CN104830734B - The method of one plant of propionibacterium freudenreichii bacterial strain and its fermented-producing bacteria element - Google Patents
The method of one plant of propionibacterium freudenreichii bacterial strain and its fermented-producing bacteria element Download PDFInfo
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Abstract
The present invention relates to the methods of one plant of propionibacterium freudenreichii bacterial strain and its fermented-producing bacteria element, belong to industrial microbial technology field.Including：（1）WithPropionibacterium freudenreichii subsp.shermaniiAs starting strain, carried out Protoplast Mutation and screen to obtain a plant mutant bacterial strain, Classification And Nomenclature isPropionibacterium freudenreichii CS1420, deposit number are CCTCC NO:M 2015050.（2）One kind is provided and improves new strains by adjusting pHPropionibacterium freudenreichii The new method of CS1420 bacteriocinogeny yield and bacteriostatic activity.The new strains of the present inventionPropionibacterium freudenreichii CS1420 is safe bacterial strain, and after which ferments under certain condition, zymotic fluid bacterium cellulose content is high, natural antiseptic agent can be used as to be applied in food and feed industry.
Description
Technical field
The present invention relates to the methods of one plant of propionibacterium freudenreichii bacterial strain and its fermented-producing bacteria element, belong to industrial microorganism
Technical field.
Background technology
Propionibacterium is widely present in nature.Early in 1909, Propionibacterium was described in detail in Orla-Jensen
Belong to, and it is divided into two major classes according to habitat, from the acne type Propionibacterium of human skin and from dairy produce
Dairy propionibacteria.
Dairy propionibacteria mainly includes propionibacterium acide-propionici, propionibacterium freudenreichii, propionibacterium jensenii, Te Shi propionic acid bars
Bacterium is a kind of Propionibacterium for the no pathogenicity separated from dairy products, ensilage, now main in the food industry to use
In production propionic acid, vitamin B12, folic acid, dairy starter etc..In addition, dairy propionibacteria can be synthesized in metabolic process it is special
Peptide or protein matter, this kind of substance（Bacteriocin）It is the special-shaped antibacterial substance that Propionibacterium generates, there is excellent characteristics, such as：
It is with extensive bacteriostasis, and fungistatic effect is better than the common chemical preservatives such as sodium benzoate, potassium sorbate, so,
Propionibacterium bacteriocin has good application prospect in food antiseptic is fresh-keeping.At present, French Rhodia has developed
A kind of New Biological Preservatives containing Propionibacterium bacteriocin are obtained, the approval of U.S. FDA have been obtained, in US and European
It sells in the market.The country produces preservative still in the laboratory research stage on Propionibacterium bacteriocin.
Propionicin PLG-1、GBZ-1、Propionicin T1、Propionicin SM1、Jenseniin G、
PAMP, Thoeniicin 447 etc. is the dairy propionibacteria bacteriocin being successively found.Wherein Propionicin PLG-1
Be first be found by dairy propionibacteria generate bacteriocin, it be byP.thoeniiWhat P127 was generated, molecule
It measures as 9328u, it is active between pH3-9, it can effectively inhibit Te Shi Propionibacteriums, propionibacterium acide-propionici and propionibacterium jensenii
And some lactic acid bacterias, yeast and mould；Propionicin T1 be byP.thoeniiThe bacteriocin that 419 bacterial strains generate, has
Preferable acid resistance can inhibit propionibacterium jensenii similar in affiliation, propionibacterium acide-propionici and some lactobacillus；
Propionicin SM1 be byP.jenseniiThe bacteriocin that DF1 is generated, pH value is respectively provided with activity when being 3-9, to fungi
With very strong inhibitory action；Jenseniin G be byP.thoenii(jensenii)What P126 was generated, pH value is in 3-12
Active, it can inhibit some other Propionibacterium and lactobacillus.As it can be seen that Propionibacterium bacteriocin is all in its optimal pH model
It encloses interior active.The research of Coventry et al., Liu W et al. show that Nisin is also active in the range of optimal pH.Have
Research also indicates that the yield and potency and medium pH of bacteriocin fermentation are closely related.
The content of the invention
It is an object of the present invention to provide one plant of propionibacterium freudenreichii CS1420（Propionibacterium freudenreichii CS1420）, in order to realize foregoing invention purpose, the technical scheme is that：
The present invention withPropionibacterium freudenreichii subsp.shermanii（It is purchased from the micro- life of China
Object Culture Collection Center）As starting strain, carried out Protoplast Mutation and screen to obtain a plant mutant bacterial strain, classification life
It is entitledPropionibacterium freudenreichii CS1420, deposit number are CCTCC NO:M 2015050.
