCO3Solid medium on, culture for 24 hours；
(4) it is obvious to choose molten calcium circle, and in convex, fine and closely woven color it is white, without mycelial bacterium colony, shown after Gram's staining
Micro mirror observes thallus and carries out preliminary judgement.
(3) the fermentation molecular biology identification of lactic acid bacteria:
(l) single bacterium genome extracts:
A. lactic acid bacteria overnight incubation step (2) screened, take the bacteria suspension 1mL of overnight incubation in 1.5mL from
Heart pipe, 10000rpm are centrifuged 2min, abandon supernatant and obtain thallus；
B. with after 1mL sterile water purging thallus, 10000rpm is centrifuged 2min, abandons supernatant and obtains thallus；
C. 200 μ LSDS lysates, 80 DEG C of water-bath 30min are added；
D. 200 μ L of phenol-chloroform solution is added in cellular lysate liquid, wherein the constituent and volume of phenol-chloroform solution
Than for Tris saturated phenol: chloroform: isoamyl alcohol=25:24:1, after being mixed by inversion, 12000rpm is centrifuged 5-10min, takes 200 μ of supernatant
L；
E. 400 μ L ice ethyl alcohol or ice isopropanol are added in 200uL supernatant, ﹣ 20 DEG C of standings 1h, 12000rpm are centrifuged 5-
10min abandons supernatant；
F. 500 μ L70% (percentage by volume) ice ethyl alcohol are added, precipitating is resuspended, 12000rpm is centrifuged 1-3min, abandons supernatant；
G.60 DEG C baking oven drying or naturally dry；
H.50 the molten precipitating of μ LddH2O weight is in case PCR；
(2)16S rDNA PCR
A. 50 μ LPCR reaction system of bacterial 16 S rDNA:
10 × Taq buffer, 5 μ L；DNTP, 5 μ L；27F, 0.5 μ L；1492R, 0.5 μ L；Taq enzyme, 0.5 μ L；Template,
0.5μL；DdH2O, 38 μ L.
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 5
μ L, 120V race 30min, then carries 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 sequencing result and be accredited as a kind of new discovery for belonging to Lactobacillus rhamnosus
Bacterial strain, -80 DEG C of preservations are spare.
Embodiment 1: Lactobacillus rhamnosus CCFM1060 has good tolerance to simulation gastro-intestinal Fluid
The Lactobacillus rhamnosus CCFM1060 of freezen protective is inoculated in mMRS culture medium (+0.05% half Guang of MRS culture medium
Propylhomoserin hydrochloride) in, in 37 DEG C of Anaerobic culturel 48h of temperature, then after mMRS culture solution secondary culture 2~3 times, take 1mL sandlwood
The culture solution of sugared lactobacillus CCFM1060, with 9.0mL pH 2.5 artificial simulation gastric juices (containing 1% pepsin, 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 agar culture
Base casting culture 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 the ratio between viable count logarithm,
It is indicated with %.Take 1mL Lactobacillus rhamnosus CCFM1060 culture solution be added 9mL artificial simulation intestinal juice (containing 0.3% cholate,
The mMRS culture medium of 1% trypsase, pH=8.0) in, the Anaerobic culturel at 37 DEG C, respectively in 0h, 0.5h, 1h, 2h, 3h and
It is sampled when 4h, carries out plate count with the casting culture of mMRS agar medium, measure viable count and calculate its survival rate.It deposits
Motility rate be viable count logarithm in the culture solution in sampling in 0h when the ratio between viable count logarithm, indicated with %.
Experimental result is as shown in Table 1 and Table 2.The result shows that Lactobacillus rhamnosus CCFM1060 is to artificial gastro-intestinal Fluid with preferable resistance to
By property.
