CN107937316B - Space lactobacillus reuteri Fullarton-9-71 and application - Google Patents
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- CN107937316B CN107937316B CN201711424823.7A CN201711424823A CN107937316B CN 107937316 B CN107937316 B CN 107937316B CN 201711424823 A CN201711424823 A CN 201711424823A CN 107937316 B CN107937316 B CN 107937316B
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Classifications
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- 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
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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- 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/173—Reuteri
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses one plant of space lactobacillus reuteri Fullarton-9-71 and applications.Lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71 provided by the present invention, it is CGMCC No.14942 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Lactobacillus reuteri Fullarton-9-71 provided by the present invention, compared with the control strain of ground, the viscosity for the skimmed milk that ferments is higher, stronger to the bacteriostasis of various bacteria, produce reuterin ability it is higher, and safety evaluatio experiment the result shows that being safe.Therefore, lactobacillus reuteri Fullarton-9-71 provided by the present invention has the potentiality and value of continual exploitation.
Description
Technical field
The invention belongs to microorganism fields, are related to one plant of space lactobacillus reuteri Fullarton-9-71 and application.
Background technique
Lactobacillus reuteri (Lactobacillus reuteri) is reported at present almost to may be present in all vertebras
Bacillus acidi lactici in animal and mammalian gut, harmless to humans and animals in the intestinal tract for often inhabiting humans and animals, tool
There is good biocompatibility, is to have to improve allergic constitution, pre- hypo-allergenic recurrent exerbation, and adjust the prebiotic of function of intestinal canal
Bacterium.In recent years, probiotics has become the research hotspot of microbiological art, and obtains extensively on health food and dairy industry
General application.Currently, China is approved as can be used for the probiotics strain reference of health food lactobacillus reuteri.
Lactobacillus reuteri not only has the main beneficial functional of lactic acid bacteria, but also is also equipped with and generates broad-spectrum antiseptic substance
Special efficacy.It is metabolized glycerol and generates a kind of special antibacterial substance --- Luo Yishi element (reuterin).Luo Yishi element is a kind of wide
Antibacterial agent is composed, can inhibit the growth of gram-positive bacteria, Gram-negative bacteria, saccharomycete, mould, protozoon, protozoan etc.,
Not only certain fungies and protozoan can also be equally acted on useful effect in bacterium.Reuterin is as antibacterial
The superiority of substance has caused people more and more to pay close attention to, also safe and non-toxic because of its unique biochemical characteristic and to humans and animals
And there is very wide application prospect.The main component of Luo Yishi element be the monomer of 3-HPA (3-HPA), hydrate and
Cyclodimerization body.Other than antibacterial, 3-HPA monomer or a kind of potential important industrial chemicals can be used as a variety of emerging chemistry
The precursor of product such as methacrylaldehyde, acrylic acid, 1,3-PD etc., is used to prepare novel polymer material；It can be with the ammonia in protein
Base reacts to form crosslinking, and the glutaraldehyde and epoxide for being expected to substituted chemistry synthesis are as new biological cross-linker.
Summary of the invention
The object of the present invention is to provide one plant of new space lactobacillus reuteri (Lactobacillus reuteri) and answer
With.
Lactobacillus reuteri (Lactobacillus reuteri) provided by the present invention is specially lactobacillus reuteri
(Lactobacillus reuteri) Fullarton-9-71, it is commonly micro- in China Committee for Culture Collection of Microorganisms
The deposit number of Bio-Centers is CGMCC No.14942.
Lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71 in the present invention is Divine Land 11
Number Spaceship Carrying lactobacillus reuteri SS23 (is compiled from Chinese industrial Microbiological Culture Collection administrative center (CICC) preservation
Number be CICC 6118 lactobacillus reuteri (Lactobacillus reuteri), 53608) which is in the number of ATCC
Into space, at too airflight 18.5 hours 31 days, after airship returns to the earth, a series of screening experiment is taken to obtain
's.China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation are preserved on November 20th, 2017
Number is CGMCC No.14942.
Correspondingly, being the lactobacillus reuteri (Lactobacillus the present invention also provides a kind of active constituent
Reuteri) the microbial inoculum of Fullarton-9-71.
