CN107974425A - Space lactobacillus reuteri Fullarton-9-79 and application - Google Patents
Space lactobacillus reuteri Fullarton-9-79 and application Download PDFInfo
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- CN107974425A CN107974425A CN201711421640.XA CN201711421640A CN107974425A CN 107974425 A CN107974425 A CN 107974425A CN 201711421640 A CN201711421640 A CN 201711421640A CN 107974425 A CN107974425 A CN 107974425A
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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
- 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
- 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
-
- 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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- 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
- A23C2220/00—Biochemical treatment
- A23C2220/20—Treatment with microorganisms
- A23C2220/204—Use of bacteria which are encapsulated, entrapped or immobilised; Fermentation with these bacteria
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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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A61K2035/11—Medicinal preparations comprising living procariotic cells
- A61K2035/115—Probiotics
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- 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
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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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- Life Sciences & Earth Sciences (AREA)
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- Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Animal Husbandry (AREA)
- Physiology (AREA)
- Nutrition Science (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses one plant of space lactobacillus reuteri Fullarton 9 79 and application.Lactobacillus reuteri (Lactobacillus reuteri) Fullarton 9 79 provided by the present invention, it is CGMCC No.14943 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Lactobacillus reuteri Fullarton 9 79 provided by the present invention, compared with the control strain of ground, the curdled milk time is shorter, the viscosity higher for the skimmed milk that ferments, hydrophobicity higher, and safety evaluatio experiment the result shows that being safe.Therefore, lactobacillus reuteri Fullarton 9 79 provided by the present invention has the potentiality and value of continual exploitation.
Description
Technical field
The invention belongs to microorganism field, is related to one plant of space lactobacillus reuteri Fullarton-9-79 and application.
Background technology
Lactobacillus reuteri (Lactobacillus reuteri) be at present reported almost 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, be 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 is obtained extensively on health food and dairy industry
General application.At present, China is approved as referring to available for the probiotics strain of health food lactobacillus reuteri.
Lactobacillus reuteri not only possesses the main beneficial functional of lactic acid bacteria, but also is also equipped with producing broad-spectrum antiseptic material
Special efficacy.It is metabolized glycerine and produces a kind of special antibacterial substance --- Luo Yishi elements (reuterin).Luo Yishi elements are a kind of wide
Antiseptic is composed, can inhibit the growth of gram-positive bacteria, Gram-negative bacteria, saccharomycete, mould, protozoon, protozoan etc.,
Not only it can also equally act on some fungies and protozoan with useful effect in bacterium.Reuterin is as antibacterial
The superiority of material has caused people more and more to pay close attention to, also because of its unique biochemical characteristic and safe and non-toxic to humans and animals
And there is very wide application prospect.The main component of Luo Yishi elements be the monomer of 3-HPA (3-HPA), hydrate and
Cyclodimerization body.In addition to antibacterial, 3-HPA monomers 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；Can be with the ammonia in protein
Base reacts to form crosslinking, be expected to substituted chemistry synthesis glutaraldehyde and epoxide as new biological cross-linker.
The content 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-9-79, it is commonly micro- in China Committee for Culture Collection of Microorganisms
The deposit number of Bio-Centers is CGMCC No.14943.
Lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-79 in the present invention is Divine Land 11
Number Spaceship Carrying lactobacillus reuteri SS23 (comes from Chinese industrial Microbiological Culture Collection administrative center (CICC) preservation volume
Number be CICC 6118 lactobacillus reuteri (Lactobacillus reuteri), 53608) which is in the numbering of ATCC
Into space, when too airflight 31 days 18.5 is small, 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
Numbering is CGMCC No.14943.
Correspondingly, it is the lactobacillus reuteri (Lactobacillus the present invention also provides a kind of active ingredient
Reuteri) the microbial inoculum of Fullarton-9-79.
Except containing the lactobacillus reuteri (Lactobacillus as active component in the microbial inoculum
Reuteri) outside Fullarton-9-79, auxiliary material is also contained.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) the Fullarton-9-79 or described microbial inoculums
Application in fermented dairy product falls within protection scope of the present invention.