Tolerance of the 1 Lactobacillus rhamnosus CCFM1060 of table in artificial simulation gastric juices
Tolerance of the 2 Lactobacillus rhamnosus CCFM1060 of table in artificial simulation intestinal juice
Embodiment 2: adjustment effect of the Lactobacillus rhamnosus CCFM1060 to NAFLD mouse intestinal flora
It takes healthy male C 57 BL/6 J mouse 48 of weight 16-20g, adapts to environment 1 week, be randomly divided into 6 groups: blank pair
According to group (NC), model control group (M), Rosiglitazone control group (RC), Simvastatin control group (SC), Lactobacillus rhamnosus
CCFM1060 intervention group (CCFM1060), Lactobacillus rhamnosus L10 intervention group (LC), every group contain mouse 8, stomach-filling bacterial strain by
3% sucrose solution is resuspended.Experimental animal grouping and processing method are shown in Table 3:
The grouping of 3 experimental animal of table
Test latter stage collects mouse fresh excreta and freezes in -80 DEG C, extracts the macro genome in excrement, and survey using two generations
Sequence instrument analyzes intestinal microflora.When off-test, mouse is deprived of food but not water 12h, and 0.5mL/10g 1% is injected intraperitoneally
Nembutal sodium solution anesthesia after, Culling heart blood, be aided with cervical dislocation execution.3000 × g of blood sample, under the conditions of 4 DEG C
It is centrifuged 15min, takes supernatant, -80 DEG C freeze for measuring associated serum index.Partial liver is immediately placed in the life of pre-cooling after collecting
Blood is removed in rinsing in reason salt water, is put into 4% neutral paraformaldehyde solution and fixes, remainder liver is quick-frozen in liquid nitrogen and shifts
It is frozen to -80 DEG C, it is subsequent that liver homogenate is made to be used to measure index of correlation, specifically the preparation method is as follows: weighing a certain amount of liver
Tissue is added physiological saline in 1:9 ratio and carries out tissue grinder, and 3000r is centrifuged 10min, takes supernatant to freeze spare in -80 DEG C.
Flora analysis experimental result is as shown in Fig. 2, the Proteus for significantly reducing High cholesterol diet induction high in fat belongs to rich
Degree increases, and reduces inflammation and urinary tract infection risk, reduces fat and immunity disease generation
Proteus category significantly rises in NAFLD stool in mice, and the intake of Lactobacillus rhamnosus CCFM1060 can incite somebody to action
Proteus belongs to abundance readjustment, and Proteus belongs to related with urinary obstruction, enteric bacteria migration and enteritis, shows tool of the present invention
It is reduced the pathogenetic functions of diseases such as urinary obstruction, enteric bacteria migration and enteritis.
Embodiment 3: Lactobacillus rhamnosus CCFM1060 significantly reduces the level of NAFLD mice serum total cholesterol (TC)
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.
Experimental result is as shown in Figure 3.Model group mice serum total cholesterol level is significantly raised, stomach-filling Lactobacillus rhamnosus
The TC that CCFM1060 significantly reduces model mice is horizontal and close to blank control group.Its reduce mice serum TC ability with
Simvastatin medicine group is similar.
Embodiment 4: it is horizontal that Lactobacillus rhamnosus CCFM1060 reduces NAFLD mice serum glutamic-pyruvic transaminase (ALT)
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.It is measured according to the detection method of ALT kit
The content of glutamic-pyruvic transaminase (ALT) in blood.
Experimental result is as shown in Figure 4.Model group mouse empty stomach ALT is significantly increased, and the intervention of rhamnose CCFM1060 is obvious
The ALT for reducing NAFLD mouse is horizontal, and the ability for reducing mouse fasting blood glucose level is similar to Simvastatin, and rhamnose
The intake of lactobacillus L10 does not reverse the raising of ALT, and the ALT level of noticeable Rosiglitazone group is significantly higher than
Model group prompts long-term use Rosiglitazone that can cause drug induced injury to liver.
Embodiment 5: Lactobacillus rhamnosus CCFM1060 reduces the level of NAFLD mice serum glutamic-oxalacetic transaminease (AST)
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.Blood is measured according to the detection method of kit
The content of middle glutamic-oxalacetic transaminease (AST).
Experimental result is as shown in Figure 5.As seen from Figure 5, model group mice serum AST content is significantly raised, stomach-filling mouse
Lee sugar lactobacillus CCFM1060 significantly reduces the content of serum AST, and its trend is consistent with ALT trend, prompts rhamnose cream bar
Bacterium CCFM1060 can alleviate hepar damnification.
Embodiment 6: Lactobacillus rhamnosus CCFM1060 reduces NAFLD mice serum low density lipoprotein cholesterol (LDL-
C level)
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.It is low close according to the detection method measurement of kit
Spend the content of lipoprotein cholesterol (LDL-C).
Experimental result is as shown in Fig. 6.It can be seen from experimental result compared with Normal group, model group mouse blood
Clear low density lipoprotein cholesterol content significantly increases, and stomach-filling Lactobacillus rhamnosus CCFM1060 can reduce serum low-density rouge egg
The content of white cholesterol, and Lactobacillus rhamnosus CCFM1060 is bright to the readjustment ability of serum LDL cholesterol level
It is aobvious to be better than Lactobacillus rhamnosus L10.
Embodiment 7: Lactobacillus rhamnosus CCFM1060 reduces the fasting blood glucose level C57BL/6J mouse point of NAFLD mouse
Group, modeling and processing method are the same as embodiment 2.
