CN101445820A - Method for preparing antibacterial metabolin of Propionibacterium and use thereof - Google Patents
Method for preparing antibacterial metabolin of Propionibacterium and use thereof Download PDFInfo
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- CN101445820A CN101445820A CNA2008102016780A CN200810201678A CN101445820A CN 101445820 A CN101445820 A CN 101445820A CN A2008102016780 A CNA2008102016780 A CN A2008102016780A CN 200810201678 A CN200810201678 A CN 200810201678A CN 101445820 A CN101445820 A CN 101445820A
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Abstract
The invention discloses a method for preparing antibacterial metabolin of Propionibacterium, the Propionibacterium (Propionibacterium.thoenii) is stored in American Type Culture Collection (ATCC) with the storing number of ATCC 4874. In the invention, the Propionibacterium is activated and fermented by microorganism fermentation technology to obtain the antibacterial metabolin containing the Propionibacterium. The antibacterial metabolin of Propionibacterium can be used in food anticorrosion and fresh-keeping, disinfector, anti-infection medicine, animal and plant disease control and the medicine field for inhibiting gram-negative bacteria, mould and microzyme. The method for preparing the antibacterial metabolin is convenient in operation, is low in production cost and has wide application prospect.
Description
Technical field
The present invention relates to the preparation method of the natural antibacterial metabolite that contains bacteriocin of Te Shi propionibacterium bacterial strain generation, and the antibiotic metabolite of above-mentioned special Propionibacterium is fresh-keeping at food antiseptic, sterilizing agent, anti-infectives, the purposes of aspects such as animals and plants disease control and medicine.
Background technology
Present modal food preservatives is Chemical Preservatives such as phenylformic acid, Sodium Benzoate, Sorbic Acid, potassium sorbate.But Chemical Preservative potential potential safety hazard more and more can not be ignored, and that biological preservative has an antibacterial substance is many, wide material sources, safety non-toxic, steady performance, in Food preservation, have huge application potential as novel natural antiseptic agent, be subjected to people's attention day by day.
Bacteriocin is that bacterium passes through a big proteinoid or the polypeptides matter that the rrna synthesis mechanism generates, and is found at first in the antagonistic action between the bacterium.(Nisin) is applied to the bacteriocin of food as first along with nisin, and people are more and more deep to the research of the bacteriocin that microbial metabolism produces, and bacteriocin is in food preservation, and aspects such as food fermentation and medical and health have demonstrated significance.Some bacteriocin lab can suppress spoilage organism and some pathogenic bacterias common in the food.Bacteriocin lab has metastable chemical property and strong bacteriostatic activity, can be widely used in the exploitation of natural food-preservative.Bacteriocin is as a kind of albumen, polypeptides matter, and bacteriostatic activity is strong, to heat, sour equistability is good, can be degraded by the enzyme effect, can be not residual in human body, and have higher security, wide in the Application in Food scope.Propionibacterium extensively is present in milk-product, soil, the silage.The Te Shi propionibacterium is that polymorphic, no gemma forms, shaft-like gram-positive microorganism, and it has been applied in the industrial fermentation of propionic acid and acetate from milk preparation, no pathogenicity.The bacteriocin of Cun Zaiing mainly contains following two kinds as the product of food preservatives in the market: one, the Nisin that is produced by milk-acid bacteria also is the most clearly bacteriocin of research at present.The cell bacteriolyze of lactococcin Nisin mediation is by changing the cell surface free energy, and then the cell permeability is changed finally cause lysis.Nisin has bacteriostatic action to gram-positive microorganism, comprises pathogenic bacteria such as listeria bacteria monocyte, also can suppress bacillus, clostridial excess growth.Nisin combines with metal chelator EDTA, tartrate, phosphoric acid salt can suppress more bacterium such as G-intestinal bacteria 0154.117., be used to preserve numerous food and comprise milk-product, cheese, canned vegetables and infant foods Europe listing several years as commodity Nisin.Be can be used for the cheese of pasteurization and the cheese of having processed by the approval of U.S. food and drug administration (FDA) in 1988, and prevented a large amount of breedings of clostridium spore and the excessive generation of clostridial toxin.Two, be the Bioprofit that obtains by Lactobaccilluus rhamnosus LC 705 and P.freudenreichii JS fermentation, it is reported that it can suppress yeast in the milk product and the genus bacillus in the acid bread, this inhibition effect is because the synergy between these two kinds of bacterium.