It is prepared by lactobacillus reuteri (Lactobacillus reuteri) the Fullarton-9-79 or described microbial inoculums
Application in leavening used in production fermented dairy product falls within protection scope of the present invention.
Further, used raw milk can be cow's milk, sheep breast, soya-bean milk during the fermented dairy product is prepared
Deng either skimmed milk or non-skimmed milk.
Further, the fermented dairy product can be Yoghourt, Kefir grains, fermentation buttermilk, Yoghourt wine, koumiss etc..
It is prepared by lactobacillus reuteri (Lactobacillus reuteri) the Fullarton-9-79 or described microbial inoculums
Application in antibacterials falls within protection scope of the present invention.
Wherein, the bacterium can be bacterium, such as gram-positive bacterium or gramnegative bacterium, or fungi.
Further, concretely Escherichia coli, staphylococcus aureus, salmonella and/or list increase Lee to the bacterium
This special bacterium etc..
Lactobacillus reuteri (Lactobacillus reuteri) the Fullarton-9-79 or described microbial inoculums are as follows
Application in any falls within protection scope of the present invention：
(a1) human or animal's the gastrointestinal tract micro ecological balance is adjusted, or prepares and is used to adjust human or animal's micro ecology of gastrointestinal tract
The product of balance；
(a2) alleviate enteritis, or prepare the product for being used for alleviating enteritis；
(a3) auxiliary protection gastric mucosa, or prepare the product for auxiliary protection gastric mucosa；
(a4) defaecation, or prepare the product for defaecation；
(a5) strengthen human or animal's immunity, or prepare the product for being used for strengthening human or animal's immunity.
Wherein, the product can be medicine, nutriment or health products etc..
Lactobacillus reuteri (Lactobacillus reuteri) the Fullarton-9-79 or described microbial inoculums are as follows
Application in any falls within protection scope of the present invention：
(b1) food additives are prepared；
(b2) animal feed additive is prepared.
Lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-79 provided by the present invention, with ground
Face control strain is compared, and the curdled milk time shortens 8.17h, and the viscosity for the skimmed milk that ferments improves 0.44 times；Hydrophobicity improves
1.21 again.Hemolytic experiment is the result is that γ-haemolysis, i.e. not haemolysis tentatively shows lactobacillus reuteri (Lactobacillus
Reuteri) Fullarton-9-79 is safe.In conclusion lactobacillus reuteri (Lactobacillus reuteri)
Fullarton-9-79 has the potentiality and value of continual exploitation.
The property of table 1Fullarton-9-79 and ground bacterial strain
Note：- represent not detect
Preservation explanation
Strain name：Lactobacillus reuteri
Latin name：Lactobacillus reuteri
Join the biomaterial (strain) of Ju：Fullarton-9-79
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On November 20th, 2017
Collection is registered on the books numbering：CGMCC No.14943
Brief description of the drawings
Fig. 1 is the bacterial strain that numbering is F-9-79 and the microscopy photo of ground control bacterium.A：The bacterial strain that numbering is F-9-79；B：
Ground compares bacterium.
Fig. 2 compares bacterium and its tolerance through each mutagenic strain obtained by space carrying to low pH and cholate for ground.
Fig. 3 is the measure that ground compares bacterium and its cell surface hydrophobicity through each mutagenic strain obtained by space carrying.
Fig. 4 is that ground compares bacterium and its ability measure of Luo Yishi elements is produced through each mutagenic strain obtained by space carrying.
Wild type in Fig. 2-4 represent ground control strain GS23.
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) are by Fuller bioengineering section
Skill (Beijing) Co., Ltd space microorganism fungus kind storehouse provides, and is related to strain and compares bacterium GS23 (Lactobacillus for ground
Reuteri FSMCC GS23) and space carrying bacterium SS23 (Lactobacillus reuteri FSMCC SS23), Liang Zhewei
Same strain bacterium, to come from the Roy that Chinese industrial Microbiological Culture Collection administrative center (CICC) deposit number is CICC6118
Family name's lactobacillus (Lactobacillus reuteri), which is 53608 in the numbering of ATCC.Wherein space carrying bacterium SS23
Into the carrying time after space for 31 days 18.5 it is small when.
