CN108094527A - Space lactobacillus reuteri Fullarton-9-87 and application - Google Patents
Space lactobacillus reuteri Fullarton-9-87 and application Download PDFInfo
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- CN108094527A CN108094527A CN201711347365.1A CN201711347365A CN108094527A CN 108094527 A CN108094527 A CN 108094527A CN 201711347365 A CN201711347365 A CN 201711347365A CN 108094527 A CN108094527 A CN 108094527A
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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
- 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
-
- 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
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- 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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/173—Reuteri
-
- 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
-
- 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
Abstract
The invention discloses one plant of space lactobacillus reuteri Fullarton 9 87 and applications.Lactobacillus reuteri (Lactobacillus reuteri) Fullarton 9 87 provided by the present invention, it is CGMCC No.14940 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.Lactobacillus reuteri Fullarton 9 87 provided by the present invention, compared with the control strain of ground, the curdled milk time is shorter, and low pH tolerances higher, hydrophobicity higher, exocellular polysaccharide is more, and safety evaluatio experiment the result shows that being safe.Therefore, lactobacillus reuteri Fullarton 9 87 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-9-87 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 with the prebiotic of improvement allergic constitution, pre- hypo-allergenic recurrent exerbation and adjusting 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 generating broad-spectrum antiseptic substance
Special efficacy.It is metabolized glycerine and generates 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 substance 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 with 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；It 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 lactobacillus reuteri (Lactobacillus reuteri) and applications.
Lactobacillus reuteri (Lactobacillus reuteri) provided by the present invention is specially lactobacillus reuteri
(Lactobacillus reuteri) Fullarton-9-87, it is commonly micro- in China Committee for Culture Collection of Microorganisms
The deposit number of Bio-Centers is CGMCC No.14940.
Lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-87 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 number 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
Number is CGMCC No.14940.
Correspondingly, it is the lactobacillus reuteri (Lactobacillus the present invention also provides a kind of active ingredient
Reuteri) the microbial inoculum of Fullarton-9-87.
Except containing the lactobacillus reuteri (Lactobacillus as active component in the microbial inoculum
Reuteri) outside Fullarton-9-87, 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-9-87 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-9-87 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-9-87 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-9-87 or described microbial inoculums are as follows
Application in any falls within protection scope of the present invention：
(a1) adjust human or animal's the gastrointestinal tract micro ecological balance or prepare to adjust human or animal's micro ecology of gastrointestinal tract
The product of balance；
(a2) alleviate enteritis or prepare to alleviate the product of 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 to enhance the product of human or animal's immunity.
Wherein, the product can be drug, nutriment or health products etc..
Lactobacillus reuteri (Lactobacillus reuteri) the Fullarton-9-87 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-9-87 provided by the present invention, with ground
Face control strain is compared, and the curdled milk time shortens 12h, and the viscosity for the skimmed milk that ferments adds 0.26 times, to the tolerance of low pH
Improve 0.43 times；Hydrophobicity improves 1.31 times；Exocellular polysaccharide adds 4 times.Hemolytic experiment is the result is that γ-haemolysis, i.e., not
Haemolysis tentatively shows that lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-87 is safe.To sum up institute
It states, lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-87 has the potentiality and value of continual exploitation.
The property of table 1Fullarton-9-87 and ground bacterial strain
Note："-" does not detect
Preservation explanation
Strain name：Lactobacillus reuteri
Latin name：Lactobacillus reuteri
Join the biomaterial (strain) of Ju：Fullarton-9-87
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 number：CGMCC No.14940
Description of the drawings
Fig. 1 is the bacterial strain that number is F-9-87 and the microscopy photo of ground control bacterium.A：The bacterial strain that number is F-9-87；B：
Ground compares bacterium.
Fig. 2 compares bacterium and its through each mutagenic strain obtained by space carrying to the tolerance of 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.
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) 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), the two is
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 number 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-9-87
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 for 24 hours, 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 number be F-9-87 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 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.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
It tests.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 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 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, 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 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
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
Strain is to the tolerance of 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.It adopts
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 variation 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.The study found that lactobacillus reuteri has low pH good tolerance, number F-9-
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.
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%
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.
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, thalline is promoted to stick, blood pressure lowering, norcholesterol, resisted
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 has chosen front research and draws 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
It crushes and stirs evenly, draw 5mL samples respectively in centrifuge tube, and the three of 5% isometric (m/v) is added in 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 are returned 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 10mL concentrated sulfuric acids mixing standing, 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
It calculates：
In formula：
m1--- sample is checked in from standard curve and measures sugar content in liquid, 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 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
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
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 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, for 24 hours, microplate reader detects OD for 37 DEG C of cultures600.To add in 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 illustrates that 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
It thanks to glycerine, generates some antipathogenic compositions.First and last, suppressions 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 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 generates the detection of ability
(1) preparation of supernatant to be measured
Lactobacillus reuteri is cultivated for 24 hours 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% ethyl alcohol, adds in 0- into 10ml volumetric flasks respectively
2ml storing solutions, the use 95% less than 2ml are supplied, and add in 1.2ml water.Then add in 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 number is that the Luo Yishi elements of F-9-87 bacterial strains are not detect.