In addition to containing the lactobacillus reuteri (Lactobacillus as active constituent in the microbial inoculum
Reuteri) outside Fullarton-9-71, also contain auxiliary material.The effect of the auxiliary material can be figuration, serves as carrier, improve and stablize
Property, solubilising, hydrotropy, sustained release etc..
It is prepared by lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71 or the microbial inoculum
Application in fermented dairy product also belongs to protection scope of the present invention.
It is prepared by lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71 or the microbial inoculum
Application in leavening used in production fermented dairy product also belongs to protection scope of the present invention.
Further, used raw milk can be cow's milk, sheep cream, soya-bean milk during preparing the fermented dairy product
Deng either skimmed milk, is also possible to non-skimmed milk.
Further, the fermented dairy product can be Yoghourt, Kefir grains, fermentation buttermilk, Yoghourt wine, koumiss etc..
Lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71 or the microbial inoculum are producing
Application in Luo Yishi element also belongs to protection scope of the present invention.
It is prepared by lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71 or the microbial inoculum
Application in antibacterials also belongs to protection scope of the present invention.
Wherein, the bacterium can also be able to be fungi such as gram-positive bacterium or gramnegative bacterium for bacterium.
Further, concretely Escherichia coli, staphylococcus aureus, salmonella and/or list increase Lee to the bacterium
This special bacterium etc..
Lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71 or the microbial inoculum are as follows
Application in any also belongs to protection scope of the present invention:
(a1) human or animal's the gastrointestinal tract micro ecological balance, or preparation are adjusted for adjusting human or animal's micro ecology of gastrointestinal tract
The product of balance；
(a2) alleviate enteritis, or prepare the product for alleviating enteritis；
(a3) auxiliary protection gastric mucosa, or preparation are used for the product of auxiliary protection gastric mucosa；
(a4) defaecation, or preparation are used for the product of defaecation；
(a5) enhance human or animal's immunity, or prepare the product for enhancing human or animal's immunity.
Wherein, the product can be drug, nutriment or health care product etc..
Lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71 or the microbial inoculum are as follows
Application in any also belongs to protection scope of the present invention:
(b1) food additives are prepared；
(b2) animal feed additive is prepared.
Lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71 provided by the present invention, with ground
Face control strain is compared, and the viscosity for the skimmed milk that ferments improves 0.58 times；Yield of extracellular polysaccharide improves 1.42 times；Fermentation supernatant
Liquid (pH=6.5) increases 0.40 times to the inhibiting rate of Escherichia coli, increases 0.12 to the inhibiting rate of staphylococcus aureus
Times, 11.81 times are improved to the bacteriostasis rate of salmonella；The ability for producing reuterin improves 0.92 times；Hemolytic experiment result
It is γ-haemolysis, i.e. not haemolysis tentatively shows lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71
It is safe.It the results are shown in Table 1.In conclusion lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71
Potentiality and value with continual exploitation.
The property of table 1 Fullarton-9-71 and ground bacterial strain
Preservation explanation
Strain name: lactobacillus reuteri
Latin name: Lactobacillus reuteri
Join the biomaterial (strain) of Ju: Fullarton-9-71
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date: on November 20th, 2017
Collection is registered on the books number: CGMCC No.14942
Detailed description of the invention
Fig. 1 is the microscopy photo of the bacterial strain that number is F-9-71 and ground control bacterium.A: the bacterial strain that number is F-9-71；B:
Ground compares bacterium.
Fig. 2 is that ground compares bacterium and its through the resulting each mutagenic strain of space carrying to the tolerance of low pH and cholate.
Fig. 3 is the measurement that ground compares bacterium and its cell surface hydrophobicity through the resulting each mutagenic strain of space carrying.
Fig. 4 is that the ability that ground compares bacterium and its produces Luo Yishi element through the resulting each mutagenic strain of space carrying measures.
Wild type in Fig. 2-4 indicates ground control strain GS23.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Lactobacillus reuteri Fuller subspecies (Lactobacillus reuteri FSMCC) is by Fuller bioengineering section
Skill (Beijing) Co., Ltd, microorganism fungus kind library, space provides, and being related to strain is that ground compares bacterium GS23
(Lactobacillusreuteri FSMCC GS23) and space carrying bacterium SS23 (Lactobacillus reuteri FSMCC
SS23), the two is same strain bacterium, to be from Chinese industrial Microbiological Culture Collection administrative center (CICC) deposit number
The lactobacillus reuteri (Lactobacillus reuteri) of CICC6118, which is 53608 in the number of ATCC.Wherein
It is 18.5 hours 31 days that space carrying bacterium SS23, which enters the carrying time after space,.