It is three parallel to survey indices sample in each experiment of following embodiments, and the data obtained is with average value ± mark
The form expression of quasi- deviation.The statistical analysis of data uses GraphPad Prism 6, using each mutagenic strain of t- check analyses
With the significance of difference of ground bacterial strain, significant difference is represented 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-9-79
First, the separation of space flight bacterial strain
After the effective MRS culture mediums activation three generations of the glycerol stocks of space carrying bacterium SS23, used on MRS solid plates
Four zoning collimation methods separate, 37 DEG C be inverted culture 24h, picking colony form compareed with ground the discrepant single bacterium colonies 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 preserved using Freezing Glycerine method.
The results show：Gram's staining is carried out to the bacterium colony isolated and purified, is gram-positive bacteria, thalli morphology is in short
Bar or stock.Fig. 1 be numbering be F-9-79 bacterial strain and ground control bacterium microscopy photo, two plants of bacterium form no significant differences.
2nd, the measure 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, see
Examine curdled milk speed and carry 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.Using ground control bacterium GS23 as control, selection carries out real in next step with the big space flight bacterial strain of control strain difference
Test.The mutagenic strain just sifted out is passed on 5 times, the fermenting property of bacterial strain is verified again, to prevent mutagenic strain back mutation
Occur.The measure of viscosity is carried out using DV-III types viscosimeter, is popped one's head in using LV3, rotating speed 200rpm, minute 2min.
The results show：100 bacterium colonies picking out are carried out with fermenting property, the bacterial strain of detection can curdled milk, it is but solidifying
The newborn time has larger difference.11 plants of the space flight bacterial strain that there is notable difference with control strain fermenting property has 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 numbering 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 the point of view of the pH of fermentation skimmed milk, this 11 plants of mutagenic strains are with compareing bacterium
The pH differences of strain are not notable.From the point of view of the viscosity of fermentation skimmed milk, numbering 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 production stick nature
| Bacterial strain | The curdled milk time/h | pH | Viscosity |
| Ground control 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* |
3rd, the gastral tolerance test of people is simulated
The precondition that probiotics plays probiotic properties in human body is the sour environment and enteron aisle for ensureing it in gastric juice
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.
1st, low pH tolerances measure
Bacterium solution is inoculated in the MRS culture mediums of pH 2.5, is placed in 37 DEG C of incubator culture 3h.Using 10 times of physiological saline
Dilution plate counting method is measured the viable count of 0h and 3h.
2nd, Bile salt resistance measures
Bacterium solution is inoculated in the MRS culture mediums that gallbladder salinity is 0.5% (m/v), is placed in 37 DEG C of incubator culture 4h.Adopt
With 10 times of dilutions 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 change represents that calculation formula is as follows：
RI=log N0/Nf；
In formula, N0Represent initial total plate count；NfRepresent final total plate count.
3rd, result
Experimental result is shown in Fig. 2.Research finds that lactobacillus reuteri has low pH good tolerance, numbering F-9-
The acid-fast ability of 87 bacterial strain is best；But it is slightly worse to the tolerance of cholate, wherein numbering 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.
4th, the measure of somatic cells surface hydrophobic
Lactobacillus reuteri is incubated overnight in MRS culture mediums, and centrifugation, 3mL PBS are washed twice, adjusted to OD600Be worth in
0.8-1.0(A0).1mL dimethylbenzene is added in 3ml bacteria suspensions, vortex oscillation 120s, 37 DEG C of standing 1h, detects the OD values of water phase
(A), using buffer solution as control.H%=[(A0-A)/A0], it is believed that perhaps there is preferable hydrophobicity, i.e. adhesion more than 50%
May also be higher.
From the point of view of hydrophobicity result, the hydrophobicity of the bacterial strain of numbering F-9-25, F-9-35, F-9-79, F-9-87 exceedes
50%, and it is significantly higher than control strain, see Fig. 3.