7th, safety evaluatio (hemolytic test)
Hemolytic test is that the lactobacillus reuteri culture 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.The study found that all surveys
Examination bacterial strain does not occur haemolysis, i.e. γ-haemolysis.It is preliminary to show that by candidate's probiotic strain of space flight be safety
's.
8th, the identification of strain
It faces 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 total 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, cycle 30 times, last 72 DEG C extend
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-9-87 in 16S
Occurs variation in rDNA sequences, 16S rDNA sequences are as shown in SEQ ID No.1.
The bacterial strain that number is F-9-87 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.14940, and the biomaterial (strain) for joining Ju is Fullarton-9-
87。
The cultivation temperature of lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-87 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 object engineering science and technology（Beijing）Co., Ltd
<120>Space lactobacillus reuteri Fullarton-9-87 and application
<130> GNCLN172071
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1390
<212> DNA
<213>Lactobacillus reuteri（Lactobacillus reuteri）
<400> 1
tgattagatg gtgcttgcac ctgattgacg atggatcacc agtgagtggc ggacgggtga 60
gtaacacgta ggtaacctgc cccggagcgg gggataacat ttggaaacag atgctaatac 120
cgcataacaa caaaagccac atggcttttg tttgaaagat ggctttggct atcactctgg 180
gatggacctg cggtgcatta gctagttggt aaggtaacgg cttaccaagg cgatgatgca 240
tagccgagtt gagagactga tcggccacaa tggaactgag acacggtcca tactcctacg 300
ggaggcagca gtagggaatc ttccacaatg ggcgcaagcc tgatggagca acaccgcgtg 360
agtgaagaag ggtttcggct cgtaaagctc tgttgttgga gaagaacgtg cgtgagagta 420
actgttcacg cagtgacggt atccaaccag aaagtcacgg ctaactacgt gccagcagcc 480
gcggtaatac gtaggtggca agcgttatcc ggatttattg ggcgtaaagc gagcgcaggc 540
ggttgcttag gtctgatgtg aaagccttcg gcttaaccga agaagtgcat cggaaaccgg 600
gcgacttgag tgcagaagag gacagtggaa ctccatgtgt agcggtggaa tgcgtagata 660
tatggaagac accagtggcg aaggcggctg tctggtctgc aactgacgct gaggctcgaa 720
agcatgggta gcgaacagga ttagataccc tggtagtcca tgccgtaaac gatgagtgct 780
aggtgttgga gggtttccgc ccttcagtgc cggagctaac gcattaagca ctccgcctgg 840
ggagtacgac cgcaaggttg aaactcaaag gaattgacgg gggcccgcac aagcggtgga 900
gcatgtggtt taattcgaag ctacgcgaag aaccttacca ggtcttgaca tcttgcgcta 960
accttagaga taaggcgttc ccttcgggga cgcaatgaca ggtggtgcat ggtcgtcgtc 1020
agctcgtgtc gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gttactagtt 1080
gccagcatta agttgggcac tctagtgaga ctgccggtga caaaccggag gaaggtgggg 1140
acgacgtcag atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggacggt 1200
acaacgagtc gcaagctcgc gagagtaagc taatctctta aagccgttct cagttcggac 1260
tgtaggctgc aactcgccta cacgaagtcg gaatcgctag taatcgcgga tcagcatgcc 1320
gcggtgaata cgttcccggg ccttgtacac accgcccgtc acaccatggg agtttgtaac 1380
gctccaaagt 1390
Claims (9)
- Lactobacillus reuteri 1. (Lactobacillus reuteri) Fullarton-9-87, it is protected in Chinese microorganism strain The deposit number for hiding administration committee's common micro-organisms center is CGMCC No.14940.
- 2. a kind of microbial inoculum, its active ingredient is lactobacillus reuteri (Lactobacillus described in claim 1 reuteri)Fullarton-9-87。
- 3. lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-87 described in claim 1 or right It is required that application of the microbial inoculum described in 2 in fermented dairy product is prepared.
- 4. lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-87 described in claim 1 or right It is required that application of the microbial inoculum in preparing to produce leavening used in acidified milk preparation described in 2.
- 5. lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-87 described in claim 1 or right 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-9-87 described in claim 1 or right It is required that microbial inoculum described in 2 it is following it is any in application：(a1) adjust human or animal's the gastrointestinal tract micro ecological balance or prepare to adjust human or animal's the gastrointestinal tract micro ecological balance Product；(a2) alleviate enteritis or prepare to alleviate the product of 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 to enhance the product of human or animal's immunity.
- 9. lactobacillus reuteri (Lactobacillus reuteri) Fullarton-9-87 described in claim 1 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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| CN109385387A (en) * | 2018-12-28 | 2019-02-26 | 上海源耀生物股份有限公司 | The lactobacillus reuteri of anti-TGEV a kind of and its application |
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