Surveyed indices sample is three parallel in each test of following embodiments, and the data obtained is with average value ± mark
The form of quasi- deviation is expressed.The statistical analysis of data uses GraphPad Prism 6, using each mutagenic strain of t- check analysis
With the significance of difference of ground bacterial strain, significant difference is indicated as p < 0.05, the level of signifiance is labeled as *；As p < 0.01,
The level of signifiance is labeled as * *, and as p < 0.001, the level of signifiance is labeled as * * *.
The separation and identification of embodiment 1, lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71
One, the separation of space flight bacterial strain
After the effective MRS culture medium activation three generations of the glycerol stocks of space carrying bacterium SS23, used on MRS solid plate
The separation of four zoning collimation methods, 37 DEG C of inversion are cultivated for 24 hours, picking colony form compareed with ground the discrepant single colonie of bacterium GS23 or
Random choosing colony selects 100 bacterium colonies altogether.Using plate streak isolated and purified three times, carry out morphological observation and
Gram's staining, and saved using Freezing Glycerine method.
As the result is shown: Gram's staining being carried out to the bacterium colony isolated and purified, is gram-positive bacteria, thalli morphology is in short
Bar or stock.Fig. 1 is the microscopy photo of the bacterial strain that number is F-9-71 and ground control bacterium, two plants of bacterium form no significant differences.
Two, the measurement of fermenting property
Isolate and purify 100 bacterial strains are connected in 12% (m/v) degreasing milk medium of sterilizing, 37 DEG C of fermentations are seen
It examines curdled milk speed and carries out primary dcreening operation.After culture to curdled milk, it is placed in 4 DEG C of refrigerators and carries out staying overnight after-ripening, measure degreasing milk medium pH value
And viscosity.It is control with ground control bacterium GS23, selection carries out real in next step with the big space flight bacterial strain of control strain difference
It tests.The mutagenic strain just sifted out is passed on 5 times, verifies the fermenting property of bacterial strain, again to prevent mutagenic strain back mutation
Occur.The measurement of viscosity is carried out using DV-III type viscosimeter, is popped one's head in using LV3, revolving speed 200rpm, minute 2min.
As the result is shown: fermenting property carried out to 100 bacterium colonies picking out, the bacterial strain of detection can curdled milk, but it is solidifying
The newborn time has larger difference.11 plants of space flight bacterial strain that there is notable difference with control strain fermenting property have been selected,
Experimental result is shown in Table 2.From the point of view of the curdled milk time, the bacterial strain curdled milk time of number F-9-20, F-9-35, F-9-79 and F-9-87
Considerably shorter than control strain, remaining bacterial strain is on the contrary.From fermentation skimmed milk pH from the point of view of, this 11 plants of mutagenic strains with compare bacterium
The pH difference of strain is not significant.From the point of view of the viscosity of fermentation skimmed milk, number F-9-17, F-9-18, F-9-35, F-9-40, F-
The viscosity and control strain of the bacterial strain of 9-58, F-9-71, F-9-79 and F-9-87 have significant difference.