5th, 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 during growth metabolism outside cell membrane
The general name of film polysaccharide.EPS has different physiological roles, including protection thalline, promotion thalline stick, blood pressure lowering, norcholesterol, resist
Oxidation, antitumor, antiulcer, antiviral, improvement intestine microenvironment and enhancing body immunity etc..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 carry
The nutrition health-care functions of high product.This research have chosen above research and draw have larger difference with control strain property 7 plants
Mutagenic strain carries out the detection of EPS.
1st, 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
Crush and stir evenly, draw 5mL samples respectively in centrifuge tube, and the three of 5% isometric (m/v) is added into zymotic fluid
Monoxone (TCA) solution, stands 30min protein precipitations at room temperature, in 4 DEG C, 10000r/min centrifugation 30min, 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 acids, are uniformly mixed, and return to zero by the use of distilled water as blank reagent, 15min are kept in boiling water bath, so
Rapid ice-water bath cooling terminates reaction afterwards.Absorbance is measured at wavelength 490nm, calculates exocellular polysaccharide content.
2nd, the measure of EPS
EPS contents are 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, to constant weight, 100mg glucose is accurately weighed after cooling in 500mL volumetric flasks
In, add distilled water to scale.Each liquor capacity of reaction system is added by table 2, and Standard glucose solution first is added to tool plug carves
Spend in test tube, add the phenol solution of 5% (v/v), be eventually adding the 10mL concentrated sulfuric acids and mix and stand, treat its cooling after
At 490nm measure absorbance, every group do 3 it is parallel.With glucose content (mg/L) for abscissa, absorbance (A490) sat to be vertical
Plotting standard curve.Obtaining equation of linear regression is：Y=1.405x-0.426, R2=0.987.With method measure EPS aqueous solutions in
Absorbance at wavelength 490nm, passes through the EPS yield of regression equation calculation bacterial strain.
In terms of mass fraction ω, unit is represented polyoses content with gram every hectogram (g/100g), is counted as follows in sample
Calculate：
In formula：
m1--- sugar content in sample measure liquid is checked in from standard curve, unit is microgram (μ g)；
V1--- sample constant volume, unit are milliliter (mL)
V2--- the volume of taking sample determination liquid is moved during colorimetric estimation, unit is milliliter (mL)；
m2--- sample quality, unit are gram (g)；
0.9 --- glucose is converted into the correction coefficient of glucose.
Result of calculation retains to 2 significant digits.
3rd, result
As a result such as table 3.Wherein numbering 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
6th, bacteriostatic test
1st, the preparation of cell-free supernatants
Lactobacillus reuteri is 37 DEG C in MRS or MRS-glycerol (MRS-g, glycerol concentration 400mM) culture medium
24h is cultivated, centrifugation, obtains supernatant.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 save backup.
2nd, 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 mediums.
96 orifice plates co-culture method：Indicator bacteria is incubated overnight, and it is 10 to take 0.1ml concentration5The instruction bacteria culture fluid of cfu/ml adds
Enter in 96 orifice plates, add 0.1ml cell free broths, 37 DEG C of culture 24h, microplate reader detection OD600.To add TSB culture mediums
Hole for control, utilize equation below carry out bacteriostasis rate calculating：
The results are shown in Table 4：The MRS supernatants of all test strains all have bacteriostasis, but during adjusting pH to 6.5
All do not have fungistatic effect, this explanation antipathogenic composition is mainly organic acid.However, supernatant (the pH=by strain fermentation MRS-g
6.5) bacteriostatic experiment discovery is carried out, many bacterial strains still have bacteriostatic activity, this bacterial strain of explanation with bacteriostasis can generation
Thank to glycerine, generate some antipathogenic compositions.First and last, suppressions of numbering 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 inhibiting rates (%) of the lactobacillus reuteri MRS-g to 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 |
3rd, reuterin produces the detection of ability
(1) preparation of supernatant to be measured
Lactobacillus reuteri cultivates 24h in MRS culture mediums, and centrifugation, PBS washes once, is resuspended in glycerine-water-soluble
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) making of standard curve
The making of methacrylaldehyde standard curve：Storing solution is diluted with 95% ethanol, adds 0- into 10ml volumetric flasks respectively
2ml storing solutions, the use 95% less than 2ml are supplied, and add 1.2ml water.Then add 0.5ml 0.01M tryptophan solution and
The dense HCl of 6.3ml, are placed in 60 DEG C of water-bath 5min, reach most dark colour.After water-bath, OD is measured560, blank is done with reagent controls.Most
The corresponding absorbance 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.