Experimental result is as shown in Figure 7.Model group mouse fasting blood-glucose significantly increases, and Lactobacillus rhamnosus CCFM1060's is dry
The fasting blood glucose level of NAFLD mouse is significantly reduced in advance, fasting blood-glucose control ability is significantly stronger than Lactobacillus rhamnosus L10
Intervention, and its reduce mouse fasting blood glucose level ability it is similar to Rosiglitazone.
Embodiment 8: Lactobacillus rhamnosus CCFM1060 alleviates the insulin resistance C57BL/6J mouse point of NAFLD mouse
Group, modeling and processing method are the same as embodiment 2.According to the content of detection method measurement insulin (INS) of kit, and combine empty
Abdomen blood sugar effects calculate insulin resistance index.
Experimental results are shown in figure 8.Compared to the blank group, High cholesterol diet high in fat is after 24 weeks, model group mouse islets
Element is resisted index and is significantly increased, and NAFLD mouse islets element resists index and decreases after Lactobacillus rhamnosus L10 intervenes, but its
Effect is not so good as Lactobacillus rhamnosus CCFM1060, and Lactobacillus rhamnosus CCFM1060 is prompted to can be improved the pancreas islet of NAFLD mouse
Plain sensibility may have certain remission effect to type-II diabetes.
Embodiment 9: Lactobacillus rhamnosus CCFM1060 reduces the level of triglycerides (TG) in the liver of NAFLD mouse
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.Glycerol is measured according to the detection method of kit
The content of three esters (TG), is corrected with liver protein concentration.
Experimental result is as shown in Figure 9.It can be seen from experimental result compared with Normal group, model group mouse liver
TG is significantly increased, and stomach-filling Lactobacillus rhamnosus CCFM1060 reduces the level of TG in NAFLD mouse liver, and Lactobacillus rhamnosus
CCFM1060 is suitable with Simvastatin to the regulating power of liver TG.
Embodiment 10: Lactobacillus rhamnosus CCFM1060 improves the superoxide dismutase from liver of NAFLD mouse
(SOD) level is corrected with liver protein concentration.
C57BL/6J mice group, modeling and processing method are the same as embodiment 2.According to superoxide dismutase (SOD) reagent
The content of SOD in the specification measurement liver of box.
Experimental result is as shown in Figure 10.The SOD level of blank control group is higher than model group it can be seen from experimental result,
But without significant difference, stomach-filling Lactobacillus rhamnosus CCFM1060 significantly improves the level of SOD in NAFLD mouse liver and rhamnose
Lactobacillus L10 does not show similar result after intervening.
Embodiment 11: the level of inflammation C57BL/6J that Lactobacillus rhamnosus CCFM1060 mitigates in NAFLD mouse liver is small
Mouse grouping, modeling and processing method are the same as embodiment 2.Liver is measured according to the specification of tumor necrosis factor-alpha (TNF-α) kit
The content of dirty middle TNF-α, is corrected with liver protein concentration.
Experimental result is as shown in figure 11.High cholesterol diet high in fat is after 24 weeks it can be seen from experimental result, model group
The horizontal significant raising of TNF-α, stomach-filling Lactobacillus rhamnosus CCFM1060 significantly reduce TNF-α level in NAFLD mouse liver, and
Its inflammation remission effect is better than Simvastatin and Rosiglitazone, and NAFLD mouse inflammation is alleviated after Lactobacillus rhamnosus L10 intervenes
Effect is general.
Embodiment 12: the liver organization that Lactobacillus rhamnosus CCFM1060 alleviates NAFLD mouse damages C57BL/6J mouse
Grouping, modeling and processing method are the same as embodiment 2.Take the liver production paraffin section that the neutral paraformaldehyde in part 4% is fixed, warp
Tissue morphology is observed under light microscopic after HE dyeing and is taken pictures, and pathological evaluation is carried out.Specific step is as follows:
(1) fixed: tissue sample is washed with physiology salt, is put into 4% neutral paraformaldehyde fixer and is fixed immediately,
The general set time is within 72h.
(2) wash: flowing water rinses or impregnates a few hours or stays overnight.
(3) be dehydrated: sample is successively dehydrated through 70%, 80%, 90% ethanol solutions at different levels, and each 30min places into 95%1
Secondary 20min, 100%2 each 10min.
(4) transparent :+1/2 dimethylbenzene mixed liquor 10min of 1/2 absolute alcohol, I 10min of dimethylbenzene, II 10min (are to transparent
Only).
(5) sample waxdip: is put into paraffin (62 DEG C) wax 2h thoroughly.