2 lactobacillus reuteri curdled milk time of table and production acid produce stick nature
| Bacterial strain | The curdled milk time/h | pH | Viscosity |
| Ground compares bacterium GS23 | 26.17±0.29 | 4.92±0.033 | 263.00±20.30 |
| F-9-04 | 34.50±0.50*** | 4.97±0.02 | 244.33±7.57 |
| F-9-17 | 114.67±0.77*** | 5.03±0.03 | 429.00±14.73*** |
| F-9-18 | 132.33±1.04*** | 5.05±0.03 | 488.67±12.50*** |
| F-9-20 | 22.67±0.58*** | 4.90±0.02 | 273.33±9.45 |
| F-9-25 | 46.50±0.05*** | 4.83±0.03 | 257.00±35.03 |
| F-9-35 | 20.67±0.58*** | 4.89±0.01 | 426±22.27*** |
| F-9-40 | 67.17±0.29*** | 4.84±0.03 | 310±13.05* |
| F-9-58 | 52.33±0.58*** | 4.85±0.03 | 359.00±40.95* |
| F-9-71 | 31.33±0.29*** | 4.83±0.02 | 415.67±16.04*** |
| F-9-79 | 18.00±0.50*** | 4.73±0.01 | 378.00±19.08** |
| F-9-87 | 14.17±0.29*** | 4.83±0.03 | 330.67±24.83* |
Three, the gastral tolerance test of people is simulated
The precondition that probiotics plays probiotic properties in human body is to guarantee it in the acidic environment and enteron aisle of gastric juice
It is survived in cholate environment.The present invention simulates the low pH of human gastrointestinal tract by experiment in vitro, and high cholate environment evaluates mutagenic bacteria
Tolerance of the strain to simulated digestive juice.
1, low pH tolerance measurement
Bacterium solution is inoculated in the MRS culture medium of pH 2.5,37 DEG C of incubator culture 3h are placed in.Using 10 times of physiological saline
Dilution plate counting method is measured the viable count of 0h and 3h.
2, Bile salt resistance measures
Bacterium solution is inoculated in the MRS culture medium that gallbladder salinity is 0.5% (m/v), is placed in 37 DEG C of incubator culture 4h.It adopts
It is diluted with 10 times of physiological saline, colony counting method is measured the viable count of 0h and 4h.The result of bacterial strain tolerance uses viable bacteria
Several variation indicates that calculation formula is as follows:
RI=log N0/Nf；
In formula, N0Indicate initial total plate count；NfIndicate final total plate count.
3, result
Experimental result is shown in Fig. 2.The study found that lactobacillus reuteri has good tolerance, number F-9- to low pH
The acid-fast ability of 87 bacterial strain is best；But it is slightly worse to the tolerance of cholate, wherein number is F-9-17, F-9-18, F-
The bacterial strain of 9-35 and F-9-58 has relatively good bile tolerance ability, and viable count is declined by less than 2 orders of magnitude.
Four, the measurement of somatic cells surface hydrophobic
Lactobacillus reuteri is incubated overnight in MRS culture medium, and centrifugation, 3mL PBS is washed twice, adjusted to OD600Be worth in
0.8-1.0(A0).1mL dimethylbenzene is added in 3ml bacteria suspension, and vortex oscillation 120s, 37 DEG C of standing 1h detect the OD value of water phase
It (A), is control with buffer.H%=[(A0-A)/A0], it is believed that perhaps be greater than 50% has preferable hydrophobicity, i.e. adhesion
It may also be higher.
From the point of view of hydrophobicity result, the hydrophobicity of the bacterial strain of number F-9-25, F-9-35, F-9-79, F-9-87 is more than
50%, and it is significantly higher than control strain, see Fig. 3.
Five, the detection of extracellular polysaccharide (EPS)
Exopolysaccharides Produced by Lactic Acid Bacteria refers to the mucilage polysaccharides or pod that lactic acid bacteria is secreted into outside cell wall during growth metabolism
The general name of film polysaccharide.EPS has different physiological roles, including protection thallus, promotion thallus stick, blood pressure lowering, norcholesterol, resist
Oxidation, antitumor, antiulcer, it is antiviral, improve and intestine microenvironment and enhance human immunity.In addition, EPS is as new
The natural food additives of type, can improve the indexs such as texture, mouthfeel, rheological properties and the flavor of food, can also further mention
The nutrition health-care functions of high product.This research has chosen front research and obtains 7 plants for having larger difference with control strain property
The detection of mutagenic strain progress EPS.
1, the extraction of EPS
Lactobacillus reuteri is inoculated in 10% (m/v) degreasing milk medium, after 37 DEG C of culture to curdled milks, by curdled milk
It is crushed and stirs evenly, draw 5mL sample respectively in centrifuge tube, and the three of 5% isometric (m/v) is added into fermentation liquid
Monoxone (TCA) solution stands 30min protein precipitation at room temperature, is centrifuged 30min in 4 DEG C, 10000r/min, is filtered with 0.45 μm
Membrane filtration obtains supernatant, dilutes 80 times with distilled water, draws every group of filtrate 1mL in tool plug test tube, be separately added into 6% (v/ of 1mL
V) phenol solution, the 5mL concentrated sulfuric acid are uniformly mixed, and are used distilled water to return to zero as blank reagent, 15min are kept in boiling water bath, so
Rapid ice-water bath is cooling afterwards terminates reaction.Absorbance value is measured at wavelength 490nm, calculates exocellular polysaccharide content.