At present, Te Shi propionibacterium (Propionibacterium.thoenii) is preserved in US mode typical case thing and collects center (ATCC), deposit number ATCC 4874.The preparation method of the antimicrobial metabolite of special Propionibacterium and application thereof are not seen in report as yet.
Summary of the invention
Technical problem to be solved by this invention provides the antibiotic metabolite preparation of compositions of a kind of Te Shi propionibacterium (Propionibacterium.thoenii) ATCC 4874 bacteriocins method, and the antimicrobial metabolite of this special Propionibacterium is fresh-keeping at food antiseptic, sterilizing agent, anti-infectives, the application of aspects such as animals and plants disease control and medicine.
Technical solution of the present invention, a kind of preparation method of antibacterial metabolin of Propionibacterium comprises the following steps:
A. cultural characteristic
Te Shi propionibacterium bacterial strain (Propionibacterium.thoenii) is preserved in US mode typical case thing and collects center (ATCC), deposit number ATCC 4874, this bacterium is incubated in the SLB solid medium, form little and the projection bacterium colony. be cream-colored, yellow, orange or scarlet, 30～37 ℃ of culture temperature;
B. the activation of Te Shi propionibacterium
Behind the ampoul tube Kaifeng, use aseptic straw, draw 0.3～0.5ml appropriate liquid substratum, splash in the ampoul tube, vibration gently makes the dissolving of Te Shi propionibacterium thalline be suspension, get 0.1～0.2ml thallus suspension liquid, inject in the SLB liquid nutrient medium, 30 ℃ of static deep layers are cultivated 48h then, and culture promptly can be used as ferment-seeded;
C. the fermentation of Te Shi propionibacterium
Inoculum size 2～8% (v/v), 29.5 ℃～30.5 ℃ of leavening temperatures, the carbon source of fermentation is made up of any in glucose, lactose, sucrose, fructose or the maltose or more than one, the carbon source consumption is 2～4% (w/v) of substratum, nitrogenous source is made up of any in Tryptones, yeast powder, corn steep liquor, yeast extract, the Sodium.alpha.-hydroxypropionate or more than one, the nitrogenous source consumption is 2～3% (w/v) of substratum, and inorganic salt are by K
2HPO
4, KH
2PO
4, MnSO
4In any or more than one compositions, the inorganic salt consumption is 0.05～0.30% (w/v) of substratum, and agar consumption is 0.5～3% (w/v) of substratum, and all the other are water, closing anaerobic fermented 4～6 days, can obtain the antibiotic metabolite of described special Propionibacterium.
The preparation method of described antibacterial metabolin of Propionibacterium, the fermented liquid of the antibiotic metabolite that contains described special Propionibacterium that its step c obtains is further rotated evaporation or ultrafiltration, make volume be concentrated to original 1/3～1/6, on freeze drier, carry out lyophilize then, the pre-freeze temperature is-45 ℃, drying temperature is made as-20 ℃, dry 12-24h is till moisture distils fully, obtain the dry powder of the antibiotic metabolite of described special Propionibacterium, 4 ℃ of sealings are preserved.
The antibiotic metabolite of described special Propionibacterium is fresh-keeping at food antiseptic, sterilizing agent, and anti-infectives is used to suppress Gram-negative bacteria in animals and plants disease control and the field of medicaments, mould and saccharomycetic application.