The specific method that Protoplast Mutation of the present invention screens to obtain Propionibacterium is：
1st, the preparation of protoplast：
The Propionibacterium that will have been activatedPropionibacterium freudenreichii subsp.shermanii
One ring of picking is inoculated into fluid nutrient medium, 30 DEG C of Anaerobic culturel 2d.Zymotic fluid is centrifuged under the conditions of 6000r/min, 4 DEG C
Abandoning supernatant after 15min washs bacterium mud with PFB buffer solutions and bacterium mud is resuspended in PFB buffer solutions afterwards twice, and adjusts thalline
Concentration is 108cfu/mL.Lysozyme and mutanolysin are added in into bacteria suspension, it is respectively 20mg/mL and 15ug/ to make its concentration
ML, under the conditions of 37 DEG C digest 2h removal cell membrane, centrifuge afterwards by precipitation PFB wash buffers termination reaction, again from
Protoplast is resuspended in spare in PFB buffer solutions by the heart.
2nd, Protoplast Mutation breeding：
UV mutagenesis：
The protoplast that 10mL has been prepared is taken to be stirred, put on magnetic stirring apparatus in the plate of a diameter of 9cm
Under 15W ultraviolet lamps, distance 25cm irradiations 120s.Period takes mutagenesis protoplast suspension 1mL every 20s, after, in red light
Lower 10 times of progress takes 0.1mL to be applied on regenerated plate respectively after being serially diluted, and is wrapped up with black cloth, is placed in 30 DEG C of anaerobic jars and detests
Oxygen culture 4 days.
Nitrosoguanidine mutagenesis：
5 equal portions protoplast suspensions are taken, add in a certain amount of nitroso guanidine solution, make mixed liquor nitrosoguanidine final concentration point
Not Wei 0.8 mg/mL, 1.0 mg/mL, 1.2 mg/mL, 1.4 mg/mL, 1.6mg/mL, shake 50min under the conditions of 30 DEG C.Phase
Between, mutagenesis protoplast 0.1mL is taken every 10min, the 4 DEG C of sterile salines termination mutagenesis for adding in 1000 times of reaction solution is anti-
Should, after, 10 times of progress takes 0.1mL to be applied on regenerated plate respectively after being serially diluted, and is placed in anaerobism in 30 DEG C of anaerobic jars
Culture 4 days.
3rd, Screening of strain with high productivity：
Single bacterium on picking mutagenesis rear plate, which is fallen within, carries out fermented and cultured in fermentation medium, by zymotic fluid 4 DEG C,
15min is centrifuged under conditions of 6000r/min, takes supernatant, pH7.0 is adjusted to 10% NaOH, then is adjusted to 10% tartaric acid
It is spare to be placed in 4 DEG C of preservations by pH5.3.
One layer of culture medium will be first poured into sterilizing plates（Element agar culture medium）, after to be solidified, Oxford cup is placed on flat
On the most central culture medium of ware, dissolved in the culture medium of upper strata and add in a certain amount of Escherichia coli bacteria liquid system when being cooled to 50 DEG C or so
It into band bacterio-agar, is then poured on rapidly on the bottom culture medium cooled down, gently rotating culture dish makes its tiling, after cooling, inhales
The fermented supernatant fluid after pH is adjusted described in 200uL epimeres is taken in Oxford cup, plate is covered and is placed on 4 DEG C of standing 3h, then move into
20h is cultivated in 37 DEG C of incubators, then moves into 4 DEG C of standing 3h, antibacterial circle diameter, the bacterium of picking inhibition zone maximum are measured with ruler
Strain, and it is named as propionibacterium freudenreichii CS1420（Propionibacterium freudenreichii CS1420）.
Another object of the present invention is to provide the new method of the bacterial strain bacteriocinogeny.
2nd, it is a kind of of the present inventionPropionibacterium freudenreichii CS1420 is by adjusting pH
To improve the new method of the bacterial strain bacteriocinogeny yield and bacteriostatic activity.
Specifically include following steps：
1) seed culture：It willPropionibacterium freudenreichii CS1420 single bacterium colonies are seeded to improvement
It is activated in PYG culture mediums, 30 DEG C of Anaerobic culturels 2.5 days.