2, the measurement of EPS
EPS content is measured using phend-sulphuric acid, with glucose as a standard product make standard curve.Take analyze in right amount it is pure
Glucose is placed in 80 DEG C of dryings in air dry oven and accurately weighs 100mg glucose in 500mL volumetric flask after cooling to constant weight
In, add distilled water to scale.Each liquor capacity of reaction system is added by table 2, and Standard glucose solution is first added to tool plug and is carved
Spend test tube in, add the phenol solution of 5% (v/v), be eventually adding the 10mL concentrated sulfuric acid mix stand, after its cooling after in
Absorbance is measured at 490nm, every group is done 3 in parallel.With glucose content (mg/L) for abscissa, absorbance (A490) it is vertical sit
Plotting standard curve.Obtain equation of linear regression are as follows: y=1.405x-0.426, R2=0.987.With method measure EPS aqueous solution in
Absorbance at wavelength 490nm passes through the EPS yield of regression equation calculation bacterial strain.
In terms of mass fraction ω, unit is indicated polyoses content with gram every hectogram (g/100g) in sample, is counted as follows
It calculates:
In formula:
m1--- from sugar content in sample measurement liquid is checked on standard curve, unit is microgram (μ g)；
V1--- sample constant volume, unit are milliliter (mL)
V2--- the volume of colorimetric estimation Shi Suoyi taking sample determination liquid, unit are milliliter (mL)；
m2--- sample quality, unit are gram (g)；
0.9 --- glucose is converted into the correction coefficient of glucose.
Calculated result retains to 2 significant digits.
3, result
As a result such as table 3.Wherein number is the mutagenic strain of F-9-17, F-9-35, F-9-58, F-9-71 and F-9-87
EPS yield will be significantly higher than control strain.
The content of the extracellular polysaccharide of 3 lactobacillus reuteri of table
Six, bacteriostatic test
1, the preparation of cell-free supernatants
Lactobacillus reuteri is 37 DEG C in MRS or MRS-glycerol (MRS-g, glycerol concentration 400mM) culture medium
For 24 hours, centrifugation obtains supernatant for culture.In order to exclude the bacteriostasis of organic acid, the pH to 6.5 of supernatant is adjusted with 1M NaOH.
By processed cell-free supernatants through 0.22 μm of membrane filtration, 4 DEG C are saved backup.
2, the detection of bacteriostatic activity
The detection of bacteriostatic activity co-cultures method using 96 orifice plates.The indicator bacteria that this experiment uses is Escherichia coli
(Escherichia coli) ATCC 8739, staphylococcus aureus (Staphylococcus aureus) ATCC 25923,
Salmonella (Salmonella enterica serovar Typhimurium) ATCC 14028, Listeria monocytogenes
(Listeria monocytogenes) ATCC 19115, these indicator bacterias 37 DEG C of shaking table cultures in TSB culture medium.
96 orifice plates co-culture method: indicator bacteria is incubated overnight, and taking 0.1ml concentration is 105The instruction bacteria culture fluid of cfu/ml adds
Enter in 96 orifice plates, adds 0.1ml cell free broth, for 24 hours, microplate reader detects OD for 37 DEG C of cultures600.TSB culture medium is added
Hole be control, utilize following formula carry out bacteriostasis rate calculating:
The results are shown in Table 4: the MRS supernatant of all test strains all has bacteriostasis, but when adjusting pH to 6.5
All do not have fungistatic effect, this illustrates that antipathogenic composition is mainly organic acid.However, by the supernatant (pH=of strain fermentation MRS-g
6.5) bacteriostatic experiment discovery is carried out, many bacterial strains still have bacteriostatic activity, this explanation has the bacterial strain of bacteriostasis can generation
It thanks to glycerol, generates certain antipathogenic compositions.First and last, suppression of number F-9-25, F-9-35, the F-9-71 to 4 kinds of pathogenic bacteria
Rate processed close to 100%, shows powerful bacteriostasis.