The beneficial effect of the invention, the present invention obtains the antibiotic metabolite of special Propionibacterium by microbial fermentation technology, bacteriocin wherein and propionic acid, acetate, other compositions such as lactic acid act synergistically mutually, the antimicrobial spectrum of this antibiotic metabolite is different from the microorganism that general bacteriocin only suppresses its nearly source, antimicrobial spectrum is wide, and it is fresh-keeping to be used for food antiseptic, sterilizing agent, anti-infectives, animals and plants disease control and medicine and other fields suppress Gram-negative bacteria, mould and saccharomycetic growth.The present invention is easy to operate, and production cost is low, has a extensive future.
Embodiment
Below by embodiment the present invention is described in further detail, a kind of preparation method of antibacterial metabolin of Propionibacterium comprises the following steps:
A. cultural characteristic
Te Shi propionibacterium bacterial strain (Propionibacterium.thoenii) is preserved in US mode typical case thing and collects center (ATCC), deposit number ATCC 4874, this bacterium is incubated in the SLB solid medium, form little and the projection bacterium colony. be cream-colored, yellow, orange or scarlet, 30～37 ℃ of culture temperature;
B. the activation of Te Shi propionibacterium
Behind the ampoul tube Kaifeng, use aseptic straw, draw 0.3～0.5ml appropriate liquid substratum, splash in the ampoul tube, vibration gently makes the dissolving of Te Shi propionibacterium thalline be suspension, get 0.1～0.2ml thallus suspension liquid, inject in the SLB liquid nutrient medium, 30 ℃ of static deep layers are cultivated 48h then, and culture promptly can be used as ferment-seeded;
C. the fermentation of Te Shi propionibacterium
Inoculum size 2～8% (v/v), 29.5 ℃～30.5 ℃ of leavening temperatures, the carbon source of fermentation is made up of any in glucose, lactose, sucrose, fructose or the maltose or more than one, the carbon source consumption is 2～4% (w/v) of substratum, nitrogenous source is made up of any in Tryptones, yeast powder, corn steep liquor, yeast extract, the Sodium.alpha.-hydroxypropionate or more than one, the nitrogenous source consumption is 2～3% (w/v) of substratum, and inorganic salt are by K
2HPO
4, KH
2PO
4, MnSO
4In any or more than one compositions, the inorganic salt consumption is 0.05～0.30% (w/v) of substratum, and agar consumption is 0.5～3% (w/v) of substratum, and all the other are water, closing anaerobic fermented 4～6 days, can obtain the antibiotic metabolite of described special Propionibacterium.
If be used for the cultivation of 10 liters of fermentor tanks, same 2～8% (w/v) inoculum size of pressing inserts activatory Te Shi propionibacterium in the fermentation culture, the composition of fermentation culture is the same, setting fermentation jar temperature is 29.5 ℃～30.5 ℃, the airtight tank pressure that makes remains between 0.04～0.05MPa after regulating air flow, cultivates the antibiotic metabolite that obtains described special Propionibacterium after 4～6 days.
The fermented liquid of the antibiotic metabolite that contains described special Propionibacterium that preparation method's step c of described antibacterial metabolin of Propionibacterium obtains can be further rotated evaporation or ultrafiltration, make volume be concentrated to original 1/3～1/6, on freeze drier, carry out lyophilize then, the pre-freeze temperature is-45 ℃, drying temperature is made as-20 ℃, dry 12～24h is till moisture distils fully, obtain the dry powder of the antibiotic metabolite of described special Propionibacterium, 4 ℃ of sealings are preserved.
The antibiotic metabolite of described special Propionibacterium is fresh-keeping at food antiseptic, sterilizing agent, and anti-infectives is used to suppress Gram-negative bacteria in animals and plants disease control and the field of medicaments, mould and saccharomycetic application.
Embodiment 1
Seed culture medium: Tryptones 10g, yeast extract 10g, 60% Sodium.alpha.-hydroxypropionate 16.7ml, K
2HPO
40.25g, MnSO
40.005g adding distil water is to 1000ml, pH7.0
Fermention medium: Tryptones 10g, yeast extract 10g, glucose 20g, K
2HPO
40.25g, MnSO
40.005g agar 20g, adding distil water be to 1000ml, pH7.0
With preserving number is that the Te Shi propionibacterium of ATCC4874 is inoculated in the seed culture medium 30 ℃ and cultivates 48h and obtain seed liquor, 4% inoculum size of seed liquor according to fermentation volume inserted in the cooled fermention medium, cultivate the antimicrobial metabolite fermented liquid that obtains containing bacteriocin behind the 5d for 30 ℃.Fermented liquid with obtaining is rotated evaporation, and it is original 1/5 that volume-diminished is arrived, and carries out lyophilize on the postlyophilization machine then, till distilling fully to moisture, obtain the dry powder of fermented liquid after 4 ℃ of sealings preserve.