2）Fermented and cultured：By above-mentioned steps 1）Obtained seed liquor is accessed according to 10% inoculum concentration in improvement PYG cultures, and 30
DEG C, Anaerobic culturel 8 days.
3）The measure of fungistatic effect：
Zymotic fluid is centrifuged into 15min under conditions of 4 DEG C, 6000r/min, supernatant is taken, is adjusted to 10% NaOH
PH7.0, then be adjusted to pH5.3 with 10% tartaric acid takes 200uL to add in described in 3, Screening of strain with high productivity in Oxford cup, covers flat
Ware is placed on 4 DEG C of standing 3h, then moves into 37 DEG C of incubators and cultivates 20h, then moves into 4 DEG C of standing 3h, and inhibition zone is measured with ruler
Diameter, antibacterial circle diameter is bigger, and fungistatic effect is better.
The step 2）In by adjusting pH be to improve the preferred embodiment of the bacterial strain bacteriocinogeny yield：The present invention will hair
Ferment culture is adjusted to 5.5,6.0,6.5,7.0,7.5 respectively with the initial pH of culture medium, in further preferred embodiment, this hair
It is bright that pH6.0 is used to be realized with this condition as the initial pH of fermentation mediumPropionibacterium freudenreichii The raising of CS1420 fermented-producing bacteria element yield.
The step 3）In by adjusting pH be to improve the scheme of bacteriocin activity：The present invention takes supernatant after fermented and cultured
Liquid is adjusted to pH7.0 with 10% NaOH, then it is respectively 5.1,5.3,5.5,5.7,5.9 to be adjusted to pH with 10% tartaric acid, further excellent
It selects in embodiment, the present invention is adjusted to pH5.5 with 10% tartaric acid, realizes with this conditionPropionibacterium freudenreichii The raising of CS1420 fermented-producing bacterias element activity.
The improvement PYG improved culture mediums are：Casein peptone 5.0g, 5.0 g of peptone, yeast extract 10.0
G, 5.0 g of beef extract, glucose 5.0 g, K2HPO42.0 g, 1.0 ml of Tween 80,1.0 ml of resazurin, salting liquid
40.0 ml, 950.0 ml of distilled water, 10.0 ml of hemin solution, vitamin K10.2 ml of solution, L-cysteine
0.5 g, pH 7.2.Salting liquid：CaCl2·2H2O 0.25g, MgSO47H2O 0.5 g, K2HPO4 1.0 g, KH2PO4 1.0
G, NaHCO3 10.0g, NaCl2.0g, distilled water 1.0L；Hemin solution：Hemin 50.0 mg, 1N
1.0 ml of NaOH, 99.0 ml of distilled water；Vitamin K1Solution：Vitamin K1 0.1 ml, 95% ethyl alcohol, 20.0 ml.
The beneficial effects of the present invention are：
The present invention'sPropionibacterium freudenreichii CS1420 bacterial strain securities are higher；The bacterial strain
In pH6.0 improves PYG culture mediums after fermented and cultured, zymotic fluid antibacterial circle diameter reaches 30mm, by this zymotic fluid with 10% NaOH
PH7.0 is adjusted to, then pH5.5 is adjusted to 10% tartaric acid, zymotic fluid antibacterial circle diameter reaches 35mm.And the bacteriocin of present invention gained
It is a kind of safe natural antiseptic agent, can be directly used in food and feed industry.
The propionibacterium freudenreichii bacterial strain of the present invention, Classification And Nomenclature arePropionibacterium freudenreichii
CS1420 submitted Chinese Typical Representative Organism Depositary on January 20th, 2015（CCTCC）, address：Chinese Wuhan, Wuhan
University, deposit number are CCTCC NO:M 2015050.
Specific embodiment
1 bacterial strain of the present invention of embodimentPropionibacterium freudenreichii The screening technique of CS1420
1st, the preparation of protoplast：
By what is activatedPropionibacterium freudenreichii subsp.shermaniiOne ring of picking
It is inoculated into fluid nutrient medium, 30 DEG C of Anaerobic culturel 2d.It is abandoned after zymotic fluid is centrifuged 15min under the conditions of 6000r/min, 4 DEG C
Supernatant is removed, washing bacterium mud with PFB buffer solutions is twice afterwards resuspended in bacterium mud in PFB buffer solutions, and adjusts cell concentration and be
108cfu/mL.Lysozyme and mutanolysin are added in into bacteria suspension, it is respectively 20mg/mL and 15ug/mL to make its concentration, in 37
2h removal cell membranes are digested under the conditions of DEG C, centrifuges to terminate precipitation PFB wash buffers afterwards and react, centrifugation will be primary again
Plastid is resuspended in spare in PFB buffer solutions.