(6) it embeds: with maximum face in bottom, making maximum shared by the covering weave face cut out.
(7) it is sliced: using hand microtome, wax stone is cut into the segment of 5 μ m thicks.
(8) it opens up piece and bonding die (fishing piece): opening water-bath, water temperature is made to maintain 42 DEG C, make to be sliced and smooth spread over the water surface
On.
(9) it bakes piece: glass slide is put into together with object slide stand to 55 DEG C of drying box, about 2h to wax melts.
(10) aquation: paraffin section dewaxes each 10min through dimethylbenzene I, II, be then placed in 100%, 95%, 90%,
80%, each 5min in 70% alcoholic solutions at different levels, places into 3min in distilled water.
(11) just contaminate: slice, which is put into hematoxylin, dyes about 20s.
(12) it washes: rinsing about 15min with tap water flowing water.Make to be sliced color and become blue, but it is noted that flowing water cannot be excessive,
It falls off to prevent slice.
(13) break up: slice being put into 1% ethanol solution hydrochloride and is faded, 7s.See that slice reddens, color is shallower.
(14) rinse: slice, which places into flushing 15-20min in tap water flowing water, makes its restore blue.
(15) it redyes: immersing eosin stain, take out be dehydrated immediately.
(16) be dehydrated: will slice successively cross 95% ethyl alcohol I, 95% ethyl alcohol II, 70% ethyl alcohol, place into 80% ethyl alcohol 50s,
Dehydrated alcohol 2min.
(17) transparent: slice is put into 1/2 dehydrated alcohol, 1min in 1/2 dimethylbenzene, each 2min in dimethylbenzene I, II.
(18) mounting: slice uses neutral gum to hide agent as envelope after dimethylbenzene is transparent, and natural gum can be diluted with dimethylbenzene
To suitable consistency.
Experimental result is as shown in figure 12.Model group mouse liver cell arranges sparse, liver fat drips it can be seen from experimental result
Quantity is more and not of uniform size, stick to each other between fat drips, lobuli hepatis have inflammatory cell infiltration, and balloon sample disease occurs for a small amount of liver cell
Become, and stomach-filling Lactobacillus rhamnosus CCFM1060 can obviously improve above-mentioned lesion, and effect is significantly better than Lactobacillus rhamnosus
L10。
Embodiment 13: Lactobacillus rhamnosus CCFM1060 improves the level of Nrf2 in fatty liver cell
By L02 cell after containing the passage three times of 10% FBS continuous-stable, it is inoculated in 6 orifice plates, in 37 DEG C, 5%
CO2It is cultivated in environment for 24 hours, after cell is adherent, gives the mouse for individually accessing 1mL after 2mg/mL palmitinic acid is incubated for for 24 hours again
Lee sugar lactobacillus CCFM1060 and Lactobacillus rhamnosus L10 (access PBS is as blank control) are incubated for for 24 hours.All incubations exist
37 DEG C, 5%CO2It is carried out in environment.Lactobacillus rhamnosus CCFM1060 stimulation test group, Lactobacillus rhamnosus L10 stimulation test
Group and each three holes of PBS control group, and in triplicate.
Culture solution is discarded, first cleans each hole with PBS buffer solution, each 1mL adds TRIZOL to crack after cleaning 3 times, into
Row cell RNA extracts.After progress qPCR measurement BA3 and fatty liver cell are incubated for altogether after being cDNA by the RNA reverse transcription of extraction
The expression of Nrf2 gene.Nrf2 primer information is as shown in table 5, as a result using GAPDH as internal reference, is expressed as 2-△△CT。
4 primer information of table
| Primer | It is positive | Reversely |
| NRF2 | CAACCCTTGTCACCATCTCA | GTGTTCTCACATTGGGCATC |
| GAPDH | AGGTCGGTGTGAACGGATTTG | TGTAGACCATGTAGTTGAGGTCA |
Experimental result is as shown in figure 13.Lactobacillus rhamnosus CCFM1060 stimulation significantly mentions it can be seen from experimental result
The high expression of the Nrf2 gene of fatty liver cell, the expression of the Nrf2 gene of Lactobacillus rhamnosus L10 stimulation group
Also there is raising but obvious not as good as Lactobacillus rhamnosus CCFM1060 stimulation group, show that Lactobacillus rhamnosus CCFM1060 may have
Certain oxidation resistance.