The antibiotic metabolite that embodiment 1 is obtained special Propionibacterium carries out the mensuration of antimicrobial spectrum.Adopt agar diffusion method respectively with intestinal bacteria, Pseudomonas aeruginosa, streptococcus aureus, Bacillus subtilus, rhodothece rubra, candiyeast etc. are measured the antimicrobial spectrum of this propionibacterium metabolite as indicator, and its result is as follows:
The antibiotic metabolite antimicrobial spectrum test-results of the special Propionibacterium of table 1
" ++ " expression bacteriostatic activity is stronger, and "+" expression has bacteriostatic activity, the no bacteriostatic activity of "-" expression.
The antibiotic metabolite of the special Propionibacterium of table 2 is to the inhibition test result of mould spores
Table 2 data show that the propionibacterium metabolite has shown very strong restraining effect to mould and aspergillus niger.The spore of blank is sprouted fully in the 8h, and has added not sprouting fully of propionibacterium metabolite; Added the mould and the aspergillus niger of propionibacterium metabolite behind the 12h, its germination rate is the highest also to have only 13.0% and 4.6% respectively, with blank extremely significant difference (P＜0.01) is arranged.
Te Shi propionibacterium derived metabolite of the present invention can suppress the part Gram-negative bacteria, and part yeast and mould can not suppress gram-positive microorganism.
Embodiment 2
Seed culture medium: Tryptones 10g, yeast powder 10g, 60% Sodium.alpha.-hydroxypropionate 16.7ml, K
2HPO
40.25g, MnSO
40.005g adding distil water is to 1000ml, pH7.0
Fermention medium: Tryptones 10g, yeast extract 10g, lactose 20g, K
2HPO
40.25g, MnSO
40.005g agar 20g, adding distil water be to 1000ml, pH7.0
With preserving number is that the Te Shi propionibacterium of ATCC 4874 is inoculated in the seed culture medium 30 ℃ and cultivates 48h and obtain seed liquor, 4% inoculum size of seed liquor according to fermentation volume inserted in the cooled fermention medium, cultivate the antimicrobial metabolite that obtains containing bacteriocin behind the 5d for 30 ℃.Fermented liquid with obtaining is rotated evaporation, and it is original 1/5 that volume-diminished is arrived, and carries out lyophilize on the postlyophilization machine then, till distilling fully to moisture, obtain the dry powder of fermented liquid after 4 ℃ of sealings preserve.
Embodiment 3
Seed culture medium: Tryptones 10g, yeast powder 10g, 60% Sodium.alpha.-hydroxypropionate 16.7ml, K
2HPO
40.25g, MnSO
40.005g adding distil water is to 1000ml, pH7.0
Fermention medium: corn steep liquor 10g, yeast extract 10g, sucrose 30g, K
2HPO
40.25g, MnSO
40.005g agar 20g, adding distil water be to 1000ml, pH7.0
With preserving number is that the Te Shi propionibacterium of ATCC 4874 is inoculated in the seed culture medium 30 ℃ and cultivates 48h and obtain seed liquor, 5% inoculum size of seed liquor according to fermentation volume inserted in the cooled fermention medium, cultivate the antimicrobial metabolite that obtains containing bacteriocin behind the 6d for 30 ℃.Fermented liquid with obtaining is rotated evaporation, and it is original 1/6 that volume-diminished is arrived, and carries out lyophilize on the postlyophilization machine then, till distilling fully to moisture, obtain the dry powder of fermented liquid after 4 ℃ of sealings preserve.