4 96 well plate method of table measures lactobacillus reuteri MRS-g to the inhibiting rate (%) of pathogen
| Strain number | E.coli | S.aureus | S.enterica | L.monocytogenes |
| Ground control strain GS23 | 70.9±0.06 | 89.2±0.87 | 7.8±0.14 | 100.1±0.97 |
| F-9-04 | 69.6±1.85 | 82.3±0.92 | 4.0±0.42 | 100.1±0.37 |
| F-9-17 | 65.6±0.96 | 34.3±1.12 | 7.6±0.43 | 0.3±0.04 |
| F-9-18 | 99.1±0.03 | 100.3±0.04 | 46.6±0.48 | 101.3±0.64 |
| F-9-20 | 99.0±0.23 | 99.8±0.10 | 46.9±8.36 | 101.3±0.85 |
| F-9-25 | 98.9±0.10 | 100.1±0.17 | 99.9±0.10 | 99.4±0.85 |
| F-9-35 | 98.9±0.10 | 99.9±0.28 | 99.9±0.05 | 100.0±0.32 |
| F-9-40 | 54.0±0.52 | 44.1±0.45 | 4.4±0.16 | 94.3±0.32 |
| F-9-58 | 73.1±2.52 | 100.3±0.11 | 58.8±1.16 | 100.4±0.21 |
| F-9-71 | 99.0±0.03 | 100.2±0.11 | 100.1±0.09 | 100.4±0.37 |
| F-9-79 | 46.8±0.03 | 8.4±0.07 | 4.0±0.10 | 87.6±0.53 |
| F-9-87 | 27.7±1.10 | 8.4±2.33 | 2.5±0.10 | 30.6±0.37 |
3, reuterin generates the detection of ability
(1) preparation of supernatant to be measured
Lactobacillus reuteri is cultivated for 24 hours in MRS culture medium, and centrifugation, PBS is washed once, and it is water-soluble to be resuspended in glycerol-
Liquid, 37 DEG C of incubation 3h, centrifugation obtain supernatant, it is to be measured to be placed in 4 DEG C of refrigerations.
(2) production of standard curve
The production of methacrylaldehyde standard curve: stock solution is diluted with 95% ethyl alcohol, 0- is added into 10ml volumetric flask respectively
1.2ml water is added less than being supplied with 95% for 2ml in 2ml stock solution.Then be added 0.5ml 0.01M tryptophan solution and
The dense HCl of 6.3ml is placed in 60 DEG C of water-bath 5min, reaches most dark colour.After water-bath, OD is measured560, blank is done with reagent controls.Most
The corresponding absorbance value of 15 μ g, 30 μ g, 45 μ g, 60 μ g, 75 μ g, 90 μ g methacrylaldehyde is established into standard curve afterwards.
As a result as shown in figure 4, compared with the control strain of ground, the reuterin production for the bacterial strain that number is F-9-71 is improved
0.92 times.
Seven, safety evaluatio (hemolytic test)
Hemolytic test is that the lactobacillus reuteri culture solution that will be incubated overnight is crossed to blood dish surface, 37 DEG C of culture 48h.
See whether haemolysis occur, i.e. beta hemolysis (the clear haemolysis circle of large area occurs in periphery of bacterial colonies), alpha hemolysis (periphery of bacterial colonies
There is light brown or grass green haemolysis circle) and γ-haemolysis (periphery of bacterial colonies does not have haemolysis circle).
The results show that carrying out hemolytic experiment to 11 plants of mutagenic strains and 1 plant of ground control strain.The study found that all surveys
Examination bacterial strain does not occur haemolysis, i.e. γ-haemolysis.Tentatively show to be safety by the candidate probiotic strain of space flight
's.