Embodiment 14: Lactobacillus rhamnosus CCFM1060 has good adsorption capacity to PFOA
Purifying and activation culture are carried out to Lactobacillus rhamnosus CCFM1060, are inoculated in MRS liquid by 1% (v/v) inoculum concentration
In body culture medium, 37 DEG C of Anaerobic culturels are for 24 hours.Then thallus is collected in 8000r/min centrifugation 5min, takes precipitating physiological saline clear
Continue to be centrifuged 5min in 8000r/min after reason, precipitating is gone to obtain viable bacteria body cell, i.e. wet thallus.Wet thallus is resuspended in 50mg/
In LPFOA solution, and so that final cell concentration is reached 1g dry mycelium/L and (wet thallus is resuspended in the ultrapure water without PFOA and is made
For blank control).The pH of the PFOA solution containing bacterium solution is adjusted rapidly to 3.0 using the NaOH or HCl solution of 0.1M, addition is few
NaOH or HCl (being less than 0.5ml) of amount its ionic strength can ignore the influence that PFOA is adsorbed.100ml sample will be then housed
The 250ml conical flask of liquid is placed in 37 DEG C, 150rpm shaking table culture, is measured by sampling after 6h, and 2 parallel tests are averaged.
The measurement of PFOA adsorbance: after adsorption experiment, sample liquid is centrifuged 5min in 8000r/min, and with 0.22 μm of moisture film
Filtering, PFOA concentration uses the UPLC-MS with Waters SYNAPT MS system to measure, using Acquity UPLC BEH c18
Column (2.1 × 100mm, 1.7 μm, Waters Co.), 35 DEG C of column temperature, 1 μ L of sample volume.Acetonitrile solution with 100% (v/v) is (molten
Liquid A) and 0.1% (v/v) aqueous formic acid (solution B) be used as eluent, carry out gradient cleaning, flow velocity is 0.3mL/min, gradient
Cleaning condition is as shown in table 5.
5 condition of gradient elution of table
| t/min | 0-0.5 | 0.5-5.0 | 5.0-7.0 | 7.0-7.5 |
| Solvent A ratio | 70% | 70-100% | 100% | 100-70% |
Mass Spectrometry Conditions: ionization source is the source ESI；MRM detection；MS+ detection；Capillary (capillary)；3.0kV；Conc
(centrum): 40.00V；Source Temperature (radiation source temperature): 120 DEG C；Desolvation (desolvation) temperature:
400℃；Conc Gas Flow:50L/h；Desolvation Gas Flow:700L/h. gas flow rate is 0.1ml/min；Matter
Son is than scanning range: 100-2000；Surface sweeping time 1s is spaced 0.061s.As a result with MassLynxV4.1 (Waters company) point
Analysis；Lactic acid bacteria is calculated to the adsorbance of PFOA according to the concentration difference of absorption front and back PFOA.Measurement result is as shown in figure 14, sandlwood
Sugared lactobacillus CCFM1060 is 70.00% ± 2.88% to the adsorption rate of 50mg/L PFOA, is significantly higher than other bacterial strains.
Embodiment 15:
Lactobacillus rhamnosus CCFM1060 microbial inoculum the preparation method comprises the following steps:
1. the preparation of strain cultures: the culture medium of Lactobacillus rhamnosus is modified MRS culture medium (mMRS), is formulated as pancreas
Peptone 10g, beef extract 10g, yeast powder 5g, glucose 20g, sodium acetate 5g, diammonium hydrogen citrate 2g, dipotassium hydrogen phosphate
2g, epsom salt 0.5g, Tween-80 1mL, Manganous sulfate monohydrate 0.25g, cysteine hydrochloride 0.5g, water 1000mL；
Adjusting its pH is 6.5 ± 0.2.
2. the protectant preparation of bacterial strain: protection agent prescription are as follows: 120g/L skimmed milk power, 120g/L maltodextrin, 150g/L
Trehalose, surplus are water, and freeze-drying obtains freeze drying protectant；
3. the inoculum concentration of the Lactobacillus rhamnosus CCFM1060 strain quality meter 2% of culture medium as described above is inoculated into
In culture medium under the conditions of 121 DEG C after sterilizing 20min, is cultivated under 37 DEG C of anaerobic conditions of temperature for 24 hours, be 6.8 phosphoric acid with pH
Salt buffer cleans 2~4 times, is resuspended with the protective agent, makes bacterium is dense to reach 1010CFU/mL；Then, allow the suspension in temperature
Preculture 60min under 37 DEG C of anaerobic conditions is spent, then in -15 DEG C of pre-freeze 12h, finally carries out vacuum freeze drying and obtain the mouse
Lee's sugar lactobacillus CCFM1060 microbial inoculum.