Embodiment 4
Seed culture medium: Tryptones 10g, yeast powder 10g, 60% Sodium.alpha.-hydroxypropionate 16.7ml, K
2HPO
40.25g, MnSO
40.005g adding distil water is to 1000ml, pH7.0
Fermention medium: corn steep liquor 10g, yeast extract 15g, lactose 35g, K
2HPO
40.25g, MnSO
40.005g agar 20g, adding distil water be to 1000ml, pH7.0
With preserving number is that the Te Shi propionibacterium of ATCC 4874 is inoculated in the seed culture medium 30 ℃ and cultivates 48h and obtain seed liquor, 5% inoculum size of seed liquor according to fermentation volume inserted in the cooled fermention medium, cultivate the antimicrobial metabolite that obtains containing bacteriocin behind the 6d for 30 ℃.Fermented liquid with obtaining is rotated evaporation, and it is original 1/6 that volume-diminished is arrived, and carries out lyophilize on the postlyophilization machine then, till distilling fully to moisture, obtain the dry powder of fermented liquid after 4 ℃ of sealings preserve.
Embodiment 5
Seed culture medium: Tryptones 10g, yeast powder 10g, 60% Sodium.alpha.-hydroxypropionate 16.7ml, K
2HPO
40.25g, MnSO
40.005g adding distil water is to 1000ml, pH7.0
Fermention medium: Tryptones 5g, yeast extract 15g, lactose 30g, K
2HPO
40.25g, MnSO
40.005g agar 20g, adding distil water be to 1000ml, pH7.0
With preserving number is that the Te Shi propionibacterium of ATCC 4874 is inoculated in the seed culture medium 30 ℃ and cultivates 48h and obtain seed liquor, seed liquor is inserted according to 5% inoculum size of fermentation volume in the 6500ml fermention medium of 10L fermentor tank, cultivate the antimicrobial metabolite that obtains containing bacteriocin behind the 4d for 30 ℃.Fermented liquid with obtaining is rotated evaporation, and it is original 1/3 that volume-diminished is arrived, and carries out lyophilize on the postlyophilization machine then, till distilling fully to moisture, obtain the dry powder of fermented liquid after 4 ℃ of sealings preserve.
Described content only is the basic explanation of the present invention under conceiving, and according to any equivalent transformation that technical scheme of the present invention is done, all should belong to protection scope of the present invention.
Claims (3)
1. the preparation method of an antibacterial metabolin of Propionibacterium comprises the following steps:
A. cultural characteristic
Te Shi propionibacterium bacterial strain (Propionibacterium.thoenii) is preserved in US mode typical case thing and collects center (ATCC), deposit number ATCC4874, this bacterium is incubated in the SLB solid medium, form little and the projection bacterium colony. be cream-colored, yellow, orange or scarlet, 30～37 ℃ of culture temperature;
B. the activation of Te Shi propionibacterium
Behind the ampoul tube Kaifeng, use aseptic straw, draw 0.3～0.5ml appropriate liquid substratum, splash in the ampoul tube, vibration gently makes the dissolving of Te Shi propionibacterium thalline be suspension, get 0.1～0.2ml thallus suspension liquid, inject in the SLB liquid nutrient medium, 30 ℃ of static deep layers are cultivated 48h then, and culture promptly can be used as ferment-seeded;
C. the fermentation of Te Shi propionibacterium
Inoculum size 2～8% (v/v), 29.5 ℃～30.5 ℃ of leavening temperatures, the carbon source of fermentation is made up of any in glucose, lactose, sucrose, fructose or the maltose or more than one, the carbon source consumption is 2～4% (w/v) of substratum, nitrogenous source is made up of any in Tryptones, yeast powder, corn steep liquor, yeast extract, the Sodium.alpha.-hydroxypropionate or more than one, the nitrogenous source consumption is 2～3% (w/v) of substratum, and inorganic salt are by K
2HPO
4, KH
2PO
4, MnSO
4In any or more than one compositions, the inorganic salt consumption is 0.05～0.30% (w/v) of substratum, and agar consumption is 0.5～3% (w/v) of substratum, and all the other are water, closing anaerobic fermented 4～6 days, can obtain the antibiotic metabolite of described special Propionibacterium.