The results are shown in Figure 4, and F-9-79 bacterial strains do not detect reuterin.
7th, safety evaluatio (hemolytic test)
Hemolytic test is that the lactobacillus reuteri nutrient solution that will be incubated overnight is rule 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 hemolytic experiment is carried out to 11 plants of mutagenic strains and 1 plant of ground control strain.Research finds, all surveys
Examination bacterial strain does not occur haemolysis, i.e. γ-haemolysis.Tentatively show, candidate's probiotic strain by space flight is safety
's.
8th, the identification of strain
Face to carry out DNA extractions respectively according to bacterium and each space carrying mutagenic strain over the ground by DNA kits, extraction is completed
Afterwards, then PCR amplification is carried out to above-mentioned DNA extracts respectively, amplification system cumulative volume is 20 μ L, includes the Taq of 2 units
Archaeal dna polymerase, PCR buffer, 2.5mM MgCl2, 500 μM of dNTP, the universal primer 1 of 100ng DNA profilings and 10pmol,
2, P1：5’-AGTTTGATCMTGGCTCAG-3’；And P2：5’-GGTTACCTTGTTACGACTT-3’.Amplification condition is：95℃
Pre-degeneration 2min, 95 DEG C of denaturation 30s, 51 DEG C of annealing (renaturation) 30s, 72 DEG C of extension 1min, are circulated 30 times, last 72 DEG C of extensions
2min, obtains final product, and product then is delivered Shanghai life work sequencing.
By 16S rDNA be sequenced as a result, and combine the morphological feature identified above, it is known that ground control bacterium respectively lures
It is lactobacillus reuteri (Lactobacillus reuteri) to become bacterial strain, and numbers the bacterial strain for being F-9-79 in 16S
Occurs variation in rDNA sequences, its 16S rDNA sequence is as shown in SEQ ID No.1.
The bacterial strain that numbering is F-9-79 was preserved in Chinese microorganism strain preservation management committee on November 20th, 2017
Member's meeting common micro-organisms center, deposit number is CGMCC No.14943, and the biomaterial (strain) for joining Ju is Fullarton-9-
79。
The cultivation temperature of lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-79 is 37 DEG C；From
Right pH；Medium component：Casein peptone 10.0g, beef extract 10.0g, dusty yeast 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 thing engineering science and technology（Beijing）Co., Ltd
<120>Space lactobacillus reuteri Fullarton-9-79 and application
<130> GNCLN172070
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1376
<212> DNA
<213>Lactobacillus reuteri（Lactobacillus reuteri）
<400> 1
cttgcacctg attgacgatg gatcaccagt gagtggcgga cgggtgagta acacgtaggt 60
aacctgcccc ggagcggggg ataacatttg gaaacagatg ctaataccgc ataacaacaa 120
aagccacatg gcttttgttt gaaagatggc tttggctatc actctgggat ggacctgcgg 180
tgcattagct agttggtaag gtaacggctt accaaggcga tgatgcatag ccgagttgag 240
agactgatcg gccacaatgg aactgagaca cggtccatac tcctacggga ggcagcagta 300
gggaatcttc cacaatgggc gcaagcctga tggagcaaca ccgcgtgagt gaagaagggt 360
ttcggctcgt aaagctctgt tgttggagaa gaacgtgcgt gagagtaact gttcacgcag 420
tgacggtatc caaccagaaa gtcacggcta actacgtgcc agcagccgcg gtaatacgta 480
ggtggcaagc gttatccgga tttattgggc gtaaagcgag cgcaggcggt tgcttaggtc 540
tgatgtgaaa gccttcggct taaccgaaga agtgcatcgg aaaccgggcg acttgagtgc 600
agaagaggac agtggaactc catgtgtagc ggtggaatgc gtagatatat ggaagacacc 660
agtgcgaagg cggctgtctg gtctgcaact gacgctgagg ctcgaaagca tgggtagcga 720
acaggattag ataccctggt agtccatgcc gtaaacgatg agtgctaggt gttggagggt 780
ttccgccctt cagtgccgga gctaacgcat taagcactcc gcctggggag tacgaccgca 840
aggttgaaac tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat 900
tcgaagctac gcgaagaacc ttaccaggtc ttgacatctt gcgctaacct tagagataag 960