2nd, Protoplast Mutation breeding：
UV mutagenesis：
The protoplast that 10mL has been prepared is taken to be stirred, put on magnetic stirring apparatus in the plate of a diameter of 9cm
Under 15W ultraviolet lamps, distance 25cm irradiations 120s.Period takes mutagenesis protoplast suspension 1mL every 20s, after, in red light
Lower 10 times of progress takes 0.1mL to be applied on regenerated plate respectively after being serially diluted, and is wrapped up with black cloth, is placed in 30 DEG C of anaerobic jars and detests
Oxygen culture 4d.
Nitrosoguanidine mutagenesis：
5 equal portions protoplast suspensions are taken, add in a certain amount of nitroso guanidine solution, make mixed liquor nitrosoguanidine final concentration point
Not Wei 0.8 mg/mL, 1.0 mg/mL, 1.2 mg/mL, 1.4 mg/mL, 1.6mg/mL, shake 50min under the conditions of 30 DEG C.Phase
Between, mutagenesis protoplast 0.1mL is taken every 10min, the 4 DEG C of sterile salines termination mutagenesis for adding in 1000 times of reaction solution is anti-
Should, after, 10 times of progress takes 0.1mL to be applied on regenerated plate respectively after being serially diluted, and is placed in anaerobism in 30 DEG C of anaerobic jars
Cultivate 4d.
3rd, Screening of strain with high productivity：
Single bacterium on picking mutagenesis rear plate, which is fallen within, carries out fermented and cultured in fermentation medium, by zymotic fluid 4 DEG C,
15min is centrifuged under conditions of 6000r/min, takes supernatant, pH7.0 is adjusted to 10% NaOH, then is adjusted to 10% tartaric acid
It is spare to be placed in 4 DEG C of preservations by pH5.3.
One layer of culture medium will be first poured into sterilizing plates（Element agar culture medium）, after to be solidified, Oxford cup is placed on flat
On the most central culture medium of ware, dissolved in the culture medium of upper strata and add in a certain amount of Escherichia coli bacteria liquid system when being cooled to 50 DEG C or so
It into band bacterio-agar, is then poured on rapidly on the bottom culture medium cooled down, gently rotating culture dish makes its tiling, after cooling, inhales
The fermented supernatant fluid after pH is adjusted described in 200uL epimeres is taken in Oxford cup, plate is covered and is placed on 4 DEG C of standing 3h, then move into
20h is cultivated in 37 DEG C of incubators, then moves into 4 DEG C of standing 3h, antibacterial circle diameter, the bacterium of picking inhibition zone maximum are measured with ruler
Strain, and be named asPropionibacterium freudenreichii CS1420。
The method that 2 bacterial strain of the present invention of embodiment produces high yield bacteriocin.
1) seed culture：It willPropionibacterium freudenreichii CS1420 single bacterium colonies are seeded to improvement
It is activated in PYG culture mediums, 30 DEG C of Anaerobic culturel 2.5d.
2）Fermented and cultured：By above-mentioned steps 1）Obtained seed liquor is accessed according to 10% inoculum concentration in improvement PYG cultures, and 30
DEG C, Anaerobic culturel 8d.
3）The measure of fungistatic effect：Zymotic fluid is centrifuged into 15min under conditions of 4 DEG C, 6000r/min, takes supernatant, is used
10% NaOH is adjusted to pH7.0, then is adjusted to pH5.3 with 10% tartaric acid, and 200uL is taken to add in embodiment 1 in Screening of strain with high productivity
In the Oxford cup, cover plate be placed on 4 DEG C standing 3h, then move into 37 DEG C of incubators and cultivate 20h, then move into 4 DEG C it is quiet
3h is put, measures antibacterial circle diameter with ruler, antibacterial circle diameter is bigger, and fungistatic effect is better.
In order to further improve mutant strainPropionibacterium freudenreichii CS1420 bacteriocinogeny
Yield, by the step 2）Middle improvement PYG medium pHs are adjusted to 5.5,6.0,6.5,7.0,7.5 progress fermented and cultureds respectively,
Zymotic fluid fungistatic effect measures（Inhibition zone）As a result it is respectively 27mm, 30mm, 28mm, 27mm, 26mm, this shows medium pH pair
Propionibacterium bacteriocinogeny has a significant impact, and most suitable pH is 6.0.