Embodiment 16:
Stripping and slicing after pear is removed the peel, impregnates 30s in the ascorbic acid solution containing 0.5%, pulls out, and juicing obtains pears
Cucumber stripping and slicing is put into blanching 30s in 90 DEG C of hot water by juice, is beaten according to solid-liquid ratio 1:4, and filtering obtains Fresh Cucumber Juice；By pears
Juice, Fresh Cucumber Juice are mixed with skimmed milk powder with the mass ratio of 5:4:1, are obtained Fresh Cucumber Juice pear juice mixed emulsion, are added 5% after homogeneous
Xylitol is dispensed into conical flask in 115 DEG C of sterilizing 20min, will be implemented after it is cooled to room temperature using aseptic technique
Lactobacillus rhamnosus CCFM1060 microbial inoculum prepared by example 15 is with 107CFU/m L is inoculated into said mixture, 37 DEG C of constant temperature incubations
Ferment 16h in case, and ice bath terminates fermentation and obtains cucumber pear juice sour milk beverage, in 4 DEG C of preservations.
Embodiment 17: Lactobacillus rhamnosus CCFM1060 belongs to the enteron aisle Desulfovibrio of type-II diabetes mouse
It influences
It takes healthy male C 57 BL/6 J mouse 40 of weight 16-20g, adapts to environment 1 week, be randomly divided into 5 groups: blank pair
According to group (NC), model control group (M), Rosiglitazone control group (RH), Lactobacillus rhamnosus CCFM1060 intervention group
(CCFM1060), containing mouse 8 for every group of Lactobacillus rhamnosus 4-1 control group (4-1), the dosage of stomach-filling bacteria suspension is 3.0 ×
109CFU/mL is resuspended in 3% sucrose solution.Experimental animal grouping and processing method are shown in Table 6:
The grouping of 6 experimental animal of table
The 2-7 weeks: normal group mouse feeding normal diet, remaining mouse feeding high lipid food.
In the 11st week 1d, all mouse were deprived of food but not water 12h, normal group injection 50mmol/L citric acid-citric acid
Sodium buffer (pH 4.5), remaining group injection according to 100mg/ (kg weight) dosage injection 50mmol/L STZ (be protected from light on ice,
It is ready-to-use), wherein the preparation of STZ is dissolved with 50mmol/L citric acid-sodium citrate buffer solution.
Test latter stage collects mouse fresh excreta and freezes in -80 DEG C, extracts the macro genome in excrement, and survey using two generations
Sequence instrument analyzes intestinal microflora.When off-test, mouse is deprived of food but not water 12h, and 0.5mL/10g 1% is injected intraperitoneally
Nembutal sodium solution anesthesia after, Culling heart blood, be aided with cervical dislocation execution.3000 × g of blood sample, under the conditions of 4 DEG C
It is centrifuged 15min, takes supernatant, -80 DEG C freeze for measuring associated serum index.Partial liver is immediately placed in the life of pre-cooling after collecting
Blood is removed in rinsing in reason salt water, is put into paraformaldehyde and fixes, remainder liver is quick-frozen in liquid nitrogen and is transferred to -80 DEG C of jellies
It deposits, 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 is added physiological saline and carries out tissue grinder, and 3000r is centrifuged 10min, takes supernatant to freeze spare in -80 DEG C.
Flora analyzes the enteron aisle that experimental result is as shown in figure 15, and Desulfovibrio belongs in type-II diabetes stool in mice
Microorganism significantly rises, and the abundance that the intake of Lactobacillus rhamnosus CCFM1060 can belong to Desulfovibrio is adjusted back, this table
The Lactobacillus rhamnosus CCFM1060 that the bright present invention screens, which has, reduces the pathogenetic functions of diseases such as Parkinson.
Embodiment 18: Lactobacillus rhamnosus CCFM1060 can promote the proliferation and Maf A of the INS-1 cell of high glucose induction
The expression of mRNA
Experiment is divided into 5 groups: normal group (the Nostoc commune Vanch liquid of the glucose containing 11.1mmol/L), and high sugar group (contains 22.2mmol/
The sugared culture solution of the height of L glucose), Rosiglitazone group (Rosiglitazones of+80 μm of ol/L of high sugar culture solution), CCFM1060 group is (high
Sugared culture solution+contain 1*109CFU/mL CCFM1060 bacterium solution) 4-1 group (high sugar culture solution+contain 1*109CFU/mL 4-1 bacterium solution).
By INS-1 cell (number: BH-AC0530) be incubated at RPMI-1640 culture solution (glucose containing 11.1mmol/L,
10%FBS, 50 μm of ol/L 2 mercapto ethanols, 1mmol/L pyruvic acid, 10mmol/L HEPES) in, and 37 DEG C are put into, 5%CO2
Incubator in.