gcgttccctt cggggacgca atgacaggtg gtgcatggtc gtcgtcagct cgtgtcgtga 1020
gatgttgggt taagtcccgc aacgagcgca acccttgtta ctagttgcca gcattaagtt 1080
gggcactcta gtgagactgc cggtgacaaa ccggaggaag gtggggacga cgtcagatca 1140
tcatgcccct tatgacctgg gctacacacg tgctacaatg gacggtacaa cgagtcgcaa 1200
gctcgcgaga gtaagctaat ctcttaaagc cgttctcagt tcggactgta ggctgcaact 1260
cgcctacacg aagtcggaat cgctagtaat cgcggatcag catgccgcgg tgaatacgtt 1320
cccgggcctt gtacacaccg cccgtcacac catgggagtt tgtaacgctc acaaag 1376
Claims (9)
- Lactobacillus reuteri 1. (Lactobacillus reuteri) Fullarton-9-79, it is protected in Chinese microorganism strain The deposit number for hiding administration committee's common micro-organisms center is CGMCC No.14943.
- 2. a kind of microbial inoculum, its active ingredient is the lactobacillus reuteri (Lactobacillus described in claim 1 reuteri)Fullarton-9-79。
- 3. lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-79 or right described in claim 1 It is required that application of the microbial inoculum described in 2 in fermented dairy product is prepared.
- 4. lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-79 or right described in claim 1 It is required that the microbial inoculum described in 2 is preparing the application in being used to produce leavening used in acidified milk preparation.
- 5. lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-79 or right described in claim 1 It is required that application of the microbial inoculum in antibacterials are prepared described in 2.
- 6. application according to claim 5, it is characterised in that：The bacterium is bacterium or fungi；Specifically, the bacterium is gram-positive bacterium or gramnegative bacterium.
- 7. application according to claim 6, it is characterised in that：The bacterium is Escherichia coli, staphylococcus aureus, sand Door Salmonella and/or Listeria monocytogenes.
- 8. lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-79 or right described in claim 1 It is required that microbial inoculum described in 2 it is following it is any in application：(a1) human or animal's the gastrointestinal tract micro ecological balance is adjusted, or prepares and is used to adjust human or animal's the gastrointestinal tract micro ecological balance Product；(a2) alleviate enteritis, or prepare the product for being used for alleviating enteritis；(a3) auxiliary protection gastric mucosa, or prepare the product for auxiliary protection gastric mucosa；(a4) defaecation, or prepare the product for defaecation；(a5) strengthen human or animal's immunity, or prepare the product for being used for strengthening human or animal's immunity.
- 9. lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-79 or right described in claim 1 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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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110447763A (en) * | 2019-07-05 | 2019-11-15 | 江西农业大学 | Bacterium is influencing the application in animal meat rate and/or fat content |
| CN111919888A (en) * | 2020-08-18 | 2020-11-13 | 富乐顿生物工程科技（北京）有限公司 | Space-induced lactobacillus reuteri and lactobacillus plantarum compound starter and application thereof in preparation of probiotic yogurt |
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| CN110447763A (en) * | 2019-07-05 | 2019-11-15 | 江西农业大学 | Bacterium is influencing the application in animal meat rate and/or fat content |
| CN111919888A (en) * | 2020-08-18 | 2020-11-13 | 富乐顿生物工程科技（北京）有限公司 | Space-induced lactobacillus reuteri and lactobacillus plantarum compound starter and application thereof in preparation of probiotic yogurt |
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| CN107974425B (en) | 2019-08-30 |
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