3 bacterial strain of the present invention of embodiment produces high-activity fine rhzomorph method.
1) seed culture：It willPropionibacterium freudenreichii CS1420 single bacterium colonies are seeded to improvement
It is activated in PYG culture mediums, 30 DEG C of Anaerobic culturel 2.5d.
2）Fermented and cultured：By above-mentioned steps 1）Obtained seed liquor is accessed according to 10% inoculum concentration in improvement PYG culture mediums,
PH6.0,30 DEG C, Anaerobic culturel 8d.
3）The measure of fungistatic effect：Zymotic fluid is centrifuged into 15min under conditions of 4 DEG C, 6000r/min, takes supernatant, is used
10% NaOH is adjusted to pH7.0, then is adjusted to pH5.3 with 10% tartaric acid, and 200uL is taken to add in institute in 1 Screening of strain with high productivity of embodiment
It states in Oxford cup, covers plate and be placed on 4 DEG C of standing 3h, then move into 37 DEG C of incubators and cultivate 20h, then move into 4 DEG C of standing 3h,
Antibacterial circle diameter is measured with ruler, antibacterial circle diameter is bigger, and fungistatic effect is better.
In order to further improve the activity of bacteriocin, the step 3）It is middle to be first adjusted to pH7.0 with 10% NaOH, then with 10%
It is respectively 5.1,5.3,5.5,5.7,5.9 that tartaric acid, which is adjusted to pH, and fungistatic effect measures（Inhibition zone）As a result be respectively 30mm,
31mm、35mm、32mm、30mm.This shows when pH is adjusted to 5.5 with 10% tartaric acid, can improve antibacterial metabolin bacteriocin
Activity.
Claims (6)
1. one plant of propionibacterium freudenreichii bacterial strain, which is characterized in that its Classification And Nomenclature is Propionibacterium
Freudenreichii CS1420, deposit number are CCTCC NO:M 2015050.
2. a kind of utilize Propionibacterium freudenreichii CS1420 fermenting and producings described in claim 1
The method of bacteriocin, which is characterized in that include the following steps：
1) seed culture：
Propionibacterium freudenreichii CS1420 single bacterium colonies are seeded in improvement PYG culture mediums and are activated,
30 DEG C of Anaerobic culturels 2.5 days；
2) fermented and cultured：
By above-mentioned steps 1) obtained seed liquor accessed according to 10% inoculum concentration in improvement PYG culture mediums, and 30 DEG C, Anaerobic culturel 8
My god, the initial pH of culture medium is 5.5-7.5.
3. Propionibacterium freudenreichii CS1420 fermented-producing bacterias according to claim 2
The method of element, which is characterized in that it further includes following steps after step 2)：
Zymotic fluid after step 2) fermented and cultured is centrifuged into 15min under conditions of 4 DEG C, 6000r/min, takes supernatant, is used
10%NaOH is adjusted to pH7.0, then is adjusted to pH5.1-5.9 with 10% tartaric acid, and 200uL is taken to carry out fungistatic effect measure.
4. method according to claim 2, which is characterized in that in the step 2), the improvement most suitable initial pH of PYG culture mediums is
6.0。
5. according to the method described in claim 2, it is characterized in that, the formula of improvement PYG culture mediums is in the step 2)：Junket
Peptone 5.0g, peptone 5.0g, yeast extract 10.0g, beef extract 5.0g, glucose 5.0g, K2HPO42.0g is spat
Temperature 80 1.0ml, resazurin 1.0ml, salting liquid 40.0ml, distilled water 950.0ml, hemin solution 10.0ml, vitamin
K1Solution 0.2ml, L-cysteine 0.5g, pH 7.2；The salting liquid formula is：CaCl2·2H2O 0.25g, MgSO4
7H2O 0.5g, K2HPO41.0g, KH2PO41.0g, NaHCO310.0g, NaCl 2.0g, distilled water 1.0L；The chlorination
Haemachrome solution：Hemin 50.0mg, 1N NaOH 1.0ml, distilled water 99.0ml；The vitamin K1Solution：Dimension
Raw element K10.1ml, 95% ethyl alcohol 20.0ml.
6. method according to claim 3, which is characterized in that the supernatant is first adjusted to pH7.0 with 10%NaOH, then uses
10% tartaric acid is adjusted to pH5.5.
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