CCK-8 method detects cell Proliferation: cell dissociation in good condition is centrifuged and is inoculated on 96 orifice plates, each hole about 5
×103A cell, the periphery hole of plate not inoculating cell, to prevent edge effect while being added PBS solution thereto.It is pasted to cell
The RPMI-1640 culture medium for containing 0.5% fetal calf serum is added in each hole for wall, and synchronization process is for 24 hours.Synchronization terminates, foundation point
Corresponding culture medium culture 48h is added to each hole in group, and every group sets three multiple holes, while zeroing hole is arranged.Pharmaceutical intervention terminates, and sucks
Old culture medium, PBS are cleaned 2 times, and 180 μ L serum free mediums and 20 μ L CCK-8 solution are added, and are incubated for 3-4h.Incubation terminates,
Each hole absorbance value is measured under 450nm using microplate reader.
Maf A mRNA expression measurement: Trizol method extract RNA, inhale abandon 6 orifice plates in original fluid, while be pre-chilled
PBS is cleaned 2 times, and 1.0mL Trizol lytic cell is separately added into each hole and celliferous lysate is gone to no enzyme EP and is managed,
Liquid-transfering gun is blown and beaten to no obvious sediment and stands 5min.0.2mL chloroform is added to each EP pipe, acutely shakes 15s, is placed at room temperature for
2-3min.4 DEG C, 12000rpm is centrifuged 15min, draws supernatant 0.4m L or so, is transferred in another no enzyme EP pipe, is added
The isopropanol of 0.5mL, is mixed by inversion, and is stored at room temperature 10min.4 DEG C, 12000rpm is centrifuged 10min, carefully discards supernatant, and is added
75% ethyl alcohol of 1.0mL is simultaneously mixed by inversion.4 DEG C, 12000rpm centrifugation 5min, abandoning supernatant, drying at room temperature 2-5 minutes.20 μ L are added
DEPC handles water dissolution, is stored in 80 DEG C for use.Measure RNA concentration and quality, and according to reverse transcription reagent box specification into
Row reverse transcription.The cDNA that reverse transcription obtains carries out q RT-PCR detection, wherein MafA specific primer: F:5'-
Atcactctgcccaccatcac-3', R:5'-atgacctcctccttgctgaa-3'.PCR system are as follows: F (10 μM), 0.50 μ
L；R(10μM),0.50μL；C DNA Template, 1.00 μ L；dd H2O, 3.00 μ L；Mix, 5.00 μ L.PCR program: 95 DEG C,
2min；(95 DEG C, 30sec；60 DEG C, 30sec；72 DEG C, 20sec) * 35；72 DEG C, 5min；Target gene passes through Real-time
After PCR detection, using 2-△△CTMethod carries out Relative gene expression analysis.First each group rat INS- is analyzed with CFX Manager software
The expression quantity of 1 cell target gene, then to organize expression quantity normally as 1, other each groups in comparison, calculate each group gene expression
It is horizontal.
CCK-8 method testing result is as shown in figure 16, compared with normal group, high sugar effect group cell growth be substantially reduced (P <
0.05), Rosiglitazone cellular control unit proliferation higher sugar group is significantly increased (P < 0.05), and CCFM1060 group is compared with high sugared group
Cell proliferative condition also obviously increases (P < 0.05).
Maf A mRNA expression such as Figure 17 shows that the expression quantity of the MafA mRNA of high sugar effect group cell is obviously low
In normal group (P < 0.05), and the Maf A mrna expression amount higher sugar of Rosiglitazone positive controls and CCFM1060 group acts on
Group is obvious to rise (P < 0.05).
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. Lactobacillus rhamnosus CCFM1060, deposit number is GDMCC No:60705.
2. the method for preparing Lactobacillus rhamnosus CCFM1060 microbial inoculum, it is characterised in that: including,
The preparation of strain cultures；
The protectant preparation of bacterial strain；
Inoculated and cultured, freeze-drying.
3. the method for preparation Lactobacillus rhamnosus CCFM1060 microbial inoculum as claimed in claim 2, it is characterised in that: the bacterial strain
Culture medium, is modified MRS culture medium, and formula is tryptone 10g, beef extract 10g, yeast powder 5g, glucose 20g, vinegar
Sour sodium 5g, diammonium hydrogen citrate 2g, dipotassium hydrogen phosphate 2g, epsom salt 0.5g, Tween-80 1mL, Manganous sulfate monohydrate
0.25g, cysteine hydrochloride 0.5g, water 1000mL；Adjusting its pH is 6.5 ± 0.2.