Eight, the identification of strain
It faces to carry out DNA extraction respectively according to bacterium and each space carrying mutagenic strain over the ground by DNA kit, extracts and complete
Afterwards, then PCR amplification is carried out to above-mentioned DNA extract respectively, amplification system total volume is 20 μ L, the Taq including 2 units
Archaeal dna polymerase, PCR buffer, 2.5mM MgCl2, 500 μM of dNTP, the universal primer 1 of 100ng DNA profiling and 10pmol,
2, P1:5 '-AGTTTGATCMTGGCTCAG-3 '；And P2:5 '-GGTTACCTTGTTACGACTT-3 '.Amplification condition are as follows: 95 DEG C
Initial denaturation 2min, 95 DEG C of denaturation 30s, 51 DEG C of annealing (renaturation) 30s, 72 DEG C of extension 1min are recycled 30 times, last 72 DEG C of extensions
2min obtains final product, and product is then delivered the raw work sequencing in Shanghai.
By 16S rDNA be sequenced as a result, and combine the morphological feature identified above, it is known that ground control bacterium respectively lures
Becoming bacterial strain is lactobacillus reuteri (Lactobacillus reuteri), and numbers the bacterial strain for being F-9-71 in 16SrDNA
Occurs variation in sequence, 16S rDNA sequence is as shown in SEQ ID No.1.
The bacterial strain that number is F-9-71 was preserved in Chinese microorganism strain preservation management committee on November 20th, 2017
Member's meeting common micro-organisms center, deposit number are CGMCC No.14942, and the biomaterial (strain) for joining Ju is Fullarton-9-
71。
The cultivation temperature of lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71 is 37 DEG C；From
Right pH；Medium component: casein peptone 10.0g, beef extract 10.0g, yeast powder 5.0g, glucose 5.0g, sodium acetate 5.0g, lemon
Lemon acid diammonium 2.0g, Tween 80 1.0g, K2HPO4 2.0g, MgSO4.7H2O 0.2g, MnSO4.H2O 0.05g, agar
15.0g, distilled water 1.0L.
<110>Fuller gives birth suddenly object engineering science and technology (Beijing) Co., Ltd
<120>space lactobacillus reuteri Fullarton-9-71 and application
<130> GNCLN172069
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1414
<212> DNA
<213>lactobacillus reuteri (Lactobacillus reuteri)
<400> 1
tactgcagtc gtacgcactg gcccaactga ttgatggtgc ttgcacctga ttgacgatgg 60
atcaccagtg agtggcggac gggtgagtaa cacgtaggta acctgccccg gagcggggga 120
taacatttgg aaacagatgc taataccgca taacaacaaa agccacatgg cttttgtttg 180
aaagatggct ttggctatca ctctgggatg gacctgcggt gcattagcta gttggtaagg 240
taacggctta ccaaggcgat gatgcatagc cgagttgaga gactgatcgg ccacaatgga 300
actgagacac ggtccatact cctacgggag gcagcagtag ggaatcttcc acaatgggcg 360
caagcctgat ggagcaacac cgcgtgagtg aagaagggtt tcggctcgta aagctctgtt 420
gttggagaag aacgtgcgtg agagtaactg ttcacgcagt gacggtatcc aaccagaaag 480
tcacggctaa ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggat 540
ttattgggcg taaagcgagc gcaggcggtt gcttaggtct gatgtgaaag ccttcggctt 600
aaccgaagaa gtgcatcgga aaccgggcga cttgagtgca gaagaggaca gtggaactcc 660
atgtgtagcg gtggaatgcg tagatatatg gaagacacca gtgcgaaggc ggctgtctgg 720
tctgcaactg acgctgaggc tcgaaagcat gggtagcgaa caggattaga taccctggta 780
gtccatgccg taaacgatga gtgctaggtg ttggagggtt tccgcccttc agtgccggag 840
ctaacgcatt aagcactccg cctggggagt acgaccgcaa ggttgaaact caaaggaatt 900
gacgggggcc cgcacaagcg gtggagcatg tggtttaatt cgaagctacg cgaagaacct 960
taccaggtct tgacatcttg cgctaacctt agagataagg cgttcccttc ggggacgcaa 1020
tgacaggtgg tgcatggtcg tcgtcagctc gtgtcgtgag atgttgggtt aagtcccgca 1080
acgagcgcaa cccttgttac tagttgccag cattaagttg ggcactctag tgagactgcc 1140
ggtgacaaac cggaggaagg tggggacgac gtcagatcat catgcccctt atgacctggg 1200
ctacacacgt gctacaatgg acggtacaac gagtcgcaag ctcgcgagag taagctaatc 1260
tcttaaagcc gttctcagtt cggactgtag gctgcaactc gcctacacga agtcggaatc 1320
gctagtaatc gcggatcagc atgccgcggt gaatacgttc ccgggccttg tacacaccgc 1380
ccgtcacacc atgggagttt gtaacgctcc aaag 1414
Claims (11)
1. lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-71, it is protected in Chinese microorganism strain
The deposit number for hiding administration committee's common micro-organisms center is CGMCC No. 14942.