2. the preparation method of the described antibacterial metabolin of Propionibacterium of claim 1, it is characterized in that: the fermented liquid of the antibiotic metabolite that contains described special Propionibacterium that step c obtains is further rotated evaporation or ultrafiltration, make volume be concentrated to original 1/3～1/6, on freeze drier, carry out lyophilize then, the pre-freeze temperature is-45 ℃, drying temperature is made as-20 ℃, dry 12-24h is till moisture distils fully, obtain the dry powder of the antibiotic metabolite of described special Propionibacterium, 4 ℃ of sealings are preserved.
3. the antibiotic metabolite of the special Propionibacterium of claim 1 or 2 preparations is fresh-keeping at food antiseptic, sterilizing agent, and anti-infectives is used to suppress Gram-negative bacteria in animals and plants disease control and the field of medicaments, mould and saccharomycetic application.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154169A (en) * | 2011-01-11 | 2011-08-17 | 常熟理工学院 | Propionibacterium strain and method for producing antibiotic metabolin by virtue of fermentation of same |
| CN102172295A (en) * | 2011-03-02 | 2011-09-07 | 华东师范大学 | Biological preservative as well as preparation method and application thereof |
| CN106688869A (en) * | 2016-12-28 | 2017-05-24 | 江苏省海洋水产研究所 | Method for preventing and treating yellow leaf blotch of shell conchocelis of porphyra yezoensis and culture apparatus |
| CN107988288A (en) * | 2017-12-11 | 2018-05-04 | 常熟理工学院 | A kind of method of high density fermentation production Propionibacterium bacteriocin |
| CN109402185A (en) * | 2018-11-23 | 2019-03-01 | 浙江华康药业股份有限公司 | A method of bacteriocin and propionic acid are produced based on xylose mother liquid raffinate |
| CN112335810A (en) * | 2020-10-20 | 2021-02-09 | 安徽绿微康生物科技有限公司 | Edible antioxidant and preparation method and application thereof |
| CN112359067A (en) * | 2020-10-19 | 2021-02-12 | 安徽绿微康生物科技有限公司 | Fermentation antioxidant and preparation method and application thereof |
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2008
- 2008-10-24 CN CNA2008102016780A patent/CN101445820A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154169A (en) * | 2011-01-11 | 2011-08-17 | 常熟理工学院 | Propionibacterium strain and method for producing antibiotic metabolin by virtue of fermentation of same |
| CN102154169B (en) * | 2011-01-11 | 2012-07-04 | 常熟理工学院 | Propionibacterium strain and method for producing antibiotic metabolin by virtue of fermentation of same |
| CN102172295A (en) * | 2011-03-02 | 2011-09-07 | 华东师范大学 | Biological preservative as well as preparation method and application thereof |
| CN106688869A (en) * | 2016-12-28 | 2017-05-24 | 江苏省海洋水产研究所 | Method for preventing and treating yellow leaf blotch of shell conchocelis of porphyra yezoensis and culture apparatus |
| CN107988288A (en) * | 2017-12-11 | 2018-05-04 | 常熟理工学院 | A kind of method of high density fermentation production Propionibacterium bacteriocin |
| CN107988288B (en) * | 2017-12-11 | 2020-05-19 | 常熟理工学院 | Method for producing propionibacterium bacteriocin through high-density fermentation |
| CN109402185A (en) * | 2018-11-23 | 2019-03-01 | 浙江华康药业股份有限公司 | A method of bacteriocin and propionic acid are produced based on xylose mother liquid raffinate |
| CN112359067A (en) * | 2020-10-19 | 2021-02-12 | 安徽绿微康生物科技有限公司 | Fermentation antioxidant and preparation method and application thereof |
| CN112335810A (en) * | 2020-10-20 | 2021-02-09 | 安徽绿微康生物科技有限公司 | Edible antioxidant and preparation method and application thereof |
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