4. the method for preparation Lactobacillus rhamnosus CCFM1060 microbial inoculum as claimed in claim 2 or claim 3, it is characterised in that: described
Bacterial strain protective agent, formula be 100g/L~150g/L skimmed milk power, 100g/L~150g/L maltodextrin, 140g/L~
160g/L trehalose, surplus are water, and freeze-drying obtains freeze drying protectant.
5. the method for preparation Lactobacillus rhamnosus CCFM1060 microbial inoculum as claimed in claim 4, it is characterised in that: the bacterial strain
Protective agent, formula be 120g/L skimmed milk power, 120g/L maltodextrin, 150g/L trehalose, surplus are water, is lyophilized, obtains
Freeze drying protectant.
6. the method for preparation Lactobacillus rhamnosus CCFM1060 microbial inoculum as claimed in claim 2, it is characterised in that: the inoculation
Culture, freeze-drying, including, Lactobacillus rhamnosus CCFM1060 is inoculated into 2% inoculum concentration and is sterilized under the conditions of 119~123 DEG C
In the strain cultures after 15~25min, under 35~39 DEG C of anaerobic conditions of temperature cultivate 24~48h, with pH be 6.8~
7.2 phosphate buffers clean 2~4 times, are resuspended with the bacterial strain protective agent, make bacterium is dense to reach 1010CFU/mL；Then, this is allowed
Bacterial strain re-suspension liquid 50~70min of preculture under 37 DEG C of anaerobic conditions of temperature, then in -15~-20 DEG C of pre-freezes 8~14h, Zhi Houzhen
Vacuum freecing-dry.
7. the method for preparation Lactobacillus rhamnosus CCFM1060 microbial inoculum as claimed in claim 6, it is characterised in that: rhamnose cream
Bacillus CCFM1060 is inoculated into culture medium under the conditions of 121 DEG C after sterilizing 20min with 2% inoculum concentration, at 37 DEG C of temperature
It is cultivated under anaerobic condition for 24 hours, is that 6.8 phosphate buffers clean 2~4 times with pH, is resuspended with the protective agent, makes bacterium is dense to reach
To 1010CFU/mL；Then, suspension preculture 60min under 37 DEG C of anaerobic conditions of temperature is allowed, then in -15 DEG C of pre-freeze 12h,
Vacuum freeze drying later.
8. a kind of fermented food, it is characterised in that: carried out using Lactobacillus rhamnosus CCFM1060 microbial inoculum as claimed in claim 2
Fermentation, the microbial inoculum contain greater than 106The active Lactobacillus rhamnosus CCFM1060 of CFU/g.
9. fermented food as claimed in claim 8, it is characterised in that: the fermented food includes dairy products, bean product and fruit
Vegetable product.
10. fermented food as claimed in claim 9, it is characterised in that: the fermented food, including cucumber pear juice lactic acid bacteria drink
Material, preparation method is to squeeze the juice pears to obtain pear juice, by cucumber stripping and slicing, is put into blanching 30s in 90 DEG C of hot water, is beaten, mistake
Filter, obtains Fresh Cucumber Juice；Pear juice, Fresh Cucumber Juice are mixed with skimmed milk powder with the mass ratio of 5:4:1, Fresh Cucumber Juice pear juice mixing cream is obtained
Liquid, 5% xylitol, packing are added after homogeneous, and the Lactobacillus rhamnosus is added in 115 DEG C of sterilizing 20min after being cooled to room temperature
CCFM1060 microbial inoculum is with 107CFU/mL is inoculated into the Fresh Cucumber Juice pear juice mixed emulsion, and ferment 16h in 37 DEG C of constant incubators.
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| CN111109359B (en) * | 2019-12-29 | 2022-09-02 | 江南大学 | Multifunctional pediococcus acidilactici CCFM1105, fermented food thereof and application |
| CN112322528A (en) * | 2020-11-03 | 2021-02-05 | 江南大学 | Lactobacillus rhamnosus capable of intervening metabolic syndrome and application thereof |
| CN112322554A (en) * | 2020-12-04 | 2021-02-05 | 光明乳业股份有限公司 | Lactobacillus rhamnosus strain, microbial inoculum, preparation method and application |
| WO2022162439A1 (en) * | 2021-01-27 | 2022-08-04 | Synbio Tech Inc. | Lactobacillus rhamnosus lrh05 isolate, and composition including the same and use thereof |
| CN113403231A (en) * | 2021-07-08 | 2021-09-17 | 江南大学 | Lactobacillus reuteri CCFM1178 capable of intervening metabolic syndrome and application thereof |
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