2. a kind of microbial inoculum, its active constituent be lactobacillus reuteri described in claim 1 (Lactobacillus reuteri) Fullarton-9-71.
3. lactobacillus reuteri described in claim 1 (Lactobacillus reuteri) Fullarton-9-71 or right
It is required that microbial inoculum described in 2 is preparing the application in fermented dairy product.
4. lactobacillus reuteri described in claim 1 (Lactobacillus reuteri) Fullarton-9-71 or right
It is required that microbial inoculum described in 2 is in preparation for producing the application in leavening used in acidified milk preparation.
5. lactobacillus reuteri described in claim 1 (Lactobacillus reuteri) Fullarton-9-71 or right
It is required that application of the microbial inoculum described in 2 in production Luo Yishi element.
6. lactobacillus reuteri described in claim 1 (Lactobacillus reuteri) Fullarton-9-71 or right
It is required that application of the microbial inoculum described in 2 in preparation antibacterials.
7. application according to claim 6, it is characterised in that: the bacterium is bacterium or fungi.
8. application according to claim 7, it is characterised in that: the bacterium is gram-positive bacterium or Gram-negative
Bacterium.
9. application according to claim 8, it is characterised in that: the bacterium is Escherichia coli, staphylococcus aureus, sand
Door Salmonella and/or Listeria monocytogenes.
10. lactobacillus reuteri described in claim 1 (Lactobacillus reuteri) Fullarton-9-71 or right
It is required that microbial inoculum described in 2 it is following it is any in application:
(a1) product for adjusting human or animal's the gastrointestinal tract micro ecological balance is prepared；
(a2) product for alleviating enteritis is prepared；
(a3) preparation is used for the product of auxiliary protection gastric mucosa；
(a4) preparation is used for the product of defaecation；
(a5) product for enhancing human or animal's immunity is prepared.
11. lactobacillus reuteri described in claim 1 (Lactobacillus reuteri) Fullarton-9-71 or right
It is required that microbial inoculum described in 2 it is following it is any in application:
(b1) food additives are prepared；
(b2) animal feed additive is prepared.
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| CN201711424823.7A CN107937316B (en) | 2017-12-25 | 2017-12-25 | Space lactobacillus reuteri Fullarton-9-71 and application |
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| CN109385387B (en) * | 2018-12-28 | 2022-04-05 | 上海源耀农牧科技有限公司 | TGEV-resistant lactobacillus reuteri and application thereof |
| CN110540950B (en) * | 2019-08-27 | 2022-05-17 | 南京农业大学 | Lactobacillus reuteri 22 and application thereof |
| CN117286083B (en) * | 2023-11-24 | 2024-03-22 | 杭州微致生物科技有限公司 | Lactobacillus reuteri VB319 and culture device and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114051A (en) * | 2012-11-15 | 2013-05-22 | 上海交大昂立股份有限公司 | Roy bacterium lacticum and application thereof |
| CN105062933A (en) * | 2015-09-11 | 2015-11-18 | 北京博锦元生物科技有限公司 | Lactobacillus reuteri and application thereof |
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| CN102304489B (en) * | 2011-09-23 | 2013-06-12 | 北京龙科方舟生物工程技术有限公司 | Lactobacillus reuteri strain and application thereof |
| CN107118975B (en) * | 2017-03-30 | 2020-08-11 | 杭州娃哈哈科技有限公司 | Lactobacillus reuteri capable of surviving in long shelf life at room temperature |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103114051A (en) * | 2012-11-15 | 2013-05-22 | 上海交大昂立股份有限公司 | Roy bacterium lacticum and application thereof |
| CN105062933A (en) * | 2015-09-11 | 2015-11-18 | 北京博锦元生物科技有限公司 | Lactobacillus reuteri and application thereof |
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